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https://openalex.org/W2053729827
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https://zenodo.org/records/1848899/files/article.pdf
|
English
| null |
<i>Contributions towards a revision of the genus</i> Lomanotus: <i>a postscript</i>
|
Annals & magazine of natural history
| 1,908
|
public-domain
| 731
|
Contributions towards
a revision of the genus
Lomanotus: a postscript
N. Colgan
Published online: 08 Sep 2009. Contributions towards
a revision of the genus
Lomanotus: a postscript
N. Colgan
Published online: 08 Sep 2009. To cite this article: N. Colgan (1908) Contributions towards a revision of
the genus Lomanotus: a postscript , Annals and Magazine of Natural History:
Series 8, 2:10, 392-392, DOI: 10.1080/00222930808692500
To link to this article: http://dx.doi.org/10.1080/00222930808692500 To cite this article: N. Colgan (1908) Contributions towards a revision of
the genus Lomanotus: a postscript , Annals and Magazine of Natural History:
Series 8, 2:10, 392-392, DOI: 10.1080/00222930808692500
To link to this article: http://dx.doi.org/10.1080/00222930808692500 To cite this article: N. Colgan (1908) Contributions towards a revision of
the genus Lomanotus: a postscript , Annals and Magazine of Natural History:
Series 8, 2:10, 392-392, DOI: 10.1080/00222930808692500 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of
all the information (the “Content”) contained in the publications
on our platform. However, Taylor & Francis, our agents, and our
licensors make no representations or warranties whatsoever as to the
accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions
and views of the authors, and are not the views of or endorsed by
Taylor & Francis. The accuracy of the Content should not be relied
upon and should be independently verified with primary sources of
information. Taylor and Francis shall not be liable for any losses,
actions, claims, proceedings, demands, costs, expenses, damages,
and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use
of the Content. This article may be used for research, teaching, and private study
purposes. Any substantial or systematic reproduction, redistribution,
reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access
and use can be found at http://www.tandfonline.com/page/terms-
and-conditions 392 Miscellaneous. P. notOera , St£1 (Brazil), is only known to me by the
description ; it is probably a true Pygiclicrana. P. caffra, Dohrn, and P. dcernell, Dohrn, are only known to
me by their description and by drawings; the position
which I have allotted them by analogy may well be
correct. P. aSnorm{s, Borm., is the ~ype of Toraopygla, Burr (1904)o t 9. biittneri, Karsch, is the type of KarseMella~ Verhoeff. I~ISCELLANEOUS. Contributions towards a t~evision of the Genus Lomanotus :
a Postscri2t. (1) L. marmoratug, Ald.& Hanc. (1845).
Z. genei, V6rany (1846).
L. hancocki, Norman (1877). Contributions towards a t~evision of the Genus Lomanotus :
a Postscri2t. I REO~ET to find that the survey of the literature of the genus
Lomanotus given in the paper which appeared under the above title
in the August issue of these ' Annals ' is incomplete, in so far as it
includes no reference to Sir C. ]~liot's valuable "Notes on some
British Nudibranehs," contributed to vol. iii. of the ' Journal of the
Marine Biological Association' in 1906. Unfortunately the exist-
ence of these "Notes" did not come to my knowledge until some
three weeks after the appearance of the August issue of the
'Annals.' Having read the section of the "Notes " dealing with
Zomanotus (pp, 348-353) I find it necessary to alter my views as to
the position of L. portlandicus. Hancock's unpublished drawings
show that this species possesses what appears to be the most
important specific character of Trinchese's L. eisigii, a fin-like caudal
process, so that the two species may be considered as identical. While still retaining two species in the genus, I desire, then, to alter
the arrangement proposed in the August number of the ' Annals '
(pp. 217-218) to the following, L. portlandis~s (1860) taking
precedence of L. eisigii (1883) :-- (1) L. marmoratug, Ald.& Hanc. (1845). Z. genei, V6rany (1846). L. hancocki, Norman (1877). (2) L. Tortlandicus, Thompson (1860},
L. eis~Tii, Trinchese (1883). (1) L. marmoratug, Ald.& Hanc. (1845). Z. genei, V6rany (1846). L. hancocki, Norman (1877). (2) L. Tortlandicus, Thompson (1860},
L. eis~Tii, Trinchese (1883). (2) L. Tortlandicus, Thompson (1860},
L. eis~Tii, Trinchese (1883). 7Whether this provisional arrangement is to stand will depend on
the value that may be conceded as a specific distinction to the
fin-like caudal process of the second species as described by
Trinehese and figured by ttancock. N. COL~A~,
|
W3210330079.txt
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https://www.mdpi.com/2076-3417/10/18/6525/pdf?version=1600428041
|
en
|
Fecal Indicator Bacteria Transport from Watersheds with Differing Wastewater Technologies and Septic System Densities
|
Applied sciences
| 2,020
|
cc-by
| 7,739
|
applied
sciences
Article
Fecal Indicator Bacteria Transport from Watersheds
with Differing Wastewater Technologies and Septic
System Densities
Guy Iverson 1, *, Christa Sanderford 1,2 , Charles P. Humphrey, Jr. 1 , J. Randall Etheridge 3
and Timothy Kelley 1
1
2
3
*
Environmental Health Sciences Program, Department Health Education & Promotion,
East Carolina University, Carol Belk Building, Mail Stop 529, Greenville, NC 27858, USA;
christa.sanderford@ncdenr.gov (C.S.); humphreyc@ecu.edu (C.P.H.J.); kelleyt@ecu.edu (T.K.)
Shellfish Sanitation Program, Division of Marine Fisheries, NC Department Environmental Quality, 3441
Arendell St., Morehead City, NC 28557, USA
Department of Engineering, Center for Sustainable Energy and Environmental Engineering, East Carolina
University, Rivers Building, Greenville, NC 27858, USA; ETHERIDGEJ15@ECU.EDU
Correspondence: iversong18@ecu.edu
Received: 22 August 2020; Accepted: 15 September 2020; Published: 18 September 2020
Abstract: Wastewater contains elevated concentrations of fecal indicator bacteria (FIB). The type of
wastewater treatment technology and septic system density may influence the FIB concentration
and exports at the watershed scale. The goal of this study was to gain a better understanding of FIB
concentrations and exports from watersheds served by conventional septic (CS) systems, sand filter
(SF) septic systems, and a municipal sewer (SEW) system. Seven watersheds (3 CS, 3 SF, and 1 SEW)
were monitored to quantify FIB concentration and export monthly from April 2015 to March 2016.
The type of wastewater treatment did not yield significant differences in FIB concentration or exports
when pooling watersheds using similar wastewater treatment. Watersheds with the highest septic
densities (approximately 0.4 systems ha−1 ) contained greater FIB concentrations and exports than
watersheds with the lowest (approximately 0.1–0.2 systems ha−1 ), but only FIB concentrations
significantly differed. These findings suggest that when the septic system density exceeds 0.4 systems
ha−1 , water quality degradation from septic leachate may be observable at the watershed scale,
especially in watersheds dominated by residential development. More research is recommended to
determine if this density threshold is similar for other water pollutants and/or in watersheds with
differing hydrogeological, land use, and wastewater characteristics.
Keywords: bacteria; transport; wastewater; sand filter; septic; fecal contamination
1. Introduction
Elevated concentrations of pathogens in water resources are one of the most commonly cited
reasons for water use impairment [1]. Fecal waste from humans and animals may contain various
types of pathogens, including viruses, protozoa, and bacteria, that can cause infections, illnesses,
or death if exposure via consumption, inhalation, or skin contact occurs [2]. Testing for each of
these pathogens in surface water or groundwater is time consuming and expensive. Instead, fecal
indicator bacteria (FIB) are often used to assess risk associated with exposure to contaminated
water, because elevated concentrations of FIB may indicate the presence of harmful pathogens [3,4].
The United States Environmental Protection Agency (US EPA) [5] recommends that geometric mean
values of Escherichia coli (E. coli) and Enterococcus spp. (henceforth called enterococcus/enterococci)
should not exceed 126 and 35 most probable number (MPN) 100 mL−1 , respectively, in recreational
Appl. Sci. 2020, 10, 6525; doi:10.3390/app10186525
www.mdpi.com/journal/applsci
Appl. Sci. 2020, 10, 6525
2 of 14
water. Furthermore, no more than 10% of samples should exceed statistical threshold values (STVs) of
410 and 130 MPN 100 mL−1 for E. coli and enterococci, respectively. Prior studies have shown that
FIB in surface waters can be linked to waste material from a variety of sources, including wildlife [6],
pets [7], livestock [8], and improperly treated domestic wastewater from cities and rural areas. In North
Carolina, regulations have been enacted to reduce non-point source pollution via implementation
of agricultural best management practices, stormwater control measures, and improvements in
centralized wastewater infrastructure [9]. Efforts to improve water quality via improvement of septic
system treatment efficiency were not initiated, partly due to a lack of information regarding their
watershed contributions.
Septic systems are commonly used in rural or suburban communities where it may not be
economically feasible to extend municipal sewer lines. In the United States, approximately 25% of
Americans rely on septic systems for their primary means of wastewater treatment and 30% of new
construction uses septic systems [10,11]. Previous studies have found greater concentrations of FIB in
waters downgradient from septic systems relative to water downgradient of municipal sewers [12–16].
Water quality and/or aquatic habitat degradation from septic-derived FIB may be exacerbated during
periods of septic system malfunction, which release partially treated or untreated wastewater directly
to groundwater or surface water [17]. Most of these studies have focused on conventional septic (CS)
systems that collect, treat, and discharge wastewater effluent to subsoils beneath drainfield trenches.
Alternative septic technologies exist that are generally used at sites that fail to meet the minimum soil
requirements for CS outlined in state regulations. Sand filter (SF) systems are one example of commonly
used alternative septic systems. SFs have a septic tank and effluent from the tank is piped to the SF
where aerobic treatment occurs, and the SF effluent is discharged directly to drainageways or streams.
Other research showed SF effluent contained FIB concentrations of up to 5000 MPN 100 mL−1 [18,19].
These studies suggest that wastewater treatment efficiency and water quality adjacent to septic systems
may be influenced by system type, but more information is needed to determine if differences in water
quality are observed at larger spatial scales.
Municipal sewer (SEW) systems may also act as a significant source of FIB to surface waters.
Elevated FIB concentrations were found in a stream that drains a watershed dominated by urban
lands compared to a stream that drains forested lands [20]. Increased FIB loads to the environment
from SEW systems can occur from excessive inflow and infiltration, which can result in poor system
performance and possibly overflow [21]. Additionally, leakage from wastewater pipes can discharge FIB
to groundwaters and/or surface waters [22]. During times of overflow or spills, the FIB concentration
in and bacterial loadings to surface water can be substantially greater than recreational water quality
standards, thus posing significant environmental and public health risks [23]. More research is needed
to quantify and compare FIB concentration and exports from watersheds that predominantly utilize SF
to watersheds that predominantly use CS and SEW.
The goal of this study was to compare the FIB concentration and exports from watersheds using
various wastewater technologies. The specific objectives were to: (i) Quantify the septic system density
within watersheds using CS and SF systems and determine at what threshold densities of systems
influence watershed-scale FIB concentrations and exports; and (ii) compare FIB concentrations and
exports from watersheds using CS, SF, and an SEW to elucidate spatiotemporal trends related to
the wastewater treatment approach. Previous research has suggested that when the septic system
density exceeds 1 system ha−1 , septic leachate can substantially influence FIB concentrations and/or
yields [12,14,16]; however, in watersheds with mixed land uses (e.g., forested and agriculture), septic
system density differences may become masked by wildlife and livestock signatures [24]. The current
study analyzed FIB concentrations and loads to determine if differences existed between watersheds
with septic system densities of <1 system ha−1 and based on wastewater treatment approach (CS, SF,
and SEW). Furthermore, few studies have included FIB transport data from SF type systems at the
watershed scale.
Appl. Sci. 2020, 10, 6525
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2. Materials and Methods
2.1. Study Area and Site Selection
The study area consisted of 7 watersheds located in Durham County that drain to Falls Lake
in the Piedmont geologic region of North Carolina (Figure 1). Each watershed was grouped and
classified according to the dominant wastewater treatment approach (Table 1). Watersheds served
by septic systems are numbered in order of increasing septic system density: 1 being the lowest
(approximately 0.2 systems ha−1 ) and 3 being the highest (approximately 0.4 systems ha−1 ). Watershed
boundaries were delineated using StreamStats 4.0, which is a web-based tool developed by the United
States Geological Survey (https://streamstats.usgs.gov/ss/), and land cover data were also complied
from StreamStats for each watershed (Table 1). The CS1, CS3, and SEW watersheds are tributaries
or segments of Lick Creek; the CS2 watershed drains the headwaters of Laurel Creek; and the SF
watersheds discharge to Little Lick Creek. Laurel Creek is considered a tributary of Lick Creek for
watershed management purposes. Each of these watersheds were analyzed independently based on
septic system densities (Table 1). Most of the septic systems in the studied watersheds were originally
permitted between the 1950s and 1970s based on public record via City of Durham’s interactive Go
Maps (https://maps.roktech.net/durhamnc_gomaps4/).
Appl. Sci. 2020, 10, x FOR PEER REVIEW
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Figure
1. Watershed
delineation map
for the 3 conventional
septicthe
(CS),33 conventional
sand filter (SF), and sewered
watershed.
LCfilter
= Lick Creek;
= Little
Lick Creek;
Figure
1. Watershed
delineation
map for
septic(SEW)
(CS),
3 sand
(SF),LLC
and
sewered
and LaC = Laurel Creek.
(SEW) watershed. LC = Lick Creek; LLC = Little Lick Creek; and LaC = Laurel Creek.
Falls Lake serves as the main water supply for the City of Raleigh and provides primary and
secondary water-based recreational activities for the region. Raleigh serves as the county seat for Wake
County, which ranks among the top 50 counties in the United States for population growth and it
gained approximately 65 new residents each day from 2010 to 2017 [25]. Sustainable management of
water resources is vital for continued growth and economic development in the region. Falls Lake
and many of its tributaries, including Lick Creek and Little Lick Creek, are designated as impaired
waters [26]. Lick Creek and Little Lick Creek have watershed restoration plans designed to improve
water quality within their respective watersheds. These plans suggested that failing septic systems
and surface discharging systems (i.e., SF-type systems) could be significant sources of wastewater
constituents (e.g., nutrients, bacteria, oxygen-demanding substances) and contributors to aquatic
habitat degradation in Lick Creek and Little Lick Creek [27,28]. The technology used for wastewater
treatment (e.g., SF, CS, SEW) varied in different sub-watersheds of Falls Lake. Some areas were served
mostly by CS, some mostly by SF, and others by SEW. Water quality monitoring data were not available
for many of the sub-watersheds served by these different technologies, so more information was needed
to determine if the wastewater treatment approach and/or septic system density affected water quality.
Appl. Sci. 2020, 10, 6525
4 of 14
Table 1. Watershed characteristics, septic system density, and proximity of septic systems to watershed outlets. CS = conventional septic; SF = sand filter; SEW= sewer.
Watershed
Stream Gradient
Imperviousness 1
(%)
Area
(ha)
Septic Systems
(#)
Septic System Density
(# ha−1 )
CS1
CS2
CS3
SF1
SF2
SF3
SEW
0.002
0.005
0.008
0.003
0.007
0.005
0.002
4
1
13
9
6
7
7
2283
184
19
335
40
83
1128
280
48
7
75
15
35
NA
0.12
0.26
0.37
0.22
0.37
0.42
NA
1
Septic System Distance to Outlet (Number of Systems)
<0.2 km
<1 km
>1 km
1
0
5
0
7
14
NA
2
3
2
20
8
21
NA
278
45
0
55
0
0
NA
= Percentages collected from StreamStats 4 based on 2011 National Land Cover Database data.
Forest
60.3%
74.6%
25.7%
45.2%
31.2%
44.7%
53.0%
Land Cover Data 1
Agriculture
Developed
7.5%
7.7%
4.5%
0.5%
0.0%
0.0%
8.0%
16.3%
7.7%
63.3%
48.9%
53.7%
52.5%
26.5%
Appl. Sci. 2020, 10, 6525
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Lick Creek drains an area of 5690 hectare (ha) comprised mostly of unmanaged rural lands (37%),
forest (21%), and protected natural area (10%). The Lick Creek watershed is currently undergoing a
transitionary period shifting from rural management and eventually to suburban/urban land uses [27].
The Little Lick Creek watershed is slightly smaller, draining an area of 4450 ha consisting mostly
of mixed density residential (49%), vacant (18%), and parks and open space (15%). Nearly half
(47%) of the watershed lies within the city limits of Durham, NC. High-density residential, industrial,
and low-density residential land uses in the watershed are expected to increase by 3, 3, and 2 times,
respectively. Meanwhile, agriculture, vacant, and parks and open spaces are expected to see declines
of up to 100%, 100%, and 22%, respectively, over the next 20 years within the Little Lick Creek
watershed [28]. Both the Lick and Little Lick Creek Watersheds are expected to see growth, which may
result in permitting of additional septic systems, thus increasing system densities in these watersheds.
The study area generally receives 122 cm of precipitation per year (https://www.usclimatedata.com/
climate/durham/north-carolina/united-states/usnc0192). Mean monthly high temperatures in warmer
months (June–September) routinely exceed 27 ◦ C. During the winter months (December–March),
mean monthly high temperatures peak at approximately 16 ◦ C and mean monthly low temperatures
of approximately −2 ◦ C. The geology of the study area is generally characterized by Triassic basin
clays and in some areas Carolina Slate Belt and felsic crystalline rock. The White Store soil series
was the dominant soil unit for subsoils of the studied sub-watersheds of Little Lick Creek (>80%),
Lick Creek (>60%), and Laurel Creek (>30%) [29].
2.2. Sampling Protocol
Water quality assessment occurred at each of the 7 watershed outlets (Figure 1). Physicochemical
parameters, including specific conductance, temperature, dissolved oxygen, and oxidation reduction
potential, were measured in the field using a YSI-556 MultiProbe meter. A Hach turbidimeter was used
to measure turbidity. Stream discharge was determined by multiplying the cross-sectional area of a
transect by the mean velocity. Stream velocity was measured using a Global Water FP101 flow meter or
the floating object method. The floating object method was used in scenarios when stream velocity was
too low or the water not deep enough to engage the flow meter propeller [30]. Water samples were
collected from watershed outlets monthly beginning in April 2015 until March 2016 (n = 84, 12 samples
per watershed). Sterile 100-mL bottles were immersed in streams until bottles reached 100 mL. Samples
were stored in an iced cooler and transported to East Carolina University where they were analyzed
for FIB concentrations within 6 hours of sampling. Additionally, 250-mL bottles were used to collect
water samples for chloride analyses. These samples were also stored on an iced cooler until they were
vacuum filtered and analyzed using a Unity SmartChem 200 autoanalyzer.
E. coli and enterococci concentrations in streams were quantified using QuantiTray2000® trays via
the IDEXX™ method (IDEXX Laboratories, Inc., Westbrook, Maine). Some samples were diluted to
allow for enumeration of FIB beyond maximum concentrations of 2419.6 MPN 100 mL−1 . Dilution
factors generally ranged from 2 to 10. FIB concentrations were determined based on the MPN 100 mL−1
associated with the number of fluorescent wells using a chart provided by IDEXX.
2.3. Statistical Analysis
The exports of FIB were estimated for each watershed by multiplying the FIB concentration
(MPN 100 mL−1 ) by stream discharge (L s−1 ). Normalization by watershed area was performed to
account for variability in discharge that may be related to differences in watershed size. Thus, stream
discharge and FIB export reported in the current study was area normalized; henceforth, “normalized
exports” and “normalized discharge” will be referred to as “exports” and “discharge”, respectively.
Concentrations and exports of FIB were compiled into comparison groups based on septic system
density and wastewater treatment approach. FIB concentrations may vary seasonally due to changes
in temperature [15,24,31–33], thus watersheds were grouped by summer (June–September) and winter
(December–March) seasons (n = 56, 8 samples per watershed) to compare data based on the largest
Appl. Sci. 2020, 10, 6525
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differences in temperatures, which may affect FIB survivability. Summer and winter months are
henceforth referred to as “warm” and “cold”, respectively. Data figures were developed using the
“ggplot2” and “cowplot” packages developed for R software [34].
A Shapiro–Wilks normality test was employed to determine if data exhibited normality. Data
were not normally distributed, even after transformation. Non-parametric statistics were employed to
determine if significant differences (p ≤ 0.05) existed between CS, SF, and SEW watersheds based on
the wastewater treatment approach or septic system density. Kruskal–Wallis H tests were employed
to test if significant differences existed in FIB concentrations and exports between: (i) pooled data
from watersheds using CS, SF, or SEW wastewater treatment; and (ii) septic system density regardless
of the type of septic system. If a significant p-value was determined via Kruskal–Wallis, post hoc
Mann–Whitney rank sum tests with Bonferroni correction were used to isolate differences among
comparison groups. Statistical tests were conducted in the R statistical framework [34].
3. Results and Discussion
3.1. Septic System Density
Concentrations of FIB were greatest in watersheds that contained the highest septic system
densities (Figure 2; Table 2). Differences in the median E. coli concentrations between the higher septic
system density watersheds (SF3 and CS3) were statistically different (E. coli: p < 0.01; enterococci:
p= 0.05) from the lower density watersheds (SF1 and CS1) but not for the intermediate density
watersheds (E. coli: p = 0.12; enterococci: p = 0.10). Watersheds served by SF systems demonstrated
clear differences in FIB concentrations based on septic system density. The SF3 watershed contained
median concentrations of E. coli and enterococci of 1203 MPN 100 mL−1 and 253 MPN 100 mL−1 ,
respectively. Median E. coli and enterococci concentrations were approximately 18 and 5 times greater
than the SF1 watershed. Both E. coli and enterococci concentrations in the SF3 watershed were
approximately 5 times greater than the SF2 watershed (Figure 2; Table 2). Furthermore, E. coli and
enterococci concentrations in the SF3 watershed exceeded concentrations in the SF1 91% and 100% of
the time, respectively. While median differences between SF3 and SF2 were not statistically significant,
concentrations of E. coli and enterococci in SF3 exceeded concentrations in SF2 73% and 82% of the
time, respectively. Watersheds served by conventional septic systems exhibited a similar trend for
E. coli but not for enterococci (Figure 2). The CS3 watershed contained the greatest median E. coli
concentration (526 MPN 100 mL−1 ) and was approximately 3 and 7 times greater than the CS1 and CS2
watersheds, respectively (Figure 2; Table 2). The CS3 and CS1 watersheds contained similar median
concentrations of enterococci of 101 and 105 MPN 100 mL−1 , respectively, and were approximately
1.3 times greater than the CS2 watershed (Table 2).
Warmer months tended to exhibit greater FIB concentrations and variability for all watersheds.
When pooling data from all watersheds based on season, warm months contained median values
of E. coli and enterococci of 725 and 253 MPN 100 mL−1 , respectively; meanwhile, the median
concentration of E. coli and enterococci was 149 and 70 MPN 100 mL−1 , respectively, during cold
months. These differences were statistically significant for both E. coli (p = 0.03) and enterococci
(p < 0.01). Differences in the FIB concentration based on septic system density were most apparent
when accounting for seasonality (Figure 2). When comparing seasonal trends based on septic system
density, the lower density watersheds (CS1 and SF1) exhibited statistically significant differences
based on season for E. coli (p = 0.04) and enterococci (p < 0.01) concentrations. Differences in median
concentrations of enterococci (p = 0.03) were statistically significant for intermediate density watersheds
(CS2 and SF2), but not for E. coli (p = 0.23). The higher density watersheds (CS3 and SF3) did not
exhibit statistical differences for either E. coli (p = 0.52) or enterococci (p = 0.69). Past studies have found
seasonal variability in FIB communities within surface waters due to wildlife and pet activity [7,35],
sediment agitation via recreation or storms [36], snowmelt [37], and/or rainfall [36,38]. In the current
study, elevated FIB concentrations in warmer months were likely due to increased wildlife and pet
Appl. Sci. 2020, 10, 6525
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activity [7,35]. The lack of a significant temporal trend in the higher density septic watersheds may
suggest that spatial differences in septic system density exhibit a stronger influence on river FIB
communities than temporal fluctuations. Past studies also found that spatial variability affected river
bacterioplankton communities more than seasonal variation [39–43]. FIB communities may be affected
by, but not limited to, changes in salinity [40], reduced river flow and sedimentation rates [42], and land
use [24,40,41,43].
Appl. Sci. 2020, 10, x FOR PEER REVIEW
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Figure 2. Concentrations of Escherichia coli and Enterococcus spp. from watersheds grouped by wastewater treatment approach (A,B) and comparison of cold (C) vs.
Figure 2. Concentrations of Escherichia coli and Enterococcus spp. from watersheds grouped by
warm (W) months showing increased fecal indicator bacteria during warmer months (C,D). Pooled data are shown for each group as CS = conventional septic; SF
wastewater
approach
(A,B) and comparison of cold (C) vs. warm (W) months showing
= sand filter; SEW
= sewer. Thetreatment
asterisks (*) denote
outliers.
increased fecal indicator bacteria during warmer months (C,D). Pooled data are shown for each group
as CS = conventional septic; SF = sand filter; SEW = sewer. The asterisks (*) denote outliers.
Land use is the most likely explanation for differences in FIB concentrations among the
studied watersheds since river flow (Table 3) and salinity (all freshwater watersheds) were similar.
Sowah et al. [24] found that watersheds with a low density of septic systems (<1 system ha−1 ) exhibited
more variability in FIB concentration due to variable sources of fecal pollution (e.g., livestock, wildlife,
and septic systems), which they attributed to an even distribution of land use between agriculture,
forested, and developed area. In the current study, forested and developed area accounted for >75%
of land cover (Table 1). Forested areas provide a habitat for wildlife, which can be a significant
non-point source of FIB to surface waters, especially during warmer months when they are most
active and FIB survivability is greater [44]. These additional microbial inputs made it more difficult to
differentiate trends in E. coli and enterococci concentrations based on septic system density during
warmer months (Figure 2). During colder months, both E. coli (median: >700 MPN 100 mL−1 )
and enterococci (median: >100 MPN 100 mL−1 ) concentrations were elevated in the higher density
watersheds (SF3 and CS3) (Figure 2). E. coli concentrations in the SF2 watershed were also elevated
during colder months (Figure 2). The SF3, CS3, and SF2 watersheds were predominantly residential
(≥53%), with lesser percentages of forested and agricultural land classes ranging from 26–45% and
0–5%, respectively (Table 1). Median values of E. coli and enterococci were <200 and <100 MPN
100 mL−1 , respectively, in the CS1, CS2, and SF1 watersheds. Forest accounted for ≥60% of land cover
in CS1 and CS2, whereas forest and residential development in SF1 was approximately equal (Table 1).
Thus, land cover data suggest that differences in FIB concentrations during colder months were likely
due to differing septic system densities when other major non-point sources of FIB were limited.
Appl. Sci. 2020, 10, 6525
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Table 2. Median and geometric mean concentrations (MPN 100 mL−1 ) and exports (MPN s−1 ha−1 )
of fecal indicator bacteria for the studied watersheds. For median values, the range is included
in parentheses. For geometric mean values, the standard deviation is included in parentheses.
Conc = concentration; CS = conventional septic; SF = sand filter; SEW = sewer.
Median
Watershed
CS1
CS2
CS3
SF1
SF2
SF3
CS
SF
SEW
E. coli
Geometric Mean
E. coli
Enterococci
Enterococci
Conc
Export
Conc
Export
Conc
Export
Conc
Export
160
(105–725)
71
(34–7068)
526
(16–12100)
67
(26–1628)
232
(21–5600)
1203
(22–12098)
160
(16–12100)
223
(21–12098)
170
(86–1153)
85
(5–486)
35
(1–1744)
47
(<1–1672)
18
(1–730)
26
(<1–2023)
151
(<1–2086)
48
(<1–1744)
41
(<1–2086)
94
(7–1117)
105
(26–1088)
80
(3–3434)
101
(19–6017)
55
(5–968)
48
(5–2176)
253
(133–12100)
96
(3–6017)
126
(5–12100)
121
(10–657)
23
(5–479)
18
(1–847)
20
(<1–855)
11
(1–434)
19
(<1–156)
66
(<1–3634)
22
(<1–855)
22
(<1–3634)
39
(4–879)
195
(207)
168
(1992)
410
(3367)
89
(449)
229
(1743)
792
(3416)
238
(2276)
245
(2256)
233
(370)
60
(149)
35
(492)
5
(723)
15
(210)
12
(597)
81
(602)
21
(524)
24
(497)
89
(319)
107
(330)
92
(963)
125
(1702)
64
(283)
75
(652)
603
(3530)
107
(1126)
139
(2134)
115
(219)
33
(133)
19
(238)
2
(267)
11
(122)
3
(64)
49
(1089)
11
(217)
12
(641)
44
(252)
Table 3. Median (range) of environmental parameters and stream discharge at watershed outlets.
SC = specific conductance; Cl− = chloride; DO = dissolved oxygen; ORP = oxidation-reduction potential.
Watershed
CS1
CS2
CS3
SF1
SF2
SF3
CS
SF
SEW
SC
µS cm−1
Cl−
mg L−1
DO
mg L−1
pH
152
(119–298)
103
(77–1069)
418
(105–598)
189
(75–818)
174
(89–585)
346
(166–774)
152
(77–1069)
203
(75–818)
204
(146–461)
15.7
(12.1–39.9)
6.8
(5.0–305.7)
50.9
(10.2–109.0)
19.0
(4.5–209.7)
17.7
(13.1–67.2)
48.1
(10.3–186.5)
15.7
(5.0–305.7)
21.5
(4.5–209.7)
18.7
(13.9–79.7)
8.5
(3.7–15.7)
7.8
(3.7–14.3)
6.3
(1.3–15.1)
5.9
(2.9–14.5)
6.0
(1.6–14.3)
5.9
(1.4–15.8)
7.5
(1.3–15.7)
5.9
(1.4–15.8)
8.7
(3.5–20.2)
7.3
(6.4–8.8)
7.3
(6.6–8.4)
7.3
(6.3–8.5)
7.1
(6.6–8.2)
7.2
(6.3–8.5)
7.3
(6.5–9.3)
7.3
(6.3–8.8)
7.2
(6.3–9.3)
7.4
(6.4–8.0)
Temperature
◦C
13.3
(1.6–25.1)
12.8
(3.2–23.1)
12.4
(1.9–23.6)
13.8
(2.7–23.9)
13.7
(3.0–22.6)
13.0
(2.0–23.5)
12.9
(1.6–25.1)
13.8
(2.0–23.9)
12.5
(1.1–26.5)
ORP
mV
Turbidity
NTU
Discharge
L min−1 ha−1
−22.0
(−136.8–163.9)
−24.5
(−118.6–193.5)
−31.7
(−143.2–141.3)
−19.4
(−128.8–187.8)
−18.4
(−137.0–163.0)
−27.0
(−154.5–164.3)
−31.6
(−143.2–193.5)
−21.3
(−154.5–187.8)
−14.7
(−120.4–132.0)
39
(9–120)
13
(5–66)
12
(4–58)
20
(5–132)
24
(5–72)
25
(6–122)
14
(4–120)
24
(5–132)
27
(12–88)
3.6
(0.1–10.2)
1.4
(0.1–4.4)
2.4
(<0.1–22.1)
1.2
(<0.1–4.0)
0.9
(<0.1–13.2)
0.7
(<0.1–6.3)
2.2
(<0.1–22.1)
0.7
(<0.1–13.2)
3.5
(0.2–10.3)
Specific conductance and chloride data in the CS3 and SF3 watersheds were also elevated
relative to the other watersheds (Figure 3; Table 3), potentially indicating wastewater effects [45,46].
These differences were statistically significant for both specific conductance and chloride at p < 0.01.
Waters downgradient from wastewater inputs tend to contain elevated specific conductance and
chloride [45,46]. Furthermore, elevated specific conductance in water may indicate a continuous source
of dissolved ions, such as septic systems, and increased septic system densities are often associated
with elevated specific conductance [47,48]. Past studies [45,46,49] reported specific conductance and
chloride values ranging from approximately 500−2000 µS cm−1 and 50–300 mg L−1 , respectively,
in wastewater, which were similar to values reported in the CS3 and SF3 watersheds (Figure 3; Table 3).
The other watersheds in the current study contained similar specific conductance (approximately
30–250 µS cm−1 ) and chloride (approximately 10 mg L−1 ) values to groundwater and surface water
unaffected by wastewater inputs reported in Birch et al. [46]. Runoff from roads treated with salt to
prevent ice accumulation during winter weather can contain specific conductance and chloride values
Appl. Sci. 2020, 10, 6525
9 of 14
up to approximately 10,000 µS cm−1 and 8900 mg L−1 , respectively [49]. Road salts are not routinely
applied throughout winters in the study area due to milder winters yielding infrequent snow and ice
storms. However, the January 2016 sampling event occurred several days after a snowstorm hit the
area when road salts were likely used, thus explaining the larger variability and sudden increase in
chloride and specific conductance in all watersheds (Figure 3).
Appl. Sci. 2020, 10, x FOR PEER REVIEW
12 of 20
Figure 3.Figure
Chloride 3.
(A,C)
and specific
conductance
(B,D) values
from watersheds
grouped
by wastewater
treatment approach
and increasing
septic system density.
Chloride
(A,C)
and specific
conductance
(B,D)
values
from watersheds
grouped
by wastewater
Pooled data are shown for each group as CS = conventional septic; SF = sand filter; SEW = sewer. The asterisks (*) denote outliers.
treatment approach and increasing septic system density. Pooled data are shown for each group as
CS = conventional septic; SF = sand filter; SEW = sewer. The asterisks (*) denote outliers.
Median E. coli and enterococci loadings increased with septic system density for the SF watersheds
(Figure 4; Table 2) but the same was not observed for the CS watersheds. Despite the observed
differences in median values, FIB loadings data were too variable to find significant differences.
When isolating density effects in the SF watersheds, statistical tests were nearly significant (p = 0.053)
between the SF3 and SF1 watershed for E. coli and enterococci loadings. These same trends were not
observed in the conventional septic watersheds, likely due in part to a prolonged period of little to
no flow in the CS3 watershed. From June–October 2015, the CS3 watershed was dry or contained
disconnected pools of stagnant water, which explains the large variability in the FIB loading data
for this watershed (Figure 4). Research in Georgia [24] and North Carolina [15,16] showed median
FIB exports typically ranged from 191–1116 and 101–721 MPN s−1 ha−1 for E. coli and enterococci,
respectively, for watersheds with ≥0.9 systems ha−1 . In the current study, watersheds with septic
systems contained densities that other studies [16,24] classified as low density (<1 system ha−1 ).
Sowah et al. [24] reported a mean density of 0.22 systems ha−1 for low density watersheds, and these
watersheds exported a mean value of approximately 200 and 450 MPN s−1 ha−1 of E. coli and enterococci,
respectively. Humphrey et al. [16] reported median values of 80 and 24 MPN s−1 ha−1 for E. coli
and enterococci from watersheds with a median septic system density of 0.15 systems ha−1 . In the
current study, median E. coli and enterococci exports ranged from 35–151 and 11–66 MPN s−1 ha−1 ,
respectively (Table 2). These exports overlapped with FIB yields (E. coli: 0.8–2559 MPN s−1 ha−1 ;
Appl. Sci. 2020, 10, 6525
10 of 14
MPN s−1 ha−1 ) reported in the aforementioned studies from watersheds with
[16,24].
2–5382
Appl. Sci. 2020, 10,enterococci:
x FOR PEER REVIEW
<0.9 systems
ha−1
15 of 20
Figure 4. BoxplotsFigure
(A,B) and
series plots
(C,D)
of area-normalized
watershed
of E. coli and Enterococcus
spp. Time
series plots
4. time
Boxplots
(A,B)
and
time series plots
(C,D)exports
of area-normalized
watershed
exports
of E.illustrate
coli median exports
of bacterial loadings. Data ranged from April 2015 to March 2016. During summer months, flow occasionally ceased, resulting in no transport of bacteria. The plus
and Enterococcus spp. Time series plots illustrate median exports of bacterial loadings. Data ranged
symbol (+) denotes outliers.
from April 2015 to March 2016. During summer months, flow occasionally ceased, resulting in no
transport of bacteria. The plus symbol (+) denotes outliers.
Previous studies have shown that watersheds with septic system densities greater than
1 system ha−1 can contain substantially elevated FIB concentrations and exports relative to watersheds
with lower septic system densities or without septic systems [15,16]. However, Sowah et al. [24]
reported similarities in FIB concentrations and exports at the watershed scale regardless of septic
system density. As noted earlier, their low-density watersheds exhibited substantial variability in
non-point sources of FIB because of land use, whereas high-density watersheds were mostly residential
developed lands (approximately 70%) with less forest and agricultural cover. In the current study,
results suggest that water quality impacts may be observed at lower densities than observed by
previous studies. Watersheds with septic system densities of 0.4 systems ha−1 contained elevated FIB
concentrations that were statistically different from watersheds containing densities of approximately
0.2 systems ha−1 . In addition to density, watershed-scale FIB concentrations could be affected by the
distance from the septic system to watershed outlets. In the SF3 and CS3 watersheds, all systems were
located within either 0.2 or 1 km to the outlet, whereas most systems were located > 1 km from outlets
in the SF1 and CS1 watersheds (Table 1). Therefore, quantifying the septic system density and distance
to watershed outlets may be strong indicators of watershed-scale concentrations and exports of FIB.
3.2. Wastewater Treatment Approach and Broader Water Quality Implications
FIB concentrations and exports were similar when aggregating FIB data based on the wastewater
treatment approach (e.g., CS vs. SF vs. SEW) (Figures 2 and 4; Table 2). SF watersheds
contained slightly elevated FIB concentrations relative to CS and SEW watersheds (Figure 2; Table 2);
Appl. Sci. 2020, 10, 6525
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however, this difference was not statistically significant for either bacteria (p > 0.84). This result was
expected since only the SF3 watershed contained median values that were greater than the SEW
watershed. Differences between the SF3 and SEW watersheds were statistically significant (p = 0.01)
for both E. coli and enterococci concentrations, whereas all other watersheds were similar to the SEW
watershed. Median monthly exports of E. coli from CS and SF watersheds were lower relative to SEW
exports 83% and 75% of the time (Figure 4), respectively. Median monthly exports of enterococci
from the SEW watershed exceeded exports from the CS and SF watersheds 75% and 67% of the time,
respectively. These differences were not significant for either E. coli (p = 0.49) or enterococci (p = 0.54)
yields. The SF3 watershed yielded median FIB that were approximately 1.6 times greater than the
SEW watershed. These differences were not significant for either E. coli (p = 0.49) or enterococci
(p = 0.44). A study by Iverson et al. [15] found that septic watersheds contained substantially elevated
FIB concentrations and yields relative to watersheds served by sewers. The current study did not find
similar results, but this was likely due to differences in the septic system density. The watersheds in the
Iverson et al. [15] study contained septic system densities of >1 system ha−1 . Findings by the current
study suggest that the wastewater treatment approach alone may not be a predictor of water quality at
the watershed scale, unless watersheds contain elevated septic system densities [12,14–16] and limited
wildlife and livestock inputs [24]. Thus, significant differences between watersheds using different
treatment approaches may be due to other factors that influence water quality (e.g., septic system
density, distance of the septic system to the outlet, land use, number of malfunctioning systems/leaking
sewer infrastructure, presence and condition of riparian buffers).
When considering the broader water quality implications of FIB transport from these watersheds,
the data suggest there is cause for concern. The US EPA [5] recommends that recreational waters
should not contain geometric mean values that exceed 126 MPN 100 mL−1 and 35 MPN 100 mL−1 for
E. coli and enterococci, respectively. If recreational water exceeds these values, there is an estimated
36 illnesses per 1000 primary contact recreationists. All watersheds, excluding SF1, exceeded the
recommended standard for both E. coli and enterococci regardless of the wastewater treatment approach
(Table 2). Furthermore, the US EPA [5] recommends that less than 10% of samples should exceed
the statistical threshold value (STV) of 410 MPN 100 mL−1 and 130 MPN 100 mL−1 for E. coli and
enterococci, respectively. All watersheds exceeded the STV for enterococci concentrations more
than 10% of the time. Excluding the SF1 watershed (8%), all watersheds also exceeded the STV for
E. coli concentrations more than 10% of the time. These data suggest that watersheds served by
septic systems and sewer systems can pose a risk to public health by exposing recreationists to FIB
concentrations high enough to indicate fecal contamination, which may be accompanied by pathogenic
microbes. Furthermore, FIB concentrations were greater and more variable during warmer months
relative to colder months in all watersheds except SF3 (Figure 2), which overlaps with the peak tourism
season when water-based recreation is more common. These findings were consistent with previous
studies showing greater FIB concentrations during warmer summer months [15,24,31–33]. While the
studied streams are not likely routinely used for recreational purposes, they are low-order streams that
discharge to Falls Lake, which is a reservoir intended for water supply and recreation.
4. Conclusions
This study found that watersheds with septic system densities of approximately 0.4 systems
ha−1 contained elevated FIB concentrations (both CS3 and SF3) and yields (SF3 only) compared to
other septic watersheds. These differences were only significant when compared to lower density
watersheds (approximately 0.2 systems ha−1 ). Past research suggests that watersheds with septic
system densities > 1 system ha−1 may have a significant effect on watershed FIB [16,24]. The current
study suggests that the septic system density threshold of watershed-scale FIB concentrations and/or
transport may be <1 system ha−1 , especially for SF systems and other surface discharging systems,
although more research is recommended to account for longer duration spatiotemporal fluctuations.
Septic system density is an important management consideration during the planning phase of
Appl. Sci. 2020, 10, 6525
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residential development and may provide guidance for retroactive water quality improvements.
Management agencies considering retrofit activities could incorporate spatial analysis to identify and
focus water quality monitoring and remediation efforts based on septic system characteristics at the
watershed scale. Tetzlaff et al. [43] suggest similar efforts as land-use-based models can be effective
predictive tools of surface water quality. While more research is recommended to determine if similar
septic system density thresholds (approximately 0.4 systems ha−1 ) are observed in watersheds with
differing hydrogeological, land use, and/or wastewater characteristics, this may be a soft threshold that
management agencies can utilize in their efforts to manage recreational and water supply watersheds.
Author Contributions: Conceptualization: C.P.H.J., C.S., J.R.E., T.K., and G.I.; methodology: G.I., C.S., and C.P.H.J.;
software: G.I. and C.S.; validation: G.I., C.S., C.P.H.J., J.R.E., and T.K.; formal analysis: G.I., C.S., and C.P.H.J.;
investigation: C.S., G.I., C.P.H.J.; resources: C.P.H.J. and J.R.E.; data curation: G.I.; writing—original draft
preparation: G.I. and C.S.; writing—reviewing and editing: G.I., C.P.H.J., J.R.E., C.S., and T.K.; visualization:
G.I. and C.S.; supervision: C.P.H.J., G.I., J.R.E., and T.K.; project administration: C.P.H.J., G.I., and C.S.; funding
acquisition: C.P.H.J., J.R.E., and T.K. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the North Carolina Department of Environmental Quality 319 Non-Point
Source Program, contract 6201.
Acknowledgments: The authors acknowledge and thank the North Carolina Department of Environmental
Quality for sponsoring this research. Further, we wish to extend a special “thank you” to the following individuals
for their assistance in project development, field support, laboratory analysis, and all other contributions made:
Eban Bean, Jordan Jernigan, Brent Serozi, and Caitlin Skibiel.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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Population exposure across central India to PM2.5 derived using remotely sensed products in a three-stage statistical model
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Scientific reports
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Prem Maheshwarkar1 & Ramya Sunder Raman1,2* Surface PM2.5 concentrations are required for exposure assessment studies. Remotely sensed Aerosol
Optical Depth (AOD) has been used to derive PM2.5 where ground data is unavailable. However, two
key challenges in estimating surface PM2.5 from AOD using statistical models are (i) Satellite data gaps,
and (ii) spatio-temporal variability in AOD-PM2.5 relationships. In this study, we estimated spatially
continuous (0.03° × 0.03°) daily surface PM2.5 concentrations using MAIAC AOD over Madhya Pradesh
(MP), central India for 2018 and 2019, and validated our results against surface measurements. Daily
MAIAC AOD gaps were filled using MERRA-2 AOD. Imputed AOD together with MERRA-2 meteorology
and land use information were then used to develop a linear mixed effect (LME) model. Finally, a
geographically weighted regression was developed using the LME output to capture spatial variability
in AOD-PM2.5 relationship. Final Cross-Validation (CV) correlation coefficient, r2, between modelled
and observed PM2.5 varied from 0.359 to 0.689 while the Root Mean Squared Error (RMSE) varied
from 15.83 to 35.85 µg m−3, over the entire study region during the study period. Strong seasonality
was observed with winter seasons (2018 and 2019) PM2.5 concentration (mean value 82.54 µg m−3)
being the highest and monsoon seasons being the lowest (mean value of 32.10 µg m−3). Our results
show that MP had a mean PM2.5 concentration of 58.19 µg m−3 and 56.32 µg m−3 for 2018 and 2019,
respectively, which likely caused total premature deaths of 0.106 million (0.086, 0.128) at the 95%
confidence interval including 0.056 million (0.045, 0.067) deaths due to Ischemic Heart Disease (IHD),
0.037 million (0.031, 0.045) due to strokes, 0.012 million (0.009, 0.014) due to Chronic Obstructive
Pulmonary Disease (COPD), and 1.2 thousand (1.0, 1.5) due to lung cancer (LNC) during this period. Increased cardiovascular and respiratory diseases in addition to a decreased life expectancy are associated with
chronic exposure to particulate matter with aerodynamic diameters < 2.5 µm, PM2.5
1. The Global Burden of
Disease (GBD) 2015 study identified air pollution as a major cause of global disease burden, with low and
middle-income countries being the worst affected2. In India, PM2.5 standards were included in the National
Ambient Air Quality Standards (NAAQS) in November 2009 and are monitored by the central and several state
pollution control boards. However, PM2.5 concentrations vary in space over sub-kilometer to continental scales3. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Population exposure across central
India to PM2.5 derived using
remotely sensed products
in a three‑stage statistical model Prem Maheshwarkar1 & Ramya Sunder Raman1,2* www.nature.com/scientificreports/ from these studies for locations in India were not properly validated, due to the lack of monitoring stations in
India at the time that these studies were conducted. Recently, a number of statistical models were developed to capture the varying relationships between AOD
and surface PM2.5 concentrations at various locations across the world, such as the linear mixed effect model,
geographically weighted regression, and generalized additive models12,13. These studies have used meteorological
parameters (height of planetary boundary layer, surface temperature, wind speed, relative humidity) and land
use information as covariates along with satellite AOD to estimate surface PM2.5 concentrations. Results from
these studies have shown that meteorological fields and land use information improve the model performance
significantly. More sophisticated models were then developed by combining two or more regression models to
hierarchically estimate the surface PM2.5 concentrations14,15. These models were usually generated by combin-
ing linear mixed-effects models in the first stage with generalized additive models or geographically weighted
regression models in the second stage to capture the spatiotemporal variation in the relationship between AOD,
PM2.5, and meteorological parameters. These hybrid models have shown a strong correlation between estimated
and measured PM2.5 mass concentration worldwide with improved performance when compared to individual
models. Another major challenge in estimating spatially continuous PM2.5 arises due to spatially non-continuous
AOD values owing to cloud coverage, rainfall, and satellite calibrations. Previous studies in the literature have
tried to fill MODIS AOD data by using random forest algorithm16,17, spatiotemporal regression kriging18 and by
using two-staged generalized additive model19. However, these studies have their own limitations and specific
pre-requisites, limiting model application to real-life situations. In India, studies to estimate surface PM2.5 concentration using satellite proxies are still in an embryonic stage
with very few national studies13,20–23 reporting low r2 values (Supplemental Table S1) between measured and
estimated PM2.5. Due to the lack of extensive ground PM2.5 measurements in India, very few studies use empirical
statistical models, of which a majority were developed for the Delhi region24,25. A recent study23 estimated PM2.5
concentration over India for January–August 2017 using spatiotemporal mixed effect models, and chose ordinary
spatial Kriging, inverse distance weighting (IDW) and spline interpolation to estimate PM2.5 concentration over
grids with no AOD values and reported that spline interpolation performed better than IDW and Kriging. www.nature.com/scientificreports/ Fur-
ther, none of these studies validated their model performances against surface PM2.5 data over Madhya Pradesh
(MP) or other states in central India. Current estimates of spatially continuous PM2.5 concentration over MP are
derived from global studies such as (van Donkelaar et al.11 and van Donkelaar et al.10), for various epidemiological
and GBD studies. These estimates from CTMs in conjunction with satellite-derived AOD are strongly influenced
by model chemistry, physical processes, and emissions inventory, all of which may in-turn fail to capture the
ground realities26. CTMs in general, do not incorporate all of the complexities in aerosol mixing states (which are
only beginning to be understood) and thus the (η) factor approach often provides biased surface PM2.5 estimates.h y
g
g
)
(η)
ppt
p
2.5
The goal of this study is to develop a three-stage statistical model to capture spatio-temporal variability in
AOD-PM2.5 relationship, in order to estimate spatially continuous surface PM2.5 concentrations over MP state,
central India, for 2018 and 2019. This endeavor is made possible by the recently available Central Pollution
Control Board (CPCB) India surface PM2.5 over several locations in the state. We take a three-step approach
to achieve our study goals. In the first step, missing Multi-Angle Implementation of Atmospheric Correction
(MAIAC) AOD values were imputed, using yearly grid-wise linear regression between MAIAC AOD and the
Modern-Era Retrospective analysis for Research and Applications (MERRA-2) derived AOD. Imputed AOD
and MEERA-2 meteorological parameters in conjunction with land use variables were then used to develop a
linear mixed effect model (LME) to capture the daily variability in the relationship between AOD, PM2.5 and
meteorological parameters. Finally, to capture the spatial variability in AOD-PM2.5 relationship a Geographically
Weighted Regression model (GWR) was developed. The annual PM2.5 concentrations thus obtained were then
compared against ground measurements and PM2.5 concentrations obtained from a recent study incorporating
advances in CTM implementation and using a GWR (Hammer et al.27). An additional objective of this study was
to utilize the derived concentrations to estimate the population exposure to PM2.5 and the associated premature
mortality over Madhya Pradesh during 2018–2019. Study area
Th
d This study was conducted over MP state (Fig. 1) in central India [27 N, 74E–21 N, 84E]. MP is the second larg-
est state in India by area with a total geographical area of 3,08,245 km2 and the fifth-largest state by population
Census 201128. The northeastern boundary of MP is lined by Indo Gangetic Plain (IGP), one of the most air-
polluted regions in the world10,11. Previous studies have shown 24 h mean PM2.5 mass concentrations of up to
170 μg m−3 in the IGP and in parts of MP23. Based on the Indian Meteorological Department classification, MP
has four distinct seasons: winter (Jan, Feb), pre-monsoon (Mar, Apr, May), monsoon (Jun, Jul, Aug, Sep) and
post-monsoon (Oct, Nov, Dec) and has a mixture of semi-arid, tropical, and subtropical climate. The annual
mean temperature over MP is 24.7 °C with an average daily high temperature of 33 °C (averages over the last
20 years). MP receives most of its rainfall in the monsoon season with a mean rainfall of 1160 mm with high
spatial variability (rainfall decreases from east to west). The mean elevation of MP ranges between 72 m amsl
and 1317 m amsl. Prem Maheshwarkar1 & Ramya Sunder Raman1,2* The current surface PM2.5 monitoring network in India is inadequate to capture this variability and to provide
adequate data for population exposure studies. Satellite retrieval proxies of PM2.5 such as Aerosol Optical Depth
(AOD), which is the measure of the overall light extinction attributed to the aerosols in the atmospheric column,
has been extensively used to estimate surface fine aerosols concentrations worldwide4–7. During the mid-2000s,
various studies estimated surface PM2.5 concentrations by establishing a linear relationship between AOD and
surface PM2.5. Another popular approach was by using η factor (the ratio of PM2.5 modelled and AOD modelled)
obtained from various global Chemical Transport Models (CTMs) such as GEOS Chem as a scaling factor to
convert satellite-derived AOD to surface PM2.5 concentration8,9. During the last decade or so, several studies
estimated the global concentrations of surface PM2.5 using satellite-derived AOD and η factor obtained from
various CTMs10,11 as this method does not require surface measurements to develop the model. However, results 1Department of Earth and Environmental Sciences, Indian Institute of Science Education and Research Bhopal,
Bhopal Bypass Road, Bhauri, Bhopal, Madhya Pradesh 462 066, India. 2Center for Research on Environment and
Sustainable Technologies, Indian Institute of Science Education and Research Bhopal, Bhopal Bypass Road, Bhauri,
Bhopal, Madhya Pradesh 462 066, India. *email: ramyasr@iiserb.ac.in | https://doi.org/10.1038/s41598-020-79229-7 Scientific Reports | (2021) 11:544 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Elevation map (amsl) of the study area and location of PM2.5 monitors in MP state. This map is
generated using QGIS 2.18.1 (http://www.qgis.org). Figure 1. Elevation map (amsl) of the study area and location of PM2.5 monitors in MP state. This map is
generated using QGIS 2.18.1 (http://www.qgis.org). Figure 1. Elevation map (amsl) of the study area and location of PM2.5 monitors in MP state. This map is
generated using QGIS 2.18.1 (http://www.qgis.org). ary 2018 and December 2019 from the CPCB database (https://app.cpcbccr.com/ccr/#/caaqm-dashboard-all/
caaqm-landing). The location of air monitoring stations are shown in Fig. 1 and data completeness over these
stations is shown in Supplemental Figure S1. MODIS AOD. The MODerate resolution Imaging Spectroradiometer (MODIS) sensors onboard Earth
Observation System (EOS) Terra and Aqua were launched to Sun-synchronous polar orbits in late 1999 and in
2002, having satellite overpass times over India at 10:30 a.m. and 01:30 p.m., respectively. They have a circular
orbit of 705 km and a swath of approximately 2330 km. Daily measurements across a wide spectral range yields
multiple datasets of AOD and a variety of other products. In this study, we have used the Multi-Angle Imple-
mentation of Atmospheric Correction (MAIAC) for a combined data product of Terra and Aqua AOD. MAIAC
algorithm was developed for MODIS data to perform the retrieval of aerosols and for atmospheric correction
over bright and dark surfaces (vegetated) utilizing image processing and time-series analysis to derive bidirec-
tional reflectance distribution function at a resolution of 1 km30. Due to clear surface characterization, MAIAC
AOD has lesser urban bias and increased spatial coverage when compared to the dark target AOD products31. Previous studies in the literature have successfully estimated daily PM2.5 concentrations by utilizing Terra and
Aqua AOD averages14,32. MAIAC files contain multiple (2–4) AOD files per day depending upon the number
of Terra and Aqua overpasses. Due to changing cloud cover during a day, there is a difference in the spatial and
temporal availability in AOD data per day. Availability of MAIAC AOD over the location of surface stations in
MP is given in Supplemental Figure S2. MERRA‑2 AOD. The Modern-Era Retrospective analysis for Research and Applications version 2 (MERRA-
2) is a NASA atmospheric reanalysis using the Goddard Earth Observing System Model, Version 5 (GEOS-5)
coupled with GOCART aerosol module with its Atmospheric Data Assimilation System (ADAS), version 5.12.4. Ground PM2.5 measurement. Ground PM2.5 measurement. The Central Pollution Control Board (CPCB) monitors ambient air quality
under a nation-wide program: National Ambient Air Quality Monitoring Programme (NAMP). The monitoring
stations report PM2.5 mass concentration in μg m−3 at 15 min resolution, measured using the tapered element
oscillating microbalance (TEOM) or the beta-attenuation method (BAM) (CPCB 2011)29. Daily mean PM2.5
concentration data were thus obtained for 12 stations in different cities across MP for the period between Janu- https://doi.org/10.1038/s41598-020-79229-7 Scientific Reports | (2021) 11:544 | www.nature.com/scientificreports/ A brief description of these datasets and their source is given in Table 1.ii pi
p
g
To maintain a reasonable file size MAIAC AOD data is stored in 1200 km × 1200 km tiled file structure. Mad-
hya Pradesh is covered by two tiles i.e. h25v06 and h24v06. These files contain multiple AOD files per day (2–4)
from multiple overpasses of Terra and Aqua platform. Daily mean AOD values from these multiple swaths were
calculated for each grid and were clipped for MP state. MAIAC AOD data was available in curvilinear projec-
tion and this had to be remapped to rectilinear projection for obtaining a consistent spatiotemporal dataset with
MERRA-2 AOD and meteorological parameters (originally in rectilinear projection). AOD re-mapping is time-
consuming and computationally expensive, therefore, to balance between spatial resolution and computational
time, the original curvilinear 1 km × 1 km MAIAC AOD was remapped to 0.03° × 0.03° rectilinear projection. Previous studies in the literature have adopted a similar remapping technique to estimate AOD at a lower reso-
lution. Ma et al.36,37 remapped MODIS C6 AOD (3 km × 3 km) to 0.1° × 0.1° and MODIS C5.1 (10 km × 10 km)
to 50 km resolution data over China, respectively. vanDonkelaar et al.11 remapped global MAIAC AOD to
regional 0.1° × 0.1° and 0.01° × 0.01° to estimate the global burden of surface PM2.5 concentrations. Meanwhile,
a bilinear-interpolation method was used for MERRA-2 AOD and meteorological parameters to match the
spatial resolution with gridded MAIAC data. For stations like Singrauli, PM2.5 mass concentration was as high
as 800–1000 μg m−3 for a few days (Supplemental Table S2). Very high episodic PM2.5 concentration days were
outliers and not reflected by unusually high AOD over these stations on such days. Therefore PM2.5 concentration
values greater than 99.9th percentile were not used in model development or validation. Gridded road density
network (Supplemental Figure S3) was obtained using the method described in “Land-cover and road-density
data” section. Finally, the number of grid cells of urban cover, grassland and cropland falling in the predefined
0.03° × 0.03° grid were calculated. Model development. Stage 1: imputing missing MAIAC AOD with MERRA‑2 AOD. MODIS data has
often missing AOD values due to cloud coverage, monsoons and satellite calibration issues. Spatial distribution
of the percentage of annual mean available data days for 2018 and 2019 are shown in Supplemental Figure S4. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 1. List of all input parameters used in the statistical model. Parameters
Temporal resolution
Resolution
Sensor
Type of data
Data period
Source
Surface PM2.5
Daily
Point data
TEOM/BAM
In-situ
2018–2019
CPCB
AOD
Daily
1 km × 1 km
MODIS
Satellite
2018–2019
LAADSDAAC
Daily
0.625° × 0.5°
MERRA-2
Reanalysis
2018–2019
GES DISC
Meteorological parameters
Daily
0.625° × 0.5°
MERRA-2
Reanalysis
2018–2019
GES DISC
Land use land cover
Yearly
300 m × 300 m
Copernicus
Satellite
2018
ECMWF
Road map
Yearly
–
Shapefile
2018 Table 1. List of all input parameters used in the statistical model. rom MERRA-2 surface flux diagnostics. These datasets were obtained hourly from 05:00 UTC to 08:00 UTC for
he study period at a spatial resolution of 0.625° × 0.5° from GES-DISC over MP. from MERRA-2 surface flux diagnostics. These datasets were obtained hourly from 05:00 UTC to 08:00 UTC for
the study period at a spatial resolution of 0.625° × 0.5° from GES-DISC over MP. Land‑cover and road‑density data. Shapefile for the major roadways map for India was obtained from
https://mapcruzin.com/. This shapefile was then clipped to match the state boundaries of MP. The density of the
road network in a grid cell was obtained by dividing the length of roads in a grid by the total area of the defined
grid cell. Land use and land cover map for 2018 over India was downloaded from European Centre for Medium-
Range Weather Forecasts (ECMWF) at a spatial resolution of 300 m × 300 m. Annual land cover classification
gridded maps are available from 1992 to 2019 and have divided land cover in 22 classes defined by the United
Nations Food and Agriculture Organization’s (UN FAO) Land Cover Classification System (LCCS). In-depth
documentation on the algorithms used to derive the land cover can be accessed via maps.elie.ucl.ac.be/CCI/
viewer/download/ESACCI-LC-Ph2-PUGv2_2.0.pdf. Out of these 22 classes available, grid cells of urban cover,
cropland and grassland cover were extracted for use in this study. Data integration. In this study, PM2.5 ground measurements were point data. Additionally, four differ-
ent gridded datasets were used: the MAIAC AOD obtained from LAADS DAAC in HDF format; daily mean
MERRA-2 AOD and meteorological parameters obtained from GES DISC originally stored in NetCDF; and land
cover classification gridded maps obtained from ECMWF. Additionally, the road map of Madhya Pradesh was in
a shapefile format. www.nature.com/scientificreports/ MERRA-2 reanalysis data is available from 1980-present, globally. MERRA-2 aerosol analysis uses the GEOS-5
Aerosol Assimilation System (GAAS) and assimilates bias-corrected AOD from the ground and satellite-based
instruments such as MODIS, MISR, AVHRR and AERONET33. MEERA-2 AOD has been validated against
MODIS, MISR AOD and in situ AOD worldwide and has shown good agreement with measured AOD33,34. In
India, daily mean MERRA-2 AOD at 550 nm compared well (r = 0.79) with AERONET AOD measured over
Kanpur during 2011–201635. In this study, we obtained hourly total aerosol extinction at 550 nm from MERRA
2 aerosol diagnostics at a resolution of 0.5° × 0.625° (latitude × longitude) over MP state for 2018 and 2019. The
dataset was then extracted for 05:00 UTC to 08:00 UTC (10:30 a.m.–01:30 p.m. IST) to match with the MAIAC
AOD data. Meteorological data. Hourly meteorological dataset: air temperature at 2 m, relative humidity as 2 m, the
eastward and northward component of wind velocity as 10 m above the surface level and surface pressure were
obtained from MERRA-2 single-level diagnostics and height of planetary boundary layer in meters was obtained https://doi.org/10.1038/s41598-020-79229-7 Scientific Reports | (2021) 11:544 | www.nature.com/scientificreports/ where Final_AODs,t is the imputed AOD over grid s on day t provided MAIACs,t is unavailable; αs and βs are the
coefficient obtained from linear regression between daily MAIAC AOD and MERRA-2 AOD over grid s; and
MERRA2_AODs,t is the MERRA-2 AOD over grid s on day t. MERRA 2 AOD was able to capture the temporal variation in MAIAC AOD with a mean temporal r2 value MERRA-2 AOD was able to capture the temporal variation in MAIAC AOD with a mean temporal r2 value
of 0.63 and 0.61 during 2018 and 2019, respectively over the MP region. Grid-wise temporal r2 between MAIAC
and MERRA-2 AODs and range of r2 values are provided in Supplemental Figures S5 and S6. However, MERRA-2
consistently under estimated AOD value throughout the study period compared to MAIAC AOD (slope and
intercept of the regression equations are provided in Supplemental Figures S7 and S8). We also fitted seasonal
linear regression between daily MAIAC and MERRA-2 AOD to estimate seasonal slope and intercept along with
the corresponding p-values (Supplemental Text S1 and Supplemental Figures S9–S12). For the monsoon season,
the p-values for the slope were greater than 0.01 for a substantial number of grid points during both 2018 and
2019 (Supplemental Figures S9 and S10) bringing down the confidence in imputed AOD. Therefore in the final
imputation, estimates of slope and intercept from yearly regression, as shown in Eq. (1) were used. Stage 2: linear mixed effect model. In the second stage, a linear mixed-effect (LME) model was developed fol-
lowing the approach proposed by Lee et al.38, over the New England region in the USA. A LME model captures
daily variability in the relationship between AOD, PM2.5, and meteorological parameters. Day-specific slopes
and intercepts for the relationship between PM2.5, AOD and meteorological parameters are calculated and incor-
porated into both fixed-effects terms and random-effects terms in a LME model. Several studies have shown
that the relationship between AOD and surface PM2.5 varies with changing meteorology due to changing PM2.5
vertical profile, hygroscopic particle growth and optical properties15,36 . Also spatially changing parameters such
as urban cover, forest cover and vegetation can also affect AOD-PM2.5 relationship due to changing sources and
chemical composition of PM2.5
36. www.nature.com/scientificreports/ Therefore, in this study, we used meteorological parameters along with land
cover variables as independent variables and surface PM2.5 as a dependent variable to develop a LME model in
the second stage, which can be written as: (2)
PM2.5 (s,t) =
a0 + a0,t
+
a1 + a1,t
AODs,t +
a2 + a2,t
Temps,t
+
a3 + a3,t
RHs,t +
a4 + a4,t
U10s,t +
a5 + a5,t
Pressures,t
+ a6UrbanCovers + a7GrassLand + εst
a0,t, a1,t, a2,t, a3,t, a4,t, a5,t
∼N[(0, 0, 0, 0, 0, 0), ] PM2.5 (s,t) =
a0 + a0,t
+
a1 + a1,t
AODs,t +
a2 + a2,t
Temps,t
+
a3 + a3,t
RHs,t +
a4 + a4,t
U10s,t +
a5 + a5,t
Pressures,t (2
+ a6UrbanCovers + a7GrassLand + εst
a0,t, a1,t, a2,t, a3,t, a4,t, a5,t
∼N[(0, 0, 0, 0, 0, 0), ] + a6UrbanCovers + a7GrassLand + εst
a0,t, a1,t, a2,t, a3,t, a4,t, a5,t
∼N[(0, 0, 0, 0, 0, 0), (2) where PM2.5(s,t) is the surface PM2.5 concentration in (µg m−3) over grid s on day t: a0 and a0,t are fixed and random
(daily varying) intercept, respectively: AODs,t is the imputed AOD (unitless) over grid s on day t. RHs,t, Temps,t,
U10s,t, Pressures,t are relative humidity and temperature (℃) at 2 m above ground level, U10 is the eastward
component of wind speed at 10 m above the ground level and Pressure is the surface pressure over grid s on day
t. UrbanCover and GrassLand are the number of grid points of urban cover and grass-land available inside the
grid s. (a1–a7) represents the fixed slopes of variables over the entire study period while (a1,t–a5,t) are the changing
slopes with day t. εst is the error term on grid s on day t and Σ is the variance–covariance matrix for the random
effects. Additional predictors such as road density, the daily height of planetary boundary layer, crop-land cover
were also included in the LME model development. But the slope estimate values were not statistically significant
therefore they were not included in the final model given by Eq. (2). Stage 3: geographically weighted regression. In the final stage, a Geographically Weighted Regression (GWR)
model was developed to capture the spatially varying relationships between AOD and PM2.5 using the output
from the LME model. www.nature.com/scientificreports/ Percentage of daily MAIAC AOD available over MP ranged from 0.0 to 73.15% with a mean value of 59.47% and
0.0% to 63.83% with a mean value of 50.56% during 2018 and 2019, respectively. There are no specific spatial
patterns in missing data, except almost no data available over large water bodies in MP. To fill daily MAIAC
AOD data gaps, first, grid-wise linear regression was fitted between daily pixel centroid values of re-sampled
0.03° MAIAC AOD and 0.03° MERRA-2 AOD for each year to obtain regression coefficients for every grid
(201 × 334 = 67,134 in total). The missing MAIAC AOD(s,t) on day “t” and grid point “s” was then filled using the
regression coefficient obtained for grid “s” from the first step and MERRA-2 AOD(s,t) over that grid point on day
“t” as shown in Eq. (1). (1) Final_AODs,t = αs + βs × MERRA2_AODs,t Final_AODs,t = αs + βs × MERRA2_AODs,t https://doi.org/10.1038/s41598-020-79229-7 Scientific Reports | (2021) 11:544 | www.nature.com/scientificreports/ Descriptive statistics. Seasons are defined following the Indian Meteorological Department (IMD) classification for this region as
winter (Jan, Feb), pre-monsoon (Mar, Apr, May), monsoon (Jun, Jul, Aug, Sep) and post-monsoon (Oct, Nov,
Dec). Seasonal variation in AOD over MP was not statistically significant (Supplemental Table S2). Further,
the 2009 NAAQS over India were revised to include the daily and annual PM2.5 mass concentration standards
with values of 60 μg m−3 and 40 μg m−3, respectively. PM2.5 mass concentrations measured over CPCB stations
in MP exceeded the daily average standard more than 33.49% of the days, for which data were available, during
the study period. Model fitting and validation. The fixed effect terms of all the variables from stage-2 after fitting the LME
model are summarized in Table 2. The PM2.5 concentration increases with increasing AOD, surface pressure,
urban cover, grassland cover and northward winds in a grid cell and decreases with increasing temperature and
relative humidity. Detailed results from the model are provided in Supplemental Table S3. ECMFW LULC classi-
fies Mosaic herbaceous cover (> 50%)/tree and shrub (< 50%) and grassland as grassland. This herbaceous cover
land becomes open/dry land during summer/dry season and could potentially contribute dust aerosol to PM2.5
loading in MP giving a positive slope with PM2.5. g
g
g
p
p
A GWR model was then developed using residual PM2.5 (LME estimated PM2.5–observed PM2.5) and AOD
as shown in “Model development” section. The model results were then compared with ground observations to
evaluate the model performances. Scatter plots between modelled and observed PM2.5 concentrations for the
model fitting and cross-validation of stage-2 and stage-3 models are shown in Fig. 2.fii i
g
g
g
g
Overall coefficient of determination (r2) values for model fitting were 0.56 and 0.60 for stage-2 and stage-3
models, respectively. The RMSE values also decreased from 22.63 to 21.63 µg m−3 from stage-2 to stage-3 indi-
cating that the overall prediction accuracy increased after using the GWR model. However, CV r2 values were
0.51 and 0.55 for stage-2 and stage-3 models while the respective CV RMSE values were 23.91 µg m−3 and
22.92 µg m−3. The r2 value decreases for CV than for model fitting and the corresponding RMSE value increases
for both models indicating the model slightly over fitted at both the stages. Also, after fitting the GWR model
slope was increased to 0.58 from 0.55 (Fig. www.nature.com/scientificreports/ A GWR model captures spatial heterogeneity by generating a continuous surface of model
parameters at every grid cell instead of universal value for all observations (predictor and response variable). We fitted a daily Gaussian GWR model using adaptive bandwidth selection to minimize the Akaike Information
Criterion (AICc) value using adaptive bi-square kernel in MGWR python39 given by Eq. (3). (3)
PM2.5_residuals,t = b0,s + b1,sAODs, t + ε1st (3) PM2.5_residuals,t = b0,s + b1,sAODs, t + ε1st where PM2.5_residuals,t is the residual PM2.5 concentration (observed–estimated) obtained after fitting the LME
model over grid s and day t, AODs,t is the imputed AOD (unitless) over grid s and day t: b0,s and b1,s are location
specific intercept and slope over grid s, respectively, which is a function of the geographic location, and ε1
st is
the error term over grid s and day t.i To assess the model fit performance, statistical indicators (r2, Root Mean Square Error (RMSE) and Mean
Absolute Error (MAE)) were used to estimate the goodness of fit for both stage-2 and stage-3 models. Further-
more, to avoid any model overfitting, a tenfold Cross-Validation (CV) approach was used after stage-2 and
stage-3 to estimate the overall model performance. In a tenfold CV, the entire dataset of 4922 observations was
divided into 10 random sub-groups (~ 492 points each), and data from 9 sub-groups were used to train the model. The remaining group was used for model validation. This validation scheme is repeated ten times until every
subgroup is validated. The metrics were then calculated by comparing PM2.5 observations and estimates that
are collected from all 10 subgroups. Furthermore, due to unavailability of AERONET stations or any campaign
mode in situ AOD measurement over MP, to estimate the performance of imputed AOD using MERRA-2 AOD
we chose days over study area where MAIAC AOD data was unavailable but surface PM2.5 data were available. We then checked final model performance on such days against surface concentration to assess the performance
of imputed AOD to estimate PM2.5 concentration. Scientific Reports | (2021) 11:544 | https://doi.org/10.1038/s41598-020-79229-7 www.nature.com/scientificreports/ Table 2. Fixed effect terms (intercept and slope estimates) for LME model after stage-2. www.nature.com/scientificreports/ Variable
Coefficient
p-value
Intercept
− 823.031
< 0.001
Imputed AOD (unitless)
34.833
< 0.001
V10 (m/s)
− 1.178
< 0.001
Temperature (℃)
− 1.104
< 0.001
RH (100)
− 23.502
< 0.001
Pressure (Pa)
0.009
< 0.001
Urban
0.100
< 0.001
Grassland
0.796
< 0.001 Variable
Coefficient
p-value
Intercept
− 823.031
< 0.001
Imputed AOD (unitless)
34.833
< 0.001
V10 (m/s)
− 1.178
< 0.001
Temperature (℃)
− 1.104
< 0.001
RH (100)
− 23.502
< 0.001
Pressure (Pa)
0.009
< 0.001
Urban
0.100
< 0.001
Grassland
0.796
< 0.001 Table 2. Fixed effect terms (intercept and slope estimates) for LME model after stage-2. Results and discussion Descriptive statistics. Percentage frequency distribution of AOD, PM2.5, and meteorological variables
used in the statistical models are summarized in Supplemental Figure S13. MAIAC AOD, MERRA-2 AOD and
PM2.5 follow a similar distribution, indicating that these three variables are indeed related to each other. The
mean MAIAC AOD over surface PM2.5 monitoring stations for the study period was 0.4034 and correspond-
ing MERRA-2 AOD and surface PM2.5 concentrations were 0.3482 and 67.89 µg m−3, respectively. MERRA-2
AOD consistently underestimated the AOD over Madhya Pradesh compared to MAIAC AOD. Temperature
and relative humidity show significant seasonality (Supplemental Figure S13) as suggested by their bimodal
distributions.ii www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. (a) LME and (b) LME are Model training and tenfold cross-validation of LME model, respectively
over Madhya Pradesh during 2018–2019 and (c) GWR is GWR model training and (d) GWR is after tenfold CV
(caxis is the point count). The blue lines are the y = 2 × and y = x/2 while black is x = y line. “m” is the slope if the
regression is forced through origin. Figure 2. (a) LME and (b) LME are Model training and tenfold cross-validation of LME model, respectively
over Madhya Pradesh during 2018–2019 and (c) GWR is GWR model training and (d) GWR is after tenfold CV
(caxis is the point count). The blue lines are the y = 2 × and y = x/2 while black is x = y line. “m” is the slope if the
regression is forced through origin. sefulness of imputed AOD to predict surface PM2.5. We also fitted stage-2 and stage-3 models using 0.03° × 0.03°
MERRA-2 AOD instead of imputed AOD and the results are discussed in Supplemental Text S2. Spatial distribution of surface PM2.5. Daily surface PM2.5 maps at a spatial resolution of (0.03° × 0.03°)
were generated using the stage-3 model over Madhya Pradesh for 2018 and 2019. This data was then used to
estimate the annual average of daily surface PM2.5 concentration and the final maps are presented in Fig. 4.h Spatial distribution of surface PM2.5. Daily surface PM2.5 maps at a spatial resolution of (0.03° × 0.03°)
were generated using the stage-3 model over Madhya Pradesh for 2018 and 2019. This data was then used to
stimate the annual average of daily surface PM2.5 concentration and the final maps are presented in Fig. 4.h g
yi
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The annual average daily PM2.5 concentration over MP varied from 22.73 to 95.24 µg m−3 with a mean value of
58.19 µg m−3 during 2018 and from 20.80 to 96.72 µg m−3 with a mean value of 56.32 µg m−3 during 2019. It was
observed that the topography of MP had a strong influence on the surface PM2.5, with the highest concentration
in northeast MP which is a part of the Indo-Gangetic Plain (IGP) and high PM2.5 mass loading downstream of
the Narmada valley, while locations at a high elevation (Dindori, Mandla, Amarkantak) had the least PM2.5 mass
loading. Descriptive statistics. 2) in model fitting and to 0.54 from 0.52 (Fig. 2) in model cross valida-
tion and reduced the intercept from 23.99 to 22.97 and from 25.38 to 24.33 in model fitting and cross validation,
respectively of the linear regression between model estimated and observed PM2.5 over MP for 2018 and 2019. Standard errors and p-values for the linear regression are provided in Supplemental Table S4. Modelled PM2.5
for days with surface PM2.5 concentration more than 100 µg m−3 were underestimated by both models in model
fitting and cross-validation, and with increasing concentration underestimation also increased. This may be
because the model was developed with most of the points below 100 µg m−3, therefore, less weighted is given
to points with such high concentration. One more possible explanation could be that such high concentrations
were local and were not reflected in a coarse resolution of 0.03° to 0.03°.i l
Station-wise r2 and RMSE for model fitting and CV for stage-2 and stage-3 models are shown as Table 3. CV r2 value between observed and modelled PM2.5 varied from 0.359 to 0.689 while RMSE varied from 15.83
to 35.85 µg m−3. Further, r2 value increased at every station after using the GWR model. In order to assess the
usefulness of imputed AOD values in estimating surface PM2.5, we selected days when MAIAC AOD was missing
but surface PM2.5 data were available. Modelled PM2.5 data on only those days were selected and compared with observed values using statistical
metrics (r2, RMSE, MAE) and a scatter plot is provided in Fig. 3. The agreement between modelled and observed
PM2.5 on such days was good (r2 = 0.54) and the overall model RMSE value was 19.42 µg m−3 clearly indicating the https://doi.org/10.1038/s41598-020-79229-7 Scientific Reports | (2021) 11:544 | www.nature.com/scientificreports/ It is also worth noting that the IGP is highly industrialized and very high population density region
potentially leading to high PM2.5 mass loading while districts of Dindori and Mandla are the least industrially
developed with huge forested cover (Kanha National Park) with negligible anthropogenic activities, potentially
leading to a cleaner environment when compared with the rest of MP. Seasonal PM2.5 maps. To examine the seasonal variation of surface PM2.5 over MP, mean seasonal maps were
generated utilizing the estimated daily PM2.5 maps for both 2018 and 2019, for a given season (Fig. 5). easonal PM2.5 maps. To examine the seasonal variation of surface PM2.5 over MP, mean seasonal maps were
enerated utilizing the estimated daily PM2.5 maps for both 2018 and 2019, for a given season (Fig. 5). PM2.5 mass was highest in the winter season (2018 and 2019 taken together) throughout MP with a mean
value of 82.54 µg m−3 and the lowest concentration was estimated during the monsoon season with a mean value
of 32.10 µg m−3. Mean PM2.5 concentrations in pre-monsoon and post-monsoon season were 60.14 µg m−3 and
71.51 µg m−3, respectively. Very high PM2.5 concentrations in northeastern MP and in Narmada valley during
the post-monsoon and winter season can be attributed to crop residue burning during these seasons and stable
atmosphere in low-lying areas. Low PM2.5 concentrations throughout MP during the monsoon season are argu-
ably due to wet deposition of atmospheric aerosols and change in synoptic meteorology fetching a lower load of
anthropogenic aerosols than during other seasons. https://doi.org/10.1038/s41598-020-79229-7 Scientific Reports | (2021) 11:544 | www.nature.com/scientificreports/ Table 3. Summary statistics of LME and LME + GWR mode (MT is model training and CV is cro
validation). www.nature.com/scientificreports/ Station
N
LME
LME + GWR
r2
RMSE (µg m−3)
MAE (µg m−3)
r2
RMSE (µg m−3)
MAE (µg m−3)
Bhopal
106
MT
0.530
23.50
20.19
0.584
22.47
19.30
CV
0.516
23.26
19.73
0.565
22.29
18.90
Damoh
353
MT
0.609
24.51
20.70
0.643
23.45
19.81
CV
0.605
27.52
23.83
0.637
26.38
22.85
Dewas
729
MT
0.385
18.57
13.21
0.424
17.75
12.63
CV
0.387
19.04
13.73
0.420
18.24
13.15
Indore
106
MT
0.450
24.67
19.95
0.498
23.55
19.05
CV
0.469
25.59
20.85
0.512
24.51
19.97
Jabalpur
104
MT
0.644
26.02
22.68
0.690
24.88
21.69
CV
0.644
31.12
26.71
0.687
29.83
25.60
Maihar
323
MT
0.343
21.05
16.68
0.364
20.14
15.96
CV
0.340
22.02
17.55
0.359
21.10
16.82
Mandideep
726
MT
0.481
20.93
14.84
0.524
20.00
14.18
CV
0.470
21.28
15.15
0.511
20.38
14.51
Pithampur
728
MT
0.524
16.08
11.78
0.558
15.37
11.25
CV
0.514
16.53
12.27
0.545
15.83
11.76
Ratlam
316
MT
0.402
18.63
14.35
0.435
17.81
13.73
CV
0.406
18.41
14.27
0.438
17.65
13.68
Singrauli
711
MT
0.652
34.32
25.99
0.701
32.80
24.84
CV
0.633
37.41
27.80
0.689
35.85
26.64
Ujjain
720
MT
0.573
19.27
12.93
0.614
18.42
12.36
CV
0.571
19.35
12.987
0.611
18.541
12.44 Table 3. Summary statistics of LME and LME + GWR mode (MT is model training and CV is cross
validation) Table 3. Summary statistics of LME and LME + GWR mode (MT is model training and CV is cross
validation). Figure 3. Comparison between the final model predicted PM2.5 using tenfold cross-validation and surface
concentration on days where MAIAC AOD was unavailable. c-axis shows the point count and the blue lines are
y = 2 × and y = x/2 while the black is x = y line. “m” is the slope if the regression is forced through origin. Figure 3. Comparison between the final model predicted PM2.5 using tenfold cross-validation and surface
concentration on days where MAIAC AOD was unavailable. c-axis shows the point count and the blue lines are
y = 2 × and y = x/2 while the black is x = y line. “m” is the slope if the regression is forced through origin. Population exposure to surface PM2.5. Integrated Exposure–response function (IER)40 has been used
widely to estimate the age and cause-specific mortality associated with exposure to PM2.5 concentrations. IER
estimates the risk function for a particular disease as a function of PM2.5 concentrations based upon previous
health studies. www.nature.com/scientificreports/ Equations (4)–(5) shows the IER framework that accounts for the dependence of relative risk
(RR) on PM2.5 concentrations, Cn. (4)
For Cn < Cncf , RRi(Cn) = 1 For Cn < Cncf , RRi(Cn) = 1 For Cn < Cncf , RRi(Cn) = 1 (4) Scientific Reports | (2021) 11:544 | https://doi.org/10.1038/s41598-020-79229-7 www.nature.com/scientificreports/ Figure 4. Spatial distribution of annual mean of daily surface PM2.5 over Madhya Pradesh for 2018 and 2019. The figure was generated using Python (version 3.7, https://www.python.org/). Figure 4. Spatial distribution of annual mean of daily surface PM2.5 over Madhya Pradesh for 2018 and 2019
The figure was generated using Python (version 3.7, https://www.python.org/). Figure 5. Season average (2018 and 2019) map of daily PM2.5 concentration over Madhya Pradesh. The figure
was generated using Python (version 3.7, https://www.python.org). Figure 5. Season average (2018 and 2019) map of daily PM2.5 concentration over Madhya Pradesh. The figure
was generated using Python (version 3.7, https://www.python.org). (5)
For Cn > Cncf , RRi(Cn) = 1 + αi
1 −exp
−γi
Cn −Cncf
δi (5) where. RR is the relative risk of ith disease for exposure to PM2.5 concentration of Cn, Cncf, counterfactual
concentration below which there is no associated risk due to PM2.5 (RR = 1) and ɑi, γi and δi are disease-spe-
cific parameters. Previous studies have reported that Lung Cancer (LNC), Ischemic Heart Disease (IHD),
Chronic Obstructive Pulmonary Disease (COPD) and Stroke deaths account for 97% of total deaths due to air
pollution41,42. Therefore in this study, we have estimated district-wise premature mortality in the adult popula-
tion (age > 25 years) due to LNC, IHD, COPD and Strokes using RR values provided in the look-up table by
Apte et al.43. In that study, age-independent RR values for LNC and COPD, and age-dependent RR values for
IHD and stroke for various PM2.5 concentrations were generated using the mean of 1000 IER curves44 and Cncf
was taken as 5.8 µg m−3. Disease-specific premature mortality for the ith district and jth age group was then
calculated using Eq. (6). https://doi.org/10.1038/s41598-020-79229-7 Scientific Reports | (2021) 11:544 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 6. Total premature deaths in MP due to exposure to ambient PM2.5 concentration during 2018–2019. This map is generated using QGIS 2.18.1 (http://www.qgis.org). Figure 6. Total premature deaths in MP due to exposure to ambient PM2.5 concentration during 2018–2019. This map is generated using QGIS 2.18.1 (http://www.qgis.org). Comparison with a CTM‑satellite AOD based global PM2.5 estimate p
g
2.5
An ancillary goal of this study was to assess the usefulness of our model in estimating surface PM2.5 compared to
other CTM output-satellite AOD approaches. In order to do so, we benchmarked annual surface PM2.5 estimated
in this study for locations with surface measurements in MP (Fig. 1) against recent estimates over the same
locations derived from Hammer et al.27. Our model results agreed better with surface measurements (r2 = 0.89)
compared to the CTM output-satellite AOD estimates (r2 = 0.55) (See Fig. 7). Also, to understand the spatial
variability in surface PM2.5 over MP estimated by the two approaches (this study and Hammer et al.27), difference
maps for 2018 and 2019 were generated (Supplemental Figure S19). These maps suggest that the CTM based
approach did not satisfactorily capture the spatial variation in surface PM2.5 over MP, while over-predicting PM2.5
over elevated regions and under-predicting its concentrations over the Narmada valley. www.nature.com/scientificreports/ (6)
M = BMj,k ×
RRi,k −1/RRi,k
× Popi,j (6) where BMj,k is the disease-specific baseline mortality rate for the jth age group for kth year obtained from GBD
India Compare Data Visualization (ICMR, PHFI, and IHME; 2019) for the year 2018 and 2019, RRi,k is rela-
tive risk for the kth year over ith district and Popi,j is the total population of the ith district in the age group “j”. Disease-wise baseline mortality rate was provided with 95% CI which was then translated to a 95% confidence
interval for disease-specific mortality over the MP. Further details on the data source and method are given in
the Supplemental Text S3. Finally, the total premature mortality was calculated by adding premature mortality
due to individual disease over MP.h District-wise population-weighted PM2.5 concentrations are shown in Supplemental Figure S17. The total
combined (2018 and 2019) premature mortality due to exposure to PM2.5 concentrations in MP is estimated to be
106,115.2 (85,717.46, 127,604.6) at 95% CI including deaths due to COPD 11,720.5 (8777.197, 14,114.21), IHD
55,501.79 (45,256.85, 66,811.22), LNC 1245.11 (1010.62, 1498.76) and strokes 37,467.7 (30,672.47, 45,180.44). IHD is the major cause of premature mortality in MP causing 52.3% of total deaths followed by Strokes (35.4%),
COPD (11.04) and LNC (1.17%). Indore city, which is the commercial capital of MP, with very high population
density, tops the number of premature deaths due to air pollution with 4853.30 (3921.40, 5836.10) total deaths
are 2018–2019 followed by Rewa, Jabalpur, Satna and Sagar. Disease-wise cause specific death for every city is
provided in Supplemental Table S8. Cause-specific deaths for the top 5 cities in MP are shown in Supplemental
Figure S18. Total premature mortality during 2018–2019 for districts in MP is shown in Fig. 6. www.nature.com/scientificreports/ Figure 7. Scatter plots between model derived annual mean concentration of daily surface PM2.5 and ground
observations (a) Hammer et al.27 model and (b) the model used in this study. The black dashed line shows x = y
and the translucent band represents 95% CI. Figure 7. Scatter plots between model derived annual mean concentration of daily surface PM2.5 and ground
observations (a) Hammer et al.27 model and (b) the model used in this study. The black dashed line shows x = y
and the translucent band represents 95% CI. seasons with a mean concentration of 32.10 µg m−3. Also, topography and land use has a strong influence on the
surface PM2.5 concentration in MP. IGP and low elevation areas were the most polluted while the high elevation
areas had the low PM2.5 concentrations. Indore city had the highest premature mortality in MP during 2018–2019
followed by Rewa, Jabalpur, Satna and Sagar illustrating the fact that air pollution and associated health burden
is not only a crowded commercial city problem in MP. This observation reiterates the need for current and future
air quality management strategies to focus on regional air quality issues and identify air quality management
districts for meaningful and effective public health protection. f
Missing AOD data is a major problem in accurately estimating the surface PM2.5 concentration for exposure
and epidemiological studies. This study has demonstrated one approach to address that problem. Although MP
is used as an illustrative example to elucidate the usefulness of the model developed in this study, the method is
robust and applicable across locations in the world. However, during the course of conducting this study, it was
observed that the rationale for choice of locations for the CPCB stations is neither clearly documented nor obvi-
ous. It appears that the choice of location is driven by considerations of determining NAAQS violations, logistics
and ease of operation. For instance, the data used in training the LME model in this study was from stations that
cannot be classified as either urban hotspots or regional background locations. Thus, a country-wide network of
ground monitoring stations, carefully situated in accordance with network design rules with sufficient density
to capture regional and/or urban aerosols are essential to effectively exploit satellite products to provide reliable
spatially continuous surface PM2.5 estimates. www.nature.com/scientificreports/ These estimates can then be used for planning both air quality
management strategies and to enhance population exposure studies and other epidemiological models that
assess the PM2.5 induced burden of disease. It is hoped that the availability of high time resolution surface PM2.5
measurements at several locations across India in conjunction with models, such as those developed in this study,
will help enhance GBD exposure assessment estimates for this region in the future. Data availabilityh y
There are no linked research data sets for this submission. All data used in this study are publicly available. W
links/citations as appropriate to the data used are listed in the manuscript. Received: 3 September 2020; Accepted: 7 December 2020 Received: 3 September 2020; Accepted: 7 December 2020 Conclusionsh This study developed a three-stage statistical model to generate full coverage daily 0.03° × 0.03° surface PM2.5
maps over Madhya Pradesh for 2018 and 2019 using MAIAC AOD, meteorological parameters and land use
information. On cross-validation, our final model was able to predict the surface PM2.5 with r2 of 0.55 and
RMSE of 22.92 µg m−3. Mean daily averaged PM2.5 concentration decreased from 58.19 µg m−3 during 2018 to
56.32 µg m−3 during 2019, over MP. Winter seasons had the highest PM2.5 loading with a mean concentration
of 82.54 µg m−3 (average of winter 2018 and 2019) and as expected the lowest loading was during the monsoon https://doi.org/10.1038/s41598-020-79229-7 Scientific Reports | (2021) 11:544 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Impact of anomalous forest fire on aerosol radiative forcing and snow cover over Himalayan
region. Atmos. Environ. 150, 264–275 (2017). 6. Ma, Z., Hu, X., Huang, L., Bi, J. & Liu, Y. Estimating ground-level PM2.5 in China using satellite remote sensing. Environ. Sci
Technol. 48, 7436–7444 (2014). 7. Ma, Z. et al. Satellite-based spatiotemporal trends in PM2.5 concentrations: China, 2004–2013. Environ. Health Perspect. 124
184–192 (2016). 8. Lee, H. J., Liu, Y., Coull, B. A., Schwartz, J. & Koutrakis, P. A novel calibration approach of MODIS AOD data to predict PM2.5
concentrations. Atmos. Chem. Phys. 11, 7991–8002 (2011). 39. Oshan, T., Li, Z., Kang, W., Wolf, L. J. & Fotheringham, A. S. mgwr: A Python implementation of multiscale geographically weighted
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40. Burnett, R. T. et al. An integrated risk function for estimating the global burden of disease attributable to ambient fine particulate
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41. Sahu, S. K. et al. Estimating ground level PM2.5 concentrations and associated health risk in India using satellite based AOD and
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42. WHO. 7 million premature deaths annually linked to air pollution (2014). 42. WHO. 7 million premature deaths annually linked to air po p
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43. Apte, J. S., Marshall, J. D., Cohen, A. J. & Brauer, M. Addressing global mortality from ambient PM2.5. Environ. Sci. Technol. 49,
8057–8066 (2015).i 4. Saini, P. & Sharma, M. Cause and age-specific premature mortality attributable to PM exposure: An analysis for Million-Plus
Indian cities. Sci. Total Environ. 710, 135230 (2020). www.nature.com/scientificreports/ www.nature.com/scientificreports/ 9. van Donkelaar, A., Martin, R. V. & Park, R. J. Estimating ground-level PM2.5using aerosol optical depth determined from satellite
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8. Liu, Y. et al. Mapping annual mean ground-level PM2.5 concentrations using Multiangle Imaging Spectroradiometer aerosol optical
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8. Liu, Y. et al. Mapping annual mean ground-level PM2.5 concentrations using Multiangle Imaging Spectroradiometer aerosol optical
thickness over the contiguous United States. J. Geophys. Res. Atmos. 109, D22206. https://doi.org/10.1029/2004JD005025 (2004). Scientific Reports | (2021) 11:544 | https://doi.org/10.1038/s41598-020-79229-7 Acknowledgementsh g
This work was carried out under the aegis of CREST at IISER Bhopal, a Center of Excellence established under
the Ministry of Human Resources (MHRD), Government of India, FAST scheme (MHRD/FAST/2016/17). The views expressed in this document are solely those of the authors and do not necessarily reflect those of the
Ministry. Neither IISER Bhopal nor the Ministry endorse any products or commercial services mentioned in this
publication. PM acknowledges DST INSPIRE for his BS-MS fellowship. The authors also gratefully acknowledge https://doi.org/10.1038/s41598-020-79229-7 Scientific Reports | (2021) 11:544 | www.nature.com/scientificreports/ Level-1 Atmosphere Archive & Distribution System (LAADS) for providing MAIAC data, Goddard Earth Sci-
ences Data and Information Services Center (GES DISC) for providing MERRA-2 data, Central Pollution Control
Board (CPCB) for providing surface PM2.5 data and the European Center for Medium-Range Weather Forecasts
(ECMWF) for land use maps. The authors would also like to thank Dr Melanie S. Hammer for providing global
annual mean PM2.5 concentrations data. Author contributions M: overall methodology, data curation, formal analysis, visualization and interpretation, writing—original draft
RSR: conceptualization, supervision, verification and interpretation, writing—reviewing and editing. Competing interests h p
g
The authors declare no competing interests. © The Author(s) 2021 Additional informationh Supplementary Information The online version contains supplementary material available at https://doi. org/10.1038/s41598-020-79229-7. Correspondence and requests for materials should be addressed to R.S.R. Correspondence and requests for materials should be addressed to R.S.R. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
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article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2021 https://doi.org/10.1038/s41598-020-79229-7 Scientific Reports | (2021) 11:544 |
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Wear evaluation of elements of V-belt transmission with the application of optical microscope
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MATEC web of conferences
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Wear
evaluation
of
elements
of
V-belt
transmission with the application of optical
microscope Piotr Krawiec1,*, Konrad Waluś1 Łukasz Warguła1, Jarosław Adamiec1 1Poznań University of Technology, Chair of Basics of MachineDesign, Piotrowo 3 60-965 Poznań,
Poland Abstract. The paper presents the analysis of reasons, form and wear of elements of
V-belt transmission. Syndromes of loss of driving properties for chosen wheels and
V-belts are specified. Methodology of failure evaluation of wheels and V-belts with
the application of optical microscope is proposed. The applied measuring set allows
for universal analysis of the measurement results. Operation principle of this device
and its measuring and analysing abilities are shown. Failures of chosen V-belts and
the influence of these failures on accuracy of kinematic features of chosen driving
units are evaluated. Keywords: V- belts, belt wear evaluation, optical measurements MATEC Web of Conferences 157, 01009 (2018)
MMS 2017 MATEC Web of Conferences 157, 01009 (2018)
MMS 2017 https://doi.org/10.1051/matecconf/201815701009 1 Introduction One of the first described applications of a rod was constructing a universal machine [1]. It
was a basis to construct many other machines in Egyptian times i.e. first versions of turning
lathe, transporting machines, etc. The beginning of using V-belts dates back to 1917 when
John Gates used them in a car powertrain (fig. 1). Fig. 1. The car in which V-belt was used for the first time [2] Fig. 1. The car in which V-belt was used for the first time [2] * Corresponding author: piotr.krawiec@put.poznan.pl * Corresponding author: piotr.krawiec@put.poznan.pl Reviewers: Andrej Czán, Juraj Gerlici © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons
Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). MATEC Web of Conferences 157, 01009 (2018)
MMS 2017 https://doi.org/10.1051/matecconf/201815701009 Due to their properties and low production costs V-belts and multi-groove belts are widely
used in many branches of industry. Their properties were also a starting point for
development research conducted by research centers and belts producers. There are a few
areas of research of V-belts and multi-groove belts such as: material, construction,
production technology [3]. The issues concerning the designing of integrated V-belts are
presented in articles [4-5]. The issues concerning the evaluation of properties of V-belts and
multi-groove belts are presented in articles [6-9]. This article tries to present the evaluation
of belt transmission used in alternator drive of engine of VW Transporter T4 1.9D. Multi-
groove belts, poly-V belt pulleys and belt tensioners. The evaluation of wear of belts,
tensioners’ pulleys was done with the use of optical microscope Bresser BIOLUX ICD
BINO 80x LED with camera CMOS 2 MP (fig. 2). Fig. 2. Optical microscope with the measurement set Fig. 2. Optical microscope with the measurement set 2 Typical causes of V-belts and Multi-groove belts failure The most common failures of V-belts and multi-groove belts are:
– Cracking of ribs. On the working surface of belt transversal cracks are visible, alongside
one or more ribs (fig. 3). This damage is probably caused by the influence of too high or
too low temperatures. Other reason of such failure may be the interaction between the belt
and pulley. It is especially important when the pulley diameter is small. If the interaction is
incorrect it may cause tensions which lead to cracks. The cracks appear on the top of the rib
and then deepen reaching down to the cord. Fig. 3. Multi-groove belt transversal cracks Fig. 3. Multi-groove belt transversal cracks 2 2 https://doi.org/10.1051/matecconf/201815701009 MATEC Web of Conferences 157, 01009 (2018)
MMS 2017 – Belt rubber flaking. When rubber flakes (fig. 4) then the belt may break immediately. Such state of the belt may be the result of many cracks cumulating in one place which are
parallel to the cord strand. Such state of belt is caused by high temperature, the duration of
using the belt and belt tensions. Fig. 4. Rubber flaking in multi-grove belt Fig. 4. Rubber flaking in multi-grove belt Fig. 4. Rubber flaking in multi-grove belt – The phenomenon of peeling. Peeling occurs (fig. 5) when the material of the belt is
rubbed off the tops of the ribs and accumulates in its grooves. One of the reasons of such
situation may be the improper tension of the belt, assembly mistakes or the wear of pulleys. This phenomenon is common for the belt drives of engines propelled by petrol or diesel oil. Fig. 5. Peeling phenomenon on multi-groove Belt
– Friction wear. The symptom of friction wear (fig. 6) is a shinny outer surface of the belt. If there is a severe wear of the belt then on its surface a piece of fabric may be visible. This
state of wear may be caused by a direct interaction of the belt with an object on its way
such as a flange or a screw. Other reason of appearing the friction wear may be the incor-
rect tension of the belt. Fig. 5. Peeling phenomenon on multi-groove Belt Fig. 5. Peeling phenomenon on multi-groove Belt Fig. 5. Peeling phenomenon on multi-groove Belt Fig. 5. Peeling phenomenon on multi-groove Belt – Friction wear. The symptom of friction wear (fig. 2 Typical causes of V-belts and Multi-groove belts failure 6) is a shinny outer surface of the belt. If there is a severe wear of the belt then on its surface a piece of fabric may be visible. This
state of wear may be caused by a direct interaction of the belt with an object on its way
such as a flange or a screw. Other reason of appearing the friction wear may be the incor-
rect tension of the belt. Fig. 6. The friction wear of multi-groove belt Fig. 6. The friction wear of multi-groove belt 3 MATEC Web of Conferences 157, 01009 (2018)
MMS 2017 https://doi.org/10.1051/matecconf/201815701009 – The damage of the external belt ribs. The symptoms of such state of belt (fig. 7) appear
on its sidewalls. The flat surface of the tie becomes shinny or in the most extreme cases the
cord on the sidewalls is ragged. The outermost belt ribs may be rubbed off or torn off. The
symptom of such failure may be the noise generated by the transmission. Such a result may
be caused by the misalignment of belt pulleys. The other symptom of such failure is bending or twisting of the belt Chile transmission
works. Fig. 7. The damage of the external belt ribs Fig. 7. The damage of the external belt ribs – The uneven wear of the belt ribs. This kind of wear (fig. 8) is indicated by the damage of
the sides of the tie, usually in a form of broken cord or ragged belt ribs. Other indicator of
this state of belt is a rumbling or scraping sound in the transmission. The cause of this form
of damage may be a foreign matter i.e. a small stone on the pulley. If nothing is done when
these symptoms occur then it may result in the uneven wear of the belt, cutting the material
of the belt ribs to the cord and eventually lead to its breaking. Fig. 8. The uneven wear of the belt ribs
– The influence of contaminations. When on the outer ( smooth) surface appear small punc-
tures or swellings ( fig. 9) and what is more, the fabric around the holes is ragged it may
suggests that some contaminations penetrate into the belt. The cause of this form of damage
is most often the situation when small stones, sand or gravel penetrate into the belt grooves
and pulley grooves. 2 Typical causes of V-belts and Multi-groove belts failure Poly – V belts are produced with different shapes of belt ribs, some of
them have sharp tops, others rounded and another ones cut off, as a result the interaction
with pulleys differs so the specks of metal are the effect of the pulley rubbing off. Fig. 8. The uneven wear of the belt ribs Fig. 8. The uneven wear of the belt ribs – The influence of contaminations. When on the outer ( smooth) surface appear small punc-
tures or swellings ( fig. 9) and what is more, the fabric around the holes is ragged it may
suggests that some contaminations penetrate into the belt. The cause of this form of damage
is most often the situation when small stones, sand or gravel penetrate into the belt grooves
and pulley grooves. Poly – V belts are produced with different shapes of belt ribs, some of
them have sharp tops, others rounded and another ones cut off, as a result the interaction
with pulleys differs so the specks of metal are the effect of the pulley rubbing off. 4 4 https://doi.org/10.1051/matecconf/201815701009 MATEC Web of Conferences 157, 01009 (2018)
MMS 2017 Fig. 9. The influence of contaminations on poly-v belt
– The band separation. This problem occurs when the outmost belt ribs start separating
from the rest of the material (fig. 10). When this process lasts longer then the whole rib
separates and causes the damage to the fabric and another belt ribs. The reason of such state
of the belt is the improper assembly of the transmission. Due to the improper assembly one
or more belt ribs is beyond the groove on the pulley what causes lack of corelation of belt
sidewalls that lowers significantly the duration of proper transmission work. Fig. 9. The influence of contaminations on poly-v belt Fig. 9. The influence of contaminations on poly-v belt – The band separation. This problem occurs when the outmost belt ribs start separating
from the rest of the material (fig. 10). When this process lasts longer then the whole rib
separates and causes the damage to the fabric and another belt ribs. The reason of such state
of the belt is the improper assembly of the transmission. 2 Typical causes of V-belts and Multi-groove belts failure Due to the improper assembly one
or more belt ribs is beyond the groove on the pulley what causes lack of corelation of belt
sidewalls that lowers significantly the duration of proper transmission work. Fig. 10. The band separation Fig. 10. The band separation – Contamination by fluids. The surface of the belt is flaking, sticky or swollen (fig. 11). This state of belt may be caused by oil, grease or chemical substances. These substances
weaken significantly the internal structure of the belt, influencing its stiffness. The belt
starts slipping on the surfaces of the pulleys. Inseparable symptom of this state is the in-
crease of the belt temperature. Fig. 11. Fluid contamination in poly-v belts
– Transversal cracking of the belt. This state of belt may be caused by a few reasons. One
of them is thrusting of a foreign matter and as a result cutting a piece of belt including the
cord. The second reason may be using too much strength when assembling the belt. Anoth-
er reason for cracking the belt may be a sudden change of the transmission load caused by a
blocked pulley. Fig. 11. Fluid contamination in poly-v belts Fig. 11. Fluid contamination in poly-v belts Fig. 11. Fluid contamination in poly-v belts – Transversal cracking of the belt. This state of belt may be caused by a few reasons. One
of them is thrusting of a foreign matter and as a result cutting a piece of belt including the
cord. The second reason may be using too much strength when assembling the belt. Anoth-
er reason for cracking the belt may be a sudden change of the transmission load caused by a
blocked pulley. 5 MATEC Web of Conferences 157, 01009 (2018)
MMS 2017 https://doi.org/10.1051/matecconf/201815701009 – Excessive noise. If the assembly is improper and the pulleys are misaligned then the ex-
cess ive noise may occur. The noisiness of belt transmission with poly-v belt may also
occur when the belt is very wide. In order to lower the noise level of transmission the belt
may be cut into two pieces decreasing its width or use two poly-v belts that work parallel. 3 The damage of poly-v belt pulleys Groove pulleys are widely use in the production of the means of transport. Cars used
nowadays have belt transmissions used i.e. to drive the alternator, power steering and air
conditioning compressor. As the cars have better performances (speed, torque) it causes
vibrations and increases the noise level in the transmission. The natural solutions to this
problem becomes applying in the car construction both silencers and transmission which
working principle limits the negative effects. The reasons for belt pulley failure (fig. 12)
may be: the assembly of the new pulley with the use of old or inappropriate screws as well
as assembly mistakes such as incorrect torque while tightening the screws. The symptoms of wear of multi-groove belt pulleys may be: The symptoms of wear of multi-groove belt pulleys may be: – loud work of the poly-v belt, – slipping of the poly-v belt, – rust on the outer part of the pulley, – the increase in the level of vibrations, – the deformation of the pulley grooves, cracks, – rubbing of the parts of belt. Fig. 12. New pulley and pulley exploited for about 250000 km Fig. 12. New pulley and pulley exploited for about 250000 km 4 The failures of multi-groove pulley tensioners The task of multi-groove pulley tensioners is to avoid excessive slippage, decrease the level
of vibrations and noise, as well as proper tension of the belt in the drive. 6 https://doi.org/10.1051/matecconf/201815701009 MATEC Web of Conferences 157, 01009 (2018)
MMS 2017 Fig. 13. The sample failure of multi-groove pulley tensioners Fig. 13. The sample failure of multi-groove pulley tensioners The most common reasons of multi-groove pulley tensioner failures (fig. 13) are: The most common reasons of multi-groove pulley tensioner failures (fig. 13) are:
– rusty dripstones and cracks. Rusty liquid flows between tensioner arm and base or it drips
from the tensioner. Rust leaks are the symptoms of internal wear of a part. Most of these
types of failure occur where the limiters and tensioner mounting screws are. yp
g
– the wear of pulley bearing. The symptom of this failure is noise, resistance while turn
the tensioner manually. – the wear of pulley bearing. The symptom of this failure is noise, resistance while turning
the tensioner manually. – the wear of the pulley. Correctly exploited pulleys of tensioners should be smooth and
without any chirps, cracks or contaminations in the grooves. The spokes on the side
surfaces of (plastic) pulleys should not be cracked or broken. – the wear of the pulley. Correctly exploited pulleys of tensioners should be smooth and
without any chirps, cracks or contaminations in the grooves. The spokes on the side
surfaces of (plastic) pulleys should not be cracked or broken. – assembly misalignment of the tensioner. The reason of this failure is lack of alignment
while assembling the tensioner. The direct reason of such failures may be misaligned base
of the tensioner, incorrect assembly of the tensioner or corrosion. – assembly misalignment of the tensioner. The reason of this failure is lack of alignment
while assembling the tensioner. The direct reason of such failures may be misaligned base
of the tensioner, incorrect assembly of the tensioner or corrosion. – noise coming from the tensioner. The noise from the tensioner is most of them caused by
the failure of the bearings. – noise coming from the tensioner. The noise from the tensioner is most of them caused by
the failure of the bearings. – misalignment of the tensioner arm. The main reason of this failure is improper belt
leading on the tensioner’s pulley. 4 The failures of multi-groove pulley tensioners The visible symptoms of this situation are shiny, smooth
strands or carves in the shield or tensioner arm. This situation may be caused by an
interaction between metal arm and metal shield of the spring. – excessive vibrations of tensioner arm. Vibrations of tensioner arm are caused by its
tendency to fall into constant vibrations. Such state is caused by an impropriate suppression
system or lowered spring tension. – excessive vibrations of tensioner arm. Vibrations of tensioner arm are caused by its
tendency to fall into constant vibrations. Such state is caused by an impropriate suppression
system or lowered spring tension. – the loss of spring properties. If there is no resistance detected while turning the tensioner,
it may mean that probably the spring lost its properties. Other symptoms of this failure are
belt noise resulting from slipping or torque speed drop of the propelled part. – the loss of spring properties. If there is no resistance detected while turning the tensioner,
it may mean that probably the spring lost its properties. Other symptoms of this failure are
belt noise resulting from slipping or torque speed drop of the propelled part. Conclusions and suggestions for designers As it results from
experimental research after one hour of work under full load, the belt lengthens by about 60
% of the total acceptable lengthiness, which is 1, 5 % of the belt length, – if one belt is broken in the belt transmission then all the belts should be exchanged,
– secure the transmission with a protective shield, – if one belt is broken in the belt transmission then all the belts should be exchanged,
– secure the transmission with a protective shield, – use proper assembly of belt tensioner and monitor its work regularly. Conclusions and suggestions for designers In order to assure proper time of failure- less work of belt pulley it is essential to: – while exchanging or assembling the belt to change the distance between the pulleys in
order to make it easier to fit the belt and then pull it to obtain a proper slippage (assuming
1,5 % tolerance of the belt total permissible lengthiness while work and 1% tolerance of the
belt length), g ),
– use only the pulleys that were manufactured in accordance with standards and catalogues
of prestigious producers. In case of poly-V belt pulleys only belt sheaves should be used
that consist of belts compiled especially in accordance with length tolerance, g )
– use only the pulleys that were manufactured in accordance with standards and catalogues
of prestigious producers. In case of poly-V belt pulleys only belt sheaves should be used
that consist of belts compiled especially in accordance with length tolerance, 7 MATEC Web of Conferences 157, 01009 (2018)
MMS 2017 https://doi.org/10.1051/matecconf/201815701009 – check if pulley and belt grooves are clean, damage less or without any anti-corrosion
substances, ,
– pay an extra attention to a proper assembly of a tie. The belt should be set in grooves in
such way that it adjoins its all sidewall with the walls of the groove, – pay an extra attention to a proper assembly of a tie. The belt should be set in grooves in
such way that it adjoins its all sidewall with the walls of the groove, – not use any substances that increase the adhesiveness of belt to the pulley, – remember that the acceptable misalignment of transmission pulley axis should not
exceed1 mm per 100 mm of axis length and the tolerance of mutual displacement of pulley
grooves should not exceed 0,25% of distance between the axis, during the first hours of its
work the belt stretches and then its tension should be corrected. References 1. F. M. Feldhaus, Maszyny w dziejach ludzkości. (PWN 1958)
2. www2.gates.com 1. F. M. Feldhaus, Maszyny w dziejach ludzkości. (PWN 1958)
2. www2.gates.com 3. M. Dudziak, Przekładnie cięgnowe. (PWN, Warszawa, 1997) 3. M. Dudziak, Przekładnie cięgnowe. (PWN, Warszawa, 1997) 4. M. Dudziak, P. Krawiec, R. Mostowski, Pasy zespolone Poli-V. Zeszyt Specjalny
Politechniki Lubelskiej, 229-234 (1995) 5. M. Dudziak, P. Krawiec, R. Mostowski, Aspekty doboru i zastosowań przekładni
pasowych z pasami zespolonymi. Zeszyt Specjalny Politechniki Warszawskiej, 121-126
(1995) 6. K. Kubas, A research stand for measuring friction parameters in a belt transmission. The Archives of Automotive Engineering 75, 69-82 (2017) 7. G. Sheng, H. Lee, V. Narravula, Experimental characterization and analysis of wet
belt friction and the vibro-acoustic behavior. Tribology International 44, 258-265
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Type I collagen promotes tumor progression of integrin β1 positive gastric cancer through a BCL9L/β-catenin signaling pathway
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ABSTRACT The mechanism of extracellular matrix induced tumor progression is poorly understood. Based on the TCGA
database and clinical tumor tissues analysis, we observed abundant type I collagen expression in tumor tissues
and poor overall survival in gastric patients with high integrin β1 (ITGB1) expression. In vitro, our study found
that 3D collagen culture promoted the capability of colony formation and growth in ITGB1 positive gastric
cancer, whereas limited colony growth was observed in ITGB1 negative gastric cancer, suggesting the role of
ITGB1 in type I collagen associated tumor progression. Mechanistically, we demonstrated that type I collagen
was capable of promoting the activation of BCL9L/β-catenin signaling pathway through ITGB1, thereby
contributing to the gastric cancer development. Subsequently, β-catenin signals further up-regulated the
expression anti-apoptosis protein BCL2, leading to the chemo-resistance in gastric cancer cells. Blockade of β-
catenin signals efficiently improved the anticancer effects of chemotherapy, providing an innovative sight for
clinical gastric cancer therapy. Research Paper
Type I collagen promotes tumor progression of integrin β1 positive
gastric cancer through a BCL9L/β-catenin signaling pathway Type I collagen promotes tumor progression of integrin β1 positive
gastric cancer through a BCL9L/β-catenin signaling pathway 1Department of Medical Oncology, The Fourth Hospital of Hebei Medical University, Hebei, China
2Department of Oncology, Cangzhou Central Hospital, Hebei, China
3Department of Immunology and Rheumatology, The Fourth Hospital of Hebei Medical University, Hebei, China
4Lifehealthcare Clinical Laboratories, Hangzhou, Zhejiang, China Correspondence to: Yalei Lv, Xiaoyun Zhang; email: lvyalei@hebmu.edu.cn, zxy20210426@hebmu.edu.cn
Keywords: type I collagen, integrin β1, BCL9L, β-catenin, gastric cancer
Abbreviations: ITGB1: integrin β1; CR: chemo-resistant; CS: chemo-sensitive; C-IN2: β-catenin inhibitor β-catenin-IN2
Received: January 1, 2021
Accepted: June 5, 2021
Published: July 28, 2021 Correspondence to: Yalei Lv, Xiaoyun Zhang; email: lvyalei@hebmu.edu.cn, zxy20210426@hebmu.edu.cn
Keywords: type I collagen, integrin β1, BCL9L, β-catenin, gastric cancer
Abbreviations: ITGB1: integrin β1; CR: chemo-resistant; CS: chemo-sensitive; C-IN2: β-catenin inhibitor β-catenin-IN2
Received: January 1, 2021
Accepted: June 5, 2021
Published: July 28, 2021 Copyright: © 2021 Lv et al. This is an open access article distributed under the terms of the Creative Commons Attribution
License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
author and source are credited. www.aging-us.com
AGING 2021, Vol. 13, No. 14
Research Paper
Type I collagen promotes tumor progression of integrin β1 positive
gastric cancer through a BCL9L/β-catenin signaling pathway
Yalei Lv1, Yujie Shan1, Lina Song2, Yufei Zhao3, Ruixue Lai3, Junyan Su4, Xiaoyun Zhang3
1Department of Medical Oncology, The Fourth Hospital of Hebei Medical University, Hebei, China
2Department of Oncology, Cangzhou Central Hospital, Hebei, China
3Department of Immunology and Rheumatology, The Fourth Hospital of Hebei Medical University, Hebei, China
4Lifehealthcare Clinical Laboratories, Hangzhou, Zhejiang, China
Correspondence to: Yalei Lv, Xiaoyun Zhang; email: lvyalei@hebmu.edu.cn, zxy20210426@hebmu.edu.cn
Keywords: type I collagen, integrin β1, BCL9L, β-catenin, gastric cancer
Abbreviations: ITGB1: integrin β1; CR: chemo-resistant; CS: chemo-sensitive; C-IN2: β-catenin inhibitor β-catenin-IN2
Received: January 1, 2021
Accepted: June 5, 2021
Published: July 28, 2021
Copyright: © 2021 Lv et al. This is an open access article distributed under the terms of the Creative Commons Attribution
License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
author and source are credited. www.aging-us.com
AGING 2021, Vol. 13, No. 14
Research Paper
Type I collagen promotes tumor progression of integrin β1 positive
gastric cancer through a BCL9L/β-catenin signaling pathway
Yalei Lv1, Yujie Shan1, Lina Song2, Yufei Zhao3, Ruixue Lai3, Junyan Su4, Xiaoyun Zhang3
1Department of Medical Oncology, The Fourth Hospital of Hebei Medical University, Hebei, China
2Department of Oncology, Cangzhou Central Hospital, Hebei, China
3Department of Immunology and Rheumatology, The Fourth Hospital of Hebei Medical University, Hebei, China
4Lifehealthcare Clinical Laboratories, Hangzhou, Zhejiang, China
Correspondence to: Yalei Lv, Xiaoyun Zhang; email: lvyalei@hebmu.edu.cn, zxy20210426@hebmu.edu.cn
Keywords: type I collagen, integrin β1, BCL9L, β-catenin, gastric cancer
Abbreviations: ITGB1: integrin β1; CR: chemo-resistant; CS: chemo-sensitive; C-IN2: β-catenin inhibitor β-catenin-IN2
Received: January 1, 2021
Accepted: June 5, 2021
Published: July 28, 2021
Copyright: © 2021 Lv et al. This is an open access article distributed under the terms of the Creative Commons Attribution
License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
author and source are credited. www.aging-us.com
AGING 2021, Vol. 13, No. 14
Research Paper
Type I collagen promotes tumor progression of integrin β1 positive
gastric cancer through a BCL9L/β-catenin signaling pathway
Yalei Lv1, Yujie Shan1, Lina Song2, Yufei Zhao3, Ruixue Lai3, Junyan Su4, Xiaoyun Zhang3
1Department of Medical Oncology, The Fourth Hospital of Hebei Medical University, Hebei, China
2Department of Oncology, Cangzhou Central Hospital, Hebei, China
3Department of Immunology and Rheumatology, The Fourth Hospital of Hebei Medical University, Hebei, China
4Lifehealthcare Clinical Laboratories, Hangzhou, Zhejiang, China
Correspondence to: Yalei Lv, Xiaoyun Zhang; email: lvyalei@hebmu.edu.cn, zxy20210426@hebmu.edu.cn
Keywords: type I collagen, integrin β1, BCL9L, β-catenin, gastric cancer
Abbreviations: ITGB1: integrin β1; CR: chemo-resistant; CS: chemo-sensitive; C-IN2: β-catenin inhibitor β-catenin-IN2
Received: January 1, 2021
Accepted: June 5, 2021
Published: July 28, 2021
Copyright: © 2021 Lv et al. This is an open access article distributed under the terms of the Creative Commons Attribution
License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
author and source are credited. AGING 2021, Vol. 13, No. 14 AGING 2021, Vol. 13, No. 14 www.aging-us.com INTRODUCTION which influence the tumor cells proliferation, integrins
associated signaling pathways activation and cellular
focal adhesion formation [4–6]. Increasing evidence has
suggested that extracellular matrix could provide
protection
against
chemotherapy-induced
cells
apoptosis, and mediate pro-survival signaling pathways
activation through bounding to integrins receptors on
tumor cells [7, 8]. Various compounds in extracellular
matrix have been suggested to be correlated with tumor
progression, including type I collagen [9] and laminin
[10]. However, the potential role of type I collagen, the
major component in extracellular matrix, still remains
controversial. Previous investigation has implicated that
type I collagen could facilitate tumor stemness and
promote the cells metastasis in a 3D collagen gel [11,
12]. However, the statistical analysis based on TCGA
database suggested a poor correlation between the type I Gastric cancer is one of the most common malignant
carcinomas with the third leading causes of cancer-
associated death worldwide [1]. Surgery combing
adjuvant chemotherapy is usually implemented for gastric
cancer patient treatment in early stage [2]. In metastatic
disease, gastric cancer patients suffered poor outcome,
with a median survival being around 1 year due to the
sustained tumor progression and developed chemo-
resistance [3]. However, the underlying mechanism of
tumor progression in gastric cancer remains controversial,
and there is urgent need to explore innovative approach to
elevate the treatment efficiency in gastric cancer therapy. Cancer development is bound up with diverse biological
process, including extracellular matrix alterations, AGING 19064 www.aging-us.com higher ITGB1 expression compared with patients in
stage I (Figure 1D). More importantly, poor overall
survival was observed in gastric cancer patients with
high ITGB1 expression (Figure 1E). Subsequently, we
further assessed the expression of type I collagen and
ITGB1 in those chemo-resistant gastric tumor tissues. Consistently, no significant difference was observed in
type I collagen expression between chemo-resistant
(CR) and chemo-sensitive (CS) tumor tissues from
gastric cancer patients (Figure 1F). As for ITGB1,
elevated expression of ITGB1 was found in CR tumor
tissues compared to CS group (Figure 1G). Together,
those results suggested elevated expression of type I
collagen and the positive correlation between ITGB1
and gastric cancer development. collagen expression and tumor prognosis in patients. Additionally, the role of matrix components in tumor
progression is currently unclear, and the underling
mechanism of cancer development induced by extra-
cellular matrix has yet to be developed. INTRODUCTION Here, our study further explored the role of type I
collagen in gastric cancer based on TCGA database and
clinical gastric tumor tissues analysis. We provided
evidence that type I collagen could mediated the
gastric cancer progression and chemotherapy resistance
through an ITGB1 dependent manner. Moreover, we
further disclosed the underlying of collagen/ITGB1
induced tumor progression, which was dependent on a
BCL9L/β-catenin/BCL2 signaling pathway. Suppression
of β-catenin signals efficiently improved the outcome of
chemotherapy, which provided an innovative approach
for gastric cancer treatment. RESULTS As ITGB1 correlated with the overall survival and
tumor progression in gastric cancer patients, we thus
questioned whether ITGB1 might regulate cells
proliferation to promote gastric cancer development. To
test this possibility, we sorted ITGB1 negative/positive
gastric cancer cells (SGC-7901 and BGC-823) and
examined
the
cells
proliferation. However,
no
significant difference was observed in cell proliferation
of ITGB1 positive/negative gastric cancer cells (Figure
2A). Given abundant expression of type I collagen in
tumor tissues, we next seeded those sorted gastric
cancer cells into 3D collagen gel. Intriguingly, ITGB1
positive SGC-7901 and BGC-823 cells revealed
significantly enhanced capability of 3D colony
formation (Figure 2B). More importantly, rapid colony
growth was observed in ITGB1 positive cancer cells
(Figure 2C), indicating that ITGB1 positive tumor cells
possessed proliferative characteristics in the presence of
collagen. Those results reminded us that type I collagen
might mediate the tumor cells proliferation through the
ITGB1 receptor. Meanwhile, the tumor progression
induced by ITGB1 was dependent on the presence of
type I collagen. To further confirm our hypothesis, we
subcutaneously injected sorted ITGB+/- SGC-7901
cells into NOD-SCID mice. The 3D collagen cultured
ITGB1+ tumor cells revealed strengthened tumorigenic
capability compared to ITGB1 negative cells or dish
cultured cells (Figure 2D). Subsequently, we further
examined the effects of ITGB1 and collagen on drugs
resistance. 5-FU is a nucleobase analogue that inhibits
DNA synthesis to slow gastric tumor growth. Here, we
sorted ITGB1+ and ITGB1- gastric cancer cells, which
were seeded into 3D collagen gels or not. After 6 days,
5-FU was used to treat those tumor cells and the
cytotoxicity of 5-FU was examined. Intriguingly, no
significant drugs resistance was observed in ITGB1+
cells or collagen cultured ITGB1- tumor cells, whereas ITGB1 expression promoted tumor progression in
gastric cancer This result indicated
that collagen could promote drugs resistance of gastric
cancer through ITGB1. Together, those results implicated
that collagen could mediate the tumor progression and
drugs resistance through the ITGB1 in gastric cancer. ITGB1 expression promoted tumor progression in
gastric cancer To explore the underlying mechanism of sustained
tumor progression in gastric cancer, we first analyzed
the TCGA database to comparing the top expression
genes between gastric tumor tissues and normal tissues
(Figure 1A). Notably, extremely high expression of
COL1A1, which encodes type I collagen, was found in
gastric cancer. To further confirm the pro-tumor effects
of COL1A1 on gastric tumor progression, we used
TCGA database to analyze the COL1A1 expression in
gastric cancer patients with different stages and the
overall survival. As shown in Figure 1B, elevated
expression of COL1A1 was observed in gastric tumor
tissues in high stage (stage II, III and IV) compared
with normal tissues or tumor tissues in stage I (Figure
1B). However, no significant difference was found in
overall survival of gastric cancer patients with high/low
COL1A1 expression (Figure 1C), reminding us that
COL1A1/type I collagen was not capable of regulating
gastric tumor progression directly. Increasing evidence
suggested that type I collagen could promote cancer
stem cells proliferation and facilitate pro-survival
signaling pathway activation through integrin receptors. Given the aberrant expression of COL1A1 in tumor
tissues, we supposed that type I collagen might play a
role through integrins signals in tumor development. ITGB1, belonging to the integrin family, has been
demonstrated to bind to type I collagen and mediate the
extracellular signals transduction [13, 14]. Intriguingly,
using TCGA database analysis, elevated expression
level of ITGB1 was found in gastric cancer tissues
compared to normal tissues. Meanwhile, gastric cancer
patients in high stage (stage II, III and IV) exhibited AGING 19065 www.aging-us.com we further examined the expression of BCL9 in sorted
ITGB+/- gastric cancer cells SGC-7901 and BGC-
823(cultured in 3D collagen gel or not). No significant
difference of BCL9 expression was observed in
collagen cultured ITGB1 positive cells. However,
upregulation of BCL9L, the paralog of BCL9 [17], was
found in collagen cultured ITGB1 positive cells (Figure
3A). To further confirm the role of BCL9L in ITBG1
induced tumor progression, we silenced BCL9L in
SGC-7901 and BGC-823 cells. As a result, blockade
of BCL9L suppressed the 3D colony formation
(Figure 3B) and tumorigenic capability (Figure 3C) of
ITGB1 positive cells in 3D collagen gels. The colony
growth of ITGB1 positive cells in 3D collagen gels
was suppressed by BCL9L silencing (Figure 3D). Meanwhile,
overexpression
of
BLC9L
efficiently ITGB1+ cells cultured in 3D collagen gels revealed
obvious 5-FU resistance (Figure 2E). Type I collagen mediated the BCL9L expression
through ITGB1 Next, we sought to investigate the underlying
mechanism of ITGB1 associated gastric cancer
progression and drugs resistance. BCL9 transcription
coactivator has been reported to mediate pro-survival
signaling pathways activation and correlated with
lymphoma and leukemia development [15, 16]. Here, Figure 1. Integrin β1 promoted gastric cancer progression. (A) Top 25 overexpression genes in gastric tumor tissues comparing with
normal tissues analyzed by TCGA database. (B) The relative expression of COL1A1 in normal tissues and gastric tumor tissues (stage I, II, III
and IV) analyzed by TCGA database. (C) The overall survival of gastric cancer patients with low/high COL1A1 expression analyzed by TCGA
database. (D) The relative expression of ITGB1 in normal tissues and gastric tumor tissues (stage I, II, III and IV) analyzed by TCGA database. (E) The overall survival of gastric cancer patients with low/high ITGB1 expression analyzed by TCGA database. (F) Relative expression of type I
collagen in chemo-sensitive (CS) and chemo-resistant (CR) tumor tissues from gastric patients, which was examined by
immunohistochemistry (n=15). The scale bar is 100 μm. (G) Relative expression of ITGB1 in chemo-sensitive (CS) and chemo-resistant (CR)
tumor tissues from gastric patients, which was examined by immunohistochemistry (n=15). The scale bar is 100 μm. *Indicates P <0.05, n.s
indicates no significant difference. Figure 1. Integrin β1 promoted gastric cancer progression. (A) Top 25 overexpression genes in gastric tumor tissues comparing with
normal tissues analyzed by TCGA database. (B) The relative expression of COL1A1 in normal tissues and gastric tumor tissues (stage I, II, III
and IV) analyzed by TCGA database. (C) The overall survival of gastric cancer patients with low/high COL1A1 expression analyzed by TCGA
database. (D) The relative expression of ITGB1 in normal tissues and gastric tumor tissues (stage I, II, III and IV) analyzed by TCGA database. (E) The overall survival of gastric cancer patients with low/high ITGB1 expression analyzed by TCGA database. (F) Relative expression of type I
collagen in chemo-sensitive (CS) and chemo-resistant (CR) tumor tissues from gastric patients, which was examined by
immunohistochemistry (n=15). The scale bar is 100 μm. (G) Relative expression of ITGB1 in chemo-sensitive (CS) and chemo-resistant (CR)
tumor tissues from gastric patients, which was examined by immunohistochemistry (n=15). The scale bar is 100 μm. *Indicates P <0.05, n.s
indicates no significant difference. AGING 19066 www.aging-us.com Figure 2. Collagen mediated gastric cancer progression through integrin β1. Type I collagen mediated the BCL9L expression
through ITGB1 (A) The proliferation of unsorted or ITGB1-/+ SGC-
7901/BGC-823 cells in 72 hours. (B) The 3D colony formation capability of unsorted or ITGB1-/+ SGC-7901/BGC-823 cells in 3D collagen gel. (C) The colony sizes of unsorted or ITGB1-/+ SGC-7901/BGC-823 cells in 3D collagen gel. The scale bar is 30 μm. (D) The ITGB1-/+ SGC-7901
cells were sorted and cultured in 3D collagen gel (6 days) or not. Then the tumorigenic capability of SGC-7901 in NOD-SCID mice were
examined. (E) The ITGB1-/+ SGC-7901 cells were sorted and cultured 3D collagen gel (6 days) or not. Then tumor cells were treated with 5-FU
(5 μg/ml) and the cell apoptosis was examined. *Indicates P <0.05, **Indicates P <0.01, n.s indicates no significant difference. Figure 2. Collagen mediated gastric cancer progression through integrin β1. (A) The proliferation of unsorted or ITGB1-/+ SGC-
7901/BGC-823 cells in 72 hours. (B) The 3D colony formation capability of unsorted or ITGB1-/+ SGC-7901/BGC-823 cells in 3D collagen gel. (C) The colony sizes of unsorted or ITGB1-/+ SGC-7901/BGC-823 cells in 3D collagen gel. The scale bar is 30 μm. (D) The ITGB1-/+ SGC-7901
cells were sorted and cultured in 3D collagen gel (6 days) or not. Then the tumorigenic capability of SGC-7901 in NOD-SCID mice were
examined. (E) The ITGB1-/+ SGC-7901 cells were sorted and cultured 3D collagen gel (6 days) or not. Then tumor cells were treated with 5-FU
(5 μg/ml) and the cell apoptosis was examined. *Indicates P <0.05, **Indicates P <0.01, n.s indicates no significant difference. Figure 3. BCL9L was up-regulated in 3D collagen cultured ITGB1+ gastric cancer. (A) The ITGB1-/+ SGC-7901 cells were sorted and
cultured in 3D collagen gel (6 days) or not. The expression of BCL9, BCL9L and β-actin was examined by western blotting. (B) The ITGB1+ SGC-
7901 cells were sorted and cultured in 3D collagen gel (6 days). Then tumor cells were treated with BCL9L shRNA or vector and the 3D colony
formation capability was examined. (C) The ITGB1+ SGC-7901 cells were sorted and cultured in 3D collagen gel (6 days). Then tumor cells
were treated with BCL9L shRNA or vector and the tumorigenic capability was examined. (D) The colony sizes of tumor cells in (C). The scale
bar is 30 μm. (E) The ITGB1+ SGC-7901 cells were sorted and cultured in 3D collagen gel (6 days). Then tumor cells were treated with BCL9L
shRNA or vector. Suppression of β-catenin signals improved outcome
of chemotherapy in gastric cancer strengthened the colony formation capability of ITGB
negative BGC-7901 and BGC-823 cells (Supplementary
Figure 1A, 1B). Next, we further examined the
association between BCL9L and drugs resistance in
gastric cancer. Silencing BCL9L efficiently reversed the
5-FU resistance in collagen cultured ITGB1 positive cells
(Figure 3E). Consistently, elevated expression of BCL9L
was observed in tumor tissues from CR patients (Figure
3F). Together, those results suggested that BCL9L is in
part relevant to the tumorigenic potential conferred by
ITGB1. Our previous results have provided evidence that
activation
of
β-catenin
signals
induced
by
collagen/ITGB1 could facilitate the chemotherapy
resistance in gastric cancer. To improve the outcome of
chemotherapeutic agents and suppress gastric cancer
progression, it might be feasible to block β-catenin
signals to improve the anticancer effects of chemo-
therapy. Here,
we
employed
C-IN2
combing
chemotherapeutic 5-FU to treat subcutaneous gastric
tumor bearing mice model. Gastric tumor bearing mice
models were constructed by subcutaneously injecting
SGC-7901 cells into NOD-SCID mice. The tumor
bearing mice were grouped randomly and received PBS,
5-FU monotherapy or combining with C-IN2 on day 14. After treatment of 2 weeks, we found that 5-FU or C-
IN2 monotherapy could suppress the tumor growth
slightly,
whereas
combination
group
revealed
significant tumor suppressive effects (Figure 5A). Additionally, the mice in combination group exhibited
obvious prolonged survival time compared to PBS or
monotherapy groups (Figure 5B and Table 1),
indicating that suppression of β-catenin signals
strengthened the anticancer effects of chemotherapy. To
further explore the anticancer effects of C-IN2 in
ITGB1 positive gastric tumor tissues, we established
subcutaneous gastric tumor bearing mice model by
injecting 3D collagen cultured ITGB1 positive SGC-
7901 cells into NOD-SCID mice, then mice were
treated mice with PBS, 5-FU monotherapy or
combining with C-IN2. Intriguingly, 5-FU monotherapy
could not suppress the tumor growth or prolonged
survival time of tumor bearing mice, which might be
due to the drugs resistance induced by ITGB1. However, 5-FU combing C-IN2 revealed obvious
anticancer effects (Figure 5C, 5D and Table 2). These
results reminded that blockade of β-catenin signals to
combine with chemotherapy could efficiently reverse
the drugs resistance induced by ITGB1 and improved
the anticancer effects, providing an innovative approach
for gastric cancer therapy. Type I collagen mediated the BCL9L expression
through ITGB1 Then tumor cells were treated with 5-FU (5 μg/ml) and the cell apoptosis was examined. (F) Western blotting of BCL9L and
β-actin in chemo-sensitive (CS) and chemo-resistant (CR) tissues from gastric patients. *Indicates P <0.05, ** Indicates P <0.01. Figure 3. BCL9L was up-regulated in 3D collagen cultured ITGB1+ gastric cancer. (A) The ITGB1-/+ Figure 3. BCL9L was up-regulated in 3D collagen cultured ITGB1+ gastric cancer. (A) The ITGB1-/+ SGC-7901 cells were sorted and
cultured in 3D collagen gel (6 days) or not. The expression of BCL9, BCL9L and β-actin was examined by western blotting. (B) The ITGB1+ SGC-
7901 cells were sorted and cultured in 3D collagen gel (6 days). Then tumor cells were treated with BCL9L shRNA or vector and the 3D colony
formation capability was examined. (C) The ITGB1+ SGC-7901 cells were sorted and cultured in 3D collagen gel (6 days). Then tumor cells
were treated with BCL9L shRNA or vector and the tumorigenic capability was examined. (D) The colony sizes of tumor cells in (C). The scale
bar is 30 μm. (E) The ITGB1+ SGC-7901 cells were sorted and cultured in 3D collagen gel (6 days). Then tumor cells were treated with BCL9L
shRNA or vector. Then tumor cells were treated with 5-FU (5 μg/ml) and the cell apoptosis was examined. (F) Western blotting of BCL9L and
β-actin in chemo-sensitive (CS) and chemo-resistant (CR) tissues from gastric patients. *Indicates P <0.05, ** Indicates P <0.01. AGING 19067 www.aging-us.com BCL9L induced activation of β-catenin/BCL2 signal
pathway to regulate gastric cancer progression Since Wnt/β-catenin served as the downstream signal of
BCL9 [18, 19], we supposed that ITGB1 might upregulate
BCL9L to mediate the activation of pro-survival β-catenin
signals in gastric cancer. Here, we found enhanced
expression of β-catenin in collagen cultured ITGB1
positive cells (Figure 4A). More importantly, suppression
of BCL9L by sh RNA also suppressed the up-regulation
of β-catenin in collagen cultured ITGB1 positive cells
(Figure 4B), indicating that BCL9L mediated the
activation of β-catenin signals in gastric cancer. Next, we
used a β-catenin inhibitor β-catenin-IN2 (C-IN2) (Ann
Marie Bode, et al. Inhibitors of beta-catenin in treatment
of colorectal cancer. Patent US20150374662A1.) to
treated collagen cultured ITGB1 positive cells. As a
result, β-catenin inhibition obviously suppressed the 3D
colony formation (Figure 4C) and tumorigenic rate
(Figure 4D) of collagen cultured ITGB1 positive cells. The colony growth was inhibited by C-IN2 as well
(Figure 4E). Subsequently, we examined the expression of
anti-apoptotic protein BCL2, which have been reported to
be up-regulated by β-catenin signals in tumor cells [20]. We found upregulation of BCL2 in collagen cultured
ITGB1 positive cells, whereas suppression of β-catenin by
C-IN2 suppressed BCL2 upregulation (Figure 4F). Subsequently, we suppressed β-catenin and BCL2
expression in collagen cultured ITGB1 positive cells, and
then examined the cytotoxicity of 5-FU to gastric cancer
cells. Consistently, suppression of β-catenin and BCL2
efficiently reversed the drugs resistance of collagen
cultured ITGB1 positive cells (Figure 4G). And
overexpression of BCL2 suppressed the effects of C-IN2
and strengthened the drugs resistance of collagen cultured
ITGB1 positive cells (Supplementary Figure 1C, 1D). To
further confirm the role of β-catenin and BCL2 in gastric
resistance development, we examined the expression of β-
catenin and BCL2 in tumor tissues from patients. Consistently, tumor tissues from CR gastric cancer
patients revealed higher expression of BCL2 (Figure 4H)
and β-catenin (Figure 4I). Those results suggested that
BCL9L mediated activation of β-catenin/BCL2 signal
pathway to regulate gastric cancer progression. DISCUSSION The mechanism of extracellular matrix induced tumor
progression is still poorly understood. In our study, we
provided evidence that type I collagen served as
fundamental component in extracellular matrix to
facilitate the tumor progression through an ITGB1
dependent manner. ITGB1 positive gastric cancer cells
possessed strengthened capability of colony formation
and proliferative characteristics in the presence of type I
collagen. More importantly, we demonstrated that type I
collagen could upregulate the expression of BCL9L AGING 19068 www.aging-us.com through ITGB1, eventually resulting in the activation β-
catenin signaling pathway. Subsequently, the intranuclear
β-catenin further up-regulated anti-apoptosis protein
BCL2, leading to the chemotherapy resistance (Figure
5E). Here, we combined β-catenin inhibitor C-IN2 with
chemotherapeutic agents to treat subcutaneous SGC-7901
tumor bearing mice. C-IN2 treatment efficiently
strengthened the anticancer effects of 5-FU and prolonged
the survival time of tumor bearing mice, which described
novel strategy for gastric cancer. as crucial participant in regulating tumor progression
[21]. A bulk of collagen associated genes have
previously been proved to be upregulated and
participated in the process of tumor development, such
as COL6A3 in pancreatic cancer [22]. Compelling
findings have implicated that the presence of type I
collagen and related signaling pathway are tightly
associated with chemo-resistance and tumor metastasis
[14, 23]. Increasing studies also suggested that the 3D
collagen culture based on type I collagen could facilitate
breast cancer cells stemness and invasion in vitro [24]. However, the correlation analysis based on TCGA
database or clinical samples implicated that the Among the components with significant correlation to
tumor progression, collagen family have been emerging Figure 4. BCL9L mediated gastric progression through downstream β-catenin and BCL2. (A) ITGB1-/+ SGC-7901/BGC-823 cells were
sorted and cultured with 3D collagen gel (6 days) or not. The expression of β-catenin and β-actin was examined by western blotting. (B) ITGB1+
SGC-7901/BGC-823 cells were cultured in 3D collagen gels (6 days) and treated with BCL9L shRNA or vector. Then the expression of β-catenin and
β-actin was examined by western blotting. (C) ITGB1+ SGC-7901/BGC-823 cells were cultured in 3D collagen gels (6 days) and treated with PBS or
C-IN2 (5 μM). Then the 3D colony formation capability was examined. (D) ITGB1+ SGC-7901/BGC-823 cells were cultured in 3D collagen gels (6
days) and treated with PBS or C-IN2 (5 μM). Then the 3D colony formation capability was examined. Then the tumorigenic capability was
examined in NOD-SCID mice. DISCUSSION (E) ITGB1+ SGC-7901/BGC-823 cells were cultured in 3D collagen gels (6 days) and treated with PBS or C-IN2 (5 μM). Then the colony sizes were examined. The scale bar is 30 μm. (F) ITGB1-/+ SGC-7901/BGC-823 cells were cultured in 3D collagen gel (6 days) and
treated with PBS or C-IN2 (5 μM). Then the expression of BCL2 and β-actin was examined by western blotting. (G) ITGB1+ SGC-7901/BGC-823
cells were cultured in 3D collagen gel (6 days) and treated with PBS, C-IN2 (5 μM) or BCL2 shRNA. Then tumor cells were treated with 5-FU (5
μg/ml) and the apoptosis was examined. (H) Immunohistochemical staining of β-catenin in chemo-sensitive (CS) and chemo-resistant (CR) tissues
from gastric patients. The scale bar is 100 μm. (I) immunohistochemical staining of BCL2 in chemo-sensitive (CS) and chemo-resistant (CR) tissues
from gastric patients. The scale bar is 100 μm. * Indicates P <0.05, ** Indicates P <0.01. Figure 4. BCL9L mediated gastric progression through downstream β-catenin and BCL2. (A) ITGB1-/+ SGC-7901/BGC-823 cells were
sorted and cultured with 3D collagen gel (6 days) or not. The expression of β-catenin and β-actin was examined by western blotting. (B) ITGB1+
SGC-7901/BGC-823 cells were cultured in 3D collagen gels (6 days) and treated with BCL9L shRNA or vector. Then the expression of β-catenin and
β-actin was examined by western blotting. (C) ITGB1+ SGC-7901/BGC-823 cells were cultured in 3D collagen gels (6 days) and treated with PBS or
C-IN2 (5 μM). Then the 3D colony formation capability was examined. (D) ITGB1+ SGC-7901/BGC-823 cells were cultured in 3D collagen gels (6
days) and treated with PBS or C-IN2 (5 μM). Then the 3D colony formation capability was examined. Then the tumorigenic capability was
examined in NOD-SCID mice. (E) ITGB1+ SGC-7901/BGC-823 cells were cultured in 3D collagen gels (6 days) and treated with PBS or C-IN2 (5 μM). Then the colony sizes were examined. The scale bar is 30 μm. (F) ITGB1-/+ SGC-7901/BGC-823 cells were cultured in 3D collagen gel (6 days) and
treated with PBS or C-IN2 (5 μM). Then the expression of BCL2 and β-actin was examined by western blotting. (G) ITGB1+ SGC-7901/BGC-823
cells were cultured in 3D collagen gel (6 days) and treated with PBS, C-IN2 (5 μM) or BCL2 shRNA. Then tumor cells were treated with 5-FU (5
μg/ml) and the apoptosis was examined. (H) Immunohistochemical staining of β-catenin in chemo-sensitive (CS) and chemo-resistant (CR) tissues
from gastric patients. DISCUSSION The scale bar is 100 μm. (I) immunohistochemical staining of BCL2 in chemo-sensitive (CS) and chemo-resistant (CR) tissues
from gastric patients. The scale bar is 100 μm. * Indicates P <0.05, ** Indicates P <0.01. AGING 19069 www.aging-us.com AKT [28] and STAT3 [29, 30], are reported to be
upregulated by integrins signals in cancer cells. Our
study further demonstrated that type I collagen could
mediate the activation of ITGB1/BCL9L/β-catenin
signaling pathway, leading to the tumor progression and
drugs resistance. Previous reports have implicated the
role of BCL9 in tumor development [31, 32] and our
study further disclosed the pro-tumor effects of BCL9
paralog BCL9L, which could promote the β-catenin
signals activation in gastric cancer. Given the crucial
role of β-catenin in chemo-resistance development, we
then suppressed the β-catenin signals by C-IN2 to
improve outcome of chemotherapy. Notably, C-IN2
treated groups revealed strengthened anticancer effects
and efficiently reversed the drugs resistance induced by
ITGB1 and collagen. More importantly, no significant
weight loss or side effects were observed in C-IN2
treated mice, suggesting the potential application of β-
catenin inhibitors in gastric cancer treatment. However,
the further application of integrin inhibitors in gastric
needed to be validated in larger cohort of gastric cancer
patients. And the systemic toxicity evaluation of
combination therapy remained to be further assessed. Meanwhile, knockdown of β-catenin by Crisp/Cas9
or shRNA to explore the role of β-catenin in gastric
cancer development might further demonstrated our
hypothesis. Our study indicated the elevated expression expression of type I collagen is prone to be not positive
correlated to tumor progression in several tumor types. Here, we observed abundant type I collagen distribution
in gastric cancer tissues in all tumor stages. No
significant difference was observed in the overall
survival between patients with high/low type I collagen
expression. However, we found the presence of type I
collagen could promote ITGB1 positive gastric cancer
cells colony formation and growth, thereby resulting in
the tumor progression. More importantly, the expression
of ITGB1 revealed a high correlation with the overall
survival of gastric cancer patients. Our results further
disclosed the role of type I collagen in tumor
progression, which is dependent on collagen binding
integrin receptor ITGB1. Given the abundant expression
of collagen in most tumor types, it might be befitting to
focus on the expression of collagen binding integrin
receptors for tumor diagnosis or prediction. DISCUSSION Increasing evidence have suggested that collagen could
mediate activation of specific pro-survival signaling
pathways in diverse tumor cells. Type VI collagen was
recently demonstrated to inhibit apoptosis through
suppressing the expression of Bax [25]. Type I collagen
could mediate the prostate cancer invasion through
RhoC GTPase and integrin α2β1 signals [26]. Moreover, some pro-survival signals, such as FAK [27], Figure 5. Blockade of β-catenin signals improved outcome of chemotherapy. (A) Tumor volume of SGC-7901 bearing mice treated
with PBS, C-IN2, 5-FU and C-IN2 combined with 5-FU. (B) Overall survival of SGC-7901 bearing mice treated with PBS, C-IN2, 5-FU and C-IN2
combined with 5-FU. (C) Tumor volume of ITGB1+ SGC-7901 bearing mice treated with PBS, C-IN2, 5-FU and C-IN2 combined with 5-FU. (D)
Overall survival of ITGB1+ SGC-7901 bearing mice treated with PBS, C-IN2, 5-FU and C-IN2 combined with 5-FU. (E) Schematic diagram of
ITGB1 induced tumor progression in gastric cancer. * indicates P <0.05, ** Indicates P <0.01, n.s indicates no significant difference. Figure 5. Blockade of β-catenin signals improved outcome of chemotherapy. (A) Tumor volume of SGC-7901 bearing mice treated
with PBS, C-IN2, 5-FU and C-IN2 combined with 5-FU. (B) Overall survival of SGC-7901 bearing mice treated with PBS, C-IN2, 5-FU and C-IN2
combined with 5-FU. (C) Tumor volume of ITGB1+ SGC-7901 bearing mice treated with PBS, C-IN2, 5-FU and C-IN2 combined with 5-FU. (D)
Overall survival of ITGB1+ SGC-7901 bearing mice treated with PBS, C-IN2, 5-FU and C-IN2 combined with 5-FU. (E) Schematic diagram of
ITGB1 induced tumor progression in gastric cancer. * indicates P <0.05, ** Indicates P <0.01, n.s indicates no significant difference. AGING 19070 www.aging-us.com Table 1. The median survival and maximum survival time of SGC-
7901 bearing mice treated with PBS, C-IN2, 5-FU and C-IN2
combined with 5-FU. Group
Median survival (days)
Maximum survival (days)
PBS
41
49
c-IN2
45
55
5-FU
50
62
C+5FU
63
67
The median survival and maximum survival time of SGC-7901 bearing mice
treated with PBS, C-IN2, 5-FU and C-IN2 combined with 5-FU. Table 1. The median survival and maximum survival time of SGC-
7901 bearing mice treated with PBS, C-IN2, 5-FU and C-IN2
combined with 5-FU. Table 1. The median survival and maximum survival time of SGC-
7901 bearing mice treated with PBS, C-IN2, 5-FU and C-IN2
combined with 5-FU. Patients’ tumor tissues collection of ITGB1 in gastric cancer and its’ association with
collagen, which might be benefit from targeted
therapies in further clinical trials. The tumor tissues of gastric cancer patients were
obtained from the Fourth Hospital of Hebei Medical
University. Samples were collected and sent to the
laboratory within 2 hours. According to the response to
chemotherapy in our follow-up study, the tumor tissues
were divided into chemo-resistant (CR) and chemo-
sensitive (CS) groups. All tumor samples were in stage
T2~T3 and the follow-op study was performed after
surgical
operation. All
samples
collection
and
processing were carried out respecting the Declaration
of Helsinki. All patients signed informed consent prior
to tumor tissues collection treatment, including allowing
their data to be used for further research. All subjects
gave written informed consent. Ethical approval was
obtained from the Committee of the Fourth Hospital of
Hebei Medical University. In conclusion, we showed that type I collagen could
mediate the tumor progression and chemotherapy
though an ITGB1 based manner, which was dependent
on
BCL9L/β-catenin/BCL2
signaling
pathway. Blockade of β-catenin strengthened the anticancer of
chemotherapy, describing an innovative approach for
gastric cancer treatment. DISCUSSION Group
Median survival (days)
Maximum survival (days)
PBS
41
49
c-IN2
45
55
5-FU
50
62
C+5FU
63
67
The median survival and maximum survival time of SGC-7901 bearing mice
treated with PBS, C-IN2, 5-FU and C-IN2 combined with 5-FU. The median survival and maximum survival time of SGC-7901 bearing mice
treated with PBS, C-IN2, 5-FU and C-IN2 combined with 5-FU. Table 2. The median survival and maximum survival time of
ITGB1+ SGC-7901 bearing mice treated with PBS, C-IN2, 5-FU and
C-IN2 combined with 5-FU. Group
Median survival (days)
Maximum survival (days)
PBS
38
48
c-IN2
51
59
5-FU
39
52
C+5FU
57
64
The median survival and maximum survival time of ITGB1+ SGC-7901 bearing
mice treated with PBS, C-IN2, 5-FU and C-IN2 combined with 5-FU. Table 2. The median survival and maximum survival time of
ITGB1+ SGC-7901 bearing mice treated with PBS, C-IN2, 5-FU and
C-IN2 combined with 5-FU. Table 2. The median survival and maximum survival time of
ITGB1+ SGC-7901 bearing mice treated with PBS, C-IN2, 5-FU and
C-IN2 combined with 5-FU. The median survival and maximum survival time of ITGB1+ SGC-7901 bearing
mice treated with PBS, C-IN2, 5-FU and C-IN2 combined with 5-FU. Cell lines and reagents Human gastric cancer cells SGC7901 and BGC-823
were purchased from the American Type Culture
Collection (Maryland, USA) and maintained in 1640
complete medium (Gibco, CA, USA) supplemented
with 10% fetal calf serum (Gibco, CA, USA) at 37° C
in 5% CO2 atmosphere. β-catenin inhibitor β-catenin-
IN2 (C-IN2) was purchased from MedChemExpress
(N.J, USA). 5-FU was purchased from Sangon
(Shanghai, China). Type I collagen and collagenase
were purchased from Thermo (MA, USA). Other
reagents were purchased from Solarbio (Beijing, China)
and of HPLC standard. 3D collagen gel culture 3D collagen gel culture was performed according
previous report [33]. Briefly, type I collagen was
diluted to 0.6 mg/ml with culture medium. 5 × 104
sorted tumor cells were added into the collagen
solution and mixed thoroughly. Next, 25 μl 10 × PBS
and 20 μl NaOH (1 M) solution were added into the
solution, and 295 μl of the mixture was seeded into AGING 19071 www.aging-us.com minutes at room temperature. The samples were then
washed
and
sorted
with
flow
cytometer
(Accuri®C6,
Becton
Dickinson,
NJ,
USA). Each
experiment
was
performed
three
times,
independently. 24-well plate. After 2 hours of 37° C incubation, the
solid clotty collagen (containing tumor cells) was
removed
into
RPMI
1640
complete
medium
supplemented with 10% fetal calf serum for cells
culture. After 4~6 days, the cells were digested by
type I collagenase and trypsin for further analysis. minutes at room temperature. The samples were then
washed
and
sorted
with
flow
cytometer
(Accuri®C6,
Becton
Dickinson,
NJ,
USA). Each
experiment
was
performed
three
times,
independently. Gene interference All constructs for functional analyses were designed
according to the lentiviral plasmid pHR-SIN-CSGW-
ΔNot, which was generated by partial NotI-digest
and subsequent fill-in reaction of one of the 2 NotI
sites originally present in the primary lentiviral
plasmid pHR-SIN-CSGW10 (Hanbio, Beijing, China). Complementary shRNA oligonucleotides directed
against either BCL9L or BCL2 were synthesized by
Hanbio (Beijing, China). Each experiment was
performed three times, independently. Cell proliferation and 3D colony formation analysis Gastric tissues dissected from patients were fixed in 4%
paraformaldehyde for 48 hours and sectioned at a
thickness
of
4
μm. The
sections
were
then
deparaffinized and rehydrated in alcohol and water. Antigen retrieval was performed in sodium citrate
buffer for five minutes at 100° C. Hydrogen peroxide
(0.3%) was used to block peroxidase. Then, the sections
were incubated with anti-collagen (1:200, Abcam,
Cambridge,
UK),
anti-ITGB1
(1:200,
Abcam,
Cambridge,
UK),
anti-BCL2
(1:200,
Abcam,
Cambridge, UK) or anti-β-catenin (1:200, Abcam,
Cambridge, UK) primary antibodies at 4° C overnight. After being washed with PBS, the samples were
incubated with goat anti-rabbit secondary antibodies
(HRP, 1:1000, Abcam, Cambridge, UK). Images were
obtained using microscope (DM4 M, Leica, Germany)
and the relative intensity of proteins was calculated by
Image-Pro Plus 6.0 software. 15 tumor samples were
calculated in each group (CS and CR groups). 20 fields
were pictured for protein intensity analysis in each
sample and at least 100 tumor cells were pictured in
each field. Using the 30 samples, each experiment was
performed at least three times, independently. The cell proliferation of SGC-7901 and BGC-823 was
performed using a MTT analysis kit (Solarbio, Beijing,
China). Briefly, 3000 sorted tumor cells were seeded
into the 96-well plates with 200 μl 1640 complete
medium supplemented with 10% fetal calf serum. After
24, 48 and 72 hours, 5 μl MMT solution was added into
the 96-well plated. After 2 hours of incubation, the
culture medium was removed and 100 μl DMSO was
added into the 96-wells. Absorbance was measured at
570 nm on a microplate reader (Bio-Rad, MA, USA). For the 3D colony formation assay, sorted tumor cells
(~200 cells/well) were seeded in the 3D collagen gel. After 4 days, the cell colonies were imaged and
counted. The colony sizes were calculated by image J
2.3 software. 50 colonies were pictured and the sizes
were calculated in each group. Each experiment was
performed three times, independently. Cells apoptosis analysis Cells apoptosis was analyzed using the FITC-Annexin
V and PE-PI apoptosis detection kit (BD, NJ, USA). Briefly, tumor cells were collected and stained with
FITC-Annexin V and PE-PI viability staining solution
for 15 min. Subsequently, apoptosis was detected
by flow cytometry on flow cytometer (Accuri®C6,
Becton Dickinson, NJ, USA). Each experiment was
performed three times, independently. Western blotting Radioimmunoprecipitation assay buffer (Beyotime,
Beijing,
China)
containing
protease
inhibitors
(Beyotime, Beijing, China) was used to lyse the treated
cells. 20 μg protein samples were separated via sodium
dodecyl sulfate polyacrylamide gel electrophoresis and
transferred
onto
nitrocellulose
membranes. The
following primary antibodies were used: anti-BCL9L
(1:500, Abcam, Cambridge, UK), anti-BCL9 (1:500,
Abcam, Cambridge, UK), anti-β-actin (1:1000, Abcam,
Cambridge, UK) and anti-β-catenin (1:5000, Abcam,
Cambridge, UK). Proteins were detected using a
chemiluminescence kit (Beyotime, Beijing, China). The
expression of β-actin was used as an internal control. Each
experiment
was
performed
three
times,
independently. CONFLICTS OF INTEREST Animal Care and Use Committee of the Fourth Hospital
of
Hebei
Medical
University,
Protocol
ID:
EMC20121128c. All
applicable
institutional
and
governmental regulations concerning the ethical use of
animals were followed. For tumorigenic analysis, 105
SGC-7901 cells were digested and resuspended in PBS
and subcutaneously injected into the NOD-SCID mice. After 25 days, the tumorigenic rate of SGC-7901 cells in
NOD-SCID mice was analyzed. 10 mice were calculated
in each group. The authors declare that they have no conflicts of
interest. Flow cytometry Female NOD-SCID mice (6–8 weeks old) were purchased
from Huafukang (Beijing, China). All mice were housed
in a specific pathogen-free facility. All animal protocols in
our experiments were approved by the Institutional SGC-7901 and BGC-823 cells were were harvested,
washed twice with cold PBS buffer and stained with a
ITGB1 antibody (eBioscience, MA, USA) for 30 AGING 19072 19072 www.aging-us.com Statistical analysis Each experiment was performed at least three times,
independently. Results were presented as the mean ±
SEM. All statistical significance between groups was
calculated by Student’s t test for two groups (Figures
1F, 1G, 4C–4E), and all statistical significance
between groups was calculated by one-way ANOVA
for more than two groups. The survival rates were
determined by Kaplan–Meier survival analysis (*p <
0.05; **p < 0.01; ***p < 0.001; ns, no significant
difference). 6. Seewaldt V. ECM stiffness paves the way for tumor
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survival and chemoresistance. Chemother Res Pract. 2012; 2012:283181. https://doi.org/10.1155/2012/283181 PMID:22567280 8. Kim SH, Turnbull J, Guimond S. Extracellular matrix and
cell signalling: the dynamic cooperation of integrin,
proteoglycan and growth factor receptor. J Endocrinol. 2011; 209:139–51. https://doi.org/10.1530/JOE-10-0377
PMID:21307119 Yalei Lv: design of this work and drafting the article;
Yujie Shan: data collection and analysis; Lina Song:
data collection; Yufei Zhao: Data collection; Ruixue
Lai: data collection; Junyan Su: data analysis;
Xiaoyun Zhang: design of this work and revise the
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PMID:32736172 30. Kesanakurti D, Chetty C, Dinh DH, Gujrati M, Rao JS. Role of MMP-2 in the regulation of IL-6/Stat3 survival AGING www.aging-us.com SUPPLEMENTARY MATERIALS Supplementary Figure 1. (A) Western blotting of BCL9L in SGC-7901/BGC-823 (Vector) and BCL9L overexpression SGC-7901/BGC-823 cells
(OE). (B) ITGB1- SGC-7901/BGC-823 (Vector) and BCL9L overexpression SGC-7901/BGC-823 cells (OE) were sorted and seeded in 3D collagen
gels. The colony formation rates were calculated on day3. (C) Western blotting of BCL2 in SGC-7901/BGC-823 (Vector) and BCL2
overexpression SGC-7901/BGC-823 cells (OE). (D) The ITGB1+ SGC-7901/BGC-823 (Vector) and BCL2 overexpression SGC-7901/BGC-823 cells
(OE) cells were sorted and cultured in 3D collagen gel (6 days). Then tumor cells were treated with 5-FU (5 μg/ml) combing C-IN2 (5 μM) and
the cell apoptosis was examined. * Indicates P <0.05, ** indicates P <0.01. Supplementary Figure 1. (A) Western blotting of BCL9L in SGC-7901/BGC-823 (Vector) and BCL9L overexpression SGC-7901/BGC-823 cells
(OE). (B) ITGB1- SGC-7901/BGC-823 (Vector) and BCL9L overexpression SGC-7901/BGC-823 cells (OE) were sorted and seeded in 3D collagen
gels. The colony formation rates were calculated on day3. (C) Western blotting of BCL2 in SGC-7901/BGC-823 (Vector) and BCL2
overexpression SGC-7901/BGC-823 cells (OE). https://doi.org/10.1038/s41467-019-08586-3
PMID:30760720 (D) The ITGB1+ SGC-7901/BGC-823 (Vector) and BCL2 overexpression SGC-7901/BGC-823 cells
(OE) cells were sorted and cultured in 3D collagen gel (6 days). Then tumor cells were treated with 5-FU (5 μg/ml) combing C-IN2 (5 μM) and
the cell apoptosis was examined. * Indicates P <0.05, ** indicates P <0.01. AGING www.aging-us.com www.aging-us.com
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English
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Effect of a Repeated Sprint Ability test on the muscle contractile properties in elite futsal players
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Scientific reports
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cc-by
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Effect of a Repeated Sprint Ability
test on the muscle contractile
properties in elite futsal players Received: 16 April 2018
Accepted: 18 October 2018
Published: xx xx xxxx Received: 16 April 2018
Accepted: 18 October 2018
Published: xx xx xxxx Javier Sánchez-Sánchez1, David Bishop 2,3, Jorge García-Unanue1, Esther Ubago-Guisado4,
Enrique Hernando4, Jorge López-Fernández4, Enrique Colino4 & Leonor Gallardo4 The aim of this study was to evaluate the effect of a repeated sprint ability (RSA) test on the contractile
properties of the muscles in elite futsal players. A total of 20 elite players completed the RSA test
(7 × 30 m), and the contractile response from the rectus femoris (RF) and biceps femoris (BF) of both
legs were analysed pre and post through tensiomyography. There was a significant increment in 30-m
times from the third sprint onwards (p < 0.05). The percent decrement in sprint ability (RSADEC) with
respect to the first sprint was significantly higher in the last sprint. The players did not show evidence
of lateral asymmetry in any of the muscle groups analysed after the RSA test (p > 0.05). Following the
RSA test there was a significant reduction in the delay time (Td) in RF, a significant decrement in half-
relaxation time (Tr) in the RF, and a significant reduction in sustain time (Ts) in the RF and BF of both
legs. The maximum radial displacement of the muscle belly (Dm) increased (1.6 mm; effect size = 0.75;
p < 0.05) in the RF after the RSA test, indicating reduced muscle stiffness and the ability to generate
strength rapidly. The decrement in performance during the RSA test was significantly correlated with
changes in contraction time (Tc) in RF and BF, Td in BF, and Dm in RF (p < 0.05). The RSA test generated
alterations in the contractile properties of the RF and BF in elite players. However, futsal players did not
present asymmetries in any muscular parameters. The baseline contractile muscle parameters could be
an important factor related to performance of players during repeated high-intensity actions. Futsal is a sport of intermittent efforts, with aerobic and dynamic components, that requires a player to be at 85%
of their maximum heart rate or above during most of the playing time1–5. Although the physical, physiological,
technical, and tactical demands have not been fully studied, researchers agree that the ability to develop and
maintain high-intensity efforts and sprint performance over time has a direct influence on the match perfor-
mance1,2,4–6. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 16 April 2018
Accepted: 18 October 2018
Published: xx xx xxxx Method
i i Participants. The sample was composed of two teams from the Spanish National Futsal League (LNFS). The
results for a total of 20 players (25.5 ± 6.1 years; 176.9 ± 5.2 cm; 74.9 ± 5.2 kg; 13.1 ± 2.4% body fat) were included
in this study. Contact with the clubs was carried out through the LNFS, with whom an agreement was signed for
the conduct of this study. The study protocol was approved by the Local Ethics Committee (Hospital of Toledo),
and was conducted in accordance with the Code of Ethics of the World Medical Association (Declaration of
Helsinki). All of the participants signed an informed consent form in which the test procedures and possible risks
were explained. In this document, the players also indicated their dominant leg. Experimental design. During the competitive period in the break for national team competitions, every
team arranged 3 days with the researchers within the given period to allow the players to perform the proposed
tests. On the first day, players performed an initial pilot test to become familiar with the tests included in the study
protocol. During the second day, players were not allowed to perform any exhaustive activity to guarantee 24 h of
rest before testing. Finally, all tests were carried out during the third day. At the beginning of the testing session,
before the warm-up, each player attached a heart rate monitor (Firstbeat Technologies Ltd., Finland). Experimental protocol. Body composition. Both fat mass (g and %) and lean mass (g) of both legs was
stimated using bioelectrical impedance (Tanita BC418-MA, Tanita Corp., Tokyo, Japan). The SECA scale (model
711; SECA GmbH & Co, KG, Hamburg, Germany) was used to measure the height of the participants. Tensiomyography (TMG). Muscle response and lateral symmetry of both the rectus femoris (RF) and biceps
femoris (BF) were assessed using tensiomyography (TMG-100 System electrostimulator, TMG-BMC d.o.o.,
Ljubljana, Slovenia). These muscles were selected because they are the most common muscles assessed in stud-
ies targeting soccer players due to their role in actions like jumping and kicking (BF) or in running and knee
stabilisation (RF)13. This assessment provided the following information: the maximum radial displacement of
the muscle belly (Dm), contraction time (Tc), delay time (Td), sustain time (Ts), and half-relaxation time (Tr) of
these muscles under basal conditions (Fig. 1). These measurements were repeated in the same room immediately
after (>1 min) the RSA test. Effect of a Repeated Sprint Ability
test on the muscle contractile
properties in elite futsal players Consequently, the use of repeated sprint ability test (RSA) is recommended to evaluate the ability of
athletes to cope with the demands of competition7,8. It has been shown that the performance of the athlete in a
RSA test offers relevant information on the explosive ability of footballers8.h f
y
The RSA test is recognized as a valid method to reproduce performance decrement and fatigue in soccer
players9. Fatigue of the lower-limb muscles appears to be an important factor elevating the risk of injury10,11. This suggests the behaviour of the lower-limb muscles after fatigue-inducing actions may be an effective way to
identify factors related to injury risk12. Some of these factors include muscle stiffness, contraction speed, or dis-
placement of the muscle belly13. These mechanical and contractile variables of the muscle can be determined by
Tensiomyography (TMG), via the application of an electrical stimulus14.h y g
y
The TMG technique has been reported to have high reproducibility and reliability to measure values like con-
traction time (Tc), half-relaxation time (Tr), delay time (Td), sustain time (Ts), and maximum radial displacement
of the muscle belly (Dm) for the medial vastus, lateral vastus, femoris rectus, and femoris biceps muscles13,15–17. Therefore, TMG has been identified as a reliable method for the identification of muscular lateral asymmetries
between dominant and non-dominant limbs in the lower-limb muscles18, which is related to the risk of injury and
the stretch-shortening cycle efficacy, especially in sports in which limb dominance plays a factor19–21. Also, TMG 1School of Sport Sciences, Universidad Europea de Madrid, Villaviciosa de Odón, (Madrid), Spain. 2Institute of Sport,
Exercise and Active Living (ISEAL), Victoria University, Melbourne, Australia. 3School of Medical & Health Science,
Edith Cowan University, Joondalup, Australia. 4IGOID Research Group, University of Castilla-La Mancha, Toledo,
Spain. Correspondence and requests for materials should be addressed to J.S.-S. (email: javiersanchezsanchez22@
gmail.com) SCIEnTIfIC REPOrts | (2018) 8:17284 | DOI:10.1038/s41598-018-35345-z SCIEnTIfIC REPOrts | (2018) 8:17284 | DOI:10.1038/s41598-018-35345-z 1 www.nature.com/scientificreports/ Figure 1. An example of how the TMG parameters were calculated (Carrasco et al.15). Td: delay time; Tc:
contraction time; Ts: sustain time; Dm: maximum radial displacement of muscle belly; Tr: half-relaxation time. Figure 1. An example of how the TMG parameters were calculated (Carrasco et al.15). Td: delay time; Tc:
contraction time; Ts: sustain time; Dm: maximum radial displacement of muscle belly; Tr: half-relaxation time. Effect of a Repeated Sprint Ability
test on the muscle contractile
properties in elite futsal players has been described as a reliable method for the identification of differences in muscle responses after completing
fatiguing efforts like the RSA test21.hf f
Thus, the aim of this study was to evaluate the effect of a RSA test on the contractile properties of the muscles
in elite futsal players. It was hypothesized that lower-limb muscle activation times (Tc, Td, and Ts) would be corre-
lated with better results in the RSA test. We also hypothesised that elite futsal players would not show muscle con-
tractile asymmetries between the dominant and non-dominant leg22,23. The results of this research will improve
understanding of the acute effects of a repeated sprint test on the muscular response of elite futsal players. Method
i i The duration of the stimulus was 1 ms at several intensities (25, 50, 75 and 100 mA),
following the protocol carried out in previous studies18. The properties of the rectus femoris were measured with
the participant in a supine position, and with the knees flexed at 120 degrees with the help of a triangular foam
cushion. The properties of the BF were measured with the participant in the prone position, and with the knee
flexed at 5 degrees with the help of a foam cushion16.h l
g
p
The muscle response was measured by placing a digital Dc-Dc transducer Trans-Tek® (GK 40, Panoptik d.o.o.,
Ljubliana, Slovenia) perpendicular to the muscle belly, along with two self-adhesive electrodes (TMG electrodes,
TMG-BMC d.o.o. Ljubljana, Slovenia) placed equidistant at a distance of 50 to 60 mm from the digital transducer,
since the distance between electrodes can vary the results24, the positions of the sensor and the electrodes were
marked with a permanent marker to ensure that all measurements were performed at the same point and between
50 to 60 mm from the digital transducer. All measurements were carried out by the same expert technician. Krizaj
et al.25 reported a low error level (0.5 to 2.0%) and a high reproducibility (ICC: 0.85–0.98) for the five parameters
measured in this study (ICC: Dm = 0.98; Tc = 0.97; Td = 0.94; Ts = 0.89; Tr = 0.86). SCIEnTIfIC REPOrts | (2018) 8:17284 | DOI:10.1038/s41598-018-35345-z 2 www.nature.com/scientificreports/ Figure 2. 5-m (A) and 30-m (B) time and performance deterioration profile; RSADEC (C) and RSACHANGE
(D) for the RSA test (7 × 30 m). RSACHANGE: ((worst time − best time)/best time) * 100). RSADEC: ((total sprint
time − best time * 7)/best time * 7) * 100). *p < 0.05; p < 0.01; ***p < 0.0015; significantly different from the 1st
sprint for 30-m times; and significantly different from the 7th sprint for RSADEC and RSACHANGE (n = 20). Data
are presented as mean and SD. Figure 2. 5-m (A) and 30-m (B) time and performance deterioration profile; RSADEC (C) and RSACHANGE
(D) for the RSA test (7 × 30 m). RSACHANGE: ((worst time − best time)/best time) * 100). RSADEC: ((total sprint
time − best time * 7)/best time * 7) * 100). Method
i i *p < 0.05; p < 0.01; ***p < 0.0015; significantly different from the 1st
sprint for 30-m times; and significantly different from the 7th sprint for RSADEC and RSACHANGE (n = 20). Data
are presented as mean and SD. Repeated sprint ability (RSA) test. The RSA test included seven repeated sprints of 30 m, with 20 s of active
recovery between each sprint. Three pairs of photocells (Witty, Microgate, Bolzano, Italy) placed at 0, 5 and 30 m
were used to assess performance in this test. This test was performed according to the methodology proposed in
previous studies26. The best sprint time (RSABEST), the mean time (RSAMEAN), the total time (RSATT), the percent
sprint decrement (RSADEC = ((total sprint time − best time*7)/best time ∗ 7) ∗ 100), and the percent difference
from best and worst sprint during the RSA test (RSACHANGE = ((worst time − best time)/best time) ∗ 100) were
also calculated27,28. Before the RSA test, participants carried out a standardised warm-up consisting of 5 minutes
of running, 5 minutes of joint mobility, and three 30-m sprints of increasing intensity. The warm-up concluded
with two 30-m sprints at maximum intensity separated by 4 minutes of active recovery (participants had to walk
during the resting time). These two previous sprints performed in the warm-up were used as a control measure
to guarantee players performed the RSA test at maximum speed. If the time of the first sprint of the RSA test was
higher (>5%) than the best individual sprint performed prior to the beginning of the test, the RSA test was not
considered valid and the player had to repeat the test after 5 min of recovery. Repeated sprint ability (RSA) test. The RSA test included seven repeated sprints of 30 m, with 20 s of active
recovery between each sprint. Three pairs of photocells (Witty, Microgate, Bolzano, Italy) placed at 0, 5 and 30 m
were used to assess performance in this test. This test was performed according to the methodology proposed in
previous studies26. The best sprint time (RSABEST), the mean time (RSAMEAN), the total time (RSATT), the percent
sprint decrement (RSADEC = ((total sprint time − best time*7)/best time ∗ 7) ∗ 100), and the percent difference
from best and worst sprint during the RSA test (RSACHANGE = ((worst time − best time)/best time) ∗ 100) were
also calculated27,28. Method
i i Before the RSA test, participants carried out a standardised warm-up consisting of 5 minutes
of running, 5 minutes of joint mobility, and three 30-m sprints of increasing intensity. The warm-up concluded
with two 30-m sprints at maximum intensity separated by 4 minutes of active recovery (participants had to walk
during the resting time). These two previous sprints performed in the warm-up were used as a control measure
to guarantee players performed the RSA test at maximum speed. If the time of the first sprint of the RSA test was
higher (>5%) than the best individual sprint performed prior to the beginning of the test, the RSA test was not
considered valid and the player had to repeat the test after 5 min of recovery. Statistical analyses. SPSS 21.0 was used for the data analysis. A descriptive analysis (mean ± SD) of the
tensiomyography test results and the performance parameters of the RSA test was performed. The Kolmogórov–
Smirnov test showed a normal distribution of the variables. Two-way analysis of variance (ANOVA) was used to
analyse the difference in the tensiomyography variables as a function of the time (pre and post) and dominance
(dominant leg and non-dominant leg). The exercise-induced change in the TMG variables (percentage of change
of the post with respect to the baseline tensiomyographic values) was also calculated. In addition, the confi-
dence interval and the effect size (ES; Cohen’s d) of the pre to post differences for all variables (CI of 95%) was
calculated. The ES was evaluated with the following criteria: 0 to 0.2 = trivial, 0.2 to 0.5 = small, 0.5 to 0.8 = mod-
erate, and >0.8 = large29. The RSA data were analysed by one-way repeated measures ANOVA. A Bonferroni
post-hoc test was used to study pairwise differences. A linear correlation (Pearson’s r) was calculated between
the results of the RSA test and the tensiomyography variables derived from the dominant leg of the futsal players. Correlations were evaluated with the following criteria: 0 to 0.1 = trivial, 0.1 to 0.3 = small, 0.3 to 0.5 = medium,
0.5 to 0.7 = large, 0.7 to 0.9 = very large and 0.9 to 1.0 = nearly perfect30. The level of significance was established
at p < 0.05. Method
i i Correlation coefficients for the baseline values of the tensiomyography in the BF and the total time
(RSATT), the best sprint time (RSABEST), the mean time (RSAMEAN), the percent sprint decrement (RSADEC),
and the percent difference from best to worst sprint (RSACHANGE) during the RSA test. *p < 0.05; **p < 0.01;
***p < 0.001; Dm = maximum radial displacement of muscle belly; Tc = contraction time, Td = delay time,
Ts = sustain time; Tr = half-relaxation time; BF = biceps femoris (n = 20). RF
RSADEC
RSACHANGE
BF
RSADEC
RSACHANGE
TdCHANGE
−0.197
−0.043
TdCHANGE
0.477*
0.578**
TcCHANGE
−0.498*
−0.263
TcCHANGE
0.497*
0.469*
TsCHANGE
−0.186
−0.217
TsCHANGE
−0.337
−0.318
TrCHANGE
−0.158
−0.232
TrCHANGE
−0.015
0.116
DmCHANGE
−0.424
−0.485*
DmCHANGE
0.184
0.018
Table 3. Correlation coefficients between the RSADEC and RSACHANGE derived from the RSA test and the
percentage change in the TMG parameters from pre to post the RSA test for the RF and BF. *p < 0.05;
**p < 0.01; ***p < 0.001; Dm = maximum radial displacement of muscle belly; Tc = contraction time,
Td = delay time, Ts = sustain time; Tr = half-relaxation time; RF = rectus femoris; BF = biceps femoris. n = 20. RF
RSADEC
RSACHANGE
BF
RSADEC
RSACHANGE
TdCHANGE
−0.197
−0.043
TdCHANGE
0.477*
0.578**
TcCHANGE
−0.498*
−0.263
TcCHANGE
0.497*
0.469*
TsCHANGE
−0.186
−0.217
TsCHANGE
−0.337
−0.318
TrCHANGE
−0.158
−0.232
TrCHANGE
−0.015
0.116
DmCHANGE
−0.424
−0.485*
DmCHANGE
0.184
0.018 Table 3. Correlation coefficients between the RSADEC and RSACHANGE derived from the RSA test and the
percentage change in the TMG parameters from pre to post the RSA test for the RF and BF. *p < 0.05;
**p < 0.01; ***p < 0.001; Dm = maximum radial displacement of muscle belly; Tc = contraction time,
Td = delay time, Ts = sustain time; Tr = half-relaxation time; RF = rectus femoris; BF = biceps femoris. n = 20. Results
Th
l The players had a RSATT of 29.9 ± 2.5 s, a RSAMEAN of 4.4 ± 0.1 s, and a RSABEST of 4.2 ± 0.2 s. There were no sig-
nificant differences between the times of the seven sprints during the first 5 m of each sprint (Fig. 2a; p > 0.05). However, compared with the first sprint, there was a significant increment in 30-m times from the third sprint
onwards (p < 0.05; Fig. 2b). The percent decrement in sprint ability (RSADEC) with respect to the first sprint was
significantly higher in the last sprint (p < 0.05; Fig. 2c). There was not a significant decrease in performance in the
seventh sprint (6.3 ± 3.3%) compared to the sixth sprint (5.9 ± 2.5%) for RSACHANGE (Fig. 2d).t p
p
p
g
In Table 1, the results of the tensiomyography for the RF and the BF (before and after the RSA test for both the
dominant (D) and non-dominant (ND) leg of the futsal players) are presented. The results reveal an absence of
significant differences between the dominant and non-dominant leg in the variables analysed (p > 0.05). However,
following the RSA test, there was a significant reduction in the Td (D: 1.1 ms [CI: 0.02–2.3]; ES: 0.62; ND: 1.3 ms [CI:
0.2–2.4]; ES: 0.77), Ts (D: 53 ms [CI: 23.5–82.4]; ES: 1.22; ND: 45.4 ms [CI: 15.9–74.8]; ES: 0.96), Tr (D: 27.5 ms [CI:
3.6–51.5]; ES: 0.76; ND: 32.2 ms [CI: 8.3–56.1]; ES: 0.84) in the RF and the Ts (D: 58.9 ms [CI: 26.2–91.6]; ES: 1.90;
ND: 53.5 ms [CI: 20.8–86.2]; ES: 0.83) in the BF in both the dominant and non-dominant legs. On the other hand, the
Dm of the RF showed higher values after the RSA test in the dominant leg (1.6 mm [CI: 0.03–3.1]; ES: 0.75; p < 0.05). In Table 1, the results of the tensiomyography for the RF and the BF (before and after the RSA test for both the
dominant (D) and non-dominant (ND) leg of the futsal players) are presented. The results reveal an absence of
significant differences between the dominant and non-dominant leg in the variables analysed (p > 0.05). Method
i i *p < 0.05; **p < 0.01;
***p < 0.001; Dm = maximum radial displacement of muscle belly; Tc = contraction time, Td = delay time,
Ts = sustain time; Tr = half-relaxation time; BF = biceps femoris (n = 20). RSATT
RSABEST
RSAMEAN
RSADEC
RSACHANGE
Td (BFpre)
0.032
−0.175
−0.007
0.487*
0.591**
Tc (BFpre)
0.254
−0.131
0.009
0.391
0.242
Ts (BFpre)
0.334
0.476*
0.528*
−0.202
−0.050
Tr (BFpre)
0.191
−0.111
−0.038
0.226
0.321
Dm (BFpre)
0.297
0.038
0.007
−0.105
−0.263
Table 2. Correlation coefficients for the baseline values of the tensiomyography in the BF and the total time
(RSATT), the best sprint time (RSABEST), the mean time (RSAMEAN), the percent sprint decrement (RSADEC),
and the percent difference from best to worst sprint (RSACHANGE) during the RSA test. *p < 0.05; **p < 0.01;
***p < 0.001; Dm = maximum radial displacement of muscle belly; Tc = contraction time, Td = delay time,
Ts = sustain time; Tr = half-relaxation time; BF = biceps femoris (n = 20). RSATT
RSABEST
RSAMEAN
RSADEC
RSACHANGE
Td (BFpre)
0.032
−0.175
−0.007
0.487*
0.591**
Tc (BFpre)
0.254
−0.131
0.009
0.391
0.242
Ts (BFpre)
0.334
0.476*
0.528*
−0.202
−0.050
Tr (BFpre)
0.191
−0.111
−0.038
0.226
0.321
Dm (BFpre)
0.297
0.038
0.007
−0.105
−0.263 Table 2. Correlation coefficients for the baseline values of the tensiomyography in the BF and the total time
(RSATT), the best sprint time (RSABEST), the mean time (RSAMEAN), the percent sprint decrement (RSADEC),
and the percent difference from best to worst sprint (RSACHANGE) during the RSA test. *p < 0.05; **p < 0.01;
***p < 0.001; Dm = maximum radial displacement of muscle belly; Tc = contraction time, Td = delay time,
Ts = sustain time; Tr = half-relaxation time; BF = biceps femoris (n = 20). Table 2. Correlation coefficients for the baseline values of the tensiomyography in the BF and the total time
(RSATT), the best sprint time (RSABEST), the mean time (RSAMEAN), the percent sprint decrement (RSADEC),
and the percent difference from best to worst sprint (RSACHANGE) during the RSA test. *p < 0.05; **p < 0.01;
***p < 0.001; Dm = maximum radial displacement of muscle belly; Tc = contraction time, Td = delay time,
Ts = sustain time; Tr = half-relaxation time; BF = biceps femoris (n = 20). Table 2. Method
i i SCIEnTIfIC REPOrts | (2018) 8:17284 | DOI:10.1038/s41598-018-35345-z 3 www.nature.com/scientificreports/ Dominant
Non-dominant
Pre
Post
Pre
Post
RF
Td (ms)
23.9 ± 2.2*
22.8 ± 1.4
23.4 ± 1.8*
22.2 ± 1.6
Tc (ms)
31 ± 8.4
27.6 ± 3.8
28.3 ± 6.1
26 ± 4.8
Ts (ms)
125 ± 53.4*
72 ± 33.2
120.8 ± 60.4*
75.5 ± 33.8
Tr (ms)
61.6 ± 40.4*
34.1 ± 31.9
66.3 ± 47.6*
34.1 ± 29.3
Dm (mm)
6.9 ± 2.5*
8.4 ± 1.7
7.4 ± 3
7.7 ± 2.5
BF
Td (ms)
23.2 ± 1.6
22.7 ± 1.6
22.4 ± 1.9
22.3 ± 1.7
Tc (ms)
26.3 ± 5.9
28.6 ± 11.6
30.6 ± 14.6
29.5 ± 13.3
Ts (ms)
205.5 ± 44.3*
146.6 ± 17.6
213.2 ± 73.1*
159.6 ± 56.3
Tr (ms)
60.4 ± 42.6
45.8 ± 27.7
65.1 ± 37.9
52.2 ± 46.2
Dm (mm)
5.8 ± 2.1
5.8 ± 2.1
5.6 ± 2.8
5.2 ± 2.5
Table 1. Results of the tensiomyography before (pre) and after (post) the RSA test for both the dominant
and non-dominant leg. *Significantly different from post (p < 0.05); Td = delay time, Tc = contraction time,
Ts = sustain time; Tr = half-relaxation time; Dm = maximum radial displacement of muscle belly; RF = rectus
femoris; BF = biceps femoris. n = 20 for all parameters. Table 1. Results of the tensiomyography before (pre) and after (post) the RSA test for both the dominant
and non-dominant leg. *Significantly different from post (p < 0.05); Td = delay time, Tc = contraction time,
Ts = sustain time; Tr = half-relaxation time; Dm = maximum radial displacement of muscle belly; RF = rectus
femoris; BF = biceps femoris. n = 20 for all parameters. RSATT
RSABEST
RSAMEAN
RSADEC
RSACHANGE
Td (BFpre)
0.032
−0.175
−0.007
0.487*
0.591**
Tc (BFpre)
0.254
−0.131
0.009
0.391
0.242
Ts (BFpre)
0.334
0.476*
0.528*
−0.202
−0.050
Tr (BFpre)
0.191
−0.111
−0.038
0.226
0.321
Dm (BFpre)
0.297
0.038
0.007
−0.105
−0.263
Table 2. Correlation coefficients for the baseline values of the tensiomyography in the BF and the total time
(RSATT), the best sprint time (RSABEST), the mean time (RSAMEAN), the percent sprint decrement (RSADEC),
and the percent difference from best to worst sprint (RSACHANGE) during the RSA test. Discussionh Futsal players showed lower Tc and Td in the RF muscle than these groups, suggesting a better
contractile ability. This means that the contractile properties of futsal players are phenotypically faster than those
found in other sports35. Tc, or contraction time, is defined as the time interval between the onset of development
of twitch force and its peak. This parameter reflects the speed of force generation and is related with the slow and
fast motor units36. Concretely, Tc is positive correlated with the proportion of type I fibres37. Therefore, athletes
from sports that need better contractile performance show lower Tc values35. In the BF, the contractile properties
were similar to elite football players13.h p y
The correlation analysis between the baseline values of the TMG for the BF and RSATT, RSABEST, and RSAMEAN
revealed a medium correlation (0.3–0.5) between the Ts and the best sprint and a large correlation (0.5–0.7)
between the Ts and the average sprint time. This suggests a better RSA (lower sprint time) in those players with
lower Ts values. However, caution is required when interpreting this result due to the possible influence of the
co-activation of other neighbouring muscles during the TMG evaluation38 and the low reliability of Ts39. There
was also a significant positive correlation between delay time of the BF and RSADEC and RSACHANGE. Td, or delay
time, is the time lapse from when the impulse is transmitted to the muscle until when the displacement of the
muscular belly reaches 10% of the maximum displacement (10% Dm). Shorter Td indicate an earlier onset of
contraction and a faster muscle reaction. Td is substantially related to the muscle fibre conduction velocity36,
being both slower in slow-twitch than in fast-twitch fibres40,41. Fast fibres have been reported to store more elas-
tic energy than slow fibers42, which enhances the speed of muscle relaxation and enables a faster realization of
consecutive contractions36. As a consequence, athletes with shorter Td have been reported to show an increased
ability to rapidly generate force during repeated muscle contractions43. Similarly, our result suggests that the futsal
players with higher Td may have a lower ability for repeated high-intensity actions which is reflected by a higher
decrement in RSA performance.hf p
The results of the present study show the effects of a RSA test on the contractile properties and the neuro-
muscular profile of elite futsal players. Discussionh The results of this study revealed that the RSA test causes an acute alteration in the mechanical muscle parameters
in the RF and the BF. This alteration is characterised by a change in the excitability of the RF in both legs (decrease
of the Td, Tc, Ts and Tr), whilst its effect on the contractile properties of the BF was less clear. Correlations were
found between the performance decrement of the RSA test and some of the tensiomyography parameters. In the
RF, we observed negative correlations between RSADEC and Tc and between RSACHANGE and Dm. In the BF, neg-
ative correlations were found between both performance decrement indices (RSADEC and RSACHANGE) and pre to
post change of Td and Tc. No significant differences in TMG parameters were found between the dominant and
non-dominant leg either before or after the RSA test. Therefore, it can be concluded that no lateral asymmetry
exists regarding the contractile properties of the RF and the BF in the professional futsal players recruited for this
study, neither at baseline nor after the RSA test.ii yt
Regarding RSA test performance, compared with the first sprint there was a significant increase in 30-m sprint
time from the third sprint onwards. This is consistent with the results of previous studies on elite football players27
that have reported a significant increase in sprint time from the second sprint. The percent sprint decrement
(4.98%) was similar to those reported in other repeated-sprint ability tests conducted with elite football31,32 and
female futsal33 players. Oliveira et al.4 observed a higher sprint decrement (6.7%) in high-level futsal players, prob-
ably due to the inclusion of changes of direction in the protocol, a greater sprint distance (40 m), and the moment
of the season (pre-season). In this study, the RSACHANGE was significant in the first five sprint, but no significant
differences were observed among sprints 6 and 7. The effect size of the differences was lower from the fourth
sprint, coinciding with Da Silva et al.31 who reported no significant differences between the last four sprints.hf p
g
p
gif
p
The baseline values of the TMG variables in elite futsal players differ from the values previously reported
for recreationally-active populations15,34, ultra-endurance athletes12, and even professional football players13,23. These outcomes show that the TMG profile of futsal players is different to that of other sports and recreationally
active populations. Results
Th
l However,
following the RSA test, there was a significant reduction in the Td (D: 1.1 ms [CI: 0.02–2.3]; ES: 0.62; ND: 1.3 ms [CI:
0.2–2.4]; ES: 0.77), Ts (D: 53 ms [CI: 23.5–82.4]; ES: 1.22; ND: 45.4 ms [CI: 15.9–74.8]; ES: 0.96), Tr (D: 27.5 ms [CI:
3.6–51.5]; ES: 0.76; ND: 32.2 ms [CI: 8.3–56.1]; ES: 0.84) in the RF and the Ts (D: 58.9 ms [CI: 26.2–91.6]; ES: 1.90;
ND: 53.5 ms [CI: 20.8–86.2]; ES: 0.83) in the BF in both the dominant and non-dominant legs. On the other hand, the
Dm of the RF showed higher values after the RSA test in the dominant leg (1.6 mm [CI: 0.03–3.1]; ES: 0.75; p < 0.05). SCIEnTIfIC REPOrts | (2018) 8:17284 | DOI:10.1038/s41598-018-35345-z 4 www.nature.com/scientificreports/ The correlational analysis did not reveal any significant relation between the tensiomyography parameters in
the RF prior to the RSA test and the results obtained in the test (p > 0.05). However, the Ts values obtained in the
BF (Table 2) showed a positive relation with the best sprint time achieved during the RSA test (r = 0.476) and the
mean sprint time (r = 0.528). There was a significant correlation between Td baseline values in BF and RSADEC
(r = 0.487) and RSACHANGE (r = 0.591). Regarding the performance decrement, the RSADEC and the RSACHANGE
were significantly correlated with the percent change of the Tc (r = −0.498) and Dm (r = −0.485), respectively,
from pre to post the RSA test in the RF (Table 3). Finally, in the BF, the percent changes in the Td and Tc values
from pre to post the RSA test were related with the deterioration in the sprint times during the test, independent
of the formula used (Table 3; p < 0.05). SCIEnTIfIC REPOrts | (2018) 8:17284 | DOI:10.1038/s41598-018-35345-z www.nature.com/scientificreports/ www.nature.com/scientificreports/ observed in the Ts of both legs. The variability of this parameter and the possible influence of the co-activation
of other neighbouring muscles during the TMG evaluation makes it difficult to interpret this behaviour38. Future
studies with different protocols47 must be performed to characterise the performance decrement in futsal. f
p
p
p
In the light of the results, we can conclude that the RSA test provokes an increase in the excitability of the RF in
both legs (lower values for Td, Tc, Ts and Tr), while its effects on the contractile properties of the BF are less clear. Also, the RF of the dominant leg seems to be the first muscle to present performance decrement symptoms, as it
is the first that starts to lose muscle tone (higher value of Dm) as a consequence of the effort. There was however,
no effect on lateral symmetry. In line with the study by Gil et al.23 in Brazilian footballers, no significant differ-
ences were found either before or after the RSA test. Although the results are hardly comparable, there are very
few studies that cover this topic in futsal players and the fact that no significant difference was found between
the dominant and non-dominant leg could be considered as a health symptom of the sample, as an imbalance is
associated with an increase in injury risk20,48. Further research is required to investigate the behaviour of these
variables in real fatigue situations as this is when there is a higher risk of muscle injury.h g
g
j
y
There were moderate negative correlations (0.3–0.5) between the performance decrement (RSADEC) and the
variation of the Tc, as well as between performance decrement (RSACHANGE) and the variation of the Dm in the the
RF. These correlations indicate that a greater decrement (identified as a significant decrease in performance) will
correspond to lower variations of the Tc and Dm in the RF. This means that the gap between the values measured
before and after the RSA test decreases as performance decrement increases. Moreover, it would be expected that,
in case of continuing the exercise until severe fatigue appears, post values will overcome pre values changing the
sense of the differences, as described earlier by other authors12,13,25. www.nature.com/scientificreports/ Regarding the BF, the results of the analysis
showed moderate-to-large positive correlations between the two performance decrement indicators (RSADEC,
RSACHANGE) and the variation of the Td and the Tc, suggesting that the evolution of these variables (Td, Tc) may
provide relevant information regarding the degree of fatigue in the BF muscles. Conclusionh The baseline contractile properties of lower-limb muscles are related to performance and the percent decrement
in repeated high-intensity actions in futsal players. Lower Ts and Td baseline values in the BF were associated with
a better performance and lower performance decrement in repeated sprint actions, respectively. Finally, greater
changes in Td and Tc after the RSA test showed a relationship with a higher decrement in RSA test performance
in futsal elite player. The behaviour of the contractile properties only describes the acute adaptations in the RF and
the BF after completing the RSA test, as the measurement was made immediately after finishing the test. It will be
necessary that future studies include neuromuscular parameter controls both 24 and 48 h after to understand the
impact of fatigue on the contractile properties of these athletes across a longer time period.i p
g
p
p
g
p
Most studies about futsal have focused on analysing both the demands of futsal fixtures and the physiological
profile of futsal players. This is the first research study to assess the effect of a repeated sprint test on the contrac-
tile muscle profile in elite futsal players. The findings suggest that muscle mechanical variables have a significant
relationship with the performance during a RSA test. Therefore, tensiomyography is a useful instrument to assess
the influence of contractile muscle ability on the physical performance and the performance decrement during
in high-intensity actions of futsal players. The contractile profile of elite futsal players from Spain provided in this
study provides knowledge about the muscle properties of healthy elite futsal players in the middle of the season. These results can be useful for physical trainers and coaches as a reference to design specific training and rehabil-
itation programs to reach the references values and an optimal muscle contractile ability. In short, this research
supports the use of tensiomyography as a tool that is sensitive enough to detect mechanical changes and to aid
in the understanding of how these changes affect the capacity of futsal players to perform intermittent efforts at
a high intensity. Discussionh Authors like De Paula Simola et al.44, or Wiewelhove et al.21 have already
investigated the effects of fatigue on the contractile properties of the muscle in different sports. Although their
results differ in many aspects, these authors generally agree that time-variables decreasing and the Dm increasing
involve a normal response following the muscle training, especially in muscles adapted to explosive exercises12. The decrease in muscle stiffness evidenced by the increment of the Dm is produced as a consequence of fatigue
and implies a loss of strength and explosive potential, reducing the ability to generate strength rapidly45. An
unusual increase of the Dm can indicate chronic fatigue35, although this was not present in this study. Regarding
the time variables (Td, Tc, Ts and Tr), the disparity between the described results of the previous research makes
it difficult to draw conclusions, although most of the studies associated muscle fatigue with increased values of
these variables. In the present study, the results show a reduction in all of the time parameters (Td, Tc, Ts and Tr)
of the RF in both legs. This indicates that our RSA test did not induce muscle fatigue in the RF muscles of our
participants but rather, it had a potentiating effect on their contractile properties14,25. The only symptom detected
is the significant increase of the Dm in the RF of the dominant leg46. Regarding the BF, a significant reduction was SCIEnTIfIC REPOrts | (2018) 8:17284 | DOI:10.1038/s41598-018-35345-z 5 References 1. Castagna, C. & Barbero Alvarez, J. C. Physiological demands of an intermittent futsal-oriented high-intensity test. J Strength Cond
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8. Cuadrado-Peñafiel, V., Párraga-Montilla, J., Ortega-Becerra, M. & Jiménez-Reyes, P. Repeated sprint ability in professional s 8. Cuadrado-Peñafiel, V., Párraga-Montilla, J., Ortega-Becerra, M. & Jiménez-Reyes, P. Repeated sprint abil
vs. professional futsal players. E-balonmano. com: Revista de Ciencias del Deporte. 10, 89–98 (2014). i
vs. professional futsal players. E-balonmano. com: Revista de Ciencias del Deporte. 10, 89–98 (2014). 9 Pau M Ibba G & Attene G Fatigue-induced balance impairment in young soccer players J Athl Train 49 454–461 (2014) 9. Pau, M., Ibba, G. & Attene, G. Fatigue-induced balance impairment in young soccer players. J Athl Train. 49, 454–461 (2014).hlil 9. Pau, M., Ibba, G. & Attene, G. Fatigue-induced balance impairment in young soccer players. J Athl Train. 49, 454–461 (2014). 10 G i
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t 9. Pau, M., Ibba, G. & Attene, G. Fatigue-induced balance impairment in young soccer players. J Athl Train. 49, 454–461 (2014). 0. Greig, M. The influence of soccer-specific fatigue on peak isokinetic torque production of the knee flexors and extensors. Am J Sport
Med. 36, 1403–1409 (2008).hfi 11. Small, K., McNaughton, L., Greig, M. & Lovell, R. The effects of multidirectional soccer-specific fatigue on markers of hamstring
injury risk. J Sci Med Sport. 13, 120–125 (2010).t 1. Small, K., McNaughton, L., Greig, M. & Lovell, R. The effects of multidirectional soccer-specific fatigue on markers of hamstring
injury risk. J Sci Med Sport. 13, 120–125 (2010).t j
y
p
2. Garcia-Manso, J. M. et al. Assessment of muscle fatigue after an ultra-endurance triathlon using tensiomyography (TMG). J Sport
Sci. 29, 619–625 (2011). 3. Rey, E., Lago-Peñas, C., Lago-Ballesteros, J. & Casáis, L. The effect of recovery strategies on contractile properties using
tensiomyography and perceived muscle soreness in professional soccer players. J Strength Cond Res. 26, 3081–3088 (2012). SCIEnTIfIC REPOrts | (2018) 8:17284 | DOI:10.1038/s41598-018-35345-z 6 Author Contributions J.S.S. and L.G. conceived of the presented idea. E.C., E.H. and E.U.G. developed the background and performed
the calibration of the different devices used in the tests. J.L.F. and J.G.U. verified the methods section. All authors
discussed the results and contributed to the final manuscript. J.S.S., E.C., E.U.G. and J.L.F. carried out the tests,
J.S.S. wrote the manuscript with support from D.B., E.H. and E.C. L.G. helped supervise the project. Both J.S.S. and D.B. contributed to the final version of the manuscript. J.G.U. and E.H. contributed to the interpretation of
the results and data analysis and they drafted the manuscript and designed the figures and tables. All authors
provided critical feedback and helped shape the research, analysis and manuscript. 7 SCIEnTIfIC REPOrts | (2018) 8:17284 | DOI:10.1038/s41598-018-35345-z www.nature.com/scientificreports/ Additional Informationh Competing Interests: The authors declare no competing interests. SCIEnTIfIC REPOrts | (2018) 8:17284 | DOI:10.1038/s41598-018-35345-z Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 © The Author(s) 2018 SCIEnTIfIC REPOrts | (2018) 8:17284 | DOI:10.1038/s41598-018-35345-z 8
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Religious identity in the Christian religion in the sources of religious teachings and sanctities, worship, feasts and holy days
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Corresponding author: E-mail: Saba.nawfal.salah@gmail.com ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161
DOI: https://doi.org/10.25130/jis.23.14.1.2.8 (
)
(
)
( )
( )
DOI: https://doi.org/10.25130/jis.23.14.1.2.8
ISSN:2073-1159 (Print) E-ISSN: 2663-8800 (Online)
ISLAMIC SCIENCES JOURNAL
Journal Homepage: http://jis.tu.edu.iq
ISJ ISJ Religious identity in the Christian religion in the
sources of religious teachings and sanctities,
worship, feasts and holy days Religious identity in the Christian religion in the
sources of religious teachings and sanctities,
worship, feasts and holy days Saba Nofal Saleh 1
Dr. Mohamed Hadi
Shehab 2
Department of Islamic
Creed and Thought,
College of Islamic
Sciences, Tikrit
University, Iraq
ISLAMIC SCIENCES JOURNAL (ISJ ISLAMIC SCIENCES JOURNAL (ISJ)
KEY WORDS:
Religious identity,
Christian religion,
religious sanctities,
worship, holy days. ARTICLE HISTORY:
Received: 25 /8 /2022
Accepted: 5 /9/ 2022
Available online: 29/1/2023
© 2022 ISLAMIC SCIENCES
JOURNAL , TIKRIT
UNIVERSITY. THIS IS AN
OPEN ACCESS ARTICLE
UNDER THE CC BY LICENSE
http://creativecommons.org/licenses
/by/4.0/ Saba Nofal Saleh 1
Dr. Mohamed Hadi
Shehab 2
Department of Islamic
Creed and Thought,
College of Islamic
Sciences, Tikrit
University, Iraq
KEY WORDS:
Religious identity,
Christian religion,
religious sanctities,
worship, holy days. ARTICLE HISTORY:
Received: 25 /8 /2022
Accepted: 5 /9/ 2022
Available online: 29/1/2023
© 2022 ISLAMIC SCIENCES
JOURNAL , TIKRIT
UNIVERSITY. THIS IS AN
OPEN ACCESS ARTICLE
UNDER THE CC BY LICENSE
http://creativecommons.org/licenses
/by/4.0/ Saba Nofal Saleh 1 اهلىية الدينية يف الدين املسيحي يف مصادر
التعاليم واملقدسات الدينية والعبادات واالعياد
واأليام املقدسة
صبا نىفل صاحل
أ.د . حممد هادي شهاب اهلىية الدينية يف الدين املسيحي يف مصادر
التعاليم واملقدسات الدينية والعبادات واالعياد
واأليام املقدسة
صبا نىفل صاحل
أ.د . حممد هادي شهاب ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161
DOI: https://doi.org/10.25130/jis.23.14.1.2.8 ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 الههية الجينية في الجين المديحي في مرادر التعاليم والمقجسات الجينية والعبادات واالعياد واأليام
المقجسة
ص
با نهفل صالح :الكلمات الجالة اليػية الجيشية، الجيغ السديحي، السقجسات الجيشية، العبادات، األيام السقجسة. ISLAMIC SCIENCES JOURNAL (ISJ ISLAMIC SCIENCES JOURNAL (ISJ) This research deals with the Christian religion, what
are its most important features, and the places where the
Christian identity is defined. It has three topics: I explained in
the first topic: the definition of the Christian religion, and its
most prominent prophets, and there are two requirements: the
first requirement: the definition of the Christian religion in
language and terminology: and the second requirement: the most
prominent prophets of religion The Christian: Then, in the
second topic, I showed: the characteristics of the Christian
religion, doctrinal, legal, and ethical, and it contains four
demands: The first requirement: religious identity in the sources
of teachings of the Christian religion: The second requirement:
religious identity in Christian sanctities, and the third
requirement: the religious gap in worship and practical rulings
Christianity and the fourth requirement: Christian religious
identity in holidays and holy days. Shehab 2
Department of Islamic
Creed and Thought,
College of Islamic
Sciences, Tikrit
University, Iraq
ISLAMIC SCIENCES JOURNAL (ISJ ISLAMIC SCIENCES JOURNAL (ISJ)
This research deals with the Christian religion, what
are its most important features, and the places where the
Christian identity is defined. It has three topics: I explained in
the first topic: the definition of the Christian religion, and its
most prominent prophets, and there are two requirements: the
first requirement: the definition of the Christian religion in
language and terminology: and the second requirement: the most
prominent prophets of religion The Christian: Then, in the
second topic, I showed: the characteristics of the Christian
religion, doctrinal, legal, and ethical, and it contains four
demands: The first requirement: religious identity in the sources
of teachings of the Christian religion: The second requirement:
religious identity in Christian sanctities, and the third
requirement: the religious gap in worship and practical rulings
Christianity and the fourth requirement: Christian religious
identity in holidays and holy days. KEY WORDS:
Religious identity,
Christian religion,
religious sanctities,
worship, holy days. ARTICLE HISTORY:
Received: 25 /8 /2022
Accepted: 5 /9/ 2022
Available online: 29/1/2023
© 2022 ISLAMIC SCIENCES
JOURNAL , TIKRIT
UNIVERSITY. THIS IS AN
OPEN ACCESS ARTICLE
UNDER THE CC BY LICENSE
http://creativecommons.org/licenses
/by/4.0/ Department of Islamic
Creed and Thought,
College of Islamic
Sciences, Tikrit
University, Iraq 144
Corresponding author: E-mail: Saba.nawfal.salah@gmail.com Corresponding author: E-mail: Saba.nawfal.salah@gmail.com 144 ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 :الخالصة ا
يتشاول ىحا البحث الجيغ السديحي، وما :ىي أىع سساتو، ومػاشغ معخفو اليُػيّة السديحية فييا وفيو ثالث مباحث
بيشت في السبحث األول: تعخيف الجيغ السديحي، وأبخز انبيائو وفيو مصمبان: السصمب األول: تعخيف الجيغ السديحي في
المغة واالصصالح: والسصمب الثاني: أبخز انبياء الجيغ السديحي: ثع بيشت في السبحث الثا ،ني: سسات الجيغ السديحي
:العقجية، والذخعية، واالخالقية، وفيو اربعة مصالب: السصمب األول: اليػية الجيشية في مرادر التعاليع لمجيغ السديحي
والسصمب الثاني: اليػية الجيشية في السقجسات السديحية والسصمب الثالث: اليػة الجيشية في العبادات واالحكام العسمية
السديحية و.السصمب الخابع: اليػية الجيشية السديحية في األعياد واأليام السقجسة :الكلمات الجالة اليػية الجيشية، الجيغ السديحي، السقجسات الجيشية، العبادات، األيام السقجسة. ٔٗ٘ ٔٗ٘ المـُـقــَـجِّمَــة ِالصاىخيغ، وعمى مَغْ سارَ عمى نيجوِ، ودعا بجعػتوِ إلى يػمِ الجيغ
:أما بعج
فإن
االديان الدساوية التي عخفت في ىحه الجنيا وكانت ليا شخائعيا السكتػبة ثالث
ة اديان ، ىي الجيغ
الييػدي
والجيغ السديحي والجيغ االسالمي ، وقبل ىحه
االديان
كان
ديغ
الشاس عمى حدب ما جاء مغ
مشيج شفػي مغ قبل االنبياء والسخسميغ، اال ما قيل عغ صحف إبخاليع، وكانت حكسا ال أحكاما
شخعية
فييا، وكانت لكل مغ ىحه
االديان
الدساوية الثالثة ما يسيد بعزيا مغ بعس، مغ سسات وخرائز
جعمت لكل واحجة مشيا ىػية تعخف بيا، فأحببت أن أكتب عغ اليػية الجيشية في
االديان
الثالثة، فكانت
رسالتي مػسػمة بو. سائال السػلى تعالى أن يػفقشي إلكساليا، أنو ولي ذلظ و.القادر عميو
ة الحسج ّللّ ربِّ العالسيغ، والرالةُ والدالمُ عمى خاتَعِ األنبياءِ والسُخسَ ميغ، وعمى آلوِ وصحبوِ الصيبيغ
. ِالصاىخيغ، وعمى مَغْ سارَ عمى نيجوِ، ودعا بجعػتوِ إلى يػمِ الجيغ
:أما بعج
فإن
االديان الدساوية التي عخفت في ىحه الجنيا وكانت ليا شخائعيا السكتػبة ثالث
ة اديان ، ىي الجيغ
الييػدي
والجيغ السديحي والجيغ االسالمي ، وقبل ىحه
االديان
كان
ديغ
الشاس عمى حدب ما جاء مغ
مشيج شفػي مغ قبل االنبياء والسخسميغ، اال ما قيل عغ صحف إبخاليع، وكانت حكسا ال أحكاما
شخعية
فييا، وكانت لكل مغ ىحه
االديان
الدساوية الثالثة ما يسيد بعزيا مغ بعس، مغ سسات وخرائز
جعمت لكل واحجة مشيا ىػية تعخف بيا، فأحببت أن أكتب عغ اليػية الجيشية في
االديان
الثالثة، فكانت
رسالتي مػسػمة بو. سائال السػلى تعالى أن يػفقشي إلكساليا، أنو ولي ذلظ و.القادر عميو
:طبيعة المهضهع
السػضػع الحي أنا برجده في رسالتي ىحه ىػ مػضػع مقارنة أديان، أدرس فييا ىػية كل ديغ سساوي
.عمى حجة، ثع أبيغ فييا ىػية كل ديغ عمى حدب السعتقج والسشيج
:أهمية المهضهع
تبخز أىسية مػضػع الخسالة بأنو يجرس ىػية
االديان الثالثة، ويبيغ ال رػاب والخصأ فييا، ويػضح أن
االديان
الدساوية كميا تجعػ الى التػحيج وتػقيخ االنبياء والسخسميغ، واالعتقاد الجازم بيػم آخخ، لكغ
اليػية الجيشية جعمت االختالف قائع بيشيا وبيغ األديان الدساوية السحفػضة مغ التحخيف، وأن االحكام
العسمية واالخالقية ىي مغ شبيعة السشيج
الجعػي
لحلظ الجيغ . المـُـقــَـجِّمَــة الحسج ّللّ ربِّ العالسيغ، والرالةُ والدالمُ عمى خاتَعِ األنبياءِ والسُخسَ ميغ، وعمى آلوِ وصحبوِ الصيبيغ
. ِالصاىخيغ، وعمى مَغْ سارَ عمى نيجوِ، ودعا بجعػتوِ إلى يػمِ الجيغ
:أما بعج
فإن
االديان الدساوية التي عخفت في ىحه الجنيا وكانت ليا شخائعيا السكتػبة ثالث
ة اديان ، ىي الجيغ
الييػدي
والجيغ السديحي والجيغ االسالمي ، وقبل ىحه
االديان
كان
ديغ
الشاس عمى حدب ما جاء مغ
مشيج شفػي مغ قبل االنبياء والسخسميغ، اال ما قيل عغ صحف إبخاليع، وكانت حكسا ال أحكاما
شخعية
فييا، وكانت لكل مغ ىحه
االديان
الدساوية الثالثة ما يسيد بعزيا مغ بعس، مغ سسات وخرائز
جعمت لكل واحجة مشيا ىػية تعخف بيا، فأحببت أن أكتب عغ اليػية الجيشية في
االديان
الثالثة، فكانت
رسالتي مػسػمة بو. سائال السػلى تعالى أن يػفقشي إلكساليا، أنو ولي ذلظ و.القادر عميو
:طبيعة المهضهع
السػضػع الحي أنا برجده في رسالتي ىحه ىػ مػضػع مقارنة أديان، أدرس فييا ىػية كل ديغ سساوي
.عمى حجة، ثع أبيغ فييا ىػية كل ديغ عمى حدب السعتقج والسشيج
:أهمية المهضهع
تبخز أىسية مػضػع الخسالة بأنو يجرس ىػية
االديان الثالثة، ويبيغ ال رػاب والخصأ فييا، ويػضح أن
االديان
الدساوية كميا تجعػ الى التػحيج وتػقيخ االنبياء والسخسميغ، واالعتقاد الجازم بيػم آخخ، لكغ
اليػية الجيشية جعمت االختالف قائع بيشيا وبيغ األديان الدساوية السحفػضة مغ التحخيف، وأن االحكام
العسمية واالخالقية ىي مغ شبيعة السشيج
الجعػي
لحلظ الجيغ . :منهج كتابة الرسالة
مشيجي في ىحه الخسالة ىػ مشيج استقخائي تحميمي يقػم عمى ذكخ السعمػمة والتفتير عغ مرادرىا، مع
.تحميل السعمػمة تحميال عمسيا قائسا عمى أساس التجخد العمسي السشزبط، والتفتير عغ الحق أيشسا كان
خصة البحث: انتطع البحث في
أربعة مصالب
:المطلب األول اليػية الجيشية في مرادر التعاليع
لمجيغ السديحي
:المطلب الثاني
اليػية الجيشية في السقجسات السديحية
:المطلب الثالث
اليػة الجيشية في العبادات واالحكام العسمية السديحية
:المطلب الرابع
اليػية الجيشية السديحية في األعياد واأليام السقجسة الحسج ّللّ ربِّ العالسيغ، والرالةُ والدالمُ عمى خاتَعِ األنبياءِ والسُخسَ ميغ، وعمى آلوِ وصحبوِ الصيبيغ
. ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 المطلب األول: الههية الجينية في مرادر التعاليم
للجين المديحي
:أوال: االناجيل المقجسة
َواالنجيل لغة مغ نجمت الذَّ يْ ء إِذا أخخجتو وَمِشْو قيل لشدل الخجل نجمو كَأَنَّوُ ىُػ
استخخجو يُقَال فتح هللا ناجميو أَي وَالِجيوِ، و (اإلنجيل) كتاب عيدى)
)
، يحكخ ويؤنث فسغ أنث أراد
الرحيفة ومغ ذكخ أراد الكتاب(
ٔ
). وفي االصصالح: يحسل اسع إنجيل كل مغ الكتب األربعة األولى فقط مغ األسفار التي وضعت ليا
الكشيدة عشػان (العيج الججيج) الحي يحتػي عمى سبع وعذخيغ رسالة، تست كتابتو بيغ أعػامٚٓ
ؤٓٓ
م
تقخيبا، األناجيل األربعة التي تعتخف بيا الكشيدة ىي (متى ومخقذ ولػقا ويػحشا) مع
أن مؤلف كل مغ
ىحه األناجيل األربعة مجيػل واالسساء ىحه تست إضافتيا في القخن الثاني السيالدي، وتعتبخ السرجر
الخئيدي لمسعمػمات عغ حياة يدػع، ومغ السؤكج تقخيبا أن أيا مشيا لع يكتبو شاىج عيان عمى حياة
يدػع(
ٕ
)، واإلجساع حاصل بيغ عمساء العرخ الحجيث ىػ أن األناجي ،ل تشتسي إلى الشػع القجيع مغ الديخ
أو الديخة الحاتية(
ٖ
). فاإلنجيل ىػ السرجر الخئيدي والسيع
لمجيغ
السديحي وىػ ما يسيد اليػية الجيشية السديحية، يزاف إلييا
.كتاب العيج القجيع وىػ التػراة :ثانياً: رسائل بهلس
وبػلذ ىػ بػلذ الصخسػسي ويعخف عشج السديحييغ بأنَّو بػ لذ الخسػل أو القجيذ
بػلذ، وكان اسسو "شاول الصخسػسي" قبل اىتجائو لمسديحية(
ٗ
)
، يقػل بػلذ: (أَنَا رَجُلٌ يَيُػدِيٌّ وُلِجْتُ فِي
َّشَخْسُ ػسَ كِيمِيكِيَّةَ، وَلكِغْ رَبَيْتُ فِي ىحِهِ الْسَجِيشَةِ مُؤَدَّبًا عِشْجَ رِجْمَيْ غَسَاالَئِيلَ عَمَى تَحْقِيقِ الش ُام) ِّػسِ األَبَػِي(
٘
)
،
ىػ أحج قادة الجيل السديحي األول ويشطخ إليو البعس عمى أنو ثاني أىع شخرية في تاريخ السديحية
"بعج يدػع نفدو، "إن كثيخاً مغ الثقات العرخيّيغ يعجونو السؤسذ الحكيقي لمسديحية(
ٙ
)
، وىػ أول مغ
حخف االنجيل(
ٚ
)، فيػ أوّل مغ غخس بحرة التثميث، فيػ قج دعا إلى تأليو السديح وبأنو ابغ هللا(
ٛ
)
، وذكخ (
ٔ
)
:يشطخ: غخيب الحجيث، أبػ دمحم عبج هللا بغ مدمع بغ قتيبة الجيشػري (الستػفىٕٚٙ
،ىــ)، السحقـق: د. عبـج هللا الجبـػري
مصبعة العاني–
،بغجاد، الصبعة: األولىٖٜٔٚ
:هٔ
/
ٕٗٙ
:، مختار الرحاحٔ
/
ٖٓ٘
. يأ
(
ٕ
)
،يشطخ: االنجيل والرميب، عبج االحج داود، جامعة دمذق، السكتبة االلكتخونيةٕٖٓٔ
:مٔ
/
ٔٛٗ
. (
ٙ) يشطخ: السديحية، د. احسج شمبي: صٜٚ
. (
ٔ
)
:يشطخ: غخيب الحجيث، أبػ دمحم عبج هللا بغ مدمع بغ قتيبة الجيشػري (الستػفىٕٚٙ
،ىــ)، السحقـق: د. عبـج هللا الجبـػري
مصبعة العاني–
،بغجاد، الصبعة: األولىٖٜٔٚ
:هٔ
/
ٕٗٙ
:، مختار الرحاحٔ
/
ٖٓ٘
.
(
ٕ
)
،يشطخ: االنجيل والرميب، عبج االحج داود، جامعة دمذق، السكتبة االلكتخونيةٕٖٓٔ
:مٔ
/
ٔٛٗ
.
(
ٖ
)
،يشطخ: مجخل إلى العيج الججيج، القذ: فييع عديد، دار الثقافة السديحية، القاىخةٜٔٛٓ: صٔٛ
.
(
ٗ
:) سفخ االعسالٜ
/
ٖٔ
.
(
٘
:) اعسال الخسلٖ
/
ٕٕ
.
(
ٙ) يشطخ: السديحية، د. احسج شمبي: صٜٚ
.
(
ٚ
)
:يشطخ: تخجيل مغ حخف التػراة واإلنجيل، صالح بغ الحديغ الجعفخي أبػ البقاء الياشسي (الستػفىٙٙٛ
:ىـ)، السحقق
محســـػد عبـــج الـــخحسغ قـــجح، مكتبـــة العبيكـــان، الخيـــاض، السسمكـــة العخبيـــة ،الدـــعػدية، الصبعـــة: األولـــىٜٔٗٔ
/ىــــٜٜٔٛ
:م
ٔ
/
ٔٓٓ
.
(
ٛ
:) روميةٕ
/
ٕٖ
-
ٕٚ
. المـُـقــَـجِّمَــة :منهج كتابة الرسالة
مشيجي في ىحه الخسالة ىػ مشيج استقخائي تحميمي يقػم عمى ذكخ السعمػمة والتفتير عغ مرادرىا، مع
.تحميل السعمػمة تحميال عمسيا قائسا عمى أساس التجخد العمسي السشزبط، والتفتير عغ الحق أيشسا كان
خصة البحث: انتطع البحث في
أربعة مصالب
:المطلب األول اليػية الجيشية في مرادر التعاليع
لمجيغ السديحي
:المطلب الثاني
اليػية الجيشية في السقجسات السديحية
:المطلب الثالث
اليػة الجيشية في العبادات واالحكام العسمية السديحية
:المطلب الرابع
اليػية الجيشية السديحية في األعياد واأليام السقجسة تبخز أىسية مػضػع الخسالة بأنو يجرس ىػية
االديان الثالثة، ويبيغ ال رػاب والخصأ فييا، ويػضح أن
االديان
الدساوية كميا تجعػ الى التػحيج وتػقيخ االنبياء والسخسميغ، واالعتقاد الجازم بيػم آخخ، لكغ
اليػية الجيشية جعمت االختالف قائع بيشيا وبيغ األديان الدساوية السحفػضة مغ التحخيف، وأن االحكام
العسمية واالخالقية ىي مغ شبيعة السشيج
الجعػي
لحلظ الجيغ . :منهج كتابة الرسالة مشيجي في ىحه الخسالة ىػ مشيج استقخائي تحميمي يقػم عمى ذكخ السعمػمة والتفتير عغ مرادرىا، مع
.تحميل السعمػمة تحميال عمسيا قائسا عمى أساس التجخد العمسي السشزبط، والتفتير عغ الحق أيشسا كان
خصة البحث: انتطع البحث في
أربعة مصالب
:المطلب األول اليػية الجيشية في مرادر التعاليع
لمجيغ السديحي
:المطلب الثاني
اليػية الجيشية في السقجسات السديحية
:المطلب الثالث
اليػة الجيشية في العبادات واالحكام العسمية السديحية
ال ا
ال طل
ة
األ ا ال ق
ا
األ
ة ف
ة ال
ة ال
ال مشيجي في ىحه الخسالة ىػ مشيج استقخائي تحميمي يقػم عمى ذكخ السعمػمة والتفتير عغ مرادرىا، مع
.تحميل السعمػمة تحميال عمسيا قائسا عمى أساس التجخد العمسي السشزبط، والتفتير عغ الحق أيشسا كان
خصة البحث: انتطع البحث في
أربعة مصالب ٔٗٙ (
ٚ
)
:رسالة بػلذ الخسػل إلى أىل غالشيةٔ
/
ٔ
.
(
ٛ
:) يشطخ ،تاريخ الكشيدة عرخ اآلباء، لػريسخ جػن، دار الثقافة، القاىخة، الصبعة األولىٕٕٓٔ
.:صٖٗٛ
.
(
ٜ
:) يشطخ
دراسات في األديان الييػدية والشرخانية: صٕٜٗ
. (
ٔ
)
:رسالتو إلى أفدذٔ
/
ٕٕ
:، ورسالتو إلى روميةٔٗ
/
ٔٓ
.
(
ٕ
)
:رسالتو إلى كػنثػسٚ
/
ٔٛ
-
ٜٔ
.
(
ٖ
)
:رسالتو إلى روميةٔ
/
٘
،
ٔٗ
-
ٔٙ
:، وإلى غالشيةٖ
/
ٕٙ
-
ٕٜ
(
ٗ
)
:يشطخ: مقجمة إنجيل بخنابأ
-
ٜ
.
(
٘
)كػرنثػس األ :ولىٙ
/
ٜ
-
ٔٔ
.
(
ٙ
:) روميةٔ
/
ٔ
.
(
ٚ
)
:رسالة بػلذ الخسػل إلى أىل غالشيةٔ
/
ٔ
.
(
ٛ
:) يشطخ ،تاريخ الكشيدة عرخ اآلباء، لػريسخ جػن، دار الثقافة، القاىخة، الصبعة األولىٕٕٓٔ
.:صٖٗٛ
.
(
ٜ
:) يشطخ
دراسات في األديان الييػدية والشرخانية: صٕٜٗ
. (
ٔ
)
:رسالتو إلى أفدذٔ
/
ٕٕ
:، ورسالتو إلى روميةٔٗ
/
ٔٓ
.
(
ٕ
)
:رسالتو إلى كػنثػسٚ
/
ٔٛ
-
ٜٔ
.
(
ٖ
)
:رسالتو إلى روميةٔ
/
٘
،
ٔٗ
-
ٔٙ
:، وإلى غالشيةٖ
/
ٕٙ
-
ٕٜ
(
ٗ
)
:يشطخ: مقجمة إنجيل بخنابأ
-
ٜ
.
(
٘
)كػرنثػس األ :ولىٙ
/
ٜ
-
ٔٔ
.
(
ٙ
:) روميةٔ
/
ٔ
.
(
ٚ
)
:رسالة بػلذ الخسػل إلى أىل غالشيةٔ
/
ٔ
.
(
ٛ
:) يشطخ
تاريخ الكشيدة عرخ اآلباء، لػريسخ جػن، دار الثقافة، القاى
(
ٜ
:) يشطخ
دراسات في األديان الييػدية والشرخانية: صٕٜٗ
. (
ٔ
)
:رسالتو إلى أفدذٔ
/
ٕٕ
:، ورسالتو إلى روميةٔٗ
/
ٔٓ
. (
ٕ
)
:رسالتو إلى كػنثػسٚ
/
ٔٛ
-
ٜٔ
. ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 (
ٚ
)
:يشطخ: تخجيل مغ حخف التػراة واإلنجيل، صالح بغ الحديغ الجعفخي أبػ البقاء الياشسي (الستػفىٙٙٛ
:ىـ)، السحقق
محســـػد عبـــج الـــخحسغ قـــجح، مكتبـــة العبيكـــان، الخيـــاض، السسمكـــة العخبيـــة ،الدـــعػدية، الصبعـــة: األولـــىٜٔٗٔ
/ىــــٜٜٔٛ
:م
ٔ
/
ٔٓٓ
. (
ٛ
:) روميةٕ
/
ٕٖ
-
ٕٚ
. ٔٗٚ ٔٗٚ (
ٖ
)
:رسالتو إلى روميةٔ
/
٘
،
ٔٗ
-
ٔٙ
:، وإلى غالشيةٖ
/
ٕٙ
-
ٕٜ ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 أن السديح سيحاسب الشاس يػم الكيامة(
ٔ
)،وندخ الختان(
ٕ
)
ً، وجعل السديحية ديشاً عالسيا(
ٖ
)
، وغيخ ذلظ
مغ االنحخافات التي أدت إلى افتخاق بخنابا الحػاري عشو في رحالتو وكتابتو إلنجيل بخنابا(
ٗ
). أما رسائل بػلذ فيي أربعة عذخ سفخًا في العيج الججيج، تتألف مغ رسائل تُشدب إلى بػلذ، مشيا رسالة
الى كػرنثػس وىي مجيشة عخفت بالفداد الذجيج(
٘
)
، ورسالة بػلذ الخسػل إلى أىل رومية يقػل في
"بجايتيا:" بػلذ، عبج ليدػع السديح، السجعػ رسػال، السفخز إلنجيل هللا(
ٙ
)
، ورسالتو الى أىل غالشية
وفييا":" بػلذ، رسػل ال مغ الشاس وال بإندان، بل بيدػع السديح وهللا اآلب الحي أقامو مغ األمػات(
ٚ
)
،
.وغيخىا مغ الخسائل أن السديح سيحاسب الشاس يػم الكيامة(
ٔ
)،وندخ الختان(
ٕ
)
ً، وجعل السديحية ديشاً عالسيا(
ٖ
)
، وغيخ ذلظ
مغ االنحخافات التي أدت إلى افتخاق بخنابا الحػاري عشو في رحالتو وكتابتو إلنجيل بخنابا(
ٗ
). أما رسائل بػلذ فيي أربعة عذخ سفخًا في العيج الججيج، تتألف مغ رسائل تُشدب إلى بػلذ، مشيا رسالة
الى كػرنثػس وىي مجيشة عخفت بالفداد الذجيج(
٘
)
، ورسالة بػلذ الخسػل إلى أىل رومية يقػل في
"بجايتيا:" بػلذ، عبج ليدػع السديح، السجعػ رسػال، السفخز إلنجيل هللا(
ٙ
)
، ورسالتو الى أىل غالشية
وفييا":" بػلذ، رسػل ال مغ الشاس وال بإندان، بل بيدػع السديح وهللا اآلب الحي أقامو مغ األمػات(
ٚ
)
،
.وغيخىا مغ الخسائل
:ثالثاً: المجاميع الكندية
لمسديحييغ يدتسجون عقائجىع وتذخيعاتيع ومعارفيع الجيشية مغ مرجريغ
أساسييغ ىسا: الكتاب السقجس، والسجامع السديحية، وىحه السجامع ثسانية مجامع ىي: مجسع نيكية سشة
(
ٖٕ٘
( ) م، ومجسع القدصشصيشية، ومجسع أفدذ سشةٖٗٔ
( ) م، ومجسع خمقيجونيو سشةٗ٘ٔ
،) م
والسجسع الدابع سشة (
ٚ٘ٗ
،
ٚٛٚ
( ) م، والسجسع الثامغ سشةٜٛٙ
) م، والسجسع الثاني عذخ الحي عقج
( سشةٕٔٔ٘
( ) م، ومجسع روما عامٜٔٚٙ
) م، والسجامع لع تكغ يػماً مغ األيام ىيئات شػريـة يتباحث
القدذ فييا اآلراء، ويتػصمػن فييا إلى الحق بأدلتو، بل كانت في األغمب تعقج لفخض رأي أو ترػر
عغ ش
خيق تمظ السجامع وبقػة الدمصان أو قػة الكشيدة(
ٛ
). ()
(4) emile boutroux، science et religion dans la philosophie contemporaine، ernest Flammarion
Flammarion editor، paris، p.9. ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 "في فتخة االباء السديحية عخفت انتذاراً واسعاً كسا عخفت التعرب معمشة ال خالص خارج الكشيدة في عجة مجن، تع استعسال كمسة "كشائذ" بذكل جساعي لإلشارة إلييع، وكانت الجساعة الػاحجة في كل بمج
تجعى كشيدة(
ٔ
). فالكشيدة ىي التي أسدت وأنذأت ورسست بفزل سمصتيا التي تجاوزت أحياناً سمصة اإلمبخاشػر، فكع
ًكان عمييا سيالً كمسا تأزم الػضع واحتجم الججال حػل مدألة مغ مدائل العقائج إال وعقجت مجسعا
،مدكػنياً تقخ مغ خاللو ما تذاء وتخفس ما تذاء وأن الفكخ السديحي مشح نذأتو، شعخ بسا لو في سخ
لثالػث، فكيف يسكغ لمسخء أن يػافق بيغ التػحيج الحي أعمشو السديحيػن األوائل مع الخسل الييػد في وجو
ًلػثشييغ وبيغ اإليسان بأنَّ السديح إلو وأن الخوح القجس إلو أيزا(
ٕ
). الثالػث، فكيف يسكغ لمسخء أن يػافق بيغ التػحيج الحي أعمشو السديحيػن األوائل مع الخسل الييػد في وجو
ًالػثشييغ وبيغ اإليسان بأنَّ السديح إلو وأن الخوح القجس إلو أيزا(
ٕ
). "فان تتبع الشطام البابػي في التجرج اليخمي الجيشي، اذ يخأس البابا الكخادلة وىع اصحاب الحق االول
واالخيخ في تشطيع الكشيدة ومشيع يتكػن السجتسع الكشائدي الحي يرجر ارادات بابػية سامية، والتي تعتبخ
في نطخىع "ارادات اليية مقجسة"، الن البابا في نطخىع ىػ تمسيح السديح االكبخ عمى األرض، ويسثل كحلظ
ارادتو الت"ي ال تقبل الججل والشقاش (
ٖ
). "فان تتبع الشطام البابػي في التجرج اليخمي الجيشي، اذ يخأس البابا الكخادلة وىع اصحاب الحق االول
واالخيخ في تشطيع الكشيدة ومشيع يتكػن السجتسع الكشائدي الحي يرجر ارادات بابػية سامية، والتي تعتبخ
في نطخىع "ارادات اليية مقجسة"، الن البابا في نطخىع ىػ تمسيح السديح االكبخ عمى األرض، ويسثل كحلظ
ارادتو الت"ي ال تقبل الججل والشقاش (
ٖ
). ا
"في فتخة االباء السديحية عخفت انتذاراً واسعاً كسا عخفت التعرب معمشة ال خالص خارج الكشيدة
الكاثػليكية، اذ مكغ سمصان الكشيدة مغ اضصياد العمع والفمدفة بعج التشكخ لحخية التفكيخ والعقيجة
والتدامح، واضصياد اآلراء السخالفة لعقيجة اال لو السرمػب السخمز، والتي فزل اصحابيا االلحاد، اذ
يرخح الكتاب الػثشيػن السمحجون بان ىحه االفكار ضج العقل ويشقل السديحيػن وجية نطخ اعجائيع لكغ
يحتجػن ضج ىحه التيسة("
ٗ
). (
ٔ
) يشطخ: االرىاب ودور العبادة السديحية في العـخاق: انسـار عبـج الجبـار جاسـع، مجمـة كميـة التخبيـة االساسـية، العـجد٘ٚ
،
ٕٜٓٓ
/ ،مٖٖٜ
. ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 فالكشيدة ىي التي أسدت وأنذأت ورسست بفزل سمصتيا التي تجاوزت أحياناً سمصة اإلمبخاشػر، فكع
ًكان عمييا سيالً كمسا تأزم الػضع واحتجم الججال حػل مدألة مغ مدائل العقائج إال وعقجت مجسعا
،مدكػنياً تقخ مغ خاللو ما تذاء وتخفس ما تذاء وأن الفكخ السديحي مشح نذأتو، شعخ بسا لو في سخ
الثالػث، فكيف يسكغ لمسخء أن يػافق بيغ التػحيج الحي أعمشو السديحيػن األوائل مع الخسل الييػد في وجو
ًالػثشييغ وبيغ اإليسان بأنَّ السديح إلو وأن الخوح القجس إلو أيزا(
ٕ
). "فان تتبع الشطام البابػي في التجرج اليخمي الجيشي، اذ يخأس البابا الكخادلة وىع اصحاب الحق االول
واالخيخ في تشطيع الكشيدة ومشيع يتكػن السجتسع الكشائدي الحي يرجر ارادات بابػية سامية، والتي تعتبخ
في نطخىع "ارادات اليية مقجسة"، الن البابا في نطخىع ىػ تمسيح السديح االكبخ عمى األرض، ويسثل كحلظ
ارادتو الت"ي ال تقبل الججل والشقاش (
ٖ
). "في فتخة االباء السديحية عخفت انتذاراً واسعاً كسا عخفت التعرب معمشة ال خالص خارج الكشيدة
الكاثػليكية، اذ مكغ سمصان الكشيدة مغ اضصياد العمع والفمدفة بعج التشكخ لحخية التفكيخ والعقيجة
والتدامح، واضصياد اآلراء السخالفة لعقيجة اال لو السرمػب السخمز، والتي فزل اصحابيا االلحاد، اذ
يرخح الكتاب الػثشيػن السمحجون بان ىحه االفكار ضج العقل ويشقل السديحيػن وجية نطخ اعجائيع لكغ
يحتجػن ضج ىحه التيسة("
ٗ
). المطلب الثاني: الههية الجينية في المقجسات المديحية
اوالً: الرليب
ىػ أبخز وأكثخ رمػز
الجيغ السديحي
إأالحي يذيخ لعسل يدػع الفجائي وفقاً لمعقائج السديحية ومع مجيء في عجة مجن، تع استعسال كمسة "كشائذ" بذكل جساعي لإلشارة إلييع، وكانت الجساعة الػاحجة في كل بمج
تجعى كشيدة(
ٔ
). فالكشيدة ىي التي أسدت وأنذأت ورسست بفزل سمصتيا التي تجاوزت أحياناً سمصة اإلمبخاشػر، فكع
ًكان عمييا سيالً كمسا تأزم الػضع واحتجم الججال حػل مدألة مغ مدائل العقائج إال وعقجت مجسعا
،مدكػنياً تقخ مغ خاللو ما تذاء وتخفس ما تذاء وأن الفكخ السديحي مشح نذأتو، شعخ بسا لو في سخ
الثالػث، فكيف يسكغ لمسخء أن يػافق بيغ التػحيج الحي أعمشو السديحيػن األوائل مع الخسل الييػد في وجو
ًالػثشييغ وبيغ اإليسان بأنَّ السديح إلو وأن الخوح القجس إلو أيزا(
ٕ
). "فان تتبع الشطام البابػي في التجرج اليخمي الجيشي، اذ يخأس البابا الكخادلة وىع اصحاب الحق االول
واالخيخ في تشطيع الكشيدة ومشيع يتكػن السجتسع الكشائدي الحي يرجر ارادات بابػية سامية، والتي تعتبخ
في نطخىع "ارادات اليية مقجسة"، الن البابا في نطخىع ىػ تمسيح السديح االكبخ عمى األرض، ويسثل كحلظ
ارادتو الت"ي ال تقبل الججل والشقاش (
ٖ
). (
ٔ
) يشطخ: االرىاب ودور العبادة السديحية في العـخاق: انسـار عبـج الجبـار جاسـع، مجمـة كميـة التخبيـة االساسـية، العـجد٘ٚ
،
ٕٜٓٓ
/ ،مٖٖٜ
.
(
ٕ
،) يشطخ: فمدفة الفكخ الجيشي بيغ االسالم والسدـيحية، لـػيذ غخديـو وجـػرج قشـػاتي، تخجسـة: صـبحي الرـالح وفخيـج جبـخ
،دار العمع لمسالييغ، بيخوتٜٜٔٚ
،مٔ
/
ٕٕٛ
.
(
ٖ) ال/ ،سديحية الرييػنية: دراسة تحميميةٔٗٙ
.
(4) emile boutroux، science et religion dans la philosophie contemporaine، ernest Flammarion ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ٔٗٛ ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 في عجة مجن، تع استعسال كمسة "كشائذ" بذكل جساعي لإلشارة إلييع، وكانت الجساعة الػاحجة في كل بمج
تجعى كشيدة(
ٔ
). فالكشيدة ىي التي أسدت وأنذأت ورسست بفزل سمصتيا التي تجاوزت أحياناً سمصة اإلمبخاشػر، فكع
ًكان عمييا سيالً كمسا تأزم الػضع واحتجم الججال حػل مدألة مغ مدائل العقائج إال وعقجت مجسعا
،مدكػنياً تقخ مغ خاللو ما تذاء وتخفس ما تذاء وأن الفكخ السديحي مشح نذأتو، شعخ بسا لو في سخ
الثالػث، فكيف يسكغ لمسخء أن يػافق بيغ التػحيج الحي أعمشو السديحيػن األوائل مع الخسل الييػد في وجو
ًالػثشييغ وبيغ اإليسان بأنَّ السديح إلو وأن الخوح القجس إلو أيزا(
ٕ
). "فان تتبع الشطام البابػي في التجرج اليخمي الجيشي، اذ يخأس البابا الكخادلة وىع اصحاب الحق االول
واالخيخ في تشطيع الكشيدة ومشيع يتكػن السجتسع الكشائدي الحي يرجر ارادات بابػية سامية، والتي تعتبخ
في نطخىع "ارادات اليية مقجسة"، الن البابا في نطخىع ىػ تمسيح السديح االكبخ عمى األرض، ويسثل كحلظ
ارادتو الت"ي ال تقبل الججل والشقاش (
ٖ
). "في فتخة االباء السديحية عخفت انتذاراً واسعاً كسا عخفت التعرب معمشة ال خالص خارج الكشيدة
الكاثػليكية، اذ مكغ سمصان الكشيدة مغ اضصياد العمع والفمدفة بعج التشكخ لحخية التفكيخ والعقيجة
والتدامح، واضصياد اآلراء السخالفة لعقيجة اال لو السرمػب السخمز، والتي فزل اصحابيا االلحاد، اذ
يرخح الكتاب الػثشيػن السمحجون بان ىحه االفكار ضج العقل ويشقل السديحيػن وجية نطخ اعجائيع لكغ
يحتجػن ضج ىحه التيسة("
ٗ
). المطلب الثاني: الههية الجينية في المقجسات المديحية
اوالً: الرليب
ىػ أبخز وأكثخ رمػز
الجيغ السديحي
الحي يذيخ لعسل يدػع الفجائي وفقاً لمعقائج السديحية ومع مجيء
يدػع السديح ليخمز العالع مغ الخصيئة وفقاً لإليسان السديحي تحػلت تمظ األداة مغ كػنيا وسيمة
تعحيب عشج العالع القجيع الى أكبخ رمد ديشي عبخ العرػر اذ ال يخمػ وجػد الرميب بيغ السديحييغ فيػ
مػج ػد معيع في كل مكان وزمان في الكشائذ والسشازل وعمى أجدادىع ويشاجػنو في صمػاتيع فالسديحييغ
:يعتبخونو تقجيدو ىػية ديشيو ليع وىي كاآلتي في عجة مجن، تع استعسال كمسة "كشائذ" بذكل جساعي لإلشارة إلييع، وكانت الجساعة الػاحجة في كل بمج
تجعى كشيدة(
ٔ
). ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ومغ ىحا السرجر يتبيغ اليػية الجيشية لمسديحية فإن السجامع السديحية ىي السرجر الحكيقي
لمجيغ
السديحي السحخف؛ ألن تمظ الفيػم التي كانت تقخر وترجر وفقيا القخارات لع تكغ تعتسج عمى نرػص
قصعية واضحة، بل أحياناً كانت
تعتسج عمى نرػص متذابية وكالم محتسل ألكثخ مغ معشى، ويكػن مغ
أقميا احتساالً السفيػم الحي تجعيو الكشيدة، كسا في دعػى ألػلية السديح عميو الدالم(
ٜ
). :رابعاً: الكنيدة البابهية تأتي الكشيدة مغ كمسة "كشذ" التي تعشي البيت، وصيغة الجسع "كشائذ" وىي
مكان عبادة السديح "ييغ، والسعبج عشج الييػدي، والكمسة اليػنانية السدتعسمة في العيج الججيج "اكميديا
"
Eklesia
"، أي مجسػعة مغ السؤمشيغ الحيغ يعتخفػن بالخب وبيدػع السديح، ومع ازدياد عجد أتباع يدػع :ثالثاً: المجاميع الكندية
لمسديحييغ يدتسجون عقائجىع وتذخيعاتيع ومعارفيع الجيشية مغ مرجريغ
أساسييغ ىسا: الكتاب السقجس، والسجامع السديحية، وىحه السجامع ثسانية مجامع ىي: مجسع نيكية سشة
(
ٖٕ٘
( ) م، ومجسع القدصشصيشية، ومجسع أفدذ سشةٖٗٔ
( ) م، ومجسع خمقيجونيو سشةٗ٘ٔ
،) م
والسجسع الدابع سشة (
ٚ٘ٗ
،
ٚٛٚ
( ) م، والسجسع الثامغ سشةٜٛٙ
) م، والسجسع الثاني عذخ الحي عقج
( سشةٕٔٔ٘
( ) م، ومجسع روما عامٜٔٚٙ
) م، والسجامع لع تكغ يػماً مغ األيام ىيئات شػريـة يتباحث
القدذ فييا اآلراء، ويتػصمػن فييا إلى الحق بأدلتو، بل كانت في األغمب تعقج لفخض رأي أو ترػر
عغ ش
خيق تمظ السجامع وبقػة الدمصان أو قػة الكشيدة(
ٛ
). ومغ ىحا السرجر يتبيغ اليػية الجيشية لمسديحية فإن السجامع السديحية ىي السرجر الحكيقي
لمجيغ
السديحي السحخف؛ ألن تمظ الفيػم التي كانت تقخر وترجر وفقيا القخارات لع تكغ تعتسج عمى نرػص
قصعية واضحة، بل أحياناً كانت
تعتسج عمى نرػص متذابية وكالم محتسل ألكثخ مغ معشى، ويكػن مغ
أقميا احتساالً السفيػم الحي تجعيو الكشيدة، كسا في دعػى ألػلية السديح عميو الدالم(
ٜ
). :رابعاً: الكنيدة البابهية تأتي الكشيدة مغ كمسة "كشذ" التي تعشي البيت، وصيغة الجسع "كشائذ" وىي
مكان عبادة السديح "ييغ، والسعبج عشج الييػدي، والكمسة اليػنانية السدتعسمة في العيج الججيج "اكميديا
"
Eklesia
"، أي مجسػعة مغ السؤمشيغ الحيغ يعتخفػن بالخب وبيدػع السديح، ومع ازدياد عجد أتباع يدػع (
ٕ
)
:رسالتو إلى كػنثػسٚ
/
ٔٛ
-
ٜٔ
. (
ٖ
)
:رسالتو إلى روميةٔ
/
٘
،
ٔٗ
-
ٔٙ
:، وإلى غالشيةٖ
/
ٕٙ
-
ٕٜ (
ٗ
)
:يشطخ: مقجمة إنجيل بخنابأ
-
ٜ
. (
٘
)كػرنثػس األ :ولىٙ
/
ٜ
-
ٔٔ
. يخو
اييغ
ع
ر
م/
(
ٖ) ال/ ،سديحية الرييػنية: دراسة تحميميةٔٗٙ
. (
ٔ
:) إنجيل لػقاٜ
:
ٕٖ
.
(
ٕ
) يشطخ: الشرخانية_ دراسة مقارنةٜٔٚ
.
(
ٖ) يشطخ:مذكالت العقيجة الشرخانية:سعج الجيغ صالح,مصبعة دار البيان, طٕ
,,القاىخةٜٖٔٛ
,مٔ٘ٗ
.
(
ٗ
. ) يشطخ: السرجر نفدو
(
٘
:) انجيل لػقاٜ
/
ٕٖ
.
(
ٙ
:) روميةٔ
/
ٕٔ
.
(
ٚ
:) غالشيةٕ
/
ٕٓ
. ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ىػ أبخز وأكثخ رمػز
الجيغ السديحي
الحي يذيخ لعسل يدػع الفجائي وفقاً لمعقائج السديحية ومع مجيء
يدػع السديح ليخمز العالع مغ الخصيئة وفقاً لإليسان السديحي تحػلت تمظ األداة مغ كػنيا وسيمة
تعحيب عشج العالع القجيع الى أكبخ رمد ديشي عبخ العرػر اذ ال يخمػ وجػد الرميب بيغ السديحييغ فيػ
مػج ػد معيع في كل مكان وزمان في الكشائذ والسشازل وعمى أجدادىع ويشاجػنو في صمػاتيع فالسديحييغ
:يعتبخونو تقجيدو ىػية ديشيو ليع وىي كاآلتي (
ٔ
) يشطخ: االرىاب ودور العبادة السديحية في العـخاق: انسـار عبـج الجبـار جاسـع، مجمـة كميـة التخبيـة االساسـية، العـجد٘ٚ
،
ٕٜٓٓ
/ ،مٖٖٜ
. اا (
ٕ
،) يشطخ: فمدفة الفكخ الجيشي بيغ االسالم والسدـيحية، لـػيذ غخديـو وجـػرج قشـػاتي، تخجسـة: صـبحي الرـالح وفخيـج جبـخ
،دار العمع لمسالييغ، بيخوتٜٜٔٚ
،مٔ
/
ٕٕٛ
. ٜٔٗ ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 إذ يخون السديحييغ أن الرميب عػد حكيقي ال يحسل أي معشى أخخ، وأنو يقجس لحاتو، حتى أنيع
يحسمػنو في أسفارىع ويتباركػن بو في حفال تيع، بل إنيع يحمػن بو ويخحمػن كذعار لعجم السباالة بالحياة
واالستعجاد لمسػت، أياً كان صػره آمميغ الشجاة فيو، ومدتشجيغ إلى القػل: " إن أراد أحج أن يأتي ورائي
" فميشكخ نفدو ويحسل صميبو ويتبعشي(
ٔ
) . وقج بمغ مغ تعطيع ىحا الفخيق لمرميب أن وججناىع يشاجػن الرميب )في صمػاتيع ويمقػن التحية (الدالم
لو قائميغ: " الدالم عميظ أييا الرميب، خمز ىحا الجسيػر السجتسع لتقجيدظ، أييا الرميب الحي أتى
" بالخالص لألشكياء(
ٕ
). :فالرمبان نساذج ثالثة ىي
أ-
( السجعػ صميب السقجس " أنجراوس " وىػ عمى شكل. ) ×
ب-
) وىػ+ ( واألخخ عمى شكل
الذائع
ت-
( والثالث عمى شكلT
)
(
ٖ
)
،، وبيحا نخى أن السديحييغ لع يختمفػا في السعشى السقرػد مغ الرميب
بل اختمفػا في رسسو أيزاً، والخالف ال يجل إال عمى عجم ثبػت األصل وفقجان السرجر ومغ ثع الذظ
في العقيجة والسعتقج(
ٗ
) . ب-
) وىػ+ ( واألخخ عمى شكل
الذائع
ت-
( والثالث عمى شكلT
)
(
ٖ
)
،، وبيحا نخى أن السديحييغ لع يختمفػا في السعشى السقرػد مغ الرميب
بل اختمفػا في رسسو أيزاً، والخالف ال يجل إال عمى عجم ثبػت األصل وفقجان السرجر ومغ ثع الذظ
في العقيجة والسعتقج(
ٗ
) . وإن تقجيذ الرميب عشج السديحييغ سبق صمب السديح نفدو، فقج ورد عغ السديح قػلو: "إن أراد أحج أن
"يأتي ورائي فميشكخ نفدو ويحسل صميبو ويتبعشي(
٘
)
، فييا إشارة الى نبػءة برمب السديح؛ حيث كانت
عادة عشج الخومان لمسحكػم عميو تُقزي ان يحسل صميبو الى مكان اإلعجام، وقج جاء في تعميع بػلذ
ِالخسػل: "إِنِّي أُناش َجُكع إِذًا، أَيُّيا اإلِخػَة، بِحَشانِ ّللاِّ أَن تُقَخِّبػا أَشْ خاصَ كع ذَبيحَةً حَيَّةً مُقَجَّسةً مَخْضِ يَّةً عِشج
"هللا. فيحِه ىي عِبادَتُكعُ الخّوحِيَّة(
ٙ
)
، ومغ ىحا السشصمق، يعتبخ حسل الرميب شخط مغ شخوط إتِّباع الخب
ونتيجة اتباع السديح يتقبل التمسيح ك ل نتائج الصاعة، ويتحسل كل العػاقب حتى الشياية، وىحه الكمسات
تُسثل أساس تعميع بػلذ الخسػل عغ اتحاد السؤمغ برميب السديح "فسا أَنا أَحْيا بَعجَ ذلِظ، بلِ السديحُ يَحْيا
ِفِيَّ. وإِذا كُشتُ أَحْيا اآلنَ حَياةً بَذَ خِيَّة، فإِنِّي أَحْياىا في اإلِيسانِ بِابغِ هللا الَّحي أَحبَّشي وجادَ بِشَفْدِ و مِغ
"أًجْمي(
ٚ
)
، ومعشى حسل الرميب عشجىع ىػ االستيانة بالحياة واالستعجاد لمسػت في أبذع صػره، أي صمبا
صمبا عمى خذبة كسا يفعل بالسجخميغ واآلثسيغ. ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 أداة تحكخ السديحييغ بالتزحية الزخسة التي قام بيا السديح مغ أجل البذخ(
ٔ
)
:، يقػل مخقذ«
ثع خخجػا
بو ليرمبػه، فدخخوا رجالً مجتازاً كان آتياً مغ الحقل، ىػ سسعان القػريشي أبػ الكدشجروس وروفذ ليحسل
صميبو»
(
ٕ
)
، "حيث أضيخ هللا قسة محبتو لمشاس فبحل ابشو الػحيج لكي ال ييمظ كل مغ يؤمغ بو بل تكػن
لو الحياة األ"بجية(
ٖ
). جاء في شخح بذارة لػقا لمقذ إبخاليع سعيج: "إن آثار قجمي السعمع تعيغ شخيق خصػات التالميح ألنو وإن
كان السديح قج صمب عشا فقال في صمبو: "قج أكسل" لكشا قج أصبحشا بحكع صمبو عشا تحت التدام شخعي
ألن نكػن شخكاء السديح الستألع، إن شخكتشا الذخعية مع السديح السرمػب يش بغي أن تخافقيا وتجعسيا
شخكة اختيارية فعمية معو، إن صمب السديح معشاه مات عشا، ولكغ صميب كل مؤمغ معشاه: "مػت الشفذ
عغ األنانية وحب الحات" وخالصة ىحه الحات ىي الشفذ األمارة بالدػء، ىي تمظ اإلرادة الستسخدة التي
يشبغي أن نخزعيا" وندتأسخىا لصاعة السديح، فقػل كل ،واحج ليذ ما أريج أنا بل ما تخيج أنت يا رب
إنو مغ أوجب واجبات كل مديحي أن يحسل صميبو مختاراً شائعاً ألن التعبيخ بحسل صميبو (
ٗ
). ثانياً: التعميج التعسيج: وىػ غسذ الجدع أو جدء مشو في الساء أو رشو، وبعزيع يكػن ذلظ عشجه بالتغصيذ ثالث
مخات، ويقػم بو الكاىغ باسع األب واالبغ والخوح القجس، وىػ مفتاح الجخػل في السديحية، فسغ لع يعسج
فميذ مديحياً عشجىع، ولػ كان مغ أبػيغ مديحييغ، ويعج التعسيج رمداً لػالدة ثانية، حيث يسػت اإلندان
«ليػلج مغ ججيج »بذكل أقخب إلى هللا(
٘
). إن يدػع كان لو مغ العسخ ثالثيغ عاماً حيغ بجأ دعػتو العمشية، وكان الكاىغ حدب شخيعة مػسى يبجأ
خجمتو في سغ الثالثيغ، وفي ذلظ الػقت تع تعسيجه، "ثع جاء يػحشا السعسجان مغ البخية وىػ يحيى بغ
زكخيا، ونادى بالتػبة والجعاء إلى الجيغ، وقج كان شعيا أخبخ أنو يخخج أيام
السديح، وجاء السديح مغ
"الشاصخة ولكيو باألردن فعسجه يػحشان وىػ ابغ ثالثيغ سشة(
ٙ
)
. التعسيج: وىػ غسذ الجدع أو جدء مشو في الساء أو رشو، وبعزيع يكػن ذلظ عشجه بالتغصيذ ثالث
مخات، ويقػم بو الكاىغ باسع
األب واالبغ والخوح القجس، وىػ مفتاح الجخػل في السديحية، فسغ لع يعسج
فميذ مديحياً عشجىع، ولػ كان مغ أبػيغ مديحييغ، ويعج التعسيج رمداً لػالدة ثانية، حيث يسػت اإلندان
«ليػلج مغ ججيج »بذكل أقخب إلى هللا(
٘
). ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 وقػيت فكخة تقجيذ الرميب بعج صمب عيدى فأصبح (
ٙ
:) روميةٔ
/
ٕٔ
. ا (
ٚ
:) غالشيةٕ
/
ٕٓ
. ٔ٘ٓ ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 (
ٔ
)يشطـــخ: العبـــادات فـــي األديـــان الدـــساوية الييػديـــة والشرـــخانية واإلســـالم، عبـــج الـــخازق عبـــج الحمـــيع صـــالح الســـػحي، دار
،األوائل، بيخوتٕٓٓٔ
:صٔ٘ٙ
.
(
ٕ
:) انجيل متي٘
/
ٙ
.
(
ٖ
)
،يشطخ: كيف نرمي، اسكشجر ججيج، دار اليجاية، استػتشغارت، الصبعة الثانيةٜٜٔ٘: صٖٗ
.
(
ٗ
)
:انجيل متيٕٙ
/
ٖٜ
.
(
٘
)
:انجيل لػقأ
/
ٔٛ
.
(
ٙ
)يشطــخ: قــامػس الكتــاب السقــجس، نخبــة مــغ األســاتحة ذوي االخترــاص والالىــػتييغ، تحخيــخ بصــخس عبــج السمــظ وجــػن
الكدانجر شسدغ وإبخاليع مصخ، دار
مكتبة العائمة، الصبعة الخابعة عذخ، بيخوتٕٓٓٔ
: صٜ٘ٗ
. ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 إن يدػع كان لو مغ العسخ ثالثيغ عاماً حيغ بجأ دعػتو العمشية، وكان الكاىغ حدب شخيعة مػسى يبجأ
خجمتو في سغ الثالثيغ، وفي ذلظ الػقت تع تعسيجه، "ثع جاء يػحشا السعسجان مغ البخية وىػ يحيى بغ
زكخيا، ونادى بالتػبة والجعاء إلى الجيغ، وقج كان شعيا أخبخ أنو يخخج أيام السديح، وجاء السديح مغ
"الشاصخة ولكيو باألردن فعسجه يػحشان وىػ ابغ ثالثيغ سشة(
ٙ
)
. (
ٔ) يشطخ: مقارنة االديان السديحية ألحسج شمبي: صٜٔٗ
. (
ٕ
)
:انجيل مخقذٔ٘
/
ٕٓ
-
ٕٖ
. (
ٖ
:) انجيل يػحشاٖ
/
ٔٙ
. (
ٗ
:) يشطخ محاضخات في الشرخانية (تبحث في األدوار التي مخَّت عمييا عقائج الشراري وفي كتبيع ومجامعيع السقجسة
:وفخقيع)، دمحم بغ أحسج بغ مرصفى بغ أحسج السعخوف بأبي زىخة (الستػفىٖٜٔٗ
ىـ)، دار الفكخ العخبي–
،القاىخة
الصبعة: الثالثةٖٔٛٔ
ىـ-
ٜٔٙٙ
م: صٔٔٓ
. (
٘) يشطخ: التعمي ،ع السديحي لمكشيدة الكاثػليكية، الكشيدة الكمجانية في بخيصانيا، لشجنٕٕٓٔ: ص٘٘
. (
ٙ
)
ديػان السبتجأ والخبخ في تاريخ العخب والبخبخ ومغ عاصخىع مغ ذوي الذأن األكبخ، عبج الخحسغ بغ دمحم بغ دمحم، ابغ
:خمجون أبػ زيج، ولي الجيغ الحزخمي اإلشبيمي (الستػفىٛٓٛىـ)، السحق :ق: خميل شحادة، دار الفكخ، بيخوت، الصبعة
،الثانيةٔٗٓٛ
ىـ-
ٜٔٛٛ
:مٕ
/
ٔٙٚ
. (
ٗ
:) يشطخ محاضخات في الشرخانية (تبحث في األدوار التي مخَّت عمييا عقائج الشراري وفي كتبيع ومجامعيع السقجسة
:وفخقيع)، دمحم بغ أحسج بغ مرصفى بغ أحسج السعخوف بأبي زىخة (الستػفىٖٜٔٗ
ىـ)، دار الفكخ العخبي–
،القاىخة
الصبعة: الثالثةٖٔٛٔ
ىـ-
ٜٔٙٙ
م: صٔٔٓ
. ٔ٘ٔ ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 المطلب الثالث: الههة الجينية في العبادات واالحكام العملية المديحية
أ
-
من اهم العبادات في المديحية
ٔ
-
:الرالة
وىي في
الجيغ
السديحي أنػاع مشيا صالة سخية ومشيا عمشية، ومشيا عائمية تؤدى في
ًالبيت، ومشيا صالة في الكشيدة، وأىع ىحه الرمػات ىي صالة يػم األحج، حيث يقخأ الكاىغ عمييع شيئا
مغ السداميخ أو مغ غيخىا مغ الكتاب السقجس، والجسيع وقػف يدتسعػن، وعشج نياية كل مقصع
يؤمشػن(
ٔ
)،" ومت ِّى صميت فال تكغ كالسخائيغ... وأما أنت فستى صميت فادخل مخجعظ وأغمق بابظ وصل
"إلى أبيظ في الخفاء(
ٕ
)
، وعجد الرمػات عشجىع سبع صمػات في اليػم والميمة، وليذ ليا كيفية محجودة
وإنسا ىي دعاء، ويختارونو في الغالب مغ األدعية السشدػبة لمسديح عميو الدالم، أو األدعية ا لسشدػبة
إلى داود عميو الدالم، كسا ذكخوىا في السداميخ مغ العيج القجيع(
ٖ
)
، مع أن الرالة الػاردة عغ الديج
"السديح كان فييا سجػد، ففي انجيل متي "ثع تقجم (السديح) قميالً وخخَّ عمى وجيو وكان يرمي(
ٗ
)
، وليا
شخشان: األول: أن تقجم الرالة باسع السديح؛ ألنيع يعتبخونو ىػ الػاسصة بيشيع وبيغ الخب، والثاني: أن
يتقجم الرالة اإليسان الكامل بالتثميث وغيخه مغ عقائجىع، وليذ ىشاك جية معيشة يتػجو الييا السرمي بل
يرمي بأي جية كانت، "يرمّي السؤمشػن بكلّ االتّجاىات، وهللا يدسعيع ويَقبل صمػاتيع إن كانت نابعة
وصادرة مغ قمػب شاىخة وعقػ"ل نكيّة(
٘
)
،عجم حرخ الرالة في أوقات محجدة وساعات معيشة تسدكا
بالقاعجة اإلنجيمية، التي أوصت بالرالة السدتسخة، فإنو ال نبغي أن تشحرخ الرالة في زمغ معيغ لكغ
مغ الالئق تعييغ أوقات معيشة لمرالة(
ٙ
). ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 فيتبيغ أن اليػية الجيشية لمسديحييغ الستجدجة في الرالة ىي التػجو
لمخب بالجعاء وشمب السغفخة، وىي
عمى زعسيع تجديج لمسحبة الستبادلة بيغ الخب والسؤمغ، كسا أن ىحه الرالة تيتع بالجانب الخوحي في
العبادة بتػجو قمبي في كل زمان ومكان باعتبار أنيا ذات شبيعة سساوية تختمف عغ صمػات الييػد التي
يعجونيا ذات شبيعة أرضية أو مادية مع أن.صالة السديحييغ تعتسج باألساس عمى مداميخ داود
ٕ
-
:الرهم يعج
الرػم في
الجيغ السديحي
بأىسية الرالة عشجىع، وىػ عشجىع شخيعة أليية، "وأَمَّا ٕٔ٘ ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 "شَ جَخَةُ مَعخِفَةِ الخَيخِ والذَّ خّ فال تَأكُلْ مِشيا، فإنَّظَ يَػمَ تأكُلُ مِشيا تَسػتُ مَػتًا(
ٔ
)
، وقج وصى الديج السديح
"بالرػم فقال: "سَ تَأتي أَيَّامٌ فييا يُخفَعْ العَخيذُ مِغ بَيْشِيع، فَحيشَئحٍ يَرػمػن(
ٕ
)
، والرػم في السفيػم
"السديحي ىػ "ىػ عجم تفكيخ اإلندان بالذخ في قمبو(
ٖ
)
، "ىحا القمب السجخوح بالخصيئة السيّال إلى الذخ
ّواألنانية، فيػ زمغ التػبة والعػدة إلى الحات بالتقذ"ف(
ٗ
)
، فإن يدػع السديح أول كسمة تفػّه بيا بعج صػمو
"صػمو األربعيشي قائالً: "فَتػبػا وآمِشػا بِالبِذارة(
٘
)
، ويقػل دانيال الشبي:"فَػَجَّيْتُ وَجْيِي إِلَى هللاِ الدَّ يِّجِ شَالِبًا
"ِبِالرَّ الَةِ وَالتَّزَ خُّعَاتِ ، بِالرَّ ػْمِ وَالْسَدْ حِ وَالخَّمَاد(
ٙ
)
، وأما عغ كيفية الرػم فيػ التبخؤ مغ الخياء والشفاق
إلى صجق اإلخالص وخالز الرفاء لمبحث عغ وجو هللا تعالى الحي يخى في الخفاء، وقج وبخ الخب
"يدػع صػم الخياء والتطاىخ قائال: "وإِذا صُ ستُع فال تُعبِّدػا كالسُخائيغ(
ٚ
)
، وقج ذكخ متي في انجيمو كالما
لمديج السديح)
)
، يقػل ِفيو:"أمَّا أَنتَ ، فإِذا صُ ستَ ، فادىُغْ رأسَ ظَ واغدِ لْ وَجيَظَ، لِكَيْال يَطْيَخَ لِمشَّاس
"أَنَّظَ صائع، بل ألَبيظَ الَّحي في الخُفْيَة، وأَبػكَ الَّحي يَخى في الخُفْيَةِ يُجازيظ(
ٛ
)
. وسبب الرػم عشج
السديحييغ ألمخيغ: االول: لصخد الذياشيغ، " الرػم ييجئ الشف ذ، يشقي الفكخ، يبعج الذياشيغ ويصخدىع
بعيجا و”
"يقخّب اإلندان إلى هللا(
ٜ
)
، والثاني: لمكيام بخسالة الجعػة: " لحا ال يسكغ أن يشفرل الرػم عغ
ذكخ الخب السحخك األساسي لو، إنشا برػمشا نتجو نحػ الخب بانفتاح جحري الحي نشتطخ مشو كل
"شيء(
ٔٓ
). ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 :والرػم يشقدع عشجىع قدسان الرػم الػاجب: ىػ ما جاء في الػصية الخابعة مغ وصايا الكشيدة: "انقصع
"عغ أكل المحع وصع الرػم في األيام التي تقخّىا الكشيدة(
ٔٔ
)
، ويقػم ىحا الرػم باالقترار عمى وجبة
ًواحجة في الشيار، مع تشاول شيء مغ الصعام صباحاً ومداء، مع االمتشاع عغ المحع بجسيع مذتقاتو، وفقا
لمعادات السحمية السػافق عمييا، مغ حيث الشػع والكسية(
ٕٔ
)
، ومغ الرػم الػاجب الرػم القخباني، "عمى (
ٔ
:) سفخ التكػيغٕ
/
ٔٚ
. (
ٕ
)
:انجيل متيٜ
/
ٔٗ
. (
ٖ
)
:سفخ زكخياٚ
/
ٔ
-
ٔٗ
. (
ٗ
:) مداميخ داودٚ
/
٘ٔ
. (
٘
:) انجيل مخقزٔ
/
ٔ٘
. (
ٙ
:) سفخ دانيالٖ
/
ٜ
. (
ٚ
:) انجيل متيٙ
/
ٔٙ
(
ٛ
)
:انجيل متيٙ
/
ٔٚ
-
ٔٛ
. (
ٜ
:) رسائل مخقذٜ
/
ٕٜ
. (
ٔٓ
:) سفخ دانيالٖ
/
ٜ
. (
ٔٔ
)
يشطخ: التعميع السديحي: بخقعٕٖٓٗ
. (
ٕٔ
:) يشطخ :مشذػر: تػبػا، البابا بػلذ الدادس القدعٖ
/
ٕ
-
ٖ
. (
ٕٔ
:) يشطخ :مشذػر: تػبػا، البابا بػلذ الدادس القدعٖ
/
ٕ
-
ٖ
. ٖٔ٘ ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 "السؤمشيغ أن يخاعػا الرػم السفخوض في كشيدتيع ليحدشػا االستعجاد لقبػل ىحا القخبان السقجس(
ٔ
)
، الرػم
الرػم السدتحب: وىػ ما يفخضو كل شخز مغ صيام عمى نفدو وعائمتو حدب ضخوفو الرحية
وعسمو،" وَانْتَخَبَا لَيُعْ قُدُ ػسً ا فِي كُلِّ كَشِيدَ ةٍ، ثُعَّ صَ مَّيَا بِأَصْ ػَامٍ وَاسْ تَػْدَعَاىُعْ لِمخَّبِّ الَّحِي كَا ِنُػا قَجْ آمَشُػا بِو("
ٕ
). (
ٔ
:) يشطخ الحق القانػني لمكشيدة الكاثػليكية الالتيشية: بخقعٖٔٛٚ
.
(
ٕ
)
:اعسال الخسلٔٗ
/
ٕٖ
.
(
ٖ
)
يشطخ: العبادات في األديان الدساويّة، عبج الخز
اق رحيع صالل، دار األوائل، دمذق-
،سػريا، الصبعة األولىٕٓٓٔ
:
ٕٓٓٔ
: صٜٔٙ
.
(
ٗ
:) أعسال الخسلٙ
/
ٕٓ
.
(
٘
:) انجيل متي٘
/
ٖٔ
.
(
ٙ
:) انجيل لػقأٙ
/
ٔٛ
. (
ٔ
:) يشطخ الحق القانػني لمكشيدة الكاثػليكية الالتيشية: بخقعٖٔٛٚ
.
(
ٕ
)
:اعسال الخسلٔٗ
/
ٕٖ
.
(
ٖ
)
يشطخ: العبادات في األديان الدساويّة، عبج الخز
اق رحيع صالل، دار األوائل، دمذق-
،سػريا، الصبعة األولىٕٓٓٔ
:
ٕٓٓٔ
: صٜٔٙ
. ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ٖ
-
:الحج
ال تػجج شقػس بعيشيا لمحج في السديحية، ألن التخكيد في بجايات ضيػر
الجيغ
كان عمى
ألػلية السديح أكثخ مغ تجديجه الجدساني، لحلظ لع تتصخق األناجيل لفخيزة الحج إلى مكان محجد، إال
إنيع يتجيػن دائسا لفكخة الحج لألماكغ السقجسة لجييع، عسال بػصية القجيذ جيخوم "إن مغ الجيغ التعبج
في السػضع الحى وشأتو قجما السديح"، مع أنّ السديح عميو الدال م لع يُشَػِّه إلى ىحا الػاجب الجيشي، إذ
يػصي السديح تالميحه في حادثة التجمي الذييخة، أال تقام مدارات أو صخوح باسسو، والشّرارى يشطخون
إلى جدج السديح عمى أنو بجيل عغ ليكل الييػد؛ لحا اقترخوا فى البجاية عمى زيارة ما يسثل رمده، وىػ
السكان الحي يسثل مكان صمب ا لسديح بدعسيع السػجػد في كل كشائديع، فيعتبخون ىحا السكان مكانًا
مُقجّساً، ويعتقجون أَنّ جدج السديح السرمػب بدعسيع يُسَثّل الحبيحة الكاممة لمخَّبِّ ، والتي تقجّس ذاتيا
بحاتيا، فيع يحىبػن شػاعيةً لمحج إلى القجس، الحي يشتيي بخؤية الشػر السقجَّس السشبعث مغ قبخ ا لديج
السديح في كشيدة السيج، في بيت لحع بفمدصيغ، لشيل البخكة وتحقيق الدعادة الخوحية(
ٖ
). ويخبخنا اإلنجيل أنّو بعج رفع السديح عميو الدالم إلى الدساء بخسذ وعذخيغ عاماً أدّى بػلذ حج
العشرخة(
ٗ
). ي
ي
ويخبخنا اإلنجيل أنّو بعج رفع السديح عميو الدالم إلى الدساء بخسذ وعذخيغ عاماً أدّى بػلذ حج
العشرخة(
ٗ
). ب
-
االحكام العملية: من اهم االحكام العملية في
الجين المديحي
ٔ
-
عجم زواج:المطلقة
:فقج حزت الذخيعة السديحية عمى عجم زواج السصمقة، ففي انجيل متي
"وقيل: مغ شمَّق امخأتو فميعصيا كتاب شالق. وأما أنا فأقػل لكع: إن مغ شمَّق امخأتو إال لعمة الدنى
"يجعميا تدني، ومغ يتدوج مصمقة فإنو يدني(
٘
)، وفي إنجيل لػقا يقػل: "كل مغ يصمِّق امخأتو ويت دوَّج بأخخى
"بأخخى يدني، وكل مغ يتدوَّج بسصمقة مغ رجل يدني(
ٙ
). ٕ
-
:عجم التعجد في الزواج
يُدسح الدواج بدوجة واحجة مع مشع التعجد الحي كان جائداً في مصمع
السديحية، ويُذتخط عشج الدواج حزػر القديذ؛ ليكيع وحجه بيغ الدوجيغ، أما القدذ والخلبان فال يجػز
ليع الدواج اقتجاء في
زعسيع بالسديح)
)
:، الحي لع يتدوج ، فقج ذكخ متي في انجيمو ىحه السدألة فقال ب
-
االحكام العملية: من اهم االحكام العملية في
الجين المديحي
ٔ
-
عجم زواج:المطلقة
:فقج حزت الذخيعة السديحية عمى عجم زواج السصمقة، ففي انجيل متي
"وقيل: مغ شمَّق امخأتو فميعصيا كتاب شالق. (
ٔ
:) يشطخ الحق القانػني لمكشيدة الكاثػليكية الالتيشية: بخقعٖٔٛٚ
. (
ٔ
:) انجيل متئٜ
/
ٖ
-
٘
.
(
ٕ)رسالة بػلذ االولى الي تيسػثاوس :
ٖ
/
ٕٔ
.
(
ٖ
)
:انجيل لػقأ
/
ٕٗ
-
ٕٙ
.
(
ٗ
:) رسالة بػلذ الى اغالشيةٗ
/
ٗ
.
(
٘
)يشطخ: الشرخانية وآدابيا بيغ عخب الجاىمية، لػيذ شيخػ: صٕٜٗ
.
(
ٙ
:) انجيل لػقإ
/
٘
-
ٚ
.
(
ٚ
)
: دسقػليةٖٔ
/ السجسػع وجوٜٚ
. ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 وأما أنا فأقػل لكع: إن مغ شمَّق امخأتو إال لعمة الدنى
"يجعميا تدني، ومغ يتدوج مصمقة فإنو يدني(
٘
)، وفي إنجيل لػقا يقػل: "كل مغ يصمِّق امخأتو ويت دوَّج بأخخى
"بأخخى يدني، وكل مغ يتدوَّج بسصمقة مغ رجل يدني(
ٙ
). ٕ
-
:عجم التعجد في الزواج
يُدسح الدواج بدوجة واحجة مع مشع التعجد الحي كان جائداً في مصمع
السديحية، ويُذتخط عشج الدواج حزػر القديذ؛ ليكيع وحجه بيغ الدوجيغ، أما القدذ والخلبان فال يجػز
ليع الدواج اقتجاء في
زعسيع بالسديح)
)
:، الحي لع يتدوج ، فقج ذكخ متي في انجيمو ىحه السدألة فقال (
ٔ
:) يشطخ الحق القانػني لمكشيدة الكاثػليكية الالتيشية: بخقعٖٔٛٚ
. (
ٕ
)
:اعسال الخسلٔٗ
/
ٕٖ
. (
ٖ
)
يشطخ: العبادات في األديان الدساويّة، عبج الخز
اق رحيع صالل، دار األوائل، دمذق-
،سػريا، الصبعة األولىٕٓٓٔ
:
ٕٓٓٔ
: صٜٔٙ
. (
٘
:) انجيل متي٘
/
ٖٔ
. (
ٙ
:) انجيل لػقأٙ
/
ٔٛ
. ٔ٘ٗ ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 " وجاء اليو الفخيديػن ليجخبػه قائميغ لو ىل يحل لمخجل ان يصمق امخأتو لكل سبب؟ فاجاب
وقال ليع أما
قخأتع ان الحي خمق مغ البجء خمقيسا ذكخا وانثى وقال: مغ اجل ىحا يتخك الخجل اباه وامو ويمترق بامخأتو
"ويكػن االثشان جدجا واحجا(
ٔ
)
، فقػلو " أما قخأتع ان الحي خمق مغ البجء خمقيسا ذكخا وانثى " يبيشيا بػلذ
في رسالتو االولى إلى تيسػثاوس:"ليكغ الذسامد"ة كل بعل امخأة واحجة مجبخيغ اوالدىع وبيػتيع حدشا(
ٕ
). (
ٔ) يشطخ: السديح لغ يغادر العالع، د. االنبا يػحشا قتمو، دار الثقافة، بيخوت: صٚٛ
.
(
ٕ
)
.وىي أحج تخاتيل عيج البذارة
(
ٖ
:) انجيل لػقإ
/
ٔٓ
-
ٔ٘
.
(
ٗ
،) مقالة بعشػان "أسبػع اآلالم.. أحج الكيامة السديح يقػم مغ قبخه بعج ثالثة أيام"، دمحم عبج الخحسغ.مجمة اليػم الدابع
(
٘
:) يشطخ :دراسات في األديان الييػدية والشرخانيةٔ
/
ٕٕٓ
.
(
ٙ
)
يشطخ: شع الشديع، شحاتة دمحم صقخ، دار الخمفاء الخاشجيغ–
:اإلسكشجريةٔ
/
ٖٔ
.
(
ٚ
)
:اعسال الخسلٖ
/
ٕ
-
ٗ
.
(
ٛ
:) سفخ الخخوجٜٔ
/
ٔٙ
. ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 فَيَا أَنَا أُبَذِّ خُكُعْ بِفَخَحٍ عَطِ يعٍ يَعُعُّ الذَّ عْبَ كُمَّوُ، فَقَجْ وُلِجَ لَكُعُ الْيَػْم
فِي مَجِيشَةِ دَاوُدَ مُخَمِّزٌ ىُػَ الْسَدِ يحُ ا لخَّبُّ ، وَىحِهِ ىِيَ الْعَالمَةُ لَكُعْ، تَجِجُونَ شِ فْالً مَمْفُػفاً بِقِسَاطٍ وَنَائِساً فِي
مِحْوَدٍ، وَفَجْأَةً ضَيَخَ مَعَ الْسَالكِ جُسْيُػرٌ مِغَ الْجُشْجِ الدَّ سَاوِيِّ ، يُدَ بِّحُػنَ هللاَ قَائِمِيغَ : السجج هلل ف ،ي االعالي
وعمى االرض الدالم، وبالش"اس السدخة(
ٖ
). فيحا العيج تطيخ فيو اليػية الجيشية السديحية واضحة السعالع، فإنيع يذاركػن فيو مخيع العحراء فخحتيا
بحسميا بالسديح عميو الدالم، وىحا العيج مغ خرػصيات
الجيغ
.السديحي
ٕ
-
:عيج يهم القيامة
ويدسى أيزا بعيج الفرح، وىػ أىع االعياد في
الجيغ
السديحي، بل أن كل
االعياد ما ىي الى تسييج ليحا العيج الكبيخ، حيث يدتحكخ فيو السديحييغ قيامة السديح مغ بيغ األمػات
بعج ثالثة أيام مغ صمبو ومػتو، وفيو يشتيي الرػم الكبيخ الحي يدتسخ عادة أربعيغ يػمًا؛ كسا يشتيي
أسبػع اآلالم، ويبجأ زمغ الكيامة السدتسخ في الدش ة الصقدية أربعيغ يػمًا حتى عيج العشرخة، ويتع في ىحا
العيج اشعال شسعة كبيخة تجل عمى قيامة السديح، وانذاد التخانيع، بعج ذلظ يتع قخاءة أجداء مغ العيج القجيع
مغ الكتاب السقجس(
ٗ
). لمبذخية كميا(
ٔ
)
،، "اليػم راس خالصشا، وضيػر الدخ الحي بيغ الجىػر، الن ابغ هللا يريخ ابغ البتػل
وجبخائيل بالشعسة يُبذخ ونحغ نرخخ نحػ والجة
"االلو: افخحي يا مستمئة الشعسة... الخب معظ(
ٕ
)
، وفي
:ُانجيل لػقا: "فَقَالَ لَيُعُ الْسَالك«
َال تَخَافُػا! فَيَا أَنَا أُبَذِّ خُكُعْ بِفَخَحٍ عَطِ يعٍ يَعُعُّ الذَّ عْبَ كُمَّوُ، فَقَجْ وُلِجَ لَكُعُ الْيَػْم
فِي مَجِيشَةِ دَاوُدَ مُخَمِّزٌ ىُػَ الْسَدِ يحُ ا لخَّبُّ ، وَىحِهِ ىِيَ الْعَالمَةُ لَكُعْ، تَجِجُونَ شِ فْالً مَمْفُػفاً بِقِسَاطٍ وَنَائِساً فِي
مِحْوَدٍ، وَفَجْأَةً ضَيَخَ مَعَ الْسَالكِ جُسْيُػرٌ مِغَ الْجُشْجِ الدَّ سَاوِيِّ ، يُدَ بِّحُػنَ هللاَ قَائِمِيغَ : السجج هلل ف ،ي االعالي
وعمى االرض الدالم، وبالش"اس السدخة(
ٖ
). ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 :المطلب الرابع: الههية الجينية المديحية في األعياد واأليام المقجسة
تمهيج : جسيع االعياد وااليام السقجسة في
الجيغ
الييػدي ىي معطسة في
الجيغ السديحي، لكغ السديحية تسيدت
عغ الييػدية بأعياد وأيام مقجسة وىي تعتبخ ىػية ديشية ليع ومغ اىع ىحه األعياد واأليام السقجسة وىي
:كالتي :أوال: األعياد
كانت في
الجيغ
السديحي أعياد تبيغ اليػية الجيشية ليع، والتي تسيدىا عغ بكية
االديان
فسغ
:ىحه االعياد ما يأتي :أوال: األعياد
كانت في
الجيغ
السديحي أعياد تبيغ اليػية الجيشية ليع، والتي تسيدىا عغ بكية
االديان
فسغ
:ىحه االعياد ما يأتي ٔ
-
:عيج البذارة
ويدسى أيزا رأس االعياد، ونبع االعياد، وىػ أول األعياد السديحية؛ ألنو لػال
البذارة وحمػل يدػع في بصغ العحراء ما كانت بكية األعياد، " كان العالع قبل مجيء الخب سالكًا في
الطمسة جالدً ا في أرض ضالل السػت، وشالسا اشتيت اآلباء أو األنبياء أن يخوا الخ ب متجدجًا، أرسل هللا
"مالكو إلى العحراء حامالً إلييا بذخى تجدج ابشو الػحيج مغ أحذائيا الشكية(
ٖ
)
، ولكغ لسا جاء ملء
الدمان(
ٗ
)، ففي بيت لحع ولج السديح، وفي الشاصخة عاش وتخعخع(
٘
)
:، وفي انجيل لػقا بيان ليحه الػالدة
َ"وَصَ عِجَ يُػسُ فُ أَيْزاً مِغْ مَجِيشَةِ الشَّاصِ خَةِ بِسِشْصَقَةِ الْجَمِيلِ إِلَى مَجِيشَةِ دَاوُدَ الْسَجْعُػَّةِ بَيْتَ ل ،ِحْعٍ بِسِشْصَقَةِ الْيَيُػدِيَّة
َّألَنَّوُ كَانَ مِغْ بَيْتِ دَاوُدَ وَعَذِ يخَتِوِ، ليَتَدَ ج َّلَ ىُشَاكَ مَعَ مَخْيَعَ الْسَخْصُػبَةِ لَوُ، وَىِيَ حُبْمَى، وَبَيْشَسَا كَانَا ىُشَاكَ، تَع
َزَمَانُيَا لِتَمِجَ، فَػَلَجَتِ ابْشَيَا الْبِكْخَ ، وَلَفَّتْوُ بِقِسَاطٍ ، وَأَنَامَتْوُ فِي مِحْوَدٍ، إِذْ لَعْ يَكُغْ لَيُس" ِا مُتَّدَ عٌ فِي الْسَشْدِل(
ٙ
)
،
وقج
تدمست الكشيدة االحتفال بيحا العيج مغ قبل الخسل أنفديع فقج جاءت في أوامخىع ما نرو "وأول األعياد
الديجية عيج البذارة مغ هللا سبحانو عمى لدان جبخائيل السالك لمديجة مخيع البتػل والجة اإللو
"والسخمز(
ٚ
)، لحلظ يحتفل السديحييغ بيحا العيج؛ ألنيع يخيجون أن يذارك ػا العحراء فخحتيا بيحه البذارة
السفخحة التي أتت إلييا مغ الدساء مغ خالل رئيذ السالئكة جبخائيل، وألن في ىحه البذارة خالص (
ٚ
)
: دسقػليةٖٔ
/ السجسػع وجوٜٚ
. ٔ٘٘ ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 لمبذخية كميا(
ٔ
)
،، "اليػم راس خالصشا، وضيػر الدخ الحي بيغ الجىػر، الن ابغ هللا يريخ ابغ البتػل
وجبخائيل بالشعسة يُبذخ ونحغ نرخخ نحػ والجة
"االلو: افخحي يا مستمئة الشعسة... الخب معظ(
ٕ
)
، وفي
:ُانجيل لػقا: "فَقَالَ لَيُعُ الْسَالك«
َال تَخَافُػا! (
ٔ) يشطخ: السديح لغ يغادر العالع، د. االنبا يػحشا قتمو، دار الثقافة، بيخوت: صٚٛ
. ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 فيحا العيج تطيخ فيو اليػية الجيشية السديحية واضحة السعالع، فإنيع يذاركػن فيو مخيع العحراء فخحتيا
بحسميا بالسديح عميو الدالم، وىحا العيج مغ خرػصيات
الجيغ
.السديحي
ٕ
-
:عيج يهم القيامة
ويدسى أيزا بعيج الفرح، وىػ أىع االعياد في
الجيغ
السديحي، بل أن كل
االعياد ما ىي الى تسييج ليحا العيج الكبيخ، حيث يدتحكخ فيو السديحييغ قيامة السديح مغ بيغ األمػات
بعج ثالثة أيام مغ صمبو ومػتو، وفيو يشتيي الرػم الكبيخ الحي يدتسخ عادة أربعيغ يػمًا؛ كسا يشتيي
أسبػع اآلالم، ويبجأ زمغ الكيامة السدتسخ في الدش ة الصقدية أربعيغ يػمًا حتى عيج العشرخة، ويتع في ىحا
العيج اشعال شسعة كبيخة تجل عمى قيامة السديح، وانذاد التخانيع، بعج ذلظ يتع قخاءة أجداء مغ العيج القجيع
مغ الكتاب السقجس(
ٗ
). ٖ
-
:عيج العنررة ولو أسساء أخخى مشيا، عيج الحمػل، وعيج الخسديغ، وعيج الرعػد(
٘
)، والعش :رخة
،ىي كمسة عبخية في األساس وتعشي " إجتساع" أو "محفل"، وىي إشارة الى اجتساع الذعب في العيج
ويأتي بسعشى: مشع او امتشع، ألن ىحا اليػم مقجَّس ويُسشع العسل فيو(
ٙ
)
، وىػ العيج الحي يحتفل بو
السديحييغ، ويقرج بو حمػل الخوح القجس عمى تالميح السديح بعج صعػد يدػع بعذخة أيام، ففي سفخ
االعسال: "ولسا حزخ يػم الخسديغ كان الجسيع بشفذ واحجه وصار بغتة مغ الدساء صػت كسا مغ
ىبػب ريح عاصفة، فسأل جػانب البيت الحي كانػا فيو، وضيخت ليع ألدشة كأنيا مغ نار قج انقدست
فػقف عمى كل مشيع لدان، فامتألوا جسيعاً مغ الخوح القجس، وأخحوا يت كمسػن بمغات غيخ لغتيع عمى ما
"وىب ليع الخوح القجس أن يتكمسػا (
ٚ
)، تذيخ العاصفة إلى ضيػر هللا في صحخاء سيشاء(
ٛ
)
،، وبيحه العالمة (
ٗ
،) مقالة بعشػان "أسبػع اآلالم.. أحج الكيامة السديح يقػم مغ قبخه بعج ثالثة أيام"، دمحم عبج الخحسغ.مجمة اليػم الدابع
(
٘
:) يشطخ :دراسات في األديان الييػدية والشرخانيةٔ
/
ٕٕٓ
. (
ٙ
)
يشطخ: شع الشديع، شحاتة دمحم صقخ، دار الخمفاء الخاشجيغ–
:اإلسكشجريةٔ
/
ٖٔ
. (
ٚ
)
:اعسال الخسلٖ
/
ٕ
-
ٗ
. (
ٛ
:) سفخ الخخوجٜٔ
/
ٔٙ
. ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ٔ٘ٙ ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 العالمة، نعخف أنّ هللا حاضخ معشا بخوحو القجوس(
ٔ
)
، والشار ىشا يخمد الى روح القجس، يقػل يػحشا
"السعسجان:" سيعسّجكع بالخوح القجس والشار(
ٕ
)
، و ،بيشسا مُشِعَ الشاس في بابل، بدبب كبخيائيع، مغ التفاىع
وما كان الػاحج يدتصيع أن يدسع صػت قخيبو، أعصت العشرخة كل واحج أن يدسع الخسل "في لغتو
"الخاصة(
ٖ
)
، لحلظ فإن االحتفال بيحا العيج يعشي الذيادة والتبذيخ بقػة مػاىب روح القجس، التي تؤىل
السحتفميغ بو لشذخ لم خسالة السديحية، وتعصييع قػة الذيادة، وتجعميا صخيحة وشجاعة، وفي انجيل يػحشا
ما يذيخ ليحا السعشى فيقػل: "وأنا سأسأل اآلب، فييب لكع مؤيجاً آخخ يكػن معكع لألبج روح الحق الحي
ال يدتصيع العالع أن يتمقاه ألنو ال يخاه وال يعخفو، أما أنتع فتعمسػن أنو يكيع عشجكع ويك"ػن فيكع(
ٗ
). فشجج ىشا أن اليػية الجيشية السديحية حاضخة بذجة في ىحا العيج مغ حيث ضيػر روح القجس عمى
شكل نار او نػر وحمػلو في الخسل الحيغ صاروا يتكمسػن بمغات عجة، لكي يػصمػا البذارة الى كل الشاس
بمغاتيع الستعجدة، وىحا يذيخ الى تحػيل الخسالة السديحية مغ رسالة خاصة لبشي
اسخائيل الى رسالة
.عالسية، يتكمع فييا السبذخون بيا لكل العالع كلٌ حدب لغتو ثانيا: األيام المقجسة
للجين المديحي
يعج يػم األحج ىػية ديشية
لمجيغ
السديحي بسا في ىحا اليػم مغ عبادة وتقخبيع الى هللا سبحانو وتعالى
وتدكية نفػسيع مغ خالل عجة قجاسات
:ديشية تحجث في ىح اليػم وىػ كاالتي ومغ السعمػم أن السديح(
)
،
مغ بشي إسخائيل، وبشػ إسخائيل يعطسػن يػم الدبت ويقجسػنو، فكان
السديح(
)
،
عمى ذلظ، إال أن السديحييغ فيسا بعج بػقت شػيل تخكػا الدبت وأخحوا يعطسػن األحج
رغبة مشيع في مخالفة الييػد الحيغ يكشػن ليع العجاء والبغس(
٘
). الخاتمة ،بعج ىحه الجػلة العمسية في رياض مػضػع الخسالة، أبيغ في ىحه الخاتسة أىع ما تػصمت إليو مغ الشتائج
:والتي سأبيشيا عمى شكل نقاط وكسا يأتي
النتائج 1
-
تبيغ لشا ان اإلنجيل ىػ السرجر الخئيدي والسيع
لمجيغ
السديحي وىػ ما يسيد اليػية الجيشية
السديحية، يزاف إلييا كتاب العيج القجيع وىػ التػراة. 2
-
:تبيغ لشا ان السديحيػن يدتسجون عقائجىع وتذخيعاتيع ومعارفيع الجيشية مغ مرجريغ أساسييغ ىسا (
ٔ
:) انجيل يػحشإٓ
/
ٕٖ
. (
ٕ
)
:انجيل لػقاٖ
/
ٖ
. (
ٖ
:) اعسال الخسلٕ
/
ٙ
. (
ٗ
:) انجيل يػحشأٗ
/
ٔٙ
-
ٔٚ
. (
٘) يشطخ: دراسات في األديان الييػ ٔ٘ٚ المرادر والمراجع ٔ
-
االرىاب ودور العبادة السديحية في العخاق: انسار عبج الجبار جاسع، مجمة كمية التخبية االساسية، العجد٘ٚ
،
ٕٜٓٓ
.م
ٕ
-
،االنجيل والرميب، عبج االحج داود، جامعة دمذق، السكتبة االلكتخونيةٕٖٓٔ
.م
ٖ
-
،تاريخ الكشيدة عرخ اآلباء، لػريسخ جػن، دار الثقافة، القاىخة، الصبعة األولىٕٕٓٔ
. ٗ
-
:تخجيل مغ حخف التػراة واإلنجيل، صالح بغ الحديغ الجعفخي أبػ البقاء الياشسي (الستـػفىٙٙٛىــ) السحقـق: محسـػد
،عبج الخحسغ قجح، مكتبة العبيكان، الخياض، السسمكة العخبية الدعػدية، الصبعة: األولىٜٔٗٔ
/ىـٜٜٔٛ
.م
٘
-
التعميع السديحي لمكشيدة الكاثػليكية، الكشيدة الكمجانية ،في بخيصانيا، لشجنٕٕٓٔ
. ٙ
-
الحق القانػني لمكشيدة الكاثػليكية الالتيشية: بخقعٖٔٛٚ
. ٚ
-
.دراسات في األديان الييػدية والشرخانية
ٛ
-
.دراسات في األديان الييػدية والشرخانية
ٜ
-
.دراسات في األديان الييػدية والشرخانية، سعػد بغ عبج العديد الخمف
ٔٓ
-
ديػان السبتجأ والخبخ في تاريخ ال
عخب والبخبخ ومغ عاصخىع مغ ذوي الذأن األكبخ، عبج الخحسغ بغ دمحم بغ دمحم، ابغ
:خمجون أبػ زيج، ولي الجيغ الحزخمي اإلشبيمي (الستػفىٛٓٛ
:ىـ)، السحقق: خميل شحادة، دار الفكخ، بيخوت، الصبعة
،الثانيةٔٗٓٛ
ىـ-
ٜٔٛٛ
.م
ٔٔ
-
شع الشديع، شحاتة دمحم صقخ، دار الخمفاء الخاشجيغ
–
.اإلسكشجرية
ٕٔ
-
،العبــادات فــي األديــان الدــساوية الييػديــة والشرــخانية واإلســالم، عبــج الــخازق عبــج الحمــيع صــالح الســػحي، دار األوائــل
،بيخوتٕٓٓٔ
. ٔ
-
االرىاب ودور العبادة السديحية في العخاق: انسار عبج الجبار جاسع، مجمة كمية التخبية االساسية، العجد٘ٚ
،
ٕٜٓٓ
.م
ٕ
-
،االنجيل والرميب، عبج االحج داود، جامعة دمذق، السكتبة االلكتخونيةٕٖٓٔ
.م
ٖ
-
،تاريخ الكشيدة عرخ اآلباء، لػريسخ جػن، دار الثقافة، القاىخة، الصبعة األولىٕٕٓٔ
. ٗ
-
:تخجيل مغ حخف التػراة واإلنجيل، صالح بغ الحديغ الجعفخي أبػ البقاء الياشسي (الستـػفىٙٙٛ
ىــ) السحقـق: محسـػد
،عبج الخحسغ قجح، مكتبة العبيكان، الخياض، السسمكة العخبية الدعػدية، الصبعة: األولىٜٔٗٔ
/ىـٜٜٔٛ
.م
٘
-
التعميع السديحي لمكشيدة الكاثػليكية، الكشيدة الكمجانية ،في بخيصانيا، لشجنٕٕٓٔ
. ٙ
-الحق القانػني لمكشيدة الكاثػليكية الالتيشية: بخقعٖٔٛٚ
. ٚ
-
.دراسات في األديان الييػدية والشرخانية ٛ
-
.دراسات في األديان الييػدية والشرخانية ٜ
-
.دراسات في األديان الييػدية والشرخانية، سعػد بغ عبج العديد الخمف
ٔٓ
-
ديػان السبتجأ والخبخ في تاريخ ال عخب والبخبخ ومغ عاصخىع مغ ذوي الذأن األكبخ، عبج الخحسغ بغ دمحم بغ دمحم، ابغ
:خمجون أبػ زيج، ولي الجيغ الحزخمي اإلشبيمي (الستػفىٛٓٛ
:ىـ)، السحقق: خميل شحادة، دار الفكخ، بيخوت، الصبعة
،الثانيةٔٗٓٛ
ىـ-
ٜٔٛٛ
.م لشديع، شحاتة دمحم صقخ، دار الخمفاء الخاشجيغ
–
.اإلسكشجرية حمد
ع
عإ
ٕٔ
-
،العبــادات فــي األديــان الدــساوية الييػديــة والشرــخانية واإلســالم، عبــج الــخازق عبــج الحمــيع صــالح الســػحي، دار األوائــل
،بيخوتٕٓٓٔ
. ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ،الكتاب السقجس، والسجامع السديحية
3
-
تبيغ لشا ان السجامع السديحية ىي السرجر الحكيقي
لمجيغ
السديحي؛ ألن تمظ الفيػم التي كانت
تقخر وترجر وفقيا القخارات لع تكغ تعتسج عمى نرػص قصعية واضحة، بل أحياناً كانت تعتسج عمى
نرػص متذابية وكالم محتسل ألكثخ مغ معشى، ويكػن مغ أقميا احتساالً السفيػم الحي تجعيو
،الكشيدة
كسا في دعػى ألػلية السديح عميو الدالم
4
-
تبيغ لشا ان الرػم في
الجيغ السديحي بأىسية الرالة عشجىع، وىػ عشجىع شخيعة أليية
5
-
تبيغ لشا ان ال تػجج شقػس بعيشيا لمحج في السديحية، ألن التخكيد في بجايات ضيػر
الجيغ
كان
عمى ألػلية السديح أكثخ مغ تجديجه الجدساني، لحلظ لع تتصخق األناجيل لفخيزة الحج إلى مكان محجد
6
-
تبيغ لشا ان جسيع االعياد وااليام السقجسة في الجيغ الييػدي ىي معطسة في
الجيغ السديحي ، لكغ
السديحية تسيدت عغ الييػدية بأعياد وأيام مقجسة وىي تعتبخ ىػية ديشية ليع
ومغ اىع ىحه األعياد واأليام
السقجسة ٔ٘ٛ ٔ٘ٛ Sources and references 1. Emile boutroux، science et religion dans la philosophie contemporaine، ernest
Flammarion editor، paris، p.9. 1. Emile boutroux، science et religion dans la philosophie contemporaine، ernest
Flammarion editor، paris، p.9. p
p
2. Terrorism and the Role of Christian Worship in Iraq: Anmar Abdul-Jabbar Jassim,
Journal of the College of Basic Education, No. 57, 2009. 2. Terrorism and the Role of Christian Worship in Iraq: Anmar Abdul-Jabbar Jassim,
Journal of the College of Basic Education, No. 57, 2009. 3. The Bible and the Cross, Abdul-Ahad Daoud, Damascus University, Electronic Library,
2013 AD. 3. The Bible and the Cross, Abdul-Ahad Daoud, Damascus University, Electronic Library,
2013 AD. 4. History of the Church, the Age of the Patriarchs, Lorimer John, House of Culture, Cairo,
first edition, 2012. 4. History of the Church, the Age of the Patriarchs, Lorimer John, House of Culture, Cairo,
first edition, 2012. 5. Shame on the Letters of the Torah and the Gospel, Saleh bin Al-Hussein Al-Jaafari Abu
Al-Baqa Al-Hashemi (deceased: 668 AH) Investigator: Mahmoud Abdel-Rahman Kadah,
Obeikan Library, Riyadh, Saudi Arabia, Edition: First, 1419 AH / 1998 AD. 5. Shame on the Letters of the Torah and the Gospel, Saleh bin Al-Hussein Al-Jaafari Abu
Al-Baqa Al-Hashemi (deceased: 668 AH) Investigator: Mahmoud Abdel-Rahman Kadah,
Obeikan Library, Riyadh, Saudi Arabia, Edition: First, 1419 AH / 1998 AD. 6. Catechism of the Catholic Church, Chaldean Church in Britain, London, 2012. 7. The legal right of the Latin Catholic Church: No. 1387. 8. Studies in the Jewish and Christian religions. 9. Studies in the Jewish and Christian religions. 10. Studies in the Jewish and Christian religions, Saud bin Abdul Aziz al-Khalaf. 11. Divan Al-Mubtada and Al-Khabar in the History of the Arabs and the Berbers, and their
contemporaries of great importance, Abd al-Rahman bin Muhammad bin Muhammad, Ibn
Khaldun Abu Zaid, Wali al-Din al-Hadrami al-Ishbili (deceased: 808 AH), investigator:
Khalil Shehadeh, Dar al-Fikr, Beirut, edition: The second, 1408 AH - 1988 AD. 12. Sham El-Nessim, Shehata Mohamed Saqr, House of the Rightly Guided Caliphs -
Alexandria. 13. Worships in the monotheistic religions of Judaism, Christianity and Islam, Abd al-Razeq
Abd al-Halim Salah al-Muhi, Dar al-Awael, Beirut, 2001. 14. Worship in the monotheistic religions, Abd al-Razzaq Rahim Salal, Dar al-Awael,
Damascus - Syria, first edition, 2001. 15. Ghareeb Al-Hadith, Abu Muhammad Abdullah bin Muslim bin Qutayba Al-Dinuri
(deceased: 276 AH), investigator: Dr. Abdullah Al-Jubouri, Al-Ani Press - Baghdad, first
edition, 1397 AH. المرادر والمراجع ٖٔ
-
العبادات في األديان الدساويّة، عبج الخزاق رحيع صالل، دار األوائل، دمذق-
،سػريا، الصبعة األولىٕٓٓٔ
. ٔٗ
-
غخيـــب الحـــجيث، أبـــػ :دمحم عبـــج هللا بـــغ مدـــمع بـــغ قتيبـــة الـــجيشػري (الستـــػفىٕٚٙ
،ىــــ)، السحقـــق: د. عبـــج هللا الجبـــػري
مصبعة العاني–
،بغجاد، الصبعة: األولىٖٜٔٚ
.ه ٔ٘
-
فمدــفة الفكــخ الــجيشي بــيغ االســالم والسدــيحية، لــػيذ غخديــو وجــػرج قشــػاتي، تخجســة: صــبحي الرــالح وفخيــج جبــخ، دار
،العمع لمسالييغ، بيخوتٜٜٔٚ
.م ٔٙ
-
قامػس الكتاب السقجس، نخبة مغ األسـاتحة ذوي االخترـاص والالىـػتييغ، تحخيـخ بصـخس عبـج السمـظ وجـػن الكدـانجر
شسدغ وإبخاليع مصخ، دار مكتبة العائمة، الصبعة الخابعة عذخ، بيخوتٕٓٓٔ
. : شسدغ وإبخاليع مصخ، دار مكتبة العائمة، الصبعة الخابعة عذخ، بيخوتٕٓٓٔ
. :
ٔٚ
-
،كيف نرمي، اسكشجر ججيج، دار اليجاية، استػتشغارت، الصبعة الثانيةٜٜٔ٘
. ٔٛ
-
محاضخات،)في الشرخانية (تبحث في األدوار التي مخَّت عمييا عقائج الشراري وفي كتبيع ومجامعيع السقجسة وفخقيع
:دمحم بغ أحسج بغ مرصفى بغ أحسج السعخوف بأبي زىخة (الستػفىٖٜٔٗ
ىـ)، دار الفكخ العخبي–
القاىخة، الصبعة: الثالثة
ٖٔٛٔ
ىـ-
ٜٔٙٙ
.م ع
إ
ٔٚ
-
،كيف نرمي، اسكشجر ججيج، دار اليجاية، استػتشغارت، الصبعة الثانيةٜٜٔ٘
. ٔٛ
-
محاضخات ،)في الشرخانية (تبحث في األدوار التي مخَّت عمييا عقائج الشراري وفي كتبيع ومجامعيع السقجسة وفخقيع
:دمحم بغ أحسج بغ مرصفى بغ أحسج السعخوف بأبي زىخة (الستػفىٖٜٔٗ
ىـ)، دار الفكخ العخبي–
القاىخة، الصبعة: الثالثة
ٖٔٛٔ
ىـ-
ٜٔٙٙ
.م ٜٔ
-
.مختار الرحاح
ٕٓ
-
مجخل إلى العيج ا ،لججيج، القذ: فييع عديد، دار الثقافة السديحية، القاىخةٜٔٛٓ
. ٕٔ
-
.السديح لغ يغادر العالع، د. االنبا يػحشا قتمو، دار الثقافة، بيخوت
ٕٕ
-
السديحية، د. احسج شمبي: صٜٚ
. ٕٖ
-
مذكالت العقيجة الشرخانية:سعج الجيغ صالح,مصبعة دار البيان, طٕ
,,القاىخةٜٖٔٛ
,م
ٕٗ
-
مقارنة االديان السديح.ية ألحسج شمبي ٜٔ
-
.مختار الرحاح
ٕٓ
-
مجخل إلى العيج ا ،لججيج، القذ: فييع عديد، دار الثقافة السديحية، القاىخةٜٔٛٓ
. ٕٔ
-
.السديح لغ يغادر العالع، د. االنبا يػحشا قتمو، دار الثقافة، بيخوت ٜٔ٘ ISLAMIC SCIENCES JOURNAL (2023) Vol ( 14) Issue (1) section(2): 144-161 ٕ٘
-
،مقالة بعشػان "أسبػع اآلالم.. أحج الكيامة السديح يقػم مغ قبخه بعج ثالثة أيام"، دمحم عبج الخحسغ.مجمة اليػم الدابع
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Effects of amount of excess solid, the type of stirring and sedimentation time on solubility of sodium phenytoin and lamotrigine
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ADMET & DMPK
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Keywords Antiepileptic drugs; equilibrium solubility; determination Abstract Solubility is the maximum quantity of a drug dissolved in a given volume of solvent at a specific
temperature. Several factors affect equilibrium solubility. Therefore, different solubility data have been
reported for a solute in a certain solvent and temperature in the literature. These variations in solubility are
one of the possible reasons for unsuccessful attempts of medicinal chemists for developing models as well
as deviation of experimental works for solubility prediction in aqueous, non-aqueous and solvent mixtures. The present research aim is to investigate the effect of the amount of excess solid and the type of stirring
on the solubility of drugs. The solubility of two antiepileptic drugs, namely sodium phenytoin and
lamotrigine was determined in water, ethanol, and HCl 0.1 M at 37 °C. Different excess amounts of drugs
were added to the constant volume of solvent. Additionally, different stirring methods such as magnetic
stirrer and shake-flask and sedimentation time were investigated on the solubility values. Saturation
solubility of drugs after dilution with water was measured using a spectrophotometer, and the
concentration was calculated according to the calibration curve. Amount of excess solid, especially when
the drug is in ionized form, and sedimentation time after 24 h have a significant effect on solubility values. Shahrzad Moattar Mohammadi1,2, Ali Shayanfar3*, Shahram Emami3, Abolghasem
Jouyban4 Shahrzad Moattar Mohammadi1,2, Ali Shayanfar3*, Shahram Emami3, Abo
Jouyban4 1Biotechnolgy Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
2Student Research Committee and Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
3Drug Applied Research Center and Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
4Pharmaceutical Analysis Research Center and Faculty of Pharmacy, Tabriz University of Medical Science, Tabriz, Iran *Corresponding Author: E-mail: shayanfara@tbzmed.ac.ir; Tel.: +984133341315; Fax: +1-111-111-112
Received: September 27, 2018; Revised: October 22, 2018; Published: November 03, 2018 ADMET & DMPK 6(4) (2018) 269-278; doi: http://dx.doi.org/10.5599/admet.621
Open Access: ISSN: 1848-7718
http://www.pub.iapchem.org/ojs/index.php/admet/index
Original scientific paper
Effects of amount of excess solid, the type of stirring and
sedimentation time on solubility of sodium phenytoin and
lamotrigine
Shahrzad Moattar Mohammadi1,2, Ali Shayanfar3*, Shahram Emami3, Abolghasem
Jouyban4
1Biotechnolgy Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
2Student Research Committee and Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
3Drug Applied Research Center and Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
4Pharmaceutical Analysis Research Center and Faculty of Pharmacy, Tabriz University of Medical Science, Tabriz, Iran
*Corresponding Author: E-mail: shayanfara@tbzmed.ac.ir; Tel.: +984133341315; Fax: +1-111-111-112
Received: September 27, 2018; Revised: October 22, 2018; Published: November 03, 2018
Abstract
Solubility is the maximum quantity of a drug dissolved in a given volume of solvent at a specific
temperature. Several factors affect equilibrium solubility. Therefore, different solubility data have been
reported for a solute in a certain solvent and temperature in the literature. These variations in solubility are
one of the possible reasons for unsuccessful attempts of medicinal chemists for developing models as well
as deviation of experimental works for solubility prediction in aqueous, non-aqueous and solvent mixtures.
The present research aim is to investigate the effect of the amount of excess solid and the type of stirring
on the solubility of drugs. The solubility of two antiepileptic drugs, namely sodium phenytoin and
lamotrigine was determined in water, ethanol, and HCl 0.1 M at 37 °C. Different excess amounts of drugs
were added to the constant volume of solvent. Additionally, different stirring methods such as magnetic
stirrer and shake-flask and sedimentation time were investigated on the solubility values. Saturation
solubility of drugs after dilution with water was measured using a spectrophotometer, and the
concentration was calculated according to the calibration curve. Amount of excess solid, especially when
the drug is in ionized form, and sedimentation time after 24 h have a significant effect on solubility values.
Keywords ADMET & DMPK 6(4) (2018) 269-278; doi: http://dx.doi.org/10.5599/admet.621 org/10.5599/admet.621
Open Access: ISSN: 1848-7718
http://www.pub.iapchem.org/ojs/index.php/admet/index Original scientific paper Introduction Solubility of drugs is an important aspect from the earliest stages of drug discovery to the latest stage of
the drug formulation [1,2]. It is defined as the maximum quantity of a drug dissolved in a given volume of a
solvent at a certain temperature [3]. Solubility data for a solute, especially for pharmaceuticals, is a
significant physicochemical property in crystallization, extraction of an analyte from different matrices,
evaluation of oral bioavailability and preparation of liquid and semi-solid dosage forms. Furthermore,
solubility in water is needed to make a solution of the drug or drug-like molecule to be tested for its
pharmacological/toxicological
activities. Despite
simple
methods
developed
for
experimental doi: 10.5599/admet.621 doi: 10.5599/admet.621 269 Ali Shayanfar et al. ADMET & DMPK 6(4) (2018) 269-278 determination of the solubility of drugs [4], various parameters can affect accuracy of the obtained data
[5]. Several different sets of solubility data have been reported for a drug at a certain solvent and
temperature in the literature [6-8]. These solubility variations are one of the possible reasons for unsuccessful attempts of medicinal
chemist in developing models for solubility prediction in water, non-aqueous solvents and solvent mixtures
with deviation in agreement with experimental works. For example, the best model for aqueous solubility
prediction has a prediction error more than 100 % [9], and estimation error for solubility in the solvent
mixture is higher than 25 % for pharmaceuticals [6]. There are various methods for solubility determination
of pharmaceuticals [10,11]. The shake-flask method is a common method for solubility determination
which an excess amount of drug is added to a certain solvent and after appropriate shaking at a constant
temperature to reach the equilibrium condition, and the saturated concentration is determined by a valid
analytical method [11]. In some cases, variation of solubility data originates from the nature of studied
compounds, such as polymorphism and formations of drug aggregates, micelles, drug-buffer complexes,
stability, solute and solvent impurities; they can affect the solubility value. Another problem in solubility
determination is invalidity of the applied method for quantification of drug concentration. Other sources of
variations, which can affect solubility values related to the solubility determination process such as time of
equilibrium, incomplete dissolution over the equilibration time and inappropriate phase separation
solution handling and adsorption to untreated surfaces, amount of residual solid, speed and stirring or
shaking patterns of the suspension [5,7,12]. Introduction Although, various sets of solubility data for chemical and
pharmaceutical materials in water and organic solvents are annually collected by different research groups
and reported in the literature, there are few investigations into the effect of different parameters on
solubility determination of drugs. Baka et al. [8] investigated the effect of various parameters such as
amount of solid excess, stirring time, temperature, and sedimentation time on solubility of
hydrochlorothiazide as a model drug. Some reports have been investigated the effect of excess solid on
solubility of drugs [13,14]; and the reported results are discussed in review articles [11,15]. Lamotrigine is a basic drug (pKa=5.7) [16] existing in unionized form in water, while it is in ionized form
in 0.1 M HCl. Sodium phenytoin is a salt form of phenytoin (an acidic compound with pKa=8.3 [16]) being
partially ionized form in water. In addition, it changes to the parent drug (un-ionized form) in acidic
medium. In this study, the effects of the amount of solid excess, shaking patterns of solution and
sedimentation time were investigated on solubility of two antiepileptic drugs, namely sodium phenytoin
and lamotrigine in water, ethanol and acidic medium (HCl 0.1 M). Samples and chemicals Sodium phenytoin (99 %) and lamotrigine (99 %) were purchased from Alhavi Pharmaceutical Company
(Tehran, Iran). Ethanol (99 %) was provided from Scharlau (Barcelona, Spain) and HCl (37 %) was supplied
from Merck (Darmstadt, Germany), and double distilled water was used as received in the lab. The effect of the excess solid amount on solubility of sodium phenytoin in water, ethanol and HCl (0.1 M) Figure 1 shows the solubility of sodium phenytoin in water at 37 °C. The experiment was investigated in
various excess amounts of phenytoin sodium. A linear relationship exists between excess solid amount of
drug and solubility. The percentage change between the maximum and minimum values of solubility is
116 %; however, the excess amount has a low effect on solubility of phenytoin in HCl 0.1 M (Figure 2) in
comparison to solubility in water (percentage change was less than 30 %). Moreover, to confirm the effect
of excess amount in ionized form, phenytoin solubility was performed in ethanol (dielectric constant of
ethanol is lower than water and the ionization in ethanol is less than water) and the results were illustrated
in Figure 3. A similar pattern to HCl (percentage difference was 21 %) was obtained for solubility of sodium
phenytoin in ethanol. Japan). The pH of the solutions was measured by a pH meter (Metrohm, Switzerland). Japan). The pH of the solutions was measured by a pH meter (Metrohm, Switzerland). Measurement of solubility and investigation of the effect of excess amount of solute and type of stirring on
solubility Excess amount of each drug was added to a small volume (e.g. 5 mL) of the studied solvent in an
Erlenmeyer flask. The amount of excess was added according to the preliminary experiments or reported
solubility of drugs in the studied solvent from the literature (solubility of phenytoin in water and ethanol
are 86 mg/ml and 19.9 mg/mL at 35 °C [17], respectively and lamotrigine solubility in water and HCl 0.1 M
are 0.19 mg/ml [18] and 2.22 mg/mL [19] at 25 °C, respectively). Therefore, in this study, the volume of
solvent was considered 5 mL in an Erlenmeyer flask, being similar to the routine experiment of solubility
determination in the pharmaceutical and chemical laboratories. For highly soluble compounds, e.g. sodium
phenytoin in water 10 %, 25 %, 100 %, 150 % and 200 % of saturated solubility of the drug was applied as
an excess solid amount. While for low solubility values, e.g. lamotrigine in water, the minimum amount of
drug could be weighted by a digital balance (the minimum amount of weighting 10-30 mg), was selected as
the lowest concentration of suspension for solubility determination and other solutions up to 5-folds of
this amount were prepared to simulate a routine solubility determination experiment. Then, the samples
were placed in an incubator equipped with a temperature-controlling system at 37 °C in 150 rpm for 48
hours. Afterward, the saturated solutions of the drugs were filtered using hydrophilic filters (0.45 µm). In
addition, magnetic stirrer and shake-flask approaches, separately, were used to saturate sodium phenytoin
and lamotrigine in water at 37 °C and 150 rpm for 48 hours. Furthermore, the concentration of samples
was measured after 24-hour sedimentation. The concentration of samples was measured by a UV
spectrophotometer. Each datum was obtained from at least three replicate independent measurements,
and the maximum relative standard deviation (RSD) of each experiment was 10 %. Apparatus A shaker incubator (Heidolph, Germany) or a magnetic stirrer (Heidolph MR Hei-Tec, Germany) was
used to mix two phases and maintain the temperature of samples at 37±0.1 °C during the solubility
measurements. Samples were filtered using a hydrophilic membrane (450 nm pore size). The
concentration of the saturated solution was measured using a UV–Vis spectrophotometer (Shimadzu, 270 Solubility of sodium phenytoin and lamotrigine ADMET & DMPK 6(4) (2018) 269-278 The effect of excess amount on solubility of sodium phenytoin and lamotrigine The study results show that excess solid amount of the studied drugs has a considerable effect on
solubility, especially in ionized form i.e. sodium phenytoin in water and lamotrigine in HCl (0.1 M). One of
the reasons for change in solubility in the presence of different values of excess solid is change in pH of
saturated solution [20]. In this study, solubility was determined in aqueous solution at own pH. However,
pH determination of final solutions (various excess amounts) showed no substantial change (pH:
11.6±0.05). A differential scanning calorimetry (DSC) study of the remaining solid of lamotrigine confirms
no possible change in crystalline phase due to a sharp peak corresponding to lamotrigine melting at 220°C,
being in agreement with the literature [19]. However, DSC thermogram sodium phenytoin in water
exhibited some new peaks resultant to phase transformation of drug to the hexahydrate form similar to
the reported study by Rubino [21], and it is not possible to judge about the polymorphic change or dimer
formation whenever the amount of excess solid is changed. Kawakami et al. [13] have reported that solubility of indomethacin increased with an increase in the
solid amount in ionizable form (pH=6.5-7) against indomethacin solubility in non-ionizable form (pH=5). They discussed the anomalous behavior in dissolution and crystallization rates at near equilibrium
conditions as the possible mechanism. However, one of the potential reasons based on Avdeef’s comment
on that study is the possible partitioning (adsorption) of the charged form of indomethacin onto the excess
solid present in the suspensions, and some ionizable compounds may be surface-active. However, further
mechanisms such as dimer formation are possible, and the results are not interpretable without analysis of
the solid form [15]. Nevertheless, a similar research study about solubility measurement of hydrochlorothiazide reported
no significant difference for solubility values in water when different excess amounts of the drug were used
[8]. Hydrochlorothiazide as an acidic compound converts to non-ionized form in saturated aqueous
solution. Another study by Mosharraf et al. [22] reported that solubility of chemical compounds (in salt
form) was affected by the amount of excess solid. Increasing the amount of excess solid of barium sulphate
or calcium carbonate can affect solubility value due to the disordering particle surface and existence of a
peripheral disordered layer. The effect of the excess solid amount on solubility of lamotrigine in water, ethanol and HCl (0.1 M) Figure 4 illustrates solubility of lamotrigine in water at 37 °C in different amounts of excess solid, and
the obtained data revealed that excess amount had no significant effect on solubility value. However, it has
a considerable effect on solubility value in HCl 0.1 M (Figure 5, the percentage difference between the
maximum and minimum amount of solubility value was higher than 40 %), which lamotrigine is in an
ionized form similar to the data obtained for phenytoin sodium. doi: 10.5599/admet.621 271 271 ADMET & DMPK 6(4) (2018) 269-278 Ali Shayanfar et al. Ali Shayanfar et al. The effect of type of stirring and sedimentation time on solubility of sodium phenytoin and lamotrigine in
water The effect of type of stirring and sedimentation time on solubility of sodium phenytoin and lamotrigine in
water Magnetic stirrer and shake-flask approaches were used to saturate sodium phenytoin and lamotrigine in
water at 37 °C. Figure 6 illustrates the effect of type of stirring on the obtained solubility value, and shown,
the type of stirring for solubility determination has no significant effect on the saturated concentration
(solubility) of phenytoin sodium. Moreover, sedimentation time after 24-hour (Figures 7 and 8) was
investigated on the solubility values, and a significant change was observed in solubility of sodium
phenytoin in both stirring instruments. Similar results were observed for solubility determination of
lamotrigine in water (Figure 9). The data indicate that sedimentation time seems to have a significant
effect on solubility value, particularly for stirring of the solution with magnet stirrer. The effect of excess amount on solubility of sodium phenytoin and lamotrigine According to the obtained study, solubility of chemical compounds can change by the amount of excess
solid, and the mutual issue between the reported studies is ionization and salt form of solute. Change in
the diffusion layer and disordering particle surface due to adsorption of the charged form of the drug and 272 ADMET & DMPK 6(4) (2018) 269-278 Solubility of sodium phenytoin and lamotrigine surface activity of solute in ionized form could be an acceptable interpretation for increasing solubility
values in the higher amount of excess solids. Similar phenomena have been proposed for increase in
saturation solubility of nanocrystals (size of particle less than 1000 nm) by the change in layer of unstirred
layer surrounding the drug particle (diffusion layer) [23]. Moreover, when drug salts are added to solution,
their solubility is expected to vary according to the amount of solid added, since the solubility product may
not be satisfied below a certain amount of added salt. The partial conversion back of sodium phenytoin to
free acid form during solubility testing reported by Chaing and Wang [24] could be reason for variability in
the solubility values. The effect of type of stirring and sedimentation time on solubility of lamotrigine and phenytoin sodium The effect of type of stirring and sedimentation time on solubility of lamotrigine and phenytoin sodium Type of stirring is a critical parameter in evaluating dissolution of pharmaceuticals [24]. However, it has
been not yet considered as an effective factor in solubility determination of solute. Magnetic stirrer and
shake-flask are common approaches in the literature to attain the solution to equilibrium. Type of stirring
could be considered as a possible reason for the variation of solubility values because of supersaturation
phenomenon. It is a state of a solution that contains more of the dissolved material than could be
dissolved in the solvent [7]. The results in Figures 6-9 show that type of stirring has a slight effect on
obtained solubility for lamotrigine and phenytoin sodium. However, sedimentation time after 24-hour, in
both types of stirring, has a considerable effect on solubility values. In this regard, Baka et al. [8] conducted
the only study about the effect of sedimentation time for solubility of hydrochlorothiazide; similar to the
results of this study, sedimentation time had a considerable effect on the aqueous solubility of drug. The results of this study indicate that although solubility determination of drugs seems a simple
experiment, the numerical values of solubility of a compound are affected by different factors. Amount of
excess solid and sedimentation time could have a significant effect on solubility values. Figure 1. The effect of excess solid amount on solubility of sodium phenytoin in water at 37 °C (added weight
of drug associated with each of the points in 5 mL of solution is 450, 500, 750, 1000, 1250 mg, respectively). Figure 1. The effect of excess solid amount on solubility of sodium phenytoin in water at 37 °C (added weight
of drug associated with each of the points in 5 mL of solution is 450, 500, 750, 1000, 1250 mg, respectively). doi: 10.5599/admet.621 273 273 ADMET & DMPK 6(4) (2018) 269-278 Ali Shayanfar et al. Figure 2. The effect of the excess solid amount on solubility of sodium phenytoin in HCl 0.1 M at 3 7 °C. (Added weight of drug associated with each of the points in 5 mL of solution is 15, 25, 50 and 75 mg,
respectively). Figure 2. The effect of the excess solid amount on solubility of sodium phenytoin in HCl 0.1 M at 3 7 °C. (Added weight of drug associated with each of the points in 5 mL of solution is 15, 25, 50 and 75 mg,
respectively). Figure 3. The effect of type of stirring and sedimentation time on solubility of lamotrigine and phenytoin sodium The effect of the excess solid amount on solubility of sodium phenytoin in ethanol at 37 °C. (Added
weight of drug associated with each of the points in 5 mL of solution is 125, 150, 175, 200, 225, 350 and 450
mg, respectively). Figure 3. The effect of the excess solid amount on solubility of sodium phenytoin in ethanol at 37 °C. (Added
weight of drug associated with each of the points in 5 mL of solution is 125, 150, 175, 200, 225, 350 and 450
mg, respectively). Figure 4. The effect of the excess solid amount on solubility of lamotrigine in water at 37 °C. (Added weight of
drug associated with each of the points in 5 mL of solution is 25, 75, 175, 250, 375, 500 mg, respectively). Figure 4. The effect of the excess solid amount on solubility of lamotrigine in water at 37 °C. (Added weight of Figure 4. The effect of the excess solid amount on solubility of lamotrigine in water at 37 °C. (Added weight of
drug associated with each of the points in 5 mL of solution is 25, 75, 175, 250, 375, 500 mg, respectively). 274 Conclusion Solubility is a classic and common physicochemical property of pharmaceuticals; however, its values are
affected by various parameters. The findings of this study indicated that the amount of excess solid,
especially in ionized form and sedimentation time after appropriate stirring, had significant effects on the
obtained solubility of sodium phenytoin and lamotrigine. The findings show that these factors are possible
reasons for variation in the reported solubility data in the literature and relatively unsuccessful attempts to
develop solubility prediction models. It seems that the proper reporting of experimental details is
necessary in solubility studies. Solubility of sodium phenytoin and lamotrigine ADMET & DMPK 6(4) (2018) 269-278 MET & DMPK 6(4) (2018) 269-278
Solubility of sodium phenytoin and lamot
Figure 5. The effect of the excess solid amount on solubility of lamotrigine in HCl (0.1M) at 37 °C. (Added
weight of drug associated with each of the points in 5 mL of solution is 25, 30, 35, 40, 50 and 75 mg,
respectively). Figure 5. The effect of the excess solid amount on solubility of lamotrigine in HCl (0.1M) at 37 °C. (Added
weight of drug associated with each of the points in 5 mL of solution is 25, 30, 35, 40, 50 and 75 mg,
respectively). Figure 6. The effect of type of stirring on sodium phenytoin solubility in water at 37 °C. (Added weight of drug
associated with each of the points in 5 mL of solution is 450 mg and 1250 mg). Figure 6. The effect of type of stirring on sodium phenytoin solubility in water at 37 °C. (Added weight of drug
associated with each of the points in 5 mL of solution is 450 mg and 1250 mg). Figure 7. The effect of sedimentation on sodium phenytoin solubility in water at 37 °C (the suspensions were
stirred with magnetic-stirrer). (Added weight of drug associated with each of the points in 5 mL of solution is
450 mg and 1250 mg, respectively). Figure 7. The effect of sedimentation on sodium phenytoin solubility in water at 37 °C (the suspensions were
stirred with magnetic-stirrer). (Added weight of drug associated with each of the points in 5 mL of solution is
450 mg and 1250 mg, respectively). doi: 10.5599/admet.621 275 275 ADMET & DMPK 6(4) (2018) 269-278 Ali Shayanfar et al. Figure 8. The effect of sedimentation on sodium phenytoin solubility in water at 37 °C (the amount of excess
solid was 450 mg/5 mL). Figure 8. The effect of sedimentation on sodium phenytoin solubility in water at 37 °C (the amount of excess
solid was 450 mg/5 mL). Figure 9. The effect of sedimentation on lamotrigine solubility in water at 37 °C (the amount of excess solid
was 175 mg/5 mL). Figure 9. The effect of sedimentation on lamotrigine solubility in water at 37 °C (the amount of excess solid
was 175 mg/5 mL). Acknowledgements This article is a part of the results of S.M’s Pharm.D thesis No. 85 registered at Faculty of Pharmacy,
Tabriz University of Medical Sciences, Tabriz, Iran. A.S. thanks the Ministry of Health and Medical Education
(grant for young assistant professors), Tehran, Iran, for financial support. 276 ADMET & DMPK 6(4) (2018) 269-278 Solubility of sodium phenytoin and lamotrigine References [1] L. Di, P.V. Fish, T. Mano. Bridging solubility between drug discovery and development. Drug
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amorphous drugs: Challenges in measurement. Pharm. Dev. Technol. 16 (2011) 187-200. [12] A. Avdeef. Suggested improvements for measurement of equilibrium solubility-pH of ionizable drugs. ADMET & DMPK 3 (2015) 84-109. [13] K. Kawakami, K. Miyoshi, Y. Ida. Impact of the amount of excess solids on apparent solubility. Pharm. Res. 22 (2005) 1537-1543. [14] Z. Wang, L.S. Burrell, W.J. Lambert. Solubility of E2050 at various pH: A case in which apparent
solubility is affected by the amount of excess solid. J. Pharm. Sci. 91 (2002) 1445-1455. [15] A. Avdeef. Solubility of sparingly-soluble ionizable drugs. Adv. Drug Deliv. Rev. 59 (2007) 568-590. [16] A.C. Moffat, M.D. Osselton, B. Widdop, J. Watts, Clarke's analysis of drugs and poisons,
Pharmaceutical Press, London, 2011. [17] A. Mabhoot, A. Jouyban. ©2018 by the authors; licensee IAPC, Zagreb, Croatia. This article is an open-access article distributed under the terms and
conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/) [23] F. Nielloud, Pharmaceutical emulsions and suspensions: revised and expanded, CRC Press, Boca
Raton, 2000. References Solubility of sodium phenytoin in ethanol + water mixtures at various
temperatures. Chem. Eng. Technol. 203 (2016) 1009-1012. [18] A. Shayanfar, M.A.A. Fakhree, W.E. Acree Jr, A. Jouyban. Solubility of lamotrigine, diazepam, and
clonazepam in ethanol + water mixtures at 298.15 K. J. Chem. Eng. Data 54 (2009) 1107-1109. [19] K. Beattie, G. Phadke, J. Novakovic, Chapter 6 - Lamotrigine, In: H.G. Brittain (Ed.) Profiles of Drug
Substances, Excipients and Related Methodology, Academic Press, 2012, pp. 245-285. [20] A. Avdeef, Absorption and drug development: solubility, permeability, and charge state, John Wiley &
Sons, Hoboken, New Jersey, 2012. [21] J.T. Rubino. Solubilities and solid state properties of the sodium salts of drugs. J. Pharm. Sci. 78
(1989) 485-489. [22] M. Mosharraf, T. Sebhatu, C. Nyström. The effects of disordered structure on the solubility and
dissolution rates of some hydrophilic, sparingly soluble drugs. Int. J. Pharm. 177 (1999) 29-51. doi: 10.5599/admet.621 277 Ali Shayanfar et al. [23] F. Nielloud, Pharmaceutical emulsions and suspensions: revised and expanded, CRC Press, Boca
Raton, 2000. [23] F. Nielloud, Pharmaceutical emulsions and suspensions: revised and expanded, CRC Press, Boca
Raton, 2000. [24] J. Bevernage, J. Brouwers, M.E. Brewster, P. Augustijns. Evaluation of gastrointestinal drug
supersaturation and precipitation: Strategies and issues. Int. J. Pharm. 453 (2013) 25-35. [24] J. Bevernage, J. Brouwers, M.E. Brewster, P. Augustijns. Evaluation of gastrointestinal drug
supersaturation and precipitation: Strategies and issues. Int. J. Pharm. 453 (2013) 25-35. 278
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THE POLYTHEISTIC PSYCHE: RE-IMAGINING SOUL AND SELF IN JAME'S HILLMAN'S ARCHETYPAL PSYCHOLOGY
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ISSN 2663-5712 Issue 9 / Part 2 Issue 9 / Part 2 Issue 9 / Part 2 Issue 9 / Part 2 SWorldJournal THE POLYTHEISTIC PSYCHE: RE-IMAGINING SOUL AND SELF IN
JAME’S HILLMAN’S ARCHETYPAL PSYCHOLOGY
King J R King J.R
PhD Candidate, Faculty of Philosophy,
University of Salamanca, Spain Abstract. This article examines the notion of psychological polytheism in the context of
Jungian depth psychology. The author focuses on the concept of the “polytheistic psyche” which
American psychologist and philosopher James Hillman, founder of the post-Jungian branch of
Archetypal Psychology, has put forth as part of a broader re-imagining of the field. Theorizing
against a backdrop of mythological metaphor, Hillman maintains that the psyche is by nature
plural, and the human personality is ultimately multiple, not unitary. Psychological wholeness of
Self means the health of diverse points of view and multiple relations within the psyche, not
wholeness in the traditional theological sense meaning a unity of “the one.” Key words: archetypal psychology, imaginal psychology, psychotherapy, C.G. Jung, post-
Jungians, James Hillman, imagination, religion, theology, polytheism, soul, psyche, soul-making. g
Introduction. Regarded as one of the most innovative heirs to the depth psychology tradition
inaugurated by C.G. Jung, the American psychologist and philosopher James Hillman
(1926-2011) is the founder and sustaining voice of Archetypal Psychology, a post-
Jungian psychology which emphasizes a movement beyond the context of clinical
analysis towards a “psychology of soul” situated within the culture of imagination,
metaphor, and Western mythology. In Archetypal Psychology we encounter the
following principal themes or main ideas: (1) a shifting of focus away from the
rational ego and towards the soul; (2) an emphasis on image and imagination as
soul’s natural language and activity; (3) the exploration of a background of
mythological metaphor which offers a vision of the soul as plural and polytheistic; (4)
and the elaboration of a process of “soul-making”– an imaginative, aesthetic response
to life that seeks to enrich the relationship between man, soul, and the world. p
As part of his program to elaborate an aesthetic psychology of soul, Hillman has
asked what idea today governs our image of the psyche, “the many or the one?” [2,
110]. His answer is that the West has made a norm out of the singleness of soul,
where unity and integration are held to be desirable advances over multiplicity and
diversity, where “health of wholeness” has meant “the one dominating the many” [2,
114-116]. Consistent with his belief that religion, or theology, influences psychology,
Hillman maintains that this is but only one consequence of a deleterious legacy that
monotheistic religion (particularly fundamentalist Christianity, which has regarded
the image literally rather than metaphorically) has inflicted upon psychology as well
as the wider cultural imagination [5]. This inheritance has so conditioned our image
of soul, says Hillman, that we remain “ruled by a bias toward the one” [2, 110]. y
y
However, instead of making a singleness out of soul, Archetypal Psychology
portrays the soul as “inherently multiple.” Affirming this conception of the psyche,
Thomas Moore has noted that an authentic psychology conceived from the point of
view of soul and the imagination necessarily “gives place to multiplicity, not
demanding integration and other forms of unity, and at the same time offer[s] a www.sworldjournal.com www.sworldjournal.com 124 Issue 9 / Part 2 SWorldJournal Issue 9 / Part 2 language adequate to a psyche that has many faces” [9, 37]. Main Text. The notion of polytheism was first employed in the context of psychology by
Jung as a means of characterizing the “objective psyche,” which he believed to
consist of a multiplicity of partial personalities [2]. Jung had already described the
psyche as polycentric and pluralistic based on his studies of the alchemical idea of the
lumen naturae, or the light of nature, which the Swiss alchemist Paracelsus had
equated to the anima mundi. Traditional descriptions of the anima mundi or soul-of-
the-world maintained that the light of nature was multiple, and when Jung in his turn
directed his psychological eye toward the lumen naturae, he wrote of it as
multiplicity of partial consciousnesses, like stars or divine sparks– “luminosities.”
With its polytheistic perspective, archetypal psychology’s understanding of the
psyche “corresponds with this description [of multiplicity] and provides its imagistic
formulation in the major traditional language of our civilization”, i.e., classical
mythology [2, 114-116]. The mythological gods are, then, the images through which
the western psyche was once manifested in ancient Greece. Hillman finds in the mythological polytheism of ancient Greece an effective
paradigm for envisioning the psyche as not only multiple, but as “a communion of
many persons each with their specific needs, fears, longings, styles and language” [9,
37]. This collective of persons echoes the many points of view that underlie what
appears to be a unified human being. The persons of dreams, for example, represent
the many personalities who play a role in the everyday dramas of the psyche. As a
polytheistic psychology, Hillman maintains that a central task of his imaginal method
is to reinvigorate this relationship with the multiple figures of psyche as beings in
their own right, beings that demand to be approached according to their own
principles, allowing each voice to be heard. A polycentric approach accepts a
multiplicity of voices without insisting on unifying them into one figure. Wholeness,
then, comes to mean the health of multiple relations within the psyche, not wholeness
in the traditional theological sense which means a unity of the one. A focus on
diversity leads to deeper insights into psychic images and their relationships to one
another. ISSN 2663-5712 g
Introduction. Yet, in describing the
nature of psyche as multiple, Hillman doesn’t choose a word like “multifaceted” to
characterize these many faces. Instead, he appropriates from religion and mythology
a more animated word, polytheism, implying an essential and profound plurality in
the soul [9, 37]. With the term polytheism, Hillman advocates a polytheistic rather
than a monotheistic psychology, and he seeks to return to, or better yet retrieve and
bring forward polytheistic mythology as a fertile metaphoric background for
imagining the psyche in its inherent multiplicity. Main Text. In keeping with Hillman’s use of the designation “archetypal” to refer to a
perspective that sees through the literal into its metaphorical background, as well as
to a move one makes which places whatever is seen in a mythic perspective, Hillman
has emphasized that archetypal psychology is neither a religion nor a theology. These
gods and goddesses are “neither believed in nor addressed directly, as they are
adjectival rather than substantive” [4, 129]. He says that it is only when these
qualities “are literalized, set apart as substances, that is, become theologized, do we www.sworldjournal.com www.sworldjournal.com 125 Issue 9 / Part 2 SWorldJournal Issue 9 / Part 2 have to imagine them through the category of belief” [ibid.]. In this respect,
Archetypal Psychology does not worship the gods. Psychological polytheism is not
about worship, but about an attitude, a way of looking at things, and the perspective
we bring to life. The myths in which the gods enact their dramas are themselves
understood “as metaphors, never as transcendental metaphysics whose categories are
divine figures” [4, 114]. Hillman emphasizes that all statements concerning myths
and the gods are to be taken metaphorically, “pre-fixed with an ‘as-if,’ just as all
speaking about the archetypal presences of the psyche “are translations from one
metaphor to another” [3, xiii]. When we keep this perspective in mind, we enable
ourselves to engage the nature of soul in an imaginative way and approach the basic
questions concerning psychology first of all by means of the imagination. The variety of perspectives portrayed in these mythical paradigms proves to be
always more psychological, that is, psyche-logical, than “scientific” psychology’s
volumes of literalized concepts and lifeless ideas. Myths, he says, “produce more
insights into emotions, images and relationships and reflect more accurately the
illusions and entanglements of the soul” [4, 114-116]. Mythical paradigms provide a
variety of perspectives and styles of thought that offer the possibility of metaphoric
insight into the events of our lives and the prospect of differentiating the
particularities of psychic phenomena. Hillman offers the example of the experience
of depression, which from this mythic point of view might be understood on the
model of Christ and his suffering and resurrection; it may through Saturn gain the
depth of melancholy and inspiration, or through Apollo serve to release the black bird
of prophetic insight. ISSN 2663-5712 Main Text. From the perspective of Demeter depression may yield
awareness of the mother-daughter mystery, or through Dionysus, we may find
depression a refuge from the excessive demands of the ruling will [4, 114]. Focusing upon the many and the different, rather than upon the one and the
same, provides multiple modes of looking at and of discovering the virtues in our
psychic conditions. Once understood metaphorically, we begin to experience these
archetypal gods as living figures within our personalities, “personifications of psychic
forces” [7, 10, par. 185]. Myths become the “backgrounds to the complexity of
human nature” [4, 114] and mythic consciousness helps us begin to understand the
variety of our own psychological dynamics and experience. When we honor psychic multiplicity, we honor the diversity and ambiguity of
our own self-definition. In this context, imaginal psychology maintains that the
personality is fundamentally multiple, rather than unitary. This is to suggest quite
radically that maybe we don’t “have” a personality, that there is no personality, “but
rather only personifications which, treated as real persons, assume the status of
autonomous personalities” [4, 114]. Proposing the relativity of all personifications,
however, is not to be mistaken with promoting “multiple personality disorder,”
although Hillman does, in fact, make the bold claim that multiple personality “is
humanity in its natural condition” [6, 40]. However, from the psychological
perspective, these many persons or personifications echo all those points of view that
underlie what appears to be a unified human being [9, 37]. The persons of a dream,
the figures in our imagination and fantasy, for example, represent the many
personalities who have a role to play in the psyche’s everyday dramas. www.sworldjournal.com 126 Issue 9 / Part 2 SWorldJournal Issue 9 / Part 2 Issue 9 / Part 2 Archetypal Psychology’s polycentric approach therefore seeks to use the
imagination in order explore the relations between all personifications and dream
figures. It does so by observing, listening, and attending carefully to each particular
image of their presentation, making sure to allow each one their due– even those
figures of the imagination that the predominant ego point of view might find
abhorrent or threatening to its position of authority. It is in this light also that Hillman
encourages us to remember that even the “I” of the ego is only one point of view, one
figure, one image, among the multiple figures of the psyche. Main Text. Viewed in this manner,
says psychologist Michael Vannoy Adams, to regard the multiplicity of personality as
a psychiatric “aberration” is simply “evidence of a cultural prejudice that erroneously
identifies one partial personality, the ego, with the personality as such” [1, 115]. As
professor of religion David Miller has articulated, polytheism and a polytheistic view
of the psyche allows a person to experience himself or herself as many different and
yet coextensive selves, each of which is felt to have autonomous power [8]. With respect to the age-old philosophical problem of “the one-and-the-many”, it
is evident that Archetypal Psychology values multiplicity over unity. It is Hillman’s
colleague Rafael López-Pedraza whose own comment most succinctly summarizes
this position. Instead of the usual formulation which proposes that unity contains
multiplicity, López-Pedraza reverses the formulation and says that “the many
contains the unity of the one without losing the possibilities of the many” (López-
Pedraza in [1, 115]. When we imagine and idea of the totality of the soul, Hillman
says, “the ‘one’ appears only as this or that image…and it appears as the unity of
each particular event, discoverable phenomenally only within eachness…[U]nity too
can be imagined polytheistically” [2, 131]. This program of cultivating polytheistic
consciousness offers a rich alternative to the monotheistic need for unity and
integration. The archetypal point of view holds that, “the one does not appear as such
but is contained as one among many within each of the many” [ibid]. g
y
y [
]
The polytheistic or polycentric approach of Archetypal Psychology seeks to
loosen the grip of what Hillman calls “our true religion,” a “monotheism of
consciousness,” and bring with it the health of a psychological polytheism that
recognizes the location of human consciousness “in multiple figures and centers” [3,
26]. In this imaginative orientation of psychology, the multiple perspectives and
persons of the psyche are personified against a mythic, metaphoric background,
obliging our normally hegemonic ego-consciousness to circulate among a field of
powers. The notion of a polytheistic or polycentric psyche means that soul has many
sources of meaning, direction, and value. As Moore notes, the richly textured images
of polytheistic mythology are themselves therapeutic because they give place to the
soul’s beauty, variety, and conflict– teaching us at the same time to find vitality in
tension, gather wisdom from ambivalence, and learn from paradox [9, 38]. ISSN 2663-5712 Main Text. Although Hillman finds in mythology an effective paradigm for his psychology,
his almost exclusive focus on the Greek gods and goddesses would seem to ignore
the ethnic and cultural diversity of the entire global pantheon. However, as Adams
has noted, Hillman justifies, or rationalizes, this election on the basis that the field of
depth psychology is historically European in origin and that Greek mythology and its
gods and goddesses are uniquely influential in that particular cultural context [1, www.sworldjournal.com www.sworldjournal.com 127 Issue 9 / Part 2 SWorldJournal Issue 9 / Part 2 116]. In sum, this insistence on the mythical polytheistic perspective is reflective of
Hillman’s belief that our psychic complexity “requires all the Gods”, and so he has
sought to defend the polytheistic diversity of the psyche from being collapsed into a
monotheistic doctrine. 116]. In sum, this insistence on the mythical polytheistic perspective is reflective of
Hillman’s belief that our psychic complexity “requires all the Gods”, and so he has
sought to defend the polytheistic diversity of the psyche from being collapsed into a
monotheistic doctrine. p
g
,
pp
5. Hillman, J. (1983a). Healing Fiction. Putnam, CT: Spring Publications. References: e e e ces:
1. Adams, M. V. (2008). The Archetypal School. In: Young-Eisendrath, P. Dawson, T. (org.): The Cambridge Companion to Jung, Cambridge: Cambridge
University Press. 2 Hill
J (1971) P
h l
M
th i ti
P l th i ti
S
i
31 1. Adams, M. V. (2008). The Archetypal School. In: Young-Eisendrath, P. Dawson, T. (org.): The Cambridge Companion to Jung, Cambridge: Cambridge
University Press. 2. Hillman, J. (1971). Psychology Monotheistic or Polytheistic. Spring, 31, pp. University Press. 2. Hillman, J. (1971). Psychology Monotheistic or Polytheistic. Spring, 31, pp. 193-207. 2. Hillman, J. (1971). Psychology Monotheistic or Polytheistic. Spring, 31, pp. 193-207. 3. Hillman, J. (1975a). Re-visioning psychology. New York, NY: Harper and
Row. 3. Hillman, J. (1975a). Re-visioning psychology. New York, NY: Harper and
Row. 4. Hillman, J. (1981). “Psychology: Monotheistic or Polytheistic.” In D.L. Miller, The New Polytheism: Rebirth of the Gods and Goddesses [1971]. Dallas,
Texas: Spring Publications, Inc. pp. 109-142. 4. Hillman, J. (1981). “Psychology: Monotheistic or Polytheistic.” In D.L. Miller, The New Polytheism: Rebirth of the Gods and Goddesses [1971]. Dallas,
Texas: Spring Publications, Inc. pp. 109-142. p
g
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pp
5. Hillman, J. (1983a). Healing Fiction. Putnam, CT: Spring Publications. 5. Hillman, J. (1983a). Healing Fiction. Putnam, CT: Spring Publications. 6. Hillman, J. (2013). Archetypal Psychology. Uniform Edition of the Writings
of James Hillman, Volume 1. Thompson, CT: Spring Publication, Inc. 6. Hillman, J. (2013). Archetypal Psychology. Uniform Edition of the Writings
of James Hillman, Volume 1. Thompson, CT: Spring Publication, Inc. 7. Jung, C. G. (1979). Collected Works of Carl Gustav Jung (R. F. C. Hull,
Trans.). Princeton, NJ: Princeton University Press. 7. Jung, C. G. (1979). Collected Works of Carl Gustav Jung (R. F. C. Hull,
Trans.). Princeton, NJ: Princeton University Press. 8. Miller, D. L. (1981). The New Polytheism: Rebirth of the Gods and
Goddesses. Dallas, Tex.: Spring Publications. 8. Miller, D. L. (1981). The New Polytheism: Rebirth of the Gods and
Goddesses. Dallas, Tex.: Spring Publications. p
g
9. Moore, T. (Ed.). (1989). Blue Fire: Selected Writings by James Hillman. New
York, NY: Harper Perennial. 9. Moore, T. (Ed.). (1989). Blue Fire: Selected Writings by James Hillman. New
York, NY: Harper Perennial. ISSN 2663-5712 www.sworldjournal.com 128
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Occupational exposure of health care personnel to SARS-CoV-2 particles in the intensive care unit of Tehran hospital
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International journal of environmental science and technology
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cc-by
| 6,404
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R. Yarahmadi1 · F. Bokharaei‑Salim2 · S. Soleimani‑Alyar3 · P. Moridi4 · O. Moradi‑Moghaddam5 · M. Niakan‑Lahiji6 ·
M.‑M. Darvishi7 · S. Golmahammadi4 · S. A. J. Mousavi3 · H. Ebrahimi8 · A. Ashtarinezad8 · A.‑A. Farshad9 ·
A. Jonidi‑Jafari10 · S. J. Kiani11 · S. Garshasbi12 · S. Mehrzadi13 R. Yarahmadi1 · F. Bokharaei‑Salim2 · S. Soleimani‑Alyar3 · P. Moridi4 · O. Moradi‑Moghaddam5 · M. Niakan‑Lahiji6 ·
M.‑M. Darvishi7 · S. Golmahammadi4 · S. A. J. Mousavi3 · H. Ebrahimi8 · A. Ashtarinezad8 · A.‑A. Farshad9 ·
A. Jonidi‑Jafari10 · S. J. Kiani11 · S. Garshasbi12 · S. Mehrzadi13 Received: 28 August 2020 / Revised: 28 September 2020 / Accepted: 17 December 2020 / Published online: 2 February 2021
© The Author(s) 2021 Abstract Anesthesiology and Critical Care Department,
Trauma and Injury Research Center, Rasool‑E‑Akram
Complex Hospital, Iran University of Medical Sciences,
Tehran, Iran
7
Department of Engineering, Payame Noor University,
Tehran, Iran
8
Air Pollution Research Center, Department of Occupational
Health, Iran University of Medical Sciences, Tehran, Iran
9
Occupational Health Research Center, Department
of Occupational Health, Iran University of Medical Sciences,
Tehran, Iran
10 Department of Environmental Health, Iran University
of Medical Sciences, Tehran, Iran
11 School of Medicine, Iran University of Medical Sciences,
Tehran, Iran
12 Vice Chancellor for Health Center, Iran University
of Medical Sciences, Tehran, Iran
13 Razi Drug Research Center, Iran University of Medical
Sciences, Tehran, Iran Editorial responsibility: Samareh Mirkia. Editorial responsibility: Samareh Mirkia. International Journal of Environmental Science and Technology (2021) 18:3739–3746
https://doi.org/10.1007/s13762-020-03095-z International Journal of Environmental Science and Technology (2021) 18:3739–3746
https://doi.org/10.1007/s13762-020-03095-z International Journal of Environmental Science and Technology (2021) 18:3739–3746
https://doi.org/10.1007/s13762-020-03095-z ORIGINAL PAPER Abstract The outbreak of SARS-CoV-2 (COVID-19) has attracted much attention to study its possible presence and airborne transmis-
sion. The possibility of COVID-19 airborne transmission in indoor environments is debatable. The present study examined
the concentration of viral RNA-containing particles produced directly or indirectly by breathing or coughing of confirmed
COVID-19 patients or by carriers without symptoms. Some studies do not accept this method of transmission (COVID-19
airborne transmission). The present study aimed to measure the possible exposure of health care personnel to SARS-CoV-2
particles that may have been suspended in the air to respond to the hypothesis of COVID-19 airborne transmission. Airborne
particle sampling was performed using impingement method based on NIOSH (chapter BA) and ASHRAE. Selection of
sampling sections was in line with the WHO guidelines. The samples were analyzed using RT-PCR technique. Based on the
given results, airborne particles of COVID-19 may present in the air and affect the health of hospital personnel. In fact, the
analysis of gene expression in ambient conditions and thereby aerosol transmission of SARS-CoV-2 through air is possible
and may lead to occupational exposure of health care personnel. Furthermore, it was found that airborne emission of COVID-
19 through the breathing zone of patients, particularly in ICU wards with confirmed cases of COVID-19, may be higher than
in other ICU wards. Also, the demonstrated results showed that there is a possibility of reaerosolization (reintroduction) of
previously airborne SARS-CoV-2 particles into the atmosphere due to health care personnel frequently walking between
different wards and stations of ICU. Keywords SARS-CoV-2 · Airborne · Health care · RT-PCR · Impingement · COVID-19 Keywords SARS-CoV-2 · Airborne · Health care · RT-PCR · Impingement · COVID-19 Vol.:(0123456789)
1 3
Keywords SARS-CoV-2 · Airborne · Health care · RT-PCR · Impingement · COVID-19
Editorial responsibility: Samareh Mirkia. * R. Yarahmadi
Yarahmadi.r@iums.ac.ir
1
Air Pollution Research Center, Department of Occupational
Health, Iran University of Medical Sciences, Tehran, Iran
2
Department of Virology, Faculty of Medicine, Iran
University of Medical Sciences, Tehran, Iran
3
Air Pollution Research Center, Iran University of Medical
Sciences, Tehran, Iran
4
Air Pollution Research Core, Pars Plasma Bonyan
(Knowledge Based Co), Tehran, Iran
5
Trauma and Injury Research Center, Critical Care Medicine
Department, Iran University of Medical Sciences, Tehran,
Iran
6
FCCM. Introduction Bioaerosols are very small airborne particles (0.001 to
100 μm) that originate from plants/animals and can contain
living organisms (Georgakopoulos, et al. 2009; Mandal and
Brandl 2011). Pathogenic or nonpathogenic dead or alive
microorganisms (e.g., viruses, bacteria, and fungi) may
exist in bioaerosols (Salthammer and Uhde 2009). Sources
of bioaerosol exposure in occupational activities are diverse
enough to include waste sorting and composting, agricul-
tural and food processing activities, the livestock industry,
etc. (Kim, et al. 2018). Health care occupational exposure
to bioaerosol can result in the deposition of the pathogen
in the respiratory tract of the host causing disease and an
immunological response. (Guzman 2020).i COVID-19, which was first reported in Wuhan, China, in
late December 2019, became a pandemic rapidly (Qiu et al. 2020), and by June 20, 2020, more than 216 countries and
territories reported a total of 8,525,042 confirmed cases and
456,973 confirmed deaths. (WHO 2020) Great efforts have
been made to enhance scientific facts related to COVID-19. (Guzman 2020; Qiu et al. 2020). COVID-19 outbreak attracted much attention to study the
possible transmission ways through bioaerosols. (Guzman
2020; WHO Organization 2020b) Respiratory droplets have
a size distribution range of between a submicron to thou-
sands of microns. Droplets with larger than 100 microns stay
in air less than for 5 s in height of 1.5 m from the ground. (Marr et al. 2019) Droplets larger than 10 microns settle
faster by gravity (Crowe et al. 1998), but droplets less than
10 microns stay in the air and spread throughout the room. (Tellier 2006) Thus, they have enough time to evaporate into
droplet nuclei, with a size of 0.74 to 2.12 microns, which
are involved in the airborne transmission of diseases. (Yang
et al. 2007) Many respiratory particles are very small at the
moment of leaving the mouth and can stay in the air for
several minutes or more before they evaporate and lose their
water content. However, some of the larger particles evapo-
rate and become smaller and can stay in the air for the same
extent of time. (Nicas et al. 2005) The size of the particle
droplets caused by sneezing or coughing generally ranges
from 1 to 5 microns. (Wang and Du 2020) Respiratory trans-
mission occurs through inhalation of viruses deposited in
the respiratory particles and sitting at the alveolar region
of the lower respiratory tract. (Spicknall et al. Editorial responsibility: Samareh Mirkia. Anesthesiology and Critical Care Department,
Trauma and Injury Research Center, Rasool‑E‑Akram
Complex Hospital, Iran University of Medical Sciences,
Tehran, Iran Vo (0123
1 23456789)
3 3740 International Journal of Environmental Science and Technology (2021) 18:3739–3746 The possibility of aerosolization of COVID-19 in the air
and its viability on the surfaces has been evaluated recently
based on the results which explained the resistance and via-
bility of COVID-19 is similar to SARS-CoV-1. Also, the
high viral load of SARS-CoV-2 in upper respiratory tract and
the potential of COVID-19 carrier persons in asymptomatic
state for disease transmission are highly important in the epi-
demiologic differences. Also, the viability of SARS-CoV-2
in the air for 3 h has been confirmed. (van Doremalen et al. 2020) However, the WHO has criticized these results. It was
argued that the study (van Doremalen Bushmaker Morris
Holbrook Gamble Williamson Tamin Harcourt, Thornburg
and Gerber 2020) was performed under laboratory condi-
tions and a 3-jet impactor nebulizer jet was used to produce
aerosols, and then, the aerosols were injected into a Gold-
berg drum. Also, it was emphasized that the high power of
this machine cannot reflect the normal sneezing or coughing
of people. (WHO Organization 2020a) A recently published
work recommended considering the hypothesis of airborne
transmission of SARS-CoV-2 through air and emphasized
the application of control devices. (Hadei et al. 2020) How-
ever, the problem of estimating the viral load emitted, which
is fundamental for the simulation of airborne transmission,
has not yet been solved. (Buonanno et al. 2020). Editorial responsibility: Samareh Mirkia. Editorial responsibility: Samareh Mirkia. * R. Yarahmadi
Yarahmadi.r@iums.ac.ir
1
Air Pollution Research Center, Department of Occupational
Health, Iran University of Medical Sciences, Tehran, Iran
2
Department of Virology, Faculty of Medicine, Iran
University of Medical Sciences, Tehran, Iran
3
Air Pollution Research Center, Iran University of Medical
Sciences, Tehran, Iran
4
Air Pollution Research Core, Pars Plasma Bonyan
(Knowledge Based Co), Tehran, Iran
5
Trauma and Injury Research Center, Critical Care Medicine
Department, Iran University of Medical Sciences, Tehran,
Iran
6
FCCM. Anesthesiology and Critical Care Department,
Trauma and Injury Research Center, Rasool‑E‑Akram
Complex Hospital, Iran University of Medical Sciences,
Tehran, Iran 7
Department of Engineering, Payame Noor University,
Tehran, Iran
8
Air Pollution Research Center, Department of Occupational
Health, Iran University of Medical Sciences, Tehran, Iran
9
Occupational Health Research Center, Department
of Occupational Health, Iran University of Medical Sciences,
Tehran, Iran
10 Department of Environmental Health, Iran University
of Medical Sciences, Tehran, Iran
11 School of Medicine, Iran University of Medical Sciences,
Tehran, Iran
12 Vice Chancellor for Health Center, Iran University
of Medical Sciences, Tehran, Iran
13 Razi Drug Research Center, Iran University of Medical
Sciences, Tehran, Iran 7
Department of Engineering, Payame Noor University,
Tehran, Iran * R. Yarahmadi
Yarahmadi.r@iums.ac.ir * R. Yarahmadi
Yarahmadi.r@iums.ac.ir 8
Air Pollution Research Center, Department of Occupational
Health, Iran University of Medical Sciences, Tehran, Iran 1
Air Pollution Research Center, Department of Occupational
Health, Iran University of Medical Sciences, Tehran, Iran 1
Air Pollution Research Center, Department of Occupational
Health, Iran University of Medical Sciences, Tehran, Iran 9
Occupational Health Research Center, Department
of Occupational Health, Iran University of Medical Sciences,
Tehran, Iran 2
Department of Virology, Faculty of Medicine, Iran
University of Medical Sciences, Tehran, Iran 10 Department of Environmental Health, Iran University
of Medical Sciences, Tehran, Iran 4
Air Pollution Research Core, Pars Plasma Bonyan
(Knowledge Based Co), Tehran, Iran 11 School of Medicine, Iran University of Medical Sciences,
Tehran, Iran 5
Trauma and Injury Research Center, Critical Care Medicine
Department, Iran University of Medical Sciences, Tehran,
Iran 12 Vice Chancellor for Health Center, Iran University
of Medical Sciences, Tehran, Iran 13 Razi Drug Research Center, Iran University of Medical
Sciences, Tehran, Iran 6
FCCM. Introduction 2010) Con-
siderable amounts of respiratory particles are aerosolized
during a usual talk. Thus, during a face to face conversation,
there is a possibility of virus transmission by a COVID-19
carrier person. (Asadi et al. 2020) Some cases of infection
by COVID-19 are reported in people without any contact
with confirmed cases; thus, COVID-19 transmission through
aerosols is possible. (Wang and Du 2020). The present study examines the following hypothesis: The
airborne transmission of SARS-CoV-2 through the presence
of the virus in the air and the possibility of occupational
exposure of health care providers to the virus. This study
was done in the intensive care unit of (ICU) of a teaching
hospital in Tehran, Iran. Air sampling (Verreault Moineau and Duchaine 2008). Based on the literature reviews, impingement method was
selected for bioaerosol sampling. This method was intro-
duced by NIOSH (capture BA) and the American Society
of Heating, Refrigerating and Air Conditioning Engineers
(ASHRAE) as one of the bioaerosol sampling techniques. (Brosseau, Vesley, Chen, Gabel, Kuehn and Goyal 1994;
Faridi et al. 2020; Girlando 2014; Lindsley, Green, Blachere,
Martin, Law, Jensen and Schafer 2017; McDermott 2004)
The sampling train consisted of a vacuum SKC personal
pump supplying 2.5 Lit/min (Brosseau, Vesley, Chen, Gabel,
Kuehn and Goyal 1994; McDermott 2004), which was cali-
brated with a standard flow meter before sampling and then
connected to a midget impinger by Tygon tubes. The midget
impinger was filled with 10 mL of HBSS. (Girlando 2014). Sampling was done at ambient temperature (24 °C) and
pressure (0.88 bar), with 34 relative humidity (% RH). The
sampling time for each sample was about 20 min, which
accounted for the total sample volume of 50 litters. After
sampling, the midget openings impinge were caped and
immediately sent to the laboratory. (Brosseau, Vesley,
Chen, Gabel, Kuehn and Goyal 1994; McDermott 2004)
The selected sampling stations are given in Fig. 1. At the
sampling time, the central air conditioner system was inten-
tionally turned off. Based on the literature reviews, impingement method was
selected for bioaerosol sampling. This method was intro-
duced by NIOSH (capture BA) and the American Society
of Heating, Refrigerating and Air Conditioning Engineers
(ASHRAE) as one of the bioaerosol sampling techniques. (Brosseau, Vesley, Chen, Gabel, Kuehn and Goyal 1994;
Faridi et al. 2020; Girlando 2014; Lindsley, Green, Blachere,
Martin, Law, Jensen and Schafer 2017; McDermott 2004)
The sampling train consisted of a vacuum SKC personal
pump supplying 2.5 Lit/min (Brosseau, Vesley, Chen, Gabel,
Kuehn and Goyal 1994; McDermott 2004), which was cali-
brated with a standard flow meter before sampling and then
connected to a midget impinger by Tygon tubes. The midget
impinger was filled with 10 mL of HBSS. (Girlando 2014). i
Sampling was done at ambient temperature (24 °C) and
pressure (0.88 bar), with 34 relative humidity (% RH). The
sampling time for each sample was about 20 min, which
accounted for the total sample volume of 50 litters. After
sampling, the midget openings impinge were caped and
immediately sent to the laboratory. Air sampling Bioaerosol samples are usually collected into liquid media
or on solid filters for measuring the particles containing
virus. (Brosseau et al. 1994; Lindsley, et al. 2017; McDer-
mott 2004; Verreault, et al. 2008) As they do not dry out
and keep their viability, the use of liquid media may cause
less stress on the bioaerosol components. (Lehtinen et al. 2013) Nonetheless, as such an application is still limited for
analysis of the effects of size segregation, it is less prefer-
able to conduct in-depth assessment of bioaerosols. Airborne
particles are collected into a liquid collection medium in
impingers method. Impingers are operated by channeling
air flow through nozzles to the collection chamber contain-
ing liquid. Hence, a number of factors (eg, the air flow rate,
distance between nozzle outlet and the surface of the liquid)
influence the size diameter of the particles to be collected. (Han and Mainelis 2012). 1 3 3741 International Journal of Environmental Science and Technology (2021) 18:3739–3746 The sampling efficiency of mentioned samplers depends
on the many environmental and methodological factors influ-
encing the integrity of the virus structure. Aerosol aerody-
namic size has a direct impact on the collection efficiency of
sampling device. (Verreault Moineau and Duchaine 2008). The sampling locations were selected to assist in evalua-
tion of the hypotheses about possible health care exposures
based on the WHO and American Society for Testing and
Materials (ASTM 2014a) (ASTM 2014; Lindsley, Green,
Blachere, Martin, Law, Jensen and Schafer 2017) recom-
mendations for surface or air sampling. (WHO Organiza-
tion 2020b) However, WHO guidelines were applied for
sampling to determine the aerosol concentration contain-
ing of COVID-19 at indoor areas. Since the aim of this
study was to determine occupational exposure of health
care personnel, the sampling area and building character-
istics of the study field were categorized (Table 1) based
on the source sampling as emission points of virus release
(breathing zone of patients) (stations code: A, H, G1, G2). Also, general areas were classified as the path of people’s
exposure (stations code: C, D, F, K, J1, J2, J3, J4) and the
breathing zone of health care personnel as the receptors
of the virus (stations code: B, E, L1, L2, G3, G4, G5, G6). Thus, a total of 20 samples were collected from air to
survey the possible occupational exposure of health care
personnel in the ICU wards (Table 2). sampling device. Air sampling (Brosseau, Vesley,
Chen, Gabel, Kuehn and Goyal 1994; McDermott 2004)
The selected sampling stations are given in Fig. 1. At the
sampling time, the central air conditioner system was inten-
tionally turned off. Choosing the number and location of sampling points
according to the variability, analytical methods used, the
variability of contaminant concentration over time at the
site, and the level of precision was required. (Figs. 1 & 2) In
addition, determining the number of locations and placement
of samplers was based on the nature of the response, ground
level, metrological conditions, site location (according to
conflicting background sources), site space, the number and Fig. Air sampling 1 The location of sampling points (English alphabet A-L) and COVID-19 Patient’s beds (blue), nurse station, and rest room ocation of sampling points (English alphabet A-L) and COVID-19 Patient’s beds (blue), nurse station, and rest room 1 3 3 International Journal of Environmental Science and Technology (2021) 18:3739–3746 3742 Table 1 Personnel and building characteristics of the study field
a All doors and windows were closed during sampling
b Air-conditioning systems off
ICU
wards
# of persons
Personnel status
Average age
(Year)
Average resistance
at ward (day/week)
Surface of
area (m2)
Open surfacea
(Door-Win) m2
Air-conditioning
b(Off/On)
COVID-19 patient
hall
13
Oxygen mask:
5,Intubated:
4PPE:4
56
5
70
6
off
General site
3
PPE
28
3
40
20
Off/with natural
ventilation
COVID-19 sus-
pected patient
room
3
Oxygen mask
58
1
24
6
off
Confirmed patient
room
3
PPE
30
0
24
6
off
Total
22
–
158
38
– Table 1 Personnel and building characteristics of the study field Table 2 Sampling and clinical characteristics of persons at risk of exposure to SARS-COV-19 Table 2 Sampling and clinical characteristics of persons at risk of exposure to SARS-COV-19
ICU
wards
Station
Chest CT
scan results
Clinical symptoms
location
Codes
Sampling
area (points)
COVID-19 patient hall
Patient room
A
Source1
Positive
fever, sneezing, coughing
B
Receiver2
Negative
–
C
General3
Negative
–
Staff rest room
D
General
Negative
–
General site
Nurse station
E
Receiver
Negative
–
Local corridor
F
General
Negative
–
COVID-19 confirmed patient room
Patients breathing zone
H
Source
Positive
–
Breathing zone of health care personnel
L1-2
Receiver
Negative
–
COVID-19 suspected patient room
Room entrance
K
General
–
–
General area
J1-J4
General
Negative
–
Patients breathing zone
G1-G2
Source
Positive
fever, sneezing, coughing
Breathing zone of Health care personnel
G3-G6
Receiver
Negative
– size and relative proximity of other sources on the site and
the above-mentioned resources. (Keith et al. 1991). impingement fluid. The preparation procedure of HBSS was
according to the Sigma-Aldrich (SAFC Biosciences 2006). Liquid media of sampling The type of liquid media in bioaerosol sampling is impor-
tant, as improper media may harm the virus RNA. (Girlando
2014) sVirus death during sampling may result from osmotic
shock, but applying sampling fluids with high osmotic pres-
sure reduces the degree of osmotic shock. Salts used in
hank’s balanced salt solution (HBSS) or phosphate-buffered
saline (PBS) minimize the osmotic shock. (Brosseau, Ves-
ley, Chen, Gabel, Kuehn and Goyal 1994) In the present
study, HBSS without calcium and magnesium was used as The viral RNA was extracted from 500 µL of the aero-
sol specimens using a QIAamp DSP Virus kit (QIAGEN
GmbH, Hilden, Germany), according to the manufacturer’s
protocols. Then, the quantity and quality of the isolated
RNAs were assessed using a NanoDrop™ (Thermo Scien-
tific, Wilmington, USA) spectrophotometer. i
The encoding region of the COVID-19 virus envelope
(E) and the RNA-dependent RNA polymerase (RdRp) 1 3 1 3 1 3 International Journal of Environmental Science and Technology (2021) 18:3739–3746 3743 Fig. 2 The schematic of the air sampling experiment setup in the
COVID-19 ICU wards stations of patient breathing zone. All positive samples were
related to COVID-19 confirmed patients (samples code: A,
and H). The stations with sample codes of G1and G2 belong
to COVID-19 suspected patient. Eight samples belonged to the breathing zone of the
health care personnel who were at higher potential risk of
infections due to airborne transmission of SARS-CoV-2
through air. (Table 5) All of the samples in this zone were
found negative except for B code. The results of this study showed that airborne emission
of COVID-19 through the breathing zone of patients, par-
ticularly in ICU wards with confirmed cases of COVID-19,
may be higher than in other ICU wards. (Tables 2–3) Almost
all patients had general symptoms of SARS-CoV-2 disease
(e.g., fever, sneezing, coughing, etc.); thus, the amount
of particle release was high. The results of Table 1 show
crowded Corona patients (60% of persons in the ICU) and
the short term of drug use by patients (maximum 2–4 days)
were the main reasons for the high potential of emission of
airborne particles due to exhale of patients. Fig. Liquid media of sampling 2 The schematic of the air sampling experiment setup in the
COVID-19 ICU wards With regards to the virus load emitted by infected indi-
viduals, currently no data are available in the scientific lit-
erature; therefore, the authors will refer to the case of SARS-
CoV-1, which has similar characteristics (Buonanno et al. 2020). genes were detected using specific primers and probes by
real-time polymerase chain reaction (RT-PCR) method
(Corman, et al. 2020), with the Rotor-Gene Q (QIAGEN,
Germany) instrument as described previously in detail. (Corman, Bleicker, Brünink, Drosten and Zambon 2020)
Also, appropriate positive and negative controls were
included in each assay. Table 4 The results of analysis for samples from the general area
Sample code Sample type
RT-PCR test result
C
General area (path of people’s
exposure)
Negative
D
Negative
F
Negative
K
Negative
J1
Positive
J2
Negative
J3
Negative
J4
Negative Table 4 The results of analysis for samples from the general area Results and discussion In this study, three zones of sampling were categorized: col-
lected samples from source of SARS-CoV-2 (patient breath-
ing zone), paths of airborne transmission (general area) and
receptor of SARS-CoV-2 particles (breathing zone of health
care personnel). A total of 20 samples were collected from
air to survey the possible occupational exposure of health
care personnel with SARS-CoV-2 particles in ambient air
of the ICU ward. The results of sample analysis are given in
Tables 3, 4 and 5. 1 3
Table 5 The results of analysis for samples from health care person-
nel breathing zone
Sample code
Sample type
RT-PCR test result
B
Breathing zone
Positive
E
Breathing zone
Negative
G3
Right side of patient bed
Negative
G4
Right side of patient bed
Negative
G5
Left side of patient bed
Negative
G6
Left side of patient bed
Negative
L1
Right side of patient bed
Negative
L2
Left side of patient bed
Negative Tables 3, 4 and 5. Results and discussion As presented in Table 3, the results of RT-PCR analy-
sis confirmed the existence of SARS-CoV-2 in air at two
Table 3 The results of analysis for samples from suspected source of
virus release
Sample code
Sample type
RT-PCR test result
A
Source (patient breathing zone)
Positive
H
Positive
G1
Negative
G2
Negative
Table 5 The results of analysis for samples from health care person-
nel breathing zone
Sample code
Sample type
RT-PCR test result
B
Breathing zone
Positive
E
Breathing zone
Negative
G3
Right side of patient bed
Negative
G4
Right side of patient bed
Negative
G5
Left side of patient bed
Negative
G6
Left side of patient bed
Negative
L1
Right side of patient bed
Negative
L2
Left side of patient bed
Negative Table 5 The results of analysis for samples from health care person-
nel breathing zone
Sample code
Sample type
RT-PCR test result Table 5 The results of analysis for samples from health care person-
nel breathing zone As presented in Table 3, the results of RT-PCR analy-
sis confirmed the existence of SARS-CoV-2 in air at two As presented in Table 3, the results of RT-PCR analy-
sis confirmed the existence of SARS-CoV-2 in air at two 3744 International Journal of Environmental Science and Technology (2021) 18:3739–3746 Also, the results revealed that if health care person-
nel or patients with suspected symptoms (with negative
or unknown PCR result) approach the breathing zone of
COVID-19 patients, there would be a high risk of expo-
sure and virus contraction. Also, the health care person-
nel without proper personal protective equipment (PPE)
who are near the breathing zone of COVID-19 patients
may be SARS-CoV-2 asymptomatic carriers and contrib-
ute to viral transmission through air or even their PPE. The result of airborne sampling at the general area of sta-
tion J1 proposes this assumption. (Table 4) Also, apply-
ing proper and standard ventilating and air-conditioning
systems in all parts of indoor spaces at hospitals will be
useful and effective in controlling transmission hazard at
general areas. In other words, it would limit the path of
airborne transmission. One of the main reasons that sam-
pling results at some stations were negative was the use
of ventilation systems and pathogenic treatment system
in clean areas of the hospital (e.g., health care staff’s rest
room) which in turn, resulted in an environment free of
airborne particles of SARS-CoV-2. Results and discussion Thus, the necessity of
using ventilation systems in closed areas was emphasized. (Janbabai et al. 2020) However, improper design or faults
in standard ventilation systems result in a contamination
source. (Abouleish 2021) Generally, the positive effect
of proper ventilation, operation of heating and air-condi-
tioning systems in reducing SARS-CoV-2 transmission is
highlighted by researchers. The relation between improper
ventilation systems and outbreak of airborne diseases has
been reported previously by WHO. (Chartier and Pessoa-
Silva 2009) It was shown that the aerodynamic diameter
of particles carrying RNA copies play an important role
in aerosolization of virus, and thus, it can be transmitted
during talking, sneezing and coughing of a carrier person
at distance of 2 m and be viable in air for about 3 h. Fine
and very fine particles that remain suspended for hours and
travel long distances may transmit SARS-CoV-2 directly
if inhaled. (Guzman 2020) Thus, keeping a safe social dis-
tance is another strategy to reduce airborne transmission. (Guzman 2020). Based on the given results from Table 4, the sampling of
general areas from different locations, SARS-CoV-2 may
have the potential to be transmitted through aerosols. The
result of J1 station proposed this fact, as it was located about
10 m from the ICU unit. The source of the aerosols in this
location may be the airborne SARS-CoV-2 from a patient
origin directly or may originally result from the direct depo-
sition of respiratory droplets or airborne SARS-CoV-2 from
a patient on the personal protective equipment worn by med-
ical staff, professors and resident students and resuspension
of virus-laden aerosols from the surface of the protective
equipment while they are walking or moving. Since the health care personnel frequently walk between
different wards and stations of ICU, there is a possibility of
reaerosolization of SARS-CoV-2 particles. (Table 4).f Therefore, the use of protective strategies and effective
control devices is highly recommended.i The final and additional results of the nasopharyngeal
swab tests of the suspected patients (cough, fever, sneezing,
shortness of breath, etc., with positive CT) were negatively
detected and released 2 days after the air sampling tests. Although aerosolization could be considered a third poten-
tial route of transmission, along with large droplets emit-
ted from sneezing or coughing and the transmission of viral
particles after touching a contaminated surface, the relative
contribution of each mode is uncertain. (Ault 2020). Results and discussion Resuspending particles of a respirable size may be dif-
ficult. However, fomites could be transmitted to the hands,
mouth, nose or eyes without requiring direct respiration into
the lungs. (Council 2020). Thus, in case of lack of adequate
protective and control devices or strategies, the suspected
COVID-19 patients are faced with the risk of exposure. Analyzing the breathing zone samples of the health
care staff was the most important part of the present study. These results can be used to investigate the relationship
between airborne particles and COVID-19, particularly the
presence of the virus in the breathing zone of the medi-
cal team in the ICU wards. Eight samples were from the
breathing zone of the health care personnel who were at
higher potential risk of virus contraction due to airborne
transmission of SARS-CoV-2. (Table 5) All the samples
in this zone were found to be negative except for B code,
which might have been due to the accumulation of parti-
cles containing inhaled coronavirus caused by 9 confirmed
COVID patients at the ICU main hall. 1 3 1 3 References World
Health Organization, Geneva Council NR (2020) Rapid expert consultation on the possibility of
bioaerosol spread of SARS-CoV-2 for the COVID-19 pandemic
(April 1, 2020). The National Academies Press, Washington, DC Acknowledgements This study was financially supported by the Air
Pollution Research Center of Iran University of Medical Sciences
(grant number 99-1-72-17719). We thank the staff of Tehran Hospital
Complex and Department of Occupational Health, School of Public
Health, Iran University of Medical Sciences. g
Crowe C, Sommerfeld M, Tsuji Y, Crowe C (1998) Multiphase Flows
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(2020) A field indoor air measurement of SARS-CoV-2 in the
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725:138401. https://doi.org/10.1016/j.scitotenv.2020.138401 The authors thank Dr. Jalil Koohpayehzadeh (the Dean of the Iran
University of Medical Sciences), Dr. Seyyed Abbas Motevalian, (the
Vice-Chancellor for Research and Technology of the Iran University
of Medical Sciences), Dr. Babak Eshrati (the Vice-Chancellor for
Healthcare of the Iran University of Medical Sciences), Dr. Fathi (the
Vice-Chancellor for Food and Drug of the Iran University of Medi-
cal Sciences), Dr. Iraj Alimohammadi (Head of Occupational Health),
Dr. Sara Minaeian (the supervisor of the Institute of Immunology and
Infectious Diseases of the Iran University of Medical Sciences), Narges
Moghadasi and Seyyed Hossein Tabatabaei (the Laboratory experts
of Occupational Health of the Iran University of Medical Sciences). Georgakopoulos D, Després V, Frohlich-Nowoisky J, Psenner R, Ariya
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Guzman MI (2020) An overview of the effect of bioaerosol size in
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of drug use by patients can be the main reasons for the high
potential of emission (of airborne particles) due to exhale
of patients. ASTM (2014) ASTM A751-14a, Standard Test Methods, Practices,
and Terminology for Chemical Analysis of Steel Products, ASTM
International, West Conshohocken, PA, 2014, www.astm.org. In:
vol. ASTM The positive result of sample in the suspected patient
room (J1 Station) may be related to the frequent move-
ment of professors and resident students from the main ICU
hall (with a high pollution load). Thus, in case of lack of
adequate protective and control devices or strategies, the
suspected COVID-19 patients are faced with the risk of
exposure. Understanding the sources and ways of possible
exposure are important in applying and selection of protec-
tive strategies and effective control technology. No detection
of SARS-CoV-2 in most ICU areas, indicating that high rates
of air exchange in the intensive care unit, is very effective
in limiting SARS-CoV-2 airborne transmission. The results
of this study highlight the necessity of applying control sys-
tems (as nonthermal plasma and UVGI process) in emis-
sion source (patient’s bed) to prevent the exposure risk of
the health care personnel and others in the ICU Wards of
hospital. Ault A (2020) Coronavirus is aerosolized through talking, exhalation,
new report says. https://www.medscape.com/viewarticle/928149. Accessed 04 Apr 2020 Brosseau L, Vesley D, Chen S, Gabel C, Kuehn T, Goyal S (1994)
Investigate and identify means of controlling virus in indoor
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(ASHRAE, RP-776). https://www.techstreet.com/standards/
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trol in health-care settings. World Health Organization, Geneva Corman V, Bleicker T, Brünink S, Drosten C, Zambon M (2020)
Diagnostic detection of 2019-nCoV by real-time RT-PCR. Conclusion At present, little is known about aerodynamic features and
SARS-CoV-2 transmission pathways in aerosols, partly
due to problems in sampling virus-containing aerosols in
real-world settings and their quantitative challenges in low
concentrations.(Liu et al. 2020) The results of this study
showed that analysis of gene expression in ambient condi-
tions (indoor air stream) and thereby aerosol transmission
of SARS-CoV-2 through air is possible and may lead to
occupational exposure of health care personnel. Although The results of this study highlight the importance of
prioritizing the application of control systems to curb
aerosol transmission primarily at the source of infection
(pollution). In other words, applying control strategies,
technologies and devices at the patient zone (especially
the breathing zone) will be highly efficient. Also, patient’s
bed can be redesigned to allow isolation, venting bed’s air
and respiratory treatment of patients. 1 3 1 3 International Journal of Environmental Science and Technology (2021) 18:3739–3746 3745 need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. there was not confirmation that the infectivity of the viruses
detected in this areas of the hospital, it is suggested that
SARS-CoV-2 may have the potential to transmit through
aerosols. In other words, the aerodynamic size of infectious
particles of SARS-CoV-2 was appropriate to remain air-
borne. (Verreault Moineau and Duchaine 2008) Also, air-
borne emission of COVID-19 through the breathing zone
of patients, particularly in ICU wards with confirmed cases
(COVID-19), may be higher than in other ICU wards. References Aerosol Air Qual Res 20(5):911–914 Open Access This article is licensed under a Creative Commons Attri-
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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
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review with special reference to residential and occupational
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item/surface-sampling-of-coronavirus-disease-(-covid-19)-a-pract
ical-how-to-protocol-for-health-care-and-public-health-profession
als. Accessed 18 Feb 2020 Nicas M, Nazaroff WW, Hubbard A (2005) Toward understanding the
risk of secondary airborne infection: emission of respirable patho-
gens. J Ooccupat Eenviron Hygiene 2(3):143–154 Qiu Y, Chen X, Shi W (2020) Impacts of social and economic factors
on the transmission of coronavirus disease 2019 (COVID-19) in
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concentration of droplets generated by coughing in human sub-
jects. J Aerosol Med 20(4):484–494 Sigma Aldrich (2006) The catalog of hanks’ Balanced salt solution. (Catalog Number:55021C). https://www.sigmaaldrich.com/catal
og/product/sigma/55021c?lang=en®ion=IR 1 3 1 3
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Improving on effective antiretroviral therapy: how good will a cure have to be?
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Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available
under the terms and conditions applicable to Other Posted Material, as set forth at http://
nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Published Version
doi:10.1136/medethics-2016-103907 Published Version
doi:10.1136/medethics-2016-103907 Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:31731892 Citation Citation
Freedberg, Kenneth A., and Paul E Sax. 2017. “Improving on effective antiretroviral therapy:
how good will a cure have to be?” Journal of Medical Ethics 43 (2): 71-73. doi:10.1136/
medethics-2016-103907. http://dx.doi.org/10.1136/medethics-2016-103907. Freedberg, Kenneth A., and Paul E Sax. 2017. “Improving on effective antiretroviral therapy:
how good will a cure have to be?” Journal of Medical Ethics 43 (2): 71-73. doi:10.1136/
medethics-2016-103907. http://dx.doi.org/10.1136/medethics-2016-103907. THE VALUE OF MATHEMATICAL AND
COST-EFFECTIVENESS MODELS One methodology that has been increas-
ingly popular over the past decade is the
use of mathematical simulation modelling
to
assess
the
clinical
impact,
cost-
effectiveness and clinical role of different
treatment strategies in HIV disease, as
well as in medicine broadly.2 Such models
provide a framework and approach for
using the best available data at any point DEFINING COST-EFFECTIVENESS How can one determine if something is
‘cost-effective’? Different countries spend
vastly different resources on health, so
what might be considered cost-effective in
one country might not be in another. While there is no consensus on what is
‘cost-effective’ in the USA, a widely cited
threshold is that strategies with an ICER
<$100 000/QALY are
considered
cost-
effective in the USA.6 The WHO historic-
ally
has
said
that
cost-effectiveness
is
likely related to the annual per person
gross
domestic
product
(GDP)
in
a
country. An intervention might be consid-
ered
‘cost-effective’
if
its
ICER
were
below three times the per capita GDP; it
might be considered ‘very cost-effective’ if
its ICER were below one times the GDP.7
More recently, it has been suggested that
most
resource-limited
countries
cannot
afford to add to their healthcare portfolio
interventions with ICERs this high, and
that ‘cost-effective’ strategies might be
those with ICERs below half the GDP or
even less.8 Regardless of the exact defin-
ition of what a country might be willing
to pay for healthcare, determining an
ICER allows one to rank the relative value
of different interventions of interest.2 Given the tremendous improvements in
ART, it is important to assess how effect-
ive and safe an HIV cure would need to
be in order to be a viable option com-
pared to ART. We argue that this should
be done prior to the availability of a cure
to provide realistic goals for researchers
and policy makers. INTRODUCTION in time, and assessing the potential impact
of emerging innovations. Further, unlike
individual trials focused on a single end-
point, these models can integrate multiple
data sources to project beyond the time-
line or outcomes of individual studies and
project
long-term
outcomes
in
many
domains,
including
virologic,
immuno-
logic, clinical and cost.3 Over the past two decades we have seen
dramatic improvements in the efficacy,
safety
and
availibity
of
antiretroviral
therapy (ART). In the USA and Europe,
life expectancy in people living with HIV
disease
approaches
that
of
the
HIV-uninfected.1 Even in regions hardest
hit by the HIV epidemic, effective HIV
therapy has reversed more than a decade
of HIV-related decreased survival. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Background UNDERSTANDING VALUE IN HEALTH:
COST ANALYSIS AND
COST-EFFECTIVENESS ANALYSIS 1Medical Practice Evaluation Center and Divisions of
General Internal Medicine and Infectious Diseases,
Massachusetts General Hospital and Harvard Medical
School, Boston, Massachusetts, USA; 2Department of
Health Policy and Management, Harvard T.H. Chan
School of Public Health, Boston, Massachusetts, USA;
3Division of Infectious Diseases, Brigham and Women's
Hospital and Harvard Medical School, Boston,
Massachusetts, USA MODELLING UNCERTAINTY AND
‘WHAT IF’ ANALYSES Despite these advances in ART, motiva-
tions to pursue HIV cure remain strong
due to the toxicity, adherence challenges,
cost and access to care issues associated
with HIV therapy, as well as the persistant
stigma associated with having HIV infec-
tion. Further and
renewed
motivation
comes from a single case of HIV cure
after stem cell transplantation for acute
leukaemia. Mathematical
models
have
particular
value in their ability to assess uncertainty
in clinical care. Examples include para-
meters determined in clinical trials or
cohort studies (such as the 95% CIs) or
the broader uncertainty over parameters
that have not or cannot readily be mea-
sured in a trial, such as stigma. Another
role of mathematical models is to estimate
changes in care that may arise in the
future; this is clearly of relevance for HIV
cure research. Where many aspects of a
potential intervention are uncertain, as is
the case with the future of cure strategies,
one can use a model to do a ‘what if’ ana-
lysis. What if a cure had an efficacy of
80% or 95%? What if the severe toxicity
rate was 0.1% or 10%? What if the cost
was $5000 or $50 000 or $500 000? By
varying multiple parameters, individually
or in combination, this type of modelling
can inform the ranges around which new
therapeutic,
or
diagnostic,
approaches
could have a major clinical impact—espe-
cially
compared
with
current
therapy. Importantly, both efficacy and costs can
be estimated.4 Improving on effective antiretroviral
therapy: how good will a cure have to
be? understanding how introducing a new
diagnostic
test
or
therapy
will
affect
healthcare
budgets
and
costs. Cost-effectiveness analysis goes further by
attempting to measure ‘value for money’
through an assessment of the costs of an
intervention, as well as the clinical bene-
fits, usually measured in changes in life
expectancy or quality-adjusted life expect-
ancy.5 The outcome of these analyses is
the
incremental
cost-effectiveness
ratio
(ICER), which is denominated in dollars/
year
of
life
saved
($/YLS),
dollars/
quality-adjusted life year ($/QALY) saved
or
dollars/disability-adjusted
life
year
($/DALY) saved. The lower the ratio, the
more cost-effective the intervention. This
is
effectively a
measure
of
value
for
money—how much are we spending to
improve outcomes—compared with other
possible interventions. Kenneth A Freedberg,1,2 Paul E Sax3 Objective and approach We set out to use mathematical modelling
to understand what characteristics an HIV
cure strategy would have to
have to
improve on currently available ART in the
USA.4 12 For our analysis, we used the
Cost-effectiveness
of
Preventing
AIDS
Complications (CEPAC) model, a vali-
dated and widely published microsimula-
tion of the natural history and treatment
of HIV disease.3 13 14 The model includes
data from multiple cohort studies, clinical
trials and economic analyses and it has
been used to assess a wide variety of treat-
ments, diagnostic tests and testing strat-
egies for HIV disease in the USA as well
as in many other countries. The model
incorporates the most currently available
data and is updated regularly. We used an initial efficacy of 10%, 20%
and 70% for gene therapy, chemotherapy
and bone marrow transplant, respectively. Fatal toxicity ranged from 0.1% to 5.0%,
with non-fatal toxicity ranging from 1%
to 55%. Relapse rates after ‘successful’
cure ranged from 0% to 0.5%/month. Cost inputs were also based, where pos-
sible, on data from similar types of therap-
ies. Cost for gene therapy was $101 000,
for
chemotherapy
$12 400/month
for
24 months, and for bone marrow trans-
plant $123 900, with additional costs of
$1000/month
for
immunosuppressive
medications. In the model, the cost of first-
line ARTwas $24 000/year on average. SETTING-SPECIFIC CARE Limited
resources
for
health,
in
the
context of increasingly effective interven-
tions for multiple acute as well as chronic
diseases, have meant that the demand for
healthcare
interventions
outstrips
the
supply of available resources. This creates
intense pressure to understand the efficacy
of new interventions, as well as the cost
and cost-effectiveness of these strategies. Cost analysis examines one outcome, cost,
and is used generally for budgeting and Current ART, and HIV care more broadly,
differs substantially in different countries. In the USA, Europe and Australia, guide-
lines for care include ART at the time
infection
is
diagnosed,
with
genotype
testing before care and at the time of viro-
logic failure, as well as virologic monitor-
ing
and
access
to
over
30
licensed
medications. While the WHO in late 2015
recommended ART at the time of HIV Correspondence to Dr Kenneth A Freedberg, MD,
MSc, Medical Practice Evaluation Center, Massachusetts
General Hospital, 50 Staniford St, Suite 901, Boston,
MA, 02114, USA; KFREEDBERG@mgh.harvard.edu Freedberg KA, Sax PE. J Med Ethics February 2017 Vol 43 No 2 71 Background How good would a cure need to be? By varying parameters in the model indi-
vidually and in combination we could
identify the ‘targets’ at which each of
these strategies might improve life expect-
ancy and be cost-effective compared with
continuing
suppressive
ART. For
gene
therapy, if the efficacy were 22%, the
ICER
dropped
below
$100 000/QALY;
with an efficacy of 34%, it became cost-
saving compared with ART. The efficacy
of chemotherapy had to be 88% to have
an ICER<$100 000/QALY and it never
became cost-saving. Bone marrow trans-
plant had an ICER<$100 000/QALY at an
efficacy of 79%, and became cost-saving
with an efficacy of 80%. For each of these
strategies, if there were no risk of relapse
—that is, the cure was completely durable
—then the efficacy needed to achieve
cost-effectiveness or cost-saving thresholds
was lower. Finally, for all of the strategies,
if an effective cure improved patients’
quality of life compared with their quality
of life on ART, then each of the strategies
became
even
more
attractive. This
depended in large part on the baseline
quality of life for patients receiving ART. If that quality of life was low (eg, based
on the psychological stressors and stigma
associated with having HIV), then the
potential benefit of cure was higher. of suppressive ART. Why cure strategies are unique in HIV
modelling analyses
d
h
h We and other investigators have examined
multiple
HIV
interventions,
including
HIV testing, first-line, second-line and
later ART, as well as diagnostic tests
including viral load and resistance geno-
type testing.13 18–21 For virtually all inter-
ventions in HIV disease—treatments or
diagnostic
tests—the
intervention
improves survival at added cost, allowing
one
to
determine
whether
it
has
an
attractive cost-effectiveness ratio. It is rare
for a strategy to actually be cost-saving,
including
even
‘win–win’
interventions
such as the use of early ART for treatment
as prevention. The primary reason these
are not cost-saving is because each of
these treatment strategies to date involves
continuing therapy lifelong. Main results of the analysis We incorporated into our analysis three
approaches studied in HIV cure research,
as detailed in Sax et al.4 These include
gene therapy,15 the ‘kick and kill’ strat-
egies, which are similar to chemotherapy
for cancer16 and stem cell transplant.17
We considered a gene therapy strategy as
potentially having lower efficacy but less
toxicity, a chemotherapy strategy having
moderate efficacy and toxicity and a bone
marrow transplant strategy as having the
highest likely efficacy as well as toxicity. Since none of these strategies are available
currently for HIV cure, in each case, we
varied parameters across a wide range in
sensitivity analyses. We incorporated into our analysis three
approaches studied in HIV cure research,
as detailed in Sax et al.4 These include
gene therapy,15 the ‘kick and kill’ strat-
egies, which are similar to chemotherapy
for cancer16 and stem cell transplant.17 y
We found that, based on the assumptions
in the model, gene therapy would offer a
modest increase in life expectancy com-
pared with ART (from 16.4 to 16.6 dis-
counted QALYs), while it would increase
lifetime costs from $591 000 to $659,000
for an ICER of $331 000/QALY. This is
well
above
the
commonly
discussed
willingness-to-pay thresholds in the USA
of
∼$100 000/QALY,
suggesting
that,
under the assumptions in the model, a
gene therapy approach would not be cost-
effective. For the chemotherapy and bone
marrow transplant approaches, the results
were even less attractive. Because of the
toxicity of both chemotherapy and bone
marrow transplant approaches, particularly
the initial toxicity, they were associated
with the same or worse life expectancy as
continued
ART and
had
substantially
higher costs. We considered a gene therapy strategy as
potentially having lower efficacy but less
toxicity, a chemotherapy strategy having
moderate efficacy and toxicity and a bone
marrow transplant strategy as having the
highest likely efficacy as well as toxicity. Since none of these strategies are available
currently for HIV cure, in each case, we
varied parameters across a wide range in
sensitivity analyses. Our cure analysis yielded qualitatively
different results. We found that across a
wide range of assumptions, strategies were
unlikely to be cost-effective compared
with ART. In a much narrower range,
there were estimates at which cure might
be cost-effective, that is, increasing life
expectancy as well as cost, with an attract-
ive ICER. But as we continued to assess
each of these parameters, whether efficacy, A ‘WHAT IF’ ANALYSIS OF HIV CURE
IN THE USA Since there are no proven effective cure
strategies, most of the inputs were hypo-
thetical; in terms of toxicity and relapse,
they were based on similar data from
other interventions with gene therapy,
chemotherapy or bone marrow transplant. SETTING-SPECIFIC CARE Second, if a cure strat-
egy failed, either initially or with later
relapse, we assumed that ART would be
resumed, with no reduction in ART effi-
cacy. Third, we did not include the possi-
bility of reinfection in those cured of HIV. Fourth, we did not adjust for the impact
of a cure strategy on the likelihood of
individuals
developing
chronic
‘non-AIDS’ specific complications, such as
heart disease and cancer. Finally, it is pos-
sible that cure could decrease the poten-
tial of people living with HIV to pass on
HIV, even though we are comparing cure
with ART in those already suppressed. This omitted factor would make a cure
more attractive relative to ART. diagnosis, based on the results of the
Temprano and START trials, in many
resource-limited settings, criteria for ART
initiation remains at a CD4 threshold of
<350 or <500 cells/μL.3 9 10 Many coun-
tries do not have routine virologic moni-
toring or genotype tests, and integrase
inhibitor-based
therapy
is
not
recom-
mended for first-line therapy, reserved
only for extraordinary cases or is not avail-
able at all.10 11 Thus, the potential use,
impact, cost and cost-effectiveness of any
cure strategy should be considered in the
context
of
currently available
therapy. Where therapy is widely available, highly
effective and safe, cure strategies would
offer less incremental value than in settings
where such therapy is not as available. Open Access This is an Open Access article
distributed in accordance with the Creative Commons
Attribution Non Commercial (CC BY-NC 4.0) license,
which permits others to distribute, remix, adapt, build
upon this work non-commercially, and license their
derivative works on different terms, provided the
original work is properly cited and the use is non-
commercial. See: http://creativecommons.org/licenses/
by-nc/4.0/ 12
Panel on Antiretroviral Guidelines for Adults and
Adolescents, Department of Health and Human
Services (DHHS). Guidelines for the use of
antiretroviral agents in HIV-1-infected adults and
adolescents. 2016. http://aidsinfo.nih.gov/
contentfiles/lvguidelines/adultandadolescentgl.pdf
(accessed 15 July 2016). 13
Freedberg KA, Losina E, Weinstein MC, et al. The cost
effectiveness of combination antiretroviral therapy for
HIV disease. N Engl J Med 2001;344:824–31. To cite Freedberg KA, Sax PE. J Med Ethics
2017;43:71–73. Received 26 August 2016
Revised 24 October 2016
Accepted 3 November 2016
Published Online First 5 December 2016
d
hi To cite Freedberg KA, Sax PE. J Med Ethics
2017;43:71–73. 14
Paltiel AD, Weinstein MC, Kimmel AD, et al. Expanded screening for HIV in the United States—an
analysis of cost-effectiveness. N Engl J Med
2005;352:586–95. 15
Tebas P, Stein D, Tang WW, et al. Gene editing of
CCR5 in autologous CD4 T cells of persons infected
with HIV. N Engl J Med 2014;370:901–10. 16
Archin NM, Liberty AL, Kashuba AD, et al. Administration
of vorinostat disrupts HIV-1 latency in patients on
antiretroviral therapy. Nature 2012;487:482–5. 17
Allers K, Hütter G, Hofmann J, et al. Evidence for the
cure of HIV infection by CCR5Delta32/Delta32 stem
cell transplantation. Blood 2011;117:2791–9. J Med Ethics 2017;43:71–73. doi:10.1136/medethics-2016-103907 J Med Ethics 2017;43:71–73. doi:10.1136/medethics-2016-103907 18
Walensky RP, Paltiel AD. Rapid HIV testing at home:
does it solve a problem or create one? Ann Intern
Med 2006;145:459–62. Limitations
h 9
World Health Organization. Guideline on when to
start antiretroviral therapy and on pre-exposure
prophylaxis for HIV. 2015. http://apps.who.int/iris/
bitstream/10665/186275/1/9789241509565_eng. pdf (accessed 15 Jul 2016). Funding Supported in part by R01 AI42006, R01
AI114617, R56 AI114617 and the Harvard University
Center for AIDS Research (NIH P30 AI060354), all from
the National Institute of Allergy and Infectious
Diseases. The content is solely the responsibility of the
authors and does not necessarily represent the official
views of the National Institutes of Health. This type of analysis is most importantly
limited by the lack of real data on cure
strategies. For that reason, we varied each
estimate across a wide range to determine
its impact on the results. Our assumption
that patients undergoing a cure strategy
already be virally suppressed—that is, suc-
cessfully treated—for a year could create a
bias against a cure, since those patients are
likely to continue to do well on ART. They are, however, the patients thought
most likely to be able to be cured at
present.22 As challenging as HIV eradica-
tion would be in a patient with virologic
suppression, it is currently scientifically
implausible
to
imagine
doing
so
in
someone with ongoing high-level viral
replication. We assumed that if a cure
failed, patients could resume ART. If ART
efficacy were reduced, either due to resist-
ance or other reasons, cure would look
worse. If we incorporated the possibility
of reinfection after cure, then cure would
be
even
less
attractive. In
terms
of
‘non-AIDS’ complications, it is possible
that a cure could decrease these by chan-
ging the factors that are increasing these
complications, or increase them, as has
been seen with chemotherapy for other
diseases. We also do not consider the
potential increased likelihood of transmis-
sion from those on ART, if they have viro-
logic failure, compared with those cured. Finally, this analysis is based on data from
the USA, including both clinical and cost
data. The results may differ substantially
in other developed as well as resource-
limited settings. Given that the cost of
ART is much lower in resource-limited
settings than in the USA, it is likely, given
increased access to ART via PEPFAR, the
Global Fund and country-specific pro-
grammes, that a cure strategy would have
to have correspondingly lower cost to be a
viable alternative to effective ART. 10
South Africa Department of Health. Assumptions going into the model We used several assumptions meant to
reflect the current state of cure research. First, we assumed that patients eligible for
cure therapy had already been on 1 year Freedberg KA, Sax PE. J Med Ethics February 2017 Vol 43 No 2 72 Background $50,000-per-QALY threshold. N Engl J Med
2014;371:796–7. $50,000-per-QALY threshold. N Engl J Med
2014;371:796–7. toxicity, relapse rate or cost, the cure strat-
egies then uniformly became cost-saving. Thus, there is only a narrow parameter
range
in
which
cure
would
be
cost-
effective rather than either cost-saving or
not cost-effective. With high efficacy, cure
could indeed be cost-saving. The primary
explanation for this finding is that an
effective cure would eliminate the need
for lifelong ART. yet known, it is possible with the use of
mathematical modelling to determine the
set of targets for these parameters under
which HIV cure would be a reasonable
alternative to ART. If novel approaches
can achieve some of these challenging
targets, cure would turn out to be cost-
saving compared with ART, as a lifelong
treatment with ART can then be avoided. toxicity, relapse rate or cost, the cure strat-
egies then uniformly became cost-saving. Thus, there is only a narrow parameter
range
in
which
cure
would
be
cost-
effective rather than either cost-saving or
not cost-effective. With high efficacy, cure
could indeed be cost-saving. The primary
explanation for this finding is that an
effective cure would eliminate the need
for lifelong ART. 7
World Health Organization. CHOosing interventions
that are cost effective (WHO-CHOICE):
cost-effectiveness thresholds. http://www.who.int/
choice/costs/CER_levels/en/ (accessed 15 Jul 2016). 8
Revill P, Walker S, Madan J, et al. Using
cost-effectiveness thresholds to determine value for
money in low- and middle-income country healthcare
systems: are current international norms fit for
purpose? CHE Research Paper 98. 2014. http://www. idsihealth.org/wp-content/uploads/2015/01/
Cost-Effectiveness-Thresholds_Value_Low-Middle-
Income-Countries_York.pdf (accessed 15 Jul 2016). Acknowledgements The authors thank Amy Zheng
for her assistance with the manuscript. Acknowledgements The authors thank Amy Zheng
for her assistance with the manuscript. Limitations
h National consolidated
guidelines for the prevention of mother-to-child
transmission of HIV (PMTCT) and the management of
HIV in children, adolescents and adults. 2015. http://
www.sahivsoc.org/upload/documents/ART%20Guidelines
%2015052015.pdf (accessed 15 Jul 2016). Provenance and peer review Commissioned;
externally peer reviewed. p
11
National AIDS Control Organisation. National
guidelines on second-line and alternative first-line
ART for adults and adolescents. 2013. http://www. naco.gov.in/upload/Policies%20&%20Guidelines/
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https://openalex.org/W3171161919
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https://figshare.com/articles/journal_contribution/Supplementary_Figure_S1_from_Gene_Expression_Signature_Correlates_with_Outcomes_in_Metastatic_Renal_Cell_Carcinoma_Patients_Treated_with_Everolimus_Alone_or_with_a_Vascular_Disrupting_Agent/22522713/1/files/39985464.pdf
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Gene Expression Signature Correlates with Outcomes in Metastatic Renal Cell Carcinoma Patients Treated with Everolimus Alone or with a Vascular Disrupting Agent
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Molecular cancer therapeutics
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Patients with Renal cell carcinoma
randomized to Everolimus +/- BNC105P
(N=107) Patients with Renal cell carcinoma
randomized to Everolimus +/- BNC105P
(N=107) Patients with Renal cell carcinoma
randomized to Everolimus +/- BNC105P
(N=107)
Patients with H&E for demarcation of
tissue and adequate RNA concentration
(N=92)
15 Excluded
6 had no available H&E slide
8 had inadequate RNA concentration
or negative A260/280 ratios
1 had low binding density
Patient with available PFS and clinical
benefit data (n=82)
10 Excluded
10 had no PFS outcome data
Computational Workflow and analysis
Supplementary Figure S1. CONSORT diagram for the phase I/II trial of everolim
with and without BNC105P 15 Excluded
6 had no available H&E slide
8 had inadequate RNA concentration
or negative A260/280 ratios
1 had low binding density Patients with H&E for demarcation of
tissue and adequate RNA concentration
(N=92) 10 Excluded
10 had no PFS outcome data Patient with available PFS and clinical
benefit data (n=82) Computational Workflow and analysis Supplementary Figure S1. CONSORT diagram for the phase I/II trial of everolimus
with and without BNC105P
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A comprehensive review on bacteriocin: Potential applications and nano based delivery systems
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Himasri Das Himasri Das
Department of Veterinary
Microbiology, College of
Veterinary Science, Khanapara,
Guwahati, Assam Agricultural
University, Assam, India Sophia Makdoh Gogoi
Department of Veterinary
Microbiology, College of
Veterinary Science, Khanapara,
Guwahati, Assam Agricultural
University, Assam, India Keywords: bacteriocins, antimicrobial peptides, nanotechnology, delivery system, therapeutics Nayanmoni Konwar
Department of Veterinary
Microbiology, College of
Veterinary Sciences and Animal
Husbandry, Aizawl, Selesih,
Central Agricultural University,
Mizoram, India Chandrani Goswami, Himasri Das, Sophia Makdoh Gogoi, Nayanmoni
Konwar, Durlav Prasad Bora and Razibuddin Ahmed Hazarika Chandrani Goswami
Department of Veterinary Public
Health, College of Veterinary
Science, Khanapara, Guwahati,
Assam Agricultural University,
Assam, India Corresponding Author
Himasri Das
Department of Veterinary
Microbiology, College of
Veterinary Science, Khanapara,
Guwahati, Assam Agricultural
University, Assam, India Razibuddin Ahmed Hazarika
Department of Veterinary Public
Health, College of Veterinary
Science, Khanapara, Guwahati,
Assam Agricultural University,
Assam, India www.thepharmajournal.com
Received: 07-10-2021
Accepted: 09-11-2021 www.thepharmajournal.com
Received: 07-10-2021
Accepted: 09-11-2021 Chandrani Goswami, Himasri Das, Sophia Makdoh Gogoi, Nayanmoni
Konwar, Durlav Prasad Bora and Razibuddin Ahmed Hazarika Abstract Bacteriocins are ribosomally synthesised group of antimicrobial peptides synthesised by almost all
groups of bacteria and demonstrates bactericidal or bacteriostatic activity, usually against members of
closely related species and different strains of similar species. Bacteriocin has gained much attention
worldwide over the last decade especially for their advantages in regards to physical stability and non-
toxicity. Various bacteriocins have been reported to act as promising food preservatives or in the health
industry as antimicrobial agents or bio-controlling agents and as so as for fight against antimicrobial drug
resistance. They are also being exploited for their anticancer properties as a new tool in fighting cancer. Bacteriocins also plays a major role in food industry by safeguarding public health and food safety as
they found their utility in chemical free preservation, enhancing shelf-life, and as well as inhibition of
food-borne pathogenic microorganisms from farm till food-processing stages. Nanotechnology has
proven to be effective drug delivery system and so as so as to avoid any existing limitation of
bacteriocins. With the increasing bacterial resistance, the evolution of nanotechnology has proven to be
an effective upgrade of traditional drug delivery systems. The incorporation of bacteriocins into
nanoparticles and site-directed delivery to areas of infection may soon become an effective method of
treatment. ISSN (E): 2277- 7695
ISSN (P): 2349-8242
NAAS Rating: 5.23
TPI 2021; SP-10(12): 993-1005
© 2021 TPI ISSN (E): 2277- 7695
ISSN (P): 2349-8242
NAAS Rating: 5.23
TPI 2021; SP-10(12): 993-1005
© 2021 TPI The Pharma Innovation Journal 2021; SP-10(12): 993-1005 The Pharma Innovation Journal 2021; SP-10(12): 993-1005 Introduction Bacteriocins are ribosomally synthesised group of antimicrobial peptides synthesised by
almost all groups of bacteria and demonstrates bactericidal or bacteriostatic activity, usually
against members of closely related species and different strains of similar species (Castellano
et al., 2012; El-Gendy et al., 2012) [1, 2]. Gratia in the year 1925 discovered the first ever
bacteriocin from Escherichia coli (Gratia, 2000) [3]. Bacteriocin-producing bacteria have been
recognised from a wide group of bacterial strains, they possess a competitive survival
advantage over other prokaryotes in the biological niche (Preciado et al., 2016) [4]. Multidrug
and even pan drug resistant strains of both gram-positive and gram-negative bacteria have
been frequently encountered in hospital settings against the most influential antibiotics. The
most dangerous being methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-
resistant Enterococci (VRE) which continues to increase the morbidity and mortality rates
(Simons et al., 2020) [5]. Many bacteriocins from bacteria showed antimicrobial efficacy
against human as well as animal microbial pathogens including MRSA and VRE without
showing toxicity (Xia et al., 2013) [16] (Calfee, 2012) [7] and thus helping to fill some gaps in
the medical sector. Emergence of multi drug resistant drug which has become one of the major
global health-concern and the need of hour requires investigation and screening of natural
novel compounds like bacteriocins with potent killing mechanisms in order to assist and
replace the existing antibiotics and against which bacteria won’t develop resistance easily. Various bacteriocins have also been reported to act as promising food preservatives
(Papagianni, 2003) [8] or in the health industry as antimicrobial agents or bio-controlling agents
(Van Heel et al., 2011; van Staden et al., 2012) [9, 10]. They are also being exploited for their
anticancer properties as a new tool in fighting cancer (Riaz et al., 2020) [11]. However, in spite
of many potential applications of bacteriocins, nisin is the only bacteriocin to get the generally
recognized as safe (GRAS) status by the Food and Drug Administration and is presently used
as a food preservative in various countries (Delves-Broughton, 1996) [12]. Mechanism of Action Different mechanisms of action have been proposed that
differ from those of antibiotics. These mechanisms can be
generally divided into those that function primarily at the cell
envelope and those that are active primarily within the cell,
affecting gene expression and protein production (Cotter et
al., 2013) [35]. The interaction of many bacteriocins to the
plasma membrane depends on their physicochemical and
structural properties (Ahmad et al., 2017)
[36]. Upon
interaction with the negatively charged cell envelope, pore-
forming positively charged bacteriocins, when in larger
concentration in the μM range insert into the plasma
membrane. Lipid II which is the key intermediate in the pep-
tidoglycan biosynthesis machinery within the bacterial cell
envelope or the membrane components of mannose
Phosphotransferase System (Man-PTS) may act as receptors
or docking molecules to promote pore formation. Lipid II
inhibits the synthesis of peptidoglycan upon binding to the
cell wall precursor, which may be combined with pore
formation or not. Hydrolysis of the peptidoglycan is
accomplished by activation of endogenous autolysins
(Martínez et al., 2016) [28]. Nisin and several lantibiotics, in
addition to some class II bacteriocins, target lipid II
(Bierbaum and Sahl, 2009; Martin and Breukink, 2007) [37, 38]
causing inhibition of peptidoglycan synthesis, and for some
this is the sole mechanism of action. As a docking molecule,
lipid II can be used by other lantibiotics to facilitate the
formation of pores in the cell membrane, resulting in a loss of
membrane potential and, thereby, cell death (Bierbaum and
Sahl, 2009; Martin and Breukink, 2007) [37, 38]. Class II contains small (less than 10 kDa) in size and they
exhibited moderate (100 °C) to high (121 °C) heat stability,
non- lantibiotics or non-modified peptides, with isoelectric
points (pIs) varying from 8.3 to 10.0. Subclass IIa were
comprised of Listeria-active peptides that contain a specific
N-terminal sequence (Tyr-Gly-Asn-Gly-Val-Xaa-Cys-), the
representative bacteriocins of this group are leucocin A,
acidocin A (Radaic et al., 2020) [23], mesentericin, pediocin
PA-1 and sakacin P (Venema et al., 1997) [24]. Class IIb
bacteriocins (two-peptide bacteriocins) require at least two
different peptides for activity e.g. thermophilin 13,
lactococcin G and lactacin F. When associated together, they
are reported to show a synergistic effect and the level of
bacterial susceptibility changes with that of individual
peptides (Nissen-Meyer et al., 2009; Perez et al., 2018; Perez
et al., 2014) [25-27]. Introduction Researches are still
under process to enhance the efficacy of bacteriocin and so as to avoid any existing limitation,
the use of nanotechnology is a novel approach to maximize the output of these peptides Durlav Prasad Bora
Department of Veterinary
Microbiology, College of
Veterinary Science, Khanapara,
Guwahati, Assam Agricultural
University, Assam, India Razibuddin Ahmed Hazarika
Department of Veterinary Public
Health, College of Veterinary
Science, Khanapara, Guwahati,
Assam Agricultural University,
Assam, India Corresponding Author
Himasri Das
Department of Veterinary
Microbiology, College of
Veterinary Science, Khanapara,
Guwahati, Assam Agricultural
University, Assam, India ~ 993 ~ http://www.thepharmajournal.com The Pharma Innovation Journal secretory mechanism of the cell; e.g. lactococcin 972
(Martínez et al., 1999) [31] and leaderless bacteriocins, such as
lacticin Q, which, significantly, has a formylated methionine
as its first residue (Fujita et al., 2007) [32]. Class III bacteriocins generally contain large (> 30 kDa) heat-
labile peptides and little is known about them e.g. colicin
produced by E. coli, classified as heat-labile lytic bacteriocins
and
heat-labile
non-lytic
bacteriocins
(Joerger
and
Klaenhammer, 1986; Vaughan, et al., 1992) [33, 34]. Another
bacteriocin of this category, dysgalacticin works by either
interfering with either glucose transport or metabolism by
binding to the phosphoenolpyruvate-dependent glucose and
mannose phosphotransferase transport system (Joerger and
Klaenhammer, 1986; Vaughan et al., 1992) [33, 34]. (Fahim et al., 2016) [13]. This review aims to discuss the
potential applications of bacteriocins in the field of
antimicrobial therapeutics including classification and mode
of action along with utilisation of nanotechnology in
enhancing the antimicrobial efficacy of bacteriocins secretory mechanism of the cell; e.g. lactococcin 972
(Martínez et al., 1999) [31] and leaderless bacteriocins, such as
lacticin Q, which, significantly, has a formylated methionine
as its first residue (Fujita et al., 2007) [32]. Class III bacteriocins generally contain large (> 30 kDa) heat-
labile peptides and little is known about them e.g. colicin
produced by E. coli, classified as heat-labile lytic bacteriocins
and
heat-labile
non-lytic
bacteriocins
(Joerger
and
Klaenhammer, 1986; Vaughan, et al., 1992) [33, 34]. Another
bacteriocin of this category, dysgalacticin works by either
interfering with either glucose transport or metabolism by
binding to the phosphoenolpyruvate-dependent glucose and
mannose phosphotransferase transport system (Joerger and
Klaenhammer, 1986; Vaughan et al., 1992) [33, 34]. Bacteriocin Classification Several approaches have been taken to classify bacteriocins. The first bacteriocin classification system was proposed by
Klaen hammer in 1993 (Zou et al., 2018; Klaenhammer,
1993) [14, 15]. Bacteriocins were initially classified into four
classes (Rea et al., 2011) [16]. However, the fourth class of
bacteriocins, consisting of large complexes with carbohydrate
or lipid moieties, has been aborted and were named as
bacteriolysins (Güllüce et al., 2013) [17]. Thus, bacteriocins
are classified mainly into three classes (Liu et al., 2014) [8]. Class I bacteriocins (composed of 19–50 amino acids) are
extensively post-translationally modified results in the
formation of unusual amino acids, such as lanthionine,
methyllanthionine, dehydrobutyrine, dehydroalanine, and
labionin. They target the skeleton of the cell wall of
pathogens, particularly Gram-positive bacteria and also are
heat-stable (González-Martínez, 2003). Class I is further
subdivided into Lantibiotics (Class Ia), these peptides are
positively-charged elongated bacteriocins that kill bacteria by
pore formation. The prototype antibiotic nisin is a member of
this group (Le Lay et al., 2016) [20], few other bacteriocins of
this class includes lanthionine and/or beta-methyllanthionine. Labyrintopeptins (Class Ib), bacteriocins, includes lacticin
481, cytolysin and salivaricin, which are characteristically
globular, inflexible, with a negative charge or with no net
charge. They inhibit various catalytic enzymes required to
complete the life-supporting processes of susceptible bacteria
(Deegan et al., 2006)
[21] and Sactibiotics (Class Ic),
bacteriocins that contain cysteine sulphur to α‑carbon linkages
(Mathur et al., 2015) [22]. Mechanism of Action Class IIc (circular bacteriocins), these are
small, heat-stable peptides that are carried by leader peptides. They contain about 58 and 70 amino acids. Their circularised
structure conferred by covalent binding between the first and
last residues, helps them to be heat resistant (some retain their
activity after treatment at 121 °C for 15 min), adopt to pH
variation, and untouched by proteolytic digestion (Martínez et
al., 2016) [28]. Finally, class IId (unmodified, linear, non-
pediocin-like
bacteriocins)
(Oppegård
et
al.,
2007;
Belguesmia et al., 2011) [30] is made out by all bacteriocins
that cannot be included in any of the first three classes
includes lineal peptides, such as lactococcin A, bacteriocins
that do not have a dedicated export system but use the general Class II (Non-lantibiotics), such as pediocin-like and the one-
peptide non-pediocin-like bacteriocins (class IIa and class
IId), binds to Man-PTS, these bacteriocins getting into the
target cell membrane causes an irreversible opening of an
intrinsic channel, which leads to the diffusion of ions through
the membrane, causing target cell death (Nissen-Meyer et al.,
2009; Diep et al., 2007; Nes et al., 2013) [25, 39, 40]. Class IIb
(two-peptide
unmodified
bacteriocins)
works
by
permeabilising the membrane of sensitive bacteria forming
pores. Circular bacteriocins (class IIc) have a positive net
charge, they interact directly with the negatively charged
bacterial membrane without requiring any receptor molecules
leading to pore formation in the cell membrane, causing ions
efflux and the dissipation of the membrane potential, leading
to cell death (Van Belkum et al., 2011; Perez et al., 2018) [41,
26]. Bacteriolysins (class IIIa bacteriocins) contribute to cell wall
hydrolysis, causing cell lysis (Sun et al., 2018; Simmonds et
al., 1996) [42, 43]. Non bacteriolytic bacteriocins (class IIIb) ~ 994 ~ http://www.thepharmajournal.com The Pharma Innovation Journal interest due to their application in the food industry as natural
bio preservatives (Zacharof and Lovitt, 2012) [59]. They can be
directly added as purified or partially purified components or
incorporated into food during cultivation with the help of
bacteriocin-producing bacterial strains (Snyder and Worobo,
2014) [60]. Mechanism of Action Bacteriocins are reported to be effective against
food spoilage microorganism, have minimal effect on human
microbiota are resistance to heat, pH and food associated
enzymes and are found to be stable in the food substrate in
which they are incorporated with no alteration of the
organoleptic properties of food, being tasteless, odorless, and
colorless; no toxicity to eukaryotic cells and simplicity to
scale-up production. (Johnson et al., 2018) [61] (Martínez et
al., 2016) [28]. Nisin is a broad-spectrum Class I lantibiotic,
produced by Lactococcus lactis subsp. lactis, Nisin was the
first antimicrobial agent in reaching the category of food-safe
additive in 1969 and most studied bacteriocin and has
received the Generally Regarded as Safe (GRAS) designation
by the FDA (Cotter et al., 2005) [62]. Enterocin AS-48 and
enterocin RM6 have been studied for their food preservatives
and sanitation efficiency against food pathogens, Listeria
monocytogenes (Espitia et al., 2013) (Barbosa et al., 2013). Gassericin A, food preservative from Lactobacillus gasseri
LA39, reported to be stable for 3 months (4 °C), 2 months (37
°C), 5 hours (60 °C) and 30 minutes (100 °C) (Ahmad et al.,
2017) [36]. exert their action by disrupting the glucose uptake by cells
and hence disturbing the membrane potential. Another
mechanism is inhibiting the biosynthesis of DNA and proteins
of target bacteria (Meade et al., 2020; Müller and Radler,
1993; Swe et al., 2009) [44-46]. The class III bacteriocin
lysostaphin kills Gram-positive Staphylococcus aureus
through cell wall lysis (Gründling and Schneewind, 2006) [47]. Colicin A exhibits DNA nuclease activity e.g. colicin E2 and
can also inhibit biosynthesis of proteins e.g. colicins E3, E5
(Ahmad et al., 2017) [36]. Moreover, Gram-negative bacteria
like Yesinia spp. and Escherichia coli are sensitive to a high
molecular weight bacteriocin (pesticin) by the mechanism of
cell wall degradation through breaking the glycosidic bonds
of the cell wall (Ahmad et al., 2017) [36] Hospital-acquired infections Bacteriocins are playing pivotal role in hospital settings where
Multi-Drug Resistant pathogens is most noticeable and many
nosocomial infections due to such MDR strains are becoming
a worldwide threat to human health. Emergence of
antimicrobial resistance against commonly used antibiotics
has become a serious issue which requires feasible alternative
to antibiotic for the safeguard of human health. Many
bacteriocin are being successfully used as probiotic in place
of antibiotic and thus boosting our immunity system (Riaz et
al., 2020) [11]. Bacteriocins have very high specificity unlike
antibiotics, and can inhibit pathogens without causing
deleterious imbalances to the host microbiota (O’Connor et
al., 2020) [48]. The main disease-causing pathogens in hospital
settings
are
Staphylococcus
aureus,
Enterococci,
Pneumococci, Acinetobacter baumannii, Citrobacter freundii,
Escherichia coli, Klebsiella pneumoniae and Proteus spp. (Ghodhbane et al., 2015; Michalet et al., 2007) [66, 67]. Reports
suggests Nisin and lacticin 3147 shows efficacy against
various pathogens in the liver, spleen and kidneys and
similarly also found to be effective against Methicillin-
resistant Staphylococcus aureus (MRSA) and Vancomycin-
resistant enterococci (VRE) (Piper et al., 2009) [68]. Knowledge of a bacteriocins’ mode of action (Cotter et al.,
2013) [35] and how it acquires resistance facilitate the
development of methodologies to minimise resistance
occurrence (Draper et al., 2015) [53]. Strategies successfully
used to reduce resistance include combining bacteriocins with
other bacteriocins with different modes of action (Dicks et al.,
2018; Algburi et al., 2017; Hols, Ledesma-García et al., 2019)
[51, 54, 55], other antimicrobials (Perales-Adán et al., 2018;
Mathur et al., 2017) [56, 57], or phages or, generating peptides
with
increased
antimicrobial
resistance
through
bioengineering (Field et al., 2019)
[58]. These hurdle
technology approaches have the added advantages of
broadening the antimicrobial spectra while reducing costs and
toxicity (Mathur et al., 2017) [57]. Pharmacokinetics and
pharmacodynamics of drugs are key factors to consider for
any in vivo therapeutics (Mathur et al., 2017) [57]. Bacteriocins Resistance Bacteriocins are considered encouraging alternatives due to
their stability, low toxicity, frequently excellent potency and
high specificity. For any antimicrobial under study, it is
important to consider the potential emergence of resistant
pathogens with respect to clinical applications. Many
bacteriocins interact electrostatically with the cell membrane
and introduce permeabilisation through interaction with
receptor or docking molecules (O’Connor et al., 2020) [48]. Bacteriocin resistance in pathogens may be innate, which is
intrinsically observed in particular genera/species, or
acquired, commonly observed with susceptible strains
(Collins et al., 2012) [49]. It can also be associated with the
pathogenic organisms’ ability to produce degradation
enzymes or the presence of immunity proteins, while acquired
resistance occurs due to horizontal gene transfer or gene
mutations that alter the cell membrane, binding receptors or
transport systems (de Freire Bastos et al., 2015; Dicks et al.,
2018) [50, 51]. Studies revealed resistance to bacteriocins that
have intracellular targets could arise through mutations in the
genes encoding the bacteriocin targets (Cotter et al., 2013)
[35]. Another mechanism called immune mimicry is indicated
which is used to describe resistance that occurs in non-
bacteriocin-producing strains which possess bacteriocin
immunity genes, or immunity as a consequence of producing
a closely related bacteriocin (Draper et al., 2009). Remarkably, antibiotic and bacteriocin resistance are
independent and, consequently, no cross-resistance has been
recorded so far (Martínez et al., 2016) [28]. Nisin Z, one of the His27Asn variant of nisin A, have a
greater solubility at higher pH and hence finds its
applicability in food industry and is also commercially
available as, for example, Nisin Z1P ultrapure nisin. By
utilising food grade techniques, nisin variants can be
bioengineered and thus nisin can be custom designed for
specific applications by increasing production, increasing
potency against specific targets or enhancing antimicrobial
spectrum of inhibition thereby increasing its commercial
potential as a food preservative (Field et al., 2018) [65]. Potential Applications of Bacteriocins (Mandal et
al., 2007) [88] a strain identified as Pediococcus acidilactici
LAB 5, isolated from vacuum-packed fermented meat,
exhibited varying degrees of antifungal activity against
Aspergillus fumigatus, A. parasiticus, Fusarium oxysporum
and Penicillium sp. Bacteriocins in veterinary medicine Bacteriocins in veterinary medicine
Mastitis, an intramammary bacterial infection caused by
Staphylococcus
aureus,
Streptococcus
uberis
and
Streptococcus dysgalactiae is common in dairy animals
against which a nisin-based udder disinfectant and an
intramammary infusion product containing nisin viz Wipe
OutR dairy wipes (Immucell, Portland, ME) and Mast OutR
(Immucell) respectively has been approved by FDA recently
(Hernández-González et al., 2021)
[98]. Lacticin 3147
produced by Lactococcus lactis DPC3147, lantibiotic having
high inhibitory potential, has been evaluated against many
mastitis-causing microorganisms as a dry cow therapy in teat
seal formulations (Dicks et al., 2018) [51]. In poultry, likewise
antimicrobial property of bacteriocins has been studied to
control and inhibit pathogenic microorganisms. In broiler
chickens, Plantaricin from Lactobacillus plantarum F1was
put forwarded as a viable replacement of antibiotics against
colibacillosis (Ogunbanwo et al., 2004) [99]. Combination of
partially purified enterocin CLE34 and Plantaricin CLP29
have wide antibacterial activity against Salmonella pullorum
and E. coli (Wang et al., 2012) [100]. Increment of body weight
have been observed in broilers when they were fed a
combination of bacteriocins divercin AS7 and nisin as an
additive in the diet (Józefiak et al., 2013) [101]. Studies Anti-cancerous activity of bacteriocins Anticancer activity of bacteriocins can be attributed to the
interaction with targeted membrane. Bacteriocins being
positively charged (cation) bind effectively with negatively
charged cell membrane of cancer cells compared to neutral
charged normal cell membranes (Hoskin and Ramamoorthy,
2008; Martín et al., 2015) [90, 91]. Presence of large number of
microvilli in the cancerous cells membranes (Chaudhary and
Munshi, 1995) [92] assist the anticancer activity of bacteriocins
by binding more efficiently to the same in opposition to
normal cells’ (Chan et al., 1998) [93]. Few of the bacteriocins
have been put forwarded as potential candidate for cancer
chemotherapy (Baumal et al., 1982; Farkas-Himsley and Yu,
1985) [94, 95] or to help in the diagnosis of some types of cancer
(Kaur and Kaur, 2015) [96] (Loiseau et al., 2016) [97]. Compared to normal cells, Pediocin PA-1, nisin and
plantaricin A are reported to show relatively increased
cytotoxicity toward cancerous cells. Bacteriocins, hence due
to this selective toxicity makes them very promising
candidates for further research and trials (Kaur and Kaur,
2015) [96]. Anti-tuberculous activity of bacteriocins Lactoferrin is an iron-binding glycoprotein present in mucosal
secretions and neutrophilic granules and considered to be
important factor for innate immunity and possess ability to
alter host reactions in M. tuberculosis infection (Actor et al.,
2009) [74]. Lactoferrin alone did not alter the growth of M. tuberculosis in either macrophages or broth culture, but
enhanced IFN-γ mediated killing of the M. tuberculosis
through the macrophages. Hence, lactoferrin suggests
potential to be used for the treatment of tuberculosis and be
helpful to minimise immune-mediated tissue damage in
infectious diseases (Welsh et al., 2011) [75]. Bacteriocin
isolated from Lactobacillus salivarius, Streptococcus cricetus
and
Enterococcus
faecalis,
have
shown
more
antimycobacterial
activity
in
comparison
to
equal
concentrations of rifampicin in an in vitro model. These
bacteriocins didn’t showed ant toxicity at a concentration of
0.1 mg/L for mouse macrophages with activity of >90 MIC
(Sosunov et al., 2007) [76]. Lantibiotics evidently possess
sufficient potential for future therapies treating tuberculosis
(Sivaraj et al., 2018) [77]. Antiviral activity of bacteriocins Many bacteriocins has shown antiviral activity against murine
norovirus S99 (MNV), influenza A Virus A/WSN/33 (H1N1),
Newcastle disease virus, Montana and feline herpes virus KS
285 (Lange-Starke et al., 2014) [79]. The safety and efficacy of
a subtilosin-based nanofibre formulation have also been
evaluated against herpes simplex virus type 1 (Torres et al.,
2013) [80]. Some bacteriocins, such as subtilosin A from
Bacillus subtilis, were reported as having anti-viral (Quintana
et al., 2014) [81]. Potential Applications of Bacteriocins Major pathogens of respiratory tract include Haemophilus
influenzae, Moraxella catarrhalis, Staphylococcus aureus, Food preservative applications of bacteriocins
Bacteriocins with promising potential have gained much ~ 995 ~ http://www.thepharmajournal.com The Pharma Innovation Journal Enterobacteriaceae, Pseudomonas aeruginosa, Streptococcus
pyogenes, Neisseria meningitidis, Pasteurella multocida and
Mycobacterium tuberculosis which cause infections such as
pneumonia, otitis, rhinitis and tuberculosis. Many reports are
suggestive of control of many by bacteriocins (Mandell et al.,
2007; Pascual et al., 2008; Ghobrial et al., 2009; Knoetze et
al., 2008) [69-72]. Two of the potent lantibiotics produced by
Streptococcus salivarius K12 are salivaricin types A and B,
both of which are used to treat infections of the upper
respiratory
tract
caused
by
streptococcal
organisms
(Balakrishnan et al., 2000) [73]. [85], are also produced by other LAB, such as P. pentosaceus
and L. sakei (Magnusson et al., 2003) [86]. [85], are also produced by other LAB, such as P. pentosaceus
and L. sakei (Magnusson et al., 2003) [86]. [85], are also produced by other LAB, such as P. pentosaceus
and L. sakei (Magnusson et al., 2003) [86]. Inhibitory activity of peptides produced by L. plantarum
strain LR/14 against A. niger, Rhizopus stolonifer, Mucor
racemosus and Penicillium chrysogenum was reported (Gupta
and Srivastava, 2014) [87]. The peptides were able to inhibit
both spore germination and hyphal growth; however, the
former
stage
exhibited
heightened,
dose-dependent
susceptibility. Strains of several Pediococcus species have
also been found to produce substances able to control the
growth of mycotoxinogenic fungi (Mandal et al., 2007; Rouse
et al., 2008) [88, 89]. In the study by Mandal et al. (Mandal et
al., 2007) [88] a strain identified as Pediococcus acidilactici
LAB 5, isolated from vacuum-packed fermented meat,
exhibited varying degrees of antifungal activity against
Aspergillus fumigatus, A. parasiticus, Fusarium oxysporum
and Penicillium sp. and L. sakei (Magnusson et al., 2003)
. Inhibitory activity of peptides produced by L. plantarum
strain LR/14 against A. niger, Rhizopus stolonifer, Mucor
racemosus and Penicillium chrysogenum was reported (Gupta
and Srivastava, 2014) [87]. The peptides were able to inhibit
both spore germination and hyphal growth; however, the
former
stage
exhibited
heightened,
dose-dependent
susceptibility. Strains of several Pediococcus species have
also been found to produce substances able to control the
growth of mycotoxinogenic fungi (Mandal et al., 2007; Rouse
et al., 2008) [88, 89]. In the study by Mandal et al. Antifungal activity of bacteriocins The application of bacteriocins for the control of fungal
growth may at first thought appear an ambitious and perhaps
illogical idea. However, if achievable this strategy could
certainly have widespread practical application. Is the
targeting of such taxonomically-distant microbes a reasonable
expectation for conventional proteinaceous bacteriocin
molecules or are the occasionally-reported observations of
anti-fungal activities by bacteria attributable to the activity of
non-bacteriocin inhibitory molecules (Todorov, et al., 2019)
[82]. The cyclic dipeptides cyclo (L-Phe-L-Pro) and cyclo (L-
Phe-trans-4-OH-L-Pro) produced by L. plantarum MiLAB
393 were identified as potential antifungal agents by Ström et
al. (Ström, et al., 2002) [83]. These small peptides cannot of
course be considered to be ‘true’ bacteriocins. Various cyclic
dipeptides have previously been shown to have both
antibacterial and antifungal activities (Graz et al., 1999) [84]
and it is likely that these substances, previously only reported
from strains of L. plantarum (Lindgren and Dobrogosz, 1990) ~ 996 ~ The Pharma Innovation Journal http://www.thepharmajournal.com reported use of colistin (polymyxin E) in piglets against
Enterotoxigenic E. coli causing diarrhea has led to the
development of a resistant strain (Bin et al., 2018) [102]
(Aguirre et al., 2020) [103]. A synergistic effect against
colistin-resistant E. coli strains isolated from pigs was
observed when a combination of nisin or enterocin DD14
along with colistin was used, the reason being the interaction
of colistin with Lipopolysaccharide which allows the entry of
bacteriocins causing loss of membrane stability to damage the
cell wall (Al Atya et al., 2016) [104]. Nisin was also reported to
prevent the formation of dento-bacterial plaque and gingivitis
in dogs, similar to action of chlorhexidine (Howell et al.,
1993; Cunha et al., 2018) [105, 106]. Incorporation of enterocin
M in the feed of horses reduced Gram-negative bacteria such
as coliforms, Campylobacter, and Clostridium spp. with no
physiological parameter being altered (Lauková et al., 2018)
[107]. Nisin when added to the feed and water of weaned
rabbits
found
to
have
reduced
harmful
intestinal
microorganism, also increase in both weight was observed
and the meat quality remained unchanged (Lauková et al.,
2014; Pogány Simonová et al., 2019) [108, 109]. Bacteriocins
have an immense potential in veterinary medicine and can be
further more investigated to exploit their use as a substitute
for antibiotics and represent a strong new antimicrobial. Antifungal activity of bacteriocins These
findings
highlight
the
immense
potential
of
bacteriocins in veterinary medicine as an excellent
antibacterial alternative against potentially pathogenic agents
that prevent could bacterial resistance as well as improved
growth performance (Schofs et al., 2020; Preciado et al.,
2016) [110, 4]. products. Bacteriocins are one of the many entity whose
benefit can be extracted most by such innovative
combination. For instance, nanoencapsulation of bacteriocins intended for
use as bio-preservatives could protect them from degradation
by proteolytic enzymes, in addition to rescuing them from
undesirable interactions with other food components, and
hence, increasing their stability for longer periods (Brandelli,
2012) [117]. Furthermore, some recent studies have shown that
encapsulation of bacteriocins in nanoparticles has enhanced
the
activity
of
these
peptides
against food-spoiling
microorganisms and multidrug-resistant bacteria (Arthur et
al., 2014; Mossallam et al., 2014) [118, 119]. In addition, the use
of nanotechnology-based materials and/or methods has, in
most cases, shown a positive impact on bacteriocin yield, thus
facilitating their commercial production (Zacharof et al.,
2013) [120]. Through this amalgamation, efficient delivery,
targeting, protection from degradation, as well as improved
drug activity and physicochemical properties, can be achieved
(Farokhzad and Langer, 2009) [121]. Nanomaterials can be developed to deliver the payload to
specific target tissue or infected sites, thus reducing the
amount of antimicrobial required for effective treatment. With
the increasing bacterial resistance, the evolution of
nanotechnology has proven to be an effective upgrade of
traditional drug delivery systems. Bacteriocins incorporated
nanoparticles and infection targeted site-directed delivery may
soon become an effective method of treatment. Bacteriocins
in a concentrated form such as nanoparticle encapsulation
delivery system, would enhance their effectivity and minimize
possible side effects (Zimina et al., 2020) [122]. Nanoformulated Bacteriocins Nanotechnological techniques and Bacteriocin delivery
system Encapsulation of bacteriocin in liposomes Along with the mentioned above, various other studies are
also under process to exploit more potentials of bacteriocins. Along with many others some includes their effectivity to
treat skin diseases. Bacteriocins from Enterococcus hirae
DCH5 and Lactococcus subsp. QU12 has successfully
showed efficacy invitro been examined vitro against
staphylococci,
enterococci,
lactobacilli
and
Listeria
monocytogenes (Sánchez et al., 2007; Sawa et al., 2009; Wu
et al., 2005) [111-113]. Immunomodulatory effect of bacteriocins
are also under study, efficaciousness depends upon the
concentration of bacteriocin used which in turns adds to the
bactericidal effect, thereby increasing host safeguard,
especially during the period of infections (Hernández-
González et al., 2021) [98]. p
p
Liposomes are biocompatible and non-toxic spherical vesicles
constituting
single
or
multiple
phospholipid
bilayer
membranes (Gómez-Hens and Manuel Fernández-Romero,
2005) [123]. They are non-toxic and biodegradable agents are
capable of encapsulating both hydrophobic and hydrophilic
compounds. Nano-sized liposomes, size varying from
micrometers to nanometers, called nano-liposomes are
emerging as promising vehicles for the encapsulation and
delivery of several bioactive components like that of
therapeutic bacteriocins to specific target cells along with
other food additives, enzymes and vitamins (Akbarzadeh et
al., 2013) [124]. Encapsulation by liposomes protects the encapsulated
compounds
from
environmental
and
physicochemical
alterations (Mozafari et al., 2008) [125]. A technique called thin
film hydration method is used to encapsulate bacteriocins in
liposomes. In this technique, the chemically synthesized lipid
film is hydrated at a higher temperature than the phase
transition temperature of lipids with bacteriocin containing
aqueous buffer leading to production of manifold population
of multilamellar vesicles of about >400 nm in size where
bacteriocins are encapsulated, which are then further
processed by sonication or heating or membrane extrusion
into homogeneous, small unilamellar vesicles of about 20–80
nm in size (Chandrakasan et al., 2019) [126]. Nanotechnological techniques and Bacteriocin delivery
system In accordance to the Science and Technology Committee of
the House of Lords of the United Kingdom, nanotechnology
is the transformation of functional materials and structures
into nanoscale sizes (with diameters from 1 to <1000 nm)
(Klaessig et al., 2011)
[114]. Drugs are loaded onto
nanomaterials to improve pharmacokinetics by altering
physical characteristics, such as solubility, half-life and
bioavailability (L. Zhang et al., 2010) [115]. This is a
completely novel technology that has several applications in
various fields of science, due to the unique properties of
synthesized nanoparticles (Chou et al., 2011) [116]. The
amalgamation of nanotechnology and biotechnology opens a
paradigm of vast set of opportunities and future perspectives
to resolve the concerns with regards to wide biological A study reported, nanoliposomes co-encapsulated with nisin
and garlic extracts showed enhanced broad spectrum activity
against
various
food-spoiling
pathogens
viz
L. monocytogenes, Salmonella enteritidis, E. coli, and S. aureus. (Pinilla and Brandelli, 2016) [127]. In another investigation,
prominent inhibitory activity against Listeria monocytogenes ~ 997 ~ http://www.thepharmajournal.com The Pharma Innovation Journal anticancer activity coupled with its ability to deliver drugs to
their specific targets is well studied (Sidhu and Nehra, 2019)
[136]. The
chitosan-bacteriocin
conjugation,
commonly
achieved by ionic gelation method, where chitosan and
bacteriocin suspension are mixed and are stirred in 1% acetic
acid at room temperature with the addition of sodium
tripolyphosphate (TPP) followed by centrifugation and the
chitosan incorporated bacteriocins are obtained as pellet
which can be lyophilized for further use has been described
(Karthick Raja Namasivayam et al., 2015) [135]. In contrast
with free nisin, higher level of antimicrobial activity was
displayed by nisin-loaded chitosan-alginate nanoparticles
against L. monocytogenes and S. aureus (Zohri et al., 2013)
[138]. Many researchers have used chitosan with bacteriocins to
obtain a material showing collaborative antibacterial activity. Synergistic antibacterial activity of chitosan–nanoconjugates
loaded with bacteriocins have been described against Listeria
monocytogenes
when
compared
with
those
of
free
bacteriocins (Karthick Raja Namasivayam et al., 2015) [137]. Incorporation of biopolymers in place of plastics in food
packaging is escalating its safety and biodegradability. Crystalline nanocellulose coated with bacteriocin and
reinforced onto starch films enhances the tensile strength of
the film by 69% and antibacterial effect by 57% (Bagde and
Vigneshwaran, 2019) [139]. Phytoglycogen nanoparticles y
g y
g
p
Novel
functional
nanoconstructs
are
prepared
from
phytoglycogen, a polysaccharide material found in plants
(Chen et al., 2015) [142]. Apart from chitosan that described
earlier, phytoglycogen and its derivatives are another
carbohydrate based nanoparticle that has been used
successfully for encapsulating nisin (Bi et al., 2011) [143, 144]. Phytoglycogen and its derivatives were studied as carriers of
nisin, and all showed antimicrobial activity against L. monocytogenes for longer period than free nisin. Among all
phytoglycogen
derivatives,
3-amylolysis
and
octenyl
succinate substitutions showed longest activity for about 21
days against common food pathogens in comparison with 7
days in case of the free nisin (Bi et al., 2011) [143, 144]. The
antibacterial activity of this nanoparticle-stabilized emulsion
has been higher than that of the free nisin during 50 days of
storage (Bi et al., 2011) [143, 144]. These findings signify that
there is an enormous scope to explore such nanomaterials as
carriers for bacteriocins which could play a great role in
public health and food safety. Nanotechnological techniques and Bacteriocin delivery
system This new class of nano-polymer
hybrid thus provides a magnitude to fight bacterial pathogens
and could therefore be an efficient weapon against food-borne
bacterial pathogens (Sharma et al., 2012; Chopra et al., 2014)
[140, 141]. was
displayed
when
nisin
was
encapsulation
into
phosphatidylcholine, incorporated into biopolymer-based
films of gelatin or cellulose indicating it to be a promising
active packaging material (Boelter and Brandelli, 2016) [128]. Moreover, no hemolytic activity on human red blood cells
was seen upon use of encapsulated bacteriocin-like
substances, thus signifying their safety as food preservatives
(Teixeira et al., 2008) [129]. (
,
)
Liposome encapsulation delivery systems have several other
advantages such as improved stability, degradation protection,
reduced doses in therapeutic applications and enhanced
antibacterial activity in terms of the time taken to exercise the
antimicrobial action and the activity spectrum. While nano-
encapsulation of bacteriocins give numerous advantages,
studies also indicated adverse effect on the antimicrobial role
of bacteriocins (da Silva Malheiros et al., 2010) [130]. Nevertheless, with appropriate selection of phospholipid
bacteriocin, this adverse effect can be prevented by
preventing unhealthy interactions between bacteriocins and
liposomes. An
appropriate
phospholipid
bacteriocin
combinations, the avoidance of adverse liposome–bacteriocin
interactions, and utmost purity of starting materials are the
key to the productive implementation of bacteriocins
encapsulated liposome (Pinilla and Brandelli, 2016) [127]. Solid lipid nanoparticles p
p
Solid lipid nanoparticle (SLN) is composed of a solid core of
triglyceride with a phospholipid coat of high-melting point,
hence maintain a solid state at room and human body
temperatures (Puri et al., 2009) [131]. In addition to the
numerous advantages of liposomes, the solid triglyceride core
of SLN makes them powerful tools for largescale production
and slow release drug formulation (Feng and Mumper, 2013)
[132]. Nisin when incorporated in SLN carriers showed
sustained release for about 25 days, depending on the pH and
the salt concentration of the buffer solution (Prombutara et
al., 2012) [133]. Moreover, nisin-loaded SLN have showed
activity against L monocytogenes DMST 2871 for up to 20
days and L plantarum TISTR 850 for up to 15 days, in
comparison with free nisin whose activity lasted only for 3
days against the former organism and for 1 day against the
latter (Prombutara et al., 2012) [133]. These studies suggested
that SLN have the potential to protect bacteriocins from
degradation, and therefore could be used to prolong their
antibacterial activity for a long duration. Though, much
studies need to be conducted before taking SLN as a delivery
system for bacteriocins as it is still in the early exploratory
phases of research. Possible expulsion of the incorporated
drug/drug-like agents from the lipid matrix and the low drug-
loading capacity are few of the challenges that need to be
overcome to fully establish the SLN as a delivery system are
the possible expulsion of the incorporated drug/drug-like
agents from the lipid matrix and the low drug-loading
capacity fna (Jenning et al., 2000; Souto et al., 2006) [134, 135]. Metallic Nanoparticles Presently, the metallic nanoparticles such as zinc, copper,
silver, and gold are being thoroughly investigated not only as
potential antimicrobials but also for their different prospective
biomedical applications (Naskar and Kim, 2020; Naskar et
al., 2020a; Naskar et al., 2020b; Naskar et al., 2020c) [145-148]. Such substantial use of these nanoparticles may be accredited
to their large surface area of positively charged nanoparticles,
which can interact with negatively charged bacterial cell
membrane (Seil and Webster, 2012) [149]. Oxidative stress
induced by the generated reactive oxygen species, together
with the toxicity of the accumulated free metal ions helps to
kill the target bacteria (Seil and Webster, 2012) [149]. Silver Conclusion And Future Prospects p
Bacteriocins are a promising tool that can be exploited for its
utilisation in various sector, may it be human or veterinary
medicine. The administration of these antimicrobial peptides
has proven to show efficacy as anti-tuberculous, antiviral as
well as antifungal agents. They have longed been used in food
industry as food preservative and so to increase shelf life of
food commodities. Bacteriocins have found their utility in
domestic animals as they eliminate potentially pathogenic
undesirable microorganisms pertaining to reduced toxicity or
no
toxicity
and
improves
productive
parameters
in
substitution of antibiotics as growth promoters. They have
also shown promising efficacy against many dangerous
nosocomial diseases known to cause maximum mortality and
morbidity. Unquestionably,
bacteriocins
may
play
a
remarkable role in combating antibiotic-resistant bacterial
strains, considering its narrow-target activity, low toxicity,
and high stability and specificity. Bacteriocins in combination
with antibiotics work synergistically and shows great potency. They also have a role in the immune response as
immunomodulators along with their other diverse field of
action. Although, they are found to have few side effect, those
can be minimised by use of newer techniques like that of
nanotechnological approaches. It is well established that
incorporation of bacteriocins into nanoparticles and specific
delivery to areas of infection may soon become an effective
treatment regime. Many instances showed combination of
nano-formulation along with free bacteriocins revealed better
stability and a broader range of antimicrobial action. Nanotechnological methods provide an interesting option
toward the formulation of these antimicrobial peptides at the
industry scale level. Similarly, gold and bacteriocins nano-conjugates shows
potential antimicrobial activity and reduced toxicity. The use
of gold nanoparticles conjugates for other biomedical
applications such as the co-delivery of nisin and doxorubicin
to treat murine skin cancer (Preet et al., 2019) [155]. A study
reported, combination of gold nanoparticles with either nisin
or a bacteriocin produced by L. plantarum ATM11 both
showed promising antibacterial effect with comparison with
free bacteriocins especially against M. luteus, B.cereus,
E.coli,
and
S.aureus
indicating
usefulness
of
such
combinations
in
inhibiting
common
food-spoilage
microorganisms and hence prolonging the shelf-life of food
products (Thirumurugan et al., 2013) [156]. Upon biochemical
examinations
and
histopathological
screening
tests,
nanoconjugate bacteriocin has been reported to be safe and
non-toxic (Mossallam et al., 2014) [119]. Nanofiber Appropriate studies need to be carried out to access their in
vivo effects, mechanism of action, interaction with the host
immune system, and large-scale production costs as well as
the emergence of bacteriocin resistance. Other several
unexplored drug delivery systems can be the next phase in
drug development process. Conclusion And Future Prospects Hence, different
nanoconjugates of bacteriocins can be used to increase the
antimicrobial array of bacteriocin alone, which can serve as
efficient weapon in the fight against food borne pathogens
and multi- drug resistant bacteria. Chitosan Chitosan is another type of nanoparticle, which is
tremendously used with bacteriocins. It is a natural
biopolymer which is synthesized by deacetylation of chitin
and
is
biodegradable,
biocompatible,
non-toxic
and
bactericidal which makes it an ideal candidate for its use in
biomedical applications and food safety. It is one of the most
extensively used polysaccharides to produce nanoparticles. Moreover, its antibacterial, antifungal, antiprotozoal and ~ 998 ~ http://www.thepharmajournal.com The Pharma Innovation Journal and gold are more commonly used and studied nanoparticles,
showing synergistic effects in biomedical applications. Many
studies indicating antibacterial effects of silver and gold
nanoparticles is being reported, making it easier to understand
the theory associated with combination of bacteriocins and
silver/gold nanoparticles. Many applications of silver
nanoparticles are well known such as coating medical
devices, wound dressing, coating textile fabrics, to water
treatment and filtration (Furno et al., 2004; Rujitanaroj et al.,
2008; Zhang et al., 2009; Dankovich and Gray, 2011) [150- 153]. This approach has been demonstrated by Sharma et. al. where
they used enterocin- capped silver nanoparticles which
showed broad-spectrum inhibition against various food-borne
pathogenic bacteria namely E.coli, L.monocytogenes, and
S.aureus along with admirable non-toxicity to red blood cells,
signifying its biocompatible nature (Sharma et al., 2012) [140]. A study also reported increased antimicrobial potential of
nisin after conjugation with silver nanoparticles associated
with food spoilage against Listeria monocytogenes, S. aureus,
Pseudomonas fluorescens, Aspergillus niger, and Fusarium
moniliforme associated with food spoilage (Pandit et al.,
2017) [154]. al., 2013) [158]. Nanofiber based bacteriocin are also used as
antibacterial and antiviral substances. A study reported
antiviral activity against Herpes simplex virus type 1 (HSV-1)
of subtilosin-loaded poly (vinyl alcohol) (PVOH) nanofiber
(Torres et al., 2013) [80]. Subtilosin-loaded nanofibers showed
potential in vitro activity without exhibiting cytotoxicity
(Torres et al., 2013) [80] with comparison to modern antiviral
treatments regime cytotoxic to nephritic tissue (Ho et al.,
2000) [159]. Bacteriocin incorporated nanofiber technology
may represent a promising novel therapeutic alternative to
current drug therapies. Nanofiber y
Considering the explicit potential of bacteriocins, it is need of
the hour to harness the full potential of bacteriocin for the
greater good. Exploration of novel bacteriocin producing
strains with unique properties and characteristics can open a
wide range of application and new prospects. The bacteriocin
pharmacokinetics,
pharmacodynamics,
toxicity,
and
immunogenicity aspects need to be taken into considering
before the products are approved for large scale consumption. Appropriate studies need to be carried out to access their in
vivo effects, mechanism of action, interaction with the host
immune system, and large-scale production costs as well as
the emergence of bacteriocin resistance. Other several
unexplored drug delivery systems can be the next phase in
drug development process. Nanofibers are extraordinarily fine threads created by
electrospinning (Fahim et al., 2016) [13]. Nanofibers have
gained popularity as carriers for direct distribution and
continuous release of many drugs such as of antimicrobials
and hemostatic agents for wound healing along with that of
bacteriocins. High encapsulation potential, larger surface area,
high physical stability, small pores size has made nanofibers
potential nanocarriers for target specific drug delivery and
sustained release of a variety of drugs (Fahim et al., 2016) [13]. In a finding, nisin and silver nanoparticles incorporated into
nanofibers has shown broad antimicrobial efficacy against a
wide range of Gram-positive and resistant Gram-negative
bacteria (Ahire et al., 2015) [157]. Nisin released from this
nanofiber dressing, maintain its antistreptococcal activity in
vitro for at least 4 days; remain active for 8 months even after
storing of the formulation at 4°C; induce nearly complete
wound healing as indicated by the formation of clear fibrotic
scar in a group of mice undergoing dressing; significantly
reduce S. aureus colonization; and as per histological analysis
of the treated group, no adverse effects reported by (Heunis et Considering the explicit potential of bacteriocins, it is need of
the hour to harness the full potential of bacteriocin for the
greater good. Exploration of novel bacteriocin producing
strains with unique properties and characteristics can open a
wide range of application and new prospects. The bacteriocin
pharmacokinetics,
pharmacodynamics,
toxicity,
and immunogenicity aspects need to be taken into considering
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Formelová Z, Lauková A. Lantibiotic Nisin Applied in
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nanoliposomes co-encapsulating nisin and garlic extract
against Gram-positive and Gram-negative bacteria in
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Hernández PE. Amino acid and nucleotide sequence, ~ 1003 ~ The Pharma Innovation Journal http://www.thepharmajournal.com Technologies 2016;36:287-93. 2014;37(1):158-64. Technologies 2016;36:287-93. 128. Boelter JF, Brandelli A. Innovative bionanocomposite
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phytoglycogen studied using β-amylolysis. Carbohydrate
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synthesis for controlling food pathogens. Food Control ~ 1004 ~ The Pharma Innovation Journal http://www.thepharmajournal.com Current Microbiology 2015;71(1):24-30. Current Microbiology 2015;71(1):24-30. Current Microbiology 2015;71(1):24-30. 158. Heunis TDJ, Smith C, Dicks LMT. Evaluation of a nisin-
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1. Journal of the American Society of Nephrology
2000;11(3):383-93. ~ 1005 ~
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Research on New Energy Grid Control Technology
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1 Introduction The output voltage of inverteris.：
c
c
c
i
C
j
i
R
U
ω
1
0
+
=
（Eq.3） The output voltage of inverteris.：
c
c
c
i
C
j
i
R
U
ω
1
0
+
=
（Eq.3） As the new energy technology is applied more and more
widely, new energy grid can be efficient used became the
focus of attention, so it is practical significance to improve
the grid-connected technology.Inverter is the core device
of the whole system grid-connected,inverter grid-
connected control technology have directly affects on the
power supply quality [1]. At present, the popular control
methods include PI control strategy and fuzzy control, etc.,
but PI control strategy can't achieve the non-static
tracking and cannot get a good results in a long time. Based on the limitation of PI control,in this paper
proposes a compound control method based on repetitive
control theory, the effectiveness of this strategy is verified
through the analysis and demonstration of compound
control strategy. （Eq.3） The output transfer function of inverter is : 0
0
0
//
)
1
(
)
(
//
)
1
(
)
(
)
(
)
(
R
R
sC
R
sL
R
R
sC
s
U
s
U
s
P
c
c
i
+
+
+
+
=
=
（Eq.4） From figure 1 knows that the inverter itself belongs to
a nonlinear circuits and have a larger output
impedance,the existence of the output impedance the
same as the nonlinear circuit will distort the system, make
the output current produces a series of spikes. RESEARCH ON NEW ENERGY GRID CONTROL TECHNOLOGY LU Zhengtong, LI Shiqiang, WU Yingjun, SUN Lu, CHEN Guozhi, PENG Weilong State Grid Zhoushan Power Supply Company
No.2-1, huiming bridge, dinghai district, zhoushan city, zhejiang province. State Grid Zhoushan Power Supply Company
No.2-1, huiming bridge, dinghai district, zhoushan city, zhejiang province. State Grid Zhoushan Power Supply Company
No.2-1, huiming bridge, dinghai district, zhoushan city, zhejiang province. Abstract: Aiming at the poor waveform quality of the new energy grid connected under the traditional PI
control strategy, based on the theory of repetitive control method, a compound control trategy is proposed. Through the analysis of the compound control module and simulation, the results show that grid harmonic
distortion rate can be reduced about 2% at the same nonlinear load, the compound control strategy can
suppress the load disturbance signal effectively, and the simulation results demonstrate the effectiveness of
the proposed method. https://doi.org/10.1051/e3sconf/20185301003 https://doi.org/10.1051/e3sconf/20185301003 E3S Web of Conferences 53, 01003 (2018)
ICAEER 2018 d by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution
ommons.org/licenses/by/4.0/). 2 Mathematical model of inverter. In order to facilitate the analysis, a single - phase inverter
circuit is used to analyze the work characteristics. It is well known that voltage inverters are used to obtain
the perfect current waveform, the PI control strategy is
used commonly in most inverter control system. The
following is a system performance of three-phase inverter
grid under the double closed loop PI control strategy[2],
the relation between the three-phase voltage under the dq
coordinate system as follows: U
i
U
0
R
R
L
c
R
C
0
U
+
−
+
−
+
−
a
b
1s
2s
3s
4s
Fi
1 Si
l
h
l
i U
i
U
0
R
R
L
c
R
C
0
U
+
−
+
−
+
−
a
b
1s
2s
3s
4s
+
+
−
+
−
=
+
−
−
+
−
=
q
q
q
q
I
p
q
d
q
d
d
I
p
d
e
Li
i
i
s
k
k
U
e
Li
i
i
s
k
k
U
ω
ω
)
)(
(
)
)(
(
*
*
(Eq.5) (Eq.5) Figure1. Single-phase voltage source inverter g
g
p
g
According to KVL and KCL law, the output current of
inverter is as follows: g
g
p
g
According to KVL and KCL law, the output current of
inverter is as follows: In order to achieve the interconnection unit power
factor, Iq * general was to be set 0, so id * to be the key
factor in the control circuit of the inverter. （Eq.1） （Eq.1） iL=ic+i0 0
0
U
Ri
L
j
U
Ri
U
U
L
L
L
i
+
+
=
+
+
=
ω Despite the current PI regulation can improve the
steady state performance of the system, the compensation （Eq.2） * Corresponding author:e-mail: 1206434337@qq.com LU Zhengtong (phone: 13587053176; fax: 0580- 5111815) © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attributio
License 4.0 (http://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 53, 01003 (2018)
ICAEER 2018 https://doi.org/10.1051/e3sconf/20185301003 effect of this integral link is not obvious when a load
mutation occurs, so the PI control can't eliminate the
steady-state error of the system perfectly . the maximum harmonic distortion and minimum damping
in no-load operation. 2 Mathematical model of inverter. The sampling frequency is 10kHZ in
system, the output capacitance of inverter C is 200uF,
inductance L is 1 mH and equivalent resistance is 1Ω, the
resonance frequency of systemis 6742.2 rad/s and
damping ratio is 0.0405. diagram 4
2
)
(
4
4
2
−
+
+
=
z
z
z
S
（Eq.10） As is shown in Figure 2, the repetitive control strategy
adopts the embedded structure where d(z) is the
interference signal, iL is the ac side current of inverter,
and P(z) is the output transfer function of the inverter
system. Thanks to PI control strategy and repetitive
control strategy work independently, so the combination
of repetitive control with PI control can realize astatic
tracking to the system, the system can get a good dynamic
response performance and inhibition to jamming signal . The following is the low-pass filter model of S1(z),the
model of the second-order low-pass filter is: 2
2
2
1
2
)
(
n
n
n
s
s
z
S
ω
ζω
ω
+
+
=
（Eq.11）
According to the parameters of this system:
33
.0
094
.1
1551
.0
1813
.0
)
(
2
1
+
−
+
=
z
z
z
z
S
（Eq.12） 2
2
2
1
2
)
(
n
n
n
s
s
z
S
ω
ζω
ω
+
+
=
（Eq.11） （Eq.11） 2
2
2
1
2
)
(
n
n
n
s
s
z
S
ω
ζω
ω
+
+
= According to the parameters of this system:
33
.0
094
.1
1551
.0
1813
.0
)
(
2
1
+
−
+
=
z
z
z
z
S
（Eq.12） 4 Design of compound controller based
on repetitive control theory. The general expression of notch filter is: a
z
a
z
z
S
m
m
+
+
+
=
−
2
)
(
2
（Eq.8）
Due to z=ejωT=ejθ,
a
a
m
S
+
+
=
2
cos
2
)
(
2
θ
θ
（Eq.9） a
z
a
z
z
S
m
m
+
+
+
=
−
2
)
(
2
（Eq.8） N
z
z
Q
−
)
(
+
+
N
z−
)
(z
C
)
(z
P
( )
Ur z
+
−
*
dc
U
+
+
)
( z
d
dc
U
)
( z
e
PI
PI
+
−
+
+
Li
pulse
+
Figure 2.Single-phase inverter compound control system a
+
2 When S2(θ) is equal to 0, the trap has the strongest
attenuation of a particular frequency.It can be obtained the
relation is 2cosmθ+a=0, when and only if a=2 meets the
conditions. At this time,there have the relation mθ =π and
θ =wT, so we can get the result m= π/θ =3.58, the value
of m is defined to 4 in here. then the trap wave filter at this
time is: Figure 2.Single-phase inverter compound control system 4 Design of compound controller based
on repetitive control theory. Therefore, the no-load transfer function of the inverter
system is: Repetitive control is based on the internal model which
function is embedded the external signal model of system
into controller[3], there is a feedback control system which
has good tracking ability and the ability to effectively
eliminate disturbance, also is a control process to achieve
zero error tracking too. According to the law of periodic
disturbance, repetitive control has the same properties as
grid harmonic signal in single cycle repeats. At this time,
the following discrete internal model is: 45454564
546
45454564
)
(
2
+
+
=
s
s
s
P
（Eq.7） （Eq.7） （Eq.7） It can be seen from Eq.8 konws that the controlled
object contains high frequency resonance peak and has a
certain phase lag too. Thanks to the controlled object has the above
properties, the model of elimination is proposed here.[7],
there have a compensation part C (z) = KrzkS2(z) S1 (z) to
be designed in the repetitive control. Thanks to the
controlled object have dead zone effect and nonlinear load
harmonic is primarily in the low frequency, S2(z) is a
notch filter which is used to offset the resonance peak of
the controlled object. S1(z) is a low pass filter used to
attenuate the high frequency harmonic signal after the
resonant peak Zk. For phase advance, the phase lag is
compensated by a certain phase lag between the filter S1
and the no-load controlled object P(s). N
N
Z
Z
z
G
−
−
−
= 1
)
(
(Eq.6) (Eq.6) T is the system sampling period, T0 is the network
voltage base wave period, f0=50Hz, and N is the sampling
number of the base wave period. Due to internal model have characteristic of single
cycle time delay, so the strategy have the results that
system can't tracking and regulating to input signal timely
when the reference signal mutation cycle,[4], it means the
system dynamic performance is poorer. Since repetitive control has the characteristics of
single cycle delay, the improvement structure is generally
adopted as follow.The improvement structure is
compound control which combine the repetitive control
with PI control strategy,the following is the structure
diagram of the compound control system. 5 Design of repetitive control structure
parameters. From the above analysis knows that both the second-
order low-pass filter S1 and the no-load controlled object
P(s) have certain phase lag[8]. In order to maintain the
stability of the system, the phase advance link zk is
designed to here. Designed by the above relationship In order to reflect the superior performance of repetitive
control[6], the following parameters are designed to be
carried out in the no-load of the inverter. The inverter has 2 2 E3S Web of Conferences 53, 01003 (2018)
ICAEER 2018 https://doi.org/10.1051/e3sconf/20185301003 and the current 2 is the waveform under PI control
strategy of grid-connected.The results shows that the
harmonic distortion rate of the grid-connected current 2 is
6.83% and the current 1 is 4.31% at the same load
condition, between trap and second-order filter can launch the beat
the relationship between k and frequency in advance, the
research shows that when k is available between 4 to 5
best compensation effect, the advanced beat is 5 in this
paper , namely the zk = z5. (2) The
following
is
the
grid-connected
simulation of the compound control under voltage
fluctuation of the power grid. Acknowledgment (1) The simulation results of nonlinear load under
double closed loop PI control and compound control
strategy. Fund project: The paper was funded by the state grid
corporation science and technology project.The project
name is research on new energy electricity generation
foundation multivoltage classes direct current collection
network
organization
and
operational
control
technology ,the project number is 5211ZS1600NP . gy
V
o… Fi
4 Th
id
lt
d
t
f
V
o… 6 The simulation of system under the
compound control strategy. Figure 5. The grid current waveform under compound control Three-phase inverter compound control system as shown
in figure 3, The dc reference voltage Udc * is 660 V, the
power grid voltage is 380V, filtering inductance
capacitance L is 1.5 mH and C is 200 u F, the output
equivalent resistance R is 1 Ω, the PI control parameters
of current loop for the Kp is 5 and Ki is 66, the PI
parameters of voltage loop for Kp is 2.1 and Ki is 1000. The switching frequency f is 10kHz, the repeated control
module parameters are consistent with the above. gure 5. The grid current waveform under compound contro The simulation results show that the harmonic
distortion rate of the compound control is reduced from
6.83% to 4.31% at the same nonlinear load, the voltage of
the power network increased by 20% from 0.02s to 0.03s
and reduced by 20% from 0.06s to 0.07s. The time of both
fluctuations was 0.01s, and the network current was
basically stable without any obvious fluctuation. +
−
+
−
*
dc
U
dc
U
PI
L
ω
PI
PI
L
ω
SVPWM
*
di
*
qi
di
qi
+
−
+
−
−
+
de
qe
d
U
q
U
+
controller
Repetitive
−
+
+
+
+
变换
2
/
3
ai
bi
ci
+
+
controller
Repetitive
controller
Repetitive
Figure 3 .Three-phase inverter system simulation diagram of
composite control structure 7 conclusion The proposed compound control strategy is based on
repetitive control theory, according to the results of
analysis and simulation we knows that the control strategy
can effectively reduce the grid current harmonic distortion
rate and the load fluctuation signal can be suppressed
effectively, the steady-state accuracy of the system has
been improved effectively under the strategy. Figure 3 .Three-phase inverter system simulation diagram of
composite control structure REFERENCES 1. Lu Sun;A Survey of Repetitive Control for
Nonlinear Systems. –Science Foundation in China
3:15-16. 2. Wenli Sun; Hong Cai; Fu Zhao: FBFN-based
adaptive
repetitive
control
of
nonlinearly
parameterized
systems. –Journal
of
Systems
Engineering and Electronics 5:115-116. Figure 4. The grid voltage and current waveform Figure 4. The grid voltage and current waveform Figure 4 shows the voltage and current waveform of
the parallel network,the voltage unit is V and current unit
is A in all the graphs. Current 1 is the current waveform
under compound control strategy of the grid-connected, 3 E3S Web of Conferences 53, 01003 (2018)
ICAEER 2018 https://doi.org/10.1051/e3sconf/20185301003 3. Edi Kurniawan; Zhen-Wei Cao; Maria Mitrevska;
Zhi-Hong Man: Design of Decentralized Multi-input
Multi-output
Repetitive
Control
Systems. –
International Journal of Automation and Computing
12:75-76. 6. Bingxi Jia , Shan Liu , Yi Liu;Visual trajectory
tracking of industrial manipulator with iterative
learning control[J].Industrial Robot,2015,42(78):12-
13. 3. Edi Kurniawan; Zhen-Wei Cao; Maria Mitrevska;
Zhi-Hong Man: Design of Decentralized Multi-input
Multi-output
Repetitive
Control
Systems. –
International Journal of Automation and Computing
12:75-76. 7. Zhang Kai,Kang Yong,Xiong Jian;Study on an
inverter with pole assignment and repetitive control
for
UPS
applications[J]. Proceedings
of
PIEMC,2012,23(2):45-48. 4. Quan Quan; Kai-Yuan Cai: Repetitive Control for
TORA
Benchmark:An
Additive-state-
decomposition-based
Approach. –International
Journal of Automation and Computing 18:45-46. 8. Andersen B R;HVDC Transmission-Opportunities
and Challenges[C].The 8#IEEE International
Conference on AC and DC Power Transmission,28-
31March 2006,24-29. 5. Wen-Long Ming,Qing-Chang Zhong;A Single-Phase
Rectifier Having Two Independent Voltage Outputs
With Reduced Fundamental Frequency Voltage
Ripples[J].IEEE
Transactions
on
Power
Electronics,2015,30(11):3662-3673. 4 4
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|
English
| null |
Impact of a Variable Speed Limit System on Driver Speed Choice and Crash Characteristics During Low Visibility Conditions
| null | 2,017
|
cc-by
| 26,643
|
Dean, School of Engineering and Applied Sciences This thesis is submitted in partial fulfillment of the
Requirements for the degree of
Master of Science (Civil Engineering) Daniela E. Gonzales Hidalgo This thesis has been read and approved by the Examining Committee: This thesis has been read and approved by the Examining Committee: Dr. Michael D. Fontaine
Thesis Advisor Dr. Michael D. Fontaine
Thesis Advisor Dr. Michael D. Fontaine Thesis Advisor Dr. Brian L. Smith Dr. Brian L. Smith Dr. T. Donna Chen Dr. T. Donna Chen Accepted for the School of Engineering and Applied Sciences: Presented to ii APPROVAL SHEET Accepted for the School of Engineering and Applied Sciences: Dean, School of Engineering and Applied Sciences iii ACKNOWLEDGEMENTS It has been profound honor and privilege to have been able to pursue my graduate
studies at the University of Virginia. I have been blessed to have met many wonderful people
who have supported, encouraged, and pushed me beyond what I thought I was capable of
achieving. First and foremost, I would like express my utmost appreciation and respect for my
advisor, Dr. Michael D. Fontaine. I was fortunate enough to be placed with a most excellent
advisor, mentor, and professor who not only instructed me in courses, guided me along the
entirety of this thesis, and spent countless hours helping me refine my work but was and is
simply an exemplary model of the kind of professional I hope one day to be. I am very grateful for the excellent professors and faculty that have instructed me,
particularly Dr. T. Donna Chen and Dr. Brian L. Smith for serving in my committee and providing
support and advice. I am especially thankful for Simona Babiceanu, Dr. Emily Parkany, Professor
Jose Gomez, and Dr. Brian L. Smith, all who I met as an undergrad and were influential in my
decision to not only continue my studies but continue them at this wonderful University. I would also like to thank Katie McCann, Mike McPherson, and Tim Martin from the
Virginia Department of Transportation for their assistance, support, and insights in the process
of this research. Their work and assistance was the foundation of this thesis. Lastly, I would like to thank my family and friends, who encouraged and supported me
the entire process, especially my parents who made innumerable sacrifices for me to pursue my
education. Support and encouragement came from many different forms yet in the end this
achievement has been made possible by grace alone, to God be all the glory. iv ABSTRACT Low visibility conditions can inhibit a driver’s ability to perceive appropriate operating speeds,
particularly during foggy conditions where the characteristics of the fog can vary
spatiotemporally. By reducing visibility and contrast in the visual field, fog obscures crucial
driving cues essential for perceiving depth and speed. Studies have shown that fog-related
crashes tend to involve more vehicles and more severe injuries. Numerous agencies have
installed countermeasures like weather advisory systems and variable speed limits (VSLs) to
mitigate these conditions, but not many studies have quantitatively analyzed the results of
these projects. In October 2016, the Virginia Department of Transportation (VDOT) activated a
VSL system on a 12-mile section of Interstate 77 that runs through mountainous terrain in
southwestern Virginia known to experience severe, recurring fog events with the objective of to
reducing the quantity and severity of crashes in the corridor. This thesis assesses how the I-77
fog VSL system in Fancy Gap, Virginia affected driver speed choice and crash characteristics
since its activation in October 2016. Prior to the installation of the VSL, drivers frequently drove
much faster than the safe speed based on the stopping sight distance during fog. The VSL
system sought to get drivers to travel closer to the safe speed based on available visibility by
posting appropriate reduced speed limits. The analysis examines the effect of the VSL system on driver speeds both before and
after at a single site and across the corridor in the after period. Effects on crashes for the entire
corridor are also examined. The results showed statistically significant reductions in mean
speeds and variances after the VSL was activated, and drivers drove closer to the safe speed
based on available visibility. Models developed to understand how the VSL system affected v v speed as a function of visibility showed that speeds are reduced by a statistically significant
amount when VSLs are active. Trends in speed by posted speed limit were examined across the
corridor, and it was found that compliance generally improved once drivers encountered
reduced visibilities. Speeds did not change as much in transition areas leading into the area
where the fog was present, however. Crash analysis revealed only two fog-related crashes in
the after period, yielding reduced crash rates during low visibility conditions and indicating
improved safety. ABSTRACT The results of this VSL implementation may be used to further refine current
VSL control algorithm to improve compliance even further and could serve as a reference for
other agencies contemplating alternatives to improve safety at fog-prone areas given the
indications that the countermeasure did have a positive effect. speed as a function of visibility showed that speeds are reduced by a statistically significant
amount when VSLs are active. Trends in speed by posted speed limit were examined across the
corridor, and it was found that compliance generally improved once drivers encountered
reduced visibilities. Speeds did not change as much in transition areas leading into the area
where the fog was present, however. Crash analysis revealed only two fog-related crashes in
the after period, yielding reduced crash rates during low visibility conditions and indicating
improved safety. The results of this VSL implementation may be used to further refine current
VSL control algorithm to improve compliance even further and could serve as a reference for
other agencies contemplating alternatives to improve safety at fog-prone areas given the
indications that the countermeasure did have a positive effect. vi vi TABLE OF CONTENTS ACKNOWLEDGEMENTS .................................................................................................................... III
ABSTRACT ........................................................................................................................................ IV
TABLE OF CONTENTS ........................................................................................................................ VI
LIST OF TABLES .............................................................................................................................. VIII
LIST OF FIGURES ................................................................................................................................ X
CHAPTER 1:
INTRODUCTION ...........................................................................................................1
1.1
PREVIOUS STUDIES OF I-77............................................................................................................ 2
1.2
PURPOSE AND SCOPE .................................................................................................................... 4
1.3
ORGANIZATION OF THESIS ............................................................................................................ 5
CHAPTER 2:
LITERATURE REVIEW ....................................................................................................6
2.1
INTRODUCTION ............................................................................................................................. 6
2.2
DRIVER BEHAVIOR DURING REDUCED VISIBILITY .......................................................................... 6
2.3
FIELD DEPLOYMENTS OF COUNTERMEASURES TO ADDRESS LIMITED VISIBILITY ......................... 8
2.3.1
ADVISORY VISIBILITY WARNING SYSTEMS ............................................................................ 9
2.3.2
SPEED ADVISORY VISIBILITY WARNING SYSTEM ................................................................. 13
2.3.3
PAST VISIBILITY BASED VSL DEPLOYMENTS ........................................................................ 17
2.4
GAPS IN RESEARCH ...................................................................................................................... 22
CHAPTER 3:
SITE DESCRIPTION AND CHARACTERISTICS .................................................................. 26
3.1
SITE DESCRIPTION ....................................................................................................................... 26
3.2
PREVIOUS WORK ......................................................................................................................... 27
3.2.1
Fog Characteristics .............................................................................................................. 28
3.2.2
Crash Analysis ..................................................................................................................... 29
3.2.3
Driver Speeds ....................................................................................................................... 30
3.3
SYSTEM DESCRIPTION ................................................................................................................. 32
3.4
VSL ALGORITHM .......................................................................................................................... 36
CHAPTER 4:
METHODOLOGY ......................................................................................................... 39
4.1
INTRODUCTION ........................................................................................................................... 39
4.2
OVERVIEW OF DATA USED IN ANALYSIS ...................................................................................... 39
4.2.1
Visibility and Weather Data ................................................................................................ 40
4.2.2
Speed and Volume Data ...................................................................................................... 42
4.2.3
VSL Posted Speed Logs ........................................................................................................ 43
4.2.4
Crash Data ........................................................................................................................... 44
4.2.5
Data Matching .................................................................................................................... 44
4.3
DATA ANALYSIS ........................................................................................................................... 45
4.3.1
Visibility ............................................................................................................................... 45
4.3.2
VSL Posted ........................................................................................................................... 46
4.3.3
Speed Choice Analysis ......................................................................................................... 46
4.3.4
Crash Analysis ..................................................................................................................... 49 ACKNOWLEDGEMENTS .................................................................................................................... III
ABSTRACT ........................................................................................................................................ IV
TABLE OF CONTENTS ........................................................................................................................ VI
LIST OF TABLES .............................................................................................................................. VIII
LIST OF FIGURES ................................................................................................................................ X
CHAPTER 1:
INTRODUCTION ...........................................................................................................1
1.1
PREVIOUS STUDIES OF I-77............................................................................................................ 2
1.2
PURPOSE AND SCOPE .................................................................................................................... 4
1.3
ORGANIZATION OF THESIS ............................................................................................................ 5
CHAPTER 2:
LITERATURE REVIEW ....................................................................................................6
2.1
INTRODUCTION ............................................................................................................................. 6
2.2
DRIVER BEHAVIOR DURING REDUCED VISIBILITY .......................................................................... 6
2.3
FIELD DEPLOYMENTS OF COUNTERMEASURES TO ADDRESS LIMITED VISIBILITY ......................... 8
2.3.1
ADVISORY VISIBILITY WARNING SYSTEMS ............................................................................ 9
2.3.2
SPEED ADVISORY VISIBILITY WARNING SYSTEM ................................................................. 13
2.3.3
PAST VISIBILITY BASED VSL DEPLOYMENTS ........................................................................ 17
2.4
GAPS IN RESEARCH ...................................................................................................................... 22
CHAPTER 3:
SITE DESCRIPTION AND CHARACTERISTICS .................................................................. 26
3.1
SITE DESCRIPTION ....................................................................................................................... 26
3.2
PREVIOUS WORK ......................................................................................................................... 27
3.2.1
Fog Characteristics .............................................................................................................. 28
3.2.2
Crash Analysis ..................................................................................................................... 29
3.2.3
Driver Speeds ....................................................................................................................... 30
3.3
SYSTEM DESCRIPTION ................................................................................................................. 32
3.4
VSL ALGORITHM .......................................................................................................................... 36
CHAPTER 4:
METHODOLOGY ......................................................................................................... 39
4.1
INTRODUCTION ........................................................................................................................... 39
4.2
OVERVIEW OF DATA USED IN ANALYSIS ...................................................................................... 39
4.2.1
Visibility and Weather Data ................................................................................................ 40
4.2.2
Speed and Volume Data ...................................................................................................... 42
4.2.3
VSL Posted Speed Logs ........................................................................................................ TABLE OF CONTENTS 43
4.2.4
Crash Data ........................................................................................................................... 44
4.2.5
Data Matching .................................................................................................................... 44
4.3
DATA ANALYSIS ........................................................................................................................... 45
4.3.1
Visibility ............................................................................................................................... 45
4.3.2
VSL Posted ........................................................................................................................... 46
4.3.3
Speed Choice Analysis ......................................................................................................... 46
4.3.4
Crash Analysis ..................................................................................................................... 49 vii 4.3.5
VSL Algorithm Assessment .................................................................................................. 50
CHAPTER 5:
RESULTS .................................................................................................................... 51
5.1
INTRODUCTION ........................................................................................................................... 51
5.2
OVERALL RESULTS ....................................................................................................................... 51
5.2.1
Visibility ............................................................................................................................... 51
5.2.2
Summary of VSLs Posted ..................................................................................................... 52
5.2.3
Case Study: Incident WX3150436 ....................................................................................... 53
5.2.4
Speed Choice ....................................................................................................................... 60
5.2.5
Crash Analysis..................................................................................................................... 68
5.3
SUMMARY OF RESULTS ............................................................................................................... 75
5.4
VSL ALGORITHM ASSESSMENT .................................................................................................... 76
CHAPTER 6:
CONCLUSIONS AND RECOMMENDATIONS .................................................................. 79
6.1
CONCLUSIONS AND DISCUSSION ................................................................................................ 79
6.1.1
Driver Speed Choice ............................................................................................................. 79
6.1.2
Crash Analysis ..................................................................................................................... 80
6.2
RECOMMENDATIONS.................................................................................................................. 81
6.3
FUTURE RESEARCH ...................................................................................................................... 82
REFERENCES .................................................................................................................................... 84
APPENDICES .................................................................................................................................... 86
APPENDIX A: PUBLICATIONS & PRESENTATIONS ............................................................................... 86
APPENDIX B: EXCERPTS FROM MCCANN & FONTAINE ....................................................................... 87
APPENDIX C: ADDITIONAL TABLES .................................................................................................... 89 viii LIST OF TABLES Table 1 CAWS CMS Messages ..................................................................................................................... 10
Table 2 CAWS Evaluation CMS Messages ................................................................................................... 11
Table 3 FDWS Messages ............................................................................................................................. 14
Table 4 Georgia DMS Messages .................................................................................................................. 15
Table 5 South Carolina DMS Messages ....................................................................................................... 16
Table 6 Utah DMS Messages....................................................................................................................... 17
Table 7 Alabama DMS and VSL Messages................................................................................................... 18
Table 8 Oregon a) Grip Factor and b) Chain Conditions Lookup Tables for Weather Subsystem (23) ....... 19
Table 9 Oregon DMS Message Lookup Table (23) ...................................................................................... 20
Table 10 Tennessee DMS and VSL Messages .............................................................................................. 21
Table 11 Summary of Visibility Warning Systems ....................................................................................... 24
Table 12 Safe Speed by Visibility Bin........................................................................................................... 28
Table 13 I-77 DMS Messages ...................................................................................................................... 35
Table 14 Summary of Data Used in this Thesis ........................................................................................... 40
Table 15 Mean Speed Before-After Comparison at MP 4.4 ....................................................................... 61
Table 16 Before-After Model Parameters .................................................................................................. 63
Table 17 Observed mean speeds by posted speed limit ............................................................................ 66
Table 18 Count of observations by posted speed limit .............................................................................. 67
Table 19 Difference from Posted Speeds by Posted Speed ........................................................................ 67
Table 20 Standard Deviations by posted Speed Limit ................................................................................ 68
Table 21 Crash Severity by Visibility Bin, updated to with 2015, 2010-2015 ............................................. 70
Table 22 Crash Severity by Visibility Bin, October 2016-August 2017 ........................................................ 70
Table 23 Number of Vehicles Involved by Visibility Bin, updated with 2015, 2010-2015 .......................... 70
Table 24 Number of Vehicles Involved by Visibility Bin, October 2016- August 2017 ............................... 71
Table 25 Crash Type by Visibility Bin, updated to include 2015, 2010-2015 .............................................. 71
Table 26 Crash Type by Visibility Bin, October 2016- August 2017 ............................................................ 71
Table 27 Updated 2010-2015 Crash Rates .................................................................................................. 74 ix Table 28 Crash Rates October 2016- August 2017 ..................................................................................... 74
Table 29 Crash Severity by Visibility Bin, 2015 ........................................................................................... 89
Table 30 Number of Vehicles Involved by Visibility Bin, 2015 .................................................................... 90
Table 31 Crash Type by Visibility Bin, 2015 ................................................................................................. 90
Table 32 2015 Crash Rates .......................................................................................................................... 90 x LIST OF FIGURES Figure 1 Diagram of CAWS System at one CMS location (12) ..................................................................... 11
Figure 2 Idaho Storm Warning System Device Layout (15) ........................................................................ 12
Figure 3 Georgia Speed Advisory Nomograph (Robinson, 2000) ............................................................... 15
Figure 4 Map of Corridor Location (to be updated).................................................................................... 27
Figure 5 Average Annual Visibility Profile, 2010-2015 (6) .......................................................................... 29
Figure 6 Corridor Diagram (28) ................................................................................................................... 33
Figure 7 VSL System Devices. a) Full Matrix DMS VSL Display, b) Speed Limit Signs with dynamic VSL
cutout, c) RWIS Station, and d) traffic sensor, CCTV, and signing at the northern end of the corridor. ............................................................................................................................................................ 34
Figure 8 VSL Algorithm Step Function ......................................................................................................... 38
Figure 9 After Period Visibility Profile ......................................................................................................... 52
Figure 10 VSL Usage by MP ......................................................................................................................... 53
Figure 11 Case Study Visibility Profile ......................................................................................................... 54
Figure 12 Case Study SB VSL Usage ............................................................................................................ 55
Figure 13 SB Speeds Over Time .................................................................................................................. 59
Figure 14 Observed mean speeds a) before and b) after VSL activation at MP 4.4. c) After Speeds Model
with VSL algorithm model for reference. ............................................................................................ 64
Figure 15 Proposed Modification to VSL..................................................................................................... 78
Figure 16 I-77 2015 Visibility Profile ........................................................................................................... 89 1 CHAPTER 1:
INTRODUCTION Driving in fog can pose additional challenges to the driving task. Fog reduces visibility
and contrast in the visual field, which are helpful for perceiving depth and speed (1). When
these crucial driving cues are obscured, a driver’s ability to judge appropriate operating speeds
may be hindered. Some studies indicate that motorists seem to compensate for these losses by
changing following distances to ensure that the taillights of a lead vehicle remain visible (1). Driving in fog can pose additional challenges to the driving task. Fog reduces visibility
and contrast in the visual field, which are helpful for perceiving depth and speed (1). When
these crucial driving cues are obscured, a driver’s ability to judge appropriate operating speeds
may be hindered. Some studies indicate that motorists seem to compensate for these losses by
changing following distances to ensure that the taillights of a lead vehicle remain visible (1). Drivers do not tend to reduce speed when driving in fog until they feel their lane keeping ability
is compromised, thus they often maintain operating speeds too great for the close following
distances and limited visibilities present under fog conditions (1). These driving behaviors in fog
conditions can increase the potential for crashes of greater severity and involving more vehicles
(2). To mitigate safety concerns, agencies sometimes install countermeasures like weather
advisory systems and variable speeds limits (VSLs) in areas where fog events are common. Weather advisory systems that include dynamic message signs (DMSs) to relay weather
information, speed advisories, and VSLs have been installed in several US states and in other
countries. However, not many quantitative evaluations of these systems have been performed. A 12-mile section of Interstate 77 that runs through mountainous terrain in
southwestern Virginia has been known to experience severe, recurring fog events. In the past
20 years, several major fog-related multi-vehicle chain reaction crashes have occurred on this
corridor. On Valentine’s Day 1997 when visibilities had dropped due to fog, a chain reaction
crash involved 56 vehicles, incurring 12 injuries (3). Another fog-related series of crashes in
September 2005 involved 50 vehicles, causing 25 injuries (4). On November 16, 2010, visibilities 2 were under 100 ft. when 70+ vehicles were involved in 10 separate crashes which resulted in 2
fatalities and 16 injuries and closed the highway for nearly 10 hours (5). One of the most severe
fog events to date happened on Easter Sunday 2013. CHAPTER 1:
INTRODUCTION When fog had limited visibilities to 167 ft. at the worst locations, a chain of 17 crashes involving 96 vehicles resulted in 3 fatalities and 25
injuries, and took almost 11 hours to clear (4). In 2002, a report on reducing fog-related crashes on I-77 suggested seeking
authorization for experimental use of VSLs (3). Other less expensive countermeasures were
employed over the years including rumble strips, delineator signs, wider pavement markings,
chevrons, and other enhanced signs (4). In 2014, the Virginia Department of Transportation
(VDOT) awarded a $7.5 million contract to build an Active Traffic and Safety Management
System (ATSMS) along 12 miles of I-77 in Fancy Gap. The system was activated in October 2016
and has experienced a full year of operations since. Now that VDOT has gained experience with
the system, there is a need to quantify its effect on traffic and safety. 1.1 PREVIOUS STUDIES OF I-77 1.1 PREVIOUS STUDIES OF I-77 1.1 PREVIOUS STUDIES OF I-77 Several safety studies have been conducted on this section of I-77 between 1995 and
2015. These studies assessed the relative safety of the corridor by quantifying the traffic
incident frequency and severity of fog-related incidents. The 2002 Crash Analysis Study
observed data from 1995-1998; the 2007 Crash Analysis and Speed Study analyzed the years
2001-2005; the 2012 Safety Analysis Update and Verification studied the years 2006-2010; and
McCann’s 2016 study examined the years 2010-2015. The later studies verified/expanded upon
the trends first identified in the 2002 study (5) (6). 3 The 2002 study found that 14 out of 139 total incidents between the mileposts 2 and 9
in a four-year period were attributable to fog, accounting for 10% of total crashes. However,
these crashes accounted for nearly 44% of all vehicles involved in crashes, averaging nearly
11.21 vehicles per crash event and 2.64 injuries per event (3). The 2007 and 2012 studies had a
broader scope and analyzed the corridor from milepost 0 to 32.5. The 2007 study found a total
of 1009 individual crashes involving 1611 vehicles, and the 2012 study identified 1118 individual
crashes involving 1718 vehicles (5). The proportion and frequency of fog-related crashes
decreased from 68 crashes (6.7% of total crashes) between 2001-2005 to 52 crashes (4.7% of
total crashes) between 2006-2010. These reductions may be due to some of the enhanced
warning and lane departure counter measures implemented following the 2002 study, or
possibly due to the fact that traffic and fog exposure was not taken into account. The most recent study in 2016, examined crash characteristics considering exposure,
and also examined driver speed choice under foggy conditions to reflect further safety
surrogate measures and aid in the development of the VSL algorithm (6). Crash analysis of
police crash reports between milepost 0 and 15 showed 524 total crashes, 58 of which occurred
under low visibility conditions. An overwhelming 84% of the fog-related crashes occurred in the
southbound direction. Five of these crashes resulted in fatalities and 23 in injuries. Although
fog-related crashes only accounted for 11% of total crashes, they accounted for 19% of fatal
and injury crashes. More than 90% of fog-related crashes involved 2 or more vehicles while this
proportion was only 47% during clear conditions. Crash rates during fog were calculated to be
about 580 crashes per 100 million vehicle miles traveled, nearly 8.5 times greater than in clear
conditions. 1.1 PREVIOUS STUDIES OF I-77 4 Another important outcome of the 2016 study reaffirmed the notion that the area
between mile posts 2 and 9 most commonly experienced severe fog events. Visibility profiles
confirmed that fog varied spatiotemporally and was concentrated most heavily near milepost
5.3 and 6.6. Speed analysis revealed that drivers traveled much faster than the stopping sight
distance (SSD) safe speed based on available visibilities. Although speed reductions were
observed during dense fog, at some of the worst visibilities mean speeds were still 25 mph or
more higher than SSD safe speeds. Increasing standard deviations of speed were also observed
as visibilities worsened. This study also developed a model of driver speeds that explained the variation of speed
due to visibility, day/night conditions, and mile marker along corridor. This model has
subsequently been used as a base in the development of the control algorithm for the I-77 VSL
system. Now that the VSL system has been activated, data from October 2016 to September
2017 is available to assess how the system has impacted driver speeds and crash
characteristics. 1.2 PURPOSE AND SCOPE 1.2 PURPOSE AND SCOPE The two primary goals of the I-77 VSL system are to reduce the quantity and severity of
crashes in the corridor. The I-77 ATSMS Concept of Operations proposed reductions in total,
fatal, property damage, and injury crashes as measures of effectiveness along with changes in
speed limit compliance (5). The purpose of this thesis is to evaluate the effectiveness of the I-77 VSL system during
its first year of operations. The specific objectives of this paper are to: 5 1. Determine the effect of the VSLs on driver speeds and compliance throughout the
corridor, 1. Determine the effect of the VSLs on driver speeds and compliance throughout the
corridor, 2. Determine changes in crash characteristics following VSL activation The work detailed in this paper uses data collected from crash reports and weather and
traffic stations across the corridor. This scope of this paper is limited: 1. Spatially to the I-77 corridor from mile post zero to mile post 12 and 2. Temporally to the first year after VSL system activation. 2. Temporally to the first year after VSL system activation. This work builds on previous safety evaluations of the site to assess if system has had
the desired effect. While prior work by McCann (6) established safety and driver behavior
trends before VSL activation, this thesis focuses on assessing whether the system created
positive changes in safety on the corridor. 1.3 ORGANIZATION OF THESIS The rest of this thesis is organized as follows: The rest of this thesis is organized as follows: The rest of this thesis is organized as follows: Chapter 2: Reviews the literature on driver behavior in fog/low visibility conditions and
past visibility based warning systems and VSL deployments. Chapter 3: Provides an overview of site, system, and previous work on the I-77 VSL
system. Chapter 4: Discusses the methodology for the evaluation of the I-77 VSL system
Ch
t
5 P
t th
lt
f th
l
i Chapter 4: Discusses the methodology for the evaluation of the I-77 VSL system
Chapter 5: Presents the results of the analysis Chapter 5: Presents the results of the analysis Chapter 6: Discusses the conclusions and recommendations of this research 6 2.1 INTRODUCTION 2.1 INTRODUCTION A literature review was done to understand driver response to fog/low visibility
conditions and countermeasures to improve safety in such conditions. This chapter examines
past visibility based warning systems and VSL deployments to discuss results and gaps in
research. 2.2 DRIVER BEHAVIOR DURING REDUCED VISIBILITY A driver’s ability to determine appropriate operating speeds relies greatly on the driver’s
visual perception. Reductions in visibility can impair drivers’ judgment and negatively impact
safety. Early research in driving in fog had focused on identifying perceptual changes that
influenced speed, often modeling fog as a uniform reduction in contrast. These studies have
considered both simulated and test-track data to understand the effects of visibility on driver
behavior and safety. In a virtual-environment driving simulation, Snowden et al (7) found that drivers’ sense
of speed decreases in fog, as drivers tend to drive faster as fog becomes more dense. Simulating fog by blending a partially transparent polygon over each pixel, RGB values where
recalculated at a 15 Hz rate decreasing the contrast at three separate levels to represent
“clear”, “misty”, and “foggy” conditions. First, the test subjects were shown two scenes that
moved at the same speed, one with “clear” conditions, another with “clear”, “misty”, or “foggy”
conditions. Foggier scenes were perceived as slower moving. In the second part of the
experiment, drivers were asked to match a certain operating speed in the different simulated 7 7 conditions. In foggier scenes, subjects drove at faster speeds, causing the study to conclude
that perceived speed depends on level of contrast, with lower contrast yielding higher speeds. However, these results consider fog as a contrast reduction evenly dispersed across the whole
visual field. A couple of years later, a similar study using a test track concluded that drivers
overestimate their actual speeds in foggy conditions (8). This study also considered fog as a
uniform contrast reduction, simulating it with plastic filters covering the windshields and
windows. When drivers in this study were asked to match a given speed, drivers’ speeds were
consistently lower than the speeds they were asked to match. While these studies had
conflicting results, neither Snowden, et al. nor Brooks, et al. addressed driving behavior which
would help to explain how drivers would actually perform in real world conditions. 2.1 INTRODUCTION A more recent study by Brooks et al (8) used driver simulator data that more accurately
coded fog as a distance dependent contrast reduction to give insight into driver behavior under
reduced visibility. The study measured the ability of participants to stay in their lane and
maintain speed. In the study, participants were assigned into one of six groups classified by a
combination of a factors including: presence or absence of auditory speed indicators, ability to
maintain speed task priorities, and speedometer availability. Participants were first given
practice sessions to get acquainted with driving in the simulator before running through six fog
scenarios. Results showed that throughout each of the groups, drivers did not appear to
significantly decrease speed as visibility decreased. In fact, results suggested that as long as
drivers were able to maintain vehicular control they would maintain high speeds while driving
in fog. The finding of the Brooks et al study, however, are limited due to small sample sizes 8 within the groups. Since the sample consisted only of college students, the results may also not
accurately represent the overall population of drivers on road. within the groups. Since the sample consisted only of college students, the results may also not
accurately represent the overall population of drivers on road. Case studies of actual traffic data have also been performed to have a better
understanding of real world driving in low visibility conditions. In a case study of the effects of
visibility and other environmental factors on driver speed by Liang et al (9), researchers studied
the speed-visibility relationship on a rural Idaho freeway prior to the installation of a storm
warning system. The data used in this study was collected from an operational test of weather
and visibility sensing systems at a spot site between December 1995 to April 1996, during which
21 days experienced extreme weather conditions. In this period where no external information
or warning signs were shown to the drivers, mean speed reductions of 8.0 km/hr. during fog
events were observed, however this was accompanied by a doubling in the variation in speeds. In this study, it was unable to be determined if trends in speed reductions were sufficient to
ensure adequate sight distances given no periods of visibility were below 528 feet. 2.1 INTRODUCTION 2.3 FIELD DEPLOYMENTS OF COUNTERMEASURES TO ADDRESS LIMITED VISIBILITY Transportation agencies have deployed countermeasures to mitigate safety concerns of fog
and low visibility conditions. Road weather management countermeasures have ranged from
simply adding enhanced signage to larger scale intelligent transportation system deployments. In this section, field deployments of three different levels of advisory and control strategies
are presented. The individual field deployment discussions include site and system descriptions
along with implementation results. The first set of field deployments presented are advisory
visibility warning systems which relay weather information and display advisory messages but
offer no control strategy. The second set of deployments discussed, speed advisory visibility Transportation agencies have deployed countermeasures to mitigate safety concerns of fog
and low visibility conditions. Road weather management countermeasures have ranged from
simply adding enhanced signage to larger scale intelligent transportation system deployments. In this section, field deployments of three different levels of advisory and control strategies
are presented. The individual field deployment discussions include site and system descriptions
along with implementation results. The first set of field deployments presented are advisory
visibility warning systems which relay weather information and display advisory messages but
offer no control strategy. The second set of deployments discussed, speed advisory visibility 9 warning systems, are like the first but do provide speed advisories as part of the warning
messages. The last set of deployments discussed are visibility based VSL deployments that set
both advisory and regulatory speed limits in response to low visibility conditions. 2.3.1 ADVISORY VISIBILITY WARNING SYSTEMS 2.3.1 ADVISORY VISIBILITY WARNING SYSTEMS This section details visibility based warning system countermeasures and their effects
on driver behavior and safety, if any. 2.3.1.1
California Automated Warning System 2.3.1.1
California Automated Warning System The California Department of Transportation (Caltrans) activated the Caltrans
Automated Warning System (CAWS) on southbound Interstate 5 and westbound State Route
120 in 1996 (10). In this corridor, visibility reductions due to wind-blown dust during the
summer and stationary, dense fog during the winter seasons pose safety concerns, as many
chain-reaction crashes have been experienced in this region (11). The system was deployed to
improve safety along the corridor and warn drivers of potential driving hazards. CAWS consists
of three primary subsystems: weather monitoring stations, traffic monitoring stations, and
changeable message signs (CMSs). Nine weather monitoring stations were deployed that
measured atmospheric conditions and visibility with a dual axis atmospheric visibility sensor. Thirty-six loop-pairs spaced approximately at half mile intervals were installed to collect traffic
count and speed data at 15-minute intervals. Nine CMS locations corresponding with weather
station locations were also installed. Thirty-six loop-pairs spaced approximately at half mile intervals were installed to collect traffic
count and speed data at 15-minute intervals. Nine CMS locations corresponding with weather
station locations were also installed. The weather station and loop detector data fed into the CMS control computer which
automatically selected warning messages to display based on traffic speed, visibility, and wind
speed. Operators can also override the system and enter messages manually. Table 1lists the 10 automatic messages that were initially used for given observed conditions. When visibilities fall
below 200 ft., the system is supplemented with vehicle guidance operations by California
Highway Patrol (CHP), which uses patrol cars to put traffic intro platoons and lead traffic at a
safe pace, typically 50 mph (11). Table 1 CAWS CMS Messages Condition
Message Displayed
Speeds 11-25 mph
“Slow Traffic Ahead”
Speeds < 11 mph
“Stopped Traffic Ahead”
Visibility 200-500 ft. “Foggy Conditions Ahead”
Visibility <200 ft. “Dense Fog Ahead”
Wind speed >35 mph
“High Wind Warning” Sometime between initial activation in 1996 and 2003, the automatic messages were
changed to those seen in Table 2 (12). A study in 2006 assessed the influence of CAWS on driver
behavior using data from traffic monitoring stations upstream and downstream of the first CMS
shown in Figure 1. Data from the 2003-2004 and 2004-2005 fog seasons showed an additional
1.1 mph speed reduction in fog conditions after encountering the CMS. Figure 1 Diagram of CAWS System at one CMS location (12) 2.3.1.1
California Automated Warning System This study also looked
at differences in potential collision speed before and after encountering the CMS and found
that these speeds actually increased by 8 mph. Crash analysis compared crash data from 1992-
1996 to 1997-2003 in 3 study areas against 5 control areas (13). Collisions during fog were
normalized to the number of collisions per 100 heavy fog days in either the before or after
period. That analysis found that for 2 of the 3 study sites reductions in rates were seen, but
one site had a crash rate that increased by 242%. Further analysis into secondary fog related
crashes found that all study areas encountered an increase in fog related crashes with 6 related
crashes in the before period and 13 in the after period. However, due to small number of Sometime between initial activation in 1996 and 2003, the automatic messages were 11 crashes and the random nature of crashes, the safety effectiveness of this system is not
definitive Table 2 CAWS Evaluation CMS Messages Visibility
2003-2004 Fog Season Message
Displayed
2004-2005 Fog Season Message
Displayed
200-500 ft. “Dense Fog Ahead, Advise 45 mph”
“Dense Fog Ahead, Advise 45
mph”
100-200 ft. “Dense Fog Ahead, Advise 30 mph”
<100 ft. No message displayed
Figure 1 Diagram of CAWS System at one CMS location (12) Figure 1 Diagram of CAWS System at one CMS location (12) Figure 1 Diagram of CAWS System at one CMS location (12) Figure 1 Diagram of CAWS System at one CMS location (12) 12 2.3.1.2
Idaho Storm Warning System 2.3.1.2
Idaho Storm Warning System In 1993, the Idaho Department of Transportation deployed the $1.2 million Idaho Storm
Warning System along a 100-mile section of Interstate 84 in response to the numerous multi-
vehicle crashes this corridor had seen in the previous 5 years (14). The system consisted of
environmental sensor stations to detect pavement conditions and collect weather data,
visibility sensors that measured visibility distance with forward-scatter detection sensors, loop
detectors for traffic data, and DMSs to display advisories. These sensors communicated sensor
data with a central computer in five minute intervals, and the computer alerted traffic
managers when visibility conditions have fallen below a 0.23-mile threshold. This allowed TMC
staff to decide on messages and manually activate DMSs. The sensor locations are shown in
Figure 2 (15). In 1993, the Idaho Department of Transportation deployed the $1.2 million Idaho Storm
Warning System along a 100-mile section of Interstate 84 in response to the numerous multi-
vehicle crashes this corridor had seen in the previous 5 years (14). The system consisted of
environmental sensor stations to detect pavement conditions and collect weather data,
visibility sensors that measured visibility distance with forward-scatter detection sensors, loop
detectors for traffic data, and DMSs to display advisories. These sensors communicated sensor
data with a central computer in five minute intervals, and the computer alerted traffic managers when visibility conditions have fallen below a 0.23-mile threshold. This allowed TMC
staff to decide on messages and manually activate DMSs. The sensor locations are shown in
Figure 2 (15). Figure 2 Idaho Storm Warning System Device Layout (15) Figure 2 Idaho Storm Warning System Device Layout (15) Figure 2 Idaho Storm Warning System Device Layout (15) 13 An evaluation of the system over the course of the first 8 years of deployment
considered the influence of the DMS messages on driver behavior by comparing traffic speeds
with and without the presence of advisories (11). An initial study in this corridor was performed
by looking at data from December 1995 to April 1996 to understand the effects of
environmental conditions on driver speeds (9). However, speed limits were increased in April
1996, rendering the results of the earlier study unsuitable for comparison (15). 2.3.1.2
Idaho Storm Warning System Since the system
relied on the judgement of the operators to choose both the content and when the messages
would be the displayed on the DMSs, there were 19 occasions in the evaluation period were
visibilities were greater than 0.23 miles and other adverse weather conditions were present
that operators did not utilize DMSs. Driving behaviors during these events were considered
baseline conditions. The report shows no indication as to the content of the messages, but
results showed that during high winds, high winds and moderate to heavy precipitation, and
snow/high wind events when DMSs were utilized to display weather conditions, speed
reductions of 23%, 12%, and 35% were seen during these types of events, respectively. During
poor visibility only periods, there was insufficient data to see if the display of warning messages
contributed to speed reductions. An evaluation of the system over the course of the first 8 years of deployment
considered the influence of the DMS messages on driver behavior by comparing traffic speeds
with and without the presence of advisories (11). An initial study in this corridor was performed
by looking at data from December 1995 to April 1996 to understand the effects of 2.3.2 SPEED ADVISORY VISIBILITY WARNING SYSTEM 2.3.2 SPEED ADVISORY VISIBILITY WARNING SYSTEM This section reviews visibility based speed advisory systems and their effects on driver
behavior and safety, if any. 2.3.2.1
California Fog Detection and Warning System (FWDS) 2.3.2.1
California Fog Detection and Warning System (FWDS) The FDWS was installed along a 13-mi corridor of Highway 99 in 2009 in a region of
California known to experience seasonal fog (16). The system consists of 22 Vaisala PWD-10 14 visibility sensors, 41 traffic sensors, 11 CCTVs, 29 CMSs, 4 Full Color CMSs, 6 portable CMSs, and
2 HAR for a total project cost of $12 million (17). Forty-percent of the field equipment is run
using solar power. Since this corridor runs through a relatively rural area, wireless
communications between field devices and TMC were chosen for this system. Should
communications with the TMC be broken, local controllers were set in place to continue system
operations. The system also included a social media outreach component and eventually would
be integrated into the state 511 system. Traffic sensors including both loops and microwave
vehicle detection sensors were installed every quarter of a mile, while visibility sensors and
CMSs where installed every half mile. This system also offers the option for vehicle guidance
operation by CHP when requested by operators. Table 3 summarizes the message sets that
were deployed as a function of visibility and traffic conditions. No effectiveness results were
available for this system. visibility sensors, 41 traffic sensors, 11 CCTVs, 29 CMSs, 4 Full Color CMSs, 6 portable CMSs, and
2 HAR for a total project cost of $12 million (17). Forty-percent of the field equipment is run
using solar power. Since this corridor runs through a relatively rural area, wireless
communications between field devices and TMC were chosen for this system. Should
communications with the TMC be broken, local controllers were set in place to continue system
operations. The system also included a social media outreach component and eventually would
be integrated into the state 511 system. Traffic sensors including both loops and microwave
vehicle detection sensors were installed every quarter of a mile, while visibility sensors and
CMSs where installed every half mile. This system also offers the option for vehicle guidance
operation by CHP when requested by operators. Table 3 summarizes the message sets that
were deployed as a function of visibility and traffic conditions. No effectiveness results were
available for this system. Visibility
Speed Condition
Warning Message
200-800 ft. >= 45 mph
“Fog Ahead”
0-200 ft. 2.3.2.1
California Fog Detection and Warning System (FWDS) “Dense Fog Ahead”
-
>= 45 mph & CHP pace speed >- 45
“Dense Fog” / “CHP Pace” / “Do Not Pass”
<800 ft. 35-45 mph
“Fog Ahead” / “Traffic Slows to 40 mph”
25-35 mph
“Fog Ahead” / “Traffic Slows to 30 mph”
15-25 mph
“Fog Ahead” / “Traffic Slows to 20 mph”
5-15 mph
“Fog Ahead” / “Traffic Slows to 10 mph”
0-5 mph
“Stopped Traffic Ahead”
2.3.2.2
Georgia Automated Adverse Visibility Warning and Control System 2.3.2.2
Georgia Automated Adverse Visibility Warning and Control System The Georgia Adverse Visibility Warning and Control System was installed along a 14-mi
section of I-75 and became operational in 2001 (18). The system consisted of 19 Vaisala
visibility sensors, 5 CCTVs, 4 DMSs, 5 sets of traffic detectors, and a weather station, with total 15 project cost of $2.4 million (19). The visibility sensors are densely spaced at approximately 1/4
mile apart. Two DMSs are installed in each direction, with the first signs giving advance warning
and second set of signs providing updated speed advisories 1 mile upstream of the fog prone
area. The system receives speed and visibility data at one minute intervals. A processing unit
installed in the middle of the corridor processes visibility and speed information to generate
warning messages. The visibility thresholds were determined using stopping sight distances for
a 2-second perception reaction time, shown visually through a nomograph in Figure 3. Table 4
shows the displayed messages for these thresholds. In 2004, preliminary studies were still
ongoing however, no study of the safety effectiveness of the system was found (20). project cost of $2.4 million (19). The visibility sensors are densely spaced at approximately 1/4
mile apart. Two DMSs are installed in each direction, with the first signs giving advance warning
and second set of signs providing updated speed advisories 1 mile upstream of the fog prone
area. The system receives speed and visibility data at one minute intervals. A processing unit
installed in the middle of the corridor processes visibility and speed information to generate
warning messages. The visibility thresholds were determined using stopping sight distances for
a 2-second perception reaction time, shown visually through a nomograph in Figure 3. Table 4
shows the displayed messages for these thresholds. In 2004, preliminary studies were still
ongoing however, no study of the safety effectiveness of the system was found (20). project cost of $2.4 million (19). 2.3.2.1
California Fog Detection and Warning System (FWDS) The visibility sensors are densely spaced at approximately 1/4
mile apart. Two DMSs are installed in each direction, with the first signs giving advance warning
and second set of signs providing updated speed advisories 1 mile upstream of the fog prone
area. The system receives speed and visibility data at one minute intervals. A processing unit
installed in the middle of the corridor processes visibility and speed information to generate
warning messages. The visibility thresholds were determined using stopping sight distances for
a 2-second perception reaction time, shown visually through a nomograph in Figure 3. Table 4
shows the displayed messages for these thresholds. In 2004, preliminary studies were still
ongoing however, no study of the safety effectiveness of the system was found (20). Table 4 Georgia DMS Messages
Visibility Level
Message Displayed
>1100 ft. No speed advisory
800 – 1100 ft. “CAUTION / FOG AHEAD” with “ADVISE 70 MPH”
500 – 800 ft. “CAUTION / FOG AHEAD” with “ADVISE 55 MPH”
300 – 500 ft. “CAUTION / FOG AHEAD” with “ADVISE 40 MPH”
< 300 ft. “CAUTION / FOG AHEAD” with “ADVISE 25 MPH”
Figure 3 Georgia Speed Advisory Nomograph (Robinson, 2000) Figure 3 Georgia Speed Advisory Nomograph (Robinson, 2000) Figure 3 Georgia Speed Advisory Nomograph (Robinson, 2000) 16 2.3.2.3
South Carolina In South Carolina, a $5 million Low Visibility Warning System was installed in 1992 on a
7-mile section of I-526 (19). It consisted of 1 environmental sensor station, 5 forward scatter
visibility sensors, 8 CCTVs, 8 DMSs, and pavement lights installed at 110 ft. spacing (11). A
central processing unit on site transmits data back to operators in district office. This system
had four levels of visibility conditions for which it would deploy an advisory message or speed
advisory. The CPU predicts/detects low visibility conditions and alerts operators who would
then accept or reject messages to display, as seen in Table 5. From 1992 until 2003, no fog-
related crashes occurred on corridor. No further results were available. Table 5 South Carolina DMS Messages
Visibility
Warning Message
700-900 ft. “Potential For Fog”/ “Light Fog Caution”
“Light Fog Trucks 45 mph”/ “Trucks Keep Right”
450-700 ft. “Fog Caution”/ “Fog Reduce Speed”
“Fog Reduce Speed 45 mph”/ “Trucks Keep Right”
300-450 ft. “Fog Caution”
“Fog Reduce Speed 35 mph”/ “Trucks Keep Right”
>300 ft. “Dense Fog Reduce Speed 25mph”/ “Trucks Keep Right”
“Prepare to Stop”/ “I-526 Bridge Closed Ahead Use I-26/US-17”/ “All Traffic Must
Exit”
2.3.2.4
Utah Adverse Visibility Information System and Evaluation In South Carolina, a $5 million Low Visibility Warning System was installed in 1992 on a
7-mile section of I-526 (19). It consisted of 1 environmental sensor station, 5 forward scatter
visibility sensors, 8 CCTVs, 8 DMSs, and pavement lights installed at 110 ft. spacing (11). A
central processing unit on site transmits data back to operators in district office. This system
had four levels of visibility conditions for which it would deploy an advisory message or speed
advisory. The CPU predicts/detects low visibility conditions and alerts operators who would
then accept or reject messages to display, as seen in Table 5. From 1992 until 2003, no fog-
related crashes occurred on corridor. No further results were available. Table 5 South Carolina DMS Messages
Visibility
Warning Message
700-900 ft. “Potential For Fog”/ “Light Fog Caution”
“Light Fog Trucks 45 mph”/ “Trucks Keep Right”
450-700 ft. “Fog Caution”/ “Fog Reduce Speed”
“Fog Reduce Speed 45 mph”/ “Trucks Keep Right”
300-450 ft. “Fog Caution”
“Fog Reduce Speed 35 mph”/ “Trucks Keep Right”
>300 ft. 2.3.2.3
South Carolina “Dense Fog Reduce Speed 25mph”/ “Trucks Keep Right”
“Prepare to Stop”/ “I-526 Bridge Closed Ahead Use I-26/US-17”/ “All Traffic Must
Exit”
2.3.2.4
Utah Adverse Visibility Information System and Evaluation 2.3.2.4
Utah Adverse Visibility Information System and Evaluation In 1995, Utah DOT’s Adverse Visibility Information System and Evaluation (ADVISE) was
deployed on a two-mile section of I-215 that lies above the Jordan River in Salt Lake City. The
system consisted of 4 forward scatter visibility sensors, 6 vehicle detection sites, and 2 DMSs. The warning system displayed warning messages with stopping sight distance based safe
speeds based on 4 different levels of visibilities as seen in Table 6 (11). Evaluations of the 17 system using data through 2000 showed that average speeds increased and speed variance
decreased from 9.5 to 7.4 mph. During this study period, however, the posted speed during
clear conditions increased from 55 mph to 65 mph, an additional lane was added, and
construction on I-15 disrupted regular traffic patterns (21). These confounding factors make it
difficult to assign these affects to the ADVISE system. Table 6 Utah DMS Messages Table 6 Utah DMS Messages Visibility Level
Message Displayed
>820 ft. No Speed advisory
656-820 ft. “Fog Ahead”
492-656 ft. “Dense Fog”/ “Advise 50 mph”
328-492 ft. “Dense Fog”/ “Advise 40 mph”
197-328 ft. “Dense Fog”/ “Advise 30 mph”
<197 ft. “Dense Fog”/ “Advise 25 mph” 2.3.3 PAST VISIBILITY BASED VSL DEPLOYMENTS 2.3.3 PAST VISIBILITY BASED VSL DEPLOYMENTS This section reviews past visibility based VSL deployments and their effects on driver
behavior and safety, if any. 2.3.3.1
Alabama Low Visibility Warning System Alabama DOT installed a Low Visibility Warning System along a 7-mile bridge on I-10 in
Mobile in 2000 (16). The system consists of 6 forward scatter visibility sensors spaced at
approximately 1-mile intervals along the bridge, 25 CCTVs, 5 DMSs, and 24 VSL signs. Operators
used the CCTVs to observe fog and confirm it with data from sensors. The system has visibility
levels with DMSs advisories and corresponding VSLs for each level. Operators manually choose
which message set to display. Operators can also request vehicle guidance operations by
highway patrol to platoon traffic together and lead at a safe speed for available visibility. Table 18 7 shows the DMS and VSL messages recommended for different visibility ranges. The speed
limits posted by VSLs are regulatory and system automatically alerts the DOT Division Office,
the Highway Patrol, and local law enforcement agencies during system activation (22). Table 7 Alabama DMS and VSL Messages
Visibility
DMS Advisory
VSLs
660-900 ft. “Fog Warning”
65 mph
450-660 ft. “Fog”/ “Slow Use Low Beams”
“Trucks Keep Right”
55 mph
280-450 ft. “Fog”/ “Slow Use Low Beams”
“Trucks Keep Right”
45 mph
175-280 ft. “Dense Fog”/ “Slow Use Low Beams”
“Trucks Keep Right”
35 mph
<175 ft. I-10 Closed, Keep Right, Exit ½ mile
Road Closed by Highway Patrol The system was later upgraded in 2008, to increase the density of radar vehicle
detection devices to every 1/3 of a mile and to use visibility sensors that could detect finer
gradations of fog (19). Since the installation of the system, Alabama DOT has reported reduced
mean speeds but no specific quantitative results are available (16). 2.3.3.2
Oregon Otherwise, speeds would be compared with suggestions from within and
across other subsystems and lowest speed limit would be posted. Additionally, operators can
choose to lower the speed limit further or have associated messages displayed shown in Table Table 8a and b. Visibility and grip factor values are gathered from weather sensors and which
then are used to determine the speed limits based on stopping sight distance safe speeds. 2.3.3.2
Oregon In 2014, Oregon DOT deployed a VSL system on Interstate 5 and US Route 97 to address
both weather and congestion on I-5 (22). The system includes pavement monitoring sensors
and visibility sensors as well as traffic sensors. VSL signs are placed 1/3 to 1/2 mile apart with
DMSs every 1.5 miles. Volume and speed data are collected in 20 second intervals. Three
subsystems, one for weather, another for congestion, and a third for operator control, were
considered but only weather subsystem has been developed so far (23). For the weather subsystem, speed limits are selected based on look up tables using
available visibility and pavement grip factor and chain conditions if applicable, as shown in 19 Table 8a and b. Visibility and grip factor values are gathered from weather sensors and which
then are used to determine the speed limits based on stopping sight distance safe speeds. ODOT’s Advanced Traffic Management System (ATMS) or Traffic Operations Center Software
(TOCS) determine which vehicles require snow chains based of gross vehicle weight, axle count,
and whether they are towing a trailer. If chain conditions are present and vehicles require
chains a second lookup table in the weather subsystem is considered to produce a second
suggested speed limit. The calculated suggested speeds from the lookup table could then be
either be absolute or recommended. If absolute, the speed limits would automatically be
displayed on the VSLs. Otherwise, speeds would be compared with suggestions from within and
across other subsystems and lowest speed limit would be posted. Additionally, operators can
choose to lower the speed limit further or have associated messages displayed shown in Table Table 8a and b. Visibility and grip factor values are gathered from weather sensors and which
then are used to determine the speed limits based on stopping sight distance safe speeds. ODOT’s Advanced Traffic Management System (ATMS) or Traffic Operations Center Software
(TOCS) determine which vehicles require snow chains based of gross vehicle weight, axle count,
and whether they are towing a trailer. If chain conditions are present and vehicles require
chains a second lookup table in the weather subsystem is considered to produce a second
suggested speed limit. The calculated suggested speeds from the lookup table could then be
either be absolute or recommended. If absolute, the speed limits would automatically be
displayed on the VSLs. 9. Currently, VSLs are advisory and no evaluations are yet available. Table 8 Oregon a) Grip Factor and b) Chain Conditions Lookup Tables for Weather Subsystem (23)
(a)
(b) Table 8 Oregon a) Grip Factor and b) Chain Conditions Lookup Tables for Weather Subsystem (23)
(a)
(b) Table 8 Oregon a) Grip Factor and b) Chain Conditions Lookup Tables for Weather Subsystem (23) (b) 20 Table 9 Oregon DMS Message Lookup Table (23) Table 9 Oregon DMS Message Lookup Table (23) Table 9 Oregon DMS Message Lookup Table (23) 2.3.3.3
Tennessee Low Visibility Warning System 2.3.3.3
Tennessee Low Visibility Warning System and data on before conditions was also not available. Table 10 Tennessee DMS and VSL Messages
Visibility
Advisory
VSL
>1320 ft. (fog detected)
“Caution”/ “Fog Ahead Turn on Low Beams”
“Fog”
480-1340 ft. “Fog Ahead”/ “Advisory Radio Tune to XXXX AM” “Reduce
Speed Turn on Low Beams” or “Speed Limit 50 mph”
“Fog”
50 mph
240-480 ft. “Fog Ahead”/ “Advisory Radio Tune to XXXX AM” “Reduce
Speed Turn on Low Beams” or “Speed Limit 35 mph”
35 mph
<240 ft. “Detour Ahead”/ “Reduce Speed Merge Right” or “I-75
Closed”/ “Detour” or “Fog Ahead”/ “Advisory Radio Tune to
XXXX AM”
“Fog”
2.3.3.4
Australia In Australia, 12 VSL signs were installed in 1993 along 11 km of the F6 Tollway (24). Each
of these signs were connected to vehicle detector loops and visibility sensors. Since the main
objective of this system was to reduce rear-end collisions in foggy conditions, the advisory VSL
was based on the visibility distance available and the speed of the preceding vehicle (25). No
evaluation results from this system were found. 2.3.3.3
Tennessee Low Visibility Warning System A Low Visibility Warning System was also installed along 19 miles of I-75 in Tennessee in
1993 for $1.2 million (16). The system included 9 forward scatter visibility sensors, 14
microwave radar vehicle detectors, 21 CCTVS, 6 static warning signs, 10 VSL signs, 10 DMSs, and
2 HAR. These systems relay their data via fiber optic cable to a central computer in the Highway
Patrol office. The system alerts operators of reduced visibilities and operators manually display
the messages based from the advisory messages suggested per visibility conditions as shown in
Table 10. Should conditions necessitate closure, ramps gates are closed and highway patrol
directs a detour. The continued communications with law enforcement suggest that VSLs are
regulatory. The system usually has one activation weekly in the months of October to March
displaying speeds of 50 mph 95% of the time when activated, 13% of these activations required
further reductions in posted speeds to 35 mph (11). From 1993 to 2012, only 1 fog-related A Low Visibility Warning System was also installed along 19 miles of I-75 in Tennessee in 21 accident was recorded with the system active, but this was not normalized relative to exposure 2.3.3.5
Netherlands In the Netherlands in 1991, a VSL was installed in the urban A16 freeway near Breda
(25). The system consisted of 20 visibility sensors and VSL signs every 700-800 m over 12 km. The system also included automatic incident detection. For visibilities above 140 m the posted
speed would remain 100 km/h, and once it dropped below 140 m the speed limit would be
reduced to 60 km/h. When incidents were detected the first upstream sign and followed by 70 22 km/h on the second one. This system reported mean speed reductions by 8-10 km/h during fog
conditions. km/h on the second one. This system reported mean speed reductions by 8-10 km/h during fog
conditions. 2.4 GAPS IN RESEARCH Table 11 summarizes the effectiveness of past field deployments. While a variety of
systems have been deployed within the past 30 years, there is a general lack of quantitative
evaluations on the results of such implementations, and even those that provide evaluations
hold several limitations. Four of the field deployments explored in this section did not provide any performance
measure on the effectiveness of the system. Another system qualitatively reported
improvements without any quantitative backing to statements. Two deployment evaluations
reported low to no fog-related crashes after activation and another reported both reductions
and increases in fog-related crashes. Presumably the South Carolina and Tennessee
deployments have achieved their objectives, but due to the rarity of the occurrence of crashes,
a simple crash frequency before after comparison does not paint a full picture of safety. Neither
of these evaluations considered exposure to fog or traffic. It could be possible that the low
visibility conditions in the after period were not representative of those in the before period. The California Motorist Warning system evaluation tried to account for fog exposure by
considering crashes per 100 heavy fog days. This measure of exposure however arguably could
have been more accurate if it used weather data from its 9 weather stations rather than
visibility conditions of nearest airport. These results point towards the expected positive
impacts but present possible bias. 23 Four deployment evaluations reported mean speed reductions during low visibility
conditions under the system and one reported mean speed increases. The mean speed
reductions in these deployments either considered reductions from clear conditions in the
same study period or from low visibility conditions prior to system activation. Classifying both
of these conditions within one deployment would more accurately characterize effects. The aim of this research is to fill in these gaps and provide a full understanding of the
problem of how VSLs impact safety in low visibility conditions. 2.4 GAPS IN RESEARCH 24 Table 11 Summary of Visibility Warning Systems Table 11 Summary of Visibility Warning Systems System
Type of System
Major Findings
Limitations
Alabama, I-10
Low Visibility Warning System
(16)
Regulatory
VSL
Manual
Reduced average speeds
Minimized crash risks in low
visibility conditions
No specific quantitative
results
Australia, F6 Tollway
(25)
Advisory VSL
Automated
No reports on results
No reports on results
California, SB I-5 & WB CA-120
Motorist Warning System
(11)
(Speed)
Advisory
Automated
(Manual
override
option)
1.1 mph reduction in mean speeds
8.0 increase in potential collision
speed
One study area experience 242%
increase in fog-related crashes
Control road used for crash
comparison
Normalized fog crashes by
“heavy fog” days not actual
visibility conditions during
collision
California, Hwy 99
Fog Detection and Warning System
(16) (17)
Speed
Advisory
Automated
No reports on results
No reports on results
Georgia, I-75
Automated Adverse Visibility Warning
and Control System
(19) (20) (26)
Speed
Advisory
Automated
No reports on results
No reports on results
Idaho, I-84
Storm Warning System
(11) (15)
Advisory
Manual
At visibilities <0.1miles and no
DMS, speed reductions 67.7 to 58.4
When DMS used with high winds,
high winds and moderate to heavy
precipitation, and snow/high wind
events, speed reductions of 23%,
12%, and 35% respectively
Insufficient data at low
visibility only conditions to
show effect
No crash analysis
No consideration to fog
exposure 25 System
Type of System
Major Findings
Limitations
Netherlands, A16
(25)
VSL
Automated
Mean speed reductions during fog
conditions approximately 8 to 10
km/h (5-6 mph)
No crash analysis
No consideration to fog
exposure
Oregon, I-5 US-97
(27)
Advisory VSL
Automated
No reports on results
No reports on results
South Carolina, I-526
Low Vis Warning System
(11)
Speed
Advisory
Semi-
Automated
No crashes 1992-2003
No speed analysis
No consideration to fog
exposure
Tennessee, I-75
Low Visibility Warning System
(16)
VSL
Automated/
Manual
Only 1 fog related incident 1993-
2012
Effective for general incident
management
No speed analysis
No consideration of fog
exposure at large
Utah, I-215
ADVISE
(11) (14)
Speed
Advisory
Automated
15% increase in speeds
22% decrease in standard deviation
of speeds
No crash analysis
Speed limit increase
implemented same year as
system
Road widened before and
after 26 3.1 SITE DESCRIPTION This section describes previous
analysis performed by McCann and Fontaine with regards to fog characteristics, crash
characteristics, and driver speeds, and compliance in the corridor. Additional “before” period Enhanced Regulatory and warning signs upgraded to new prismatic sheeting Enhanced Regulatory and warning signs upgraded to new prismatic sheeting
Regulatory signs dual indicated
Regulatory signs dual indicated Figure 4 Map of Corridor Location (to be updated) Figure 4 Map of Corridor Location (to be updated) 3.1 SITE DESCRIPTION Interstate 77 runs 68 miles through the mountainous southwestern region of Virginia,
connecting northbound and southbound traffic between North Carolina and West Virginia. The
I-77 ATSMS project is located in the southernmost section of this interstate from mile post zero
at the North Carolina border to mile post 12, just south of Route 702 as shown in Figure 4. Interstate 77 runs 68 miles through the mountainous southwestern region of Virginia,
connecting northbound and southbound traffic between North Carolina and West Virginia. The
I-77 ATSMS project is located in the southernmost section of this interstate from mile post zero
at the North Carolina border to mile post 12, just south of Route 702 as shown in Figure 4. While grades vary across the site, there is a relatively constant +4% grade between the North
Carolina state line and approximately milepost 6, with southbound traffic traveling downhill (5). This section of I-77 is a four-lane divided freeway, with an additional truck climbing lane from
mile post 0 to 7 in the northbound direction. There are also two runaway truck ramps in the
southbound direction. The base posted speed limit during clear conditions is 65 mph. As of
2016, average annual daily traffic (AADT) is over 19,000 vehicles in each direction with over
25% truck traffic. Shoulders widths along the corridor range from 4-6 ft. for left shoulders and
10-12 ft. for right shoulders (4). In addition to steep grades, there are also 11 horizontal curves
throughout the site. Prior to VSL implementation, VDOT had already implemented the following
countermeasures (4) (5): 5 DMSs Safety Service Patrol Safety Service Patrol 11 RWIS stations installed by Fall 2009 Shoulder rumble strips installed in Fall 2012 on majority of the roadway sections 27 Delineation signs added behind guardrail Wider 8” pavement markings added Chevron signs (MUTCD Sign W1-8) added in all curves Enhanced Regulatory and warning signs upgraded to new prismatic sheeting
Regulatory signs dual indicated
Figure 4 Map of Corridor Location (to be updated)
3.2 PREVIOUS WORK
McCann and Fontaine (6) sought to determine the relationship between speed and
visibility and other possible environmental factors on I-77 to better understand the conditions
prior to VSL activation. Their work provides most of the “before” period analysis that is used as
baseline comparisons for the analysis portion of this thesis. 3.2 PREVIOUS WORK McCann and Fontaine (6) sought to determine the relationship between speed and
visibility and other possible environmental factors on I-77 to better understand the conditions
prior to VSL activation. Their work provides most of the “before” period analysis that is used as
baseline comparisons for the analysis portion of this thesis. This section describes previous
analysis performed by McCann and Fontaine with regards to fog characteristics, crash
characteristics, and driver speeds, and compliance in the corridor. Additional “before” period 28 data analyzed for use as baseline comparisons for this thesis will be discussed in the
Methodology section. data analyzed for use as baseline comparisons for this thesis will be discussed in the
Methodology section. 3.2.1 Fog Characteristics 3.2.1 Fog Characteristics McCann and Fontaine used a criterion for visibilities based on stopping sight distance
(SSD) to better compare actual operating speeds to a theoretical safe speed (6). Using the
stopping sight distance equation assuming a flat grade and a 2.5 second reaction time, the
maximum safe speed for a given visibility could be determined. Table 12 lists the bins used for
analysis. Visibility Bin Range
Safe Speed
>645 ft. 65 mph
Clear
Conditions
495-645 ft. 55 mph
Low Visibility
360-495 ft. 45 mph
250-360 ft. 35 mph
155-250 ft. 25 mph
<155 ft. < 25 mph Visibility profiles were constructed using available visibility sensor data and confirmed
that fog varied spatially. Although there was variability in the amount of fog year to year, the
distribution of the fog was fairly consistent with a concentration of recurring low visibility
conditions occurring between mile posts 4.4 and 7.3. In the 2010-2015 study years, milepost
6.6 experienced reduced visibility between 4% to 7% of the year, making it the location along
the corridor experiencing the highest average yearly low visibility conditions. The average
annual visibility profile is depicted in Figure 5. 29 Figure 5 Average Annual Visibility Profile, 2010-2015 (6) Figure 5 Average Annual Visibility Profile, 2010-2015 (6) Figure 5 Average Annual Visibility Profile, 2010-2015 (6) 3.2.2 Crash Analysis Prior to McCann and Fontaine, safety studies analyzing the characteristics of fog-related
crashes did not account for exposure to low visibility conditions or volume changes and simply
considered crash frequency and characteristics (5). Using visibility readings from nearby RWIS
stations McCann and Fontaine were able to estimate visibilities present during the crash rather
relying simply on crashes coded as “fog” in police crash reports (6). Crashes were then
compared by crash type, severity, numbers of vehicles involved, and visibility condition. The
research found that low-visibility conditions were correlated with increased crash severity, had
more vehicles involved, and were more likely to be rear end crashes than crashes during clear
conditions. Crashes during low visibility were much more common in the southbound
(downhill) direction. 30 McCann and Fontaine also calculated crash rates per 100 million vehicle miles traveled
(VMT) by visibility bin (6). This analysis indicated the disparity between crash rates in clear vs. low visibility conditions, with increasing crash rates as visibilities decreased. For clear conditions
in the north and southbound directions crash rates were 66.8 and 69.1 crashes per 100 million
VMT, respectively. For all low visibility conditions, crash rates worsened to between 175.3 and
1000.5 crashes per 100 million VMT depending on the density of fog. Although this crash rate
analysis was limited by a lack of real-time volume and the accuracy of times and locations
indicated by police reports, the analysis shows that there is indeed an association between fog
and higher crash rates. 3.2.3 Driver Speeds 3.2.3 Driver Speeds Speed data at three southbound locations were used to summarize driver behavior in
terms of mean speeds by visibility bin and speed differentials between lanes by visibility bin. McCann and Fontaine found that although there is an overall trend towards speed reduction
with decreasing visibility, the speeds do not decrease to the corresponding SSD safe speeds. Speed data at three southbound locations were used to summarize driver behavior in
terms of mean speeds by visibility bin and speed differentials between lanes by visibility bin. McCann and Fontaine found that although there is an overall trend towards speed reduction
with decreasing visibility, the speeds do not decrease to the corresponding SSD safe speeds. Differentials between observed speeds and the SSD safe speed tended to increase as visibilities
worsened. Trends in speed differentials between lanes were inconsistent by milepost, but there
seemed to be an increase at the lowest visibilities, potentially indicating an increased potential
for crashes. Across mile posts, mean speeds and speed differentials between lanes by visibility
at MP 6.6 were consistently greater than at MP 5.3 and MP 7.3. Differentials between observed speeds and the SSD safe speed tended to increase as visibilities
worsened. Trends in speed differentials between lanes were inconsistent by milepost, but there
seemed to be an increase at the lowest visibilities, potentially indicating an increased potential
for crashes. Across mile posts, mean speeds and speed differentials between lanes by visibility
at MP 6.6 were consistently greater than at MP 5.3 and MP 7.3. Speed profiles by visibility bin were created for MP 6.6. Coefficients of variation nearly
doubled at the lowest visibility from those during clear conditions, another sign of increased 31 safety concerns. The percentage of vehicles traveling at speeds above the SSD safe speed
increased from 74% at visibilities between 495 and 645 ft. to 99% at visibilities below 155 ft. safety concerns. The percentage of vehicles traveling at speeds above the SSD safe speed
increased from 74% at visibilities between 495 and 645 ft. to 99% at visibilities below 155 ft. A baseline model for driver behavior in fog prior to VSL installation was also developed. McCann and Fontaine found that driver speeds are inversely related to visibility, vary for day
and night times, and by mile post. Though some temperature and precipitation data were
available, McCann and Fontaine’s model did not find those variables statistically significant. 3.2.3 Driver Speeds The
final model shown below yielded an adjusted R2 value of 0.451. 𝑆= 64.6 −4204
𝑉𝑖𝑠+ (1.13 ∗𝐷𝑎𝑦𝑁𝑖𝑔ℎ𝑡) + (6.07 ∗𝑆𝐵6) −(2.67 ∗𝑆𝐵7) Where: S = Mean speed per 5 minutes (mph) Vis = Visibility distance (ft.) DayNight = Day or night dummy variable, with 1 indicating day and 0 indicating night y
g
y
g
y
,
g
y
g
g
SB6 = Dummy variable, with 1 indicating site Southbound MP 6.6 SB6 = Dummy variable, with 1 indicating site Southbound MP 6.6 SB7 = Dummy variable, with 1 indicating site Southbound MP 7.3 Although the R2 fit was low, the model is useful to explain the overall trend observed. The coefficients for the dummy variables for mile posts suggested that for the same visibilities,
driver speeds vary significantly by site with means speeds 6 mph higher at MP 6.6 or 2.67 mph
lower at MP than at MP 5.3, raising another concern for driver safety at MP 6.6. The results
from McCann and Fontaine’s research was crucial to the development of the initial VSL
algorithm. 32 3.3 SYSTEM DESCRIPTION A $7.5 million contract to construct the I-77 ATSMS was awarded to G4S Technologies in
February 2014. Before construction of the system began, twelve miles of power and 14 miles
of fiber optic communications infrastructure was installed beginning in July 2011 to support the
system installation (4). The system was initially set to be in operation by the summer of 2015,
but due to construction delays it was not operational until October 2016. When construction
was completed, the project added 13 DMSs, 36 full matrix VSL displays, 8 speed limit signs with
dynamic VSL cutouts, 25 CCTV cameras, 22 traffic sensors, and 14 road weather information
system (RWIS) stations. The locations of these devices are shown in Figure 6. Examples of
devices are shown in Figure 7. 33 34 (a)
(b)
(c)
(d)
Figure 7 VSL System Devices. a) Full Matrix DMS VSL Display, b) Speed Limit Signs with dynamic VSL cutout, c) RWIS Station, and
d) traffic sensor, CCTV, and signing at the northern end of the corridor. Prior to entering corridor, static signs reading “Speed Limit May Vary Next 12 Miles”
were posted, along with static warning signs with flashers reading “Reduced Speed When (c) (a)
(b) (b) (a) (c) (a) (b) (a)
(b)
(c)
(d)
Figure 7 VSL System Devices. a) Full Matrix DMS VSL Display, b) Speed Limit Signs with dynamic VSL cutout, c) RWIS Station, and
d) traffic sensor, CCTV, and signing at the northern end of the corridor. ( )
( )
( )
(d)
Figure 7 VSL System Devices. a) Full Matrix DMS VSL Display, b) Speed Limit Signs with dynamic VSL cutout, c) RWIS Station, and
d) traffic sensor, CCTV, and signing at the northern end of the corridor. (d) ( )
Figure 7 VSL System Devices. a) Full Matrix DMS VSL Display, b) Speed Limit Signs with dynamic VSL cutout, c) RWIS Station, and
d) traffic sensor, CCTV, and signing at the northern end of the corridor. Prior to entering corridor, static signs reading “Speed Limit May Vary Next 12 Miles”
were posted, along with static warning signs with flashers reading “Reduced Speed When
Flashing” that are also scattered throughout the corridor in both directions (28). DMSs were
installed at locations at the start of the corridor in both directions and at various intervals
throughout the site to warn users of fog conditions ahead and to reduce speeds downstream. 3.3 SYSTEM DESCRIPTION 35 ng on conditions throughout the corridor, the DMSs as encountered in NB and SB Depending on conditions throughout the corridor, the DMSs as encountered in NB and SB
directions will display the messages in Table 13. Other weather-related messages may also be
posted depending on conditions (27). directions will display the messages in Table 13. Other weather-related messages may also be
posted depending on conditions (27). Table 13 I-77 DMS Messages
Fog
Level
(Visibility
in ft.)
Messages Displayed
1st DMS
2nd DMS
3rd, 4th, and 5th
DMS
6th DMS
(exit
condition)
DMS at Exit 1
1
≥ 650 ft. “Fog On Mountain | Use Caution”
2
496 to
650 ft. “Fog Ahead |
Use Caution /
Adjust Speed |
To Conditions”
“Fog Ahead |
Use Caution /
Reduce |
Speed”
“Fog Ahead |
Reduce
Speed”
“Fog Present |
Use Caution”
“Fog Ahead |
Use Caution”
3
361 to
496 ft. “Fog Ahead |
Use Caution /
Adjust Speed |
To Conditions”
“Moderate
Fog | Ahead |
Use Caution /
Reduce |
Speed”
“Moderate
Fog | Ahead |
Reduce
Speed”
“Fog Present |
Use Caution”
“Moderate
Fog | Ahead |
Use Caution”
4
<361 ft. “Dense Fog
Ahead | Use
Caution /
Adjust Speed |
To Conditions”
“Dense Fog |
Ahead | Use
Caution /
Reduce |
Speed”
“Dense Fog |
Ahead |
Reduce
Speed”
“Fog Present |
Use Caution”
“Dense Fog |
Ahead | Use
Caution” VSL messages are displayed on the full matrix VSL displays and speed limit signs with
dynamic VSL cutouts, shown in Figure 7, all of which are dual mounted in each direction and
spaced no more than 1.5 miles apart. During clear conditions, VSLs post the base regulatory
speed of 65 mph. Speed limits can be posted as low as 30 mph when conditions dictate. VSL
speeds are set based off of the visibility data collected from the RWIS stations and traffic data
from traffic detection stations. A more detailed description of the VSL algorithm can be found in
the following sections. 36 Vaisala RWIS stations are spaced within 1.7 miles of each other and more densely within
the fog prone area near mile post 6.6. These stations contain equipment to collect pavement
temperature and condition, air temperature, humidity, pressure, precipitation type and
intensity, wind speed and direction, and visibility. The visibility sensors at each station are
mounted 20 ft. 3.3 SYSTEM DESCRIPTION in the air and use forward scatter techniques to estimate visibility distance. Near each RWIS station there are also corresponding Wavetronix side-fire radar devices to
collect traffic data. Although speeds posted by VSLs are regulatory, speed enforcement during
low visibility conditions is limited. Automated speed enforcement of speed limits is not legally
permitted in Virginia. Due to safety concerns, enforcement by Virginia State Police (VSP) during
low visibility conditions is selective, so as to not put enforcement officers at excessive risk
during limited visibility conditions. 3.4 VSL ALGORITHM As part of a collaborative effort between VDOT Southwest Region Operations (SWRO),
VDOT Traffic Engineering Division, VDOT Operations Division, and the Virginia Transportation
Research Council, Kimley-Horn prepared a methodology for operating the I-77 VSLs. In light of
the poor compliance to SSD safe speeds revealed by the work performed by McCann and
Fontaine, VDOT was concerned that simply posting these speeds would not adequately alter
driver behavior and instead would further increase speed variance and interactions between
vehicles under low visibilities. Thus, the initial VSL algorithm considered intermediate speeds
between the pre-VSL driving behavior model and the SSD safe speed, which resulted in a step
function of visibility to determine the posted speed. 37 The step function was based off of the driver behavior model. However, the equation
used is a modification of the McCann and Fontaine’s driver behavior model equation, requiring
less parameters as it serves to model of speeds throughout the entire site, both northbound
and southbound. This model mean speed was represented by the equation (28): 𝑆= 64.6 −4204
𝑉𝑖𝑠+ (2.15 ∗𝐷𝑎𝑦𝑁𝑖𝑔ℎ𝑡) Where: S = Mean speed per 5 minutes (mph) S = Mean speed per 5 minutes (mph) = Visibility distance (ft.) Vis = Visibility distance (ft.) DayNight = Day or night dummy variable, with 1 indicating day and 0 indicating night
The two cut off points for the step functions are when SSD safe speed equals 50 and 40 mph. The SSD safe speed is determined directionally due to uphill/downhill grades. When SSD safe
speeds are greater than 50 mph, the model mean speed was used. When SSD safe speeds were
between 40 and 50 mph, the model mean speed is reduced by 5 mph. When SSD safe speeds
were below 40 mph, the model mean speed was reduced by 10 mph. Additionally the algorithm
considers a day/night variable, therefore there are 4 step-functions considered for both day
and night with different cutoff points for steps for north and south directions. A graphical
representation is shown below in Figure 8 for the southbound direction. At each VSL location,
depending on observed average speeds over an interval, the algorithm would determine what
the posted VSL should be based on the minimum value of either the observed mean speed or
the step-adjusted model fit. 3.4 VSL ALGORITHM VSLs would not post values below 30 mph, and an additional
smoothing algorithm would adjust VSLs over the corridor to have a smooth transition between
posted VSLs as vehicles traveled into and out of fog zones. Speed limits between successive VSL 38 signs cannot decrease by more than 15 mph, but could return to 65 mph as quickly as possible
after exiting the fog zone, provided no additional visibility impacts follow. For example, SB VSL
signs approaching fog zone would transition 60 to 45 to 30 mph at the worst fog locations. As
soon as drivers exit the fog zone, the next VSL sign could read 65 mph if visibilities were clear in
the remained of the corridor. VSL speeds at individual locations were also subject to a step
range that would not allow them to vary by more than 15 mph over successive 5 minute
intervals. Figure 8 VSL Algorithm Step Function Figure 8 VSL Algorithm Step Function Figure 8 VSL Algorithm Step Function 39 4.1 INTRODUCTION This chapter discusses the data used for this thesis and how that data was analyzed in
order to evaluate the effectiveness of the I-77 VSL system. Additional data prior to VSL
activation, not available during McCann and Fontaine’s study, is also presented here. This
additional before period data and new after period data are discussed in this chapter. OVERVIEW OF DATA USED IN ANALYSIS 4.2 OVERVIEW OF DATA USED IN ANALYSIS Data was available at a limited number of locations along the corridor prior to VSL
activation from both permanent and temporary data collection stations. After VSL installation,
some stations were relocated and additional permanent data collection sites were installed. Data prior to VSL activation was obtained primarily through querying existing databases. For
the after period, SWRO provided logs of speed limits, detected speed, and visibility during fog
events. Although data was available for both directions of travel, the after analysis of this thesis
focuses on the southbound (downhill) direction given that past work indicated this direction
was responsible for the vast majority of safety concerns. Table 14 summarizes the data used in
this thesis. Subsequent sections describe the data used in more detail. 4.1 INTRODUCTION 40 Table 14 Summary of Data Used in this Thesis Data
Type
Data
Source
Period
Location
Aggregation
Interval
Visibility
Vaisala
Jan 2015 - Dec
2015
MPs 1.2, 1.8, 2.7, 3.0, 4.4, 5.3, 6.6,
7.3, 9.0, 9.6, 11.3, and 16.9
10 min
SWRO Logs
Oct 2016 - Sep
2017
MPs 1.3, 1.9, 2.7, 3.1, 3.5, 4.4, 5.4,
5.6, 6.5, 7.2, 8.1, 9, 9.5, and 11.4
~6 min
Weather Vaisala
Jan 2015 - Jul 2016
MP 4.4 and 6.6
10 min
Speed
VDOT
Portable
Speed
Detection
Jan 2015 - Jul 2016
SB 4.4, NB 6.6
15 min
SWRO Logs
Oct 2016 - Sep
2017
SB 1.0, 3.3, 4.3, 4.4, 5.3, 6.2, 6.6,
7.5, 8.1, 8.8, 9.7, 10.2, and 11.3
~6 min
VSLs
SWRO Logs
Oct 2016 - Sep
2017
SB 1.8, 3.4, 4.5, 5.6, 6.5, 7.2, 8.1,
9.5, 10.2, and 11.6
~6 min
Volume
VDOT
Traffic
Monitoring
System
Jan 2015 - Aug
2016
MP 0 – 15
5, 15 min
Oct 2016 - Aug
2017
MP 0 – 12
Crash
VDOT
Roadway
Network
System
Jan 2015 - Dec
2015
MP 0 – 15
N/A
Oct 2016 - Aug
2017
MP 0 – 12
4.2.1 Visibility and Weather Data 4.2.1 Visibility and Weather Data Visibility data was collected from Vaisala PWD10/12 visibility sensors at each RWIS
station. These sensors use forward scatter technology to measure visibility over a short range
and extrapolate it out to estimate visibility in feet. Additional weather data collected at RWIS
stations included pavement temperature and condition, air temperature, humidity, pressure,
precipitation type and intensity, and wind speed and direction. This data was stored on a
Vaisala external website that could be queried. 41 Before weather data consisted of two types of information. This visibility data consisted
of visibility readings collected every 10 minutes from RWIS stations located at mile posts 1.2,
1.8, 2.7, 3.0, 4.4, 5.3, 6.6, 7.3, 9.0, 9.6, 11.3, and 16.9 from January 2015 to December 2015. Additional visibility and weather data was retrieved for the RWIS station at MP 4.4 for the
period of January 2015 until July 2016 when data was no longer available through the Vaisala
external site. This additional data was used to further enhance the before condition model of
speed behavior. For the after period analysis, three additional RWIS stations installed at mile posts 3.5,
5.6, and 8.1 were available, and several stations were relocated. 4.1 INTRODUCTION The RWIS station at MP 16.9
was outside scope of study for after period, and thus was not used. The stations used for the
after analysis were located at mile posts: 1.3, 1.9, 2.7, 3.1, 3.5, 4.4, 5.4, 5.6, 6.5, 7.2, 8.1, 9, 9.5,
and 11.4. Although RWIS Stations were located either on the NB shoulder, SB shoulder, or in
the median, most stations had the ability to provide visibility readings for both directions
regardless of location. RWIS stations at MP 4.4, 5.4, and 11.4 were on the SB shoulder; the
station at MP 5.4 provided data only for the SB direction. RWIS stations at MP 1.9, 2.7, 3.5, and
8.1 were on the median. The remainder of the stations were located on the NB shoulder of
which RWIS stations at MP 1.3, 5.6, and 9.0 provided data only for the NB direction. After
system activation, visibility data was acquired from SWRO logs for fog events between October
2016 to end of September 2017. These visibility readings were updated at an average rate of
every 6.5 minutes. Outside of SWRO logs, no visibility data is available but it is assumed to be
clear conditions since low visibilities trigger activation of the system. 42 Additionally, sunrise and sunset times for the years 2015-2017 were also acquired from
the US Naval Observatory to determine day/nighttime conditions. 4.2.2 Speed and Volume Data 4.2.2 Speed and Volume Data Prior to VSL activation, newly available continuous speed data at MP 6.6 NB and 4.4 SB
was available from the VDOT Traffic Monitoring System for the period January 2015 to August
2016. The detectors recorded the count of vehicles in 5 mph bins in 15-minute intervals. The
15-minute volume was taken as the sum of the vehicle counts in each 5 mph bin for the 15-
minute interval. In order to match speeds to visibility readings, these 15 minute intervals were
converted into 10 minute intervals. This conversion involved first evenly splitting 15 minute
intervals into 5 minute intervals assuming a linear distribution of data during each 15-minute
interval. In this way a ##:15 15-minute interval would become ##:05, ##:10, and ##:15 5-minute
interval, a ##:30 15-minute interval would become ##:20, ##:25, and ##:30 5-minute intervals,
and so on. 4.1 INTRODUCTION Then these 5-minute intervals would then be recombined to form 10-minute
intervals where ##:05 and ##:10 5-minute intervals would combine to make ##:10 10-minute
intervals, and ##:15 and ##:20 5-minute interval would combine to make ##:20 10-minute
interval, etc. Average speeds for the new 10-minute intervals were calculated assuming all
vehicles were traveling at the midpoint of each 5 mph bin and finding the volume weighted
average speed. In the after period, SWRO provided logs with mean observed speeds and the posted
speed limit during every fog event for every VSL location. The recording interval averaged
approximately 6.5 minutes. Mean observed speeds for each interval were collected by
Wavetronix speed detectors at MPs 1.0, 3.3, 4.3, 4.4, 5.3, 6.2, 6.6, 7.5, 8.1, 8.8, 9.7, 10.2, and 43 11.3 in the SB direction. Posted speeds were recorded from each VSL sign locations at MPs 1.8,
3.4, 4.5, 5.6, 6.5, 7.2, 8.1, 9.5, 10.2, and 11.6. Since SWRO logs only provided mean speeds,
volume data needed to be retrieved from the VDOT Traffic Monitoring System for the entire
after period. 11.3 in the SB direction. Posted speeds were recorded from each VSL sign locations at MPs 1.8,
3.4, 4.5, 5.6, 6.5, 7.2, 8.1, 9.5, 10.2, and 11.6. Since SWRO logs only provided mean speeds,
volume data needed to be retrieved from the VDOT Traffic Monitoring System for the entire
after period. Volume data for the entire corridor was retrieved from VDOT Traffic Monitoring System
for the 2015 before period and for the October 2016 to June 2017 after period. In the NB
direction, volume data for links MP 0-0.94, MP 0.94-8.57, and MP 14.85-19.03 were retrieved. The link for MP 8.57-14.85 did not have continuous data available, but volumes were estimated
as the average of the links directly upstream and downstream of it. In the SB direction, MP 0-
1.07, MP 1.07-8.99, and 15.22-19.53 were retrieved. Similarly, the link for MP 8.99-15.22 did
not have any data and was estimated as the average of the links directly upstream and
downstream of it. The volume data were recorded in 5- and 15-minute intervals. 15-minute
intervals were converted into 5-minute intervals assuming a linear distribution of data during
15-minute intervals. Before data was further converted to 10-minute data to match visibility
data. 4.2.3 VSL Posted Speed Logs 4.2.3 VSL Posted Speed Logs VSL signs for the southbound direction were located at MPs: 1.8, 3.4, 4.5, 5.6, 6.5, 7.2,
8.1, 9.5, 10.2, and 11.6. In the after period, posted VSLs at each VSL location were recorded in
the SWRO logs at approximate 6.5 minute intervals. Posted speed values were: 30, 35, 40, 45,
50, 55, 60, and 65 mph. Periods when VSLs were offline were recorded in SWRO logs as “Blank”
and were discarded from analysis. 44 4.2.4 Crash Data 4.2.4 Crash Data Police crash reports for 2015 were retrieved from VDOT’s Roadway Network System
(RNS) to understand crash characteristics and frequencies for the year immediately prior VSL
activation. Crash data from October 2016 up until August 2017 (the latest date when crash data
were closed out in RNS) were also collected to perform a preliminary crash analysis following
VSL activation. For both data sets, crashes coded as fog were then matched with visibility data
to confirm low visibility conditions. 4.2.5 Data Matching Data sets came in several different reporting intervals, and data was matched across
different sets in a number of ways. For crash rate calculations, visibility and volumes needed to
be matched, and crashes and visibilities needed to be matched. For the before data, volume
data from the entire corridor was converted to 10-minute bins to match visibility data. For after
data, volume data was converted to 5-minute intervals since visibility readings in the after
period were roughly 6 minutes to maintain as much granularity as possible. Visibility readings
were then matched to 5-minute volume readings by linearly interpolating between visibility
readings. Visibility readings for times outside of SWRO logs, were considered as clear
conditions. For the post-VSL during activation logs, if intervals between visibility readings were
ever more than 30 minutes apart, the 5-minute intervals to be matched in between periods
were marked as missing. To match visibility to crash records, visibilities at the nearest visibility stations and
timestamps closest to crash record times were determined. Then visibilities were linearly
interpolated between nearest RWIS station and visibility readings. 45 4.3 DATA ANALYSIS 4.3 DATA ANALYSIS The following section presents the analysis performed in this thesis. The analysis
examines the effect of the VSL system on driver speeds both before and after at a single site
and across the corridor in the after period and on crashes for the entire corridor. To be consistent with prior work, visibility conditions were categorized into groups
corresponding to a range of stopping sight distance safe speeds. Boundaries for each of these
visibility bins were calculated by solving for speeds of 65, 55, 45, 35, and 25 mph using the
equation for stopping sight distance below: 𝑆𝑆𝐷= 1.468 × 𝑉× 𝑡𝑟+ 2.155 × 𝑉2
2 × 𝑎 𝑆𝑆𝐷= 1.468 × 𝑉× 𝑡𝑟+ 2.155 × 𝑉2
2 × 𝑎 SSD = Stopping sight distance (ft.)
V = Speed (mph)
tr = reaction time (sec)
a = deceleration rate (ft. /sec2) SSD = Stopping sight distance (ft.) tr = reaction time (sec) a = deceleration rate (ft. /sec2) The SSD was calculated assuming flat grades, a reaction time of 2.5 sec, and a
deceleration rate of 11.2 ft. /sec2 as specified in the AASHTO Policy on the Geometric Design of
Highways and Streets. The SSD calculated from each of the above speeds was rounded up to
the nearest multiple of 5. This resulted in the following visibility bins: >645 ft. = >65 mph >645 ft. = >65 mph 495-645 ft. = 55-65 mph 495-645 ft. = 55-65 mph 46 360-495 ft. = 45-55 mph 250-360 ft. = 35-45 mph 155-250 ft. = 25-35 mph <155 ft. = <25 mph <155 ft. = <25 mph From this a visibility profile of the corridor was created to examine the frequency of
reduced visibility across the corridor. The visibility profile served to indicate if fog exposure
during both the before and after were representative of each other and to McCann and
Fontaine’s initial study. This frequency of reduced visibility served to create crash rates. 4 3 2 VSL Posted To present how often the system is posting reduced speeds, VSLs were summarized by
how often each PSL was posted at each VSL sign. Average posted speeds per SB VSL sign
location were also calculated. 4.3.3 Speed Choice Analysis .1
Before-After Evaluation at MP 4.4 4.3.3.1
Before-After Evaluation at MP 4.4 There were only two sites along the corridor that provided continuous speed and
visibility data both before and after system activation, those being SB 4.4 and NB 6.6. Since the
focus of this thesis is on the SB direction, only SB 4.4 was analyzed before-after to compare
changes in driver behavior and compliance. Speed data was summarized into counts, mean
speeds, and standard deviations by visibility bin for both periods. Mean speeds and standard
deviations by visibility bin were also calculated for both before and after periods. Z tests were
used to test if mean speeds in the after period were statistically significantly different from the 47 before period. F tests were also performed to assess if the variance in speeds by visibility
changed for the periods tested. before period. F tests were also performed to assess if the variance in speeds by visibility
changed for the periods tested. Weather data and VSL data were considered to develop driver speed choice models at
the singular comparison site MP 4.4. Several different types of speed choice models were
attempted: a before model to compare to McCann’s original model/VSL algorithm model, an
after model to compare to before model, and a combined before-after model at MP 4.4. Stepwise linear regression was performed in order to describe speeds as a function of
visibility, weather conditions, and/or VSL factors for each of the models considered. For the
before model, independent variables considered were: Available visibility Weather data (pavement temperature and condition, air temperature, humidity,
pressure, precipitation type and intensity, and wind speed and direction) Day/night conditions Day/night conditions In the after period, the only data available from RWIS stations available was visibility, thus
weather data was not considered. In addition to visibility and day/night condition variables, the
after period also considered posted speed limits as an independent variable. The before-after
model considered the same variables as the after model with the addition of a VSL In the after period, the only data available from RWIS stations available was visibility, thus
weather data was not considered. In addition to visibility and day/night condition variables, the
after period also considered posted speed limits as an independent variable. 4.3.3.1
Before-After Evaluation at MP 4.4 The before-after
model considered the same variables as the after model with the addition of a VSL
absence/presence indicator variable which used the value of 0 to indicate the before period or
of 1 for the after period. absence/presence indicator variable which used the value of 0 to indicate the before period or
of 1 for the after period. absence/presence indicator variable which used the value of 0 to indicate the before period or
of 1 for the after period. Transformations and interactions of all these variables within their respective models
were also taken into consideration. Periods with visibilities over 645 ft. (clear conditions) and of
zero feet were discarded from the model. Theoretically, visibilities of 645 ft. and above should 48 provide adequate SSD for the base speed limit of 65 mph. Based on feedback from VDOT,
visibility readings of zero feet were taken to be errors and were discarded from the data set. Furthermore, speed data during crash events and their aftermath were ignored as this analysis’
intent was to understand speed under undisturbed low visibility conditions and not during
congestion. Models were further refined using Cook’s Distance value to exclude outlier data
points whose values were excessively influencing the models. To check if regression
assumptions were satisfied, residual plots and probability-probability (P-P) plots were reviewed. Models accepted showed no curvilinear or systematic trends in residual plots and had errors
that were homoscedastic, uncorrelated, and normally distributed. Adjusted R2 values and
average absolute error and bias were also checked. adequate SSD for the base speed limit of 65 mph. Based on feedback from VDOT, 4.3.3.2
Speed Changes During Fog Along Corridor Although speed data throughout the entire corridor was not available in the before
period, it was available in the after period and was used to characterize driver behavior in
response to VSLs. To understand speed changes during fog along the corridor, speed data was
summarized in several forms: posted speeds by MP, observed speeds for posted speeds by mile
post, and speed differentials for posted speeds by mile post. The frequency of different reduced speeds was mapped by milepost. For periods of
activation, average posted speed was also calculated by mileposts. Observed mean speeds and
differentials between observed mean speed and posted speeds were summarized by posted
speed and mileposts. To analyze the effect of the posted speed on the mean speed, paired t-
tests were performed on observed speeds to test if means speeds for a set posted speed at a
mile post were significantly different than posted speeds. 49 4.3.4 Crash Analysis 4.3.4 Crash Analysis 4.3.4.1
Crash Frequency and Characteristics Crashes matched with low visibilities and coded as fog in police reports were
summarized by crash type, severity, and number of vehicles involved. These were summarized
also by visibility bin and direction to compare with decreased visibility. All other crashes were
considered “clear conditions” and were summarized similarly. For the before period, the newly
acquired 2015 data was combined with the crash characteristics previously summarized by
McCann and Fontaine to create a crash characteristics of the corridor for the 5 full years
immediately prior to VSL activation. Due to the limited amount of crash that has been reported
since VSL system has been activated, analysis is discussed more qualitatively although similar
summaries are provided. 4.3.4.2
Crash Rates Crash rates along the corridor were calculated using visibility and crash data during this
period. After visibility and volume data had been matched, vehicle miles traveled (VMT) per
visibility bin needed to be estimated. First, the corridor was broken into segments according to
the number of RWIS stations corresponding to each direction. The NB direction was broken into
13 segments with the midpoints between RWIS station MPs 1.3, 1.9, 2.7, 3.1, 3.5, 4.4, 5.6, 6.5,
7.2, 8.1, 9, 9.5, and 11.4 as the segment boundaries. Similarly, the SB direction was broken into
11 segments with the midpoints between RWIS stations 1.9, 2.7, 3.1, 3.5, 4.4, 5.4, 6.5, 7.2, 8.1,
9.5, and 11.4 as segment boundaries. For each segment the vehicle miles traveled would be
determined by the multiplying segment length and with corresponding link volume. This was 50 done for all intervals. The sum of VMT per visibility bin was found and crashes per 100 million
VMT per visibility bin were calculated. The crash rate for all combined low visibilities was also
calculated. VMT per visibility bin was also summarized into percentages to get a picture of the
traffic actually on roads during periods of reduced visibility. 4.3.5 VSL Algorithm Assessment 4.3.5 VSL Algorithm Assessment CHAPTER 5:
RESULTS CHAPTER 5:
RESULTS 4.3.5 VSL Algorithm Assessment Based on driver responses to the system, modifications to the current VSL algorithm
may be warranted to increase system’s effectiveness. To assess the VSL algorithm,
modifications to different constraints in the algorithm were examined. Modifications to
algorithm could entail altering spatial and temporal step ranges or altering the (input) values
used in the algorithm to determine the recommended speed. Other alternatives to improve
system effectiveness beyond control algorithm itself were also discussed. 51 5.1 INTRODUCTION This section describes the results of the analysis described in the previous chapter
beginning with a case study to illustrate system operations. Whenever tables and charts list
MPs for the SB direction, they are listed in descending order to reflect the order in which
drivers encounter MPs as they enter the corridor from the north at MP 12 and exit at the south
at MP 0. 5.2 OVERALL RESULTS 5.2.1 Visibility 5.2.1 Visibility Altogether, there were 106 VSL separate activations from October 2016 to September
2017, which resulted in at least some portion of the corridor having a reduced VSL for a total of
702.5 hours. From the VSL activation logs, visibilities were retrieved to construct the visibility
profile for the after period, as shown Figure 9. The figure shows a concentration of low visibility
conditions between MP 7.3 and 5.3, consistent with previous findings that this section Altogether, there were 106 VSL separate activations from October 2016 to September
2017, which resulted in at least some portion of the corridor having a reduced VSL for a total of
702.5 hours. From the VSL activation logs, visibilities were retrieved to construct the visibility
profile for the after period, as shown Figure 9. The figure shows a concentration of low visibility
conditions between MP 7.3 and 5.3, consistent with previous findings that this section
experiences the most amount of time of reduced visibilities in the corridor yearly. For the after
VSL activation period, RWIS stations at MP 6.6 and 5.6 experienced the most reduced visibility
with 386 and 275 hours(4.4 and 3.1% of the time in the after period, respectively) of reduced
visibilities of less than 645 feet available. Although this is about 1% of the time less than the
before period shown in Figure 5, visibilities less than 360 ft. occur approximately 3.1% of the
total time for both periods at MP 6.6. experiences the most amount of time of reduced visibilities in the corridor yearly. For the after
VSL activation period, RWIS stations at MP 6.6 and 5.6 experienced the most reduced visibility
with 386 and 275 hours(4.4 and 3.1% of the time in the after period, respectively) of reduced
visibilities of less than 645 feet available. Although this is about 1% of the time less than the
before period shown in Figure 5, visibilities less than 360 ft. occur approximately 3.1% of the
total time for both periods at MP 6.6. 52 In the after period, 10 stations experienced no more than 3 hours of missing data during
activations. Visibility data was only available during VSL activations, and any time outside of
those periods available visibility readings are assumed to be clear conditions at all RWIS
stations. RWIS stations at MPs 9.6, 7.3, and 1.8 had the most missing hours of data with 34-42
hours of missing data. 5.2.1 Visibility Since RWIS stations at MPs 9.6 and 1.8 fall outside the worst fog zone
area, this missing data likely does not greatly affect the overall visibility profile. At MP 7.3 there
is a sudden drop, which can probably attributed to the missing data. Overall, however, the
visibility in the corridor for the after period was found to be spatially similar in distribution to
the average low visibility conditions summarized by McCann and Fontaine for the 2010-2015
period, with slightly shorter durations of about 1% less total time in reduced visibilities across
all stations. Figure 9 After Period Visibility Profile
5.2.2 Summary of VSLs Posted Figure 9 After Period Visibility Profile Figure 9 After Period Visibility Profile 5.2.2 Summary of VSLs Posted 5.2.2 Summary of VSLs Posted Figure 10 shows the amount of time and overall percentage of the after period in which
reduced speed limits were posted in the southbound direction. This figure parallels the visibility 53 profile in Figure 9 showing the most usage of reduced speeds between MPs 7.2 and 5.6 posting
reduced speeds for more than 5% of the after period. In general, this figure displays how during
fog events the VSL system will post speeds that gradually reduce as drivers encounter the first
VSL sign at 11.6 until the worst fog area between 7.2 and 5.6 where reduced speeds are the
lowest. After drivers traversed the worst fog zone, posted speeds quickly increase back up to
base speed limit. Figure 10 VSL Usage by MP Figure 10 VSL Usage by MP
5.2.3 Case Study: Incident WX3150436 Figure 10 VSL Usage by MP 5.2.3 Case Study: Incident WX3150436 5.2.3 Case Study: Incident WX3150436 Before discussing overall trends in the results, a case study is presented to illustrate how
the system works over the course of an event. This allows for a more granular presentation of
results, and helps illustrate common performance trends. Incident number WX3150436 was initiated on 12/17/2016 at 8:38:39 PM and lasted
until 12/18/2016 at 11:01:46 AM, for a total duration of 14 hours and 23 minutes. During this
event, all RWIS stations and VSL signs were operational with no missing data. All SB speed 54 sensors were operational with no missing data as well except for those at MPs 8.1, 6.2, and 4.4
which were offline for the entirety of the event. At its most critical point, visibilities were as low
as 177 ft. at MP 5.6, with reduced visibilities extending from MP 2.7 to 8.1. On average, there
was a median spread of fog of 2.2 mile centered about MP 6.6, with a visibility profile as shown
in Figure 11. The VSLs were used as shown in Figure 12. From this figure, we can see that the
VSLs used at MPs 7.3, 6.6, and 5.6 are almost identical, posting reduced speeds of 30 MPH over
60% of the event duration. Although visibilities at MP 7.3 fared better than the preceding
station at MP 8.1 due to the worsened condition ahead, the trooping in the VSL algorithm
caused all speeds in this region to be equal, and VSLs at 8.1 were posted at 45 mph a majority
of the time. Figure 11 Case Study Visibility Profile Figure 11 Case Study Visibility Profile Figure 11 Case Study Visibility Profile 55 Figure 12 Case Study SB VSL Usage Figure 12 Case Study SB VSL Usage Figure 12 Case Study SB VSL Usage Figure 12 Case Study SB VSL Usage Figure 13 shows how speeds and visibilities vary over time at successive MPs for this
event. Only visibilities less than 1000 ft. are graphed. From this, it can be seen that upon
entering the corridor observed speeds closely follow the posted speeds that for almost 99% of
the time remained at 65 mph. When drivers approach the first reduced speed limits at MP 9.5,
observed speeds still remain about the posted speeds +5 mph or so. 5.2.3 Case Study: Incident WX3150436 The lowest posted speeds
are first seen at MP 7.2, and downstream speeds are first recorded at MP 6.6 where the most
severe fog was concentrated. Here we see the greatest differentials between posted speeds
and observed speeds. Posted speeds over time at this location fluctuate between 45 mph and
30 mph, but as time progresses the vehicles passing through this section maintain speeds in the
50s. Speeds remain far above posted speeds until drivers encounter the VSL at MP 4.5, where
there are even instances where average speeds are below the posted speeds. Here the delayed
reaction to VSLs after the fog is seen most prominently. 56 This delayed effect that causes an increase of compliances at MP 4.4 may suggest that
the before-after site analysis at this location may be the best-case scenario/location to conduct
a before-after study, thus effects at this location cannot be generalized over the entire corridor
but perhaps only to the locations downstream MP 4.4. 57 58 58 59 Figure 13 SB Speeds Over Time Figure 13 SB Speeds Over Time 60 5.2.4 Speed Choice 5.2.4.1
Before-After Evaluation at MP 4.4 Table 15 compares mean speeds and standard deviations by visibility range for the
before and after periods. Although the aggregation rate for both periods differ, the number of
intervals for both periods are given to establish the number of observations to make these
conclusions. Note that the number of intervals available for visibilities greater than 645 ft. in
the before period is substantially great than in the after period. In the after period, visibilities
greater than 645 ft. were only recorded if there was fog elsewhere on the mountain. Hypothesis testing at a confidence interval α=0.05 showed that all mean speeds showed
reductions across every visibility range available in the after period. For every, visibility range
available mean speed reductions of 2-5 mph were seen. At a 95% confidence interval, there
were also reductions of standard deviation across all visibility ranges available. Reductions in
speed and variance suggest the safety has improved after the VSL has been activated. Though,
no periods of visibilities below 250 ft. were recorded in the after period, if the trend continues
reductions could be expected for lower visibilities as well. These reductions point toward the
overall positive impact by the VSL system. 61 Table 15 Mean Speed Before-After Comparison at MP 4.4 Visibility
Bin (ft.)
SSD
Safe
Speed
(mph)
Before
After
p-values
No. of
Intervals
(10 min
intervals)
Mean
Speed
(mph)
Standard
Deviation
(mph)
No. of
Intervals
(5 min
intervals)
Mean
Speed
(mph)
Standard
Deviation
(mph)
Mean Variance
>645
65
69307
67.07
7.31
5158
64.34
5.41
0.000
0.000
495-645 55-65
513
59.88
8.45
526
55.12
6.33
0.000
0.000
360-495 45-55
524
56.63
9.03
561
51.83
5.40
0.000
0.000
250-360 35-45
297
52.43
8.83
73
50.49
5.04
0.018
0.000
155-250 25-35
22
49.75
7.96
0
-
-
-
-
<155
<25
0
-
-
0
-
-
-
- The model building sought to explain the trends in speeds as a function of visibility and
additional variables discussed in the Methodology chapter. Of the several different types of
speed choice models attempted, the combined before-after model at MP 4.4 was found to be
most useful for understanding the changes in speeds as a result of the VSL system. Models were developed using data that was commonly available across both the before
and after periods. 5.2.4.1
Before-After Evaluation at MP 4.4 For the after period, posted speed limit and the interaction of posted speed
limit and visibility were found to be the best predictors of speed. For the before period the only
value for posted speed limit was the base posted speed of 65 mph. Although these models
yielded high R2 values greater than 0.7, these models were not useful in relating how speeds
changed from before period. The combined before-after model was best equipped to help understand how the VSL
system affected SB driver speeds in the corridor. The results of the model building showed that
posted speed limits (PSL) showed stronger correlations with observed mean speed than with
visibility variables. However, since the pre-VSL activation period only had one posted speed
limit of 65 mph and posted speed limits in the post-activation period were highly correlated 62 with visibility, the posted speed limit variable and interactions were dropped from model
building. Unlike the McCann and Fontaine’s original model and the VSL algorithm model, the
day/night indicator variable did not appear as significant in the after data. After comparing different variations of the models without variables that were highly
correlated the model equation below was selected: After comparing different variations of the models without variables that were highly
correlated the model equation below was selected: he model equation below was selected: 𝑆𝑝𝑒𝑒𝑑= 67.236 −4242.723
𝑉𝑖𝑠
−2518.621 ∗𝑉𝑆𝐿
𝑉𝑖𝑠 Where: Speed = Mean speed per time interval (mph) Speed = Mean speed per time interval (mph) Vis = Visibility distance (feet) VSL = indicator variable, with 1 indicating VSL is active and 0 VSL is inactive. VSL = indicator variable, with 1 indicating VSL is active and 0 VSL is inactive. Table 16 shows that all model parameters were highly significant. With the exception of
the day/night variable, the magnitude of the constant and the coefficient of the inverse
visibility variable found in this model are similar to that found in McCann and Fontaine’s
original model and the VSL algorithm model. The greatest departure from these models is the
inclusion the indicator variable that VSL indicator variable that indicates that there is no longer
as big of a difference between daytime and nighttime speeds as much as there is a difference
between pre-VSL and post-VSL speeds. Although the adjusted R2 value was only 0.32, this is
similar to earlier efforts used to define the VSL algorithm for previous models (6). 5.2.4.1
Before-After Evaluation at MP 4.4 The data in
the after period appears more varied about the model fit than in the before period. The smaller
aggregation intervals in the after period (6 minutes versus 10 minutes) may contribute to the
noise seen in the after period that weaken the R2 values. This model implies that when active,
the presence of the VSL produces and additional 60% reduction in speeds over what would 63 have occurred based on visibility alone, showing that the VSL does in fact positively affect driver
speed reductions. Table 16 Before-After Model Parameters Table 16 Before-After Model Parameters Model Elements
Coefficients
t-statistic
p-value
Unstandardized
Standardized
Constant
67.235978
149.382
.000
Inverse Visibility
-4242.722554
-0.367
-22.578
.000
VSL × Inverse Visibility
-2518.621476
-0.439
-27.069
.000 Figure 14 shows the observed mean speeds during reduced visibility conditions before
and after VSL activation at milepost 4.4 southbound. Although observed mean speeds are
somewhat noisy as a function of visibility, a downward trend in mean speeds as visibilities
decrease can be seen for both the pre- and post- VSL conditions at milepost 4.4. Figure 14b
shows that speeds have generally shifted lower for comparable visibilities in the after period,
indicating the VSL is having a positive effect. The SSD safe speed line drawn for reference also
suggests that speeds in the post VSL activation period align more closely to the safe speeds for
a given available visibility. Figure 14c also shows the general trend in post-VSL activation
average speeds falls below the VSL Algorithm model, which may suggest that VSL algorithm can
be further refined to align mean speeds more closely to SSD safe speeds. 64 Figure 14 Observed mean speeds a) before and b) after VSL activation at MP 4 4 c) After Speeds Model with VSL algorithm model for reference
0
10
20
30
40
50
60
70
80
0
100
200
300
400
500
600
700
Mean Speed (mph)
Visibility (ft.)
After Predicted
SSD Safe Speed
VSL Model Fit Night
VSL Model Fit DAY 65 5.2.4.2
Speed Changes During Fog Along Corridor Table 17, Table 18, Table 19, and Table 20 provide a summary of how observed speeds
differ for various posted speeds summarizing observed mean speeds , count of intervals posted,
differences between the posted speed and observed mean speed, and standard deviations of
observed speeds vary by mile marker and posted speed along the corridor. 5.2.4.1
Before-After Evaluation at MP 4.4 In the absence of
individual speed data to show compliance, differentials between observed and posted speeds
were included to provide a surrogate picture of compliance. Table 19 is color coded to visualize
the degree of compliance, with green showing high relative compliance and red showing large
non-compliance based on the differential between posted and observed speeds. Entering the corridor from the north, at every posted speed limit, mean observed
speeds exceed the posted speeds, with differences increasing to the highest levels at MP 6.6,
after which downstream speeds start to decrease to speeds below those entering corridor. This
may suggest that drivers do not follow the reduced speed as closely until they enter the actual
fog zone. The reaction to the VSL speeds seems more pronounced right before exiting the thick
of the fog zone and after which drivers resume their regular speeds. In general, the difference
in observed speeds and posted speeds increases with decreasing posted speed at every mile
post, especially at posted speeds below 50 mph. However, with this apparent delayed reaction
to fog, and compliance with lower limits improves past MP 5.3. This implies that drivers will not
immediately reduce speeds upon seeing the VSLs, and that they must experience some reduced
visibility before altering speeds. ntering the corridor from the north, at every posted speed limit, mean observed eed the posted speeds, with differences increasing to the highest levels at MP 6.6, In Table 17 the speeds in bold are not significantly different from the posted speed limit
at a 95% confidence. With a few exceptions, all mean speeds are greater than the posted 66 speeds. Excluding posted speeds of 50 mph, for the first couple miles entering the corridor, the
average speeds tend to be no more than 5 mph over the posted speed limits. When posted
speeds are below 50 mph, for the first couple miles the average speeds tend to be about 10
mph higher than posted speeds. As drivers traverse the worst fog zone between mile posts 7.5
and 4.4, the difference between means speeds and posted speeds increases to almost 10 mph
above the posted speed limits, and even more so for posted speeds below 50 mph. 5.2.4.1
Before-After Evaluation at MP 4.4 After exiting
the fog zone, compliance seems to improve even for posted speeds below 50 mph as means
speeds return to being within 6 mph of the posted speed, with a few exceptions at the lowest
posted speeds limit that are still within 10 mph of the posted speed. Though speeds are still
higher than posted, the VSL does seem to have a positive effect on reducing speeds based on
model results and table summary. 5.2.4.1
Before-After Evaluation at MP 4.4 Table 17 Observed mean speeds by posted speed limit
Location (Mile Post)
Posted Speed Limit
VSL
RWIS
Station
Downstream
Speed
Sensor
65
60
55
50
45
40
35
30
11.6
11.3
11.3
65.0
64.8
57.0
59.0
57.4
-
-
-
10.2
9.6
9.7
67.6
62.5
63.5
59.4
-
-
-
-
9.5
9.0
8.8
68.0
64.7
59.2
57.9
49.5
-
-
-
8.1
7.3
7.5
65.7
62.4
60.7
56.4
56.3
50.3
-
42.1
7.2
6.6
6.6
69.1
67.2
62.6
62.2
58.6
57.5
57.3
52.9
5.6
5.3
5.3
66.0
65.5
59.8
60.2
55.8
55.3
52.2
51.3
4.5
4.4
4.4
65.1
62.7
58.5
55.6
49.9
48.1
41.4
-
4.5
4.4
4.3
65.5
63.3
58.5
55.1
48.8
45.8
40.3
-
3.4
3.1
3.3
67.9
64.8
58.5
57.1
50.6
49.6
-
-
1.8
1.0
1.0
68.4
64.7
-
-
-
-
-
- 67 Table 18 Count of observations by posted speed limit Location (Mile Post)
Posted Speed Limit
VSL
RWIS
Station
Downstream
Speed
Sensor
65
60
55
50
45
40
35
30
11.6
11.3
11.3
6209
45
23
28
10
-
-
-
10.2
9.7
9.7
6153
92
18
29
-
-
-
-
9.5
8.8
8.8
4625
1403
142
31
54
-
-
-
8.1
7.5
7.5
3498
429
577
248
1368
132
-
56
7.2
6.6
6.6
1924
649
178
1029
384
621
24
1508
5.6
5.3
5.3
1949
561
191
1072
388
619
24
1510
4.5
4.4
4.4
3130
242
304
427
199
38
12
-
4.5
4.3
4.3
4173
452
442
763
356
66
21
-
3.4
3.3
3.3
4450
765
297
688
86
26
-
-
1.8
1.0
1.0
6204
100
-
-
-
-
-
-
Table 19 Difference from Posted Speeds by Posted Speed
Location (Mile Post)
Posted Speed Limit
VSL
RWIS
Station
Downstream
Speed
Sensor
65
60
55
50
45
40
35
30
11.6
11.3
11.3
0.0
4.8
2.0
9.0
12.4
-
-
-
10.2
9.7
9.7
2.6
2.5
8.5
9.4
-
-
-
-
9.5
8.8
8.8
3.0
4.7
4.2
7.9
4.5
-
-
-
8.1
7.5
7.5
0.7
2.4
5.7
6.4
11.3
10.3
-
12.1
7.2
6.6
6.6
4.1
7.2
7.6
12.2
13.6
17.5
22.3
22.9
5.6
5.3
5.3
1.0
5.5
4.8
10.2
10.8
15.3
17.2
21.3
4.5
4.4
4.4
0.1
2.7
3.5
5.6
4.9
8.1
6.4
-
4.5
4.3
4.3
0.5
3.3
3.5
5.1
3.8
5.8
5.3
-
3.4
3.3
3.3
2.9
4.8
3.5
7.1
5.6
9.6
-
-
1.8
1.0
1.0
3.4
4.7
-
-
-
-
-
- Location (Mile Post)
Posted Speed Limit
VSL
RWIS
Station
Downstream
Speed
Sensor
65
60
55
50
45
40
35
30
11.6
11.3
11.3
6209
45
23
28
10
-
-
-
10.2
9.7
9.7
6153
92
18
29
-
-
-
-
9.5
8.8
8.8
4625
1403
142
31
54
-
-
-
8.1
7.5
7.5
3498
429
577
248
1368
132
-
56
7.2
6.6
6.6
1924
649
178
1029
384
621
24
1508
5.6
5.3
5.3
1949
561
191
1072
388
619
24
1510
4.5
4.4
4.4
3130
242
304
427
199
38
12
-
4.5
4.3
4.3
4173
452
442
763
356
66
21
-
3.4
3.3
3.3
4450
765
297
688
86
26
-
-
1.8
1.0
1.0
6204
100
-
-
-
-
-
- Posted Speed Limit Table 19 Difference from Posted Speeds by Posted Speed
Location (Mile Post)
Posted Speed Limit
VSL
RWIS
Station
Downstream
Speed
Sensor
65
60
55
50
45
40
35
30
11.6
11.3
11.3
0.0
4.8
2.0
9.0
12.4
-
-
-
10.2
9.7
9.7
2.6
2.5
8.5
9.4
-
-
-
-
9.5
8.8
8.8
3.0
4.7
4.2
7.9
4.5
-
-
-
8.1
7.5
7.5
0.7
2.4
5.7
6.4
11.3
10.3
-
12.1
7.2
6.6
6.6
4.1
7.2
7.6
12.2
13.6
17.5
22.3
22.9
5.6
5.3
5.3
1.0
5.5
4.8
10.2
10.8
15.3
17.2
21.3
4.5
4.4
4.4
0.1
2.7
3.5
5.6
4.9
8.1
6.4
-
4.5
4.3
4.3
0.5
3.3
3.5
5.1
3.8
5.8
5.3
-
3.4
3.3
3.3
2.9
4.8
3.5
7.1
5.6
9.6
-
-
1.8
1.0
1.0
3.4
4.7
-
-
-
-
-
- Table 19 Difference from Posted Speeds by Posted Speed 68 Table 20 Standard Deviations by posted Speed Limit Location (Mile Post)
Posted Speed Limit
VSL
RWIS
Station
Downstream
Speed
Sensor
65
60
55
50
45
40
35
30
11.6
11.3
11.3
5.4
2.8
1.5
6.6
9.3
-
-
-
10.2
9.7
9.7
4.8
12.0
4.0
12.5
-
-
-
-
9.5
8.8
8.8
6.0
6.2
5.7
10.4
17.5
-
-
-
8.1
7.5
7.5
5.5
5.5
4.4
4.7
5.8
5.8
-
13.7
7.2
6.6
6.6
6.8
4.5
8.7
6.1
6.4
6.0
6.5
7.1
5.6
5.3
5.3
4.7
3.4
3.4
5.2
5.8
6.2
14.1
6.5
4.5
4.4
4.4
3.7
2.5
2.9
3.6
3.3
5.9
8.9
-
4.5
4.3
4.3
4.6
4.0
3.5
4.2
3.3
5.6
7.6
-
3.4
3.3
3.3
4.6
3.6
3.4
5.2
4.4
7.4
-
-
1.8
1.0
1.0
2.8
3.5
-
-
-
-
-
-
5.2.5 Crash Analysis
5.2.5.1
Crash Frequency and Characteristics Posted Speed Limit 5.2.5 Crash Analysis 5.2.5.1
Crash Frequency and Characteristics 5.2.5.1
Crash Frequency and Characteristics Crash analysis for 2015, the year prior to VSL activation, was performed. Although
McCann and Fontaine had already performed a 5-year crash analysis with 2010-2014 data, this
2015 had some new data available to help validate previous findings. For this year, out of the
108 crash reports retrieved, 5 crashes were coded as fog but only 4 were found to occur during
low visibility conditions. One crash was an injury crash, and 3 were PDOs. One crash involved 1
vehicle, 2 involved 2, and the fourth involved 3+. Two were rear-end crashes, one was a
sideswipe same direction, and the fourth was a non-collision. Overall, however, the addition of
2015 data to the 2010-2014 average minimally shifted average values, making it a fairly
representative year of crash characteristics for pre-VSL activation. Updated crash characteristics 69 for the 2010-2015 period are shown below in Table 21, Table 23, and Table 25. Appendix C
includes all 2015 charts used to update tables. Crash analysis for the after period was limited to a 11 months of crash data from
October 2016 to August 2017. Out of 89 crashes that occurred in the corridor in this time, 12
were reported to have occurred during times when VSL was active. There were 6 crash reports
that had weather condition types labeled as fog, one of which occurred outside of a VSL
activation time. After matching visibility with corresponding times and location of these
crashes, only two crashes appeared to have actually occurred in conditions where visibilities
were less than 645 ft. These two crashes occurred on 12/26/2016 when visibilities were
between 155-200 ft. during a 54-hour event, one of the longest continuous VSL activations to
date. Of these crashes, the first was an injury crash and involved 6 vehicles, including a tractor
trailer. The second crash was only a property damage crash which reported that vehicles “were
stopped in traffic due to dense fog and a separate crash ahead” when a rear-end crash
occurred. This crash and secondary crash are the common types expected in reduced visibility
conditions, but given that these two are the only ones that were coded as fog and matched low
visibility may be an indication in that the VSL has been influential in reducing the frequency of
crashes. Crash characteristics for the after period are summarized in Table 22, Table 24, and
Table 26. 5.2.5.1
Crash Frequency and Characteristics >645
1
1%
20
23%
21
24%
66
76%
87
All Low Visibility
0
0%
1
50%
1
50%
1
50%
2
2. 495-645
0
0%
0
0%
0
0%
0
0%
0
3. 360-495
0
0%
0
0%
0
0%
0
0%
0
4. 250-360
0
0%
0
0%
0
0%
0
0%
0
5. 155-250
0
0%
1
50%
1
50%
1
50%
2
6. <155
0
0%
0
0%
0
0%
0
0%
0
ERROR, NO VISIBILITY READING
0
0%
0
0%
0
0%
0
0%
0
All Conditions
1
1%
21
24%
22
25%
67
75%
89
Table 23 Number of Vehicles Involved by Visibility Bin, updated with 2015, 2010-2015
Visibility Bin
1
2
3+
Total
1. >645
302
53%
209
37%
55
10%
566
All Low Visibility
6
10%
29
47%
27
44%
62
2. 495-645
2
20%
5
50%
3
30%
10
3. 360-495
2
29%
4
57%
1
14%
7
4. 250-360
0
0%
1
14%
6
86%
7
5. 155-250
1
3%
19
51%
17
46%
37
6. <155
1
100%
0
0%
0
0%
1
ERROR, NO VISIBILITY READING
2
50%
2
50%
0
0%
4
All Conditions
310
49%
240
38%
82
13%
632 Table 22 Crash Severity by Visibility Bin, October 2016-August 2017
Visibility Bin
Fatal
Injury
Fatal +
Injury
Property
Damage Only
Total
1. >645
1
1%
20
23%
21
24%
66
76%
87
All Low Visibility
0
0%
1
50%
1
50%
1
50%
2
2. 495-645
0
0%
0
0%
0
0%
0
0%
0
3. 360-495
0
0%
0
0%
0
0%
0
0%
0
4. 250-360
0
0%
0
0%
0
0%
0
0%
0
5. 155-250
0
0%
1
50%
1
50%
1
50%
2
6. <155
0
0%
0
0%
0
0%
0
0%
0
ERROR, NO VISIBILITY READING
0
0%
0
0%
0
0%
0
0%
0
All Conditions
1
1%
21
24%
22
25%
67
75%
89 Table 23 Number of Vehicles Involved by Visibility Bin, updated with 2015, 2010-2015
Visibility Bin
1
2
3+
Total
1. >645
302
53%
209
37%
55
10%
566
All Low Visibility
6
10%
29
47%
27
44%
62
2. 495-645
2
20%
5
50%
3
30%
10
3. 360-495
2
29%
4
57%
1
14%
7
4. 250-360
0
0%
1
14%
6
86%
7
5. 5.2.5.1
Crash Frequency and Characteristics 70 Table 21 Crash Severity by Visibility Bin, updated to with 2015, 2010-2015 Visibility Bin
Fatal
Injury
Fatal +
Injury
Property
Damage Only
Total
1. >645
9
2%
124
22%
133
23%
433
77%
566
All Low Visibility
5
8%
24
39%
29
47%
33
53%
62
2. 495-645
2
20%
4
40%
6
60%
4
40%
10
3. 360-495
0
0%
2
29%
2
29%
5
71%
7
4. 250-360
1
14%
3
43%
4
57%
3
43%
7
5. 155-250
2
5%
15
41%
17
46%
20
54%
37
6. <155
0
0%
0
0%
0
0%
1 100%
1
ERROR, NO VISIBILITY READING
0
0%
2
50%
2
50%
2
50%
4
All Conditions
14
2%
150
24%
164
26%
468
74%
632
Table 22 Crash Severity by Visibility Bin, October 2016-August 2017
Visibility Bin
Fatal
Injury
Fatal +
Injury
Property
Damage Only
Total
1. >645
1
1%
20
23%
21
24%
66
76%
87
All Low Visibility
0
0%
1
50%
1
50%
1
50%
2
2. 495-645
0
0%
0
0%
0
0%
0
0%
0
3. 360-495
0
0%
0
0%
0
0%
0
0%
0
4. 250-360
0
0%
0
0%
0
0%
0
0%
0
5. 155-250
0
0%
1
50%
1
50%
1
50%
2
6. <155
0
0%
0
0%
0
0%
0
0%
0
ERROR, NO VISIBILITY READING
0
0%
0
0%
0
0%
0
0%
0
All Conditions
1
1%
21
24%
22
25%
67
75%
89 Visibility Bin
Fatal
Injury
Fatal +
Injury
Property
Damage Only
Total
1. >645
9
2%
124
22%
133
23%
433
77%
566
All Low Visibility
5
8%
24
39%
29
47%
33
53%
62
2. 495-645
2
20%
4
40%
6
60%
4
40%
10
3. 360-495
0
0%
2
29%
2
29%
5
71%
7
4. 250-360
1
14%
3
43%
4
57%
3
43%
7
5. 155-250
2
5%
15
41%
17
46%
20
54%
37
6. <155
0
0%
0
0%
0
0%
1 100%
1
ERROR, NO VISIBILITY READING
0
0%
2
50%
2
50%
2
50%
4
All Conditions
14
2%
150
24%
164
26%
468
74%
632
Table 22 Crash Severity by Visibility Bin, October 2016-August 2017
Visibility Bin
Fatal
Injury
Fatal +
Injury
Property
Damage Only
Total
1. 5.2.5.1
Crash Frequency and Characteristics 155-250
1
3%
19
51%
17
46%
37
6. <155
1
100%
0
0%
0
0%
1
ERROR, NO VISIBILITY READING
2
50%
2
50%
0
0%
4
All Conditions
310
49%
240
38%
82
13%
632 71 Table 24 Number of Vehicles Involved by Visibility Bin, October 2016- August 2017 Table 24 Number of Vehicles Involved by Visibility Bin, October 2016- August 2017
Visibility Bin
1
2
3+
Total
1. >645
37
43%
39
45%
11
13%
87
All Low Visibility
0
0%
0
0%
2
100%
2
2. 495-645
0
0%
0
0%
0
0%
0
3. 360-495
0
0%
0
0%
0
0%
0
4. 250-360
0
0%
0
0%
0
0%
0
5. 155-250
0
0%
0
0%
2
100%
2
6. <155
0
0%
0
0%
0
0%
0
ERROR, NO VISIBILITY READING
0
0%
0
0%
0
0%
0
All Conditions
37
42%
39
44%
13
15%
89
Table 25 Crash Type by Visibility Bin, updated to include 2015, 2010-2015
Visibility Bin
Rear End
Fixed
Object- Off
Road
Angle
SideSwipe
- Same
Direction
Other
Total
1. >645
138
24% 213
38% 30
5%
59
10% 126
22%
566
All Low Visibility
39
63%
3
5% 10 16%
7
11%
3
5%
62
2. 495-645
4
40%
1
10%
4 40%
0
0%
1
10%
10
3. 360-495
2
29%
0
0%
1 14%
2
29%
2
29%
7
4. 250-360
7
100%
0
0%
0
0%
0
0%
0
0%
7
5. 155-250
26
70%
1
3%
5 14%
5
14%
0
0%
37
6. <155
0
0%
1 100%
0
0%
0
0%
0
0%
1
ERROR, NO VISIBILITY
READING
1
25%
3
75%
0
0%
0
0%
0
0%
4
All Conditions
178
28% 219
35% 40
6%
66
10% 129
20%
632
Table 26 Crash Type by Visibility Bin, October 2016- August 2017
Visibility Bin
Rear End
Fixed
Object- Off
Road
Angle
SideSwipe
- Same
Direction
Other
Total
1. >645
29
33%
28
32%
2
2%
16
18%
12
14%
87
All Low Visibility
2
100%
0
0%
0
0%
0
0%
0
0%
2
2. 495-645
0
0%
0
0%
0
0%
0
0%
0
0%
0
3. 360-495
0
0%
0
0%
0
0%
0
0%
0
0%
0
4. 250-360
0
0%
0
0%
0
0%
0
0%
0
0%
0
5. 5.2.5.1
Crash Frequency and Characteristics 155-250
2
100%
0
0%
0
0%
0
0%
0
0%
2
6. <155
0
0%
0
0%
0
0%
0
0%
0
0%
0
ERROR, NO VISIBILITY
READING
0
0%
0
0%
0
0%
0
0%
0
0%
0
All Conditions
31
35%
28
31%
2
2%
16
18%
12
13%
89 72 Comparing Table 21 and Table 22, crash severity by visibility bin during low visibility
conditions show about an even split between fatal and injury crashes and PDOs in both the
before and after periods. Although proportions are similar, the low frequency in the after
period as compared to the 10 crashes per year average in the before conditions suggest
reductions in overall frequency. Crash severity by visibility bin for clear conditions show crash
severity percentages in the after period remained within 1% of the before period indicating
that overall conditions in the after period are characteristic of corridor. Thus, changes observed
for low visibility conditions must be related to the activation of VSL system. Percentages of crashes with different number of vehicles involved during low visibility
conditions in Table 23 and Table 24 show that in the before period 44% of crashes involved 3+
vehicles versus 100% of crashes in the after period. Similarly, a shift in crash type to all rear-end
crashes in the after period also occurred. Given that only two crashes occurred in the after
period during low visibility, it is difficult to draw conclusions from this data. 5.2.5.2
Crash Rates Crash rates were updated for 2015 with continuous count data and new crash rates
were developed for the after period. Table 27 provides updated crash rates by visibility for the
now including 2015 period. In 2015, there were 10% missing visibility data. The 2015 crash rates
for all visibility conditions of 47.1 and 58.5 crashes per 100 million VMT for NB and SB,
respectively, are lower than for the 2010-2014 average. When combined these rates lower the
overall average, the new 2010-2015 average drops from 63 and 74 crashes per 100 million VMT
to 49.3 and 58.8 crashes per 100 million VMT for NB and SB, respectively. The crash rates for all
low visibility conditions also show rates of the same magnitude as those found as those of Crash rates were updated for 2015 with continuous count data and new crash rates
were developed for the after period. 5.2.5.1
Crash Frequency and Characteristics Table 27 provides updated crash rates by visibility for the
now including 2015 period. In 2015, there were 10% missing visibility data. The 2015 crash rates
for all visibility conditions of 47.1 and 58.5 crashes per 100 million VMT for NB and SB, respectively, are lower than for the 2010-2014 average. When combined these rates lower the
overall average, the new 2010-2015 average drops from 63 and 74 crashes per 100 million VMT
to 49.3 and 58.8 crashes per 100 million VMT for NB and SB, respectively. The crash rates for all
low visibility conditions also show rates of the same magnitude as those found as those of low visibility conditions also show rates of the same magnitude as those found as those of 73 2010-2014, with updated values of 181 and 854 crashes per 100 million VMT for low visibility
conditions for the NB and SB directions respectively. 2010-2014, with updated values of 181 and 854 crashes per 100 million VMT for low visibility
conditions for the NB and SB directions respectively. Table 28 provides crash rates for the after period. Since all times outside VSL activations
are considered clear conditions, there are less missing variables. For the individual 155-200 ft. visibility bin that the two crashes were found in, the crash rate of 2226.6 crashes per 100
million VMT is nearly as large as the 2010-2015 crash rate for the same bin of 2779.7 crashes
per 100 million VMT. However, for the All Low Visibilities crash rate is lower in the post-VSL
period than 2010-2015, dropping from 854.1 to 366.8 crashes per 100 million VMT. The overall
crash rates for combined directions remain consistent in about 59 crashes per 100 million VMT,
but crash rate per direction shifts. While as expected the crash rate for southbound direction
lowered to 48.7, the northbound direction saw an increase in crashes to 72.3 crashes per 100
million VMT. The reduction of crash rate during low visibility conditions agrees with the notion of
increased safety first indicated in the crash characteristics discussion. Considering VMTs and
exposure to low visibility conditions, this reduction can be interpreted to mean that without the
VSL system 2.3 times more crashes can be expected for every 100 VMT travel under low
visibility conditions. 5.2.5.1
Crash Frequency and Characteristics Thus if results are transferrable, for VSL systems for low visibility conditions
implemented in corridors with larger AADTs, longer corridor lengths, or with more severe fog
exposure, greater reduction in frequency of crashes could be expected. The primary limitation of this crash rate calculation lies within the police reporting. Due
to the varied spatiotemporal nature of fog, should time and location in the be inaccurate,
crashes cannot be correctly matched to appropriate visibility reading. Though having 74 continuous volume data is an advantage over the crash rates from 2010-2014, the visibility
readings come at a different rate and are matched assuming linear relationship between
visibility readings and visibility stations. The interval between visibility readings are close
enough in time and stations in the most fog prone are close enough together that this
assumption is not expected to be of major concern. Table 27 Updated 2010-2015 Crash Rates
2010-2015
Number of Crashes
Crash Rate
% of Total VMT
North
South
Both
North
South
Both
North
South
Both
>645
278
288
566
52.9
56.4
54.6
45%
44%
89%
ALL LOW VISIBILITY
12
54
66
181.0
854.1
509.5
0.57%
0.54%
1.11%
495-645
0
10
10
0.0
619.9
302.8
0.14%
0.14%
0.28%
360-495
2
5
7
107.4
281.6
192.4
0.16%
0.15%
0.31%
250-360
1
6
7
60.0
375.7
214.4
0.14%
0.14%
0.28%
155-250
6
31
37
514.3
2795.7
1626.0
0.10%
0.09%
0.19%
<155
1
0
1
409.1
0.0
211.7
0.02%
0.02%
0.04%
Error/No Reading
2
2
4
3.5
3.1
3.3
4.83%
5.51%
10.34%
ALL VISIBILITY
290
342
632
49.3
58.8
54.0
50%
50%
100%
Table 28 Crash Rates October 2016- August 2017
Oct 2016- Aug 2017
Visibility Bin
Number of Crashes
Crash Rate
% of Total VMT
North South Both
North
South
Both
North
South
Both
>645
55
33
88
75.7
45.4
60.6
48%
48%
96%
ALL LOW VISIBILITY
0
2
2
0.0
366.8
165.2
0.44%
0.36%
0.80%
495-645
0
0
0
0.0
0.0
0.0
0.11%
0.12%
0.23%
360-495
0
0
0
0.0
0.0
0.0
0.12%
0.10%
0.22%
250-360
0
0
0
0.0
0.0
0.0
0.12%
0.08%
0.19%
155-250
0
2
2
0.0
2226.6
885.7
0.09%
0.06%
0.15%
<155
0
0
0
0.0
0.0
0.0
0.00%
0.00%
0.00%
Error/No Reading
0
0
0
0.0
0.0
0.0
1.78%
1.86%
3.64%
ALL VISIBILITY
55
35
90
72.3
46.0
59.2
50%
50%
100% Table 28 Crash Rates October 2016- August 2017 75 5.3 SUMMARY OF RESULTS Overall, with the introduction of the VSL, positive effects have been seen in driver
speeds and limited insights into crash characteristics. Before-after analysis at one site along the
corridor, showed statistically significant reductions in speeds across all visibility ranges of
approximately 2 to 5 mph for every range. Reductions in variance across all visibility ranges
suggest that safety has improved. Speed models reconfirmed speed is inversely correlated with
visibility and that the VSL has had a role in further reducing speeds. Though R2 values are not as
strong as the models before, there is a trend in decreasing speeds visibility and the speeds post-
VSL activation adhere more closely SSD safe speeds. Exploring speeds throughout the corridor post-VSL activation also showed reductions in
speed with reduced posted speed limits. While observed speeds varied by location and posted
speed throughout the corridor, a general trend arises where drivers do not appear to comply as
strongly with reduced speeds until they experience fog. There appears to be a delayed reaction
to VSL until drivers traverse fog and see the present need for reduced speeds. Additionally,
though speeds do decrease with decreasing posted speeds, compliance decreases as posted
speed limits are reduced. Crash analysis for the year immediately prior to VSL resulted in similar crash
characteristics as in the previous years. Crash analysis for the post-VSL data showed signs of
improvement. The only two crashes that were matched to fog conditions, occurred back-to-
back during a fog event within 3 months of first activation. Though this contributed to the crash
rate, the southbound crash rate for all low visibility conditions was cut by more than half and
the overall crash rate decreased to less than 45.4 crashes per 100 million VMT. More 76 interestingly crash rates for all low visibility conditions in the SB direction were reduced 2.3
times. Taking exposure into consideration, this figure alone suggests the system has been
successful in improving safety. 5.4 VSL ALGORITHM ASSESSMENT 5.4 VSL ALGORITHM ASSESSMENT The positive initial results from this analysis beg the question if compliance can be
further improved. It is possible that the VSL control algorithm could be modified to attain better
results. Currently, the VSL algorithm uses a step-adjusted model fit of pre-VSL observed speeds
to generate proposed VSLs and smooths speeds throughout the corridor subject to a step range
where successive VSL signs cannot decrease by more than 15 mph. The VSL step-adjusted
model fit set as was an intermediate step between previously observed speeds and SSD safe
speeds due to a concern that simply posting SSD safe speeds would not adequately alter driver
behavior and instead would further increase speed variance and interactions between vehicles
under low visibilities. The 15 mph step range between VSL signs was chosen over 10 mph as it
was thought to ensure the message remained credible to the motorists (Kimley-Horn, I-77
Variable Speed Limit: Methodology for Establishing Variable Speed Limits, 2015). The analysis
has shown, however, that the trend line for mean observed speeds falls below SSD safe speeds
until about 50 mph and is consistently below the VSL step-adjusted model fit for the available
visibilities. To improve these promising initial findings, compliance could be improved by
changing some of the VSL algorithm parameters. It appears drivers do not sense the need to slow down until they encounter the fog and
by then difference in observed and posted speeds is too great to slow down to posted speeds. By the time drivers have slowed enough, posted speeds raise back up but by now drivers have 77 slowed down enough to maintain more constant speeds. Based on this apparent delayed
reaction to VSL until drivers encounter the fog, the key to achieving increased compliance
across the whole corridor may lie in improving compliance prior to worst fog area. One way to
do so is by changing the step range between VSL signs. Lowering the step range to 10 mph
would cause to lower the speeds well in advance of actual fog. Though the concern of
credibility is still present, perhaps having a more seeing a more gradual decrease in speed will
give drivers time to really consider taking heed of the speed limits rather than encountering a
more sudden drop in speeds. 5.4 VSL ALGORITHM ASSESSMENT For example, from 60 to 45 to 30 mph from MPs 9.0 to 8.1 to 7.3
to could instead 60 to 50 to 40 to 30 starting at MP 10.2 instead. This may lead to decreased
compliance in the entering region but improved compliance in the worst fog region where it
matters most. However, unless VSL signs are closer this may not be a viable option given the
distance between VSL signs would cause speed reductions to need to be posted far upstream. An intermediate route to maintain credibility but have step ranges that more readily adjust to
declining conditions is to have variable step ranges depending on severity of fog. Should fog
conditions warrant the lowest VSLs of 30 mph, step ranges of 10 mph begin to gradually
decrease far in advance but for reduced visibilities were a step range of 15 mph could still
adequately decrease posted speeds, the step range remain at 15 mph. slowed down enough to maintain more constant speeds. Based on this apparent delayed
reaction to VSL until drivers encounter the fog, the key to achieving increased compliance
across the whole corridor may lie in improving compliance prior to worst fog area. One way to
do so is by changing the step range between VSL signs. Lowering the step range to 10 mph
would cause to lower the speeds well in advance of actual fog. Though the concern of
credibility is still present, perhaps having a more seeing a more gradual decrease in speed will
give drivers time to really consider taking heed of the speed limits rather than encountering a
more sudden drop in speeds. For example, from 60 to 45 to 30 mph from MPs 9.0 to 8.1 to 7.3
to could instead 60 to 50 to 40 to 30 starting at MP 10.2 instead. This may lead to decreased
compliance in the entering region but improved compliance in the worst fog region where it
matters most. However, unless VSL signs are closer this may not be a viable option given the
distance between VSL signs would cause speed reductions to need to be posted far upstream. An intermediate route to maintain credibility but have step ranges that more readily adjust to
declining conditions is to have variable step ranges depending on severity of fog. 5.4 VSL ALGORITHM ASSESSMENT Should fog
conditions warrant the lowest VSLs of 30 mph, step ranges of 10 mph begin to gradually
decrease far in advance but for reduced visibilities were a step range of 15 mph could still
adequately decrease posted speeds, the step range remain at 15 mph. Another possible way to improve the model is readjust the steps to follow VSL more
closely. Currently the model has a 5 mph decrease from the model fit when SSD safe speeds
equal 50 and a 10 mph decrease from model fit when SSD Safe speeds equal 40 mph. One close
alternate could be to have an additional step and shift step boundaries, so instead of stepping
down 5 mph starting SSD safe speeds of 50 mph, start the shift at 55 mph, with additional 5 78 mph reductions at SSD safe speeds of 45 and 35. Figure 15 depicts this adjusted model. This
adjusted model fit would follow yield speeds much closer to SSD safe speeds and at lower
visibilities would yield speeds closer to the observed mean speeds in the post-VSL period. Seeing that average observed speeds are falling below the current VSL step-adjusted model,
this proposed readjusted model should continue to have observed speeds lower if not equal to
the model fit. Though likely unfeasible at this stage of system deployment, perhaps having more
densely distributed VSL signs may achieve the improve compliance desired. System evaluation
of CalTrans’ FDWS with its VSLs spaced ¼ mile apart would shed light on how effective this is. Apart from changes to the physical system, improved compliance in the system will likely have
to involve VSL algorithm modifications. Apart from changes to the physical system, improved compliance in the system will likely have
to involve VSL algorithm modifications. Figure 15 Proposed Modification to VSL Figure 15 Proposed Modification to VSL 79 6.1.1 Driver Speed Choice Before-after analysis at a single site showed statistically significant reductions in speeds
of 5 mph and standard deviations of 1-2 mph for reduced visibilities of 250 – 645 ft. While
ideally a before-after comparison at all locations throughout the corridor would show a clearer
picture of how speeds changed as a result of the VSLs, having data for the southbound location
at the edge of the worst fog area gives an indication of how drivers may have reacted at the
rest of the locations. Though speeds are still higher than SSD safe speeds at MP 4.4 , the
combination of reduced speeds and reduced variances imply increased safety in the corridor. Regression models related speeds as function of visibility showing that when VSL is active,
visibility’s influence to decrease speeds increased by 60%. The development of a model similar
to McCann and Fontaine’s suggests that incorporating the driver behavior model into the VSL
algorithm had an effect on compliance. There were no periods of reduced visibilities below 250
ft. to speak of safety at the periods of worst visibility, but trend lines suggest improvements in
those visibilities are likely. Due to limitations in the way data were reported, compliance figures could not be
determined. However, a surrogate measure of compliance could be inferred from the
differentials in observed and posted speeds. Analysis of speeds along the corridor revealed an
apparent lag in reaction to the VSLs, with drivers continuing to travel above the posted speed
limit until they were amid the fog and could visually confirm the need for reduced speeds. Upon 80 entering the corridor, differences in observed mean speeds and posted speeds were within 10
mph and increased to as much as 23 mph over the posted speeds in the thick of the fog, until
leveling out again and returning to between within 10 mph of posted speeds. Understanding
what causes this delay in compliance will be key to knowing how achieve higher compliances. Additionally, analysis of speed along corridor also revealed that at the lowest posted speeds
compliance decreases. This is seen more prominently with posted speed under 50 mph
between mileposts 7.3 and 4.4. If greater compliance can be achieved upstream the worst fog
zone, it is possible that compliance in the lower speed limits can also be improved. 6.1.1 Driver Speed Choice Overall, reductions in means speeds are an indication that the VSL is achieving its
desired effect and could be modified to maximize results. There is an apparent lack of response
to VSLs until drivers can visually confirm the need for slower speeds, however. The 11 months’ worth of crash data is not sufficient enough to make definitive claims as
to the system’s effectiveness in improving safety by reducing the overall frequency and severity
of crashes. However, given that the only fog-related crashes occurred early in the system’s
activation lifetime and that these event were correlated, suggests the reductions in crash rates
are indicative of the improved safety under the system. It is possible that with the system in
place the severity and number of vehicles involved in these multi-vehicle crashes was
minimized. The crash rate of all low visibility bins for the southbound direction of 366.8 crashes
per 100 million VMT, while still greater during reduced visibilities than in clear conditions, is less
than half of the crash rate of 854 crashes per 100 million VMT seen for the years 2010-2015. If 81 not the frequency alone, the crash rates for the post-VSL period for low visibilities indicate that
safety has improved in a tangible way. not the frequency alone, the crash rates for the post-VSL period for low visibilities indicate that
safety has improved in a tangible way. 6.2 RECOMMENDATIONS 6.2 RECOMMENDATIONS Based on this study several recommendations can be made: 1. Pursue modifications to VSL control algorithm. The methodology for the VSL control
algorithm allows for modifications as the operators see fit. Now that almost a full
year of VSL operations has passed, these initial findings indicate that the VSL
algorithm could be altered to see improved compliances. Considering alternate step
ranges between successive VSL signs or even step ranges that vary by fog severity
may trade off compliance entering the corridor for improved compliance in fog zone. Alternatively, the step ranges of the VSL model fit could be altered to more closely
reflect SSD safe speeds by shifting step boundaries and adding one more step. 2. Decrease distances between VSL devices in future deployments. Although not
necessarily applicable for this system, more closely spaced together VSL signs may
be part of the answer to understand the lag in speed reductions before entering fog
zones. Agencies looking to implement similar systems should consider densely
placing VSL signs to give greater advanced warning, if feasible within budget. The
spatial variance of fog prompts RWIS stations to be as densely spaced as possible,
also. If an existing system already has static (signage and lane departure) countermeasures implemented as I-77 did and traffic conditions are similar, then the
addition of a VSL system is likely to have positive results. countermeasures implemented as I-77 did and traffic conditions are similar, then the
addition of a VSL system is likely to have positive results. 82 3. Reevaluate safety analysis in future. This study was limited to the first year of
operations of the system. As time elapses and more data accumulates, regression to
the mean bias will adjust for extreme scenarios that may have taken place and larger
sample sizes will give more weight to analysis results. If less aggregated data is
available for future studies, more conclusive measures can be derived. A follow-up
evaluation will confirm if early emerged trends continue. 6.3 FUTURE RESEARCH The positive initial findings of the effectiveness of the I-77 VSLs suggest that the system
has been successful in influencing driver behavior. Of greatest concern for future research is
understanding why the lag in speed reductions prior to entering the fog zone. Investigating how
to better reduce speeds in advance of the densest fog zones to achieve greater compliance
would be most beneficial to the system. Modifications to VSL algorithm may shed light into this
issue. Since the southbound direction was found to be of greater concern during low visibility
conditions, this analysis focused mainly on the effects of the system on driver speeds and
behavior on it. Future research would also entail analyzing how the system affected the
northbound direction and additional investigation as to the increased crash rates for this
direction. Although this project applies to one particular implementation of a low visibility
activated VSL system, the benefits observed at this site may serve as a reference for other 83 agencies contemplating alternatives for fog mitigation. Compliance figures based on individual
vehicle speed data would be needed if recommendations are to be made on this. With the advent of connected vehicle (CV) technology, other methods to achieve speed
harmonization could affect the value of a system like this. If the connected vehicles need only
communicate with each other, the risks associated with limited visibilities would be lessened
since vehicles would know others’ positions and speeds and real time visibility readings at
ground level. Should connected vehicles be communicating with infrastructure that sets the
speed limits, compliance is expected to increase. CVs themselves could be used as probes
providing visibility reading at ground level throughout the entirety of corridor. The more CVs in
the corridor the more accurate the speed recommendations. However, assuming not all
vehicles in the corridor are connected, VSL signage would still be required. Depending on how
non-CV compliance in the corridor is, recommended speeds would still have to be closer to
observed speeds and not just SSD speeds, as variances are prone to more potential conflicts
which could be riskier than just non-compliance. Future research in this area could involve
simulating driver speeds based on current/proposed VSL algorithms. Benefits of connected
vehicle environments versus infrastructure based approaches are emerging research areas. 84 REFERENCES 1. Hamilton, B., B. Tefft, L. Arnold, and J. Grabowski. Hidden Highways: Fog and Traffic Crashes on
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Fog and Smoke. Lisbon, 2010. 3. Lynn, C., C. Schreiner, and R. Campbell. Reducing Fog-Related Crashes on The Afton and Fancy Gap
Mountain Sections of I-64 and I-77 in Virginia. Charlottesville, VA, 2002. 4. McDonald, C. Active Traffic and Safety Management System for Interstate 77 in Virginia. July 2015. 5. URS. Concept of Operations for the I-77 Corridor at Fancy Gap. 2012. pt of Operations for the I-77 Corridor at Fancy Gap. 2012. 6. McCann, K., and M. D.P.E.P.D. Fontaine. Investigation of Driver Speed Choice and Crash
Characteristics During Low Visibility Events. Charlottesville, VA, 2016. 7. Snowden, R. J., N. Stimpson, and R. A. Ruddle. Speed Perception Fogs Up As Visibility Drops. Nature,
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Performance in SImulated Foggy Conditions. Accident Analysis and Prevention, 2011, pp. 698-705. 9. Liang, W. L., M. Kyte, F. Kitchener, and P. Shannon. Effect of Environmental Factors on Driver Speed:
A Case Study. Transportation Research Record, 1998. 10. MacCarley, A. Evaluation of Caltrans District 10 Automated Warning System: Year Two Progress
Report. 1999. 11. Goodwin, L. Best Practices for Road Weather Management, Version 2.0. Washington, DC, 2003. 12. MacCarley, C.A., C. Ackles, and T. Watts. Highway Traffic Response to Dynamic Fod Warning and
Speed Advisory Messages. Transportation Research Record: Journal of the Transportation Research
Board, 2006, pp. 95-104. 13. Final Report, Instrumentation and Evaluation of District 10 CalTrans Automated Warning System
(CAWS). San Luis Obispo, California, 2005. 14. Traffic Control in Adverse Weather. ITS Decision, 2005. http://fresno.ts.odu.edu/newitsd/
ITS_Serv_Tech/weather_app/weather_applications_weather_traffic_control_report5.html. 15. Kyte, M., P. Shannon, and F. Kitchener. Idaho Storm Warning System Operational Test. 2000. 16. Murphy, R., R. Swick, and G. Guevara. Best Practices for Road Weather Management, Version 3.0. 85 Washington DC, 2012. Washington DC, 2012. g Detection and Warning System. January 30, 2013. 17. Liu, J. Fog Detection and Warning System. January 30, 2013. 18. Balke, K., P. Songchitruksa, H. Liu, R. Brydia, D. Jasek, and R. Benz. Concept For Managing Freeway
Operations During Weather Events. 2007. 19. Abdek-Aty, M., M. Ahmed, J. Lee, Q. Shi, and M. Abuzwidah. Synthesis of Visibility Detection
Systems. Orlando, 2012. 20. Gimmestad, G. Development of a Prototype Development of a Prototype Adverse Visibility Warning
Adverse Visibility Warning and Control System for and Control System for Operational Evaluation
Operational Evaluation. 2004. 21. Perrin, J., P. T. Martin, and B. Coleman. Testing the Adverse Visibility Information System Evaluation
(ADVISE) - Safer Driving in Fog. 2000. 22. Kimley-Horn. Synthesis of Practice for Weather-Related Variable Speed Limit Systems in the United
States. 2014. 23. Olson, C. S., J. Peters, and J. Crain. Oregon DOT Weather-Responsive Active Traffic Management. in
Joint Western/Midwestern District ITE Annual Meeting, 2014. 24. Han, C., J. Luk, V. Pyta, and P. Cairney. Best Practice for Variable Speed Limits: Literature Review. 2009. 25. Robinson, M. Examples of Variable Speed Limit Applications. in TRB 79th Annual Meeting, 2000. 26. California Center for Innovative Transportation. Weather Applications - Traffic Control in Adverse
Weather. ITS Decision, 2005. http://fresno.ts.odu.edu/newitsd/ITS_Serv_Tech/weather_app/
weather_applications_weather_traffic_control_report.html. 27. Kimley-Horn. I-77 Variable Speed Limit Standard Operating Procedures. 2015. 28. Kimley-Horn. I-77 Variable Speed Limit: Methodology for Establishing Variable Speed Limits. 2015. 86 Presentations Presentations
“Effectiveness of I-77 Fancy Gap Variable Speed Limits”, ITSVA/VASITE Joint Conference,
Richmond, VA, 2017. “Impact of a Visibility Based Variable Speed Limit on Driver Speeds”, SHPE National Conference,
Kansas City MO, 2017. “Impact of a Variable Speed Limit System on Driver Speeds During Low Visibility Conditions”,
Accepted for Poster Presentation, Publication Decision Pending. TRB Annual Meeting,
Washington D.C., 2018. “Effectiveness of I-77 Fancy Gap Variable Speed Limits”, ITSVA/VASITE Joint Conference,
Richmond, VA, 2017. “Impact of a Visibility Based Variable Speed Limit on Driver Speeds”, SHPE National Conference,
Kansas City MO, 2017. “Impact of a Variable Speed Limit System on Driver Speeds During Low Visibility Conditions”,
Accepted for Poster Presentation, Publication Decision Pending. TRB Annual Meeting,
Washington D.C., 2018. 87 APPENDIX B: EXCERPTS FROM MCCANN & FONTAINE 88 89 APPENDIX C: ADDITIONAL TABLES APPENDIX C: ADDITIONAL TABLES Updated 2015 charts
-0.5%
0.5%
1.5%
2.5%
3.5%
4.5%
5.5%
0
50
100
150
200
250
300
350
400
450
500
1.2
1.8
2.7
3
4.4
5.3
6.6
7.3
9
9.6
11.3
16.9
Percentage of Time During the Year
Hours
Mile Post
495-645
360-495
250-360
155-250
<155
% Updated 2015 charts
Figure 16 I-77 2015 Visibility Profile
Table 29 Crash Severity by Visibility Bin, 2015
Visibility Bin
Fatal
Injury
Fatal +
Injury
Property
Damage Only
Total
1. >645
0
0%
19
18%
19
18%
85
82%
104
All Low Visibility
0
0%
1
25%
1
25%
3
75%
4
2. 495-645
0
0%
0
0%
0
0%
0
0%
0
3. 360-495
0
0%
0
0%
0
0%
2 100%
2
4. 250-360
0
0%
1
50%
1
50%
1
50%
2
5. 155-250
0
0%
0
0%
0
0%
0
0%
0
6. <155
0
0%
0
0%
0
0%
0
0%
0
ERROR, NO VISIBILITY
READING
0
0%
0
0%
0
0%
0
0%
0
All Conditions
0
0%
20
19%
20
19%
88
81%
108
-0.5%
0.5%
1.5%
2.5%
3.5%
4.5%
5.5%
0
50
100
150
200
250
300
350
400
450
500
1.2
1.8
2.7
3
4.4
5.3
6.6
7.3
9
9.6
11.3
16.9
Percentage of Time During the Year
Hours
Mile Post
495-645
360-495
250-360
155-250
<155
% Updated 2015 charts 90 Table 30 Number of Vehicles Involved by Visibility Bin, 2015 Table 30 Number of Vehicles Involved by Visibility Bin, 2015
Visibility Bin
1
2
3+
Total
1. >645
56
54%
37
36%
11
11%
104
All Low Visibility
1
25%
2
50%
1
25%
4
2. 495-645
0
0%
0
0%
0
0%
0
3. 360-495
1
50%
1
50%
0
0%
2
4. 250-360
0
0%
1
50%
1
50%
2
5. 155-250
0
0%
0
0%
0
0%
0
6. <155
0
0%
0
0%
0
0%
0
ERROR, NO VISIBILITY READING
0
0%
0
0%
0
0%
0
All Conditions
57
53%
39
36%
12
11%
108
Table 31 Crash Type by Visibility Bin, 2015
Visibility Bin
Rear End
Fixed
Object-
Off Road
Angle
SideSwipe -
Same
Direction
Other
Total
1. >645
22
21%
35
34%
7
7%
14
13%
26
25%
104
All Low Visibility
2
50%
0
0%
0
0%
1
25%
1
25%
4
2. APPENDIX C: ADDITIONAL TABLES 495-645
0
0%
0
0%
0
0%
0
0%
0
0%
0
3. 360-495
0
0%
0
0%
0
0%
1
50%
1
50%
2
4. 250-360
2
100%
0
0%
0
0%
0
0%
0
0%
2
5. 155-250
0
0%
0
0%
0
0%
0
0%
0
0%
0
6. <155
0
0%
0
0%
0
0%
0
0%
0
0%
0
ERROR, NO VISIBILITY
READING
0
0%
0
0%
0
0%
0
0%
0
0%
0
All Conditions
24
22%
35
32%
7
6%
15
14%
27
25%
108
Table 32 2015 Crash Rates
Visibility Bin
Number of Crashes
Crash Rate
% of Total VMT
North
South
Both
North
South
Both
North
South
Both
>645
47
57
104
51.9
62.8
57.4
44%
44%
89%
ALL LOW VISIBILITY
1
3
4
127.5
393.0
258.4
0.38%
0.37%
0.76%
495-645
0
0
0
0.0
0.0
0.0
0.12%
0.12%
0.24%
360-495
1
1
2
384.8
398.9
391.8
0.13%
0.12%
0.25%
250-360
0
2
2
0.0
1076.0
531.4
0.09%
0.09%
0.18%
155-250
0
0
0
0.0
0.0
0.0
0.04%
0.04%
0.08%
<155
0
0
0
0.0
0.0
0.0
0.00%
0.00%
0.00%
Error/No Reading
0
0
0
0.0
0.0
0.0
5.16%
5.39%
10.54%
ALL VISIBILITY
48
60
108
47.1
58.5
52.8
50%
50%
100%
|
https://openalex.org/W4312124059
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https://www.mdpi.com/2076-3417/12/24/12674/pdf?version=1670985006
|
English
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Off-Axis Holographic Interferometer with Ensemble Deep Learning for Biological Tissues Identification
|
Applied sciences
| 2,022
|
cc-by
| 5,322
|
Keywords: digital hologram; holographic scanner; deep learning; biological specimen recognition;
wavefront recognition; complex object wavefront; interferometer Citation: Lam, H.; Zhu, Y.;
Buranasiri, P. Off-Axis Holographic
Interferometer with Ensemble Deep
Learning for Biological Tissues
Identification. Appl. Sci. 2022, 12,
12674. https://doi.org/10.3390/
app122412674 Communication
Off-Axis Holographic Interferometer with Ensemble Deep
Learning for Biological Tissues Identification Hoson Lam 1,*
, Yanmin Zhu 2
and Prathan Buranasiri 3 1
Department of Electrical Engineering, City University of Hong Kong, Hong Kong SAR, China
2
Department of Electrical and Electronic Engineering, The University of Hong Kong, Hong Kong SAR, China
3
Physics Department, School of Science, King Mongkut’s Institute of Technology Ladkrabang,
Bangkok 10520, Thailand
*
Correspondence: hoson@live.hk 1
Department of Electrical Engineering, City University of Hong Kong, Hong Kong SAR, China
2
Department of Electrical and Electronic Engineering, The University of Hong Kong, Hong Kong SAR, China
3
Physics Department, School of Science, King Mongkut’s Institute of Technology Ladkrabang,
Bangkok 10520, Thailand
*
Correspondence: hoson@live.hk Abstract: This paper proposes a method with an off-axis interferometer and an ensemble deep
learning (I-EDL) hologram-classifier to interpret noisy digital holograms captured from the tissues
of flawed biological specimens. The holograms are captured by an interferometer, which serves as
a digital holographic scanner to scan the tissue with 3D information. The method achieves a high
success rate of 99.60% in identifying the specimens through the tissue holograms. It is found that
the ensemble deep learning hologram-classifier can effectively adapt to optical aberration coming
from dust on mirrors and optical lens aberrations such as the Airy-plaque-like rings out-turn from
the lenses in the interferometer. The deep learning network effectively adapts to these irregularities
during the training stage and performs well in the later recognition stage without prior optical
background compensations. The method does not require an intact sample with a full outline shape
of the specimens or the organs to understand the objects’ identities. It demonstrates a new paradigm
in object identification by ensemble deep learning through a direct wavefront recognition technique. 1. Introduction The advancements in optics and computing technologies have enabled digital holo-
grams of physical three-dimensional (3D) objects to be captured and analyzed at high speed
and achieve close to real-time response performance. Holograms can be displayed with a
spatial light modulator to reconstruct a visible image and is an ideal solution for recording,
storing, and displaying 3D objects in the digital world. However, a hologram comprises
high-frequency fringe patterns and is almost impossible to recognize with traditional com-
puter vision methods. Furthermore, in many practical situations, intact extraction of a
biological specimen or organ is not feasible, and therefore the object’s identity cannot be
inferred directly from its outline shape. However, a digital holographic interferometer is
an effective hologram-capturing device to examine the microstructure inside a specimen. Furthermore, the off-axis configuration simplifies the difficulties of separating a hologram’s
zero-order image from the two conjugate virtual and real images in Fourier space. Academic Editor: Maria Antonietta
Ferrara applied
sciences applied
sciences Academic Editor: Maria Antonietta
Ferrara Received: 6 November 2022
Accepted: 8 December 2022
Published: 10 December 2022 Received: 6 November 2022
Accepted: 8 December 2022
Published: 10 December 2022 Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. During the early stage of optical hologram classification methods, almost all methods
were based on correlation, where a targeted hologram is matched against a library of
reference hologram templates. The matching is generally realized with optical correlation
and only results in a high matching score if a pair of objects have similar poses and depth. Most of these approaches are sensitive to positional shifts and deformations. The methods
and the problems are described in detail by VanderLugt works in [1] and the articles in [2–5]. The hologram classifier employed in this paper is EDL-IOHC [6], in which a modern
deep learning approach is employed. The feature extraction process of the deep learning Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). https://www.mdpi.com/journal/applsci Appl. Sci. 2022, 12, 12674. https://doi.org/10.3390/app122412674 Appl. Sci. 2022, 12, 12674 2 of 10 approach is fully automated. The method solves most of the shift-invariant problems
within a single framework with the capability to handle occlusion problems effectively. Nowadays, some researchers are using the common term Hologram interchangeably
with the terms ‘Raw Hologram’ and ‘Digital Hologram’. In order to avoid potential
confusion, the following definitions explicitly state the difference in their meanings. ‘Raw
Hologram’ is the intensity map recorded on a camera or a film, and usually, this intensity
map will be used for the object wavefront reconstruction, as Dennis Gabor showed in his
works in 1948 [7]. ‘Digital Hologram’ is a representation of the complex optical wavefront
diffracted from an object. The complex wavefront is either generated directly by a computer
or reconstructed digitally from a ‘Raw Hologram’. A computer-generated hologram is often
referred to as wavefront-based CGH [8], Computer Generated Hologram, starting from the
works by J.W. Goodman and R.W. Lawrence [9] during the period of the mid-1960s. y
g
p
A basic tool in two-dimensional digital signal processing used in digital holography
is DFT (discrete Fourier transform) and the corresponding IDFT (inverse discrete Fourier
transform), making fast computation possible. Academic Editor: Maria Antonietta
Ferrara For raw holograms captured by a modern
digital camera, their corresponding frequency spectrums are also discrete. The DFT trans-
forms the raw hologram from the spatial domain to the frequency domain in a discrete
manner. The raw hologram spectrum and the corresponding object wavefront extraction
methods have been reported in detail in numerous works in the literature, such as [10–13]. In the frequency domain of a raw hologram, the spectrums of the zero-order image, virtual
image, and real image are shifted and separated by the off-axis configuration. As a result,
the object wavefront (digital hologram) can be extracted relatively easily. In this paper, we have proposed a novel technique for identifying biological samples
based on their tissues’ digital holograms. The paper is organized as follows. In Section 2, a
detailed optical setup of a single-shot off-axis digital holographic interferometer, an outline
of the digital hologram-classifier EDL-IOHC in [14], and the workflow of converting a
raw hologram to a digital hologram are described. Experimental results are presented in
Section 3. A conclusion summarizing the essential findings is given in Section 4. 2.1. Single-Shot Off-Axis Digital Holographic Interferometer Our proposed interferometer and ensemble deep learning (I-EDL) method consisted
of a single-shot off-axis digital holographic interferometer and an ensemble deep learn-
ing system for raw hologram capturing and complex object wavefront recognition. The
interferometer provides off-axis holograms with a shifted spectrum of the object’s real
image that can be easily extracted and processed by the fast Fourier transform method,
which is explained in detail by [10,11]. The optical setup is based on the principle of spatial
coherence and is installed on a curtain-enclosed optical table [15]. The coherent light source
is a red He-Ne laser with a wavelength of 632.8 nm [14], and the laser beam is approxi-
mately 2 mm in diameter. The object light formed by a laser beam passes through the tissue
specimen and records the object information related to it. Change the specimen positions
in x-y directions and record hundreds of experimental data images. The holograms are
captured by a CMOS camera equipped with a Nikon Plan microscope objective [16]. The
following Figure 1 shows a schematic diagram of the optical setup in detail, where MO is a
microscopic objective, and NDF is a neutral density filter. The reference light is separated
by several beam splitters (BS2, BS3) into three light beams and polarized by three linear
polarizers (LP0◦, LP45◦, and LP90◦) under 0◦, 45◦, and 90◦, respectively. Then, they are
combined by BS4 and BS5 and interfere with the object light to form the fringe image. The
system employs two neutral density filters in the object light beam (Lo) and references
light beam (Lr) to adjust the light intensity from overexposure. The raw hologram of the
object is recorded by the camera. Compared with a standard off-axis system, the setup can
get raw holograms with different polarization states in a single shot, reduces image degra-
dation due to light scatterings, and has higher imaging efficiency. The extra polarization
information is capable of image denoising and more details can be found in [17]. Appl. Sci. 2022, 12, 12674 3 of 10 Figure 1. Schematic diagram of the single-shot off-axis digital holographic interferometer. Figure 1. Schematic diagram of the single-shot off-axis digital holographic interferometer. 2.2. Ensemble Deep-Learning Network The hologram-classifier employed in this paper is EDL-IOHC. The approach in the
articles [18,19] opens a new paradigm for digital hologram recognition directly by wave-
front analysis. 2.1. Single-Shot Off-Axis Digital Holographic Interferometer The choice of EDL-IOHC is due to its robustness and high accuracy under
noisy conditions with occlusion compared to its predecessors in [18,19]. For clarity of
explanation, the hologram-classifier EDL-IOHC, a complex wavefront recognition system,
is outlined below. In reference to Figure 2a, which shows the structure of EDL-IOHC in [6] for recognizing
digital holograms with the powerful capability to handle occluded objects contaminated
with speckle noise. The input of the network is the reconstructed hologram with both
magnitude and phase information, as shown in Figure 2a. The first and second CNN,
known as the Magnitude CNN and the Phase CNN, accept the magnitude and phase
components of the digital hologram, which is structurally the same but trained with
different components’ information. The architecture of both CNNs, as shown in Figure 2b,
can be divided into three sections. Sections 1 and 2 have identical structures but different
hyper-parameters, containing a convolution layer for local feature extraction, max-pooling,
and dropout layers. Section 3 is a shared section for both the CNNs, and it is a “Concatenate
Unit” to ensemble output information from the two CNNs. The concatenate unit ensembles
all the extracted phase features and magnitude features into a combined flatten features
vector before fitting into the “Output Dense Layer” for the decision unit to output the
identity of the input digital hologram. This study employs the hologram-classifier EDL-IHC to identify the tissue object
wavefronts (digital holograms) reconstructed from the raw intensity fringe patterns. Fringe pattern intensity is referred to as a raw hologram; Γ is a real number quan-
tity and can be obtained as the result of measuring the intensity that results from the
linear superposition of a diffracted object wavefront ‘O’ and a reference wavefront ‘R’. Mathematically, the recorded intensity image can be expressed as follows: Γ(m, n) = ∥R(m, n) + O(m, n)∥2
(1) (1) where Γ(m, n) is the intensity of the captured hologram with a size of M columns × N rows. R(m, n) is the reference wavefront, and O(m, n) is the object wavefront. E
di
E
ti
(1) i
f ll Γ(m, n) =∥R(m, n)∥2 + ∥O(m, n)∥2
+ O(m, n)R∗(m, n) + O∗(m, n)R(m, n)
(2) (2) where ∗is the complex conjugate operation for complex numbers, ∥R(m, n)∥2 is the square
magnitude of the reference wavefront, and ∥O(m, n)∥2 is the square magnitude of the object Appl. Sci. 2.1. Single-Shot Off-Axis Digital Holographic Interferometer 2022, 12, 12674 4 of 10 wavefront. Γ is a set of dark and bright fringes that embeds the amplitude and the phase
information of the corresponding complex object wavefront. wavefront. Γ is a set of dark and bright fringes that embeds the amplitude and the phase
information of the corresponding complex object wavefront. Figure 2. The structure of (a) EDL-IOHC and (b) the expansion structure of CNN components and
their connection with the concatenate unit. Discrete Fourier Transform (DFT) is performed on the off-axis raw hologram and
generates the four terms in the frequency domain. The DFT transforms the raw hologram
from the spatial domain to the frequency domain in a discrete manner. After performing
DFT on Equation (2) and getting Equation (3), as below. H(u, v) =A2MNδ(u, v) + DFT
n
∥O(m, n)∥2o
+ DFT{O(m, n)R∗(m, n) + O∗(m, n)R(m, n)}
(3) (3) where u, v are the frequency axis, δ(∗) is the delta function, and A is the reference wave’s
amplitude. In the frequency domain, the spectral locations of the frequency components sepa-
rated by the recorded off-axis hologram provide easy means to separate specific wavefront
information in the Fourier space. The spectrum in the third term is extracted by a mask-
ing method, and the zero-order low-frequency spectrum and the twin image spectrum
are removed. The third term extracted spectrum DFT{O(m, n)R∗(m, n)}, as shown in
Equation (3), is centered (the masking method and centering algorithm is introduced
in [10] with great detail), and then inverse Fourier transform is performed and get the
scaled complex object wavefront AO(m, n), which is the object wavefront multiplied by
the reference wave with amplitude A. Then, a ‘min-max’ normalization algorithm [20]
is applied to A. This method of normalization algorithm used in the machine learning Appl. Sci. 2022, 12, 12674 5 of 10 5 of 10 community scales the values in a data array from [minimum value, maximum value] to
[−1, 1] through a linear mapping. It normalizes the effect of the scalar multiplication by
the reference wave for recognition. The normalization provides a robust pre-processing
method for recognition purposes. In the following Figure 3 illustrates the procedures to
get the object wavefront (the digital hologram) for training the EDL-IOHC deep learning
network. Figure 3. The workflow diagram for the processing steps from the biological samples to get the object
wavefront. Figure 3. 3. Experiments The system consisted of a computer equipped with an i-7 Intel processor, Nvidia
RTX 2080 Super GPU with 384 Tensor cores, the interferometer, a microscope objective,
and a CMOS camera. The hologram-classifier uses the same set of hyperparameters of
the EDL-IOHC reported in [6]. The new optical parameters for the digital holographic
interferometric system are shown in Table 2. Table 2. Optical parameters of the digital holographic interferometer system. Table 2. Optical parameters of the digital holographic interferometer system. Optical Parameters
Values
Wavelength of light
632.8 nm
Pixel size
3.45 µm
Size of hologram
2056 rows × 2546 columns
Off-axis angle
1.5 degrees Table 2. Optical parameters of the digital holographic interferometer system. Table 2. Optical parameters of the digital holographic interferometer system. Optical Parameters
Values
Wavelength of light
632.8 nm
Pixel size
3.45 µm
Size of hologram
2056 rows × 2546 columns
Off-axis angle
1.5 degrees Wavelength of light
Pixel size
Size of hologram
Off-axis angle As illustrations, Figures 4–8 show examples from the human chromosome and the
house fly wing datasets. Figures 4 and 6 are the corresponding images. Figures 5 and 7
are 3D plots of the spectrums and complex wavefront as a better means of visualization
as they are not typical interpretable visual images but are a 2D array of complex phasors. The chromosome is close to transparent, and it is one of the most challenging samples to be
separated if not using a hologram-classifier. The house fly wing is semitransparent, and
the phase information can complement the magnitude information to build better decision
boundaries for the deep learning network. p
g
From the full dataset of size 5000 capture raw holograms, 5000 digital holograms
(complex object wavefront) are extracted. Then 4000 out of the 5000 are taken as the in-
training dataset, while the remaining out-training dataset is used as a test set. The ensemble
CNN is trained with 3200 in-training set data, and the remaining 800 are used as a validation
set to stop the training process by an early stopping mechanism. Finally, both the in-training
dataset and the out-training dataset are used to evaluate the hologram-classifier. Training is
stopped by the validation set when the change of the validating accuracy is less than 0.01%. (a) The specimen photo. (b) Raw hologram. (c) Reconstructed tissue image. Figure 4. Sample image and experimental results for human chromosome sample. (c) Reconstructed tissue image. 2.1. Single-Shot Off-Axis Digital Holographic Interferometer The workflow diagram for the processing steps from the biological samples to get the object
wavefront. Figure 3. The workflow diagram for the processing steps from the biological samples to get the object
wavefront. Raw holograms of the tissue are captured from the biological samples. Fast DFT
transforms the raw holograms from the spatial domain to the frequency domain. The
spectrums of the object wavefronts are extracted, and fast IDFT restores the spectrums
into the object wavefronts. The object wavefront extraction methods from raw holograms
have been reported in [10–13] with details. The object wavefront extracted from the above
process is a complex quantity that contains both the magnitude and phase components
of the object wave O(m, n), a digital hologram. The full dataset is split into an in-training
train set and an out-training test set. The EDL-IOHC is trained with the train set and tested
by the test set. It is found that aberrations have come from dust on optical lenses and
mirrors, Airy-plaque-like rings [21] out-turn from the system’s lenses. However, the deep
learning network can adapt to these background irregularities during the first training
stage and continue to perform well in the later recognition stage without any necessary
background compensation. g
p
Ten different types of tissues are captured from ten different types of flawed biological
specimens, which are Cucurbita Stem, Pine Stem, Corn (Zea Mays) Seed, House Fly Wing,
Honeybee Wing, Bird Feather, Corpus Ventriculi, Liver Section, Lymph Node and Human
Chromosome with their class labels shown in Table 1. Table 1. The class labels for different specimens. Table 1. The class labels for different specimens. Table 1. The class labels for different specimens. Specimen Class
Class Label
Cucurbita Stem
0
Pine Stem
1
Corn (Zea mays) Seed
2
House Fly Wing
3
Honeybee Wing
4
Bird Feather
5
Corpus Ventriculi
6
Liver Section
7
Lymph Node
8
Human Chromosome
9 Appl. Sci. 2022, 12, 12674 6 of 10 6 of 10 Five hundred raw holograms are captured from tissues of each class of the ten biologi-
cal specimens and result in a total dataset size of 5000 digital holograms (object wavefront). They are used to train the hologram-classifier EDL-IOHC. Then, the trained hologram-
classifier is used to identify the type of biological specimens by recognizing the tissues’
digital holograms. 3. Experiments (a) The specimen photo. (b) Raw hologram. (a) The specimen photo. (b) Raw hologram. (c) Reconstructed tissue image. Figure 4. Sample image and experimental results for human chromosome sample. Appl. Sci. 2022, 12, 12674 7 of 10 7 of 10 (b) Clipped frequency spectrum plot (in dB). (b) Clipped frequency spectrum plot (in dB). (a) Full frequency spectrum plot (in dB). (b) Clipped frequency spectrum plot (in dB). (c) Magnitude plot of the complex wavefront. (d) Phase plot of the complex wavefront. Figure 5. Frequency spectrum and reconstructed wavefront images of human chromosome sample. (a) Full frequency spectrum plot (in dB). (b) Clipped frequency spectrum plot (in dB). (c) Magnitude plot of the complex wavefront. (c) Magnitude plot of the complex wavefront. (d) Phase plot of the complex wavefront. (c) Magnitude plot of the complex wavefront. (d) Phase plot of the complex wavefront. Figure 5. Frequency spectrum and reconstructed wavefront images of human chromosome sample. Figure 5. Frequency spectrum and reconstructed wavefront images of human chromosome sample. (a) The specimen photo. (b) Raw hologram. (c) Reconstructed tissue image. Figure 6. Sample image and experimental results for house fly wing sample. (b) Raw hologram. (c) Reconstructed tissue image. (a) The specimen photo. (a) The specimen photo. (b) Raw hologram. (c) Reconstructed tissue image. Figure 6. Sample image and experimental results for house fly wing sample. Appl. Sci. 2022, 12, 12674 8 of 10 8 of 10 (b) Clipped frequency spectrum plot (in dB). (b) Clipped frequency spectrum plot (in dB). (a) Full frequency spectrum plot (in dB). (b) Clipped frequency spectrum plot (in dB). (c) Magnitude plot of the complex wavefront. (d) Phase plot of the complex wavefront. Figure 7. Frequency spectrum and reconstructed wavefront images of house fly wing sample. (a) Full frequency spectrum plot (in dB). (b) Clipped frequency spectrum plot (in dB). (c) Magnitude plot of the complex wavefront. (d) Phase plot of the complex wavefront. (c) Magnitude plot of the complex wavefront. (d) Phase plot of the complex wavefront. Figure 7. Frequency spectrum and reconstructed wavefront images of house fly wing sample. Figure 7. Frequency spectrum and reconstructed wavefront images of house fly wing sample. Figure 8. The classification confusion matrix labels 0–9 correspond to the ten specimen classes as
shown in Table 1 ‘Class Label’ column. Figure 8. 3. Experiments The classification confusion matrix labels 0–9 correspond to the ten specimen classes as
shown in Table 1 ‘Class Label’ column. The ensemble CNN is trained by the dataset with cosine smoothing on the phase
components, the validating set stops the training epoch, and the actual epoch run is 16. In each epoch, the holograms in the in-training set of the dataset are used to train the
deep learning structure. The trained structure is then applied to classify the datasets. The Appl. Sci. 2022, 12, 12674 9 of 10 9 of 10 following confusion matrix in Figure 8 shows that eight out of the ten classes are correct
with very high overall classification accuracy, as shown in Table 3. following confusion matrix in Figure 8 shows that eight out of the ten classes are correct
with very high overall classification accuracy, as shown in Table 3. following confusion matrix in Figure 8 shows that eight out of the ten classes are correct
with very high overall classification accuracy, as shown in Table 3. Table 3. Success rates for classifying the out-training test set and the complete dataset by the EDL-
IOHC hologram-classifier. Table 3. Success rates for classifying the out-training test set and the complete dataset by the EDL-
IOHC hologram-classifier. Dataset
Success Rate
Test set
99.60%
Complete set (both out- and in-training sets)
99.82% Dataset
Success Rate
Test set
99.60%
Complete set (both out- and in-training sets)
99.82% The results in Table 3 reflect that in classifying the object, EDL-IOHC can maintain a
high success rate of 99.60% for the out-training test set and 99.82% for the entire dataset. The performance is better than the benchmarking experiment conducted in [6] on partially
occluded digit objects with speckle noise contamination. 4. Conclusions This paper proposes a hologram-classifier on the digital hologram obtained by an off-
axis interferometer from biological specimen tissues. The method does not require an intact
sample with a complete outline shape of the specimens or the organs to understand the
objects’ identities and achieves a high success rate of 99.60% on the out-training test set, and
is able to adapt for optical aberrations from lenses and mirrors without prior background
compensations. The result demonstrates that the hologram-classifier EDL-IOHC is robust
under noisy and imperfect holography optical processes, and the off-axis interferometer is
effective for scanning the microstructure of the tissues from the specimens. The simplicity
of direct application in recognition of biological specimen tissue by EDL-IOHC, without
any changes in architectural or hyperparameters, verified the generality of EDL-IOHC. In passing, more potential applications such as pollutant plastics, defective glasses, or
identification of infected red blood cells from normal cells will be feasible by using EDL-
IHC with a similar interferometer. Intuitively the biological specimen tissue hologram
looks noisy and challenging. However, it is numerically not more challenging than the
occluded simple digit patterns with speckle noise tested in the EDL-IOHC. Furthermore,
for potential applications that require more compact size and lower cost but could accept
lower performance, a smaller on-axis inline interferometer could also be considered. The
method demonstrates a new paradigm in object identification by ensemble deep learning
through the EDL-IOHC wavefront recognition technique. Author Contributions: Conceptualization, project administration, and writing-original draft prepa-
ration, H.L. and Y.Z.; software, investigation and methodology, H.L.; validation, visualization and
writing—review and editing, Y.Z., P.B. and H.L. All authors have read and agreed to the published
version of the manuscript. Funding: This research received no external funding. Data Availability Statement: The data that support the findings of this study are openly available in
github at https://github.com/lamhoson/Hologram-Dataset (accessed on 2 December 2021). Data Availability Statement: The data that support the findings of this study are openly available in
github at https://github.com/lamhoson/Hologram-Dataset (accessed on 2 December 2021). Acknowledgments: We acknowledge the support provided by the staff of the Imaging Systems
Laboratory at the University of Hong Kong. Conflicts of Interest: The authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a potential conflict of interest. Appl. Sci. 4. Conclusions 2022, 12, 12674 10 of 10 Abbreviations
The following abbreviations are used in this manuscript:
I-EDL
Interferometer and an ensemble deep learning
3D
Three-dimensional
EDL-IOHC
Ensemble deep learning invariant hologram classification
CGH
Computer generated hologram
DFT
Discrete Fourier transform
IDFT
Inverse discrete Fourier transform
CMOS
Complementary metal–oxide–semiconductor
MO
Microscope objective
NDF
Neural density filter
BS
Beam splitter
CNN
Convolutional neural network
GPU
Graphics processing unit References 1967, 11, 77–79. [CrossRef] [
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English
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Raman-Scattering Spectroscopy of Epitaxial Graphene Formed on SiC Film on Si Substrate
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E-journal of surface science and nanotechnology
| 2,009
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cc-by
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I.
INTRODUCTION In the formation of GOS structure, it is of highest prior-
ity to form a qualified SiC film on top of the Si substrate. In this respect, the large lattice mismatch (>20%) be-
tween Si and 3C-SiC crystals (Si>SiC) is a big challenge,
which might be one of the reasons for the absence of pre-
ceding studies on the GOS structure. We have solved
the problem by using a unique growth properties of the
3C-SiC/Si hetero-system; a 3C-SiC(111) film grows on
the Si(110) substrate. In this combination, the strain in
the SiC film is significantly reduced from that in the 3C-
SiC(111)/Si(111) system, as evidenced by a factor-of-four
reduction in the anisotropy of the lattice constants be-
tween in-plane and out-of-plane directions. This orien-
tational rotation in the heteroepitaxy is consistent with
a recent potential-energy calculation [11], which accounts
for this phenomenon as due to the reduction of the strain
energy in the film. This reduction overwhelms the in-
crease in the interfacial energy caused by deviation of the
interfacial coordination number from the ideal number of
four. The (111)-oriented 3C-SiC surface is desirable to
form graphene on it because its Si face has, up to four bi-
layers from the surface, exactly the identical layer stacking
as of the 6H-SiC(0001) Si surface used in conventional EG
process. From its extremely high electron mobility [1] as well
as from its unique optical [2] and spin [3] properties,
graphene is attracting skyrocketing attention as a new
electronic material. Although the mechanical exfoliation
method using highly oriented graphite crystals [4] is most
popular and widespread, the epitaxial graphene (EG)
method that utilizes a high-temperature (>∼1250◦C) an-
nealing of hexagonal (4H or 6H) SiC bulk crystals in vac-
uum [5–9] is technologically by far more practical. The
EG method, however, has its own problems of the limita-
tion in the size and the cost-effectiveness of the SiC wafers. To introduce graphene into the prevailing Si technology,
an innovative technology that realizes growth of graphene
on top of Si substrates must be developed. In this respect,
we have recently succeeded in forming a graphene layer
on a Si substrate [10]. The essence of this graphene-on-
silicon (GOS) technology is the use of an ultrathin SiC
film formed on a Si substrate, instead of SiC bulk crystals
in EG. By annealing the SiC film in vacuum, a graphene
layer is formed. Hideki Nakazawa Dept. of Materials Science and Technology, Hirosaki University, Hirosaki 036-8561, Japan Dept. of Materials Science and Technology, Hirosaki University, Hirosaki 036-8561, Japan Tetsuo Endoh
Center for Interdisciplinary Research, Tohoku University,
6-3 Aramaki aza-Aoba, Aoba-ku, Sendai 980-8578, Japan (Received 2 December 2008; Accepted 14 January 2009; Published 21 February 2009) By conducting a 1200◦C vacuum annealing of a 3C-SiC(111) ultrathin film preformed on a Si(110) surface,
we have succeeded in forming a graphene layer on a Si substrate. Raman-scattering spectrum from this surface
presents a distinct 2D band, whose deconvolution into four subcomponents indicates that the film mostly consists
of a two-layer graphene. The peak position is blue-shifted from that of a free-standing graphene formed by a
mechanical exfoliation method, suggesting a compressive stress in the film. [DOI: 10.1380/ejssnt.2009.107] Keywords: Silicon carbide; Heteroepitaxy; Organosilane; Gas-source MBE; Graphene ∗Corresponding author: y-miyamo@riec.tohoku.ac.jp ISSN 1348-0391
c⃝2009 The Surface Science Society of Japan (http://www.sssj.org/ejssnt) e-Journal of Surface Science and Nanotechnology
e-J. Surf. Sci. Nanotech. Vol. 7 (2009) 107-109 e-Journal of Surface Science and Nanotechnology
e-J. Surf. Sci. Nanotech. Vol. 7 (2009) 107-109 21 February 2009 Regular Paper Raman-Scattering Spectroscopy of Epitaxial Graphene Formed on
SiC Film on Si Substrate
Yu Miyamoto,∗Hiroyuki Handa, Eiji Saito, and Atsushi Konno
Research Institute of Electrical Communication, Tohoku University, Sendai 980-8577, Japan
Yuzuru Narita
Department of Electrical Engineering, Yamagata University, 4-3-16 Jonan, Yonezawa, 992-8510, Japan
Maki Suemitsu
Research Institute of Electrical Communication, Tohoku University, Sendai 980-8577, Japan
and
CREST, Japan Science and Technology Agency, Tokyo 107-0075, Japan
Hirokazu Fukidome and Takashi Ito
Center for Interdisciplinary Research, Tohoku University,
6-3 Aramaki aza-Aoba, Aoba-ku, Sendai 980-8578, Japan,
Kanji Yasui
Dept. of Electrical Engineering, Nagaoka University of Technology, Nagaoka 940-2188, Japan
Hideki Nakazawa
Dept. of Materials Science and Technology, Hirosaki University, Hirosaki 036-8561, Japan
Tetsuo Endoh
Center for Interdisciplinary Research, Tohoku University,
6-3 Aramaki aza-Aoba, Aoba-ku, Sendai 980-8578, Japan
(Received 2 December 2008; Accepted 14 January 2009; Published 21 February 2009) I.
INTRODUCTION In this paper, we will report our detailed
Raman scattering analysis of this graphene layer. The graphene was characterized with the Raman scat-
tering spectroscopy, which presents several important
peaks. The G band, appearing at around 1580 cm−1, 009 The Surface Science Society of Japan (http://www.sssj.org/ejssnt) 107 Volume 7 (2009) Miyamoto, et al. 2000
3000
Raman shift(cm-1)
Raman Intensity (arb.unit)
Fig. 1 Raman spectra from GOS (top) and mechanically exfoliated graphene (middle) and graphite
D G
2D
FIG. 1: Raman spectra from GOS (top) and mechanically
exfoliated graphene (middle) and graphite (bottom). arises from a pair of LO and TO phonons degenerated
at the Γ point. The D (1350 cm−1) and the 2D (2700
cm−1) bands arise from a double resonant Raman scat-
tering in the vicinity of the K point with the latter being
the overtone of the former [12]. The D band, however, is
in principle Raman inactive in graphene, and its presence
suggests presence of defects within the layer. From their
origins, the D and 2D bands should be excitation-energy
dependent while the G band should be independent. As
FIG. 2: The excitation-energy dependence of the Raman spec-
tra. 2000
3000
Raman shift(cm-1)
Raman Intensity (arb.unit)
Fig. 1 Raman spectra from GOS (top) and mechanically exfoliated graphene (middle) and graphite
D G
2D
FIG. 1: Raman spectra from GOS (top) and mechanically
exfoliated graphene (middle) and graphite (bottom). 2000
3000
Raman shift(cm-1)
Raman Intensity (arb.unit)
Fig. 1 Raman spectra from GOS (top) and mechanically exfoliated graphene (middle) and graphite
D G
2D
FIG. 1: Raman spectra from GOS (top) and mechanically
exfoliated graphene (middle) and graphite (bottom). 2000
3000
Raman shift(cm-1)
Raman Intensity (arb.unit)
n spectra from GOS (top) and mechanically exfoliated graphene (middle) and
D G
2D FIG. 2: The excitation-energy dependence of the Raman spec-
tra. FIG. 1: Raman spectra from GOS (top) and mechanically
exfoliated graphene (middle) and graphite (bottom). arises from a pair of LO and TO phonons degenerated
at the Γ point. The D (1350 cm−1) and the 2D (2700
cm−1) bands arise from a double resonant Raman scat-
tering in the vicinity of the K point with the latter being
the overtone of the former [12]. The D band, however, is
in principle Raman inactive in graphene, and its presence
suggests presence of defects within the layer. III.
RESULTS AND DISCUSSIONS The middle (bottom) Raman spectrum in Fig. 1 is
from a free-standing graphene (graphite), which has been
transferred from a HOPG crystal onto a SiO2 film by a
mechanical exfoliation method. The one in the top is from
the UHV-annealed 3C-SiC film in our GOS structure. We
can see distinct G and 2D bands in the spectrum, which FIG. 3: The excitation-energy dependence of the D and 2D
bands. I.
INTRODUCTION From their
origins, the D and 2D bands should be excitation-energy
dependent while the G band should be independent. As
the excitation energy increases, the resonant energy of the
π-band electrons in the vicinity of the K point increases,
which shifts the energy of the relevant phonons involved in
the scattering. The peak position of the 2D band, there-
fore, should be sensitive to minute modifications in the
electronic band structure at the K point. In other words,
the line-shape analysis of the 2D band can be a good tool
to characterize the number of layers in the graphene layer. In this study, we have evaluated the graphene layer in our
GOS structure by using a Raman scattering spectroscopy. FIG. 2: The excitation-energy dependence of the Raman spec-
tra. FIG. 2: The excitation-energy dependence of the Raman spec-
tra. II.
EXPERIMENTAL Boron-doped, p-type Si(110) wafers were used in the
experiment, which were cut to 7 × 35 mm2 in size. Af-
ter a wet clean, samples were introduced into the vacuum
chamber, where flash annealings were conducted for sev-
eral times at 1200◦C to yield a clean surface. Sample
heating was conducted by passing a DC current through
the sample. 3C-SiC film has been grown with a gas-source
molecular beam epitaxy (GSMBE) using monomethyl-
silane (MMS, 99.999%) at 3.0 × 10−2 Pa as a single
source [13]. The growth consists of a pair of stages: the
buffer layer formation at 600◦C [14] and the SiC growth
at 1050◦C. After the growth, MMS was evacuated from
the chamber, and a 1200◦C annealing was conducted for
90 min to form graphene on top of the 3C-SiC film. The
graphene layer was evaluated with a micro-Raman spec-
troscopy (Renishaw) with the laser-spot diameter of 1 µm
and the excitation photon wavelength of 514 nm. For
measurements of the excitation-energy dependence, an-
other Raman setup [15] was used. FIG. 3: The excitation-energy dependence of the D and 2D
bands. http://www.sssj.org/ejssnt (J-Stage: http://www.jstage.jst.go.jp/browse/ejssnt/) 108 e-Journal of Surface Science and Nanotechnology Volume 7 (2009) Volume 7 (2009) 2600
2700
2800
2900
Ram an shift (cm -1)
Raman Intensity (arb.unit)
FIG. 4: The 2D band fitted with four components. tion of the G band is blue-shifted from those from the
free-standing graphene (graphite). This kind of blue
shifts have also been observed in epitaxial graphene lay-
ers formed on hexagonal SiC substrates, which has been
related to compressive strains due either to the lattice mis-
match at the film/substrate interface [18] or to the differ-
ence in the thermal expansion coefficients [19]. Since our
SiC film is highly relaxed after a high-temperature anneal-
ing [20], the latter possibility still holds in our GOS struc-
ture. As the number of graphene layers increases, the blue
shifts in EG tend to disappear [18]. From these consider-
ations, it is highly likely that the present GOS film con-
sists of few layer graphene with compressive strain. The
2D band was best fitted with four components (Fig. 4),
which implies that the current GOS film consists mainly
of two-layer graphene [21]. FIG. 4: The 2D band fitted with four components. indicates presence of graphitic layers including graphene. The presence of the D band, however, suggests presence
of defects as well. Acknowledgments Acknowledgments This work has been supported by JST-CREST as well
as by the Specially Promoted Research in Center for In-
terdisciplinary Research, Tohoku University. This work
was partly supported by Grant-in-Aid for Young Scien-
tists (B) (20760485). The peak position of the G band suggests presence of
a compressive strain in the graphene layer in the present
GOS structure. As we notice in Fig. 1, the peak posi- [1] S. V. Morozov, K. S. Novoselov, M. I. Katsnelson, F. Schedin, D. C. Elias, J. A. Jaszczak, and A. K. Geim,
Phys. Rev. Lett. 100, 016602 (2008). [2] F. Wang, Y. Zhang, C. Tian, C. Girit, A. Zettl, M. Crom-
mie, and Y. R. Shen, Science 320, 206 (2008). [1] S. V. Morozov, K. S. Novoselov, M. I. Katsnelson, F. Schedin, D. C. Elias, J. A. Jaszczak, and A. K. Geim,
Phys. Rev. Lett. 100, 016602 (2008). and M. Suemitsu, Appl. Phys. Express 1, 111201 (2008). [
] [12] R. Saito, A. Gr¨uneis, G. G. Samsonidze, V. W. Brar, G. Dresselhaus, M. S. Dresselhaus, A. Jorio, L. G. Cancado,
C. Fantini, M. A. Pimenta, and A. G. Souza Filho, New. J. Phys. 5, 157 (2003). (
)
[2] F. Wang, Y. Zhang, C. Tian, C. Girit, A. Zettl, M. Crom-
mie, and Y. R. Shen, Science 320, 206 (2008). [13] H. Nakazawa, M. Suemitsu, and S. Asami, Thin Solid
Films 369, 269 (2000). [3] M. Ohishi, M. Shiraishi, R. Nouchi, T. Nozaki, T. Shinjo,
and Y. Suzuki, Jpn. J. Appl. Phys. 46, L605 (2007). [4] K. S. Novoselov, A. K. Geim, S. V. Morozov, D. Jiang, Y. Zhang, S. V. Dubonos, I. V. Grigorieva, and A. A. Firsov,
Science 306, 666 (2004). [14] H. Nakazawa and M. Suemitsu, Appl. Phys. Lett. 79, 755
(2001). [15] T. Itoh and R. L. McCreery, Anal. Bioanal. Chem. 388,
131 (2007). ,
(
)
[5] C. Riedl, U. Starke, J. Bernhardt, M. Franke, and K. Heinz, Phys. Rev. B 76, 245406 (2007). (
)
Riedl, U. Starke, J. Bernhardt, M. Franke, and [16] A. C. Ferrari, Solid State Commun. 143, 47 (2007). [
] [6] C. Faugeras, A. Nerri`ere, M. Potemski, A. Mahmood, E. Dujardin, C. Berger, and W. A. de Heer, Appl. Phys. Lett. 92, 011914 (2008). [17] A. C. Ferrari, J. C. Meyer, V. Scardaci, C. Casiraghi, M. Lazzeri, F. Mauri, S. Piscanec, D. Jiang, K. S. Novoselov,
S. Roth, and A. K. II.
EXPERIMENTAL The spike at 2300 cm−1, as well as
the inclination of the baseline from 1200 to 1700 cm−1,
is related to amorphous carbon [16] although there are
portions where no spikes are observed. IV.
CONCLUSION Graphene layer on Si substrate, or GOS, has been fab-
ricated by GSMBE of 3C-SiC(111) on Si(110) substrate
followed by a vacuum annealing. Raman-scattering spec-
troscopy indicates that the film contains compressively
strained two-layer graphene. The excitation-energy dependence of the peak positions
of the D, G, and 2D bands also supports the presence of
graphene (Fig. 2). As we increase the photon energy from
2.41 to 2.54 or to 2.71 eV, the D and 2D bands show a
shift towards high wavenumbers with gradients of 62 and
109 cm−1/eV, respectively (Fig. 3). The G band stays
unchanged. These results are consistent with the band
structure of graphene and with previous results [16, 17] as
well, suggesting that our GOS film is highly crystallized. Acknowledgments Geim, Phys. Rev. Lett. 97, 187401
(2006). [7] E. Rolling, G. H. Gweon, S. Y. Zhou, B. S. Mun, J. L. McChesney, B. S. Hussain, An. Fedorov, P. N. First, W. A. de Heer, and A. Lanzara, J. Phys. Chem. Solids 67,
2172 (2006). [18] Z. H. Ni, W. Chen, X. F. Fan, J. L. Kuo, T. Yu, A. T. S. Wee, and Z. X. Shen, Phys. Rev. B 77, 115416 (2008). [19] J. Rohrl, M. Hundhausen, K. V. Emtsev, Th. Seyller, and
L. Ley, Appl. Phys. Lett. 92, 201918 (2008). (
)
[8] P. Mallet, F. Varchon, C. Naud, L. Magaud, C. Berger,
and J.-Y. Veuillen, Phys. Rev. B 76, 041403 (2007). [20] A. Konno, M. Suemitsu, Y. Narita, T. Ito, K. Yasui, H. Nakazawa, and T. Endoh, International Conference on
Silicon Epitaxy and Heterostructures, Marseille, May 20
to 24, 2007. [9] H. Hibino, H. Kageshima, F. Maeda, M. Nagase, Y. Kobayashi, and H. Yamaguchi, Phys. Rev. B 77, 075413
(2008). [21] D. Graf, F. Molitor, K. Ensslin, C. Stampfer, A. Jungen,
C. Hierold, and L. Wirtz, Nano Lett. 7, 238 (2007). [10] M. Suemitsu, Y. Miyamoto, H. Handa, and A. Konno,
e-J. Surf. Sci. Nanotech., in press. [11] T. Ito, T. Kanno, T. Akiyama, K. Nakamura, A. Konno, http://www.sssj.org/ejssnt (J-Stage: http://www.jstage.jst.go.jp/browse/ejssnt/) 109
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CATSCaNS: Co-Axial Tomography of the Solar Corona and Near Sun environment
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Bulletin of the AAS
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Synopsis The STEREO mission gave us a tantalizing glimpse of the capabilities of
multi-spacecraft imaging to resolve coronal structures and extract information vital
to modeling and forecasting solar events. However, it also demonstrated the
limitations posed by a limited number of spacecraft. While significant progress has
been made over the past two decades in detecting, analyzing and understanding
CMEs and associated energetic charged particles, another breakthrough is needed
in our observing capability of solar transients and their heliospheric consequences
to facilitate the next generation science needed to uncover the complex three-
dimensional (3D) internal structure of CMEs. Advances in SmallSat
communications and miniaturization of coronagraphs make feasible multi-
spacecraft missions to image solar transients from multiple viewpoints at
reasonable cost. CATSCaNS (Co-Axial Tomography of the Solar Corona and Near
Sun environment) will be the first to establish true 3-D reconstruction of CMEs
enabling a revolution in predictions of the space weather effects of CMEs, based
solely on remote observations. These advances can be used to safeguard space
assets and the journey to Mars. 1. Introduction Our current understanding of CME propagation and evolution is insufficient for the
reliable prediction of space weather based on remote observations. Even if our understanding of
and ability to simulate the physics was perfect, our predictions would still be confounded by our
limited ability to provide inputs that describe the 3-dimensional structure of these CMEs. In the
recent past, the STEREO mission, combined with SOHO, provided as many as 3 viewpoints for
observing CMEs. However, while these missions brought about a transition in the capacity to derive 3D
kinematic characterization of transients via forward modeling and triangulation-type analyses,
due to the very complex structure of most CMEs, three viewing angles does not suffice for full
3D tomographic imaging. Resolving the internal structure of CMEs requires more viewpoints. Incorporating more detailed 3D inputs to heliospheric models would help reveal why the models
are in error and help improve both the models and our understanding of the underlying physics. 2. Science Traceability
Threshold STM Science
Goal
Threshold Science
Objectives
Measurement
Req
Instrument Req
Mission Req. Accurately
predict the
transport and
evolution of solar
wind structures
and their
associated
energetic
particles based
only on remote
imaging
Determine the 3-dimensional
multiscale plasma properties
of CMEs and shocks
Tomographic imaging of
coronal density structures in
3D space
-2.5-15 Rs FOV
-100 arcsec image resolution
-15 min time cadence
- ≧5 heliospheric
spacecraft at 1 AU with
total longitudinal range
between 100º-140º
- 10 arscec pointing with
1 arcmin tracking of the
sun
- Selected 3 hours
(event) of remote
sensing data downlink
over each 24 hour
window; Continuous
coverage for in-situ
measurements
- Two-year main science
Determine how the dynamic
inner heliosphere controls the
transit of transients to Earth
and beyond
Tomographic imaging of the
near sun environment
See above
Constrain observational
models with ground truth
measurements of solar wind
properties. Plasmag suite consisting of
a thermal plasma instrument
(<~30keV), an energetic
particle instrument (30 KeV-
2MeV), and a
magnetometer (1nT
accuracy)
• Bulk density range 1–100
particles/cm³. • Bulk speed range 200–1500 km/s. Velocity vector measured. • Proton temperature range 40K–2M
Kelvin
• 60 second sampling
• MAG Dynamic range 1-20 Hz ±200
nT Tomographic imaging of
coronal density structures in
3D space Determine the 3-dimensional
multiscale plasma properties
of CMEs and shocks Tomographic imaging of the
near sun environment Determine how the dynamic
inner heliosphere controls the
transit of transients to Earth
and beyond Plasmag suite consisting of
a thermal plasma instrument
(<~30keV), an energetic
particle instrument (30 KeV-
2MeV), and a
magnetometer (1nT
accuracy) Additions for Baseline STM Additions for Baseline STM
Science
Goal
Additional Science
Objectives for
baseline mission
Measurement Req
Instrument Req
Additional Mission
Req. Accurately predict
the transport and
evolution of solar
wind structures
and their
associated
energetic particles
based only on
remote imaging
Determine the solar
photospheric magnetic field
360 degrees of vector
magnetograph
measurements
See SOHO/MDI or HINODE/SP
specifications
2 spacecraft separated by 180
degrees in heliographic longitude
carrying vector magnetograph
Track and monitor 3-D evolution
of ICME and shocks
Tomographic imaging with
heliospheric imagers
See STEREO/HI specifications
All spacecraft carrying Heliospheric
imagers
Track and monitor CMEs
initiation
EUV imager off the sun-earth
line combined with existing
measurements on the sun-
earth line (e.g. 2. Science Traceability
Threshold STM SDO/EVE)
See SunCET specifications
One spacecraft separated from
Earth by 20-40 degrees in
heliographic longitude carrying the
EUV imager With the goal of accurately predicting the transport and evolution of solar wind structures
in mind, a draft science traceability matrix can be developed for a Threshold mission and
Baseline mission. These are shown above. Note that instrument specifications are being used
here as shorthand for a longer list of instrument requirements for the sake of brevity and clarity. 3. Solar Tomography and the CATSCaNS concept Solar Tomography using Thompson scattering can provide the needed 3-D input to
address 2 of the 3 science objectives needed to reach closure. Its accuracy is only limited by the
number of viewpoints and the resolution of the imagers. The CATSCaNS concept provides these
multiple viewpoints by arranging the spacecraft in a rough formation shown on the left, using
electric propulsion for formation maintenance, and laser
communications for data return. Davila [1994] and references
therein provide an excellent summary of the technique and, most
importantly, the benefits of additional viewpoints spread over a
broad range of angles. Figure 4 of that paper demonstrates the
technique in a simple 3X3 array. Figure 6 of that paper
demonstrates that a large range of angles is required for the
reconstruction, and Figure 7 shows that the fidelity of the
reconstruction increases dramatically with increasing numbers of observation points. More
recently, Vourlidas et al. [2020] details the added benefits of a solar polar viewpoint. However,
the propulsion requirements of such a mission drive up cost. p
p
q
p
Figure 1 (left) depicts an artist’s rendition of such a concept using multiple white-light
images to reconstruct CME electron densities from remote locations providing these data at least
12 hours in advance of any terrestrial effects even for the fastest CMEs. Figure 1: Tomographic reconstruction from multiple viewpoints can provide detailed 3-
dimensional electron density reconstructions as well as their evolution in time. These detailed
inputs are necessary to move space weather modeling to an operational, predictive capability. They are enabled by near term advances in CubeSat scale optical communications and white-
light coronagraph miniaturization technologies. The potential benefits of this tomography cannot be overstated. The ability to resolve the
internal structure of CMEs is critical both to our understanding of these structures and our ability Figure 1: Tomographic reconstruction from multiple viewpoints can provide detailed 3-
dimensional electron density reconstructions as well as their evolution in time. These detailed
inputs are necessary to move space weather modeling to an operational, predictive capability. They are enabled by near term advances in CubeSat scale optical communications and white-
light coronagraph miniaturization technologies. Figure 1: Tomographic reconstruction from multiple viewpoints can provide detailed 3-
dimensional electron density reconstructions as well as their evolution in time. These detailed
inputs are necessary to move space weather modeling to an operational, predictive capability. 3. Solar Tomography and the CATSCaNS concept They are enabled by near term advances in CubeSat scale optical communications and white-
light coronagraph miniaturization technologies. The potential benefits of this tomography cannot be overstated. The ability to resolve the
internal structure of CMEs is critical both to our understanding of these structures and our ability Figure 2: (left) Original synthetic images of a modeled CME from viewpoints at -70, -42, and -
14 degrees respectively in heliographic longitude with respect to the Earth. (right) Synthetic
images of the CME after tomographic reconstruction based solely on the images on the left. Figure 2: (left) Original synthetic images of a modeled CME from viewpoints at -70, -42, and -
14 degrees respectively in heliographic longitude with respect to the Earth. (right) Synthetic
images of the CME after tomographic reconstruction based solely on the images on the left. Figure 2: (left) Original synthetic images of a modeled CME from viewpoints at -70, -42, and -
14 degrees respectively in heliographic longitude with respect to the Earth. (right) Synthetic
images of the CME after tomographic reconstruction based solely on the images on the left. Figure 3: (left) Original synthetic images of a modeled CME from viewpoints at 14, 42, and 70
degrees respectively in heliographic longitude with respect to the Earth. (right) Synthetic images
of the CME after tomographic reconstruction based solely on the images on the left. Figure 3: (left) Original synthetic images of a modeled CME from viewpoints at 14, 42, and 70
degrees respectively in heliographic longitude with respect to the Earth. (right) Synthetic images
of the CME after tomographic reconstruction based solely on the images on the left. of the CME after tomographic reconstruction based solely on the images on the left. to model their evolution and the energetic particles they create as they propagate to 1 AU. Figures 2 and 3 demonstrate the practical potential for these reconstructions. In these figures, a
complete tomographic process is done on synthetic images of a modeled CME. In each figure,
the original on the left is an image from one of 6 viewpoints of the modeled CME. This image as
well as those from the other 5 viewpoints are then used to tomographically reconstruct the
electron density map of CME with a modernized version of the Davila [1994] algorithm and an
understanding of Thompson scattering. 3. Solar Tomography and the CATSCaNS concept The image on the right is taken from the same viewpoint,
but it is taken of the reconstructed CME based on the derived electron density map. The
reconstructed images preserve the overall structure and most of the important features of the
event, but in the case of the reconstruction, we have the underlying 3-dimensional electron
density map and know precisely where the electrons being excited by the Thomas Scattering are
in 3D space. Imagine being able to input such detailed structures into models of space weather or
models of CME evolution in the Heliosphere. Every additional spacecraft added to the formation
enhances the fidelity of the reconstruction and out-of-ecliptic viewpoints can remove the artifacts
seen in the ecliptic plane. 4. Feasibility With 2020 technology, such a mission concept using CubeSats would be infeasible
primarily due to limitations on the communications system. Solar tomography was, in fact,
proposed in the original STEREO mission concept which consisted of 4 spacecraft rather than 2. However, it had to be descoped to 2 spacecraft because of cost. The technology now exists to do
what STEREO was meant to do. Recent advances in optical communications for CubeSats that only level state-of-the-art
pointing requirements on the bus [Mathason et al., 2019] will likely reach TRL 9 by 2025 and
SmallSat scale propulsion system for both delta-H control (e.g. the MarCO cold-gas thrusters
system) and high delta-V propulsions systems (see the Busek portfolio for example) are reaching
maturity y
Figure 4: (left) JPL’s OCTL showing a 1-meter optical aperture and (right) JPL’s DSS-13, a
34m RF antenna, showing a 1.3-meter optical aperture in its center. Credit: NASA JPL. Optical ground stations are also coming online that can support missions like CATSCaNS. In
2022 a small prototype RF-Optical system, including the mirror and cameras/backend has been
installed into DSS-13 at the Goldstone Deep Space Communications Complex. DSS-13 is the
R&D 34m BWG antenna at Goldstone. The combination of seven small (0.5m) mirrors
comprises a synthesized prototype optical aperture of about 1.3m diameter. As of 2022, the
seven-segment prototype mirror system has been undergoing alignment and test/checkout. y t) JPL’s OCTL showing a 1-meter optical aperture and (right) JPL’s DSS-13, a
na, showing a 1.3-meter optical aperture in its center. Credit: NASA JPL. Figure 4: (left) JPL’s OCTL showing a 1-meter optical aperture and (right) JPL’s DSS-13, a
34m RF antenna, showing a 1.3-meter optical aperture in its center. Credit: NASA JPL. Optical ground stations are also coming online that can support missions like CATSCaNS. In
2022 a small prototype RF-Optical system, including the mirror and cameras/backend has been
installed into DSS-13 at the Goldstone Deep Space Communications Complex. DSS-13 is the
R&D 34m BWG antenna at Goldstone. The combination of seven small (0.5m) mirrors
comprises a synthesized prototype optical aperture of about 1.3m diameter. As of 2022, the
seven-segment prototype mirror system has been undergoing alignment and test/checkout. Figure 4: (left) JPL’s OCTL showing a 1-meter optical aperture and (right) JPL’s DSS-13, a
34m RF antenna, showing a 1.3-meter optical aperture in its center. Credit: NASA JPL. 5. Instrumentation With the focus on miniaturization of instruments, versions of most instruments now fit in
a CubeSat form factor as well, providing capability comparable to the previous generation of
instruments in a much smaller form factor. The MiniCor concept [Korendyke et al., 2015]
demonstrates the feasibility of a miniaturized white-light coronagraph capable of providing
STEREO quality imagery in a CubeSat form factor. Figure 1 (right) shows the MiniCor CubeSat
design. These two advances combined enable a swarm of CubeSats capable of returning white-
light coronagraph data from deep space for the cost of a single SMEX mission. g
g p
p p
g
Figure 5: (left) Busek electric propulsion modules can provide the large delta-V needed for
maintaining the CATSCaNS formation. (right) The Compact Laser Communications System
enables long distance communications in a 2U volume. Figure 5: (left) Busek electric propulsion modules can provide the large delta-V needed for
maintaining the CATSCaNS formation. (right) The Compact Laser Communications System
enables long distance communications in a 2U volume. However, WL coronagraphs are not the only measurements needed to achieve our science
goals. Additional instruments provide critical information in support of these goals and also benefit
from multiple viewpoints spread over 180 degrees in heliographic longitude. The L4/L5 magnetograph
has long been a goal to provide better characterization of photospheric and coronal magnetic fields. The Compact Doppler Magnetograph shown in Figure 6 (left) is an example of a CubeSat form factor
instrument that can deliver those measurements. EUV imagers also have a desire for at least one
additional view point off of the Sun-Earth line for triangulation of events. SunCET, shown in Figure 6
(right) is a CubeSat form factor instrument that can provide this capability. However, WL coronagraphs are not the only measurements needed to achieve our science
goals. Additional instruments provide critical information in support of these goals and also benefit
from multiple viewpoints spread over 180 degrees in heliographic longitude. The L4/L5 magnetograph
has long been a goal to provide better characterization of photospheric and coronal magnetic fields. The Compact Doppler Magnetograph shown in Figure 6 (left) is an example of a CubeSat form factor
instrument that can deliver those measurements. EUV imagers also have a desire for at least one
additional view point off of the Sun-Earth line for triangulation of events. SunCET, shown in Figure 6
(right) is a CubeSat form factor instrument that can provide this capability. 4. Feasibility Optical ground stations are also coming online that can support missions like CATSCaNS. In
2022 a small prototype RF-Optical system, including the mirror and cameras/backend has been
installed into DSS-13 at the Goldstone Deep Space Communications Complex. DSS-13 is the
R&D 34m BWG antenna at Goldstone. The combination of seven small (0.5m) mirrors
comprises a synthesized prototype optical aperture of about 1.3m diameter. As of 2022, the
seven-segment prototype mirror system has been undergoing alignment and test/checkout. Control was verified to maintain segment position to <1 microradian, with first light
successfully received from natural light sources. 1550 nm light was measured through the
100-meter fiber at the pedestal. JPL was able to track multiple sources across the sky from 20-
80 degrees elevation. The communications detector and receiver planned for the spacecraft
demo with the Psyche spacecraft have been installed and tested in DSS-13 as well. Control was verified to maintain segment position to <1 microradian, with first light
successfully received from natural light sources. 1550 nm light was measured through the
100-meter fiber at the pedestal. JPL was able to track multiple sources across the sky from 20-
80 degrees elevation. The communications detector and receiver planned for the spacecraft
demo with the Psyche spacecraft have been installed and tested in DSS-13 as well. The operational RF-Optical hybrid will ultimately include 64 mirrors each of 1
m diameter, installed as a segmented 8 m optical receive aperture/mirror physically inside one of
the new DSN 34- m radio frequency ground terminals (DSS-23, in California). The operational RF-Optical hybrid will ultimately include 64 mirrors each of 1
m diameter, installed as a segmented 8 m optical receive aperture/mirror physically inside one of
the new DSN 34- m radio frequency ground terminals (DSS-23, in California). 5. Instrumentation Heliospheric Imagers in a CubeSat form factor are more difficult to find. WISPR managed a
volume of ~30U, but this is dramatically more than other instruments in the suite and drives the scale
size of the bus. This is a potential descope if suitable instrumentation in a CubeSat form factor cannot
be found. Finally, a PlasMag suite is an essential component of such a mission because the in-situ
measurements provide a ground truth measurement against which to test models of CME evolution
and energetic particle generation at multiple points in the heliosphere. For this suite, example
instruments that meet measurement requirements in a CubeSat form factor are Co-Dice and mini-mag (Figure 7). Co-Dice provides both thermal and energetic particle measurements with a single
instrument and mini-mag is a very low SWAP magnetometer that can measure to 0.1 nT accuracy in a
magnetically clean environment. It can also be combined with body-mounted magnetometers and a
cleaning algorithm (Sheinker and Moldwin [2016],Deshmuk et al. [2019]) to provide accurate
magnetic field measurements in a noisy environment. (Figure 7). Co-Dice provides both thermal and energetic particle measurements with a single
instrument and mini-mag is a very low SWAP magnetometer that can measure to 0.1 nT accuracy in a
magnetically clean environment. It can also be combined with body-mounted magnetometers and a
cleaning algorithm (Sheinker and Moldwin [2016],Deshmuk et al. [2019]) to provide accurate
magnetic field measurements in a noisy environment. Figure 6: (left) The Compact Doppler Magnetograph can provide characterization of the solar
magnetic field from points off the Sun-Earth line. It’s small form factor enables it to fit on a SmallSat. (right) SunCet is an EUV imager with a CubeSat form factor. NISP/GONG R&D work
• NASA SOLARIS Phase A (over-the-pole, PI: SwRI, NSO: Co-Is, Compact
Doppler Magnetograph (CDM)
• 1-2 additional tests will be performed at TC (Boulder)
Future GONG? CDM magnetogram (17 Nov.)
Top view of CDM on optical table in TC Figure 6: (left) The Compact Doppler Magnetograph can provide characterization of the solar
magnetic field from points off the Sun-Earth line. It’s small form factor enables it to fit on a SmallSat. (right) SunCet is an EUV imager with a CubeSat form factor. CDM magnetogram (17 Nov.)
p
p Figure 7: (left) The GSFC Mini-mag has space flight heritage and very low SWAP while producing
better than 1 nT accuracy measurements. 5. Instrumentation (right) CoDice provides particle measurements from 10 eV
to 10 MeV in a single 2U instrument. Figure 7: (left) The GSFC Mini-mag has space flight heritage and very low SWAP while producing
better than 1 nT accuracy measurements. (right) CoDice provides particle measurements from 10 eV
to 10 MeV in a single 2U instrument. 6. Conclusion CATSCaNS is a concept that would revolutionize our understanding of CMEs in the Corona,
their evolution in the heliosphere, and consequently, the energetic particles they generate. Additionally, recent advances in CubeSat instrumentation, propulsion, and communication
technologies, have made a concept that can close on its science questions feasible. CATSCaNS,
or a mission like it, should be included in the list of recommended missions in the Heliophysics
Decadal Survey. Decadal Survey. References Joseph M. Davila. Solar Tomography. ApJ, 423:871, March 1994. https://doi.org/10.1086/173864. Joseph M. Davila. Solar Tomography. ApJ, 423:871, March 1994.
https://doi.org/10.1086/173864. Deshmukh, A. A., Sharma, S., Cutler, J. W., Moldwin, M., Scott, C., Simple Regret
Minimization for Contextual Bandits, Proceedings of the 2nd Exploration in Reinforcement
Learning Workshop at the 36th International Conference on Maching Learning, 2019. Jonathan Kolbeck, Andre ́ Anders, Isak I. Beilis, and Michael Keidar. Micro-propulsion based on
vacuum arcs. Journal of Applied Physics, 125(22):220902, 2019. https://doi.org/10.1063/1.5081096. C. Korendyke, D. Chua, R. Howard, S. Plunkett, D. Socker, A. Thernisien, P. Liewer, David
Redding, J. Cutler, J. Forbes, and A. Vourlidas. Minicor: A miniature coronagraph for an
interplanetary cubesat. 2015. C. Korendyke, D. Chua, R. Howard, S. Plunkett, D. Socker, A. Thernisien, P. Liewer, David
Redding, J. Cutler, J. Forbes, and A. Vourlidas. Minicor: A miniature coronagraph for an
interplanetary cubesat. 2015. Brian Mathason, Michael M. Albert, Doruk Engin, He Cao, Keith G. Petrillo, Jacob Hwang,
Khoa Le, Kent Puffenberger, Slava Litvinovitch, Mark Storm, and Richard Utano. CubeSat
lasercom optical terminals for near-Earth to deep space communications. In Hamid Hemmati and
Don M. Boroson, editors, Free-Space Laser Communications XXXI, volume 10910, pages 24 –
29. International Society for Optics and Photonics, SPIE, 2019. doi: 10.1117/12.2508047. URL
https://doi.org/10.1117/12.2508047. Sheinker, A., and M. B. Moldwin, "Adaptive interference cancelation using a pair of
magnetometers," in IEEE Transactions on Aerospace and Electronic Systems, vol. 52, no. 1, pp. 307-318, February 2016. https://doi.org/10.1109/TAES.2015.150192 Sheinker, A., and M. B. Moldwin, "Adaptive interference cancelation using a pair of
magnetometers," in IEEE Transactions on Aerospace and Electronic Systems, vol. 52, no. 1, pp. 307-318, February 2016. https://doi.org/10.1109/TAES.2015.150192 A. Vourlidas, S. Gibson, D. Hassler, T. Hoeksema, M. Linton, N. Lugaz, and J. Newmark. The
science case for the 4π perspective: A polar/global view for studying the evolution &
propagation of the solar wind and solar transients, 2020.
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https://openalex.org/W1553324314
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https://www.biodiversitylibrary.org/itempdf/16493
|
English
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The home life of wild birds; a new method of the study and photography of birds
| null | 1,901
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public-domain
| 80,861
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^
RICK
PPPHP ^
RICK '. i-n
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Marine
Biological
Laboratory
Library
Woods
Hole,
Mass. Presented by
the estate of
Dr. Herbert W. Rand
Jan. 9,
1964
)
I
I
I
I
[
[
1 Robin
Family Cock Robin, who stands
at the back, has just brought
and delivered a load of wild red cherries. His mate, who
was brooding at the moment, did not leave the
nest, but
hopped to one
side, and
presently returned
to her post,
where you see her in the picture. "Cbo IRmcfccrbocfecr
Ipccsa, IRcw THE HOME LIFE
OF WILD BIRDS /
:, COPYRIGHT, MAY,
igoi
BY
FRANCIS HOBART HERRICK
Set up, electrotyped, and printed May, 1901
Reprinted August, 1901
; January, 1902 TO THE MEMORY
OF
MY FATHER AND MOTHER TO THE MEMORY
OF
MY FATHER AND MOTHER PREFACE. PREFACE. IN
studying the habits of wild birds two important problems arc
at once encountered,
that of approach and the control of the position of the
nest. My
first experiments
were made with Redwing
Blackbirds and Cedar Waxwings, and
I soon
perceived
that an important principle was involved, which every subsequent experiment tended to
confirm. Wishing to test
its value as fully
as possible, every available nest which came
to hand was utilized, without
the exercise
of choice
in
regard
to
species. IN The observations were made
for the most
part
in
central New Hampshire,
in
the
towns of Northfield and
Tilton, and pertain
to the common
birds of the country. I am
indebted
to my sister for many practical and
valuable suggestions. FKANVIS
HOP.AKT HERRITK. FKANVIS
HOP.AKT HERRITK. WESTERN RESERVE UNIVERSITY,
ADELBERT COLLEGE, CLEVELAND, Oni", April,
n.iui. CONTENTS. CO
S
CIIAI'TKK
IV. I. PREFACE
vii
LlM
OF ILLUSTRATIONS ... . ix
INTKOUUCTK 'N..... ... XV
I. A NEW METHOD OF BIRD STUDY AND PHOTOGRAPHY
i
II. ILLUSTRATIONS OF THE METHOD: THE CEDAR-BIRD; THE BALTIMORE
ORIOLE; THE REDWING BLACKBIRD, AND THE KINGBIRD ... 17
III. TENT AND CAMERA: THE TOOLS OF BIRD-PHOTOGRAPHY
. . 29
IV. THE
ROBIN- AT ARM'S LENGTH; A STUDY OF INDIVIDUALITY
. . 36
V. THE CEDAR-BIRD
. . . ... 52
VI. RED-EYED VIREOS
. . . . .... 64
VII
THE NEST-HOLE OF THE BLUEBIRD
... . . 71
VIII. MINUTE OBSERVATIONS ON CATBIRDS
. . . 76
IX. THE REARING OF THE NIGHT HA\VK
. . .... So
X. THE
KINGFISHERS AND THEIR KING Row
86
XL
CARE OF YOUNG AND NEST
. . . .... 94
I. BROODING, AND FEEDING THE VOUNG. II. CLEANING THE NEST. XII. THE FORCE OF HABIT ... ... . . in
XIII. FEAR
IN BIRDS
. . . 117
XIV. TAMING WILD BIRDS \YITHOUT A CAGE
125
INDEX
. . . . . . 139 ILLUSTRATIONS. Rubin Family. Cock
Robin, who stands
at
the back, has
just brought and
delivered
a
load
of
wild
red
cherries. His
mate, who was brooding
at the moment, did
not leave the nest, but hopped to one side, and presently returned to her post,
where you see her in the
picture. Lens 9^ inch
focus
; speed
5-
;
stop
32
;
time
1 second;
plate, Seeds'
Xo. 27
"gilt edge"
(which should
be
under-
stood
as
generally used
in
this
work)
;
distance
of object
4
feet
;
full sun
;
July
28, 1900
... Frontispiece g
FIG. 4. Truncated elm, riddled by Woodpeckers, the lower nest-hole recently occupied
by Bluebirds
6 by
FIG. 5. Tent and Bluebird's nest. Compare Figs. 10, 59-65
. .... 7 p
FIG. 6. Kingbird's nesting tree, and nesting branch
removed and mounted on stakes
with tent. The tent-cloth
is laid
in position
at one end of peak, and ready
to be drawn over frame. The Kingbird pictures were all made on this spot
. gb
d p
p
Fir,. 7. Female Kingbird astride nest,
the later brooding attitude .... g
FIG. S. Kingbird family, the female partly hidden at the back. It was an easy matter
to focus directly upon the head
of the standing or brooding bird
. . 9 directly upon
g
g
FIG. 9. Cedar-bird about
to
feed young by regurgitation. Photographed
at the
nest
shown in Figs,
i,
12, and
13. Zeiss
Anastigmat, Ser. ii
a,
61- inch; speed
\\
distance about 30 inches,
in
full sun
. . . . . . . .11 Illustrations PAGE
FIG. 10. Female Bluebird with cricket at converted nest-hole of Flicker
. . 13
FIG. ii. Female Chestnut-sided Warbler standing oxer young. Compare Fig. 3
. . 14
FIG. 12. Cedar-bird's nest in pine,
15 feet from the ground. Upstretched neck of
the
old bird could be seen at a point just beneath the upper arrow-head. Nesting
bough carried to field beyond, and mounted as shown
in Figs,
i and 13
. 18
p JG
I3
Nesting branch of Cedar-bird set up in field and
tent pitched beside
it. Com-
pare Figs,
i and 12 ... 19
FIG. 14. Oriole inspecting young. Still timid to a degree ... .20
FIG. 15. Oriole inspecting nest. Behavior freer than in last
... . 2t
FIG. 16. ILLUSTRATIONS. Tent in swamp, fronting Redwing Blackbird's nest
22
FIG. 17. Kingbird feeding young, and balancing herself with uplifted wings
. . 23
FIG. 18. 'Male Kingbird serving a Cicada or harvest-fly, which a youngster is striving to
master. Its efforts were not in vain
. . 23
FIG. 19. Unequal contest between Kingbirds and a dragon-fly. This insect was crushed
and served up piecemeal
. . 2 5
FIG. 20
Kingbirds serving a
dragon-fly, whose wings and
stick-like body are seen pro-
truding from the mouth of one of the young
. . 25
FIG. 21. Male Redwing Blackbird feeding a young one
. . 27
FIG. 22. Female "
bristling
"
to keep cool, while shielding the young on morning of a
hot day. July n, 1900
. 2 7
FIG. 23
Kingbird
out
of
its nest at age of eighteen
days, with power of
flight well de-
veloped. July
13,
1900 .... FIG. 24. Tent, folded for carrying, cameras, and plate-bag
the tools of bird-photography
30
FIG. 25. Brown Thrush entering her nest to brood ..... -33
FIG. 26. Robin
in
an April
snow. Wade
Park, Cleveland, Ohio, April
4,
1900. X 3
FIG. 27. Head of Cock Robin,
life-size X 4s
.... -37
FIG. 28. Head
of
female
Robin,
life-size X
4^. The
slime on
her
bill
is from
the
throat of a young bird ..... -37
FIG. 29. Female Robin brooding
.... -4
FIG. 30. Female Robin inspecting nest
.... 4 1
FIG. 31. Cock feeding cluster of earthworms
. ... -43
FIG. 32. Cock standing at nest immediately after serving food, and ready for the duty of
inspection and cleaning
.... . 46
FIG. 33. Female Robin cleaning nest ... -49
FIG. 34. Head of brooding female,
life-size X
zj-
. . -5
FIG. 35. Cedar-bird
chorus. The young, with wings spread and
a-quiver,
with open
mouth and upstretched necks are calling
to the silent mother. Life-size X 3
53
FIG. 36. Cedar-bird family group, the male with
full throat and black cherry in
bill
. 54
FIG. 37. Cedar-bird approaching nest of young which are nearly ready to
fly
. 55
FIG. 38. The same bird standing
at nest with
full
gullet,
a little later in day after one
of the young had left ...... 5^
FIG. 39. The Cedar-bird approaches with closed bill but
full throat
. 57
FIG. 40. ILLUSTRATIONS. After feeding she inspects the young and
in
this instance appears to be sitting
with
tail resting on the branch, but this
is probably not the case
. 57
FIG. 41. She tosses up her head, and produces a cherry
FIG. 42.'
She
is startled at a strange sound ... 5$
FIG. 43. She looks curiously at the tent while inspecting nest
59 FIG. 10. Female Bluebird with cricket at converted nest-hole of Flicker FIG. ii. Female Chestnut-sided Warbler standing oxer young. Compare Fig. 3 Illustrations xi PAGI
I'H'.. 44. She stands like a statuette while inspecting her family ..... 59
IMC. 45. She devours what
is sometimes removed from the nest ..... 60
FIG. 46. The sac
is taken directly from the cloaca of the young bird
. . .61
FIG. 47
A young Waxwing from this nest on
the morning of
flight,
in natural attitude
expressive of fear. July
19,
1900
........ 62
FIG. 48. Cedar-bird,
thirty-six
hours
old. Typical
instinctive
response
to sound
or
vibration of nest. The stub- wings are used for suppoit
.... 62
Cedar-bird standing at nest after delivery of food
. ....... 63
FIG. 49. Male Red-eyed Vireo standing over nest. Life-size X 3 ..... 65
FIG. 50
Female Red-eyed Vireo with a neuropterous (?) insect in
bill .... 66
F'IG. 51. Feeding a nestling ............ 66
FIG. 52. Female inspecting the nest in the characteristic manner ..... 66
FIG. 53. Male standing over the young .......... 67
FIG. 54. F'emale Red-eyed Vireo
cautiously
inspecting
nest. Compare with
the
freer
manner illustrated in Fig. 52. July 5,
1900 ....... 67
FIG. 55. Approaching to inspect the nest and drawing back
. . . -67
FIG. 56. Cautiously feeding the young
. . .68
FIG. 57. Inspecting nest at a distance
. . . .69
FIG. 58. Young birds from this nest just before flight ... -7
FIG. 59. Female Bluebird on point of leaving nest-hole
. . 71
lie. 60. Taking grasshopper to young ........ 72
FIG. 61. Standing at entrance with green insect-larva in
bill
. . -73
FIG. 62. Female Bluebird cleaning the nest
.... ... 74
FIG. 63. Cleaning the nest
. . . . . . 74
FIG. 64. Cleaning the nest ...... -74
FIG. 65. Taking a final glance around before entering nest-hole with grasshopper
. . 75
FIG. 66. ILLUSTRATIONS. Female Catbird bringing
to young a limp
dragon-fly, the large .-Eschna heros,
which has just issued from
its pupa-skin ....... 76
FIG. 67. Catbird inspecting her young .......... 77
FIG. 68
Catbird cleaning the nest .......... 79
FIG. 69. Night Hawk on
bare ground, and eggshells from which
it emerged three days
before
. . ..... . .80
FIG. 70. Night Hawk approximately three days old
. . Si
IMG. 71. Night Hawk about nine days old
. . .81
I
ic. 72. Night Hawk about twelve days old
. . . . 82
FIG. 73. Night Hawk about sixteen days old
... . . .82
FIG. 74. Front-face view of bird shown
in
Fig. 72
....... 83
''"
75'
Young Night Hawk in enclosure where
it remained until able to
fly
. . 83
FIG. 76.'
Tunnel of Kingfisher (on the right) in sand-bank overgrown with pines, beside
country road. Northfield, New Hampshire. August,
1899
. . 86
FIG. 77. Nest of 1900 in same bank and
probably of same pair. Kingfisher taking fish
to young. Lens 9 T\ inch; speed \\
stop 8;
time
-j s second;
distance 9
ft. 8
in. ;
full sun. July 24, 1900
. . . . .87
FIG. 78. Kingfisher backing out of tunnel. A stream of sand is started from tne opening
at every entrance and exit ....... .87
FIG. 79
Five Kingfishers from chamber
at end of tunnel
approximately nine days old. I'H'.. 44. She stands like a statuette while inspecting her family p
g
y
IMC. 45. She devours what
is sometimes removed from the nest p
g
y
59
IMC. 45. She devours what
is sometimes removed from the nest ..... 60 IG. 46. The sac
is taken directly from the cloaca of the young bird
. . y
g
FIG. 47
A young Waxwing from this nest on
the morning of
flight,
in natural attitude
expressive of fear. July
19,
1900
........ 62 p
y
,
FIG. 48. Cedar-bird,
thirty-six
hours
old. Typical
instinctive
response
to sound
or
vibration of nest. The stub- wings are used for suppoit
.... 62 g
FIG. 73. Night Hawk about sixteen days old
... . g
y
FIG. 74. Front-face view of bird shown
in
Fig. 72
.... ''"
75'
Young Night Hawk in enclosure where
it remained until able to
fly g
g
y
FIG. ILLUSTRATIONS. 76.'
Tunnel of Kingfisher (on the right) in sand-bank overgrown with pines, beside
country road. Northfield, New Hampshire. August,
1899
. . 86 y
p
g
FIG. 77. Nest of 1900 in same bank and
probably of same pair. Kingfisher taking fish
to young. Lens 9 T\ inch; speed \\
stop 8;
time
-j s second;
distance 9
ft. 8
in. ;
full sun. July 24, 1900
. . . . .87 July
, 1900
.8
FIG. 78. Kingfisher backing out of tunnel. A stream of sand is started from tne opening
at every entrance and exit ....... .87 Illustrations xii PACE
FIG. So. Posed in
line, biting and pulling ... 89
FIG. Si. Posed
in row to illustrate habit of sitting still
. .... 90
FIG. 82. King-row at a later stage
birds thirteen days old
90
FIG. 83. Kingfisher at nine days, showing feather tubes and tracts
.... 91
FIG. 84. At thirteen days. The wing-quills show one half inch of the blue-black, white-
tipped feather-shafts ..... 9 1
Fie.. 85. Kingfisher at fifteen days. Nearly
all feathers partly unsheathed ... 92
FIG. 86. Kingfishers eighteen days
old. The bright blue
of the upper parts and
the
white and chestnut bands across the breast are now very prominent
. . 92
F, G
gy
Kingfishers twenty-two days old. To illustrate how they break ranks and walk
backwards, when placed in line. The second on the left has already taken a
few backward steps
. . . -93
FIG. 88. Female Brown Thrush brooding. Lens 9T\ inch; speed {;
stop 32;
time | sec-
ond;
distance 4 feet in full sun. July
13,
1900
. . 95
FIG. 89. Female Robin brooding. Attitude of keen attention
. 97
FIG. 90. Female Redwing Blackbird feeding a young bird ... .98
FIG. 9:. The same bird awaiting the reflex response of the throat and gullet of young. If not forthcoming, the food
is withdrawn, and another is tested
. . 98
FIG. 92. Female Kingbird standing over young with drooping wings to ward off the sun. Typical brooding attitude during last days of life at nest
. . 99
FIG. 93. Kingbirds rending an unruly grasshopper
99
FIG. 94
Helping a grampus down the throat of a Kingbird ... . 101
FIG. 95. The male grampus,
Corydaliis cornutus. Full size, from life
. . 101
FIG. 96. ILLUSTRATIONS. Female Chestnut-sided Warbler bristling
to keep cool while brooding on a hot
June day
. . ....... . 102
FIG. 97. The same bird in the more common attitude of brooding during the early life of
the
young. Lens
Zeiss Anastigmat,
Ser. ii
a;
6i-
inch; speed
|;
stop 32;
time \ second;
distance 3 feet in
full sun. June 23, 1900 .... IDJ
FIG. 98. Male Chestnut-sided Warbler brings food for his little children. His mate, who
is brooding, receives
it into her own bill, but does not taste a particle herself
. 103
FIG. 99. Female
Chestnut-sided
Warbler brooding with
throat
puffed out and head-
feathers erect ............ i3
FIG. 100. Female Brown Thrush placing food well down
in the throat. Point of bill
is
on level with external ear of young .... '4
FIG. 101. The same bird cleaning the nest ...... 105
FIG. 102. Cedar-bird taking sac from cloaca of young
. . . . . 106
FIG. 103. Female Kingbird cleaning the nest
... i7
FIG. 104. Baltimore Oriole feeding
its young
.... 108
FIG. 105
The same bird in another attitude ...... I09
A Hatful of Kingfishers
.no
FIG. 106. Male Redwing Blackbird inspecting nest ..... . 112
FIG. 107. The same bird engaged
in
the same occupation. To illustrate the formation
of habits in the daily routine ...... 112
FIG. 108. Cock with a large grasshopper .... i>3
FIG. 109.-
Cock "
taking aim " ..... . ''3
FIG. 1 10. Cock ready to inspect the nest .... i'4
FIG. in. Female Robin inspecting the nest in a typical attitude ... .114 Illustrations PACK
Fi<:. iiJ. Female Kingbird inserting an insect in the throat of a fledgling
. . 115
FIG. 11,5. Cedar-bird's eggs with two blind and naked young thirty-six hours old
. . ii.S
FIG. 114. The same ......... .118
FIG. 115. The same,
illustrating different phases of instinctive behavior
. .118
FIG. 1
1 6. Kingfishers twenty-four days old, posed to illustrate fearlessness, when capable
of flight .... 119
FIG. 117. Young Cedar-birds at nest,
in characteristic attitude. Under the influence of
fear and ready for flight. For description, see page 60
.120
FIG. 118. Brown Thrush startled on nest
. . .... . 121
FIG. 119. Cock Robin startled by alarm call of his male ...... 122
FIG. 120. Red-tailed Hawk worried. I. TO
describe and illustrate a new means of studying animal behavior, anil
to record
what has been learned by
its aid concerning the lives of some
of our common
birds
is the main purpose of this volume. It is a popular study of birds in action
and
is chiefly concerned with the homes or nests and their occupants. TO y
p
While the desire has been present to make these pages readable, no effort has been
spared
to render them
accurate. Many
of
the
observations
are new
;
nearly
all
are
original, and every statement
of fact
is believed
to be true as
it stands. The wish to give a human interest to every phase of animal activity
is of very ancient
origin and has done too much already in spreading the seeds of popular error and super-
stition concerning animal life and lore. Animals should be studied as animals which they
are, and not
as human
beings which they have never been and
are
not
likely ever to
become. The constant reading of human attributes into the activities of animals is to begin at
the wrong end, and
is a drag on the progress of accurate knowledge. We should first study
the animal as far as possible from
its own standpoint, and learn with exactness the
facts
of its
life, taking care not to press analogies farther than the observed
facts will warrant. Ignorance of anatomy as well as of physiology, and the desire to find
in the doings of ani-
mals a marvelous counterpart
of human powers of
intelligence and reason have already
stocked our libraries with
fables, anecdotes, and
stories, many of which make delightful
reading, but possess
little value for the nvidern student. g,
p
The
first duty of the narrator of natural as well as
civil
history
is to
tell the truth,
and to the naturalist belongs also the privilege of showing that the lives of the higher ani-
mals, when fully and clearly revealed, possess a more viUl interest than the puppet dressed
in human clothes, however admirable the latter may be as a work
<.f art. y
I
trust
that
the
reader
will
not misunderstand
these
remarks. Is
it denied
that
animals
possess
intelligence or any powers of reason? Not at all! Such questions de-
pend largely upon our
definitions of words, and without
fresh
observations arc
usually
fruitless of result. ILLUSTRATIONS. Instinctive attitude expressive of fear, and serving
to inspire fear ... ..... . 123
FIG. 121. Young Cowbird standing at nest of Magnolia Warblers,
its foster parents
. 124
Fu;. 122. Young Cowbird, as
it appeared when found, completely filling the nest, having
smothered
its rightful occupants ...... .124
FIG. 123. Male Kingbird standing at nest, and young
in characteristic attitude
. .126
FIG. 124. Female Robin, engaged in nest-cleaning
127
FIG. 125. Female Red-eyed Vireo feeding the young
. . 128
FIG. 126. Her mate ready to inspect and clean the nest
. 129
FIG. 127. Offering food to a Chestnut-sided Warbler, tamed without a cage
. . . 132
FIG. 128. Chestnut-sided Warbler
family,
the
male above the
nest on which
sits
his
brooding mate .... '33
FIG. 129. Female Chestnut-sided Warbler taking a peep at the nest, which then contained
eggs or young birds barely hatched
. 134
FIG. 130. The same bird inspecting her young after feeding them
. 135 I. What
is criticized
is the gross anthropomorphism which characterizes
much that
is written upon the actions of animals. If
I am an offender
in this direction,
I hope
it
is only in a minor degree. I am anxious to attribute to the animal every power
which
it
is actually known to possess, and look for the roots of human instinct and
intel- Introduction. xvi licence all alone the
line of animal
evolution. It tends only to confusion, however, to
o
o
call those
acts
of association which
lead
to acquired
habits, instincts, or the
countless
mechanical
or chemical
reactions
of organisms to external
stimuli,
the
expressions
of
intelligence and thought. " Go to the ant thou sluggard
! "
is good advice, but one should
bring from the ant a trustworthy account of how
it performs its wonderful works. It is im-
portant to distinguish the root from the bud, as well as from the perfected flower and fruit. portant
distinguish
p
Although this
is not a treatise on animal behavior, a general working theory has been
adopted and will now be given. Every animal at birth inherits with
its bodily organs the
power to use them
in a more or less definite way, and all but the lowest animals, of which
the Protozoa, jelly-fishes, and possibly the worms may be taken as representatives, acquire
some power of learning to do things in the course of
their
lives. Their equipment thus
consists of (i) unlearned or inherited powers, and (2) of learned or acquired abilities, which
are the results of experience
often very bitter. The term " instinct
" when used in a very
broad sense may be given to all inherited or ingrained tendencies, and " habit
" reserved
for what is acquired or learned through a process
of
association
of
certain
things with
certain acts. An animal's powers thus
consist
of
free
gifts
at
its start in
life, and later
acquisitions gained through its own efforts in the struggle for existence. acquisitions g
g
gg
The catalogue of
instinctive acts
even in the narrower sense of involving a number
of
different organs
is surprisingly great in an animal standing so high in the scale as the
bird, but examples drawn from a single species will suffice. I. When the spring comes the
young bird, who returns to the place of its birth,
is prompted to find a mate, and with her
soon begins to build a nest. Though unattended by instructors and unprepared by prac-
tice,
it uses the inherited tools of its guild
bill, breast, and feet
with a nice precision,
and be
it Oriole, Robin, Flycatcher, or Vireo, follows with wonderful closeness the type of
architecture which
its ancestors have used for ages. g
Why does the Robin in
its first attempt at nest-building begin by laying a foundation
of dry grass or stubble, and add to this mud softened with water and made into a mortar,
which
it then heaps about its breast and molds into a symmetrical cup, often selecting a
rainy day for the work? One might as well ask why the Robin lays blue eggs, or why it
utters its well known
call. It acts in these ways because
it must, because Robins have
been doing these things for hundreds of generations. It not only inherits tools, but a cer-
tain aptitude for their use. Its organization compels or determines its actions. aptitude
g
p
No learning of such initial actions is required or even possible since all this has been
attended to, as one might say, centuries before
the animal was born. These instinctive
responses are spontaneous, and when the right button
is pressed or the right stimulus ap-
plied from without or within, the reaction follows as a matter of
course. Of course the
Robin must make a mortar of mud and straw;
of course
it must lay blue eggs, and after
incubating them, carefully rear and feed
its young. To do otherwise would not only be
absurd, but very uncomfortable. Had
its ancestors been Cowbirds
it would have made
no nest at all, but filched another's, and foisting its eggs upon some simple minded nurse,
shirked the duties of parents to their offspring. The Cowbird was thus very early to enter
the field of experimental psychology. p
p y
gy
Every bird must follow the laws of its nature, and
its inherited instincts are no more
wonderful than
its
inherited
organs,
its vocal
cords,
its keen
eyes, and
its marvelous
feathers. Introduction. Introduction. xvii The higher animals thus start
in
life with
a definite equipment,
a body tuned
to
respond to the world in which they are placed, and this ingrained ability for action may
be
called
instinct. In speaking of the "habits" of animals we usually mean the manner of
their
life
in
general, while a " habit
"
in thetechnic.il sense may be regarded as a mode of action which
the animal has learned or acquired. It
is associated with pleasure, and
in
the course of
repetition may become more
or
less
fixed
or "stereotyped."
In
this sense
habits are
formed out of the raw material which heredity provides. The young bird learns to
eat
certain things, to avoid certain enemies, to
start
at
certain sounds, to
ignore
others, to
approach
its
nest
in
a
certain
way. Thus also the vertebrate sometimes acquires
tin-
habit of walking backward, while
its instinct leads
it to walk forward. g
Habits must
in time take the place of instincts in a very large measure, and
it would
not be strange
if a Robin's second nest were more nearly perfect than
its
first, or
if the
third were better than the second, but this would also depend upon other conditions. depe d upo
The power of forming habits
is a sign of intelligence, but
not
necessarily of reason. The intelligence may be a small grain and never destined to grow into a
flourishing tree
of knowledge, but
it must exist along with the power of profiting by experience. g ,
g
p
p
g
y
p
The mental faculties of birds seem to exhibit a wide range of gradation from exces-
sive stupidity to a
fair degree
of
intelligence, with strong associative powers,
rarely
if
ever the
association
of
ideas, but
of
things
with
actions,
and
often
with
wonderful
powers
of
imitation. p
The habits acquired by one generation are probably never handed
on
to
the
next,
but this is a subject from which the dust of argument has not yet cleared away. II. That a bird in the hand
is worth two in the bush may be a good motto
for the
an-
atomist or epicure, but for the observer of living animals a bird within reach of the hand
and
still in
the bush
is
of
far greater worth. The problem
is how to
see and
not be
seen. If a bird
is actually caught and kept
in a cage or put under restraint in any way,
its behavior is no longer perfectly natural and
free, at
least
not
until
all
fear has been
subdued and
it
is no longer wild but tame. What
is needed
is an invisible chain which
shall hold the animals to some fixed and chosen spot which
can then be approached
in
disguise. disguise. Fortunately for the student of bird-habit and instinct all these conditions are fulfilled
for a most important and interesting period,
that of
life at the
nest. The
nest
is the
given fixed point, and parental instinct
is the
invisible
chain. The
wild
bird, however,
is bound not merely to the nest, but to
its young. Wherever the young go, the old birds
follow. By using the nearly fledged young as a
lure, some species could,
I
believe, be
led across country for
a
mile
or more. I have taken them two hundred
feet without
special
effort. p
Hitherto the bird-photographer has had
to
rely mainly upon chance
in
getting a
picture of the nesting scenes. Most land birds depend upon concealment for protection
from
their enemies
during the season
of young. Their nests
are apt to be shrouded Introduction. xviii in grass or foliage, and,
if easily approached, are
usually inaccessible
to the camera. If
the nest
is in a high bush or tree, the difficulties of the position and light
are
usually an
effectual
bar
to
obtaining good
pictures, to
say
nothing
of
seeing what
takes
place. When the nest
is on or near the ground and in a
well-lighted spot, conditions which
are
only rarely fulfilled,
it has been customary to
set up the camera, and
attaching a long
rubber tube or thread to the
shutter, to
retire
to
a distance and
wait
for the birds to
appear. II. When one of them
is seen to go to the nest, the plate
is exposed by pulling the
thread or pressing the pneumatic bulb, and,
if
in
luck, a
picture may thus be obtained. Many plates, however, are sure to be spoiled
;
little can be seen, and the observer has no
control over the course of events. In the pages which follow, a method
is described by
which nesting birds can, in many cases, be successfully approached and studied with ease
whatever the
position
of the
nest. If p
It
is a comparatively easy matter to examine and photograph the
nest, the eggs, or
the young of such species whose dwellings are
accessible
to
all, but
to portray the
free
behavior of the adult bird of the shy land species is quite another question. y
q
q
The method
is limited in
its application from the necessities of the case. It is based
on the solid ground of animal instinct, and may confidently be expected to have a wide
application
; but how wide or general
its use may become can only be determined by
well-directed experiment. III. Nearly all the illustrations of this volume are from photographs of adult land
birds,
and the reader will observe that they are
in many cases arranged
in
series, and portray
certain actions which are performed in a kind of routine. With very few exceptions
all
were made by means of the method, that
is to say, the photographs were taken
deliber-
ately and not by chance. My plan was to watch the life at the nest very closely, hour by
hour, and day by
day, and
I
often made a
large number
of photographs
to
illustrate
typical and unusual scenes at a nest. The observer has the advantage of being on
the
spot,
of
being
able to see every act performed and to seize
every
opportunity which
may arise. Many of the photographs here shown could not have been obtained by any
other means. What
is offered now represents but a beginning in the attempt to portray the whole
life of birds at the nest. The
first furrow only has been struck in an old and
still fallow
field. These pictures
will possibly seem crude when compared with those which
the
future will yield, but there
is this to be said about
all
really good photographs
of
wild
animals, that they possess a permanent interest and value, since within their limits they
represent the truth, vigor, and freshness of nature. When this method comes to be ap-
plied to some of the water birds, the Terns, Gulls, and
their congeners along the
coast,
which are more
easily approached than
the shyer land
species, serial
pictures
will
be
obtained of far greater perfection and beauty than anything which has yet appeared. g
p
y
y
g
y
pp
For the portrayal of animals in action the camera is of supreme value, and
if
I have
emphasized
its use,
it
is only as a means
to an
end. Scientific books dealing with
the
anatomy and development of animals will always require good drawings for the illustra- Introduction. XIX tion of their subjects, and these are preferable to poor photographs, but for the study of
animal behavior in both
the
invertebrates and
vertebrates the camera
is immeasurably
superior to brush or pencil. III. Popular natural history books have already a large body of
invaluable material to draw upon for illustrative purposes, and the often
crude, impossi-
ble, or imperfect drawings, which have so long done
service
in
the
past,
will
gradually
give place to truthful delineations uf anim.iU at home, and
in the midst of that nature of
which they form a part. A NEW METHOD OF BIRD STUDY AND PHOTOGRAPHY. TH THE
method of studying the habits of wild birds which
this volume
illustrates con-
sists
in
bringing the birds to you and
then camping beside them,
in watching
their behavior
at arm's length and
in
recording with the camera
their varied
activities. By means of such a method
one may live with the birds for days at a time,
and watch the play of their most interesting habits and instincts. The actors are not con-
fined in cages; they suffer indeed no
restraint, excepting that only which
their nature
imposes. They come and go at will, and their life is as free and untrammeled as ever. p
y
g
The method enables one to see with his own eyes at a distance of a few inches or
feet, more or less, what birds do in and about their nests, and
at the same time affords
the rare opportunity of making photographs, not a single picture or a chance shot now
and then, but an unlimited series of pictures to illustrate the behavior of birds in the
full-
est manner and at the most interesting period of their lives. It
is often an easy matter
to focus your camera
directly upon the bird
itself and
to
give a time exposure when
desired. Moreover, you can approach as near as you wish, and make photographs of any
required size. q
I will now give the reader a less enigmatical account of the method, first considering
its psychological basis or the scientific principles on which
it rests, and then recording
in
a separate chapter, as practical examples of
its working, the exact history of a few of the
cases in which
it has been applied. applied. The method in use depends mainly upon two conditions: p
y
p
(1) The control of the nesting
site, and p
y
p
(1) The control of the nesting
site, and (2) The concealment of the observer. By nesting
site
is meant the nest and
its immediate surroundings, such as a twig,
branch, hollow trunk, stem, or whatever part of a tree the nest may occupy, a bush, stub,
strip of sod, or tussock of sedge, that
is
the nest with
its immediate
settings. Wild
Birds. The nesting bough
is
carried
to a convenient distance
from
the
tree, and
firmly
fastened to two stakes, driven into the ground and placed in a good light. If the nest
is
in
a tussock
in
a shaded swamp, the whole
is
cut out and
taken
to
the
nearest
well-
lighted place
;
if in the woods,
it
is carried to a clearing where the
light
is
favorable for
study. Again, when a nest like that of the Brown Thrush occupies the center of a dense
thorn bush which no human eye can penetrate and much less that of the camera, its main
supports are cut off, and the essential parts are removed to the outside of the clump or to
any favorable point close at hand. If the nest
is but five or ten feet up, the main stem is
severed, and the nesting branch
lowered
to the four-foot mark,
a convenient working height. I
wish
to
emphasize
the
fact that the nest itself is usually
not moved or disturbed, or rath-
er
that
it
is moved
only with
its
supports. The
change
is
one
of
space
relations, which
may
change
with
every
pass-
ing breeze, but the
relation
of
nest
to support remains undis-
turbed. s
Fig. i. Tent in front of Cedar-bird's nest, shown in its original position
in Fig. 12. One of the birds is feeding its young. turbed. This sudden
displacement
of the
nesting bough
is
of no
special importance to either old
or young, provided
certain pre-
cautions are taken
to be dwelt
upon a little later. It is as if an
apartment or living room were
removed from
the
fourth story
of some human
abode
to the
ground
floor,
or
in
the case of
the ground
building
birds as
if
the
first
story were
raised
to
a
level with the second. The
immediate surroundings
of the
nest remain the same in any case. The nest might indeed be taken
from
its bough
or
from the sward, but this would be
inadvisable, chiefly because
it would destroy the na-
tural site or the exact conditions selected and
in some measure determined by the birds
themselves. s
Fig. i. Tent in front of Cedar-bird's nest, shown in its original position
in Fig. 12. One of the birds is feeding its young. Fig. i. A NEW METHOD OF BIRD STUDY AND PHOTOGRAPHY. If the
nest,
like that
of an
Oriole,
is fastened
to
the
leafy
Nestin ^ite
6
branch of a tree, the nesting bough
is cut off, and the whole is then care-
fully lowered to the ground and set up in a good light, so that the branch with the nest
shall occupy the same
relative
positions which they did
before. The nest, however, is
now but four instead of forty or more feet from the ground. Nestin ^ite
6 i i Wild
Birds. Tent in front of Cedar-bird's nest, shown in its original position
in Fig. 12. One of the birds is feeding its young. nest remain the same in any case. The nest might indeed be taken
from
its bough
or
from the sward, but this would be
inadvisable, chiefly because
it would destroy the na-
tural site or the exact conditions selected and
in some measure determined by the birds
themselves. For an observatory I have adopted a green tent which effectually conceals the student
together with
his camera and
entire
outfit. The reader will find this fully described in
the chapter on the tools of the bird-photographer. The tent
is pitched
beside
the
nest, and when
in
operation,
is
open
only
at
one
point,
marked by a small square window, in
line with the photographic lens
Concealment of
the Observer. A Nc\v Method of Bird Study and Photography. 3 It seems
.it
first
thought
strange and
almost
incredible
that one
may
take
sucli
liberties with
wild
birds, without wreaking
destruction
upon
the young or introducing
such unnatural conditions as would be
intolerable to every true student
Principles which
and
lover
of
birds, but
this
is by
no means
the
case. No
injury
is
underlie the
wrought upon
old
or young. The
former nesting conditions are soon
Method,
forgotten, while
the new
are quickly adopted and defended
with
all
the boldness and
persistence of which birds are capable. p
p
This method of studying birds depends mainly upon the strength of the parental
in-
stincts which bind
old
to young by an invisible
chain, and upon the ease with which a
bird K-arns to adapt itself to new conditions. Upon more complete analysis we recognize
the following psychological principles: following psychological principles:
(a) The strength of an instinct increases through exercise, and may be rt-On forced by
habit
; following psychological p
c p es:
(a) The strength of an instinct increases through exercise, and may be rt-On forced by
habit
; An instinctive impulse may be blocked or suppressed by any contrary impu ;
(b) An instinctive impulse may be blocked or suppressed by any contra (c) The
instinct
of
fear
is
often temporarily suppressed by the fighting instinct, or
permanently overcome by
the
repetition
of any experience leading to the formation of
new habits or associations. Concealment of
the Observer. and the nest. and the nest. A Nc\v Method of Bird Study and Photography A Nc\v Method of Bird Study and Photography. We may also add
: may
(d) New habits are readily formed and rcenforce or supplant those
of older growth
;
(c) Abstract ideas, if they form any part of the furniture of the average bird-mind, are may
(d) New habits are readily formed and rcenforce or supplant those
of older growth
;
Ab t
t
f
f th
f
it
f th
bi d
i d y
(d) New habits are readily formed and rcenforce or supplant those
of older growth
; (d) New habits are readily formed and rcenforce or supplant those
of older growth
;
(c) Abstract ideas, if they form any part of the furniture of the average bird-mind, are
extremely hazy and fleeting
; (c) Abstract ideas, if they form any part of the furniture of the average bird-mind, are
extremely hazy and fleeting
; y
y
g
(f)
Finally we must recall the physiological fact that birds are guided in most of their
operations by sight and hearing, not by scent. Their olfactory organ is very rudimentary
at best, and avails them neither in finding food, nor in avoiding enemies. y
y
(f)
Finally we must recall the physiological fact that birds are guided in most of their
operations by sight and hearing, not by scent. Their olfactory organ is very rudimentary
at best, and avails them neither in finding food, nor in avoiding enemies. g
g
After a
brief
analysis
of
the
parental
instincts we
will
endeavor to show how the
principles
just
given
are
applied
to
the problem of approaching wild birds in the way
described. The parental instincts begin to control the
life of the adult with the
periodic revival
of the reproductive functions, and vary greatly in their scope and intensity at the different
stages of their reign as well as in different species of birds. They are periodic, recurring
at definite jntervals during sexual
life and
in serial form, one kind of act
usually
leading
to the next
in sequence, and so on until the series
is complete. A Nc\v Method of Bird Study and Photography. q
,
p
When more than one
litter
is produced in a season, the
series of
events
is repeated
with minor changes
If we include the typical migratory movements, the principal terms
of the reproductive cycle may be expressed more fully as follows
: ep oduct
e
y
y
p
y
(1) Spring migration of the summer residents to the place of birth
; p
y
y
p
y
(1) Spring migration of the summer residents to the place of birth
;
(2) Mating
; p
y
y
p
(1) Spring migration of the summer residents to the place of birth
;
Mating ( )
p
g
(2) Mating
; ) Mating
)
Selection of nesting site and construction of the nest
; (2)
at
g
(3)
Selection of nesting site and construction of the nest
; (4)
Egg-laying
; (5) Incubation
; (5)
(6) Care of the young in the nest, including feeding, and cleaning nest and young; (5)
(6) Care of the young in the nest, including feeding, and cleaning nest and y
g
(7) Care and education of young from time of flight
; y
g
(8)
Fall migration to winter quarters. (8)
migration
quarters. Birds seem to follow one line of conduct, whether
it be sitting over the eggs, brood-
ing, or tending the young, until their instinct in that particular direction has been satisfied, ( )
g
q
Birds seem to follow one line of conduct, whether
it be sitting over the eggs, brood-
ing, or tending the young, until their instinct in that particular direction has been satisfied, Wild
Birds. thus normally completing one term of the series before passing to the next in sequence. The machinery, however, rarely works with absolute precision. Perturbations are sure to
arise whenever
a
contrary impulse
comes into the field, and either blocks
the path or struggles for supremacy. Fig. 2. Tent
in bushy pasture
beside nest
of Chestnut-sided
Warbler, shown in detail in Fig. 3. p
gg
p
y
The surge
of parental feeling is
often marked by an inbred pugnacity,
which begins to show itself in certain
species
at the very beginning of the
breeding season. This fighting mood,
which is an adaptation for the protec-
tion of the home and
all that
it con-
tains,
is by no means a measure
of
the other parental impulses. A Nc\v Method of Bird Study and Photography. It has
a gradual
rise,
reaches a maximum
when the young are ready to leave the
nest,
at
a time when
protection
is
most needed, and then gradually sub-
sides. When a pair are robbed during
the breeding season, or
in any way
disturbed in mind or property, three
courses are open to them, either to
desert
and
begin operations
anew,
to stay by the nest and save what
is
left,
or, having done
this,
to
fill up
the gap by laying more eggs. The
ture
of the
bird, or upon the relative When a pair are robbed during
the breeding season, or
in any way
disturbed in mind or property, three
courses are open to them, either to
desert
and
begin operations
anew,
to stay by the nest and save what
is
left,
or, having done
this,
to
fill up
the gap by laying more eggs. The
course
eventually
followed
depends upon the nature
of the
bird, or upon the relative
strength of fear, the parental instincts, and habit. Fig. 2. Tent
in bushy pasture
beside nest
of Chestnut-sided
Warbler, shown in detail in Fig. 3. Fig. 2. Tent
in bushy pasture
beside nest
of Chestnut-sided
Warbler, shown in detail in Fig. 3. left,
or, having done
this,
to
fill up
the gap by laying more eggs. The
course
eventually
followed
depends upon the nature
of the
bird, or upon the relative
strength of fear, the parental instincts, and habit. Fig. 2. Tent
in bushy pasture
beside nest
of Chestnut-sided
Warbler, shown in detail in Fig. 3. g p
y
y
g
gg
course
eventually
followed
depends upon the nature
of the
bird, or upon the relative
strength of fear, the parental instincts, and habit. g
p
The parental instinct,
1 reenforced by habit, gradually increases
until
the young
are
reared. It
is therefore
safest
to change the nesting surroundings when this instinct is
approaching its culmination. pp
g
The general feeling of fear is gradually or quickly suppressed according to the value
of the different factors in the equation, by the parental instinct, which impels a bird at all
hazards to go to its young wherever placed. This impulse though
it be weak
at
first,
is strengthened by
exercise, or what amounts
to the same
thing,
by
the growth
of
habits or associations. 1 This phrase
is sometimes used (or the sake of brevity and convenience in nearly the same sense
as parental
attachment or parental love. A New Method of Bird Study and Photography. At
its
first
report when two
feet away, many a
bird
will jump
as
if shot, give an angry
scream,
and even
fly at the tent as
if to exorcise an evil spirit, while after a few hours, or on
the
second
day, they will only wince
;
finally they will not budge
a
feather
at
this or any
other often repeated sound, whether from shutter, steam whistle, locomotive, or the human
voice. This illustrates the effect of the alarm clock over again. At our
first experience
with this nerve-wracking machine, we start from deep sleep and promptly heed
its sum-
mons
;
then we are apt to mind
it less and
less
until we sleep on serenely in spite of
it. If we were to place an alarm clock on or near the nesting bough, and let
it off at regular
but not too frequent intervals, the birds would soon learn to disregard
it as we do, and as
some of them disregard the babel of a city
stn.-'-t. p
Possibly
the
fears
of
the
old
birds are renewed at sight of the win-
dow which
is now opened in the tent-
front,
and
of
the
glass
eye
of
the
camera gleaming through
it, but the
lens
is also silent and motionless, and
soon becomes a familiar object to be
finally disregarded. Again
there
is
the fear which the SOUnd of the shut-
ter, a sharp metallic click,
at
first
in-
spires,
unless you
are the fortunate
ter, an instrument which
is unknown
report when two
feet away, many a
and even
fly at the tent as
if to exorci
second
day, they will only wince
;
fin
other often repeated sound, whether f
voice. This illustrates the effect of t
with this nerve-wracking machine, we
mons
;
then we are apt to mind
it les
If we were to place an alarm clock on
but not too frequent intervals, the bir
some of them disregard the babel of a Fig. 3. Tent beside nest of Chestnut-sided Warbler. The female
broods, while the male is foraging. possessor
of an
absolutely
silent and
rapid
shut-
to the
trade, at
least
in this country. A New Method of Bird Study and Photography. and they may keep away for a time or advance with caution. If very shy. like most Cat-
birds, they will sometimes skirmish about the tent for two hours or more before touching
the nest. The ice
is
usually broken
however
in
from twenty minutes to
an hour, and
I have known a Chip-
ping Sparrow and Red-eyed Vireo to
feed
their young
in
three minutes
after the tent was in
l and they may keep away for a time or advance with caution. If very shy. like most Cat-
birds, they will sometimes skirmish about the tent for two hours or more before touching
the nest. The ice
is
usually broken
however
in
from twenty minutes to
an hour, and
I have known a Chip-
ping Sparrow and Red-eyed Vireo to
feed
their young
in
three minutes
after the tent was in place. Fig. 3. Tent beside nest of Chestnut-sided Warbler. The female
broods, while the male is foraging. p
At every approach
to the
nest
in
its new position, the birds see the
same objects which work them no ill. The
tent stands
silent and
motion-
less,
unless
it happens to be windy,
but the young are close by, and fear
of the new
objects gradually
wears
away. Parental
instinct,
or
in
this
case maternal
love,
for the
instinct
to
cherish
the
young
is
usually
stronger in the mother, wins the day. The mother
bird comes to the nest
and feeds her clamoring brood. The
spell
is broken
;
she
comes
again. The male
also approaches, and their
visits are thereafter repeated. p
Possibly
the
fears
of
the
old
birds are renewed at sight of the win-
dow which
is now opened in the tent-
front,
and
of
the
glass
eye
of
the
camera gleaming through
it, but the
lens
is also silent and motionless, and
soon becomes a familiar object to be
finally disregarded. Again
there
is
Fig. 3. Tent beside nest of Chestnut-sided Warbler. The female
the fear which the SOUnd of the shut-
broods, while the male is foraging. ter, a sharp metallic click,
at
first
in-
spires,
unless you
are the fortunate
possessor
of an
absolutely
silent and
rapid
shut-
ter, an instrument which
is unknown
to the
trade, at
least
in this country. A Nc\v Method of Bird Study and Photography. After a bird once visits the nest in
its new position,
it returns again and
again, and
in proportion as its visits to the old nesting place diminish and finally cease, its approaches
to the new position become more frequent, until a new habit has been formed, or
if you
will, until the old habit is reinstated. When the birds approach the nest any strange objects like the stakes which support
the bough, or the tent which
is pitched beside
it arouse
their sense of
fear or suspicion, A New Method of Bird Study and Photography. Wild
Birds. It
is the young, the young, always THE YOUNG in whom the interest of the old birds
is centered, and about whom their lives revolve. They are the strong lure, the talisman,
the magnet to which the
parent
is
irresistibly
drawn. The
tree,
the
branch,
the
nest
itself,
what are these in compari-
son
with
the young
for
whom alone they exist ? It
is the young, the young, always THE YOUNG in whom the interest of the old birds
is centered, and about whom their lives revolve. They are the strong lure, the talisman,
the magnet to which the
parent
is
irresistibly
drawn. The
tree,
the
branch,
the
nest
itself,
what are these in compari-
son
with
the young
for
whom alone they exist ? is centered, and about whom their lives revolve. They are t
Fig. 4. Truncated elm with nest-holes
of \Voodpecker, the lowermost
re-
cently occupied by Bluebirds. To bring down the
nest, the trunk may be cut
from below or in line with arrow. With some species
it
is possible
to
make
the
necessary change without
evil
consequences
when
there are eggs in the nest
;
with others we must wait
until
the young are from
When to
f
ur to nine day s
Change
old. It
is
all
a
the Nest- question
of
the
ing Site "
strength
of
the
parental
instinct, and
this
varies between wide limits
in
different
species,
and
very considerably between
different individuals. From
the
nature
of
the
case
there can be no
infallible
rule. If we know
little of
the
habits
of the birds in
question
it is safest to wait
until
the
seventh
to
the
ninth day after the young
are
hatched,
or when
in
many
passerine
birds,
as
g-quills begin to appear in
half inch beyond the feather
ximum, and, what is equally Fig. 4. Truncated elm with nest-holes
of \Voodpecker, the lowermost
re-
cently occupied by Bluebirds. To bring down the
nest, the trunk may be cut
from below or in line with arrow. many
passerine
Robins, Orioles, and Waxwings, the feather-shafts of the
wing-quills begin to appear in
the young, or better when they project from one quarter to one half inch beyond the feather
tubes. At this period the parental instinct
is reaching its maximum, and, what is equally
important, the sense of fear has not appeared in the young. A New Method of Bird Study and Photography. At
its
first
bird
will jump
as
if shot, give an angry
scream,
se an evil spirit, while after a few hours, or on
the
ally they will not budge
a
feather
at
this or any
rom shutter, steam whistle, locomotive, or the human
he alarm clock over again. At our
first experience
start from deep sleep and promptly heed
its sum-
and
less
until we sleep on serenely in spite of
it. or near the nesting bough, and let
it off at regular
ds would soon learn to disregard
it as we do, and as
city
stn.-'-t. Wild
Birds. The tent
is then
to be placed
in position, or
it may be pitched and
left
overnight beside the
nest.'
In other words, operations
may
begin
at
once
or
be
postponed until
the follow-
ing day, the better plan for
a
beginner
until
he
has
mastered minor
difficulties,
which,
though
small
in
themselves, are far from un-
important. When the tent
is
closed
absolute
silence
must
be
maintained,
for
while
this
is
not
always
necessary,
it
is the best rule
to
follow
during
the
first
days of observation. Th g
y
Either a dark foliage or
the desire
of the operator o
natural background
it would
bough
or
to
use
reflected
light for softening the shad-
ows,
but
no
experiments
have yet been made in this
direction. The tent
is then
to be placed
in position, or
it may be pitched and
left
overnight beside the
nest.'
In other words, operations
may
begin
at
once
or
be
postponed until
the follow-
ing day, the better plan for
a
beginner
until
he
has
mastered minor
difficulties,
which,
though
small
in
themselves, are far from un-
important. When the tent
is
closed
absolute
silence
must
be
maintained,
for
while
this
is
not
always
necessary,
it
is the best rule
to
follow
during
the
first
days of observation. Fig. 5. Nest-hole of Flicker, used by Bluebird. Trunk removed from tree,
and mounted on pivot
so that
it can be turned to any angle with sun. See
No. 15 of table, p. 12, Fig. 10, and Chapter VII. y
The best time to begin
is from eight to nine o'clock
in the morning, because the
young
will then have been
fed,
and
the
sun
will be
getting
high
enough
for
the most rapid photograph-
ic
work. One may
spend
as many hours a day, and as many days at one nest, as time permits or inclination decides. 1 Directions fur use of the tent are yivcn in Chapter III. Fig. 5. Nest-hole of Flicker, used by Bluebird. Trunk removed from tree,
and mounted on pivot
so that
it can be turned to any angle with sun. See
No. 15 of table, p. 12, Fig. 10, and Chapter VII. Fig. 5. Nest-hole of Flicker, used by Bluebird. Trunk removed from tree,
and mounted on pivot
so that
it can be turned to any angle with sun. See
No. 15 of table, p. 12, Fig. Wild
Birds. p
,
pp
y
g
When we try to formulate a rule, however, we at once encounter numerous excep-
tions. Thus in Cuckoos the
feathers do not shed
their envelopes gradually as in most
birds, but remain sheathed up to the last day in the nest. Of greater importance
is the
understanding of the principles involved, and with these in mind and judiciously applied
very few mistakes should be made. y
At the beginning of observations a
nest with eggs should be watched, but not dis-
turbed. When the period of incubation has been determined, and the time of hatching A New Method of Bird Study and Photography. kno\vn, the young may be examined and photographed
if
it
is desired. At
all
events
they
should be
watched
until the
critical time
arises
for
closer study. Mode of
This decided upon
in the manner already suggested, circumstances must
Procedure,
determine the course to be followed. kno\vn, the young may be examined and photographed
if
it
is desired. At
all
events
they
should be
watched
until the
critical time
arises
for
closer study. Mode of
This decided upon
in the manner already suggested, circumstances must
Procedure,
determine the course to be followed. If the nest, like that of a Robin or Kingbird,
is saddled to the branch of a tree, saw
off the whole limb and nail
it to stakes driven into the ground, so placed as always to give
the best light. The nesting bough, in case there is one, should be set with
its long axis
parallel with the course of the sun, but the position of the bough or tent may be changed
during the day when exceptional conditions render
it necessary. during
day
exceptional
y
Either a dark foliage or a sky background may be chosen
for the
nest, according
the desire
of the operator or the
possibilities of the
situation. If not
satisfied with
natural background
it would be possible to place dark or light screens behind the nesti
bough
or
to
use
reflected
light for softening the shad-
ows,
but
no
experiments
have yet been made in this
direction. Precautions
to be
Observed. Wild
Birds. 10, and Chapter VII. Wild
Birds. 8 I will only suggest that the second day
is always better than the
first, and that the third
or fourth
is always sure to bring something new. If one would learn the nesting habits
of any species thoroughly, it will hardly do to rely upon one nest. The more you
see
of
different nests and
different
birds the better. I usually spend five or six hours in the tent, from nine
in the morning until three in
the afternoon, when the weather is fine. If the camping ground
is near my house, as
it
usually is, I leave the tent for
half an hour at noon, but
if
it
is
far,
I carry a lunch and
spend the day. When possi-
ble, I am always on hand dur-
ing the last day of life at the
nest, to
see the young leave
it, usually one at a time, and
to witness
the
manoeuvres
of
the parents in conducting
them to the nearest trees. I usually spend five or six hours in the tent, from nine
in the morning until three in
the afternoon, when the weather is fine. If the camping ground
is near my house, as
it Fig. 6. Nesting bough of Kingbird removed from apple tree in background
at a point where extended arrows meet, and fixed to upright stakes. Tent-
cloth thrown over frame which is set in position. Precautions
to be
Observed. Young birds from one to
five
days
old
cannot,
as a
rule, stand exces-
sive heat. Even
when
fed
and
brooded they will
sometimes succumb, and here
lies the serious danger to be
guarded against. A
nest
of
very young birds well shaded
by
foliage
cannot be safely
carried
into the
direct
sun-
shine of a hot summer's day,
hence the importance
of be-
ginning
operations
at
the
proper time when the weath-
er is suitable, and
further of
not allowingyour enthusiasm
to
get
the
better
of
your
judgment. Precautions
to be
Observed. Fig. 6. Nesting bough of Kingbird removed from apple tree in background
at a point where extended arrows meet, and fixed to upright stakes. Tent-
cloth thrown over frame which is set in position. wenty-six
nests
Extent of Appli-
cation of the
Method, Wild
Birds. j
g
The morning of a clear, mild day is preferable, but since we cannot order the weather,
it
is better to leave the birds to themselves,
if
it promises to be excessively hot or windy. The morning of a clear, mild day is preferable, but since we cannot order the weather,
it
is better to leave the birds to themselves,
if
it promises to be excessively hot or windy. The young may be fed or handled as much
as one
wishes, provided they have not
acquired the instinct of fear. If you are uncertain as to this and your aim
is to study the
nesting habits,
it
is better to avoid approaching, touching, or
in any way disturbing the
young after the
flight
feathers have appeared. The cutting of leaves
or twigs which
obstruct the light or cast undesirable shadows should be done before this time. g
On the other hand,
investigations
of the young which require accurate weighing, Fig. 7. Female Kingbird astride nest, protecting young from heat. This and the following
from photographs made at nest shown on facing page. Fig. 7. Female Kingbird astride nest, protecting young from heat. This and the following
from photographs made at nest shown on facing page. Fig. 8. Kingbird family. The male with grasshopper in
bill,
his mate, partly hidden,
behind him. 9 Fig. 8. Kingbird family. The male with grasshopper in
bill,
his mate, partly hidden,
behind him. 9 Fig. 8. Kingbird family. The male with grasshopper in
bill,
his mate, partly hidden,
behind him. 9 A New Method of Bird Study and Photography. 1
1 measurements, or photographs
of the
birds themselves, place the matter
in a different
light. With these objects in view the nest must be frequently approached and the young
taken out, and
for such studies the change of
the nesting site offers such obvious advan-
tages that
it
is needless
to dwell upon
them. In
taking down
the
nesting bough
it
is
often necessary to touch the nest, and this docs no harm. measurements, or photographs
of the
birds themselves, place the matter
in a different
light. With these objects in view the nest must be frequently approached and the young
taken out, and
for such studies the change of
the nesting site offers such obvious advan-
tages that
it
is needless
to dwell upon
them. Wild
Birds. In
taking down
the
nesting bough
it
is
often necessary to touch the nest, and this docs no harm. y
Young birds eight or nine days old stand the heat well, provided they are fed, but on
very hot days they should
not be allowed to go with-
out
food
for
more
than
two hours
at the
longest. Should
the
parents bring
no food during this time, it
is better to feed the young
in the nest, and to suspend
operations
until
the next
day. Fig. 9. Cedar-bird at nest shown in Figs,
i, 12, and 13, prepared to feed young
by regurgitation
: a characteristic attitude. The
parallel outlines of the neck
show that the gullet Is full. As has
been
already-
said, the old birds may be
expected
to come
to
the
nest
in from twenty
min-
utes to an
hour, when the
tent
is brought into imme-
diate use after removal
of
the
nesting bough. It
is
naturally
impossible
to
predict
exactly
what
will
happen
in any given
case
until
the
experiment
is
tried,
since
the
personal
equation
or
individuality
of the
birds themselves
is
an unknown and
variable
factor. One thing only
is
certain,
that the parental
instincts,
reen forced
In-
habit,
will win
in the end,
that they will cast out fear,
and draw the birds to their
young Fig. 9. Cedar-bird at nest shown in Figs,
i, 12, and 13, prepared to feed young
by regurgitation
: a characteristic attitude. The
parallel outlines of the neck
show that the gullet Is full. young. I have used the
tent and
altered
the
nesting
site
in the
case
of twenty-six
nests
belonging
to
fifteen
different
species
of
birds. The experiments were
Extent of Appli-
made
in
the course
of two seasons, and
the entire
list
is tabulated
as
cation of the
follows, the age of the young
in most
cases being only approximately
Method,
accurate
: young. I have used the
tent and
altered
the
nesting
site
in the
case
of twenty-six
nests
belonging
to
fifteen
different
species
of
birds. The experiments were
Extent of Appli-
made
in
the course
of two seasons, and
the entire
list
is tabulated
as
cation of the
follows, the age of the young
in most
cases being only approximately
Method,
accurate
: Wild
Birds. 12 EXPERIMENTS
IN THE USE OF THE OBSERVATION TENT AND IN CHANGE OF NESTING
SITE. A New Method of Bird Study and Photography. A New Method of Bird Study and Photography. In one or two instances
I had serious trouble from cutting away too much foliage about
a nest in very hot weather, but such accidents are really needless,
if one follows the
rule
of leaving the birds to their own devices on days of excessive heat and humidity. In
all
the other cases, everything went well, and the young left the nest in due course. In one or two instances
I had serious trouble from cutting away too much foliage about
a nest in very hot weather, but such accidents are really needless,
if one follows the
rule
of leaving the birds to their own devices on days of excessive heat and humidity. In
all
the other cases, everything went well, and the young left the nest in due course. y
g
Kingbirds have remained
in the
nest eleven days
after the
change,
Robins a week, Cedar-birds six days. A glance at the table
will show that
in one instance, that of the Chestnut-
sided Warbler (16), observations were
begun
while there were
eggs, and
I
have no doubt that in many species
the whole period
of
life
in
the
nest
from hatching to the time of
flight
may be watched from the
tent, but
the subject is yet open to experiment. It
is all a question of the strength of
the parental
instincts
at the period
in question. Where this attachment
to nest and eggs is strong as in Owls,
Fish
Hawks,
Flickers,
Kingbirds,
and the Chipping Sparrows, to men-
tion
a
few
cases, we may look
for
success. Fig. 10. Female Bluebird with cricket in
bill, ready to enter
nest-hole. See Fig. 5. I am confident that the movable
tent has a great future as an obser-
vatory for the study of bird-habit, and
that
it will be possible to watch the
building of the nest in such species as
have a strong attachment to chosen
sites, and whose plans are not easily disturbed by trifles. Here
is certainly a fallow field
which has been scratched only here and there by the plow, and where attempts to
culti-
vate
it
fail, no harm
is done. In making experiments
in
this direction care should be
taken not to approach too near with the tent, at least on the first day. Wild
Birds. EXPERIMENTS
IN THE USE OF THE OBSERVATION TENT AND IN CHANGE OF NESTING
SITE. A New Method of Bird Study and Photography. Again
it
is pro-
bable that many kinds of birds may be attracted by food and other lures, but the possible
rewards of sedentary experiments in this direction are too uncertain
to arouse much en-
thusiasm in the mind of the active bird student. Fig. 10. Female Bluebird with cricket in
bill, ready to enter
nest-hole. See Fig. 5. Fig. 10. Female Bluebird with cricket in
bill, ready to enter
nest-hole. See Fig. 5. I have no
desire
to
anticipate every objection
which might be
raised against the
method, were
it possible to do so, but
after testing
it to the best of my ability with the
opportunities of two summers,
I am confident of
its value and am ready
to stand sponsor for
it in judicious hands. It
is hardly necessary to insist
the Method
that
it
is not designed
for exhibition
purposes, and
that
its successful
practice requires some knowledge, with more patience and time. ice requires
g ,
p
To the trained naturalist patience has long ceased to be a virtue. He
is acc practice
equ
es
g ,
p
To the trained naturalist patience has long ceased to be a virtue. He
is accustomed Wild
Birds. to work
in the
field or laboratory for weeks or months to attain a well-defined end, and
that end he will attain, provided
it can be compassed by intelligence, industry, and
skill. Patience
is the
naturalist's stock
in
trade, and
while no
success may come because
of
it alone, none can be assured without
it. In the ten days or two
weeks or more
of
life
at
the nest events move rap-
idly
and
the
question
of
time
is important. Any
interruptions are therefore
opportunities
for the
dis-
play
of
patience
rather
than
for
the
increase
of
knowledge. Fig. n. Female Chestnut-sided Warbler shielding the young on a warm day. Photographed from tent shown in Figs. 2 and 3. Fig. n. Female Chestnut-sided Warbler shielding the young on a warm day. Photographed from tent shown in Figs. 2 and 3. t
g
is of no practical consequence, but this should not be carried too far, both on account of
the young which need the protection
of shade, and for the sake
of natural appearances
which we wish to preserve. ' When the nesting branch is vertical and not too large, it can be easily kept fresh by placing
it in a jug or can
<>f \vater which should be set in the ground. A New Method of Bird Study and Photography. A New Method of Bird Study and Photography. 15 young need no protection from this source. As to this point, however, the
illustrations
in this book will speak for themselves. young need no protection from this source. As to this point, however, the
illustrations
in this book will speak for themselves. p
Evergreens
like the
pine and
spruce hold
their
leaves
bright for a long time after
cutting, and
in this respect the various deciduous trees and shrubs
differ
greatly, those
with a hard, close grain keeping fresh the longer. As to any injury to trees which the method may be supposed to entail,
it
is not worth
considering, since no
valuable
tree should be mutilated
without
first obtaining the per-
mission of the owner, for however
trifling the damage may appear, his point of view
is
likely to be different from your own. The cutting of an occasional twig or branch, even
if
it does not trim
the
tree,
is not
regarded
as an
important event
in
this country at
present. If every farmer who owns orchards and woodlands did his duty, he would cut out
more useless wood
in a year than a student of birds would need to do in a decade. It
is
possibly unnecessary to add that no one should
set up
a
nest
in
a
field, and
leave the
trouble of removing
it to the owner of the land. g
A more serious objection
is likely to occur to the ornithologist, namely the
liability
of exposing the
birds
to new
enemies. I feared lest prowling cats should discover the
young ones whose nest and branch had been brought down from the tree top, and set up
again
in
plain
sight
within easy
reach from
the ground, but
I was happily mistaken. Predacious animals of all kinds seem to avoid such nests as
if they were new devices to
entrap or slay them. p
y
As to the weather, barring heat which must be guarded against
in the way described,
the nesting bough
is more secure when
fixed
firmly
to supports than
it could
possibly
have been
before. The only depredator of whom
I
stand
in fear is the irresponsible or
malicious small boy, and to anticipate his possibilities for evil,
I take a look at the nest now
and then when not encamped beside
it. A New Method of Bird Study and Photography. p
It might be supposed
that when a branch
is lopped
off,
its
foliage would
at once
wither, and unduly expose the nest or detract from
the
artistic value of a picture. The
fact
is, however, that there
is commonly enough sap in a hard wood bough
of moderate
size to keep the leaves fresh for several days,
1 and towards the close of life at the nest the ' When the nesting branch is vertical and not too large, it can be easily kept fresh by placing
it in a jug or can
<>f \vater which should be set in the ground. A New Method of Bird Study and Photography. p
When the nest
is completely exposed and the weather
is very hot, the young may be
tempted to forsake
it a day or two earlier than they would naturally do, but this does not
usually happen and
is not necessarily serious. Some Kingbirds, already referred to, spent
eighteen days in the nest, and were a week old when
it was moved. This was probably
longer than common, and certainly longer than necessary. g
y
g
The tent not only conceals the observer but protects his camera, an
important con-
sideration, since the prolonged action of the sun
is liable to spring a leak
in the bellows. As to the portability and general convenience of the tent
I shall speak elsewhere. portability
general
p
With notebook in hand you can sit in your tent, and see and record everything which
transpires at the nest, the mode of approach, the kind of food brought, the varied
activi-
ties of the old and young, the visits of intruders, and their combats with
.. ... . Advantages of
the owners
of the
t
the
of
which sometimes
on With notebook in hand you can sit in your tent, and see and record everything which
transpires at the nest, the mode of approach, the kind of food brought, the varied
activi-
ties of the old and young, the visits of intruders, and their combats with
.. ... . Advantages of
the owners
of the
nest, the capture
of prey which sometimes goes on
the Method
under your
eye. No
better position could be chosen
for hearing the
songs, responsive calls, and alarm notes of the birds. You
can
thus gather materials for
an exact and minute history of life at the nest, and of the behavior of
birds during
this
important period. More than this, you can photograph the birds at will, under the most
perfect conditions, recording what no naturalist has ever seen, and what no
artist could
ever hope to
portray. The
birds come and
go
close to your
eye, but
unconscious of
being observed. Advantages of
the Method g
I have watched
the Night Hawk
feed
her chick with
fireflies
barely fifteen
inches Wild
Birds. 1 6 from my hand, the Kingfisher carrying live fish to its brood whose muffled
rattles issued
from their subterranean gallery a few feet away. ILLUSTRATIONS OF THE METHOD. IT IT
is always interesting to see how birds actually behave when put to the
test, and
as
illustrations of the method applied
I have selected
four common
birds, the Cedar
\Vaxwing, the Baltimore
Oriole, the Redwing
Blackbird, and
the
Kingbird. The
choice might
have
fallen, however, upon any others
in my
list, for the principles are in
every case the same. y
Since the breeding habits of these birds will be described more
fully at a later time,
the change of their nesting site and their behavior in the face of new surroundings need
only concern us for the present. only
present. On the third day of July a Cedar-bird's nest (No. 10 of table on page 12) was discovered
in an unusually attractivesituation. It was fastened to the horizontal branch of a white pine
about
fifteen
feet
up,
in
the
line of an old stone wall that bounded an
open
field. In passing beneath the
tree almost daily during the follow-
ing week,
I was sure to find one of the old birds, the female as
I supposed, always on the
nc>t and
sitting
in
the same
alert
attitude, engaged
either
in
incubation
or brooding. \Yith upstretched neck she would
sit motionless and
silent
as a statue, as
if
listening
intently, her dark eye shining
like a
jet black bead
against
the
background
of
pine
needles. I was waiting for the propitious time to move this nest to the open field. This
time arrived on July I4th, when the heads of
the young began
to appear over the rim
of their nest. The bough was then sawn
off, carried
fifty feet from the
tree, and
set up
in the newly mown
field, in an east to west line at a height of four feet from the ground,
and in such a way that the birds could be " skyed," and the light would be good from nine
o'clock in the morning
until
three in
the afternoon. The
tent was then
pitched and
closed
;
the whole operation lasted longer than usual owing to some
difficulty in getting
stakes of the right height. Fifteen minutes
is usually long enough for this work. g
From peep holes the old birds could be seen in the nesting tree, and you began to
hear their faint z-e-e-e-e-e-t', in response to calls from the young. A New Method of Bird Study and Photography. When near enough to count her respi-
rations
accurately,
I have seen the Redwing Blackbird leave her nest on a hot day, hop
down to the cool water of the swamp, and
after taking a sip, bathe
in
full view, within
reach of the hand
;
then, shaking the water from her plumage, she would return refreshed
to the nest. I have seen
the male Kingbird come to
his nesting bough with feathers
drenched from his midday bath in the river, the Orioles flash their brilliant colors all day
long before the eye, and Chestnut-sided Warblers become so tame after several days that
the female would allow you to approach and stroke her back with the hand. y
pp
It
is
difficult
to describe the
fascination which
this method
of study
affords
the
student of animal
life. New discoveries, or unexpected sights wait on the minutes, for
while there
is
a well-ordered
routine
in the actions
of many
birds the most charming
pictures occur at odd moments, and there
is an endless variety of detail. It
is like a suc-
cession of scenes in a drama, only this
is real
life, not an
imitation, and there
is no need
of introducing tragedy. g
g
y
He who runs may sometimes read and observe a few things, and so may he who per-
forms gymnastic feats
in the branches of
tall trees or does penance
in a hundred other
ways, but from the tent one may read the life of the nesting bird as out of an open book. The
^Baltimore
Oriole. ILLUSTRATIONS OF THE METHOD. They
flew on
the morning
of the
nineteenth
of
July, when thirteen days old, seeking the cover of a
thicket
of birches
close
by, where
they were cared for by their parents
until ready to leave the neighborhood. They were
scattered
over
an
area
of
several
square
rods, and
kept
calling
in
their monotonous
way, s-c-e-c-e-t ! z-e-e-c-c-t ! One
of
their number, shown in a photograph (Fig. 47), was
not touched or posed, but occupied
a natural perch chosen by himself in his flight from
tree to tree. Fig. 13. Cedar-bird's nest
No. 10 of table
in original position marked by
arrows. See Figs. 9 and 13. Fig. 13. Cedar-bird's nest
No. 10 of table
in original position marked by
arrows. See Figs. 9 and 13. ruption. The young now sat or stood with heads upturned in the
characteristic attitude
shown in
one
of
the
illustrations. They
flew on
the morning
of the
nineteenth
of
July, when thirteen days old, seeking the cover of a
thicket
of birches
close
by, where
they were cared for by their parents
until ready to leave the neighborhood. They were
scattered
over
an
area
of
several
square
rods, and
kept
calling
in
their monotonous
way, s-c-e-c-e-t ! z-e-e-c-c-t ! One
of
their number, shown in a photograph (Fig. 47), was
not touched or posed, but occupied
a natural perch chosen by himself in his flight from
tree to tree. ruption. The young now sat or stood with heads upturned in the
characteristic attitud
shown in
one
of
the
illustrations. They
flew on
the morning
of the
nineteenth
o
July, when thirteen days old, seeking the cover of a
thicket
of birches
close
by, wher
they were cared for by their parents
until ready to leave the neighborhood. They wer
scattered
over
an
area
of
several
square
rods, and
kept
calling
in
their monotonou
way, s-c-e-c-e-t ! z-e-e-c-c-t ! One
of
their number, shown in a photograph (Fig. 47), wa
not touched or posed, but occupied
a natural perch chosen by himself in his flight from
tree to tree. This Oriole's 'nest (No. 19 of table) was fortunately placed
in an apple tree scarcely
twenty feet up, so that no gymnastic feat was needed to bring the branch to the ground. ILLUSTRATIONS OF THE METHOD. In twenty-four minutes
the female was on the bough and fed her brood with red
bird
cherries by regurgitation. At
this point
I was obliged to leave the tent and
request some curious boys to keep
a\\,iy, but the mother bird was back in a moment. In a short time the old
birds began
to alight on the peak of the tent, which was an observatory for them
as well as
for the
person inside. Taking a look about, they would drop down to the nest only a step away. This was done more than
ten
times
in the course
of the day. Observations began at
8.40 in the morning and closed at 4.40, so that with an
intermission at noon, they lasted
nearly seven hours and twenty minutes. During this interval the young were
fed with 17 17 Wild
Birds. i8 wild red cherries, blueberries and insects, mainly grasshoppers, and nearly always by regur-
gitation. The nest and young were regularly cleaned, and the new conditions seemed to
have been completely adopt-
ed. The young, whose wing-
quills now
showed
half
an
inch
of
the
feather -shaft,
were entirely fearless. Fig. 13. Cedar-bird's nest
No. 10 of table
in original position marked by
arrows. See Figs. 9 and 13. On July i6th, the second
day
of
observation
and the
third after the removal of the
nesting bough, the
old
birds
beean the work of feeding
in
o
o
exactly twelve minutes after
the tent was in place. I will
add here that
I have always
removed the
tent at the end
of the day's work, although
in some species
it would be
of undoubted
advantage
to
leave
it overnight. In a little
more than
three
hours
the
old
birds came
to
the
nest
eighteen
time s,
bringing
abundant stores
of
fruit and
insects. On July
ijth,
the
third
day
at
this
nest, feeding be-
gan
in
three
minutes
after
closure
of the
tent. It was
the hottest day
of the sum-
mer, but life at the nest went
on without accident or
inter-
ruption. The young now sat or stood with heads upturned in the
characteristic attitude
shown in
one
of
the
illustrations. ILLUSTRATIONS OF THE METHOD. The noisy young calling
with
incessant
reiteration,
ivick-ick-ick-ick-ick
.'
The
^Baltimore
ad vert i se d their nest to every passer by, and
it was surprising that
it had
remained
unmolested. Oriole. Illustrations of the Method. Illustrations of the Method. This beautiful nest with
the
entire bough
to which
it was strung was moved eight
yards from the tree, set up in the way described, and the tent was closed at a quarter past
eight o'clock. After repeated visits to the apple tree both birds disappeared, but did not
go out of hearing of their young, who
in a half-hour's time began giving their id, -X-- /</(-
/</[-/<
/(
/
with an emphasis sure to evoke response. p
p
The old
birds began to approach, sounding now and then their peculiar
rattle, and
the female could be seen exploring the foliage of a neighboring tree. At fifteen minutes
past
nine one
of them was skirmishing about
the
tent, and
in
five minutes alighted
above the nest with a green larva
in
bill. This larva however had another destiny that
was apparent at the moment, for a puff of wind frightened the bird away. At her next
visit
a strawberry was brought and safely delivered, in exactly one hour and seventeen
minutes from the beginning of operations. g
g
p
Observations were continued until 4.25
P.M. or, allowing for the noon
intermission,
during seven and a quarter hours. In this period the parents were at the nest fifty times
bringing insects and fruit. Sometimes the feedings would follow at two or three minute
intervals; then longer lapses would occur. On the second da
was up, and during t
1 ast ed
, the young
were
fed
one hund-
red and sixteen times
by both birds. I
left
the tent and entered
it again several times
during the day, and
once moved
both
bough
and
tent
to
improve
the
light. By this time the Ori-
oles showed no
fear,
but came to the nest-
ing
branch
in from
one
to two
minutes
after
I
had
entered
the tent. During an
interval
of ten
min-
utes, the young were
fed eleven times. Fig. 13. Cedar-bird's nest in its
Compare Figs,
i, 9, and 12. Nesting bough moved fifty feet to open field. The
tent
was
closed at 8.30 on the
morning of the third
day, June
27th,
and
the first feeding came
off in five minutes. In two hours and twenty-five minutes the old birds made forty-four
visits to the nest bringing strawberries and insects, and towards eleven o'clock one of the
Fig. 13. Illustrations of the Method. Cedar-bird's nest in its
Compare Figs,
i, 9, and 12. Nesting bough moved fifty feet to open field. Fig. 13. Cedar-bird's nest in its
Compare Figs,
i, 9, and 12. Nesting bough moved fifty feet to open field. day, June
27th,
and
the first feeding came
off in five minutes. In two hours and twenty-five minutes the old birds made forty-four
visits to the nest bringing strawberries and insects, and towards eleven o'clock one of the
Compare Figs,
i, 9, and 12. the first feeding came
off in five minutes. In two hours and twenty-five minutes the old birds made forty-four
visits to the nest bringing strawberries and insects, and towards eleven o'clock one of the Wild
Birds. 2O young who for a long time had been exercising his wing- and leg-muscles by climbing to
the rim of the pouch, took his first flight, making a neighboring
tree. Not long
after, a
second bird climbed out
of the sack
&^
and was off, lured away by its parents. The
third and
last bird
left a
little
later, and towards evening the young
were
calling
from
trees down
the
hillside. &^
Fig. 14. them. Baltimore
Oriole
inspecting young after having fed On the fifth day of July a nest of
three young Blackbirds (No. 2 of the
table), aged five days,
The Redwing
/
Blackbird. was found on the edge
of what was once an
alder swamp, close
to the town and
the " Cove
" made by
the
Winnipi-
seogee
River
in Northfield. It was
fixed
to
several
slender
stems
of
Spiraea,
amid
a
dense
tangle
of
Cephalthus,
wild
roses,
and
purple
milkweeds. The
situation
was
so
attractive and
offered such
fine
op-
portunities
for studying these birds
that, notwithstanding the water and
mud,
I determined
to make careful
preparations. A
space
four
feet
square was at once cleared of bushes
at one side
of
the
nest. In
order
to sky the
birds,
the nesting
twigs
were
slightly
raised,
but
none
of
these were severed
or otherwise dis-
placed. The Redwing
Blackbird. Fig. 14. them. Baltimore
Oriole
inspecting young after having fed or platform on the cleared
area, and
painted it green, possibly an unnecessary precaution. When weighted with the observer and
his apparatus, the flooring was barely clear of the water. On the following day, the tent was
pitched
over
this
stranded
raft and guyed
to the bushes, the tent poles having been
previously lengthened to suit the depth of mud and water. Everything was ready for
observations at half-past nine o'clock. At first the birds fluttered around the nest clinch-
ing and
whistling
incessantly, but
in
less than an hour the warble
of the male was
heard, which
is a sure sign of growing confidence. Then both birds went
off
for food,
returned, reconnoitred the tent and nest, and after precisely one hour and twenty-three
minutes from the beginning of observations the female came and fed her clamoring young. Again she was off and back three times in rapid succession. Three minutes later she was
brooding, and remained on the nest thirteen minutes. Leaving
it again, she examined
the tent anew, then brooded ten minutes more. A little later the young were fed and the
nest cleaned with great care. platform
painted it green, possibly an unnecessary precaution. When weighted with the observer and
his apparatus, the flooring was barely clear of the water. On the following day, the tent was
pitched
over
this
stranded
raft and guyed
to the bushes, the tent poles having been
previously lengthened to suit the depth of mud and water. Everything was ready for
observations at half-past nine o'clock. At first the birds fluttered around the nest clinch-
ing and
whistling
incessantly, but
in
less than an hour the warble
of the male was
heard, which
is a sure sign of growing confidence. Then both birds went
off
for food,
returned, reconnoitred the tent and nest, and after precisely one hour and twenty-three
minutes from the beginning of observations the female came and fed her clamoring young. Again she was off and back three times in rapid succession. Three minutes later she was
brooding, and remained on the nest thirteen minutes. Leaving
it again, she examined
the tent anew, then brooded ten minutes more. A little later the young were fed and the
nest cleaned with great care. p at o
painted it green, possibly an unnecessary precaution. Illustrations of the Method. 21 The male was more cautious and did not actually feed his young until twenty-seven
minutes after eleven. His fears were then dispelled and
life at the nest went on without
interruption. At about noon the old
birds were
using the
tent
as
a half-w?y
house,
alighting on
its peak and guys, and foraging about it for food. In the space of four hours
on the
first day, during which the birds were watched at a distance of about twenty-seven
inches,
fifty-four
visits were made and
the young were
fed
forty
times. The female
brooded
her young over an
hour,
fed them twenty-nine
times, and
cleaned
the
nest
thirteen times. The male made eleven
visits, attending to sanitary matters but twice. This example illustrates as well as any which could be given the advantage which attends
the use of the observation tent. On the following day, July nth, the female was at the nest and brooding her young
in
five minutes
after the
tent was
in position. Presently she
left
to hunt
for
insects,
alighted on the tent, and five minutes later was feeding her young and cleaning the nest. In the course of nearly three and one half hours, fifty-five visits were made and the young
were fed collectively or singly forty-three times. At about half-past eleven
o'clock one
of the fledglings left the nest and was fed by the old
birds in the surrounding bushes of
the swamp. The female brought food thirty-two times, cleaned the nest eight times, and
brooded eighteen times
for intervals varying from
thirty seconds to eighteen
minutes. On the following day, July nth, the
in
five minutes
after the
tent was
in p
alighted on the tent, and five minutes la
In the course of nearly three and one hal
were fed collectively or singly forty-thre
of the fledglings left the nest and was fe
the swamp. The female brought food t
brooded eighteen times
for intervals va
This bird cut a queer figure while stand-
ing or sitting
in the
sun,
with
wings
spread and
bristling
like a turkey-cock
with
every
feather
erect,
and
with
mouth agape, trying to keep cool while
shielding her family from the heat. Her
breathings were
at the
rate
of
1 50 to
160 times a
minute. The male
bird
served food eleven times and attended
to sanitary matters once. Illustrations of the Method. In the course
of forty-two minutes
the
first
young
bird
to leave
the
nest
was
fed
eight
times, seven times by the mother and
once by the
father. Three days later
the swamp was visited at just after sun-
down, when the young birds suddenly
arose from
the
nest and
flew
off with
ease and precision. On the following day, July nth, the female was at the nest and brooding her young
in
five minutes
after the
tent was
in position. Presently she
left
to hunt
for
insects,
alighted on the tent, and five minutes later was feeding her young and cleaning the nest. In the course of nearly three and one half hours, fifty-five visits were made and the young
were fed collectively or singly forty-three times. At about half-past eleven
o'clock one
of the fledglings left the nest and was fed by the old
birds in the surrounding bushes of
the swamp. The female brought food thirty-two times, cleaned the nest eight times, and
brooded eighteen times
for intervals varying from
thirty seconds to eighteen
minutes. This bird cut a queer figure while stand-
ing or sitting
in the
sun,
with
wings
spread and
bristling
like a turkey-cock
with
every
feather
erect,
and
with
mouth agape, trying to keep cool while
shielding her family from the heat. Her
breathings were
at the
rate
of
1 50 to
160 times a
minute. The male
bird
served food eleven times and attended
to sanitary matters once. In the course
of forty-two minutes
the
first
young
bird
to leave
the
nest
was
fed
eight
times, seven times by the mother and
once by the
father. Three days later
the swamp was visited at just after sun-
down, when the young birds suddenly
arose from
the
nest and
flew
off with
ease and precision. Kingbirds pose so
well,
especially
about their nests, that
I was anxious to
see how th
would stand Fig. 15. Baltimore Oriole inspecting nest when behavior has
become more free. p
Kingbirds pose so
well,
especially
about their nests, that
I was anxious to
see how they would stand
the test of a sudden change
in
their surroundings. Accordingly,
I
watched
with
unusual care two nests
which were found near my house. On
the thirteenth day of June one had two and the other four eggs all freshly laid, and these
appeared to be the full complement. The Redwing
Blackbird. When weighted with the observer and
his apparatus, the flooring was barely clear of the water. On the following day, the tent was
pitched
over
this
stranded
raft and guyed
to the bushes, the tent poles having been
previously lengthened to suit the depth of mud and water. Everything was ready for
observations at half-past nine o'clock. At first the birds fluttered around the nest clinch-
ing and
whistling
incessantly, but
in
less than an hour the warble
of the male was
heard, which
is a sure sign of growing confidence. Then both birds went
off
for food,
returned, reconnoitred the tent and nest, and after precisely one hour and twenty-three
minutes from the beginning of observations the female came and fed her clamoring young. Again she was off and back three times in rapid succession. Three minutes later she was
brooding, and remained on the nest thirteen minutes. Leaving
it again, she examined
the tent anew, then brooded ten minutes more. A little later the young were fed and the
nest cleaned with great care. Illustrations of the Method. Illustrations of the Method. Young were hatched
in each
nest on
or near the
twenty-fifth of the month. The Kingbird. Fig. 15. Baltimore Oriole inspecting nest when behavior has
become more free. Fig. 15. Baltimore Oriole inspecting nest when behavior has
become more free. Wild
Birds. 22 The first nest was built at the top of a hill, about a rod from the Oriole's nest already
described, on the horizontal limb of a small apple tree twelve feet up, and was a conspicu-
ous object to all who passed that way. The nesting bough was removed and mounted in
a good position on the morning
of July 2d, and
the tent was
closed
at
half-past
eight
o'clock. At this time the two young were six days old and covered with light gray down. While the operation was in progress the old birds hovered over the nest, and with their
usual boldness, swooped down
close to my head, snapping their
bills and uttering their piercing
alarms. Fig. 16. Tent over raft in water of swamp beside Redwing Blackbird's
nest. See Figs, 21 and
22. After the
tent was closed,
much to my surprise all became
quiet, and
I could see both birds
the female with
insect in bill
exploring
the
nesting
tree
twenty
feet away. She would
fly to that point in space which
the nest formerly occupied, and
hover over
it repeatedly, a char-
acteristic action
of many birds
under such circumstances. Ten
minutes
later the
female
was
again
at the nesting
tree with
insects. For an hour afterwards
all was quiet. The
old
birds
were sitting by in
silence, prob-
ably not far away. At ten min-
utes before eleven
o'clock one
of the pair, probably the female,
came with a swoop to the nest-
ing branch, and I believe fed her
young. In thiscase the observer
had to wait two hours and twen-
ty
minutes
before having the
birds close to his eye, but he was
minute the mother had returned,
hours and six minutes (from 10.50
minutes when
the observer was
st. Not only had
they become
nything about
it, and one could
ooding or standing bird. After
I
e minutes
or even less time, and
asionally both birds were at the
ale was brooding. After the
tent was closed,
much to my surprise all became
quiet, and
I could see both birds
the female with
insect in bill
exploring
the
nesting
tree
twenty
feet away. She would
fly to that point in space which
the nest formerly occupied, and
hover over
it repeatedly, a char-
acteristic action
of many birds
under such circumstances. Wild
Birds. Ten
minutes
later the
female
was
again
at the nesting
tree with
insects. For an hour afterwards
all was quiet. The
old
birds
were sitting by in
silence, prob-
ably not far away. At ten min-
utes before eleven
o'clock one
of the pair, probably the female,
came with a swoop to the nest-
ing branch, and I believe fed her
young. In thiscase the observer
had to wait two hours and twen-
ty
minutes
before having the
birds close to his eye, but he was
he sequel will show. In one minute the mother had returned,
up for lost time. In five hours and six minutes (from 10.50
r an intermission
of
forty minutes when
the observer was
venty-five
visits to the
nest. Not only had
they become
soon paid
little heed
to anything about
it, and one could
using directly upon the brooding or standing bird. After
I
ould be at the
nest
in
five minutes
or even less time, and
half-minute
intervals. Occasionally both birds were at the
m happened unless the female was brooding. wamp beside Redwing Blackbird's Fig. 16. Tent over raft in water of swamp beside Redwing Blackbird's
nest. See Figs, 21 and
22. y ,
well repaid for the delay as the sequel will show. In one minute the mother had returned,
and now both began to make up for lost time. In five hours and six minutes (from 10.50
A.M. to 4.36 P.M., allowing
for an intermission
of
forty minutes when
the observer was
away), the old birds made seventy-five
visits to the
nest. Not only had
they become
accustomed to the tent, but soon paid
little heed
to anything about
it, and one could
photograph them at will, focusing directly upon the brooding or standing bird. After
I
had entered the tent, they would be at the
nest
in
five minutes
or even less time, and
the young were often fed
at half-minute
intervals. Occasionally both birds were at the
nest together, but this seldom happened unless the female was brooding. y ,
well repaid for the delay as the sequel will show. In one minute the mother had returned,
and now both began to make up for lost time. In five hours and six minutes (from 10.50
A.M. Wild
Birds. to 4.36 P.M., allowing
for an intermission
of
forty minutes when
the observer was
away), the old birds made seventy-five
visits to the
nest. Not only had
they become
accustomed to the tent, but soon paid
little heed
to anything about
it, and one could
photograph them at will, focusing directly upon the brooding or standing bird. After
I
had entered the tent, they would be at the
nest
in
five minutes
or even less time, and
the young were often fed
at half-minute
intervals. Occasionally both birds were at the
nest together, but this seldom happened unless the female was brooding. g
pp
g
On the second day the male came to the nesting branch in twelve minutes after the Fig. 17. Female Kingbird balancing herself with raised wings while feeding young. Fig. 17. Female Kingbird balancing herself with raised wings while feeding young. Fig. 17. Female Kingbird balancing herself with raised wings while feeding young. Fig. 18. Male Kingbird seeing a cicada safely down a hungry throat. Fig. 18. Male Kingbird seeing a cicada safely down a hungry throat. Fig. 19. Kingbirds rending an unruly dragon-fly. The female, who stands in front, was brooding when
the prey was brought by the male. Fig. 19. Kingbirds rending an unruly dragon-fly. The female, who stands in front, was brooding when
the prey was brought by the male. Fig. 20. Kingbird family. The male
to the right
has captured a dragon-fly, whose stick of a body
is seen projecting from the mouth of a young bird. 25 Fig. 20. Kingbird family. The male
to the right
has captured a dragon-fly, whose stick of a body
is seen projecting from the mouth of a young bird. 25 Fig. 20. Kingbird family. The male
to the right
has captured a dragon-fly, whose stick of a body
is seen projecting from the mouth of a young bird. 25 Illustrations of the Method. 27 i'ig. 21. Male Redwing Blackbird feeding young. tent was in position, and the panora-
mic scenes of
life at this nest went on
without disturbance
for the
rest
of
that
day. The
birds were
before
your eye,
literally
at hand, and
the
observer
had only
to watch and
re-
cord the rapidly
shifting scenes with
pencil and camera. Wild
Birds. If the female happened to be brooding at the time, she would seize the
struggling insect and try to start
it down one of the hungry throats. If she failed in this
the male would snatch
it from her to try his skill, and usually with as
little success. In
this way the prey would be passed back and
forth, until
it was crushed between
the
bills
of
the two
birds, or
torn
limb
from
limb. Some of these unequal contests between birds
and
insects
are
illustrated
by
the
photo-
graphs. When the male brought a moth
miller
and accidentally dropped it close to the tent,
he went after
it like a flash, and
to
place
its
security beyond doubt swallowed
it himself. Again, one of the birds while perched near by
was seen to disgorge the indigestible parts of
its insect food, a common practice with
fly-
catchers both old and young. I
have added the foregoing
details
in
order to show with what harmony life at the
new
nesting
site
proceeds when
once
the
severed threads have been united. A knowl-
Fig. 23. Young Kingbird eighteen daysold. "The last
to leave flew easily two hundred feet down the hillside." y
g
p
Whenever the male brought a large dragon-fly to the young an exciting scene was
sure to follow. If the female happened to be brooding at the time, she would seize the
struggling insect and try to start
it down one of the hungry throats. If she failed in this
the male would snatch
it from her to try his skill, and usually with as
little success. In
this way the prey would be passed back and
forth, until
it was crushed between
the
bills
of
the two
birds, or
torn
limb
from
limb. Some of these unequal contests between birds
and
insects
are
illustrated
by
the
photo-
graphs. Fig. 23. Young Kingbird eighteen daysold. "The last
to leave flew easily two hundred feet down the hillside." g
p
When the male brought a moth
miller
and accidentally dropped it close to the tent,
he went after
it like a flash, and
to
place
its
security beyond doubt swallowed
it himself. Again, one of the birds while perched near by
was seen to disgorge the indigestible parts of
its insect food, a common practice with
fly-
catchers both old and young. Wild
Birds. p
On
the third day, July 4th, the
female was on the bough
in six min-
utes, and
in
six and
a half minutes
from the beginning of operations fed
her brood. The fourth day
of study at this
nest, or the sixth from
the time
of
displacement, was
the most interest-
ing of all. There were now two foster
children
in
addition to the two born
in
the house,
for
I had
transferred
two birds from a former nest (No. 21
of table). No
protest was made
at Fig. 21. Female Redwing Blackbird with feathers erect, keep-
ing cool while shielding young from heat. i'ig. 21. Male Redwing Blackbird feeding young. i'ig. 21. Male Redwing Blackbird feeding young. this intrusion, but the strangers were
adopted almost immediately and fed
and guarded with
all
the care given
to their own offspring. p
g
In the space of
four hours (8.54
A.M. to
12.50
I'.M.) the parents made
one hundred and
eight
visits to the
nest and fed
their brood
ninety-one
times. In this task the female bore
the larger share, bringing food more
than
fifty times, although
the male
made a good showing, having a rec-
ord of thirty-seven visits to his credit. During this long
interval the young
were thus fed on the average of once
in two and one half minutes. At each
feeding usually one and
but
rarely
two birds were served. During the
first hour the young were fed on an
average
of once
in one and
a
half
minutes. The observer was kept on Fig. 21. Female Redwing Blackbird with feathers erect, keep-
ing cool while shielding young from heat. Wild
Birds. 28 the alert in recording what took place, and the scenes would often shift so quickly that
it
was difficult to decide which bird came to the nest. The mother brooded eighteen times,
and altogether for the space of one hour and twenty minutes. The nest was cleaned
seven
times, and
the nest
and young were constantly
inspected and
picked
all
over
by both birds, although the female was the more scrupulous in her attentions. by
, a t oug
p
Whenever the male brought a large dragon-fly to the young an exciting scene was
sure to follow. TENT AND CAMERA:
THE TOOLS OF I'-IRD-PHOTOGRAPHY. The eaves
of the
tent
consist
of
a double
fold
of cloth
projecting
half an
inch, to each corner
of which
is sewn a covered wire
ring. When
in
position
the
tent
is
firmly guyed by
small cords fastened to each ring. The flaps are placed at one of the corners, and may be
pinned together when in use. The free lower border of the tent
is fixed to the ground
by wire pins, which may be pushed through the cloth at convenient
places. From four
to eight of these pins are needed, and each should
be seven or eight
inches
long, and
have a large soldered loop at one end. j
,
p
The
tent which
I
have used
for two seasons and
will now
describe,
meets these
requirements
fairly
well. It
is made
of stout grass-green
' denim, and with the frame
weighs only six and one half pounds. It can be pitched
in ten minutes almost anywhere,
and may be compactly rolled, and carried for miles without serious inconvenience. It
is
6i
ft. tall, 4^ ft. long, and 3^
ft wide, dimensions which will be found suitable for a person
not much above the average height. One may spend any number of hours in
it by day or
night, and with a
fair degree of comfort, excepting
in very hot or sultry weather, when
exposed
to
the sun on
all
sides. I have suspended
operations
but once on account
of the heat, but there have been occasions when to have done so might have been better. g
The tent frame
is in three pieces, two upright poles or stakes with folding cross-bars,
and an adjustable ridge-pole. The stakes should be from six to six and a half feet long,
and may be easily lengthened at any time, as when the tent
is to be pitched
in a swamp
or over mud and
water. They
are
pointed
at
the
lower ends which
are
set
in
the
ground, and capped above with an arch
of sheet
zinc or iron to receive the
ridge-pole. The latter
is held in place with two pins or wire
nails which are pressed through a hole
in the zinc cap, and through the end of the ridge-pole into the upright stake. CHAPTER
III. CHAPTER
III. Wild
Birds. y
g
I
have added the foregoing
details
in
order to show with what harmony life at the
new
nesting
site
proceeds when
once
the
severed threads have been united. A knowl-
edge
of former conditions seemed
to have
been completely effaced. The nesting bough was defended with the same bold spirit for
which
this
bird
is
celebrated. The young were brooded night and day, while
birds
of
other species were constantly assailed and driven from the premises. Fig. 23. Young Kingbird eighteen daysold. "The last
to leave flew easily two hundred feet down the hillside." y
g
I
have added the foregoing
details
in
order to show with what harmony life at the
new
nesting
site
proceeds when
once
the
severed threads have been united. A knowl-
edge
of former conditions seemed
to have
gh was defended with the same bold spirit for
were brooded night and day, while
birds
of
riven from the premises. Fig. 23. Young Kingbird eighteen daysold. "The last
to leave flew easily two hundred feet down the hillside." p
y
premises. At noon on the ninth day of July one Kingbird, then
full-fledged, was standing on
the branch
beside the
nest. When touched he was
off
like a
shot, and
at
this
signal
the
others
tried
their wings
for the
first time and landed
in
the
grass. After being
replaced many times, two consented to remain, and spent that night in the old home, but
forsook
it the next morning, when two weeks old. The
first
nest, which had been
dis-
placed in a similar way and which as we have seen eventually contained two birds, was
occupied eighteen days. The last to leave flew easily two hundred
feet down
the
hill-
side on the thirteenth
of
July. After taking
this one home
to secure a photograph,
I carried him to the hilltop and tossed him
in the
air. In
his second
flight which was
long and good, he made a distant apple tree. Both old and young birds remained in the
neighborhood for several weeks, and were still there when
I went away in early August. 1 Brown
or gray might answer as well.
The green color serves to render the tent inconspicuous to both ani-
mals and men. or gray might answer as well.
The green color serves to render the tent inconspicuous to both ani TENT AND CAMERA:
THE TOOLS OF I'-IRD-PHOTOGRAPHY. PHOTOGRAPHY
has become
so
essential
to
the
practice of the other
arts and
sciences, that the student need not suffer from lack of advice, or of detailed man-
uals which treat every branch of the subject. PHO e e y
subject. In the notes which follow I shall confine myself mainly to the results of personal ex-
perience in working with the tent. p
g
The Observation
Tent. To satisfy the student and photographer of
birds, the tent
must not only afford a perfect means of concealment, but must be light, portable, easily
adjusted, and to the fastidious, a most important consideration,
comfortable to work in. must not only afford a perfect means of concealment, but must be light, portable, easily
adjusted, and to the fastidious, a most important consideration,
comfortable to work in. The
tent which
I
have used
for two seasons and
will now
describe,
meets these
requirements
fairly
well. It
is made
of stout grass-green
' denim, and with the frame
weighs only six and one half pounds. It can be pitched
in ten minutes almost anywhere,
and may be compactly rolled, and carried for miles without serious inconvenience. It
is
6i
ft. tall, 4^ ft. long, and 3^
ft wide, dimensions which will be found suitable for a person
not much above the average height. One may spend any number of hours in
it by day or
night, and with a
fair degree of comfort, excepting
in very hot or sultry weather, when
exposed
to
the sun on
all
sides. I have suspended
operations
but once on account
of the heat, but there have been occasions when to have done so might have been better. The tent frame
is in three pieces, two upright poles or stakes with folding cross-bars,
and an adjustable ridge-pole. The stakes should be from six to six and a half feet long,
and may be easily lengthened at any time, as when the tent
is to be pitched
in a swamp
or over mud and
water. They
are
pointed
at
the
lower ends which
are
set
in
the
ground, and capped above with an arch
of sheet
zinc or iron to receive the
ridge-pole. The latter
is held in place with two pins or wire
nails which are pressed through a hole
in the zinc cap, and through the end of the ridge-pole into the upright stake. 1
Wild Life at Home
:
Hotu to Study and Photograph It.
By R. Kearton, illustrated by C. Kearton.
Cassell
& Company, l8qq. Wild
Birds. cutting out a large flap on each side of the roof, extending this a foot or
less, and then
guying each corner separately, at such an angle as to admit a free passage of
air under
the peak. For convenience
I prefer the simpler form. first time the interesting work
ind
is used. They devised an
imitation tree-trunk, having a
skeleton
of bamboo rods and
a covering
of galvanized wire
and green cloth, large enough
to
hold
the
photographer
standing erect with his camera. The
outside
was
painted
in
imitation of bark and decorat-
ed with moss and leaves. This
was
used
in
cases
of
nests
placed on or near the ground
in
favorable
situations. Mr. Kearton says
it would hardly
do to set this up beside an ex-
posed
nest
like
a
lark's
'' in
the middle of a bare ten-acre
field," and to
suit such a case
they
constructed
an
artificial
rubbish heap, from which pho-
tographs
were
successfully
made. After working one summer with the tent I saw for the first time the interesting work
of the brothers Kearton,' in which a different kind
of blind
is used. They devised an
imitation tree-trunk, having a
skeleton
of bamboo rods and
a covering
of galvanized wire
and green cloth, large enough
to
hold
the
photographer
standing erect with his camera. The
outside
was
painted
in After working one summer with the tent I saw for the first time the interesting work
of the brothers Kearton,' in which a different kind
of blind
is used. They devised an Fig. 24. The tools of bird-photography:
the tent
rolled up
in portable
form at right. Such devices are of course
unnecessary when the nesting
site
is brought under control,
since
in
this
case
the
birds
must become accustomed to a
changed environment, and the
addition of the
tent
is
a
fac-
tor
of
no
great
importance. Then again the great heat of
summer would prohibit their use in most parts of this country. Aside from the question
of comfort however, the advantages of the tent lie in
its convenience and portability. It
is a simple means of attaining what
is chiefly sought, perfect concealment. The reason it
has not been adopted before possibly arises from the fact that the readiness with which
many birds become accustomed to strange objects, or form new
habits, has not hitherto
been
appreciated. TENT AND CAMERA:
THE TOOLS OF I'-IRD-PHOTOGRAPHY. The eaves
of the
tent
consist
of
a double
fold
of cloth
projecting
half an
inch, to each corner
of which
is sewn a covered wire
ring. When
in
position
the
tent
is
firmly guyed by
small cords fastened to each ring. The flaps are placed at one of the corners, and may be
pinned together when in use. The free lower border of the tent
is fixed to the ground
by wire pins, which may be pushed through the cloth at convenient
places. From four
to eight of these pins are needed, and each should
be seven or eight
inches
long, and
have a large soldered loop at one end. loop
may be ventilated from above and made more comfortable on hot days by large
p
The tent may be ventilated from above and made more comfortable on ight answer as well. The green color serves to render the tent inconspicuous to both ani- 1 Brown
or gray might answer as well. The green color serves to render the tent inconspicuous to both ani-
mals and men. 29 Wild
Birds. 1
Wild Life at Home
:
Hotu to Study and Photograph It.
By R. Kearton, illustrated by C. Kearton.
Cassell
& Company
l8qq Wild
Birds. Since
individual and
specific
differences
are so great in the class of
birds, whose distribution
is world-wide, one should
not be
surprised
if there
are many-
cases in which the tent or any similar blind would not work with success. Fig. 24. The tools of bird-photography:
the tent
rolled up
in portable
form at right. Fig. 24. The tools of bird-photography:
the tent
rolled up
in portable
form at right. g
g
summer would prohibit their use in most parts of this country. Aside from the question
of comfort however, the advantages of the tent lie in
its convenience and portability. It
is a simple means of attaining what
is chiefly sought, perfect concealment. The reason it
has not been adopted before possibly arises from the fact that the readiness with which
many birds become accustomed to strange objects, or form new
habits, has not hitherto
been
appreciated. Since
individual and
specific
differences
are so great in the class of
birds, whose distribution
is world-wide, one should
not be
surprised
if there
are many-
cases in which the tent or any similar blind would not work with success. g
g
summer would prohibit their use in most parts of this country. Aside from the questio
of comfort however, the advantages of the tent lie in
its convenience and portability. I
is a simple means of attaining what
is chiefly sought, perfect concealment. The reason i
has not been adopted before possibly arises from the fact that the readiness with whic
many birds become accustomed to strange objects, or form new
habits, has not hithert
been
appreciated. Since
individual and
specific
differences
are so great in the class o
birds, whose distribution
is world-wide, one should
not be
surprised
if there
are many
cases in which the tent or any similar blind would not work with success. g
g
summer would prohibit their use in most parts of this country. Aside from the question
of comfort however, the advantages of the tent lie in
its convenience and portability. It
is a simple means of attaining what
is chiefly sought, perfect concealment. The reason it
has not been adopted before possibly arises from the fact that the readiness with which
many birds become accustomed to strange objects, or form new
habits, has not hitherto
been
appreciated. Tent and Camera: The Tools
of Bird-Photography. 31 Tlic Tent in
L'sc. Some difficulty may be experienced in pitching the tent in exactly
;he right
position with
reference to the
nest, without the
necessity
of
further change. The factors to be borne in mind are the height of the sun, the
focal length
of the
lens,
and the position of the window to be made
in the tent-front directly opposite the
nest. The front of the tent should be parallel with the nesting bough (when there
is onci, and
the long axis of the latter should be parallel with the sun's course. The tent
is so placed
that the nest
is in direct line, not with the middle
of the
tent, but
with
the window to
one side. When the observer stands within, facing the nest, the window lies to his left, at
one side of the vertical stake, and either just over the cross-piece or somewhere below
it,
depending on the height of the nest from the ground. The tent will not overshadow the
nesting bough when once
it
is in proper position. g
g
p
p
p
If the focal length nf the lens be 61 inches, and the nest that of a Cedar Waxwing, which
is mounted at the height of four feet, and the tent be so placed that the front of the lens
is
twenty-eight inches from the rim of the nest, we shall get a picture with adequate setting on
34x5 plate, like many shown
in the engravings. With lenses of longer focus, which
it
is advisable to use
if possible,
it
is not necessary to approach
so
near. The large Robin
pictures were made with a 9-^ inch lens on a 5x7 plate, at a distance of about four feet. p
9
5
plate,
When the position has been determined the tent-poles are set firmly into the ground,
the
ridge-pole adjusted and the tent-cloth thrown over
it. It saves time to lay one end
of the peak in position and draw the other over to its proper place. The cross-pieces are
then lowered from the inside and the guys loosely set. A flap about six inches square is
then cut with scissors in the front of the tent, to the
left
of the pole opposite the
nest,
which can be viewed through the opening. Wild
Birds. Since
individual and
specific
differences
are so great in the class of
birds, whose distribution
is world-wide, one should
not be
surprised
if there
are many-
cases in which the tent or any similar blind would not work with success. Tent and Camera: The Tools
of Bird-Photography. Wild
Birds. 32 Camera. Any good long
focus camera with
reversible back will answer, the
size
and weight being the
considerations of greatest moment. Most naturalists and
sports-
men, who travel long distances and
carry their own traps, find a camera which takes a
4x5 plate the most convenient and
economical. I have used
this, but
for work with
the tent prefer the 5x7
size because
it gives a larger and
better picture
of the object
sought. The large camera with a heavy lens may be a drag on the mind and body of the
most
enthusiastic
pedestrian, but one
is usually amply repaid
for the greater trouble
involved. For long journeys however the lightest possible outfit
is decidedly preferable. g j
y
g
p
y p
In working at short range with lenses of moderate focus the long bellows is a necessity,
and at the same time enables one to take full sized pictures of small objects, as well as to
use the telephoto lens should
this be
desired. The
reversible back, making
it
possible
to reverse the position of the plate without moving the camera and often without disturb-
ing the bird,
is an adjunct of the greatest convenience. g
j
g
While the best tools are always to be desired, excellent pictures can be made with a
cheap outfit, provided the lens
is rapid enough. Nearly
all
of my own work has been
done in the tent with the birds at hand, but in taking quick shots of birds or quadrupeds
when there
is no lure to chain them to a given spot a hand-box camera is needed. The
lens should be of long focus, and the adjustments such as to enable the operator to focus
and expose as nearly simultaneously as possible. To meet these requirements the twin-
lens and reflecting cameras, both of which are old inventions,
1 have
in recent years been
placed on the market in improved and serviceable forms. p
p
The
" twin-lens "
consists
of two cameras, set one above the other, the bellows of
which move as one. The lower takes the picture, while the upper gives the image which
is reflected on a glass
plate
set
in
the top
of the
box. Besides being expensive and
heavy, the trade
sizes
of these cameras
are apt
to be of too short focus to be of much
service to the animal photographer. 1 The principle of the reflecting camera was applied as early as 1860, and various forms of the reflex type were
devised during the next thirty years.
In 1891 Dr. Kriigener of Frankfort brought out his " Normal Reflex-Camera,"
in which the construction, though somewhat complicated, was much improved.
The
principles
are
essentially the
same in the later designs
: see AusfUhrliches Handbucli dcr Photographic, by Josef Maria Eder, Halle, 1891.
For an
account of the reflecting camera with focal plane shutter, by Mr. John Rowley, see Bird Lore, April, 1900.
'* Manufactured by the Reflex Camera Co., Yonkers, N. Y. Tent and Camera: The Tools
of Bird-Photography. Should the position subsequently prove to be
wrong, the poles may be raised both together and reset. When everything
is
right
the
guys are tightened, and the free edges fixed to the ground with wire pins, which will hold
the walls taut and prevent excessive flapping when there
is wind. It
is often convenient
to have the flap at the front on the operator's left so that one leg of the tripod may pro-
ject through
it. j
g
The proper adjustment of the camera follows, the nest being the object focused until
the old birds appear. I have found
it advantageous
to
pin the focusing cloth
firmly
around the camera so that
it
is always in position for use, and to stretch a piece of green
denim on the side of the camera next the observer, fixing
it between the front fold of the
focusing cloth and the tent so that it hangs vertical, and effectually conceals the operator
when standing upright and
setting the
shutter. Peep-holes are made
to command
all
directions, and of course the nesting bough to which attention
is mainly given. It
is con-
venient to make small V-shaped openings which can be pinned up or down. A bird will
sometimes detect some movement of the eye when close to such
openings, so
that they
should not be made larger or more numerous than necessary. g
y
When a photograph has been made and the shutter
is to be reset, the vertical flap
is
released from the focusing cloth and carefully drawn over the window,
if the birds happen
to be at the nest as when the female is brooding. Otherwise if timid or unaccustomed to the
new conditions, the movement of the hand may be a source of alarm. I have successively
photographed family groups without disturbing them, when at a distance of twenty-eight
to thirty-six inches, after they had learned to disregard the click of the shutter. When a
window in a different position
is wanted, the old one
is patched up and a new one made. 3 Wild
Birds. 1 These lenses arc made
l>y the llausch \
I.nnili
i
>|.tn:il Co.,
Kmhi-ster. N. V. Wild
Birds. p
g
p
The
reflecting
camera
2
does the work
of the two
lenses
with a single
lens and
bellows, and in the recent designs
is provided with a focal plane shutter, which
is one of
the best for exposures quicker than the -j-^ second mark of ordinary shutters. Like the
upper half of the " twin-lens "
it has a movable mirror, set at an angle of 45
, which casts
the image made by the lens on a plate of ground glass set in the top of the box and shielded
by an adjustable hood. The mirror
is so placed between the plate and lens that the dis-
tance from lens to sensitive plate equals the distance traversed by light in passing from lens
to mirror and ground glass. When the object is focused, a lever is pressed which raises the
mirror and automatically releases the shutter. One must expect to find the image on the
ground glass somewhat dimmer than when no interposing mirror
is used. To be most
serviceable this camera should have a long bellows. long
The Lens. In animal photography short and
long
focus, and telephoto
lenses are
available. My own experience has been mainly limited to the following
:
Zeiss Anastigmat Tent and Camera
: The Tools
of Bird-Photography. 33 Series
ii a, 6
inch, speed i
:
Convertible Anastigmat, Series
vii
a, combined
equivalent
focus 8 inches, speed
r
'
3
;
Extra Rapid Universal Lens, Series D, 9
7
, ;
inch, speed
.' The
convertible
anastigmats
are
convertible
in
two
or
three
lenses
of
different
foci, according as the single anastigmats are of equal or different focus. They thus com-
bine in a single lens the possibilities of working with
short and
long
focus, the
greatest
speed being obtained when each system of the doublet has the same focus. Fig. 25. Female Brown Thrush stepping into her nest to brood. Fig. 25. Female Brown Thrush stepping into her nest to brood. In photographing animals close at hand the anastigmatic qualities of a lens count for
little. It
is depth
of
focus combined
with high speed which
arc most needed, conse-
quently any lens possessing these qualities will answer. quently any lens possessing these qualities will answer. One of the most difficult problems in bird-photography has hitherto been that of ap-
proach within " shooting
" distance. Wild
Birds. ing
slipping
The " Graphic
" ball and
socket clamp, used as a camera holder
for the bicycle, has
been strongly recommended
as a substitute
for the tripod or as an adjunct
to
it, as
in
photographing nests in trees, when the clamp which
is screwed to the camera is fastened
to a convenient limb, but since my own work has been of another kind,
I have had
little
occasion for its use. The Shutter. In photographing birds whose sense of hearing
is well known to be
acute, next to a good lens, a silent shutter
is most needed, especially when the camera is
less than three feet away. The shutter which
is silent not only in name but in actual use,
and at all speeds, is at present one of the greatest needs in the photography of animals,
and especially of birds. p
y
Birds will often jump into the air as
if shot, at the first click of the metallic shutter. Fortunately, however, the force of habit now comes to our aid, since they gradually learn
that
it
is harmless, and may be safely disregarded. y
y
g
The " iris diaphragm shutter," which
I have mainly used, is often troublesome in that
some part of
the sound
arises at the very beginning of the exposure, so
that a startled
bird in the course of ^
of a second may be all over your
plate. The marks on
all such
shutters, which are conventional rather than exact time measurements, differ in different
shutters
of
the
same
or different make, and
their limit
of rapidity does not exceed
"yfj. second."
For greater speeds the
focal
plane
or some other very rapid
shutter
must be used. Plates. For animal photography the most rapid plates are none too fast, and any of
the best brands can be recommended. It
is always a good plan to adhere
to one kind
which has proved satisfactory. One piece of advice should not come amiss, which
is to
always use fresh plates, and all of the same emulsion
if
possible, and
if any doubt as to
their age exists, to test them before starting on an expedition. Old plates blacken along
their edges in a characteristic manner, when placed in the developer, and
if deterioration
passes this stage the whole plate will fog. Wild
Birds. 34 In photographing birds sitting, brooding, or standing at the nest there is no difficulty
with a lens of speed ^, which requires ^V second to
fully expose the
plate, at a distance
of twenty-eight
inches with full lens and strong light. With scenes in which the actors
are
in constant motion, however, we
require a much
faster
lens, which
will reduce
the
exposure to at least -fa of a second. exposure
fa
For photographing inaccessible nests, and birds which pose well but are unapproach-
able under ordinary conditions, we must
resort to the long focus and telephoto lenses. The long exposure required for the telephoto lenses now on the market, from one half a
second to a second or more, restricts their use to comparatively rare and lucky chances
The
Tripod. When two cameras are carried
of
the 4x5 and 5x7 size, a
single
tripod will answer for both, provided
it
is moderately stiff about the head. A two-length
tripod of medium weight will serve most purposes, but a shorter one
is also required for
nests on or near the ground. This
is best made by cutting down one of the ordinary
kind, rather than resorting to those of the multifolding type, which are constantly spread-
ing and slipping at critical moments. p
For photographing inaccessible nests, and birds which pose well but are unapproach-
able under ordinary conditions, we must
resort to the long focus and telephoto lenses. The long exposure required for the telephoto lenses now on the market, from one half a
second to a second or more, restricts their use to comparatively rare and lucky chances ,
p
y
y
The
Tripod. When two cameras are carried
of
the 4x5 and 5x7 size, a
single
tripod will answer for both, provided
it
is moderately stiff about the head. A two-length
tripod of medium weight will serve most purposes, but a shorter one
is also required for
nests on or near the ground. This
is best made by cutting down one of the ordinary
kind, rather than resorting to those of the multifolding type, which are constantly spread-
ing and slipping at critical moments. Wild
Birds. The control of the nesting site, and the use of the tent
offer a solution so
far as
life
at the nest
is concerned, in
at
least many species, and the
tent
in
its general
use does away with
the need
of
the very
long
focus
or telephoto
lenses. Wild
Birds. The dusting of plates, slides, and holders be-
fore reloading, and the carriage of
all unused plates in a dust proof bag, are as much a
necessity now as ever. necessity
Much
of my own work has been done
in
the country with dark room and
base of Tent and Camera
: The Tools
of Bird-Photography. 35 supplies close at hand. Under these conditions
it
is not necessary to carry more than t\\
>
or three dozen plates at a time. By developing on the day of exposure
it
is possible
ti>
correct errors or
fill up the gaps on the day following. supplies close at hand. Under these conditions
it
is not necessary to carry more than t\\
>
or three dozen plates at a time. By developing on the day of exposure
it
is possible
ti>
correct errors or
fill up the gaps on the day following. p
g p
y
g
Orthochromatic
plates require careful treatment, but
in
skillful hands
offer advan-
tages which should not be neglected. This
is well illustrated in the case of birds of bril-
liant
colors
like the Orioles, which on ordinary plates appear as "Blackbirds" (compare
figures
14,
15). g
Accessories. The minor articles which
are needed to complete
the photographer's
outfit,
all of which can be rolled up with the tent or better carried
in a hand bag, will be
suggested by a little experience in the field. A saw, hatchet and nails are often required,
as well as scissors, pins, the supply of which
is always liable to run out, and a small hand
mirror
for use in setting the shutter from the rear. A
toilet
hand mirror which can be
turned at any angle
is a convenient means of inspecting the interior of nests inaccessible
to the hand, but within reach of the mirror attached to a pole. Accessories. The minor articles which
are needed to complete
the photographer's
outfit,
all of which can be rolled up with the tent or better carried
in a hand bag, will be
suggested by a little experience in the field. A saw, hatchet and nails are often required,
as well as scissors, pins, the supply of which
is always liable to run out, and a small hand
mirror
for use in setting the shutter from the rear. THE ROBIN AT ARM'S LENGTH. It would he hard to find a better symbol of cheerfulness than the Robin singing through the rain. The green
grass pricking through the April snow is a pleasant sight because
it
is the sign of
spring. For
the same reason the
snow-laden twigs of
the apple tree on the lawn take on a new
interest when a Robin alights in them and
turns
its
bright breast to your window. NO NO
bird
is better known in America than the Robin who annually visits nearly every
part of the continent. Upon the whole
it shuns the
forest and comes
to
the
haunts of man, to the farm, the village and the city street, with
their
attractive
orchards and parks, their long lines of shade trees and green lawns. g
Is
it
possible
to
say
anything new
about such
a familiar personality? Not
much, you may think, yet
it will be inter-
esting to study our friend at a closer range
than
is usually possible. In this case we
shall " make the mountain come to Maho-
met," or bring the nest from the treetop to
a point nearer the ground, where there
is
no foliage to obscure our vision, and where
we
can
see every
thing
that
transpires,
within reach of the hand. Fig. 26. " Robin snow " in April X 3. Birds differ slightly
in
every bodily
character, as well as
in every mental trait,
and while we commonly meet with average
types, extremes of temperament are by no
means rare. This fact
is illustrated by the
Robins whose history follows. sto y
One pair dwelt in the woods and were
exceedingly wary, while the other was com-
fortably
settled
in town, and
lived on
a
familiar
footing
with
man. The
town
Robins had,
I suspected, already led forth
a brood from a pine tree on the bank close
to my house, but at all events there was a
new nest
in
the
apple
tree on
the top
of the
hill, and on the twenty-fifth
of
July the
mother
bird was sitting on three blue eggs. Incubation lasted about two weeks, and life
at the nest about twelve days. Fig. 26. " Robin snow " in April X 3. y
One pair dwelt in the woods and were
exceedingly wary, while the other was com-
fortably
settled
in town, and
lived on
a
familiar
footing
with
man. Wild
Birds. A
toilet
hand mirror which can be
turned at any angle
is a convenient means of inspecting the interior of nests inaccessible
to the hand, but within reach of the mirror attached to a pole. . The Robin
at Arm's
Length. 39 When the young were three days old the mother passed some moments
of great
suspense. A small flock of Crow Blackbirds alighted on her tree, but either did not dis-
cover the nest, or thought better of disturbing
it after seeing its guardian. The wily old
Robin stood alert on the rim of the nest, but said never a word, a plan which good sense
and
intelligence could not have improved upon. When the young were eight days old,
the entire bough was sawn
off, carefully lowered to the ground and set up on the hillside. g
y
g
p
In exactly fifty-five minutes from the beginning of operations the mother appeared
with a large grasshopper, which she gave to the young, and afterwards cleaned the nest. The male came
also, when
the comparative safety of the new conditions had become
apparent, but approached with more caution. At
first both
birds flew
to the
tree by
their accustomed paths, and examined the place where their bough had been lopped
off,
and in their admirable and fearless manner blustered about for a while, taking no pains to
conceal their anger. Of course they knew where their young were
all the time, for
in
certain directions their vision
is keener than any man's. y
We know
well
with what confidence the Robin
flies
direct
to
its
nest, when
no
danger threatens, but under the present circumstances their suspicions might
well have
been aroused. The absence of sound and motion in strange objects
is always reassuring,
and soon Mother Robin could be seen perched on the top of an apple
tree, surveying
the
field. She called
sect ! sect ! while the grasshopper in her bill squirmed to get free,
and the young chirped loudly in reply. y
g
p
y
p y
When
their behavior
is
free and spontaneous
it
is
pleasant
to see these birds act
promptly without apparent
hesitation. They haggle over nothing but
follow the bent
of their strongest
instincts. In
the present case the
fear which
controls them
for a
time, and overpowers their strong
parental
love,
is
gradually worn away. Suddenly
down comes one
of the
old
birds with
all
its weight on
the limb. The young have
felt
similar
vibrations
before
and know what
to expect. THE ROBIN AT ARM'S LENGTH. The
town
Robins had,
I suspected, already led forth
a brood from a pine tree on the bank close
to my house, but at all events there was a
he
hill, and on the twenty-fifth
of
July the
Incubation lasted about two weeks, and life Fig. 26. " Robin snow " in April X 3. Fig. 27. Head of Cock Robin, life-size X 4i- Fig. 27. Head of Cock Robin, life-size X 4i- Fig. 28. Head of female Robin, life-size
;' 4}. Photographed at nest immediately after the
young were fed. The slime from their throats sticks to her bill. 37 Fig. 28. Head of female Robin, life-size
;' 4}. Photographed at nest immediately after the
young were fed. The slime from their throats sticks to her bill. 37 The Robin
at Arm's
Length. The Robin
at Arm's
Length. Up go
the three heads
at
once, each mounted on a slender stalk, and each bearing at its apex what might suggest
a full-blown, brilliant flower, for as
is well known, the extent of their gape
is extraordin-
ary and the inside of the mouth has a bright orange hue. The young tremble with violent
emotions as they jostle, struggle, and call with undiminished zeal even after being fed. y j
,
gg
,
g
After the
first
visit had proved successful, confidence was established
at
once, the
female and later the male coming to the young at intervals of about five minutes, bring-
ing grasshoppers, and
occasionally removing the excreta or devouring
it on
the spot. They frequently carried five or six insects at one
load, when their
bills would suggest
a
solid stalk of grasshoppers,
all struggling to get free. g
pp
,
gg
g
g
The mother did not touch the nest with her feet at the time
of her
first
visit, but
clasped a small vertical branch, and bent clown over her young, but ever after both birds
would alight on the broad rim of the nest, and from this vantage point feed, inspect, and
clean the young, one at a time. They suffer nothing to waste, and rarely allow a cricket
or grasshopper to escape, but releasing one at a time see
it safely down an open mouth. Then after inspection
is over they fly to the nearest perch, and make haste to clean their
bills and set their dress in order. This done, there is often a pause of a few moments as if
in doubt whether to hunt more grasshoppers, to dig angleworms in yonder cornfield, or to
try the cherry trees along the fence-row. They will take everything which
their sharp
eye discerns, and often pick up an insect close to the nest. Wild
Birds. Accordingly, after feeding the young,
I decided to strike
tent and wait
until next
day. There was
a heavy thunder
storm
in the
afternoon,
but when
I visited the nest towards evening
I was pleased to
find the young as lively as
ever, and the old birds on guard with their usual spirit and tenacity unimpaired. gua d
spirit
tenacity unimpaired. The next morning they stormed vigorously about the tent and the male even came
to the nest while
I was standing near. After closing the tent
I was under the cross-fire
of their wrath for seven or eight minutes, when
the alarm
calls suddenly ceased, and
in
two minutes more the mother was on the nesting bough. The female actually came to
the nest or to the branch which held
it eight times in
succession, in the space
of twelve
minutes, with insect ready but without delivering
it. Matters did not altogether please
her yet, and with a
shrill sect
.'
sec! ! away she would go, but only to return a half min-
ute later. Finally she came boldly to the nest's brim, uttered a sound
like cuck ! aick ! which means "Open wide!" and produced
a number
of sturdy looking grasshoppers. Two minutes later the mother came again, and after feeding the young, picked them
all
over, spending a minute and a half in the duties of inspecting and cleaning. It was a hard
task
to conquer these
birds, but they had
to submit
to
the
inevitable, and
I have no
doubt but a few days more would have brought them to the hand. y
g
The relative strength of the parental instinct was well illustrated by the behavior of
these Robins. The female was always the
first
at the nest, and came
at
forty minutes
after nine o'clock on the second day. The male though constantly skirmishing about with
bill loaded, was not on the branch with food until two hours and ten minutes later. Mean-
time the mother had been giving the young her constant attention. The cock, though at
the nest or on the bough several times, did not actually have the courage to feed his little
ones
until
long
past noon. In
the
performance
of this duty he was three hours and
four minutes behind his mate. Wild
Birds. 4o One Robin at the age of eleven days left the family circle early on August I3th, and
at nine o'clock the two which remained were standing up and flapping their wings. The
old birds would come near, displaying tempting morsels in their bills, but with no intention
of feeding their young so long as they remained on the nest. By such tantalizing meth-
ods they soon drew them away. Both old and young hung about the apple trees for sev-
eral days, when they disappeared and were not seen again. Fig. 29. Female Robin brooding on a hot day
her left wing pushed up by a young bird. Fig. 29. Female Robin brooding on a hot day
her left wing pushed up by a young bird. At the stage of
flight the young Robins have several
distinct
call and alarm notes
like those of the adult birds. They can take short, low
flights, can hop briskly, and go
to cover instinctively whether with or without warnings. They will also
lie quiet
in the
grass, as in hiding, a common instinctive act. grass,
d
g,
The second
family of Robins nested
high
in
an
oak, and whenever they were ap-
proached the old birds made an admirable show of pugnacity, scolding, screaming, erect-
ing their feathers, snapping their bills and darting straight at your head. Their nesting
branch was taken
from
the woods
to
a
bare, open
field, and
set
up
sixty
feet from
the tree
in the way already described. The
first
morning's experience was
rather
dis-
couraging, for neither bird would come to
its nest while the tent was in front of
it. They Fig. 30. Female Robin inspecting her household immediately after the young have been fed
:
L characteristic attitude. Fig. 30. Female Robin inspecting her household immediately after the young have been fed
:
L characteristic attitude. Fig. 3i. Male Robin serving a cluster of angleworms and a grasshopper. Notice his position
here on the right as in all other pictures of this nest. See Chapter XII. Fig. 3i. Male Robin serving a cluster of angleworms and a grasshopper. Notice his position
here on the right as in all other pictures of this nest. See Chapter XII. 43 43 The Robin
at Arm's
Length. 45 called plaintively from the
trees, and
circled about the nesting bough again and
again,
but always kept at a distance. Wild
Birds. When the male did come at last and deliver food, he gave the nest a good cleaning,
and flew off to a corn patch
a hundred yards away. In
thirteen minutes, during which
interval the female had brought grasshoppers twice, the male returned triumphantly with
a great cluster of writhing angleworms. After safely dispensing them, he went the rounds of
inspection, devoured the excreta, then stood for a full minute on the rim of his nest and
with
crest
erect
called, wit ! wit ! wit ! as
if to
celebrate a
victory and announce
his bravery to the world. Now and again the cock came to the nesting bough but without
food. He wished only to take a look and see that all was well. At one of these visits
he stood on silent guard for full ten minutes, then sped away calling loudly, wit ! wit ! wit ! In
the
course
of
the same day
a Robin,
possibly
a young
bird,
alighted on
the
peak
of the
tent, surveyed the situation, and passed
on. When
eight
days
old, on
July 26th, the young began to present their spotted
breasts over the walls of the nest
and
to
spread,
stretch, and
flap
their wings, the
quills
of which now showed
half an
inch
of
feather
at the
tips. At every
visit
of their elders the whole brood went wild
with
excitement,
but soon
quieted down,
and
the
intervals were
spent
in
preening
their sprouting
feathers,
calling
for more
food,
or
dozing
with
heads
hanging down
over the edge
of the
nest. g
The third day opened warm and clear, and towards noon became very hot. Mother Wild
Birds. 46 Robin began
to brood at twelve o'clock and for the space of three hours was on and off
the
nest
constantly, rarely remaining longer than
ten minutes
at
a time
either at
her
post or away from
it. On the fourth day, July 28th, which was destined
to be hotter
still, brooding began
at
exactly eighteen minutes
before
ten
o'clock
and
the mother,
was
quietly
sitting over the
little ones when
the
tent was struck long past
noon. Fig. 32. Cock Robin standing at inspection, after having fed his young. Fig. 32. Cock Robin standing at inspection, after having fed his young. Wild
Birds. Another visit from the male, who comes quickly, delivers a grasshopper or two
and
departs,
while
his
faithful mate resumes her post
of duty. open
g
g
y
pp
10.30. Another visit from the male, who comes quickly, delivers a grasshopper or two
and
departs,
while
his
faithful mate resumes her post
of duty. departs,
p
y
10.45. The cock brings another coil of angleworms, and the hen, leaving her charge just
long enough
for the business
of
feeding, drops back on
the
nest. depa ts,
p
y
10.45. The cock brings another coil of angleworms, and the hen, leaving her charge just
long enough
for the business
of
feeding, drops back on
the
nest. o g
g
g
p
10.55. The male
is taking
it
easy. This time he has an
unusually large grasshopper,
which
is not cut
in twain but delivered whole. At the signal
of
his approach the
mother
leaves, having brooded
forty minutes by the watch. g
g
10.55. The male
is taking
it
easy. This time he has an
unusually large grasshopper,
which
is not cut
in twain but delivered whole. At the signal
of
his approach the
mother
leaves, having brooded
forty minutes by the watch. g
y
10.57. Two minutes elapse. Back comes the alma mater, loaded
to the muzzle with
blueberries, which
are shot out one by one, and
strike the yellow targets in the
bull's eye every time. She comes to the farther side and broods
at the moment
the preliminary work
of
feeding and
inspection
is over. preliminary
feeding
inspection
1 1. 16. The male has now brought a load of bright red choke cherries. He hops down
the branch by the usual path and up to the nest, but the female, who
is brooding,
strangely keeps
her
position
and, whether from
absent-mindedness or
capricCj
refuses to budge. When the male gives an impatient cuck ! cuck ! the mother can
keep her position no longer, for the young upset her equilibrium in their struggle,
and she hops to one side. Resuming her place she
sits there
in the bright sun-
shine, with back to the tent, mouth agape, and crest erect. Twenty inches away
are the tent, the camera, and the eye of the observer, but for none of these things
does she now care a straw. They have been thoroughly tested and found harmless. 11.43. Wild
Birds. Many charming scenes were enacted at this nest during the day, but colored phrases
or colorless pictures do them
scant
justice. You must
fill
in the backgrounds of soft
blues and
greens, and add the touch of
life and
color to the
actors on the stage. g
g
The following extracts from my notes of this day may give some idea of the panoramic
character
of the
scenes,
in which the element
of repetition
is not
wanting. p
g
July 28, 4th day in tent. 10 A.M. The female comes to the back of the
nest,
delivers
food and goes the rounds of inspection and cleaning, devouring the excreta on the
spot, then settles down on the margin of the nest, steps in and gradually tucks the
young under her breast and wings. y
g
gs. 10.12. A whirring sound announces the coming of the male. He approaches always on
the observer's right, and deliberately hops down to the nest. He is bringing a big
cluster
of earthworms. The young get the message the moment the branch
is
touched, and poke their heads out from under their mother's tail, wings, and head,
sometimes raising her bodily, and almost tipping her over. However, she holds The Robin
at Arm's
Length. 47 her place until her mate
is close
by, then hops up and stands to one side, finally
leaving him to deliver what he has brought. her place until her mate
is close
by, then hops up and stands to one side, finally
leaving him to deliver what he has brought. leaving
g
10.15. The mother
is back with food, but
it was down the throat of a young one before
I could
tell what
it was. Cleaning and brooding them followed in due course as
before. ea
g
g
10.15. The mother
is back with food, but
it was down the throat of a young one before
I could
tell what
it was. Cleaning and brooding them followed in due course as
before. 10. 18. Cock Robin comes again, but my eye was again
off the
nest, and
in a moment
the business was done. Mother Robin
stays and broods. I change the shutter,
open and close the tent window without giving her any apparent anxiety. open
giving
y
pp
y
10.30. Wild
Birds. preliminary
g
p
1 1. 16. The male has now brought a load of bright red choke cherries. He hops down
the branch by the usual path and up to the nest, but the female, who
is brooding,
strangely keeps
her
position
and, whether from
absent-mindedness or
capricCj
refuses to budge. When the male gives an impatient cuck ! cuck ! the mother can
keep her position no longer, for the young upset her equilibrium in their struggle,
and she hops to one side. Resuming her place she
sits there
in the bright sun-
shine, with back to the tent, mouth agape, and crest erect. Twenty inches away
are the tent, the camera, and the eye of the observer, but for none of these things
does she now care a straw. They have been thoroughly tested and found harmless. are the tent, the camera, and the eye of the observer, but
o e
things
does she now care a straw. They have been thoroughly tested and found harmless. 11.43. Cock Robin is on hand with a beak full of grasshoppers coming, as is now his invar-
iable custom, to the right side. On this occasion the mother hopped up promptly
and received a part of the food into her own
bill. Did she eat it ? Not a particle. The young got
it all. The male then retired, followed closely by his mate. In one
minute she has captured prey and
is back to
her brood. The young erect their
crests like their elders, and flapping their
half-fledged wings, try to climb
to the
edge of the nest, but without
success. The last day of July opened hot and
sultry, and when
I approached the nest one
young Robin was already out, and making for the highest point of the nesting bough. He cheeped aloud for food, and looked uncomfortable, for the heat was already strong. The male only was in attendance as on the previous day, the female being occupied, as
I
suspected, in starting a new nest. It was difficult to get any food past this enterprising fledgling, who stood
in the path
and
took everything that was brought. Several times the bird would make a mo%'e
as
if intending to fly to the peak of the tent, and might have done so, had
I not decided
to replace him in his nest. Wild
Birds. The expected certainly happened, for all tumbled out shriek-
and
Put them back and out th
would
i
and fl
down on the y
g
y
11.43. Cock Robin is on hand with a beak full of grasshoppers coming, as is now his invar-
iable custom, to the right side. On this occasion the mother hopped up promptly
and received a part of the food into her own
bill. Did she eat it ? Not a particle. The young got
it all. The male then retired, followed closely by his mate. In one
minute she has captured prey and
is back to
her brood. The young erect their
crests like their elders, and flapping their
half-fledged wings, try to climb
to the
edge of the nest, but without
success. edge
The last day of July opened hot and
sultry, and when
I approached the nest one
young Robin was already out, and making for the highest point of the nesting bough. He cheeped aloud for food, and looked uncomfortable, for the heat was already strong. The male only was in attendance as on the previous day, the female being occupied, as
I
suspected, in starting a new nest. p
,
g
It was difficult to get any food past this enterprising fledgling, who stood
in the path
and
took everything that was brought. Several times the bird would make a mo%'e
as
if intending to fly to the peak of the tent, and might have done so, had
I not decided
to replace him in his nest. The expected certainly happened, for all tumbled out shriek-
ing and squealing. Put them back and out they would go again, and flop down on the Wild
Birds. 48 grass. At last two birds consented
to remain
for a few
minutes, when
the male came
with an angleworm and a large green
katydid. He paused
a moment while
I photo-
graphed him, and this proved to be the closing scene. The curtain dropped suddenly
when
first one bird and then the other left their home forever, not even waiting to get the
katydid. The old bird at once led his brood to the woods, and being able to take short
flights, they had no difficulty in finding safe quarters. grass. At last two birds consented
to remain
for a few
minutes, when
the male came
with an angleworm and a large green
katydid. horizontal supports,
is generally chosen, but they
also
resort
to the
leafy elm, the ever-
green, the dense and remote woods, or
like the
Phoebe, accept
the
hospitality of
barn,
porch, or shed.
In the course of one afternoon
in Sanbornton, New Hampshire,
I once
found six nests all under cover.
One was
fixed
to
a beam
inside an
old
barn, already
occupied by Swallows, the only means of entrance and egress being cracks between the
boards of the gable above the haymow.
The Swallows shot with unerring aim through
these cracks, but one of
their full-fledged young, which lay dead on the
hay, had
appar-
ently dashed
its brains out
in attempting
this
feat.
In
a dilapidated shed of another
barn, then abandoned, were three nests, two of which set in line and close together, were
doubtless the work of the same builders.
Where the nest has already begun to crumble into ruins by the time the young
fly, Wild
Birds. He paused
a moment while
I photo-
graphed him, and this proved to be the closing scene. The curtain dropped suddenly
when
first one bird and then the other left their home forever, not even waiting to get the
katydid. The old bird at once led his brood to the woods, and being able to take short
flights, they had no difficulty in finding safe quarters. g
,
y
y
g
q
The number of times the young are fed in the course of the day depends upon their
age and the weather. The older they are the more food they require. At this nest the
labor of feeding and cleaning was shared about equally by both
birds, but on hot days
the female was necessarily less active since there was much brooding to be done. y
g
The following table illustrates the relative
activities of this pair
in caring
for their
young,
the
time
of
observation being approximately from
nine
o'clock
until three
in
the afternoon. PERIOD OF OBSERVATION. The
Robin
at Arm's
Length. 49 lington, Vermont, on March 3Oth. A few Bluebirds are usually reported on the same d.iy. In 1900, Robins were heard or seen in different parts of Cleveland on the ninth of March,
a mild, bright day, while but a week before the country was
in the grip of one
of the
worst ice-storms ever known in this region. Every exposed object was incased
in
solid
ice for days and the birds fasted or starved. y
In the choice
of
a nesting
site, the Robin,
as we have
seen, obeys no
law. The
apple
tree,
which
from
its mode
of
branching
yields
wide,
open
crotches
and
safe Fig. 33. Female Robin in act of cleaning the nest. Fig. 33. Female Robin in act of cleaning the nest. horizontal supports,
is generally chosen, but they
also
resort
to the
leafy elm, the ever-
green, the dense and remote woods, or
like the
Phoebe, accept
the
hospitality of
barn,
porch, or shed. In the course of one afternoon
in Sanbornton, New Hampshire,
I once
found six nests all under cover. One was
fixed
to
a beam
inside an
old
barn, already
occupied by Swallows, the only means of entrance and egress being cracks between the
boards of the gable above the haymow. horizontal supports,
is generally chosen, but they
also
resort
to the
leafy elm, the ever-
green, the dense and remote woods, or
like the
Phoebe, accept
the
hospitality of
barn,
porch, or shed.
In the course of one afternoon
in Sanbornton, New Hampshire,
I once
found six nests all under cover.
One was
fixed
to
a beam
inside an
old
barn, already
occupied by Swallows, the only means of entrance and egress being cracks between the
boards of the gable above the haymow.
The Swallows shot with unerring aim through
these cracks, but one of
their full-fledged young, which lay dead on the
hay, had
appar-
ently dashed
its brains out
in attempting
this
feat.
In
a dilapidated shed of another
barn, then abandoned, were three nests, two of which set in line and close together, were
doubtless the work of the same builders.
Where the nest has already begun to crumble into ruins by the time the young
fly, Wild
Birds. round
Many
wild
birds,
such as Robins, Orioles,
Wrens, Woodpeckers, to
mention
only
a
feu-
species, breed within the
confines
of
cities,
and
the
question
naturally
arises,
do
the
birds
come
to town,
or does
the town
go
to them ? We know how strong
is
the
instinct
for young
birds
to
return
to
the
place
of
their
birth,
if
not to the selfsame spot,
at
least
to
the
same
neighborhood, and they
continue to do this until
driven off by enemies or
by
hard
times. My
house in Cleveland hap-
pens to be placed in the
midst of what
was
an
apple orchard of a large
farm
a generation
ago,
and a few of
its ancient
trees
still remain
in the
which
nest in them to-day
the
descendants
of
the
birds
d farm ? Possibly,
for the birds will return, so long as the
ood which their presence insures remain. In this way many
wn into
city
life. As the farm became a part of the
village
d by the town, the migratory
species, true to their old asso-
rmer haunts each spring. I have known two illustrations of
d-headed Woodpeckers
clung
to
the
ancestral
tree
until
streets, and
indeed
until
their
old home
was
actually
ale Robin
life size. X
J. Many
wild
birds,
such as Robins, Orioles,
Wrens, Woodpeckers, to
mention
only
a
feu-
species, breed within the
confines
of
cities,
and
the
question
naturally
arises,
do
the
birds
come
to town,
or does
the town
go
to them ? We know how strong
is
the
instinct
for young
birds
to
return
to
the
place
of
their
birth,
if
not to the selfsame spot,
at
least
to
the
same
neighborhood, and they
continue to do this until
driven off by enemies or
by
hard
times. My
house in Cleveland hap-
pens to be placed in the
midst of what
was
an
apple orchard of a large
farm
a generation
ago,
and a few of
its ancient
trees
still remain
in the
escendants
of
the
birds
ll return, so long as the
main. In this way many
came a part of the
village
es, true to their old asso-
known two illustrations of
the
ancestral
tree
until
old home
was
actually
often leave their seclusion Fig. 34. Head of female Robin
life size. X
J. Fig. 34. Head of female Robin
life size. X
J. back yard. Wild
Birds. The Swallows shot with unerring aim through
these cracks, but one of
their full-fledged young, which lay dead on the
hay, had
appar-
ently dashed
its brains out
in attempting
this
feat. In
a dilapidated shed of another
barn, then abandoned, were three nests, two of which set in line and close together, were
doubtless the work of the same builders. Where the nest has already begun to crumble into ruins by the time the young
fly, horizontal supports,
is generally chosen, but they
also
resort
to the
leafy elm, the ever-
green, the dense and remote woods, or
like the
Phoebe, accept
the
hospitality of
barn,
porch, or shed. In the course of one afternoon
in Sanbornton, New Hampshire,
I once
found six nests all under cover. One was
fixed
to
a beam
inside an
old
barn, already
occupied by Swallows, the only means of entrance and egress being cracks between the
boards of the gable above the haymow. The Swallows shot with unerring aim through
these cracks, but one of
their full-fledged young, which lay dead on the
hay, had
appar-
ently dashed
its brains out
in attempting
this
feat. In
a dilapidated shed of another
barn, then abandoned, were three nests, two of which set in line and close together, were
doubtless the work of the same builders. Where the nest has already begun to crumble into ruins by the time the young
fly, Where the nest has already begun to crumble into ruins by the time the young
fly, Wild
Birds. it
is often abandoned and a new one built
for the second brood, but whether a new nest
shall be built
or not depends rather upon habit or caprice than actual need. The old
nest
is sometimes repaired, or even occupied without change during the same season. On the other hand, three nests are sometimes built
in line and under cover, where a sin-
gle one
if put in good
repair would have answered the purpose. I once saw a Robin's
nest fixed to the end of a stick of wood that leaned
against the side
of a barn, and the
stone-gray color of the background formed an excellent screen for its concealment. Wild
Birds. Are the Robins which
nest in them to-day
the
descendants
of
the
birds
which used to come to the old farm ? Possibly,
for the birds will return, so long as the
human inhabitants and the food which their presence insures remain. In this way many
birds have undoubtedly grown into
city
life. As the farm became a part of the
village
and the village was swallowed by the town, the migratory
species, true to their old asso-
ciations, returned to their former haunts each spring. I have known two illustrations of
this
in
Cleveland, where
Red-headed Woodpeckers
clung
to
the
ancestral
tree
until
enveloped
by
miles
of
city
streets, and
indeed
until
their
old home
was
actually
destroyed. dest oyed. On the other hand
it
is true that many shy and timid birds often leave their seclusion
and come to the haunts of man, and this is not remarkable when we remember how much The Robin
at Arm's Length 51 individuals
differ
in
relative
lameness and
wildness, and how
rapidly new
habits
arc-
formed. As to the abundance of food on which bird-life depends, some species, like the Robin,
would seem
to
fare
equally
well
in
the
country, and
as
to
protection, much
better. Young Robins have no more
persistent and
fatal enemy than
cats, and every one who
has possessed a city yard knows to what
extent
it
is overrun by tommies and
tabbies. In
the
city
also
one
has
to
reckon
with
the
large floating
population
of
famished
vagrants, which the biological laboratory
is never able to fully claim. They are also on
hand to rake the young broods out
of the
nests, and pick up the
fledglings which
arc-
frightened off prematurely and drop to the ground. Though forced to build high, city
Robins find
it
impossible to get beyond
the reach of some rough-and-ready climbers
of
whom Jan Steen was a shining example, and were every thomas as fearless and expert in
tree-climbing
as
he,
this
race
of
birds
would
soon
be
driven
out
or
exterminated. Some Robins used
to nest
in the very top of a neighboring apple
tree, but Jan found
them out and watched their actions attentively from day to day. One fine afternoon he
decided to bring down the whole brood. 1
The Auk, vol.
vii., October,
1890. THE CEDAR-BIRD. ON ON
the twenty-seventh of May,
I saw a small company of birds settling in the top-
most branches of an elm. You might infer from
their behavior that they were
new
arrivals. They keep
together,
sit prim and
erect, and move about
as
if
under discipline. With a glass you can see their erected crests, their sleek drab plumage,
and recognize at once the familiar Cedar or Cherry Bird. g
y
At Northfield, New Hampshire, the earliest nests have eggs by the
first
or second
week in June, but the breeding season
is not at
its height until the last of July or August. A few
still have young in the nest in
early September, when many are flocking or have
already started southward. Professor Baird speaks of finding these birds sitting on their
unhatched eggs as late as the twelfth day of October. gg
y
The winter flocks of Cedar Waxwings, which are occasionally seen in Northern New
England, are probably migrants whose summer home
is farther north. The Cedar-birds borrow no trouble from their neighbors, and seem to lead a life of
ease and pleasure, lessening their denominator when the times are hard, but
living high
when cherries are ripe. The nesting season, which
brings much that
is sweet and
bitter
to the lives of most birds, appears to give them
the
least anxiety. The immaturity
of
their eggs
at
a time when most
of our birds have already reared their first broods
is a
striking fact, and
is due to some unknown cause which retards the growth of the
ovaries. It
is evidently not caused by a lack of suitable food
as some have supposed, since the
case of the Goldfinch
is similar. The young Cedar-bird gets about the same kind of food
as the young Robin or Oriole, and
it
is not likely that a greater or less amount of fruit in
the diet of old or young would sensibly alter their condition. So quiet and retired is the
Cedar-bird,
it may live in comparative seclusion although not three rods from your house,
and may remain on your grounds for the whole summer unnoticed, unless some one is on
the watch, so that the name "chatterer" formerly
applied to the family,
1 can
have only
an ironical significance in this least garrulous of birds. 1 This epithet
is said to have been first applied to the Bohemian Waxwing, because
of
its Latin name, Amfelis
gar nilus, the specific term garniltis having been suggested by the crest and slight resemblance in the color of
this
bird
to
the European
Jay, Garrulus glaiidariiis.
See Schufeldt,
Chapters on the Xatural History of
///<
l'>iit,-i!
States. Wild
Birds. He had climbed to the tree top and was claw-
ing at the
nest, when
fortunately
his plans and equilibrium were
upset
in the
nick
of
time by a well-directed missile. y
Although the Robin
is one of our most common
birds its gregarious habits seem
to
have
attracted
little
attention
until
Mr. Brewster's
account
appeared
in
1890. His
record
for Cambridge,
Massachusetts,
extends back
to
1867. At
one
roost
he
esti-
mated the
number
of
birds
at
25,000
(August
4,
1875). The
old
males
and
first
broods in spotted plumage compose these assemblages during the second and third weeks
of June. By the middle of July the movement becomes more general and by August
ist, the
roost
is made up of young and old of both
sexes and
of
all conditions. Mr. Faxon saw a male after feeding its young fly off to
its roost one and one fourth miles away
at 7.30
P.M., while the female apparently remained for the night and brooded her young. pp
y
g
y
g
These
local
associations seem
to
be based
upon
the
instinct
of
protection and
sociability, and
it
is important to observe that
the
old
lead
the way while the young
follow,
suggesting,
as Mr. Brewster
remarks, what
usually
takes
place
in
the annual
migration. 1 g A winter Robin roost, in a swamp of matted reeds, resorted to at night by thousands
of
birds, has also been described
in
Missouri. At daybreak the
host
dispersed
in
all
directions, some going fifty miles to their feeding grounds." O. Widmann
; The Auk, vol. xii.,
1805. THE CEDAR-BIRD. The fondness of this bird for the
berries of the red cedar and for cherries
is responsible
for two
of
its commonest names,
while the term " waxwing
" has reference to the
peculiar horny scales of the secondary
wing-quills, which look as
if tipped with red sealing-wax. Less commonly, the
tail
also
bears similar appendages, but there
is much variation in their appearance in both old and 52 The Cedar Bird. 53 young. Most of the birds \vhicli
I have .studied at the nest have been entirely lacking in
appendages of this kind. Late in spring the Cedar-birds are seen coursing about in small squads, selecting some
treetop for an observatory, and always showing the most marked uniformity, there being
little to distinguish the sexes either in size or color. Their plump
oval forms and
easy. undulating flight are characteristic, and their manner of flying and
perching
in compact
bodies as one bird should not escape the observer. Apple
trees
of moderate size are in
high
favor,
since
they
afford
such
fine
opportunities
for
nest-building, and
are
usually
surrounded
by good
feeding
grounds. Fig. 35. Cedar-bird chorus at the most exciting moment just before food
is served, August 6,
1899, two days before
flight and the development of the
sense of fear. Life-size X 3. Two summers
ago
some
\Vaxwings
built on
the
hori-
zontal bough
of
a
pine
tree,
just
above
a
Robin's
nest. Song Sparrows and Chipping
Sparrows
also
occupied
the
same
tree. They usually
fre-
quent scrubby pastures, select-
ing the
witch-hazel,
or thorn-
apple
bushes
by
preference,
and
occasionally
a small
sap-
ling oak or maple. The
nest
is either
set
in
a fork or sad-
dled to a spreading branch, at
a height of from five to twenty
feet. It is nicely wrought from
vegetable and animal material
such as dead
grass, roots,
fine
twigs, weed-stems, pine
need-
les, wool, yarn, and twine. A
nest
built
in
an
orchard was
composed of dead clover stems,
witch
grass, with
thistle-down
and the fluffy heads of the In-
dian tobacco, a plant growing
close
by, worked
over
its rim
and interior. Fig. 35. Cedar-bird chorus at the most exciting moment just before food
is served, August 6,
1899, two days before
flight and the development of the
sense of fear. Life-size X 3. Four
or
five eggs
are
ordinarily
laid,
but
the
total
product
of ten nests which
I
examined
in
1899 was only
thirty-six
eggs, out of which about twenty-five young were
hatched and from sixteen to twenty reared.
The parental instincts during the early days of nest-building and incubation are often
weak, and this
is shown to a marked degree
in
the Cedar-bird, who
is easily
robbed and
ever ready to take fright and abandon
its eggs. Wild
Birds. 54 During the month of July a pair began to collect nesting material in an apple tree in
full view from our porch, and
I frequently watched them at work through an opera-glass,
and once or twice passed under their tree. This inspection of their private affairs pleased
them so little that they left their completed nest, and moved to the adjoining field a few
rods away, where there was less publicity, and where five eggs hatched out on the twenty-
sixth of August. A nest built
in a young oak
tree in a remote
clearing was discovered
on
August
7th, when
it
contained a single egg. I
did not see the old
birds
on this occasion and heard
but
a
faint sound,
which
was evidently a murmur of
remonstrance
since
their
nest
was
promptly
for-
saken. During the month of July a pair began to collect nesting material in an apple tree in
full view from our porch, and
I frequently watched them at work through an opera-glass,
and once or twice passed under their tree. This inspection of their private affairs pleased
them so little that they left their completed nest, and moved to the adjoining field a few
rods away, where there was less publicity, and where five eggs hatched out on the twenty-
sixth of August. A nest built
in a young oak
tree in a remote
clearing was discovered
on
August
7th, when
it
contained a single egg. I
did not see the old
birds
on this occasion and heard
but
a
faint sound,
which
was evidently a murmur of
remonstrance
since
their
nest
was
promptly
for-
saken. During the month of July a pair began to collect nesting material in an apple tree in
full view from our porch, and
I frequently watched them at work through an opera-glass,
and once or twice passed under their tree. This inspection of their private affairs pleased
them so little that they left their completed nest, and moved to the adjoining field a few
rods away, where there was less publicity, and where five eggs hatched out on the twenty-
sixth of August. A nest built
in a young oak
tree in a remote
clearing was discovered
on
when
i Fig. 36. The female Cedar-bird broods, while
the male passes the
cherries
around. He stands at the back with his gullet loaded and a berry in bill. THE CEDAR-BIRD. Four
or
five eggs
are
ordinarily
laid,
but
the
total
product
of ten nests which
I
examined
in
1899 was only
thirty-six
eggs, out of which about twenty-five young were
hatched and from sixteen to twenty reared. Wild
Birds. Approaching cautiously with throat loaded to the brim with choke
cherries, the
mother bird delivered them one by one, and then inspected and cleaned her household. three tii four feet. ( >u returning to the spot two days later,
gone well. After yetting the tent up
it was not many m
tr-c-c-c-c-k ! or zc-f-c-c-t ! was heard, to which the young
strain. Approaching cautiously with throat loaded to th
mother bird delivered them one by one, and then inspec
After a longer interval the pair came and stood on t
nothing
in
their
bills, but
their gullets were crammed
full
of
blueberries,
and
after tantalizing the suppli-
cating
young
for
a
mo-
&
J
o
ment, up went a head, and
presto! out came a berry,
which was quickly placed
in
an
open
throat,
and
passed around
until
it was
promptly swallowed. Up
went the head
again, and
the
performance
was
re-
peated. It was like a ma-
gician shaking eggs from a
bag, and there seemed to
be no limit to its capacity. Many who have witnessed
such actions have supposed
that
the
old
birds were
attempting
to
distribute
the food without partiality
to
their hungry
children,
but this is not the case. It
is all a question of nervous
reaction. The food is not
simply placed in the mouth
but pressed well down into
the sensitive throat, which
promptly responds
unless
the gullet
is already
full. The old
bird watches the result intently
and
if the food
Fig- 37-
Tantalizing the young. but hesitates to advance and deliver y
p
he pair came and stood on the edge of the nest. There was y
p
After a longer interval the pair came and stood on the edge of the nest. There was After a longer interval
nothing
in
their
bills, but
their gullets were crammed
full
of
blueberries,
and
after tantalizing the suppli-
cating
young
for
a
mo-
&
J
o
ment, up went a head, and
presto! out came a berry,
which was quickly placed
in
an
open
throat,
and
passed around
until
it was
promptly swallowed. Up
went the head
again, and
the
performance
was
re-
peated. It was like a ma-
gician shaking eggs from a
bag, and there seemed to
be no limit to its capacity. Wild
Birds. I have camped beside
four different
nests of the
Cedar Waxwings, and after
having spent nearly a week
in watching
the behavior
of
both
old
and
young
birds
at short
range,
feel
that I know by heart most
of their nesting habits. There is a certain rou-
tine or etiquette which
is
observed by all birds at the
nests. Certain duties must
be
performed
over
and
over, such as the capture
of prey, bringing
it and dis-
tributing
it
to the young,
inspecting
and
cleaning
the
household,
besides
brooding
the
young,
es-
pecially
during
the early
days of life in the nest. To
record each visit made and every recurring act performed by the birds would make tedi-
ous reading, but
strange
to
say
it
never seems monotonous
to the observer. As the
young birds grow older, and begin to stand on
the
rim
of the
nest, they
furnish ample
excitement, and
while
their theme
is always
the same,
it
is delivered with innumerable
i
i
Fig. 36. The female Cedar-bird broods, while
the male passes the
cherries
around. He stands at the back with his gullet loaded and a berry in bill. Fig. 36. The female Cedar-bird broods, while
the male passes the
cherries
around. He stands at the back with his gullet loaded and a berry in bill. record each visit made and every recurring act performed by the birds would make tedi-
ous reading, but
strange
to
say
it
never seems monotonous
to the observer. As the
young birds grow older, and begin to stand on
the
rim
of the
nest, they
furnish ample
excitement, and
while
their theme
is always
the same,
it
is delivered with innumerable
variations. The method of controlling the nesting site was
first suggested by some Cedar-birds,
\\ hose nest of four eggs was in a thorn-apple bush, and about seven feet from the ground. The main stem supporting the nest was cut
off, and fixed firmly in the
soil at
a height
of The Cedar
Hircl. 55 three tii four feet. ( >u returning to the spot two days later,
I \vas pleased to find that all had
gone well. After yetting the tent up
it was not many minutes before
a low-murmured
tr-c-c-c-c-k ! or zc-f-c-c-t ! was heard, to which the young always responded
in a
similar
strain. Wild
Birds. Many who have witnessed
such actions have supposed
that
the
old
birds were
attempting
to
distribute
the food without partiality
to
their hungry
children,
but this is not the case. It
is all a question of nervous
reaction. The food is not
simply placed in the mouth
but pressed well down into
the sensitive throat, which
promptly responds
unless
the gullet
is already
full. The old
bird watches the r
from one to another
until a
ments of the bird are so
rap
difficult
to make an accura
as many choke cherries are
sibility of
the
gullet
of
th
a time, thought
that
it serv
and
insects
it
is true
often Fig- 37-
Tantalizing the young. The mother Cedar-bird has come with
food,
but hesitates to advance and deliver it. Compare with Fig. 38. Fig- 37-
Tantalizing the young. The mother Cedar-bird has come with
food,
but hesitates to advance and deliver it. Compare with Fig. 38. gu
et
y
The old
bird watches the result intently, and
if the food
is not taken at once
it
is passed
from one to another
until a throat with the proper reaction time
is found. The move-
ments of the bird are so
rapid, and
the berry
is
so
often
quickly withdrawn, that
it
is
difficult
to make an accurate count. Usually from six to eleven
blueberries and almost
as many choke cherries are thus carried in the
gullet. Wilson, who noticed the disten-
sibility of
the
gullet
of
this
bird, which will take from twelve to fifteen cedar berries at
a time, thought
that
it served as a crop to prepare the food for digestion. The
berries
and
insects,
it
is true, often come up crushed to a pulp and
reeking with slime, but
it
is Wild
Birds. not likely that the (esophagus serves any other purpose than a temporary
receptacle for
the food. When the berries had gone the rounds, both birds would suddenly leave the
nest
with a whisk. Again one would hear their murmuring call, tr-c-e-c-e-k ! growing more dis-
tinct as they came nearer. Then both would alight on the nest rim, and stand there
a
moment like statuettes with heads erect. After regurgitating the food and
distributing
it, they keenly eye everything in the nest, snap up the excreta from each
bird
in
turn, d suddenly leave the
nest
c-e-c-e-k ! Wild
Birds. I
had
sat down but
a moment when
the male flew past, and gave an alarm
which brought off his mate in a flash. Both
then
alighted
in
the
tops
of
neighboring trees, and standing erect,
uttered
their
low
responsive
call-
notes. After a short
interval, during
which
I went to get a notebook and pencil,
this bird was back
again, and once
more her jet-black eye and clean-cut
profile appeared
above the
nest. I
had
sat down but
a moment when
the male flew past, and gave an alarm
which brought off his mate in a flash. Both
then
alighted
in
the
tops
of
neighboring trees, and standing erect,
uttered
their
low
responsive
call-
notes. Fig. 39. Female Cedar-bird ready to feed young
by regurgitation
gullet stuffed with cherries. Six days
later
August
2 1st
the
bush was removed
a
rod away
and the tent placed beside
it at nine
o'clock. The familiar
calls of both
birds
were now
heard and
in
just
thirty-five minutes from
the time of
closing the tent
a
soft
whirring
of
wings announced
the
mother
bird,
who
alighted
near
the
nest. She Six days
later
August
2 1st
the
bush was removed
a
rod away
and the tent placed beside
it at nine
o'clock. The familiar
calls of both
birds
were now
heard and
in
just
thirty-five minutes from
the time of
closing the tent
a
soft
whirring
of
wings announced
the
mother
bird,
who
alighted
near
the
nest. She Fig. 39. Female Cedar-bird ready to feed young
by regurgitation
gullet stuffed with cherries. Fig. 40. After feeding the young
the gullet empty. Notice
the "sitting" posture, and com pare curves of throat in Fig. 39. approached cautiously, as an intelligent
bird should do, surveying the situation
at every step, and finally landed on the
nest. After a momentary pause she be-
gan
tossing up her head and producing
black cherries
which were
judiciously
placed, one at a time,
in the throats
of
her nestlings. Then a thorough inspec-
tion followed, and the sanitary condition
of the establishment was insured by the
method already
described, after which
the mother remained a full minute
; then
with a low whistle she sped away. At
her next
visit she began
to
shield her
young from
the growing
heat. Wild
Birds. growing more dis-
nest rim, and stand there
a
the food and
distributing
a from each
bird
in
turn,
swallow
it,
and
are
off. The young sat or stood on
the nest with heads up and
all pointed oneway. Pres-
ently,
every
black
bead-
like
eye was
alert
;
four
scarlet-orange
mouths
opened
at
the same mo-
ment, and four necks were
stretched now to this side,
now to that, whence came
the
least
sound. When
their parents
actually
ap-
proached with
their
low-
whispered
call, they would
huddle
together
and
stretch
their
legs,
wings,
and whole
bodies
to
the
utmost. Then would arise
such a chorus of supplicat-
ing cries as no parent could
resist. Touch but a twig
and
the
nest
presents an
even
livelier
spectacle. The young
fairly tumble
over
each
other,
while
their
wings,
heads,
and
bodies
vibrate
with
an
intensity
of desire
which
their eager voices can only
feebly express. Two days
ago these young lay quietly
t to-day the instinct of fear
e nest and hid in the grass. h, in the crotch of a witch-
yed Vireos whose history
is
to the branch brought
off
t
t
th Fig. 38. Female Cedar-bird prepared to regurgitate tood from the gullet. Notice the outlines of the neck, which mark the full throat. "Twenty min-
utes later, the last fledgling had left the nest."
August 25,
1890,. Fig. 38. Female Cedar-bird prepared to regurgitate tood from the gullet. Notice the outlines of the neck, which mark the full throat. "Twenty min-
utes later, the last fledgling had left the nest."
August 25,
1890,. in their nest, and when touched showed absolutely no fear, but to-day the instinct of fear
had possessed them, and when approached, all hopped off the nest and hid in the grass. Another Waxwing family was discovered on August
I5th, in the crotch of a witch-
hazel bush seven feet up, in the same pasture with the Red-eyed Vireos whose history
is
yet to be told, and not many rods from their nest. A touch to the branch brought
off
the mother, who was brooding three tender young barely two days out
of the
shell- The Cedar
Bird. 57 After a short
interval, during
which
I went to get a notebook and pencil,
this bird was back
again, and once
more her jet-black eye and clean-cut
profile appeared
above the
nest. Fig. 42.
She hears a suspicious sound. Wild
Birds. With
half-spread wings and with back to the
sun the mother protected her little ones
for a
full hour from
the broiling
sun. while
her mate came
repeatedly
and
handed out the cherries. Fig. 40. After feeding the young
the gullet empty. Notice
the "sitting" posture, and com pare curves of throat in Fig. 39. The
Cedar-bird
will
jxint
with Wild
Birds. mouth
agape
when
uncomfortably
warm, but
is never seen
to erect the
feathers generally, as many birds do
in order to keep
cool. Nothing
es-
caped that came within range of their
sharp eyes and
bills. One
of the
photographs shows the male on the
farther side of the nest with cherry in
beak and
full neck, while the mother,
with back
to the camera,
gives her
neck a peculiar twist and
looks be-
hind her. While
I was watching the
performance, a bird of another spe-
cies, which I was unable to recognize,
dashed up, alighted for a moment on
the top of my tent, and
giving out a
harsh chatter, disappeared. Fig. 41. Regurgitating food. Up goes the head,
and presto
! out comes a berry. pp
One day in July
I happened to
see a Cedar-bird tugging at the frayed
ends of a cord which had been
fast-
ened to a branch
of one
of
the
fir Fig. 41. Regurgitating food. Up goes the head,
and presto
! out comes a berry. trees,
close by our house. Taking
the
hint
I placed
a quantity of red
and blue yarn on the branches, and
on some bean poles near the nesting
site. Every
thread was taken from
the fir and worked into what became
a very gay mansion. It was placed
on
a
spreading apple bough,
at
a
fork in the limb and between upright
shoots, fifteen feet from the ground. The blue yarn was
in
excess of the
red, but I am sure this meant nothing
to the birds. They simply took what
was provided, and had
all been
red,
it would have been accepted. Fig. 42. She hears a suspicious sound. These birds were most expedi-
tious, for in two days the last straw
was in
place, and
in
six days from
the start four eggs had been laid and
incubation begun. Ten
days
later
three of these eggs had hatched into Fig. 42. She hears a suspicious sound. The Cedar
Bird. 59 Fig. Wild
Birds. 6o the young gave the call-notes repeatedly, but the old birds usually approached without a
sound, and were never both at the nest at the same time. On the next day the mother
bird was feeding the young before
I
could
set
up the
tent. Both
birds
came frequently bringing black cher-
ries and grasshoppers. At each feed-
ing the following order of events was
usually observed
:
the
parent sounds
the call-note at a distance,
to
which
the young
reply, but
observes
strict
silence
in drawing near;
the
young
are
fed,
inspected, and
cleaned
;
the
old
bird
flies
to
a convenient
perch,
rubs the bill clean, plumes, and speeds
off to the nearest cherry
trees. Fig. 45. Devouring the excreta
:
an unusual attitude. In
the
course
of
the
forenoon
these fledglings became very
restless,
and as the heat from the sun increased,
one crawled
out,
sat
in
the shadow
of the leaves, and
finally dropped to
the
grass. Here
it was immediately
fed, and then hopped away surprisingly
fast. The male enticed
it
along, and
thereafter took care
of
it,
while the
mother returned to her remaining nest-
ling. Twenty minutes
later, the last
ng had
left the
nest,
never
to
return, and
the
curtain
was immediately rung
The young had spent exactly two weeks in their temporary home, and had the
r been cooler they might have tarried at least two days longer. . 45. Devouring the excreta
:
an unusual attitude. Fig. 45. Devouring the excreta
:
an unusual attitude. fledgling had
left the
nest,
never
to
return, and
the
curtain
was immediately rung
down. The young had spent exactly two weeks in their temporary home, and had the
weather been cooler they might have tarried at least two days longer. y
g
g
At this age the crest
is not very prominent, and
instead of the jet-black, triangular
band which surrounds the eye
in an
old
bird, the crown
of the head
is encircled by a
light band, passing above the eye. At the age of ten days, or a little earlier, the tubes
of the wing-quills burst, and the red wax-like tips of the secondaries, when present at
all,
also appear, or at least did appear in the young from this nest. Wild
Birds. 43. Cedar-bird listening intently while inspecting nest. young birds, while one was addled. Born
blind, naked, and helpless, the Cedar-bird
begins to see when three days old, through
narrow
slits which
gradually
open,
and
expose the eyes to full light. When this
nest was touched
the young would
raise
their tremulous heads
aloft, and with red
mouths opened
wide, express
in
silence
the simple sign language of newly hatched
birds. One of the brood mysteriously dis-
appeared,
so
that
eventually
only
two
were raised, and
this
recalls the
loss of a
young bird from
the
first
nest which was
built by the same pair. When evil befalls
a nestling,
the parents
either remove
its
body or abandon the whole
family. The
latter course
is seldom,
if
ever,
followed
after the eggs have
all been
hatched. gg
Bough and nest in
this case were
re-
moved on August
23d, when
the young
were between
eight
and
nine days
old. Fig. 43. Cedar-bird listening intently while inspecting nest. They were set up on a hillside, in an
exposed
position,
with
a
house on
one
hand
and
a
public
drive
and
monument on the other, but the birds
stood
it
well,
as
the
photographic
record shows. (Figs. 37, 38.) They were set up on a hillside, in an
exposed
position,
with
a
house on
one
hand
and
a
public
drive
and
monument on the other, but the birds
stood
it
well,
as
the
photographic
record shows. (Figs. 37, 38.) - 44-
Standing at inspection
:
a characteristic pose. (
g
,
)
Owing to
unfavorable
weather
the tent was not used until the after-
noon of August 25th. In a few min-
utes, the female was on
the nesting
bough, coming and going, but finally
kept
her
perch
and
examined
the
situation
critically. Something
un-
usual had
happened
full
of
signifi-
cance to
herself and
family, but
it
was an enigma hard to solve. Silence
at last brought assurance, as it usually
does in such cases. She approached
nearer,
pausing
at every
step, until
she could
no
longer resist the mag-
netic influence
of the calling young-
sters, who
fairly palpitated
in
their
eager desire for food. At
this
nest - 44-
Standing at inspection
:
a characteristic pose. Wild
Birds. The Cedar
Bird. 61 feathers has appeared
in
front of the eye and
replaces the fawn-colored
fillet
already
mentioned. This change takes place in about four days. g
The fourth and in many ways the
most
interesting nest was
built in a
pine, some account
of which lias
al-
ready been
given,
in
illustrating the
change of the nesting site. I watched
these birds over ten hours from
the
tent, saw
a
great many
interesting
sights, and made a long series of pic-
tures. Fig. 46. Cleaning the nest,
the old bird
is but half done. en the young are fed, the duty of sights, and made a long series of pic-
tures. The young at this nest were vis-
ited and fed forty-seven times during
an
interval of exactly ten hours and
forty-seven minutes, on
three
differ-
ent days. On the last day they were
fed on the average once
in
ten min-
utes. The food
consisted
of choke
cherries and red bird cherries, varied
with
raspberries,
blackberries,
and
blueberries,
together
with
insects
which, during the last days of life at
the nest, constituted about one quar
ter
of
the
fare. At
one
half
the
number of visits recorded, fruit alone
was
served. From
six
to
ten cher-
ries were brought
in
the gullet at a
time, and
once
by
count
eleven
blueberries. Feeding
was
effected
almost
always
by
regurgitation
in
whole
or
part, and
rarely was any
food
visible
when
the
birds
came to the nest. Now and then, however, a bird would approach loaded to the muzzle,
with a berry or insect in the
bill to round out the measure. Soft
fruits
like
raspberries
were
crushed
to
a
pulp, and
insects
which
are commonly served
with
the
berries,
came up covered with saliva, and
often
in
an
unrecognizable
state. The
staple animal
food was grasshoppers and
I have seen
the
large cicada or
harvest-fly brought
to
the
nest, but never
dragon-flies,
butterflies,
or moths. The cicada made
a
lively
struggle
for a few minutes;
it was placed in one open throat after another and withdrawn eight
different times, before a gullet was found capable of the proper reaction time. If a bird
was slow he lost his chance, and another was
tried. The key was
at
last
fitted
to the
lock, and the bruised and battered cicada was taken
in, but the old bird had not finished
her task. Wild
Birds. pp
,
pp
y
g
When about ready to fly and waiting to be
fed
the young have the peculiar habit
already
noticed
of standing
erect
with
upturned
heads. A
nest
of
these
birds,
in
this
attitude
makes
a
curious
picture. Any
danger
signal
is now
likely
to
bring
them off in an instant. This particular brood had their abode in a pine tree close to our
house. On July
1 7th, shortly before the picture was made, the family of five was stand-
ing bolt upright, all facing one way, as
if under military discipline. When
their branch
was touched all but the two shown in Fig. 1 17 gained the nearest trees in their first flight
and escaped. This pair came to the ground, and were replaced in the nest. In their second
attempt made ten minutes later, the larger of the two birds was more successful. It flew
to the roof of the barn, not far above it, and
after hopping
to
the
ridge-pole, made the
upper branches of a tall elm. In the larger of the two birds the black band
of
velvety The Cedar
Bird. The Cedar
Bird. The black cherry tree is a pleas-
ant sight, when laden with the pendant racemes of black cher-
ries, its tremulous foliage shining in the sun, with Robins and
Cedar-birds fluttering about
it. Every good tree is an aviary
when its fruit
is ripe in late summer and early autumn. Both
old and young are on hand. Then you may see one sidle along
a bough, stretch
its neck, wag
its
tail, and fondle another
with
its
bill. Their
fine
breezy
call-notes
suggest
the
bleating of the insects in the grass below. Tent
caterpil-
lars
spin
large
nests
in
these
trees,
but the
birds
prefer the
acid-bitter
fruit
to
the
insects. Occasionally a bird will leave its perch, and dive for an insect in the air with the
1 For this note I am indebted to Mr. Robert J. Sim, of Jefferson, Ohio. Fig. 47. Young Cedar-bird from
nest shown in
Figs. 39-46
: photographed on the morning of flight,
July 19, 1900. The bird was not touched, but occu-
pies a natural perch, chosen by himself. Fig. 48. Cedar-bird about thirty-six
hours old, blind, naked, and helpless
:
characteristic instinctive response to
any sound or vibration, as when the
parent
brings
food,
or
the
nest or
branch
is
tapped. Notice
that
the
bird rests on
its
pot-belly,
and uses
both wings and legs for support. En-
larged to life. g
Towards the
last
of
August,
small
flocks
of Cedar-birds
are
moving
about
in
search
of
food,
the low murmur of
their call-notes be-
i
audible
for
a Fig. 48. Cedar-bird about thirty-six
hours old, blind, naked, and helpless
:
characteristic instinctive response to
any sound or vibration, as when the
parent
brings
food,
or
the
nest or
branch
is
tapped. Notice
that
the
bird rests on
its
pot-belly,
and uses
both wings and legs for support. En-
larged to life. Fig. 47. Young Cedar-bird from
nest shown in
Figs. 39-46
: photographed on the morning of flight,
July 19, 1900. The bird was not touched, but occu-
pies a natural perch, chosen by himself. Fig. 48. Cedar-bird about thirty-six
hours old, blind, naked, and helpless
:
characteristic instinctive response to
any sound or vibration, as when the
parent
brings
food,
or
the
nest or
branch
is
tapped. Notice
that
the
bird rests on
its
pot-belly,
and uses
both wings and legs for support. En-
larged to life. Fig. 48. The Cedar
Bird. She began tossing up her head and producing
bird
cherries. Then
she gave
the nest a thorough renovation. In doing this the mother often
walks around
the
rim,
and attends to each nestling in succession, sometimes even inspecting one bird more than
once. Fig. 46. Cleaning the nest,
the old bird
is but half done. en the young are fed, the duty of Fig. 46. Cleaning the nest,
the old bird
is but half done. en the young are fed, the duty of At
first
I
found
it
difficult
to
tell
the
old
birds
apart
until
I
noticed
a
dis-
tinguishing mark
on
the
female, who had
a
little bare spot where the
feathers had Wild
Birds. 62 come out, on the
right
side
in
front
of the wing. This shows
plainly in many of the
photographs. come out, on the
right
side
in
front
of the wing. This shows
plainly in many of the
photographs. A As
I have said in another place, the female
would
often
fly direct to the tent and alight on
the end
of
the
ridge-pole
just above
the nest. Here she would pause a moment, then go to her
young. Should they
fail
to respond promptly,
she gives a peculiar clucking sound, a habit com-
mon
to many species, which
is the stimulus ap-
plied as a last resort. At this signal every mouth
is opened wide, even
if the gullet
is already
full. Indigestible substances pass through the alimen-
tary
canal, and
are never regurgitated in either
young or adults. Fig. 47. Young Cedar-bird from
nest shown in
Figs. 39-46
: photographed on the morning of flight,
July 19, 1900. The bird was not touched, but occu-
pies a natural perch, chosen by himself. y
g
Cedar Waxwings have been seen
in
the
act
of sipping maple sap
in March, either standing
near a broken
twig and reaching round
to pick
off the
drops from
the
underside
or hovering
over
the
spot
and
taking
sips
while
on
the
wing.' wing. Towards the
last
of
August,
small
flocks
of Cedar-birds
are
moving
about
in
search
of
food,
the low murmur of
their call-notes be-
ing
audible
for
a
moment
only
as
they
pass
over-
head. They know
when the wild cher-
ries
are
ripe, and
never
fail to
visit
the trees skirting the fields. The Cedar
Bird. Cedar-bird about thirty-six
hours old, blind, naked, and helpless
:
characteristic instinctive response to
any sound or vibration, as when the
parent
brings
food,
or
the
nest or
branch
is
tapped. Notice
that
the
bird rests on
its
pot-belly,
and uses
both wings and legs for support. En-
larged to life. RED-EYED VIREOS. TH THE
moment
I touched the spreading branch of a witch-hazel bush out
flew a bird,
and the next instant my eye rested on
the
nest
of
a Red-eyed
Vireo. It was
suspended between the forks of a twig about six feet from
the ground, and was
well protected and concealed by the leaves. It then contained two young birds, four or
five days old. After examining it carefully
I retired, but before doing so fixed a cord to
the branch and drew down the nest so that its brim was horizontal, and the whole about
four feet from the ground, a convenient height for future study. g
,
g
y
The young were quite naked, save for a sprinkling of light down on
their heads and
backs. They had yellow-rimmed
bills, bright yellow
throats, and were
just beginning
to see through the narrow vertical
slits, which admit light gradually to the eyes. The old
birds betrayed no unusual anxiety, but uttered
their unobtrusive piort ! piort ! and the
female soon approached with an insect. This nest was surrounded by
tall bushes with
barely space to pitch the tent in front of
it, and as
I decided to make no further changes,
a somewhat spotted leafy background was unavoidable in the pictures. Coming again on
July 3 1st, the tent was soon in place. The female, who was brooding
at
the
time, flew
off quickly, but returned
in a few moments. q
y,
These Vireos soon became quite unconscious of being observed, although literally as
near the eye as one would hold a book to read. I spent parts of three days on this spot
watching a most fascinating panorama of bird-life. On the third day the tent was moved
up to within eighteen inches of the nest, but my lack of experience at this time in photo-
graphing moving objects at such close range was the cause of many failures. g
p
g
g
j
g
y
On the first day
it required forty minutes to restore perfect confidence, or before the
affairs of the nest were conducted with
their
usual
regularity. The young
raised
their
heads aloft and called loudly for attention, or hung drowsily over the brim
of the
nest. The Cedar
Bird. 63 ease and precision of a professional fly catcher. I have seen the Cedar-bird either taking
the spider from his \veb or possibly robbing him of his prey. The birds peck at the cher-
ries, pull them off, suck up the juicy pulp, but drop the hard stone. The ground under
the trees, as well as beneath their favorite perches,
is covered with
cherry stones. Sud-
denly there is a swirl of wings, and the band moves off
rapidly to try the
fruit
in some
other quarter. ease and precision of a professional fly catcher. I have seen the Cedar-bird either taking
the spider from his \veb or possibly robbing him of his prey. The birds peck at the cher-
ries, pull them off, suck up the juicy pulp, but drop the hard stone. The ground under
the trees, as well as beneath their favorite perches,
is covered with
cherry stones. Sud-
denly there is a swirl of wings, and the band moves off
rapidly to try the
fruit
in some
other quarter. RED-EYED VIREOS. At this time their skin was dotted with the fine rapidly growing
feathers, and
the wing-
quills looked like slender paint brushes, having just burst the tips of the cylindrical horny
tubes in which they grow. y g
The old birds examined the situation carefully. Their mournful piort ! piort ! was
heard
again
and
again, the male answering
his mate
as she
deliberately approached
the
nest. After
advancing many
times, and
turning
back
as
often
through
fear
or
distrust,
the
mother hopped up
briskly with a bee
in
her beak. Her
instinct to
care for her young was stronger than the
male's, and she almost
invariably approached
in the same way, by the path of the twig in the fork of which hung the nest. A smaller
division in the
fork gave off a still smaller branch
close to the
nest, and upon
this the 64 Reel-Eyed
Vireos. birds always perched, and
thus stood
directly over
their brood. Any vibration of the
nest, as when the feet of the
old
bird touched
the main stem
to which
it was
fixed, or
any sound above or below electrified the young, and up popped their heads like two jacks
in a box. With mouths wide agape, they would clamor and quaver, expressing their emo-
tions not only by the
vibration
of the
wings but by the shaking of the whole body. But
the young at this tender age are unable
to discriminate with any exactness. The
f
or the birds always perched, and
thus st
nest, as when the feet of the
old
b
any sound above or below electrifie
in a box. With mouths wide agape
tions not only by the
vibration
o
But
the young at this tender age
quivering of a leaf, or the
stirring
"f a twig close
at
hand, a puff
of
wind, the
flutter of a wing or the
voice
of any
passing
bird would
throw them into the same state of
excitement. But
this was
only
for a moment. Their heads would
again drop
listlessly over the wall
of the nest, and with open mouths,
they would
cloze
in the sunshine. Something
would
then suddenly
arouse them, when they would in-
stinctively go
to preening them-
selves just like old birds, although
they had
at
this time no feathers
which seemed
to
need this atten-
tion. Fig. 49. RED-EYED VIREOS. Male Red-eyed Vireo standing
at nest after feeding
the
young. Life-Size x 3. Quite
often you
would
hear
a
/i it it' ! liuii
.'
which
always
aroused
the
young,
who
would
/.v//
1 / back in
earnest. While the
ni< "tlier was
again coming
slowly
towards the nest with a bee in her
mouth, another bee happened
to
cross her path. She darted after
it but missed her aim. Then, dis-
posing
of
the
first
insect,
she
watched
her young intently fora
moment,
stooped,
picked
up
a small white package, and hurried away. At one o'clock the
old
birds took a midday rest, and
it was
full twenty
minutes
before that reassuring piort ! piort ! was heard. Then as, step by step, the mother came
nearer the magnet, the drawing power of which was
irresistible, her
livelier huic ! hiiic ! awoke the young, who started and replied swit ! sicit ! Thereupon the old bird quickly
hopped along the branch, straddled the fork, and tucked a large grasshopper into one of
the open mouths. In
three minutes
she was back
with another, this time stopping to
clean the nest again. Five minutes by the watch had
passed when
she returned with a
brown gray-winged insect over an inch long, which an entomologist might be able to name
from the photograph. She paused
for
a moment while the young
called
eagerly and
stretched their necks to the utmost
; then she helped
the
insect down the throat of the
Fig. 49. Male Red-eyed Vireo standing
at nest after feeding
the
young. Life-Size x 3. Quite
often you
would
hear
a
/i it it' ! liuii
.'
which
always
aroused
the
young,
who
would
/.v//
1 / back in
earnest. While the
ni< "tlier was
again coming
slowly
towards the nest with a bee in her
mouth, another bee happened
to
cross her path. She darted after
it but missed her aim. Then, dis-
posing
of
the
first
insect,
she
watched
her young intently fora
moment,
stooped,
picked
up
a Fig. 49. Male Red-eyed Vireo standing
at nest after feeding
the
young. Life-Size x 3. moment,
stooped,
picked
up
a small white package, and hurried away. At one o'clock the
old
birds took a midday rest, and
it was
full twenty
minutes
before that reassuring piort ! piort ! was heard. RED-EYED VIREOS. Then as, step by step, the mother came
nearer the magnet, the drawing power of which was
irresistible, her
livelier huic ! hiiic ! awoke the young, who started and replied swit ! sicit ! Thereupon the old bird quickly
hopped along the branch, straddled the fork, and tucked a large grasshopper into one of
the open mouths. In
three minutes
she was back
with another, this time stopping to
clean the nest again. Five minutes by the watch had
passed when
she returned with a
brown gray-winged insect over an inch long, which an entomologist might be able to name
from the photograph. She paused
for
a moment while the young
called
eagerly and
stretched their necks to the utmost
; then she helped
the
insect down the throat of the ,
stooped,
picked
up
package, and hurried away. At one o'clock the
old
birds took a midday rest, and
it was
full twenty
minutes
before that reassuring piort ! piort ! was heard. Then as, step by step, the mother came
nearer the magnet, the drawing power of which was
irresistible, her
livelier huic ! hiiic ! awoke the young, who started and replied swit ! sicit ! Thereupon the old bird quickly
hopped along the branch, straddled the fork, and tucked a large grasshopper into one of
the open mouths. In
three minutes
she was back
with another, this time stopping to
clean the nest again. Five minutes by the watch had
passed when
she returned with a
brown gray-winged insect over an inch long, which an entomologist might be able to name
from the photograph. She paused
for
a moment while the young
called
eagerly and
stretched their necks to the utmost
; then she helped
the
insect down the throat of the Wild
Birds. 66 lucky bird. However,
it
stuck
at the gullet, and the
little one
gulped hard before its protrud-
ing wings had disappeared. Fig
. 50. Female Red-eyed Vireo ready to deliver a large insect. g
g
As is well known the young
bird
has
wonderful powers
of
digestion and
assimilation, and
after the first week the rapidity
of
its
development
becomes
even more striking. A lapse of
twenty-four hours now means a
great stride in growth. It takes
food almost constantly through-
out the day, and digests it quick-
ly, though
imperfectly. RED-EYED VIREOS. The
adult Vireo
like the Flycatcher
is
said
to
regurgitate the indi-
gestible
parts
of
its
food
in
pellets. Fig
. 50. Female Red-eyed Vireo ready to deliver a large insect. Fig. 51. Placing
it well down in a hungry throat. pellets. The
male
Vireo
seldom
came
with
food,
and then
al-
ways with an extra degreeof cau-
tion. Twice he followed swiftly
after his mate, acting as herguar-
dian while she quickly went the
rounds. The
role
of
the
old
birds
in
feeding was almost
in-
variably the same, as
I have in
part
described. They
trace
a
zigzag line to the nest, a straight
one
from
it. You
hear
first
their responsive call-notes. The
mother
bird with
insect ready
is
in a bush
a rod away;
then
she comes a step nearer, and pau-
ses
; her piort ! is now more dis-
tinct. She slowly advances, until
the twig which
holds the
nest
is touched. Up go the heads
of the young
; they call
aloud,
stretch
their
necks
to
every
side, gaze up to the clouds and
around upon the leaves. Then
as
the
mother
hops
nimbly
along the twig, and Stands OVCr Fig. 51. Placing
it well down in a hungry throat. rig. 52. Standing in characteristic prone attitude of inspection. rig. 52. Standing in characteristic prone attitude of inspection. rig. 52. Standing in characteristic prone attitude of inspection. Red-Eyed
Vircos. 67 them, what
a picture
of
desire,
tremulous
impatience,
and keen
rivalry they present
! The food
is sometimes quickly
placed
in the throat of one, and
as quickly withdrawn to be giv-
en to another, and when
there
are more than two
it may go
tlu- rounds
before
it
is allowed
to
remain,
a common practice
the
true meaning of which we
have already seen. rig. 53. Male Red-eyed Vireo who
is less preoccupied
in performing
the same duty. After
inspection
is
com-
pleted and the nest cleaned, the
parent bird
flies to any conven-
ient
spot,
carefully
wipes
the
slime
from
her
bill,
stretches
her wings, and smoothes out all
the ruffles in her dress. These
birds always look as sleek as a
new silk hat, every feather lying
smooth in
its place. rig. 53. Male Red-eyed Vireo who
is less preoccupied
in performing
the same duty. p
g
the same duty. Fig. 54-
Female Red-eyed Vireo approaching the young. RED-EYED VIREOS. The male warbled his
pleasant strains from a branch
hard
by,
while
the
mother
hunted
for
insects
in
the
grass below. A large brown
locust with yellow and black wings was soon
brought
in. The adult Vireos glean most
of their animal food from the foliage and, as might be expected, are great caterpillar de-
stroyers, but while feeding their young, I frequently saw them exploring the grass as any
Robin or Song Sparrow might do, snapping up every insect which came in their path. Fig. 56. Bending over to feed young. Fig. 56. Bending over to feed young. Fig. 56. Bending over to feed young. grass
large
locust with yellow and black wings was soon
brought
in. The adult Vireos glean most
of their animal food from the foliage and, as might be expected, are great caterpillar de-
stroyers, but while feeding their young, I frequently saw them exploring the grass as any
Robin or Song Sparrow might do, snapping up every insect which came in their path. g ass
a ge
locust with yellow and black wings was soon
brought
in. The adult Vireos glean most
of their animal food from the foliage and, as might be expected, are great caterpillar de-
stroyers, but while feeding their young, I frequently saw them exploring the grass as any
Robin or Song Sparrow might do, snapping up every insect which came in their path. g
p
g
pp
g
p
y
p
On the third day, when my tent was but eighteen inches from the nest, the old birds
came to
it even more
readily than
before. They would
still occasionally start
at the
click of the shutter, but they did not mind
the shrill scream of a locomotive across the
river, or the rumble and
splash
of logs which were momentarily being set free and sent
tumbling headlong down a steep slide
into the
river below. They had become used to
these sounds and had learned from experience that they were harmless. On
this day, a
great change seemed
to have come over the young. They had become almost
trans-
formed
in appearance, and were very restless. Their bodies were now well covered with
feathers, and they were beginning to show the
first
traces
of
fear. Their snow-white
breasts gleamed through the
thin
walls
of
their cup-shaped
nest, or from over its rim. RED-EYED VIREOS. One day while in my tent,
a small bird
of another species
suddenly
darted
down
upon
this nest. There was a momen-
tary
flutter,
a
clash
of
beaks
and claws, and the intruder was
promptly driven away. Fig. 54-
Female Red-eyed Vireo approaching the young. Fig. 54-
Female Red-eyed Vireo approaching the young. p
p
y
y
It
was
always
interesting
to watch the behavior
of
the
young between the intervals of
feeding. The moisture would
fairly glisten in their wide-open
mouths. They snapped at every
ant and flying insect which came
within
their
reach, but
I never
saw a single capture. The prey-
ing instinct
is undoubtedly one
of
the most ancient amontj an-
o
imals,
and
young
birds
peck
instinctively at all kinds of small
objects,
but
precision
of
aim
which
leads
to
success
in cap-
turing
their prey must
be
ac- y
pp
g
y
g
Fig. 55. Drawing back through timidity. Fig. 55. Drawing back through timidity. Fig. 55. Drawing back through timidity. Wild
Birds. 68 quired
by practice. These young Vireos would
often hang their heads down over the
nest, and doze until aroused by the piping of the Robin, or by the call of some other bird. Then the mother would appear, with a huge green katydid,
its wings crumpled and
held
tightly in her sharp bill. It was surprising how quickly and gently
it was assisted down
one of the hungry throats. hungry
At one
of
his
visits the
male, after cleaning the
nest and young with
great
care,
stepped
in and
settled down
to brood. In a moment two downy heads shot up from
under
his
breast,
and
I
regretted
that my camera was
not loaded
at
the moment. He showed
unmistakable
signs
of
displeasure
or uneasiness,
repeatedly
erecting and
lowering his crest, and puffing out his throat. With mouth wide open he gazed keenly
about him, and after a few moments dashed off as
if in pursuit of an enemy. h When
a
large grasshop-
per which had been given to
a young bird had made good
its escape, the mother darted
after it, seized
it before it had
touched the ground, and you
may be
sure
that
there was
no possibility
of escape
this
time. A
grasshopper
was
sometimes
divided
between
the two young, but usually a
single bird only was fed
at a
time. RED-EYED VIREOS. Grasshoppers, katydids, green larvae, beetles, and bugs of many kinds were served again
and again, but
it would be a mistake to suppose that there was no fruit to vary this diet. Upon the
third day the mother brought a ripe red
raspberry,
its juice
fairly streaming Red-Eyed
Vireos. 69 down her
bill, and
after a few beetles had been
taken, she appeared with a large black-
berry. Fruit was served to the young about
half
a dozen times
in the course of
four
hours during which watch was kept on
this
particular day, but
I had
not seen a single
berry brought to the young before this time. down her
bill, and
after a few beetles had been
taken, she appeared with a large black-
berry. Fruit was served to the young about
half
a dozen times
in the course of
four
hours during which watch was kept on
this
particular day, but
I had
not seen a single
berry brought to the young before this time. y
g
y
g
On the first two days of observation the young were fed on
the average of once
in
fifteen minutes, but upon the third day food was brought every nine minutes. p
y
g
Hitherto
I had taken
pains not
to touch the nest, but
as
I approached
for a
final
look at the young at about two o'clock they immediately took alarm, and popped out one
at a time. The
larger of the two disappeared, and was never seen
again by me, and
although
I replaced the smaller bird in
its
nest
time
after
time,
it
positively refused
to
stay. Like the young of so many wild birds, when once they have tasted the freedom of
the world, they seem to look with disdain upon
their old home. Although
these
birds
could only
flutter
in
their
first
attempts
at
flight,
they
could
hop
nimbly
from
branch
to
branch, and thus ascend readily
to the tops of high bushes. fig- 57. Inspecting cautiously,
which express no fear. Compare such attitudes with Figs. 50-53, Upon
visiting
the
site
of
this nest on
the
following day
one of the young birds was dis-
covered
in
the
grass
less than
two
rods from
its empty
nest. It was
calling loudly
for
food,
and the old birds were tending
it. RED-EYED VIREOS. A few hours later I returned
in the
nick
of time
to save
its
life by the
capture
of
a
large
garter snake which in some way
had discovered
its opportunity. fig- 57. Inspecting cautiously,
which express no fear. Compare such attitudes with Figs. 50-53, pp
y
During the
past
summer,
a
Yireo's
nest was found on
the twenty-eighth of June, when the
female was
incubat-
ing two eggs. Her plans were, however, suddenly interrupted, apparently through her
own carelessness. A storm soon ripped up the nest, the
walls
of which were unusually
weak and
fragile, and
the eggs were
spilled. This nest was apparently the first
of the
season, and might have
represented
the
first attempt
of
a young
bird. There
is the
possibility, however,
that this was really a second and hurried attempt
at
nest-building. due to a former accident. p The snow and storms of winter usually knock the bottom out of the Vireos' pendant
.nests, but some remain whole for over a year. Wilson speaks of finding the
nest
of
the
Yellow Warbler built inside of an old Vireo's
nest. The deer mouse sometimes
takes
possession
of an abandoned
nest in
fall, and converts
it into a snug
globular house
for
itself and young. I remember the feeling of astonishment which the discovery of one of
these converted nests gave me when a boy at school, and of wondering
to what
animal
those black lustrous eyes, which appeared at the entrance, could belong. In this case the
original framework was concealed
by a symmetrical dome
of
thistle-down, a substance Wild
Birds. Wild
Birds. ;o used also in lining and covering the original walls. There was a small round hole or side
entrance, just above the old rim. When
disturbed this sleek little mouse left its warm
house, ran down the branch and disappeared. used also in lining and covering the original walls. There was a small round hole or side
entrance, just above the old rim. When
disturbed this sleek little mouse left its warm
house, ran down the branch and disappeared. Fig. 58-
Young Red-eyed
Vireos from the nest shown on page 68. No. 12 of table, p. ii. Fig. 58-
Young Red-eyed
Vireos from the nest shown on page 68. No. 12 of table, p. ii. THE NEST-HOLE OF THE BLUEBIRD. Wrens and
Martins
are easily driven off, but the
pugnacity
of
the
Sparrow, and
the
greater
numbers
which
he
can
usually muster
^^
^^
render
all
resistance
hopeless. An
f
-^JfJjH^VJy ^$B&
abandoned Woodpecker's hole
is not
m&*'
\&'' JK
'
disdained since
it forms a safe, cozy
H^^JH
B^i^^SHi
^
house which needs
little
furnishing. 7
JtiflttKi
This
snug cavern
is
sheltered
from
^Bfll
,
sun and
rain, and
secure from most
Jim
'
birds and beasts of prey. The rotten
fence-post, and the many holes in the
decayed apple trees may also contain
the secret of the Bluebird's nest. f
-^JfJjH^VJy ^$B&
m&*'
\&'' JK
'
H^^JH
B^i^^SHi
^
7
JtiflttKi
^Bfll
,
Jim
'
Fig. 60. Female Bluebird carrying grasshopper to young. On August
1
1,
1899,
I saw a pair
of
Bluebirds
paying
marked
atten-
tions
to an
old "auger-hole"
in an
apple
tree, made by Golden-winged
Woodpeckers. It was plainly a case
of nest within nest. The female was
carrying
insects
to
her
invisible
young, which
I supposed at this late
date were ready
to
fly,
but, as
it
af-
terwards
appeared, they were
only
five days
old. This
hole had
been
nicely
drilled beneath the springing
branch of a truncated and now dead
prong of the
tree,
fifteen
feet
from
the ground. g
When the opportunity first offered
on August
1 5th,
I sawed off the limb,
two feet from the opening, and set
it
up
in
a
convenient
spot
fifty
feet
away. It was so arranged
that
the whole
trunk
could
be
rotated, and
the
circular
entrance
of
this
nest
turned
directly
to
the sun
at any time
of
day. I had
barely
left
the
place
to
fetch
the
tent when
the mother
bird
flew
from
the
apple
tree
to
the stump, entered the
hole, and having
fed the young, came
out with a small, white
parcel
in her
bill. This bird had her eye on the nest, and was ready to
visit
it
in
its
new
situation, when
free
to do
so. The
tent was
placed
two
feet
away,
but
later
drawn up
to a distance of about eighteen
inches. After concluding these operations,
I had
to wait
longer
for the parent
bird
to come
again. When one
considers
that
the
nesting
branch
was
suddenly
moved
fifty
feet
from
its
original
position
and
1 Bulletin Xuttall Ornithological
Cliit',
vol. THE NEST-HOLE OF THE BLUEBIRD. THE
mellow note of the Bluebird
is a welcome sound on March mornings when the
air
is yet wintry, and the snow stands deep in the woods. Its meaning is unmis-
takable, but to
appreciate
it, one must
live
in
the North where
spring means
literally
'' turning over a new
leaf," a new order of
existence. Should
cold weather or
heavy snows
return, the
birds
retire
for
a
time,
but promptly
re-appear
with better days. TH Fig- 59-
Female Bluebird taking a look outside, as if hesitating,
before going in search of food. y
Robins,
Song
Sparrows,
Blue-
birds, and Phcebes all arrive from the
South during the latter part of March,
and
the
personalities
of these
birds
are too well marked
to be mistaken. On
March
24th,
I
heard
a bird call-
ing
from
a
distant
apple
orchard,
when
it
presently
flew
in my direc-
tion, alighted on
an elm beside the
road, and repeated
its low sweet call-
notes again and again. Through the
mist
not a feather could be seen, but
there was no mistaking this
plaintive
voice. Five days earlier in the month
the
Bluebird was seen at
Northfield,
thirty miles to the south. The males
are first to arrive, coining singly or in
small
straggling companies. As we
walk
along
the
desolate
country
roads, they rise from wall and fence-
row,
displaying
their
brilliant
azure
wings,
or when
flying
overhead
the
cinnamon brown and white of their und
note which
is heard as they fly
is not p Fig- 59-
Female Bluebird taking a look outside, as if hesitating,
before going in search of food. y
y
p
y
When the females come
a
little
later, the males are
in
full song, and the period
of
courtship, which
is very ardent in the
Bluebird, begins. The
affection and
gallantry
of Wild
Birds. the
Bluebird have aroused
the enthusiasm
of many observers. Unfortunately, we
are
obliged to add that a case of polygamy in this species has been reported.' obliged
polygamy
spec es
p
The choice of a nesting site is made with great care and deliberation. If they accept
the house or box prepared
for them, they often have to defend
it against the Wren,
the Martin, and the House Sparrow. THE NEST-HOLE OF THE BLUEBIRD. Female Bluebird about
to
enter nest-hole with green y
them. These actions were repeated by
both
Bluebirds many
times,
while
they
ultered
their
responsive phee-
nr
note. Again,
calling
eagerly,
both would
fly towards the new po-
sition
of the
nest. Finally, the
fe-
male, who
in
this case assumed the
whole
task
of
feeding
the
brood,
came to the
stump,
paused
a
mo-
ment, quickly entered
the hole and
came out in hot haste. The absolute
stillness, however, had restored confi-
dence,
for
in
five
minutes
she
re-
turned with a huge green grasshopper
and
in
ten minutes was back
again
with
another. In
the
course
of
each
visit
the
plaintive
call would
announce
her
presence
as
she
ap-
proached
with
insect
in
bill,
and
alighted on
a
half-dead peach
tree
close by. After a momentary survey
of the situation she would
flit to the
stump, sit for a few seconds on a dead branch at one side, then hop down, fly to the hole and
catch on the bark or cling to the rough edge of the circular opening with her sharp claws,
pausing there a tenth of a second, or long enough to
cast a swift glance backwards. In
this position she was photographed many times, with grasshoppers, crickets, green larvae,
katydids, and once with a large robber fly in her beak, the profile of her head being sharply
vignetted by the dark circular entrance. The young must have been all a-quaver at the
sound of their mother's wings, for the old stump seemed to become suddenly
alive with
brisk chirping sounds the moment she touched any part of
it. The bird used her tail to
Fig. 61. Female Bluebird about
to
enter nest-hole with green These actions were repeated by
both
Bluebirds many
times,
while
they
ultered
their
responsive phee-
nr
note. Again,
calling
eagerly,
both would
fly towards the new po-
sition
of the
nest. Finally, the
fe-
male, who
in
this case assumed the
whole
task
of
feeding
the
brood,
came to the
stump,
paused
a
mo-
ment, quickly entered
the hole and
came out in hot haste. The absolute
stillness, however, had restored confi-
dence,
for
in
five
minutes
she
re-
turned with a huge green grasshopper
and
in
ten minutes was back
again
with
another. THE NEST-HOLE OF THE BLUEBIRD. viii
p
63
Fig. 60. Female Bluebird carrying grasshopper to young. Fig. 60. Female Bluebird carrying grasshopper to young. The Nest-Hole of the Bluebird. fixed
on
the ground, and
that
a
tent
was then
pitched
so
close to
it
that the
birds
could
not
fly straight
to the entrance but had
to
flit
first
to
the
trunk, and then go
around to the hole,
it
is not surprising that they held aloof. I
waited
exactly one hour
and twenty-five minutes before the mother again brought food to her young. Meanwhile
it was interesting to see what was happening, from a peep-hole
of the
tent. Both
birds
would fly to the tree which they had known as their home, and mechanically go through
their usual
motions
in
approaching
the nest, hopping first to this branch,
then to that, following a well-defined
path, which they had
traveled hun-
dreds of times, and finally hover over
the spot which was once occupied by
the
nest,
as
if
to
become
assured
that
their
eyes
had
not
deceived
them. fixed
on
the ground, and
that
a
tent
was then
pitched
so
close to
it
that the
birds
could
not
fly straight
to the entrance but had
to
flit
first
to
the
trunk, and then go
around to the hole,
it
is not surprising that they held aloof. I
waited
exactly one hour
and twenty-five minutes before the mother again brought food to her young. Meanwhile
it was interesting to see what was happening, from a peep-hole
of the
tent. Both
birds
would fly to the tree which they had known as their home, and mechanically go through fixed
on
the ground, and
that
a
tent
was then
pitched
could
not
fly straight
to the entrance but had
to
flit
f
around to the hole,
it
is not surprising that they held aloo
and twenty-five minutes before the mother again brought
it was interesting to see what was happening, from a peep-
would fly to the tree which they had known as their home
their usual
motions
in
approaching
the nest, hopping first to this branch,
then to that, following a well-defined
path, which they had
traveled hun-
dreds of times, and finally hover over
the spot which was once occupied by
the
nest,
as
if
to
become
assured
that
their
eyes
had
not
deceived
them. Fig. 61. THE NEST-HOLE OF THE BLUEBIRD. In
the
course
of
each
visit
the
plaintive
call would
announce
her
presence
as
she
ap-
proached
with
insect
in
bill,
and
alighted on
a
half-dead peach
tree
close by. After a momentary survey
of the situation she would
flit to the
stump, sit for a few seconds on a dead b
catch on the bark or cling to the roug
pausing there a tenth of a second, or
this position she was photographed m
katydids, and once with a large robber
vignetted by the dark circular entranc
sound of their mother's wings, for the
brisk chirping sounds the moment she Fig. 61. Female Bluebird about
to
enter nest-hole with green Wild
Birds. 74 help support
her weight against the
side of the
tree, like a Woodpecker,
and
I
noticed
that
the tail feathers
were frayed and worn at the points. were frayed and worn at the points. The male during the numerous
visits which
followed came two
or
three times and
sat above the
door,
but
never actually
entered
it, and
never brought
to the young a single
morsel of food
in the course of the
entire day. He would warble very
sweetly, however, and
probably
en-
couraged the exertions of
his mate. The
next time
this
bird
appeared
with a grasshopper she did not trust
herself
inside,
but stood
at the
en-
trance, put her head in and as quickly
drew back
to
take another
glance
around, then leaned far down and fed
her clamoring brood. When she came
again,
I made a picture of her as she
stood
at the
hole, and
in
so doing
frightened her off, but she was back
in an instant, and another picture was
secured as she left the nest. At this
moment a flock
of Goldfinches
flew
overhead, and were heard
calling
/;<-
b? ! he-be f,
at which the young Blue-
birds
were
instantly
aroused,
and
made the old stump resound
again
with theircries. After many grasshop-
pers andcrickets had been dispatched,
a hairy robber
fly, or Asilus already
mentioned,
was brought
in. Then
another bright green katydid, with its
wings half spread in
its vain effort to
get free, was served to the young. If
frightened in an attempt to enter the
nest
this
bird
invariably
returned
shortly, and
after the
feeding
was
over, would take the excreta, and
fly
some distance before dropping it. THE NEST-HOLE OF THE BLUEBIRD. In
no case was it known to be eaten at
the nest. During the afternoon, when
these birds had become more at ease Figs. 62, 63, 64. This series represents the Bluebird engaged
in
cleaning her nest on three distinct
visits, at each of which food
was served. Nearly one half
life size. Figs. 62, 63, 64. This series represents the Bluebird engaged
in
cleaning her nest on three distinct
visits, at each of which food
was served. Nearly one half
life size. Figs. 62, 63, 64. This series represents the Bluebird engaged
in
cleaning her nest on three distinct
visits, at each of which food
was served. Nearly one half
life size. The Nest-Hole of the Bluebird. The Nest-Hole of the Bluebird. 75 in their new surroundings, the nest was cleaned six
times
in
two hours. I saw this bird
bring
to
her young no
less than twenty grasshoppers, four cone-headed katydids, two
black
crickets, besides larvae and many small
insects. During the forenoon, in the space
of nearly three hours, the young were fed on the average of once in six
minutes, and for
two hours in the afternoon once in nine and a half minutes. in their new surroundings, the nest was cleaned six
times
in
two hours. I saw this bird
bring
to
her young no
less than twenty grasshoppers, four cone-headed katydids, two
black
crickets, besides larvae and many small
insects. During the forenoon, in the space
of nearly three hours, the young were fed on the average of once in six
minutes, and for
two hours in the afternoon once in nine and a half minutes. The history of this interesting nest came to an unfortunate close, though through no
fault of mine. The old
birds were subsequently frightened away, and
their
five young
ones left to perish. The young were not quite three inches
long, and
less than a week
old. They had yellow
skins, and
bright yellow mouths, and
there was a sprinkling of
plumbeous down on the head, back, and shoulders. p
Toward evening on March 22d of the present year
I
saw a male Bluebird
sitting
comfortably in an old Robin's nest, having apparently settled down to spend
the
night
there. The Bluebird
is one of the most unobtrusive of wild
birds. It goes about
its busi-
ness quietly, and seems never to fight, except in defense of its home. According to one
authority, there are usually three broods, and
before the
first
set
of young can
shift for
themselves the female repairs the nest and gets ready for the second. The male continues
to care
for the
first brood
after the second has appeared, will
feed
his mate, and even
take her place at the nest. Fig. 65. Standing at entrance with large grasshopper in bill. Fig. 65. Standing at entrance with large grasshopper in bill. MINUTE OBSERVATIONS ON CATBIRDS. W W
and hard
HILE the Catbird has a strong attachment for its young, especially during later
days of life at the
nest, when any intrusion will arouse its fighting instinct to
the highest pitch, it is under ordinary conditions exceedingly wary, suspicious,
to approach. In the account which follows
I shall describe only what was seen
while camping beside two nests of these
birds. Fig. 66. -the Aesc
Female Catbird bringing in a large limp dragon-fly
na 'hens The
first
of
these
attractive
nests
rested on a spray of the sweet viburnum,
in a little clearing
in dense bushes, and
about four feet from the ground, so that
no change in
its position was necessary. It contained
a
single addle egg and two
young with the feather-shafts of the wings
barely exposed. y
p
For an hour or more
after the tent
was in position, the old
birds kept up a
perpetual din, in which
their exasperat-
ing tsliaying note was most pronounced. They would circle round and round the
tent, often coming close as
if to discover
the way
in, or fluttering and screaming
at
it, as
if
it were a demon
to be exor-
cised. After this they gradually became
more quiet, and began
to
alight on the
tent's guys and roof. At last the female
was
seen
stealthily
to
approach
and
quickly feed her young. After a fresh re-
connaissance both birds went to the nest
together and
with
rapid,
jerky
move-
ments stuffed
red cherries into the
hun-
gry
throats, inspected and cleaned each
young bird, and then darted away. While in a
state of mind wavering
,
r
. ,
/-
.. ,
between
fear and
the Catbird y
g
y
While in a
state of mind wavering
,
r
. ,
/-
.. ,
between
fear and assurance, the Catbird Fig. 66. -the Aesc
Female Catbird bringing in a large limp dragon-fly
na 'hens 76 76 Catbirds. ing the
tail pauses
in atbirds. g the
tail pauses
in Minute Observations on
Catbirds. 77 Minute Observations on
Catbirds. 77 passes
rapidly
to
a branch, and spreading and pumping the
tail pauses
in an attitude
of attention before making another movement. g
Both birds no\v began to bring a
lost time. The female came with two
cherries in her bill and promptly gave
one to each of the two
birds. MINUTE OBSERVATIONS ON CATBIRDS. bc-be of the Goldfinches could
be heard as these birds passed leisurely overhead. The
conditions were all reassuring, and presently the Catbirds became silent, and went off for
food. In a few moments a rustling of leaves was heard
close
to the tent and the male
could be seen coining boldly in its direction. bc-be of the Goldfinches could
be heard as these birds passed leisurely overhead. The
conditions were all reassuring, and presently the Catbirds became silent, and went off for
food. In a few moments a rustling of leaves was heard
close
to the tent and the male
could be seen coining boldly in its direction. g
y
Up
to this time the young lay quietly in
the
nest, but were
alert to every sound,
whether from the wind or any passing bird. Their wing-quills had become exposed in
the course of two days to a length of three quarters of an inch. y
g
q
Suddenly a jubilant song burst forth from the throat of the male, and his mate thus
encouraged approached the nest with
insect
in
bill, but her
fears were not allayed, for
after beating about she swallowed the insect herself and went in search of another. g
The young now began to yip in earnest and to stretch their scantily feathered trans-
parent necks. One of the lustiest of the
four even climbed to the edge and
sat in the
shade. They would erect their scanty crest-feathers and pant in the sun, which though
not
excessively
hot,
was with the added
feeling
of hunger,
beginning to make them
restless. The sense of fear was at last overcome in the mother, who came, fed and cleaned the
young, and flew off again. After another pause a huge dragon-fly was brought to the
nest. The observer had to wait long at the beginning, but his reward was now quick in
coming. The young were then fed every five or six minutes, but the male only rarely
went to the nest himself. Still cautious to a
degree, he would follow after the female,
but stop a few feet short of the nest. Then after delivering her insect she would go at
once to her mate, take the food from his bill, and bear
it to the young. MINUTE OBSERVATIONS ON CATBIRDS. Then
a grasshopper was
served,
and
still
again
a dragon-fly, with
blue body
and
spotted
wings
(the
Libcllula
pitlcliclla
}. The insect was swallowed
wings and
all,
but
only
after pro-
longed
efforts. As confidence was
gradually regained,
the
birds would
remain longer and longer at the nest,
pick
the young
all
over,
and
clean
everything
with
care
and
delibera-
tion. F ' g
' 67 '
Female Catb ' rd insP"Un e aftcr havine fed the >"> un e- At this time (July 23d) the young
were about eight days old, and could
be easily approached. Two days later
when
their nest was
touched, they
tumbled out
in an instant, disappear-
ing as
if by magic amid the leaves. I
succeeded
in
finding one of them,
but
it refused
to remain
in
the old
nest. Its wing-quills now showed a
half inch of the
feather-shaft, which
represented two days' growth, while
the tail feathers were still in the stub-
brush stage. There were
four young
in the
second nest, which was discovered in
some bushes
close to the river bank
F ' g
' 67 '
Female Catb ' rd insP"Un e aftcr havine fed the >"> un e-
on the
nineteenth
of June. It
rested
in the crotch formed by the crossing of shoots of
the dogwood and alder. The young were in pin-feathers, but not a tube had burst. Both
old birds happened to be off
foraging, but quickly returned with
food
in their mouths,
and besjan to alarm the neighborhood. O
C3 F ' g
' 67 '
Female Catb ' rd insP"Un e aftcr havine fed the >"> un e- The tent was pitched in front of this nest at eight o'clock on the morning of June 23d. After
it was closed both birds began their cautious explorations in the vicinity, tschaving
incessantly
and
with
nerve-rasping vehemence. A male Redwing Blackbird was soon
attracted to the spot, and added his note of alarm to the general outcry, but after finding
that the matter did not concern him, returned to his nest in the flags farther away. g
y
In twenty minutes the Catbirds had become more
quiet, and began
to pay close
attention
to
the tent. The Redwing was heard
con-quer-eeing in
the
distance. Song
Sparrows were singing merrily. Veeries called from
the woods
close
at hand, and
the Wild
Birds. MINUTE OBSERVATIONS ON CATBIRDS. y
g
The following table gives the number
of
visits
at which food was brought during
eight consecutive hours from 8 A.M. to 4 P.M., and
illustrates how the parental instincts,
nided by habit, gradually overcome the feeling of fear in a very shy and suspicious animal. HOUR. Minute Observations on
Catbirds. 79 ing to the utmost limit
tliL'ir transparent red necks display the yellow target of the open
mouth as they tsit ! tsit ! to the approaching mother, who sounds her well-known
call. ing to the utmost limit
tliL'ir transparent red necks display the yellow target of the open
mouth as they tsit ! tsit ! to the approaching mother, who sounds her well-known
call. y
pp
g
On one occasion
I saw
the female deliver a blac
from the
bill of the male, who was standing near, a carri
Then
keenly
eying her brood, she
deliberately
bent
over, and
as the
body of one was raised took from
it
a small white package and flew away. Many
of the photographs show the
birds performing this
sanitary
act, a
practice common to many other spe-
cies. During her first visits the female
ate the excreta, but thereafter
it was
invariably removed from the nest. h'lg. 68. Female Catbird cleaning the nest. y
The food served to these young
Catbirds
consisted
of
dragon-flies,
which were brought to the nest thir-
teen
times,
insect
larvae,
beetles,
moth
millers, and
a great variety of
smaller
insects,
varied
with
liberal
courses of strawberries. At
first the
old
birds
approached
quietly,
fed
theiryoung hurriedly from the farth-
er side, and were off in a few seconds,
but as confidence
in their surround-
ings
was
gradually
restored,
they
would come
to
the nest-front, with
the camera but three
feet away,
re-
main there for a full minute, and after
assisting the young to dispose of their
harder
subjects,
inspect
everything
with the greatest care. h'lg. 68. Female Catbird cleaning the nest. g eatest
When
this
nest was visited two
days
later the young looked bright
and hearty. They were now in
full feather, and about ready for flight. When the tent had
been cautiously set up,
I noticed that
a number
of
leaves
cast undesirable shadows on
the nest. Though knowing well what to expect,
I decided
to take the risk, and reached
out to cut them
off. MINUTE OBSERVATIONS ON CATBIRDS. This was the fatal spark which fires the train of gunpowder, for all
went off in an instant in a panic of
fear, and
the game was up, for Catbirds when
well
out of their nest at this stage are out for good. h'lg. 68. Female Catbird cleaning the nest. THE REARING OF THE NIGHT HAWK. IN
crossing a clearing one day
in June
I flushed a Night Hawk, who showed by her
behavior that the
little depression from which she rose contained something of great
interest to both the bird and
myself. She was indeed incubating a single marbled
gray egg, which lay on a marbled gray patch of earth still covered with ashes and cinder. The bird retired quietly, dropping with a thud to the ground a few feet away. IN d
away. ght Hawk cracked
his
shell
seen on the twenty-sixth
of
attened ball of fluffy worsted,
of a dark cream
color mot-
tled
with
brown,
colors
which harmonize well with
the usual
tints of the
soil. You had
to
look
a second
time to detect
the stub
of
a beak at the base of which
the
large
round
nostrils
were sufficiently prominent. Whenever
this
bird
was
aroused
from
its
all-day
slumbers the eyelids would
gradually open and disclose
a
pair
of
large,
soft, deep
blue
eyes,
the
lower
lids
showing
decided
angular
contours
which
became
more
striking
as
the
bird
grew q
y,
pp
g
g
y
Two days
later,
if my estimate
is
correct, a young Night Hawk cracked
his
shell
neatly in two and emerged to the light of day. When
first seen on the twenty-sixth
of
June, he was well clothed in down, and looked like a little flattened ball of fluffy worsted,
of a dark cream
color mot-
tled
with
brown,
colors
which harmonize well with
the usual
tints of the
soil. You had
to
look
a second
time to detect
the stub
of q
y,
pp
g
g
y
Two days
later,
if my estimate
is
correct, a young Night Hawk cracked
his
shell
neatly in two and emerged to the light of day. When
first seen on the twenty-sixth
of
June, he was well clothed in down, and looked like a little flattened ball of fluffy worsted,
of a dark cream
color mot-
tled
with
brown,
colors
which harmonize well with
the usual
tints of the
soil. You had
to
look
a second
time to detect
the stub
of
a beak at the base of which
the
large
round
nostrils
were sufficiently prominent. The Rearing of the
Night Hawk. When the young Night Hawk is exposed to a hot sun, its lower jaw also begins to
vibrate
but
at a much higher rate of speed, when
it
will toddle off and crouch in the shade of
a leaf. It begins to walk when
three or four days old, but
rarely emits a sound, except
under circumstances which will be presently described. Fearing lest the old bird should
entice
it away,
I coralled
it
in a small
enclosure of wattled twigs on
July 3d. In
this
pen
it
remained
a week
longer
or
until able to
fly
at
the age of about
eighteen days. Fig. 70. Night Hawk three days old. Nearly life size. g
y
Wishintr to witness the feeding
S>
O
habits of these birds, which
I believe
have never
been
described,
I
spent
parts of three days and nights camped
beside the enclosure and was the wit-
ness of some interesting and
curious
sights. On the
first day
I set up the
tent at three o'clock in the afternoon,
but heard no sound for an hour, when
the young began
to pe-iip! At
five
o'clock the />/.</
of the male sound-
ed
for
the
first
time. During
the
interval a single incident occurred to
vary the monotony. A green snake
in the course of
his rambles had dis-
covered the young Night Hawk, and
when first seen was watching the bird
intently from a stump
close to the
tent. The snake after remaining with
elevated head keenly eying the bird
for a long time, slowly advanced, put-
ting out
his tongue, but when a few
inches away hesitated again, and as
if
deciding not
to experiment
further,
turned to one
side and disappeared. The bird paid no attention whatever
to the advances of the snake. At this
juncture
I
left the tent
for an
hour,
returning as the sun was
setting
at
half-past seven o'clock. Fig. 70. Night Hawk three days old. Nearly life size. pjg
y]
Njght Hawk njne
days
old
July
Jd . Lcngth
in
sit. ting posture, 3J inches. p
At dark a change begins to come
over the Night Hawk family. The
young bird shows signs of
life, moves
pjg
y]
Njght Hawk njne
days
old
July
Jd . Lcngth
in
sit. about
Calling
for food,
and grOWS
ting posture, 3J inches. THE REARING OF THE NIGHT HAWK. Whenever
this
bird
was
aroused
from
its
all-day
slumbers the eyelids would
gradually open and disclose
a
pair
of
large,
soft, deep
blue
eyes,
the
lower
lids
showing
decided
angular
contours
which
became
more
striking
as
the
bird
Fig. 69. Night Hawk and eggshells from which
it emerged,
old, June 27, 1900. Three days Fig. 69. Night Hawk and eggshells from which
it emerged,
old, June 27, 1900. Three days Fig. 69. Night Hawk and eggshells from which
it emerged,
old, June 27, 1900. Three days grew. The mother brooded during the heat of the day or sat as if dozing beside her charge. When surprised at such times she rose and with feathers erect and
tail spread
fluttered
off in a slow shambling manner as
if to encourage pursuit. With her feathers raised and
her huge mouth wide open or the mandible vibrating up and down, with an audible snap-
ping sound, as
if
set on
springs, this bird presented a curious appearance, recalling the
not wholly dissimilar behavior which
eagles display when
stirred by similar emotions. 80 81 The Rearing of the
Night Hawk. The Rearing of the
Night Hawk. livelier
as
the
darkness
increases,
making a sound like /><-?-.' pe-up ! Both old birds are now alert and gyrating overhead. You hear their pisk ! pisk ! and the startling sound caused by the vibration of the wings pjg
y]
Njght Hawk njne
days
old
July
Jd . Lcngth
in
sit. ting posture, 3J inches. Wild
Birds. Wild
Birds. 82 as an old bird descends like a bolt toward the earth. As these sounds increase with their
nearer approach, the nervous excitement of the young
is curious
to
behold. He
is
all
a-tremor, moves now
in one
direction, now in
another and
\\\s fc-iir
.' note
reaches
a
nkno n b f nds increase with their
to
behold. He
is
all
fc-iir
.' note
reaches
a
pitch unknown before. Presently you
hear
a
thud
as
if
a
clod
of
earth
had
dropped. Then the mother bird,
crawling
over the
leaves,
begins
calling
o
o
ke-ark ! ke-ark ! This
sound
however
un-
couth
to
the human
ear, corresponds to the
cluck ! of
the hen
to
her
chicks, and awak-
ens an
immediate
re-
sponse
in
the young
Night Hawk. He does
his
best to go
to
his
mother,
but
the
ob-
stacles
being
insur-
mountable, she comes
to him. She
is
load-
ed
with
fireflies, and
as
her
great
mouth
opens, you behold the
wide jaws and
throat
brilliantly
illuminated
like
a spacious
apart-
ment
all
aglow
with
electricity. With wings
erect
and
full-spread,
the
old bird
a p-
proached
to
within
fifteen
inches
of
un-
hand, making an
elec-
tric
display
at
every
utterance of her harsh
ke-ark! Then
stand-
bill well down
into
his
ead and a-quiver. In this
es. When the feeding
brood. It was not long
rformance was repeated, Fig. 72. Night Hawk twelve days old, July 6th. Fig. 72. Night Hawk twelve days old, July 6th. Fig. 73. Night Hawk sixteen days old, July loth. Length in sitting posture, 4j inches, Fig. 73. Night Hawk sixteen days old, July loth. Length in sitting posture, 4j inches, ing over her young, with raised and quivering wings, she put her bill well down
into
his
throat and pumped him full. His down-covered wings were also spread and a-quiver. In this
position they remained interlocked and silent for one or two minutes. When the feeding
was over she tucked the little one under her breast and began to brood. It was not long
before she was off again in the darkness, and upon returning the performance was repeated, >
J
__
Fig. 74. Front view of bird shown in Fig. 72. Fig. 74. Front view of bird shown in Fig. 72. Fig. 74. Front view of bird shown in Fig. 72. Wild
Birds. F'g- 75-
Young Night Hawk in enclosure on spot where
it was born, and where
it remained
until able to fly when eighteen days old. F'g- 75-
Young Night Hawk in enclosure on spot where
it was born, and where
it remained
until able to fly when eighteen days old. The Rearing
of the
Night Hawk. The Rearing
of the
Night Hawk. ^5 after which she settled down to brood as if for the night. This young bird was fed but twice
each evening between the hours of eight and nine o'clock, and always, as I believe, by the
female. It
is probable that another feeding time also occurs at dawn. During the earlier
hours the male would sometimes swoop down with
terrific wing-blast as
if
to drive away
intruders, and he once came and
sat by
his chick
for ten minutes
after dusk without
causing any excitement. The task of feeding was borne by the mother, and her presence
never failed to excite the young. y
g
I tried to make a flash-light picture of the old and young bird interlocked in the feed-
ing process, and could easily have succeeded had my lamp been
of a kind which showed
no light before the flash. In two weeks the mottled down of the Night Hawk chick has given place to mottled
feathers,
in which the tints range from dark to light brown or buff. The wing-quills
arc-
almost black with buff edges. The fifth quill or primary has a pure white transverse spot
near the point of emergence from the feather tube, the first trace of what becomes a con-
spicuous mark on the wings of an adult bird. The
fledgling
is more
lively
in the day-
time, runs about easily, will utter his /<-////
note, and can fly short distances. The
Kingfishers and
their King Row. The
Kingfishers and
their King Row. 89 already acquired some curious
habits. They, like the adult birds, stand not on the toes
.simply, but on the whole tarsus, which corresponds to the scaly part of the leg of a fowl,
so that the " drum-stick
" rises from the heel. They can be posed in any position like toy
soldiers, but
if placed in line they will soon break ranks and walk backwards, even mov-
ing
up
inclined
planes
or
against
obstacles set
in
their
paths. They
are rarely seen to take
a
single
for-
ward step for many days after reach-
ing this stage. already acquired some curious
habits. They, like the adult birds, stand not on the toes
.simply, but on the whole tarsus, which corresponds to the scaly part of the leg of a fowl,
so that the " drum-stick
" rises from the heel. They can be posed in any position like toy
soldiers, but
if placed in line they will soon break ranks and walk backwards, even mov-
ing
up
inclined
planes
or
against
obstacles set
in
their
paths. They
are rarely seen to take
a
single
for-
ward step for many days after reach-
ing this stage. Fig. 79. Five Kingfishers from chamber at end of tunnel
;
nine days
old. July 19. 1900. The human
infant
and
verte-
brated animals generally instinctive-
ly walk
forward
;
how then does
it
happen that the young
Kingfisher
early
acquires the grotesque
habit
of walking backwards ? The anom-
aly
is
readily
understood. From
the time of birth the young lie hud-
dled
in
a
cluster in their dark
un-
derground
chamber,
which
opens
to the outside by means
of a single
narrow
tunnel. As
they grow
in
size and strength the monotony
of
sitting
still,
often
with
legs
and
wings
interlocked,
must
become
o
very great, and whether
for
diver-
sion or not, at
all events they soon
begin to bite and tease one another
like young puppies. Should one be
hard-pressed,
the only way
of
es-
cape lies along the narrow
passage,
which they naturally traverse head
first
; but
the
instinct to
return to
the warm
family
cluster
is
strong,
and to do this they are obliged
to
walk backwards. THE KINGFISHERS AND THEIR KING ROW. THE
Kingfisher has
a
strong attachment
for
particular
nesting
places, and
will
occupy the same bank for years,
if unmolested, and sometimes even when robbed. The Belted
Kingfisher, though widely
distributed, seems
to be nowhere very
abundant. In New Hampshire one
rarely finds more than a single pair nesting
in the TH Fig. 76. Tunnel
of Kingfisher
the opening seen at the right
ID sand bank
overgrown with pines, beside country road. August, 1899. 86 The nest now to be de-
scribed
was
drilled
into
a
sand bank beside a country
road. It had a straight four-
inch
bore,
which
four
feet
from the opening expanded
into a low-vaulted chamber
six
inches
high
and
ten
inches
across. When
this
dark
subterranean
abode
was opened
at the
rear, on
the nineteenth day of July,
1900,
I put in my hand and
drew forth in succession five
very
strange looking creat-
ures. They had huge coni-
cal bills, short legs, and
fat
squatty
bodies, which
bris-
tled
all over with steel gray
"
quills," the feather tubes,
which
had
not
yet
burst,
suggesting an
antediluvian
monster or reptilian bird on
a reduced plan. p
These five young King-
fishers
which
were
then
. . . ,
,
about
mile
days
Old
had Fig. 76. Tunnel
of Kingfisher
the opening seen at the right
ID sand bank
overgrown with pines, beside country road. August, 1899. 86 86 Fig. 77. Nest in same bank as shown in Fig. 76, and probably belonging to same pair. Tak-
ing fish to young. July, 1900. Fig. 77. Nest in same bank as shown in Fig. 76, and probably belonging to same pair. Tak-
ing fish to young. July, 1900. *
^
*%,
-
r
Fig. 78. Kingfisher backing out of tunnel. The sand streams from the opening at every en-
trance and exit. Fig. 78. Kingfisher backing out of tunnel. The sand streams from the opening at every en-
trance and exit. 8? The
Kingfishers and
their King Row. Again when
the
rattle of the alma mater announcing
the capture of another fish
is heard,
each
struggles to get down the nar-
row passage-way
first, but when the p
each backward movement the young
food and warmth. Thus the habit
i Fig. 79. Five Kingfishers from chamber at end of tunnel
;
nine days
old. July 19. 1900. Fig. 80. Posed in line,
biting, pulling, and crowding one another. Fig. 80. Posed in line,
biting, pulling, and crowding one another. Fig. 80. Posed in line,
biting, pulling, and crowding one another. p
y
Wishing to see these birds take
fish to
their young,
I decided
to
try the
tent, al-
though
it was impossible to get nearer than eight feet, and the hole was
in
full light for
only a part of the forenoon
;
besides, being situated on the roadside, one was in constant
danger of
interruption. The experiment succeeded, however, even better than
I
had Wild
Birds. 9o anticipated;
ten visits were recorded, and the old
birds were photographed
in the act
..f
both
entering and
leaving their tunnel. They brought
a single fish each time, usually
what appeared to be a small chub or dace, and
I once recognized a good-sized sunfish. what appeared
,
g
g
When the tent and camera were ready at nine o'clock on the morning of July 23d,
the parent birds were away on a fishing excursion, and
did
not
return for half an hour. At last a series of warning
rattles, at
first
faint, but momentarily becoming more
shrill,
announced the approaching
bird, who came
at
full
tilt
with
fish
in
bill. Hesitat-
ing at sight of the
tent she
perched
on
the dead
limb
of
a
pine, flew
to
and
fro
from one side of the road to on the morning of July 23d,
not
return for half an hour. arily becoming more
shrill,
announced the approaching
bird, who came
at
full
tilt
with
fish
in
bill. Hesitat-
ing at sight of the
tent she
perched
on
the dead
limb
of
a
pine, flew
to
and
fro
from one side of the road to
the
other,
and
made
the
woods resound as never be-
fore. Even the depths of the
earth
seemed
to
respond,
as
the
muffled
rattles
of
the
five young Kingfishers
issued
from
their
subter-
ranean abode. The
Kingfishers and
their King Row. The longest
visit recorded
lasted
three and a half minutes. When
a youngster
was encountered near the mouth of the tunnel he was driven back to the chamber, where
th
f
d
di
ib
d
O except on one occasion when
1 saw the bird
turn
nea
first. The longest
visit recorded
lasted
three and a
was encountered near the mouth of the tunnel he was
the food was distributed. Once
only did I see an old bird pause
at
the
entrance
for
a
hasty
glance
backward,
and
thus
give
a
good
profile
view
of
head with fish in
bill. Unfor-
tunately the plate had already
been exposed,
and
before
it
could be changed, the oppor-
tunity was lost. The old birds,
however,
must
have
often
turned about
at the entrance
on
both
entering and leaving
the hole, as shown by the deep
furrows plowed by the bill
at
either side of the opening. except on one occasion when
1
first. The longest
visit recor
was encountered near the mout
the food was distributed. Once
only did I see an old bird pause
at
the
entrance
for
a
hasty
glance
backward,
and
thus
give
a
good
profile
view
of
head with fish in
bill. Unfor-
tunately the plate had already
been exposed,
and
before
it
could be changed, the oppor-
tunity was lost. The old birds,
however,
must
have
often
turned about
at the entrance
on
both
entering and leaving
the hole, as shown by the deep
furrows plowed by the bill
at
either side of the opening. K 'S- 83-
Kingfisher nine days old, showing feather tubes and tracts. p
g
When the young are ten
days
old,
the
feather
tubes
have be"Un tO burst at the tips,
and
their horny substance
is
gradually shed in the form of
powdery scales. The feathers
grow slowly, but at the age of
two
weeks
the
characteristic
colors of the adult are becom-
ing apparent,
the
slaty-blue
of the upper
parts,
and
the
white
of the
breast
which
is
traversed
by
a
bluish-brown
belt, with
rusty brown
along
the sides. As they rattle when
taken
from
the
nest
their
whole body
quavers. They
will hiss, bite one another, hud-
dle
together, and
erect
their
crests
of
long
stiff
feathers. The
Kingfishers and
their King Row. From what-
ever point of view we regard
this
singular
note,
it
cer-
tainly carries well and is ad-
mirably
adapted
to arouse
the fish under water and the
young bird under ground. Fig. 81. The " King Row." Five Kingfishers in line, illustrating habit of sit-
ting still. July 19, 1900. Fig. 81. The " King Row." Five Kingfishers in line, illustrating habit of sit-
ting still. July 19, 1900. tainly
mirably
adapted
to arouse
the fish under water and the
young bird under ground. When the wriggling fish
nearly
slipped
from
her
grasp, the bird would
shift
it about
until
her
forceps
had a firmer grip at a point
just back
of
its head. At
every reel of the rattle, each
of which seemed more shrill
and
more
impatient
than
the last, she would
start
as
if
to go
to her nest a few
yards
away. Occasionally
a peculiar creaking sound es-
caped her, suggesting the grating of dead limbs when swayed by the wind. Suddenly with
rattle in shrillest crescendo she bolted straight into the hole, delivered the fish, remained for
half a minute, then came out backwards, turning in the air as she dropped from the entrance,
and with a parting rattle was off to the river. During these visits the Kingfishers usually
remained but a quarter or half a minute
in the tunnel, and always came out backwards,
Fig. 82. The " King Row " at a later period
; thirteen days old, July 23, 1900. Fig. 82. The " King Row " at a later period
; thirteen days old, July 23, 1900. Fig. 82. The " King Row " at a later period
; thirteen days old, July 23, 1900. peculiar
g
caped her, suggesting the grating of dead limbs when swayed by the wind. Suddenly wit
rattle in shrillest crescendo she bolted straight into the hole, delivered the fish, remained f
half a minute, then came out backwards, turning in the air as she dropped from the entrance
and with a parting rattle was off to the river. During these visits the Kingfishers usual
remained but a quarter or half a minute
in the tunnel, and always came out backwards The
Kingfishers and
their King Row. 9 r except on one occasion when
1 saw the bird
turn
near the entrance, and shoot out head
first. The
Kingfishers and
their King Row. They attain to full plumage or
nearly
so
when
three weeks
old, at which time their bright
They can fly but
little, and sho
a peculiar sardonic grin. K 'S- 83-
Kingfisher nine days old, showing feather tubes and tracts. Fig. 84. At thirteen days
; many of the feather tubes burst. The blue-
black, white-tipped wing-quills project half an
inch. Notice that these
birds always stand, not on the toes only, but on the short shank or tarsus. Fig. 84. At thirteen days
; many of the feather tubes burst. The blue-
black, white-tipped wing-quills project half an
inch. Notice that these
birds always stand, not on the toes only, but on the short shank or tarsus. peculiar
grin. On the fourth of August
I took these birds home in a basket, when twenty-five days peculiar
g
On the fourth of August
I took these birds home in a basket, when twe Wild
Birds. 9 2 old,
if their age was correctly estimated. They were about ready to
fly and would have
voluntarily
left
their
nest
in
a short
time. The nesting chamber had been
gradually
opened up in front and
filled at the rear, until
it had advanced a foot and a half toward
the mouth of the tunnel. At this time fear was possessing them, and a day later
it was
impossible to handle them with-
out throwing them into a panic. When
quiet
they would
still
pose
well,
would
strike
with
open
bill, and walk backwards. Fig. 85. sheathed. Kingfisher fifteen days old, with nearly
all feathers partly un-
July 25, igoo. During captivity
I fed them
on
fish
which, however,
they
would
never
seize of their own
accord. It was
necessary
to
open
their
bills and
press the
food well down
into
their
dis-
tensible
throats. They
would
perch on a branch placed in their
cage, drink water and
sit
in
it
by
the
half-hour,
but
never
touch the most tempting mor-
sels
of
food. Raw meat was
rejected,
but
they
throve
on
fish
if fed by the hand. When
perched they stood as before on
the whole tarsus or shank, and
would
sit
together
and
in
si-
lence, with
breasts thrown out,
for hours. You
heard only an
occasional
rattle, and that usu-
ally in the morning. The
Kingfishers and
their King Row. The King-
fisher's ossophagus
is very
dis-
tensible and the throat
is lined
with inwardly projecting papil-
lae, so that when a
fish
is once
taken in the throat,
it
is impos-
sible for
it to escape. Fig. 85. sheathed. Kingfisher fifteen days old, with nearly
all feathers partly un-
July 25, igoo. Fig. 86. At eighteen days. The bright blue tints of the upper parts, and
the white and chestnut bands around
the neck and
breast
are now very
prominent. July 28, 1900. Fig. 86. At eighteen days. The bright blue tints of the upper parts, and
the white and chestnut bands around
the neck and
breast
are now very
prominent. July 28, 1900. ting edges
gripe
the prey. A
fish once seized rarely makes its escape, to prevent which the bird has other
resources. I once saw a curious trick performed by a Kingfisher, who having made a good
capture, was perched on
a dead
tree over the water. In the course of its struggles the
fish nearly got
free, and for a moment was held only by
its tail. The
bird
with
a quick
movement of the head tossed the fish in the air, and as it descended caught
it by the head
and proceeded to swallow
it. BROODING AND FEEDING THE YOUNG. WHE WHEN
the callow young are hatched, brooding
is the order
of the day as well as
of the night, and in some species the young seem to require this kind of
pro-
tection as much as food. During the
first days
of
life
in
the
nest
it
is
not
easy to distinguish a brooding from a sitting bird, but
this
is
not
the
case when a
little
later the mother begins to rest her wings over the rim, or spreading wings and tail stands
astride the nest with back to the sun. The young must be protected
from
heat, cold,
and
rain, and the
instinct
to perform
this duty
is
as strong with old
birds
as
that
of
bringing food. g
g
Cedar Waxwings and Kingbirds which
I have watched, brooded
regularly
at
night,
but
I have known young Robins to be left alone in the nest. Should the day be cloudy
but with no
rain,
or sunny but not too warm
little
or no brooding has been observed
among the various species which
I have
studied, but
let the sun beat relentlessly upon
the young, or the air become laden with moisture, and the faithful mother
is promptly at
her post. I have seen the Robin brood the young when eleven days old for forty minutes
at a time, while her mate brought
an abundance
of
food. As he approached with an
insect or cluster of worms, she would step aside, but immediately settle back on the
nest
when the food had been safely disposed. As a rule, however, she would brood for five or
ten minutes, leave at the approach
of the
male, return promptly with
food, and brood
until her mate again appeared. I have on several occasions seen
a brooding
bird
leave
the nest when the sun became temporarily obscured and
return when
the clouds
lifted. It
was
not
quite
certain, however,
that
the
element
of chance
did
not
vitiate
the
observation. While camped beside a nest of Brown Thrushes whose photographs
are shown, and
whose young were approximately four days old, the female came to the nest
for
inspec-
tion frequently on the
first day of observation, and brooded
intermittently, but
fed
her
young only once in
the space
of three and
a
half hours. The
Kingfishers and
their King
Ro\v. 93 When
liberated on August
I2th, at the age of
thirty-three days, the young King-
fishers were suddenly thrown upon their own
resources, and
it was questionable whether
they would be able
to recover the
instinct
to seek and capture
prey. However, they
were strong and healthy, and
I hope that nature came to their aid not only in prompting
them to find food, but in starting them south later in the autumn. Fig. 87. Kingfishers twenty-two days old. Placed in line to illustrate habit of walking back-
wards. The second bird at the left has already broken ranks and taken a few backward steps. August
i, 1900. Fig. 87. Kingfishers twenty-two days old. Placed in line to illustrate habit of walking back-
wards. The second bird at the left has already broken ranks and taken a few backward steps. August
i, 1900. BROODING AND FEEDING THE YOUNG. When
I frightened this bird
off with the hand stretched through the tent-window, she would
dart
at
it, scold em-
phatically, but in a few moments return to her brooding again, as
if her young required
this attention more than food. The Chestnut-sided Warbler who
is
represented
in
main-
characteristic
attitudes The Chestnut-sided Warbler who
is
represented
in
main-
characteristic
attitudes
94 94 Fig. 88. Female Brown Thrush brooding her young. Lens, Extra Rapid gjn inch
; speed,
;
stop, 32
;
time,
J second
;
plate, Seeds' No. 27 "gilt edge"
;
distance, four feet
;
in
full sun. July 13, 1900. Fig. 88. Female Brown Thrush brooding her young. Lens, Extra Rapid gjn inch
; speed,
;
stop, 32
;
time,
J second
;
plate, Seeds' No. 27 "gilt edge"
;
distance, four feet
;
in
full sun. July 13, 1900. 95 95 the nest, bend far down and deliver the insects into the mouth of the brooding hen, who
would promptly hop up and give every morsel to the young.
This little warbler would sometimes sit well down in the nest, and erect some of her
feathers and apparently inflate the throat so that the bird's head appeared as
if swollen
to twice its natural
size.
She made the most comical
picture, however, when on a hot
day she stood or sat over the young, with every feather erect, striving to keep them cool
and to be comfortable herself meanwhile.
Th
f
l
b
d
h
th
h
t
d
h Care of Young and Nest. Care of Young and Nest. 97 about the nest by a long series of photographs, only a few of which can be shown, was a
most devoted brooder
for days. She would stick to her charge until driven off by sheer
force or by hunger. I have often seen
her drop down
in the grass, pick up a morsel on
her own account, and be back to the nest
in a fraction
of
a second before the insect was
fairly swallowed. Again she might leave the
nest twenty times in the course of an hour
to procure food either for
herself
or
her
children. Her mate would often alight above Fig. 89. Female Robin brooding
: a characteristic attitude when alert, or listening to any unusual sound. Fig. 89. Female Robin brooding
: a characteristic attitude when alert, or listening to any unusual sound the nest, bend far down and deliver the insects into the mouth of the brooding hen, who
would promptly hop up and give every morsel to the young. p
p
y
p
p
g
y
y
g
This little warbler would sometimes sit well down in the nest, and erect some of her
feathers and apparently inflate the throat so that the bird's head appeared as
if swollen
to twice its natural
size. She made the most comical
picture, however, when on a hot
day she stood or sat over the young, with every feather erect, striving to keep them cool
and to be comfortable herself meanwhile. The female Kingbird broods constantly when the heat
is severe, and at the approach Wild
Birds. 98 Fig. 90. Female Redwing Blackbird placing food in the throat of
a nestling. of the male
will
often
assist
in
dis-
patching unruly insects and in seeing
them safely down a responsive throat. The persistence of the Redwing Star-
ling in this line
of conduct
is admir-
able. I have seen one of these birds
stand
with
drooping
wings,
erect
feathers,
and
mouth agape,
in
the
strong heat of a July day
for hours
though not continuously, for she
in-
variably left at the approach
of
her
mate for a few moments' respite, and
then usually returned with food. y
The
Cedar-bird
gapes
persist-
ently
when
uncomfortably
warm,
but
only the
crest feathers are ever
erected, and then not to the extent
usually shown
in
drawings
of
this
species. Care of Young and Nest. Both Robins and Catbirds
bristle up when their nests and well-
fledged young are assailed, but I have
never seen this habit in the brooding
bird, although their emotion
is often
expressed by raising the
feathers
of
the crown. Fig. 90. Female Redwing Blackbird placing food in the throat of
a nestling. Fig. 91. The same bird watching the food in the throat. If not
immediately
swallowed,
the
insect
is withdrawn
and
passed
around until a bird with the proper reaction time is found. The
duty
of
brooding
rests
mainly with
the female in our com-
mon land birds, but the male in some
species either regularly or intermit-
tently takes his turn at the nest. y
Passerine birds feed their young
at brief intervals from early morning
until
nightfall,
but
apparently
sel-
dom
if ever after dark. The Night
Hawk, as has been
seen, broods by
day, and feeds its young
at dusk, or
just
after
dark, and probably
again
at dawn. Both sexes
usually share
in
bringing
food
to
the
nest, but
this
rule
is by no means
universal. The young require animal food
during the
early
days
of
life, and
in the
interior
of the
country
this
consists
mainly
of
insects
in
the
larval
or
mature
stages,
spiders, y
Passerine birds feed their young
at brief intervals from early morning
until
nightfall,
but
apparently
sel-
dom
if ever after dark. The Night
Hawk, as has been
seen, broods by
day, and feeds its young
at dusk, or
just
after
dark, and probably
again
at dawn. Both sexes
usually share
in
bringing
food
to
the
nest, but
this
rule
is by no means
universal. y
The young require animal food
during the
early
days
of
life, and
in the
interior
of the
country
this
consists
mainly
of
insects
in
the
larval
or
mature
stages,
spiders, Fig. 91. The same bird watching the food in the throat. If not
immediately
swallowed,
the
insect
is withdrawn
and
passed
around until a bird with the proper reaction time is found. Fig. 92. Female
Kingbird
astride
nest with
drooping
half-spread wings,
shielding
her
brood from the hot sun. Notice the characteristic attitude of the young. Fig. 92. Female
Kingbird
astride
nest with
drooping
half-spread wings,
shielding
her
brood from the hot sun. Notice the characteristic attitude of the young. Fig. 93. Care of Young and Nest. Kingbirds bruising a too active grasshopper
between their
bills preparatory
to
serving it to the young
: the female in front with tail full-spread. 99 Fig. 93. Kingbirds bruising a too active grasshopper
between their
bills preparatory
to
serving it to the young
: the female in front with tail full-spread. Fig. 93. Kingbirds bruising a too active grasshopper
between their
bills preparatory
to
serving it to the young
: the female in front with tail full-spread. Care of Young and Nest. 101 earthworms (at least in the Robin) and possibly slugs. Aside from the habits of the adult
the nature of the food brought depends much upon the character of the supply. When the
Kingfisher finds crayfish abundant they are carried to the nest, and
this species has also
been known to go to the fields for insects. Along the coast various other invertebrates un-
doubtedly contribute to the food supply of both young and adult birds of many species. Birds which never taste of fruit themselves naturally do not give
it to their young, while
Robins, Orioles, Vireos, and Waxwings, to mention but a few of the berry-pickers, vary
the diet of their fledglings with a liberal supply of fruits of various kinds. Fig. 95. Male grampus,
from life. full
size, Kig. 94. Female Kingbird assisting a grampus down
the
throat of a nestling. The long gray wings of this insect are
still protruding from the mouth. Fig. 95. Male grampus,
from life. full
size, Kig. 94. Female Kingbird assisting a grampus down
the
throat of a nestling. The long gray wings of this insect are
still protruding from the mouth. The food
is placed not simply
in
the mouth
of the young but well down into the
sensitive throat, and
if the bird does not immediately respond, it is withdrawn and passed
to another, and
often
to a third, until a throat
is found which has the proper
reaction
time. If the gullet
is already
full, the swallowing power
is inhibited, and the bird mu>t
wait. If the experiment
of feeding a young bird like a Robin at the nest
is tried, it will
be found that the
food
passes slowly down the oesophagus, and when
this
is
filled no
more can
be taken
until
the channel
is
clear. Care of Young and Nest. The
gullet
thus
acts as a brake to the
tendency of the greedy young bird
to gorge
itself
to
suffocation. According to Audu- Wild
Birds. 102 bon,
Cedar-birds
will
sometimes
gorge
themselves to such excess with berries as
to
be
unable
to
fly, and
a number
of
wounded
birds
of
this species
which
he
kept in a cage ate of apples until suffocated. When opened they were found to be filled
to the mouth. Fig. 96. Female Chestnut-sided Warbler bristling
to keep
cool while brooding on a hot June day. The automatic response given by the
young is the signal awaited by the old bird,
though often with impatience. The insect
is watched
after being placed in a respon-
sive throat, until
it disappears. Should
it
stick
at
the gullet
it
is withdrawn and
re-
placed time and
again, or given a gentle
pull, until
it
is
safely down. Sometimes
the insect
is bruised against a twig, beaten
into a pulp
or crushed and
torn
asunder
between the bills of the parent birds before
it can be safely delivered. As has already
been seen, many birds utter a peculiar note
as a
special stimulus to the young. At
such times even the silent Cedar-bird finds
a voice and
gives an impatient died ! If
this call passes unheeded
it often becomes
extremely
shrill, especially
in
Kingbirds,
with whom
failure on
part of their young
to quick response seems
to be
peculiarly
exasperating. Fig. 96. Female Chestnut-sided Warbler bristling
to keep
cool while brooding on a hot June day. Fig.g?. The same bird in the more common brooding attitude. p
g
While
watching
a
Kingbird's
nest
from
the
tent,
a moth
miller
was once
brought in by the male. It was passed to
each one of the young
in
turn, but even
under the spur of his
shrill
chitter, they
were unresponsive, and he devoured the
prey himself. This sharp economy is often
practiced at the nest, and I have even seen
the
leg
of a grasshopper
picked up and
eaten by an
old
bird. Not
a crumb
is
allowed to go to waste. If an insect gets
away
it
is
usually pursued
and immedi-
ately snapped up. Once, however, I saw a
female Kingbird fooled by a fly who owed
its life to
its small
size. Care of Young and Nest. As she opened
her
bill
in her attempt
to land
it
safely
i"
ail opcil throat, the fly darted
off. The Fig.g?. The same bird in the more common brooding attitude. Care of Young and Nest. 103 Fig. 98. A common scene
at this nest. The male brings
food, while
his mate, who
is brooding, receives
it into her
own bill and passes it on to the children. bird
seemed
dazed
for
a
moment, and
stood gazing at the departing fly
as
if in
mute astonishment. Exciting scenes usually follow
at the
nest of the Kingbird when a large dragon-
fly, cicada, or grampus
is brought
to
the
family circle. The
insect
often struggles
hard, but escape
is out
of the
question,
especially with both birds at the nest, who
at once begin
to rend and crush
it with
their bills. The male grampus
(Corydalus
corn-
iitiis) better known as the larval hellgamite
of
which
black
bass
are sometimes
ex-
tremely
fond, has long gray wings folded
back over the body when at rest, and the
head is armed with horns an inch long but
formidable
only
in
their
appearance. I
have seen these huge insects measuring four
inches from tip of the jaws to the extrem-
ities
of the
folded wings
fed
to
a
single
bird, and they were swallowed
wings and
all. The operation
is shown at an incom-
plete
stage
in
one
of
the
illustrations,
where the wings
of the grampus can
be
seen
projecting an inch or more from the
mouth
of the struggling bird. Fig. 98. A common scene
at this nest. The male brings
food, while
his mate, who
is brooding, receives
it into her
own bill and passes it on to the children. Fig 99. The same brooding bird, with feathers erect and
throat inflated. gg
g
The cicada is even tougher and harder
to manage but
is beaten
into subjection,
and served up in a limp condition. Last
August,
I
witnessed
a
street combat be-
tween one of these cicadae and an
House
Sparrow. The
insect
was
bounding up
and down on the ground and sounding its
crescendo at an alarming
rate, but unable
to avoid the blows which rained from
the
Sparrow's bill. As the music of the dying
cicada
finally ceased, the Sparrow picked
up his victim and bore
it off to his brood CLEANING THE NEST. The
sanitary condition of the young
is a matter of great concern to most birds, Fig 99. The same brooding bird, with feathers erect and
throat inflated. Wild
Birds. 104 who
as
a
class
are
extremely
neat
and
clean. This
is
especially
true
of
many
species who
breed in
holes
or
cavities
of
any kind
like the Woodpeckers and Chick-
adees, the young
of which are crowded
in close quarters or even
piled up
in more than
one layer. The Woodpecker's hole and the Bluebird's nest are always sweet and clean,
and the nestlings immaculate. t-cleaning follows each
feeding with
clock-like
regularity, and
is one of
the most
characteristic and import-
ant activities to be observed
in the
nesting habits of a
large number of
o
o
the smaller land birds, yet apparently
it
is not mentioned
in the standard
treatises of ornithology, and
I have
found
but
few references
to
it
in
works of any kind. Audubon, who
has probably recorded more facts on
the behavior of American birds than
any other writer, does not,
I believe,
mention
this
important
function. The reason
is
not
far
to
seek,
for
without the possibility of close
ap-
proach to the nest, and the use of a
convenient
blind,
such
acts
are
difficult or impossible to observe. Fig. 100. Brown Thrush feeding a nestling. " The food is placed
not simply in the mouth of the young, but well down into the sensi-
tive throat." p
The
instinct
of inspecting and
cleaning the nest
is mainly confined
to the great passerine and
picarian
orders
represented
in
this country
by
hundreds
of
species. It
is
a
well-marked trait
in Thrushes, Wax-
wings,
Vireos,
Warblers,
Orioles,
Blackbirds,
and
Woodpeckers,
to
mention
those
families
in which
it
has been observed. has been observed. The excreta of the young leave
the
cloaca
in
the
form
of white,
opaque
or transparent, mucous sacs. The sac is probably secreted at the lower end of the alimentary canal, and
is
sufficiently
consistent to admit of being picked up without soiling bill or fingers. The parent birds
often leave the nest hurriedly bearing one of these small white packages
in bill, an action
full of significance to every member
of the
family. I have seen the Oriole carry these
packages
a few rods from the nest and drop them
before alighting. CLEANING THE NEST. The
Robin
has
undoubtedly
been seen by many
in the character-
istic pose shown
in a number of the
photographs standing on the rim of
the nest with the head
erect,
or
in-
clined
as
if
doting
on
her young
ones and thinking what fine children
they were, whereas
this
attitude
is
really
one
of
sanitary
inspection. When
an
old
bird
of
any
of
the
species mentioned above has fed one
of the
broo.d,
its
duty
is
but
half
done;
it
pauses,
bends
over,
and
keenly scrutinizes each young
bird
in
turn and
every part
of the
nest. Shortly after
being
fed, the
nestling becomes very
uneasy, and
raises
its body as
if
to drop the sac
over
the
edge
of
the
nest. The
old
bird
follows
every
movement,
snaps
up
the
package
as
it
leaves
the
body, and
either swallows
it immediately
or
carries
it
off. When
seen
flying
from
the
nest
with head
depressed, the Robin
is usually engaged
in errands of this kind. The Robins and Cedar-birds have
frequently been seen to take
the sacs from two
or three birds
in rapid succession, in which
case they
are always
de-
voured
on
the
spot. The
Robin
will
often
convey the
package
to
any convenient
perch, and after examining
it, devour a part, or reject the whole. While watching Rob-
ins from the tent
I have seen them carry the excreta thirty rods away before letting
it
Fig. 101
. Brown Thrush cleaning the nest. The
Robin
has
undoubtedly
been seen by many
in the character-
istic pose shown
in a number of the
photographs standing on the rim of
the nest with the head
erect,
or
in-
clined
as
if
doting
on
her young
ones and thinking what fine children
they were, whereas
this
attitude
is
really
one
of
sanitary
inspection. When
an
old
bird
of
any
of
the
species mentioned above has fed one
of the
broo.d,
its
duty
is
but
half
done;
it
pauses,
bends
over,
and
keenly scrutinizes each young
bird
in
turn and
every part
of the
nest. Shortly after
being
fed, the
nestling becomes very
uneasy, and
raises
its body as
if
to drop the sac
over
the
edge
of
the
nest. The
old
bird
follows
every
movement,
snaps
up
the
package
as
it
leaves
the
body, and
either swallows
it immediately
or
carries
it
off. CLEANING THE NEST. The Bluebird and
Redwing Blackbird take them a long distance before letting them
fall. Fig. 100. Brown Thrush feeding a nestling. " The food is placed
not simply in the mouth of the young, but well down into the sensi-
tive throat." Fig. 100. Brown Thrush feeding a nestling. " The food is placed
not simply in the mouth of the young, but well down into the sensi-
tive throat." Redwing
g
g
Some Crow Blackbirds which
I watched last spring had their young in the top
of
a
fir tree beside a small pond, which lay between me and their nest. In approaching with ('arc of Youn^ ami
Nest. 105 food they would stealthily cater the tree on the farther side and after a few moments
fly
over the pond and drop what looked like a small white marble in the water below. ThN
effected,
they would
veer and
fly
off
to the feeding ground. The
same
action
was
repeated by birds from other nests. p
y
Removing the excreta piecemeal and dropping
it
at a safe
distance,
is the common
instinctive method
not only
of insuring the sanitary
condition
of the
nest
itself, but
what
is
even
more
important,
of
keeping the grass and
leaves below
free from any
sign which might be-
tray them to an enemy. Fig. 101
. Brown Thrush cleaning the nest. Many
other
birds,
of which
I
can now certify the Robin, Catbird,
Cedar Waxwing,
Red-eyed
Vireo,
Kingbird, Redwing Blackbird, Brown
Thrush, and Chestnut-sided Warbler,
devour a part and often
the
major
part
of
the
excreta
at
the
nest. This
is
a
very common
practice
with the Warbler, Robin, Waxwing,
and
Yireo,
but
was
only
casually
observed
in
Catbirds
and
Brown
Tli rushes. Many
other
birds,
of which
I
can now certify the Robin, Catbird,
Cedar Waxwing,
Red-eyed
Vireo,
Kingbird, Redwing Blackbird, Brown
Thrush, and Chestnut-sided Warbler,
devour a part and often
the
major
part
of
the
excreta
at
the
nest. This
is
a
very common
practice
with the Warbler, Robin, Waxwing,
and
Yireo,
but
was
only
casually
observed
in
Catbirds
and
Brown
Tli rushes. CLEANING THE NEST. When
seen
flying
from
the
nest
with head
depressed, the Robin
is usually engaged
in errands of this kind. The Robins and Cedar-birds have
frequently been seen to take
the sacs from two
or three birds
in rapid succession, in which
case they
are always
de-
voured
on
the
spot. The
Robin
will
often
convey the
package
to
any convenient
perch, and after examining
it, devour a part, or reject the whole. While watching Rob-
ins from the tent
I have seen them carry the excreta thirty rods away before letting
it
Fig. 101
. Brown Thrush cleaning the nest. Fig. 101
. Brown Thrush cleaning the nest. Wild
Birds. io6 fall or alighting to examine
it, and have tried to find the sac but usually without success. One day I saw a male Robin drop the " white marble " in the grass about fifty feet from
the nest, and proceed to peck at
it. Upon going to the spot a little later
I found the sac
covered with dirt but not opened. It had a tenacious opaque white wall, was
perfectly
odorless, and contained besides a few small pellets, a whole blueberry which had survived
the digestive process. The
actions of the
old bird were thus explained. He was look-
ing for food on his own account, but in this case missed
it. g
On another occasion the mother Robin devoured
all the excreta which soiled the
nest, and a moment later took
it directly from the young and carried
it away. Again on
a later day, the same bird after swal-
lowing
all
the
excreta
available,
dropped on
the
nest
and
brooded
her young
twenty
minutes by the
watch,
without
showing
the
least
desire to reject anything which had
been eaten. Fig. 102. Cedar-bird cleaning the nest. Compare
this common
attitude with that shown in Figs. 45 and 46. The
female
Cedar-bird
in
her
usual
round
of
domestic
duties
comes
to
her
nest
of
half-fledged
young,
regurgitates
cherries,
and
after distributing them
in the usual
fashion, inspects her household with
the closest attention, picking up and
swallowing every particle which
it
is
necessary to remove. This
accom-
plished
the
mother bird
has
been
seen
to spread
her wings
over her
brood, and shield them from
a hot
August sun for over an hour. Mean-
time her mate came
repeatedly, and
passed the cherries around. CLEANING THE NEST. The
fe-
male
who
stood
erect
astride
the
nest, would
frequently
inspect
and
clean
the
household. She
would
also snap at every passing insect, and
I saw her catch a large red ant, and quickly transfer
it to the mouth
of a young bird. She would erect and lower her crest and stand with
mouth agape for long intervals, but there was never
a sign
of ejecting anything which
had been
eaten. Fig. 102. Cedar-bird cleaning the nest. Compare
this common
attitude with that shown in Figs. 45 and 46. Fig. 102. Cedar-bird cleaning the nest. Compare
this common
attitude with that shown in Figs. 45 and 46. also snap at every passing insect, and
I saw her catch a large red ant, and quickly transfer
it to the mouth
of a young bird. She would erect and lower her crest and stand with
mouth agape for long intervals, but there was never
a sign
of ejecting anything which
had been
eaten. At still another nest of the Waxwing
I saw the female after feeding cherries, inspect,
and walking around the rim of the nest, take the sacs from four young birds in succession,
direct from the body, and after swallowing them
all, look for more. She then flew to a
neighboring tree and cleaned her
bill. In performing the sanitary act
this bird bends
over, and reaching forward with head turned
slightly to one
side, takes the sac
rather
gingerly as
it
leaves the cloaca, and quickly disposes
of
it. In the course of
forty-four
visits
to
their young of which exact
record was made, this nest was cleaned eighteen Care of Young and Nest. 107 times
; once a part of the excreta was taken away and a part eaten
:
five times
it was re-
moved from the nest, and on eleven visits all was devoured. After watching such behavior, which
I have seen repeated with slight variations hun-
dreds of times,
I am convinced that
the excreta in such cases
is actually eaten, and
not
merely taken into the gullet to be later regurgitated. It
is true that the Cedar-bird uses
its distensible gullet as a temporary receptacle for the food destined
for the young, and
it might seem probable that the excreta went no farther than the oesophagus, from which
it was later ejected. CLEANING THE NEST. The actions
of the birds
just described and
in many similar cases
observed do not support this idea. r Fig. 103. Female Kingbird attending sanitation
of nest. pp
Not only
are
the
young
care-
fully tended
in
the way
explained,
but the old birds often put the head
down
in
the
nest
and
rummage
about for any stray
particle of food
or fragments of any kind which
it
is
desirable
to
remove. While
stand-
ing at the nest they
will sometimes
pick energetically their own legs and
toes, and the heads and bodies of the
young,
a
very
important
function
where the nest is infested with those
minute swarming particles known as
lice
and
mites. Every
straw
and
fiber in
the Cedar-bird's
nest shown
in one of thephotographs(Fig. 38) was
literally covered with parasites, in this
c.ise a species of mite which is a poor
and degenerate relation of the spider. When the nest or anything
in
it was
touched
they would swarm up
the
hand
by
hundreds,
but
they
are
Fig. 103. Female Kingbird attending sanitation
of nest
barely visible
to the
eye, and
apart
from a slight tickling sensation between the fingers are scarcely
felt. They do not
to trouble the old birds much, but must give discomfort to the young, especially
if
any other cause they happen to be weakly. pp
Not only
are
the
young
care-
fully tended
in
the way
explained,
but the old birds often put the head
down
in
the
nest
and
rummage
about for any stray
particle of food
or fragments of any kind which
it
is
desirable
to
remove. While
stand-
ing at the nest they
will sometimes
pick energetically their own legs and
toes, and the heads and bodies of the
young,
a
very
important
function
where the nest is infested with those
minute swarming particles known as
lice
and
mites. Every
straw
and
fiber in
the Cedar-bird's
nest shown
in one of thephotographs(Fig. 38) was
literally covered with parasites, in this
c.ise a species of mite which is a poor
and degenerate relation of the spider. When the nest or anything
in
it was
touched
they would swarm up
the
hand
by
hundreds,
but
they
are
barely visible
to the
eye, and
apart
from a slight tickling sensation betwee
to trouble the old birds much, but mu
any other cause they happen to be we Fig. 103. Wild
Birds. loS solitude on
or near the ground to remove every
particle of
litter which would
whiten
the
grass
or
foliage and thus advertise the
nest
to
their enemies, even
to those who
prowl at night. p
g
When a
Red-eyed Vireo whose actions
I was watching
at
close
range dropped one
of the
sacs by accident, she would
dart
after
it and snap
it up before
it reached the
ground not four feet from the nest. I have also witnessed the same performance in the
Kingbirds. Not a trace of defilement
is
ever seen about
the dwellings
of
any bird
possessed
of
the cleaning
instinct. Fig. 104. Baltimore Oriole hurriedly feeding her young before
all fear has been subdued and behavior is free. On
the other hand predaceous
birds like Eagles and Hawks pay no
attention to such
matters. The ex-
creta of the young as of the adult
is
voided
in a semi-fluid
state and
in
a
peculiar
manner. With
tail
up-
turned over the edge of the
nest
it
is shot to a distance of several
feet,
and may
strike
the ground two or
more yards from
the
nesting
tree. In this way the eyry at least
is kept
clean. These
bold
and
persistent
robbers have few enemies to reckon
with, and their nests may be as open
to view as a castle on a hill. In this way the eyry at least
is kept
clean. These
bold
and
persistent
robbers have few enemies to reckon
with, and their nests may be as open
to view as a castle on a hill. Owls, which breed
in
holes
in
trees,
are
reported
to
have
filthy
nests, especially when the cavity has
been occupied for several successive
years, but this seems to be due mainly
to the remains of their quarry or to
the
accumulation
of
the
rejected
food-pellets. The haunts
of certain
sea fowl
are often
reeking with filth
Fig. 104. Baltimore Oriole hurriedly feeding her young before
during the breeding SCaSOIl, and the
all fear has been subdued and behavior is free. guano-beds
of
the South American
coast mark the breeding grounds of myriads of sea fowl. However, the birds themselves
both old and young seem to manage to keep clean, and any other condition would soon
become intolerable. young and nest
is
instinctive
in a very large number of
birds,
1 Ornithological Biography,
vol.
ii.,
p. 43. CLEANING THE NEST. Female Kingbird attending sanitation
of nest. y
y
pp
One would suppose that cleanliness must be an imperative instinct with such a bird
as
the
Kingfisher, whose
nest
is underground,
but
the
semi-fluid
excreta
is
not
re-
moved from
the
tunnel, which according
to some
observers, becomes
fouled
in
con-
sequence. This was not
true
of the nest which
I had under observation
last summer. In
the course
of seventeen days the
nesting chamber was moved forward more than
a
foot, so that
it always presented a clean surface. y
p
The Barn Swallow, the House Sparrow, and
the wild
Passenger Pigeon represent
a
considerable number of birds which secure protection
in
their breeding haunts by
other
means than by concealing the nest. While their nests may be
clean, this
is not
true
of
the ground
beneath. It
is
plainly advantageous
for
the
smaller
birds which breed
in Wild
Birds. Wild
Birds. Owls, which breed
in
holes
in
trees,
are
reported
to
have
filthy
nests, especially when the cavity has
been occupied for several successive
years, but this seems to be due mainly
to the remains of their quarry or to
the
accumulation
of
the
rejected
food-pellets. The haunts
of certain
sea fowl
are often
reeking with filth
Baltimore Oriole hurriedly feeding her young before
during the breeding SCaSOIl, and the
been subdued and behavior is free. guano-beds
of
the South American
rk the breeding grounds of myriads of sea fowl. However, the birds themselves
and young seem to manage to keep clean, and any other condition would soon
ntolerable. Fig. 104. Baltimore Oriole hurriedly feeding her young before
all fear has been subdued and behavior is free. guano beds
coast mark the breeding grounds of myriads of sea fowl. However, the birds themselves
both old and young seem to manage to keep clean, and any other condition would soon
become intolerable. The Turkey Buzzard seems to have touched the lowest depths of squalor to which
any bird can descend and
live. In speaking of their abodes, Audubon says that before
the final departure of the young, a person,
if forced to remain in their vicinity for half an
hour, would be in danger of suffocation.' g
he cleaning of the young and nest
is
instinctive
in a very large number o
1 g
ning of the young and nest
is
instinctive
in a very large number of
birds 1 Ornithological Biography,
vol. ii.,
p. 43. 1 Ornithological Biography,
vol. ii.,
p. 43. Care of Young and Nest. 109 and so
is also the care with which they avoid any defilement of the nesting site. The use
of the excreta as food, however,
is to be regarded
in
a different
light. If
it should be
proved that in the Robin, for instance, some individuals never eat the excreta, while others
as we know do, we should
regard
the
action as an acquired
habit. When the pellicle
breaks in the mouth, an accident which
I have seen happen
in the case of the Robin,
t he-
bird
is obliged to swallow a part in order to get
rid of
it. and so
is also the care with which they avoid any defilement of the nesting site. The use
of the excreta as food, however,
is to be regarded
in
a different
light. Wild
Birds. If
it should be
proved that in the Robin, for instance, some individuals never eat the excreta, while others
as we know do, we should
regard
the
action as an acquired
habit. When the pellicle
breaks in the mouth, an accident which
I have seen happen
in the case of the Robin,
t he-
bird
is obliged to swallow a part in order to get
rid of
it. g
p
Much
light
is thrown
on
this
question
by
the
behavior
of
the
Chestnut-sided
Warblers, whose
habits will be
referred
to
again
in
the concluding chapter. Both sexes
in
this
case
fed,
brooded,
and
cleaned the young and
nest. The
male regularly removed the excreta
but was never seen to eat
it. The
female on the contrary often ate of
it, and brooded
so constantly
that
she was obliged
to
leave the
nest
to
satisfy
her own
hunger. She
would often be back in half a minute,
having taken only a bite as it were. \Vhen
the
female
had
received
the
food
which
her
mate
sup-
plied and had seen
it
safely
deliv-
ered,
she
would
inspect,
devour
everything which needed
removal,
and then continue to brood. If a
sac
should
accidentally
fall,
she
would snap
it off the ground, return
to the
nest, and brood
as
before. At other times when
the male ap-
proached
she
would
stand
aside
and allow him
to deliver the food
and
make
the
inspection. Twice
I saw the male take a sac to
carry
it away,
and
the female snatch
it
from him, swallow
it, and settle dow
and each
bird went
off with a half
without bringing food, showing that
only proves that the
excreta is use
may be forced upon a hungry brood
Since digestion in the young
is
how any kind
of pre-digested
or pa Fig. 105. Serving a black cricket to a fledgling who has climbed to
the rim of the nest and is struggling to maintain his position. Fig. 105. Serving a black cricket to a fledgling who has climbed to
the rim of the nest and is struggling to maintain his position. y
p
g y
g
Since digestion in the young
is an imperfect process at best,
it
is easy to understand
how any kind
of pre-digested
or partly digested
food might be acceptable
in times of
stress when the staple article was not easy to procure. Wild
Birds. The fact that a bird only casually
devours a pellet or swallows one and removes another is easy to understand. It
is a ques- Wild
Birds. Wild
Birds. I IO tion of the hunger of the moment, and another illustration of the economy which birds dis-
play in
all such matters. tion of the hunger of the moment, and another illustration of the economy which birds dis-
play in
all such matters. p
y
While the removal of the excreta
is an
instinctive
act, the use of
it as food
is pro-
bably an acquired
habit, the strength of which depends on the
force of circumstances,
and may be limited in some cases to one sex alone. THE FORCE OF HABIT. UNDER
some conditions habits are formed with surprising quickness. The
habit
ma}- be
of
trifling significance and have only
a
brief
reign, but
no
habits are
absolutely rigid, and the genesis of
all
is probably the same,
pleasurable conse-
quences following repeated actions which may be forced or accidental. The
result
is in
all cases similar,
a mental association of certain things with certain actions. UND g
While watching hour bv hour the Robins described
in Chapter IV, and
recording
their
visits to
their young,
I began
to notice on
the
third day that the male usually
approached on the right side of the nest, that
is on the observer's right as he stood facing
it, while the female frequently came to the back or on the left. From that time I recorded
the manner of each approach, and found that the male invariably came to the
right side,
and hopped down the limb to his nest. pp
In the table given below in which the visits of both
birds are recorded
for two con-
secutive days, R
is
for the
right, L for the
left, and B
for the
far side
of the nest with
reference to the tent, while the dashes represent visits the character of which was unde-
termined. Each sign represents a visit to the nest, at which
food was usually served. g
p
( Female RR
RRLBRRRRRBRL
- L R -
July 27th. Third day of Ob- J
- L -
- R
servation
6i hours
I Male RBRRRRRRRR
RRB
RRR-
R
R- g
p
( Female RR
RRLBRRRRRBRL
- L R -
July 27th. Third day of Ob- J
- L -
- R
servation
6i hours
I Male RBRRRRRRRR
RRB
RRR-
R
R- | Male RRRRRRRRRRRRR
RRR For the fourth day
I have no record of the female approaching by the right side, and
no record of the male coming in any other way. On the two following days the female
did
not appear, and
as
I had
reason
to
believe, was engaged
in
building
a new
nest. The
male
at
this
period always approached
his
nest
in
the
habitual manner. Now
whether the male bird had formed
this habit shortly after the nest was
built or shortly
after the nesting bough was removed
is of
little consequence. At
all events a definite
mode of behavior had developed in a short space of time, in one case in two weeks or in
the other in two days. On the fourth day the young had to be brooded often, owing to
the heat, which accounts for the apparent inactivity of the female in providing food. pp
y
p
g
Probably most birds form
definite
habits
in
the manner
of approach
to
the
nest,
entering on a certain side, or flying to a certain
twig, following the path suggested
in the
first instance by convenience or dictated by caution. A
pair
of Red-eyed Vireos with
whom
I spent parts of three days followed
a definite course
in approach with surprising Wild
Birds. I 12 y
Figs. 106, 107. Male Redwing Blackbird cleaning nest on two
distinct visits
; photographed under similar conditions, and illus-
trating the formation of habit in the daily routine. y y regularity. They would
fly
to the
main branch, hop along toward the
fork in which the nest was suspended,
and
finally
perch on
a
small
con-
venient
twig just over
their young. Out of sixty recorded visits they de-
viated from this habitual method but
three
times, and
then
only
before
they had
recovered from their
first
feelings of fear. In this case the nest-
ing
branch
had been drawn
down
about a foot by means of a cord, but
was not otherwise disturbed. In cleaning the nest the attitude
is frequently the same
in
successive
visits,
the
birds
often
clasping
the
same twigs, so that a number of pho-
tographs of the
act
taken
without
moving the camera may be so nearly
identical that
only
the most careful
inspection will reveal the least differ-
ence in pose or position. Figs. 106, 107. | Male RRRRRRRRRRRRR
RRR Male Redwing Blackbird cleaning nest on two
distinct visits
; photographed under similar conditions, and illus-
trating the formation of habit in the daily routine. p
p
While engaged in studying some
Redwing
Blackbirds
last
July
the
weather was hot, and the young had
to
be
brooded
almost
constantly. The
female would
sit
on the
nest,
often
with
back
to
the
tent,
with
feathers erect and mouth open in her
efforts to keep
cool. Suddenly the
shriek of a steam whistle sounded the
hour of noon at a mill scarcely three
rods away. It
startled me, but the
bird did not budge a feather. It
is
not difficult to imagine that her
first
experience with
this
instrument
of
torture was quite
different
in
its re-
sult, but the case
illustrates the ease
with which birds become quickly ac-
customed
to
strange
and
uncouth
sounds, when, as sometimes happens,
they place
their nests
in a
saw-mill
a few
feet from
the buzzing saw or
above the grinding trolley cars
of a
city street. Figs. 106, 107. Male Redwing Blackbird cleaning nest on two
distinct visits
; photographed under similar conditions, and illus-
trating the formation of habit in the daily routine. The Force
of Habit. Every animal must adapt itself
in some
measure
to
changes
in
its
surroundings,
and
with
birds
this
power
is well expressed
in
the
nest,
the position, materials, and construc-
tion of which are subject to incessant
change. The change may be slight
or
of
a
very marked
character,
as
when
the
common
type
of
archi-
tecture
is
abandoned,
or
a
distinct
nest-structure wanting. Only a few
examples of change in nesting habits
need be
considered
since
the
facts
are matters of common observation. K- 108. Cock Robin with large grasshopper ready. Fig. 109. The same bird taking aim. The Swift of this country is often
quoted as one of the most remarkable
examples
of
birds
whose
nesting
habits have changed
in recent
times. Formerly breeding
in
hollow
trees
and
still doing
so
in
places
remote
from
mankind,
it
now
attaches
its
little wicker
crates
to
the
inside
of
chimneys. From
the standpoint
of
the Swift the change has really been
very slight, and had
it not become so
widespread
it would
have
attracted
little attention. | Male RRRRRRRRRRRRR
RRR This bird was proba-
bly drawn to the town and open coun-
try by the greater abundance
of
its
insect
prey, and to the mind of the
Swift a chimney cannot be very differ-
ent from
a hollow tree. Its instinct
probably does not lead
it to select a
dead
tree
for
its
roosts
or nests be-
cause
it
is
a
tree, any more than
it
leads
it
to prefer
a sycamore to an
oak. What
is probably inherited
is
the tendency
to seek
a dark or cav-
ernous
place
with easy entrance and
exit. The chimney which emits no
smoke in summer and usually stands
in the open,
fulfils every requirement
in
places
where
hollow
trees
arc-
scarce. K- 108. Cock Robin with large grasshopper ready. Fig. 109. The same bird taking aim. K- 108. Cock Robin with large grasshopper ready. Fig. 109. The same bird taking aim. The
Swift
is
yet
capable
<>f Wild
Birds. adapting
its needs to conditions far
more
unlike those
of
the
ancestral
tree, and
has been known
to enter
a
barn
and
nest
with
the
Barn
Swallows. This happened in Dorset,
Ohio,
1 where some
Swifts
fastened
their
nest
to
the
vertical
boards
near a
hole made
for
the
conven-
ience of the Swallows, and just below
the peak
of
the
roof. Five young
were hatched and were seen clinging
to the boards just beneath the nest. The old birds would sometimes enter
by the open door, fly straight
to the
nest and
cling to the wall beside
it. The quavering
voices
of
the
little
Swifts would then drown every other
sound about the
place. Fig. no. the right s
Ready to inspect. Notice that he invariably comes to
ide by force of habit. p
In
still another
case,
2 a pair of
Swifts nested in the dim interior of a
shed
beside an old saw-mill
at Dor-
chester, New
Hampshire,
in
June,
1899. This nest was fastened to the
boards, well up towards the roof, and
an open door formed easy entrance
and egress. Fig. no. the right s
Ready to inspect. Notice that he invariably comes to
ide by force of habit. Fig. in. Inspecting the household. The female approaches on
the lef:. g
In
at
least
one
respect
birds
resemble men in their ordinary build-
ing operations. 1 This account
was
given
to me
by
Mr.
Robert
I. Sim. 1 This account
was
given
to me
by
Mr.
Robert
I. Sim.
4 Observed by Professor William Patten. 4 Observed by Professor William Patten. | Male RRRRRRRRRRRRR
RRR This
Hawk will nest on the ground,
on
rocks, in low or high trees,
in woods or
in the open, on a
chimney, a pile of rails, a rock-
ing
buoy,
or
a
dilapidated
windmill. It will even
suffer
its nest to be displaced, and at
Bristol, Rhode Island, it eager-
ly appropriates the cart wheel
which the
hospitable farmers
raise aloft on the tops of poles
for the benefit of these birds. The Osprey
is not only on
wonderful adept
in
making
th
mountain
of a
nest,
it seems
Apparently its controlling ambi
nothing comes amiss which can
ing operations this bird seems
some of
its eyries formed
on
rocky crags have
possibly ex-
isted for more than a hundred
years, or might
last so long
if
undisturbed
by
man. This
Hawk will nest on the ground,
on
rocks, in low or high trees,
in woods or
in the open, on a
chimney, a pile of rails, a rock-
ing
buoy,
or
a
dilapidated
windmill. It will even
suffer
its nest to be displaced, and at
Bristol, Rhode Island, it eager-
ly appropriates the cart wheel
which the
hospitable farmers
raise aloft on the tops of poles
for the benefit of these birds. Fig. 112. Female Kingbird inserting an insect into the throat of a fledgling. At
Plum
Island,
New
York, which was formerly col-
onized
by
hundreds
of
Os-
preys, Mr. Allen
found
their
nests in almost every conceivable situation, about thirty or forty per cent, of them being
on the ground. " High rocks on the shore, and low rocks far out
in the water, scarcely
above high tide and swept by the autumn storms, were chosen as situations for the nests. A
large buoy, with a
lattice work top, near the west
end
of
Fisher's
Island, was
also
occupied for many years by a nest of these birds, greatly to the advantage of sailors and
fishermen, who were warned in thick weather of the position of the buoy by the screaming
of
the Fish Hawks."
Fig. 112. Female Kingbird inserting an insect into the throat of a fledgling. Fig. 112. Female Kingbird inserting an insect into the throat of a fledgling. | Male RRRRRRRRRRRRR
RRR They make
use
of
the materials at hand, but
in
the se-
lection of the site for the nest many
seem to obey no
rule, being ever on
the alert to adapt themselves to their
lot, and
a habit once formed
often
leads to a steady line of conduct. y
The English Sparrow has even
found
a
convenient
shelf
in
the
hood of the
electric
arc lamps, and
although these are lowered
daily to
the
street,
it
sticks
to
its
nest over
the light. I have seen this impudent
little
wretch
dispossess
the
Eaves Fig. in. Inspecting the household. The female approaches on
the lef:. The
Force
of
Habit. Swallow and convert its mud retort into a grass-lined nest of
its own. This occurred at
Basin Harbor, Vermont, in 1883, before the Sparrows were so generally condemned. The
nests were in line under the eaves of a farmer's barn, and the Swallows were still fighting
for possession. About every other nest was then occupied by the Sparrows. possession. y
p
y
Spa
o s. The Osprey
is not only one of the most remarkable nest-builders in the world, but a
wonderful adept
in
making
the most
of
its opportunities. In
selecting
a
site
for
its
mountain
of a
nest,
it seems
at times
to
exercise
little
choice, taking whatever
offers. Apparently its controlling ambition
is to raise a huge edifice in the construction of which
nothing comes amiss which can be seized and carried
in
its powerful talons. In
its build-
ing operations this bird seems to have an eye for the centuries rather than the years, and p
y
p
y
p
The Osprey
is not only one of the most remarkable nest-builders in the worl
wonderful adept
in
making
the most
of
its opportunities. In
selecting
a
sit
mountain
of a
nest,
it seems
at times
to
exercise
little
choice, taking whateve
Apparently its controlling ambition
is to raise a huge edifice in the construction
nothing comes amiss which can be seized and carried
in
its powerful talons. In
ing operations this bird seems to have an eye for the centuries rather than the ye
some of
its eyries formed
on
rocky crags have
possibly ex-
isted for more than a hundred
years, or might
last so long
if
undisturbed
by
man. g
* Thf
Osprtv,
vol.
ii.,
p.
55.
Many
interesting
pictures of
nests
of
the Osprey have
been published
in
this
magazine. | Male RRRRRRRRRRRRR
RRR An observer who described a nest on
an
old windmill
said that while the fan of the
mill was gone "the rudder remained, and the wind catching this would swing the nest
part way round, and then the wind changing slightly would swing
it back again
; the sit-
ting female
not seeming
to mind
the movement
in
the
K'.i-st." ting
g
On the shore of Narragansett Bay, Rhode Island, which has been colon g
g
On the shore of Narragansett Bay, Rhode Island, which has been colonized by Fish
1 " Breeding Habits of
I lie Fish Hawk on Plum Island New York."
('. S. Allen, Th< Auk, vol. ix. p. 315. 1892. * Thf
Osprtv,
vol. ii.,
p. 55. Many
interesting
pictures of
nests
of
the Osprey have
been published
in
this 1 " Breeding Habits of
I lie Fish Hawk on Plum Island New York."
('. S. Allen, Th< Auk, vol. ix. p. 315. 1892. Wild
Birds. ii6 Hawks from an
early period, the birds are not only protected by
law, but are offered
every inducement to make them feel at home. 1
When a dead
tree containing a nest
is
blown down the owner of the land will sometimes erect a tall pole, with a carriage wheel
laid
flat
on
top. The
birds
readily accept the new wheel
of fortune, which becomes
their home. In selecting a bare tree or a wheel on top of a pole the hawk makes a nice choice, for
owing to its great extent of wing, as with the eagle,
it
is convenient to have the path to
the
nest
free from
obstructions. When an Osprey loses its mate its actions seem to depend on its character. A case is
reported where two birds were seen to pair on the second day after each had lost a mate,
while another who was bereft by a stroke
of
lightning, which destroyed both
the
nest
and the sitting
bird,
is said
to have lingered about the spot
for the remainder
of the
summer, and to have even returned the next year still unmated to his solitary vigil.' ,
y
y
The diet of an insectivorous bird
is extremely varied
at
all times, depending much
upon the locality and the season of the year. 1 For an interesting r.ccount of the nesting habits of the Osprey see Forest ami Stream, July
7,
1900.
2 The Osprey, vol.
ii,,
p.
50.
3 Nature,
vol.
!xii.,
p. 366, 1900. 3 Nature,
vol.
!xii.,
p. 366, 1900. 1 For an interesting r.ccount of the nesting habits of the Osprey see Forest ami Stream, July
7,
1900.
2 The Osprey, vol.
ii,,
p.
50.
3 FEAR IN BIRDS. BIRDS
as a rule are possessed of
fear which
is primarily an
instinct, but as we shall
see
later on many species
in their
natural
adult state
are entirely devoid of
this
sense. With others
it may \vax or wane according to their environment or indi-
vidual experiences. Again
the nature of the
fear
manifested
varies
with
age
or
the
period of life. It
is a generalized sense of fear, or fear of the strange and unusual, which
comes over the young bird, while later it learns to dread particular objects or sounds with
which some bitter experience
is associated. Furthermore, the time of the appearance of
the instinct varies in different species, coming late in some and early in others. Generally
speaking the manifestation of
fear
is well timed, and
is an adaptation for the good of
its
possessor. BIR p
Let us
first
see how fear enters
into the
life
of the young. Birds are sometimes
roughly classified
into altricial
species, which feed
their young
for days
or weeks at the
nest, and praecocial
birds, whose young are born clad
in soft down, and are able to walk,
run, or swim at once or very soon
after hatching. The Altrices
like the
Robin, Wood-
pecker, and
Humming-bird are hatched from
eggs which are small in relation to the si/.e
of the
parent, and
the young
are
at
first
blind, helpless, and more or
less completely
naked. In
all such the nest
is only a temporary home, but
is often very elaborate, while
the instinct of fear
is delayed or deferred until the time of flight, a period varying from a
few days
to three weeks
or more. The
Praecoces
lay eggs with big yolks, upon
the
stored energy of which the unhatched young subsist until they step forth into the world,
seeing, able to walk
or swim, and
in some degree their own
masters. The common do-
mestic fowls, Partridges, Ostriches, Geese, Loons, Plover, and Snipe, are some of the better
known
representatives
of
this group, but the
dividing
line
is never sharply drawn, and
there are innumerable gradations between the extremes in either class. g
When
I
first camped beside
a
Catbird's
nest (No. 6 of table. Chap.
I.) last summer,
the young, who were then about a week
old, were incapable of
fear.
They would
shift
about the nest to get into the shade, pant, and erect their growing head-feathers.
When
a breeze rocked
the
cradle, or a Redwing Blackbird sang
his
conquer-ce, or
the parent | Male RRRRRRRRRRRRR
RRR While a few kinds of insects may be avoided
because of a repugnant odor or
taste, they capture
as
a
rule whatever comes
in
their
way. The Robin commonly
brings
to
its
nest
grasshoppers,
crickets,
katydids,
and
angleworms, because in
its customary manner of search
it finds and
is able to secure these
forms in abundance. The Kingbird, which takes most of its prey on the wing, discovers a
far greater variety. When certain species of
insects are abundant they are often eaten
by many birds who under ordinary conditions would never touch them. Thus during a
plague of Rocky Mountain
locusts which
visited
the Western States, these
insects are
reported
to have been
eaten by nearly every
bird
in
the
region, and
to have
served
as a staple
for most
of
the
species. Birds
of
prey such
as
the
smaller hawks and
owls devoured them
eagerly. The food
habits
of most
birds
are
exceedingly
plastic
and
liable
to sudden change under the spur
of
necessity. g
p
y
A good illustration of a change in feeding habits has been recently given. 3
It appears
that the Rhinoceros-bird (Buphaga crythnrpync/ia) was until lately regarded as so valuable a
scavenger that
it was accorded special protection by law in British East Africa. Its habit
was to feed on the ticks and other parasites which infest wild and domestic animals. " Since
the cattle plague," says Captain Hinde, " destroyed the immense herds in Ukambani, and
nearly all the sheep and goats were eaten during the
late famine, the
birds, deprived
of
their food, have become carnivorous, and now any domestic animal not constantly watched
is killed by them. Perfectly healthy animals have
their
ears eaten down
to
the bone,
holes torn in their backs and in the femoral regions."
The new conditions introduced by
man have thus converted a useful animal into a dangerous pest. FEAR IN BIRDS. In
the pr.tcocial
birds the feeling of fear is either present at birth, or appears in a very few hours or days, g
p
pp
y
y ,
As an illustration of the development of
fear in the
altricial kinds one might select
any of the common passerine birds. Thrushes, Warblers, Finches, or Flycatchers, but we
should bear in mind that the development of this instinct
is not always uniformly timed,
even in the same species. We will choose the Catbird, the Chestnut-sided Warbler, and
the Kingfisher. g
When
I
first camped beside
a
Catbird's
nest (No. 6 of table. Chap. I.) last summer,
the young, who were then about a week
old, were incapable of
fear. They would
shift
about the nest to get into the shade, pant, and erect their growing head-feathers. When
a breeze rocked
the
cradle, or a Redwing Blackbird sang
his
conquer-ce, or
the parent "7 Wild
Birds. iiS Fig. 113. Eggs and young of altricial Cedar-bird. Young about
thirty-six hours old
blind and naked. came
with
meat
or
fruit
they
stretched
necks,
opened
mouths,
each struggling to get some advant-
age over the other, and uttered their
sharp
tsit ! (sit ! notes. You could
handle them at
will
; they were ab-
solutely
fearless. If such
a
nest
is
overturned
they will cling
to
it but
will never cower or crouch. As we have seen, the clipping of
a leaf at this nest two days later sent
them
off
in a
panic, and
all hurried
to
the
nearest
cover. Should you
succeed in catching them under such
circumstances, which is doubtful, and
try to replace them
in the nest, they
will pop out repeatedly as if mounted
on
springs, and
if you
try
to hold
them
in the hand they will struggle,
squeal and
fairly
shriek
in their
en-
deavors
to
escape. They
are now
covered with a
coat of
slate-colored
feathers,
but
fly
with
difficulty. When
placed on open ground they
hop
off
at once toward
the nearest
bush. No
greater
change
in
the
behavior of a wild
bird
is ever
wit-
nessed than that which the sense of
fear brings to pass. Fig. 113. Eggs and young of altricial Cedar-bird. Young about
thirty-six hours old
blind and naked. Fig. 114. Bird on
left shows instinctive response
to sound
or
vibratory movement, and the use of wings for support. Fig. 114. FEAR IN BIRDS. Bird on
left shows instinctive response
to sound
or
vibratory movement, and the use of wings for support. I have seen
a young Chestnut-
sided Warbler jump out
of
its nest,
when
unable
to
stand
erect
and
much less to
use
its wings. In this
case
the
pin-feathers
of
the wings
had
barely burst, and
the body was
nearly
naked. When
the
bird was
returned to its nest,
it refused
to re-
main
until the operation was many
times
repeated
and
it
was
finally
overcome by fatigue. I have known
the young of the Redstart
to
leave
the nest remarkably
early, but
the
case
just
recorded
appears
to
be
somewhat exceptional. Fig. 115. Bird to the left
in reptilian crouching attitude
; at the
right, as
in Fig. 113, the characteristic response of the new-born
bird is seen, and the use of the pot-belly as a foot. About
^ life -size. Fig. 115. Bird to the left
in reptilian crouching attitude
; at the
right, as
in Fig. 113, the characteristic response of the new-born
bird is seen, and the use of the pot-belly as a foot. About
^ life -size. The instinct
of
fear comes with Fear in Birds. 119 a certain maturity of the nervous system, with comparative suddenness, as we have just
seen, but
is
usually timed
to correspond with the development
of the wing-quills and
the power of flight. p
g
At the age of twenty-four days the
Kingfisher
is in full
feather, but shows no fear. He will perch comfortably on your hand
or shoulder, and pose in any desired position,
as the photographs made at this period will show, but the instinct soon appears after this
stage is passed. In from twenty-four to forty-eight hours later when these birds not only
possess the power of flight, but use
it at the first intimation of danger, their docile nature
has completely changed. With them the late development of this instinct
is most oppor- Fig. 116. Young Kingfishers twenty-four days old. They are capable of flight, but show
no fear. Fig. 116. Young Kingfishers twenty-four days old. They are capable of flight, but show
no fear tune, since they are not tempted to leave the security of their tunnel in the ground until
they can make long excursions and follow their parents to the favorite fishing grounds. FEAR IN BIRDS. On the other hand when the ducklings have been led to
the water no birds show a keener sense of
fear than they or respond more promptly to
the alarm signals of their parents. I was greatly impressed when a boy at the sight of a
Black Duck leading her trim
little fleet of yellow sail up the mouth of a small sedge-
h on of
fear could hardly come
he ducklings have been led to
or respond more promptly to
d when a boy at the sight of a
the mouth of a small sedge-
bordered
stream. The
old
bird
quickly gave the alarm,
rose,
veered,
and
flew
to-
wards
the
river,
while
the
young scrambled to the bank
and
hid
in
the
rushes. I
hunted
long
but
succeeded
in finding only one who
lay
flat
in the marsh and
kept
perfectly
still, true to
its
in-
herited
instinct. These
ducklings
had
not
been
afloat many
hours, and had
this action been repeated be-
fore,
the
lesson
could
not
have been
taught,
since,
as
we
have
seen,
the
young
under
such
circumstances
are left to their own devices. I
have
seen
a
young
chick
while
feeding
quietly
close to the house suddenly
turn
its
head,
look
straight
at the
zenith, and then
run
off in a panic of fear. Look-
ing up
also
I
saw
a Hen
Hawk sailing aloft like a toy
kite,
a
mere
speck
against
the
blue
heavens. I
think
it probable that the bird got
an
alarm
signal
from some
ook, and its response was not
so had ample time
to learn
per kite
it
is not
likely that
we
have
seen, with
the de-
, thus
indirectly causing the Fig. 117. Young Cedar-birds in displaced nest standing in characteristic at-
titude with upturned heads. Photographed on day of flight,
July
17, 1899,
when possessed of fear. For account, see page 60. Fig. 117. Young Cedar-birds in displaced nest standing in characteristic at-
titude with upturned heads. Photographed on day of flight,
July
17, 1899,
when possessed of fear. For account, see page 60. signal
other fowls in the yard
;
at
all events
it knew where
to
look, and its response was not
slow. This chicken may have been three weeks
old, and
so had ample time
to learn
its lesson,
if such
it was. FEAR IN BIRDS. tune, since they are not tempted to leave the security of their tunnel in the ground until
they can make long excursions and follow their parents to the favorite fishing grounds. y
g
p
Turning now to the
prascocial
birds, according to the best
testimony,
fear
in
the
domestic chick hatched in an incubator
is
at
first very slight and
is soon checked by con-
trary impulses such
as to
nestle
in a warm
place,
unless the
instinct be brought
into
immediate exercise. Mr. Charles A. Allen
says that the newly hatched young of the
Black Duck (Anas
obscura) show no fear, but will " cuddle under one's hand very confidingly."
1 once saw
a nest of this species on the shore of Lake Champlain, near Burlington, Vermont, on the
very verge of a high, overhanging cliff. It was set against the stems of a slender shrub,
the pulling of which would doubtless have precipitated the
entire clutch fifty feet into Wild
Birds. I2O on of
fear could hardly come
he ducklings have been led to
or respond more promptly to
d when a boy at the sight of a
the mouth of a small sedge-
bordered
stream. The
old
bird
quickly gave the alarm,
rose,
veered,
and
flew
to-
wards
the
river,
while
the
young scrambled to the bank
and
hid
in
the
rushes. I
hunted
long
but
succeeded
in finding only one who
lay
flat
in the marsh and
kept
perfectly
still, true to
its
in-
herited
instinct. These
ducklings
had
not
been
afloat many
hours, and had
this action been repeated be-
fore,
the
lesson
could
not
have been
taught,
since,
as
we
have
seen,
the
young
under
such
circumstances
are left to their own devices. I
have
seen
a
young
chick
while
feeding
quietly
close to the house suddenly
turn
its
head,
look
straight
at the
zenith, and then
run
off in a panic of fear. Look-
ing up
also
I
saw
a Hen
Hawk sailing aloft like a toy
kite,
a
mere
speck
against
the
blue
heavens. I
think
it probable that the bird got
an
alarm
signal
from some
ook, and its response was not
so had ample time
to learn
per kite
it
is not
likely that the water below. A
little delay in
the
instinctive
reaction of
fear could hardly come
amiss to young in such a nest. FEAR IN BIRDS. Had the dark object been a paper kite
it
is not
likely that
the fear evoked would have been appreciably less. pp
y
In
altricial
birds
the
sense
of fear
usually comes, as we
have
seen, with
the de-
velopment of the
flight feathers, but
it
is often premature, thus
indirectly causing the
death of thousands of birds every year. In July and August how many helpless spar-
rows and thrushes are found on the ground, having
left their nests too early
! Some-
times they tumble out by accident, are drawn off by hunger, or are blown out in a gale, i'Yur in Birds. but
I believe that by far the greater number of such strays are driven forth by fright, and
when
this perilous step has once been taken
it can seldom be retraced. The young of
such
birds
as the Wilson
thrushes, whose
nests
are on
or near the ground
out of the
reach of storms, are often found
in this predicament.' Many immature birds w
discrimination in any direc-
tion. You
will
see
them
respond as promptly to the
flutter of a
leaf or the call-
note of any passing bird as
to their own mother's voice
but
a more curious specta-
cle may be witnessed when
a
fledgling
of
one
of
our
common
species
like,
the
Baltimore Oriole climbs
to
the top of its nest. All the
others immediately salute it
as
if
it
were
an
old
bird,
and with open mouths beg
vainly to be fed. If a young
bird within a day of taking
flight
cannot
distinguish
one of its brothers from
its
mother,
it
can
hardly
be
expected to " know a hawk
from
a
handsaw,"
or
an
enemy from a friend. Fig. 118. Brown Thrush startled while at nest
: attitude of keen attention. After taking
flight the
young of altricial birds
are
fed by one or both parents
for
a
period
of
days
or
weeks, and much
is quickly
learned by imitation and
in-
dividual experience. Their
ingrained sense
of fear becomes
in the course
of time
gradually
specialized
in
certain
directions. Fear of man, guns, hawks, snakes, cats and the various agents of destruction
with which each species must contend
in
the course
of
its
life, seems
in every ca>e
t
be acquired or learned
ratln-r than inherited. Fig. 118. 1 The huge pot-belly of the young altricial bird has a
U-.L- <|uiu- apart from the function of
digestion.
It anchors
it to the nest, and as in the modern " Brownie" keeps
it right
side
up.
The
pliant viscera conform to every move-
ment, and form a central supporting
pillar long before the legs can
sustain
the weight of
the
body.
(See young
CeJar-birds in Figs.
113-115.) FEAR IN BIRDS. Brown Thrush startled while at nest
: attitude of keen attention. Fig. 118. Brown Thrush startled while at nest
: attitude of keen attention. experience. ingrained sense
of fear becomes
in the course
of time
gradually
specialized
in
certain
directions. Fear of man, guns, hawks, snakes, cats and the various agents of destruction
with which each species must contend
in
the course
of
its
life, seems
in every ca>e
t
be acquired or learned
ratln-r than inherited. acquired
On the hist day of June
I found a Cowbird nearly full-fledged but either unable or
disinclined
to
fly. He occupied the nest of a warbler, apparently the species known as Wild
Birds. I 22 the Black and Yellow or Magnolia Warbler, and as
his photograph shows, filled
it com-
pletely. He would stand on the rim of the nest and, with raised feathers, squeak and call
vehemently for his foster parents. I took from beneath him the dried mummy of a little
warbler and one addled egg, which illustrates the advantage nature gives this
bird over
his competitors
in
early life. He showed no fear, but clung like a monkey to the
nest,
while
I carried the branch several hundred feet to find a quiet place out of the wind. I
regret that
I cannot show the nurse feeding this monster, but unfortunately the day was
stormy and the bird was soon gone. ch
attract
or arouse other
ry. I remember seeing an
unusually striking exhibi-
tion
of
this
fact
while
watching
unobserved
some
Red
Crossbills
en-
gaged in picking the seeds
out
of pine
cones. They
were on
the ground
in
a
run where
it was impossi-
ble
for
the
birds
to
see
out
on
either
side. A
Crow espied me at
a
dis-
tance, gave his short quick
alarm car ! car ! when the
Crossbills
went
off
as
if
carried
in
a
whirlwind. They had apparently seen
nothing to awaken suspic-
ion, and
the crow
is
not
their enemy so far as
I am
aware. stormy
g
Many
birds have alarm
calls
or
signals
of
distress, which
attract
or arouse other
species, as every one knows who has studied birds in the country. I remember seeing an
unusually striking exhibi-
tion
of
this
fact
while
watching
unobserved
some
Red
Crossbills
en-
gaged in picking the seeds
out
of pine
cones. FEAR IN BIRDS. Thereupon going to the
place,
I
almost
stepped on what
looked
at
first
like a coil of rubber hose
in the grass. It proved to
be
a
large
black
snake,
whose
head
was distorted
in the act of swal-
lowing a young bird. The crisis
for
the
unfortunate
bird being
past,
I stood by and watched the
proceedings. The
snake
had
taken
his victim head
first, and
its body was slowly disappearing
between his distended jaws. As
I
disturbed
his
meal, he
folded
Fig. 120. Red-tailed Hawk, four m
,
. i
11
uu
VI
u
J
*. his own fear, and well calculated to
his
dull,
rubber-like body into a
bird at the
nest will spread
its win
coil and
his
gleaming
eyes
be-
ft-m, and hiss at intruders,
trayed
an
unpleasant
frame
of
mind. When
I approached
nearer, he lifted his swollen head
glided off to enjoy his spoils in peace; but his enemy followed. O
serpent at a disadvantage, but he did not remain muzzled long
that bird had to go down, notwithstanding the throes of deglut
the salivary glands, but they were equal to the task, and the pl
their victim. What
a
picture of
stealth this animal made
as
darting angry glances, he stole through the grass
! The first act o
it was time to add the final touches of the second
As
I struck y
On a warm July
da)- while
c
golden-rod and sweet-fern, my att
grasshopper
in
its beak. It was t
was clearly intended
for her youn
about, uttering her sharpest monosy
prey. Thinking that her young w
ently several Buntings dashed up
the
ground,
and
then
at
me,
emitting such a
flow
of
incisive
protests
as
to
suggest
the
at-
tempt
to
draw
attention
from
their nest. This was plainly not
the
case when
some
Kingbirds
left their young
in a neighboring
tree,
and
raising
their
war-cry,
hovered over the spot and darted
at some
object
on
the
ground. Thereupon going to the
place,
I
almost
stepped on what
looked
at
first
like a coil of rubber hose
in the grass. It proved to
be
a
large
black
snake,
whose
head
was distorted
in the act of swal-
lowing a young bird. The crisis
for
the
unfortunate
bird being
past,
I stood by and watched the
proceedings. FEAR IN BIRDS. y
On a warm July
da)- while
golden-rod and sweet-fern, my at
grasshopper
in
its beak. It was t
was clearly intended
for her youn
about, uttering her sharpest monosy
prey. Thinking that her young w
ently several Buntings dashed up
the
ground,
and
then
at
me,
emitting such a
flow
of
incisive
protests
as
to
suggest
the
at-
tempt
to
draw
attention
from
their nest. This was plainly not
the
case when
some
Kingbirds
left their young
in a neighboring
tree,
and
raising
their
war-cry,
hovered over the spot and darted
at some
object
on
the
ground. Thereupon going to the
place,
I
almost
stepped on what
looked
at
first
like a coil of rubber hose
in the grass. It proved to
be
a
large
black
snake,
whose
head
was distorted
in the act of swal-
lowing a young bird. The crisis
for
the
unfortunate
bird being
past,
I stood by and watched the
proceedings. The
snake
had
taken
his victim head
first, and
its body was slowly disappearing
between his distended jaws. As
I
disturbed
his
meal, he
folded
,
. i
11
uu
VI
u
J
*. his
dull,
rubber-like body into a
coil and
his
gleaming
eyes
be-
trayed
an
unpleasant
frame
of
mind. When
I approached
nearer
glided off to enjoy his spoils in peac
serpent at a disadvantage, but he di
that bird had to go down, notwith
the salivary glands, but they were
their victim. What
a
picture of
darting angry glances, he stole thro
was ti
t
add the fi
l touche y
joined
general outcr
On a warm July
da)- while
crossing a barren
strip of
la
golden-rod and sweet-fern, my attention was called
to a smal
grasshopper
in
its beak. It was the Bay-winged Bunting or
was clearly intended
for her young, but
instead
of deliverin
about, uttering her sharpest monosyllables, in the course of whi
prey. Thinking that her young were at hand,
I sat down to a
ently several Buntings dashed up to the spot a few yards away
the
ground,
and
then
at
me,
emitting such a
flow
of
incisive
protests
as
to
suggest
the
at-
tempt
to
draw
attention
from
their nest. This was plainly not
the
case when
some
Kingbirds
left their young
in a neighboring
tree,
and
raising
their
war-cry,
hovered over the spot and darted
at some
object
on
the
ground. FEAR IN BIRDS. They
were on
the ground
in
a
run where
it was i
i y
g
Many
birds have alarm
calls
or
signals
of
distress, which
attract
or arouse other
species, as every one knows who has studied birds in the country. I remember seeing an
hibi Fig. 119. Cock Robin startled while at nest by a quick,
decisive alarm
call
from his mate. His head shot up like a flash, and in a moment he was off. When
a robin
hears
the alarm call of his mate,
his head goes up instantly,
and
he
stands
for a mo-
ment with outstretched neck, listening intently to see
if he
is needed. I was
fortunate
in catching the male bird
at the nest
in just
this attitude, expressive
of attention and
wariness, bordering on fear. Fig. 119. Cock Robin startled while at nest by a quick,
decisive alarm
call
from his mate. His head shot up like a flash, and in a moment he was off. Fig. 119. Cock Robin startled while at nest by a quick,
decisive alarm
call
from his mate. His head shot up like a flash, and in a moment he was off. ment with outstretched neck, listening intently to see
if he
is needed. I was
fortunate
in catching the male bird
at the nest
in just
this attitude, expressive
of attention and
wariness, bordering on fear. g
A hawk, owl, crow, cat, snake, or any well-known or dreaded enemy of birds will set the
community in a hubbub in a very short time. Birds of other species hurry to the scene out
of sympathy or curiosity, as some would say, but probably more from instinct of a different
character. The smallest spark often kindtes the largest blaze. Thus while passing through
a pasture last June
I happened to encounter a Robin with mouth stuffed with food, as if on
the way to her nest. She at once set up a loud cry, and mounting the bare branch of a dead
apple tree, in five minutes drummed up eleven different birds, among which
I recognized
the Baltimore
Oriole, Brown Thrush, two Catbirds, Chestnut-sided Warbler, Red-eyed Fear in Birds. 123 Vireo, Maryland Yellow Throat, Song Sparrow, Chickadee, the Redstart and a Goldfinch,
many of which became excited and joined in the general outcry. FEAR IN BIRDS. The
snake
had
taken
his victim head
first, and
its body was slowly disappearing
between his distended jaws. As
I
disturbed
his
meal, he
folded
,
. i
11
uu
VI
u
J
*. his
dull,
rubber-like body into a
coil and
his
gleaming
eyes
be-
trayed
an
unpleasant
frame
of
mind. When
I approached
nearer
glided off to enjoy his spoils in peace
serpent at a disadvantage, but he di
that bird had to go down, notwiths
the salivary glands, but they were
their victim. What
a
picture of
darting angry glances, he stole thro
it was time to add the final touche
he shot through the grass, and
it r Fig. 120. Red-tailed Hawk, four months old, in attitude expressive of
his own fear, and well calculated to inspire fear in others. The young
bird at the
nest will spread
its wings ls well as ercct its Elizab
y
ethan
ft-m, and hiss at intruders, Fig. 120. Red-tailed Hawk, four months old, in attitude expressive of
his own fear, and well calculated to inspire fear in others. The young
bird at the
nest will spread
its wings ls well as ercct its Elizab
y
ethan
ft-m, and hiss at intruders, coil and
his
gleaming
eyes
be-
ft-m, and hiss at intruders,
trayed
an
unpleasant
frame
of
mind. When
I approached
nearer, he lifted his swollen head high in the
air, and slowly
glided off to enjoy his spoils in peace; but his enemy followed. On this occasion we had the
serpent at a disadvantage, but he did not remain muzzled long
Having proceeded thus far,
that bird had to go down, notwithstanding the throes of deglutition. It was a tax upnn
the salivary glands, but they were equal to the task, and the pliant jaws soon closed over
their victim. What
a
picture of
stealth this animal made
as with head erect, and eyes
darting angry glances, he stole through the grass
! The first act of the tragedy being closed,
it was time to add the final touches of the second. As
I struck at him with my cane how
he shot through the grass, and
it required no
little speed to reach him for the fatal blow
! TAMING WILD BIRDS WITHOUT A CAGE. M
illustrations could be given of
birds which in most parts of
their range are
wild or shy while in others they are very tame r and the same principle underlies
them
all. Wildness
is due to fear which
is partly inherited and partly learned
by experience with
this wicked
world. Tameness, on
the other hand, comes with the
casting out of
fear, and may be brought about by the formation of new habits which are
either spontaneous or forced. M p
The House Sparrows
of the
Tuileries, and the pious Stork of
Holland, Germany
and
France, are familiar examples of
birds whose near or remote ancestors are shy and
wary. The Stork
is said to be excessively wild
in the woods and marshes, yet
it comes
with confidence to the village and town, builds
its nests upon house tops and
steeples,
and struts about the streets and door-yards in search of food. y
It would be interesting to know how long the Doves of Venice have enjoyed the
freedom of the Piazza del Marco. They are probably the best
fed pigeons in the world,
and few hours pass
in the course of the day when
their guardian, the vendor of sacks of
corn,
is not surrounded by his flock. They will alight
all over you, and take the grain
from hand or mouth. The
Pigeon,
it
is true, has been long domesticated and responds
more readily to friendly influences than the wild stock from which
it has sprung. g
Strange and possibly true stories are told of persons who have won the confidence
of beast or bird. The wild
bird responds to their
call and the quadruped comes forth
from his den and takes food from their hand. Such persons are popularly supposed to
possess a mysterious power of
fascination
or a superior knowledge of woodcraft, but
all
this belongs in the catalogue of vulgar errors. It depends less upon the individuality of
the person than that of the animal. Individual variation knows hardly a limit, whether
in man
or beast. Some birds are
naturally tame and confiding, while
their next door
neighbors of the same kin and
living
in the same
field may possess a temperament
of
such an opposite character as to baffle every attempt to dispel their fears. Wild
Birds. 124 We have seen that the instinct of
fear
is inherited, and often delayed, where
it
is a
special adaptation, not only leading the young, as Lloyd Morgan remarks, to accept a
foster parent and not to shrink from her, but what
is more important, keeping the young
in the
nest, barring accidents, until they can
in some degree help themselves. Fear of
particular objects
is learned, or becomes grafted on
to the original stock. The
instinct
may gather
force
or disappear,
at
least
in
adult
life, according to the
nature
of
the
environment and the new habits formed
in consequence. The instinctive basis of fear is
apparently handed down from generation to generation, but in the life of the full-grown
bird,
it
is probably
largely replaced by
habit,
or the formation
of
associations. The
innate or latent
capacity remains, but
the definite
association of
certain
actions with
particular objects or experiences
is probably handed down by tradition
rather than by
heredity. Fig. 122. Young Cowbird comfortably
filling the nest
of its foster parent, whose children
it smothered:
fearless,
though nearly ready to fly. Fig. ill. Young Cowbird and nest of Magnolia Warbler
Fig. 122. Young Cowbird comfortably
filling the nest
in which it was reared. of its foster parent, whose children
it smothered:
fearless,
though nearly ready to fly. Fig. ill. Young Cowbird and nest of Magnolia Warbler
in which it was reared. Fig. ill. Young Cowbird and nest of Magnolia Warbler
in which it was reared. Fig. ill. Young Cowbird and nest of Magnolia Warbler
Fig. 122. Young Cowbird comfortably
filling the nest
in which it was reared. of its foster parent, whose children
it smothered:
fearless,
though nearly ready to fly. Fig. ill. Young Cowbird and nest of Magnolia Warbler
Fig. 122. Young Cowbird comfortably
filling the nest
in which it was reared. of its foster parent, whose children
it smothered:
fearless,
though nearly ready to fly. Fig. 122. Young Cowbird comfortably
filling the nest
of its foster parent, whose children
it smothered:
fearless,
though nearly ready to fly. Wild
Birds. 126 It
is easy to conceive a state
in which
all animals would be tame, but
it would not
be the state
of nature known to us which has developed under the laws
of
battle, the
survival of the strongest, the wariest, the best protected or concealed, or the most intelli-
gent. The higher animals either prey on one another or on the helpless invertebrates, or
are preyed upon, and with most, tameness would soon lead to extinction. Wildness or
wariness
is not only the law of
their nature, but
the very condition of
their
existence. The animal which fails to
profit by experience,
or
at
least to the extent
of
learning caution, and thus
displaying the rudiments
of
intelligence, must
go
to
the
wall,
unless
the
conditions of
its
life
are
exceptional
or nature
grants
it some extraordi-
nary favor such as protec-
tive or deceptivecoloring. It
is easy to conceive a state
in which
all animals would be tame, but
it would not
be the state
of nature known to us which has developed under the laws
of
battle, the
survival of the strongest, the wariest, the best protected or concealed, or the most intelli-
gent. The higher animals either prey on one another or on the helpless invertebrates, or
are preyed upon, and with most, tameness would soon lead to extinction. Wildness or
wariness
is not only the law of
their nature, but
the very condition of
their
existence. The animal which fails to Fig. 123. Male standing at nest after having fed his young. Notice the character-
istic instinctive pose of one of the fledglings. p
g
While most animals
are wild
in
the
state of
nature and many are
al-
most untamable,
a com-
paratively
large number
submit to the taming pro-
cess, and
a
few become
tame in the natural state. The principle of the sur-
vival of the strongest or
the
fittest
as a result of
the struggle for existence
is so general and so primi-
tive
that
when we
find
animals
already tame
in
nature, we
must
regard
them as the descendants
of wild ancestors Fig. 123. Male standing at nest after having fed his young. Notice the character-
istic instinctive pose of one of the fledglings. As
a
rule
no
wild
beast or bird approaches man without some inducement. TAMING WILD BIRDS WITHOUT A CAGE. pp
y
p
p
The power
of remaining
motionless
like
a
stone
or stump
in the woods
is
often
enough to win the temporary confidence of both mammal and bird, and many will doubt-
less
recall
illustrations of
this
fact
from
their own
experience. This suggests an early
episode which impressed
itself
rather strongly at the time. With
raised
fishing-pole in
hand
I was sitting
quietly by the river, possibly watching the common sunfish or bream
standing guard
over
their
nests, which they defend
with such
fiery pugnacity, when
I
suddenly had a " bite."
Looking up,
I saw a Kingbird comfortably perched on the end
of my rod. He doubtless had a nest in the alders close by. 125 125 Wild
Birds. Unless some other instinct be
aroused,
it comes,
if
at
all,
to defend
or
feed
its
offspring,
to appease
its hunger, or
in
very
rare
cases
to
find
protection
froin
danger. The taming
process
depends,
as
we have
just
seen, upon
the
ability
to form
new
associations and may
be
brought
about
artificially by
restraint
as when
a
wild animal
is caged and new habits
are,
as
it were,
forced upon
it,
or by means
of strong
lures. Of the
latter, one
of the
best
is
food
in the presence
of
hunger,
but
the strongest
of
all
are the young
at
a
cer-
tain
stage
of growth. In
order
to tame
a wild animal without recourse
to
restraint beast or bird approaches man without some inducement. Unless some other instinct be
aroused,
it comes,
if
at
all,
to defend
or
feed
its
offspring,
to appease
its hunger, or
in
very
rare
cases
to
find
protection
froin
danger. The taming
process
depends,
as
we have
just
seen, upon
the
ability
to form
new
associations and may
be
brought
about
artificially by
restraint
as when
a
wild animal
is caged and new habits
are,
as
it were,
forced upon
it,
or by means
of strong
lures. Of the
latter, one
of the
best
is
food
in the presence
of
hunger,
but
the strongest
of
all
are the young
at
a
cer-
tain
stage
of growth. In
order
to tame
a wild animal without recourse
to
restraint Turning Wild Birds without
;i Cas^c. 127 there must be
sonic means
of breaking
the
ice,
or beginning a
course
of
instruction,
by
chaining
it
to
a
fixed
point. In
case
of
birds
with young the
invisible chain
is
parental
instinct, which inhibits fear and
holds
the animal
to a given spot. We will
at-
tempt to analyze the taming process by
the
ust;
of
food and young birds as lures, and
finally consider the
similar experiments which nature
occasionally conducts independ-
ently ami on a
larger
scale. I throw some
cracked
cor
ubiquitous Sparrows. When
they
.see me standing behind
the
pane they
are
afraid
to
approach,
but
they
are
also
hungry. At
last the impulse
to
get
the
food
overcomes
their
fears, and
they are
re-
warded by the feeling of pleas-
ure and
satisfaction. y
g
the experiment with success in any reasonable time.
There
are many species which respond more rapidly than
the
wily Sparrow.
Of
these,
I will mention
the Chickadee, Nuthatch, Canada Jay, and Goose.
The Chickadee
has
to work harder
for a living
in
winter than
the Sparrow,
is
far
less gregarious and
wary by nature, and
is seemingly endowed with a keen sense of curiosity.
Mr. Chapman
thus speaks
of
the behavior
of some of these
birds
in
Central
Park, New York
City,
in February: "they would often flutter before one's
face and plainly give expression to
their desire
for
food, which they took from
one's hand without the
slightest evidence
of fear.
Sometimes they even remained
to pick the nut from a shell while perched on Wild
Birds. Early in the winter of 1899, a Red-breasted Nuthatch formed the habit of going to
certain yard
in Jefferson, Ohio, for
food."
At
first
it stayed among the trees like the
rown Creepers, but at length came
to
the
window-sill
for
scraps
of
suet which were
placed
there. This window
happened
to
be
opposite
a
pump and
sink, but the Nut-
hatch
soon
showed
no
fear
even when one stood close by
and worked the pump. Blue
Jays, Downyand Hairy Wood-
peckers, Chickadees, and Eng-
lish Sparrows also came to the
garden for food. After several
weeks
of
this
kind
of
treat-
ment
Mr. Sim went
outside,
placed some suet on his palm
and
rested
his
hand
on
the
window-sill. The
Nuthatch
came to the
lure, picked up a
piece
of the
food, and appar-
ently tried
to hide
it between
his thumb and
finger. After
the Red-breasted Nuthatch de-
arted a Whitebreast came down, helped himself to the suet and was off. After this the
uthatches often came and alighted on somebody's hand, head, or shoulder, but the Red-
east was much the tamest. When she was up in the big elm tree, she would swoop down
call, not
touching a twig between her
lofty perch and the hand. Hickory nuts were
fered and
preferred to the suet, but the seeds of the Norway spruce were
still more to
er taste. She would
fly to a branch with a seed, rub off
its wing, and
after placing
it
a suitable notch or crack, eat
it leisurely. The Red-breasted
Nuthatch
would
drink
rom
a dish
held
in
the
hand, would
take
the
proffered
food while perched near the
round, and once even settled down in the hand as
if going to sleep. Fig. 125
Female Red-eyed Vireo feeding young. In these birds the be-
avior was perfectly free after the first day of study. Fig. 125
Female Red-eyed Vireo feeding young. In these birds the be-
havior was perfectly free after the first day of study. Fig. 125
Female Red-eyed Vireo feeding young. In these birds the be-
havior was perfectly free after the first day of study. parted a Whitebreast came down, helped himself to the suet and was off. After this the
Nuthatches often came and alighted on somebody's hand, head, or shoulder, but the Red-
breast was much the tamest. 1 Bud Studies with a Camera,
p.
4<).
* For this account
I am indebted to Mr. Robert J. Sim of Jefferson, Ohio. Wild
Birds. When
they
come
repeatedly,
each
time reaping a reward without
evil consequences, a new habit
is
gradually
formed
by
the
repetition
of
the
act. The
pleasure
of
getting
food
is
gradually associated
with
fly-
ing
to
a
certain
spot
in
the
presence
of objects
which
in
the
course
of
time
become
familiar. If the contrary
im-
pulse, due
in this case to hun-
ger,
is
sufficiently
strong, the
process may be carried forward
step by
step
until
the
birds
come
to
the
hand
for
food. With the gregarious Sparrow,
however,
life
in
a
populous
town
is
usually
too
compli-
cated to admit of carrying out
the experiment with success in Fig. 124. Female Robin
in act of nest-cleaning. She approaches at the
back. See Chapter XII. Fig. 124. Female Robin
in act of nest-cleaning. She approaches at the
back. See Chapter XII. p
y
There
are many species which respond more rapidly than
the
wily Sparrow. Of
these,
I will mention
the Chickadee, Nuthatch, Canada Jay, and Goose. The Chickadee
has
to work harder
for a living
in
winter than
the Sparrow,
is
far
less gregarious and
wary by nature, and
is seemingly endowed with a keen sense of curiosity. Mr. Chapman
thus speaks
of
the behavior
of some of these
birds
in
Central
Park, New York
City,
in February: "they would often flutter before one's
face and plainly give expression to
their desire
for
food, which they took from
one's hand without the
slightest evidence
of fear. Sometimes they even remained
to pick the nut from a shell while perched on Wild
Birds. 128 one's finger."
'
They become equally tame when hard
pressed by hunger in the remote
woods, and I have no doubt
that
the
following account
which was
given
to me by a
man who worked
at
a
woodchopper's camp
in New
Hampshire
during
the
winter
is strictly true. He said that at meal times
the Chickadees would come about and pick
up any crumbs that were left over or were thrown
to them, and
that they soon became
so bold as to
alight on the
hand, or
hat, and
even
to
take
pieces
of bread
from
the
mouth
;
that he would
often amuse himself by
trying to " close
over them " with his
hand, and that while they were
usually too
quick
for
him, he had caught them in this
manner. anner. ,
p.
4 ).
For this account
I am indebted to Mr. Robert J. Sim of Jefferson, Ohio. Wild
Birds. Im-
mediately on seeing the tempting
morsel, the Jays alight on
it, and
while
they
are
busily engaged
in devouring
it, the woodcutter
gives a smart blow to the end of
the
pole within
the
hut, which
seldom
fails to
drive
the
birds
high
in
the
air, and
not
infre-
quently
kills them. They even
enter the camps and would fain eat from the hands of the men whi
Possibly no bird
has keener vision or sharper ears than the C
its wild state is said to be vigilant, suspicious, and hard to be surpris
Fig. 126. Male Red-eyed Vireo prepare
Notice that
in
this series
Figs. 50-57, 125
the same perch. Detail of bird shown in The
familiarity
of
the Canada Jay or Meat
Bird
is known to everybody who has
hunted or camped in the northern woods;
its fear is allayed by hunger even more promptly
than
in Chickadees and
Nuthatches. Audubon
says of
these
birds
that " when
their
appetite
is satisfied, they become shy, and
are in
the habit of
hiding themselves among
close woods or thickets
; but when hungry they show no alarm
at the approach of man."
While his friend was fishing in a canoe on one of the Maine lakes in the summer of
1833, The
familiarity
of
the Canada Jay
hunted or camped in the northern woods;
than
in Chickadees and
Nuthatches. A
appetite
is satisfied, they become shy, an
close woods or thickets
; but when hungr
While his friend was fishing in a canoe on
"the Jays were
so
fearless
as to
alight
in
one
end
of
his
bark,
while
he
sat
in
the
other, and
help themselves to his bait. . . . The
lumberers
or
woodcutters
of
this
state,
. . . frequently
amuse themselves
in their camp
during the eating hour with what
they
call
' transporting
the
car-
rion bird.'
This is done by cut-
ting
a pole eight or ten
feet
in
length, and
balancing
it on
the
sill
of
their hut, the end outside
of the entrance being baited with
a piece of flesh of any kind. Wild
Birds. When she was up in the big elm tree, she would swoop down
at call, not
touching a twig between her
lofty perch and the hand. Hickory nuts were
offered and
preferred to the suet, but the seeds of the Norway spruce were
still more to
her taste. She would
fly to a branch with a seed, rub off
its wing, and
after placing
it
in a suitable notch or crack, eat
it leisurely. The Red-breasted
Nuthatch
would
drink
from
a dish
held
in
the
hand, would
take
the
proffered
food while perched near the
ground, and once even settled down in the hand as
if going to sleep. g
,
going
p
These
birds were seen
to
eat
snow, and Chickadees would
frequently
cling
to an
icicle on the roof and catch the drops of water as they fell from a shorter
icicle near by. Taming \Vild Birds without a Cage. 129 Three or four Downy and Hairy Woodpeckers c.une to the window-Mil, and would some-
times peck the fingers of persons
feeding them. The Hrown Creeper was far more cau-
tious, and never came to the hand. The
familiarity
of
the Canada Jay or Meat
Bird
is known
hunted or camped in the northern woods;
its fear is allayed by hunge
than
in Chickadees and
Nuthatches. Audubon
says of
these
b
appetite
is satisfied, they become shy, and
are in
the habit of
hid
close woods or thickets
; but when hungry they show no alarm
at
While his friend was fishing in a canoe on one of the Maine lakes i
"the Jays were
so
fearless
as to
alight
in
one
end
of
his
bark,
while
he
sat
in
the
other, and
help themselves to his bait. . . . The
lumberers
or
woodcutters
of
this
state,
. . . frequently
amuse themselves
in their camp
during the eating hour with what
they
call
' transporting
the
car-
rion bird.'
This is done by cut-
ting
a pole eight or ten
feet
in
length, and
balancing
it on
the
sill
of
their hut, the end outside
of the entrance being baited with
a piece of flesh of any kind. Wild
Birds. They once had the habit of alighting on the roof
of a tall building
near Wade
Park, but after one of
their number met with the mishap of
falling down a
ventilating shaft this practice seems to have been abandoned. g
Audubon speaks of a pair of geese which bred for three years near the mouth of the
Green River in Kentucky,' and of
his experience in feeding them at the nest. The male
was at
first very pugnacious, and once dealt him such a blow on the arm that he thought it
was broken. In the course of a week both birds would take the proffered corn, but never
allowed him to touch them. " Whenever I attempted this," says Audubon,
" the male met
my fingers with his bill, and bit me so severely that
I gave
it up."
Later he trapped the
entire family of
eleven, pinioned them and turned them loose
in his garden. He kept
the whole
flock three years. The old
birds did not breed again, but two pairs
of
the
young reared new broods. young
On one
of
his shooting excursions Audubon shot a wild
goose, and on
his
return
sent
it to the kitchen to be prepared for the table. The cook brought him an egg ready
to be laid. This was placed under a hen, and in due time produced a bird, which became
very gentle and would
feed
from the hand. When two years old
it mated
with a male
and reared a family. y
We have seen how fear may vanish before the surge of
the parental impulse which
impels a bird to seek, nourish, and defend
its offspring, even at the risk of life itself, and will
now consider how
this
instinct may be
used
in taming wild birds
at
the
nest and
in
bringing them to the hand. g
g
If young birds of those species
in which the parental
instincts are very strong, are
taken from the
nest when nearly ready to
fly, the old birds, especially
if they be among
the
class
of tamer individuals, may be brought
direct
to the hand
in a short space of
time. To their excited
vision men are as walking trees. Their attention
is riveted on
the young, and
the man
is
nothing
to them, providing
he remains
quiet,
or moves
about with caution. Wild
Birds. Im-
mediately on seeing the tempting
morsel, the Jays alight on
it, and
while
they
are
busily engaged
in devouring
it, the woodcutter
gives a smart blow to the end of
the
pole within
the
hut, which
seldom
fails to
drive
the
birds
high
in
the
air, and
not
infre-
quently
kills them. They even
enter the camps and would fain eat from
Fig. Notice
the sa Fig. 126. Male Red-eyed Vireo prepared
to inspect and clean
nest. Notice that
in
this series
Figs. 50-57, 125
the birds uniformly occupy
the same perch. Detail of bird shown in Fig. 49. Fig. 126. Male Red-eyed Vireo prepared
to inspect and clean
nest. Notice that
in
this series
Figs. 50-57, 125
the birds uniformly occupy
the same perch. Detail of bird shown in Fig. 49. quently
They
enter the camps and would fain eat from the hands of the men while at their meals." p
Possibly no bird
has keener vision or sharper ears than the Canada Goose, which in
its wild state is said to be vigilant, suspicious, and hard to be surprised, yet it
is often easily
and quickly tamed. There are in Cleveland nearly forty of these geese, which are descended
from a smaller number introduced about twenty-five years ago. Their migratory impulse
has been completely
lost, and their sense of
fear subdued, but their other wild instincts
remain. They
live mostly
in the parks, going from one to another as the spirit moves
them, and
breed
on
the small
artificial
islands
in
artificial
ponds. I sometimes hear
their honk ! as they fly over the city at night or in early morning, and see their" harrow"
or "triangle" which plows the air by day often within bow-shot from
Euclid Avenue. g
p
y
y
When the birds are feeding on a lawn you can walk among them and drive them
like a flock of tame geese, but they hate dogs and take to wing or water the moment one Wild
Birds. 130 is seen to approach. They once had the habit of alighting on the roof
of a tall building
near Wade
Park, but after one of
their number met with the mishap of
falling down a
ventilating shaft this practice seems to have been abandoned. is seen to approach. y
1 Ornithological Biography, vol.
Hi., pp.
S,
9. Wild
Birds. Whatever
fear remains
is blocked by the stronger
instinct
to go
to their young. y
g
Every occasion on which the tent described
in these pages
is brought up to a nest
of young birds
is a direct experiment
in the taming
process. No matter how
far the
discipline
is carried or how little permanency
it may possess, the principle
is always the
same. By this method wild birds, while the parental instincts are at their height, can be
tamed to a degree without use of a cage. In illustration of the process, we will select the
Robin and Chestnut-sided Warbler, although the experiments to be described were not
carried out with this end
especially
in view. In any case parental
instinct
is the chief
agent employed. g
p
y
The Robins now referred to have served
so often
in
these pages as a text
for the
illustration of habit and instinct that
I need only say that they nested high in an oak tree
in some woods, and that the entire branch with the
nest was carried to a perfectly bare
field on July 25th, when the young were a week old. At this new site the young passed
another week, taking their first flight
at noon on the
last day of the month. I was en-
camped beside them for parts of six days, and spent altogether twenty-four hours at their
nest. Although the familiar Robin
is usually an easy mark for the bird-photographer, this
particular
pair were extremely wary. They showed a bold front when openly assailed, y
y
1 Ornithological Biography, vol. Hi., 1 Ornithological Biography, vol. Hi., pp. S,
9. Taming Wild Birds without a Cage. 13' but
succumbed
to
fear
completely,
the moment the tent was
closed, and
refused
to
approach
the
nest. On
the
second day the female was on the
nesting bough
in
ten
minutes,
but
hesitated and made seven consecutive
visits before
actually
feeding the
y<>ung. After several hours their fear had become so well subdued that
the wary male
brought and delivered food while
I was engaged
in taking down the tent and stood close
by. On the third day the young were
fed while the
tent was going up, but
a
full half
hour had
elapsed before
their
behavior was
perfectly
free and spontaneous. Wild
Birds. On
the
fourth day the birds came as
before, and
life at
the
nest was resumed with perfect con-
fidence after the space
of twenty minutes. The
female would now
sit
placidly on the
nest
in face of the tent and the window in
its front, across which the hand was frequently
drawn to adjust the shutter that was clicking at random intervals but twenty-eight inches
from her ears. At the close of the day's observations,
I took the camera outside the tent, and photo-
graphed the male as he came
to the nest. The moment
I
entered
the tent
to take
it
down he was back again with a mouth
full of cherries. When after striking the tent and
rolling
it up
I
stood quietly by the nest for a few moments, the cock came
for the third
time and delivered a large grasshopper to his never-to-be-satisfied brood. g
g
pp
On the
first day four hours failed to bring these birds to their needy children, while
in the ninth and
last the male, the more suspicious
of the two, was on hand with food
in seven minutes. With the new objects
in constant
view, new associations had been
formed. The strong parental
instinct supported by habit had banished most of
their
former fear. The
first steps in the taming process had been taken, and were carried
fur-
ther in the case now to be described. Two nests of the Chestnut-sided Warbler, each containing fresh eggs, were found in
a pasture on the twelfth day of June. The behavior of the
birds
at both
nests was
at
first essentially the same, so
far as
it was
tested. While the eggs were
still
fresh, the
nests were often visited without seeing or hearing a bird, but during incubation the female,
who is a close sitter, would allow me to approach within a yard or two feet. Then as
I
extended my hand slowly toward her she would hop out and cling with head down on the
farther side of the nest, so that only her
little
tail was
visible over
its
rim. Any one
prone to discover protective mimicry in such cases would find a striking example of
it
in
this attitude,
the
little gray
tail of the bird simulating so well one of the twigs which
helped to support the gray wall of the
nest. Wild
Birds. It was rather the case of an alert animal
lying
still or in hiding until a present danger might be past. If you kept your position
long enough the bird would drop to the ground, where joined by her mate, both would
hop about
in the grass chipping nervously, but keeping well out
of sight. On approach-
ing one of the nests still later when there were young, the female was usually overtaken
in the act of brooding. At such
times
it was easy to walk slowly up and
place your
hand
close to the brooding
bird. But before allowing you
actually to touch
her, she
would
flit to the grass, and with spread wings and
tail practice that "art of feigning" as
it is usually called, although
it
is not an art or anything learned or practiced for the occa-
sion, but an inherited
instinct, the end and advantage of which
is to distract your atten-
tion from the nest to the moving bird. One day
I stood by and watched the little mother
to see how long her antics would
last. She would come within a yard
of my feet when
I remained perfectly quiet, and
trail her wings along the ground, making repeated sallies Wild
Birds. 132 back and forth, flying only when close pressed, and then always away from her nest. On
one occasion
this was kept up from ten
to
fifteen minutes, and
did
not
cease
until
I
withdrew. My experiments at
the
first
nest were begun on June
I2th, by clearing away the
bushes
in
front. The
tent
was
set up two
feet away on the morning
of the
I5th,
while the
little hen was
still
sitting over the
eggs. She would
dart out
of the
nest,
return and take a peep inside, sit for a few minutes and be
off. When all was quiet she
could be
seen jumping
in and
out
repeatedly,
as
if
equally
uncomfortable
whether
away from her treasures or hugging them
close. In
the course
of half an hour
it was
easy
to photograph
the
sitting
bird,
who
now-
paid
little heed
to
the
shutter, and remained un-
disturbed
on
the
nest
during my
preparations
for leaving. On th My experiments at
the
first
nest were begun on June
I2th, by clearing away the
bushes
in
front. Wild
Birds. The
tent
was
set up two
feet away on the morning
of the
I5th,
while the
little hen was
still
sitting over the
eggs. She would
dart out
of the
nest,
return and take a peep inside, sit for a few minutes and be
off. When all was quiet she
could be
seen jumping
in and
out
repeatedly,
as
if
equally
uncomfortable
whether
away from her treasures or hugging them
close. In
the course
of half an hour
it was Fig. 127. Offering grasshopper to a Chestnut-sided Warbler who has been tamed
without use of a cage. It was possible to approach this bird and stroke her back
with the hand, without giving alarm. g
On the following day
the old bird was still per-
sistently sitting, and even
allowed me to erect the
tent
close
beside
her
without budging. When
finally driven
off by the
hand, she uttered
a few
tseeps and
returned
in a
moment. Once the male
came, and as I supposed,
placed an
insect
in
the
nest, when his mate, who
stood
close
by, hopped
ler who has been tamed
to the brim, put dOWII her
bird and stroke her back
head
and
3S
I thought
ate the food, but no, she
now a mother. Four young
birds,
scarcely
d
from
their
shells. They must have been g
On the following day
the old bird was still per-
sistently sitting, and even
allowed me to erect the
tent
close
beside
her
without budging. When
finally driven
off by the
hand, she uttered
a few
tseeps and
returned
in a
moment. Once the male
came, and as I supposed,
placed an
insect
in
the
nest, when his mate, who
stood
close
by, hopped
to the brim, put dOWII her
head
and
3S
I thought
t
th
b t
she On the following day
the old bird was still per-
sistently sitting, and even
allowed me to erect the
tent
close
beside
her
without budging. When
finally driven
off by the
hand, she uttered
a few
tseeps and
returned
in a
moment. Once the male
came, and as I supposed,
placed an
insect
in
the
nest, when his mate, who
stood
close
by, hopped
127. Offering grasshopper to a Chestnut-sided Warbler who has been tamed
to the brim, put dOWII her
ut use of a cage. Wild
Birds. It was possible to approach this bird and stroke her back
head
and
3S
I thought
he hand, without giving alarm. ate the food, but no, she
feeding the
little
ones,
for she was now a mother. Four young
birds,
scarcely
r than
bumblebees, had
just emerged
from
their
shells. They must have been
ed since noon of the previous day. Fig. 127. Offering grasshopper to a Chestnut-sided Warbler who has been tamed
without use of a cage. It was possible to approach this bird and stroke her back
with the hand, without giving alarm. was feeding the
little
ones,
for she was now a mother. Four young
birds,
scarcely
bigger than
bumblebees, had
just emerged
from
their
shells. They must have been
hatched since noon of the previous day. On the third day these Warblers paid no attention to either the tent or the operator,
and before going away
I was able
to touch
the
bird on
the
nest, though
not without
sending her
off. The
fourth day found
their confidence undiminished, for the sitting
bird eagerly seized a grasshopper which
I offered from the hand stretched through the
tent window. Four days later still
I spent nearly seven hours with these Warblers, and
in the afternoon began to test more systematically the strength of the intimacy which we
had cultivated. Taking a long twig in the hand and reaching through the window in the
front of the tent, I touched the old bird. She resented this but
little and when
her back
was scratched seemed to like the sensation. Then
I left the tent to look for insects, and Taming \Yild Birds without a Cage. '00 after a long search returned
with
a few small
grasshoppers. When one
of
these was
offered the
bird would eye the squirming
insect and
try to
seize
it when
held within
reach. Wishing to economize,
I held on to the
insect and nearly pulled the bird off the
nest. after a long search returned
with
a few small
grasshoppers. When one
of
these was
offered the
bird would eye the squirming
insect and
try to
seize
it when
held within
reach. Wishing to economize,
I held on to the
insect and nearly pulled the bird off the
nest. Wild
Birds. After discarding the tent
I was able to walk up to this bird and stroke her back with
my hand without disturbing her in the least. Setting up the camera outside and attach-
ing a tube with pneumatic bulb at the end,
I made a number of photographs which show
the Warbler sharply eying an
insect and
prepared
to
seize
it when
held
a few
inches
away. It
would have been an
easy
matter to take her
in the hand, though
possibly
not without injury to the young. Their
early flight from the nest cut short any
further
experiments,
but
what
could
not have been
done with
a
bird who
had become so tame and
confiding
in
the course of a few days ? Fig
128. Chestnut-sided Warbler family. The male, perched
above, has just delivered an
insect to
his mate, who quickly
passed it to the young and continued to brood. The same nest
is shown in FiBS - 3 ' " " 7 ' I29 ' and
' 3
- y
The
foregoing
account does
not
necessarily imply that
a wild
bird
can
be
induced
to
remain
docile
in
the
presence
of man
for any
great
length
of
time
while
still
enjoying
the
free-
dom
of
its
wild
life. If
the
lesson
learned
is
to be
a permanent
acquisi-
tion,
it must be often repeated, and no
other teachers allowed to interfere. To
effect
this
the animal must
as
a
rule
be placed under
restraint
or
in a cage,
where its experiences are more uniform,
more limited and under perfect control. In free
life a new habit must strug-
gle with other competitors and
is liable
to be suppressed quickly. However,
1
think
it has been clearly shown that
in
the beginnings
of the
tamill"
process
Fig
128. Chestnut-sided Warbler family. The male, perched
above, has just delivered an
insect to
his mate, who quickly
which have been
illustrated, where no
passed it to the young and continued to brood. The same nest
physical restraint
is used, the sense
of
is shown in FiBS - 3 ' " " 7 ' I29 ' and
' 3
-
fear must
be combated by
a
stronger and
contrary
impulse, such
as
hunger or the
parental
instincts, which will
lead
the
bird
to undergo new experiences, and
finally
to
adopt new habits. Fig
128. Chestnut-sided Warbler family. 1 Fur an account of a pair of Bald Eagles nesting on the ground in
the New York Zoological Park and incubat-
ing a goud-si/ed stone which was placed
in the improvised nest,
see Hir,i Lore,
vol.
iii..
p.
34.
1901.
; The Auk.
vol.
ix.,
p. 313,
iS(j2 Wild
Birds. The male, perched
above, has just delivered an
insect to
his mate, who quickly
passed it to the young and continued to brood. The same nest
is shown in FiBS - 3 ' " " 7 ' I29 ' and
' 3
- p
Audubon has given an interesting account of some Phcebes or Pewees which nested
in a cave on his plantation in Pennsylvania, and became the subject of some of his earliest
studies and experiments
in
ornithology. It
admirably
illustrates the taming process
under the spur of natural instinct. ' p
"On my
first going into the cave," he says,
" the male flew violently towards the en-
trance, snapped his bill sharply and repeatedly, accompanying this action with a tremulous 1 Ornithological Kivgraphy,
vol. ii
. p. 122. 1 Ornithological Kivgraphy,
vol. ii
. p. 122. Wild
Birds. 134 rolling note, the import of which
I soon guessed. . . . Several days
in succession
I
went to the
spot, and saw with pleasure that as my visits increased
in
frequency, the
birds became more
familiarized
to me, and, before a week had elapsed, the Pewees and
myself were quite on terms of intimacy. It was now the tenth of April. . . . The
Pewees, I observed, began working
at
their old
nest. My presence no longer alarmed
either of them."
He was soon able to put his hand close to the sitting bird without dis-
turbing
it. rolling note, the import of which
I soon guessed. . . . Several days
in succession
I
went to the
spot, and saw with pleasure that as my visits increased
in
frequency, the
birds became more
familiarized
to me, and, before a week had elapsed, the Pewees and
myself were quite on terms of intimacy. It was now the tenth of April. . . . The
Pewees, I observed, began working
at
their old
nest. My presence no longer alarmed
either of them."
He was soon able to put his hand close to the sitting bird without dis-
turbing
it. it many birds, as
is well known, are indifferent
to danger and will hug their eggs at any cost. In
this
respect
few
can
excel
the
" tame
villagic fowl," who displays greater stupidity
than most wild
birds, who
rarely
sit on an
empty nest,
1 and have been known to reject
strange eggs. Wild
Birds. In this state
birds cannot be
considered tame although the sense
of
fear
may be temporarily
dulled, and one
of the
conditions
of
the taming
process
fulfilled. The
hen
will
peck
vigorously
at
the
in-
truder, and
if hustled
tiff the
nest will soon
return. Some
birds
like
Song
Sparrows
and
Brown
Thrushes
will
remain immov-
able as
if hiding until you come dangerously
near, when they glide off silently, but usually
remain
quiet
for
a
moment
only. The
Robin
flies
off
in
a
passion. The
Tropic
Bird
fights
but
sticks
to
her
egg. The
Woodpeckers
are
close
sitters
and
may
sometimes be taken
in
the hand. A Chick-
adee which
I worried with a straw
would
peck angrily at
it, but remained on the nest. The Cedar-birds
retire
in
silence. In
this
state birds become passive merely through
. the temporary
suppression
of
the sense
of
fear. it many birds, as
is well known, are indifferent
to danger and will hug their eggs at any cost. In
this
respect
few
can
excel
the
" tame
villagic fowl," who displays greater stupidity
than most wild
birds, who
rarely
sit on an
empty nest,
1 and have been known to reject
strange eggs. In this state
birds cannot be
considered tame although the sense
of
fear
may be temporarily
dulled, and one
of the
conditions
of
the taming
process
fulfilled. The
hen
will
peck
vigorously
at
the
in-
truder, and
if hustled
tiff the
nest will soon
return. Some
birds
like
Song
Sparrows
and
Brown
Thrushes
will
remain immov-
able as
if hiding until you come dangerously
near, when they glide off silently, but usually
remain
quiet
for
a
moment
only. The
Robin
flies
off
in
a
passion. The
Tropic
Bird
fights
but
sticks
to
her
egg. The
Woodpeckers
are
close
sitters
and
may
sometimes be taken
in
the hand. A Chick-
adee which
I worried with a straw
would
peck angrily at
it, but remained on the nest. The Cedar-birds
retire
in
silence. In
this
state birds become passive merely through
. the temporary
suppression
of
the sense
of
fear. bing
While possessed by the incubation spirit many birds, as
is well known, are indifferent
to danger and will hug their eggs at any cost. Wild
Birds. In
this
respect
few
can
excel
the
" tame
villagic fowl," who displays greater stupidity
than most wild
birds, who
rarely
sit on an
empty nest,
1 and have been known to reject
I
thi
t t
bi d
t be Fig. 129. Female Chestnut-sided Warbler approaching
nest and looking in. At this time there were eggs, or the
young had barely pipped the sheii. Fig. 129. Female Chestnut-sided Warbler approaching
nest and looking in. At this time there were eggs, or the
young had barely pipped the sheii. Fish Hawks
used to nest on Plum Island, New York, where according to Mr. C. S. Allen,
2 they had been
zealously protected by the owner of the island
for upwards of
thirty years previous to
1885. The first nest shown to him by Mr. Jerome, the faithful
guardian
of the
birds, was
"
fairly
in
his door-yard,
close by
his
front
gate, and
only
about fifty yards from his home. It was placed upon an old pile of
fence rails, rotted to
black mould in the center, but kept up by the yearly addition of fresh rails. Mr. Jerome
said that to
his knowledge this nest had been occupied every year for forty years."
It
had been added to yearly until its huge bulk of sticks and miscellaneous materials would Turning \Yild Birds without
;i '35 make three
cart
loads. It was but seven
or
eight
feet from
the ground,
so that by
stepping on
a projecting
rail the beautifully spotted eggs within could be seen. " Mr. Jerome could
pass
close
to
the
pile
of
rails without the birds leaving the
nest, while
I could
not
get nearer than
thirty
or
forty
feet."
At other places on
the
island, the
birds would alight on one nest while he was examining another near by. This illustrates
how a shy bird may become relatively tame during the breeding season, and shows clearly
how some
learn
to
discriminate. That many birds become tame in a stat
full of
interest. The Pine Grosbeak
is as
found
in
this
part
of
the
world. Pine
Grosbeaks make their summer home in
tin-
vast forests
of eversireens which cover the
o
continent
from
Labrador
to
Alaska. A
few,
it
is said, have been found breeding in
latitude 47
in New Brunswick, and they
have even been recorded in summer on Mt. LaFayette, New
Hampshire. Wild
Birds. They
are
irregular winter
visitors
to
the
Northern
States, sometimes
going
so
far
south
as
Maryland
and
Kansas. In the winter of
1884, they were very common
at
Holder-
ness, New Hampshire, beginning to appear
in
small
flocks about the middle
of Feb-
ruary
and
finally
disappearing
after
the
eighteenth
of March. At
first they were
tame
and
could
be
approached
without
difficulty, while later they became shy and
timid. They
frequented
the white
pines. on
the buds
of which they
fed, but
occa-
sionally came into the open, and sang loud
and merrily. That many birds become tame in a state of nature
is well known and the su
full of
interest. The Pine Grosbeak
is as good an
illustration
of the
fact
as m
found
in
this
part
of
the
world. Pine
Grosbeaks make their summer home in
tin-
vast forests
of eversireens which cover the
o
continent
from
Labrador
to
Alaska. A
few,
it
is said, have been found breeding in
latitude 47
in New Brunswick, and they
have even been recorded in summer on Mt. LaFayette, New
Hampshire. They
are
irregular winter
visitors
to
the
Northern
States, sometimes
going
so
far
south
as
Maryland
and
Kansas. In the winter of
1884, they were very common
at
Holder-
ness, New Hampshire, beginning to appear
in
small
flocks about the middle
of Feb-
ruary
and
finally
disappearing
after
the
eighteenth
of March. At
first they were
tame
and
could
be
approached
without
difficulty, while later they became shy and
timid. They
frequented
the white
pines. on
the buds
of which they
fed, but
occa-
sionally came into the open, and sang loud
and merrily. Fig. 130. Female Chestnut-sided Warbler inspecting her
young afur having served food
' Fig. 130. Female Chestnut-sided Warbler inspecting her
young afur having served food
' I remember meeting a
flock of these
Fig. 130. Female Chestnut-sided Warbler inspecting her
plump, stalwart looking birds
in a grove of
young afur having served food
'
sapling pines on the last day of February. The woods on every side were hoary with snow
which had been
falling
for hours. When
a young pine drooping under
its weight of
snow suddenly blossomed
with a bright company of these
birds, you might
travel
far
to
find a more
attractive winter picture. A bird would sometimes drop on a branch,
and
settle
down
as
if
going to
sleep. Wild
Birds. Then
suddenly
aroused
by
the
desire
for
food he would
sidle
to
the
end
of
the bough, pick out the terminal
or
largest bud,
twirl
it
between
his
stout
cone-shaped
mandibles to get
rid
of the scales and then
swallow
the
resinous
morsel. After seeing
this experiment performed
a good many
times,
I selected a handsome male, walked up to him, and caught him with my hat, as
if
he were a butterfly. When
I stooped to pick him off the snow, he squeaked and struck
violently with his beak, uttering a peculiar car-r-r-r-r ! When placed on the snow again
he flapped about for a few moments resisting every attempt to take him, and finally rose I remember meeting a
flock of these
plump, stalwart looking birds
in a grove of
y
sapling pines on the last day of February. The
which had been
falling
for hours. When
a y
snow suddenly blossomed
with a bright com
to
find a more
attractive winter picture. A
and
settle
down
as
if
going to
sleep. Th
food he would
sidle
to
the
end
of
the boug
twirl
it
between
his
stout
cone-shaped
man
swallow
the
resinous
morsel. After seeing
t
times,
I selected a handsome male, walked up
he were a butterfly. When
I stooped to pick
violently with his beak, uttering a peculiar car-
he flapped about for a few moments resisting e The woods on every side were hoary with snow
a young pine drooping under
its weight of
company of these
birds, you might
travel
far
A bird would sometimes drop on a branch,
Then
suddenly
aroused
by
the
desire
for
bough, pick out the terminal
or
largest bud,
mandibles to get
rid
of the scales and then
ng
this experiment performed
a good many
up to him, and caught him with my hat, as
if
ick him off the snow, he squeaked and struck
car-r-r-r-r ! When placed on the snow again
ing every attempt to take him, and finally rose Wild
Birds. 136 and disappeared among the snow-laden trees. There were about fifty birds in this flock
and the grove resounded with their clear whistled
notes. They were
easily approached
at all times and in
all weathers, during the early weeks of
their visit, agreeing in this
re-
spect with the Bohemian Waxwing, the Arctic representative of the Cedar-bird. Wild
Birds. Two
small
flocks
of these
birds
visited
Burlington, Vermont, November 24 and January 21,
1882. A low plaintive
call-note
first
attracted my attention, when a party of eight of
these
fine
birds came
into
view. They were
leisurely preening their feathers on the
lower branches of a red cedar tree. When close upon them, they paid no attention, and
finally wishing to
see them
fly,
I had almost to shake them from the branches. They
went
off
in
a
compact body
like
their
smaller
relative,
giving a
"see,
zci\ zee-ze ! "
call-note. Audubon
speaks
of
the
familiarity
of
Crossbills
which
he
observed
while
on
a
moose hunt
in the summer
of
1833. They alighted on
his head, showing no
fear, and
five or
six were caught
at one time under a snowshoe. 1 g
This tameness found among many Arctic species has been met with on a much wider
scale in remote oceanic islands, where man
is almost unknown and where the conditions
of life are very different from those of the mainland. The inhabitants of the Galapagos
Islands, which
lie under the equator between five and six hundred
miles from
the west
coast of South America, offer
a most
striking example of this anomaly. Their natural
history which has been told in one of Darwin's interesting chapters, first led him to reflect
on the origin of species. 2
He says that many of the animals and plants are aboriginal, and
found nowhere else, that " there
is even a difference between the inhabitants of the differ-
ent islands
; yet
all show
a marked
relationship with
those
of America. . . . The
archipelago
is a little world within itself, or rather a satellite attached to America, whence
it has derived a few stray colonists, and has received the general character of
its
indi-
genous productions."
He
found
twenty-six species
of
land
birds, all
peculiar to the
islands excepting only one, the Bobolink, whose summer range extends
as
far north
as
Labrador. All the common terrestrial
birds
of these volcanic
islands were very tame, and
all
says Darwin, " often approached sufficiently near to be
killed with a switch, and some-
times,
as
I
myself
tried,
with
a
cap
or
hat. A
gun
is here
almost superfluous
;
for with the muzzle
I pushed a hawk
off the branch
of
a
tree. Ornitli,<l^'i::il
A';,I-;-,I///_V, vol.
ii., p. 436. Wild
Birds. One day whilst lying
down, a mocking-thrush alighted on the edge of a pitcher, made of the shell of a tortoise,
which
I held in my hand, and began very quickly to sip the water
;
it allowed me to
lift
it from
the ground
whilst
seated on the
vessel:
I
often
tried, and
very
nearly
suc-
ceeded
in catching these
birds by
their
legs. g
" These birds, although now
still more persecuted, do not
readily become
wild
:
in
Charles Island, which had then been colonized about six years,
I saw a boy sitting by a
well with a switch
in his hand, with which he killed the doves and finches as they came to
drink. He had already procured a little heap of them for his dinner; and he said
that
he had constantly been in the habit of waiting by this well for the same purpose." y
Darwin remarks that the most anomalous fact on this subject which he had met was
the wildness of certain small birds in the Arctic portions of North America, while some
of the same species were said to be tame in their winter quarters
in the United
States. Taming Wild Birds without a Cage. 137 " How strange it is," says he, "that the
English wood-pigeon, generally
so
wild
a
bird,
should very
frequently
rear
its young
in
shrubberies
close to houses
! " y
q
y
y
g
Respecting the wildness which birds exhibit towards man, Darwin could find no way
of accounting for
it except as inherited habit, but
in another work, he thus refers to the
same subject
'
:
"If we
look to successive generations, or to the race, there
is no doubt
that birds and other animals gradually both acquire and lose caution
in
relation
to man
and other enemies
; and this caution
is certainly in chief part an inherited habit or instinct,
but in part the
result
of individual experience." p
p
The observations which have been made on the behavior of old and young birds do
not support any theory of the inheritance of habits to account
for lameness
in
animals,
but as already shown afford a betler clue of how this has been brought
about. A. Abdomen,
size and secondary use of, in young,
121. y
y
g,
Abstract ideas, the nature of,
if present in birds,
3. y
y
g
Abstract ideas, the nature of,
if present in birds,
3. p
Accessories, or bird-photographer's outfit enumerated, 35. p
Accessories, or bird-photographer's outfit enumerated, 35. ,
bird photographer s
, 35. Alarm calls, in Catbird, 76, 77;
effect of, upon birds of other species,
77, 122-125;
in Rbin,
123. Alarm clock, illustration of,
5 p
g
p
Alarm calls, in Catbird, 76, 77;
effect of, upon birds of other species,
77, 122-125;
in
l p
g
p
Alarm calls, in Catbird, 76, 77;
effect of, upon birds of other p
g
p
Alarm calls, in Catbird, 76, 77;
effect of, upon birds of other species,
77, 122-125;
in Rbin,
123. ;
Alarm clock, illustration of,
5. Allen, Charles A.,
in/. Allen,
C. S.,
115,
134. ,
,
115,
134. Alt rices,
definition
of. 117;
development of fear
in young
of,
120;
fate of young due
to premature
development of fear in, 120-121;
care and education of young in, 121;
specialization of fear in
young
of,
121. Ampclis garrulus (Bohemian Waxwing), origin of specific name in,
52. Animal photography, a desideratum of, 34. photography,
, 34. Animals, the evil of anthropomorphism in study of, xv; duty and privilege of student
of, xv;
the nai ive
equipment
of, xvi; vulgar error concerning, 125; variable personalties of, 12^; winning confi-
dence of,
125-127; origin of natural wildness and tameness
of,
125,
126,
137. conditions of
taming,
126,
127. g,
Anthropomorphism, evil
of, in study of animal behavior, xv. Approach to the wild bird, the problem of, and its solution, 3 Anthropomorphism, evil
of, in study of animal behavior, xv. pp
p
Audubon, John James,
104,
108,
129,
130,
133,
136. Audubon, John James,
104,
108,
129,
130,
133,
136. Wild
Birds. Let us
go back
to the
Pine
Grosbeak
which, when
fresh
from
his
sub-Arctic home, can be
approached and caught with your hat as could many of the birds in the Galapagos Islands
when Darwin visited them in
1835. So far as
I know, no one has studied the young of
this species in the nest and ascertained whether they show the same instincts of fear in
general toward strange sights and sounds, as we
find
in passerine birds nesting farther
south. Assuming
that they do, and
there
can be
little doubt
<>f
it,
the
instinct
has
lapsed through
disuse
in adult
life, although the capacity
of
expressing
fear
remains
and may be quickly aroused and
directed towards particular objects. The timidity of
this bird
in March
after a
brief experience with the ways of men
is therefore virtually
an acquired character, and there
is no evidence that
it
is handed down by inheritance. acquired
,
by
The breeding range of many northern
birds
covers
a
vast
area,
and
in
different
sections there
is reason to expect much variation in the habits of the same species. The
timidity of the Arctic birds referred
to may have been due to local conditions affecting
a relatively small number, or the birds may have been young individuals whose
intuitive
fear had not been worn away, or old ones possessed of a wisdom derived from extensive
travel southward. Among
birds
which
are reputed
to be shy, tamer
individuals
are
to be found, and many acquire the habit of nesting in gardens and often close lo houses. In the Galapagos
Islands, where
birds had
lived
in comparative security for ages with
no fierce and
relentless enemies to mar
their tranquillity,
the
instinct of
fear had
not
only lapsed, but the power of forming new habits had weakened. It
is therefore not
surprising that they should be slow
in acquiring a fear
of man, but any animal which
finally
fails
in
the
face
of constant
persecution
to
profit
by experience
has
touched
the
lowest depths
of
stupidity, and
its days are numbered. 1 Tht n,-se,'iit cf Man,
]>. So. 1 Tht n,-se,'iit cf Man,
]>. So. INDEX. C. C. Call-notes,
of Cedar-bird,
17,
56;
of young of Baltimore Oriole,
18;
of Robin, 45;
of Red-eyed Vireo,
64. 65;
of Bluebird, 73;
of Redwing Blackbird, 77;
of Night Hawk, Si, 82;
of Kingfisher, 90;
of Canada Goose,
129;
of Bohemian Waxwing,
136. Call-notes,
of Cedar-bird,
17,
56;
of young of Baltimore Oriole,
18;
of Robin, 45;
of Red-eyed Vireo,
64. 65;
of Bluebird, 73;
of Redwing Blackbird, 77;
of Night Hawk, Si, 82;
of Kingfisher, 90;
of Canada Goose,
129;
of Bohemian Waxwing,
136. ,
129;
g,
Camera, value of, in portrayal of animals in action, xviii;
size and construction
of, 32;
the twin lens,
32;
the reflecting, invention and history of, 32. ,
;
g,
Camera, value of, in portrayal of animals in action, xviii;
size and construction
of, 32;
the twin lens,
32;
the reflecting, invention and history of, 32. ;
Catbird (Galci>Si\>ftcs
CLirolincnsis. Linn.)
, shyness
of, 5; minute observations on, 76-79;
parental and
lighting instincts of,
76, 77;
description of nests
of,
76,
77; feeding and care of young in. 77-
79;
development of wing-quills in young of. 77, 78; capture of dragon-flies by,
77. 78;
alarm
notes
of. 76. 77;
suppression
of fear
in, 76-78;
rate
of feeding young
in,
78;
behavior
of
young
of,
77. 79,
117,
118;
eating of excreta of young by. 105;
attracted by alarm of Robin. 122. Cats, and young after removal of nesting bough,
15;
as enemies of young birds, 51
; exemplified in Jan
Stcen,
51. Cedar-bird, Cedar Waxwing
(Aiiifclis ccdivriim. ViclD,
life at nest
of, after change of
site,
13, 61;
tin-
nesting of,
17, 52, 53;
removal of
nesting bough of,
17. 54,
57, 59;
call-notes of,
17, 56;
care
of young by,
18,
54-62, 94;
flight of young
of,
iS,
60;
spring and
fall behavior
of,
52,
53;
winter flocks
of,
52;
late breeding of,
52;
breeding season at
Northfield
(N. H.),
52;
quiet
nature of, 52;
origin of names of, 52;
appendages to feathers of wings and tails in young and
adult of, 52-53;
food
of, 62, 63;
position, materials, and construction of nests of, 53, 58;
pro-
portion of young of, reared,
53;
early weakness of
parental instincts of,
53,
54;
desertion of
nests in,
54;
the
hatching of, 54;
routine in nesting habits of, 54;
favorite nesting trees and
bushes of. B. Bag, for accessories of bird-photography, 35;
for plates, 34. Baltimore Oriole (MV Oriole). Basin Harbor (Yt
|,
[{avis Swallow dispossessed by House Sparrows at,
114,
115. Bird-photography (MV Photography). Bird photography
Photography). Birds, mental faculties of, xvii; instincts of, xvi; problem of approaching, xvii, 33
; strongest lure for. xvii
;
guiding senses of, 3
;
rudimentary condition of olfactory organ
in,
3;
actions
of, when
in
i
is
robbed, 4; effect of noise of photographic shutter upon, 5. 34; effect of sounds upon, 5. (i.S. 112;
appearance of feather-shafts in young of, 6; behavior of, after change of nesting site,
i
i,
_>.?,
;.),
73;
attachment to nest, eggs, and young in, 6,
13;
individualities of, 36;
attractions in haunts
of man
for,
5
i
; routine in home life of, 54; interest in watching nesting habits of, at short range,
15. 16, 54;
brooding in Cedar-birds,
17,
56;
maternal instincts of. 5;
digestion, assimilation,
and growth of young of, 66;
care of young in nest
of, 04;
brooding attitudes
of, 94, 9798;
automatic response in gullet of young of,
101, 102;
inspection and cleaning of nest in, 103-1 10;
economy of food in,
102;
struggles with insects at nests of,
103;
cleaning or sanitary instinct
in,
103,
104;
disposal of excreta of young of, 104110;
use of excreta of young as food by,
105-110;
force of
habit displayed
in. 111;
cleanliness of
nesting
site
in. 107,
108;
adapta-
bility
of, 113; change of diet
in. i 16:
classification
of, based on early condition of young
in,
117;
fear in old and young of,
117;
fate of young of, due to premature development of fear,
120, 121;
lack of discrimination in young of,
121;
specialization of fear in young of, i2i;use
of pot-belly in young of,
121;
effect of alarm calls of, on birds of other species,
77,
122,
123;
in winter at Jefferson
(O.),
128;
behavior
of, during incubation,
134;
taming
of, 125137;
tameness of, in nature, 135-137;
of Galapagos Islands. 136, 137;
of Charles Island, 136;
wild-
ness of, in Arctic America, 136-137. (>'<< under names of species.) 139 Index. 140 Blackbird, Crow or Purple Crackle
(Quiscalus qniscala. Linn.),
nest-cleaning instincts
of,
104,
105;
and brooding Robin,
39. Blackbird, Crow or Purple Crackle
(Quiscalus qniscala. Linn.),
nest-cleaning instincts
of,
104,
105;
and brooding Robin,
39. Blackbird, Crow or Purple Crackle
(Quiscalus qniscala. B. Bunting, Bay winged (
g
Burlington
(Vt.),
arrival of Robins at, 48-49;
nest of
Black Duck
at,
119,
120;
records of visits of
Bohemian Waxwings at,
136. g,
y
g
Burlington
(Vt.),
arrival of Robins at, 48-49;
nest of
Black Duck
at,
119,
120;
records of visits of
Bohemian Waxwings at,
136. g
Buzzard, Turkey, filthiness of nests of,
108. B. Linn.),
nest-cleaning instincts
of,
104,
105;
and brooding Robin,
39. g
Blackbird, Redwing
(Agelaius
phccniccus. Linn.),
the
bathing
of,
16;
an
attractive
nest
of,
20;
preparation of nesting site
of, for use of tent,
20;
behavior of,
20; care of young of,
20,
21;
brooding of,
20,
21,
112;
respiration
of,
21;
flight of young from nest
of,
21;
nest-cleaning
instinct of,
104,
105,
112;
eating excreta of young by,
105;
erection of feathers in female of,
2 i
;
force of habit displayed in. 1 12. p
y
Blackbirds, nest-cleaning instincts
of,
104. g
Black cherry tree as an aviary in late summer, 62. cherry
a
a y
Bluebird
(Sialia
sialis. Linn.),
arrival
of, at Cleveland
(O.),
49: nest-hole
of,
71;
general habits of,
71-75; arrival at Holderness (N. H), 71;
at Northfield (N. H.), 71; call-notes of, 71, 73; court-
ship of, 71; polygamy in, 72;
choice and care of
nesting
site,
72;
defense of
nest
of,
72;
in
converted nest of Flicker,
72;
removal of nest
of, 72; adjustment of vertical tree-trunk with
nest
of,
72;
behavior of, after removal of nest, 73;
feeding young in, 73;
use of
tail
for sup-
port in, 73;
strength of parental
instincts
in, 73-75;
response of young in, 73, 74;
nest-clean-
ing of, 74, 7^, 104; young of , and their food, 75;
rate of feeding young in,
75;
in old Robin's
nest,
75
; pugnacity of,
75
; number of broods of, 75
;
repair of nest in, 75. p g
y
Breeding season, lateness of, in Cedar-bird, 52;
in Goldfinch,
52. g
Brewster, William,
51. g
Brewster, William,
51. Bristol (R. I), nesting of Osprey at,
115,
116. (R. I), nesting
Osprey
,
115,
Brooding, in Redwing Blackbird, 20, 21.112:
in Kingbird, 28, 04. 97;
in Robin. 46. 47, 94, 98;
in Cedar-
bird. 17. 57;
in Red-eyed Vireo, 68;
in Night Hawk,
So, 82, 85;
necessity
of, 94;
in Brown
Thrush,
04. (
g
p
y
Brooding, in Redwing Blackbird, 20, 21.112:
in Kingbird, 28, 04. 97;
in Robin. 46. 47, 94, 98;
in Cedar-
bird. 17. 57;
in Red-eyed Vireo, 68;
in Night Hawk,
So, 82, 85;
necessity
of, 94;
in Brown
Thrush,
04. ,
0 . Brown Thrush
(sec Thrush). ,
Brown Thrush
(sec Thrush). ,
Brown Thrush
(sec Thrush). (sec
)
Bunting, Bay-winged (Pooccelcs graininctis, Gmcl.), and black snake,
123. C. 53
; regurgitation of food for young in, 55,61; number of berries carried in gullet of
55;
tuiK-tion of gullet in,
55,
56,
101;
habits of
nestlings of,
56;
sudden appearance of sense
< if fear in nestlings of. 56. 60;
timidity
of,
54;
response
to alarm signal
in,
57;
brooding in,
17, 57. 94;
supplying colored yarn for nest of, 58;
time occupied in building nest in, 58;
laying Index. y
Eggs, of Kingbird,
21;
birds strongly attached to,
13;
incubation
of, in Robin, 36;
lateness of laying
of, in Cedar-bird and American Goldfinch,
52;
proportion of y
roung reared to number of,
in
Cedar-bird, 53;
laying and incubation of, in Cedar-bird, 58;
destruction of, in Red-eyed Vireos,
69;
hatching of, in Night Hawk, So;
relation of size
of, to condition of young at birth,
117;
of Canada. Goose hatched under hen,
130;
behavior of Chestnut-sided
Warblers, with,
131;
behavior of birds when incubating,
134. D. Darwin, Charles. 136,
137. Darwin, Charles. 136,
137. Dorchester (X. II.), nesting of Swift in shed at,
i 14. Dorchester (X. II.), nesting of Swift in shed at,
i 14. Dorchester (X. II.), nesting of Swift in shed at,
i 14. g
Dorset (O.)
, nesting of Swift in barn at, 114. g
Dorset (O.)
, nesting of Swift in barn at, 114. g
Dorset (O.)
, nesting of Swift in barn at, 114. (O.)
, nesting
, 114. Dragon-fly, capture and killing of, by Kingbirds, 28, 103;
as food of young Catbirds, 77-79. g
Dragon-fly, capture and killing of, by Kingbirds, 28, 103;
as food of young Catbirds, 77-79. Dragon-fly, capture and killing of, by Kingbirds, 28, 103;
as food of young Catbirds, 77-79
Duck
Black
(
1 nas obscura
Giticl )
absence of fear in newly hatched young
> if
i
i n
nesti Duck, Black
(.-1 nas obscura, Giticl.)
, absence of fear in newly hatched young
> if. i
i n
'. nesting of, 119,
1 20
;
behavior of old and young of. when latter are possessed of fear,
i 20. Duck,
(. 1
obscura,
,
newly
young
nesting
, 119,
;
behavior of old and young of. when latter are possessed of fear,
i 20. uc ,
(. 1
obscura,
newly
young
ne
behavior of old and young of. when latter are possessed of fear,
i 20. Index. Color, discrimination of, in Cedar-bird, 58;
development of, in Kingfisher,
Sf>, gi. ,
Creeper, Brown (Certhiafamiliarisamericana, Bonap.) , shyness of,
128,
129. (Certhiafamiliarisamericana,
p )
shyness
,
merican (Lo.via tiin-iroslra
iniiii'r, Ijrclun.), effect of alarm of Crow upon, 122. Creeper,
(
,
p )
y
Crossbills, American (Lo.via tiin-iroslra
iniiii'r, Ijrclun.), effect of alarm of Crow upon, 12 Cn iw, effect of alarms of, upon other birds,
i 22. pp
Cycle, the reproductive, in birds, 3. Index. Cedar-bird
Cellar Waxwing
(~ t nitiin/:\l
ami incubating, and hatching of eggs in. 58,
i<>:
young of, at birth,. 50:
age of young of, when
eyes
open,
59; disappearance of young in nest of, 59; behavior of,
in approaching
nest with
food, 60;
development
of eolor marks
in
fledglings
of, 60, 61;
appearance of
feather-shafts
and wax-like tips in wings of, 60; habits of young of, when ready to fly, 60;
rate of feeding of
young in, in
;
the feeding, food, and eare of young of. 55,
61,
101;
similarity in sexes of, 61,
iu
: peculiar signals of, at nest, 62,
102; habit of sipping maple sap
in,
(12, Hocking of. in Au-
guM,f)2; and the black cherry tree, 62; eating spiders or robbing them of their prey, 63
; taking
insects on the wing, 62, 63;
gaping habit
in,
ijS;
gluttony
of,
102;
inspection and cleaning
the nest
by. 104,
105-107; eating excreta of young by,
105,
io<>;
parasites
in
nest
of,
107;
habits of, during incubation,
134. g
Central Park, Chickadees in,
127. Central Park, Chickadees in,
127. Chapman. F. M
. 127. Chapman. F. M
. 127. Chapman. F. M
. 127. p
Charles Island, lameness of birds of,
136. Chatterer, the origin of name as applied to the Waxwings, 52. Cherry
(see
Chick, domestic, instinct of fear in,
ii<>;
behavior of. when Hawk passes overhead,
120. y
(
Chick, domestic, instinct of fear in,
ii<>;
behavior of. when Hawk passes overhead,
120. Chickadee (l\
tit i
Linn )
cleanliness
f
104
ultracte 1 b
Robin's alarm
122
12
habit Chickadee (l\n 'tis titrica pilliis, Linn.)
. cleanliness of,
104;
ultracte.1 by Robin's alarm,
122. 12;;
habits
and lameness of. 127. 128;
during incubation,
134. Cicada, eaten by young of Cedar-bird, 61:
struggles of Kingbirds with,
103;
combat
of, with House
Sparrow. 103. Sparrow. 103. City life, possible origin of, in many birds, 50. Sparrow. 103. City life, possible origin of, in many birds, 50. City
p
y
Clamp, the "Graphic" ball and socket,
34. City
p
y
Clamp, the "Graphic" ball and socket,
34. y
p
y
Clamp, the "Graphic" ball and socket,
34. p,
p
Cleaning instinct, 103-110
(sec Instinetsi. p,
p
Cleaning instinct, 103-110
(sec Instinetsi. Cleaning
Cleveland (O.), spring arrival of Robins and Bluebirds at, 49;
Robins in, 50;
Red-headed Woodpeckers
in,
50 in,
50. Eagle, behavior of, when nesting, compared with that of Night Hawk. So:
improvised nest of, 134.
Earthworms, fed to young Robin, 46, 47. Fauna of Galapagos Islands, peculiar character of, 136,
137. Fauna of Galapagos Islands, peculiar character of, 136,
137. ,
,
51. Fear, the instinct
of,
3;
the suppression
of,
4;
development
of,
in relation to appearance of feather-
shafts of wings, 6;
suppression of, in Cedar-bird,
17, 57, 59;
in Oriole,
19;
in Redwing Black-
birds, 20, 21;
in Kingbird, 22, 27;
in Robin, 39, 40, 45;
appearance of, in young Cedar-birds,
56, 60;
in young of Red-eyed Vireo, 69;
suppression
of, in adult Bluebird, 72, 73;
in Catbird,
76-78;
development of, in young Catbirds,
77, 79;
nature, time of appearance, sudden mani-
festation, and adaptive value of, 117-124;
instinct of,
in domestic
chick,
119;
in
ducklings
of Black Duck,
120;
of Hawk in sky, expressed by chick,
120:
distinction between inherited
and acquired. 121, 137;
expression
of, in Robin,
122;
checked by hunger, 126128. q
p
Feathers, development of, as guide in controlling nesting site, 6;
development of, in Cuckoos, 6;
down,
in Bluebird,
75;
development
of, in Red-eyed Vircos, 64, 68:
in Catbirds,
77,
78;
in King-
fisher,
86,
91;
in Night Hawk,
So,
85;
condition
of, at birth as basis for classification,
117;
development of, relation to fear, 6, 117-124. development
,
Feather-shafts, appendages of, in Cedar-bird, 52, 60
(sec Feathers). p
Feather-shafts, appendages of, in Cedar-bird, ,
pp
g
Fireflies as food of young Night Hawk, 82. pp
g
Fireflies as food of young Night Hawk, 82. g
Fireflies as food of young Night Hawk, 82. Fish
captured by Kingfisher
90;
resources of Kingfisher to prevent escape of, 92 Fireflies as food of young Night Hawk, 82. Fish, captured by Kingfisher, 90;
resources of Kingfisher to prevent escape of, 92. , captu ed
y
g
,
;
g
p
p
Fly, robber (Asilns), fed to young of Bluebird,
73, 74;
escape of, from grasp of Kingbird,
102,
103. F
i
l th
adj stment
of
in tent
31 p
y
g
,
Fly, robber (Asilns), fed to young of Bluebird,
73, 74;
escape of, from grasp of King Fly, robber (Asilns), fed to young of Bluebird,
73, 74;
escape of, from grasp of Kingbird,
102,
103. Focusing-cloth, adjustment
of, in tent,
31. y,
,
y
g
Focusing-cloth, adjustment
of, in tent,
31. Index. 142 Excreta, character of, in young of passerine birds,
104;
disposal of, by parents, 104-107;
character of,
in young Kingfisher, 107;
use of, as food by adult birds, 105-107, 109;
character of, in Eagles
and Hawks, 108;
actions of Cedar-birds in taking, devouring, or removing, from nest, 106, 107;
use of, as food in Chestnut-sided Warblers, 109. Excreta, character of, in young of passerine birds,
104;
disposal of, by parents, 104-107;
character of,
in young Kingfisher, 107;
use of, as food by adult birds, 105-107, 109;
character of, in Eagles
and Hawks, 108;
actions of Cedar-birds in taking, devouring, or removing, from nest, 106, 107;
use of, as food in Chestnut-sided Warblers, 109. ,
Eyelids, angular contoxir of, in young Night Hawk, So. ,
Eyelids, angular contoxir of, in young Night Hawk, So. Eyelids, angular
y
g
g
Eyes, opening of, in young of Cedar-bird, 59;
in young of Red-eyed Vireo, 64. Fauna of Galapagos Islands, peculiar character of, 136,
137. Focusing cloth, adjustment
,
,
Food, of young Cedar-birds,
17,
18,
55, 61;
of young Baltimore Orioles,
19;
of Kingbird,
28,
102,
103,
116;
of young Robins, 39, 48,
1 16;
of Robin in summer and winter, 48;
of Cedar-bird, 52, 62,
63;
economy
of,
in Kingbird,
28,
102;
in Red-eyed Vireos, 68;
in Chestnut-sided Warbler,
109;
distribution
of,
to young Cedar-birds explained,
55;
and
its distribution
in Red-eyed
Vireos,
67-69;
of young Bluebirds,
75;
of young Catbirds,
78,
79;
of young Kingfishers,
90-92;
of young Night Hawk, 82;
of Hawks, Owls, and other birds under exceptional con-
ditions,
116. ,
Foster-children, treatment by Kingbird,
27. y
g
,
Fowl, domestic, stupidity and pugnacity of, 134. stup d ty
pug ac ty
Fruits, fed to young by Cedar-birds,
17, 61; by Orioles. 19;
by Robins, 48;
eaten by Robin in winter,
48;
cultivated, eaten as makeshift, 48;
served to young of Red-eyed Vireos, 68, 69;
of Cat-
bird,
76,
77,
79. E. ,
you g
,
my of food, in Robin, 39;
in the Kingbird, 28, 102;
in the Red-eve. 1 Yireo, 68. Index. p
g,
,
;
p
Gullet, distcnsibility of, in the Cedar-bird, 55, 61;
effect of full, in young,
101; automatic response of,
in young birds, 55, 101, 102. Gluttony
Cedar birds,
101,
Goldfinch, American (Spinus Iristis, Linn.), lateness of breeding of, 52;
attracted by Robin's alarm, 123.
Goose, Canada (Branta
canadensis, Linn.), young
of,
117;
tamability
of.
127,
129,
130;
habits and
breeding
of,
in captivity,
129.
130. Grasshoppers brought to nest by Robin, 39. g
,
captivity,
Grampus (Corydalus cornntus)
, feeding
of,
to. young Kingbirds,
103;
formidable appearance and size
of,
101,
103. g ,
spring, 135, 136;
capture of male of, with hat, 135. Grasshoppers brought
by
, 39.
Grosbeak,
Pine
(Pinicola
ennclcator,
Linn.),
range,
habits, and
relative lameness
of,
in winter and
l Kearton, the brothers, blinds designed by, 30.
Kingbird (Tyrannus tyranniis. Linn.). the time spent by young of, in nest after change of site,
13:
the
breeding of, 21; displacement of the nesting bough of, 22;
fighting instinct of,
22:
suppression
of fear in.
22, 23;
rate of feeding young of,
22,
27;
foster children
of,
27;
crushing prey by,
28;
the rejection of indigestible parts of food by, 28;
flight from m-st
of.
...S;
brooding in. 94,
97; economy of food in, 102;
escape of prey from bill of,
102.
103;
exciting scenes at nest of,
105:
feeding of large insects to young of.
103;
eating excreta of young of,
105;
attracted by
alarms of other birds, 123;
perching on fishing-rod, 125. I. Incubation, in Kingbird, 21;
in Robin, 36;
in Cedar-bird, 58;
of Eagle in captivity. 134;
behavior of
birds during,
134 during,
Insivts, struggles of, when brought to nest,
102,
103. g
Insivts, struggles of, when brought to nest,
102,
103. d
th
and
l
under p
)
Instincts, defined in broad and narrow sense, xvi:
illustration of, in the Robin, xvi;
substitution of, by
habits, xvii, 4;
the great number of, xvi; determining cause of, xvi;
the parental, analysis of,
3;
culmination
of, 4; strength
of, how increased,
3, 4;
suppression
of,
3;
periodic and serial
nature
of, 3;
the
fighting,
3, 4
(sec Pugnacity);
parental, relative strength of, in birds,
5,
13;
in Orioles,
19;
in Kingbirds,
22;
in Redwing Blackbirds,
20, 21;
in Robins, 39, 45;
in Cedar-
birds,
17,
18, 55, 57, 59;
in Red-eyed Vireo, 64, 65;
in Bluebirds, 72-75;
in \ight Hawks, So,
82,85;
in Kingfishers, 86, 90, 91
; use of parental, in taming birds, 130133;
instinctive reactions
of young
of Cedar-birds, 55;
of
fear,
suppression
of,
in
Red-eyed
Vireos,
68;
appear-
ance
of, in young Red-eyed Vireos,
68;
of
fear, suppression
of,
in
Bluebirds,
72. 73;
walk-
ing, in vertebrates, 89; of inspection of young and nest, 103-106; cleaning or sanitary, in birds,
103-110;
dearth of observations upon cleaning,
104;
of cleaning nesting site, 107-110;
prey-
ing, in young Red-eyed Vireos, 67;
of preening in young, 65;
of Chimney Swift in nest-build-
ing. 113;
of fear in old and young,
1 17-124;
of fear in domestic chick,
1 19;
of Canada Goose
i
modified in captivity, 129, 130;
of "
feigning" in Chestnut-sided Warbler. 131, 132. p
y,
g
g
Intelligence, human, the roots of, xv. xvi;
the sign of. xvii. G. Galapagos Islands, observations of Darwin on fauna of,
136,
137. Gluttony in Cedar birds,
101,
102. Goldfinch, American (Spinus Iristis, Linn.), lateness of breeding of, 52;
attracted by Robin's alarm, 123. Goose, Canada (Branta
canadensis, Linn.), young
of,
117;
tamability
of. 127,
129,
130;
habits and
breeding
of,
in captivity,
129. 130. g
,
captivity,
Grampus (Corydalus cornntus)
, feeding
of,
to. young Kingbirds,
103;
formidable appearance and size
of,
101,
103. g
p
y,
Grampus (Corydalus cornntus)
, feeding
of,
to. young Kingbirds,
103;
formidable appearance and size
of,
101,
103. ,
03. Grasshoppers brought to nest by Robin, 39. p
g,
;
p
Gullet, distcnsibility of, in the Cedar-bird, 55, 61;
effect of full, in young,
101; automatic response of,
in young birds, 55, 101, 102. Index. H3 lay. Canada, Moosebird (Pen'soreits
< anadensis, Linn.), lameness and habits of,
129. (cllerson (O.), winter birds at, 128. H. Habit, definition
of. as distinguished from
Itiii'il.-. in the popular sense,
xvii:
the formation
of. 3,
4,
5,
in,
116;
of Cedar-bird
in alighting on tent. 62;
of sipping maple sap. in Cedar-bird, 62;
in
manner of approach to nest
in Red-eyed Vireo,
111-112;
of walking backward and sitting
still in young Kingfishers explained, 89;
of entering and leaving tunnel, in Kingfisher, 90, 9
i
;
of Kingfisher to prevent escape of prey, 92;
of eating excreta, how acquired in a hungry bird,
109, no;
illustration
of, in nest-cleaning,
112;
change of food, in Rhinoceros-bird,
116;
force
and variation of, in nesting of Osprcv. 115, 116;
plasticity of, in reference to food, 116;
of fear,
of special objects,
121. p
j
,
Hawk, the Fish (sec Osprey)
;
Hen, the effect of, in sky upon chick in dooryard,
120. Haks feeding on locusts,
i 16. g
Hellgamitc,
larva; of grampus,
103. Hinde, Captain,
Holderness (N. H.), spring arrival of Bluebirds at,
71:
habits of Pine Grosbeaks
in winter and spring
at,
135. Humming-bird, eggs and young of,
117. g
gg
y
g
Hunger in relation to fear, 126-128. lay. Canada, Moosebird (Pen'soreits
< anadensis, Linn.), lameness and habits of,
129.
(cllerson (O.), winter birds at, 128. M. Maple sap, sipping of, by Cedar-birds, 62. p
p,
pp
g
y
Methods of bird-photography, the old, xvii, xviii;
the new. xviii. 1-16;
analysis of new, 3-6;
applica-
tion
of,
7, 8;
precautions to be observed in use of. 8
11;
extent of application of, 1113;
table
of experiments in, 12;
objections to, 13-15; advantages of, 1516;
fascination of. 16;
illustra-
tion of,
17-18;
original suggestion of, 54, 55. (Sec also table, page
12, and under names of
species.) p
)
Mirrors, use of, 35. Mites, parasitic, on young of Cedarrbird,
107. p
,
y
g
Mount La Fayette (N. H.), Pine Grosbeak in summer at,
135. p
,
y
g
Mount La Fayette (N. H.), Pine Grosbeak in summer at,
135. y
Mouse, Deer- or White-footed (Hcspcromys laicofns,
]<>:/, LcC.), nest of Red-eyed Vireo occupied by,
N K. Index. 144 Kingfisher, the Belted (Ceryle alcyon, Linn.), general habits
of, 86-93:
attachment
of, to nesting site,
86;
subterranean nest of , 86
;
dimensions of nest
of, 86
; young of, 86, 89, 91-93;
use of tarsus
in foot
of, 89;
habits of young of,
89, 91,
92;
habit of
walking backward, how acquired in
young of, 89, 90; pugnacity in young of, 89; use of tent before tunnel of, 89, 90; photographing
adult, 90;
the feeding of young, by parents, 90, 91;
feeding of captive young, by hand, 92;
moving nesting chamber by, 92;
habits of young of, in captivity, 92;
structure of oesophagus
and bill of, 92;
trick-like performance of, 92;
notes of adult and young of, 90, 91, 92;
visits
of, to nest, and manner of entering and leaving tunnel by, 90, 91
;
colors in young of , 86, 91, 92;
peculiar expression in young of, 91;
liberation of captive young of, 93; parental
instincts
in,
86, 90, 9 1
; character of excreta in young of, 107
;
sanitary condition of nest in, 107
;
develop-
ment of fear in young of,
1 19. L. Leaves, plucking or cutting
of, about a nest, 8, 14;
keeping, fresh on branches cut from trees of various
kinds,
14, 15;
result of cutting of, at Catbird's nest, 79. Leaves, plucking or cutting
of, about a nest, 8, 14;
keeping, fresh on branches cut from trees of various
kinds,
14, 15;
result of cutting of, at Catbird's nest, 79. ,
,
;
g
Lenses, kinds
of, available for photographing wild animals, 32-34;
the Anastigmat,
32, 33;
qualities
of, most needed in animal photography, 33;
long focus, 32, 34;
telephoto, 32, 34. ;
g
Lenses, kinds
of, available for photographing wild animals, 32-34;
the Anastigmat,
32, 33;
qualities
of, most needed in animal photography, 33;
long focus, 32, 34;
telephoto, 32, 34. ,
photography,
;
g
,
;
p
,
,
Locust, Rocky Mountain, eaten by birds during plague. 116. Loon, eggs and young of,
117. Locust, Rocky Mountain, eaten by birds during plague. 116. Loon, eggs and young of,
117. y
Loon, eggs and young of,
117. Nesting
Nesting O. Objections
i<> method eonsidered,
13-15. j
Observations from tent, the best time for,
7, S. ,
Oriole, Baltimore
(Icterus g^il/nila,
I. inn.}, nest
of,
iS;
call-notes of young of,
iS;
removal of nesttn<:
bough of,
ii):
behavior of,
19;
food brought 1" young by,
icj;
rate of feeding at nest of,
iq;
exercise of the fledglings of, 20;
flight of young of, from the nest, 20;
use of tent at nest of, 19;
cleaning instinct in, 104; lack of discrimination in young of , 121; summoned by alarm of Robin,
122. Oriole, Baltimore
(Icterus g^il/nila,
I. inn.}, nest
of,
iS;
call-notes of young of,
iS;
removal of nesttn<:
bough of,
ii):
behavior of,
19;
food brought 1" young by,
icj;
rate of feeding at nest of,
iq;
exercise of the fledglings of, 20;
flight of young of, from the nest, 20;
use of tent at nest of, 19;
cleaning instinct in, 104; lack of discrimination in young of , 121; summoned by alarm of Robin,
122. Osprey, the American, or Fish Hawk
(7\?;;, //<>>; haliaftus
Ctimlinrnsis,
Gnicl.), nests and nesting habits
of, at Plum Island (X. Y.),
115,
134,
15,;
at Bristol (R. I.),
115,
116;
nest of, on cart-wheel
on top of pole, 116;
actions of, upon loss of mate, 116; tameness of,
1.34, 135. N. Narragansett Bay (R. I.)
, Fish Hawks on shores of, 115,
1 16. g
y
Naturalist, duty and privilege of, xv;
patience of,
13. 14. Nest,
inspection and sanitation
of,
103-110;
displacement
of,
2
(see Nesting
site);
photographing,
when inaccessible to tent, 34;
of Cedar-bird,
17,
53,
54,
56,
58,
61;
of Baltimore Oriole,
18;
of Robin, 36, 40, 49,
50;
of Red-eyed Vireo,
64, 69;
of Bluebird,
72;
of Catbird,
76,
77; of
Night Hawk, So;
of Kingfisher, 86;
of Chimney Swift,
113, 114;
of House Sparrow, 114;
of
Osprey, 115,
116, 134, 135;
of Black Duck, 119;
of Magnolia Warbler, 121;
of Chestnut-sided
Warbler,
131;
of Phoebe, 133;
of Red-eyed Vireo, occupied by Yellow Warbler, 69;
of same,
used by Deer-mouse, 69,
70;
destruction
of, in Red-eyed Vireo, 69;
Bluebird in old Robin's,
75;
movement of nesting chamber in Kingfisher, 92;
filthiness
of, in Turkey Buzzard,
roS;
parasites
of,
in Cedar-bird,
107;
sanitary condition
of,
in
Kingfisher,
107;
filthiness
of,
in
Owls,
108;
habit in approaching, in Robins, in;
in Red-eyed Vireos, in,
112;
in Redwing
Blackbird,
112;
adaptation
in character and position
of,
113,
114;
unusual position
of,
in
Chimney Swift,
113, 114;
in House Sparrow, 114;
of Bay-winged Bunting despoiled by black
snake,
123. Nesting bough, removal and mounting of,
7
(see Nesting site). Nesting site, control of,
i;
'-'hen to change, 6;
experiments in change of, tabulated,
12;
accidents due
to change of, guarded against,
12,
13;
change of, in Cedar-bird,
17,
54,
55,
57,
59;
in Oriole,
19;
in Robin, 39, 40;
in Red-eyed Vireo, 64;
in Bluebird,
72;
in Catbird,
76,
77;
in Night
Hawk, So;
in Kingfisher, 86, 89, 90;
the choice of new, by operator,
7;
attachment of birds
to, 13;
of Kingfisher to, 86;
of Osprey to,
1 1 5
;
importance of cleanliness of, in passerine birds,
105; unusual, in Chimney Swift, 113, 114; in Osprey, 115; lack of cleanliness of,in birds, 107, 108. Index. Index. 145 Night Hawk, Bull-ba1
(Chordeiles virginianus,Gmel.),
the feeding of, 15, 16;
the rearing of, 80-85
;
'
and incubation
of. So;
behavior of, during incubation,
.so:
hatching
of. So:
the yoir
So-,s'5;
expression of fear in old and young of,
So,
Sr:
eorallinir the young
of, Si;
eyes and
eyelids in young of. So;
brooding habits of. So, 82, 85;
call and alarm notes of. Si, 82;
feeding
habits in y< >ung ol. Si. 82
;
illumination of throat of, by phosphorescent insects, 82
;
encounter
of young of, with snake, Si
;
Hedging of. 85. y
g
g
g
Xorthticl.l (X. II. i, breeding of Cedar-birds at, 52; spring Bluebirds at, 71; nesting of Kingfisher at, 86. (See
Preface
i (See
Xuthatehes, lameness and habits of,
127,
128. (
Xuthatehes, lameness and habits of,
127,
128. (
Xuthatehes, lameness and habits of,
127,
128. P. Parasites on nest and young of Cedar-bird,
107. y
g
Partridges, eggs and young of,
i 17. gg
y
g
Patience required in the naturalist's work,
13,
14. 1 attcn,
William,
114. Peep-holes in observation tent, the form and size of, 31. ,
,
. Peep-holes in observation tent, the form and size of, 31. Phoebe, taming of, 133,
134 g
,
Phosphorescence, display of, in Xight Hawk, 82. p
g
Photography of birds, method of, xvii;
the future of. xviii;
a new method of. based on animal instinct,
xviii;
new method described,
1-16;
its conditions,
i;
its principles analyzed,
3;
mode
of
procedure in,
7;
precautions in use of, S;
extent of application
of, 11-13;
objections to, con-
sidered,
13-15;
advantages
of,
15-16;
illustrations
of,
17-28;
the tools
of, 29-35;
of birds
after they have been tamed,
in Robin, 131,
in Chestnut-sided Warbler,
i ^. Pigeon, English Wood, habit of, 137; domestic, tameability of,
125;
wild passenger, condition of nest-
ing site
in,
107. ing
,
107. Plates, photographic, exposure of, 34;
deterioration of, 34
;
carriage and care of,
34. g
Plates, photographic, exposure of, 34;
deterioration of, 34
;
carriage and care of,
34. l
and p
g
p
, exposure
,
Plover, eggs and young of,
117. , eggs
young
,
117. Plum Island (N. Y.), In-ceding and habits of Osprey at,
i 15,
134,
135. Polygamy in Bluebirds,
72. Polygamy
,
72. Popular natural history, defects of, xv, xvi;
illustrations of, xviii, xix. yg
y
,
72. Popular natural history, defects of, xv, xvi;
illustrations of, xviii, xix. P Precautions to be observed in change' of nesting site, 8 Precautions to be observed in change' of nesting site, 8. g
nesting s te,
Precision in instinctive acts of young birds, 67. g
nest
Precision in instinctive acts of young birds, 6 Precision in instinctive acts of young birds, 67. young
Preening instinct
in young Vireos, 65. g
y
g
65. Principles of new method of bird-study, 3. c p es
bird study, 3. Pugnacity, the instinct
of, 4;
in Kingbirds, 22;
in Robins. 40,
134;
in Red-eyed Vireos, 67;
in young
of Kingfisher, 89;
in domestic fowl, 134;
in Tropic Bird,
134. R. young
food,
55.
Redstart, flight of young of, from nest.
118;
attraeled by Robin's alarm, 123. Reaction of young bird to stimulus of food,
55. Osprey, the American, or Fish Hawk
(7\?;;, //<>>; haliaftus
Ctimlinrnsis,
Gnicl.), nests and nesting habits
of, at Plum Island (X. Y.),
115,
134,
15,;
at Bristol (R.
I.),
115,
116;
nest of, on cart-wheel
on top of pole, 116;
actions of, upon loss of mate, 116; tameness of,
1.34, 135. S. Sac of excreta in young,
104;
disposition
of, by parents,
104-110;
character
of, in Robin and inspec-
tion
of, after removal from nest,
105,
106;
bursting of, in mouth of Robin,
109;
seizing and
devouring of, by Chestnut-sided Warbler,
109. Sac of excreta in young,
104;
disposition
of, by parents,
104-110;
character
of, in Robin and inspec-
tion
of, after removal from nest,
105,
106;
bursting of, in mouth of Robin,
109;
seizing and
devouring of, by Chestnut-sided Warbler,
109. g
y
,
Sanbornton (N. H.), Robin's nest under cover at, 49. g
y
,
Sanbornton (N. H.), Robin's nest under cover at, 49. (
),
, 49. Sanitation
of
nest,
103110;
in Woodpeckers,
Chickadees, Thrushes, Waxwings,
Vireos,
Warblers,
Orioles, Blackbirds, Bluebirds,
104;
in Crow Blackbird,
104,
105. (See also under names
of
species.) Sanitation
of
nest,
103110;
in Woodpeckers,
Chickadees, Thrushes, Waxwings,
Vireos,
Warblers,
Orioles, Blackbirds, Bluebirds,
104;
in Crow Blackbird,
104,
105. (See also under names
of
species.) species.)
Shutter, concealment of observer while setting, 31; iris diaphragm, 34;
focal plane, p
)
Shutter, concealment of observer while setting, 31; iris diaphragm, 34;
focal plane, 34;
time marks of,
34;
rapidity of, 34;
a desideratum in,
5, 34. J.,
,
,
Snake, rescue of Vireo from, 69;
encounter of Night Hawk with, Si;
black, in act of swallowing young
bird,
123. ,
,
Snake, rescue of Vireo from, 69;
encounter of Night Hawk with, Si;
black, in act of swallowing young
bird,
123. ,
123. Snipe, eggs and young of,
117. Snipe, eggs and young of,
117. p ,
gg
y
g
Snow eaten by Chickadees,
128. y
Sounds, effect of, upon birds,
5, 68,
112. p
Sparrow, Chipping, suppression of fear in, 5; House (Passer domestic-us. Linn.), pugnacity of, 72; com-
bat
of,
with
cicada,
103;
condition
of nesting
site
in,
107;
nesting
of, in hood of electric
street-lamps,
114;
nest of Eaves Swallow appropriated by, 114, 115; tameability of. 125,127;
Song (Mclospiza
fasciata, GincL), nest of,
12;
attracted by alarm of Robin,
123;
habits of,
during incubation, 134. during
, 134. Spiders or their prey eaten by Cedar-birds, 63. p
p
y
Stork, habits of,
125. (
y), d spossessed
y
p
,
Swift, Chimney (Chcctura pelagica. Linn.), significance in change of nesting habits of
instinct of, 113;
nesting of, in barn and shed,
114. R. 46-48;
economy in food in, 39;
neatness
of,
39;
spontaneous behavior
in,
39;
parental instincts
of,
39,
40, 45;
panoramic scenes at
nest of, 46, 47;
flight from nest of, 40, 47, 48;
keenness of vision
of, 39;
habits and instincts
of fledglings of, 40, 47, 48; peculiar notes of, for arousing the young at nest, 45;
in winter, 48;
fruits eaten by, in summer and winter, 48;
nests of, under cover, 49, 50;
nest of second brood of,
50;
in city
life,
50,
51;
gregarious habits of, in summer and winter,
51;
Bluebird in nest of,
75; eating excreta of young by, 39, 105, 106, 109;
characteristic attitudes of, 105; actions
of, in
cleaning the nest,
105,
106;
formation of habits in,
111;
food brought to nest
of,
116;
eggs
and young of,
117;
effect of alarm of
female
of, upon cock at nest,
122;
as an exponent of
taming process, 130-131;
display of pugnacity in, 40,
134. R. 46-48;
economy in food in, 39;
neatness
of,
39;
spontaneous behavior
in,
39;
parental instincts
of,
39,
40, 45;
panoramic scenes at
nest of, 46, 47;
flight from nest of, 40, 47, 48;
keenness of vision
of, 39;
habits and instincts
of fledglings of, 40, 47, 48; peculiar notes of, for arousing the young at nest, 45;
in winter, 48;
fruits eaten by, in summer and winter, 48;
nests of, under cover, 49, 50;
nest of second brood of,
50;
in city
life,
50,
51;
gregarious habits of, in summer and winter,
51;
Bluebird in nest of,
75; eating excreta of young by, 39, 105, 106, 109;
characteristic attitudes of, 105; actions
of, in
cleaning the nest,
105,
106;
formation of habits in,
111;
food brought to nest
of,
116;
eggs
and young of,
117;
effect of alarm of
female
of, upon cock at nest,
122;
as an exponent of
taming process, 130-131;
display of pugnacity in, 40,
134. Rhinoceros-bird (Bnpliaga erytkmpyncha) , change in food habits of, 116. Robin
(.1/Vn/Ai inigratoria. Linn.), the
instincts
of, displayed in migration and nest building, xvi;
no
learning of instinctive responses required or possible,
xvi;
time spent by young
of,
in non-
nesting site,
13;
as symbol of cheerfulness, 36;
history of, 3651;
spring arrival
of,
48, 49;
incubation in, 36;
choice of nesting site in, 49;
behavior of, when nesting bough is moved, 39,
40, 45;
call-notes of, 39, 45;
feeding young in, 39. R. Reaction of young bird to stimulus of food,
55. Index. 146 Redwing (sec Blackbird). Redwing (sec Blackbird). Regurgitation, of indigested food in Kingbirds, 28;
of food for young in Cedar-birds, 55, 61;
in Vireo, 66. d
i
l
f Redwing (sec Blackbird). Regurgitation, of indigested food in Kingbirds, 28;
of food for young in Cedar-birds, 55, 61;
in Vireo, 66. Reproduction, cycle
of,
3,
4. p
,
Respiration in Redwing Blackbird,
21. Respiration
Redwing Blackbird,
21. Response, of throat and gullet of young, 55, 101, 102
;
in young of Baltimore Oriole to one of their num-
ber, 12 1
;
of young Red-eyed Vireos to notes of other birds, 68;
of young of Bluebirds, 74;
of
young of Catbirds,
77,
78. Respiration
ed
g
,
Response, of throat and gullet of young, 55, 101, 102
;
in young of Baltimore Oriole to one of their num-
ber, 12 1
;
of young Red-eyed Vireos to notes of other birds, 68;
of young of Bluebirds, 74;
of
young of Catbirds,
77,
78. young
,
77,
78. Rhinoceros-bird (Bnpliaga erytkmpyncha) , change in food habits of, 116. Rhinoceros-bird (Bnpliaga erytkmpyncha) , change in food habits of, 116. Robin
(.1/Vn/Ai inigratoria. Linn.), the
instincts
of, displayed in migration and nest building, xvi;
no
learning of instinctive responses required or possible,
xvi;
time spent by young
of,
in non-
nesting site,
13;
as symbol of cheerfulness, 36;
history of, 3651;
spring arrival
of,
48, 49;
incubation in, 36;
choice of nesting site in, 49;
behavior of, when nesting bough is moved, 39,
40, 45;
call-notes of, 39, 45;
feeding young in, 39. Tail of Bluebird used for support. 73-74. support. 73 74.
Tamcness, of birds in nature,
125,
127,
128, 135-137:
of the Pine Grosbeak,
135:
of Bohemian Wax-
wing, 136;
of Crossbills, 136;
of birds of Galapagos Islands, 136. T. Tail of Bluebird used for support. 73-74. a
o
Bluebird used for support. 73-74. Tamcness, of birds in nature,
125,
127,
128, 135-137:
of the Pine Grosbeak,
135:
of Bohemian Wax-
wing, 136;
of Crossbills, 136;
of birds of Galapagos Islands, 136. Index. 147 Taming birds without a cage. 125-157. Taming birds without a cage. 125-157. Taming process, conditions and unal\
:is
of,
126,
127;
use of tent
in,
130;
Robins and Chestnut-sided
Warblers as exponents
..f, 1^0-133;
Phoebe as illustration of, 133, 134. ,
g
s e ,
Tent, as an observatory, 2,
i;. id:
time required for birds to become accustomed to, 5,
IT: window of,
5,
31
:
tin- time to use,
7;
]ireeautions in use of,
S;
experiments in
us,-
of, tabulated,
12;
the
future of, as an observatory for the study of birds,
13;
protection afforded by,
i
,
:
advantages
of position of,
15; before
l.'ecl.ir- bird's nest,
17, 5;. 57,
51;; as an observati >ry
tor the birds,
12,
21, 4;,
=;S:
In-fore nest of Redwing Blaekbird,
20;
befon- nest
of Oriole,
iy;
construction
o|_
29:
convenience of, 30;
instructions for use of, 31
;
before nest of Red-eyed Vireo. d.[;
betore
nest-hole of Bluebird. 7.-;
beside nest of Catbird,
7'.. 77;
before voting of Night Hawk, Si;
before tunnel of KingtiMier,
NI), 90;
use of, in lamina; birds,
130;
before nest of Chestnut-sided
Warbler. 132. i
$3. $
Ti-nt-eloth, material anil color of,
29. $
Ti-nt-eloth, material anil color of,
29. Tent-frame, dimensions and construction
of,
29. Tent-frame, dimensions and construction
of,
29. Tent-pins, form and use of, 29. 31. Tent-pins, form and use of, 29. 31. Tent-pins, form and use of, 29. 31. Tent-pins, form and use of, 29. 31. p
Tent-window
. po
ition
-f. 3 i. Tent-window
. po
ition
-f. 3 i. Tent window
. po
ition
f. 3 i. Throat, response of,
in young birds. ^5;
eolorof,
in young Robin, 30;
as target for the parent. 49:
in
\oiuig Cedar-bird, 56;
inflation of, in Red-eyed Yirco, 66;
in Chestnut-sided Warbler, 97. Thrush, Brown
(I larporltynilius rnjus,
I. inn.), camping beside nest
of, 94;
brooding of young
in, 04:
eating excreta of young by,
105;
attracted by alarm of Robin, 122;
Wilson's or Veery (Tnrdn:-
/H SYOWH V
Sicph )
. young of, 12: premature development of fear in young of, 121. T. po
Throat, response of,
in young birds. ^5;
eolorof,
in young Robin, 30;
as target for the parent. 49:
in
\oiuig Cedar-bird, 56;
inflation of, in Red-eyed Yirco, 66;
in Chestnut-sided Warbler, 97. y
Thrush, Brown
(I larporltynilius rnjus,
I. inn.), camping beside nest
of, 94;
brooding of young
in, 04:
eating excreta of young by,
105;
attracted by alarm of Robin, 122;
Wilson's or Veery (Tnrdn:-
/H.SYOWH.V, Sicph.)
. young of, 12: premature development of fear in young of, 121. /H.SYOWH.V,
p
)
young
Thrushes, cleaning instinct of. 104. g
Trees, keeping fresh leaves of cut branches of, 15;
mutilation of,
15. g
Trees, keeping fresh leaves of cut branches of, 15;
mutilation of,
15. Tripod, best form of. 34. Tripod, best form of. 34. p
,
Tropic Bird, pugnacity of, during incubation,
134. V. Venice, "doves" or pigeons of,
125. p g
Vireo, Red-eyed (l"m\> oliraci-iix, Li tin.}, coming to tent, 5;
nest and young of, 64;
call-notes of, d;
:
be-
havior of nestlings of, 64, 65, 67, 68;
digestion and assimilation in young of, 66;
feeding the
young in, 65-68;
inspection and cleaning the young in, 65,
(17, dS;
sleekness and neatness of,
67;
preying instinct in young of. 67;
young of, aroused by notes of other birds, 68;
capture
of prey by, 68;
indifference of, to customary sounds. 68;
signs of emotion in, 68;
supprc^i, >n
of fear in, 68;
appearance of sense of fear in young of, 69;
rate of feeding at nest of. 69;
old
nest of, utilized by Yellow Warblers, 69;
old nest of, used by deer-mouse, 69, 70; flight from
nest
of. 69;
rescue
of young of, from snake, 60;
destruction of nest
of, 6q;
fragility of
old
nests of,
69;
carelessness in construction of nest
in,
69;
eating of excreta of young by. 105;
cleanliness of nesting site
in. 108;
habit of approaching the nest
in. in,
112;
attracted by
alarm of Robin, 122,
123. Woodpecker, use of old nest-hole of, by Bluebird,
72;
cleanliness of nest in,
104;
Downy and Hairy,
lameness of, in winter,
128, 129;
eggs and young of, 117;
habits of, during incubation,
13;. Waxwing, origin of name of, 52; Bohemian, habits and record of, 136 (see Cedar-bird)
.
\Vildness, of birds, origin of,
125, 126, 137 Y. Young, as strong lure, xvii, 6, 126, 127, 130; exposure of , to intense heat, 8; study of , at nest, 8; danger
to, from insufficient food, 10; proper age of, when nesting site is changed, 6;
necessity of shade
to,
8,
13;
of Cedar-bird,
18, 60;
of Baltimore Oriole,
18, 19;
frequency of feeding, in Orioles,
19;
call-notes of, in Oriole,
18;
exercise
of, in
Oriole,
20;
flight from nest
of, in Oriole,
20;
feeding of, in Redwing Blackbird,
2 i
;
hatching of, in Kingbird,
2 1
;
change of, in nest of King-
bird, 27;
feeding, in Kingbird,
27,
28,
103;
brooding in
Kingbird,
28, 94, 97;
flight
of, from
nest in Kingbird, 28;
gape, color of mouth, and behavior of, in Robin,
39, 40, 47;
flight of,
from nest, in Robin, 40, 47, 48;
cats as enemies of, 51;
instinctive reaction of, to food in Cedar-
bird,
55;
hatching of, in Cedar-bird, 58, 59;
opening of eyes of, in Cedar-bird, 59;
of Cedar-
bird leaving the nest,
18, 60;
time spent in nest by, in Cedar-bird, 60;
appearance of feather-
shafts and wax-like appendages to wings of, in Cedar-bird, 60;
development of color-marks of,
in Cedar-bird, 60, 61; habits of, in Cedar-bird, 60; food and care of , in Cedar-bird, 55-62; cleaning
of, in Cedar-bird, 56, 105-107;
digestion, assimilation, and growth of, in Red-eyed Vireo, 66;
behavior of, in Red-eyed Vireo, 64-68;
preying instinct
of, in Red-eyed Vireos, 67;
aroused
by notes of other birds, 68;
appearance of
fear
in Red-eyed
Vireos, 69;
rate
of feeding
of,
in Red-eyed Vireos, 69;
response and feeding of, in Bluebird, 73; food and rate of feeding, in
Bluebird,
75;
feeding and care
of, in Catbird,
77,
78,
79;
rate of feeding of, in Catbirds, 78;
behavior of, in Catbirds, 78, 79;
of Night Hawk, So;
hatching of, in Night Hawk, So;
color of,
in Night Hawk, So, 85; eyes of, in Night Hawk, So; behavior of, in Night Hawk, 80-82; walking
of, in Night Hawk, Si;
coralling of, in Night Hawk, Si;
call-notes of, in Night Hawk, Si, 82;
feeding habits in Night Hawk, Si, 82, 85;
fledgling stage in Night Hawk, 85;
call-notes of, in
Kingfisher, 90, 91, 92;
colors of, in Kingfisher, 86, 91, 92;
peculiar expression of, in Kingfisher,
91;
development of feathers
of, in Kingfisher, 86, 91;
function of tarsus of, in foot of King-
fisher. W. instinct of, in vertebrates, 89;
habit of, in young of Kin gfishcr. 89. y
g
g
Warbler. Yellow
(Daiilr. -i,
.1
tiva,
Gmel.), using nest
of Red-eyed
Yireo,
69:
Chestnut-sided
i
droica pennsylvanica, f.inn.). excreta of young of, eaten by, 105, 109; nesting habits of, 131-133;
taming of,
132-133;
photographing, without
tent,
133;
attracted by alarm of Robin,
122;
development of fear in young of. 118;
Magnolia
(Dcn<!i,>i,
<i maculosa,
tiincl.}, as foster parent
to Cowbird. 121. 122;
fate of rightful young of,
122;
Maryland Yellow Throat, attracted by
alarm of Robin,
123. Waxwing, origin of name of, 52; Bohemian, habits and record of, 136 (see Cedar-bird)
\Vildness
of bi d
i i
f
126 \Vildness, of birds, origin of,
125, 126, 137. Index. 148 Y. 89;
general habits
of, in Kingfisher, 89, 91, 92;
habit of walking backward in, of King-
fisher, how acquired, 89;
habit of sitting still, in Kingfisher, 89, 90;
pugnacity of, in Kingfisher,
89;
feeding of, in Kingfisher, 90, 91: habits of, in captive Kingfishers, 92; careof,94; brooding
and feeding of, 94-103;
diet of, in Cedar-bird, 101;
automatic response of gullet of,
101, 102;
inspection and cleaning of, 103-110;
character of excreta in,
104;
disposal of excreta of, by
parents, 104-107;
character of excreta in Kingfisher, 107;
use of excreta of, as food by adults,
105,
107,
109;
development of fear of, in Catbird,
117,
118,
in Chestnut-sided Warbler,
118,
in Kingfisher, 119;
imperfect digestion' of food in, 109;
fear in,
1 17-122;
condition of, at time
of hatching, as basis for classification,
117;
fear in Black Ducklings,
120;
death
of, due to
premature development of fear,
120,
121;
lack of discrimination in,
121;
behavior of, in Bal-
timore Orioles, 121;
care and education of, 121;
acquisition of fear of special objects in, 121;
use of pot-belly of, 121;
of Bay-winged Bunting attacked by black snake, 123. '
:
'
; >
. . - LAWNS AND GARDENS How
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"The authors of this excellent work offer it, not in competition with scientific botanies,
but with the hope that by their drawings and descriptions they may make it easy to become
acquainted with the wild flowers of the northeastern portion oi the United States'. Anybody
who can read English can use the work and make his identifications, and, in the case of some
of the flowers, the drawings alone furnish all that
is necessary. . . . The descriptions are
as good of their kind as the drawings are of theirs."
N. Y. Times.
|
https://openalex.org/W4205205669
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https://ahlimedia.com/jurnal/index.php/jira/article/download/240/216
|
Indonesian
| null |
Penggunaan Media Pop Up Book (Gambar Buku Tiga Dimensi) untuk Meningkatkan Kompetensi Membaca Literasi Buku Fiksi dan Non-Fiksi Siswa SMP
|
Jurnal Inovasi dan Riset Akademik
| 2,022
|
cc-by-sa
| 2,833
|
Vol.2 No.11 2021
ISSN: 2745-6056 | e-ISSN: 2745-7036
https://doi.org/10.47387/jira.v2i11.240
Received
: 12-09-2021
Revised
: 25-10-2021
Published
: 30-11-2021 Vol.2 No.11 2021
ISSN: 2745-6056 | e-ISSN: 2745-7036
https://doi.org/10.47387/jira.v2i11.240
Received
: 12-09-2021
Revised
: 25-10-2021
Published
: 30-11-2021 Vol.2 No.11 2021
ISSN: 2745-6056 | e-ISSN: 2745-7036
https://doi.org/10.47387/jira.v2i11.240 Received
: 12-09-2021
Revised
: 25-10-2021
Published
: 30-11-2021 Penggunaan Media Pop Up Book (Gambar Buku Tiga
Dimensi) untuk Meningkatkan Kompetensi Membaca
Literasi Buku Fiksi dan Non-Fiksi Siswa SMP Sulistyaningsih
SMP Negeri 1 Pakisaji Kabupaten Malang, Indonesia
sulistyaning130@gmail.com Vol.2 No.11 2021
ISSN: 2745-6056 | e-ISSN: 2745-7036
https://doi.org/10.47387/jira.v2i11.240 PENDAHULUAN PENDAHULUAN Berbagai Macam Upaya Pendidkan untuk menyempurnakan strategi pembelajaran
dalam meningkatkan mutu pendidikan nasional. Rendahnya minet baca merupakan salah satu
foktor rendahnya informasi pengetahuan yang didapat sehingga ketertinggalan akan
perkembangan teknologi. Pada abad ini hampir di segala sektor kehidupan terjadi perubahan
yang sangat cepat, bahkan hampir tak terduga.Supaya semua perubahan tersebut segera dapat
diketahui seseorang harus memperoleh informasi dari sumber manapun.Kemampuan yang
harus dimiliki untuk melakukan itu semua adalah kemampuan membaca. Kemampuan
membaca tersebut bukan sekadar dapat membaca, melainkan membaca secara cepat, apalagi
sumber informasi digital dan elektronis yang sekarang ini semakin pesat g
y
g
g
p
Menurut Baldridge (1979), setiap calon cendekiawan abad modern ini dituntut untuk
membaca 850.000 kata/menit. Jika seseorang hanya mampu membaca 250 kata/menit, dalam
seminggu ia harus membaca kira-kira 56 jam, artinya 8 jam/hari. Sungguh dramatis, bukankah
hidup ini tidak hanya diabdikan untuk membaca? Masih banyak tugas lain yang lebih penting
daripada itu. Agar seseorang dapat memanfaatkan waktu dengan efisien, sekali lagi seseorang
perlu memiliki keterampilan membaca cepat. Kemampuan membaca cepat ini dapat
digunakan untuk berbagai keperluan sesuai dengan tujuan dan manfaat yang ditetapkan Kenyataan menunjukkan bahwa semakin berkembang karier seseorang tuntutan untuk
membaca juga semakin besar, padahal waktu yang tersedia semakin terbatas.Semua harus
berpacu dengan informasi dan gagasan yang setiap hari membanjiri meja kerjanya. Informasi
yang membanjir akan memperbudaknya apabila ia tidak terampil membaca cepat. Sementara
itu, masih terdengar keluhan bahwa kemampuan membaca buku-buku para mahasiswa
Indonesia terlalu lemah.Mereka terlalu lama menyelesaikan pembacaan buku-buku, bahkan
buku-buku yang tipis sekalipun.Hal itu terjadi bukan hanya karena kesalahan mereka.Sewaktu
bersekolah di Taman Kanak-Kanak dan Sekolah Dasar mereka memang diajari membaca,
mengenali kata, mengejanya dan seterusnya. Ketika duduk di bangku sekolah menengah
pertama mereka tidak lagi diajari cara membaca yang benar. Salah satunya adlah cara
membaca ceoat yang benar. Tampaknya terdapat berbagai sebab mengapa kemampuan membaca para siswa kita
rendah Faktor yang dimaksud dapat berasal dari dalam maupun dari luar siswa.Faktor dari
dalam berarti faktor dari siswa.Mereka mempunyai kebiasaan 'menunda atau interupsi,
mengulangi pembacaan, vokalisasi dan subvokalisasi.Sedangkan faktor dari luar misalnya
dari guru. Guru kurang tepat dalam memilih dan menggunakan media pembelajaran. Secara
tidak langsung hal tersebut akan semakin membuat kemampuan membaca para siswa semakin
rendah dan ini berarti semakin memperbesar ketidak berhasilan pembelajaran membaca cepat. ABSTRAK Melalui media buku pop up merupakan suatu media yang memiliki unsur tiga
demensi buku pop up dapat digunakan untuk media pembelajaran dengan
mengunakan kertas warna sebagi penunjang bentuk-bentuk unik dan kreatif yang
bisa kembangkan jadi kerya unik tempat untuk mengreasikan ide-ide siswa yang
didapat dari analisis buku-buku fiksi dan non fiksi yang dibaca oleh siswa Tujuan
dari peneliti adalah untuk meningkatkan kompetensi membaca dalam menemukan
isi buku saat berliterasi baik buku fiksi maupun non fiksi Bentuk penelitian ini
adalah penelitian tindakan kelas (PTK) dengan menggunakan dua siklus dengan
teknik pengumpulan data dari penilaian portofolio pertunjukan saat proses belajar
mengajar dan presentasi hasil dimana siswa mempresentasikan hasil berliterasi
sedangkan guru menilai hasil kerja siswa serta menentukan hasil kerja siswa yang
terbaik ,Siswa yang belum sempurna hasil kerjanya akan di sempurnakan bersama-
sama dengan cara memberi masukkanMedia pop up digunakan siswa sebagai media
berliterasi.Hasil penelitian ini berupa 1) produk media pembelajaran pop up book
kombinasi dengan kartas warna yang berisikan analisis buku fiksi dan nonfiksi
sebagai bahan penguasaan kompetensi membaca pada siswa kelas VIII SMP Negeri
1 Pakisaji Media yang dihasilkan siswa 92% dapat di terima siswa, serta dapat
menambah karakter rasa percaya diri untuk mencipta, tanggung jawab dengan tugas
yang di berikan berani untuk mengemukakan hasil kerja yang dihasilkan sendiri,dan
mampu bersaing dengan karya-karya lain sebagai inpiriksi. Sehingga dengan mudah
memahami buku-buku fiksi dan non fiksi. Kata kunci: media popup book; literasi; fiksi non-fiksi 1555 METODE Dalam penelitian tindakan kelas ini peneliti menginformasikan salah satu penggunaan
media pembelajaran yang dapat meningkatkan kemampuan membaca cepat.Media yang
dimaksud adalah Media Olah kertas warna dalam menuangkan ide dan kreatifitas siswa. Dengan media POP UP diaharapkan siswa semakin berminat membaca buku baik fiksi mau
pun buku non fiksi. p
Data penelitian yang diperoleh berupa hasil uji coba item butir soal, data observasi
berupa pengamatan pengelolaan pembelajaran thinks pair share dan pengamatan aktivitas
siswa dan guru pada akhir pembelajaran, dan data tes formatif siswa pada setiap siklus. 1556 Vol.2 No.11 2021
ISSN: 2745-6056 | e-ISSN: 2745-7036
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ISSN: 2745-6056 | e-ISSN: 2745-7036
https://doi.org/10.47387/jira.v2i11.240 Data hasil uji coba item butir soal digunakan untuk mendapatkan tes yang betul-betul
mewakili apa yang diinginka. Data ini selanjutnya dianalisis tingkat validitas, reliabilitas,
taraf kesukaran, dan daya pembeda Data lembar observasi diambil dari dua pengamatan yaitu data pengamatan penglolaan
pembelajaran thinks pair share yang digunakan untuk mengetahui pengaruh penerapan
metode pembelajaran thinks pair share dalam meningkatkan prestasi Data tes formatif untuk mengetahui peningkatan prestasi belajar siswa setelah
diterapkan pembelajaran thinks pair share. Analisis Item Butir Soal Sebelum melaksanakan pengambilan data melalui instrumen penelitian berupa tes dan
mendapatkan tes yang baik, maka data tes tersebut diuji dan dianalisi.Uji coba dilakukan
pada siswa di luar sasaran penelitian. Analisis tes yang dilakukan meliputi: 1. Validitas
Validitas butir soal dimaksudkan untuk mengetahui kelayakan tes sehingga dapat
digunakan sebagai instrument dalam penelitian ini.Dari perhitungan 5 soal diperoleh 1
soal tidak valid dan 4 soal valid.Hasil dari validits soal-soal dirangkum dalam tabel di
bawah ini. 1. Validitas
Validitas butir soal dimaksudkan untuk mengetahui kelayakan tes sehingga dapat
digunakan sebagai instrument dalam penelitian ini.Dari perhitungan 5 soal diperoleh 1
soal tidak valid dan 4 soal valid.Hasil dari validits soal-soal dirangkum dalam tabel di
bawah ini. Tabe1. Ketuntasan siswa masing-masing siklus
Tes I siklus 1
Banyak siswa
Soal no 1
16 tuntas 15 remidi
Soal no 2
11 tuntas 20 remidi
Soal no 3
28 tuntas 3 remidi
Soal no 4
22 tuntas 9 remidi
Soal no 5
14 tuntas 17 remidi
Tes I siklus 2
Banyak siswa
Soal no 1
25 tuntas 7 remidi
Soal no 2
17 tuntas 14 remidi
Soal no 3
28 tuntas 3 remidi
Soal no 4
22 tuntas 9 remidi
Soal no 5
14 tuntas 17 remidi 2. Reliabilitas
Reliable nilai portopolio dengan menggunakan penilaian proses yang di pandu
dengan kisi-kisi soa dan rubrik penilan proses untuk portofolio. 3
f
k
(
) 2. Reliabilitas
Reliable nilai portopolio dengan menggunakan penilaian proses yang di pandu
dengan kisi-kisi soa dan rubrik penilan proses untuk portofolio. 3
Taraf Kesukaran (TK ) g
p
p
p
3. Taraf Kesukaran (TK )
Tarap kesukaran soal dengan menggunakan penilaian portopolio siswa dituntut
untuk membandingkan menemukan gagasa mengembangkan ide untuk berpendapat
tentang buku fiksi dan non fiksi dengan berfikir tingkat tinggi. Dengan menggunakan
rubrik penilaian siswa tidak di rugikan saat penilaian karena setiap ide dan gagasan
yang siswa kemukakan akan mendapat hasil sesuai dengan rubrik yang di beri bobot
sesuai dengan tingkat kesukaran butir soal. 1557 Vol.2 No.11 2021
ISSN: 2745-6056 | e-ISSN: 2745-7036
https://doi.org/10.47387/jira.v2i11.240 Vol.2 No.11 2021
ISSN: 2745-6056 | e-ISSN: 2745-7036
https://doi.org/10.47387/jira.v2i11.240 Vol.2 No.11 2021
ISSN: 2745-6056 | e-ISSN: 2745-7036
https://doi.org/10.47387/jira.v2i11.240 Analisis Data Penelitian Persiklus 1. Siklus I 1. Siklus I a. Tahap Perencanaan
Pada tahap ini peneliti mempersiapkan perangkat pembelajaran yang terdiri
dari rencana pelajaran 1. LKS 1, soal tes formatif 1, dan alat-alat pengajaran yang
mendukung. b. Tahap Kegiatan dan Pelaksanaan
Pelaksanaan kegiatan belajar mengajar untuk siklus I dilaksanakan pada
tanggal 4 Nopember 2019 di kelas VIIIG dengan jumlah siswa 31 siswa.Dalam hal
ini peneliti bertindak sebagai guru. Adapun proses belajar mengajar mengacu pada
rencana pelajaran yang telah dipersiapkan. Pengamatan (observasi) dilaksanakan
bersamaan dengan pelaksanaan belajar mengajar. Pada akhir proses belajar mengajar siswa diberi tes formatif I dengan tujuan
untuk mengetahui tingkat keberhasilan siswa dalam proses belajar mengajar yang
telah dilakukan. Adapun data hasil penelitian pada siklus I adalah sebagai berikut Pada akhir proses belajar mengajar siswa diberi tes formatif I dengan tujuan
untuk mengetahui tingkat keberhasilan siswa dalam proses belajar mengajar yang
telah dilakukan. Adapun data hasil penelitian pada siklus I adalah sebagai berikut B. Hasil tes dalam siklus 1 dan 2 Tabel 3. Rekap hasil Pengerjaan Tes II
Tes I
Banyak siswa
Siswa yang tuntas belajar
18
Siswa yang belum tuntas belajar
13 Tabel 3. Rekap hasil Pengerjaan Tes II
Tes I
Banyak siswa
Siswa yang tuntas belajar
18
Siswa yang belum tuntas belajar
13 Tabel 3. Rekap hasil Pengerjaan Tes II Tabel 3. Rekap hasil Pengerjaan Tes II Dari tabel di atas terlihat bahwa 18 atau 60,61% siswa telah mampu memenuhi kriteria
keberhasilan belajar yang telah ditetapkan sebelumnya. Sementara sisanya, 13 atau 39,39%
siswa, belum memenuhi kriteria keberhasilan belajar. Siswa masih kesulitan mengkreasika
data dalam bentuk pop up dengan cerita yang sesuai dengan struktur teks cerita Tabel 5. Rekap hasil Pengerjaan Tes II Tabel 5. Rekap hasil Pengerjaan Tes II
Tes II
Banyak siswa
Siswa yang tuntas belajar
27
Siswa yang belum tuntas belajar
6 Tabel 5. Rekap hasil Pengerjaan Tes II Dari tabel di atas terlihat bahwa 27 atau 90,28 % siswa telah mampu memenuhi kriteria
keberhasilan belajar yang telah ditetapkan sebelumnya. Sementara sisanya, 6 atau 18,18%
siswa, belum memenuhi kriteria keberhasilan belajar. Dengan demikian, hasil ini telah mampu memenuhi kriteria keberhasilan belajar yang
ditetapkan. Sehingga penelitian ini tidak dilanjutkan penelitian pada siklus selanjutnya 1) Pelaksanana dan hasil siklus 1 dan 2
A. Catatan lapangan Selama pelaksanaan pembelajaran, observer memberikan catatan lapangan sebagai
berikut. Tabel 2. Ringkasan Catatan Lapangan
Pertemuan ke-
Observer
Catatan Lapangan
1 dalam siklus 1
1
•
Pembelajaran cukup menyenangkan
•
Siswa
tampak
aktif
mengikuti
kegiatan
pembelajaran
•
Penggunaan waktu sudah cukup efektif
•
Siswa belum bekerjasama dengan baik dalam
kelompok karena belum faham cara
•
Karakter Bersahabat/Komunikatif yang diharapkan
muncul pada KBM belum tampak
•
Guru masih banyak membantu
2 dalam siklus 2
1
•
Pembelajaran cukup menyenangkan, siswa aktif
dan guru juga cukup kreatif dalam memberikan
bimbingan pada tiap tahapan model POP UP
•
Guru telah memberikan reward bagi yang aktif
dalam pembelajaran, namun kurang (belum
tampak) dalam memberikan penguatan secara
verbal hasil karya projek siswa
•
Siswa sudah lebih bisa bekerja kelompok tetapi
masih perlu bantuan guru dalam segi bentuk
•
Karakter Bersahabat/Komunikatif dalam KBM
sedikit lebih muncul dengan adanya bimbingan
dari guru untuk saling bekerjasama dan menjadi
tutor sebaya
•
Kresi siswa membuat media pup up namapak
•
Menuangkan ide dan menemukan struktur tek
lebih cepat
•
Siswa antusias belajar karena ada persaingan
kelompok dalam kreatifitas •
Pembelajaran cukup menyenangkan, siswa aktif
dan guru juga cukup kreatif dalam memberikan
bimbingan pada tiap tahapan model POP UP •
Guru telah memberikan reward bagi yang aktif
dalam pembelajaran, namun kurang (belum
tampak) dalam memberikan penguatan secara
verbal hasil karya projek siswa •
Siswa sudah lebih bisa bekerja kelompok tetapi
masih perlu bantuan guru dalam segi bentuk •
Karakter Bersahabat/Komunikatif dalam KBM
sedikit lebih muncul dengan adanya bimbingan
dari guru untuk saling bekerjasama dan menjadi
tutor sebaya •
Kresi siswa membuat media pup up namapak •
Menuangkan ide dan menemukan struktur tek
lebih cepat •
Siswa antusias belajar karena ada persaingan
kelompok dalam kreatifitas 1558 Vol.2 No.11 2021
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https://doi.org/10.47387/jira.v2i11.240 Keterangan Penilaian
4 = Sangat baik
3 = Baik
2 = Cukup
1 = Kurang Analisis Identifikasi Kebutuhan Tahap analisis awal kebutuhan peneliti melakukan observasi terhadap kegiatan
pembelajaran bahasa Indonesia kelas VIIIG SMPN 1 Pakisaji selain observasi peneliti
memahami konsep kebutuhan siswa yang sering ada masalah keterkaitan dengan kejenuhan
belajar dan variasi belajar. Seperti yang sering dilakukan siswa saat pembelajaran, mereka
akan malas mengerjakan karena cara guru menyampaikan materi selalu monoton, siswa hanya
mendengarkan dan mengerjakan LK sehingga hasil akhir siswa belum maksimal. Setelah
adanya media yang berbentuk Pop Up dua dan tiga dimensi yang dapat digerakkan dapat
menambah motivasi belajar siswa dalam memahami materi literasi buku fisi dan non fiksi
pada kompetensi dasar membaca. Tabel 6. Hasil Pengamatan Guru Siklus I dan 2
Unsur Pengamatan
Penilaian
Siklus 1
Penilaian
Siklus 2
Menyampaikan Tujuan Pembelajaran
2
3
Mengorganisasikan siswa dalam belajar
3
3
Membimbing siswa dalam belajar
2
4
Menghubugkan dengan materi sebelumnya
3
4
Memberikan penugasan
3
4
Jumlah
13
18
Rata-rata
2.6
3,6 Tabel 6. Hasil Pengamatan Guru Siklus I dan 2 1559 Analisis karakteristik siswa nalisis karakteristik siswa
Analisis karakteristik siswa dalam mengikuti kegiatan pembelajaran bahasa Indonesia alisis karakteristik siswa
Analisis karakteristik siswa dalam mengikuti kegiatan pembelajaran bahasa Indonesia Analisis karakteristik siswa dalam mengikuti kegiatan pembelajaran bahasa Indonesia Tabel 8. Hasil Pengamatan Guru Siklus I dan 2 Unsur Pengamatan
Penilaian
Siklus 1
Penilaian
Siklus 2
Tanggung jawab
2
3
Jujul
3
4
Berani
3
4
Jumlah
8
11
Rata-rata
2.6
3,6
Keterangan Penilaian
4 = Sangat baik
3 = Baik
2 = Cukup
1 = Kurang Gambar 1. Foto kegiatan Gambar 1. Foto kegiatan Gambar 1. Foto kegiatan Gambar 1. Foto kegiatan Analisis Kurikulum Analisis Kurikulum Analisis kurikulum dilakukan dengan cara mengkaji kurikulum 2013 karena SMPN 1
Pakisaji sudah menggunakan Kurikulum 2013 sebagai pedoman kegiatan belajar mengajar. Hal ini dilakukan agar media pembelajaran Pop Up dapat dikembangkan dan tidak
menyimpang dari tujuan pembelajaran yang terdapat pada standar kompetensi. Kompetensi
dasar yang digunakan dalam penelitian ini adalah 4.18 menyajikan tanggapan terhadap buku
fiksi dan nonfiksi yang dibaca secara lisan dan tertulis yang memiliki indicator pembelajaran
menanggapi isi buku fisi dan nonfiksi serta siswa dituntut untuk memiliki karakter jujur,
kreatif, komunikatif, dan kritis dalam membuat tanggapan isi teks baik judul, isi buku,
kelemahan dan kelebihan teks tersebut. Yang nantinya akan diimplementasikan dalam bentuk
RPP yang merujuk pada tujuan pembelajaran yang kompetitif sesuai dengan media
pembelajaran dalam membantu proses belajar. Tabel 7. Hasil Pengamatan Guru Siklus I dan 2
Unsur Pengamatan
Penilaian
Siklus 1
Penilaian
Siklus 2
Aktif
2
4
kreatif
2
4
Kritis
3
3
kolaboratif
3
4
Komunikatif
2
4
Jumlah
12
19
Rata-rata
2.4
3,8
Keterangan Penilaian
4 = Sangat baik
3 = Baik
2 = Cukup
1 = Kurang Tabel 7. Hasil Pengamatan Guru Siklus I dan 2
Unsur Pengamatan
Penilaian
Siklus 1
Penilaian
Siklus 2
Aktif
2
4
kreatif
2
4
Kritis
3
3
kolaboratif
3
4
Komunikatif
2
4
Jumlah
12
19
Rata-rata
2.4
3,8
Keterangan Penilaian
4 = Sangat baik
3 = Baik
2 = Cukup
1 = Kurang Tabel 7. Hasil Pengamatan Guru Siklus I dan 2 1560 Vol.2 No.11 2021
ISSN: 2745-6056 | e-ISSN: 2745-7036
https://doi.org/10.47387/jira.v2i11.240 Vol.2 No.11 2021
ISSN: 2745-6056 | e-ISSN: 2745-7036
https://doi.org/10.47387/jira.v2i11.240 Vol.2 No.11 2021
ISSN: 2745-6056 | e-ISSN: 2745-7036
https://doi.org/10.47387/jira.v2i11.240 DAFTAR RUJUKAN Aminudin. 1984. Pengantar Memahami Unsur-unsur Dalam Karya Sastra. Malang: FPBS
IKIP Malang. SIMPULAN 1. Kemampuan membaca siswa rendah karena kurang tertariknya penyajian perencanaan
pembelajaran di kelas sehingga siswa bosan menerima pelajara berkaitan dengan
pemahaman bacaan fiksi non fiksi apalagi harus memberikan pendapat atau komentar
buku yang di baca 2. Meningkatkan membaca dengan literasi bu fiksi dan non fiksi dengan menggunakan
media POP UP hasil karya siswa 3. Dengan menggunakan media POP UP yang kreatif siswa dapat mengebangkan krestifita
dan mampu mgng eksplor karya dalam bentuk pop up 4. Media POP UP dapat meningkatkan berliterasi dalam buku fiksi dan non fiksi karena
dengan media ini siswa dapat mengeksplor semua apa yang dia inginkan dalam bentuk
ketrampilan membentuk tiga dimensi. 5. Kesederhanaan PTK yang penulis buat semata karena berorientasi pada pengalaman
penulis dengan menggunakan berbagai media yang menarik agar siswa dapat
menggunakan materi pembelajaran dengan menggunakan media yang tepat sesuai
tujuan pembelajaran yang guru sampaikan. 1561 IKIP Malang. Buku Workshop di Batu Malang. 2005. Pedoman Pelaksanaan penelitian Tindakan kelas. Dinas Pendidikan dan Kebudayaan Propinsi Jawa Timur. Depdikbud, 1993. Kurikulum Pendidikan Dasar, Landasan, Program dan pengembangan. Jakarta: Depdikbud. Depdikbud, 1999. Bahan Pelatihan Penelitian Tindakan (Action Research). Jakarta: Dirjen
Dikdasmen dan Dikmenum. Depdiknas, 2003.Garis-Garis Besar Program Pengajaran Mata Pelajaran Bahasa Indonesia
untuk SMP/MTs. Jakarta: Depdikbud. Depdiknas, 2006. Lampiran peraturan menteri pendidikan Nasional No. 22 tahun 2006
tanggal 23 mei 2006 (Perment 22-23,2006) Effendi, Aep.2003. Bina Bahasa dan Sastra Idonesia, Jakarta: Erlangga. , Aep.2003. Bina Bahasa dan Sastra Idonesia, Jakarta: Erlangga. Ibrahin, Muslimin, dkk. 2000. Pembelajaran Kooperatif. Surabaya: University Press Moody, H.L.B. 1971. The Teaching of Literature. London: Longman Group LTD. Moody, H.L.B. 1971. The Teaching of Literature. London: Longman Group LTD. Nurhadi 2004 Pembelajaran Contextual dan penerapannya dalam KBK Universitas Negeri y,
g
g
p
Nurhadi. 2004. Pembelajaran Contextual dan penerapannya dalam KBK.Universitas Negeri
Malang. h
d
b l
f
b Nur, Mohamad. 2005. Pembelajaran Kooperatif. Surabaya: Unesa. Mohamad. 2005. Pembelajaran Kooperatif. Surabaya: Unesa. Oka, I Gusti Nyoman, 1983. Pengantar Membaca dan pengajarannya. Surabaya: Usaha
nasional Priyanti Endah Tri. 2002. Konsep dan Penerapan Penelitian Tindakan Kelas. Malang
Fakultas Sastra Rusyana, Yus. 1982. Metode Pengajaran Sastra. Bandung: Gunung Larang. Musfiqon. 2012. Pengembangan media dan sumber pembelajaran. Jakarta: PT. Prestasi
Pustaka Oemar, Eko Agus Basuki . 2016 Perancangan Buku POP UP sebagai media Pembelajaran. Jurnal pendidikan Seni Rupa Vol.4 No.03 Surabaya 1562 1562
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English
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The Genomic Landscape of Male Breast Cancers
|
Clinical cancer research
| 2,016
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cc-by
| 1,466
|
plification in ER-positive/HER2-negative male breast cancers and ER-positive/HER2-ne
emale breast cancers. comparisons of amplifications in ER-positive/HER2-negative male breast cancers (MaBCs) com
st cancers (FBCs) (A) and post-menopausal ER-positive/HER2-negative FBCs (B). The freque
plotted on the y-axis, according to their genomic position on the x-axis. Inverse Log10 values
ding to genomic location (x-axis). Copy number data of FBCs were retrieved from The Cancer Ge sitive/HER2-negative male breast cancers and ER-positive/HER2-ne
. cations in ER-positive/HER2-negative male breast cancers (MaBCs) com
and post-menopausal ER-positive/HER2-negative FBCs (B). The frequ
according to their genomic position on the x-axis. Inverse Log10 values
n (x-axis). Copy number data of FBCs were retrieved from The Cancer G ance fication in ER-pos
ale breast cancers
mparisons of amplif
cancers (FBCs) (A)
otted on the y-axis,
g to genomic locatio R-po
ncers
amplif
s) (A)
-axis,
ocatio lification in ER-po
male breast cancers
omparisons of amplif
cancers (FBCs) (A
otted on the y-axis
ng to genomic locatio fication in
ale breast c
mparisons of
cancers (FBC
otted on the
g to genomic
ations in ER-positive/HER2-negative male breast cancers and non-lobular ER
pausal female breast cancers. positive/HER2-negative male breast cancers (MaBCs) compared to non-lobular ER
d post-menopausal non-lobular ER-positive/HER2-negative FBCs (B). The frequency
plotted on the y-axis, according to their genomic position on the x-axis. Inverse Log1
g to genomic location (x-axis). Copy number data of FBCs were retrieved from The
HER2-negative male breast cancers and non-lobular E
cancers. male breast cancers (MaBCs) compared to non-lobular E
lobular ER positive/HER2 negative FBCs (B) The frequen lobular ER positive/HER2 negative FBCs (B). The frequen
ccording to their genomic position on the x-axis. Inverse Lo
(x-axis). Copy number data of FBCs were retrieved from T ( )
axis. Inv position ations in ER-positive
pausal female breast
positive/HER2-negative
post-menopausal non
plotted on the y-axis, a
g to genomic location breast
egative
sal non
-axis, a
ocation tions in ER-positive
ausal female breast
ositive/HER2-negativ
post-menopausal non
lotted on the y-axis, a
g to genomic location ons in ER-po
usal female b
itive/HER2-n
ost-menopaus
ted on the y-
o genomic lo
of copy number alterations in male breast cancers and female breast cancers/ post-menopaus
nd B subtypes. ct test comparisons of chromosomal gains and losses in 17 luminal A-like male breast cancers (MaBCs
BCs) (A) and post-menopausal FBCs (C) of luminal A subtype, and in 42 luminal B-like MaBCs, compare
BCs (D) of luminal B subtype. The frequency of gains (green bars) or losses (purple bars) for each gene
genomic position on the x-axis. Inverse Log10 values of the Fisher’s exact test p-values are plotted accordin
ber data of FBCs were retrieved from The Cancer Genome Atlas (13).
d female breast cancers/ post-menopau
luminal A-like male breast cancers (MaBC
pe, and in 42 luminal B-like MaBCs, compa
bars) or losses (purple bars) for each gene
sher’s exact test p-values are plotted accord
as (13).
rations in male breast cancers and female breast cancers/ post-menopau
chromosomal gains and losses in 17 luminal A-like male breast cancers (MaBC
nopausal FBCs (C) of luminal A subtype, and in 42 luminal B-like MaBCs, compa
ubtype. The frequency of gains (green bars) or losses (purple bars) for each gen
e x-axis. Inverse Log10 values of the Fisher’s exact test p-values are plotted accord
retrieved from The Cancer Genome Atlas (13).
ations in male breast cancers and female breast cancers/ post-menopau
chromosomal gains and losses in 17 luminal A-like male breast cancers (MaB
opausal FBCs (C) of luminal A subtype, and in 42 luminal B-like MaBCs, compa
btype. The frequency of gains (green bars) or losses (purple bars) for each gen
x-axis. Inverse Log10 values of the Fisher’s exact test p-values are plotted accord
etrieved from The Cancer Genome Atlas (13).
erations in male breast cancers and
chromosomal gains and losses in 17
nopausal FBCs (C) of luminal A subtyp
ubtype. The frequency of gains (green
e x-axis. Inverse Log10 values of the Fis
retrieved from The Cancer Genome Atla er alt
ons o
ost-m
al B
on t
were umber alt
pes. parisons o
nd post-m
uminal B
sition on t
FBCs were f copy number alt
d B subtypes. test comparisons o
Cs) (A) and post-m
Cs (D) of luminal B
enomic position on t
r data of FBCs were of copy numbe
d B subtypes. ct test compariso
BCs) (A) and po
Cs (D) of lumina
enomic position
er data of FBCs of copy numb
d B subtypes
t test compari
BCs) (A) and p
Cs (D) of lumi
enomic positio
er data of FBC of copy number alterations in male breast cancers and non-lobular female breast cancers/ po
f luminal A and B subtypes. ct test comparisons of chromosomal gains and losses in 17 luminal A-like male breast cancers (MaBC
st cancer (FBCs) (A) and post-menopausal non-lobular FBCs (C) of luminal A subtype, and in 42 lumina
FBCs (B) and post-menopausal non-lobular FBCs (D) of luminal B subtype. The frequency of gains (gr
gene is plotted on the y-axis, according to their genomic position on the x-axis. Inverse Log10 values of
according to genomic location (x-axis). Copy number data of FBCs were retrieved from The Cancer Geno and non-lobular female breast cancers/
17 luminal A-like male breast cancers (Ma
Cs (C) of luminal A subtype, and in 42 lumi
uminal B subtype. The frequency of gains (
osition on the x-axis. Inverse Log10 values
of FBCs were retrieved from The Cancer Ge erations in male breast cancers a
btypes. f chromosomal gains and losses in 1
and post-menopausal non-lobular FBC
enopausal non-lobular FBCs (D) of lu
y-axis, according to their genomic po
ocation (x-axis). Copy number data o mber alte
and B sub
parisons o
BCs) (A) a
nd post-m
ed on the
genomic er alt
d B su
sons o
s) (A) a
post-m
on the
nomic of copy numbe
luminal A and
t test compariso
t cancer (FBCs)
FBCs (B) and p
ene is plotted on
ccording to geno of copy numb
luminal A and
t test comparis
t cancer (FBCs
FBCs (B) and p
ene is plotted o
ccording to gen s of amplifications in male breast cancers and female breast cancers/ post-menopausal female breast
s. xact test comparisons of amplifications in 17 luminal A-like male breast cancers (MaBCs), compared with
post-menopausal FBCs (C) of luminal A subtype, and in 42 luminal B-like MaBCs, compared with FBCs (B)
inal B subtype. The frequency of amplifications (green bars) for each gene is plotted on the y-axis, according
. Inverse Log10 values of the Fisher’s exact test p-values are plotted according to genomic location (x-axis). ved from The Cancer Genome Atlas (13). erns of amplifications in male breast cancers and female breast cancers/ post-menopausal female breast
ypes. s exact test comparisons of amplifications in 17 luminal A-like male breast cancers (MaBCs), compared with
and post-menopausal FBCs (C) of luminal A subtype, and in 42 luminal B-like MaBCs, compared with FBCs (B)
luminal B subtype. The frequency of amplifications (green bars) for each gene is plotted on the y-axis, according
-axis. Inverse Log10 values of the Fisher’s exact test p-values are plotted according to genomic location (x-axis)
etrieved from The Cancer Genome Atlas (13). st cancers/ post-menopausal female breas
male breast cancers (MaBCs), compared wit
minal B-like MaBCs, compared with FBCs (B
r each gene is plotted on the y-axis, accordin
plotted according to genomic location (x-axis plifications in male breast cancers and female breast cancers/ post-menopa
comparisons of amplifications in 17 luminal A-like male breast cancers (MaB
enopausal FBCs (C) of luminal A subtype, and in 42 luminal B-like MaBCs, comp
ubtype. The frequency of amplifications (green bars) for each gene is plotted on t
e Log10 values of the Fisher’s exact test p-values are plotted according to genom
m The Cancer Genome Atlas (13). in male breast cancers and female breas
ns of amplifications in 17 luminal A-like ma
BCs (C) of luminal A subtype, and in 42 lum
frequency of amplifications (green bars) for
es of the Fisher’s exact test p-values are p
er Genome Atlas (13). n male breast cancers and female breas
s of amplifications in 17 luminal A-like ma
BCs (C) of luminal A subtype, and in 42 lum
requency of amplifications (green bars) for
es of the Fisher’s exact test p-values are p
r Genome Atlas (13). of amplifications in
ct test comparisons
post-menopausal FB
nal B subtype. The f
Inverse Log10 value
ed from The Cancer ns in
sons
al FB
The f
value
ncer ns in
sons
al FB
The f
value
ncer ns i
son
al FB
The f
value
nce of amplificatio
s. act test compar
post-menopaus
nal B subtype. T
Inverse Log10 v
ved from The Ca
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W1998640218.txt
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https://www.ajol.info/index.php/gjas/article/download/2106/10926
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en
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Relative time of planting and spatial arrangement for soybean/maize intercropping
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Ghana journal of agricultural science
| 2,006
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cc-by
| 0
| ||
https://openalex.org/W3033230026
|
https://iris.uniroma1.it/bitstream/11573/1466328/1/Aaij_Observation_2020.pdf
|
English
| null |
Observation of New <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:msubsup><mml:mi mathvariant="normal">Ξ</mml:mi><mml:mi>c</mml:mi><mml:mn>0</mml:mn></mml:msubsup></mml:math> Baryons Decaying to <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:msubsup><mml:mi mathvariant="normal">Λ</mml:mi><mml:mi>c</mml:mi><mml:mo>+</mml:mo></mml:msubsup><mml:msup><mml:mi>K</mml:mi><mml:mo>−</mml:mo></mml:msup></mml:math>
|
Physical review letters
| 2,020
|
cc-by
| 10,685
|
DOI: 10.1103/PhysRevLett.124.222001 DOI: 10.1103/PhysRevLett.124.222001 natural units with ℏ¼ c ¼ 1 are used throughout. Later
that year, another analysis was published [21], looking at
strongly interacting prompt decays of charm-strange bary-
ons to several final states, one of which was Λþc K−. No
resonances were reported in the Λþc K−mass spectrum. The
Belle Collaboration also reported the study of B−→
K−Λþc ¯Λ−c decays [22]. A peaking structure was observed
in the Λþc K−mass spectrum compatible with the results of
Ref. [20] and interpreted as a new Ξ0c baryon, dubbed
Ξcð2930Þ0. Similarly, evidence of the isospin partner
Ξcð2930Þþ
in
¯B0 →¯K0Λþc ¯Λ−c
decays
has
been
claimed [23]. Singly charmed baryons are composed of a charm quark
and two light quarks. Because of the large mass difference
between the charm and the lighter quarks, these baryons
provide an insight into the spectrum of states using
symmetries described by the heavy quark effective theory
[1,2]. Numerous theoretical predictions of the properties of
heavy baryons, containing either a charm or a beauty quark,
have been made in recent years [3–13]. In many of these
models, the heavy quark interacts with a lighter diquark,
which is treated as a single object. Other predictions are
based on lattice QCD calculations [14]. In 2017, the LHCb Collaboration reported the observa-
tion of five new narrow Ω0c baryons decaying to the Ξþc K−
final state [15], four of which were later confirmed by the
Belle Collaboration [16]. It is currently not understood why
the natural widths of these resonances are small [17,18],
although a similar trend has recently been observed in the
excited Ω−
b states decaying to Ξ0
bK−[19]. Investigating a
different charmed mass spectrum could lead to a better
understanding of this feature. This Letter presents a search for excited Ξ0c baryons,
hereafter referred to as Ξ0
c , in the Λþc K−spectrum in a
mass region around the Ξcð2930Þ0 state, with the Λþc
baryons reconstructed in the pK−πþ final state. Defining
ΔM ≡mðΛþc K−Þ −mðΛþc Þ −mðK−Þ, the region consid-
ered is ΔM < 300 MeV. The data are collected in pp
collisions with the LHCb detector at a center-of-mass
energy of 13 TeV, corresponding to an integrated lumi-
nosity of 5.6 fb−1. A natural extension to the Ξþc K−analysis is the study of
the Λþc K−spectrum. Published by the American Physical Society under the terms of
the Creative Commons Attribution 4.0 International license.
Further distribution of this work must maintain attribution to
the author(s) and the published article’s title, journal citation,
and DOI. Funded by SCOAP3. *Full author list given at the end of the article. PHYSICAL REVIEW LETTERS 124, 222001 (2020)
Editors' Suggestion
Featured in Physics PHYSICAL REVIEW LETTERS 124, 222001 (2020) Observation of New Ξ0c Baryons Decaying to Λ +c K −
R. Aaij et al.*
(LHCb Collaboration) The Λþc K−mass spectrum is studied with a data sample of pp collisions at a center-of-mass energy of
13 TeV corresponding to an integrated luminosity of 5.6 fb−1 collected by the LHCb experiment. Three Ξ0c states are observed with a large significance and their masses and natural widths are measured
to
be
m½Ξcð2923Þ0 ¼ 2923.04 0.25 0.20 0.14 MeV,
Γ½Ξcð2923Þ0 ¼ 7.1 0.8 1.8 MeV,
m½Ξcð2939Þ0 ¼ 2938.55 0.21 0.17 0.14 MeV,
Γ½Ξcð2939Þ0 ¼ 10.2 0.8 1.1 MeV,
m½Ξcð2965Þ0 ¼ 2964.88 0.26 0.14 0.14 MeV,
Γ½Ξcð2965Þ0 ¼ 14.1 0.9 1.3 MeV,
where
the uncertainties are statistical, systematic, and due to the limited knowledge of the Λþc mass. The
Ξcð2923Þ0 and Ξcð2939Þ0 baryons are new states. The Ξcð2965Þ0 state is in the vicinity of the known
Ξcð2970Þ0 baryon; however, their masses and natural widths differ significantly. DOI: 10.1103/PhysRevLett.124.222001 The BABAR Collaboration was the
first to observe a structure in the Λþc K−mass spectrum in
B−→K−Λþc ¯Λ−c decays peaking at 2.93 GeV in 2007 [20]. However, it was not interpreted as a new state due to the
absence of an amplitude analysis. Unless otherwise stated,
charge-conjugate processes are implicitly included, and The LHCb detector [24,25] is a single-arm forward
spectrometer covering the pseudorapidity range 2 < η < 5,
designed for the study of particles containing b or c quarks. The detector elements that are particularly relevant to this
analysis are a silicon-strip vertex detector surrounding the
pp interaction region that allows c and b hadrons to be
identified from their characteristically long flight distance;
a tracking system that provides a measurement of the
momentum of charged particles; and two ring-imaging
Cherenkov detectors that are able to discriminate between
different species of charged hadrons. The online event
selection is performed by a trigger, which consists of a
hardware stage, based on information from the calorimeter 222001-1 © 2020 CERN, for the LHCb Collaboration 0031-9007=20=124(22)=222001(11) PHYSICAL REVIEW LETTERS 124, 222001 (2020) 2250
2300
2350
) [MeV]
+
−
K
p
(
m
0
200
400
600
800
1000
3
10
×
Candidates / (0.5 MeV)
LHCb
FIG. 1. Distribution of the reconstructed invariant mass
mðpK−πþÞ for 20% of the candidates in the Λþc sample passing
the selection described in the text. The solid blue curve shows the
result of the fit, and the dashed blue line indicates the background
component of the fit. 2250
2300
2350
) [MeV]
+
−
K
p
(
m
0
200
400
600
800
1000
3
10
×
Candidates / (0.5 MeV)
LHCb and muon systems, followed by a two-level software stage,
which applies a full event reconstruction [26,27]. Simulated
data samples are produced with the software packages
described in Refs. [28–32] and are used to optimize the
selection requirements, to quantify the invariant-mass
resolution, and to model physics processes which may
constitute peaking backgrounds in the analysis. p
g
g
y
Candidate Λþc baryons are formed from the combination
of three tracks of good quality which are inconsistent with
originating from any primary proton-proton interaction
vertex (PV) and have large transverse momentum (pT). Particle identification (PID) requirements are imposed on
all three tracks to suppress combinatorial background and
misidentified charm-meson decays. DOI: 10.1103/PhysRevLett.124.222001 The Λþc candidates are
required to have pT > 2 GeV and are constrained to
originate from the associated PV by requiring a small
χ2
IP, defined as the difference between the vertex fit χ2 of the
PV reconstructed with and without the candidate in ques-
tion. The Λþc vertex must also be displaced from the
associated PV such that the Λþc decay time is longer than
0.3 ps. A multivariate classifier based on a boosted decision
tree (BDT) algorithm [33,34] implemented in the TMVA
toolkit [35] is used to further improve the Λþc signal purity. The input variables given to the BDT are the χ2 value of the
Λþc decay-vertex fit, the Λþc flight distance between the
production and decay vertex, the angle between the Λþc
momentum vector and the line that joins the Λþc decay
vertex with its associated PV, the χ2
IP and pT of the Λþc
candidate, and the χ2
IP and PID responses of the Λþc decay
particles. The background sample used in the BDT training
consists of the lower and upper sidebands of the pK−πþ
invariant
mass
distribution,
2230–2250
and
2320–
2340 MeV, respectively. The signal sample used is the
Λþc sample in the data after subtracting the background by
means of the sPlot technique [36], exploiting mðpK−πþÞ as
a discriminating variable. The training of the multivariate
algorithm is carried out by using 20 000 candidates of the
reconstructed Λþc candidates from the data recorded in
2016. The requirement on the BDT response is determined
using 200 000 Λþc candidates by maximizing the figure of
merit S=
ffiffiffiffiffiffiffiffiffiffiffiffi
S þ B
p
, where S is the Λþc signal yield extracted
from a fit to the mass spectrum of Λþc candidates passing a
given BDT requirement and B is the expected background
yield. The value for B is extrapolated by scaling the
background yield over the full mass range of the fit to a
15 MeV mass range around the Λþc peak. FIG. 1. Distribution of the reconstructed invariant mass
mðpK−πþÞ for 20% of the candidates in the Λþc sample passing
the selection described in the text. The solid blue curve shows the
result of the fit, and the dashed blue line indicates the background
component of the fit. would result in a large loss of signal efficiency and,
therefore, is not implemented. DOI: 10.1103/PhysRevLett.124.222001 The ΔM distribution also shows a broad structure to the left
of the three narrow structures consistent with being
partially reconstructed Ξcð3055Þ →Σcð2455Þð→Λþc πÞK−
and Ξcð3080Þ →Σcð2455Þð→Λþc πÞK−decays, where the
pion is not reconstructed. An
unbinned
maximum-likelihood
fit,
henceforth
denoted the reference fit, is performed to the ΔM distri-
bution to measure the parameters of each peak. The
background is modeled by an empirical function of the
form ΔMa × expð−b × ΔMÞ, where a and b vary freely. Each signal peak is described by an S-wave relativistic
Breit-Wigner function convolved with a mass-resolution
function. The experimental mass resolution is determined
using simulated Ξ0
c
→Λþc K−decays at several Ξ0
c
masses. In the ΔM interval where the three narrow peaks
occur, the mass resolution varies between 1.7 and 2.2 MeV. Simulated data are also generated to determine the shape of
partially reconstructed Ξcð3055Þ and Ξcð3080Þ decays. The
shapes of these contributions are allowed to shift in ΔM by
the uncertainties in the decay-product masses, where the
shift is Gaussian constrained. From isospin symmetry, the
yields of the Ξcð3055Þþ and Ξcð3080Þþ components are
constrained to be twice as large as the corresponding
Ξcð3055Þ0 and Ξcð3080Þ0 components. The fit model
outlined so far does not accurately describe the data in
the mass region close to the kinematic threshold, and, thus, The ΔM distribution with the fit to the data super-
imposed is shown in Fig. 2(a). The goodness-of-fit value is
χ2=ndof ¼ 301=ð300 −19Þ ¼ 1.07, where ndof is the
number of degrees of freedom. Table I shows the results
for the parameters of the signal peaks of the reference fit,
hereafter named Ξcð2923Þ0, Ξcð2939Þ0, and Ξcð2965Þ0. To validate the presence of the signal components and
test the stability of the fit parameters, several additional
checks are performed. The data are fitted in samples
according to the year of data taking and to different
data-taking conditions depending on the LHCb magnet
configuration. The Λþc K−sample and its charge conjugate
are also studied separately. The results are consistent
among all samples. g
p
The data and the reference fit show the least compati-
bility in the region around ΔM ≃100 MeV. This may be
due to a mismodeling of the partially reconstructed dis-
tributions, but it could also be due to the presence of further
new Ξ0
c
baryon states. DOI: 10.1103/PhysRevLett.124.222001 0
0
100
200
300
) [MeV]
−
K
(
m
) -
+
c
Λ
(
m
) -
−
K
+
c
Λ
(
m
0
500
1000
1500
2000
Candidates / (1 MeV)
LHCb
(b)
−
K
+
c
Λ
0
(2923)
c
Ξ
−
K
+
c
Λ
0
(2939)
c
Ξ
−
K
+
c
Λ
0
(2965)
c
Ξ
+
π
−
K
+
c
Λ
+
(2923)
c
Ξ
−
K
)
+
π
+
c
Λ
→
(
++
c
Σ
+
(3055)
c
Ξ
−
K
)
0
π
+
c
Λ
→
(
+
c
Σ
0
(3055)
c
Ξ
−
K
)
+
π
+
c
Λ
→
(
++
c
Σ
+
(3080)
c
Ξ
−
K
)
0
π
+
c
Λ
→
(
+
c
Σ
0
(3080)
c
Ξ
Background
Additional component 0
100
200
300
) [MeV]
−
K
(
m
) -
+
c
Λ
(
m
) -
−
K
+
c
Λ
(
m
0
500
1000
1500
2000
Candidates / (1 MeV)
LHCb
(a) FIG. 2. Distributions of the reconstructed invariant-mass difference ΔM ¼ mðΛþc K−Þ −mðΛþc Þ −mðK−Þ for all candidates passing
the selection requirements described in the text. The black symbols show the selected signal candidates. The result of a fit, described in
the text, is overlaid (solid blue line). In (a), the reference fit is shown. (b) shows an alternative description to the data, where an additional
Gaussian component given by the cyan dot-dashed line is added to the fit model around ΔM ≃100 MeV. The missing child particles in
the reconstruction are indicated in gray in the legend. an additional component is considered. There are no known
decays of Σcð2455Þð→Λþc πÞK−or Σcð2520Þð→Λþc πÞK−
which could enter the sample as partially reconstructed
components at ΔM ≃0. It is observed that the missing
component is consistent with being due to the partial
reconstruction of the state that peaks around ΔM ≃
140 MeV when it decays directly to the Λþc K−πþ final
state without any intermediate resonance. The shape of
these partially reconstructed decays is taken from simulated
samples generated using the RapidSim package [39], and
the yield is a free parameter in the fit. wrong-sign Λþc Kþ candidates or Λþc sideband distributions. DOI: 10.1103/PhysRevLett.124.222001 However, it is checked that
the results of the analysis are stable when these background
components are removed fully. About 125 million Λþc
signal decays are selected for further analysis with a purity
of 93%. The invariant-mass distribution of 20% of the Λþc
candidates satisfying these selection requirements is shown
in Fig. 1. The Ξ0
c
candidates are formed from Λþc K−combina-
tions, where the Λþc candidate mass is required to be within
20 MeVof the known Λþc mass [37]. Each Λþc candidate is
combined with a K−candidate that is consistent with
originating from the associated PV. The Λþc
and K−
particles are fitted to a common vertex, which is required
to be consistent with the associated PV. The main contribution to the combinatorial background
in the Λþc K−mass spectrum is due to the large number of
kaon candidates from the PV. The signal to background
ratio is improved by optimising the PID criteria of the K−
candidates and the pT requirement on the Ξ0
c
candidates
using the figure of merit ϵ=ð
ffiffiffiffiffiffi
BP
p
þ 5=2Þ [38]. Here, ϵ is the
efficiency determined using simulated Ξcð2930Þ0 →Λþc K−
decays, and BP is the number of Λþc K−candidates in the
mass region 260 < ΔM < 290 MeV, corresponding to the
background expected in a mass window around the
expected Ξcð2930Þ0 signal, with width Γ½Ξcð2930Þ0 ¼
26 8 MeV [37]. Based on the optimization above, the pT
of the Ξ0c candidates is required to be larger than 7350 MeV,
and the kaon PID is required to satisfy a tight criterion. The
fraction of events with multiple candidates is found to be
0.88% in the entire ΔM range. All candidates are included
in the analysis. Misidentified Dþ →K−πþπþ, Dþ →KþK−πþ, and
Dþs →KþK−πþ background decays are observed after
changing the mass hypothesis of the proton into a kaon
or a pion. These background components are reduced by
employing a tighter PID selection and requiring the
invariant mass mðKþK−Þ to differ by at least 10 MeV
from the known ϕð1020Þ mass [37]. Removing all candi-
dates in mass windows around the Dþ
ðsÞ mass distributions The resulting ΔM distribution of the signal candidates is
shown in Fig. 2, where a fit to the data is superimposed. Three narrow structures are observed in the Λþc K−candi-
date spectrum. DOI: 10.1103/PhysRevLett.124.222001 These peaking structures are not seen in the 222001-2 PHYSICAL REVIEW LETTERS 124, 222001 (2020) 0
100
200
300
) [MeV]
−
K
(
m
) -
+
c
Λ
(
m
) -
−
K
+
c
Λ
(
m
0
500
1000
1500
2000
Candidates / (1 MeV)
LHCb
(a)
0
100
200
300
) [MeV]
−
K
(
m
) -
+
c
Λ
(
m
) -
−
K
+
c
Λ
(
m
0
500
1000
1500
2000
Candidates / (1 MeV)
LHCb
(b)
−
K
+
c
Λ
0
(2923)
c
Ξ
−
K
+
c
Λ
0
(2939)
c
Ξ
−
K
+
c
Λ
0
(2965)
c
Ξ
+
π
−
K
+
c
Λ
+
(2923)
c
Ξ
−
K
)
+
π
+
c
Λ
→
(
++
c
Σ
+
(3055)
c
Ξ
−
K
)
0
π
+
c
Λ
→
(
+
c
Σ
0
(3055)
c
Ξ
−
K
)
+
π
+
c
Λ
→
(
++
c
Σ
+
(3080)
c
Ξ
−
K
)
0
π
+
c
Λ
→
(
+
c
Σ
0
(3080)
c
Ξ
Background
Additional component
FIG. 2. Distributions of the reconstructed invariant-mass difference ΔM ¼ mðΛþc K−Þ −mðΛþc Þ −mðK−Þ for all candidates passing
the selection requirements described in the text. The black symbols show the selected signal candidates. The result of a fit, described in
the text, is overlaid (solid blue line). In (a), the reference fit is shown. (b) shows an alternative description to the data, where an additional
Gaussian component given by the cyan dot-dashed line is added to the fit model around ΔM ≃100 MeV. The missing child particles in
the reconstruction are indicated in gray in the legend. DOI: 10.1103/PhysRevLett.124.222001 More data are required to understand th
of this additional structure. It is accounted fo
calculating the systematic uncertainties. Several sources of systematic uncertainty may a
measured parameters. The fit model uncertainty is ev
by replacing the background model by an alternati
tion, consisting of a combination of the wro
mðΛþc KþÞ invariant-mass distribution shape and th
obtainedfromcandidatesintheΛþc sideband.Inaddi
choice of the relativistic Breit-Wigner model is cha
setting the values of the angular momentum L betw
child particles to L ¼ 1, 2 and separately varying th
Weisskopf factors [40] from 2 to 4 GeV−1. Furtherm
fit is adapted to include any partially reconstructed
Ξ
c →Σcð2455=2520Þð→Λþc πÞK−that are found
contribute significantly to the reference fit. Finally
tions in fit parameters between the reference fit an
shown in Fig. 2(b) are included in the fit model unc
The largest deviation from the reference fit is quote
systematicuncertaintyforthefit model.Resonances
same spin parity that are close in mass can interf
interference term is introduced between neighbori
nances, for one pair of resonances at a time. W
interference
term,
the
line
shape
takes
th
A ¼ jcjBWj þ ckBWkeiϕj2, where j and k denote
resonances,BWj;k areBreit-Wignerfunctions,andc
are free real parameters. The largest difference betw
referencefitandafitwhereresonanceinterferenceis
isusedasthesystematicuncertainty.Inaddition,seve
sources of systematic uncertainty affect only th
measurement. These include the momentum-scale
tainty, evaluated by shifting the momentum scale of
TABLE III. Summary of the parameters for t
the natural widths, where the first uncertainty
measurement, the third uncertainty denotes th
Resonance
Peak of ΔM [MeV]
Ξcð2923Þ0
142.91 0.25 0.20
Ξcð2939Þ0
158.45 0.21 0.17
Ξcð2965Þ0
184.75 0.26 0.14 g
y
Several sources of systematic uncertainty may affect the
measured parameters. The fit model uncertainty is evaluated
by replacing the background model by an alternative func-
tion, consisting of a combination of the wrong-sign
mðΛþc KþÞ invariant-mass distribution shape and the shape
obtainedfromcandidatesintheΛþc sideband.Inaddition,the
choice of the relativistic Breit-Wigner model is changed by
setting the values of the angular momentum L between the
child particles to L ¼ 1, 2 and separately varying the Blatt-
Weisskopf factors [40] from 2 to 4 GeV−1. Furthermore, the
fit is adapted to include any partially reconstructed decays
Ξ
c →Σcð2455=2520Þð→Λþc πÞK−that are found to not
contribute significantly to the reference fit. Finally, devia-
tions in fit parameters between the reference fit and the fit
shown in Fig. 2(b) are included in the fit model uncertainty. DOI: 10.1103/PhysRevLett.124.222001 The largest deviation from the reference fit is quoted as the
systematicuncertaintyforthefit model.Resonanceswiththe
same spin parity that are close in mass can interfere. An
interference term is introduced between neighboring reso-
nances, for one pair of resonances at a time. With the
interference
term,
the
line
shape
takes
the
form
A ¼ jcjBWj þ ckBWkeiϕj2, where j and k denote the two
resonances,BWj;k areBreit-Wignerfunctions,andcj;k andϕ
are free real parameters. The largest difference between the
referencefitandafitwhereresonanceinterferenceisallowed
isusedasthesystematicuncertainty.Inaddition,severalother
sources of systematic uncertainty affect only the mass
measurement. These include the momentum-scale uncer-
tainty, evaluated by shifting the momentum scale of charged The observations described in this Letter and the lack of
anyΞcð2930Þ0 signalindicatesthatthebroadbumpobserved
in B−→K−Λþc ¯Λ−c decays [20,22] might be due to the
overlap of two narrower states, such as the Ξcð2923Þ0 and
Ξcð2939Þ0 baryons. The Ξcð2965Þ0 baryon is in the vicinity
of the known Ξcð2970Þ0 baryon, which has been observed in
different decay modes: Σcð2455Þ0K0
S [21], Ξ0cþπ−[44], and
Ξcð2645Þþπ−[45]. Furthermore, the Ξcð2965Þ0 resonance
has a natural width and mass which differ significantly from
those
of
the
Ξcð2970Þ0
baryon:
Γ½Ξcð2970Þ0 ¼
28.1þ3.4
−4.0 MeV and m½Ξcð2970Þ0 ¼ 2967.8þ0.9
−0.7 MeV [37]. Further studies are required to establish whether the
Ξcð2965Þ0 state is indeed a different baryon. The equal
spacing rule [46,47] succeeded to predict the mass of the Ω
baryonandholdsforotherflavormultipletssuchasthesextet
of the JP ¼ 3=2þ charmed ground states: m½Ωcð2770Þ0 −m½Ξcð2645Þ0
≃m½Ξcð2645Þ0 −m½Σcð2520Þ0 ≃125 MeV: TABLE III. Summary of the parameters for the studied states, showing the measured ΔM values, the masses, and
the natural widths, where the first uncertainty is statistical and the second uncertainty is systematic. For the mass
measurement, the third uncertainty denotes the uncertainty on the known Λþc mass [37]. TABLE III. Summary of the parameters for the studied states, showing the measured ΔM values, the masses, and
the natural widths, where the first uncertainty is statistical and the second uncertainty is systematic. For the mass
measurement, the third uncertainty denotes the uncertainty on the known Λþc mass [37]. DOI: 10.1103/PhysRevLett.124.222001 Figure 2(b) shows the ΔM
distribution for the signal sample where an additional
component, parametrized by an empirical Gaussian func-
tion, has been added to the reference fit. The fit has a
goodness-of-fit value of χ2=ndof ¼ 278=ð300 −22Þ ¼
1.00. As a cross-check, this structure is tested in subsam-
ples of the dataset divided by data-taking year and showed
an inconsistency in the scaling of the yield with respect to
the integrated luminosity. Furthermore, the feed-down
components are highly suppressed when this contribution TABLE I. Peak positions in the invariant-mass difference
distribution ΔM, natural widths Γ, signal yields, and local
significances of the three mass peaks obtained from the fit to
the Λþc K−mass spectrum, where the systematic uncertainties are
statistical. Peak of ΔM [MeV]
Γ [MeV]
Signal yields
142.91 0.25
7.1 0.8
5400 400
158.45 0.21
10.2 0.8
10400 600
184.75 0.26
14.1 0.9
11700 600 222001-3 PHYSICAL REVIEW LETTERS 124, 222001 (2020) TABLE II. Summary of the contributions to the systematic uncertainties on the resonance parameters. Absolute
deviations from the nominal fit are quoted. Source
Ξcð2923Þ0
Ξcð2939Þ0
Ξcð2965Þ0
m½MeV
Γ½MeV
m½MeV
Γ½MeV
m½MeV
Γ½MeV
Alternative fit model
0.15
1.6
0.14
0.4
0.04
1.1
Resonance interferences
0.08
0.7
0.06
1.0
0.11
0.7
Momentum scale
0.04
0.05
0.06
Energy losses
0.04
0.04
0.04
Resolution calibration
0.6
0.2
0.3
Total
0.20
1.8
0.17
1.1
0.14
1.3 TABLE II. Summary of the contributions to the systematic uncertainties on the resonance parameters. Absolute
deviations from the nominal fit are quoted. tracksby0.03%[41]insimulateddecays,andtheimperfect
modelingoftheenergylossinthedetectormaterial,resulting
in a systematic uncertainty of 0.04 MeV [42]. Finally, a
systematic uncertainty is attributed to the width measure-
ment, to account for the fact that the simulation may not
reproduce the absolute mass resolution perfectly. The cor-
responding systematic uncertainty is obtained by the change
in the width when the value of the resolution, determined on
simulated data, is varied by 10% [43]. The systematic
uncertainties are summarized in Table II, and in Table III
their measured masses and natural widths are summarized. is included. More data are required to understand the cause
of this additional structure. It is accounted for when
calculating the systematic uncertainties. is included. PHYSICAL REVIEW LETTERS 124, 222001 (2020) PHYSICAL REVIEW LETTERS 124, 222001 (2020) [1] A. G. Grozin, Introduction to the heavy quark effective
theory. Part 1, arXiv:hep-ph/9908366. It is noted that the rule also seems to hold for the Ξcð2923Þ0,
Ξcð2939Þ0, and Ξcð2965Þ0 baryons within a precision of a
few MeV: [2] T. Mannel, Effective theory for heavy quarks, Lect. Notes
Phys. 479, 387 (1997). y
[3] D. Ebert, R. N. Faustov, and V. O. Galkin, Masses of excited
heavy baryons in the relativistic quark-diquark model, Phys. Lett. B 659, 612 (2008). m½Ωcð3050Þ0 −m½Ξcð2923Þ0
≃m½Ξcð2923Þ0 −m½Σcð2800Þ0 ≃125 MeV;
m½Ωcð3065Þ0 −m½Ξcð2939Þ0 ≃125 MeV;
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analysis are related to the excited Ω0c baryons observed in the
Ξþc K−spectrum. Measurements of spin parities will be
crucial to confirm whether they belong to the same flavor
multiplets. This pattern may indicate that the new states reported in this
analysis are related to the excited Ω0c baryons observed in the
Ξþc K−spectrum. Measurements of spin parities will be
crucial to confirm whether they belong to the same flavor
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spectrum. Three
different
Ξ0c
baryons,
Ξcð2923Þ0,
Ξcð2939Þ0, and Ξcð2965Þ0, are unambiguously observed. The two baryons at lower mass are observed for the first
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CERN and from the national agencies: CAPES, CNPq,
FAPERJ, and FINEP (Brazil); MOST and NSFC (China);
CNRS/IN2P3
(France);
BMBF,
DFG,
and
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(Germany); INFN (Italy); NWO (Netherlands); MNiSW
and
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(Poland);
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(Romania);
MSHE
(Russia);
MinECo
(Spain);
SNSF
and
SER
(Switzerland);
NASU
(Ukraine);
STFC
(United
Kingdom); DOE NP and NSF (USA). We acknowledge
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(Netherlands),
PIC
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Peak of ΔM [MeV]
Mass [MeV]
Γ [MeV]
Ξcð2923Þ0
142.91 0.25 0.20
2923.04 0.25 0.20 0.14
7.1 0.8 1.8
Ξcð2939Þ0
158.45 0.21 0.17
2938.55 0.21 0.17 0.14
10.2 0.8 1.1
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14.1 0.9 1.3 Resonance
Peak of ΔM [MeV]
Mass [MeV]
Γ [MeV]
Ξcð2923Þ0
142.91 0.25 0.20
2923.04 0.25 0.20 0.14
7.1 0.8 1.8
Ξcð2939Þ0
158.45 0.21 0.17
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U. Uwer,16 A. Vagner,80 V. Vagnoni,19 A. Valassi,47 G. Valenti,19 M. van Beuzekom,31 H. Van Hecke,66 E. van Herwijnen,47
C. B. Van Hulse,17 M. van Veghel,75 R. Vazquez Gomez,44 P. Vazquez Regueiro,45 C. Vázquez Sierra,31 S. Vecchi,20
J. J. Velthuis,53 M. Veltri,21,q A. Venkateswaran,67 M. Veronesi,31 M. Vesterinen,55 J. V. Viana Barbosa,47 D. Vieira,64
M. Vieites Diaz,48 H. Viemann,74 X. Vilasis-Cardona,44,l G. Vitali,28 A. Vitkovskiy,31 A. Vollhardt,49 D. Vom Bruch,12
A. Vorobyev,37 V. Vorobyev,42,w N. Voropaev,37 R. Waldi,74 J. Walsh,28 J. Wang,3 J. Wang,72 J. Wang,6 M. Wang,3 Y. Wang,7
Z. Wang,49 D. R. Ward,54 H. M. Wark,59 N. K. Watson,52 D. Websdale,60 A. Weiden,49 C. Weisser,63 B. D. C. Westhenry,53
D. J. White,61 M. Whitehead,53 D. Wiedner,14 G. Wilkinson,62 M. Wilkinson,67 I. Williams,54 M. Williams,63
M R J Willi
61 T Willi
52 F F Wil
56 W Wi li ki 35 M Wi k 33 L Wi l
16 G W
11 S A W
54 222001-7 PHYSICAL REVIEW LETTERS 124, 222001 (2020) (LHCb Collaboration) Wills Physics Laboratory, University of Bristol, Bristol, United Kingdom
222001-9 alisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany
17 17School of Physics, University College Dublin, Dublin, Ireland
18 18INFN Sezione di Bari, Bari, Italy 19INFN Sezione di Bologna, Bologna, Italy
20 20INFN Sezione di Ferrara, Ferrara, Italy
21 21INFN Sezione di Firenze, Firenze, Italy 22INFN Laboratori Nazionali di Frascati, Frascati, Italy
23 23INFN Sezione di Genova, Genova, Italy 24INFN Sezione di Milano-Bicocca, Milano, Italy
25 25INFN Sezione di Milano, Milano, Italy 26INFN Sezione di Cagliari, Monserrato, Italy
27 27INFN Sezione di Padova, Padova, Italy 28INFN Sezione di Pisa, Pisa, Italy 29INFN Sezione di Roma Tor Vergata, Roma, Italy
30 30INFN Sezione di Roma La Sapienza, Roma, Italy 31Nikhef National Institute for Subatomic Physics, Amsterdam, Netherlands of Science and Technology, Faculty of Physics and Applied Computer Science, Kraków, Poland
35 40Institute for Nuclear Research of the Russian Academy of Sciences (INR RAS), Moscow, Russi
41 41Yandex School of Data Analysis, Moscow, Russia 42Budker Institute of Nuclear Physics (SB RAS), Novosibirsk, Russia High Energy Physics NRC Kurchatov Institute (IHEP NRC KI), Protvino, Russia, Protvino, Russia
44 Instituto Galego de Física de Altas Enerxías (IGFAE), Universidade de Santiago de Compostela, Santiago de Compostela, Spain
46Instituto de Fisica Corpuscular Centro Mixto Universidad de Valencia
CSIC Valencia Spain 47European Organization for Nuclear Research (CERN), Geneva, Switzerland 49Physik-Institut, Universität Zürich, Zürich, Switzerland 52University of Birmingham, Birmingham, United Kingdom 53H. H. Wills Physics Laboratory, University of Bristol, Bristol, United Kingdom 222001-9 M. Zavertyaev,15,c M. Zdybal,33 M. Zeng,3 D. Zhang,7 L. Zhang,3 S. Zhang,4 W. C. Zhang,3,y Y. Zhang,47 A. Zhelezov,16
Y. Zheng,5 X. Zhou,5 Y. Zhou,5 X. Zhu,3 V. Zhukov,13,39 J. B. Zonneveld,57 and S. Zucchelli19,e M. Zavertyaev,15,c M. Zdybal,33 M. Zeng,3 D. Zhang,7 L. Zhang,3 S. Zhang,4 W. C. Zhang,3,y Y. Zhang,47 A. Zhelezov,16
Y. Zheng,5 X. Zhou,5 Y. Zhou,5 X. Zhu,3 V. Zhukov,13,39 J. B. Zonneveld,57 and S. Zucchelli19,e PHYSICAL REVIEW LETTERS 124, 222001 (2020) 222001-8 PHYSICAL REVIEW LETTERS 124, 222001 (2020) (LHCb Collaboration) 1Centro Brasileiro de Pesquisas Físicas (CBPF), Rio de Janeiro, Brazil
2Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
3Center for High Energy Physics, Tsinghua University, Beijing, China
4School of Physics State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing, China
5University of Chinese Academy of Sciences, Beijing, China
6Institute of High Energy Physics (IHEP), Beijing, China
7Institute of Particle Physics, Central China Normal University, Wuhan, Hubei, China
8Universit´e Grenoble Alpes, Universit´e Savoie Mont Blanc, CNRS, IN2P3-LAPP, Annecy, France
9Universit´e Clermont Auvergne, CNRS/IN2P3, LPC, Clermont-Ferrand, France
10Aix Marseille Universit´e, CNRS/IN2P3, CPPM, Marseille, France
11Universit´e Paris-Saclay, CNRS/IN2P3, IJCLab, Orsay, France
12LPNHE, Sorbonne Universit´e, Paris Diderot Sorbonne Paris Cit´e, CNRS/IN2P3, Paris, France
13I. (LHCb Collaboration) Physikalisches Institut, RWTH Aachen University, Aachen, Germany
14Fakultät Physik, Technische Universität Dortmund, Dortmund, Germany
15Max-Planck-Institut für Kernphysik (MPIK), Heidelberg, Germany
16Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany
17School of Physics, University College Dublin, Dublin, Ireland
18INFN Sezione di Bari, Bari, Italy
19INFN Sezione di Bologna, Bologna, Italy
20INFN Sezione di Ferrara, Ferrara, Italy
21INFN Sezione di Firenze, Firenze, Italy
22INFN Laboratori Nazionali di Frascati, Frascati, Italy
23INFN Sezione di Genova, Genova, Italy
24INFN Sezione di Milano-Bicocca, Milano, Italy
25INFN Sezione di Milano, Milano, Italy
26INFN Sezione di Cagliari, Monserrato, Italy
27INFN Sezione di Padova, Padova, Italy
28INFN Sezione di Pisa, Pisa, Italy
29INFN Sezione di Roma Tor Vergata, Roma, Italy
30INFN Sezione di Roma La Sapienza, Roma, Italy
31Nikhef National Institute for Subatomic Physics, Amsterdam, Netherlands
32Nikhef National Institute for Subatomic Physics and VU University Amsterdam, Amsterdam, Netherlands
33Henryk Niewodniczanski Institute of Nuclear Physics Polish Academy of Sciences, Kraków, Poland
34AGH—University of Science and Technology, Faculty of Physics and Applied Computer Science, Kraków, Poland
35National Center for Nuclear Research (NCBJ), Warsaw, Poland
36Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest-Magurele, Romania
37Petersburg Nuclear Physics Institute NRC Kurchatov Institute (PNPI NRC KI), Gatchina, Russia
38Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP NRC KI), Moscow, Russia
39Institute of Nuclear Physics, Moscow State University (SINP MSU), Moscow, Russia
40Institute for Nuclear Research of the Russian Academy of Sciences (INR RAS), Moscow, Russia
41Yandex School of Data Analysis, Moscow, Russia
42Budker Institute of Nuclear Physics (SB RAS), Novosibirsk, Russia
43Institute for High Energy Physics NRC Kurchatov Institute (IHEP NRC KI), Protvino, Russia, Protvino, Russia
44ICCUB, Universitat de Barcelona, Barcelona, Spain
nstituto Galego de Física de Altas Enerxías (IGFAE), Universidade de Santiago de Compostela, Santiago de Compostela, Spain
46Instituto de Fisica Corpuscular, Centro Mixto Universidad de Valencia—CSIC, Valencia, Spain
47European Organization for Nuclear Research (CERN), Geneva, Switzerland
48Institute of Physics, Ecole Polytechnique F´ed´erale de Lausanne (EPFL), Lausanne, Switzerland
49Physik-Institut, Universität Zürich, Zürich, Switzerland
50NSC Kharkiv Institute of Physics and Technology (NSC KIPT), Kharkiv, Ukraine
51Institute for Nuclear Research of the National Academy of Sciences (KINR), Kyiv, Ukraine
52University of Birmingham, Birmingham, United Kingdom
53H. H. M. Zavertyaev,15,c M. Zdybal,33 M. Zeng,3 D. Zhang,7 L. Zhang,3 S. Zhang,4 W. C. Zhang,3,y Y. Zhang,47 A. Zhelezov,16
Y. Zheng,5 X. Zhou,5 Y. Zhou,5 X. Zhu,3 V. Zhukov,13,39 J. B. Zonneveld,57 and S. Zucchelli19,e PHYSICAL REVIEW LETTERS 124, 222001 (2020) cAlso at PN Lebedev Physical Institute Russian Academy of Science (LPI RAS) Moscow Russia 54Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom
55Department of Physics, University of Warwick, Coventry, United Kingdom
56STFC Rutherford Appleton Laboratory, Didcot, United Kingdom
57School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom
58School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom
59Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom
60Imperial College London, London, United Kingdom
61Department of Physics and Astronomy, University of Manchester, Manchester, United Kingdom
62Department of Physics, University of Oxford, Oxford, United Kingdom
63Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
64University of Cincinnati, Cincinnati, Ohio, USA
65University of Maryland, College Park, Maryland, USA
66Los Alamos National Laboratory (LANL), Los Alamos, New Mexico, USA
67Syracuse University, Syracuse, New York, USA
68Laboratory of Mathematical and Subatomic Physics, Constantine, Algeria
[associated with Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil]
69School of Physics and Astronomy, Monash University, Melbourne, Australia
(associated with Department of Physics, University of Warwick, Coventry, United Kingdom)
70Pontifícia Universidade Católica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, Brazil
[associated with Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil]
71Guangdong Provencial Key Laboratory of Nuclear Science, Institute of Quantum Matter,
South China Normal University, Guangzhou, China
(associated with Center for High Energy Physics, Tsinghua University, Beijing, China)
72School of Physics and Technology, Wuhan University, Wuhan, China
(associated with Center for High Energy Physics, Tsinghua University, Beijing, China)
73Departamento de Fisica, Universidad Nacional de Colombia, Bogota, Colombia
(associated with LPNHE, Sorbonne Universit´e, Paris Diderot Sorbonne Paris Cit´e, CNRS/IN2P3, Paris, France)
74Institut für Physik, Universität Rostock, Rostock, Germany
(associated with Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany)
75Van Swinderen Institute, University of Groningen, Groningen, Netherlands
(associated with Nikhef National Institute for Subatomic Physics, Amsterdam, Netherlands)
76Universiteit Maastricht, Maastricht, Netherlands
(associated with Nikhef National Institute for Subatomic Physics, Amsterdam, Netherlands)
77National Research Centre Kurchatov Institute, Moscow, Russia
[associated with Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP NRC KI), Moscow, Russia]
78National University of Science and Technology “MISIS,” Moscow, Russia
[associated with Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP NRC KI), Moscow, Russia]
79National Research University Higher School of Economics, Moscow, Russia
(associated with Yandex School of Data Analysis, Moscow, Russia)
80National Research Tomsk Polytechnic University, Tomsk, Russia
[associated with Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP NRC KI), Moscow, Russia]
81University of Michigan, Ann Arbor, Michigan, USA
(associated with Syracuse University, Syracuse, New York, USA)
aAlso at Universidade Federal do Triângulo Mineiro (UFTM) Uberaba-MG Brazil 75Van Swinderen Institute, University of Groningen, Groningen, Netherlands (associated with Nikhef National Institute for Subatomic Physics, Amsterdam, Netherlands)
76 6Universiteit Maastricht, Maastricht, Netherlands (associated with Nikhef National Institute for Subatomic Physics, Amsterdam, Netherlands)
77 77National Research Centre Kurchatov Institute, Moscow, Russia [associated with Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP N
78 [associated with Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP
78National University of Science and Technology “MISIS,” Moscow, Russ th Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP NRC KI), Mosc
78 f
p
y
78National University of Science and Technology “MISIS,” Moscow, Russia 78National University of Science and Technology “MISIS,” Moscow, Russia [associated with Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP NRC KI), Moscow, Russia]
79National Research University Higher School of Economics, Moscow, Russia [associated with Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP NRC KI), Moscow, Russia]
79National Research University Higher School of Economics, Moscow, Russia [associated with Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP NRC KI), Moscow, Russia]
79National Research University Higher School of Economics, Moscow, Russia (associated with Yandex School of Data Analysis, Moscow, Russia)
80 80National Research Tomsk Polytechnic University, Tomsk, Russia Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP NRC KI), Moscow, Russia]
81 [associated with Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP
81 81University of Michigan, Ann Arbor, Michigan, USA (associated with Syracuse University, Syracuse, New York, USA) aAlso at Universidade Federal do Triângulo Mineiro (UFTM), Uberaba-MG, Brazil. Kraków, Poland. lAlso at DS4DS, La Salle, Universitat Ramon Llull, Barcelona, Sp lAlso at DS4DS, La Salle, Universitat Ramon Llull, Barcelona, Spain. t DS4DS, La Salle, Universitat Ramon Llull, Barc mAlso at Hanoi University of Science, Hanoi, Vietnam. mAlso at Hanoi University of Science, Hanoi, Vietnam. nAlso at Universit`a di Padova, Padova, Italy. nAlso at Universit`a di Padova, Padova, Italy. oAlso at Universit`a di Pisa, Pisa, Italy. oAlso at Universit`a di Pisa, Pisa, Italy. 222001-10 PHYSICAL REVIEW LETTERS 124, 222001 (2020) bAlso at Laboratoire Leprince-Ringuet, Palaiseau, France. so at U ve s ta d
o a
o Ve gata,
o a, ta y.
kAlso at AGH—University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications,
Kraków, Poland.
l PHYSICAL REVIEW LETTERS 124, 222001 (2020) b bAlso at Laboratoire Leprince-Ringuet, Palaiseau, France. bAlso at Laboratoire Leprince-Ringuet, Palaiseau, France. cAlso at P.N. Lebedev Physical Institute, Russian Academy of Science (LPI RAS), Moscow, Russia. d cAlso at P.N. Lebedev Physical Institute, Russian Academy of Scien
d dAlso at Universit`a di Bari, Bari, Italy. eAlso at Universit`a di Bologna, Bologna, Italy. f eAlso at Universit`a di Bologna, Bologna, Italy. f fAlso at Universit`a di Cagliari, Cagliari, Italy. fAlso at Universit`a di Cagliari, Cagliari, Italy. gAlso at Universit`a di Ferrara, Ferrara, Italy. h hAlso at Universit`a di Genova, Genova, Italy. i hAlso at Universit`a di Genova, Genova, Italy. i iAlso at Universit`a di Milano Bicocca, Milano, Italy. j iAlso at Universit`a di Milano Bicocca, Milano, Italy. j jAlso at Universit`a di Roma Tor Vergata, Roma, Italy. jAlso at Universit`a di Roma Tor Vergata, Roma, Italy. k g
,
,
y
kAlso at AGH—University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications,
Kraków, Poland. kAlso at AGH—University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications,
Kraków, Poland. PHYSICAL REVIEW LETTERS 124, 222001 (2020) PHYSICAL REVIEW LETTERS 124, 222001 (2020) 54Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom
55Department of Physics, University of Warwick, Coventry, United Kingdom
56STFC Rutherford Appleton Laboratory, Didcot, United Kingdom
57School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom
58School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom
59Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom
60Imperial College London, London, United Kingdom
61Department of Physics and Astronomy, University of Manchester, Manchester, United Kingdom
62Department of Physics, University of Oxford, Oxford, United Kingdom
63Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
64University of Cincinnati, Cincinnati, Ohio, USA
65University of Maryland, College Park, Maryland, USA
66Los Alamos National Laboratory (LANL), Los Alamos, New Mexico, USA
67Syracuse University, Syracuse, New York, USA
68Laboratory of Mathematical and Subatomic Physics, Constantine, Algeria
[associated with Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil]
69School of Physics and Astronomy, Monash University, Melbourne, Australia
(associated with Department of Physics, University of Warwick, Coventry, United Kingdom)
70Pontifícia Universidade Católica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, Brazil
[associated with Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil]
71Guangdong Provencial Key Laboratory of Nuclear Science, Institute of Quantum Matter,
South China Normal University, Guangzhou, China
(associated with Center for High Energy Physics, Tsinghua University, Beijing, China)
72School of Physics and Technology, Wuhan University, Wuhan, China
(associated with Center for High Energy Physics, Tsinghua University, Beijing, China)
73Departamento de Fisica, Universidad Nacional de Colombia, Bogota, Colombia
(associated with LPNHE, Sorbonne Universit´e, Paris Diderot Sorbonne Paris Cit´e, CNRS/IN2P3, Paris, France)
74Institut für Physik, Universität Rostock, Rostock, Germany
(associated with Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany)
75Van Swinderen Institute, University of Groningen, Groningen, Netherlands
(associated with Nikhef National Institute for Subatomic Physics, Amsterdam, Netherlands)
76Universiteit Maastricht, Maastricht, Netherlands
(associated with Nikhef National Institute for Subatomic Physics, Amsterdam, Netherlands)
77National Research Centre Kurchatov Institute, Moscow, Russia
[associated with Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP NRC KI), Moscow, Russia]
78National University of Science and Technology “MISIS,” Moscow, Russia
[associated with Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP NRC KI), Moscow, Russia]
79National Research University Higher School of Economics, Moscow, Russia
(associated with Yandex School of Data Analysis, Moscow, Russia)
80National Research Tomsk Polytechnic University, Tomsk, Russia
[associated with Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP NRC KI), Moscow, Russia]
81University of Michigan, Ann Arbor, Michigan, USA
(associated with Syracuse University, Syracuse, New York, USA)
aAlso at Universidade Federal do Triângulo Mineiro (UFTM), Uberaba-MG, Brazil. 222001-10 PHYSICAL REVIEW LETTERS 124, 222001 (2020) pAlso at Universit`a degli Studi di Milano, Milano, Italy. PHYSICAL REVIEW LETTERS 124, 222001 (2020) pAlso at Universit`a degli Studi di Milano, Milano, Italy. qAlso at Universit`a di Urbino, Urbino, Italy. rAlso at Universit`a della Basilicata, Potenza, Italy. sAlso at Scuola Normale Superiore, Pisa, Italy. sAlso at Scuola Normale Superiore, Pisa, Italy. tAlso at Universit`a di Modena e Reggio Emilia, Modena, Italy. uAlso at Universit`a di Siena, Siena, Italy. vAlso at MSU—Iligan Institute of Technology (MSU-IIT), Iligan, Philippines. y
xAlso at INFN Sezione di Trieste, Trieste, Italy. yAlso at School of Physics and Information Technology, Shaanxi Normal University (SNNU), Xi’
z zAlso at Universidad Nacional Autonoma de Honduras, Tegucigalpa, Honduras. 222001-11
|
https://openalex.org/W2330667642
|
https://www.biodiversitylibrary.org/partpdf/86440
|
English
| null |
Notes on Some American Ducks
|
The Auk/The auk
| 1,920
|
public-domain
| 4,656
|
Juny,
1 VOL. XXX VOL. XXX Marila
marila.
Scavp. The A. O. U. ‘Check-List’ gives this species as breeding in south-
ern British Columbia; I can find no reliable record of this, and
consider it in the highest degree improbable, as I have never
seen the species in summer even as far north as I have been in
central
British
Columbia,
(lat. 54°),
except
for
a
few
crippled
birds. Marlia
affinis. Lesser
Scaup. Marlia
affinis. Lesser
Scaup. Marila
americana.
RepHEAD. Marila
americana. RepHEAD. There is a very frequent tendency to albinism in the female
Red-head,
not
in
the
male. Adult
females
are
almost
always
plentifully sprinkled with white feathers on the back of the head
and neck; this is accompanied by a varying amount of white in
the down. I have carefully plucked the outer feathers from a
number of females, the down on the lower surface may or may
not
be
white. The
amount
of
white
in
the
down
seems
to
parallel
the
amount
of
white
in
the
feathers
of
the
head,
very rarely is the down continuously white but is usually marbled
with
patches
of
dusky
colored
down. The
fattest
birds
very
often have the largest areas of white in the down on the lower
surface, it may be that the down may not be properly pigmented
because of the heavy layer of fat. On the lake in front of my house at the present moment among
the hundreds of Redheads is a female with an almost entirely
white head, the body being quite normal. The variation in the numerical strength of the sexes accord-
ing to season is probably as pronounced in this species as in any
duck. At
present
(November),
the
proportion
of
females
to
males is about 2 to 3, in midwinter, (January), one hardly sees a
female in the large flocks of males, and not until the end of Feb-
ruary
are
the
proportions
anything
like
equal. A
similar
se-
quence occurs in nearly every species of duck at this latitude,
with the possible exception of the Mallard, in which the sexes
are usually proportionate throughout the year. Marila
marila. Scavp. Plates
XV-XVI This
contribution
has
been
stimulated
by
the
many
valuable
papers by American ornithologists on these hitherto rather neg-
lected birds. Mr. Hollister’s paper on the Ringneck in “The Auk’
for October 1919 is especially welcome, expressing as it does the
first appreciation of the real affinities of this species. The changes that he proposes for the next A. O. U. ‘Check-List’
regarding the position of this duck are quite in order, but a more
important one is to put the Ruddy Sheldrake where it belongs,
its
present
position
in
the
‘Check-List’
being
quite
impossible. There is a great deal of work to be done yet even on the
commonest
of
North
American
ducks
especially
with
the
plum-
ages of the females. The variation in these is considerable. In
the
surface-feeding
ducks
it
consists
largely
of
a
decrease
in
the
spotting
of
the
lower
surface
in
many
species. I
always
put this down to age but since I have found that a similar con-
dition found in the larger Falcons, is really an individual variation
without any change through successive moults, I am inclined to
wait until observations are recorded of female ducks in captivity. The variation in the females of the diving ducks is not as
a rule so pronounced, but it occurs in many species. 353 304 te
July Notes
on
American Marlia
affinis.
Lesser
Scaup. This
is
a
common
breeder
in
central
British
Columbia
(the
region between Quesnelle Lake and Lac la Hache), but a scarce
one in the southern portion of the province. _
el,
oo. v _
el,
oo. v 355 Brooks,
Notes
on
America Unlike the larger species the females are very variable, in many
fully adult and breeding birds there is no white at the base of
the bill, the whole head being light brown; in others the head is
very dark brown with a conspicuous white patch on the face, as
in
the
Greater
Scaup. These
dark
birds
very
often
have
the
back freckled with white, a character I have not noticed in the
light
brown
headed
birds,
in
which
the
whole
body
plumage,
except
the
breast
and
belly,
is
uniform
light
brown. Marila
yvalisineria.
CANVAS-BACK. Breeding range exactly as in the Lesser Scaup. Southern breed-
ing
records
are
Lumby—one
pair
in
1902,
and
Grand
Forks—
three pairs in 1919. Marila
collaris.
Rinc-NeckED
Duck. I
can
completely
endorse
all
that
Mr. Hollister
says
about
this
duck. One
other
point
of
similarity
between
it
and
the
Redhead is the color of the downy young, exactly the same in
both birds and quite different from the dusky ducklings of the
Seaups. When
in
England
I
frequently
watched
the
Tufted
Ducks
very
closely,
to
see
points
of
affinity
to
our
Ringneck. The full plumaged males certainly look very much alike, espec-
ially when one sees them diving in shallow water, the whole body
being almost enveloped in the light colored flank feathers. They,
like the male Ringnecks, are very conspicuous as they dart about
along
the
bottom
rising
like
corks
after
a
short
immersion. But the females are not so much alike, and the young are utterly
different, the downy young of the Tufted Duck being the most
dusky colored ducklings I know of. And the females and young
are a far better indication of affinities than the males. The fe-
male of the old world Pochard (Marila ferina) is extraordinarily
like
a
female
Canvas-back,
a
female
Redhead
in
the
London
Zoo ponds alongside of the Pochards looked utterly unlike, both
in form and color, but strangely like a couple of female Rosy-
billed
ducks
(Metopiana
peposaca)
which
often
came
alongside
of her. And
yet
a
certain
well-known
ornithologist
in
England
pro-
poses to make our Redhead a subspecies of the Old World Pochard! 356 Auk
Tiily :
Notes
on
American Clangula
islandica.
Barrow’s
GOLDEN-EYE. This is an attempt to find a reliable method for separating
two
perfectly
distinct
species. For
Barrow’s
Golden-eye
is
a
perfectly distinct species and has always been recognized as such,
yet it would be more than difficult for an ordinary man to identify
a series of specimens by the aid of any of the works of reference
I have come across. Even the best authorities themselves seem uncertain as to the
reliability of their points for distinguishing the females and young
of the two species. This has probably led to the slighting references and inadequate
descriptions by minor authors. Barrow’s Golden-eye is not a ‘‘perpetuated accident of varia-
tion’ except to those who know nothing of the bird. The adult
males are not only easily distinguishable in the hand by at least
eight
points
of
difference
(including
structural)
but
are
readily
identified in the field as far as one can separate one species of
duck
from
another. | Millais in his ‘British Diving Ducks’ is the only author who
recognizes this, probably because he is the only one familiar with
the species in life. The crescentic cheek mark, the purplish head, the black wing
bar, and the spotted scapulars, are the marks usually given for
field identification of the adult male; but the most striking differ-
ence is the very black appearance. Adult
males
of
the
Common
and
American
Golden-eyes
are
very white birds, the body looks almost altogether white, just as
a
male
Bufflehead’s
does,
especially
when
sitting. The
adult
male Barrow’s on the other hand looks to have a body more black
than white. The most conspicuous feature of a duck at rest is
the flank. Whatever color the flank feathers are, they will domi-
nate the mass of the bird, as they overlap the whole wing and
sometimes even a portion of the back. Thus a fully plumaged Ringneck drake looks to be almost as
white as a Scaup, a Black Brant looks more white than black,
and so on. The flank feathers in Barrow’s Golden-eye (adult male) are
heavily
margined
with
black,
fully
two-thirds
of
an
inch
wide, THE
Auk,
VoL. XXXVII PLATE
XVI. THE
Auk,
VoL. XXXVII
PLATE
1. BaRRow’s GOLDEN-EYE. Mates Prun.na. FEMALE ASLEEP. Barrow’s GOLDEN-EYE COURTING. MALE IN SWALLOWING ACTION. FEMALE BosBBING Up Aanp Down. XVI. 1. BaRRow’s GOLDEN-EYE. Mates Prun.na. FEMALE ASLEEP. Barrow’s GOLDEN-EYE COURTING. MALE IN SWALLOWING ACTION. Clangula
islandica.
Barrow’s
GOLDEN-EYE. FEMALE BosBBING Up Aanp Down. 1. BaRRow’s GOLDEN-EYE. Mates Prun.na. FEMALE ASLEEP. Barrow’s GOLDEN-EYE COURTING. MALE IN SWALLOWING ACTION. FEMALE BosBBING Up Aanp Down. 307 rs
Mee
Brooks,
Noles
on
America Brooks,
Noles
on
America and the black comes almost, or quite, to the water line in front
of the wing. But while a child could distinguish the adult males of the two
species, it is a very different matter when it comes to the females
and young. The
case
of
the
Cinnamon
and
Blue-winged
Teal
is
similar,
and there are many others where two utterly dissimilar males
have females that are almost identical. In the Golden-eyes this
has been complicated by the oft-quoted recognition marks that
have no value, as they are common to both species. Perhaps it may be as well to go over the accepted and proffered
distinctions for separating islandica from americana. 1. The wing bar. This is the most often quoted distinction. In the adult male of Barrow’s there is certainly a constant and
conspicuous black bar separating the white patch on the wing,
this is caused by the bases of the greater coverts being black. But the bar formed by the black tipping of these feathers in the
females
and
young
is,
as
pointed
out
by
Mr. Brewster
(Auk,
Vol. XXVI,
p. 159),
an
utterly
valueless
distinction,
as
both
species may or may not have it in different individuals. Five
adult
females
of
Barrow’s
in
my
collection
have
these
feathers as follows: No. 1. Solid black, no bar. No. 1. Solid black, no bar. No. 2. Base black, terminal half white with small black tips,
forming a slight bar. No. 3. Tips black, well-defined bar. No. 3. Tips black, well-defined bar. No. 4. Trace of spots on tips of two feathers, no bar. No. 5. Slight bar. No. 5. Slight bar. All of these are absolutely identified, being taken in the spring
when paired. The most pronounced bar in a female in my collection belongs
to an otherwise typical americana, which has all the coverts tipped
black, while another has a trace of a bar. 2. Deeper coloring of head and neck in female Barrow’s. This
is a fairly reliable distinction but it is a comparative one. Clangula
islandica.
Barrow’s
GOLDEN-EYE. Ridg-
way in his manual says of zslandica, “brown of head descending
to middle of neck all round.”’ I can see no difference in amount
of brown in fresh specimens of the two species; the above dis-
tinction probably depends on the make-up of the skin. 358 A
hare é
Notes
on
American 3. Wider
gray
pectoral
band. Probably
a
sound
distinction
but one which is dependent, in the skin, on a uniform method of
make-up. 3. Wider
gray
pectoral
band. Probably
a
sound
distinction
but one which is dependent, in the skin, on a uniform method of
make-up. 3. Wider
gray
pectoral
band. Probably
a
sound
distinction
but one which is dependent, in the skin, on a uniform method of
make-up. 4. Shape and proportions of bill. The more tapering bill of
islandica is a thoroughly good distinction, but as given by all
authorities it is a comparative one, just as is “ bill more goose-like.”’ How can a man who has only one Golden-eye tell whether the
bill is tapering when no measurements are given, or that it is
goose-like if he has no example of the other species to compare
with
it?)
Ridgway’s
‘Manual’
however
has
a
definite
formula
as the best distinction between the two species. “Al. Height of upper mandible at base, measured from point of frontal
angle to nearest point on cutting edge, less than distance from anterior
edge of loral feathering to anterior end of nostril, and usually little if any
greater than distance from latter point to tip of upper mandible G. clangula. 151. G. clangula americana.” “A?. Height of upper mandible at base, measured from extremity of
frontal angle to nearest point on cutting edge, equal to distance from an-
terior point of loral feathering to anterior end of nostril, and much greater
than from latter point to tip of upper mandible
”’ 152. G. islandica.”’ 152. G. islandica.”’ 152. G. islandica.”’ I have carefully tested this with the following results: If the first measurement is taken by placing one point of the
dividers on the frontal angle, and the other on the cutting edge,
i. e., the chord of the distance around the bill, the results are
wrong
in
one-fourth
of
my
specimens
of
islandica,
including
one adult male, and wrong in two-thirds of my specimens of
americana including two adult males. If this measurement is taken by holding one point of the dividers
level with the frontal angle in line immediately above the nearest
point of the cutting edge, 7. e., the actual height of the former
above the latter, the results are hopelessly out in nearly all my
islandica, but correct for all but one of my americana. If
the
first
portion
of
the
proposition
is
taken,
eliminating
the distance from nostril to end of bill, measurements taken by
first method, the results are correct in all my americana, but
wrong in four out of eleven islandica. So this distinction, which
I had great hopes of, proved a “no thoroughfare.” tha
on
o 309 tha
on
oad
Brooks,
Notes
on
America Brooks,
Notes
on
America 5. Nail
of
bill. Ridgway
gives
width
in
americana
female
“not more than .20.” I have a female with a nail width of .22. In
islandica
female
“not
less
than
.23,”
this
holds
good
even
when a very young islandica is included, it has a nail .26 wide. Brewster
says
he
is
unable
to
verify
this
distinction
with
his
series, so it must be reckoned uncertain. Nail
larger
and
more
hooked
at
tip
(‘Game
birds
of
Cali-
fornia’), also Munro in ‘The Condor,’ No. 1, Vol. XV. “Nail is
wider at the front, projects further over tip of bill, and is slightly
raised above the bill forming a noticeable lump.” All of these distinctions hold good but then again, as given,
they are mostly comparative. 6. Color of bill. The yellow color of the bill in zslandica is an
oft-quoted
distinction
and
one
to
which
Mr. 151. G. clangula americana.” Brewster
attached
great faith. The following facts I can vouch for: Adult females of americana
usually have a yellow or dull orange bar on the terminal third
of the upper mandible, sometimes more than one-third of the
bill is yellow and in one instance (and here I speak from memory
only) the entire bill orange yellow. The young of this species have always (?) an olive bill—no
yellow. The
young
females
of
islandica
have
an
olive,
brownish,
or
blackish bill, no yellow. In the adult females it is wholly orange
or cheese-colored, or else the same with the base more or less
flecked with dusky. But this is a seasonal feature only. During
the past summer (1919) I kept a number of breeding females
under
observation. When
pairing
with
the
males
in
the
latter
part of April and early in May, all had orange bills, even some
of the unmated immatures of the previous year had the bill more
or less orange. In July,
when these
same
females were
each
leading a brood on their respective ponds, all had dusky or black-
ish bills, showing no yellow at all. So the yellow bill, which can only apply to adult females at
best, cannot be relied on at all seasons. 7. Skull. The difference in the shape of the frontal bones has
been noticed by several authorities, it is pronounced enough in
adult males, but a rather subtle distinction when applied to young
birds (see figures p. 362). pa 360 Brooks, Notes on American Ducks. Brooks, Notes on American Ducks. am
WW
and
and
and
and
7
3
to]
Barrow’s Golden-eye 2 ad. March 21, 1914. American Golden-eye 2 ad. April 7, 1914. Barrow’s Golden-eye o ad. April 15, 1911. American Golden-eye o ad. March 17, 1891. 3 nek
Pana nek
Pana 361 Brooks,
Notes
on
America Now the sum of all of this discussion seems to be that it would
be easy to wrongly identify a bird of either species from the works
of reference available in America, nor do ‘those of European authors,
up to and including Millais’ very elaborate work, give a really
reliable method of distinguishing the two species at all stages. As their main distinction, that of the greater size of zslandica,
only applies when it is compared with the smaller old world sub-
species,
C. clangula
clangula. 151. G. clangula americana.” The
following
attempt
to
differ-
entiate them may also prove abortive, but it works out well with
my series. This series is small, as events of recent years have interfered
with my plan of making a really good series of both species. Still
I have eleven thoroughly identified specimens of the rarer bird,
and
have
had
exceptional
opportunities
of
identifying
these,
by taking the paired female of an undoubted male islandica, or
the young of the same species after watching them through the
summer. Also I have had a very much larger number through my hands,
and
since
my
first
introduction
to
Barrow’s
Golden-eye
some
twenty-three years ago, I have lived in a region where it is the
commonest breeding duck, for the greater part of my time. This fact must be my excuse for attempting what is apparently
a
rather
difficult
undertaking. To
get
correct
measurements,
and ones that properly illustrate the differences, has not been
easy. First,
the
nail
has
ill-defined
boundaries
in
many
cases. I have found it advisable to wet the bill to more clearly define
these. Second, it has been difficult to get a measurement that
shows the very pronounced taper of the bill in islandica; if the
width is taken near the tip, where it is most prominent, there
must be a definite point. Through the base of the nail would
seem to be the best, but the longer nail of islandica brings this
measurement further back in that species, and so makes them
more nearly alike than the actual shape of the bill would indi-
cate. Half way between nostrils and base of nail suffers from
the same cause, so I have had to take the measurement across
the anterior angle of the nostrils although the taper is not so
pronounced there. As Mr. Brewster has done (loc. cit. p. 159), I shall place the
characters in what I consider the order of their importance. As Mr. Brewster has done (loc. cit. p. 159), I shall place the
characters in what I consider the order of their importance. Gaes 362 Notes
on
American (
I
is
7. 6. "4
Ms:
MELE
=
{
=
5
6
—
1. Barrow’s Golden-eye o ad. April 15, 1911. 2. American Golden-eye o& ad. March 17, 1891. 3 and 5. Barrow’s Golden-eye o’ ad. October 15, 1913. 4 and 6. Barrow’s Golden-eye @ ad. October 15, 1913. 151. G. clangula americana.” 1. Barrow’s Golden-eye o ad. April 15, 1911. 2. American Golden-eye o& ad. March 17, 1891. 3 and 5. Barrow’s Golden-eye o’ ad. October 15, 1913. 4 and 6. Barrow’s Golden-eye @ ad. October 15, 1913. 363 ee
rs
se Brooks,
Notes
on
American Those relating only to adult males are disregarded as these
can be separated by any one without difficulty. 1. Nail. Americana—Nail flattened or depressed, not conspicuously
raised above the contour of the bill when viewed from the side, and not
arched in its transverse section towards base. Length of nail, o, less than .46 in., longest .44, shortest .38, average
.406; @, less than .40, average .37. Length of nail, o, less than .46 in., longest .44, shortest .38, average
.406; @, less than .40, average .37. g
Islandica—Nail arched in both longitudinal and transverse sections,
showing as a conspicuous hump above the contour of the bill. Islandica—Nail arched in both longitudinal and transverse sections,
showing as a conspicuous hump above the contour of the bill. Length of nail, o’, over .46, longest .53, shortest .48, average 508;
2 , over .40, longest .46, shortest .41, average .43. Length of nail, o’, over .46, longest .53, shortest .48, average 508;
2 , over .40, longest .46, shortest .41, average .43. g
g
2. Shape of bill. Americana—Bill not conspicuously tapered when
viewed from above. g
g
2. Shape of bill. Americana—Bill not conspicuously tapered when
viewed from above. Width at a point through anterior angle of nostril, co’, over .69, widest
.82, narrowest .70, average .74; Q , over .60, average .69. Width at a point through anterior angle of nostril, co’, over .69, widest
.82, narrowest .70, average .74; Q , over .60, average .69. Tslandica—Bill conspicuously tapered, width taken as above, ©”, less
than .69, widest .68, narrowest .63, average .65; Q , less than .60, widest
.59, narrowest .55, average .57. 3. Color of head in Q. Americana— Hair brown or grayish amber.”
Islandica—“ Deep sepia or purplish snuff brown.” (Ridgway.) 4. Shape of Skull. Americana—Frontals continuing the slope of cul-
men, without trace of a bulge. 4. Shape of Skull. Americana—Frontals continuing the slope of cul-
men, without trace of a bulge. Islandica—Frontals conspicuously bulging in adult males, hardly less
so in immature males of second year, bulge distinctly noticeable to the
touch in adult females, and faintly so in juvenals. 151. G. clangula americana.” Something might be made out of the amount of black at base
of
greater
coverts. This
is
decidedly
more
in
islandica,
usually
showing beyond the overlap of the lesser coverts, and sometimes
covering the entire feather. In young males commencing to show
the white cheek patch a crescentic formation is sometimes ap-
parent in americana, this is due to the fact that the white feathers
commence to come in along the edge of the bill, following the
curve of its base, but it may be noted that this crescent is con-
fined to the lower portion of the bill, in islandica it continues up
to the mental angle, even in its first stages. The diagrams of bills appended are drawn from dried speci-
mens, as this will be the condition of most of the birds to be com-
pared with them. The tomia is apt to roll up and enclose the under mandible
in drying, sometimes more on one side than the other, giving the
bill a lop-sided appearance. 292
364 Auk
July =
:
Notes
on
American
Du The top views of the bills of the juvenal male and female tslandica
show a greater length as these are made from skulls, where there
is no feathering to cut off the extreme base when viewed in this
manner. The differences in the trachea have been commented on before,
but it is as well to include them here, the figures illustrate these. The sketches of courting antics are from a notebook, and were
made in the field. (Plates NV and XVI.) The most common form of display in the drake is the ‘swallow-
ing’ or ‘gulping’ action, this may or may not be followed by a kick
which
throws
the
water
up
behind. ‘Chasing,’
with
the
head
close to or level with the water, and the body sunk, always occurs
when
one
male
invades
another’s
territory. The
pursuer
often
dives and comes up under the intruder who then makes off at
great speed. 1. Trachea of American Golden-eye. la. Barrow’s Golden-eye. 1. Trachea of American Golden-eye. la. Barrow’s Golden-eye. Continually between these antics the males preen themselves,
frequently turning over on the back. 151. G. clangula americana.” The
females
pay
little
attention
to
their
lords,
occasionally
they approach bobbing or pumping their heads up and down,
and turning the bill from side to side; more rarely they will join
in the chase of an intruding male. Vol. cpa
1920 Vol. cpa
1920 365 Brooks,
Notes
on
America Quite often a female will turn her head around, tuck her bill
away in the back feathers and calmly go to sleep, oblivious of
the display of the drake who seemingly does not care whether
his spouse looks at him or not. p
Once in November I watched a lone drake going through the
whole
performance
by
himself—water-kick
and
all. When
asleep
the tail is held up at a good angle, though not such a conspicuous
cant as is affected by Ruddy Ducks and Scoters when resting. Barrow’s Golden-eye is a common breeder throughout the arid
interior
of
British
Columbia,
from
the
southern
boundary
north
at least to lat. 54 , and from 1000 ft. altitude up to at least 6000
ft., wherever the mountain lakes are sufficiently clear of ice to
allow them to rear a brood. I have only twice seen the common
Golden-eye breeding in this region. Once in November I watched a lone drake going through the
whole
performance
by
himself—water-kick
and
all. When
asleep
the tail is held up at a good angle, though not such a conspicuous
cant as is affected by Ruddy Ducks and Scoters when resting. cant as is affected by Ruddy Ducks and Scoters when resting. Barrow’s Golden-eye is a common breeder throughout the arid
interior
of
British
Columbia,
from
the
southern
boundary
north
at least to lat. 54 , and from 1000 ft. altitude up to at least 6000
ft., wherever the mountain lakes are sufficiently clear of ice to
allow them to rear a brood. I have only twice seen the common
Golden-eye breeding in this region. All the drakes, including those of the preceding year (which
do not acquire the full plumage until their second autumn), leave
for the coast before the middle of June, and before the young are
hatched. The bulk of the females and young follow them about the
middle of September as in the case of the Harlequin Duck. I have so far been unable to actually verify that they do so
to the salt water, but hope to during the coming summer. 151. G. clangula americana.” A few birds may be seen throughout the fall and winter, includ-
ing adult males. I am inclined to believe these are not the birds
that have bred here, but rather migrants from the northeastern
districts. I have found Barrow’s Golden-eye fairly common at the coast
in the winter, and much tamer than the common Golden-eye. This file was generated 1 April 2024 at 12:06 UTC Brooks, Allan. 1920. "Notes on Some American Ducks." The Auk 37, 353–367. https://doi.org/10.2307/4073263. Oidemia
americana.
AmpRICAN
ScoTsr. Oidemia
americana. AmpRICAN
ScoTsr. In
British
Columbia
this
Scoter
is
an
exclusively
maritime
duck, at least I have not come across a single reliable inland
record. Not only is it a maritime bird, but it is seldom found
in the small bays and inlets where the other species swarm, but
frequents the exposed shores and outer reefs together with the
Harlequin. It
has
many
points
in
common
with
that
duck,
rising
easily
from
the
water
and
doing
much
flying
about
in
small lots of four or five—mostly males—seemingly for the pleas-
ure of flying, usually returning to the point they started from. Copyright & Reuse Copyright Status: Public domain. The BHL considers that this work is no longer under
copyright protection. This document was created from content at the Biodiversity Heritage Library, the world's
largest open access digital library for biodiversity literature and archives. Visit BHL at
https://www.biodiversitylibrary.org. This file was generated 1 April 2024 at 12:06 UTC
|
https://openalex.org/W3124706646
|
https://sciforum.net/paper/download/8984/manuscript
|
English
| null |
Identification of vulnerable zone of surface water epidemiology using Remote Sensing and GIS techniques
|
Proceedings of The 3rd International Electronic Conference on Environmental Research and Public Health —Public Health Issues in the Context of the COVID-19 Pandemic
| 2,021
|
cc-by
| 4,963
|
Chandramohan Karuppaiah1, Vijaya Ramachandran1 and Sangu Muthuraju2 Chandramohan Karuppaiah1, Vijaya Ramachandran1 and Sangu Muthuraju2 Chandramohan Karuppaiah1, Vijaya Ramachandran1 and Sangu Muthuraju2 1 Department of Environmental Remote Sensing And Cartography, School of Earth and Atmospheric Scienc-
es, Madurai Kamaraj University, Madurai-625021, India j
y
2 Department of pharmacology and pharmaceutical, 4849 Calhoun Rd, University of Houston, Texas, 77204,
U.S.A. 2 Department of pharmacology and pharmaceutical, 4849 Calhoun Rd, University of Houston, Texas, 77204,
U.S.A. * Correspondence: drcmresearchlab@gmail.com Abstract ：The maintaining of drinking water quality, access to clean urban surface wa-
ter are the most challenge resulting in water-borne diseases. Although, the developing
countries, the Geographical Information System (GIS) technology has not been yet sys-
tematically utilized. Therefore, the present study aimed to establish the parameters for
assessing surface water quality and how they would cause health issues in the study ar-
ea. This study reveals the surface water quality variations by using principal component
analysis (PCA) techniques with the help of Landsat 8 Operational Land Imager (OLI)
satellite imagery. Regular monitoring of environmental quality database produced by
(GIS) could manage information from various sources such as, domestic, industrial, rec-
reational activities, point and non-points sources etc. This monitoring activity could
make spatial correlations with epidemiology data about time and space distribution of
water-borne diseases. The spatial correlation of water contamination tank and it’s sur-
roundings of land use / land cover (LULC) activities were calculated by the technique of
buffer analysis. Using Medical GIS, could easily detect the circulation and spread of dis-
ease across the geographic regions which could be used for planning, policy making,
water resource protection and avoid contamination. The study area also included the in-
dustrial and residential areas of Madurai urban region, Tamil Nadu, India, to validate
ground truth verification. The study would help to reveal the disease monitoring, sur-
veillance systems, improving the distribution of health resources by predicting available
health care accessibility, the source of pollution and it impacts on public health. Especial-
ly in Madurai need more attention to epidemiology. Because, the 11,132 epidemic dis-
ease affected cases were recorded in 2019[1]. In every year government of India spends
more money for this type of epidemiological spread; especially in Tamil Nadu spend
8868 lakhs of money. WHO also revealed that the developing countries are highly af-
fected by the epidemic diseases. Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional
claims in published maps and insti-
tutional affiliations. Chandramohan Karuppaiah1, Vijaya Ramachandran1 and Sangu Muthuraju2 Keywords: Remote Sensing; Medical geographical information system (GIS), Surface water; epi-
demiology; vulnerability; NDWI; Principal Component Analysis (PCA), LULC; Buffer Copyright: © 2021 by the authors. Submitted for possible open access
publication under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(http://creativecommons.org/licenses
/by/4.0/). 1. Introduction In 2016, according to the Global Health Observatory of World Health Organization
(WHO) reported, 1,32,121 cholera cases and 2420 deaths were recorded worldwide. Health & family welfare Department reported the Tamil Nadu is endemic for Acute Di-
arrhoeal Diseases with sporadic outbreak of cholera in most of the districts throughout
the year, and in epidemic proportions during the rainy seasons and peak summer peri- The 3rd International Electronic Conference on Environmental Research and Public Health The 3rd International Electronic Conference on Environmental Research and Public Health The 3rd International Electronic Conference on Environmental Research and Public Health The 3rd International Electronic Conference on Environmental Research and Public Health 2 of 11 ods. Epidemiology is the scientific study [3] of disease spread by particular environmen-
tal issues like waterborne and airborne diseases, etc. Remote Sensing (RS) and GIS are
very effective tools for analyzing the spatio temporal variations [4] to control of diseases. Remote Sensing is an excellent technology to observe and detect the changes occurs on
earth surface features [5, 6] which are associated with diseases. Geographic information system (GIS) is a Database Management System (DBMS)
tool for the collection of data from many sources by various methods [7], which is also
accomplishing of organizing, storing, retrieving, analyzing and presenting the spatial
data [8].The ERDAS Imagine 9.1v image processing software and the Arc GIS 10.1v spa-
tial analysis software are used for mapping, spatial analysis and image processing of the
both non-spatial and spatial data [9]. 2. Study Area and Data Study area of Madurai city, Tamil Nadu, India, lay between the aerial extensions of
78°2'50.733"E 9°59'38.597"N and 78°11'55.885"E 9°49'23.73"N.(Figure 1-A). According to
the Survey of India (SOI) toposheet showing in and around the study area of Madurai
having more than 100 tanks, from these tanks 70 tanks are having greater in size of 15
acre. Less than 15 acres of tank already encroached by various construction activities. Remote sensing techniques of satellite image represented the tank with /without water. The urban settlement occupied by 25020 acres. The population of the city was 1,734,000
(Population Census 2011). The domestic waste water directly joins to the tank and
stream. The 3rd International Electronic Conference on Environmental Research and Public Health 3 of 11 Figure.1 (A) Location of study area derived from India, Tamil Nadu, Madurai District and Madurai city boundary also
viewed through satellite imagery with 30m resolution.(B) Land Use / Land Cover map using Digital Image Processing
(DIP) of unsupervised classification techniques for area calculations of features.(C) Drainage network of Urban region. Blue colour shows the tank and red colour shows the canal connective to the tanks as well as open canal travel through
the dense residential area. Source: Google Earth pro – 2020. (D) Showing Normalized Differentiate Water Index using Spa-
tial Analyst Tools of Raster Calculation techniques for differentiate the water density and other features. (E) (left image)
PCA having band combinations of 5, 4, and 3 of tank showing the availability of water (dark blue) and vegetation (pink). € (right image) The same image PCA with the band combinations of 5, 4, and 3 indicate the quality variations of surface
water fully reflect the similar pattern of vegetation because of the growth of algal bloom and other water living plants. Figure.1 (A) Location of study area derived from India, Tamil Nadu, Madurai District and Madurai city boundary also
viewed through satellite imagery with 30m resolution.(B) Land Use / Land Cover map using Digital Image Processing
(DIP) of unsupervised classification techniques for area calculations of features.(C) Drainage network of Urban region. Blue colour shows the tank and red colour shows the canal connective to the tanks as well as open canal travel through
the dense residential area. Source: Google Earth pro – 2020. (D) Showing Normalized Differentiate Water Index using Spa-
tial Analyst Tools of Raster Calculation techniques for differentiate the water density and other features. 2. Study Area and Data (E) (left image)
PCA having band combinations of 5, 4, and 3 of tank showing the availability of water (dark blue) and vegetation (pink). € (right image) The same image PCA with the band combinations of 5, 4, and 3 indicate the quality variations of surface
water fully reflect the similar pattern of vegetation because of the growth of algal bloom and other water living plants. Satellite Sensor and Image Selection: Collection of four-band multispectral Landsat 8 OLI imagery acquired on 26 July
2020 was downloaded from the Earth explorer, USGS (United States of Geological Sur- The 3rd International Electronic Conference on Environmental Research and Public Health 4 of 11 vey) which are free of cost. The image acquisition date was selected to coincide for the
post monsoon period of November collecting more amount water through rainfall. In
each band or combinations of bands having unique characters of earth features based on
it’s observing and reflecting capacity of Ultra Violet (UV) visible, Near Infrared (NIR),
and Infrared (IR) spectrum. Blue, Green, and Red band combination were used to classi-
fying the LULC classification, band 3 and band 5 used for calculating NDWI, band 1 to
band 5 used for calculating the PCA. Table.A shows clear explanations of the spatial res-
olutions and spectral characters of the image. Table.1 Table: 1 (A) shows the details of sensor and bands, (B) shows the feature classification and its area calculation,
(3). Physiographical characters and its aerial extension cover. Table.1 Table: 1 (A) shows the details of sensor and bands, (B) shows the feature classification and its area calculation,
(3). Physiographical characters and its aerial extension cover. Table.1 Table: 1 (A) shows the details of sensor and bands, (B) shows the feature classification and its area calculation,
(3). Physiographical characters and its aerial extension cover. 5 of 11 The 3rd International Electronic Conference on Environmental Research and Public Health g
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To identify the water quality index by NDWI To identify the water quality index by NDWI y
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To identify the vulnerable zone of epidemiology of waterborne diseases y
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To identify the vulnerable zone of epidemiology of waterborne disease y
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To identify the vulnerable zone of epidemiology of waterborne diseases 5.1. Water Bodies and Settlement Density using Image Classification Method: 5.1. Water Bodies and Settlement Density using Image Classification Method: The study area of Madurai urban region having 8 major tank and 20 small tanks of
surface water bodies identified by the satellite imagery cross verification to SOI toposh-
eet. There is high efficiency with the water index approaches when multispectral image
classification of 30 m resolution, the water indexes is Normalized Difference Water In-
dex (NDWI) [10], Modified NDWI (MNDWI) [11]. These two methods used to calculate
the water index, distinguish between water and building, the optimal threshold to ex-
tract water is highly subjective, and also varies with region and time. But in that the eve-
ry water index has its main problem is the NDWI was poor at distinguishing between
water and buildings, mountain shadows [12]. Statistical methods of unsupervised classi-
fications are used for identifying the water bodies. These methods are more accurate
than other methods, because they are derived from the real satellite imagery. Prior
knowledge is applied in the unsupervised classification makes classifications (figure 1-B)
by learning from image interpretation key. Every feature’s aerial extension calculates
from unsupervised classification methods and its calculated values given table.B. NDWI = (Green – NIR) / (Green + NIR)) According to the table.A Landsat 8 OLI Band 3 (green) – Band 5 (NIR) According to the table.A Landsat 8 OLI Band 3 (green) – Band 5 (NIR) Data Sets and Methodology The availability of various data of primary and secondary data sets sources such as
the primary data collected were the SOI toposheets of 1:50,000 scale for the consistent
region, Landsat- 8 consistent path -154, row-053 LOR for year November 2020 from
Earth Landsat 8 Operational Land Imager (OLI) and Thermal Infrared Sensor (TIRS) im-
ages consist of nine spectral bands with a spatial resolution of 30 meters for Bands 1 to 7
and 9. NIR band of Landsat 8 as water index was found more satisfactory in extracting
water bodies compared to the multi-band water indexes. For this study, the satellite im-
agery was used with remote sensing techniques to identify the settlement density distri-
bution and water spread area, NDWI method used to calculate the water index for sur-
face quality variations within the tank, buffer analysis studied to identify the settlement
or urban buildup arrangements in surrounding of tank/waterbody area. 3. Objectives To trace out the tanks / surface water bodies near by the settlement using satellite
imagery 5.2. Calculation of NDWI The image statistical calculation of NDWI (figure 1-D) useful to analyze the distin-
guishing of feature based on the pixel values. It was calculated by the band 3 and band 5
of the imagery using the formula of: NDWI = (Green – NIR) / (Green + NIR))
(1) (1) 5.3. Identifying the contamination and it’s source: The study area settlement located nearby the major tanks which store rain water for
ground water recharge as well as every home having at least one borehole for domestic
water consumption. The septic tank leaching create the Giardiasis, typhoid, Toxigenic
E.coli gastroenteritis, salmonellosis, etc [13 & 14 ]. Canal and nearest settlement, com-
mercial, industrial, recreational activities are involving the major contamination sources. When the contamination increases it support profuse growth of weed plants on the sur- The 3rd International Electronic Conference on Environmental Research and Public Health 6 of 11 face water body [15 & 16]. The canal path and residential or buildup identified (figure 1-
C) easily around the water source using satellite digital image processing (DIP) tech-
niques (figure 1-B). 5.3.1. Principle Component Analysis (PCA) Principle Component method usually capture all the spectral information from the
bands, and compressed/merged it all into principle components. Technically two meth-
ods followed to firstly extract the future most of spectral information followed by sec-
ondly the rest of the spectral information which can't be stored in first calculation. PCA
is widely used in Machine Learning task. PC is new variables that are constructed as lin-
ear combinations of the initial variables. The figure 1-E (left image) PCA1 having band
combinations of 5,4, and 3 of tank showing the availability of water (dark blue) and veg-
etation (pink). The same image of figure 1-E (right image) with the band combinations of
5, 4, and 3 indicate the quality variations of surface water fully reflect the similar pattern
of vegetation because of the growth of algal bloom and other water living plants. From
this PCA generate eigenvalues follows. 5.4. Physiography of Study area: Elevation, Geological (figure 2-A), geomorphological (figure 2-B) parameters and
also physiological characters of precipitation, wind is important to study the water con-
tamination as well as epidemiological intensity [17]. Some soils (figure 2-C), such as
clays, absorb less water at a slower rate than sandy soils. Soils absorbing less water re-
sult in more runoff overland into streams. Generally hard rock does not have primary
porosity. Secondary porosity in the form of fractures and weathering only allow
groundwater occurrence [18]. The extent of weathering was identified based on VES
which indicated weathering up to a depth of 40 m from the ground surface. High rank is
assigned for meta-basalt rock (Table. C), which has high amount of porosity and good
groundwater potential. The 3rd International Electronic Conference on Environmental Research and Public Health 7 of 11 Figure 2. Figure: (A) Shows the type of geological characters available in the study area are Intrusive Rocks (Archaean -
Pre-cambrian) and Crystalline Rocks (Archaean - Pre-cambrian). The aerial extension of these geological types intrusive
is one of the two ways igneous rock can form. Igneous rocks tend to have low porosity and low permeability unless they
are highly fractured by tectonic processes. Which cover maximum of 66% of study area. But the crystal or crystalline
rock is a solid are arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all direc-
tions cover 34% of total study area. Crystalline rocks are having more capacity of water permeability rate when compare
with the intrusive rock type as well as the Crystallinerocks spread major tank and river lying area. Which also increase
the ground water contamination level. (B) shows the varied geomorphic characters in this study area Pediplain (68%),
Denudational Hills (1%), Alluvial Plain (25%), Flood Plain (3%), Structural Hills (1%), and Upland (2%). From this ob-
servation alluvial plain is having more surface water permeability then the other geomorphic characters. Meanwhile al- Figure 2. Figure: (A) Shows the type of geological characters available in the study area are Intrusive Rocks (Archaean
Pre-cambrian) and Crystalline Rocks (Archaean - Pre-cambrian). The aerial extension of these geological types intrusiv
is one of the two ways igneous rock can form. Igneous rocks tend to have low porosity and low permeability unless the
are highly fractured by tectonic processes. Which cover maximum of 66% of study area. But the crystal or crystallin Figure 2. 5.4. Physiography of Study area: (C) showing the soil types of clay (26.62%), clay loam (51.95%),
sandy clay loam (9.74%) and water body (18%) are present in the study area. Northern part of the study area fully occu-
pied by clay loam and sandy clay lo.am which are highly favorable to water percolation and the southern part of the
study area less percolation of water than the other area. When conclude this northern part of the study area is highly
vulnerable than the other parts and settlements/build-ups are densely spread over in northern part. Table.3 gives an ex-
act level of depth of the soil layer from the earth surface. (D) Shows the major tank and river present in the study area
which they are closely relate with the residential area. 5.4. Physiography of Study area: Figure: (A) Shows the type of geological characters available in the study area are Intrusive Rocks (Archaean -
Pre-cambrian) and Crystalline Rocks (Archaean - Pre-cambrian). The aerial extension of these geological types intrusive
is one of the two ways igneous rock can form. Igneous rocks tend to have low porosity and low permeability unless they
are highly fractured by tectonic processes. Which cover maximum of 66% of study area. But the crystal or crystalline
rock is a solid are arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all direc-
tions cover 34% of total study area. Crystalline rocks are having more capacity of water permeability rate when compare
with the intrusive rock type as well as the Crystallinerocks spread major tank and river lying area. Which also increase
the ground water contamination level. (B) shows the varied geomorphic characters in this study area Pediplain (68%),
Denudational Hills (1%), Alluvial Plain (25%), Flood Plain (3%), Structural Hills (1%), and Upland (2%). From this ob-
servation alluvial plain is having more surface water permeability then the other geomorphic characters. Meanwhile al- 8 of 11 The 3rd International Electronic Conference on Environmental Research and Public Health luvial plain cover 38 sqkm of 25% total study area and also present in the major tank and river lying area. Due to this
reason the contamination possibilities are very high. (C) showing the soil types of clay (26.62%), clay loam (51.95%),
sandy clay loam (9.74%) and water body (18%) are present in the study area. Northern part of the study area fully occu-
pied by clay loam and sandy clay lo.am which are highly favorable to water percolation and the southern part of the
study area less percolation of water than the other area. When conclude this northern part of the study area is highly
vulnerable than the other parts and settlements/build-ups are densely spread over in northern part. Table.3 gives an ex-
act level of depth of the soil layer from the earth surface. (D) Shows the major tank and river present in the study area
which they are closely relate with the residential area. luvial plain cover 38 sqkm of 25% total study area and also present in the major tank and river lying area. Due to this
reason the contamination possibilities are very high. 5.6. Epidemiology of Water-Borne Diseases and their Impact The contaminated water and their toxic exudates generate the pathogenic microor-
ganisms, cause serious conditions such as cholera, diarrhea, typhoid, amebiasis, hepati-
tis, Diptheria, gastroenteritis, giardiasis, campylobacteriosis, scabies, and worm infec-
tions, etc [19]. Drinking or swimming in contaminated water could be dangerous to
health [20]. The Landsat 8 OLI image of NDWI and PCA methods were indicate the clear
identifications of surface water quality variation. Within the range of 200m to 400 m dis-
tance from the tank buffered zone living people are using contaminated ground water. Where, the buffered regions are considering as epidemiological vulnerable zone. Bore
well water contaminants are associated with several illnesses including gastrointestinal
illnesses, cancer, reproductive issues, and neurological disorders [21 & 22]. The collec-
tion of waste material and sewage water mixed to the tank in rainy season and due to
the biological processes it will pollute and contaminate gradually. 5.5. Buffering Analysis. The study area tanks were extracted from the survey of India toposheet. Buffer
analysis is one of the main techniques to identify the surrounding area calculation with
specific distance from the center or outer of the features. Buffer builds a new object by
identifying all areas that are within a certain specified distance of the original objects. In
raster, buffers can be spread outwards from objects to create friction surface. The figure
2-D, shows the major water bodies selected for this study, figure 3-A is a villapuram
tank more than 80% of the tank occupied by settlement, figure3-B of Vandiyur tank and
it’s surroundings for 200m buffer area generated from the center of the tank and for
400m buffer area generated. The main source of contamination identified as point source
such as domestic solid waste and waste water collection through open urban sewage
system (Canal) connectivity as well as direct waste dumping from the nearest residential
and commercial. The ground water contamination process is more possible because the
physiographical characters such as soil, geomorphology and geology are highly support
the infiltration of contaminate surface water storage tank. The 3rd International Electronic Conference on Environmental Research and Public Health 9 of 11 Figure 3. (A) Villapuram tank buffer analysis of 200m and 400m (B) Vandiyur tank buffer analysis of 200m and 400m. Figure 3. (A) Villapuram tank buffer analysis of 200m and 400m (B) Vandiyur tank buffer analysis of 200m and 400m. Figure 3. (A) Villapuram tank buffer analysis of 200m and 400m (B) Vandiyur tank buffer analysis of 200m and 400m. 5. Limitation of the study At this COVID-19 pandemic situation is making difficult to connect the every house
around the tank within the range of 200m and 400m because of the reason for collecting The 3rd International Electronic Conference on Environmental Research and Public Health 10 of 11 10 of 11 primary data. The remote sensing data of high resolution imagery (e.g. 1m) give more
information of LULC than the low resolution of 30m of Landsat 8 OLI. But high resolu-
tion imagery is not available freely, cost is very high. Based on the time consumption the
research might not carry out all major tanks in the study area. 6. Conclusion This study reveals, the people are consuming contaminated water leads into more
possibilities of epidemiology. The Remote Sensing techniques of PCA, NDWI, DIP and
GIS techniques of buffer analysis, spatial anlyst tool, and mapping were highly effective
to identifying the possible area of ground water contamination. As well as focusing the
large spatial distribution of population of epidemiological spread vulnerable zone. Based on this study result we would prepare decision making and early warning system
of epidemiological activities to the society. In future, this study extends further to reveal
the disease type, epidemiological measurements of exposure and disease status, other
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nar river basin around Pavagada, Karnataka, India using remote sensing and GIS techniques. J Indian Soc Remote Sens
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https://openalex.org/W4205939476
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https://discovery.ucl.ac.uk/10152385/1/Lorencatto_A%20Systematic%20Review%20of%20Mental%20Health%20Professionals%2C%20Patients%2C%20and%20Carers%27%20Perceived%20Barriers%20and%20Enablers%20to%20Supporting%20Smoking%20Cessation%20in%20Mental%20Health%20Settings_VoR.pdf
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English
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A Systematic Review of Mental Health Professionals, Patients, and Carers’ Perceived Barriers and Enablers to Supporting Smoking Cessation in Mental Health Settings
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Nicotine & tobacco research
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Received: May 19, 2021. Revised: October 20, 2021. Accepted: January 5, 2022
© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nicotine and Tobacco Research, 2022, 24, 945–954
https://doi.org/10.1093/ntr/ntac004
Advance access publication 8 January 2022 Review Review Abstract Introduction: Evidence-based smoking cessation and temporary abstinence interventions to address smoking in mental health settings are
available, but the impact of these interventions is limited. Introduction: Evidence-based smoking cessation and temporary abstinence interventions to address smoki
available, but the impact of these interventions is limited. c.oup.com/ntr/article/24/7/945/6501336 by UCL Library Services user on 19 July 2022 Aims and Methods: We aimed to identify and synthesize the perceived barriers and enablers to supporting smoking cessation in mental health
settings. Six databases were searched for articles reporting the investigation of perceived barriers and enablers to supporting smoking cessation
in mental health settings. Data were extracted and coded using a mixed inductive/deductive method to the theoretical domains framework, key
barriers and enablers were identified through the combining of coding frequency, elaboration, and expressed importance. Results: Of 31 included articles, 56 barriers/enablers were reported from the perspectives of mental healthcare professionals (MHPs), 48 from
patient perspectives, 21 from mixed perspectives, and 0 from relatives/carers. Barriers to supporting smoking cessation or temporary abstin-
ence in mental health settings mainly fell within the domains: environmental context and resources (eg, MHPs lack of time); knowledge (eg,
interactions around smoking that did occur were ill informed); social influences (eg, smoking norms within social network); and intentions (eg,
MHPs lack positive intentions to deliver support). Enablers mainly fell within the domains: environmental context and resources (eg, use of ap-
propriate support materials) and social influences (eg, pro-quitting social norms). Conclusions: The importance of overcoming competing demands on staff time and resources, the inclusion of tailored, personalized support,
the exploitation of patients wider social support networks, and enhancing knowledge and awareness around the benefits smoking cessation is
highlighted. Implications: Identified barriers and enablers represent targets for future interventions to improve the support of smoking cessation in mental
health settings. Future research needs to examine the perceptions of the carers and family/friends of patients in relation to the smoking behavior
change support delivered to patients. A Systematic Review of Mental Health Professionals,
Patients, and Carers’ Perceived Barriers and Enablers to
Supporting Smoking Cessation in Mental Health Settings
Lisa Huddlestone PhD1, Emily Shoesmith PhD1,
, Jodi Pervin BSc1, Fabiana Lorencatto PhD2,
Jude Watson PhD1, Elena Ratschen PhD1 1Department of Health Sciences, University of York, York, UK
2Centre for Behaviour Change, University College London, London, UK 1Department of Health Sciences, University of York, York, UK
2Centre for Behaviour Change, University College London, London, UK Corresponding Author: Emily Shoesmith, PhD, Department of Health Sciences, Faculty of Sciences, University of York, Heslington, York Y010 5DD, UK. Telephone: 01904 321765; E mail: emily shoesmith@york ac uk Corresponding Author: Emily Shoesmith, PhD, Department of Health Sciences, Faculty of Sciences, University of York, Heslingto
Telephone: 01904 321765; E-mail: emily.shoesmith@york.ac.uk mily Shoesmith, PhD, Department of Health Sciences, Faculty of Sciences, University of York, Heslington, York Y010 5DD, UK. E-mail: emily.shoesmith@york.ac.uk Introduction to 70% of inpatients smoking.13,14 Yet, smokers with mental
health conditions are equally, or more motivated to quit smok-
ing than those without mental health conditions.15 However,
they are less likely to receive support compared with the general
population.16 There are many reasons for this, including the
smoking culture within mental health services,16,17 often driven
by misconceptions, for example, relating to the “therapeutic”
function of smoking in this population.18 There are substantial inequalities in morbidity and premature
mortality between individuals with mental health problems
and those without.1 One of the largest contributory factors to
early mortality in this population is smoking.2 Among indi-
viduals with a common mental health condition in England,
smoking prevalence is over 50%,3 compared with 14% in the
general population,4 and this difference increases further for
more severe mental health conditions.5 Despite this, the World Health Organisation recommends
that all healthcare facilities are smokefree, a policy that is
increasingly being adopted internationally.19 However, the
healthcare system and the respective development and imple-
mentation of tobacco control policies can vary substantially
across countries.20 These differences may present various con-
textual, political, and economic barriers that may impact on
the success of quitting behaviors that require separate con-
sideration. For example, economic barriers may limit the
potential to implement evidence-based smoking cessation While the number of smokers in the general population
has been steadily declining over recent decades,6 the number
of people with mental health conditions who smoke have not
been declining at the same rate.7 Those with mental health con-
ditions are more likely to display patterns of heavy smoking,3,5,8
greater dependence on nicotine, and more severe withdrawal
symptoms when quitting, and lower quite rates.9–12 Previous
research has estimated the percentage of smokers with mental
health conditions vary dependent on setting, but can reach up 946 Huddlestone et al. interventions in healthcare settings,21 and there is a wide vari-
ation in the provision of smoking cessation advice offered by
healthcare professionals dependent on setting.22 Help-seeking
behavior may also differ by setting and influence smoking-
related outcomes. Previous literature has reported individuals
living in higher-income countries were more likely to seek ad-
vice from a healthcare professional to quit, and have higher
use of quit smoking medications, compared with those in
low- and middle-income countries.22 As such, there are con-
siderable differences between settings in regard to quitting be-
haviors and type of support used. Introduction Such variation reflects the
differences in tobacco control implementation, the capacity
of the country and the priority given to specific policies (eg,
regulatory measures and the provision of cessation support).22 inform appropriate policy, and to facilitate the development
of more effective tailored smoking cessation interventions. Furthermore, due to the increased prevalence and overall
reduced rates of successful cessation success among those
with mental health conditions,9–12 a need identify the barriers
and enablers to quitting smoking in mental health settings
from the perspective of people with mental illness and men-
tal health professionals and those providing mental health
services is required. Effective behavior change interventions require an under-
standing of the broader context of the problem (eg, the so-
cial and environmental context, and noncontextual influences
on behavior such as knowledge consequences and motiv-
ation).39 The theoretical domains framework (TDF) is an in-
tegrative theoretical model that synthesizes main behavior
change constructs across key theories into 14 domains, such
as knowledge or goals.40 The TDF is helpful for investigating
implementation barriers and enablers, and provides a use-
ful conceptual basis for assessing implementation problems,
designing interventions to enhance healthcare practice, and
understanding behavior change processes.29 Regardless of setting, the evidence base reports that there
are factors that can influence the success of quitting behav-
iors among vulnerable groups.23,24 Within health behavior
literature, factors that hinder an individual from making a
health behavior change have been referred to as barriers, and
factors that facilitate an individual to make a change are re-
ferred to as enablers. Barriers and enablers can be conceptu-
alized as either individual or structural psychosocial factors.25
Individual factors refer to subjective experience, and can
be nonmodifiable (eg, age, ethnicity, nicotine dependence),
whilst others are modifiable and thus, potentially amenable
to intervention (eg, plans to not smoke or a desire to quit).26
Structural factors include organizations and the relationship
between these organizations and individuals. Likewise, some
are nonmodifiable (eg, pharmacist’s behavioral control of rec-
onciling medications),27 whilst others are modifiable (eg, ac-
cessibility to smoking cessation interventions).28 Therefore, the aim of this systematic review was to identify
and synthesize the evidence relating to the barriers and en-
ablers that influence smoking abstinence, and the delivery of
smoking cessation or temporary abstinence interventions in
mental health settings from the perspective of those delivering
and receiving such interventions. Specifically, the research
questions are: 1. Introduction What are the modifiable barriers and enablers that in-
fluence smoking cessation or temporary abstinence for
patients in mental health settings?i Despite a growing evidence base in relation to barriers and
enablers to the implementation of behavior change interven-
tions by healthcare professionals,29 there remains a lack of
focus on those factors that are shared across professional
groups.30 Given this limitation, it is important to differentiate
between the roles that individuals involved in the delivering
and receiving of smoking cessation interventions may play. For example, clinical staff are likely to be involved in the im-
plementation and delivery of the intervention, and thus, the
perceived lack of time is a frequently reported barrier,31,32
whereas nonclinical staff may report barriers at the commis-
sioning and policy level (eg, lack of adequate information on
the cost, volume, and quality of healthcare services).33 2. What are the modifiable barriers and enablers that in-
fluence the delivery of smoking cessation or temporary
abstinence interventions for mental health professionals
(MHPs) in mental health settings?il 3. What are the modifiable barriers and enablers that influ-
ence the support of smoking cessation for relatives/carers
in mental health settings? Search Strategy The systematic review was conducted in accordance
with PRISMA guidelines and registered on PROSPERO
(CRD42020193125). In addition to individual and organizational factors, bar-
riers and enablers may also be conceptualized as socially in-
fluenced. For example, the family is an influential context in
which smoking behavior occurs.34 Such social networks may
play an important role in the individual’s quit attempt, since
cohabitants smoking status is a known major determinant for
adult smoking behavior change.35,36 Indeed, previous research
reports cases of family members actively discouraging quit at-
tempts by people with mental illness, as well as encouraging
the maintenance of smoking due to concerns about cessa-
tion adversely impacting the individual’s mental health37 or
because smoking was perceived to be the individual’s only
source of enjoyment.38 However, and somewhat paradox-
ically, research also reports that family relationships are a
prime motivator to quit,38 indicating that family may also be
a crucial enabler for smoking cessation. Searches were conducted in four bibliographic databases
(MEDLINE, EMBASE, PsycInfo, and CINAHL), as well as
the Cochrane Central Register of Clinical Trials, and the UK
Clinical Research Network Portfolio database. The search
strategy included search terms relating to the population
(eg, inpatients, mental health nurses, relatives/carers), inter-
vention (smoking cessation or temporary abstinence), out-
come (eg, barriers, enablers), and relevant settings (eg, mental
health services). Supplementary Table 1 provides details of the
search terms. Searches were limited to papers published in
English, and from 1990 onwards due to pharmacological, be-
havioral, and other counseling approaches not being widely
available prior to 1990.41 Methods Search Strategy Quality Assessment Article quality was assessed using the Mixed Methods
Appraisal Tool (MMAT).42 Two authors (ES and JP) rated in-
dependently rated included studies, and a third author (LH)
independently assessed a random sample of 11 (35%) studies. Minor differences in opinion relating to the quality of studies
were resolved through discussion. Inclusion and Exclusion Criteria Understanding these perceived barriers and enablers to
quitting is important in order to facilitate our understand-
ing of smoking, relapse and quitting-related behaviors, to Article inclusion were based on the population, intervention,
comparator, outcome (PICO) method for eligibility, shown in 947 Nicotine & Tobacco Research, 2022, Vol. 24, No. 7 Table 1. Articles utilizing quantitative experimental (including
randomized control trials) or observational methods, qualita-
tive methods, or mixed-methods were eligible for inclusion. Systematic reviews, conference papers, or those articles that
were not original research were excluded. which domain they were judged to best represent. For
example, the extracted data point barriers that were
notably endorsed by psychiatrists were “lack of time
(49%)” 44 would be coded to the domain “environmen-
tal context and resources,” and “social norms, attitudes
and behaviors toward smoking as an undesirable social
behavior helped some participants in the quitting pro-
cess” 45 would be coded to the domain “social influences.”
Coding was guided by the definitions of the TDF do-
mains outlined by Cane et al.40 Three authors (LH, ES,
and JP) reviewed and verified each coded item. Data Screening Endnote X9 was used to record publications at all stages of
the selection process. After removal of duplicates, two mem-
bers of the research team (LH and ES) independently screened
all identified titles and abstracts against the inclusion and
exclusion criteria to ensure consensus. A third author (JP)
rescreened 100 titles and abstracts to ensure reliability. Where
disagreements arose, these were settled by discussion. Where
exclusion could not be determined from the abstract, articles
were included for full-text review. Full-text copies of poten-
tially eligible studies were obtained and a final decision was
made on inclusion by consensus among the review team. 2. 2. Inductive thematic synthesis was conducted to combine
similar data points coded to the same domain, and in-
ductively generating a summary theme label and corres-
ponding subthemes. Coding was conducted independ-
ently by two authors (LH and ES), with discussion to
identify consistency in the development of themes and
subthemes. Discrepancies between coders were resolved
through discussion with a third author (JP). Themes were
then categorized as either a barrier, enabler, or mixed in-
fluence, and as relating to the perception of patient, carer,
family member, friend, MHP, or organization. 2. Inductive thematic synthesis was conducted to combine
similar data points coded to the same domain, and in-
ductively generating a summary theme label and corres-
ponding subthemes. Coding was conducted independ-
ently by two authors (LH and ES), with discussion to
identify consistency in the development of themes and
subthemes. Discrepancies between coders were resolved
through discussion with a third author (JP). Themes were
then categorized as either a barrier, enabler, or mixed in-
fluence, and as relating to the perception of patient, carer,
family member, friend, MHP, or organization. Data Extraction Data were extracted using a customized spreadsheet by three
authors (LH, ES, and JP). The extracted study characteristics
were country, research design, methods, setting (inpatient, out-
patient, community), participants, and target behavior (smok-
ing cessation or temporary abstinence for patients; delivering
smoking cessation support for MHPs). Authors identified and
extracted quantitative and qualitative data reporting perceived
barriers and enablers associated with target behaviors. 3. Key barriers and enablers were then identified by ranking
TDF domains in terms of importance using established
criteria46: (1) frequency (number of studies that identi-
fied each domain); (2) elaboration (number of thematic
subthemes and themes) within each domain; and (3) ex-
pressed importance (a statement from the authors’ dis-
cussion or direct quotations from the study participants
expressing importance). The frequencies from each of the
three categories were combined and a median frequency
with standard deviation was calculated. TDF domains
exceeding this calculated mean frequency were con-
sidered as being of importance. 3. Key barriers and enablers were then identified by ranking
TDF domains in terms of importance using established
criteria46: (1) frequency (number of studies that identi-
fied each domain); (2) elaboration (number of thematic
subthemes and themes) within each domain; and (3) ex-
pressed importance (a statement from the authors’ dis-
cussion or direct quotations from the study participants
expressing importance). The frequencies from each of the
three categories were combined and a median frequency
with standard deviation was calculated. TDF domains
exceeding this calculated mean frequency were con-
sidered as being of importance. up.com/ntr/article/24/7/945/6501336 by UCL Library Services user on 19 July 2022 • Adult smokers using community, outpatient, and acute inpatient mental health services and their family, friends,
carers, and visitors Description of Studies Database searches yielded a total of 11 445 articles. After
the removal of duplicates and screening of titles, abstracts,
and full-text articles, 31 papers were included in the re-
view31,44,45,47–74 (Figure 1). In order to identify and understand the context of barriers and
enablers to smoking behavior change, the TDF was utilized. The approach to data analysis followed the combined three-
step method reported by Graham-Rowe et al.,43 in which con-
tent and framework analysis approaches are combined: Fourteen studies were observational quantitative studies,
eight utilized a qualitative methodology, six were random-
ized control trials, and three adopted a mixed-methods
design. A total of 8626 participants were recruited across 30
of the included studies, with one quantitative study not re- 1. Deductive content analysis was conducted by coding the
extracted data to the TDF. Two authors (LH and ES)
coded the extracted data from all studies according to 1. Deductive content analysis was conducted by coding the
extracted data to the TDF. Two authors (LH and ES)
coded the extracted data from all studies according to Table 1. Description of Studies Criteria for Article Inclusion Based on the PICO Method for Eligibility g
y
Population
• Adult smokers using community, outpatient, and acute inpatient mental health services and their family, friends,
carers, and visitors
• Members of staff working inpatient and outpatient, or community mental health settings
Intervention
• Smoking cessation (including cutting down to quit)
• Temporary abstinence (in the context of an inpatient admission)
• Interventions aimed at promoting cessation or preventing relapse after temporary abstinence/quitting (eg, in the
context of discharge from an inpatient admission)
Comparator
Not applicable
Outcome
• Reported barriers to and enablers of the use, implementation, and delivery of evidence-based smoking cessation
interventions
• Other influences on the use and uptake of interventions may include type of provider, specification of pathways,
type of intervention (eg, frequency, duration), and intended and unintended consequences of interventions • Members of staff working inpatient and outpatient, or community mental health settings • Smoking cessation (including cutting down to quit) • Temporary abstinence (in the context of an inpatient admission) • Interventions aimed at promoting cessation or preventing relapse after temporary abstinence/quitting (eg, in the
context of discharge from an inpatient admission) • Reported barriers to and enablers of the use, implementation, and delivery of evidence-based smoking cessation
interventions • Other influences on the use and uptake of interventions may include type of provider, specification of pathways,
type of intervention (eg, frequency, duration), and intended and unintended consequences of interventions • Other influences on the use and uptake of interventions may include type of provider, specification of pathways,
type of intervention (eg, frequency, duration), and intended and unintended consequences of interventions 948 Huddlestone et al. Figure 1. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram. Downloaded from https://academic.oup.com/ntr/article/24/7/945/6501336 by UCL Library Downloaded from https://academic.oup.com/ntr/article/24/7/945/6501336 by UCL Library Services user on 19 July 2022 mic.oup.com/ntr/article/24/7/945/6501336 by UCL Library Services user on 19 July 2022 Figure 1. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram. questions. Full detail of the included studies is provided in
Supplementary Table 3. porting a sample size.69 Most studies were conducted in com-
munity mental health settings (n = 12), followed by inpatient
settings (n = 9), and outpatient clinics (n = 5). A number of
studies gathered data in mixed mental health settings (n = 5). Description of Studies Seventeen studies recruited only patients, seven studies re-
cruited a range of clinical and nonclinical MHPs, five included
both patients and MHPs, and two obtained the perceptions
of mental health service managers and directors). Studies re-
cruiting carers, family members, or friends of individuals with
mental health problems could not be identified. Full study
characteristics are presented in Supplementary Table 2. Frequency of Identified Barriers and Enablers to the
Delivery and Receipt of Smoking Behavior Change
Interventions A total of 75 barriers and 50 enablers were identified across
the included articles. Fifty-six barriers and enablers were eluci-
dated from the perspectives of MHPs or organizations (44 bar-
riers; 12 enablers), and 48 from the perspective of patients (17
barriers; 31 enablers). Twenty-one were from a mixed (patient/
MHP/organizational) perspectives (14 barriers; 7 enablers).i Importance Expressed by Study Authors c.oup.com/ntr/article/24/7/945/6501336 by UCL Library Services user on 19 July 2022 Fifteen authors interpreted study findings as identifying im-
portant influences. Important domains were: environmental
context and resources (15 items in 16 studies); knowledge (9
items in 12 studies); social influences (10 items in 10 studies);
intentions (8 items in 9 studies); and emotion (7 items in 7
studies). Ranking Criteria Convergence Finally, the theme of “task rich and time poor” exempli-
fied the perceived competing demands on MHP’s time and
resources as a barrier to the delivery of smoking cessation
interventions. Competing demands included: limited clin-
ical time to address mental health needs and tobacco use, the
need to prioritize the support offered individually to patients,
and immovable organizational and service-level responsibilit
ies.44,50,60,61,63 Domains ranked according to the importance criteria of fre-
quency, elaboration, and expressed importance are presented
in Figure 2. Accordingly, the most important domains were:
environmental context and resources, knowledge, social in-
fluences, intentions, and emotion. These are summarized nar-
ratively below. Figure 2. Domains ranked according to the importance criteria of
frequency, elaboration, and expressed importance. ECR = environmental
context and resources; K = knowledge; SI = social influence;
I = intentions; E = emotions; BCap = beliefs about capabilities;
R = reinforcement; SK = skills; SPRI = social/professional role identity;
G = goals; BCon = beliefs about consequences; MADP = memory,
attention, and decision processes; O = optimism. Level of Elaboration The theme of “presence or absence of available support”
related to the availability and accessibility of a range of
preference-based support, and the materials and format of
the support. For example, these barriers included access to
nicotine replacement therapy while admitted to a smokefree
mental health setting, as well as the inaccessibility of nico-
tine replacement therapy due to financial costs following dis-
charge.55,61,62 Moreover, MHPs reported that resources were
not adequate (eg, lack of referral and/or clinical resources),
and this negatively impacted the implementation of either
the smokefree policy or the smoking cessation support avail-
able.44,56,61,70 In terms of the support materials, MHPs and
patients perceived the format of the support materials as an
enabler, if they were easy to use, colorful, and incorporated
useful information.52 However, a barrier would include po-
tential literacy issues for some patients, but this could be over-
come with the additional use of technology, if resources were
available.52 The level of elaboration was calculated from the number of
themes and subthemes generated within each domain iden-
tified in the inductive analysis. Environmental context and
resources had the highest number of themes (n = 3) and
subthemes (n = 23), followed by intentions and emotion
(n = 2 themes; 9 subthemes in each domain), and social in-
fluences and knowledge (n = 2 themes; 8 subthemes in each
domain). Frequency of Coding to Domains Data were coded most frequently to the domains of: envir-
onmental context and resources (n = 16 articles); knowledge
(n = 12 articles); social influences (n = 10 articles); intentions
(n = 9 articles); beliefs about capabilities (n = 8 articles); and
emotion (n = 7 articles). Quality Assessment Barriers and enablers were identified across 13 of the 14
TDF domains. The majority of these fell within the domains
environmental context and resources (n = 20 barriers; 9 en-
ablers); knowledge (n = 15 barriers; 5 enablers); intentions
(n = 10 barriers; 5 enablers); and social influences (n = 7 bar-
riers; 7 enablers). All studies clearly stated their research questions or research
objectives. The majority provided the requisite information
required by the MMAT. Those which lacked the required in-
formation included four randomized control trials, where it
was not possible to ascertain the appropriateness of random-
ization73 and blinding procedures,48,51,59,73 and three quantita-
tive descriptive studies lacked sufficient information to assess
the risk of nonresponse bias.61,62,69 All studies used estab-
lished methods that were appropriate to answer the research All studies clearly stated their research questions or research
objectives. The majority provided the requisite information
required by the MMAT. Those which lacked the required in-
formation included four randomized control trials, where it
was not possible to ascertain the appropriateness of random-
ization73 and blinding procedures,48,51,59,73 and three quantita-
d
d
l
k d
ffi
f Supplementary Table 4 presents the themes inductively
generated for each TDF domain, organized by perspective
(patient, MHP, organization, mixed), and influence (barrier,
enabler, mixed). Supplementary Table 5 also presents all the Nicotine & Tobacco Research, 2022, Vol. 24, No. 7 949 Environmental Context and Resources themes and subthemes generated in the 13 identified domains
of the TDF, organized by perspective (patient, MHP, organiza-
tion, mixed), and influence (barrier, enabler, mixed). Overall, environmental context and resources appeared to
have a mixed influence on the delivery and receipt of interven-
tions to support smoking cessation or temporary abstinence
following discharge from a mental health setting. The theme
“integration of services” related to the organization and co-
operation within and between mental health and other health
services, and was reported by both patients, MHPs, and or-
ganizations. For many of these participants, “integration of
services” identified barriers concerning the absence or cohe-
sion of referral and smoking cessation support pathways, and
the availability of resources.31,44,47,63 Social Influences In addition, the theme “lack of meaningful activities” was
predominantly identified as a barrier by patients, and fre-
quently referred to boredom, inactivity and time alone that
would subsequently lead to smoking behavior as an activity
to fill time or manage cravings.52,55–57 Furthermore, one study
reported that boredom was common among patients in both
hospital and community settings, and individuals maintained
they smoked in the absence of other meaningful daily activ-
ities.56 A mixture of barriers and enablers were identified within the
domain of social influence, all from the perspective of the pa-
tients. The theme “influence of social network members” cap-
tures the smoking norms, attitudes and behaviors of social
network members, and how these impacts on the individual. A number of patients reported that smoking was normative
in many social contexts, and as a result, quit attempts were
challenging due to their peers and family smoking around
them.45,55,58 Conversely, when social network members con-
sidered smoking an undesirable behavior, this helped some
patients in their quit attempt.45,55,57 One study identified that
almost all patients (92%) could identify a key support person
in their life on whom they could rely on to provide assistance
and general support, and 70% of participants with a partner
believed the partner would be supportive of them making a
quit attempt.66 Knowledge Several studies reported a lack of awareness about tobacco
use, its links to mental illness, and treatment both during ad-
mission and within the community as barriers to both the
delivery and receipt of interventions.31,47,52,55,56,60,62,63,70 Lack of knowledge and misinformation was widespread across both
groups. For example, one study identified that interactions
around smoking that did occur were ill informed in rela-
tion to inaccurate advice.52 Another study identified a lack of
knowledge and information with regard to strategies to sup-
port stopping smoking, especially the use of nicotine replace-
ment therapy products.55 Lastly, MHPs were found to actively
discourage smoking cessation attempts due to concerns about
the impact on patients’ mental health or due to a perception
that stop smoking medications are unsuitable for people with
a mental health condition.63 Conversely, when MHPs were perceived to have a greater
awareness and knowledge regarding tobacco use and its links
to mental illness, this was perceived as an enabler to patient
engagement.63 Additionally, patients identified access to a wide
range of information as an enabler (eg, more detailed infor-
mation about the health consequences, social impact of smok-
ing, and pharmacological support).49,51,52 Thus, providing Figure 2. Domains ranked according to the importance criteria of
frequency, elaboration, and expressed importance. ECR = environmental
context and resources; K = knowledge; SI = social influence;
I = intentions; E = emotions; BCap = beliefs about capabilities;
R = reinforcement; SK = skills; SPRI = social/professional role identity;
G = goals; BCon = beliefs about consequences; MADP = memory,
attention, and decision processes; O = optimism. 950 Huddlestone et al. “coping mechanisms for stress” highlighted that smoking was
often used to cope with acute stressors (eg, health scares, be-
reavements), everyday stresses of life, and also as a coping
mechanism specifically in relation to one’s mental health diag-
nosis.52,56–58,62 One study reported that the majority of MHPs
agreed at least in part with the statement that “smoking re-
lieves efficiently from daily tensions or stress.” 62 training and education was identified as a crucial compo-
nent by both MHPs and patients, including evidence-based
pharmacotherapies and behavioral interventions.56 One study
reported that both patients and MHPs acknowledged the im-
portance of education about the harmful effects of tobacco
use versus the potential benefit of symptom control.56 Discussion This paper presents a systematic, theoretically informed ap-
proach to the identification of perceived barriers and enablers
to supporting smoking cessation in mental health settings. Our findings identify five TDF domains as being important
influences on delivery or receipt of smoking cessation or tem-
porary abstinence support: (1) environmental context and re-
sources, (2) knowledge, (3) social influences, (4) intentions,
and (5) emotion. c.oup.com/ntr/article/24/7/945/6501336 by UCL Library Services user on 19 July 2022 The theme “smoking culture within a mental health con-
text” highlights that many patients identified the smoking
culture as a barrier. For example, frequently observing to-
bacco use among MHPs and other patients challenged one’s
own quit attempt.52,56 One study reported that when social
activities were available, these reinforced smoking behaviors. Indeed, patients frequently commented on how difficult it was
to consider quitting when those around them smoked. Both
MHPs and patients viewed smoking as a social event, and a
way to connect with family, peers, and staff.56 However, an-
other study reported that some MHPs acknowledged how
smoking was once an activity shared between staff and pa-
tients, but the Trust had progressed in denormalizing the so-
cial culture that was once ingrained into the mental health
context.31 This systematic review emphasizes the need for smoking
cessation support for people with mental health conditions
to be integrated within and between mental health and other
health services. Many of the factors identified by MHPs as
barriers to addressing smoking in mental health settings
link directly to the environmental context and resources. For example, the importance of integration of services, and
overcoming competing demands on staff time and resour
ces.44,50,60,61,63 These findings emphasize the importance of
a protected space with allocated time to focus on smoking
cessation support outside of routine work in mental health
settings.63 Furthermore, such findings are also acknowledged
by other researchers who highlight that people with mental
health conditions can be disadvantaged by fragmented care.75
Likewise, authors have also demonstrated that if MHPs were
provided with protected space and time to focus on smoking
cessation, they were able to effectively liaise between primary
and secondary services.63i Intentions The theme “stability of intentions and stages of change” re-
lates to the patient’s intentions and their readiness to quit. Many patients were determined and motivated to quit, and
had intentions to do so, despite a potential lack of self-belief
in their ability.57 One study did report that measures of motiv-
ation (stages of change, thoughts about abstinence scales) pre-
dicted abstinence status significantly,59 indicating that positive
intentions are an enabler for smoking cessation. A number of
studies identified that MHPs perceived patient’s lack of inten-
tion or interest to quit as a barrier for the individual to engage
with smoking cessation support.44,50,60,61 Similarly, through the identification of the barriers concern-
ing the absence or cohesion of referral and smoking cessation
support pathways, the need to consider the additional chal-
lenges that people with mental health conditions encounter
when undertaking cessation attempts is indicated. Indeed,
authors note that the variability and complexity of mental
health service provision may result in confusion for patients,
particularly when they are required to self-refer to cessation
services following discharge from a mental health inpatient
setting.76 Lastly, attention is drawn to the importance of
available and accessible preference-based support. Moreover,
in alignment with previous research, the personalization of
support within a mental health context has been shown to en-
able changes in smoking behavior.73 Indeed, results from the
SCIMITAR+ trial confirm the positive influence of bespoke
smoking cessation interventions in this population, finding a
doubling of the likelihood of quitting at 6 months in compari-
son to the control group.77 Lastly, one study reported on the lack of intention of MHPs
to deliver smoking cessation interventions. For example,
nursing staff had lower scores than medical staff with regard
to the intention to provide tobacco treatment.67 The findings
reported that staff attitudes were independently associated
with intentions to provide tobacco treatment.67 Limitations and Strengths This review included studies comprising various methodo-
logical designs, and which included the perspectives of a
range of stakeholders in variety of mental health settings. Even with this diversity of mental health settings, there ap-
peared to be consistency in the findings across these contexts. Therefore, this review offers a comprehensive overview of the
barriers and enablers to addressing smoking in mental health
settings. However, a number of limitations should be acknow-
ledged. This review only included studies from high-income
countries, which limits the generalizability of our conclusions,
since low- and middle-income countries may present differ-
ent contextual, political, and economic barriers that were
not explored. The data analyzed were obtained from the in-
terpretation of the of the study findings from the article au-
thors. Therefore, the potential for reporting bias cannot be
excluded. Importantly, authors may have selectively reported
findings on barriers and enablers, potentially drawing conclu-
sions from those that aligned neatly with the research ques-
tion, or which were perceived as controversial or interesting. The TDF domain of social influence appeared to have
mixed consequences on smoking cessation in mental health
settings, and patients frequently identified support networks
as either a barrier or an enabler. These findings are consistent
with a social norms perspective on health behavior change,
whereby individual choices are significantly influenced by the
behaviors and opinions of important others.80 Awareness of
ex-smokers and those within a patient’s social network who
are also undertaking quit attempts may be particularly im-
portant for populations that experience a high prevalence of
smoking.28,81 Thus, the exploitation of patients wider social
support networks may be an effective strategy for support-
ing smoking cessation among individuals with mental health
conditions. .oup.com/ntr/article/24/7/945/6501336 by UCL Library Services user on 19 July 2022 Despite intentions of the authors to understand the barriers
and enablers to addressing smoking in people with mental
health conditions from the perspective of their carers, family
members, or friends, the included studies did not yield evi-
dence on this. To date, there has been little attempt to under-
stand how family and friends of those with mental health
conditions understand, experience, and respond to the smok-
ing behaviors of those they support. Limitations and Strengths Although informal carers
can provide a strong source of emotional and practical sup-
port for their relative, family members, and friends can lack
awareness of available resources and fear social stigma.82
Therefore, they tend to adapt negatively to the individual’s
smoking-related behavior,82 possibly providing an explanation
for the dearth of literature within this population. However,
such an absence of evidence highlights the need for further
investigation into the role of informal support networks and
the needs of informal carers, to increase their involvement in
supporting attempts at changing smoking behavior.i Emotion All of the data coded to the domain of emotion was identified
as barriers to the delivery and receipt of interventions to sup-
port smoking cessation or temporary abstinence. The theme Nicotine & Tobacco Research, 2022, Vol. 24, No. 7 951 Nicotine & Tobacco Research, 2022, Vol. 24, No. 7 Nicotine & Tobacco Research, 2022, Vol. 24, No. 7 Within the TDF domain of “knowledge,” a lack of aware-
ness and comprehension was frequently reported in relation
to tobacco use, its links to mental illness, and the support
available.31,47,52,55,56,60,62,63,70 What is more, MHPs who receive
specialist training to offer services designed to improve an
individual’s mental health have a crucial role in reducing to-
bacco smoking among people with mental health conditions,
as they are best placed to encourage and support smokers
to quit.78 However, the findings from this review highlight
a need for increased specialist training in smoking cessation
interventions, as well as broader education to challenge mis-
conceptions about smoking cessation in the context of men-
tal illness and mental health services. Additionally, improved
access to flexibly delivered mandatory training (with peri-
odic refreshers) for MHPs should improve the consistency
of smoking-related health messages delivered to patients. Correspondingly, previous research advocates for additional
training of smoking cessation advisors in the United Kingdom
working with people with mental health conditions.79l Within the TDF domain of “knowledge,” a lack of aware-
ness and comprehension was frequently reported in relation
to tobacco use, its links to mental illness, and the support
available.31,47,52,55,56,60,62,63,70 What is more, MHPs who receive
l
ff
d
d Finally, our findings indicate that many of the factors iden-
tified by patients as barriers to smoking cessation or tempor-
ary abstinence related to the TDF domain of “emotion.” It
was frequently reported that smoking was used as a coping
strategy for everyday stressors and in relation to one’s men-
tal health diagnosis.52,56–58,62 Similarly, psychosocial stressors
have been implicated as risk factors for tobacco use in a range
of populations, including people living with other health
conditions,83 those from disadvantaged communities,84,85
those in the military,86 and those in the general population.87
Accordingly, this indicates the need to develop tailored inter-
ventions that target the identification and implementation
of alternative coping strategies for individuals with mental
health conditions. Conclusion Environmental context and resources, knowledge, social in-
fluences, intentions, and emotion are key factors influencing
smoking cessation in mental health settings. Specific bar-
riers to the delivery of intentions by MHPs include compet-
ing demands on time and resources and limited knowledge
in relation to tobacco use and its links with mental health. Enablers to enhance patients’ engagement with smoking ces-
sation support include tailored, personalized support, and the
teaching of alternative coping strategies, and the inclusion of
social networks with pro-quitting social norms. Targeting or
exploiting these factors are more likely to result in success-
ful interventions. Future research should explore the enablers
and barriers to smoking cessation in low- and middle-income
countries to identify contextual differences that may have an
impact on smoking-related behaviors. Lastly, further research
is required to seek the perception of the informal carers and
patients’ social networks in relation to the support offered to
address smoking in mental health settings. The domain of intentions was also identified as
influencing changes in smoking behaviors. In particular, the
patient’s intentions and lack of interest in smoking cessa-
tion was identified as a barrier to engagement.44,50,61 Despite
this, compelling evidence exists that indicates most people
with mental health conditions do want to quit and intend
to do so, and that smoking cessation interventions targeting
this population are effective.15 It is important therefore, that
fluctuations in motivation or intentions are not equated
with wanting to disengage with support, but rather to allow
the flexibility for individual’s to reengage when they wish
to do so.63 References Theories of behaviour change
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On the Non-Gaussianity of Sea Surface Elevations
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Journal of marine science and engineering
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1. Introduction Much attention in the literature is dedicated to the study of the sea surface height [1–3], a
function of the sea surface elevation which is generally obtained by making use of the zero-up
or down crossing methodology. The sea surface height is relevant because of design and
analysis of off-shore structures [4] and ships [5] and, therefore, the literature is large in terms
of studying its distribution [6–10]. The sea surface height has been modeled, for instance, as Academic Editors: Angelica Lo Duca
and Andrea Marchetti •
a Rayleigh distribution [11,12], •
a Rayleigh distribution [11,12],
•
a, more general, Weibull distribution [13],
•
a Forristall distribution [1],
•
a Naess distribution [14],
•
a Boccotti distribution [15],
•
a Klopman distribution [16],
•
a van Vledder distribution [17],
•
a Battjes–Groenendijk distribution [18],
•
a Mendez distribution [19], or
•
a LoWiSh II distribution [20]. •
a Rayleigh distribution [11,12],
•
a, more general, Weibull distribution [13],
•
a Forristall distribution [1],
•
a Naess distribution [14],
•
a Boccotti distribution [15],
•
a Klopman distribution [16],
•
a van Vledder distribution [17],
•
a Battjes–Groenendijk distribution [18],
•
a Mendez distribution [19], or
•
a LoWiSh II distribution [20]. •
a Rayleigh distribution [11,12],
•
a, more general, Weibull distribution [13],
•
a Forristall distribution [1],
•
a Naess distribution [14],
•
a Boccotti distribution [15],
•
a Klopman distribution [16],
•
a van Vledder distribution [17],
•
a Battjes–Groenendijk distribution [18],
•
a Mendez distribution [19], or
•
a LoWiSh II distribution [20]. •
a, more general, Weibull distribution [13], •
a Forristall distribution [1], Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. a Battjes–Groenendijk distribution [18], •
a Mendez distribution [19], or •
a LoWiSh II distribution [20]. Copyright:
© 2022 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). In [21] it is experimentally proved that the sea heights do not follow a Gaussian distribution. In [21] it is experimentally proved that the sea heights do not follow a Gaussian distribution. This study is dedicated, however, to the analysis of the sea surface elevation, as op-
posed to the sea heights. Journal of
Marine Science
and Engineering Journal of
Marine Science
and Engineering Journal of
Marine Science
and Engineering Journal of
Marine Science
and Engineering Citation: Nieto-Reyes, A. On the
Non-Gaussianity of Sea Surface
Elevations. J. Mar. Sci. Eng. 2022, 10,
1303. https://doi.org/10.3390/
jmse10091303 Article
On the Non-Gaussianity of Sea Surface Elevations Alicia Nieto-Reyes † Alicia Nieto-Reyes † Department of Mathematics, Statistics and Computer Science, Universidad de Cantabria, 39005 Santander, Spain;
alicia.nieto@unican.es
† Current address: Faculty of Science, Universidad de Cantabria, Avd. Los Castros s/n, 39005 Santander, Spain. Abstract: The sea surface elevations are generally stated as non-Gaussian processes in the current
literature, being considered Gaussian for short periods of relatively low wave heights. The objective
here is to study the evolution of the distribution of the sea surface elevation from Gaussian to non-
Gaussian as the period of time in which the associated time series is recorded increases. To do this,
an empirical study based on the measurements of the buoys in the US coast downloaded at a casual
day is performed. This study results in rejecting the null hypothesis of Gaussianity in below 25% of
the cases for short periods of time and in over 95% of the cases for long periods of time. The analysis
pursued relates to a recent one by the author in which the heights of sea waves are proved to be
non-Gaussian. It is similar in that the Gaussianity of the process is studied as a whole and not just
of its one-dimensional marginal, as it is common in the literature. It differs, however, in that the
analysis of the sea surface elevations is harder from a statistical point of view, as the one-dimensional
marginals can be Gaussian, which is observed throughout the study and in that a longitudinal study
is performed here. Keywords: Gaussian process; normal distribution; nortsTest R package; random projections; stationarity;
time series analysis Citation: Nieto-Reyes, A. On the
Non-Gaussianity of Sea Surface
Elevations. J. Mar. Sci. Eng. 2022, 10,
1303. https://doi.org/10.3390/
jmse10091303
Academic Editors: Angelica Lo Duca
and Andrea Marchetti
Received: 10 August 2022
Accepted: 9 September 2022
Published: 15 September 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Copyright:
© 2022 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). 1. Introduction The measurements of sea surface elevation are obtained by buoys
throughout the sea, which are later preprocessed to obtain the sea heights. Sea surface
elevations have been studied from a statistical point of view, studying its distribution [22],
the skewness of the distribution [23], and the modellization of the process [24,25]. Con-
sideration has also being given to how to measure [26] and record the data [27]. From an https://www.mdpi.com/journal/jmse J. Mar. Sci. Eng. 2022, 10, 1303. https://doi.org/10.3390/jmse10091303 J. Mar. Sci. Eng. 2022, 10, 1303 2 of 19 applied perspective, the literature contains works on sea surface elevations to, for instance,
ship motion forecasting [28] and the development of sea surface elevation maps [29]. Fur-
thermore, ref. [30] studies that for certain wave groups the periods of stationarity are
short and [31] that the sea surface elevation is only Gaussian in short periods of relatively
low wave heights. For many sea states the process is nonlinear due to its second order
structure [32], and even to much higher order structures for high waves [33]. This work goes beyond the existing literature and it is dedicated to empirically study
how the Gaussianity of the distribution of the sea surface elevation evolves along an
increase of the time period of the associated time series. For that, the measurements of
59 buoys along the US coast are studied. It is obtained that over 50% of the cases are
non-Gaussian with a length time period corresponding to 2 × 104 observations. This results
increases to over 95% of the cases if the length time period increases to correspond to 105
observations. From a statistical point of view, the importance of studying the sea surface
elevation is high and lies in that it is a raw measurement. While experimental studies show
that the distribution of sea heights are clearly non-Gaussian, having a non-Gaussian one
dimensional marginal, the non-Gaussianity of the sea surface elevation is not so obvious;
which makes the problem more interesting. In fact, in proving the non-Gaussianity, it is here demonstrated that some cases that
common hypothesis tests consider as Gaussian correspond to non-Gaussian processes with
Gaussian one-dimensional marginals. 1. Introduction It is worth saying that out of a 22.03% rejection
rate for a length time period corresponding to 103 observations, a 6.78% correspond to
non-Gaussian processes with Gaussian one-dimensional marginals, the Guassianity of
which would not have been rejected but for the use of the methodology applied here. This
methodology is based on the random projection test [34], a goodness of fit test that checks
the Gaussianity of the process as a whole and not just of a finite order marginal, as other
established test in the literature do; see, for instance [35,36]. The obtained findings are
important due to the cases that the literature considered as Gaussian are numerous. These
cases include very large waves and, in fact, according to [37], very large waves might be
much more frequent than commonly assumed. The rest of the manuscript includes: The description of the studied dataset in Section 2
and of the applied methodology in Section 3. The results of the analysis are described in
Section 4. The analysis makes use of the nortsTest package of the R software. Section 5
contains the obtained conclusions. 2. Datasets The Coastal Data Information Program (https://cdip.ucsd.edu (accessed on 20 August
2022)) contains surface elevations measured by buoys that are along the cost of the US. For the
present study, these measurement where downloaded on the 20 August 2022 from the web
page https://thredds.cdip.ucsd.edu/thredds/catalog/cdip/realtime/catalog.html (accessed
on 20 August 2022). In particular, the variable downloaded is that named xyzZDisplace-
ment. The set of data used here differs from that in [21] and it has not being used in the
literature before. There are a total of 59 datasets, each corresponding to the collected time series of a
station (buoy). Each buoy has an identification number, which is displayed in the first
and fourth columns of Table 1. The latitude and longitude coordinates of these buoys are
displayed in Figure 1 and, rounded to 2 decimal values, also included in the table. The top
plot of Figure 1 contains a world map where the coordinates have been drawn. The bottom
plot is a zoom of the top one that contains only the coordinate of the buoys that are close to
the US mainland coast. In Table A1, in Appendix A, it can be observed the time period of the time series
associated to each of the 59 buoys, under the columns designated with the names Start Time
(columns 2 and 3) and End Time (columns 4 and 5). The shortest time period is depicted in
bold, which is that of station 244. This is the buoy with the shortest time period because
the length of the associated time series is the smallest among that of the 59 time series. This J. Mar. Sci. Eng. 2022, 10, 1303 3 of 19 information is presented in Table A2, also in Appendix A, under the columns designated
with the name length. information is presented in Table A2, also in Appendix A, under the columns designated
with the name length. information is presented in Table A2, also in Appendix A, under the columns designated
with the name length. information is presented in Table A2, also in Appendix A, under the columns designated
with the name length. Table 1. Buoys identification number, with associated longitud and latitude coordinates, whose
surface elevation measurements constitute the datasets analyzed in this paper. Buoy
Latitude
Longitude
Buoy
Latitude
Longitude
028
33.86◦
−118.64◦
185
36.7◦
−122.34◦
029
37.94◦
−123.46◦
188
19.78◦
−154.97◦
036
46.86◦
−124.24◦
189
−14.27◦
−170.5◦
045
33.18◦
−117.47◦
191
32.52◦
−117.43◦
067
33.22◦
−119.87◦
192
35.75◦
−75.33◦
071
34.45◦
−120.78◦
194
30◦
−81.08◦
076
35.2◦
−120.86◦
196
13.68◦
144.81◦
092
33.62◦
−118.32◦
197
15.27◦
145.66◦
094
40.29◦
−124.73◦
198
21.48◦
−157.75◦
098
21.41◦
−157.68◦
201
32.87◦
−117.27◦
100
32.93◦
−117.39◦
202
22.28◦
−159.57◦
106
21.67◦
−158.12◦
203
33.77◦
−119.56◦
121
13.35◦
144.79◦
204
59.6◦
−151.83◦
132
30.71◦
−81.29◦
209
39.77◦
−73.77◦
134
27.55◦
−80.22◦
213
33.58◦
−118.18◦
139
43.77◦
−124.55◦
214
27.59◦
−82.93◦
142
37.79◦
−122.63◦
215
33.7◦
−118.2◦
143
28.4◦
−80.53◦
217
34.21◦
−76.95◦
144
27.34◦
−84.27◦
220
32.75◦
−117.5◦
147
36.92◦
−75.72◦
222
34.77◦
−121.5◦
150
34.14◦
−77.72◦
224
37.75◦
−75.33◦
154
40.97◦
−71.13◦
230
48.03◦
−87.73◦
157
36.33◦
−122.1◦
239
20.75◦
−157◦
158
36.63◦
−121.91◦
240
37.02◦
−76.15◦
160
42.8◦
−70.17◦
241
64.47◦
−165.48◦
162
46.22◦
−124.13◦
243
36◦
−75.42◦
166
50.03◦
−145.2◦
244
24.41◦
−81.97◦
168
40.9◦
−124.36◦
430
36.26◦
−75.59◦
171
36.61◦
−74.84◦
433
36.2◦
−75.71◦
181
18.38◦
−67.28◦ Each of the 59 datasets under study is restricted to a time series of length 5 × 105. The first 5 × 105 are the ones selected. The datasets consist of raw data, which contains
unknown, unobserved, values. After taking out those from the first 5 × 105 selected, the
length of the time series associated to each of the 59 buoys can also be observed from
Table A2, in Appendix A. It is designated by the name studied. It can be observed from
Table A2, in Appendix A, that there is one buoy for which the whole 5 × 105 first elements
have been observed: buoy 249. There, it can also be observed that there are two buoys, 188
and 202, that have the minimum value under the label studied . J. Mar. Sci. Eng. information is presented in Table A2, also in Appendix A, under the columns designated
with the name length. 2022, 10, 1303 4 of 19 36
76
100
92
94
241
430
198
201
244
191
98
28
2945
67
71
106
121
132
134
139
142
143
144
147
150
154
157
158
160
162
166
168
171
181
185
188
189
192
194
196
197
202
203
204
209
213
214
215
217
220
222
224
230
239
240
243
433
28
29
36
45
67
71
76
92
94
100
132
134
139
142
143
144
147
150
154
157
158
160
162
168
171
185
191
192
194
201
203
209
213
214
215
217
220
222
224
230
240
243
244
430
433
Figure 1. Top panel: World map with the coordinates of the 59 buoys whose measurements constitute
the datasets analyzed in this paper. Bottom panel: Zoom of the top panel that shows the US mainland
with the coordinates of the buoys close to it. The identification number is that in Table 1. 244
Figure 1. Top panel: World map with the coordinates of the 59 buoys whose measurements constitute
the datasets analyzed in this paper. Bottom panel: Zoom of the top panel that shows the US mainland
with the coordinates of the buoys close to it. The identification number is that in Table 1. 181
In the left panel of Figure 2, it is displayed part of the recorded data for buoy 433. The
displayed data results from restricting the 1,622,186 observations stored for buoy 433 and
taking the ones corresponding to the first 105 time points. As it is obvious from the plot,
the first 105 time points contain unobserved elements. Indeed, 410,144 observations have
been made out of the 5 × 105 selected for the study (see Table A2, in Appendix A). From
the left panel of the figure, it is also observable that the unobserved data splits the time
series in ten parts. The right panel of Figure 2 is a zoom of the left panel containing solely
the part to the left of the time series. Greenwich mean time (GMT) in the format year-month-day and hours:minutes:seconds
is used for the x-axis of the plots in the figure. 5 of 19 J. Mar. Sci. Eng. (Xt1, . . . , Xtn) is a Gaussian random vector for all n ∈N. It occurs that a stationary Gaussian process is strictly stationarity. X is strictly stationary if It occurs that a stationary Gaussian process is strictly stationarity. X is strictly stationary if (Xt1, . . . , Xtn) and (Xt1+k, . . . , Xtn+k) are equally distributed for all n ∈N and k, t1, . . . , tn ∈Z. Consequently, given a stationary
process X, it is Gaussian if are equally distributed for all n ∈N and k, t1, . . . , tn ∈Z. Consequently, given a stationary
process X, it is Gaussian if (Xt, . . . , Xt) is a Gaussian random vector for all t ∈N. (1) (1) information is presented in Table A2, also in Appendix A, under the columns designated
with the name length. 2022, 10, 1303 time
elevation
2022−08−05 17:00:22
2022−08−06 07:28:24
−0.4
0.0
0.2
0.4 time
elevation
2022−08−05 17:00:22
2022−08−06 07:28:24
−0.4
0.0
0.2
0.4
time
elevation
2022−08−05 17:00:22
2022−08−05 19:29:58
−0.3
−0.1
0.1
0.3
Figure 2. Left panel: representation of the 105 first recordings, including unobserved (missing) data,
of the time series associated to buoy 433. The nine observed voids represent unobserved data. Right
panel: representation of the first segment of the time series in the left panel. Sea surface elevation in
meters and time in seconds GMT with the format year-month-day and hours:minutes:seconds. time
elevation
2022−08−05 17:00:22
2022−08−05 19:29:58
−0.3
−0.1
0.1
0.3 elevation Figure 2. Left panel: representation of the 105 first recordings, including unobserved (missing) data,
of the time series associated to buoy 433. The nine observed voids represent unobserved data. Right
panel: representation of the first segment of the time series in the left panel. Sea surface elevation in
meters and time in seconds GMT with the format year-month-day and hours:minutes:seconds. Figure 2. Left panel: representation of the 105 first recordings, including unobserved (missing) data,
of the time series associated to buoy 433. The nine observed voids represent unobserved data. Right
panel: representation of the first segment of the time series in the left panel. Sea surface elevation in
meters and time in seconds GMT with the format year-month-day and hours:minutes:seconds. 3. Methodology 3. Methodology Given Xt a real valued random variable for each t ∈Z, Given Xt a real valued random variable for each t ∈Z, Given Xt a real valued random variable for each t ∈Z, X := {Xt}t∈Z is a stochastic process [38]. Most common hypotheses on stochastic processes are those of
stationarity [39] and Gaussianity [40]. X is stationary if is a stochastic process [38]. Most common hypotheses on stochastic processes are those of
stationarity [39] and Gaussianity [40]. X is stationary if •
E[Xt] = E[Xt+k] for all k, t ∈Z, where E denotes the expectation function,
•
Cov(Xt, Xk) = Cov(Xt−k, X0) for all k, t ∈Z, where Cov denotes the covariance
function and •
E[Xt] = E[Xt+k] for all k, t ∈Z, where E denotes the expectation function,
•
Cov(Xt, Xk) = Cov(Xt−k, X0) for all k, t ∈Z, where Cov denotes the covariance
function and [
t]
[
t+k]
,
,
p
,
•
Cov(Xt, Xk) = Cov(Xt−k, X0) for all k, t ∈Z, where Cov denotes the covariance
function and •
Var[Xt] < ∞for all t ∈Z, where Var denotes the variance. •
Var[Xt] < ∞for all t ∈Z, where Var denotes the variance. X is Gaussian if •
Var[Xt] < ∞for all t ∈Z, where Var denotes the variance. X is Gaussian if •
Var[Xt] < ∞for all t ∈Z, where Var denotes the variance. X is Gaussian if [
t] <
∈
,
X is Gaussian if 3.1. Tests for Stationarity This manuscript is about testing the Guassianity of stocastic processes. Typically, those
tests assume that the process is stationary. Thus, this assumption has to be previously
checked. For that, the most common tests in the literature are 1. Ljung-Box test [41],
2
Augmented Dickey Fuller test [42] 2. Augmented Dickey-Fuller test [42], 2. Augmented Dickey-Fuller test [42], 3. Phillips-Perron test [43] and
4. kpps test [44]. 3. Phillips-Perron test [43] and 4. kpps test [44]. For the first three tests, the tests can be simplified as contrasting the null hypothesis For the first three tests, the tests can be simplified as contrasting the null hypothesis H0,1 : X is non stationary
(2) H0,1 : X is non stationary
(2) (2) against the alternative Ha,1 : X is stationary J. Mar. Sci. Eng. 2022, 10, 1303 6 of 19 while the kpps test results in the null hypothesis Ha,2 : X is non stationary. The hypotheses are tested in different ways. For instance, Ljung-Box test makes use of
the autocorrelation function, which, at lag k for a stationary process is Cov(Xt, Xt+k)
Var(Xt)
. Cov(Xt, Xt+k)
Var(Xt)
. This is observable from its statistic: n(n + 2)
h
∑
k=1
ˆρ2
k
n −k, where ˆρk denotes the sample autocorrelation at lag k and n the sample size. Note that it
depends on a constant h. where ˆρk denotes the sample autocorrelation at lag k and n the sample size. Note that it
depends on a constant h. 3.2. Tests for Gaussianity Most tests for Gaussianity of stochastic processes assume the process is stationary
and test whether a finite marginal distribution of the process is Gaussian, generally, the
one-dimensional marginal. That is, instead of testing whether (1) is satisfied, these tests
contrast the null hypothesis H0,3 : Xt is a Gaussian random variable
(4) (4) H0,3 : Xt is a Gaussian random variable against the alternative while the kpps test results in the null hypothesis while the kpps test results in the null hypothesis H0,2 : X is stationary
(3) (3) H0,2 : X is stationary against the alternative Ha,2 : X is non stationary. Ha,2 : X is non stationary. Ha,3 : Xt is not a Gaussian random variable by checking whether Xt is a Gaussian random variable. Let us reflect that, because of the
stationarity, the distribution of Xt is the same for all t ∈Z; that is, it is independent of t. by checking whether Xt is a Gaussian random variable. Let us reflect that, because of the
stationarity, the distribution of Xt is the same for all t ∈Z; that is, it is independent of t. Common tests to check the Gaussianity of a real valued random variable require a
sample of independent and identically distributed random variables [45]. As this work
deals with stochastic processes, the independence assumption is not verified. However,
there are also many tests for this situation. Here, it is made use of the Epps test [35], which
checks that the characteristic function of the one-dimensional distribution of the process is
that of a Gaussian distribution, and of the Lobato and Velasco test [36], which checks that
the third and fourth order moments of the one-dimensional distribution of the process are
those of a Gaussian distribution. If the null hypothesis H0,3 is rejected, with the above mentioned tests, the null hypothesis (5) is rejected against the alternative Ha,4 : X is not a Gaussian process. However, it may occur that Xt is a Gaussian random variable Xt is a Gaussian random variable while while X is not a Gaussian process. X is not a Gaussian process. J. Mar. Sci. Eng. 2022, 10, 1303 7 of 19 7 of 19 The above mentioned tests are at nominal level again this type of alternatives. For
this, it is used here the random projection test [34], which test the Gaussianity of the whole
distribution of the process and not just of a finite dimensional marginal. For elaboration on
it, see Section 3.2. 4. Results of the Analysis This section analyzes whether each of the 59 datasets provided in Section 2, one per
buoy, is drawn from a Gaussian process as the length of the time series increases. For that,
the tests described in Section 3.2 are used here. As commented in Section 2, our datasets
have been restricted to 5 × 105 observations that include missing data; with the amount
of non-missing observations being recorded in Table A2 of Appendix A under the label
studied. The length of the time series along which this longitudinal study is performed,
makes use of the following values Length ∈{103, 104, 2 × 104, 4 × 104, 6 × 104, 8 × 104, 105}
(6) (6) and is computed by selecting that amount of non-missing observations from the buoys that
have them. To relate the above length quantities to time, it is important to take into account that
the time in seconds UTC (Coordinated universal time) associated to an observation t ∈N is
computed as Tt := T0 + t −1
r
−d where T0 is the time at which the recording starts, r is the sample rate and d is the filter
delay. r takes value 1.28 and d 133.3 but for buoys in where T0 is the time at which the recording starts, r is the sample rate and d is the filter
delay. r takes value 1.28 and d 133.3 but for buoys in B := {132, 142, 171, 194, 204 and 244} B := {132, 142, 171, 194, 204 and 244} that r takes value 2.56 and d value 130. Thus, the amount of recorded time, Tt −T0, for
t = 103 observations corresponds to 647.1688 s in UTC time in general and to 260.2344 for
the buoys in B. This results in GMT time in 10.79 min and 4.34 min, respectively. Table 2
displays these results, rounded to two decimal values, for each t equal to the length in (6). The table also includes the translation of seconds UTC into GMT time. Thus, it is observable
from the table that the analysis we pursue here is of time series that have been recorded
for a period that varies between 4 min and 21 h. Random Projection Test 2022, 10, 1303 8 of 19 8 of 19 result in projected processes different from X while providing an effective method. result in projected processes different from X while providing an effective method. 3.3. False Discovery Rate When multiple tests are performed, the multiplicity has to be taken into account. For
that it is used here the false discovery rate [48]. The false discovery rate aims at controlling
the expected proportion of falsely rejected hypothesis. It was first introduced in [49] to
take into account the multiplicity of independent tests. In [48] it was established that the
definition in [49] remains valid for certain types of dependency. However, for general
dependent cases [48] has to be applied. Random Projection Test The random projection test was introduced in [34] as a tool to test the Gaussianity
of stationary processes that is able to reject the null hypothesis of Gaussianity (5) against
alternatives with Gaussian finite-dimensional marginals. The procedure is based on a result
in [46] that implies that if ⟨{Xj}j≤t, d⟩, ⟨{Xj}j≤t, d⟩, p
⟨{Xj}j≤t, d⟩, with d drawn from a Dirichlet distribution [47], is Gaussian, then with d drawn from a Dirichlet distribution [47], is Gaussian, then {Xj}j≤t {Xj}j≤t is Gaussian. Note that due to the stationarity assumption, the Gaussianity of {Xj}j≤t is
equivalent to (1). In what follows, the procedure is explained in detail. Let λ1, λ2 > 0 be two parameters. Making use of the following stick-breaking procedure, a Dirichlet
distribution is considered: 1. Let 1. Let β(λ1, λ2) p
1
2
2. Let d0 be drawn from the distribution β(λ1, λ2). Note that d0 ∈[0, 1]. 3. For any k ∈N, the natural numbers, let dk be the result of multiplying 3. For any k ∈N, the natural numbers, let dk be the result of multiplying 3. For any k ∈N, the natural numbers, let dk be the result of multiplying 1 −
k−1
∑
i=0
di and an element drawn independently from he distribution β(λ1, λ2). Note that and an element drawn independently from he distribution β(λ1, λ2). Note that dk ∈[0, 1 −
k−1
∑
i=0
di]. dk ∈[0, 1 −
k−1
∑
i=0
di]. Let X be a stationary process. The associated projected process based on {dk}k∈N is Let X be a stationary process. The associated projected process based on {dk}k∈N is Y := {Yt}t∈Z with Yt :=
∞
∑
i=0
diXt−i. Then, making use of this randomly projected process, it suffices to apply to it a test for
the null hypothesis of Gaussianity (4). yp
y
The selection of the parameters λ1, λ2 is important. It is explained in [34] that values
ch as The selection of the parameters λ1, λ2 is important. It is explained in [34] that values
such as λ1 = 100 and λ2 = 1 result in an projected process Y similar to X. However, values such as result in an projected process Y similar to X. However, values such as λ1 = 2 and λ2 = 7 λ1 = 2 and λ2 = 7 J. Mar. Sci. Eng. 4. Results of the Analysis In fact, in general, for a length of 103 the
periods are close to 11 min while the are just abobe 2 h for a length of 104 and of more
than 4 h for a length of 2 × 104. Let us select, for instance, a time period of 2.13 h in general and of 1.05 h for the
buoys in B. Then, for each of the buoys (but for buoys 188, 189, 202 and 204), the first
non-missing observations corresponding to those time periods would be selected. Buoys
188, 189, 202 and 204 are not studied for these time periods because, as reported in Table A2
under the label studied, their non-missing observations are less than 104. Thus, 55 out of the
59 buoys are studied in this scenario. This is not the case, however, when we study buoys
recorded for 4.34 min as the time series duration associated to all the buoys is larger than
that time period. p
First, despite the time series associated to each buoy consists of non iid (independent
identically distributed) drawn observations, in Figure 3 it is plotted the histogram and
kernel density estimated curve associated to each of the 55 time series for a time period
of 2.13 h in general and of 1.05 h for the buoys in B. For each histogram 70 cells have
been used; but for buoy 241 that is plotted based on 40 cells. A Gaussian kernel is used J. Mar. Sci. Eng. 2022, 10, 1303 9 of 19 9 of 19 for the density estimations with the bandwidth resulting of applying least squares cross
validation [50,51]. The obtained bandwidths are reported in Table A3, in Appendix A. These plots are done to display descriptive information on the datasets. Note that the null
hypothesis of Gaussianity in (5) cannot be rejected by just inspecting the plots. This is
due to the fact that when testing the null hypothesis of Gaussianity it is assumed X is a
Gaussian process and the objective is to reject such assumption by the evidence provided
by the data. This is done at certain significance level. Here, it is used 0.05. By inspection of
the plots in Figure 3, one cannot guarantee whether there is enough evidence for such a
rejection as it cannot be quantified. Table 2. Time period in UTC and GMT associated to each length in (6). 4. Results of the Analysis The times are divided in
those for the 6 buoys in B and the rest of 53 buoys, which are labelled by general. Length
UTC
GMT
General
B
General
B
103
647.17 s
260.23 s
10.79 min
4.34 min
2.5 × 103
1819.04 s
846.1719 s
30.32 min
14.10 min
104
7678.42 s
3775.86 s
2.13 h
1.05 h
2 × 104
15,490.92 s
7682.11 s
4.30 h
2.13 h
4 × 104
31,115.92 s
15,494.61 s
8.64 h
4.30 h
6 × 104
46,740.92 s
23,307.11 s
12.98 h
6.47 h
8 × 104
62,365.92 s
31,119.61 s
17.32 h
8.64 h
105
77,990.92 s
38,932.11 s
21.66 h
10.81 h
433
density
−0.3
0.0
0.2
0
3
430
−0.6
0.0
0.4
0.0
2.5
244
−3
−1
1
3
0.0
243
−0.4
0.0
0.4
0.0
241
−0.15
0.00
0.15
0
8
240
density
−0.3
0.0
0.2
0
4
239
−3
−1
1
0.0
230
−0.3
−0.1
0.1
0
4
224
−0.5
0.5
0.0
222
−1.5
0.0
1.0
0.0
1.0
220
density
−1.5
0.0
1.5
0.0
217
−1.0
0.0
0.0
215
−1.0
0.0
1.0
0.0
1.5
214
−0.2
0.2
0
3
213
−1.0
0.0
1.0
0.0
1.4
209
density
−0.5
0.5
0.0
203
−1.0
0.0
1.0
0.0
201
−0.5
0.5
0.0
198
−1.0
0.0
1.0
0.0
1.2
197
−0.5
0.5
0.0
196
density
−0.5
0.5
0.0
1.5
194
−3
−1
1
3
0.0
0.6
192
−0.6
0.0
0.4
0.0
2.5
191
−0.5
0.5
0.0
185
−1.0
0.0
1.0
0.0
1.2
181
density
−0.6
−0.2
0.2
0.0
3.0
171
−3
−1
1
3
0.0
0.5
168
−1.0
0.0
1.0
0.0
1.2
166
−1.5
0.0
1.0
0.0
162
−1.5
0.0
1.0
0.0
Figure 3. Cont. Table 2. Time period in UTC and GMT associated to each length in (6). The times are divided in
those for the 6 buoys in B and the rest of 53 buoys, which are labelled by general. 4. Results of the Analysis 150
−0.6
0.0
0.4
0.0
2.5 154
−0.5
0.5
0.0 142
−2
0
1
2
3
0.0
0.5 139
−2
−1
0
1
2
0.0
0.8 132
−2
0
1
2
3
0.0 121
−3
−1
1
3
0.0
0.5 134
density
−0.6
0.0
0.4
0.0
2.5 106
−3
−1
1
2
0.0
0.6 100
−0.5
0.5
0.0 076
−1.5
0.0
1.0
0.0
1.0
029
elevation
−3
−1
1
2
0.0 076
−1.5
0.0
1.0
0.0
1.0
029 094
−1.0
0.0
1.0
0.0
1.2
045
elevation
−0.6
0.0
0.6
0.0
2.0 094
−1.0
0.0
1.0
0.0
1.2 092
−1.0
0.0
1.0
0.0
036
1 0
0 0
1 0
0.0
1.4 092
−1.0
0.0
1.0
0.0 098
density
−1.5
0.0
1.5
0.0
067
elevation
density
−1.5
0.0
1.0
0.0
0.8 045
elevation
−0.6
0.0
0.6
0.0
2.0 036
elevation
−1.0
0.0
1.0
0.0
1.4 Figure 3. Histogram, based on 70 cells (but for buoy 241 on 40 cells), and kernel density estimated
curve associated to each of the 55 buoys (time series) analyzed for a time period of 2.13 h in general
and of 1.05 h for the buoys in B. As the tests for testing the null hypothesis of Gaussianity reported in Section 3.2
require the stationarity assumption for the process, making use of the tests provided in
Section 3.1, it is first checked whether each of the datasets, when making used of each of
the time periods in Table 2, is drawn from a stationary process. The results obtained from
checking the stationarity are displayed in Table 3. Only one result is provided by test and
time period because the maximum of the obtained p-values is reported for the Augmented
Dickey-Fuller, Phillips-Perron and Ljung-Box tests while the minimum is reported for the
kpps test. Note that in the first three tests the null hypothesis of non-stationarity is tested,
as in (2), and in the fourth it is the null hypothesis of stationarity, as in (3). Thus, p-values
smaller than 0.01 are obtained for the Augmented Dickey-Fuller test and the Phillips-Perron
test and larger than 0.1 for the kpps test. g
pp
p-Values that are close to zero are obtained for the Ljung-Box test, but for time periods
of 17.32 h and 21.66 h in general, and of 8.64 h and 10.81 h for buoys in B. 4. Results of the Analysis 433
density
−0.3
0.0
0.2
0
3 241
−0.15
0.00
0.15
0
8 222
−1.5
0.0
1.0
0.0
1.0 224
−0.5
0.5
0.0 240
density
−0.3
0.0
0.2
0
4 215
−1.0
0.0
1.0
0.0
1.5 217
−1.0
0.0
0.0 213
−1.0
0.0
1.0
0.0
1.4 214
−0.2
0.2
0
3 220
density
−1.5
0.0
1.5
0.0 197
−0.5
0.5
0.0 198
−1.0
0.0
1.0
0.0
1.2 209
density
−0.5
0.5
0.0 201
−0.5
0.5
0.0 194
−3
−1
1
3
0.0
0.6
192
−0.6
0.0
0.4
0.0
2.5 192
−0.6
0.0
0.4
0.0
2.5 191
−0.5
0.5
0.0 196
density
−0.5
0.5
0.0
1.5 185
−1.0
0.0
1.0
0.0
1.2 166
−1.5
0.0
1.0
0.0
162
−1.5
0.0
1.0
0.0 Figure 3. Cont. Figure 3. Cont. J. Mar. Sci. Eng. 2022, 10, 1303 10 of 19 10 of 19 160
density
−0.6
0.0
0.4
0.0
158
−0.2
0.0
0.2
0
4
157
−1.5
0.0
1.0
0.0
154
−0.5
0.5
0.0
150
−0.6
0.0
0.4
0.0
2.5
147
density
−0.6
0.0
0.4
0.0
3.0
144
−0.5
0.0
0.5
0.0
2.0
143
−0.3
0.0
0.3
0
3
142
−2
0
1
2
3
0.0
0.5
139
−2
−1
0
1
2
0.0
0.8
134
density
−0.6
0.0
0.4
0.0
2.5
132
−2
0
1
2
3
0.0
121
−3
−1
1
3
0.0
0.5
106
−3
−1
1
2
0.0
0.6
100
−0.5
0.5
0.0
098
density
−1.5
0.0
1.5
0.0
094
−1.0
0.0
1.0
0.0
1.2
092
−1.0
0.0
1.0
0.0
076
−1.5
0.0
1.0
0.0
1.0
071
−1.5
0.0
1.0
0.0
067
elevation
density
−1.5
0.0
1.0
0.0
0.8
045
elevation
−0.6
0.0
0.6
0.0
2.0
036
elevation
−1.0
0.0
1.0
0.0
1.4
029
elevation
−3
−1
1
2
0.0
028
elevation
−0.5
0.5
0.0
Figure 3. Histogram, based on 70 cells (but for buoy 241 on 40 cells), and kernel density estimated
curve associated to each of the 55 buoys (time series) analyzed for a time period of 2.13 h in general
and of 1.05 h for the buoys in B. 4. Results of the Analysis For a time period
of 17.32 h in general, and of 8.64 h for buoys in B, the minimum p-value is 0.18 in the
Ljung-Box test and is obtained for buoy 241. The other p-values of that test for that time
period are technically zero. It is also for buoy 241 that the Ljung-Box test gives a p-value
of 0.36 when the time period is of 21.66 h in general, and of 10.81 h for buoys in B. For
that time period and test, the p-values associated to the rest of buoys are smaller than 0.01. When considering low time periods, the ultimate case being 4.34 min, all the tests provides
the aimed result. Thus, it can be assumed (with the possible exception of buoy 241 for time
periods of 17.32 h and 21.66 h in general and of 8.64 h and 10.81 h for buoys in B) that the
studied datasets are drawn from stationary processes, and check their Gaussianity under
the mentioned assumption. J. Mar. Sci. Eng. 2022, 10, 1303 11 of 19 11 of 19 Table 3. Summary of the obtained p-values, for different time periods, for each of the 59 studied
datasets under four different stationarity tests: Augmented Dickey-Fuller (first column), Phillips-Perron
(second column), Ljung-Box test (third column) and kpps (fourth column). The null hypothesis is of
stationarity for the kpps test and of non-stationarity for the other three. For each of the lengths, the
minimum p-value value over the studied buoys is displayed for the kpps test and the maximum in the
other three cases. Time Period
Tests
General
B
Augmented Dickey-Fuller
Phillips-Perron
Ljung-Box
kpps
10.79 min
4.34 min
<0.01
<0.01
8.66 × 10−8
>0.1
30.32 min
14.10 min
<0.01
<0.01
6.09 × 10−10
>0.1
2.13 h
1.05 h
<0.01
<0.01
1.48 × 10−6
>0.1
4.30 h
2.13 h
<0.01
<0.01
0
>0.1
8.64 h
4.30 h
<0.01
<0.01
2.60 × 10−7
>0.1
12.98 h
6.47 h
<0.01
<0.01
0
>0.1
17.32 h
8.64 h
<0.01
<0.01
0.18
>0.1
21.66 h
10.81 h
<0.01
<0.01
0.36
>0.1 In order to study the Gaussianity of the datasets under study, it is first analyzed the
one-dimensional marginal distribution of the process. This is because a rejection of the
null hypothesis (4) implies the sought rejection of the whole distribution of the process,
in (5). 4. Results of the Analysis For analyzing the one-dimensional marginal distribution, it is made use of the Epps
and Lobato and Velasco tests commented in Section 3. The results are displayed in Table 4
for the particular case of a time period of 2.13 h in general and of 1.05 h for the buoys
in B. There, for each dataset, associated to a buoy (columns 1 and 5), it can be observed
the p-values resulting from applying the Epps test (columns 2 and 6) and the Lobato and
Velasco test (columns 3 and 7). As multiplicity has to be taken into account, columns 4
and 8 display the FDR values. It can be observed from the table that 20 of the 55 FDR
values are smaller than 0.05. They have been highlighted in bold. If the less conservative
FDR introduced in [49] had been used, the number of rejections would have increased. If
multiplicity had not been taken into account at all and the null hypothesis (4) were rejected
when the minimum of the two p-values was smaller than 0.05, the number of rejections
would have increased to 22. Table 4. p-Values resulting from applying the Epps test (columns 2 and 6) and the Lobato and Velasco
test (columns 3 and 7) per dataset associated to each of the 55 buoys (columns 1 and 5) studied under
a time period of 2.13 h in general and of 1.05 h for the buoys in B. FDR (columns 4 and 8) combination,
for dependent p-values, of the two p-values per buoy, with the ones smaller than 0.05 highlighted
in bold. Buoy
Epps
L.-V. FDR
Buoy
Epps
L.-V. FDR
028
0.91
0.05
0.09
171
0.66
7.14 × 10−11
1.43 × 10−10
029
0.42
0.61
0.61
181
1.61 × 10−3
1.54 × 10−4
3.07 × 10−4
036
0.15
0.14
0.15
185
0.91
0.6
0.91
045
0.97
0.28
0.56
191
0.52
0.31
0.52
067
0.28
0.15
0.28
192
0.88
0.07
0.14
071
0.47
0.61
0.61
194
8.07 × 10−3
2.76 × 10−9
5.53 × 10−9
076
0.20
0.24
0.24
196
0.07
0.35
0.15
092
0.09
0.01
0.03
197
0.81
0.16
0.33
094
0.87
0.27
0.54
198
0.49
1.58 × 10−4
3.17 × 10−4
098
0.39
0.79
0.77
201
0.01
0.31
0.02 12 of 19 12 of 19 J. Mar. Sci. Eng. 2022, 10, 1303 Table 4. Cont. Buoy
Epps
L.-V. FDR
Buoy
Epps
L.-V. 4. Results of the Analysis FDR
100
0.58
0.99
0.99
203
0.93
0.74
0.93
106
3.29 × 10−4
0.02
6.58 × 10−4
209
0.73
0.01
0.01
121
0.41
1.43 × 10−6
2.86 × 10−6
213
0.43
0.42
0.43
132
0.08
3.08 ×
10−11
6.16 ×
10−11
214
0.04
6.35 × 10−6
1.27 × 10−5
134
0.76
0.76
0.76
215
0.81
0.04
0.09
139
0.82
0.22
0.43
217
0.03
0.00
0.01
142
3.56 × 10−4
1.59 × 10−8
3.18 × 10−8
220
0.56
0.77
0.77
143
0.02
1.49 × 10−4
2.98 × 10−4
222
0.86
0.60
0.86
144
0.01
5.51 × 10−4
1.10 × 10−3
224
0.01
3.13 × 10−9
6.27 × 10−9
147
0.48
0.36
0.48
230
0.66
0.04
0.09
150
0.33
0.47
0.47
239
0.22
0.77
0.44
154
0.63
0.06
0.11
240
0.74
0.64
0.74
157
0.16
0.45
0.33
241
0.10
0.20
0.19
158
0.60
0.67
0.67
243
0.06
0.01
0.01
160
0.18
0.02
0.04
244
0.51
0.02
0.04
162
0.92
0.20
0.40
430
0.17
0.32
0.32
166
0.68
6.87 × 10−4
1.37 × 10−3
433
0.19
0.08
0.15
168
0.72
0.96
0.96 To better illustrate the findings, the results of Table 4 are summarized in the left plot of
Figure 4. The x-axis represents the buoy’s identification number while the y-axis displays
the obtained FDR for dependent tests. A grey line at y = 0.05 is drawn to show what buoys
have a FDR above or below that value, which result in a rejection of the null hypothesis (4). It can observe that there are three FDR that are just above 0.05. They correspond to buoys
028, 215 and 230. 100
200
300
400
0.0
0.2
0.4
0.6
0.8
1.0
buoy identification number
FDR: no projection
100
200
300
400
0.2
0.4
0.6
0.8
1.0
buoy identification number
FDR: random projection
Figure 4. Left panel: FDR values corresponding to the studied buoys in Table 4. Right panel: FDR
values corresponding to the buoys studied in Table 5. The line y = 0.05 is displayed in both panels in
color grey. 100
200
300
400
0.0
0.2
0.4
0.6
0.8
1.0
buoy identification number
FDR: no projection 100
200
300
400
0.2
0.4
0.6
0.8
1.0
buoy identification number
FDR: random projection buoy identification number buoy identification number Figure 4. Left panel: FDR values corresponding to the studied buoys in Table 4. Right panel: FDR
values corresponding to the buoys studied in Table 5. 4. Results of the Analysis The line y = 0.05 is displayed in both panels in
color grey. In what follows it is pursued a further study in the 35 buoys for which there is yet no
evidence to reject the null hypothesis of Gaussianity, displayed in (5). This further study
consists in applying the random projection test based on the Epps and Lobato and Velasco
tests with parameters (100,1) and (2,7). The results of applying the random projection test are
reported in Table 5. There it can be observed that the random projection test is able to reject the
null hypothesis of Gaussianity in 2 out of the 35 buoys, which results in a total of 22 rejections
out of 55 (the 40%). The FDR values that result in a rejection are highlighted in bold. J. Mar. Sci. Eng. 2022, 10, 1303 13 of 19 13 of 19 Table 5. FDR values (column 6) resulting of applying the random projection test for each buoy
(column 1) with a FDR adjusted p-value larger than 0.05 in Table 4 (time period of 2.13 h in general
and of 1.05 h for the buoys in B). The parameters and the one-dimensional tests used in performing
the random projection test are included for each buoy. Buoy
Epps
(100,1)
Epps
(2,7)
L.-V. (100,1)
L.-V. The results in Table 5 have been summarized in the right plot of Figure 4. There, the
FDR values larger and smaller than 0.05 can be clearly observed; and that there is a p-value
just above 0.05, the one corresponding to buoy 230. 4. Results of the Analysis (2,7)
FDR
028
0.89
0.62
0.04
0.01
0.04
029
0.42
0.46
0.61
0.65
0.65
036
0.20
0.26
0.16
0.26
0.26
045
0.98
0.98
0.27
0.26
0.81
067
0.34
0.26
0.15
0.16
0.34
071
0.46
0.53
0.62
0.78
0.78
076
0.21
0.73
0.24
0.62
0.73
094
0.87
0.81
0.27
0.76
0.87
098
0.39
0.56
0.79
0.85
0.85
100
0.62
0.84
1.00
1.00
1.00
134
0.72
0.24
0.76
0.32
0.76
139
0.80
0.56
0.24
0.39
0.80
147
0.54
0.75
0.36
0.55
0.75
150
0.19
0.53
0.48
0.61
0.61
154
0.60
0.50
0.07
0.29
0.30
157
0.19
0.16
0.43
0.52
0.52
158
0.59
0.51
0.69
0.76
0.76
162
0.91
0.98
0.19
0.24
0.72
168
0.73
0.76
0.96
0.87
0.96
185
0.91
0.77
0.60
0.60
0.91
191
0.50
0.47
0.33
0.63
0.63
192
0.69
0.09
0.08
0.08
0.18
196
0.10
0.32
0.36
0.45
0.39
197
0.58
0.07
0.14
0.01
0.04
203
0.93
0.95
0.74
0.72
0.95
213
0.43
0.65
0.42
0.87
0.87
215
0.67
0.50
0.05
0.11
0.19
220
0.60
0.88
0.78
0.35
0.88
222
0.84
0.62
0.59
0.64
0.84
230
0.79
0.83
0.02
0.08
0.08
239
0.22
0.29
0.77
0.79
0.79
240
0.78
0.93
0.76
0.96
0.96
241
0.11
0.18
0.26
0.11
0.26
430
0.21
0.41
0.34
0.63
0.63
433
0.31
0.72
0.14
0.65
0.58
The results in Table 5 have been summarized in the right plot of Figure 4 There the The results in Table 5 have been summarized in the right plot of Figure 4. There, the
FDR values larger and smaller than 0.05 can be clearly observed; and that there is a p-value
just above 0.05, the one corresponding to buoy 230. J. Mar. Sci. Eng. 2022, 10, 1303 14 of 19 This procedure that we have exemplified through the use of a time period of 2.13 h
in general and of 1.05 h for the buoys in B, has been performed for all the time periods
displayed in Table 2. This has resulted in a rejection rate displayed in Table 6 with the
label with projection. Thus, if it is used a time period of 4.34 min for the buoys in B and
of 10.79 min in general, the Gaussianity of 22.03% of the processes is rejected. This rate
increases to 58.49% and 82.35% for time periods of 2.13 h and 4.30 h in general and of 1.05 h
and 2.13 h for buoys in B, respectively. 4. Results of the Analysis It reaches 96.08% for a time period of 21.66 h in
general and 10.81 h for buoys in B. Table 6. Rejection rates along different length periods when no projections are used (first row), when
the proposed projection procedure is used (second row) and when no multiplicity is taken into
account (third row), i.e., the minimum of the p-values is used for the rejection. m stands for minutes
and h for hours. Table 6. Rejection rates along different length periods when no projections are used (first row), when
the proposed projection procedure is used (second row) and when no multiplicity is taken into
account (third row), i.e., the minimum of the p-values is used for the rejection. m stands for minutes
and h for hours. General
10.79 m
30.32 m
2.13 h
4.30 h
8.64 h
12.98 h
17.32 h
21.66 h
Time Period
B
4.34 m
14.10 m
1.05 h
2.13 h
4.30 h
6.47 h
8.64 h
10.81 h
no projection
15.25
19.30
36.36
58.49
78.43
80.39
90.20
96.08
with projection
22.03
22.81
40.00
58.49
78.43
82.35
92.16
96.08
minimum
30.51
24.56
43.64
66.04
80.39
82.35
92.16
96.08 If the random projection was not applied, that is, if only the Epps and Lovato and
Velasco tests were applied, the rejection rates would have been just below for time periods
of 10.79 min, 30.32 min, 2.13 h, 12.98 h and 17.32 h in general and of 4.34 min, 14.10 min,
1.05 h, 6.47 h and 8.64 h for buoys in B. The corresponding rejection rates are in Table 6
with the label no projection. Meanwhile, if the multiplicity would have not been taken into
account altogether and the minimum p-value would have been selected, the rejection rates
would also have been similar, just a bit over as the time period decreases, separately in
general and in B. These values are displayed in Table 6 with the label minimum. To better
illustrate all these rejections, we have plotted them in Figure 5. There, the rejections rates of
the proposed procedure are plotted with a back plus symbol, those obtained without using
the random procedure are plotted with green triangles and the ones where the minimum
of the p-values is computed are plotted with blue circles. It can observed from the figure
that the three values get closer as the time period increases. 4. Results of the Analysis 0
5
10
15
20
20
40
60
80
time period (hours)
rejection rate
Figure 5. Display of the rejection rates in Table 6: the values labelled by no projection are in green
triangles, those by with projection in back plus symbols and those by minimum with blue circles. The
time period in the x-axis corresponds to the buoys in general. For the buoys in B, it is 4.34 min,
14.10 min, 1.05 h, 2.13 h, 4.30 h, 6.47 h, 8.64 h and 10.81 h. 0
5
10
15
20
20
40
60
80
time period (hours)
rejection rate Figure 5. Display of the rejection rates in Table 6: the values labelled by no projection are in green
triangles, those by with projection in back plus symbols and those by minimum with blue circles. The
time period in the x-axis corresponds to the buoys in general. For the buoys in B, it is 4.34 min,
14.10 min, 1.05 h, 2.13 h, 4.30 h, 6.47 h, 8.64 h and 10.81 h. J. Mar. Sci. Eng. 2022, 10, 1303 15 of 19 15 of 19 5. Conclusions For the analysis, the sea surface elevations provided by the measurements of the
buoys along the US coast have been analyzed along different time periods. It has been
obtained in the analysis section that the rejection rates increase when the time peri-
ods increase. Thus, while less than 25% of the studied processes can be considered
as non-Gaussian when the time period consists of less than 11 min, it reaches over
the 95% of the cases when the time period is larger than 21 h in general and of 10 h
for the buoys in B. Thus, it is clear the non-Gaussianity of the sea surface elevation
and that only enough information (in terms of the time period) is need for its rejection. If we had only used non-randomized tests, as it is common in the literature, the obtained
rejection rates would have been a bit lower, with the larger differences being obtained as
the time period decreases. For the obtained results, a more complex and new goodness of
fit test has been required. This is that known as random projection test and requires of the
selection of a distribution used to draw a vector in which to project the data and a good-
ness of fit test for the one-dimensional marginal distribution of the process to apply on the
projected data. This leads to the conclusion that the sea surface elevations are generally non-
Gaussian despite that their one-dimensional distribution is Gaussian in a few of the cases. The obtained results are significant as they indicate two important aspects: For the analysis, the sea surface elevations provided by the measurements of the
buoys along the US coast have been analyzed along different time periods. It has been
obtained in the analysis section that the rejection rates increase when the time peri-
ods increase. Thus, while less than 25% of the studied processes can be considered
as non-Gaussian when the time period consists of less than 11 min, it reaches over
the 95% of the cases when the time period is larger than 21 h in general and of 10 h
for the buoys in B. Thus, it is clear the non-Gaussianity of the sea surface elevation
and that only enough information (in terms of the time period) is need for its rejection. 5. Conclusions •
The sea surface elevations are generally not Gaussian as the time period increases,
from time series recorded in just 4 to 10 min to time series recorded in 10 to 21 h. j
•
In a few of the cases the one-dimensional marginal is Gaussian for small and moderate
time periods. For instance, for time series recorded in just 4 to 10 min, in 14 to 30 min
and those recorded in 1 to 2 h. j
•
In a few of the cases the one-dimensional marginal is Gaussian for small and moderate
time periods. For instance, for time series recorded in just 4 to 10 min, in 14 to 30 min
and those recorded in 1 to 2 h. These two facts result in that sea surface elevations cannot be modeled as Gaussian
processes but in a few of the cases can be modeled as non-Gaussian processes with one-
dimensional Gaussian marginals. Note that this is not a contradiction due to, as explained
in Section 3, there a non-Gaussian processes with one-dimensional Gaussian marginals. In testing the non-Gaussianity, multiple tests are performed and consequently multi-
plicity has to be taken into account. However, if it was not to be taken into account, the
same results would have been obtained when all the buoys are studied for time periods of
6.47 h and over; with the rejection rates getting larger than those based on the FDR rate
when the length period decreases. Funding: A.N.-R. was supported by grant MTM2017-86061-C2-2-P funded by MCIN/AEI/10.13039/
501100011033 and “ERDF A way of making Europe”. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Coastal Data Information Program at https://thredds.cdip.ucsd.edu/
thredds/catalog/cdip/realtime/catalog.html (accessed on 20 August 2022). Conflicts of Interest: The author declares no conflict of interest. Conflicts of Interest: The author declares no conflict of interest. Abbreviations The following abbreviations are used in this manuscript: FDR
False discovery rate UTC
Coordinated universal time J. Mar. Sci. Eng. 2022, 10, 1303 16 of 19 16 of 19 Appendix A
Table A1. Start and end times for which the measurements have been recorded by each of the 59 buoys. These are GMT times which are reported in the format year-month-day and hours-minutes-seconds. In bold, the buoy whose measurements have been recorded for a smaller period of time. Table A1. Start and end times for which the measurements have been recorded by each of the 59 buoys. These are GMT times which are reported in the format year-month-day and hours-minutes-seconds. In bold, the buoy whose measurements have been recorded for a smaller period of time. Abbreviations Buoy
Start Time (GMT)
End Time (GMT)
Date (yyyy-mm-dd)
Time
Date (yyyy-mm-dd)
Time
028
2021-04-29
19:00:00
2022-08-20
08:59:57
029
2022-01-26
21:00:00
2022-08-20
08:59:58
036
2022-05-25
23:00:00
2022-08-20
08:59:58
045
2022-03-01
20:00:00
2022-08-20
08:59:58
067
2020-12-02
21:00:00
2022-08-20
08:59:57
071
2022-08-02
19:32:13
2022-08-20
08:59:58
076
2022-06-02
18:00:00
2022-08-20
08:59:58
092
2021-08-04
21:00:00
2022-08-20
08:59:57
094
2020-10-08
21:00:00
2022-08-20
08:59:57
098
2022-02-04
21:00:00
2022-08-20
08:59:58
100
2021-02-19
18:00:00
2022-08-20
08:59:57
106
2022-01-20
23:00:00
2022-08-20
08:59:58
121
2021-06-21
13:32:13
2022-04-10
22:59:58
132
2020-09-15
22:30:00
2022-03-01
01:22:48
134
2022-01-15
15:00:00
2022-08-20
08:59:58
139
2021-11-14
20:00:00
2022-08-20
08:59:58
142
2022-03-02
15:00:00
2022-05-20
08:22:49
143
2022-08-16
23:00:00
2022-08-20
08:59:58
144
2021-11-11
17:00:00
2022-08-20
09:29:58
147
2022-03-21
15:00:00
2022-08-20
08:59:58
150
2021-11-18
15:00:00
2022-08-20
08:59:58
154
2021-01-12
17:00:00
2022-06-27
21:59:57
157
2020-06-20
20:00:00
2022-08-20
08:59:57
158
2022-07-06
02:02:13
2022-08-20
08:59:58
160
2022-05-02
21:02:13
2022-08-20
08:59:58
162
2021-11-20
18:00:00
2022-08-20
08:59:58
166
2022-05-16
21:00:00
2022-05-18
03:29:58
168
2022-03-25
20:00:00
2022-08-20
08:59:58
171
2022-01-14
16:00:00
2022-05-12
07:52:49
181
2022-08-11
21:02:13
2022-08-20
08:59:58
185
2022-08-06
02:00:00
2022-08-20
09:29:58
188
2021-11-03
10:23:10
2022-08-20
09:20:55
189
2021-10-20
20:02:13
2022-08-20
08:59:58
191
2021-12-13
20:00:00
2022-08-20
08:59:58
192
2022-08-04
17:00:00
2022-08-20
08:59:58
194
2021-11-05
15:00:00
2022-02-06
23:52:49
196
2022-07-19
04:02:13
2022-08-20
09:29:58
197
2022-07-19
04:02:13
2022-08-20
08:59:58
198
2021-06-16
21:00:00
2022-08-20
08:59:57
201
2021-11-10
21:00:00
2022-08-20
08:59:58
202
2021-07-21
08:02:13
2022-05-04
23:59:58
203
2021-05-12
19:00:00
2022-08-20
08:59:57
204
2021-08-20
23:00:00
2022-05-10
02:52:49
209
2022-06-10
18:32:13
2022-08-20
08:59:58
213
2022-03-15
17:00:00
2022-08-20
08:59:58
214
2022-06-28
04:02:13
2022-08-20
08:59:58
215
2022-04-14
19:00:00
2022-08-20
08:59:58
217
2022-08-08
23:02:13
2022-08-20
08:59:58
220
2022-04-26
22:00:00
2022-08-20
08:59:58
222
2021-11-15
15:00:00
2022-08-20
08:59:58
224
2020-07-16
15:00:00
2022-08-20
08:59:57
230
2022-05-19
17:00:00
2022-08-20
08:59:58
239
2022-07-17
19:05:10
2022-08-20
09:03:56
240
2022-07-22
19:02:13
2022-08-20
08:59:58
241
2022-08-02
00:00:00
2022-08-20
08:29:58
243
2021-09-13
20:00:00
2022-08-20
08:59:57
244
2022-03-31
04:30:00
2022-03-31
10:12:49
430
2022-07-15
18:02:13
2022-08-20
08:59:58
433
2022-08-05
17:00:00
2022-08-20
08:59:58 J. Mar. Sci. Eng. 2022, 10, 1303 17 of 19 17 of 19 Table A2. The 59 available buoys are labelled by an identification number in descending order. The
length of the associated time series is also reported, the smallest being highlighted in bold. The studied
label represents the length of the studied time series after selecting the first 5 × 105 time points and
eliminating the unobserved, missing, values. Abbreviations Buoy
Length
Studied
Buoy
Length
Studied
028
52,817,065
412,278
185
1,583,018
216,438
029
22,722,218
423,968
188
32,062,463
2304
036
9,557,162
458,528
189
33,569,279
6912
045
18,966,698
488,480
191
27,592,874
419,360
067
69,170,857
334,112
192
1,732,778
451,754
071
1,942,272
394,016
194
20,652,288
162,047
076
8,695,466
317,984
196
3,564,288
396,288
092
42,080,425
500,000
197
3,561,984
463,136
094
75,258,025
412,448
198
47,499,433
306,294
098
21,726,890
444,704
201
31,237,802
476,960
100
60,447,913
453,920
202
31,813,631
2304
106
23,376,554
145,322
203
51,379,369
403,232
121
32,447,231
29,952
204
57,986,303
6912
132
117,484,797
14,592
209
7,808,256
426,272
134
23,966,378
474,656
213
17,432,234
446,838
139
30,800,042
347,766
214
5,884,416
301,856
142
17,403,648
10,752
215
14,109,866
481,855
143
378,026
336,554
217
1,262,592
403,232
144
31,147,946
447,008
220
12,768,938
449,142
147
16,777,898
476,960
222
30,712,490
430,880
150
30,380,714
412,448
224
84,575,400
449,312
154
58,742,953
479,264
230
10,248,362
481,568
157
87,427,752
467,574
239
3,714,125
352,544
158
5,008,896
479,264
240
3,161,088
375,584
160
12,109,824
467,744
241
2,029,994
456,224
162
30,145,706
435,488
243
37,661,353
467,744
166
140,714
126,890
244
52,992
29,952
168
16,312,490
453,920
430
3,939,840
426,272
171
26,015,999
205,088
433
1,622,186
410,144
181
940,032
493,088
Table A3. Bandwidth, per buoy, rounded to 2 decimal values used for the kernel density estimated
curves in Figure 3. buoy
028
029
036
045
067
071
076
092
094
098
100
bandwidth
0.04
0.12
0.05
0.04
0.06
0.08
0.07
0.04
0.06
0.08
0.04
buoy
106
121
132
134
139
142
143
144
147
150
154
bandwidth
0.1
0.14
0.08
0.03
0.09
0.13
0.02
0.03
0.01
0.03
0.04
buoy
157
158
160
162
166
168
171
181
185
191
192
bandwidth
0.04
0.01
0.03
0.05
0.07
0.05
0.11
0.02
0.06
0.04
0.03
buoy
194
196
197
198
201
203
209
213
214
215
217
bandwidth
0.13
0.04
0.04
0.05
0.04
0.04
0.04
0.03
0.02
0.04
0.04
buoy
220
222
224
230
239
240
241
243
244
430
433
bandwidth
0.08
0.07
0.04
0.01
0.13
0.01
0.01
0.02
0.13
0.03
0.02 Table A3. Bandwidth, per buoy, rounded to 2 decimal values used for the kernel density estimated
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Benjamini, Y.; Hochberg, Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J. R.
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J. Am. Stat. Assoc. 1970, 65, 1509–1526. [CrossRef] J. Am. Stat. Assoc. 1970, 65, 1509–1526. [CrossRef] 42. Said, S.E.; Dickey, D.A. Testing for Unit Roots in Autoregressive-Moving Average Models of Unknown Order. Biometrika 1984,
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1992 54 159 178 [C
R f] 44. Kwiatkowski, D.; Phillips, P.C.; Schmidt, P.; Shin, Y. Testing the Null Hypothesis of Stationarity Against
Root: How sure Are We that Economic Time Series Have a Unit Root? J. Econom. 1992, 54, 159–178. [C wiatkowski, D.; Phillips, P.C.; Schmidt, P.; Shin, Y. Testing the Null Hypothesis of Stationarity Against the Al 45. D’Agostino, R.B.; Stephens, M.A. Goodness-of-fit techniques. Qual. Reliab. Eng. Int. 1986, 3, 71. [CrossRef] 46. Cuesta-Albertos, J.; del Barrio, E.; Fraiman, R.; Matrán, C. The Random Projection Method in Goodness of Fit for Functional Data. Comput. Stat. Data Anal. 2007, 51, 4814–4831. [CrossRef] J. Mar. Sci. Eng. 2022, 10, 1303 19 of 19 19 of 19
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https://openalex.org/W2528510938
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https://journals.urfu.ru/index.php/r-economy/article/view/2941/2525
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English
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How to Assess Advantages of Economic-Geographical Position for Russian Regions
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R-Economy
| 2,016
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cc-by
| 12,598
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S. P. Zemtsov a), V. L. Baburin b) a) Institute for Applied Economic Research (Moscow, Russian Federation)
b) Lomonosov Moscow State University (Moscow, Russian Federation) a) Institute for Applied Economic Research (Moscow, Russian Federation)
b) Lomonosov Moscow State University (Moscow, Russian Federation) HOW TO ASSESS ADVANTAGES
OF ECONOMIC-GEOGRAPHICAL POSITION FOR RUSSIAN REGIONS?
1 The category of economic-geographical position (EGP) was formalized based on a review of the scientific
literature. The developed method of international and interregional EGP potential assessment was based
on the use of gravity models; it can further be widely used in regional studies to explore the benefits of the
spatial location of objects (countries, regions, cities, etc.). These calculations for Russia's regions showed
significant spatial differentiation. The maximum potential of interregional EGP potential have the regions
located near Moscow and St. Petersburg agglomerations, the potential decreases uniformly to the east. The
maximum international EGP potential concentrated in regions on the coast of the Black Sea, the Baltic
Sea and the Sea of Japan. The potential of the Kaliningrad region 5.6 times higher than it is for the Tyva
Republic. In addition, it was revealed a significant increase in the total EGP potential in the 2000s, and its
shift to the southern regions of the Far East due to the growth of the Asia-Pacific economies. The results were
also used to identify connections between the EGP potential and indicators of socio-economic development. It was found that favourable EGP is one of the factors for GRP growth, investment, foreign trade, migration
growth and spread of new technologies. Formalizing EGP as a category allows using it to predict the spatial
changes in the socio-economic development of Russia. Keywords: economic-geographical position, market potential, Russian regions, gravity models, regional d 385
R-Economy Vol. 2, Issue 3, 2016
1 Original Russian Text © Zemtsov S. P., Baburin V. L., published in Ekonomika regiona [Economy of Region]. — 2016. — Vol. 12,
Issue 1. — 117–138.
2 Geographical position includes physical-geographical characteristics of a region, so the EGP is often associated with agro-climatic
resources, coastal location and the availability of natural resources, which is inconsistent with the original understanding of this category.
3 In most cases, we are talking about a major transport route passing through regional territory, or proximity to major markets.
4 F. Cairncross [6] describes in detail the processes in his book "The Death of Distance."
5 Determining the EGP category we can impose an additional constraint associated with the need to use an object in space in a ratio of
only those that would have the characteristics of the system, and were similar in terms of their system characteristics. That is geographical 1 Original Russian Text © Zemtsov S. P., Baburin V. L., published in Ekonomika regiona [Economy of Region]. — 2016. — Vol. 12,
Issue 1. — 117–138.
2
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l S. P. Zemtsov, V. L. Baburin S. P. Zemtsov, V. L. Baburin doi 10.15826/recon.2016.2.3.035
UDC 332.1 h
5 Determining the EGP category we can impose an additional constraint associated with the need to use an object in space in a ratio of
only those that would have the characteristics of the system, and were similar in terms of their system characteristics. That is geographical Introduction “Economic-geographical position” (EGP) is one of the basic categories of regional studies in Russia. Moreover, we can assume that this is one of the few concepts originally appeared and developed in
Russia and it is rarely used in other countries.i Numerous recent studies of regional inequality in Russia (e.g. [1–3]) refer to significant differences
between the regions in their geographical position. The strategies of socio-economic development in
some regions have a special item about its geographical position 2. These documents mainly provide
a qualitative characteristic of a "favourable" or "unfavourable" economic-geographical position of a
region 3. However, there is still a lack of a formal model for quantitative assessment of the potential of
an EGP. Nowadays, costs of interactions between economic agents are rapidly falling with the acceleration
of transport and information technology development 4 [4]. Nevertheless, there is still a strong
differentiation in living conditions in regions and countries, and remote and underdeveloped areas are
still less attractive to migrants and investors [5]. One of the factors of spatial differentiation is an EGP. One of the main tasks of regional studies is to establish connections between different objects
in space. Therefore, the EGP concept is one of the keys to a system of regional science (economic
geography, regional and spatial economy), as it allows to explain many properties of spatial objects
and predict their development. p
p
An economic-geographical position of a region is a historically evolved, but varying set of spatial
relationships between economic agents of this region and external factors potentially influencing
regional development 5. The spatial relationship between objects, in this case, is primarily associated
with a distance between them. ph
5 Determining the EGP category we can impose an additional constraint associated with the need to use an object in space in a ratio of
only those that would have the characteristics of the system, and were similar in terms of their system characteristics. That is geographical R-Economy Vol. 2, Issue 3, 2016 385 S. P. Zemtsov, V. L. Baburin Any object as part of the space-time continuum is able to change its position, striving to achieve the
most favourable location in space 6, in other words, it is trying to reach the point, where the potential
impact of external conditions would be the most favourable for its development 7. position of economic agents, such as firms, should be considered only in relation to economic entities (companies, markets, etc.). In this
case, methodologically wrong to regard the average temperature as a component of the EGP for an enterprise, which often can be seen
in economic researches. Estimation of the EGP potential may indirectly include environmental characteristics through such economic
categories as income, expenses, damages, efficiency, etc. Introduction The actions of the
object can be modelled as a movement in phase space to a certain stable point, which is called an
attractor. In this case, a favourable EGP is an attracting set of economic agents’ positions in space,
where their relationship is optimal. Thus, a regional EGP is a dynamic category.i Changes of an object location may have a significant impact on it. Novosibirsk was founded on
the intersection between the Trans-Siberian Railway and the Ob River and became the largest city
in Siberia due to the substantial gains of the location on the route between the European part of
Russia and the Far East [7]. As a modern example, the new automobile factories in Russia are located
in the regions (Kaluga, Leningrad region) close to the largest and growing in 2000th regional consumer
markets (Moscow and St. Petersburg, respectively). Poor development of distant Russian regions, as the
Republic of Altai and Tuva Republic, is related to their unfavourable landlocked position away from the
main traffic flows and major economic centres. It is common for other large countries, e.g. in China,
economic activity is concentrated mainly in the coastal zone, where export costs are lower.i The aim of this work is to formalize and assess the benefits (potential) of the economic-geographical
position of the Russian regions.i Thus, “EGP” is a probabilistic category, and its potential benefits can be or can be not realized
depending on the regional policy, development of infrastructure and other factors. In fact, it is
important to assess what the advantages of location are in a particular region for economic agents
(firms, employees, regional economies as a whole, etc.); first, these advantages are associated with the
proximity and the availability of large markets. An empirical assessment of an EGP potential of a region should take into consideration dynamic
features, because an EGP is depending on dynamics of economic processes outside the region, e.g. changes in traffic and trade flows as a result of the new roads construction. Giving a dynamic component
to the concept will significantly expand the scope of its application. 7 This condition can be carried out directly to cities at early stages of their development, for example, Orenburg three times changed its
location [7], but moving for large areas such as countries and regions is limited. This raises the question of the category applicability to them.
Changes of EGP potential of specific localities (districts) within a region affect economic agents, which by moving transform the internal
structure of a region. That is, a region (as a socio-economic system) does not change its location and configuration, but optimizes the
internal territorial structure. For example, according to W. Christaller, regional settlement system strives for optimum hexagonal structure
[9]. g
gfi
y
6 B. Rodoman [8] proposed a "positional pressure principle", meaning the force that causes the object to move, if its position is not
optimal. R-Economy Vol. 2, Issue 3, 2016 [ ]
8 A similar definition: "EGP is a kind of geographical location, defined as a set of spatial relations of companies, localities, areas,
regions, individual countries and its groups to external sites of economic importance to them," [18]. fi
6 B. Rodoman [8] proposed a "positional pressure principle", meaning the force that causes the object to mo
optimal.h Theoretical Basis According to one of the authors of the term N. N. Baranskiy, the economic-geographical position is
"an attitude of any place, area or city to other outside lying givens, which have a particular economic
significance... It is extremely important to a country (or region, or city) to be in a short distance to the
main routes, markets and large centres (industrial, commercial, administrative, cultural)" [10, p. 129] 8. N. N. Baranskiy allocated three following EGP levels [10]: micro- (within the region, city), meso- (within
the country) and macrolocation (between countries). Initially, the concept was applied to studies of
cities’ position at the mesolevel. This work is devoted to the research of meso- and macrolocation of
the Russian regions. g
I. M. Maergoiz [11–13], Yu. G. Saushkin [14] Ya. G. Mashbits [15], Ye. Ye. Leizerovich [16, 17] and
many other Soviet and Russian scientists made a significant contribution to the concept. i
“Geographical position” as a qualitative characteristic of an object can be central, peripheral and
neighbour. [11] A central position of an object within a larger territorial system brings additional
economic and social benefits: lower transport costs, trade and migration flows concentration, and so
on. Moscow position in the centre of the transport and the settlement system of the European part of R-Economy Vol. 2, Issue 3, 2016 386 S. P. Zemtsov, V. L. Baburin Fig. 1. Scheme of the major channel bends of the Volga river with straight lines that indicate the potential interaction zones
[19] f the major channel bends of the Volga river with straight lines that indicate the potential interaction zones
[19] Russia can be a good example. Otherwise, a peripheral 9 position of economic agents, which is deep and
remote from a centre, in general carries additional costs. Russia can be a good example. Otherwise, a peripheral 9 position of economic agents, which is deep and
remote from a centre, in general carries additional costs. Central position, in this case, is rather a number of potential connections than a location in the
geographical centre of any territory. A historical development of many cities along the Volga River
was determined by their advantageous position in the bend of the river. Convex portions of any river
(Fig. 1), ceteris paribus, are more profitable for a city, because such position is able to serve more vast
territory, and therefore have a greater number of potential connections. 9 Considering the global trade flows, which are carried out mainly by sea, we should consider landlocked position as a peripheral and
seaside as central. Theoretical Basis During the period of water
transport dominance, this location was one of the cities’ competitive advantages. The rarely used, but the highly formalized method of a geographical position analysis is a technique
from the theory of central places, developed by V. Kristaller and A. Lösch (see [9, 20]), which allows to
determine a position of a city in the hierarchy of a settlement system. The neighbouring position of two territorial systems, ceteris paribus, usually acts as a favourable
factor for their development. Proximity to a large neighbour can bring benefits of cooperation,
economic transfer and new technologies diffusion, e.g. location of shopping centres in the Moscow
region near Moscow [21]. However, this proximity may inhibit a development of various sectors of
weaker neighbours. Less developed regions, in this case, can be converted into agrarian and raw
material appendages and suppliers of labour resources. A classic example is an inner periphery with low
population density between Moscow and St. Petersburg. [22] By using the concept of "neighbourhood"
[12], it is important to distinguish neighbours of first (direct), the second (neighbours of neighbours)
and subsequent orders.i “Geographical position” can be classified by functions [15]: geodesic, environmental, economic-
geographical (EGP), politico-geographical, geopolitical, etc. In this paper, we examine only an economic
component (as the first step towards an integrated estimation), which itself can be divided into market-,
transport-, industrial-, agricultural-geographical position, and so on. An EGP of a region is associated
with its proximity to markets, traffic flows, industrial centres and other facilities, which affecting or
potentially able to exert influence on it. the global trade flows, which are carried out mainly by sea, we should consider landlocked position as a peripheral and R-Economy Vol. 2, Issue 3, 2016 387 S. P. Zemtsov, V. L. Baburin “Proximity” as a category can be estimated using various distance measures [23] 10. The most simple
is a measurement of the geodesic distance in a straight line. More economically viable is to measure an
actual distance by the length of railways, motor roads, navigable rivers, etc. In addition, the distance
can be measured based on the time interval, which is widely used in isochrones maps [21] 11. Theoretical Basis Transport-geographical position (TGP) is measured in the cases, when benefits of regional
geographical position are related to a remoteness or a proximity of a region to main routes, a position
of a region in a transport system and associated costs for goods and people delivery [23]. A significant
number of works is dedicated to TGP studies of regions and cities (e.g. [17, 24]). One of the most
operational and developed methods of a TGP assessment is to measure an economic distance in terms
of transport costs (tariffs) [25–27]. Topological distance is an often-used method for assessment of an object centrality in a transport
network, where a distance is measured by a sum of the Koenig numbers from one centre to all others:
the lower the number of edges connecting the centre with others, the more neutral and the more
favourable position it has in a system. There are other applied methods of the graph theory [28, 29]. However, these estimations not always provide an information on potential economic benefits. y p
pi
Many empirical works about “EGP” in Russia are devoted to studies of cities [11, 12, 30–32], and
most of them are descriptive and are not sufficiently formalized [33–35]. In [35], a method of EGP scoring was devoted to an assessment of integration potential of the
Russian Far Eastern regions in the Asia-Pacific Region. Studied variables of the EGP index were
land areas, distances to other Russian regions and Asia-Pacific countries, coastlines and indicators
of transport infrastructure. The disadvantages of this approach are weak formalization, a subjective
selection of the indicators and weights. g
In [36, 37], a method for an EGP potential calculation, associated with a proximity to major
innovation centres, was proposed: (1) _
,
i
i
i
i
i
i
EGP
Capital
Agglom
Coast
Moscow
Fed distr
=
+
+
+
+
(1) _
,
i
i
i
i
i
i
EGP
Capital
Agglom
Coast
Moscow
Fed distr
=
+
+
+
+
(1) where i — is a region, Capital, Agglom, Coast, Moscow, Fed_distr are binary variables (0 or 1), describing
respectively special status of a region (Moscow, St. Petersburg, Moscow and Leningrad Region), presence
of an agglomeration with over 1 million residents, presence of ice-free ports, proximity to the Moscow
and presence of a Federal District capital. According to the calculations 12, the EGP potential (Fig. 12 On the map, the contours of the EGP index was offset in the northern direction, as there are no points for extrapolation. Schematic
map serves only the purposes of visualization, technical features of a software, will not reflect on calculations. 10 In the current regional studies, ‘proximity’ can be determined not only as s geographical phenomena, but also institutional,
organizational, cognitive, etc. [38]. [
]
is a line connecting the point of concurrency of any phenomenon.f g
g
[
]
11 Isochronous is a line connecting the point of concurrency of any phenomenon.f 11 Isochronous is a line connecting the point of concurrency of any phenomenon 13 The model is ‘induced’, as it allows to measure the potential of a particular region, in contrast to the model J. Stewart [51], which was
applied for mapping spatial distribution of "settlement-field potential" [53, 55]. Theoretical Basis Assessment of the potential of economic-geographical position of the Russian regions [35] An actual number of interactions (V), for example, trade or migration flows, patent citations, etc. can be used to calculate the empirical coefficients. Then, we obtain the logarithm of (1): ln
ln
ln
,
ij
i
j
i j
V
const
P
P
aR
=
+ α
+ β
-
+ ε (3) (3) ere const is a constant, and ε is a random variable, or unexplained residue. For the purposes of our work, the market potential models, estimating proximity to potential
markets, can be used. The classical model of cooperation between two regions was developed by
Charles Harris in 1954 [43]: ,
j
ij
ij
MV
V
R
=
(4) (4) where Vij is a trade turnover between regions i and j; MVj is a market volume indicator, such as gross
regional product (GRP) in a j-th region; Rij is a distance between regions. However, this form does
not allow for “multilateral resistance" [60; 67], when trade between two regions is affected by market
volumes of all other regions. where Vij is a trade turnover between regions i and j; MVj is a market volume indicator, such as gross
regional product (GRP) in a j-th region; Rij is a distance between regions. However, this form does
not allow for “multilateral resistance" [60; 67], when trade between two regions is affected by market
volumes of all other regions. g
Another model to determine the market potential of a region in Russian literature was called
"induced potential" [57] 13: ,
j
i
i
ij
MV
MP
MV
R
=
+∑
(5) (5) where MPi is a market potential of a region i, MVi is a market volume indicator. It was used to calculate the market potential of Russian regions [49], taking into account that where MPi is a market potential of a region i, MVi is a market volume indicator. d
l
l
h
k
i l f
i
i
ki
i where MPi is a market potential of a region i, MVi is a market volume indicator. i
sed to calculate the market potential of Russian regions [49], taking into account that It was used to calculate the market potential of Russian regions [49], taking into acc 2
,
3
i
ii
S
R =
×
π (6) (6) where S is an area of a region i. Theoretical Basis 2) is concentrated in the largest metropolitan
areas and on the coast. The study also confirmed the importance of the favorable EGP position for
regional innovation potential. Proximity and availability of large markets of innovative products is an
important factor for new technologies development. The disadvantages of the approach are the same
as described above for the scoring. Gravity models, which serve to analyse potential social and economic interactions, were applied
in many studies to assess the benefits of the geographical position. A prerequisite for this model usage
has come from physics law, which stats that an interaction between two objects depends on their size
and a degree of closeness [39–41]. Applications of gravity models include an assessment of market [42–50], demographic [32, 51–
57] and innovation potential [58–59], trade [60–67] and migration flows [68–73]. The model may be
described by the following relation: ,
i
j
i j
a
ij
P
P
V
R
α
β
×
=∑
(2), (2), where V is a number of potential interactions between regions i and j, P is a size of a region, for example,
gross regional product, population, number of scientists, etc., Rij is a distance between regions, α, β are
empirical coefficients, a is a coefficient of proportionality, showing a speed of interaction decrease
between regions, caused by increasing distance between them. where V is a number of potential interactions between regions i and j, P is a size of a region, for example,
gross regional product, population, number of scientists, etc., Rij is a distance between regions, α, β are
empirical coefficients, a is a coefficient of proportionality, showing a speed of interaction decrease
between regions, caused by increasing distance between them. 10 In the current regional studies, ‘proximity’ can be determined not only as s geographical phenomena, but also institutional,
organizational, cognitive, etc. [38]. 11 I
h
i
li
ti
th p i t f
f
ph 12 On the map, the contours of the EGP index was offset in the northern direction, as there are no points for extrapolation. Schematic
map serves only the purposes of visualization, technical features of a software, will not reflect on calculations. R-Economy Vol. 2, Issue 3, 2016 388 S. P. Zemtsov, V. L. Baburin Fig. 2. Assessment of the potential of economic-geographical position of the Russian regions [35] Fig. 2. R-Economy Vol. 2, Issue 3, 2016 389
R-Economy Vol. 2, Issue 3, 2016 389
R-Economy Vol. 2, Issue 3, 2016 Theoretical Basis As a variable describing the size of a market, gross regional product
(GRP) was used. Unfortunately, the result of the calculation is virtually identical to GRP because of the
large distances between the Russian regions. R-Economy Vol. 2, Issue 3, 2016 389 S. P. Zemtsov, V. L. Baburin The mentioned models can also be used to solve very specific problems. In particular, the authors
used it to assess [59] the innovation potential of the Russian regions. The number of patents per 100
thousand citizens was used as a "market" variable 14. In [62, 63], a model was derived from a type of Cobb — Douglas utility functions [74]: . ij
R
i
j
j J
MP
MV
e
-
ε
=
×
∑
(7) (7) In [45–48], it was showed based on (7) that profitability of firms in a region i depends on its market
potential and classical production factors (capital and labour). In addition, production factors according
to the Cobb-Douglas model [74] have declining returns to scale, while a market potential has growing
returns: 1
ln
(1
)ln
(
1)
ln
,
i
i
i
i
i
Profit
w
v
MP
A
-
= -α
-
- α
+ σ -
+
(8), (8), where Profit is revenues of firms in a region i; w is a cost of labour; v is other factors of production; MPi
is a market potential of a region i; A is a total factor productivity; α is a share of labour costs; σ is a
single product elasticity of substitution [75]. In [50], it was shown that a high percentage of fast-growing manufacturing companies in Russia
depends positively on the market potential of the regions. All the described research methods of EGP assessment can be reduced to four main approaches
(Table 1). T bl 1 Table 1
The basic approaches of the EGP analysis
Approach
Methods
Disadvantages
Analysis of location in
space or network
Topological distance, proximity
matrix, methods of the theory of
the central places
The approach do not give quantitative characteristics of
potential economic benefits. ‘Centrality’ is not always
beneficial
Calculation of
economic distances
Calculation of transport costs,
isochronous
The approach considers transportation costs, but does
not take into account potential benefits of cooperation
Calculation of an
integral index
Construction of indices
The subjective evaluation of a set of variables and
weights. Theoretical Basis The approach assesses relative capacities of EGP
Calculation of
potential interactions
Gravity models
The approach often do not take into account the actual
distance, specific types of positions (such as the seaside) 14 Regional innovation potential depends on the possible interregional flows of knowledge, or knowledge spillovers [76].
15 In [67], distance coefficient a, which influence on product import in a region i from country j by maritime transport, is about 2 if
coefficient for GRP of a region i is 0.6 and for the GDP of country j is 0.34. Methods and data P is an equivalent of a market potential. (
)
,
j
a
crit
Mean P
Dist
≤δ (10)
(
)
,
j
a
crit
Mean P
Dist
≤
δ
(11)
(
)
ln
ln
,
j
crit
Mean P
a
Dist
≤
δ
(12)
(
)
ln
. ln
j
crit
Mean P
a
Dist
δ
≥
(13) (
)
,
j
a
crit
Mean P
Dist
≤δ (10)
(
)
,
j
a
crit
Mean P
Dist
≤
δ
(11)
(
)
ln
ln
,
j
crit
Mean P
a
Dist
≤
δ
(12) (10) (
)
,
j
a
crit
Mean P
Dist
≤
δ
(11) (11) (
)
ln
ln
,
j
crit
Mean P
a
Dist
≤
δ
(12) (12) (
)
ln
. ln
j
crit
Mean P
a
Dist
δ
≥
(13) (13) Here is an example of the coefficient calculation bases on an average GRP in 2000-th in Russia:
an average GRP 17 was nearly 64,597,810,000 rubles. And an average transport distance of one tonne
of goods by rail (the most used in Russia) to the end of the period has reached 1.5 thousand km. We
assumed that a minimal interaction between two distant regions, situated at a distance of 8000 km
from each other (for example, the Amur and Arkhangelsk region), that can occur in a year is 1000
rubles, then ln64597810
2. ln8000
a =
≈
(14) (14) If we do the calculation for the countries in the same period, an average GDP 18 amount was
10,779,445,240,000 rubles, and the maximum distance, which still allows the carriage of goods
by sea (the main form of transport for international trade.) is about 25 thousand km (for example,
Dudinka — Melbourne). If we assume, that a minimum necessary for communication at this distance
should cost about one million rubles, then: ln10779445
1,6. Methods and data In this paper, we used the last of the discussed approaches (table 1), since it allows us to take into
account potential benefits for economic agents in a region, derived from possible interactions. Our
approach is based on the formula 5. The calculation of the EGP potential (EGP) of a region includes
an assessment of the potential of inter-regional, or national (EGP Reg), and international, or external
(EGP World), geographical position: 1
,
n
j
All
Reg
World
i
i
i
a
j
ij
MV
EGP
EGP
EGP
R
=
=
+
=∑
(9) (9) where MVj is gross regional product of a region j, or gross domestic product of a country j; Rij is an actual
distance between the capital of a region i and capitals in other regions or countries j; a is an empirical
coefficient, showing a speed of potential socio-economic interaction decrease between regions with
increasing distance between them. The higher the EGP potential is, the more intensive interactions
can be and the higher benefits for regions will be. where MVj is gross regional product of a region j, or gross domestic product of a country j; Rij is an actual
distance between the capital of a region i and capitals in other regions or countries j; a is an empirical
coefficient, showing a speed of potential socio-economic interaction decrease between regions with
increasing distance between them. The higher the EGP potential is, the more intensive interactions
can be and the higher benefits for regions will be.i The calculations required to determine the value of the coefficient a, which will be different
within the country and between countries. Unfortunately, accurate estimation of the coefficients for
inter-regional and international trade does not fully reflect the potential of the EGP, and will include
effects of trade barriers and other factors 15. Obtaining such estimates is time-consuming [67]. Besides, R-Economy Vol. 2, Issue 3, 2016 390 S. P. Zemtsov, V. L. Baburin a favourable EGP includes externalities of many economic relations, not only trade. That is why we
proposed another approach. a favourable EGP includes externalities of many economic relations, not only trade. That is why we
proposed another approach. Let us assume there is a critical distance Dist crit, after which an interaction between the regions
becomes insignificant, and δ is the threshold number of interactions, for example, a single interaction 16. 16 For example, it is known that the number of patent citations decreases rapidly with increasing distance between its inventors. In [77],
it was shown that after 120–150 miles researchers cite each other in patents rarely, most likely, they do not interact. For Russia, this critical
distance presumably is lower due to less mobility and greater isolation of scientific schools. 19 Regions surrounding the Arctic Ocean, as well as Sakhalin, Kamchatka and Magadan region have been excluded due to the above
conditions. 17 GRP is in prices of 1998, adjusted for inter-regional price index. t
distance presumably is lower due to less mobility and greater isolation of scientific schools.
17 GRP is in prices of 1998, adjusted for inter-regional price index.
18 GDP is calculated by purchasing power parity, translated into rubles using the official data on exchange rates of the Russian Central
Bank
19 17 GRP is in prices of 1998, adjusted for inter-regional price index.
18 GDP is calculated by purchasing power parity, translated into rubles using the official data on exchange rates of the Russian Central
Bank 391
R-Economy Vol. 2, Issue 3, 2016
16 For example, it is known that the number of patent citations decreases rapidly with increasing distance between its inventors. In [77],
it was shown that after 120–150 miles researchers cite each other in patents rarely, most likely, they do not interact. For Russia, this critical
distance presumably is lower due to less mobility and greater isolation of scientific schools.
17 GRP is in prices of 1998, adjusted for inter-regional price index.
18 GDP is calculated by purchasing power parity, translated into rubles using the official data on exchange rates of the Russian Central
Bank
19 Regions surrounding the Arctic Ocean, as well as Sakhalin, Kamchatka and Magadan region have been excluded due to the above
conditions. i
ices of 1998, adjusted for inter-regional price index.fi 18 GDP is calculated by purchasing power parity, translated into rubles using the official data on exchange rate
Bank Methods and data The general
formula for calculating the potential of an external EGP 21: 2
1,5
2
2
,
,
,
,
,
min(
)
(
)
q
World
n
i
i p
p q
i e
e n
GDP
GDP
EGP
R
R
R
R
=
+
+
+
∑
∑
(17) (17) where i is a region; GDP is gross domestic product (million rubles); q is a distant country (170); Ri,p is
a distance from a region i to the Russian port region p (km); Rp,q is the distance from a port region p to
the distant country q (km); n is a border country: economic interrelations with n are carried out mainly
by land through the regions e (Table 2). Table 2 Ta
Countries and regions, which are preferential for land interaction
Country (n)
Regions (e)
Armenia
The Republic of North Ossetia — Alania
Azerbaijan
The Republic of Dagestan
Belarus
Smolensk and Pskov regions
Estonia
Pskov and Leningrad oblast
Finland
The Republic of Karelia and Leningrad region
Georgia
The Republic of North Ossetia — Alania
Kazakhstan
Orenburg and Astrakhan region
Kyrgyzstan
Orenburg and Astrakhan region
Lithuania
Smolensk region
Latvia
Smolensk and Pskov regions
Mongolia
The Republic of Buryatia
Tajikistan
Orenburg and Astrakhan region
Turkmenistan
Astrakhan region
Ukraine
Kursk, Bryansk and Belgorod region
Uzbekistan
Orenburg and Astrakhan region
Compiled by the authors according to the Federal Statistical Service of Russia. Countries and regions, which are preferential for land interaction
Country (n)
Regions (e)
Armenia
The Republic of North Ossetia — Alania
Azerbaijan
The Republic of Dagestan
Belarus
Smolensk and Pskov regions
Estonia
Pskov and Leningrad oblast
Finland
The Republic of Karelia and Leningrad region
Georgia
The Republic of North Ossetia — Alania
Kazakhstan
Orenburg and Astrakhan region
Kyrgyzstan
Orenburg and Astrakhan region
Lithuania
Smolensk region
Latvia
Smolensk and Pskov regions
Mongolia
The Republic of Buryatia
Tajikistan
Orenburg and Astrakhan region
Turkmenistan
Astrakhan region
Ukraine
Kursk, Bryansk and Belgorod region
Uzbekistan
Orenburg and Astrakhan region
Compiled by the authors according to the Federal Statistical Service of Russia. Countries and regions, which are preferential for land interaction Countries and regions, which are preferential for land interaction The data of the Russian Federal Statistics Service was used for calculations. Methods and data ln25000
a =
≈
(15) (15) The potential of an interregional EGP can be calculated by the following formula: The potential of an interregional EGP can be calculated by the following formula: 2
j
Reg
i
ij
GRP
EGP
R
=∑
(16), (16), where i –is a region, GRP is gross regional product (calculated by the index of physical volume) (million
rubles), j is other regions (83), R is a distance (km) by rail; for regions where there are no railways, we
used data on automobile road and river routes. To calculate the potential of an international EGP we have identified nine Russian regions, where
foreign trade activity is concentrated. It is regions (p) with nonfreezing major ports, which have
connected to other regions by year-round infrastructure 19: Arkhangelsk, Kaliningrad, Leningrad,
Murmansk, Rostov, St. Petersburg, Krasnodar and Primorsky regions. Other Russian regions may carry 16 For example, it is known that the number of patent citations decreases rapidly with increasing distance between its inventors. In [77],
it was shown that after 120–150 miles researchers cite each other in patents rarely, most likely, they do not interact. For Russia, this critical
distance presumably is lower due to less mobility and greater isolation of scientific schools. rchasing power parity, translated into rubles using the official data on exchange rates of the Russian Central 19 Regions surrounding the Arctic Ocean, as well as Sakhalin, Kamchatka and Magadan region have been excluded due to the above
conditions. R-Economy Vol. 2, Issue 3, 2016 391 S. P. Zemtsov, V. L. Baburin out foreign trade relations with distant countries mainly through these regions. This does not exclude
the possibility of foreign trade with neighbouring countries. out foreign trade relations with distant countries mainly through these regions. This does not exclude
the possibility of foreign trade with neighbouring countries. y
g
g
g
Economic ties on land are less intense than ones on the sea due to higher transport costs [78]. Therefore, the coefficient a is lower for international relations than for inter-regional 20. Methods and data The data on GDP by
purchasing power parity was collected from the statistics of the International Monetary Fund (http://
www.imf.org/). p
y
gi
y
p
g
21 The EGP potential, as measured by the proposed method, allows to calculate the potential volume of foreign trade activities in the
case of the maximum development of infrastructure and the development of adequate institutions (investment climate, trade barriers, etc.). 20 Company significantly increases its revenues when it starts exporting [79].h Results 1. Assessment of a potential of economic-geographical position of the Russia g
g
g
The maximum potential of an interregional EGP in 2012 (Fig. 3) was in regions near the Moscow
and St. Petersburg agglomerations. The EGP potential decreases from them in all directions with a
significant excess in the Ural regions (Yekaterinburg, Perm, Ufa, Chelyabinsk agglomerations) and in
the highly profitable Tyumen region. The Russian Far East (Magadan, Yakutsk, Kamchatka regions),
the most remote from the central regions, have the least potential for interregional cooperation.i The EGP potential is an estimation of possible benefits (in terms of value), which regional economy
can receive due to its proximity to other major markets through inter-regional interactions (exchange
of goods, services, investment, etc.). This natural advantage of a particular region is not directly related
to activities of economic agents in that region. R-Economy Vol. 2, Issue 3, 2016 392 S. P. Zemtsov, V. L. Baburin Fig. 3. Interregional potential of EGP of Russian regions in 2012 Fig. 3. Interregional potential of EGP of Russian regions in 2012 Fig. 3. Interregional potential of EGP of Russian regions in 2012 Fig. 3. Interregional potential of EGP of Russian regions in 2012 For interpretation reasons, we assumed that there is a company with revenues of 64 billion
rubles per year 22 (average GRP in Russia) in the Moscow region. It could earn by exporting products
to neighbouring regions to 484 million rubles a year more than the same company located in the
Chukotka Autonomous District 23 only because of its location. The benefit from the EGP will be 0.75 %
of the revenue of the enterprise, but small and medium enterprises can realize their EGP more easily,
and this proportion may be higher. p
p
y
g
If we assume that all enterprises, located in the Moscow region, will concentrate in one conventional
point, and the Moscow region GRP 4.3 times more than the average GRP, then the absolute value of
the benefits of economic agents can be more than 2 billion rubles per year. But the potential of an
EGP is calculated for the capital city of each region, and the economic agents are located in different
settlements. Therefore, the combined effect can be a little lower for the Moscow region, as there are
remote from Moscow businesses. There is a divergence of the potential of interregional EGP (Fig. 4). 23 It is also possible that we are talking about the industry average enterprise, which has all industries as well as the average for
the regions of Russia, but it is clear that the EGP potential will also be different for enterprises of different specialization. It is higher for
enterprises producing consumer products. problem with the dimension (million rubles / sq. km), it is assumed that it is located on an area of 1 sq. km. 22 To solve the problem with the dimension (million rubles / sq. km), it is assumed that it is located on an area Results If the potential in a region i in
1998 was 1 % higher than it is in the region j, this region had 0.4 % higher rate of increase during 1998–
2012. The potential of an interregional EGP grew up more than 2.5 times in St. Petersburg, Moscow,
Leningrad, Novgorod, Moscow, Tver region due to the growth of GRP of two largest agglomerations and
in the Krasnodar region due to the significant growth of GRP of the North Caucasian republics. The
lowest growth rate (less than 1.8 times) was observed in Sverdlovsk (city Yekaterinburg), Novosibirsk
and Omsk regions due to the high base effect and low growth of GRP in Siberian oil and gas producing
regions. According to our methodology, there are two main reasons of the EGP potential dynamics:
the growth of GRP in the neighbouring regions or GDP of neighbouring countries (which is almost
independent from any activities of regional authorities) and the construction of highways and port
facilities, allowing essentially to bring foreign markets. For example, if the plan to build the Northern
latitudinal line, which will connect Tomsk and Khanty-Mansiysk, was implemented in 2012, the R-Economy Vol. 2, Issue 3, 2016 393 S. P. Zemtsov, V. L. Baburin Fig. 4. The relationship between the potential of an interregional EGP in 1998 and its growth over the period 2012–1998 Fig. 4. The relationship between the potential of an interregional EGP in 1998 and its growth over the period 2012–1998 elationship between the potential of an interregional EGP in 1998 and its growth over the period 2012–199 Fig. 5. The total potential of interregional and international EGP of the Russian regions in 2012 Fig. 5. The total potential of interregional and international EGP of the Russian regions in 2012 Fig. 5. The total potential of interregional and international EGP of the Russian regions in 2012 Fig. 5. The total potential of interregional and international EGP of the Russian regions in 2012 potential of an interregional EGP of Tomsk region have been already increased from 3.05 up to 5
million rubles i e 60 % potential of an interregional EGP of Tomsk region have been already increased from 3.05 up to 5
million rubles, i.e. 60 %. The maximum potential of the international EGP (more than 10 billion rubles) is concentrated
in the regions of the Baltic Sea (Kaliningrad and Leningrad region, St. R-Economy Vol. 2, Issue 3, 2016 Results Petersburg), the Black See
(Krasnodar Territory and Rostov Region), the Sea of Japan (Primorsky region) and in close proximity to R-Economy Vol. 2, Issue 3, 2016 394 S. P. Zemtsov, V. L. Baburin Fig. 6. The relationship between the total potential of EGP in 1998, the base and its growth over the period 2012–1998. Note: The numbers on the graph show 1 — Jewish Autonomous Region, 2 — Orel region, 3 — the Republic of Dagestan,
4 — Amur Oblast, 5 — Sakhalin region, 6 — Krasnodar region, 7 — Rostov region, 8 — Novgorod region, 9 — Murmansk
region, 10 — Arkhangelsk region Fig. 6. The relationship between the total potential of EGP in 1998, the base and its growth over the period 2012–1998. Note: The numbers on the graph show 1 — Jewish Autonomous Region, 2 — Orel region, 3 — the Republic of Dagestan,
4 — Amur Oblast, 5 — Sakhalin region, 6 — Krasnodar region, 7 — Rostov region, 8 — Novgorod region, 9 — Murmansk
region, 10 — Arkhangelsk region them (Novgorod region). The worst international EGP (capacity less than 50 million rubles) are in th
Chukotka Autonomous District and the Republic of Tyva. The maximum international EGP potential growth (more than 3.5 times) was observed in the
southern regions of the Far East: the Primorsky and Khabarovsk regions and the Jewish Autonomous
Region. The uniqueness of the Russian Far Eastern regions position is that they are on the periphery of
economic activity in Russia (Fig. 3) but close to the rapidly growing markets of Asia-Pacific countries
[13, 80]. The lowest growth rates (less than 2.5) were demonstrated by the regions close to the slow-
growing Nordic countries: St. Petersburg, Leningrad and Kaliningrad regions. The potential of an interregional EGP increased by 2.2 times in 1998–2012, and the potential of an
international EGP — by 3 times 24. In other words, it was more profitable for economic agents to focus
on a foreign trade in this period. The total potential of EGP comprises interregional and international components (Fig. 4). The
total potential is mainly concentrated near the major port centres of Russia. The leaders in 2012 were
Primorsky, Krasnodar, Leningrad, Rostov region and St. Petersburg. Moscow region, the leader of the
interregional EGP potential, ranks only 21st place on the total potential. g
y
There is a weak convergence of the total EGP potential (Fig. 24 Note that in dollar terms inflation is not taken into account for the countries of the world. 25 We note that the EGP potential does not include infrastructure constraints of mountainous and remote areas.
26 A detailed description of Kaliningrad region EGP is given in [33]. Results 6) with a strong division of trends
between Far Eastern regions and the regions of the European part of Russia. If the first is characterized
by the divergence (the higher the capacity in 1998, the faster it will grow due to increased economies
of Asia-Pacific countries), the situation is reversed for the second (regions with smaller potential grew
slower). In the southern regions of the European part of Russia, the EGP potential has increased more
significant in spite of the negative trends in the economies of the southern countries of the European
Union (EU). Comparing of the total EGP potential with an existing GRP of regions can show how they realize
the advantages of their positions (Fig. 7).i g
(
g
)
The potential benefits of economic cooperation with other regions and countries for the Republic of
Ingushetia, the Jewish Autonomous Region, Republic of Kalmykia, the Kaliningrad region, Karachaevo-
Circassian Republic are higher than their real GRP. The Republic of Ingushetia underutilized benefits
from its position near the port facilities in the Krasnodar region and the main traffic arteries in the
Caucasus because of the weak development of the economy, institutional and social barriers and poor Note that in dollar terms inflation is not taken into account for the countries of the world. R-Economy Vol. 2, Issue 3, 2016 395 S. P. Zemtsov, V. L. Baburin Fig. 7. The ratio of the total potential of EGP and GRP of Russian regions in 2012 Fig. 7. The ratio of the total potential of EGP and GRP of Russian regions in 2012 Fig. 7. The ratio of the total potential of EGP and GRP of Russian regions in 2012 infrastructure 25. Kaliningrad region 26is located near the largest European market, but the EGP potential
is not fully realized because of high trade barriers and isolation from the rest of Russia. The total EGP potential of Kaluga region amounted to about 2.6 billion rubles, or about 20 % of the
GRP, despite the rapid growth of auto industry cluster, the EGP potential is not fully used. At the same time, the regions of Siberia, the Urals, the Republic of Tatarstan and Bashkortostan, as
well as Moscow and St. Petersburg is almost fully utilized the potential. 2. The relationship between the EGP potential and indicators of socio-economic development
of regions 2. The relationship between the EGP potential and indicators of socio-economic development
of regions Baburin
Table 3
The correlation coefficients between the EGP potentials and indicators of socio-economic development of the
Russian regions in the period 1998–2012
Indicators of socio-economic development of the Russian
regions
Total potential of EGP,
million rubles
Potential of interregional
EGP, million rubles
GRP, million rubles
0,21
Growth of GRP, %
0,06
Investments in fixed assets, million rubles
0,14
0,33
Ratio of fixed investment to GRP, %
0,15
Export, million rubles
0,18
Ratio of imports to the GRP, %
0,56
0,18
Foreign direct investments, million rubles
0,33
Number of enterprises per 1,000 employees
0,24
0,2
Share of employment in wholesale and retail trade, %
0,24
0,15
Population density, persons per km2
0,11
0,41
Urbanization, %
0,07
0,16
Net migration, migrants per 10 thousand citizens
0,11
0,16
Technology export, million rubles
0,1
0,24
Technology and technical services import, million rubles
0,23
0,23
Number of mobile phones per 1000 citizens
0,26
0,18
Number of personal computers with Internet access per 100
employees
0,22
0,12
Note: all the coefficients are significant at the 5 % p-value. Fig. 8. Dynamics of the correlation coefficient between the potential of an interregional EGP and several indicators of socio-
economic development of the Russian regions
Conclusions
Based on the review of the literature the category of economic-geographical position (EGP) was
formalized, which is a historically evolved, but varying set of spatial relations between economic agents
in a region and external factors potentially influencing the regional development S. P. Zemtsov, V. L. 2. The relationship between the EGP potential and indicators of socio-economic development
of regions Baburin Table 3 Table 3
The correlation coefficients between the EGP potentials and indicators of socio-economic development of the
Russian regions in the period 1998–2012
Indicators of socio-economic development of the Russian
regions
Total potential of EGP,
million rubles
Potential of interregional
EGP, million rubles
GRP, million rubles
0,21
Growth of GRP, %
0,06
Investments in fixed assets, million rubles
0,14
0,33
Ratio of fixed investment to GRP, %
0,15
Export, million rubles
0,18
Ratio of imports to the GRP, %
0,56
0,18
Foreign direct investments, million rubles
0,33
Number of enterprises per 1,000 employees
0,24
0,2
Share of employment in wholesale and retail trade, %
0,24
0,15
Population density, persons per km2
0,11
0,41
Urbanization, %
0,07
0,16
Net migration, migrants per 10 thousand citizens
0,11
0,16
Technology export, million rubles
0,1
0,24
Technology and technical services import, million rubles
0,23
0,23
Number of mobile phones per 1000 citizens
0,26
0,18
Number of personal computers with Internet access per 100
employees
0,22
0,12
Note: all the coefficients are significant at the 5 % p-value Note: all the coefficients are significant at the 5 % p-value. Note: all the coefficients are significant at the 5 % p-value. Fig. 8. Dynamics of the correlation coefficient between the potential of an interregional EGP and several indicators of socio-
economic development of the Russian regions Fig. 8. Dynamics of the correlation coefficient between the potential of an interregional EGP and several indicators of socio-
economic development of the Russian regions R-Economy Vol. 2, Issue 3, 2016 2. The relationship between the EGP potential and indicators of socio-economic development
of regions To test the hypothesis about the influence of the EGP potential on socio-economic development
of Russian regions, the correlation coefficients with a number of indicators were calculated (Table. 4,
Fig. 8–9). The higher total EGP potential is in the region, the higher proportion of imports in GRP is, as well
as technology import, a number of enterprises per 1,000 employees, a share of employment in trade,
mobile communication and the Internet development. The high interregional EGP potential is related
to higher population density, GRP, investment, including FDI, and export of goods and technologies. e e at o s
p betwee
t e
G
pote t a a d ot e
d cato s c a ged ove t
e (
g. 8 9). In the early period, the proportion of people employed in trade was significantly higher in regions
with high total EGP potential, that is close to the foreign markets in a period of “shuttle” trade, but
then the connection with the potential of interregional EGP grew up. In the late 90's, the migration
indicator was higher in regions with the high total EGP potential, but the regions of the European
part of Russia with a high potential of interregional EGP became more attractive due to its economic
development. At the end of the 2000s, the implementation of large investment projects in seaside
regions (Sochi Olympic games 2014, APEC Summit 2012 and others) have led to an increase in the
correlation coefficient between the total EGP potential and shares of investments in the GRP. R-Economy Vol. 2, Issue 3, 2016 396
S. P. Zemtsov, V. L. Conclusions Based on the review of the literature the category of economic-geographical position (EGP) was
formalized, which is a historically evolved, but varying set of spatial relations between economic agents
in a region and external factors, potentially influencing the regional development. g
, p
yl
g
g
p
The developed method the EGP potential assessment, based on the use of gravity models, can
further be widely used in regional studies to explore the benefits of the different locations of spatial R-Economy Vol. 2, Issue 3, 2016 397 S. P. Zemtsov, V. L. Baburin Fig. 9. Dynamics of e the correlation coefficient between the total potential of EGP and several indicators of socio-economic
development of the Russian regions Fig. 9. Dynamics of e the correlation coefficient between the total potential of EGP and several indicators of socio-economic
development of the Russian regions objects (countries, regions, cities, etc.). Calculations of the potential of the international EGP of the
Russian regions have been carried out for the first time. i
These calculations for the Russian regions showed a significant spatial differentiation. The
maximum potential of interregional EGP have the regions located near the Moscow and St. Petersburg
agglomerations, the potential decreases uniformly to the east. The maximum potential of the
international EGP is concentrated in the regions on the coast of the Black Sea, the Baltic Sea and the
Sea of Japan. The total EGP potential of the Kaliningrad region is 5.6 times higher than in distant inland
region — the Republic of Tyva, that is 5.6 times more profitable for economic agents located on the
Baltic coast near the large EU market than within the continent away from the sea and major markets.i A significant change in the total EGP potential was found in the 2000s. It shifts towards the
southern regions of the Far East due to the growth of the economies of the Asia-Pacific region. There
is a divergence of the potential of the interregional EGP, while the potential of the international EGP
has two trajectories for the Far Eastern regional group, which is characterized by divergence and other
regions with weak convergence. g
g
When comparing total EGP potential with the existing GRP, regions with high and low utilization
efficiency of its EGP were identified. The Kaliningrad region, the North Caucasus republics have more
opportunities to build the regional economy by harnessing the benefits of their position. 1. Drobyshevskiy, S., Lugovoy, O., Astafyeva, E., Polevoy, D., Kozlovskaya, A., Trunin, P. & Lederman, L. (20065). Faktory ekonomicheskogo
rosta v regionakh RF [Factors of economic growth in Russia’s regions]. Moscow: IEPP Publ., 278.
2. Lugovoy, O., Dashkeev, V., Mazaev, I., Fomchenko, D., Polyakov, E. & Khekht, A. (2007). Ekonomiko-geograficheskie i institutsionalnyye
aspekty ekonomicheskogo rosta v regionakh [Analysis of economic growth in regions: economic-geographical and institutional aspects]. Moscow:
IEPP Publ., 164.
3. Grigoryev, L., Zubarevich, N. & Urozhaeva, Yu. (2008). Stsilla i Kharibda regionalnoy politiki [Scylla and Charybdis of regional pol-
icy]. Voprosy ekonomiki [Questions of economy], 2, 83–98.
4. Combes, P. P., Mayer, T. & Thisse, J. F. (2008). Economic geography: The integration of regions and nations. Princeton University Press,
399. g
[
f
g
g
]
,
2. Lugovoy, O., Dashkeev, V., Mazaev, I., Fomchenko, D., Polyakov, E. & Khekht, A. (2007). Ekonomiko-geograficheskie i institutsionalnyye
aspekty ekonomicheskogo rosta v regionakh [Analysis of economic growth in regions: economic-geographical and institutional aspects]. Moscow:
IEPP Publ., 164.
3. Grigoryev, L., Zubarevich, N. & Urozhaeva, Yu. (2008). Stsilla i Kharibda regionalnoy politiki [Scylla and Charybdis of regional pol-
icy]. Voprosy ekonomiki [Questions of economy], 2, 83–98.
4 C
b
P P M
T & Thi
J F (2008) E
i
h
Th i t
ti
f
i
d
ti
P i
t
U i
it P Conclusions The regions of
Siberia, the Urals, the Republic of Tatarstan and Bashkortostan, as well as Moscow and St. Petersburg,
is almost completely using their EGP potential. p
y
g
p
It was found that the favourable EGP is one of the factors of GRP growth, investment growth,
foreign trade, migration increase and diffusion of new technologies. Formalizing EGP allows using it as a method for externalities evaluation of infrastructural projects
and to predict the spatial changes in the socio-economic development of Russia. S. P. Zemtsov, V. L. Baburin Ekonomicheskaya geografiya: istoriya, teoriya, metody, praktika [Economic geography: history, the
ctice]. Moscow: Mysl Publ., 362.i p
]
y
15. Mashbits, Ya. G. (1998). Geograficheskoye polozhenie [Geographic location]. Kompleksnoye stranovedenie [Complex regional
geography]. Moscow: Smolensk Publ., 101–112.i p
y
15. Mashbits, Ya. G. (1998). Geograficheskoye polozhenie [Geographic location]. Kompleksnoye stranovedenie [Complex regional
geography]. Moscow: Smolensk Publ., 101–112. g
g
p y
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297–308. 76. Audretsch, D. B. & Feldman, M. P. (2004). Knowledge spillovers and the geography of innovation. Handbook of regional and urban
economics, 4, 2713–2739.f Trajtenberg, M. Authors Stepan Petrovich Zemtsov — PhD in Geography, Senior Research Associate, Russian Presidential Academy of National Economy and
Public Administration, Institute for Applied Economic Research (82/1, Vernandskogo Ave., Moscow, 119571, Russian Federation; e-mail:
spzemtsov@gmail.com). p
g
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Vyacheslav Leonidovich Baburin — Doctor of Geography, Professor, Head of the Department of Economic and Social Geography of
Russia, Lomonosov Moscow State University (1, Leninskie Gory St., Moscow, 119991, Russian Federation). Vyacheslav Leonidovich Baburin — Doctor of Geography, Professor, Head of the Department of Economic and Social Geography of
Russia, Lomonosov Moscow State University (1, Leninskie Gory St., Moscow, 119991, Russian Federation). 401 R-Economy Vol. 2, Issue 3, 2016
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Mammalian Epidermis: A Compendium of Lipid Functionality
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Frontiers in physiology
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Mammalian Epidermis: A
Compendium of Lipid Functionality Matteo Vietri Rudan and Fiona M. Watt* Mammalian epidermis is a striking example of the role of lipids in tissue biology. In
this stratified epithelium, highly specialized structures are formed that leverage the
hydrophobic properties of lipids to form an impermeable barrier and protect the humid
internal environment of the body from the dry outside. This is achieved through tightly
regulated lipid synthesis that generates the molecular species unique to the tissue. Beyond their fundamental structural role, lipids are involved in the active protection of
the body from external insults. Lipid species present on the surface of the body possess
antimicrobial activity and directly contribute to shaping the commensal microbiota. Lipids belonging to a variety of classes are also involved in the signaling events that
modulate the immune responses to environmental stress as well as differentiation of the
epidermal keratinocytes themselves. Recently, high-resolution methods are beginning
to provide evidence for the involvement of newly identified specific lipid molecules in
the regulation of epidermal homeostasis. In this review we give an overview of the wide
range of biological functions of mammalian epidermal lipids. Edited by: Edited by:
Pasquale Simeone,
University of Studies G. d’Annunzio
Chieti and Pescara, Italy Reviewed by:
Christoph Reinhardt,
Johannes Gutenberg University
Mainz, Germany
Sandra Iden,
Saarland University, Germany REVIEW
published: 12 January 2022
doi: 10.3389/fphys.2021.804824 REVIEW
published: 12 January 2022
doi: 10.3389/fphys.2021.804824 INTRODUCTION The evolution of an impermeable barrier that could preserve the internal aqueous environment of
the body away from water in ancestral reptiles was one of the most important events that allowed
the colonization of dry land by vertebrates. That barrier, the “cornified” or “horny” epidermis,
leveraged the physical properties of hydrophobic lipid and protein molecules to achieve the
separation between the “wet” inside and the “dry” outside. Since then, over the course of evolution,
the epidermis has acquired numerous additional structures and adaptations. However, the core
physical mechanisms through which it exerts its most fundamental functions as well as its basic
architecture have remained similar in all terrestrial vertebrates (reptiles, birds and mammals). *Correspondence:
Fiona M. Watt
fiona.watt@kcl.ac.uk *Correspondence:
Fiona M. Watt
fiona.watt@kcl.ac.uk Specialty section:
This article was submitted to
Lipid and Fatty Acid Research,
a section of the journal
Frontiers in Physiology
Received: 29 October 2021
Accepted: 13 December 2021
Published: 12 January 2022
Citation:
Vietri Rudan M and Watt FM
(2022) Mammalian Epidermis:
A Compendium of Lipid Functionality. Front. Physiol. 12:804824. Specialty section:
This article was submitted to
Lipid and Fatty Acid Research,
a section of the journal
Frontiers in Physiology In mammals, the epidermis is the outermost part of the skin, overlying the connective tissue,
the dermis. Mammalian epidermis is a stratified epithelium comprising a series of layers of
progressively more differentiated cells, called keratinocytes. At the dermal interface, resting on
a basement membrane, the basal keratinocyte layer contains cycling cells, responsible for the
renewal of the tissue. Basal cells undergo a phase of commitment and then start the process of
differentiation, exiting the cell cycle and beginning to migrate upward toward the body surface. As the cells progress through differentiation, they move through the spinous layer and the
granular layer, all the while accumulating specific lipids and proteins and building functional
ultrastructures. The keratinocytes ultimately lose their nucleus and become flattened “bricks” of
insoluble protein called corneocytes, surrounded by lipid “mortar” to hold them together in the Received: 29 October 2021
Accepted: 13 December 2021
Published: 12 January 2022 Keywords: lipids, epidermis, keratinocytes, ceramides, signaling, fatty acids, lipidomics Granular Layer As keratinocytes progress through differentiation and become
more specialized, their lipid and protein makeup change. Starting
from the upper spinous layer and in the granular layer the
keratinocytes begin forming cross-linked bundles of keratin
fibers and filaggrin in their cytoplasm. Their organelles begin
degenerating and they assemble lamellar bodies (LB), also known
as membrane-coating granules, lamellar granules, or Odland
bodies (Selby, 1957; Odland, 1960; Proksch et al., 2008; Wertz,
2018). LB have been traditionally described as membrane-bound
organelles of ∼200 nm in diameter containing a series of bilayer
membranes 6–7 nm thick that are closely stacked together
(Matoltsy and Parakkal, 1965; Figure 1), although more recent
evidence suggests that they form a tubuloreticular network
derived from the trans-Golgi apparatus (Matoltsy and Parakkal,
1965; Elias et al., 1998; Norlén et al., 2003). Lipids are essential for the fundamental function of the
epidermis since they are a key element in the water-insulating
properties of the cornified layer. The relative abundance of
different lipid species changes across the various layers of the
epidermis, underscoring how the correct establishment of a
functional barrier requires tightly controlled regulation of lipid
production (Lampe et al., 1983). Indeed, dysregulation of the
lipid balance can cause a number of pathological conditions,
notably ichthyoses (Akiyama, 2017; Vahlquist and Törmä,
2020). Beyond their structural role in the formation of the physical
barrier of the epidermis, several studies have unraveled the
participation of lipid molecules in the active protection of
the body from external harmful agents, such as pathogenic
microbes and damaging UV-radiation. Moreover, several studies
have shown how specific lipids may be directly involved
in the regulation of keratinocyte differentiation itself, and
new techniques are now allowing the exploration of this
field of research. Early characterization of the contents of LBs revealed that
they are mostly made up of lipids and are specifically rich
in glycolipids (mostly glucosylceramides), phospholipids and
cholesterol as well as smaller amounts of sterol esters, ceramides,
and fatty acids (Wertz et al., 1984; Grayson et al., 1985). An
especially remarkable lipid species found uniquely in LB is
an acylglucosylceramide comprising a linoleic acid (C18:2ω6)
molecule esterified to the ω-hydroxy group of a very long chained
(30–34 carbons) fatty acid moiety of the glucosylceramide (Wertz
et al., 1984; Bowser et al., 1985). Granular Layer A specific role has been proposed
for this particular lipid in the correct structural assembly of LBs,
whereby its very long ω-hydroxyacid chain is able to span an
entire lipid bilayer while the linoleate tail can insert itself in a
neighboring bilayer, thus acting as a “molecular rivet” holding
together the tightly packed series of membranes found in LBs
(Wertz and Downing, 1982; Wertz, 2018). Importantly, besides
its potential role in structuring the LB’s contents, the linoleic
acid-containing acylglucosylceramide is an essential component
of the LB’s bounding membrane, where the majority of it is found
(Wertz, 2000, 2018). This review covers the multi-faceted roles, from structural
to regulatory, that lipids play in epidermal homeostasis and
protection from the external environment. The epidermis
is a model of the versatility of this class of molecules,
highlighting their important, often understated, and still
potentially uncharacterized activities. We first describe the
central role of lipids in the formation of the epidermal water
permeability barrier. Then we detail the antimicrobial and
immunomodulatory activity of certain epidermal lipids. Finally,
we analyze how epidermal lipids of many different classes can
influence epidermal cell signaling. This review will not, however,
cover lipid vitamins. For details of the extremely important part
vitamin A, vitamin D and their derivatives play in the biology of
the epidermis, the reader is referred to recent reviews (Piotrowska
et al., 2016; Szyma´nski et al., 2020). Besides lipid molecules, LBs contain several hydrolytic
enzymes such as carboxypepdidase, cathepsin B, acid hydrolase,
as
well
as
acid
lipase,
β-glucosidases,
phospholipase
A,
sphingomyelinase,
ceramidases,
and
steroid
sulfatase
that
play an important role during the subsequent differentiation
steps (Freinkel and Traczyk, 1985; Grayson et al., 1985). Cornified Layer (Stratum corneum) Cornified Layer (Stratum corneum)
The boundary between the granular and cornified layers
represents the water-permeability barrier of the body, as
demonstrated by dermal injection of water-soluble tracers (Elias
and Friend, 1975). As the cells move toward and through this
boundary, they become corneocytes, the “bricks” of the stratum
corneum: they form an extremely resistant protein shell, the
cornified cell envelope, just beneath their plasma membrane,
made of crosslinked involucrin, loricrin, small proline-rich
proteins and other proteins. The nucleus starts to degenerate
and ultimately disappears, as the cytoplasm becomes filled
with keratin bundles and the cell becomes flattened. The LBs
approach the apical plasma membrane of the cell, their bounding Long, 1970). Citation: Vietri Rudan M and Watt FM
(2022) Mammalian Epidermis:
A Compendium of Lipid Functionality. Front. Physiol. 12:804824. doi: 10.3389/fphys.2021.804824 January 2022 | Volume 12 | Article 804824 1 Frontiers in Physiology | www.frontiersin.org Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt outermost impermeable cornified layer or stratum corneum
(Nemes and Steinert, 1999; Watt, 2014; Figure 1). THE BODY’S OUTER WALL – THE
FUNDAMENTAL STRUCTURAL ROLE OF
EPIDERMAL LIPIDS The lipid composition varies dramatically along the thickness of
mammalian epidermis (Figure 1). Early dissections of the lipid
makeup of the epidermis of different mammals revealed striking
differences between the basal and spinous layers, the granular
layer and the cornified layer (stratum corneum) (Kooyman, 1932;
Long, 1970). Basal and Spinous Layers In the basal and lower spinous layers, phospholipids are
most common, with high levels of phosphatidylcholines,
phosphatidylethanolamines,
phosphatidylserines
and
sphingomyelins
(Gray
et
al.,
1980). This
phospholipid
predominance likely underlies the main roles that lipids
have
in
these
cells:
membrane
maintenance,
energy
production and signaling. January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 2 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt FIGURE 1 | Role of lipids in the formation of the epidermal water permeability barrier. Illustration of the structure of the skin with the main lipid components of the
various layers indicated on the right. The left inset shows the microstructure at the boundary between the granular layer and the stratum corneum. The right inset
displays the possible molecular arrangement of the main stratum corneum lipids in the intercellular lamellae according to the “sandwich model.” Alternative models
have also been proposed. FIGURE 1 | Role of lipids in the formation of the epidermal water permeability barrier. Illustration of the structure of the skin with the main lipid components of the
various layers indicated on the right. The left inset shows the microstructure at the boundary between the granular layer and the stratum corneum. The right inset FIGURE 1 | Role of lipids in the formation of the epidermal water permeability barrier. Illustration of the structure of the skin with the main lipid components of the
various layers indicated on the right. The left inset shows the microstructure at the boundary between the granular layer and the stratum corneum. The right inset
displays the possible molecular arrangement of the main stratum corneum lipids in the intercellular lamellae according to the “sandwich model.” Alternative models
have also been proposed. membrane finally fusing with it, releasing the LB contents
in the extracellular space (Wertz, 1996; Proksch et al., 2008;
Figure 1). Upon extrusion, the lipids contained in the LBs undergo
dramatic changes, likely becoming substrates for the lipid
hydrolases that convert them into ceramides (∼50%), cholesterol
(∼25%), and fatty acids (∼10%), the main constituents of the
intercellular lipid “mortar” of the cornified layer (Figure 2),
which also comprises smaller amounts of cholesterol sulfate and
cholesterol esters (Nicolaides, 1974; Wertz and van den Bergh,
1998). Basal and Spinous Layers Notably, the release of fatty acids from phospholipids
by phospholipase A2 contributes to the creation of an acidic
environment in the stratum corneum, which in turn regulates
the activity of other enzymes, such as β-glucocerebrosidase, acid
sphingomyelinase as well as serine proteases. This is necessary The
linoleate-containing
acylglucosylceramide
in
the
bounding
membrane
is
processed
to
become
ω-hydroxyceramide, which is covalently bound to the cornified
cell envelope’s proteins, notably involucrin, forming an additional
layer around the cell, known as the corneocyte lipid envelope
(Swartzendruber et al., 1987; Wertz and Downing, 1987; Nemes
et al., 1999; Grond et al., 2017). Together, the cornified cell
envelope and the lipid envelope replace the plasma membrane
of the corneocyte. January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 3 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt FIGURE 2 | Main lipids of the stratum corneum. Ceramide classes are labeled both according to the classical nomenclature (Cer1-9) when applicable and by the
letter code indicating the sphingoid base-fatty acyl moiety combination. In the example structures of the ceramides, all sphingoid bases and fatty acid moieties are
shown as C18, but can be substituted by other chain lengths according to what is indicated at the top of the figure or described in the main text. Cer[EO] structures
are presented as C26 fatty acids esterified to linoleic acid (as that is what is most commonly found in the epidermis). Both ceramide fatty acyl groups and stratum
corneum free fatty acids are generally saturated or monounsaturated. FIGURE 2 | Main lipids of the stratum corneum. Ceramide classes are labeled both according to the classical nomenclature (Cer1-9) when applicable and by the
letter code indicating the sphingoid base-fatty acyl moiety combination. In the example structures of the ceramides, all sphingoid bases and fatty acid moieties are
shown as C18, but can be substituted by other chain lengths according to what is indicated at the top of the figure or described in the main text. Cer[EO] structures
are presented as C26 fatty acids esterified to linoleic acid (as that is what is most commonly found in the epidermis). Both ceramide fatty acyl groups and stratum
corneum free fatty acids are generally saturated or monounsaturated. to ensure the correct formation of the epidermal barrier and its
homeostatic regulation (Hachem et al., 2003). chains of the acylceramides acting as a zipper (Swartzendruber
et al., 1989). Frontiers in Physiology | www.frontiersin.org Basal and Spinous Layers X-ray diffraction studies of the human cornified
layer’s intercellular lipid matrix instead describe two lamellar
phases, referred to as the long-periodicity phase (LPP, ∼13 nm)
and the short-periodicity phase (SPP, ∼5 nm), with the former
likely to be relevant for the barrier function, having been
identified in multiple animal species (Bouwstra et al., 1991,
1992). The LPP has in fact been equated to one Landmann unit,
in which the ceramides, cholesterol and fatty acids in the outer
bilayers arrange themselves as a dense crystalline orthorhombic
lattice, responsible for hindering the movement of compounds
through the outer epidermis, and form the impermeable barrier
that lines the body, while the intervening monolayer made up
of the ω-esterified unsaturated fatty acid (mostly linoleate) and The
chemical
changes
in
lipids
are
accompanied
by
morphological ones, as the disk-like bilayers seen in the
LBs fuse to become a series of broad lamellar sheets (Elias
et al., 1977; Landmann, 1986; Madison et al., 1987; Figure 1). The corneocyte lipid envelope plays a fundamental role in
the assembly of these lamellae, acting as a template for their
orientation with respect to the corneocytes (Behne et al., 2000). More specifically, lipids arrange themselves into repeating units
that appear as a series of broad-narrow-broad electron-lucent
bands in electron micrographs. Each of these units (sometimes
referred to as Landmann units) are thought to be juxtaposed
lipid bilayers with an intervening monolayer formed by the long January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt cholesterol forms a liquid phase that could provide flexibility and
resistance to shear stress. This is referred to as the “sandwich
model” (Bouwstra et al., 2000, 2002; Figure 1). the epidermis also actively participates in the protection of the
organism against potential threats from opportunistic pathogenic
micro-organisms (Natsuga et al., 2016; Fischer, 2020). Given its
position as the outer boundary of the organism, the epidermal
surface is host to a diverse set of commensal microbes, whose
composition varies at different body sites, depending on certain
physiological characteristics such as degree of moisture or
abundance of sebaceous secretions (Table 1; Grice et al., 2009;
Grice and Segre, 2011). Free Sphingoid Bases One source of antimicrobial lipids comes from the stratum
corneum lipids themselves. Among the enzymes contained in
LBs are ceramidases, which cleave stratum corneum ceramides
into a sphingosine base and a fatty acid. The fatty acids
generated from these ceramides and more generally all stratum
corneum fatty acids synthesized by keratinocytes have very
long, mostly saturated carbon chains and do not possess any
antiseptic activity (Rothman et al., 1946; Kabara et al., 1972,
1977; Zheng et al., 2005; Brogden et al., 2011). However,
multiple studies have demonstrated the antimicrobial potency
of sphingosine, dihydrosphingosine, and 6-hydroxysphingosine, The functional importance of such lipid complexity is
underscored by several studies that attempt to replicate the
ultrastructure of cornified layer lipid lamellae in chemically
defined model membranes (Bouwstra et al., 2002; de Jager et al.,
2003; Opálka et al., 2016; Školová et al., 2017; Schmitt et al.,
2019; Beddoes et al., 2021). It is further revealed by a variety of
ichthyotic skin conditions in which different lipid biosynthetic
pathways are perturbed (Akiyama, 2017; Vahlquist and Törmä,
2020). TABLE 1 | Main members of epidermal surface bacterial communities at different
body sites grouped based on their microenvironmental characteristics. Moist (e.g., armpit)
Sebaceous (e.g., glabella)
Dry (e.g., forearm)
Corynebacterium spp. (28%)
Cutibacterium spp. (46%)
Betaproteobacteria (32%)
Betaproteobacteria (22%)
Staphylococcus spp. (16%)
Corynebacterium spp. (15%)
Staphylococcus spp. (21%)
Corynebacterium spp. (10%)
Flavobacteriales (14%)
Flavobacteriales (9%)
Betaproteobacteria (9%)
Cutibacterium spp. (13%)
Cutibacterium spp. (7%)
Flavobacteriales (3%)
Gammaproteobacteria (7%)
Gammaproteobacteria
Lactobacillales (3%)
Staphylococcus spp. Lactobacillales
Clostridiales
Lactobacillales
Bacteria are in order of abundance, with approximate average percentages in each
microenvironment indicated. Data adapted from Grice et al. (2009). TABLE 1 | Main members of epidermal surface bacterial communities at different
body sites grouped based on their microenvironmental characteristics. Moist (e.g., armpit)
Sebaceous (e.g., glabella)
Dry (e.g., forearm)
Corynebacterium spp. (28%)
Cutibacterium spp. (46%)
Betaproteobacteria (32%)
Betaproteobacteria (22%)
Staphylococcus spp. (16%)
Corynebacterium spp. (15%)
Staphylococcus spp. (21%)
Corynebacterium spp. (10%)
Flavobacteriales (14%)
Flavobacteriales (9%)
Betaproteobacteria (9%)
Cutibacterium spp. (13%)
Cutibacterium spp. (7%)
Flavobacteriales (3%)
Gammaproteobacteria (7%)
Gammaproteobacteria
Lactobacillales (3%)
Staphylococcus spp. Lactobacillales
Clostridiales
Lactobacillales
Bacteria are in order of abundance, with approximate average percentages in each
microenvironment indicated. Data adapted from Grice et al. (2009). Basal and Spinous Layers Lipids produced in the epidermis
can directly target surface-dwelling microbes to shape the
commensal microbiota and quell the growth of potentially
pathogenic species as well as participate in regulation of the
innate and adaptive immune responses that follow after invasion
of foreign pathogens. Once the corneocytes reach the surface of the body, they are
detached from the epidermis by desquamation. This process is
mediated by the action of certain serine proteases that attack
the desmosomes that connect corneocytes together (Suzuki et al.,
1993). Cholesterol sulfate present in the stratum corneum acts as
an inhibitor of these proteases. The concentration of cholesterol
sulfate is highest in the granular layer, and from there it gradually
decreases along the thickness of the cornified layer due to the
action of steroid sulfatase. The lower concentration of cholesterol
sulfate near the surface thus allows the proteases to become active
and enables the eventual desquamation of the corneocytes (Elias
et al., 1984; Sato et al., 1998). Heterogeneity of Stratum corneum
Lipids The surface of the epidermis is a generally inhospitable
environment for microbes owing to its mild acidity (pH 4-5.5),
scarcity of water and poor availability of essential metabolites
like phosphate (Aly et al., 1972, 1975; Forslind et al., 1995). Moreover, during differentiation, keratinocytes produce and
secrete peptides and proteins that possess antimicrobial activity,
such as defensins, cathelicidins (LL-37 in humans) and RNAse 7. Production of these peptides is stimulated upon damage to the
epidermis (Fulton et al., 1997; Dorschner et al., 2001; Falconer
et al., 2001). While the acylceramides, uniquely found in the epidermis, are
crucial for the establishment of the water permeability barrier,
there is considerable heterogeneity in the pool of stratum
corneum ceramides and fatty acids, which is likely to contribute
to the proper architecture of the lipid matrix (Figure 2). Ceramides show a remarkably wide range of diversity due
to heterogeneity in each of their sub-components. One of four
different sphingoid bases – sphingosine, dihydrosphingosine,
phytosphingosine, 6-hydroxysphingosine, usually C18 or C20
in length – can be paired with fatty acids that vary in carbon
chain size (C15 to C34, though most are between C20 to C30),
hydroxylation profile (nonhydroxy, α-hydroxy- or ω-hydroxy-),
and presence of an ω-esterified fatty acid moiety (almost always
linoleic acid) (Lampe et al., 1983; Masukawa et al., 2009; van
Smeden et al., 2011). Based on the different combinations of
these components, epidermal ceramides have been categorized
into several classes (Figure 2). Free fatty acids of the cornified
layer are nearly all saturated but their carbon chain length can
vary between C18 and C28, with the most abundant species being
C22 and C24 (Nicolaides, 1974; Wertz et al., 1987; Wertz and van
den Bergh, 1998). In addition to peptides, one of the main antimicrobial
components of the epidermis resides in its lipid fraction
(Burtenshaw, 1942). Indeed, two major components of lipid-
mediated immunity have been identified: free sphingoid bases in
the stratum corneum and free fatty acids in the sebum (Figure 3). BEYOND THE MORTAR – LIPIDS AS
ACTIVE PROTECTORS OF EPIDERMAL
INTEGRITY Besides its role as an impermeable physical barrier between
the internal environment of the body and the outside world, January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 5 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt FIGURE 3 | Main antimicrobial lipids of the epidermis. Rough spectra of activity are indicated on the right with a few examples. The lists of target micro-organisms
are not exhaustive and efficacy can vary among the different stratum corneum sphingoid bases or the various sebaceous fatty acids. FIGURE 3 | Main antimicrobial lipids of the epidermis. Rough spectra of activity are indicated on the right with a few examples. The lists of target micro-organisms
are not exhaustive and efficacy can vary among the different stratum corneum sphingoid bases or the various sebaceous fatty acids. FIGURE 3 | Main antimicrobial lipids of the epidermis. Rough spectra of activity are indicated on the right with a few examples. The lists of target micro-organisms
are not exhaustive and efficacy can vary among the different stratum corneum sphingoid bases or the various sebaceous fatty acids. which are found in the outer epidermis (Figure 3; Bibel
et al., 1992). Phytospingosine also displays similar properties
(Pavicic et al., 2007), but it is not found in free form in
human stratum corneum (Wertz and Downing, 1990). The
spectrum of antimicrobial activity of sphingosines is quite broad,
including activity against numerous Gram-positive bacteria such
as Cutibacterium acnes, Staphylococcus aureus, and Streptococcus
pyogenes; some Gram-negative bacteria are also affected, such as
Escherichia coli and Porphyromonas gingivalis; finally, fungi such
as Candida albicans can also be targeted by these compounds
(Figure 3; Bibel et al., 1992, 1993; Fischer et al., 2012b). The
varying degrees of efficacy exhibited by sphingoid bases against
different micro-organisms point to a certain level of specificity in
their mode of action. In addition to their direct effect on bacterial
growth and survival, sphingosines have proven very effective at
interfering with bacterial biofilm formation (Seitz et al., 2019;
Beck et al., 2020). in which the cell wall is not synthesized, are more resistant
to sphingoid base treatment. Interestingly, the cell wall of the
Gram-negative E. Coli is not affected, implying that the effect
on cell wall biosynthesis is likely a secondary consequence of the
treatment. BEYOND THE MORTAR – LIPIDS AS
ACTIVE PROTECTORS OF EPIDERMAL
INTEGRITY Given that the sphingoid bases become incorporated
into the bacteria, one possibility is that they may insert themselves
into the bacterial envelope/plasma membranes and render these
structures non-functional. Alternatively, they may enter the
cytoplasm and accumulate intracellularly where they might
interfere with cellular metabolism (Bibel et al., 1993; Fischer
et al., 2012b). This latter possibility is supported by the fact that
sphingosines can participate in cellular signaling by inhibiting
protein kinase C (PKC) in mammalian cells (Hannun et al., 1986). PKC is an important hub of cellular signaling; the different PKC
isoforms vary in their regulation and can play numerous roles in
influencing cellular behavior. Some PKC isoforms can be directly
regulated by lipids such as diacylglycerol, as described below. The mechanisms through which sphingoid bases enact their
antimicrobial activity have not been fully elucidated. Treatment
of S. aureus and E. Coli with sphingosine or dihydrosphingosine
causes shrinking and distortion of the cells with alterations
of cell membranes and emergence of inclusion bodies. In the
Gram-positive S. aureus, the cell wall is lost and L-form strains, Frontiers in Physiology | www.frontiersin.org Sebaceous Free Fatty Acids been implicated in helping to maintain the integrity of the
epidermal barrier, in thermoregulation, photoprotection, and
helping to deliver vitamin E to the skin (Zouboulis, 2004). Alterations in sebaceous secretions can lead to imbalances in
the skin microbiome and are linked to pathological conditions. More specifically, the mutual influence between sebum and the
commensal microbiota is exemplified by the fact that sebum-
rich areas of the body host a specific subset of microbial species
(Table 1), by the direct influence of bacterial lipases on sebum
composition, and by the changes in sebum composition observed
in acne patients (Scheimann et al., 1960; Lovászi et al., 2017;
Dréno et al., 2020; Oulès et al., 2020). Sebum composition is species-specific. The main components
in humans are triglycerides (∼45%), wax esters (∼25%), squalene
(∼12%), and fatty acids (∼10%). In contrast, mouse sebum is
mostly made up of wax esters (∼70%), with a much smaller
fraction of triglycerides (∼6%) and no fatty acids or squalene
(Wilkinson and Karasek, 1966; Nicolaides et al., 1968; Nikkari,
1974; Pappas, 2009). Among human sebum components, fatty
acids are the ones responsible for the antiseptic action of sebum. As sebum flows through the pilosebaceous duct, fatty acids are
released from sebaceous triglycerides by the action of bacterial
and possibly host lipases (Nicolaides and Wells, 1957; Scheimann
et al., 1960; Marples et al., 1971; Götz et al., 1998; Zouboulis et al.,
1999; Drake et al., 2008). When compared to those found in the
stratum corneum, sebum fatty acids have shorter, in some cases
odd-numbered, carbon chains, a higher degree of unsaturation,
and display potent antimicrobial activity (Weitkamp et al., 1947). Among these, enanthic (C7:0), pelargonic (C9:0), undecylic acid
(C11:0), and tridecylic acids (C13:0) extracted from human hair
fat have antifungal activity that is implicated in protection against
ringworm of the scalp (Rothman et al., 1946). Undecylenic acid
(C11:1) is found in sweat and has widespread use as an antifungal
treatment (Landau, 1983). Oleic (C18:1) and linoleic (C18:2)
acid are abundant in sebum and have efficacy as antibacterials
(Weitkamp et al., 1947; Kabara et al., 1972). Some of the most
potent sebaceous fatty acids include lauric acid (C12:0), present
in relatively minor amounts, and sapienic acid (C16:1ω10), thus
named because it is the most abundant fatty acid found in Homo
sapiens sebum (Figure 3; Weitkamp et al., 1947; Downing and
Strauss, 1974; Stewart and Downing, 1991). Sebaceous Free Fatty Acids Another important source of antimicrobial lipids is sebum,
a liquid mixture of neutral lipids assembled and secreted by
sebaceous glands that coats and lubricates the outer epidermis. The function of sebum is to a degree still debated, but it has January 2022 | Volume 12 | Article 804824 6 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt The
molecular
basis
of
the
antimicrobial
activity
of
sebaceous fatty acids is not understood completely (Desbois
and Smith, 2010). The amphipathic nature of fatty acids
is necessary for their antibacterial action, as replacing the
-OH group at one end of the molecule with a methyl
group abolishes any effect (Kodicek and Worden, 1945;
Kabara et al., 1972; Zheng et al., 2005). The shape of the
fatty acid molecule appears to affect its activity, with key
determinants being carbon chain length as well as presence,
number, position, and orientation of unsaturations. More
specifically, for saturated species there is a tendency for
antimicrobial potency to be highest at C10–12 and decrease
with either longer or shorter carbon chains (Kabara et al.,
1972; Bergsson et al., 2001; Wille and Kydonieus, 2003). The presence and number of unsaturations tend to increase
efficacy at a given carbon length, with the cis- orientation
(which is most often found in endogenous compounds)
most effective in boosting activity (Kabara et al., 1972;
Galbraith and Miller, 1973; Saito et al., 1984; Knapp and
Melly, 1986). For example, despite its relatively long carbon
chain, the double bond in sapienic acid is an unusual
cis C-6 unsaturation that allows the molecule to adopt
a
conformation
resembling
a
shorter-chained
fatty
acid
(Fischer, 2020). been implicated in helping to maintain the integrity of the
epidermal barrier, in thermoregulation, photoprotection, and
helping to deliver vitamin E to the skin (Zouboulis, 2004). Alterations in sebaceous secretions can lead to imbalances in
the skin microbiome and are linked to pathological conditions. More specifically, the mutual influence between sebum and the
commensal microbiota is exemplified by the fact that sebum-
rich areas of the body host a specific subset of microbial species
(Table 1), by the direct influence of bacterial lipases on sebum
composition, and by the changes in sebum composition observed
in acne patients (Scheimann et al., 1960; Lovászi et al., 2017;
Dréno et al., 2020; Oulès et al., 2020). Sebaceous Free Fatty Acids The principal target of sebum fatty acids seems to be
the bacterial cytoplasmic membrane, where they are thought
to interfere with oxidative phosphorylation-mediated energy
production by way of disruption of the electron transport
chain or dissipation of the membrane potential necessary
for ATP synthesis (Sheu and Freese, 1972; Galbraith and
Miller, 1973; Greenway and Dyke, 1979). One hypothesis
is that the shorter-chained or cis- unsaturated fatty acids
cause membrane fluidification and destabilization of electron
transport proteins (Greenway and Dyke, 1979; Chamberlain
et al., 1991). Other proposals are that they increase membrane
permeability to protons (Borst et al., 1962; Greenway and
Dyke, 1979; Gutknecht, 1988), or inhibit components of
the ATP synthase machinery (Wojtczak and Załuska, 1967). Besides their action at the cell membrane, unsaturated fatty
acids can inhibit cellular enzymes such as those responsible
for nutrient uptake (Galbraith and Miller, 1973), and can
inhibit fatty acid biosynthesis (Zheng et al., 2005; Sado-
Kamdem et al., 2009). They can increase oxidative stress
after undergoing peroxidation (Knapp and Melly, 1986) or
undergo auto-oxidation yielding other antibacterial compounds
(Gutteridge et al., 1974). Finally, fatty acids can cause leakage
of intracellular components and cell lysis (Galbraith and
Miller, 1973; Greenway and Dyke, 1979; Carson and Daneo-
Moore, 1980). Understanding which of these mechanisms
is responsible for the antimicrobial action of fatty acids is
challenging, because some are connected (e.g., the decrease
in energy production and the suppression of nutrient uptake,
or the effect on membrane fluidity and the inhibition of
fatty acid biosynthesis) and because different mechanisms
may involve different fatty acid-microbe pairings and different
environmental conditions. Sebum fatty acids tend to be more active toward Gram-
positive bacteria (e.g., C. acnes, S. aureus) and lack efficacy
against Gram-negative bacteria (e.g., E. coli, P. aeruginosa). This
has been associated with the fact that the lipopolysaccharide-
coated outer membrane of Gram-negative bacteria represents an
effective barrier against penetration of hydrophobic compounds
(Nikaido, 1976; Greenway and Dyke, 1979). Sebaceous fatty acid
species can also be effective against certain viruses (Figure 3). Interestingly, the antimicrobial spectra of lauric and sapienic
acids do not completely overlap (Kabara et al., 1972, 1977;
Bergsson et al., 2001; Wille and Kydonieus, 2003; Thormar and
Hilmarsson, 2007; Nakatsuji et al., 2009; Fischer et al., 2012a;
Huang et al., 2014). As is the case with sphingosines, the variation
in effectiveness of different compound-bacterium pairings may
indicate some specificity in their action. Eicosanoids and Related Lipids Eicosanoids are a heterogeneous group of lipids derived
from the metabolism of C20 poly-unsaturated fatty acids
(PUFAs)
such
as
arachidonic
acid
(C20:4ω6),
dihomo-
γ-linolenic
acid
(C20:3ω6),
and
eicosapentaenoic
acid
(C20:5ω3). These
molecules,
together
with
some
related
PUFAs, can be found in the epidermis and they can become FIGURE 4 | Impact of epidermal bioactive lipids on the immune response. 9-HODE, 9-hydroxyoctadecadienoic acid; 12-HETE,
12 h d
i
t t
i
id 15 HET E 15 h d
i
t i
i
id In addition to their direct antimicrobial activity, fatty acids
contribute to the acidification of the surface of the skin – the so-
called “acid mantle” (Schade and Marchionini, 1928) – which in
turn influences which micro-organisms can successfully colonize
the skin and contributes to epidermal integrity (Puhvel et al.,
1975; Fluhr et al., 2001; Hachem et al., 2003). A fascinating example of the crosstalk between the immune
system and the epidermal “shield” is the ability of epidermis-
resident innate lymphoid cells to limit the growth of sebocytes
and influence the presence of antimicrobial fatty acid in the
sebum. To be retained in the tissue, innate lymphoid cells require
secretion of chemokines and cytokines by the epithelium. If
the lymphoid cells are lost, the epidermis reacts by producing
more sebum and increasing sebaceous antimicrobial lipid
content, in turn regulating the composition of the commensal
microbiota (Kobayashi et al., 2019). In another instance that
underscores the close relationship between the regulation of
lipid production and immune response, overexpression of the
transcription factor GATA6 in cultured sebocytes leads to
alterations in the expression of lipid-modifying enzymes and
reduced accumulation of lipids after stimulation with the PPARγ
agonist troglitazone, while at the same time increasing the
levels of anti-inflammatory mediators such as PD-L1 and IL10
(Oulès et al., 2020). Some
antimicrobial
lipids
have
anti-inflammatory
activity. Sapienic
acid
inhibits
gene
expression
resulting
from lipopolysaccharide stimulation in murine macrophages
(Astudillo et al., 2018). Though a relatively high concentration
of the fatty acid was necessary to see the effect (25 µM), it
is still consistent with an anti-inflammatory action in skin,
given the abundance of sapienic acid in sebum (>100 µM). Phytosphingosine
demonstrates
effective
inhibition
of
interleukin-1α (IL-1α) secretion and PKC activation in skin
explants and artificial human epidermis (Pavicic et al., 2007). Similar effects have been reported on a broader range of cytokines
and chemokines (Klee et al., 2007; Brogden et al., 2012). Sebaceous Free Fatty Acids January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 7 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt Lipids as Part of Epidermal Innate and
Adaptive Immunity the physiological equilibrium of the skin or to intervene in the
regulation of epidermal cell behavior (Figure 4). The active role of epidermal lipids in the protection of the
organism should be considered in the wider context of the innate
immune response. The combination of environmental (acidity,
humidity) and biochemical – either protein or lipid – factors of
both host and microbial origin shapes the composition of the skin
microbiota (Grice et al., 2009; Grice and Segre, 2011). All these
components can directly interact with one another; for example,
lipid and proteinaceous antimicrobial factors can have synergistic
action, as in the case of sphingosine and LL-37 or sebaceous fatty
acids and the antimicrobial histone H4 (Lee et al., 2009; Brogden
et al., 2012). Frontiers in Physiology | www.frontiersin.org Eicosanoids and Related Lipids In atopic
dermatitis, a skin condition with an inflammatory component,
levels of both sapienic acid and sphingosines are found to be
downregulated (Arikawa et al., 2002; Takigawa et al., 2005). FIGURE 4 | Impact of epidermal bioactive lipids on the immune response. 9-HODE, 9-hydroxyoctadecadienoic acid; 12-HETE,
12-hydroxyeicosatetraenoic acid; 15-HETrE, 15-hydroxyeicosatrienoic acid;
cysLT, cysteinyl-leukotrienes; PGD2, prostaglandin D2; PGE2, prostaglandin
E2; S1P, sphingosine-1-phosphate; SPC, sphingosylphosphorylcholine. FIGURE 4 | Impact of epidermal bioactive lipids on the immune response. 9-HODE, 9-hydroxyoctadecadienoic acid; 12-HETE,
12-hydroxyeicosatetraenoic acid; 15-HETrE, 15-hydroxyeicosatrienoic acid;
cysLT, cysteinyl-leukotrienes; PGD2, prostaglandin D2; PGE2, prostaglandin
E2; S1P, sphingosine-1-phosphate; SPC, sphingosylphosphorylcholine. Lipids from numerous different classes and cellular origins
can work as paracrine or autocrine mediators through cell-
surface receptors to help determine the type of adaptive immune
response that is triggered after an external stimulus perturbs January 2022 | Volume 12 | Article 804824 8 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt proposed to lead to cytoskeletal rearrangements and inhibition
of cellular movement (Angeli et al., 2001, 2004). substrates of a set of different enzymes. Cyclooxygenases
can generate prostanoids (prostaglandins, prostacyclins, and
thromboxanes). Lipoxygenases can give rise among others
to leukotrienes, a variety of hydroxy-fatty acids [namely
hydroxyeicosatrienoic acids (HETrEs), hydroxyeicosatetraenoic
acids (HETEs), and hydroxyoctadecadienoic acids (HODEs)],
and resolvins. In addition, cytochrome P450 enzymes lead to
the production of such molecules as dihydro-eicosatetraenoic
acids (DHETs) and epoxyeicosatetraenoic acid (EET). All
these lipid species are known autocrine or paracrine biological
mediators, with some of them being active in the epidermis
(Kendall and Nicolaou, 2013). While there is some evidence that resident epidermal cells
express 5-lipoxygenase (5-LOX) – one of the key enzymes for
the generation of leukotrienes – the major source of leukotriene
production in the epidermis is thought to be the infiltrating
immune cells (Janssen-Timmen et al., 1995; Breton et al., 1996). Leukotrienes, particularly cysteinyl-leukotrienes such as LTC4,
have been implicated in the migration of Langerhans cells
toward the lymph nodes, too, as mice lacking 5-LOX display
a strongly reduced Langerhans cell movement (Robbiani et al.,
2000; Doepping et al., 2007). Among prostanoids, prostaglandin E2 is one of the main
mediators of inflammation in skin. PGE2 exerts its action by
binding to four different G-protein-coupled receptors (GDPR),
termed EP1−4 (Tober et al., 2007). In the context of normal
epidermis, PGE2 has been mostly implicated as a vasodilation-
promoting agent in the first phase of the sunburn response
to acute UVB radiation. Eicosanoids and Related Lipids In human epidermis, levels of PGE2
increase between 6 and 24 h after UVB irradiation, leading to the
development of erythema within 48 h (Black et al., 1978; Rhodes
et al., 2001, 2009). In mice, it has been shown that PGE2 produced
in the epidermis exerts its vasodilatory action by binding to EP2
and EP4 receptors on blood vessels (Kabashima et al., 2007). In
addition to its effects following UVB irradiation, PGE2 possesses
activity in Langerhans cells (epidermal dendritic cells). In a
murine model, ablation of EP4 receptor impaired Langerhans cell
migration toward the lymph nodes following antigen stimulation
and the ability of Langerhans cells to stimulate T lymphocytes,
suggesting a positive modulation of these processes by PGE2
(Kabashima et al., 2003). Epidermal cells can also express 12-LOX and 15-LOX. While
12-HETE is able to attract neutrophils and monocytes to the
epidermis (Dowd et al., 1985), 15-LOX downstream products
appear to have anti-inflammatory activity. 15-HETE and 15-
HETrE can inhibit the release of inflammatory mediators such
as leukotriene B4 (LTB4) from immune cells (Ziboh et al., 2000). Mice deficient for 15-LOX exhibit dramatic inflammatory skin
phenotypes, including extensive immune infiltrates in the skin,
epidermal hyperproliferation and compromised barrier function. These effects are at least partially due to a deficiency in resolvin
D2 (Kim et al., 2018). Oxidative stresses such as UVB radiation can cause oxidation
of linoleic acid in the stratum corneum to the eicosanoid-related
species 9-HODE. This mediator can bind the G2A receptor on
keratinocytes, leading to the release of inflammatory cytokines
and inhibition of proliferation through cell cycle arrest and
DNA synthesis suppression. G2A is also induced by oxidative
stress, making for a co-ordinated ligand/receptor response to
an external insult (Hattori et al., 2008). This mechanism is
particularly interesting, as it shows that an essential component
of the epidermal barrier structure also functions as a sensor for
damage to the barrier itself, highlighting how lipids can have
multi-faceted functions within the epidermis. PGD2 can be produced in the epidermis by Langerhans cells
or by sub-epidermal mast cells (Lewis et al., 1982; Maciejewski-
Lenoir et al., 2006). PGD2 can skew the profile of the T-cell
response induced by antigen presentation toward Th2 cell
activation by inhibiting the secretion of IL-12 from dendritic
cells (Theiner et al., 2006). Accordingly, PGD2 promotes
the recruitment and activation of mast cells and eosinophils
characteristic of a Th2-type immune response. Eicosanoids and Related Lipids This effect is
mediated by PGD2 binding to prostaglandin receptor DP2, also
known as CRTH2, a GPCR that triggers inhibition of adenylate
cyclase and lowers cAMP levels (Gαi signaling), ultimately
leading to cellular mobilization (Hirai et al., 2001; Spik et al.,
2005). PGD2 is able to induce expression of β-defensin-3 (hBD-
3) in keratinocytes by engaging a src/MEK/ERK/c-Fos signaling
axis, through DP2, and can thus bolster the antimicrobial defense
of the epidermis. Moreover, since hBD-3 can trigger mast
cell activation and further release of PGD2, this leads to the
establishment of a positive feedback loop of communication
between mast cells and keratinocytes (Kanda et al., 2010). Endocannabinoids Consistent with this inflammatory phenotype, SPC can also
induce keratinocytes to express intercellular adhesion molecule-1
(ICAM-1), a surface protein necessary for leukocyte recruitment
and retention, as well as the secretion of cytokines TNFα
and IL-6. These phenotypes are in line with the remarkable
increase in SPC levels seen in atopic dermatitis patients
(Imokawa et al., 1999). Endocannabinoids Endocannabinoids are endogenous lipids that can bind and
activate GPC cannabinoid receptors (CB1 and CB2). The most
prominent members of this class of compounds are arachidonic
acid metabolites N-arachidonoyl-ethanolamine (anandamide)
and 2-arachdonoylglycerol (2-AG). Anandamide can work as a
partial agonist for CB1 and CB2 receptors, while 2-AG functions
as a full ligand for both receptors (Sugiura et al., 2006; Smita et al.,
2007). Biosynthesis of these compounds happens “on demand”
in cell membranes through the action of N-acyltransferase
and N-acyl-phosphatidylethanolamine phospholipase D in the
case of anandamide, or the activity of phospholipase C
and diacylglycerol lipase in the case of 2-AG (Wang and
Ueda, 2009). The production of these two endocannabinoids
occurs in epidermal keratinocytes and melanocytes, which
also express both CB1 and CB2 as well as transient receptor
potential vanilloid-1 (TRPV-1) which can also be activated by
endocannabinoids (Maccarrone et al., 2003; Tóth et al., 2011;
Pucci et al., 2012). PGD2
can also be released by the helminth parasite
Schistosoma mansoni to inhibit the migration of Langerhans cells
to the lymph nodes following tumor necrosis factor-α (TNF-
α) stimulation. In this case, the release of Langerhans cells is
impaired by PGD2 binding to prostaglandin receptor DP1 and
is dependent on the subsequent adenylate cyclase activation and
rise in cAMP levels (Gαs signaling). Increased Gαs signaling is Generally,
endocannabinoids
possess
anti-inflammatory
activity, being able to suppress T cell receptor signaling Frontiers in Physiology | www.frontiersin.org January 2022 | Volume 12 | Article 804824 9 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt and
inhibit
dendritic
cell-mediated
immune
stimulation
(Wacnik et al., 2008; Börner et al., 2009). In skin, the
endocannabinoid system has been implicated in the attenuation
of the inflammatory response during contact hypersensitivity
reactions. When challenged with an obligate contact allergen,
mice lacking cannabinoid receptors or treated with receptor
antagonists show increased signs of inflammation and immune
infiltration. Conversely, mice that accumulate anandamide due
to the lack of its catabolic enzyme fatty acid amide hydrolase
(FAAH) have reduced inflammatory responses after allergen
treatment. Interestingly, stimulation by the contact allergen
produces an increase in the levels of anandamide and 2-AG. Monocyte chemotactic protein 2 (MCP2) is one of the genes
most upregulated in the absence of cannabinoid receptors
(Karsak et al., 2007). Consistent with this, anandamide can lower
the levels of IL-6 and MCP1 released by keratinocytes following
TNF-α stimulation (Leonti et al., 2010). Sphingosine-1-Phosphate The hydrolysis of ceramides by ceramidases can release
sphingosine bases that become substrates for sphingosine kinases,
yielding sphingosine-1-phosphate (S1P). Due to its relatively low
hydrophobicity and presence of a polar headgroup, S1P can
exit the membrane space and move into solution but is not
able to readily flip-flop across membrane leaflets. S1P is mostly
found in serum and interstitial fluid, is strongly released during
platelet degranulation, and exerts its actions in a paracrine or
autocrine manner through GPCRs (termed S1P1−5) as well as
intracellularly (Hannun and Obeid, 2008; van Meer et al., 2008;
Gomez-Larrauri et al., 2020). Lipid Modulation of Epidermal Cell
Behavior Sphingosine-1-phosphate generally behaves as a mitogen
and a migration-stimulatory agent. However, some of its
effects in skin go against this trend. S1P interferes with the
migration of Langerhans cells toward the lymph node (Reines
et al., 2009). Additionally, it affects how these dendritic cells
stimulate T lymphocytes by promoting Th2 cell activation
(Müller et al., 2005). The journey of a keratinocyte from basal layer stem cell to
stratum corneum corneocyte is influenced by several cellular
signaling pathways that control the balance between proliferation
and differentiation, exit from the stem cell compartment
and progression through the epidermal layers. Some of the
prominent signaling molecules involved in these processes
include β-catenin, extracellular-regulated kinase (ERK) and p38
mitogen-activated protein kinases (MAPK), phosphoinositide-3-
kinase (PI3K)/RACα serine/threonine-protein kinase (Akt) and
PKC. More specifically, accumulation of β-catenin in the nucleus
promotes expansion of the stem cell compartment (Zhu and
Watt, 1999; Watt and Collins, 2008) and high ERK activity
is associated with more proliferative/stem-like keratinocytes
(Hiratsuka et al., 2020). Conversely, p38 MAPK inhibition blocks
the induction of differentiation of cultured human keratinocytes
(Connelly et al., 2011). Activation of the PI3K/Akt pathway has
been linked with the stimulation of keratinocyte differentiation
(Janes et al., 2004, 2009). PKC is required for keratinocyte
differentiation in response to a range of stimuli; for example
blocking PKC can prevent cells from undergoing suspension-
induced commitment and differentiation (Adhikary et al., 2010;
Mishra et al., 2017). Sphingosine-1-phosphate is also involved in the response to
bacterial invasion. Secretions from P. aeruginosa and S. aureus
contain ceramidase or sphingomyelinase, respectively, that can
stimulate the production of S1P. Subsequent binding to S1P1 and
S1P2 receptors on the surface of keratinocytes can trigger the
expression and release of inflammatory cytokines such as TNFα,
IL-8, and IL-36γ, making S1P function as an “alarm signal” for
the immune response (Oizumi et al., 2014; Igawa et al., 2019). UNDER-RECOGNIZED ACTORS – THE
ROLE OF LIPID SIGNALING IN
EPIDERMAL HOMEOSTASIS For nearly a century of research, lipids have played a
center-stage role in studies of the structural integrity and
protection of the skin barrier. However, recent evidence
is beginning to reveal an even more dynamic role for
lipid mediators in signaling events that are key to the
maintenance of epidermal homeostasis. Lipid molecules can
act in an autocrine, paracrine or intracellular fashion to
modulate several aspects of epidermal cell behavior, particularly
keratinocyte differentiation, and the extent of their action
has likely been underappreciated so far. In this respect, it
is important to understand the enormous diversity of lipid
species present in the epidermis and how this impacts the
biology of the skin. Sphingosylphosphorylcholine Another sphingolipid, sphingosylphosphorylcholine (SPC, also
known as lysosphingomyelin), arising from the action of
sphingomyelin deacylase on sphingomyelin, has biological
activity in epidermal cells. SPC is sufficiently polar to be able
to move between different membranes and enter solution; it is
thought to exert its effects through low-affinity binding to S1P
receptors as well as intracellularly (Nixon et al., 2008; van Meer
et al., 2008). SPC has pro-inflammatory effects on keratinocytes,
increasing production of reactive oxygen species, activating
COX-2 and increasing PGE2 production (Choi et al., 2010). Complementing our understanding of these well characterized
signaling proteins, various different classes of lipids play
a role in the regulation and maintenance of epidermal
homeostasis (Figure 5). January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 10 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt n bioactive lipid species active in the regulation of keratinocyte differentiation. Use of the indeterminate article in front of compound name denotes if
es for a certain compound are possible; in these cases, all “variable” fatty acid moieties are shown as C18:0 but can be substituted by alternative
ng in chain length and degree of unsaturation. Parentheses denote abbreviations, while brackets indicate the specific name of the structure
applicable. FIGURE 5 | Main bioactive lipid species active in the regulation of keratinocyte differentiation. Use of the indeterminate article in front of compound name denotes if
multiple structures for a certain compound are possible; in these cases, all “variable” fatty acid moieties are shown as C18:0 but can be substituted by alternative
fatty acids differing in chain length and degree of unsaturation. Parentheses denote abbreviations, while brackets indicate the specific name of the structure
displayed, when applicable. Eicosanoids and Related Lipids in this context does not affect adenylate cyclase activity but
results in an increase in the levels of intracellular diacylglycerol
and ceramide, both of which can promote keratinocyte
differentiation. This suggests the tantalizing possibility of a
lipid-mediated coupling mechanism between proliferation and
differentiation in basal keratinocytes (Konger et al., 2005b;
Figure 6). Besides their role in the modulation of the immune response,
eicosanoids and related lipids also intervene in the regulation of
keratinocyte differentiation. Epidermal keratinocytes express all
four prostaglandin EP receptors bound by PGE2. The different
receptors possess varying affinity for PGE2, with EP1 and
EP2 having affinities in the nanomolar range, while EP3 and
EP4 bind to PGE2 in the sub-nanomolar range. The lower-
affinity EP2 receptor, expressed both basally and suprabasally,
is responsible for stimulating keratinocyte proliferation, possibly
in response to increased PGE2 levels during inflammatory
events. This mitogenic effect is mediated by Gαs signaling
and activation of adenylate cyclase (Konger et al., 1998,
2005a; Tober et al., 2007). In contrast, binding of PGE2
to the high affinity, basally expressed, EP3 receptor leads
to keratinocyte growth inhibition, indicating a potential role
for PGE2 in the limitation of basal cell proliferation in
homeostatic conditions. Interestingly, EP3 receptor activation The
linoleic
acid
derivative
13-HODE
can
promote
keratinocyte
differentiation
through
activation
of
NF-κB
(Ogawa et al., 2011). While the NF-kB pathway is mainly
associated with inhibition of proliferation and protection form
apoptosis in keratinocytes (Seitz et al., 2000), it was also shown to
promote expression of differentiation-associated proteins such
as Keratin 10 and Keratin 1. Treatment of keratinocytes with 13-
HODE activates IκB kinase (IKK), which in turn phosphorylates
and inactivates Inhibitor of κB (IκB) and stimulates NF-κB
activity, leading to an increase in the expression of Keratin 1
(Ogawa et al., 2011). January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 11 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt FIGURE 6 | Receptor-mediated lipid signaling in keratinocytes. Yellow halos around proteins denote activation, while gray halos denote inhibition. Arrows next to
metabolites/proteins denote increase or decrease in levels/expression. cAMP, cyclic adenosine monophosphate; LPA, lysophosphatidic acid; PGE2, prostaglandin
E2; S1P, sphingosine-1-phosphate. FIGURE 6 | Receptor-mediated lipid signaling in keratinocytes. Yellow halos around proteins denote activation, while gray halos denote inhibition. Arrows next to
metabolites/proteins denote increase or decrease in levels/expression. cAMP, cyclic adenosine monophosphate; LPA, lysophosphatidic acid; PGE2, prostaglandin
E2; S1P, sphingosine-1-phosphate. Diacylglycerols and the Protein Kinase C Pathway Diacylglycerols and the Protein Kinase C Pathway
As mentioned above, the PKC pathway can be directly
regulated by lipids. Depending on the isoform, PKC activity is
modulated by both intracellular calcium levels and presence of
diacylglycerol/lipids (-α -β -γ), by diacylglycerol/lipids alone (-
δ, -ε, -η, -θ), or by allosteric interaction with other proteins (-ζ
-ι/λ) (Nishizuka, 1992; Rosse et al., 2010). The two PKC isoforms
that are most relevant to keratinocyte differentiation – PKCδ and
PKCη – are insensitive to calcium but activated by the presence
of phosphatidylserine and diacylglycerol (Adhikary et al., 2010). Moreover, lysophosphatidylcholines and unsaturated fatty acids
can further enhance the activation of PKC (Bronfman et al.,
1988; Shinomura et al., 1991; Nishizuka, 1992). Presence and
activation of PKCδ and η, and the subsequent triggering of
specific MAPK enzymes (MEKK, MEK-6, MEK-3, and p38δ),
are necessary for the induction of keratinocyte differentiation in
response to a number of stimuli (Figure 7; Adhikary et al., 2010;
Mishra et al., 2017). Eicosanoids and Related Lipids endocannabinoid system potentially represents a direct link
between exposure to damaging sunlight and melanin production
by melanocytes (Pucci et al., 2012). CYP2B19, expressed in the granular layer of mouse epidermis,
produces 14,15-EET, which enhances the local activity of
transglutaminases in the formation of the cornified cell envelope. The mechanisms through which this is achieved remain unclear,
but no receptors for 14,15-EET have been robustly identified,
suggesting that this mediator may be acting intracellularly on
the complex regulation of transglutaminase activity (Figure 7;
Gibson et al., 1996; Kalinin et al., 2002; Ladd et al., 2003). Frontiers in Physiology | www.frontiersin.org Endocannabinoids Anandamide
inhibits
keratinocyte
differentiation
through
interaction with CB1, suppression of PKC and subsequent
suppression of activator protein 1 (AP-1) transcription factor
action (Figure 6). Keratinocytes treated with anandamide
show decreased expression of cornified cell envelope proteins
(such as involucrin and loricrin) and reduced transglutaminase
activity. Keratinocyte anandamide levels are regulated during
differentiation by increasing their degradation through FAAH
(Maccarrone et al., 2003). In melanocytes, endocannabinoids (likely from neighboring
keratinocytes) can enhance melanin production by increasing
tyrosinase expression following p38/ERK1/ERK2 and CREB
activation downstream of CB1. Since keratinocytes can increase
their anandamide synthesis in response to UVB radiation, the The specific acyl residues contained in diacylglycerol, as well
as its hydrophilicity/hydrophobicity profile, can influence the January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 12 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt FIGURE 7 | Intracellular lipid signaling in keratinocytes. Arrows denote activation, while “T” symbols denote inhibition. Arrows crossing the DNA symbol indicate
transcription, while dotted lines represent translation. Arrows going through PPARs are depicted with different shadings for visual clarity. Protein colors indicate
approximate function: transcription factors in dark blue, signaling mediators in green, and structural/differentiation-associated proteins in dark red. 13-HODE,
13-hydroxyoctadecadienoic acid; 14,15-EET, 14,15-epoxyeicosatrienoic acid; 15d-PGJ2, 15-deoxy-prostaglanding J2; 15-HETrE, 15-hydroxyeicosatrienoic acid;
C1P, ceramide-1-phopshate; DAG, diacylglycerol; S1P, sphingosine-1-phosphate; SPC, sphingosylphosphorylcholine. FIGURE 7 | Intracellular lipid signaling in keratinocytes. Arrows denote activation, while “T” symbols denote inhibition. Arrows crossing the DNA symbol indicate
transcription, while dotted lines represent translation. Arrows going through PPARs are depicted with different shadings for visual clarity. Protein colors indicate
approximate function: transcription factors in dark blue, signaling mediators in green, and structural/differentiation-associated proteins in dark red. 13-HODE,
13-hydroxyoctadecadienoic acid; 14,15-EET, 14,15-epoxyeicosatrienoic acid; 15d-PGJ2, 15-deoxy-prostaglanding J2; 15-HETrE, 15-hydroxyeicosatrienoic acid;
C1P, ceramide-1-phopshate; DAG, diacylglycerol; S1P, sphingosine-1-phosphate; SPC, sphingosylphosphorylcholine. Similar findings were also reported for diacylglycerol containing
the
anti-inflammatory
eicosanoid-related
lipid
15-HETrE
(Cho and Ziboh, 1997). degree to which PKC becomes regulated (Mori et al., 1982;
Molleyres and Rando, 1988). In support of this, the eicosanoid-
related
molecule
13-HODE,
a
hydroxylated
linoleic
acid
metabolite produced in the epidermis, becomes incorporated
into membrane phosphatidylcholines and phosphatidylinositols,
which in turn can be converted to 13-HODE-containing
diacylglycerol
(Cho
and
Ziboh,
1994b). This
particular
diacylglycerol is able to downmodulate the activity and
expression of PKCβ (Figure 7). Endocannabinoids Interestingly, inhibition of
this PKC isoform by 13-HODE has been correlated with
the rescue of a keratinocyte hyperproliferation phenotype in
guinea pigs (Cho and Ziboh, 1994a, 1995; Ziboh et al., 2000). Frontiers in Physiology | www.frontiersin.org Sphingolipids Sphingolipids are one of the most functionally multifaceted
lipid classes in the epidermis. They play a fundamental role in
the formation and the structure of the epidermal barrier and
also participate in the protection of the epidermis. Additionally,
several sphingolipid species take part in epidermal signaling
(Hannun and Obeid, 2008; Gomez-Larrauri et al., 2020). These
include ceramides, glycosylceramides, ceramides-1-phosphate, January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 13 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt produces an expansion of the hair follicle stem cell compartment
(Peters et al., 2015). The different effects of alkaline ceramidase
and CerS4 deletion can be reconciled by the fact that while the
absence of alkaline ceramidase produces a generalized increase
of all ceramide species, lack of CerS4 produces a decrease of
some ceramide species, but an increase in others, indicating that
only specific ceramide subspecies might influence the behavior
of murine epidermal stem cells. In addition to its effect on the
stem cell compartment, epidermal deletion of CerS4 impairs
maintenance of the epidermal barrier in adult mice, leading
to increased trans-epidermal water loss, hyperkeratosis and
accumulation of immune cells. This effect is notable because
CerS4 catalyzes the synthesis of shorter-chained ceramide species
that are not directly involved in the structure of the barrier. These
species or other lipids indirectly affected by CerS4 knockdown
may thus be involved in signaling events that are necessary for
epidermal homeostasis (Peters et al., 2020). sphingosine-1-phosphate,
and
sphingosylphosphorylcholine. The metabolism of sphingolipids is remarkably interconnected,
with interconversion between different species occurring readily
depending on the relevant enzymes’ presence and abundance
(Hannun, 1994; Hannun et al., 2001; Hannun and Obeid, 2008). Ceramides can be formed de novo by the condensation of
palmitate and serine catalyzed by serine palmitoyl transferase
and the subsequent activity of ceramide synthase. Alternatively,
they can be derived from either sphingomyelin through the
action of sphingomyelinases or glucosylceramides through the
action of glucocerebrosidase. They can be mainly found in the
ER membranes (where their de novo synthesis occurs) or at
the plasma membrane. Due to their physico-chemical properties
they are unable to transfer freely between different cellular
compartments, needing either vesicular transport or protein
mediators to move across membranes. They can, however,
flip-flop between membrane leaflets, though this may too be
subject to regulation (Hannun and Obeid, 2008; van Meer
et al., 2008). Sphingolipids Ceramides act as signaling molecules in a variety
of settings and are mostly known for inducing apoptosis and
responding to cellular stress (Uchida, 2014; Uchida and Park,
2021). In vitro, treatment of keratinocytes with exogenous short-
chained (C2–C8) ceramides can inhibit their proliferation and
induce differentiation (Wakita et al., 1994). In contrast to ceramides, glucosylceramide has mitogenic
effects
on
epidermal
keratinocytes
when
administered
subcutaneously in mice (Figure 7; Marsh et al., 1995). Conversely, certain glycosylceramides such as the ganglioside
GM3, which are present on the outer leaflet of keratinocyte
plasma membranes, have an antiproliferative effect when
supplemented
in
the
medium
of
cultured
keratinocytes
(Paller et al., 1993). Gangliosides (ceramides conjugated to
oligosaccharides containing sialic acid) have been implicated
in some important membrane dynamics in the early phases
of
keratinocyte
differentiation. When
basal
keratinocytes
commit to differentiation, the adherens junctions connecting
the basal cells to one another must relax to allow the cells to
delaminate to the suprabasal layers. In order for this process to
occur, desmosomes containing the differentiation-dependent
cadherin desmoglein 1 (Dsg1) must be localized within insoluble
membrane compartments enriched in gangliosides. Interfering
with the compartmentalization of Dsg1 in these lipid domains
impairs the release of tension at the adherens junctions and
leads to reduced keratinocyte delamination from monolayers
(Figure 7; Nekrasova et al., 2018). Both exogenous short-chained (C2 and C6) ceramides and
endogenous ceramides can directly regulate the expression
of certain differentiation-related genes in keratinocytes. One
example is glucosylceramide transporter ABCA12, involved in
the translocation of glucosylceramides into lamellar bodies,
whose transcription is enhanced by ceramide through increased
PPARβ/δ expression (Jiang et al., 2009). Another case is the
expression of caspase-14 – a protein present in differentiated
keratinocytes and responsible for the processing of profilaggrin
into filaggrin, important for the appropriate hydration of
the stratum corneum. Caspase-14 expression can be directly
stimulated by ceramides, although the mechanism does not
involve PPARβ/δ (Jiang et al., 2013). C2 ceramide administered
in vitro has been shown to strongly reduce Akt activity in
melanocytes, to inhibit their growth, and to decrease tyrosinase
activity and consequent melanin production (Kim et al., 2001). Ceramides can also intervene in the response of epidermal
keratinocytes to UVB radiation. Exposing keratinocytes to UVB
increases the de novo synthesis of ceramides, which in turn
participate in the subsequent induction of apoptosis by a caspase-
independent mechanism (Uchida et al., 2003; Figure 7). Frontiers in Physiology | www.frontiersin.org Lysophosphatidic Acid Besides its activity through GPCRs, S1P can act intracellularly
to influence the behavior of keratinocytes. Activation of
sphingosine kinase 1, one of the two enzymes catalyzing S1P
biosynthesis, mobilizes calcium from intracellular stores, leading
to inhibition of keratinocyte proliferation and stimulation of
differentiation (Hong et al., 2008). Similarly, inhibition of
S1P lyase, the main enzyme responsible for S1P catabolism,
increases intracellular S1P levels and causes cell cycle arrest
and expression of differentiation markers (Jeon et al., 2020). In response to ER-stress, which can be caused for example
by perturbation of the epidermal barrier, S1P levels rise and
promote NF-κB activation via assembly of an intracellular
signaling complex, causing the stimulation of C/EBPα-dependent
transcription, and ultimately leading to increased expression
of the cathelicidin LL-37 and enhanced epidermal protection
(Park et al., 2013, 2016; Figure 7). The effect of ER-stress-
induced S1P on cathelicidin LL-37 mimics that of ER-stress-
induced C1P on β-defensins (Kim et al., 2014), resulting
in a synergistic response to external injury via two related
sphingolipid metabolites. Lysophosphatidic acids (LPAs) are the simplest phospholipid,
formed by a glycerol backbone attached to a phosphate group,
a fatty acid, and a hydroxyl moiety. LPA can be synthesized
intracellularly by glycerophosphate acyltransferase, acylglycerol
kinase or phospholipase A. Alternatively, it can be generated
extracellularly by the secreted lysophospholipase D autotaxin. Degradation of LPA is mainly carried out by lipid phosphate
phosphatases. The single aliphatic chain of LPA makes it
sufficiently soluble to leave membranes (van Meer et al., 2008; Lei
et al., 2018). In addition to being a precursor in phospholipid biosynthesis,
LPA is a ligand of a series of GPCRs – LPA1−6 – through which
it exerts its biological activity. Some of these receptors belong
to the same family as S1P receptors, and S1P and LPA share
some structural similarity (Geraldo et al., 2021). A dependence
of the biological activity of LPA on its acyl-chain length has been
reported (van Corven et al., 1992). During wound repair, LPA is delivered to the injury site mainly
through platelet degranulation and its effects are akin to S1P:
keratinocyte migration is enhanced, with increases in expression
of laminin-332, TGF-α and TGF-β. Unlike S1P, however, LPA
appears to stimulate keratinocyte proliferation, although this may
be an indirect consequence of the induction of TGF-α expression
rather than direct LPA-mediated signaling (Figure 6; Piazza et al.,
1995; Demoyer et al., 2000; Amano et al., 2004). Sphingolipids Ceramides-1-phosphate (C1Ps) are sphingolipid mediators
that generally exert opposing effects to those of ceramides
(e.g., increased cell survival, increased proliferation), and
phosphorylation by ceramide kinase is usually considered a
way in which ceramides can be inactivated. C1Ps can be
found in the Golgi apparatus and the plasma membrane. They
are limited in their mobility between membranes, and owing
to their polar head group are unlikely to flip-flop between
membrane leaflets (Hannun and Obeid, 2008; Uchida, 2014;
Gomez-Larrauri et al., 2020). In keratinocytes, a role for C1P
has been described during cellular stress. Serum starvation of
mouse keratinocytes induces apoptosis which is mitigated by
the activity of PPARβ/δ. Among its targets is ceramide kinase,
whose activity is necessary to reduce the levels of intracellular
ceramides and prevent excessive apoptosis (Tsuji et al., 2008). C1P produced in response to endoplasmic reticulum stress leads
to an increase in the expression of β-defensin 2 and 3 (hBD-
2/3). This suggests a model whereby C1P activates cellular
phospholipase A2, causing the release of arachidonic acid from Interfering with sphingolipid metabolism in vivo not only,
as predicted, leads to disruption of epidermal homeostasis but
has also a surprising impact on keratinocyte differentiation. Eliminating alkaline ceramidase, an enzyme responsible for
ceramide degradation, in mouse skin leads to an abnormal
accumulation of ceramides, which in turn compromise the
barrier function of the epidermis and cause alterations in
the final stages of keratinocyte differentiation and an increase
in the epidermal stem cell population of the hair follicle
(Liakath-Ali et al., 2016). In apparent contrast, knockout of ceramide synthase 4
(CerS4), an enzyme responsible for ceramide synthesis, in mice January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 14 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt urokinase-type plasminogen activator (uPA) and its receptor
(uPA-R) (Figure 7; Wakita et al., 1998). phospholipids and an increase of 15-deoxy-PGJ2; this leads to
activation of PPARα or -β/δ, which promotes expression of Src
and stimulates STAT1/3-mediated expression of the β-defensin
genes (Figure 7; Kim et al., 2014). phospholipids and an increase of 15-deoxy-PGJ2; this leads to
activation of PPARα or -β/δ, which promotes expression of Src
and stimulates STAT1/3-mediated expression of the β-defensin
genes (Figure 7; Kim et al., 2014). Sphingomyelins (SMs) are synthesized in the Golgi apparatus
and found at the plasma membrane. Sphingolipids Their double aliphatic chain
makes them insoluble in water and their polar phosphocholine
group
impairs
flip-flopping
between
membrane
leaflets
(Hannun and Obeid, 2008; van Meer et al., 2008). Unlike
other sphingolipids, SMs are a major component of cellular
membranes, which makes them less likely to be able to act as
bioactive mediators. In fact, SMs are often considered a reservoir
for the generation of bioactive sphingolipids (Hannun, 1994;
Hannun et al., 2001; Wanner et al., 2004). However, variation
in SM levels can influence the biophysical characteristics of
the membrane such as, for example, fluidity, stiffness and
mechanosensory properties, which in turn can drive epidermal
cellular responses (Mobasseri et al., 2019). Moreover, by virtue
of the mostly saturated nature of its acyl chains, SMs can form
lipid aggregates with cholesterol within membranes which can
influence protein interaction dynamics. While the existence
of such lipid “rafts” in biological membranes in vivo has been
controversial, they have been observed in artificial bilayers
mimicking the lipid composition of the plasma membrane
(Dietrich et al., 2001; Samsonov et al., 2001; Simons and Sampaio,
2011; Levental et al., 2020). Early studies showed that epidermal
desmosome preparations are enriched in cholesterol, SM and
gangliosides (Drochmans et al., 1978); more recently, rafts have
been implicated in desmosome assembly and dismantling in
keratinocytes (Stahley et al., 2014). Along with its function in immunity, spiingosine-1-phosphate
(S1P) also inhibits proliferation and promotes differentiation
of keratinocytes (Vogler et al., 2003). Binding of S1P to S1P2
antagonizes the proliferative signals coming from the insulin
receptors by activating PKCδ, causing dephosphorylation of Akt
and leading to inhibition of cyclin D2 and upregulation of cyclin-
dependent kinase inhibitors p21 and p27Kip1 (Kim et al., 2004;
Schüppel et al., 2008). Moreover, signals mediated through S1P3
are reported to increase the intracellular calcium concentration
in keratinocytes, which has been linked to differentiation (Watt
et al., 1984; Lichte et al., 2008; Figure 6). In addition to proliferation inhibition and in sharp contrast
to its role in Langerhans cells, S1P stimulates keratinocyte
migration. This effect is enacted by S1P receptors through
transforming growth factor-β (TGF-β)-receptor I-mediated
phosphorylation and activation of Smad3 (Figure 6). In fact, S1P
and TGF-β treatment cause similar phenotypes in keratinocytes,
both in terms of general cellular behavior and in terms of
specific gene expression changes; for example, both ligands
can increase laminin 5 production (Amano et al., 2004; Sauer
et al., 2004). Sphingolipids The overlap between S1P and TGF-β signaling is
likely important during the complex orchestration of the wound
healing process, when both these ligands as well as multiple
other mediators are abundantly produced (Vogler et al., 2003;
Rognoni and Watt, 2018). Frontiers in Physiology | www.frontiersin.org An Unbiased Screen for Lipid Regulators
of Differentiation While we have presented evidence that separate lipid classes
play a role in signaling to impact keratinocyte proliferation,
differentiation and other processes, this is inevitably an
oversimplification. The lipid species produced in the cell and the
enzymes that modify them form an extremely intricate network
and it is difficult to predict the multiple perturbations that ensue
from a specific intervention, such as depletion of a single lipid
modifying enzyme (Muro et al., 2014). Lipid subspecies can
influence cellular biology in ways that go beyond their direct
interaction with proteins as ligands or allosteric modulators, by
for example working as ensembles that can facilitate (or impede)
the activity of groups of molecular partners (Muro et al., 2014). Activation of all PPAR isoforms or LXRs pushes keratinocytes
to differentiate both in vitro in human keratinocytes and in vivo
in mouse epidermis (Hanley et al., 1998, 2000b,a; Mao-Qiang
et al., 2004; Schmuth et al., 2004). More specifically, while all
receptors stimulate expression of differentiation markers, PPARα
and LXR also cause inhibition of keratinocyte proliferation
(Hanley et al., 1998, 2000b). PPARs or LXR activation induce
accumulation of lipids as well as expression of lipid metabolism
enzymes, including ones involved in lamellar body biogenesis,
and stimulate lamellar body secretion when activated topically
in mouse epidermis (Rivier et al., 2000; Man et al., 2006; Jiang
et al., 2008, 2010; Lu et al., 2010). While the expression of
lipid-modifying enzymes appears to be directly regulated by the
receptors’ transcriptional activity, the kinetics of induction of
differentiation markers suggests an indirect regulation (Hanley
et al., 1998; Schmuth et al., 2004). Despite these challenges, newly developed techniques or
refinement of established methods are beginning to shed more
light on the potential contribution of endogenous cellular
lipid subspecies to a variety of cellular processes. Prompted
by the finding that the alteration of the ceramide profile in
mouse epidermis could lead to an effect on the stem cell
compartment (Peters et al., 2015; Liakath-Ali et al., 2016),
our lab recently performed lipidomics in a well characterized
in vitro keratinocyte differentiation system combined with a
lipid-modifying enzyme siRNA screen to evaluate the changes in
cellular lipid composition occurring during differentiation and to
try to understand which changes might be important for the early
phases of this process. Our study identified several ceramide and glucosylceramide
subspecies that could induce keratinocyte differentiation when
administered exogenously in vitro (Vietri Rudan et al., 2020). Peroxisome Proliferator-Activated Receptors
Signaling g
g
Other lipid-sensitive proteins that have been shown to be
involved in the regulation of keratinocyte differentiation are
peroxisome proliferator-activated receptors (PPARs) and liver
X receptors (LXRs). When activated by a lipid ligand, PPARs
or LXR heterodimerize with retinoid X receptor (RXR) and
regulate gene expression. Three PPAR isoforms (α-, β/δ-, γ-)
exist, all of which are expressed in keratinocytes: while PPARβ/δ
expression is constitutive, PPARα and PPARγ levels increase with
differentiation (Rivier et al., 1998). Both LXR isoforms – α and
β – are constitutively expressed in human keratinocytes (Russell
et al., 2007). PPAR endogenous ligands are fatty acids and their
metabolites, including eicosanoids, while LXRs are activated by
the cholesterol metabolite oxysterol (Bocos et al., 1995; Janowski
et al., 1996). Cholesterol Sulfate Cholesterol metabolites can also participate in the modulation
of keratinocyte differentiation. An example of this is cholesterol
sulfate, whose levels progressively increase over the course
of differentiation, peaking in the granular layer. Treatment
of
human
keratinocytes
with
cholesterol
sulfate
causes
induction of the expression of the retinoic acid receptor-
related orphan receptor α (RORα), which in turn is able to
elicit transcription of the profilaggrin gene (Hanyu et al., 2012;
Figure 7). This mechanism provides another link between the
constantly changing protein and lipid landscape of differentiating
keratinocytes, further underscoring the functional connection of
these changes in the creation of the epidermal barrier. Lysophosphatidic Acid An interesting
study examining skin blisters showed that LPA production is
increased in blister fluid by autotaxin activity and expression of Sphingosylphosphorylcholine (SPC) appears to promote an
abnormal route of differentiation, with increased intracellular
calcium levels and activation of transglutaminase 1, at the same
time as inhibiting the expression of cornified cell envelope
proteins such as profilaggrin and loricrin (Higuchi et al.,
2001; Choi et al., 2010). SPC also promotes re-epithelialization
during wound healing by acting as a keratinocyte mitogen and
increasing migration through stimulation of the expression of January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 15 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt LPA1 is also enhanced, pointing to the relevance of this signaling
in vivo (Mazereeuw-Hautier et al., 2005). transcription of differentiation proteins has been proposed as
a coupling mechanism to coordinate the changes in lipids and
proteins occurring during epidermal maturation (Figure 7; Rivier
et al., 2000; Man et al., 2006; Jiang et al., 2008, 2010; Lu et al.,
2010). When treating keratinocytes with LPA, an induction of
differentiation can be observed, potentially through multiple
pathways. LPA induces an increase of intracellular calcium
concentration, likely through binding to LPA2 (Lichte et al.,
2008). Binding to LPA1 or LPA5 instead leads to an increase in
the differentiation marker pro-filaggrin and alteration of cellular
morphology, signaling via Gα12/13 to RhoA-ROCK and activating
Serum Response Factor (SRF) (Figure 6). The SRF pathway is
involved in keratinocyte differentiation induced by restricted
spreading on micropatterned substrates (Connelly et al., 2010;
Sumitomo et al., 2019). The increase in filaggrin triggered
by LPA treatment provides a likely molecular explanation
for the beneficial effects of LPA on skin moisturization
(Yahagi et al., 2011). Frontiers in Physiology | www.frontiersin.org An Unbiased Screen for Lipid Regulators
of Differentiation Using such a combined approach it was possible to leverage
the high specificity of protein-targeting techniques while keeping
the focus on the lipid changes. This is key because of the
cascading effects of lipid enzyme perturbation and the potential
presence of compensatory mechanisms. In line with this, while
the enzymes that emerged from our siRNA screen (ELOVL1 and In the case of PPARα and LXL, differentiation proceeds
through the activation of AP-1 transcription factors (Hanley
et al., 2000a,b). While there is overlap between the consequences
of activating different PPAR isoforms, the different isoforms
are able to exert their effect independently of one another,
which suggests a certain degree of redundancy. The fact that
PPARs and LXRs are sensitive to endogenous lipid mediators
generated during keratinocyte differentiation and can affect the January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 16 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt FATP1) were directly associated with generation of specific chain-
length saturated and mono-unsaturated fatty acids and fatty acyl
CoAs, the bioactive lipids identified belonged to various different
phospholipid and sphingolipid classes. Conversely, the relative abundance of sebaceous secretions can
determine the abundance of different bacterial species at specific
body sites (Table 1; Grice et al., 2009; Grice and Segre, 2011). Some parasites can secrete lipids to interfere with the host’s
immune response, as is the case for PGD2 secreted by S. Mansoni
(Angeli et al., 2001). The full range of lipid mediators of
microbial origin that participate in their interaction with the host
remains to be explored. While our approach demonstrates the power of an unbiased
lipidomic screen, it is important to note that our lipidomic panel
did not include a number of potentially relevant lipid classes. For example, we did not include fatty acids and fatty acyl-
CoAs, which may regulate keratinocyte differentiation through,
for example, PPAR or PKC signaling (Bronfman et al., 1988;
Bocos et al., 1995). In addition, the size of the effect of bioactive
lipids we observed experimentally will depend as how efficiently
they reach the appropriate subcellular destination. Furthermore,
lipids operate by affecting the functionality of whole assemblies
of molecular partners, including proteins and carbohydrates,
which makes it harder to evaluate their role using the linear
approaches that are typically employed when studying protein
signaling pathways. DISCUSSION With lipids playing such an important functional role
in the epidermis, it is not surprising that they intervene
in the regulation of keratinocyte differentiation and in the
maintenance of epidermal homeostasis. In this review, we
have highlighted numerous examples of how bioactive lipid
mediators from several different classes can affect in these
processes, whether by influencing membrane characteristics or
participating in cellular signaling. The importance of membrane
properties in keratinocytes is highlighted for example by the
interaction between key proteins involved in their differentiation,
desmosomal cadherins, and membrane lipid domains to ensure
the appropriate assembly and disassembly of these functional
ultrastructures and in turn guarantee proper delamination from
the basal layer (Stahley et al., 2014; Nekrasova et al., 2018). We have described how in the epidermis the physical, chemical
and biological characteristics of lipids are all functionally
leveraged to build, protect, and maintain the outer barrier
of the organism. The hydrophobic and fluidity properties of
ceramides, cholesterol and fatty acids are exploited to create
an impermeable, resistant, yet flexible, dynamic structure in
the stratum corneum. Nevertheless, despite nearly one hundred
years of research into the physiology of the body’s outer
boundary, questions still remain as to how exactly different
ceramide species participate in the generation of the exquisite
biophysical properties of the cornified layer. This is not
surprising, considering that the composition of the stratum
corneum can vary quite extensively in different species (e.g., in
birds), while maintaining its fundamental microstructure and
functional characteristics (Champagne et al., 2012). Similarly,
mammals living in cold environments, such as arctic foxes
or reindeer, have dramatically lower melting points for the
lipid mixtures isolated from their extremities as compared
to the ones obtained from the body core, reflecting the
fact that their peripheral tissues can be up to 40◦C colder
than their central tissues (Irving et al., 1957). How this
adaptation can influence the properties, microstructure, and
composition of the epidermis of these animals is yet to be
fully elucidated. More generally, the apico-basal polarity of the plasma
membrane is a fundamental feature of epithelial cells, with
keratinocytes being no exception; the relevance of lipids in the
establishment of such oriented cell membranes remains poorly
understood and represents a major avenue for future research
(Ikenouchi, 2018). Moreover, lipids can influence membrane
stiffness, fluidity and mechanosensitivity, which are known to
have an effect on keratinocyte behavior (Mobasseri et al., 2019). An Unbiased Screen for Lipid Regulators
of Differentiation The participation of secreted lipid species of keratinocyte or
sebaceous origin n innate immunity provides a fascinating insight
into how different systems in the body can act in unison to ensure
the preservation of homeostasis and the appropriate response
to external perturbations, and how even foreign micro-organism
colonization can be used as a means to maintain and protect the
outer limit of the body (Gallo and Nakatsuji, 2011; Kobayashi
et al., 2019). Gaining a fuller picture of how antimicrobial lipids
act on their targets, intervene in the inflammatory response and
communicate with the innate and adaptive immune system may
help us in the development of new, more effective, treatments
against pathogens or inflammatory disorders. Frontiers in Physiology | www.frontiersin.org January 2022 | Volume 12 | Article 804824 DISCUSSION However, the extent to which lipids may contribute to the
regulation of these membrane properties, the mechanisms and
species involved, still needs to be fully characterized. There are many factors that can contribute to the biological
activity of lipids. While variation in levels can be a good
indication of the involvement in a biological process – as for
example in the case of cholesterol sulfate in the stratum corneum
(Elias et al., 1984; Sato et al., 1998; Hanyu et al., 2012) – there
are other layers of regulation that likely influence lipid-mediated
regulation of cellular behavior. A characteristic of several lipid
classes is their limited ability, owing to physical and chemical
constraints, to move between different cellular environments or
even across the two leaflets of the same membrane (Hannun and
Obeid, 2008; van Meer et al., 2008). Therefore, cells can selectively
target the activity of specific lipid players to certain subcellular
locales by modulating the expression and activation of transport Far from being only passive building blocks, lipids also
protect the epidermis by way of their chemical features, as in
the fatty acid contribution to the “acid mantle,” and biological
activity against microbes, significantly contributing to shape the
makeup of the commensal microbiota. The crosstalk between
skin surface resident microbes and host factors is extensive. Numerous lines of research have shown that the normal skin
microflora can modulate the expression of antimicrobial peptides
to protect from opportunistic pathogens (Gallo and Nakatsuji,
2011). Lipases of microbial origin are mainly responsible for
the presence of fatty acids in the sebum, and disruption of
the normal microbiome can lead to altered sebum composition. January 2022 | Volume 12 | Article 804824 17 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt molecules and vesicular trafficking processes. The importance
of this level of regulation in the epidermis is underscored by
the diversity of lipid transporters expressed in keratinocytes and
the emergence of severe pathological conditions associated with
transporter loss or dysfunction (Elias et al., 2008; Khnykin et al.,
2011; Vietri Rudan et al., 2020). membranous organelles may play into their functionality, or
how much individual lipid species or lipid features matter
for epidermal homeostasis in vivo. Technological advances will
be key to answering these complex questions. ACKNOWLEDGMENTS We are grateful for funding from the Department of Health
via the National Institute for Health Research comprehensive
Biomedical Research Centre award to Guy’s and St Thomas’
National Health Service Foundation Trust in partnership with
King’s College London and King’s College Hospital NHS
Foundation Trust. Challenges still remain to explore some of the more
elusive aspects of lipid biology, for example how the cellular
distribution and/or traffic of bioactive lipids among the various Challenges still remain to explore some of the more
elusive aspects of lipid biology, for example how the cellular
distribution and/or traffic of bioactive lipids among the various DISCUSSION In this respect,
implementation of recently developed, sophisticated techniques,
like single-cell lipidomics (Capolupo et al., 2021; Li et al., 2021),
already promises more exciting developments in unraveling the
finer details of the roles played by lipids in the regulation of
cellular behavior (Züllig et al., 2020; Capolupo et al., 2021; Li et al.,
2021). We are confident that in the future the application of these
new technologies will provide a clearer picture of the rich and
essential contribution of lipid molecules to epidermal biology. In the case of the modulation of keratinocyte differentiation, it
often remains unclear to what degree the activity of endogenously
generated individual lipid mediators is important and at what
stage of the in vivo differentiation process they might come into
play. An example of this is the fatty acid-mediated regulation
of PPARs: while it is established that fatty acids can regulate
PPAR activity and that they are abundant in keratinocytes, it is
still not clear whether they do so endogenously, at what stage
of the differentiation process, what PPAR isoforms are involved
and, importantly, what fatty acids. This last question points
toward another key fact to be considered: the cell produces
a vast amount of lipid subspecies (varying for example in
carbon chain length, number, and location of unsaturations, and
hydroxylation profile) whose influence on cellular behavior is still
poorly understood and has only recently begun to be investigated. A combination of lipid enzyme targeting and lipidomics has
identified individual lipid subspecies from multiple classes that
can influence keratinocyte differentiation in vitro, underscoring
the still little-explored regulatory potential of the cellular lipid
repertoire (Vietri Rudan et al., 2020). Such comprehensive
strategies can pinpoint individual lipid actors that can take
part in signaling events or provide indications as to what lipid
properties matter most within a certain process. Indeed, studies
are beginning to shed light on the influence of this diverse
collection of lipid molecules on several cellular and biological
activities, such as cell division or immune cell activation (Atilla-
Gokcumen et al., 2014; Köberlin et al., 2015). FUNDING This work was funded by grants to FW from the United Kingdom
Medical Research Council (MR/PO18823/1) and the Wellcome
Trust (206439/Z/17/Z). AUTHOR CONTRIBUTIONS MV and FW wrote and edited the text. MV designed the
figures. Both authors contributed to the article and approved the
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Integrated genomic and metabolomic profiling of ISC1, an emerging Leishmania donovani population in the Indian subcontinent
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Infection, genetics and evolution
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A R T I C L E I N F O Keywords:
Leishmania donovani
Visceral leishmaniasis
Genome diversity
Metabolomics
Genomics
Multi-omics integration
Virulence Keywords:
Leishmania donovani
Visceral leishmaniasis
Genome diversity
Metabolomics
Genomics
Multi-omics integration
Virulence Leishmania donovani is the responsible agent for visceral leishmaniasis (VL) in the Indian subcontinent (ISC). The
disease is lethal without treatment and causes 0.2 to 0.4 million cases each year. Recently, reports of VL in
Nepalese hilly districts have increased as well as VL cases caused by L. donovani from the ISC1 genetic group, a
new and emerging genotype. In this study, we perform for the first time an integrated, untargeted genomics and
metabolomics approach to characterize ISC1, in comparison with the Core Group (CG), main population that
drove the most recent outbreak of VL in the ISC. We show that the ISC1 population is very different from the CG,
both at genome and metabolome levels. The genomic differences include SNPs, CNV and small indels in genes
coding for known virulence factors, immunogens and surface proteins. Both genomic and metabolic approaches
highlighted dissimilarities related to membrane lipids, the nucleotide salvage pathway and the urea cycle in ISC1
versus CG. Many of these pathways and molecules are important for the interaction with the host/extracellular
environment. Altogether, our data predict major functional differences in ISC1 versus CG parasites, including
virulence. Therefore, particular attention is required to monitor the fate of this emerging ISC1 population in the
ISC, especially in a post-VL elimination context. Before 2000, sporadic VL cases were reported in the hilly regions of
Nepal (1 km above sea level) and were considered the result of travel,
rather than a local transmission cycle. However, more recently reports
of VL have been increasing in these Nepalese highlands and transmis-
sion has been shown to occur locally (Ostyn et al., 2015). Interestingly,
some isolates derived from VL patients in this region belong to a ge-
netically distinct population of L. donovani (called ISC1), which di-
verged much earlier than 1850 (Imamura et al., 2016). Rai et al. (2017)
investigated the geographic spread of different L. donovani genotypes
over a period of 12 years (2002–2014) in Nepal and showed that the
frequency of ISC1 was increasing in the country and that this emerging
genotype was also present in the lowlands. Integrated genomic and metabolomic profiling of ISC1, an emerging
Leishmania donovani population in the Indian subcontinent Bart Cuypersa,b, Maya Berga,1, Hideo Imamuraa, Franck Dumetza, Géraldine De Muyldera,
Malgorzata A. Domagalskaa, Suman Rijalc, Narayan Raj Bhattaraic, Ilse Maesa, Mandy Sandersd,
James A. Cottond, Pieter Meysmanb, Kris Laukensb,2, Jean-Claude Dujardina,e,⁎,2 a Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium
b Department of Mathematics and Computer Science, University of Antwerp, Antwerp, Belgium
c BP Koirala Institute of Health Sciences, Dharan, Nepal
d Wellcome Trust Sanger Institute, Hinxton, United Kingdom
e Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium a Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium
b Department of Mathematics and Computer Science, University of Antwerp, Antwerp, Belgium
c BP Koirala Institute of Health Sciences, Dharan, Nepal
d Wellcome Trust Sanger Institute, Hinxton, United Kingdom
e Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium a Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium
b Department of Mathematics and Computer Science, University of Antwerp, Antwerp, Belgium
c BP Koirala Institute of Health Sciences, Dharan, Nepal
d Wellcome Trust Sanger Institute, Hinxton, United Kingdom
e Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium A R T I C L E I N F O Our earlier study showed
that out of all sampled ISC1 isolates, 75% of them came from patients
relapsing to miltefosine treatment, vs 50% for the CG strains (Imamura
et al., 2016). We have also demonstrated experimentally that ISC1 https://doi.org/10.1016/j.meegid.2018.04.021
Received 5 December 2017; Received in revised form 14 April 2018; Accepted 17 April 2018
⁎ Corresponding author.
1 Current address: Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium
2 Shared senior authors
E-mail address: jcdujardin@itg.be (J.-C. Dujardin).
Available online 19 April 2018
1567-1348/ ©
2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecom
m
ons.org/licenses/BY/4.0/). https://doi.org/10.1016/j.meegid.2018.04.021
Received 5 December 2017; Received in revised form 14 April 2018; Accepted 17 April 2018
Available online 19 April 2018
1567-1348/ ©
2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecom
m
ons.org/licenses/BY/4.0/). Infection, Genetics and Evolution 62 (2018) 170–178 Infection, Genetics and Evolution 62 (2018) 170–178 Contents lists available at ScienceDirect 1. Introduction Visceral Leishmaniasis (VL) or kala-azar is a neglected tropical dis-
ease globally responsible for 0.2 to 0.4 million cases each year (Alvar
et al., 2012). The disease is lethal without treatment and caused by
protozoan parasites of the Leishmania donovani complex. Transmission
occurs by sand flies of the Phlebotomus genus. In the Indian subcontinent
(ISC), VL is of major concern for public health with > 200 million people
at risk (Ostyn et al., 2011). The recent whole-genome sequencing and
phylogenomic analysis of 204 Leishmania strains from the ISC (India,
Nepal and Bangladesh), revealed that the most recent VL epidemic in this
region were essentially driven by one large L. donovani population that
emerged around 1850 and spread across the lowlands in the Ganges
plain. Given its recent origin, this population is genetically very homo-
geneous (Imamura et al., 2016) and was called the Core Group (CG). B. Cuypers et al. Infection, Genetics and Evolution 62 (2018) 170–178 and small indels were called using the GATK haplotypecaller v3.4
(McKenna et al., 2010). Low quality SNPs were marked using GATK
Variant Filtration with paramers: “QD < 2.0 || MQ < 40 || FS > 60.0 ||
ReadPosRankSum < -8.0” and removed from further analysis. The
same tool was used for filtering indels, but with parameters “QD < 2.0
|| FS > 200.0 || ReadPosRankSum < -20.0 || SOR > 200”. Variants
were annotated with SnpEffv4.1 (Cingolani et al., 2012). SNPs and
small indels were considered significantly different between CG and
ISC1, when the allele shift difference was at least 0.25 (Tihon et al.,
2017) and Mann–Whitney U test p-value < 0.05. Allele shifts larger
than 0.80 were considered homozygous variants (Tihon et al., 2017). Local copy number variation and chromosomal somy were determined
according to Downing et al. (2011). For somy estimation, initially, the
median read depth of each chromosome was calculated (di). All posi-
tions with a read depth > 1 standard deviation away from this initial
median were then removed and di was recalculated. This approach
removed depth outliers by assembly errors, local CNVs or spurious high
coverage regions impacting the final median. Subsequently, the median
depth of the 36 chromosomes dm was calculated and the somy (s-value)
of each chromosome was obtained with the following formula s = 2*di/
dm. 2.2.1. Library preparation and sequencing 2.2.1. Library preparation and sequencing Library preparation and sequencing for BPK026, BPK604, BPK612,
BPK512, BPK406 was performed at the Wellcome Trust Sanger Institute
(Hinxton, United Kingdom). Genomic DNA was sheared into 400–600-
base pair fragments by focused ultrasonication (Covaris Adaptive
Focused Acoustics technology (AFA Inc., Woburn, USA)) and standard
Illumina libraries were prepared. 125 base pair paired end reads were
generated on the HiSeq 2000 v4 according to the manufacturer's
standard sequencing protocol (Bronner et al., 2014). Libraries of
BPK275, BPK282, BHU568 and BHU573 were prepared and sequenced
at the Beijing Genomics Institute (BGI). Briefly, mechanical shearing
with the Covaris (Illumina) was used to create 250-350 bp fragments,
after which the fragment ends were repaired and 3′ A-tailed. Adapters
were subsequently ligated and the target fragments were recovered
from a 2% agarose gel after electrophoresis. Finally, the libraries were
PCR amplified and 2 × 151 BP sequenced on an Hiseq 4000. All
sequencing data of this study was submitted to the Sequence Read
Archive (SRA) under accession number SRP125482. Individual acces-
sion numbers for each sample are available in Supplementary Table S2. 2.1. Parasites Nine L. donovani strains cloned from seven Nepalese and two Indian
clinical isolates were analyzed in this study (Supplementary Table
S1.1). Among these are five ISC1 strains (BPK026/0, BPK604/0,
BPK612/0, BPK519/12, BPK406/6) and two Nepalese (BPK275/0,
BPK282/0) as well as two Indian (BHU568/0, BHU573/0) strains from
the CG. Promastigotes of all strains were grown in modified Eagle's
medium (Invitrogen) supplemented with 20% (v/v) heat inactivated
fetal calf serum (PAA Laboratories GmbH, Linz, Austria) pH 7.5 at
26 °C. At each passage, all parasite lines were inoculated to a starting
concentration of 5∙105 parasites/mL and growth was followed for two
passages enabling synchronized sampling of the different cultures and
minimizing the effect of differences in growth rate between strains. For
each strain, four growth replicates were cultured for metabolomics
analysis and one for genome sequencing. Metabolites were extracted in
stationary growth phase according to t'Kindt et al. (2010) and DNA with
the QIAamp DNA Blood Mini Kit (Qiagen). 1. Introduction Haploid gene copy number was defined to be the median depth of a
gene divided by the median depth of the chromosome on which it is
located. We used two criteria to evaluate whether a gene or chromo-
some copy number difference between the CG and ISC1 strains was
biologically meaningful and statistically significant. The first require-
ment was that the absolute difference in gene/chromosome copy
number between CG and ISC1 should be at least 0.5 (Dumetz et al.,
2017). Secondly, the FDR adjusted p-value (Student t-test + Benjamini
Hochberg correction) had to be lower than 0.05. parasites need more time and molecular changes to develop antimonial
(Sb) resistance in vitro than CG strains, which led to the hypothesis of
Sb-resistance pre-adaptation of the CG, but not of ICS1 (Dumetz et al.,
2018). Since 2005, the kala-azar elimination programme (KAEP) has been
running in India, Nepal and Bangladesh, based on vector control and
detection/treatment of VL cases. In such a programme, it is essential to
track parasites and in particular to characterize emerging populations
like ISC1 or the parasites recently reported in Sri Lanka (Zhang et al.,
2014), as these could react differently to control tools like diagnostics,
drugs or vaccines. In this context, we report here the results of a deep
molecular comparison between ISC1 and CG parasites. In the present
study, we focused on parallel untargeted genomics and metabolomics,
hereby undertaking the first Leishmania diversity study integrating
these two ‘omics layers. 2.3.2. Data preprocessing Data analysis was performed using the XCMS 1.42.0 (Smith et al.,
2006) and mzMatch 1.0.1 (Scheltema et al., 2011) packages in R as
described previously (Berg et al., 2013b). In summary, mzXML files
were first subjected to retention time alignment using ObiWarp (Prince
and Marcotte, 2006) and peak detection with the centWave algorithm
(Tautenhahn et al., 2008). Corresponding peaks from the 4 biological
replicates were subsequently combined and filtered on a maximum
allowed reproducibility standard deviation (RSD) of 0.5. Peaksets from
all samples were then combined and filtered further, requiring for each
peak a minimal scaled CoDA-DW quality value of 0.8 (mzMatch
noisefilter), detection in at least 3 out of 4 replicates and a minimal
peak intensity of 3000. The gapfiller tool was used to fetch peaks from
the mzXML files that were missing in one or more samples and deri-
vative peaks were annotated (Scheltema et al., 2009). The peaks were
then putatively identified allowing 2 ppm of mass deviation using se-
quentially the LeishCyc database (Doyle et al., 2009), LipidMAPS (Fahy
et al., 2007), KEGG (Kanehisa et al., 2017), the peptide database 2.3. Metabolomics Samples were analyzed with LC-MS using an Orbitrap Exactive mass
spectrometer (Thermo Fisher) coupled to a 2.1 mm ZIC-HILIC column at
Glasgow Polyomics (University of Glasgow, Scotland) (Sequant), ex-
actly as previously described (Berg et al., 2015). Both negative and
positive ion modes were run in parallel with rapid polarity switching. Several quality control samples were run at the start, middle and end of
the LC-MS run to aid accurate metabolite identification and verify LC-
MS stability (Berg et al., 2013a). These included: 1) Authentic standard
mixes containing in total 217 metabolites (50–400 Da) representing a
wide array of metabolic classes and pathways. 2) An amino acid stan-
dard mix (Sigma Product No. A9906). 3) Serial dilutions (Undiluted, 1/
2, 1/4, 1/8 and 1/16) of a pooled sample of all extracts to filter out
spurious signals that do not follow the dilution trend (Jankevics et al.,
2012). 2.2. Genomics 2.2. Genomics 2.3.3. Data exploration and statistics For the initial data exploration, principal component analysis (PCA)
was performed in R directly on the metabolite intensity values of each
sample. Subsequently, the relative expression between the CG and ISC1
strains was calculated for each individual metabolite (Jara et al., 2017). Briefly, first the average metabolite intensity was calculated per strain. Then, the obtained values were again averaged over the ISC1 strains
and over the CG strains. Finally, these strain averages were converted
into base-2 logarithm ratios between the two populations, henceforth
called the ‘Log2 Fold Change’ (Log2FC). A two-sample t-test assuming
unequal variance was applied to the strain averages to calculate whe-
ther or not this Log2FC was significant (p < 0.05). In total, we detected 53,101 SNPs between the CG and ISC1 clusters
(allele shift > 0.25 & pval < 0.05) of which 51,668 were homozygous
(allele shift > 0.8) and of which 19,776 were located in protein coding
regions (Supplementary Table S3). 10,550 of these SNPs caused non-
synonymous codon changes and 24 were flagged by SnpEfffor having a
probable high impact on the final protein: fifteen premature stop co-
dons were gained, seven stop codons were lost, and two start codons
were gained in ISC1 strains compared to CG ones (Supplementary Table
S1.2). Fourteen of these high impact variants concerned hypothetical
proteins of which the function and impact is unclear. However, of the
remaining ten genes (with homozygous SNPs), three play key roles in
Leishmania virulence (autophagy protein Atg8, trypanothione synthe-
tase and amastin-like surfaceprotein) and two are involved in phos-
phatidylinositol metabolism (CDP-diacylglycerol-inositol 3-phospha-
tidyl transferase and phosphatidylinositol 3- and 4-kinase). While a
high impact is predicted by SnpEff, these genes could certainly still be
functional, as for many of them (including trypanothione synthase,
autophagy protein Atg8 and the amastin-like surface protein) the SNP
was found towards the end of the gene. To investigate the overall correlation between genome and meta-
bolome, we calculated three types of pairwise distances between all
strains: 1) The SNP distance (S-distance) was defined as the total
number of SNP allele changes between a strain pair. In this calculation,
a heterozygous SNP was counted as a distance of 1, while a homozygous
SNP was counted as a distance of 2. 2.2.2. Data analysis Genomic analysis was performed as described in Dumetz et al. (2017). Briefly, reads were mapped to the L. donovani LdBPKv2 re-
ference genome (9) using Smalt v7.4 (www.sanger.ac.uk/resources/
software/smalt/) and the options exhaustive searching (−x), read
identity threshold of 80% (−y 0.8) and random mapping of multiple
hits reads. Duplicate reads were marked with Picard v1.85 and SNPs 171 Fig. 1. Neighbour joining tree based on whole genome SNP loci. Bootstrap support was calculated on 1000 bootstrap replicates. LdonLV9 = East African L. donovani
LV9, SRI_CLB = Sri Lankan L. donovani SRI_CLB. B. Cuypers et al. Infection, Genetics and Evolution 62 (2018) 170–178 B. Cuypers et al. Infection, Genetics and Evolution 62 (2018) 170–178 Fig. 1. Neighbour joining tree based on whole genome SNP loci. Bootstrap support was calculated on 1000 bootstrap replicates. LdonLV9 = East African L. donovani
LV9, SRI_CLB = Sri Lankan L. donovani SRI_CLB. ed on whole genome SNP loci. Bootstrap support was calculated on 1000 bootstrap replicates. LdonLV9 = East African L. dono
novani SRI_CLB. Fig. 1. Neighbour joining tree based on whole genome SNP loci. Bootstrap support was calculated on 1000 bootstrap replicate
LV9, SRI_CLB = Sri Lankan L. donovani SRI_CLB. distances in a distance matrix are not independent from one other. R2
values were determined by linear regression analysis with the lm
function in R. included in mzMatch and the Human Metabolome Database (Wishart
et al., 2007). Each time, only unmatched peaks were aligned against the
next database, reducing the number of potential identifications to the
most likely candidates (Scalbert et al., 2009). Peaks of which the in-
tensity did not follow the dilution pattern in the pooled sample dilution
series (Pearson correlation > 0.8 or p-value > .05) were removed from
the analysis with the mzMatch Dilution Trend Filter tool (Jankevics
et al., 2012). The remaining peaks were normalized for total ion count
and visually verified before exporting them to a csv file containing all
peak heights, retention times and relevant database annotations. The
full analysis script and detailed parameter settings are available in
Supplementary Table S5. The csv files from the positive and negative
ionization mode were then merged. When metabolites were identified
in both modes, only the results from the most intense peak were kept. 3.1. Genome sequence diversity Promastigotes of five ISC1 (BPK026/0, BPK604/0, BPK612/0,
BPK519/12, BPK406/6) and four CG strains (BPK275/0, BPK282/0,
BHU568/0, BHU573/0) were sequenced with an average read depth of
at least 38.5× and 97.1% of the genome was covered at least 20×. Detailed mapping to BPK282v2 (Dumetz et al., 2017) and coverage
statistics can be found in Supplementary Table S2. Using new SNP data
of our strains and published ones of East African L. donovani LV9 and Sri
Lankan L. donovani SRI-CLB, we performed a phylogenomic analysis
(Fig. 1). In correspondence with Imamura et al. (2016) (Imamura et al.,
2016), this showed that (i) ISC1 parasites cluster at a larger distance
from CG than the Sri Lanka strain SRI-CLB and (ii) in an intermediate
position with respect to East African L. donovani LV9. 2.3.3. Data exploration and statistics With respect to tandem
arrays of the same gene family (Supplementary Table S1.5), a large
proportion (37%) of the genes with known functions were also coding
for surface proteins, virulence factors or immunogens. More specifi-
cally, we found CNVs of genes coding for amastins, proteophophogly-
cans, HSP70, HSP83, GP63, p1/s1 nucleases, lorien proteins, cysteine
peptidase B and surface antigen 2 proteins. Interestingly, some of these
CNVs were linked to the observations at sequence diversity level. Indeed, frameshift indels were found in both LdBPK_300020700 coding
for a p1/s1 nuclease, and in LdBPK_120014400 coding for a surface
antigen protein 2. For both genes the adjacent loci contained genes of
the same gene family involved in a CNV. Other families that were
targeted by both sequence and structure variants were amastins and
autophagy protein Atg8. Finally, amplifications concerning groups of
genes with different functions (Supplementary Table S1.6) are ex-
tensively reported to occur as circular episomes (Grondin et al., 1993;
Mukherjee et al., 2007; Chiquero et al., 1994), but also as intra-chro-
mosomal amplicons (Imamura et al., 2016; Olmo et al., 1995). For in-
stance, the H- and M-loci are amplified in CG strains but have only a
single copy per haploid genome in ISC1. Noteworthy, the H-locus
contains among others the gene encoding for arginine-succinate syn-
thase (ASS, see metabolomics section). Reciprocally, the R-locus (Ubeda
et al., 2014) has a single copy per haploid genome in CG strains, but has
2.15 copies per haploid genome in ISC1 strains. Both the H- and R-
locus have been associated with drug resistance (Grondin et al., 1993;
Kundig et al., 1999). Five of the other amplified gene groups are as-
sociated with virulence, including proteophosphoglycan, amastins,
surface antigen protein 2 and acid phosphatases. While all three de-
scribed types of CNVs concerned a large proportion of genes coding for
surface proteins, virulence factors and immunogens there was no clear
directionality to these CNVs since we observed both deletions (60%)
and duplications (40%) in ISC1 versus CG. regions and 83 concerned frameshift mutations, which are often highly
disruptive. 2.3.3. Data exploration and statistics 2) The gene dosage distance (GD-
distance) was determined by taking the Euclidean distance between two
strains based on all absolute gene copy differences; as such, it combined
the effect of CNV and aneuploidy. 3) The metabolic distance (M-dis-
tance) was defined as the Euclidean distance based on all metabolite
intensities, however, since these intensities have a large, metabolite-
specific dynamic range, they were first transformed into Z-scores. Analysis of the correlation between the respective distances was per-
formed with a non-parametric Mantel test (Mantel, 1967) with 999
permutations, using the QIIME2 package in Python. Contrary to the
classical correlation test, the Mantel test deals with the problem that Also at the indel level major differences were observed between
ISC1 and CG with a total of 6435 heterozygous and 5780 homozygous
indels (allele shift > 0.25 and
> 0.80 respectively, p-value < 0.05,
Supplementary Table S4). 442 of these indels were located in coding 172 B. Cuypers et al. Infection, Genetics and Evolution 62 (2018) 170–178 Fig. 2. Somy levels in ISC1 and Core Group strains (CG). ISC1 strains are depicted in shades of red and yellow, while CG strains in shades of blue. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.)
B. Cuypers et al. Infection, Genetics and Evolution 62 (2018) 170178 Fig. 2. Somy levels in ISC1 and Core Group strains (CG). ISC1 strains are depicted in shades of red and yellow, while CG strain
of the references to colour in this figure legend, the reader is referred to the web version of this article.) d Core Group strains (CG). ISC1 strains are depicted in shades of red and yellow, while CG strains in shades of blue. (For interp
this figure legend, the reader is referred to the web version of this article.) Fig. 2. Somy levels in ISC1 and Core Group strains (CG). ISC1 strains are depicted in shades of red and yellow, while CG strains in shades of blue. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.) virulence factors or immunogens, including GP63, amastins, 60S acidic
ribosomal protein P2 and surface antigen 2. 3.2. Genome structure diversity The genome structure was first investigated at chromosome level
(Fig. 2). Chromosomal somy estimates do not always correspond to
integer values as these represent the average of a population of cells
that do not necessarily show identical karyotypes (mosaicism). Many
somy differences were found between chromosomes of different strains,
but only two of these were consistent between ISC1 and CG populations
(p-value < 0.05 & |somy difference| > 0.5). The ISC1 strains had 0.8
copies less of chromosome 8 and 0.7 copies less of chromosome 23. p
p
Secondly, we analyzed local gene copy number variations (CNVs)
between ISC1 and CG isolates. These differences are the result of gene
amplification or deletion events since the most recent common ancestor
of ISC1 and CG. For every gene and sample, copy numbers per haploid
genome are available in Supplementary Table S4. By expressing copy
number differences per haploid genome, we can describe these changes
independently of the aneuploidy phenomenon. As Leishmania is diploid
for most chromosomes, we used 0.5 copies/haploid genome as a cut-off,
which corresponds with a single copy for diploid chromosomes. In total
we found 62 loci containing genes with CNVs: for 29 of these loci, genes
were more abundant in ISC1, while for the 33 remaining loci, genes
were more abundant in CG. 29 loci concerned single gene duplications
or deletions (Supplementary Table S1.4). The remaining 33 loci con-
tained multiple genes showing CNVs: (i) 22 contained tandem arrays of
the same gene family, expanding or contracting (Supplementary Table
S1.5); (ii) 11 loci contained sets of genes of a different family, all am-
plified/deleted together (Supplementary Table S1.6). 2.3.3. Data exploration and statistics A large proportion (58%) of the frameshift indels were lo-
cated in hypothetical proteins, while the 33 functionally annotated
indels (Supplementary Table S1.3) concerned several important biolo-
gical functions, including, but not limited to, transmembrane transport
(Imamura et al., 2016), translation (Ostyn et al., 2011), nucleic acid
binding (Imamura et al., 2016) and nucleotide salvage (Ostyn et al.,
2015). 21 of these annotated indels were homozygous mutations. Re-
markably, two genes encoding for (i) an AAAfamily ATPAse and (ii) a
dihydrouridine synthase domain protein respectively each had two in-
dels. In addition, phosphatidylinositol metabolism was highlighted in
both SNP and INDEL analysis: Phosphatidylinositol-specific phospholi-
pase C,X/Y/C2 domain containing protein (indel), CDP-diacylglycerol-
inositol 3-phosphatidyl transferase (SNP) and phosphatidylinositol 3-
and 4-kinase (SNP). 4. Discussion In contrast to aneuploidy, these were shown not to
be affected during the life cycle of the parasite (Dumetz et al., 2017),
and CNVs have been shown to be excellent markers for studies on
Leishmania evolution (Dujardin, 2009), both in experimental (for in-
stance, amplification of specific loci during drug resistance selection
(Ubeda et al., 2014; Leprohon et al., 2009)) or in natural settings (for
instance, decrease in copy number of gp63, mini-exon and rDNA genes
in Leishmania peruviana (Victoir et al., 2009; Kebede et al., 1999; Inga
et al., 1998)). In the absence of gene expression regulation at initiation
(Günzl, 2012), amplification/deletion of specific genes is a genomic
solution to modulate the level of transcripts and corresponding pro-
ducts. As such, these CNVs may have an important functional sig-
nificance. In this context, it is striking to observe that many of the CNVs
concerned genes that code for surface proteins, virulence factors or
immunogens: most notably, lower copy numbers of amastins, surface
antigens and GP63 genes were encountered in ISC1. Interestingly, many
Leishmania virulence genes were the target of highly impactful non-
synonymous SNPs (removed/introduced stop codons), such as autop-
hagy protein Atg8, trypanothione synthetase and amastin-like surface
proteins. Although it is impossible to predict now the exact con-
sequences of these SNPs, they are likely to have a high impact at the 33 metabolites were more abundant in ISC1 strains (Log2FC > 1, p-
value < 0.05),
while
12
were
more
abundant
in
CG
strains
(Log2FC > 1, p-value < 0.05). Interestingly, 19 of these metabolites
were glycerophospholipids (GPLs), providing evidence for major dif-
ferences in lipid metabolism between CG and ISC1 (Fig. 4). In addition,
ISC1
parasites
contained
higher
metabolite
levels
of
phosphor-
ylethanolamine,
glycerylphosphorylethanolamine,
and
glyceropho-
sphoinositol pointing further towards lipid changes between both
groups. Secondly, changes were observed in the urea cycle in ISC1 vs
CG. ISC1 had a 2.1 fold higher amounts of citrulline and 3.6 fold lower
amounts of argininosuccinate. The reaction that involves these meta-
bolites, namely citrulline + aspartate + ATP - > argininosuccinate +
AMP + PPi, is catalyzed by argininosuccinate synthase (ASS). These
metabolite differences therefore indicate a lower ASS activity in ISC1,
which is in correspondence with the lower copy number of the re-
spective gene in ISC1 (as described in the genome structure diversity
section). 4. Discussion In this work we have performed a genomic and metabolic profiling
study of strains representing ISC1, a new Leishmania donovani popula-
tion that is currently emerging in Nepal. We have demonstrated that
these parasites are genomically and metabolically very distinct from the
main population (CG) of L. donovani, which was the driver of the most
recent VL epidemic in the Indian subcontinent (Imamura et al., 2016). BHU568 and BPK282) from ISC1 strains (BPK026, BPK406, BPK519,
BPK604, BPK612). The second principal component explained 23% of
the variation, which separated CG strains in 3 groups: BPK275 (Nepal,
SSG-resistant), BHU573-BHU568 (India, SSG-resistant) and BPK282
(Nepal, SSG-sensitive). p
(
,
)
From a genomic point of view, the differences between ISC1 and CG
strongly contrast with the homogeneity of the latter group and ex-
plained by its young evolutionary history (Imamura et al., 2016). First,
we report a large number of SNPs and indels, many of them with a
strong impact (stop codons gained/lost and frameshifts) on several
important biological functions. Secondly, we also encountered differ-
ences in the karyotypes and more particularly two chromosomes
(chromosome 8 and chromosome 23) that showed a lower somy in
ISC1. Somy variation is an adaptive strategy well known in Leishmania,
for instance in response to experimental drug resistance selection
(Prieto Barja et al., 2017), but its interpretation in clinical isolates
needs to be done very carefully. Indeed, we showed that aneuploidy is
strongly dependent on the environment and more abundant in pro-
mastigotes in vitro than in amastigotes within the vertebrate host
(Dumetz et al., 2017). Strictly speaking, we can thus only conclude that
cultivated promastigotes of ISC1 show significant karyotypic differ-
ences to those of CG. Further extrapolation of these karyotype differ-
ences to the situation in vivo should be tested by direct sequencing of
clinical samples or in experimental animal infections. A third major
class of genomic changes here observed concerned local copy number
variations (CNVs). 3.3. Metabolomics In this PCA plot, average values are shown for each strain based on
four biological replicates. For each principal component, the proportion of
variation of the total dataset it explains is mentioned. 3.3. Metabolomics In total, 290 metabolites were identified with a mass accuracy <
2 ppm, covering a broad diversity of metabolic pathways and re-
presenting the following classes (in order of quantitative representa-
tion): glycerophospholipids (GPLs), amino acids and derivatives, fatty
acyls and carbohydrates, nucleobases and nucleosides, sphingolipids,
steroids and derivatives (Supplementary Table S5 Supplementary Table
S1.4). As a first data exploration of the variation in our dataset, we
performed Principal Component Analysis (PCA, Fig. 3). The first prin-
cipal component explained 34% of the total variation in our metabo-
lomics experiment and clearly separated the CG (BPK275, BHU573, Duplication/deletions of single copy genes concerned a wide array
of biological functions (Supplementary Table S1.4). Noteworthy, 43%
of the genes with known functions were coding for surface proteins, 173 Infection, Genetics and Evolution 62 (2018) 170–178 B. Cuypers et al. Fig. 3. Principal component analysis of the metabolic profiles of the 5
Leishmania donovani ISC1 isolates and 4 CG strains in this study. This analysis
was based on the quantitative measurements of all 290 putatively identified
metabolites. In this PCA plot, average values are shown for each strain based on
four biological replicates. For each principal component, the proportion of
variation of the total dataset it explains is mentioned. metabolome variation within the CG is present, there are clear and
consistent differences with ISC1 on the level of GPLs, salvage pathway
and urea cycle. Finally, we compared the metabolomic and genomic polymorphism
to quantify the relationship between both data sets and assess if/how
metabolomic variation could be explained by genomic variation. Therefore, we computed distances between each pair of isolates, based
on M-distance, S-distance and GD-distance, as explained in the Methods
section. Unfortunately, for the S-distance the intra group variance was
far smaller than the inter group variance, which biases the results. Therefore we did not analyse S-distance any further. However, this
problem was absent for GD-distance and a Mantel test established a
significant positive correlation between M-distance and GD-distance (p-
value = 0.005, Spearman rho = 0.64). This means that GD-distance
explains a significant proportion of the metabolic distance, but the low
spearman rho indicates that much other variation is present as well. Fig. 3. Principal component analysis of the metabolic profiles of the 5
Leishmania donovani ISC1 isolates and 4 CG strains in this study. This analysis
was based on the quantitative measurements of all 290 putatively identified
metabolites. 4. Discussion Interestingly, the metabolite levels also indicate a difference
within the nucleotide salvage pathway between both groups, as we
found increased levels of guanine, uracil, thymine, AMP in ISC1 vs CG. Finally, concentrations of basic amino acids lysine (up) and histidine
(down) were altered in ISC1 vs CG. We have previously shown that significant metabolic variation is
present between SSG-resistant strains (BPK275, BHU573 and BHU568:
CG-R) and SSG sensitive strains (BPK282: CG-S) (Berg et al., 2013b). Considering these metabolic differences, we also made individual
comparisons of ISC1 vs CG-R, ISC1 vs CG-S and CG-S vs CG-R. As ex-
pected, this confirmed substantial differences in the metabolome be-
tween CG-S and CG-R, with 52 metabolites showing a significant
(Log2FC > 1, p-value < 0.05) difference in concentration in only one
of both groups. Consequently, when comparing CG-R and CG-S to ISC1
individually, we found many metabolites only differential in one of
both comparisons (62 and 51 for CG-R and CG-S respectively), that
were not observed in the original ISC1 vs CG in comparison. However,
when looking in detail to the functions of these newly identified dif-
ferential metabolites, these related towards the same functional classes
of metabolites as those found when comparing ISC1 to all CG strains
(Supplementary Table S1.7). This shows that although significant 174 B. Cuypers et al. Infection, Genetics and Evolution 62 (2018) 170–178 BHU568
BHU573
BPK282
BPK275
BPK026
BPK406
BPK604
BPK519
BPK612
GPC(35:3/2)
GPC(O−32:3/2)
GPC(39:6/2)
GPP(18:1/1)
GPE(18:1/1)
GPC(18:1/1)
GPC(17:1/1)
GPC(15:1/1)
GPC(33:1/2)
GPC(36:4/2)
GPE(36:4/2)
GPE(36:3/2)
GPE(36:6/2)
GPC(33:3/2)
GPE(36:5/2)
GPI(18:1/1)
GPC(19:3/1)
GPE(18:2/1)
GPE(18:3/1)
GPC(18:3/1)
GPS(14:4/1)
GPC(O−18:1/1)
GPC(O−15:1/1)
GPC(O−16:1/1)
GPE(O−36:4/2)
GPC(38:3/2)
GPC(38:4/2)
GPC(38:5/2)
GPP(43:6/2)
GPC(O−14:1/1)
GPC(38:6/2)
GPE(41:6/2)
GPC(19:1/1)
GPC(20:3/1)
GPC(20:1/1)
GPC(20:2/1)
GPC(18:2/1)
GPC(22:5/1)
GPC(22:6/1)
GPC(16:1/1)
−2
0
1
2
Value
Color Key
Fig. 4. The 40 differentially expressed glycerophospholipids (|Log2FC| > 1 & p < 0.05) between ISC1 and CG parasites. Infection, Genetics and Evolution 62 B
B
B
B
B
B
B
B
B
Fig. 4. The 40 differentially expressed glycerophospholipids (|Log2FC| > 1 & p < 0.05) between ISC1 and CG parasites. Fig. 4. The 40 differentially expressed glycerophospholipids (|Log2FC| > 1 & p < 0.05) between ISC1 and CG parasites protein level by extending/shortening the protein. Altogether, genomic
differences between ISC1 and CG suggest a potential for differences in
virulence, which should be explored by further work. 4. Discussion A first experi-
mental verification of this hypothesis was made the context of antimony
susceptibility showing CG isolates were pre-adapted to antimony while
ISC1 ones were not. This particular feature could be linked to the
presence of the H-locus amplification in the CG (Dumetz et al., 2018). argininosuccinate and arginine deprivation have been linked with re-
duced thiol content (Sardar et al., 2016; Mandal et al., 2016) and ASS
mutants have shown a lower virulence than WT parasites (Lakhal-
Naouar et al., 2012). Therefore, the importance of ASS should be fur-
ther analyzed. Finally, a metabolic change that also came forward in
our comparisons between ISC1 and CG strains was the nucleotide sal-
vage pathway. This pathway is essential, since Leishmania cannot syn-
thesize the purine ring de novo and is therefore dependent on salvaging
these from host purines (Boitz et al., 2012). Our results suggest that
ISC1 parasites might be better at salvaging nucleotides from their en-
vironment. Altogether, ISC1 and CG parasites appeared to show dif-
ferent levels of activity among different metabolic pathways, all of them
susceptible to predict differences in virulence, which should be tested
by further work. Notably, our observations were made in stationary
phase axenic promastigotes, which contain a high degree of metacyclic,
infectious promastigotes (Jamdhade et al., 2015). This is of particular
interest, since virulence is of essential importance to this life stage. However, it is certainly possible that more differences between ISC1
and CG might come forward when studying additional parasite life
stages in follow-up studies. Ultimately, the rapidly evolving metabo-
lomics technology will most likely allow metabolite quantifications of
low amounts of cells in vivo, or even single-cells in the future
(Madhubala et al., 2008). argininosuccinate and arginine deprivation have been linked with re-
duced thiol content (Sardar et al., 2016; Mandal et al., 2016) and ASS
mutants have shown a lower virulence than WT parasites (Lakhal-
Naouar et al., 2012). Therefore, the importance of ASS should be fur-
ther analyzed. Finally, a metabolic change that also came forward in
our comparisons between ISC1 and CG strains was the nucleotide sal-
vage pathway. This pathway is essential, since Leishmania cannot syn-
thesize the purine ring de novo and is therefore dependent on salvaging
these from host purines (Boitz et al., 2012). Our results suggest that
ISC1 parasites might be better at salvaging nucleotides from their en-
vironment. 1. Argininosuccinate Synthase ↘ ASS ↘
argininosuccinate
ornithine
arginine
citrulline
putrescine
Trypanothione
Biosynthesis
1. Argininosuccinate Synthase ↘
Urea Cycle DNA / RNA p1/s1 nuclease ↗ arginine ornithine Purine Salvage Pyrimidine Salvage Purine Salvage Trypanothione
Biosynthesis CG ISC1 Fig. 5. The 3 key differences between ISC1 and CG that are shared between genome and metabolome. 1. Argininosuccinate (ASS) has a lower genomic copy number
in ISC1 vs CG. More citrulline and less argininosuccinate were detected in ISC1, suggesting a corresponding downregulation of the ASS protein. Although the urea
cycle might be incomplete in Leishmania, links have been established with Trypanothione biosynthesis and virulence. 2. Several glycerophospholipid (or precursor)
biosynthetic enzymes had higher gene copy numbers in ISC1 which could be linked to increased GPL amounts (See also Fig. 4). 3. More copies of a p1/s1 nuclease
gene were found in ISC1, associated with higher concentrations of several salvage pathway metabolites. Genomic copy number information is underlined and in bold. Pathway names are in italics. Arrows in grey represent potentially missing enzymes in the urea cycle of Leishmania. Blue = down in ISC1, Red = up in ISC1. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) The parallel untargeted analysis of genome and metabolome of
different L. donovani strains also allowed us to undertake a first in-
tegration of both datasets. A quantitative approach was attempted by
directly correlating the pairwise GD-distance and metabolic distances
between strains. Although this correlation was found to be significant,
they also demonstrate that the genomic distances only explain a limited
part of the variation observed at the metabolome level. This is not
unexpected, since regulation of gene expression in Trypanosomatids is
essentially post-transcriptional. Qualitative analysis of our results
showed a few correspondences between genomic and metabolic mar-
kers (Fig. 5). Correspondence could concern either an enzyme and the
specific metabolites of the reaction it catalyses, or this enzyme and
metabolites from the same pathway. The former is best illustrated by
the argininosuccinate synthase (ASS) and argininosuccinate, the gene
encoding ASS is located on the H-locus and had a 6.7 fold lower ex-
pression in ISC1 by the combined effect of somy and local copy number
and argininosuccinate is 3.6 times less abundant in the metabolome of
ISC1. 1. Argininosuccinate Synthase ↘ Two other examples of convergence between genomics and me-
tabolomics concern pathways: genes and metabolites pointing towards
a same altered pathway, even if they did not point towards the same
product. Firstly, the major changes revealed in the lipid metabolism by
metabolomics were mirrored by CNVs. We found 1.55 copies per hap-
loid genome more in ISC1 for diacyl glycerol acyltransferase, which is
part of the GPC biosynthesis pathway. Interestingly, we found pre-
dominantly higher GPC levels in ISC1. A CNV was also found in a fatty
acid elongase (1.65 copies per haploid genome more in ISC1), which is
an enzyme responsible for the extension of fatty acids that are later used
to build GPLs. Another CNV related to GPL building blocks was found in
the glycerol uptake protein. Finally, a CNV was found in 3-ketoacyl-CoA
thiolase, which is located in the glycosome and an enzyme of the β-
oxidation pathway (breakdown of fatty acids) (Jamdhade et al., 2015). We also detected high impact SNPs in the ISC1 group for two enzymes that are important for lipid signaling: a premature stop codon was in
phosphatidylinositol 3&4-kinase and a stop codon was removed from
CDP-diacylglycerol-inositol 3-phosphatidyl transferase. Secondly, the
different activity of the salvage pathway revealed by metabolomics was
also mirrored by genomics. We found indeed that ISC1 parasites have
four copies more of the p1/s1 nuclease, which is part of the salvage
pathway and responsible for digesting the ssRNA and ssDNA to nu-
cleotides. This was accompanied by an increase of guanine, uracil,
thymine and AMP in ISC1. In summary, our data strongly suggest that
gene copy number variation may be an important adaptation strategy of
the parasite as shown by the gene dosage changes related to metabolic
pathways where metabolites were altered (Fig. 5). This implies corre-
sponding changes in protein levels, which is somewhat surprising as
transcript levels (driven by gene dosage) are generally assumed to
match poorly with protein levels (Madhubala et al., 2008). However,
these observations were made in context of differentiation, rather than
inter-strains comparisons as we have performed here. Our data suggest
that between strains, gene dosage levels might in fact drive protein
levels, as was also found in yeast, however, further work is required to
define the extent of agreement between variation of genome and pro-
teome. The evolutionary history and origin of L. donovani ISC1 are still
unclear. 4. Discussion Altogether, ISC1 and CG parasites appeared to show dif-
ferent levels of activity among different metabolic pathways, all of them
susceptible to predict differences in virulence, which should be tested
by further work. Notably, our observations were made in stationary
phase axenic promastigotes, which contain a high degree of metacyclic,
infectious promastigotes (Jamdhade et al., 2015). This is of particular
interest, since virulence is of essential importance to this life stage. However, it is certainly possible that more differences between ISC1
and CG might come forward when studying additional parasite life
stages in follow-up studies. Ultimately, the rapidly evolving metabo-
lomics technology will most likely allow metabolite quantifications of
low amounts of cells in vivo, or even single-cells in the future
(Madhubala et al., 2008). The metabolome being the molecular expression of the parasite's
phenotype, its study is extremely relevant to detect important func-
tional differences between organisms. Metabolic differences between
ISC1 and CG were found in several functional groups and pathways. First, the most notable changes were found in the lipid metabolism,
with 19 GPLs (predominantly GPCs), showing significantly different
levels between both groups. Apart from their structural function, GPLs
are involved in a wide array of cellular functions including protein and
glycoconjugate anchoring, host cell infection, and apoptosis-like pro-
cesses (Pulido et al., 2017). A second notable metabolic difference
concerned the urea cycle. In ISC1 versus CG we detected a higher
concentration of citrulline and a lower concentration of arginino-
succinate. In organisms with a complete urea cycle, argininosuccinate is
then further converted to arginine and fumarate by argininosuccinate
lyase (ASL), however, in Leishmania this enzyme is missing (Sardar
et al., 2016). This difference is of particular interest since both 175 Infection, Genetics and Evolution 62 (2018) 170–178 B. Cuypers et al. Key differences ISC1 vs CG shared between genome and metabolome Key differences ISC1 vs CG shared between genome and metabolome ASS ↘
argininosuccinate
ornithine
arginine
citrulline
putrescine
Trypanothione
Biosynthesis
1. Argininosuccinate Synthase ↘
2. Glycerophospholipids ↗
3. Salvage Pathway ↗
CG
ISC1
GPL biosynthec enzymes ↗
Urea Cycle
p1/s1 nuclease ↗
DNA / RNA
Purine Salvage
Pyrimidine Salvage
guanine
AMP
uracil
thymine
Fig. 5. The 3 key differences between ISC1 and CG that are shared between genome and metabolome. 1. Argininosuccinate (ASS) has a lower genomic copy number
in ISC1 vs CG. More citrulline and less argininosuccinate were detected in ISC1, suggesting a corresponding downregulation of the ASS protein. Although the urea
cycle might be incomplete in Leishmania, links have been established with Trypanothione biosynthesis and virulence. 2. Several glycerophospholipid (or precursor)
biosynthetic enzymes had higher gene copy numbers in ISC1 which could be linked to increased GPL amounts (See also Fig. 4). 3. More copies of a p1/s1 nuclease
gene were found in ISC1, associated with higher concentrations of several salvage pathway metabolites. Genomic copy number information is underlined and in bold. Pathway names are in italics. Arrows in grey represent potentially missing enzymes in the urea cycle of Leishmania. Blue = down in ISC1, Red = up in ISC1. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 2. Glycerophospholipids ↗
CG
ISC1
GPL biosynthec enzymes ↗ 2. Glycerophospholipids ↗
3. Salvage Pathway ↗
CG
ISC1
GPL biosynthec enzymes ↗
p1/s1 nucleas
DNA / RNA
Purine Salvage
Pyrimidine
guanine
AMP
uracil
thymine 3. Salvage Pathway ↗
p1/s1 nuclease ↗
DNA / RNA
Purine Salvage
Pyrimidine Salvage
guanine
AMP
uracil
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é
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d
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b il
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sistant Leishmania. Nucleic Acids Res. 21, 1895–1901. sistant Leishmania. Nucleic Acids Res. 21, 1895–1901. Günzl, A., 2012. In: Bindereif, Albrecht (Ed.), RNA Metabolism in Trypanosomes. Springer, Berlin Heidelberg. Imamura, H., Downing, T., Van den Broeck, F., Sanders, M.J., Rijal, S., Sundar, S.,
Mannaert, A., Vanaerschot, M., Berg, M., De Muylder, G., et al., 2016. Evolutionary
genomics of epidemic visceral leishmaniasis in the Indian subcontinent. elife 5. References Ostyn, B., Gidwani, K., Khanal, B., Picado, A., Chappuis, F., Singh, S.P., Rijal, S., Sundar,
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Pacanowska, D., Gamarro, F., 1994. Amplification of the H locus in Leishmania in-
fantum. Biochim. Biophys. Acta 1227, 188–194. Cingolani, P., Platts, A., Wang le, L., Coon, M., Nguyen, T., Wang, L., Land, S.J., Lu, X.,
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L., Marasini, B., Van der Auwera, G., et al., 2015. Transmission of Leishmania 177 B. Cuypers et al. Infection, Genetics and Evolution 62 (2018) 170–178 donovani in the hills of eastern Nepal, an outbreak investigation in Okhaldhunga and
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P., Sirimanna, G., Wickremasinghe, R., Myler, P., Matlashewski, G., 2014. Genetic
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2-[(2-Hydroxynaphthalen-1-yl)methylideneamino]-5,6,7,8-tetrahydro-4<i>H</i>-cyclohepta[<i>b</i>]thiophene-3-carbonitrile
|
Acta crystallographica. Section E
| 2,013
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cc-by
| 4,534
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2-[(2-Hydroxynaphthalen-1-yl)methyl-
ideneamino]-5,6,7,8-tetrahydro-4H-
cyclohepta[b]thiophene-3-carbonitrile 28490 measured reflections
7066 independent reflections
5288 reflections with I > 2(I)
Rint = 0.035 Agilent SuperNova (Dual, Cu at
zero, Atlas, CCD) diffractometer
Absorption correction: multi-scan
(CrysAlis PRO; Agilent, 2012)
Tmin = 0.858, Tmax = 1.000 organic compounds Experimental
Crystal data
C21H18N2OS
Mr = 346.43
Orthorhombic, Pbca
a = 13.5472 (2) A˚
b = 14.4747 (4) A˚
c = 35.7902 (6) A˚
V = 7018.2 (2) A˚ 3
Z = 16
Cu K radiation
= 1.71 mm1
T = 296 K
0.37 0.21 0.14 mm
Data collection
Agilent SuperNova (Dual, Cu at
zero, Atlas, CCD) diffractometer
Absorption correction: multi-scan
(CrysAlis PRO; Agilent, 2012)
Tmin = 0.858, Tmax = 1.000
28490 measured reflections
7066 independent reflections
5288 reflections with I > 2(I)
Rint = 0.035
Refinement
R[F 2 > 2(F 2)] = 0.054
wR(F 2) = 0.161
S = 1.02
7066 reflections
457 parameters
H atoms treated by a mixture of
independent and constrained
refinement
max = 0.52 e A˚ 3
min = 0.25 e A˚ 3 Acta Crystallographica Section E
Structure Reports
Online
ISSN 1600-5368 Acta Crystallographica Section E
Structure Reports
O li V = 7018.2 (2) A˚ 3
Z = 16
Cu K radiation
= 1.71 mm1
T = 296 K
0.37 0.21 0.14 mm ISSN 1600-5368 Table 1
Hydrogen-bond geometry (A˚ , ). D—H A
D—H
H A
D A
D—H A
O1—H1O N1
0.82 (3)
1.84 (3)
2.578 (2)
150 (3)
O2—H2O N3
0.82 (3)
1.84 (3)
2.582 (2)
151 (3) Two independent molecules, A and B, comprise the asym-
metric unit of the title compound, C21H18N2OS, with the
difference in the angle of orientation between the naphthalene
ring system and the mean plane of the cycloheptyl ring
[16.13 (1) in A and 11.48 (5) in B], being evident. The
cycloheptyl ring adopts a distorted chair conformation in each
molecule
with
r.m.s. deviations
of
0.2345 (4)
(A)
and
0.2302 (4) A˚
(B). Intramolecular
O—H N
hydrogen
bonding generates planar six-membered S(6) loops with
r.m.s. deviations of 0.0099 (1) (A) and 0.0286 (1) A˚ (B). Data collection: CrysAlis PRO (Agilent, 2012); 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); molecular graphics:
PLATON (Spek, 2009); software used to prepare material for
publication: WinGX (Farrugia, 2012) and X-SEED (Barbour, 2001). The authors would like to thank the Deanship of Scientific
Research at King Abdulaziz University for the support of this
research via Research Group Track grant No. 3–102/428. Related literature For the synthesis and related structures, see: Asiri et al. (2011a,b). For graph-set notation, see: Bernstein et al. (1995). Supplementary data and figures for this paper are available from the
IUCr electronic archives (Reference: TK5186). Agilent (2012). CrysAlis PRO. Agilent Technologies, Yarnton, England.
Asiri, A. M., Khan, S. A. & Tahir, M. N. (2011a). Acta Cryst. E67, o2254.
Asiri, A. M., Khan, S. A. & Tahir, M. N. (2011b). Acta Cryst. E67, o2355.
Barbour, L. J. (2001). J. Supramol. Chem. 1, 189–191.
Bernstein, J., Davis, R. E., Shimoni, L. & Chang, N.-L. (1995). Angew. Chem.
Int. Ed. Engl. 34, 1555–1573.
Farrugia, L. J. (2012). J. Appl. Cryst. 45, 849–854.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Spek, A. L. (2009). Acta Cryst. D65, 148–155. Supplementary data and figures for this paper are available from the
IUCr electronic archives (Reference: TK5186). Table 1 Key indicators: single-crystal X-ray study; T = 296 K; mean (C–C) = 0.004 A˚;
R factor = 0.054; wR factor = 0.161; data-to-parameter ratio = 15.5. Abdullah M. Asiri,a,b* Muhammad Nadeem Arshad,a,b
Tariq R. Sobahia and Ghulam Mustafac* aChemistry Department, Faculty of Science, King Abdulaziz University, PO Box
80203, Jeddah 21589, Saudi Arabia, bCenter of Excellence for Advanced Materials
Research (CEAMR), Faculty of Science, King Abdulaziz University, PO Box 80203,
Jeddah 21589, Saudi Arabia, and cDepartment of Chemistry, University of Gujrat,
Gujrat, Pakistan H atoms treated by a mixture of
independent and constrained
refinement
max = 0.52 e A˚ 3
min = 0.25 e A˚ 3 Correspondence e-mail: aasiri2@kau.edu.sa, ghulam.mustafa@uog.edu.pk Correspondence e-mail: aasiri2@kau.edu.sa, ghulam.mustafa@uog.edu.pk Received 27 December 2012; accepted 2 January 2013 Table 1
Hydrogen-bond geometry (A˚ , ). D—H A
D—H
H A
D A
D—H A
O1—H1O N1
0.82 (3)
1.84 (3)
2.578 (2)
150 (3)
O2—H2O N3
0.82 (3)
1.84 (3)
2.582 (2)
151 (3) Acta Cryst. (2013). E69, o193 Refinement The C—H H-atoms were positioned with idealized geometry with C—H = 0.93 Å for aromatic-H and C—H = 0.97 Å for
methylene groups. H-atoms were refined as riding with Uiso(H) = 1.2Ueq(C). The O—H atoms were refined with Uiso(H) =
1.5Ueq(O). Comment In extension of synthesis of Schiff bases containing a thiophene (Asiri et al. 2011a; 2011b) residue, we herein report the
crystal structure of title compound. The title compound (I), Fig. 1, crystallized with two molecules per asymmetric unit. The cycloheptyl ring adopted a
chair conformation in each molecule with the r. m. s. deviations being 0.2345 (4) & 0.2302 (4) Å, respectively. The
naphthalene ring system is inclined at dihedral angle of 4.97 (4) & 16.13 (1)° with respect to the planes produced from
best fitted atoms of thiophene and cycloheptyl rings, respectively in molecule A while the corresponding inclination
angles in molecule B are 4.16 (1) & 11.48 (5)°. The thiophene and cycloheptyl rings planes are oriented at dihedral angles
of 16.10 (7)° and 15.22 (6)° with respect to each other in molecules A & B. Only intramolecular O—H···N classical
hydrogen bonding is observed in both molecules which generates planar six-membered ring motifs S(6) (Bernstein et al.,
1995) with r. m. s. deviations of 0.0099 (1) & 0.0286 (1) Å from the least-squares planes of member atoms, see Table 1
for details. Experimental The title compound was prepared following literature methods (Asiri et al. 2011a; 2011b) and recrystallized from its
methanol solution by slow evaporation. References Asiri et al. o193 Acta Cryst. (2013). E69, o193 doi:10.1107/S160053681300007X supplementary materials Acta Cryst. (2013). E69, o193 [doi:10.1107/S160053681300007X] Acta Cryst. (2013). E69, o193 [doi:10.1107/S160053681300007X] Acta Cryst. (2013). E69, o193 [doi:10.1107/S160053681300007X] Figure 1 E69, o193
Crystal data
C21H18N2OS
Mr = 346.43
Orthorhombic, Pbca
Hall symbol: -P 2ac 2ab
a = 13.5472 (2) Å
b = 14.4747 (4) Å
c = 35.7902 (6) Å
V = 7018.2 (2) Å3
Z = 16
F(000) = 2912
Dx = 1.311 Mg m−3
Cu Kα radiation, λ = 1.54184 Å
Cell parameters from 7977 reflections
θ = 3.3–74.7°
µ = 1.71 mm−1
T = 296 K
Prismatic, dark red
0.37 × 0.21 × 0.14 mm
Data collection
Agilent SuperNova (Dual, Cu at zero, Atlas,
CCD)
diffractometer
Radiation source: SuperNova (Cu) X-ray
Source
Mirror monochromator
ω scans
Absorption correction: multi-scan
(CrysAlis PRO; Agilent, 2012)
Tmin = 0.858, Tmax = 1.000
28490 measured reflections
7066 independent reflections
5288 reflections with I > 2σ(I)
Rint = 0.035
θmax = 74.9°, θmin = 4.1°
h = −16→14
k = −17→13
l = −44→44
Refinement
Refinement on F2
Least-squares matrix: full
R[F2 > 2σ(F2)] = 0.054
wR(F2) = 0.161
S = 1.02
7066 reflections
457 parameters
0 restraints
Primary atom site location: structure-invariant
direct methods
Secondary atom site location: difference Fourier
map
Hydrogen site location: inferred from
neighbouring sites
H atoms treated by a mixture of independent
and constrained refinement Crystal data
C21H18N2OS
Mr = 346.43
Orthorhombic, Pbca
Hall symbol: -P 2ac 2ab
a = 13.5472 (2) Å
b = 14.4747 (4) Å
c = 35.7902 (6) Å
V = 7018.2 (2) Å3
Z = 16
F(000) = 2912
Dx = 1.311 Mg m−3
Cu Kα radiation, λ = 1.54184 Å
Cell parameters from 7977 reflections
θ = 3.3–74.7°
µ = 1.71 mm−1
T = 296 K
Prismatic, dark red
0.37 × 0.21 × 0.14 mm
Data collection
Agilent SuperNova (Dual, Cu at zero, Atlas,
CCD)
diffractometer
Radiation source: SuperNova (Cu) X-ray
Source
Mirror monochromator
ω scans
Absorption correction: multi-scan
(CrysAlis PRO; Agilent, 2012)
Tmin = 0.858, Tmax = 1.000
28490 measured reflections
7066 independent reflections
5288 reflections with I > 2σ(I)
Rint = 0.035
θmax = 74.9°, θmin = 4.1°
h = −16→14
k = −17→13
l = −44→44
Refinement
Refinement on F2
Least-squares matrix: full
R[F2 > 2σ(F2)] = 0.054
wR(F2) = 0.161
S = 1.02
7066 reflections
457 parameters
0 restraints
Primary atom site location: structure-invariant
direct methods
Secondary atom site location: difference Fourier
map
Hydrogen site location: inferred from
neighbouring sites
H atoms treated by a mixture of independent
and constrained refinement F(000) = 2912
Dx = 1.311 Mg m−3
Cu Kα radiation, λ = 1.54184 Å
Cell parameters from 7977 reflections
θ = 3.3–74.7°
µ = 1.71 mm−1
T = 296 K
Prismatic, dark red
0.37 × 0.21 × 0.14 mm sup-2 Acta Cryst. Computing details Data collection: CrysAlis PRO (Agilent, 2012); cell refinement: CrysAlis PRO (Agilent, 2012); data reduction: CrysAlis
PRO (Agilent, 2012); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine
structure: SHELXL97 (Sheldrick, 2008); molecular graphics: PLATON (Spek, 2009); software used to prepare material
for publication: WinGX (Farrugia, 2012) and X-SEED (Barbour, 2001). sup-1 Acta Cryst. (2013). E69, o193 supplementary materials Figure 1 g
The labelled molecular structures of the two independent molecules comprising the asymmetric unit of (I) with
displacement ellipsoids drawn at the 50% probability level. The intramolecular hydrogen bonding shown as dashed line 2-[(2-Hydroxynaphthalen-1-yl)methylideneamino]-5,6,7,8-tetrahydro-4H- cyclohepta[b]thiophene-3-
carbonitrile 2-[(2-Hydroxynaphthalen-1-yl)methylideneamino]-5,6,7,8-tetrahydro-4H- cyclohepta[b]thiophene-3-
b
it il sup-2
Acta Cryst. (2013). 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,
conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > 2sigma(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. supplementary materials w = 1/[σ2(Fo2) + (0.0753P)2 + 3.2292P]
where P = (Fo2 + 2Fc2)/3
(Δ/σ)max = 0.001 Δρmax = 0.52 e Å−3
Δρmin = −0.25 e Å−3 Figure 1 (2013). E69, o193 supplementary materials Special details Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x
y
z
Uiso*/Ueq
S1
0.24913 (4)
0.21343 (5)
0.314590 (15)
0.05676 (19)
S2
0.45786 (4)
0.50708 (5)
0.564135 (16)
0.05789 (19)
O1
0.30203 (12)
0.26884 (15)
0.46051 (5)
0.0657 (5)
O2
0.51390 (13)
0.57956 (16)
0.70915 (5)
0.0684 (5)
N1
0.27388 (13)
0.23563 (13)
0.39056 (5)
0.0472 (4)
N2
0.0201 (2)
0.2458 (3)
0.42460 (7)
0.0989 (10)
N3
0.48552 (13)
0.54653 (14)
0.63907 (5)
0.0510 (5)
N4
0.2337 (2)
0.5752 (3)
0.67298 (7)
0.0986 (10)
C1
0.43541 (16)
0.24092 (16)
0.41754 (6)
0.0459 (5)
C2
0.54027 (16)
0.23125 (16)
0.41241 (6)
0.0499 (5)
C3
0.58494 (18)
0.2128 (2)
0.37759 (8)
0.0645 (7)
H3
0.5456
0.2070
0.3564
0.077*
C4
0.6851 (2)
0.2034 (2)
0.37445 (10)
0.0812 (9)
H4
0.7130
0.1908
0.3513
0.097*
C5
0.7461 (2)
0.2124 (2)
0.40592 (11)
0.0816 (9)
H5
0.8141
0.2064
0.4035
0.098*
C6
0.7060 (2)
0.2298 (2)
0.43951 (9)
0.0713 (8)
H6
0.7470
0.2356
0.4602
0.086*
C7
0.60312 (17)
0.23961 (17)
0.44399 (7)
0.0557 (6)
C8
0.56125 (19)
0.25724 (19)
0.47943 (7)
0.0625 (7)
H8
0.6025
0.2626
0.5001
0.075*
C9
0.46320 (19)
0.2665 (2)
0.48411 (7)
0.0611 (6)
H9
0.4377
0.2780
0.5078
0.073*
C10
0.39935 (17)
0.25896 (17)
0.45341 (6)
0.0511 (5)
C11
0.36858 (16)
0.23066 (16)
0.38672 (6)
0.0475 (5)
H11
0.3944
0.2200
0.3630
0.057*
C12
0.21176 (16)
0.22661 (16)
0.36021 (6)
0.0465 (5)
C13
0.11097 (16)
0.22964 (19)
0.36215 (6)
0.0531 (6)
C14
0.06278 (18)
0.2232 (2)
0.32699 (7)
0.0670 (7)
C15
0.12889 (19)
0.2154 (2)
0.29865 (7)
0.0673 (7)
C16
0.1107 (3)
0.2146 (3)
0.25696 (8)
0.0941 (11)
H16A
0.0941
0.2768
0.2492
0.113*
H16B
0.1718
0.1978
0.2446
0.113* Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x
y
z
Uiso*/Ueq Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Acta Cryst. (2013). Special details E69, o193 sup-3 supplementary materials C17
0.0333 (3)
0.1525 (3)
0.24402 (9)
0.1011 (12)
H17A
0.0490
0.0907
0.2527
0.121*
H17B
0.0352
0.1512
0.2169
0.121*
C18
−0.0719 (3)
0.1746 (3)
0.25595 (9)
0.0967 (11)
H18A
−0.0866
0.2377
0.2487
0.116*
H18B
−0.1165
0.1346
0.2422
0.116*
C19
−0.0935 (2)
0.1643 (3)
0.29717 (11)
0.1069 (13)
H19A
−0.1644
0.1677
0.3006
0.128*
H19B
−0.0728
0.1030
0.3048
0.128*
C20
−0.0477 (2)
0.2311 (3)
0.32213 (10)
0.1028 (13)
H20A
−0.0783
0.2254
0.3465
0.123*
H20B
−0.0624
0.2926
0.3129
0.123*
C21
0.06072 (18)
0.2395 (2)
0.39699 (7)
0.0646 (7)
C22
0.64645 (16)
0.54730 (16)
0.66648 (6)
0.0489 (5)
C23
0.75161 (17)
0.53553 (17)
0.66162 (6)
0.0508 (5)
C24
0.79674 (19)
0.5194 (2)
0.62665 (7)
0.0635 (7)
H24
0.7578
0.5162
0.6053
0.076*
C25
0.8967 (2)
0.5085 (2)
0.62369 (9)
0.0763 (8)
H25
0.9245
0.4973
0.6004
0.092*
C26
0.9576 (2)
0.5139 (2)
0.65491 (10)
0.0767 (8)
H26
1.0255
0.5066
0.6525
0.092*
C27
0.9170 (2)
0.5298 (2)
0.68894 (9)
0.0699 (8)
H27
0.9577
0.5332
0.7098
0.084*
C28
0.81442 (18)
0.54127 (18)
0.69330 (7)
0.0557 (6)
C29
0.77185 (19)
0.55757 (19)
0.72897 (7)
0.0621 (7)
H29
0.8127
0.5596
0.7498
0.075*
C30
0.6739 (2)
0.57016 (19)
0.73347 (7)
0.0622 (6)
H30
0.6483
0.5815
0.7571
0.075*
C31
0.61033 (18)
0.56608 (18)
0.70233 (6)
0.0535 (6)
C32
0.57975 (16)
0.53732 (17)
0.63555 (6)
0.0508 (5)
H32
0.6055
0.5237
0.6121
0.061*
C33
0.42264 (16)
0.53346 (17)
0.60903 (6)
0.0489 (5)
C34
0.32167 (16)
0.54044 (17)
0.61095 (6)
0.0500 (5)
C35
0.27183 (17)
0.52450 (18)
0.57632 (6)
0.0526 (6)
C36
0.33707 (18)
0.50529 (19)
0.54855 (7)
0.0563 (6)
C37
0.3190 (2)
0.4775 (3)
0.50862 (7)
0.0785 (9)
H37A
0.3811
0.4810
0.4952
0.094*
H37B
0.2980
0.4134
0.5083
0.094*
C38
0.2441 (3)
0.5336 (3)
0.48794 (8)
0.0933 (11)
H38A
0.2462
0.5159
0.4618
0.112*
H38B
0.2633
0.5981
0.4894
0.112*
C39
0.1380 (2)
0.5250 (3)
0.50151 (9)
0.0816 (9)
H39A
0.0947
0.5523
0.4830
0.098*
H39B
0.1215
0.4600
0.5034
0.098*
C40
0.1183 (2)
0.5701 (2)
0.53854 (8)
0.0778 (8)
H40A
0.1429
0.6329
0.5373
0.093*
H40B
0.0473
0.5737
0.5418
0.093*
C41
0.16098 (18)
0.5257 (2)
0.57281 (8)
0.0686 (7)
H41A
0.1378
0.4623
0.5738
0.082* sup-4 Acta Cryst. (2013). E69, o193 supplementary materials sup
Acta Cryst. (2013). Special details E69, o193
H41B
0.1344
0.5572
0.5945
0.082*
C42
0.27270 (18)
0.5608 (2)
0.64544 (7)
0.0624 (7)
H1O
0.272 (3)
0.261 (2)
0.4409 (9)
0.094*
H2O
0.486 (3)
0.577 (3)
0.6889 (9)
0.094*
Atomic displacement parameters (Å2)
U11
U22
U33
U12
U13
U23
S1
0.0477 (3)
0.0840 (5)
0.0386 (3)
0.0013 (3)
0.0038 (2)
−0.0040 (3)
S2
0.0453 (3)
0.0822 (5)
0.0461 (3)
0.0019 (3)
0.0062 (2)
−0.0034 (3)
O1
0.0496 (10)
0.1030 (15)
0.0445 (9)
−0.0027 (9)
0.0032 (7)
−0.0078 (9)
O2
0.0559 (10)
0.1014 (15)
0.0480 (9)
0.0012 (10)
0.0052 (8)
−0.0017 (10)
N1
0.0428 (9)
0.0580 (11)
0.0408 (9)
0.0009 (8)
−0.0020 (7)
−0.0018 (8)
N2
0.0766 (17)
0.156 (3)
0.0644 (15)
0.0023 (18)
0.0201 (14)
−0.0130 (17)
N3
0.0453 (10)
0.0644 (13)
0.0432 (9)
0.0002 (9)
−0.0012 (8)
0.0046 (9)
N4
0.0770 (17)
0.167 (3)
0.0524 (13)
0.0185 (18)
0.0161 (12)
−0.0012 (17)
C1
0.0453 (11)
0.0495 (13)
0.0430 (11)
−0.0007 (9)
−0.0035 (9)
0.0009 (9)
C2
0.0451 (11)
0.0516 (13)
0.0528 (12)
−0.0020 (10)
−0.0029 (10)
0.0047 (11)
C3
0.0458 (13)
0.0838 (19)
0.0640 (15)
−0.0004 (12)
0.0018 (11)
−0.0035 (14)
C4
0.0553 (15)
0.101 (2)
0.087 (2)
0.0032 (15)
0.0154 (15)
−0.0028 (18)
C5
0.0381 (13)
0.091 (2)
0.116 (3)
0.0000 (13)
−0.0038 (15)
0.005 (2)
C6
0.0500 (14)
0.0738 (19)
0.090 (2)
−0.0047 (13)
−0.0174 (14)
0.0081 (16)
C7
0.0485 (12)
0.0513 (14)
0.0673 (15)
−0.0053 (10)
−0.0132 (11)
0.0071 (12)
C8
0.0644 (16)
0.0688 (17)
0.0543 (13)
−0.0093 (13)
−0.0190 (12)
0.0073 (12)
C9
0.0662 (15)
0.0746 (18)
0.0424 (11)
−0.0086 (13)
−0.0081 (11)
0.0022 (12)
C10
0.0504 (12)
0.0585 (14)
0.0444 (11)
−0.0042 (10)
−0.0018 (9)
0.0021 (10)
C11
0.0442 (11)
0.0569 (14)
0.0413 (10)
0.0013 (9)
0.0005 (9)
−0.0016 (10)
C12
0.0447 (11)
0.0569 (14)
0.0378 (10)
0.0019 (9)
−0.0003 (8)
−0.0013 (10)
C13
0.0435 (11)
0.0726 (16)
0.0432 (11)
0.0011 (10)
0.0010 (9)
−0.0052 (11)
C14
0.0486 (13)
0.102 (2)
0.0505 (13)
0.0009 (13)
−0.0074 (11)
−0.0089 (14)
C15
0.0577 (14)
0.101 (2)
0.0428 (12)
−0.0007 (14)
−0.0074 (11)
−0.0055 (13)
C16
0.091 (2)
0.147 (3)
0.0446 (14)
−0.017 (2)
−0.0099 (14)
−0.0047 (18)
C17
0.109 (3)
0.137 (3)
0.0577 (17)
−0.005 (2)
−0.0232 (17)
−0.011 (2)
C18
0.086 (2)
0.121 (3)
0.084 (2)
0.008 (2)
−0.0430 (18)
−0.014 (2)
C19
0.0621 (19)
0.157 (4)
0.102 (3)
0.001 (2)
−0.0229 (18)
−0.015 (3)
C20
0.0498 (16)
0.177 (4)
0.082 (2)
0.008 (2)
−0.0145 (15)
−0.027 (2)
C21
0.0475 (13)
0.097 (2)
0.0490 (13)
0.0010 (13)
0.0024 (11)
−0.0069 (13)
C22
0.0480 (12)
0.0530 (13)
0.0457 (11)
−0.0017 (10)
−0.0027 (9)
0.0044 (10)
C23
0.0476 (12)
0.0535 (13)
0.0511 (12)
−0.0008 (10)
−0.0028 (10)
0.0020 (11)
C24
0.0524 (14)
0.0793 (18)
0.0587 (14)
0.0019 (12)
−0.0005 (11)
−0.0054 (13)
C25
0.0558 (15)
0.091 (2)
0.0816 (19)
0.0042 (14)
0.0106 (14)
−0.0116 (17)
C26
0.0463 (14)
0.083 (2)
0.101 (2)
0.0052 (13)
−0.0027 (15)
−0.0004 (18)
C27
0.0543 (15)
0.0727 (18)
0.0826 (19)
−0.0016 (13)
−0.0186 (14)
0.0064 (15)
C28
0.0542 (13)
0.0544 (14)
0.0584 (13)
−0.0011 (11)
−0.0112 (11)
0.0046 (11)
C29
0.0654 (16)
0.0699 (17)
0.0512 (13)
−0.0060 (13)
−0.0144 (11)
0.0074 (12)
C30
0.0710 (16)
0.0732 (17)
0.0424 (11)
−0.0082 (13)
−0.0028 (11)
0.0042 (12)
C31
0.0523 (13)
0.0619 (15)
0.0463 (12)
−0.0036 (11)
0.0012 (10)
0.0037 (11)
C32
0.0486 (12)
0.0598 (14)
0.0441 (11)
−0.0012 (10)
0.0005 (9)
0.0043 (10)
C33
0.0463 (12)
0.0581 (14)
0.0422 (11)
0.0008 (10)
0.0015 (9)
0.0038 (10)
C34
0.0461 (11)
0.0604 (14)
0.0434 (11)
−0.0004 (10)
0.0031 (9)
0.0021 (10) sup-5 Acta Cryst. Special details (2013). Acta Cryst. (2013). E69, o193 Special details E69, o193 supplementary materials
C35
0.0450 (12)
0.0667 (15)
0.0460 (11)
−0.0010 (10)
−0.0003 (9)
0.0011 (11)
C36
0.0508 (13)
0.0720 (16)
0.0462 (12)
−0.0018 (11)
0.0014 (10)
−0.0043 (11)
C37
0.0730 (18)
0.113 (3)
0.0492 (14)
−0.0037 (17)
0.0027 (13)
−0.0168 (15)
C38
0.102 (2)
0.130 (3)
0.0482 (15)
−0.006 (2)
−0.0155 (16)
−0.0017 (17)
C39
0.0742 (19)
0.101 (2)
0.0699 (18)
−0.0038 (17)
−0.0273 (15)
−0.0072 (17)
C40
0.0606 (16)
0.090 (2)
0.083 (2)
0.0038 (15)
−0.0208 (14)
−0.0045 (17)
C41
0.0473 (13)
0.095 (2)
0.0639 (15)
0.0006 (13)
−0.0045 (12)
0.0006 (15)
C42
0.0485 (13)
0.093 (2)
0.0461 (12)
0.0072 (13)
0.0028 (10)
0.0027 (13)
Geometric parameters (Å, º)
S1—C12
1.720 (2)
C18—H18A
0.9700
S1—C15
1.726 (3)
C18—H18B
0.9700
S2—C33
1.719 (2)
C19—C20
1.456 (5)
S2—C36
1.729 (2)
C19—H19A
0.9700
O1—C10
1.350 (3)
C19—H19B
0.9700
O1—H1O
0.82 (3)
C20—H20A
0.9700
O2—C31
1.343 (3)
C20—H20B
0.9700
O2—H2O
0.82 (3)
C22—C31
1.400 (3)
N1—C11
1.292 (3)
C22—C32
1.436 (3)
N1—C12
1.380 (3)
C22—C23
1.445 (3)
N2—C21
1.135 (3)
C23—C24
1.412 (3)
N3—C32
1.290 (3)
C23—C28
1.420 (3)
N3—C33
1.385 (3)
C24—C25
1.368 (4)
N4—C42
1.137 (3)
C24—H24
0.9300
C1—C10
1.398 (3)
C25—C26
1.391 (4)
C1—C11
1.435 (3)
C25—H25
0.9300
C1—C2
1.439 (3)
C26—C27
1.356 (4)
C2—C3
1.411 (3)
C26—H26
0.9300
C2—C7
1.420 (3)
C27—C28
1.408 (4)
C3—C4
1.368 (4)
C27—H27
0.9300
C3—H3
0.9300
C28—C29
1.420 (4)
C4—C5
1.403 (4)
C29—C30
1.349 (4)
C4—H4
0.9300
C29—H29
0.9300
C5—C6
1.343 (4)
C30—C31
1.410 (3)
C5—H5
0.9300
C30—H30
0.9300
C6—C7
1.410 (4)
C32—H32
0.9300
C6—H6
0.9300
C33—C34
1.373 (3)
C7—C8
1.413 (4)
C34—C35
1.430 (3)
C8—C9
1.346 (4)
C34—C42
1.432 (3)
C8—H8
0.9300
C35—C36
1.359 (3)
C9—C10
1.403 (3)
C35—C41
1.507 (3)
C9—H9
0.9300
C36—C37
1.505 (3)
C11—H11
0.9300
C37—C38
1.495 (5)
C12—C13
1.368 (3)
C37—H37A
0.9700
C13—C14
1.421 (3)
C37—H37B
0.9700
C13—C21
1.428 (3)
C38—C39
1.522 (5)
C14—C15
1.358 (4)
C38—H38A
0.9700
C14—C20
1.511 (4)
C38—H38B
0.9700
C15
C16
1 513 (3)
C39
C40
1 501 (4) supplementary materials C35
0.0450 (12)
0.0667 (15)
0.0460 (11)
−0.0010 (10)
−0.0003 (9)
0.0011 (11)
C36
0.0508 (13)
0.0720 (16)
0.0462 (12)
−0.0018 (11)
0.0014 (10)
−0.0043 (11)
C37
0.0730 (18)
0.113 (3)
0.0492 (14)
−0.0037 (17)
0.0027 (13)
−0.0168 (15)
C38
0.102 (2)
0.130 (3)
0.0482 (15)
−0.006 (2)
−0.0155 (16)
−0.0017 (17)
C39
0.0742 (19)
0.101 (2)
0.0699 (18)
−0.0038 (17)
−0.0273 (15)
−0.0072 (17)
C40
0.0606 (16)
0.090 (2)
0.083 (2)
0.0038 (15)
−0.0208 (14)
−0.0045 (17)
C41
0.0473 (13)
0.095 (2)
0.0639 (15)
0.0006 (13)
−0.0045 (12)
0.0006 (15)
C42
0.0485 (13)
0.093 (2)
0.0461 (12)
0.0072 (13)
0.0028 (10)
0.0027 (13) Geometric parameters (Å, º) sup-6 Acta Cryst. supplementary materials s
Acta Cryst (2013) E69 o193
C16—C17
1.457 (5)
C39—H39A
0.9700
C16—H16A
0.9700
C39—H39B
0.9700
C16—H16B
0.9700
C40—C41
1.500 (4)
C17—C18
1.522 (5)
C40—H40A
0.9700
C17—H17A
0.9700
C40—H40B
0.9700
C17—H17B
0.9700
C41—H41A
0.9700
C18—C19
1.511 (5)
C41—H41B
0.9700
C12—S1—C15
91.95 (11)
C14—C20—H20B
108.2
C33—S2—C36
92.41 (11)
H20A—C20—H20B
107.4
C10—O1—H1O
108 (2)
N2—C21—C13
178.8 (4)
C31—O2—H2O
106 (2)
C31—C22—C32
120.4 (2)
C11—N1—C12
121.09 (19)
C31—C22—C23
118.5 (2)
C32—N3—C33
121.3 (2)
C32—C22—C23
121.0 (2)
C10—C1—C11
120.3 (2)
C24—C23—C28
117.3 (2)
C10—C1—C2
118.7 (2)
C24—C23—C22
123.5 (2)
C11—C1—C2
121.0 (2)
C28—C23—C22
119.2 (2)
C3—C2—C7
117.5 (2)
C25—C24—C23
121.1 (3)
C3—C2—C1
123.7 (2)
C25—C24—H24
119.5
C7—C2—C1
118.8 (2)
C23—C24—H24
119.5
C4—C3—C2
121.1 (3)
C24—C25—C26
121.2 (3)
C4—C3—H3
119.4
C24—C25—H25
119.4
C2—C3—H3
119.4
C26—C25—H25
119.4
C3—C4—C5
120.6 (3)
C27—C26—C25
119.3 (3)
C3—C4—H4
119.7
C27—C26—H26
120.3
C5—C4—H4
119.7
C25—C26—H26
120.3
C6—C5—C4
119.9 (3)
C26—C27—C28
121.4 (3)
C6—C5—H5
120.0
C26—C27—H27
119.3
C4—C5—H5
120.0
C28—C27—H27
119.3
C5—C6—C7
121.3 (3)
C27—C28—C23
119.7 (2)
C5—C6—H6
119.3
C27—C28—C29
121.3 (2)
C7—C6—H6
119.3
C23—C28—C29
118.9 (2)
C6—C7—C8
121.1 (2)
C30—C29—C28
122.0 (2)
C6—C7—C2
119.6 (3)
C30—C29—H29
119.0
C8—C7—C2
119.3 (2)
C28—C29—H29
119.0
C9—C8—C7
121.8 (2)
C29—C30—C31
120.0 (2)
C9—C8—H8
119.1
C29—C30—H30
120.0
C7—C8—H8
119.1
C31—C30—H30
120.0
C8—C9—C10
120.2 (2)
O2—C31—C22
122.3 (2)
C8—C9—H9
119.9
O2—C31—C30
116.4 (2)
C10—C9—H9
119.9
C22—C31—C30
121.3 (2)
O1—C10—C1
122.3 (2)
N3—C32—C22
122.5 (2)
O1—C10—C9
116.5 (2)
N3—C32—H32
118.8
C1—C10—C9
121.2 (2)
C22—C32—H32
118.8
N1—C11—C1
122.6 (2)
C34—C33—N3
124.3 (2)
N1—C11—H11
118.7
C34—C33—S2
109.85 (17)
C1—C11—H11
118.7
N3—C33—S2
125.82 (17)
C13—C12—N1
124.44 (19)
C33—C34—C35
114.5 (2)
C13—C12—S1
110.22 (16)
C33—C34—C42
121.3 (2) sup-7
1.457 (5)
C39—H39A
0.9700
0.9700
C39—H39B
0.9700
0.9700
C40—C41
1.500 (4)
1.522 (5)
C40—H40A
0.9700
0.9700
C40—H40B
0.9700
0.9700
C41—H41A
0.9700
1.511 (5)
C41—H41B
0.9700
91.95 (11)
C14—C20—H20B
108.2
92.41 (11)
H20A—C20—H20B
107.4
108 (2)
N2—C21—C13
178.8 (4)
106 (2)
C31—C22—C32
120.4 (2)
121.09 (19)
C31—C22—C23
118.5 (2)
121.3 (2)
C32—C22—C23
121.0 (2)
120.3 (2)
C24—C23—C28
117.3 (2)
118.7 (2)
C24—C23—C22
123.5 (2)
121.0 (2)
C28—C23—C22
119.2 (2)
117.5 (2)
C25—C24—C23
121.1 (3)
123.7 (2)
C25—C24—H24
119.5
118.8 (2)
C23—C24—H24
119.5
121.1 (3)
C24—C25—C26
121.2 (3)
119.4
C24—C25—H25
119.4
119.4
C26—C25—H25
119.4
120.6 (3)
C27—C26—C25
119.3 (3)
119.7
C27—C26—H26
120.3
119.7
C25—C26—H26
120.3
119.9 (3)
C26—C27—C28
121.4 (3)
120.0
C26—C27—H27
119.3
120.0
C28—C27—H27
119.3
121.3 (3)
C27—C28—C23
119.7 (2)
119.3
C27—C28—C29
121.3 (2)
119.3
C23—C28—C29
118.9 (2)
121.1 (2)
C30—C29—C28
122.0 (2)
119.6 (3)
C30—C29—H29
119.0
119.3 (2)
C28—C29—H29
119.0
121.8 (2)
C29—C30—C31
120.0 (2)
119.1
C29—C30—H30
120.0
119.1
C31—C30—H30
120.0
120.2 (2)
O2—C31—C22
122.3 (2)
119.9
O2—C31—C30
116.4 (2)
119.9
C22—C31—C30
121.3 (2)
122.3 (2)
N3—C32—C22
122.5 (2)
116.5 (2)
N3—C32—H32
118.8
121.2 (2)
C22—C32—H32
118.8
122.6 (2)
C34—C33—N3
124.3 (2)
118.7
C34—C33—S2
109.85 (17)
118.7
N3—C33—S2
125.82 (17)
124.44 (19)
C33—C34—C35
114.5 (2)
110.22 (16)
C33—C34—C42
121.3 (2) sup-7 Acta Cryst. Special details (2013). E69, o193 supplementary materials supplementary materials (2013). E69, o193 supplementary materials Acta Cryst. (2013). E69, o193
N1—C12—S1
125.31 (16)
C35—C34—C42
124.2 (2)
C12—C13—C14
114.3 (2)
C36—C35—C34
111.1 (2)
C12—C13—C21
121.6 (2)
C36—C35—C41
126.1 (2)
C14—C13—C21
124.1 (2)
C34—C35—C41
122.7 (2)
C15—C14—C13
111.3 (2)
C35—C36—C37
130.1 (2)
C15—C14—C20
125.0 (3)
C35—C36—S2
112.13 (18)
C13—C14—C20
123.5 (2)
C37—C36—S2
117.67 (19)
C14—C15—C16
129.1 (3)
C38—C37—C36
115.8 (3)
C14—C15—S1
112.15 (18)
C38—C37—H37A
108.3
C16—C15—S1
118.6 (2)
C36—C37—H37A
108.3
C17—C16—C15
115.8 (3)
C38—C37—H37B
108.3
C17—C16—H16A
108.3
C36—C37—H37B
108.3
C15—C16—H16A
108.3
H37A—C37—H37B
107.4
C17—C16—H16B
108.3
C37—C38—C39
116.0 (3)
C15—C16—H16B
108.3
C37—C38—H38A
108.3
H16A—C16—H16B
107.4
C39—C38—H38A
108.3
C16—C17—C18
117.1 (3)
C37—C38—H38B
108.3
C16—C17—H17A
108.0
C39—C38—H38B
108.3
C18—C17—H17A
108.0
H38A—C38—H38B
107.4
C16—C17—H17B
108.0
C40—C39—C38
114.5 (2)
C18—C17—H17B
108.0
C40—C39—H39A
108.6
H17A—C17—H17B
107.3
C38—C39—H39A
108.6
C19—C18—C17
115.7 (3)
C40—C39—H39B
108.6
C19—C18—H18A
108.3
C38—C39—H39B
108.6
C17—C18—H18A
108.3
H39A—C39—H39B
107.6
C19—C18—H18B
108.3
C41—C40—C39
117.8 (3)
C17—C18—H18B
108.3
C41—C40—H40A
107.8
H18A—C18—H18B
107.4
C39—C40—H40A
107.8
C20—C19—C18
116.8 (4)
C41—C40—H40B
107.8
C20—C19—H19A
108.1
C39—C40—H40B
107.8
C18—C19—H19A
108.1
H40A—C40—H40B
107.2
C20—C19—H19B
108.1
C40—C41—C35
117.2 (2)
C18—C19—H19B
108.1
C40—C41—H41A
108.0
H19A—C19—H19B
107.3
C35—C41—H41A
108.0
C19—C20—C14
116.3 (3)
C40—C41—H41B
108.0
C19—C20—H20A
108.2
C35—C41—H41B
108.0
C14—C20—H20A
108.2
H41A—C41—H41B
107.2
C19—C20—H20B
108.2
N4—C42—C34
178.7 (4)
C10—C1—C2—C3
179.8 (2)
C31—C22—C23—C24
−177.8 (2)
C11—C1—C2—C3
1.0 (4)
C32—C22—C23—C24
4.0 (4)
C10—C1—C2—C7
0.5 (3)
C31—C22—C23—C28
1.4 (4)
C11—C1—C2—C7
−178.3 (2)
C32—C22—C23—C28
−176.8 (2)
C7—C2—C3—C4
0.0 (4)
C28—C23—C24—C25
1.0 (4)
C1—C2—C3—C4
−179.3 (3)
C22—C23—C24—C25
−179.8 (3)
C2—C3—C4—C5
−0.4 (5)
C23—C24—C25—C26
−0.7 (5)
C3—C4—C5—C6
0.5 (5)
C24—C25—C26—C27
0.3 (5)
C4—C5—C6—C7
−0.2 (5)
C25—C26—C27—C28
−0.2 (5)
C5—C6—C7—C8
179.5 (3)
C26—C27—C28—C23
0.4 (4) sup-8 Acta Cryst. (2013). supplementary materials E69, o193 supplementary materials C5—C6—C7—C2
−0.2 (4)
C26—C27—C28—C29
179.7 (3)
C3—C2—C7—C6
0.3 (4)
C24—C23—C28—C27
−0.8 (4)
C1—C2—C7—C6
179.7 (2)
C22—C23—C28—C27
179.9 (2)
C3—C2—C7—C8
−179.5 (2)
C24—C23—C28—C29
179.9 (2)
C1—C2—C7—C8
−0.1 (4)
C22—C23—C28—C29
0.6 (4)
C6—C7—C8—C9
−179.9 (3)
C27—C28—C29—C30
178.9 (3)
C2—C7—C8—C9
−0.1 (4)
C23—C28—C29—C30
−1.8 (4)
C7—C8—C9—C10
−0.1 (4)
C28—C29—C30—C31
0.9 (4)
C11—C1—C10—O1
−1.1 (4)
C32—C22—C31—O2
−3.0 (4)
C2—C1—C10—O1
−179.9 (2)
C23—C22—C31—O2
178.8 (2)
C11—C1—C10—C9
178.1 (2)
C32—C22—C31—C30
175.8 (2)
C2—C1—C10—C9
−0.8 (4)
C23—C22—C31—C30
−2.4 (4)
C8—C9—C10—O1
179.8 (2)
C29—C30—C31—O2
−179.9 (2)
C8—C9—C10—C1
0.6 (4)
C29—C30—C31—C22
1.3 (4)
C12—N1—C11—C1
179.2 (2)
C33—N3—C32—C22
−177.8 (2)
C10—C1—C11—N1
−1.4 (4)
C31—C22—C32—N3
1.2 (4)
C2—C1—C11—N1
177.5 (2)
C23—C22—C32—N3
179.3 (2)
C11—N1—C12—C13
178.9 (2)
C32—N3—C33—C34
178.7 (2)
C11—N1—C12—S1
−3.1 (3)
C32—N3—C33—S2
−1.7 (3)
C15—S1—C12—C13
1.5 (2)
C36—S2—C33—C34
−0.2 (2)
C15—S1—C12—N1
−176.7 (2)
C36—S2—C33—N3
−179.8 (2)
N1—C12—C13—C14
177.1 (2)
N3—C33—C34—C35
179.7 (2)
S1—C12—C13—C14
−1.1 (3)
S2—C33—C34—C35
0.1 (3)
N1—C12—C13—C21
−2.2 (4)
N3—C33—C34—C42
−1.2 (4)
S1—C12—C13—C21
179.5 (2)
S2—C33—C34—C42
179.2 (2)
C12—C13—C14—C15
−0.1 (4)
C33—C34—C35—C36
0.2 (3)
C21—C13—C14—C15
179.3 (3)
C42—C34—C35—C36
−178.9 (3)
C12—C13—C14—C20
−176.0 (3)
C33—C34—C35—C41
177.8 (2)
C21—C13—C14—C20
3.4 (5)
C42—C34—C35—C41
−1.3 (4)
C13—C14—C15—C16
−174.5 (3)
C34—C35—C36—C37
175.1 (3)
C20—C14—C15—C16
1.3 (6)
C41—C35—C36—C37
−2.5 (5)
C13—C14—C15—S1
1.3 (4)
C34—C35—C36—S2
−0.3 (3)
C20—C14—C15—S1
177.1 (3)
C41—C35—C36—S2
−177.9 (2)
C12—S1—C15—C14
−1.6 (3)
C33—S2—C36—C35
0.3 (2)
C12—S1—C15—C16
174.6 (3)
C33—S2—C36—C37
−175.7 (2)
C14—C15—C16—C17
−48.8 (6)
C35—C36—C37—C38
47.0 (5)
S1—C15—C16—C17
135.7 (3)
S2—C36—C37—C38
−137.9 (3)
C15—C16—C17—C18
66.1 (5)
C36—C37—C38—C39
−66.9 (4)
C16—C17—C18—C19
−67.5 (5)
C37—C38—C39—C40
71.3 (4)
C17—C18—C19—C20
69.4 (5)
C38—C39—C40—C41
−71.0 (4)
C18—C19—C20—C14
−70.2 (5)
C39—C40—C41—C35
65.8 (4)
C15—C14—C20—C19
48.6 (6)
C36—C35—C41—C40
−42.6 (4)
C13—C14—C20—C19
−136.1 (4)
C34—C35—C41—C40
140.1 (3)
Hydrogen-bond geometry (Å, º)
D—H···A
D—H
H···A
D···A
D—H···A
O1
H1O N1
0 82 (3)
1 84 (3)
2 578 (2)
150 (3) Hydrogen-bond geometry (Å, º) sup-9
Acta Cryst. (2013). E69, o193
Hydrogen-bond geometry (Å, º)
D—H···A
D—H
H···A
D···A
D—H···A
O1—H1O···N1
0.82 (3)
1.84 (3)
2.578 (2)
150 (3)
O2—H2O···N3
0.82 (3)
1.84 (3)
2.582 (2)
151 (3) Acta Cryst. (2013). E69, o193 sup-9
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https://openalex.org/W4392433841
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https://bsj.uobaghdad.edu.iq/index.php/BSJ/article/download/1409/1340
|
Arabic
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The Effect of Aqueous Extract of Citrullius colocynthis Seeds on Cellular Immunity
|
Mağallaẗ baġdād li-l-ʿulūm
| 2,012
|
cc-by
| 3,616
|
Baghdad Science Journal Baghdad Science Journal Vol.9(4)2012 The Effect of Aqueous Extract of Citrullius colocynthis Seeds
on Cellular Immunity Ali Hafedh Abbas*
Received 7, October, 2011
Accepted 21, February, 2012 Ali Hafedh Abbas* Received 7, October, 2011
Accepted 21, February, 2012 Abstract: The aqueous extract of Citrullius colocynthis dried seeds (160 μg/ml) was in vitro
evaluated for its effect on phagocytic index (PI) and lymphocyte transformation index
(LTI) of blood cells obtained from 30 apparently healthy blood donors (15 males and
15 females). The PI was further in vivo evaluated in cells of peritone, spleen and liver
of mice treated with the extract at a dose of 0.64 mg/kg. The results revealed that in in
vitro study, phagocytic cells treated with the extract showed a significant increased
percentage as compared with untreated cells (60.0 vs. 44.1%). Phagocytes obtained
from peritone (44.1 vs. 30.0%) and spleen (45.6 vs. 39.6 %) of treated and untreated
mice behaved in a similar manner, while liver phagocytes showed no significant
difference between PI of immunological function of the investigated cells, and may
use as therapeutic agent. treated and untreated mice. For LTI, cultures I and II shared
an approximated mean (70.0 and 68.0%, respectively), but both indices were
significantly higher than the recorded LTI in culture III (54.0%). These findings
suggest that the plant extract is effective in enhancing the Keywords: Citrullius colocynthis, Bitter apple, Aqueous extract of seeds,
Lymphocyte transformation. Introduction: Citrulluscolocynthis
(Cucurbitaceae),
commonly known as ‘bitter apple’,
‘colosynth’,
‘vine-of-Sodom’
and
‘tumba’ is a tropical plant that grows
abundantly in the Arabian countries
and widely in other parts of the world
[1]. In traditional medicine, this plant
has been used to treat constipation [2],
diabetes [3], odema, fever, jaundice,
bacterial infections and cancers, and it
is also used as an abortifacient [1]. Further studies have implanted that C. colocynthis is rich in compounds that
have antioxidant and free radical
scavenging
potentials
[4,5]. Preliminary phytochemical screening
of the plant showed the presence of
large
amounts
of
phenolic
and
flavonoids
[6]. Flavonoids
and
phenolic
compounds
are
widely distributed in plants, and have been
reported to exert multiple biological
effects, including antioxidant, free
radical
scavenging
abilities,
anti-
inflammatory, anti-carcinogenic, and
others [7]. Accordingly, the present
study was planned with aims to
evaluate
the
cellular
immunity
potentials of C. colocynthis seed
aqueous extract in vitro and in vivo. *Biological Tropical Researches Unit/College of Science/University of Baghdad Materials and Methods: The extract was then
evaporated on a rotary evaporator [8]. For in vitro evaluation, a concentration
of 160 μg/ml was used, while for in
vivo evaluation, a dose of 0.64 mg/kg
was employed. p y
In vitro Evaluation: Such evaluation
included phagocytic index (PI) and
lymphocyte transformation index (LTI)
of blood cells obtained from 30
apparently healthy blood donors (15
males and 15 females) with an age
range of 34.4 ±2.8 year. From each
subject, 5 ml of venous blood were
collected in heparinized tube under
aseptic conditions. For PI, 0.5 ml blood
was mixed with equal volumes of C. colocynthis seed extract (160 μg/ml)
and suspension of Staphylococcus
aureus at a concentration of 1 x 106
cell/ml (the bacterial isolate was
obtained from the Department of
Biology, College of Science) and
incubated for 30 minutes at 37°C. After incubation, a thin blood film was
made and stained with Leishman stain. The slide was examined under oil
immersion lens and the percentage of
phagocytic cells (PI) was recorded. For
each subject, a control evaluation was
also
made
and
included
similar
conditions but without the seed extract
[9, 10]. y
Lymphocyte transformation test: In
the assessment of LTI, three cultures
were set-up for each subject. Culture I
included mixing 2.5 ml RPMI-1640
medium, 0.5 ml extract (160 μg/ml),
0.3 ml PHA and 0.2 ml blood. In
culture II, similar components were
mixed
but
without
the
extract
(Control), while in culture III, the PHA
was replaced by the extract. The three
cultures were incubated at 37°C for 72
hours, and after that, they were
processed with a hypotonic solution
(0.075M KCl) and the obtained cells
were fixed (3 parts of absolute
methanol with 1 part of glacial acetic
acid), and then, 4-5 drops of the cell
suspension were dropped on a clean
slide from a height of about two feet. The slide was air-dried at room
temperature, and by then it was stained
with Giemsa stain for 15 minutes and
rinsed with distilled water. The slide
was examined under oil emersion lens
(100X), and at least 500 cells were
examined, and percentage of blast and
dividing cells (LTI) was recorded [12]. Statistical
Analysis:
Data
are
expressed as mean ± standard error
(S.E.), t-test was used to determine the
differences between the groups by
using the computer program SPSS
version 13.0. Statistical significance
was considered at P≤ 0.05. Materials and Methods: Plant Seed Collection and Extraction:
Dried seeds of C. colocynthis were
collected from a local store of herbal
medicine, and they were certified by
the Herbarium of the College of
Science (University of Baghdad). The
seeds were powdered using coffee
grinder, and 100 grams of the powder 156 Vol.9(4)2012 Baghdad Science Journal Staphylococcus
aureus
at
a
concentration of 1 × 106 cell/ml, and
after 24 hours, the animals were
sacrificed,
dissected
and
the
phagocytes were obtained from the
peritone, spleen and liver. These cells
were
processed
as
in
in
vitro
evaluation to record the PI [9, 10]. All
studies were conducted accordance
with National Institute of Health's
Guide for the Care and Use of
Laboratory Animals [11]
Lymphocyte transformation test: In
the assessment of LTI, three cultures
were set-up for each subject. Culture I
included mixing 2.5 ml RPMI-1640
medium, 0.5 ml extract (160 μg/ml),
0.3 ml PHA and 0.2 ml blood. In
culture II, similar components were
mixed
but
without
the
extract
(Control), while in culture III, the PHA
was replaced by the extract. The three
cultures were incubated at 37°C for 72
hours, and after that, they were
processed with a hypotonic solution
(0.075M KCl) and the obtained cells
were fixed (3 parts of absolute
methanol with 1 part of glacial acetic
acid), and then, 4-5 drops of the cell
suspension were dropped on a clean
slide from a height of about two feet. The slide was air-dried at room
temperature, and by then it was stained
with Giemsa stain for 15 minutes and
rinsed with distilled water. The slide
was examined under oil emersion lens
(100X), and at least 500 cells were
examined, and percentage of blast and
dividing cells (LTI) was recorded [12]. Statistical
Analysis:
Data
are
expressed as mean ± standard error
(S.E.), t-test was used to determine the
differences between the groups by
i
h
SPSS Staphylococcus
aureus
at
a
concentration of 1 × 106 cell/ml, and
after 24 hours, the animals were
sacrificed,
dissected
and
the
phagocytes were obtained from the
peritone, spleen and liver. These cells
were
processed
as
in
in
vitro
evaluation to record the PI [9, 10]. All
studies were conducted accordance
with National Institute of Health's
Guide for the Care and Use of
Laboratory Animals [11] was subjected to successive extraction
(four hours) in a Soxhlet apparatus,
using 500 ml of distilled water as
solvent at 50°C. Materials and Methods: Phagocyte
Sources
Phagocytic Index (Mean ± S.E.; %)
ANOVA
Probability ≤ 0.05
Treated
Untreated
Peritone
44.1 ± 2.1
30.0 ± 1.7
0.05
Spleen
45.6 ± 1.3
39.6 ± 0.7
0.05
Liver
35.3 ± 3.2
33.2 ± 2.4
Not Significant Table 2: Phagocytic index of phagocytes obtained from peritone, spleen and liver of mice
treated with 25 mg/kg of Citrullus colocynthis aqueous seed extract. Phagocyte
S
Phagocytic Index (Mean ± S.E.; %)
ANOVA
P
b bilit ≤0 05
T
t d
U t
t d Table 2: Phagocytic index of phagocytes obtained from peritone, spleen and liver of mice
reated with 25 mg/kg of Citrullus colocynthis aqueous seed extract. antimicrobial properties is Alkaloids,
tannins, steroids, pigments and iridoids
[18].In
agreement
with
such
augmentation [6, 13, 14, 15, 16, 17]
were able to demonstrate that these
constituents have a wide range of
biological effects, C. colocynthis is an
Iranian
medicinal
plant
that
has
traditionally
been
used
as
an
abortifacient and to treat constipation,
oedema, bacterial infections, cancer
and diabetes [6], the plant extract had
decrease the formation of granuloma
tissue in chronic inflammation model
in male albino rats this ynvestigation
support that C. colocynthis is used as
anti-inflammatory
agent
[15]. including treatment of diabetic people
in Mediterranean countries, and used
for
treatment
many
inflammatory
disease, constipation, odema, fever,
jaundice,
leukemia,
bacterial
infections, cancer, used as abortifacient antimicrobial properties is Alkaloids,
tannins, steroids, pigments and iridoids
[18].In
agreement
with
such
augmentation [6, 13, 14, 15, 16, 17]
were able to demonstrate that these
constituents have a wide range of
biological effects, C. colocynthis is an
Iranian
medicinal
plant
that
has
traditionally
been
used
as
an
abortifacient and to treat constipation,
oedema, bacterial infections, cancer
and diabetes [6], the plant extract had
decrease the formation of granuloma
tissue in chronic inflammation model
in male albino rats this ynvestigation
support that C. colocynthis is used as
anti-inflammatory
agent
[15]. including treatment of diabetic people
in Mediterranean countries, and used
for
treatment
many
inflammatory
disease, constipation, odema, fever,
jaundice,
leukemia,
bacterial
infections, cancer, used as abortifacient the present results demonstrated the
plant extract was significantly effective
in enhancing phagocytosis in both in
vitro and in vivo, and accordingly it is
possible to suggest that the extract may
contain chemical
compounds
that
enhanced the non-specific cellular
immunity of phagocytes reviewing the
literature revealed that C. Materials and Methods: In vivo Evaluation: In such evaluation,
the PI was recorded for phagocytes
obtained from the peritone, spleen and
liver of albino male mice (8-9 weeks
old), which were distributed into two
groups. The first group (5 mice) was
administrated orally with 0.25 ml of
the extract (25 mg/kg) daily for two
weeks, while the second group (5
mice) was treated with distilled water
in a similar manner. In both cases and
at the end of treatment period, each
mouse was injected intraperitoneally
with
0.1
ml
of
formalin-killed Results and Discussion:
Phagocytic index (PI): In in vitro
study, phagocytic cells were treated Phagocytic index (PI): In in vitro
study, phagocytic cells were treated 156 Baghdad Science Journal Vol.9(4)2012 Lymphocyte Transformation Index
(LTI): Cultures I and II shared an
approximated mean of LTI (70.0 and
68.0%, respectively), but both indices
were significantly higher than the
recorded LTI in culture III (54.0%)
(Table 1). Lymphocyte Transformation Index
(LTI): Cultures I and II shared an
approximated mean of LTI (70.0 and
68.0%, respectively), but both indices
were significantly higher than the
recorded LTI in culture III (54.0%)
(Table 1). with the extract showed a significant
increased percentage as compared with
untreated cells (60.0 vs. 44.1%) (Table
1). Phagocytes obtained from peritone
(44.1 vs. 30.0%) and spleen (45.6 vs. 39.6 %) of treated and untreated mice
behaved in a similar manner, while
liver phagocytes showed no significant
difference between PI of treated and
untreated mice (Table 2). Table 1: Phagocytic index and lymphocyte transformation index of blood cells treated with
160 μg/ml of Citrullus colocynthis aqueous seed extract. Parameter
Mean ± S.E. (%)
ANOVA
Probability ≤
0.05
Treated
Untreated
Phagocytic Index
60.0 ± 4.0
44.1 ± 2.5
0.05
Lymphocyte
Transformation Index
Culture I
Culture II
Culture III
I vs. III P ≤ 0.05
II vs. III p ≤ 0.05
70.0 ± 5.6
68.0 ± 3.2
54.0 ± 3.0 Table 1: Phagocytic index and lymphocyte transformation index of blood cells treated with
60 μg/ml of Citrullus colocynthis aqueous seed extract. Table 2: Phagocytic index of phagocytes obtained from peritone, spleen and liver of mice
treated with 25 mg/kg of Citrullus colocynthis aqueous seed extract. Materials and Methods: سللم، ه اءلل س و سللمياه ري،لل براللم و للر ا، ه
. لمللل،ل لم،للل2006
. رر سلللن عء ،لللن لم للل م
أور ق يبللل ل لوا للليOleo europea
. عجملللن
)أ ح ث ل ق ين لح،يان ه لمجمم لث عن ( لعمر ألول
:
75
-
83
. 9. سللم، ه اءلل س و سللمياه ري،لل براللم و للر ا، ه
. لمللل،ل لم،للل2006
. رر سلللن عء ،لللن لم للل م
أور ق يبللل ل لوا للليOleo europea
. عجملللن
)أ ح ث ل ق ين لح،يان ه لمجمم لث عن ( لعمر ألول
:
75
-
83
. 9. 10. Furth, R. V.; Theda, L. V. and
Leijilt,
P. C. 1985. In
vitro
Determination
Phagocytosis
and
Intranuclear
Killing
by
Polymorphnuclear and Mononuclear
Phagocytosis
"
Hand
book
of
experimental immunology " Black
Well Scientific Publication (3rd ed.), 2:
1 – 14. Materials and Methods: colocynthis
is rich in three flavonoids glycosides
(isosaponarin,
isoviterxin
and
isoorientin 3'-Ο-methyl ether), protein
( rich in isoleusine, leusine and
tryptophan
amino
acids),
lipids,
saponins, flavonoids (such as apigenin
and quercetin and leuteolin), and a
considerable amount of ions
( potassium, phosphorus and iron) such
constituents have been demonstrated to
have immunomodulatory effects [6,
13, 14, 15, 16, 17]. Another active
material that founded in the C. colocynthis seed extract that have 156 Baghdad Science Journal Vol.9(4)2012 Methanolic fruit extract. Acta. Pharm.,
58:215 – 220. and it had antibacterial and antifungal
properties, which have confirmed by
others [1, 2, 3, 18, 19].Furthermore,
the Folkloric application of the plant
(for instance anti-oxidant, antibacterial,
antifungal and anti-leukemic activity)
can also be interpreted on such ground,
especially it use consider the anti-
oxidant potential of the plant or these
active ingredient [4, 5, 6, 7, 20, 21, 21,
23]. With respect to LTI, the extract
was less effective than PHA in cross-
linking the lymphocyte receptors, and
therefore the LTI value came lower in
culture III as compared with culture II. However a value of 54 % for LTI in
culture III cannot be ignored and some
potential effects in enhancing the
cellular response of T lymphocyte can
be suggested and it might also be
related to the chemical constituents of
the plant. 5. Dallak, M. and Bin-Jaliah, I. 2010. Antioxidant activity of Citrullus
colocynthis pulp extract n the RBC's of
alloxan-induced diabetic rats. Pak. J. Physiol., 6(1):1 – 5. 6. y
( )
6. Delazar,
A.;
Gibbons,
S.;
Kosari,
A. R.;
Nazemiyeh,
H.;
Modarresi, M. Nahar, L. and Sarker, S. D. 2006. Flavone C-glycosides and
cucurbitacin glycosides from Citrullus
colocynthis. DARU. , 14:109 – 114. 7. Miller, A. L. 1996. Antioxidant
flavonoids: structure, function and
clinical usage. Alt. Med. Rev., 1: 103 –
111. 8. Marzouk, B.;
Marzouk, Z.;
Haloui, E.; Fenina, N.; Bouraoui, A. and Aouni, M. 2010. Screening of
analgesic
and
anti-inflammatory
activities of Citrullus colocynthis from
southern Tunisia. J. Ethnopharmacol.,
128: 15-19. From the findings of both indices, it is
possible to suggest that the plant may
be
characterized
with
immunomodulation potentials; but it is
too early to reach a final conclusion,
and further investigations are required
to cover such subject. From the findings of both indices, it is
possible to suggest that the plant may
be
characterized
with 9. References 1.Madari, H. and Jacobes, R. S. 2004. An analysis of cytotoxic botanical
formulation used in the traditional
medicine
of
ancient
Persia
as
abortifacient. J. Nat. Prods., 67:1204 –
1210. 2. Alkofahi, A.; Batshoun, R.;
Owis, W.; Najib, N. 1996. Biological
activity of some Jordanian plants
extracts. Fitoterapia. , 5:435 – 442. 11. National Institute of Health
1996. Guide for the care and use of
laboratory animals. Revised. DHEW
Publication (NIH), Office of Science
and
Health
Reports,
DRR/NIH,
Bethesda, MD. 11. National Institute of Health
1996. Guide for the care and use of
laboratory animals. Revised. DHEW
Publication (NIH), Office of Science
and
Health
Reports,
DRR/NIH,
Bethesda, MD. 3. Ziyyat, A. and Legssyer, A. 1997. Phytotherapy of hypertension
and diabetes in oriental Morocco. J. Ethanopharmacol., 58:45 – 54. 12. Shubber, E. K. and Allak, B. M. 1985. Spontaneous
chromosomal
aberration
in
human
lymphocyte. Effect of Cellular Condition. The
Nucleus, 29: 92 – 98. 12. Shubber, E. K. and Allak, B. M. 1985. Spontaneous
chromosomal
aberration
in
human
lymphocyte. Effect of Cellular Condition. The
Nucleus, 29: 92 – 98. 4. Kumar,
S. ;
Kumar,
D. Manjusha ; Saroha, K. Singh, N. and
Vashishta, B. 2008. Antioxidant and
free radical scavenging potential of
Citrullus
colocynthis
(L.)
Schrad. 156 Baghdad Science Journal Vol.9(4)2012 13. Gruenwald, J.; Brenler, T. and
Jaenicke, C. 1999. Sage in PDR for
Herbal Medicine.1st Ed. Montvale:
Medical Economics Company. New
Jersey.pp:425 – 426, 1113 – 1114. 19. Sebbagh,
N.;
Cruciani-
Guglielmacci, C.; Ouali, F.; Berthault,
M.-F.; Rouch, C.; Chabane Sari, D. and Magnan, C. 2009. Comparative
effects
of
Citrullus
colocynthis,
sunflower and olive oil-enriched diet in
streptozotocin-induced diabetes in rats. Diab. Metabol., 35(3): 178-184. 14. Duke, J. A. 1983. Citrullius
colocynthis (L.) SCHARD. Handbook
of energy crops. gy
p
15. Rajamanickam, E.; Gurudeeban,
S.; Ramanathan, I. and Satyavani, K. 2010. Evaluation of anti-inflammatory
activity of Citrullius colocynthis. Int. J. Curr. Res., 2:67 – 69. 20. Nerurkar, P. and Ray, R. B. 2010. Bitter melon: Antagonist to
cancer. Pharm. Res. J., 27: 1049-1053. 21. Marzouk, B.;
Marzouk, Z.;
Mastouri, M.; fenina, N. and Aouni,
M. 2011. Comparative evaluation of
the antimicrobial activity of Citrullius
colocynthis immature fruit and seed
organic extracts. Afr. J. Biotechnol.,
10(10):2130 – 2134. 16. El-Fergani, S. O. and Buatig, R. H. 2008. Gross composition, mineral
content
and
nutritional
value
of
colocynth fruit seeds. Alex. J. Fd. Sci. and
Technol.,
Special
volume
conference: 41 – 47. 22. References Phate, A. R.; Wayal, S. R. and
Oswal,
R. J. 2011. Study
of
antimicrobial activity on Citrullius
colocynthis Linn. Imperial. Pharma. Cosmetol., 1(1):14 – 18. 17. Lucas, E. A.; Dumancas, G. G.;
Smith, B. J.; Clarke, S. L. and
Arjmandi, D. H. 2010. Chapter 35-
Health benefits of bitter melon. Fruits
and Vegetable, PP.:525 – 549. 23. Takemoto, D. J.; Dunfoerd, C. and McMurray, M. M. 1982. The
cytotoxic and cytostatic effect of the
bitter melon on human lymphocytes. Toxicon, 20(3): 593 – 599. 18. Marzouk, B.;
Marzouk, Z.;
Décor, R.; Edziri, H.; Haloui, E.;
Fenina, N. and Aouni, M. 2009. Antibacterial
and
anticandidal
screening
of
Tunisian
Citrullus
colocynthis Schrad. From medicine. J. Ethnopharmacol., 125: 344-349. 18. Marzouk, B.;
Marzouk, Z.;
Décor, R.; Edziri, H.; Haloui, E.;
Fenina, N. and Aouni, M. 2009. Antibacterial
and
anticandidal
screening
of
Tunisian
Citrullus
colocynthis Schrad. From medicine. J. Ethnopharmacol., 125: 344-349. تأثير المستخلص المائي لبذور نبات الحنظلCitrullius colocynthis
على المناعة الخلوية
علي حافظ عباس*
*
وحدة األبحاث البايولوجية للمناطق الحارة–
كلية العلوم– جامعة بغداد
الخالصة :
ت
،،تق تأث،ر لم م لم ئي لبذور
يب ل لحءظ
Citrullius colocynthis
لج ب
ن
رك،و611
/ ع،كروغر ا عم،م ر
ل رج لج لحي عن لالل عع ع لبمعمن (
PI) وعع ع ل حيل لممف وي
LTI)) لـ30
،ءن را
ً لم بر ،ن صح س ظ ارا
(
15
ذكر و15
.)أيثى وكذلك ت تق،، تأث،ر لم م ر ل لج لحي عن لالل عع ع لبمعمن
ل الا لغش س لبرا ييي
ه لط
ح ل و لكبم
لفئر عع عمن رك،و ع م25
. عمغ / كغ وأظهرل لء ئج ل ي أجرات ل رج لج لحي يجير
زا رة ععءيان ،ن ل الا لبمعم،ن
لمع عمن لم م لمق رين
عع ل الا غ،ر لمع عمن
(60.0 vs. 44.1%)
. كم ك يت
ي بن عع ع لبمعمن لم الا لبمعم،ن ل ي
ت لحصيل م،ه عن لغش س لبرا ييي(44.1 vs. 30.0%)
و لطح ل
(45.6
vs. References 39.6 %)
لمفئر لمع عمن لم م وغ،ر لمع عمن ع م ثمنه بي ح،ن
ل تظهر بروق ععءيان لمع ع لبمعمن لم الا
لبمعم،ن ل ي ت لحصي
ل م،ه عن لكبم
عن لفئر لمع عمن وغ،ر لمع عمن لم م ه أع ي ئج ل حيل و أليق ا
لممف وي بك ع يسطي لورع ألول و لث يي ع ق ر ،ن(70.0 and 68.0%,)
و لمذ أظهر زا رة ععءيان لمق رين عع
. لورع لث لث وتش،ر اذه لء ئج
لى أ لمم م لءب تي أث،ر ت بي تحف،و لجه ز لمء ي و عك ي،ن
أس م عه كع ع
. الجي تأثير المستخلص المائي لبذور نبات الحنظلCitrullius colocynthis
على المناعة الخلوية
علي حافظ عباس*
ألبحاث البايولوجية للمناطق الحارة–
كلية العلوم– جامعة بغداد
ة ا
ت
،،تق تأث،ر لم م لم ئي لبذور
يب ل لحءظ
Citrullius colocynthis
لج ب
ن
رك،و611
/ ع،كروغر ا
عم،م ر
ل رج لج لحي عن لالل عع ع لبمعمن (
PI) وعع ع ل حيل لممف وي
LTI)) لـ30
،ءن را
ً لم بر ،ن صح س ظ ارا
(
15
ذكر و15
.)أيثى وكذلك ت تق،، تأث،ر لم م ر ل لج لحي عن لالل عع ع لبمعمن
ل الا
لغش س لبرا ييي
ه لط
ح ل و لكبم
لفئر عع عمن رك،و ع م25
. عمغ / كغ وأظهرل لء ئج ل ي
أجرات ل رج لج لحي يجير
زا رة ععءيان ،ن ل الا لبمعم،ن
لمع عمن لم م لمق رين
عع ل الا غ،ر لمع عمن
(60.0 vs. 44.1%)
. كم ك يت
ي بن عع ع لبمعمن لم الا لبمعم،ن ل ي
ت لحصيل م،ه عن لغش س لبرا ييي(44.1 vs. 30.0%)
و لطح ل
(45.6
vs. 39.6 %)
لمفئر لمع عمن لم م وغ،ر لمع عمن ع م ثمنه بي ح،ن
ل تظهر بروق ععءيان لمع ع لبمعمن لم الا
لبمعم،ن ل ي ت لحصي
ل م،ه عن لكبم
عن لفئر لمع عمن وغ،ر لمع عمن لم م ه
أع ي ئج ل حيل و أليق ا
لممف وي بك ع يسطي لورع ألول و لث يي ع ق ر ،ن(70.0 and 68.0%,)
و لمذ أظهر زا رة ععءيان لمق رين عع
. لورع لث لث وتش،ر اذه لء ئج
لى أ لمم م لءب تي أث،ر ت بي تحف،و لجه ز لمء ي و عك ي،ن
أس م عه كع ع
. الجي 155
|
https://openalex.org/W3082008050
|
https://www.compadre.org/per/items/5342.pdf
|
English
| null |
Investigating student understanding of the stationary state wavefunction for a system of identical particles
|
2020 Physics Education Research Conference Proceedings
| 2,020
|
cc-by
| 4,979
|
2020 PERC Proceedings edited by Wolf, Bennett, and Frank; Peer-reviewed, doi.org/10.1119/perc.2020.pr.Keebaugh
Published by the American Association of Physics Teachers under a Creative Commons Attribution 4.0 license.
Further distribution must maintain the cover page and attribution to the article's authors. Investigating student understanding of the stationary state wavefunction for a system of identical
particles Christof Keebaugh
Department of Physics and Astronomy, Franklin and Marshall College, Lancaster, PA 17603 Emily Marshman
Department of Physics and Astronomy, Community College of Allegheny County, Pittsburgh, PA 15260 Chandralekha Singh
Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA 15260 Chandralekha Singh
Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA 15260 I.
INTRODUCTION AND BACKGROUND two non-interacting identical particles has terms such as
ψna(xi)ψnb(xj), where ψna(xi) and ψnb(xj) are the single-
particle wavefunction for the ith particle with coordinate xi
in the state na and for the jth particle with coordinate xj in
the state nb, respectively. Quantum mechanics (QM) is a particularly challenging
subject for upper-level undergraduate and graduate students
in physics as evidenced by many investigations, e.g., see Refs. [1–20]. We have been involved in a number of research
studies aimed at investigating student reasoning in QM and
improving student understanding of QM [21–35]. However,
there have been few investigations into student difficulties
with fundamental concepts involving a system of identical
particles. Through researching students’ understanding and
reasoning about a system of identical particles, we have
found many common student difficulties that can hinder
the development of a consistent and coherent knowledge
structure pertaining to these concepts. If we have a system of two non-interacting identical
fermions, the two-particle stationary state wavefunction must
be completely antisymmetric. There are two ways to
construct a completely antisymmetric wavefunction:
the
spatial part of the wavefunction could be completely
symmetric and the spin part of the wavefunction could
be completely antisymmetric or the spatial part of the
wavefunction could be completely antisymmetric and the spin
part of the wavefunction could be completely symmetric. If we have a system of two non-interacting identical
bosons, the two-particle stationary state wavefunction must
be completely symmetric. There are two ways to construct
a completely symmetric wavefunction:
the spatial and
spin parts of the wavefunction could both be completely
symmetric or the spatial and spin parts of the wavefunction
could both be completely antisymmetric. In nature, there are two general types of particles: fermions
with a half-integer spin quantum number (e.g., electrons and
protons) and bosons with an integer spin quantum number
(e.g., photons and mesons). A system of N identical particles
consists of N particles of the same type (e.g., electrons). For
a system of identical particles in classical mechanics (e.g.,
five identical tennis balls), each particle can be distinguished
from all the other particles. In contrast, in quantum
mechanics, identical particles are indistinguishable and there
is no measurement that can be performed to distinguish
these identical particles from one another. For example, if
the coordinates of two identical particles are interchanged,
there is no physical observable that would reflect this
interchange. I.
INTRODUCTION AND BACKGROUND Furthermore, one property that distinguishes
these two types of particles is that two or more bosons can
occupy the same single-particle quantum state, but two or
more fermions can never occupy the same single-particle
quantum state. The restriction for fermions is known as the
Pauli exclusion principle and is consistent with a system of
fermions having a completely antisymmetric wavefunction. To reflect the indistinguishability of these identical particles
and make the statistical properties of fermions and bosons
consistent with observations, the wavefunction for a system
of identical fermions must be completely antisymmetric and
the wavefunction for a system of identical bosons must be
completely symmetric. Here we focus on student difficulties
with the many-particle stationary state wavefunction that is
a solution to the Time-Independent Schrödinger Equation
(TISE) for a system of non-interacting identical particles. Unless otherwise stated, throughout, we refer to the stationary
state wavefunction as the wavefunction. When considering the spin part of the wavefunction for a
single-particle, we use the notation |si, msi⟩(in which si and
msi are the quantum numbers corresponding to the total spin
and z-component of the spin for the ith particle, respectively). The states |s1, ms1⟩are eigenstates of ˆS2
1 and ˆS1z and the
states |s2, ms2⟩are eigenstates of ˆS2
2 and ˆS2z. We use the
following abbreviated notation for a spin-1/2 particle, e.g.,
an electron: |↑⟩1 = |s1, ms1⟩= |1/2, 1/2⟩1 and |↓⟩1 =
|s1, ms1⟩= |1/2, −1/2⟩1 for electron 1 in the “spin up"
and “spin down" state, respectively, and |↑⟩2 = |s2, ms2⟩=
|1/2, 1/2⟩2, and |↓⟩2 = |s2, ms2⟩= |1/2, −1/2⟩2 for
electron 2 in the “spin up" and “spin down" state, respectively. When considering the spin part of the wavefunction for
the two spin-1/2 particles in the uncoupled representation in
the product space, we will use the notation |↑↑⟩= |↑⟩1|↑⟩2,
|↑↓⟩= |↑⟩1|↓⟩2, |↓↑⟩= |↓⟩1|↑⟩2, and |↓↓⟩= |↓⟩1|↓⟩2
for the basis states. In order to satisfy the symmetrization
requirement for a system of two spin-1/2 particles, the
spin part of the wavefunction must be either completely
symmetric or antisymmetric so that the overall wavefunction
is antisymmetric. In the case of two spin-1/2 particles,
|↑↑⟩, |↓↓⟩,
1
√
2 (|↑↓⟩+ |↓↑⟩) are completely symmetric spin
states of the two-fermion wavefunction and often referred
to as the “triplet" states. Chandralekha Singh We discuss an investigation of student difficulties with concepts related to the many-particle stationary state
wavefunction for a system of non-interacting fermions or bosons in cases in which the many-particle stationary
state wavefunction can be written as the product of the spatial and spin parts. The investigation was carried
out in advanced quantum mechanics courses by administering free-response and multiple-choice questions and
conducting individual interviews with students. We find that students share many common difficulties related to
these concepts. Many students struggled to write a many-particle stationary state wavefunction consistent with
the symmetrization requirements for the system, i.e., a completely antisymmetric wavefunction for a system of
fermions or a completely symmetric wavefunction for a system of bosons. We discuss the common difficulties
pertaining to these concepts that can be used as a guide to develop research-validated learning tools. 266 II.
METHODOLOGY Q3 below was posed as an in-class clicker question to 16
undergraduate students in a junior/senior level undergraduate
quantum mechanics course following instruction on identical
particles. The
students
first
answered
the
question
individually and then answered it a second time after
discussing the question with their peers in small groups. Student difficulties with determining the many-particle
stationary state wavefunction for a system of identical
fermions or bosons were first investigated using three years
of data involving responses to open-ended and multiple-
choice questions administered after traditional instruction in
relevant concepts from 57 upper-level undergraduate students
in a junior/senior level QM course and 30 graduate students
in the second semester of the graduate core QM course. Additional insight concerning these difficulties was gained
from responses of 14 students during a total of 81 hours of
individual “think-aloud" interviews. Q3. Choose all of the following statements that are correct
about bosons. (1) The spin of a boson is an integer. (2)
The overall wavefunction of identical bosons can be anti-
symmetric. (3) Two bosons cannot occupy the same single-
particle state. Only option (1) is correct for question Q3. Option (2)
is incorrect because the overall wavefunction for a system
of identical bosons MUST be symmetric and option (3) is
incorrect because two or more bosons can occupy the same
single-particle state. We discuss student responses to several questions that were
posed either as in class clicker questions or as open-ended
quiz questions. Additional insight into these difficulties
was gleaned during the individual think-aloud interviews
in which students were asked questions pertaining to these
issues. To probe whether students are able to identify
and generate a many-particle stationary state wavefunction
including spin, the following two questions were posed to
the students. Questions Q1 and Q2 were posed on a quiz
following traditional instruction on concepts involving a
system of identical particles to 30 graduate students and 25
undergraduate students. Students were told that the particles
are confined in one spatial dimension and that ψn1, ψn2,
etc., are the single-particle stationary state wavefunctions. Below, we discuss the questions students were asked: Q1. Write one possible spatial part of the wavefunction for
two indistinguishable spin-1 bosons if the spin part of
the wavefunction (expressed in terms of the uncoupled
representation) is χ(ms1, ms2)
=
1
√
2[|1 1⟩1|1 0⟩2 +
|1 0⟩1|1 1⟩2]. I.
INTRODUCTION AND BACKGROUND The state
1
√
2 (|↑↓⟩−|↓↑⟩) is the
completely antisymmetric normalized spin state of the two-
fermion wavefunction, often referred to as the “singlet" state. Even
though
the
spatial
and
spin
parts
of
the
wavefunction
can
be
entangled
in
many
situations,
we
will
only
consider
many-particle
wavefunctions
Ψ(x1, x2, x3, . . . , ms1, ms2, ms3, . . .)
=
ψ(x1, x2, x3, . . .)χ(ms1, ms2, ms3, . . .)
in
one
spatial
dimension that can be written as the product of the spatial
part of the wavefunction ψ(x1, x2, x3, . . .) and the spin
part of the wavefunction χ(ms1, ms2, ms3, . . .), in which
xi denotes the spatial coordinate and msi denotes the
z-component of spin quantum number of the ith particle. The following are examples of completely antisymmetric
normalized many-particle stationary state wavefunctions
for a system of two spin-1/2 fermions in which the spatial
part of the wavefunction is antisymmetric and the spin part
of the wavefunction is symmetric (or vice-versa, assume
n1 ̸= n2): Ψ(x1, x2, ms1, ms2) =
1
√
2{ψn1(x1)ψn2(x2) −
ψn2(x1)ψn1(x2)}|↑⟩1|↑⟩2
and
Ψ(x1, x2, ms1, ms2)
=
ψn1(x1)ψn1(x2) 1
√
2{|↑⟩1|↓⟩2 −|↓⟩1|↑2⟩}. The spatial part of the wavefunction of a system of 267 For a spin-1 boson, |si, msi⟩= {|1, −1⟩, |1, 0⟩, |1, 1⟩}
for each particle. When considering the spin part of the
wavefunction for two spin-1 particles in the uncoupled
representation
in
the
product
space
(3 × 3
=
9
dimensional),
we will use the notation |1, 1⟩1|1,
1⟩2,
|1, 1⟩1|1, 0⟩2, |1, 1⟩1|1, −1⟩2, |1, 0⟩1|1, 1⟩2, |1, 0⟩1|1, 0⟩2,
|1, 0⟩1|1,
−1⟩2,
|1, −1⟩1|1,
1⟩2,
|1, −1⟩1|1,
0⟩2,
and
|1, −1⟩1|1, −1⟩2 for the basis states. bosons must be completely symmetric. Since the spin part
of the wavefunction given in Q1 is symmetric, the spatial
part of the wavefunction must also be symmetric to ensure
that the overall wavefunction is completely symmetric. Two
possible symmetric spatial states for the two spin-1 bosons
are ψ(x1, x2) = ψn1(x1)ψn1(x2) and (assume n1 ̸= n2)
ψ(x1, x2) =
1
√
2[ψn1(x1)ψn2(x2) + ψn2(x1)ψn1(x2)]. Q2. Write one possible spin part of the wavefunction
for two electrons if the spatial part of the wavefunction is
ψ(x1, x2) =
1
√
2[ψn1(x1)ψn2(x2)+ψn2(x1)ψn1(x2)]. I.
INTRODUCTION AND BACKGROUND If it is
not possible to write a spin part of the wavefunction with the
given spatial part of the wavefunction, write “not possible"
and state the reason. The wavefunction for a system of identical bosons
must
be
completely
symmetric. The
spatial
and
spin
parts
of
the
many-particle
stationary
state
wavefunction
can
either
be
both
symmetric
or
both
antisymmetric. For example, the following are examples
of completely symmetric many-particle stationary state
wavefunctions
for
a
system
of
two
spin-1
bosons:
Ψ(x1, x2, ms1, ms2)
=
ψn1(x1)ψn1(x2)|1,
1⟩1|1,
1⟩2
and
Ψ(x1, x2, ms1, ms2)
=
1
√
2{ψn1(x1)ψn2(x2) −
ψn2(x1)ψn1(x2)} 1
√
2{|1, 1⟩1|1, 0⟩2 −|1, 0⟩1|1, 1⟩2} in
which both the spatial and spin parts of the wavefunction are
symmetric or antisymmetric (assume n1 ̸= n2), respectively. The overall wavefunction for the two electrons must be
completely antisymmetric. Since the spatial part of the
wavefunction given in Q2 is symmetric, the spin part of
the wavefunction must be antisymmetric to ensure that the
overall wavefunction is completely antisymmetric. The
antisymmetric spin state for the two spin-1/2 fermions is
χ(ms1, ms2) =
1
√
2[|↑↓⟩−|↓↑⟩]. II.
METHODOLOGY If it is not possible to write a spatial part of
the wavefunction with the given spin part of the wavefunction,
write “not possible" and state the reason. Question Q4 was posed during the think-aloud interviews
to investigate students’ proficiency at identifying whether the
spin part of a wavefunction is a symmetric or antisymmetric
wavefunction. The question focuses on a system of two
spin-1/2 particles (s1 = 1/2, s2 = 1/2). The students
were familiar with the shorthand notation |↑↑⟩= |↑⟩1|↑⟩2,
|↑↓⟩= |↑⟩1|↓⟩2, |↓↑⟩= |↓⟩1|↑⟩2, and |↓↓⟩= |↓⟩1|↓⟩2. Q4. For the spin part of the wavefunction (spin state) of a
two-particle system given below, identify whether the spin
state is symmetric, antisymmetric, or neither symmetric nor
antisymmetric with respect to exchange of the two particles. Explain your reasoning. (a) |↑↑⟩(b) |↓↓⟩(c) |↑↓⟩(d)
1
√
2 (|↑↓⟩+ |↓↑⟩) (e) 1
√
2 (|↑↓⟩−|↓↑⟩) Question Q4 was posed during the think-aloud interviews
to investigate students’ proficiency at identifying whether the
spin part of a wavefunction is a symmetric or antisymmetric
wavefunction. The question focuses on a system of two
spin-1/2 particles (s1 = 1/2, s2 = 1/2). The students
were familiar with the shorthand notation |↑↑⟩= |↑⟩1|↑⟩2,
|↑↓⟩= |↑⟩1|↓⟩2, |↓↑⟩= |↓⟩1|↑⟩2, and |↓↓⟩= |↓⟩1|↓⟩2. Q4. For the spin part of the wavefunction (spin state) of a
two-particle system given below, identify whether the spin
state is symmetric, antisymmetric, or neither symmetric nor
antisymmetric with respect to exchange of the two particles. Explain your reasoning. (a) |↑↑⟩(b) |↓↓⟩(c) |↑↓⟩(d)
1
√
2 (|↑↓⟩+ |↓↑⟩) (e) 1
√
2 (|↑↓⟩−|↓↑⟩) In Q4, options (a), (b), and (d) are symmetric spin states
(triplet states) since exchanging the two particles results in
the same state. Option (e) in Q4 is an antisymmetric spin state
(singlet state) since exchanging the two particles results in the
original state multiplied by -1. Option (c) in Q4 is neither a
symmetric nor antisymmetric spin state. The overall wavefunction for the two indistinguishable 268 a two-particle state consisting of product states of spatial
states involving anti-symmetric spatial wavefunction and
symmetric spin states. TABLE I. The percentages of graduate (N = 30) and undergraduate
(N = 25) students who correctly answered questions Q1 and Q2
after traditional instruction. B. III.
STUDENT DIFFICULTIES Many students struggled to recognize and generate the
completely symmetric many-particle wavefunction for a
system of indistinguishable bosons in question Q1 and
the completely antisymmetric many-particle wavefunction
for a system of indistinguishable fermions in question
Q2. Table I summarizes the percentages of students who
answered questions Q1 and Q2 correctly for a system of
two indistinguishable fermions or bosons after traditional
instruction. Below, we discuss several of difficulties students had
that interfered with their ability to write the completely
symmetric/antisymmetric
many-particle
stationary
state
wavefunction for a system of indistinguishable particles. A. Difficulty
applying
Pauli’s
exclusion
principle
correctly for a system of identical fermions: Some students
struggled to realize that Pauli’s exclusion principle states
that no two fermions can be in the same single-particle
state. Students with this type of difficulty often incorrectly
overgeneralized the Pauli exclusion principle to state that
no two fermions can occupy the same spatial state or the
same spin state. Often students had difficulty realizing that
two fermions can be in the same spatial state if they are
in different spin states or vice-versa, so that the overall
wavefunction is antisymmetric. During the interview, several
students incorrectly applied the Pauli exclusion principle
when considering a separable many-particle stationary state
wavefunction that can be expressed as the direct product
of the spatial and the spin parts of the wavefunction. For
example, one interviewed student correctly stated that no
two fermions can be in the same single-particle state, but
then went on to incorrectly claim that “this means two
fermions could not exist in the same single-particle stationary
state (pointing to a case in which they were in the same
spatial state)". This student and others with this type of
difficulty often had difficulty realizing that if the two spin-
1/2 fermions are in different spin states then it is possible
for the two fermions to be in the same single-particle spatial
state, i.e. ψn1(x1)ψn1(x2) is a possible spatial part of the
wavefunction. C. Difficulty identifying the correct symmetrization
requirement for a system of identical particles: Students
also
had
difficulty
identifying
that
the
many-particle
wavefunction for a system of identical bosons must be
completely symmetric. For example, in response to Q3,
43% of the undergraduate students incorrectly answered that
the overall wavefunction of identical bosons can be anti-
symmetric (option (2)). Even after peer discussion, 31%
again incorrectly selected option (2) as correct. II.
METHODOLOGY Difficulty with the symmetrization requirements for
the overall many-particle stationary state wavefunction:
Nature
demands
that
the
many-particle
wavefunction
for a system of indistinguishable bosons be completely
symmetric and the many-particle wavefunction for a system
of indistinguishable fermions be completely antisymmetric. Therefore, in order to identify and generate a many-
particle wavefunction for a system of indistinguishable
particles, students must be able to determine a completely
symmetric/antisymmetric
wavefunction
involving
both
spatial and spin degrees of freedom. However, some students
struggled to correctly identify the appropriate symmetrization
requirement for a system of identical particles. Other students
had difficulty determining whether the spatial part of the
wavefunction or the spin part of the wavefunction is
symmetric or antisymmetric when considering each part of
the wavefunction separately. In interviews, students with this
type of difficulty often were not able to correctly identify
the symmetry of the overall wavefunction that included both
the spatial and spin parts of the wavefunction. Lastly, some
students struggled to construct the overall many-particle
stationary state wavefunction due to the fact that it is the
product of the spatial and spin parts. Question Type of Particle Graduate (%) Undergraduate (%)
Q1
Bosons
33
48
Q2
Electrons
30
44 III.
STUDENT DIFFICULTIES Written
explanations and interviews suggest that these students
knew that the many-particle wavefunction for a system
of identical particles (bosons or fermions) must obey a
symmetrization requirement, but could not correctly identify
which symmetrization requirement corresponds to which
particle. For example, one interviewed student in response to
questions Q1 and Q2 stated that “there is a symmetrization
requirement for fermions and a different symmetrization
requirement for bosons." But the student was unable to
recognize the appropriate symmetrization requirements and
wrote the same symmetric wavefunction for both a system
of indistinguishable fermions and indistinguishable bosons. In response to Q1, some students attempted to generate
a completely antisymmetric wavefunction for a system of
identical bosons. Similarly, in response to Q2, some students
generated a completely symmetric wavefunction for a system
of identical fermions. Additionally, 67% of the graduates
and 28% of undergraduate students incorrectly provided a
symmetric spin part of the wavefunction in response to Q2
resulting in an overall symmetric many-particle wavefunction
for the two electrons. By a similar argument, several students incorrectly claimed
that two fermions could not exist in the same spin state as
this too would violate the Pauli exclusion principle. For
example, some interviewed students claimed that |↑↑⟩and
|↓↓⟩were not possible spin states for the system of two
spin-1/2 fermions regardless of whether the two fermions
were in different single-particle spatial states. These students
did not consider the fact that the two fermions could be in 269 Identifying the correct symmetrization requirement for
the overall many-particle stationary state wavefunction is
challenging due in part to the fact that one must consider
the symmetry of both the spatial and spin parts of the
wavefunction before determining the overall symmetry of
the many-particle stationary state wavefunction. This
can be confusing for students who simply memorized the
appropriate combinations of the spatial and spin parts of the
wavefunction rather than developing an understanding of the
symmetry of a wavefunction comprised of two separate parts. wavefunctions is a completely symmetric wavefunction. During the interviews, some students incorrectly claimed
that in order for the overall many-particle stationary state
wavefunction to be antisymmetric, both the spatial and spin
parts of the wavefunction must be antisymmetric. F. III.
STUDENT DIFFICULTIES Difficulty identifying that the spatial and spin
parts of the wavefunction can both be antisymmetric
for a system of identical bosons:
Some students had
difficulty realizing that both the spatial and spin parts of the
wavefunction can be antisymmetric to produce an overall
symmetric wavefunction for a system of identical bosons. A number of students correctly reasoned that the overall
many-particle wavefunction for a system of indistinguishable
bosons must be completely symmetric. However, some
of these students went on to incorrectly claim that both
the spatial part and spin part of the wavefunction must be
symmetric. These students did not realize that a many-
particle stationary state wavefunction in which both the
spatial part and spin part are antisymmetric is completely
symmetric. D. Incorrectly determining the symmetry based on
the appearance of a +/- sign in the many-particle
wavefunction: Some students incorrectly applied a heuristic
by which they claimed that a wavefunction is symmetric
if the wavefunction is written in terms of a sum. These
students simply looked for all “+" signs to determine that
a wavefunction is symmetric. They claimed that any
wavefunction written as terms added together is a symmetric
wavefunction. By a similar logic, these same students looked
for a “-" sign to determine whether a given wavefunction is
antisymmetric. Some claimed that any wavefunction that had
at least one negative sign was antisymmetric. For example,
in response to question Q4(a), one interviewed student
incorrectly claimed that the spin part of the wavefunction
given by |↑↑⟩is neither symmetric nor antisymmetric as “the
wavefunction is not a sum so it can’t be symmetric and there
is not a minus sign, so it can’t be antisymmetric." However,
the spin part of the wavefunction given by |↑↑⟩is symmetric
as the exchange of the two particles results in the same
wavefunction, thus there need not be a plus sign in order for
a wavefunction to be symmetric. Other students used similar
reasoning when determining the symmetry of the spin part of
the wavefunction. For example, students were asked to construct the spin part
of a two-particle stationary state wavefunction for two spin-
1 bosons whose spatial part of the wavefunction is given by
1
√
2[ψn1(x1)ψn2(x2) −ψn2(x1)ψn1(x2)]. One interviewed
student incorrectly claimed that “it is not possible to write
the spin part since the spatial part is antisymmetric. III.
STUDENT DIFFICULTIES There
is no way to make the whole wavefunction symmetric." This
student and many others with this type of difficulty did not
realize that by choosing an antisymmetric spin part of the
wavefunction, the overall two-particle wavefunction would
be completely symmetric. IV.
SUMMARY AND FUTURE OUTLOOK Singh, Student understanding of quantum mechanics, Am. J. Phys. 69, 885 (2001). [21] C. Singh, M. Belloni, and W. Christian, Improving students’
understanding of quantum mechanics, Physics Today 59, 43
(2006). [3] D. Zollman, N. S. Rebello, and K. Hogg, Quantum mechanics
for everyone: Hands-on activities integrated with technology,
Am. J. Phys. 70, 252 (2002). [22] C. Singh, Interactive learning tutorials on quantum mechanics,
Am. J. Phys. 76, 400 (2008). [4] R. Muller and H. Wiesner, Teaching quantum mechanics on an
introductory level, Am. J. Phys. 70, 200 (2002). [5] M. Wittmann et al. Investigating student understanding of
quantum physics: Spontaneous models of conductivity, Am.J. Phys. 70, 218 (2002). [23] G. Zhu and C. Singh, Improving students’ understanding of
quantum measurement: I. Investigation of difficulties, Phys. Rev. ST PER 8, 010117 (2012). [6] D. Domert, C. Linder, and A. Ingerman, Probability as a
conceptual hurdle to understanding one-dimensional quantum
scattering and tunnelling, Eur. J. Phys. 26, 47 (2004). [24] G. Zhu and C. Singh, Improving students’ understanding of
quantum mechanics via the Stern-Gerlach experiment, Am. J. Phys. 79, 499 (2011). [25] G. Zhu and C. Singh, Improving students’ understanding of
quantum measurement:
II. Development of research-based
learning tools, Phys. Rev. ST PER 8, 010118 (2012). [7] C. Singh, Student understanding of quantum mechanics at the
beginning of graduate instruction, Am. J. Phys. 76, 277 (2008). [8] S. Y. Lin and C. Singh, Categorization of quantum mechanics
problems by professors and students, Euro. J. Phys. 31, 57
(2010). [26] G. Zhu and C. Singh, Improving student understanding of
addition of angular momentum in quantum mechanics, Phys. Rev. ST PER 9, 010101 (2013). [9] A. Kohnle et al., Developing and evaluating animations for
teaching quantum mechanics concepts, Eur. J. Phys. 31, 1441
(2010). [27] B. Brown, A. Mason, and C. Singh, Improving performance
in quantum mechanics with explicit incentives to correct
mistakes, Phys. Rev. PER 12, 010121 (2016). [10] A. J. Mason and C. Singh, Do advanced students learn from
their mistakes without explicit intervention?, Am. J. Phys. 78,
760 (2010). [28] E. Marshman and C. Singh, Investigating and improving
student understanding of quantum mechanics in the context of
single photon interference, Phys. Rev. PER 13, 010117 (2017). [11] G. Zhu and C. Singh, Surveying students’ understanding of
quantum mechanics in one spatial dimension, Am. J. Phys. 80,
252 (2012). [29] R. Sayer, A. Maries and C. IV.
SUMMARY AND FUTURE OUTLOOK Singh, A quantum interactive
learning tutorial on the double-slit experiment to improve
student understanding of quantum mechanics, Phys Rev PER
13, 010123 (2017). [12] A. Kohnle, I. Bozhinova, D. Browne, M. Everitt, A. Fomins, P. Kok, G. Kulaitis, M. Prokopas, D. Raine and E. Swinbank, A
new introductory quantum mechanics curriculum, Eur. J. Phys. 35, 015001 (2014). [30] A. Maries, R. Sayer and C. Singh, Effectiveness of interactive
tutorials in promoting “which-path" information reasoning in
advanced quantum mechanics, Phys. Rev. PER 13, 020115
(2017). [13] E. Gire and E. Price, The structural features of algebraic
quantum notations, Phys. Rev. ST PER 11, 020109 (2015). [14] E. Marshman and C. Singh, Framework for understanding the
patterns of student difficulties in quantum mechanics, Phys. Rev. ST PER 11, 020119 (2015). [31] C. Keebaugh, E. Marshman and C. Singh, Investigating and
addressing student difficulties with the corrections to the
energies of the hydrogen atom for the strong and weak field
Zeeman effect, Eur. J. Phys. 39, 045701 (2018). [15] C. Singh and E. Marshman, Review of student difficulties
in upper-level quantum mechanics, Phys. Rev. ST PER 11,
020117 (2015). [32] C. Keebaugh, E. Marshman and C. Singh, Investigating
and addressing student difficulties with a good basis for
finding perturbative corrections in the context of degenerate
perturbation theory, Eur. J. Phys. 39, 055701 (2018). [16] G. Passante, P. Emigh, and P. Shaffer, Examining student
ideas about energy measurements on quantum states across
undergraduate and graduate levels, Phys. Rev. ST PER 11,
020111 (2015). [33] C. Keebaugh, E. Marshman and C. Singh, Improving student
understanding of corrections to the energy spectrum of the
hydrogen atom for the Zeeman effect, Phys. Rev. Phys. Educ. Res. 15, 010113 (2018). [17] E. Marshman and C. Singh, Investigating and improving
student understanding of the expectation values of observables
in quantum mechanics, Eur. J. Phys. 38, 045701 (2017). [34] C. Keebaugh, E. Marshman and C. Singh, Improving student
understanding of fine structure corrections to the energy
spectrum of the hydrogen atom, Am. J. Phys. 87, 594 (2019). [18] V. Dini and D. Hammer, Case study of a successful learner’s
epistemological framings of quantum mechanics, Phys. Rev. Phys. Educ. Res. 13, 010124 (2017). [35] C. Keebaugh, E. Marshman and C. Singh, Improving student
understanding of a system of identical particles with a fixed
total energy, Am. J. Phys. 87, 583 (2019). [19] E. Marshman and C. IV.
SUMMARY AND FUTURE OUTLOOK Our investigation suggests that upper-level undergraduate
and graduate students have many common difficulties with
fundamental concepts involving a system of non-interacting
identical particles. We are using the difficulties found
in this context as a guide to develop and validate a
Quantum Interactive Learning Tutorial (QuILT). The QuILT
focuses on helping students learn that the wavefunction for
a system of fermions must be completely antisymmetric
and the wavefunction for a system of bosons must be
completely symmetric and then construct the many-particle
stationary state wavefunction for a system of non-interacting
identical particles (fermions or bosons) consistent with the
symmetrization requirements. In particular, the QuILT strives
to help students understand and operationalize completely
symmetric or completely anti-symmetric functions with
respect to the exchange of any two particles’ coordinates
and use this knowledge to construct a many-particle wave
function consisting of both spatial and spin parts. Students with this type of difficulty often struggled to
write the overall many-particle stationary state wavefunction
for a system of identical particles with the appropriate
symmetrization requirement. For example, students who
incorrectly claimed that the spin part of the wavefunction |↑↑⟩
is neither symmetric nor antisymmetric often also incorrectly
claimed that it is not possible to write a many-particle
stationary state wavefunction for two fermions with this spin
state. E. Difficulty identifying that the spatial part is
antisymmetric and the spin part is symmetric (or vice
versa) for the wavefunction for a system of identical
fermions:
Some students correctly identified that the
many-particle stationary state wavefunction for a system
of identical fermions must be completely antisymmetric,
but incorrectly claimed that both the spatial and spin parts
of the wavefunction must be completely antisymmetric. For example, when asked to determine the spatial part
of the wavefunction for two fermions if the spin part
of the wavefunction was the singlet state, some students
incorrectly claimed that the spatial part of the wavefunction
is
1
√
2[ψn1(x1)ψn2(x2) −ψn2(x1)ψn1(x2)]. They struggled
to realize that the product of two completely antisymmetric The initial in-class evaluation of the validated QuILT is
encouraging. ACKNOWLEDGEMENTS
We thank the NSF for award PHY-1806691. 270 [20] E. Marshman and C. Singh, Investigating and improving
student understanding of quantum mechanical observables and
their corresponding operators in Dirac notation, Eur. J. Phys. 39, 015707 (2017). [1] P. Jolly, D. Zollman, S. Rebello and A. Dimitrova. Visualizing
potential energy diagrams, Am. J. Phys. 66, 57 (1998). [2] C. IV.
SUMMARY AND FUTURE OUTLOOK Singh, Investigating and improving
student understanding of the probability distributions for
measuring physical observables in quantum mechanics, Eur. J. Phys. 38, 025705 (2017). 271
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ANALISIS SENTIMEN APLIKASI BRIMO PADA ULASAN PENGGUNA DI GOOGLE PLAY MENGGUNAKAN ALGORITMA NAIVE BAYES
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JATI (Jurnal Mahasiswa Teknik Informatika)
Vol. 7 No. 1, Februari 2023
ANALISIS SENTIMEN APLIKASI BRIMO PADA ULASAN PENGGUNA DI
GOOGLE PLAY MENGGUNAKAN ALGORITMA NAIVE BAYES
Moh Khoirul Insan, Umi Hayati, Odi Nurdiawan
Program Studi Teknik Informatika S1, Fakultas Teknik Infomatika
Stmik Ikmi Cirebon, Jl. Perjuangan No. 10 B Majasem Kec. Kesambi Kota Cirebon
Khoirulinsan0105@gmail.com
ABSTRAK
Perkembangan tekonologi saat ini semakin cepat berkembang termasuk aplikasi dalam bidang perbankan
yang saat ini banyak digunakan untuk bertrasnsaksi secara mobile tanpa perlu ke bank. Tentunya sangat mudah
bagi pegguna nasabah dalam bertransaksi menggunakan aplikasi mobile bangking.Ulasan menjadi sumber
informasi penting bagi pengembang untuk mengetahui keluhan dari para pengguna atau nasabah. ulasan pengguna
yang berisikan komentar atau ulasan dan rating pengguna sangat dibutuhkan guna meningkatkan pengembangan
terhadap kinerja aplikasi. Namun tidak semua ulasan pengguna itu mewakili pendapat pada apliksi tesebut
Penelitian ini bertujuan untuk menganalisis sentimen terhadap pengguna yang menggunakan aplikasi brimo di
google play dengan pengambilan data melalui teknik web scraping. Analisis sentimen merupakan proses
mengevaluasi apakah suatu teks memiliki sentimen positif atau negatif. Pengguna aplikasi brimo di google play
merupakan salah satu platform yang banyak digunakan nasabah BRI untuk transaksi perbankan. Metode yang
digunakan dalam analisis sentimen menggunakan algoritma Naive Bayes. dari hasil penelitian terhadap analisis
sentimen pengguna aplikasi brimo ke dalam kategori positif dan negatif yang mana data diambil melalui hasil
scraping dari bulan Agustus 2022 hingga Januari 2023 sebanyak 1550 data teks didapatkan hasil bahwa pengguna
aplikasi memberikan ulasan positif dimana dari jumlah prediksi hasil klasifikasi sentimen positif sebanyak 1012
dan sentimen negatif sebanyak 894 data teks artinya pengguna banyak memberikan komentar positif terhadap
aplikasi brimo, namun tidak sedikit juga pengguna memberikan komentar negatif terhadap aplikasi brimo. Dan
hasil klasifikasi menggunakan metode naive bayes didapatkan hasil acuracy sebesar 84,52% precision 82,51%
dan recall 87,62%.
.
Kata kunci: Analisis sentimen. Naive Bayes, google play, Rapid Miner
1.
PENDAHULUAN
Perkembangan tekonologi saat ini semakin cepat
berkembang termasuk aplikasi dalam bidang
perbankan yang saat ini banyak digunakan untuk
bertrasnsaksi secara mobile tanpa perlu ke bank.
Brimo merupakan salah satu aplikasi berbasis mobile
yang dimiliki oleh Bank Rakyat Indonesia ( BRI )
untuk memudahkan nasabah melakukan transaksi
perbankan. Menurut peraturan Bank Indonesia Nomor
: 9/15/PBI/2007 Layanan Perbankan Melalui Media
Elektronik atau selanjutnya disebut Electronic
Banking adalah layanan yang memungkinkan nasabah
Bank untuk memperoleh informasi, melakukan
komunikasi, dan melakukan transaksi perbankan
melalui media elektronik antara lain ATM, phone
banking, electronic fund transfer, internet banking,
mobile phone .berdasarkan data pada situs Google
Play aplikasi brimo telah di download kurang lebih 10
juta unduhan dengan rating 4,5 dan tercatat 1 juta
ulasan komentar pengguna aplikasi brimo pada situs
google playClick or tap here to enter text.[1]. Menurut
Winarko (2015) Ada dua aspek review yang
dipertimbangkan, yaitu rating nilai dan komentar lebih
mendalam. Rating nilai menunjukkan suatu nilai
angka, sedangkan komentar lebih tekstual berfokus
pada pendapat lebih mendalam. [2]. oleh karena itu
dibutuhkan analisis sentimen terdahap ulasan
pengguna aplikasi brimo dengan algoritma naive
bayes. Analisis sentimen atau opinion mining adalah
proses mengolah data tekstual secara otomatis untuk
mengetahui apakah opini yang terkandung dalam suatu
kalimat adalah positif atau negatif dan Algoritma
Naive Bayes Classifier dianggap cocok untuk
digunakan dalam analisis sentimen karena bertujuan
sebagai metode klasifikasi kedalam kategori positif
dan negatif [3]
Menurut penelitian yang dilakukan oleh Irfandi
Ricky Afandi, Firman Noor Hasan, Ali Abdul Rizki,
Nunik Prartiwi, Zuhri Halim pada Jurnal Linguistik
Komutasional Tahun 2022 yang berjudul Analisis
Sentimen Opini Masyarakat Terkait Pelayanan Jasa
Ekspedisi Anteraja Dengan metode Naive Bayes
Permasalahan pada penelitian ini terkait suatu
perusahaan ekspedisi terhadap Customer Service yang
memberikan pelayanan serta keluhan yang dihadapi
oleh penggunanya dalam menggunakan jasa tersebut
banyak pengguna layanan Anteraja memepunyai
pendapat yang beragam terhadap layanan Anteraja
serta megetahui sikap masyarakat terhadap pelayanan
Anteraja. Dengan menerapkan metode Algoritma
Naive Bayes. Dengan pengumpulan data pada media
sosial Twitter yang di dapatkan sebanyak 1180 data
dengan hasil akurasi menggunakan algoritma Naive
Bayes di peroleh sebesar 85.06%.[4]
Berdasarkan peneliti terdahulu diatas yang
dilakukan oleh Irfandi Ricky Afandi Dkk melakukan
478
JATI (Jurnal Mahasiswa Teknik Informatika)
analisis sentimen opini masyarakat terhadap pelayanan
ekspedisi melalui media sosial Twitter namun
perbedaan Objek peneliti yang akan dilakukan pada
penelitian ini melalui situs Google Play Pada aplikasi
Brimo dengan metode yang sama yaitu Algoritma
Naive Bayes. Diharapkan dari penelitian yang akan
dilakukan dengan metode yang sama evaluai dari
penerapan algortima naive bayes dapat menghasilkan
dierapkan dalam analisis sentimen dengan baik.
Adapun Kajian penelitian yang diusulkan yaitu
menerapkan sistem analisis sentimen atau disebut
opinion mining adalah studi komputasi tentang
perasaan, pandangan, dan emosi yang terkandung
dalam entitas dan atribut yang diekspresikan dalam
bentuk teks, secara umum analisis sentimen dibagi
menjadi lima tahapan yaitu Crawling data, Preprocesing, feature selection, clasification dan
evaluation. Manfaat analisis sentimen yaitu sebagai
sebuah ide atau evaluasi pada berbagai bidang.
Analisis sentimen ini memungkinkan pengelompokan
polaritas teks dalam kalimat atau dokumen untuk
memahami apakah opini pada teks tersebut bersifat
positif atau negatif [5]
Tujuan penelitian ini mengananlisis setimen
terhadap pengguna aplikasi Brimo pada ulasan atau
komentar di Google Play. untuk mengtahui sentimen
pengguna terhadap layanan aplikasi dengan kategori
postif dan negatif sehingga nantinya pengembang
aplikasi dapat mengambil langkah yang diperlukan
untuk meningkatkan layanan sehingga hasil penelitian
juga diharapkan memberikan informasi yang berguna
kepada pengembang terhadap aplikasi Brimo serta
mengevaluasi apa yang harus di benahi dalam
meningkatkan layanan pada aplikasi tersebut.
2. TINJAUAN PUSTAKA
2.1. Penelitian Terdahulu
Pada penelian yang dilakukan Safitri Juanita
pada jurnal Media Informatika Budidarma tahun 2020
dengan judul Anlisis Sentimen Persepsi Masyarakat
Terhadap Pemilu 2019 Pada Media Sosial Twitter
Menggunakan Algoritma Naive Bayes mengenai
masalah rendahnya partisipasi masyarakat dalam
pemilu. Penelitian ini bertujuan analisis sentiman
bagaimana persepsi masyarakat di media sosial twitter
pada pemilu 2019 dengan pengambilan data di twitter
sebanyak 221 data tweet dan hasil menggunakan
algortima naive bayes dengan data 221 persepsi
negatif sebesar 52% lebih besar dari persepsi positif
sebesar 18% dan netral sebesar 31% dengan data
training 81% dan data testing 76% dengan rata-rata
precision sentimen positif 86,65% sentimen negatif
77,5%, dan sentimen netral 80,95%, sedangkan ratarata recall sentimen positif 36%, sentimen negatif
93,2% dan sentimen netral 86,8% pengujian ini
menggunakan tols Weka.[6]
Pada penelitian yang dilakukan George Kenneth
Lacarso pada jurnal Teknik Informatika Kaputama
tahun 2022 dengan judul Analisis Sentimen Review
Aplikasi Pedulilindungi Pada Google Playstore
Vol. 7 No. 1, Februari 2023
Menggunakan Nbc terkait masalah opini masyarakat
mengenai kehadiran aplikasi pedulilindungi yang
diterbitkan pemerintahan untuk melacak penyebaran
covid-19 yang menimbulkan opini dan kritikan dari
masyarakat. Dari hasil penelitian terebut data yang
dikumpulkan memlalui ulasan aplikasi pedulilindungi
di google playstore sebanyak 1179. Bedasarkan hasil
pengujian menggunakan algoritma naive bayes dengan
memberikan label positif, negatif, dan netral
mendapatkan akurasi 83,3% dimana algoritma NBC
dapat melakukan klasifikasi dengan baik. [7]
Pada peneltian yang dilakukan Mochamad Daffa
Rhajendra dan Nurvita Trianasari pada jurna eProceeding of Management tahun 2021 yang berjudul
Analisis Sentimen Ulasan Spotify Untuk Peningkatan
Layanan Menggunakan Algoritma Naive Bayes.
terkait permasalahan Mengenai orang-orang banyak
beralih pada perangkat digital untuk mendengarkan
musik serta memberikan insight mengenai kebutuhan
dan keinginan pengguna terhadap kepuasan aplikasi
spotify. Dalam penelitian ini data yang dikumpulkan
pada ulasan aplikasi spotify di google play sebanyak
3600 data. Dan algoritma yang digunakan adalah
Naive Bayes dengan hasil acurasi performance vektor
sebesar 74,85%[8]
2.2. Naive Bayes
Naive Bayes adalah metode yang tidak memiliki
aturan dan menggunakan cabang matematika yang
disebut teori probabilitas untuk mendapatkan peluang
setinggi mungkin dengan melihat frekuensi atau
jumlah kemunculan setiap klasifikasi dalam data
pelatihan. Dalam pengembangan basis data, Naive
Bayes melibatkan pembelajaran yang diawasi jenis
pembelajaran mesin yang membutuhkan sampel
sebagai data pelatihan label. dikelompokkan menjadi
dua bagian, yaitu. Klasifikasi dan regresi. Klasifikasi
saat variabel menjadi kategori, panas dan dingin, sakit
atau tidak sakit dll. Variabel berupa nilai riil seperti
bobot, nilai uang dll contoh lainnya yaitu Support
Vector Machine (SVM), K-Nearst Neighbor (KNN),
Artificial Neural Network (ANN). Naive Bayes juga
merupakan metode yang menggunakan teknik
probabilistik, dimana satu fitur dan fitur lainnya dalam
data yang sama tidak saling berhubungan[9]. Adapaun
rumus dari Teroma Bayes sebagai berikut:
𝑃(𝑋|𝐻 )𝑃(𝐻)
𝑃(𝑋)
Keterangan :
X
: Data yang belum diketahui.
H
: Hipotesis data X merupakan suatu class
spesifik.
P(H|X) : Probabilitas hipotesis H berdasarkan kondisi
X ( Posterior Probability).
P(H)
: Probabilitas hipotesis H (Prior Probability).
P(X|H) : Probabilitas X berdasarkan Hipotesis H.
P(X)
: Probabilitas X.
𝑃 (𝐻 |𝑋 ) =
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JATI (Jurnal Mahasiswa Teknik Informatika)
3.
METODE PENELITIAN
Vol. 7 No. 1, Februari 2023
mengunjungi situs resmi google play dengan keyword
Brimo lalu kemudian dilakukan scraping dengan target
link pada aplikasi brimo.
Gambar 1. Alur Penelitian KDD
Metode penelitian yang digunakan yaitu
Knowledge Discover in Database (KDD) dimana
metode KDD ini merupakan cara untuk mengektraksi
data dalam mendapatkan informasi atau pola dari suatu
data yang telah dipilih sebelumnya [10]. Tahapan
dalam metode KDD meliputi Data Selection,
Preprocessing, Tranformation, Data Mining, dan
Evaluation.
3.1. Data Selection
Pada tahap ini merupakan tahapan KDD dimana
akan dilakukan seleksi atribut dan pengumpulan data.
Data dikumpulkan dari ulasan pengguna pada aplikasi
brimo di google play pada tanggal 22 November 2022
sampai tanggal 18 Januari 2023
3.2. Preprocessing
Preprocessing adalah tahap pembersihan data
dan perbaikan. Pada Saat mengumpulkan data, yang
didapat biasanya merupakan data yang tidak
terstruktur dan mengandung banyak karakter. Tujuan
dari preprocessing adalah untuk menghilangkan noise
tersebut. Dalam tahap preprocessing terdapat
beberapa langkah yang harus dilakukan, seperti
cleaning, case folding, tokenize, filtering yang
dibawah ini
3.3. Tranformation
Pada tahap tranformation adalah tahap mengubah
data menjadi bentuk yang dapat diolah pada tahapan
data mining
Gambar 2. Aplikasi Brimo
Data yang berisi ulasan pengguna aplikasi
diambil sejak tamggal tanggal 22 November 2022
sampai tanggal 18 Januari 2023 data diambil sebanyak
1550 data. Terdapat empat atribut pada saat
pengambilan data melalui hasil scrapping, dimana
terdapat atribut username, score, at dan content.
Berikut adalah dataset hasil scarping dapa dilihat pada
tabel 3
Gambar 3. Data Hasil Scrapping
Kemudian Pada tahap ini dilakukan pemilihan
atribut yang akan digunakan, sebelumnya pada tahap
penagambilan data melalui scrapping. Terdapat empat
atribut yaitu username, score, at dan content.
Selanjutnya dilakukan penghupasan atribut yang
digunakan yaitu hanya content
3.4. Data Mining
Pada tahap data mining ini dimana proses
klasifikasi analisis sentimen pada data ulasan
menggunakan algoritma naive bayes
3.5. Evaluation
Pada tahap evaluasi ini akan menggunakan
confusion matriks untuk mengetahui hasil perfroma
akurasi dari algoritma naive bayes dengan hasil
evaluasi berupa accuracy, precicion, dan recall.
Dimana Confusion Matrix merupakan salah satu
metode untuk mengukur kinerja dari model klasifikasi
4. HASIL DAN PEMBAHASAN
4.1. Data Selection
Tahap pengumpulan data ini dilakukan dengan
teknik scraping dengan bantuan bahasa program
pyhton di google colab adapun langkah awal
Gambar 4. Atribut Content
4.2. Cleansing
Berikut adalah proses cleansing yang digunakan
pada beberapa operator untuk pengahapusan simbol,
karakter, angka.
Pada pengahapusan simbol ini operator yang
digunakan yaitu operator replace. Pada operator
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JATI (Jurnal Mahasiswa Teknik Informatika)
replace pertama dinamakan replace simbol satu
digunakan untuk menghapus simbol-simbol.
Vol. 7 No. 1, Februari 2023
Tabel 3. Tahap Tokenize
Input Proses
Aplikasi bagus
memudahkan saya untuk
transaksi elektronik
bintang bagus dipersulit
proses registrasinya
kecewa
Gambar 5. Proses Cleansing
Berikut adalah contoh hasil pada proses
cleansing dapat dilihat pada tabel di bawah ini.
Tabel 1. Tahap Cleansing
Input Proses
Mau daftar susah sekali
gagal terus bgg mau bgmn
cara daftarnyaŸ‘ðŸ sudah
sesuai petunjuk tetap gk
bisa gk seperti Mbanking
sebelah yg gk ribet cara
daftarnyaauto unistal dan
tutup rekeningð‘ðŸ‘ðŸ‘
Perfect nampol mah ini the
bestlah pokoknya🤗
😙
Output Proses
Mau daftar susah
sekaligagal terusbgg mau
bgmn cara daftarnya
sudah sesuai petunjuk
tetap gk bisa gk seperti
Mbanking sebelah yg gk
ribet cara daftarnya auto
unistal dan tutup
rekening
Perfect nampol mah ini
the bestlah pokoknya
4.3. Labeling
Setelah semua data di bersihkan melalui text
procesing diatas tahap selanjutnya yaitu melakukan
pelabelan data secara manual. dimana proses yang
dilakukan dengan membaca satu persatu dari masingmasing ulasan dengan melihat kata-kata yang
mengandung emosi maka akan dilabeli sentimen
negatif begitu sebaliknya jika terdapat kata-kata pujian
maka akan dilabeli sentimen positif. Berikut data yang
sudah dibersihkan dan dilabeli secara manual.
Tabel 2. hasil pelabelan
Ulasan
Sangat membantu bertransaksi tanpa harus
ke Bank
Klo pgn bintang bagus jgn dipersulit proses
registrasinyakecewa
Sentimen
Positif
Negatif
4.4. Tokenize
Pada tahap selanjutnya dilakukan tokenisasi
dimana proses pemecah dokumen menjadi bagianbagian kata Berikut adalah contoh sebelum dan
sesudah proses tokenize dapat dilihat pada tabel di
bawah ini
Output Proses
Aplikasibagus memudahkan - saya untuk – transaksi elektronik
Bintang - bagus - Dipersulit
proses – egistrasin - kecewa
4.5. Tranform Case
Pada tahap transform case atau disebut case
folding yaitu proses mengubah teks menjadi huruf
kecil (lowercase) dari dokumen yang awalan berupa
(uppercase) huruf besar. Dimana tujuan dari proses ini
untuk menghilangkan perbedaan huruf kecil dan besar
dalam suatu dokumen teks. Berikut contoh hasil dari
Trasnform Case atau Case folding dilihat pada Tabel
di bawah ini.
Tabel 4. Tahap Transform Case
Input Proses
Aplikasi bagus
memudahkan saya untuk
transaksi elektronik
Bintang bagus dipersulit
proses registrasinya
kecewa.
Output Proses
aplikasi bagus
memudahkan saya untuk
transaksi elektronik
bintang bagus dipersulit
proses registrasinya
kecewa.
4.6. Stopword Removal
Selanjutnya pada tahap filter stopword biasa
disebut stopword removal dimana tahapan pada text
procesing dilakukan penghapusan kata-kata yang tidak
memiliki makna namun tidak berpengaruh pada suatu
kalimat seperti “ada” “yang” “aku”. Berikut di bawah
ini contoh hasil Stopword Removal
.
Tabel 5. Tahap Stopword Removal
Input proses
pada saat ingin upload video
selalu gagal dan selalu
kembali ke tampilan awal
untuk membuat video sudah
di coba beberapa kali tetap
gagal
saya mau registrasi ambil
photo saja susah nya minta
ampun tolong di perbaiki
apk nya biar mudah dalam
pendaftaran terima kasih.
Output Proses
upload video gagal
tampilan video coba kali
gagal
registrasi ambil photo
susah nya minta ampun
tolong perbaiki apk nya
biar mudah pendaftaran
terima kasih.
4.7. Filter token (by length)
Filter token (by length) pada tahap ini dilakukan
penghapusan sejumlah kata atau kata singkatan yang
miniamal karakternya sudah ditentukan sebelum
digunakan , pada penelitian ini peneliti menggunakan
panjang minimal 4 karakter dan maksimal 25 karakter
Berikut adalah contoh tabel sebelum dan sesudah
dilakukan filter token by length. Dapat dilihat pada
tabel di bawah ini.
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JATI (Jurnal Mahasiswa Teknik Informatika)
Tabel 6. Tahap Filter (by length)
Input Proses
brimo mantap okk
registrasi ambil photo susah
nya minta ampun tolong
perbaiki apk nya biar
mudah pendaftaran terima
kasih.
Output Proses
brimo mantap
registrasi ambil photo susah
ampun tolong perbaiki biar
mudah pendaftaran terima
kasih.
4.8. Tranformation
Pada tahap tranformation merupakan tahap
mengubah data menjadi bentuk yang dapat diolah
pada tahapan data mining yaitu mengubah data
nominal ke teks, adalah nominal to text
4.9. Data Mining
pada tahap ini dilakukan model klasifikasi naive
bayes yang mana terdapat beberapa operator
diantaranya Read Excel yang berfungsi menampung
data train lalu dihubungkan dengan Set Role yang
mana untuk mengubah target role nya menjadi label
kemudian dihubungkan denggan Nominal to Text
untuk mengubah mominal ke text lalu ke proses
document from data yang didalamnya terdapat sub
proses sperti casefolding, tokenize. Kemudian
upsampling untuk menyeimbangkan data kemudian
cross validation untuk menguji model naive bayes
dengan pengujian 1-10 kali.
Gambar 6. Proses Model Naive Bayes
Berdasarkan hasil dari klasifikasi Naive Bayes
didapatkan klasifikasi prediksi sentimen positif
sebesar 1,012 teks dan klasfikasi prediksi sentimen
negatif sebesar 894 teks dari total ulasan 1.906
Vol. 7 No. 1, Februari 2023
dibagi seluruh dokumen dan sentimen negatif dibagi
seluruh dokumen. Berikut persentasi sentimen negatif
dan positif dibawah ini.
Persentasi sentimen positif
1012
𝑥 100% = 53,09 %
1906
Persentasi sentimen negatif
894
𝑥100% = 46,90 %
1906
Diperoleh hasil persentasi sentimen dengan
jumlah ulasan positif lebih banyak daripada jumlah
ulasan negatif. Jumlah sentimen positif sebesar
53,09% , dan jumlah sentimen negatif sebesar 46,90%.
Artinya dari keseluruhan hasil klasifikasi terdapat hasil
prediksi sentimen positif lebih besar dari pada
sentimen negatif
4.10. Evaluation
Tahap ini dlakukan untuk menghitung evaluasi
dari model naive bayes dengan cross validation yang
didalamnya dihubungkan dengan apply model dan
perfomance untuk dihitung terhadap hasil dari naive
bayes
Gambar 8. Proses Validation Naive Bayes
Berdasarkan hasil evaluasi model. bahwa hasil
klasfikasi terhadap model naive bayes didaptkan hasil
acuracy sebesar 84.52%, bisa dilihat pada gambar
dibawah ini
Gambar 9. hasil accuracy Naive Bayes
Gambar 7. diagram sentimen
Berikut peresentasi sentimen positif dan ngatif
dari hasil klasifikasi dengan hasil sentimen positif
Langkah selanjutnya peneliti menggunakan
perhitungan manual
Confusion Matrix dimana
diakukan untuk menghitung acuracy, precision, dan
482
JATI (Jurnal Mahasiswa Teknik Informatika)
recall. Adapun rumus Confusion Matrix dilihat pada
rumus dibawah ini.
𝑇𝑃+ 𝑇𝑁
Acuracy 𝑇𝑃+𝑇𝑁+𝐹𝑃+𝐹𝑁 =
835+776
835+776+177+118
=
1611
𝑋 100% = 84,52%
1906
𝑇𝑃
835
835
Precision 𝑇𝑃+𝐹𝑃 = 835 + 177 1012 𝑥100% = 82,51%
𝑇𝑃
835
Recall 𝑇𝑃+𝐹𝑁 = 835 +118 =
835
953
𝑥 100% = 87,62 %
Keterangan :
TP : True Positive
TN : True Negatif
FP : False Positif
FN : False Negatif
Berdasarkan pada tabel Confusion Matrix bahwa
terdapat data yang terklasifikasi benar sebanyak 835
data dan banyak juga data yang benar namun
terklasifikasi atau prediksi negatif sebanyak 776 data,
dan salah prediksi terdapat 177 data yang masuk
dalam kategori negatif yang seharusnya positif,
kemudian terdapat data 118 data salah prediksi atau
klasifikasi yang masuk dalam kategori positif namun
seharusnya negatif.
Pada hasil perhitungan manual confusion Matrik
diatas menjukan hasil akurasi sebesar 84,52 %
precision 82,51% dan recall sebesar 87,62%. Dari hasil
perhitungan tersebut sama dengan hasil perhitungan
dari rapidminer diatas
5.
KESIMPULAN DAN SARAN
Berdasarkan kesimpulan dari hasil penelitian
terhadap analisis sentimen pengguna aplikasi brimo ke
dalam kategori positif dan negatif yang mana data
diambil melalui hasil scraping dari bulan November
2022 hingga Januari 2023 sebanyak 1550 data teks
didapatkan hasil
bahwa pengguna aplikasi
memberikan ulasan positif dimana dari jumlah
prediksi hasil klasifikasi sentimen positif sebanyak
1012 atau 53,09% dan sentimen negatif sebanyak 894
atau 46,90% data teks artinya pengguna banyak
memberikan komentar positif terhadap aplikasi brimo,
namun tidak sedikit juga pengguna memberikan
komentar negatif terhadap aplikasi brimo Dan hasil
klasifikasi menggunakan metode naive bayes
didapatkan hasil acuracy menggunakan algoritma
naive bayes mendapatkan tingkat acuracy sebesar
84,52% precision 82,51% dan recall 87,62%. Adapun
saran dari peneliti yaitu dapat Menggunakan algoritma
klasifikasi yang berbeda untuk dapat memberikan hasil
perbandingan serta model untuk mendapatkan hasil
yang lebih baik dan menerapkan analsis sentimen
dengan menggunakan tols seperti mechine learning
lainya.
Vol. 7 No. 1, Februari 2023
DAFTAR PUSTAKA
[1] Google
Play,
“Google
Play
Store,”
https://play.google.com/store/apps/details?id=i
d.co.bri.brimo. Diakses pada tanggal 08 januari
2023, Jan. 08, 2023.
[2] R. Wahyudi et al., “Analisis Sentimen pada
review Aplikasi Grab di Google Play Store
Menggunakan Support Vector Machine,”
JURNAL INFORMATIKA, vol. 8, no. 2, 2021,
[Online].
Available:
http://ejournal.bsi.ac.id/ejurnal/index.php/ji
[3] A. V. Sudiantoro and E. Zuliarso, “Analisis
Sentimen Twitter Menggunakan Text Mining
Dengan Algoritma Naïve Bayes Classifier
ANALISIS
SENTIMEN
TWITTER
MENGGUNAKAN TEXT MINING DENGAN
ALGORITMA NAÏVE BAYES CLASSIFIER,”
vol. 10, no. 2, pp. 69–73, 2018.
[4] I. R. Afandi, F. Noor, H. #2, A. A. Rizki, N.
Pratiwi, and Z. Halim, “Analisis Sentimen Opini
Masyarakat Terkait Pelayanan Jasa Ekspedisi
Anteraja Dengan Metode Naive Bayes,” 2022.
[Online]. Available: https://t.co/2HAdwg1drL
[5] R. S. Perdana and M. A. Fauzi, “Analisis
Sentimen Tingkat Kepuasan Pengguna Penyedia
Layanan Telekomunikasi Seluler Indonesia Pada
Twitter dengan Metode Support Vector Machine
dan Lexicon Based Features Smart Mobile
Navigation System View project Sentiment
Analysis View project,” 2017. [Online].
Available:
https://www.researchgate.net/publication/32023
4928
[6] S. Juanita, “Analisis Sentimen Persepsi
Masyarakat Terhadap Pemilu 2019 Pada Media
Sosial Twitter Menggunakan Naive Bayes,”
JURNAL
MEDIA
INFORMATIKA
BUDIDARMA, vol. 4, no. 3, p. 552, Jul. 2020,
doi: 10.30865/mib.v4i3.2140.
[7] G. K. Locarso, “Analisis Sentimen Review
Aplikasi Pedulilindungi Pada Google Play Store
Menggunakan NBC,” Jurnal Teknik Informatika
Kaputama (JTIK), vol. 6, no. 2, 2022.
[8] M. Daffa Rhajendral and N. Trianasari, “Analisis
Sentimen Ulasan Aplikasi Spotify Untuk
Peningkatan Layanan Menggunakan Algoritma
Naive Bayes Sentiment Analysis of Spotify
Application Reviews for Service Improvement
Using Naive Bayes Algorithm,” 2021.
[9] B. K. Widodo, N. H. Matondang, and D. S.
Prasvita, “Penerapan Algoritma Naive Bayes
Untuk Analisis Sentimen Penggunaan Aplikasi
Jobstreet,” Techno.Com, vol. 21, no. 3, pp. 523–
533, 2022, doi: 10.33633/tc.v21i3.6361.
[10] S. M. Siroj, I. Arwani, and D. E. Ratnawati,
“Analisis Sentimen Opini Publik pada Twitter
terhadap Efek Pembelajaran Daring di
Universitas Brawijaya menggunakan Metode KNearest Neighbor,” 2021. [Online]. Available:
http://j-ptiik.ub.ac.id
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Read Aloud Strategy for Reading Literacy of Elementary Pupils: Basis for Enhancing the Reading Program
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Zenodo (CERN European Organization for Nuclear Research)
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cc-by
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I. INTRODUCTION The Governance of Basic Education Act of 2001 requires the Department of Education (DepEd) to adopt national educational
policies to improve service delivery and basic education results. The Department of Education (DepEd) established Republic Act
No. 10533 (RA No. 10533), also known as the K–12 Program, to equip Filipino students with 21st-century skills and competencies,
including improved literacy skills. g
p
y
Literacy is needed to attend school and participate in society, according to [1] Caroll and Breadmore (2019). Reading and writing
difficulties make it hard for kids to learn anything. Teachers must help students develop literacy skills. Reading and comprehension
help students succeed in other subjects. This suggests that reading literacy is crucial to completing a curriculum. The Philippines participated in the Program for International Student Assessment (PISA) after implementing its new kindergarten
through twelfth grade curriculum in 2018. Filipino students had an average reading literacy score of 340, compared to 487 for the
[2] Organization for Economic Co-operation and Development (OECD). When the reading literacy scores were announced, the
nation ranked last. The PISA findings inspired [3] DepEd Memo No. 17, s. 2019, Hamon: Bawat Bata Bumabasa. This document
requires schools nationwide to help students become proficient readers. Literacy is needed to attend school and participate in society, according to [1] Caroll and Breadmore (2019). Reading and writing
difficulties make it hard for kids to learn anything. Teachers must help students develop literacy skills. Reading and comprehension
help students succeed in other subjects. This suggests that reading literacy is crucial to completing a curriculum. The Philippines participated in the Program for International Student Assessment (PISA) after implementing its new kindergarten
through twelfth grade curriculum in 2018. Filipino students had an average reading literacy score of 340, compared to 487 for the
[2] Organization for Economic Co-operation and Development (OECD). When the reading literacy scores were announced, the
nation ranked last. The PISA findings inspired [3] DepEd Memo No. 17, s. 2019, Hamon: Bawat Bata Bumabasa. This document
requires schools nationwide to help students become proficient readers. In relation to that, studies on reading literacy have remained a focus of attention, which is particularly relevant in light of the fact
that the ongoing pandemic has disrupted conventional school structures over the course of the past two years. INTERNATIONAL JOURNAL OF SOCIAL SCIENCE HUMANITY & MANAGEMENT RESEARCH
ISSN (print) 2833-2172, ISSN (online) 2833-2180
Volume 02 Issue 06 June 2023
DOI: 10.58806/ijsshmr.2023.v2i6n22
Page No. 454-460 INTERNATIONAL JOURNAL OF SOCIAL SCIENCE HUMANITY & MANAGEMENT RESEARCH
ISSN (print) 2833-2172, ISSN (online) 2833-2180
Volume 02 Issue 06 June 2023
DOI: 10.58806/ijsshmr.2023.v2i6n22
Page No. 454-460 INTERNATIONAL JOURNAL OF SOCIAL SCIENCE HUMANITY & MANAGEMENT RESEARCH
ISSN (print) 2833-2172, ISSN (online) 2833-2180
Volume 02 Issue 06 June 2023
DOI: 10.58806/ijsshmr.2023.v2i6n22
Page No. 454-460 INTERNATIONAL JOURNAL OF SOCIAL SCIENCE HUM
ISSN (print) 2833-2172, ISSN (online) 2833-2180
Volume 02 Issue 06 June 2023
DOI: 10.58806/ijsshmr.2023.v2i6n22
Page No. 454-460 Read Aloud Strategy for Reading Literacy of Elementary Pupils: Basis for
Enhancing the Reading Program Karla Mae J. Silva1, Jasper M. Del Valle, EdD, PhD2, Cecilia Q. Velasco, EdD3
1Academic Coordinator of Bixby Knolls Preparatory, Inc.- San Antonio, Quezon, Philippines
2Faculty of San Pablo City Integrated High School-San Pablo City, Philippines
3Faculty of Laguna State Polytechnic University-San Pablo City Campus, Philippines ABSTRACT: The study aimed to use the read aloud strategy in the enhancement of the Reading Literacy Program of the school. It
specifically determined the reading levels of the pupils based on their Lexile scores using the Scholastic Reading Inventory (SRI);
their mean scores in terms of comprehension, vocabulary, and fluency using the researcher-made test; their Lexile growth, and the
significant difference between the mean scores from the researcher-made pre-test and post-test. A one-group pre-test and post-test
design was utilized, wherein a total of 44 students in grades 4-6 were used as participants. The instruments used were the Scholastic
Reading Inventory (SRI) assessments adopted from the Reading Literacy Program and the researcher-made tests for comprehension,
vocabulary, and fluency. Results revealed that there is a significant difference between the mean pre-test and post-test scores from
the researcher-made tests, indicating that the use of reading aloud as a strategy is effective in enhancing the Reading Program. Thus,
the hypothesis “there is no significant difference between the mean percentage scores of pre-test and post-test of pupils” is not
sustained. KEYWORDS: Read Aloud Strategy, Lexile scores, Lexile growth, Scholastic Reading Inventory, Literac II. METHOD The study utilized the one-group pretest-posttest research design. According to [4] Yazon, Callo, and Buenvinida (2019), it is the
easiest type of experiment because it does not have a control group. In other words, researchers often only look at one group and do
not compare it to a group that does not get any treatment. This design's use of a pre-test to ascertain baseline scores was an advantage
because the scores in the pre-test serve as the basis for improvement. There was a total of 44 upper elementary school students at Bixby Knolls Preparatory Academy, Inc. (BKPA) who were chosen
through the complete enumeration method, which simply includes all the students in the elementary grade levels from 4 to 6 who
were identified as Basic and Below Basic readers after taking the pre-test. Specifically, there were 16 students from grade 4, 20
students from grade 5, and 8 students in grade 6. The researcher chose them as her respondents because it is one of the departments that she directly supervises. She chose this level
because she believes that a strong foundation in reading and comprehension must be fully established at the early stages of children’s
education to prepare them for the more complex disciplines that they will encounter when they enter high school. There were two instruments used in the study. The first instrument was the pretest and posttest through the Scholastic Reading
Inventory (SRI), adopted from the Reading Literacy Program. The researcher decided to adopt the instrument because the school is
in contract with Scholastic Asia and has been using the reading platform since 2017. The [5] Scholastic Reading Inventory (SRI) is a research-based reading assessment that tests and reports on students' reading
comprehension using the Lexile Framework® for Reading. Its electronic edition is an online adaptive test that responds to students'
responses. The difficulty levels of the questions alter depending on how well students perform once they begin the test. The exam
ends when the SRI assessment software has gathered enough data to generate a Lexile score. Adaptive testing reduces test
preparation time, improves test accuracy, and ensures that no two students are given the same test. It is based on passages from
children's literature, including fiction and nonfiction, as well as excerpts from young adult and classic literature, newspapers,
journals, and periodicals, and features a test bank of 5,119 questions. Read Aloud Strategy for Reading Literacy of Elementary Pupils: Basis for Enhancing the Reading Program and develop students into either Proficient or Advanced readers by the end of the academic year (AY). With these, the school has
implemented the Independent Reading Program (IRP), which was soon renamed the Reading Literacy Program (RLP). Under this
program, students choose and read the books from Scholastic collection that they want to read on their own, and then they are
evaluated on their reading comprehension. Despite this, it would appear that the program's intended purpose has not been achieved over the course of the last few years. The
pretest results in the Upper Elementary Grade Levels (Grades 4-6) the Academic Year (AY) 2022-2023, has shown that 54.41% of
the pupils are still categorized as Basic and Below Basic readers. She would like to address this problem while simultaneously
concentrating on the enhancement of the program by making an effort to include the read-aloud strategy in the reading program. She believes that as an Educational Management student, she must also look at the programs being implemented in the school,
especially when it is for the students’ academic progress. The researcher believes that the findings of her study can be of great assistance to students in improving their post-test reading
literacy levels in comparison to the results of their pretest reading literacy levels. English teachers and those in charge of
implementing the program can make use of the data to conduct careful evaluations and figure out what else could be done while the
program is being carried out to assist them in accomplishing their goal. I. INTRODUCTION The researcher notes
that now that students are beginning to physically return to school, the learning crisis in reading is evident in a certain number of
students who are enrolled in classes. This is the case for some of the students, especially in the early childhood and elementary
grades. This paves the way for the researcher's interest in focusing the study on the underlying topic of reading literacy. The
researcher is the Academic and reading program coordinator at the school, so this paved the way for her interest. The researcher is
of the opinion that one of the best ways to measure the reading literacy of learners is by using an appropriate assessment tool that is
guided by an appropriate reading strategy. In this particular instance, the "Read Aloud Strategy" was used to assist her in keeping
track of the books that her students have read. Bixby Knolls Preparatory Academy, Inc. (BKPA) is a private school that has been operating in the San Antonio, Quezon for the
past six years. Scholastic Inc. has been the school's literacy partner for the past five (5) academic years. The purpose of the
partnership is to foster a community of readers, improve the reading literacy skills of students in Kindergarten through Grade 10, IJSSHMR, Volume 2 Issue 06 June 2023 www.ijsshmr.com Page 454 Read Aloud Strategy for Reading Literacy of Elementary Pupils: Basis for Enhancing the Reading Program pretation of the gathered data, the study employed the following: For the analysis and interpretation of the gathered data, the study employed the following: For the analysis and interpretation of the gathered data, the study employed the following:
Simple descriptive statistics was used including frequency counting and percentage in determining the reading levels of the students
based on their Lexile scores in the pre-test and post-test results. Simple descriptive statistics was used including frequency counting and percentage in determining the reading levels of the students
based on their Lexile scores in the pre-test and post-test results. Two-tailed T-test was employed in determining the significant difference between the reading levels of the students based on the
scores in the pre-test and post-test results from the researcher-made test. Two-tailed T-test was employed in determining the significant difference between the reading levels of the students based on the
scores in the pre-test and post-test results from the researcher-made test. II. METHOD Students respond to fill-in-the-blank or cloze questions on the
SRI, which are similar to those found on many standardized tests and are used to assess students' comprehension of the texts they
read. To interpret the Lexile scores, the following data was used: Table I. Interpretation of Lexile Scores and Reading Levels
Lexile Scores
Reading Levels
901-1700+
Advanced
600-900
Proficient
350-599
Basic
BR-349
Below Basic Table I. Interpretation of Lexile Scores and Reading Levels The second instrument was the researcher-made pre- and post-test. The researcher selected books from Scholastic collections that
she read to the students aloud every week during the intervention, then she formulated pre-tests and post-tests from the chosen books
to test their vocabulary, fluency, and comprehension. To interpret the scores, the following data was used: IJSSHMR Volume 2 Issue 06 June 2023
www ijsshmr com
Page 455
Table II. Interpretation of Scores
SCORES
INTERPRETATION
15-20
Advanced
10-14
Proficient
5-9
Needs improvement
0-4
Did not meet expectation IJSSHMR, Volume 2 Issue 06 June 2023 www.ijsshmr.com Page 455 Read Aloud Strategy for Reading Literacy of Elementary Pupils: Basis for Enhancing the Reading Program In September, the participants took their pre-test through the online Reading Literacy Program. The pre-test results were then
interpreted through the SRI, which helped generate the learners’ lexical and reading levels. The results of the pre-test were
interpreted by the Scholastic representative for the researcher, who is also the Reading and Academic Coordinator of the school. It
was found out that there were 44 upper elementary pupils (Grades 4-6) who needed intervention and were identified as Basic and
Below Basic readers. After the results interpretation, the TOS for the pre-test and post-test focusing on comprehension, vocabulary, and fluency
assessments created by the researcher have been prepared together with the tests and program matrix. The researcher-made tests
were validated through pilot testing with proficient and advanced readers in grades 4 to 6. Upon retrieval of scores, the results were
subjected to item analysis, and revisions were made in the needed areas. In light of ethical considerations, the students’ participation was based on their agreement with the researcher. Prior to the conduct
of the study, she ensured that all data would be treated confidentially and would only be used for research purposes by sending
letters of permission, consent, and assent forms. Participants were also assured that they have the right to withdraw from the study
at any stage if they wish to do so. The program officially took off after the approval of the proposed intervention, validation of the researcher-made tests, and securing
letters of permission from the Head of School, the participants, and their parents. The Project Read Aloud was scheduled four times a week. Students also took short, researcher-made quizzes to assess their
understanding of each story. The evaluations focused on comprehension, vocabulary, and fluency. She continued to monitor their
reading progress through the course of the year. The Scholastic Reading program ran for eight (8) months, but the study’s
implemented treatment was only observed for one (1) month. After a month-long intervention, students took their post-tests. The pupils’ Lexile scores from pre-test and post-test results generated
through SRI and their scores in Comprehension, Vocabulary, and Fluency tests generated through the researcher-made test were
then compared and analyzed in order to identify whether the treatment is effective or not in enhancing the reading literacy of the
students. III. RESULTS AND DISCUSSIONS III. RESULTS AND DISCUSSIONS III. RESULTS AND DISCUSSIONS
Table III. Distribution of the Respondents based on the Reading Proficiency Levels using Lexile Scores before using the
Read Aloud Strategy
Legend:
901-1700+ - Advanced
600-900 - Proficient
350-599 - Basic
BR-349 - Below Basic
Scores
Grade 4
Grade 5
Grade 6
Verbal Interpretation
f
%
f
%
f
%
BR-349
8
50
11
55
4
50
Below basic
350-599
8
50
9
45
4
50
Basic
600-900
0
0
0
0
0
0
Proficient
901-1700+
0
0
0
0
0
0
Advanced
Total
16
100
20
100
8
100 III. RESULTS AND DISCUSSIONS
Table III. Distribution of the Respondents based on the Reading Proficiency Levels using Lexile Scores before using the
Read Aloud Strategy
Scores
Grade 4
Grade 5
Grade 6
Verbal Interpretation
f
%
f
%
f
%
BR-349
8
50
11
55
4
50
Below basic
350-599
8
50
9
45
4
50
Basic
600-900
0
0
0
0
0
0
Proficient
901-1700+
0
0
0
0
0
0
Advanced
Total
16
100
20
100
8
100 Table III. Distribution of the Respondents based on the Reading Proficiency Levels using Lexile Scores before using the
Read Aloud Strategy of the Respondents based on the Reading Proficiency Levels using Lexile Scores before using the Legend:
901-1700+ - Advanced
600-900 - Proficient
350-599 - Basic
BR-349 - Below Basic Table 3 shows the reading levels of the students based on their Lexile scores before the intervention program. From the Lexile scores
in their pre-test, it was shown that the respondents were equally distributed between below basic and Basic reading levels. The poor reading performance can be attributed to the two years of no face-to-face schooling due to the pandemic, where students’
performance in the reading program was not closely monitored and they got used to reading books with low Lexile levels merely
for leisure reading. When students were asked as to why they prefer to read books with a lower Lexile, they said that it is easier for
them to meet the required number of books to be read as those books with low Lexiles are also usually shorter and more engaging
to read. IJSSHMR, Volume 2 Issue 06 June 2023 www.ijsshmr.com Page 456
Table IV. III. RESULTS AND DISCUSSIONS Distribution of the Respondents based on the Reading Proficiency Levels using Lexile Scores after using the Read
Aloud Strategy of the Respondents based on the Reading Proficiency Levels using Lexile Scores after using the Read Table IV. Distribution of the Respondents based on the Reading Proficiency Levels using Lexile Scores after using the Read
Aloud Strategy IJSSHMR, Volume 2 Issue 06 June 2023 www.ijsshmr.com Page 456 IJSSHMR, Volume 2 Issue 06 June 2023 www.ijsshmr.com Page 456 Read Aloud Strategy for Reading Literacy of Elementary Pupils: Basis for Enhancing the Reading Program
Scores
Grade 4
Grade 5
Grade 6
Verbal
Interpretation
f
%
f
%
f
%
BR-349
8
50
11
55
4
50
Below basic
350-599
8
50
9
45
4
50
Basic
600-900
0
0
0
0
0
0
Proficient
901-1700+
0
0
0
0
0
0
Advanced
Total
16
100
20
100
8
100 Read Aloud Strategy for Reading Literacy of Elementary Pupils: Basis for Enhancing the Reading Program
Legend:
901-1700+ - Advanced
600-900 - Proficient
350-599 - Basic
BR-349 - Below Basic
Table 4 shows the distribution of reading levels among respondents after using the read-aloud strategy. It can be seen from the table
that the percentage of below-basic and Basic readers decreased, and four (4) of them became proficient readers. The increase can
be attributed to the read-aloud strategy that served as the intervention in the current Scholastic Reading Program at the school A
Scores
Grade 4
Grade 5
Grade 6
Verbal
Interpretation
f
%
f
%
f
%
BR-349
8
50
11
55
4
50
Below basic
350-599
8
50
9
45
4
50
Basic
600-900
0
0
0
0
0
0
Proficient
901-1700+
0
0
0
0
0
0
Advanced
Total
16
100
20
100
8
100 for Reading Literacy of Elementary Pupils: Basis for Enhancing the Reading Program Read Aloud Strategy for Reading Literacy of Elementary Pupils: Basis for Enhan Table 4 shows the distribution of reading levels among respondents after using the read-aloud strategy. It can be seen from the table
that the percentage of below-basic and Basic readers decreased, and four (4) of them became proficient readers. The increase can
be attributed to the read-aloud strategy that served as the intervention in the current Scholastic Reading Program at the school. A
9% increase in the proficient readers from 0% was particularly noticeable. III. RESULTS AND DISCUSSIONS The read-aloud sessions have helped them be guided on how they can further understand the stories they are reading and also assisted
them in decoding the meanings of the vocabulary words used in the stories, which is why the results improved during the post-test. According to Trelease (2013), interactive reading aloud makes sure that students have fun listening to the book and that their
curiosity about the book is sparked. Most importantly, interactive reading aloud builds vocabulary, creates conditions for the child's
brain to enjoy reading, builds on the child's prior knowledge, shows them how to read, and gets them interested in reading. With
this in mind, in the read-aloud sessions during the conduct of the study, the researcher used books that were applicable to the students
who took part in the study. She ensured that there was a proper matching of books according to their lexical levels so that the
students could also start to gain interest in reading the books within their lexical range with or without the teacher’s supervision. Though there was an increase in the number of proficient readers, the number of below-basic and Basic readers combined is still
higher. This can be rooted in the amount of time spent reading aloud. If the read-aloud strategy was implemented for a longer period
of time, there might be a higher percentage of proficient readers after the post-test. g
g
g
p
Though there was an increase in the number of proficient readers, the number of below-basic and Basic readers combined is still
higher. This can be rooted in the amount of time spent reading aloud. If the read-aloud strategy was implemented for a longer period
of time, there might be a higher percentage of proficient readers after the post-test. Table V. Mean Scores of Respondents in the Comprehension, Vocabulary, and Fluency Tests before using the Read Aloud
Strategy
Legend for Mean Scores Interpretation:
15-20 - Advanced
10-14 - Proficient
5-9 - Needs Improvement
0-4 - Did not meet expectation
Mean Scores
SD
Interpretation
comprehension
8.89
3.00
Needs Improvement
Vocabulary
11.02
4.44
Proficient
Fluency
19.73
1.09
Advanced Table V. Mean Scores of Respondents in the Comprehension, Vocabulary, and Fluency Tests before using the Read Aloud
Strategy of Respondents in the Comprehension, Vocabulary, and Fluency Tests before using the Read Aloud Table V. Read Aloud Strategy for Reading Literacy of Elementary Pupils: Basis for Enhancing the Reading Program difficult for them to comprehend the stories, mainly because they were tasked with reading them independently. Given these, an
intervention should be conducted to improve the students’ comprehension skills. It is usually empirical that when students understand vocabulary terms in what they are reading, their comprehension skills are
expected to be good as well, but the results presented in Table 5 do not match this idea. It can be attributed to the way the tests in
different areas of literacy skills were constructed. The test for the vocabulary skill is formed through context clues. On the other
hand, the test for the comprehension skill is based on their understanding of the stories they read. Table VI. Mean Scores of Respondents in the Comprehension, Vocabulary, and Fluency Tests afte Table VI. Mean Scores of Respondents in the Comprehension, Vocabulary, and Fluency Tests after using the Read Aloud Table VI. Mean Scores of Respondents in the Comprehension, Vocabulary, and Fluency Tests after using the Read Aloud
Strategy
Legend for Mean Scores Interpretation:
15-20 - Advanced
10-14 - Proficient
5-9 - Needs Improvement
0-4 - Did not meet expectation
Mean Scores
SD
comprehension
16.89
1.54
Advanced
Vocabulary
17.25
1.53
Advanced
Fluency
19.93
0.45
Advanced of Respondents in the Comprehension, Vocabulary, and Fluency Tests after using the Read Aloud Legend for Mean Scores Interpretation: Legend for Mean Scores Interpretation
15-20 - Advanced
10-14 - Proficient
5-9 - Needs Improvement
0-4 - Did not meet expectation Legend for Mean Scores Interpretation:
15-20 - Advanced
10-14 - Proficient
5-9 - Needs Improvement
0-4 - Did not meet expectation Table 6 shows the mean scores of the respondents in the pre-test in terms of comprehension, vocabulary, and fluency. The mean
scores and the low standard deviation in all three areas indicate that the results are better compared to the pre-test in Table 5. The
mean scores became higher, and the scores were not dispersed. g
p
It can be seen from the results that the reading comprehension, vocabulary, and fluency skills of students improved after the
implementation of read-aloud. The researcher observed that the students who attended the read-aloud were able to comprehend the content of the books that were
read to them. Thus, they were able to consistently pass the short assessments or quizzes given to them after every session of read-
aloud. III. RESULTS AND DISCUSSIONS Mean Scores of Respondents in the Comprehension, Vocabulary, and Fluency Tests befor Legend for Mean Scores Interpretation:
15-20 - Advanced
10-14 - Proficient
5-9 - Needs Improvement
0-4 - Did not meet expectation
Fluency
19.73
1.09
Advanced Legend for Mean Scores Interpretation: Legend for Mean Scores Interpretation
15-20 - Advanced
10-14 - Proficient
5-9 - Needs Improvement
0-4 - Did not meet expectation Shown in Table 5 above are the mean scores of the respondents in the pre-test in terms of comprehension, vocabulary, and fluency. The highest mean score of 19.73 with a standard deviation of 1.09 has been recorded in the fluency test, which means that the scores
are not dispersed and students can read. However, their mean score in terms of comprehension is the lowest, interpreted as needing improvement, and scores were dispersed
at a 3.00 standard deviation. The results imply that although students can read, they do not fully comprehend what they are reading. The low results in comprehension and vocabulary are alarming, given the idea that poor comprehension may also result in poor
academic performance. The claim is supported by [7] San Juan (2019), who stated that reading is, without a doubt, the actual foundation of most learning. As children progress through school, more reading is normally necessary as subjects get denser and more difficult. Not the other
way around; the difficulty level simply rises. If a student’s reading comprehension is poor, his or her performance in other disciplines
is likely to suffer as a result. When students were asked as to why they thought they performed poorly in the pre-test, most of the answers were that they found
it difficult to understand the stories they read, even if they were given a week to read them. Since some stories are long, it was also IJSSHMR, Volume 2 Issue 06 June 2023 www.ijsshmr.com Page 457 www.ijsshmr.com Table VIII. Test of Difference between the Mean Scores of the Pupils before and after the Read Aloud Implementation
Legend: Sig (2-tailed) ≤ .05 (Significant); Sig (2-tailed) ≥ .05 (Not significant) (2016), who emphasized the educational benefits of reading aloud, including helping
children understand the structure and norms of texts and fostering the development of linguistic requirements in other subjects, such
as mathematics. In addition, reading aloud to children provides a crucial opportunity for focused interaction, thereby transforming
reading into a valued social practice. During read-aloud sessions, embedded social behaviors such as inquiring skills, dialogic
participation, and inquiry-based learning are instilled in children. The result shows that reading aloud is an effective intervention strategy to improve the students’ reading literacy skills. It helps the
children have a better understanding of the stories and texts they are reading. In the read-aloud implementation, questions before,
during, and after reading have been posted before they answer the short quizzes. The process helped them recall significant events
from the stories, guided them in decoding the meanings of vocabulary words, and also made them more engaged in the stories. The statement is supported by [11] Canoy et al. (2016), who emphasized the educational benefits of reading aloud, including helping IV. CONCLUSIONS A significant difference is found to exist between the pre-test and the post-test scores of the pupils in terms of comprehension and
vocabulary, while there was none in fluency which means that the hypothesis “there is no significant difference between the mean
percentage scores of the pre-test and post-test of pupils” is not sustained. ACKNOWLEDGMENT The researcher would like to express her profound gratitude to the following institutions: Laguna State Polytechnic University-SPC,
for providing a high-quality education that enabled her to expand her knowledge for professional progress, and for granting
permission to conduct this study. She is particularly grateful to the administration at Bixby Knolls Preparatory Academy, Inc. for
all of their help. Read Aloud Strategy for Reading Literacy of Elementary Pupils: Basis for Enhancing the Reading Program This is mainly because the researcher was able to process and ask questions before, during, and even after the read-aloud
sessions to check and ensure the students’ understanding and comprehension. The process guided the students on how they could
further understand the stories or texts that they were reading. This claim is supported by a respondent in the study of Albright and [8] Ariail (2011), who stated that she reads aloud to her students
for them to focus on comprehension rather than pronunciation. Table VII. Lexile Growth of the Respondents Based on SRI after the Read Aloud Implementation Table VII. Lexile Growth of the Respondents Based on SRI after the Read Aloud Implementation
Legend: 901-1700+ - Advanced
600-900 - Proficient
350-599 - Basic
BR-349 - Below Basic
Lexile/SRI
Test
Mean
SD
Mean
difference
Pre-test
387.86
189.412
132.27
Post test
520.14
182.983 Legend: 901-1700+ - Advanced
600-900 - Proficient
350-599 - Basic
BR-349 - Below Basic As seen in Table 7 above, the computed mean difference is 132.27, which means that there is a huge increase in the scores from
pre-test to post-test. This notes that the scores improved, which can be rooted in the read-aloud implementation. The improvement in the scores due to read-aloud implementation matches the claim of [9] Kalb and Ours (2014), who considered
reading out loud to be the single most important factor in the development of young children’s literacy levels. This was found to be
the case following the discovery that reading aloud was the most important factor in the development of children’s literacy. They
also stated that it has numerous benefits as a reading intervention technique, such as encouraging children to read and improving
basic literacy comprehension and development. Reading aloud was defined as the shared reading experience that occurs between a
child and a parent, guardian, or teacher in the context of the research. However, even if the scores improved, it can also be seen from the table that, based on the mean of 520.14 from the post-test, the
students in general are still identified as Basic readers. The range for each level is high, which makes it challenging for students to
move up in terms of levels. Their scores increased, but their levels stayed at Basic. IJSSHMR, Volume 2 Issue 06 June 2023 www.ijsshmr.com IJSSHMR, Volume 2 Issue 06 June 2023 www.ijsshmr.com Page 458 Read Aloud Strategy for Reading Literacy of Elementary Pupils: Basis for Enhancing the Reading Program The [10] National Assessment of Educational Progress (NAEP), 2013 defined Basic and below-basic readers as those who can
discern the meaning of familiar words within the same sentence or paragraph by using context; identify a specific detail to make a
simple inference about a character's actions, motivations, or emotions, using a single point or multiple points in close proximity;
organize or categorize the story's events; provide some support for ideas related to the plot or characters; find meaning or evidence
from one of the texts when making a comparison across texts; identify explicit details from the text; and state an opinion with general
support from one section of the text. Table VIII. Test of Difference between the Mean Scores of the Pupils before and after the Read Aloud Implementation
Legend: Sig (2-tailed) ≤ .05 (Significant); Sig (2-tailed) ≥ .05 (Not significant) Table VIII. Test of Difference between the Mean Scores of the Pupils before and after the Read Aloud Implementation
Legend: Sig (2-tailed) ≤ .05 (Significant); Sig (2-tailed) ≥ .05 (Not significant)
The post-test and the pre-test scores showed significant differences based on t values of -18.927 and -11.388 for comprehension and
vocabulary, respectively, which are all less than the critical value with a 0.05 level of significance. Pre-test
Post Test
t
df
Sig. (2-tailed)
Verbal Interpretation
Mean
SD
Mean
SD
Comprehension
8.89
3.00
16.89
1.54
-18.927
43
0.000
Significant
Vocabulary
11.02
4.44
17.25
1.53
-11.388
43
0.000
Significant
Fluency
19.73
1.09
19.93
0.45
-1.594
43
0.118
Not significant Legend: Sig (2-tailed) ≤ .05 (Significant); Sig (2-tailed) ≥ .05 (Not significa The post-test and the pre-test scores showed significant differences based on t values of -18.927 and -11.38 The post-test and the pre-test scores showed significant differences based on t v vocabulary, respectively, which are all less than the critical value with a 0.05 level of significance. Pre-test
Post Test
t
df
Sig. (2-tailed)
Verbal Interpretation
Mean
SD
Mean
SD
Comprehension
8.89
3.00
16.89
1.54
-18.927
43
0.000
Significant
Vocabulary
11.02
4.44
17.25
1.53
-11.388
43
0.000
Significant
Fluency
19.73
1.09
19.93
0.45
-1.594
43
0.118
Not significant Fluency, on the other hand, was found to have no significant difference between the pre-test and post-test, as it only had a minimal
difference of 0.23 in their mean scores. It can be recalled that during the pre-test, students were already categorized as advanced in
terms of fluency which means to say that they can read even before the read aloud implementation, but they cannot fully comprehend
the stories they were reading. The slight increase in their post-test mean score indicates that there was minimal increase in their
reading fluency as well. The result shows that reading aloud is an effective intervention strategy to improve the students’ reading literacy skills. It helps the
children have a better understanding of the stories and texts they are reading. In the read-aloud implementation, questions before,
during, and after reading have been posted before they answer the short quizzes. The process helped them recall significant events
from the stories, guided them in decoding the meanings of vocabulary words, and also made them more engaged in the stories. The statement is supported by [11] Canoy et al. REFERENCES 1) Carroll, J. and Breadmore, H. (2019). Theories of Early Literacy Development.https://theeducationhub.org.nz/theories-of-
early-literacy-development/ 2) OECD. (2014). Reading for Change: Performance and Engagement across Countries: Results from PISA 2000. Paris:
OECD. 3) Department of Education (2018). PISA 2018 National Report of the Philippines.Retrieved Nov 22, 2021 from
https://www.deped.gov.ph/wp-content/uploads/2019/12/PISA-2018-Philippine-National-Report.pdf 4) Yazon, A., Callo, E., Buenvinida, L. (2019). Learning Guide in Methods of Research 4) Yazon, A., Callo, E., Buenvinida, L. (2019). Learning Guide in Methods of Research IJSSHMR, Volume 2 Issue 06 June 2023 www.ijsshmr.com IJSSHMR, Volume 2 Issue 06 June 2023 Page 459 Page 459 Read Aloud Strategy for Reading Literacy of Elementary Pupils: Basis for Enhancing the Reading Program 5) Scholastic Asia. (2015). Instructions for Administering the Electronic (Online) Scholastic Reading Inventory (SRI)
Assessment.https://www.ousd.org/cms/lib07/ca01001176/centricity/domain/93/instructions_forgiving_sri_assessment-
v2-0-revised-september2015.pdf 5) Scholastic Asia. (2015). Instructions for Administering the Electronic (Online) Scholastic Reading Inventory (SRI
Assessment.https://www.ousd.org/cms/lib07/ca01001176/centricity/domain/93/instructions_forgiving_sri_assessment-
v2-0-revised-september2015.pdf p
p
6) Trelease, J. (2016). The Read-Aloud Handbook. UK: Penguin.www.trelease-on-reading.com 6) Trelease, J. (2016). The Read-Aloud Handbook. UK: Penguin.www.trelease-on-reading.com 7) San Juan, R. (2019). Philippines lowest in reading comprehension among 79 countries. https://www philstar com/headlines/2019/12/03/1974002/philippines-lowest-reading-compreh 7) San Juan, R. (2019). Philippines lowest in reading comprehension among 79 countries. https://www.philstar.com/headlines/2019/12/03/1974002/philippines-lowest-reading-comprehension-among-79-countrie 7) San Juan, R. (2019). Philippines lowest in reading comprehension among 79 countries. https://www.philstar.com/headlines/2019/12/03/1974002/philippines-lowest-reading-comprehension-among-79-countries
8) Albright, L. K., & Ariail, M. (2015). Tapping the potential of teacher read alouds in middle schools. Journal of Adolescent )
,
(
)
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p
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https://www.philstar.com/headlines/2019/12/03/1974002/philippines-lowest-reading-comprehension-among-79-countries
8) Albright, L. K., & Ariail, M. (2015). Tapping the potential of teacher read alouds in middle schools. Journal of Adolescent
& Adult Literacy,vol/issue: 48(7), pp. 582-591. p
p
p
pp
g
p
g
8) Albright, L. K., & Ariail, M. (2015). Tapping the potential of teacher read alouds in middle schools. Journal of Adolescent
& Adult Literacy,vol/issue: 48(7), pp. 582-591. 9) Kalb, G., & Van Ours, J. (2014). Reading to young children: a head-start in life. Economic of Education Review 40, 1-24. http://doi.org.libproxy.murdoch.edu.au/10.1016/j.econedurev.2014.01.002 9) Kalb, G., & Van Ours, J. (2014). Reading to young children: a head-start in life. Economic of Education Review 40, 1-24. http://doi.org.libproxy.murdoch.edu.au/10.1016/j.econedurev.2014.01.002 10) National Center for Education Statistics. (2014). NAEP s
http://nces.ed.gov/nationsreportcard/statecomparisons/ 10) National Center for Education Statistics. (2014). NAEP state comparisons .Retrieved from
http://nces.ed.gov/nationsreportcard/statecomparisons/ 11) Canoy, M., J.C. van Ours, & F. van der Ploeg. (2016). The economics of books. in Ginsburgh, A., & Throsby, D. (Eds.),
Handbook of the Economics of Art and Culture, (721-761). Amsterdam: Elsevier. IJSSHMR, Volume 2 Issue 06 June 2023 IJSSHMR, Volume 2 Issue 06 June 2023 www.ijsshmr.com www.ijsshmr.com Page 460
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Discovery of Potent c-MET Inhibitors with New Scaffold Having Different Quinazoline, Pyridine and Tetrahydro-Pyridothienopyrimidine Headgroups
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Molecules/Molecules online/Molecules annual
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Discovery of Potent c-MET Inhibitors with New
Scaffold Having Different Quinazoline, Pyridine and
Tetrahydro-Pyridothienopyrimidine Headgroups
Discovery of Potent c-MET Inhibitors with New
Scaffold Having Different Quinazoline, Pyridine and
Tetrahydro-Pyridothienopyrimidine Headgroups * Correspondence: profwjh@126.com (J.W.); syzyclx@163.com (L.C.); Tel./Fax: +86-24-23986479 (J.W. & L.C
* Correspondence: profwjh@126.com (J.W.); syzyclx@163.com (L.C.); Tel./Fax: +86-24-23986479 (J.W. & L.C Abstract: Cellular mesenchymal-epithelial transition factor (c-MET) is closely linked to human
malignancies, which makes it an important target for treatment of cancer. In this study, a
series of 3-methoxy-N-phenylbenzamide derivatives, N-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)
benzamide derivatives and N1-(3-fluoro-4-methoxyphenyl)-N3-(4-fluorophenyl) malonamide
derivatives were designed and synthesized, some of them were identified as c-MET inhibitors. Among these compounds with new scaffolds having different quinazoline, pyridine and
tetrahydro-pyridothienopyrimidine head groups, compound 11c, 11i, 13b, 13h exhibited both potent
inhibitory activities against c-MET and high anticancer activity against tested cancer cell lines
in vitro. In addition, kinase selectivity assay further demonstrated that both 13b and 13h are potent
and selective c-MET inhibitors. Molecular docking supported that they bound well to c-MET and
VEGFR2, which demonstrates that they are potential c-MET RTK inhibitors for cancer therapy. Abstract: Cellular mesenchymal-epithelial transition factor (c-MET) is closely linked to human
malignancies, which makes it an important target for treatment of cancer. In this study, a series of
3-methoxy-N-phenylbenzamide derivatives, N-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl) benzamide
derivatives and N1-(3-fluoro-4-methoxyphenyl)-N3-(4-fluorophenyl) malonamide derivatives were
designed and synthesized, some of them were identified as c-MET inhibitors. Among these compounds
with new scaffolds having different quinazoline, pyridine and tetrahydro-pyridothienopyrimidine
head groups, compound 11c, 11i, 13b, 13h exhibited both potent inhibitory activities against c-MET
and high anticancer activity against tested cancer cell lines in vitro. In addition, kinase selectivity assay
further demonstrated that both 13b and 13h are potent and selective c-MET inhibitors. Molecular
docking supported that they bound well to c-MET and VEGFR2, which demonstrates that they are
potential c-MET RTK inhibitors for cancer therapy. Keywords: quinazoline; pyridine; tetrahydro-pyridothienopyrimidine; MET inhibitor; cancer therapy
Keywords: quinazoline; pyridine; tetrahydro-pyridothienopyrimidine; MET inhibitor; cancer therapy molecules molecules molecules Molecules 2016, 21, 612; doi:10.3390/molecules21050612
Molecules 2016, 21, 612; doi:10.3390/molecules21050612 molecules molecules 1. Introduction
1. Introduction Binding of Hepatocyte Growth
Factor/Scatter Factor (HGF/SF) to c-MET induces phosphorylation of tyrosine residues on c-MET and
activates its downstream signaling pathway [6,7], which is associated with cell proliferation, migration,
invasion and survival and is essential for normal embryonic development and wound healing [8,9]. However, it is reported that the c-MET/HGF axis is also involved in the development of various
human malignancies. Aberrant or mutated expression of c-MET/HGF axis has been observed in a
number of malignancies such as breast, gastric, bladder and lung cancers, which is closely linked
to tumorigenesis and metastasis [10–12]. Moreover, it is reported that dysregulation of c-MET also
correlated with a poor prognosis in clinical studies [13]. Therefore, c-MET shows high potential as a
therapeutic target for human cancer. Well-known agents for targeting the c-MET/HGF axis include anti-HGF antibodies, anti-c-MET
antibodies, and c-MET tyrosine kinase inhibitors (TKIs). Among them, c-MET TKIs are the most
attractive means to target c-MET pathway, because they are thought to be effective against both
ligand-dependent and ligand-independent activation of c-MET [14]. In recent years, a number of c-MET
inhibitors have been reported or have entered clinical trials, and many of them are c-MET/VEGFR-2
(vascular endothelial growth factor receptor 2) dual inhibitors (see Figure 1) [15–17]. For example,
Cabozantinib (XL184, BMS-907351) is a potent c-MET inhibitor with IC50 of 1.3 nM and also inhibits
VEGFR2, Ret, Kit, Flt-1/3/4, Tie2, and AXL in cell-free assays, respectively. Compounds 2 and 4
represent potential lead compounds with c-MET inhibitory effect in high throughput screening [18,19]. There are two classes of c-MET inhibitors based on their chemical structures or binding modes, but
they both have some shortcomings [20,21]. Class I inhibitors bind in a U-shaped conformation to the
ATP-binding site at the entrance of a kinase pocket, wrap around Met1211 and bind to a hinge-block,
and thus specifically inhibiting MET kinases, such as Crizotinib. Class II inhibitors bind to a region of
MET that extends from the ATP binding site to Ile1145 near the C-C-spiral block, such as Cabozantinib. Studies suggested that class II inhibitors maybe more effective than class I inhibitors, but they exhibited
off-target effects of other protein kinases, and clinical use of class II inhibitors have shown that these
agents cause serious toxic effects in many organs [22–25]. Therefore, potent c-MET inhibitors with
improved selectivity and minimal side effects should be developed. 1. Introduction
1. Introduction Tyrosine kinase is an enzyme that transfers a phosphate group from ATP to a protein, and it
functions as an “on” or “off” switch in many cellular functions. They become potent oncogene that
has the potential to cause cancer, when they are often mutated or expressed at high levels [1]. Several
receptor tyrosine kinases (RTKs) inhibitors have been found to have effective anti-tumor activity and
some of them have been approved or are in clinical trials. Recent FDA approved drugs Sorafenib
(Nexavar) [2] is such example of multi-targeted agents (Figure 1). Tyrosine kinase is an enzyme that transfers a phosphate group from ATP to a protein, and it
functions as an “on” or “off” switch in many cellular functions. They become potent oncogene that
has the potential to cause cancer, when they are often mutated or expressed at high levels [1]. Several
receptor tyrosine kinases (RTKs) inhibitors have been found to have effective anti-tumor activity and
some of them have been approved or are in clinical trials. Recent FDA approved drugs Sorafenib
(Nexavar) [2] is such example of multi-targeted agents (Figure 1). Figure 1. Representative c-MET inhibitor (1, XL184) and Multi-kinase inhibitor (2–4). HN
O
N
H
N
N
N
O
O
O
H
N
O
H
N
O
F
1, XL184
2
N
H
N
O
O
N
H
N
H
O
Cl
CF3
3, Sorafenib
N
N
S
BocN
HN
F
4
Figure 1. Representative c-MET inhibitor (1, XL184) and Multi-kinase inhibitor (2–4). HN
O
N
H
N
N
N
O
O
O
H
N
O
H
N
O
F
1, XL184
2
N
H
N
O
O
N
H
N
H
O
Cl
CF3
3, Sorafenib
N
N
S
BocN
HN
F
4 2 3, Sorafenib Figure 1. Representative c-MET inhibitor (1, XL184) and Multi-kinase inhibitor (2–4). Figure 1. Representative c-MET inhibitor (1, XL184) and Multi-kinase inhibitor (2–4). www.mdpi.com/journal/molecules
www.mdpi.com/journal/molecules www.mdpi.com/journal/molecules
www.mdpi.com/journal/molecules 2 of 16 Molecules 2016, 21, 612 Cellular mesenchymal-epithelial transition factor (c-MET), the hepatocyte growth factor receptor
(HGFR), belongs to a subfamily of RTK that are composed of an extracellular a chain and a
membrane-spanning b chain connected through a disulfide bond [3–5]. 1. Introduction
1. Introduction In this study, we disclose our efforts towards the design and synthesis of new c-MET inhibitors. To
further understand the structure–activity relationship (SAR) of this novel series of compounds, different
quinazoline, pyridine and tetrahydro-pyridothienopyrimidine fragments were investigated. A series
of 3-methoxy-N-phenylbenzamide derivatives, N-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl) benzamide
derivatives and N1-(3-fluoro-4-methoxyphenyl)-N3-(4-fluorophenyl) malonamide derivatives were
synthesized, and their inhibitory activities against c-MET and three different cancer cell lines were
evaluated. We were interested to see if such modifications were possible within our new classes of
molecules compared to the parent compounds. 2.1. Chemistry Reagents and conditions: (i)
Formamidine acetate, CH3OCH2CH2OH, 125 °C, 16 h; (ii) THF, SOCl2, reflux, 3 h; (iii) DMF, SOCl2, 120 °C,
6 h; (iv) THF, EDCI, CH3NH2, rt., 3 h; (v) S, NCCH2CO2Et, EtOH, Et3N, rt, 16 h; (vi) Formamidine
acetate DMF 120 °C 16 h; (vii) POCl3 DIPEA toluene 80 °C 12 h The chemistry described in Scheme 2 shows compound libraries could be made simply by using the
reaction protocol with N-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)-3-hydroxybenzamide (Compound 8)
and different head groups. A cyclization reaction with nitrile and hydrazine was carried to afford
3-(tert-butyl)-1-phenyl-1H-pyrazol-5-amine. Compound 8 was synthesized by a conventional peptide
synthesis method using ClCOCOCl at 0 °C [28]. The chemistry described in Scheme 2 shows compound libraries could be made simply by
using the reaction protocol with N-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)-3-hydroxybenzamide
(Compound 8) and different head groups. A cyclization reaction with nitrile and hydrazine was
carried to afford 3-(tert-butyl)-1-phenyl-1H-pyrazol-5-amine. Compound 8 was synthesized by a
conventional peptide synthesis method using ClCOCOCl at 0 ˝C [28]. The chemistry described in Scheme 2 shows compound libraries could be made simply by using the
reaction protocol with N-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)-3-hydroxybenzamide (Compound 8)
and different head groups. A cyclization reaction with nitrile and hydrazine was carried to afford
3-(tert-butyl)-1-phenyl-1H-pyrazol-5-amine. Compound 8 was synthesized by a conventional peptide
synthesis method using ClCOCOCl at 0 °C [28]. Scheme 2. The synthesis procedure of Compound 9a–c. Reagents and conditions: (i) EtOH, reflux, 16 h;
(ii) THF, 3-hydroxybenzoic acid, ClCOCOCl, 0 °C, 3 h; (iii) 5, 6 or 7, DMF, K2CO3, 80 °C, 24 h. Inspired by the structure of the lead compound 4, we have also designed a series of
N-phenylbenzamide derivatives. The synthetic route is shown in Scheme 3. Compounds 10 were
Scheme 2. The synthesis procedure of Compound 9a–c. Reagents and conditions: (i) EtOH, reflux, 16 h;
(ii) THF, 3-hydroxybenzoic acid, ClCOCOCl, 0 °C, 3 h; (iii) 5, 6 or 7, DMF, K2CO3, 80 °C, 24 h. Scheme 2. The synthesis procedure of Compound 9a–c. Reagents and conditions: (i) EtOH, reflux, 16 h;
(ii) THF, 3-hydroxybenzoic acid, ClCOCOCl, 0 ˝C, 3 h; (iii) 5, 6 or 7, DMF, K2CO3, 80 ˝C, 24 h. nd 9a–c. Reagents and conditions: (i) EtOH, reflu
°C 3 h; (iii) 5 6 or 7 DMF K2CO3 80 °C 24 h Inspired by the structure of the lead compound 4, we have also designed a series of
-phenylbenzamide derivatives. 2.1. Chemistry The chemistry described in Scheme 1 shows the synthetic route chosen to obtain quinazolines
(Compound 5), pyridines (Compound 6) and tetrahydro-pyridothienopyrimidines (Compound 7)
fragments. The reaction of 2-amino-4,5-dimethoxybenzoic acid with formamidine acetate afforded
intermediate 6,7-dimethoxyquinazolin-4-ol, which upon reaction with SOCl2 afforded compound 5. The synthetic method of compound 7 is similar to the above process [26,27]. 3 of 16 Molecules 2016, 21, 612 Scheme 1. The synthesis procedure of the three different headgroups. Reagents and conditions: (i)
Formamidine acetate, CH3OCH2CH2OH, 125 °C, 16 h; (ii) THF, SOCl2, reflux, 3 h; (iii) DMF, SOCl2, 120 °C,
6 h; (iv) THF, EDCI, CH3NH2, rt., 3 h; (v) S, NCCH2CO2Et, EtOH, Et3N, rt, 16 h; (vi) Formamidine
acetate, DMF, 120 °C, 16 h; (vii) POCl3, DIPEA, toluene, 80 °C, 12 h. Scheme 1. The synthesis procedure of the three different headgroups. Reagents and conditions:
(i) Formamidine acetate, CH3OCH2CH2OH, 125 ˝C, 16 h; (ii) THF, SOCl2, reflux, 3 h; (iii) DMF,
SOCl2, 120 ˝C, 6 h; (iv) THF, EDCI, CH3NH2, rt., 3 h; (v) S, NCCH2CO2Et, EtOH, Et3N, rt, 16 h;
(vi) Formamidine acetate, DMF, 120 ˝C, 16 h; (vii) POCl3, DIPEA, toluene, 80 ˝C, 12 h. ,
,
Scheme 1. The synthesis procedure of the three different headgroups. Reagents and conditions: (i)
Formamidine acetate, CH3OCH2CH2OH, 125 °C, 16 h; (ii) THF, SOCl2, reflux, 3 h; (iii) DMF, SOCl2, 120 °C,
6 h; (iv) THF, EDCI, CH3NH2, rt., 3 h; (v) S, NCCH2CO2Et, EtOH, Et3N, rt, 16 h; (vi) Formamidine
acetate, DMF, 120 °C, 16 h; (vii) POCl3, DIPEA, toluene, 80 °C, 12 h. Scheme 1. The synthesis procedure of the three different headgroups. Reagents and conditions: (i)
Formamidine acetate, CH3OCH2CH2OH, 125 °C, 16 h; (ii) THF, SOCl2, reflux, 3 h; (iii) DMF, SOCl2, 120 °C,
6 h; (iv) THF, EDCI, CH3NH2, rt., 3 h; (v) S, NCCH2CO2Et, EtOH, Et3N, rt, 16 h; (vi) Formamidine
acetate, DMF, 120 °C, 16 h; (vii) POCl3, DIPEA, toluene, 80 °C, 12 h. Scheme 1. The synthesis procedure of the three different headgroups. Reagents and conditions:
(i) Formamidine acetate, CH3OCH2CH2OH, 125 ˝C, 16 h; (ii) THF, SOCl2, reflux, 3 h; (iii) DMF,
SOCl2, 120 ˝C, 6 h; (iv) THF, EDCI, CH3NH2, rt., 3 h; (v) S, NCCH2CO2Et, EtOH, Et3N, rt, 16 h;
(vi) Formamidine acetate, DMF, 120 ˝C, 16 h; (vii) POCl3, DIPEA, toluene, 80 ˝C, 12 h. Scheme 1. The synthesis procedure of the three different headgroups. 2.1. Chemistry The synthesis procedure of Compound 11a–i. Reagents and conditions: (i) THF, ClCOCOCl, synthetic route is shown in Scheme 4. After multi-step protection and de-protection reaction, a series
ethyl hydrogen malonate derivative was obtained. Then, Compounds 12 were synthesized by a
conventional peptide synthesis method using isobutyl chloroformate and 4-Methylmorpholine. After
that, a typical Williamson ether synthesis was carried on to afford compound 13a–i [30]. We also combined different head groups to the side chain of lead compound 1, XL184. The
synthetic route is shown in Scheme 4. After multi-step protection and de-protection reaction, a series
ethyl hydrogen malonate derivative was obtained. Then, Compounds 12 were synthesized by a
conventional peptide synthesis method using isobutyl chloroformate and 4-Methylmorpholine. After
that, a typical Williamson ether synthesis was carried on to afford compound 13a–i [30]. 0 C, 3 h; (ii) 5, 6 or 7, DMF, K2CO3, 80 C, 24 h. We also combined different head groups to the side chain of lead compound 1, XL184. The
synthetic route is shown in Scheme 4. After multi-step protection and de-protection reaction, a series
ethyl hydrogen malonate derivative was obtained. Then, Compounds 12 were synthesized by a
conventional peptide synthesis method using isobutyl chloroformate and 4-Methylmorpholine. After
that, a typical Williamson ether synthesis was carried on to afford compound 13a–i [30]. Scheme 4. The synthesis procedure of Compound 13a–i. Reagents and conditions: (i) EtONa, CH3I,
0 °C, 5 h, or 1,2-dibromoethane or 1,3-dibromopropane, Bu4NBr, K2CO3, DMF, rt., 12 h; (ii) KOH, EtOH,
0 °C, 5 h; (iii) 4-amino-2-fluorophenol, IBCF, NMM, THF, −15 °C, 5 h; (iv) KOH, EtOH, 0 °C, 5 h; (v)
4-fluoroaniline, IBCF, NMM, THF, −15 °C, 3 h; (vi) 5, 6 or 7, DMF, K2CO3, 80 °C, 24 h. 2.2. Biology
2.1.1. Kinase Inhibitory Assay
Scheme 4. The synthesis procedure of Compound 13a–i. Reagents and conditions: (i) EtONa, CH3I,
0 °C, 5 h, or 1,2-dibromoethane or 1,3-dibromopropane, Bu4NBr, K2CO3, DMF, rt., 12 h; (ii) KOH, EtOH,
0 °C, 5 h; (iii) 4-amino-2-fluorophenol, IBCF, NMM, THF, −15 °C, 5 h; (iv) KOH, EtOH, 0 °C, 5 h; (v)
4-fluoroaniline, IBCF, NMM, THF, −15 °C, 3 h; (vi) 5, 6 or 7, DMF, K2CO3, 80 °C, 24 h. 2.2. Biology
Scheme 4. The synthesis procedure of Compound 13a–i. 2.1. Chemistry The synthetic route is shown in Scheme 3. Compounds 10 were
Scheme 2. The synthesis procedure of Compound 9a–c. Reagents and conditions: (i) EtOH, reflux, 16 h;
(ii) THF, 3-hydroxybenzoic acid, ClCOCOCl, 0 °C, 3 h; (iii) 5, 6 or 7, DMF, K2CO3, 80 °C, 24 h. Scheme 2. The synthesis procedure of Compound 9a–c. Reagents and conditions: (i) EtOH, reflux, 16 h;
(ii) THF, 3-hydroxybenzoic acid, ClCOCOCl, 0 ˝C, 3 h; (iii) 5, 6 or 7, DMF, K2CO3, 80 ˝C, 24 h. synthesized by a conventional peptide synthesis method with aniline and benzoic acid using
ClCOCOCl at 0 °C. Then, a typical Williamson ether synthesis was carried on to afford compound
11a–i [29]. Inspired by the structure of the lead compound 4, we have also designed a series of
N-phenylbenzamide derivatives. The synthetic route is shown in Scheme 3. Compounds 10 were
synthesized by a conventional peptide synthesis method with aniline and benzoic acid using
ClCOCOCl at 0 °C. Then, a typical Williamson ether synthesis was carried on to afford compound
11a–i [29]. Inspired by the structure of the lead compound 4, we have also designed a series of
N-phenylbenzamide derivatives. The synthetic route is shown in Scheme 3. Compounds 10
were synthesized by a conventional peptide synthesis method with aniline and benzoic acid using
ClCOCOCl at 0 ˝C. Then, a typical Williamson ether synthesis was carried on to afford compound
11a–i [29]. 4 of 16 Molecules 2016, 21, 612 Scheme 3. The synthesis procedure of Compound 11a–i. Reagents and conditions: (i) THF, ClCOCOCl,
0 °C, 3 h; (ii) 5, 6 or 7, DMF, K2CO3, 80 °C, 24 h. l
b
d d ff
h
d
h
d
h
f l
d
d
h
Scheme 3. The synthesis procedure of Compound 11a–i. Reagents and conditions: (i) THF, ClCOCOCl,
0 ˝C, 3 h; (ii) 5, 6 or 7, DMF, K2CO3, 80 ˝C, 24 h. Molecules 2016, 21, 612
4 of 16
Scheme 3. The synthesis procedure of Compound 11a–i. Reagents and conditions: (i) THF, ClCOCOCl, Scheme 3. The synthesis procedure of Compound 11a–i. Reagents and conditions: (i) THF, ClCOCOCl,
0 °C, 3 h; (ii) 5, 6 or 7, DMF, K2CO3, 80 °C, 24 h. Scheme 3. The synthesis procedure of Compound 11a–i. Reagents and conditions: (i) THF, ClCOCOCl,
0 ˝C, 3 h; (ii) 5, 6 or 7, DMF, K2CO3, 80 ˝C, 24 h. Scheme 3. A
results
2.1.1. K
2.2. Biology A
results
2.1.1. K
2.2. Biology A
results
2.1.1. K
2.2. Biology 2.1. Chemistry Reagents and conditions: (i) EtONa, CH3I, 0 ˝C,
5 h, or 1,2-dibromoethane or 1,3-dibromopropane, Bu4NBr, K2CO3, DMF, rt., 12 h; (ii) KOH, EtOH,
0 ˝C, 5 h; (iii) 4-amino-2-fluorophenol, IBCF, NMM, THF, ´15 ˝C, 5 h; (iv) KOH, EtOH, 0 ˝C, 5 h;
(v) 4-fluoroaniline, IBCF, NMM, THF, ´15 ˝C, 3 h; (vi) 5, 6 or 7, DMF, K2CO3, 80 ˝C, 24 h. 13a–i. Reagents and conditions: (i) EtONa
ne, Bu4NBr, K2CO3, DMF, rt., 12 h; (ii) KOH ,
; (
)
p
,
,
,
,
,
; (
)
,
,
,
; ( )
4-fluoroaniline, IBCF, NMM, THF, −15 °C, 3 h; (vi) 5, 6 or 7, DMF, K2CO3, 80 °C, 24 h. 2.2. Biology
2.1.1. Kinase Inhibitory Assay
Scheme 4. The synthesis procedure of Compound 13a–i. Reagents and conditions: (i) EtONa, CH3I,
0 °C, 5 h, or 1,2-dibromoethane or 1,3-dibromopropane, Bu4NBr, K2CO3, DMF, rt., 12 h; (ii) KOH, EtOH,
0 °C, 5 h; (iii) 4-amino-2-fluorophenol, IBCF, NMM, THF, −15 °C, 5 h; (iv) KOH, EtOH, 0 °C, 5 h; (v)
4-fluoroaniline, IBCF, NMM, THF, −15 °C, 3 h; (vi) 5, 6 or 7, DMF, K2CO3, 80 °C, 24 h. 2 2 Biology
Scheme 4. The synthesis procedure of Compound 13a–i. Reagents and conditions: (i) EtONa, CH3I, 0 ˝C,
5 h, or 1,2-dibromoethane or 1,3-dibromopropane, Bu4NBr, K2CO3, DMF, rt., 12 h; (ii) KOH, EtOH,
0 ˝C, 5 h; (iii) 4-amino-2-fluorophenol, IBCF, NMM, THF, ´15 ˝C, 5 h; (iv) KOH, EtOH, 0 ˝C, 5 h;
(v) 4-fluoroaniline, IBCF, NMM, THF, ´15 ˝C, 3 h; (vi) 5, 6 or 7, DMF, K2CO3, 80 ˝C, 24 h. A
results
2.1.1. K
2.2. Biology Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay results were summarized in Tables 1–3. Also included was the representative c-MET inhibitor (XL184). Among these compounds, compound 11c, 11i, 13b, 13h showed potent inhibitory activity against
All the synthesized compounds were assayed with the enzymatic activity against c-MET. The
results were summarized in Tables 1–3. Also included was the representative c-MET inhibitor (XL184). Among these compounds, compound 11c, 11i, 13b, 13h showed potent inhibitory activity against
c-MET, with IC50 of 0.08 µM (11c), 0.05 µM (11i), 0.02 µM (13b), 0.05 µM (13h), which is compared to
XL184 (IC50 = 0.03 µM against c-MET). 5 of 16
16 Molecules 2016, 21, 612
c-MET, with IC
Molecules 2016 21 612
Molecules 2016, 21, 61 Table 1. Enzymatic and cellular results for compound 9a–c. Table 1. Enzymatic and cellular results for compound 9a–c. µM (11c), 0.05 µM (11i), 0.02 µM (13b), 0.05 µM (13h), which is co
gainst c-MET). µM (11c), 0.05 µM (11i), 0.02 µM (13b), 0.05 µM (13h), which is com
i
MET)
8 µM (11c), 0.05 µM (11i), 0.02 µM (13b), 0.05 µM (13h), which is c
0.08 µM (11c), 0.05 µM (11i), 0.02 µM (13b), 0.05 µM (13h), wh
µM against c-MET). Table 1. Enzymatic and cellular results for compound 9a–c. Table 1. Enzymatic and cellular results for compound 9a–c. µM (11c), 0.05 µM (11i), 0.02 µM (13b), 0.05 µM (13h), which is co
gainst c-MET). µM (11c), 0.05 µM (11i), 0.02 µM (13b), 0.05 µM (13h), which is com
i
MET)
8 µM (11c), 0.05 µM (11i), 0.02 µM (13b), 0.05 µM (13h), which is c
0.08 µM (11c), 0.05 µM (11i), 0.02 µM (13b), 0.05 µM (13h), wh
µM against c-MET). Table 1. Enzymatic and cellular results for compound 9a c. Compound
A
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
9a
8.4 ± 0.6
28.7 ± 1.1
38.5 ± 1.3
46.1 ± 0.8
9b
>10
>100
>100
59.7 ± 1.3
9c
>10
>100
>100
66.7 ± 1.5
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. Table 2. Enzymatic and cellular results for compound 11a–c. Compound
A
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
9a
XL184 (IC50 0.03 µM against c MET). Table 1. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Enzymatic and cellular results for compound 9a–c. Compound
A
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
9a
8.4 ± 0.6
28.7 ± 1.1
38.5 ± 1.3
46.1 ± 0.8
9b
>10
>100
>100
59.7 ± 1.3
9c
>10
>100
>100
66.7 ± 1.5
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. 8.4 ˘ 0.6
28.7 ˘ 1.1
38.5 ˘ 1.3
46.1 ˘ 0.8
9b
XL184 (IC50 = 0.03 µM against c-MET). Table 1. Enzymatic and cellular results for compound 9a–c. Compound
A
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
9a
8.4 ± 0.6
28.7 ± 1.1
38.5 ± 1.3
46.1 ± 0.8
9b
>10
>100
>100
59.7 ± 1.3
9c
>10
>100
>100
66.7 ± 1.5
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. >10
>100
>100
59.7 ˘ 1.3
9c
c MET, with IC50 of 0.08 µM (11c), 0.05 µM (11i), 0.02 µM (13b), 0.05 µM (13h), which is compared to
XL184 (IC50 = 0.03 µM against c-MET). Table 1. Enzymatic and cellular results for compound 9a–c. Compound
A
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
9a
8.4 ± 0.6
28.7 ± 1.1
38.5 ± 1.3
46.1 ± 0.8
9b
>10
>100
>100
59.7 ± 1.3
9c
>10
>100
>100
66.7 ± 1.5
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations
>10
>100
>100
66.7 ˘ 1.5
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were incubated
for 30 min. b IC50 values obtained with viability assays. Each value is representative of three independent determinations. XL184 (IC50 0.03 µM against c MET). Table 1. Enzymatic and cellular results for compound 9a–c. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Compound
A
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
9a
8.4 ± 0.6
28.7 ± 1.1
38.5 ± 1.3
46.1 ± 0.8
9b
>10
>100
>100
59.7 ± 1.3
9c
>10
>100
>100
66.7 ± 1.5
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
i d
d
t d t
i
ti
c-MET, with IC50 of 0.08 µM (11c), 0.05 µM (11i), 0.02 µM (13b), 0.05 µM (13h), which is compared to
XL184 (IC50 = 0.03 µM against c-MET). Table 1. Enzymatic and cellular results for compound 9a–c. Compound
A
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
9a
8.4 ± 0.6
28.7 ± 1.1
38.5 ± 1.3
46.1 ± 0.8
9b
>10
>100
>100
59.7 ± 1.3
9c
>10
>100
>100
66.7 ± 1.5
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
c-MET, with IC50 of 0.08 µM (11c), 0.05 µM (11i), 0.02 µM (13b), 0.05 µM (13h), which is compared to
XL184 (IC50 = 0.03 µM against c-MET). Table 1. Enzymatic and cellular results for compound 9a–c. Compound
A
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
9a
8.4 ± 0.6
28.7 ± 1.1
38.5 ± 1.3
46.1 ± 0.8
9b
>10
>100
>100
59.7 ± 1.3
9c
>10
>100
>100
66.7 ± 1.5
Molecules 2016, 21, 612
5 of 16
c-MET, with IC50 of 0.08 µM (11c), 0.05 µM (11i), 0.02 µM (13b), 0.05 µM (13h), which is compared to
XL184 (IC50 = 0.03 µM against c-MET). Table 1. Enzymatic and cellular results for compound 9a–c. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Table 2 Enzymatic and cellular results for compound 11a c
tio
o t e ki ase
eactio
wit
A
( 0 µM), co
pou ds a d e
y
e we e
n. b IC50 values obtained with viability assays. Each value is representative of three
tion of the kinase reaction with ATP (10 µM), compounds and enzyme were able 2. Enzymatic and cellular results for compound 11a–c. able 2. Enzymatic and cellular results for compound 11a–c. minations. Table 2. Enzymatic and cellular results for compound 11a–c. Table 2. Enzymatic and cellular results for compound 11a–c. terminations. Table 2 Enzymatic and cellular results for compound 11a c
a io
o
e
i a e
ea io
i
A
(
µ
),
o
pou
a
e
y
n. b IC50 values obtained with viability assays. Each value is representativ
ation of the kinase reaction with ATP (10 µM), compounds and enzy Table 2. Enzymatic and cellular results for compound 11a–c. Table 2. Enzymatic and cellular results for compound 11a–c. Table 2. Enzymatic and cellular results for compound 11a–c. Table 2. Enzymatic and cellular results for compound 11a–c. eterminations. Table 2 Enzymatic and cellular results for compound 11a c
(
µ
),
p
n. b IC50 values obtained with viability assays. Each value is representat
ation of the kinase reaction with ATP (10 µM), compounds and enz Enzymatic Inhibition (IC50,
c-MET
Me
0.52 ± 0.05
zymatic Inhibition (IC50, µM) a,b
c-MET
zymatic Inhibition (IC50, µM) a,b
c-MET
nzymatic Inhibition (IC50, µM) a,b
nd cellular results for comp
y
y
nd cellular results for comp
(
µ
)
ed with viability assays. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Ea
nd cellular results for com Compound
A
R
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
11a
3,4-diOMe
0.52 ± 0.05
14.1 ± 0.16
>100
3.4 ± 0.21
11b
4-Cl
0.12 ± 0.03
1.8 ± 0.06
9.2 ± 0.17
7.8 ± 0.14
11c
4-F
0.08 ± 0.04
0.9 ± 0.12
10.1 ± 0.20
5.2 ± 0.36
11d
3,4-diOMe
>10
>100
>100
71.2 ± 0.34
11e
4-Cl
7.4 ± 0.07
26.4 ± 0.20
>100
34.5 ± 0.36
11f
4-F
5.3 ± 0.05
27.8 ± 0.34
>100
42.1 ± 0.27
11g
3,4-diOMe
1.4 ± 0.10
4.4 ± 0.26
>100
17.3 ± 0.38
11h
4-Cl
0.25 ± 0.08
10.7 ± 0.18
61.9 ± 0.23
22.9 ± 0.22
11i
4-F
0.05 ± 0.02
1.5 ± 0.11
23.2 ± 0.36
1.1 ± 0.31
XL184
0.03 ± 0.02
2.6 ± 0.07
49.1 ± 0.25
6.6 ± 0.25
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. Compound
A
R
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
11a
3,4-diOMe
0.52 ± 0.05
14.1 ± 0.16
>100
3.4 ± 0.21
11b
4-Cl
0.12 ± 0.03
1.8 ± 0.06
9.2 ± 0.17
7.8 ± 0.14
11c
4-F
0.08 ± 0.04
0.9 ± 0.12
10.1 ± 0.20
5.2 ± 0.36
11d
3,4-diOMe
>10
>100
>100
71.2 ± 0.34
11e
4-Cl
7.4 ± 0.07
26.4 ± 0.20
>100
34.5 ± 0.36
11f
4-F
5.3 ± 0.05
27.8 ± 0.34
>100
42.1 ± 0.27
11g
3,4-diOMe
1.4 ± 0.10
4.4 ± 0.26
>100
17.3 ± 0.38
11h
4-Cl
0.25 ± 0.08
10.7 ± 0.18
61.9 ± 0.23
22.9 ± 0.22
11i
4-F
0.05 ± 0.02
1.5 ± 0.11
23.2 ± 0.36
1.1 ± 0.31
XL184
0.03 ± 0.02
2.6 ± 0.07
49.1 ± 0.25
6.6 ± 0.25
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Compound
A
R
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
11a
3,4-diOMe
0.52 ± 0.05
14.1 ± 0.16
>100
3.4 ± 0.21
11b
4-Cl
0.12 ± 0.03
1.8 ± 0.06
9.2 ± 0.17
7.8 ± 0.14
11c
4-F
0.08 ± 0.04
0.9 ± 0.12
10.1 ± 0.20
5.2 ± 0.36
11d
3,4-diOMe
>10
>100
>100
71.2 ± 0.34
11e
4-Cl
7.4 ± 0.07
26.4 ± 0.20
>100
34.5 ± 0.36
11f
4-F
5.3 ± 0.05
27.8 ± 0.34
>100
42.1 ± 0.27
11g
3,4-diOMe
1.4 ± 0.10
4.4 ± 0.26
>100
17.3 ± 0.38
11h
4-Cl
0.25 ± 0.08
10.7 ± 0.18
61.9 ± 0.23
22.9 ± 0.22
11i
4-F
0.05 ± 0.02
1.5 ± 0.11
23.2 ± 0.36
1.1 ± 0.31
XL184
0.03 ± 0.02
2.6 ± 0.07
49.1 ± 0.25
6.6 ± 0.25
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. Compound
A
R
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
11a
3,4-diOMe
0.52 ± 0.05
14.1 ± 0.16
>100
3.4 ± 0.21
11b
4-Cl
0.12 ± 0.03
1.8 ± 0.06
9.2 ± 0.17
7.8 ± 0.14
11c
4-F
0.08 ± 0.04
0.9 ± 0.12
10.1 ± 0.20
5.2 ± 0.36
11d
3,4-diOMe
>10
>100
>100
71.2 ± 0.34
11e
4-Cl
7.4 ± 0.07
26.4 ± 0.20
>100
34.5 ± 0.36
11f
4-F
5.3 ± 0.05
27.8 ± 0.34
>100
42.1 ± 0.27
11g
3,4-diOMe
1.4 ± 0.10
4.4 ± 0.26
>100
17.3 ± 0.38
11h
4-Cl
0.25 ± 0.08
10.7 ± 0.18
61.9 ± 0.23
22.9 ± 0.22
11i
4-F
0.05 ± 0.02
1.5 ± 0.11
23.2 ± 0.36
1.1 ± 0.31
XL184
0.03 ± 0.02
2.6 ± 0.07
49.1 ± 0.25
6.6 ± 0.25
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Compound
A
R
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
11a
3,4-diOMe
0.52 ± 0.05
14.1 ± 0.16
>100
3.4 ± 0.21
11b
4-Cl
0.12 ± 0.03
1.8 ± 0.06
9.2 ± 0.17
7.8 ± 0.14
11c
4-F
0.08 ± 0.04
0.9 ± 0.12
10.1 ± 0.20
5.2 ± 0.36
11d
3,4-diOMe
>10
>100
>100
71.2 ± 0.34
11e
4-Cl
7.4 ± 0.07
26.4 ± 0.20
>100
34.5 ± 0.36
11f
4-F
5.3 ± 0.05
27.8 ± 0.34
>100
42.1 ± 0.27
11g
3,4-diOMe
1.4 ± 0.10
4.4 ± 0.26
>100
17.3 ± 0.38
11h
4-Cl
0.25 ± 0.08
10.7 ± 0.18
61.9 ± 0.23
22.9 ± 0.22
11i
4-F
0.05 ± 0.02
1.5 ± 0.11
23.2 ± 0.36
1.1 ± 0.31
XL184
0.03 ± 0.02
2.6 ± 0.07
49.1 ± 0.25
6.6 ± 0.25
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. Compound
A
R
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
11a
Table 2. Enzymatic and cellular results for compound 11a–c. Compound
A
R
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
11a
3,4-diOMe
0.52 ± 0.05
14.1 ± 0.16
>100
3.4 ± 0.21
11b
4-Cl
0.12 ± 0.03
1.8 ± 0.06
9.2 ± 0.17
7.8 ± 0.14
11c
4-F
0.08 ± 0.04
0.9 ± 0.12
10.1 ± 0.20
5.2 ± 0.36
11d
3,4-diOMe
>10
>100
>100
71.2 ± 0.34
11e
4-Cl
7.4 ± 0.07
26.4 ± 0.20
>100
34.5 ± 0.36
11f
4-F
5.3 ± 0.05
27.8 ± 0.34
>100
42.1 ± 0.27
11g
3,4-diOMe
1.4 ± 0.10
4.4 ± 0.26
>100
17.3 ± 0.38
11h
4-Cl
0.25 ± 0.08
10.7 ± 0.18
61.9 ± 0.23
22.9 ± 0.22
11i
4-F
0.05 ± 0.02
1.5 ± 0.11
23.2 ± 0.36
1.1 ± 0.31
XL184
0.03 ± 0.02
2.6 ± 0.07
49.1 ± 0.25
6.6 ± 0.25
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay 3,4-diOMe
0.52 ˘ 0.05
14.1 ˘ 0.16
>100
3.4 ˘ 0.21
11b
4-Cl
0.12 ˘ 0.03
1.8 ˘ 0.06
9.2 ˘ 0.17
7.8 ˘ 0.14
11c
4-F
0.08 ˘ 0.04
0.9 ˘ 0.12
10.1 ˘ 0.20
5.2 ˘ 0.36
11d
i
epe
e
e e
i a io
Table 2. Enzymatic and cellular results for compound 11a–c. Compound
A
R
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
11a
3,4-diOMe
0.52 ± 0.05
14.1 ± 0.16
>100
3.4 ± 0.21
11b
4-Cl
0.12 ± 0.03
1.8 ± 0.06
9.2 ± 0.17
7.8 ± 0.14
11c
4-F
0.08 ± 0.04
0.9 ± 0.12
10.1 ± 0.20
5.2 ± 0.36
11d
3,4-diOMe
>10
>100
>100
71.2 ± 0.34
11e
4-Cl
7.4 ± 0.07
26.4 ± 0.20
>100
34.5 ± 0.36
11f
4-F
5.3 ± 0.05
27.8 ± 0.34
>100
42.1 ± 0.27
11g
3,4-diOMe
1.4 ± 0.10
4.4 ± 0.26
>100
17.3 ± 0.38
11h
4-Cl
0.25 ± 0.08
10.7 ± 0.18
61.9 ± 0.23
22.9 ± 0.22
11i
4-F
0.05 ± 0.02
1.5 ± 0.11
23.2 ± 0.36
1.1 ± 0.31
XL184
0.03 ± 0.02
2.6 ± 0.07
49.1 ± 0.25
6.6 ± 0.25
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. 3,4-diOMe
>10
>100
>100
71.2 ˘ 0.34
11e
4-Cl
7.4 ˘ 0.07
26.4 ˘ 0.20
>100
34.5 ˘ 0.36
11f
4-F
5.3 ˘ 0.05
27.8 ˘ 0.34
>100
42.1 ˘ 0.27
11g
incubated for 30 min. IC50 values obtained with viability assays. Each value is representative of three
independent determinations. Table 2. Enzymatic and cellular results for compound 11a–c. independent determinations.
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations.
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were incubated
for 30 min.
b IC50 values obtained with viability assays.
Each value is representative of three
independent determinations. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Compound
A
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
9a
8.4 ± 0.6
28.7 ± 1.1
38.5 ± 1.3
46.1 ± 0.8
9b
>10
>100
>100
59.7 ± 1.3
9c
>10
>100
>100
66.7 ± 1.5 nd cellular results for compound
nd cellular results for compound
and cellular results for compoun
matic and cellular results for co
µM (11i), 0.02 µM (13b), 0.05
c and cellular results for compo
µM (11i), 0.02 µM (13b), 0.05
c and cellular results for compo
µM (11i), 0.02 µM (13b), 0.05
c and cellular results for comp A
A
A A a Prior to the initiation of the k
incubated for 30 min. b IC50 values
independent determinations
9c
to the initiation of the kinase re
9c
t
th
i iti ti
f th
ki
9c
9c
9b
9b Table 2. Enzymatic and cellular results for compound 11a–c. incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
i d
d
d
i
i
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were incubated
for 30 min. b IC50 values obtained with viability assays. Each value is representative of three independent determinations. a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
9c
>10
>100
>100
66.7 ± 1.5
a P i
t
th
i iti ti
f th
ki
ti
ith ATP (10
M)
d
d
9c
>10
>100
>100
66.7 ± 1.5
9c
>10
>100
>100
66 7 ± 1 5 able 2. Enzymatic and cellular results for compound 11a–c. able 2. Enzymatic and cellular results for compound 11a–c. inations. Table 2. Enzymatic and cellular results for compound 11a–c. Table 2. Enzymatic and cellular results for compound 11a–c. terminations. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay It is clear that compo
y
p
human cervical carcinoma), Hep-G2 (human liver cancer) a
sults were also summarized in Table1 3 It is clear that compo
Anti-proliferation against Tumor Cells Assay
ll h
h
i
d
d
l
d f
h i
All the synthesized compounds were evaluated for their
uman cervical carcinoma), Hep-G2 (human liver cancer) a
All the synthesized compounds were evaluated for their
human cervical carcinoma) Hep G2 (human liver cancer) a
p
g
y
All the synthesized compounds were evaluated for thei
Anti-proliferation against Tumor Cells Assay
1.2. Anti-proliferation against Tumor Cells Assay
All the synthesized compounds were evaluated for their
1.2. Anti-proliferation against Tumor Cells Assay
2.2.2. Anti-proliferation against Tumor Cells Assay All the synthesized compounds were evaluated
(human cervical carcinoma), Hep-G2 (human liver
sults were also summarized in Table1-3. It is clear that compo
nticancer activity against tested cancer cell lines. Compared
All the synthesized compounds were evaluated for thei
),
p
(
)
sults were also summarized in Table1-3. It is clear that compo
ti
ti it
i
t t
t d
ll li
C
d
uman cervical carcinoma), Hep-G2 (human liver cancer) a
sults were also summarized in Table1-3. It is clear that compo
y
p
uman cervical carcinoma), Hep-G2 (human liver cancer) a
sults were also summarized in Table1 3 It is clear that compo
Anti-proliferation against Tumor Cells Assay
ll h
h
i
d
d
l
d f
h i
All the synthesized compounds were evaluated for their
uman cervical carcinoma), Hep-G2 (human liver cancer) a
All the synthesized compounds were evaluated for their
uman cervical carcinoma) Hep G2 (human liver cancer) a
p
g
y
All the synthesized compounds were evaluated for thei
Anti-proliferation against Tumor Cells Assay
1.2. Anti-proliferation against Tumor Cells Assay
All the synthesized compounds were evaluated for their
1.2. Anti-proliferation against Tumor Cells Assay
2.2.2. Anti-proliferation against Tumor Cells Assay results were also summarized in Table1-3. It is clear that compound 11c, 11i, 13b, 13h all exhibited good
anticancer activity against tested cancer cell lines. Compared to XL184 (IC50 = 6.6 µM against HeLa),
these four compounds all showed better anti-proliferation activity against HeLa with compound 11c
(IC50 = 0.9 µM against HeLa) exhibiting the highest activity. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Similarly, they also showed better anti-
proliferation activity against Hep-G2 than XL184 (IC50 = 49.1 µM), and it is obvious that compound 13h
(IC50 = 1.7 µM) showed the highest activity, which is far better than XL184. In addition, compound 11i
(IC50 = 1.1 µM) and 13b (IC50 = 1.2 µM) had better results against MCF-7 than XL184 (IC50 = 2.6 µM). 2.1.3. Structure Activity Relationship Analysis
Preliminary structure–activity SAR study appeared that different head groups showed great
hese four compounds all showed better anti-proliferation activity against HeLa with compound 11c
C50 = 0.9 µM against HeLa) exhibiting the highest activity. Similarly, they also showed better anti-
roliferation activity against Hep-G2 than XL184 (IC50 = 49.1 µM), and it is obvious that compound 13h
C50 = 1.7 µM) showed the highest activity, which is far better than XL184. In addition, compound 11i
C50 = 1.1 µM) and 13b (IC50 = 1.2 µM) had better results against MCF-7 than XL184 (IC50 = 2.6 µM). 1.3. Structure Activity Relationship Analysis
Preliminary structure–activity SAR study appeared that different head groups showed great
fferences in cell and enzymatic activity. Among them, quinazolines (compound 9a, 11a–c, 13a–c),
nd tetrahydro-pyridothienopyrimidines (compound 9c, 11g–i, 13g–i) head groups provided much
etter activity than pyridines (compound 9b, 11d–f, 13d–f). For the side chain, N-(3-(tert-butyl)-1-
an cervical carcinoma), Hep-G2 (human liver cancer) and MCF-7 (human breast cancer). The
were also summarized in Table1-3. It is clear that compound 11c, 11i, 13b, 13h all exhibited good
ncer activity against tested cancer cell lines. Compared to XL184 (IC50 = 6.6 µM against HeLa),
our compounds all showed better anti-proliferation activity against HeLa with compound 11c
0.9 µM against HeLa) exhibiting the highest activity. Similarly, they also showed better anti-
ration activity against Hep-G2 than XL184 (IC50 = 49.1 µM), and it is obvious that compound 13h
1.7 µM) showed the highest activity, which is far better than XL184. In addition, compound 11i
1.1 µM) and 13b (IC50 = 1.2 µM) had better results against MCF-7 than XL184 (IC50 = 2.6 µM). Structure Activity Relationship Analysis
nticancer activity against tested cancer cell lines. Compared to XL184 (IC50 = 6.6 µM against HeLa),
ese four compounds all showed better anti-proliferation activity against HeLa with compound 11c
C50 = 0.9 µM against HeLa) exhibiting the highest activity. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Similarly, they also showed better anti-
roliferation activity against Hep-G2 than XL184 (IC50 = 49.1 µM), and it is obvious that compound 13h
C50 = 1.7 µM) showed the highest activity, which is far better than XL184. In addition, compound 11i
C50 = 1.1 µM) and 13b (IC50 = 1.2 µM) had better results against MCF-7 than XL184 (IC50 = 2.6 µM). 1.3. Structure Activity Relationship Analysis
Preliminary structure–activity SAR study appeared that different head groups showed great
fferences in cell and enzymatic activity. Among them, quinazolines (compound 9a, 11a–c, 13a–c),
nd tetrahydro-pyridothienopyrimidines (compound 9c, 11g–i, 13g–i) head groups provided much
etter activity than pyridines (compound 9b 11d f 13d f) For the side chain N (3 (tert butyl) 1
nticancer activity against tested cancer cell lines. Compared to XL184 (IC50 = 6.6 µM against HeLa),
ese four compounds all showed better anti-proliferation activity against HeLa with compound 11c
C50 = 0.9 µM against HeLa) exhibiting the highest activity. Similarly, they also showed better anti-
roliferation activity against Hep-G2 than XL184 (IC50 = 49.1 µM), and it is obvious that compound 13h
C50 = 1.7 µM) showed the highest activity, which is far better than XL184. In addition, compound 11i
C50 = 1.1 µM) and 13b (IC50 = 1.2 µM) had better results against MCF-7 than XL184 (IC50 = 2.6 µM). 1.3. Structure Activity Relationship Analysis
Preliminary structure–activity SAR study appeared that different head groups showed great
fferences in cell and enzymatic activity. Among them, quinazolines (compound 9a, 11a–c, 13a–c),
nd tetrahydro-pyridothienopyrimidines (compound 9c, 11g–i, 13g–i) head groups provided much
sults were also summarized in Table1 3. It is clear that compound 11c, 11i, 13b, 13h all exhibited good
nticancer activity against tested cancer cell lines. Compared to XL184 (IC50 = 6.6 µM against HeLa),
ese four compounds all showed better anti-proliferation activity against HeLa with compound 11c
C50 = 0.9 µM against HeLa) exhibiting the highest activity. Similarly, they also showed better anti-
roliferation activity against Hep-G2 than XL184 (IC50 = 49.1 µM), and it is obvious that compound 13h
C50 = 1.7 µM) showed the highest activity, which is far better than XL184. In addition, compound 11i
C50 = 1.1 µM) and 13b (IC50 = 1.2 µM) had better results against MCF-7 than XL184 (IC50 = 2.6 µM). 1.3. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Structure Activity Relationship Analysis
Preliminary structure–activity SAR study appeared that different head groups showed great
fferences in cell and enzymatic activity. Among them, quinazolines (compound 9a, 11a–c, 13a–c),
nd tetrahydro-pyridothienopyrimidines (compound 9c 11g–i 13g–i) head groups provided much
ll the synthesized compounds were evaluated for their anticancer activity in vitro against HeLa
n cervical carcinoma), Hep-G2 (human liver cancer) and MCF-7 (human breast cancer). The
were also summarized in Table1-3. It is clear that compound 11c, 11i, 13b, 13h all exhibited good
ncer activity against tested cancer cell lines. Compared to XL184 (IC50 = 6.6 µM against HeLa),
our compounds all showed better anti-proliferation activity against HeLa with compound 11c
0.9 µM against HeLa) exhibiting the highest activity. Similarly, they also showed better anti-
ration activity against Hep-G2 than XL184 (IC50 = 49.1 µM), and it is obvious that compound 13h
1.7 µM) showed the highest activity, which is far better than XL184. In addition, compound 11i
1.1 µM) and 13b (IC50 = 1.2 µM) had better results against MCF-7 than XL184 (IC50 = 2.6 µM). Structure Activity Relationship Analysis
sults were also summarized in Table1-3. It is clear that compound 11c, 11i, 13b, 13h all exhibited good
nticancer activity against tested cancer cell lines. Compared to XL184 (IC50 = 6.6 µM against HeLa),
ese four compounds all showed better anti-proliferation activity against HeLa with compound 11c
C50 = 0.9 µM against HeLa) exhibiting the highest activity. Similarly, they also showed better anti-
oliferation activity against Hep-G2 than XL184 (IC50 = 49.1 µM), and it is obvious that compound 13h
C50 = 1.7 µM) showed the highest activity, which is far better than XL184. In addition, compound 11i
C50 = 1.1 µM) and 13b (IC50 = 1.2 µM) had better results against MCF-7 than XL184 (IC50 = 2.6 µM). 1.3. Structure Activity Relationship Analysis
Preliminary structure–activity SAR study appeared that different head groups showed great
fferences in cell and enzymatic activity. Among them, quinazolines (compound 9a, 11a–c, 13a–c),
uman cervical carcinoma), Hep G2 (human liver cancer) and MCF 7 (human breast cancer). The
sults were also summarized in Table1-3. It is clear that compound 11c, 11i, 13b, 13h all exhibited good
nticancer activity against tested cancer cell lines. Compared to XL184 (IC50 = 6.6 µM against HeLa),
ese four compounds all showed better anti-proliferation activity against HeLa with compound 11c
C50 = 0.9 µM against HeLa) exhibiting the highest activity. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Similarly, they also showed better anti-
roliferation activity against Hep-G2 than XL184 (IC50 = 49.1 µM), and it is obvious that compound 13h
C50 = 1.7 µM) showed the highest activity, which is far better than XL184. In addition, compound 11i
C50 = 1.1 µM) and 13b (IC50 = 1.2 µM) had better results against MCF-7 than XL184 (IC50 = 2.6 µM). 1.3. Structure Activity Relationship Analysis
Preliminary structure–activity SAR study appeared that different head groups showed great
fferences in cell and enzymatic activity Among them quinazolines (compound 9a 11a–c 13a–c)
(human cervical carcinoma), Hep-G2 (human liver cancer) and MCF-7 (human breast cancer). The
results were also summarized in Table1-3. It is clear that compound 11c, 11i, 13b, 13h all exhibited good
anticancer activity against tested cancer cell lines. Compared to XL184 (IC50 = 6.6 µM against HeLa),
these four compounds all showed better anti-proliferation activity against HeLa with compound 11c
(IC50 = 0.9 µM against HeLa) exhibiting the highest activity. Similarly, they also showed better anti-
proliferation activity against Hep-G2 than XL184 (IC50 = 49.1 µM), and it is obvious that compound 13h
(IC50 = 1.7 µM) showed the highest activity, which is far better than XL184. In addition, compound 11i
(IC50 = 1.1 µM) and 13b (IC50 = 1.2 µM) had better results against MCF-7 than XL184 (IC50 = 2.6 µM). 2.1.3. Structure Activity Relationship Analysis
Preliminary structure–activity SAR study appeared that different head groups showed great
ll the synthesized compounds were evaluated for their anticancer activity in vitro against HeLa
n cervical carcinoma), Hep-G2 (human liver cancer) and MCF-7 (human breast cancer). The
were also summarized in Table1-3. It is clear that compound 11c, 11i, 13b, 13h all exhibited good
ncer activity against tested cancer cell lines. Compared to XL184 (IC50 = 6.6 µM against HeLa),
our compounds all showed better anti-proliferation activity against HeLa with compound 11c
0.9 µM against HeLa) exhibiting the highest activity. Similarly, they also showed better anti-
ration activity against Hep-G2 than XL184 (IC50 = 49.1 µM), and it is obvious that compound 13h
1.7 µM) showed the highest activity, which is far better than XL184. In addition, compound 11i
1.1 µM) and 13b (IC50 = 1.2 µM) had better results against MCF-7 than XL184 (IC50 = 2.6 µM). Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay All the synthesized compounds were evaluated for their anticancer activity in vitro against HeLa
uman cervical carcinoma), Hep-G2 (human liver cancer) and MCF-7 (human breast cancer). The
sults were also summarized in Table1-3. It is clear that compound 11c, 11i, 13b, 13h all exhibited good
nticancer activity against tested cancer cell lines. Compared to XL184 (IC50 = 6.6 µM against HeLa),
ese four compounds all showed better anti-proliferation activity against HeLa with compound 11c
C50 = 0.9 µM against HeLa) exhibiting the highest activity. Similarly, they also showed better anti-
oliferation activity against Hep-G2 than XL184 (IC50 = 49.1 µM), and it is obvious that compound 13h
C50 = 1.7 µM) showed the highest activity, which is far better than XL184. In addition, compound 11i
C50 = 1.1 µM) and 13b (IC50 = 1.2 µM) had better results against MCF-7 than XL184 (IC50 = 2.6 µM). 1.3. Structure Activity Relationship Analysis
Preliminary structure–activity SAR study appeared that different head groups showed great
All the synthesized compounds were evaluated for their anticancer activity in vitro against HeLa
uman cervical carcinoma), Hep-G2 (human liver cancer) and MCF-7 (human breast cancer). The
sults were also summarized in Table1-3. It is clear that compound 11c, 11i, 13b, 13h all exhibited good
nticancer activity against tested cancer cell lines. Compared to XL184 (IC50 = 6.6 µM against HeLa),
ese four compounds all showed better anti-proliferation activity against HeLa with compound 11c
C50 = 0.9 µM against HeLa) exhibiting the highest activity. Similarly, they also showed better anti-
oliferation activity against Hep-G2 than XL184 (IC50 = 49.1 µM), and it is obvious that compound 13h
C50 = 1.7 µM) showed the highest activity, which is far better than XL184. In addition, compound 11i
C50 = 1.1 µM) and 13b (IC50 = 1.2 µM) had better results against MCF-7 than XL184 (IC50 = 2.6 µM). 1.3. Structure Activity Relationship Analysis
All the synthesized compounds were evaluated for their anticancer activity in vitro against
HeLa (human cervical carcinoma), Hep-G2 (human liver cancer) and MCF-7 (human breast cancer). The results were also summarized in Tables 1–3. It is clear that compound 11c, 11i, 13b, 13h all
exhibited good anticancer activity against tested cancer cell lines. Compared to XL184 (IC50 = 6.6 µM
against HeLa), these four compounds all showed better anti-proliferation activity against HeLa with
compound 11c (IC50 = 0.9 µM against HeLa) exhibiting the highest activity. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Compound
A
R
Enzymatic Inhibition (IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
11a
3,4-diOMe
0.52 ± 0.05
14.1 ± 0.16
>100
3.4 ± 0.21
11b
4-Cl
0.12 ± 0.03
1.8 ± 0.06
9.2 ± 0.17
7.8 ± 0.14
11c
4-F
0.08 ± 0.04
0.9 ± 0.12
10.1 ± 0.20
5.2 ± 0.36
11d
3,4-diOMe
>10
>100
>100
71.2 ± 0.34
11e
4-Cl
7.4 ± 0.07
26.4 ± 0.20
>100
34.5 ± 0.36
11f
4-F
5.3 ± 0.05
27.8 ± 0.34
>100
42.1 ± 0.27
11g
3,4-diOMe
1.4 ± 0.10
4.4 ± 0.26
>100
17.3 ± 0.38
11h
4-Cl
0.25 ± 0.08
10.7 ± 0.18
61.9 ± 0.23
22.9 ± 0.22
11i
4-F
0.05 ± 0.02
1.5 ± 0.11
23.2 ± 0.36
1.1 ± 0.31
XL184
0.03 ± 0.02
2.6 ± 0.07
49.1 ± 0.25
6.6 ± 0.25
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. 3,4-diOMe
1.4 ˘ 0.10
4.4 ˘ 0.26
>100
17.3 ˘ 0.38
11h
4-Cl
0.25 ˘ 0.08
10.7 ˘ 0.18
61.9 ˘ 0.23
22.9 ˘ 0.22
11i
4-F
0.05 ˘ 0.02
1.5 ˘ 0.11
23.2 ˘ 0.36
1.1 ˘ 0.31
XL184
0.03 ˘ 0.02
2.6 ˘ 0.07
49.1 ˘ 0.25
6.6 ˘ 0.25
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were incubated
for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations IC50, µM)
,
IC50, µM) a,
(IC
M) A
A 6 of 16 Molecules 2016, 21, 612 Table 3. Enzymatic and cellular results for compound 13a–c. Table 3. Enzymatic and cellular results for compound 13a–c. Table 3. Enzymatic and cellular results for compound 13a–c. Table 3. Enzymatic and cellular results for compound 13a–c. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Compound
A
R
Enzymatic Inhibition
(IC50, µM) a,b
Proliferative Inhibition
(IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
13a
7.1 ± 0.18
>100
>100
71.8 ± 0.31
13b
0.02 ± 0.09
4.7 ± 0.11
12.8 ± 0.28
1.2 ± 0.21
13c
1.2 ± 0.06
50.1 ± 0.25
49.1 ± 0.30
>100
13d
>10
>100
>100
91.2 ± 0.26
13e
>10
66.7 ± 0.36
>100
>100
13f
>10
87.8 ± 0.13
>100
>100
13g
9.4 ± 0.6
>100
>100
27.7 ± 0.35
13h
0.05 ± 0.01
4.6 ± 0.03
1.7 ± 0.04
2.8 ± 0.23
13i
0.25 ± 0.08
4.2 ± 0.31
43.2 ± 0.26
21.7 ± 0.16
XL184
0.03 ± 0.02
6.6 ± 0.11
49.1 ± 0.13
2.6 ± 0.03
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. 2.1.2. Anti-proliferation against Tumor Cells Assay
Compound
A
R
Enzymatic Inhibition
(IC50, µM) a,b
Proliferative Inhibition (IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
13a
olecules 2016, 21, 612
6 of 16
Table 3. Enzymatic and cellular results for compound 13a–c. Compound
A
R
Enzymatic Inhibition
(IC50, µM) a,b
Proliferative Inhibition
(IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
13a
7.1 ± 0.18
>100
>100
71.8 ± 0.31
13b
0.02 ± 0.09
4.7 ± 0.11
12.8 ± 0.28
1.2 ± 0.21
13c
1.2 ± 0.06
50.1 ± 0.25
49.1 ± 0.30
>100
13d
>10
>100
>100
91.2 ± 0.26
13e
>10
66.7 ± 0.36
>100
>100
13f
>10
87.8 ± 0.13
>100
>100
13g
9.4 ± 0.6
>100
>100
27.7 ± 0.35
13h
0.05 ± 0.01
4.6 ± 0.03
1.7 ± 0.04
2.8 ± 0.23
13i
0.25 ± 0.08
4.2 ± 0.31
43.2 ± 0.26
21.7 ± 0.16
XL184
0.03 ± 0.02
6.6 ± 0.11
49.1 ± 0.13
2.6 ± 0.03
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. 1.2. Anti-proliferation against Tumor Cells Assay
All the synthesized compounds were evaluated for their anticancer activity in vitro against HeLa
uman cervical carcinoma), Hep-G2 (human liver cancer) and MCF-7 (human breast cancer). The
Table 3. Enzymatic and cellular results for compound 13a–c. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay ompound
A
R
Enzymatic Inhibition
(IC50, µM) a,b
Proliferative Inhibition
(IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
13a
7.1 ± 0.18
>100
>100
71.8 ± 0.31
13b
0.02 ± 0.09
4.7 ± 0.11
12.8 ± 0.28
1.2 ± 0.21
13c
1.2 ± 0.06
50.1 ± 0.25
49.1 ± 0.30
>100
13d
>10
>100
>100
91.2 ± 0.26
13e
>10
66.7 ± 0.36
>100
>100
13f
>10
87.8 ± 0.13
>100
>100
13g
9.4 ± 0.6
>100
>100
27.7 ± 0.35
13h
0.05 ± 0.01
4.6 ± 0.03
1.7 ± 0.04
2.8 ± 0.23
13i
0.25 ± 0.08
4.2 ± 0.31
43.2 ± 0.26
21.7 ± 0.16
XL184
0.03 ± 0.02
6.6 ± 0.11
49.1 ± 0.13
2.6 ± 0.03
Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
cubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
dependent determinations. Anti proliferation against Tumor Cells Assay
7.1 ˘ 0.18
>100
>100
71.8 ˘ 0.31
13b
lecules 2016, 21, 612
6 of 16
Table 3. Enzymatic and cellular results for compound 13a–c. Compound
A
R
Enzymatic Inhibition
(IC50, µM) a,b
Proliferative Inhibition
(IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
13a
7.1 ± 0.18
>100
>100
71.8 ± 0.31
13b
0.02 ± 0.09
4.7 ± 0.11
12.8 ± 0.28
1.2 ± 0.21
13c
1.2 ± 0.06
50.1 ± 0.25
49.1 ± 0.30
>100
13d
>10
>100
>100
91.2 ± 0.26
13e
>10
66.7 ± 0.36
>100
>100
13f
>10
87.8 ± 0.13
>100
>100
13g
9.4 ± 0.6
>100
>100
27.7 ± 0.35
13h
0.05 ± 0.01
4.6 ± 0.03
1.7 ± 0.04
2.8 ± 0.23
13i
0.25 ± 0.08
4.2 ± 0.31
43.2 ± 0.26
21.7 ± 0.16
XL184
0.03 ± 0.02
6.6 ± 0.11
49.1 ± 0.13
2.6 ± 0.03
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. 1.2. Anti-proliferation against Tumor Cells Assay
All the synthesized compounds were evaluated for their anticancer activity in vitro against HeLa
0.02 ˘ 0.09
4.7 ˘ 0.11
12.8 ˘ 0.28
1.2 ˘ 0.21
13c
lecules 2016, 21, 612
6 of 16
Table 3. Enzymatic and cellular results for compound 13a–c. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Compound
A
R
Enzymatic Inhibition
(IC50, µM) a,b
Proliferative Inhibition
(IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
13a
7.1 ± 0.18
>100
>100
71.8 ± 0.31
13b
0.02 ± 0.09
4.7 ± 0.11
12.8 ± 0.28
1.2 ± 0.21
13c
1.2 ± 0.06
50.1 ± 0.25
49.1 ± 0.30
>100
13d
>10
>100
>100
91.2 ± 0.26
13e
>10
66.7 ± 0.36
>100
>100
13f
>10
87.8 ± 0.13
>100
>100
13g
9.4 ± 0.6
>100
>100
27.7 ± 0.35
13h
0.05 ± 0.01
4.6 ± 0.03
1.7 ± 0.04
2.8 ± 0.23
13i
0.25 ± 0.08
4.2 ± 0.31
43.2 ± 0.26
21.7 ± 0.16
XL184
0.03 ± 0.02
6.6 ± 0.11
49.1 ± 0.13
2.6 ± 0.03
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. 1.2. Anti-proliferation against Tumor Cells Assay
All the synthesized compounds were evaluated for their anticancer activity in vitro against HeLa
1.2 ˘ 0.06
50.1 ˘ 0.25
49.1 ˘ 0.30
>100
13d
lecules 2016, 21, 612
6 of 16
Table 3. Enzymatic and cellular results for compound 13a–c. Compound
A
R
Enzymatic Inhibition
(IC50, µM) a,b
Proliferative Inhibition
(IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
13a
7.1 ± 0.18
>100
>100
71.8 ± 0.31
13b
0.02 ± 0.09
4.7 ± 0.11
12.8 ± 0.28
1.2 ± 0.21
13c
1.2 ± 0.06
50.1 ± 0.25
49.1 ± 0.30
>100
13d
>10
>100
>100
91.2 ± 0.26
13e
>10
66.7 ± 0.36
>100
>100
13f
>10
87.8 ± 0.13
>100
>100
13g
9.4 ± 0.6
>100
>100
27.7 ± 0.35
13h
0.05 ± 0.01
4.6 ± 0.03
1.7 ± 0.04
2.8 ± 0.23
13i
0.25 ± 0.08
4.2 ± 0.31
43.2 ± 0.26
21.7 ± 0.16
XL184
0.03 ± 0.02
6.6 ± 0.11
49.1 ± 0.13
2.6 ± 0.03
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. 1.2. Anti-proliferation against Tumor Cells Assay
All h
h
i
d
d
l
d f
h i
i
i i
i
i
i
H L
s 2016, 21, 612
6 of 16
Table 3. Enzymatic and cellular results for compound 13a–c. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay ompound
A
R
Enzymatic Inhibition
(IC50, µM) a,b
Proliferative Inhibition
(IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
13a
7.1 ± 0.18
>100
>100
71.8 ± 0.31
13b
0.02 ± 0.09
4.7 ± 0.11
12.8 ± 0.28
1.2 ± 0.21
13c
1.2 ± 0.06
50.1 ± 0.25
49.1 ± 0.30
>100
13d
>10
>100
>100
91.2 ± 0.26
13e
>10
66.7 ± 0.36
>100
>100
13f
>10
87.8 ± 0.13
>100
>100
13g
9.4 ± 0.6
>100
>100
27.7 ± 0.35
13h
0.05 ± 0.01
4.6 ± 0.03
1.7 ± 0.04
2.8 ± 0.23
13i
0.25 ± 0.08
4.2 ± 0.31
43.2 ± 0.26
21.7 ± 0.16
XL184
0.03 ± 0.02
6.6 ± 0.11
49.1 ± 0.13
2.6 ± 0.03
Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
cubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
dependent determinations. >10
>100
>100
91.2 ˘ 0.26
13e
lecules 2016, 21, 612
6 of 16
Table 3. Enzymatic and cellular results for compound 13a–c. Compound
A
R
Enzymatic Inhibition
(IC50, µM) a,b
Proliferative Inhibition
(IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
13a
7.1 ± 0.18
>100
>100
71.8 ± 0.31
13b
0.02 ± 0.09
4.7 ± 0.11
12.8 ± 0.28
1.2 ± 0.21
13c
1.2 ± 0.06
50.1 ± 0.25
49.1 ± 0.30
>100
13d
>10
>100
>100
91.2 ± 0.26
13e
>10
66.7 ± 0.36
>100
>100
13f
>10
87.8 ± 0.13
>100
>100
13g
9.4 ± 0.6
>100
>100
27.7 ± 0.35
13h
0.05 ± 0.01
4.6 ± 0.03
1.7 ± 0.04
2.8 ± 0.23
13i
0.25 ± 0.08
4.2 ± 0.31
43.2 ± 0.26
21.7 ± 0.16
XL184
0.03 ± 0.02
6.6 ± 0.11
49.1 ± 0.13
2.6 ± 0.03
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. .2. Anti-proliferation against Tumor Cells Assay
>10
66.7 ˘ 0.36
>100
>100
13f
lecules 2016, 21, 612
6 of 16
Table 3. Enzymatic and cellular results for compound 13a–c. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Compound
A
R
Enzymatic Inhibition
(IC50, µM) a,b
Proliferative Inhibition
(IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
13a
7.1 ± 0.18
>100
>100
71.8 ± 0.31
13b
0.02 ± 0.09
4.7 ± 0.11
12.8 ± 0.28
1.2 ± 0.21
13c
1.2 ± 0.06
50.1 ± 0.25
49.1 ± 0.30
>100
13d
>10
>100
>100
91.2 ± 0.26
13e
>10
66.7 ± 0.36
>100
>100
13f
>10
87.8 ± 0.13
>100
>100
13g
9.4 ± 0.6
>100
>100
27.7 ± 0.35
13h
0.05 ± 0.01
4.6 ± 0.03
1.7 ± 0.04
2.8 ± 0.23
13i
0.25 ± 0.08
4.2 ± 0.31
43.2 ± 0.26
21.7 ± 0.16
XL184
0.03 ± 0.02
6.6 ± 0.11
49.1 ± 0.13
2.6 ± 0.03
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. 1.2. Anti-proliferation against Tumor Cells Assay
>10
87.8 ˘ 0.13
>100
>100
13g
Molecules 2016, 21, 612
6 of 16
Table 3. Enzymatic and cellular results for compound 13a–c. Compound
A
R
Enzymatic Inhibition
(IC50, µM) a,b
Proliferative Inhibition
(IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
13a
7.1 ± 0.18
>100
>100
71.8 ± 0.31
13b
0.02 ± 0.09
4.7 ± 0.11
12.8 ± 0.28
1.2 ± 0.21
13c
1.2 ± 0.06
50.1 ± 0.25
49.1 ± 0.30
>100
13d
>10
>100
>100
91.2 ± 0.26
13e
>10
66.7 ± 0.36
>100
>100
13f
>10
87.8 ± 0.13
>100
>100
13g
9.4 ± 0.6
>100
>100
27.7 ± 0.35
13h
0.05 ± 0.01
4.6 ± 0.03
1.7 ± 0.04
2.8 ± 0.23
13i
0.25 ± 0.08
4.2 ± 0.31
43.2 ± 0.26
21.7 ± 0.16
XL184
0.03 ± 0.02
6.6 ± 0.11
49.1 ± 0.13
2.6 ± 0.03
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. 2 1 2 A ti
lif
ti
i
t T
C ll A
s 2016, 21, 612
6 of 16
Table 3. Enzymatic and cellular results for compound 13a–c. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay ompound
A
R
Enzymatic Inhibition
(IC50, µM) a,b
Proliferative Inhibition
(IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
13a
7.1 ± 0.18
>100
>100
71.8 ± 0.31
13b
0.02 ± 0.09
4.7 ± 0.11
12.8 ± 0.28
1.2 ± 0.21
13c
1.2 ± 0.06
50.1 ± 0.25
49.1 ± 0.30
>100
13d
>10
>100
>100
91.2 ± 0.26
13e
>10
66.7 ± 0.36
>100
>100
13f
>10
87.8 ± 0.13
>100
>100
13g
9.4 ± 0.6
>100
>100
27.7 ± 0.35
13h
0.05 ± 0.01
4.6 ± 0.03
1.7 ± 0.04
2.8 ± 0.23
13i
0.25 ± 0.08
4.2 ± 0.31
43.2 ± 0.26
21.7 ± 0.16
XL184
0.03 ± 0.02
6.6 ± 0.11
49.1 ± 0.13
2.6 ± 0.03
Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
cubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
dependent determinations. 9.4 ˘ 0.6
>100
>100
27.7 ˘ 0.35
13h
lecules 2016, 21, 612
6 of 16
Table 3. Enzymatic and cellular results for compound 13a–c. Compound
A
R
Enzymatic Inhibition
(IC50, µM) a,b
Proliferative Inhibition
(IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
13a
7.1 ± 0.18
>100
>100
71.8 ± 0.31
13b
0.02 ± 0.09
4.7 ± 0.11
12.8 ± 0.28
1.2 ± 0.21
13c
1.2 ± 0.06
50.1 ± 0.25
49.1 ± 0.30
>100
13d
>10
>100
>100
91.2 ± 0.26
13e
>10
66.7 ± 0.36
>100
>100
13f
>10
87.8 ± 0.13
>100
>100
13g
9.4 ± 0.6
>100
>100
27.7 ± 0.35
13h
0.05 ± 0.01
4.6 ± 0.03
1.7 ± 0.04
2.8 ± 0.23
13i
0.25 ± 0.08
4.2 ± 0.31
43.2 ± 0.26
21.7 ± 0.16
XL184
0.03 ± 0.02
6.6 ± 0.11
49.1 ± 0.13
2.6 ± 0.03
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. 0.05 ˘ 0.01
4.6 ˘ 0.03
1.7 ˘ 0.04
2.8 ˘ 0.23
13i
lecules 2016, 21, 612
6 of 16
Table 3. Enzymatic and cellular results for compound 13a–c. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Compound
A
R
Enzymatic Inhibition
(IC50, µM) a,b
Proliferative Inhibition
(IC50, µM) b
c-MET
HeLa
Hep-G2
MCF-7
13a
7.1 ± 0.18
>100
>100
71.8 ± 0.31
13b
0.02 ± 0.09
4.7 ± 0.11
12.8 ± 0.28
1.2 ± 0.21
13c
1.2 ± 0.06
50.1 ± 0.25
49.1 ± 0.30
>100
13d
>10
>100
>100
91.2 ± 0.26
13e
>10
66.7 ± 0.36
>100
>100
13f
>10
87.8 ± 0.13
>100
>100
13g
9.4 ± 0.6
>100
>100
27.7 ± 0.35
13h
0.05 ± 0.01
4.6 ± 0.03
1.7 ± 0.04
2.8 ± 0.23
13i
0.25 ± 0.08
4.2 ± 0.31
43.2 ± 0.26
21.7 ± 0.16
XL184
0.03 ± 0.02
6.6 ± 0.11
49.1 ± 0.13
2.6 ± 0.03
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were
incubated for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. 0.25 ˘ 0.08
4.2 ˘ 0.31
43.2 ˘ 0.26
21.7 ˘ 0.16
XL184
0.03 ˘ 0.02
6.6 ˘ 0.11
49.1 ˘ 0.13
2.6 ˘ 0.03
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were incubated
for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. c and cellular results for compound 13a–c. and cellular results for compound 13a–
c and cellular results for compound 13a–c. c and cellular results for compound 13a–c. c and cellular results for compound 13a–c. and cellular results for compound 13a–
c and cellular results for compound 13a–c. c and cellular results for compound 13a–c. tic and cellular results for compound 13a–c
and cellular results for compound 13a–
c and cellular results for compound 13a–c. c and cellular results for compound 13a–c. A
A
A
A
A
A
A R
R
R
R
R
R d
e ti All the synthesized compounds were evaluated
(human cervical carcinoma), Hep-G2 (human liver
esults were also summarized in Table1-3. It is clear that compo
nticancer activity against tested cancer cell lines. Compared
All the synthesized compounds were evaluated for thei
),
p
(
)
sults were also summarized in Table1-3. It is clear that compo
ti
ti it
i
t t
t d
ll li
C
d
human cervical carcinoma), Hep-G2 (human liver cancer) a
sults were also summarized in Table1-3. 2.2.3. Structure Activity Relationship Analysis Preliminary structure–activity SAR study appeared that different head groups showed
great differences in cell and enzymatic activity. Among them, quinazolines (compound 9a,
11a–c, 13a–c), and tetrahydro-pyridothienopyrimidines (compound 9c, 11g–i, 13g–i) head groups
provided much better activity than pyridines (compound 9b, 11d–f, 13d–f). For the side chain,
N-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)-3-methoxybenzamide derivatives had lowest activities
while malonamide derivatives had lowest activities. For compound 13a–i, different R groups provided
different activity, when R group was cyclopropane (compound 13b and 13h), the best activity
was observed. Among these compounds,
All the synthesized co
l
i
d i
2.2.1. Kinase Inhibitory Assay Similarly, they also showed
better anti-proliferation activity against Hep-G2 than XL184 (IC50 = 49.1 µM), and it is obvious that
compound 13h (IC50 = 1.7 µM) showed the highest activity, which is far better than XL184. In addition,
compound 11i (IC50 = 1.1 µM) and 13b (IC50 = 1.2 µM) had better results against MCF-7 than XL184
(IC50 = 2.6 µM). 7 of 16 Molecules 2016, 21, 612 7 of 16 2.2.4. Enzymatic Selectivity Assay Compound 13b and 13h were further assayed with enzymatic activities against VEGFR-2, c-Kit,
PDGFR-b and EGFR to test their kinase selectivity [31]. The results were summarized in Table 4. Compound 13b demonstrated extraordinary selectivity against c-Kit (215 fold), PDGFR-b (>500 fold)
and EGFR (>500 fold), but it showed some inhibitory activity against VEGFR-2 (IC50 = 0.1 µM). Compound 13h exhibited extraordinary selectivity against c-Kit (104 fold), PDGFR-b (144 fold) and
EGFR (>200 fold), and it also showed some inhibitory activity against VEGFR-2 (IC50 = 0.25 µM). Table 4. Kinase selectivity of compounds 13b and 13h. Compound
Enzyme, IC50, µM a,b
VEGFR-2
c-Kit
PDGFR-b
EGFR
13b
0.1 ˘ 0.02
4.3 ˘ 0.11
>10
>10
13h
0.25 ˘ 0.02
5.2 ˘ 0.09
7.2 ˘ 0.10
>10
a Prior to the initiation of the kinase reaction with ATP (10 µM), compounds and enzyme were incubated
for 30 min. b IC50 values obtained with viability assays. Each value is representative of three
independent determinations. Table 4. Kinase selectivity of compounds 13b and 13h. 2.3. Molecular Docking and Molecular Dynamics Simulation Study 2.3. Molecular Docking and Molecular Dynamics Simulation Study University of Groningen, Groningen, The Netherlands), the root-mean-square deviation (RMSD)
fluctuations is a principal criterion to evaluate the stability of the protein-ligand system. very well. In addition, they can form hydrophobic interaction in the ATP-binding sites of c-MET and
VEGFR-2. Compounds formed hydrophobic interaction with residues ILE-1084, ALA-1108, LEU-1157,
MET-1160 and ALA-1221 of c-MET. Figure 2. Binding poses of compound 11c (A) and 11i (B) with c-MET. Compound 11c and 11i were
displayed by blue and red, respectively. The important residues and their critical interactions (dash lines)
with compounds in the binding site were depicted, hydrogen bond, hydrophobic interactions were
colored green and carnation, respectively. Figure 2. Binding poses of compound 11c (A) and 11i (B) with c-MET. Compound 11c and 11i were
displayed by blue and red, respectively. The important residues and their critical interactions (dash
lines) with compounds in the binding site were depicted, hydrogen bond, hydrophobic interactions
were colored green and carnation, respectively. lecules 2016, 21, 612
8 o
LA-866, LYS-868, LEU-889, VAL-899, VAL-916, LEU-1035 to VEGFR2. Binding energy of compou
b is much better than compound 11c and 11i, the dock interaction energy of compound 13b w
MET, compound 11c and 11i with c-MET is −70.33 kJ/mol, −44.8 kJ/mol and −47.6 kJ/mol, respective
he nice binding model of compound 13b and 13h with c-MET and VEGFR-2 was consistent w
nase assay data, which indicates that compounds were potent dual c-MET/VEGFR-2 inhibitors. olecules 2016, 21, 612
LA 866 LYS 868 LEU 889 VAL 899 VAL 916 LEU 8 o
EU 1035
VEGFR2 Bi di
f Figure 2. Binding poses of compound 11c (A) and 11i (B) with c-MET. Compound 11c and 11i were
displayed by blue and red, respectively. The important residues and their critical interactions (dash lines)
with compounds in the binding site were depicted, hydrogen bond, hydrophobic interactions were
colored green and carnation, respectively. Figure 2. Binding poses of compound 11c (A) and 11i (B) with c-MET. Compound 11c and 11i were
displayed by blue and red, respectively. The important residues and their critical interactions (dash
lines) with compounds in the binding site were depicted, hydrogen bond, hydrophobic interactions
were colored green and carnation, respectively. A 866, LYS 868, LEU 889, VAL 899, VAL 916, LEU 1035 to VEGFR2. 2.3. Molecular Docking and Molecular Dynamics Simulation Study Binding energy of compou
b is much better than compound 11c and 11i, the dock interaction energy of compound 13b w
MET, compound 11c and 11i with c-MET is −70.33 kJ/mol, −44.8 kJ/mol and −47.6 kJ/mol, respectiv
e nice binding model of compound 13b and 13h with c-MET and VEGFR-2 was consistent w
ase assay data, which indicates that compounds were potent dual c-MET/VEGFR-2 inhibitors For the better active compounds 13b and 13h, We demonstrated in Figure 3 the compound 1
d 13h docking into the binding site of c-MET kinase (PDB: 4MXC) and VEGFR-2 kinase (PDB: 4AS
r compound 13b, there are two hydrogen bonds formed by residue MET-1160 and ASP-12
ditional, compound 13b could form hydrophobic interactions with ILE-1084, ALA-1108, MET-1
EU-1157, ALA-1221 to c-MET. Compound 13b can form three hydrogen bonds by residue GLU-8
YS-919, and ASP-1046, there also exist hydrophobic interaction with residue LEU-840, VAL-8
Figure 3. Binding poses of compound 13b (A,C) and 13h (B,D) with c-MET and VEGFR2. Compound
13b and 13h were displayed by blue and red, respectively. The important residues and their critical
interactions (dash lines) with compounds in the binding site were depicted, hydrogen bond,
hydrophobic interactions were colored green and carnation, respectively. Figure 3. Binding poses of compound 13b (A,C) and 13h (B,D) with c-MET and VEGFR2. Compound
13b and 13h were displayed by blue and red, respectively. The important residues and their
critical interactions (dash lines) with compounds in the binding site were depicted, hydrogen bond,
hydrophobic interactions were colored green and carnation, respectively. For the better active compounds 13b and 13h, W
13h docking into the binding site of c-MET kinase
compound 13b, there are two hydrogen bonds
itional, compound 13b could form hydrophobic in
U-1157, ALA-1221 to c-MET. Compound 13b can fo
S-919, and ASP-1046, there also exist hydrophob We demonstrated in Figure 3 the compound
(PDB: 4MXC) and VEGFR-2 kinase (PDB: 4A
s formed by residue MET-1160 and ASP-12
nteractions with ILE-1084, ALA-1108, MET-1
orm three hydrogen bonds by residue GLU-8
bic interaction with residue LEU-840, VAL-8 h, W
ase Figure 3. Binding poses of compound 13b (A,C) and 13h (B,D) with c-MET and VEGFR2. Compound
13b and 13h were displayed by blue and red, respectively. The important residues and their critical
interactions (dash lines) with compounds in the binding site were depicted, hydrogen bond,
hydrophobic interactions were colored green and carnation, respectively. Figure 3. 2.3. Molecular Docking and Molecular Dynamics Simulation Study In order to better understand the interaction between compounds and kinases, molecular
docking studies on the potent compound 11c and 11i were performed using the Discovery Studio
3.1/CDOCKER protocol [32]. In Figure 2, we showed that compound 11c and 11i could bind to c-MET kinase (PDB: 4MXC)
very well. In addition, they can form hydrophobic interaction in the ATP-binding sites of c-MET and
VEGFR-2. Compounds formed hydrophobic interaction with residues ILE-1084, ALA-1108, LEU-1157,
MET-1160 and ALA-1221 of c-MET. In Figure 2, we showed that compound 11c and 11i could bind to c-MET kinase (PDB: 4MXC)
very well. In addition, they can form hydrophobic interaction in the ATP-binding sites of c-MET and
VEGFR-2. Compounds formed hydrophobic interaction with residues ILE-1084, ALA-1108, LEU-1157,
MET-1160 and ALA-1221 of c-MET. For the better active compounds 13b and 13h, We demonstrated in Figure 3 the compound 13b and
13h docking into the binding site of c-MET kinase (PDB: 4MXC) and VEGFR-2 kinase (PDB: 4ASE). For
compound 13b, there are two hydrogen bonds formed by residue MET-1160 and ASP-1222, additional,
compound 13b could form hydrophobic interactions with ILE-1084, ALA-1108, MET-1131, LEU-1157,
ALA-1221 to c-MET. Compound 13b can form three hydrogen bonds by residue GLU-885, CYS-919,
and ASP-1046, there also exist hydrophobic interaction with residue LEU-840, VAL-848, ALA-866,
LYS-868, LEU-889, VAL-899, VAL-916, LEU-1035 to VEGFR2. Binding energy of compound 13b is
much better than compound 11c and 11i, the dock interaction energy of compound 13b with c-MET,
compound 11c and 11i with c-MET is ´70.33 kJ/mol, ´44.8 kJ/mol and ´47.6 kJ/mol, respectively. The nice binding model of compound 13b and 13h with c-MET and VEGFR-2 was consistent with
kinase assay data, which indicates that compounds were potent dual c-MET/VEGFR-2 inhibitors. y
p
p
To further evaluate the binding affinity between compound 13b and c-MET/VEGFR-2. The
molecular dynamics (MD) simulations were performed by using GROMACS package (version 4.5, 8 of 16
4MXC) 8 of 16
4MXC) Molecules 2016, 21, 612
3.1/CDOCKER prot
I
Fi
2 University of Groningen, Groningen, The Netherlands), the root-mean-square deviation (RMSD)
fluctuations is a principal criterion to evaluate the stability of the protein-ligand system. very well. In addition, they can form hydrophobic interaction in the ATP-binding sites of c-MET and
VEGFR-2. Compounds formed hydrophobic interaction with residues ILE-1084, ALA-1108, LEU-1157,
MET-1160 and ALA-1221 of c-MET. 2.3. Molecular Docking and Molecular Dynamics Simulation Study Binding poses of compound 13b (A,C) and 13h (B,D) with c-MET and VEGFR2. Compound
13b and 13h were displayed by blue and red, respectively. The important residues and their
critical interactions (dash lines) with compounds in the binding site were depicted, hydrogen bond,
hydrophobic interactions were colored green and carnation, respectively. Figure 3. Binding poses of compound 13b (A,C) and 13h (B,D) with c-MET and VEGFR2. Compound
13b and 13h were displayed by blue and red, respectively. The important residues and their critical
interactions (dash lines) with compounds in the binding site were depicted, hydrogen bond,
hydrophobic interactions were colored green and carnation, respectively. Figure 3. Binding poses of compound 13b (A,C) and 13h (B,D) with c-MET and VEGFR2. Compound
13b and 13h were displayed by blue and red, respectively. The important residues and their
critical interactions (dash lines) with compounds in the binding site were depicted, hydrogen bond,
hydrophobic interactions were colored green and carnation, respectively. To further evaluate the binding affinity between compound 13b and c MET/VEGFR 2. The
molecular dynamics (MD) simulations were performed by using GROMACS package (version 4.5,
University of Groningen, Groningen, The Netherlands), the root-mean-square deviation (RMSD)
fluctuations is a principal criterion to evaluate the stability of the protein-ligand system. As shown in Figure 4, although the RMSD values for compound 13b fluctuated in a narrow
range around 2700 ps, they remained stable for most of the simulation in c-MET complex. RMSD of
compound 13b reach the equilibrium state after about 500 ps and kept stable among the rest of simulation
in VEGFR-2 complex, indicating a stabilities of the dynamics equilibriums. In general, the maximum
As shown in Figure 4, although the RMSD values for compound 13b fluctuated in a narrow
range around 2700 ps, they remained stable for most of the simulation in c-MET complex. RMSD
of compound 13b reach the equilibrium state after about 500 ps and kept stable among the rest of
simulation in VEGFR-2 complex, indicating a stabilities of the dynamics equilibriums. In general, the
maximum RMSD for each case was lower than 0.2 nm, suggesting that c-MET and VEGFR-2 complexes
are reliable and the low RMSD fluctuations of system observed indicated stable binding models of
compound 13b with c-MET and VEGFR-2, respectively. 9 of 16
13b Molecules 2016, 21, 612
and the low RMSD c ME
a d
EG
, espec i e y p
y
Figure 4. 2.3. Molecular Docking and Molecular Dynamics Simulation Study RMSD of compound 13b with c-MET (A) and VEGFR-2 (B), respectively. Figure 4. RMSD of compound 13b with c-MET (A) and VEGFR-2 (B), respectively. Figure 4. RMSD of compound 13b with c-MET (A) and VEGFR-2 (B), respectively. Figure 4. RMSD of compound 13b with c-MET (A) and VEGFR-2 (B), respectively. 3.1.2. General Procedure for the Preparation of {tert-Butyl,4-chloro-5,6-dihydropyrido(4’,3’:4,5)thieno
(2,3-d)pyrimidine-7(8H)-carboxylate} (Compound 7) The N-(tert-Butoxycarbonyl)-4-piperidone, NCCH2CO2Et and Et3N was mixed at room
temperature then stirring for 16 h, and then heated to 120 ˝C for 16 h in DMF with the formamidine
acetate. Then, the intermediate was reacted with POCl3 and DIPEA in toluene to obtain the
compound 7 (yield 89%). 3.1. Chemical Synthesis All reagents were purchased from commercial sources and used without further purification. Melting points are corrected. 1H-NMR spectra were determined on a Bruker Avance III 400 MHz
spectrometer (Bruker, Billerica, MA, USA) in CDCl3 or DMSO-d6 solution. J values were in Hz. Chemical shifts were expressed in ppm downfield from internal standard TMS. HRMS data were
obtained using Bruker micro TOF-Q instrument (Bruker) or TOF-MS instrument (Bruker). 3.1.1. General Procedure for the Preparation of 4-Chloro-6,7-dimethoxyquinazoline (Compound 5) 2-amino-dimethyl aminobenzoic acid 2.02 g (10 mmol) and acetic acid formamidine 2.10 g
(20 mmol) were added in 2-methoxy ethanol. The mixture was reflux in 125 ˝C. After evaporation of
solvent, the residue was added in the 10% ammonia solution, stirring, then after filtering, the solid
was washed by water to get the brown solid powder (yield 89%). 1.82 g (8.8 mmol) product was
dissolved in 25 mL SOCl2, and 30 drops of DMF was added. After heating 7 h to reflux, the solvent
was evaporated, then after filtering, the solid was washed by water to get the brown solid powder
4-chloro-6,7-dimethoxyquinazoline (yield 93%). 3.1.2. General Procedure for the Preparation of {tert-Butyl,4-chloro-5,6-dihydropyrido(4’,3’:4,5)thieno
(2,3-d)pyrimidine-7(8H)-carboxylate} (Compound 7) 3.1.4. General Procedure for the Preparation of 3-Methoxy-N-phenylbenzamide Derivatives
Compound 11a–i 138.6 mg (1 mmol) hydroxybenzoic acid in 5 mL THF was added three drops of DMF and reacted
with oxalyl chloride 0.5 mL in 0 ˝C. After 1.5 h reaction, the reaction liquid was dissolved in THF
and HCl, which was dissolved in THF solution of aniline derivatives. After 3 h, the reaction mixture
was evaporated; the crude product was purified by column chromatography to obtain compounds 10. Compounds 10 were further reacted with compounds 5, 6, 7 in the presence of K2CO3 to obtain the
final products 11a–i. Compound 11a: N-(3,4-dimethoxyphenyl)-3-((6,7-dimethoxyquinazolin-4-yl)oxy)benzamide. Yellow
solid, yield 86%, m.p. 220–222 ˝C. 1H-NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 7.90 (s, 1H), 7.81 (d,
J = 7.4 Hz, 2H), 7.60 (t, J = 8.2 Hz, 1H), 7.53 (s, 1H), 7.47 (t, 2H), 7.33 (s, 1H), 6.99 (dd, J = 8.6, 2.3 Hz, 1H),
6.84 (d, J = 8.6 Hz, 1H), 4.07 (d, J = 2.8 Hz, 6H), 3.90 (d, J = 5.2 Hz, 3H), 3.87 (s, 3H). 13C-NMR (100 MHz,
CDCl3) δ 166.79, 165.05, 154.75, 153.84, 153.57, 149.95, 148.67, 148.21, 138.69, 132.68, 129.19, 126.27,
122.57, 122.29, 118.41, 114.98, 113.43, 111.44, 108.20, 107.06, 56.83.HRMS (m/z): calcd. for 462.1660
([M + H]+), obsd.462.1665. calcd. for 484.11478 ([M + Na]+), obsd.484.1490. Compound 11b: N-(4-chlorophenyl)-3-((6,7-dimethoxyquinazolin-4-yl)oxy)benzamide. Faint yellow
solid, yield 89%, m.p. 203–205 ˝C. 1H-NMR (400 MHz, DMSO) δ 10.42 (s, 1H), 8.58 (s, 1H), 7.94 (s, 1H),
7.92 (s, 1H), 7.83 (s, 1H), 7.80 (s, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.60 (s, 1H), 7.58 (s, 1H), 7.41 (t, 3H), 4.00
(d, J = 5.4 Hz, 6H). 13C-NMR (100 MHz, DMSO) δ 165.19, 162.11, 155.85, 154.74, 153.57, 147.81, 137.54,
136.45, 130.37, 129.25, 128.67, 127.37, 122.88, 122.46, 121.79, 114.79, 110.88, 107.91, 57.24.HRMS (m/z):
calcd. for 420.1354 ([M + H]+), obsd.436.1059. calcd. for 458. 0878 ([M + Na]+), obsd.458.0883. Compound 11c: 3-((6,7-dimethoxyquinazolin-4-yl)oxy)-N-(4-fluorophenyl)benzamide. Yellow solid,
yield 86%, m.p. 178–180 ˝C. 1H-NMR (400 MHz, DMSO) δ 10.35 (s, 1H), 8.58 (s, 1H), 7.94 (s, 2H),
7.92 (d, J = 2.3 Hz, 2H), 7.79 (q, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.62–7.56 (m, 2H), 7.42 (s, 1H), 7.20
(t, J = 7.2 Hz, 2H), 4.00 (d, J = 5.2 Hz, 6H). 13C-NMR (100 MHz, DMSO) δ 167.33, 166.05, 162.07,
159.55, 154.65, 153.84, 153.12, 148.21, 138.69, 134.01, 129.03, 125.77, 122.62, 122.03, 115.11, 111.14,
108.30, 55.43.HRMS (m/z): calcd. for 420.1354 ([M + H]+), obsd.420.1356. calcd. 3.1.3. General Procedure for the Preparation of {N-(3-(tert-Butyl)-1-phenyl-1H-pyrazol-5-yl)-3-
((6,7-dimethoxyquinazolin-4-yl)oxy)benzamide} Derivatives Compound 9a–c 3.1.3. General Procedure for the Preparation of {N-(3-(tert-Butyl)-1-phenyl-1H-pyrazol-5-yl)-3-
((6,7-dimethoxyquinazolin-4-yl)oxy)benzamide} Derivatives Compound 9a–c Pivaloylacetonitrile 4.07 g (32 mmol) and Phenylhydrazine 3.51 g (32 mmol) were added to
40 mL ethanol and the mixture was heated to reflux for 15 h. After the solvent was evaporated,
[3-(tert-butyl)-1-phenyl-1H-pyrazol-5-amine] was obtained. 138.6 mg (1 mmol) hydroxybenzoic acid in
5 mL THF was added three drops of DMF and reacted with oxalyl chloride 0.5 mL in 0 ˝C. After 1.5 h
reaction, the reaction liquid was dissolved in THF and HCl, which was dissolved in THF solution of
3-(tert-butyl)-1-phenyl-1H-pyrazol-5-amine. After 3 h, the reaction mixture was evaporated; the crude
product was purified by column chromatography to obtain compound 8. Compound 8 was further
reacted with compounds 5, 6, 7 in the presence of K2CO3 to obtain the final products 9a–c. Compound
9a:
N-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)-3-((6,7-dimethoxyquinazolin-
4-yl)oxy)benzamide. White solid, yield 88%, m.p. 217–219 ˝C. 1H-NMR (400 MHz, DMSO) δ 10.40 10 of 16 10 of 16 Molecules 2016, 21, 612 (s, 1H), 8.57 (s, 1H), 7.83 (d, J = 7.3 Hz, 1H), 7.79 (s, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.60 (s, 1H), 7.57
(s, 1H), 7.54 (s, 2H), 7.52 (s, 2H), 7.44 (t, J = 7.8 Hz, 2H), 7.40 (s, 1H), 7.31 (t, J = 7.3 Hz, 1H), 6.40 (s, 1H),
4.01(s, 3H), 3.97(s, 3H), 1.31 (s, 9H). HRMS (m/z): calcd. for 524.2292 ([M + H]+), obsd. 524.2294. Compound 9b: 4-{3-((3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)carbamoylphenoxy}-N-methylpicolinamide:
yellow solid, yield 71%. 1H-NMR (400 MHz, CDCl3) δ 8.78 (d, 5.6 Hz, 1H), 8.35–7.95 (m, 2H), 7.94–7.67
(m, 5H), 7.62–7.42 (m, 2H), 7.39–7.05 (m, 2H), 6.74 (s, 1H), 2.85 (s, 3H), 1.29 (s, 9H). 13C-NMR (100 MHz,
CDCl3 ) δ 169.26, 167.39, 166.32, 158.34, 153.42, 151.87, 148.57, 146.98, 139.89, 135.40, 129.43, 128.96,
128.93, 125.18, 123.62, 118.50, 113.11, 112.47, 95.64, 34.41, 28.32, 26.37.HRMS (m/z): calcd. for 470.2192
([M + H]+), obsd. 470.2181. Compound 9c:
tert-butyl 4-{3-((3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)carbamoyl)phenoxy}-5,
6-dihydropyrido(4’,3’:4,5)thieno(2,3-d)pyrimidine-7(8H)-carboxylate, yellow solid, yield 71%. 1H-NMR
(400 MHz, CDCl3) δ 8.01–7.67 (m, 5H), 7.67–7.42 (m, 2H), 7.43–7.02 (m, 2H), 4.65–4.55 (m, 2H), 3.70–3.40
(m, 2H), 3.20–3.15 (m, 2H), 1.42 (s, 9H), 1.29 (s, 9H). 13C-NMR (100 MHz, CDCl3) δ 170.28, 169.27,
162.31, 157.47, 154.66, 153.42, 151.87, 148.98, 139.89, 138.69, 129.19, 128.96, 128.93, 126.61, 126.27, 125.18,
122.57, 122.30, 119.22, 117.01, 95.64, 81.20, 43.81, 42.71, 34.41, 28.32, 23.62. HRMS (m/z): calcd. for
625.2597 ([M + H]+), obsd. 625.2611. 3.1.4. General Procedure for the Preparation of 3-Methoxy-N-phenylbenzamide Derivatives
Compound 11a–i for 442.1174
([M + Na]+), obsd.442.1185. Compound 11d: 4-{3-((3,4-dimethoxyphenyl)carbamoyl)phenoxy}-N-methylpicolinamide: yellow
solid, yield 82%, 1H-NMR (400 MHz, CDCl3 ) δ 8.78 (d, J = 5.6 Hz, 1H), 8.22–7.89 (m, 2H), 7.67–7.37
(m, 2H), 7.34 (s, 1H), 7.27–7.04 (m, 3H), 6.91 (d, J = 15.4 Hz, 1H), 6.48 (s, 1H), 3.83 (s, 3H), 3.76 (s, 3H), 11 of 16 11 of 16 Molecules 2016, 21, 612 2.85 (s, 3H). 13C-NMR (100 MHz, CDCl3) δ 168.12, 167.12, 164.25, 157.14, 149.75, 148.47, 147.57, 146.98,
135.32, 132.18, 129.23, 123.62, 118.50, 117.10, 113.43, 113.11, 112.47, 107.06, 56.83, 26.37. HRMS (m/z):
calcd. for 407.1481 ([M + H]+), obsd. 407.1499. 2.85 (s, 3H). 13C-NMR (100 MHz, CDCl3) δ 168.12, 167.12, 164.25, 157.14, 149.75, 148.47, 147.57, 146.98,
135.32, 132.18, 129.23, 123.62, 118.50, 117.10, 113.43, 113.11, 112.47, 107.06, 56.83, 26.37. HRMS (m/z):
calcd. for 407.1481 ([M + H]+), obsd. 407.1499. 2.85 (s, 3H). 13C-NMR (100 MHz, CDCl3) δ 168.12, 167.12, 164.25, 157.14, 149.75, 148.47, 147.57, 146.98,
135.32, 132.18, 129.23, 123.62, 118.50, 117.10, 113.43, 113.11, 112.47, 107.06, 56.83, 26.37. HRMS (m/z):
calcd. for 407.1481 ([M + H]+), obsd. 407.1499. Compound 11e: 4-{3-((4-chlorophenyl)carbamoyl)phenoxy}-N-methylpicolinamide: yellow solid, yield
68%. 1H-NMR (400 MHz, CDCl3) δ 8.62 (d, J = 5.6 Hz, 1H), 8.01–7.78 (m, 2H), 7.81–7.62 (m, 2H),
7.65–7.42 (m, 2H), 7.48–7.29 (m, 3H), 7.16 (m, 1H), 6.52 (s, 1H), 2.85 (s, 3H). 13C-NMR (100 MHz, CDCl3)
δ 171.77, 169.22, 165.15, 159.14, 149.17, 147.28, 136.46, 135.21, 131.37, 129.44, 127.45, 124.57, 121.78,
118.60, 114.31, 113.17, 24.77. HRMS (m/z): calcd. for 382.0958 ([M + H]+), obsd. 382.0961. Compound 11f: 4-{3-((4-fluorophenyl)carbamoyl)phenoxy}-N-methylpicolinamide: yellow solid, yield
82%, 1H-NMR (400 MHz, CDCl3 ) δ 8.74 (d, J = 5.6 Hz, 1H), 8.02–7.76 (m, 2H), 7.72–7.46 (m, 4H), 7.34
(m, 1H), 7.21–7.01 (m, 3H), 6.50 (s, 1H), 2.85 (s, 3H). 13C-NMR (100 MHz, CDCl3) δ 169.39, 168.32,
167.05, 163.07, 159.55, 158.34, 148.57, 146.98, 135.40, 134.03, 132.01, 128.43, 123.62, 122.66, 121.58, 118.51,
115.10, 113.90, 112.17, 110.47, 28.37. HRMS (m/z): calcd. for 407.1481 ([M + H]+), obsd. 407.1499. Compound 11g:
tert-butyl 4-{3-((3,4-dimethoxyphenyl)carbamoyl)phenoxy}-5,6-dihydropyrido
(4’,3’:4,5)thieno(2,3-d)pyrimidine-7(8H)-carboxylate: yellow solid, yield 71%, m.p. 103–105 ˝C. 1H-NMR (400 MHz, CDCl3 ) δ 8.92 (s, 1H), 8.50 (s, 1H), 7.65–7.40 (m, 2H), 7.34 (m, 1H), 7.29–7.07
(m, 3H), 6.91 (m, 1H), 4.70 (s, 2H), 3.81 (s, 3H), 3.78 (s, 3H), 3.59–3.43 (m, 2H), 3.17 (m, 2H), 1.42
(s, 9H). 3.1.6. General Procedure for the Preparation of N1-(3-Fluoro-4-methoxyphenyl)-N3-(4-fluorophenyl)malonamide Derivatives Compound 13a–i Compounds 12 were further reacted with compounds 5, 6, 7 in the presence of K2CO3 to obtain
the final products 13a–i as mentioned above. Compound 13a: N1-{4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl}-N3-(4-fluorophenyl)-2,
2-dimethylmalonamide. White solid, yield 85%, m.p. 210–212 ˝C. 1H-NMR (400 MHz, CDCl3) δ 8.84
(s, 1H), 8.51 (s, 1H), 8.22 (s, 1H), 7.70 (m, 1H), 7.48 (s, 1H), 7.44–7.23 (m, 2H), 7.23–7.15 (m, 1H), 6.97
(t, J = 8.6 Hz, 1H), 3.99 (d, J = 4.3 Hz, 1H), 1.62 (s, 1H). 13C-NMR (100 MHz, CDCl3) δ 172.19, 171.92,
165.66, 162.17, 159.64, 156.11, 155.44, 153.27, 152.81, 150.33, 149.47, 136.40, 136.30, 136.20, 136.07, 133.02,
124.10, 122.59, 115.91, 110.17, 109.39, 109.15, 106.81, 100.93, 56.36, 50.76, 24.18. HRMS (m/z): calcd. for
523.1788 ([M + H]+), obsd.535.1791. Compound
13b:
N-{4-[(6,7-dimethoxyquinazolin-4-yl)oxy]-3-fluorophenyl}-N-(4-fluorophenyl)
cyclopropane-1,1-dicarboxamide. Yellow solid, yield 86%, m.p. 180–182 ˝C. 1H-NMR (400 MHz,
CDCl3) δ 9.66 (s, 1H), 8.52 (s, 1H), 8.47 (s, 1H), 7.67 (d, J = 11.1 Hz, 1H), 7.48 (s, 1H), 7.38 (dd, J = 9.0, 4.8
Hz, 1H), 7.20 (d, J = 5.5 Hz, 1H), 6.98 (t, J = 8.6 Hz, 1H), 3.99 (d, J = 4.5 Hz, 3H), 1.66 (t, J = 6.1 Hz, 1H),
1.58–1.51 (m, 1H). 13C-NMR (100 MHz, CDCl3) δ 169.43, 168.43, 164.68, 156.04, 152.69, 150.36, 149.45,
136.42, 136.03, 124.07, 122.95, 122.87, 116.10, 115.96, 115.73, 110.20, 109.63, 109.40, 106.78, 100.96, 56.37,
31.93, 29.70, 29.29, 22.69, 17.61, 14.12. HRMS (m/z): calcd. for 521.1631 ([M + H]+), obsd.521.1634. Compound
13c:
N-{4-((6,7-dimethoxyquinazolin-4-yl)oxy)-3-fluorophenyl}-N-(4-fluorophenyl)
cyclobutane-1,1-dicarboxamide. White solid, yield 80%, m.p. 250–252 ˝C. 1H-NMR (400 MHz, CDCl3)
δ 8.58 (s, 1H), 8.53 (d, J = 7.2 Hz, 1H), 8.16 (d, J = 7.5 Hz, 1H), 7.80 (dd, J = 11.9, 2.1 Hz, 1H), 7.57–7.48
(m, 1H), 7.35–7.30 (m, 1H), 7.28 (d, J = 3.4 Hz, 1H), 7.03 (t, J = 8.5 Hz, 1H), 4.06 (d, J = 4.5 Hz, 1H),
2.06–1.90 (m, 1H), 1.28–1.23 (m, 1H). 13C-NMR (101 MHz, CDCl3) δ 170.54, 170.40, 164.67, 160.91,
158.47, 156.02, 155.52, 153.05, 152.69, 150.34, 149.44, 136.64, 136.12, 133.38, 124.14, 122.02, 121.94, 115.88,
115.65, 110.16, 109.09, 108.86, 106.78, 100.93, 56.36, 56.02, 29.76, 15.67. HRMS (m/z): calcd. for 535.1788
([M + H]+), obsd.535.1793. Compound 13d: N1-(4-fluorophenyl)-2,2-dimethyl-N3-{4-((2-(methylcarbamoyl)pyridin-4-yl)oxy]
phenyl}malonamide. Faint yellow solid, yield 82%, m.p. 113–115 ˝C. 3.1.4. General Procedure for the Preparation of 3-Methoxy-N-phenylbenzamide Derivatives
Compound 11a–i 13C-NMR (100 MHz, CDCl3) δ 170.28, 166.05, 162.31, 157.47, 154.66, 149.95, 148.98, 148.67,
138.69, 132.68, 129.19, 126.61, 126.27, 122.57, 122.30, 119.22, 118.40, 117.01, 113.43, 107.06, 81.20, 56.83,
43.81, 42.71, 28.33, 23.62. HRMS (m/z): calcd. for 563.1964 ([M + H]+), obsd. 563.1921. Compound 11h: tert-butyl 4-{3-((4-chlorophenyl)carbamoyl)phenoxy}-5,6-dihydropyrido(4’,3’:4,5)
thieno(2,3-d)pyrimidine-7(8H)-carboxylate: yellow solid, yield 74%, 1H-NMR (400 MHz, CDCl3) δ 8.50
(s, 1H), 7.81–7.67 (m, 2H), 7.61–7.42 (m, 2H), 7.42–7.16 (m, 4H), 4.65 (s, 2H), 3.67–3.40 (m, 2H), 3.17
(m, 2H), 1.42 (s, 9H). 13C-NMR (100 MHz, CDCl3 ) δ 173.11, 168.12, 164.45, 159.26, 155.78, 149.12, 139.57,
136.46, 130.37, 129.26, 128.01, 127.74, 126.77, 123.78, 122.86, 121.41, 118.12, 116.21, 79.31, 44.25, 43.78,
27.32, 24.42. HRMS (m/z): calcd. for 537.1363 ([M + H]+), obsd. 537.1378. Compound 11i: tert-butyl 4-{3-((4-fluorophenyl)carbamoyl)phenoxy}-5,6-dihydropyrido(4’,3’:4,5)
thieno(2,3-d)pyrimidine-7(8H)-carboxylate: yellow solid, yield 77%, 1H-NMR (400 MHz, CDCl3)
δ 8.50 (s, 1H), 7.73–7.43 (m, 4H), 7.34–7.15 (m, 4H), 4.65 (s, 2H), 3.65–3.42 (m, 2H), 3.18 (m, 2H), 1.42
(s, 9H). 13C-NMR (100 MHz, CDCl3 ) δ 171.01, 167.25, 163.42, 162.01, 159.45, 158.27, 155.84, 147.88,
137.32, 135.05, 134.00, 130.12, 127.42, 125.77, 123.66, 122.57, 121.70, 119.62, 118.71, 116.10, 114.93, 82.18,
44.28, 43.54, 27.43, 24.55. HRMS (m/z): calcd. for 521.1659 ([M + H]+), obsd. 521.1711. 3.1.5. General Procedure for the Preparation of
1 3.1.5. General Procedure for the Preparation of 3.1.5. General Procedure for the Preparation of p
N1-(3-Fluoro-4-hydroxyphenyl)-N3-(4-fluorophenyl)malonamide Derivatives Compounds 12 N1-(3-Fluoro-4-hydroxyphenyl)-N3-(4-fluorophenyl)malonamide Derivatives Compounds 12 Diethyl malonate 2.53 mL (17 mmol), iodomethane or 1,2-dibromoethane or 1,3-dibromoethane
(22 mmol), K2CO3 5.7 g (42 mmol) and tetrabutylammonium bromide 0.27 g (0.08 mmol) were added
in DMF. The mixture is stirred for 16 h at room temperature. After evaporation of solvent, the residue
was extracted with ethyl acetate 100 mL three times, and the organic layer was washed with water. After the concentration of the solvent, colorless oil diethyl cyclopropane-1,1-dicarboxylatederivatives
were obtained (yield 89%). Diethyl cyclopropane-1,1-dicarboxylatederivatives (1.0 eq.) were dissolved in EtOH. KOH (1.0 eq.)
in ethanol solution was added and stirred for 5 h. After the completion of the reaction, the intermediate
was dissolved in THF solution at ´15 ˝C. 4-Methylmorpholine (1.2 eq.), isobutyl chloroformate
(1.0 eq.) and 4-amino-2-fluorophenol (1.0 eq.) were added. The crude product was purified by column
chromatography, and the above process was repeated. The final product compounds 12 were obtained. 12 of 16 Molecules 2016, 21, 612 3.1.6. General Procedure for the Preparation of 3.1.6. General Procedure for the Preparation of 1H-NMR (400 MHz, CDCl3) δ
8.72 (s, 1H), 8.54 (s, 1H), 8.37 (d, J = 5.6 Hz, 1H), 8.02 (d, J = 4.0 Hz, 1H), 7.66 (d, J = 1.9 Hz, 1H), 7.60–7.50
(m, 3H), 7.09–6.99 (m, 4H), 6.94 (dd, J = 5.6, 2.5 Hz, 1H), 2.99 (d, J = 5.1 Hz, 3H), 1.70 (s, 6H). 13C-NMR
(100 MHz, CDCl3) δ 171.71, 166.36, 164.46, 160.93, 158.50, 152.26, 150.23, 149.67, 135.10, 133.32, 122.47,
122.39, 122.32, 121.43, 115.81, 115.59, 114.10, 110.12, 50.69, 29.70, 26.15, 25.36, 24.24. HRMS (m/z): calcd. for 451.1776 ([M + H]+), obsd.451.1779. Calcd. for 473.1596 ([M + Na]+), obsd.473.1602. Compound 13e: N-(4-fluorophenyl)-N-{4-((2-(methylcarbamoyl)pyridin-4-yl)oxy)phenyl}cyclopropane-1,
1-dicarboxamide. White solid, yield 82%, m.p. 138–140 ˝C. 1H-NMR (400 MHz, CDCl3) δ 9.46 (s, 2H),
8.31 (d, J = 5.6 Hz, 1H), 7.99 (d, J = 4.9 Hz, 1H), 7.54–7.50 (m, 2H), 7.49 (s, 1H), 7.43–7.36 (m, 1H), 6.99
(s, 1H), 6.96 (d, J = 2.6 Hz, 1H), 6.94 (s, 1H), 6.91 (4, 1H), 4.03 (q, 1H), 2.88 (d, J = 5.1 Hz, 3H), 1.19
(t, 2H), 1.13 (d, J = 6.1 Hz, 2H). 13C-NMR (100 MHz, CDCl3) δ 169.65, 169.34, 166.39, 164.64, 152.09,
150.24, 149.81, 135.18, 133.43, 123.21, 122.91, 121.42, 115.72, 115.50, 114.39, 109.72, 64.43, 60.43, 28.89,
26.18, 25.35, 21.05, 17.91, 14.19. HRMS (m/z): calcd. for 624.2087 ([M + H]+), obsd.449.1620. calcd. for
471.1439 ([M + Na]+), obsd.471.1464. Compound 13f: N-(4-fluorophenyl)-N-{4-((2-(methylcarbamoyl)pyridin-4-yl)oxy)phenyl}cyclobutane-1,
1-dicarboxamide. White solid, yield 77%, m.p. 192–194 ˝C. 1H-NMR (400 MHz, CDCl3) δ 8.29 (s, 1H),
8.27 (d, J = 5.1 Hz, 1H), 8.14 (s, 1H), 7.94 (d, J = 4.5 Hz, 1H), 7.60–7.56 (m, 1H), 7.54 (s, 1H), 7.49–7.38
(m, 1H), 6.96 (dd, J = 8.6, 4.5 Hz, 1H), 6.92 (s, 1H), 6.86 (dd, J = 5.5, 2.5 Hz, 1H), 2.92 (d, J = 5.1 Hz,
1H), 2.68 (t, J = 7.9 Hz, 1H), 2.01–1.88 (m, 1H). 13C-NMR (100 MHz, CDCl3) δ 170.49, 170.45, 166.31,
164.53, 152.28, 150.10, 149.71, 135.33, 121.96, 121.88, 121.81, 121.43, 115.80, 115.57, 114.09, 110.09, 64.42, 13 of 16 13 of 16 Molecules 2016, 21, 612 55.91, 29.78, 26.14, 25.35, 15.68. HRMS (m/z): calcd. for 463.1776 ([M + H]+), obsd.463.1779. calcd. for
485.1596 ([M + Na]+), obsd.485.1602. 55.91, 29.78, 26.14, 25.35, 15.68. HRMS (m/z): calcd. for 463.1776 ([M + H]+), obsd.463.1779. calcd. for
485.1596 ([M + Na]+), obsd.485.1602. Compound 13g: tert-butyl-4-{2-fluoro-4-(3-((4-fluorophenyl)amino)-2,2-dimethyl-3-oxopropanamido)
phenoxy}-5,6-dihydropyrido(4’,3’:4,5)thieno(2,3-d)pyrimidine-7(8H)-carboxylate. Faint yellow solid,
yield 75%, m.p. 126–128 ˝C. 3.2. Cell Proliferative Assay The anti-proliferative activities of synthesized compounds against HeLa, Hep-G2 and MCF-7
cells were evaluated by MTT assay [10]. HeLa cells were grown to log phase in RPMI 1640 medium
supplemented with 10% fetal bovine serum. Hep-G2 and MCF-7 cells were grown to log phase in
DMEM medium supplemented with 10% fetal bovine serum. After diluting to 5 ˆ 105 cells¨L´1 with
the complete medium, 100 µL of the obtained cell suspension was added to each well of 96-well culture
plates. The subsequent incubation was permitted at 37 ˝C, 5% CO2 atmosphere for 48 h before the
anti-proliferative assessments. Tested samples at pre-set concentrations were added to 6 wells. Before
the anti-proliferative assessments, 20 µL of PBS containing 2.5 mg¨mL´1 of MTT was added to each
well. Four h later, 100 µL extraction solution (DMSO) was added to each well and the optical density
was measured at a wavelength of 570 nm on an ELISA microplate reader. 3.1.6. General Procedure for the Preparation of 1H-NMR (400 MHz, CDCl3) δ 8.92 (s, 1H), 8.48 (s, 1H), 8.24 (s, 1H), 7.75
(dd, 1H), 7.53–7.45 (m, 2H), 7.25–7.18 (m, 2H), 7.05 (t, J = 8.6 Hz, 2H), 4.74 (s, 2H), 3.80 (t, J = 5.4 Hz,
2H), 3.18 (s, 2H), 1.69 (s, 6H), 1.51 (s, 9H). 13C-NMR (100 MHz, CDCl3) δ 171.94, 171.13, 169.06, 162.68,
159.13, 158.67, 154.55, 152.49, 136.38, 135.74, 135.61, 132.99, 124.02, 122.59, 122.51, 118.09, 115.93, 115.81,
115.71, 109.35, 109.11, 80.57, 50.74, 31.93, 29.70, 29.66, 29.37, 28.43, 24.20, 22.70, 19.55, 14.12. HRMS
(m/z): calcd. for 624.2087 ([M + H]+), obsd.624.2091. Calcd. for 646.1906 ([M + Na]+), obsd.646.1913. Compound 13h: tert-butyl-4-{2-fluoro-4-(1-((4-fluorophenyl)carbamoyl)cyclopropanecarboxamido)
phenoxy}-5,6-dihydropyrido(4’,3’:4,5)thieno(2,3-d)pyrimidine-7(8H)-carboxylate. White solid, yield
82%, m.p. 171–173 ˝C. 1H-NMR (400 MHz, CDCl3) δ 9.76 (s, 1H), 8.52 (s, 1H), 8.41 (s, 1H), 7.65–7.34
(m, 3H), 7.22–7.12 (m, 2H), 6.97 (t, J = 8.6 Hz, 2H), 4.66 (s, 2H), 3.72 (t, J = 5.6 Hz, 2H), 3.11 (s, 2H),
1.54 (dd, J = 7.7, 4.9 Hz, 2H), 1.43 (s, 9H), 1.18 (t, J = 7.1 Hz, 4H). 13C-NMR (100 MHz, CDCl3) δ
169.56, 169.03, 168.49, 162.68, 161.13, 158.69, 155.43, 154.58, 152.97, 152.49, 136.55, 136.45, 135.69, 135.56,
132.87, 123.95, 123.02, 118.21, 116.19, 115.93, 115.81, 109.70, 109.47, 80.70, 60.54, 29.80, 29.34, 28.53,
21.16, 17.65, 14.25. HRMS (m/z): calcd. for 622.1930 ([M+H]+), obsd.622.1930. Calcd. for 644.1750
([M + Na]+), obsd.644.1793. Compound 13i:
tert-butyl-4-{2-fluoro-4-(1-((4-fluorophenyl)carbamoyl)cyclobutanecarboxamido)
phenoxy}-5,6-dihydropyrido(4’,3’:4,5)thieno(2,3-d)pyrimidine-7(8H)-carboxylate. Yellow solid, yield
83%, m.p. 101–103 ˝C. 1H-NMR (400 MHz, CDCl3) δ 8.48 (s, 1H), 8.36 (s, 1H), 7.96 (s, 1H), 7.78
(dd, J = 11.9, 2.1 Hz, 1H), 7.56–7.41 (m, 2H), 7.27–7.18 (m, 2H), 7.04 (t, J = 8.6 Hz, 2H), 4.74 (s, 2H), 3.80
(t, J = 5.4 Hz, 2H), 3.18 (s, 2H), 2.85–2.65 (m, 4H), 2.05–1.99 (m, 2H), 1.51 (s, 9H). 13C-NMR (100 MHz,
CDCl3) δ 170.48, 170.16, 169.07, 162.68, 160.91, 158.51, 155.46, 154.54, 152.99, 152.47, 136.60, 135.79,
135.55, 134.32, 133.24, 131.70, 124.05, 121.99, 121.91, 118.09, 115.91, 115.68, 115.47, 109.04, 108.81, 80.56,
61.84, 55.94, 31.93, 29.75, 29.70, 28.43, 15.68. HRMS (m/z): calcd. for 636.2087 ([M + H]+), obsd.636.2068. Calcd. for 658.1906 ([M + Na]+), obsd.658.1912. 3.4. Molecular Dockingand Dynamics Simulation Molecular docking of compounds into the three dimensional X-ray structure of c-MET (PDB code:
4MXC) and VEGFR-2 (PDB code: 4ASE) was carried out using the Discovery Studio (version3.1) as
implemented through the graphical user interface Discovery Studio CDOCKER protocol [32]. The three-dimensional structure of all compounds was constructed using ChemBio 3D Ultra
11.0 software (Chemical Structure Drawing Standard; Cambridge Soft corporation, Waltham, MA,
USA), then it was energetically minimized by using MMFF94 with 5000iterations and minimum RMS
gradient of 0.10. The crystal structures of VEGFR-2 kinase were retrieved from the RCSB Protein Data
Bank (http://www.rcsb.org/pdb/home). All bound waters and ligands were eliminated from the
protein and the polar hydrogen was added. The whole protein structure was defined as are acceptor
and the site sphere was selected based on ATP binding site of 4ASE or 4MXC. Compounds were placed
during the molecular docking procedure. Types of interactions of the docked protein with ligand were
analyzed after the end of molecular docking. To further confirm the binding model of compound 13b with c-MET and VEGFR-2, respectively,
MD simulations was performed using the GROMACS package (version 4.5, University of
Groningen) [33]. By using the AMBER99 force field and general AMBER force field (gaff), receptor
and ligands were charged, respectively [34]. The simulations study were carried out under periodic
boundary conditions (PBC) with minimum distance (0.1 nm) between the kinase and the edge of the
box. The complex of compound 13b with protein were solvated in a cubic box of TIP3P water molecules
and neutralized by adding Na+ and Cl´ to mimic physiological NaCl concentration of 0.15 M. Prior
to MD simulations, energy minimization were performed to remove bad contacts, 100-ps NVT and
100-ps NPT ensembles with protein and ligand position restraints were carried out to equilibrate each
system. Finally, 3-ns MD production simulations were carried out for each system with a 2-fs time step
at constant pressure (1 atm) and temperature (300 K). 3.3. In Vitro Kinase Assay In vitro kinase inhibitory ability was determined using HTScan MET Kinase Assay Kit, HTScan
VEGF Receptor 2 Kinase Assay Kit, HTScan c-Kit Kinase Assay Kit, HTScan PDGF Receptor β
Kinase Assay Kit and HTScan EGFR Kinase Assay Kit(purchased from Cell Signaling Technology, Inc. Danvers, MA, USA) by colorimetric ELISA assay according to the manufacturer’s instructions. Briefly,
reaction cocktail containing recombinant human MET kinase or other kinases were incubated with
various concentrations of tested compounds or DMSO (0.1%) for 5 min at room temperature, and then
ATP/substrate peptide cocktail was added to the pre-incubated reaction cocktail. After incubation at 14 of 16 Molecules 2016, 21, 612 room temperature for 30 min, the reaction was stopped and transferred to a 96-wellstreptavidin-coated
plate, and incubated for 1 h at room temperature. Primary antibody (phosphorylated tyrosine
monoclonal antibody (pTyr-100), 1:1000 in PBS/T with 1% bovine serum albumin (BSA)) was added
into per well until the wells were washed thrice with PBS/T. After incubated at room temperature
for 1 h, phosphorylation of the substrate was monitored with HRP-labeled anti-mouse IgG antibody
(1:500 in PBS/T with 1% BSA), followed by a chromogenic reaction. Finally, the kinase assay was
detected at 450 nm with microplate reader. The reaction processed with only DMSO (0.1%) served as a
vehicle control. The results were expressed as percent kinase activity of the vehicle control, and IC50
was defined as the compound concentration that resulted in 50% inhibition of enzyme activity. The
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inhibitors that bind to active and inactive kinase conformations. J. Am. Chem. Soc. 2010, 132, 4152–4160. [CrossRef] [PubMed] 18. Simard, J.R.; Getlik, M.; Grütter, C. 4. Conclusions In this study, a series of 3-methoxy-N-phenylbenzamide derivatives, N-(3-(tert-butyl)-1-phenyl-
1H-pyrazol-5-yl) benzamide derivatives and N1-(3-fluoro-4-methoxyphenyl)-N3-(4-fluorophenyl)
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c-MET inhibitors. Compound 13b showed the most potent inhibitory activity against c-MET with
IC50 of 0.02 µM, and it exhibited good anticancer activity against tested cancer cell lines. In addition,
compound 11c, 11i, 13h also demonstrated potent c-MET inhibitory activity and excellent anticancer
activity in vitro. Importantly, compound 13h showed the highest anti-proliferation activity against
Hep-G2 with IC50 of 1.7 µM, which is better than the c-MET inhibitor XL184, suggesting that compound
13h may have great potential for liver cancer therapy. Kinase selectivity assay suggested that compound
13b and 13h also showed a little inhibitory activity against VEGFR-2. Moreover, molecular docking
results disclosed binding modes of these potent inhibitors and c-MET. Therefore, compound 11c, 11i,
13b, 13h are potent c-MET inhibitors and could be potential anticancer agents. 15 of 16 15 of 16 Molecules 2016, 21, 612 Acknowledgments: We are grateful to TANG Bioscience (Shanghai, China) for their help in kinase assay. This work is supported by grants from Key Projects of the National Science and Technology Pillar Program
(2012BAI30B02), National Natural Science Foundation (Nos. 81260628, 81303270, and U1303124) and Liaoning
Science and Technology Project (2013226027-4). Author Contributions:
J.-H.W. and L.-X.C. conceived and designed the experiments;
Y.-N.J.,
K.Z.,
S.-Y.G. and G.-H.W. performed the experiments; Y.-N.J. and K.Z. analyzed the data; J.H. contributed
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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/). © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
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English
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Effect of zinc oxide nanoparticles synthesized from Carya illinoinensis leaf extract on growth and antioxidant properties of mustard (Brassica juncea)
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Frontiers in plant science
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cc-by
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OPEN ACCESS EDITED BY
Kanchan Vishwakarma,
Swedish University of Agricultural Sciences,
Sweden
REVIEWED BY
Faisal Zulfiqar,
The Islamia University of Bahawalpur,
Pakistan
Hassan Ragab El-Ramady,
Kafrelsheikh University, Egypt
*CORRESPONDENCE
Laura Carson
lecarson@pvamu.edu
SPECIALTY SECTION
This article was submitted to
Plant Nutrition,
a section of the journal
Frontiers in Plant Science
RECEIVED 25 November 2022
ACCEPTED 02 January 2023
PUBLISHED 23 January 2023
CITATION
Geremew A, Carson L, Woldesenbet S,
Wang H, Reeves S, Brooks N Jr., Saganti P,
Weerasooriya A and Peace E (2023) Effect
of zinc oxide nanoparticles synthesized
from Carya illinoinensis leaf extract on
growth and antioxidant properties of
mustard (Brassica juncea). Front. Plant Sci. 14:1108186. doi: 10.3389/fpls.2023.1108186 EDITED BY
Kanchan Vishwakarma,
Swedish University of Agricultural Sciences,
Sweden Addisie Geremew 1, Laura Carson 1*, Selamawit Woldesenbet 1,
Huichen Wang 2, Sheena Reeves 3, Nigel Brooks Jr.3,
Premkumar Saganti 2, Aruna Weerasooriya 1 and Elisha Peace 1 Addisie Geremew 1, Laura Carson 1*, Selamawit Woldesenbet 1,
Huichen Wang 2, Sheena Reeves 3, Nigel Brooks Jr.3,
Premkumar Saganti 2, Aruna Weerasooriya 1 and Elisha Peace 1 1Cooperative Agricultural Research Center, Prairie View A&M University, Prairie View, TX, United States,
2Department of Chemistry and Physics, College of Arts and Sciences, Prairie View A&M University,
Prairie View, TX, United States, 3Department of Chemical Engineering, College of Engineering, Prairie
View A&M University, Prairie View, TX, United States CITATION
Geremew A, Carson L, Woldesenbet S,
Wang H, Reeves S, Brooks N Jr., Saganti P,
Weerasooriya A and Peace E (2023) Effect
of zinc oxide nanoparticles synthesized
from Carya illinoinensis leaf extract on
growth and antioxidant properties of
mustard (Brassica juncea). Front. Plant Sci. 14:1108186. doi: 10.3389/fpls.2023.1108186 Background: The sustainability of crop production is impacted by climate change
and land degradation, and the advanced application of nanotechnology is of
paramount importance to overcome this challenge. The development of
nanomaterials based on essential nutrients like zinc could serve as a basis for
nanofertilizers and nanocomposite synthesis for broader agricultural applications
and quality human nutrition. Therefore, this study aimed to synthesize zinc oxide
nanoparticles (ZnO NPs) using pecan (Carya illinoinensis) leaf extract and
investigate their effect on the growth, physiology, nutrient content, and
antioxidant properties of mustard (Brassica juncea). COPYRIGHT
© 2023 Geremew, Carson, Woldesenbet,
Wang, Reeves, Brooks, Saganti, Weerasooriya
and Peace. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). TYPE Original Research
PUBLISHED 23 January 2023
DOI 10.3389/fpls.2023.1108186 TYPE Original Research
PUBLISHED 23 January 2023
DOI 10.3389/fpls.2023.1108186 COPYRIGHT
© 2023 Geremew, Carson, Woldesenbet,
Wang, Reeves, Brooks, Saganti, Weerasooriya
and Peace. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The
use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms. 1 Introduction enzymatically or non-enzymatically (Hasanuzzaman et al., 2020;
Lukacova et al., 2021). The enzymatic antioxidants defense involves
the production of diverse enzymes such as superoxide dismutase,
catalase, ascorbate peroxidase, glutathione reductase,
monodehydroascorbate reductase, dehydroascorbate reductase,
glutathione peroxidase, glutathione, peroxiredoxins, ferritin,
thioredoxins and glutaredoxin (Gill and Tuteja, 2010; Kaur et al.,
2019). Whereas the nonenzymatic antioxidant mechanism
encompasses the production of ascorbic acid, glutathione, phenolic
acids, alkaloids, flavonoids, carotenoids, alpha-tocopherol,
nonprotein amino acids, etc. (Gill and Tuteja, 2010; Zulfiqar and
Ashraf, 2022a). To overcome oxidative stress, plants have also
employed osmolyte accumulation like proline as endogenous
strategies (Zulfiqar and Ashraf, 2022b). Proline can scavenge free
radicals generated through osmoprotection, osmoregulation, ROS
quenching, metal chelation, and buffering of cellular redox potential
of plants under various stressors (Zulfiqar and Ashraf, 2022b). Zulfiqar et al. (2019) have also highlighted the role of
osmoprotectants such as amino acids, polyamines, quaternary
ammonium compounds and sugars in mitigating the negative effect
of abiotic stress by scavenging ROS, acting as metabolic signals and
stabilizing cellular structures and enzymes. Moreover, a recent study
has also shown that foliar application of ascorbic acid mitigates the
adverse effects of salinity on lettuce (Lactuca sativa) by reducing
oxidative injury (Naz et al., 2022). Different plants have varied
capacities to tolerate oxidative stress that depends on the ability of
their antioxidant machinery. Research towards increasing the
antioxidant defense in plants is vital. These days, the applications of
metallic NPs are thought to be sound solutions for ameliorating
different stresses through increasing antioxidant enzymes (Faizan and
Hayat, 2019; Hasanuzzaman et al., 2020; Alabdallah and
Alzahrani, 2020). Globally, the sustainability of crop production is impacted by
several factors including climate change and land degradation (Webb
et al., 2017; Jiang et al., 2021). To maintain sustainable agriculture and
food production, the advanced application of nanotechnology is of
paramount importance (Fraceto et al., 2016; Wang et al., 2018; Neme
et al., 2021). Its application improves agricultural production by
reducing losses and enhancing the efficiency of inputs (Manjunatha
et al., 2016; Shang et al., 2019; Neme et al., 2021) and crop yields and
productivity (Noohpisheh et al., 2021). Nanoparticles are
nanomaterials with peculiar physicochemical characteristics
including enhanced reactivity, typical surface structure, and high
surface-to-volume ratio (Noohpisheh et al., 2021; Badawy et al.,
2021). OPEN ACCESS 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. Methods: The ZnO NPs were characterized by UV-Vis spectrophotometry,
Dynamic Light Scattering (DLS), X-ray diffractometer (XRD), Scanning Electron
Microscopy (SEM), and Fourier Transform Infra-Red Spectroscopy (FTIR). Mustard
plants were subjected to different concentrations of ZnONPs (0, 20, 40, 60, 80,
100 and 200 mg L-1) during the vegetative growth stage. Results: The UV-Vis spectra of ZnO NPs revealed the absorption maxima at 362
nm and FTIR identified numerous functional groups that are responsible for
capping and stabilizing ZnO NPs. DLS analysis presented monodispersed ZnO
NPs of 84.5 nm size and highly negative zeta potential (-22.4 mV). Overall, the
application of ZnO NPs enhanced the growth, chlorophyll content (by 53 %),
relative water content (by 46 %), shoot biomass, membrane stability (by 54 %) and
net photosynthesis significantly in a dose-dependent manner. In addition, the
supplement of the ZnO NPs augmented K, Fe, Zn and flavonoid contents as well as
overcome the effect of reactive oxygen species by increasing antioxidant capacity
in mustard leaves up to 97 %. 01 Frontiers in Plant Science frontiersin.org Geremew et al. 10.3389/fpls.2023.1108186 10.3389/fpls.2023.1108186 Conclusions: In conclusion, ZnO NPs can be potentially used as a plant growth
stimulant and as a novel soil amendment for enhancing crop yields. Besides, the
biofortification of B. juncea plants with ZnO NPs helps to improve the nutritional
quality of the crop and perhaps potentiates its pharmaceutical effects. zeta potential, flavonoids, net photosynthesis, chlorophyll content, macronutrients,
micronutrients, reactive oxygen species (ROS) Frontiers in Plant Science Geremew et al. 2.2 Plant sample collection and extraction the zinc conventional fertilizer and increase the availability of Zn for
plants (Rajput et al., 2021; Awan et al., 2021). ZnO NPs application has enhanced plants growth, photosynthesis
and development of corn, onion, tomato, olive, capsicum, cucumber,
wheat, and zucchini (Stampoulis et al., 2009; Zhao et al., 2013; Munir
et al., 2018; Wang et al., 2018; Neto et al., 2020; Awan et al., 2021;
Regni et al., 2022) in a dose-dependent manner. Nevertheless, the
response of plants to ZnO NPs application is a function of genotypes,
the stage of the plant, and the concentration of nanoparticles provided
(Salama et al., 2019). On the other hand, the application of chemically
synthesized ZnO NPs has been criticized compared to the
biosynthesized counters (Rai et al., 2018; Jamkhande et al., 2019). In fact, the latter is regarded as environmentally friendly, safer and
potentially more efficiently obtained using plant extracts (Jamkhande
et al., 2019; Jangannanavar et al., 2021; Regni et al., 2022). However,
the use of biologically synthesized ZnO NPs as nanofertilizers to
enhance zinc content and improve morpho-physiological traits and
antioxidant properties of leafy vegetables at early vegetative stages is
limited (Iziy et al., 2019; Salama et al., 2019; Regni et al., 2022). Green leaves of pecan (Carya illinoinensis (Wangenh). K. Koch)
were collected from the Bill and Vara Daniel Farm and Ranch located
at Prairie View A&M University (PVAMU) and the sample was
identified and the voucher specimen was stored at the Cooperative
Agricultural Research Center (CARC) at Prairie View A&M
University. The collected leaves were cleaned by rinsing in distilled
water several times to remove debris. Subsequently, the leaves were
freeze-dried using BenchTop Pro with Omnitronics™freeze dryer
(BTP-8ZL00W, SP Scientific, PA, USA) and grounded manually
using mortar and pestle. Next, the fine leave powder (15 g) was
added to 400 mL deionized water and shaken with an orbital shaker
(IKA Basic Variable-Speed Digital Orbital Shaker, model, 115 V) at
200 rpm at 30°C for 48 hrs. The extract solution was filtered using
Stericup® Quick Release Vacuum driven disposable filter (integrated
with Millipore Express® Plus 0.22 µm PES System (Sigma Aldrich). The filtrate was kept at 4°C pending the synthesis of ZnO NPs. Mustard (Brassica juncea (L.) Czern) belongs to the Brassicaceae
family. 2.2 Plant sample collection and extraction The green vegetables and seeds of mustard are economically
valuable and widely consumed by humans due to their astonishing
provision of several health-promoting metabolites and nutrients
(Sharma et al., 2018; Majdoub et al., 2020; Geremew et al., 2021). Overall, studies have shown that high consumption of mustard is
linked with the prevention of several cancers (Kwak et al., 2016),
antioxidant activities and inhibition of fat increase (Kim et al., 2003). Despite these economic and health benefits, poor soil fertility during its
vital stages such as seed germination, growth, flowering, and pod filling
severely impacts crop yield (Geremew et al., 2021). Owing to these dietary
and economic values, mustard plants need special interest to boost their
production and their nutritional value under limited soil fertility. 2.3 Biosynthesis of ZnO nanoparticles ZnO NPs were synthesized using a modified method suggested by
Jayachandran et al. (2021). Zinc nitrate hexahydrate (Zn (NO3)
2.6H2O) was used as a precursor for the synthesis of the ZnO NPs. Ninety (90) mL of 1 mM Zn (NO3)2.6H2O was poured into 10 mL of
pecan leaf extract in a 200 mL flask. The mixture was stirred at 65°C
for 25 min until light yellow colloidal suspension formed. This
colloidal suspension was further centrifuged at 10,000 RPM for 10
min twice. The pellet was retained and washed with ethanol to remove
the remaining organic matter and centrifuged again at the same
speed. The pellet was completely dried and calcinated at 600°C for 2
hrs under furnace (Thermo Fisher). We further removed organic
matter (ash) from calcination by washing the powder in ethanol and
centrifuging at 10,000 RPM for 10 min. Pending the characterization
of the nanoparticles, the collected pellet was dried, crushed and stored
in dark glass under a desiccator. To fulfill this ever-increasing need for nutrients, only soil is not
adequate, the micronutrients and macronutrients should be
supplemented in the soil in very small quantities (Iris et al., 2018)
in the form of nanoparticles (Iziy et al., 2019). Therefore, in the
present study, we synthesized ZnO NPs using pecan (Carya
illinoinensis) leaf extract and investigated their effect on the growth,
physiology, nutrient content, and antioxidant properties of mustard
(B. juncea). Specifically, we asked the following questions: (i) Do ZnO
NPs affect the morpho-physiological traits of mustard plants? (ii) Do
ZnO NPs enhance the macro- and micronutrient contents of mustard
leaves in a concentration-dependent manner? (iii) How ZnO NPs
affect the antioxidant properties of mustard plants? 1 Introduction Owing to these attributes, NPs are used as nanofertilizers
(Jiang et al., 2021; Noohpisheh et al., 2021; Awan et al., 2021) and
reduce nutrient deficiency (Etienne et al., 2018). Thus, supplying
controlled and targeted mineral nutrient release to plants (Salama
et al., 2019) and then resulting in increased crop growth and
development (Wang et al., 2018; Rajput et al., 2021; Srivastava
et al., 2021). However, recent studies have revealed that NPs may
show both positive or negative impact on plants which mainly
depends on the chemical structure, size, reactivity, and dose
(Elizabeth et al., 2017; Salama et al., 2019) that vary according to
plant species (Rastogi et al., 2017; Regni et al., 2022). Plants face uninterrupted fluxes of environmental conditions and
are frequently subjected to associated abiotic stresses such as drought,
salinity, heavy metals, waterlogging, extreme temperatures, and
oxygen deprivation, which influence plant growth, and
development, ultimately impacting yield and quality (Kapoor et al.,
2020; Zulfiqar and Ashraf, 2022a). Plants exposed to abiotic stress,
singularly or in combination, produce excess reactive oxygen species
(ROS) which leads to oxidative stress and impaired redox
homeostasis. (Noctor et al., 2018; Hasanuzzaman et al., 2020;
Zulfiqar and Ashraf, 2021). In addition to their negative impact,
ROS play a significant function as secondary messengers or signaling
molecules in different cellular mechanisms to increase tolerance
against various abiotic stresses (Singh et al., 2019; Hasanuzzaman
et al., 2020), specifically during the acclimation processes (Antoniou
et al., 2016). The balance between ROS generation and the antioxidant
defense system protects plants from the impact of stress. However, to
withstand oxidative stress caused by ROS over-accumulation, plants
activate their endogenous antioxidant defense mechanisms, either Zinc is an essential micronutrient required for a broader range of
plants’ key functions such as improving water use efficiency,
photosynthesis, protein synthesis, regulation of reactive oxygen species,
antioxidant function, maintenance of membranes integrity, growth
regulation, and gene expression (Bagci et al., 2007; Wang et al., 2018;
Noohpisheh et al., 2021). In addition to these functions, from the
consumer perspective considering the basic benefits of Zn in human
health, the bio-fortification of crops withsuch essential nutrients through
the application of nanomaterials has recently gained attention (Iziy et al.,
2019; Salama et al., 2019). To comply with the zinc demands of plants,
ZnO NPs have been reported as the smartestdelivery tools that substitute Frontiers in Plant Science 02 frontiersin.org Geremew et al. 10.3389/fpls.2023.1108186 Frontiers in Plant Science 2.4 Characterization of ZnO NPs The sample of ZnO NPs (6 mg) was dissolved in 10 mL of double
distilled water for characterization. To measure the optical
parameters, the synthesized ZnO NPs were dispersed in deionized
water. The absorption spectrum of the ZnO NPs was determined
using UV-VIS Spectrophotometer (SpectraMax® PLUS 384,
England) in a spectrum range between 200-800 nm. Deionized
water was used as a reference. The surface chemistry of functional
groups and biomolecules attached to the ZnO NPs was analyzed by
FTIR spectrometer (JASCO/FTIR-6300, Japan) with a resolution of 4
cm-1 at a frequency of 4,000-500 cm-1. The particle size distribution
and zeta potential of the samples were obtained through dynamic
light scattering (DLS) procedure operated using Litesizer™500
(Anton Paar, Austria) coupled with a 10 mW He-Ne laser (633
nm) running at an angle of 90° and temperature of 25°C. Water was
used as a dispersant to measure the zeta potential. In addition, the
evaluation of the morphology of ZnO NPs was performed using frontiersin.org (2) where 0.72 is the correction factor for leaf area in mustard plants adopted
from Ramil and Sulaiman (2021). Individual plants that were separated
into roots and shoots and leaves and were dried in an oven at 70°C until
their constant weight acquired 60 days after germination. These dried
weights were their respective biomass values (below-ground biomass and
above ground biomass, respectively). 2.6.4 Photosynthetic pigments content The overall chlorophyll contents in the intact leaves of mustard
plants were measured using Chlorophyll meter, SPAD-502 (Minolta Co.,
Ltd., Osaka, Japan). The SPAD values were taken at the leaf lamina and
towards the tip. The observations were made early in the morning
between 10:00 and11.00 a.m. To further analyze the various components
of photosynthetic pigments, 200 mg mustard leaves of ZnO NPs treated
and untreated plants were extracted in 20 mL of chilled acetone: ethanol
(1:1, v/v) and kept in dark for 24 hrs under room temperature. This
extract was centrifuged at 8,000 RPM for 10 min, and the supernatant
was collected. After centrifugation, the absorbance of the supernatant
was taken at 663, 645 and 480 nm. Chlorophyll a, chlorophyll b, and
carotenoid contents were estimated in mg, per g offresh weight following
methods by Ulhassan et al. (2019). 2.6.3 Relative water content Dp = 0:9l=bCosq
(1) (1) Dp = 0:9l=bCosq Relative water content (RWC)was quantified by applying the method
of Barrs and Weatherley (1962). Healthy and fully expanded leaves were
collected from individual plants 45 days after germination and cut into 6 x
6 mm2 discs. Fresh weight (FW) of these discs was measured and then
immersed in distilled water for 12 hrs. Afterward, the exterior of the discs
was dried using tissue paper and then turgid weight (SW) was recorded. Thereafter, the discs were dried in an oven at 70°C for 24 hrs and dry
weight (DW) was recorded. RWC in percent was then calculated as: Where Dp represents the average crystallite size, l stands for the
wavelength (1.5406 Å for Cu Ka), b designates the full width at half
maximum (FWHM) of main intensity peak after subtraction of the
equipment broadening and q is used as a diffraction angle in radians. Frontiers in Plant Science 2.5 Plant material and growth conditions RWC =
FW −DW
SW −DW
X 100
(4) The experiment was carried out in a plant growth chamber at
CARC, PVAMU, Texas, USA during the Summer of 2022. Seeds of
the Indian mustard (Brassica juncea (L.) Czern) were acquired from
Twilley seed company (Hodges, SC, USA). Ten seeds were sown in
plastic pots (8-inch size) containing 2 kg of sieved clay soil, electrical
conductivity, and pH of 0.7925 dS m−1, and 7.65, respectively. After
germination, seven seedlings were thinned to ensure that every pot
comprised three plants of the same vigor. Pots with mustard seedlings
thoroughly received 200 mL suspensions of 0 (deionized water), 20,
40, 60, 80, 100, and 200 mg L-1 ZnO NPs directly on the soil after 20
and 40 days of germination. A completely randomized design with
four replications of each treatment was applied. All pots were
irrigated with distilled water twice a week. The time and list of
measurements carried out are summarized in Supporting Figure 1. (4) 2.1 Chemicals used All chemicals used were of analytical grades. Zinc nitrate
hexahydrate (99%), methanol, DPPH (99%), acetone, ethanol,
ascorbic acid (99%), sodium hydroxide (99%), aluminum trichloride,
potassium acetate and quercetin were purchased from Sigma Aldrich
(Burlington, MA). MitoSOX™Mitochondrial Superoxide Indicators
(Invitrogen™, M36008), and Propidium iodide in 1mg/ml aqueous
solution (Thermo Scientific™, J66584.AB) were used. Frontiers in Plant Science 03 frontiersin.org 10.3389/fpls.2023.1108186 Geremew et al. Geremew et al. gram of sample containing 5 leaf portions, 4 cm long each, was
immersed in a test tube with 15 mL of distilled water. The
submersed samples were incubated for 24 hrs at 20 °C. Subsequently,
the electrical conductivity of the water (C1) was measured using
conductivity meter HI198129 (Hanna Instruments Inc., Woonsocket,
Rhode Island). Then, we boiled the samples at 100 °C for 10 min and
conductance was noted (C2). Membrane stability was computed as: scanning electron microscopy (SEM) coupled with an energy-
dispersive x-ray spectroscopy (EDX) system (JOEL JSM-6010LA,
Japan). The EDX spectrometry particularly was run to identify and
quantify the elemental composition of the nanoparticles. X-ray
diffractometer (XRD-7000, Shimadzu, Japan) run at 40 kV and 30
mA was used to examine the surface morphology, size and crystalline
nature of ZnO NPs. The diffraction pattern was recorded by CuKa
radiation with a wavelength of l = 1.541 Å. The scanning was carried
out in 2q value range of 10° to 80° at 0.02 min-1 and 1 second time
constant. Scherrer’s equation was used to compute the average
crystalline size of synthesized ZnO NPs as: MSI ¼ 1 −
c1
c2
*100
(3) (3) 2.6.5 Photosynthetic pigments content Plant height was measured from the stem base of mustard plant to
the tips of its shoot using a meter 45 days after treatment. Forty-five
days after germination leaf area (LA) was determined from
measurements of leaf length and width using the equation: Net photosynthetic rate (Pn), leaf stomatal conductance (gs),
intercellular CO2 concentration (Ci), and transpiration rate (E) of the
second young and fully expanded three mustard leaves were recorded
using a portable photosynthesis system (Li-Cor 6400XT, Lincoln, NE,
USA). The measurements were conducted under the conditions of
photosynthetically active radiation of 1000 mmol m-2 s-1, an ambient
CO2 concentration of 360 ± 10 mmol mol-1, air temperature of 22°C, and
relative humidity of 50%. Three leaves per pot were measured two times
per leaf. LA = 0:72 x length x width
(2) 2.6.2 Membrane stability index Membrane stability index (MSI) was determined 45 days after
germination following the method suggested by Sairam (1994). One Young leaves of the mustard plant were obtained for macro- and
micronutrient analysis after 60 days of treatment application. The leaves 04 frontiersin.org 10.3389/fpls.2023.1108186 Geremew et al. were freeze-dried using BenchTop Pro with Omnitronics™freeze dryer
(BTP-8ZL00W,SP Scientific, PA, USA) for 12 hrs to constant weight and
were ground manually using mortar and pestle. For microwave
digestion, about 250 mg of each mustard leaves sample were directly
placed into a microwave closed vessel. Then, 2 mL of 30% H2O2 and 7.0
mL of 65% (m/m) HNO3 solutions were poured into each vessel. Digestion was run with a high-pressure microwave oven (Milestone
Ethos UP 1600, Sorisole, Italy) at a frequency of 2450 Hz. The digested
samples were filtered through a 0.45 µm nylon membrane (Millipore
Sigma™Millex™-GP Sterile Syringe Filters,Burlington,Massachusetts). The concentration of P, K, Ca, Cu, Mg, Fe, Mn, Na, and Zn in each
sample were analyzed using radial view of Inductive Coupled Plasma
Optical Emission Spectrometer (ICP-OES, Agilent ICP-5100) equipped
with Agilent SP4 autosampler. picrylhydrazyl (DPPH) assay was performed with a modification of
Choi et al. (2002). Fifty (50) mL of each ZnO NPs treated mustard
plant leaf methanolic extracts were mixed with DPPH radical solution
in methanol (0.1 mM, 150 mL) in 150 mL flasks. Each flask was
covered with aluminum foil and incubated at room temperature in
the dark for 30 min. Then the absorbance was recorded at 517 nm
using a UV-Vis spectrophotometer (SpectraMax® PLUS 384). DPPH
methanol reagent without the leave extract was used as control and
percentage radical scavenging activity was determined as: Radical Scanging activity( % ) =
A0-A1
A0
* 100
(5) (5) where A0 and A1 represent the OD of the ascorbic acid and the
ZnO NPs treated leaf extracts, respectively. where A0 and A1 represent the OD of the ascorbic acid and the
ZnO NPs treated leaf extracts, respectively. Frontiers in Plant Science 3.1 Zinc oxide nanoparticles characteristics The leaves were first cut in 1 in square and dissected into a small slice
under a stereomicrosopy (Motic SMZ-168 Series; Motic, Hong-Kong,
China). The slide was put on glass slide. For superoxide analysis, samples
were incubated in 5 mM MitoSOX Red in darkness for 30 min at room
temperature. After three washes, the plant tissues were immediately
imaged with a Leica SP8 confocal laser-scanning microscope (SP8) with
the excitation/emission at 405/516-580 nm (Leica Microsystems,
Wetzlar, Germany) equipped with an HC PL CS2 20×/0.75. The focus of the present study was to test the hypothesis if the
biologically synthesized ZnO NPs using pecan (C. illinoinensis) leaf
extract could be used as an environmentally friendly alternative
nanofertilizer or growth stimulating agent for enhanced
physiological performance, nutrient content, and antioxidant
properties of mustard (B. juncea). While the reaction between Zn
(NO3)2.6H2O and pecan extract progressed, the color transformation
of the reaction mixture from light green to creamy yellow after the
incubation period indicated the biosynthesis of ZnO NPs. The optical
absorption band of ZnO NPs was analyzed by UV-vis spectrometer to
monitor and confirm the formation and stability of the nanoparticles
(Figure 1). The absorption spectra of the green synthesized ZnO NPs
showed a maximum optical absorption peak at 362 nm. The peak
recorded between 320 and 380 nm could also be associated with
phenolic compounds (Ozsoy et al., 2008), which are involved in the
reduction and stabilization of ZnO NPs. The peak pattern observed
matches the typical characteristic of ZnO NPs (Sharmila et al., 2018). Despite the surface plasmon resistance (SPR) is a function of the
diameter, shape and size distribution of ZnO NPs (Narayana et al.,
2020) other studies also reported plasmon peak appears between 320
to 380 nm (Zare et al., 2017; Salama et al., 2019). Similarly, free
electrons present SPR. 2.12 Statistical analysis The experiment was carried out in four replicates and the data
was subjected to one-way analysis of variance (ANOVA) using R v 3.5
(http://www.R-project.org) and the agricolae package (Mendiburu,
2013) and expressed as mean values ± standard error. Tukey multiple
comparison test (significance level 5%) was used to calculate the
differences between each concentration level of ZnO NPs. Sigma Plot
Software (Version 14.5) was used for graphical presentation. The leaves obtained after 60 days after treatment were also subjected
to SEM to detect the accumulation of ZnO NPs in the leaf. Briefly, the
freeze-dried leaf samples (24 hrs at 50°C) were sectioned and sputter-
coated with carbon and affixed on an aluminum stub. Then the samples
were then imaged with SEM with EDX on a JEOL JSM-6610 (Oxford
Instruments). Percent zinc and other nutrients were calculated. 2.10 Total flavonoids content in the leaves The total flavonoid content in B. juncea leaves was determined by
the colorimetric method of aluminum trichloride as described by
Chang et al. (2002). A 0.5 mL aliquot of the ethanol extract of B. juncea leaves was mixed with 2.8 mL of water, 1.5 mL of 95% ethanol,
0.1 mL of 10% aluminum trichloride and 0.1 mL of potassium acetate
(1 M). The mixture was vortexed and allowed to stand for 30 min. The
absorbance was measured with a UV-Vis spectrophotometer
(SpectraMax® PLUS 384) at 424 nm. Quercetin was used as a
standard solution. The total flavonoid content was expressed as
quercetin equivalents (mg QE g-1 dry leaf). 2.11 Antioxidant activity FTIR analysis was carried out to analyze the composition, the
nature of the functional groups, and the purity as well as identify the
potential mechanism of the ZnO NPs synthesis. The observed FTIR
spectrum for ZnO NPs showed peaks at 3742, 3215, 2765, 1641, 1251 To assess the effect of ZnO NPs treatment on the antioxidant
potentials of mustard plants the radical 2, 2-diphenyl-1- 05 frontiersin.org 10.3389/fpls.2023.1108186 Geremew et al. FIGURE 1
UV-Vis absorbance spectra of ZnO NPs synthesized using pecan leaf
extracts. According to the XRD pattern, ZnO NPs displayed sharp peaks at
2q values of 31.61°, 33.92°, 36.74°, 47.61°, 56.67°, 62.95°, 68.03°, and
71.65° (Figure 3). These peaks, respectively, correspond to the
diffraction planes 100, 002, 101, 102, 110, 103, 112 and 201, which
confirmed the hexagonal wurtzite ZnO NPs structure (Bala et al.,
2015). These values of ZnO NPs were in good agreement with the
standard value (JCPDS No. 36-1415) (Jabeen et al., 2017), reflecting
the phase purity of ZnO NPs. All peaks were appropriated to the ZnO
NPs structure reported by Chen et al. (2008) and by Archana et al. (2016). The crystallite size was about 53.2 nm as computed by the
Scherrer formula. DLS technique was applied to determine the hydrodynamic
diameter of ZnO NPs in the aqueous suspension. The particle
diameter distribution showed a stable colloidal suspension of ZnO
NPs with a mean size of 84.5 nm (Figure 4A). This size is relatively
larger than the theoretical size of the ZnO NPs computed using XRD,
indicating the agglomeration of the nanoparticles attributable to the
presence of ions and phytochemicals such as capping and stabilizing
agents attached to surfaces of ZnO NPs in aqueous suspension
(Jamdagni et al., 2018). However, the polydispersity index (PDI) of
0.37 in the present study exhibited monodispersion and homogeneity
of the NPs in the medium. As a proxy of the stability of biologically
synthesized ZnO NPs, negative zeta potential of -22.4 mV was
recorded (Figure 4B), ascertaining the efficacy of phytochemicals in
pecan leaf extract as capping agents in the stabilization of the
particles. The negative zeta potential value of ZnO NPs could be
ascribed to negatively charged capping agents attached to the surface
of nanoparticles (Wei et al., 2020). 2.11 Antioxidant activity Conventionally, the zeta potential
values between + 25 and −25 mV mark a stable suspension of
nanoparticles (Kuznetsova and Rempela, 2015; Murali et al., 2017),
which validates the high stability (−22.4 mV) of synthesized ZnO NPs
colloidal suspension in this study. and 704 cm-1 (Figure 2). Pecan leaf extract exhibited various
functional group stretches between 3511 cm-1 and 652 cm-1. However, the peaks detected in the extract were observed to shift in
the ZnO NPs inferring the role of different functional groups in the
bioreduction and stabilization of ZnO NPs (Geremew et al., 2022). The ZnO NPs exhibited strong transmittance spectra at 3742 cm-1
representing the N-H stretch strong amines group (Umamaheswari
et al., 2021; Geremew et al., 2022), 3215 cm-1 associated with the
hydroxyl group stretching vibration of phenol or flavonoids (Adil
et al., 2019). In addition, the 1641 cm-1, 2765 cm-1 and 1251 cm- 1
bands linked to a carbonyl group (C=O) (Rastogi et al., 2011), alkyl
methylene group (C=H) and C-O group bonded with strong alcohols,
respectively. Furthermore, 1377 cm-1 representing C=C strong stretch
assigned to aromatic group and 712 cm-1 for C-H bond assigned to
strong mono-substituted aromatic benzene group (Geremew
et al., 2022). Additionally, the shape and surface morphology of pecan leaf
extract-mediated ZnO NPs were assessed by SEM. SEM images of
ZnO NPs at different magnifications are shown in Figures 5A, B. The
SEM analysis depicted star-shaped ZnO NPs with slight
agglomeration and regular morphology. The elemental composition
and chemical purity of ZnO NPs were studied by EDX Spectroscopy. EDX analysis revealed that zinc is the primary constituent (45%) with
strong peaks at 1, 8.6 and 9.6 keV due to the SPR effect of ZnO NPs The purity, crystalline nature and size of the fabricated ZnO NPs
were measured by XRD analysis in the scanning angle (2q). FIGURE 2
Comparison of the FTIR spectra of the pecan leaf extract and ZnO
NPs. Each peak in the ZnO NPs indicates the functional group of the
phytochemical involved in nanoparticle synthesis. FIGURE 2
Comparison of the FTIR spectra of the pecan leaf extract and ZnO
NPs. Each peak in the ZnO NPs indicates the functional group of the
phytochemical involved in nanoparticle synthesis. FIGURE 3
X-ray diffractometer patterns for biosynthesized ZnO NPs using pecan
leaf extract. FIGURE 3
X-ray diffractometer patterns for biosynthesized ZnO NPs using pecan
leaf extract. 2.11 Antioxidant activity FIGURE 3
X-ray diffractometer patterns for biosynthesized ZnO NPs using pecan
leaf extract. FIGURE 2
Comparison of the FTIR spectra of the pecan leaf extract and ZnO
NPs. Each peak in the ZnO NPs indicates the functional group of the
phytochemical involved in nanoparticle synthesis. 06 Frontiers in Plant Science frontiersin.org Geremew et al. 10.3389/fpls.2023.1108186 B
A
FIGURE 4
Size distribution (A) and zeta potential (B) of ZnO NPs obtained using aqueous extracts of pecan leaves. B
C
A
FIGURE 5
SEM micrographs 1102X (A) and 2340X (B) and, EDX spectra (C) of synthesized ZnO NPs using pecan leaf extract as a reducing agent. The inset bar plot
of the EDX showed the percent weight of the proportion of Zn, Al, O, C, Na and Mg. F
ti
i
Pl
t S i
f
ti
i
07 B
A FIGURE 4
Size distribution (A) and zeta potential (B) of ZnO NPs obtained using aqueous extracts of pecan leaves. FIGURE 4
Size distribution (A) and zeta potential (B) of ZnO NPs obtained using aqueous extracts of pecan leaves. B
C
A
FIGURE 5
SEM micrographs 1102X (A) and 2340X (B) and, EDX spectra (C) of synthesized ZnO NPs using pecan leaf extract as a reducing agent. The inset bar plot
of the EDX showed the percent weight of the proportion of Zn, Al, O, C, Na and Mg. B
C
A B FIGURE 5
SEM micrographs 1102X (A) and 2340X (B) and, EDX spectra (C) of synthesized ZnO NPs using pecan leaf extract as a reducing agent. The inset bar plot
of the EDX showed the percent weight of the proportion of Zn, Al, O, C, Na and Mg. 07 Frontiers in Plant Science frontiersin.org 10.3389/fpls.2023.1108186 Geremew et al. (Figure 5C). In addition, carbon, oxygen, sodium and magnesium
were detected as elemental components that might be associated with
the pecan leaf extract used for the synthesis of the nanoparticles. Aluminum was detected as a major element due to the grid used. that growth of the Oryza sativa and Solanum lycopersicum plants,
respectively, significantly decreased at 100 and 200 ppm of ZnO NPs
treatment. This suggests that the effect of ZnO NPs on plant growth
may strongly depend on the plant species (Garcı́a-Gómez et al., 2017)
and its dose. 3.2 Effect of ZnO NPs on mustard
plant growth Understanding the effect of nanoparticles synthesized on plant
growth and development is a significant metric of toxicity and
evaluation tool prior to agricultural application at a large scale. Plant growth traits such as height, leaf area and biomass are
extensively used as biomarkers for phytotoxicity (Ali et al., 2015). In the present study B. juncea plants exposed to the different
concentrations of ZnO NPs (20, 40, 60, 80, 100 or 200 mg L-1),
enhanced the values for all the growth traits such as an increase in
height, leaf area, and shoot dry weight compared to the control
(distilled water) at early vegetative stage (Table 1). Phytotoxicity test
on mustard percent seed germination is provided in Supplementary
Figures 2 A-C. Maximum leaf area (85.5 cm2), height (77.4 cm), and
shoot dry weight (46.5 g) were found to be 52%, 45% and 78%,
respectively, higher than the control (40.91 cm2, 42.91 cm and 10.32
g) in plants treated with 200 mg L_1 ZnO NPs (Table 1). At 100 and
200 mg L-1 these traits were significantly higher than plants in the
control (P < 0.05). However, a significant reduction in root dry weight
at higher concentrations (100 and 200 mg L-1 ZnO NPs) was
recorded. Though the function of nanoparticles depends on their
properties and methods of synthesis (Liu et al., 2020), with the
application of chemically synthesized ZnO NPs Rao and Shekhawat
(2014) reported a dose-dependent significant reduction in shoot
length of B. juncea under higher concentrations (1000 and 1500 mg
L-1). Similarly, other research showed that the addition of ZnO NPs
enhanced the growth of Lolium perenne (Lin and Xing, 2007), Allium
cepa (Laware and Raskar, 2014), Olea europaea (Micheli et al., 2018),
Brassica nigra (Zafar et al., 2016), Cicer arietinum (Burman et al.,
2013), Capsicum annuum (Deore et al., 2010), Capsicum annuum
(Datir et al., 2012), Zea mays (Neto et al., 2020), Gossypium hirsutum
(Venkatachalam et al., 2017), Triticum aestivum (Munir et al., 2018),
Brassica oleracea (Awan et al., 2021) and Cucumis sativus (Zaho et al.,
2013). In contrast, Chen et al. (2018) and Wang et al. (2018) found 2.11 Antioxidant activity The increase in plant height and leaf area observed in response to
the nanoparticles might be associated with nutritional behavior of
particles or dissociated ions as well as the intricate function of Zn on
crucial processes such as plant growth and development (Mukherjee
et al., 2016). Studies have also highlighted that the increase in
vegetative growth is linked to the role of zinc in controlling
enzymes, protein synthesis, cell elongation, structural stability of
cell membrane (Cakmak, 2008; Boonchuay et al., 2013) and
speeding up metabolism (Singh et al., 2013). Overall, our results
support the growth-promoting potential of ZnO NPs (Dimkpa et al.,
2017; Neto et al., 2020) due to dissociated Zn+2 which can play a
prominent role in the synthesis of tryptophan a precursor for the
biosynthesis of auxin a plant growth hormone (Brennan, 2005; Faizan
and Hayat, 2019; Srivastava et al., 2021). Furthermore, the observed
improved mustard plant growth could emanate from the positive
effect of ZnO NPs on photosynthesis (Xu et al., 2018). Different letters denote significant differences (p ≤0.05) among ZnO NPs concentrations. RWC, relative water content; LAI, leaf area Index; MSI, membrane stability index; SDW= shoot dry weight;
RDW= root dry weight; Pn, net photosynthesis; E, transpiration rate; gs, stomatal conductance and Ci, internal carbon dioxide concentration. Frontiers in Plant Science Frontiers in Plant Science
08 3.3 Effects of ZnO NPs on physiological
traits of B. juncea In contrast, Wang et al. (2016) have shown a decrease in the
expression of chlorophyll biosynthesis and photosystem-associated
genes that eventually reduced chlorophyll a and b in Arabidopsis
plants that received ZnO NPs. In addition to the effect of ZnO NPs on gas exchange measures,
effects on membrane stability index (MSI) and leaf relative water
content (RWC) were determined. Provision of 200 mg L-1 ZnO NPs
overall increased MSI and RWC significantly by 55% and 47%,
respectively, followed by 42% and 38% correspondingly with 100
mg L-1 (P < 0.05; Table 1) over the control. The increase in MSI could
be explained by the fundamental roles of zinc in the maintenance of
membranes integrity and in reducing the effect of lipid peroxidation
due to the accumulation of reactive oxygen species (Bagci et al., 2007;
Wang et al., 2018; Rai-Kalal and Jajoo, 2021; Noohpisheh et al., 2021). Similarly, the addition of ZnO NPs improved membrane stability and
plant water status of eggplant (Semida et al., 2021). On the other
hand, the augment in RWC might be associated with water potential
adjustment owing to the increased uptake of water and macro- and
micro-nutrients (Semida et al., 2021), particularly Fe, K, and Zn
accumulation in the presence of ZnO NPs (Table 2). Furthermore,
higher RWC in ZnO NPs treated mustard plants might be due to
improved acquisition of osmolytes as supported by the increased MSI
(Geremew et al., 2021). 3.3 Effects of ZnO NPs on physiological
traits of B. juncea Several studies have shown that nanoparticle exposure
significantly altered the total chlorophyll content and
photosynthetic performance in various plants in concentration
gradients (Baskar et al., 2018; Wang et al., 2018). Contrastingly, in
our study, the treatment of mustard plants with ZnO NPs revealed an
increase in the total chlorophyll content and their response was
concentration-dependent (Table 1; Figure 6). The augment in the
SPAD values significantly varied between concentrations (P < 0.05). Among the different tested concentrations of ZnO NPs, the
application of 200 mg L-1 of ZnO NPs proved to be most effective
and increased the SPAD by 53% over the control. Spectrometric
analysis was applied to further examine the effect of ZnO NPs on the
different photosynthetic pigments content (chlorophyll a, chlorophyll
b, and carotenoid). The data showed an overall significant increase in
chlorophyll a, chlorophyll b, and carotenoids (P < 0.05) in the TABLE 1 Effects of ZnO NPs application on morpho-physiological traits of B. juncea. Treatment
(mg L-1)
Height
(cm)
LAI
(cm2)
RWC
(%)
MSI
(%)
SPAD
SDW
(g)
RDW
(g)
Pn (µmol (CO2)
m-2s-1)
E (mmol
m−2 s−1)
gs (mmol
m−2 s−1)
Ci
(ppm)
Control (0)
42.91d
40.91de
51.82e
40.59d
35.75e
10.32e
35.33a
16.64d
0.57c
115.74e
455.6de
20
41.25d
39.54e
49.01e
37.37d
38.19e
12.25e
24.28b
14.38d
0.52c
120.47e
378.3e
40
43.37d
45.29de
50.44e
40.92cd
41.95de
13.51de
20.48b
18.57d
0.73b
125.11e
470.4d
60
52.45 c
48.97d
64.31d
50.82c
46.68d
16.25d
19.12c
20.33cd
0.96ab
144.86d
488.2cd
80
54.41 c
58.58c
76.75c
60.84bc
52.26c
25.71c
18.59c
21.35c
1.01a
180.82c
502.4c
100
61.47 b
68.62b
83.03b
70.21b
60.87b
33.45b
7.73d
30.22b
1.33a
203.68b
602.2b
200
77.44 a
85.46a
97.18a
89.88a
76.66a
46.53a
4.92d
39.21a
1.38a
288.2a
1089.4a TABLE 1 Effects of ZnO NPs application on morpho-physiological traits of B. juncea. TABLE 1 Effects of ZnO NPs application on morpho-physiological traits of B. juncea. Frontiers in Plant Science 08 frontiersin.org 10.3389/fpls.2023.1108186 Geremew et al. mustard plants treated with ZnO NPs (Table 1). The mustard plants
that received ZnO NPs (200 mg L–1) revealed the highest values of Pn
(58%), gs (60%), Ci (58%), and E (59%) in comparison with control
plants. In contrast to our results, Faizan and Hayat (2019) have
reported the maximum decrease in photosynthetic parameters even at
200 ppm of ZnO NPs. Frontiers in Plant Science 3.3 Effects of ZnO NPs on physiological
traits of B. juncea The improvement in photosynthetic traits
following the exposure to ZnO NPs may be due to the boost in light
acquisition that further helps to shield the chloroplast from aging and
eventually leads to enhanced photosynthesis (Yang et al., 2006; Xu
et al., 2018). Metallic oxide nanoparticles can stimulate the net
photosynthesis rate in photosynthetic systems either directly or
indirectly affecting photosynthetic machinery in plants (Govorov
and Carmeli, 2007). ZnO NPs improve stability and photosynthetic
efficiency by enhancing antioxidant systems and boosting proline
accumulation (Faizan et al., 2018). It is claimed that enhanced
photosynthetic efficiency after the application of ZnO NPs could be
caused by the improved activity of the water-splitting system during
the light reaction, photochemical extinction, non-photosynthetic
quenching, maximum PSII efficiency, and heightened rubisco
activity (Yu et al., 2004; Siddiqui et al., 2018). High records of
transpiration rate following exposure to ZnO NPs can be related to
increased stomatal conductance (Table 1). FIGURE 6
Variation in photosynthetic pigments content of mustard leaves under
different concentrations of ZnO NPs. Values are mean standard error. Different letters denote significant differences (p ≤0.05) among
concentrations for each pigment. FIGURE 6
Variation in photosynthetic pigments content of mustard leaves under
different concentrations of ZnO NPs. Values are mean standard error. Different letters denote significant differences (p ≤0.05) among
concentrations for each pigment. mustard plant along with an increased ZnO NPs concentration than
the control (Figure 6). The maximum increase in chlorophyll a,
chlorophyll b, and carotenoids contents (51%, 46% and 56%,
respectively) were recorded in the mustard plants treated with 200
mg L−1 ZnO NPs (p < 0.05). The present results corroborate with the
findings of Prasad et al. (2012); Raliya et al. (2015); Venkatachalam
et al. (2017); Samreen et al. (2017) and Narendhran et al. (2016) who
studied the effect of ZnO NPs on the content of photosynthetic
pigments in cotton, peanut, tomato, mung bean and sesame,
respectively. This increase in photosynthetic pigments can be
rationalized based on the contribution of zinc in chlorophyll
synthesis and development and protochlorophyllide biosynthesis
(Faizan and Hayat, 2019). Also, this might be due to metal
nanoparticles being powerful amplifiers of photosynthetic
effectiveness that in parallel can cause light absorption by
chlorophyll, as it causes the transfer of energy from chlorophyll to
nanoparticles (Mohsenzadeh and Moosavian, 2017). 3.3 Effects of ZnO NPs on physiological
traits of B. juncea The addition of
ZnO NPs leads to more nitrogen uptake and subtly stimulates
nitrogen metabolism invaluable for chlorophyll molecules synthesis
(Dimkpa et al., 2019). The underlining mechanism in the increased
chlorophyll content as well could be linked to the role of Zn as a vital
nutrient in the biosynthesis of chlorophyll (Faizan and Hayat, 2019). Furthermore, progress in the translation of chlorophyll biosynthetic
genes, rate of chlorophyll aging, and associated proteins in the
photosystem antenna complex could be ascribed to the rise in
chlorophyll content with the addition of ZnO NPs (Bajguz and
Asami, 2005; Sadeghi and Shekafandeh, 2014; Faizan et al., 2021). In contrast, Wang et al. (2016) have shown a decrease in the
expression of chlorophyll biosynthesis and photosystem-associated
genes that eventually reduced chlorophyll a and b in Arabidopsis
plants that received ZnO NPs. mustard plant along with an increased ZnO NPs concentration than
the control (Figure 6). The maximum increase in chlorophyll a,
chlorophyll b, and carotenoids contents (51%, 46% and 56%,
respectively) were recorded in the mustard plants treated with 200
mg L−1 ZnO NPs (p < 0.05). The present results corroborate with the
findings of Prasad et al. (2012); Raliya et al. (2015); Venkatachalam
et al. (2017); Samreen et al. (2017) and Narendhran et al. (2016) who
studied the effect of ZnO NPs on the content of photosynthetic
pigments in cotton, peanut, tomato, mung bean and sesame,
respectively. This increase in photosynthetic pigments can be
rationalized based on the contribution of zinc in chlorophyll
synthesis and development and protochlorophyllide biosynthesis
(Faizan and Hayat, 2019). Also, this might be due to metal
nanoparticles being powerful amplifiers of photosynthetic
effectiveness that in parallel can cause light absorption by
chlorophyll, as it causes the transfer of energy from chlorophyll to
nanoparticles (Mohsenzadeh and Moosavian, 2017). The addition of
ZnO NPs leads to more nitrogen uptake and subtly stimulates
nitrogen metabolism invaluable for chlorophyll molecules synthesis
(Dimkpa et al., 2019). The underlining mechanism in the increased
chlorophyll content as well could be linked to the role of Zn as a vital
nutrient in the biosynthesis of chlorophyll (Faizan and Hayat, 2019). Furthermore, progress in the translation of chlorophyll biosynthetic
genes, rate of chlorophyll aging, and associated proteins in the
photosystem antenna complex could be ascribed to the rise in
chlorophyll content with the addition of ZnO NPs (Bajguz and
Asami, 2005; Sadeghi and Shekafandeh, 2014; Faizan et al., 2021). frontiersin.org 3.4 Flavonoid content and total
antioxidant capacity Treatment (mg/
L)
Macronutrients (ppm)
Micronutrients (ppm)
Ca
K
Mg
Na
P
B
Cu
Fe
Mn
Zn
Control (0)
25221 ± 11
21302.0 ± 47
2777.7 ±
11
7761.7 ± 9.21
12491.5 ± 2
53.1 ± 0.4
6.9 ±
0.2
65.4 ± 0.34
15.93 ±
0.1
58.5 ± 0.4
20
23054.9 ±
26
23769.7 ± 38.4
2842 ± 4.8
7665 ± 14.7
8787.8 ± 18
51.3 ± 0.4
5.2 ±
0.4
69.92 ±
0.66
15.7 ± 0.2
76.1 ± 0.2
40
22564.43+7
24474.6 ±
26.26
2470.7 ±
3.1
7336.75 ±
5.11
10008.1 ±
15
41.2± 0.2
4.6 ±
0.1
75.66 ±
0.66
10.4 ± 0.1
93.8 ± 0.3
60
21889.6 ±
26
25426.5 ± 44.3
2363.1 ±
4.9
6790.2 ±
10.88
9613 ±
21.74
47.6 ± 0.4
5.5 ±
0.1
88.1 ± 0.6
16.7 ± 0.3
101 ± 0.5
80
21810.4 ±
25
26684.6 ±
34.34
2576.71 ±
3
6529.2 ±
13.22
9139.1 ± 33
39.8 ± 0.2
4.8 ±
0.3
89.5 ± 0.4
11.56 ±
0.3
149.9 ±
0.3
100
21790.8 ±
25
30652.8 ±
26.26
2537.58 ±
7
5459.5 ± 4.99
9411.5 ±
17.4
41.1 ± 0.4
5.9 ±
0.3
115.3+0.9
13.04 ±
0.1
247.5 ±
1.3
200
18927 ± 19
32145.9 ±
54.54
2305.7 ±
3.7
4364 ±
7.1111
7549.6 ±
12.3
40.23 ±
0.1
3.9 ±
0.1
152.7 ±
0.88
11.7 ± 0.1
338.9 ±
0.4
Values are mean ± standard error. findings (Ushahra et al., 2014; Singh et al., 2018; Iziy et al., 2019) that
reported a positive impact of ZnO NPs on antioxidant activities of
different plant species. The antioxidant activity in the mustard leaves
of plants provided with different concentrations of biosynthesized
ZnO NPs confirms that the addition of ZnO NPs promotes the
biosynthesis of compounds with antioxidant activity like flavonoids
and phenols. Nevertheless, the augment or decline of antioxidant
activity is a function of the balance between the antioxidant activity of
metabolites and the degree of oxidative stress (Baskar et al., 2018;
Srivastava et al., 2021). On the other hand, our results contradict the
findings of Javed et al. (2017) who studied the effect of ZnO NPs on
the TFC of Stevia rebaudiana and demonstrated a reduction in the
TFC of the plants treated with 100 and 1,000 mg L−1 of ZnO NPs,
compared to the control plants. Zhu et al. 3.4 Flavonoid content and total
antioxidant capacity Total flavonoid contents (TFC) in response to all six ZnO NPs
concentrations are shown in Figure 7. This study indicated that the
application of ZnO NPs significantly induced total flavonoid synthesis
in B. juncea leaves in a concentration-dependent pattern compared to
the control (P < 0.05). To overcome oxidative stress due to the
metallic nanoparticles, plants activate their antioxidant defense
system encompassing phenols and flavonoids which serve as metal
chelators (through electron donation) and natural scavengers of ROS
(Ilboudo et al., 2012; Gupta and Pandey, 2020; Hussain et al., 2021). In the current study, maximum TFC in mustard leaves (58 µg g-1) was
recorded at the 200 mg L-1 ZnO NPs treatment followed by a 100 mg ZnO NPs amplify the photosynthetic efficiency by increasing the
chlorophyll capacity to absorb light through energy transfer from
ZnO NPs to chlorophyll molecules, in turn, triggers the boost in
photosynthetic pigment contents (Faizan and Hayat, 2019). In the
present study, consistent with an increase in chlorophyll content,
measures of gas exchange parameters such as net photosynthetic rate
(PN), stomatal conductance (gs), internal CO2 concentration (Ci),
and transpiration rate (E) were increased significantly (P < 0.05) in 09 frontiersin.org 10.3389/fpls.2023.1108186 Geremew et al. TABLE 2 Effects of ZnO NPs application on macro- and micronutrients of B. juncea leaves. 3.4 Flavonoid content and total
antioxidant capacity (2013) highlighted that the
antioxidant capacity could be affected by the levels of metals like zinc,
which function as cofactors for enzymes, signaling molecules and
transcription factors. Overall, the application of ZnO NPs resulted in L-1 with TFC of 43 µg g-1 (Figure 7). In support of our results, Zafar
et al. (2016) have also reported increase in TFC in one of the closely
related species of B. juncea, namely B. nigra seedlings treated with
ZnO NPs. Comparably, the ZnO NPs treatment was shown to
increase the contents of TFC in Glycyrrhiza glabra seedlings
(Oloumi et al., 2015), Persicaria hydropiper (Hussain et al., 2021)
Raphanus sativus (Mahmoud et al., 2019) and Vicia faba (Mogazy
and Hanafy, 2022) plants. The use of nanoparticles as oxidative stress
producers resulted in the production of secondary metabolites like
flavonoids and phenols that function as ROS scavengers (Javed et al.,
2017; Vela´zquez-Gamboa et al., 2021; Mogazy and Hanafy, 2022). The antioxidant potential was measured using DPPH free radical
scavenging assay in the B. juncea leaves under the different ZnO NPs
treatments. Amendment of B. juncea plants using ZnO NPs revealed
that antioxidant activity in leaves was significantly boosted from 54%
(in 20 mg L-1 of ZnO NPs) to 97% (in 200 mg L-1 of ZnO NPs) relative
to the control (35%) (Figure 8). These results corroborated previous FIGURE 8
The antioxidant activity of ZnO NPs measured using DPPH radical
scavenging assay. AA indicates ascorbic acid. Different letters denote
significant differences (p ≤0.05) across different concentrations for a
particular antioxidant. Values are mean standard error. FIGURE 7
Total flavonoid contents of mustard leaves under different
concentrations of ZnO NPs. Values are mean standard error. Different
letters denote significant differences (p ≤0.05) in total flavonoid
content among ZnO NPs concentrations. FIGURE 7
Total flavonoid contents of mustard leaves under different
concentrations of ZnO NPs. Values are mean standard error. Different
letters denote significant differences (p ≤0.05) in total flavonoid
content among ZnO NPs concentrations. FIGURE 8
The antioxidant activity of ZnO NPs measured using DPPH radical
scavenging assay. AA indicates ascorbic acid. Different letters denote
significant differences (p ≤0.05) across different concentrations for a
particular antioxidant. Values are mean standard error. FIGURE 7
Total flavonoid contents of mustard leaves under different
concentrations of ZnO NPs. Values are mean standard error. Different
letters denote significant differences (p ≤0.05) in total flavonoid
content among ZnO NPs concentrations. 3.5 Macro and micro-nutrient content An ICP-OES analysis was carried out to measure the amount of P,
K, Ca, B, Cu, Mg, Fe, Mn, Na, and Zn content in ZnO NPs exposed
mustard plant leaf samples. The application of ZnO NPs did
significantly affect the mustard green concentration of some
essential and beneficial nutrients both positively and negatively
including Zn (Table 2). The application of ZnO NPs had
significantly increased the K, Fe and Zn accumulation and in
contrast, significantly decreased Ca and Na content following dose-
dependent (P < 0.05). Besides, the ZnO NPs had no significant effect
on Mg, P, Cu, B and Mn (P > 0.05). The K, Fe and Zn accumulation in
plants subjected to 200 mg L-1 ZnO NPs was 34%, 17% and 83%
higher than that of the control group, respectively. Compared with
the control, the content of Ca and Na significantly decreased by 33%
and 67%, respectively, in mustard plants exposed to the highest
concentration of the nanoparticles (200 mg L-1). The lowest P
content was observed under the highest ZnO NPs concentration
(200 mg L-1). This could be due to the toxic effect of the nanoparticles
on P-solubilizing microorganisms and decreased enzyme activity and
consequently impacting P uptake of plants (Chai et al., 2015; Raliya
et al., 2016). In agreement with the present study, the application of
ZnO NPs increased K, Fe and Zn in different plants (Dimkpa et al.,
2019; Grangah et al., 2020; Semida et al., 2021). Therefore,
amendment with Zn ONPs improved the nutritional status of
B. juncea. Considering the discrepancy in magnitude of Zn content between
the ICP and SEM analyses as well as the size of the ZnO NPs used
(84.5 nm), the dissolution of ZnO NPs to preferentially absorbed Zn
ions or directly adsorbed ZnO NPs could result in the higher Zn
content in plant leaves (Xu et al., 2018; Pejam et al., 2020). Despite the
mechanism of absorption and translocation of nanoparticles from the
soil to the different plant tissues are still the subject of research. The
absorption and translocation of metallic and metallic oxide
nanoparticles counterparts occur in a similar way as micro and
micronutrients (Fraceto et al., 2016). 3.4 Flavonoid content and total
antioxidant capacity FIGURE 7
Total flavonoid contents of mustard leaves under different
concentrations of ZnO NPs. Values are mean standard error. Different
letters denote significant differences (p ≤0.05) in total flavonoid
content among ZnO NPs concentrations. 10 Frontiers in Plant Science frontiersin.org Geremew et al. 10.3389/fpls.2023.1108186 3.6 Zn accumulation and ROS in
mustard leaves 3.6 Zn accumulation and ROS in
mustard leaves the enrichment of Zn2+ which could increase the nutritional quality
and antioxidant activity for human consumption as a functional food. Mustard leaves were examined under SEM to detect the
bioaccumulation of ZnO NPs in the leaves. In support of the ICP
analysis, the SEM coupled with the EDX analysis revealed that Zn
content in B. juncea leaf increased with a dose of ZnO NPs
supplemented from 3% to 65% weight (Figure 9). However, the
amounts of Zn detected at the low concentrations of nanoparticles
were not significant (P > 0.05). On the other hand, the maximum Zn
accumulation (65%) was observed in 200 mg L-1 ZnO NPs treated
mustard leaves. The aggregation of ZnO NPs in the leaves was evident
in several spots of Zn in the SEM images. The high accumulation of
Zn has also been reported in plant tissues by various recent findings
performed with Zn-based NPs (Singh and Kumar, 2016; Zoufan et al.,
2020; Alsuwayyid et al., 2022). The SEM images also provide
supporting evidence on how ZnO NPs can improve the nutritional
status or zinc content of mustard plants by strengthening the vascular
system (Pejam et al., 2021) and enhancing nutrient uptake efficiency
by regulating nanoscale plant pores (Abd El-Aziz et al., 2019;
Srivastava et al., 2021). 3.5 Macro and micro-nutrient content It has been thought that
nanoparticles are assimilated by the root hairs and further proceed
through cellular pores following either the symplastic or apoplastic or
a combination of both pathways (Rico et al., 2011; Lambreva et al.,
2015; Rajput et al., 2018; Usman et al., 2020). It is apparent from
recent studies that Zn may be accumulated in plant tissues and
cellular and sub-cellular organelles and regulate cellular
organizations (Bradfield et al., 2017; Wang et al., 2018; Radi et al.,
2018). The uptake of NPs by several plants led to their accretion in Though the mechanisms of how ZnO NPs affect the content of
other nutrients have not been clearly established Haynes (1980) and
Dimkpa et al. (2019) suggested that divalent metal ions (Zn2+) in the
root change potential across the cell membrane thus enabling the
uptake of monovalent cations such as K. Alternatively, the influence
of Zn from the nanoparticles on the content of particular nutrients
may be linked to synergistic or antagonistic interactions and which
varies strongly with nutrient ratios (Dimkpa et al., 2017; Rietra et al.,
2017; Dimkpa et al., 2019; Geremew et al., 2021). FIGURE 9
SEM analysis showing Zn accumulation in mustard leaves from plants treated with different ZnO NPs concentrations. The bar plot from EDX shows the
percent weight of Zn detected from each leaf. FIGURE 9
SEM analysis showing Zn accumulation in mustard leaves from plants treated with different ZnO NPs concentrations. The bar plot from EDX shows the
percent weight of Zn detected from each leaf. 11 11 Frontiers in Plant Science frontiersin.org 10.3389/fpls.2023.1108186 Geremew et al. subcellular locations (Schwab et al., 2016; Faizan et al., 2018), Overall,
the uptake of the nanoparticles relies on the plant anatomy and shape,
composition and size of NPs (Wang et al., 2018). subcellular locations (Schwab et al., 2016; Faizan et al., 2018), Overall,
the uptake of the nanoparticles relies on the plant anatomy and shape,
composition and size of NPs (Wang et al., 2018). et al., 2020; Aazami et al., 2021). Phenols and flavonoid
accumulation recorded in this study is part of the adaptive response
acting as ROS scavengers either in conjunction with or individually of
antioxidative enzymes (Mogazy and Hanafy, 2022). 3.5 Macro and micro-nutrient content Despite the
accumulation of Zn in plant tissue that results in ROS increase, the
enhanced production of flavonoids with radical scavenging potential,
overall, in this current study indicated that no ZnO NPs toxicity was
noted in the mustard plants in terms of growth and physiological
performance. This suggests that ZnO NPs stimulate ROS signaling to
enhance the defense mechanism in mustard plants. p
g
The major forms of reactive oxygen species (ROS) includes
hydrogen peroxide (H2O2), superoxide (O2
.-
), singlet oxygen (1O2)
and the hydroxyl radical (HO-), mainly produced in the chloroplast,
mitochondria and peroxisomes during environmental stress in plants
(Das and Roychoudhury, 2014; Faizan et al., 2021). ROS signaling
from plant stomata plays a critical role in innate immunity and
defense mechanism (Castro et al., 2021; Mittler et al., 2022). In
stomatal closure, NADPH oxidase catalyzes the transfer of electrons
from NADPH to 1O2 form O2
.-, then to H2O2 (Sierla et al., 2016). Mitosox staining for superoxide showed that ROS are accumulated in
stomata (Figure 10A). When treated with 200 mg L-1, the level of ROS
is slightly enhanced. ROS is also accumulated on the leaf midrib and
minor lamina veins (Figure 10B). Fluorescent microscopy images
showed the ROS in the leaf vein coil structure of plants treated with
ZnO NPs. However, their effect on the disruption of physiological
processes such as chlorophyll content and photosynthesis were not
observed. Like other plant species, B. juncea might overcome such
effects of ROS with an intricate non-enzymatic and enzymatic
antioxidant system (Ali et al., 2008; Faizan et al., 2018; Faizan and
Hayat, 2019). According to Faizan and Hayat (2019) exogenous
application of ZnO NPs elevated the enzymatic defense
mechanisms by increasing the synthesis of catalase, peroxidase and
superoxide dismutase. On the other hand, zinc ions from the ZnO
NPs benefit in raising the expression of antioxidant genes in plants by
supporting non-enzymatic antioxidant production and eventually
overcoming the influence of ROS (Hassan et al., 2020; Adhikari 4 Conclusions This study highlights the effect of biosynthesized ZnO-NPs using
pecan leaves at different concentrations on B. juncea (mustard) plant
growth, chlorophyll content, relative water contents, membrane
stability, and net photosynthesis rate. Application of ZnO NPs up
to 200 mg L-1 enhances nutrient accumulation including Zn, Fe and
K, flavonoids and antioxidant potentials in mustard leaves and then
reduces the effect of ROS. Therefore, ZnO NPs can be potentially used
as a plant growth stimulant and as a novel soil amendment for
enhancing crop yields. Besides, the biofortification of B. juncea plants
with ZnO NPs helps to improve the nutritional quality of the crop and
perhaps potentiates its pharmaceutical effects. Moreover, further
investigations are required to examine the effect of ZnO NPs on
different secondary metabolites and the mechanisms at a molecular
level for extensible applications as nanofertilizers and the synthesis
of nanocomposites. B
A
FIGURE 10
Increase in reactive oxygen species (superoxide) in mustard leaves treated with ZnO NPs and stained with Mitosox. (A) Confocal images of the structure
of chloroplast and stomata. The boxed region represents the magnified region. (B) Bright field fluorescent images of the leaf veins. The intensity of the
dye represents the high accumulation of superoxide in the leaf veins. B xygen species (superoxide) in mustard leaves treated with ZnO NPs and stained with Mitosox. (A) Confocal images of the structure
mata. The boxed region represents the magnified region. (B) Bright field fluorescent images of the leaf veins. The intensity of the
gh accumulation of superoxide in the leaf veins. FIGURE 10
Increase in reactive oxygen species (superoxide) in mustard leaves treated with ZnO NPs and stained with Mitosox. (A) Confocal images of the structure
of chloroplast and stomata. The boxed region represents the magnified region. (B) Bright field fluorescent images of the leaf veins. The intensity of the
dye represents the high accumulation of superoxide in the leaf veins. 12 Frontiers in Plant Science frontiersin.org Geremew et al. 10.3389/fpls.2023.1108186 Supplementary material This work was partially funded by the United States Department
of Agriculture National Institute of Food and Agriculture (USDA-
NIFA) Evans-Allen Grant 180835-82601. The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fpls.2023.1108186/
full#supplementary-material SUPPLEMENTARY FIGURE 1 Data availability statement at Prairie View A&M University, United States for providing research
facilities to carry out the entire scientific investigation. Authors are
also thankful for the Surface Characterization Facility in the
Department of Chemistry and Physics. The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation. SUPPLEMENTARY FIGURE 2 SUPPLEMENTARY FIGURE 2
Phytotoxicity test of ZnO NPs on mustard seeds germination (A), partial view of
seedlings vary in height (B) and percent germination (C). Authors highly acknowledge the Cooperative Agricultural
Research Center in the College of Agriculture and Human Sciences Conflict of interest AG developed the concept including the methods, data curation,
formal statistical analysis, and writing of the original draft. LC
contributed to resources, funding acquisition, project administration,
evaluation of the experimental approach, assisted in the development
of the project idea and the review of the manuscript. SW conducted
ICP-OES analysis of nutrients and review. HW performed the ROS
analysis. NB and SR carried out the XRD analysis and verification of the
data as well as a review of the manuscript. EP gathered data related to
RWC, biomass and LA as well as participated in the synthesis of ZnO
NPs. PS provided the facility to carry out inflorescence analysis of ROS. AW verify the nomenclature of the plants used for the synthesis of ZnO
NPs as well as reviewed the manuscript. All authors contributed to the
article and approved the submitted version. 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 All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations,
or those of the publisher, the editors and the reviewers. Any product
that may be evaluated in this article, or claim that may be made by its
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Dopamine-induced calcium signaling in olfactory bulb astrocytes
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OPEN Timo Fischer*, Paula Scheffler & Christian Lohr* It is well established that astrocytes respond to the major neurotransmitters glutamate and GABA
with cytosolic calcium rises, whereas less is known about the effect of dopamine on astroglial cells. In the present study, we used confocal calcium imaging in mouse brain slices of the olfactory bulb, a
brain region with a large population of dopaminergic neurons, to investigate calcium signaling evoked
by dopamine in astrocytes. Our results show that application of dopamine leads to a dose-dependent
cytosolic calcium rise in astrocytes (EC50 = 76 µM) which is independent of neuronal activity and
mainly mediated by PLC/IP3-dependent internal calcium release. Antagonists of both D1- and D2-class
dopamine receptors partly reduce the dopaminergic calcium response, indicating that both receptor
classes contribute to dopamine-induced calcium transients in olfactory bulb astrocytes. Dopamine (DA) is one of the most important modulatory neurotransmitters in the mammalian brain. It has sev-
eral physiological functions in the central nervous system (CNS) including modulation of voluntary movement,
reward, sleep regulation, feeding, affect, attention, cognitive function and olfaction1. Besides that, dopamine is
involved in diverse diseases, such as schizophrenia, Parkinson’s disease and depression. To accomplish all these
functions, there are 11 populations of dopaminergic neurons distributed in the CNS2. Dopaminergic neurons in
the olfactory bulb (OB) are the most numerous among these cell groups3 and are subdivided in two subgroups:
the short axon (SA) cells and a subpopulation of external tufted (ET) cells4. Within the OB, dopaminergic neu-
rons have been reported to be found mainly in the glomerular layer, which is populated by a variety of differ-
ent periglomerular interneurons. 10–16% of them are supposed to be dopaminergic5,6. A common property of
the OB dopaminergic neurons is the ability to co-release dopamine and GABA from separate vesicle pools7–9. Dopaminergic neurons are the only catecholaminergic neurons located in the OB, hence they could be recog-
nized by the expression of tyrosine hydroxylase (TH), a rate-limiting enzyme in the biosynthesis of dopamine10,11. Dopamine acts on dopamine receptors that are metabotropic G protein-coupled receptors containing seven
transmembrane domains. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports University of Hamburg, Division of Neurophysiology, Hamburg, 20146, Germany. *email: timo.fischer@uni-
hamburg.de; christian.lohr@uni-hamburg.de Scientific Reports | (2020) 10:631 | https://doi.org/10.1038/s41598-020-57462-4 Dopamine induces dose-dependent calcium transients in olfactory bulb astrocytes.
l
d
b
h
l
l
d
h
d
fi
f Hence, all cells responding to dopamine were identified as astrocytes, while Fluo-8-loaded neurons
did not respond to bath application of dopamine with calcium signals (Fig. 2A,B). p
pp
p
g
( g
)
As shown in Fig. 2D, bath application of 100 µM dopamine led to transient monophasic or oscillating eleva-
tions in cytosolic calcium in both layers. Dopamine application for 30 seconds evoked a calcium response with an
amplitude of 163.9+/− 3.5% ΔF and an area of 2000.6+/− 69.1 ΔF*s (n = 328) in the external plexiform layer
and 169.1+/− 5.8% ΔF and an area of 1957.4+/− 137.7 ΔF*s (n = 130) in the glomerular layer (Fig. 2E). No
significant differences in amplitude and area of the responses were observed between the glomerular layer and
the external plexiform layer. Hence, in all following experiments, data of both layers were pooled. Application of
100 µM dopamine for several minutes evoked long-lasting calcium oscillations (Fig. 2F). For the following exper-
iments, we opted for a relatively short application of 30 s. To identify the optimal concentration of dopamine, we
established a dose-response curve for bath application of dopamine (Fig. 2G–I), starting with the lowest con-
centration of 3 µM dopamine and increasing stepwise up to 1000 µM. We monitored the amount of responding
astrocytes for every concentration. It became apparent that using 3 µM dopamine (n = 9) none of the astrocytes
showed any change in intracellular calcium, while at 10 µM dopamine (n = 41) one of 41 cells displayed a small
calcium response. When using 30 µM dopamine (n = 86), 41.6% of the astrocytes showed a response. At 100 µM
dopamine (n = 86) and above, every monitored astrocyte generated a prominent calcium elevation, indicating
that 100 µM dopamine is sufficient to activate all astrocytes containing dopamine receptors. Furthermore, using
a concentration of 100 µM is consistent with the experimentally determined EC50 of 76 µM dopamine (Fig. 2H). Dopamine-induced calcium transients are independent of neuronal influence and mediated
by internal calcium stores. The former experiments were performed in the presence of TTX to suppress
action potential firing and hence indirect effects by neurons. However, it cannot be excluded that dopamine elic-
ited action potential-independent local calcium rises in neurons that could lead to release of neurotransmitters
such as glutamate and GABA. Dopamine induces dose-dependent calcium transients in olfactory bulb astrocytes.
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f Dopamine induces dose-dependent calcium transients in olfactory bulb astrocytes. A
general issue in studies about astrocytes using chemical calcium indicators is the identification of astrocytes. Sulforhodamine 101 has been used to identify astrocytes in brain regions such as hippocampus and cortex but
fails to label astrocytes in the brain stem19,20. We found that astrocytes in the OB also fail to accumulate sulfor-
hodamine 101, however, a well-established approach for OB astrocyte identification is based on their response
to ATP, ADP and low concentrations of potassium21,22. Removal of external potassium induces calcium influx
into astrocytes, but not into neurons (Fig. 2A,B). Hence, the cells that did not respond to K+-free saline were pre-
sumed to be neurons. These cells also responded to 50 mM K+ with a large calcium transient, while 100 µM ADP
had no effect on the calcium concentration. Astrocytes, in contrast, not only responded to K+-free saline with a
calcium increase, but also to application of ADP due to expression of P2Y1 receptors21 (Fig. 2A,B). According to
the results mentioned above, we subsequently used the application of ADP for the identification of astrocytes and
to test their viability. y
In the present study, we aimed to establish whether olfactory bulb astrocytes in situ respond to dopamine
application. Results were collected in the glomerular layer and the external plexiform layer that comprise most of
the synapses involved in odor information processing in the olfactory bulb23. Experiments were performed in the
presence of TTX to suppress action potential firing and, hence, neuronal effects on astrocytic calcium. Both short
pressure application (500 µM dopamine, 2 s) and bath application (100 µM dopamine, 30 s) led to transient eleva-
tion in cytosolic calcium in OB astrocytes (Fig. 2C). Pressure application is a more local stimulation and enables
shorter application, followed by short elevations in cytosolic calcium. However, this procedure has considerable
disadvantages with regard to the comparability of the experiments. It is not possible to adjust a constant concen-
tration, as the dopamine-containing solution applied to the cells mixes with the bath solution with increasing
distance. For this reason we decided to perform all subsequent experiments using bath application. All cells that
responded to dopamine with calcium transients did also respond to ADP, which was used to identify astrocytes
(see above). OPEN Based on coupling to either Gαs,olf proteins or Gαi/o proteins to stimulate or inhibit the
production of the second messenger cAMP, respectively, dopamine receptors are classified as D1-class receptors
(including D1R and D5R) or D2-class receptors (including D2R, D3R and D4R)12–14.l g
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Although the actions of dopamine on neurons are well described, its influence on astroglia remains poorly
understood. There has been evidence for the presence of dopamine receptors leading to cAMP production and
subsequent PKA activation as well as NADH modulation and calcium signaling mediated by D1-class receptors in
cultured astrocytes of different brain regions15–17. In addition to the studies above describing dopamine-induced
calcium transients in cultured astrocytes, Jennings et al. (2017) and Xin et al. (2019) could demonstrate that
hippocampal and midbrain astrocytes in acute brain slices respond to dopamine with a cytosolic calcium rise. However, the effect of dopamine on astrocytes in other brain regions such as the olfactory bulb remains unknown
and has been investigated in the present study. We intended to elucidate whether olfactory bulb astrocytes respond to dopamine with calcium signaling and
to identify the receptor subtypes involved as well as the origin of the intracellular calcium. Our results show that
astrocytes respond to bath application of dopamine with calcium release from internal stores by activation of both
D1-class and D2-class receptors. Scientific Reports | (2020) 10:631 | https://doi.org/10.1038/s41598-020-57462-4 www.nature.com/scientificreports/ Results Distribution of olfactory bulb astrocytes and tyrosine hydroxylase-expressing neurons. To
analyze the distribution of OB astrocytes and TH-expressing neurons in the GL and EPL by immunohistochemis-
try, we used TH-Cre × tdTomatofl/fl mice and aimed to visualize astrocytes by anti-GFAP staining. GFAP-positive
astrocytes were located throughout the GL, which can be distinguished from the ONL by glomerular structures,
and the EPL, in which GFAP-expression was weaker compared to the GL (Fig. 1A). TH expression also exhibits its
highest density in the GL, but the EPL also contains some TH-positive neurons and neuronal processes (Fig. 1A). In the glomerular layer, dendrites of tdTomato-positive SA cells are closely intermingled with astrocyte processes,
suggesting a possible physiological interaction such as neurotransmitter release by SA cells and subsequently
activation of astrocytic dopamine receptors (Fig. 1B,C). Close proximity of dendrites of TH-positive neurons and
astrocyte processes could also be observed in the EPL, albeit at a lower density (Fig. 1D). In both layers, astrocyte
processes were in direct vicinity of tdTomato-positive varicosities, presumptive dopamine release sites18. Dopamine induces dose-dependent calcium transients in olfactory bulb astrocytes.
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f Since glutamate and GABA have been shown to trigger calcium signals in olfactory
bulb astrocytes22,24, indirect neuronal effects might contribute to the calcium transients in astrocytes evoked by
dopamine application. To elucidate whether dopamine-evoked neurotransmitter release contributed to the cal-
cium transients in astrocytes, we investigated dopamine-induced calcium transients in synaptic isolation. We first
tested whether repetitive application of dopamine results in rundown of the calcium response, e.g. due to receptor
desensitization. After multiple applications of dopamine, a rundown of the dopamine-induced calcium transients
could be observed (Fig. 3A). Compared to the first application, the second application evoked calcium transients
that were reduced by 8.0+/− 4.6% (n = 79; p < 0.001), whereas calcium transients evoked by the third applica-
tion were attenuated by 21.6+/− 3.4% (n = 79; p < 0.001; Fig. 3A,C). We then compared responses evoked by
dopamine application either in the presence of TTX (1 µM; Suppl. Fig. 1) or in a mix of GABAergic, glutamatergic Scientific Reports | (2020) 10:631 | https://doi.org/10.1038/s41598-020-57462-4 www.nature.com/scientificreports/ and purinergic antagonists (GABAA: Gabazine 10 µM; GABAB: CGP55845 10 µM; AMPA receptors: NBQX
10 µM; NMDA receptors: D-APV 50 µM; mGluR5: MPEP 2 µM; P2Y1: MRS2179 50 µM; A2A: ZM241385 0.5 µM;
Figure 1. Distribution of astrocytes and tyrosine hydroxylase-expressing neurons in the olfactory bulb. (A) GL, glomerular layer; ONL, olfactory nerve layer. Anti-GFAP staining (green) shows astrocytes. Cell bodies
of astrocytes are located in the GL and EPL. TH+ neurons are labeled by tdTomato in THCre × tdTomatofl/
fl mice (red) and are located mainly in the GL, but single neurons expressing tdTomato can be found in the
EPL. Scale bar: 50 μm. (B) Magnified view from A, highlighting an interglomerular connecting short axon
cell (arrowhead) in close proximity to astrocyte processes. Scale bar: 20 µm. (C) Magnified view from B,
highlighting short axon cell dendrites, varicosities (arrowheads) and astrocytic processes in detail. Scale bar:
10 µm. (D) Magnified view from A, highlighting localization of astrocytes and TH+ varicosities (arrowheads). Scale bar: 20 µm. Figure 1. Distribution of astrocytes and tyrosine hydroxylase-expressing neurons in the olfactory bulb. (A) GL, glomerular layer; ONL, olfactory nerve layer. Anti-GFAP staining (green) shows astrocytes. Cell bodies
of astrocytes are located in the GL and EPL. and purinergic antagonists (GABAA: Gabazine 10 µM; GABAB: CGP55845 10 µM; AMPA receptors: NBQX
10 µM; NMDA receptors: D-APV 50 µM; mGluR5: MPEP 2 µM; P2Y1: MRS2179 50 µM; A2A: ZM241385 0.5 µM;
sodium channels: TTX 1 µM) with the corresponding control application in the absence of receptor antagonists
to assess the contribution of these receptors to the dopamine-evoked response (blockermix, BM; Fig. 3B,C). In Dopamine induces dose-dependent calcium transients in olfactory bulb astrocytes.
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f TH+ neurons are labeled by tdTomato in THCre × tdTomatofl/
fl mice (red) and are located mainly in the GL, but single neurons expressing tdTomato can be found in the
EPL. Scale bar: 50 μm. (B) Magnified view from A, highlighting an interglomerular connecting short axon
cell (arrowhead) in close proximity to astrocyte processes. Scale bar: 20 µm. (C) Magnified view from B,
highlighting short axon cell dendrites, varicosities (arrowheads) and astrocytic processes in detail. Scale bar:
10 µm. (D) Magnified view from A, highlighting localization of astrocytes and TH+ varicosities (arrowheads). Scale bar: 20 µm. and purinergic antagonists (GABAA: Gabazine 10 µM; GABAB: CGP55845 10 µM; AMPA receptors: NBQX
10 µM; NMDA receptors: D-APV 50 µM; mGluR5: MPEP 2 µM; P2Y1: MRS2179 50 µM; A2A: ZM241385 0.5 µM;
sodium channels: TTX 1 µM) with the corresponding control application in the absence of receptor antagonists
to assess the contribution of these receptors to the dopamine-evoked response (blockermix, BM; Fig. 3B,C). In and purinergic antagonists (GABAA: Gabazine 10 µM; GABAB: CGP55845 10 µM; AMPA receptors: NBQX
10 µM; NMDA receptors: D-APV 50 µM; mGluR5: MPEP 2 µM; P2Y1: MRS2179 50 µM; A2A: ZM241385 0.5 µM;
sodium channels: TTX 1 µM) with the corresponding control application in the absence of receptor antagonists
to assess the contribution of these receptors to the dopamine-evoked response (blockermix, BM; Fig. 3B,C). In Scientific Reports | (2020) 10:631 | https://doi.org/10.1038/s41598-020-57462-4 www.nature.com/scientificreports/ www.nature.com/scientificreports
re.com/scientificreports/
Figure 2. Dopamine-triggered calcium response in olfactory bulb (OB) astrocytes. (A) Fluo-8 staining of
acute OB slices with example ROIs shown in idle state (left), in the presence of 100 µM ADP (middle) and
high potassium ACSF (right). (B) Example traces of cells that are considered to be a neuron (ROI1) and
an astrocyte (ROI2). (C) Dopamine-induced calcium responses of astrocytes by short pressure application
(500 µM, left) and bath application (100 µM, right). (D) Calcium responses of astrocytes evoked by application
of ADP (30 s, 100 µM) and dopamine (DA; 30 s, 100 µM) in both EPL (black trace) and GL (blue trace). (E)
Averaged amplitudes (left bars) and area (right bars) of DA-induced calcium responses in EPL and GL (error
bars: SEM). Sample size as specified in the bars: cells/slices/animals. n.s.: not significant. (F) Calcium response
of an astrocyte monitored during a 10-min application of DA (100 µM). (G) Calcium responses induced by
application of dopamine at different concentrations. Dopamine induces dose-dependent calcium transients in olfactory bulb astrocytes.
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f (H) Dose-response curve of dopamine-induced astroglial
calcium responses (area), normalized to application of 100 µM DA (+/−SEM). (I) Percentage of astrocytes
responding to dopamine application. Figure 2. Dopamine-triggered calcium response in olfactory bulb (OB) astrocytes. (A) Fluo-8 staining of
acute OB slices with example ROIs shown in idle state (left), in the presence of 100 µM ADP (middle) and
high potassium ACSF (right). (B) Example traces of cells that are considered to be a neuron (ROI1) and
an astrocyte (ROI2). (C) Dopamine-induced calcium responses of astrocytes by short pressure application
(500 µM, left) and bath application (100 µM, right). (D) Calcium responses of astrocytes evoked by application
of ADP (30 s, 100 µM) and dopamine (DA; 30 s, 100 µM) in both EPL (black trace) and GL (blue trace). (E)
Averaged amplitudes (left bars) and area (right bars) of DA-induced calcium responses in EPL and GL (error
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SEM) S
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(F) C l i Figure 2. Dopamine-triggered calcium response in olfactory bulb (OB) astrocytes. (A) Fluo-8 staining of
acute OB slices with example ROIs shown in idle state (left), in the presence of 100 µM ADP (middle) and
high potassium ACSF (right). (B) Example traces of cells that are considered to be a neuron (ROI1) and
an astrocyte (ROI2). (C) Dopamine-induced calcium responses of astrocytes by short pressure application
(500 µM, left) and bath application (100 µM, right). (D) Calcium responses of astrocytes evoked by application
of ADP (30 s, 100 µM) and dopamine (DA; 30 s, 100 µM) in both EPL (black trace) and GL (blue trace). (E)
Averaged amplitudes (left bars) and area (right bars) of DA-induced calcium responses in EPL and GL (error
bars: SEM). Sample size as specified in the bars: cells/slices/animals. n.s.: not significant. (F) Calcium response
of an astrocyte monitored during a 10-min application of DA (100 µM). (G) Calcium responses induced by
application of dopamine at different concentrations. (H) Dose-response curve of dopamine-induced astroglial
calcium responses (area), normalized to application of 100 µM DA (+/−SEM). (I) Percentage of astrocytes
responding to dopamine application. Dopamine induces dose-dependent calcium transients in olfactory bulb astrocytes.
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f the presence of the blocker mix, the dopamine-induced calcium transient was slightly decreases by 12.1+/−
3.6% (n = 55; p < 0.05) in amplitude and 9.7+/− 7.4% (n = 55; p < 0.05) in area as compared to the second
application of the control experiment (rundown). No significant differences between the values in the control
experiments and in experiments using the blockermix were found, indicating that the neuronal contribution to
dopamine-evoked calcium transients in OB astrocytes is negligible (Fig. 3C). p
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To test whether dopamine-evoked calcium transients are mediated by calcium release from internal stores,
we applied dopamine before and after calcium stores were depleted by incubation with 20 μM cyclopiazonic
acid (CPA). Depletion of internal stores led to a long-lasting elevation in intracellular calcium and suppressed
dopamine-evoked calcium responses. The mean amplitude of dopamine-induced calcium transient was reduced
by 93.8+/− 2.5% (n = 50; p < 0.001) and the area by 88.2+/− 2.9% (n = 50; p < 0.001) (Fig. 4A,D), indicating that
the calcium rise is mainly mediated by internal calcium release. The canonical pathways downstream of dopa-
mine receptors are stimulation or inhibition of adenylate cyclase via Gαs and Gαi proteins, however, intracellular
signaling via Gαq/11 proteins and phospholipase C (PLC) has also been described by both D1-class and D2-class
dopamine receptors25–29. To ascertain whether the calcium rise depends on the PLC/IP3 signaling pathway, we Scientific Reports | (2020) 10:631 | https://doi.org/10.1038/s41598-020-57462-4 www.nature.com/scientificreports/ Figure 3. Dopamine-induced calcium transients in OB astrocytes in synaptic isolation. (A) Example of
multiple applications of DA (100 µM) with a 10-min interval. (B) Calcium transients were not affected in
presence of GABAergic, glutamatergic und purinergic antagonists (Blockermix contains: NBQX 10 µM, D-APV
50 µM, gabazine 10 µM, CGP55845 10 µM, MPEP 2 µM, MRS2179 50 µM, ZM241385 0.5 µM, TTX 1 µM). (C) Normalized averaged amplitudes (light grey, +/−SEM) and area (dark grey, +/−SEM) of calcium responses
under control conditions and after application of Blockermix (BM). Results of BM are additionally compared to
rundown (RD) experiment as depicted in (A) (error bars: SEM). *P < 0.05, **P < 0.01, ***P < 0.005. Figure 3. Dopamine-induced calcium transients in OB astrocytes in synaptic isolation. (A) Example of
multiple applications of DA (100 µM) with a 10-min interval. Dopamine induces dose-dependent calcium transients in olfactory bulb astrocytes.
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f (B) Calcium transients were not affected in
presence of GABAergic, glutamatergic und purinergic antagonists (Blockermix contains: NBQX 10 µM, D-APV
50 µM, gabazine 10 µM, CGP55845 10 µM, MPEP 2 µM, MRS2179 50 µM, ZM241385 0.5 µM, TTX 1 µM). (C) Normalized averaged amplitudes (light grey, +/−SEM) and area (dark grey, +/−SEM) of calcium responses
under control conditions and after application of Blockermix (BM). Results of BM are additionally compared to
rundown (RD) experiment as depicted in (A) (error bars: SEM). *P < 0.05, **P < 0.01, ***P < 0.005. Figure 4. Internal calcium release mediates dopamine-induced calcium transients in OB astrocytes. (A) Dopamine-induced calcium transients are entirely suppressed in the presence of SERCA inhibitor
cyclopiazonic acid (CPA, 20 µM). (B) Calcium transients are partly diminished in the presence of IP3 receptor
antagonist 2-APB (50 µM). (C) Attenuated calcium transients in the presence of PLC inhibitor U73122 (50 µM). (D) Normalized averaged amplitudes (left, +/−SEM) and area (right, +/−SEM) of calcium responses under
control conditions and after application of CPA, 2-APB and U73122. Results are additionally compared to
rundown (RD) experiment as depicted in Fig. 3A. *P < 0.05, **P < 0.01, ***P < 0.005. Figure 4. Internal calcium release mediates dopamine-induced calcium transients in OB astrocytes. Figure 4. Internal calcium release mediates dopamine-induced calcium transients in OB astrocytes. (A) Dopamine-induced calcium transients are entirely suppressed in the presence of SERCA inhibitor
cyclopiazonic acid (CPA, 20 µM). (B) Calcium transients are partly diminished in the presence of IP3 receptor
antagonist 2-APB (50 µM). (C) Attenuated calcium transients in the presence of PLC inhibitor U73122 (50 µM). (D) Normalized averaged amplitudes (left, +/−SEM) and area (right, +/−SEM) of calcium responses under
control conditions and after application of CPA, 2-APB and U73122. Results are additionally compared to
rundown (RD) experiment as depicted in Fig. 3A. *P < 0.05, **P < 0.01, ***P < 0.005. Figure 4. Internal calcium release mediates dopamine-induced calcium transients in OB astrocytes. (A) Dopamine-induced calcium transients are entirely suppressed in the presence of SERCA inhibitor
cyclopiazonic acid (CPA, 20 µM). (B) Calcium transients are partly diminished in the presence of IP3 receptor
antagonist 2-APB (50 µM). (C) Attenuated calcium transients in the presence of PLC inhibitor U73122 (50 µM). (D) Normalized averaged amplitudes (left, +/−SEM) and area (right, +/−SEM) of calcium responses under
control conditions and after application of CPA, 2-APB and U73122. Discussion
D
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i Dopamine induces calcium transients in olfactory bulb astrocytes. In the present study we showed
that dopamine induced an increase in intracellular calcium concentration in olfactory bulb astrocytes in situ. We could demonstrate that the dopamine-evoked calcium rise was entirely blocked in the presence of SERCA
inhibitor CPA, indicating that dopamine triggered calcium release from internal stores such as the endoplas-
mic reticulum (ER). The most common mechanism to liberate calcium from the ER is the PLC/IP3 signaling
pathway, which has previously been shown in OB astrocytes to be activated by P2Y1 receptors, A2A receptors
and mGluR5
21,30. Although dopamine receptors are mainly considered to be linked to adenylate cyclases, sev-
eral instances have shown stimulation of PLC by both D1-class and D2-class dopamine receptors (reviewed by
Beaulieu et al.14). Hence, it is likely that dopamine releases calcium from the ER by stimulation of PLC. Based on
this assumption, we performed experiments in the presence of phospholipase C inhibitor U73122 and the IP3
receptor antagonist 2-APB, which both resulted in a considerable decrease in dopamine-induced calcium rise. Furthermore, the kinetics of the calcium response was altered in the presence of both 2-APB and U73122. The
property of 2-APB acting not only as an IP3 receptor antagonist, but also as an inhibitor of store-operated calcium
entry (SOCE), may provide an explanation for the rapid decay of the calcium rise after reaching its peak, since
inhibition of SOCE channels suppresses the late phase of the calcium response31. This result may be considered as
a first indication of the involvement of SOCE in dopamine-induced calcium transients in astrocytes. In contrast,
in the presence of the PLC inhibitor U73122, despite a significant reduction in mean amplitude, a prolongation of
the dopamine-induced calcium transient could be observed. This might be due to the U73122-mediated attenu-
ation of the PKC signaling pathway, which acts downstream of PLC activation as a negative feedback mechanism
to terminate calcium elevations evoked by G protein-coupled receptors32,33. Our results suggest major contribu-
tion of the PLC/IP3 pathway to dopaminergic calcium responses in OB astrocytes, however, both U73122 and
2-APB failed to entirely block dopamine-induced calcium transients. Discussion
D
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i We cannot exclude that the concentrations
of the compounds at their targets were not sufficient to totally block PLC-mediated IP3 receptor activation, since
U73122 does not easily diffuse within tissue and, in order to avoid activation of TRPV channels, the concentra-
tion of 2-APB could not be raised to values needed for complete block of IP3 receptors24. D1- and D2-class receptors mediate calcium signaling in olfactory bulb astrocytes. The
dopamine-induced calcium elevation was decreased by both D1- and D2-class receptor antagonists, thus both
classes of dopamine receptors appear to contribute to the dopamine-evoked calcium transients. This is in line
with observations suggesting that both D1- and D2-class receptors elicit release of IP3 and calcium signaling in
astrocytes25–29. Whether this effect in OB astrocytes is mediated by D1-D2 heteromers, as shown in striatal neu-
rons34, or by independent D1 and D2 receptors is not known and needs further investigation. In contrary to our
results, studies in the hippocampus and ventral midbrain have shown that activation of D2 receptors leads to a
decrease in cytosolic calcium, whereas D1 receptors mediate a calcium rise29,35. The D2-class receptor-mediated
decrease in calcium in hippocampal astrocytes presumably results from modulation of L-type voltage-gated
calcium channels29, which has also been shown in nucleus accumbens neurons36. Apparently, this mechanism
does not exist in astrocytes of the olfactory bulb. However, it must me mentioned that, due to the limitations
of two-dimensional confocal imaging and lack of visibility of small cell processes after bulk loading of calcium
indicators, it is possible that other small-scaled but functionally important calcium signals in fine astrocyte pro-
cesses may have been unexploited. Furthermore, in comparison to the work of Jennings et al.29, there are some
considerable differences in experimental design, such as our method of application differed in terms of duration. While Jennings et al. used a puff application of 3 minutes and bath application of 10 minutes in most trials, it was
sufficient to confine the dopamine application to 2 seconds (puff application) and 30 seconds (bath application)
in our study, suggesting a high dopamine sensitivity in astrocytes of the OB, but only moderate sensitivity in the
hippocampus. This is confirmed by the long delay in onset of the calcium response often seen in hippocampal
astrocytes29. Dopamine-induced calcium transients depend on D1 and D2-like receptors.
To accomplish Dopamine-induced calcium transients depend on D1 and D2-like receptors. To accomplish
the characteristics of astrocytic dopamine signaling, we were interested which class of dopamine receptor is
expressed and mediates calcium signaling in OB astrocytes. Therefore, we tested the effects of D1-class as well
as D2-class dopaminergic receptor antagonists on dopamine-induced calcium transients. In the presence of
the D1-antagonist SCH23390, the dopamine-induced calcium transient was reduced by 47.2+/− 5.0% (n = 54;
p < 0.001) in amplitude and 64.1+/− 6.2% (n = 54; p < 0.001) in area (Fig. 5A,B). The D2-antagonist sulpiride
resulted in an attenuation of the dopamine-induced calcium transient by 37.9+/− 4.1% (n = 124; p < 0.001) in
amplitude and 48.2+/− 6.9% (n = 124; p < 0.001) in area (Fig. 5C,D). In the presence of both SCH23390 and
sulpiride, dopamine-dependent calcium signaling was nearly completely abolished with a reduction by 85.9+/−
2.8% (n = 96; p < 0.001) in amplitude and 93.1+/− 1.7% (n = 96; p < 0.001) in area, respectively (Fig. 5E,F). In
all experiments, the antagonist-dependent decrease in calcium rise was at least partly reversible. These results
indicate a participation of both D1-class and D2-class receptors in dopamine-induced calcium signaling in OB
astrocytes. Scientific Reports | (2020) 10:631 | https://doi.org/10.1038/s41598-020-57462-4 Dopamine induces dose-dependent calcium transients in olfactory bulb astrocytes.
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f Results are additionally compared to
rundown (RD) experiment as depicted in Fig. 3A. *P < 0.05, **P < 0.01, ***P < 0.005. applied dopamine in the presence of the IP3 receptor antagonist 2-APB (Fig. 4B). A relatively low concentration
of 50 µM 2-APB was used, since higher concentrations activate transient receptor potential channels TRPV and
lead to calcium oscillations in OB astrocytes24. 50 µM 2-APB attenuated dopamine-induced calcium responses
by 36.8+/− 6.6% (n = 49; p < 0.001) in amplitude and 71.3+/− 3.1% (n = 49; p < 0.001) in area, suggesting the
involvement of IP3 receptors (Fig. 4B,D). In addition, the sustained phase of the calcium response was entirely applied dopamine in the presence of the IP3 receptor antagonist 2-APB (Fig. 4B). A relatively low concentration
of 50 µM 2-APB was used, since higher concentrations activate transient receptor potential channels TRPV and
lead to calcium oscillations in OB astrocytes24. 50 µM 2-APB attenuated dopamine-induced calcium responses
by 36.8+/− 6.6% (n = 49; p < 0.001) in amplitude and 71.3+/− 3.1% (n = 49; p < 0.001) in area, suggesting the
involvement of IP3 receptors (Fig. 4B,D). In addition, the sustained phase of the calcium response was entirely Scientific Reports | (2020) 10:631 | https://doi.org/10.1038/s41598-020-57462-4 www.nature.com/scientificreports/ blocked by 2-APB (Fig. 4B). In order to obtain further indication for the participation of this signaling pathway,
dopamine was applied after 30 minutes of incubation with the PLC inhibitor U73122 (50 µM). Under these con-
ditions, the dopamine-induced calcium response was reduced by 61.6+/− 5.2% (n = 22; p < 0.001) in amplitude
and by 46.2+/− 5.0% (n = 22; p < 0.001) in area (Fig. 4C,D). Interestingly, the dopamine-evoked calcium tran-
sient appeared to be sustained in the presence of U73122 as compared to the control response (Fig. 4C). Discussion
D
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i The dopamine sensitivity of OB astrocytes might even be underestimated, since bulk-loading of
calcium indicators results in significantly smaller calcium-dependent fluorescence changes compared to loading
single cells (and gap junction-coupled cells) via a patch pipette29. Scientific Reports | (2020) 10:631 | https://doi.org/10.1038/s41598-020-57462-4 www.nature.com/scientificreports
cientificreports/
Figure 5. Effect of DA receptor antagonists on dopamine-induced calcium signaling in OB astrocytes. (A) Calcium transients evoked by DA (30 s, 100 µM) where reduced in amplitude and integral in the presence
of D1-class antagonist SCH23390 (50 µM). (B) Normalized averaged amplitude and area of calcium responses
in presence of SCH23390. (C) Calcium transients where reduced in the presence of D2-class antagonist
sulpiride (50 µM). (D) Normalized averaged amplitude and area of calcium responses in presence of sulpiride. (E) Effect of both SCH23390 and sulpiride on dopamine-evoked calcium responses. (F) Normalized averaged
amplitude and area (+/−SEM) of calcium responses in the presence of both antagonists. *P < 0.05, **P < 0.01,
***P < 0.005. www.nature.com/scientificreports/ Figure 5. Effect of DA receptor antagonists on dopamine-induced calcium signaling in OB astrocytes. Figure 5. Effect of DA receptor antagonists on dopamine-induced calcium signaling in OB astrocytes. (A) Calcium transients evoked by DA (30 s, 100 µM) where reduced in amplitude and integral in the presence
of D1-class antagonist SCH23390 (50 µM). (B) Normalized averaged amplitude and area of calcium responses
in presence of SCH23390. (C) Calcium transients where reduced in the presence of D2-class antagonist
sulpiride (50 µM). (D) Normalized averaged amplitude and area of calcium responses in presence of sulpiride. (E) Effect of both SCH23390 and sulpiride on dopamine-evoked calcium responses. (F) Normalized averaged
amplitude and area (+/−SEM) of calcium responses in the presence of both antagonists. *P < 0.05, **P < 0.01,
***P < 0.005. Figure 5. Effect of DA receptor antagonists on dopamine-induced calcium signaling in OB astrocytes. (A) Calcium transients evoked by DA (30 s, 100 µM) where reduced in amplitude and integral in the presence
of D1-class antagonist SCH23390 (50 µM). (B) Normalized averaged amplitude and area of calcium responses
in presence of SCH23390. (C) Calcium transients where reduced in the presence of D2-class antagonist
sulpiride (50 µM). (D) Normalized averaged amplitude and area of calcium responses in presence of sulpiride. (E) Effect of both SCH23390 and sulpiride on dopamine-evoked calcium responses. (F) Normalized averaged
amplitude and area (+/−SEM) of calcium responses in the presence of both antagonists. Methods
i
l Animals used for slice preparation. Naval Medical Research Institute (NMRI) outbred mice from post-
natal day 7 (p7) to p21 were used for calcium imaging experiments. The mice were bred in the institute’s animal
facility at the University of Hamburg. This study was carried out in accordance with the recommendations of the
European Union’s and local animal welfare guidelines. Mice were anesthetized using isoflurane (5% mixed with
1 L/min O2) and decapitated before using for experiments. The permission to despatch mice for the purpose of
organ extraction was obtained by the Hamburg Authority of Health and Consumer Protection (GZ G21305/591-
00.33; Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany). According to the local laws, an
additional ethical approval for animal experiments in the current study was not required. Olfactory bulb slices
were prepared as described before60. Slices were quickly transferred into a chilled artificial cerebrospinal fluid
(ACSF, see below). 200 µm thick horizontal slices of the bulbs were cut using a vibratome (Leica VT1200S,
Bensheim, Germany). Brain slices were stored in ACSF for 30 min at 30 °C and 15 min at room temperature
before starting experiments. ACSF was continuously gassed with carbogen (95% O2/5% CO2; buffered to pH 7.4
with CO2/bicarbonate). Solutions and chemicals. The standard ACSF for acute brain slices contained (in mM): NaCl 125,
KCl 2.5, CaCl2 2, MgCl2 1, D-glucose 25, NaHCO3 26, NaHPO4 1.25, gassed during the entire experiment
with carbogen to adjust the pH to 7.4. MRS 2179 (2′-deoxy-N6-methyladenosine 3′, 5′-bisphosphate tetra-
sodium salt) and ZM241385 (4-(2-[7-Amino-2-(2-furyl)[1, 2, 4]triazolo[2, 3-a][1, 3, 5]triazin-5-ylamino]
ethyl)phenol) was obtained from Tocris (Bristol, UK). D-APV (D-2-amino-5-phosphonopentanoic acid),
NBQX (2, 3-dioxo-6-nitro-1, 2, 3, 4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide), gabazine (6-Imino-
3-(4-methoxyphenyl)-1(6 H)-pyridazinebutanoic acid hydrobromide), TTX (tetrodotoxin, Octahydro-
12-(hydroxymethyl)-2-imino-5, 9:7, 10a-dimethano-10aH-[1, 3]dioxocino[6, 5-d] pyrimidine-4, 7, 10,
11, 12-pentol + citrate buffer), CPA (cyclopiazonic acid; (6aR, 11aS, 11bR)-10-acetyl-2, 6, 6a, 7, 11a,
11b-hexahydro-11-hydroxy-7, 7-dimethyl-9H-pyrrolo[1′, 2′:2, 3]isoindolo[4, 5, 6-cd]indol-9-one, SCH23390
((R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2, 3, 4, 5-tetrahydro-1H-3-benzazepine) and sulpiride ((R, S)-
(±)-5-Aminosulfonyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxybenzamide) were obtained from Abcam
(Cambridge, United Kingdom). All substances were stored as stock solutions according to the manufacturers’
description. Immunohistochemistry. Immunohistochemistry on olfactory bulbs of TH-Cre (B6.Cg-7630403G23RikT
g(Th-cre)1Tmd/J) × tdTomatofl/fl mice (P28) was performed as described22,61,62. These mice express the red fluores-
cent reporter tdTomato under control of Cre recombinase, which is expressed only by cells with active tyrosine
hydroxylase promoter. Putative functional roles of dopamine-evoked calcium signaling in astrocytes.
It is well known Putative functional roles of dopamine-evoked calcium signaling in astrocytes. It is well known
that astrocytes are involved in a variety of processes in the CNS. Apart from well-established supportive astro-
cytic functions such as metabolic supply and potassium homeostasis, more recent studies revealed that astrocytes
are tightly integrated into neural networks in a functional manner. As a result of astroglial calcium excitability,
astrocytes are capable of “gliotransmission”, i.e. release of transmitters which can lead to signaling to neighbor-
ing neurons and blood vessels47,48. In the main olfactory bulb, several studies provided evidence for important
functions of astrocytes, e.g. gliotransmission49. Kozlov et al. demonstrated that astrocytes in the rat olfactory bulb
release two classical transmitters, GABA and glutamate, upon mechanical stimulation to evoke synchronous
currents in mitral and granule cells50. Another gliotransmitter released by OB astrocytes is ATP that is degraded
to adenosine, a potent neuromodulator in the OB23,51. In mitral cells, adenosine activates two-pore domain potas-
sium channels and inhibits presynaptic calcium channels, resulting in reduced spontaneous firing and attenu-
ated reciprocal dendro-dendritic inhibition52,53. OB astrocytes also play a key role in neurovascular coupling,
since it has been shown that the physiological activation of olfactory sensory neurons by odors reliably triggers
calcium increases in astrocyte processes in the glomerular layer and subsequent dilation of blood vessels30,54. Neurovascular coupling is not restricted to the GL, since astrocytes transmit panglial calcium waves to olfactory
ensheathing glial cells in the nerve layer, which there contribute to regulation of blood vessel diameter55,56. Which
role dopaminergic calcium signaling in astrocytes plays in neuronal performance and neurovascular coupling in
the OB is not known so far and needs further investigation. However, tuning of olfactory information processing
and odor perception by neuromodulators such as dopamine, noradrenaline, acetylcholine and serotonin has
moved into the focus of research on olfaction, in particular in recent years23,57–59, and it is becoming increasingly
clear that astrocytes have to be considered to take actively part in these processes. This hypothesis appears to
be supported by findings showing interactions of dopamine and astrocytes in other regions of the CNS, such as
ventral midbrain and hippocampus29,35. Discussion
D
i
i *P < 0.05, **P < 0.01,
***P < 0.005. Although the localization of tyrosine hydroxylase and thus dopamine-containing neuronal elements has its
highest density in the glomerular layer (Fig. 1A)10, the distribution of the D1-receptor is widely spread over all
OB layers except of the nerve layer37–42. This indicates a functional role of dopamine in the entire OB, including
the glomerular layer as well as in external plexiform layer, which is consistent with our results. The distribution
of the D2 receptor has its highest density in the GL and nerve layer, while D2 receptor expression in the EPL is
sparse40,43–45. Despite that, the dopamine-induced calcium transients where strongly reduced in the presence of
D2 antagonist sulpiride in both the GL and EPL, indicating expression of D2 receptors in astrocytes of the EPL. Previous studies by Pignatelli et al.37 showed that during the process of maturation of adult-born dopaminergic
neurons, two populations of TH+ neurons outside the glomerular layer exist. One of these populations represents
dopaminergic neurons that undergo the last step in maturation and can be found in the mitral cell layer and Scientific Reports | (2020) 10:631 | https://doi.org/10.1038/s41598-020-57462-4 www.nature.com/scientificreports/ external plexiform layer37. These cells project numerous processes within the EPL, suggesting release of dopa-
mine not only in the GL but also in deeper layers. As shown in Fig. 1, our histological analysis of TH-expressing
neurons supports this observation. Hence, astrocytes in the EPL might detect dopamine released by immature
adult-born neurons with calcium signaling and, in turn, provide growth factors and components of the extracel-
lular matrix to support neuronal maturation as shown, e.g., in the cerebellum46. Methods
i
l After dissection, the olfactory bulbs were kept for 1 h at room temperature (RT) in for-
malin (4% paraformaldehyde in PBS, pH 7.4). Subsequently, 120 μm thick sagittal slices were prepared with a
vibratome (VT1000S, Leica, Nussloch, Germany) and incubated for 1 h in blocking solution (10% normal goat
serum [NGS], 0.5% Triton X-100 in PBS) at RT. Afterwards, the slices were incubated for 48 h at 4 °C with a
chicken anti-GFAP antibody (Synaptic Systems, Göttingen, Germany; 1:1000). The antibody was diluted in 1%
NGS, 0.05% Triton X-100 in PBS. Slices were incubated in PBS with the goat anti-chicken Alexa 488 secondary Scientific Reports | (2020) 10:631 | https://doi.org/10.1038/s41598-020-57462-4 www.nature.com/scientificreports/ antibody (Abcam; 1:1000) for 24 h at 4 °C. Subsequently, the slices were mounted on slides using self-hardening
embedding medium (Immu-Mount, Thermo Fisher). Immunohistological stainings were analyzed using a confo-
cal microscope (C1 Eclipse, Nikon, Düsseldorf, Germany). Confocal images were deconvolved using a theoretical
point-spread function (Huygens, SVI, Hilversum, Netherlands) and enhanced for brightness and contrast using
ImageJ and Adobe Photoshop CS2. antibody (Abcam; 1:1000) for 24 h at 4 °C. Subsequently, the slices were mounted on slides using self-hardening
embedding medium (Immu-Mount, Thermo Fisher). Immunohistological stainings were analyzed using a confo-
cal microscope (C1 Eclipse, Nikon, Düsseldorf, Germany). Confocal images were deconvolved using a theoretical
point-spread function (Huygens, SVI, Hilversum, Netherlands) and enhanced for brightness and contrast using
ImageJ and Adobe Photoshop CS2. Calcium imaging. Slices were incubated with the membrane-permeable form of the calcium indicator Fluo-8
(Fluo-8-AM; 2 µM in ACSF) made from a 2 mM stock solution (dissolved in DMSO and 20% pluronic acid) for
30 min. Brain slices were then placed in the recording chamber and fixed with a U-shaped platinum wire with
nylon strings. Brain slices were continuously superfused at a rate of 2 ml/min with ACSF that was gassed with
carbogen (95 O2/5% CO2). Bath perfusion with ACSF was accomplished using a peristaltic pump (Vario, Ismatec,
Germany). Drugs were applied via the perfusion system. If not stated otherwise, ACSF contained 1 µM TTX to
suppress neuronal activity. Changes in intracellular calcium levels in olfactory bulb astrocytes were recorded by
confocal microscopy (C1 Eclipse, Nikon, Düsseldorf, Germany). An excitation laser wavelength of 488 nm and a
frame rate of 0.75 fps were used and the fluorescence was collected through a 500–530 nm bandpass filter. Data analysis and statistic. Data availability Data availability
All data are available from the authors upon request. y
All data are available from the authors upon request. Received: 27 September 2019; Accepted: 30 December 2019;
Published: xx xx xxxx Received: 27 September 2019; Accepted: 30 December 2019;
Published: xx xx xxxx Received: 27 September 2019; Accepted: 30 December 2019;
Published: xx xx xxxx Received: 27 September 2019; Accepted: 30 December 2019;
Published: xx xx xxxx Methods
i
l The data were evaluated with Nikon EZ-C1 Viewer (Nikon) and statistical tests
have been applied with Origin Pro 9.1 (OriginLab Corporation, Northampton, USA). To analyze changes of the
calcium level in astrocytes, cell somata were marked as regions of interests (ROIs). Cells located in the glomer-
ular layer and external plexiform layer that showed a calcium response to ADP were identified as astrocytes21,22. This method of cell identification has been verified by using K+-free and 50 mM K+-containing ACSF, in which
K+ was exchanged for Na+. Astrocytes, but not neurons, respond to K+-free ACSF with calcium signaling31. The
astrocytic calcium elevations have been observed in both glomerular layer and external plexiform layer, without
a significant difference in amplitude or duration, therefore most of the results in the present study are pooled data
of both layers. To evaluate changes of calcium levels over time, Fluo-8 fluorescence intensity (F) was recorded
during the course of the experiment and normalized to the basal fluorescence intensity in absence of pharma-
cological stimuli. Changes in calcium are given by ΔF as the percentage changes in fluorescence with respect to
the basal fluorescence. Calcium responses were assessed as amplitude (baseline to peak) and area (integral of
the curve from beginning of the response until baseline was reached again). All values are given as mean values
(+/−) standard error of the mean with “n” representing the number of cells analyzed for a certain set of experi-
ment. In the figures, sample size is given as “number of cells/number of brain slices/number of animals”. The proof
of statistical significance was done by Wilcoxon signed-rank test at an error probability p (*p < 0.05; **p < 0.01;
***p < 0.001). In case of independent data the Mann-Whitney-U test was applied. The dose-response curve was
generated using the response to 100 µM dopamine, a concentration used in all measurements analyzed for the
dose-response relationship, as reference. The values of all responses to 100 µM dopamine were averaged, resulting
in 85.2+/− 5.1% ΔF, and ΔF values of all individual cells were normalized to this value. Normalized values were
pooled and the dose-response curve was generated using Origin Pro 9.1. References
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2. Savitt, J. M., Jang, S. S., Mu, W., Dawson, V. L. & Dawson, T. M. Bcl-x is required for proper development of the mouse substantia
nigra. J. Neurosci. 25, 6721–6728, https://doi.org/10.1523/JNEUROSCI.0760-05.2005 (2005). Acknowledgements Acknowledgements
We thank A.C. Rakete and M. Fink for technical assistance and S. Wiegert for providing TH-Cre mice. This study
was supported by grants from the Deutsche Forschungsgemeinschaft (LO 779/10 and SFB 1328 A07 to C.L.). g
We thank A.C. Rakete and M. Fink for technical assistance and S. Wiegert for providing TH-Cre mice. This study
was supported by grants from the Deutsche Forschungsgemeinschaft (LO 779/10 and SFB 1328 A07 to C.L.). Author contributions
T.F. and C.L. conceived the experiments, T.F. and P.S. conducted the experiments, T.F. and P.S. analyzed the
results. All authors reviewed the manuscript. Author contributions
T.F. and C.L. conceived the experiments, T.F. and P.S. conducted the experiments, T.F. and P.S. analyzed the
results. All authors reviewed the manuscript. Competing interests
The authors declare no competing interests. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-57462-4. Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-57462-4 Correspondence and requests for materials should be addressed to T.F. or C.L. Correspondence and requests for materials should be addressed to T.F. or C.L. Correspondence and requests for materials should be addressed to T.F. or C.L. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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Revealing the abnormal meiosis and the variation of the functional female gametes of aneuploid lily (Lilium) using genomic in situ hybridization (GISH)
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Revealing the abnormal meiosis and the variation of
the functional female gametes of aneuploid lily
(Lilium) using genomic in situ hybridization (GISH) Kongzhong Xiao
Jiangxi Agricultural University
Yanni Sun
Jiangxi Agricultural University
Shujun Zhou
(
zhou2014@jxau.edu.cn
)
Jiangxi Agricultural University Research Article Keywords: Aneuploid, Abnormal meiosis, Fritillaria-type embryo sac, Kinetochore, Chromatid
Posted Date: August 7th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-3223598/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Version of Record: A version of this preprint was published at Euphytica on September 22nd, 2023. See
the published version at https://doi.org/10.1007/s10681-023-03238-6. Page 1/18 Page 1/18 Abstract Aneuploid lilies (Lilium) could be obtained from the LAA/LAAA × AA/AAAA hybridization; however, the
characteristics of their meiosis and fertility has not been reported. In this study, an aneuploid lily, J1614,
was extensively investigated for its microsporogenesis, fertility and functional eggs using conventional
and modern cytogenetic methods. The results indicated that J1614 was an aneuploid Longiflorum-
Asiatic (LA) lily (2n = 48 = 7L + 39A + 2L/A) while ‘Pearl Jason’ was an autotetraploid Asiatic lily (2n =
48A); L-chromosomes of J1614 usually formed univalent while A-chromosomes associated not only
predominantly trivalents, but also tetravalents, bivalents, and even univalent at metaphase I as well;
clearly, both univalents and other associated chromosomes were separated and moved to opposite poles
at anaphase I; besides, lagging chromosomes and micronuclei were observed during microsporogenesis. Hybridization showed that J1614, regardless of its male sterility, had better partial female fertility when
tetraploid a lily was used as male than when a diploid as male. The nine seedlings of J1614 × AAAA were
all aneuploid with variable total chromosomes ranging from 46 to 53, meaning that the functional eggs
produced by J1614 contained variable chromosomes ranging from 22 to 29. Based on the present
results, we concluded that once good lines are selected from aneuploid lilies, they may not only become
cultivars through vegetative propagation, but also become parents to breed new aneuploids and realize
introgression breeding. In addition, the mechanism of abnormal meiosis of aneuploid lilies was
hypothesized. Introduction Lily, one of the most important bulb flowers worldwide, refers to the genus Lilium of the family Liliaceae
and its derived cultivars. The genus consists of 100 wild species, and they are classified into seven
sections (De Jong 1974). Modern lily cultivars, such as Asiatic (A), Longiflorum (L), Oriental (O), and
Trumpet lily (T) are breed from intra-section hybridization; meanwhile the cultivar groups, like LA, LO, OA,
OT etc., are obtained from inter-section hybridization (Van Tuyl et al. 2000; Van Tuyl and Arens 2011). Most intra-sectional lilies are diploid (2n = 2x = 24), while most inter-sectional lilies are allotriploid (2n = 3x
= 36), such as LAA, LOO, OTO, etc., and a few are odd-allotetraploids (2n = 4x = 48), like LAAA and LLLO
(Zhang et al. 2012; Zhou et al. 2013). Although triploid and odd-allotetraploid lilies have abnormal
meiosis during gametogenesis and they are male sterile, they could be used as female to cross with
suitable males to produce aneuploid progenies (Lim et al. 2003; Barba-Gonzalez et al. 2006; Zhou 2007;
Khan et al. 2009, Zhou et al. 2011, 2012; Xiao et al. 2019). Most aneuploid lilies grow weak or die early
because of chromosomal or gene unbalanced, fortunately some are vigor and can be as cultivars since
their genes are rebalanced in the aneuploid lilies (Zhou 2011-12; 2014). They are also used for lily
introgression breeding (Zhong et al. 2022). However, their characteristics of meiosis and functional
gametes have not been systematically studied. In this study, an aneuploid lily was extensively investigated for its microsporogenesis, fertility, and
genome composition of its female functional gametes, and the significance of aneuploid on lily
introgression breeding was discussed. Page 2/18 Plant materials An aneuploid lily, coded J1614, was used as female parent. The aneuploid was obtained by ‘Honesty’
(LAAA) × ‘Nello’ (AAAA). Two Asiatic lilies were as males – one is diploid, J1707 (AA), the other is a
tetraploid, ‘Pearl Jason’ (AAAA). A Longiflorum lily ‘White Fox’ (LL) was used to extract its genomic DNA
as probe for genomic in situ hybridization (GISH). All lilies were grown in the greenhouse of Jiangxi
Agricultural University. Microsporogenesis Microsporogenesis was analyzed referring to Xiao et al. (2019). When the flower buds were 28–32 mm,
their anthers were treated with a fixative, containing three parts ethanol and one acetic acid by volume, at
least 30 min, then, some pollen mother cells (PMCs) were mixed with a drop of 2% Carbol Fuchsin
(Beijing Solarbio Science & Technology Co. Ltd.) on a slide, and then covered with a square cover glass
for check under a light microscope (ZEISS Scope. A1). Chromosome preparation at metaphase I and anaphase I The meiotic chromosome slides were prepared almost the same as described above, except that a drop
of 2% Carbol Fuchsin was replaced by a drop of 45% acetic acid. A gentle squash was done with thumb
after the slides were covered; then, they were stored at -80 ℃ for about 1 h, following their covers were
removed with a knife, and then immersed in ethanol for 1 min, and then dried on a slide rack. The slides
were checked with a phase contrast microscope (ZEISS Scope. A1). The types and germination of pollen grains Fresh pollen grains were scattered on a medium, containing100 g/L sucrose, 5 g/L bacteriological agar,
20 mg/L H3BO3, and 200 mg/L Ca(NO3)2, and cultured at 25 ℃ for 4h, then observed under microscope
(ZEISS Scope. A1). Mitotic chromosome preparation Mitotic chromosome preparation The mitotic chromosomes were prepared according to Wu et al. (2021). The root tips were cut off; and
incubated in 0.7 mM cycloheximide (Duchefa Biochemie) at room temperature for 4 h, then stored in a
fixative at least 30 min. The root tips were treated with 1% (w/v) cellulase RS (Duchefa Biochemie) and
1% (w/v) pectinase Y23 (Duchefa Biochemie) mix, at 37 ℃ for 1 h. Their meristem was mixed with 16 µL
45% acetic acid on a glass slide, covered with a cover glass and then gently squashed. The slides were
checked with a phase contrast microscope (ZEISS Scope. A1). In situ hybridization 5S rDNA and 45S rDNA were used as probe to analyze mitotic chromosomes of ‘Pearl Jason’ using
fluorescence in situ hybridization (FISH) according to Lan et al. (2018). 5S rDNA and 45S rDNA were used as probe to analyze mitotic chromosomes of ‘Pearl Jason’ using
fluorescence in situ hybridization (FISH) according to Lan et al. (2018). The genomic DNA of ‘White Fox’ (LL) was extracted with CTAB method, and then labeled with biotin using
nick translation kit (Roche 11745824910) as probe, meanwhile, HS (herring sperm) DNA, cooked for 30
min at 100 ℃, as block for genomic in situ hybridization (GISH). The hybridization mix (40µL) consisted of 50% deionized formamide, 10% dextran sulphate (Amresco
0198), 2x SSC (0.3M NaCl plus 30 mM sodium citrate, pH 7.0), 0.25% SDS, 25-50ng probe DNA, 25-50ng
probe and 1–3µg block DNA. In situ hybridization was performed according to the method described by
Barba-Gonzalez et al. (2004). Biotin signal was detected and amplified with Streptavidin-CY3 (Invitrogen,
Camarillo, CA) and Biotinylated anti-Streptavidin (Vector Laboratories, Burlingame, CA). After mounted
with VECTASHIELD (H-1200, Vector Laboratories, Burlingame, CA), the slides were observed under a
fluorescence microscope (ZEISS Scope. A1). Results Genome composition of J1614 and ‘Pearl Jason’ J1614 grew well and its flowers were deep red (Fig. 1a). It had seven L-chromosomes, two L/A
recombinant chromosomes and 39 A-chromosomes, totally 48 chromosomes (Fig. 1b). The results
showed that the lily was an aneuploid or pseudotetraploid as its total chromosome number was 48. Its
formula was written as 2n = 48 = 7L + 39A + 2L/A. As shown in Fig. 2, ‘Pearl Jason’ had orange-yellow flowers with strong reflex tepals and 48 A-
chromosomes (2n = 4x = 48A), and FISH karyotype clearly indicated that it was autotetraploid Asiatic lily. Pollination and embryo rescue Pollination and embryo rescue The pollination and embryo rescue were referred to Zhou et al. (2012). When the flowers of J1614 were
open, they were pollinated with fresh pollen of ‘Pearl Jason’ and J1707 using normal pollination. Having
pollinated, the stigmas were wrapped with aluminum foil. When the fruits matured, they were harvested. In a fume hood, their embryo sacs or developed ovules were selected out and put on lily embryo rescue
medium (pH = 5.8), containing 2.2 g·L− 1 MS (Duchefa Biochemie), 60 g·L− 1 sucrose and 4 g·L− 1 gelrite
(Duchefa Biochemie). They were stored in paper cases at 25 ℃ for 40–60 d and then transferred to lily
propagation medium (pH = 5.8), containing 2.2 g·L− 1 MS, 50 g·L− 1 sucrose and 4 g·L− 1 gelrite at 25 ℃
and 2000 lx light intensity for about 10 weeks. Page 3/18 Page 3/18 Page 3/18 Microsporogenesis of J1614 As shown in Fig. 3, its meiosis was abnormal at metaphase I and anaphase I. (1) At metaphase I (Fig. 3a
& b), the A-chromosomes were predominantly associated into trivalents (white “III”), and bivalents (white
“II”), tetravalents (white “IV”) and even univalents (white “I”) were also rarely possible; however, most of Page 4/18 Page 4/18 the L-chromosomes did not pair with any other L- or A- chromosomes and exist as univalents (red “I”),
except that a few formed bivalent or multivalents with L- or A- chromosomes (white “I” + red “I” or “II’). (2)
At anaphase I (Fig. 3c-e), clearly, most of the sister L-chromosomes were separated and move to two
opposite poles; seemingly, most of the associated A-chromosomes also disjoined and moved to two
poles; a few recombinant chromosomes were also observed, conforming that a few L- and A-
chromosomes associated at metaphase I. (3) Microspores contained different free micronuclei (Fig. 4a
and b) which be consequences of lagging chromosomes or acentric fragments. The types and sizes of
their pollen grains were variable and near all of them did not germinate (Fig. 4c and d), showing that
J1614 was highly male sterile. Female fertility of J1614 J1614 was male highly sterile; however, its fruits developed well when it was used as female to cross with
diploid or tetraploid Asiatic lily. The developed fruits between the two crosses seemingly similar, but their
compatibilities were different. 12.5 embryo sacs were isolated from J1614 × AA per fruit and no seedling
was obtained, while 60 embryo sacs were isolated from J1614 × AAAA and 9.5 seedlings were obtained
per fruit. The results indicated that the aneuploid lily, J1614, regardless of its male sterility, had partial
female fertile and its compatibility with tetraploid Asiatic lily was much better than that with diploid. Genome composition of functional eggs of J1614 GISH showed that the nine progenies of J1614 × AAAA had variable total chromosomes ranging from 46
to 53, and they consisted of variable L-, A-, and L/A recombinant chromosomes (Fig. 5, Table 1). L-
chromosomes or L-fragments in the progenies were less than those in J1614, suggesting that aneuploid
lily would be a way to realize lily introgression breeding. Since tetraploid Asiatic lily ‘Pearl Jason’
contributed 24 A-chromosomes, the genome composition of functional egg cells produced by aneuploid
J1614 were deduced and shown in Table 1. Obviously, the recombinant chromosome numbers in the
functional eggs were agreement with the association between L- and A-chromosomes at metaphase I
during microsporogenesis. The results indicated that aneuploid lily can produce functional aneuploid
eggs regardless of its male sterility. Page 5/18 Page 5/18 Page 5/18 Table 1 Table 1 The genome compositions of the progenies of J1614 × AAAA and those of functional
eggs produced by J1614. The number of L-chromosomes, A-chromosomes, and L/A
bi
h
d b L A
d L/A
i
l recombinant chromosomes are represented by L , A , and L/A , respectively. Progenies
Chromosomes of progenies
Chromosomes of functional eggs
Total
L-
A-
L/A-
Total
L-
A-
L/A-
20106-1
53
3
47
3
29
3
23
3
20106-4
49
3
43
3
25
3
19
3
20106-5
47
3
42
2
23
3
18
2
20106-7
50
2
48
0
26
2
24
0
20106-8
49
5
42
2
25
5
18
2
20106-10
51
5
45
1
27
5
21
1
20106-13
49
2
45
2
25
2
21
2
20106-15
46
1
44
1
22
1
20
1
20106-17
51
5
44
2
27
5
20
2
Average
49.44
3
44.66
1.78
25.44
3
20.66
1.78 Kinetochore and chromosome separation during abnormal meiosis However, according to GISH analysis on lily abnormal meiosis, it is
more plausible to explain the abnormal meiosis I in a modified way shown as in Fig. 6(2). This is because
a centromere on each chromosome has two kinetochores which are attached to spindle microtubules in
normal mitosis; however, during normal meiosis, a centromere on each chromosome of a bivalent only
has one kinetochore attached to spindle microtubules (www.wikipedia.org). Possibly, one chromosome
has one kinetochore in its centromere, and kinetochore is possibly duplicated accompanying DNA
replicating in mitosis (Fig. 6(2d)); however, during normal meiosis I, kinetochore duplication is hindered by
synapses (Fig. 6(2e)) or the two duplicated kinetochores on sister chromatids are so close and function
as one due to cohesion and chiasmata; and during abnormal meiosis I (Fig. 6(2f)), some bivalents
premature and become univalent, or some chromosomes do not pair and remain univalents, and their
kinetochores could replicate because no chiasmata or cohesion suppress kinetochore duplication. Since
then, it is reasonable that both univalents and bivalents are disjoined and move to opposite poles during
abnormal meiosis I in F1 distant lily hybrids (Zhou et al. 2008). 2) Bivalents are also common in
allotriploid lilies, and are also formed in F1 distant LA or OA hybrids at metaphase I. They are usually
disjoined at anaphase I as normal meiosis (Zhou et al. 2008; Xiao et al. 2022; Cui et al. 2022). The similar
result is also reported in allotriploid Alstroemeria (Kamstra et al. 2004). 3) Trivalents or other multivalents
are often found in odd-allotetraploid (Xiao et al. 2022), and occasionally occur in allotriploid (Cui et al.,
2022) and distant hybrids (Zhou et al., 2008). They are disjoined evenly or unevenly at anaphase I (Zhou
et al. 2015). Besides, lagging chromosomes and micronuclei are common in abnormal meiosis not only
in lily (Zhou et al. 2008; Zhang et al., 2017; Cui et al. 2022; Xiao et al. 2022), but also in rice hybrids (Liu et
al. 2021), Saccharum hybrids (Li et al. 2021), Populus hybrids (Wang et al. 2015), and Musa (Ahmad et
al. 2021). Interestingly, the sister-chromatid separations of univalents, which are formed by premature separation of
bivalents at meiosis I, are observed in aging-related oocytes of mice and human, and the cohesion loss
and splitting of sister kinetochores are regarded as the reasons for the abnormal phenomenon (Zielinska
et al. 2015; Nakagawa and FitzHarris 2017). Kinetochore and chromosome separation during abnormal meiosis Kinetochore and chromosome separation during abnormal meiosis The present study and previous reports show that all the abnormal meiosis have interesting features
although each have some its own characteristics in distant F1 hybrids (Zhou et al. 2008; Luo et al. 2013),
allotriploid (Cui et al. 2022) and odd-allotetraploid lilies (Xiao et al. 2022). 1) Univalents are quite
common at metaphase I and their sister-chromatids usually separated and moved to the two opposite
poles at anaphase I. At metaphase I, in distant F1 hybrids, such as LA and OA, most homoeologous
chromosomes do not pair and thus form univalent (Barba-Gonzalez et al. 2004; Zhou et al. 2008); In
allotriploid (LLO), all the homologous L-chromosomes prefer to pair each other and O-chromosomes tend
to form univalent (Cui et al. 2022); Similarly, odd-allotetraploid (LAAA), all the homologous A-
chromosomes prefer to associate multivalents and L-chromosomes tend to be univalent (Xiao et al. 2022). Intriguingly, the sister-chromatids of such univalents are clearly separated and moved to the
opposite poles at anaphase I. The same phenomenon is also observed in allotriploid Alstroemeria
(Kamstra et al. 2004). This is totally different from normal meiosis I, in which the chromosomes of
bivalents separate rather than sister-chromatids. Univalents are commonly reported in plant F1 hybrids,
and they are the reason for 2n gametes resulting from an abnormal meiosis in many plants (Ramanna
and Jacobsen 2003), such as Trifolium (Ansari et al. 2022), wheat/rye hybrids (Silkova et al. 2013), etc. Page 6/18 Page 6/18 Interestingly, the sister-chromatid separations of univalents, which are formed by premature separation of
bivalents at meiosis I, are observed in aging-related oocytes of mice and human, and the cohesion loss
and splitting of sister kinetochores are regarded as the reasons for the abnormal phenomenon (Zielinska
et al. 2015; Nakagawa and FitzHarris 2017). Akera and Lampson (2016) suggested that sister Interestingly, the sister-chromatid separations of univalents, which are formed by premature separation of
bivalents at meiosis I, are observed in aging-related oocytes of mice and human, and the cohesion loss
and splitting of sister kinetochores are regarded as the reasons for the abnormal phenomenon (Zielinska
et al. 2015; Nakagawa and FitzHarris 2017). Akera and Lampson (2016) suggested that sister
kinetochores should have been fused during normal meiosis I and sister kinetochores of univalents were
split at abnormal meiosis I (Fig. 6(1)). Kinetochore and chromosome separation during abnormal meiosis Akera and Lampson (2016) suggested that sister
kinetochores should have been fused during normal meiosis I and sister kinetochores of univalents were
split at abnormal meiosis I (Fig. 6(1)). However, according to GISH analysis on lily abnormal meiosis, it is
more plausible to explain the abnormal meiosis I in a modified way shown as in Fig. 6(2). This is because
a centromere on each chromosome has two kinetochores which are attached to spindle microtubules in
normal mitosis; however, during normal meiosis, a centromere on each chromosome of a bivalent only
has one kinetochore attached to spindle microtubules (www.wikipedia.org). Possibly, one chromosome
has one kinetochore in its centromere, and kinetochore is possibly duplicated accompanying DNA
replicating in mitosis (Fig. 6(2d)); however, during normal meiosis I, kinetochore duplication is hindered by
synapses (Fig. 6(2e)) or the two duplicated kinetochores on sister chromatids are so close and function
as one due to cohesion and chiasmata; and during abnormal meiosis I (Fig. 6(2f)), some bivalents
premature and become univalent, or some chromosomes do not pair and remain univalents, and their
kinetochores could replicate because no chiasmata or cohesion suppress kinetochore duplication. Since
then, it is reasonable that both univalents and bivalents are disjoined and move to opposite poles during
abnormal meiosis I in F1 distant lily hybrids (Zhou et al. 2008). 2) Bivalents are also common in
allotriploid lilies, and are also formed in F1 distant LA or OA hybrids at metaphase I. They are usually
disjoined at anaphase I as normal meiosis (Zhou et al. 2008; Xiao et al. 2022; Cui et al. 2022). The similar
result is also reported in allotriploid Alstroemeria (Kamstra et al. 2004). 3) Trivalents or other multivalents
are often found in odd-allotetraploid (Xiao et al. 2022), and occasionally occur in allotriploid (Cui et al.,
2022) and distant hybrids (Zhou et al., 2008). They are disjoined evenly or unevenly at anaphase I (Zhou
et al. 2015). Besides, lagging chromosomes and micronuclei are common in abnormal meiosis not only
in lily (Zhou et al. 2008; Zhang et al., 2017; Cui et al. 2022; Xiao et al. 2022), but also in rice hybrids (Liu et
al. 2021), Saccharum hybrids (Li et al. 2021), Populus hybrids (Wang et al. 2015), and Musa (Ahmad et
al. 2021). The partial female fertile of aneuploid lilies The partial female fertile of aneuploid lilies It is confirmed that F1 distant hybrids can be female parents to be backcrossed and produce allotriploid
lilies regardless their male sterility (Zhou 2007; Liu et al. 2021); Similarly, triploid, allotriploid and odd-
allotetraploid can be female parents to hybridize with appropriate males though they are highly male
sterile (Lim et al. 2000; 2003; Khan et al. 2009; Natenapit et al. 2010; Xie et al. 2010; Zhou et al. 2011,
2012, 2014; Chung et al. 2013; Wang et al. 2015; Suzuki and Yamagishi 2015; Xi et al. 2015; Xiao et al. 2019; Cui et al. 2022). The phenomena seemingly look strange, not only because triploids are usually
seedless, but also one plant’s male fertility should be similar to its female fertility due to same genetic
materials, i.e., same meiosis of gametogenesis. So, why are there such big difference between male and
female fertility in these lilies? The question was well explained by comparative analysis between
Fritillaria embryo sac and Polygonum embryo sac (Zhou 2007). According to megasporogenesis, in a Page 7/18 Page 7/18 polygonum embryo sac, the nucleic DNA of its central cell is twice that of its egg cell, while in a fritillaria
embryo sac, the nucleic DNA of its central cell is twice that its somatic cell (Fig. 7) (Zhou 2007; Zhou et al
2011, 2012). So, for triploid watermelon as an example of polygonum-type plant, in its embryo sac, both
egg and central cell are aneuploid, once double fertilized with a diploid, both embryo and endosperm are
aneuploid; thus, triploid watermelon is seedless. By contrast, for a triploid lily, in its fritillaria-type embryo
sac, its egg is aneuploid, but its central cell is hexaploidy; Once double fertilized with a diploid or
tetraploid, endosperm is euploid and it develops well, and then make some aneuploid embryos survival
(Zhou 2007; Zhou et al. 2011, 2012). This is the reason why all the male sterile lilies have some partial
female fertility. In the present study, the fertility of the aneuploid lily is similar to that of triploid or odd-
allotetraploid lilies. Once embryo sacs of an aneuploid lily are double fertilized by diploid or tetraploid
male, both embryo and endosperm are aneuploid; surprisingly, some seeds develop well rather than
seedless in triploid watermelon. How to explain its partial female fertility? The partial female fertile of aneuploid lilies Aneuploid lilies are usually less
vigor and die early, however some grow well, like J1614 in the present research, indicating that it has
balanced genes though it is an aneuploid. The nucleic DNA of each central cell in its embryo sac is
invariably twice that of a somatic cell, meaning that all its central cells have balanced genes. Once
fertilized with tetraploid male, its endosperm has the balanced genes and could develop and make
aneuploid embryos survival (Fig. 7). hromosome numbers of progenies or functional gametes of aneuploid lilies In the present study, J1614, containing 48 chromosomes, is aneuploid because of its unbalanced
chromosomal composition. Its progenies have variable chromosomes, ranging from 46–53, when
tetraploid as male. This is like aneuploid progenies obtained from 2x/4x × aneuploid (Zhong et al. 2022)
and 3x × 2x/4x hybridizations (Lim et al. 2000, 2003; Khan et al. 2009; Xie et al. 2010; Natenapit et al. 2010; Zhou et al. 2011, 2012, 2014; Chung et al. 2013; Wang et al. 2015; Suzuki and Yamagishi 2015; Xi
et al. 2015; Cui et al. 2022). All of them indicate that functional gametes produced by aneuploid or triploid
lilies usually have much higher chromosome numbers when tetraploid used as male than when diploid
used as male (Lim et al. 2003; Khan et al. 2009; Zhou et al. 2011, 2012; Wang et al. 2015; Xi et al. 2015). Besides, 3x × 4x is much more successful than 3x × 2x in Lilium (Zhou et al. 2011, 2012). The present
study also shows that aneuploid × 4x is much more successful than aneuploid × 2x in Lilium. Conclusions Based on the present research and above discussion, it is concluded that lilies have huge chromosomes
because of large genomes, which make them ideal to do cytogenetics research; and they produce
Fritillaria embryo sacs, which makes F1 distant hybrids, triploids, odd-allotetraploids; and aneuploid lilies
are partial female fertile regardless of their male sterility and thus produce aneuploid again. In addition,
the most important for lily breeding: once good lines are selected from aneuploid lines, they may not only
become cultivars through vegetative propagation, but also become parents to breed new aneuploids. Declarations Page 8/18 Acknowledgments This work was supported by the National Natural Science Foundation of China for financial supports (No. 32260783 and 31572154). References 1. Ahmad F, Poerba YS, Kema GHJ, De Jong H (2021) Male meiosis and pollen morphology in diploid
Indonesian wild bananas and cultivars. Nucleus 64:181-191. https://doi.org/10.1007/s13237-021-
00350-7 2. Akera T, Lampson MA (2016) Chromosome segregation: freewheeling sisters cause problems. eLife
5: e13788. https://doi.org/10.7554/eLife.13788 2. Akera T, Lampson MA (2016) Chromosome segregation: freewheeling sisters cause problems. eLife
5: e13788. https://doi.org/10.7554/eLife.13788 2. Akera T, Lampson MA (2016) Chromosome segregation: freewheeling sisters cause problems. eLife
5: e13788. https://doi.org/10.7554/eLife.13788 3. Ansari HA, Ellison NW, Verry IM, Williams WM (2022) Asynapsis and unreduced gamete formation in
a Trifolium interspecific hybrid. BMC Plant Biol 22:1-14. https://doi.org/10.1186/s12870-021-03403- p
y
p
g
w w 4. Barba-Gonzalez R, Lokker AC, Lim KB, Ramanna MS, Van Tuyl JM (2004) Use of 2n gametes for the
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Applied Genetics 109(6): 1125-1132. https://doi.org/ 10.1007/s00122-004-1739-0 5. Barba-Gonzalez R, Van Silfhout AA, Ramanna MS, Visser RGF, Van Tuyl JM (2006) Progenies of
allotriploids of Oriental × Asiatic lilies (Lilium) examined by GISH analysis. Euphytica 151: 243-250. https://doi.org/10.1007/s10681-006-9148-x 6. Chung MY, Chung JD, Ramanna MS, Van Tuyl JM, Lim KB (2013) Production of polyploids and
unreduced gametes in Lilium auratum × L. henryi hybrid. Int J Biol Sci 9:693-701. https://doi.org/10.7150/ijbs.6427 7. Cui L, Sun Y, Xiao K, Wan L, Zhong J, Liu Y, Zhou S (2022) Analysis on the abnormal chromosomal
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exceptional to the hypothesis of lily interploid hybridizations. Sci Hortic 293:110746. https://doi.org/10.1016/j.scienta.2021.110746 8. De Jong PC (1974) Some notes on the evolution of lilies. North Amer Lily Yearb 27: 23-28. 9. Kamstra SA, De Jong JH, Jacobsen E, Ramanna MS, Kuipers A (2004) Meiotic behaviour of
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segregation in interspecific hybrids of rice by genomic in situ hybridization. Rice Science 28(1):69-80. https://doi.org/10.1016/j.rsci.2020.11.008 15. Liu MS, Tseng SH, Tseng TC, Chung MC (2021) Visualizing meiotic chromosome pairing and
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and its significance on lily breeding: a case of analyzing OT hybrids. Euphytica 217:203-214. https://doi.org/10.1007/s10681-021-02935-4 17. Luo J, Van Tuyl JM, Arens P, Niu L (2013) Cytogenetic studies on meiotic chromosome behaviors in
sterile Oriental × Trumpet lily. Genet. Mol Res 12 (4):6673-6684. http://dx.doi.org/10.4238/2013 17. Luo J, Van Tuyl JM, Arens P, Niu L (2013) Cytogenetic studies on meiotic chromosome behaviors in
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related chromosome segregation rrrors in mouse cocyte meiosis-I. Current Biology 27(7):1040-1047. https://doi.org/10.1016/j.cub.2017.02.025 18. Nakagawa S, FitzHarris G (2017) Intrinsically defective microtubule dynamics contribute to age-
related chromosome segregation rrrors in mouse cocyte meiosis-I. Current Biology 27(7):1040-1047. https://doi.org/10.1016/j.cub.2017.02.025 19. Natenapit J, Taketa S, Narumi T, Fukai S (2010) Crossing of allotriploid LLO hybrid and Asiatic lilies
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progenies of an odd-allotetraploid Lilium ‘Honesty’. Sci Hortic 253:316-321. https://doi.org/10.1016/j.scienta.2019.04.012. 31. References Xie S, Ramanna MS, Van Tuyl JM (2010) Simultaneous identification of three different genomes in
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of tetraploid Asiatic lilies (Lilium) using FISH. Genome 11: 453-461. https://doi.org/10.1139/gen-
2015-0057 43. Zhou S, Zhong L, Zhang L, Xu Z, Liu X, Li K, Zhou G (2015) Study on the homology of the genomes
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2015-0057 44. Zielinska AP, Holubcova Z, Blayney M, Elder K, Schuh M (2015) Sister kinetochore splitting and
precocious disintegration of bivalents could explain the maternal age effect. eLife 4:e11389. https://doi.org/10.7554/eLife.11389 Figures P
12/18
Figure 1
J1614: (a) flowers and (b) metaphase chromosomes, in which A-chromosomes are dyed blue with DAPI
while L-chromosomes red with Cy3 using GISH. Arrows indicate break points of L/A-recombinant Figure 1 J1614: (a) flowers and (b) metaphase chromosomes, in which A-chromosomes are dyed blue with DAPI
while L-chromosomes red with Cy3 using GISH. Arrows indicate break points of L/A-recombinant Page 12/18 Page 12/18 chromosomes. Bar = 20μm. chromosomes. Bar 20μm. Figure 2
Asiatic lily ‘Pearl Jason’: (a) flower, (b) metaphase chromosomes of FISH painting with 5S rDNA (red) and
45S rDNA green), and c) FISH karyotype. Bar = 20μm. Figure 2
Asiatic lily ‘Pearl Jason’: (a) flower, (b) metaphase chromosomes of FISH painting with 5S rDNA (red) and
)
d )
k Figure 2 Asiatic lily ‘Pearl Jason’: (a) flower, (b) metaphase chromosomes of FISH painting with 5S rDNA (red) and
45S rDNA green), and c) FISH karyotype. Bar = 20μm. Page 13/18 Figure 3
The microsporogenesis of J1614: (a-b) metaphase I, (c-e) anaphase I, in which A-chromosomes are blue
and L-chromosomes are red; “I”, “II”, “III”and “IV”means univalent, bivalent, trivalent and tetravalent,
respectively；the arrow heads indicate break points of L/A recombinant chromosomes. Bar = 20μm. Figure 3 The microsporogenesis of J1614: (a-b) metaphase I, (c-e) anaphase I, in which A-chromosomes are blue
and L-chromosomes are red; “I”, “II”, “III”and “IV”means univalent, bivalent, trivalent and tetravalent,
respectively；the arrow heads indicate break points of L/A recombinant chromosomes. Bar = 20μm. Page 14/18 Figure 4
Microspores and pollen grains formed from abnormal meiosis. In (a) and (b), the arrows indicate
micronuclei and Bar = 20μm and in (c) and (d) bar = 200μm Figure 4
Microspores and pollen grains formed from abnormal meiosis. In (a) and (b), the arrows indicate
micronuclei, and Bar = 20μm, and in (c) and (d), bar = 200μm. Figure 4 Microspores and pollen grains formed from abnormal meiosis. In (a) and (b), the arrows indicate
micronuclei, and Bar = 20μm, and in (c) and (d), bar = 200μm. Page 15/18 Page 15/18 Figure 5 Figure 5
The metaphase chromosomes of root tips of 9 seedlings obtained from J1614 × AAAApainted with G
(a) 20106-1, (b) 20106-4, (c) 20106-5, (d) 20106-7, (e) 20106-8, (f) 20106-10, (g) 20106-13, (h) 20106-
and (i) 20106-17. L-chromosomes are dyed red with Cy3 while A-chromosomes blue with DAPI. The a
heads indicate the break points of L/A recombinant chromosomes. Figures Bar = 20 μm. Figure 5 The metaphase chromosomes of root tips of 9 seedlings obtained from J1614 × AAAApainted with GISH:
(a) 20106-1, (b) 20106-4, (c) 20106-5, (d) 20106-7, (e) 20106-8, (f) 20106-10, (g) 20106-13, (h) 20106-15,
and (i) 20106-17. L-chromosomes are dyed red with Cy3 while A-chromosomes blue with DAPI. The arrow
heads indicate the break points of L/A recombinant chromosomes. Bar = 20 μm. Page 16/18 Page 16/18 Page 16/18 Figure 6
The differences of relationship of kinetochore and chromosome separation between Akera and Lampson
(2016)’s mechanism and ours based on the findings in Lilium. (1) The diagram is redrawn referring to
Akera and Lampson (2016): for all chromosomes, their kinetochores (red dots) are split or duplicate
accompanying their DNA replicate; in normal mitosis (a) or in abnormal meiosis I (c) in which univalent
are formed by premature separation of bivalents, the two kinetochores per chromosome attach to
microtubules (blue arrows) in opposite direction and thus the two sister chromatids are pulled to two
opposite poles; in normal meiosis I (b), the kinetochores on sister chromatids are fused together and
become attached to microtubules from the same poles (double arrows) and then two homologous
chromosomes (bivalents) are disjoined to the opposite poles. (2) one chromosome has one kinetochore
(red dot) in its centromere, and the kinetochore is duplicated accompanying DNA replicates in mitosis (d);
however, in normal meiosis I (e), kinetochore duplication is hindered by synapses and cohesion in
bivalents; and in abnormal meiosis I (f), their kinetochores could replicate because no chiasmata or
cohesion suppress kinetochore duplication. Figure 6 Figure 6 All of them are abnormal meiosis; However, in each embryo sac of polyogonum type
plants (a), the nucleic DNA of its central cell is twice that of its egg cell, while in each embryo sac of
Fritillaria-type plants (b & c), the nucleic DNA of its central cell is twice that of a somatic cell. Figure 6 The differences of relationship of kinetochore and chromosome separation between Akera and Lampson
(2016)’s mechanism and ours based on the findings in Lilium. (1) The diagram is redrawn referring to
Akera and Lampson (2016): for all chromosomes, their kinetochores (red dots) are split or duplicate
accompanying their DNA replicate; in normal mitosis (a) or in abnormal meiosis I (c) in which univalent
are formed by premature separation of bivalents, the two kinetochores per chromosome attach to
microtubules (blue arrows) in opposite direction and thus the two sister chromatids are pulled to two
opposite poles; in normal meiosis I (b), the kinetochores on sister chromatids are fused together and
become attached to microtubules from the same poles (double arrows) and then two homologous
chromosomes (bivalents) are disjoined to the opposite poles. (2) one chromosome has one kinetochore
(red dot) in its centromere, and the kinetochore is duplicated accompanying DNA replicates in mitosis (d);
however, in normal meiosis I (e), kinetochore duplication is hindered by synapses and cohesion in
bivalents; and in abnormal meiosis I (f), their kinetochores could replicate because no chiasmata or
cohesion suppress kinetochore duplication. The differences of relationship of kinetochore and chromosome separation between Akera and Lampson
(2016)’s mechanism and ours based on the findings in Lilium. (1) The diagram is redrawn referring to
Akera and Lampson (2016): for all chromosomes, their kinetochores (red dots) are split or duplicate
accompanying their DNA replicate; in normal mitosis (a) or in abnormal meiosis I (c) in which univalent
are formed by premature separation of bivalents, the two kinetochores per chromosome attach to
microtubules (blue arrows) in opposite direction and thus the two sister chromatids are pulled to two
opposite poles; in normal meiosis I (b), the kinetochores on sister chromatids are fused together and
become attached to microtubules from the same poles (double arrows) and then two homologous
chromosomes (bivalents) are disjoined to the opposite poles. (2) one chromosome has one kinetochore
(red dot) in its centromere, and the kinetochore is duplicated accompanying DNA replicates in mitosis (d);
however, in normal meiosis I (e), kinetochore duplication is hindered by synapses and cohesion in
bi
l
d i
b
l
i
i I (f) h i ki
h
ld
li
b
hi Page 17/18 Figure 7
Comparison of megasporogenesis of triploid polygonum-type plants (3x), triploid (3x) and aneuploid (A*)
Fritllaria-type Lilium. Figure 7 Comparison of megasporogenesis of triploid polygonum-type plants (3x), triploid (3x) and aneuploid (A*)
Fritllaria-type Lilium. All of them are abnormal meiosis; However, in each embryo sac of polyogonum type
plants (a), the nucleic DNA of its central cell is twice that of its egg cell, while in each embryo sac of
Fritillaria-type plants (b & c), the nucleic DNA of its central cell is twice that of a somatic cell. Page 18/18
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"Letting myself go forward past wrongs": How regulatory modes affect self-forgiveness
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PloS one
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Antonio Pierro1☯, Gennaro Pica1☯*, Anna Maria Giannini1‡, E. Tory Higgins2‡, Arie
W. Kruglanski3‡ 1 University of Rome “La Sapienza”, Rome, Italy, 2 Columbia University, New York, NY, United States of
America, 3 University of Maryland, College Park, MD, United States of America ☯These authors contributed equally to this work. ‡ These authors also contributed equally to this work. * gennaro.pica@uniroma1.it a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 Editor: Danilo Garcia, Landstinget Blekinge,
SWEDEN Received: May 13, 2017
Accepted: February 2, 2018
Published: March 12, 2018 RESEARCH ARTICLE OPEN ACCESS Citation: Pierro A, Pica G, Giannini AM, Higgins ET,
Kruglanski AW (2018) "Letting myself go forward
past wrongs": How regulatory modes affect self-
forgiveness. PLoS ONE 13(3): e0193357. https://
doi.org/10.1371/journal.pone.0193357 Editor: Danilo Garcia, Landstinget Blekinge,
SWEDEN Editor: Danilo Garcia, Landstinget Blekinge,
SWEDEN Abstract The present research addresses the question of whether regulatory-mode orientations
affect self-forgiveness. We expected that people with a strong locomotion orientation would
be more inclined to self-forgiveness because of their tendencies toward movement and
change, which focus them on the future, whereas people with a strong assessment orienta-
tion would refrain from self-forgiveness due to their evaluative tendencies which focus them
on the past. These hypotheses were supported by the results in four studies that tested the
relation between regulatory modes and self-forgiveness by measuring (Studies 1, 3 and 4)
and manipulating (Study 2) regulatory-mode-orientations. Finally, in Study 4 we examined
more closely our hypothesis that the relation between self-forgiveness and regulatory
modes is mediated by past and future temporal foci. The implications of the results for regu-
latory mode theory are also discussed. OPEN ACCESS
Citation: Pierro A, Pica G, Giannini AM, Higgins ET,
Kruglanski AW (2018) "Letting myself go forward
past wrongs": How regulatory modes affect self-
forgiveness. PLoS ONE 13(3): e0193357. https://
doi.org/10.1371/journal.pone.0193357 Self-forgiveness: Antecedents, correlates and consequences Self-forgiveness has been defined as a positive attitudinal shift in the feelings, actions, and
beliefs about the self, following a self-perceived transgression or wrongdoing committed by
the self [3–5]. Thus, forgiving the self can be considered as an adaptive mechanism of humans
that helps them to restore a positive sense of the self and safeguards their overall well-being
against the toxic effects of guilt, shame and regret [6–8]. A transgression from normative rules
or offences toward other people with unwanted consequences may in fact elicit psychological
distress that needs to be reduced. Self-forgiveness may help to achieve such a restoration by
limiting self-punishment, self-condamnation, and, instead, increasing benevolence towards
the self [8]. Consistent with this reasoning, research has found that self-forgiveness is associ-
ated with value reaffirmation [9] and self-acceptance [10]. Empirical evidence suggests that self-forgiveness is linked with high self-esteem, low neu-
roticism and low levels of anxiety and depression [11–12]. Similarly, it has been found to be
positively linked with positive emotions, and with a lack of shame [13]. Numerous studies
have consistently demontrated that self-forgiveness has a positive impact on overall well-being
[14–18]. For instance, when people self-forgive their feelings, attitudes and beliefs toward the
self become more positive and this in turn leads to lower levels of depressive affect [19]. Fur-
thermore, self-forgiveness has been found to reduce procrastination [20]. More specifically,
among students who reported high levels of self-forgiveness for procrastinating studying for
the first examination, procrastination on preparing for the subsequent examination was
reduced. The above finding suggests that self-forgiveness for past wrongs allows for forward
movement toward goal pursuit by reducing procrastination tendencies. g
y
g
Because of the correlational nature of the findings in many of the past studies, it is not fully
clear what the motivational antecedents of self-forgiveness might be. It seems, however, that
the purpose of self-forgiveness is to protect one’s well-being (self-image and self-concept) in
order to move forward optimistically, avoiding being stopped by undermining feelings of
guilt, blame and regret. This view of self-forgiveness is consistent with McCullough and colle-
gues‘[21–22] definition of forgiveness as a suite of motivational changes that occurs following a
transgression whereby the victim becomes less motivated by revenge and more motivated
toward benevolence. Regulatory mode and self-forgiveness versus assessment [2] may relate to differences in self-forgiveness. Specifically, locomotors’
tendency to change from a state to state and move forward leads to a greater inclination to be
self-forgiving. In contrast, assessors‘ tendency to critically evaluate and compare their current
state with previous experiences leads to a lesser inclination to be self-forgiving. Competing interests: The authors have declared
that no competing interests exist. Self-forgiveness: Antecedents, correlates and consequences Even though this definition refers to interpersonal forgiveiness, it is fairly
likely to assume that the same motivational changes occur in the process of self-forgiveness
whereby the person shifts from feeling guilty to benevolence toward the self. If this is the case,
then self-forgiveness would be supported by a motivational orientation that encourages
change, thus allowing movement towards the future. As we will see next, this describes the
motivational orientation of the locomotion mode. Introduction Copyright: © 2018 Pierro 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. All of us, at least once in our life, has behaved badly and felt guilty later. For instance, you may
have offended someone or hurt their feelings, made mistakes that harmed others, or did some-
thing you knew was wrong at the time. All such situations have the potential to produce nega-
tive thoughts and feelings toward the self [1]. For example, people may begin to dislike
themselves and believe themselves not worthy of trust and love, may reduce their self-esteem,
or may even suffer affective disorders. In order to cope with these destructive consequences of
wrongful actions and to move on, people may need to forgive themselves. However, not all of
us forgive ourselves and turn our gaze to the future, and, if we do, we don’t do it all of the time. How do we deal with past wrongdoings and what are the antecedents of forgiving the self? The
aim of the present research is to address these questions and increase our understanding of the
antecedents of self-forgiveness. Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. Funding: This work was supported by the
University of Rome “La Sapienza” (2015) prot. C26A15CT93 to Prof. Antonio Pierro and
“Fondazione Ania per la sicurezza stradale,
Progetto ANIACARES” to Prof. Anna Maria
Giannini. The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript. Given that self-forgiveness involves some sort of motivational change that involves self-reg-
ulation, it should be useful to consider self-forgiveness from the perspective of self-regulatory
mechanisms. We propose that differences in the self-regulatory orientations of locomotion 1 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 Regulatory mode and self-forgiveness in a straightforward and direct manner, without undue distractions and delays” (p. 794). In
other words, the assessment function is to select the right or best course of action in goal pur-
suit, whereas the locomotion function is to move on, effect change, and manage action towards
the desired end-state. Locomotion and assessment modes can operate as individual difference variables, but they
can also be situationally induced [25]. Thus, although assessment and locomotion can work
together as parts of the same goal pursuit orientation [26], they are also conceived as essentially
orthogonal dimensions, each of which can receive differential emphasis to same phenomena
[23]. Empirical evidence shows that assessment positively correlates with fear of invalidity, dis-
comfort with ambiguity, neuroticism, low self-esteem, and negative mood [2]. Locomotion, on
the other hand, positively correlates with psychological vitality, self-esteem, optimism, and
being decisive, and it negatively correlates with social anxiety and depression [2]. Furthermore, assessors, compared to locomotors, are more inclined to experience nostalgia
[27] and suffer more from counterfactual thinking after failure and from experiencing regret
about their choices [28]. More specifically, Pierro and colleagues [28] for example, found in a
series of studies that high (vs. low) assessors were more likely to engage in counterfactual
thinking and this produced more regret about a previous negative decision (e.g., purchasing a
faulty product with poor customer service). In contrast, high locomotors were less likely to
engage in counterfactual thinking or experience regret from a past mistake. Whereas assessors’
concern with making the right choice through critical evaluation orients them toward past
experiences, considering and reconsidering the consequences of past actions to understand
the right way to proceed [27–28], locomotors’ concern with effecting change and moving
focuses them on present and future possibilities [2, 24, 29]. The above regulatory concerns can
have secondary consequences: assessment may leave people confined in the current state, eval-
uating the past and comparing it with the present, potentially creating repercussions for self-
forgiveness; whereas locomotion may help to overcome the past mistakes and effectively move
forward, potentially more easily leading to self-forgiveness. Consistent with this difference,
assessment has been found to be positively related to procrastination whereas locomotion has
been found to be negatively related [30]. Notably, procrastination is negatively linked to self-
forgiveness [20]. All of this suggests that there could be a positive link between locomotion
and self-forgiveness and a negative link between assessment and self-forgiveness. PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 Locomotion and assessment orientations Regulatory mode theory [2, 23–24]captures two critical dimensions of goal-directed behavior,
assessment and locomotion. The assessment dimension refers to “the comparative aspect of self-
regulation concerned with critically evaluating entities or states, such as goals or means, in
relation to alternatives in order to judge relative quality” [2] (p.794). The locomotion dimen-
sion refers to “the aspect of self-regulation concerned with movement from state to state and
with committing the psychological resources that will initiate and keep goal-related movement PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 2 / 20 Participants On a volunteer basis, a total of 323 Italian people (192 women) participated in the study. Their
Mage was 30.06 (SDage = 10.93). The total sample consisted of two separate sub-samples. Sample
1 consisted of 168 university students (103 women). Their Mage was 23.73 (SDage = 2.89). Sample 2 consisted of 155 non-students (89 women), with Mage of 36.92 (SDage = 12.24). This
second non-student sample comprised 81 (52.3%) white-collar employees, 52 (33.5%) profes-
sionals, 4 (2.6%) entrepreneurs, 10 (6.5%) unemployed, and 8 (5.2%) pensioners. Other than
the differences on student and professional status, the two sub-samples also significantly differ
on age, non-students sample being older than the students sample, t(321) = -13.56, p < .001. As previous research showed that older adults are more forgiving than younger adults [31], we
decided to run the analyses separately on each sub-sample as well as on the total sample, con-
trolling for gender and age. Procedure All participants filled out the Locomotion and Assessment scales. They then completed a mea-
sure designed to assess self-forgiveness. The present research The purpose of the present research was to test whether individuals’ self-regulatory mode ori-
entation affects self-forgiveness. The theory and the findings described above suggest that peo-
ple with a strong locomotion orientation are inclined to shift their attention away from what
happened in the past, effect change and move forward to attain goals in the future. We hypoth-
esize that these locomotion tendencies toward change and movement promotes self-forgive-
ness. In contrast, people with a strong assessment orientation are inclined to evaluate their
past experience in order to make the right choice in the present. A consequence of this is that
they may keep in mind the past and thus enhance their tendencies toward stasis over dynamic
action and change. Thus, we hypothesize these assessment concerns would reduce self-forgive-
ness. These predictions should hold true whether individuals’ heightened locomotion or
assessment states derive from chronic predispositions to be in these states or from situational
forces that momentarily induce them. To test these hypotheses, we conducted a series of four studies whereby we tested the gen-
eral association between regulatory modes and self-forgiveness, using different methods and
measures. Specifically, we measured (in Studies 1, 3 and 4) and manipulated (in Study 2) PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 3 / 20 Regulatory mode and self-forgiveness regulatory mode and then we measured self-forgiveness. Finally, in Study 4 we examined more
closely our hypothesis that the relation between self-forgiveness and the regulatory modes is
mediated by the different attention locomotors and assessors give to thinking about the future
or the past. This set of studies was approved by the Ethical Committee of the department of
developmental and socialization processes (University of Rome “La Sapienza”) under protocol
63-11/23, titled: “The influence of Regulatory Mode Orientations on Self-Forgiveness”. Study 1 Our first study examined the basic relationships between chronic locomotion and assessment
orientations and self-forgiveness. We hypothesized that people with a strong locomotion ori-
entation would be more inclined to self-forgive their past misdeeds; while people with a strong
assessment orientation would be more resistant to self-forgive their own wrongs. PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 Assessing self-forgiveness All participants responded to 4 items derived from the feeling subscale of Wohl and colleagues’
[19] State Self-Forgiveness Scale (SFS). This state scale was created to target self-forgiveness for
specific transgressions against the self as opposed to more general, or global forgiveness of the
self. Specifically, following the Wohl and colleagues’ [19] procedure, participants were told to
think of the most significant experience in which they did something they believe to have been
wrong. To the point, participants were asked to think about an incident in which they took
responsibility for committing a wrongful behavior. Instructions also specified that the event
should involve an incident that was hurtful to someone else. Participants were told to take a
moment to consider the circumstances of that event and recall as much detail about what they
did that was wrong. They were asked not to write what the incident entailed but instead to
think about the incident and the feelings that the incident arouses in them and then complete
the scale while thinking how they feel about themselves “right now” regarding the wrongful
event. The 4-item scale asked for the respondents’ current feelings about their wrongdoing: I
feel. . . rejecting of myself (Item Reversed, R), accepting of myself, dislike toward myself (R),
forgiving myself. Ratings were made on a 4-point scale with the response alternatives anchored
at the ends with 1 (Not at all) to 4 (Completely). The 4 items were averaged to create a compos-
ite score (Total sample Cronbach’s α = .72). Higher scores reflected greater self-forgiveness. Locomotion and assessment orientations The Italian versions of the Locomotion and Assessment Scales [2] constitute of two separate
12-item self-report measures designed to tap individual differences in these tendencies. Specif-
ically, respondents rated the extent to which they agree with self-descriptive statements reflect-
ing locomotion (e.g., "By the time I accomplish a task, I already have the next one in mind")
and assessment (e.g., I spend a great deal of time taking inventory of my positive and negative
characteristics"). Ratings were made on a 6-point Likert type scale with the response alterna-
tives anchored at the ends with 1 (Strongly Disagree) to 6 (Strongly Agree). We computed two
composite scores (one for Locomotion and one for Assessment) by averaging across responses
to each item. For the total sample the Cronbach ‘s α for the locomotion scale was .78 and the α
for the assessment scale was .75. In this total sample, the two scales were not correlated (r =
-.08; n.s.), consistent with previous studies [2] PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 4 / 20 Regulatory mode and self-forgiveness https://doi.org/10.1371/journal.pone.0193357.t001 Results Descriptive statistics and correlations among variables for total sample and for each sub-sam-
ple are reported in Table 1. Note that in each sub-sample and in the total sample the self-for-
giveness measure was positively and significantly related to locomotion, but negatively and
significantly related to assessment. Also note that the relationship between locomotion and
self-forgiveness as well as between assessment and self-forgiveness in the two sub-samples did
not significantly differ (Z = 1.042, n.s.; and Z = .382, n.s., respectively). Predictions regarding the differential and unique effects of locomotion and assessment ori-
entations on self-forgiveness were tested by means of three separate multiple regression Table 1. Descriptive and correlations between variables (Study 1). M
SD
1
2
3
Sample 1 (Students, N = 168)
Locomotion
4.50
.70
(.81)
Assessment
3.74
.79
-.20
(.76)
Self-Forgiveness
2.94
.68
.30
-.28
(.73)
Sample 2 (Non-Students, N = 155)
Locomotion
4.76
.64
(.73)
Assessment
3.65
.78
.08
(.74)
Self-Forgiveness
3.13
.62
.19
-.24
(.70)
Total Sample (N = 323)
Locomotion
4.62
.68
(.78)
Assessment
3.70
.79
-.08
(.75)
Self-Forgiveness
3.03
.66
.27
-.27
(.72) Table 1. Descriptive and correlations between variables (Study 1). M
SD
1
2
3
Sample 1 (Students, N = 168)
Locomotion
4.50
.70
(.81)
Assessment
3.74
.79
-.20
(.76)
Self-Forgiveness
2.94
.68
.30
-.28
(.73)
Sample 2 (Non-Students, N = 155)
Locomotion
4.76
.64
(.73)
Assessment
3.65
.78
.08
(.74)
Self-Forgiveness
3.13
.62
.19
-.24
(.70)
Total Sample (N = 323)
Locomotion
4.62
.68
(.78)
Assessment
3.70
.79
-.08
(.75)
Self-Forgiveness
3.03
.66
.27
-.27
(.72)
p < .05
p < .01
p < .001. In bracket (Cronbach’s α). https://doi.org/10.1371/journal.pone.0193357.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 5 / 20 Regulatory mode and self-forgiveness analyses: one for each sub-sample and one for the total sample. In these analyses we regressed
the self-forgiveness scores on both the locomotion and assessment indices as predictors. Gen-
der (dummy coded; Male = 0; Female = 1) and age were entered as control variables. For the
total sample we also entered sample (dummy coded; Student = 0; non-Student = 1) as control
variable. Summary of results of these analyses are reported in Table 2. As Table 2 shows, for each sub-sample, as well as for total sample, the pattern of results was
the same. Participants 97 university students (55 women) in Italy participated in the study on a volunteer basis. Their
Mage was 23.07 (SDage = 2.91). Results Specifically, as expected, self-forgiveness was significantly and positively related to
locomotion (sample 1, β = .25, p < .001; sample 2, β = .21, p < .01; Total sample, β = .23, p <
.001, respectively) and significantly and negatively related to assessment (sample1, β = -.23,
p < .001; sample 2, β = -.26, p < .001; Total sample, β = -.25, p < .001, respectively). Study 2 The purpose of the second study was to replicate the results of Study 1, this time using an
experimental manipulation of locomotion and assessment. This replication is important
because regulatory mode theory assumes the functional equivalence of individual difference
measures and situational inductions of the locomotion and assessment orientations. Addition-
ally, the manipulation of regulatory mode allows for a stronger causal inference to be drawn
between assessment/locomotion and subsequent experience of self-forgiveness. PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 Procedure and materials Participants were randomly assigned to the locomotion, assessment, and control conditions. Following Avnet and Higgins’ [25] procedure, locomotion and assessment were manipulated
by asking participants to complete a ‘‘personal memories” questionnaire by which participants
will be asked to think of three different situations in which they personally exemplified either
high locomotion or high assessment behaviors and to write them down. Specifically, to manip-
ulate locomotion, participants were asked to: ‘‘Think of a day when you made many different
things”; ‘‘Think of a time when you finished one project and did not wait long before you
started a new one”; ‘‘Think of a time when you decided to do something and you could not Table 2. Summary of multiple regression analyses (Study 1). Sample 1
(Students)
Sample 2
(Non-Students)
Total Sample
β
β
β
Locomotion
.25
.21
.23
Assessment
-.23
-.26
-.25
Gender
.02
-.01
.01
Age
.03
-.02
-.01
Sample
-
-
.09
p < .01
p < .001. Gender (Male = 0; Female = 1); Sample (Students = 0; Non-students = 1). Table 2. Summary of multiple regression analyses (Study 1). Sample 1
(Students)
Sample 2
(Non-Students)
Total Sample
β
β
β
Locomotion
.25
.21
.23
Assessment
-.23
-.26
-.25
Gender
.02
-.01
.01
Age
.03
-.02
-.01
Sample
-
-
.09
p < .01
p < .001. Gender (Male = 0; Female = 1); Sample (Students = 0; Non-students = 1). PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 6 / 20 Regulatory mode and self-forgiveness wait to get started.” For assessment, they will be asked to: ‘‘Think of some occasion in which
you compared yourself with other people”; ‘‘Think of some occasion in which you thought
about your positive and negative characteristics”; ‘‘Think of some occasion in which you cri-
tiqued work done by others or yourself.” These situations were taken from items in the loco-
motion and assessment scales of the regulatory mode questionnaire [2]. Avnet and Higgins
[25] have shown that this experimental manipulation of locomotion and assessment states is
effective in inducing the corresponding psychological states. After completing the “personal memories” questionnaire, which experimentally induced
the locomotion and assessment tendencies, participants completed the same 4-item scale of
self-forgiveness used in study 1. Control group participants did not complete the personal
memories questionnaire and instead were immediately asked to complete the self-forgiveness
scale. Procedure and materials In this study the self-forgiveness Cronbach ‘s α was .72 (M = 2.84, SD = .66). Results As predicted, a one-way ANOVA, with age and gender (dummy coded; Men = 0; Women = 1)
as covariates, yielded a significant effect of experimental conditions on self-forgiveness, F(2,
92) = 7.90, p = .001, pη2 = .15, while age and gender did not affect self-forgiveness [F (1,92) =
.54, p = .46, pη2 = .01; F (1,92) = .75, p = .39, pη2 = .01, respectively]. Simple contrasts revealed
that participants in the locomotion condition showed greater self-forgiveness (N = 33,
M = 3.15, SD = .48) than participants in either the assessment orientation condition (N = 33,
M = 2.55, SD = .57), t (94) = 3.95, p = .00, Cohen’s d = 1.14, or the control group condition
(N = 31, M = 2.83, SD = .77), t (94) = 2.08, p = .04, Cohen’s d = .50. Furthermore, participants
in the assessment orientation condition (though only approaching significance) showed lower
self-forgiveness than participants in the control group condition, t (94) = 1.80, p = .07, Cohen’s
d = .41. PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 Study 3 In Study 1, we found that assessment was negatively related and locomotion positively related
to self-forgiveness. In Study 2, we induced locomotion or assessment regulatory mode and we
found that the individuals under condition of high locomotion were more likely to experience
self-forgiveness than those under high assessment. A possible limit of the first two studies is
that the effects of regulatory mode orientations on self-forgiveness were not controlled for pos-
sible effects of secondary variables possibly having a role in the process. For instance, we rea-
soned that locomotion influences self-forgiveness because of its tendency to effect change and
move forward. However, one may suspect that locomotors, in order to go head and quickly
move on, may recall less severe transgressions or accept less responsibility with respect of the
fact, thus enhancing the ease of the self-forgiveness process. In other words, it might be possi-
ble that people with strong locomotion concerns forgive themselves more also because they do
not fully admit their responsibility for the wrongful episode. On the other hand, we reasoned
that assessment should block self-forgiveness because of its evaluative tendencies that lead peo-
ple to remain anchored to past errors. Again, this logic may lead one to expect that assessors,
being focused on past mistakes by their comparative and evaluative tendencies, may recall
more severe transgressions and accept more responsibility for the wrongs recalled thus reduc-
ing the ease of the self-forgiveness process. Furthermore, as it is possible that forgive the self
with respect to errors of the remote past may be easier (because these errors may lose their
power and may be already overcome), we controlled for the time passed from the recalled epi-
sode and for positive and negative affect that may be induced by the recalled transgressions
which both may influence self-forgiveness. PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 7 / 20 Regulatory mode and self-forgiveness To rule out the above possibilities, in the present study we asked participants to assess their
assumption of responsibility and the severity of the recalled transgressions, and we assessed
the objective severity of the participant’s recalled transgressions by having two independent
raters evaluating the written descriptions of the event. Participants Eighty-five students (60 women; Mage = 23.16, SDage = 3.92) participated in the experiment. The same measure of self-forgiveness used in previous two studies was the dependent variable. Procedure The study proceeded in two phases. During the first phase, participants completed in class the
same locomotion and assessment scales used in Study 1. In the second phase, approximately 1
month later, participants were asked to think back to an episode where they had offended or
hurt someone and to briefly describe it. The instructions were as follows: “Every now and
then, most or all people have hurt somebody else. We ask you to think about an episode where
you offended or hurt someone”. After receiving these instructions, participants were asked to
write a paragraph about the offense. The writing part served to induce participants to bring to
mind the episode and their feelings about it. The written descriptions of offenses reflected a
wide variety of interpersonal situations (e.g., hurt the partner, a family member, a friend). After describing the offense, participants were carefully instructed to answer the PANAS scale,
the same 4 items assessing state self-forgiveness used in Study 1, the transgression severity, the
time passed since the episode occurred and the acceptance of responsibility for the wrong
recalled. At the end of this task, they were debriefed, thanked and dismissed. Study 3 We expected that the relation between
regulatory mode orientations and self-forgiveness should be maintained regardless of partici-
pants’ acceptance of responsibility, the severity of the transgressions recalled, the time passed
since the episode occurred and regardless participants’ affect elicited by the recalled transgres-
sion. The purpose of Study 3 was to confirm the relationship between regulatory modes and
self-forgiveness using a different recall task and by controlling for these possible variables. Locomotion and assessment orientations Participants’ locomotion and assessment orientations were measured with the same Italian
version of the regulatory mode scale [21] used in Study 1. The Cronbach ‘s α for the locomo-
tion scale was .83 and the α for the assessment scale was .71. Consistent with Study 1 and previ-
ous studies [2], the two scales were not correlated (r = .12; p = .25). Assessing subjective and objective trasgression severity Participants were asked to rate using a ten point scale (1 = not at all; 10 = completely) the extent
to which they saw the described offense as: (a) serious and (b) harmful or damaging to the
other person [7]. The two items were correlated, r = .34, p = .002, and were averaged to assess
subjective transgression severity. In order to also assess the objective transgression severity, two
independent raters, naïve to the study’s hypotheses, were asked to rate the written offenses
using the same two items described above. Within-rater internal consistency (Pearson’s rs)
was .63 for rater 1 and .71 for rater 2, and averaged .73. The inter-rater agreement (Pearson’s
rs) for the two scales was .53 for “serious”, and .52 for “harmful”, and averaged .59. Furthermore, the correlation between the subjective and objective indexes of transgression
severity were highly correlated (r = .54; p < .001). In addition, we used the rwg(j) as an index
of inter-rater (subjective score, and objective raters 1 and 2 scores) agreement for multiple
items [33]. The value obtained of .93, calculated using a normal distribution, suggested high
inter-rater agreement. Thus, we calculated a total index of transgression severity by adding the
subjective and objective transgression severity scores. Assessing self-forgiveness All participants responded to the same 4 items used in Study 1 (M = 3.14, SD = .59, α = .74). Responsibility and the time passed since the incident occurred Five items [11], rated from 0 (completely disagree) to 10 (completely agree), assessed the degree to
which participants felt responsible for the offense (α = .82). Statements included: ‘‘I feel I was
responsible for what happened,” ‘‘I wasn’t really to blame for this” (Reverse scored), ‘‘I was in the
wrong in the situation,” ‘‘This was clearly my fault,” and ‘‘I did not really do anything wrong” (R). They also reported how long ago the incident occurred (i.e., in months; M = 24.14; SD = 34.61). Panas The Panas (Positive and Negative Affect Schedule) [32] was used to gauge positive (e.g., active,
proud, determined) and negative (e.g., upset, guilty, distressed) emotional responses to the
transgression reported. Specifically, participants used a Likert-type scale ranging from 1 (not
at all) to 7 (extremely) to endorse each of 20 adjectives in response to the question: “How do
you feel currently about this transgression?”. These were then separately collapsed into the
positive (M = 2.61, SD = .72, α = .83) and negative (M = 2.42, SD = .82, α = .90) affectivity sub-
scales used in our analysis. 8 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 Regulatory mode and self-forgiveness PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 Regulatory mode and self-forgiveness Table 3. Descriptive and correlations between variables (Study 3). M
SD
1
2
3
4
5
6
7
8
9
10
(N = 85)
Self-Forgiveness
3.14
.59
(.74)
Locomotion
4.36
.64
.28
(.83)
Assessment
3.76
.63
-.24
.12
(.71)
Transgression Severity ^
7.15
0.97
-.08
.03
-.10
(.93+)
Acceptance of Responsibility
7.45
1.63
-.16
-.05
.07
.34
(.82)
Positive affect
2.61
.72
.31
.09
.04
.07
.05
(.83)
Negative affect
2.42
.82
-.39
-.19
.05
.26
.07
-.03
(.90)
Time
24.14
34.62
.07
.16
.05
.20
.06
-.23
-.15
-
Age
23.16
3.92
.23
.13
-.11
.03
-.01
.07
-.22
.52
-
Gender
-
-
.07
.10
.02
-.13
.02
.04
-.03
.01
-.07
- Table 3. Descriptive and correlations between variables (Study 3). M
SD
1
2
3
4
5
6
7
8
9
10
(N = 85)
Self-Forgiveness
3.14
.59
(.74)
Locomotion
4.36
.64
.28
(.83)
Assessment
3.76
.63
-.24
.12
(.71)
Transgression Severity ^
7.15
0.97
-.08
.03
-.10
(.93+)
Acceptance of Responsibility
7.45
1.63
-.16
-.05
.07
.34
(.82)
Positive affect
2.61
.72
.31
.09
.04
.07
.05
(.83)
Negative affect
2.42
.82
-.39
-.19
.05
.26
.07
-.03
(.90)
Time
24.14
34.62
.07
.16
.05
.20
.06
-.23
-.15
-
Age
23.16
3.92
.23
.13
-.11
.03
-.01
.07
-.22
.52
-
Gender
-
-
.07
.10
.02
-.13
.02
.04
-.03
.01
-.07
-
p < .001
p < .01
p < .05. In bracket (Cronbach’s α)
+rwg(j) index. ^ Total (subjective and objective) transgression severity index. https://doi.org/10.1371/journal.pone.0193357.t003 Table 3. Descriptive and correlations between variables (Study 3). above variables: self-forgiveness was positively related to locomotion (β = .20, t = 2.01, p =
.048) and negatively related to assessment (β = -.25, t = -2.59, p = .01). We also run two sepa-
rate regression analyses as described in the text, one controlling for subjective and one control-
ling for objective indexes of transgression severity. The pattern of results was the same for both
the objective and subjective indexes as well as for the total severity transgression index. Results Descriptive statistics and correlations among variables are reported in Table 3. Note that the
self-forgiveness measure was positively and significantly related to locomotion, but negatively
and significantly related to assessment. Furthermore, transgression severity, time passed since
the transgression occurred and acceptance of responsibility of the wrong were not related to
regulatory mode orientations, helping to rule out the possibility that high-locomotors simply
recall less severe transgressions and do not fully accept their responsibility for the past wrong;
and that high-assessors simply recall more severe transgressions and attribute to the self a
greater responsibility for the offense reported. Predictions regarding the differential and unique effects of locomotion and assessment ori-
entations on self-forgiveness were tested by means of a multiple regression analyses. In this
analysis we regressed the self-forgiveness scores on both the locomotion and assessment indi-
ces as predictors. Gender (dummy coded; Men = 0; Women = 1), age, transgression severity,
assumption of responsibility, time occurred since the transgression, positive and negative
mood were entered as control variables. Nor the effects of transgression severity (β = -.02, t =
-.19, p = .85), the time passed since the episode occurred (β = .06, t = .47, p = .64) and the
acceptance of responsibility (β = -.12, t = -1.24, p = .22), neither the effects of age (β = .07, t =
.59, p = .56) or gender (β = .03, t = .34, p = .74) were significant. As expected, self-forgiveness
was positively related to positive affect (β = .31, t = 3.06, p = .003) and negatively related to neg-
ative affect (β = -.30, t = -2.93, p = .004). More importantly, the relation between self-forgive-
ness and the two regulatory mode orientations remain significant after controlling for all the PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 9 / 20 Study 4 The previous three studies, using different methods, offer consistent evidence that locomotion
and assessment are associated with self-forgiveness in opposite directions: locomotion is posi-
tively associated with self-forgiveness whereas assessment is negatively associated with self-for-
giveness. Notably, our proposal was that these two relations are mediated by different forces;
specifically a difference in temporal focus. The purpose of Study 4 was to investigate this differ-
ence. We hypothesized that the general association between regulatory modes and self-forgive-
ness is mediated by the attention locomotors and assessors give to thinking about the future or
the past: Locomotion should be positively associated with self-forgiveness because of the loco-
motors’ tendency to focus on the future, whereas assessment should be negatively associated
with self-forgiveness because of assessors’ tendency to focus on the past. In summary, the purpose of the fourth study was to replicate the results of Study 1 and
Study 3, this time using a dispositional self-forgiveness scale, and, more important, to verify
the mediating role of temporal focus on the relations between regulatory modes and self-
forgiveness. On a volunteer basis, a total of 189 students from University of Rome “La Sapienza” (133
women) participated in the study. Their Mage was 24.53 (SDage = 2.81). Assessing temporal focus Participants completed the 12-item Temporal Focus Scale (TFS) (four items each for the past,
present, and future), which was developed by Shipp, Edwards and Lambert [34]. Illustrative
items include ‘‘I replay memories of the past in my mind” for past temporal focus; ‘‘My mind
is on the here and now” for present temporal focus; and ‘‘I focus on my future” for future tem-
poral focus. The TFS items were rated on a 7-point scale describing the frequency with which
the respondent thought about the time frame indicated by the item (1 = Never; 7 = Constantly). We computed three composite scores (one for each temporal focus subscale) by averaging
across responses to each item. The Cronbach ‘s αfor the past temporal focus scale was .91, the
αfor the present temporal focus scale was .85, and the α for the future temporal focus scale was
.92. Procedure All participants filled out the Locomotion and Assessment scales followed by temporal focus
scale. They then completed a measure designed to assess dispositional self-forgiveness
tendency. Assessing self-forgiveness All participants responded to the 6 items derived from the dispositional Self-Forgiveness sub-
scale of the Heartland Forgiveness Scale (HFS) developed by Thompson et al. [35]. Specifically,
following the Thompson et al. [35] procedure, participants were told that “in the course of our
lives negative things may occur because of our own actions. For some time after these events,
we may have negative thoughts or feelings about ourselves.” To the point, participants were
asked to think about how they typically respond to such negative events and, then, to complete
the following 6 items: “Although I feel bad at first when I mess up, over time I can give myself
some slack”; “I hold grudges against myself for negative things I’ve done” (R); “Learning from
bad things that I’ve done helps me get over them”; “It is really hard for me to accept myself
once I’ve messed up” (R); “With time I am understanding of myself for mistakes I’ve made”; “I
don’t stop criticizing myself for negative things I’ve felt, thought, said, or done” (R). Ratings were made on a 7-point scale with the response alternatives anchored at the ends
with 1 (Almost Always False of Me) to 7 (Almost Always True of Me). The 6 items were averaged
to create a composite score (α = .70; M = 4.43, SD = .94). Higher scores reflect greater disposi-
tional self-forgiveness tendency. Participants On a volunteer basis, a total of 189 students from University of Rome “La Sapienza” (133
women) participated in the study. Their Mage was 24.53 (SDage = 2.81). 10 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 Regulatory mode and self-forgiveness Locomotion and assessment orientations Participants responded to the same Italian versions of the Locomotion and Assessment Scales
used in previous studies. For the present sample the Cronbach ‘s α for the locomotion scale
was .83, and the α for the assessment scale was .77. M of the locomotion score was 4.37 (SD =
.62) and M of the assessment score was 3.65 (SD = .67). Again, in this sample, the two scales
were not correlated (r = -.05; p = .53), consistent with previous studies [2]. Regulatory mode and self-forgiveness Table 4. Descriptive and correlations between variables (Study 4). M
SD
1
2
3
4
5
6
(N = 189)
Locomotion
4.37
.62
(.83)
Assessment
3.65
.67
-.05
(.77)
Past temporal focus
5.10
1.09
-.12+
.41
(.91)
Present temporal focus
5.12
1.00
.25
.003
-.09
(.85)
Future temporal focus
5.54
1.05
.37
.02
.004
.33
(.92)
Self-Forgiveness
4.43
.94
.22
-.27
-.40
.19
.25
(.70) Table 4. Descriptive and correlations between variables (Study 4). In bracket (Cronbach’s α). https://doi.org/10.1371/journal.pone.0193357.t004 locomotion and assessment indices as predictors. Gender (dummy coded; Men = 0;
Women = 1) and age were entered as control variables. As expected, self-forgiveness, after controlling for gender (β = -.25, p < .001) and age (β =
-.07, p = .30), was significantly and positively related to locomotion (β = 23, p < .001) and sig-
nificantly and negatively related to assessment (β = -.28, p < .001). Results Descriptive statistics and correlations among variables are reported in Table 4. As in previous studies, predictions regarding the differential and unique effects of locomo-
tion and assessment orientations on self-forgiveness tendency were tested by means of a multi-
ple regression analysis. In this analysis we regressed the self-forgiveness scores on both the PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 11 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 A mediational analysis: Multiple mediator model As anticipated, we also tested the hypothesized mediating role of temporal focus in the rela-
tionship between regulatory modes and self-forgiveness. We expected the positive relation
between locomotion and self-forgiveness to be mediated by the future temporal focus, and the
negative relation between assessment and self-forgiveness, by the past temporal focus. To test these mediational hypotheses we estimated a model where the potential mediators
(past, present and future temporal focus in our case) controlled for one another and that
includes multiple independent variables (locomotion and assessment modes in our case). To
examine the proposed mediation model, we employed Preacher and Hayes’ [36–37] procedure
to extrapolate estimates of direct and indirect effects. Preacher and Hayes’ strategy employs
the use of bootstrapping, a non-parametric re-sampling procedure, to estimate the size of indi-
rect effects. The present analysis was performed using PROCESS program (Model 4) [38]. As
specified by Hayes, PROCESS (Model 4) can be used to estimate the coefficients in a multiple
mediation model with multiple independent variables, although it provides no information
that can be used to test a combined indirect effect involving all independent variables. Never-
theless, covariates are mathematically treated exactly like independent variables in the estima-
tion, with paths to all mediators and the outcome, so if the desired model has k independent
variables, PROCESS can be run k times, each time listing one variable as the independent vari-
able and treating remaining k– 1 independent variables as covariates. Each run of PROCESS
generates the effects (including indirect effects) for the variable currently listed as independent
variable. Then, following this procedure to extrapolate estimates of direct and indirect effects,
we performed two multiple mediation analyses testing a model with the mediating role of past,
present and future temporal focus: one for the effect of locomotion on self-forgiveness and one
for the effect of assessment on self-forgiveness. For each of the regulatory mode effects, we
controlled for the alternative mode’s effect, included in the model as a covariate. Also gender
and age were included in the models as control variables. Ninety-five percent confidence inter-
vals (CI) were employed and 1000 bootstrapping re-samples were run. Confidence intervals PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 12 / 20 Regulatory mode and self-forgiveness were adjusted for bias (bias corrected, BC). The results obtained from the two analyses are
summarized in Table 5 and in Fig 1. A mediational analysis: Multiple mediator model As Fig 1 shows, of the three potential mediators, the future temporal focus was significantly
and positively related to self-forgiveness and the past temporal focus was significantly and neg-
atively related to self-forgiveness. No significant effect was found for present temporal focus. The results obtained from the analysis regarding locomotion mediated effect shows that the
total effect of locomotion on self-forgiveness was significant and positive, b = .35, SE = .10, p <
.001. Moreover, as Fig 1 (Panel A) shows, the direct effect of locomotion become non-signifi-
cant (b = .17, SE = .10, p = .11) after controlling for the mediators. Concerning the relationship
between locomotion and the three potential mediators, locomotion was significantly and posi-
tively associated with future and present temporal focus and negatively (although only margin-
ally significant) with past temporal focus. Finally, as Table 5 shows, the total indirect effect of
locomotion on self-forgiveness was significant. More importantly, of the three indirect effects,
only the effect of locomotion on self-forgiveness through the future temporal focus was signifi-
cant (indirect effect = .11, with BC 95% CI of .0368 to .2112, not containing zero). The results obtained from the analysis regarding assessment mediated effect shows that the
total effect of assessment on self-forgiveness was significant and negative (b = -.38, SE = .09,
p < .001). Moreover, as Fig 1 (Panel B) shows, the direct effect of assessment remain significant
(b = -.20, SE = .10, p = .04) although significantly reducted when controlling for the mediators. Assessment was significantly and positively associated only with the mediating variable of past
temporal focus. Finally, and more importantly, of the three indirect effects, only the effect of
assessment on self-forgiveness through past temporal focus was significant (indirect effect =
-.19, with BC 95% CI of -.3086 to -.1046, not containing zero). Overall, the multiple mediator
model was highly significant, F (7,181) = 11.47, p < .001, with R2 = .31. These results are highly
consistent with our mediational hypotheses. PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 General discussion The major purpose of the present research was to contribute to the understanding of the self-
regulatory antecedents of self-forgiveness. Based on recent findings linking regulatory mode
with the construct of time [29, 39], where the concept of self-forgiveness can be included as it
refers to dealing with past memories (i.e., time as a flow from past, present and future), it was Table 5. Indirect effects of regulatory mode orientations on self-forgiveness through proposed mediators (Study
4). Bias-corrected bootstrap CIs
Mediator
Bootstrap effect
SE
Lower
Upper
Mediated effect of locomotion
Total indirect effect
.19.05.03.11
.06
.09
.32
Past temporal focus
.04
-.01
.13
Present temporal focus
.03
-.02
.08
Future temporal focus
.04
.04
.21
Mediated effect of assessment
Total indirect effect
-.18-.19.00.00
.05
-.30
-.09
Past temporal focus
.05
-.31
-.10
Present temporal focus
.01
-.02
.03
Future temporal focus
.02
-.03
.04
Note. CI = Confidence Interval. https://doi.org/10.1371/journal.pone.0193357.t005 Table 5. Indirect effects of regulatory mode orientations on self-forgiveness through proposed mediators (Study
4). 13 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 Regulatory mode and self-forgiveness Fig 1. Coefficients (b values) representing effects of locomotion (Panel A) and assessment (Panel B) orientations
on mediators and self-forgiveness (Study 4). Note: The effects of Gender and Age were omitted for a better clarity of
the figure. The value in parentheses represents the direct effect of regulatory modes on self-forgiveness after controlling
for mediating variables. p < .01; p < .001; +p = .13. https://doi.org/10.1371/journal.pone.0193357.g001 Fig 1. Coefficients (b values) representing effects of locomotion (Panel A) and assessment (Panel B) orientations
on mediators and self-forgiveness (Study 4). Note: The effects of Gender and Age were omitted for a better clarity of
the figure. The value in parentheses represents the direct effect of regulatory modes on self-forgiveness after controlling
for mediating variables. p < .01; p < .001; +p = .13. https://doi.org/10.1371/journal.pone.0193357.g001 https://doi.org/10.1371/journal.pone.0193357.g001 https://doi.org/10.1371/journal.pone.0193357.g001 predicted that locomotion and assessment would impact self-forgiveness differently. We rea-
soned that intrinsic locomotion tendencies towards change and movement helps to move for-
ward into the future effectively, overcoming the past and enhancing the possibilities of self-
forgiveness, while assessment tendencies to appraisals and comparisons leads to a stasis state
where the past is evaluated in the present, thus reducing the easy of self-forgiveness. The results of four studies provided strong support for our hypotheses. PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 Regulatory mode and self-forgiveness Furthermore, with the aim of examine the mediating role of temporal focus in the relation
between self-forgiveness and regulatory modes, the results of Study 4 also showed that the pos-
itive relation between locomotion and dispositional self-forgiveness was mediated by the
future temporal focus, and the negative relation between assessment and self-forgiveness was
mediated by the past temporal focus. It should be noted, however, that the mediation analysis
was conducted using cross-sectional data, thus giving problems to inferring the causality
between the variables [36]. However, both theoretical and methodological issues make us con-
fident that the findings of Study 4 are consistent with (or do not contradict) our mediation
hypothesis. Theoretically speaking, consistent with the suggestion that theory might help to determine
the proper order of the variables in a proposed mediation model [37, 41], our mediation
hypothesis was built upon previous work showing that locomotion and assessment influence
how individuals differently focus on time [29]. In particular, due to their predominant motiva-
tion towards change and movement which focuses them towards the future, high-locomotors
have been found to be less nostalgic, while due to their predominant motivation towards
appraisal which focuses them on assessing and comparing the past with the present high-asses-
sors have been found to be more nostalgic [27]. In the same vein, high assessors have been
found to experience more regret and counterfactual thinking, whereas high locomotors have
been found to experience less counterfactuals and less regret about past decisions [28]. Fur-
thermore, high-locomotors procrastinate less and focus on goal pursuit, while high-assessors
procrastinate more [30]. Importantly, procrastination and self-forgiveness has been shown to
be negatively related [20], suggesting that people who forgive themselves reduce their procras-
tination tendencies by focusing on immediate goal pursuing, thus allowing for movement. Methodologically speaking, three indicators play a supportive role of our hypotheses. First,
we know from Study 2 that regulatory mode orientations (being manipulated) influence self-
forgiveness. Second, in Study 3 we measured the locomotion and assessment scales approxi-
mately 1 month before participants were asked to think back to answer the self-forgiveness
questionnaire. Third, in Study 1 we first measured regulatory mode orientations and then self-
forgiveness; and in Study 4 again we first measure regulatory mode orientations and then we
measured temporal focus and self-forgiveness (in this exact temporal order). PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 General discussion In Study 1 we mea-
sured locomotion, assessment [2] and state self-forgiveness by means of a self-report scale, and
we found a positive association between locomotion and self-forgiveness and a negative associ-
ation between assessment and self-forgiveness. In Study 2 regulatory modes were manipulated
and, consistent with the hypotheses, it was found that participants in the locomotion condition
exhibited higher tendency towards self-forgiveness compared to participants in both assess-
ment and control conditions; participants in the assessment condition exhibited lower ten-
dency towards self-forgiveness compared to participants in the control condition. Study 3
confirmed the results of Study 1 helping us to rule out the possibility that, in order to move on
and easily change the current state, locomotors simply referred to less severe transgressions
and present less acceptance of responsibility than assessors, thus resulting in an ease of self-for-
giveness. In fact, since self-forgiveness involves the process of admitting one’s responsibility of
the wrong, controlling for the above variables we can exclude that we are not referring to what
has been called pseudo-self-forgiveness, that is when people do not accept their responsibility
for the error [40]. It was shown that the hypothesized relations between regulatory mode ori-
entations and self-forgiveness remained even controlling for transgression severity, acceptance
of responsibility, time passed since the recalled transgressions occurred and positive and nega-
tive mood. PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 14 / 20 Notwithstanding, as suggested in Preacher and Hayes [37], in order to corroborate the
validity of our mediation hypothesis, and to rule out possible alternative causal models (e.g.,
temporal foci influence regulatory mode orientations which in turn influence self-forgiveness),
might be adopted two different strategies (1) implement longitudinal designs, also including
covariates (e.g., self-acceptance) to help eliminate source of spurious correlation between tem-
poral foci (M) and self-forgiveness (Y); and (2) use the experimental-causal-chain strategy [42]
to experimentally test the hypothesized mediation model by (a) manipulating regulatory mode
and then measuring temporal foci, and by (b) manipulating temporal foci and then measuring
self-forgiveness. Future studies may implement the above designs to corroborate the validity of
the proposed mediation hypothesis. Taken together, the four studies are consistent with the
proposition that locomotion inclinations lead to effectively overcoming past mistakes and
move on, focusing on future goals, thus enhancing the inclination to forgive the self, while
assessment inclinations lead to dig deeper to understand better what happened in past wrongs
and compare that situations with the present, producing a state of stasis that obstacles the self-
forgiveness process. Although the above results are quite clear, other variables, not considered in the present
studies, might potentially play a role in the described process (e.g., self-acceptance as conceptu-
alized in the construct of self-directedness, [43–44]). The concept of self-acceptance, in fact, is
closely related to self-forgiveness as it implies the ability to let go of inner criticism and PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 15 / 20 Regulatory mode and self-forgiveness struggles [43–44]. Therefore, it might be possible that high locomotion concerns may help
self-acceptance of past wrongs by enhancing, through the movement toward the future, the
ease to let go of inner criticism which in turn increases self-forgiveness. This possibility may
deserve to be investigated by future studies. The findings of the present research also support recent dual-process models of self-forgive-
ness [45–46], which assert that self-forgiveness entails both (1) making a decision to accept
responsibility for wrongs, and (2) replace of self-condemning emotions with self-affirming
emotions. In fact, locomotion was positively associated with self-forgiveness, being perhaps
positively associated with both components of self-forgiveness. On the other hand, assessment
was negatively associated with self-forgiveness, being perhaps negatively associated with emo-
tional restoration of personal esteem. p
This all sounds good for high locomotion, but we need to mention a potential fly in the
ointment. It is possible that high locomotors’ urge for movement and change could lead them
to take action for future goals irrespective of past wrongs and mistakes. Yes, they are more self-
forgiving but, at the same time, they could be more susceptible to repeat errors and mistakes. In this vein, research has suggested that self-forgiveness for ongoing, wrongful behavior (e.g.,
smoking; gambling) may function as an alleviating strategy that reduce negative feelings asso-
ciated to the wrongs committed by the self, thereby decreasing any behavioral learning from
these wrongs [13, 40, 47–49]. Thus, locomotion tendencies to move forward enhances self-for-
giveness, but may also lead to an underestimation of past errors and, as a consequence, the rep-
etition of wrongs. This possibility needs to be investigated in future research. Furthermore,
considering that the two regulatory mode orientations (locomotion and assessment) are
orthogonal dimensions and may work together [26] as part of the same goal pursuit (self-for-
giveness in this case), we might further develop the idea expressed above. Specifically, even
though self-forgiveness has taken place, and thus the impact of negative emotions of guilt and
shame are considerably reduced, high assessment concerns may lead individuals to still con-
sider past errors in order to not repeat them in the future. Therefore, it is possible that locomo-
tion concerns help individuals to forgive themselves, reducing the negative impact of guilt and
shame allowing for movement toward the future, while in a second moment the assessment
concerns help to keep in mind the teaching from past wrongs in order to not repeat them in
the future. The investigation of the above hypothesis may be interesting not only for its theo-
retical implications, but also because it may have notable and relevant applicability. For
instance, knowledge on how the two regulatory mode orientations work together (in predict-
ing self-forgiving and learning by mistakes) can be beneficial when working with youths. A
problem with young people, in fact, is that they are typically highly impulsive, and display low
levels of self-control [50], which lead them to an higher insorgence of risky and uncontrolled
behaviors [51–52], and to lower self-forgiveness tendencies [53–54]. PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 S1 File. Dataset of Study1.
(SAV) S2 File. Dataset of Study2. (SAV) S2 File. Dataset of Study2. (SAV) S3 File. Dataset of Study3. (SAV) S3 File. Dataset of Study3. (SAV) S4 File. Dataset of Study4. (SAV) Author Contributions Conceptualization: Antonio Pierro, Gennaro Pica, E. Tory Higgins, Arie W. Kruglanski. Data curation: Antonio Pierro, Gennaro Pica. Conceptualization: Antonio Pierro, Gennaro Pica, E. Tory Higgins, Arie W. Kruglanski. Data curation: Antonio Pierro, Gennaro Pica. Formal analysis: Antonio Pierro, Gennaro Pica. Formal analysis: Antonio Pierro, Gennaro Pica. Funding acquisition: Antonio Pierro, Anna Maria Giannini. Investigation: Antonio Pierro, Gennaro Pica. Investigation: Antonio Pierro, Gennaro Pica. Methodology: Antonio Pierro, Gennaro Pica. Project administration: Antonio Pierro, Gennaro Pica. Project administration: Antonio Pierro, Gennaro Pica. Supervision: Antonio Pierro, Gennaro Pica, Anna Maria Giannini. Writing – original draft: Antonio Pierro, Gennaro Pica. Writing – review & editing: Antonio Pierro, Gennaro Pica, Anna Maria Giannini, E. Tory
Higgins, Arie W. Kruglanski. A concomitant combina-
tion of high locomotion and high assessment might allow highly impulsive young students to
face their everyday duties at school in the best way, by both helping self-forgiveness for the
occurrence of possible mistakes and learning from them, thereby reducing the reiteration of
wrongful behaviors in the future. Also this possibility might be considered in future research. Another potential area for future research would be to test the same hypotheses as the pres-
ent research but for other kinds of forgiveness, such as forgiving others rather than self-for-
giveness. In this regard, research has found that locomotion increases the motivation to
reconcile and decreases the influence on reconciliation of the feelings of negativity from the
conflict. In contrast, assessment decreases the motivation to reconcile and increases the influ-
ence of the negative feelings from the conflict [55]. These forgiveness-of-others findings are
consistent with what we found for self-forgiveness. Given the results of above studies, however,
there may be cases where the assessment orientation and self-forgiveness become positively PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 16 / 20 Regulatory mode and self-forgiveness related. For instance, because high-assessors tend to feel guilty for their past wrongs they may
more likely attempt to make amends by offering an apology, thereby possibly receiving for-
giveness from their victims and ultimately increasing self-forgiveness [56]. On the contrary,
assessment orientation may even prompt perpetrators to punish themselves, perhaps if
attempts to making amends are improbable both because (a) victims deny their forgiveness to
offenders and because (b) perpetrators perceive victims’ positive reactions towards them
improbable. These hypotheses should be considered as hints for future research. Supporting information Supporting information
S1 File. Dataset of Study1. (SAV)
S2 File. Dataset of Study2. (SAV)
S3 File. Dataset of Study3. (SAV)
S4 File. Dataset of Study4. (SAV) S1 File. Dataset of Study1. (SAV) PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
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or to “just do it”: Locomotion and assessment as distinct self-regulatory imperatives. J Pers Soc Psy-
chol. 2000; 79: 793–815. PMID: 11079242 3. Enright RD. Counseling within the forgiveness triad: On forgiving, receiving forgiveness, and self–for-
giveness. Counseling Values. 1996; 40: 107–126. 4. Hall JH, Fincham FD. Self-forgiveness: The stepchild of forgiveness research. J Soc Clin Psychol. 2005; 24: 621–637. 5. Worthington EL Jr. Forgiveness and reconciliation: Theory and application. 1st ed. New York: Rout-
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March 12, 2018 Regulatory mode and self-forgiveness 7. Fisher ML, Exline JJ. Moving toward self-forgiveness: Removing barriers related to shame, guilt, and
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hol problems. Substance Abuse. 2013; 34: 396–404. https://doi.org/10.1080/08897077.2013.781564
PMID: 24159911 11. Mauger PA, Perry JE, Freeman T, Grove DC, McBride AG, McKinney KE. The measurement of forgive-
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other-forgiveness in the Five Factor Model. J Pers Assessment. 2004; 82: 207–214. 13. Tangney JP, Boone AL, Dearing R. Forgiving the self: Conceptual issues and empirical findings. In:
Worthington EL Jr, editor. Handbook of Forgiveness. New York: Brunner-Routledge; 2005. pp. 143–
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Clin Psychol. 2000; 19: 43–55. 16. Seybold KS, Hill PC, Neumann JK, Chi DS. Physiological and psychological correlates of forgiveness. J
Psychol Christianity. PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
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doi.org/10.1037/a0025943 PMID: 22103579 18 / 20 PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 Regulatory mode and self-forgiveness 31. Cheng S-T, Yim Y-K. Age Differences in Forgiveness: The Role of Future Time Perspective. Psych
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tive experience of past, present, and future. Org behav and human decis processes. 2009; 1: 1–22. 35. Thompson LY, Snyder CR, Hoffman L, Michael ST, Rasmussen HN, Billings LS, et al. Dispositional for-
giveness of self, others, and situations. J Pers. 2005; 73: 313–360. https://doi.org/10.1111/j.1467-
6494.2005.00311.x PMID: 15745433 36. Preacher KJ, Hayes AF. Asymptotic and resampling strategies for assessing and comparing indirect
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and assessors plan and perceive time. Int J Psych. 2014; 49: 192–199. 40. Wohl MJA, McLaughlin KJ. Self-forgiveness: The good, the bad, the ugly. Soc Pers Psychol Compass. 2014; 8: 422–435. 41. Hoyle RH, Robinson JC. Mediated and moderated effects in social psychological research: Measure-
ment, design, and analysis issues. In: Sansone C, Morf CC, Panter AT, editors. The Sage handbook of
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tive than mediational analyses in examining psychological processes. J Pers Soc Psychol. 2005; 89:
845–851. https://doi.org/10.1037/0022-3514.89.6.845 PMID: 16393019 43. Cloninger CR. Feeling Good: The Science of Well-Being. 1st ed. New York: Oxford University Press;
2004. 44. Cloninger CR. PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
March 12, 2018 55.
Webb CE, Coleman PT, Tomasulo LR, Higgins ET. Moving On or Digging Deeper: Regulatory Mode
Effects on Conflict Resolution. J Pers Soc Psychol. 2017; 112: 621–641. https://doi.org/10.1037/
pspp0000131 PMID: 28095008 56.
Carpenter TP, Carlisle RD, Tsang JA. Tipping the scales: Conciliatory behavior and the morality of self-
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March 12, 2018 References What makes people healthy, happy, and fulfilled in the face of current world challenges? Mens Sana Monographs. 2013; 11: 16–24. https://doi.org/10.4103/0973-1229.109288 PMID:
23678235 45. Griffin BJ, Moloney JM, Green JD, Worthington EL, Cork B, Tangney JP, et al. Perpetrators‘ reactions
to perceived interpersonal wrongdoing: The associations of guilt and shame with forgiving, punishing,
and excusing oneself. Self Ident. 2016; 15: 650–661. 46. Woodyatt L, Wenzel M. Self-forgiveness and restoration of an offender following an interpersonal trans-
gression. J Soc Clinic Psych. 2013; 32: 225–259. 47. Squires EC, Sztainert T, Gillen NR, Caouette J, Wohl MJA. The Problem with Self-Forgiveness: Forgiv-
ing the Self Deters Readiness to Change Among Gamblers. J Gaml Stud. 2012; 28: 337–350. 48. Wohl MJA, Thompson A. A dark side to self-forgiveness: Forgiving the self and its association with
chronic unhealthy behaviour. British J Soc Psychol. 2011; 50: 354–364. 49. Wohl MJA, Salmon MM, Hollingshead SJ, Lidstone SK, Tabri N. The Dark Side of Self-Forgiveness:
Forgiving the Self Can Impede Change for Ongoing, Harmful Behavior. In: Woodyatt L, Worthington EL
Jr, Wenzel M, Griffin BJ, editors. Handbook of the Psychology of Self-Forgiveness; 2017. pp. 147–159. 50. Steinberg L, Albert D, Cauffman E, Banich M, Graham S, Woolard J. Age Differences in Sensation
Seeking and Impulsivity as Indexed by Behavior and Self-Report: Evidence for a Dual Systems Model. Develop Psychol. 2008; 44: 1764–1778. 51. Lowenstein G. Out of control: Visceral influences on behavior. Org Behav Hum Dec Proc. 1996; 65:
272–292 52. Tangney JP, Baumeister RF, Boone AL. High Self-Control Predicts Good Adjustment, Less Pathology,
Better Grades, and Interpersonal Success. J Pers. 2004; 72: 271:324. PMID: 15016066 53. Burnette JL, Davisson EK, Finkel EJ, Van Tongeren DR, Hui CM, Hoyle RH. Self-control and Forgive-
ness: A meta-analytic review. Soc Psychol Pers Sci. 2015; 5: 443–450. 54. Lucas T, Young JD, Zhdanova L, Alexander S. Self and other justice beliefs, impulsivity, rumination and
forgiveness: Justice beliefs can both prevent and promote forgiveness. Pers Indiv Diff. 2010; 49: 851–
856. PLOS ONE | https://doi.org/10.1371/journal.pone.0193357
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Investigating the uptake, effectiveness and safety of COVID-19 vaccines: protocol for an observational study using linked UK national data
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BMJ open
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Strengths and limitations of this study Introduction The novel coronavirus SARS-CoV-2, which
emerged in December 2019, has caused millions of
deaths and severe illness worldwide. Numerous vaccines
are currently under development of which a few have
now been authorised for population-level administration
by several countries. As of 20 September 2021, over
48 million people have received their first vaccine dose
and over 44 million people have received their second
vaccine dose across the UK. We aim to assess the uptake
rates, effectiveness, and safety of all currently approved
COVID-19 vaccines in the UK. ►We will use national data for each UK nation and
across the UK general population. ►Rapid and robust real-time estimates on uptake, ef-
fectiveness and safety of COVID-19 vaccines will be
provided using data from existing national pandemic
platforms in the UK. ►This is an observational study and analyses are,
therefore, potentially susceptible to residual or un-
measured confounders. ►This is an observational study and analyses are,
therefore, potentially susceptible to residual or un-
measured confounders. ►Prepublication history and
additional supplemental material
for this paper are available
online. To view these files,
please visit the journal online
(http://dx.doi.org/10.1136/
bmjopen-2021-050062). on February 22, 2022 by guest. Protected by copyright. tp://bmjopen.bmj.com/ Methods and analysis We will use prospective cohort
study designs to assess vaccine uptake, effectiveness
and safety against clinical outcomes and deaths. Test-
negative case–control study design will be used to assess
vaccine effectiveness (VE) against laboratory confirmed
SARS-CoV-2 infection. Self-controlled case series and
retrospective cohort study designs will be carried out
to assess vaccine safety against mild-to-moderate and
severe adverse events, respectively. Individual-level
pseudonymised data from primary care, secondary care,
laboratory test and death records will be linked and
analysed in secure research environments in each UK
nation. Univariate and multivariate logistic regression
models will be carried out to estimate vaccine uptake
levels in relation to various population characteristics. VE estimates against laboratory confirmed SARS-CoV-2
infection will be generated using a generalised additive
logistic model. Time-dependent Cox models will be used
to estimate the VE against clinical outcomes and deaths. The safety of the vaccines will be assessed using logistic
regression models with an offset for the length of the risk
period. Where possible, data will be meta-analysed across
the UK nations. Ethics and dissemination We obtained approvals
from the National Research Ethics Service Committee,
Southeast Scotland 02 (12/SS/0201), the Secure
Anonymised Information Linkage independent Information
Governance Review Panel project number 0911. BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2 BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2 Protocol Open access Investigating the uptake, effectiveness
and safety of COVID-19 vaccines:
protocol for an observational study
using linked UK national data To cite: Vasileiou E, Shi T,
Kerr S, et al. Investigating
the uptake, effectiveness and
safety of COVID-19 vaccines:
protocol for an observational
study using linked UK
national data. BMJ Open
2022;12:e050062. doi:10.1136/
bmjopen-2021-050062 Strengths and limitations of this study Concerning English data, University of Oxford is compliant
with the General Data Protection Regulation and the
National Health Service (NHS) Digital Data Security and
Protection Policy. This is an approved study (Integrated
Research Application ID 301740, Health Research
Authority (HRA) Research Ethics Committee 21/HRA/2786). The Oxford-Royal College of General Practitioners
Clinical Informatics Digital Hub meets NHS Digital’s Data
Security and Protection Toolkit requirements. In Northern
Ireland, the project was approved by the Honest Broker
Governance Board, project number 0064. Findings will be
made available to national policy-makers, presented at
conferences and published in peer-reviewed journals. Received 09 February 2021
Accepted 19 January 2022 For numbered affiliations see
end of article. Correspondence to
Dr Eleftheria Vasileiou;
eleftheria.vasileiou@ed.ac.uk
© Author(s) (or their
employer(s)) 2022. Re-use
permitted under CC BY. Published by BMJ. Eleftheria Vasileiou ,1 Ting Shi ,1 Steven Kerr,1 Chris Robertson,2,3 Mark Joy,4
Ruby Tsang ,4 Dylan McGagh,4 John Williams,4 Richard Hobbs,4
Simon de Lusignan ,4 Declan Bradley,5 Dermot OReilly,5 Siobhan Murphy,5
Antony Chuter,6 Jillian Beggs,6 David Ford,7 Chris Orton ,7 Ashley Akbari ,7
Stuart Bedston,7 Gareth Davies,7 Lucy J Griffiths ,7 Rowena Griffiths,7
Emily Lowthian ,7 Jane Lyons ,7 Ronan A Lyons ,7 Laura North,7
Malorie Perry,8 Fatemeh Torabi,7 James Pickett,9 Jim McMenamin,3
Colin McCowan,10 Utkarsh Agrawal,10 Rachael Wood ,1,11
Sarah Jane Stock ,1,11 Emily Moore,3 Paul Henery ,12 Colin R Simpson,1,13
Aziz Sheikh
1 Investigating the uptake, effectiveness
and safety of COVID-19 vaccines:
protocol for an observational study
using linked UK national data on February 22, 2022 by guest. Protected by copyright. http://bmjopen.bmj.com/
st published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Eleftheria Vasileiou ,1 Ting Shi ,1 Steven Kerr,1 Chris Robertson,2,3 Mark Joy,4
Ruby Tsang ,4 Dylan McGagh,4 John Williams,4 Richard Hobbs,4
Simon de Lusignan ,4 Declan Bradley,5 Dermot OReilly,5 Siobhan Murphy,5
Antony Chuter,6 Jillian Beggs,6 David Ford,7 Chris Orton ,7 Ashley Akbari ,7
Stuart Bedston,7 Gareth Davies,7 Lucy J Griffiths ,7 Rowena Griffiths,7
Emily Lowthian ,7 Jane Lyons ,7 Ronan A Lyons ,7 Laura North,7
Malorie Perry,8 Fatemeh Torabi,7 James Pickett,9 Jim McMenamin,3
Colin McCowan,10 Utkarsh Agrawal,10 Rachael Wood ,1,11
Sarah Jane Stock ,1,11 Emily Moore,3 Paul Henery ,12 Colin R Simpson,1,13
Aziz Sheikh 1 INTRODUCTION In the UK, the ‘Understanding Society’
COVID-19 survey asked 12 035 participants (in November
2020) their likelihood of vaccine uptake and reason for
hesitancy.19 High levels of hesitancy were found in women
(21%), younger adults aged 16–24 years old (27%), those
with lower education levels (19%) and in black (72%)
and Pakistani/Bangladeshi (42%) ethnic groups.19 This is
particularly concerning given that ethnic minority groups
are some of the subgroups of the population that have
been most at risk in this pandemic.19 The identification
of key factors related to vaccine uptake may be useful in
efforts to increase uptake and subsequently maximise the
impact of the COVID-19 vaccination programme in the
UK. detected in Wuhan, China in December 2019.1 On 11
March 2020, WHO declared the COVID-19 as a global
pandemic, which as of 20 September 2021 has caused
more than 228 million infections and four million deaths
worldwide.1 The need for vaccines against this novel
virus triggered an emergency response by governments,
pharmaceutical companies and research institutions to
develop, licence and manufacture COVID-19 vaccines at
scale. INTRODUCTION Dozens of COVID-19 vaccines are currently under
development with some vaccines now having successfully
completed their prelicensure clinical trials and been
approved for population vaccine administration.2 3 The
speed with which the world’s first vaccines3 were available
for mass administration at the end of 2020 is unprece-
dented given that typically it takes years for a vaccine to
be available for use at a population level.4 p p
The Pfizer-BioNTech and Oxford-AstraZeneca were
among the first vaccines approved by national regulatory
authorities such as the UK’s Medicines and Healthcare
products Regulatory Agency.5 Based on prelicensure clin-
ical trials, the Pfizer-BioNTech vaccine was 95% effective
at preventing laboratory-confirmed COVID-19 illness in
individuals without evidence of previous infection.6 The
Oxford-AstraZeneca vaccine has also reported significant
efficacy of 64% and 70% after one and two doses, respec-
tively, against symptomatic disease.7 As of the 8 January
2021, the UK has also approved a third COVID-19 vaccine
manufactured by Moderna which has shown 94% efficacy
against confirmed SARS-CoV-2 infection after receipt
of second dose.8 All three vaccines were well tolerated
with mild-to-moderate side effects mostly reported.6–8 In
March 2021, the European Medicines Agency reported
extremely rare but serious side effects including blood
clots and bleeding following administration of the
Oxford-AstraZeneca vaccine.9 10 As a result, the Joint
Committee on Vaccination and Immunisation (JCVI)
of the UK Government has recommended that healthy
adults aged 18–39 years old should not be offered the
Oxford-AstraZeneca vaccine but the Pfizer-BioNTech or
Moderna vaccines instead.11 Rare cases of cardiac inflam-
mation including myocarditis and pericarditis following
immunisation with the Pfizer-BioNTech and Moderna
vaccines have also been reported12 and are thus closely
monitored by national and international regulatory
agencies. on February 22, 2022 by guest. Protected by copyright. mjopen.bmj.com/ ary 22, 2022 by guest. Protected by copyright. As of 20 September 2021, over 48 million people have
received their first vaccine dose and over 44 million
people have received their second vaccine dose across
the UK13 based on JCVI’s vaccination priority list which
targets those most at risk of severe COVID-19 illness (eg,
older adults and people with comorbidities).14 guest. Protected by copyright. INTRODUCTION Correspondence to
Dr Eleftheria Vasileiou;
eleftheria.vasileiou@ed.ac.uk The first human cases infected by the novel
coronavirus SARS-CoV-2 pathogen were Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 1 Open access events. These postmarketing observational studies will
add additional value to the prelicensure clinical trials as
they can assess real-life effects of the COVID-19 vaccines
and the impact of the vaccination programme at popula-
tion levels.15 All UK-licensed vaccines have demonstrated
high efficacy in clinical trials; however, more evidence is
needed about the type, level and duration of protection
for different segments of the population. The recom-
mended time period between administration of the first
and second doses of the Pfizer-BioNTech6 and Oxford-
AstraZeneca7 vaccines are 3 and 6 weeks, respectively. The
UK government decided to lengthen this gap to up to 12
weeks for both vaccines.14 This is because of the desire to
provide some degree of protection to as many people as
possible and the decision was supported by findings from
the Oxford-AstraZeneca vaccine trial, which have shown
that vaccine efficacy was higher (65%) in the group that
was administered the second dose more than 6 weeks after
the first dose, compared with the group given the second
dose less than 6 weeks after the first dose (53%).7 16 A simu-
lation study has also shown that high vaccine coverage
even with less efficacious vaccines (due to partial immu-
nisation) can lead to a greater reduction of SARS-CoV-2
infection levels compared with a lower vaccine coverage
with more efficacious vaccines.17 On the other hand, some
experts have expressed their concerns that delay between
doses could increase the risk of vaccine-resistant strains
emerging due to a partially immunised population.16 The
provision of timely estimates on the protection conferred
between doses is thus urgently needed. The assessment
of vaccine-induced adverse events also needs to be
carried out, particularly for rare adverse events that are
usually only detectable in large population studies.18 The
rate of vaccine receipt by demographic, socioeconomic
and other epidemiological characteristics also needs to
be measured. Overview of linked databases and study population We will use pseudonymised individual level data routinely
collected at primary and secondary care visits, linked with
mortality, laboratory and vaccination data across the UK. Unique national datasets will be developed and hosted
within secure research environments in each UK nation
with standardised individual-level analyses run across data-
sets. Pooled estimates across the UK nations will also be
calculated. For England, data from the Royal College of
General Practitioners Research and Surveillance Centre
(RCGP RSC) (approximately 5.4 million people)23 will be
used. We will also access and analyse national coverage
data from Northern Ireland (approximately 1.9 million
people). For Scotland, data will be derived from the
Early Pandemic Evaluation and Enhanced Surveillance
of COVID-19 (approximately 5.4 million people).24 For
Wales, data from the Controlling COVID-19 through
enhanced population surveillance and intervention
(ConCOV) (approximately 3.2 million people)25 26 and
the Secure Anonymised Information Linkage (SAIL)
Databank27 will be used. See table 1 for details on data
sources from each UK nation. Aim and objectives Timely postlicensure monitoring of coverage, protec-
tion and safety of these newly introduced vaccines is
imperative.15 Specifically, robust observational epidemi-
ological studies are required to measure coverage rates
in the population in relation to demographic and other
population characteristics, assess effectiveness against
infection, severe illness and deaths, and to detect adverse The aims of this study are to assess the uptake, effec-
tiveness, and safety of the currently licensed COVID-19
vaccines (Pfizer-BioNTech, Oxford-AstraZeneca and
Moderna) in the UK using linked healthcare and admin-
istrative data. We will also seek to assess any additional
licensed vaccines during the course of this study. Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 2 Open access on February 22, 2022 by
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from on February 22, 2022 by guest. Protected by
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from (NHS) Digital Data Security and Protection approved
method.29 30 NHS Digital will link additional data to these
pseudonyms. (NHS) Digital Data Security and Protection approved
method.29 30 NHS Digital will link additional data to these
pseudonyms. Our primary objectives are to: (1) measure variation
in vaccine uptake in relation to population characteris-
tics; (2) assess vaccine effectiveness (VE) against infec-
tion, transmission,20 severe outcomes and deaths; and (3)
identify the risk of adverse events following immunisation
(AEFIs) in each UK nation. Our secondary objectives are
to provide UK-wide pooled estimates of each primary
objective. Scotland For the Scottish data, Public Health Scotland (PHS) will
carry out the data linkage and offer a secure environment
for researchers to access and analyse the pseudonymised
individual level data.24 The Community Health Index
number (a unique identifier for each resident receiving
healthcare) will be used to link individuals’ data which
will be replaced by a study ID. Wales Withing SAIL and in collaboration with the ConCOV
project,25 26 data will be linked and anonymised from
NHS sources via a mature split-file system. All identifiable
data will stay within the NHS, and will be linked to the
Welsh Demographic Service Dataset (WDSD), pseudony-
mised and encrypted within NHS Wales by Digital Health
and Care Wales, with pseudonymised demographics
supplied to SAIL at Swansea University,27 where the non-
identifiable clinical data are held. SAIL will link the demo-
graphic data to relevant clinical data, and then further
encrypt the linked data before presenting to research
teams within a secure virtual desktop. Exposure (vaccination) data
England on February 22, 2022 by guest. Protected by copyright. mjopen.bmj.com/ In England, data on vaccination will derive from general
practitioners (GPs) and the National Immunisation
Management Service (NIMS).31 NIMS is the System of
Record for the NHS COVID-19 vaccination programme
in England developed by NHS Digital.31 NIMS will collect
any demographic, GP and employee (for NHS) data to
identify groups of the population eligible for vaccination. Data collected from NIMS will also feed back into GP
systems so that an individual’s electronic health record is
updated regarding to their vaccination history.31 Vaccina-
tion data in GP records will be recorded using the System-
atised Nomenclature of Medicine (SNOMED) Clinical
Terms (CT).32 RCGP RSC now uses SNOMED CT for all
its key variables including vaccine data.33 A key part of
these curated variables are those for COVID-19; there
have been three iterations of these and we have carefully
curated case definitions.34–36 y 22, 2022 by guest. Protected by copyright. guest. Protected by copyright. METHODS
Study design A prospective cohort study design will be used to measure
variations in vaccine uptake and assess VE against severe
illness, deaths, secondary SARS-CoV-2 infection due to
household transmission. A test-negative design (TND)
case-control study will be carried out to assess VE against
laboratory confirmed COVID-19 infection. In the TND,
cases and controls will be those with a positive and nega-
tive test for COVID-19, respectively.21 A self-controlled
case series (SCCS) study design will be used to assess
the risk of AEFI. The SCCS study will be used to deter-
mine the relative incidence of adverse events for exposed
time periods (periods following vaccine administration)
compared with unexposed time periods (prevaccination
or postvaccination periods unrelated to vaccination) in
individuals who present with the outcome of interest.22
For more severe and event dependent safety outcomes, a
retrospective cohort study will be considered. England English primary care data will be held in the Oxford-
Royal College of General Practitioners Clinical Infor-
matics Digital Hub.28 Data will be pseudonymised as close
to source as possible using an National Health Service Northern Ireland For the Northern Irish data, the Honest Broker Service
(HBS) will carry out the data linkage and offer a secure
environment for researchers to access and analyse the
pseudonymised individual-level data. The Healthcare
Number will be used to link individuals’ data though
replaced with an anonymous study ID in the analysis
dataset. on February 22, 2022 by
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from ADBE, Annual District Birth Extract (ONS Births); ADDE, Annual District Death Extract (ONS Deaths); CDDS, Critical Care DataSet; CHESS, COVID-19 Hospitalisation in England
Surveillance System; COPS, COVID-19 in Pregnancy in Scotland; CVSP, COVID-19 Shielded People list; CVVD, COVID-19 Vaccine Data; ECDS, Emergency Care Data Set; ECOSS,
Electronic Communication of Surveillance in Scotland; EDDD, Emergency Department Data Daily; EDDS, Emergency Department Dataset; EPD, Electronic Prescribing Database;
GP, general practitioner; HEPMA, Hospital Electronic Prescribing and Medicines Administration; HES, Hospital Episode Statistics; ICU, intensive care unit; IMD, Index of Multiple
Deprivation; LIS, Laboratory Information System; MIDS, Maternal Indicators DataSet; MSDS, Maternity Services Dataset; NCCH, National Community Child Health database; NHAIS,
National Health Applications and Infrastructure Services; NHS, National Health Service; NIMATS, Northern Ireland Maternity System; NIMS, National Immunisation Management
Service; NIRAES, Northern Ireland Regional Accident and Emergency System; NRS, National Records of Scotland; ONS, Office for National Statistics; PATD, Pathology data COVID-19
Daily; PEDW, Patient Episode Database for Wales; PHE, Public Health England; PHS, Public Health Scotland; PIS, Prescribing Information System; RCGP RSC, Royal College of
General Practitioners Research and Surveillance Centre; SGSS, Second Generation Surveillance System; SICSAG, Scottish Intensive Care Society Audit Group; SIRS, Scottish
Immunisation and Recall System; SMR01, Scottish Morbidity Record 01; SUS, Secondary Users Service; TVMT, Turas Vaccination Management Tool; VMS, Vaccine Management
System; WDSD, Welsh Demographic Service Dataset; WLGP, Welsh Longitudinal General Practice. on February 22, 2022 by
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from on February 22, 2022 by guest. Protected by copyright. http://bmjopen.bmj.com/
shed as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from ADBE, Annual District Birth Extract (ONS Births); ADDE, Annual District Death Extract (ONS Deaths); CDDS, Critical Care DataSet; CHESS, COVID-19 Hospitalisation in England
Surveillance System; COPS, COVID-19 in Pregnancy in Scotland; CVSP, COVID-19 Shielded People list; CVVD, COVID-19 Vaccine Data; ECDS, Emergency Care Data Set; ECOSS,
Electronic Communication of Surveillance in Scotland; EDDD, Emergency Department Data Daily; EDDS, Emergency Department Dataset; EPD, Electronic Prescribing Database;
GP, general practitioner; HEPMA, Hospital Electronic Prescribing and Medicines Administration; HES, Hospital Episode Statistics; ICU, intensive care unit; IMD, Index of Multiple
Deprivation; LIS, Laboratory Information System; MIDS, Maternal Indicators DataSet; MSDS, Maternity Services Dataset; NCCH, National Community Child Health database; NHAIS,
National Health Applications and Infrastructure Services; NHS, National Health Service; NIMATS, Northern Ireland Maternity System; NIMS, National Immunisation Management
Service; NIRAES, Northern Ireland Regional Accident and Emergency System; NRS, National Records of Scotland; ONS, Office for National Statistics; PATD, Pathology data COVID-19
Daily; PEDW, Patient Episode Database for Wales; PHE, Public Health England; PHS, Public Health Scotland; PIS, Prescribing Information System; RCGP RSC, Royal College of
General Practitioners Research and Surveillance Centre; SGSS, Second Generation Surveillance System; SICSAG, Scottish Intensive Care Society Audit Group; SIRS, Scottish
Immunisation and Recall System; SMR01, Scottish Morbidity Record 01; SUS, Secondary Users Service; TVMT, Turas Vaccination Management Tool; VMS, Vaccine Management
System; WDSD, Welsh Demographic Service Dataset; WLGP, Welsh Longitudinal General Practice. Northern Ireland In Northern Ireland, GP practices and Trust vaccination
sites are currently delivering the COVID-19 vaccination 3 Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 on February 22, 2022 by guest. Protected
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from on February 22, 2022 by
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from p
Table 1 Available UK datasets for each data item of interest
Data item
England
Northern Ireland
Scotland
Wales
Exposures
Pfizer-BioNTech vaccine
GP, NIMS
VMS
GP, TVMT/PHS,
SIRS
CVVD
Oxford-AstraZeneca vaccine
GP, NIMS
VMS
GP, TVMT/PHS,
SIRS
CVVD
Moderna vaccine
GP, NIMS
VMS
GP, TVMT/PHS,
SIRS
CVVD
Outcomes
Laboratory confirmed SARS-CoV-2 infection
GP, Pillar 1 and 2
SGSS, PHE
Pillar 1 and Pillar 2
ECOSS
PATD (Pillar 1, 2 and 3 data from all
NHS and private labs), CVLF testing
and results data
COVID-19-related GP consultation
GP
NA
GP
WLGP
COVID-19-related emergency department
consultation
GP, ECDS
Symphony, NIRAES
SMR01
EDDD and EDDS
COVID-19-related hospital admission
GP, SUS
Admissions and discharge
dataset
SMR01
PEDW
COVID-19-related ICU admission
GP, CHESS
Admissions and discharge
dataset
SICSAG
CDDS, ICCD and ICNC
COVID-19-related death
GP, ONS, SSRS
NHAIS
NRS
ADDE and ADDE (ONS mortality),
CDDS and WDSD
Secondary SARS-CoV-2 infection due to
household transmission
RCGP RSC
household key
Pillar 1 and Pillar 2 dataset
ECOSS
PATD (Pillar 1, 2 and 3 data from all
NHS and private labs), CVLF testing
and results data
Maternity outcomes
GP, MSDS
NIMATS
COPS study
ADBE (ONS births), MIDS and NCCH
Patient characteristics and confounders
Age
GP
NHAIS
GP
C19_COHORT20
Sex
GP
NHAIS
GP
C19_COHORT20
Socioeconomic status
Postal code to IMD
NHAIS
GP
C19_COHORT20
Ethnicity
GP, SUS
VMS
Census 2011
National ethnicity spine (made up of
20 EHR data sources and the ONS
Census 2011)
Underlying medical condition
GP
EPD
GP
GP, WLGP, PEDW, CVSP
Type of settlement (urban/rural)
GP
NHAIS
GP
C19_COHORT20
Type of settlement (eg, private home, care
home or social housing)
GP
NHAIS
GP
C19_COHORT20, CARE
Smoking status
GP
NA
GP
WLGP
Body Mass Index
GP
NA
GP
WLGP
Prescribed medications
GP
EPD
GP, PIS, HEPMA
WLGP, WDDS
Other non-COVID-19 vaccines (eg, influenza,
pneumococcal)
GP
VMS
GP
WLGP, NCCH
Occupation (eg, healthcare workers, front-
line workers, essential workers)
GP where recorded
Pillar 1 and 2
To be confirmed
HWRA, SWAC
History of healthcare utilisation (eg, GP
consultations, hospital admissions)
GP, SUS
Admissions and discharge
dataset
GP, SMR01
PEDW, WLGP
ADBE, Annual District Birth Extract (ONS Births); ADDE, Annual District Death Extract (ONS Deaths); CDDS, Critical Care DataSet; CHESS, COVID-19 Hospitalisation in England
Surveillance System; COPS, COVID-19 in Pregnancy in Scotland; CVSP, COVID-19 Shielded People list; CVVD, COVID-19 Vaccine Data; ECDS, Emergency Care Data Set; ECOSS,
Electronic Communication of Surveillance in Scotland; EDDD, Emergency Department Data Daily; EDDS, Emergency Department Dataset; EPD, Electronic Prescribing Database;
GP, general practitioner; HEPMA, Hospital Electronic Prescribing and Medicines Administration; HES, Hospital Episode Statistics; ICU, intensive care unit; IMD, Index of Multiple
Deprivation; LIS, Laboratory Information System; MIDS, Maternal Indicators DataSet; MSDS, Maternity Services Dataset; NCCH, National Community Child Health database; NHAIS,
National Health Applications and Infrastructure Services; NHS, National Health Service; NIMATS, Northern Ireland Maternity System; NIMS, National Immunisation Management
Service; NIRAES, Northern Ireland Regional Accident and Emergency System; NRS, National Records of Scotland; ONS, Office for National Statistics; PATD, Pathology data COVID-19
Daily; PEDW, Patient Episode Database for Wales; PHE, Public Health England; PHS, Public Health Scotland; PIS, Prescribing Information System; RCGP RSC, Royal College of
General Practitioners Research and Surveillance Centre; SGSS, Second Generation Surveillance System; SICSAG, Scottish Intensive Care Society Audit Group; SIRS, Scottish
Immunisation and Recall System; SMR01, Scottish Morbidity Record 01; SUS, Secondary Users Service; TVMT, Turas Vaccination Management Tool; VMS, Vaccine Management
System; WDSD, Welsh Demographic Service Dataset; WLGP, Welsh Longitudinal General Practice. Laboratory confirmed outcomes
England In Northern Ireland, the Admissions and Discharge
dataset will be used to collate data relating to admitted
patient care delivered by HSC hospitals in Northern
Ireland, generated by the patient administration systems
within each hospital. These data are held centrally by the
HSC Regional Data Warehouse.43 For England, most community testing (called Pillar 2)
is in the GP record, though for this study we will addi-
tionally link to resources held by NHS Digital this is
the Second Generation Surveillance System, this also
included hospital tests.39 Open access Clinical outcomes arising from a patient consultation.24 Additional data on
vaccination will also be available via the Turas Vaccination
Management Tool (TVMT), which is a web-based tool
that enables front-line vaccinators to capture and create
real-time patient vaccination records. PHS is currently
collating vaccination data from Turas.38 Scheduled vacci-
nations for children may also be recorded in the Scottish
Immunisation Recall System (SIRS) database. Data on
vaccinations administered at schools and not in GPs may
thus derive from SIRS.24 arising from a patient consultation.24 Additional data on
vaccination will also be available via the Turas Vaccination
Management Tool (TVMT), which is a web-based tool
that enables front-line vaccinators to capture and create
real-time patient vaccination records. PHS is currently
collating vaccination data from Turas.38 Scheduled vacci-
nations for children may also be recorded in the Scottish
Immunisation Recall System (SIRS) database. Data on
vaccinations administered at schools and not in GPs may
thus derive from SIRS.24 Data on primary care consultations for COVID-19 illness
will be accessed via GP electronic health records in each
UK nation. on February 22, 2022 by guest. Protected by copyright. http://bmjopen.bmj.com/
published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Scotland In Scotland, data on patients receiving care in general
or acute hospitals are recorded in the Scottish Morbidity
Record 01 (SMR01).24 The International Statistical Clas-
sification of Diseases and Related Health Problems, 10th
Revision (ICD-10) codes are used to index any diagnoses
recorded in the patient’s medical notes by a clinician.24
Consistent and high level (>90%) of data accuracy have
been shown in recent data quality reports for the SMR01
database.20 Data on adult patients admitted to general
intensive care units (ICUs) will derive from the Scottish
Intensive Care Society Audit Group database.24 For Northern Ireland, the Pillar 1 dataset is extracted from
the Laboratory Information Systems in Northern Ireland
hospitals on a daily basis into a central repository in the
Health and Social Care (HSC) Regional Data Warehouse,
which is maintained by the HSC Business Services Organ-
isation.39 It contains details of COVID-19 antigen tests
carried out in each of the hospital laboratories, including
those processed on behalf of primary care, social care and
community settings. Pillar 2 data are processed by NHS
Digital and extracts for NI residents are sent to the NI
HSC Regional Data Warehouse.39 on February 22, 2022 by guest. Protected by copyright. bmjopen.bmj.com/ England In England, we will link to secondary care data through
collections held by NHS Digital. Hospital data are held
in two forms: Hospital Episode Statistics (HES), which
are the long-term validated record; and Secondary Uses
Services which is an extract of contemporary operational
data which after validation will become HES. We plan
to use HES and also have an extract of intensive care
data. We will access these data through NHS Digital’s
Data Access Request Service.40 We also have SARS-CoV-2
virology data that were collected through the sentinel
surveillance system.41 The English primary care sentinel
system within RCGP RSC has a strong working relation-
ship with Public Health England (PHE), with whom we
have worked closely for over half a century.42 Wales In Wales, the vaccine programme is administered and
recorded nationally in the all Wales Immunisation System
(WIS) and is available in SAIL through the COVID-19
Vaccine Data. This is a separate independent system and
data source to the GP data, which is also available in SAIL
with vaccination recorded using Read codes.27 Scotland In Wales, data on all interactions with secondary care
including Accident and Emergency (A&E) events (Emer-
gency Department Dataset), inpatient hospital admis-
sions (Patient Episode Database for Wales), intensive
care (Critical Care DataSet—CDDS and Intensive Care
National Audit and Research Centre) and outpatient
appointments and activity (Outpatient Database for
Wales) are all available within SAIL Databank.27 There
are also a collection of specialised services and condition
specific secondary care such as cancer which are available
within SAIL.27 For Scotland, laboratory results from Scottish diagnostic
and reference laboratories are captured by the Electronic
Communication of Surveillance in Scotland (ECOSS)
database, which can be used for surveillance and research
purposes.24 Laboratory results from NHS and community
(Lighthouse laboratory) testing centres will also be avail-
able through ECOSS. y guest. Protected by copyright. Scotland programme. Vaccination data from GP practices and
Trust vaccination sites are stored in a central Vaccination
Management System and made available via the HBS
secure research environment.37 In Scotland, GPs usually facilitate vaccination programmes
and record any data related to vaccine administration. GPs
use Read codes to code and record relevant information Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 4 Wales For Wales, national coverage of Pillar 1, 2 and 3 data from
all NHS and private laboratories will be available, as well
as national lateral flow testing data.39 We will also pursue to access genome sequencing data
in a proportion of laboratory tests positive for SARS-
CoV-2 available from national sequencing centres where
possible. Deaths
England
In England, NHS Digital’s Personal Demographic Service
flags the date of death in the GP record. We have previously Deaths
England
In England, NHS Digital’s Personal Demographic Service
flags the date of death in the GP record. We have previously 5 Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 on February 22, 2
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Open access Community Child Health and Maternity Indicators
DataSet. Data sources for Wales will be sought via the
SAIL Databank.27 Community Child Health and Maternity Indicators
DataSet. Data sources for Wales will be sought via the
SAIL Databank.27 Community Child Health and Maternity Indicators
DataSet. Data sources for Wales will be sought via the
SAIL Databank.27 used these data of death to report excess mortality, both
overall44 45 and in people with known COVID-19 status.46
We will augment these data with the certificated cause
of death provided by the Office for National Statistics
(ONS), which will be linked for us at individual pseud-
onymised patient level by NHS Digital. used these data of death to report excess mortality, both
overall44 45 and in people with known COVID-19 status.46
We will augment these data with the certificated cause
of death provided by the Office for National Statistics
(ONS), which will be linked for us at individual pseud-
onymised patient level by NHS Digital. Northern Ireland In Northern Ireland, the National Health Applications
and Infrastructure Services (NHAIS) will be used for
mortality data.47 NHAIS receives regular updates from
General Register Office on fact and cause of death. ICD-10
codes on deaths records derive from diagnoses recorded
by the certifying doctor on the death certificate.47 Exposure definitions i
Data on the currently licensed COVID-19 vaccines,
including Pfizer-BioNTech, Oxford-AstraZeneca and
Moderna, will be derived from GPs, NIMS, TVMT/PHS,
WIS and HSC Trusts databases. For the first vaccine dose
(partial vaccination), an individual will be defined as
exposed or vaccinated from day 14 after receiving the first
dose between the period of 8 December 2020 and until
the end of follow-up. For the second vaccine dose (full
vaccination), an individual will be defined as exposed
or vaccinated from day 14 after receiving the second
dose during the study period. Exposed or vaccinated
groups will be stratified by the following time intervals:
(1) 0–13 days after dose 1; (2) 14–20 days after dose 1;
(3) 21–27 days after dose 1; (4) 28–34 days after dose 1;
(5) 35–41 days after dose 1; (6) >42 days after dose 1; (7)
0–13 days after dose 2 and (8) >14 days after dose 2. England In England, we will use a customised ‘sliding window’ to
capture pregnancy data.49 Specifically, an algorithm that
accurately inferred pregnancies will be used by adopting
an ontological approach for case finding.49 The ontolog-
ical approach will thus be used to identify pregnancies
and associated complications using a systematic approach
to derive this information from routinely collected admin-
istrative health data which will be available via the RCGP
RSC.49 Northern Ireland 2022 by guest. Protected by copyright. In Northern Ireland, data will be accessed via the
Northern Ireland Maternity System.50 Pregnancy and neonatal outcomes Capturing vaccine exposure in pregnancy is important.48
As no trial to date has included pregnant women this
type of study is the only opportunity to explore safety and
effectiveness in pregnant women and their babies. Scotland In Scotland, the death registry within National Records
of Scotland records information included in the death
certificates.24 ICD-10 codes on deaths records derive from
diagnoses recorded by the certifying doctor on the death
certificate.24 y
y
Controls or unvaccinated will be defined as those
who have not yet received a COVID-19 vaccine or have
only received one vaccine dose. Controls who become
vaccinated with any vaccine (ie, including one dose of
Moderna) or receive a second vaccine will then be assigned
within the exposure group. As a result, follow-up of the
exposure period will be censored for both the vaccinated
and control recipient if the control meets the criteria
to be classified as exposed (receiving a first dose when
compared with the unvaccinated group and receiving
a second dose when compared with the partially/one
dose vaccinated group). Maximum follow-up period will
correspond to the latest event date depending on the
outcome of interest. Similar vaccinated and unvaccinated
groups and periods will be determined for the Moderna
vaccine.52 Scotland In Scotland, pregnancy and neonatal outcomes in Scot-
tish participants will be identified through the COVID-19
in Pregnancy in Scotland study.51 Adverse events following immunisation Adverse events to be monitored are derived from the
safety results of the prelicensure vaccine clinical trials,
common side effects related to influenza vaccines and an
unpublished study protocol of an ongoing observational
study.6 7 53 54 These include use of health services such as
GP or out-of-hours GP consultation, A&E department
attendance, inpatient hospital admission and admission
to ICU for suspected adverse events. Safety of vaccines in
pregnant women will also be considered once vaccines
are widely administered in this group of the population. AEFI by specific vaccine type will also be considered. A
full list of candidate AEFI is available in in online supple-
mental material, appendices 1 and 2. Northern Ireland
In Northern Ireland, data will be accessed via the
Northern Ireland Maternity System.50 Northern Ireland Wales In Wales, multiple sources of mortality data including
information from the National Population Spine
(WDSD), ONS death data (ADDE and ADDD) and a
national NHS master patient index record (Consolidated
Death Data Source—CDDS) will be accessed to retrieve
complete, harmonised details on cause and associated
mortality details.27 Pregnancy and neonatal outcomes History of COVID-19 infection Any suspected or confirmed COVID-19 infections in the
previous last 6 months (prior to 1 December 2020) will be
included. Wales Protective effects of the vaccines will be assessed against
the following outcomes: (1) RT-PCR laboratory confirmed
COVID-19 infection; (2) GP consultations related to In Wales, pregnancy and neonatal outcomes will be iden-
tified from linked Annual District Birth Extract, National Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 6 Open access social/council housing for each UK nation if data are
available. social/council housing for each UK nation if data are
available. suspected or confirmed COVID-19 illness; (3) A&E
attendance related to suspected or confirmed COVID-19
illness at presentation; (4) hospital admissions related to
confirmed COVID-19 illness; (5) ICU admissions related
to confirmed COVID-19 illness and (6) deaths related to
suspected or confirmed COVID-19 illness. We will also
explore the effects of vaccines on secondary SARS-CoV-2
infection due to household transmission. VE against
maternal and neonatal COVID-19 related outcomes will
be explored once the vaccines are more widely available
to pregnant women. suspected or confirmed COVID-19 illness; (3) A&E
attendance related to suspected or confirmed COVID-19
illness at presentation; (4) hospital admissions related to
confirmed COVID-19 illness; (5) ICU admissions related
to confirmed COVID-19 illness and (6) deaths related to
suspected or confirmed COVID-19 illness. We will also
explore the effects of vaccines on secondary SARS-CoV-2
infection due to household transmission. VE against
maternal and neonatal COVID-19 related outcomes will
be explored once the vaccines are more widely available
to pregnant women. At-risk underlying medical conditions Based on the QCOVID algorithm,63 we will consider
the following conditions: (1) cardiovascular condi-
tions (atrial fibrillation, heart failure, stroke, peripheral
vascular disease, coronary heart disease, congenital heart
disease); (2) diabetes (type 1 and type 2); (3) respiratory
conditions (asthma, rare respiratory conditions (cystic
fibrosis, bronchiectasis or alveolitis), chronic obstructive
pulmonary disease, pulmonary hypertension or pulmo-
nary fibrosis); (4) cancer (blood cancer, chemotherapy,
lung or oral cancer, marrow transplant, radiotherapy);
(5) neurological conditions (cerebral palsy, Parkinson’s
disease, rare neurological conditions (motor neuron
disease, multiple sclerosis, myasthenia, Huntington’s
chorea), epilepsy, dementia, learning disability, severe
mental illness) and (6) other conditions (liver cirrhosis,
osteoporotic fracture, rheumatoid arthritis or systemic
lupus erythematosus, sickle cell disease, venous thrombo-
embolism, solid organ transplant, renal failure (chronic
kidney disease stages 3–5 with or without dialysis or trans-
plant).63 Body mass index will also be considered.63 p
g
A COVID-19 hospital or ICU admission will be defined
based on either a RT-PCR confirmed positive test for
SARS-CoV-2 in the 28 days prior to admission or based on
an ICD-10 code for COVID-19 (U07.1 or U07.2) in any
diagnostic position. A COVID-19 death will be defined
as COVID-19 as the underling ICD-10 cause of death
recorded on the death certificate, or death from any cause
within 28 days of a positive RT-PCR test for SARS-CoV-2. Population characteristics, confounding factors and effect
modifiers A number of key characteristics that could explain vari-
ation in vaccine uptake will be considered. In addition,
these characteristics could confound our planned anal-
yses. We will determine and include these characteristics
or potential confounders at the baseline of our study’s
cohort which include (see table 1 for details on data
sources for each UK nation): History of prescribed medications Based on the QCOVID algorithm,63 we will measure
number (>4) of prescriptions from general practices for
oral steroids, long acting β agonists or leukotrienes, immu-
nosuppressants in previous 6 months prior to the start of
the study cohort (1 December 2020). Prior or concomi-
tant usage of the novel oral anticoagulants, warfarin and
heparin will also be measured. Geographic In England, the RSC will use the ONS data on population
density and classify households as rural, urban (town and
city) or conurbation.60 The RSC also has a unique house-
hold key, so we can report household incidence of respi-
ratory and other infectious conditions.61 In Scotland,
settlement type will be included using the urban/rural
sixfold classification (UR6).24 In Wales, urban and rural
household classification that is based on the Lower-layer
Super Output Area of the person’s residence information
will be used.26 In Northern Ireland, NISRA’s Statistical
Classification and Delineation of Settlements will be used
to determine settlement type.62 Type of residence will also
be considered such as private residence, care home and History of healthcare utilisation Sex at birth will be included in a binary format (females
and males). Age will be included in bands that will be
determined based on available vaccination data. Socio-
economic status will be assessed by the following national
versions of area level deprivation indices: (1) Index of
Multiple Deprivation (IMD), 2019 version for England55 56
; (2) the Scottish IMD (SIMD)24; (3) the Welsh IMD57 and
(4) the Northern Ireland Multiple Deprivation Measure.58
For England, we will maximise the capture of ethnicity
data through the use of a customised ontology.59 Ethnicity
data for the other UK nations will also be included if
available. Number of GP consultations and hospital admissions
in the 6 months before the start of the study cohort
(December 1, 2020) will also be measured as a proxy of
severity of pre-existing medical conditions. on February 22, 2022 by guest. Protected by copyright. bmjopen.bmj.com/ History of non-COVID-19 vaccination Receipt of influenza or pneumococcal vaccination during
the 2020–2021 season (1 September to 30 November 2020)
available in a binary format (yes or no) will be included
which could be predictive of COVID-19 vaccination. History of prescribed medications Smoking status Smoking status will be included through four catego-
ries: smoker, ex-smoker, non-smoker and ‘not recorded’. Smoking status will not be determined for Northern
Ireland due to no access to primary care data. on February 22, 2022 by guest. Protected by copyright. http://bmjopen.bmj.com/
ed as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from At-risk underlying medical conditions Vaccine effectiveness
TND case–control study for laboratory-confirmed outcomes Vaccine effectiveness
TND case–control study for laboratory-confirmed outcomes TND case control study for laboratory confirmed outcomes
Vaccine status will be compared between cases (patients
with a positive test for COVID-19) and controls (patients
with a negative test for COVID-19) using a TND case–
control study.24 The main advantage of the TND studies
compared with traditional case–control studies is that it
minimises confounding factors from health care-seeking
behaviour, which means both cases and controls have
similar likelihood of seeking healthcare when having
symptoms indicative of COVID-19 illness.64 Selection
bias can still arise if study participants are not recruited
based on predefined criteria (eg, signs/symptoms
indicative to COVID-19 illness) but based on clinician-
ordered test.64 In this scenario, clinicians may be more
likely to carried out a test on patients that are more
likely to have COVID-19 illness (outcome) or not being
vaccinated.64 This will result to biased sampling (non-
representativeness) of the study participants from the
source population which could lead to overestimation
of the VE estimates.64 VE is estimated based on the OR
using the formula VE=1 OR. OR is defined as the odds
of a SARS-CoV-2 infection among the vaccinated group
divided by the odds of a SARS-CoV-2 infection among
the unvaccinated group. A generalised additive logistic Additional sensitivity or post hoc analyses such as using
different time intervals following administration of the
vaccine to define exposure will also be explored for all
study outcomes related to VE. Prospective cohort study for vaccine uptake Overall proportion of individuals that receive the vaccine,
stratified by sociodemographic, medical and other char-
acteristics will be reported using a prospective cohort
study. We will also consider reporting uptake levels within
certain subgroups of the population where possible (eg,
ethnic minorities and healthcare and other front-line
workers, care home residents and pregnant women). We
will also seek to examine patterns related to the number
of eligible individuals that were offered vaccine but
refused to be vaccinated if data are available. Univari-
able and multivariable logistic regression models will be
carried out to estimate the coefficient of each predictor
variable in the model and their 95% CIs, as well as the OR
for vaccine uptake. Prospective cohort study for clinical outcomes and deaths
A prospective cohort study will be used to estimate VE
against clinical outcomes and deaths in vaccinated and
unvaccinated individuals respectively. Time-dependent
Cox models will provide the adjusted rate ratios for these
outcomes. VE will be calculated according to VE=1 RR. VE
estimates will be adjusted for potential confounders and
effect modifiers including age, sex, underlying medical
condition, SES, history of healthcare utilisation, medi-
cation and non-COVID-19 vaccination. Other potential
confounders and effect modifiers may also be explored. Recall and misclassification bias will be minimised in our
planned prospective cohort studies as we will use data
from national linked datasets which allow rapid analysis
of vaccination and clinical outcomes data derived from
electronic health records. Nevertheless, unmeasured
confounding can still influence the VE estimates (given
the observational nature of these study designs) despite
attempts to provide VE adjusted for potential confounders
as mentioned above. Vaccine effectiveness on February 22, 2022 by guest. Protected by copyright. ttp://bmjopen.bmj.com/ Individual patient-level analyses
Vaccine uptake Prospective cohort study for vaccine uptake Statistical analysis We will report summary statistics of baseline character-
istics for the individuals in the study as of 1 December Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 7 Open access 2020. These will be also stratified by vaccine status and
respective study outcome of interest. Relevant measures
of tendency (eg, mean, median, proportion) and vari-
ability (eg, SD, IQR) will be calculated. Relative estimates
(eg, OR and rate ratio (RR)) and their respective 95% CI
for risk of outcomes of interest will also be included for
different population strata. Missing data will be reported
as percentages of total, imputation will be considered
if possible, and sensitivity analysis will be carried out to
examine if the nature of the missing data mechanism
affects the study findings. All statistical hypotheses tests
will be two tailed with significance level set at 5% for all
outcomes of interest. Statistical computing language R
will be used to conduct all planned statistical analyses. model will be used to estimate the OR and VE. Strati-
fied VE estimates by vaccine type, dose, dosing schedules,
COVID-19 strain, age group and underlying medical
condition will also be considered. The analyses in English databases will be carried out in
collaboration with PHE, following their guidance, as the
RSC is a major data and sampling source for PHE.65 It
will be important to avoid the confusion that might arise
from different approaches to analysing RSC data, and to
benefit from shared expertise in monitoring VE.66 67 We
have a shared protocol for this season’s analysis.68 Like-
wise, in Northern Ireland, we will work closely with the
Public Health Agency. In Scotland, all planned analyses
will also be carried out in collaboration with PHS. In
Wales, analyses will be carried out in collaboration with
PHW, who are involved with other colleagues in Wales
in the national rollout and evaluation of the vaccine
deployment, and are unique placed to provide expertise,
and are key contributors as part of the existing ConCOV
project in Wales.26 Pooled analyses In our study, we will initially provide estimates on vaccine
uptake, effectiveness, and safety for each UK nation. We
will also provide pooled estimates across the UK nations. A generic inverse variance method for meta-analysis will
be used. Heterogeneity of our pooled estimates will be
assessed using the standard χ2 and the I2 statistic.71 Forest
plots will be used to visualise any statistical heterogeneity
in our pooled estimates for the four nations. Individual
nation’s ORs or RRs and their 95% CIs will be used to
estimate the pooled VE estimates. Measured and unmea-
sured heterogeneity is highly probable across the UK
nations. Effect estimates from random-effect models will
thus only be considered. 2022 by guest. Protected by copyright. ETHICS AND DISSEMINATION For Scotland, approvals have been obtained by the
National Research Ethics Service Committee (REC),
South East Scotland 02 (REC number: 12/SS/0201) and
the Public Benefit and Privacy Panel for Health and Social
Care (reference number: 1920–0279). For Wales, the data
used in this study are available in the SAIL Databank at
Swansea University, Swansea, UK. All proposals to use
SAIL data are subject to review by an independent Infor-
mation Governance Review Panel (IGRP). Before any
data can be accessed, approval must be given by the IGRP. The IGRP gives careful consideration to each project to
ensure proper and appropriate use of SAIL data. When
access has been approved, it is gained through a privacy-
protecting safe haven and remote access system referred
to as the SAIL Gateway. SAIL has established an applica-
tion process to be followed by anyone who would like to
access data via SAIL.27 Similarly, in Northern Ireland, the
project was approved by the HSC HBS Governance Board
(project number 0064), and accessed through the online
secure research platform. Findings will be presented at
conferences, published in peer-reviewed journals and to
the funders and government COVID-19 advisory bodies
as appropriate. Strengthening the Reporting of Observa-
tional Studies in Epidemiology and Reporting of studies
Conducted using Observational Routinely-collected Data
(via the COVID-19 extension) checklists will guide our
study findings reporting.75 76 We will also consider using
the European Network of Centres for Pharmacoepidemi-
ology and Pharmacovigilance checklist.77 A sensitivity analysis by previous history of SARS-CoV-2
infection will also be considered. We will explore if
previous SARS-CoV-2 infection is associated with any AEs
observed following a COVID-19 vaccination. Patient and public involvement and engagement The exact duration (in days) of the risk and control
intervals will be determined for each AEFI outcome based
on severity level (mild-to-moderate, severe and typical
onset) and vaccine type separately (see online supple-
mental material, appendix 3). We will use the Benjamini-
Hochberg procedure to control the False Discovery rate
of testing a large number of hypotheses related to each
prespecified adverse events of interest.70 Subgroup anal-
yses by vaccine type, dose and dosing schedules will also
be considered. Patient and public involvement and engagement (PPIE)
members (Antony Chuter, Alex Brownrigg and Jillian
Beggs) have been involved since the beginning of this
project. The research proposal for the Wales analysis
has also been reviewed by members of the public. Their
contribution includes defining research questions, inter-
pretation and dissemination of study findings. As part
of the ConCOV project26 in Wales, PPIE members have
been involved in the design and evaluation of findings,
as well as presentations made to the independent SAIL
consumer panel group made up of lay members. It is possible that sample selection bias could be induced
in the SCCS if inclusion in the study is related nontrivially
to the adverse outcome of interest. This may be partic-
ularly true for severe adverse events. For example, if an
individual has a cardiac arrest then they are less likely to
be vaccinated and thus less likely to be included in the
study. We will therefore carry out a retrospective cohort
or an event-dependent exposure version of SCCS study
for severe outcomes. Vaccine safety
SCCS and retrospective cohort studies for adverse events The risk of any vaccine-related adverse events will be
assessed using a SCCS study design.24 This study design
tests whether the risk of an adverse event is higher at post-
vaccination period compared with other periods that are
temporarily unrelated to vaccine administration.69 The
main advantage of this case series method over other
methods of analysis is that it only includes individuals
who have been vaccinated. As a result, adequate statistical
power can often be obtained with relatively small sample
sizes. guest. Protected by copyright. Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 8 Open access on February 22, 20
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from on February 22, 2022 by guest. Protec
http://bmjopen.bmj.com/
BMJ Open: first published as 10.1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from In addition, all confounders (eg, sex, genetics, SES,
location, underlying condition) that do not vary with
time over the observation period are implicitly controlled
for.69 The number of adverse events in a pre-defined
risk interval will be compared with predefined control
intervals. Risk interval refers to postvaccine administra-
tion period over the observation period of the study and
control intervals refer to prevaccine and postvaccine
administration over the study’s observation period. Risk
and control intervals will also be determined in relation
to vaccine dose administration (eg, between first and
second doses of the vaccines). A clearance or wash-out
interval between the risk and control intervals will also
be applied. 0.89 against COVID-19 hospitalisation at 28–34 days post-
vaccination, with a SD of 0.06. Assuming our VE estimates
are asymptotically normally distributed, this gives almost
100% power to detect a VE of over 0.5. The number of COVID-10 vaccines doses required to
detect a relative risk of 5.0 is at least 10 000 doses for a
relatively common adverse outcome (eg, myocardial
infraction with a background incidence rate of 1400 per
100 000 person years in men older than 85 years old) and
more than a million doses are needed to detect a rela-
tive risk of 1.5 for a rare adverse outcome (eg, myocardial
infraction with a background incidence rate of 28 per
100 000 person years among those 18–34 years).73 74 http://bmjopen.bmj.com/
1136/bmjopen-2021-050062 on 14 February 2022. Downloaded from Author affiliations p
y p
Supplemental material This content has been supplied by the author(s). It has
not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been
peer-reviewed. Any opinions or recommendations discussed are solely those
of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and
responsibility arising from any reliance placed on the content. Where the content
includes any translated material, BMJ does not warrant the accuracy and reliability
of the translations (including but not limited to local regulations, clinical guidelines,
terminology, drug names and drug dosages), and is not responsible for any error
and/or omissions arising from translation and adaptation or otherwise. Acknowledgements England: Patients and general practices who provide samples
and agree to share data with the Oxford-RCGP RSC. EMIS, TPP, In-Practice Systems
Wellbeing software for supporting the RSC data extraction. Public Health England
for support and collaboration. Wales: This work uses data provided by patients
and collected by the NHS as part of their care and support. We would also like to
acknowledge all data providers who make anonymised data available for research. We wish to acknowledge the collaborative partnership that enabled acquisition and
access to the de-identified data, which led to this output. The collaboration was led
by the Swansea University Health Data Research UK team under the direction of the
Welsh Government Technical Advisory Cell (TAC) and includes the following groups
and organisations: the Secure Anonymised Information Linkage (SAIL) Databank,
Administrative Data Research (ADR) Wales, NHS Wales Informatics Service (NWIS),
Public Health Wales, NHS Shared Services Partnership and the Welsh Ambulance
Service Trust (WAST). All research conducted has been completed under the
permission and approval of the SAIL independent Information Governance Review
Panel (IGRP) project number 0911. Northern Ireland: The authors would like to
acknowledge the help provided by the staff of the Honest Broker Service (HBS)
within the Business Services Organisation Northern Ireland (BSO). The HBS is
funded by the BSO and the Department of Health (DoH). The authors alone are
responsible for the interpretation of the data and any views or opinions presented
are solely those of the author and do not necessarily represent those of the BSO. on February 22, 2022 by guest. Prote
http://bmjopen.bmj.com/
ary 2022. Author affiliations Division (Welsh Government), Public Health Agency (Northern Ireland), British
Heart Foundation (BHF) and the Wellcome Trust; and Administrative Data Research
UK which is funded by the Economic and Social Research Council (grant ES/
S007393/1). Division (Welsh Government), Public Health Agency (Northern Ireland), British
Heart Foundation (BHF) and the Wellcome Trust; and Administrative Data Research
UK which is funded by the Economic and Social Research Council (grant ES/
S007393/1). 2Department of Mathematics and Statistics, University of Strathclyde, Glasgow, UK
3Public Health Scotland, Glasgow, UK 4Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford,
UK Competing interests AS is a member of the Scottish Government Chief Medical
Officer’s COVID-19 Advisory Group. RAL reports grants from MRC during the
conduct of the study. SJS reports grants from Wellcome Trust, during the conduct
of the study; grants from National Institute of Healthcare Research HTA, grants
from Tommy's Charity and grants from Chief Scientist for Scotland, outside the
submitted work. SdeL is Director of the Royal College of General Practitioners
Research and Surveillance Centre. He has received grant funding through his
University from AstraZeneca, Eli Lilly, GSK MSD, Seqirus and Takeda. He has
been members of advisory boards for AstraZeneca, Sanofi, and Seqirus. DB is
jointly employed by Queen’s University Belfast, the Public Health Agency and the
Department of Health (Northern Ireland), and he is currently or has been a member
of COVID-19 government advisory groups, including the Scientific Advisory Group
for Emergencies (SAGE), its subgroups, and the UK Vaccine Effectiveness Expert
Panel. All other authors report no conflicts of interest. 6BREATHE – The Health Data Research Hub for Respiratory Health, London, UK
7Population Data Science, Swansea University Medical School, Swansea, UK
8Vaccine Preventable Disease Programme, Public Health Wales, Cardiff, UK ,
,
10School of Medicine, University of St Andrews, St Andrews, UK 10School of Medicine, University of St Andrews, St Andrews, UK 10School of Medicine, University of St Andrews, St Andrews, UK 11Public Health Scotland, Edinburgh, UK 12MRC/CSO Social and Public Health Sciences Unit, University of Glasgow, Glasgow,
UK
13 13Wellington School of Health, Faculty of Health, Victoria University of Wellington,
Wellington, New Zealand Patient consent for publication Not applicable. Twitter Eleftheria Vasileiou @elvasileiou, Simon de Lusignan @Lusignan_S, Chris
Orton @chrisortonSUHIG, Ashley Akbari @AshleyAkbari, Sarah Jane Stock @
sarahjanestock and Aziz Sheikh @DrAzizSheikh Provenance and peer review Not commissioned; externally peer reviewed. ORCID iDs Eleftheria Vasileiou http://orcid.org/0000-0001-6850-7578
Ting Shi http://orcid.org/0000-0002-4101-4535
Ruby Tsang http://orcid.org/0000-0002-2520-526X
Simon de Lusignan http://orcid.org/0000-0001-5613-6810
Chris Orton http://orcid.org/0000-0002-9561-2493
Ashley Akbari http://orcid.org/0000-0003-0814-0801
Lucy J Griffiths http://orcid.org/0000-0001-9230-624X
Emily Lowthian http://orcid.org/0000-0001-9362-0046
Jane Lyons http://orcid.org/0000-0002-4407-770X
Ronan A Lyons http://orcid.org/0000-0001-5225-000X
Rachael Wood http://orcid.org/0000-0003-4453-623X
Sarah Jane Stock http://orcid.org/0000-0003-4308-856X
Paul Henery http://orcid.org/0000-0003-0380-738X
Aziz Sheikh http://orcid.org/0000-0001-7022-3056 Contributors AS conceived this study and commented on several drafts of the
protocol. EV wrote the first draft of the protocol with assistance from TS and SK. SdeL, RT, MJ and DMcC, JW and RH contributed about the English sentinel system
and commented on the research methods. DB, DO and SM contributed about the
Northern Ireland data and commented on the research methods. AC and JB were
involved in the study design and commented on introduction and research methods
from a patient and public perspective. DF, CO, AA, SB, GD, LJG, RG, EL, JL, RAL,
LN, MP and FT contributed about the Wales data and commented on the research
methods. JP contributed on the study design and commented on several drafts. JM, CM, UA, RW, SJS, EM, PH, CRS and CR contributed about the Scotland data and
comments on research methods. All authors contributed to the study design. All
authors contributed to drafting the protocol. All authors revised the manuscript for
important intellectual content. All authors gave final approval of the version to be
published. Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 Author affiliations Downloaded from 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/. Sample size We are basing sample size calculations on Scottish testing
and vaccination data because it is currently the only UK
nation with full national data coverage at the time of
writing. Based on previous work,72 we estimated a VE of 9 Vasileiou E, et al. BMJ Open 2022;12:e050062. doi:10.1136/bmjopen-2021-050062 Open access Author affiliations
1The University of Edinburgh, Usher Institute, Edinburgh, UK
2Department of Mathematics and Statistics, University of Strathclyde, Glasgow, UK
3Public Health Scotland, Glasgow, UK
4Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford,
UK
5School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast,
Belfast, UK
6BREATHE – The Health Data Research Hub for Respiratory Health, London, UK
7Population Data Science, Swansea University Medical School, Swansea, UK
8Vaccine Preventable Disease Programme, Public Health Wales, Cardiff, UK
9Health Data Research, London, UK
10School of Medicine, University of St Andrews, St Andrews, UK
11Public Health Scotland, Edinburgh, UK
12MRC/CSO Social and Public Health Sciences Unit, University of Glasgow, Glasgow,
UK
13Wellington School of Health, Faculty of Health, Victoria University of Wellington,
Wellington, New Zealand Author affiliations
1The University of Edinburgh, Usher Institute, Edinburgh, UK
2Department of Mathematics and Statistics, University of Strathclyde, Glasgow, UK
3Public Health Scotland, Glasgow, UK
4Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford,
UK
5School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast,
Belfast, UK
6BREATHE – The Health Data Research Hub for Respiratory Health, London, UK
7Population Data Science, Swansea University Medical School, Swansea, UK
8Vaccine Preventable Disease Programme, Public Health Wales, Cardiff, UK
9Health Data Research, London, UK
10School of Medicine, University of St Andrews, St Andrews, UK
11Public Health Scotland, Edinburgh, UK
12MRC/CSO Social and Public Health Sciences Unit, University of Glasgow, Glasgow,
UK
13Wellington School of Health, Faculty of Health, Victoria University of Wellington,
Wellington, New Zealand on February 22, 2022 by
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Lead expansion and virtual screening of Indinavir derivate HIV-1 protease inhibitors using pharmacophoric - shape similarity scoring function
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Hypothesis www.bioinformation.net Background : g
The HIV-1 (human immunodeficiency virus type 1) protease is a C2
symmetric and an aspartic acid homodimeric enzyme, where aspartate
25 plays a pivotal role in binding the substrate [1]. The HIV-1 protease
does not have a homologue in mammalian cells and has a function to
cleave the Gag-Pol polyprotein precursor into shorter pieces to create
the active protein components for viral packaging and maturation. This
proteolysis occurs late in the viral life cycle and is essential for viral
infectivity [2]. The protease active site is located in the buried area
(tunnel), where the two subunits meet each other. Highly active
antiretroviral therapy (HAART), using protease inhibitors (PI), is
commonly used in management of HIV infection. These inhibitors are
able to irreversibly bind the HIV-1 protease to block its function. Among these compounds, Indinavir (Crixivan, MK-639, or IDV) [3, 4]
is a potent and selective protease inhibitor that had approval for AIDS
therapy. But genotypic analyses of the viral populations during the
course of protease inhibitor therapy had shown various mutations that
can occur in as many as 20 amino acids within the protease gene [5,
6]. 13.7 % of IDV failure were identified for 1021 new viral genotypes
from HIV patients [7]. In case of HIV-1 subtype C (African strain), it
had been shown that the most common primary mutation observed in
PI treated patients was L90M [8], a main course in multi-PI resistance
establishment [9]. Even naturally occurring polymorphism, such as
L89M and I93L, located in the hydrophobic core of the enzyme would
possibly change the shape of the substrate-binding cleft and diminish
the potency of IDV [10]. Only flexibility of the newly synthesized
compounds seems to overcome this effect. Here, we describe the
designing scheme for possible HIV-1 protease inhibitors using a lead
expansion protocol with a pharmacophoric-shape similarity scoring
function. Abstract: Indinavir (Crivaxan®) is a potent inhibitor of the HIV (human immunodeficiency virus) protease. This enzyme has an important role in viral
replication and is considered to be very attractive target for new antiretroviral drugs. However, it becomes less effective due to highly resistant
new viral strains of HIV, which have multiple mutations in their proteases. For this reason, we used a lead expansion method to create a new set
of compounds with a new mode of action to protease binding site. 1300 compounds chemically diverse from the initial hit were generated and
screened to determine their ability to interact with protease and establish their QSAR properties. Further computational analyses revealed one
unique compound with different protease binding ability from the initial hit and its role for possible new class of protease inhibitors is discussed
in this report. Keywords: protease; Indinavir; lead expansion; docking; pharmacophore 5362440). IDV initial refinement was performed by means of the
MarvinSketch program provided by ChemAxon [12]. 5362440). IDV initial refinement was performed by means of the
MarvinSketch program provided by ChemAxon [12]. ceived October 16, 2009; Revised November 07, 2009; Accepted November 16, 2009; Published January 20, 2010 Received October 16, 2009; Revised November 07, 2009; Accepted November 16, 2009; Published January 20, 2010 Compound library generation: Compound library generation:
Pharmacophoric - shape similarity scoring function was used for
compound library generation. This is build-in function integrated in
the Muse™ molecular design workflow to accelerate the identification
and optimization of lead candidates. This function seeks molecules
that have fairly different structures but similar 3D pharmacophores and
shapes. It is appropriate for lead expansion – exploration around an
initial hit. Indinavir compound was used as a reference molecule and
all similarities were measured with respect to it. The reference
structure was also used as the initial population for invention. In our
case a default set of compounds was used if no any other structures
were given. Such generation of multiple compounds with similar
shape but different 3D structure organization would provide more
interesting results in the invention and library screening afterwards. For lead expansion method we did not specified any seed structures
and preserved cores. 99 undesirable and 35 questionable substructures
were excluded from to be present in invented structures. Structural
ranking was done by means of the Pareto-Borda method to maximize
pharmacophoric-shape similarity in preferential shape similarity
specified range (from 0.35 to 0.50 score units). ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 4(7): 295-299 (2010) Department of Bioinformatics, Biocenter of the University of Würzburg, 97074 Würzburg, Germany; Sergey Shityakov - E-mail:
shityakov@vim.uni-wuerzburg.de, * Corresponding Author ent of Bioinformatics, Biocenter of the University of Würzburg, 97074 Würzburg, Germany; Sergey Shityakov - E-mail:
shityakov@vim.uni-wuerzburg.de, * Corresponding Author Protease active site detection: Protein binding surfaces could have very complicated and irregular
structures. Atoms could form pockets, cavities and tunnels. Solvent
molecules can get into these tunnels from outer environment and move
through them. Buried shape and volume of such tunnels vary in time
due to protein dynamics and kinetics. Here, we analyzed buried
tunnels of the protease using the CAVER module, to identify certain
atomic positions of hidden binding moieties [13]. Dijkstra’s algorithm
was implemented in searching process and started from source node
(starting point), which is located deeply in the protein pocket. Before
the search procedure, 136 water molecules, chloride and sodium ions
were removed from IDV-bound protease (liganded holo-structure)
protein database file. All calculations were performed on 32612 grid
points. Catalytic tunnel (key interaction site) was detected in the
protease structure (Figure 2(A)). The tunnel coordinates to specify the
starting point in x, y and z axis are 2.5, 6.5, -7.5 respectively. These
coordinates were taken from the AutoDock manual [14].IDV Lead expansion and virtual screening of Indinavir derivate
HIV-1 protease inhibitors using pharmacophoric - shape
similarity scoring function Sergey Shityakov*, Thomas Dandekar Department of Bioinformatics, Biocenter of the University of Würzburg, 97074 Würzburg, Germany; Sergey Shityakov - E-mail:
shityakov@vim.uni-wuerzburg.de, * Corresponding Author Methodology:
HIV 1 subtype Methodology:
HIV-1 subtype C protease and Indinavir structures: The three dimensional structure of the HIV-1 protease - IDV complex
(PDB code: 2R5P) was retrieved from Protein Data Bank at 2.3 Å
RMSD resolution [10]. The VADAR (volume, area, dihedral angle
reporter) server [11], which is an improved version of the
PROCHECK software, was used for stereochemical validation of
HIV-1 protease. Altogether, 95 % of all residues were in φ-ψ core
areas. Indinavir was obtained from PubChem database (CID: 295 ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 4(7): 295-299 (2010) © 2010 Biomedical Informatics © 2010 Biomedical Informatics Bioinformation open access
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ISSN 0973 2063 (online) 0973 8894 (print)
296
Figure 1: Chemical structure of IDV and its ‘derivate’- novel hit (N'-cyano-N-{3-[(1S, 2S)-1,2-diamino-2-(4-oxo-3, 4-dihydroquinazolin-7-
yl)ethyl]-5-[(2R)-oxolan-2-yl]pyridin-2-yl}benzenecarboximidamide)
Figure 2: (A) HIV protease catalytic tunnel (binding site) was predicted by PyMol CAVER module. IDV (B) and novel hit (C) interactions wi
the HIV-1 protease are shown. H-bonds are depicted as dashed lines. IDV – protease complex was analyzed as a crystal structure. 3D alignme
and ‘fuzzy’ model (D) of the hit ‘native’ conformation (gray) together with its ‘functional’ conformation (yellow) at 2.0 Å RMSD. The show
potential pharmacophore points are color-coded as follows: lipophilic areas are green, H-bond donors and acceptors are colored in blue and re
respectively. Bioinformation
open access
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Hypothesis
Figure 1: Chemical structure of IDV and its ‘derivate’- novel hit (N'-cyano-N-{3-[(1S, 2S)-1,2-diamino-2-(4-oxo-3, 4-dihydroquinazolin-7-
yl)ethyl]-5-[(2R)-oxolan-2-yl]pyridin-2-yl}benzenecarboximidamide) www.bioinformation.net www.bioinformation.net
Hypothesis
Figure 1: Chemical structure of IDV and its ‘derivate’- novel hit (N'-cyano-N-{3-[(1S, 2S)-1,2-diamino-2-(4-oxo-3, 4-dihydroquinazolin-7-
yl)ethyl]-5-[(2R)-oxolan-2-yl]pyridin-2-yl}benzenecarboximidamide) Figure 1: Chemical structure of IDV and its ‘derivate’- novel hit (N'-cyano-N-{3-[(1S, 2S)-1,2-diamino-2-(4-oxo-3, 4-d
yl)ethyl]-5-[(2R)-oxolan-2-yl]pyridin-2-yl}benzenecarboximidamide) Figure 2: (A) HIV protease catalytic tunnel (binding site) was predicted by PyMol CAVER module. IDV (B) and novel hit (C) interactions with
the HIV-1 protease are shown. H-bonds are depicted as dashed lines. IDV – protease complex was analyzed as a crystal structure. 3D alignment
and ‘fuzzy’ model (D) of the hit ‘native’ conformation (gray) together with its ‘functional’ conformation (yellow) at 2.0 Å RMSD. The shown
potential pharmacophore points are color-coded as follows: lipophilic areas are green, H-bond donors and acceptors are colored in blue and red
respectively. Figure 2: (A) HIV protease catalytic tunnel (binding site) was predicted by PyMol CAVER module. IDV (B) and novel hit (C) interactions with
the HIV-1 protease are shown. H-bonds are depicted as dashed lines. open access
Hypothesis ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 4(7): 295-299 (2010) Protein-ligand docking The Molegro Virtual Docker [16] and the AutoDock software were
implemented to analyze ligand interactions with the HIV-1 protease
binding site. The Molegro Virtual Docker and the AutoDock combine
a rapid energy evaluation through pre-calculated grids of affinity
potentials with a variety of search algorithms to find appropriate
binding positions. In Molegro Virtual Docker docking experiment we
used MolDock scoring function, which is based on a piecewise linear
potential and a re-ranking procedure was applied to the highest ranked
poses to increase docking accuracy. Affinity grid resolution was set to
0.3 Å. Ligand evaluations were based on internal energy of binding,
internal H-bonds formation, Sp2-Sp2 (trigonal planar electron domain
geometry) torsion angles. Binding site was previously detected by
CAVER module and further measured for cavity volume (282.1 Å3),
surface (681.0 Å2) and radius (15.0 Å). Customized search algorithm
was set to MolDock SE (simplex evolution). Number of runs was 10. Parameter settings were set to 1500 iterations, 50 population sizes,
100.0 kcal/mol of energy threshold for pose generation, 300 simplex
evolution steps and 1.0 neighbor distance factor. All dockings were
performed at 1.0 Å RMSD threshold. For preparing the AutoDock
docking parameter file we used default settings (genetic algorithm
parameters: population size = 150, number of energy evaluations =
2500000, rate of gene mutation = 0.02, rate of crossover = 0.8,
maximum number of generations = 27000, number of GA runs = 10,
initial dihedrals were randomly specified, elitism value was set to 1). By default, clustering of docked results was done at 0.5 Å RMSD. Prior to docking, total Kollman and Gasteiger charges were added to
the protein and the ligand. Probably this acceptor nitrile group along with the tetrahydropyridine
ring might have some influence on the ligand conformational shift
with subsequent change in binding affinity mode. In both docking
cases, the catalytic function of the Asp 25 residues is completely
blocked. Subsequently, we performed the AutoDock experiments to
validate the above data and revealed reduction in hydrogen bonds
formation presumably due to differences in affinity grid resolution of
Molegro Virtual Docker and AutoDock (0.3 vs 0.375 Å). However,
consistent with previous observation, all crucial amino acids obtained
from Molegro docking experiments are also present in the AutoDock
docking profiles (Figure 3(A, B)).The IDV spatial conformation
recruits less docking energy (-15.1 kcal/mol) in comparison to novel
hit and forms H-bonds with amino acid residues of chain B. Bioinformation open access
Hypothesis open access
Hypothesis Protein-ligand docking In this
orientation, the amide nitrogen donor together with carboxyl group
(hydroxyl moiety) of the Asp 29 residue, the Asp 25 carboxyl group
(hydroxyl moiety) and the Gly 27 amide oxygen acceptor make
hydrogen bonds with two hydroxyl functional groups and one amide
nitrogen donor of the ligand molecule. Discussion: Genetic algorithm produced 1300 compounds (70 generations) of
diverse chemical structure. 500 of top molecules were selected
according to their shape similarity (0.7-0.9 functional scores). These
molecules, which had pharmacophoric score less than 0.8 were
excluded from the list. 227 out of 500 compounds satisfied all that
criteria and were tested for Lipinski’s Rule of Five using the
LigandScout pharmacophore software [18]. Only one compound
(novel hit) did not have rule violations, in contrast to IDV, which had
one violation of Lipinski’s rule. A useful parameter, such as
topological polar surface area (TPSA), was defined for these two
molecules as the surface sum over all polar atoms to evaluate
parameters for the prediction of cell permeability and drug transport
properties (Table 1 in supplementary material). This parameter shows
good drug transport, even in ability to penetrate the blood-brain
barrier. In this case TPSA should not exceed 600 Å2 threshold. The
novel hit was analyzed for Cramer rules, carcinogenicity and
biodegradability. It also showed a strong affinity (in comparison with
reference molecule) to the HIV-1 protease binding site. Docking
results revealed the interaction modes for this compound with the
HIV-1 protease. Novel hit adopts IDV binding mode (Asp 25, 29 and
Gly 27) and also has the additional interacting residues (Gly 49-Ile 50,
Asp 29-Ala 28, Asp 30). Moreover, it exceeds IDV in H-bond
formations (10 instead of 9). New compound’s nitrile group is creating
H-bonds with donor nitrogen of Gly 49 - Ile 50 amide group (Figure
2(B, C)). Available decision tree algorithms such as: Benigni-Rossa rulebase,
Verhaar scheme, Cramer rules and START (structural alerts for
reactivity in toxtree) biodegradability were used for the analysis. Benigni-Rossa rule base predicts the possibility of carcinogenicity and
mutagenecity by discriminant analysis and structural rules [15]. START biodegradability estimates biodegradation potential of the
chemical compound based on structural alerts compiled from the
Canadian Environmental Protection Agency. Methodology:
HIV 1 subtype IDV – protease complex was analyzed as a crystal structure. 3D alignment
and ‘fuzzy’ model (D) of the hit ‘native’ conformation (gray) together with its ‘functional’ conformation (yellow) at 2.0 Å RMSD. The shown
potential pharmacophore points are color-coded as follows: lipophilic areas are green, H-bond donors and acceptors are colored in blue and red
respectively. 296 ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 4(7): 295-299 (2010) © 2010 Biomedical Informatics Bioinformation open access
Hypothesis www.bioinformation.net towards the rational design of HIV-1 protease inhibitors and their
effectiveness. towards the rational design of HIV-1 protease inhibitors and their
effectiveness. Although, novel hit conformation interacts with both chains of the
HIV protease but its global minimum in docked energy (-13.8
kcal/mol) with the protease binding site is more than that of IDV,
resulting in decrease of binding affinity and intermolecular force
between the ligand and its receptor. The Asp 29, Ile 50 amide nitrogen
donors form H-bonds with N1 of the tetrahydropyrimidine ring and
nitrogen acceptor of the aminoformonitrile residue. Carbonyl and
hydroxyl moieties of both Asp 25 carboxyl groups are interacting with
amine functional donor groups of novel hit. Asp 25, Asp 29, Gly 27
residues are located in the ‘eye’ areas, whereas Ile 50 residue is
localized in the ‘flap’ region. The ‘flap’ region of the protease plays an
important role in the enzyme “opening” and ligand binding to the
‘flap’ β-hairpins could block “opening” process.Finally, we measured
the hydrogen bond length by measuring the distance between the
donor and acceptor atoms. Usually, longest H-bond length is about 3.5
Å. Anything longer of this parameter would be considered a pure
dipole-dipole interaction. It is well established that hydrogen bonds
have a typical length around 2.5 Å. In our case, all H-bond distances
were in the range from 1.71 to 2.11 Å. Interestingly, one bond in IDV
and two bonds in the hit molecule were detected as ultra-short
hydrogen bonds with donor and acceptor distances of less than 2.0 Å. This observation supports further that identified the novel hit
compound should have a tendency to form strong hydrogen bonds to
the protease catalytic center.One drawback to IDV and novel hit is
that, they both have a heterocyclic rings with complex substituents. Those features are responsible for the third class of toxicity (highly
toxic). Both molecules belong to the second class of biodegradability,
which means no alerts for notifying an easily degradable chemical
were found and there was one alert notifying a persistent chemical the
compound under investigation is declared as “persistent chemical”. Acknowledgments: g
The authors are grateful to the DFG (SFB630/C6, Da 208/11-1) and
the IZKF (Interdisziplinäres Zentrum für Klinische Forschung der
Universität Würzburg) for the support of this work. LIQUID-fuzzy pharmacophore models The PyMol LIQUID module was utilized to create a pharmacophore
‘fuzzy’ model. At the first model computation LIQUID used standard
cluster radii to calculate cluster sizes at 4.0 Å RMSD for lipophilic
areas and 1.9 Å RMSD for H-bond donors and acceptors [17]. In our
experiment we set up default cluster radius (2.0 Å) for each potential
pharmacophore point type (Figure 2(D)). Figure 3: (A) Indinavir and (B) novel hit docking profiles calculated by AutoDock software. Both ligands are gray. HIV protease chain A and B
amino acids are painted in green and magenta respectively. The hydrogen bonds are shown for each of both molecules; see text for details. Figure 3: (A) Indinavir and (B) novel hit docking profiles calculated by AutoDock software. Both ligands are gray. HIV protease chain A and B
amino acids are painted in green and magenta respectively. The hydrogen bonds are shown for each of both molecules; see text for details. 297 ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 4(7): 295-299 (2010) © 2010 Biomedical Informatics © 2010 Biomedical Informatics ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 4(7): 295-299 (2010) References: References: [1]
D. M. York, et al., Biochemistry., 32:3196 (1993) [PMID:
8431424] [2]
N. E. Kohl, et al., Proc. Natl. Acad. Sci., 85:4686 (1988) [PMID:
3290901] [3]
B. D. Dorsey, et al., J. Med. Chem., 37:3443 (1994) [PMID:
7932573] [4]
J. P. Vacca, et al., Proc. Natl. Acad. Sci., 91:4096 (1994)
[PMID: 8171040] [5]
J. H. Condra, et al., J. Virol., 70:8270 (1996) [PMID: 8970946] [6]
J. H. Condra, et al., Nature., 374:569 (1995) [PMID: 7700387] and two bonds in the hit molecule were detected as ultra-short
hydrogen bonds with donor and acceptor distances of less than 2.0 Å. This observation supports further that identified the novel hit
compound should have a tendency to form strong hydrogen bonds to
the protease catalytic center.One drawback to IDV and novel hit is
that, they both have a heterocyclic rings with complex substituents. Those features are responsible for the third class of toxicity (highly
toxic). Both molecules belong to the second class of biodegradability,
which means no alerts for notifying an easily degradable chemical
were found and there was one alert notifying a persistent chemical the
compound under investigation is declared as “persistent chemical”. [7]
E. Poveda, et al., J. Antimicrob. Chemother., 60:885 (2007)
[PMID: 17646201] [8]
P. A. Cane, et al., J. Clin. Microbiol., 39:2652 (2001) [PMID:
11427587] [9]
D. J. Kempf, et al., J. Virol., 75:7462 (2001) [PMID: 11462018]
10
4
(
) [10] R. M. Coman, et al., Biochemistry., 47:731 (2008) [PMID:
18092815] [11] L. Willard, et al., Nucleic. Acid. Res., 31:3316 (2003) [PMID:
12824316] [12] G. Pirok, et al., J. Chem. Inf. Model., 46:563 (2006) [PMID:
16562984] [13] M. Petrek, et al., BMC Bioinformatics., 7:316 (2006) [PMID:
16792811] Citation: Shityakov & Dandekar, Bioinformation 4(7): 295 299 (2010)
License statement: This is an open-access article, which permits unrestricted use, distribution, and reproduction in any medium, for non-
commercial purposes, provided the original author and source are credited. ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 4(7): 295-299 (2010) Conclusion:
I
h [14] http://autodock.scripps.edu/faqs-help/manual [14] http://autodock.scripps.edu/faqs-help/manual In the present study, we generated library of diverse chemical
compounds on the bases of Indinavir and screened them for possible
anti-protease activity. However, mutagenic efficiency of HIV is very
high in generating new viral strains and could be bypassed by creating
drugs with different binding properties and strong affinity to the target
protein. The results are shown in this report, indicate that at least one
compound has binding residues different from the initial hit, with
improved affinity to the protease as well as its QSAR properties. Further studies on this compound will provide useful information [15] G. M. Cramer, et al., Food. Cosmet. Toxicol., 16:255 (1978)
[PMID: 357272] [16] R. Thomsen & M. N. Christensen., J. Med. Chem., 49:3315
(2006) [PMID: 16722650] [17] Y. Tanrikuli, et al., Biochemistry., 8:1932 (2007) [PMID:
17896338] [18] G. Wolber & T. Langer., J. Chem. Inf. Model., 45:160 (2005)
[PMID: 15667141] Edited by P. Kangueane
Citation: Shityakov & Dandekar, Bioinformation 4(7): 295-299 (2010)
mits unrestricted use, distribution, and reproduction in any medium, for non-
commercial purposes, provided the original author and source are credited. Edited by P. Kangueane
Citation: Shityakov & Dandekar, Bioinformation 4(7): 295-299 (2010)
mits unrestricted use, distribution, and reproduction in any medium, for non-
commercial purposes, provided the original author and source are credited. Citation: Shityakov & Dandekar, Bioinformation 4(7): 295-299 (2010)
License statement: This is an open-access article, which permits unrestricted use, distribution, and reproduction in any medium, for non-
commercial purposes, provided the original author and source are credited. open access
Hypothesis Conclusion:
I
h 298 ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 4(7): 295-299 (2010) © 2010 Biomedical Informatics Bioinformation
open access
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Hypothesis
Supplementary material
Table 1: Comparative characteristics of IDV and novel hit
Molecular properties
IDV
Novel hit
Atoms
92
63
MW (≤ 500)
613.8↑
494.6
TPSA (Å2)
118
181
cLogP (≤ 5)
2.9
2.4
HBD (≤ 5)
4
4
HBA (≤ 10)
7
6
NI1
0
0
PI2
2
3
Docking results
MolDock Score
-
-185.7
Rerank Score
-
-148.5
Docking Score
-
-191.6
H-bond energy
-
-12.8
Flexible torsions
Energy Minimum
-
-15.1
8
-13.8
ADME/Tox
Cramer rules
Class 3
Class 3
Biodegradability
Class 2
Class 2
Carcinogenicity
no
no
1negative ionizable groups; 2positive ionizable groups Bioinformation Bioinformation
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Hypothesis open access
Hypothesis Supplementary material Supplementary material
Table 1: Comparative characteristics of IDV and novel hit
Molecular properties
IDV
Novel hit
Atoms
92
63
MW (≤ 500)
613.8↑
494.6
TPSA (Å2)
118
181
cLogP (≤ 5)
2.9
2.4
HBD (≤ 5)
4
4
HBA (≤ 10)
7
6
NI1
0
0
PI2
2
3
Docking results
MolDock Score
-
-185.7
Rerank Score
-
-148.5
Docking Score
-
-191.6
H-bond energy
-
-12.8
Flexible torsions
Energy Minimum
-
-15.1
8
-13.8
ADME/Tox
Cramer rules
Class 3
Class 3
Biodegradability
Class 2
Class 2
Carcinogenicity
no
no
1negative ionizable groups; 2positive ionizable groups Table 1: Comparative characteristics of IDV and novel hit 299 ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 4(7): 295-299 (2010) © 2010 Biomedical Informatics
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https://openalex.org/W4312315425
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https://www.revistas.usp.br/sausoc/article/download/204018/187670
|
English
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Vulnerabilidade e saúde de mulheres quilombolas em uma área de mineração na Amazônia
|
Saúde e Sociedade
| 2,022
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cc-by
| 7,573
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Vulnerability analysis and quilombola women’s
health in a mining area in the Amazon
Vulnerabilidade e saúde de mulheres quilombolas em uma
área de mineração na Amazônia Veridiana Barreto do Nascimentoa
https://orcid.org/0000-0003-4655-9670
E-mail: veridianaiespes@gmail.com
Ana Carolina Vitorio Arantesa
https://orcid.org/0000-0002-3867-6327
E-mail: anacv.arantes@gmail.com
Luciana Gonçalves de Carvalhob
https://orcid.org/0000-0001-7916-9092
E-mail: luciana.carvalho@ufopa.edu.br
aUniversidade Federal do Oeste do Pará. Instituto de
Biodiversidade e Florestas. Santarém, PA, Brasil. bUniversidade Federal do Oeste do Pará. Instituto de Ciências
da Sociedade. Santarém, PA, Brasil. Original articles Original articles Veridiana Barreto do Nascimentoa Vulnerability encompass different factors that
contribute to exposing individuals and groups to
the illness processes. In this study, this concept was
used to assess the health situation of 139 women
living in eight quilombola communities located in
a mining area, on the banks of the Trombetas River,
in Oriximiná, Pará, Brazil. Data were collected
by semi-structured individual interviews, which
underwent quantitative analysis and descriptive
statistics. Although quilombola communities
are historically vulnerable, women are further
weakened by individual, social, and programmatic
factors, revealed in the poor access to education,
information, work, income and health. Hence, public
policies specifically suited to their gender and socio-
cultural profile are needed. Keywords: Women’s Health; Vulnerability Analysis;
Quilombola Communities; Quilomboa’s Health. Abstract Veridiana Barreto do Nascimentoa
https://orcid.org/0000-0003-4655-9670
E-mail: veridianaiespes@gmail.com
Ana Carolina Vitorio Arantesa
https://orcid.org/0000-0002-3867-6327
E-mail: anacv.arantes@gmail.com
Luciana Gonçalves de Carvalhob
https://orcid.org/0000-0001-7916-9092
E-mail: luciana.carvalho@ufopa.edu.br
aUniversidade Federal do Oeste do Pará. Instituto de
Biodiversidade e Florestas. Santarém, PA, Brasil. bUniversidade Federal do Oeste do Pará. Instituto de Ciências
da Sociedade. Santarém, PA, Brasil. Introduction Resumo The concept of vulnerability is important
in public health because it allows the interpretation
of the onset of communicable diseases from
alternative approaches to those based on the
concept of risk, typical of epidemiology, as well as
to individualizing approaches to disease management
(Oviedo; Czeresnia, 2015; Sanchez, 2015). The use
of this concept, especially in the study of sexually
transmitted infections, has been highly effective
in revealing indicators of social inequities and
inequalities in gender, race and class, which are
relevant factors of exposure to illness (Andrade, 2017;
Guilhem; Azevedo, 2008). O conceito de vulnerabilidade considera diferentes
fatores que concorrem para a exposição de indivíduos
e grupos a processos de adoecimento. Neste estudo,
o referido conceito foi utilizado com o objetivo
de avaliar a situação da saúde de 139 mulheres
residentes em oito comunidades quilombolas,
localizadas em uma área de exploração mineral,
nas margens do Rio Trombetas, em Oriximiná
(PA). O estudo serviu-se de entrevistas individuais
semiestruturadas para coleta de dados, os quais
foram submetidos a uma abordagem quantitativa
e analisados por meio de estatística descritiva. Embora as comunidades quilombolas sejam
historicamente vulneráveis, os dados evidenciaram
que as mulheres são ainda mais fragilizadas por
fatores individuais, sociais e programáticos que
se revelam nas dificuldades de acesso à educação,
à informação, ao trabalho, à renda e à saúde. Elas necessitam, portanto, de políticas públicas
especialmente adequadas à sua condição de gênero
e ao seu perfil sociocultural. Perceived by Oviedo and Czeresnia (2015, p. 238)
as a “constitutive and constituent ontological
dimension of human life”, vulnerability is a state
that compromises, temporarily or permanently,
the capacity of individuals or groups to make
decisions and act, exposing them to diseases
and other damage as a result of this condition
(Guerrero, 2010). However, several factors
geographic, economic, environmental, social,
cultural and political, go beyond the individual
dimension and converge to determine vulnerability,
in social and programmatic levels. Palavras-chave: Saúde da Mulher; Análise
de Vulnerabilidade; Comunidades Quilombolas;
Saúde da População Quilombola. Thus, individual choices and behaviors, moral
systems and cultural traditions, public policies
and government programs, among other elements
contribute, at different levels, to the health of
individuals and groups of individuals. In other words,
vulnerability is linked to the living conditions of
a population and reflects its social inequalities
(Garcia et al., 2008; Oliveira et al., 2015; Taquetti, 2010). Correspondence Veridiana Barreto do Nascimento
Rua São Jorge, 358, Santarenzinho, PA, Brasil. CEP: 68035135. Veridiana Barreto do Nascimento
Rua São Jorge, 358, Santarenzinho, PA, Brasil. CEP: 68035135. DOI 10.1590/S0104-12902022210024en DOI 10.1590/S0104-12902022210024en Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 1 Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 2 Introduction This research also attests that, in the close
contact with mining routines, the susceptibility of
the researched quilombola communities, particularly
women, is exacerbated by restrictions on access to
knowledge, means and health care services, that
make up structural dynamics of exclusion of this
social segment from public policies on education,
housing, employment and income. Although the
provision of health services through projects in the
communities and emergency care at MRN’s hospital
facilities contribute to meet some of the needs of
the quilombolas, as a rule, they are left with the
difficult and insufficient recourse to public health
services offered at the municipal headquarters of
Oriximiná (Castro, 2019). In four decades, MRN has become one of the
world’s largest producers of bauxite, attracting
to Porto Trombetas and surroundings countless
workers - mostly men – from various cities of Pará,
other Brazilian states and even other countries. In On the other hand, the cumulative socio-
environmental impacts of mining activity have
worsened significantly the historic vulnerability of
quilombola communities and produced changes in
their ways of life, including reflections on the health
of individuals, such as attest to studies carried out
in the region. According to Fidelis (2019), the recent expansion
of access of members of certain quilombola
communities to some health services maintained
by MRN, in the village of Porto Trombetas,
results from claims that gained momentum after
publication, in 2017, of the technical reports on
the identification and delimitation of the Alto
Trombetas I and II territories. The publication of
these reports constituted a significant advance in
the process of recognition of the territorial right of
those communities and gave them more power to
negotiate with the company, especially in the context
of environmental licensing processes. The quality of
the services offered in the communities, however,
is a frequent target of complaints. Its restricted focus
on prevention, the lack of specialized treatments, and
even racial discrimination practices were complaints
noted by Fidelis (2019), especially among women. The Quilombola Component Study (ECQ)
conducted in 2016, as part of the environmental
licensing of new bauxite mines in the region,
recorded among the main negative impacts of
mining the increase in the rate of occurrence
of diseases, whose significance was considered
very high (Cumbuca Norte, 2016). Introduction The concept of vulnerability was used in this
study to assess the health conditions of quilombola
women living in eight communities located on the
banks of the Trombetas River, in the municipality
of Oriximiná, in the northwest of the state of Pará. It is a historically marginalized social group, whose
trajectory dates to Brazil’s colonial and slave past. Specificaly in this region, quilombola occupation
dates to the flight of slaves from rural properties
in the inland of the Amazon region situated in the
state of Pará where Africans and their descendants
were systematically enslaved until the end of the urinary tract infections, gastritis and intestinal
infections, among others. 19th century (Funes, 2000; Salles, 2005). This
population earns their living from traditional and
family-based agro-extractive activities, and even
today it is not adequately supported by health,
education, housing, sanitation, communication,
work, and income policies. In a study later conducted by the Comissão
Pró-Índio de São Paulo, focusing specifically on
the water-mining interaction, which involves from
the exploration of the deposit to the processing
of the ore, Andrade (2018) corroborated the ECQ
records regarding the health conditions of the local
population. The author pointed out that, in the
perception of quilombolas, water and air pollution,
resulting from mining, causes diseases that “did
not exist before” (Andrade, 2018, p. 38). Besides the historical context of marginalization,
more recently, in the 1970s, black rural or quilombola
communities in the region were unexpectedly
faced with the installation of a mining company
in the area they inhabited. With support from the
Brazilian government, the mining company Mineração
Rio do Norte (MRN) set, in the middle of the forest,
a complex industrial plant, connecting dozens
of mines to a river port, which is the main gateway
to village Porto Trombetas, established in 1976,
together with the first bauxite shipment abroad. This village is, in fact, a company town restricted to
MRN employees and guests, equipped with hospital,
sanitation services, school, bank, commerce, club,
bars, churches and cleaning and private security
services, among others (Cumbuca Norte, 2016). The infrastructure of MRN is much better than in several
cities in the Lower Amazon region, where it is located. Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 3 Methodology Throughout the 20th century, after the abolition
of slavery, much of the population of the mocambos
moved to areas of easier access and closer to urban
centers where their production was commercialized. The Blacks dispersed throughout the Trombetas
Basin, forming various population centers. From the 1980s to the 1990s, these settlements
became associated in the form of 37 communities
self- identified as quilombola communities. Distributed in eight territories, these communities
have currently around ten thousand individuals
(Comissão Pró- Índio de São Paulo, 2020). Introduction The diversity of
diseases that communities associate with the mining
activity is also significant: AIDS and other sexually
transmitted infections, obesity, cancer, itchy skin,
hypertension, respiratory problems, allergies, In this context, assuming that quilombola
women present greater vulnerability to illness, this Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 3 dense forests, fertile lands and fishy waters, somewhat
protected by waterfalls that hindered the recapture
expeditions. In the mocambos, the blacks developed
a relatively autonomous way of life, based primarily
on extractivism and agricultural production for own
consumption and exchange with local traders, as well
as with the natives themselves (Funes, 2000). study seeks to evaluate the health conditions of
women from eight communities in the Trombetas
region. Thus, the study asks: What are and how do
the vulnerabilities of quilombola women living on
the banks of the Trombetas River, in Oriximiná (PA)? Study area The occupation of lands by black communities
in Oriximiná began in the 19th century after
successive escapes of enslaved Africans who
worked in farms located on the Amazon River bed
(Acevedo; Castro, 1998; Funes, 2000). The source
of the Trombetas River, which crosses Oriximiná
from north to south, as well as the course of other
tributaries on the left bank of the Amazon River,
was the preferred destination of black individuals
who sought freedom in the forests of the region,
due to its specific geographic characteristics and
the network of collaboration established with
indigenous peoples who inhabited the region. Eight communities belonging to three quilombola
territories were defined for this study, due to their
proximity to Port Trombetas (Figure 1). There are
378 families living there, with an average number of
six members each, representing an estimated total
population of 2,268 people (Chart 1). With the support of the natives, Africans and their
descendants formed so-called mocambos in areas of Figure 1 – Map of the Trombetas River region
Source: L. Andrade, 2011. Figure 1 – Map of the Trombetas River region Figure 1 – Map of the Trombetas River region Source: L. Andrade, 2011. Source: L. Andrade, 2011. Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 4 Chart 1 – Characterization of communities
COMMUNITY
TERRITORY
TITRATION
AREA (ha)
FAMILIES
Boa Vista
Boa Vista
1995
1.125,0341
150
Água Fria
Água Fria
1996
5.571.355
23
Terra Preta, Serrinha, Arancuan de Cima,
Arancuan do Meio, Arancuan de Baixo e Bacabal
Trombetas
1997
80.887,0941
205
Source: Comissão Pró-Índio de São Paulo (2020) adaptado. Chart 1 – Characterization of communities Source: Comissão Pró-Índio de São Paulo (2020) adaptado. of pursuing their studies in order to have more
opportunities for professional qualification, young
people turn to the activities traditionally carried
out in the communities or, more recently, to mining
and logging. Access to quilombola communities is exclusively
by river. Small community or private boats depart
from the municipal headquarters of Oriximiná to the
communities. The trips take five to eight hours,
depending on the distance from the community. Consequently, traveling to seek basic services
entails a considerable expenditure of time and
financial resources. The main traditional economic activities are
agriculture, the production of manioc flour and the
extraction of non-wood forest products, in particular,
Brazil nuts, whose sale is converted into monetary
income. Fishing and hunting are exclusively for own
consumption, being essential sources of protein
in the diet of quilombola families. In addition
to these practices, wage labor and temporary work
as self-employed persons or through cooperatives
have become increasingly common. In the Boa Vista
quilombola Territory (TQ), for example, at least one
member of almost all families works in the mining
sector. In Trombetas TQ, several residents are
employed in activities of management, extraction
and sale of wood. Also, there is a lack of Basic Health Units (UBS)
in the territories. Therefore, most of the quilombola
population does not receive regular health care. This
population relies mainly on medicinal plants and
on traditional knowledge to cure common diseases
such as colds, diarrhea, fever, wounds and localized
pain. Another widely used resource is the search for
local specialists such as chiropractors and prayer
healers. The residents of these communities rarely
seek the official health care system and therefore,
they are rarely given prescriptions for the use of
industrialized drugs. The infrastructure of these communities
is precarious. Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 5 Figure 1 – Map of the Trombetas River region Devoid of a power supply system,
they use generators powered by diesel oil, which
are turned on for about three hours at night,
or for a longer time on commemorative dates and
days of meetings of the residents. The water used
by residents is obtained directly from rivers, lakes
and streams, either manually or through community
microsystems, and is boiled and/or treated
with hypochlorite solution when it is supplied
by Community Health Agents (ACS). According to the prevalent sexual division
of labor, common in rural areas, men are in charge of
services considered “heavy”, either in traditional
activities or in mining and logging companies. Some of their activities include clearing the land for
planting, hunting, working in mines, cutting wood
and in areas for reforestation. Women, in turn, are mainly dedicated to
domestic work, maintenance of the farming
land, and fishing for family consumption. When
they enter the labor market, they usually provide
general services, gardening and cleaning services,
both on the company’s premises and in the
homes of middle and high-ranking employees
(Maini, 2018). Finally, although the topic is not
openly discussed in the communities, some women
work as sex workers in Vila Paraíso, which is close There is only one elementary school in each
territory. Therefore, young people are forced to
migrate to Oriximiná or other larger cities to attend
high school. As this migration to Oriximiná entails
costs, most quilombola families cannot send their
children to study in that city. Due to the impossibility Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 5 to Port Trombetas and is frequented by mining
workers (Madeira- Filho et al., 2012). Also known
as Brega 45, the village is usually associated with
an increase in the cases of unwanted pregnancies
and sexually transmitted infections in the region. Subsequently, the researchers made home
visits for the administration of a form composed
of 56 closed-ended and open-ended questions, for
the characterization of the group’s socioeconomic
profile and identification of the vulnerabilities in
the local health system. All visits were accompanied
by ACS or local leaders, but the forms were applied
individually, and in the locations chosen by the
participants, inside or outside the residence, so that
the meetings were private and the participants felt
free to answer the questions. Data collection and analysis This study has an exploratory, descriptive,
prospective, cross-sectional methodology,
with a quantitative approach. Participants
were 139 women contacted with the aid of five
community health agents (ACS) and community
leaders, who also provided logistical support during
the field research. The following criteria were used
for the selection of the participants: they should
have permanent residence in the communities
and be 15-55 years old, as it has been previously
agreed with the Association of Remaining Quilombo
Communities of the Municipality of Oriximiná
(ARQMO) and local leaders. The data obtained in the field research were
stored in an Excel spreadsheet and transferred to
SPSS version 20 and Bioestat version 5.3 programs
for statistical treatment. The results were described
by absolute and relative frequencies, and sample
weighing was considered in relative frequency. Results and discussion The sample consisted of 139 quilombola women,
and the results were stratified according to age groups:
16 ┤20 years; 21 ┤30 years, 31 ┤40 years, 41 ┤50 years
of 51 ┤60 years and distributed by territory as follows:
44 (31.7%) in TQ Boa Vista; 16 (11.6%) in TQ Água Fria;
and 79 (56.7%) in TQ Trombetas. The average age of
the participants was 30 years old (sd = 10.31), ranging
from 16 to 55 years. In the age range of 21-30, young
adults, there were 51 women (36.8%). They make up the
largest group of respondents, of 115 (82.7%) women aged
up to 40 years, therefore, in their reproductive stage. In accordance with CNS Resolution No. 466/2012,
the study was evaluated and approved by the
Research Ethics Committee. A Free and Informed
Consent Form was signed by all participants, and for
participants under 18, it was signed by parents/
guardians, and a Term of Assent, signed by the
adolescents was included. The field research was developed in two stages,
both in October 2016. First, meetings were held with
the employees and the women appointed by them,
for the presentation of the proposal, its objectives,
possible risks and benefits. The meetings were held
in community shacks, church halls and schools,
and lasted about 20 to 30 minutes. Regarding color/race, 88 women declared
themselves black (63.3%), and 51 (36.7%) said they were
brown, corroborating data related to the history of black
occupation and racial affirmation in the region (Table 1). Table 1 – Demographic characteristics of quilombola women from the Trombetas River (Pará), 2016
VARIABLES
AGE (N = 139)
16┤20 years
21 ┤30 years
31 ┤40 years
41 ┤50 years
51 ┤60 years
TOTAL
N
%
N
%
N
%
N
%
N
%
N
%
COLOR/RACE
Black
15
55,5
32
62,7
23
62,2
8
66,7
10
83,3
88
63,3
Brown
12
44,5
19
37,3
14
37,8
4
33,3
2
16,7
51
36,7
continue... ble 1 – Demographic characteristics of quilombola women from the Trombetas River (Pará Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 6 VARIABLES
AGE (N = 139)
16┤20 years
21 ┤30 years
31 ┤40 years
41 ┤50 years
51 ┤60 years
TOTAL
N
%
N
%
N
%
N
%
N
%
N
%
EDUCATION
Never studied
0
-
0
-
0
-
1
8,3
4
33,3
5
3,6
Inc. Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 7 Results and discussion Regarding individual monthly income,
32 (60.4%) of them received less than the minimum
wage in force at the time; 19 (35.8%) received one to
two minimum wages; and only 2 (3.8%) received more
than two minimum wages. In this group of women,
36 (67.9%) lived in brick houses, while 17 (32.1%) had
wooden houses; 34 (64.2%) consumed water distributed
by a community microsystem; and 19 (35.8%) had a
private well (Table 2). and did not earn a regular income. These women were
usually supported by their companions or sometimes
would earn money by providing general services in
Vila de Porto Trombetas, selling agricultural products
or even handicraft products. In this group, 26 (30.2%)
lived in brick houses; 51 (59.3%) in wooden houses; and
9 (10.5%) in straw houses. Regarding water supply,
26 women (30.2%) obtained it directly from the river;
51 (59.3%) were supplied by a community microsystem;
and 9 (10.5%) had wells. Table 2 – Working conditions of quilombola women, Rio Trombetas (Pará), 2016
VARIABLES
YES
NO
TOTAL
N
%
N
%
N
%
PAID ACTIVITY
53
38,1
86
61,9
139
100
INDIVIDUAL MONTHLY INCOME
< 1 salary
32
60,4
-
-
32
60,4
1 to 2 salaries
19
35,8
-
-
19
35,8
> 2 salaries
2
3,8
-
-
2
3,8
TYPE OF HOUSING
Brick houses
36
67,9
26
30,2
62
44,6
Wooden houses
17
32,1
51
59,3
68
48,9
Straw houses
0
-
9
10,5
9
6,5
WATER FOR CONSUMPTION
Community microsystem
34
64,2
51
59,3
85
61,2
Private well
19
35,8
9
10,5
28
20,1
River
0
-
26
30,2
26
18,7 Table 2 – Working conditions of quilombola women, Rio Trombetas (Pará), 2016 ble 2 – Working conditions of quilombola women, Rio Trombetas (Pará), 2016 Among the parameters used to characterize
the socioeconomic profile of the participants,
the following deserve mention: a) affirmation
of blackness as an identification criterion and
ethnic- racial belonging consistent with the history
of occupation and the quilombola social organization
in Oriximiná; b) low education due to the lack of
educational services in the quilombos; and c) labor
market exclusion and the low income earned by
women, highlighting their economic dependence
on their partners/spouses. The last two factors are strongly interconnected
and contribute to increasing women’s individual
and social vulnerability to illness, deepening
gender inequalities in the quilombos. Results and discussion elementary
school
1
3,7
5
9,8
11
29,7
6
50,0
6
50,0
29
20,9
Comp. elementary
school
11
40,7
22
43,1
13
35,1
2
16,7
0
-
48
34,5
Inc. high
school
0
-
3
5,9
0
-
0
-
0
-
3
2,2
Comp. high
school
14
51,9
15
29,4
11
29,7
1
8,3
2
16,7
43
30,9
Inc. higher
education
1
3,7
3
5,9
0
-
1
8,3
0
-
5
3,6
Comp. higher
education
0
-
3
5,9
2
5,4
1
8,3
0
-
6
4,3
MARITAL STATUS
Single
16
59,3
13
25,5
6
16,2
1
8,3
3
25,0
39
28,0
Married/
Stable union
11
40,7
37
72,5
28
75,7
11
91,7
9
75,0
96
69,1
Separated
0
-
1
2,0
3
8,1
0
-
0
-
4
2,9
Table 1 – Continuation Table 1 – Continuation Regarding education, 48 (34.5%) had completed
elementary school, and 43 (30.9%) had
completed high school. Women over 41 years had
the lowest levels of education, which is consistent
with the late establishment of school units in the
communities. Only six (4.3%) women aged 21-50
years had attended higher education, within
the scope of the National Teacher Training Plan
(Parfor), implemented in 2009, by the Ministry of
Education to improve the training of teachers in
the public basic education network (Table 1). Low
educational level is a common trait in quilombola
communities in Pará, since most of the population
has completed only primary education, and modular
classes are common. As for the marital status of the participants,
96 (69.1%) were married or lived in a stable
relationship; 39 (28.0%) were single and 4 (2.9%) were
separated (Table 1). The prevalence of married women
is explained by cultural and economic reasons and
by aspects related to the quilombola way of life, in
which the family is the fundamental productive unit. In fact, in the local communities, women marry very
often before reaching adulthood and their husbands
are often older men who earn income from farming
their own cleared lands, wage labor or even retirement. To describe the living conditions of the respondents
data related to income, housing and access to water
were crossed (Table 2). The largest group consisted
of 86 (61.9%) women who did not perform paid work Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 7 Only 53 (38.1%) women said they performed paid
activities. Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 8 Results and discussion On the other
hand, structural aspects of the municipality of
Oriximiná and, particularly, of Trombetas river,
express specific geographical, social, economic,
and political conditions, which contribute to
characterize the vulnerability of the quilombola
population as a whole, and particularly of women,
in Brazil (Silva, 2011). Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 8 The precarious health assistance in TQ Boa Vista,
Água Fria and Trombetas clearly corroborates
a routine situation in quilombola communities
in Pará (Cavalcante, 2011). None of the eight
communities covered in the study had a UBS,
although five had community health agents (ACS) -
which is not quite different from the percentage of
57.0% of communities in Pará that count on ACS
services (Oshai; Silva, 2013). The absence of UBS,
and in some of them, even of ACS, signals the
situation of marked programmatic vulnerability
of the quilombola population, recurrently neglected
in public health policies (Guerrero, 2015). VARIABLE
(n = 139)
%
PUBLIC HEALTH SERVICE
AVAILABLE IN THE COMMUNITY
No
139
100,0
TRANSPORT FOR ACCESS TO
HEALTH SERVICE
Motorized canoe
43
30,9
Community boat
59
42,4
Family boat
21
15,2
Others
16
11,5
CONDUCT WHEN YOU ARE SICK
Take home remedy
81
58,3
Go to the hospital in the city
(Oriximiná)
51
36,7
Go to the pharmacy in the
city (Oriximiná)
7
5,0
Table 3 – Continuation Table 3 – Continuation Given the local conditions and the importance
of cultural traditions, women often resort to
homemade remedies when they fall ill. As is usual in
black rural communities, they are preferably treated
with preparations derived from barks, roots, leaves
and oils, vegetables or animals, whose production
is based on knowledge passed from generation to
generation (Sales; Albuquerque; Cavalcanti, 2009). In fact, this is the option of 81 (58.3%) respondents,
while the search for hospitals and pharmacies
is preferred by 51 (35.7%) and 7 (5.0%) women,
respectively (Table 3). The low demand for assistance in the units of
the official healthcare system is partly explained
by the geographical characteristics of the Trombetas
Basin, which, in general, are typical of Northern
Brazil. The lowest percentages of health service use in
the country are concentrated in this region (Stopa et
al., 2013). For example, people who live near the
Trombetas River need to travel to Porto Trombetas
or to the city of Oriximiná to reach a hospital, that
is, long, exhausting, and costly trips are necessary. Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 9 Results and discussion For this purpose, 59 (42.4%) women use the
community boat; 43 (30.9%) use their own motorized
canoe; and 21 (15.2%) use the family boat (Table 3). Table 3 – Educational activities to prevent diseases
developed in quilombola communities, Rio Trombetas
(Pará), 2016
VARIABLE
(n = 139)
%
PREVENTION ACTIVITY
Yes
68
48,9
FREQUENCY OF ACTIVITIES
Yearly
56
82,3
Monthly
8
11,8
Semiannual
4
5,9
RESPONSIBLE FOR THE ACTIVITY
(n = 68)
ACS
17
25,0
Nurses
25
36,8
Others (interns)
26
38,2
continue... Table 3 – Educational activities to prevent diseases
developed in quilombola communities, Rio Trombetas
(Pará), 2016 Due to environmental constraints associated
to the mining activity, quilombola women from TQ
Boa Vista are registered as users at the hospital of
the mining company (MRN), however, this option
is not available to residents of all communities
surrounding the company, except in urgent
and emergency cases. In these specific cases,
the patient’s condition can be stabilized at the Porto
Trombetas hospital, but the continuity of treatment
must be given at the municipal hospital of Oriximiná,
which demands from the patients and their eventual Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 9 companions resources for the displacement. Taking
into account the regional geography, Castro (2019)
points out that the costs of boat fuel, food, lodging,
and transportation in the city are not compatible
with the means of most quilombola families, given
their situation of social vulnerability. Therefore, it is
not uncommon for treatments to be discontinued. VARIABLE
N
%
PLACE OF PERFORMANCE
(n = 51)
Public Service-City
44
86,3
Public-Community Service
2
3,9
Hospital-Vila Service
3
5,9
Others
2
3,9
CONSULTATION TYPE
Routine
16
31,4
Urgency
35
68,6
DIFFICULTY IN SERVICE
Yes
45
88,2%
No
6
11,8%
EXAMS (last 3 years)
Yes
10
74,1%
EXAM PLACE
Health center-city
88
85,4%
Community actions
4
3,9%
Others (private)
11
10,75
Table 4 – Continuation In this scenario, preventive actions must be taken
to improve the health conditions of the quilombola
population. However, these actions are usually carried
out through lectures, and local women do not seem to
be interested in them. Only 68 (48.9%) respondents
participated in any action of this type. According to 56
(82.3%) participants, these activities are carried out
annually and are itinerant. Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 10 Results and discussion It was found that 88 (85.4%) of the women who
participated in the study underwent exams at a
UBS in the city of Oriximiná; of these, 4 (3.9%)
underwent exams within the scope of health actions
in the TQ; and 11 (10.7%) underwent the exams in
private services (Table 4). According to the author, the Brazilian Institute
of the Environment and Renewable Natural
Resources (Ibama), which is responsible for the
environmental licensing of the project, notified
the mining company that pollution in water
courses used by the Boa Vista community had
reached levels capable of causing damage to the
health of the quilombolas (Andrade, 2018). In fact,
the increasing difficulty in accessing drinking
water has been reported by the population as
one of the main negative impacts of mining
(Cumbuca Norte, 2016). Therefore, most of the
residents stopped using natural sources and
began to collect water from the taps available
in Vila de Porto Trombetas, using recycled plastic
bottles. Many women reported that, as with
children, who play in the river and in streams, they
are also more affected by waterborne diseases,
due to the daily activities of washing clothes,
collecting and using the liquid for cooking
(Cumbuca Norte, 2016). Regarding the most common diseases among
the respondents, 107 (74.8%) reported diseases
of the integumentary system, such as itching and
skin wounds. Flu and pneumonia were reported
by 86 (61.9%) women. Gynecological problems, body
pains and chronic diseases, such as hypertension
and diabetes, were less mentioned (Table 5). Table 5 – Most common health problems reported
by quilombola women, Rio Trombetas (Pará), 2016
VARIABLE
N
%
PROBLEMS
(n = 139)
Integumentary system-itching,
wounds
107
74,8%
Respiratory-influenza,
pneumonia
86
61,9
Gynecological-Discharge
24
17,3
Pains-lumbar, tooth
18
12,9
Hypertension
15
10,8
Diabetes
7
5,1
Accidents (snake bite, injuries)
16
11,5
Others (cancer, rheumatism)
2
1,4 Table 5 – Most common health problems reported
by quilombola women, Rio Trombetas (Pará), 2016 The set of factors identified in this study
converges to exacerbate the vulnerability
of quilombola women in the programmatic
dimension. First, there is a notable lack of
primary health care services in the areas surveyed,
characterized by the lack of basic health units
(UBS) and the insufficient care provided by
community health agents (ACS). Results and discussion The speakers are mainly
nursing professionals from Oriximiná, as reported
by 25 (36.8%) women; ACS, according to 17 others
(25.0%); and interns in technical course, according
to 26 (38.2%) women (Table 3). Thus, investments in prevention actions based
on diversified and emancipatory methodologies are
considered, which take into account the traditional
knowledge of quilombola communities and pay
special attention to the most appropriate ways of
approaching women, in order to make them play
an active role in the development of individual and
collective health care strategies, thus reducing the
individual vulnerability of these women. Problems of access to healthcare services by the
target population of this study is shown on the
data obtained. Associated with the ineffectiveness
of these services, this factor is one of the
most relevant components of the vulnerability
of quilombola communities to illnesses and their
consequences (Garcia et al., 2008). Due to the
lack of primary care services in the communities,
and because there was no resizing of the services
available in the city, this population, historically
marginalized and culturally discriminated, tends
to remain excluded from the official health care
system (Cavalcante, 2011). Regarding medical appointments, the participants
were asked whether they sought assistance to deal
with a health problem in the six months preceding the
interviews. Only 51 (36.7%) women answered yes to the
question, and 45 (88.2%) reported that it was difficult
to obtain medical assistance. Among them, 44 (86.3%)
sought public health services in the city of Oriximiná,
and 35 (68.6%) did so in urgent situations (Table 4). Table 4 – Type of health care for quilombola women,
Rio Trombetas (Pará), 2016
VARIABLE
N
%
CONSULTATION (last 6 months)
(n = 139)
Yes
51
36,7
No
88
63,3
continue... Table 4 – Type of health care for quilombola women,
Rio Trombetas (Pará), 2016 The ineffectiveness of the health services,
in turn, may be related to the slowness and/or
insufficiency of diagnostic services, which are
essential for the definition of appropriate and
timely treatments (Santos, 2016). In this regard,
the study showed that 103 (74.1%) participants
underwent health tests at some point in their lives. Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 10 clean and healthy, but now they are contaminated
and cause diseases. However, considering only the three years before
the study, this number dropped to 70 (67.9%). Final considerations Prevention, which could be a powerful means
of intervention in this scenario, comprises
actions that are not very attractive or effective,
usually carried out in the form of lectures, given
by professionals from the city of Oriximiná, who are
probably not very familiar with the reality, way of life
and culture of quilombola communities. Quilombola communities are a segment
of Brazilian society whose educational, work
and health opportunities have historically been
very limited. Therefore, this population lives
in a continuous state of vulnerability (Silva,
2011). In the Amazon, the susceptibility of
quilombola communities is aggravated by the
dimensions and geographic characteristics of
the region, as residents of rural and riverside
areas need to travel great distances to urban
centers where public services and facilities are
concentrated. (Cavalcante, 2011; Oshai; Silva, 2013;
Guerrero, 2016). Thus, such communities tend
to remain outside the inclusive processes of the
country’s health network. Paradoxically, despite being the most important
and most used local health strategy, the wide
collection of traditional knowledge associated with
natural resources applicable to the maintenance
of well-being and the cure of diseases is often
ignored in actions targeted to the communities. Thus, the endogenous potential of quilombola
territories for the development of individual and
collective health practices is little explored, both in
terms of prevention and in terms of the diagnosis
and treatment of diseases. In the region of the Trombetas river, which is far
from the municipal headquarters in Oriximiná and
has suffered the impacts of mineral exploitation
for four decades, quilombola communities
are affected by vulnerability factors in three
dimensions: individual, social and programmatic. The precarious living, working, and housing
conditions of this population, always associated
with the absence or insufficiency of public
policies that address their needs, contribute
significantly to the increase of their fragility. Due
to the lack of health services in their territories,
quilombolas depend on the assistance provided
at MRN facilities, in emergency situations, and
on the routine health care services provided
by the city of Oriximiná, which are difficult to
access, expensive and ineffective. Moreover,
health promotion and disease prevention
actions carried out in quilombola territories
are sporadic and inefficient. Results and discussion The referred
lack of health services, added to the fact that
quilombola communities are far from urban
centers, which concentrate public services, makes
access to health services exceedingly difficult. This difficulty translates into the repeated
denial of a constitutional right to the quilombola
population of the Trombetas River region,
who cannot access hospitals, pharmacies and
other health equipment and services. Regarding the activities of the bauxite mining
company in the surrounding communities,
it is estimated that skin and respiratory problems
may be associated with water and air pollution
by residues from ore washing, drying and
transport activities. This situation is more
serious in the Boa Vista community, closer to
the mining company’s port, where quilombolas
interviewed by Andrade (2018) claim that, before
the establishment of MRN, the waters of the
Trombetas river and the Água Fria stream were Second, the unavailability of essential resources
for the early diagnosis of diseases favors the
worsening of health conditions that, otherwise,
could be more easily controlled. These factors
contribute to the inability of health services Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 11 health policies is exacerbated by the ineffectiveness
of the environmental policy in mitigating the
harmful effects of mining, intensifying the
programmatic vulnerability of the quilombola
population of the region of Trombetas river. to solve the health problems of the quilombola
population. These individuals also find it difficult
to schedule their laboratory tests, wait a long
time for the results of the tests and often do not
find any specialist to explain the results to them. These factors contribute to the ineffectiveness of
the treatments adopted. Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 12 Final considerations Regarding all the aspects reported, the situation
in TQ Boa Vista, Água Fria and Trombetas is similar
to the one described by Cavalcante (2011) in most
quilombola communities in the Amazon region
in the state of Pará, where health services are
concentrated in large cities. Thus, the unassisted
population can only rely on sporadic campaigns or
actions that do not take in consideration the real
needs of the users. However, some characteristics
of the Trombetas river region aggravate the
vulnerability of the communities surveyed. The environmental damage caused directly
or indirectly by the mining activities developed in
the surroundings of the said quilombola territories
threatens the residents’ health. As it has been
shown, the frequent and inevitable contacts with
water and air polluted by bauxite residues affect
the integumentary and respiratory systems and are
related to the diseases most commonly identified
among the women interviewed. Thus, the lack of With regard to women, vulnerability to
diseases is more significant in the three Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 12 transmissíveis. Acta Paulista de Enfermagem,
São Paulo, v. 10, n. 30, p. 8-15, 2017. DOI:10.1590/1982-0194201700003 dimensions cited, in all quilombola communities
surveyed. In the individual dimension, the
low level of education and restricted access
to specialized health information make it
difficult for them to incorporate preventive
knowledge and attitudes into everyday
actions. In the social dimension, the historical
gender inequality makes the precarious
living conditions of quilombola families more
harmful to women, as they have less access
to job and income opportunities than men,
and, consequently, less decision-making power. In the programmatic dimension, the absence
and/or ineffectiveness of public health policies
reinforces women’s dependence on men,
on the mining company and on the services
provided in Oriximiná. ANDRADE, L. M. M. Antes a água era cristalina,
pura e sadia: percepções quilombolas e
ribeirinhas dos impactos e riscos da mineração
em Oriximiná, Pará. São Paulo:
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em Oriximiná: pressões e ameaças. São Paulo:
Comissão Pró-Índio de São Paulo, 2011. CASTRO, N. J. C. Medicina popular
e desenvolvimento regional: registros e
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Socioambiental) – Núcleo de Altos Estudos
Amazônicos, Universidade Federal do Pará,
Belém, 2019. Final considerations In this context, there is a clear need for new public
policies tailored to the needs of quilombola women
from the region of Trombetas river, their gender
condition and their ethnic-cultural profile. These
issues have been repeatedly neglected in the health
actions carried out in the communities surveyed. In addition to regularly providing opportunities for
health care and assistance to these women, these
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aos serviços de saúde em três quilombos na
Amazônia paraense: um olhar antropológico. 2011. Dissertação (Mestrado em Saúde e Sociedade) –
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e Endemias na Amazônia, Universidade Federal
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e Endemias na Amazônia, Universidade Federal
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culturally appropriate mechanisms to encourage the
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are inserted in the communities of the Trombetas
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Resubmitted: 04/01/2022
Approved: 28/05/2022 OVIEDO, R. A. M.; CZERESNIA, D. O conceito
de vulnerabilidade e seu caráter biossocial. Saúde Soc. São Paulo, v.31, n.3, e210024en, 2022 14
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Pengaruh Skeptisisme Profesional dan Independensi terhadap Ketepatan Pemberian Opini oleh Auditor
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Bandung Conference Series: Accountancy Bandung Conference Series: Accountancy https://doi.org/10.29313/bcsa.v2i2.2987 Kata Kunci: Skeptisisme Profesional, Independensi, Ketepatan Pemberian Opini
oleh Auditor. Shafa Astari Azzahra*, Pupung Purnamasari, Nopi Hernawati
Prodi Akuntansi, Fakultas Ekonomi dan Bisnis, Universitas Islam
Bandung, Indonesia. Shafa Astari Azzahra*, Pupung Purnamasari, Nopi Hernawati
Prodi Akuntansi, Fakultas Ekonomi dan Bisnis, Universitas Islam
Bandung, Indonesia. *shafastaria@gmail.com, p_purnamasari@yahoo.co.id, nopihernawati@unisba.ac.id Corresponding Author
Email: p_purnamasari@yahoo.co.id A. Profesional merupakan kompetensi seseorang yang menekuni suatu profesi tertentu yang
berkaitan dan dipengaruhi oleh bidang pendidikan dan keahlian. Menjadi profesional berarti
seorang individu harus bertanggung jawab dan memahami tugas yang diberikan, serta
konsisten dan fokus terhadap tujuan organisasi. Sikap profesional tersebut wajib dimiliki
setiap individu dalam melaksanakan pekerjaan atau profesinya. Salah satu profesi yang
dituntut untuk mempunyai sikap profesional yaitu akuntan pubik atau auditor eksternal. Akuntan publik sendiri merupakan akuntan yang pekerjaannya bertugas dalam memberikan
dan melakukan jasa auditing profesional kepada klien di bawah satuan kantor akuntan publik
(Abdul Halim, 2008). Dalam melakukukan audit seorang akuntan publik dapat memberikan laporan
keuangan perusahaan sebagai nilai tambahnya tersendiri, hal tersebut dikarenakan akuntan
publik yang merupakan pihak ahli serta independen, di akhir pemeriksaannya akan
memberikan masukan mengenai kewajaran hasil usahaha, posisi keuangan, perubahan ekuitas
dan laporan arus kas. Sehingga laporan audit menjadi tujuan akhir dari proses audit itu sendiri. Laporan audit yang nantinya dijadikan acuan bagi pemakai laporan keuangan, disampaikan
auditor melalui pernyataan atau pendapatnya (opini) kepada para pemakai (Alvin A. Arens,
2012). Agar perumusan opininya tepat, maka bukti audit harus dinilai, sehingga dibutuhkan
sikap skeptisisme profesional. Menurut Alvin. A Arens (2011) yang dimuat dalam Herman
Wibowo, menjelaskan bahwa skeptisisme profesional merupakan suatu sikap yang dilihat dari
auditor yang tidak mengedepankan asumsi manajemen tidak jujur (berbohong) tetapi juga
tidak mengasumsikan kejujuran yang absolut. Dengan diterapkannya sikap skeptisisme
profesional, maka akan membantu auditor dalam mengungkap atau mengetahui potensi salah
saji material untuk meminimalisir risiko pengambilan keputusan yang salah serta informasi
yang menyesatkan bagi pengguna laporan keuangan. Sebagaimana penelitian sebelumnya yang dilakukan oleh Isnaini Oktalina (2021)
memperlihatkan bahwa terdapat pengaruh signifikan antara skeptisisme profesional terhadap
keakuratan atau ketepatan pemberian opini oleh auditor. Selain itu, Tania Kautsarrahmelia
(2013) mengatakan skeptisisme profesional mempunyai pengaruh yang signifikan terhadap
keakuratan atau ketepatan pemberian opini oleh akuntan publik. Akuntan publik berperan sebagai profesi yang dipercaya masyarakat atas kualitas atau
mutu jasa audit. Masyarakat sebagai public menginginkan penilaian independen dalam laporan
keuangannya atas informasi yang disajikan (Agus, 2014). Independensi sendiri merupakan
keadaan seseorang dimana seseorang tersebut tidak terikat atau terkait dengan pihak manapun,
atau keadaan seseorang yang bebas, mandiri, dan tidak bergantung kepada pihak lain
(Sihotang, 2016). Pengguna laporan keuangan akan sulit untuk percaya kembali jika auditor
tidak bisa menerapkan sikap independen. Sehingga hasil laporan keuangan yang diperiksa
menjadi tidak bernilai jika tidak menerapkan sikap tersebut. Hal tersebut berdampak juga
terhadap kehandalan dan opini yang dikeluarkannya juga akan dipertanyakan. *shafastaria@gmail.com, p_purnamasari@yahoo.co.id, nopihernawati@unisba.ac.id Abstract. This study aims to determine the effect of professional skepticism and
independence on the accuracy of giving opinions by auditors at the Public
Accounting Firm (KAP) in Bandung. The population of this study are all auditors
who work at the Public Accounting Firm in Bandung City who are active and obtain
the permit from the Minister of Finance 2022. The number of auditors who were
sampled in the study were 37 respondents from 10 Public Accounting Firms in
Bandung City. This study uses a verification method with a quantitative approach. The data collected is primary data and data collection techniques using a
questionnaire. Hypothesis testing in this study used multiple regression analysis
with SPSS version 23 statistical tool. The results showed that partially professional
skepticism had no effect on the accuracy of giving an opinion by the auditor. Meanwhile, independence has a significant effect on the accuracy of giving an
opinion by the auditor. Meanwhile, it simultaneously shows that professional
skepticism and independence have a significant effect on the accuracy of giving an
opinion by the auditor. Keywords: Professional Skepticism, Independence, Accuracy of Public
Accountant Opinion. Keywords: Professional Skepticism, Independence, Accuracy of Public
Accountant Opinion. Abstrak. Penelitian ini bertujuan untuk mengetahui adanya pengaruh skeptisisme
profesional dan independensi terhadap ketepatan pemberian opini oleh auditor pada
Kantor Akuntan Publik (KAP) di Kota Bandung. Populasi penelitian ini adalah
seluruh auditor yang bekerja pada Kantor Akuntan Publik di Kota Bandung yang
aktif dan memperoleh izin Menteri Keuangan 2022. Jumlah auditor yang menjadi
sampel dalam penelitian sebanyak 37 responden dari 10 Kantor Akuntan Publik di
Kota Bandung. Penelitian ini menggunakan metode verifikatif dengan pendekatan
kuantitatif. Data yang dikumpulkan merupakan data primer dan teknik
pengumpulan data menggunakan kuesioner. Pengujian hipotesis dalam penelitian
ini menggunakan analisis regresi berganda dengan alat statistik SPSS versi 23. Hasil
penelitian menunjukkan bahwa secara parsial skeptisisme profesional tidak
berpengaruh terhadap ketepatan pemberian opini oleh auditor. Sedangkan,
independensi berpengaruh signifikan terhadap ketepatan pemberian opini oleh
auditor. Sementara, secara simultan menunjukkan bahwa skeptisisme profesional
dan independensi berpengaruh signifikan terhadap ketepatan pemberian opini oleh
auditor. Corresponding Author
Email: p_purnamasari@yahoo.co.id Corresponding Author
Email: p_purnamasari@yahoo.co.id 1063 1064 | Shafa Astari Azzahra, et al. Vol. 2 No. 2 (2022), Hal: 1063-1072 A. Independensi
merupakan hal penting yang harus dijaga oleh auditor secara rutin. Dalam penelitian sebelumnya, menurut oleh Aprilia Ratna Sari (2017) menunjukkan
bahwa independensi memiliki pengaruh yang signifikan terhadap keakuratan atau ketepatan
opini auditor. Penelitian lain yang dilakukan oleh Lastri Nofitri (2020) juga memperlihatkan
bahwa independensi berpengaruh positif signifikan terhadap keakuratan atau ketepatan opini
auditor. Berdasarkan penjelasan sebelumnya di atas, maka dapat dirumuskan beberapa masala
sebagai berikut: 1. Apakah terdapat pengaruh antara skeptisisme profesional terhadap ketetapan pemberian
opini oleh auditor? 1. Apakah terdapat pengaruh antara skeptisisme profesional terhadap ketetapan pemberian
opini oleh auditor? 2. Apakah terdapat pengaruh antara independensi auditor terhadap ketepatan pemberian
opini oleh auditor? Selanjutnya, terkait tujuan dari penelitian ini dijelaskan dalam poin-poin penting
rikut: Vol. 2 No. 2 (2022), Hal: 1063-1072 ISSN: 2828-254X ISSN: 2828-254X 1065 Pengaruh Skeptisisme Profesional dan Independensi terhadap Ketepatan Pemberian Opini oleh Auditor | 1065 1. Untuk mengindentifikasi dan menganalisis pengaruh skeptisisme professional terhadap
ketepatan atau keakuratan pemberian opini audit oleh auditor yang bekerja pada kantor
akuntan public (KAP) di Kota Bandung. 1. Untuk mengindentifikasi dan menganalisis pengaruh skeptisisme professional terhadap
ketepatan atau keakuratan pemberian opini audit oleh auditor yang bekerja pada kantor
akuntan public (KAP) di Kota Bandung. 1. Untuk mengindentifikasi dan menganalisis pengaruh skeptisisme professional terhadap
ketepatan atau keakuratan pemberian opini audit oleh auditor yang bekerja pada kantor
akuntan public (KAP) di Kota Bandung. p
(
)
g
2. Untuk mengindentifikasi dan menganalisis pengaruh independensi auditor terhadap
ketepatan atau keakuratan opini audit yang diungkapkan oleh auditor yang bekerja pada
kantor akuntan public (KAP) di Kota Bandung. p
g
2. Untuk mengindentifikasi dan menganalisis pengaruh independensi auditor terhadap
ketepatan atau keakuratan opini audit yang diungkapkan oleh auditor yang bekerja pada
kantor akuntan public (KAP) di Kota Bandung. Vol. 2 No. 2 (2022), Hal: 1063-1072
ISSN: 282 Vol. 2 No. 2 (2022), Hal: 1063-1072 B.
Metodologi Penelitian Metode yang dipilih dan digunakan oleh peneliti yaitu metode verifikatif dengan pendekatan
kuantitatif. Variabel independen yang dipakai oleh peneliti yaitu skeptisisme profesional dan
independensi. Variabel skeptisisme profesional menggunakan aspek pikiran yang selalu
bertanya, suspensi atau penundaan pada penilaian, mencari pengetahuan, pemahaman
interpersonal, percaya diri, penentuan sendiri (keteguhan hati). Sedangkan, variabel
independensi menggunakan aspek-aspek seperti independensi dalam program audit,
independensi dalam verifikasi, independensi dalam pelaporan. Selain itu, variabel ketepatan
opini oleh auditor menggunakan aspek opini wajar tanpa pengecualian, opini wajar dengan
pengecualian, opini wajar tanpa pengecualian dengan bahasa penjelas, pernyataan tidak
memberikan pendapat, dan pendapat tidak wajar. Sumber data yang diterapkan peneliti dalam penelitian ini adalah data primer. Data
primer adalah data yang didapatkan langsung dari objek penelitian, dikumpulkan langsung
dari responden. Teknik pengumpulan data yang digunakan menggunakan kuesioner yang telah
disusun secara terstruktur yang kemudian akan diisi oleh responden. Adapun populasi yang
dipilih sebagai objek penelitian yaitu Kantor Akuntan Publik (KAP) di Kota Bandung yang
beroperasi dan disahkan oleh Menteri Keuangan. Sedangkan, untuk pengambilan sampel
menggunakan teknik purposive sampling. Uji validitas dan uji reliabilitas merupakan instrumen atau alat uji dalam penelitian ini. Sedangkan, untuk mengubah data dari data ordinal menjadi data interval digunakan Metode
Succesive Interval (MSI). Selanjutnya, uji asumsi klasik atau pengujian hipotesis klasik
penelitian meliputi uji normalitas, uji multikolinearitas, dan uji heteroskedastisitas. Berikut
merupakan persamaan model dalam analisis regresi berganda sebagai berikut: KPO = 11.637 + (-0,141) SP + 0,204 I + e
Keterangan: KPO = Ketepatan Pemberian Opini
α = Konstanta KPO = Ketepatan Pemberian Opini KPO = Ketepatan Pemberian Opini
α = Konstanta
β1 dan β2 = Koefisien Regresi SP = Skeptisisme Profesional I = Independensi e = Standar Eror Untuk menguji hipotesis dilakukan uji simultan (Uji F), uji parsial (Uji t), dan
koefisien determinasi. Accountancy 1066 | Shafa Astari Azzahra, et al. C.
Hasil Penelitian dan Pembahasan
Uji Validitas C. Hasil Penelitian dan Pembahasan
Uji Validitas j
Tabel 1. Rekapitulasi Hasil Pengujian Validitas Variabel Skeptisisme Profesional (X1) Tabel 1. Rekapitulasi Hasil Pengujian Validitas Variabel Skeptisisme Profesional (X1) Berdasarkan tabel 1 diatas, rekapitulasi atau ringkasan menunjukkan bahwa
pernyataan variabel skeptisisme profesional memiliki nilai r hitung lebih besar dari nilai r
tabel sebesar 0,334. Selain itu, nilai signifikansi dalam tabel tersebut < 0,05. Hasil ini
menunjukkan bahwa seluruh butir atau item pernyataan dikatakan valid. Tabel 2. Rekapitulasi Hasil Pengujian Validitas Variabel Independensi (X2) Tabel 2. Rekapitulasi Hasil Pengujian Validitas Variabel Independensi (X2) Tabel 2. Rekapitulasi Hasil Pengujian Validitas Variabel Independensi (X2) Pengaruh Skeptisisme Profesional dan Independensi terhadap Ketepatan Pemberian Opini oleh Auditor | 1067 Berdasarkan tabel 2 terkait rekapitulasi diatas menunjukkan bahwa pernyataan-
pernyataan variabel independensi memiliki nilai r hitung lebih besar dari nilai r tabel 0,334. Selain itu, nilai signifikansi pada tabel tersebut kurang dari 0,05. Hasil tersebut menunjukkan
bahwa seluruh butir atau item pernyataan dikatakan valid. Tabel 3. Rekapitulasi Hasil Pengujian Validitas Variabel Ketepatan Pemberian Opini oleh
Auditor (Y) Tabel 3. Rekapitulasi Hasil Pengujian Validitas Variabel Ketepatan Pemberian O
Auditor (Y) Berdasarkan tabel 3 rekapitulasi di atas menunjukkan bahwa pernyataan-pernyataan
variabel ketepatan pemberian opini auditor mempunyai nilai r hitung lebih besar dari r tabel
0,334. Selain itu, nilai signifikansi dalam tabel tersebut kurang dari 0,05. Berdasarkan tabel 3
maka butir pernyataan dikatakan valid dan dapat digunakan sebagai alat ukur penelitian. Vol. 2 No. 2 (2022), Hal: 1063-1072 Uji Reliabilitas Tabel 4. Hasil Perhitungan Uji Reliabilitas Kuesioner Data Penelitian Tabel 4. Hasil Perhitungan Uji Reliabilitas Kuesioner Data Penelitian Berdasarkan Tabel 4 di atas menunjukkan variabel skeptisisme profesional,
independensi, dan ketepatan pemberian opini auditor masing-masing nilai cronbach’s alpha
sebesar 0,797 untuk variabel skeptisisme professional, lalu 0,779 untuk variabel independensi,
dan 0,636 untuk variabel ketepatan pemberian opini oleh auditor. Oleh karena itu, dapat
disimpulkan bahwa seluruh pernyataan dalam kuisioner ini dikatakan reliabel karena memiliki
nilai Cronbach’s Alpha lebih besar dari 0,60, sehingga setiap butir pernyataan yang digunakan
dapat memperoleh data yang konsisten serta dapat digunakan dalam pengujian hipotesis. Accountancy 1068 | Shafa Astari Azzahra, et al. Uji Normalitas Data Uji Normalitas Data
Tabel 5. Hasil Uji Normalitas Data Tabel 5. Hasil Uji Normalitas Data Tabel 5. Hasil Uji Normalitas Data Berdasarkan tabel 5 di atas dapat dilihat bahwa variabel skeptisisme profesional,
independensi, dan ketepatan pemberian opini auditor yang setiap masing-masingnya memiliki
nilai Asymp. Sig. (2-tailed) sebesar 0,200 untuk variabel skeptisisme profesional, 0,138 untuk
variabel independensi, dan 0,120 untuk variabel ketepatan pemberian opini. Oleh karena itu,
dapat disimpulkan bahwa data dalam penelitian ini terdistribusi secara normal, karena
mempunyai nilai Asymp. Sig. (2-tailed) lebih besar dari 0,05. Vol. 2 No. 2 (2022), Hal: 1063-1072 Pengaruh Skeptisisme Profesional dan Independensi terhadap Ketepatan Pemberian Opini oleh Auditor | 1069 1069 Uji Multikolinearitas Data
Tabel 6. Hasil Uji Multikolinearitas Berdasarkan tabel 6 di atas, semua variabel dalam penelitian ini diketahui memiliki
nilai tolerance lebih dari 0,1 (0,906 > 0,1) dan nilai VIF (variance inflation factor) kurang dari
10 (1.104 < 10). Oleh karena itu, dapat disimpulkan bahwa data dalam penelitian ini terbebas
dari gangguan multikolinearitas. Vol. 2 No. 2 (2022), Hal: 1063-1072 Uji Heteroskedastisitas Data Uji Heteroskedastisitas Data Sumber: Hasil Pengolahan SPSS Versi 23, 2022
Gambar 1. Uji Heteroskedastisitas Sumber: Hasil Pengolahan SPSS Versi 23, 2022
Gambar 1. Uji Heteroskedastisitas Sumber: Hasil Pengolahan SPSS Versi 23, 2022 Berdasarkan gambar di atas, grafik Scatterplot memperlihatkan bahwa tidak ada pola
tertentu yang jelas atau bulir-bulir yang ada tersebar (terdistribusi) di atas dan di bawah angka
nol pada sumbu Y. Sehingga dapat disimpulkan bahwa di dalam model regresi ini tidak terjadi
heteroskedastisitas, maka dari itu dapat digunakan untuk memprediksi ketepatan atau
keakuratan pemberian opini auditor berdasarkan variabel yang mempengaruhi opini tersebut
yaitu skeptisisme profesional dan independensi. Accountancy 1070 | Shafa Astari Azzahra, et al. Analisis Regresi Berganda
Tabel 7. Hasil Regresi Linear Berganda Analisis Regresi Berganda Tabel 7. Hasil Regresi Linear Berganda Berdasarkan Tabel 7 di atas, diketahui bahwa model persamaan untuk analisis regres
berganda adalah sebagai berikut: Berdasarkan Tabel 7 di atas, diketahui bahwa model persamaan untuk analisis regresi
berganda adalah sebagai berikut:
KPO = 11.637 + (-0,141) SP + 0,204 I + e Berdasarkan Tabel 7 di atas, diketahui bahwa model persamaan untuk analisis regresi
berganda adalah sebagai berikut:
KPO = 11.637 + (-0,141) SP + 0,204 I + e Uji Simultan (F) Uji Simultan (F)
Tabel 8. Hasil Pengujian Simultan Tabel 8. Hasil Pengujian Simultan Berdasarkan Tabel 8 di atas dapat dapat diketahui bahwa nilai F sebesar 3.336 dengan
nilai signifikansi sebesar 0,048 lebih kecil dari 0,05. Dengan demikian, dapat disimpulkan
bahwa X1 (skeptisisme profesional) dan X2 (independensi) keduanya berpengaruh terhadap
ketepatan dalam pemberian opini oleh auditor. Vol. 2 No. 2 (2022), Hal: 1063-1072 ISSN: 2828-254X Pengaruh Skeptisisme Profesional dan Independensi terhadap Ketepatan Pemberian Opini oleh Auditor | 1071 Uji Parsial (Uji t)
Tabel 9. Hasil Pengujian Parsial Uji Parsial (Uji t) 1. Pengaruh Skeptisisme Profesional terhadap Ketepatan Pemberian Opini oleh Auditor
Berdasarkan Tabel 9 di atas dapat diketahui bahwa skeptisisme profesional (X1) tidak
berpengaruh terhadap ketepatan pemberian opini auditor (Y). Nilai signifikansi untuk
variabel skeptisisme profesional sebesar 0,071 yang dimana lebih besar dari 0,05. Artinya Ha ditolak dan Ho diterima, sehingga dapat disimpulkan bahwa variabel
skeptisisme profesional tidak berpengaruh secara signifikan terhadap ketepatan
pemberian opini auditor. p
p
2. Pengaruh Independensi terhadap Ketepatan Pemberian Opini oleh Auditor
Berdasarkan Tabel 9 di atas, dapat diketahui bahwa variabel independensi (X2)
berpengaruh terhadap ketepatan pemberian opini auditor (Y). Kemudian, nilai
signifikansi variabel independensi adalah 0,029 yang dimana lebih kecil atau kurang
dari 0,05. Artinya Ha diterima dan Ho ditolak. Dengan demikan, dapat disimpulkan
bahwa variabel independensi berpengaruh positif dan signifikan terhadap ketepatan
atau keakuratan pemberian opini oleh auditor. p
p
2. Pengaruh Independensi terhadap Ketepatan Pemberian Opini oleh Auditor
Berdasarkan Tabel 9 di atas, dapat diketahui bahwa variabel independensi (X2)
berpengaruh terhadap ketepatan pemberian opini auditor (Y). Kemudian, nilai
signifikansi variabel independensi adalah 0,029 yang dimana lebih kecil atau kurang
dari 0,05. Artinya Ha diterima dan Ho ditolak. Dengan demikan, dapat disimpulkan
bahwa variabel independensi berpengaruh positif dan signifikan terhadap ketepatan
atau keakuratan pemberian opini oleh auditor. D.
Kesimpulan Berdasarkan pada data yang terkumpul serta hasil pengujian yang dilakukan oleh peneliti,
maka dapat ditarik kesimpulan sebagai berikut: Berdasarkan pada data yang terkumpul serta hasil pengujian yang dilakukan oleh peneliti,
maka dapat ditarik kesimpulan sebagai berikut: 1. Skeptisisme profesional tidak berpengaruh signifikan terhadap ketepatan ata
keakuratan pemberian opini oleh auditor. keakuratan pemberian opini oleh auditor. 2. Independensi berpengaruh positif signifikan terhadap ketepatan atau keakuratan dalam
pemberian opini yang diungkapkan oleh auditor di Kantor Akuntan Publik (KAP) Kota
Bandung. 2. Independensi berpengaruh positif signifikan terhadap ketepatan atau keakuratan dalam
pemberian opini yang diungkapkan oleh auditor di Kantor Akuntan Publik (KAP) Kota
Bandung. Koefisien Determinasi Koefisien Determinasi Accountancy
Tabel 10. Hasil Uji Koefisien Determinasi Tabel 10. Hasil Uji Koefisien Determinasi Tabel 10. Hasil Uji Koefisien Determinasi Tabel 10. Hasil Uji Koefisien Determinasi Accountancy Accountancy 1072 | Shafa Astari Azzahra, et al. 1072 | Shafa Astari Azzahra, et al. Berdasarkan Tabel 10 di atas dapat diketahui bahwa nilai koefisien angka R-square
sebesar 0,164 atau secara persentase sebesar 16,4%. Artinya skeptisisme profesional dan
independensi memiliki dan memberikan pengaruh sebesar 16,4% sedangkan sebesar 83,6%
dipengaruhi oleh variabel lain yang tidak diteliti atau diuji dalam penelitian ini. [4]
Hasna Iftinan, Syifa. 2022. Pengaruh Pengalaman Auditor dan Kompetensi terhadap
Pendeteksian Kecurangan Laporan Keuangan, Jurnal Riset Akuntansi, 2(1). [5]
Arens, Alvin A., et al. 2011. Auditing dan Jasa Assurance. Jakarta: Erlangga. Alih
Bahasa: Herman Wibowo. Editor: Wibi Hardani, dan Suryadi Saat. Vol. 2 No. 2 (2022), Hal: 1063-1072 Acknowledge g
Penulis mengucapkan dan menyampaikan terima kasih sebesar-besarnya kepada dosen
pembimbing yang telah bersedia untuk meluangkan waktu, tenaga serta pikiran untuk
memberikan nasihat dan bimbingan selama penyusunan dan penulisan laporan penelitian ini. Selain itu, peneliti mengucapkan terima kasih kepada semua pihak yang telah memberikan
doa, dukungan, bantuan, motivasi dan semangatnya kepada peneliti dalam menyelesaikan
skripsi ini, yang tidak dapat peneliti sebutkan satu persatu. Berdasarkan hal tersebut, semoga
semua doa, dukungan, dan bantuannya mendapatkan balasannya menjadi amal baik serta
mendapat pahala dan berkah dari-Nya. Besar harapan penulis agar laporan penelitian ini dapat
bermanfaat bagi pembaca, walaupun penulis menyadari bahwa dalam laporan skripsi ini masih
jauh dari sempurna. Namun penulis menyadari tiada gading yang tak retak, begitu pula dengan
hasil dari penyusunan laporan skripsi ini. Untuk itu, penulis sangat mengharapkan kritik dan
saran yang dapat membangun dan menjadi bahan pengetahuan bagi penulis agar dapat
menyelesaikan tugas selanjutnya jauh lebih baik. Daftar Pustaka
[1]
Sihotang, K. 2016. Etika Profesi Akuntansi. Yogyakarta: PT. Kanisius. [2]
Halim, Abdul. 2008. Auditing I. Dasar-dasar Audit Laporan Keuangan, Edisi Ketiga. Yogyakarta. UPP STIM YKPN. [3]
Agus triyanto, 2014. Pengaruh skeptisisme profesional auditor, situasi audit,
independensi, etika, keahlian dan pengalaman terhadap ketepatan pemberian opini
auditor di Kantor Akuntan Publik di wilayah Yogyakarta. Jurnal Akuntansi
Muhamadiyah. 1(3): h: 1-17
[4]
Hasna Iftinan, Syifa. 2022. Pengaruh Pengalaman Auditor dan Kompetensi terhadap
Pendeteksian Kecurangan Laporan Keuangan, Jurnal Riset Akuntansi, 2(1). [5]
Arens, Alvin A., et al. 2011. Auditing dan Jasa Assurance. Jakarta: Erlangga. Alih
Bahasa: Herman Wibowo. Editor: Wibi Hardani, dan Suryadi Saat. Daftar Pustaka Daftar Pustaka
[1]
Sihotang, K. 2016. Etika Profesi Akuntansi. Yogyakarta: PT. Kanisius. [2]
Halim, Abdul. 2008. Auditing I. Dasar-dasar Audit Laporan Keuangan, Edisi Ketig
Yogyakarta. UPP STIM YKPN. [3]
Agus triyanto, 2014. Pengaruh skeptisisme profesional auditor, situasi audit,
independensi, etika, keahlian dan pengalaman terhadap ketepatan pemberian opini
auditor di Kantor Akuntan Publik di wilayah Yogyakarta. Jurnal Akuntansi
Muhamadiyah. 1(3): h: 1-17 [3]
Agus triyanto, 2014. Pengaruh skeptisisme profesional auditor, situasi audit,
independensi, etika, keahlian dan pengalaman terhadap ketepatan pemberian opini
auditor di Kantor Akuntan Publik di wilayah Yogyakarta. Jurnal Akuntansi
Muhamadiyah. 1(3): h: 1-17 [4]
Hasna Iftinan, Syifa. 2022. Pengaruh Pengalaman Auditor dan Kompetensi terhadap
Pendeteksian Kecurangan Laporan Keuangan, Jurnal Riset Akuntansi, 2(1). [5]
Arens, Alvin A., et al. 2011. Auditing dan Jasa Assurance. Jakarta: Erlangga. Alih
Bahasa: Herman Wibowo. Editor: Wibi Hardani, dan Suryadi Saat. Vol. 2 No. 2 (2022), Hal: 1063-1072 ISSN: 2828-254X ISSN: 2828-254X
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TINGKAT KEPERCAYAAN PADA SISWA TERHADAP PELAYANAN GURU BK (BIMBINGAN KONSELING) DI SMP NEGERI 01 TURIKALE MAROS
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Isdiana Holida Bahary1, Mansyur Radjab2, Suparman Abdullah3 1Mahasiswa Sosiologi Fisip Universitas Hasanuddin, Makassar, Indonesia, email: isdianahb@gmail.com
2Dosen Pascasarjana Sosiologi Fisip Universitas Hasanuddin, Makassar, Indonesia, email: radjabmansyur@gmail.com
3Dosen Pascasarjana Sosiologi Fisip Universitas Hasanuddin, Makassar, Indonesia, email: mansosio87@yahoo.com A R T I C L E I N F O The background of this research was the existence of the Child Protection
Law (UU No. 23 of 2002 about Child Protection) creating a serious
dilemma for teachers in giving punishment to students who violate the
school rules. The impact of this dilemma was the teacher became
indecisive towards naughty students or those who disobeyed the school
rules. Teacher indecisiveness caused the teacher’s authority became lower
in front of students, especially among the naughty students. They
increasingly broke the school rules. This affected the trust of other students
to their teacher became very susceptible. This study aimed to analyze the
forms of the trust of respondent and the service of conseling teachers, the
perceptions of respondents’ trust towards conseling teachers, the influence
of students’ trust towards conseling teachers. This research used univarat
and bivarat analysis by using computer program SPSS version 24. The
students in this study were 78 students. The sampling technique used
disproportionately stratified random sampling method. The data collection
techniques used were questionnaires, observation, semi-structured
interviews and documentation study. Based on the calculation results
obtained there was a positive and significant relationship between service
and students’ trust. This means that the better the service variable, the
higher the student's trust, and vice versa, the less the service variable, the
lower the student's trust. How to Cite:
Bahary, I. H., Radjab, M., &
Abdullah, S. (2020).Tingkat
Kepercayaan Pada Siswa
Terhadap Pelayanan Guru BK
(Bimbingan Konseling) di
SMP Neheri 01 Turikale
Maros. Hasanuddin Journal of
Sociology (HJS), 2(1), 50-60. Kata Kunci :
Kepercayaan, Siswa,
Pelayanan Tingkat Kepercayaan Pada Siswa Terhadap Pelayanan Guru BK (Bimbingan
Konseling) di SMP Negeri 01 Turikale Maros The Level of Students’ Trust Towards Conseling Teacher Services (Conseling Guidelines) In SMP
Negeri 01 Turikale Maros Available online at journal.unhas.ac.id/index.php/HJS A B S T R A K Penelitian ini dilatarbelakangi olehadanya Undang Undang Perlindungan
Anak (UU No. 23 Tahun 2002 tentang Perlindungan Anak) menjadikan
dilema yang berat bagi para guru dalam memberikan hukuman bagi para
siswa yang melanggar aturan-aturan di sekolah. Dampak dari dilema HASANUDDIN JOURNAL OF SOCIOLOGY tersebut, akhirnya guru menjadi kurang tegas terhadap siswa yang nakal
atau melanggar tata tertib yang ada di sekolah. Ketidaktegasan guru
berdampak terhadap semakin rendahnya wibawa guru di hadapan
parasiswa, khususnya di kalangan siswa-siswa yang nakal. Mereka
semakin seenaknya melanggar tata tertib sekolah. Sehingga trust atau
kepercayaan siswa yang lain terhadap gurunya menjadi sangat
rentan.Penelitian ini bertujuan untukmenganalisis tentang bentuk
kepercayaan responden dan pelayanan guru BK, persepsi kepercayaan
responden terhadap pelayanan guru BK, pengaruh antara kepercayaan
siswa terhadap pelayanan guru BK. Penelitian ini menggunakan
analisisunivaratdanbivaratmenggunakan
bantuan
software
aplikasi
computer program SPSS versi 24.Siswa dalam penelitian ini berjumlah
78siswa. Teknik Sampling menggunakan metode disproportionate
stratified random sampling. Teknik pengumpulan data menggunakan
teknik melalui kuesioner (angket), observasi, wawancara semiterstruktur
dan studi dokumentasi. Berdasarkan hasil perhitungan diperoleh ada
pengaruh positif dan signifikan antara pelayanan dan kepercayaan siswa. Artinya apabila variabel pelayanan semakin baik, maka kepercayaan siswa
juga akan semakin tinggi, demikian pula sebaliknya, apabila variabel
pelayanan semakin kurang, maka kepercayaan siswa juga akan semakin
berkurang. 1. PENDAHULUAN Fenomena pendidikan berkembang seiring dengan kemajuan teknologi, informasi, ekonomi, dan
sosial budaya masyarakat. Pada masa lampau, hubungan antara guru dan murid dapat dikatakan
berjarak, sementara pada saat ini guru tidak hanya berperan sebagai orang dewasa yang mendidik, tapi
juga berperan sebagai orang tua yang mengasihi, dan sering kali juga menjadi tempat mencurahkan
persoalan yang dihadapi oleh para siswa. Apalagi dengan adanya guru BK (Bimbingan Konseling) yang
setiap harinya menangani siswa. Guru BK di tuntut untuk dapat menangani permasalahan-permasalahan
dalam perilaku menyimpang yang terjadi padasiswa di sekolah. Dengan begitu, kepercayaansiswa
adalah salah satu penunjang bagi guru BK untuk menanggapi dan membantu siswa untuk
menyelesaikan masalah-masalah yang terkait dengan perilaku siswa di sekolah. Menurut Falcone & Castelfranci, kepercayaanmerupakan suatu fenomena yang dinamis, hal ini
karena kepercayaan terjadi secara intrinsik pada suatu keadaan yang alamiah, dimana
kepercayaanmerupakan hal yang menyangkut masalah mental yang didasarkan oleh situasi seseorang
dan konteks sosialnya, misalnya ketika seseorang harus mengambil suatu keputusan, maka ia akan lebih
memilih keputusan berdasarkan pilihan dari orang-orang yang lebih dapat ia percayai dari pada yang
kurang ia percayai. Berbeda dengan jaman dulu, dimana guru BK adalah guru yang sangat ditakuti oleh setiap siswa
karena kegalakannya dalam mendisiplinkan murid-muridnya. Mereka takut untuk berbuat salah karena
hukuman yang diberikan biasanya berupa cubitan, pukulan, bahkan tamparan. Jikapun mereka
diberikan hukuman, mereka tidak berani untuk mengadu kepada orang tua bukan karena diancam oleh
guru tetapi karena takut mendapatkan hukuman tambahan. 51 P-ISSN: 2685-5348, E-ISSN: 2685-4333 Saat ini semenjak adanya Undang Undang Perlindungan Anak (UU No. 23 Tahun 2002 tentang
Perlindungan Anak) menjadikan dilema yang berat bagi para guru dalam memberikan hukuman bagi
para siswa yang melanggar aturan-aturan di sekolah. Karena hukuman berupa cubitan atau pukulan jika
dilihat pada saat ini, dapat dianggap sebagai bentuk penganiayaan kepada siswa. Tindakan hukuman
disiplin yang dilakukan oleh guru, yang pada dahulu kala dianggap biasa-biasa saja, kini mulai bergeser
dan malah dinilai sebagai bentuk pelanggaran Hak Asasi Manusia (HAM). Hal ini bisa dilihat dari
beberapa kasus yang sering diberitakan di beberapa media massa Indonesia, tentang guru yang dianggap
terlalu keras pada siswa. Guru harus menegakkan disiplin kepada para siswa, namun saat ini oleh pihak luar dalam hal ini
bisa saja dari orang tua siswa atau dari LSM pembela anak untuk tidak melakukan sentuhan fisik kepada
si anak, sentuhan fisik ini cenderung disalah artikan sebagai kekerasan pada anak. Hal tersebut diperkuat
dengan adanya guru yang terjerat kasus penganiayaan terhadap siswanya. 1. PENDAHULUAN Contohnya lagi kasus
Muhammad Samhudi, seorang guru SMP di Sidoarjo, Jawa Timur, yang memberi hukuman pada
siswanya dengan memukul dan mencubit, divonis 3 bulan dengan masa percobaan 6 bulan. Hakim
Pengadilan Negeri Sidoarjo, Jawa Timur menjatuhkan vonis terhadap Samhudi pada sidang yang
digelar Kamis, 4 Agustus 2016. Hakim menjatuhkan hukuman tiga bulan penjara dengan enam bulan
masa
percobaan. (sumber: https://regional.kompas.com/read/2016/07/01/17403801/sambudi.pak.guru.yang.disidang.karena.men
cubit.siswanya?page=all) Dampak dari dilema tersebut, akhirnya guru menjadi kurang tegas terhadap siswa yang nakal atau
melanggar tata tertib yang ada di sekolah. Ketidaktegasan guru berdampak terhadap semakin rendahnya
wibawa guru di hadapan parasiswa, khususnya di kalangan siswa-siswa yang nakal. Mereka semakin
seenaknya melanggar tata tertib sekolah. Sehingga trust atau kepercayaan siswa yang lain terhadap
gurunya menjadi sangat rentan. Tidak dapat dipungkiri bahwa banyaknya kasus kriminalisasi terhadap guru membuat guru menjadi
was-was ketika akan memberikan sanksi pelanggaran disiplin kepada siswa karena khawatir melanggar
Undang-undang Perlindungan anak. Akibatnya guru menjadi masa bodoh ketika melihat ada siswa yang
melanggar disiplin. Jika hal ini terus dibiarkan, maka akan menghambat pencapaian daritujuan pendidikan nasional,
yaitu berkembangnya potensi peserta didik agar menjadi manusia yang beriman dan bertakwa kepada
Tuhan Yang Maha Esa, berakhlak mulia, sehat, berilmu, cakap, kreatif, mandiri, dan menjadi warga
negara yang demokratis serta bertanggung jawab. 52 HASANUDDIN JOURNAL OF SOCIOLOGY Tetapi, ketika kita melihat lebih jelas peran dan fungsi guru BK sebenarnya selama ini kita keliru
bahwa guru BK haruslah orang yang tegas dan menyeramkan. Tetapi, ketika kita melihat lebih jelas peran dan fungsi guru BK sebenarnya selama ini kita keliru
bahwa guru BK haruslah orang yang tegas dan menyeramkan. Fenti (2010) mengungkapkan bahwa pendekatan guru BK sebenarnya lebih kepada Bimbingan dan
Konseling, dimana pendekatan melalui Bimbingan dan Konseling berbeda dengan pendekatan disiplin
yang memungkinkan pemberian sanksi untuk menghasilkan efek jera, pananganan siswa bermasalah
melalui Bimbingan Konseling sama sekali tidak menggunakan bentuk sangsi apapun, tetapi lebih
mengandalkan pada terjadinya kualitas hubungan interpersonal yang saling percaya di antara konselor
dan siswa yang bermasalah, sehingga setahap demi setahap siswa tersebut dapat memahami dan
menerima diri dan lingkungannya, serta dapat mengarahkan diri guna tercapainya penyesuaian diri yang
lebih baik. Bila hukuman tidak lagi menyentuh tubuh, lalu apa yang disentuh? Menurut Foucault (1975),
jawabannya sangat jelas, yakni jiwa.Hukuman harus menyentuh kedalam hati, pemikiran, kehendak,
dan kecenderungan. Seseorang yang bersalah dihukum dengan sistem hukuman baru, yakni hukuman
internal yang disesuaikan dengan perkembangan individu. Hukuman dimaksudkan untuk mengawasi individu, menetralkan bahayanya dan mengubah
kecendrungan buruknya. 2.2. Populasi dna Sampel Populasi adalah siswakelastiga di SMP Negeri 01 TurikaleMaros. Sampel sebanyak 78
siswayang dipilih secara disproportionate stratified random sampling. Siswakelastiga dipilih
sebagai sampel karena siswakelastigasudahlebihbanyakmendapatkanpelayanan yang diberikan
guru BK mulai dari baru masuk sekolah hingga akan mengikuti ujian akhir sekolah dan nasional. 2.3. Metode Pengumpulan Data Metode pengumpulan data yang digunakan dalam penelitian ini adalah dengan menyebar
kuesioner atau angket kepada 78 siswa, kemudian melakukan studi dokumentasi untuk mengetahui
informasi dan data-data yang mendukung penelitian ini. Dan peneliti melakukan observasi
pengamatan langsung kepada siswaSMP Negeri 01 TurikaleMaros. 2.4. Analisis Data Teknik analisis data dalam penelitian ini menggunakan analisis data kuantitatif secara univariat
dan bivariat menggunakan bantuan software aplikasi computer program SPSS versi 24. Teknik analisis data dalam penelitian ini menggunakan analisis data kuantitatif secara univariat
dan bivariat menggunakan bantuan software aplikasi computer program SPSS versi 24. 1. PENDAHULUAN Melalui pengetahuan individu tersebut, mekanisme hukuman yang sah
dilengkapi dengan pembenaran yang berdasarkan bukan hanya pada kesalahan, tetapi juga pada
individu, bukan hanya pada apa yang diperbuat, tetapi pada individu itu sendiri. Foucault menganjurkan agar analisis atas penghukuman dibersihkan dari anggapan umum bahwa
hukuman berfungsi untuk mengurangi kesalahan. Hukuman harus ditempatkan bukan hanya sebagai
mekanisme negatif yang menjadikan mampu menekan, menghalangi, mencegah, menghilangkan
kejahatan, tetapi harus dikaitkan juga dengan serangkaian mekanisme pelatihan, pengontrolan, yang
membawa akibat positif dan berguna. Demi tujuan itu, dikembangkan teknik pendisiplinan.disiplin
yang dimaksud adalah mengoreksi dan mendidik. Sehingga dengan adanya UU Perlindungan Anak ( UU No. 23 Tahun 2002 tentang Perlindungan
Anak) guru masih dapat mendisiplinkan siswa bermasalah tanpa memberikan sangsi fisik. Berdasarkan masalah yang telah penulis paparkan diatas, maka penulis tertarik untuk melakukan
penelitian dengan judul “Tingkat Kepercayaan Siswa Terhadap Pelayanan Guru BK (Bimbingan
Konseling) di Sekolah SMP Negeri 01 Turikale Maros”. 53 P-ISSN: 2685-5348, E-ISSN: 2685-4333 2.1. Lokasi dan Rancangan Penelitian Penelitian ini dilaksanakan di SMP Negeri 01 Turikale Maros yang berada di Kabupaten
Maros.Jenis Penelitian ini menggunakan metodekuantitatif. Penelitian ini dilaksanakan di SMP Negeri 01 Turikale Maros yang berada di Kabupaten
Maros.Jenis Penelitian ini menggunakan metodekuantitatif. 2. METODE PENELITIAN 2.1. Lokasi dan Rancangan Penelitian 2.1. Lokasi dan Rancangan Penelitian 3. HASIL PENELITIAN 3.1. Bentuk Kepercayaan Responden dan Pelayanan Guru BK 3.1. Bentuk Kepercayaan Responden dan Pelayanan Guru BK 3.1. Bentuk Kepercayaan Responden dan Pelayanan Guru BK Menurut Bryk dan Schneider, (2003) orang yang memiliki trust ditandai dengan: 1. Consistency, yaitu adanya ketetapan dalam memberikan pesan kepada orang lain tanpa
membedakan satu sama lain. Dengan demikian tingkat keyakinan seseorang akan semakin
besar karena adanya rasa aman dari ketetapan tersebut yang menghasilkan suatu
kepercayaan. 2. Compassion, yaitu kepedulian yang tinggi penting dalam hubungan saling percaya. Dengan
saling berkasih saying menyisaratkan bentuk perlindungan sehingga tidak akan muncul
perasaan merugikan orang lain. 3. Communication, yaitu berfokus pada bagaimana berbagi informasi yang mana informasi
tersebut tidakakan dieksploitasi bebas. Dengan kata lain, hal ini mengacu pada keterbukaan
sebagai strategi dalam menjaga kerahasiaan yang bersifat pribadi. 4. Competency, yaitu adanya tanggung jawab dan konsitensi seseorang dalam suatu pekerjaan 54 HASANUDDIN JOURNAL OF SOCIOLOGY dan seberapa baik hasil yang diperoleh. dan seberapa baik hasil yang diperoleh. Menurut David Wijaya (2012) mengutip pernyataan Parasuraman dkk (1998), terdapat lima
dimensi SERVQUAL yang dikenal dengan istilah RATER. RATER ini dapat diterapkan dalam
dunia pendidikan dan terdiri atas unsur-unsur berikut ini:
1. Reliability (Keandalan) Menurut David Wijaya (2012) mengutip pernyataan Parasuraman dkk (1998), terdapat lima
dimensi SERVQUAL yang dikenal dengan istilah RATER. RATER ini dapat diterapkan dalam
dunia pendidikan dan terdiri atas unsur-unsur berikut ini:
1. Reliability (Keandalan) Reliability yaitu keandalan/ kemampuan sekolah untuk menyediakan jasa Pendidikan
sesuai dengan janji secara akurat dan terpercaya. Kinerja sesuai harapan pelanggan jasa
Pendidikan berupa ketepatan waktu, pelayanan yang seragam untuk setiap pelanggan jasa
Pendidikan tanpa kesalahan, sikap simpatik dan keakuratan yang tinggi. 2. Assurance (Jaminan) Assurance yaitu pengetahuan, kesopansantunan serta kemampuan karyawan sekolah untuk
menumbuhkan rasa percaya pelanggan jasa Pendidikan pada sekolah, meliputi komunikasi,
kepercayaan, keamanan, kompotensi dan sopan santun. Tangible adalah kemampuan sekolah untuk menunjukkan keberadaan dirinya pada pihak
eksternal sekolah, meliputi fasilitas fisik (gedung, Gudang dan lain-lain), perlengkapan dan
peralatan pendidikan yang digunakan serta penampilan karyawan sekolah. 4. Empathy (Empati) Sekolah mampu memberikan perhatian yang tulus dan pribadi kepada pelanggan jasa
Pendidikan dengan memahami keinginan pelanggan jasa pendidikan. Sekolah juga diharapkan
memiliki pengertian dan pengetahuan tentang pelanggan jasa pendidikan, memahami
kebutuhan pelanggan jasa pendidikan secara khusus, sertamemilikiwaktuoperasijasapendidikan
yang nyaman bagi pelanggan jasa pendidikan. Responsivines (Ketanggapan), Responsivines yaitu kebijakan untuk membantu serta memberikan jasa kependidikan yang
cepat dan tepat kepada pelanggan jasa pendidikan. Responsivines yaitu kebijakan untuk membantu serta memberikan jasa kependidikan yang
cepat dan tepat kepada pelanggan jasa pendidikan. 3.2. Persepsi Kepercayaan Responden terhadap Pelayanan Guru BK 3.2. 3.1. Bentuk Kepercayaan Responden dan Pelayanan Guru BK Persepsi Kepercayaan Responden terhadap Pelayanan Guru BK 3.2. Persepsi Kepercayaan Responden terhadap Pelayanan Guru BK Berikut tabel yang memuat hasil rekapitulasi skor total persepsi responden tentang keempat
aspek kepercayaan: konsistensi, kepedulian, komunikasi, dan kompetensi. Berikut tabel yang memuat hasil rekapitulasi skor total persepsi responden tentang keempat
aspek kepercayaan: konsistensi, kepedulian, komunikasi, dan kompetensi. Tabel 1. Rekapitulasi Skor Total Persepsi Responden tentang Kepercayaan
Siswa terhadap Guru BK di SMP Negeri 01 Turikale Maros`
No
Aspek Kepercayaan
JumlahSkor
%
1
Konsistensi
2113
25,0
2
Kepedulian
2208
26,2
3
Komunikasi
1919
22,8 55 P-ISSN: 2685-5348, E-ISSN: 2685-4333 4
Kompetensi
2195
26,0
Jumlah
8435
100,00 Berdasarkan Tabel 4.5 terlihat bahwa skor total persepsi responden tentang kepercayaan siswa
terhadap guru BK sebesar 8435 (100,00%). Kemudian dibuat kategorisasinya : sangat tinggi, tinggi,
rendah, dan sangat rendah. Bahwa hal ini menunjukkan dari empat aspek konsistensi, kepedulian,
komunikasi, dan kompetensi menghasilkan tinggkat kepercayaan yang tinggi. Responden
berpersepsi tentang kepercayaan dengan menghitung interval tiap-tiap kategori dengan cara
menghitung : Skor Tertinggi
= Nilai Tertinggi x Jumlah Soal x Jumlah Responden
= 4 x 40 x 78 = 12.480 Skor Tertinggi
= Nilai Tertinggi x Jumlah Soal x Jumlah Responden Skor Terendah
= Nilai Terendah x Jumlah Soal x Jumlah Responden Skor Terendah
= Nilai Terendah x Jumlah Soal x Jumlah Responden
= 1 x 40 x 78 = 3120 = (nilai tertinggi- nilai terendah) : Kelas Interval = (nilai tertinggi- nilai terendah) : Kelas Interval = (12.480 – 3120) : 4 = 2340 = (12.480 – 3120) : 4 = 2340 = (12.480 – 3120) : 4 = 2340 = (12.480 – 3120) : 4 = 2340 Berdasarkan rumus di atas, didapatkan hasil skor interval kepercayaan siswa adalah 2340. Dari
hasil interval tersebut, maka: 10.140 – 12.480 = Sangat Tinggi
7800 – 10.139
= Tinggi
5460 – 7799
= Rendah
3120 - 5459
= Sangat Rendah Secara keseluruhan, rekapitulasi responden tentang kepercayaan siswa terhadap guru BK di
SMP Negeri 01 Turikale Maros adalah 8435 yang berarti tingkat kepercayaan siswa tinggi terhadap
guru BK di SMP Negeri 01 Turikale Maros. engaruh antara Kepercayaan Siswa Terhadap Pelayanan Guru BK 3.3. Pengaruh antara Kepercayaan Siswa Terhadap Pelayanan Guru BK Berdasarkan analisis regresi yang dilakukan dengan menggunakan bantuan SPSS versi 24
diperoleh output sebagai berikut: Tabel 2. Hasil Uji Korelasi Variabel Pelayanan dan Kepercayaan Tabel 2. Hasil Uji Korelasi Variabel Pelayanan dan Kepercayaan Tabel 2. Hasil Uji Korelasi Variabel Pelayanan dan Kepercayaan
Correlations
Pelayanan
Kepercayaan
Pelayanan
Pearson Correlation
1
.622**
Sig. (2-tailed)
.000
N
78
78
Kepercayaan
Pearson Correlation
.622**
1
Sig. (2-tailed)
.000
N
78
78
**. Correlation is significant at the 0.01 level (2-tailed). Berdasarkan hasil perhitungan diperoleh nilai koefisien korelasi R = 0,622, dan Probabilitas (P) =
0,000 (dimana P < 0,01), maka dapat disimpulkan bahwa ada pengaruh positif dan signifikan antara
pelayanan dan kepercayaan siswa. Artinya apabila variabel pelayanan semakin baik, maka kepercayaan
siswa juga akan semakin tinggi, demikian pula sebaliknya, apabila variabel pelayanan semakin kurang,
maka kepercayaan siswa juga akan semakin berkurang. 3.2. Persepsi Kepercayaan Responden terhadap Pelayanan Guru BK Persentase posisi skor total kepercayaan responden berdasarkan aspek kompetensi pada
diagram tabel diperoleh melalui perhitungan berikut : Persentase posisi skor total kepercayaan responden berdasarkan aspek kompetensi pada
diagram tabel diperoleh melalui perhitungan berikut : (%) =
Total skor pada Kepercayaan
skor maksimum
x 100%
(%) =
8435
12480 x 100%
(%) = 67,6 % (%) =
Total skor pada Kepercayaan
skor maksimum
x 100%
(%) =
8435
12480 x 100%
(%) = 67,6 % (%) =
Total skor pada Kepercayaan x 100 Proses perhitungan yang dilakukan, menghasilkan skor jawaban responden. Secara keseluruhan
skor jawaban responden terhadap kepercayaan sebesar 8435 (67,6 %) termasuk dalam kategori
positif maksudnya bahwa responden cukup percaya kepada guru BK. Tetapi masih ada 32,,4% 56 HASANUDDIN JOURNAL OF SOCIOLOGY responden termasuk dalam kategori negatif, ini menunjukkan bahwa masih ada Sebagian responden
yang kurang percaya terhadap guru BK. Hal ini, dapat diartikan bahwa kepercayaan siswa terhadap guru BK dikategorikan tinggi (67,6
%). Meskipun demikian, masih ada Sebagian kecil responden yang tidak percaya terhadap guru BK
(32,4 %). Jika dilihat dari persentase negatif, kita sebaiknya tidak mengabaikan hasil tersebut
karena kepercayaan responden terhadap guru BK apa lagi jika di dalamnya terdapat responden yang
bermasalah. 4. KESIMPULAN Berdasarkan hasil perhitungan diperoleh nilai koefisien korelasi R = 0,622, dan Probabilitas (P) =
0,000 (dimana P < 0,01), maka dapat disimpulkan bahwa ada pengaruh positif dan signifikan antara
pelayanan dan kepercayaan siswa. Artinya apabila variabel pelayanan semakin baik, maka kepercayaan
siswa juga akan semakin tinggi, demikian pula sebaliknya, apabila variabel pelayanan semakin kurang,
maka kepercayaan siswa juga akan semakin berkurang. Ada beberapa siswa yang ditemukan masih kurang dalam melihat pelayanan BK. takut dalam
menyampaikan pendapat, tidak berani mengungakapkan pendapat.Pelaksanaan layanan bimbingan
konseling tersebut sudah berjalan namun belum dapat dikatakan sempurna dikarenakan kurangnya 57 P-ISSN: 2685-5348, E-ISSN: 2685-4333 fasilitas yang mampu menunjang keberhasilan pelaksanaan layanan tersebut, dan kurangnya jam BK
disekolah tersebut. Pelaksanaan layanan tersebut dapat dilakukan apabila adanya waktu luang dan hal
itu menjadi hal yang urgensi dan mesti diselesaikan dengan cepat. Layanan bimbingan konseling yang diberikan oleh guru pembimbing kepada siswa,dengan
mengikuti tahapan-tahapan bimbingan kelompok, meskipun adanya terjadi hambatan-hambatan seperti
sering ditemui siswa kurang fokus mengikuti atau mendengarkan bimbingan yang disampaikan. Wijaya, David. (2012). Pemasaran Jasa Pendidikan. Jakarta: Salemba Empat. 5. SARAN Peneliti akan memberikan saran yang akan menjadi masukan dan pertimbangan untuk
peserta didik dalam tingkat Kepercayaan Siswa SMP 01 Turikale Maros terhadap pelayanan BK,
antara lain : 1. Kepala sekolah selaku pemegang kebijakan otonomi sekolah hendaknya: a. Memberikan ruang yang cukup kepada konselor untuk mengembangkan diri dengan
usaha-usaha peningkatan kompetensinya masing-masing dan meningkatkan pengadaan
sarana prasarana BK b. Menyelenggarakan
kegiatan-kegiatan
workshop
untuk
konselor
dalam
rangka
pengembangan dan peningkatan kompetensi yang berdampak meningkatnya mutu dan
efektititas layanan bimbingan konseling. 2. Konselor selaku pembimbing konseling SMP 1 Turikale Maros, diharapkan: a. Selalu meningkatkan kompetensi, mengikuti perkembangan layanan konseling dengan
mengikuti pelatihan atau workshop yang terkait dengan efektifitas bimbinga nkonseling. b. Terus termotivasi untuk selalu memberi layanan bimbingan dan konseling demi tercapainya
tujuan pembelajaran serta meningkatnya mutu pendidikan. 3. Kepada siswa dan siswi di SMP 1 Turikale Maros Hendaknya mempunyai perhatian terhadap dirinya sendiri misalnya apabila ada masalah
yang sulit dipecahkan secara sendirilah hendaknya berkonsultasi pada guru atau orang lain yang
dapat membantu memecahkan masalah yang dihadapinya. Selain itu siswa dan siswi di
harapkan dapat lebih memotivasi diri untuk lebih giat belajar dan selalu berusaha semaksimal
mungkin dalam belajar baik belajar sendiri maupun belajar kelompok. 58 58 HASANUDDIN JOURNAL OF SOCIOLOGY DAFTAR PUSTAKA Abdullah Idi. (2011). Sosiologi Pendidikan. Jakarta: PT Raja Grafindo Persada. Alfitri. (2011). Community Development: Teori dan Aplikasi. Yogyakarta: Pustaka Pelajar
Anthony S. Bryk and Barbara Schneider. (2003) Trust in School: A Core Resource for School Reform
Astrid S. (2003). Pengantar Sosiologi dan Perubahan Sosial . Grafindo Persada. Jakarta
Coleman J. S. 1988. Social Capital in The Creation of Human Capital. Amercan Journal of Sociology
94 (supplement): S95-S120
D
P
t
T
i S
i l
i J k t
F j
D
i Abdullah Idi. (2011). Sosiologi Pendidikan. Jakarta: PT Raja Grafindo Persada. Alfitri. (2011). Community Development: Teori dan Aplikasi. Yogyakarta: Pustaka Pelajar
Anthony S. Bryk and Barbara Schneider. (2003) Trust in School: A Core Resource for School Reform
Astrid S. (2003). Pengantar Sosiologi dan Perubahan Sosial . Grafindo Persada. Jakarta
Coleman J. S. 1988. Social Capital in The Creation of Human Capital. Amercan Journal of Sociology
94 (supplement): S95-S120 Astrid S. (2003). Pengantar Sosiologi dan Perubahan Sosial . Grafindo Persada. Jakarta Coleman J. S. 1988. Social Capital in The Creation of Human Capital. Amercan Journal of Sociology
94 (supplement): S95-S120 Damsar. Pengantar Teori Sosiologi. Jakarta: Fajar Dunia Fadjar. (1999). Reorientasi Pendidikan Islam. Jakarta: Fajar Dunia enti Hikmawati. (2010). Woolfolk hoy, A. &Tschannen-Moran (1999). Implications of cognitive approaches to peer learning
for teacher education.In A.M. O’Donnell & A. King (Eds.) Cognitive Perspectives on peer
learning.Mahwah, NJ: Lawrence Erlbaum. Zulganef dan Murni.(2008). Hubungan Kepuasan dan Kepercayaan Mahasiswa terhadap Lembaga
Pendidikan Tinggi dengan Keinginan untuk Membujuk Calon Mahasiswa melanjutkan Studi ke
Perguruan Tinggi. Woolfolk Hoy, A. &Tschannen-Moran (2001). Collaboration and the need for trust. Journal of
educational Administration, 39, 308-331. Zainuddin Maliki. (2008). Sosiologi Pendidikan. Yogyakarta: Gajah Mada University Pre 5. SARAN Bimbingan dan Konseling.Jakarta: PT Raja Grafindo Persada. (07 http://regional.liputan6.com/read/2575357/5-konflik-pelik-guru-versus-siswa-berujung-pidana
(07
September 2017) http://www.ascd.org/publications/educational-leadership/mar03/vol60/num06/Trust-in-School@-A-
Core-Resource-for-School-Reform.aspx (06 September 2017) Jousairi Hasbullah. (2006). Social Capital. Jakarta: MR-United Press Jakarta Laksamana, Arsono. (2002). Pengaruh Saling Ketergantungan, Kepercayaan, dan Keselarasan Tujuan
Terhadap Kooperasi dan Kinerja Perusahaan Manufaktur pada Hubungan Kontraktual dengan
Pemasoknya”. Jurnal Akuntansi & Keuangan. Mansyur Radjab. (2014). Bahan Ajar Metode Penelitian Kualitatif. Jurusan Sosiologi FISIP UNHAS. Makassar. Mansyur Radjab. (2014). Bahan Ajar Sosiologi Pendidikan. Jurusan Sosiologi FISIP UNHAS. Makassar Moh Nazir. (2009). Metode Penelitian Kuantitatif. Bandung: Alfabeta. Moh Nazir. (2009). Metode Penelitian Kuantitatif. Bandung: Alfabeta. Muhammad Noor Syam. (1986). Filsafat Pendidikan dan DasarPendidikan Pancasila. Surabaya:
Usaha Nasional. Nanang Martono. (2010). Metode PENELITIAN Kuantitatif. Jakarta: Karisma Putra Utama Offset. Ojha Amithb, M.P. Gupta. (1998). Does E-Government Enhance Trust ingovernment. Computer
Society of India. Ratnasari,
Murni. Optimalisasi
Modal
Sosialdalam
Pembangunan
KesejahteraanPekerjaOutsourchingdi PT. Aneka Tambang Unit BisnisPertambanganNikelPomala
Sulawesi Tenggara.(2014). TesisSosiologi.UniversitasHasanuddin. Sugiyono. (2007). Metode Penelitian Kuantitatif Kualitatif dan R&D. Bandung: Alfabeta Sugiyono. (2008). Metode Penelitian Kuantitatif Kualitatif dan R&D. Bandung: Alfabeta Sugiyono.(2011). Metode Penelitian Kuantitatif Kualitatif dan R&D. Bandung: Alafabeta Tschannen-Moran, M., Hoy, A. W., & Hoy, W.K, (1998).Teacher efficacy: Its meaning and measure. Review of Educational research, 68(2), 189-190 Wijaya, David. (2012). Pemasaran Jasa Pendidikan. Jakarta: Salemba Empat. 59 P-ISSN: 2685-5348, E-ISSN: 2685-4333 uddin Maliki. (2008). Sosiologi Pendidikan. Yogyakarta: Gajah Mada University Press. Zulganef dan Murni.(2008). Hubungan Kepuasan dan Kepercayaan Mahasiswa terhadap Lembaga
Pendidikan Tinggi dengan Keinginan untuk Membujuk Calon Mahasiswa melanjutkan Studi ke
Perguruan Tinggi. Zulganef dan Murni.(2008). Hubungan Kepuasan dan Kepercayaan Mahasiswa terhadap Lembaga
Pendidikan Tinggi dengan Keinginan untuk Membujuk Calon Mahasiswa melanjutkan Studi ke
Perguruan Tinggi. 60
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PENINGKATAN KOMPETENSI TENAGA PENDIDIK MUSLIMAT NU SURABAYA DALAM PENGUASAAN TEKNOLOGI INFORMASI DAN KOMUNIKASI (TIK)
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PENINGKATAN KOMPETENSI TENAGA PENDIDIK MUSLIMAT NU SURABAYA
DALAM PENGUASAAN TEKNOLOGI INFORMASI DAN KOMUNIKASI (TIK) Udik Pudjianto1*, Anang Kukuh Adisusilo2, Lestari Retnawati3, Nia Saurina4 1BPPAUD & DIKMAS Jawa Timur
2,3,4Universitas Wijaya Kusuma Surabaya
Jalan Gebang Putih No. 10 Keputih, Surabaya, 60117
*Email: udik.its@gmail.com Abstrak Seorang tenaga pendidik harus memiliki kompetensi karena seorang tenaga pendidik memiliki
kewajiban untuk mencerdaskan anak bangsa, bukan hanya cerdas secara fisik tetapi secara
emosional juga. Sehingga tugas tenaga pendidik adalah mendidik bukan hanya mengajar, karena
mendidik memilki makna yang lebih luas dan lebih kompleks dari pada mengajar. Lembaga
Muslimat NU Surabaya merupakan salah satu pilar yang menunjang pendidikan dengan
memberikan tenaga pendidik yang professional. Program kegiatan Masyarakat (PkM) kali ini
adalah melaksanakan peningkatan kompetensi tenaga pendidik Muslimat NU Surabaya melalui
aplikasi FIlmora untuk menunjang Kegiatan Belajar Mengajar (KBM). Adapun metode yang
digunakan adalah: ceramah, diskusi, dan praktek/latihan melakukan pendokumentasian KBM
menggunakan Filmora. Setelah dilakukan PkM diharapkan dapat menumbuhkan motivasi besar dari
para tenaga pendidik ditengah beragam kesulitan yang mereka hadapi untuk dapat menguasai TIK
bagi proses belajar mengajar di kelas.. Tumbuh motivasi besar dari para tenaga pendidik ditengah
beragam kesulitan yang mereka hadapi untuk dapat menguasai TIK bagi proses belajar mengajar
di kelas. Adapun lima indikator yang dijadikan indikator kualitas pembelajaran adalah: suasana
pembelajaran; kemampuan tim PkM dalam menyampaikan pelajaran; penyajian materi;
pembelajaran bersifat riil; dan menggunakan teknologi pembelajaran untuk kegiatan KbM, dimana
kriteria yang didapatkan mayoritas bersifat Sangat Baik. Kata kunci: Muslimat NU Surabaya, tenaga pendidik, penguasaan TIK Jurnal ADIMAS Jurnal ADIMAS Jurnal ADIMAS Keywords: Muslimat NU Surabaya, educator, Be Advance in ICT, TLA ADIMAS 2020| Jurnal Pengabdian Masyarakat 1. PENDAHULUAN Teknologi informasi dan komunikasi merupakan suatu padanan yang tidak terpisahkan yang
mengandung pengertian luas tentang segala kegiatan yang terkait dengan pemrosesan, manipulasi,
pengelolaan, dan transfer/pemindahan informasi antar media. Adapun menurut Munir (Munir, 2009)
bahwa teknologi informasi dan komunikasi meliputi berbagai aspek yang melibatkan teknologi,
rekayasa dan teknik pengelolaan yang digunakan dalam pengendalian dan pemrosesan informasi
serta penggunaanya, komputer dan hubungan mesin (komputer) dan manusia, dan hal yang berkaitan
dengan sosial, ekonomi dan kebudayaan. Kemajuan ilmu dan teknologi informasi telah banyak mengubah cara pandang dan gaya hidup
masyarakat Indonesia dalam menjalankan aktivitas dan kegiatannya. Keberadaan dan peranan
teknologi informasi dalam sistem pendidikan telah membawa era baru perkembangan dunia
pendidikan, tetapi perkembangan tersebut belum diimbangi dengan peningkatan sumber daya
manusia yang menentukan keberhasilan dunia pendidikan di Indonesia pada umumnya. Hal ini lebih
desebabkan masih tertinggalnya sumber daya manusia kita untuk memanfaatkan teknologi informasi
dalam proses pendidikan tersebut (Santoso, 2014). Institusi pendidikan di Indonesia mulai berlomba-
lomba mememanfaatkan Teknologi Informasi dan Komunikasi (TIK) untuk pendidikan dengan
membangun infrastruktur hardware, jaringan internet, pengadaan sofware dan lain sebagainya, yang
semua itu dilakukan dalam usaha memenuhi kebutuhan akan metode pembelajaran yang lebih efektif
dan efisien. Pelatihan-pelatihan dengan pemanfaatan aplikasi komputer pun sering diselenggarakan
seperti; Intelligent Tutoring System (ITS), Computer Basad Training (CBT), dan e-Learning System
(Hariningsih, 2005) Beragam kemampuan TIK yang luar biasa sudah seharusnya dimanfaatkan dalam dunia
pendidikan dalam kerangka melahirkan sistem pendidikan yang lebih baik, baik dalam aspek sarana
prasarana, peningkatan profesionalisme kualitas sumber daya manusia pendidik (guru) maupun
menghasilkan anak didik yang berkualitas (Elston, 2007) Pelaksanaan pembelajaran berbasis TIK tentunya harus didukung adanya ketersediaan fasilitas
TIK. Oleh sebab itu di dalam peraturan pemerintah No. 19 Tahun 2005 tentang standar sarana dan
prasarana ditetapkan harus menggunakan sarana berbasis TIK dalam proses pembelajaran. Menurut
Pustekkom (Pustekkom, 2009), sarana TIK dibedakan atas: (1) komponen TIK, dan (2) fasilitas TIK. Ketersediaan komponen serta fasilitas TIK ini mendapat dukungan dari pemerintah dalam upaya
meningkatkan kualitas dan akses terhadap layanan pendidikan. Kebijakan Departemen Pendidikan
memprioritaskan bagi Sekolah Menengah Atas dan ditargetkan 1000 sekolah SMA di Indonesia pada
tahun 2010 sudah terhubung dengan jaringan internet dengan nama Schoolnet. Kompetensi adalah salah satu tujuan utama program pendidikan profesional. Menurut Parry
(Dikutip dalam Applin, 2011) mendefinisikan kompetensi sebagai kumpulan pengetahuan, sikap,
dan keterampilan terkait (kompetensi) yang terkait dengan kinerja. di tempat kerja dan dapat diukur
terhadap standar yang diterima dengan baik. Abstract An educator must have competence because an educator has an interest in educating the nation's
children, not only physically intelligent but also emotionally. The task of educators is to educate not
only to teach, because educating are more complex meaning than teaching. Muslimat NU Surabaya
is one of the pillars that support education by providing professional teaching staff. The Community
Activity Program (CAP) this time is increasing the competence of educators of Muslimat NU
Surabaya through the FIlmora application to support Teaching and Learning Activities (TLA). The
method used is: lectures, discussions, and practices / exercises to document KBM using Filmora. This activity expected a great motivation of the teaching staff in the midst of the various difficulties
they face in be advance in ICT for teaching and learning in the classroom. Growing great motivation
from the teaching staff amid diverse teaching in the classroom. While the five indicators made
indicators of learning quality are: learning space; the ability of the CAP team in preparing lessons;
presentation material; real learning; and use learning technology for TLA activities, where those
criteria obtained Very Good. ADIMAS 2020| Jurnal Pengabdian Masyarakat 22 Jurnal ADIMAS Jurnal ADIMAS Jurnal Pengabdian Masyarakat| ADIMAS 2020 2. METODE Kegiatan PKM menggunakan metode dalam bentuk pelatihan keterampilan melalui ceramah,
demonstrasi dan tanya jawab serta praktek yang dilaksanakan selama 1 hari. Adapun tahapan-tahapan
dalam pelaksanaan kegiatannya : a. Ceramah digunakan untuk menyampaikan pengetahuan secara umum tentang pengenalan aplikasi
video editing, perkenalan FilmoraGo, bagaimana pengaturan konten video, bagaimana
mempublikasikan video. a. Ceramah digunakan untuk menyampaikan pengetahuan secara umum tentang pengenalan aplikasi
video editing, perkenalan FilmoraGo, bagaimana pengaturan konten video, bagaimana
mempublikasikan video. b. Praktek digunakan untuk memberikan ketrampilan langsung kepada peserta untuk membuat video
dari bahan yang sudah disiapkan sebelumnya. Peserta dapat membuat dari foto kegiatan
pembelajaran dan video singkat pembelajaran yang mereka lakukan. Konten yang mereka buat
ini diharapkan dapat menjadi bahan untuk mempromosikan lembaganya. c. Tanya jawab dapat langsung digunakan untuk melengkapi hal-hal yang belum terakomodasi pada
saat kegiatan pelatihan tsersebut. d. Presentasi hasil editing video kegiatan belajar mengajar yang telah dibuat dilakukan dengan
melibatkan sebagian peserta pelatihan sebagai bahan diskusi. e. Kegiatan evaluasi hasil akhir bertujuan untuk mengetahui sejauhmana kemampuan peserta dalam
menyerap materi yang telah diberikan. Kegiatan PKM telah diikuti oleh tujuh puluh lima orang tenaga pendidik Muslimat NU
Surabaya. 1. PENDAHULUAN demikian juga Menurut Alfaro-LeFevre (Dikutip dalam
Haraldseid, 2015), mengembangkan konsep kompetensi dengan mengemukakan bahwa kompetensi
tercermin dalam pengetahuan, pemikiran kritis, keterampilan teknis dan interpersonal yang
tunjukkan seorang profesional kepada situasi praktik profesional. Dalam menunjang pencapaian
kompetensi banyak cara yang digunakan oleh institusi pendidikan dalam melaksanakan
pembelajaran di kelas seperti ceramah, walaupun pada akhir-akhir ini pendidikan profesi yang sangat
menekankan pencapaian kompetensi yang terukur telah banyak dikurangi dengan menggantinya
dengan metode clinical skill laboratorium oleh Haraldseid (Haraldseid. 2015) yaitu peserta didik
dibawah pada pengalaman nyata pada pembelajaran berbasis studi kasus dalam menyelesaikan kasus
– kasus seputar penguasaan teknologi informasi dan komunikasi. Lembaga Muslimat NU Surabaya merupakan salah satu pilar yang menunjang pendidikan
dengan memberikan tenaga pendidik yang professional. Program kegiatan Masyarakat (PkM) kali
ini membahas peningkatan kompetensi tenaga pendidik Muslimat NU Surabaya melalui aplikasi
FIlmora untuk menunjang Kegiatan Belajar Mengajar (KBM). Filmora sebagai salah satu aplikasi editing video yang cukup ringan dari file yang dibawanya
artinya tidak membutuhkan ruang (space) hardisk yang besar. Menggunakan filmora ini
membutuhkan waktu yang relatif cepat dalam melakukan editing video dari video proses kegiatan
pembelajaran yang dilakukan lembaga pendidikan. Filmora juga memiliki fitur-fitur yang ringkas Jurnal Pengabdian Masyarakat| ADIMAS 2020 23 23 Jurnal ADIMAS dan mudah dioperasionalkan. Selain itu juga aplikasi ini memili efek-efek yang membuat video hasil
editing user menjadi semakin menarik. dan mudah dioperasionalkan. Selain itu juga aplikasi ini memili efek-efek yang membuat video hasil
editing user menjadi semakin menarik. ADIMAS 2020| Jurnal Pengabdian Masyarakat 3. HASIL DAN PEMBAHASAN Kegiatan ini bertujuan untuk memberikan pengetahuan dan keterampilan berupa pemanfaatan
TIK dalam Pembelajaran bagi tenaga pendidik Muslimat NU Surabaya dilaksanakan dengan
meminjam ruangan pertemuan Muslimat NU Surabaya. Melalui kegiatan pelatihan ini diharapkan
para tenaga pendidik dapat memperoleh pengetahuan, pemahaman dan keterampilan menggunakan
teknologi informasi dan komunikasi bagi optimalisasi proses belajar mengajar di kelas. Kegiatan
pelatihan ini direncanakan diikuti sekitar 65 orang tenaga pendidik. Selain itu hadir pula Pimpinan
organisasi Muslimat NU Surabaya, Kepala Sub Bidang Pendidikan, yang merangkap menjadi
peserta. Kegiatan pelatihan ini diawali dengan penyampaian materi melalui metode ceramah
mengenai pengenalan TIK serta bagaimana TIK dapat dimanfaatkan bagi proses pembelajaran serta
ragam aplikasi yang dapat digunakan untuk membantu para tenaga pendidik dalam pembelajarannya. P d
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Pada pemaparan materi ini, sudah terlihat bagaimana peserta merespon dengan positif, dimana
terkadang disela-sela pemaparan ada pertanyaaan yang muncul dari peserta pelatihan. Hal ini
dikarenakan sebagian peserta memaksimalkan aplikasi-aplikasi tersebut, tetapi mereka menunjukan
keinginan belajarnya dengan sesekali bertanya. Pada sesi ini juga ditawarkan aplikasi yang akan di
fokuskan untuk diperdalam, karena tidak mungkin seluruh aplikasi dapat diberikan dan dikuasai oleh
kegiatan yang singkat seperti ini. Aplikasi yang ditawarkan adalah Filmora sebagai alat bantu
mendokumentasikan Kegiatan Belajar Mengajar (KBM). g
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Di sepakati oleh seluruh peserta bahwa untuk Filmora merupakan alat bantu yang memiliki
fitur lengkap dan mudah untuk pembuatan video sebagai dokumentasi KBM. Kegiatan dilanjutkan
dengan pemaparan pengenalan aplikasi Filmora bagi pembelajaran, pada materi ini peserta diberikan
pengantar dan beberapa contoh video yang dapat meningkatkan minat tenaga pendidik pada belajar
dikarenakan tampilan yang menarik sehingga tidak menimbulkan kebosanan dalam belajar yang
dilanjutkan dengan membuka sesi tanya jawab. Ketika sesi ini tanya jawab terlihat antusiasme
peserta dengan banyaknya pertanyaan khususnya berkenaan dengan menyisipkan teks dan gambar
untuk pembuatan opening dan closing video. Materi dilanjutkan mengenai pengenalan fitur
pemotongan video dan penggabungan beberapa video kegiatan, yang dimana pada kegiatan ini
tenaga pendidik perlu mengulang beberapa lengkah dalam pembuatan satu video yang utuh dan
menarik. Kegiatan dilanjutkan dengan materi penyisipan suara atau audio, dimana aplikasi ini
memungkinkan para tenaga pendidik untuk mengelola lagu pendukung, mulai dari opening video, 24 Jurnal ADIMAS Jurnal ADIMAS video inti, menampilkan prestasi Lembaga sampai pada closing video. 3. HASIL DAN PEMBAHASAN Hal ini menjadi sesuatu yang
menarik bagi para tenaga pendidik dan menjadi pengetahuan berharga, akan tetapi mereka menyadari
kembali pada kemampuan SDM dan sarana bahwa hal ini butuh waktu untuk mereka dapat
menguasai dan melaksanakannya. Sedangkan materi praktek atau latihan dititik beratkan pada
optimalisasi penyisipan suara dan penggabungan video dalam pembuatan video dokumentasi KBM. Hal ini dapat dilakukan karena Filmora memiliki fitur dan Bahasa yang mudah dipahami untuk
pembuatan video, tetapi bagaimana mengoptimalkannya mereka tidak mengetahui dan
menguasainya. Sehingga ketika materi ini dilakukan langsung fokus pada dasar-dasar Filmora dan
bagaimana tools tersebut dapat digunakan, serta diberikan latihan untuk prakteknya. Tampilan fitur
Filmora dalam melakukan memasukkan suara untuk menambahkan lagu sebagai theme song, dapat
dilihat pada Gambar 2. Gambar 2. Tampilan Fitur Filmora dalam Memasukkan Suara
Pada gambar 3 menjelaskan grafik mengenai hasil kegiatan pelaksanaan PkM. Fitur Filmora
yang menjadi Materi pada kegiatan pelaksanaan PkM kali ini adalah kemampuan peserta didik dalam
melakukan pengambilan gambar dari HP peserta didik masing-masing untuk dimasukkan ke dalam
aplikasi Filmora, dimana prosentase keberhasilan yang diraih adalah 85% dari peserta didik berhasil
melakukannya. Kemudian Fitur Filmora yang lain adalah memilih Gambar, baik dari template
Filmora maupun gambar koleksi HP/laptop peserta didik, dimana prosentase keberhasilannya adalah
95%. Selain itu fitur Filmora yang lain adalah memberikan efek transisi pada text maupun gambar
yang telah ditampilkan oleh peserta didik dimana prosentase keberhasilannya adalah 78%. Kemudian
fitur Filmora untuk memasukkan suara dari koleksi pribadi maupun template Filmora yang
mendapatkan prosentase keberhasilan 83%. Kemudian fitur Filmora export video untuk dijadikan
file dengan berekstensi MP4, AVI dimana prosentase keberhasilannya adalah 81%. Gambar 2. Tampilan Fitur Filmora dalam Memasukkan Suara Gambar 2. Tampilan Fitur Filmora dalam Memasukkan Suara Pada gambar 3 menjelaskan grafik mengenai hasil kegiatan pelaksanaan PkM. Fitur Filmora
yang menjadi Materi pada kegiatan pelaksanaan PkM kali ini adalah kemampuan peserta didik dalam
melakukan pengambilan gambar dari HP peserta didik masing-masing untuk dimasukkan ke dalam
aplikasi Filmora, dimana prosentase keberhasilan yang diraih adalah 85% dari peserta didik berhasil
melakukannya. Kemudian Fitur Filmora yang lain adalah memilih Gambar, baik dari template
Filmora maupun gambar koleksi HP/laptop peserta didik, dimana prosentase keberhasilannya adalah
95%. Selain itu fitur Filmora yang lain adalah memberikan efek transisi pada text maupun gambar
yang telah ditampilkan oleh peserta didik dimana prosentase keberhasilannya adalah 78%. Kemudian
fitur Filmora untuk memasukkan suara dari koleksi pribadi maupun template Filmora yang
mendapatkan prosentase keberhasilan 83%. 3. HASIL DAN PEMBAHASAN Kemudian fitur Filmora export video untuk dijadikan
file dengan berekstensi MP4, AVI dimana prosentase keberhasilannya adalah 81%. Gambar 3. Prosentase Keberhasilan Peserta Didik dalam menguasai Fitur Filmora
0
20
40
60
80
100
Pengambilan
Gambar
Memilih
Gambar
Memberikan
Efek Transisi
Memasukkan
Suara
Export Suara
Prosentase Keberhasilan
Prosentase Keberhasilan Gambar 3. Prosentase Keberhasilan Peserta Didik dalam menguasai Fitur Filmora Jurnal Pengabdian Masyarakat| ADIMAS 2020 Jurnal Pengabdian Masyarakat| ADIMAS 2020 ADIMAS 2020| Jurnal Pengabdian Masyarakat Jurnal ADIMAS Coherent, focused instruction (Tim PkM menyampaikan pelajaran secara sistematis dan
terfokus); c. Coherent, focused instruction (Tim PkM menyampaikan pelajaran secara sistematis dan
terfokus); Thoughtful discourse (Tim PkM menyajikan materi dengan bijaksana); d. Thoughtful discourse (Tim PkM menyajikan materi dengan bijaksana); e. Authentic learning (pembelajaran bersifat riil (autentik dengan permasalahan yang dihadapi
masyarakat dan siswa); e. Authentic learning (pembelajaran bersifat riil (autentik dengan permasalahan yang dihadapi
masyarakat dan siswa); y
)
f. Effective use of technology (menggunakan teknologi pembelajaran, baik untuk mengajar maupun
kegiatan belajar). y
f. Effective use of technology (menggunakan teknologi pembelajaran, baik untuk mengajar maupun
kegiatan belajar). g
j
Tabel 1 menjelaskan hasil indicator keberhasilan pelaksanaan PkM yang telah diikuti oleh
75 orang tenaga pendidik Muslimat NU Surabaya. Indikator yang digunakan adalah suasana
pembelajaran kondusif untuk belajar dengan mendapatkan prosentase keberhasilan 87% dengan
kriteria Sangat Baik, Indikator yang lain yang dijadikan kualitas pembelajaran adalah Tim PkM
menyampaikan pelajaran dengan jelas dan semua Peserta Didik mempunyai keinginan untuk
berhasil, mendapatkan prosentase keberhasilan sebesar 88% dengan kriteria Sangat Baik. Kemudian
indikator kualitas pembelajaran yang lain adalah Tim PkM menyajikan materi dengan bijaksana
mendapatkan prosentase keberhasilan 78% dengan kriteria Baik. Indikator kualitas pembelajaran
yang lain adalah pembelajaran bersifat riil (autentik dengan permasalahan yang dihadapi bersifat riil
dimana mendapatkan prosentase keberhasilan 75% dengan kriteria Baik. Untuk yang terakhir
indikator kualitas pembelajarannya yang digunakan adalah menggunakan teknologi pembelajaran,
untuk kegiatan KbM mendapatkan prosentase keberhasilan 92% dengan kriteria Sangat Baik. Tabel 1. Indikator Kualitas Pembelajaran
No
Indikator
Prosentase Keberhasilan
Kriteria
1
Suasana pembelajaran kondusif untuk
belajar
87%
Sangat Baik
2
Tim PkM menyampaikan pelajaran dengan
jelas dan semua Peserta Didik mempunyai
keinginan untuk berhasil
88%
Sangat Baik
3
Tim PkM menyajikan materi dengan
bijaksana
78%
Baik
4
Pembelajaran bersifat riil (autentik dengan
permasalahan yang dihadapi Peserta Didik)
75%
Baik
5
Menggunakan teknologi pembelajaran,
untuk kegiatan KbM
92%
Sangat Baik Tabel 1. Indikator Kualitas Pembelajaran Jurnal ADIMAS Terlihat para tenaga pendidik begitu senang ketika mengetahui banyak sekali fitur yang selama
ini mereka tidak mengetahui cara menggunakannya tetapi sangat membantu dalam pembuatan video
kegiatan, seperti pembuatan video profile lembaga, pembuatan prestasi lembaga dan lain sebagainya. Secara umum para tenaga pendidik peserta pelatihan terlihat antusias mengikuti kegiatan pelatihan
ini, terlebih bagi tenaga pendidik yang menyadari betul bahwa TIK sangat berguna dalam membantu
pembelajaran, yang dapat dilihat pada Gambar 4. Mereka merasa terbantu dengan adanya kegiatan
pelatihan ini sebagai sarana untuk menambah motivasi, pengetahuan, pemahaman dan keterampilan
dalam memanfaatkan TIK bagi pembelajaran. Semua pihak, khususnya Para Tenaga Pendidik
Muslimat NU Surabaya peserta pelatihan menyadari bahwa dengan pelatihan singkat semacam ini
tidak mungkin para tenaga pendidik dapat menguasai TIK dalam pembelajaran dengan baik, terutama
bagi mereka yang sama sekali tidak pernah menggunakan TIK, akan tetapi setidak nya pelatihan ini
menumbuhkan motivasi dan pengetahuan akan manfaat TIK bagi pembelajaran, yang dapat di
tularkan sebagai motivasi kepada para tenaga pendidik untuk melek teknologi. Gambar 4. Kegiatan PkM Gambar 4. Kegiatan PkM Gambar 4. Kegiatan PkM Untuk mengetahui tingkat kualitas pembelajaran dalam kegiatan belajar mengajar, maka perlu
diketahui dan dirumuskan indikator-indikator kualitas pembelajaran. Morrison, Mokashi & Cotter
(Morrison, 2006) dalam risetnya telah merumuskan 44 indikator kualitas pembelajaran yang reduksi
ke dalam 10 indikator. Kesepuluh indikator tersebut adalah: a. Rich and stimulating physical environment (lingkungan fisik mampu menumbuhkan semangat
siswa untuk belajar); j );
Classroom climate condusive to learning (suasana pembelajaran kondusif untuk belajar); c. Clear and high expectation for all students (guru menyampaikan pelajaran dengan jelas dan
semua siswa mempunyai keinginan untuk berhasil); p
y
g
Coherent, focused instruction (guru menyampaikan pelajaran secara sistematis dan terfokus); e. Thoughtful discourse (guru menyajikan materi dengan bijaksana); Authentic learning (pembelajaran bersifat riil (autentik dengan permasalahan yang dihadap
masyarakat dan siswa); g. Regular diagnostic assessment for learning (ada penilaian diagnostik yang dilakukan secara
periodic); h. Reading and writing as essential activities (membaca dan menulis sebagai kegiatan yang esensial
dalam pembelajaran); i. Mathematical reasoning (menggunakan pertimbangan yang rasional dalam memecahkan
masalah); j. Effective use of technology (menggunakan teknologi pembelajaran, baik untuk mengajar maupun
kegiatan belajar). Dari kesepuluh indikator tersebut tim PkM menggunakan enam indikator yang sesuai dengan
pelaksanaan PkM yaitu: y
Classroom climate condusive to learning (suasana pembelajaran kondusif untuk belajar); b. Clear and high expectation for all students (guru menyampaikan pelajaran dengan jelas dan semua
siswa mempunyai keinginan untuk berhasil); ADIMAS 2020| Jurnal Pengabdian Masyarakat Jurnal ADIMAS c. Jurnal ADIMAS Berdasarkan hasil PkM yang telah dilakukan maka penulis memiliki beberapa saran yang
kiranya bermanfaat bagi pihak-pihak yang terkait, diantaranya: a. Tenaga pendidik Muslimat NU Surabaya memiliki literasi TIK yang cukup baik, sehingga
kegiatan PkM terkait integrasi TIK dapat dilakukan di Muslimat NU Surabaya. a. Tenaga pendidik Muslimat NU Surabaya memiliki literasi TIK yang cukup baik, sehingga
kegiatan PkM terkait integrasi TIK dapat dilakukan di Muslimat NU Surabaya. b. PkM lebih lanjut diharapkan dapat menggali keterampilan tenaga pendidik Muslimat NU
Surabaya dalam membuat media pembelajaran dan memanfaatkan sumber belajar berbasis TIK. b. PkM lebih lanjut diharapkan dapat menggali keterampilan tenaga pendidik Muslimat NU
Surabaya dalam membuat media pembelajaran dan memanfaatkan sumber belajar berbasis TIK. 4. KESIMPULAN Setelah dilakukan kegiatan PkM kepada tenaga pendidik Muslimat NU Surabaya berupa
pemanfaatan TIK bagi pembelajaran diperoleh beberapa kesimpulan sebagai berikut: a. Tumbuh motivasi besar dari para tenaga pendidik ditengah beragam kesulitan yang mereka hadapi
untuk dapat menguasai TIK bagi proses belajar mengajar di kelas. Adapun lima indikator yang
dijadikan indikator kualitas pembelajaran adalah: suasana pembelajaran; kemampuan tim PkM
dalam menyampaikan pelajaran; penyajian materi; pembelajaran bersifat riil; dan menggunakan
teknologi pembelajaran untuk kegiatan KbM, dimana kriteria yang didapatkan mayoritas bersifat
Sangat Baik. a. Tumbuh motivasi besar dari para tenaga pendidik ditengah beragam kesulitan yang mereka hadapi
untuk dapat menguasai TIK bagi proses belajar mengajar di kelas. Adapun lima indikator yang
dijadikan indikator kualitas pembelajaran adalah: suasana pembelajaran; kemampuan tim PkM
dalam menyampaikan pelajaran; penyajian materi; pembelajaran bersifat riil; dan menggunakan
teknologi pembelajaran untuk kegiatan KbM, dimana kriteria yang didapatkan mayoritas bersifat
Sangat Baik. g
b. Pengetahuan dan pemahaman para tenaga pendidik mengenai ragam aplikasi TIK yang dapat
digunakan bagi pembelajaran mengalami peningkatan. Adapun fitur Filmora yang telah dikuasai
oleh peserta didik Muslimat NU adalah pengambilan gambar, memilih gambar, memberikan efek
transisi, memasukkan suara dan export suara, dimana prosentase keberhasilannya mendapatkan
prosentase lebih dari 75%. p
c. Pemanfaatan TIK dapat mendukung peningkatan kualitas pembelajaran, baik dari peningkatan
kemampuan tenaga pendidik dalam menyiapkan media pembelajaran; kesadaran dan kemampuan
tenaga pendidik dalam menggunakan teknologi untuk mendukung belajar kegiatan belajar
mengajar. Jurnal Pengabdian Masyarakat| ADIMAS 2020 27 DAFTAR PUSTAKA Applin, H. et al., 2011. A comparison of competencies between problem-based learning and non-problem-
based graduate nurses. Nurse Education Today, 31(2), hal.129– 134. Elston, Carol, 2007, Using ICT in the Primary School, Sage Publications. Haraldseid, C., Friberg, F. & Aase, K., 2015. Nursing students’ perceptions of factors influencing their learning
environment in a clinical skills laboratory: A qualitative study. Nurse Education Today, 35(9), hal.e1–
e6. Hariningsih, S. 2005. Teknologi Informasi. Yogyakarta: Graha Ilmu Morrison, D.M. dan Mokashi K. & Cotter, K. Instructional Quality Indicators, (Cambridge: Research
Foundations, 2006), hlm. 4-21 Munir. 2009. Kontribusi Teknologi Informasi dan Komunikasi (TIK) dalam Pendidikan di Era Globalisasi
Pendidikan Indonesia. Jurnal Pendidikan Teknologi Informasi dan Komunikasi, Vol. 2, No. 2, hal.1-
4. Pustekkom, 2009. TIK untuk Pembelajaran (On-line) (Pustekkom, Jardiknas http://www.scribd. com diakses:
15 oktober 2019). Santoso, D. H. 2014. Pemanfaatan Teknologi Komunikasi dan Informasi pada Erupsi Merapi 2010 di Sleman
Yogyakarta. Jurnal Pekommas, 17(3), 181–188. Seels, Barbara B, dan Richey. 1994. Instructional Technology: the Definitions and Domains of the Field,
Washington DC: AECT. Wijayanti, Inggit Dyaning. 2011. Peningkatan Pendidikan Berbasis ICT. UIN Sunan Kalijaga: Yogyakarta. ADIMAS 2020| Jurnal Pengabdian Masyarakat ADIMAS 2020| Jurnal Pengabdian Masyarakat 28
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The pros and cons of utilizing crude herbal preparations as opposed to purified active ingredients, with emphasis on the COVID pandemic
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Qeios, CC-BY 4.0 · Article, July 31, 2023 Open Peer Review on Qeios The pros and cons of utilizing crude herbal preparations as
opposed to purified active ingredients, with emphasis on the
COVID pandemic The pros and cons of utilizing crude herbal preparations as
opposed to purified active ingredients, with emphasis on the
COVID pandemic The pros and cons of utilizing crude herbal preparations as
opposed to purified active ingredients, with emphasis on the
COVID pandemic Michal Haran1, Alain Berrebi1
1 Hebrew University of Jerusalem Michal Haran1, Alain Berrebi1 1 Hebrew University of Jerusalem Funding: No specific funding was received for this work. Potential competing interests: No potential competing interests to declare. Abstract It is well recognized that many plants contain substances with pharmacological activities, and there is a large body of
traditional knowledge regarding their use in different clinical situations. Yet, most physicians are reluctant to use herbal
preparations, because there are no large-scale randomized controlled trials to support their use. In this paper, we
challenge this approach and propose that herbal treatments should be added to our armamentarium, even if cautiously. This pertains to situations in which there is no existing well-studied evidence-based approach; the known
pharmacological properties of the plant or plants being used are relevant to the pathophysiology of the disease and the
safety profile of the herbal preparation is well established. Specifically, we share our knowledge and experience
regarding the treatment of patients with COVID with the plant Artemisia. Michal Haran1,* and Alain Berrebi1
1Hematology Institute Kaplan Medical Center and Faculty of Medicine, Hebrew University, Jerusalem. *Corresponding author: Michal.haran@gmail.com
Qeios ID: 43I4RU.2 · https://doi.org/10.32388/43I4RU.2 Michal Haran1,* and Alain Berrebi1 Michal Haran1,* and Alain Berrebi1 Michal Haran1,* and Alain Berrebi1 ematology Institute Kaplan Medical Center and Faculty of Medicine, Hebrew University, Jerusalem. *Corresponding author: Michal.haran@gmail.com Qeios ID: 43I4RU.2 · https://doi.org/10.32388/43I4RU.2 1/8 Qeios, CC-BY 4.0 · Article, July 31, 2023 It is well recognized that many plants contain substances that have pharmacological activities [1][2]. For thousands of
years, crude preparations of those plants were used as treatment for human diseases. There are numerous examples of
plant-based treatments in ancient Western medicine and in traditional Chinese medicine[3]. Some of these are being
further studied and used as complementary treatments to this day[4]. In contrast to this traditional approach, Modern
Medicine is based on the concept that pharmacological treatments should be precise. Accordingly, medications are
manufactured in a reproducible manner and contain one or at most a small number of purified active ingredients, of which
the concentration is known and clearly written on the package. The optimal dosing of each medication is found in well-
designed clinical trials. Using a crude herbal preparation that contains numerous pharmacological compounds, some of
which we know very little about, is not compatible with this concept. Furthermore, when crude herbal preparations are
employed, the concentrations of the pharmacologically active ingredients may vary from one plant to the other and may
depend on numerous environmental factors. These could include, for example, the soil in which a plant was grown, the
amount of water it had received, etc. [5][6] Although there are many levels of evidence, such as case series, small trials, and even case reports and in vitro studies,
evidence-based medicine has become synonymous with large-scale randomized controlled trials (RCT). While the
significant advantages of RCTs are obvious, is the precision-based foundation of Modern Medicine truly feasible, given
that biological systems like the human body are far from being precise? Can our adoption of precise dosing of a given
medication guarantee that we provide each patient with an optimal dose? We know from our practice that this is often not
the case. First, a given medication may have numerous possible interactions with other medications, as well as with food
and supplements[7]. Second, the metabolism of drugs may vary between different patients[8]. These factors are rarely
being taken into account in RCTs. It is generally assumed that the maximal dose that has been tolerated in a clinical trial is
suitable for all patients. Yet, often times more is not better. Qeios ID: 43I4RU.2 · https://doi.org/10.32388/43I4RU.2 Michal Haran1,* and Alain Berrebi1 In fact, the NIH created a list of such food supplements, with their safety profile and
limited evidence for their efficacy based on small case series and clinical trials, adding that Data are insufficient to support
recommendations for or against the use of any vitamin, mineral, herb or other botanical, fatty acid, or other dietary
supplement ingredient to prevent or treat COVID-19. Thus, making the information available to the public while deterring
physicians from recommending them to their patients. In this paper, we would like to focus on the plant Artemisia, as an
example of a highly studied plant with well-established pharmacological activities which was used mostly in developing
countries. The genus Artemisia is one of the largest and most widely distributed genera of the family Astraceae (Compositae). It is a
heterogenous genus, consisting of over 500 diverse species distributed mainly in the temperate zones of Europe, Asia,
and North America. Its different species have been used in traditional medicine world-wide for many years [15]. All
Artemisia species contain pharmacologically active substances such as terpenoids, flavonoids, coumarins, caffeoylquinic
acid, sterols, and acetylenes [16][17]. Numerous in vitro studies, as well as small-scale clinical trials, have shown their
activity in infectious, malignant, respiratory, and immune diseases[15]. The effect of A. annua specifically against malaria
was elucidated by Y. Tu, who then purified one of its many active compounds, artemisinin. She eventually received a
Nobel Prize for her discovery. In the early days of the pandemic, Artemisia extracts were specifically shown to have an in
vitro effect on the replication of SARS-CoV-2. . Yet, due to the lack of exact dosing and solid scientific proof of efficacy in
RCTs, its employment was widely discouraged in many Western countries[18]. These authors’ own clinical and academic experience with Artemisia and appreciation of its safety (it is being used as
herbal tea in many households in Israel) has led one of them to recommend it to multiple patients, together with breathing
exercises. The rational for this management approach was that by that time it was quite clear that the severity of the
disease was an interplay between the virus and the immune system, and that the cytokine storm was a result of a
dysregulated immune system[19]. It was also noticed that the rheological properties of the sputum of the patients was very
similar to the viscous secretions seen in patients with cystic fibrosis[20]. Michal Haran1,* and Alain Berrebi1 It should also be taken into account that a randomized clinical
trial for a specific medicine does not necessarily mean that it is safe and effective for a given patient. Moreover, the
concept of using pure pharmacologically active substances does not take into account the complexity of the human
organism and can often inadvertently turn the patient into a jigsaw puzzle of seemingly unrelated problems. It is true that most herbal preparations have not been tested in large-scale RCTs. Nevertheless, their safety has been
assessed for hundreds of years[4]. Furthermore, there are certain clinical situations under which the utilization of crude
plant extracts may be beneficial, even in modern countries. Indeed, such plants are readily available and may be a
constituent in most kitchens or gardens, and there are no inactive ingredients that may cause adverse reactions[9]. Under which clinical situation is there a place for herbal treatments? Clearly, in the case of a life-threatening disease for
which there is a proven and well-established treatment approach, we should always prefer using that treatment. However,
when faced with a patient with an indolent disease, which does not yet require treatment, rather than taking the “wait and
see” approach, it is conceivable to attempt treatment with an appropriate herbal preparation, based on the mode of action
of its active ingredients. In addition, in the case of a refractory disease, one might try an experimental treatment (if it is
available) or use an herbal preparation as part of the supportive care[10]. This may apply even to a new disease when it 2/8 Qeios, CC-BY 4.0 · Article, July 31, 2023 lacks a known treatment. lacks a known treatment. A good example for such a situation is a pandemic caused by a novel virus, such as SARS-CoV-2. It was realized quite
early in the pandemic that the rather insidious onset of the disease in most patients creates a possible window of
opportunity to intervene at home when the patient has minimal symptoms [11]. Some physicians around the world
attempted to use repurposed drugs, food supplements, and traditional herbal preparations in various combinations as a
means to alter the course of the disease[12][13][14] Furthermore, many people around the world used food supplements
and herbal treatments without any guidance from their physicians, who were reluctant to recommend such treatments
without solid evidence-based data. Qeios ID: 43I4RU.2 · https://doi.org/10.32388/43I4RU.2 Michal Haran1,* and Alain Berrebi1 Thus, the combination of the antiviral activity of
Artemisia with its effect on the immune system seemed to be an optimal “package” for treating COVID. A short video
tutorial and a brochure with suggestions for supportive care in the early stages of the illness were prepared and
distributed via social media. Data regarding the effect of artemisia on patients with COVID was extracted from A patient-
reported study on COVID outcomes (approved by the institutional review board of Kaplan Medical Center, the results of
which will be published separately). Qeios ID: 43I4RU.2 · https://doi.org/10.32388/43I4RU.2 3/8 Qeios, CC-BY 4.0 · Article, July 31, 2023 Overall, 75 patients reported using Artemisia extracts either in the form of a lukewarm extract for drinking or steam
inhalation, typically in conjunction with breathing exercises. None of these patients required hospitalization. Most were
young with no underlying conditions, though20 patients were older than 50 and 16 had at least one underlying disease. All
had a relatively mild clinical course. 15 patients reported that they felt improvement in their respiratory symptoms and
could expel disease-related phlegm more readily. The other patients used additional treatment modalities, such as
vitamins and food supplements and therefore could not validate what helped. None had significant post-COVID symptoms
and all returned to their baseline condition. Below we will briefly describe the clinical course of a few patients who demonstrated early signs of deterioration in their
condition, such as a drop of O2 saturation or significant dyspnea and reported significant improvement when using
Artemisia. -A 60-year-old woman who had undergone a lung transplant due to pulmonary fibrosis and was accordingly on
immunosuppressive treatment, had mild to moderate dyspnea and a drop in her O2 saturation to 90 within 2 days of
contracting COVID. She started using food supplements and Artemisia as luke-worm tea and steam inhalation in
combination with breathing exercises. Within a few hours, she started coughing a significant amount of copious phlegm
with significant improvement in her respiratory symptoms and O2 saturation. She continued to use Artemisia a few times a
day and every time she felt it was hard to expel the phlegm. Under this treatment, she continued to have respiratory
symptoms and intermittent drops in her O2 saturation, but was stable and did not require any other treatment or
hospitalization. She fully recovered back to her baseline within a few months. Qeios ID: 43I4RU.2 · https://doi.org/10.32388/43I4RU.2 Michal Haran1,* and Alain Berrebi1 Within a week, she was fully recovered and able to
participate in strenuous physical activities. The patients described above showed evidence for significant improvement in both objective (O2 saturation and ease of
sputum expulsion) and subjective (feeling of well-being) parameters, which were consistent with the mode of action of
Artemisia as an anti-inflammatory and antiviral pharmacological agent. This treatment approach was well-tolerated also in
a much larger group of patients, not described here in detail. Many of these patients reported experiencing a subjective
improvement in their condition, although objective measures were lacking. Medical practice should always be based on the assessment of risk vs. benefit for each specific patient, as well as each
specific treatment modality. Based on this edict, and given that there was no known proposed treatment at the early
stages of COVID, the application of herbal preparations possessing a very high safety profile and a potentially significant
beneficial effect was a very reasonable approach. There are over 1000 papers in the medical literature regarding the
clinical or in-vitro effects of different types of artemisia on SARS-CoV-2. Thus, although this treatment modality was not
tested in large-scale RCTs, there was enough evidence to support its use. Why then have such treatments not seen a more wide-spread use in the Western world? We believe that the reason for
that was mostly the concern that they are not “evidence-based”, meaning that they were not tested in large-scale
RCTs[18] and that physicians would be considered as promoting “primitive” treatments and even quackery, instead of well-
established modern treatments. The debate regarding the role of RCT driven evidence-based medicine (EBM) in shaping
our clinical decisions is not new[21][22], but the pendulum seems to be going strongly towards the EBM approach. We
propose that the careful and thoughtful incorporation of herbal preparations into our practice, when deemed appropriate,
will not lead to quackery, but rather to the exact opposite. Indeed, physicians who are ready to incorporate traditional
treatment modalities, assessed in the light of modern science and medicine, are more likely to be able to provide their
patients with a wider armamentarium and thus gain their trust, decreasing their need to search for alternative and less
well-studied treatments elsewhere and feel abandoned by their physicians at a time of crisis[23]. Michal Haran1,* and Alain Berrebi1 -An 84-year-old man with multiple co-morbidities chose comfort care at home when he was advised to be hospitalized, as
he had significant hypoxemia and bilateral consolidation on CXR. His daughter took care of him and started doing
respiratory physiotherapy in combination with steam inhalation of Artemisia, every hour. Within a few days, there was
significant improvement in his condition. He fully recovered back to his baseline within a few weeks. -A 75-year-old woman, with no underlying diseases started using artemisia and breathing exercises after a few days of her
illness when she started even mild to moderate dyspnea, significant weakness, and her oxygen saturation dropped to
93%. Within hours she reported that her dry cough was changed to a productive cough, and she started expelling copious
sputum. Her oxygen saturation returned to normal-97%, but dropped again to the low 90s, within a few hours. She
continued with hourly breathing exercises and Artemisia steam for the next few days, with a gradual stabilization of her
respiratory condition. She fully recovered within a few months. She did not require hospitalization, supplemental oxygen,
or respiratory support. -A 59-year-old woman with neuromuscular disease. She had significant dyspnea and worsening of her respiratory muscle
weakness within a day of getting COVID, necessitating NIV support at home. She started using Artemisa, both as a
lukeworm tea and steam inhalation, combined with respiratory physiotherapy by her caregiver a few times a day. She had
intermittent drops in her oxygen saturation in the low 90s, which improved with the treatment. She did not require
increased respiratory pressure and did not require hospitalization. She had significant improvement within 10 days and Qeios ID: 43I4RU.2 · https://doi.org/10.32388/43I4RU.2 4/8 Qeios, CC-BY 4.0 · Article, July 31, 2023 fully recovered back to her baseline after a few months. fully recovered back to her baseline after a few months. A 58-year-old woman who started to have respiratory symptoms a few days after contracting COVID. She started
treatment with artemisia and breathing exercises and called an ambulance which took her to the nearby hospital where
she was found to have an abnormal chest X-ray and oxygen saturation in the low 90s. She was started on steroids and
continued with breathing exercises and Artemisia tea and steam inhalation. Within 2 days, there was a significant and
unexpected improvement in her condition, and she was discharged. Qeios ID: 43I4RU.2 · https://doi.org/10.32388/43I4RU.2 Michal Haran1,* and Alain Berrebi1 In conclusion, we have argued in this article that although using RCT driven evidence-based approaches have no doubt
improved the quality of medicine, one should be cautious that the means does not become the end and that this excellent
tool that aids us in our clinical practice does not become a procrustean bed. Understanding the limitations of precision-
driven medicine and carefully adding herbal treatments and food supplements, when there is no better management
approach available, is not only desirable but can benefit both physicians and patients. We stress that the decision to use
herbal preparations should be based on a sound knowledge regarding their safety and contraindications to their use, as
well as knowledge of their clinical use in traditional medicine and a good understanding of their pharmacological activity in 5/8 Qeios, CC-BY 4.0 · Article, July 31, 2023 various medical situations. In case of doubt, it is always beneficial to consult a qualified herbal specialist regarding the
amount and mode of administration. It is also important to verify the source of the plants and, when using ready-made
extracts, it is important that those will be obtained from an herbal pharmacy, in order to ensure their quality, purity, and a
reasonable level of standardization. It is also important to understand that utilizing herbal treatment approaches is not
mutually exclusive to sound scientifically proven evidence-based medicine. There is a growing body of knowledge which
combines the tools of modern medicine and science with traditional knowledge. There are also numerous studies
regarding the interactions of various plants with commonly used medications. We call for more studies, including large
randomized clinical trials that assess both the safety and efficacy of specific herbal preparations in various clinical
situations, so that we can more readily incorporate them into the routine care of our patients. We also think that during a pandemic with a novel virus for which there is no proven treatment, it is important to explore all
possible management approaches, as long as they are known to be safe, even if, at the time, there are no large-scale
RCTs to prove their efficacy. Finally, we have to remember that “primum non-nocere” was coined long before the era of RCTs and that even many
commonly used medications were not tested by this approach (such as mestinon for myasthenia gravis and steroids for
many clinical situations). Michal Haran1,* and Alain Berrebi1 This famous dictum implies that one should always be cautious when treating patients and be
aware of any signs that suggest more harm than good. However, it does not imply that one should only give patients only
medications that have gone through large-scale clinical trials and push aside all other human experience and knowledge. Acknowledgements The authors would like to thank Prof. Gilad Haran for careful reading and useful comments on the manuscript. References 1. ^Aye, M. M., Aung, H. T., Sein, M. M., & Armijos, C. (2019). A review on the phytochemistry, medicinal properties and
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https://doi.org/10.3390/molecules24020293. 2. ^Chen, Y., et al. (2022). Dietary Supplements and Natural Products: An Update on Their Clinical Effectiveness and
Molecular Mechanisms of Action During Accelerated Biological Aging. Frontiers in Genetics, 13. Preprint at
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Qeios ID: 43I4RU.2 · https://doi.org/10.32388/43I4RU.2
6/8 1. ^Aye, M. M., Aung, H. T., Sein, M. M., & Armijos, C. (2019). A review on the phytochemistry, medicinal properties and
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Increasing Lateralized Motor Activity in Younger and Older Adults using Real-time fMRI during Executed Movements
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Neuroscience
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Increasing Lateralized Motor Activity in Younger and Older Adults using
Real-time fMRI during Executed Movements
Heather F. Neyedli, a,b* Cassandra Sampaio-Baptista, a Matthew A. Kirkman, a David Havard, a Michael Lu¨ hrs, c
Katie Ramsden, a David D. Flitney, a Stuart Clare, a Rainer Goebel c and Heidi Johansen-Berg a
a Centre for Functional MRI of the Brain, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK
b Heather F. Neyedli, a,b* Cassandra Sampaio-Baptista, a Matthew A. Kirkman, a David Havard, a Michael Lu¨ hrs, c
Katie Ramsden, a David D. Flitney, a Stuart Clare, a Rainer Goebel c and Heidi Johansen-Berg a
a Centre for Functional MRI of the Brain, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK
b School of Health and Human Performance, Dalhousie University, Halifax, Nova Scotia, Canada
c Department of Cognitive Neuroscience, Maastricht University, The Netherlands Brain Innovation B.V., Maastricht, The Netherlands Heather F. Neyedli, a,b* Cassandra Sampaio-Baptista, a Matthew A. Kirkman, a David Havard, a Michael Lu¨ hrs, c
Katie Ramsden, a David D. Flitney, a Stuart Clare, a Rainer Goebel c and Heidi Johansen-Berg a
a Centre for Functional MRI of the Brain, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK
b School of Health and Human Performance, Dalhousie University, Halifax, Nova Scotia, Canada
c Department of Cognitive Neuroscience, Maastricht University, The Netherlands Brain Innovation B.V., Maastricht, The Netherlands Abstract—Neurofeedback training involves presenting an individual with a representation of their brain activity
and instructing them to alter the activity using the feedback. One potential application of neurofeedback is for
patients to alter neural activity to improve function. For example, there is evidence that greater laterality of
movement-related activity is associated with better motor outcomes after stroke; so using neurofeedback to
increase laterality may provide a novel route for improving outcomes. However, we must demonstrate that indi-
viduals can control relevant neurofeedback signals. Here, we performed two proof-of-concept studies, one in
younger (median age: 26 years) and one in older healthy volunteers (median age: 67.5 years). The purpose was
to determine if participants could manipulate laterality of activity between the motor cortices using real-time fMRI
neurofeedback while performing simple hand movements. The younger cohort trained using their left and right
hand, the older group trained using their left hand only. In both studies participants in a neurofeedback group
were able to achieve more lateralized activity than those in a sham group (younger adults: F(1,23) = 4.37,
p < 0.05; older adults: F(1,15) = 9.08, p < 0.01). Moreover, the younger cohort was able to maintain the lateralized
activity for right hand movements once neurofeedback was removed. The older cohort did not maintain lateralized
activity upon feedback removal, with the limitation being that they did not train with their right hand. E-mail address: heather.neyedli@ndcn.ox.ac.uk (H. F. Neyedli).
Abbreviations:
BOLD,
Blood-oxygen-level-dependent;
FWHM,
full
width at half maximum; GLM, general linear model; LI, laterality
index; NF, Neurofeedback; PSC, percent signal change; ROI, region of
interest;
TR,
repetition
time;
tDCS,
transcranial
direct
current
stimulation; TCP, transmission control protocol. *Corresponding author. Address: FMRIB Centre, Nuffield Dept. of
Clinical Neurosciences, University of Oxford, John Radcliffe Hospital,
Headington, Oxford OX3 9DU, UK. Increasing Lateralized Motor Activity in Younger and Older Adults using
Real-time fMRI during Executed Movements
Heather F. Neyedli, a,b* Cassandra Sampaio-Baptista, a Matthew A. Kirkman, a David Havard, a Michael Lu¨ hrs, c
Katie Ramsden, a David D. Flitney, a Stuart Clare, a Rainer Goebel c and Heidi Johansen-Berg a
a Centre for Functional MRI of the Brain, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK
b The results
provide evidence that neurofeedback can be used with executed movements to promote lateralized brain activity
and thus is amenable for testing as a therapeutic intervention for patients following stroke. This article is part of a Special Issue entitled: Neurofeedback and Functional Enhancement: Mechanisms, Methodology, Beha- g
p
p
g
This article is part of a Special Issue entitled: Neurofeedback and Functional Enhancement: Mechanisms, Methodology, Beha-
vioural and Clinical Applications. 2017 The Authors. Published by Elsevier Ltd on behalf of IBRO. This is an open access article
under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Key words: neurofeedback, motor cortex, ageing, stroke, real-time fMRI. https://doi.org/10.1016/j.neuroscience.2017.02.010
0306-4522/ 2017 The Authors. Published by Elsevier Ltd on behalf of IBRO.
This is an open access article under the CC BY license (http://creativecommons NEUROSCIENCE
RESEARCH ARTICLE H. F. Neyedli et al. / Neuroscience 378 (2018) 165–174 H. F. Neyedli et al. / Neuroscience 378 (2018) 165–174 INTRODUCTION et al., 1999). Furthermore, worse motor function is asso-
ciated
with
a
more
bilateral
activation
pattern
(Johansen-Berg et al., 2002; Ward et al., 2003) thus reha-
bilitation interventions that rebalance brain activity to a
more lateralized or contralateral pattern may enhance
therapy outcomes (Ward and Cohen, 2004). For example,
improvements in motor function with transcranial direct
current stimulation (tDCS) are accompanied by increased
activity in contralateral (ipsilesional) sensorimotor cortex
(Stagg et al., 2012; Allman et al., 2016). An alternative
intervention that may promote the lateralization of brain
activity and thus lead to beneficial plasticity following
stroke is neurofeedback. Neurofeedback involves mea-
suring an individual’s brain activity and displaying it back
to them in an intuitive format. The individual can then be
asked to use this feedback display to alter their brain
activity with the aim of inducing plasticity and improved
function. Thus, there is potential for stroke patients to
use neurofeedback training to lateralize brain activity fol-
lowing stroke. There are a range of neurorehabilitation techniques that
have been developed to facilitate motor recovery after
stroke such as physiotherapy, motor imagery and non-
invasive brain stimulation (Allman et al., 2016; Barclay-
Goddard et al., 2011; French et al., 2007; Hao et al.,
2013; Pollock et al., 2007; Sirtori et al., 2009; Thieme
et al., 2013). Following stroke, movement of the affected
limb is associated with increased activity in the unaffected
motor cortex and hence bilateral activation of motor
regions (Cramer et al., 1997; Gerloffet al., 2006; Nelles E-mail address: heather.neyedli@ndcn.ox.ac.uk (H. F. Neyedli). Abbreviations:
BOLD,
Blood-oxygen-level-dependent;
FWHM,
full
width at half maximum; GLM, general linear model; LI, laterality
index; NF, Neurofeedback; PSC, percent signal change; ROI, region of
interest;
TR,
repetition
time;
tDCS,
transcranial
direct
current
stimulation; TCP, transmission control protocol. 165 H. F. Neyedli et al. / Neuroscience 378 (2018) 165–174 166 stroke patients and thus further supportive data are
required. Neurofeedback
can
be
used
in
rehabilitation
to
enhance
motor
imagery
which
may
promote
more
lateralized activity (e.g., Auer et al., 2015; Chiew et al.,
2012; deCharms et al., 2004; Hampson et al., 2011;
Yoo et al., 2008). For example, previous research using
motor imagery has shown that healthy participants can
use
neurofeedback
from
real-time
fMRI
(rtfMRI)
to
increase activation in the hemisphere contralateral to
imagined movement (deCharms et al., 2004; Yoo et al.,
2008). However, there is variability in efficacy: Chiew
et al. EXPERIMENT I METHODS Relatively few studies to date have analyzed the
benefits of neurofeedback in conjunction with executed
movement to promote lateralization. This is important
because, although more than 75% of stroke patients
have motor disability following stroke (Lawrence et al.,
2001), many retain at least some movement in the
affected limbs and much of the movement rehabilitation
received after stroke focuses on executed movements. This group could benefit from neurofeedback combined
with physical practice, which may be more effective than
neurofeedback with motor imagery. The association
between real hand movements and increased spread of
cortical activity with neurofeedback has been demon-
strated elsewhere (Yoo and Jolesz, 2002) but in this study
no comparison was made to a no feedback or sham con-
dition. Overall, more evidence is needed on capacity to
modulate
neurofeedback
signals
during
executed
movement. Participants Twenty-six (seven male), right-handed participants aged
20–32, median age 26, were recruited from the Oxford
community. All participants provided informed consent in
accordance
with
the
Declaration
of
Helsinki
and
University of Oxford ethics committee approved the
protocol (MSD-IDREC-C1-2012-151). Participants were
randomly assigned to a neurofeedback (NF) group
(n = 13, age range: 20–31, median age: 23, four male)
or a Sham group (n = 13, age range: 20–32, median
age: 29, three male) and were not aware of their group
assignment. Further, all instructions to the participants
were provided by an experimenter who was blinded to
the group assignment. One participant in the NF group
(male) was removed from analysis due to excessive
head motion in the feedback scans. When considering the application of neurofeedback to
patients with stroke, it is important to note that most (c.f.,
Sitaram et al., 2012) prior rtfMRI neurofeedback studies
were performed in healthy younger adults, however the
condition largely affects older individuals
(Lawrence
et al., 2001). There is evidence that age affects factors
such as the hemodynamic response, neuronal and glial
responsiveness, and scope for plasticity (Di et al., 2014;
Freitas et al., 2011; Gauthier et al., 2013; Leal and
Yassa, 2013; Thomas et al., 2014), which means that
older individuals may respond differently to rtfMRI tasks. Indeed, it is known that older individuals tend to perform
worse on cognitive tasks than their younger counterparts,
with fMRI data supporting the theory that age influences
brain activity, and that brain activity is related to task per-
formance (Steffener et al., 2014). Further, older adults
also show a decline in motor performance and may show
decreased motor learning of fine or complex motor skills
(Voelcker-Rehage, 2008). Although there is some case
evidence that older stroke patients can control imagery-
related ventral pre-motor activity using neurofeedback
(Sitaram et al., 2012) this is based on a study of two INTRODUCTION (2012) showed that only a sub-set of their healthy
participants (6 of 13 participants) could modulate the lat-
erality of activity between hemispheres using motor ima-
gery. With a longer training period of twelve sessions,
Auer et al. (2015) demonstrated that 25 of 32 participants
could control a laterality index using motor imagery. Although motor imagery provides a useful paradigm for
neurofeedback, some people struggle to carry out motor
imagery tasks and it is difficult to assess neurofeedback
performance which may be confounded with motor ima-
gery ability. The aim of the current study was to determine whether
adults can increase the laterality of activation between the
motor
cortices
while
executing
movements
when
presented with a visual representation of a laterality
index (LI) measured through rtfMRI neurofeedback. Two
experiments were conducted, one with healthy younger
adults (age range 20–32 years) and one in healthy older
adults (age range 50–77 years). In each experiment,
participants
were
split
into
two
groups:
one
which
received real neurofeedback and one which received
sham feedback. If participants are able to lateralize
brain activity while performing physical movements,
participants in the neurofeedback group should have a
larger LI magnitude than participants in the sham group. Following training, a scan where no neurofeedback was
present was also included to determine if participants
could maintain the increased laterality in the absence of
feedback. Procedure A transmission control proto-
col (TCP) based network interface plug in for Turbo-
BrainVoyager was used to transfer the processed region
of interest (ROI) time course data to a custom-made soft-
ware tool, Turbo-Feedback, which performed neurofeed-
back signal calculation and presented the feedback
signal back to the participants. The feedback was pro-
jected
to
the
participant
in
the
scanner
using
a
1042 768 pixel screen with a 75-Hz refresh rate. Participants were given button boxes to hold in each
hand, each with four buttons corresponding to the four
fingers (excluding the thumb). The pre-feedback (pre-
FB) scan (150 volumes) provided a functional localizer
and consisted of eight 12-second tapping blocks, four
blocks for each hand, alternating hands between blocks,
interspersed with 24-second rest. The first block of
tapping started after a 14-second rest period and a 22-
second rest period followed the final tapping block. The
participants saw the instructions ‘Right Tap’, ‘Left Tap’
and ‘Rest’ displayed in white on a black background. During the tap instructions participants were told to use
each finger in sequence starting with their index finger
and moving outward toward the little finger to press and
release the button under their finger on the button box
at a rate of approximately 1 Hz, and to repeat the
sequence until they saw the rest instruction. Participants next took part in four FB scans during
which they were instructed to perform sequential button
presses with the fingers on their right or left hand, with
the order of hands alternated and counterbalanced
between participants. Each FB scan (180 volumes)
consisted of six, 30-second blocks of finger tapping
interspersed with blocks of 30-second rest. The first
block of tapping started after a 20-second rest period
and a 10-second rest period followed the final tapping
block. Participants saw a horizontal red bar with a
vertical line delineating the center point (Fig. 1A). The
calculation of bar width (see below) occurred within one
TR and thus the width of the red bar was updated in
response
to
Blood-oxygen-level-dependent
(BOLD)
signal every TR (i.e. every 2000 ms) (Fig. Procedure Imaging was performed on a 7.0T Siemens Magnetom
MRI system (Siemens, Erlangen, Germany). A structural
image was acquired using a T1 weighted, MPRAGE
sequence with 1 1 1 mm3 isotropic voxels (repetition
time = 2200 ms; echo time = 2.2 ms; flip angle 7, field
of view, 192 192; matrix = 192 192). All fMRI scans
were performed using a 16 slice (2 mm, no slice gap)
axial plane, gradient echo planar image acquisition, with
2 2 mm2
in
plane
resolution
(repetition
time = 2000 ms; echo time = 25 ms; flip angle = 90
field of view, 220 220 mm; matrix = 110 110). A
limited field of view was used so the data could be
analyzed within a single repetition time (TR) to be fed
back to the participant. The field of view, a slab of 32-
mm depth, was placed angled parallel with the top of
the brain and was deep enough to cover sensorimotor
and pre-motor cortex. Turbo-BrainVoyager
software
(Brain
Innovation,
Maastricht, The Netherlands) was used to preprocess H. F. Neyedli et al. / Neuroscience 378 (2018) 165–174 167 the motor ROIs were only used for data analysis. For
participants in the Sham group, two sham ROIs, the
same size as the motor ROIs, were also selected
aligned along the anterior-posterior axis centered along
the midline, toward the posterior of the brain. These
sham ROIs were used to provide sham feedback to the
participants in the Sham group in order to provide the
same visual and motivational environment as the NF
group. These regions were selected to avoid voxels
activated during the motor task (Fig. 1B). Thus our goal
was to select regions that were not involved in our task
of interest (e.g., tapping). Note that the experimenter
operating the real-time set up and selecting the ROIs
was necessarily aware of the group assignment (NF or
Sham) of the participant but the experimenter providing
instructions
and
interacting
with
the
participant
throughout the experiment did not interact with the
neurofeedback software or other components of the
real-time set up and therefore was not aware of group
assignment
thus
maintaining
the
double-blind
study
design. (including online 3D motion correction) and analyze the
fMRI data in real time using a recursive general linear
model (Pollock et al., 2007). Procedure 1A); however,
for the visual feedback display the bar width smoothly The results from the real-time general linear model
(GLM) analysis of the localizer scan were used to select
two motor ROIs (18 18 10 mm) for each participant
(regardless of group assignment), each centered over
the peak of activation in the region of the hand knob in
the right or left hemisphere (Fig. 1B). If the participant
was in the NF group, the motor ROIs would be used for
neurofeedback during the experiment and subsequent
data analysis. If participants were in the Sham group, Fig. 1. (A) Feedback display shown to participants. The bar updates in width according to the participant’s LI (see text for details). The left image
presents a typical frame during the left tapping block (where the bar growing leftward represents right hemisphere lateralized activity) and the right
image presents a typical frame during a rest block (where a bar close to the center represents LI close to 0 as activity is similar between the two
hemispheres) (B) Image on the left shows the motor ROIs from an example participant (blue boxes) located over sensorimotor cortex. Image on the
right shows sham ROIs from an example participant (blue boxes). (C) Online data processing pipeline (D) Offline data processing pipeline. Please
refer to the text for details on the calculations. Fig. 1. (A) Feedback display shown to participants. The bar updates in width according to the participant’s LI (see text for details). The left image
presents a typical frame during the left tapping block (where the bar growing leftward represents right hemisphere lateralized activity) and the right
image presents a typical frame during a rest block (where a bar close to the center represents LI close to 0 as activity is similar between the two
hemispheres) (B) Image on the left shows the motor ROIs from an example participant (blue boxes) located over sensorimotor cortex. Image on the
right shows sham ROIs from an example participant (blue boxes). (C) Online data processing pipeline (D) Offline data processing pipeline. Please
refer to the text for details on the calculations Fig. 1. (A) Feedback display shown to participants. The bar updates in width according to the participant’s LI (see text for details). Offline data analysis Note that this
equation
focuses
on
contralateral
vs. ipsilateral
activation instead of the left vs. right activation used for
the NF display. The left vs. right calculation was used
for the display to make the feedback bar intuitive for
the participant (i.e., moves more to the left during left-
handed movements). The maximum magnitude the bar could move in either
direction was initially set at one percent signal change
(PSC) unit higher than the participant achieved in the
most active feedback ROI during the Pre-FB scan. PSC
is the difference in activation in the ROI between the
tapping blocks and rest. In other words, if the PSC
signal change was 1.5% in one ROI and 2% in the other
ROI, the maximum bar width on each side of the bar
would be set at 3%. During each feedback run, if the
participant
was
consistently
achieving
close
to
the
maximum bar width, the maximum width value would be
set 1% higher on the next feedback scan. This was
done to attempt to make the task challenging for the
participant across all training blocks. Thus, the LI given
in the equation above was scaled by the maximum bar
width in order to display the feedback. During
the
tapping
instruction,
participants
were
required to perform the tapping sequence as described
for the functional localizer scan and to make the bar
grow as far to the tapping side as possible (i.e., to grow
to the right during the right-handed tapping scans and to
the left during the left-handed tapping scans). Before
the
scan,
a
number
of
example
strategies
were
suggested to participants (by the experimenter reading
a standardized set of instructions), such as increasing
the rate, force and amplitude of the movement as well
as focusing more on the moving hand and focusing less
on the non-moving hand. During the rest period, the
participants were required to stop moving their hand, lie
still and let the bar shrink toward the center. For the feedback scans, contralateral and ipsilateral
PSC and the magnitude of the LI were submitted to
separate
two
Group
(NF,
Sham), 4
Scan
mixed
ANOVA, with Group as the between subjects factor. Offline data analysis where ROIact = BOLD signal in the motor ROI on the
previous volume; ROIrest = mean BOLD signal in the
motor ROI during the previous rest block. BOLD fMRI data for each subject were analyzed off-line
using tools from the FMRIB software library (http://www. fmrib.ox.ac.uk/fsl). Pre-processing
of
the
images
included, motion correction spatial smoothing using a
Gaussian kernel of 5-mm full width at half maximum
(FWHM), and slice-timing correction. Note that for the Sham group, the midline sham ROIs
were used in place of the Right and Left ROIs in the above
equation. Thus as activity became more right hemisphere
lateralized during left-handed movement, the bar would
grow further to the left and vice versa for the right-
handed
movement. As
the
difference
in
activation
between hemispheres decreased (for instance while the
participant was at rest, or if there was more bilateral
activation during movements), the bar shrunk toward the
center. We calculated an offline LI for each block of each
scan. Note that there are slight differences from the
calculation of LI for bar width which are detailed in the
following paragraph. We first extracted the average
time series across voxels within each of the motor
ROIs for each participant in both the NF and Sham
groups. The PSC associated with tapping in each
block was calculated by taking the difference between
the average signal over the central eight volumes of
the tapping block and the average signal during the
central eight samples of all of the rest blocks and
scaling by the activity during all of the rest blocks in
the
scan. (Fig. 2A). We
started
the
sample
eight
volumes after the initial task instruction. The first four
volume shift accounted for the hemodynamic delay and
the
further
four
volume
shift
was
done
to
ensure
sampling of the central eight volumes of the 15 volume
tapping block. The central eight volumes of each block
were selected because this was the most stable point
in the signal (i.e., the activation was not in the process
of changing from rest to active or vice versa). The
average ipsilateral and contralateral PSC and LI was
calculated for each of the FB scans (or for each hand
in the scans in the case of the Pre and Post NF
scans). The
PSC
from
ROIs
contralateral
and
ipsilateral to the hand moved was used to calculate an
LI (Contralateral PSC – Ipsilateral PSC). Procedure The left image
presents a typical frame during the left tapping block (where the bar growing leftward represents right hemisphere lateralized activity) and the right
image presents a typical frame during a rest block (where a bar close to the center represents LI close to 0 as activity is similar between the two
hemispheres) (B) Image on the left shows the motor ROIs from an example participant (blue boxes) located over sensorimotor cortex. Image on the
right shows sham ROIs from an example participant (blue boxes). (C) Online data processing pipeline (D) Offline data processing pipeline. Please
refer to the text for details on the calculations. Fig. 1. (A) Feedback display shown to participants. The bar updates in width according to the participant’s LI (see text for details). The left image
presents a typical frame during the left tapping block (where the bar growing leftward represents right hemisphere lateralized activity) and the right
image presents a typical frame during a rest block (where a bar close to the center represents LI close to 0 as activity is similar between the two
hemispheres) (B) Image on the left shows the motor ROIs from an example participant (blue boxes) located over sensorimotor cortex. Image on the
right shows sham ROIs from an example participant (blue boxes). (C) Online data processing pipeline (D) Offline data processing pipeline. Please
refer to the text for details on the calculations. H. F. Neyedli et al. / Neuroscience 378 (2018) 165–174 168 on 5-point Likert scale (Likert, 1932) how much control
they felt they had over the bar. The questionnaire also
presented a number of strategies (e.g., focusing more
on the moving hand, moving faster) and asked the partic-
ipant to report whether they used those strategies and
how effective they felt each strategy was on a 5-point Lik-
ert scale. increased or decreased over the course of the TR to the
new value. Thus the edge of the bar appeared to
smoothly and continuously move back and forth of the
block over the course of the entire block rather than
updating in a single jump each TR. The equation used to calculate LI for feedback bar
width during feedback was as follows: LI for Bar Width ¼ ½Left ROIact Left ROIrest=Left ROIrest
½Right ROIact Right ROIrest=Right ROIrest EXPERIMENT II METHODS Methods are broadly similar to Experiment 1 with a few
key changes. First, we tested older adults, who are
more similar in age to typical stroke patients. Second,
participants only trained with one hand, allowing for an
increased amount of neurofeedback training for that one
hand. Further, training with a single limb is more similar
to the paradigm that would be used with stroke patients
who would only be training with their affected limb. We
chose the left hand because Experiment I showed no
transfer for left hand movements following NF removal
thus it was of interest to determine whether more
training could induce transfer in the left hand. To localize the ROIs for feedback, participants were
instructed to tap their fingers in sequence from index to
little finger during tapping blocks (12 s) which were
interspersed
with
rest
blocks
(24 s). Participants
completed three tapping blocks for each hand, first
three blocks for the right hand then three blocks for the
left
hand
(Pre-NF
Scan)
(rather
than
alternating
between hands for each tapping block as was done in
Experiment I). 15 15 9 mm ROIs were centered
over the peak of activation in left and right hand knob. In the NF blocks, participants only used their left hand
and completed four feedback scans with the rest and
tapping blocks having similar timing to Experiment I. For
participants in the Sham Group, a replay of a matched
participant’s data was presented to the participants
using the custom-made plug in for Turbo-Brain Voyager
combined with developed stimulus presentation software
which allowed the Sham Feedback to be triggered in a
similar manner to the real NF. The Post-NF scan was
identical to the Pre-NF scan. The way sham feedback was implemented was also
changed in Experiment II. The use of control brain
areas to provide sham feedback may have frustrated
participants in the sham group in Experiment I given
that they reported feeling as if they had less control over
the feedback than participants in the NF group (detailed
in following results section). In Experiment II, the Sham
feedback consisted of a replay of the feedback from a
yoked participant in the NF group. EXPERIMENT II METHODS Finally, the study was
conducted on a 3T scanner (whereas Experiment I used
7T) as the increased bore size and less stringent safety
requirements of the 3T scanner would make it more
amenable for NF training in a stroke patient population. The older adults had increased head motion with hand
movement, therefore, prior to the offline PSC and LI
calculations, FIX, a FSL based tool to autoclassify noise
components was used to correct for noise components
including motion in the data (Griffanti et al., 2014;
Salimi-Khorshidi et al., 2014). For the feedback scans,
contralateral and ipsilateral PSC and the magnitude of
the LI were submitted to separate two Group (NF,
Sham), 4 Scan mixed ANOVA, with Group as the
between subjects factor. One participant only completed
three NF scans thus was removed from the repeated
measures analysis of training effects. For the Pre and Procedure and offline data analysis Unless
otherwise
specified
in
the
following section, the procedure and
analysis
were
the
same
as
Experiment
I. Imaging
was
performed on a 3.0T Siemens Verio
MRI
system
(Siemens,
Erlangen,
Germany). All
fMRI
scans
were
performed using a 35-slice (3-mm)
axial plane, gradient EPI acquisition,
with
3 3 mm2
in
plane
resolution
(TR = 2000 ms;
TE = 30 ms;
flip
angle = 90
FOV = 192 192 mm;
matrix = 64 64). The feedback was projected to the
participant in the scanner using a 1920 1080 pixel
screen with a 60-Hz refresh rate. al and ipsilateral ROI
cross participants and
ed from the rest block
ed to compute PSC in
ns for movement with
ars are standard error Fig. 2. Experiment I. (A) Timeseries of difference between the contralateral and ipsilateral ROI
(i.e., LI) for the Sham (gray line) and NF (black line) groups averaged across participants and
scans. The red bars along the x-axis indicate the volumes that were sampled from the rest block
and the green bars indicate the samples from the tapping blocks that were used to compute PSC in
each block (see text for details). (B) LI values averaged over all four FB scans for movement with
the left and right hands. There was a significant main effect of group. Error bars are standard error
of the mean. *Significant effect of Group. Offline data analysis For
the
Pre
and
Post-NF
test,
the
contralateral
and
ipsilateral PSC and the magnitude of the LI were
submitted to separate two Group (NF, Sham), 2 Hand
(Left, Right) 2 Scan (Pre, Post-NF) mixed ANOVA,
with Group as the between subjects factor. The Post-FB scan (150 volumes) was identical to the
pre-FB scan. No feedback was provided and only the
instructions ‘Right Tap’, ‘Left Tap’ and ‘Rest’ were
displayed. Participants
were
instructed
to
use
the
strategy that they found most successful during the
preceding FB blocks at increasing the bar magnitude. Mann–Whitney
non-parametric
tests
(Mann
&
Whitney, 1947) were used for comparisons between the
NF and Sham group on the questionnaire scores. Following removal from the scanner, participants
completed a brief questionnaire. Participants indicated H. F. Neyedli et al. / Neuroscience 378 (2018) 165–174 169 Fig. 2. Experiment I. (A) Timeseries of difference between the contralateral and ipsilateral ROI
(i.e., LI) for the Sham (gray line) and NF (black line) groups averaged across participants and
scans. The red bars along the x-axis indicate the volumes that were sampled from the rest block
and the green bars indicate the samples from the tapping blocks that were used to compute PSC in
each block (see text for details). (B) LI values averaged over all four FB scans for movement with
the left and right hands. There was a significant main effect of group. Error bars are standard error
of the mean. *Significant effect of Group. Participants
were
equally
distributed between a neurofeedback
(NF) group (age range: 50–75 years;
median age 64 years, seven males)
or a Sham group (age range: 52–
77 years; median age: 69 years, four
males). There
was
no
significant
difference in age between the NF
and Sham groups (p > 0.10). Similar
to Experiment I, participants were
not aware of their group assignment
and a blinded experimenter provided
all instructions to the participant. Neurofeedback scans Consistent with our hypotheses, the older NF group had a
significantly larger magnitude LI than the Sham group
during neurofeedback scans, F(1,15) = 9.08, p < 0.01,
(Fig. 4A). No other main effects or interactions were
significant for LI. Questionnaire Participants in the Feedback group felt they had more
control over the feedback than participants in the Sham
group, U(24) = 27.0, Z = 2.92, p < 0.05, (NF Mean:
3.41, Range: 1–4; Sham Mean: 2.15, Range: 1–4). Further,
more
participants
in
the
Feedback
group
reported having greater control over one hand than the
other (9/12 participants) than participants in the Sham
group (5/13 participants), a difference that approached
significance,
Z = 1.98,
two-tailed
p < 0.07). The
majority of participants reported they felt more in control
of the feedback when using their dominant (right) hand. With regards to participants’ rankings on how useful the
strategies they tried were, there was no difference in the
rankings between the questions for participants in the
NF group (ps > 0.05). Pre and post test scans Also important for our hypotheses, in the Pre and Post-FB
scans there was a significant interaction between Time,
Hand
and
Group,
F(1,23) = 4.55,
p < 0.05
on
LI
(Fig. 3). The young NF group had a significantly larger
magnitude LI during the right hand tapping blocks in the
Post-feedback scan (Tukey’s CV = 0.57) indicating that
there was some transfer from the feedback training
once feedback had been removed for the right hand. For LI, no main effects or other interactions were
significant. No significant main effects or interactions were found
for
the
neurofeedback
scans
for
the
PSC
in
the
contralateral ROI. In the ipsilateral ROI, the NF group
had a significantly smaller PSC than the Sham group, F
(1,15) = 4.77,
p < 0.05,
(Fig. 4B)
indicating
the
difference in the LI may have been driven by lower
activation in the ipsilateral hemisphere. No other main
effects or interactions were significant for PSC in the
ipsilateral ROI. g
For PSC, we did not find clear evidence for effects of
feedback. In the contralateral ROI, no effects involving
Group were found. In the ipsilateral ROI, a three-way
interaction between Time, Hand and Group, F(1,23)
= 4.68, p < 0.05, was found, but this was driven by
significantly higher ipsilateral activation for the left hand
in
the
Sham
group
during
the
Pre-feedback
scan
(Tukey’s
CV = 0.49). No
other
main
effects
or
interactions were significant. Participants Eighteen healthy older adults over 50 years old were
recruited from the community (age range: 50–77 years;
median age: 67.5 years; 11 males, 1 left handed). All
participants
provided
informed
consent
and
the
procedures were approved by the local ethics board
(University
of
Oxford
Central
University
Research
Ethics Committee, approval reference: MSD-IDREC-C1-
2012-151). H. F. Neyedli et al. / Neuroscience 378 (2018) 165–174 170 Post-NF test, the contralateral and ipsilateral PSC and the
magnitude of the LI were submitted to separate two
Group (NF, Sham), 2 Hand (Left, Right) Scan (Pre,
Post-NF) mixed ANOVA, with Group as the between sub-
jects factor. Neurofeedback scans Consistent with our hypothesis, the young NF group had a
larger magnitude LI than participants in the Sham group
during
the
FB
scans,
F(1,23) = 4.37,
p < 0.05
(Fig. 2A,
B). No
other
main
effects
or
interactions
involving Group were significant for LI. No main effects
or
interactions
were
significant
for
PSC
in
the
contralateral ROI or ipsilateral ROI during the feedback
scans. Thus there is only a difference between the two
groups when the two ROIs are considered together as a
LI, the metric that was fed back to the participants. Pre- and post-test scans erest in comparing Pre- and Post-NF
est if the increased laterality achieved
ning
persisted
after
the
removal
of
ould predict this to be evident by either
a Group (NF, Sham) by Scan (Pre,
Post) interaction where the NF group
would
have
a
larger
LI
following
training in the Post-NF scan or a
Group (NF, Sham) by Scan (Pre,
Post)
by
Hand
(Left,
Right)
interaction
where
an
improvement
would only be seen in the trained,
left
hand. The
Group
by
Scan
interaction
was
not
significant
F
(1,16) = 0.002,
p > 0.10
for
LI
suggesting that the neurofeedback
effect seen during training did not
persist to the Post-NF scan (Fig. 4C)
nor was the three-way interaction F
(1,16) = 0.62, p > 0.10. Our primary interest in comparing Pre- and Post-NF
scans was to test if the increased laterality achieved
during
NF
training
persisted
after
the
removal
of
feedback. We would predict this to be evident by either
a Group (NF, Sham) by Scan (Pre,
Post) interaction where the NF group
would
have
a
larger
LI
following
training in the Post-NF scan or a
Group (NF, Sham) by Scan (Pre,
Post)
by
Hand
(Left,
Right)
interaction
where
an
improvement
would only be seen in the trained,
left
hand. The
Group
by
Scan
interaction
was
not
significant
F
(1,16) = 0.002,
p > 0.10
for
LI
suggesting that the neurofeedback
effect seen during training did not
persist to the Post-NF scan (Fig. 4C)
nor was the three-way interaction F
(1,16) = 0.62, p > 0.10. Our primary interest in comparing Pre- and Post-NF
scans was to test if the increased laterality achieved
during
NF
training
persisted
after
the
removal
of
feedback. We would predict this to be evident by either Fig. 3. Experiment I. LI values for the Pre and Post-NF scans for the left and right hand, Error bars
are standard error of the mean. *Significant effect of Group. There were no significant main
effects or interactions for LI or the
ipsilateral ROI PSC. There was a
significant interaction between Group Fig. 3. Experiment I. LI values for the Pre and Post-NF scans for the left and right hand, Error bars
are standard error of the mean. *Significant effect of Group. H. F. Neyedli et al. / Neuroscience 378 (2018) 165–174 171 Fig. 4. Experiment II. Neurofeedback training In both experiments, participants who received real NF
had more lateralized motor activity during NF scans
than
those
in
the
Sham
group. The
finding
that
participants can modulate the activity in motor cortex
using neurofeedback is consistent with the findings of a
number of previous studies. However, the majority of
previous studies have used motor imagery tasks (e.g.,
Auer et al., 2015; Chiew et al., 2012; deCharms et al.,
2004; Yoo et al., 2008). By contrast, the present study required participants to
physically perform the movements rather than engage in
motor imagery. The use of physical movements may
have made it more difficult to find a difference between
the two groups as the participants in the Sham group
would be expected to and did have strongly lateralized
activity
in
motor
ROIs
while
moving
their
hand
compared to rest. Thus participants in the NF group
were able to further alter the activity in the motor ROIs
beyond
the
Sham
group’s
already
high
level
of
activation, even though both groups had been given the
same instructions for movement strategies by a blinded
experimenter. In contrast, motor imagery is a more
difficult
task
to
perform,
and
many
studies
have
suggested that this task does not typically engage the
primary motor cortex (e.g., Hanakawa et al., 2005). Thus
a proportion of participants are not able to increase activa-
tion in the motor regions of the brain significantly above
rest levels during motor imagery, with or without feedback
(e.g., Auer et al., 2015; Berman et al., 2012; Chiew et al.,
2012; c.f., Bray et al., 2007). The task used in the present
study therefore allowed for a strong test of participants’
ability to use neurofeedback as all participants were able
to perform the task and activate motor cortex, but with the
participants in the NF group showing better performance
at manipulating the LI than those in the Sham group. Fig. 4. Experiment II. (A) Significant difference in laterality index (LI)
between the Sham and NF groups during NF training. (B) LI for each
group during the Pre/Post-NF scans. (C) Significantly larger ipsilat-
eral activation in the Sham group during NF training. (D) Contralateral
activation in Sham compare to NF group. All error bars are standard
error of the mean. Questionnaires There was no significant difference between the NF and
Sham group in how much control they felt they had over
the feedback bar (NF Mean: 2.72, Range: 2–4; Sham
Mean: 2.5, Range: 1–3.5) or between the effectiveness
of any of the strategies queried on the questionnaire (all
ps > 0.10). Pre- and post-test scans (A) Significant difference in laterality index (LI)
between the Sham and NF groups during NF training. (B) LI for each
group during the Pre/Post-NF scans. (C) Significantly larger ipsilat-
eral activation in the Sham group during NF training. (D) Contralateral
activation in Sham compare to NF group. All error bars are standard
error of the mean. Note that PSC and LI values are lower in
Experiment II compared to Experiment I because a 3T (instead of 7T)
scanner was used and FIX (see text for details) was used to auto-
classify and remove noise components from the data. the NF effect did not consistently persist following
removal of the NF. Each of these results will be
discussed in turn. Neurofeedback training Note that PSC and LI values are lower in
Experiment II compared to Experiment I because a 3T (instead of 7T)
scanner was used and FIX (see text for details) was used to auto-
classify and remove noise components from the data. xperiment II. (A) Significant difference in laterality index (LI and Hand for the PSC in the contralateral ROI, F(1,16)
= 5.80, p < 0.05. Post hoc testing revealed greater
activation
for
the
left
(M = 0.31,
SEM = 0.10)
compared to right hand (M = 0.18, SEM = 0.10) in the
NF
group,
t(8) = 4.34,
p < 0.01
but
no
significant
difference between hands for the Sham group, t(8)
= 0.20, p > 0.10 (left, M = 0.40, SEM = 0.10; right,
M = 0.41, SEM = 0.10). Reasons for this particular
interaction are unclear but given that the absence of any
interaction with scan, and the lack of any baseline
differences
in
activity
between
groups,
we
do
not
consider the group hand interaction relevant to the
neurofeedback effect or its generalization. at manipulating the LI than those in the Sham group. Note that there was no effect of scan of any of the
brain activity measures in the current study, indicating
that NF group was quickly able to use the feedback
signal to alter their brain activity and that no further
improvements were seen across scans. This lack of any
apparent learning over scans may have been due to
ceiling effect, or the task may not have been challenging
enough. For
instance,
the
percent
signal
change
associated with the maximum bar width could have
been increased at a greater rate to make it more difficult
for participants to increase the bar width. Finally, further
improvement may have been seen with more training
such as increasing the number of session across days,
an issue that will be discussed more in the following
section regarding transfer to the Post-NF scans. GENERAL DISCUSSION Of note,
Berman et al. (2012) found no difference between a NF
and no NF group during motor execution when the feed-
back derived only from the signal in the contralateral
hemisphere. Moving forward, displaying activity in the
contra and ipsilateral hemisphere separately may encour-
age strategies that both increase activity in the contralat-
eral hemisphere and decrease activity in the ipsilateral
hemisphere, leading to even greater lateralization. A pos-
sible reason for reduction in ipsilateral activity being par-
ticularly evident in the older sample is that older adults
tend to have a more bilateral pattern of activation com-
pared to younger adults with increased activation in the
ipsilateral motor regions (Ward and Frackowiak, 2003). Similarly,
we
are
limited
in
direct,
statistical,
comparisons of the laterality index and PSC values
between the younger and older adults because different
strength scanners (7T for the younger adults and 3T for
the
older
adults)
were
used
between
the
two
Experiments and therefore different levels of signal
change, and therefore LI, would be expected (e.g. LI
values in Figs. 2B and 4A differ substantially). Previous
work comparing EPI signal in the motor cortex has
shown that and increase field strength increases signal-
to-noise ratio (SNR) (e.g., van der Zwaag et al., 2009). An increase in SNR with increased field strength could
provide improved rt-FMRI NF; however, the decreased
bore size and more stringent exclusion criterion at higher
field strengths may not be amenable for a stroke patient
population. Thus it was encouraging that our older adult
population was able to use the NF from the 3T scanner. p
g
(
)
Maintaining a difference in activation following NF
removal is a key requirement in a therapeutic context. We
found
mixed
evidence
for
such
transfer,
with
evidence for transfer for the right hand only for the
younger adults. Older adults only trained with their left
hand and similar to the younger adults, saw no transfer
of altered brain activity following NF removal. All but one
of the participants were right handed, thus it may be
that transfer occurs more readily for the dominant hand;
however, we are unable to conclude this in the older
adult population as NF training with the right hand was
not undertaken for this group. As we will elaborate on
further below, the use of a 3T vs. GENERAL DISCUSSION a 7T scanner for the
older adults is a limitation to comparison between the
two studies. The use of the 3T scanner may have
reduced our ability to detect a transfer effect in the older
adults compared to the younger adults. Similar to other studies (e.g., Bray et al., 2007; Chiew
et al., 2012; deCharms et al., 2004; Yoo et al., 2008) the
present study used sham control groups. Compared to a
‘no feedback’ control group, sham feedback allowed the
experimenter to recreate a similar visual environment
and to provide the same instructions and possible modu-
lation strategies. In the first experiment the use of control
brain areas to provide sham feedback may have frus-
trated participants in the Sham group. Consistent with
this, participants in the sham group for Experiment 1
reported lower feelings of control over the feedback signal
than the NF group. The second experiment used a differ-
ent control condition, presenting NF from a yoked partici-
pant as Sham. In this case, participants in the Sham
group were seeing the same stimuli (and receiving the
same impression of improving performance) as the NF
group, but the changes in the NF display did not match
the changes in their brain activity (and their brain LI did
not increase). In Experiment 2, both the NF and the Sham
groups reported a similar level of control of the NF signal
thus yoked feedback may better match feelings of feed-
back control between the two groups. As noted previ-
ously, in both experiments the NF had more lateralized
activity than the Sham group thus the type of Sham
may play a minimal role in the determining the effects of
neurofeedback on LI. This observation is consistent with
the finding from a study that used RT-fMRI neurofeed-
back to down-regulate the rostral anterior cingulate cortex
to reduce pain (deCharms et al., 2005). A feedback group
was compared to four different control groups included a
no-feedback group, a yoked-sham group where the feed-
back was based on another participant’s feedback and
feedback from an unrelated brain area. The feedback y
g
Regardless of hand dominance, more neurofeedback
training may be necessary for participants to maintain the
altered activation patterns. To minimize fatigue this could
be delivered over multiple sessions rather than longer
sessions. GENERAL DISCUSSION Taken together, we have shown in two different samples,
that participants can use a NF signal to control the
laterality
of
their
brain
activity
while
executing
movements. Further, this effect was present across two
different age ranges, and importantly was present in a
cohort of participants that had a similar age range as
many people who have had a stroke. With regards to
potential clinical application, it was less encouraging that Our results show that both younger and older adults
were able to increase LI through neurofeedback. LI
contrasts activity across the two hemispheres so we
aimed to unpack this effect by investigating signal
change within the ipsilateral or contralateral ROIs. For
the younger adults in Experiment I no significant effects H. F. Neyedli et al. / Neuroscience 378 (2018) 165–174 172 the worst performance in transfer tests results from
blocked-schedule practice and mixed-schedule transfer
(e.g., Brady, 2004; Magill and Hall, 1990). In addition, in
both experiments, the tapping–rest block cycle in the
Post-NF scan was shorter (12 s tapping/ 24 s rest) com-
pared to that used in training (30 s tapping/ 30 s rest). The difference in timing between the NF scans and the
Pre and Post NF scans is a limitation in the design
because it restricts to comparisons and conclusions that
can be drawn from comparing between NF training and
after NF removal. were found, whereas for the older adults in Experiment II,
there was reduced activation in the ipsilateral ROI in the
NF group compared to the Sham Group. Of note,
Berman et al. (2012) found no difference between a NF
and no NF group during motor execution when the feed-
back derived only from the signal in the contralateral
hemisphere. Moving forward, displaying activity in the
contra and ipsilateral hemisphere separately may encour-
age strategies that both increase activity in the contralat-
eral hemisphere and decrease activity in the ipsilateral
hemisphere, leading to even greater lateralization. A pos-
sible reason for reduction in ipsilateral activity being par-
ticularly evident in the older sample is that older adults
tend to have a more bilateral pattern of activation com-
pared to younger adults with increased activation in the
ipsilateral motor regions (Ward and Frackowiak, 2003). were found, whereas for the older adults in Experiment II,
there was reduced activation in the ipsilateral ROI in the
NF group compared to the Sham Group. GENERAL DISCUSSION For instance, Auer et al., 2015 found that partic-
ipants were able to maintain increased lateralization fol-
lowing NF removal after 12 sessions spread over four
weeks. Another possibility is for patients to practice the
task outside of the scanner following an initial NF session. For example, Yoo et al. (2008), provided young adults
with neurofeedback from contralateral motor cortex while
performing motor imagery. Participants then practiced the
motor imagery task daily at home for two weeks and par-
ticipants were able to maintain the level of activation seen
in the presence of neurofeedback after the neurofeedback
was removed. Another factor that may have limited our ability to
detect transfer effects was differences in the timing and
movement conditions between the Pre/Post-NF scans
compared to the NF scans. For instance, in Experiment
I, a blocked practice schedule was used where each
scan consisted of participants only using one hand. The
Post-test however had a mixed test schedule with
participants alternating between using their right and left
hand on each tapping block within the scan. Previous
learning
research
has
demonstrated
that
the
best
performance in transfer test results from mixed-schedule
practice with a blocked-schedule transfer test whereas H. F. Neyedli et al. / Neuroscience 378 (2018) 165–174 173 (28):7498–7507. https://doi.org/10.1523/JNEUROSCI.2118-
07.2007. (28):7498–7507. https://doi.org/10.1523/JNEUROSCI.2118-
07.2007. https://doi.org/10.1523/JNEUROSCI.2118- group had better performance than all of the control
groups, which performed similarly. Chiew M, Laconte SM, Graham SJ (2012) NeuroImage Investigation
of fMRI neurofeedback of differential primary motor cortex activity
using
kinesthetic
motor
imagery. Neuroimage
61(1):21–31. https://doi.org/10.1016/j.neuroimage.2012.02.053. ACKNOWLEDGMENTS The research leading to these results has received
funding
from
the
People
Programme
(Marie
Curie
Actions) of the European Union’s Seventh Framework
Programme
FP7/2007-2013/under
REA
grant
agreement
no. PITN-GA-2011-290011. HJB
is
a
Wellcome Trust Principal Research Fellow (110027/
Z/15/Z). MAK is funded by a National Institute for Health
Research
(NIHR)
Academic
Clinical
Fellowship
in
Neurosurgery. We acknowledge support from the NIHR
Oxford Biomedical Research Centre. p
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Cortical
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changes underlying stimulation-induced behavioural gains in (Received 27 July 2016, Accepted 7 February 2017)
(Available online 15 February 2017)
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THE ROLE OF THE RELIGIOUS SCHOLARS OF FAQIH ALI DATO’ MAHARAJA’S DESCENT IN UPHOLDING SHARIA IMPLEMENTATION IN TERENGGANU
|
Malaysian journal of syariah and law/Malaysian Journal of Syariah and Law
| 2,021
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cc-by-sa
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Vol. 12, No. 1, pp. 216-227 | APRIL 2024
DOI: https://doi.org/10.33102/mjsl.vol12no1.674 i,ii,*Wan Ahmad Fauzi Wan Husain, iiiWan Rohaida Wan Husain & ivHanif Md Lateh iInternational Institute of Islamic Thought and Civilization (ISTAC-IIUM), 50480 Kuala Lumpur, Malaysia
iiFaculty of Industrial Management, Universiti Malaysia Pahang Al-Sultan Abdullah, Persiaran Tun Khalil Yaakob, 26300
Kuantan, Pahang, Malaysia g
y
iiiDepartment of Business Administration, Kuliyah of Economics & Management Sciences, International Islamic University
Malaysia, 53100 Kuala Lumpur, Malaysia g
y
epartment of Business Administration, Kuliyah of Economics & Management Sciences, International Islamic Universi
Malaysia, 53100 Kuala Lumpur, Malaysia y
p
y
esearch Institute for Islamic Products and Malay Civilization (INSPIRE), Universiti Sultan Zainal Abidin, Gong Bada
Campus, 21300 Kuala Nerus, Terengganu, Malaysia ivResearch Institute for Islamic Products and Malay Civilization (INSPIRE), Universiti Sultan Zainal Abidin, Gong Badak
Campus, 21300 Kuala Nerus, Terengganu, Malaysia *(Corresponding author) e-mail: wanfauzi@iium.edu.my ABSTRACT Article history:
Submission date: 15 Dec 2023
Received in revised form: 22 March 2024
Acceptance date: 27 March 2024
Available online: 30 April 2024 Faqih Ali Dato’ Maharaja is a well-known figure among local
historians, particularly among those who study prominent religious
scholars in Southeast Asia. His descendants contributed significantly
to promoting the sustainability of Islamic teaching in Terengganu. Islam was upheld as the principle of Terengganu's sovereignty,
forming the basis of its constitutional system, thus legitimizing the
position of Sharia as the law of the land. This was manifested on the
Inscribed Stone dated 4 Rejab 702H. Nevertheless, the role of the
descendants of Faqih Ali have not been a focal point in academic
writing. The paucity of literature and elucidation within this domain
serves as the basis for this study. This article aims to investigate the
role of prominent religious scholars of Faqih Ali Dato’ Maharaja's
descent in upholding the implementation of Sharia in the Terengganu
Sultanate until the Independence Day of Tanah Melayu in 1957. This
study is qualitative in nature and employs historical research methods. Data were obtained from archival documents, government
publications, field research, and secondary sources. Findings show that
the preservation of the Sharia legacy in Terengganu since the discovery
of the Inscribed Stone was influenced significantly by the substantial
contributions of religious scholars who were also the administrators of
Faqih Ali Dato’ Maharaja’s descent, evidenced by the legislation of
Itqanul Muluk bi Ta'dil al-Suluk in 1911 and its extension, the
Terengganu State Constitution. Hence, the practice of Sharia before
1957, was not confined to personal affairs as understood by many; by
virtue of the Federal Constitution, the public affairs of Sharia are now
entrusted to public authorities at the federal level unless those matters
are retained in the state administration. Keywords:
Faqih Ali Dato’ Maharaja, Terengganu State
Constitution, Wan lineage, religious scholars,
Joint-court Keywords:
Faqih Ali Dato’ Maharaja, Terengganu State
Constitution, Wan lineage, religious scholars,
Joint-court Funding: Funding:
International Institute of Islamic Thought and
Civilization (ISTAC) Research Grant Scheme
(grant number: ISTAC23-013-0015). Funding:
International Institute of Islamic Thought and
Civilization (ISTAC) Research Grant Scheme
(grant number: ISTAC23-013-0015). Competing interest:
The author(s) have declared that no
competing interests exist. Introduction Faqih Ali Dato’ Maharaja is an imminent figure among local historians, particularly among those who
study prominent religious scholars in Southeast Asia, including Sheikh Ahmad al-Fathani, Sheikh Daud
al-Fathani and Sheikh Abdul Kadir al-Fathani. Those great Muslim scholars are the descendants of Faqih
Ali Dato’ Maharaja. He was also known as Faqih Nik Ali al-Malbari or Andik Ali. Reliable sources
claimed that Faqih Ali Dato’ Maharaja had two wives, Che Dewi binti Dato’ Seri Bija diRaja and Wan
Teja binti Paduka Raja Tun Abdul Jamil. However, according to Dato’ Perdana Menteri Paduka Raja
Kelantan Haji Nik Mahmud bin Nik Ismail, Wan Bidah binti Wan Ibrahim @ Tok Mekong was the third
wife of Faqih Ali Dato’ Maharaja. His descendants could be traced from genealogical trees maintained
by credible figures and typically carry the surnames of Wan, Nik and Tengku (Nik Mansor, 2023; Nik
Abdul Rahman, 2019). Tun Zainal Abidin, the founding Sultan of the modern Terengganu Sultanate in 1708 CE, was the adopted
son of Paduka Raja Tun Abdul Jamil, the father-in-law of Faqih Ali Dato’ Maharaja. He is the son of
Bendahara Seri Maharaja Tun Habib Abdul Majid. Before his installation as the Sultan of the modern
Terengganu, he was brought to Patani because Paduka Raja’s daughter, Wan Teja, had married Faqih Ali
Dato’ Maharaja, and resided in Jambu, Patani. However, Paduka Raja was killed in Seberang Takir, Kuala
Terengganu, en route to Patani. The above family background explains why Tun Zainal Abidin arrived in
Terengganu as part of an entourage of 80 families from Patani under the command of the Patani Queen,
Nang Chayam. Religious scholars played a fundamentally important role in the state of Terengganu from
the very beginning of the establishment of the Sultanate. For instance, the coronation of Tun Zainal Abidin
as the first Sultan of Terengganu proclaimed as Sultan Zainal Abidin Shah I, was performed by Qadi Wan
Imam Mahmud, a representative from Patani, in Tanjung Baru, Hulu Terengganu, marking the
establishment of a new dynasty. Interestingly, Qadi Wan Imam Mahmud bin Faqih Wan Hasan was the
grandson of Faqih Ali Dato’ Maharaja (Mohamed Anwar et al., 2011). In historical terms, the Terengganu Inscribed Stone is a significant piece of evidence that demonstrates
the early Islamisation of Terengganu, preceding the process in Melaka and even much earlier than the
founding of the modern Terengganu Sultanate itself. Cite as: Wan Husain, W. A. F., Wan Husain, W. R., &
Md Lateh, H. (2024). The role of the religious
scholars of Faqih Ali Dato’ Maharaja’s
descent in sustaining Sharia implementation
in Terengganu. Malaysian Journal of Syariah
and Law, 12(1), 216-227. https://doi.org/10.33102/mjsl.vol12no1.674 © The authors (2024). This is an Open
Access article distributed under the terms of
the Creative Commons Attribution (CC BY
NC) (http://creativecommons.org/licenses/by- ) (
p
g
y
nc/4.0/), which permits non-commercial re-
use, distribution, and reproduction in any
medium, provided the original work is
properly cited. For commercial re-use, please
contact usimpress@usim.edu.my. ) (
p
g
y
nc/4.0/), which permits non-commercial re-
use, distribution, and reproduction in any
medium, provided the original work is
properly cited. For commercial re-use, please
contact usimpress@usim.edu.my. 216 3. Judiciary function. 3. Judiciary function. Accordingly, the role played by the descendants of Faqih Ali Dato’ Maharaja in sustaining the legacy of
Syariah in the Terengganu Sultanate from its establishment until Independence Day is investigated from
the above framework. Sharia and Islamic legislation are two related aspects as opposed to the Westminster concept of legislation. Sharia is the revelation sent to the Holy Prophets and the Messengers of Allah the Almighty. Whilst Al-
Qur’an is the final revelation from Allah, Hadith or the Tradition of the Holy Prophet Muhammad SAW
is the manifestation of Al-Qur’an. Al-Qur’an and Hadith embody the Islamic laws and legal principles
underpinning juristic opinions (ijtihad) and legislation. Those legal principles are the fundamentals of
legislating statutes, and no laws shall contradict the Sharia. The role of religious scholars is critical in interpreting those religious injunctions and addressing the
contemporary needs and challenges in public lives that rely upon the legal principles for legislative,
executive and judicial actions. Meanwhile, the Westminster concept of legislation manifested in
parliamentary democracy recognises the general will of the people they represent. The members of the
Parliament can make and amend law beyond religious sanctions as long as its legislative procedure is
adhered to. Hence, the role of religious scholars is significant before and after independence day (Husain,
2018; Husain, 2021; Husain, 2022). In the traditional political system, the three branches of sovereign rule are performed by authorities as
follows: 1. Religious advisors, mufti, sheikhul ulama, khadimu as-syar’ie, and members of the State Council
undertook legislative functions. 1. Religious advisors, mufti, sheikhul ulama, khadimu as-syar’ie, and members of the State Council
undertook legislative functions. 2. Ministers, mufti, commissioners, chiefs and officers performed executive functions. 3. Mufti, judges and qadis administered judiciary functions. The above practice indicates no apparent separation of powers but the distribution of the three branches
under sovereign rule since the ultimate authority is vested in Allah the Almighty alone. This study
investigates the role of the religious scholars of Faqih Ali Dato’ Maharaja’s descent in the above functions
in the stipulated period. Methodology This is a qualitative study employing historical research methods. Data was extracted from archives,
government publications and secondary sources. This study uses thematic analysis to extract historical
facts in the local context. The roles of religious scholars are defined based on the three branches of a
sovereign rule, consisting of the following: 1. Legislative function. 2. Executive function. 3. Judiciary function. Introduction 216-227 | April 2024 Persekutuan Tanah Melayu or, in English, Malaya (Federation of Malaya Independence Act, 1957, 5 & 6
ELIZ. 2, 31st. July 1957 & Federation of Malaya Agreement, 1957). Persekutuan Tanah Melayu or, in English, Malaya (Federation of Malaya Independence Act, 1957, 5 & 6
ELIZ. 2, 31st. July 1957 & Federation of Malaya Agreement, 1957). Introduction The process of Islamisation in Terengganu carries a
significant theme in the history of Islam in this region. This Sultanate holds a vital treasure trove of
information regarding the earliest Islamisation in the Malay archipelago, known as the Terengganu
Inscribed Stone, dated 702 Hijrah, corresponding to 1303 CE. Historians, both within and outside the
country, who have conducted specific research on historical sources unanimously conclude that the
Terengganu Inscribed Stone serves as evidence of the beginning of the spread of Islam in this region. It
is the oldest text written in the Malay language using Arabic script (Jawi). Casparis (1980), a respected
scholar of ancient Malay Archipelago (Nusantara) history, particularly in deciphering ancient inscriptions
in the region, stated that the Terengganu Inscribed Stone is the first known text in Jawi script. The
inscription originates from the Malay Peninsula, and its tradition is likely shared with the island of
Sumatra as well. The above statement contains two crucial points: Firstly, it validates the Terengganu Inscribed Stone,
prepared by a highly respected regional historian, as the earliest Malay-language Islamic inscription in
the Malay Archipelago region. Many other scholars have also echoed this validation. Secondly, although
the first Malay-language inscription created in the Malay Archipelago was carved in the Malay Peninsula,
its roots can be traced back to Sumatra (Mohamed Anwar, 2023; Kozok, 2006). According to Kozok
(2006), who studied the ancient texts of the Kerinci tribe, specifically the "Kitab Undang-undang Tanjung
Tanah”, the Malay calligraphic tradition peaked in popularity when Arabic-Malay script evolved into
post-Pallava writing in various places since the 14th century, as evidenced by the oldest Jawi inscription,
the Terengganu Inscribed Stone. Therefore, it has its roots in the authentic Islamic calligraphic tradition
instead of being an imitation of the writing of other nations. This article aims to investigate the role of prominent religious scholars of Faqih Ali Dato’ Maharaja’s
descent in sustaining the implementation of Sharia in the Terengganu Sultanate until Independence Day
on the 31st of August 1957. Independence Day was a significant timeline for this study as it marked the
establishment of a new sovereign and independent federation within the Commonwealth. The Federation
consists of nine sovereign Malay states that accepted two strait settlements bearing the name of 217 Malaysian Journal of Syariah and Law | مجلة الشريعة والقانون بماليزيا | Vol. 12, No. 1, pp. Legacy of the Sharia in Terengganu The current Sharia legacy in Terengganu indicates the vigorous enforcement of Islamic legislation no
later than 4 Rejab 702H (which corresponds to February 22, 1303, CE), as documented on the Terengganu
Inscribed Stone. The Terengganu Inscribed Stone was discovered on the banks of the Tersat River in
Kuala Berang, Terengganu, in 1887. The content of the Inscribed Stone shows that Islamic legislation had
already become the foundation of the local legal system, although not all Sharia laws were recorded. On the front face of the Inscribed Stone, the following is recorded: Rasulullah dengan yang arwah santabi mereka Asa pada Dewata Mulia Raya beri hamba
meneguhkan agama Islam. Dengan benar bicara derma mereka bagi sekalian hamba
Dewata Mulia Raya. Di Benuaku ini penentu agama RasululLah sallalLahi wassalama
raja Mandalika yang benar bicara sebelah Dewata Mulia Raya di dalam Bumi penentuan
itu fardhu pada sekalian Raja Mandalika Islam menurut setitah Dewata Mulia Raya
dengan benar. Bicara berbajiki benua penentuan itu maka titah Seri Paduka. Tuhan
menduduki Tamra ini di Benua Terengganu adi pertama ada. Translation: The Prophet, along with the departed saints, strengthens the faith in Islam
for us through the grace of the Noble and Great Deity. Their truthful speech is a charity
for all of us, the servants of the Noble and Great Deity. In my land, the determination of
the Prophet's religion, peace be upon him, is the true speech of the Mandalika king, who
sides with the Noble and Great Deity on Earth. This determination is obligatory for all
Islamic Mandalika Kings according to the decree of the Noble and Great Deity with truth. The speech filled with virtue marks this land of determination, thus decreed by His
Majesty. The Lord occupies this Tamra in the land of Terengganu, where the first nobility
exists. Jumaat di bulan Rajab di tahun saratan disasanakala. Baginda RasululLah telah lalu
tujuh ratus dua Translation: On a Friday in the month of Rajab, in the year of Saratan according to the
calendar. The Messenger of Allah passed away in the year seven hundred and two. On the back face of the Inscribed Stone, the following is recorded: On the back face of the Inscribed Stone, the following is recorded: Keluarga di Benua jauhkan (Datang berikan. Keempat orang berpiutang. Jangan mengambil … (a)mbil hilangkan emas. Kelima derma barang orang …
(mar)dika. Jangan mengambil (tugas buat) temasnya Jika ia ambil hilangkan emas. THE ROLE OF THE RELIGIOUS SCHOLARS OF FAQIH ALI DATO’ MAHARAJA’S DESCENT IN SUSTAINING SHARIA IMPLEMENTATION IN
TERENGGANU THE ROLE OF THE RELIGIOUS SCHOLARS OF FAQIH ALI DATO’ MAHARAJA’S DESCENT IN SUSTAINING SHARIA IMPLEMENTATION IN
TERENGGANU command of Sultan Muhammad Shah (A. Samad, 2015). Subsequently, from the mid-16th century to the
early 18th century, Terengganu was ruled interchangeably by two Sultanates, Johor and Patani. command of Sultan Muhammad Shah (A. Samad, 2015). Subsequently, from the mid-16th century to the
early 18th century, Terengganu was ruled interchangeably by two Sultanates, Johor and Patani. Sultan Zainal Abidin is a half-brother of Sultan Abdul Jalil Riayat Shah IV. Sultan Abdul Jalil Riayat
Shah IV ascended to the Johor-Riau Lingga-Pahang Sultanate's throne after Sultan Mahmud II died in
1699. His prince, Bendahara Seri Maharaja Tun Abas, was assigned to rule Pahang, where his grandson,
Temenggong Seri Maharaja Tun Jamal, is the predecessor of the present-day Sultanate of Johor. Sultan
Zainal Abidin Shah ruled until his passing in 1733. The current Sultan of Terengganu is His Royal
Highness Al-Wathiqu Billah Sultan Mizan Zainal Abidin, the prince of the late Sultan Mahmud Al-
Muktafi Billah Shah, is the 16th Sultan through the lineage of Sultan Zainal Abidin III. He has been
reigning since May 15, 1998 (Mohamed & Nik, 2009). The Sultanate of Terengganu According to Chao Ju Kua's (1967) records from the 12th or 13th century, Terengganu is the name of an
ancient kingdom once under the influence of Srivijaya. The record indicates the existence of the
Terengganu kingdom predating Melaka (Buyong, A., 1974). However, during the reign of Sultan
Muhammad Shah (1424-1444 CE), Terengganu was part of the Malacca Sultanate's dominion. The
Hikayat Hang Tuah recounts an event in which the Terengganu prince, Megat Panji Alam, was killed by
Hang Jebat and Hang Kasturi in Istana Inderapura during the time of Sultan Mansor Shah of Melaka
(1456-1477 CE) (Kassim, A., 1997). Besides, records from the era of Sultan Alauddin Shah of Melaka
(1477-1488 CE) associated the killing of Telanai Terengganu by Seri Akar Raja in 1478 with the 218 On the left side of the Inscribed Stone, the following is recorded: … tiada benar dendanya setahil sepaha kesembilan derma. … Seri Paduka Tuhan (Tuan)
siapa tiada harta dendanya … Ke sepuluh derma jika anakku atawa pemainku atawa
cucuku atawa keluarga ku atawa anak. … tamra ini segala isi tamra ini barang siapa
tiada menurut tamra ini laknat Dewata Mulia Raya. … dijadikan Dewata Mulia Raya
bagi yang langgar acara tamra ini. … tiada benar dendanya setahil sepaha kesembilan derma. … Seri Paduka Tuhan (Tuan)
siapa tiada harta dendanya … Ke sepuluh derma jika anakku atawa pemainku atawa
cucuku atawa keluarga ku atawa anak. … tamra ini segala isi tamra ini barang siapa
tiada menurut tamra ini laknat Dewata Mulia Raya. … dijadikan Dewata Mulia Raya
bagi yang langgar acara tamra ini. Translation: ... not allowed, the fine is one tahil and three-quarters for the ninth donation. ... His Majesty the Lord (Master) for those without wealth, their fine... The tenth donation,
if my child or my dependant or my grandchild or my family or child... of this Tamra, all
contents of this Tamra, whoever does not follow this Tamra, cursed by the Noble and
Great Deity. ... made by the Noble and Great Deity for those who violate the rules of this
Tamra. Excerpt: “Rasulullah dengan yang arwah santabi mereka Asa pada Dewata Mulia Raya beri hamba
meneguhkan agama Islam. Dengan benar bicara derma mereka bagi sekalian hamba Dewata Mulia
Raya. Di Benuaku ini penentu agama RasululLah sallalLahi wassalama raja Mandalika yang benar
bicara sebelah Dewata Mulia Raya di dalam Bumi penentuan itu fardhu pada sekalian Raja Mandalika
Islam menurut setitah Dewata Mulia Raya dengan benar." This statement emphasizes the function of
Islam as the state religion vis-à-vis the principle of sovereignty, which defines the ruler’s responsibility
to uphold Islamic principles as conveyed by the Prophet Muhammad (peace be upon him). According to
Husain (2018), sovereignty is the main element to be examined when articulating a legal system in the
study of constitutional systems. Its principle of sovereignty serves as the source of authority that
legitimizes legislative, executive, and judicial powers. Jabatan Warisan Negara (2023) confirms that the Terengganu Inscribed Stone is the earliest evidence of
the Jawi script based on the Arabic alphabet in the Malay Islamic world of Southeast Asia. Legacy of the Sharia in Terengganu Keenam derma
barang orang berbuat bala cara laki-laki perempuan satitah Dewata Mulia Raya jika
merdeka bujang palu. Seratus ratun jika merdeka beristeri. Atawa perempuan bersuami
ditanam hinggakan pinggang dihambalang dengan batu matikan. Jika inkar (bala cara)
hambalang jika anak Mandalika … 219 Malaysian Journal of Syariah and Law | مجلة الشريعة والقانون بماليزيا | Vol. 12, No. 1, pp. 216-227 | April 2024 Translation: Families in the distant lands (bring offerings. Four people are indebted. Do not take... (take) away the gold. The fifth is to donate items from free people. Do not undertake (the task of creating) its problems if it involves taking away gold. The
sixth is to donate items from those who cause trouble, in the manner of both men and
women, according to the decree of the Noble and Great Deity, if free and single, a fine of
one hundred ratun; if free and married, or a woman with a husband, to be buried up to the
waist and stoned to death. If denying (the manner of causing trouble) stoning if a child of
Mandalika... On the right side of the Inscribed Stone, the following is recorded: On the right side of the Inscribed Stone, the following is recorded: Bujang dandanya sepuluh tengah tiga jika ia … Menteri Bujang dandanya tujuh tahil
se(paha … Tengah tiga, jika tetua bujang dandanya lima (tahil … Tujuh tahil sepaha
masuk bendara. Jika O(rang) … Merdeka. Ketujuh derma barang perempuan hendak …
Tiada dapat bersuami, jika ia berbuat balabicara …
Translation: The fine for a single man is ten and three-quarters if he... The fine for a
Minister who is single is seven tahils (thigh... Three-quarters, if an elder who is single,
the fine is five (tahils... Seven tahils are required for entering into the ranks. If a (person)... is free. The seventh is to donate items for a woman who wishes to... cannot find a
husband, if she engages in misconduct... Bujang dandanya sepuluh tengah tiga jika ia … Menteri Bujang dandanya tujuh tahil
se(paha … Tengah tiga, jika tetua bujang dandanya lima (tahil … Tujuh tahil sepaha
masuk bendara. Jika O(rang) … Merdeka. Ketujuh derma barang perempuan hendak …
Tiada dapat bersuami, jika ia berbuat balabicara … Bujang dandanya sepuluh tengah tiga jika ia … Menteri Bujang dandanya tujuh tahil
se(paha … Tengah tiga, jika tetua bujang dandanya lima (tahil … Tujuh tahil sepaha
masuk bendara. Jika O(rang) … Merdeka. Ketujuh derma barang perempuan hendak …
Tiada dapat bersuami, jika ia berbuat balabicara … Translation: The fine for a single man is ten and three-quarters if he... The fine for a
Minister who is single is seven tahils (thigh... Three-quarters, if an elder who is single,
the fine is five (tahils... Seven tahils are required for entering into the ranks. If a (person)... is free. The seventh is to donate items for a woman who wishes to... cannot find a
husband, if she engages in misconduct... On the left side of the Inscribed Stone, the following is recorded: On the left side of the Inscribed Stone, the following is recorded: THE ROLE OF THE RELIGIOUS SCHOLARS OF FAQIH ALI DATO’ MAHARAJA’S DESCENT IN SUSTAINING SHARIA IMPLEMENTATION IN
TERENGGANU THE ROLE OF THE RELIGIOUS SCHOLARS OF FAQIH ALI DATO’ MAHARAJA’S DESCENT IN SUSTAINING SHARIA IMPLEMENTATION IN
TERENGGANU Inscribed Stone additionally documents the regional trade development during the process of Islamisation
and portrays the patterns of trade and community movements at that time. Inscribed Stone additionally documents the regional trade development during the process of Islamisation
and portrays the patterns of trade and community movements at that time. During the reign of Sultan Zainal Abidin Shah, the Terengganu political system was very much influenced
by the Sultanate of Melaka, possibly due to the close relationship it had with Melaka, Johor, and Patani
(Mohamed, 2009). However, after the fall of the port city of Melaka, the constitutional law, namely the
Melaka Legal Code, was extended to suit local needs. The code was later renamed after Johor, Pahang,
and Perak. The Pahang Legal Code stipulates that the code is applicable in Pahang, Johor, and Perak
(Husain, 2022). From that era until 1948, the Mufti held a more critical position and played a more significant role than
today because they administered justice that encompassed Sharia's public and personal affairs. The Mufti
also served as a royal advisor in all matters of the Sultanate rule. During the Sultanate of Baginda (Sultan)
Umar, Mufti played challenging roles as the former imposed a centralization policy and was actively
involved in the administration. Terengganu has two written constitutions, Itqanul Muluk 1911 and its
extension, Undang-Undang bagi Diri Kerajaan Terengganu (the Terengganu State Constitution). A
comparison between the two was made by Husain (2017; 2018) to illustrate the continuation of the
principle of sovereignty based on the following aspects: 1. The ruler is of an independent sovereign government. 2. The ruler embraces Islam. 2. The ruler embraces Islam. 3. The ruler ascends to the throne through consultation and an oath of allegiance (bai'ah). 4. The application of the local principle of sovereignty. Sultan Ismail Nasiruddin Shah Ibni Almarhum Sultan Zainal Abidin III, on February 1, 1948, decreed the
Terengganu State Constitution (Part One) as an addition to Itqanul Muluk 1911, by the advice and
consensus of the members of the Council of Ministers and the State Council. Subsequently, on August 9,
1950, the Terengganu State Constitution (Part Two) was proclaimed. The institutionalization of the
Terengganu State Constitution (Part One) was in line with the Terengganu State Agreement of 1948, dated
January 21, 1948. On the left side of the Inscribed Stone, the following is recorded: Since the
arrival of Islam in the region in the 10th or 11th century, the way of life-based on the Quranic and the
Hadiths, as practised by the Prophet Muhammad (peace be upon him), has flourished in Southeast Asia. It marked the increasing use of the Jawi script, gradually replacing Sanskrit writing. This artefact further
reflects the development of a culture based on Islamic teachings that grew parallel with the expansion of
maritime trade centred around Kuala Berang, where the Terengganu Inscribed Stone was discovered. The 220 THE ROLE OF THE RELIGIOUS SCHOLARS OF FAQIH ALI DATO’ MAHARAJA’S DESCENT IN SUSTAINING SHARIA IMPLEMENTATION IN
TERENGGANU Among the commandments stipulating the principle of sovereignty and position of Sharia is Article 51 of
Itqan al-Muluk 1911 and Article 5 of the Terengganu State Constitution (Part One). Article 51 reads as
follows: Sesungguhnya telah disifatkan dan disebutkan dan ditetapkan selama-lamanya
Kerajaan Terengganu ini kerajaan Islamiyyah Malayuwiyyah ialah yang dikatakan
ugama negeri dan kerajaan maka tiadalah boleh sekali-kali sebarang ugama lain dijadi
atau disebut ugama negeri sekalipun banyak segala bangsa dan ugama diamankan dan
dibenarkan masuk diam di dalam negeri dan jajahan Terengganu dibenarkan masuk
diam di dalam negeri dan jajahan Terengganu. Translation: Indeed, it has been described, mentioned, and established that the Malay
State of Terengganu is genuinely an Islamic Malay government, proclaimed as the state
religion and government. Therefore, no other religion shall ever be made or proclaimed
as the state religion, even if various races and religions are allowed to live in and enter
the state and its territories of Terengganu. Article 5 of the Terengganu State Constitution (Part One) requires the Sultan of Terengganu to rule jus
and by the law. Article 5 reads as follows: Duli Yang Maha Mulia hendaklah melaku membawa diri Baginda kepada semua rakyat
Baginda dan kepada semua orang yang duduk di dalam Negeri dengan keadaan adil dan
memerintah menurut undang-undang. Tiadalah boleh seseorang itu dihilangkan
kemerdekaannya, ditahan atau dipenjara akan dia melainkan dengan mengikut
perjalanan undang-undang. Kebebasan dan kemerdekaan segala orang yang tidak 221 Malaysian Journal of Syariah and Law | مجلة الشريعة والقانون بماليزيا | Vol. 12, No. 1, pp. 216-227 | April 2024 ditegah oleh perbuatan benar dan adil bagi undang-undang itu adalah asas bagi
kesemua Kerajaan yang baik. ditegah oleh perbuatan benar dan adil bagi undang-undang itu adalah asas bagi
kesemua Kerajaan yang baik. Translation: His Royal Highness shall conduct himself to his subjects and all those who
reside in the State with justice and rule by the law. No person shall be deprived of his
freedom, detained, or imprisoned except by the procedures of the law. Freedom and
liberty of every person not prohibited by the just and lawful actions of the law are the
foundation of a good government. THE ROLE OF THE RELIGIOUS SCHOLARS OF FAQIH ALI DATO’ MAHARAJA’S DESCENT IN SUSTAINING SHARIA IMPLEMENTATION IN
TERENGGANU The Islamic features of the Terengganu State Constitution (Part One) are also found in its preamble that
reads, “In the name of God, the Compassionate, the Merciful, PRAISE be to God, the Lord of the
Universe, and may the benediction and peace of God be upon Our Leader Muhammad and all his Relations
and Friends”. Itqan al-Muluk bi al-Ta‘dil al-Suluk 1911 was a constitution that was enacted, signed, and promulgated
as the constitution of the Malay Sultanate of Terengganu on 11 Zul Kaedah 1329H (corresponding to
November 2, 1911M) by Sultan Zainal Abidin Shah III and the state dignitaries, the majority of the
members were from the local religious scholars. Itqan al-Muluk bi al-Ta‘dil al-Suluk is translated as "The
King's Dedication to Upholding Just Government". Two prominent rulers in Terengganu's history are
often associated with drafting Itqan al-Muluk 1911. The first was Sultan Omar (1831, 1839-1876), and
the second was Sultan Zainal Abidin III (1881-1918). The institutionalization of Itqan al-Muluk 1911, according to Lateh (2017), where its form was modelled
after Undang-Undang Tubuh Kerajaan Johor 1895 to serve various purposes, mainly overcoming the
British intervention and foreign influence in Terengganu and the following: 1. Expressing Islam as the state religion and Terengganu as a Malay government. 1. Expressing Islam as the state religion and Terengganu as a Malay government. 2. Designating the royal lineage of Sultan Zainal Abidin III as the sultans and rulers of the
Terengganu government. 2. Designating the royal lineage of Sultan Zainal Abidin III as the sultans and rulers of the
Terengganu government. 3. Preserving the sovereignty of the Malay State of Terengganu. 4. Affirming the concept of indigenous sovereignty. 4. Affirming the concept of indigenous sovereignty. 5. Recording the foundational principles of Terengganu's government that cannot be destroyed or
amended (Mohamed & Nik, 2009). THE ROLE OF THE RELIGIOUS SCHOLARS OF FAQIH ALI DATO’ MAHARAJA’S DESCENT IN SUSTAINING SHARIA IMPLEMENTATION IN
TERENGGANU THE ROLE OF THE RELIGIOUS SCHOLARS OF FAQIH ALI DATO’ MAHARAJA’S DESCENT IN SUSTAINING SHARIA IMPLEMENTATION IN
TERENGGANU In addition to Qadi Wan Imam Mahmud, during his sovereign, Sultan Zainal Abidin Shah I also assigned
important portfolios to the scholars and religious scholars in the administration of the Sultanate, for
example, Tok Pulau Manis Sheikh Abdul Malek bin Syed Abdullah and Che Buang (Muhammad, 1991). In addition to Qadi Wan Imam Mahmud, during his sovereign, Sultan Zainal Abidin Shah I also assigned
important portfolios to the scholars and religious scholars in the administration of the Sultanate, for
example, Tok Pulau Manis Sheikh Abdul Malek bin Syed Abdullah and Che Buang (Muhammad, 1991). During the era of Baginda Umar (1839-1876), various reforms in the local political system were
implemented, including establishing multiple institutions and the Balai Court (Mohamed Anwar, 2009). Baginda Umar further established the position of Mufti and appointed Tok Sheikh Bukit Bayas Haji Wan
Abdul Kadir bin Wan Abdul Rahim to this position. Tok Sheikh Bukit Bayas served as Mufti and headed
the judicial department (Hashim, 2023). Below the Mufti, there were Qadis who assisted in judicial
matters. The influence of Tok Sheikh Bukit Bayas on Sultan Umar was apparent because he served as the Sultan's
guru and primary advisor. Sultan Umar's second reign extended from 1839 to 1876; it corresponded with
Tok Sheikh Bukit Bayas's 1832M arrival in Terengganu until his passing in 1864, providing ample time
for a close relationship to flourish between them. However, some sources claimed that Tok Sheikh Bukit
Bayas died in 1853 instead. Regardless, due to their closeness, it is unsurprising that Sultan Umar affixed
his royal seal on Tok Sheikh Bukit Bayas's will and a guarantee of royal protection for his family and
descendants. Baginda Umar also established other positions, including ministers and aristocrats, aligning with the
introduction of a new policy in the Sultanate's administration, as follows: 1. Implementing a policy of centralizing the Sultanate's power by reducing the autonomy of regional
nobles. 1. Implementing a policy of centralizing the Sultanate's power by reducing the autonomy of regional
nobles. 2. Granting the title of "Orang-Orang Besar" (nobles) to individuals outside the royal family 3. Developing infrastructure to symbolize the unity of the sovereign Sultanate's institutions. In terms of amenities, Baginda Umar constructed Istana Hijau (the Green Palace), Abidin Mosque, Awang
Senda Firehouse, the cannon at Bukit Puteri, and several roadways. The Role of Islamic Scholars A religious scholar possesses at least the knowledge of compulsory religious obligations (fardhu ain) and
communal religious obligations (fardhu kifayah) with the ability to analyze, dissect, and respond to related
issues based on authentic sources. They hold positions within the government or the administration of the
Sultanate. According to Robert (1977), Malay scholars in the administration of the Terengganu government are
categorized as the third element in the Terengganu ruling class. However, this is not an entirely accurate
view because such a ranked position indicates the respect given to scholars who do not hold official
positions within the Sultanate's administration. On the contrary, religious scholars cum administrators
hold a higher status as they consist of royal family members and aristocrats. The influence of religious scholars of Faqih Ali Dato’ Maharaja's descent in the administration of the
Terengganu Sultanate cannot be denied. It began with Qadi Wan Imam Mahmud and continued with each
successive generation, producing renowned religious scholars and administrators who held senior
positions in legislative, executive, and judiciary while upholding the principles of Sharia in both public
and personal aspects of life. 222 THE ROLE OF THE RELIGIOUS SCHOLARS OF FAQIH ALI DATO’ MAHARAJA’S DESCENT IN SUSTAINING SHARIA IMPLEMENTATION IN
TERENGGANU 1, pp. 216-227 | April 2024 References can be found in the lists of individuals involved in enacting Itqanul Muluk 1911 and Undang-
Undang Bagi Diri Kerajaan Terengganu. Official documents also list the Muftis and grand Qadis of
Terengganu. References can be found in the lists of individuals involved in enacting Itqanul Muluk 1911 and Undang-
Undang Bagi Diri Kerajaan Terengganu. Official documents also list the Muftis and grand Qadis of
Terengganu. Itqanul Muluk 1911 enlisted the names of state dignitaries who were the member of the State Council and
were of Faqih Ali Dato’ Maharaja’s descent, for example, Haji Wan Muhammad Salleh bin Muhammad
(Mufti), Haji Wan Muhammad Salleh bin Ismail (Judge), Haji Wan Sulaiman bin Daud (Judge), and Encik
Nik Muhammad bin Hitam (Deputy Judge). Besides, the document also recorded administrators, such as
Dato' Panglima Dalam Wan Mohamed bin Ibrahim (Minister) and Dato' Sangsura Pahlawan Wan Abdul
Hamid (Official). The Terengganu State Constitution also recorded the names of state officials who were
religious scholars cum administrators and were of Faqih Ali Dato’ Maharaja’s descent. This includes
Dato' Biji Sura Dato' Haji Wan Abdullah bin Dato' Kamal Wangsa and Dato' Sangsura Pahlawan Dato'
Haji Wan Long Muhammad Soleh bin Haji Wan Ahmad. They were members of the Government
Advisory Council, responsible for advising the Sultan of Terengganu in the formulation of the Undang-
Undang Bagi Diri Kerajaan Terengganu for the first and second parts after the enforcement of the
Federation of Malaya Agreement in 1948. The Chief Qadis and Qadis who served from Sultan Zainal Abidin Shah I reign until Independence Day
include individuals like Qadi Wan Imam Mahmud, Khadimus Syar’ie Haji Wan Abdul Latif bin Haji Wan
Endut al-Hafiz Losong, Qadi Haji Wan Abdullah bin Haji Wan Abdul Latif, Qadi Haji Wan Taib bin Wan
Abdul Rahman, Chief Qadi Haji Wan Endut bin Hakim Haji Wan Long @ Muhammad and Chief Qadi
Dato’ Perba diRaja Haji Wan Abdul Rahman bin Long. The book "Ulama Terengganu: Suatu Sorotan" (New Edition) discusses several religious scholars cum
administrators mentioned in this article. They include Tok Sheikh Bukit Bayas, Tok Sheikh Duyong,
Khadimu as-Syar’ie Haji Wan Abdul Latif bin Haji Wan Endut al-Hafiz, and Hakim Haji Wan Long
Muhammad bin Qadi Haji Wan Abdullah. THE ROLE OF THE RELIGIOUS SCHOLARS OF FAQIH ALI DATO’ MAHARAJA’S DESCENT IN SUSTAINING SHARIA IMPLEMENTATION IN
TERENGGANU Terengganu earned the nickname
'Birmingham of the Peninsula' for its success in advancing various industries such as silk, cotton, songket
fabric, weaponry, blacksmithing, and carpentry (Mohamed, 2009). In terms of amenities, Baginda Umar constructed Istana Hijau (the Green Palace), Abidin Mosque, Awang
Senda Firehouse, the cannon at Bukit Puteri, and several roadways. Terengganu earned the nickname
'Birmingham of the Peninsula' for its success in advancing various industries such as silk, cotton, songket
fabric, weaponry, blacksmithing, and carpentry (Mohamed, 2009). During Baginda Umar's reign, the position of Chief Minister (Menteri Besar) already existed, he appointed
Syed Muhammad Zain bin Syed Muhammad al-Idrus with the title Engku Sayyid Seri Perdana (Omar,
2014: 37). He also selected Tok Ku Tuan Besar Syed Muhammad Zainal Abidin bin Syed Muhammad
al-Idrus and Tok Sheikh Duyong Haji Wan Abdullah bin Tok Sheikh Qadi Haji Wan Muhammad Amin
to his administration. Before being appointed as Mufti, Tok Sheikh Duyong Haji Wan Abdullah served
as a Qadi. He was also selected as a district aristocrat with the title Orang Kaya Duyong. Both prominent
figures were also students of Tok Sheikh Bukit Bayas (Mohamad, 2023). In 1860, Tok Ku Tuan Besar was appointed to a new position as Sheikhul Ulama’ (Musa, O., 2014). The
title Sheikhul Ulama’ was also given to a Mufti. The Mufti who held the title Sheikhul Ulama’ included
Tok Sheikh Duyong and his son Haji Wan Muhammad (Mohamad, 2023). Sultan Zainal Abidin III was the ruler who completed the drafting of Itqan al-Muluk in 1911 based on the
framework introduced by Sultan Omar (Mohamed, 2009). His chief advisor, Tok Sheikh Duyong, had
previously served under Sultan Umar. After Tok Sheikh Duyong passed away in February 1889, a new
generation of religious scholar cum administrators emerged, including Sheikhul Ulama’ Haji Wan
Muhammad, Sheikhul Islam Tok Ku Paloh Syed Abdul Rahman, Khadimu as-Syar'ie Haji Wan Abdul
Latif bin Haji Wan Endut and Qadi Haji Wan Abdullah bin Haji Wan Abdul Latif. Many religious scholars from the noble class held positions in the administration of the Sultanate,
covering legislative, executive and judiciary arms. Sultan Zainal Abidin III also consulted some religious
scholars who did not hold official positions. Among them was Tuan Guru Haji Wan Ahmad bin Haji Wan
Endut al-Hafiz Bukit Bayas, who was popularly known as Haji Mat Keramat. 223 Malaysian Journal of Syariah and Law | مجلة الشريعة والقانون بماليزيا | Vol. 12, No. Overcoming the Impact of the British Advisory System Terengganu, since the coronation of Sultan Zainal Abidin Shah I in 1708, has been a sovereign sultanate. Islamic law and Malay customs constitute the law of the land, covering both public and personal affairs
that are enforced on the people of the Sultan of Terengganu. This is consistent with the other Malay rulers
who inherited the laws from the Hukum Kanun Melaka and subsequent legal codes such as the Hukum
Kanun Johor, Hukum Kanun Pahang, and Undang-Undang Sungei Ujong, the Sultan of Terengganu also
assumed the position of a Khalifah. Before the reign of Sultan Zainal Abidin Shah I, the people of Terengganu, who were under the rule of
the Sultanates of Johor-Pahang-Riau Lingga and Patani, had already accepted the traditional Malay
political system based on the teachings of Islam. Baginda Umar established the Courts to preside over
legal disputes based on Islamic law under his administration. By 1909, two courts were established in
Kuala Terengganu: the Balai Court (Mahkamah Balai) and the Syariah Court (Mahkamah Syariah). The
Balai Court hosted hearings for criminal and civil cases, while the Syariah Court hosted hearings
associated with religious rituals and family law matters. Both of these courts ruled based on Islamic law. Only after the Terengganu Agreement of 1910 was a special court named the Joint Court set up to
adjudicate cases involving British subjects. The Joint Court was presided over by a Malay judge and a
British officer serving as an assessor. Since the establishment of the Joint Court, the British judicial system
began to influence the functions of the Balai Court, eventually leading to its renaming. The influence of the British judicial system continued to evolve in the local law with the passing of the
Undang-Undang Tertib Mahkamah (Court Procedure Law) in 1916. This law re-structure the organization
of the courts in Terengganu into six levels: Court of Appeal, High Court (Mahkamah Besar), First-Class
Magistrates Court, Second-Class Magistrates Court, Qadi's Court, and Penghulu's Court. Despite this
reorganization, the local judicial systems maintained Islamic law and Malay customs as the sources of
law with the introduction of the English common laws and the rules of equity therein. Under the Federation of Malaya Agreement of 1948, the distribution of legislative, executive, and judicial
powers between the Federation and the Malay States was carried out with the consent of the Malay rulers. THE ROLE OF THE RELIGIOUS SCHOLARS OF FAQIH ALI DATO’ MAHARAJA’S DESCENT IN SUSTAINING SHARIA IMPLEMENTATION IN
TERENGGANU Several other imminent figures of Faqih Ali Dato’ Maharaja’s descent introduced in this article are
recorded in the book titled "Pentadbiran Hal Ehwal Islam Negeri Terengganu: Sejarah dan
Perkembangan Sehingga Hari Ini (2013)” traced from the government service records and other
additional sources. Many held positions such as Commissioner of Islamic Affairs and Mufti of
Terengganu. Some of them include: 1. Dato’ Biji Sura Haji Nik Muhammad: He served as Deputy Judge and State Council Member
(1911-1913), Judge of the Sharia Court (1913-1915), Judge of the Balai and Joint Court (1915-
1928), Judge of the Supreme Court (1928-1937), Acting Mufti (1937-1940), and Commissioner
of Islamic Affairs of Terengganu (1940-1942). He is the grandson of Tok Syeikh Duyong through
his mother. 2. Dato’ Sangsura Pahlawan Dato’ Haji Wan Long @ Muhammad Soleh: He served as the Grand
Commissioner of West Terengganu (Besut and Setiu), Commissioner of Islamic Affairs of
Terengganu, as well as a member of the State Cabinet and later the Deputy President II of the
Terengganu Royal Council. His expertise in Islamic jurisprudence was well-recognized, and he
was called to serve as the State Mufti of Terengganu after he retired from government service. 3. Dato’ Biji Sura Haji Wan Abdullah bin Dato’ Kamal Wangsa: He served as the State Secretary,
a member of the State Cabinet, and a member of the Terengganu Royal Council. 3. Dato’ Biji Sura Haji Wan Abdullah bin Dato’ Kamal Wangsa: He served as the State Secretary,
a member of the State Cabinet, and a member of the Terengganu Royal Council. 4. Dato’ Perba diRaja Haji Wan Abdul Rahman bin Long; He served as the Chief Qadi,
Commissioner of Islamic Affairs of Terengganu, acting Mufti and later the president of the
Terengganu Royal Council. 4. Dato’ Perba diRaja Haji Wan Abdul Rahman bin Long; He served as the Chief Qadi,
Commissioner of Islamic Affairs of Terengganu, acting Mufti and later the president of the
Terengganu Royal Council. The above dignitaries of the Terengganu sultanate held significant posts in government administration
and Islamic personal affairs; hence, their contributions played an important role in preserving the
implementation of Sharia. 224 Conclusion Islamic scholarship fortified the glory within the Sultanate of Terengganu, which consisted of the ruling
monarch and aristocrats. It gave birth to a modern written constitution under the rulership of two great
Sultans of Terengganu, strengthening its local legal systems. The enacting of Itqan al-Muluk 1911, which
evolved into the Terengganu State Constitution, was a manifestation of the role of the religious scholars
cum administrators and such role continued until Independence Day. This connection can be traced back
to the strong relationship between the ruling monarch and the descendants of Faqih Ali Dato’ Maharaja. This collaborative effort preserved Islam's and Malay's identities, which were incorporated into a written
constitution and later enshrined in the Federal Constitution, upholding the principle of Sharia compliance
in public administration. The influential roles played by the descendants of Faqih Ali Dato’ Maharaja were not only in the realm
of legislation but also in judicial and political administration, thus preserving Islam as the law of the land
in Terengganu. Hence, the practice of Sharia before Independence Day is not confined to personal affairs
as assumed by many; by the Federal Constitution, the public aspects of Sharia are entrusted to the public
authority at the federal level unless those matters are retained in the state administration. This study has specifically proven the contributions of the Faqih Ali lineage in the public administration
of Terengganu. It does not in any way deny the contributions of existing scholars highlighted in current
academic writings. However, the lack of related literature has resulted in the contributions of the Faqih
Ali lineage not being thoroughly discussed and duly recognized. Furthermore, this study has implications
for the profiling of the roles of scholars, not merely adding to biographical data but also opening avenues
for further research. Subsequent studies could be conducted in the context of Terengganu in-depth and
even expanded to other governments as well. Overcoming the Impact of the British Advisory System The Federation of Malaya Agreement of 1957 eventually overrode this interim agreement and enacted of
the Federal Constitution of Malaya 1957. Nevertheless, the sovereignty of the Malay rulers, as it existed
before the Japanese occupation and the Malayan Union, was preserved through Article 181(1) of the
Federal Constitution and the modern written state constitutions, such as Undang-undang Bagi Diri
Kerajaan Terengganu [the State Constitution of Terengganu] found in Part I (enacted in 1948) and Part II
(enacted in 1950). In the Terengganu legal system, English common law and rules of equity principles were adopted under
certain conditions according to Section 3 of the Civil Law Act 1956. English common law and rules of
equity principles could not be adopted without necessary modification to preserve local circumstances. In
other words, despite the adoption of English common law and rules of equity in the judicial administration
of public law in Terengganu by judges and lawyers trained therein, Islamic law remains as the law of the
land. The sovereign power, rooted in Islamic teachings, remained with the Sultan of Terengganu as the
ruling monarch, encompassing the public and personal aspects of the people's lives with the practice of
consultation (shura). Public affairs are the rulings enforced on all the people and inhabitants of Terengganu, regardless of
religion. The principles of Sharia are preserved because the religious scholars are directly involved in the
public administration of the Sultanate. Besides, the enacted modern written constitution further
strengthened the position of Sharia in controlling British intervention throughout its period. According to
the Federal Constitution passed by the Terengganu State Council in August 1957, the public aspects of
Sharia have been delegated into federal jurisdiction unless such matters are retained under the state
administration. 225 Malaysian Journal of Syariah and Law | مجلة الشريعة والقانون بماليزيا | Vol. 12, No. 1, pp. 216-227 | April 2024 Malaysian Journal of Syariah and Law | مجلة الشريعة والقانون بماليزيا | Vol. 12, No. 1, pp. 216-227 | April 2024 Acknowledgement This work was funded by the International Institute of Islamic Thought and Civilization (ISTAC)
Research Grant Scheme (grant number: ISTAC23-013-0015). This work was funded by the International Institute of Islamic Thought and Civilization (ISTAC)
Research Grant Scheme (grant number: ISTAC23-013-0015). THE ROLE OF THE RELIGIOUS SCHOLARS OF FAQIH ALI DATO’ MAHARAJA’S DESCENT IN SUSTAINING SHARIA IMPLEMENTATION IN
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Asymmetric glaciation, divide migration, and postglacial fluvial response times in the Qilian Shan
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Jingtao Lai1 and Kimberly Huppert1, 2
3 1Earth Surface Process Modelling, GFZ German Research Centre for Geosciences, 14473
4
Potsdam, Germany
5 2Department of Earth & Atmospheric Sciences, City College of New York, NY 10031, USA
6 ABSTRACT
7 Glacial-interglacial cycles have repeatedly perturbed climate and topography in many
8
mid-latitude mountain ranges during the Quaternary. Glacial erosion can move drainage divides
9
and induce fluvial adjustments downstream, yet the timescale over which these adjustments
10
occur remains unclear. We examine landscape evolution in the northwest-southeast trending
11
Qilian Shan, where the contrast in solar insolation between north- and south-facing slopes has
12
generated larger glaciers on the northern range crest. Our analyses suggest that this asymmetric
13
glaciation has caused southward migration of the main drainage divide, prompting river channels
14
below the extents of ice on north-facing slopes to become oversteepened for their drainage area
15
and channels on south-facing slopes to become analogously understeepened. These changes in
16
steepness should accelerate or slow down postglacial fluvial incision, even in these regions
17
where topography has not been directly modified by glacial erosion. Numerical modeling
18
suggests these discrepancies persist for millions of years – much longer than the duration of
19
recent glacial-interglacial cycles – implying a widespread and enduring influence of intermittent
20
glaciations on landscape evolution in glaciated mountain ranges during the Quaternary. 21 Glacial-interglacial cycles have repeatedly perturbed climate and topography in many
8
mid-latitude mountain ranges during the Quaternary. Glacial erosion can move drainage divides
9
and induce fluvial adjustments downstream, yet the timescale over which these adjustments
10
occur remains unclear. We examine landscape evolution in the northwest-southeast trending
11
Qilian Shan, where the contrast in solar insolation between north- and south-facing slopes has
12
generated larger glaciers on the northern range crest. Our analyses suggest that this asymmetric
13
glaciation has caused southward migration of the main drainage divide, prompting river channels
14
below the extents of ice on north-facing slopes to become oversteepened for their drainage area
15
and channels on south-facing slopes to become analogously understeepened. These changes in
16
steepness should accelerate or slow down postglacial fluvial incision, even in these regions
17
where topography has not been directly modified by glacial erosion. Numerical modeling
18
suggests these discrepancies persist for millions of years – much longer than the duration of
19
recent glacial-interglacial cycles – implying a widespread and enduring influence of intermittent
20
glaciations on landscape evolution in glaciated mountain ranges during the Quaternary. 21 INTRODUCTION
22 INTRODUCTION
22
Quaternary glaciations have influenced the topography of mid-latitude mountain ranges
23
worldwide (e.g., Ehlers et al., 2018; Herman et al., 2021). During glacial-interglacial cycles, the
24
dominant geomorphic processes in these regions shift between glacial and fluvial erosion. 25
During glacial periods, glacial erosion can outpace rock uplift below warm-based glaciers, and
26
cold-based glaciers can buffer bedrock from erosion (Herman et al., 2021). After glaciers recede,
27
rivers become the dominant geomorphic agents, and they progressively adjust their bed
28
elevations and slopes to sustain incision rates that approximately balance rock uplift rates
29
(Whipple and Tucker, 1999; Dadson and Church, 2005; Hobley et al., 2010). Constraining the
30
timescale over which these adjustments occur is crucial for understanding the interactions
31
between climate, topography, and erosion in intermittently glaciated mountain ranges. In
32
particular, if postglacial response times exceed tens of thousands of years, Quaternary glaciations
33
that occur every ~40-100 kyr will create persistent dynamic adjustments in these mountain
34
ranges (Hobley et al., 2010; Moon et al., 2015). Or, if response times are shorter than interglacial
35
periods, Quaternary glaciations may only fleetingly disrupt long-term, tectonically driven
36
landscape evolution. 37 Quaternary glaciations have influenced the topography of mid-latitude mountain ranges
23
worldwide (e.g., Ehlers et al., 2018; Herman et al., 2021). During glacial-interglacial cycles, the
24
dominant geomorphic processes in these regions shift between glacial and fluvial erosion. 25 Quaternary glaciations have influenced the topography of mid-latitude mountain ranges
23
worldwide (e.g., Ehlers et al., 2018; Herman et al., 2021). During glacial-interglacial cycles, the
24
dominant geomorphic processes in these regions shift between glacial and fluvial erosion. 25
During glacial periods, glacial erosion can outpace rock uplift below warm-based glaciers, and
26
cold-based glaciers can buffer bedrock from erosion (Herman et al., 2021). After glaciers recede,
27
rivers become the dominant geomorphic agents, and they progressively adjust their bed
28
elevations and slopes to sustain incision rates that approximately balance rock uplift rates
29
(Whipple and Tucker, 1999; Dadson and Church, 2005; Hobley et al., 2010). Constraining the
30
timescale over which these adjustments occur is crucial for understanding the interactions
31
between climate, topography, and erosion in intermittently glaciated mountain ranges. INTRODUCTION
22 In
32
particular, if postglacial response times exceed tens of thousands of years, Quaternary glaciations
33
that occur every ~40-100 kyr will create persistent dynamic adjustments in these mountain
34
ranges (Hobley et al., 2010; Moon et al., 2015). Or, if response times are shorter than interglacial
35
periods, Quaternary glaciations may only fleetingly disrupt long-term, tectonically driven
36
landscape evolution. 37 Despite the potentially dominant role that glacial-interglacial transitions can have on
38
landscape evolution, postglacial response times remain poorly quantified. This is largely because
39
glacial erosion can modify mountain topography and affect postglacial erosion in diverse ways. 40
In some instances, glacial erosion can steepen hillslopes and promote landsliding (Moon et al.,
41
2015). Glacial erosion can also accelerate postglacial river incision by steepening channel slopes
42
in previously glaciated terrains (Norton et al., 2010), or it can slow down incision by flattening
43
valley bottoms (Hobley et al., 2010; Johnson et al., 2022). Glaciers also directly erode the
44 ridgelines and affect the geometry and drainage area of river networks (Oskin and Burbank,
45
2005). Especially in mountain ranges with asymmetric glaciation, glacial erosion can create
46
topographic asymmetry across drainage divides, promoting divide migration (e.g., Gilbert, 1904;
47
Evans, 1977; Naylor and Gabet, 2007; Foster et al., 2010). Such divide migration can change a
48
basin’s drainage area and induce changes in river incision rates downstream (Willett et al.,
49
2014). Despite the ubiquity of river network reorganization by glacial erosion, few studies have
50
assessed the implications of these drainage area changes on the pace of postglacial fluvial
51
incision and the timescales over which rivers regrade their slopes to balance rock uplift rates. 52
The Qilian Shan (Fig 1A), a northwest-southeast trending mountain range on the
53
northeast margin of the Tibetan Plateau, provides an excellent opportunity to assess the
54
implications of glacial drainage reorganization on river incision because it experiences highly
55
asymmetric glaciation. North-facing slopes receive more summer precipitation from the East
56
Asian Monsoon and less solar insolation than south-facing slopes, so the majority of modern
57
glaciers occupy north-facing slopes (Fig. 1C), and their equilibrium line altitudes (ELAs) are
58
~200 m lower than south-facing glaciers (Guo et al., 2021). Because the East Asian Monsoon has
59
been relatively stable since the late Oligocene (Wu et al., 2022), this asymmetric pattern has
60
likely persisted throughout the Quaternary. INTRODUCTION
22 61
We combine topographic analyses with numerical simulations to examine the impact of
62
asymmetric glaciation on postglacial fluvial incision in the Qilian Shan. Our results show that
63 We combine topographic analyses with numerical simulations to examine the impact of
62
asymmetric glaciation on postglacial fluvial incision in the Qilian Shan. Our results show that
63
divide migration has caused transient changes of erosion rates in rivers below past ice cover, and
64
rivers adjust to these glacial modifications over millions of years – a timescale much longer than
65
~40-100 kyr glacial-interglacial cycles. 66 We combine topographic analyses with numerical simulations to examine the impact of
62
asymmetric glaciation on postglacial fluvial incision in the Qilian Shan. Our results show that
63
divide migration has caused transient changes of erosion rates in rivers below past ice cover, and
64
rivers adjust to these glacial modifications over millions of years – a timescale much longer than
65
~40-100 kyr glacial-interglacial cycles. 66 TOPOGRAPHIC EFFECT OF ASYMMETRIC GLACIATION
67 Numerical modeling
68
Using a 1D profile model crossing head-to-head glaciated valleys, we first explore the
69
influence of asymmetric glaciation on topography. We construct pre-glacial fluvial profiles in
70
steady state using the stream power model (Howard, 1994) and Hack’s Law (Hack, 1957). We
71
then simulate glaciation over these fluvial profiles using the shallow ice approximation (Hutter,
72
1983), with different ELAs on each side of the divide yielding asymmetric glaciation. We
73
assume glacial erosion rates scale linearly with glacier sliding velocity (Humphrey and
74
Raymond, 1994; Braun et al., 1999). In this set of experiments, we focus on exploring the
75
influence of asymmetric glacial erosion on divide mobility and do not model the evolution of
76
downstream fluvial topography. 77
Our simulations show that asymmetric glaciation is capable of moving the divide towards
78
the mountain side with higher ELA (Fig 2A). In cases with larger ELA differences between the
79
two sides, greater glacial asymmetry and erosion leads to farther divide migration. We also find
80
that divide migration rates are faster when the ELA is closer to the mountain peak because ELA
81
contrasts at higher elevations focus glacial erosion asymmetry near the ridgeline, directly
82
impacting ridgeline location (Fig. S2). 83
To quantify the topographic effect of asymmetric glacial erosion on our modeled valleys,
84
we calculate a normalized channel steepness index, 𝑘!" = 𝑆𝐴#, where 𝑆 is slope, 𝐴 is drainage
85
area, and 𝜃 is a concavity index that is calibrated based on fluvial profiles in the Qilian Shan
86
(Fig. S1; Perron and Royden, 2013). In fluvial landscapes, erosion rates typically scale with 𝑘!"
87
(e.g., Ouimet et al., 2009). We also calculate a normalized channel distance metric, 𝜒, to
88
highlight along-stream variations in 𝑘!", which manifest as changes in slope in 𝜒-elevation
89 Using a 1D profile model crossing head-to-head glaciated valleys, we first explore the
69
influence of asymmetric glaciation on topography. We construct pre-glacial fluvial profiles in
70
steady state using the stream power model (Howard, 1994) and Hack’s Law (Hack, 1957). We
71
then simulate glaciation over these fluvial profiles using the shallow ice approximation (Hutter,
72
1983), with different ELAs on each side of the divide yielding asymmetric glaciation. We
73
assume glacial erosion rates scale linearly with glacier sliding velocity (Humphrey and
74
Raymond, 1994; Braun et al., 1999). TOPOGRAPHIC EFFECT OF ASYMMETRIC GLACIATION
67 In this set of experiments, we focus on exploring the
75
influence of asymmetric glacial erosion on divide mobility and do not model the evolution of
76
downstream fluvial topography. 77
Our simulations show that asymmetric glaciation is capable of moving the divide towards
78
the mountain side with higher ELA (Fig 2A). In cases with larger ELA differences between the
79
two sides, greater glacial asymmetry and erosion leads to farther divide migration. We also find
80
that divide migration rates are faster when the ELA is closer to the mountain peak because ELA
81
contrasts at higher elevations focus glacial erosion asymmetry near the ridgeline, directly
82
impacting ridgeline location (Fig. S2). 83
To quantify the topographic effect of asymmetric glacial erosion on our modeled valleys,
84 To quantify the topographic effect of asymmetric glacial erosion on our modeled valleys,
84
we calculate a normalized channel steepness index, 𝑘!" = 𝑆𝐴#, where 𝑆 is slope, 𝐴 is drainage
85
area, and 𝜃 is a concavity index that is calibrated based on fluvial profiles in the Qilian Shan
86
(Fig. S1; Perron and Royden, 2013). In fluvial landscapes, erosion rates typically scale with 𝑘!"
87
(e.g., Ouimet et al., 2009). We also calculate a normalized channel distance metric, 𝜒, to
88
highlight along-stream variations in 𝑘!", which manifest as changes in slope in 𝜒-elevation
89 profiles (Perron and Royden, 2013). We update post-migration drainage area based on new
90
along-stream distance from the drainage divide and Hack’s Law and calculate 𝜒 and 𝑘!". 91
Glacial erosion causes changes in 𝑘!" in both glaciated and non-glaciated parts of the
92
landscape. At high, glaciated elevations, glacial erosion lowers the mountain peak and carves
93
gentle valley floors with low 𝑘!" above the ELA (Fig. 2). At lower, non-glaciated elevations,
94
even though glacial erosion does not directly modify topography, it still changes 𝑘!" by inducing
95
divide migration and drainage area changes upstream. Specifically, river segments on the range
96
flank that gains drainage area (north in Fig. 2) become oversteepened for their drainage area with
97
high 𝑘!" because their channel slopes remain more steeply graded to the smaller contributing
98
areas they had before divide migration (Fig. 2C). In contrast, river segments on the range flank
99
that loses drainage area (south in Fig. 2) become understeepened relative to their drainage area,
100
with consequently lower 𝑘!" (Fig. TOPOGRAPHIC EFFECT OF ASYMMETRIC GLACIATION
67 2C). 101
Steepness patterns in the Qilian Shan
102 profiles (Perron and Royden, 2013). We update post-migration drainage area based on new
90
along-stream distance from the drainage divide and Hack’s Law and calculate 𝜒 and 𝑘!". 91
Glacial erosion causes changes in 𝑘!" in both glaciated and non-glaciated parts of the
92
landscape. At high, glaciated elevations, glacial erosion lowers the mountain peak and carves
93
gentle valley floors with low 𝑘!" above the ELA (Fig. 2). At lower, non-glaciated elevations,
94
even though glacial erosion does not directly modify topography, it still changes 𝑘!" by inducing
95
divide migration and drainage area changes upstream. Specifically, river segments on the range
96
flank that gains drainage area (north in Fig. 2) become oversteepened for their drainage area with
97
high 𝑘!" because their channel slopes remain more steeply graded to the smaller contributing
98
areas they had before divide migration (Fig. 2C). In contrast, river segments on the range flank
99
that loses drainage area (south in Fig. 2) become understeepened relative to their drainage area,
100
with consequently lower 𝑘!" (Fig. 2C). 101
Steepness patterns in the Qilian Shan
102 Steepness patterns in the Qilian Shan
102 We observe similar spatial patterns of enhanced and reduced 𝑘!" below the extents of
103
glaciation on the north and south flanks of the Qilian Shan. We select a group of channels
104
originating from previously glaciated ridges in the study area (Owen and Dortch, 2014) and
105
calculate 𝑘!" using the SRTM 30m Digital Elevation Model (USGS, 2018). Our analysis shows
106
that both north- and south-facing channels have low 𝑘!" above ~4000 m – the Last Glacial
107
Maximum (LGM) ELA in this region (Fig. 3A, B; Owen and Benn, 2005). Below the LGM
108
ELA, north-facing channels have higher 𝑘!" than south-facing channels (Fig. 3A, B). This spatial
109
pattern of 𝑘!" is consistent with the predictions of our models. We do not observe a clear
110
correlation between rock type and 𝑘!", nor a correlation between fault location and 𝑘!" in the
111
Qilian Shan (Fig. S5). Therefore, we suggest that the observed 𝑘!" contrasts between north- and
112 We observe similar spatial patterns of enhanced and reduced 𝑘!" below the extents of
103
glaciation on the north and south flanks of the Qilian Shan. We select a group of channels
104
originating from previously glaciated ridges in the study area (Owen and Dortch, 2014) and
105
calculate 𝑘!" using the SRTM 30m Digital Elevation Model (USGS, 2018). Our analysis shows
106
that both north- and south-facing channels have low 𝑘!" above ~4000 m – the Last Glacial
107
Maximum (LGM) ELA in this region (Fig. 3A, B; Owen and Benn, 2005). Below the LGM
108
ELA, north-facing channels have higher 𝑘!" than south-facing channels (Fig. 3A, B). This spatial
109
pattern of 𝑘!" is consistent with the predictions of our models. We do not observe a clear
110
correlation between rock type and 𝑘!", nor a correlation between fault location and 𝑘!" in the
111
Qilian Shan (Fig. S5). Therefore, we suggest that the observed 𝑘!" contrasts between north- and
112 south-facing channels in our study area are likely the consequence of divide migration caused by
113
asymmetric glacial erosion. 114
We select a small divide to show that the magnitude of 𝑘!" contrasts we observe can be
115
attributed to divide migration (Fig. 3C). Below the LGM ELA, the north-facing channels here
116
have maximum 𝑘!" nearly three times higher than the south-facing channels (Fig. 3D). Steepness patterns in the Qilian Shan
102 The
117
range crest juts conspicuously south in the glaciated basins of this region, suggesting that higher
118
glacial erosion rates on the north-facing slopes have caused southward migration of the main
119
drainage divide (Fig. 3C). We estimate their pre-glaciation drainage divide location by extending
120
the non-glaciated ridgeline into the glaciated region. We then calculate the drainage area
121
difference between the reconstructed and current divide location. 122
To hypothetically undo this apparent drainage reorganization, we remove the captured
123
drainage area from the observed drainage area of north-facing channels and we add it back to the
124
south-facing channels. With this exchange, the contrast in 𝑘!" between the north- and south-
125
facing channels is significantly reduced, and the channels on both sides collapse to a similar 𝜒-
126
elevation profile, implying drainage network equilibrium (Fig. 3E; Willett et al., 2014). These
127
calculations suggest that the observed contrasts in 𝑘!" between north- and south-facing channels
128
can be predominately attributed to drainage area change caused by asymmetric glaciation. 129
POSTGLACIAL RESPONSE TIME
130
Because drainage reorganization arising from asymmetric glaciation modifies channel
131
steepness, it can also create transient imbalances between erosion and rock uplift rates. Erosion
132
rates typically scale with 𝑘!" in fluvial landscapes (Ouimet et al., 2009), so the high 𝑘!" north-
133
facing reaches in the Qilian Shan likely erode faster than other parts of the landscape during
134 south-facing channels in our study area are likely the consequence of divide migration caused by
113
asymmetric glacial erosion. 114
We select a small divide to show that the magnitude of 𝑘!" contrasts we observe can be
115
attributed to divide migration (Fig. 3C). Below the LGM ELA, the north-facing channels here
116
have maximum 𝑘!" nearly three times higher than the south-facing channels (Fig. 3D). The
117
range crest juts conspicuously south in the glaciated basins of this region, suggesting that higher
118
glacial erosion rates on the north-facing slopes have caused southward migration of the main
119
drainage divide (Fig. 3C). We estimate their pre-glaciation drainage divide location by extending
120
the non-glaciated ridgeline into the glaciated region. We then calculate the drainage area
121
difference between the reconstructed and current divide location. 122 south-facing channels in our study area are likely the consequence of divide migration caused by
113
asymmetric glacial erosion. Steepness patterns in the Qilian Shan
102 114 south-facing channels in our study area are likely the consequence of divide migration caused by
113
asymmetric glacial erosion. 114 asymmetric glacial erosion. 114
We select a small divide to show that the magnitude of 𝑘!" contrasts we observe can be
115
attributed to divide migration (Fig. 3C). Below the LGM ELA, the north-facing channels here
116
have maximum 𝑘!" nearly three times higher than the south-facing channels (Fig. 3D). The
117
range crest juts conspicuously south in the glaciated basins of this region, suggesting that higher
118
glacial erosion rates on the north-facing slopes have caused southward migration of the main
119
drainage divide (Fig. 3C). We estimate their pre-glaciation drainage divide location by extending
120
the non-glaciated ridgeline into the glaciated region. We then calculate the drainage area
121
difference between the reconstructed and current divide location. 122 To hypothetically undo this apparent drainage reorganization, we remove the captured
123
drainage area from the observed drainage area of north-facing channels and we add it back to the
124
south-facing channels. With this exchange, the contrast in 𝑘!" between the north- and south-
125
facing channels is significantly reduced, and the channels on both sides collapse to a similar 𝜒-
126
elevation profile, implying drainage network equilibrium (Fig. 3E; Willett et al., 2014). These
127
calculations suggest that the observed contrasts in 𝑘!" between north- and south-facing channels
128
can be predominately attributed to drainage area change caused by asymmetric glaciation. 129 interglacials. Over time, they should become gentler and eventually sustain erosion rates in
135
quasi-equilibrium with uplift rates if climatic and tectonic conditions remain constant. 136
To quantify the timescales over which these adjustments occur, we model the evolution
137
of deglaciated topography (after 50 kyr of glaciation in Fig 2a) by rock uplift and fluvial incision
138
using the stream power model. We calibrate parameters based on river profiles and CRN erosion
139
rates in the Qilian Shan (Palumbo et al., 2011). We focus on north-facing channels that gained
140
drainage area due to divide migration, and we assume these channels experience no further
141
drainage area change following deglaciation. 142
Our simulations show that channel reaches with high 𝑘!" migrate upstream gradually, so
143
it takes >6 Myr for the entire channel profile to regrade to an unchanging geometry with erosion
144
rates everywhere in balance with rock uplift rates (Fig 4A). Steepness patterns in the Qilian Shan
102 We quantify the time required for
145
erosion rates everywhere along the profile to reach within 5% of the rock uplift rate, considering
146
different initial extents of drainage area gain and divide migration distance (Figs 4, S3, and S4). 147
Even for migration distances <500 m, these response times are >3 Myr. Greater drainage area
148
gains lead to longer response times (Fig. 4B). Fluvial readjustment is slow even in parts of the
149
channel that were never glaciated nor modified directly by glacial erosion; where divides have
150
migrated at least 500 m, response times of just these portions still exceed 2 Myr (open dots in
151
Fig. 4B) – an order of magnitude longer than the ~40-100 kyr durations of Quaternary glacial-
152
interglacial cycles. 153
DISCUSSION
154
Cosmogenic erosion rates measured in the northeast Qilian Shan are higher in north-
155 interglacials. Over time, they should become gentler and eventually sustain erosion rates in
135
quasi-equilibrium with uplift rates if climatic and tectonic conditions remain constant. 136 To quantify the timescales over which these adjustments occur, we model the evolution
137
of deglaciated topography (after 50 kyr of glaciation in Fig 2a) by rock uplift and fluvial incision
138
using the stream power model. We calibrate parameters based on river profiles and CRN erosion
139
rates in the Qilian Shan (Palumbo et al., 2011). We focus on north-facing channels that gained
140
drainage area due to divide migration, and we assume these channels experience no further
141
drainage area change following deglaciation. 142 Our simulations show that channel reaches with high 𝑘!" migrate upstream gradually, so
143
it takes >6 Myr for the entire channel profile to regrade to an unchanging geometry with erosion
144
rates everywhere in balance with rock uplift rates (Fig 4A). We quantify the time required for
145
erosion rates everywhere along the profile to reach within 5% of the rock uplift rate, considering
146
different initial extents of drainage area gain and divide migration distance (Figs 4, S3, and S4). 147
Even for migration distances <500 m, these response times are >3 Myr. Greater drainage area
148
gains lead to longer response times (Fig. 4B). Steepness patterns in the Qilian Shan
102 161 the west Himalaya, where most modern glaciers are in north-facing basins, cosmogenic erosion
158
rates are also higher in north-facing basins than in south-facing basins (Dortch et al., 2011). This
159
ongoing fluvial erosion asymmetry may drive continued divide migration (Hu et al., 2021) and
160
further prolong response times. 161 p
g
p
Drainage reorganization can create disequilibrium and decouple topography from
162
climatic, tectonic, and lithologic conditions (Willett et al., 2014). The impact of divide migration
163
on landscape evolution depends on how quickly divides migrate vs. how quickly river profiles
164
respond to changes in drainage area (Whipple et al., 2017). If divide migration outpaces channel
165
profile response, drainage reorganization can create disequilibrium that persists for millions or
166
even tens of millions of years (Beeson et al., 2017). Concordantly, in our models, we find that
167
postglacial adjustment times are on the order of a million to a few million years – a hundred-fold
168
longer than the O(10 kyr) glacial periods over which this topographic disequilibrium may arise. 169
Although our model uses idealized topography and simplified erosion laws, the ~2 order-
170
of-magnitude difference in the timescales of postglacial response and divide migration strongly
171
suggests that divide migration outpaces fluvial adjustment in the Qilian Shan. This order of
172
magnitude difference persists regardless of the erodibility coefficient or slope exponent we use in
173
our stream power modeling (Figs. S3 and S4). Therefore, glacial drainage reorganization likely
174
plays an enduring role in landscape evolution of glaciated mountain ranges worldwide,
175
particularly along east-west trending ridgelines with cross-divide differences in solar insolation
176
(e.g., Tian Shan; Oskin and Burbank, 2005) and in mountain ranges impacted by unidirectional
177
wind regimes and asymmetric precipitation patterns (e.g., Sierra Nevada; Brocklehurst and
178
Whipple, 2002). 179 Drainage reorganization can create disequilibrium and decouple topography from
162
climatic, tectonic, and lithologic conditions (Willett et al., 2014). The impact of divide migration
163
on landscape evolution depends on how quickly divides migrate vs. how quickly river profiles
164
respond to changes in drainage area (Whipple et al., 2017). If divide migration outpaces channel
165
profile response, drainage reorganization can create disequilibrium that persists for millions or
166
even tens of millions of years (Beeson et al., 2017). Steepness patterns in the Qilian Shan
102 Fluvial readjustment is slow even in parts of the
149
channel that were never glaciated nor modified directly by glacial erosion; where divides have
150
migrated at least 500 m, response times of just these portions still exceed 2 Myr (open dots in
151
Fig. 4B) – an order of magnitude longer than the ~40-100 kyr durations of Quaternary glacial-
152
interglacial cycles. 153
DISCUSSION
154
Cosmogenic erosion rates measured in the northeast Qilian Shan are higher in north-
155
facing basins than the south-facing basins (Hu et al., 2021), supporting our finding that
156
asymmetric glaciation leads to postglacial erosion asymmetry. Similarly, in the Ladakh Range in
157 Our simulations show that channel reaches with high 𝑘!" migrate upstream gradually, so
143
it takes >6 Myr for the entire channel profile to regrade to an unchanging geometry with erosion
144
rates everywhere in balance with rock uplift rates (Fig 4A). We quantify the time required for
145
erosion rates everywhere along the profile to reach within 5% of the rock uplift rate, considering
146
different initial extents of drainage area gain and divide migration distance (Figs 4, S3, and S4). 147
Even for migration distances <500 m, these response times are >3 Myr. Greater drainage area
148
gains lead to longer response times (Fig. 4B). Fluvial readjustment is slow even in parts of the
149
channel that were never glaciated nor modified directly by glacial erosion; where divides have
150
migrated at least 500 m, response times of just these portions still exceed 2 Myr (open dots in
151
Fig. 4B) – an order of magnitude longer than the ~40-100 kyr durations of Quaternary glacial-
152
interglacial cycles. 153 Cosmogenic erosion rates measured in the northeast Qilian Shan are higher in north-
155
facing basins than the south-facing basins (Hu et al., 2021), supporting our finding that
156
asymmetric glaciation leads to postglacial erosion asymmetry. Similarly, in the Ladakh Range in
157 the west Himalaya, where most modern glaciers are in north-facing basins, cosmogenic erosion
158
rates are also higher in north-facing basins than in south-facing basins (Dortch et al., 2011). This
159
ongoing fluvial erosion asymmetry may drive continued divide migration (Hu et al., 2021) and
160
further prolong response times. Steepness patterns in the Qilian Shan
102 Concordantly, in our models, we find that
167
postglacial adjustment times are on the order of a million to a few million years – a hundred-fold
168
longer than the O(10 kyr) glacial periods over which this topographic disequilibrium may arise. 169
Although our model uses idealized topography and simplified erosion laws, the ~2 order-
170
of-magnitude difference in the timescales of postglacial response and divide migration strongly
171
suggests that divide migration outpaces fluvial adjustment in the Qilian Shan. This order of
172
magnitude difference persists regardless of the erodibility coefficient or slope exponent we use in
173
our stream power modeling (Figs. S3 and S4). Therefore, glacial drainage reorganization likely
174
plays an enduring role in landscape evolution of glaciated mountain ranges worldwide,
175 particularly along east-west trending ridgelines with cross-divide differences in solar insolation
176
(e.g., Tian Shan; Oskin and Burbank, 2005) and in mountain ranges impacted by unidirectional
177
wind regimes and asymmetric precipitation patterns (e.g., Sierra Nevada; Brocklehurst and
178
Whipple, 2002). 179 The Myr postglacial response times we find – like comparable response times estimated
180
in the deglaciated Washington Cascades (Moon et al., 2015), northwest Himalaya (Hobley et al.,
181
2010), and Alpine Rhone Valley tributaries (Leith et al., 2018) – are on the order of or longer
182
than Quaternary glacial-interglacial periods. Thus, our findings add to a growing consensus that
183
glaciated mountain ranges worldwide have not evolved to a fluvial steady state during the
184
current, nor previous, interglacial periods. Rather, periodic climatic disturbances in the
185
Quaternary have persistently disrupted long-term, tectonically driven landscape evolution in
186
these settings. Importantly, we show that this prolonged impact of glaciation extends even
187
beyond the extent of past ice cover, suggesting a more spatially pervasive legacy of glaciation on
188
topography. 189 180 CONCLUSIONS
190
Our analyses indicate that asymmetric glaciation in the Qilian Shan has caused transient
191
changes of erosion rates in rivers below the extents of past glaciation. These transient changes
192
last over Myr timescales – much longer than ~40-100 kyr glacial-interglacial cycles. This
193
suggests that periodic climatic perturbations in the Quaternary can create persistence imbalance
194
between tectonics and topography, and this impact can extend even beyond the bounds of past
195
ice cover. 196
ACKNOWLEDGMENTS
197
J. Lai is supported by funding from the European Union’s Marie Skłodowska-Curie
198
Actions Postdoctoral Fellowship No. 101064307. Steepness patterns in the Qilian Shan
102 We are grateful to J. Braun for helpful
199
discussions. We thank S.F. Gallen, M. Morriss, and an anonymous reviewer for their
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286
doi:10.1029/1999JB900120. 287 Willett, S.D., McCoy, S.W., Perron, J.T., Goren, L., and Chen, C.-Y., 2014, Dynamic
288
Reorganization of River Basins: Science, v. 343, p. 1248765–1248765,
289
doi:10.1126/science.1248765. 290 Wu, F., Fang, X., Yang, Y., Dupont-Nivet, G., Nie, J., Fluteau, F., Zhang, T., and Han, W., 2022,
291
Reorganization of Asian climate in relation to Tibetan Plateau uplift: Nature Reviews
292
Earth & Environment, p. 1–17, doi:10.1038/s43017-022-00331-7. 293 Earth & Environment, p. 1–17, doi:10.1038/s43017-022-00331-7. 293
FIGURES
294
295 FIGURES
294
295 295 Figure 1: (A) Topography, modern glaciers (white), and study area (white box) in the Qilian
296 Shan. The star shows the location of the divide in Fig. 3C. (B) Location of the Qilian Shan on the
297
Tibetan Plateau. (C) Aspect distribution of modern glaciers (RGI Consortium, 2017). 298 299 300
Figure 2: (A) Modeled evolution of a 1D mountain profile during 50-kyr asymmetric glaciation. 301
The left-hand side, representing north-facing slopes in the Qilian Shan, has lower ELA than the
302
right-hand side, which is the south-facing slopes. (B) 𝜒-elevation profile and (C) 𝑘!" distribution
303
at different elevations of the modeled north- (blue) and south-facing (green) profiles at 50 kyr. and after the captured drainage area i REFERENCES
202 304
O
d
h d li
i di
t
l
i l
diti
305 300
Fi
2 (A) M d l d
l ti
f
1D
t i
fil d
i
50 k
t i
l
i ti
301 300 Figure 2: (A) Modeled evolution of a 1D mountain profile during 50-kyr asymmetric glaciation. 301
The left-hand side, representing north-facing slopes in the Qilian Shan, has lower ELA than the
302
right-hand side, which is the south-facing slopes. (B) 𝜒-elevation profile and (C) 𝑘!" distribution
303
at different elevations of the modeled north- (blue) and south-facing (green) profiles at 50 kyr. 304
Orange dashed lines indicate preglacial conditions. 305 Figure 2: (A) Modeled evolution of a 1D mountain profile during 50-kyr asymmetric glaciation. 301 Figure 2: (A) Modeled evolution of a 1D mountain profile during 50-kyr asymmetric glaciation. 301
The left-hand side, representing north-facing slopes in the Qilian Shan, has lower ELA than the
302
right-hand side, which is the south-facing slopes. (B) 𝜒-elevation profile and (C) 𝑘!" distribution
303
at different elevations of the modeled north- (blue) and south-facing (green) profiles at 50 kyr. 304
Orange dashed lines indicate preglacial conditions. 305 Figure 2: (A) Modeled evolution of a 1D mountain profile during 50-kyr as
301 6 307
Figure 3: (A, B) 𝑘!" at different elevations on the north- and south-facing slopes of the study
308
area (Fig. 1). Crosses represent the average value of evenly spaced elevation bins. (C) The spatial
309 307 Figure 3: (A, B) 𝑘!" at different elevations on the north- and south-facing slopes of the study
308
area (Fig. 1). Crosses represent the average value of evenly spaced elevation bins. (C) The spatial
309
pattern of 𝑘!" across a sample divide. White lines represent the current divide (solid) and
310
inferred past divide location (dashed). (D, E) Binned average 𝑘!" at different elevations before
311
and after the captured drainage area is restored in Fig.3C, with inset (F, G) 𝜒-elevation profiles. 312 Figure 3: (A, B) 𝑘!" at different elevations on the north- and south-facing slopes 313 314
Figure 4: (A) 𝑘!" at different elevations of the modeled north-facing river following
315 14 north-facing river following Figure 4: (A) 𝑘!" at different elevations of the modeled north-facing river following deglaciation. 0 Myr line is the blue profile in Fig.2A. (B) Postglacial response time of the whole
316 deglaciation. REFERENCES
202 0 Myr line is the blue profile in Fig.2A. (B) Postglacial response time of the whole
316 profile (closed dots) and only the non-glaciated portion (open dots) for cases with different
317 divide migration distance after glaciation. Grey shaded area indicates response times shorter than
318 divide migration distance after glaciation. Grey shaded area indicates response times shorter than
318
100 kyr
319 divide migration distance after glaciation. Grey shaded area indicates response times shorter than
318
100 kyr. 319 g
g
100 kyr. 319
320
321 Supplemental Material. A supplemental text provides details of the numerical model and
322
simulation set-ups. 323
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ТЕОРЕТИЧЕСКИЕ ОСНОВЫ РЕКОНСТРУКЦИИ ЖИЛЬЯ В ФОРМИРОВАНИЯ ТЕРРИТОРИЙ ИСТОРИЧЕСКИХ МАХАЛЛИ
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Zenodo (CERN European Organization for Nuclear Research)
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cc-by
| 1,124
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МЕЖДУРОДНАЯ КОНФЕРЕНЦИЯ
АКАДЕМИЧЕСКИХ НАУК ТЕОРЕТИЧЕСКИЕ ОСНОВЫ РЕКОНСТРУКЦИИ ЖИЛЬЯ В
ФОРМИРОВАНИЯ ТЕРРИТОРИЙ ИСТОРИЧЕСКИХ МАХАЛЛИ
Сереева Гулжазира Адилбаевна
старший преподователь, кафедра “Градостроительства
и ландшафтная архитектуры”
Ташкентского архитектурно-строительного института
https://doi.org/10.5281/zenodo.6590478
Аннотация: В статье рассмотрена, современные тенденция сохранения и
реставрации
жилья,
особенности
инновационной
модуляции
в
архитектуре
в
будущем,
проблемы
философии
своего
времени,
представление о будущем архитектуры и ее прошлом, ее значение в
современных
отношениях,
изменения
в
исторической
городской
архитектуре. Комплексное улучшение жилищных условий в городах и сельской
местности Республики Узбекистан является делом большой социально-
экономической значимости и одним из важных направлений политики,
проводимой нашим государством. В современном развитии крупных исторических городов Республики
Узбекистан
решается
ряд
социально-экономических
вопросов
градостроительства. Гузары,
махалли,
являющиеся
гражданским
обществом,
представляют
собой
самое
справедливое
социальное
пространство, в полной мере учитывающее интересы и права человека,
реализующее его извечную мечту - его надежды. В то же время развитие
городов в Узбекистане ставит ряд актуальных вопросов, связанных с
формированием структуры современных поселений. Это расширение функциональных и планировочных структур жилых и
общественных
зданий,
жилых
кварталов,
махаллей,
оформление
архитектурно-художественных
аспектов
на
основе
многовековых
градостроительных традиций, а также сосуществование нашего народа
как общности, соседства, приумножение национальных традиций и их
действенность
связаны
с
учетом
градостроительных,
природно-
климатических условий и других особенностей Узбекистана. Известно, что махалля издавна была формой правления. Институт
местничества проводился на общинной основе и имел свои неписаные
внутренние процедуры, одинаково законные для всех. Современные
тенденции сохранения и реставрации жилья, особенности инновационной
модуляции в архитектуре в будущем, проблемы философии своего
времени, представление о будущем архитектуры и ее прошлом, ее 47
р
р
у у
р
ур
р МЕЖДУРОДНАЯ КОНФЕРЕНЦИЯ
АКАДЕМИЧЕСКИХ НАУК
значение в современных отношениях, изменения в исторической
й МЕЖДУРОДНАЯ КОНФЕРЕНЦИЯ
АКАДЕМИЧЕСКИХ НАУК
значение в современных отношениях, изменения в исторической МЕЖДУРОДНАЯ КОНФЕРЕНЦИЯ
АКАДЕМИЧЕСКИХ НАУК значение в современных отношениях, изменения в исторической
городской архитектуре. значение в современных отношениях, изменения в исторической
городской архитектуре. Изучение путей развития кварталов, их композиционно-средовых качеств
и других архитектурных особенностей позволяет сделать вывод о том, что
изучаемые объекты являются не только уникальными объектами в
восточной архитектуре, но и играют важную роль в современной
градостроительной практике. Анализ современных способов формирования микрорайонов выявил, что
многие из них не отвечают спросу. Прежде всего, это требования
архитектурно-градостроительные и функциональные особенности, не
учитываемые в регионах и местные социальные условия, что в конечном
итоге привело к произвольному строительству зданий. Для них
характерна деградация архитектурного наследия. Поэтому для приведения районных центров и исторических улиц города,
традиционной жилой застройки к совершенно единому решению
предложено проводить их комплексную архитектурно-функциональную
модернизацию в следующие этапы. На первом этапе модернизации прежде всего учитывалась сочетаемость
здания или сооружения с окружающей средой, его близость к памятникам
архитектуры
(индивидуальное
состояние,
расположение,
функция
здания). Он требует, чтобы общественные здания, торговые центры
располагались на исторической территории города, не портили красоту
архитектурных памятников, композиционную масштабность квартальных
ансамблей, адаптируя каждое здание к квартальным ансамблям и
размещая их соразмерно зданиям данного района. Второй этап модернизации включает в себя реконструкцию жилых домов
микрорайона. Это реставрация памятников архитектуры, парков и
скверов, модернизация элементов искусства и жилищ, представляющих
историческую ценность. В
результате
исследований
установлено,
что
градостроительное
планирование находит отражение в индивидуальности городской среды,
стилевой и ландшафтной гармонии кварталов, решении исходной
пространственной среды, топографических особенностей застройки,
традиционных
художественно-исторических
композициях,
малых
архитектурные ансамбли. Проблема
сохранения
исторического
наследия,
особенно
старых
городских
поселений,
является
одной
из
актуальнейших
задач ия
исторического
наследия,
особенно
старых
й,
является
одной
из
актуальнейших
задач 48 МЕЖДУРОДНАЯ КОНФЕРЕНЦИЯ
АКАДЕМИЧЕСКИХ НАУК МЕЖДУРОДНАЯ КОНФЕРЕНЦИЯ
АКАДЕМИЧЕСКИХ НАУК МЕЖДУРОДНАЯ КОНФЕРЕНЦИЯ
АКАДЕМИЧЕСКИХ НАУК Выявлены следующие факторы и проблемы, влияющие на стоимость
исторического городского жилья: экономическая незащищенность семьи,
увеличение количества семей в здании (снос и увеличение площади),
пренебрежение пожилыми людьми в жилище, непонимание культурная
ценность здания, подражание европейским домам, их подход, с точки
зрения градостроительства, сохраняется под влиянием соседних зданий. Предприниматели и строители скупают старые дома и строят на их месте
сложные, отсутствие коммуникаций (канализация, газ и вода) в
большинстве
исторических
домов,
использование
экономически
выгодными домовладельцами зарубежных строительных материалов,
воздействие природных факторов (землетрясение, осадки), грунтовые
воды), влияние дорожно-транспортной системы, отсутствие единой
приоритетной системы строительных работ в исторических городах,
снижение контроля за историческими поселениями. Для
развития
и
восстановления
исторической
городской
жизни
разработаны
следующие
комплексные
решения
и
программы:
рассмотрение
правительством
программы
развития
исторического
города, разработка управленческого плана развития исторического
города, разработка генерального плана должно быть организовано
развитие исторического города. Вводить предметы, связанные с реконструкцией жилья в исторических
городах, прививать в наших школах материальные и нематериальные
ценности, наши культурные ценности и преподавать их как учебники,
проводить
разъяснительную
работу
с
населением,
оказывать
материальную и моральную поддержку населению, обеспечивать его
долгосрочные льготные микрокредиты, отделение, организация ремесел в
исторических домах, восстановление исторических поселений на основе
традиций. МЕЖДУРОДНАЯ КОНФЕРЕНЦИЯ
АКАДЕМИЧЕСКИХ НАУК современного
градостроительства. «Исторической
основой»
города
является район с многоэтажной застройкой, причем к наиболее
общественным зданиям этой застройки относятся традиционные жилые
дома. Именно они впоследствии формируют городскую структуру, к
которой добавляются общественные здания и памятники прошлого. Наиболее распространенным типом построек являются жилые дома в
исторических
районах,
которые
часто
трудно
обслуживать
из-за
неустойчивости устаревших строительных материалов и нерешенных
технических и инженерных задач. В настоящее время основные здания в исторических городах остаются
незащищенными
как
необходимый
«исторический
элемент»
градостроительства. Это приводит к негативным последствиям, нарушая
достоинство и самобытность национального зодчества. С учетом этого в
ходе исследования были решены следующие практические задачи
модернизации традиционного жилья: - теоретические решения проблемы сохранения, реконструкции и
организации
ремесленных
и
частных
гостиниц
в
исторических
поселениях, преимущественно туристических маршрутах; - теоретические решения проблемы сохранения, реконструкции и
организации
ремесленных
и
частных
гостиниц
в
исторических
поселениях, преимущественно туристических маршрутах; -
практические
рекомендации
и
выводы
для
проектировщиков,
строителей
и
исследователей
по
реставрации
и
сохранению
традиционных домов исторических городов; - Проекты реконструкции поврежденной части города Самарканда,
махаллей Багимайдон в районе мечетей Гори Амир, Рухабад и Тиллакори,
Бибиханим, улица 8 Марта, Ювелирная; -
Рекомендации
и
проектные
предложения
по
применению
и
использованию современных инновационных технологий в традиционном
жилище, реализации функции умного дома. Модернизация исторически сложившихся традиционных жилых зданий
означает не их полную перестройку, придание им облика современных
новых домов, а ремонт их старых, поврежденных и поврежденных частей и
частей, приспособление к их структуре современных инженерных
коммуникаций. Это связано с тем, что другие современные потребности,
отвечающие функции этого жилого дома, могут быть преобразованы в
мастерскую народных художественных промыслов или домашнюю
гостиницу. В любом случае модернизируемый жилой дом не должен
терять свою историческую ценность и национальное архитектурное
качество. 49 МЕЖДУРОДНАЯ КОНФЕРЕНЦИЯ
АКАДЕМИЧЕСКИХ НАУК Список используемой литературы: 1. Мусурманкулов Ф.В. Махалля – важный субъект самоуправления
граждан // Молодой ученый. - 2013. № 7. 2. Аҳмедов М. К. Ўрта Осиё меъморчилиги тарихи: Олий ўқув юрт учун
ўқув қўлланмаси.— Т.: Ўзбекистон, 1995. 3. Аскаров Ш.Д. Генезис архитектуры Узбекистана. -Т.: 2014. – С. 10. 4. Журнал «Строительство и архитектура» Узбекистана №10. овская Л.Ю. Типологические основы зодчества Средней Азии (IX– 50
5. Маньковская Л.Ю. Типологические основы зодчества Средней Азии (IX–
начало ХХ в.) Т.: ―Фан, 1980. 5. Маньковская Л.Ю. Типологические основы зодчества Средней Азии (IX–
начало ХХ в.) Т.: ―Фан, 1980. 50
5. Маньковская Л.Ю. Типологические основы зодчества Средней Азии (IX–
начало ХХ в.) Т.: ―Фан, 1980.
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https://europepmc.org/articles/pmc5604368?pdf=render
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English
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Conduction Electrohydrodynamics with Mobile Electrodes: A Novel Actuation System for Untethered Robots
|
Advanced science
| 2,017
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cc-by
| 5,112
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Conduction Electrohydrodynamics with Mobile Electrodes:
A Novel Actuation System for Untethered Robots to Cacucciolo,* Hiroki Shigemune, Matteo Cianchetti, Cecilia Laschi,
d Shingo Maeda* Vito Cacucciolo,* Hiroki Shigemune, Matteo Cianchetti, Cecilia Laschi,
and Shingo Maeda* providing the energy from the external
environment. Electrical power supplies
and transduction components cannot be
easily scaled down due to technological
limitations, thus requiring a large size
increasing in the robot.[2] For this reason,
some researchers proposed the direct use
of chemical energy to obtain mechan-
ical actuation.[1,4,9] This solution, while
advanced and attractive, still presents
drawbacks that limit its applicability, such
as the difficult interfacing with digital con-
trol. Harvesting the energy from the envi-
ronment can be an effective alternative
way to activate a miniaturized robot. In
this framework, magnetic field actuation
is one of the most common solutions,
however usually it requires the robot to be at microscale while
the magnets at macroscale.[10] This scale limitation follows the
fact that the area of activation depends on the strength of the
magnetic field, so higher current and more coils are required
to activate a wider area. Miyashita et al. developed an unteth-
ered robot activated by magnetic field at millimeter scale, using
high currents (≈10 A) and powers (≈100 W) while their opera-
tional area is only a small portion of the whole system.[11] An
alternative solution consists in the use of electrohydrodynamic
(EHD) forces. EHD refers to the direct conversion of electrical
energy into mechanical energy of a fluid through the interaction
between the electric and the flow fields.[12–14] The main applica-
tion of EHD in the literature is in the development of simple
pumping devices for miniaturized cooling systems, proving its
feasibility and robustness.[15–17] Chang et al. in 2007 proposed
to use the reaction body forces exerted by the fluid on the
electrodes to employ EHD as a propulsion method for minia-
ture diodes.[18] They applied an AC medium-intensity electric
field (≈104 V m−1) to a liquid with relatively high conductivity
having particle diodes suspended on its free surface. The cur-
rent is rectified by the diodes, resulting in local DC fields that
produced the EHD forces by electro-osmosis, pushing the fluid
and the diodes in opposite directions. What we propose in this
work is to expand the idea of using EHD reaction forces as a
propulsion method for untethered devices by exploiting a dif-
ferent physical mechanism called conduction EHD. COMMUNICATION COMMUNICATION Electrohydrodynamics www.advancedscience.com Conduction Electrohydrodynamics with Mobile Electrodes:
A Novel Actuation System for Untethered Robots This is an open access article under the terms of the Creative
Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly cited. Adv. Sci. 2017, 4, 1600495 Conduction Electrohydrodynamics with Mobile Electrodes:
A Novel Actuation System for Untethered Robots In conduc-
tion EHD, Coulomb forces are the dominant ones; it applies to
dielectric fluids that ionize in the proximity of the electrodes
in domains called heterocharge layers (Figure 1a), if the inten-
sity of the applied DC electric field exceeds a certain threshold
(≈105 V m−1).[12,14]
electrical
mobile elec-
ng and testing
ng the energy
erted sail-boat,
The diffusion
t its movement
nd dynamic
results show
ng untethered
of this actua-
soft robotics. Electrohydrodynamics (EHD) refers to the direct conversion of electrical
energy into mechanical energy of a fluid. Through the use of mobile elec-
trodes, this principle is exploited in a novel fashion for designing and testing
a millimeter-scale untethered robot, which is powered harvesting the energy
from an external electric field. The robot is designed as an inverted sail-boat,
with the thrust generated on the sail submerged in the liquid. The diffusion
constant of the robot is experimentally computed, proving that its movement
is not driven by thermal fluctuations, and then its kinematic and dynamic
responses are characterized for different applied voltages. The results show
the feasibility of using EHD with mobile electrodes for powering untethered
robots and provide new evidences for the further development of this actua-
tion system for both mobile robots and compliant actuators in soft robotics. Untethered became a fundamental keyword in the mobile
robotics area, representing one of the most challenging bottle-
necks in the development of the field.[1–4] Especially looking at
robotics branches featuring innovative actuation technologies,
such as soft robotics, we see that untethered solutions are quite
rare.[5–8] There are two main ways to avoid tethering: loading
the power supply and transduction elements onto the robot or 1600495 (1 of 6)
Dr. V. Cacucciolo
The BioRobotics Institute
Scuola Superiore Sant’Anna
Viale Rinaldo Piaggio 34, Pontedera (Pisa) 56025, Italy
E-mail: vito.cacucciolo@santannapisa.it
H. Shigemune
Department of Applied Physics
Graduate School of Science and Engineering
Waseda University
3-4-1 Okubo Shinjuku-ku, Tokyo 169-8555, Japan
Dr. M. Cianchetti, Prof. C. Laschi
The BioRobotics Institute
Scuola Superiore Sant’Anna
Viale Rinaldo Piaggio 34, Pontedera (Pisa) 56025, Italy
Prof. S. Maeda
Department of Engineering Science and Mechanics
Shibaura Institute of Technology
3-7-5 Toyosu, Koto-ku, Tokyo 135-8548, Japan
E-mail: maeshin@shibaura-it.ac.jp
DOI: 10.1002/advs.201600495
© 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA,
Weinheim. www.advancedscience.com www.advancedsciencenews.com Figure 1. Schematics and picture of the experiment, with the illustration of the EHD phenomenon and the robot sailing in the two fluids. a) The robot
is designed as an inverted sail-boat; the high-intensity electric field is generated between the sail and a fixed external electrode, in a volume totally
immersed in the dielectric fluid; the EHD forces are exerted between the sail electrode and the ions in the heterocharge layer. In order to keep the robot
untethered, we designed the electrical connection between the sail electrode and the ground by means of the conductive liquid that floats in contact
with the hull, on the top of the dielectric one. b) Illustration of the robot with all the components and dimensions. c) Top view of the acceleration
experiment, with the tracking of the path of the Center of Mass (COM) of the robot (see also Movie S1, Supporting Information). Figure 1. Schematics and picture of the experiment, with the illustration of the EHD phenomenon and the robot sailing in the two fluids. a) The robot
is designed as an inverted sail-boat; the high-intensity electric field is generated between the sail and a fixed external electrode, in a volume totally
immersed in the dielectric fluid; the EHD forces are exerted between the sail electrode and the ions in the heterocharge layer. In order to keep the robot
untethered, we designed the electrical connection between the sail electrode and the ground by means of the conductive liquid that floats in contact
with the hull, on the top of the dielectric one. b) Illustration of the robot with all the components and dimensions. c) Top view of the acceleration
experiment, with the tracking of the path of the Center of Mass (COM) of the robot (see also Movie S1, Supporting Information). produced an independent self-sailing robot powered through the
external environment. On the one hand, it represents a feasibility
study for the use of conductive EHD with mobile electrodes, on
the other it is an attractive application in the fields of mobile
robotics and autonomous systems. In this short report our main
contribution is to verify the feasibility and robustness of the use
of EHD pumping as an actuation method for mobile electrodes
by: (1) proposing a set-up consisting of a millimeter scale self-
propelled robot; (2) proving that the motion of the robot was
driven by EHD rather than by thermal fluctuations in the fluid;
(3) measuring the response of the robot in terms of velocity;
(4) computing the performance in terms of thrust produced;
(5) highlighting a power-law correlation between the delay time
and maximum thrust power. We believe that this technology will
find wide application in the field of untethered mobile robots,
once further studies will be conducted about controlling the
direction of the robot, studying the different kind of fluids that
can be used and optimizing the thrust produced. The conduction EHD has been understood only recently
and still a few applications have been proposed, mostly about
pumping a dielectric fluid by means of two fixed electrodes.[13,15,19]
The use of conduction EHD with mobile electrodes, which we
propose here for the first time, presents several advantages com-
pared to other actuation methods for untethered robots: (1) it
does not require bulky external hardware (e.g., coils); (2) it can
work with very small electrical currents (≈10 µA) and powers ≈10
mW), allowing the use of portable power sources such as min-
iaturized batteries; (3) although a high intensity electric field is
required (≈105 Vm−1), it is confined in a small volume between
the electrodes and it moves with them (Figure 1a), thus removing
any limitation about the operational area of the robot. Addition-
ally, EHD is widely scalable: it has been successfully applied in
the MEMS area, but we show here that it can work robustly also
at the millimeter scale. Respect to the electro-osmosis proposed
by Chang et al., our system does not require diodes but simple
electrodes, which in our case consist of thin copper sheets with
a dielectric adhesive layer on one side.[18] Moreover, the fluids
involved are dielectric, opening to the potential use of this tech-
nology in different industrial applications such as inspections
in pipelines containing oils or coolants, while keeping the elec-
trical currents at very low values. In order to test the feasibility
of the system and to propose a possible application, we designed
an untethered robot as an inverted sail-boat (Figure 1a,b). DOI: 10.1002/advs.201600495 1600495 (1 of 6) Adv. Sci. 2017, 4, 1600495 © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim The
sail, i.e., the mobile electrode where the thrust is generated, is
submerged in the liquid and fabricated in conductive material. The hull is on the top of it and floats on the liquid. This design The proof that the motion of the robot, considered as a sus-
pended particle, is a consequence of the thrust induced by
EHD rather than by thermal fluctuations, can be obtained
computing its diffusion constant.[20] We conducted an experi-
ment to measure the diffusion constant when there is no
voltage applied (V0 condition) and applying a voltage of
1250 V (VI condition). For details see Section S1 in the Sup-
porting Information. The results are DV0 = 0.0272 cm2 s−1 and
DVI = 0.582 cm2 s−1. It is possible to observe that, when the
voltage is applied, the diffusion constant increases over one order 1600495 (2 of 6) Adv. Sci. 2017, 4, 1600495 © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedscience.com www.advancedsciencenews.com i
d
h
d
d b
h
l fl
Th
j
d
ib d b
h
b
ll
. Kinematics and dynamics behavior of the robot. a) Time evolution of the x and y positions of the COM of the robot for different app
Tangential speed of the COM of the robot for different voltages (solid line), fitted with the trilinear model in Equation (1). c) Estimat
roduced by EHD pumping on the robot, for different voltages. d) Log–log plot of the maximum thrust power versus delay time t1. Figure 2. Kinematics and dynamics behavior of the robot. a) Time evolution of the x and y positions of the COM of the robot for different applied volt-
ages. b) Tangential speed of the COM of the robot for different voltages (solid line), fitted with the trilinear model in Equation (1). c) Estimation of the
thrust produced by EHD pumping on the robot, for different voltages. d) Log–log plot of the maximum thrust power versus delay time t1. of magnitude respect to the one produced by thermal fluctua-
tions, confirming that the motion of the robot is a consequence
of the thrust induced by EHD. The second experiment had the
scope of characterizing the kinematic and dynamic responses of
the robot for different applied voltages. © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedscience.com In this work we designed and tested an untethered sailing ( )
if
if
if
0
0
1
1
0
1
2
1
2
2
1
1
2
2
v t
v
v
a
t
t
v
a
t
t
a
t
t
t
t
t
t
t
t
t
(
)
(
)
(
)
=
+
−
+
−
+
−
≤
< ≤
≥
(1) (1) where t1 is the delay time, t2 is the time threshold for the
change in the acceleration, a1 and a2 are the first and second
accelerations, respectively, and v0 is the average initial speed
driven by thermal fluctuations. The fitting was realized through
a least-squares curve fitting with the trust-region-reflective algo-
rithm, using as parameters p = [v0,t1, t2, a1, a2].[22] The results
are shown in Table 1 and in Figure 2b, where it is possible to
observe that the model accurately fitted the data. We can notice that, with VH = 196 V, the EHD thrust was
negligible and the robot moved driven by thermal fluctua-
tions in the fluid. This was reflected in the fitting, as the accel-
erations a1 and a2 converged to 0, reducing to a motion at a
constant average speed of v0. Such a behavior is explained by
the fact that the correspondent field intensity (0.46 kV cm−1)
is lower than the general threshold of the EHD phenomenon
(≈1 kV cm−1). In the second voltage increment (VH = 1058 V),
the transition to the third region was never observed: the best
fitting was represented by a constantly accelerated motion. From the third to the sixth increments it was instead possible
to clearly identify the three regions. It is noticeable that the
acceleration in the second phase a1 increased monotonically
with the voltage, as shown in Table 1, while a2 was almost
constant and very small up to VH = 2263 V. The acceleration
a2 was always smaller than a1. From the kinematics data, we
estimated the thrust FT(t) generated by EHD on the robot
(see Section S2 in the Supporting Information for details In this work we designed and tested an untethered sailing
robot powered by conduction EHD. We demonstrated the fea-
sibility of using conduction EHD with mobile electrodes for
untethered sailing of centimeter-scale robots, captured the
dynamic response of the robot and quantified the thrust pro-
duced. www.advancedscience.com It is possible to observe
that the two quantities are related by a same power law for
1058 V ≤ VH ≤ 2029 V, while there is a discontinuity for
VH ≥ 2029 V. The presence of a delay time t1 in the EHD motion
has been reported in transient experiments by Tobazeon.[23]
They observed that t1 follows a power law respect to the applied
voltage t1 = aV−k, where both a and k stay constant varying the
voltage in a wide range (101 to 104 V), while the variation of the
gap between the electrodes affects a, shifting the relationship
between parallel lines in a log–log plot similar to the one in
Figure 2d.[23] The power law found in our experiments relates
t1 and the maximum power rather than the applied voltage. The two phenomena are arguably strictly connected while the
difference can be due to differences in the electrodes geom-
etries, in the fluids and to the presence in our case of one
mobile electrode. From the evidence presented by Tobazeon,
it appears that the discontinuity shown for VH ≥ 2029 V in
our experiments can be motivated by the fact that the electro-
static force between the electrodes slightly reduced the gap
between them, since the robot is not vertically constrained
(see Figure 1). Therefore, we expect the point correspondent
to 2263 V to belong to a power law parallel to the one shown
for 1058 V ≤ VH ≤ 2029 V, as represented by the dashed line in
Figure 2d. A further investigation of the physical mechanisms
observed in the proposed experiments would require a multi-
domain model describing: the interaction between the EHD
force and the ions in the heterocharge layers; the unsteady
fluid flow driven by the motion of the ions; the perturbations
in the electric field and etherocharge layers due to the move-
ment of one of the electrodes (electrode sail). Such a model
requires a dedicated study and lies outside the scope of this
paper. Nevertheless, the experimental results obtained in this
work and the simple model proposed in (1) provide informa-
tion for a further understanding of the phenomenon, which
will be pursued as future work. It consisted in placing
the robot on one side of the tank, oriented toward the center,
turning on the voltage up to the reaching of the boundary in a
different point (Figure 1c; Movie S1, Supporting Information). The trajectory described by the robot was usually curvilinear
due to small asymmetries in its body and to boundary effects
and convective motions of the fluid inside the tank. However,
the curvatures of the trajectories were small and the robot
moved mostly along its axis. Figure 2a shows the x and y time evolution of the Center of
Mass (COM) of the robot actuated with different voltages. All
the data were aligned such that time t = 0 coincides with the 1600495 (3 of 6) Adv. Sci. 2017, 4, 1600495 © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedscience.com A further investigation of the physical mechanisms
observed in the proposed experiments would require a multi-
domain model describing: the interaction between the EHD
force and the ions in the heterocharge layers; the unsteady
fluid flow driven by the motion of the ions; the perturbations
in the electric field and etherocharge layers due to the move-
ment of one of the electrodes (electrode sail). Such a model
requires a dedicated study and lies outside the scope of this
paper. Nevertheless, the experimental results obtained in this
work and the simple model proposed in (1) provide informa-
tion for a further understanding of the phenomenon, which
will be pursued as future work. application of the voltage at the electrodes and the origin of the
reference frame is the position occupied by the COM at that
instant. Because the data were still noisy after the sampling,
we computed the time derivatives of the x and y positions
using the smooth derivation with noise filtering proposed by
Holoborodko, using a filter length of N = 7.[21] From the speed
response, it is possible to distinguish three main regions in the
behavior of the robot (Figure 2b). The first one shows latency in
the EHD-induced thrust: the robot moves with random oscilla-
tions typical of thermal fluctuations. The second one is charac-
terized by a steep acceleration that then decreases in the third
one, eventually becoming zero. In order to capture these trends
and estimate the accelerations, we fitted the data with a simple
trilinear model of the kind about the computation). Coherently with what observed in
the acceleration (Figure 2b), the thrust presents latency in
the beginning. The second region is dominated by inertial
effects, since the robot presents here the higher acceleration,
right after the appearance of the EHD phenomenon. The third
region is instead characterized by the balance between drag
and produced thrust (FT ∼ f(v)). The presence of a residual
acceleration, significant for VH = 2263 V, can be explained
with the transient effects characteristic of EHD, which can
be peculiar in the novel case of one moving electrode pro-
posed in this work.[23] Finally, we investigated the correlation
between the delay time t1 (Equation (1) and Table 1) and the
maximum thrust power generated, for each applied voltage. The results are shown in Figure 2d. www.advancedsciencenews.com www.advancedscience.com www.advancedscience.com about the computation). Coherently with what observed in
the acceleration (Figure 2b), the thrust presents latency in
the beginning. The second region is dominated by inertial
effects, since the robot presents here the higher acceleration,
right after the appearance of the EHD phenomenon. The third
region is instead characterized by the balance between drag
and produced thrust (FT ∼ f(v)). The presence of a residual
acceleration, significant for VH = 2263 V, can be explained
with the transient effects characteristic of EHD, which can
be peculiar in the novel case of one moving electrode pro-
posed in this work.[23] Finally, we investigated the correlation
between the delay time t1 (Equation (1) and Table 1) and the
maximum thrust power generated, for each applied voltage. The results are shown in Figure 2d. It is possible to observe
that the two quantities are related by a same power law for
1058 V ≤ VH ≤ 2029 V, while there is a discontinuity for
VH ≥ 2029 V. The presence of a delay time t1 in the EHD motion
has been reported in transient experiments by Tobazeon.[23]
They observed that t1 follows a power law respect to the applied
voltage t1 = aV−k, where both a and k stay constant varying the
voltage in a wide range (101 to 104 V), while the variation of the
gap between the electrodes affects a, shifting the relationship
between parallel lines in a log–log plot similar to the one in
Figure 2d.[23] The power law found in our experiments relates
t1 and the maximum power rather than the applied voltage. The two phenomena are arguably strictly connected while the
difference can be due to differences in the electrodes geom-
etries, in the fluids and to the presence in our case of one
mobile electrode. From the evidence presented by Tobazeon,
it appears that the discontinuity shown for VH ≥ 2029 V in
our experiments can be motivated by the fact that the electro-
static force between the electrodes slightly reduced the gap
between them, since the robot is not vertically constrained
(see Figure 1). Therefore, we expect the point correspondent
to 2263 V to belong to a power law parallel to the one shown
for 1058 V ≤ VH ≤ 2029 V, as represented by the dashed line in
Figure 2d. © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Experimental Section Received: December 6, 2016
Revised: March 25, 2017
Published online: May 22, 2017 Received: December 6, 2016
Revised: March 25, 2017
Published online: May 22, 2017 The experiments were conducted in a cylindrical glass tank with a
diameter of 135 mm and height of 74 mm, filled with a layer of dielectric
liquid topped by a layer of conductive solution (Figure 1a). The liquids
that were used were Novec 7000 (1-methoxyheptafluoropropane
(C3F7OCH3), density ρN7 = 1400 kg m−3, dynamic viscosity
μN7 = 0.450 g m−1 s−1, volume resistivity 108 Ω cm) by 3 m as a dielectric
and a solution of sodium chloride in water (density ρs = 1120 kg m−3,
dynamic viscosity μs = 1.30 g m−1 s−1, resistivity 4.4 Ω cm) as conductive
fluid. The Novec 7000 was chosen for the following reasons: (1) its
density is higher than that of the NaCl solution and (2) the two fluids
are not miscible (solubility ≤ 60 ppm by weight); (3) it is chemically
stable; (4) its conductivity is very low, allowing to establish the high-
intensity electric field required by EHD and increasing the efficiency of
the mechanism.[15] On the bottom of the glass container, a copper sheet
electrode with a thickness of 40 μm was glued, connected to the voltage
source (+VH) through a conductive wire insulated for high voltages. As for the ground electrode, a tin wire with a diameter of 0.7 mm was
inserted into the NaCl solution. In this set-up, the positive electrode
was in contact only with the dielectric fluid, while the NaCl solution only
was connected to the ground (Figure 1a,c). The robot was composed
by three elements (Figure 1b): (1) a hollow cylinder of polyurethane for
the hull; (2) two fins made of poly(ethylene terephthalate) (PET) acting
as stabilizers; (3) one submerged sail electrode, which generates the
thrust, made by a square copper sheet. The extremes of the hollow
cylinder were corked with hot-melt adhesive to increase the buoyancy
by sealing the internal chamber. The weight of this component was of
1.10 g, while the one of the fins 0.25 g. The electrode sail was conductive
on one side only, while the other one was covered by a thin layer of
dielectric glue. Its total weight was 0.20 g. www.advancedscience.com This work shows a novel possible application of EHD
actuation and provides data for a better understanding of
this complex phenomenon. In particular, the presence of one
mobile electrode in the proposed scenario introduces peculiar
phenomena since the boundary conditions, which have a fun-
damental importance in EHD, are different from the generally Table 1. Fitting parameters for the model in Equation (1). 1600495 (4 of 6)
Adv. Sci. 2017, 4, 1600495
Table 1. Fitting parameters for the model in Equation (1). VH [V]
196
1058
1342
1696
2029
2263
v0 [cm s−1]
0.117
0.087
0.145
0.132
0.121
0.094
t1 [s]
∞
0.713
0.600
0.541
0.437
0.270
t2 [s]
∞
∞
1.378
1.051
0.673
0.458
a1 [cm s−2]
0
0.334
1.160
2.148
7.744
8.831
a2 [cm s−2]
0
0
0.119
0.105
0.118
0.791
MSE [cm s−1]
0.0017
0.0142
0.0088
0.0087
0.0284
0.0658 © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim The authors declare no conflict of interest. The authors declare no conflict of interest. Supporting Information Supporting Information is available from the Wiley Online Library or
from the author. www.advancedscience.com www.advancedscience.com studied case consisting in two fixed electrodes.[24] One example
is the possible coupling with electrostatic attraction between
the electrodes, which can vary the gap as a consequence of
the applied voltage, as discussed in the previous paragraph. Experiments consisting in connecting the robot to a horizontal
slider at different fixed distances from the bottom electrode can
contribute in quantifying this effect. On the other hand, the role
of the mobility of the electrode can be investigated by moving
the robot at fixed velocities and measuring the thrust produced. Although the requirements of our system, e.g., two separated
layers of fluids, one conductive and the other one dielectric,
high voltage, an electrode on the bottom of the tank, may
seem restrictive, future improvements of this primitive set-up
will be able to remove some of these constraints and expand
its applicability. One possible method for eliminating the need
of the fixed bottom electrode and the conducting liquid on the
top is to embed the power source and the HV converter on
board and to use two electrodes both connected to the robot. The low power absorbed by EHD propulsion (≈10 mW) allows
the use of miniaturized lithium batteries, while HV converters
for VH ≤ 5 kV weight ≈6 g, therefore requiring only a slight
increase in the size of the robot.[25] Furthermore, replacing
mobile electrodes with compliant ones, a similar system could
find application in the development of compliant actuators for
soft robotics. robot was tracked through automatic tracking software (Kinovea 0.8.15)
from movies acquired at 20 fps. The raw data were sampled at 5 ms with
a spline interpolation. robot was tracked through automatic tracking software (Kinovea 0.8.15)
from movies acquired at 20 fps. The raw data were sampled at 5 ms with
a spline interpolation. Keywords electrohydrodynamics, mobile robots, soft actuators, soft robotics,
untethered Acknowledgements This work was supported by RoboSoft – A Coordination Action for
Soft Robotics (FP7-ICT-2013-C # 619319). The authors would like to
thank the Italian Ministry of Foreign Affairs, General Directorate for the
Promotion of the “Country System,” Bilateral and Multilateral Scientific
and Technological Cooperation Unit, for the support through the Joint
Laboratory on Biorobotics Engineering project. www.advancedsciencenews.com Experimental Section All the experiments were
performed using the same gap of 4.3 mm between the plane electrode
on the bottom of the tank and the sail electrode on the mobile robot. This gap was chosen as a balance between obtaining enough thrust,
which in EHD has been shown to depend on the gap squared and
avoiding the breakdown.[13] In all the experiments, the position of the [1] S. Maeda, Y. Hara, T. Sakai, R. Yoshida, S. Hashimoto, Adv. Mater. 2007, 19, 3480. [2] M. T. Tolley, R. F. Shepherd, B. Mosadegh, K. C. Galloway,
M. Wehner, M. Karpelson, R. J. Wood, G. M. Whitesides, Soft Rob. 2014, 1, 213. [3] C. Majidi, Soft Rob. 2014, 1, 5. [4] M. Wehner, R. L. Truby, D. J. Fitzgerald, B. Mosadegh,
G. M. Whitesides, J. A. Lewis, R. J. Wood, Nature 2016, 536, 451. [5] S. Kim, C. Laschi, B. Trimmer, Trends Biotechnol. 2013, 31, 287. [6] V. Cacucciolo, F. Renda, E. Poccia, C. Laschi, M. Cianchetti,
Smart Mater. Struct. 2016, 25, 105020. [7] H. Shigemune, S. Maeda, V. Cacucciolo, Y. Iwata, E. Iwase,
S. Hashimoto, S. Sugano, IEEE Rob. Autom. Lett. 2017, 2, 1001. [8] J. Shintake, S. Rosset, B. E. Schubert, D. Floreano, H. Shea, IEEE/
ASME Trans. Mechatronics 2015, 20, 1997. [9] S. Maeda, Y. Hara, R. Yoshida, S. Hashimoto, Angew. Chem., Int. Ed. 2008, 47, 6690. [10] C. Pawashe, S. Floyd, M. Sitti, Int. J. Rob. Res. 2009, 28, 1077. [11] S. Miyashita, S. Guitron, M. Ludersdorfer, C. R. Sung, D. Rus, IEEE
Int. Conf. Robotics and Automation, IEEE, Seattle, WA, USA, 2015,
p. 1490. [12] J. R. Melcher, Continuum Electromechanics, MIT Press, Cambridge 1981. [13] M. Pearson, J. Seyed-Yagoobi, IEEE Trans. Dielectr. Electr. Insul. 2009, 16, 424. [14] A. Ramos, Electrokinetics and Electrohydrodynamics in Microsystems,
Springer, Vienna 2011. [15] P. Atten, J. Seyed-Yagoobi, IEEE Trans. Dielectr. Electr. Insul. 2003,
10, 27. 1600495 (5 of 6) Adv. Sci. 2017, 4, 1600495 © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedscience.com www.advancedscience.com [20] A. Einstein, Ann. Phys. 1905, 17, 549. [16] S. Liang, L. Weng, S. Tan, J. Xu, X. Zhang, L. Zhang, Appl. Phys. Lett. 2007, 90, 153506. [21] P. Holoborodko, Smooth Noise Robust Differentiators, http://www. holoborodko.com/pavel/numerical-methods/numerical-derivative/
smooth-low-noise-differentiators/ (accessed: March 2017). [17] J. W. Kim, T. Suzuki, S. Yokota, K. Edamura, Sens. Actuators, A 2012,
174, 155. [22] T. F. Coleman, Y. Li, SIAM J. Optim. 1996, 6, 418. [18] S. T. Chang, V. N. Paunov, D. N. Petsev, O. D. Velev, Nat. Mater. 2007, 6, 235. [23] R. Tobazeon, J. Electrostat. 1984, 15, 359. [24] A. Castellanos, IEEE Trans. Electr. Insul. 1991, 26, 1201. [19] Tasuku Sato, Yoko Yamanishi, Vito Cacucciolo, Yu Kuwajima,
Hiroki
Shigemune,
Matteo
Cianchetti,
Cecilia
Laschi,
Shingo Maeda, Chemistry Letters 2017, DOI: 10.1246/cl.170217. [25] EMCO A series, http://www.emcohighvoltage.com/proportional/
aseries.php (accessed: March 2017). Adv. Sci. 2017, 4, 1600495 1600495 (6 of 6) © 2017 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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http://old.scielo.br/pdf/cta/v39n3/0101-2061-cta-fst06118.pdf
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English
| null |
Lipid profile and quality of meat from finishing pig supplemented with minerals
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Food Science and Technology
| 2,019
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cc-by
| 7,725
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Received 01 Mar., 2018
Accepted 04 Mar., 2019
1Departamento de Medicina Veterinária, Universidade Federal de Lavras, Lavras, MG, Brasil
2Departamento de Ciência dos Alimentos, Universidade Federal de Lavras, Lavras, MG, Brasil
*Corresponding author: peterbfvet@yahoo.com.br Abstract This study aimed evaluate the effects of the associated supplementation of: chromium more iron (CrFe), magnesium more
selenium (MgSe) and the four minerals (CrFeMgSe) on the parameters related to the pork quality. Supplementation with
MgSe reduced the ether extract of the meat and changed the fatty acids profile, increasing the poly-unsaturated, n-3, n-6, the
polyunsaturated: saturated rate and the activity of the enzyme Thioesterase index, besides reducing the total number of saturated
fatty acids and the Atherogenicity index. It promoted a reduction in a*, b* and C* indices and increased h* of the chilled meat
stored. Over the storage days under refrigeration, there was linear drop for L* and a* and an increase to C*. The associated
use of magnesium and selenium promotes changes in lipid profile without changing the meat quality, and they may be used in
order to obtain meat with more appropriate nutritional aspects. Keywords: chromium; fatty acid; iron; magnesium; selenium. Practical application: Magnesium and selenium reduce the saturated fatty acid and increase n-3 levels of the pork. Practical application: Magnesium and selenium reduce the saturated fatty acid and increas 1 Introduction Pork represents the largest source of animal protein
consumed globally and the demand for this product grows each
day (United States Department of Agriculture, 2018). However,
consumers have become more demanding in relation to the
quality of the pork and the inherent health benefits it can provide. To meet this demand, nutritional strategies applied during the
animals’ production, arise as a tool to improve the meat quality
characteristics, as well as to reduce the fat content, making it
leaner and with a lipid profile better suited for consumption as
recommended by the World Health Organization (Food and
Agriculture Organization of United Nations, 2010). loss of color and an unpleasant aspect to the consumer during its
frozen exposure. Thus, the swine’s supplementation with iron can
increase the iron-heme levels in the muscle (Yu et al., 2000), an
integral part of the pigment myoglobin, promoting improvement
in the meat color, turning it redder and more intense. In addition,
the supplementation with Selenium can contribute for a better
meat stability due to its protective action in the membranes,
preserving it against the oxidizing agents (Calvo et al., 2016). The color of the pigment myoglobin is also dependent on the
lipid oxidation degree in the meat (Apple et al., 2007). The studies reported in the literature relate to the supplementation
with these minerals in isolation and many of these are in their
inorganic sources, which has a lower bioavailability. The aim
of this study was to verify the influence of supplementation
associated with organic sources of chromium, iron, magnesium
and selenium, on the physical and chemical properties and
centesimal composition of pork from finishing pig. Supplementation with minerals such as chromium and
magnesium, can contribute to an increase in the amount of
tissue muscle and reduction of the fat deposition in the meat
due to the effect of the nutrients repartition, which act on
the carbohydrates and lipids metabolism (Apple et al., 2000). Magnesium supplementation pre-slaughter has also been shown
to reduce the effects of stressors, reducing the catecholamines
and cortisol levels release, muscle relaxation and reduced
neuromuscular stimulation, preventing the sudden drop in muscle
pH post‑slaughter, reduces the L* indices (Swigert et al., 2004),
affect the activity of the enzyme Δ-6 desaturase, participating in
the metabolism of long-chain fatty acids, and it may increase the
levels of fatty acids n-3 in the meat (Mahfouz & Kummerow, 1989). Lipid profile and quality of meat from finishing pig supplemented with minerals Lipid profile and quality of meat from finishing pig supplemented with minerals
Tatiane Mendonça Nogueira Carneiro de ALBUQUERQUE1 , Eduardo Mendes RAMOS2,
Isabella Fiche da Matta MACHADO1, Paula Caixeta BORGES1, Ana Gabriella BOLLETA1, Joanna Oliveira MARÇAL1,
Fernanda Paul de CARVALHO1, Peter Bitencourt FARIA1* Tatiane Mendonça Nogueira Carneiro de ALBUQUERQUE1 , Eduardo Mendes RAMOS2,
Isabella Fiche da Matta MACHADO1, Paula Caixeta BORGES1, Ana Gabriella BOLLETA1, Joanna Oliveira MARÇAL1,
Fernanda Paul de CARVALHO1, Peter Bitencourt FARIA1* ISSN 0101-2061 (Print)
ISSN 1678-457X (Online) ISSN 0101-2061 (Print)
ISSN 1678-457X (Online) Food Science and Technology DOI:
D
https://doi.org/10.1590/fst.06118 Lipid profile and quality of meat from finishing pig supplemented with minerals
Tatiane Mendonça Nogueira Carneiro de ALBUQUERQUE1 , Eduardo Mendes RAMOS2, Lipid profile and quality of meat from finishing pig supplemented with minerals
Tatiane Mendonça Nogueira Carneiro de ALBUQUERQUE1 , Eduardo Mendes RAMOS2,
Isabella Fiche da Matta MACHADO1, Paula Caixeta BORGES1, Ana Gabriella BOLLETA1, Joanna Oliveira MARÇAL1,
Fernanda Paul de CARVALHO1, Peter Bitencourt FARIA1* 2.2 Physical and chemical parameters At the time of the slaughter, pH and initial temperature
were measured at 45 minutes post-slaughter in the Longissimus
thoracis muscle (LT) of left half carcasses, at the 12th rib height,
using a stem digital thermometer and pHmeter with penetration
probe (Hanna Instruments, HI 99163, Romania). The carcasses
were kept in a chiller for 24 h. The temperature and the final
pH were measured again and a portion of the LT muscle was
removed for the evaluations of physical and chemical parameters,
centesimal composition and lipid profile. Table 1. Composition of basal diet provided to animals during the
termination period. Table 1. Composition of basal diet provided to animals during the
termination period. Ingredients
kg per 100 kg of diet*
Corn
75.70
Soybean meal
20.00
Soybean oil
1.00
Dicalcium Phosphate
0.77
Limestone
0.56
Salt
0.35
Premix Mineral1
0.18
Premix Vitaminic2
0.30
L- Lysine 50,7%
0.47
DL- Methionine 99%
0.09
L- Threonine 98%
0.10
Ractopamine
0.05
Kaolin**
0.45
Iron3
6.12 × 10-4
Chromium4
3.4 × 10-6
Magnesium5
3.22 × 10-3
Selenium6
3.06 × 10-4
TOTAL
100.00
Calculated nutritional values
Crude protein (%)
15.01
Metabolizable energy (kcal/kg)
3240
Calcium (%)
0.47
Available phosphorus (%)
0.23
Sodium (%)
0.16
Digestible Lysine (%)
0.88
Digestible methionine (%)
0.31
Methionine + Cystine (%)
0.54
Digestible threonine (%)
0.61 The objective evaluation of the muscle color was performed at
24 hours post mortem, using a colorimeter (Konica Minolta CM‑700,
Singapore), operating in the system CIELAB, with illuminant
D65, observer angle of 10º and Specular Component Excluded,
(SCE), to obtain the indices of luminosity (L*), red (a*), yellow
(b*), oxygen saturation
(
)
/
*
*
1 2
2
2
C
a
b
=
+
and tonality angle
(
)
(
,
*
*/ *
1
h
tan
b
a
−
=
in degrees), according Ramos & Gomide (2012). The percentages of metmyoglobin (MMb), reduced myoglobin
(Mb+) and oxymyoglobin (O2Mb), were also calculated using
the following equations:
(
) (
)
(
)
–
,
. –
/
–
572
730
525
730
MMb
1 395
A
A
A
A
=
(
) (
)
(
)
(
)
. –
–
/
–
473
730
525
730
Mb
2 375
1
A
A
A
A
=
×
and
(
)
–
2
O Mb
1
MMb
Mb+
=
+
,
respectively, according to the methodology of Krzywicki (1979). 2.2 Physical and chemical parameters Visual assessment of color and marbling was conducted by five
evaluators through comparison with a standard National Pork
Producers Council (National Pork Producers Council, 1999) and
meat pigment content by the equation:
(
)
(
)
(
)
. –
. *
409
730
3 249
A
2 68 A
×
according to Ramos & Gomide (2012). The determination of the drip loss was made by the
suspension method for 48 hours, being expressed as a percentage
of the initial weight, according to Honikel (1998). The cooking
loss, also expressed as a percentage of the initial weight, was
made involving the samples in aluminum foil and broiled on a
preheated electric grill (Mega Grill; Britânia, Curitiba, PR, Brazil),
to an internal temperature of 72 °C, according to methodology
described by Ramos & Gomide (2012). The analysis of shear force was performed based on methodology
by Silva et al. (2015), using six square cross-section cores with
1.0 cm × 1.0 cm and the samples cross-cutting sectioned through
Warner Bratzler probe coupled to a texturometer (Extralab,
TA.XT Plus, UK) and the shear force expressed in Newtons (N). *Diets: 1) Control: basal diet without mineral supplementation; 2) CrFe: basal diet
supplemented with 3.4 mg/kg of Cr and 612.4 mg/kg of Fe; 3) MgSe: basal diet
supplemented with 3215.4 mg/kg of Mg and 306.1 mg/kg of Se; 4) CrFeMgSe: basal
diet supplemented with 3.4 mg/kg of Cr, 612.4 mg/kg of Fe, 3215.4 mg/kg of Mg and
306.1 mg/kg of Se. **the minerals were included in place of kaolin. 1Composition per kg
of product: cobalt, 299.7 mg; copper, 9.000 mg; iron, 48 g; iodine, 659.7 mg; manganese,
21 g; zinc, 78.3 g; selenium, 240.3 mg; 2Composition per kg of product: folic acid, 144 mg;
pantothenic acid, 2.160 mg; biotin, 21.60 mg; niacin, 3.960 mg; choline, 36.02 g; Vit. A,
1.440.000 U.I.; Vit.B1, 288 mg; Vit.B12, 3.960 mcg; Vit.B2, 720 mg; Vit.B6, 540 mg;
Vit. D3, 540.000 U.I.; Vit. E, 7.200 U.I.; Vit. K3, 540 mg. 3Biometal Chromium: chromium
picolinate (11.68% of Cr); 4Biometal Iron: 16.33% of Fe; 5Biometal magnesium:
magnesium bisglycinate (9.33% Mg); 6Biometal selenium: selenium glycinate (0.98% Se). L: levorotatory monomer; DL: racemic mixture of monomers. The centesimal composition was assessed according to the
methodology of the Association of Official Analytical Chemists
(Horwitz, 1990). 2.1 Experiment design and diet For the experiment, 44 barrows were used (crossbreeding
between DanBred females - DB90 × males PIC - AGPIC337),
with an average weight of 81.6 ± 5.22 kg, housed in finishing
swine-house, with concreted floor pens (2.3 × 1.5 m), with
semi-automatic feeders and nipple type waterer. The total
experimental period was 28 days. The experimental design was
in randomized complete block design (RCBD), according to the Currently, due to the genetic breeding focused on increasing
lean tissue and predominance of polyunsaturated fatty acids, pork
has become pale. This, combined with a higher propensity to
lipid oxidation and hence of the pigments of such meat, favor the 721 Food Sci. Technol, Campinas, 39(3): 721-728, July-Sept. 2019 Meat quality of pigs receiving minerals live weight, with four treatments (diets) and 11 repetitions with
each experimental plot represented by an animal. and 100 ppm of iron during 28 days + 300 ppm of magnesium
and 3 ppm of selenium in the last seven days. The diets were formulated based on corn, soybean meal, vitamins
and minerals, amino acids, and ractopamine, formulated to meet
the minimum requirements of finishing pigs and supplemented
with chromium, iron, magnesium and selenium from organic
source (Biometal, NPA - Núcleo de Pesquisas Aplicadas Ltda,
Jaboticabal, SP, Brazil), being the minerals included replacing
kaolin (Table 1). The animals were randomly divided into four
groups, receiving the following treatments: 1) Control: basal
diet for 28 days; 2) CrFe: basal diet + 400 ppb of chromium and
100 ppm of iron during 28 days; 3) MgSe: basal diet for 28 days
+ 300 ppm of magnesium and 3 ppm of selenium in the last
seven days; 4) CrFeMgSe: basal diet + 400 ppb of chromium Food and water were provided ad libitum daily and at the
end of the experiment, animals were slaughtered with an average
weight of 111.55 ± 9.53 kg, after 12 hours of rest and fasting,
in commercial slaughterhouse according to current standards. Food Sci. Technol, Campinas, 39(3): 721-728, July-Sept. 2019 3.1 Physical-chemical characteristics The supplementation with different minerals did not influence
the values of pH and initial temperature (45 minutes) and final
(24 hours post mortem), cooking loss and shear force (Table 2). Table 2. Evaluation of the physical-chemical parameters and centesimal composition of the Longissimus thoracis (LT) muscle of finishing swines
supplemented with different associations between minerals. al-chemical parameters and centesimal composition of the Longissimus thoracis (LT) muscle of finishing swines
ociations between minerals. Table 2. Evaluation of the physical-chemical parameters and centesimal composition of the Longissimus thoracis (LT) muscle of finishing swines
supplemented with different associations between minerals. Parameters
Diets
P-value5
Control1
CrFe2
MgSe3
CrFeMgSe4
Physicochemical
Initial pH
6.58 ± 0.23
6.35 ± 0.34
6.51 ± 0.30
6.37 ± 0.25
0.16
Final pH
5.79 ± 0.11
5.82 ± 0.09
5.86 ± 0.13
5.83 ± 0.08
0.59
Initial temperature (ºC)
36.73 ± 2.00
38.09 ± 1.45
36.50 ± 1.86
36.91 ± 1.81
0.18
Final temperature (ºC)
10.96 ± 0.77
10.59 ± 1.04
10.57 ± 0.82
10.42 ± 1.29
0.55
Drip loss (%)
8.28 ± 2.42ab
10.99 ± 3.11a
7.04 ± 2.12b
8.13 ± 3.12ab
0.01
Cooking loss (%)
29.40 ± 4.36
29.63 ± 4.95
26.95 ± 3.91
31.06 ± 3.86
0.11
Shear force (N)
70.58 ± 1.04
68.92 ± 1.36
69.05 ± 1.22
76.13 ± 1.36
0.48
Subjective marbling6
2.41 ± 0.66
2.25 ± 0.52
2.04 ± 0.27
1.96 ± 0.44
0.11
Subjective color 6
2.95 ± 0.18
2.98 ± 0.53
3.34 ± 0.74
2.83 ± 0.41
0.14
Total pigments (mg/g)
0.73 ± 0.26
0.75 ± 0.13
0.81 ± 0.20
0.67 ± 0.12
0.42
Centesimal composition
Moisture (%)
72.95 ± 1.85
72.73 ± 1.15
73.04 ± 1.98
72.30 ± 1.87
0.76
Protein (%)
23.51 ± 1.12
24.13 ± 1.28
24.45 ± 2.04
23.16 ± 1.45
0.15
Ash (%)
1.23 ± 0.22
1.21 ± 0.13
1.28 ± 0.20
1.26 ± 0.13
0.79
Ethereal extract (%)
2.38 ± 0.60a
2.03 ± 0.72ab
1.32 ± 0.43c
1.66 ± 0.43bc
0.01
1Control: basal diet; 2CrFe: supplementation with chromium and iron; 3MgSe: supplementation with magnesium and selenium; 4CrFeMgSe: supplementation with chromium, iron,
magnesium and selenium; 5Tukey’s test (α = 0.05); 6Evaluated by comparison with standard National Pork Producers Council (1999) ranging from 1 (pale pinkish gray to white) to
6 (dark purple red) for color and from 1 (light) to 10 (abundant) for marbling; a,b,c: Different letters indicate statistically significant differences between treatments. 2.2 Physical and chemical parameters The moisture content was determined by
oven-dried the samples at 105 °C for 24 hours and ether extract
was determined by Soxhlet extractor and results expressed
as a percentage of the initial weight of sample. The amount
of protein in the samples was determinate by digestion,
distillation and titration with HCl of the samples, being 722 Food Sci. Technol, Campinas, 39(3): 721-728, July-Sept. 2019 Albuquerque et al. Albuquerque et al. monounsaturated fatty acids (MUFA), total polyunsaturated
fatty acids (PUFA), total fatty acids omega 6 (n-6) and omega 3
(n-3) and their connections. The activities of the enzymes Δ9-
desaturase, elongase and thioesterase were estimated according
to Malau-Aduli et al. (1998) and Kazala et al. (1999) through
the following equations:
9
16
desaturase C
enzyme activity index
∆−
=
(
) (
)
:
/
:
:
100
C16 n7
C16 1n7
C16 0
+
; 9
18
desaturase C
enzyme activity index
∆−
=
(
) (
)
:
/
:
:
100
C18 1n9c
C18 1n9c
C18 0
+
;
16
18
C
a C
elongase enzyme activity index =
(
) (
)
:
:
/
:
:
:
:
100
C18 0
C18 1n9c
C16 0
C16 1n7
C18 0
C18 1n9c
+
+
+
+
;
C16 a C14 thioesterase enzyme activity index =
(
) (
)
:
/
:
:
100
C16 0
C16 0
C14 0
+
. Still, the atherogenicity and thrombogenicity indices were calculated,
considered as indicators of health, related to the risk of
cardiovascular disease, according to Ulbricht & Southgate (1991). The cholesterol content in its turn was quantified by colorimetric
method, described by Bragagnolo & Rodriguez-Amaya (2002),
and the results were expressed in mg/100g of meat. expressed in percentage of protein calculated by the equation:
(
)
(
)
%
. /
. protein
0 02
14
fc
100
sample weight
6 25
=
×
×
×
×
. The ashes
amount was assessed through dry ashing procedures in muffle
furnace at 550 °C, being the ash content expressed on initial
weight of sample. 2.3 Retail display For evaluation of retail display, steaks of 2.5 cm in thickness
were stored in polypropylene trays, covered with film of polyvinyl
chloride (PVC) permeable to oxygen and they were kept frozen
(4 °C), under constant light (24 watts), for six days. Along this
period of time, the CIELAB color indexes were measured daily,
and the readings were performed through the PVC film. 2.4 Lipid profile The analysis of the composition of the meat fatty acids and
cholesterol was performed by the extraction method described
by Folch et al. (1957) and esterification by Hartman & Lago
(1973). The extracts for fatty acids profile were subjected to gas
chromatography in Shimatzu chromatograph GC 2010 (Agilent
Technologies Inc., Palo Alto, CA, USA) equipped with a flame
ionization detector, split injector at the rate of 1:50 and capillary
column of Supelco SPTM-2560, 100 m × 0.25 mm × 0.20 μm (Supelco
Inc., Bellefonte, PA, USA). The chromatographic conditions were
initial temperature of the column of 140 °C/5 minutes; increased
4 °C/minute to 240 °C and kept for 30 minutes, amounting to
60 minutes. The injector temperature was 260 °C. Helium was
the carrier gas utilized (Faria et al., 2015). The identification
of fatty acids was carried out through comparison with the
retention times presented by the standard chromatogram
SupelcoTM37 FAME mix (Supelco Inc., Bellefonte, PA, USA)
and expressed in percentage of total fatty acids identified and
subsequently grouped into: total saturated fatty acids (SFA), total 1Control: basal diet; 2CrFe: supplementation with chromium and iron; 3MgSe: supplementation with magnesium and selenium; 4CrFeMgSe: supplementation with chromium, iron,
magnesium and selenium; 5Tukey’s test (α = 0.05); 6Evaluated by comparison with standard National Pork Producers Council (1999) ranging from 1 (pale pinkish gray to white) to
6 (dark purple red) for color and from 1 (light) to 10 (abundant) for marbling; a,b,c: Different letters indicate statistically significant differences between treatments. Number of replicates
per parameters = 11 2.5 Statistical analysis All the measured variables were tested for normality by
Shapiro-Wilk Test, and those that did not show normal distribution
were transformed by the procedure RANK of SAS (SAS 9.3
Intit. Inc., Cary, NC, USA). PROC RANK with NORMAL
option was used to produce a transformed standard variable. All data were submitted to analysis of variance ANOVA, being
that the variables that showed significant differences at a level of
significance of 5% of probability, were submitted to the Tukey’s
test and regression for quantitative variables. 3.1 Physical-chemical characteristics Number of replicates
per parameters = 11. Table 2. Evaluation of the physical-chemical parameters and centesimal composition of the Longissimus thoraci
supplemented with different associations between minerals. Food Sci. Technol, Campinas, 39(3): 721-728, July-Sept. 2019 723 Meat quality of pigs receiving minerals (C14:0) (p = 0.003) and palmitic acid (C16:0) (p = 0.008) and
increase in heptadecanoic acid (C17:1) (p = 0.013), linoleic acid
(C18:2n‑6c) (p = 0.008), eicosadienoic acid (C20:2n-6) (p = 0.027),
eicosatrienoic acid (C20:3n-6) (p = 0.032), arachidonic acid
(C20:4n-6) (p = 0.030) and behenic acid (C22:0) (p = 0.031). The increase in the levels of these fatty acids, associated with an
increase in the PUFA and reduction of C14:0 and C16:0, being
these last two considered fatty atherogenic acids, contributed
to the reduction in the atherogenicity rate, when compared to
the control group. The magnesium deficiency was associated
with a change in the atherogenic lipid composition of patients
with heart disease (Rasmussen et al., 1989), and its use in diet
caused an increase in the apoliprotein A: apoliprotein B, as a
protective mechanism against atherosclerosis. The results found in the literature regarding the supplementation
with chromium, magnesium and selenium in general, did not
influence the pH values of the meat (Frederick et al., 2006;
Jin et al., 2018; Peres et al., 2014; Wojtasik-Kalinowska et al.,
2018), although other studies indicated an improvement with
the use of magnesium (Swigert et al., 2004). The drip loss at 48 hours was lower (p = 0.013) for the
animals’ meat that received MgSe than those supplemented
with CrFe, however, both groups did not differ from the control
group and this presented similar means to CrFeMgSe (Table 2). Khan et al. (2018) demonstrated that supplementation of broilers
with 0.30 mg Se/kg in the form of sodium selenite, increased the
water hold capacity and the glutathione peroxidase (GSH-Px)
activity. Selenium is an integral part of various selenoproteins,
and it is possible to mention the selenoprotein W (Sel W) which
has demonstrated present antioxidant activity dependent on
GSH-Px and contrary to the other selenoproteins, its expression
in the muscle is increased even in cases of excessive selenium
consumption (Jeong et al., 2004). In a study carried out by Li et al. 3.1 Physical-chemical characteristics (2011), the supplementation with selenium (0.3 and 3 mg/kg)
promoted an increase of gene expression Sepw1 related to Sel
W and, it was shown a high and negative correlation (-0.90)
between the expression of this gene and the weight drop loss,
and this is the crucial point of improvement in water retention
capacity in the meat. In addition to selenium, magnesium has
also contributed to the reduction of water loss by the meat
(Lisiak et al., 2014), indicating the use of these minerals as an
alternative therapy in reducing the PSE occurrence. Still, higher levels of docosahexaenoic acid (DHA)
(p = 0.002), whereas the control group showed the lowest
level. The docosahexaenoic acid (DHA) has beneficial effects
on health including antiatherogenic, antithrombotic and
anti‑inflammatory action and its synthesis from α-linolenic
acid in adult humans is limited. The magnesium, due to being
enzymes cofactor responsible for desaturation of long-chain
fatty acids (Nakamura & Nara, 2004), may have influenced
the highest production of DHA. The other fatty acids were not
influenced by the treatments applied. The PUFA: SFA ratio was 43% higher for the group MgSe
(p = 0.007) as compared to the control group, while the groups
CrFe and CrFeMgSe did not differ from the others. There was
also an increase of 41% and 38% in the content of essential fatty
acids n-3 (p = 0.018) and n-6 (p = 0.009), respectively, for the
group that received MgSe when compared to the control group
without, however, affecting the ratio n-6: n-3 and the other
groups showed intermediate values. The scores of subjective colors and the values of total pigments
were not influenced by the supply of minerals, as well as the
marbling (Table 2). Similar results were found by Apple et al. (2007) working with iron and Tarsitano et al. (2013) evaluating
the supplementation with magnesium. The increase, particularly in the content of fatty acids n-3
in the meat is an aspect of paramount importance, due to its
antithrombotic and anti-atheromatous role, contributing to
the prevention of cardiovascular diseases. Thus, the pork due
to being the largest source of animal protein consumed in the
world, can contribute to the reduction of the ratio n-6: n-3
consumed. 3.1 Physical-chemical characteristics The increase in the content of essential fatty acids
in the group supplemented with magnesium and selenium
can be explained by the fact that the first is a cofactor for
enzymes Δ5 and Δ6‑desaturase, responsible for the reactions of
long‑chain fatty acids desaturation (Nakamura & Nara, 2004). It was demonstrated that the magnesium deficiency led to a
change in the rat’s lipid profile, due to decreased activity of the
enzyme Δ6-desaturase (Mahfouz & Kummerow, 1989), showing
its importance in lipid metabolism. Selenium deficiency also
demonstrated to interfere in the rats’ lipid profile, reducing
the fatty acids n-3 and low levels of these have been associated
with inhibition of the Δ6-desaturase activity, acting indirectly
in the process of fatty acids oxygen desaturation, altering the
lipid profile (Schäfer et al., 2004). The levels of moisture, protein and ash content were not
altered in function of the treatments (Table 2). On the other hand,
the percentage of ether extract was lower (p = 0.01) for the group
that received MgSe compared to the control group. The group
CrFeMgSe did not differ between the groups MgSe and CrFe
and the latter was similar to the control group. The reduction
in total lipids as observed in this study could explain because
magnesium may impair the glucose uptake stimulated by
insulin, in addition to reducing the concentration of lipids in
the bloodstream (Günther, 2010). This mineral forms chelates
with the free fatty acids, deviating them to be eliminated along
with the feces, damaging indirectly the lipid synthesis in the
tissues (Apple et al., 2000). 3.2 Lipid profile The use of minerals showed effects on the meat lipid
profile (Table 3), and in general, the group supplemented with
MgSe showed a reduction in the levels of saturated fatty acids
(SFA) (p = 0.035) and increase in poly-unsaturated (PUFA)
(p = 0,009), when compared to the control group. The CrFe
and CrFeMgSe groups showed intermediate values for these
fatty acids summations. The use of MgSe in the finishing
pigs’ diets also promoted reduction of levels of myristic acid The activity of the enzyme Thioesterases C16-14 was
higher (p = 0.010) for MgSe regarding the control, and that
the groups CrFe and CrFeMgSe, showed similar averages
to the others. In spite of the MgSe having presented a
reduction of 3.6% in the level of C16:0 as compared to the 724 Food Sci. Technol, Campinas, 39(3): 721-728, July-Sept. 2019 Albuquerque et al. Table 3. 3.2 Lipid profile Lipid profile of Longissimus thoracis (LT) muscle of finishing swines supplemented with different associations between mineral
Fatty acids
Diets
P-value5
Control1
CrFe2
MgSe3
CrFeMgSe4
C10:0
0.04 ± 0.02
0.05 ± 0.02
0.03 ± 0.02
0.04 ± 0.02
0.15
C12:0
0.11 ± 0.04
0.17 ± 0.16
0.11 ± 0.09
0.14 ± 0.09
0.78
C13:0
0.00 ± 0.01
0.01 ± 0.05
0.01 ± 0.01
0.01 ± 0.01
0.39
C14:0
1.14 ± 0.12a
1.03 ± 0.11ab
0.94 ± 0.08b
1.06 ± 0.14ab
0.01
C14:1
0.01 ± 0.01
0.01 ± 0.01
0.01 ± 0.01
0.01 ± 0.01
0.87
C15:0
0.05 ± 0.02
0.06 ± 0.02
0.07 ± 0.03
0.07 ± 0.03
0.41
C16:0
25.71 ± 0.61a
25.21 ± 0.72ab
24.75 ± 0.38b
25.36 ± 0.84ab
0.01
C16:1
2.73 ± 0.47
2.66 ± 0.24
2.42 ± 0.30
2.56 ± 0.34
0.22
C17:0
0.27 ± 0.07
0.30 ± 0.06
0.33 ± 0.09
0.30 ± 0.08
0.30
C17:1
0.54 ± 0.13b
0.66 ± 0.16ab
0.76 ± 0.13a
0.69 ± 0.16ab
0.01
C18:0
11.79 ± 1.20
11.01 ± 0.80
11.23 ± 0.75
11.44 ± 1.03
0.22
C18:1n-9t
0.12 ± 0.01
0.13 ± 0.06
0.16 ± 0.09
0.13 ± 0.04
0.65
C18:1 n-9c
45.09 ± 2.24
43.37 ± 2.51
42.32 ± 2.40
43.24 ± 2.78
0.09
C18:2 n-6c
9.19 ± 1.92b
11.35 ± 1.85ab
12.35 ± 2.11a
11.10 ± 2.21ab
0.09
C20:0
0.12 ± 0.03
0.10 ± 0.12
0.10 ± 0.02
0.11 ± 0.02
0.10
C18:3 n-6
0.05 ± 0.01
0.06 ± 0.02
0.06 ± 0.01
0.06 ± 0.02
0.08
C20:1 n-9
0.55 ± 0.07
0.50 ± 0.08
0.51 ± 0.09
0.49 ± 0.08
0.36
C18:3 n-3
0.09 ± 0.02
0.10 ± 0.02
0.10 ± 0.03
0.09 ± 0.02
0.43
C21:0
0.01 ± 0.01
0.01 ± 0.01
0.01 ± 0.01
0.01 ± 0.01
0.59
C20:2 n-6
0.19 ± 0.04b
0.22 ± 0.03ab
0.25 ± 0.05a
0.20 ± 0.04ab
0.03
C22:0
0.06 ± 0.01b
0.08 ± 0.02ab
0.09 ± 0.02a
0.08 ± 0.03ab
0.03
C20:3 n-6
0.17 ± 0.07
0.24 ± 0.08ab
0.26 ± 0.05a
0.22 ± 0.06ab
0.03
C22:1 n-9
0.10 ± 0.11
0.07 ± 0.05
0.06 ± 0.06
0.12 ± 0.08
0.32
C20:3 n-3
0.03 ± 0.01
0.03 ± 0.01
0.04 ± 0.02
0.03 ± 0.01
0.28
C20:4 n-6
1.88 ± 0.78b
2.60 ± 0.85ab
2.92 ± 0.64a
2.47 ± 0.84ab
0.03
C20:5 n-3
0.04 ± 0.03
0.05 ± 0.02
0.06 ± 0.02
0.06 ± 0.04
0.17
C22:6 n-3
0.02 ± 0.01b
0.03 ± 0.01ab
0.04 ± 0.01a
0.03 ± 0.02ab
0.01
Parameters
SFA
39.35 ± 1.74a
38.08 ± 1.42ab
37.66 ± 0.97b
38.66 ± 1.71ab
0.04
MUFA
49.02 ± 2.43
47.27 ± 2.55
46.07 ± 2.52
47.10 ± 2.92
0.09
PUFA
11.78 ± 2.79b
14.81 ± 2.74ab
16.23 ± 2.77a
14.39 ± 3.15ab
0.01
Σ n-3
0.17 ± 0.03b
0.21 ± 0.04ab
0.24 ± 0.04a
0.22 ± 0.07ab
0.02
Σ n-6
11.48 ± 2.76b
14.47 ± 2.71ab
15.83 ± 2.70a
14.04 ± 3.09ab
0.01
Σ n-6/Σ n-3
67.32 ± 9.89
68.73 ± 12.15
67.19 ± 9.34
66.22 ± 11.97
0.96
PUFA/SFA
0.30 ± 0.08b
0.39 ± 0.08ab
0.43 ± 0.09a
0.38 ± 0.09ab
0.01
∆9-desaturase C16
9.54 ± 1.58
9.53 ± 0.73
8.93 ± 1.01
9.14 ± 1.16
0.50
∆9-desaturase C18
79.22 ± 1.86
79.78 ± 1.47
79.08 ± 1.23
78.99 ± 2.02
0.62
Elongase C16-C18
66.64 ± 0.89
66.09 ± 1.16
66.30 ± 1.12
66.15 ± 1.09
0.64
Thioesterase C16-14
95.77 ± 0.32b
96.08 ± 0.40ab
96.34 ± 0.27a
96.01 ± 0.44ab
0.01
Atherogenicity
0.59 ± 0.04a
0.56 ± 0.03ab
0.53 ± 0.03b
0.56 ± 0.04ab
0.01
Thrombogenicity
0.40 ± 0.05
0.38 ± 0.05
0.38 ± 0.04
0.40 ± 0.05
0.66
Cholesterol (mg/100g)
75.41 ± 23.30
118.14 ± 27.84
101.83 ± 22.21
101.88 ± 22.31
0.52
1Control: basal diet; 2CrFe: supplementation with chromium and iron; 3MgSe: supplementation with magnesium and selenium; 4CrFeMgSe: supplementation with chromiu
magnesium and selenium; SFA: total of saturated fatty acids; MUFA: total of monounsaturated fatty acids; PUFA: total of unsaturated fatty acids; PUFA/SFA: unsaturated: s
ratio; 5Tukey’s test (α = 0.05). Food Sci. Technol, Campinas, 39(3): 721-728, July-Sept. 2019 3.3 Display life The color parameters evaluated during the storage time at
4 °C, were affected by the treatments with minerals, although
interaction between the diets and the time was not observed
(Table 4). The values of a* (p = 0.001) and b* (p = 0.001) were
higher in the group CrFeMgSe and lower for MgSe. The control
group and the CrFe did not differ among themselves, being
that the first had a mean similar to CrFeMgSe and the second a
mean similar to MgSe. There was a reduction of C* (p = 0.001)
for CrFe and MgSe compared with the control group and the
latter had mean similar to CrFeMgSe. On the contrary, CrFe and
MgSe presented higher values for h* (p = 0.003) compared to the
control and CrFeMgSe, being that these last two did not differ
among themselves. There was no influence of minerals in L*. There was also an influence of time on the color parameters
(Table 4) with linear drop to L* (p = 0.011) and a* (p < 0.001)
and linear increase for h* (p < 0.001) (Figure 1). There was no
effect of storage time in b* and C*. Table 4. Color and percentage of pigments of Longissimus thoracis (LT) muscle of finishing swines supplemented with different associations
between minerals, stored at 4 °C for six days. Meat quality of pigs receiving minerals These results are in disagreement with the studies of
Frederick et al. (2004) who observed no influence of magnesium
supplied through drinking water during 6 days before slaughter,
in the parameters a* and b* of pork stored at 4 ºC during 8 days. However, these same authors did not also observe effect of the
mineral in L* as well as in the present study. Regarding the
effect of iron, Wallis et al. (2003) did not observe any change in
L*, a*, b*, C* and h* when they used different concentrations
of organic iron, contradicting the findings of the present study,
except for the L* that was also not changed by the treatments. Jin et al. (2018) also did not observe the effect of chromium
methionine (200 µg/kg) in parameters of color. On the other
hand, Khan, et al. (2018), using 0.3 mg/kg of sodium selenite,
observed increased in a* and b* in poultry, in discordance with
our finds. The cholesterol levels ranged from 73.97 to 116.88 (mean of
98.08 mg/100g of meat), not being altered by supplementation
with minerals used and getting close to those obtained by
Faria et al. (2015) (84.76 mg/100g). 3.3 Display life Parameter
Diets (D)
P-value5
Control1
CrFe2
MgSe3
CrFeMgSe4
D
T
D×T
Color
L*
56.26 ± 3.18
55.47 ± 4.89
54.40 ± 6.97
56.68 ± 2.68
0.09
0.01
1.00
a*
0.74 ± 0.90ab
0.31 ± 0.94bc
0.21 ± 0.32c
1.18 ± 0.69a
0.01
0.01
0.95
b*
10.34 ± 1.01ab
9.78 ± 0.99bc
9.31 ± 1.42c
10.37 ± 0.89a
0.01
0.07
0.10
C*
10.46 ± 0.96a
9.88 ± 0.90b
9.36 ± 1.48b
10.51 ± 0.89a
0.01
0.07
0.99
h*
86.05 ± 5.36b
88.35 ± 5.60a
88.87 ± 2.18a
85.50 ± 3.97b
0.01
0.01
0.99
% Pigments
O2Mb
48.59 ± 2.56
46.02 ± 3.16
46.21 ± 3.78
47.40 ± 4.01
0.31
0.01
0.73
MMb
34.00 ± 1.85
33.90 ± 3.79
33.35 ± 4.25
33.66 ± 2.24
0.73
0.01
0.70
Mb+
17.31 ± 3.99
20.11 ± 6.58
20.52 ± 7.96
18.90 ± 5.93
0.52
0.01
0.97
1Control: basal diet; 2CrFe: supplementation with chromium and iron; 3MgSe: supplementation with magnesium and selenium; 4CrFeMgSe: supplementation with chromium, iron,
magnesium and selenium; T: time; D×T: Interaction between diet and time; L*: lightness; a*: redness; b* yellowness; C*: Chroma; h*: hue angle; O2Mb: oxymyoglobin; MMb: metmyoglobin;
Mb+: reduced myoglobin. 5Tukey’s test (α = 0.05). Number of replicates per parameters = 11. a,b,c: Different letters indicate statistically significant differences between treatments. Table 4. Color and percentage of pigments of Longissimus thoracis (LT) muscle of finishing swines supplemented with different associations
between minerals, stored at 4 °C for six days. of pigments of Longissimus thoracis (LT) muscle of finishing swines supplemented with different associations
for six days. 1Control: basal diet; 2CrFe: supplementation with chromium and iron; 3MgSe: supplementation with magnesium and selenium; 4CrFeMgSe: supplementation with chromium, iron,
magnesium and selenium; T: time; D×T: Interaction between diet and time; L*: lightness; a*: redness; b* yellowness; C*: Chroma; h*: hue angle; O2Mb: oxymyoglobin; MMb: metmyoglobin;
Mb+: reduced myoglobin. 5Tukey’s test (α = 0.05). Number of replicates per parameters = 11. a,b,c: Different letters indicate statistically significant differences between treatments. Figure 1. Regression equations for lightness (A), redness (B) and hue angle (C) of LT muscle of finishing swines supplemented with different
mineral associations stored at 4 °C for six days. *: CIELAB System. Figure 1. Regression equations for lightness (A), redness (B) and hue angle (C) of LT muscle of finishing swines supplemented with different
mineral associations stored at 4 °C for six days. *: CIELAB System. Food Sci. Food Sci. Technol, Campinas, 39(3): 721-728, July-Sept. 2019 3.2 Lipid profile Number of replicates per parameters = 11. a,b,c: Different letters indicate statistically significant differences between treatments. Table 3. Lipid profile of Longissimus thoracis (LT) muscle of finishing swines supplemented with different associations between minerals. mus thoracis (LT) muscle of finishing swines supplemented with different associations between minerals. Table 3. Lipid profile of Longissimus thoracis (LT) muscle of finishing swines supplemented with different asso 1Control: basal diet; 2CrFe: supplementation with chromium and iron; 3MgSe: supplementation with magnesium and selenium; 4CrFeMgSe: supplementation with chromium, iron,
magnesium and selenium; SFA: total of saturated fatty acids; MUFA: total of monounsaturated fatty acids; PUFA: total of unsaturated fatty acids; PUFA/SFA: unsaturated: saturated
ratio; 5Tukey’s test (α = 0.05). Number of replicates per parameters = 11. a,b,c: Different letters indicate statistically significant differences between treatments. that the tissue (fat and/or meat) has a better profile of
antiatherogenic fatty acids, having then, increased capacity
for prevention of coronary heart disease (Arruda et al.,
2012). Thus, the reduction in the atherogenicity index in the
meat of those animals that received MgSe demonstrated that
supplementation resulted in a meat with lipid profile more
beneficial to health. The thrombogenicity index, however,
showed similar values for all treatments. control group, there was also a more pronounced reduction
of C14:00 (17.22%), which increased the ratio C16:0:C14:0. There was a reduction (p = 0.002) in the atherogenicity index
of 10.6% for the meat of those animals supplemented with
MgSe, when compared to the control group, while the other
groups showed intermediate values. The atherogenicity and
thrombogenicity indexes indicate the potential to stimulate
platelet aggregation, being that the lower their values means 725 Food Sci. Technol, Campinas, 39(3): 721-728, July-Sept. 2019 Meat quality of pigs receiving minerals 3.3 Display life Technol, Campinas, 39(3): 721-728, July-Sept. 2019 726 Albuquerque et al. Figure 2. Regression equations for the relative percentage of metmyoglobin
(MMb), reduced myoglobin (Mb+) and oxymyoglobin (O2Mb) of the
LT muscle of finishing swines supplemented with different mineral
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Santa Inês alimentados com diferentes níveis energéticos. Semina:
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0359.2012v33n3p1229. Fo. (2012). Perfil de ácidos graxos no Longissimus dorsi de cordeiros
Santa Inês alimentados com diferentes níveis energéticos. Semina:
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different sources of chromium on performance and meat quality. Revista Brasileira de Zootecnia, 43(7), 369-375. http://dx.doi. org/10.1590/S1516-35982014000700005. Yu, B., Huang, W. J., & Chiou, P. W. S. (2000). Bioavailability of iron
from amino acid complex in weanling pigs. Animal Feed Science
and Technology, 86(1-2), 39-52. http://dx.doi.org/10.1016/S0377-
8401(00)00154-1. Ramos, E. M., & Gomide, L. A. M. (2012). Avaliação da qualidade de
carnes: fundamentos e metodologias. Viçosa: Editora UFV. 728 Food Sci. Technol, Campinas, 39(3): 721-728, July-Sept. 2019
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Porous perovskite films integrated with Au–Pt nanowire-based electrodes for highly flexible large-area photodetectors
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INTRODUCTION Large-area, cost-effective, and flexible photodetectors are required
in
multiple
applications
such
as
next-generation
wearable
optoelectronics, robotics, bio-imaging, illumination monitoring
systems, and remote sensing1–5. Organometal halide perovskites,
specifically CH3NH3PbI3 (or MAPbI3) has emerged as outstanding
light-harvesting material in the optoelectronic field due to its long
charge carrier diffusion length, low exciton binding energy,
broadband
absorption,
and
direct
bandgap6–9. The
added
advantage of their solution based low-temperature fabrication
process makes them suitable for application in flexible electronics
and photonic devices10,11. Flexibility is primarily defined by the
ability to bend these devices to achieve a low form factor or easy
integration with a curved surface for mounting on non-traditional
spaces. The common strategy in perovskite photodetectors (and
others) for reducing the maximum strain on bending has been
based on two common themes, 1. By making nano/micro-wire
arrays to limit the dimensions of the material12–14, or 2. Using thin
perovskite films to reduce the thickness4,15,16. Combined with the
inherent challenge of stability in perovskites, either of these
strategies has limited success and as a result, most of the previous
reports on flexible perovskite photodetectors, either conducted
only a few hundred bending cycles test at large bending radii or
suffer from poor environmental stability4,12,15,16. Progress has
been made recently in improving stability by encapsulation of the
perovskite layer, along with achieving ultra-fast response time in
photodetectors17,18. It is also worth noting that most of the
reported performances for flexible perovskite photodetectors are
on the very small area < 0.4 cm2 and very few reports exist on the
large area (>1 cm2) devices4,19,20. Therefore, there is a need to
perform further studies and improve on the repeatable bending
durability, while stabilizing the device performance. Further to
ensure cost-effectiveness facile manufacturing processes resulting p
q
Here, we demonstrate a highly bendable (10,000 cycles), stable,
and large-area (3 cm2) flexible polystyrene (PS) incorporated
MAPbI3 photodetectors. The high bendability is achieved by
having a thin porous film of perovskite made by spin casting. This
combines the effect of lowering the bending strain due to, the
small thickness of the film and a lower modulus from the porous
structure. The specific interaction between PbI2 and MAI in the
perovskite precursor solution and PS chains results in the
formation of a cross-linked polymer-perovskite network, which
assists in enhancing the performance and improving the stability
of
the
PS-MAPbI3
photodetectors. www.nature.com/npjflexelectron ARTICLE
OPEN
Porous perovskite films integrated with Au–Pt nanowire-based
electrodes for highly flexible large-area photodetectors Rohit Saraf1, Hua Fan1 and Vivek Maheshwari
1✉ Flexible, large-area, and stable perovskite photodetectors have drawn increasing widespread research attention for next-generation
wearable and portable optoelectronic devices. However, high mechanical durability coupled with large device area and enhanced
environmental stability has not been demonstrated yet to attain practical viability. Herein, a highly bendable, stable, and large-area
(3 cm2) flexible polystyrene incorporated perovskite photodetector is presented. Due to the formation of a porous polystyrene-
perovskite composite film in a single step it allows unprecedented mechanical stability, maintaining 85% of its original
photocurrent value after 10,000 bending cycles at a bending angle of 120°. Equally crucial, the solution-processed self-assembled
Pt–Au nanochains were developed to provide a simple and fast method of patterning the conductive and flexible electrodes onto
the filter substrate. The optimized polystyrene-perovskite photodetector exhibits a high responsivity up to 2.73 A W−1, a maximum
specific detectivity of 6.2 × 1013 Jones, and a superior switching ratio of 1.0 × 104. In addition, the polystyrene-perovskite
photodetector yields excellent stability under the combined stresses of moisture, ambient air, and room light, and retains 92% of its
original performance for over 30 days. All these results demonstrate that this work provides a facile and cost-effective approach
that paves the way to develop high-performance, stable, and highly flexible optoelectronic devices. npj Flexible Electronics (2020) 4:30 ; https://doi.org/10.1038/s41528-020-00094-5 in large-area photodetectors, without significantly affecting the
performance characteristics of the material are required. 1Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, ON N2L 3G1, Canada. ✉email: vmaheshw@uwaterloo.ca R. Saraf et al. 2 1.0 × 104 as compared to plain MAPbI3 device. Furthermore, the 1
wt % PS-MAPbI3 flexible photodetector shows outstanding
bending stability and retains a photocurrent of 85% of its initial
value after 10,000 bending cycles at a bending angle of 120°. This
also illustrates the robustness of the nanoparticle electrodes for
bending. Under the same bending conditions, a substantial
decrease in photocurrent to 50% was observed for plain MAPbI3
device. Finally, the 1 wt % PS-MAPbI3 flexible photodetector
shows excellent stability and retains 92% of its original value even
after 30 days (720 h) of continuous exposure of moisture, ambient
air, and under room light, which was far better than that of the
plain MAPbI3 photodetector whose performance significantly
deteriorates to almost zero after 8 days (192 h). This facile
fabrication process of large-area flexible photodetectors with
superior bending robustness coupled with improved environ-
mental stability and reduced cost will pave the way for next-
generation wearable and smart optoelectronics. The photocurrent generation from the flexible PS-MAPbI3
photodetector is schematically depicted in Fig. 2a. Our photo-
detector is based on the photoelectric effect in which the applied
voltage results in an electric field that leads to the effective
separation of electrons and holes generated on light illumination. Due to the close matching of the work function of Pt–Au (5.2 eV)
with the highest occupied molecular orbital (HOMO) of PS-MAPbI3
perovskite (5.4 eV), the collection of holes are favorable by the
negative Pt–Au electrode in the symmetric Pt–Au/PS-MAPbI3 (or
MAPbI3)/Pt–Au device. Whereas, the electrons are not easily
collected (or get trapped) due to the large energy level difference
between the Lowest Unoccupied Molecular Orbital (LUMO) of PS-
MAPbI3 (3.9 eV) and work function of Pt–Au (5.2 eV). Figure 2b
presents the photoresponse from both plain MAPbI3 and PS-
MAPbI3 devices (with varying concentrations of PS) at 2 V and
under the illumination of 1.0 sun. The plain MAPbI3 device showed
a photocurrent of 2.8 µA, which enhanced with the incorporation
of PS. Specifically, the photocurrent for 0.5 wt % PS-MAPbI3 device
is 4.8 µA, and it increased substantially to 11.2 µA for 1 wt % PS-
MAPbI3 device. We attribute this to the fewer defects and
suppressed charge carrier recombination by the cross-linking of
PS within MAPbI3
22. Further, the addition of PS to 3 and 7 wt %
caused a drop in photocurrent to 6.9 and 1.1 µA, respectively. Fabrication and opto-electronic performance Fabrication and opto-electronic performance A schematic representation of the fabrication process of flexible
PS-MAPbI3 photodetector is shown in Fig. 1a, b. First, the highly
conductive Pt–Au electrodes were deposited on the flexible
polyamide filter membrane (see I–V curves of Pt–Au electrodes in
Supplementary Fig. 1) by solution filtration of micron size long-
chain networks of Au nanoparticles self-assembled in a solution
using Pt4+ ions and then reduced to form continuous Pt–Au nano-
networks. The TEM and high-resolution TEM (HRTEM) images of
Pt–Au nanochains are presented in Supplementary Fig. 2. The TEM
image illustrates the typical Pt–Au nanoparticles (~microns in size,
with local nanowire width of ~5–10 nm) chain structure. The pore
size of the polyamide filter is around 1–2 µm as observed in the
field emission scanning electron microscopy (FESEM) image
(Fig. 1a). Then, the PS containing MAPbI3 (or plain MAPbI3)
precursor solution was spin-coated and annealed on 2.0 × 1.5 cm2
area Pt–Au-patterned polyamide substrate (shown in Fig. 1b). The
cross-sectional FESEM images shown in Supplementary Fig. 3
illustrate that the thickness of the plain MAPbI3 and 1 wt % PS-
MAPbI3 films are around 1.010 and 1.038 µm, respectively. The
porous
structure
of
the
polyamide
filter
would
lead
to
the formation of a porous perovskite structure on the filter during
the spin-coating process. A FESEM image of the resulting porous
PS-MAPbI3 films is shown in Fig. 1b. A similar approach was used
to fabricate the porous plain MAPbI3 (without PS) films on the
polyamide filter. A photograph of the Pt–Au electrodes on the
flexible polyamide filter is displayed in Fig. 1c. Figure 1d and e
shows the photograph of the resulting PS-MAPbI3 device and its
flexibility. The details of the fabrication process are in the
experimental section. The Lewis-acid characteristic of PbI2 leads
to the interaction between PS and MAPbI3 precursors and cross-
linking of the polymer chains. While the cation-π electron
interactions are the basis of coupling between the MAI and PS
chains. We have confirmed and reported these interactions in our
previous work22,23. The impact of PS incorporation into the
perovskite was evaluated by Xray diffraction (XRD), and UV–Vis
absorption spectroscopy. The diffraction patterns (Fig. 1f) showed
no obvious changes with and without PS, and all the samples
exhibit the same tetragonal perovskite crystal structure with the
dominant (110) lattice plane. As displayed in Fig. R. Saraf et al. The
corresponding light switching on/off ratios is illustrated in
Supplementary Fig. 4, and it also follows a similar trend. It is
worth noting that the light switching ratio for 1 wt % PS-MAPbI3
device (1.0 × 104) is 1 order of magnitude higher than that of plain
MAPbI3 device (1.0 × 103). The comparison reveals that among all
the above PS concentrations, 1 wt % PS device gives the best
performance due to the highest carrier lifetime and mobility, and
simultaneously lowest, charge recombination effects, the density
of defect traps, and ion migration,
and therefore it was
characterized in detail22. The dark currents and photocurrents
were measured to quantitatively compare the performance of
photodetectors made with both plain MAPbI3 and 1 wt % PS-
MAPbI3. For both the photodetectors, with increasing voltage the
dark currents and photocurrents increase (Fig. 2c) due to greater
strength of the electric fields, as it leads to more effective charge
separation and collection. The 1 wt % PS-MAPbI3 device exhibits a
better performance with ~3 times lower dark current and ~4 times
higher photocurrent than the plain MAPbI3 devices. This can be
attributed to the presence of PS (insulating material), reduced ion-
migration effects, and lower carrier recombination in PS-MAPbI3
photodetectors22. The photoresponse curves of the optimized 1
wt % PS-MAPbI3 photodetector as a function of different
wavelengths (410–710 nm) at a low bias of 2 V are presented in
Fig. 2d. The photocurrent of the device was measured to be 26, 22,
28, 40, 45, and 22 nA under 410, 475, 520, 585, 650, and 710 nm
light irradiation, respectively. The device shows a consistent and
stable response over the cycles under monochromatic light. To
quantitatively evaluate the output performance of plain MAPbI3
and 1 wt % PS-MAPbI3 photodetectors, their spectral responsivity
(Fig. 2e), detectivity (Fig. 2e), and external quantum efficiency
(Supplementary Fig. 5) were measured. The responsivity (R) and INTRODUCTION In
this
work,
we
used
microporous polyamide filters as the flexible substrate for
fabricating large-area photodetectors, owing to their excellent
bendability, durability, good recyclability, and low-cost. The other
significant challenge that needs to be addressed for making
flexible devices is the requirement of equally durable and flexible
high-performance electrodes21. The mechanical and electrical
properties
of
electrodes
fabricated
on
soft
and
bendable
substrates degrade gradually due to fatigue damage during
repeated deformations. To address this key issue, we synthesized
self-assembled platinum–gold (Pt–Au) nanoparticle chains and
deposited them on the polyamide filters by simple vacuum
filtration to fabricate the patterned Pt–Au electrodes. This
approach shows significant advantages in terms of conductivity,
ease of fabrication, cost-effectiveness, and compatibility with the
soft and flexible substrates used for the devices. In addition, the
solution-processed self-assembled Pt–Au nanoparticles chains
patterned as electrodes are highly bendable and provide a simple
and fast method for fabricating flexible metal electrodes. The optimum 1 wt % PS-MAPbI3 device exhibits a low dark
current of 1.1 nA at 2 V, a high responsivity of 2.73 A W−1, a large
detectivity of 6.2 × 1013 Jones, and a superior switching ratio of Nanotechnology, University of Waterloo, Waterloo, ON N2L 3G1, Canada. ✉email: vmaheshw@uwaterloo.ca Published in partnership with Nanjing Tech University R. Saraf et al. Published in partnership with Nanjing Tech University Fabrication and opto-electronic performance 1g, the UV–Vis
absorption spectra show a broad absorption in the range of
300–780 nm, indicating high light-harvesting capabilities over the
ultraviolet to the visible spectrum. There is a small difference in
the absorption value that can be due to the slight variation (±50
nanometers, Supplementary Fig. 3) in the thickness of the
perovskite films, which revealed that the absorption properties
of MAPbI3 films with and without PS are preserved. R ¼ Iph Id
Pin
D ¼
R
ffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2qId=A
p R ¼ Iph Id
Pin
(1)
D ¼
R
ffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2qId=A
p
(2) (1) (2) where Iph is the photocurrent, Id is the dark current, Pin is the
incident light power, q is the electron charge, and A is the effective
area of the photodetector. The responsivity and detectivity of the
1 wt % PS-MAPbI3 photodetector (Fig. 2e) in the spectral range
from 400 to 750 nm are 5 and 7.5 times higher than the plain
MAPbI3 photodetector, respectively. We observe that the 1 wt %
PS-MAPbI3 device attains a maximum responsivity of 0.25 A W−1 where Iph is the photocurrent, Id is the dark current, Pin is the
incident light power, q is the electron charge, and A is the effective
area of the photodetector. The responsivity and detectivity of the
1 wt % PS-MAPbI3 photodetector (Fig. 2e) in the spectral range
from 400 to 750 nm are 5 and 7.5 times higher than the plain
MAPbI3 photodetector, respectively. We observe that the 1 wt %
PS-MAPbI3 device attains a maximum responsivity of 0.25 A W−1 npj Flexible Electronics (2020) 30 R. Saraf et al. Fig. 1
Fabrication process and characterization of flexible PS-MAPbI3 photodetectors. a Deposition of Pt–Au electrodes using self-
assembled Pt–Au nanoparticles chains solution on polyamide filter membrane, with the FESEM image (on the right) shows the Pt–Au
nanoparticles (~10 nm) chain structure with the 200 nm scale bar and the 1–2 µm pore size of the polyamide filter with the scale bars
represent 10 µm (left image) and 1 µm (right image). b Deposition of PS-MAPbI3 film on Pt–Au-patterned polyamide filter. The lower resolution
(left image with 2 µm scale bar) and higher resolution (right image with 1 µm scale bar) FESEM images show the porous PS-MAPbI3 film. Fabrication and opto-electronic performance Photograph of the c Pt–Au electrodes on the flexible substrate with the 1 cm scale bar, d final PS-MAPbI3 device with a 4 mm scale bar, and
e bending (or flexibility) of the device. f XRD patterns and g UV–Vis absorption spectra of plain MAPbI3, 0.5, 1, 3, and 7 wt % PS-MAPbI3 films. 3 Fig. 1
Fabrication process and characterization of flexible PS-MAPbI3 photodetectors. a Deposition of Pt–Au electrodes using self-
assembled Pt–Au nanoparticles chains solution on polyamide filter membrane, with the FESEM image (on the right) shows the Pt–Au
nanoparticles (~10 nm) chain structure with the 200 nm scale bar and the 1–2 µm pore size of the polyamide filter with the scale bars
represent 10 µm (left image) and 1 µm (right image). b Deposition of PS-MAPbI3 film on Pt–Au-patterned polyamide filter. The lower resolution
(left image with 2 µm scale bar) and higher resolution (right image with 1 µm scale bar) FESEM images show the porous PS-MAPbI3 film. Photograph of the c Pt–Au electrodes on the flexible substrate with the 1 cm scale bar, d final PS-MAPbI3 device with a 4 mm scale bar, and
e bending (or flexibility) of the device. f XRD patterns and g UV–Vis absorption spectra of plain MAPbI3, 0.5, 1, 3, and 7 wt % PS-MAPbI3 films. and detectivity of 5.7 × 1012 Jones under illumination by mono-
chromatic light with a wavelength of 650 nm. The corresponding
external quantum efficiency (EQE) of 1 wt % PS-MAPbI3 device is
greater than that of the plain MAPbI3 device as shown in
Supplementary Fig. 5. The photoresponse of 1 wt % PS-MAPbI3
device
irradiated
by
650 nm
light
of
different
intensities
(0.001–10 mW cm−2) is shown in Fig. 2f. The photocurrent
increases with the light intensity, which can be ascribed to the
increased number of photogenerated charge carriers in response
to the higher photon flux26. It should be noted that the
photocurrent at very low intensity (0.001 mW cm−2 or 1 µW
cm−2) of 650 nm light is 45 nA, which is more than 1 order of
magnitude higher than that under dark conditions (1.1 nA). The
corresponding responsivity and detectivity are shown in Supple-
mentary Fig. 6. The response time of the 1 wt % PS-MAPbI3
photodetector was also determined and found to be <5 ms, which
is the detection limit of our setup (Supplementary Fig. 7). Published in partnership with Nanjing Tech University Fabrication and opto-electronic performance The
responsivity and detectivity of plain MAPbI3 and 1 wt % PS-
MAPbI3 photodetectors using 2 V at different light intensities by
attenuating 1.0 sun illumination (from solar simulator) using
neutral density filters is presented in Fig. 2g. The maximum value of R and D* for 1 wt % PS-MAPbI3 device is 2.73 A W−1 and 6.2 ×
1013 Jones at illumination intensity of 0.001 mW cm−2, which is
comparable and even higher than the previous reports on the
flexible photodetectors (Table 1)3,12,13,20,27–38. In contrast, the plain of R and D* for 1 wt % PS-MAPbI3 device is 2.73 A W−1 and 6.2 ×
1013 Jones at illumination intensity of 0.001 mW cm−2, which is
comparable and even higher than the previous reports on the
flexible photodetectors (Table 1)3,12,13,20,27–38. In contrast, the plain
MAPbI3 device achieves only R (0.61 A W−1) and D* (0.86 × 1013
Jones), which implies that the incorporation of 1 wt % PS in
MAPbI3 greatly improved the photodetector performance. The
higher R and D* observed under illumination from the solar
simulator (a broad-spectrum source) compared to illumination at
650 nm is attributed to the broad absorption spectrum of MAPbI3
which leads to greater absorption of photons of higher energies. and detectivity of 5.7 × 1012 Jones under illumination by mono-
chromatic light with a wavelength of 650 nm. The corresponding
external quantum efficiency (EQE) of 1 wt % PS-MAPbI3 device is
greater than that of the plain MAPbI3 device as shown in
Supplementary Fig. 5. The photoresponse of 1 wt % PS-MAPbI3
device
irradiated
by
650 nm
light
of
different
intensities
(0.001–10 mW cm−2) is shown in Fig. 2f. The photocurrent
increases with the light intensity, which can be ascribed to the
increased number of photogenerated charge carriers in response
to the higher photon flux26. It should be noted that the
photocurrent at very low intensity (0.001 mW cm−2 or 1 µW
cm−2) of 650 nm light is 45 nA, which is more than 1 order of
magnitude higher than that under dark conditions (1.1 nA). The
corresponding responsivity and detectivity are shown in Supple-
mentary Fig. 6. The response time of the 1 wt % PS-MAPbI3
photodetector was also determined and found to be <5 ms, which
is the detection limit of our setup (Supplementary Fig. 7). npj Flexible Electronics (2020) 30 Fabrication and opto-electronic performance The
responsivity and detectivity of plain MAPbI3 and 1 wt % PS-
MAPbI3 photodetectors using 2 V at different light intensities by
attenuating 1.0 sun illumination (from solar simulator) using
neutral density filters is presented in Fig. 2g. The maximum value MAPbI3 device achieves only R (0.61 A W−1) and D* (0.86 × 1013
Jones), which implies that the incorporation of 1 wt % PS in
MAPbI3 greatly improved the photodetector performance. The
higher R and D* observed under illumination from the solar
simulator (a broad-spectrum source) compared to illumination at
650 nm is attributed to the broad absorption spectrum of MAPbI3
which leads to greater absorption of photons of higher energies. Flexibility and operational stability Flexibility and operational stability As mechanical stability is the major concern for the practical
feasibility of flexible photodetectors for wearable electronics, we
studied the bending durability of both plain MAPbI3 and 1 wt %
PS-MAPbI3 photodetectors. Figure 3a shows
the
schematic
representation of the bending of the flexible PS-MAPbI3 photo-
detector. The device was bent at varied angles as shown in the
inset of Fig. 3b, and the photocurrent was measured during the npj Flexible Electronics (2020) 30 R. Saraf et al. R. Saraf et al. 4 Fig. 2
Device configuration, operation mechanism, and its performance. a Schematic diagram showing the current generation by li
irradiation from the flexible PS-MAPbI3 photodetector. b Photoresponse cycles of the plain MAPbI3 and different concentrations of PS-MAP
devices at 2 V under 1.0 sun illumination. c Dark and photocurrent of the devices at different voltages under one sun illuminati
d Photoresponse of the 1 wt % PS-MAPbI3 device under the illumination of different wavelengths at 2 V. e Responsivity and detectivity of
devices at 2 V and under fourteen different wavelengths ranging from 410 to 710 nm. f Photoresponse curves of the 1 wt % PS-MAPbI3 dev
at 2 V and under 650 nm light wavelength at various power intensities. g Spectral responsivity and detectivity of the devices as a function
different light intensities at 2 V under white light. Typical measurements were conducted on multiple devices (at least 4) and yield an er
range of 3−5% for all the samples. Fig. 2
Device configuration, operation mechanism, and its performance. a Schematic diagram showing the current generation by light
irradiation from the flexible PS-MAPbI3 photodetector. b Photoresponse cycles of the plain MAPbI3 and different concentrations of PS-MAPbI3
devices at 2 V under 1.0 sun illumination. c Dark and photocurrent of the devices at different voltages under one sun illumination. d Photoresponse of the 1 wt % PS-MAPbI3 device under the illumination of different wavelengths at 2 V. e Responsivity and detectivity of the
devices at 2 V and under fourteen different wavelengths ranging from 410 to 710 nm. f Photoresponse curves of the 1 wt % PS-MAPbI3 device
at 2 V and under 650 nm light wavelength at various power intensities. g Spectral responsivity and detectivity of the devices as a function of
different light intensities at 2 V under white light. Published in partnership with Nanjing Tech University METHODS Preparation of Pt–Au nanoparticles chains
Hundred and thirty microliters of 4.5 mg mL−1 platinum (IV) tetrachloride
(PtCl4, Sigma Aldrich) were mixed with 1 mL citrate capped gold
nanoparticles (~10 nm) solution, purchased from BBI Solutions OEM
Limited, and then left on the shaker until the solution color changed to
dark blue due to the self-assembly of Au nanoparticles. Seventy-five
microliters of 4 mg mL−1 sodium borohydride (NaBH4) was then added to
reduce the Pt4+ ions, the color of the solution changes from initial dark
blue to black due to the formation of Pt metal. In summary, we have demonstrated the feasibility of using PS-
incorporated MAPbI3 networks to develop a porous film on a
flexible filter membrane for fabricating large-area ultra-flexible
photodetector. Low-cost flexible Pt–Au electrodes were patterned Flexibility and operational stability METHODS
Preparation of Pt–Au nanoparticles chains
Hundred and thirty microliters of 4.5 mg mL−1 platinum (IV) tetrachloride
(PtCl4, Sigma Aldrich) were mixed with 1 mL citrate capped gold
nanoparticles (~10 nm) solution, purchased from BBI Solutions OEM
Limited, and then left on the shaker until the solution color changed to
dark blue due to the self-assembly of Au nanoparticles. Seventy-five
microliters of 4 mg mL−1 sodium borohydride (NaBH4) was then added to
reduce the Pt4+ ions, the color of the solution changes from initial dark
blue to black due to the formation of Pt metal. Preparation of MAI precursor
Methylammonium iodide (MAI) was synthesized by dropwise addition of
30 mL of hydroiodic acid (Sigma-Aldrich 57 wt % in water) to 27 8 mL of
Table 1. Performance comparison with some previously reported flexible perovskite-based photodetectors. Flexible substrate
Device structure
Bias (V)
Response range
R (A W−1)
D* (× 1012
Jones)
Bending cycles (bending
radius or angle)
Ref. Polyamide filter
Pt–Au/PS-MAPbI3/Pt–Au
2
White light
2.73
62
10,000 (120°)
This work
650 nm
0.25
5.7
PEN
ITO/NIO SnO2/ perovskite/Spiro-
OMeTAD/Ag
0
720 nm
0.473
13.5
500 (R = 4 mm)
3
PEN
Au/P(VDF-TrFE)/MAPbI3
nanowires/Au
0
650 nm
0.012
7.3
200 (180°)
12
Kapton
Au/MAPbI3−x(SCN)x/PMMA/Au
10
White light
0.62
7.3
12000 (R = 1 cm)
13
PEN
Ag/Spiro-OMeTAD/MAPbI3/
In2S3/ITO
0
720 nm
0.451
0.11
500 (R = 2.5 mm)
20
Polyimide
Au/MAPbI3/Au
0
300 nm
0.002
0.176
(R = 3.7 mm)
27
PET
Au/CsPbBr3/Au
2
254 nm
0.00024
0.019
220 (R = 19.94 mm)
28
365 nm
0.00055
0.044
540 nm
0.00002
0.0016
PET
Ni/Au/MAPbI3−x Clx/Ni/Au
5
650 nm
2.17
0.94
500 (150°)
29
PET
ITO/CsPbBr3/ITO
10
517 nm
0.64
–
10,000 (80°)
30
PET
Au/PbPc/MAPbI3−xClx/Au
50
655 nm
0.152
0.85
1000 (R = 6.5 mm)
31
PEN
Au/MAPbI3/RhB/Au
5
550 nm
0.0436
–
1000 (R = 9 mm)
32
Common paper
Ti3C2Tx/CsPbBr3 nanosheets/
Ti3C2Tx
10
450 nm
0.0449
0.00064
1500 (160°)
33
PEN
Au/P(VDF-TrFE) /MAPbI3/Au
0
650 nm
0.02
14
200 (180°)
34
PET
Au/CsPbBr3/ZnO/Au
210
515 nm
<1
4.25
–
10,000 (R = 2.5 mm)
35
PET
Ag/CsPbBr3 nanosheets/CNTs/
Ag
10
442 nm
31.1
–
10,000
36
PET
MAPbI3/C8BTBT bulk
heterojunction
3
532 nm
8.1
2.17
10,000 (R = 7.5 mm)
37
PET
Au/FA(1−x)CsxPb[BryI(1−y)]3/Au
1
450 nm
1.1
28
10000 (R = 3 mm)
38 5 Table 1. Performance comparison with some previously reported flexible perovskite-based photodetectors. Flexibility and operational stability Further to ensure the
reliability of the photoresponse measurement, the photoresponse
of the plain MAPbI3 and 1 wt % PS-MAPbI3 devices were recorded
as a function of time at 2 V under 650 nm light irradiation (0.1 mW
cm−2 intensity), as presented in Fig. 3e. Both the devices exhibited
very stable performances. Measured for 60 min, the photocurrent
of the plain MAPbI3 device was measured to be at 37 nA, whereas
the 1 wt % PS-MAPbI3 device exhibited a photocurrent of 169 nA. Published in partnership with Nanjing Tech University Flexibility and operational stability by using the facile solution-processed self-assembled nanochains. The optimum 1 wt % PS-MAPbI3 device showed extremely high
bending stabilities, maintaining a photocurrent of 85% of their
original values after 10,000 bending cycles under a large bending
angle (θ = 120°). The responsivity and detectivity of 1 wt % PS-
MAPbI3 flexible photodetector reached 2.73 A W−1 and 6.2 × 1013
Jones, respectively. This is among the best-reported performance
with high mechanical durability when compared to other flexible
perovskite photodetectors. Moreover, the PS incorporation in
MAPbI3 showed long-term device stability for more than 30 days
in the ambient environment. The presented low-cost, ultra-
flexible, highly stable, and large-area photodetectors will lay a
strong foundation for developing next-generation wearable and
portable optoelectronic devices. and reduced ion-migration effects22,39. Further to ensure the
reliability of the photoresponse measurement, the photoresponse
of the plain MAPbI3 and 1 wt % PS-MAPbI3 devices were recorded
as a function of time at 2 V under 650 nm light irradiation (0.1 mW
cm−2 intensity), as presented in Fig. 3e. Both the devices exhibited
very stable performances. Measured for 60 min, the photocurrent
of the plain MAPbI3 device was measured to be at 37 nA, whereas
the 1 wt % PS-MAPbI3 device exhibited a photocurrent of 169 nA. These results demonstrate that our 1 wt % PS-MAPbI3 photo-
detector has excellent mechanical flexibility and durability as well
as outstanding environmental stability. The mechanical stability is
also attributed to the porous and chain-like structure of the Pt–Au
electrode (Supplementary Fig. 2) which can endure repeated
bending without failure. It also shows that the Pt–Au electrode
remains chemically stable for days and under device operating
conditions, without any degradation with the perovskite layer. Ag
nanowires in comparison can undergo redox reactions with the
perovskite layer with relative ease impacting stability40–42. Ag
nanowires also have the challenge of mechanical and thermal
stability and junction resistance43. Further the Pt–Au electrodes
are made using a room temperature based self-assembly process
for synthesis and simple vacuum filtration for control of the
deposition process. The conductivity/cost ratio of the Pt–Au
electrodes is ~63,000 Sm−1/US$, while for Ag nanowire-based
electrodes this can range from 8000–123,000 Sm−1/US $, making
them comparable in cost. It is also higher than pure Au electrodes
(1023 Sm−1/US $)21,44. and reduced ion-migration effects22,39. Flexibility and operational stability Flexible substrate
Device structure
Bias (V)
Response range
R (A W−1)
D* (× 1012
Jones)
Bending cycles (bending
radius or angle)
Ref. Polyamide filter
Pt–Au/PS-MAPbI3/Pt–Au
2
White light
2.73
62
10,000 (120°)
This work
650 nm
0.25
5.7
PEN
ITO/NIO SnO2/ perovskite/Spiro-
OMeTAD/Ag
0
720 nm
0.473
13.5
500 (R = 4 mm)
3
PEN
Au/P(VDF-TrFE)/MAPbI3
nanowires/Au
0
650 nm
0.012
7.3
200 (180°)
12
Kapton
Au/MAPbI3−x(SCN)x/PMMA/Au
10
White light
0.62
7.3
12000 (R = 1 cm)
13
PEN
Ag/Spiro-OMeTAD/MAPbI3/
In2S3/ITO
0
720 nm
0.451
0.11
500 (R = 2.5 mm)
20
Polyimide
Au/MAPbI3/Au
0
300 nm
0.002
0.176
(R = 3.7 mm)
27
PET
Au/CsPbBr3/Au
2
254 nm
0.00024
0.019
220 (R = 19.94 mm)
28
365 nm
0.00055
0.044
540 nm
0.00002
0.0016
PET
Ni/Au/MAPbI3−x Clx/Ni/Au
5
650 nm
2.17
0.94
500 (150°)
29
PET
ITO/CsPbBr3/ITO
10
517 nm
0.64
–
10,000 (80°)
30
PET
Au/PbPc/MAPbI3−xClx/Au
50
655 nm
0.152
0.85
1000 (R = 6.5 mm)
31
PEN
Au/MAPbI3/RhB/Au
5
550 nm
0.0436
–
1000 (R = 9 mm)
32
Common paper
Ti3C2Tx/CsPbBr3 nanosheets/
Ti3C2Tx
10
450 nm
0.0449
0.00064
1500 (160°)
33
PEN
Au/P(VDF-TrFE) /MAPbI3/Au
0
650 nm
0.02
14
200 (180°)
34
PET
Au/CsPbBr3/ZnO/Au
210
515 nm
<1
4.25
–
10,000 (R = 2.5 mm)
35
PET
Ag/CsPbBr3 nanosheets/CNTs/
Ag
10
442 nm
31.1
–
10,000
36
PET
MAPbI3/C8BTBT bulk
heterojunction
3
532 nm
8.1
2.17
10,000 (R = 7.5 mm)
37
PET
Au/FA(1−x)CsxPb[BryI(1−y)]3/Au
1
450 nm
1.1
28
10000 (R = 3 mm)
38 omparison with some previously reported flexible perovskite-based photodetectors. eviously reported flexible perovskite-based photodetector Table 1. Performance comparison with some previously reported flexible perovskite-based photodetectors. by using the facile solution-processed self-assembled nanochains. The optimum 1 wt % PS-MAPbI3 device showed extremely high
bending stabilities, maintaining a photocurrent of 85% of their
original values after 10,000 bending cycles under a large bending
angle (θ = 120°). The responsivity and detectivity of 1 wt % PS-
MAPbI3 flexible photodetector reached 2.73 A W−1 and 6.2 × 1013
Jones, respectively. This is among the best-reported performance
with high mechanical durability when compared to other flexible
perovskite photodetectors. Moreover, the PS incorporation in
MAPbI3 showed long-term device stability for more than 30 days
in the ambient environment. The presented low-cost, ultra-
flexible, highly stable, and large-area photodetectors will lay a
strong foundation for developing next-generation wearable and
portable optoelectronic devices. Flexibility and operational stability Typical measurements were conducted on multiple devices (at least 4) and yield an error
range of 3−5% for all the samples. photocurrent dropped to 90, 68, and 50% of its initial value. The
photodetectors are also evaluated for their long-term stability in
operation. The plain MAPbI3 and 1 wt % PS-MAPbI3 devices with
and without an adhesive layer were tested upon exposure to the
ambient environment (in the air at a relative humidity of 35–40%
under room light). Figure 3d shows the 1 wt % PS-MAPbI3 devices
maintain 92% of its initial photocurrent value after 720 h (30 days). In contrast, the plain MAPbI3 devices completely degrade in just
192 h (8 days) with more than 60% loss within 3 days. Similarly, we
also achieved a better stability for the 1 wt % PS-MAPbI3 device
without an adhesive layer in the ambient environment as
compared to the plain MAPbI3 device (Supplementary Fig. 8). The improved stability of 1 wt % PS-MAPbI3 devices can be
attributed to the combined effects of the presence of PS
(hydrophobic in nature) which leads to passivation of defects, bending to ensure the performance reliability of the flexible
device. The photocurrent of both flexible devices remains
unchanged with the bending angle varying from 0° to 120°
(Fig. 3b). Even at the maximum bending angle of 180°, there is a
slight decrease in photocurrent which can be due to the reduction
of the light-receiving area at the bending state. The photocurrent
retained to its original value as the device brings back to zero
bending angles. Further, we measured the photocurrent of both
devices after bending for 10,000 cycles at a fixed bending angle of
120° and a bending radius of 1.5 mm. For clarity, all the
photocurrents were normalized to their respective initial values. As can be seen in Fig. 3c, the photocurrent of the 1 wt % PS-
MAPbI3 device maintained 100, 91, and 85% of its initial value
after 1000, 5000, and 10000 bending cycles, whereas under the
same
bending
conditions
for
plain
MAPbI3
devices
the Published in partnership with Nanjing Tech University npj Flexible Electronics (2020) 30 R. Saraf et al. and reduced ion-migration effects22,39. Flexibility and operational stability Further to ensure the
reliability of the photoresponse measurement, the photoresponse
of the plain MAPbI3 and 1 wt % PS-MAPbI3 devices were recorded
as a function of time at 2 V under 650 nm light irradiation (0.1 mW
cm−2 intensity), as presented in Fig. 3e. Both the devices exhibited
very stable performances. Measured for 60 min, the photocurrent
of the plain MAPbI3 device was measured to be at 37 nA, whereas
the 1 wt % PS-MAPbI3 device exhibited a photocurrent of 169 nA. These results demonstrate that our 1 wt % PS-MAPbI3 photo-
detector has excellent mechanical flexibility and durability as well
as outstanding environmental stability. The mechanical stability is
also attributed to the porous and chain-like structure of the Pt–Au
electrode (Supplementary Fig. 2) which can endure repeated
bending without failure. It also shows that the Pt–Au electrode
remains chemically stable for days and under device operating
conditions, without any degradation with the perovskite layer. Ag
nanowires in comparison can undergo redox reactions with the
perovskite layer with relative ease impacting stability40–42. Ag
nanowires also have the challenge of mechanical and thermal
stability and junction resistance43. Further the Pt–Au electrodes
are made using a room temperature based self-assembly process
for synthesis and simple vacuum filtration for control of the
deposition process. The conductivity/cost ratio of the Pt–Au
electrodes is ~63,000 Sm−1/US$, while for Ag nanowire-based
electrodes this can range from 8000–123,000 Sm−1/US $, making
them comparable in cost. It is also higher than pure Au electrodes
(1023 Sm−1/US $)21,44. In summary, we have demonstrated the feasibility of using PS-
incorporated MAPbI3 networks to develop a porous film on a
flexible filter membrane for fabricating large-area ultra-flexible
by using the facile solution-processed self-assembled nanochains. The optimum 1 wt % PS-MAPbI3 device showed extremely high
bending stabilities, maintaining a photocurrent of 85% of their
original values after 10,000 bending cycles under a large bending
angle (θ = 120°). The responsivity and detectivity of 1 wt % PS-
MAPbI3 flexible photodetector reached 2.73 A W−1 and 6.2 × 1013
Jones, respectively. This is among the best-reported performance
with high mechanical durability when compared to other flexible
perovskite photodetectors. Moreover, the PS incorporation in
MAPbI3 showed long-term device stability for more than 30 days
in the ambient environment. The presented low-cost, ultra-
flexible, highly stable, and large-area photodetectors will lay a
strong foundation for developing next-generation wearable and
portable optoelectronic devices. Preparation of MAI precursor Methylammonium iodide (MAI) was synthesized by dropwise addition of
30 mL of hydroiodic acid (Sigma-Aldrich, 57 wt. % in water) to 27.8 mL of
methylamine (Sigma-Aldrich, 33 wt. % in absolute ethanol) under constant npj Flexible Electronics (2020) 30 Fig. 3
Bending and stability test of the flexible photodetectors. a Schematic showing the bending of the flexible PS-MAPbI3 photodetector. b Photocurrent response of the plain MAPbI3 and 1 wt % PS-MAPbI3 devices under 650 nm light illumination at 0.1 mW cm−2 intensity during
the bending process. The inset shows the corresponding optical images of the device under different bending angles. c Normalized
photocurrent of the flexible devices after different bending cycles at a bending angle of 120° showing the mechanical stability and durability
of 1 wt % PS-MAPbI3 device even after 10000 bending cycles. d Normalized photocurrent of the devices with an adhesive layer after aging
continuously in ambient air, 35–40% relative humidity, and under room light for 720 h (30 days). e Operational stability of the devices at a
constant bias of 2 V under 650 nm light irradiation of 0.1 mW cm−2 intensity. Typical measurements were conducted on multiple devices (at
least 4) and yield an error range of 3–5% for all the samples. R. Saraf et al. R. Saraf et al. 6 Fig. 3
Bending and stability test of the flexible photodetectors. a Schematic showing the bending of the flexible PS-MAPbI3 photodetector. b Photocurrent response of the plain MAPbI3 and 1 wt % PS-MAPbI3 devices under 650 nm light illumination at 0.1 mW cm−2 intensity during
the bending process. The inset shows the corresponding optical images of the device under different bending angles. c Normalized
photocurrent of the flexible devices after different bending cycles at a bending angle of 120° showing the mechanical stability and durability
of 1 wt % PS-MAPbI3 device even after 10000 bending cycles. d Normalized photocurrent of the devices with an adhesive layer after aging
continuously in ambient air, 35–40% relative humidity, and under room light for 720 h (30 days). e Operational stability of the devices at a
constant bias of 2 V under 650 nm light irradiation of 0.1 mW cm−2 intensity. Typical measurements were conducted on multiple devices (at
least 4) and yield an error range of 3–5% for all the samples. configuration was Pt–Au/PS-MAPbI3 (or MAPbI3)/Pt–Au on the polyamide
filter. stirring at 0 °C for 2 h. Characterization and measurement The surface morphology of the self-assembled Pt–Au nanoparticles,
polyamide filter, and perovskite films was observed by FESEM (FESEM,
Zeiss Ultraplus). Transmission electron microscopy (TEM) images were
obtained with a LEO 912AB transmission electron microscope. The phase
of the perovskite samples was characterized by X-ray diffraction (XRD)
using a PANalytical Empyrean diffractometer with Cu-Kα radiation (λ =
1.54 Å). The optical absorption spectra were recorded using a UV–Visible
spectrophotometer (Perkin Elmer Lambda 750). The photoelectrical
measurements of the device under different light wavelengths and
intensities, including I–V, photocurrent, dark current, spectra responsivity,
detectivity, and EQE were recorded by a Keysight 3458 A Digital
multimeter combined with 6614 C 50 Watt system power supply from
Agilent Technologies in ambient condition. All the measurements on
Pt–Au/PS-MAPbI3 (or MAPbI3)/Pt–Au devices were conducted on the
probing station by using a two-probe method. The voltage was applied on
the plain MAPbI3 or PS-MAPbI3 porous films by connecting one probe to
the first Pt–Au electrode on the filter and another probe connected to the
second Pt–Au electrode. Simulated AM 1.5 G irradiation (100 mW cm−2)
with a xenon-lamp based solar simulator (Newport Oriel Instrument 67005,
150 W Solar Simulator) was used as the illumination source. The Newport
optical filters were used to produce incident light at various intensities. The
light intensity was calibrated by an NREL calibrated KG5 silicon reference
cell to minimize spectral mismatch. All measurements were conducted at
room temperature and in ambient air. The active area of the devices was
1.2 cm2. For the bending test, an MFA motorized miniature linear stage was
used to bend the flexible device at a particular angle and to perform
10,000 bending cycles at a constant speed. The long-term stability test of
the devices was tested by directly exposing the devices in ambient air with
35−40% relative humidity and under room light. The humidity was Preparation of plain MAPbI3 and PS-MAPbI3 solutions Preparation of plain MAPbI3 and PS-MAPbI3 solutions
The 1.35 M of plain MAPbI3 solution (without PS) was prepared by mixing
230.5 mg of lead iodide (PbI2), 79.5 mg of MAI in 53.3 µL of dimethyl
sulfoxide (DMSO) and 317.5 µL of dimethylformamide (DMF) for 1 h. To
prepare the 0.5, 1, 3, and 7 wt % PS-MAPbI3 solutions, the required amount
of PS (PS, Alpha Chemistry, Mw = 45,000) was dissolved in DMSO and DMF
and stirred constantly for 30 min. Characterization and measurement Then, MAI and PbI2 (1/1 by molar) were
added to the above PS solution under constant stirring for 1 h (time for
crosslinking) at room temperature. The entire process was carried out in
ambient conditions. Published in partnership with Nanjing Tech University Preparation of MAI precursor A dark yellow precipitate was recovered using a
rotary evaporator at 60 °C for 1 h. The precipitate was then washed and
recrystallized with a copious amount of diethyl ether and ethanol,
respectively, until it turned white. The resultant white precipitate was
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flow rate of the carrier gas (dry N2). measured by a portable RH sensor and was controlled by adjusting the
flow rate of the carrier gas (dry N2). 25. Zhou, J. & Huang, J. Photodetectors based on organic-inorganic hybrid lead
halide perovskites. Adv. Sci. 5, 1700256 (2018). Published in partnership with Nanjing Tech University AUTHOR CONTRIBUTIONS R.S. and H.F. contributed equally to this work. V.M. and R.S. planned the idea, R.S. and
H.F. conducted the experiments, and all authors analyzed and discussed the results
and contributed to the writing of the manuscript. R.S. and H.F. contributed equally to this work. V.M. and R.S. planned the idea, R.S. and
H.F. conducted the experiments, and all authors analyzed and discussed the results
and contributed to the writing of the manuscript. 23. Saraf, R., Tsui, T. & Maheshwari, V. Modulation of mechanical properties and
stable light energy harvesting by poling in polymer integrated perovskite films: a
wide range, linear and highly sensitive tactile sensor. J. Mater. Chem. A 7,
14192–14198 (2019). Fabrication of flexible photodetectors The polyamide filter membranes with a 25 mm diameter were purchased
from GE Healthcare. The self-assembled Pt–Au nanochains solution was
used to deposit the flexible and highly conductive Pt–Au electrodes on the
polyamide membrane through the vacuum-filtration process. The PDMS
mask was used during the vacuum filtration to obtain a certain Pt–Au
patterned electrode. The distance between the Pt–Au electrodes was
1.6 mm, the thickness of Pt–Au electrodes was around 550 nm and was
kept constant for all samples. 90 µL of plain MAPbI3 or PS-MAPbI3 solution
was then spin-coated on the polyamide filter membrane patterned with
Pt–Au electrodes at 4000 rpm for 30 s. After 8 s of rotation, 200 μL of
diethyl ether was dropped onto the center of the flexible polyamide
substrate. The obtained films were then heated at 100 °C for 10 min to
form the perovskite phase. The thickness of the MAPbI3 perovskite films
with and without PS was around 1 µm (±50 nm). Finally, transparent self-
adhesive tape was used to cover the perovskite films. The device Published in partnership with Nanjing Tech University npj Flexible Electronics (2020) 30 COMPETING INTERESTS 24. Li, D. et al. Plasmonic photonic crystals induced two‐order fluorescence
enhancement
of
blue
perovskite
nanocrystals
and
its
application
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English
| null |
On the convergence of certain integrals
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Annales Universitatis Mariae Curie-Skłodowska. Sectio A, Mathematica
| 2,019
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cc-by
| 3,214
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2010 Mathematics Subject Classification. 32A15, 32A22. 2010 Mathematics Subject Classification. 32A15, 32A22.
Key words and phrases. Entire functions, Hadamard’s three-circles theorem, infinite
integrals. Key words and phrases. Entire functions, Hadamard’s three-circles theorem, infinite
integrals. On the convergence of certain integrals Abstract. Let M(r) := max|z|=r |f(z)|, where f(z) is an entire function. Also let α > 0 and β > 1. We discuss the behavior of the integrand
M(r)e−α(log r)β as r →∞if
R ∞
1
M(r)e−α(log r)βdr is convergent. 1. Convergence of integrals vis-`a-vis convergence of series. There is
one fundamental property of a convergent infinite series in regard to which
the analogy between infinite series and infinite integrals breaks down. If
P∞
n=1 θ(n) is convergent, then θ(n) →0 as n →∞; but it is not always true,
even when θ(r) is always positive, that if
R ∞
a θ(r) dr, a > 0, is convergent,
then θ(r) →0 as r →∞. As a counterexample, we can consider the function
given by θp(r) :=
∞
X
n=0
{fn(r, p) + gn(r, p)}
(p > 1), where the functions fn(r, p) and gn(r, p) of the real variable r are defined by
fn(r, p) := {(n + 1)pr + 1 −n(n + 1)p} 1h
n−
1
(n+1)p ;n
h(r) where the functions fn(r, p) and gn(r, p) of the real variable r are defined by
fn(r, p) := {(n + 1)pr + 1 −n(n + 1)p} 1h
n−
1
(n+1)p ;n
h(r) ere the functions fn(r, p) and gn(r, p) of the real variable r are define
fn(r, p) := {(n + 1)pr + 1 −n(n + 1)p} 1h
n−
1
(n+1)p ;n
h(r) fn(r, p) := {(n + 1)pr + 1 −n(n + 1)p} 1h
n−
1
(n+1)p ;n
h(r) and gn(r, p) := {−(n + 1)pr + 1 + n(n + 1)p} 1h
n;n+
1
(n+1)p
i(r). 2010 Mathematics Subject Classification. 32A15, 32A22. Key words and phrases. Entire functions, Hadamard’s three-circles theorem, infinite
integrals. 20 M. A. Hachani Then, for every positive x, Then, for every positive x, Z x
0
θp(r)dr ≤
∞
X
n=0
1
(n + 1)p < ∞, while θp(r) does not tend to 0 as r →∞. while θp(r) does not tend to 0 as r →∞. while θp(r) does not tend to 0 as r →∞. p
It is however true that if
R ∞
a θ(r) dr converges and θ(r) is non-negative,
then lim inf
r→∞r (log r)(log log r) · · · (ℓkr) θ(r) = 0 , where ℓkr is the k-th iterate of log r. On the convergence of certain integrals Thus r2QM(r) = o (eπr)
was not expected to be all that the convergence of
R ∞
1 r2QM(r) e−πr dr
would imply. Recently, Qazi [5] has proved the following stronger result,
which is “essentially” best possible. 2. A special kind of integrands. Let M(r) := max|z|=r |f(z)|, where
f(z) is an entire function. In his work on Carlson’s theorem ([1, Chapter
9]) for entire functions of exponential type, Rahman ([7, Theorem 7]) had
a situation where the integral
R ∞
1 r2QM(r) e−πr dr was convergent and he
wanted to know the behavior of M(r) for large values of r. He noted ([7,
Lemma 6]) that r2QM(r) e−πr →0 as r →∞. In order to prove it he does
not require anything more than the fact that M(r) is a non-decreasing func-
tion of r. However, M(r) is not just a non-decreasing function of r but also
log M(r) is a downward convex function of log r. Thus r2QM(r) = o (eπr)
was not expected to be all that the convergence of
R ∞
1 r2QM(r) e−πr dr
would imply. Recently, Qazi [5] has proved the following stronger result,
which is “essentially” best possible. 2. A special kind of integrands. Let M(r) := max|z|=r |f(z)|, where
f(z) is an entire function. In his work on Carlson’s theorem ([1, Chapter
9]) for entire functions of exponential type, Rahman ([7, Theorem 7]) had
a situation where the integral
R ∞
1 r2QM(r) e−πr dr was convergent and he
wanted to know the behavior of M(r) for large values of r. He noted ([7,
Lemma 6]) that r2QM(r) e−πr →0 as r →∞. In order to prove it he does
not require anything more than the fact that M(r) is a non-decreasing func-
tion of r. However, M(r) is not just a non-decreasing function of r but also
log M(r) is a downward convex function of log r. Thus r2QM(r) = o (eπr)
was not expected to be all that the convergence of
R ∞
1 r2QM(r) e−πr dr
would imply. Recently, Qazi [5] has proved the following stronger result,
which is “essentially” best possible. Theorem 2.1. Let M(r) := max|z|=r |f(z)|, where f is an entire function
and suppose that
R ∞
0 rα M(r) e−βr dr < ∞for some α > 0 and some β > 0. Then √r · rαM(r) e−βr = O(1) as r →∞. 3. On the convergence of certain integrals If this was not true, then there would
exist positive numbers c and R0 such that for all R > R0, we would have Z eR
R
θ(r) dr >
Z eR
R
c
r (log r)(log log r) · · · (ℓkr) dr = c (ℓkR −ℓk+1R) and then
R eR
R θ(r) dr could not be made arbitrarily small by taking R suf-
ficiently large ([2, p. 376]), contradicting the convergence of the integral
R ∞
a θ(r) dr. On the other hand, it is well known that if θ(r) is positive and
non-increasing, then
R ∞
a θ(r) dr can converge only if r θ(r) →0 as r →∞. The same conclusion can be drawn if θ(r) is the product of a monotonic
function ϕ(r) and a non-negative function L(r) which is continuous and
L(cr) ∼L(r) as r →∞(i.e. limr→+∞
L(cr)
L(r) = 1). This can be explained as
follows. Let ε be any given positive number. Then for all sufficiently large
values of u, we have ε >
Z 2u
u
ϕ(r) L(r) dr
≥min {|ϕ(u)| , |ϕ(2u)|}
Z 2u
u
L(r) dr
= |ϕ(2u)|
Z 2u
u
L(r) dr, = |ϕ(2u)|
Z 2u
u
L(r) dr, say. That uθ(u) →0 as u →∞, now follows from the fact (see Lemma 1.1
below) that Z u
a
L(r) dr ∼uL(u) . Lemma 1.1 (see [4, Lemma 4]). The condition Lemma 1.1 (see [4, Lemma 4]). The condition ϕ1(t) =
Z t
1
ϕ(u)du ∼tϕ(t) is equivalent to ϕ(kt)
∼
t→∞ϕ(t) for every fixed positive k. On the convergence of certain integrals 21 2. A special kind of integrands. Let M(r) := max|z|=r |f(z)|, where
f(z) is an entire function. In his work on Carlson’s theorem ([1, Chapter
9]) for entire functions of exponential type, Rahman ([7, Theorem 7]) had
a situation where the integral
R ∞
1 r2QM(r) e−πr dr was convergent and he
wanted to know the behavior of M(r) for large values of r. He noted ([7,
Lemma 6]) that r2QM(r) e−πr →0 as r →∞. In order to prove it he does
not require anything more than the fact that M(r) is a non-decreasing func-
tion of r. However, M(r) is not just a non-decreasing function of r but also
log M(r) is a downward convex function of log r. On the convergence of certain integrals The main result. An entire function f is a polynomial if and only if
there exists a positive number k such that M(r) := max|z|=r |f(z)| = O(rk)
as r →∞. The degree n of f is the infimum of all such numbers k. In this
case, we have lim
r→∞
log M(r)
log r
= n. If f is a transcendental entire function, then (see a remark following Theo-
rem 3.1) log M(r)
log r
−→∞as r →∞; however, M(r) e−α(log r)β may tend to zero as r →∞for some α > 0 and
some β > 1. This can happen if f is an entire function of order 0, that is, if lim sup
r→∞
log log M(r)
log r
= 0 . In connection with Theorem 2.1, one may then ask the following question:
What can we say about the behavior of M(r) as r →∞if f is an entire
function such that
R ∞
1 M(r) e−α(log r)β dr converges for some α > 0 and
some β > 1? We give an answer to this question. The proof of Theorem 2.1 as given by
Qazi [5] is based on the use of the well-known Stirling’s formula for Euler’s
Gamma function. This was somehow natural because of the integrand in
R ∞
0 rα M(r) e−βr dr having e−βr as a factor. Since the integrand does not
anymore have such a factor, the use of Stirling’s formula is more or less
out of the question. So, we have to use some other ideas. In addition 22 M. A. Hachani to Stirling’s formula, Qazi’s proof of Theorem 2.1 uses Hadamard’s three-
circles theorem. That remains available to us and we have tried to use it as
efficiently as we could. to Stirling’s formula, Qazi’s proof of Theorem 2.1 uses Hadamard’s three-
circles theorem. That remains available to us and we have tried to use it as
efficiently as we could. y
Hadamard’s three-circles theorem [8, p. 172] can be stated as follows: Theorem 3.1. Let f(z) be an analytic function, regular for r1 ≤|z| ≤r3. Furthermore, let r1 < r2 < r3, and let M1, M2, M3 be the maxima of |f(z)|
on the three circles |z| = r1, r2, r3, respectively. Then (3.1)
Mlog(r3/r1)
2
≤Mlog(r3/r2)
1
Mlog(r2/r1)
3
. On the convergence of certain integrals (3.1) Since we may write (3.1) in the form (3.2)
log M(r2) ≤log r3 −log r2
log r3 −log r1
log M(r1) + log r2 −log r1
log r3 −log r1
log M(r3), Hadamard’s three-circles theorem may be interpreted by saying that
log M(r) is a convex function of log r. If f is a transcendental entire func-
tion, then inequality (3.2) leads to the existence of a positive number r0
such that r 7→log M(r)
log r
is a strictly increasing and unbounded function of r,
for r ≥r0. Now we can state our theorem: Now we can state our theorem: Theorem 3.2. Let M(r) := max|z|=r |f(z)|, where f is an entire function
and suppose that
R ∞
1 M(r) e−α (log r)β dr < ∞for some α > 0 and some
β > 1. Then, for any ε > 0, Theorem 3.2. Let M(r) := max|z|=r |f(z)|, where f is an entire function
and suppose that
R ∞
1 M(r) e−α (log r)β dr < ∞for some α > 0 and some
β > 1. Then, for any ε > 0, lim
r→∞r (log r)−γ−ε · M(r) e−α (log r)β = 0 , where γ := max {0 , (β −2)/2} . where γ := max {0 , (β −2)/2} . 4. Proof of Theorem 3.2. We present the proof in several steps. Step I. First we prove that 4. Proof of Theorem 3.2. We present the proof in several steps. Step I. First we prove that (4.1)
R
(log R)β−1 M(R) e−α(log R)β →0 as R →∞. (4.1) Take any ε > 0 and note that M(r)
(log r)β−1(1/r) M(r)
(log r)β−1(1/r) is an increasing function of r for all large r. Hence, if R is large enough,
then R M(R)
αβ(log R)β−1
Z R2
R
α β (log r)β−1
1
r
e−α (log r)β dr
≤
Z R2
R
M(r) e−α (log r)β dr < ε , R M(R)
αβ(log R)β−1
Z R2
R
α β (log r)β−1
1
r
e−α (log r)β dr 23 On the convergence of certain integrals that is, R
(log R)β−1 M(R)
e−α (log R)β −e−α (2 log R)β
< αβε , which implies (4.1). which implies (4.1). p
(
)
Step II. Next, we prove that for all large r, p
(
)
Step II. On the convergence of certain integrals Next, we prove that for all large r, (4.2) M(S) e−α (log S)β < (log S)γ
S
for some S = S(r)∈
r , r+
r
(log r)γ
. If this was not true, then for all t ∈(r , r + r/(log r)γ), which in the case
where 1 < β ≤2 means “for all t ∈(r , 2r)”, we would have M(t) e−α (log t)β ≥(log t)γ
t
. This would imply that This would imply that
Z r+r/(log r)γ Z r+r/(log r)γ
M )γ
M(t) e−α (log t)β dt ≥
Z r+r/(log r)γ
r
(log t)γ
t
dt
=
1
γ + 1
(
log
r +
r
(log r)γ
γ+1
−(log r)γ+1
)
. It is easily checked that the last expression is equal to log 2 if γ is zero and
is 1 + o (1) if γ is positive. Thus the integral
R ∞
1 M(t) e−α (log t)β dt would
not be convergent, contradicting our hypothesis. Hence (4.2) holds. This
means that for all large r, (4.3)
M(λr) < (log λr)γ
λr
eα (log λr)β for some λ ∈
1 , 1 +
1
(log r)γ
. Step III. Since log M(r) is a convex function of log r, we have (4.4)
(M(r))2 ≤M
r
λ
M(λr)
(λ > 0) . (4.4) This is our main tool. We use (4.1) and (4.3) in (4.4) to conclude that
(4.5)
lim
r→∞r (log r)−(γ+β−1)/2 M(r) e−α (log r)β = 0 . This is our main tool. We use (4.1) and (4.3) in (4.4) to conclude that This is our main tool. We use (4.1) and (4.3) in (4.4) to conclude that
(4 5)
li
(l
)−(γ+β−1)/2 M( )
−α (log r)β
0 This is our main tool. We use (4.1) and (4.3) in (4.4) to conclude that (4.5)
lim
r→∞r (log r)−(γ+β−1)/2 M(r) e−α (log r)β = 0 . (4.5)
lim
r→∞r (log r)−(γ+β−1)/2 M(r) e−α (log r)β = 0 . On the convergence of certain integrals If r is sufficiently large and (4.5) If r is sufficiently large and If r is sufficiently large and (4.6)
λ ∈
1 , 1 +
1
(log r)γ
(4.6) is chosen such that (possible by (4.3)) M(λr) < (log λr)γ
λr
eα (log λr)β, en using this and (4.1) in (4.4), we obtain
(M(r))2 ≤c1(r) (log(r/λ))β−1
r/λ
eα (log(r/λ))β · (log(λr))γ
λr
eα (log(λr))β, (M(r))2 ≤c1(r) (log(r/λ))β−1
r/λ
eα (log(r/λ))β · (log(λr))γ
λr
eα (log(λr))β, 24 M. A. Hachani where c1(r) = o(1) as r →∞. where c1(r) = o(1) as r →∞. Now note that ( )
( )
Now, note that ( )
( )
Now, note that )β−1 (log(λr))γ
= (log r)γ+β−1
(
1 −log λ
log r
β−1
1 + log λ
log r
γ)
≤(log r)γ+β−1
(
1 −log λ
log r
β−1
1 + log λ
log r
β−1)
< (log r)γ+β−1 (log(r/λ))β−1 (log(λr))γ because γ < β −1. Hence because γ < β −1. Hence (M(r))2 ≤c1(r)(log r)γ+β−1
r2
exp
(
α (log r)β
1 −log λ
log r
β
+
1 + log λ
log r
β!)
= c1(r)(log r)γ+β−1
r2
exp
(
α (log r)β
2 + (β (β −1) + c2(r))
log λ
log r
2!)
= c1(r)(log r)γ+β−1
r2
exp
n
α (log r)β 2 + (β (β −1) + c2(r)) (log r)−2γ−2o
, where c2(r) = o(1) as r →∞, and where we have used (4.6) in the last line. Note that β −2γ −2 is negative if 1 < β < 2 and zero if β ≥2. Hence
(log r)β−2γ−2 = O(1) as r →∞. This allows us to conclude that where c2(r) = o(1) as r →∞, and where we have used (4.6) in the last line. Note that β −2γ −2 is negative if 1 < β < 2 and zero if β ≥2. Hence
(log r)β−2γ−2 = O(1) as r →∞. This allows us to conclude that M(r) ≤c3(r) (log r)(γ+β−1)/2
r
eα (log r)β = c3(r) (log r)γ+(β−1−γ)/2
r
eα (log r)β, where c3(r) = o(1) as r →∞, which is equivalent to (4.5). where c3(r) = o(1) as r →∞, which is equivalent to (4.5). Inequality (4.5) is considerably stronger than (4.1) and provides a better
estimate for M(r/λ) in (4.4). On the convergence of certain integrals Using (4.5) and (4.3) in (4.4) the way (4.1)
and (4.3) were used above in (4.4), we obtain (4.7)
lim
r→∞r (log r)−γ−(β−1−γ)/22 M(r) e−α (log r)β = 0 , (4.7) which may in turn be used to conclude that (4.8)
lim
r→∞r (log r)−γ−(β−1−γ)/23 M(r) e−α (log r)β = 0 . (4.8) Clearly, (4.8) is stronger than (4.7). Since this process can go on indefinitely,
we see that for any positive integer k, we have lim
r→∞r (log r)−γ−(β−1−γ)/2k M(r) e−α (log r)β = 0 , 2 2 from which the desired result follows. 25 On the convergence of certain integrals Remark. The property of the function M(r) by which log M(r) is a con-
vex function of log r is shared by some other functions associated with an
entire function f, which makes the proof of Theorem 3.2 applicable to these
associated functions. In fact, let f(z) := P∞
ν=0 aνzν be an entire function;
for any r > 0, we define the function Mp(r) = Mp(f; r) by Mp(f; r) :=
1
2π
Z 2π
0
f(r eiθ)
p
dθ
1/p
,
p > 0, and the maximum term of f, denoted by µ(r), is given by the maximum of
|aν|rν for ν ∈{0, 1, 2, . . .}. Then, log µ(r) and log Mp(r) (for any p > 0)
are two convex functions of log r. The proof of this statement was given
by G. Valiron ([9, pp. 30–31]) for the function log µ(r) and by G. H. Hardy
[3] for log Mp(r). The reader might find [6] to be of some interest in this
connection. References [1] Boas, Jr., R. P., Entire Functions, Academic Press, New York, 1954. [2] Hardy, G. H., A Course of Pure Mathematics, Cambridge University Press, London,
1921. [3] Hardy, G. H., The mean value of the modulus of an analytic function, Proc. London
Math. Soc. 14 (1915), 269–277. [4] Hardy, G. H., Rogosinski, W. W., Notes on Fourier series (III), Q. J. Math. 16 (1)
(1945), 49–58. [5] Qazi, M. A., Application of the Euler’s gamma function to a problem related to F. Carl-
son’s uniqueness theorem, Ann. Univ. Mariae Curie-Skłodowska Sect. A 70 (1) (2016),
75–80. [6] Rahman, Q. I., On means of entire functions, Q. J. Math. 7 (1) (1956), 192–195. [7] Rahman, Q. I., Interpolation of entire functions, Amer. J. Math. 87 (1965), 1029–1076 [8] Titchmarsh, E. C., The Theory of Functions, 2nd Edition, Oxford University Press,
London, 1939. [9] Valiron, G., Lectures on the General Theory of Integral Functions, Chelsea Publishing
Company, New York, 1949. Mohamed Amine Hachani Mohamed Amine Hachani D´epartement de Math´ematiques et de Statistique D´epartement de Math´ematiques et de Statistique Universit´e de Montr´eal Montr´eal, Qu´ebec H3C 3J7 Montr´eal, Qu´ebec H3C 3J7 Canada e-mail: hachani@dms.umontreal.ca e-mail: hachani@dms.umontreal.ca Received January 30, 2019
|
https://openalex.org/W4386766339
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https://www.researchsquare.com/article/rs-3345655/latest.pdf
|
English
| null |
GOIZ ZAINDU trial: a FINGER-like multidomain lifestyle intervention feasibility study to prevent dementia in Southern Europe.
|
Research Square (Research Square)
| 2,023
|
cc-by
| 7,528
|
GOIZ ZAINDU trial: a FINGER-like multidomain
lifestyle intervention feasibility study to prevent
dementia in Southern Europe. Mikel Tainta Mikel Tainta Fundacion CITA Alzheimer
Mirian Ecay-Torres
Fundacion CITA Alzheimer
Maria de Arriba
Fundacion CITA Alzheimer
Myriam Barandiaran
Fundacion CITA Alzheimer
Ane Otaegui-Arrazola
Fundacion CITA Alzheimer
Ane Iriondo
Fundacion CITA Alzheimer
Maite Garcia-Sebastian
Fundacion CITA Alzheimer
Ainara Estanga
Fundacion CITA Alzheimer
Jon Saldias
Fundacion CITA Alzheimer
Montserrat Clerigue
Fundacion CITA Alzheimer
Alazne Gabilondo
Biodonostia
Naia Ros
University of the Basque Country
Justo Mugica
Osakidetza
Aitziber Barandiaran
Osakidetza
Francesca Mangialasche Page 1/22 Karolinska Institutet
Miia Kivipelto
Karolinska Institutet
Arantzazu Arrospide
Biodonostia
Javier Mar
Biodonostia
Pablo Martinez-Lage
Fundacion CITA Alzheimer Conclusions: The GOIZ-ZAINDU study has proved that the FINGER methodology is adaptable and feasible in a different
socio-cultural environment. Exploratory efficacy results show a lower risk of decline in executive function
and processing speed. These results support the design of a large-scale efficacy trial. Background: GOIZ ZAINDU ("caring early" in Basque) is a pilot study to adapt the Finnish Geriatric Intervention Study to
Prevent Cognitive Impairment and Disability (FINGER) methodology to the Basque population and
evaluate feasibility and adherence to a FINGER-like multidomain intervention program. Additional aims
included the assessment of efficacy on cognition and collecting data to design a large efficacy trial. Results: One hundred twenty-five participants were recruited (mean age: 75.64 (±6.46); 58% women). MD-Int
(n=61) and RHA (n=64) groups were balanced in demographics and cognition. 52 (85%) participants
from RHA and 56 (88%) from the MD-Int group completed the study. More than 70% of participants
presented good overall adherence to intervention activities. The risk of cognitive decline was higher in the
RHA group compared with MD-Int regarding executive function (p=.019) and processing speed scores
(p=.026). Method: GOIZ ZAINDU is a one-year, randomized, controlled trial of a multidomain intervention in persons aged
60+ years, with Cardiovascular Risk Factors, Aging and Dementia (CAIDE) risk score ≥ 6, no dementia,
and below-than-expected performance in at least one of three cognitive screening tests. Randomization
to a Multidomain intervention (MD-Int) or Regular Health Advice (RHA) was stratified by sex, age (>/≤ 75),
and cognitive status (Mild Cognitive Impairment (MCI)/normal cognition). MD-Int included cardiovascular
risk factors control, nutritional counseling, physical activity, and cognitive training. The primary outcome
was retention rate and adherence to the intervention program. Exploratory cognitive outcomes included
Neuropsychological Test Battery z-scores change. Analyses were carried out by intention to treat. Research Article Keywords: Cognitive impairment, Dementia prevention, Lifestyle, Multidomain intervention, Randomized
trial Posted Date: September 15th, 2023 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Additional Declarations: Competing interest reported. MK is Editor-in-Chief of the journal “Alzheimer’s
Research and Therapy”. Additional Declarations: Competing interest reported. MK is Editor-in-Chief of the journal “Alzheimer’s
Research and Therapy”. Version of Record: A version of this preprint was published at Alzheimer's Research & Therapy on
February 27th, 2024. See the published version at https://doi.org/10.1186/s13195-024-01393-z. Page 2/22 Page 2/22 Page 2/22 BACKGROUND To fully understand the impact of such preventive interventions, their feasibility and efficacy need to be
tested worldwide. To this aim, the World-Wide FINGERS network of multidomain trials for dementia risk
reduction and prevention was established to explore the feasibility and efficacy of multidomain lifestyle
intervention [16] in different populations, regions, and social contexts worldwide. Previous To fully understand the impact of such preventive interventions, their feasibility and efficacy need to be
tested worldwide. To this aim, the World-Wide FINGERS network of multidomain trials for dementia risk
reduction and prevention was established to explore the feasibility and efficacy of multidomain lifestyle
intervention [16] in different populations, regions, and social contexts worldwide. Previous
experiences[17] has shown the importance of conducting a pilot study to adapt the “FINGER-like”
methodology and obtain data on which to base the design of a large-scale efficacy study. Here, we
present the GOIZ ZAINDU ("caring early" in Basque) multidomain intervention pilot study results. GOIZ
ZAINDU is a feasibility study to adapt the FINGER trial methodology to a southern European context, in a
real clinical practice setting. We have evaluated the applicability and adherence to a FINGER-like
intervention and, as an exploratory outcome, the effect on cognitive performance in older adults after one
year. Study design and participants: Study design and participants: BACKGROUND Aging of the population is one of the most remarkable counterparts of medicine and social progress. However, since aging is the main risk factor for dementia, the growth in numbers of older adults is linked
to the increase of people living with disabilities, including dementia. According to the last Global Burden
of Disease Study[1], people living with dementia more than doubled from 1990 to 2016. The World Health
Organization (WHO) in the “Global status report on the public health response to dementia” has estimated
that people living with dementia could be about 139 million by 2050. In this sense, forecasting models for Page 3/22 Page 3/22 the future burden of dementia in many countries predict an unmanageable growth of the number of
cases if efficacious prevention initiatives are not developed [2]. Dementia is a multifactorial process influenced by genetic and environmental conditions and results from
life-long interactions between protective and risk factors[3]. Midlife modifiable dementia risk factors such
as cardiovascular health, physical inactivity, depression, and low education may count for up to a third of
cases of dementia worldwide [4]. Since different degrees of exposure to these factors can modify the
plastic trajectories of aging [3, 5] a window of opportunity for research on prevention is open[5, 6]. Achievements in control and promotion of cardiovascular health along with improvements in the
population education levels are probably behind the apparent reduction of dementia occurrence in some
developed countries[2, 7, 8]. Different scores and indexes have been proposed to estimate individual dementia risks based on risk
factors. The Cardiovascular Risk Factors, Aging and Dementia (CAIDE) risk score [9] is a well-known
validated tool able to predict cognitive trajectories, neurodegeneration, and amyloid deposition [10, 11]. The CAIDE score has also been used to identify and enrol in prevention initiatives at-risk individuals with
modifiable conditions. 12]. The Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and
Disability (FINGER, ClinicalTrials.gov Identifier: NCT01041989) [12] is the first randomized clinical trial
showing that a multidomain, lifestyle-based intervention benefits cognition in persons with increased
CAIDE risk score. Despite their primary negative results, other multidomain European trials (the French
Multidomain Alzheimer Preventive Trial - MAPT, and the Dutch Prevention of Dementia by Intensive
Vascular care - PreDIVA) have confirmed that interventions on risk and protective factors represent a
window of opportunity for dementia prevention in participants with increased risk and frailty [13–15]. Screening evaluation: Subject’s demographic information included age, sex, race/ethnicity, native language, and years of
education. During the screening visits, assessments were conducted to ensure fulfillment of inclusion
criteria and the absence of exclusion criteria. The participants' general practitioners (GP) collected
medical conditions and current medication. CAIDE dementia Risk score was calculated, and T@M,
Fototest, and AD8 questionnaires were administered. Page 4/22
The GOIZ ZAINDU pilot trial is a one-year controlled, randomized, multidomain intervention trial, for
prevention of cognitive decline, carried out in the municipality of Beasain in the Basque Country (Spain). Page 4/22
The GOIZ ZAINDU pilot trial is a one-year controlled, randomized, multidomain intervention trial, for
prevention of cognitive decline, carried out in the municipality of Beasain in the Basque Country (Spain). Page 4/22 Participants were recruited in collaboration with the primary care center health providers from Beasain
Municipality after an informative prevention campaign on lifestyle and dementia. Participants were at least 60 years of age, had a CAIDE score ≥ 6 points and performed below than
expected[18] in at least one of two brief cognitive tests - Memory alteration test, T@M [19] and Fototest
[20] or had a score of 2 or higher in the AD8 informant's questionnaire [21] of cognitive symptoms. Exclusion criteria included the presence of not well controlled cardiovascular or respiratory disease,
previous diagnosis of dementia, ongoing neurological disorders, unstable psychiatric disease, evidence of
any other severe disease of any etiology, or any situation in the investigator's opinion that could
compromise safe engagement in the intervention. GOIZ ZAINDU feasibility trial was approved by the Euskadi Drug Research Ethics Committee
(ID: PI2017134). All participants signed the corresponding informed consent. Baseline visit and diagnostic workout evaluation: Pre-selected participants in the screening phase performed a clinical evaluation and a physical, cognitive,
and behavioral assessment to ensure the completion of the study assessment. Dementia cases, defined
by DSM-IV, were excluded from the study, and the diagnosis of Mild Cognitive Impairment (MCI)[22] cases
were ascertained. Randomization and masking: After the baseline evaluation, all participants received in person verbal information on the potential
benefits of caring for vascular risk factors, adhering to Mediterranean diet, and having good cognitive and
physical activity routines. Participants were later randomly assigned to a standard health advice control
group (RHA, control) or the multidomain intervention group (MD-Int). Random assignment followed a
proportion of 1:1 and was stratified by age (<75 years vs.≥75 years), sex, and cognitive status (normal
cognition vs. MCI). Double blinding is challenging to achieve in this type of studies. Nevertheless, participants were not informed about which group they were assigned. They were urged not
to comment on program details during the evaluation sessions. Nevertheless, participants were not informed about which group they were assigned. They were urged not
to comment on program details during the evaluation sessions. INTERVENTION PERIOD (summarized in figure 1) INTERVENTION PERIOD (summarized in figure 1) Page 5/22 Page 5/22 1. Regular health advice control (RHA) group. Participants randomized to the control group followed
preventive programs already ongoing at their Primary Care Unit regarding physical activity, socialization,
and smoking and alcohol usage. Visits to the GP and nurse depended on personal demands and
necessities; nevertheless, annual consultation was recommended for all patients over 60 years old. 2. Multidomain intervention (MD-Int) group. The MD-Int program was designed to provide tools and
routines that participants could incorporate into daily living activities and reinforce the social
environment. Although the program included standardized guidelines and exercises, each participant was
considered individually, adapting nutritional requirements and physical and cognitive activity according
to individual needs and abilities. This methodology is based on the FINGER trial design[23] but has been
adapted to local resources and the healthcare system. GPs and nurses were involved in the follow-up
visits. Most intervention activities were carried out at the local Primary Care Centre. Local town hall
resources such as group activities for older adults at the municipality sports centre and current outdoor
sports activities were incorporated in the study. The MD-Int program included: 1) individual follow-up
visits every three months for cardiovascular risk factor monitoring and nutritional counselling with
primary health care providers; 2) two nutritional workshops led by a nutritionist; 3) 20 hours of cognitive
stimulation delivered through group sessions and 4) 40 hours of individual cognitive training exercises. Randomization and masking: Participants in the MD-Int group received recommendations to practice 2 to 6 hours of physical exercise
per week and get involved in sports activities. Social stimulation was promoted through group activities. A. Intensive control and monitoring of cardiovascular risk factors: Every three months, a follow-up visit was performed individually at the Primary Care Unit for
cardiovascular risk factors check-up, including measures of blood pressure, pulse, height, weight, hip, and
waist circumference. During these visits, participants were reminded of the study objectives and
motivated to adhere to them. Whenever a poorly controlled or new detected risk factor was detected,
advice and recommendations for adequate control or initiation or adjustment of pharmacological
treatments were provided by the participant’s GP. B. Nutritional counseling was based on the Mediterranean Diet pattern [24,25]. Two workshops were
driven by the nutritionist of the study at the beginning and in the middle of the intervention period. Individual sessions with verbal and written counseling were administered during follow-up visits. Materials given to participants included example menus to increase adherence to the Mediterranean diet. At baseline and 12 month visits and every three months, a 14-itmes dietary questionnaire[26] to assess
adherence to Mediterranean diet was recorded. C. Physical activity and exercise: Participants were encouraged to stay physically active during baseline
and every follow-up visit. Recommendations were based on the American Heart Association Guidelines
and the ICOPE (Integrated Care for Older People) Guidelines from the WHO. Participants were encouraged
to sign up for the city council's outdoor aerobic physical activity programs like hiking or Nordic walking. Indoor group activities were organized and guided by personnel from the Municipal Sports Center twice a
week during the intervention period of nine months. Page 6/22 D. Cognitive intervention was divided into individual sessions and 13 group activities. The main goal was
to incorporate cognitively stimulant daily habits and routines and emphasize the family and social
environment. Group sessions lasted 90 min. and were driven by a neuropsychologist and included several
topics such as age-related cognitive changes, learning strategies for activities of daily living, or
knowledge of self-cognition. For ten months of the intervention period, individual work was designed to
be completed by subjects within 20 minutes, three times per week. This paper material was based on the
NeuronUP© platform and was specially designed for non-demented people and adapted to every
participant according to three characteristics: cognitive status, education, and current or past (if retired)
occupational level. The objective is to train and reinforce executive function, visuospatial skills, language,
episodic memory and working memory. A. Intensive control and monitoring of cardiovascular risk factors: This material was completed with EXERCITA© cognitive training
materials that were developed considering the cultural and linguistic context of the Basque Country
population. Efficacy exploratory outcomes: Cognitive performance was assessed at baseline and 12 months with the modified Neuropsychological
Test Battery (NTBm)[23,27] which includes the following test: Wechler Memory Scale-III Logical Memory,
Consortium to Establish a Registry for Alzheimer's Disease (CERAD) Word List, WMS-R Visual Paired
Associates, Category Fluency, Wechler Adult Inteligence Scale-III Digit Span, Concept Shifting Test, Trail
Making Test, a shortened 40-stimuli version of Stroop Test, and Letter Digit Substitution Test. Feasibility outcomes: Feasibility was the study primary objective. Therefore, adherence to the intervention and retention rates
were the primary outcomes. Retention rate was defined as the proportion of participants who completed
the 12-month trial period. We considered a successful rate if less than 20% of participants dropped out. Adherence to each intervention component is based on participation in offered activities in the
intervention group. Study coordinators assessed the adherence to intervention activities by recording the
number of workshops and follow-up visits attended and by checking the cognitive training workbook. Self-reported information on weekly physical activity and attendance to group activities at the sports
center was recorded for physical exercise. In order to evaluate overall adherence to intervention activities we defined a semi-quantitative scale. Table
1 describes how every intervention component is weighted. We considered both the degree of adherence
to each intervention component and the degree of attendance simultaneously to each intervention. "Very
good" overall adherence was considered when attendance to all intervention components was higher
than 50%. "Good" adherence was defined by attendance to at least 30% of activities of all intervention
components. "Bad" overall adherence was considered when attending one of the intervention
components was lower than 30%. Attendance of less than 30% to two or more intervention components
was considered "Very bad" overall adherence. Table 1. Semi-quantitative scale to estimate overall adherence. Page 7/22 Adherence rates were calculated for the entire follow-up period, including external factors to the
intervention program, such as medical and family issues and COVID-19 outbreak social distancing
measures, to obtain a realistic picture of the potential implementation and maintenance of this type of
intervention in a real scenario. Demographics and cognitive status at baseline were analyzed as
predictors of participant adherence. Additional information: At baseline and final evaluations, additional information was collected on global cognition measured
with the Mini-Mental State Examination (MMSE)[28], occupation level was classified according to
Hollingshead Four Factor Index of Social Status, depression and anxiety symptoms were assessed with
the Hospital Anxiety and Depression Scale (HAD) [29] and physical fitness was measured with the 6
minutes walking test[30]. Safety assessments: Information was obtained and confirmed from participant GPs regarding current diagnoses, medication,
and laboratory values (blood count, cholesterol, glucose, renal and liver function, thyroid hormones, B12
vitamin, folic acid) before the study interventions started. A structured interview for adverse events was
performed at every follow-up visit. Statistical analysis: Variables were checked for normal distribution. Independent samples t-test, Mann-Whitney test, and χ2 as
appropriate were conducted to compare demographics, psychological symptoms, and cognitive
performance between MD-Int and RHA groups at pre-intervention and post-intervention visits. Page 8/22 Page 8/22 NTBm z-scores were calculated [12] and standardized to the baseline mean and SD, with higher scores
suggesting better performance. Five cognitive domain indexes were created: NTBm total score was based
on the 14 tests (table 2S); Executive Functioning domain based on five tests; Processing Speed domain
based on three tests; and Memory domain based on six tests (Memory global). The minimum number of
necessary NTB components was set to eight of 14 for calculating NTB total score, three of five for
executive functioning, two of three for processing speed, and three of six for memory. As an exploratory objective, mean change in z-scores between pre-intervention and post-intervention visits
were calculated for each group and compared between both groups applying independent samples t-test. Mixed models of repeated measures were conducted considering the two evaluations made on the study
subjects to assess the intervention effect on the z-scores. Binary logistic analyses were carried out to
analyze the risk of cognitive decline in the Standard Health Advice control group compared to the
Multidomain Intervention Group. Cognitive decline was defined as a decrease in NTBm scores between
the assessments at pre-intervention and post-intervention. Mixed models and logistic binary regressions
were repeated, introducing level of education, and any variables showing significant differences on group
comparisons as covariables. Analyses were carried out by intention to treat. All statistical analyses were
conducted with SPSS version 20 (SPSS Inc, Chicago, IL). Mixed models were carried out with STATA. RESULTS In early 2017, the Beasain municipality had over 4100 people aged 60+ years. GOIZ-ZAINDU study
recruitment period started in March 2017. 509 people were screened until March 2018. 180 fulfilled
inclusion criteria, 23 declined to participate, and 32 met at least one exclusion criteria, mostly the
presence of dementia. The intervention period lasted from October 2018 to November 2019. 125 subjects
were randomized to the MD-Int. group (n:64) or the RHA group (n:61). (Figure 2). Of these, 108 (86%)
participants completed the post-intervention assessment (retention rate by group was 88% in the MD-Int
arm, and 85% in the RHA arm). Due to the COVID outbreak and lockdown period in Spain (which started in
March 2020), 14 post-evaluation assessments were delayed five months from February to July 2020, 5
from the RHA group and 9 in the MD-Int group. As shown in figure 2, dropout rates were similar in both
groups. The main reasons were lack of time or difficulties in participation (10 participants), health-related
reasons (4 subjects), and one subject declined to perform post-intervention evaluation because of fear of
COVID-19. Two persons, one in each group, died during the study. The mean age was 75.64 (SD 6.46), range 60 to 90 years. Years of education and the distribution of
employment levels were those expected for a population of this generation in an industrial town in our
country. Both groups were balanced in demographics, distribution of dementia risk factors, cognitive
performance, and the presence of MCI (tables 2 and 2S). Adherence scores to Mediterranean Diet and
physical fitness (distance walked in six minutes) were slightly higher in the MD-Int group (p<0.05). Table 2. Pre-intervention characteristics per group. Page 9/22 Mean (SD) median [IQR] of measures unless they are categorical variables which are giv Mean (SD), median [IQR] of measures unless they are categorical variables, which are given in number
(%). Independent-samples t-test, Mann-Whitney test, and χ2 tests were conducted. HADS: Hospital Anxiety
and Depression Scale, NTBm: Neuropsychological Test Battery modified. Mean (SD), median [IQR] of measures unless they are categorical variables, which are given in number
(%). Independent-samples t-test, Mann-Whitney test, and χ2 tests were conducted. HADS: Hospital Anxiety
and Depression Scale, NTBm: Neuropsychological Test Battery modified. Figure 3 shows the mean adherence to each intervention component for participants in the MD-Int. group. The mean attendance to cardiovascular monitoring visits, nutritional workshops, and physical program
activities was more than 70%. RESULTS Adherence to cognitive intervention workshops and completion of
individual cognitive training materials was 64,8% and 55,5%, respectively. Figure 3 shows the mean adherence to each intervention component for participants in the MD-Int. group. The mean attendance to cardiovascular monitoring visits, nutritional workshops, and physical program
activities was more than 70%. Adherence to cognitive intervention workshops and completion of
individual cognitive training materials was 64,8% and 55,5%, respectively. Adherence rates to individual components of the intervention are presented in Table 1S. 67,2% and 73,4%
of the subjects completed at least 2/3 of the cardiovascular monitoring and nutritional counseling visits, Page 10/22 respectively. 64,1% of the participants completed more than 50% of the cognitive training individual
materials, and 70% attended more than half of the cognition workshops. Over 75% of the subjects during
the intervention period reported practicing physical exercise at least twice a week. respectively. 64,1% of the participants completed more than 50% of the cognitive training individual
materials, and 70% attended more than half of the cognition workshops. Over 75% of the subjects during
the intervention period reported practicing physical exercise at least twice a week. Overall adherence to all intervention activities was at least "good" in 71,9% of the participants (figure 3). Thirty-five participants (54,7%) showed "very good" adherence to the intervention plan. "Bad" overall
adherence was observed in 12 participants, mostly because of logistic and health issues. Due to a lack of
motivation, 6 participants did not adhere to intervention activities. At the final visit, the mean 12-month change for the NTB executive z-scores was significantly different
between groups favoring the intervention (MD-Int group 0.11 (SD 0.43); RHA group -0.13 (SD 0.48);
p=0,009) with a moderate Cohen's d size effect of 0.52 (table 3). This finding remains after adjustment
for education and post-intervention anxiety levels in mixed models and logistics regression as
covariables (Table 5S). At the post-intervention visit, the anxiety level was higher in the MD-Int group than
in the RHA group, but still in the normal range (< cut off of 11) (Table 3S). There was no difference in cognitive performance between groups in the mixed models of repeated
measures (Table 6S and 7S). However, 32 (64%) participants in the RHA and 22 (40%) in the MD-Int group
declined in NTB executive function z-scores; 30 persons (61%) in the RHA and 22 (39%) in the MD-Int
declined in NTB processing speed z-score (Table 4). RESULTS Risk of decline was higher in the RHA group
compared with the MD-Int. group for NTB executive function score (p = .019; odds ratio 2.57, 95% CI 1.13-
5.84) and NTB processing speed score (p = .026, odds ratio 2.3, 95% CI 1.03-5.16) (Table 4S). Table 3. Mean change between baseline visit and final visit. Table 3. Mean change between baseline visit and final visit. NTBm: Neuropsychological Test Battery modified. Mean (SD) difference between z scores at pre-
intervention and post-intervention visits in each group is shown. NTBm: Neuropsychological Test Battery modified. Mean (SD) difference between z scores at pre-
intervention and post-intervention visits in each group is shown. NTBm: Neuropsychological Test Battery modified. Mean (SD) difference between z scores at pre-
intervention and post-intervention visits in each group is shown. Table 4. Percentage of cognitive decline. Page 11/22 NTBm: Neuropsychological Test Battery modified. Decline = Diff (z post – z pre) < 0; No decline = Diff (z
post – z pre) ≥ 0 NTBm: Neuropsychological Test Battery modified. Decline = Diff (z post – z pre) < 0; No decline = Diff (z
post – z pre) ≥ 0 DISCUSSION For the GOIZ ZAINDU trial study, public and private resources have been invested in this
project, and rational usage has played a crucial role in its consecution. Beasain town hall and Primary
Health Care center involvement in the study activities ensure the sustainability of this health prevention
initiative in the future. As an exploratory analysis, we observed a benefit in executive function, and
processing speed is consistent with previously reported data from multidomain intervention. Nevertheless these data should be considered cautiously as the study design is not oriented to evaluate Together with the feasibility analysis, we must consider the institution's implication and economic
sustainability. For the GOIZ ZAINDU trial study, public and private resources have been invested in this
project, and rational usage has played a crucial role in its consecution. Beasain town hall and Primary
Health Care center involvement in the study activities ensure the sustainability of this health prevention
initiative in the future. As an exploratory analysis, we observed a benefit in executive function, and
processing speed is consistent with previously reported data from multidomain intervention. Nevertheless, these data should be considered cautiously as the study design is not oriented to evaluate
the efficacy of the intervention. Nevertheless, these data should be considered cautiously as the study design is not oriented to evaluate
the efficacy of the intervention. Lifestyle intervention trials have some limitations inherent to their nature. Thus, previously healthier
individuals usually adhere more to the prescribed activities[34]. Thereby, even in the RHA group, healthier
people can follow the recommendations given better than the participants in the MD-Int. with poorer
adherence. Regarding this "healthy adherer" effect, we have observed that the subgroup of MD-Int. participants with better adherence have a lower CAIDE dementia risk score, better MMSE score, and lower
proportion of MCI (table 8S). Likewise, the control group also received recommendations on vascular risk
factors, diet, and physical activity for ethical reasons. Therefore, this may have led to underestimating the
intervention effect. With this pilot experience, we have noticed that other needs and aspects may be addressed in a
multidomain lifestyle intervention. Observational studies suggest the benefits in healthy aging, promoting
social engagement and psychological well-being [35]. Although it has been reported spontaneously, in our
pilot experience, many subjects have also experienced a well-being sensation by participating in group
activities and increasing their previous degree of socialization. DISCUSSION GOIZ ZAINDU pilot trial shows that a multidomain lifestyle and risk factor monitoring intervention to
prevent cognitive decline in older adults at high risk of dementia is feasible and reproducible. GOIZ
ZAINDU design has successfully adapted the FINGER trial methodology to Southern European conditions,
including diet, exercise habits, and healthcare system, to ensure external validity and acceptance. Secondary efficacy analysis supports previous findings suggesting a protective effect on cognition
related to simultaneous intervention in different domains [12]. The feasibility concept is not as common in a clinical research context as in an economic or business
management environment. We define feasibility for a clinical setting as the capacity to carry out the
protocol and the degree of commitment by all the implicated institutions and participants. We argue that
with the GOIZ ZAINDU study, we have accomplished enough to conclude that this multidomain lifestyle
intervention can be adapted and performed in our social, cultural, and institutional framework. As
described in Fig. 2, we observed a high degree of interest in any activity regarding the cognitive decline
and its prevention in older adults. In less than a month, almost 20% of the total population older than 60
years old in the Beasain Municipality (n: 850) participated in the informative campaign performed by the
local institutions. More than half of the participants in the informative sessions attended the screening
invitation (n:509). This is a meaningful result, considering data from multinational surveys indicating a
low level of awareness, among citizens, on the possibility to ameliorate lifestyle and vascular health in
order to prevent dementia [44]. The proportions of dropouts in the GOIZ ZAINDU pilot study are similar to previous data reported [12–14]. Local adaptation of these kind of multidomain intervention protocol may help participant adherence[17,
31]. Participants adherence is also essential to ensure intervention program acceptance and efficacy. Furthermore, overall adherence was better than in previous multidomain intervention trials [32, 33]. Besides the adherence, compliance degree measured by changes in individuals' lifestyles and correction
of previous modifiable risk factors should be assessed. In the GOIZ ZAINDU study, due to the small
sample size, we could not measure it, but we propose to address this aspect within a larger efficacy study. Page 12/22 Together with the feasibility analysis, we must consider the institution's implication and economic
sustainability. DISCUSSION Therefore, it may be considered in the
design of future lifestyle interventions. GOIZ ZAINDU study results are consistent with previous data [12–14] and reinforce the importance of
selecting an adequate window of opportunity and an at-risk population for this intervention. These
participants are at a unique momentum of the cognitive decline continuum. These subjects are older
adults with some modifiable dementia risk factors that have already experienced slight changes in
cognition. In the GZ study, we replicated FINGER trial participant characteristics regarding cognitive
variables by including brief cognitive testing in the screening period and then excluding participants
showing dementia symptoms. [36] It is never too early or too late in the lifespan to begin any initiative to
promote cognition and healthy aging, especially in at-risk populations. Beyond present and future drugs against dementia pathophysiological targets, a holistic approach to
dementia care is needed, especially in primary/secondary prevention. Sufficient evidence suggests that
dementia should be addressed as a multifactorial entity [4], and a holistic approach is needed to promote
healthy aging and dementia prevention (Geneva 2017) [37]. The WHO published in 2019 the Guidelines
for cognitive decline risk reduction based on a multicomponent approach. While these Guidelines are
data-driven to date, they have not been consistently proven in appropriately designed trials. FINGER
results hold the promise that healthy eating, exercise, and cognitive and social activity may have Page 13/22 Page 13/22 Page 13/22 favorable effects on cognition, functional independence [38, 39], and health-related quality of life [40] and
reduce the need for health care services [41] in older adults. The GOID ZAINDU pilot study experience
provides us with data to base an efficacy study, the CITA GO-ON trial (ClinicalTrials.gov Identifier:
NCT04840030), which is currently part of the initiatives coordinated by WW-FINGER[42, 43] to deepen our
knowledge of the mechanisms to prevent cognitive deterioration and promote healthy aging. favorable effects on cognition, functional independence [38, 39], and health-related quality of life [40] and
reduce the need for health care services [41] in older adults. The GOID ZAINDU pilot study experience
provides us with data to base an efficacy study, the CITA GO-ON trial (ClinicalTrials.gov Identifier:
NCT04840030), which is currently part of the initiatives coordinated by WW-FINGER[42, 43] to deepen our
knowledge of the mechanisms to prevent cognitive deterioration and promote healthy aging. Abbreviations FINGER: Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability
GZ: GOIZ ZAINDU Declarations Page 14/22 Ethics approval and consent to participate. GOIZ ZAINDU feasibility trial was approved by the Euskadi
Drug Research Ethics Committee (CEIm-E) (ID: PI2017134). All participants signed the corresponding
informed consent. Consent for publication. All participants signed the corresponding informed consent which included an
explicit consent for publication the study results and experience. Consent for publication. All participants signed the corresponding informed consent which included an
explicit consent for publication the study results and experience. Availability of data and materials. The datasets used and/or analyzed during the current study are
available from the corresponding author on reasonable request. Availability of data and materials. The datasets used and/or analyzed during the current study are
available from the corresponding author on reasonable request. Competing interests. MK is Editor-in-Chief of the journal “Alzheimer’s Research and Therapy”. The rest of
the authors declare that they have no competing interests. Funding. This project has been promoted by CITA-alzheimer Foundation and has received a grant of the
Health Department of the Basque Government (File No. 2017111120). The research is part of the World-
Wide FINGERS Network, and we acknowledge the WW-FINGERS Network and its Global Scientific
Coordinating Center for their contributions. The GOIZ ZAINDU study is also part of the EU-FINGERS
consortium, a Project funded by the Joint Program of Neurodegenerative Disorders project (EU-FINGERS:
multimodal precision prevention toolbox for dementia in Alzheimer’s disease), through the following
funding organizations under the aegis of JPND - www.jpnd.eu: Finland, Academy of Finland; Germany,
Federal Ministry of Education and Research; Spain, National Institute of Health Carlos III; Luxemburg,
National Research Fund; Hungary, National Research, Development and Innovation Office; The
Netherlands, Netherlands Organization for Health Research and Development. Authors' contributions. MT: Conception or design of the work, Data collection; Data analysis and interpretation, Drafting the
article Page 15/22
MT: Conception or design of the work, Data collection; Data analysis and interpretation
article
ME: Data analysis and interpretation, Critical revision of the article
MA: Data collection
MB: Conception or design of the work, Final approval of the version to be published
AO: Conception or design of the work
AI: Data collection
MG-S: Critical revision of the article
AE: Conception or design of the work
JS: Data collection ME: Data analysis and interpretation, Critical revision of the article Page 15/22 MC: Data collection
AG: Data collection
NR: Data collection
JM: Data collection
AB: Data collection FM: Conception or design of the work, Critical revision of the article
MK: Conception or design of the work, Critical revision of the article
AA: Data analysis and interpretation JM: Data analysis and interpretation, Critical revision of the article JM: Data analysis and interpretation, Critical revision of the article PML: Conception or design of the work, Data analysis and interpretation, Critical revision of the article
All authors read and approved the final manuscript. Acknowledgements. All the authors of this manuscript and the components of the GOIZ ZAINDU study
group would like to acknowledge to all the participants that take part of this initiative. GOIZ ZAINDU study
group: Aquizu I.; Arrondo M.A.; Baztarrika E.; Etxeberria L.; García-Arrea E.; García-Domínguez M.; Imaz E.;
Iparragirre M.; Iridoy M.; Larrea A.; López M.D.; Martin F.; Olaskoaga A.; Pacheco P.; Pérez-Rodiguez A.M.;
Porres Y.; Ruibal M.; San Juan B.; Tilves M.J.; Zapirain E. References 1. Collaborators G 2016 D, Nichols E, Szoeke CEI, Vollset SE, Abbasi N, Abd-Allah F, et al. Global,
regional, and national burden of Alzheimer’s disease and other dementias, 1990–2016: a systematic
analysis for the Global Burden of Disease Study 2016. Lancet Neurology. 2019;18:88–106. 1. Collaborators G 2016 D, Nichols E, Szoeke CEI, Vollset SE, Abbasi N, Abd-Allah F, et al. Global,
regional, and national burden of Alzheimer’s disease and other dementias, 1990–2016: a systematic
analysis for the Global Burden of Disease Study 2016. Lancet Neurology. 2019;18:88–106. 2. Soto-Gordoa M, Arrospide A, Moreno-Izco F, Martínez-Lage P, Castilla I, Mar J. Projecting Burden of
Dementia in Spain, 2010–2050: Impact of Modifying Risk Factors. Journal of Alzheimer’s Disease. 2015;48:721–30. 2. Soto-Gordoa M, Arrospide A, Moreno-Izco F, Martínez-Lage P, Castilla I, Mar J. Projecting Burden of
Dementia in Spain, 2010–2050: Impact of Modifying Risk Factors. Journal of Alzheimer’s Disease. 2015;48:721–30. 3. Lisko I, Kulmala J, Annetorp M, Ngandu T, Mangialasche F, Kivipelto M. How can dementia and
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disability be prevented in older adults: where are we today and where are we going? J Intern Med. 2021; 4. Livingston G, Huntley J, Sommerlad A, Ames D, Ballard C, Banerjee S, et al. Dementia prevention,
intervention, and care: 2020 report of the Lancet Commission. Lancet. 2020;396:413–46. 4. Livingston G, Huntley J, Sommerlad A, Ames D, Ballard C, Banerjee S, et al. Dementia prevention,
intervention, and care: 2020 report of the Lancet Commission. Lancet. 2020;396:413–46. 5. Solomon A, Kivipelto M, Soininen H. Prevention of Alzheimer’s disease: moving backward through
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prediction of dementia risk in 20 years among middle aged people: a longitudinal, population-based
study. The Lancet Neurology. 2006;5:735–41. 10. Ecay-Torres M, Estanga A, Tainta M, Izagirre A, Garcia-Sebastian M, Villanua J, et al. Increased CAIDE
dementia risk, cognition, CSF biomarkers, and vascular burden in healthy adults. Neurology. 2018;91:e217–26. 11. O’Brien JT, Firbank MJ, Ritchie K, Wells K, Williams GB, Ritchie CW, et al. Association between midlife
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intervention of diet, exercise, cognitive training, and vascular risk monitoring versus control to prevent
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cluster-randomised controlled trial. The Lancet. 2016;388:797–805. 14. Andrieu S, Guyonnet S, Coley N, Cantet C, Bonnefoy M, Bordes S, et al. Effect of long-term omega 3
polyunsaturated fatty acid supplementation with or without multidomain intervention on cognitive
function in elderly adults with memory complaints (MAPT): a randomised, placebo-controlled trial. The Lancet Neurology. 2017;16:377–89. 15. Delrieu J, Payoux P, Carrié I, Cantet C, Weiner M, Vellas B, et al. Multidomain intervention and/or
omega‐3 in nondemented elderly subjects according to amyloid status. Alzheimer’s Dementia. 2019;15:1392–401. 15. Delrieu J, Payoux P, Carrié I, Cantet C, Weiner M, Vellas B, et al. Multidomain intervention and/or
omega‐3 in nondemented elderly subjects according to amyloid status. Alzheimer’s Dementia. 2019;15:1392–401. 16. Kivipelto M, Mangialasche F, Snyder HM, Allegri R, Andrieu S, Arai H, et al. World‐Wide FINGERS
Network: A global approach to risk reduction and prevention of dementia. Alzheimer’s Dementia J
Alzheimer’s Assoc. 2020;16:1078–94. 16. Kivipelto M, Mangialasche F, Snyder HM, Allegri R, Andrieu S, Arai H, et al. World‐Wide FINGERS
Network: A global approach to risk reduction and prevention of dementia. Alzheimer’s Dementia J
Alzheimer’s Assoc. 2020;16:1078–94. 17. Chew KA, Xu X, Siongco P, Villaraza S, Phua AKS, Wong ZX, et al. SINgapore GERiatric intervention
study to reduce physical frailty and cognitive decline (SINGER)–pilot: A feasibility study. Alzheimer’s
Dementia Transl Res Clin Interventions. 2021;7:e12141. 18. Olazarán J, Hoyos-Alonso MC, Ser T del, Barral GA, Conde-Sala JL, Bermejo-Pareja F, et al. References Aplicación
práctica de los test cognitivos breves. Neurología. 2016;31:183–94. Page 17/22 Page 17/22 19. Rami L, Molinuevo J, Sanchez‐Valle R, Bosch B, Villar A. Screening for amnestic mild cognitive
impairment and early Alzheimer’s disease with M@T (Memory Alteration Test) in the primary care
population. International Journal of Geriatric Psychiatry. 2007;22:294–304. 20. Carnero-Pardo C, Espejo-Martinez B, Lopez-Alcalde S, Espinosa-Garcia M, Saez-Zea C, Vilchez-Carrillo
R, et al. Effectiveness and costs of phototest in dementia and cognitive impairment screening. BMC
Neurology. 2011;11:92. 21. Galvin JE, Roe CM, Powlishta KK, Coats MA, Muich SJ, Grant E, et al. The AD8 A brief informant
interview to detect dementia. Neurology. 2005;65:559–64. 22. Petersen R. Mild cognitive impairment as a diagnostic entity. Journal of Internal Medicine. 2004;256:183–94. 23. Kivipelto M, Solomon A, Ahtiluoto S, Ngandu T, Lehtisalo J, Antikainen R, et al. The Finnish Geriatric
Intervention Study to Prevent Cognitive Impairment and Disability (FINGER): Study design and
progress. Alzheimer’s & Dementia. 2013;9:657–65. 24. Estruch R, Ros E, Salas-Salvadó J, Covas M-I, Corella D, Arós F, et al. Primary Prevention of
Cardiovascular Disease with a Mediterranean Diet Supplemented with Extra-Virgin Olive Oil or Nuts. New Engl J Med. 2018;378:e34. 25. Rest O van de, Berendsen AA, Haveman-Nies A, Groot LC de. Dietary Patterns, Cognitive Decline, and
Dementia: A Systematic Review. Adv Nutr. 2015;6:154–68. 26. Estruch R, Martínez-González MA, Corella D, Salas-Salvadó J, Ruiz-Gutiérrez V, Covas MI, et al. Effects of a Mediterranean Style Diet on Cardiovascular Risk Factors: A Randomized Trial Ann Intern 26. Estruch R, Martínez-González MA, Corella D, Salas-Salvadó J, Ruiz-Gutiérrez V, Covas MI, et al. 26. Estruch R, Martínez-González MA, Corella D, Salas-Salvadó J, Ruiz-Gutiérrez V, Covas MI, et al. Effects of a Mediterranean-Style Diet on Cardiovascular Risk Factors: A Randomized Trial. Ann Intern
Med. 2006;145:1. Effects of a Mediterranean-Style Diet on Cardiovascular Risk Factors: A Randomized Trial. Ann Intern
Med. 2006;145:1. 27. Harrison J, Minassian SL, Jenkins L, Black RS, Koller M, Grundman M. A Neuropsychological Test
Battery for Use in Alzheimer Disease Clinical Trials. Arch Neurol-chicago. 2007;64:1323–9. 28. Folstein MF, Folstein SE, McHugh PR. “Mini-mental state” A practical method for grading the
cognitive state of patients for the clinician. Journal of Psychiatric Research. 1975;12:189–98. 29. Julian LJ. Measures of anxiety: State‐Trait Anxiety Inventory (STAI), Beck Anxiety Inventory (BAI),
and Hospital Anxiety and Depression Scale‐Anxiety (HADS‐A). Arthrit Care Res. 2011;63:S467–72. 30. Agarwala P, Salzman SH. Six-Minute Walk Test Clinical Role, Technique, Coding, and Reimbursement. References Chest. 2020;157:603–11. 30. Agarwala P, Salzman SH. Six-Minute Walk Test Clinical Role, Technique, Coding, and Reimbursement. Chest. 2020;157:603–11. 31. Xu X, Chew KA, Wong ZX, Phua AKS, Chong EJY, Teo CKL, et al. The SINgapore GERiatric Intervention
Study to Reduce Cognitive Decline and Physical Frailty (SINGER): Study Design and Protocol. J Prev
Alzheimer’s Dis. 2022;9:40–8. 31. Xu X, Chew KA, Wong ZX, Phua AKS, Chong EJY, Teo CKL, et al. The SINgapore GERiatric Intervention
Study to Reduce Cognitive Decline and Physical Frailty (SINGER): Study Design and Protocol. J Prev
Alzheimer’s Dis. 2022;9:40–8. 32. Coley N, Ngandu T, Lehtisalo J, Soininen H, Vellas B, Richard E, et al. Adherence to multidomain
interventions for dementia prevention: Data from the FINGER and MAPT trials. Alzheimer’s Dementia. 2019; 33. Ngandu T, Lehtisalo J, Korkki S, Solomon A, Coley N, Antikainen R, et al. The effect of adherence on
cognition in a multidomain lifestyle intervention (FINGER). Alzheimer’s Dementia. 2021; 33. Ngandu T, Lehtisalo J, Korkki S, Solomon A, Coley N, Antikainen R, et al. The effect of adherence on
cognition in a multidomain lifestyle intervention (FINGER). Alzheimer’s Dementia. 2021; Page 18/22 Page 18/22 34. Simpson SH, Eurich DT, Majumdar SR, Padwal RS, Tsuyuki RT, Varney J, et al. A meta-analysis of the
association between adherence to drug therapy and mortality. Bmj. 2006;333:15. 35. Fratiglioni L, Marseglia A, Dekhtyar S. Ageing without dementia: can stimulating psychosocial and
lifestyle experiences make a difference? Lancet Neurology. 2020;19:533–43. 35. Fratiglioni L, Marseglia A, Dekhtyar S. Ageing without dementia: can stimulating psychosocial and
lifestyle experiences make a difference? Lancet Neurology. 2020;19:533–43. 36. Ngandu T, Lehtisalo J, Levälahti E, Laatikainen T, Lindström J, Peltonen M, et al. Recruitment and
Baseline Characteristics of Participants in the Finnish Geriatric Intervention Study to Prevent
Cognitive Impairment and Disability (FINGER)—A Randomized Controlled Lifestyle Trial †. Int J
Environ Res Pu. 2014;11:9345–60. 37. Kivipelto M, Mangialasche F, Ngandu T. Lifestyle interventions to prevent cognitive impairment,
dementia and Alzheimer disease. Nat Rev Neurol. 2018;14:1. 38. Kulmala J, Ngandu T, Havulinna S, Levälahti E, Lehtisalo J, Solomon A, et al. The Effect of
Multidomain Lifestyle Intervention on Daily Functioning in Older People. J Am Geriatr Soc. 2019;67:1138–44. 39. Kulmala J, Ngandu T, Kivipelto M. Prevention Matters: Time for Global Action and Effective
Implementation. Journal of Alzheimer’s Disease. 2018;Preprint:S191–8. 39. Kulmala J, Ngandu T, Kivipelto M. Prevention Matters: Time for Global Action and Effective
Implementation. Journal of Alzheimer’s Disease. References 2018;Preprint:S191–8. 40. Strandberg TE, Levälahti E, Ngandu T, Solomon A, Kivipelto M, Group F, et al. Health-related quality of
life in a multidomain intervention trial to prevent cognitive decline (FINGER). Eur Geriatr Med. 2017;8:164–7. 41. USE OF HEALTH CARE SERVICES AMONG OLDER ADULTS PARTICIPATING IN A MULTIDOMAIN
LIFESTYLE INTERVENTION TO PREVENT COGNITIVE IMPAIRMENT (FINGER). Cochrane Central
Register Control Trials Central. 2018;2018. 42. Rosenberg A, Mangialasche F, Ngandu T, Solomon A, Kivipelto M. Multidomain Interventions to
Prevent Cognitive Impairment, Alzheimer’s Disease, and Dementia: From FINGER to World-Wide
FINGERS. J Prev Alzheimer’s Dis. 2019;1–8. 43. Kivipelto M, Mangialasche F, Ngandu T, Network W, Martín E, Kivipelto M, et al. World Wide Fingers
will advance dementia prevention. Lancet Neurology. 2018;17:27. 44. Alzheimer’s Disease International. 2019. World Alzheimer Report 2019: Attitudes to dementia. London: Alzheimer’s Disease International. Figures Page 19/22 Page 19/22 Figure 3 Mean adherence to each intervention component in the MD-Int group. Page 20/22
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GOIZ ZAINDU study design Figure 1 GOIZ ZAINDU study design Page 20/22 Page 20/22 igure 2
GOIZ ZAINDU study participants flowchart. GOIZ ZAINDU study participants flowchart. Page 21/22 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. his is a list of supplementary files associated with this preprint. Click to dow GZSupplementarymaterialART.docx Page 22/22 Page 22/22
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Confessando o estranho
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Resumo O artigo propõe uma nova leitura dos documentos conhecidos como Confissões da
Bahia, produzidos pela Inquisição Portuguesa na América no final do século XVI. Tal
reinterpretação tem como recorte principal as chamadas “santidades” ou “heresias
indígenas”, movimentos religiosos/sociais da tradição nativo americana surgidos na
América Colonial durante o final do século XVI e início do XVII. A nova análise aqui
proposta matiza e leva a cabo uma leitura crítica inicial da já clássica interpretação do
historiador Ronaldo Vainfas, na obra A Heresia dos Índios. Deste modo, se propõe a noção,
ainda não totalmente desenvolvida, de éthos indígena em contraposição à categoria de
“disjunção social” proposta pelo último autor na análise da referida massa documental. Essa releitura visa, principalmente, enxergar nos documentos uma agência indígena que
não seja apenas de cunho revoltoso ou reativo ao colonialismo. Palavras-chave: Indígenas americanos; inquisição; colonização portuguesa. Confessando o estranho: hegemonia, éthos americano e a
colonização lusitana a partir de documentos do Santo Ofício da
Inquisição de Lisboa no findar do século XVI Martiniano Sardeiro de Alcântara Neto
Pesquisador Colaborador do Departamento de Antropologia da Universidade de Brasília
martiniano.neto@gmail.com Abstract The article proposes a new reading of the documents known as Confessions of Bahia,
produced by the Portuguese Inquisition in America at the end of the 16th century. Such
reinterpretation has as its main feature the so-called “sanctities” or “indigenous heresies”,
religious / social movements of the Native tradition, which emerged in Colonial America
during the late 16th and early 17th centuries. This new reading nuances and proposes
to make a critical overview of the already classic interpretation of the historian Ronaldo Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 A história indígena não se subsume à história indigenista. (Manuela Carneiro da Cunha) Manuela Carneiro da Cunha, em um texto introdutório ao conjunto de artigos que
compõem a coletânea História dos Índios no Brasil (Carneiro da Cunha 1992), trabalha
parte de seu argumento a partir da diferenciação entre uma política indígena e uma
política indigenista. A última é usada como referência a uma ação política propriamente
estatal, seja ela abertamente colonial ou, em tempos mais recentes, não-assimilacionista – ao menos no que se refere explicitamente à letra da lei da última e ainda parcialmente
vigente Constituição Federal. A primeira, por outro lado, é a ação política gestada e posta em
prática por atores sociais convencionalmente chamados de índios ou nativos americanos. Historicamente, por vezes, essas vertentes políticas se aproximaram: Carneiro da Cunha
usa o exemplo dos portugueses se alinhando aos Tupiniquins para combater os Tamoios,
que se aliaram aos franceses, por sua vez, ainda nas primeiras levas de colonos do Velho
Continente à América (Carneiro da Cunha 1992: 18). – ao menos no que se refere explicitamente à letra da lei da última e ainda parcialmente
vigente Constituição Federal. A primeira, por outro lado, é a ação política gestada e posta em
prática por atores sociais convencionalmente chamados de índios ou nativos americanos. Historicamente, por vezes, essas vertentes políticas se aproximaram: Carneiro da Cunha
usa o exemplo dos portugueses se alinhando aos Tupiniquins para combater os Tamoios,
que se aliaram aos franceses, por sua vez, ainda nas primeiras levas de colonos do Velho
Continente à América (Carneiro da Cunha 1992: 18). Contudo, mesmo que próximas, há de se frisar que essas duas políticas são
patentemente diferentes. Cada uma delas, portanto, tem uma trajetória histórica
e sociológica própria, ainda que existam momentos históricos de embate, alianças
conjunturais, de isolamento ou disputas entre elas. Tal diferenciação visa, em última
instância, deixar claro que os índios “não são vítimas de uma fatalidade mas agentes
de seu destino”, passagem em que Carneiro da Cunha resume certas facetas dos mitos
ameríndios do surgimento dos brancos ou não-indígenas dentro de diversas sociedades
indígenas (Carneiro da Cunha 1992: 19). O artigo que se segue é uma tentativa de enxergar uma política indígena a partir
de documentos produzidos por e para uma política indigenista. Confessando o estranho 190 Vainfas, in the work A Heresia dos Índios. In this way, the category of American ethos, not
here completely developed, is suggested in opposition to the notion of “social disjunction”
proposed by the last author in the analysis of the referred documentary mass. This
reinterpretation aims, mainly, to see in the documents an indigenous agency that is not
only reactive to colonialism. Keywords: Native Americans; inquisition court; Portuguese colonization. A história indígena não se subsume à história indigenista. (Manuela Carneiro da Cunha) A história indígena não se subsume à história indigenista. Assim, irei me dedicar a
uma reinterpretação das chamadas Confissões da Bahia (Vainfas 1997 e, anteriormente,
Abreu 1922 [ambos 1592]), conjunto de documentos produzidos na primeira incursão da Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Martiniano Sardeiro de Alcântara Neto Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 1
Kawerêtxikô foi fundada no ano de 2008 no território tradicional dos Mebengôkrê, que não é a terra
original em que os Tapayúna criaram fortes laços simbólicos e materiais. Estes últimos, que habitavam
tradicionalmente a região entre o rio do Sangue e do rio Arinos (MT), sofreram diversas tentativas de
genocídio até conseguirem se refugiar na Terra Indígena Kapoto Jarinã, onde se localiza atualmente
Kawerêtxikô, às margens do rio Xingu, extremo norte do Mato Grosso. 2
Este é o termo que os habitantes de língua Jê, como os Mebengôkrê (Kayapó), os Panará e os Tapayúna
(Kaykwakhratxi), no Território Indígena do Xingu (atual TIX, antigo Parque Indígena do Xingu, PIX)
usam para se referir, de forma genérica, a todos os não indígenas. 3
“Dados parciais dos gestores da saúde indígena e relatos das Equipes Multidisciplinares de Saúde
Indígena indicavam extrema relevância das doenças respiratórias agudas no padrão de morbimortalidade Martiniano Sardeiro de Alcântara Neto 191 Inquisição de Lisboa às Terras Baixas Sul-Americanas no findar do século XVI. Inquisição de Lisboa às Terras Baixas Sul-Americanas no findar do século XVI. Antes de iniciar a análise das fontes históricas em questão, gostaria de esclarecer
que, apesar de seu autor ser bacharel em História e ter alguma experiência na análise
de fontes documentais (Alcantara Neto 2007), o texto que se segue visa dar uma
interpretação antropológica (e não exatamente histórica ou historiográfica) ao conjunto
de documentos conhecidos mais recentemente como Confissões da Bahia (Vainfas 1997). A tradição analítica à qual me filio é a da Antropologia Social ou Cultural, disciplina que
vem ocupando minhas reflexões acadêmicas há quase vinte anos e que deu base à (quase)
toda minha inspiração interpretativa das Confissões. De todo modo, como penso em
deixar claro abaixo, não ignoro por completo os historiadores: o diálogo com eles se dá
mais como contraponto à análise do que como base teórico-metodológica. É preciso dizer, também, que essa não era minha primeira opção de pesquisa ao
me filiar ao Laboratório de Indigenismo e Etnologia da Universidade de Brasília (LINDE –
UnB). Como Pesquisador Colaborador da referida Instituição de Pesquisa e Laboratório,
eu tinha em mente retomar questões anteriormente tratadas em minha tese de doutorado
(Alcantara Neto 2016). Deste modo, visava basicamente voltar ao trabalho de campo
participativo, face-a-face, na aldeia Kawerêtxikô dos Tapayúna1, continuando a pesquisa
que havia iniciado no meu doutorado. Contudo meu primeiro planejamento se viu frustrado em ao menos duas frentes
principais. Inicialmente eu não tinha as condições econômicas necessárias para viajar e
me manter numa distante e relativamente dispendiosa nova incursão a campo. Isto se
deve, em parte, às novas prioridades (se realmente alguma há) no fomento à pesquisa
adotada pelo Executivo Federal eleito no ano de 2018. Além disso, vivemos atualmente (junho/julho de 2020) a pandemia de uma
nova enfermidade, altamente infecciosa. Essa nova pandemia me fez perder qualquer
esperança de, ao menos no médio prazo, rever os amigos Tapayúna. Há fortes indícios
de que as populações nativas da América são especialmente sensíveis às novas doenças
dos kuben2, principalmente àquelas ligadas ao trato respiratório3. Sabe-se também que o 1
Kawerêtxikô foi fundada no ano de 2008 no território tradicional dos Mebengôkrê, que não é a terra
original em que os Tapayúna criaram fortes laços simbólicos e materiais. 4
“Em relação ao estudo das doenças emergentes e reemergentes, as análises das alterações ambientais
incluem as mobilizações populacionais na era da globalização como fatores importantes na disseminação
de patógenos e a existência de ambientes modificados e degradados propícios ao aparecimento de
novas doenças.” (Pignatti 2004 :137) Confessando o estranho contato com novos agentes patológicos, como o novo vírus COVID-19, possivelmente tem
alguma relação com o intenso desmatamento que os mesmos kuben vem promovendo
sistematicamente já há alguns séculos4. Desta feita, dediquei os últimos meses à leitura e análise do já citado material
histórico e seus correlatos. Descobri, inicialmente, que não há como falar da primeira
“visitação” (o termo é dos próprios inquisidores) da Inquisição de Lisboa à América
Portuguesa sem fazer referência ao já clássico trabalho do historiador Ronaldo Vainfas,
A Heresia dos Índios: catolicismo e rebeldia no Brasil Colonial. (Vainfas 1995). O próximo
tópico é dedicado a uma breve contextualização e leitura crítica desta obra. de crianças indígenas Guarani nos litorais do Sul e Sudeste do Brasil, tal como referido para povos
indígenas de diversas outras localidades ao redor do mundo e para grupos infantis não indígenas na
maioria dos países em desenvolvimento.” (Cardoso 2010 :133). Martiniano Sardeiro de Alcântara Neto Estes últimos, que habitavam
tradicionalmente a região entre o rio do Sangue e do rio Arinos (MT), sofreram diversas tentativas de
genocídio até conseguirem se refugiar na Terra Indígena Kapoto Jarinã, onde se localiza atualmente
Kawerêtxikô, às margens do rio Xingu, extremo norte do Mato Grosso. 1
Kawerêtxikô foi fundada no ano de 2008 no território tradicional dos Mebengôkrê, que não é a terra
original em que os Tapayúna criaram fortes laços simbólicos e materiais. Estes últimos, que habitavam
tradicionalmente a região entre o rio do Sangue e do rio Arinos (MT), sofreram diversas tentativas de
genocídio até conseguirem se refugiar na Terra Indígena Kapoto Jarinã, onde se localiza atualmente
Kawerêtxikô, às margens do rio Xingu, extremo norte do Mato Grosso. 2
Este é o termo que os habitantes de língua Jê, como os Mebengôkrê (Kayapó), os Panará e os Tapayúna
(Kaykwakhratxi), no Território Indígena do Xingu (atual TIX, antigo Parque Indígena do Xingu, PIX)
usam para se referir, de forma genérica, a todos os não indígenas. 2
Este é o termo que os habitantes de língua Jê, como os Mebengôkrê (Kayapó), os Panará e os Tapayúna
(Kaykwakhratxi), no Território Indígena do Xingu (atual TIX, antigo Parque Indígena do Xingu, PIX)
usam para se referir, de forma genérica, a todos os não indígenas. 3
“Dados parciais dos gestores da saúde indígena e relatos das Equipes Multidisciplinares de Saúde
Indígena indicavam extrema relevância das doenças respiratórias agudas no padrão de morbimortalidade 3
“Dados parciais dos gestores da saúde indígena e relatos das Equipes Multidisciplinares de Saúde
Indígena indicavam extrema relevância das doenças respiratórias agudas no padrão de morbimortalidade Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 192 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 5
A partir daqui, visando não poluir o texto de Abreu com a expressão latina “sic erat scriptum” (“assim
estava escrito”, sic, abreviação do latim), escolhi por transcrevê-lo sempre na íntegra e mantendo a
grafia original imprensa no documento de 1922. Martiniano Sardeiro de Alcântara Neto 193 possuem inúmeras vantagens no estreitamento dos diálogos acadêmicos. Antes de Vainfas, havia apenas uma rara edição de Capistrano de Abreu (Abreu
1922) que dava algum acesso público aos documentos em questão. Quando comparada à
de Vainfas, esta última publicação imprimiu o material produzido pelos inquisidores não
só na cidade de Salvador e Recôncavo, mas também em Pernambuco e outras localidades. Esta informação é trazida pelo próprio Vainfas (1997), já que eu só pude ter acesso, até
o presente momento, à “Separata da Serie (sic) Eduardo Prado para melhor conhecer
o Brasil”, que não contempla a totalidade dos documentos editados por Capistrano: há
apenas a Introdução do autor, o “Monitorio (sic) do Inquisidor Geral” e, por fim, algumas
determinações do Inquisidor Heitor Furtado de Mendonça, uma espécie de ‘pós-Monitório’
(Abreu 1922). Sobre o texto de Capistrano, o que me interessa, quando comparado à interpretação
dada por Vainfas, é que o primeiro autor mostra que a proeminência do cristianismo
católico estava em crise. Na América portuguesa, que na época se encontrava restrita
praticamente ao litoral atlântico, se argumenta que Em 1587 escrevia Gabriel Soares, a respeito dos francezes que “muitos
se amancebaram na terra, onde morreram, sem se quererem tornar
para França, e viveram como gentios com muitas mulheres, dos quaes e
dos que vinham todos os annos a Bahia e ao rio de Sergipe em naus da
França [...], viveram e morreram como gentios; dos quais ha hoje muitos
seus descendentes, que são louros, alvos e sardos, e havidos por índios
tupinambás e são mais bárbaros que elles”5 (Souza apud Abreu 1922: 28). Inicialmente, há de se dizer que Vainfas ‘pinta’ o litoral das Terras Baixas de maneira
um tanto diferente de Capistrano. Além de praticamente ignorar a disputa colonial entre
portugueses e franceses no litoral, o primeiro autor classifica as “santidades” indígenas
como [...] fenômeno histórico-cultural de resistência indígena, a idolatria pode se
referir a um domínio em que a resistência ou a renovação de antigos ritos
e crenças se mesclava com a luta social, com a busca de uma identidade
cada vez mais destroçada pelo colonialismo, com a reestruturação ou
inovação das relações de poder e, inclusive, com certas estratégias de
sobrevivência no plano da vida material dos índios (Vainfas 1995 :31). O colonialismo católico-lusitano na análise de Vainfas O trabalho de Vainfas, não só na última obra citada, foi o que deu visibilidade
contemporânea às chamadas “santidades” (categoria luso-americana apropriada pelo
séquito inquisitorial) indígenas. Isto porque foi ele que organizou o já citado livreto com
parte das confissões inquisitoriais, além de, anos antes, ter publicado sua tese de livre
docência exatamente sobre este material (Vainfas 1995). Há de se ressaltar aqui o fato
raro, tanto entre antropólogos quanto entre historiadores, de se efetivamente trabalhar
na publicação do material que deu origem às pesquisas. Assim, se não fosse o empenho de
Ronaldo Vainfas em republicar tal massa documental e contextualizá-la para os leitores
contemporâneos não-iniciados no tema, eu possivelmente não teria tido contato com ela
no início de minha graduação em História, já no final da década de 90 do século XX. Além disso, a obra de Vainfas pode ser vista no contexto das primeiras aproximações
contemporâneas mais efetivas entre os estudos antropológicos com fontes e, por vezes,
métodos que eram típicos da disciplina histórica, por um lado, e as análises históricas
dessas mesmas fontes com uma inspiração ou leitura crítica na/da Antropologia Social. Deste modo, Manuela Carneiro da Cunha, Jonh Monteiro e outros estudiosos que
escreveram artigos para a já citada coletânea da História dos Índios no Brasil (Carneiro
da Cunha 1992) receberam com acertado entusiasmo a obra de Vainfas (1995). Ela é tida
como exemplo da interface possível e necessária entre duas áreas do conhecimento que Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Martiniano Sardeiro de Alcântara Neto 5
A partir daqui, visando não poluir o texto de Abreu com a expressão latina “sic erat scriptum” (“assim
estava escrito”, sic, abreviação do latim), escolhi por transcrevê-lo sempre na íntegra e mantendo a
grafia original imprensa no documento de 1922. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Confessando o estranho Confessando o estranho 194 Não só na passagem acima, mas em toda sua análise, Vainfas se esforça para mostrar
o peso do colonialismo na desestruturação dos índios. As “santidades”, nesse sentido, são
reiteradamente referidas como movimentos “rebeldes”, reativos a um sistema colonial
imposto, em última instância, pela força bélica do que talvez tenha sido o primeiro Estado
Absolutista na Idade Moderna. Um dos problemas analíticos claros, já notado à época da publicação de A Heresia
dos Índios: catolicismo e rebeldia no Brasil colonial (ênfase minha), é que Vainfas não se
preocupa em diferenciar as categorias analíticas dele próprio – usa-se reiteradamente
“idolatria”, como já citado, ou “sincretismo” (Vainfas 1995:68, 1995: 159) – daquelas do
Santo Ofício. Como argumenta o antropólogo Sérgio da Mata, Vainfas utiliza, ao longo de todo seu livro, a categoria “seita” para se referir
às santidades. Não foi uma boa escolha. Revela, neste particular, absorção
acrítica (em que pesem todos os cuidados tomados) da linguagem
inquisitorial. Nesta, como nos meios cristãos em geral, “seita” assume um
significado diverso do sociológico. Onde o senso comum eclesiástico vê
“heresia”, “desvio”, “erro” (e daí a sua repressão), a sociologia da religião
vê um tipo de comunidade religiosa com um padrão configuracional
próprio. (Mata 1996 :173) A resenha do antropólogo foi publicada, é necessário notar, um ano depois da
divulgação da tese de livre docência do historiador. Após a leitura cuidadosa da obra de
Vainfas, foi-me impossível discordar da crítica de Mata, ao menos neste ponto específico. Penso que outras categorias, como as já citadas “sincretismo” ou “idolatria”, também não
são escolhas completamente apropriadas. Isto pelo fato de não ser possível, por exemplo,
encontrar em Vainfas uma definição de “sincrético” que extrapole a noção, muito comum
dentro da religiosidade cristã-católica, de um conjunto de crenças e rituais que são uma
espécie de cópia-não-completa daquela desenvolvida no Velho Continente. Esse uso está
intimamente relacionado à noção de “hibridismo”, do mesmo autor, que será discutida
adiante. No mesmo ano da publicação da interpretação de Vainfas, o também antropólogo
Marco Antonio Gonçalves assevera, numa detida leitura, que [apesar do livro estar] amplamente ancorado em sua “etnografia”, em
determinados momentos, talvez por força do contexto histórico [d]o
“colonialismo” e [d]a “conquista”, apresenta um caráter generalizante,
deixando de tratar de acontecimentos históricos específicos e situações
etnográficas particulares e assumindo um tom que condena índios Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 6
Outro sinal de que o texto de Vainfas não passou por qualquer modificação recente é o uso que ele faz
do artigo “O mármore e a murta: sobre a inconstância da alma selvagem”, do antropólogo Eduardo
Viveiros de Castro. É desnecessário argumentar sobre a importância que a teoria do Perspectivismo
Ameríndio teve e continua tendo no campo das Ciências Sociais de maneira geral, valendo-lhe traduções
para outras línguas e, além disso, uma edição conjunta com outros artigos na língua portuguesa
(Viveiros de Castro 2002). Vainfas cita o artigo, ainda na reimpressão de 2010, como em mimeo. Em
uma comunicação via correio eletrônico que consegui manter com Eduardo Viveiros de Castro, ele
ressaltou que, possivelmente, a versão usada por Vainfas seria a mesma que a citada acima, ainda que
para o ano de 1995. Não consegui efetivar qualquer contato com Ronaldo Vainfas, nem mesmo por meio
eletrônico. De todo modo, é explícito o afastamento (consciente ou não) de Vainfas não só das novas
edições do texto de Viveiros de Castro, mas sobre a teoria do Perspectivismo em si. Confessando o estranho 2020 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 195 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 7
Além disso, este é o projeto de pesquisa principal de diversos outros pesquisadores mais experientes
que eu no estudo da história indígena e indigenista, como a própria Manuela Carneiro da Cunha ou John Martiniano Sardeiro de Alcântara Neto sem rosto à “exploração” e à “miséria” causadas pelo também genérico
“colonialismo” (Gonçalves 1995 :163). É exatamente esse “colonialismo genérico” que pretendo melhor matizar nas
próximas páginas. Esclareço que não tenho notícia de qualquer nova edição da análise
de Vainfas e que a reimpressão de 2010, aqui utilizada, não debate ou incorpora qualquer
crítica referente às questões agora (re)analisadas6. Na leitura que faço, reitero as críticas supracitadas de Mata e Gonçalves e proponho
algumas novas. No mesmo mote, deixo claro também que Vainfas, por vezes, chega a
dar alguma ‘voz’ para questões que, na minha leitura, mereciam um peso maior ou uma
análise mais detida – assim como existem outros pontos a que o historiador dedica grande
atenção e que são marginais na leitura que proponho. Por outro lado, a contextualização histórica e historiográfica é, além da excelente
Introdução de Vainfas ao conjunto de documentos (Vainfas 1995, 1997), como veremos,
de grande importância e não pode ser descartada na análise que agora proponho. Há
um outro ponto em que é impossível de divergir de A Heresia dos Índios: as confissões
inquisitoriais são documentos pontualmente plurais, que admitem várias leituras, a
depender, entre outras coisas, da bagagem acadêmica que o potencial leitor traz e dos
problemas que ele coloca. Ainda assim não há como negar que existem profundas consequências teórico-
metodológicas no uso que Vainfas faz das categorias inquisitoriais. Contudo uma análise
mais pormenorizada dessas ‘sequelas’ pediria a escrita de um novo artigo, voltado
exclusivamente para a inserção historiográfica e antropológica da obra – e, sem dúvida, a
construção de uma análise crítica de como os historiadores da América colonial se referem
aos nativos americanos nas histórias que escrevem. Como já dito, este não é o objetivo do
presente texto7. 7
Além disso, este é o projeto de pesquisa principal de diversos outros pesquisadores mais experientes
que eu no estudo da história indígena e indigenista, como a própria Manuela Carneiro da Cunha ou John Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Manuel Monteiro. Para o último autor pode-se conferir, por exemplo, o estudo de Monteiro (1992) para
os Guarani sul-meridionais no mesmo período histórico que aqui trato. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Confessando o estranho 196 Irei me focar, portanto, exclusivamente na já citada questão do sobrepeso do
sistema colonial na análise de Vainfas, da qual já dei um exemplo a partir de Capistrano
de Abreu. A Introdução deste último autor possui mais pontos interessantes sobre tal
questão. Capistrano argumenta, sobre a origem da Inquisição e seu contexto no Velho
Continente, que O Santo Officio surgiu em terras de herejes notáveis pelo número e
pelo poderio; os denunciantes arriscavam a vida no caso de serem
identificados; mais de um inquisidor sucumbiu á vindicta popular; urgia o
maior segredo. Agora [época do 1º Regimento de D. Henrique] a situação
mudara; os réus eram os escorraçados e os indefesos (Abreu 1922: 17-
18). Pretendo insistir aqui na preponderância apenas relativa do cristianismo católico,
mesmo quando se fala da Inquisição na Europa. O século XVI marca, por exemplo, o
surgimento e desenvolvimento do chamado “luteranismo” (categoria dos inquisidores),
um dos movimentos fundadores da conhecida Reforma Protestante. A Inquisição, sabe-
se, é uma instituição da chamada Contrarreforma Católica – e visava, basicamente,
combater o avanço de outros cristianismos recém-fundados no Velho Continente. Ou seja:
a proeminência católica de vários séculos sobre o cristianismo não estava em risco apenas
no Novo Mundo. Com isso, se o colonialismo se faz, neste momento, pela justificação
divina (católica, para o caso em questão) do Estado Absolutista, que deveria espalhar a
palavra de Cristo e do Deus único entre as almas americanas que a ignoravam, a posição
do Santo Ofício como o cristianismo único e hegemônico é mais um dado dentro de uma
complexa disputa política pela legitimação de uma tradição cristã específica. Sua força não
está exatamente na manutenção do exercício de um monopólio da religiosidade cristã na
Europa, mas na tentativa de manutenção de uma antiga hegemonia, agora cada vez mais
relativa. Esse contexto não tem quase nenhum peso na análise de Vainfas das confissões. Voltando o olhar para a América quinhentista, meu objetivo principal é deixar claro
que os documentos da Primeira Visitação da Inquisição ao Brasil podem ser interpretados
não somente como uma “rebeldia”, “hibridismo” ou “seita” indígena numa América
“destroçada” pelo colonialismo (Vainfas 1995 :227). Penso, portanto, na existência de
um éthos americano, e não exatamente de uma cultura ou sociedade específica que se
contrapunha ao colonialismo lusitano. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 8
Se certamente conformavam sociedades ou culturas, “nações” ou “países” não parecem ser termos
completamente adequados para se referir à organização política dos grupos nativos americanos das
Terras Baixas no Período Colonial. Além de uma ocupação caracteristicamente não centralizada e, o mais
importante, não exclusiva, Monteiro argumenta que “não muito distante do exemplo Tupi, o constante
abandono e regeneração de aldeias, o quadro mutável de alianças e hostilidades e as migrações de
longa distância mobilizadas por carismáticos profetas são fatores que se contrapõem a qualquer visão
monolítica de uma ‘nação’ Guarani” (Monteiro 1992 :477). Explicando melhor o uso que faço da noção de éthos, não uso o termo com base Explicando melhor o uso que faço da noção de éthos, não uso o termo com base Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Martiniano Sardeiro de Alcântara Neto 9
Neste ponto, deixo de lado os africanos por pura e simples ignorância dos contextos históricos e
sociológicos que o êxodo transcontinental forçado de pessoas da África teve, certamente, na conformação
dessa nova população americana. Confessando o estranho inadequação dos termos acima entre aspas e o passo inicial de tentar gestar, ainda que
preliminarmente, os primeiros passos para uma noção mais adequada podem justificar a
leitura do argumento que se segue. inadequação dos termos acima entre aspas e o passo inicial de tentar gestar, ainda que
preliminarmente, os primeiros passos para uma noção mais adequada podem justificar a
leitura do argumento que se segue. Há de se notar, de todo modo, que a influência diretamente ‘etnografável’ da
Santidade de Jaguaripe possui, sem dúvida, um forte componente Tupi. Neste sentido,
concordo com Cristina Pompa no que tange aos problemas de se tentar especular sobre
uma grande ‘sociedade’ ou ‘cultura’ Tupi-Guarani, principalmente a partir de fontes tão
dispersas quanto o relato dos cronistas do XVI e, ao mesmo tempo, as descrições dos
grupos Guarani no XIX (Pompa 2004). De todo modo, estou menos interessado em mostrar
como as tradições Tupi e Guarani, em separado, podem ter se desdobrado historicamente
em séculos de colonização europeia: como já dito, minha intenção é tentar enxergar uma
agência não reativa dentro de um fenômeno histórico bastante determinado que é a
Santidade de Jaguaripe. Voltando à Vainfas, aproximo-me de sua análise no que tange ao argumento de que
a interação entre indígenas e europeus deu condições para o surgimento de uma cultura ou
sociedade muito específica no Novo Continente, nem completamente indígena, tão pouco
lusitana9. Contudo, no momento em que a Inquisição eufemicamente ‘visitava’ a América,
este “hibridismo” estava em pleno processo de formação e há, em minha leitura, espaço
para enxergar mais que “disjunção” ou uma simples reação (“rebeldia”) ao colonialismo,
reação esta muitas vezes com o ‘tom’ de já-fadada-ao-fracasso no estudo de Vainfas (1995). Assim, tal éthos nativo, ainda que pouco definido em sua substância, era
compartilhado, as Confissões Inquisitoriais não deixam dúvidas, entre habitantes
portugueses, “híbridos”, indígenas e até mesmo os “negros da Guiné” (categoria inquisitorial
para os escravos africanos). Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Martiniano Sardeiro de Alcântara Neto 197 na tradição analítica da Antropologia estadunidense, famosa na Escola de Cultura e
Personalidade (cf., por exemplo, Benedict 1960). Faço uso como contraposição às noções
mais complexas de sociedade ou cultura, que pressupõem, muitas vezes, o compartilhamento
de um histórico de organização social comum que remontaria a vários séculos. Ou, mais
particularmente para o caso da Antropologia estadunidense, de um fundo psicossocial
compartilhado que daria a base de um “padrão cultural” (Benedict 1960, tradução livre). Não é este, penso eu, exatamente o caso da Santidade de Jaguaripe, ao menos em sua
relação com os colonizadores lusitanos, como deixarei claro no desenvolvimento do meu
argumento. Isto não quer dizer que os nativos americanos não possuíssem uma cultura
ou sociedade, mas apenas que as várias culturas ou sociedades indígenas ‘reverberam’
no litoral de ocupação lusitana menos com o peso de uma tradição étnica ou cultural
específica e mais como uma influência difusa8, mas decisiva e característica do cotidiano
colonial da segunda metade do século XVI. Essa é, não tenho dúvidas, uma leitura possível
da massa documental das confissões. Essa é uma definição negativa de éthos. Afirmar positiva e pedagogicamente
quais seriam as características próprias desta noção é uma empreitada que extrapola em
muito os limites das reflexões que pude tecer até agora sobre os documentos em questão. Se é certo que grupos da tradição Tupi, como argumenta Vainfas, estavam presentes e
efetivamente se relacionavam com os portugueses na época da chegada da Inquisição, não
é possível determinar, por outro lado, que outros grupos ou tradições diferentes também
não estivessem presentes (mesmo que diretamente em contato com os Tupi e, portanto,
em contato indireto com os portugueses). Isso inviabiliza, como já dito, o uso das categorias
de sociedade ou cultura determinadas historicamente (ao menos na tradição histórica
do Ocidente). Assim, essa (não) definição de éthos como uma influência social nativa,
difusa, mas sem dúvida presente no processo colonizador, como procurarei mostrar, é até
onde os documentos me deixaram caminhar até o presente momento. Não há, ao menos
por enquanto, a possibilidade de substituir as noções de “seita”, “heresia”, “idolatria” ou
qualquer outra usada por Vainfas por uma categoria mais teoricamente rebuscada que a de
éthos como uma influência sociológica difusa, mas efetivamente presente nos primórdios
da colonização lusitana na América. Contudo, penso que a indicação e justificação da Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 198 Confessando o estranho Martiniano Sardeiro de Alcântara Neto E confessando suas culpas, disse que de idade de dezoito anos até
idade de trinta e seis anos viveu como homem gentio, não rezando,
nem se encomendando a Deus, cuidando que não havia de morrer nem
tendo conhecimento de Deus, como verdadeiro cristão, e posto que se
confessava pelas Quaresmas, era por cumprir com a obrigação, e sua vida
no dito tempo foi mais de gentio que de cristão, porém nunca deixou a fé
de Cristo e essa teve sempre em seu coração10 (Vainfas 1997: 346-347). Não há dúvida que, confessadamente, o cristianismo foi, durante boa parte da vida
de Tomacaúna, uma religiosidade externa, com ritos levados a cabo “para cumprir com a
obrigação”. Não há dúvida que, confessadamente, o cristianismo foi, durante boa parte da vida
de Tomacaúna, uma religiosidade externa, com ritos levados a cabo “para cumprir com a
obrigação”. O confessante havia procurado o visitador inquisitorial para, no “período” ou
“tempo” da graça”, confessar seus desvios de fé e pedir perdão pelos erros de doutrina que
havia cometido contra a fé católica. O “tempo da graça” se fazia num intervalo de trinta
dias concedido pelo visitador Heitor Furtado de Mendonça, no qual quem fizesse total
confissão de seus erros teria a vantagem de, logo de antemão, não ter a pena de morte
como sentença possível, além de diminuídas outras penas, como açoites ou autos-de-fé. É necessário esclarecer, ainda, que a confissão inquisitorial não se confunde com
a confissão auricular tradicional até hoje comum no cristianismo católico. A primeira
é uma confissão pública de erros estruturais contra a doutrina católica, como apoiar o
“luteranismo” ou ser um criptojudeu – como dito, a Inquisição é uma instituição da
Contrarreforma. A segunda faz parte, ao contrário da primeira, da própria estrutura da
religiosidade e da espiritualidade cristã-católica: é uma confissão íntima dos pecados da
alma, comuns a todo e qualquer cristão, e do arrependimento de tê-los cometidos. Sem
pecados, arrependimento e (para os cristãos católicos) a mediação da absolvição pelo
sacerdote, não há como se falar em cristianismo, lato sensu11. Tomacaúna é figura central na saga da Santidade de Jaguaripe. Uma nova leitura das confissões e da Santidade de Jaguaripe Tomacaúna é um “mestiço”, filho de pai “branco” e mãe “negra do gentio deste Brasil”
(categorias dos inquisidores e também utilizadas por Vainfas), que faz sua confissão no
último dia do “tempo da Graça”. O notário do Santo Ofício coloca as palavras de Tomacaúna
no papel da seguinte maneira: 9
Neste ponto, deixo de lado os africanos por pura e simples ignorância dos contextos históricos e
sociológicos que o êxodo transcontinental forçado de pessoas da África teve, certamente, na conformação
dessa nova população americana. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 199 Martiniano Sardeiro de Alcântara Neto 11 As explicações para o Período ou Tempo da Graça e para as Confissões Inquisitoriais, bem como para
as classificações típicas do Santo Oficio que se seguem, são dadas por Vainfas na excelente Introdução
e notas de pé de página ao conjunto de fontes que ele organiza. As digressões sobre o que compõe o
cristianismo católico e o que não coincide com ele são de minha própria autoria. 10 Ao contrário de Capistrano, Vainfas moderniza toda a grafia de época dos documentos. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Confessando o estranho Confessou que haverá vinte e dois anos pouco mais ou menos que, em
Pernambuco, pecou no pecado da carne com duas moças suas afilhadas, das
quais ele foi padrinho quando, sendo elas gentias, as batizaram e fizeram
cristãs, parecendo-lhe que tanto pecado em dormir com elas sendo suas
afilhadas como se não o foram (Vainfas 1995: 347). O “pecado da carne” não é exclusividade da confissão de Tomacaúna. Nos
distanciando brevemente da primeira confissão aqui analisada, é importante notar que
nele incorreu, por exemplo, o vigário de Matoim, o padre Frutuoso Álvares. Isso nos ajudará
a mostrar que pecados gravíssimos entre os cristãos-católicos não eram exclusividade de
“mestiços” ou nativos. A diferença em relação à confissão do “mestiço” é que o sacerdote católico pecou não
exatamente com suas afilhadas, mas com outros homens de diferentes idades e até mesmo
com um menino de 12 ou 13 anos (Vainfas 1997: 45-51). No mesmo mote, o “pecado da
carne” também confessa a “cristã-velha” (em contraposição aos “cristãos-novos”, judeus
ou filhos de judeus recentemente convertidos ao cristianismo católico, cf. nota 08) Paula
Siqueira, que, “ajuntando seus vasos naturais [o dela e o da viúva Felipa de Souza] tendo
deleitação [gozo sexual, cf. nota 08] e consumado com efeito o cumprimento natural de
ambas as partes como se propriamente foram homem com mulher” (Vainfas 1997: 106),
também reconhece seus erros frente à mesa do Santo Ofício. Voltemos a Tomacaúna. Ele confessa ainda que [...] haverá vinte anos pouco mais ou menos que ele foi ao sertão de Porto
Seguro em companhia de Antônio Dias Adorno, à conquista de ouro, e no
dito sertão ele usou dos usos e costumes dos gentios, tingindo-se pelas
pernas com uma tinta chamada urucum e outra jenipapo, e empenando-
se pela cabeça de penas, e tangendo os pandeiros dos gentios, que são
uns cabeços com pedras dentro [maracás, cf. nota 08], bailando com eles,
cantando suas cantigas gentílicas pela língua gentílica que ele bem sabe,
o que estas coisas fez por dar a entender aos gentios do dito sertão que
ele era valente e não os temia, por andaram sempre em guerra (Vainfas
1995: 347). Poder-se-ia dizer que tais costumes e o próprio conhecimento da língua nativa
seriam exclusividades de Tomacaúna, sertanista experiente, inclusive em “descer” ou
“resgatar” (eufemismo do vocabulário do português quinhentista para “capturar”) índios
para servirem de escravos para senhores de engenho do litoral. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Martiniano Sardeiro de Alcântara Neto Foi ele que, por
ordem do nobre e rico senhor de engenho Fernão Cabral de Taíde, liderou a comitiva que
conseguiu negociar pacificamente a saída da Santidade do “sertão” (sinônimo de mata
do interior, no português quinhentista, o contrário ao litoral parcialmente ocupado por
colonos lusitanos e seus aliados e escravos) e sua ida para o engenho de Jaguaripe, de
Taíde. Continuemos seguindo de perto a confissão de Tomacaúna: Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 200 Martiniano Sardeiro de Alcântara Neto 201 praticar os costumes gentios, como participar dos rituais da Santidade de Jaguaripe, por
exemplo, Fernão Cabral de Taíde também seria acusado e, por fim, confessaria esta culpa,
sendo preso e degredado para fora do litoral de ocupação lusitana. Sobre a sinceridade da devoção de Taíde aos rituais da Santidade, Vainfas (1995)
gasta muitas dezenas de páginas para concluir, por fim, que o nobre senhor de engenho
queria, na verdade, os índios da Santidade em sua localidade para, a posteriori, deles
fazerem escravos. Na minha interpretação, vejo tal empreitada de Vainfas como uma
digressão apenas marginal e, portanto, de pouca importância. De que interessa se a crença
de Taíde tinha motivos mais materialistas se diversos outros cristão-católicos, “novos”
e “velhos”, “mestiços” ou não “mestiços”, “negros da terra” (indígenas) ou “da Guiné”
(africanos) também tomaram parte efetivamente nos rituais dos “gentios”? Este ponto pode ser melhor ilustrado a partir da confissão da esposa de Fernão
Taíde, a senhora Margarida da Costa. Ela, como nos informa o notário no início da confissão,
era “cristã-velha”, nascida na cidade portuguesa de Moura. A cônjuge de Taíde declara que […] haverá cinco anos, pouco mais ou menos, que, na dita sua fazenda de
Jaguaripe, se aposentaram [...] uns gentios da terra que faziam a abusão
chamada de Santidade, tendo um ídolo de pedra que não tinha figura
humana a qual ídolo chamavam a Santidade, e faziam suas reverências e
suas cerimônias gentílicas. E no dito tempo, duas negras e três negros do dito gentio da terra da dita
abusão vieram da casa a que estavam aposentados dentro, na sua fazenda,
ter às casas do aposento dela confessante, que será da distância de quase
meia légua, tudo dentro da dita sua fazenda, e a choraram ao seu modo
gentílico como costumam fazer quando querem reverenciar e festejar
alguma pessoa, e ela confessante, por obra de uma hora que aí estiveram,
os mandou agasalhar dando-lhes peixe e farinha, e uma das ditas negras
era a que chamavam de mãe de Deus na dita abusão, e a essa deu ela
confessante umas fitas, dizendo-lhe que fosse com elas mais honrada. Confessando o estranho Não seria verdade. Sobre Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Confessando o estranho saudação lacrimal – facetas que poderiam diretamente incriminá-la frente à Inquisição. É
claro que Margarida nega, ao fim da confissão, que tenha algum dia realmente crido nos
rituais “gentílicos”. Mas esta espécie de confusão (ou confissão?) inicial de Margarida e a
negação final coincidem com o argumento de Tomacaúna e com todos os interpelados pela
Inquisição: eles tomaram parte dos rituais efetivamente, mas negam, ao fim, que tenham
realmente crido no ídolo indígena, sua ritualística ou espiritualidade. Tal posicionamento
é, sem dúvida, decorrente do lugar-de-fala dos confessores frente à uma mesa inquisitorial
com poderes reais de desestruturação de suas vidas, como multas, abjurações, auto-de-fé
e até mesmo deportações forçadas, como no caso de Fernão Taíde. No que tange ao conhecimento da língua indígena, há, inicialmente, o exemplo de
Fernão Ribeiro, “índio do Brasil” que, para se confessar, precisou da tradução do padre
Francisco Lemos, da Companhia de Jesus (Vainfas 1997:81-83). Os jesuítas, é sabido,
tiveram que inicialmente aprender as línguas dos indígenas do litoral e, mais tarde, adaptá-
las às narrativas bíblicas a fim de tentar evangelizar os nativos americanos na fé católica. Vainfas (1995) usa este fato reiteradamente para justificar o caráter “sincrético” das
Santidades: elas não eram tipicamente indígenas, mas uma junção de momento, rebelde,
da tradição Tupi-Guarani que se insubordinava contra o colonialismo. Este argumento
está presente em toda a tese de livre docência e será a base para a noção de “disjunção
social” (Vainfas 1995). Aqui, vejo-me em outro ponto de discordância com o historiador, este mais sutil
e de maior importância que o anterior. A questão da língua e da conversão jesuítica do
“gentio” à fé católica é central para o peso que Vainfas dá ao colonialismo lusitano e
extrapola a simples interpretação mais direta das confissões, tendo uma relação estreita
com os estudos da Sociolinguística. Sobre a questão, José Ribamar de Bessa Freire assevera que Durante todo o período colonial, no entanto, a língua portuguesa – cujas
categorias não davam inteligibilidade à realidade cultural e ecológica da
região – permaneceu minoritária, como língua exclusiva da administração,
mas não da população (Freire 2004: 16). Trabalhando na construção de um éthos americano para este período, não nos
parece absurdo supor que a língua franca, mesmo no litoral de ocupação lusitana, não
era o português. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Martiniano Sardeiro de Alcântara Neto Confessou mais, que no dito tempo que a dita abusão esteve na dita sua
fazenda, que poderia ser de dois meses pouco mais ou menos, ela tinha
para si, e dizia, que não podia ser aquilo demônio, senão alguma coisa
santa de Deus, pois traziam cruzes que o demônio foge, e pois faziam
grandes reverências às cruzes e traziam contas, e nomeavam Santa Maria
(Vainfas 1997: 170-171). Não há motivos para descreditar a confissão acima: além de não pesarem
diretamente sobre a confessora os interesses puramente materialistas do marido, ela
narra facetas que sabidamente compunham a ritualística indígena, como a famosa Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 202 Confessando o estranho Martiniano Sardeiro de Alcântara Neto Lusitana na América. Há de se lembrar, também, a dificuldade da Coroa Portuguesa em
trazer colonizadores cristãos de origem ibérica para o Novo Mundo: sem contar os de
caráter ‘empreendedor’, não é absurdo supor que boa parte deles não vinha de vontade
própria e eram degredados12. Lusitana na América. Há de se lembrar, também, a dificuldade da Coroa Portuguesa em
trazer colonizadores cristãos de origem ibérica para o Novo Mundo: sem contar os de
caráter ‘empreendedor’, não é absurdo supor que boa parte deles não vinha de vontade
própria e eram degredados12. Não estou argumentando, contudo, que o português não era falado no período
da Santidade. O que tento apontar é que havia uma diversidade linguística variadíssima
em todo o subcontinente hoje referido como Terras Baixas Sul-Americanas. O português
foi apenas, durante vários séculos, mais uma língua dentro dessa “babel” colonial (a
metáfora é do próprio Freire). E, é preciso ressaltar, não foi a língua franca do cotidiano,
principalmente quando tratamos das relações dos colonizadores lusitanos com as
populações nativas americanas no final do século XVI. Se o conhecimento e prática de uma língua é, como argumentam os linguistas,
algo que dá “inteligibilidade” ao mundo, uma complexa rede de classificação simbólica
própria e original, então os jesuítas é que foram obrigados a se submeter ao sistema
nativo americano de pensamento – e não o contrário. Sobre a questão, Vainfas argumenta,
por exemplo, que “no coração do culto [da Santidade], pontificava o ídolo, que só não era
totalmente indígena porque Tupanasu era uma invenção jesuítica expressa em ‘língua
geral’” (Vainfas 1995 :134). Assim, não considero nem Tupanasu, muito menos a língua
geral, uma “invenção” dos padres da Companhia de Jesus. Inicialmente porque as línguas,
em termos sociolinguísticos, não podem ser simplesmente inventadas, nem por um
indivíduo nem por um grupo de indivíduos determinados, como os jesuítas. Além disso
as línguas se adaptam (e, portanto, os sistemas de pensamentos e classificação do mundo
em geral se adaptam) e ‘fagocitam’ componentes exógenos, sejam eles novas palavras ou
mesmo novas séries de categorias de pensamento – sem, necessariamente, tornarem-se
outras coisas como “híbridos” ou “dilacerados”. Porém é preciso notar que Vainfas não ignora por completo tal dinâmica: Refiro-me, portanto, a um processo aculturador de mão dupla, e não à
simples assimilação dos valores ocidentais pelos nativos, tendência que
Nathan Watchel atribuiu a situações de aculturação imposta. 12 Uma excelente análise do degredo e da Inquisição lusitana no período colonial pode ser encontrada na
obra Os Excluídos do Reino, do historiador Geraldo Pieroni (2000). Confessando o estranho O Marquês de Pombal ainda não havia nascido e não existia uma política
organizada para a imposição desta variante do latim como idioma comum na Colônia Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Martiniano Sardeiro de Alcântara Neto 203 13 O Tupi, de maneira mais abrangente, é um tronco linguístico, do qual derivam várias famílias linguísticas
e, portanto, várias tradições de classificação do mundo ou línguas. Freire (2004) é enfático em dizer,
em toda sua análise, que não houve uma única língua geral, mas várias, diretamente relacionadas às
tradições nativas locais que os indígenas desenvolveram durante pelo menos 15 mil anos de ocupação
da América. Confessando o estranho Ainda que pese o uso não completamente adequado, ao menos no campo da
antropologia social atual, da categoria de cultura e seus correlatos (principalmente o termo
“aculturação”), Vainfas reconhece, nesta passagem, a complexidade do tema. Contudo,
sua análise não só dá maior peso à “doutrina e sacramento” da religiosidade católica,
praticamente ignorando o lastro linguístico nativo que torna o processo evangelizador
possível, mas principalmente descreve o uso de termos católicos na Santidade como signo
último do “hibridismo” ou “sincretismo” que seriam típicos à “rebeldia” americana. Aqui
está a caracterização final da Santidade de Jaguaripe na pena de Vainfas: Formação cultural híbrida, resultado da “colonização da língua tupi” pelos
jesuítas, e da superposição de imagens cristãs aos heróis indígenas no dia-
a-dia da catequese. Formação cultural híbrida pela adesão interesseira
e irrefreável dos mamelucos. Foram eles, sem dúvida, a ponte e o nexo
entre o mundo dos índios e dos brancos – além de serem exemplos
privilegiados da disjunção cultural que o colonialismo era capaz de gerar. Afinal, convém lembrar, os mamelucos eram a um só tempo seres híbridos
e dilacerados, homens que podem apresar índios para os escravocratas,
mas também podiam combatê-los (e até comê-los), como demonstram
as incisões de jenipapo que traziam em seus corpos (Vainfas 1995: 227). Antes de voltarmos à confissão de Tomacaúna, cabe comentar mais
pormenorizadamente esta polêmica passagem. Ainda que fosse factível a “colonização”
jesuítica da “língua tupi”13, Vainfas acaba se esquecendo, por fim, da “mão dupla” do
processo “aculturador”, ignorando a americanização relativa dos jesuítas e, penso eu,
dos habitantes lusitanos ou não-indígenas na América no seu quadro da Santidade como
movimento “rebelde”. Tal americanização é, sem dúvida, um dos componentes mais
explícitos da Santidade – ao menos na leitura que agora proponho. Voltemos, neste ponto, à confissão de Tomacaúna. Martiniano Sardeiro de Alcântara Neto No contexto
da catequese, não resta dúvida de que os nativos assumiram mensagens
e símbolos religiosos cristãos, sobretudo por meio das imagens, mas é
também certo que os jesuítas foram forçados a moldar sua doutrina e
sacramentos conforme as tradições tupis (Vainfas 1995:110). 12 Uma excelente análise do degredo e da Inquisição lusitana no período colonial pode ser encontrada na
obra Os Excluídos do Reino, do historiador Geraldo Pieroni (2000). Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 204 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 14 Vainfas se dedica a tentar provar que a tese de Pierre e Hèlene Clastres sobre a origem nativa do
profetismo tupi não tem amparo histórico e que os profetas nativos à procura da Terra Sem Mal seriam
resultado do “hibridismo” ou “sincretismo” com o cristianismo católico. Discordo em parte desta
interpretação, mas a refutação parcial deste argumento do historiador pediria um novo artigo – e uma
análise específica da teoria do Perspectivismo Ameríndio, da qual Vainfas se afasta (cf. nota 05). Martiniano Sardeiro de Alcântara Neto uns pós pretos, e depois de sarado, ficam os lavores pretos impressos nos
braços e nádegas, ou onde os põem, como ferretes, para sempre (Vainfas
1997 :348). uns pós pretos, e depois de sarado, ficam os lavores pretos impressos nos
braços e nádegas, ou onde os põem, como ferretes, para sempre (Vainfas
1997 :348). A passagem acima se dá no momento em que o confessor narra uma das suas
incursões para capturar índios no sertão no intuito de fazê-los escravos no litoral. O
“mestiço” havia passado cerca de meio ano em tal empreitada e as escarificações eram
plenamente visíveis à época de sua confissão. Vainfas acertadamente nota que tais
marcas indeléveis eram típicas daqueles que mataram outros guerreiros em batalha e,
possivelmente, ritualmente se alimentaram de seus corpos – o que era ignorado pelo
Santo Ofício. Ao que o historiador não dá a devida importância é que essas tatuagens
arcaicas eram comuns não só à Tomacaúna, mas a praticamente todos os homens que iam
ao sertão e tinham relações mais diretas com o “gentio da terra”. E não eram somente “mestiços” ou “mamelucos” que se dedicavam a esse serviço
e que, na empreitada, se “riscaram” (categoria dos inquisidores) para o resto da vida: o
ferreiro Gaspar Nunes Barreto, filho de pai cristão-velho e mãe que pensava ser também
cristã-velha, ex-senhor de engenho e morador de Itaparica, é um exemplo. Ele “estando em
Itaparica, se mandou riscar por um negro da terra na perna esquerda [...] o qual riscado
consentiu e mandou fazer em si sem nenhuma má intenção gentílica [...] o qual riscado é
que, com um dente agudo de um bicho que se fazem uns lavores rasgados na carne [...]”
(Vainfas 1997: 208-208). João Gonçalves, sob o qual parecia não existirem dúvidas sobre a condição de
cristão-velho, alfaiate e morador do Recôncavo, confessa que “haverá três anos que [...]
se fez riscar em um braço e logo mostrou o braço esquerdo, entre o cotovelo e o ombro,
riscado de lavores cortados na carne [...]” (Vainfas 1997: 263-267). Ferreiros, alfaiates
e possivelmente outros profissionais citadinos do litoral e imediações se riscaram
permanentemente, seguindo o costume dos nativos americanos. Assim, penso que, tanto
na cidade de Salvador, quanto também no Recôncavo baiano, não seria incomum achar
“gentios”, “mamelucos”, “mestiços”, mas também “brancos” riscados à moda indígena. Aqui cabe uma breve digressão da etnologia sobre a inscrição dos e nos corpos. Confessando o estranho [...] ele [Tomacaúna] tinha mulheres, duas, ao modo gentílico, as quais
eram gentias filhas de gentios que lhas davam por mulheres, e se tingia
ao seu uso gentílico, e bailava e cantava e tangia com os gentios ao seu
uso gentílico, e se riscou pelas coxas, nádegas e braços ao modo gentílico,
o qual riscado se faz rasgando com um dente de um bicho chama paca, e
depois de rasgar a carne levemente pelo couro, esfregam por cima com 13 O Tupi, de maneira mais abrangente, é um tronco linguístico, do qual derivam várias famílias linguísticas
e, portanto, várias tradições de classificação do mundo ou línguas. Freire (2004) é enfático em dizer,
em toda sua análise, que não houve uma única língua geral, mas várias, diretamente relacionadas às
tradições nativas locais que os indígenas desenvolveram durante pelo menos 15 mil anos de ocupação
da América. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 205 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Martiniano Sardeiro de Alcântara Neto Pierre Clastres14 argumenta que Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 206 Confessando o estranho Ora, uma cicatriz, um sulco, uma marca são indeléveis. Inscritos na
profundidade da pele, atestarão para sempre que, se por um lado a dor
pode não ser mais do que uma recordação desagradável, ela foi sentida num
contexto de medo e de terror. A marca é um obstáculo ao esquecimento,
o próprio corpo traz impressos em si os sulcos da lembrança – o corpo é
uma memória (Clastres 1978: 128, itálicos no original). Não quero dizer que todos os homens “riscados” que se confessaram na mesa
do inquisidor chefe Heitor Furtado de Mendonça compartilhassem desse profundo
simbolismo das escarifações. Mas não seria absurdo supor que alguns deles tivessem certa
ideia do que representavam as tatuagens que faziam, já que eles, além de se tatuarem,
potencialmente também dominavam (parcial ou fluentemente) a linguagem falada nativa. Tal faceta das escarificações como memória ‘impressa’ no corpo também escapa à Vainfas,
que as vê somente como símbolo do canibalismo do guerreiro tupi e, consequentemente,
da ignorância de Furtado de Mendonça desta última prática “gentílica”. Voltemos à Tomacaúna. Ele explica, na mesa do Santo Ofício, que os riscados [...] costumam fazer os gentios em si quando querem mostrar que são
valentes e que têm já mortos a homens, e por ele confessante se ver então
em um aperto dos gentios, que se levantavam contra ele, se fez riscar por
um negro do dito modo para se mostrar valente e assim escapou, porque
vendo isso os gentios lhe fugiram, e então se riscou com ele pela dita
maneira Francisco Afonse Capara, morador em Pirajá, termo desta cidade
(Vainfas 1997 :348). Há aqui não apenas indícios de que, novamente, foram os europeus que, ao menos
nos primórdios da colonização, se adaptaram, ainda que temporariamente, aos sistemas
de guerras vigentes na América – e não o contrário. A conhecida e já citada refrega entre
franceses aliados aos Tamoios e portugueses juntando armas aos Tupiniquim é o exemplo
histórico mais conhecido dessa dinâmica. Tomacaúna, então, fala de outras incursões ao sertão, onde novamente obteve
mulheres como esposas, fumava a “erva-santa” (tabaco, cf. nota 08) dos nativos, bebia
e dançava com os indígenas, tudo “para os gentios lhe darem bom tratamento” (Vainfas
1997: 349). Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Martiniano Sardeiro de Alcântara Neto Ele confessa também que deu armas aos “gentios”, inclusive para aqueles que
faziam guerra contra os colonos lusitanos (Vainfas 1997 :350). Como já dito, Domingos Fernandes (Tomacaúna) foi o líder da incursão de vários
homens que trouxe a Santidade ao engenho do nobre e rico Taíde. Ele fez, então, contato Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Martiniano Sardeiro de Alcântara Neto Martiniano Sardeiro de Alcântara Neto 207 pacífico com a maior liderança da “erronia” indígena: Antonio, referido como “papa” ou
mesmo “Deus” entre os “gentios” da própria Santidade (Vainfas 1997: 354). Tomacaúna
foi rebatizado e era chamado pelos nativos de “São Luís” (Vainfas 1997: 355). Encaminhando-me para uma conclusão, retomo aqui o argumento de que não
é impossível ver todos os exemplos aqui trazidos das confissões como imagens de um
cristianismo-católico hegemônico aculturando uma tradição de organização social nativa
e gerando um produto social “híbrido” ou “sincrético”. Contudo, não é essa a conclusão que
tive da leitura destas passagens acima e da documentação que a elas se correlacionam. Tais
passagens podem ser vistas no contrapelo da leitura de Vainfas e conformariam, assim,
exemplos explícitos da influência difusa ou éthos indígena sobre aqueles que estavam lá
para, ao menos teoricamente, lhes civilizarem ou colonizarem a partir do que, para os
últimos, inicialmente, era a verdadeira sociabilidade ou religião. Finalizando a presente seção, gostaria de aproveitar alguns férteis exemplos de
outros tipos de cristianismo, todos não católicos, que pululam nas confissões. Antônio
Guedes, “cristão-velho”, confessa que o tomaram os ingleses luteranos [categoria geral para o cristianismo
não-católico, cf. nota 08] no mar [...] e o trouxeram consigo [...] E ele
confessante, com medo dos ditos ingleses, algumas quatro ou cinco vezes
[...] se ajoelhou também e tirou o chapéu da cabeça (Vainfas 1997: 115). 15 Agradeço às antropólogas Alcida Rita Ramos e Nádia Farage a indicação e o seminal debate desta
e de uma bibliografia mais vasta sobre como levar a sério a agência nativa dentro de processos de
colonização. Os textos foram trabalhados durante a disciplina de Antropologia da Conquista, no PPGAS-
UnB, no ano de 2009. As conclusões, é claro, são de minha inteira responsabilidade. Confessando o estranho ocupação portuguesa. Vejo a Santidade de Jaguaripe como exemplo dessa ‘reverberação’ –
e não apenas como uma “rebeldia”. Tal influência nativo americana na sociabilidade geral
do subcontinente, inclusive entre os colonos lusitanos, não pode ser descartada em uma
análise sociológica mais densa dos primórdios do período colonial na História do Brasil. ocupação portuguesa. Vejo a Santidade de Jaguaripe como exemplo dessa ‘reverberação’ –
e não apenas como uma “rebeldia”. Tal influência nativo americana na sociabilidade geral
do subcontinente, inclusive entre os colonos lusitanos, não pode ser descartada em uma
análise sociológica mais densa dos primórdios do período colonial na História do Brasil. No mesmo mote, Nicolau Luís, francês, filho de franceses católicos, confessa que [...] haverá vinte e quatro anos, pouco mais ou menos que, indo ele
confessante em uma nau de seu pai de Bordéus para sua terra, em que
não havia portugueses senão todos franceses, foram tomados no mar
pelos franceses luteranos, os quais costumavam fazer suas salvas pela
manhã e à tarde luteranas na nau, e, espaço de mês e meio que com eles
andou, constrangido e com medo deles, ele confessante se ajoelhava e
desbarretava e estava com eles ditos luteranos quando eles faziam as
ditas salvas luteranas, porém nunca ele confessante as aprovou em seu
coração, nem lhes pareceu em, mas com medo dos ditos luteranos se
punha com eles no tempo que eles as faziam (Vainfas 1997 :156). Esse é o contexto da colonização lusitana no final do XVI, ao menos o que podemos
inferir a partir dos documentos inquisitoriais: um cristianismo católico não hegemônico,
não só pela constante competição com outros colonizadores europeus, mas também pela
franca e direta influência nativa no sertão, que ‘reverberava’ constantemente no litoral de Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 208 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Martiniano Sardeiro de Alcântara Neto 209 sendo interpelado e cobrado a dizer explicitamente onde realmente jazia sua fé, mas isso
só quando em contato com a Inquisição lusitana em 1592. Relembro, ainda neste ponto, a
descrição que faz Capistrano sobre o fato de vários colonos europeus terem preferido se
‘indianizar’ a retomar o contato com suas nações de origem, citada acima. Este último fato
pode ser visto como uma negação de se reincorporar ao cristianismo e aos seus dogmas
específicos. Por fim, cito como dado básico o fato de que a noção de “heresia” como
desvio de fé é típica das religiões de conversão: um caraíba tupi dificilmente seria um
heresiarca quando tomado como pano de fundo seu próprio contexto sociocultural, onde
potencialmente a própria divisão entre corpo e alma não opera como no cristianismo. Argumento novamente que essa faceta escapa à análise de Vainfas. O profeta
maior da Santidade, conhecido como Antonio, fugiu de um aldeamento da Companhia de
Jesus. Ele era chamado de “papa” por seus seguidores, renomeou Tomacaúna como “São
Luís” – e sua companheira, uma co-liderança, lhe era a “Virgem Maria”. A Santidade de
Jaguaripe realizava o rebatismo daqueles que a seguiam e dizia que os desviantes haviam
de se transformar em paus e pedras. Isso não é, necessariamente, um signo de “disjunção
social”: é, ao meu ver, um exemplo clássico do ‘laboratório sociológico’ nativo, que tentava
experimentar o cristianismo católico a seu modo – ainda que, aqui seja impossível delimitar
exatamente este modo. Como movimento histórico determinado, concordo com Vainfas que a Santidade
de Jaguaripe se finda com a destruição do ídolo de pedra (mas não dos ídolos-maracás)
e do barracão onde fora instalada na fazenda de Fernão Taíde. Os índios fugidos, que
seriam certamente escravizados por esse ignóbil senhor de engenho, foram devolvidos
aos seus “proprietários” de origem. Sobre sua fama de péssima pessoa, Taíde queimou,
ainda viva, uma mulher indígena grávida, sua escrava, no forno de seu engenho, entre
outras atrocidades (Vainfas 1997). Ronaldo Vainfas interpreta isso tudo como “o triunfo
do colonialismo, ao menos do seu “sentido mercantil” (Vainfas 1995 :228). Onde o historiador vê “triunfo” ou a “máxima vitória do sistema colonial” (Vainfas
1995: 229), prefiro pensar na existência de uma disputa hegemônica em curso. As confissões como agência indígena O casal de antropólogos Jean and John Comaroff15, ainda que analisando um
contexto colonial diferente deste sobre o qual agora me debruço, argumentam que a
hegemonia ou a proeminência social de determinado grupo humano sobre outro, em
contextos de colonização, tem sua força “in what it silences, what it prevents people from
thinking and saying, what it puts beyond the limits of rational and credible” (Comaroff &
Comaroff 1991: 23). Assim, a luta por hegemonia, pela dominação ou colonização, não é
tão somente uma luta bélica ou de controle das condições materiais de reprodução social:
é uma batalha pelo domínio eficaz dos símbolos, do vocabulário, da linguagem em que
o embate de tradições sociais diferentes e conflituosas se dão. Neste sentido, como bem
lembra Vainfas em toda sua análise das confissões, a última coisa que os inquisidores
pensaram ao “visitar” a América seria uma interpretação indígena do paradigma cristão-
católico, com “papas”, “santos” e até mesmo uma própria “Virgem Maria”. Essa faceta da
Santidade de Jaguaripe pode ser vista, portanto, como a inserção do vocabulário cristão-
católico dentro de um sistema de significação não europeu. A justificação simbólica, como já dito na primeira seção, era responsabilidade
última da Igreja Católica para o caso lusitano. O caso indígena é muito mais complexo e
diverso, já que a tradição de organização social nativo-americana muitas vezes não se fez
e, até hoje, não se faz afeita às centralizações típicas dos processos colonizadores do Velho
Continente (nem tão pouco por registrar isto em códigos escritos). Contudo, é possível
dizer que a gana pela reforma completa da alma e do corpo, o arrependimento e a fundação
de uma nova vida característicos das religiões comumente chamadas de religiões de
conversão, como os cristianismos e islamismos, parecem não estar presentes no contexto
indígena, ao menos com a mesma força ou fundamento religioso. A maior prova desta
faceta está na própria capacidade de, por exemplo, uma pessoa como Tomacaúna, entre
outros, poder circular entre as aldeias indígenas e a colônia lusitana de Salvador. Ele acaba Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Martiniano Sardeiro de Alcântara Neto Para os
indígenas, a tentativa de impor o que e como se deveria pensar ou fazer, a construção
e a obrigação de respeitar os limites (recentemente) pré-determinados, fossem eles
geográficos (as capitanias, por exemplo) ou de pensamento (a filosofia platônica embutida
no cristianismo, por exemplo), parecia não ser minimamente aceitável. Então, o caraíba
Antonio perfazia seus batismos, se auto-denominava “papa” ou “Deus”, renomeava seus
companheiros com o panteão católico e, enfim, colocava a ortodoxia do Velho Continente
de pernas para o ar. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 210 Confessando o estranho A reverberação desse éthos americano no litoral não se finda com o desbaratamento
da Santidade de Jaguaripe, o julgamento e consequente degredo de Taíde e a devolução
dos nativos à escravidão. Dois anos depois da destruição da “igreja dos índios”, tem-se
notícia que haverá mais de três mil índios que se têm feito fortes e fazem insultos
e danos nas fazendas de meus vassalos daquelas partes , recolhendo a
si todos os negros da Guiné que andam alevantados e impedem poder-
se caminhar por terra de umas capitanias a outras, vos encomendo
que, podendo desarraigar daquele lugar este gentio e dar-lhe o castigo
que se merece, pelos portugueses e mais gente que mataram, o façais...”
(Francisco Giraldes apud Vainfas 1995 :222). Não há como negar, portanto, que as santidades, de maneira geral, continuaram
sendo um forte empecilho à colonização litorânea que os portugueses gostariam de ver
realizada ou acabada. Contudo, uma tentativa de colonizar corpos e mentes a partir de
uma tradição cristã específica não é uma “vitória máxima do colonialismo”, ao menos para
o período histórico e a região geográfica em questão. Se é possível ler uma massa documental de diversas maneiras, como apontado
anteriormente, tentei interpretar as confissões inquisitoriais dando maior ‘luz’ à ação
política específica dos nativos americanos. Era o sistema guerreiro nativo em operação
que fazia com que os homens se marcassem, pelo resto da vida, com os “lavores” indígenas. Era dentro de uma religiosidade e espiritualidade não católicas que os colonos, mesmo os
nobres senhores de engenho, visitavam a “Virgem Maria” e pagavam respeito aos ídolos
indígenas (que só seriam demonizados mais tarde, pela Inquisição). Enfim, não foi o
cristianismo-católico europeu que fez surgir, em plena América quinhentista, um “papa”
ou os “santos” do Velho Mundo incorporados nos indígenas da Santidade. Todos esses
exemplos são, portanto, influências diretas da sociabilidade indígena dentro da colônia
lusitana pensada como exemplarmente cristã-católica. Uma das consequências ou das críticas que potencialmente podem ser feitas
dessa leitura é que, por exemplo, não dou a devida importância à violência bélica trazida
pelos lusitanos, ignorando também o impacto dos microrganismos que, em conjunto,
prenunciaram uma espécie de apocalipse na América indígena. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Martiniano Sardeiro de Alcântara Neto Martiniano Sardeiro de Alcântara Neto 211 uma espécie de crônica de sua extinção. Se é verdade que o impacto do
contato sobre as populações nativas foi negativo em todos os quadrantes
das Américas, o problema central não deve se limitar à dizimação
(Monteiro 1992 :479). Finalizando, o próprio Santo Ofício não ignorou o que chamo de reverberação de
um éthos americano. Assim, Capistrano de Abreu nos dá acesso à seguinte informação,
adicionada ao Monitório Inquisitorial depois que Heitor Furtado e sua comitiva já estavam
em terras americanas: — Segue-se algumas Determinaçãis q. se asentaram nesta mesa alguns
casos q nella se trataram “Tratando se nesta Mesa de incurrião na
Excomunhão da Bulla da Cea os que dão Arinas a Estes gentios Brazis
deste Brazil que tem guerra com os brancos e com os jndios Xpãos. Asentou se que não se comiprehendem na dita Bulla estes gentios, por
quanto não são inimigos do nome de Xpo como são os turcos & mouros
etc. E não faze guerra aos Xpão por respeito de serem Xpãos em ódio do
nome Xpão senão por outros Respeitos, differetes na Baya, 29 de julho
de 1593. — O Bispo — Heitor furtado de mendoça. — fernão Cardim. — lionardo Arminio. [...]” [Xpo: abreviação, do grego, para Cristo. Xpão:
cristão, por derivação no português.] (Abreu 1922: 46). — Segue-se algumas Determinaçãis q. se asentaram nesta mesa alguns
casos q nella se trataram “Tratando se nesta Mesa de incurrião na
Excomunhão da Bulla da Cea os que dão Arinas a Estes gentios Brazis
deste Brazil que tem guerra com os brancos e com os jndios Xpãos. Asentou se que não se comiprehendem na dita Bulla estes gentios, por
quanto não são inimigos do nome de Xpo como são os turcos & mouros
etc. E não faze guerra aos Xpão por respeito de serem Xpãos em ódio do
nome Xpão senão por outros Respeitos, differetes na Baya, 29 de julho
de 1593. — O Bispo — Heitor furtado de mendoça. — fernão Cardim. — lionardo Arminio. [...]” [Xpo: abreviação, do grego, para Cristo. Xpão:
cristão, por derivação no português.] (Abreu 1922: 46). Ou seja, o Santo Ofício é obrigado a reconhecer que os nativos americanos não se
enquadravam exatamente na categoria clássica de “herege”, como o “luteranismo” ou os da
“seita de Bafomé” (categoria inquisitorial para os muçulmanos). Confessando o estranho São escolhas que fazemos
e eu justifico a minha a partir de uma reflexão de John Manuel Monteiro: Ao adotar esta perspectiva [de uma “pavorosa tragédia demográfica” de
John Hemming], corre-se o risco de apresentar a história dos índios como Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Confessando o estranho de engenho, El Rei, ou mesmo um influente caraíba tupi. Finalizando, o ‘enrijecimento’
desse colonialismo se deu, não há dúvida, em contraposição à ‘reverberação’ do éthos
indígena. Referências ABREU, João Capistrano de. 1922. “Um visitador do Santo Officio á cidade de Salvador e ao
Reconcavo da Bahia de Todos os Santos (1591-1592)”, “Monitiorio [..]” e determinações
pós-Monitório” . Separata da Serie Eduardo Prado para melhor conhecer o Brasil. Rio de
Janeiro: Jornal do Commercio. ALCANTARA NETO, 2007. O Caso Haximu: a construção do crime de genocídio em um
processo criminal. Dissertação de Mestrado. Programa de Pós-Graduação em Antropologia
Social do Museu Nacional, Universidade Federal do Rio de Janeiro. ______. 2016. Educação Formal e Saúde Alopática entre os Tapayúna (Kaykwkhratxi). Tese de
doutorado. Programa de Pós-Graduação em Antropologia Social, Universidade de Brasília. ______. 2016. Educação Formal e Saúde Alopática entre os Tapayúna (Kaykwkhratxi). Tese de
doutorado. Programa de Pós-Graduação em Antropologia Social, Universidade de Brasília. CARNEIRO DA CUNHA, Manuela. 1992 . “Introdução a uma história indígena”. In: M. Carneiro da Cunha (ed.), História dos índios no Brasil. São Paulo: Companhia das Letras/
SMC/FAPESP. pp. 9-24. CARNEIRO DA CUNHA, Manuela. 1992 . “Introdução a uma história indígena”. In: M. Carneiro da Cunha (ed.), História dos índios no Brasil. São Paulo: Companhia das Letras/
SMC/FAPESP. pp. 9-24. CLASTRES, Pierre. 1978. A sociedade contra o Estado: pesquisas de antropologia política. Rio de Janeiro: Francisco Alves. CLASTRES, Pierre. 1978. A sociedade contra o Estado: pesquisas de antropologia política. Rio de Janeiro: Francisco Alves. ENEDICT, Ruth. 1960. Patterns of culture. New York: The New American Library. COMAROFF, Jean; COMAROFF, John. 1991. Of revelation and revolution. Chicago: University
of Chicago Press. COMAROFF, Jean; COMAROFF, John. 1991. Of revelation and revolution. Chicago: University
of Chicago Press. CARDOSO, Andrey Moreira. 2010. Doença respiratória aguda em indígenas Guarani no
Sul e Sudeste do Brasil. Tese de doutorado. Escola Nacional de Saúde Pública, Fundação
Oswaldo Cruz do Rio de Janeiro. FREIRE, José Ribamar Bessa. 2004. Rio Babel. Rio de Janeiro: Atlântica. GONÇALVES, Marco Antonio. 1995. “Etno-história de uma resistência”. Revista Estudos
Históricos, 8(15):161-169. MATA, Sérgio da. 1996. “Resenha de A Heresia dos Índios”. Revista Varia História, 2(16):171-
177. MONTEIRO, John Manuel. 1992 “Os Guarani e a História do Brasil Meridional”. In: M. Carneiro da Cunha (ed.), História dos Índios no Brasil. São Paulo: Companhia das Letras/
SMC/FAPESP. pp. 475-498. POMPA, Cristina. 2004. “O profetismo tupi-guarani: a construção de um objeto
antropológico”. Revista de Índias, LXIV(230):141-174. PIERONI, Geraldo. 2000. Os Excluídos do Reino. Brasília: Universidade de Brasília. PIGNATTI, Marta G. 2004. “Saúde e ambiente: as doenças emergentes no Brasil”. Martiniano Sardeiro de Alcântara Neto Estes últimos conheciam
o cristianismo católico há alguns séculos e, ainda assim, não o reconheciam como doutrina
ideal. Tal fato não deve ser menosprezado: ele é uma ‘confissão’, ainda que parcial, dos
próprios inquisidores sobre a alteridade americana e suas especificidades chocantes. Os “caboclos” ou “mamelucos”, Ronaldo Vainfas classifica como “interesseiros”,
“irrefreáveis” e “dilacerados” pelo colonialismo (Vainfas 1995: 227), já que eles capturavam
índios para servirem como escravos nos engenhos do litoral e, ao mesmo tempo,
mostravam-se típicos guerreiros nativos, tatuando-se, comendo carne dos vencidos e
dando armas aos seus pares do sertão. Talvez esses “dilacerados” agissem de tal forma
não porque estivessem perdidos pela força do colonialismo em curso, mas justamente
pelo fato de que o próprio colonialismo, com seus determinismos característicos, ainda
não tivesse fincado raízes fortes na América quinhentista. Possuíam, portanto, a liberdade
de não servir exclusivamente a potentado nenhum, fosse ele um poderoso e nobre senhor Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 212 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 Referências Revista
Ambiente e Sociedade, VII(1):133-147. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020 VAINFAS, Ronaldo. 1995. A heresia dos índios: catolicismo e rebeldia no Brasil colonial. São
Paulo: Companhia das Letras. VAINFAS, Ronaldo (org.). 1997. Confissões da Bahia: o Santo Ofício da Inquisição de Lisboa. São Paulo: Companhia das Letras. VIVEIROS DE CASTRO, Eduardo. 2002. A inconstância da alma selvagem e outros ensaios
de antropologia. São Paulo: Cosac Naify. Recebido em 22 de julho de 2019. Aceito em 08 de dezembro de 2020. Martiniano Sardeiro de Alcântara Neto Martiniano Sardeiro de Alcântara Neto 213 Recebido em 22 de julho de 2019. Aceito em 08 de dezembro de 2020. Revista de @ntropologia da UFSCar, 12 (2), jul./dez. 2020
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Predictive Value of Adiposity Level, Metabolic Syndrome, and Insulin Resistance for the Risk of Nonalcoholic Fatty Liver Disease Diagnosis in Obese Children
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Canadian journal of gastroenterology & hepatology
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Hindawi
Canadian Journal of Gastroenterology and Hepatology
Volume 2018, Article ID 9465784, 8 pages
https://doi.org/10.1155/2018/9465784 Hindawi
Canadian Journal of Gastroenterology and Hepatology
Volume 2018, Article ID 9465784, 8 pages
https://doi.org/10.1155/2018/9465784 Hindawi
Canadian Journal of Gastroenterology and Hepatology
Volume 2018, Article ID 9465784, 8 pages
https://doi.org/10.1155/2018/9465784 School of Medicine in Katowice, Department of Pediatrics and Pediatric Endocrinology, Medical University of Silesia, Katowice, Poland School of Medicine in Katowice, Department of Pediatrics and Pediatric Endocrinology, Medical University of Silesi School of Medicine in Katowice, Department of Pediatrics and Pediatric Endocrinology, Medical University of Silesia, Katowice, Poland Correspondence should be addressed to Pawel Matusik; endocrin@wp.pl Received 14 January 2018; Accepted 8 April 2018; Published 26 April 2018 Academic Editor: Branka Filipovi´c Copyright © 2018 Zofia Prokopowicz 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. Background. Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in obese children. Early
diagnosis and treatment are essential for curing or slowing down the disease progression. The aim of the study was to assess the
prevalence of NAFLD in this population and to identify anthropometrical and metabolic risk factors for NAFLD prediction and
its development. Material and Methods. The study included 108 obese children. Anthropometric measurements, NAFLD diagnosis
(based on ALT level and/or liver ultrasound), and metabolic syndrome (MS) components were assessed in all patients. Patients
were divided into groups with and without NAFLD. Results. NAFLD was diagnosed in 49 (45%) patients with similar prevalence
in boys (27; 55.10%) and girls [22 (44.9%), 𝑝= 0.089]. NAFLD patients had significantly greater waist circumference, WHR, and
WHtR and significantly higher total cholesterol, triglyceride, and fasting insulin concentrations as well as higher glucose and insulin
concentrations in 120 minutes of OGTT and higher HOMA-IR levels compared to group of patients without NAFLD. In NAFLD
patients, MS was significantly more likely to be diagnosed than in group without NAFLD (40.82% versus 22.81%, 𝑝= 0.04), but
among the MS components only hypertriglyceridemia was significantly more frequently diagnosed in the group with NAFLD
(𝑝= 0.002). Among analysed parameters the best independent risk factor for NAFLD was fasting insulin concentration with the
cut-off point = 18,9 uIU/ml (AUC = 0.829). Conclusions. NAFLD is a very common disease in obese children. NAFLD predictive risk
factors include increased waist circumference, elevated WHR and WHtR, and elevated total cholesterol, triglycerides, and fasting
insulin as well as elevated glucose and insulin concentration in the OGTT and HOMA-IR index. NAFLD increases the risk of
potential cardiovascular complications expressed by diagnosis of metabolic syndrome. The best independent predictive risk factor
for diagnosing NAFLD in obese children is fasting insulin > 18.9 uIU/ml. 2. Materials and Methods 2.1. Study Population. The study was prospective and includ-
ed 108 children aged 6 to 18, hospitalized in our department
between years 2012 and 2014, whose BMI exceeded 97 pc
for sex and age, after informed consent. Children with acute
infectious disease, chronic hepatitis of known etiology, using
hepatotoxic drugs, or consuming alcohol were excluded from
the study. A general medical examination, anthropometric
measurements, laboratory tests, blood pressure measure-
ments, and abdomen ultrasound imaging were performed in
all subjects. 2.2. Anthropometric Evaluation and Body Composition. All
anthropometric measurements were made in the morning,
with fasting with empty bladder in the upright position. Subjects were barefoot and lightly dressed. Height and weight
were measured to the nearest 0,1 cm and 0,1 kg, respectively,
by using calibrated scale and Harpenden stadiometer. Waist
and hip circumference were measured to the nearest 0,5 cm
using standard technique with nonelastic tape, at the end of
normal expiration. Waist circumference (WC) was measured
at a point midway between the lower border of the ribs
and the iliac crest, and hip circumference was measured at
the widest part of the hip. BMI, waist-hip ratio (WHR),
and waist-to-height ratio (WHtR) were calculated. Stan-
dard deviations scores for height and BMI were calculated
using the LMS method based on Polish reference values
[17]. 2.2. Anthropometric Evaluation and Body Composition. All
anthropometric measurements were made in the morning,
with fasting with empty bladder in the upright position. Subjects were barefoot and lightly dressed. Height and weight
were measured to the nearest 0,1 cm and 0,1 kg, respectively,
by using calibrated scale and Harpenden stadiometer. Waist
and hip circumference were measured to the nearest 0,5 cm
using standard technique with nonelastic tape, at the end of
normal expiration. Waist circumference (WC) was measured
at a point midway between the lower border of the ribs
and the iliac crest, and hip circumference was measured at
the widest part of the hip. BMI, waist-hip ratio (WHR),
and waist-to-height ratio (WHtR) were calculated. Stan-
dard deviations scores for height and BMI were calculated
using the LMS method based on Polish reference values
[17]. 2.5. Blood Pressure Measurements. Blood pressure (BP) was
measured three times daily by the Korotkoff method using
sphygmomanometer, on the right arm after 5–10 minutes
of rest in the sitting position. The results were referred
to Polish children reference values [20]. Canadian Journal of Gastroenterology and Hepatology 2 metabolic rate per kilogram body weight (BMR/kg), and
standard deviations scores for fat percentage (fat%𝑍-score =
2 ∗(fat% −50 pc fat%)/(98 pc fat% −50 pc fat%)) based on
children reference values [18]. liver disease can precede with type 2 diabetes and metabolic
syndrome and may even be a risk factor for their develop-
ment [14, 15]. Due to biopsy limitations, hepatic steatosis is
also diagnosed with biochemical tests like elevated alanine
transaminase, or imaging methods, especially ultrasound. Despite the limitations, ultrasonography is characterized
by high sensitivity (60–94%) and specificity (84–100%) for
NAFLD detection [16]. Early diagnosis in the asymptomatic
period and effective therapy implementation enable curing
or, at least, slow down the progression of the disease.h 2.3. Liver Ultrasonography. Liver ultrasound examination
was performed by two radiologists with Siemens Acuson
Antares, convex transducer 5 MHz. Fatty liver was diagnosed
on the basis of increased echogenicity of the liver parenchyma
compared to the right kidney echogenicity. The aim of the study was to assess the prevalence of
NAFLD, as well as identifying additional predictive anthro-
pometrical and metabolic risk factors for NAFLD develop-
ment in the obese children. 2.4. Laboratory Assessment. Fasting venous blood sample was
taken in the second day of hospitalization. Blood chemistry
analyses, alanine transaminase (ALT), gamma glutamyltrans-
ferase (GGT), total cholesterol (Tch), high density lipoprotein
(HDL), low density lipoprotein (LDL), triglycerides (TG),
and bilirubin, were performed in hospital laboratory by
using standard methods. Oral glucose tolerance test (OGTT;
1.75 g/kg body weight, up to 75 g) was performed in all
patients. Glucose and insulin concentration were measured
during fasting and in 120 minutes of the test. In this study,
elevated ALT levels were identified for ALT > 35 U/l in
children aged 3–11 years and ALT > 40 U/l in children aged ≥
11 years. Hypertriglyceridemia was defined as fasting triglyc-
erides ≥150 mg/dl, and low HDL cholesterol as fasting HDL <
40 mg/dl in children aged < 16 years and <40 mg/dl for boys
or <50 mg/dl for girls aged > 16 years [19]. Impaired fasting
glucose (IFG) was defined as fasting glucose ≥100 mg/dl. 1. Introduction and its progression are complex process, called multiple-
hit theory combining environmental (dietary habits, phys-
ical activity [3, 4]), molecular (lipotoxicity [5]), endoplas-
mic reticulum stress [6], mitochondrial dysfunction [7],
organokines effect [8, 9], genetic (polymorphism involved in
the onset and progression of the disease), and other factors
like dysbiosis [7, 10, 11]. Nonalcoholic fatty liver disease (NAFLD) is the most com-
mon cause of chronic liver disease in children [1]. Due to the
growing number of obese children the prevalence of fatty liver
increases, and it was assessed in around one-third of clinical
population [2]. NAFLD is defined as hepatic fat infiltration
> 5% of hepatocytes, assessed by liver biopsy, after exclusion
of excessive alcohol intake and other liver pathologies. In
children NAFLD is, usually at the time of diagnosis, a
simple steatosis, which is reversible, but also can initiate
further stages of disease, such as nonalcoholic steatohepatitis
(NASH), fibrosis, and cirrhosis. The pathogenesis of NAFLD NAFLD not only in adults but also in children is asso-
ciated with severe metabolic disorders as hypertension, dys-
lipidaemia, increased risk of type 2 diabetes, metabolic syn-
drome, and cardiovascular diseases [12, 13]. For many years,
NAFLD has been perceived as a hepatic consequence of
insulin resistance, but recent studies have shown that fatty Canadian Journal of Gastroenterology and Hepatology 2. Materials and Methods Variable
All (𝑁= 108)
Males (𝑁= 50)
Females (𝑁= 58)
𝑡test
Mean
SD
Mean
SD
Mean
SD
𝑝value
BMI 𝑧-score
2.40
0.48
2.36
0.47
2.43
0.50
0.463
height 𝑧-score
0.42
1.4
0.60
1.55
0.27
1.26
0.230
WC (cm)
103.9
12.9
108.17
12.06
100.16
12.49
0.001
Hip circumference (cm)
108
11.3
108.12
11.47
107.90
11.29
0.922
WHR
0.96
0.07
1.0
0.06
0.93
0.07
<0.001
WHtR
0.63
0.06
0.64
0.06
0.62
0.06
0.073
Fat%
39.27
6.79
37.17
7.36
41.08
5.71
0.002
Fat% 𝑧-score
3.06
1.03
2.84
0.87
3.31
1.14
0.017
Visceral fat/fat%
0.46
0.45
0.44
0.45
0.48
0.45
0.769
BMR (kJ)
7851
1233
8681
1056
7135
878
<0.001
BMR/kg (kJ/kg)
92
13.94
96.4
13.7
88.5
13.17
0.003 Table 1: Anthropometric characteristic of studied population. 3
2
4
8
18
23
2
2
7
8
19
12
0
5
10
15
20
25
6–8
8–10
10–12
12–14
(years)
14–16
16–18
Age structure of the studied population
p < 0,05 2.7. NAFLD Diagnosis. NAFLD was diagnosed based on
ultrasound examination and/or elevated ALT. 2.8. Statistical Analysis. The statistical software (Statistica
v. 12.5 and Microsoft Excel) was used in data analysis. Descriptive data were expressed as median, mean values, and
standard deviations (SD) for continuous variables. Patients
were divided into two groups: with and without NAFLD,
which were then compared for anthropometric features,
biochemical parameters, and metabolic syndrome compo-
nents. Mann–Whitney U, ANOVA Kruskal-Wallis, Spearman
rank correlation tests were used to compare continuous
variables. Chi-square test was used for qualitative variables. The receiver operating characteristics (ROC) analysis was
used to verify the characteristics of the independent variables
and to assess an appropriate cut-off. Statistical significance
was set at 𝑝value less than 0.05. Figure 1: Sex distribution in particular age groups. diagnosed in 34 patients (16 girls and 18 boys) and elevated
ALT in 30 patients (8 girls and 22 boys). In 15 patients (2 girls
and 13 boys) both were reported. Based on proposed criteria,
two groups were identified: (I) NAFLD patients (𝑁= 49;
45.4%) and (II) non-NAFLD patients with normal liver and
normal ALT concentration (𝑁= 59; 54.6%).t 2. Materials and Methods Variable
All (𝑁= 108)
Males (𝑁= 50)
Females (𝑁= 58)
𝑡test
Mean
SD
Mean
SD
Mean
SD
𝑝value
BMI 𝑧-score
2.40
0.48
2.36
0.47
2.43
0.50
0.463
height 𝑧-score
0.42
1.4
0.60
1.55
0.27
1.26
0.230
WC (cm)
103.9
12.9
108.17
12.06
100.16
12.49
0.001
Hip circumference (cm)
108
11.3
108.12
11.47
107.90
11.29
0.922
WHR
0.96
0.07
1.0
0.06
0.93
0.07
<0.001
WHtR
0.63
0.06
0.64
0.06
0.62
0.06
0.073
Fat%
39.27
6.79
37.17
7.36
41.08
5.71
0.002
Fat% 𝑧-score
3.06
1.03
2.84
0.87
3.31
1.14
0.017
Visceral fat/fat%
0.46
0.45
0.44
0.45
0.48
0.45
0.769
BMR (kJ)
7851
1233
8681
1056
7135
878
<0.001
BMR/kg (kJ/kg)
92
13.94
96.4
13.7
88.5
13.17
0.003
2.7. NAFLD Diagnosis. NAFLD was diagnosed based on
ultrasound examination and/or elevated ALT. 2.8. Statistical Analysis. The statistical software (Statistica
v. 12.5 and Microsoft Excel) was used in data analysis. Descriptive data were expressed as median, mean values, and
standard deviations (SD) for continuous variables. Patients
were divided into two groups: with and without NAFLD,
which were then compared for anthropometric features,
biochemical parameters, and metabolic syndrome compo-
nents. Mann–Whitney U, ANOVA Kruskal-Wallis, Spearman
rank correlation tests were used to compare continuous
variables. Chi-square test was used for qualitative variables. The receiver operating characteristics (ROC) analysis was
used to verify the characteristics of the independent variables
and to assess an appropriate cut-off. Statistical significance
t t
l
l
th
0 05
3
2
4
8
18
23
2
2
7
8
19
12
0
5
10
15
20
25
6–8
8–10
10–12
12–14
(years)
14–16
16–18
Age structure of the studied population
number of girls
number of boys
p < 0,05
Figure 1: Sex distribution in particular age groups. Table 1: Anthropometric characteristic of studied population. 2. Materials and Methods Patients with at
least two measurements > 90 pc (high normal pressure) and
patients with previously treated hypertension performed 24-
hour ambulatory blood pressure monitoring using the Space-
labs ABP. Hypertension was diagnosed when the systolic
blood pressure was above 95 pc for more than 25% of the
measurements. 2.6. Insulin Resistance and Metabolic Syndrome (MS). Insulin
resistance was measured by homeostasis model assessment of
insulin resistance (HOMA-IR) described by Matthews et al. [21], defined as follows: HOMA-IR = fasting glucose (mg/dl)
× fasting insulin (uIU/ml)/405. The outcomes were referred
to Caucasian obese children reference values [22].i Body composition was evaluated by bioelectrical impe-
dance using the Tanita MC 980 MA device-multifrequency
segmental analyser. Estimates of body composition were
obtained from the equipped software. Patient stand barefoot
on the marked electrodes and in straightened hands held the
handles equipped with electrodes. The measurement of the
tissue impedance through which the six-frequency (1 kHz,
5 kHz, 50 kHz, 250 kHz, 500 kHz, and 1000 kHz) and low
intensity (<90 𝜇A) current was applied is painless and lasts
for several seconds. Of the many variables, the following
results were analysed: total body water in kg and % (TBW),
fat mass in kg and fat%, fat-free mass in kg (FFM), muscle
mass in kg, visceral fat in %, and basal metabolic rate in kJ
(BMR). Moreover the following indicators were calculated:
visceral fat to total body fat (visceral fat%/fat%), basal We used the definition of the International Diabetes
Federation (IDF) for the metabolic syndrome in children and
adolescents [19]. The first criterion reached in all our patients
was central obesity defined as WC > 90 pc for age and sex
or above adult norms (>80 cm for women and >94 cm for
men). If patients reached at least 2 of the other 4 criteria (TG
concentration ≥150 mg/dl or hypolipemic treatment; HDL
levels < 40 mg/dl (for girls > 16 years, HDL < 50 mg/dl);
elevated blood pressure (systolic > 130 mmHg or diastolic
> 85 mm Hg) or hypertension; fasting glucose ≥100 mg/dl;
or type 2 diabetes) then the metabolic syndrome was diag-
nosed. Canadian Journal of Gastroenterology and Hepatology Canadian Journal of Gastroenterology and Hepatology 3 Table 1: Anthropometric characteristic of studied population. 3. Results The study included 108 obese children (50 boys) aged 6
years and 2 months to 17 years and 10 months. Mean
age was 14.24 ± 2.73 SD. Mean age of girls was 14.75 ±
2.79 SD and of boys 13.9 ± 2.64 SD. Girls were about one
year older than boys, but the difference was not statistically
significant (𝑝= 0.074). The age distribution of the examined
group is shown in Figure 1. The only statistically significant
difference was observed in the 16–18-year age group, where
girls predominated. Table 1 gives the anthropometric and
body composition characteristic of the study population
stratified by gender. Males (𝑛
=
50) and females (𝑛
=
58) had similar BMI and height 𝑍-scores while males had
significantly higher mean WC and WHR. Among important
anthropometrical data for obesity diagnosis, parameters of
body composition males had significantly higher fat% and
fat%𝑍-score; however no difference in visceral fat/fat% was
observed. NAFLD was diagnosed more often in boys than in girls;
however the difference was insignificant (55.1% versus 44.9%;
𝑝= 0.089). 3.2. Anthropometry and Body Composition. NAFLD patients
did not differ significantly from patients without NAFLD in
age, height, body weight, BMI, or BMI 𝑍-score, while NAFLD
was associated with a significantly greater waist circumfer-
ence, WHR, and WHtR. Body composition analysis did not
show statistically significant differences between groups in
key obesity parameters such as fat mass, fat%, fat% 𝑍-score,
or visceral fat%. Groups differed significantly only in fat-free
mass (FFM), total body water (TBW), and basal metabolic
rate (BMR); however BMR per kilogram remained similar in
both groups. The comparison of the two groups in terms of
basic anthropometric and body composition parameters is
presented in Table 2. 3.1. NAFLD Prevalence. NAFLD was diagnosed in patients
with hyperechogenic liver on ultrasound and/or elevated ALT
concentration. Ultrasonographic features of fatty liver were Canadian Journal of Gastroenterology and Hepatology Canadian Journal of Gastroenterology and Hepatology 4 Table 2: Anthropometry and body composition. Comparison of patients with and without NAFLD. 3. Results Variable
NAFLD (𝑁= 49)
Patients without NAFLD (𝑁= 59)
𝑝value (Mann–Whitney 𝑈test)
Mean
SD
Median
Mean
SD
Median
Age (years)
14.40
2.49
14.83
14.17
2.94
14.92
0.828
Waist (cm)
107.70
12.44
107.00
100.95
12.60
100.50
0.017
Hip (cm)
108.78
11.50
109
107.53
10.97
108
0.794
WHR
0.99
0.06
0.98
0.94
0.08
0.94
<0.001
WHtR
0.65
0.06
0.65
0.62
0.06
0.62
0.041
Height (cm)
166.10
11.95
166.40
162.70
12.41
161.50
0.127
Weight (kg)
92.22
22.78
89.00
84.53
19.74
84.80
0.126
BMI (kg/m2)
32.97
5.29
32.00
31.49
4.82
31.60
0.243
BMI 𝑍-score
2.45
0.48
2.38
2.37
0.48
2.36
0.479
Height 𝑍-score
0.53
1.44
0.53
−0.573
0.566
−0.573
0.566
Fat%
39.40
6.34
38.90
39.15
7.23
39.60
0.897
Fat mass (kg)
36.61
11.58
36.50
33.44
10.91
34.60
0.214
FFM (kg)
55.62
14.01
54.60
51.08
12.68
48.90
0.047
FFM%
60.61
6.35
61.07
60.83
7.23
60.35
0.879
Muscle mass (kg)
52.40
13.72
51.80
48.57
12.13
46.60
0.094
TBW
40.72
10.26
40.00
37.38
9.28
35.80
0.047
TBW%
44.33
4.67
44.70
44.53
5.28
44.20
0.919
Trunk fat%
35.39
7.08
35.00
34.84
8.07
35.80
0.886
Fat% 𝑍-score
3.15
0.99
3.04
2.98
1.07
2.92
0.577
Trunk/total
0.89
0.06
0.88
0.88
0.07
0.89
0.986
BMR
8158
1293
7880
7617
1144
7512
0.028
BMR/kg
90.89
12.33
86.17
92.80
14.85
88.57
0.597 Table 2: Anthropometry and body composition. Comparison of patients with and without NAFLD. Table 3: Biochemical analysis. Comparison of patients with and without NAFLD. Variable
NAFLD patients (𝑁= 49)
Patients without NAFLD (𝑁= 59)
𝑝value
Mean
SD
Median
Mean
SD
Median
(Mann–Whitney 𝑈test)
Tch (mg/dl)
185.12
28.64
185.00
169.02
30.01
167
0.004
HDL (mg/dl)
45.34
10.26
43.90
47.77
11.38
45.85
0.298
LDL (mg/dl)
103.16
25.69
99.90
96.26
28.34
62.03
0.153
TG (mg/dl)
183.08
79.69
173.00
129.15
62.03
114.00
<0.001
glu 0 (mg/dl)
90.88
10.00
90.00
88.95
7.74
88.50
0.403
glu 120 (mg/dl)
122.04
22.68
121.00
109.91
19.84
109.00
0.007
ins 0 (𝜇IU/ml)
28.48
21.54
23.15
12.86
6.76
12.00
<0.001
ins 120 (𝜇IU/ml)
130.38
98.18
93.80
81.36
44.03
70.70
0.004
HOMA-IR
6.44
4.83
4.93
2.86
1.67
2.58
<0.001 Table 3: Biochemical analysis. Comparison of patients with and without NAFLD. established a threshold of each variable that showed the
highest accuracy for identifying children at high risk of
NAFLD. Figure 2 shows a comparison of the ROC curves for
the evaluated parameters. 3. Results Variable
Symbol
AUC (95% CI)
Cut point
Sensitivity
Specificity
SE
Total cholesterol
Tch
0.660 (0.557–0.764)
197 mg/dl
44.9%
80.7%
0.053
Triglycerides
TG
0.713 (0.614–0.813)
161 mg/dl
61.2%
75.4%
0.051
Glucose in 120 min of OGTT
glu 120
0.654 (0.548–0.759)
112 mg/dl
70.2%
56.4%
0.054
Fasting insulin
ins 0
0.829 (0.746–0.911)
18.9 𝜇IU/ml
75%
87.3 %
0.042
Insulin in 120 min of OGTT
ins 120
0.666 (0.559–0.772)
129 𝜇IU/ml
38.3%
90.9%
0.054
Insulin resistance
HOMA-IR
0.817 (0.733–0.901)
4.089
70.8%
83.6%
0.043 Table 4: Diagnostic value of analysed variables in NAFLD prediction. Table 5: Prevalence of metabolic factors in NAFLD and non-NAFLD group. Table 5: Prevalence of metabolic factors in NAFLD and non-NAFLD group. MS components
Total (%)
NAFLD patients (𝑁= 49)
Patients without NAFLD (𝑁= 59)
𝑝
WC > 90 pc
108 (100%)
49 (100%)
59 (100%)
NS
TG > 150 mg/dl or hypolipemic treatment
49 (45.37%)
30 (61.22%)
19 (32.20%)
0.002
HDL < 40 mg/dl (girls > 16 years; HDL < 50 mg/dl) 39 (36.11%)
20 (40.82%)
19 (32.20%)
NS
Hypertension
21 (19.44%)
11 (22.45%)
10 (16.95%)
NS
IFG or diabetes 2
13 (12.04%)
6 (12.24%)
7 (11.86%)
NS
MS
34 (31.48%)
20 (40.82%)
14 (23.73%)
0.04 WC > 90 pc
108 (100%)
TG > 150 mg/dl or hypolipemic treatment
49 (45.37%)
HDL < 40 mg/dl (girls > 16 years; HDL < 50 mg/dl) 39 (36.11%)
Hypertension
21 (19.44%)
IFG or diabetes 2
13 (12.04%)
MS
34 (31.48%)
0,0
0,2
0,4
0,6
0,8
1,0
1 − specificity
0,0
0,2
0,4
0,6
0,8
1,0
sensitivity
Total cholesterol (Tch)
Triglycerides (TG)
OGTT 2-h glucose (glu 120)
Insulin (ins 0)
OGTT 2-h insulin (ins 120)
HOMA-IR
Figure 2: Comparison of ROC curves for parameters significantly
differentiating patients with NAFLD from non-NAFLD patients. 0,0
0,2
0,4
0,6
0,8
1,0
1 −specificity
0,0
0,2
0,4
0,6
0,8
1,0
sensitivity 3. Results The largest area under the ROC
curve was obtained for fasting insulin (AUC = 0.829, 95%
CI 0.746–0.911) and for HOMA-IR (AUC = 0.817; 95% CI
0,733–0,901). The optimal cut-off point of the insulin level for
diagnosing NAFLD was 18,9 uIU/l with the sensitivity of 75%
and specificity of 87,3%. The performance of these variables
in NAFLD prediction is summarized in Table 4. 3.3. Biochemical Analysis. Of the biochemical parameters,
the serum concentration of total cholesterol, triglycerides,
fasting insulin, glucose, and insulin in 120 minutes of OGTT
and HOMA-IR were significantly higher in patients with
NAFLD.it established a threshold of each variable that showed the
highest accuracy for identifying children at high risk of
NAFLD. Figure 2 shows a comparison of the ROC curves for
the evaluated parameters. The largest area under the ROC
curve was obtained for fasting insulin (AUC = 0.829, 95%
CI 0.746–0.911) and for HOMA-IR (AUC = 0.817; 95% CI
0,733–0,901). The optimal cut-off point of the insulin level for
diagnosing NAFLD was 18,9 uIU/l with the sensitivity of 75%
and specificity of 87,3%. The performance of these variables
in NAFLD prediction is summarized in Table 4. NAFLD was significantly more often diagnosed in
patients with HOMA-IR exceeding reference values [22] than
in children with the normal range of HOMA-IR (79% versus
28%, 𝑝= 0.00 for HOMA-IR > 90 percentile; 85% versus 15%,
𝑝= 0.00 for HOMA-IR > 97 percentile). A detailed summary
of the biochemical parameters is presented in Table 3.h 3.4. Metabolic Syndrome. In the study population 34 (31.48%)
patients were diagnosed with metabolic syndrome (MS). Of
the metabolic syndrome components abdominal obesity and The ROC analysis was performed on variables that dif-
ferentiated NAFLD patients from non-NAFLD ones. Based
on the area under receiver operating curves (AUC) we Canadian Journal of Gastroenterology and Hepatology 5 Table 4: Diagnostic value of analysed variables in NAFLD prediction. Canadian Journal of Gastroenterology and Hepatology Canadian Journal of Gastroenterology and Hepatology 6 Canadian Journal of Gastroenterology and Hepatology Canadian Journal of Gastroenterology and Hepatology In our survey in diagnostics of carbohydrate metabolism
fasting glucose remained useless, which was confirmed in
other studies [31, 36]. Significant differences between groups
were observed in fasting insulin concentration and glucose
and insulin concentration in 120 minutes of OGTT. In our
study, the highest diagnostic value for detecting NAFLD
achieved fasting insulin with AUC = 0.829, and the cut-off
point of 18.9 𝜇IU/ml with 75% sensitivity and 87.3% specificity
diagnosed NAFLD. Studies in obese patients [31, 36, 37]
confirm correlation of fasting insulin concentrations with
NAFLD diagnosis; however there are no clear standards for
this parameter, which makes it difficult to apply. In Shashaj
et al. study [22] mean fasting insulin concentration in obese
patients was 13.8 𝜇IU/ml ± 9.4, while in Pacifico et al. it
was 10.6 𝜇IU/ml in obese children without steatosis and
15.6 𝜇IU/ml in obese children with NAFLD [36]. Much
higher mean concentration of fasting insulin was observed
by Korean researchers, 15.1 𝜇IU/ml ± 6.0 in obese children
without NAFLD and 24.8 𝜇IU/ml + 14.6 in children with
NAFLD [37]. These results are comparable to our study
(12, 86 𝜇IU/ml ± 6.76 in children without steatosis versus
28.48 𝜇IU/ml ± 21.53 in children with NAFLD).i researchers [23], which is caused by the inclusion criteria:
abdominal obesity defined as WC > 75 pc and significantly
higher ALT (52 U/L for girls and ALT > 72 U/L for boys)
as well as ethnic differences (only 29% of the children were
Caucasian). As much as 83.1% of patients were diagnosed
with NAFLD in the American study [29]; however, the group
consisted of children who were qualified to bariatric surgery. The mean prevalence of NAFLD diagnosed on ultrasound
in the previously cited meta-analysis was 41%, while that
diagnosed on the basis of elevated alanine aminotransferase
was only 13.7% [2]. Males have higher prevalence of NAFLD than females,
which is confirmed by most studies [2, 24, 30], but not in
the Italian study [31] where NAFLD was similar in both sexes
(58% of boys and 46% of girls, 𝑝= 0.31). However, in that
study prepubertal population was analysed, which was signif-
icantly younger than in other studies. Canadian Journal of Gastroenterology and Hepatology In our study prevalence
of NAFLD was similar in males and females, which could
have come from the sex distribution in age groups (there were
significantly more girls in the 16–18 age group, 𝑝< 0.05). gi
y
g
g g
𝑝
In our study NAFLD patients were similar to non-
NAFLD ones in age and basic anthropometric parameters
such as height, weight and even BMI, and BMI 𝑍-score. There
was also no difference in the most important parameters
of the body composition (fat mass, fat%, fat% 𝑍-score, or
visceral fat%). Children with NAFLD had only significantly
higher fat-free mass, total body water content, and BMR (but
not BMR/kg); however, these parameters, due to their weight,
age, or gender dependence, have little diagnostic value.h NAFLD patients had significantly higher HOMA-IR val-
ues compared to non-NAFLD ones. Similar observations
have been made by Denzer et al. [30] and D’Adamo et al.,
who confirmed that insulin resistance indexes were signifi-
cantly associated with NAFLD independently of BMI [31]. In
contrast G¨okc¸e et al. concluded that neither mean HOMA-IR
values nor the prevalence of insulin resistance was higher in
NAFLD group [12]. g
g
p
g
The only anthropometric parameters discriminating
NAFLD patients from non-NAFLD, in our study, are waist
circumference (WC) and the indicators associated with WC,
WHR and WHtR. These are important, practical pieces of
information indicating that BMI, even referred to reference
values, is not sufficient for obese patients, while WC, which is
still not a routine medical examination, may be an effective
tool for detecting patients at risk of metabolic disorders,
including NAFLD. Many studies have shown that waist
circumference may be used in central obesity screening, car-
diovascular risk assessment in children [32], and NAFLD
diagnosis [33–35]. It is proven that insulin resistance increases in obese
patients; therefore separate standards are developed for the
population of obese children [22]. Authors of this study
emphasize that in obese children HOMA-IR > 75 pc is
associated with an increased cardiometabolic risk defined as
at least one of the following: hypercholesterolemia, hyper-
triglyceridemia, reduced HDL levels, and ALT > 40 U/l. They have also observed that HOMA-IR > 3.42 (AUC =
0.71) with 48.8% sensitivity and 81.3% specificity identifies
cardiovascular risk in obese patients. 4. Discussion We diagnosed NAFLD on the basis of elevated ALT and/or
presence of steatosis on ultrasound. Both of these criteria
are often used in noninvasive diagnosis of fatty liver disease. High sensitivity (60–94%) and specificity (84–100%) of ultra-
sonography make this examination suitable for screening
[16]. Alanine aminotransferase (ALT) concentration due to
wide availability and low cost is the basic marker used in
liver diseases screening, including NAFLD. Interpretation
difficulties are caused by different cut-off points proposed by
each investigator like ALT > 52 U/L for girls and >72 U/L for
boys in the Brazilian study [23], ALT > 50 U/L the in the
Wiegand et al. study [24], or ALT > the laboratory standard in
most authors’ studies. On the other hand, we should be aware
of low sensitivity of ALT at standard cut-off point (45 U/l);
despite liver steatosis confirmed by imaging studies, in most
pediatric patients, ALT levels remain normal. Therefore, a
number of population studies suggest introducing new, sex-
specific thresholds for ALT [25]: in children ALT < 25.8 IU/l
for boys and ALT < 22.1 IU/l for girls in American study
[26], ALT < 33 IU/l for boys and <25 IU/l for girls in Korean
study [27], or ALT < 30 IU/l and <21 IU/l in Iranian study,
respectively [28]. sensitivity In this study we diagnosed NAFLD in 45% of patients. Such a high prevalence is caused by both group selection
(obese children hospitalized for obesity) and diagnostic cri-
teria. Fatty liver on ultrasound was reported in 34 (31.48%)
patients, and elevated ALT levels were reported in 30 (27.78%)
patients. Figure 2: Comparison of ROC curves for parameters significantly
differentiating patients with NAFLD from non-NAFLD patients. hypertriglyceridemia were most often recognised. MS was
diagnosed significantly more frequently in NAFLD group
than in patients without fatty liver (40.82% versus 23.73%, 𝑝=
0.04), but of the MS components only hypertriglyceridemia
was significantly more often diagnosed in NAFLD patients. The frequency of the MS and its components in the study
population is presented in Table 5. The authors of meta-analysis from 2015 [2], based on 56
clinical trials, assessed the prevalence of NAFLD in obese
children on 34.2% with a prediction range from 5.2% to
83.1%. Researchers, however, emphasize significant differ-
ences in the methodology, diagnostic criteria, or accepted
standards for the laboratory tests results. With the lowest fre-
quency NAFLD was diagnosed in 2,3% patients by Brazilian 5. Conclusions [5] K. Cusi, “Role of obesity and lipotoxicity in the development
of nonalcoholic steatohepatitis: pathophysiology and clinical
implications,” Gastroenterology, vol. 142, no. 4, pp. 711.e6–
725.e6, 2012. NAFLD is a very common disease in obese children. NAFLD
risk factors include increased waist circumference, elevated
WHR and WHtR, and elevated total cholesterol, triglycerides,
and fasting insulin as well as glucose and insulin concen-
tration in 120 min of OGTT and HOMA-IR index. NAFLD
increases the risk of potential cardiovascular complications
expressed by diagnosis of metabolic syndrome. The best
independent risk factor for diagnosing NAFLD in obese
children is fasting insulin concentration > 18.9 uIU/ml. [6] L. Ozcan and I. Tabas, “Role of endoplasmic reticulum stress
in metabolic disease and other disorders,” Annual Review of
Medicine, vol. 63, pp. 317–328, 2012. [7] J. Yu, S. Marsh, J. Hu, W. Feng, and C. Wu, “The pathogenesis
of nonalcoholic fatty liver disease: interplay between diet,
gut microbiota, and genetic background,” Gastroenterology
Research and Practice, vol. 2016, Article ID 2862173, 13 pages,
2016. g
In our study, we focused on the diagnosis of obese
children in the context of metabolic disorders, especially
related to obesity and fatty liver disease. The prevalence
of NAFLD and other metabolic disorders in the study
population indicates the need to improve diagnostics in obese
children already at primary health care level. Simple diag-
nostic methods such as waist circumference measurement
and fasting plasma insulin concentration may contribute to
the early identification and prediction of patients at risk
of NAFLD and other metabolic complications. Appropriate
therapy and lifestyle change in these patients and their
families will help to prevent the negative effects of obesity in
the future. [8] M. Kłusek-Oksiuta, I. Bialokoz-Kalinowska, E. Taras´ow, M. Wojtkowska, I. Werpachowska, and D. M. Lebensztejn, “Chem-
erin as a novel non-invasive serum marker of intrahepatic lipid
content in obese children,” Italian Journal of Pediatrics, vol. 40,
article 84, 2014. [9] D. M. Lebensztejn, M. Flisiak-Jackiewicz, I. Bialokoz-Kalinow-
ska, A. Bobrus-Chociej, and I. Kowalska, “Hepatokines and
non-alcoholic fatty liver disease,” Acta Biochimica Polonica, vol. 63, no. 3, pp. 459–467, 2016. [10] K. Miura and H. Ohnishi, “Role of gut microbiota and Toll-like
receptors in nonalcoholic fatty liver disease,” World Journal of
Gastroenterology, vol. 20, no. 23, pp. 7381–7391, 2014. [11] E. Buzzetti, M. Pinzani, and E. A. Tsochatzis, “The multiple-
hit pathogenesis of non-alcoholic fatty liver disease (NAFLD),”
Metabolism - Clinical and Experimental, vol. Canadian Journal of Gastroenterology and Hepatology Referring our results
to the proposed standards [22] we proved that NAFLD
is significantly more common in patients with increased
HOMA-IR compared to obese patients with normal HOMA-
IR (79% versus 28%, 𝑝= 0.00 for HOMA-IR > 90 pc; 85%
versus 15%, 𝑝= 0.00 for HOMA-IR > 97 pc), and HOMA-
IR > 4.089 is a good indicator of NAFLD (AUROC = 0.817,
sensitivity = 70.8%, specificity = 83.6%, and 95% CI = 0.733). Similar results were obtained by Italian and Taiwanese
researchers who did not observe differences in BMI and body
composition between the groups with and without NAFLD,
but waist circumference was significantly higher in NAFLD
group [31, 34]. Denzer et al. also observed significantly greater
waist circumference in patients with NAFLD (mean 111 cm
versus 101 cm in patients without steatosis, 𝑝< 0.0001) but,
in contrast to our results, NAFLD patients had significantly
higher BMI-𝑍score (mean 2, 78 versus 2.56 in non-NAFLD,
𝑝< 0.0001) [30]. i
Metabolic syndrome (MS) in adult patients increases the
risk of cardiovascular disease. In the pediatric population
such conclusions are not clear. Due to both lack of long-term
follow-up of pediatric patients and different criteria for MS,
some authors suggest that the prevalence of individual com-
ponents of metabolic syndrome in the pediatric population
would be more relevant for assessment of the cardiovascular
risk than the diagnosis of metabolic syndrome [38, 39]. In the
studied population, based on IDF criteria [19], 34 (31.48%)
patients were diagnosed with MS, which is comparable to
other studies using the same criteria (Strojny et al., 29%
[40]). In contrast, Manco et al. diagnosed MS only in 10% Considering the complexity of links between NAFLD,
insulin resistance (IR), and type 2 diabetes, it is extremely
difficult to find out whether NAFLD is the cause or the
effect of insulin resistance. For many years, NAFLD has been
treated as the hepatic consequence of IR, but recent studies
suggest that hepatic steatosis may precede type 2 diabetes and
metabolic syndrome and may even be a risk factor for their
development [14, 15]. 7 Canadian Journal of Gastroenterology and Hepatology of children with NAFLD, which may be because only 65%
of the children in the study group were obese [35]. Canadian Journal of Gastroenterology and Hepatology In our
study, patients with MS were more likely to have NAFLD than
patients without MS (60.6% versus 39.19%, 𝑝< 0.05), but
no correlation was found between the number of metabolic
syndrome criteria and the prevalence NAFLD (𝑝= 0.052),
which may be caused by the fact that only fasting glucose but
not the insulin resistance is considered in the IDF criteria for
MS. fatty liver disease in children and adolescents: A systematic
review and meta-analysis,” PLoS ONE, vol. 10, no. 10, Article ID
e0140908, 2015. [3] A. A. Bremer, M. Mietus-Snyder, and R. H. Lustig, “Toward a
unifying hypothesis of metabolic syndrome,” Pediatrics, vol. 129,
no. 3, pp. 557–570, 2012. [4] M. F. Abdelmalek, A. Suzuki, C. Guy et al., “Increased fructose
consumption is associated with fibrosis severity in patients with
nonalcoholic fatty liver disease,” Hepatology, vol. 51, no. 6, pp. 1961–1971, 2010. 5. Conclusions 65, no. 8, pp. 1038–
1048, 2016. Conflicts of Interest The authors declare that there are no conflicts of interest
regarding the publication of this paper. [13] R. Kelishadi, S. R. Cook, A. Adibi et al., “Association of the
components of the metabolic syndrome with non- alcoholic
fatty liver disease among normal-weight, overweight and obese
children and adolescents,” Diabetology & Metabolic Syndrome,
vol. 1, no. 1, p. 29, 2009. Data Availability The data that support the findings of this study are available
from the corresponding author [Pawel Matusik], upon rea-
sonable request. [12] S. G¨okc¸e, Z. Atbinici, Z. Aycan, H. G. C¸inar, and P. Zorlu,
“The relationship between pediatric nonalcoholic fatty liver
disease and cardiovascular risk factors and increased risk of
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no. 2, pp. 308–315, 2013. Acknowledgments [14] A. Lonardo, S. Ballestri, G. Marchesini, P. Angulo, and P. Loria,
“Nonalcoholic fatty liver disease: a precursor of the metabolic
syndrome,” Digestive and Liver Disease, vol. 47, no. 3, pp. 181–
190, 2015. The study was financially supported by the Medical Univer-
sity of Silesia Grant (KNW-2-033/D/3/N), founded by Polish
Ministry of Science. [15] A. Kasturiratne, S. Weerasinghe, A. S. Dassanayake et al., “Influ-
ence of non-alcoholic fatty liver disease on the development of
diabetes mellitus,” Journal of Gastroenterology and Hepatology,
vol. 28, no. 1, pp. 142–147, 2013. Canadian Journal of Gastroenterology and Hepatology 8 [33] P. A. Monteiro, B. de Moura Mello Antunes, L. S. Silveira, D. G. D. Christofaro, R. A. Fernandes, and I. F. Freitas Junior, “Body
composition variables as predictors of NAFLD by ultrasound in
obese children and adolescents,” BMC Pediatrics, vol. 14, no. 1,
article no. 25, 2014. [17] S. K. Hourigan, S. Abrams, K. Yates et al., “Relation between
vitamin D status and nonalcoholic fatty liver disease in chil-
dren,” Journal of Pediatric Gastroenterology and Nutrition, vol. 60, no. 3, pp. 396–404, 2015. [18] H. D. McCarthy, T. J. Cole, T. Fry, S. A. Jebb, and A. M. Prentice,
“Body fat reference curves for children,” International Journal of
Obesity, vol. 30, no. 4, pp. 598–602, 2006. [34] Y.-C. Lin, P.-F. Chang, S.-J. Yeh, K. Liu, and H.-C. Chen, “Risk
factors for liver steatosis in obese children and adolescents,”
Pediatrics and Neonatology, vol. 51, no. 3, pp. 149–154, 2010. [19] “The IDF consensus definition of the Metabolic Syndrome in
children and adolescents,” International Diabetes Federation,
2007. [35] M. Manco, G. Bedogni, M. Marcellini et al., “Waist circumfer-
ence correlates with liver fibrosis in children with non-alcoholic
steatohepatitis,” Gut, vol. 57, no. 9, pp. 1283–1287, 2008. [20] Z. Kułaga, M. Litwin, A. Grajda, B. Gurzkowska, E. Napieralska,
and K. I. Kułaga, “Rozklady wartosci cisnienia krwi w populacji
referencyjnej dzieci i mlodziezy w wieku szkolnym,” Standardy
Medyczne - Pediatria, vol. 7, pp. 100–111, 2010. [36] L. Pacifico, C. Anania, F. Martino et al., “Functional and
morphological vascular changes in pediatric nonalcoholic fatty
liver disease,” Hepatology, vol. 52, no. 5, pp. 1643–1651, 2010. [37] H. R. Yang and E. J. Chang, “Insulin resistance, body compo-
sition, and fat distribution in obese children with nonalcoholic
fatty liver disease,” Asia Pacific Journal of Clinical Nutrition, vol. 25, no. 1, pp. 126–133, 2016. [21] D. R. Matthews, J. P. Hosker, A. S. Rudenski, B. A. Naylor, D. F. Treacher, and R. C. Turner, “Homeostasis model assessment:
insulin resistance and 𝛽-cell function from fasting plasma
glucose and insulin concentrations in man,” Diabetologia, vol. 28, no. 7, pp. 412–419, 1985. [38] G. J. Paz-Filho, “Metabolic syndrome in children and teenagers:
Worth assessing it, but how?” Archives of Endocrinology and
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between carotid intima-media thickness and metabolic syn-
drome in adolescents,” Journal of Pediatrics, vol. 163, no. 2, pp. 327–332, 2013. [23] R. Rocha, H. P. Cotrim, A. G. V. Bitencourt et al., “Nonalcoholic
fatty liver disease in asymptomatic Brazilian adolescents,” World
Journal of Gastroenterology, vol. 15, no. 4, pp. 473–477, 2009. [40] W. Strojny, D. Drozdz, K. Fijorek et al., “Looking for new diag-
nostic tools and biomarkers of hypertension in obese pediatric
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The Importance of Image when Developing a Powerful Political Brand
|
Postmodern Openings
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cc-by
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Postmodern Openings
ISSN: 2068-0236 | e-ISSN: 2069-9387
Covered in: Web of Sciences (WOS); EBSCO; ERIH+; Google Scholar; Index Copernicus; Ideas RePeC; Econpapers;
Socionet; CEEOL; Ulrich ProQuest; Cabell, Journalseek; Scipio; Philpapers; SHERPA/RoMEO repositories; KVK;
WorldCat; CrossRef; CrossCheck Postmodern Openings
ISSN: 2068-0236 | e-ISSN: 2069-9387
Covered in: Web of Sciences (WOS); EBSCO; ERIH+; Google Scholar; Index Copernicus; Ideas RePeC; Econpapers;
Socionet; CEEOL; Ulrich ProQuest; Cabell, Journalseek; Scipio; Philpapers; SHERPA/RoMEO repositories; KVK;
WorldCat; CrossRef; CrossCheck Postmodern Openings
ISSN: 2068-0236 | e-ISSN: 2069-9387
Covered in: Web of Sciences (WOS); EBSCO; ERIH+; Google Scholar; Index Copernicus; Ideas RePeC; Econpapers;
Socionet; CEEOL; Ulrich ProQuest; Cabell, Journalseek; Scipio; Philpapers; SHERPA/RoMEO repositories; KVK;
WorldCat; CrossRef; CrossCheck 2019, Volume 10, Issue 3, pages: 72-85 | doi:10.18662/po/82 Abstract: A brand represents a name, a sign or a symbol that
has the purpose of identifying goods (set to the disposal of
customers) and to highlight them from the competitors. There are many similarities between the construction process
of a brand in the commercial and political domain. The key
elements of a succesfull brand, either commercial, personal or
political, must fullfill the following qualities: to be authentic,
to create an emotional bond between the brand and the
customers or followers, to set well defined values and to be a
step ahead other brands on the market. A political brand
becomes powerful if it possesses good communication
aptitudes with the electorate to expose the desired message. In
the construction process of a brand, an essential role is the
image, that needs to be a combination of qualities meant to
put aside the other candidates. Popularity, prestige, authority
and competence are key qualities to allure the attention and
the interest of masses. The created image must be very closed
to reality, so that the people don’t lose their confidence in the
candidate. Nowadays, new media and technology play a very
important role in the construction of political people
presenting both advantages and disadvantages for them. The Importance of
Image when
Developing a
Powerful Political
Brand Octavia Cristina BORŞ1 1 Babes-Bolyai University, Department
of Communication, Public Relations,
and Advertising, Faculty of Political,
Administrative and Communication
Sciences, Cluj-Napoca, Romania. Email: octaviacristina.bors@gmail.com Keywords: brand; political brand; political communication; politician;
image; media; social media. How to cite: Borş, C. (2019). The Importance of Image
when Developing a Powerful Political Brand. Postmodern
Openings, 10(3), 72-85. doi:10.18662/po/82 Postmodern September, 2019
Openings Volume 10, Issue 3 Postmodern
Openings September, 2019
Volume 10, Issue 3 September, 2019
Volume 10, Issue 3 September, 2019
Volume 10, Issue 3 Introduction This paper attempts to show that there is a relation between the
personal brand and the political one as well as the image is an important
component in the political system, playing a key role for the success of a
political campaign. I have chosen this topic because I wanted to bring more
knowledge in this field and because I consider that in the political
communication area there are so far too few studies that have been
conducted to bring the phenomenon of political image and political brand
into question. 1. From brand to personal brand Brand is a marketing-specific notion, but nowadays knows a great
rising in other areas, such as sports, culture, and politics. There are many
similarities between the commercial brand and the personal brand, in terms
of definition, traits or purpose. Political marketing shares much in common
with marketing in the business world; candidates and parties can be
compared to consumer products in terms of promotion. There are many
definitions for the term, however none of them is unanimously accepted. According to the American Marketing Association a brand represents "a
term, sign, symbol, or design, or a combination of them, intended to identify
the goods and services of one seller or group of sellers and to differentiate
them from the competitors'' (Kotler, 2008, p. 592). According to another
definition a brand is a company's promise to deliver a specific set of features,
benefits, services and experiences consistently to buyers. The message
communicated by a brand can have up to six types of meanings: attributes,
advantages, value, cultural symbols, personality and use (Kotler, 2008, p. 593). )
Another definition of the brand would be that a brand is nothing but
a name that remains in the mind of the consumer as a result of creating an
emotional bond between the product and the customer. A brand lives on in
the mind of the consumer and is the sum of all the experiences, perceptions
and expectations the customer has about a product, a service or a company
(Manea, 2011, pp. 10-11). If we consider the three definitions above, we observe that there is a
common element in all attempts of definining it, namely the need to identify,
to singularize a product or a service. The brand seems to be perceived as an
empty concept that fills with meaning over time, along with the numerous
and repeated experiences of the consumers (Corbu, 2013, p. 59). If we 73 The Importance of Image when Developing a Powerful Political Brand
Octavia Cristina BORŞ The Importance of Image when Developing a Powerful Political Brand
Octavia Cristina BORŞ expand the above-mentioned definitions to the personal brand, we can see
that they are also valid in this area. The candidate or politician can be seen as
a service provider and the parties can be compared to the companies that
offer certain services. 1. From brand to personal brand This involves identifying those elements that make that brand 74 September, 2019
Volume 10, Issue 3 Postmodern
Openings better than the others (Temporal, 2015, p. 70). What is interesting in this
regard is the external dimension of politicians, which is composed or created
from a set of messages, emotional components or politico-democratic
powers. In this sense, it is emphasized that both the external and internal
developement of the politicians is in fact a process by which the political life
and the reality of the civil society must be compatible. Thus, personal
branding and its use can increase the visibility and notoriety of politicians. An effective brand strategy provides a central unifying idea around
which all behavior, actions, and communications are aligned. It works
beyond products and services and is effective over time. Brand strategy is
based on a vision that aligns business strategy and reflects a deep
understanding of customer needs and perceptions. A strong brand strategy
must resonate with all stakeholders, with all the interested parties. Usually
there is a team that develops it, no one does it on its own, some companies
even bring specialists in this field. Companies often survive and prosper
because they have a clear and effective brand strategy and some companies
are not successful just because they do not have a good brand strategy
(Wheeler, 2013, pp. 12-13). Therefore, regarding the above statement, the
political brand is represented by all the signs and personality traits for the
care of the media includes in the political audience. In politics, in order to be successful, it is necessary for politicians to
understand their market, meaining their voters, their needs and aspirations,
in order to be able to represent them in an efficient way, but also to gain
their trust. Market orientation requires a candidate to recognize the nature of
the exchange process when asking for the vote. If the promises made before
taking up the desired position are met, then the candidate will enjoy the
sympathy and, most importantly, the confidence of the voters and the
public. Also, another important feature of a successful political brand is its
recognition by the public. Moreover, it is essential that the brand has what is
called "equity", that is, a certain quality that the public regards as
synonymous with the brand in question and which ensures trust. 1. From brand to personal brand Upon further examination, it becomes clear that for a brand to be
successful, it must go through a complex developing process. As can be
noted from Jean-Noël Kapferer's analysis, political communication uses
mainly two brand building tools: brand identity, which mentions the brand's
uniqueness and value, and brand positioning (Kapferer, 2012, p. 150). Brand
identity is tangible so it appeals to the senses. Brand identity amplifies
differentiation, takes disparate elements and unifies them into whole systems
(Wheeler, 2013, p. 4). Investing in brand identity has its benefits, making it
easier for the customer to buy and making the sales force bigger. Competition always creates innumerable options, so companies are looking
for ways to connect emotionally with customers, become irreplaceable, as
well as to create lifelong relationships with them. A strong brand stands out
in a densely crowded marketplace. People fall in love with brands, trust
them, and believe in their superiority. How a brand is perceived affects its
success, regardless of whether it's a start-up, a nonprofit, or a product. The brand identity process demands a combination of investigation,
strategic thinking, design excellence, and project management skills. It
requires an extraordinary amount of patience, an obsession with getting it
right, and endpoints, which facilitate decision making at the appropriate
intervals. In this process, ideals are an essential element; the ideals of a brand
must be the following: vision, differentiation, flexibility, significance,
sustainability, engagement, authenticity, consistency, value (Wheeler, 2013, p. 28). In the end, the brand strategies used by politicians are actually aimed at
investigating and analyzing the wishes of the public, respectively of the
voters, with the purpose of developing voter oriented public policies. That is
why, all this structure considers the application of market research,
segmentation targeting and positioning, but also the strengthening of
relations with the voters. g
p
p
p There are three main steps in developing a brand strategy. The first
step is to ensure identification of the brand with customers, to create a
brand based on emotion, this is a long-term perspective that summarizes
what the brand means. The second step is to create a well-defined set of
brand values, preferably framed as personality traits.This allows the brand to
stand out and differentiate itself from competitors. The third step is to
establish a market position that makes the brand stand out from its
competitors. 1. From brand to personal brand y
y
q
Another concept worth mentioning when talking about brand is
branding. Branding can be defined as a disciplined process used to build
awareness and extend customer loyalty. Branding also means taking
advantage of every opportunity to state why people should choose a brand
over another (Wheeler, 2013, p. 6). In other words, branding is the process
by which certain attributes and values, consistent, distinct and attractive to a
brand-related consumer are developed and maintained. Branding creates
identity, structures myths, because the brand is a myth performer and bearer
of an identity myth (Corbu, 2013, p. 60). The overall purpose of the brand 75 The Importance of Image when Developing a Powerful Political Brand
Octavia Cristina BORŞ management process is to increase its value over time. Management is a
process that tries to take control over everything a brand does and says. Therefore, in order for people to see what the brand wishes to convey, it is
necessary to influence the perceptions of the targeted target audience. One
of the most difficult parts of brand management is achieving a balance
between the short-term quantitative targets and long-term growth of the
brand and its value (Temporal, 2015, pp. 147-148). This highlights the fact
that every promise made by a political actor must reflect his desire and
ability to meet the needs and wishes of voters at a certain time, but at the
same time he must leave the impression that this is lasting. In politics, branding refers to the way in which a political
organization or a politician is perceived by the electorate. The frequent use
of branding techniques is due to the professionalisation of electoral
campaigns and voter behaviour, which can be likened to "consumers" in a
political market. Considering that we live in a world where brands are
everywhere we look, it is important that they use every opportunity to
position themselves in the minds of customers, respectively of the voters. 2. The personal and political brand Branding plays an increasingly important part in politics sports,
culture, the public sector, or voluntary activities. Those who use branding
know that a strong brand can impress people on an emotional level (Olins,
2010, p. 12). Public sector brands are found in every country of the world,
whether intentionally created or not. Institutions and organizations in the
public sector have relationships with individual consumers, conpanies, other
countries and governments, and so on, all of which have images of a certain
kind. Because stakeholder and customer perceptions are so important in
achieving success for the public sector, the need to control, manage and
develop a brand image is of fundamental importance. Not being able to
undertake this activity could lead to negative perceptions and insignificant
fruition of national targets. Public sector branding has become a necessity. The increased competition in every sector makes branding an imperative. From nations to non-profit organizations, from public services to cities,
from sectors to services, there is no escape from the harsh demands of the
need to create a differentiation point and a positive image. Competition is
ubiquitous and every country or entity in the public sector is fighting for
political support and funding. In an era when resources are scarce and
insufficient and there is a constant change, the only way to persuade
customers that any public sector or organization institution is different and 76 September, 2019
Volume 10, Issue 3 Postmodern
Openings better than others is through the development of a strong brand.Anything
else, be it services, products, processes, systems or technologies, can be
copied. The only thing that any public sector entity can create and cannot be
copied is a strong image of the brand Until recently, branding has been seen
by many as a private sector activity, admiration for top brands in the world
being a global phenomenon. But the public sector, including politicians, has
now come to realize that the same techniques that have created successful
brands of enormous financial value can be used in exactly the same way to
achieve their own goals. Publicity in the public sector is a global trend in
constant rise and growth (Temporal, 2015, pp. 7-8). 2. The personal and political brand g
(
p
pp
)
In his book, Branding For The Public Sector, author Paul Temporal lays
out the reasons why the public sector needs branding.The same reasons can
apply to politicians.These include the issue of differentiation; strong brands
differ from common ones and draw people towards them. The public sector
has entered the branding world with the aim of differentiattion and
uniqueness and attracting people towards it. A second reason for the public
sector's interest in branding is represented by the survival in a changing
world.Another reason is that the public sector has noticed that brands can
bring both strength and financial rewards. Fourthly and most importantly,
the public sector already has an image, whether it is a good one or not. Some
of the perceptions that make up this image may be negative or positive, but
it is better for the public sector to be one step ahead and to manage and
control these images than to allow others to formulate opinions in a way that
might be counterproductive (Temporal, 2015, pp. 10-11). The public sector
has rapidly adopted the techniques of private sector brands worldwide to
develop a strong brand vision that targets both the emotional and the
rational side. Using emotion is essential for building public sector brands. The concept of a political brand has not yet been defined in a clear
and conclusive way. Although there is no universal definition of this
concept, there are some partial definitions that cover different aspects of the
problem. One of the starting points may be the claim that it identifies itself
with "a name that has the power to influence” (Kapferer, 2012, p. 8). The
fact that there is an ability to influence leads the brand into the sphere of
political communication and, at the same time, turns it into a tool of
persuasion. In the political field, creating a brand is a specific process. This is
because the product in this case is represented by a political man or the
political ideas and conceptions he has. Thus, the product to be transformed
into a brand already has a number of specific attributes: the voice stamp, the
features of the face, the way of speaking, the style of clothing. Based on 77 The Importance of Image when Developing a Powerful Political Brand
Octavia Cristina BORŞ these factors, the voters formulate an opinion on the political product. 2. The personal and political brand So,
when creating a political brand, must first and foremost to focus on the
existing attributes. At the same time, belonging to a certain political party
can also automatically determine the target audience, meaning that segment
of the electorate who is among the supporters of that party. However, it
should also be borne in mind that the existing attributes and meanings can
not be omitted, and in the process of creating the brand they must be taken
into account. Nowadays the personal brand is a growing phenomenon due
to factors such as new technologies and social networks. In a world
dominated by a stunning and fierce competition where rational choice has
become
almost
impossible,
brands
represent
clarity,
reassurance,
consistency, belonging - everything that helps people define themselves. Brands represent identity (Wheeler, 2013, p. 90). But more interesting and
useful to look at than a brand definition are its features and manifestations. As far as the process of instrumentalization is concerned, the brand becomes
a complex construction that is used in, but also for persuasive
communication. Personal brands in the political sphere benefit primarily from a
symbolic dimension in which religious or spiritual elements play an
important role. Through the roles they have to assume, the personal brands
are closest to the symbolic game brought by the persona that underlies the
idea of a person. Political personalities must play a role in a script. Being the
instrument of such a persuasive action, the brand becomes useful in
situations where risk reduction is required. Finally, "perceived risk could be
functional (performance-related), experience (related to our own self-
concept) or social (related to our social image)" (Kapferer, 2012, p. 9). In
this case, the symbolic interactionism call for research into the process of
image construction, which has been neglected in the past. One feature of a strong political brand is that it has a distinct,
different way of communication through which it effectively transmits the
desired message, regardless of the medium in which it is transmitted:
speeches, television appearances, press conferences etc.Communication
must be memorable, identifiable and accessible to all the interested parties. Voice and tone play a very important role in transmitting the message to the
audience; every word can be a source of information or inspiration. Messages sent by brands are effective if they become repetitive (Wheeler,
2013, p. 26). 2. The personal and political brand The repetition of the same message often has long-term effects,
meaning acceptance of the message. Goebbels said that a repeated lie ends
by becoming a great truth (Manea, 2011, p. 45). The language used must
resonate with meaning. Receptors will complete the message with elements 78 September, 2019
Volume 10, Issue 3 Postmodern
Openings of their own experiences. The goal must be clarity, accuracy and precision so
people who are busy and have only a few free minutes can quickly
understand what is relevant. Each sentence will reveal new and interesting
aspects for those who listen (Wheeler, 2013, p. 27). In other words, the
development of a strong political brand may represent the distinct element
against the competition, while at the same time providing the most efficient
and quick way to convey the message to the public. First of all, the personal
brand is a promise to those with whom the political man interacts. Ensuring
its qualities and conduct will help people understand it better. A personal
brand creates expectations in people's minds, so promises must be respected
so that voters are not disappointed and dissatisfied (Manea, 2011, pp. 29-30). On the same note it should be mentioned that in politics the electorate
chooses not between simple individuals but between ideas and personalities. The ultimate goal of creating and promoting the political brand lies in the
fact that voters must be able to identify themselves with the political
product. An effective strategy for creating a personal brand is to influence the
public by talking about what they want to hear. However, it is not necessary
to construct an unrealistic, overly positive self-image because this can
generate very high expectations regarding future performances. The
simplicity and quality of the information transmitted, the consistency with
cultural norms, the consensual validation, the reputation and the persuasive
qualities of the person are the variables that facilitate the acceptance of the
information provided by the audience (Manea, 2011, pp. 45-46). Thus, we
must also emphasize that is necessary to differentiate between the image
projected by a political actor and the image perceived by the voters. One of
the most effective tools for maintaining continuity between transmission and
perception is the message. Interaction with voters is a relationship based on meanings. 2. The personal and political brand The
meanings are the result of a dialogue, they often involve a structure of
reciprocity in which the political brand and electorate are continually
enriched in the process of electoral communication. From the reflections on
electoral communication, we can infer that this communication is not a mere
transmission of messages from politician to possible voters. ,, It is a rather
complex connection in which the message once sent to voters returns to a
transmitter enriched by the receiver's experience, and only then becomes a
comprehensive message of authentic significance received from the
perspective of the values that the receiver of the message embodies. At the
basis of this reciprocity relationship lies a type of transfer that we find in the
consumption of commercial brands, a transfer that is more visible to 79 The Importance of Image when Developing a Powerful Political Brand
Octavia Cristina BORŞ The Importance of Image when Developing a Powerful Political Brand
Octavia Cristina BORŞ political brands and is favored by the fact that the brand is in this situation
either an organization composed of individuals or a person who can be the
bearer of values, powers and experiences specific to building the brand"
(Medveschi, Frunză, 2018, p. 144). (
p
)
If for commercial brands, the actual purchasing decision tends to be
taken at an emotional level in most cases, the decision to support a particular
candidate is also taken at an emotional level. A brief incursion into medical
science tells us why this happens. The idea that the rational, conscious part
of the brain dominates the nonracial parts was rejected. Medical research has
shown that making decisions is largely quick and emotional, often
subconscious, and is more intuitive than previously thought.It is currently
widely accepted among medical experts that emotion tends to drive reason
rather than vice versa. This is mainly due to the fact that the emotional side
of the brain is considerably higher than the rational side and it exceeds it in
terms of intensity, sending ten times more signals to the rational brain
(Temporal, 2015, p. 80). The goal of branding in the commercial field is to
add value to an entity, be it a company, a product or an idea; Instead, the
personal brand is aimed at achieving interests such as getting more fans,
success and personal benefits (Bogdan, 2011, p. 149). 2. The personal and political brand On the other hand,
regarding the political environment, the attributes of the candidates are used
to outline a profile of a public person. The communication style of the
politician which includes the use of the press and the skills of the speaker, as
well as personal attributes such as honesty and credibility, are part of the
stylistic role of the political actor. Both the speeches and slogans, as well as
the electoral programs allow him to build an image as he wishes. 3. The role of the image in developing a strong political brand The image plays a very important role in building a strong and
effective political brand. The truth is that whenever political people come
into contact with a target audience they want to influence and the image
counts.Abraham Lincoln said that: ''With public sentiment, nothing can fail;
without it nothing can succeed” (Manea, 2011, p. 45). His statement remains
valid today, the public being the one who decides to give or not to give
credit to promises made by politicians. Image is very important in politics,
but not because we live in a media age obsessed with it. The widespread idea
that the issue replaces the substance in politics is not always valid.This has
much more to do with professionalism than with aesthetic reasons. The
image helps politicians to win the trust of the interlocutor. The ultimate goal
of communication is not to win votes but to try to solve people's 80 September, 2019
Volume 10, Issue 3 Postmodern
Openings problems.In this context, the first priority of the political people is to design
a professional image so that voters will have confidence that the politicians
will solve their problems (Archetti, 2014, pp. 43-44). In this sense, in the
political environment it is assumed that some candidates assessments are
based on the previous experience of voters in relation to the image of the
candidates. Due to the fact that the image is a construct made by the people,
the political actors can place and build their image so that it is molded
according to the wishes of the electorate, but also with the purpose of
changing the old preferences of the voters. g g
p
In political and electoral marketing, the political image of a candidate
does not relate to the totality of the elements that give the perfect image of a
personality, but to the combination of those elements that achieve the
symbolic profile of the candidate. Success consists in adapting the image to
the wishes of the public, meaning to symbolically identify it with the role of
the politician. 3. The role of the image in developing a strong political brand Advertising techniques aim to adapt the product to basic
human needs so that potential customers would have the impression that the
product meets these needs.The political image resembles that of a factory
brand, that is, it helps to individualize the product for its promotion and sale
under more advanced conditions.In our case, politicians need to have a well-
defined picture to differentiate themselves from others because this image,
once formed, will accompany the politician further. The continuity of the
created image can only be made by adapting political behavior to the
electorate's expectations (Frigioiu, 2013, pp. 24-25). The need for exemplary
models and, in particular, the need to imitate and follow them, fuel the way
in which the dynamics of the political brand can be perceived. Thus, first of
all, we must note that in the electoral campaigns, the communication process
focuses on building the image in a complex approach. Both the
characteristics of the personality that embodies the political brand and those
built to enhance the already existing qualities are taken into account. Secondly, communication and brand building and developing must respond
to a personal ideal that is close to potential voters. The image of the
politician must correspond with the voters' expectations. Thirdly, we need to
take into account the fact that it is necessary to harmonize the politician's
personal qualities with his own brand and, on the other hand, to reduce the
distance between what the politician represents as a personal presence and as
a public presence " (Medveschi, Frunză, 2018, pp. 143-144). Thus, if we look
in more detail, we can see that the political arena is driven by a tendency to
personalize the vote and also to underline the importance of the personal
image of the political actor. 81 The Importance of Image when Developing a Powerful Political Brand
Octavia Cristina BORŞ Among the qualities that a personality has to have to capture
people's attention and interest include popularity, prestige, authority and
competence. Competence means a general ability to predict, negotiate,
temporize or dare. For the electorate, the first levels of image creation
techniques in political marketing, civil and professional identity, are more
interesting. What matters to humans is the outer and superficial form of the
image, not the deep and prodound one. 3. The role of the image in developing a strong political brand Based on information such as family
life, completed studies, and titles obtained, the electorate forms an opinion
on the candidates and decides whether or not to give them their vote
(Frigioiu, 2014, pp. 25-26). The personal brand is the mental image of others
when they think of a certain political personality. The values, personality and
qualities that offer uniqueness compared to others. That is why it's very
important to keep authenticity after creating your own brand. People do not
want to see an artificially created marketing image, so the outlined image
must reflect reality as much as possible (Manea, 2011, p. 30). The image, in
fact, must reach a certain balance: on the one hand, the politician must look
professional enough to inspire confidence in his ability to address a problem;
on the other hand he wants to avoid intimidation (Archetti, 2014, p. 46). Nowadays, it is necessary that the specialists in political communication,
together with the political leaders, find a way to best combine the strategies
of development of the political brand with three important elements: the
political candidate, the political organization and the political environment. p
p
g
p
There are some mistakes that politicians do when developing a
brand.Identifying can sometimes be more difficult in the political field,
because it has not only a personal but also a party ideology; this can greatly
dilute the authenticity of a political character. As we have already mentioned,
communication has a very important role to play in building the brand, so it
is important for politicians to have qualities in this respect. Another
disadvantage that may arise is the party's image, which may or may not help
in building the brand.Authenticity and charisma matter a great deal. A
common mistake for Romanian politicians is the proiection of a rich man
image at the helm of a poor country. They display their cars, watches and
expensive shoes while they are sending messages about the economic
precariousness of the country they are leading. These situations are
characteristic of undeveloped countries, where a small group of people seize
the wealth of a country and the rest of the population live in poverty
(Bogdan, 2011, pp. 154-155). Postmodern
Openings In current politics, the media has become omnipresent.Gianpietro
Mazzoleni and Winfried Schulz call this phenomenon the "mediatization" of
politics and explains that "politics has lost its autonomy, has become
dependent on the media and is continuously modeled by interacting with the
media" (Archetti, 2014, p. 10). Such a vision affects the perspective of the
political actors, the communication techniques used and the content of the
political discourse.'' These aspects seem to materialize in the mask of the
politicians who turn themselves into artists who often recite repeated scripts
on a skillfully arranged stage" (Archetti, 2014, p. 11). It is not difficult to see how personal image becomes crucial in a
context where direct contact with a politician is lost, voters are more volatile,
and parties have to rely on the media, mainly television, to convey their
message. This trend leads to a personalization of policy where the campaign
is centered on candidates. „The personal image of the politician can be seen
as a reflection of the identity of the elected representative.His image can be
built by face to face relationships or mediated ones (through communication
or mediation technologies) " (Archetti, 2014, p. 83). Rulers are using the new
media to promote their policies and decisions they take and give them a
positive image. The growing and increasingly massive use of the internet and
its services, especially social media, has had a major impact on branding in
the public sector, especially in terms of risk management and
communications crisis. Global boundaries have disappeared and everyone
can now be both a reporter and a commentator, which may be useful or, on
the contrary, totally useless for governments and public sector organizations. Of course, there is a strong need for any public sector brand to develop a
solid online communication strategy that must be permanently working. The
emergence of social media has brought many challenges for both the public
and the private sector. Companies such as Facebook, which had more than
1.3 billion monthly active users on January 1, 2014, have become very
important in this area. Brand managers need to understand that they can no
longer rely on one-way communication, such as advertising to get into
contact with customers (Temporal, 2015, p. 189). 3. The role of the image in developing a strong political brand An image can be preserved and fixed in public
memory by positioning and consolidating it, which means reducing the
image to a few essential features, easy to be perceived by the public, and
focusing the public attention on this image (Frigioiu, 2014, p. 27). 82 Postmodern September, 2019
Openings Volume 10, Issue 3 September, 2019
Volume 10, Issue 3 Postmodern
Openings 4. Personal contributions The main purpose of this research was to add value to the area of
political communication, presenting several conceptual approaches to
political branding and political image. Through this study I support the idea
that the brand is an extremely powerful tool when we talk about the
meaning and the role of the political image, the brand being formed by a 83 The Importance of Image when Developing a Powerful Political Brand
Octavia Cristina BORŞ The Importance of Image when Developing a Powerful Political Brand
Octavia Cristina BORŞ combination of aestethetic, cultural values and rational choices. Also, this
topic that I have approached brings new informations and increase
knowledge even in the field of political marketing, the last one mentioned
normally omitting the sphere of political communication, and abstracting the
personal traits in politics. Scientifically speaking, the chosen topic is a relatively new one, and
this is due to the fact that at present, regarding the political brand, the
activity of political communication is focusing exclusively on filtering the
political brand through the perspective of political marketing. 5. In lieu of a conclusion Nowadays, branding seems unstoppable. Irrespective of the field,
brands have become a social and cultural phenomenon with extremely high
power. The power of brands and branding will continue to grow, so it is
important for us to understand how we can manage and control them. p
g
Political communication was guided by a series of theoretical-
ideological directions that were universally valid until the mid-20th century,
and then attempted to target the use of instruments with a greater and faster
range of action among the masses. For this reason, the political man of
today understood that in order to be remarked, to distinguish himself in any
action of political communication, he must say things simple and plain, so
that everyone understands what he says, easily, in order to consume as little
effort as possible in transmitting information. The brand itself can be
considered as the measure of the success of a campaign by which a politician
can build and develop his image. At its foundation, the notion of brand
refers to the image and reputation a product has, and this notion appears in
many contexts today. Whether we talk about products, celebrities, cities,
companies or even politicians, we are always urged to consider them as a
brand. Certainly, trade brands have evolved in consumers' minds much
faster than political brands, but the idea of expanding branding to areas
other than commercial is quite recent. Political branding bases its foundation
on people and their personality. This makes it much harder to control it than
service or commercial branding. Today it is relatively easy to control the
image and standard of a product, but as far as the branding of a political
personality is concerned, it is practically impossible to control your mind and
soul. In a context represented by a complexity of the political space and
political offers present in the electoral campaign, the voters will have to 84 September, 2019
Volume 10, Issue 3 Postmodern
Openings identify and choose their favorite rather easily. Clearly, the completion of the
process of building the political image, but also its consolidation until the
moment of the achievement of the political brand effect, is strictly related to
the use of the persuasive instrument. One of the decisive factors of the
persuasive dimension in this relational process is precisely the political
branding. 5. In lieu of a conclusion By addressing this article, we have tried to highlight the premises
and circumstances in which a political leader gradually turns into a political
brand. Identity, in its most different forms of manifestation, has captured
our minds and hearts, because we are keen to express our need to belong,
but also to separate ourselves and our aspirations from those around us. If
identity is the one that marks the 21st century, branding can pass it on. However, the fundamental idea of the political brand is that, through
everything it does and what it produces, the political man has to project a
very clear picture of himself and his goals. References References Archetti, C. (2014). Politicians, personal image and the construction of political identity:
A comparative study of the UK and Italy. Basingstoke, UK: Palgrave
Macmillan. Bogdan, A. (2011). Branding on the East front: About reputation, against the current. Bucharest, Romania: Brandient Publishing. Corbu, N. (2013). Global brands: A cross-cultural perspective in a media context (2nd
ed.). Bucharest, Romania: Tritonic. gioiu, N. (2013). Power and political imaginary. Bucharest, Romania: Tritonic. Kapferer, J. N. (2012). The new strategic brand management: Advanced insights and
strategic thinking. London, UK: KoganPage. Kotler, P. (2008). Marketing management. Bucharest, Romania: Teora. Manea, A. D. (2011). Personal brand. From ecstasy to agony. Bucharest, Romania:
Tritonic. Medveschi, I., & Frunză, S. (2018). Political brand, symbolic construction
and public image communication. Journal for the Study of Religions and
Ideologies, 17(49), 137-152. doi:10.1057/9781137353429_2 ins W. (2010). About branding. Bucharest, Romania: Comunicare.ro Temporal, P. (2015). Branding for the public sector: Creating, building and managing
brands people will value. Chichester, UK: John Wiley and Sons Ltd. Wheeler, A. (2012). Designing brand identity (4th ed.). Hoboken, USA: John
Wiley & Sons. 85
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Unilateral versus bilateral thyroarytenoid Botulinum toxin injections in adductor spasmodic dysphonia: a prospective study
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Unilateral versus bilateral thyroarytenoid Botulinum toxin
injections in adductor spasmodic dysphonia: a prospective study
Tahwinder Upile*1,2,3, Behrad Elmiyeh1, Waseem Jerjes2,3, Vyas Prasad1,
Panagiotis Kafas4, Jesuloba Abiola5, Bryan Youl1, Ruth Epstein1,
Colin Hopper2,3, Holger Sudhoff6 and John Rubin1 Unilateral versus bilateral thyroarytenoid Botulinum toxin
injections in adductor spasmodic dysphonia: a prospective study
Tahwinder Upile*1,2,3, Behrad Elmiyeh1, Waseem Jerjes2,3, Vyas Prasad1,
Panagiotis Kafas4, Jesuloba Abiola5, Bryan Youl1, Ruth Epstein1,
Colin Hopper2,3, Holger Sudhoff6 and John Rubin1 Address: 1The Royal National Throat, Nose and Ear Hospital, London, UK, 2UCLH Head & Neck Centre, London, UK, 3Department of Surgery,
University College London Medical School, London, UK, 4Department of Oral Surgery and Radiology, School of Dentistry, Aristotle University,
Greece, 5Department of Medicine, University College London Medical School, London, UK and 6Department of Otorhinolaryngology, Head and
Neck Surgery, Klinikum Mitte, Bielefeld, Germany Email: Tahwinder Upile* - mrtupile@yahoo.com; Behrad Elmiyeh - belmiyeh@doctors.org.uk; Waseem Jerjes - waseem_wk1@yahoo.co.uk;
Vyas Prasad - vyasprasad@hotmail.com; Panagiotis Kafas - pankafas@yahoo.com; Jesuloba Abiola - ja.abiola@googlemail.com;
Bryan Youl - bryan.youl@royalfree.nhs.uk; Ruth Epstein - ruth.epstein@royalfree.nhs.uk; Colin Hopper - c.hopper@ucl.ac.uk;
Holger Sudhoff - holger.sudhoff@rub.de; John Rubin - jsrubin@compuserve.com * Corresponding author Received: 21 October 2008
Accepted: 24 October 2009 Published: 24 October 2009 d & Face Medicine 2009, 5:20
doi:10.1186/1746-160X-5-20 This article is available from: http://www.head-face-med.com/content/5/1/20 This article is available from: http://www.head-face-med.com/content/5/1/20
© 2009 Upile et al; licensee BioMed Central Ltd. © 2009 Upile 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 Upile et al; licensee BioMed Central Ltd. p
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BioMed Central BioMed Central BioMed Central Head & Face Medicine Open Access Page 1 of 11
(page number not for citation purposes) Introduction units/vial of frozen lyophilized toxin (stored at -5°C,
$281.22). The toxin is shipped from the distributor in dry
ice and is stored in a freezer at -5°C until reconstitution. Dysport® is available as 500 units/vial (stored at 2-8°C,
$308.13). Dysport® is the formulation generally used in
the UK hospitals. Adductor spasmodic dysphonia (ADSD) is a focal dysto-
nia of the laryngeal musculature, causing abrupt, intermit-
tent and involuntary vocal folds spasms producing a
strained and strangled speech pattern. It is idiopathic in
nature and may reflect abnormalities in central motor
processing [1]. A randomised controlled trial of Botox® and Dysport® sug-
gested that Dysport® tends to have higher efficacy, longer
duration, and hence higher frequency of adverse effects
[15]. The exact conversion factor for equivalence between
the preparations is varied (site specific) and remains con-
troversial. In clinical use in the larynx it is suggested that
1 Unit of Botox® is approximately equal to 3 Units of Dys-
port® [15]. The cardinal signs of ADSD are effortful vocal straining
and harshness, quaver and voice arrest due to laryngos-
pasm in the midst of non-effortful phonatory periods. It is
described as "speaking whilst being strangled". Examina-
tion of the larynx may reveal true and false vocal folds
hyper-adduction with laryngeal elevation and its attend-
ant effects on speech. ADSD, a disabling disorder of voice,
is characterised by involuntary disruption of phonation
with functional, social and emotional consequences [1]. In this preliminary study, we prospectively captured data
and compared unilateral and bilateral thyroarytenoid
muscle injections of Botulinum toxin in 31 patients with
adductor spasmodic dysphonia, who had undergone
more than 5 consecutive Dysport® injections (either uni-
lateral or bilateral) and had completed 5 concomitant
self-rated efficacy and complication scores questionnaires
related to the previous injections. We also attempted to
address whether treatment should be administered unilat-
erally or bilaterally and also the dose of Dysport® that pro-
duces
the
optimal
clinical
benefit. To
improve
communication between disciplines (in this case neuro-
physiology and ENT) we developed a Neurophysiological
Scoring (NPS) system which, for us, had utility in the
treatment administration. This, however, was not under
research in this article. The Dysport infiltrations were
done under EMG-guidance. This scale was used on a non-
inferential basis to assess optimum needle placement
within the muscle before administration of the injection. Abstract Objectives: In this preliminary prospective study, we compared unilateral and bilateral
thyroarytenoid muscle injections of Botulinum toxin (Dysport) in 31 patients with adductor
spasmodic dysphonia, who had undergone more than 5 consecutive Dysport injections (either
unilateral or bilateral) and had completed 5 concomitant self-rated efficacy and complication scores
questionnaires related to the previous injections. We also developed a Neurophysiological Scoring
(NPS) system which has utility in the treatment administration. Method and materials: Data were gathered prospectively on voice improvement (self-rated 6
point scale), length of response and duration of complications (breathiness, cough, dysphagia and
total voice loss). Injections were performed under electromyography (EMG) guidance. NPS scale
was used to describe the EMG response. Dose and unilateral/bilateral injections were determined
by clinical judgment based on previous response. Time intervals between injections were patient
driven. Results: Low dose unilateral Dysport injection was associated with no significant difference in the
patient's outcome in terms of duration of action, voice score (VS) and complication rate when
compared to bilateral injections. Unilateral injections were not associated with any post treatment
total voice loss unlike the bilateral injections. Conclusion: Unilateral low dose Dysport injections are recommended in the treatment of
adductor spasmodic dysphonia. Page 1 of 11
(page number not for citation purposes) http://www.head-face-med.com/content/5/1/20 Head & Face Medicine 2009, 5:20 http://www.head-face-med.com/content/5/1/20 Introduction Botulinum toxin is the treatment of choice for ADSD and
has been in use since the late 1980's [2-5]. It improves the
patients' perception of dysphonia, mental health and
their social function [6]. The American Academy of
Otolaryngology-Head and Neck Surgery recognizes treat-
ment with Botulinum toxin as the primary treatment for
the ADSD (Policy statement: Botulinum Toxin; Reaf-
firmed March 1st, 1999). Botulinum toxin inhibits the release of acetylcholine at
the neuromuscular junction, causing a chemical denerva-
tion, thus resulting in muscle weakness or even paralysis
in a reversible but long standing manner. The toxin has
seven serotypes (A-G) [7,8] of which type A is commer-
cially available and used as Botox® and Dysport® formula-
tions. Over the past two decades, in the absence of standardized
guidelines, the dosing requirements for Botulinum toxin
therapy for unilateral and or bilateral injections has varied
significantly, both between patients and between injec-
tions in any one patient. Unilateral Botulinum toxin
doses reported to vary between 2.5 mouse units (mu) [6],
4.0-4.5 mu [9,10], 5 mu [11], 15-16 mu [10,12,13], and
30 mu [14]. The technique q
Botulinum type A in the formulation of Dysport® was used
in all patients. Sequential dilutions with saline were per-
formed until 2.5 mu were present in each 0.1 ml saline. There was no variation in the drug concentration used. All
Dysport® injections were performed using an insulated
low profile fine bore 27-gauge mono-polar needle by or
under the direct supervision of the laryngologist. No local
anesthetic was used. The diagnosis was made in a joint laryngology-neurology
clinic. The patients were the major factor in influencing
the choice of the course of their treatment regime. Since
both the dosage and the side(s) of the injections were
determined by clinical judgment and patient choice based
on their previous injection clinical response and side we
again point out that this was not a randomized controlled
study. Clinical judgment mainly affected needle place-
ment and dosage. The time intervals between injections
were also patient driven since patients attended when
their subjective voice quality deteriorated. The time inter-
val between injections was determined by comparing
dates of the previous and current Dysport® injections. We
reviewed 5 injection episodes with 4 time intervals and 5
pre-injection voice and neuro-physiological scores. Assessment was immediately before the next potential
treatment episode. Our technique is to insert the needle through the cricothy-
roid membrane and then angle it upwards and laterally to
enter the thyroarytenoid muscle. The accuracy of the nee-
dle placement was confirmed by electromyographic
(EMG) evidence of the characteristic waveforms during
sustained phonation of a vowel sound (Figures 1 and 2). In order to improve communication between the neuro-
physiologist and the injecting surgeon we have developed
a 10-point subjective Neurophysiological Scoring (NPS)
system to describe the amplitude and specificity of the
EMG response (Table 3), where the NPS 0 to 5 (Figure 1)
represents increase in distant motor activity and 5 to 10
(Figure 2) represents rise in the local muscle field motor
activity on EMG. The NPS was recorded for each injection;
all the NPS recording were carried out by the same team,
in order to maintain consistency and a standardized inter-
pretation of the EMG waveform. In summary: the study includes patients who received five
consecutive unilateral or bilateral injections. Once thera-
peutically stabilised outside of these five injections, no
patient crossed-over treatment regimes. These five injec-
tions were not recorded from the first ever Botulinum
toxin injection that each patient received. The technique The initial dose
and side per patient determined based upon previous
responses and patient wishes. Materials and methods This prospective study was carried out in the Royal
National Throat, Ear and Nose Hospital, London, UK. Data were collected prospectively on a specific proforma
from 1998 to 2006; 68 patients (42F/26M) with ADSD
who had more than 5 consecutive Dysport® injections
were identified. However only 31 of those patients had
either unilateral or bilateral thyroarytenoid muscle injec-
tions; the rest (37 patients) had a combination of both
unilateral and bilateral injections (cross over). All diag-
noses were made by a multi-spectral analysis (including
laryngoscopy, voice analysis and speech therapist assess-
ment) by the multidisciplinary team with due considera-
tion to differential diagnoses. Bilateral doses reported to vary between 2.0 mu [10] and
2.5 mu [6,9,11,13] for each side. Both unilateral and bilat-
eral thyroarytenoid muscle injections have been reported
to be successful. To date, published literature has been
inconclusive in comparing their effectiveness [2-5,10,14]. Commercially Botulinum toxin A is available as Botox®
and Dysport®. Botox® is manufactured in the US by Aller-
gan Pharmaceuticals (Irvine, California, USA) and Dys-
port® is manufactured in the UK by Ipsen Products
(Maidenhead, Berkshire, UK). Botox® is available as 100 An information sheet explaining the procedure in simple
non-scientific terms was given to each of the patients. Each patient was asked to sign a consent form prior to Page 2 of 11
(page number not for citation purposes) http://www.head-face-med.com/content/5/1/20 http://www.head-face-med.com/content/5/1/20 Head & Face Medicine 2009, 5:20 Table 2: Patient self-assessment of Voice Score
0
No improvement
1
Very slight improvement
2
Slight improvement
3
Moderate improvement
4
Marked improvement
5
Extreme/near normal treatment. The trial protocol was approved by the local
committee of the ethics for human research. Table 2: Patient self-assessment of Voice Score Inclusion criteria were patients who have had only unilat-
eral or bilateral Dysport® injections for ADSD and contin-
ued on the same regimen for at least five consecutive
treatments. Also, patients needed to be more than 18
years of age. Patients were excluded if they crossed over in
treatment regimes (received unilateral and bilateral injec-
tions), had ADSD with vocal tremor or had previous
laryngeal surgery or trauma. Pregnant women were not
included in this study. Statistical analysis SPSS version 14 and Graph Pad Prism 4.0 statistical soft-
ware packages were used. Student t, Chi Squared and
ANOVA tests were used where appropriate with a signifi-
cance level of p < 0.05. Data were gathered on the response to treatment and the
side effects to the previous injection (Table 1) on a stand-
ardised proforma developed by the authors. Each patient
was asked to rate their best vocal quality following the
previous injection on a six-point scale (Table 2). We recognize a number of statistical assumptions that
were made:- • A sufficient washout period occurred between each
patient consultation. Table 1: Complications of Botulinum toxin injection
Side effects
Total voice loss
Breathy voice "whispery"
Cough
Dysphagia Table 1: Complications of Botulinum toxin injection
Side effects
Total voice loss
Breathy voice "whispery"
Cough
Dysphagia Table 1: Complications of Botulinum toxin injection • There was no cumulative effect of treatment either in
local drug accumulation, motor end plate loss, needle
track scarring or fibrosis from previous haematoma
formation. • There was no cumulative effect of treatment either in
local drug accumulation, motor end plate loss, needle
track scarring or fibrosis from previous haematoma
formation. Page 3 of 11
(page number not for citation purposes) Page 3 of 11
(page number not for citation purposes) Head & Face Medicine 2009, 5:20 http://www.head-face-med.com/content/5/1/20 Monitor showing NPS score of 2
Figure 1
Monitor showing NPS score of 2. Monitor showing NPS score of 2
Figure 1
Monitor showing NPS score of 2. Our study revealed greater mean dysphagia duration and
a shorter breathiness with the bilateral injections (Figure
4) but the difference in duration of these complications
between unilateral and bilateral treatments were not sta-
tistically significant. Page 4 of 11
(page number not for citation purposes) Results Thirty-one patients (16 females/15 males) with ADSD,
who had undergone at least 5 consecutive Dysport® injec-
tions were included in our study (Table 4); those patients
had completed 5 concomitant self-rated efficacy and com-
plication scores questionnaires (Figures 3, 4, 5, 6). There was no significant statistical difference between uni-
lateral and bilateral groups, which both had similar rates
of complications. However, the unilateral group has a
trend to lower complication rate as would be expected. Despite our assumptions the data has passed normality
testing with an alpha score of less than "0.05". There was
a significant difference in the mean voice score (VS)
between unilateral and bilateral groups with the unilateral
groups doing better (Student t test p < 0.05). This is also
confirmed on one way ANOVA test. Even though there is
a trend with unilateral injections having a longer duration
of action this is not statistically significant. The total voice
loss may be dose related since the dosing in bilateral
patients was double the dose in unilateral patients (6.6 vs. 3.6), which could easily account for the 9 episodes of total
voice loss in the bilateral group with none in the unilat-
eral group. There were total of 151 injection episodes, 56 unilateral
and 95 bilateral. Eleven patients received unilateral treat-
ment and 20 bilateral treatments. No patient had their
first injection of Dysport® within our study (Table 4). The mean Dysport® dose was 3.6 ± 0.02 units (~1.2 Botox®
units) for unilateral injection group compared to the
mean total dose of 6.6 ± 0.02 units in bilateral injection
group. The mean interval between injections for the uni-
lateral group was 136 days as compared to 122 in the
bilateral group. No patient expressed any deterioration in
their voice. All the patients in the unilateral group and
90% of the patients in the bilateral group had a voice
score of more than 2. The average voice score of 94% is
consistent with other published results reported [1-
4,14,16]. Page 4 of 11
(page number not for citation purposes) Page 4 of 11
(page number not for citation purposes) Head & Face Medicine 2009, 5:20 http://www.head-face-med.com/content/5/1/20 Monitor showing NPS score of 9
Figure 2
Monitor showing NPS score of 9. Monitor showing NPS score of 9
Figure 2
Monitor showing NPS score of 9. Monitor s
Figure 2 Page 5 of 11
(page number not for citation purposes) Discussion There are disadvantages of giving any injection;
these include discomfort and scarring of the injection
tract. This may later have adverse effects on voice, which
intuitively would be worse as the numbers of injections
increases. Patients' subjective perception of treatment suc-
cess was a significant indicator of outcome in this study. Patients' prime concern is less likely to be the instrumen-
tal measurements and more likely to be about their expe-
rience in daily life. It would have been desirable to also
have an objective quantification of vocal changes. How-
ever, within the constraint of our study this was not feasi-
ble. Patients may have under or over reported their best
voice score and complication duration due to recall bias. Using patient diaries can partially tackle this problem
however we will then face the problem with patient com-
pliance with diary completion as it drops over time [13]. As treatment was to a large extent patient driven and it is
not feasible to blind them to treatment, patient reporting
bias can not be totally eliminated. We used a lower equivalent dose of Botox® preparation
(Dysport®) than in many published studies but with sim-
ilar effectiveness [1-4,14,16]. It is possible that this is due
to more exact needle placement guided by the electro-
physiological waveform of the laryngeal adductors but we
must assume that this is also carried out in other centres
and the differences may simply be a statistical aberration. Whereas some studies have shown a greater voice
improvement and duration of response with the bilateral
injections [9,11]; there have also been studies suggesting
that unilateral thyroarytenoid muscle injections are more
effective with a consistent treatment effect/side effect pro-
file than the bilateral injections [10,16]. Low dose Botuli-
num toxin, especially for the unilateral treatment in our
institution compared favorably to those reported by other
centers. This supports the cumulative dose theory propa-
gated by others [17,18] and can also decrease the likeli-
hood of developing resistance to the medication. Bilateral
Botox® doses reported generally vary between 2 & 2.5 mu
per side, whilst for the unilateral injections has been
much higher (2.5 to 30 mu per site). The amount of Dys-
port® we used was considerably below that of published
data with mean equivalent dose of 1.2 mu of Botox ® com- The efficacy/benefit and complications profile were not
significantly different. Discussion sent those who were not satisfied with their previous treat-
ment. Hence our analysis group may reflect a satisfaction
bias. In this study, our patients had either unilateral or bilateral
thyroarytenoid muscle injections. As expected the 'real
life' situation is reflected by the fact that we had a crosso-
ver group who had a combination of unilateral & bilateral
injections; those patients were excluded from this study. Exclusion was considered necessary in order to compare
pure injections groups. The patients in this group repre- The unilateral injections compared to bilateral injections
have less discomfort, less voice loss and reduced total drug
used. However, historically, bilateral injections were used
because the unilateral injections at that time were not sat- Table 3: The neurophysiological score (NPS) and its electromyographic pattern
NPS
Electromyographic pattern
0
No motor activity
1
No local motor unit activity, few distant motor units
2
No local motor unit activity, moderate distant motor units
3
No local motor unit activity, abundant distant motor units
4
Occasional local activity, abundant distal motor units
5
Few low amplitude low interference pattern local field motor unit activity. moderate distal motor units
6
Moderate low amplitude low interference pattern local field motor unit activity, few distal motor units
7
Abundant low amplitude low interference pattern local field motor unit activity, no distal motor units
8
Half amplitude half interference pattern local field motor unit activity, no distal motor units
9
Near full amplitude near full interference pattern local field motor unit activity, no distal motor units
10
Full amplitude, full interference pattern local field motor unit activity, no distal motor units Table 3: The neurophysiological score (NPS) and its electromyographic pattern Page 5 of 11
(page number not for citation purposes) Head & Face Medicine 2009, 5:20 http://www.head-face-med.com/content/5/1/20 Graph of voice score and laterality
Figure 3
Graph of voice score and laterality. Showing a statistically significant trend for the unilateral injection group to have a bet-
ter voice score over the interval between visits. Graph of voice score and laterality
Figure 3
Graph of voice score and laterality. Showing a statistically significant trend for the unilateral injection group to have a bet-
ter voice score over the interval between visits. efferent signaling to the contra-lateral larynx, however we
cannot support this rationale hence essentially the find-
ings remain unexplained. We feel that this would be the
subject of further physiological experimentation. isfactory. Page 6 of 11
(page number not for citation purposes) Discussion The unilateral injection involved
only a single needle puncture and as would be expected
from a single sided treatment is not associated with total
voice loss. Furthermore it should be noted that the total
volume of Dysport® is increased in bilateral injections
without a commensurate improvement in outcome. The
equivalence of bilateral and unilateral injection outcomes
may be explained in neurophysiological terms. Reduced
afferent feedback may cause a compensatory reduction in Page 6 of 11
(page number not for citation purposes) Head & Face Medicine 2009, 5:20 http://www.head-face-med.com/content/5/1/20 Graph of episodes of complications and laterality of injections
Figure 4
Graph of episodes of complications and laterality of injections. Showing that the unilateral injections group experience
no total voice loss and are otherwise not statistically significantly different to the bilateral injection group. Graph of episodes of complications and laterality of injections
Figure 4
Graph of episodes of complications and laterality of injections. Showing that the unilateral injections group experience
no total voice loss and are otherwise not statistically significantly different to the bilateral injection group. Graph of episodes of complications and laterality of injections
Figure 4
Graph of episodes of complications and laterality of injections. Showing that the unilateral injections group experience
no total voice loss and are otherwise not statistically significantly different to the bilateral injection group. pared to the range of 4-30 mu in the literature for unilat-
eral therapy. thyroarytenoid muscle, which results in scarring and
fibrosis. Another plausible theory is the changes in the
central pathophysiology and or possible effect of the toxin
in the presynaptic neuron [7,20]. Unilateral low dosage
injections of Dysport® proved as successful as low dose
bilateral injections in the treatment of ADSD in this lim-
ited patient sample. However, as treatment was to a large
extent patient driven, there may be a patient reporting
bias. This, however, does not invalidate this empirical
study and is in line with other published series
[2,6,9,10,13]. No improvement (VS = 0) was seen in 8.3%
of bilateral injections compared to 2.2% in the unilateral. Patients who did not improve were more likely to be
retreated with either higher dose or, if treated previously
with unilateral dose, bilateral dose. Although not specifi-
cally investigated, these patients are likely to be those with
more 'difficult' technical aspects to the injections. This
undoubtedly led to the slightly higher incidence of no
response in bilateral vs. unilateral injections. Page 7 of 11
(page number not for citation purposes) Discussion It also may
have caused a slight bias towards better results in unilat-
eral injections. Boutsen et al. suggested that the unilateral
injection method is associated with a better side effect
profile [21]. In the literature, bilateral injections were
found to cause more dysphagia adverse effect [11-14] and
breathiness [9,11]. A systematic review of 22 studies, involving Botulinum
toxin for treatment of spasmodic dysphonia showed no
significant difference in magnitude of effect between the
unilateral and bilateral injections [19]. Liu et al. and
Zwirner et al. have also shown that unilateral and bilateral
injections did not differ in their efficacy or duration in
relieving spasms [13,14]. There have also been studies
that showed no significant difference in magnitude of
effect between the unilateral and bilateral injections but
in comparison much higher doses were used for the uni-
lateral treatments [7,13,14]. The unilateral injection reduces the duration of the proce-
dure and therefore the discomfort; it also makes the use of
local anaesthesia which may interfere with EMG unneces-
sary. Knowing that the unilateral group had a high NPS,
this may empower the patient and may also have a psy-
chological effect, though this study did not specifically
look at the psychological state of the patient. Another advantage of the unilateral injections is the
reduction in discomfort and cumulative needle injury to Page 7 of 11
(page number not for citation purposes) Page 7 of 11
(page number not for citation purposes) Head & Face Medicine 2009, 5:20 http://www.head-face-med.com/content/5/1/20 Graph showing the average interval between presentations and the laterality of injections
Figure 5
Graph showing the average interval between presentations and the laterality of injections. The unilateral injection
group has a trend to a longer period between visits. Graph showing the average interval between presentations and the laterality of injections
Figure 5
Graph showing the average interval between presentations and the laterality of injections. The unilateral injection
group has a trend to a longer period between visits. due to a cumulative effect of toxin over time, requiring
smaller dosage for similar efficacy [24,27]. The total mean dose Dysport® injected in our study was
3.6 mu for the unilateral and 6.6 mu (3.3 mu each side)
for the bilateral injections. Clinically this corresponds to
1.2 mu Botox® for the unilateral and the total of 2.2 mu
(1.1 mu each side) for the bilateral treatments [15,22]. Discussion One advantage to unilateral injections is the reduction in
cumulative needle injury to the thyroarytenoid muscle,
which results to scarring and fibrosis and possibly transac-
tion denervation. The effects of this therefore require fur-
ther investigation. In an experimental model progressive
muscle atrophy was noted [28]. Thus it appears that a relatively lower dose Botulinum
toxin A administered for the unilateral injection episodes
in our institution, compared to those reported by other
centres to produce comparable results. Our results may
support the cumulative dose theory propagated by others
[18,23,24]. Another intuitive advantage to low dosage is decreased
likelihood of development of resistance. This may just be
theoretical in importance as such low cumulative dosage
is given to the larynx. In torticollis, where much higher
dosages are required, it is a more significant issue. Greene
et al. found about 10% of patients treated for torticollis
developed resistance to Botulinum toxin type A [17]. Although to date this has not shown in cases of ADSD, it
has led others to look at other types of Botulinum Toxin
[18]. Several reported series have examined the difference in
outcomes between unilateral and bilateral injections,
both in terms of voice improvement and side effects [25]. Many show a longer duration of the treatment effect with
the bilateral [9-11], compared to the unilateral injections
but some show the opposite [10]. Bielamowicz et al. showed that the unilateral injection has more optimal
and consistent treatment effect/side effect profile [10] and
reduced the spasmodic muscle bursts in both the injected
and non-injected muscles significantly. This is related
closely to improvements in the speech symptoms [26]. Anatomically, there is no muscle fibre communication
between the thyroarytenoid muscles on one side with the
opposite side [26]. Therefore it is unlikely that changes are
result of diffusion of Botulinum toxin to the non-injected
side. The results suggest that changes in the central patho-
physiology may play a role in changes in speech symp-
toms following treatment [26]. There is evidence that in
dystonia, Botulinum toxin transiently changes mapping
of muscle representation areas in the motor cortex, and In our study, we were surprised to find that so many of our
patients improved with a very low dose especially on
injections to only one side. Interestingly, many centers are
now using smaller dosages of Botulinum toxin for injec-
tions than were first used. Page 8 of 11
(page number not for citation purposes) Discussion This has been postulated to be Page 8 of 11
(page number not for citation purposes) Page 8 of 11
(page number not for citation purposes) http://www.head-face-med.com/content/5/1/20 Head & Face Medicine 2009, 5:20 Schematic summary showing average values for voice score (VS), neurophysiological score (NPS) and time intervals betwee
attendance for injection in the unilateral and bilateral injection group
Figure 6
Schematic summary showing average values for voice score (VS), neurophysiological score (NPS) and time
intervals between attendance for injection in the unilateral and bilateral injection group. Schematic summary showing average values for voice score (VS), neurophysiological score (NPS) and time intervals between
attendance for injection in the unilateral and bilateral injection group
Figure 6
Schematic summary showing average values for voice score (VS), neurophysiological score (NPS) and time
intervals between attendance for injection in the unilateral and bilateral injection group. Schematic summary showing average values for voice score (VS), neurophysiological score (NPS) and time intervals between
attendance for injection in the unilateral and bilateral injection group
Figure 6
Schematic summary showing average values for voice score (VS), neurophysiological score (NPS) and time
intervals between attendance for injection in the unilateral and bilateral injection group. nature of the EMG response. This provided an immediate
feedback to the injecting surgeon, reducing the time taken
for each injection and hence patient discomfort. Thus we
found the use of local anaesthesia unnecessary, especially
as it may interfere with the EMG signals [31]. However,
the use of the NPS does not explain why there was no sta-
tistical difference in the mean complication duration in
the unilateral and bilateral injection groups. reorganizes inhibitory and excitatory intracortical path-
ways, probably through peripheral mechanisms [29]. Moreno-Lopez et al. indicated that intracellular retrograde
axonal transport of Botulinum toxin in motor neurons
may occur, suggesting a possible effect of the toxin on the
pre-synaptic neuron. Interestingly the number of bursts in
the non-injected side was reduced to a greater degree in
patients receiving smaller dosages [26]. This correlates
with the good response to treatment to low dose unilat-
eral injection in our study. The NPS can help treatment planning as a disappointing
result from one injection, in a patient who previously
responded well, may be due to poor needle placement(s)
[32]. Page 9 of 11
(page number not for citation purposes) Discussion There are certainly some patients whose anatomy
makes injection technically more challenging, and a poor
result from the last injection combined with a relatively
low neurophysiological score at that injection might lead
one to suspect technical failure [33]. Although our scoring
system is largely subjective, by working consistently with
the same neurophysiologist a level of reproducibility can
be reached which allows for meaningful longitudinal Some studies including a review of head-to-head, rand-
omized, controlled trials of Botox® and Dysport® in pri-
mary palmar hyperhidrosis suggest that Dysport® tends to
have higher efficacy, longer duration, but higher fre-
quency of adverse effects [22,30]. Conversion factors
between the preparations are varied and remain contro-
versial. The neurophysiological score (NPS), a 10-point subjective
scoring system, was used to describe the amplitude and Page 9 of 11
(page number not for citation purposes) http://www.head-face-med.com/content/5/1/20 Head & Face Medicine 2009, 5:20 Table 4: Profile of treated cases (31 patients)
Category
Unilateral group
Bilateral group Table 4: Profile of treated cases (31 patients)
Category
Unilateral group
Bilateral group
Gender
Male
6
9
Female
5
11
Age (years)
Mean
55.65
58.52
Median
63.52
60.54
SD
15.45
16.03
Injection episodes
56
95
Mean interval between injections (days)
136.05 ± 56.2
122.68 ± 52.04
Mean voice score
4.24
3.93
Complications
26 episodes
59 episodes
Total voice loss
0
9 ± 5.73 (n = 6)
Breathy voice "whispery"
9.31 ± 7.76 (n = 16)
6.57 ± 4.88 (n = 35)
Cough
2.4 ± 0.89 (n = 5)
5.45 ± 6.23 (n = 11)
Dysphagia
4.8 ± 5.26 (n = 5)
6 ± 4.65 (n = 7) Table 4: Profile of treated cases (31 patients) Unilateral group comparison. An inter-observer reliability test is being car-
ried out as part of another study. Other critiques of this study include the effect of the learn-
ing curve on the technique which may skew towards uni-
lateral injections over the study period. The subjective
nature of the decision of unilateral vs. bilateral was heav-
ily influenced by patient choice. This, however, does
reflect the current vogue of patient centered care and deci-
sion making and has been similarly reflected by Liu et al
[13]. Further negative skew could be attributed by self
selection of patients who required further treatment and
hence attendance for injection therapy. Discussion Further, it may be
inferred that patients may have preferred one injection
episode to two; however, this inference is not without
clinical significance. The use of established objective
voice-related quality of life questionnaires (i.e. V-RQOL,
VHI...etc.) would help. Our study has assumed that there was a sufficient washout
period such that one injection methodology did not affect
the following. However this is not necessary so, as the
cumulative dose theory propagated [18,24]. Since
patients made their appointment for repeat treatments
when their subjective quality of voice deteriorated, we
assumed that the time intervals between the injection epi-
sodes were the effective duration of the therapy. However
patients may have prolonged their injection intervals due
to personal circumstances. The use of Dysport® preparation of Botulinum toxin A in
our study may have been a confounding factor in obtain-
ing acceptable voice scores with lower clinically equiva-
lent dose compared to Botox® [22,23,34]. Several studies
have shown a greater efficacy for Dysport® injections than
Botox® injections however with increased complication
duration. In this application, this may have been a con-
founding factor and more research into pharmaco-biol-
ogy of different Botulinum toxin A preparations is
warranted. Furthermore, the type of injection (unilateral
vs. bilateral), dosing, and toxin type (Dysport vs. Botox)
are not the only variables that may influence outcomes
after injection. Volumes of reconstitution as well as con-
centration are also factors that may influence outcome as
well and adds to the difficulty in comparing different
research studies. Conclusion We recommend the use of unilateral injections for the
treatment of ADSD. The advantages of unilateral injec-
tions are that there is no total voice loss so the patient can
phonate and less drug dose is used. Treatment planning
should be tailored to individuals keeping an acceptable
balance between symptom relief and side effects. Obvi-
ously, further prospective studies are warranted, perhaps
incorporating the use of voice related quality of life V-
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DL: Comparison of Botulinum toxin injection procedures in
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(page number not for citation purposes) References Foster KA, Bigalke H, Aoki KR: Botulinum neurotoxin - from lab-
oratory to bedside. Neurotox Res 2006, 9(2-3):133-40. Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
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BioMedcentral
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(page number not for citation purposes) Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
Submit your manuscript here:
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BioMedcentral Publish with BioMed Central and every
scientist can read your work free of charge Publish with BioMed Central and every
scientist can read your work free of charge y
(
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eral Botulinum toxin injections in spasmodic dysphonia:
acoustic and perceptual results. J Otolaryngol 1993, 22(3):171-5. p
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18. Blitzer A: Botulinum toxin A and B: a comparative dosing
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133(6):836-8. 19. Whurr R, Nye C, Lorch M: Meta-analysis of Botulinum toxin
treatment of spasmodic dysphonia: a review of 22 studies. Int
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20. Watts C, Nye C, Whurr R: Botulinum toxin for treating spas-
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(page number not for citation purposes)
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Quality improvement project to reduce medicare 1-day write-offs due to inappropriate admission orders
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BMC Health Services Research BMC Health Services Research Oke et al. BMC Health Services Research (2024) 24:204
https://doi.org/10.1186/s12913-024-10594-z Open Access Quality improvement project
to reduce medicare 1-day write-offs due
to inappropriate admission orders Olufolarin Oke1*, K. Michaela Sullivan2, Jason Hom3, David Svec3, Yingjie Weng4 and Lisa Shieh3 Abstract Background We identified that Stanford Health Care had a significant number of patients who after discharge are
found by the utilization review committee not to meet Center for Mediare and Medicaid Services (CMS) 2-midnight
benchmark for inpatient status. Some of the charges incurred during the care of these patients are written-off and
known as Medicare 1-day write-offs. This study which aims to evaluate the use of a Best Practice Alert (BPA) feature
on the electronic medical record, EPIC, to ensure appropriate designation of a patient’s hospitalization status as either
inpatient or outpatient in accordance with Center for Medicare and Medicaid services (CMS) 2 midnight length of stay
benchmark thereby reducing the number of associated write-offs. Method We incorporated a best practice alert (BPA) into the Epic Electronic Medical Record (EMR) that would
prompt the discharging provider and the case manager to review the patients’ inpatient designation prior to
discharge and change the patient’s designation to observation when deemed appropriate. Patients who met the
inclusion criteria (Patients must have Medicare fee-for-service insurance, inpatient length of stay (LOS) less than 2
midnights, inpatient designation as hospitalization status at time of discharge, was hospitalized to an acute level of
care and belonged to one of 37 listed hospital services at the time of signing of the discharge order) were randomized
to have the BPA either silent or active over a three-month period from July 18, 2019, to October 18, 2019. Result A total of 88 patients were included in this study: 40 in the control arm and 48 in the intervention arm. In
the intervention arm, 8 (8/48, 16.7%) had an inpatient status designation despite potentially meeting Medicare
guidelines for an observation stay, comparing to 23 patients (23/40, 57.5%) patients in the control group (p = 0.001). The estimated number of write-offs in the control arm was 17 (73.9%, out of 23 inpatient patients) while in the
intervention arm was 1 (12.5%, out of 8 inpatient patient) after accounting for patients who may have met inpatient
criteria for other reasons based on case manager note review. Conclusion This is the first time to our knowledge that a BPA has been used in this manner to reduce the number of
Medicare 1-day write-offs. Abstract Keywords Utilization review, Center for medicare and medicaid services (CMS), Best practice alert (BPA), Medicare
1-day write offs, Inpatient, Outpatient, Observation, CMS 2-midnight benchmark 2Stanford Health Care, Palo Alto, California, USA
3Stanford University School of Medicine, Palo Alto, California, USA
4Quantitative Sciences Unit, Stanford University School of Medicine, Palo
Alto, California, USA *Correspondence:
Olufolarin Oke
Olufolarin.Oke@UTSouthwestern.edu
1UT Southwestern Medical Center, Dallas, Texas, USA *Correspondence:
Olufolarin Oke
Olufolarin.Oke@UTSouthwestern.edu
1UT Southwestern Medical Center, Dallas, Texas, USA Background Background
Designation of a patient’s hospital encounter at time of
discharge as either outpatient or inpatient, also known
as status determination, is important for all parts of the
health system including hospitals, insurer, and patients. Outpatient is defined by CMS as a person who has not
been admitted as an inpatient but who is registered on
the hospital or critical access hospital (CAH) records as
an outpatient and receives services (rather than supplies
alone) directly from the hospital or CAH [1]. Another
designation that is used for patients hospitalized for
acute care is called observation. Observation care is care
that is provided when additional time for patient test
ing, monitoring and treatment is needed to help deter
mine if inpatient care is needed and according to CMS,
it should be very rare that observation services exceed
48 h and are usually less than 24 h (New referencence)
[2]. Observation status is considered outpatient for CMS
billing purposes. As a hospital system, status deter
mination has an impact on the amount billed for ser
vices provided. This is due to the higher reimbursement
rates by the Center for Medicaid and Medicare services
(CMS) for encounters designated as inpatient which is
billed under Medicare part A and usually reimbursed at
a higher rate than services designated as outpatient ser
vices which are billed under Medicafare part B [3, 4]. Although services provided to patients hospitalized for
inpatient services are almost always of a longer duration
than those provided for patients designateted as outpa
tient, the higher reimbursement rates for inpatient ser
vices remains true even when these services are similar
to those assigned an outpatient designation. Six of the
10 most common reasons for short inpatient stays were
also among the 10 most common reasons for observation
stays [3]. However, short inpatient stays were far more
costly to Medicare than observation stays [3]. Medicare
paid an average of $5,142 per short inpatient stay, but
it paid an average of $1,741 per observation stay [3]. To
deter hospitals from designating a patient as inpatient
when an observation stay may have been appropriate,
CMS established the recovery audit program tasked with
finding and correcting improper claims to the Medicare
program [4]. Data available after creation of this program
showed that a large amount of money is being recouped
yearly. Background The amount of money recouped by Medicare
based on these programs increased from $939 million in
2011, to $2.4 billion in 2012, to $3.8 billion in 2013 [3]. RAC recoupment reduced in subsequent years for sev
eral reasons including hospital’s increased use of appeals
and increased compliance but primarily was a result of
RAC’s change to the program in 2014 due to industry
feedback about the overzealous nature of the RAC pro
gram in its previous state [5] RAC recoupment dropped Inpatient versus outpatient status designation also has
financial consequences for the patients too. Patients are
often responsible for higher payments under Medicare
part B as they may be liable for up to 20% co-insurance
for expenses incurred during their stay [9]. Medicare
fee-for-service and Medicare advantage enrollees must
be provided with the Medicare Outpatient Observation
Notice (MOON) according to CMS rules [10]. This noti
fies patients that they are outpatient receiving observa
tion services and not inpatient.hi The classification of a patient as either inpatient or out
patient is made by the patient’s admitting physician but
CMS has established a rule to guide physicians known as. CMS has established a rule to guide physicians known as. a length of stay benchmark [2, 11]. Providers bill
ing Medicare for services are encouraged to follow this
benchmark in determining a patient’s status at time of
hospitalization as either inpatient or Observation. This
information can be found on the Inpatient Prospective
Payment System (IPPS-2014) final rule and states that a
provider should designate patients whose hospitalization
are expected to span less than two midnights as outpa
tient based on medical necessity with two notable exemp
tions: Procedures appearing on the CMS’s inpatient-only
procedure list and a “rare and unusual” circumstance in
which inpatient admission would be reasonable regard
less of length of stay [12]. Due to the complexity of status
determinations and the monetary advantage an inpatient
designation confers, it is not surprising to see large vari
ability between hospitals in the application of outpa
tient versus inpatient status [3, 6]. To assist physicians
with making this occasionally complex decision and as
required by Medicare Conditions of Participation, most
hospital systems establish a utilization review (UR) team
which often comprises of physicians, nurse case man
agers and physician advisors who have higher levels of
expertise with the insurance related rules. © 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://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. Oke et al. BMC Health Services Research (2024) 24:204 Oke et al. BMC Health Services Research (2024) 24:204 Page 2 of 8 Page 2 of 8 to $24.33 million in 2017, $73.03 million in 2018 and to
$162.03 million in 2019 [6–8]. Background Commercial
tools such as InterQual (McKesson Corporation, San
Francisco, CA) and MCG (formally known as Milliman
Care Guidelines; MCG Health, LLC, Seattle, WA) are
also available to help define inpatients versus outpatients
[13, 14] but CMS guidelines including the 2-midnight
LOS benchmark takes precedence over these tools. At Stanford Health Care, ensuring compliance with
CMS guidelines on hospitalization status (Inpatient ver
sus outpatient) is monitored by the UR team which com
prises of nurse case managers, providers and physician
advisors with expertise in insurance and CMS guidelines. Nurse case Managers review each patient’s designation
within twenty-four hours of hospitalization and prior to
discharge for compliance with CMS criteria for an inpa
tient stay with a physician advisor readily available for
more complex cases. If a patient is admitted as inpatient Oke et al. BMC Health Services Research (2024) 24:204 Page 3 of 8 Page 3 of 8 Oke et al. BMC Health Services Research cost savings was about $72,543 over 17 months in the
post-intervention period [17]. and does not meet length of stay criteria or any of the
exceptions listed on Medicare guidelines, the UR team
including the nurse case manager can then make recom
mendations to the patient’s treating provider who ulti
mately is responsible for making the decision on whether
the patient should remain in inpatient admission status
or if the patient should be changed to an Observation sta
tus.” The billing code associated with this change in sta
tus is known by CMS as a “condition code 44” [1]. After a
condition code 44 is performed, SHC bills for outpatient
services and is reimbursed based on Medicare fee service
for outpatient services (Medicare Outpatient Prospective
Payment Schedule). Unless the patient requires observa
tion services for at least 8 h, the inpatient part B charges
are not captured or billed.f A prospective study done in a University of Virginia
surgical/trauma/burn ICU using a BPA aimed at identify
ing with patients with septic shock and promoting timely
administration of antibiotics [19]. The study showed
that there was a trend towards decreased time-to-anti
biotics following implementation of the BPA (7.4vs4.2 h,
p = 0.057) [18]. Another study done at eight UCLA primary care set
ting in 2013 aimed to assess provider responses to a
focused BPA alert for the intensification of blood pres
sure medications before versus after implementation
of the chart closure hard stop (20). Methods
Settingh The randomized control phase of the study took place at
Stanford Health Care (SHC) which comprises of SHC-
Palo Alto located in Stanford, California, and SHC-Val
ley Care (SHC-VA) located in Pleasanton, California,
between July 18, 2019, and October 18, 2019. The BPA
was non-randomized to all non-surgical services after
the BPA was completed. Independent of this study, SHC
collects data regarding 1-day Medicare write-offs and
reported Medicare 1-day write-offs in charge dollars for
every fiscal year from 2019 to 2023 as part of our BPA
effect confirmation. f
These Medicare 1-day write-offs lead to significant
annual income loss for Stanford Health Care. Charge data
for write-offs for fiscal year 2016–2018 were $10.8 M,
$3.4 M, and $2.6 M. It should be noted that Charge data
is often significantly higher than Medicare allowable
charges/re-imbursement. To reduce these Medicare 1-day write-offs, we sought
to take advantage of the electronic health record system,
in our case (EPIC), to incorporate a tool called best prac
tice alert (BPA) that has been shown to be effective in the
past by other healthcare systems [17–19] in both inpa
tient and outpatient setting for a variety of interventions. BPA has been used successfully to Improve efficient utili
zation use of resources within healthcare system, change
patient clinical outcomes for specific conditions such as
sepsis and increase appropriate medication use in pri
mary care clinic setting for common medical co-morbidi
ties such as hypertension and diabetes. fi
Patients included in the study must meet all the inclu
sion criteria which were: Patients must have Medicare fee
for service insurance, inpatient length of stay (LOS) less
than 2 midnights (Zero to one day), inpatient status des
ignation at time of discharge, was admitted to an acute
level of care and belonged to one of 37 listed hospital ser
vices at the time of signing of the discharge order. Inpa
tient length of stay spans the time from admission order
placement by ED provider to discharge order placement. The names of the 37 non-surgical services are listed in
Appendix 1. An IRB waiver (Protocol number 70,191)
was obtained on a retroactive basis on 4/26/23 based on
the classification of this study as quality improvement. Researchers at University of Florida conducted a three-
year study from 2014 to 2017 that examined the efficacy
of EMR BPA in reducing repetitive laboratory test and
hospital cost [11]. Background Results showed that
among BPA that represent clear opportunities for treat
ment, providers ordered the indicated medication more
often (41% vs. 75%) after the “chart closure” hard stop
was implemented (P = 0.01) [19]. Despite the efforts of our nurse case managers and UR
team, there are often cases of patients who are admitted
and discharged in an inpatient admission status but upon
retrospective review prior to billing CMS are found not
to meet the medical necessity or the length of stay bench
mark for an inpatient stay. CMS can deny these claims if
billed under Medicare Part A for inpatient services if they
also find them not reasonable and necessary [11]. Based on the success of these studies, we aimed to
incorporate a BPA tool into Epic with the goal of improv
ing compliance with Medicare rules regarding appro
priate inpatient status designation to Medicare 1 day
write-off at Stanford Health Care. i
In accordance with CMS rules, SHC does not bill
Medicare under Part A for these patients. Instead, Stan
ford, under a process known as self-denial, bills for part
B services for some ancillary services that qualify per
Medicare rules called “Type of bill-121” [15, 16]. The
remaining charges for services provided but not billed to
Medicare are considered a write-off. Methods
Settingh The intervention reduced the over
all duplicates by 18% (OR = 0.82, standard error + 0.16,
P-value < 0.000). In addition, in this study, the estimated Problem definitioni BMC Health Services Research (2024) 24:204 2019, and October 18, 2019, to SHC-Palo Alto and SHC
Valley Care Medical Center were randomized to either
the intervention arm (BPA fires) or control (BPA silent). A total of 88 patients met this inclusion criteria. We identified several human factors, process/policy,
equipment/supplies, and environmental/cultural issues
in the Stanford healthcare system that contributed to
the failure to designate a patient the appropriate hospi
talization status. After evaluating each one of these fac
tors, the intervention we identified that is most likely to
achieve our goal of improving compliance with Medicare
2 midnight rule regarding the appropriate designation of
patients in an inpatient status thus reducing Medicare
write-offs in the Stanford health care system in the inpa
tient setting was the introduction of a best practice alert
(BPA) in the EPIC electronic medical record system. The
EPIC BPA would alert providers to resolve any discrep
ancy between a patient’s length of stay, the complexity
of primary hospital problem and the designated hospi
talization status during the discharge process but prior
to completing discharge in compliance with Medicare
guidelines. Chart review was then conducted and information
regarding each patient’s designation as either inpatient or
outpatient at time of discharge was documented. Nurse
case manager notes were also reviewed for each patient
to obtain information regarding whether inpatient desig
nation was appropriate based on other criteria that may
not have been known by the discharging provider. The
BPA was then activated in EPIC for all patients in fiscal
year 2019 and income loss attributable to Medicare 1-day
write-off was obtained.h f
The primary outcome was the estimated number of
write-offs in both intervention and control arm while the
secondary outcome was the number of patients assigned
observation status at time of discharge compared to
those assigned an inpatient status. Statistical analysis
d h f i
BPA Efficacy was calculated as the percentage of
patients in the intervention group who were appropri
ately designated correctly either as inpatient or observa
tion at the end of the study. Effect confirmation Review Medicare write-offs at the end of the fiscal year
2019 and 2020 to determine if the BPA has the effect that
would be expected based on the study results above. Statistical analysis
d h f A best practice advisory (BPA) intervention was co-devel
oped the help of our EPIC EMR and utilization review
team including a member who was an expert in Medicare
compliance. Pre-specified inclusion criteria are: Patients
must have Medicare fee for service insurance, inpatient
length of stay (LOS) less than 2 midnights, inpatient sta
tus designation at time of discharge, hospitalization to an
acute level of care and belonged to one of 37 listed hospi
tal services listed in Appendix 1 at the time of signing of
the discharge order. We reported the frequencies and proportions of patients
who were ultimately assigned to inpatient vs. observation
status at discharge by the intervention (Epic BPA) and
control (non-Epic BPA) groups respectively. The differ
ence of the proportions of patients assigned to observa
tion status was compared by the intervention group to
the control group and reported along with the 95% confi
dence interval. We further reported frequencies and pro
portions write-off in patients who were assigned to the
inpatient status by the intervention and control group. Fisher’s exact tests were performed to evaluate the dif
ferences between two groups in both discharge status
assignment and write-offs, using a free online tool which
can be found here (https://www.socscistatistics.com/
tests/fisher/default2.aspx). p < 0.05 is considered as statis
tically significant.fi The BPA which can be seen in Figure S1 fires when a
provider places a discharge order for a patient who meets
the inclusion criteria as stated above. The BPA would
prompt the provider to discuss the patient’s inpatient sta
tus with the utilization review nurse case manager who
also receives an alert. The utilization review nurse case
manager consults with the utilization management team
who then reviews the patient’s hospital stay for medical
necessity to ensure they meet CMS’s established criteria
for inpatient status designation. The utilization review’s
recommendation is then passed on to the provider who
attempted to place the discharge order. If the recom
mendation of the UR team is a change in status from
inpatient to observation and the primary team agrees,
the discharge order is cancelled, and the condition Code
44 process would occur. The previous inpatient order is
cancelled, and an observation order would be placed
followed by a new discharge order. Given that the total
hospitalization time in observation status is essentnially
zero, SHC does not bill CMS for comprehensive observa
tion services. Problem definitioni We identified that Stanford Health Care had a significant
number of patients who after discharge are found by the Page 4 of 8 Oke et al. BMC Health Services Research (2024) 24:204 Oke et al. BMC Health Services Research UR committee not to meet CMS 2-midnight benchmark
for inpatient status. Some of the charges incurred dur
ing the care of these patients are written-off and known
as Medicare 1-day write-offs. We began by performing
a root cause analysis using A3 methodology and a fish
bone analysis as seen in Fig. 1 to identify what interven
tion would best address the issue of income loss due to
Medicare 1- day write-offs. The primary initial event was
incorrect designation of a patient at time of hospitaliza
tion as either observation or inpatient. The two key driv
ers identified in this process were the primary provider
team and the nurse case manager responsible for the
patient as shown in Fig. 2. If neither the primary team
nor the nurse case manager intervened in changing the
ti
t ’ d
i
ti
i
t di
h
thi
ld lik l
Fig. 2 Key drivers and interventions
Fig. 1 Fishbone Analysis Fig. 2 Key drivers and interventions
Fig. 1 Fishbone Analysis Fig. 2 Key drivers and interventions
Fig. 1 Fishbone Analysis Fig. 1 Fishbone Analysis UR committee not to meet CMS 2-midnight benchmark
for inpatient status. Some of the charges incurred dur
ing the care of these patients are written-off and known
as Medicare 1-day write-offs. We began by performing
a root cause analysis using A3 methodology and a fish
bone analysis as seen in Fig. 1 to identify what interven
tion would best address the issue of income loss due to
Medicare 1- day write-offs. The primary initial event was
incorrect designation of a patient at time of hospitaliza
tion as either observation or inpatient. The two key driv
ers identified in this process were the primary provider
team and the nurse case manager responsible for the
patient as shown in Fig. 2. If neither the primary team
nor the nurse case manager intervened in changing the
patients’ designations prior to discharge, this would likely
result in a Medicare write-off. Fig. 2 Key drivers and interventions Fig. 2 Key drivers and interventions Page 5 of 8 Page 5 of 8 Oke et al. BMC Health Services Research (2024) 24:204 Oke et al. Sustain plan BPA efficacy in the intervention group was 98%
On chart review, provider for the one patient n
verted to observation bypassed the BPA with co
“Will discuss with CM” but no nurse case manag
was seen so it was unclear the reason why patient
in an inpatient status. Data from SHC in the subsequent years show
Medicare 1-day stay write-off charge dollars wer
lower compared to the years before the BPA. The
off charge data were $1.0 M for FY 2019, 1.07 M
2020, $792K for FY 2021, $551K for FY 2022 and
for FY23 comapred to $10.8 M, $3.4 M, and $2.6
fiscal years 2016, 2017 and 2018 respeectively. O
charge data is often much higher than allowable
care charges which would represent actual M
1-day write-off amount in dollars. Discussion
We incorporated a BPA to supplement the ef
patient’s treatment provider, nurse case manage
UR team in ensuring compliance with CMS 2 m
rule benchmark for designation of patients as inpa
observation (Outpatient) status thereby reducin
Medicare write-offs. Like other prior studies that have shown the ef
ness of BPA as a tool with diverse impact on im
several aspects of the functioning of a healthcare
[17–19], our study showed that a BPA can be effe
a supplementary tool to improve compliance with
lines in our case Medicare 2 midnight benchm
Table 1 The number of patients with observation status versus inpatient status in intervention and interventional arm
Intervention
(n = 48)
Control
(n = 40)
Difference in % Observation Patient (Intervention-Control)
[95% Confidence Interval]
P
Inpatient
8 (16.7%)
23 (57.5%)
40.8% [22.4%, 59.4%]
0
Observation
40 (83.3%)
17 (42.5%)
*P value based on fisher’s exact test
Fig. 3 Flow chart showing estimated number of write offs in the interven
tion and control arms of the study Fig. 3 Flow chart showing estimated number of write offs in the interven
tion and control arms of the study Fig. 3 Flow chart showing estimated number of write offs in the interven
tion and control arms of the study The estimated value of the 1-day Medicare write-
off averted because of the BPA is estimated to be about
$329,088($5,142*16*4). This calculation is based on his
torical data from CMS who paid an average of $5,142 for
short inpatient stays. BPA efficacy in the intervention group was 98% (47/48). Discussion We incorporated a BPA to supplement the efforts of
patient’s treatment provider, nurse case managers and
UR team in ensuring compliance with CMS 2 midnight
rule benchmark for designation of patients as inpatient or
observation (Outpatient) status thereby reducing 1-day
Medicare write-offs.f i
The estimated number of write-offs in the control arm
was 17 (73.9%, out of 23 inpatient patients) while in the
intervention arm was 1 (12.5%, out of 8 inpatient patient)
after accounting for patients who may have met inpatient
criteria for other reasons based on nurse case manager
note review (Fig. 3).f f
Like other prior studies that have shown the effective
ness of BPA as a tool with diverse impact on improving
several aspects of the functioning of a healthcare system
[17–19], our study showed that a BPA can be effective as
a supplementary tool to improve compliance with guide
lines in our case Medicare 2 midnight benchmark for
appropriate status designation. In addition, the effects of
the BPA have been sustained for several years since com
pletion of the study in 2019 as Medicare 1-day write-offs
post intervention (FY 2019 onwards) has remained con
sistently lower than years before the BPA was instituted. Estimated number of write-offs was calculated using
the number of patients that were not converted from
inpatient to observation as a surrogate after deduct
ing the number of patients who the nurse case manager
stated met inpatient criteria based on chart review.h The percentage of patients who met inpatient criteria
in the intervention arm based on chart review was used
to calculate the number of patients who would have met
such criteria in the control arm if all patients had been
reviewed by the nurse case manager. This is because the
active BPA in the intervention arm ensured that nurse
case managers reviewed the patients prior to discharge. The percentage of patients who met inpatient criteria in
the intervention arm (7/48 = 14.58%). Therefore, about 6
patients (0.1458*40) was calculated to have met inpatient
criteria in the observation group leading to 17 (23 − 6)
estimated number of write-offs in the control group. In addition, this BPA also provides an opportunity to
improve patient satisfaction in our health care system. Sustain plan On chart review, provider for the one patient not con
verted to observation bypassed the BPA with comment
“Will discuss with CM” but no nurse case manager note
was seen so it was unclear the reason why patient was left
in an inpatient status. Data from SHC in the subsequent years showed that
Medicare 1-day stay write-off charge dollars were much
lower compared to the years before the BPA. The write-
off charge data were $1.0 M for FY 2019, 1.07 M for FY
2020, $792K for FY 2021, $551K for FY 2022 and $571K
for FY23 comapred to $10.8 M, $3.4 M, and $2.6 M for
fiscal years 2016, 2017 and 2018 respeectively. Of note,
charge data is often much higher than allowable Medi
care charges which would represent actual Medicare
1-day write-off amount in dollars. Fig. 3 Flow chart showing estimated number of write offs in the interven
tion and control arms of the study Results A total of 88 patients were included in this study: 40 in
the control arm and 48 in the intervention arm. In the
intervention arm, 8 (8/48, 16.7%) had an inpatient status
designation despite potentially meeting Medicare guide
lines for an observation stay, comparing to 23 patients
(23/40, 57.5%) patients in the control group, which is sta
tistically significant (p = 0.001) (Fig. 3; Table 1).hf Sustain plan Utilization management team’s monthly review of BPA
data to ensure that providers are being reminded by
nurse case managers to change patient’s designation from
inpatient to observation when deemed appropriate. Of note, the primary team has the option to bypass the
BPA and the reason for BPA override reason would be
noted. All patients that were admitted between July 18, Oke et al. BMC Health Services Research (2024) 24:204 Page 6 of 8 Table 1 The number of patients with observation status versus inpatient status in intervention and interventional arm
Intervention
(n = 48)
Control
(n = 40)
Difference in % Observation Patient (Intervention-Control)
[95% Confidence Interval]
P value*
Inpatient
8 (16.7%)
23 (57.5%)
40.8% [22.4%, 59.4%]
0.0001
Observation
40 (83.3%)
17 (42.5%)
*P value based on fisher’s exact test Table 1 The number of patients with observation status versus inpatient status in intervention and interventional arm
Intervention
(n = 48)
Control
(n = 40)
Difference in % Observation Patient (Intervention-Control)
[95% Confidence Interval]
P value*
Inpatient
8 (16.7%)
23 (57.5%)
40.8% [22.4%, 59.4%]
0.0001
Observation
40 (83.3%)
17 (42.5%)
*P value based on fisher’s exact test Results
A total of 88 patients were included in this study: 40 in
the control arm and 48 in the intervention arm. In the
intervention arm, 8 (8/48, 16.7%) had an inpatient status
designation despite potentially meeting Medicare guide
lines for an observation stay, comparing to 23 patients
(23/40, 57.5%) patients in the control group, which is sta
tistically significant (p = 0.001) (Fig. 3; Table 1). The estimated number of write-offs in the control arm
was 17 (73.9%, out of 23 inpatient patients) while in the
intervention arm was 1 (12.5%, out of 8 inpatient patient)
after accounting for patients who may have met inpatient
criteria for other reasons based on nurse case manager
note review (Fig. 3). Estimated number of write-offs was calculated using
the number of patients that were not converted from
inpatient to observation as a surrogate after deduct
ing the number of patients who the nurse case manager
stated met inpatient criteria based on chart review. The estimated value of the 1-day Medicare
off averted because of the BPA is estimated to b
$329,088($5,142*16*4). This calculation is based
torical data from CMS who paid an average of $5
short inpatient stays. Data availability
Th d t
t
d Data availability
The datasets used and/or analyzed during the current study are available from
the corresponding author on reasonable request. Discussion Given that patients billed under Medicare part B often
have a higher out of pocket medical bill than those billed
under Medicare part A [7], using the condition code 44
process prior to discharge and providing patients with
the “MOON” prepares patients as opposed to a surprise Oke et al. BMC Health Services Research (2024) 24:204 Oke et al. BMC Health Services Research (2 Page 7 of 8 Page 7 of 8 bill they may receive in the mail if a Part A self-denial is
done by the hospital with retrospective billing for Medi
care part B services. not get the alert or be able to get in touch with provid
ers in time prior to patient discharge. This can be allevi
ated by also alerting a dedicated utilization management
team member simultaneously to follow up in real time if
the case manager is unable to do so but this may strain
resources and increase workload. Condition code 44 billing process provides additional
benefit such as the concurrent review process falls
within the natural workflow of the case management
and UR team workflow process as opposed to a retro
spective review. One of the most important aspects of
utilizing a condition code 44 is to increase compliance
with CMS guidelines which can lessen the risk of CMS
audit for inpatient services given that condition code 44
is billed as outpatient as opposed to inpatient. Another
advantage that is gained is that there is a shorter time to
reimbursement when a condition code 44 process is uti
lized allowing hospitals to be able to allocate resources
and complete other necessary projects more efficiently. Lastly, there is no effect on re-admission data for patients
appropriately placed in observation status. Acknowledgements g
Paul A. Heindenreich for aiding and advise in the statistical analysis of the data
results. Benjamin Leung, for providing data generated from the EPIC BPA alert
in a simple excel format for further analysis. Frederick III, William, MD, PhD for
providing guidance on Medicare claims processing guidelines and referral to
Medicare online resources cited in this paper. Abbreviations The BPA can be potentially modified and expanded to
other status determination scenarios such as the captur
ing of patients in observation status who may qualify for
inpatient status and medically complex patients desig
nated as hospitalized surgical outpatient who spend at
least one night in the hospital for routine recovery who
may qualify for inpatient. At SHC, these cases would be
referred to our physician advisor and UR team for further
review. Supplementary Information Supplementary Information
The online version contains supplementary material available at https://doi. org/10.1186/s12913-024-10594-z. Supplementary Material 1: Figure S1: Screen shot of Epic BPA
Supplementary Material 2: Appendix 1: List of 37 non-surgical hospital
services included in the study The success of the BPA and its continued effectiveness
relies greatly on full participation from all the members
involved in the process which included admitting provid
ers, nurse case managers, physician advisors and other
members of the UR team. Other healthcare systems who
wish to incorporate similar BPA should ensure that all
the team members are fully invested and willing to par
ticipate in the intervention otherwise the efficacy rate of
the intervention will likely decrease. In the future, there
is potential for other types of BPA to be used in address
ing other issues faced within SHC. Author contributions L.S., K.M.S., J.H., D.S. conceived and designed the study. O.O. and L.S. Performed
the study. O.O. wrote the main manuscript text and prepared all figures and
tables and YW wrote the statistics section and portion of the result section. O.O., L.S., J.H., D.S. and K.M.S. performed critical revision of the manuscripts. L.S. and O.O. were the lead investigator. Limitations One main limitation of the study is that the estimated
1-day Medicare write-off is an over-estimation. This is
because charge data as reported in this study is often
much higher than Medicare allowable charges which
would represent the true write-off dollars. However,
our finance department does apply a formula based on
expected payment from that financial class to “value”
the adjustment. Nonetheless, the improved compliance
with Medicare 2 midnight benchmark guidelines is our
desired outcome and remains a benefit. Funding Not applicable. Ethics approval and consent to participate A retrospective human subject determination was submitted to Stanford
university’s IRB for review, and it was determined that this project does
not meet the definition of human subject research as defined in federal
regulations 45 CFR 46.102 or 21 CFR 50.3 and no further IRB review was
required. The need for ethical approval and informed consent was waived
by Stanford university’s IRB. All methods were carried out in accordance with
relevant guidelines and regulations. i
BPA efficacy rate was less than 100% (98%) as the BPA
is not a hard stop. Providers under the pressure of time
may choose to bypass the Epic BPA and if case manag
ers are not available nor assigned to the team, they may Conclusioni We are the first to incorporate this Epic BPA tool into
the discharge workflow and show its positive sustained
effect in reducing Medicare 1-day write-offs. We have
expanded the alerting process to a dedicated UR member
for all services and health insurances. Abbreviations
BPA
Best Practice Alert
SHC
Stanford Health Care
CMS
Center for Medicaid and Medicare Services
IPPS
Inpatient Prospective Payment System
EMR
Electronic Medical Record
RAC
Recovery Audit Program
Supplementary Information
The online version contains supplementary material available at https://doi. org/10.1186/s12913-024-10594-z. Supplementary Material 1: Figure S1: Screen shot of Epic BPA
Supplementary Material 2: Appendix 1: List of 37 non-surgical hospital
services included in the study Abbreviations
BPA
Best Practice Alert
SHC
Stanford Health Care
CMS
Center for Medicaid and Medicare Services
IPPS
Inpatient Prospective Payment System
EMR
Electronic Medical Record
RAC
Recovery Audit Program
Supplementary Information
The online version contains supplementary material available at https://doi. org/10.1186/s12913-024-10594-z. Supplementary Material 1: Figure S1: Screen shot of Epic BPA
Supplementary Material 2: Appendix 1: List of 37 non-surgical hospital
services included in the study Abbreviations
BPA
Best Practice Alert
SHC
Stanford Health Care
CMS
Center for Medicaid and Medicare Services
IPPS
Inpatient Prospective Payment System
EMR
Electronic Medical Record
RAC
Recovery Audit Program References 1. DHHS C. Use of Condition Code 44, Inpatient admission changed to outpa
tient September 10, 2004 Available from: https://www.cms.gov/regulations-
and-guidance/guidance/transmittals/downloads/r299cp.pdf. 1. DHHS C. Use of Condition Code 44, Inpatient admission changed to outpa
tient September 10, 2004 Available from: https://www.cms.gov/regulations-
and-guidance/guidance/transmittals/downloads/r299cp.pdf. 2. CMS. Billing and coding: Acute care: Inpatient, observation and treatment
room services March 17, 2023 Available from: https://www.cms.gov/medi
care-coverage-database/view/article.aspx?articleid=52985. 2. CMS. Billing and coding: Acute care: Inpatient, observation and treatment
room services March 17, 2023 Available from: https://www.cms.gov/medi
care-coverage-database/view/article.aspx?articleid=52985. 13. Corp M. InterQual evidence-based criteria portfolio. 2023 Available from:
https://www.changehealthcare.com/clinical-decision-support/interqual. 14. Health M, Indicia. Evidence-based clinical decision support for provid
ers. 2023 Available from: https://www.mcg.com/how-we-help/providers/
indicia-evidence-based-clinical-decision-support-for-providers/. 3. DHHS, Testimony of Jodi D, Nudelman. Regional Inspector General for the
Office of Evaluation and Inspections, Office of the Inspector General, US
Department of Health and Human Services, Hearing: Current Hospital Issues
in the Medicare Program, House Committee on Ways and Means, Subcom
mittee on Health. May 20, 2014 Available from: https://oig.hhs.gov/docu
ments/testimony/137/20140520_-_Nudelman.pdf. 15. CMS. Billing for Hospital Part B Inpatient Services March 22., 2019 Available
from: https://www.cms.gov/Outreach-and-Education/Medicare-Learning-
Network-MLN/MLNMattersArticles/downloads/MM11181.pdf. 16. Department of Health & Human Services (DHHS) CfMMsC. CMS Manual Sys
tem wPub. 100-04 Medicare Claims Processing September 17, 2004 Available
from: https://www.cms.gov/Regulations-and-Guidance/Guidance/Transmit
tals/downloads/R301CP.pdf. 4. Locke C, Sheehy AM, Deutschendorf A, Mackowiak S, Flansbaum BE, Petty
B. Changes to inpatient versus outpatient hospitalization: Medicare’s 2-mid
night rule. J Hosp Med. 2015;10(3):194–201. 5. CMS. Recovery Audit Program Improvements. 2014 Available from: https://
www.cms.gov/Research-Statistics-Data-and-Systems/Monitoring-Programs/
Medicare-FFS-Compliance-Programs/Recovery-Audit-Program/Downloads/
RAC-Program-Improvements.pdf. 17. Bejjanki H, Mramba LK, Beal SG, Radhakrishnan N, Bishnoi R, Shah C, et al. The role of a best practice alert in the electronic medical record in reducing
repetitive lab tests. Clinicoecon Outcomes Res. 2018;10:611–8. 17. Bejjanki H, Mramba LK, Beal SG, Radhakrishnan N, Bishnoi R, Shah C, et al. The role of a best practice alert in the electronic medical record in reducing
repetitive lab tests. Clinicoecon Outcomes Res. 2018;10:611–8. 18. Chanas T, Volles D, Sawyer R, Mallow-Corbett S. Analysis of a new best-prac
tice advisory on time to initiation of antibiotics in surgical intensive care unit
patients with septic shock. J Intensive Care Soc. 2019;20(1):34–9. 18. Chanas T, Volles D, Sawyer R, Mallow-Corbett S. Analysis of a new best-prac
tice advisory on time to initiation of antibiotics in surgical intensive care unit
patients with septic shock. J Intensive Care Soc. 2019;20(1):34–9. 6. CMS, Appendix B. References Dollar amounts (in Millions) returned to medicare trust
fund in FY 2017 2017 Available from: https://www.cms.gov/files/document/
fy-2017-medicare-ffs-rac-report-congress-appendices.pdf. 19. Ramirez M, Maranon R, Fu J, Chon JS, Chen K, Mangione CM, et al. Primary
care provider adherence to an alert for intensification of diabetes blood pres
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Am Med Inform Assoc. 2018;25(9):1167–74. 19. Ramirez M, Maranon R, Fu J, Chon JS, Chen K, Mangione CM, et al. Primary
care provider adherence to an alert for intensification of diabetes blood pres
sure medications before and after the addition of a chart closure hard stop. J
Am Med Inform Assoc. 2018;25(9):1167–74. 7. CMS, Appendix B. Dollar amounts (in Millions) returned to medicare trust
fund in FY 2018. FY 2018 2018 Available from: https://www.cms.gov/files/
document/fy-2018-medicare-ffs-rac-report-congress-appendices.pdf. 8. CMS. Dollar amounts (in Millions) returned to medicare trust fund in FY
2019. FY 2019 2019 Available from: https://www.cms.gov/files/document/
fy-2019-medicare-ffs-rac-report-congress-appendices.pdf.f Received: 13 April 2023 / Accepted: 11 January 2024 12. CMS. Medicare Program; Hospital inpatient prospective payment systems for
acute care hospitals and the long-term care hospital prospective payment
system and fiscal. Year 2014 Rates; Quality Reporting Requirements for Spe
cific Providers; Hospital Conditions of Participation; Payment Policies Related
to Patient August 19, 2013 Available from: https://www.federalregister.gov/
documents/2013/08/19/2013-18956/medicare-program-hospital-inpatient-
prospective-payment-systems-for-acute-care-hospitals-and-the. Consent for publication
Not applicable. Consent for publication
Not applicable. Page 8 of 8 Page 8 of 8 (2024) 24:204 Oke et al. BMC Health Services Research (2024) 24:204 Oke et al. BMC Health Services Research The authors declare that they have no competing interests. The authors declare that they have no competing interests. 11. DHHS C. CMS Administrator’s Ruling: Part A to Part B rebilling of denied hos
pital inpatient claims March 22, 2013 Available from: https://www.cms.gov/
Regulations-and-Guidance/Guidance/Transmittals/downloads/R1203OTN. pdf. Received: 13 April 2023 / Accepted: 11 January 2024 Competing interests
h
h
d
l
h 10. DHHS C. Medicare Outpatient observation Notice (MOON) instructions Janu
ary 20, 2017. Publisher’s Note 9. CMS. How will my cost be affected by inpatient or observation status
August 25, 2022 Available from: https://www.medicareresources.org/faqs/
how-will-my-costs-be-affected-by-inpatient-or-observation-status/. 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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English
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microRNA-21 regulates astrocytic reaction post-acute phase of spinal cord injury through modulating TGF-β signaling
|
Aging
| 2,018
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cc-by
| 8,877
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ceived: May 3, 2018
Accepted: June 14, 2018 Published: June 23, 2018 Received: May 3, 2018
Accepted: June 14, 2018 Published: June 23, 2018 Copyright: Liu et al. This is an open‐access article distributed under the terms of the Creative Commons Attribution License
(CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and
source are credited. AGING 2018, Vol. 10, No. 6 AGING 2018, Vol. 10, No. 6 ABSTRACT Astrogliosis following spinal cord injury (SCI) was considered as a negative factor for neural regeneration. We
found that miR-21 was significantly upregulated after SCI. So, we aim to determine whether miR-21 acts in a
positive manner post SCI. In vitro, we measured the proliferation, apoptosis and cytokine secretion of primary
cultured astrocytes after modulating the expression of miR-21 by western blot, RT-PCR and
immunofluorescence. In vivo, we performed a modified Allen’s weight drop model. Manipulation of the miR-21
expression level was achieved by interfering with antagomir and agomir. Clinic score was evaluated and
recorded every day. Then, western blot, immunohistochemistry, TUNEL assay and ELISA were performed to
detect pathological and functional alterations. Our results demonstrate that miR-21 can modulate the
secretion, proliferation and apoptosis of astrocytes to promote recovery after SCI both in vivo and in vitro. These effects are likely mediated through transforming growth factor beta mediated targeting of the
PI3K/Akt/mTOR pathway. These data suggest that miR-21 can regulate astrocytic function, then promote the
functional recovery after SCI. We therefore highlight the positive effects of miR-21 after SCI. AGING 2018, Vol. 10, No. 6 www.aging-us.com Research Paper
microRNA-21 regulates astrocytic reaction post-acute phase of spinal
cord injury through modulating TGF-β signaling microRNA-21 regulates astrocytic reaction post-acute phase of spinal
cord injury through modulating TGF-β signaling onghan Liu1, Wenzhao Wang1, Shuya Wang1, Wei Xie2, Hongfei Li1, Bin Ning1 1Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong 250013, China
2Affiliated Hospital of Taishan Medical University, Taian, Shandong 271000, China Correspondence to: Bin Ning; email: ningbin@sdu.edu.cn Correspondence to: Bin Ning; email: ningbin@sdu.edu.cn Keywords: spinal cord injury, microRNA-21, astrocytic scar, fibrotic scar, astrogliosis, TGF-β, glial f
apoptosis Altered miR and mRNA expression after SCI in
vivo. To gain the expression profiling of miR and mRNA
after SCI, we performed the microarray analysis
between tissues the sham (normal) group and day 3
post-SCI group. This screening identified 1213
upregulated mRNAs and 2008 downregulated mRNAs
in SCI group as compared to the sham group. In
addition,
we
found
93
upregulated
and
189
downregulated miRs in the SCI group compared with
the sham group (Fig. 1A and B). After verification and
we hypothesized that miR-21 might serve as an
important candidate of astrogliosis regulator, as it was
significantly upregulated after SCI. The upregulation of
miR-21 expression in tissues 3 days after SCI was
further confirmed by qRT-PCR analysis as compared
with the sham group (Fig. 1C). Meanwhile, the mRNA
screening also suggested many altered gene expressions
such as TGF-β1, GFAP, and CSPGs (Fig. 1B). Furthermore,
the
immunohistochemistry
analysis
confirmed the significantly elevated protein levels of
TGF-β1 (Fig. 1D), GFAP (Fig. 1E), and CSPGs (Fig. 1F) in the SCI sections compared to the sham
counterparts. First,
we
found
that
miR-21
expression
was
significantly upregulated after SCI. Considering the
pathological process within the lesion area after SCI is
mainly fibrotic, we postulated miR-21 as a key factor
for regulating astrogliosis and astrocytic scar formation,
as well as fibrotic scar formation. Even though miR-21
was already reported to regulate the astrocytic response
[29], the mechanisms underlying its response remain
unknown. Further, the in vivo effects of miR-21 are still
yet to be defined. Also the effects of miR-21 in TGF-β
signaling still need further explanation. In the current
study, we investigated the potential role of miR-21 in
astrocytic scar formation during the early stage of SCI
in vitro and in vivo. Although both astrocytic scarring
and miR-21 are controversial in this area, we identified
miR-21 as a main regulator controlling fibrosis and
astrocytic scar formation during the early stage of SCI. INTRODUCTION the loss of the positive effect of astrocytic scars. Moreover, astrocytes play a critical role in the process
of astrocytic scar formation. After injury, astrocytes
become activated, characterized by increased expression
of glial fibrillary acidic protein (GFAP). They become
hypertrophic and hyperplasia occurs around the lesion
site. Subsequently, they acquire stronger secretory
functions,
especially
for
chondroitin
sulfate
proteoglycans (CSPGs), which is a potent inhibitor of
axonal regeneration [2, 6-8]. However, beyond
astrocytes, fibroblasts, pericytes and other inflammatory
cells [9] could also produce CSPGs [10]. Additionally,
secretion of neurotrophins such as brain-derived
neurotrophic factor (BDNF) and nerve growth factor
(NGF) has been identified in astrocytes [11]. In this
regard, we hypothesized that astrocytes might play a
dual role in axon regeneration during both acute and
chronic stages of SCI. Spinal cord injury (SCI) is the most severe complication
of spine trauma because it causes serious physical and
psychological harm. Many studies have suggested that
astrogliosis following SCI resulted in local tissue
hypertrophic response, limited inflammation, and
subsequential formation of glial scars that may act as a
physical barrier for axon regeneration [1, 2]. However,
more recent investigations indicated that astrogliosis
might act positively after SCI [3]. Glial scar not only
prevented inflammation, but could also provide
favorable conditions for axon regeneration [3, 4]. In the
chronic phase after SCI, astrocytic scarring was
suggested to act as a barrier because of its interaction
with fibroblasts [5], although recent studies found that
removal of astrocytic scars from the lesion area did not
improve axon regeneration [3], which probably due to AGING 1474 www.aging-us.com Phosphatase
and
tensin
homologue
deleted
on
chromosome 10 (PTEN) is a reported target protein of
miR-21 and an inhibitor of the phosphoinositide 3-
kinase/protein kinase B/mechanistic target of rapamycin
(PI3K/Akt/mTOR)
signaling
pathway
[27,
30]. Interestingly, TGF-β1 can activate PI3K/Akt/mTOR
signaling. Thus, we hypothesized that TGF-β1 may
activate this pathway by regulating miR-21 expression
and the interaction between miR-21 and TGF-β1 could
be important after SCI. In our study, we found that miR-
21 regulated astrocyte proliferation, secretion, and
activation, and promoted nerve function recovery after
SCI in vitro and in vivo. Although miR-21 can promote
secretion of CSPGs, which inhibit axon regeneration, it
can also promote the secretion of potentially beneficial
factors such as BDNF and NGF. Additionally, we
verified that miR-21 modulated astrocytic scarring
through the PI3K/Akt/mTOR signaling pathway. RESULTS MicroRNAs (miRs) are small (18–22 nucleotides) non-
coding RNA molecules that can regulate gene
expression at the post-transcriptional level by inhibiting
mRNA translation and/or destroying the complete
structure of mRNA [17, 18]. miRs exert various
biological functions in a cell context dependent manner,
including cell proliferation, growth, and differentiation
[17, 18]. Up-regulation or down-regulations of certain
miRs have been implicated in various disease settings
[19-21], and some miRs may serve as key factors for
pathogenesis. miR-21 was reported to stimulate the
fibrogenic effects of hepatic stellate cells in liver,
cardiac tissue and cancer-related fibrosis [22-24]. It is
worth noting that miR-21 could be a key regulator of
fibrosis of several organs [22, 23, 25-28]. INTRODUCTION Collectively, we demonstrate that miR-21 acts as a
positive factor for SCI recovery in the acute phase by
regulating astrocyte function through PTEN-mediate
targeting of PI3K/Akt/mTOR signaling. In our current study, we first measured total mRNA and
microRNA (miR) expression in spinal cord samples
from the 3 days, when the glial scar formed, after SCI
group, and compared them with a control group. After a
microarray screen of miRs and mRNA, and statistical
analysis, we found that many miRs and mRNA
expression were changed. Among these, we found that
miR-21
showed
a
significant
up-regulation. Transforming
growth
factor-beta1
(TGF-β1),
its
receptor and its related proteins were upregulated after
SCI, as previously demonstrated [12]. We also noticed
the expression changes regarding the TGF-β1 related
protein family and TGF-β1 could be produced by many
types of cells, and is believed to promote cell
proliferation and migration [13]. For instance, TGF-β1
activates glial cells and phagocytes to form connective
tissue and extracellular matrix, which may play a key
role during tissue repair post SCI [14, 15]. We thus
speculate that TGF-β1 might play critical roles in
astrocyte hypertrophy and proliferation, and therefore,
are important for both astrogliosis and astrocytic scar
formation [16]. In our current study, we first measured total mRNA and
microRNA (miR) expression in spinal cord samples
from the 3 days, when the glial scar formed, after SCI
group, and compared them with a control group. After a
microarray screen of miRs and mRNA, and statistical
analysis, we found that many miRs and mRNA
expression were changed. Among these, we found that
miR-21
showed
a
significant
up-regulation. Transforming
growth
factor-beta1
(TGF-β1),
its
receptor and its related proteins were upregulated after
SCI, as previously demonstrated [12]. We also noticed
the expression changes regarding the TGF-β1 related
protein family and TGF-β1 could be produced by many
types of cells, and is believed to promote cell
proliferation and migration [13]. For instance, TGF-β1
activates glial cells and phagocytes to form connective
tissue and extracellular matrix, which may play a key
role during tissue repair post SCI [14, 15]. We thus
speculate that TGF-β1 might play critical roles in
astrocyte hypertrophy and proliferation, and therefore,
are important for both astrogliosis and astrocytic scar
formation [16]. miR-21 regulates astrocyte activation and function
in vitro. As previously suggested, TGF-β1 plays a key role
during astrogliosis. After treatment with TGF-β1 during AGING 1475 www.aging-us.com www.aging-us.com Figure 1. Astrocytes were activated and miR-21, TGF-β1, and CSPGs were upregulated after SCI. (A) Heat map of miRs with
significantly altered expression in 3 days after SCI group compared with the sham group (n = 4). (B) Heat map of mRNAs with
significantly altered expression in the 3 days after SCI group compared with sham group (n = 4). (C) qRT-PCR for miR-21 expression in
sham and 3 days after SCI groups (n = 3). Data are expressed as mean ± SD. **P < 0.01 compared with sham group. Expression of TGF-
β1 (scale bar: low magnification, 100μm; high magnification, 20μm) (D), GFAP (scale bar: low magnification, 100μm; high magnification,
20μm) (E) and CSPGs (scale bar: low magnification, 50μm; high magnification, 20μm) (F) in sham and 3 days after SCI group (n = 3). CSPGs, chondroitin sulfate proteoglycans; GFAP, glial fibrillary acidic protein; miR, microRNA; qRT-PCR, quantitative real-time
polymerase chain reaction; SCI, spinal cord injury; SD, standard deviation; TGF-β1, transforming growth factor beta 1. Figure 1. Astrocytes were activated and miR-21, TGF-β1, and CSPGs were upregulated after SCI. (A) Heat map of miRs with
significantly altered expression in 3 days after SCI group compared with the sham group (n = 4). (B) Heat map of mRNAs with
significantly altered expression in the 3 days after SCI group compared with sham group (n = 4). (C) qRT-PCR for miR-21 expression in
sham and 3 days after SCI groups (n = 3). Data are expressed as mean ± SD. **P < 0.01 compared with sham group. Expression of TGF-
β1 (scale bar: low magnification, 100μm; high magnification, 20μm) (D), GFAP (scale bar: low magnification, 100μm; high magnification,
20μm) (E) and CSPGs (scale bar: low magnification, 50μm; high magnification, 20μm) (F) in sham and 3 days after SCI group (n = 3). CSPGs, chondroitin sulfate proteoglycans; GFAP, glial fibrillary acidic protein; miR, microRNA; qRT-PCR, quantitative real-time
polymerase chain reaction; SCI, spinal cord injury; SD, standard deviation; TGF-β1, transforming growth factor beta 1. the astrocytes culture, significantly upregulated GFAP
expression was observed (Fig. 2A and B). In addition,
astrocytes stimulated by TGF-β1 exhibited increased
miR-21 expression compared with the control group
(Fig. 2C). To determine whether miR-21 can regulate
astrocyte activation and function, lentiviral vectors were
used to manipulate miR-21 expression levels in
astrocytes. miR-21 regulates astrocyte activation and function
in vitro. After transfection, we first confirmed that
miR-21 lentiviral vectors worked efficiently as
compared with the negative control (NC) group (Fig. 2C). We observed that TGF-β1 can promote miR-21
expression, with the highest miR-21 expression
observed in the group treated with a combination of
TGF-β1 and miR-21 overexpression (OE) (Fig. 2C). Under these conditions, GFAP was also examined by
western blot and immunofluorescence to evaluate the astrocyte activation and hypertrophy. Indeed, we
observed the upregulated GFAP expression upon
stimulation with TGF-β1 or miR-21 OE (Fig. 2A and
B). In contrast, GFAP expression was downregulated
upon knockdown (KD) of miR-21 expression, whether
or not astrocytes were treated with TGF-β1 (Fig. 2B). Altogether, these findings strongly suggested that miR-
21 could be a downstream regulator of TGF-β1
signaling and the activation of astrocytes. Additionally,
qRT-PCR analysis indicated that the expression of
CSPGs, BDNF, and NGF could be enhanced by miR-21
and reduced by inhibiting miR-21 expression (Fig. 2E). Thus, we speculated that increased CSPGs might result
from enhanced secretion by activated astrocytes. Moreover, BDNF and NGF showed significant
correlation with miR-21, indicating miR-21 might AGING AGING 1476 www.aging-us.com Figure 2. miR-21 regulates astrocyte activation and secretion of CSPGs, NGF, and BDNF. To determine the effects of miR-21
and TGF-β1 on astrocytes, we examined GFAP expression with or without miR-21 and TGF-β1 (10 ng/mL). (A) Astrocytes were treated
with PBS alone or in combination with transfection of miR-21 OE, miR-21 KD, or NC (n = 3). (B) Astrocytes were treated with TGF-β1
alone or in combination with transfection of miR-21 OE, miR-21 KD, or NC. GFAP protein expression was examined by western blot (n =
3). (C) Effects of transfection were verified by qRT-PCR (n = 3). To determine changes in secretory function influenced by miR-21 and
TGF-β1, astrocytes were treated with PBS alone or in combination with transfection of miR-21 OE, miR-21 KD, and NC (n = 3). RNA
expression levels of BDNF (D), CSPGs (E), and NGF (F) were detected by qRT-PCR (n = 3). Data are expressed as mean ± SD. *P < 0.05,
**P < 0.01, ***P < 0.001 compared with NC or TGF-β1 groups; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with NC group. miR-21 regulates astrocyte activation and function
in vitro. BDNF,
brain-derived neurotrophic factor; CSPGs, chondroitin sulfate proteoglycans; GFAP, glial fibrillary acidic protein; miR, microRNA; NGF,
nerve growth factor; miR-21 KD, LV-mmu-miR-21a-inhibition; miR-21 OE, LV-mmu-miR-21a; NC, negative control; qRT-PCR, quantitative
real-time polymerase chain reaction; SCI, spinal cord injury; SD, standard deviation; TGF-β1, transforming growth factor beta 1. Figure 2. miR-21 regulates astrocyte activation and secretion of CSPGs, NGF, and BDNF. To determine the effects of miR-21
and TGF-β1 on astrocytes, we examined GFAP expression with or without miR-21 and TGF-β1 (10 ng/mL). (A) Astrocytes were treated
with PBS alone or in combination with transfection of miR-21 OE, miR-21 KD, or NC (n = 3). (B) Astrocytes were treated with TGF-β1
alone or in combination with transfection of miR-21 OE, miR-21 KD, or NC. GFAP protein expression was examined by western blot (n =
3). (C) Effects of transfection were verified by qRT-PCR (n = 3). To determine changes in secretory function influenced by miR-21 and
TGF-β1, astrocytes were treated with PBS alone or in combination with transfection of miR-21 OE, miR-21 KD, and NC (n = 3). RNA
expression levels of BDNF (D), CSPGs (E), and NGF (F) were detected by qRT-PCR (n = 3). Data are expressed as mean ± SD. *P < 0.05,
**P < 0.01, ***P < 0.001 compared with NC or TGF-β1 groups; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with NC group. BDNF,
brain-derived neurotrophic factor; CSPGs, chondroitin sulfate proteoglycans; GFAP, glial fibrillary acidic protein; miR, microRNA; NGF,
nerve growth factor; miR-21 KD, LV-mmu-miR-21a-inhibition; miR-21 OE, LV-mmu-miR-21a; NC, negative control; qRT-PCR, quantitative
real-time polymerase chain reaction; SCI, spinal cord injury; SD, standard deviation; TGF-β1, transforming growth factor beta 1. control the secretion of these nerve-related factors (Fig. 2D and F). and TGF-β1 promoted proliferation and inhibited the
apoptosis of astrocytes. In this regard, they play a
positive role after SCI, similar to previous studies
examining the effect of TGF-β1 on neural regeneration
[31, 32]. Notably, the proliferation induced by TGF-β1
could be reduced by miR-21 KD, whereas the apoptosis
can be enhanced by miR-21 KD. Similarly, the
expression of proliferation related protein Ki-67, as
detected by immunofluorescence (Fig. 3H), was also
increased which further confirmed the results of western
blots (Fig. 3I and J). Taken together, miR-21 and TGF-
β1 act as important regulators of astrogliosis, and miR-
21 may be a key factor for TGF-β1–induced
astrogliosis. miR-21
regulates
astrocyte
proliferation
and
apoptosis in vitro. While we established that miR-21 could control
astrocyte activation and secretion, whether miR-21 can
regulate their proliferation and apoptosis remained
unknown. Western blot and immunofluorescence were
performed to examine the expression of the apoptotic
proteins Bax, Bcl2, PCNA, caspase 3 and cleaved
caspase 3, as well as GAPDH (Fig. 3A), which was
analyzed by Image J (Fig. 3B–G). Indeed, both miR-21 AGING 1477 www.aging-us.com Figure 3. miR-21 regulates the proliferation and apoptosis of astrocytes. Western blot and immunofluorescence of cells
divided into two groups: one treated with PBS alone, and transfected with miR-21 OE or NC; and a second treated with TGF-β1 alone in
combination with transfection of miR-21 KD or NC. (A) Bax, Bcl2, PCNA, caspase 3, cleaved caspase 3, and GAPDH were examined by
western blot (n = 5). (B–G) Results were analyzed by ImageJ. (H) Ki-67 was examined with immunofluorescence. (I-J) The results of
immunofluorescence were analyzed by imageJ and SPSS. Data are expressed as mean ± SD. *P < 0 .05, **P < 0.01, ***P < 0.001
compared with normal, TGF-β1, or NC group. Bcl2, B-cell lymphoma 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; miR,
microRNA; miR-21 KD, LV-mmu-miR-21a-inhibition; miR-21 OE, LV-mmu-miR-21a; NC, negative control; PBS, phosphate-buffered
saline; PCNA, proliferating cell nuclear antigen; SD, standard deviation; TGF-β1, transforming growth factor beta 1. Fi
3
iR 21
l
h
lif
i
d
i
f
bl
d
fl
f
ll Figure 3. miR-21 regulates the proliferation and apoptosis of astrocytes. Western blot and immunofluorescence of cells
divided into two groups: one treated with PBS alone, and transfected with miR-21 OE or NC; and a second treated with TGF-β1 alone in
combination with transfection of miR-21 KD or NC. (A) Bax, Bcl2, PCNA, caspase 3, cleaved caspase 3, and GAPDH were examined by
western blot (n = 5). (B–G) Results were analyzed by ImageJ. (H) Ki-67 was examined with immunofluorescence. (I-J) The results of
immunofluorescence were analyzed by imageJ and SPSS. Data are expressed as mean ± SD. *P < 0 .05, **P < 0.01, ***P < 0.001
compared with normal, TGF-β1, or NC group. Bcl2, B-cell lymphoma 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; miR,
microRNA; miR-21 KD, LV-mmu-miR-21a-inhibition; miR-21 OE, LV-mmu-miR-21a; NC, negative control; PBS, phosphate-buffered
saline; PCNA, proliferating cell nuclear antigen; SD, standard deviation; TGF-β1, transforming growth factor beta 1. Upregulated miR-21 expression promoted functional
recovery in vivo. appeared to be more significant 7 days after SCI (Fig. 4A). Univariate analysis of variance showed that the
functional recovery was closely related with treatment. To specify the influence of miR-21 on the secretory
functions after SCI, qRT-PCR and Elisa were used to
measure NGF, and BDNF. Indeed, both NGF and
BDNF expression could be inhibited by treatment with
antagomir-21 and induced by agomir-21; whereas
ELISA analysis showed the same tendency (Fig. 4C–F). In addition, we measured the GFAP and CSPGs
expression 7 days after SCI by immunohistochemistry
(Fig. 4G and H), which showed that GFAP and CSPGs
were downregulated after inhibiting miR-21 expression
and upregulated by agomir-21. Altogether, these data To determine whether miR-21 can act positively after
SCI in vivo, antagomir-21, agomir-21, and its negative
control (NC) were used to modulate the miR-21
expression level in a mouse SCI model. We first
confirmed an efficient up or down regulation of miR-21
expression by qRT-PCR (Fig. 4B). Basso mouse scale
(BMS) score was also recorded every day, the scores
indicated that agomir-21 promoted functional recovery
compared with its NC, whereas the antagomir-21
showed the opposite effect. The agomir-21 group
started to show a higher score at 5 days after SCI, which AGING 1478 www.aging-us.com Figure 4. miR-21 has a positive effect on SCI in vivo. To determine whether miR-21 could regulate astrocytes in vivo, expression
of miR-21 was interrupted in a SCI mouse model. Mice were divided into four groups: sham, agomir-21, antagomir-21 and NC. (A) BMS
score for each group (n = 8). Unpaired t-test was used for comparison of agomir-21 and antagomir-21 groups with the antagomir NC
group. Univariate analysis of variance also used to analyze this result showed that miR-21 had significant correlation with BMS scores (P
< 0.001). (B) The expression of miR-21 was detected by qRT-PCR. BDNF (D, F), and NGF (C, E) were detected by qRT-PCR and Elisa (n =
3). Immunohistochemistry was performed to examine GFAP (scale bar: low magnification, 100μm; high magnification, 20μm) (G) and
CSPGs (scale bar: low magnification, 50μm; high magnification, 20μm) (H) and the results were analyzed by ImageJ and SPSS (I and J). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the NC group. Upregulated miR-21 expression promoted functional
recovery in vivo. BDNF, brain-derived neurotrophic
factor; BMS, Basso Motor Score, CSPGs, chondroitin sulfate proteoglycans; GFAP, glial fibrillary acidic protein; miR, microRNA; NGF,
nerve growth factor; NC, negative control; qRT-PCR, quantitative real-time polymerase chain reaction; Elisa, enzyme-linked
immunosorbent assay; SCI, spinal cord injury. Figure 4. miR-21 has a positive effect on SCI in vivo. To determine whether miR-21 could regulate astrocytes in vivo, expression
of miR-21 was interrupted in a SCI mouse model. Mice were divided into four groups: sham, agomir-21, antagomir-21 and NC. (A) BMS
score for each group (n = 8). Unpaired t-test was used for comparison of agomir-21 and antagomir-21 groups with the antagomir NC
group. Univariate analysis of variance also used to analyze this result showed that miR-21 had significant correlation with BMS scores (P
< 0.001). (B) The expression of miR-21 was detected by qRT-PCR. BDNF (D, F), and NGF (C, E) were detected by qRT-PCR and Elisa (n =
3). Immunohistochemistry was performed to examine GFAP (scale bar: low magnification, 100μm; high magnification, 20μm) (G) and
CSPGs (scale bar: low magnification, 50μm; high magnification, 20μm) (H) and the results were analyzed by ImageJ and SPSS (I and J). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the NC group. BDNF, brain-derived neurotrophic
factor; BMS, Basso Motor Score, CSPGs, chondroitin sulfate proteoglycans; GFAP, glial fibrillary acidic protein; miR, microRNA; NGF,
nerve growth factor; NC, negative control; qRT-PCR, quantitative real-time polymerase chain reaction; Elisa, enzyme-linked
immunosorbent assay; SCI, spinal cord injury. indicate that miR-21 could regulate functional recovery
after SCI by controlling astrocyte secretion and
astrogliosis in vivo. /mTOR pathway was involved in the regulatory
mechanisms of miR-21 for astrogliosis, we first
examined the protein expression of key proteins in the
PI3K/Akt/mTOR pathway by western blot (Fig. 5A)
and the expression level was quantified with Image J
(Fig. 5B–K). This signaling pathway as considered as a
major signaling for cell proliferation and growth. Indeed, the phosphorylation of the key components of
the PI3K/Akt/mTOR pathway was upregulated with
miR-21 overexpression. Collectively, these results
showed that miR-21 regulated the expression/activity of miR-21
regulates
astrogliosis
through
the
PI3K/Akt/mTOR pathway. The
PI3K/Akt/mTOR
pathway
has
been
well
documented to play critical roles in regulating cell
proliferation, growth and apoptosis in multiple organ
systems [33, 34]. To explore whether the PI3K/Akt AGING 1479 www.aging-us.com igure 5. miR-21 regulates astrogliosis through the PI3K/Akt/mTOR signaling pathway. Western blot was performed t
xamine changes in the expression of key proteins in the PI3K/Akt/mTOR signaling pathway; an Akt signaling inhibitor was used t
onfirm results. Two groups were examined: one treated with TGF-β1 in addition to transfection with miR-21 KD or NC, and a secon
ansfected with miR-21 OE or NC alone. (A) PTEN, PI3K, p-Akt, Akt, p-mTOR, GAPDH were examined (n = 5). (B–K) Results wer
nalyzed by ImageJ and SPSS. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with NC group. (L) Tw
roups were analyzed: one transfected with miR-21 OE or NC with or without LY294002; and a second treated with TGF-β1 or PBS wit
r without LY294002. p-Akt, Akt, p-mTOR and GAPDH were examined by western blot (n=5). (M–P) Results were analyzed by Image
nd SPSS. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Akt, protein kinase B; GAPDH, glyceraldehyde 3
hosphate dehydrogenase; miR, microRNA; miR-21 KD, LV-mmu-miR-21a-inhibition; miR-21 OE, LV-mmu-miR-21a; mTOR, mechanist
arget of rapamycin; NC, negative control; PBS, phosphate-buffered saline; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase an
ensin homolog deleted on chromosome ten; SD, standard deviation; TGF-β1, transforming growth factor beta 1. Figure 5. miR-21 regulates astrogliosis through the PI3K/Akt/mTOR signaling pathway. Western blot was performed t
examine changes in the expression of key proteins in the PI3K/Akt/mTOR signaling pathway; an Akt signaling inhibitor was used t
confirm results. Two groups were examined: one treated with TGF-β1 in addition to transfection with miR-21 KD or NC, and a secon
transfected with miR-21 OE or NC alone. (A) PTEN, PI3K, p-Akt, Akt, p-mTOR, GAPDH were examined (n = 5). (B–K) Results we
analyzed by ImageJ and SPSS. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with NC group. (L) Tw
groups were analyzed: one transfected with miR-21 OE or NC with or without LY294002; and a second treated with TGF-β1 or PBS wit
or without LY294002. p-Akt, Akt, p-mTOR and GAPDH were examined by western blot (n=5). (M–P) Results were analyzed by Imag
and SPSS. miR-21
regulates
astrogliosis
through
the
PI3K/Akt/mTOR pathway. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Akt, protein kinase B; GAPDH, glyceraldehyde
h
h t d h d
iR
i
RNA
iR 21 KD LV
iR 21 i hibiti
iR 21 OE LV
iR 21
TOR
h
i t Figure 5. miR-21 regulates astrogliosis through the PI3K/Akt/mTOR signaling pathway. Western blot was performed to
examine changes in the expression of key proteins in the PI3K/Akt/mTOR signaling pathway; an Akt signaling inhibitor was used to
confirm results. Two groups were examined: one treated with TGF-β1 in addition to transfection with miR-21 KD or NC, and a second
transfected with miR-21 OE or NC alone. (A) PTEN, PI3K, p-Akt, Akt, p-mTOR, GAPDH were examined (n = 5). (B–K) Results were
analyzed by ImageJ and SPSS. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with NC group. (L) Two
groups were analyzed: one transfected with miR-21 OE or NC with or without LY294002; and a second treated with TGF-β1 or PBS with
or without LY294002. p-Akt, Akt, p-mTOR and GAPDH were examined by western blot (n=5). (M–P) Results were analyzed by ImageJ
and SPSS. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Akt, protein kinase B; GAPDH, glyceraldehyde 3-
phosphate dehydrogenase; miR, microRNA; miR-21 KD, LV-mmu-miR-21a-inhibition; miR-21 OE, LV-mmu-miR-21a; mTOR, mechanistic
target of rapamycin; NC, negative control; PBS, phosphate-buffered saline; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and
tensin homolog deleted on chromosome ten; SD, standard deviation; TGF-β1, transforming growth factor beta 1. AGING 1480 www.aging-us.com Figure 6. Astrogliosis could be regulated though intervention of PI3K/Akt/mTOR signaling. Western blot and
immunofluorescence two groups: one transfected with miR-21 OE or NC with or without LY294002; and a second treated with TGF-β1
or PBS with or without LY294002. (A) Bax, Bcl2, PCNA, caspase 3, and GAPDH were evaluated (B–G) and analyzed by ImageJ and SPSS. (H and I) Immunofluorescence was used for the detection of Ki-67 (H). (J-K) The results were analyzed by ImageJ and SPSS. Data are
expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Akt, protein kinase B; Bcl2, B-cell lymphoma 2; GAPDH, glyceraldehyde 3-
phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; mTOR, mechanistic target of rapamycin; PCNA, proliferating cell nuclear
antigen; PI3K, phosphoinositide 3-kinase; SD, standard deviation. Figure 6. miR-21
regulates
astrogliosis
through
the
PI3K/Akt/mTOR pathway. Astrogliosis could be regulated though intervention of PI3K/Akt/mTOR signaling. Western blot and
immunofluorescence two groups: one transfected with miR-21 OE or NC with or without LY294002; and a second treated with TGF-β1
or PBS with or without LY294002. (A) Bax, Bcl2, PCNA, caspase 3, and GAPDH were evaluated (B–G) and analyzed by ImageJ and SPSS. (H and I) Immunofluorescence was used for the detection of Ki-67 (H). (J-K) The results were analyzed by ImageJ and SPSS. Data are
expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Akt, protein kinase B; Bcl2, B-cell lymphoma 2; GAPDH, glyceraldehyde 3-
phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; mTOR, mechanistic target of rapamycin; PCNA, proliferating cell nuclear
antigen; PI3K, phosphoinositide 3-kinase; SD, standard deviation. miR-21
regulates
astrocyte
proliferation
and
apoptosis in vivo through AKT/mTOR pathway. the key proteins of the PI3K/Akt/mTOR pathway,
which is highly involved in both cell proliferation and
apoptosis. To further confirm whether this signaling
pathway was affected by miR-21 and/or involved in
astrogliosis, the Akt signaling inhibitor LY294002[30]
was used to decrease p-Akt expression (Fig. 5L–P). Western blot and immunofluorescence experiments
demonstrated that LY294002 could reduce miR-21¬–
and
TGF-β1–induced
proliferation
and
promote
apoptosis, as indicated by Bax, Bcl2, PCNA, and
caspase 3 expression (Fig. 6A-G). Finally, the results of
immunofluorescence showed that LY294002 could also
inhibit Ki-67 expression (Fig. 6H), indicating that the
PI3K/Akt/mTOR pathway takes part in the regulation of
miR-21. To confirm these in vitro findings, Western blot was
carried out to detect the protein expression of AKT, p-
mTOR, Bax and bcl2 in astrocytes isolated from lesion
tissue (Fig. 7A). These results were then analyzed by
Image J (Fig. 7B-D). The agomir-21 can active the
AKT and mTOR signaling and the antagomir-21
showed an opposite effect. Accordingly, antagomir-21
can inhibit the proliferation of the cells from tissue. On
the
other
hand,
Ki-67
was
detected
by
immunohistochemistry (Fig. 7E) and the analysis
showed an apparent difference. TUNEL assay also
showed the same trend with in vitro studies (Fig. 7F and AGING 1481 www.aging-us.com Figure 7. miR-21 regulates astrogliosis through the Akt/mTOR signaling pathway. To determine the effects of miR-21 in the
regulation of proliferation and apoptosis in vivo, expression of miR-21 was interrupted in a SCI mouse model. Mice were divided into
four groups: sham, agomir-21, antagomir-21 and NC. (A) p-AKT, AKT, p-mTOR, Bax and Bcl2 were detected by Western blot and
analyzed by ImageJ and SPSS (B-D). (E) the expression of Ki-67 was detected by immunohistochemistry (scale bar: low magnification,
100μm; high magnification, 20μm) and analyzed by ImageJ and SPSS. (G). (F) TUNEL assay (scale bar: low magnification, 100μm; high
magnification, 20μm) was performed and analyzed by ImageJ and SPSS (H). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01,
***P < 0.001 compared with the NC group. miR, microRNA; NGF, nerve growth factor; NC, negative control; SCI, spinal cord injury. Figure 7. miR-21 regulates astrogliosis through the Akt/mTOR signaling pathway. To determine the effects of miR-21 in the
regulation of proliferation and apoptosis in vivo, expression of miR-21 was interrupted in a SCI mouse model. Mice were divided into
four groups: sham, agomir-21, antagomir-21 and NC. miR-21
regulates
astrocyte
proliferation
and
apoptosis in vivo through AKT/mTOR pathway. (A) p-AKT, AKT, p-mTOR, Bax and Bcl2 were detected by Western blot and
analyzed by ImageJ and SPSS (B-D). (E) the expression of Ki-67 was detected by immunohistochemistry (scale bar: low magnification,
100μm; high magnification, 20μm) and analyzed by ImageJ and SPSS. (G). (F) TUNEL assay (scale bar: low magnification, 100μm; high
magnification, 20μm) was performed and analyzed by ImageJ and SPSS (H). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01,
***P < 0.001 compared with the NC group. miR, microRNA; NGF, nerve growth factor; NC, negative control; SCI, spinal cord injury. several nerve growth factors; these features were
positively correlated with neural regeneration. Even
though our data indicated that astrocytes could also
secrete CSPGs, which are proven inhibitors of neural
regeneration [2]. The recent research proved that
astrocytes are not responsible for main accumulation of
CSPGs[3]. So we believed that astrocytes could act as a
good role in the acute phase of SCI. H). These data clearly support the notion that agomir-21
can promote the proliferation and inhibit the apoptosis
of the cells in spinal cord tissue through modulating the
AKT/mTOR signaling. DISCUSSION Axonal regeneration and functional recovery are
important for the prognosis of SCI. During this process,
astrocytes have been well demonstrated to play a
significant role [3]. In the present study, we
demonstrated that astrocytes became proliferating, and
hypertrophied after SCI, and concurrently secreted In our study, we found that miR-21 could act as an
important regulatory factor in the acute phase of SCI. A
recent study also indicated the elevated expression of
miR-21 during the subacute and chronic phases [28]. AGING 1482 www.aging-us.com Figure 8. The signaling pathway which miR-21 can regulate multiple functions in astrocytes. TGF-β1 can up-regulate the
miR-21 level through binding to TGF-β receptor and SMADs signaling. SMAD2/3 can translocate into nucleus and promote the
transcription and maturation of miR-21. After the miR-21 come into the cytoplasm, it can activate the PI3K/Akt/mTOR signaling through
inhibiting the PTEN expression. Also TGF-β1 can activate the PI3K/Akt/mTOR signaling, but this can be regulated by miR-21. Figure 8. The signaling pathway which miR-21 can regulate multiple functions in astrocytes. TGF-β1 can up-regulate the
miR-21 level through binding to TGF-β receptor and SMADs signaling. SMAD2/3 can translocate into nucleus and promote the
transcription and maturation of miR-21. After the miR-21 come into the cytoplasm, it can activate the PI3K/Akt/mTOR signaling through
inhibiting the PTEN expression. Also TGF-β1 can activate the PI3K/Akt/mTOR signaling, but this can be regulated by miR-21. shown in Fig.8). Since we have only examined the acute
phase of SCI in vivo, additional studies are definitely
needed to verify its potential role in promoting neural
regeneration. TGF-β1, a key cytokine for fibrosis with increased
expression after SCI, promoted astrocyte activation and
miR-21 expression. We further demonstrated that the
increased miR-21 expression was mediated by TGF-β1/
SMADs signaling [26], which is also highly relevant in
fibrosis (as shown in Fig.8). By inhibiting miR-21
expression, the astrocyte activation induced by SCI or
TGF-β1 could be suppressed. Thus, miR-21 could be a
downstream factor of TGF-β1 signaling and might play
a vital role in regulating the astrocyte activation,
proliferation, hypertrophy and secretion capacity. The
PI3K/Akt/mTOR pathway, which is implicated in cell
growth, autophagy, proliferation, and other cellular
functions, was involved in this process. Although a
previous study found that miR-21 regulated the
proliferation and apoptosis of spinal cord neurons
through the PTEN/mTOR pathway [28], they did not
examine the detailed underlying mechanism. DISCUSSION By
inhibiting this signaling, the proliferation and secretion
induced by miR-21 and TGF-β1 could be weakened. PTEN, which can suppress this signaling, was proven to
be a target gene of miR-21 [27, 30]. Thus, we proposed
that miR-21 could regulate astrogliosis through the
PI3K/Akt/mTOR pathway by targeting PTEN (as After the acute phase of SCI, several studies have
indicated that fibrotic scarring might be an important
factor inhibiting axonal regeneration [16, 35]. Fibrotic
scar was formed by fibroblasts, and the astrocytes may
promote this formation, Interestingly, our findings
suggest that disrupting astrocytic scarring could regulate
fibrotic scar formation, such that astrocytic scarring
could be an important point in functional recovery after
SCI [4, 36]. Even though in the subacute and chronic
phases, fibrotic scarring becomes the main barrier for
axonal regeneration, it is likely to inhibit astrocytic scar
formation by regulating miR-21 expression. Thus, we
conclude that the astrocytic scar formation by regulating
miR-21 expression during certain phases of SCI may be
vital for neural regeneration. It is known that the pathological processes after SCI are
extremely complicated. In the present study, we
conclude that miR-21 can regulate astrocytic scarring AGING 1483 www.aging-us.com Part 2. Thirty-two mice were randomly divided into
four groups: sham, antagomir, agomir, and negative
control (NC) groups (n = 8 per group). In the negative
control group, mice were subjected to SCI and treated
intrathecally with miR-21 NC (50 μL/d, 100 nmol/mL;
RiboBio, Guangzhou, China) for 3 days (0, 1, and 2
days). In antagomir and agomir groups, mice were
subjected to SCI and treated intrathecally with
antagomir-21 or agomir-21 (50 μL/d, 100 nmol/mL;
RiboBio) for 3 days. In the sham group, mice were
subjected to laminectomy without any treatment. through modulating the PI3K/Akt/mTOR activity by
targeting PTEN. Herein, we highlighted the effect of
miR-21 in promoting neural regeneration after SCI. During the acute phase, astrocytic scarring was
suggested as a positive factor for neural regeneration
[3]. In the subacute phase, astrocytic scarring can also
be beneficial by promoting the secretion of BDNF and
NGF [11], however it can promote fibrotic scar
formation. Thus, promoting astrogliosis by solely
increasing miR-21 expression is an effective treatment
for SCI in the acute phase, but this may not be the case
after that. Cell culture and transfection The primary cultured astrocyte from mouse (M1800-57)
was purchased from ScienCell Research Laboratories
(San Diego, CA). Cells were cultured in RPMI-1640
Medium (Gibco; Thermo Fisher Scientific, Shanghai,
China) supplemented with 10% fetal bovine serum
(Gibco; Thermo Fisher Scientific, Brisbane, Australia)
and
100
IU/mL
penicillin-streptomycin
(Beijing
Solarbio Science & Technology, Beijing, China), and
were incubated in a humidified chamber supplemented
with 5% CO2 at 37ºC. Mice were anesthetized with intraperitoneal 10%
chloral hydrate (3 mg/kg). Laminectomy was performed
at T8–T10 of the thoracic vertebra without any SCI. The
moderate collision injury was caused by a modified
Allen’s weight drop apparatus (8-g weight at 50 mm, 8
g × 50 mm) knocking on the exposed spinal cord. The
sham group was only subjected to laminectomy without
the collision injury. Animals and SCI model Male C57BL/6 background adult mice, provided by the
animal center of Shandong University (Jinan, China),
were maintained in individual cages under controlled
conditions of 22–24°C, relative humidity of 40–60%,
with a 12-h light/dark cycle and free access to food and
water. The operation of this study was performed in
accordance with National Institutes of Health (NIH)
guidelines and regulations of the Shandong University
Committee for the Care and Use of Laboratory
Animals. DISCUSSION Considering the positive effects of astrocytes
in the acute phase and its negative effects in the
subacute phase, and the positive role of miR-21 on
astrocytes, We may speculate to develop the strategies
that promoting the miR-21 expression in the acute phase
and suppressing miR-21 expression in the right time,
may be after the acute phase of SCI, to inhibit
astrogliosis, or possibly even fibroblasts, will be the
main focus of our future studies. Locomotor activity was evaluated for Basso Motor
Score (BMS) for 7 days. Two independent and well-
trained investigators observed movement and scored
locomotor function according to the BMS standard. Treatments were single-blind for the investigators. The
final score of each animal was obtained by averaging
values from both investigators. Mice were sacrificed on
the seventh day by euthanasia with an overdose of 10%
chloral hydrate. The injured segment was exposed and a
10-mm long spinal cord section at the injury lesion was
harvested for further analysis. The mouse's blood is
extracted directly from the heart and centrifuge to
collect the serum for further analysis. miR and mRNA microarray detection After samples were harvested and fresh-frozen in liquid
nitrogen, total RNA was extracted using TRIzol™
(Invitrogen; Thermo Fisher Scientific, Waltham, MA). miR and mRNA microarray detection were performed
by Shanghai Genechem (Shanghai, China). Immunohistochemistry Spinal cord sections were deparaffinized with xylene,
and then hydrated using a series of 100%, 95%, and
85% ethanol solutions before heating in citrate buffer
for 10 min for antigen retrieval. After washing with
PBS twice, sections were incubated for 2 h at 37ºC with
primary
antibodies. Subsequently,
sections
were
incubated in horseradish peroxidase-labeled goat anti-
rabbit IgG secondary antibody for 30 min at 37ºC and
stained with 3,3'-diaminobenzidine tetrahydrochloride
(DAB). After washing with PBS for three times, Animal
experimental
design
and
behavioral
observation LV-mmu-miR-21a (miR-21 OE) and its negative
control (OE NC), as well as LV-mmu-miR-21a-
inhibition (miR-21 KD) and its negative control (KD
NC) were synthesized by Shanghai Genechem. The
transfection was performed by adding polybrene (50
μg/mL; Shanghai Genechem) to the complete medium,
which was combined with 1 x 107 TU of lentiviral
vectors. Part 1. Sham and 3 days post-SCI mice groups were
used for miR and mRNA microarray analysis. Twenty
mice were randomly divided into two groups: sham and
3 days post-SCI (n = 4). Subsequently, microarray data
were confirmed by quantitative real-time polymerase
chain
reaction
(qRT-PCR)
(n
=
3)
and
immunohistochemistry (n = 3). AGING 1484 www.aging-us.com Drop 2000/2000c Spectrometer. Total RNA (1 μg) was
used for cDNA synthesis with Takara PrimeScript RT
Reagent kit (Takara Biotechnology, Dalian, China). Subsequently, expression of GFAP, CSPGs, BDNF, and
NGF were detected by qPCR with a SYBR® Premix Ex
Taq (Takara), using an Applied Biosystems 7300 Fast
RT-PCR system (Thermo Fisher Scientific). mRNA
expression levels of GFAP, CSPGs, BDNF, and NGF
were normalized to GAPDH. miR-21–related qRT-PCR
was performed with a Bulge-Loop™ miRNA qRT-PCR
Starter Kit with miR-21 primers and U6 as an internal
reference for miR-21. (RiboBio, Guangzhou, China). In addition, the fibrosis related factor TGF-β1 (10
ng/mL; Proteintech, Wuhan, China) was used to
activate astrocytes, and the specific PI3K/Akt inhibitor
LY294002 (50 μM; Selleck Chemicals, Houston, TX)
was used to test the role of the Akt pathway for
observed effects of miR-21. Protein isolation and western blot analysis Total protein was isolated from treated astrocytes using
Cell
Lysate
Buffer
(Beyotime
Institute
of
Biotechnology, Beijing, China) containing 1 mM
phenylmethane
sulfonyl
fluoride
and
protein
phosphatase inhibitor (1×; Solarbio Life Sciences,
Beijing, China). Protein was collected after washing,
grinding,
lysis,
and
centrifugation. Protein
concentrations were measured with a NanoDrop™
2000/2000c Spectrometer (Thermo Fisher Scientific,
Wilmington, DE). After mixing with 5× Loading Buffer
(Beyotime), the mixtures were boiled in water at 100ºC
for 10 min and stored at -80ºC. Oligonucleotide primers were as follows: mouse GFAP,
forward
5'-ACCAGCTTACGGCCAACAG-3'
and
reverse 5'-CAGCCTCAGGTTGGTTTCATC-3'; mouse
CSPGs forward 5'-AGTTGGCTTCGTCAGGCACA-3'
and reverse 5'-CACGCACATCACCTGGAAGTC-3';
mouse
BDNF
forward
5'-
TCAAGTTGGAAGCCTGAATGAATG-3' and reverse
5'-CTGATGCTCAGGAACCCAGGA-3'; and mouse
NGF forward 5'-TGCCAAGGACGCAGCTTTC-3' and
reverse 5'-TGAAGTTTAGTCCAGTGGGCTTCAG-3'. Protein samples (100 μg) were separated by SDS-PAGE
and
transferred
onto
polyvinylidene
difluoride
membranes (Millipore, Billerica, MA). After blocking
in 5% milk at room temperature for 1 h, membranes
were incubated with primary antibodies at 4ºC
overnight. The following day, membranes were
incubated with secondary antibody for 1 h at room
temperature. Images were detected by FluorChem E
(ProteinSimple, San Jose, CA) and analyzed by ImageJ
software (version 2.1.4.7; NIH, Bethesda, MD). Immunofluorescence Briefly, cells in 24-well plates were rinsed twice with
phosphate-buffered saline (PBS), fixed for 15 min with
4% paraformaldehyde, permeabilized for 10 min with
0.2% Triton X-100, and then blocked for 30 min with
normal goat serum (Beijing Zhongshan Jinqiao
Biotechnology, Beijing, China). Subsequently, cells
were incubated overnight with primary antibodies at
4ºC. The following day, cells were incubated with
Alexa Fluor® 594 goat anti-rabbit immunoglobulin G
(IgG) secondary antibody (Thermo Fisher Scientific) for
30 min at 37ºC. After washing three times with PBS,
nuclei were stained for 5 min with 4',6-diamidino-2-
phenylindole
(DAPI;
Invitrogen;
Thermo
Fisher
Scientific). Images were acquired with an inverted
fluorescence microscope (Olympus Corporation, Tokyo,
Japan). Primary antibodies were as follows: GFAP
(1:1,000; Abcam) and Ki-67 (1:250; Abcam). Primary and secondary antibodies were as follows:
GFAP (1:1,000; Cell Signaling Technology, Danvers,
MA);
glyceraldehyde
3-phosphate
dehydrogenase
(GAPDH; 1:10,000; Wuhan Sanying Biotechnology,
Wuhan, China), PTEN (1:5,000; Abcam, Cambridge,
UK), PI3K P85 (1:1,000; Abcam), PI3K P110 (1:1,000;
Abcam), phospho-Akt (p-Akt; 1:2,000; Cell Signaling
Technology), Akt (1:1,000; Cell Signaling Technology),
phospho-mTOR (p-mTOR; 1:1,000; Abcam), Bax
(1:1,000; Cell Signaling Technology), B-cell lymphoma
2
(Bcl2;
1:1,000;
Cell
Signaling
Technology),
proliferating cell nuclear antigen (PCNA; 1:1,000; Cell
Signaling Technology), caspase 3 (1:1,000; Cell
Signaling Technology), goat anti-rabbit secondary
antibody (1:5,000; Proteintech, Wuhan, China), and
goat
anti-rabbit
secondary
antibody
(1:5,000;
Proteintech). FUNDING Cleavage of genomic DNA during apoptosis may yield
double stranded, low molecular weight DNA fragments
(mono- and oligonucleosomes) as well as single strand
breaks (“nicks”) in high molecular weight DNA, which
can be measured using In Situ Cell Death Detection kit
(Roche) according to the manufacturer's instructions. Briefly, the paraffin-embedded tissue sections were
permeabilized after dewaxation, rehydration, protease
treatment with 0.1% Triton1) X-100 in 0.1% sodium
citrate. Then the samples were added in TUNEL
reaction mixture and Converter-POD, Substrate solution
in order. The apoptosis was then analyzed using light
microscopy. Grant support was provided by the National Natural
Science Funds of China (Nos. 81401014, 81771346),
the Chinese Postdoctoral Science Foundation (No. 2014M561935), and the Chinese Postdoctoral Science
Foundation (No. 2015T80725), Technology Research
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https://openalex.org/W4390774753
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https://europeanjournaloftaxonomy.eu/index.php/ejt/article/download/2391/10549
|
English
| null |
Morphological and genetic analyses verify the occurrence of the butterfly Graphium chironides (Lepidoptera: Papilionidae) in Peninsular Malaysia and resolve the confusion on the validity of its subspecies malayanum Eliot, 1982
|
European Journal of Taxonomy
| 2,024
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cc-by
| 13,504
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Phon C.-K., Kirton L.G. & Kuah M.-K. 2024. Morphological and genetic analyses verify the occurrence of the
butterfly Graphium chironides (Lepidoptera: Papilionidae) in Peninsular Malaysia and resolve the confusion
on the validity of its subspecies malayanum Eliot, 1982. European Journal of Taxonomy 917: 94–121.
https://doi.org/10.5852/ejt.2024.917.2391 Keywords. Papilionidae, Graphium bathycles bathycloides, Graphium chironides malayanum,
morphology, DNA barcode. European Journal of Taxonomy 917: 94–121 ISSN 2118-9773
https://doi.org/10.5852/ejt.2024.917.2391 www.europeanjournaloftaxonomy.eu
2024 · Phon C.-K. et al..
his work is licensed under a Creative Commons Attribution License (CC BY 4.0).
R e s e a r c h a r t i c l e
urn:lsid:zoobank.org:pub:9BC36596-2248-4A84-B514-938054D32D57
Morphological and genetic analyses verify the occurrence of the
butterfly Graphium chironides (Lepidoptera: Papilionidae) in
Peninsular Malaysia and resolve the confusion
on the validity of its subspecies malayanum Eliot, 1982
Chooi-Khim PHON 1,*,§, Laurence G. KIRTON 2,§ & Meng-Kiat KUAH 3
1,2 Entomology Branch, Forest Research Institute Malaysia (FRIM), 52109 Kepong,
Selangor, Malaysia.
3 Lab-Ind Resource Pte. Ltd., 57–59 Jalan Adenium 2G/6, Pusat Perniagaan Adenium,
48300 Bandar Bukit Beruntung, Selangor, Malaysia.
* Corresponding author: phonchooikhim@frim.gov.my
2 Email: kirton.frim@gmail.com
3 Email: kuahmk@gmail.com
§ Joint first authors
1 urn:lsid:zoobank.org:author:4AC10597-7AA4-401B-8444-3F46E3F0EE44
2 urn:lsid:zoobank.org:author:40CFD67F-FEF4-4490-9C49-F17CE622DD7C
3 urn:lsid:zoobank.org:author:D65D5963-575B-4AEE-AB64-C4698FE86EC6
Abstract. Graphium chironides malayanum Eliot, 1982 was described as a taxon occurring sympatrically
with G. bathycles bathycloides in Peninsular Malaysia. However, the validity of the subspecies has been
uestioned in a recent publication that was based on a study of DNA and morphology, implying that
G. c. malayanum is a synonym of G. b. bathycloides and G. chironides is absent from the Peninsula.
A re-examination of male wing morphology, genitalia and DNA shows that G. c. malayanum is a valid
axon distinguished from G. b. bathycloides by wider discal markings, a less falcate forewing, distinct
ifferences in the arms of the harpe in the male genitalia and clearly divergent mtDNA COI genes. In
he DNA analysis, G. c. malayanum formed a monophyletic clade closely related to G. chironides from
hina, and both were well-separated from the G. b. bathycloides clade. An examination of characters used
n the previous study showed that the conclusions reached were due to misinterpretation of diagnostic
haracters, misidentification of specimens and the absence of G. c. malayanum among the specimens European Journal of Taxonomy 917: 94–121
https://doi.org/10.5852/ejt.2024.917.2391 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
urn:lsid:zoobank.org:pub:9BC36596-2248-4A84-B514-938054D32D57 urn:lsid:zoobank.org:pub:9BC36596-2248-4A84-B514-938054D32D57 Morphological and genetic analyses verify the occurrence of the
butterfly Graphium chironides (Lepidoptera: Papilionidae) in
Peninsular Malaysia and resolve the confusion
on the validity of its subspecies malayanum Eliot, 1982 Morphological and genetic analyses verify the occurrence of the
butterfly Graphium chironides (Lepidoptera: Papilionidae) in
Peninsular Malaysia and resolve the confusion
on the validity of its subspecies malayanum Eliot, 1982 Chooi-Khim PHON 1,*,§, Laurence G. KIRTON 2,§ & Meng-Kiat KUAH 3
1,2 Entomology Branch, Forest Research Institute Malaysia (FRIM), 52109 Kepong,
Selangor, Malaysia. 3 Lab-Ind Resource Pte. Ltd., 57–59 Jalan Adenium 2G/6, Pusat Perniagaan Adenium,
48300 Bandar Bukit Beruntung, Selangor, Malaysia. Chooi-Khim PHON 1,*,§, Laurence G. KIRTON 2,§ & Meng-Kiat KUAH 3
1,2 Entomology Branch, Forest Research Institute Malaysia (FRIM), 52109 Kepong,
Selangor, Malaysia. 3 Lab-Ind Resource Pte. Ltd., 57–59 Jalan Adenium 2G/6, Pusat Perniagaan Adenium,
48300 Bandar Bukit Beruntung, Selangor, Malaysia. Specimens examined We examined and measured 54 specimens of the bathycloides-malayanum group from the collections
and repositories listed below, including all of the rarer broad-banded phenotypes. All specimens were
males, which is the more commonly encountered sex because of its habit of puddling and patrolling. Introduction Graphium bathycles (Zinken, 1831) and G. chironides (Honrath, [1884]) are closely related species of
swallowtail butterflies that are distinguished from other similar black-and-blue species in their genus by
having the discal series of bluish spots on the hindwing well-divided by dark lines along the upper disco-
cellular vein and the cubitus. In addition, the bluish discal spots in the hindwing cell and at the bases of
spaces 7 and 8 form a broken or continuous, curved band. Graphium bathycles is a Sundaic species, while Graphium chironides is primarily a continental species. The latter was long considered a subspecies of G. bathycles, following Rothschild (1895), until Saigusa
et al. (1977) showed that G. bathycles and G. chironides (“chiron,” name preoccupied) have very
different male genitalia and are sympatric in Peninsular Malaysia. The Peninsular Malaysian phenotype
of G. chironides, which was later described as a new subspecies, malayanum Eliot, 1982, has a broader
forewing bluish band than G. b. bathycloides, the Peninsular Malaysian race of G. bathycles. A recent study on the DNA barcode sequences of thirteen species of Graphium Scopoli, 1777 and
the morphology of these two taxa and their type specimens (Wilson et al. 2014) casts doubt on the
validity of the taxon malayanum as a subspecies of chironides that can be differentiated from the taxon
bathycloides. It suggests that the nominal taxon malayanum was based on doubtful taxonomic characters
and, by implication, is a synonym of bathycloides (type locality: Peninsular Malaysia and Borneo). This
would also imply that G. chironides is confined to the continent and does not occur in the Peninsula. The resulting uncertainty over the status of the taxon in Peninsular Malaysia is perhaps reflected in the
choice of the nomenclatural combination “Graphium bathycles malayanum” for the holotype and one of
the paratypes of malayanum in the Data Portal of the Natural History Museum, London (Natural History
Museum 2023a, 2023b, respectively), which suggests that malayanum is an infraspecific taxon under
G. bathycles rather than G. chironides. As a result of these uncertainties, the joint first authors of the present study have in the past received
several enquiries about the status of G. chironides in Peninsular Malaysia. Therefore, in this paper, we
address the question of whether there are two separable phenotypes that correspond to two different
species in the Peninsula using the morphology of the wings and genitalia, and genetic sequences. § Joint first authors Abstract. Graphium chironides malayanum Eliot, 1982 was described as a taxon occurring sympatrically
with G. bathycles bathycloides in Peninsular Malaysia. However, the validity of the subspecies has been
questioned in a recent publication that was based on a study of DNA and morphology, implying that
G. c. malayanum is a synonym of G. b. bathycloides and G. chironides is absent from the Peninsula. A re-examination of male wing morphology, genitalia and DNA shows that G. c. malayanum is a valid
taxon distinguished from G. b. bathycloides by wider discal markings, a less falcate forewing, distinct
differences in the arms of the harpe in the male genitalia and clearly divergent mtDNA COI genes. In
the DNA analysis, G. c. malayanum formed a monophyletic clade closely related to G. chironides from
China, and both were well-separated from the G. b. bathycloides clade. An examination of characters used
in the previous study showed that the conclusions reached were due to misinterpretation of diagnostic
characters, misidentification of specimens and the absence of G. c. malayanum among the specimens
examined. When these characters were correctly interpreted, each specimen was readily assigned to the
correct taxon. Diagnostic morphological characters are reclarified based on the current data. Keywords. Papilionidae, Graphium bathycles bathycloides, Graphium chironides malayanum,
morphology, DNA barcode. Phon C.-K., Kirton L.G. & Kuah M.-K. 2024. Morphological and genetic analyses verify the occurrence of the
butterfly Graphium chironides (Lepidoptera: Papilionidae) in Peninsular Malaysia and resolve the confusion
on the validity of its subspecies malayanum Eliot, 1982. European Journal of Taxonomy 917: 94–121. https://doi.org/10.5852/ejt.2024.917.2391 94 PHON C.-.K. et al., Graphium chironides (Lepidoptera) in Peninsular Malaysia Material and methods To determine whether there are two phenotypes in the Peninsula, we measured wing characters, including
those described by Eliot (1982). Principal components analysis (PCA) of key characters combined with
bivariate ordination was used to identify differing phenotypes. Male genitalia and genetic sequences
were used to associate phenotypes to known taxa outside the Peninsula. The interpretation of characters
by Wilson et al. (2014) was examined, and the reliability of all measured wing characters was determined
by critical quantitative analysis. Collections and repositories Chong-Arshad = The joint private collection of Chow-Yang Chong and Sabri John Arshad
FRIM
= Entomological Reference Collection, Forest Research Institute Malaysia, Kepong
Kirton
= The joint private collection of Eric, Colin and Laurence Kirton
Liew
= The private collection of Nyok-Lin Liew g-Arshad = The joint private collection of Chow-Yang Chong and Sabri John Arshad g
y
p
g
= The joint private collection of Eric, Colin and Laurence Kirton = The private collection of Nyok-Lin Liew 95 European Journal of Taxonomy 917: 94–121 (2024) MNM
= Natural History Museum of the Malaysian National Museum, Department of Museums,
Kuala Lumpur
MZUM
= Museum of Zoology, University of Malaya, Kuala Lumpur (abbreviated as UMKL
in Wilson et al. 2014)
ZRC
= Zoological Reference Collection, Lee Kong Chian Natural History Museum, National
University of Singapore MNM
= Natural History Museum of the Malaysian National Museum, Department of Museums,
Kuala Lumpur
MZUM
= Museum of Zoology, University of Malaya, Kuala Lumpur (abbreviated as UMKL
in Wilson et al. 2014)
ZRC
= Zoological Reference Collection, Lee Kong Chian Natural History Museum, National
University of Singapore In addition, we examined photographs of types and their labels in the Data Portal of the Natural History
Museum, London: a specimen labelled and identifiable as the holotype of G. c. malayanum with
accession number BMNH(E)#149669 (Natural History Museum 2023a), and a specimen said to be
a syntype of G. b. bathycloides with accession number BMNH(E)#149397 (Natural History Museum
2023c). These are likely to be the same photographs said to be provided by John Chainey of the Natural
History Museum, London to Wilson et al. (2014), as they have the same accession numbers mentioned
above. Two specimens figured in Wilson et al. (2014) that could not be traced in the MZUM and a
specimen seen in the wild at Fraser’s Hill, Pahang by Michael Soh (Soh 2023) were also examined only
from photographs. Wing characters and measurements Wing characters measured are listed and described in Table 1 and illustrated in Fig. 1. Several of the
characters (nos. 1 to 5 and 8 to 13) are based on Eliot’s (Eliot 1982; Corbet & Pendlebury 1992), while
two (nos. 6 and 14) were based on suggestions by C.Y. Chong (pers. com.) and five (nos. 7, and 15 to
18) were our own. Characters on which nos. 14 and 18 are based were originally pointed out by Wallace
(1865) in his original description of Graphium chironides (“Papilio chiron”). We did not attempt to
quantify the colour characters described by Eliot, as we deemed these to be inconsistent and affected by
specimen age. Specimens were usually photographed against a calibrated ruler (GEI International) with an error of
less than ± 0.0083 mm per cm over a scale measurement length of 15.24 cm. When a different ruler was
used, it was calibrated against the former wherever possible. The right wing was measured, or the left if
the right was too damaged. Linear measurements were made using image analysis software (Digimizer®
ver. 4.6.1.), while area measurements were made using Adobe Photoshop® ver. 6.0.1 with the aid of the
magic wand tool. Where specimens were too damaged or could only be analysed based on available
photographs that lacked a scale, measurements were restricted to ratio and angle characters that were
independent of scale. Genitalia The genitalia of six males of each phenotype that was identified by the ordination procedure described
above were dissected, examined, and matched with described taxa. Since a detailed comparison of
the male genitalia of the two taxa, G. c. malayanum and G. b. bathycloides, has not previously been
published, we have figured and described the genitalia in detail with an emphasis on key differences
between the taxa and individual variation. An explanation of the methods used in imaging the genitalia
is given in Supp. file 1 (Part A). Ordination of phenotypes Since the width of the blue spots on the forewing upperside was a key character used by Eliot (1982) in
distinguishing G. c. malayanum from G. b. bathycloides, we used principal components analysis (PCA)
of the correlation matrix in Minitab® ver. 19.2020.1 to reduce the individual widths of the blue spots in
spaces 1b to 5 to a linear combination of these widths that represented the greatest variance among the
specimens (component 1 of the PCA). Scores for principal component 1 were plotted against forewing
length as a standardising variable for size, and against forewing angle because a less falcate forewing
has been said to be a character of Graphium chironides (Wallace 1865). In addition, we plotted the width
of the forewing spot in space 5 relative to the length of vein 5 (a ratio) against the forewing angle and
included other specimens such as the types and specimens for which only images without a scale were
available. Representative specimens from the natural groups (phenotypes) that could be seen in the
scatter plots were examined for differences in their genitalia and DNA barcodes. 96 96 PHON C.-.K. et al., Graphium chironides (Lepidoptera) in Peninsular Malaysia DNA barcodes Genetic analysis was used to further investigate the phenotypes obtained by ordination and to validate
the identities that were obtained by comparison of genitalia morphology. Mitochondrial DNA (mtDNA)
from the cytochrome oxidase subunit I gene (COI) was used since ribosomal RNA (28S rRNA) did not
adequately separate species of Graphium in Wilson et al. (2014). Two specimens of each phenotype
that was identified by the ordination were sequenced. In addition, we used three Peninsular Malaysian
specimen sequences from Wilson et al. (2014) and two available sequences of specimens from China. Fig. 1. Illustrations of characters measured on the right wings of specimens of Graphium Scopoli, 1777,
as described and numbered in Table 1. Area measurements are not illustrated but were measured for the
corresponding spots on which linear measurements are shown. Left: upperside wing spot measurements. Middle: underside hindwing spot measurements for two phenotypes. Right: wing dimension, vein and
angle measurements (diagrammatic, showing wing outline, veins and vein numbers). Abbreviations:
1B′ = straight-line length representing vein 1b as measured for calculation of ratio 1B; 3B′ = example
straight-line vein length as measured for calculation of ratio 3B; C = cubitus; R = radius; UD, MD and
LD = upper, middle and lower discocellular veins, respectively. Fig. 1. Illustrations of characters measured on the right wings of specimens of Graphium Scopoli, 1777,
as described and numbered in Table 1. Area measurements are not illustrated but were measured for the
corresponding spots on which linear measurements are shown. Left: upperside wing spot measurements. Middle: underside hindwing spot measurements for two phenotypes. Right: wing dimension, vein and
angle measurements (diagrammatic, showing wing outline, veins and vein numbers). Abbreviations:
1B′ = straight-line length representing vein 1b as measured for calculation of ratio 1B; 3B′ = example
straight-line vein length as measured for calculation of ratio 3B; C = cubitus; R = radius; UD, MD and
LD = upper, middle and lower discocellular veins, respectively. Fig. 1. Illustrations of characters measured on the right wings of specimens of Graphium Scopoli, 1777,
as described and numbered in Table 1. Area measurements are not illustrated but were measured for the
corresponding spots on which linear measurements are shown. Left: upperside wing spot measurements. Middle: underside hindwing spot measurements for two phenotypes. Right: wing dimension, vein and
angle measurements (diagrammatic, showing wing outline, veins and vein numbers). DNA barcodes 98 PHON C.-.K. et al., Graphium chironides (Lepidoptera) in Peninsular Malaysia Type of character
No. Character abbreviation
Character description§
Hindwing underside orange
spots *
10A
UnHwSp5SA
Size (area) of the spot at the base
of space 5, if present. 10B
UnHwSp5SL
Maximum length of the spot at
the base of space 5, if present. 11A
12A
13A
UnHwSp2OSA
UnHwSp3OSA
UnHwSp4OSA
Size (area) of the spots in spaces
2, 3 and 4, respectively. 11B
12B
13B
UnHwSp2OSW
UnHwSp3OSW
UnHwSp4OSW
Width of the spots in spaces 2,
3 and 4, respectively, measured
midway between and parallel to
the bounding veins. Wing shape and size
14
FwAng
Angle formed between two lines
originating at the end of vein
7 on the forewing margin, one
extending to the base of vein
7 at the cell-end and the other
extending to the end of vein 5 on
the forewing margin. 15
16
FwWV2
FwWV5
The widths of the forewing
between the costa and termen on
straight lines that cross the bases
of veins 2 and 5, respectively,
and terminate at the end of the
respective vein. 17
FwL
Forewing length, measured from
the base of the cell to the wing
apex. 18
HwAng
Angle formed between two lines
originating at the end of vein 2
on the apical hindwing margin,
one extending to the base of the
cubitus and the other extending
to the end of vein 8 on the wing
margin. § Units for measurements: mm (linear), mm2 (area), degrees (angle). * Hindwing underside orange spot in space 1b
omitted because the hindwing tends to fold near the dorsum, partially occluding the spot. Table 1 (continued). Descriptions of wing characters measured. Characters are illustrated with their
numerical codes in Fig. 1. Table 1 (continued). Descriptions of wing characters measured. Characters are illustrated with their
numerical codes in Fig. 1. § Units for measurements: mm (linear), mm2 (area), degrees (angle). * Hindwing underside orange spot in space 1b
omitted because the hindwing tends to fold near the dorsum, partially occluding the spot. Graphium agamemnon agammemnon (Linnaeus, 1758) and G. evemon eventus (Fruhstorfer, 1908) were
used as outgroups. All specimens were examined except those from China. Graphium agamemnon agammemnon (Linnaeus, 1758) and G. evemon eventus (Fruhstorfer, 1908) were
used as outgroups. All specimens were examined except those from China. DNA barcodes Abbreviations:
1B′ = straight-line length representing vein 1b as measured for calculation of ratio 1B; 3B′ = example
straight-line vein length as measured for calculation of ratio 3B; C = cubitus; R = radius; UD, MD and
LD = upper, middle and lower discocellular veins, respectively. 97 European Journal of Taxonomy 917: 94–121 (2024) Table 1 (continued on next page). Descriptions of wing characters measured. Characters are illustrated
with their numerical codes in Fig. 1. Type of character
No. Character abbreviation
Character description§
Forewing upperside band
1A
2A
3A
4A
5A
UpFwSp1bBW
UpFwSp2BW
UpFwSp3BW
UpFwSp4BW
UpFwSp5BW
The width of the discal band
between the innermost and
outermost edges of the pale blue
spot in space 1b, 2, 3, 4 and 5,
respectively. 1B
2B
3B
4B
5B
UpFwSp1bBWR
UpFwSp2BWR
UpFwSp3BWR
UpFwSp4BWR
UpFwSp5BWR
The ratio formed by dividing
characters 1A–5A above by a
straight-line length representing
the vein beneath the spots: in 1B,
the straight-line being from the
cubitus along vein 1b bisecting
the spots in spaces 1a and 1b and
terminating at the end of vein 1b;
in 2B–5B, the straight lines being
from the origins to the ends of
the respective veins. Hindwing upperside bluish
spots
6A
UpHwSp1bSA
Size (area) of the streak at the
base of space 1b, if present. 6B
UpHwSp1bSL
Length of the streak at the base
of space 1b, if present, from its
furthest basal extent to its most
apical end. 7A
UpHwSp2SA
Size (area) of the spot in the
basal half of space 2. 7B
UpHwSp2SL
Length of the spot in the basal
half of space 2 from its furthest
basal extent to its most apical
end. Hindwing underside bluish
spots
8A
UnHwSp1bSA
Size (area) of the subdiscal streak
in space 1b, if present. 8B
UnHwSp1bSL
Length of the subdiscal streak
in space 1b, if present, from its
furthest basal extent to its most
apical end, even if the streak is
broken. 9A
UnHwSp3SA
Size (area) of the spot at the base
of space 3, if present. 9B
UnHwSp3SL
Length of the spot at the base
of space 3, if present, from its
furthest basal extent to its most
apical end. Table 1 (continued on next page). Descriptions of wing characters measured. Characters are illustrated
with their numerical codes in Fig. 1. Table 1 (continued on next page). Descriptions of wing characters measured. Characters are illustrated
with their numerical codes in Fig. 1. DNA barcodes DNA was extracted from a single hindleg of each specimen using the QIAamp DNA Mini Kit (Qiagen
2016: 32–35, 53–54). The primer pair described by Wilson (2012), LepF1 and LepR1, was used to
amplify a 658-bp fragment from the COI gene. PCR was performed on 3 µl of extracted DNA in a Q5®
Hot Start High-Fidelity Master Mix (New England Biolabs, USA) following the thermocycles described
by Wilson (2012). The resulting PCR products were then gel purified and sequenced in both forward and
reverse directions. 99 European Journal of Taxonomy 917: 94–121 (2024) Sequences were aligned using ClustalW with default parameters in MEGA ver. 11.0.11. Base pairs
outside of the target 658-bp region were trimmed manually. A maximum-likelihood (ML) tree was
constructed using IQ-TREE ver. 2.2.0 (Minh et al. 2020) in which ModelFinder (Kalyaanamoorthy
et al. 2017) was applied to determine the best-fit model, and ultrafast bootstrap (Hoang et al. 2018) and
an SH-like approximate likelihood ratio test (SH-aLRT) were used in the analysis. ModelFinder selected
the model TIM2+F+I with the highest Bayesian Information Criterion (BIC) weight. The analysis
produced one maximum likelihood tree and one consensus tree. Support values were determined from
1000 re-samplings. Examination of characters used by Wilson et al. Specimens used by Wilson et al. (2014) were re-examined as far as possible, and the characters they
used were also examined in detail with reference to their illustrations and descriptions so as to determine
reasons for their perceived ambiguity. Separability of taxa based on each wing character Quantified characters were compared to determine those that differ on average between taxa and those
that can reliably separate them. Median, mean, inter-quartile range (IQR), 1.5 × IQR and outliers were
compared graphically between the taxa. Nonparametric multivariate tests (NPMV) in R ver. 4.2.0 were
used to determine whether there were significant differences between taxa after grouping variables by
type (structural characters and wing markings were tested separately). Where width and a width ratio
were measured for the same wing marking, only width was tested to avoid redundancy. Similarly, where
area and length or width were measured for the same wing marking, only area was tested. Based on the
results, we clarify the diagnostic characters of the taxa concerned. Ordination of phenotypes Component 1 of the PCA of widths of the forewing blue spots in spaces 1b to 5 represented 86.4% of
the variance. Eigenvectors were 0.392, 0.464, 0.467, 0.459, and 0.449, respectively, indicating all spots
but especially those from space 2–5 contributed in the same direction and in nearly the same magnitude
to this component. Scores of the first component of PCA were plotted against forewing length and forewing angle, and the
resulting ordinations revealed two clusters, one comprising 11 specimens with markedly wider wing
spots and the other comprising 43 specimens with narrower wing spots with respect to wing length and
forewing angle (Fig. 2). The cluster with wider wing markings corresponded exactly with the cluster
with a larger forewing angle or less falcate forewing shape. Similar separation into two groups was observed when the ratio of the width of the forewing spot in
space five to vein length was plotted against forewing angle (Fig. 3). Measurements made on the images
of the holotype of G. chironides malayanum and a syntype of G. bathycles bathycloides enabled them
to be included in the ordination, and they clustered with the wide- and narrow-spotted phenotypes,
respectively (Fig. 3). Three specimens used by Wilson et al. (2014) that we were either unable to trace
(KC0002 and KC0003) or that had broken wing apices (JJW0119) clustered with the narrow-spotted
phenotype as did the other specimens they used (Fig. 3), while a specimen photographed in the wild
(Soh 2023) clustered with the wide-spotted phenotype (Fig. 3). All measurements and specimen data are
given in Supp. file 2 (Part A). Georeferencing of the collection localities from the data labels (excluding
two imprecisely labelled specimens indicated in the supplement) showed that the two phenotypes
occurred sympatrically and sometimes at the exact same location in Peninsular Malaysia (Fig. 4). 100 PHON C.-.K. et al., Graphium chironides (Lepidoptera) in Peninsular Malaysia Fig. 2. Scatterplots of scores of PCA component 1 (a linear combination representing forewing
band width) against (a) forewing length and (b) forewing angle. The two resulting widely-separated
clusters are indicated by square and round markers, and two clusters correspond in specimens
between graphs. Fig. 2. Scatterplots of scores of PCA component 1 (a linear combination representing forewing
band width) against (a) forewing length and (b) forewing angle. The two resulting widely-separated
clusters are indicated by square and round markers, and two clusters correspond in specimens
between graphs. Fig. Ordination of phenotypes 3. Scatterplot of the width ratio of the forewing spot in space five against the forewing angle, for
both the current dataset and specimens examined in images without an included scale. Fig. 3. Scatterplot of the width ratio of the forewing spot in space five against the forewing angle, for
both the current dataset and specimens examined in images without an included scale. 101 European Journal of Taxonomy 917: 94–121 (2024) Differences in the structure of the genitalia When six representative specimens from each of the two clusters in the scatter plots (Figs 2–3) were
dissected, clear differences could be seen between them, and they were readily recognisable as the two
taxa, G. bathycles and G. chironides. An overview of the lateral profile of the genitalia of the Peninsular
Malaysian races of both taxa is shown in Fig. 5, with greater detail of the different parts of the genitalia
shown in Fig. 6. The general structure of the genitalia of both taxa is discussed in Supp. file 1 (Part B). The genitalia differ between the two taxa mainly in the harpe, which is the highly sclerotised, armed
structure in the middle of the inner surface of the valva (Fig. 5b, d). The harpe comprises three arms. One
arm (hereafter referred to as the lower arm) is situated posteroventrally, and the arm on the opposite end
of the harpe (upper arm) is situated anterodorsally, while the other arm (middle arm) is located between
them (Figs 5, 6d, h). Differences between the harpe of the two taxa are illustrated most clearly by images of the arms of the
harpe shown in Fig. 7, which were taken at different angles. The harpe of the narrow-spotted phenotype Fig. 4. Distribution of G. c. malayanum Eliot, 1982 (wide-spotted) and G. b. bathycloides (Honrath,
[1884]) (narrow-spotted) phenotypes in Peninsular Malaysia based on georeferenceable locality data of
specimens used in Fig. 3. Fig. 4. Distribution of G. c. malayanum Eliot, 1982 (wide-spotted) and G. b. bathycloides (Honrath,
[1884]) (narrow-spotted) phenotypes in Peninsular Malaysia based on georeferenceable locality data of
specimens used in Fig. 3. 102 PHON C.-.K. et al., Graphium chironides (Lepidoptera) in Peninsular Malaysia agrees with the illustrations of the harpe of G. bathycles in Saigusa et al. (1977) (locality unspecified),
Eliot (1982) (Peninsular Malaysia) and Tsukada & Nishiyama (1982) (Sumatra) in having the middle
arm close to the lower arm and joined to it at its base (Fig. 7c–d). This phenotype is therefore G. b. bathycloides, the Peninsular Malaysian subspecies of G. bathycles. The harpe of the wide-spotted
phenotype agrees with the illustrations of G. chironides shown in Saigusa et al. (1977) (“chiron,” locality
unspecified) and Racheli & Cotton (2009) (Fujian, China) in having the middle arm located almost
midway between the upper and lower arms (Fig. 7h–i). This phenotype is, therefore, G. c. malayanum, Fig. 5. Genitalia. a–b. G. b. Differences in the structure of the genitalia bathycloides (Honrath, [1884]). c–d. G. c. malayanum Eliot, 1982. a,
c. Left lateral view of genitalia with left valva removed and aedeagus angled posteroventrally to show
harpe on right valva. b, d. Inner lateral view of dissected left valva with valvula and hairs that surround
the aedeagus dissected and upturned to expose inner surface of valva. Conventions used are defined
here for all subsequent genitalia figures. All images for each taxon are based on the same specimen,
unless otherwise stated. Scales for images produced by EFI are based on the lowermost focal plane
unless otherwise stated and show a compensation factor for measurements in the uppermost focal
plane (positive percentage). Where the scale is not based on the lowermost focal plane, an additional
compensation factor is provided for this focal plane (negative percentage). Magnifications shown are
objective × tube factor. Axes show the orientation of structures. Abbreviations: ∠ = angle of inclination
stated in degrees (with ‘+’ indicating upward tilt, and ‘-’ indicating downward tilt); A = antero/anterior;
D = dorso/dorsal; I = inner; L = lateral; O = outer; P = postero/posterior; V = ventro/ventral. Where
not otherwise indicated, all scales, magnifications, orientations and angles of inclination are identical
between corresponding images on the same row. Fig. 5. Genitalia. a–b. G. b. bathycloides (Honrath, [1884]). c–d. G. c. malayanum Eliot, 1982. a,
c. Left lateral view of genitalia with left valva removed and aedeagus angled posteroventrally to show
harpe on right valva. b, d. Inner lateral view of dissected left valva with valvula and hairs that surround
the aedeagus dissected and upturned to expose inner surface of valva. Conventions used are defined
here for all subsequent genitalia figures. All images for each taxon are based on the same specimen,
unless otherwise stated. Scales for images produced by EFI are based on the lowermost focal plane
unless otherwise stated and show a compensation factor for measurements in the uppermost focal
plane (positive percentage). Where the scale is not based on the lowermost focal plane, an additional
compensation factor is provided for this focal plane (negative percentage). Magnifications shown are
objective × tube factor. Axes show the orientation of structures. Abbreviations: ∠ = angle of inclination
stated in degrees (with ‘+’ indicating upward tilt, and ‘-’ indicating downward tilt); A = antero/anterior;
D = dorso/dorsal; I = inner; L = lateral; O = outer; P = postero/posterior; V = ventro/ventral. Differences in the structure of the genitalia Where
not otherwise indicated, all scales, magnifications, orientations and angles of inclination are identical
between corresponding images on the same row. 103 European Journal of Taxonomy 917: 94–121 (2024) the Peninsular Malaysian race of G. chironides, and the structure of its harpe also agrees with the
illustration for malayanum in the original description by Eliot (1982). the Peninsular Malaysian race of G. chironides, and the structure of its harpe also agrees with the
illustration for malayanum in the original description by Eliot (1982). The setae on the less sclerotised surfaces of the harpe are more numerous and denser in G. b. bathycloides
(Fig. 7c–d) than in G. c. malayanum (Fig. 7h–i). There are also clear differences between the taxa in the
shape and size of the arms. In G. b. bathycloides the upper arm of the harpe is trihedral, large, toothed,
hump-like in anterolateral profile, and usually with a prominent pointed apex (Fig. 7c–e). In G. c. malayanum, the upper arm follows a similar trihedral pattern but it is very small and untoothed, being
reduced almost to a tooth itself (Fig. 7h–j). The middle arm of the harpe is relatively narrow in G. b. bathycloides in lateral profile, expanding slightly and becoming highly sclerotised at its toothed distal
end before tapering to a pointed apex (Fig. 7c–e). In contrast, the middle arm of G. c. malayanum is very
broad, moderately sclerotised and somewhat quadrate in shape, with its distal margin bearing teeth and
serrations of varying size that are most prominent on its dorsal and ventral apical corners (Fig. 7h–j). The lower arm of the harpe is directed posteriorly in G. b. bathycloides (Figs 5a–b, 7a–d) and is strongly
incurved (Figs 6d, 7e). It may also be slightly expanded towards the apex (Fig. 7c). In G. c. malayanum,
the lower arm is straighter and longer, and is slenderer towards the apex (Figs 5c–d, 7h–j). The most
obvious difference is that it is less strongly incurved (Fig. 6h) than in G. b. bathycloides (Fig. 6d) and is Fig. 6. Structure of the uncus, saccus and paired valvae. a–d. G. b. bathycloides (Honrath, [1884]). e–h. G. c. malayanum Eliot, 1982. a, e. Left lateral view of uncus. b, f. Posterodorsal view of uncus. c, g. Ventral view of saccus and sacculus (within the saccus) seen through the abdominal sternites. d, h. Differences in the structure of the genitalia Posterodorsal view of entire genitalia showing paired valvae with the arms of the harpe curving
inwards towards each other. Scales, magnifications, orientation axes and angles as explained in Fig. 5. Image (d) is not based on the same specimen of G. b. bathycloides as the other images. Fig. 6. Structure of the uncus, saccus and paired valvae. a–d. G. b. bathycloides (Honrath, [1884]). e–h. G. c. malayanum Eliot, 1982. a, e. Left lateral view of uncus. b, f. Posterodorsal view of uncus. c, g. Ventral view of saccus and sacculus (within the saccus) seen through the abdominal sternites. d, h. Posterodorsal view of entire genitalia showing paired valvae with the arms of the harpe curving
inwards towards each other. Scales, magnifications, orientation axes and angles as explained in Fig. 5. Image (d) is not based on the same specimen of G. b. bathycloides as the other images. 104 PHON C.-.K. et al., Graphium chironides (Lepidoptera) in Peninsular Malaysia 105
Fig. 7. Structure of the left harpe. a–e. G. b. bathycloides (Honrath, [1884]). f–j. G. c. malayanum Eliot,
1982. a–b, f–g. Inner dorsolateral view. c, h. Inner lateral view. d, i. Outer lateral view. e, j. Posterior
view. The harpe is shown within the entire valva in images (a) and (f), and after dissection from the valva
in all other images. Scales, magnifications and orientation axes as explained in Fig. 5. The scale shown
in (g) applies to the tip of the ventral arm of the harpe (also for all images in the same row). Fig. 7. Structure of the left harpe. a–e. G. b. bathycloides (Honrath, [1884]). f–j. G. c. malayanum Eliot,
1982. a–b, f–g. Inner dorsolateral view. c, h. Inner lateral view. d, i. Outer lateral view. e, j. Posterior
view. The harpe is shown within the entire valva in images (a) and (f), and after dissection from the valva
in all other images. Scales, magnifications and orientation axes as explained in Fig. 5. The scale shown
in (g) applies to the tip of the ventral arm of the harpe (also for all images in the same row). 105 European Journal of Taxonomy 917: 94–121 (2024) Fig. 8. Variation in the harpe of the left valva seen at an inner anterolateral angle of 45° in five specimens
each. a–e. G. b. bathycloides (Honrath, [1884]). f–j. G. c. malayanum Eliot, 1982. Affinity of DNA barcodes The gene analysis showed strong agreement between clades, ordination clusters and genitalia type. The
ML tree separated the nine samples into two distinct clades (Fig. 10), with individuals from different
clades separated by 18–23 base pairs and ML distances of 10.17–13.49%. The two clades corresponded
with the two clusters in the ordination and the genitalia morphology of the two taxa, G. bathycles
and G. chironides (Fig. 10). Specimens from China formed a subclade within the G. chironides clade,
sister to the Peninsular Malaysian subclade (Fig. 10). Specimens from these two geographically distinct
subclades were separated by 6–7 base-pair differences and ML distances of 3.21–3.78%, lending further
support to the inferred identity of the chironides clade. Within the G. bathycles clade, specimens differed
by at most one base pair. Similar topologies for the bathycles-chironides clade were obtained when our
sequences were integrated into a re-analysis of the sequences used by Wilson et al. (2014), using both
MP and ML (Supp. file 3). Support values for the bathycles-chironides node increased in the full ML
tree. Differences in the structure of the genitalia Scales, magnification,
orientation axis and angles as explained in Fig. 5. Fig. 8. Variation in the harpe of the left valva seen at an inner anterolateral angle of 45° in five specimens
each. a–e. G. b. bathycloides (Honrath, [1884]). f–j. G. c. malayanum Eliot, 1982. Scales, magnification,
orientation axis and angles as explained in Fig. 5. 106 PHON C.-.K. et al., Graphium chironides (Lepidoptera) in Peninsular Malaysia directed posteroventrally (Fig. 7f–j) instead of posteriorly. Additional differences between the genitalia
of the two taxa are given in Supp. file 1 (Part C). Variation in the shape, size, and curvature of the arms of the harpe and their teeth occurs within the two
taxa (Fig. 8). In G. b. bathycloides, the upper arm differs the most between specimens in width, shape,
and size of serrations (Fig. 8a–e), which range from small (Fig. 8b) to large and tooth-like (Fig. 8e). In
G. c. malayanum, the greatest variation occurs in the middle arm (Fig. 8f–j), which varies from being
laterally wide and short (Fig. 8f) to being longer and narrower (Fig. 8h). The serrations and teeth on its
apical margin vary from relatively small (Fig. 8f) to very large (Fig. 8h). The drawing by Eliot (1982),
which is partly re-illustrated in Fig. 9, shows that the middle arm can be bifurcated at its apex. Although
none of the specimens we dissected had a bifurcated middle arm, one specimen had a notch that formed
a slight cleft on the apical margin, which might suggest a trace of a bifurcation (Fig. 8j). Examination of characters used by Wilson et al. An examination of the characters used by Wilson et al. (2014) reveals that the characters on the forewing
upperside that differentiate G. c. malayanum from G. b. bathycloides were misinterpreted (Table 2,
characters A and B). These characters that were explained by Eliot (1982) are the widths of the pale
blue discal markings on the forewing upperside (character A) and in particular the spot in space 5
(Table 2, character B). The illustrations of Wilson et al. (2014), in which they circle the character they
used as the widths of the markings, show that they misinterpreted it to be the small post basal bluish
streak-like marking that is joined to the inner edge of the bluish discal spot in space 1a (Fig. 11). As a
result, they scored most of their specimens as having broad markings as in G. c. malayanum, when the
actual markings were narrower than in the photograph of the type of this taxon. The forewing upperside
spot in space 5 (character B) is a single spot on the disc (Fig. 11). However, Wilson et al. (2014)
again misinterpreted this character (Fig. 11), as they circled both the discal and submarginal spot in
their illustrations. In one specimen (JJW0017), spots in space 6 instead of space 5 were circled. It is
therefore uncertain what was actually measured or scored for this character. Although Eliot (1982) did
not specifically state the width of the pale blue discal spot in space 5 in G. b. bathycloides, referring
to it as “little more than a dot,” he specified a range for the spot width in G. c. malayanum. In the six
specimens examined by Wilson et al. (2014), the widths were stated to be “around 1 mm wide in all.”
Despite this being much smaller than Eliot’s stated range of 2.0–2.5 mm for G. c. malayanum, they
scored all but one of their six specimens as having the character state of this taxon. Using the ratios of
spot widths to vein lengths measured from the illustrations of Wilson et al. (2014) and the smallest vein
lengths of both taxa in our samples, we calculate the maximum likely spot widths to be less than 1.5 mm
wide in those of their specimens that we were unable to examine physically (Supp. file 2, Part B), while
its width was 0.94 in the specimen we excluded due to broken wing apices. Examination of characters used by Wilson et al. 107 European Journal of Taxonomy 917: 94–121 (2024) Fig. 9. Harpe of G. c. malayanum Eliot, 1982 based on the illustration by Eliot (1982). a. Outline of
harpe in Eliot’s original line drawing. b. An approximate predicted projection if the valva is viewed from
the same angle as the illustrations in Fig. 8. Fig. 9. Harpe of G. c. malayanum Eliot, 1982 based on the illustration by Eliot (1982). a. Outline of
harpe in Eliot’s original line drawing. b. An approximate predicted projection if the valva is viewed from
the same angle as the illustrations in Fig. 8. Fig. 10. Maximum likelihood tree for COI mtDNA barcodes in G. bathycles bathycloides (Honrath,
[1884]) and two subspecies of G. chironides (Honrath, [1884]), i.e., subspecies malayanum Eliot, 1982 Fig. 10. Maximum likelihood tree for COI mtDNA barcodes in G. bathycles bathycloides (Honrath,
[1884]) and two subspecies of G. chironides (Honrath, [1884]), i.e., subspecies malayanum Eliot, 1982
and the nominate subspecies chironides (Honrath, [1884]), with two other species of Graphium Scopoli,
1777 as outgroups. Fig. 10. Maximum likelihood tree for COI mtDNA barcodes in G. bathycles bathycloides (Honrath,
[1884]) and two subspecies of G. chironides (Honrath, [1884]), i.e., subspecies malayanum Eliot, 1982
and the nominate subspecies chironides (Honrath, [1884]), with two other species of Graphium Scopoli,
1777 as outgroups. Fig. 10. Maximum likelihood tree for COI mtDNA barcodes in G. bathycles bathycloides (Honrath,
[1884]) and two subspecies of G. chironides (Honrath, [1884]), i.e., subspecies malayanum Eliot, 1982
and the nominate subspecies chironides (Honrath, [1884]), with two other species of Graphium Scopoli,
1777 as outgroups. 108 PHON C.-.K. et al., Graphium chironides (Lepidoptera) in Peninsular Malaysia Table 2. Characters for the differentiation of G. c. malayanum Eliot, 1982 (M) and G. b. bathycloides
(Honrath, [1884]) (B) as given by Eliot (1982), and as interpretated by Wilson et al. (2014) for the six
specimens of Graphium Scopoli, 1777 they examined, shown alongside the correct interpretation for
the same specimens. Character
defined by Eliot
Character
state (Eliot)
Interpretation
of character by
Wilson et al. Character state
as interpreted by
Wilson et al. Examination of characters used by Wilson et al. Correct
interpretation of
character state
Forewing upperside markings:
A
Pale blue discal
markings (five
spots)
M: wider
B: narrower
Misinterpreted as
postbasal bluish
streak in space 1a
Four wider; two
narrower
All six narrower
(than in the type of
malayanum)
B
Discal spot in
space 5
M: wider
(2.0–2.5 mm)
B: little more
than a dot
Misinterpreted
as discal and
submarginal spots in
space 5
Five wider; one little
more than a dot (all
around 1 mm wide)
All six narrow,
being under
1.5 mm wide
Hindwing underside markings:
C
Basal and
subbasal spots in
space 8
M: pale blue,
faintly yellowish
B: pale blue
Correctly interpreted
Five faintly
yellowish; one pale
blue
All six faintly
yellowish
D
Postdiscal
orange markings
in spaces 1b to 4
M: orange-
yellow, narrow
B: richer orange,
much wider
Colour and width of
the spots combined
as one character
Four orange-yellow,
narrow; two richer
orange, wider
Applying one state
to two characters
results in conflicts
E
Basal spot in
space 3
M: prominent
B: absent or
vestigial
Correctly interpreted
Two prominent; four
absent or vestigial
Two vestigial, four
absent
F
Orange stria in
space 5
M: absent
B: present
Uncertain—question
mark for all
specimens
No character state
specified
Five present; one
absent
G
Pale blue streak
(“window”) in
space 1b
M: present, long
B: virtually
obliterated by
black
Correctly interpreted
Three long; three
obliterated
Three present, much
shorter than in
malayanum type Hindwing underside markings:
C
Basal and
subbasal spots in
space 8
M: pale blue,
faintly yellowish
B: pale blue
Correctly interpreted
Five faintly
yellowish; one pale
blue
All six faintly
yellowish
D
Postdiscal
orange markings
in spaces 1b to 4
M: orange-
yellow, narrow
B: richer orange,
much wider
Colour and width of
the spots combined
as one character
Four orange-yellow,
narrow; two richer
orange, wider
Applying one state
to two characters
results in conflicts
E
Basal spot in
space 3
M: prominent
B: absent or
vestigial
Correctly interpreted
Two prominent; four
absent or vestigial
Two vestigial, four
absent
F
Orange stria in
space 5
M: absent
B: present
Uncertain—question
mark for all
specimens
No character state
specified
Five present; one
absent
G
Pale blue streak
(“window”) in
space 1b
M: present, long
B: virtually
obliterated by
black
Correctly interpreted
Three long; three
obliterated
Three present, much
shorter than in
malayanum type Hindwing underside markings: Besides misinterpreting the characters on the forewing upperside, Wilson et al. Examination of characters used by Wilson et al. (2014) also misassigned
some of the character states for the hindwing underside or were unable to understand the character
(Table 2, characters C–G). They scored one of their specimens as having the basal and subbasal spots in
space 8 pale blue when all are faintly yellowish (Table 2, character C). They applied the same character
states to two characters, width and colour of the orange markings in spaces 1b to 4 (Table 2, character
D), which resulted in the states conflicting with some of the characters. Thus, the two specimens they
considered to have wide spots have spots that are no wider than in some of the others they figure but 109 European Journal of Taxonomy 917: 94–121 (2024) appear to be scored as wide only because of their more orange spot colour. The two specimens that they
scored as having prominent spots at the base of space 3 on the hindwing underside (Table 2, character E)
have very small spots that are much smaller than in the photograph of the type of G. c. malayanum that
they examined. They appear to have been uncertain what the additional stria in space 5 was (Table 2,
character F), as they used a question mark for this character for the types and all their specimens. The
stria is visible in all but one of their specimens and in the syntype of G. b. bathycloides. It is just barely
visible in the holotype of G. c. malayanum. They scored the pale blue subdiscal streak (or window)
in space 1b of the hindwing underside (Table 2, character G) as long in three specimens although the
streaks are much shorter than in the type of G. c. malayanum. Separability of taxa based on each wing character Boxplots for the individual wing characters measured for G. b. bathycloides and G. c. malayanum are
shown in Fig. 12 based on measurement data given in Supp. file 2 (Part A). Table 3 summarises the
degree of overlap between measurement ranges of the two taxa and shows whether the medians differed
significantly between them. The only characters that did not differ significantly between taxa in the
NPMV tests in R were the area measurements of the hindwing upperside blue spot in space 2 (character
#7A in Tables 1 and 3) and the hindwing underside orange spots in space 5 (#10A) and space 2 (#11A). Although the significant differences in other characters tested indicate differences in the population
medians, their usefulness as diagnostic characters for the two taxa depends on the degree of overlap in
their respective ranges. Fig. 11. Diagrammatic representation of the forewing upperside and its venation and markings in G. c. malayanum Eliot, 1982, showing characters mistakenly used by Wilson et al. (2014) (left) compared
with the actual characters used by Eliot (1982) (right). Fig. 11. Diagrammatic representation of the forewing upperside and its venation and markings in G. c. malayanum Eliot, 1982, showing characters mistakenly used by Wilson et al. (2014) (left) compared
with the actual characters used by Eliot (1982) (right). Fig. 11. Diagrammatic representation of the forewing upperside and its venation and markings in G. c. malayanum Eliot, 1982, showing characters mistakenly used by Wilson et al. (2014) (left) compared
with the actual characters used by Eliot (1982) (right). Fig. 11. Diagrammatic representation of the forewing upperside and its venation and markings in G. c. malayanum Eliot, 1982, showing characters mistakenly used by Wilson et al. (2014) (left) compared
with the actual characters used by Eliot (1982) (right). 110 PHON C.-.K. et al., Graphium chironides (Lepidoptera) in Peninsular Malaysia Fig. 12. Box and whisker plots for all wing characters measured for G. b. bathycloides (Honrath, [1884])
nd G. c. malayanum Eliot, 1982. Bars are interquartile ranges (IQR) and whiskers represent 1.5 × IQR,
eyond which are outliers (point markers). Fig. 12. Box and whisker plots for all wing characters measured for G. b. bathycloides (Honrath, [1884])
and G. c. malayanum Eliot, 1982. Bars are interquartile ranges (IQR) and whiskers represent 1.5 × IQR,
beyond which are outliers (point markers). 111 European Journal of Taxonomy 917: 94–121 (2024) Table 3. NPMV test results: significant difference (+), no significant difference (–), not tested (blank). Separability of taxa based on each wing character Levels of usefulness of different characters in differentiating G. c. malayanum Eliot, 1982
from G. b. bathycloides (Honrath, [1884]) in Peninsular Malaysia, based on non-parametric multivariate
(NPMV) test results and degree of overlap between measurement ranges. Level of overlap in character
Level of usefulness
Character
No. Character code
NPMV test
result
No overlap between ranges
Diagnostic
3A
UpFwSp3BW
+
3B
UpFwSp3BWR
4A
UpFwSp4BW
+
4B
UpFwSp4BWR
5A
UpFwSp5BW
+
5B
UpFwSp5BWR
14
FwAng
+
One or more outliers in a taxon are
within the whiskers or IQR of the
other taxon or exceed its median
Usually reliable
2A
UpFwSp2BW
+
2B
UpFwSp2BWR
6A
UpHwSp1bSA
+
6B
UpHwSp1bSL
A whisker of one taxon reaches the
whisker or IQR of the other taxon
Sometimes usable
9A
UnHwSp3SA
+
9B
UnHwSp3SL
8A
UnHwSp1bSA
+
8B
UnHwSp1bSL
12A
UnHwSp3OSA
+
12B
UnHwSp3OSW
The median of one taxon is within
the IQR or whiskers of the other
taxon, or their IQRs overlap
Not useful
13A
UnHwSp4OSA
+
13B
UnHwSp4OSW
1A
UpFwSp1bBW
+
1B
UpFwSp1bBWR
15
FwWV2
+
16
FwWV5
+
17
FwL
+
18
HwAng
+
7A
UpHwSp2SA
–
7B
UpHwSp2SL
10A
UnHwSp5SA
–
10B
UnHwSp5SL
11A
UnHwSp2OSA
–
11B
UnHwSp2OSW
NPMV test results: significant difference (+), no significant difference (–), not tested (blank). 112 PHON C.-.K. et al., Graphium chironides (Lepidoptera) in Peninsular Malaysia The most diagnostic wing markings in which the range did not overlap between taxa were the widths of
the blue spots in spaces 3 to 5 (#3A, 4A, and 5A) and their respective ratios with respect to vein length
(#3B, 4B, and 5B). The only structural character that was diagnostic was the forewing angle (#14). Two other characters, both on the upperside, were usually reliable, with only occasional outliers in one
taxon infringing into the range of the other. They were the width and ratio of the forewing spot in space
2 (#2A and 2B) and the area and length of the hindwing spot in space 1b (#6A and 6B). A few underside characters on the hindwing were sometimes usable in that they did not overlap between
taxa very often. In these characters, the whisker of one taxon overlapped with the whisker or IQR of the
other taxon but did not reach its median. Revised differential diagnoses for G. c. malayanum and G. b. bathycloides On the basis of the analysis of wing characters and differences in genitalia, we provide the following
improved diagnoses of the two taxa. Separability of taxa based on each wing character They were the area measurements and lengths of the bluish
spots in spaces 1b (#8A and 8B) and 3 (#9A and 9B) and the area and width of the orange spot in space
3 (#12A and 12B). The remaining characters were not useful in separating the taxa because of frequent overlap in
measurements between taxa, with the median of one taxon being encompassed by the whiskers or even
IQR of the other taxon, or with their IQRs overlapping. They included the three characters that did not
differ significantly between taxa. Graphium chironides malayanum (male) Wings (Fig. 13a): upperside forewing with the pale blue discal markings comparatively broad, especially
in spaces 3 to 5, the spot in space 5 at least 2.0 mm wide at its widest extent. Spot width in space 4 at
least 3.5 mm, and spot in space 3 exceeding 5.0 mm. Forewing apex less falcate and less pointed than
in G. b. bathycloides. Hindwing upperside and underside usually with a bluish streak at base of space
1b. Hindwing underside pale blue spot at the base of space 3 always present, 1.0–2.5 mm long (but see
measurements for G. b. bathycloides). Genitalia (Figs 5c–d, 6e–h, 7f–j, 8f–j): harpe with middle arm
located about midway between the upper and lower arms. Upper arm of harpe very small, being reduced
to almost a tooth. Middle arm of harpe very broad and somewhat quadrate with teeth and serrations on
its distal margin. Lower arm of harpe relatively long, moderately incurved and directed posteroventrally,
tapering to a point towards the apex. Graphium bathycles bathycloides (male) Wings (Fig. 13b): upperside forewing with the pale blue discal markings comparatively narrow, the
spot in space 5 not more than 1.5 mm wide at its widest extent and sometimes absent. Spot in space 4
not more than 3.0 mm wide, and the spot in space 3 rarely exceeding 5.0 mm in width. Forewing apex
more falcate and more pointed than in G. c. malayanum. Hindwing upperside and underside usually
without a bluish streak at base of space 1b, especially on the upperside. Hindwing underside pale blue
spot at the base of space 3 sometimes absent, and when present usually relatively small, up to 1.5 mm
long (but see measurements for G. c. malayanum). Genitalia (Figs 5a–b, 6a–d, 7a–e, 8a–e): harpe with
middle arm located close to the lower arm and joined to its base. Upper arm of harpe large and hump-
like in anterolateral profile, usually with a pointed apex and slightly serrated margins. Middle arm of
harpe relatively narrow, expanding at its toothed distal end and tapering to a point. Lower arm of harpe
relatively short, strongly incurved, and directed posteriorly, with a blunt and slightly serrated apex. The differences described above apply well to G. c. malayanum and G. b. bathycloides from Peninsular
Malaysia but not always to subspecies that occur outside this region. Illustrated specimens of the 113 European Journal of Taxonomy 917: 94–121 (2024) nominate subspecies of G. bathycles from Java (Tsukada & Nishiyama 1982; Page & Treadaway 2014)
resemble G. c. malayanum in its broad discal band. Throughout its range, however, G. chironides usually
has a touch of blue in space 1b on the hindwing upperside, which is absent in G. bathycles. Specimens of
continental subspecies of G. chironides illustrated by various authors (Saigusa et al. 1977; Chou 1994;
Gu & Chen 1997; Osada et al. 1999; Monastyrskii 2007; Racheli & Cotton 2009; Kimura et al. 2011;
Page & Treadaway 2014; Inayoshi 2023a) vary in forewing discal band width and many have a slightly
narrower band than in subspecies malayanum, resembling that in G. b. bathycloides. However, the
spot in space 5 is usually wider than in G. b. bathycloides, the veins across all the pale bands are often
blackened, and there is sometimes a pale postdiscal spot in space 3 on the hindwing upperside. Fig. 13. Upperside (left) and underside (right) of the two taxa. a. Graphium chironides malayanum
Eliot, 1982. b. G. Revised differential diagnoses for G. c. malayanum as a subspecies Graphium c. malayanum is the only non-continental subspecies of G. chironides. It has been recorded
from southernmost Thailand (Inayoshi 2023b) to Peninsular Malaysia. Other subspecies (Page &
Treadaway 2014) are G. chironides chironides, which occurs from northeast India to central and south
China, G. chironides tereus (Fruhstorfer, [1908]), which occurs in Hainan, and G. chironides clanis
Jordan, 1909, which occurs in Southeast China. A fifth nominal subspecies, G. chironides punctatus
Page & Treadaway, 2014, said by its describers to occur in Thailand, Laos and Vietnam, is of uncertain
validity as a subspecies because the characters used to diagnose it, such as an additional blue hindwing
spot in space 3, can sometimes be found on specimens of subspecies chironides (e.g., Kehimkar 2008:
147, fig. 17UP) and may be absent in specimens from within the stated geographical range of punctatus
(e.g., Ek-Amnuay 2012: pl. 33, P65, first row, right). Furthermore, the stated range of punctatus divides
the range of the nominate subspecies, making the distribution of the latter somewhat disjunct. In the continental subspecies, the forewing pale bluish discal band is very often crossed by black scaling
along the upperside of veins 1b and 2, which may be so extensive as to form wide black borders between
well-separated pale spots. The hindwing band is also usually crossed by black scaling that may be
very prominent too. The forewing band is variable in width but is often narrower than in subspecies
malayanum. Continental subspecies sometimes have a bluish white spot in space 3 at the cell-end on the
hindwing upperside. The following is a more accurate differential diagnosis for subspecies malayanum with respect to the
continental subspecies of G. chironides, based on Eliot’s (1982) original diagnosis and specimens we
were able to examine: forewing upperside lacks black scaling along vein 1b on the pale bluish discal
band, and usually also lacks black scaling across the band along vein 2, or the latter may be very
narrowly black-scaled. Hindwing upperside lacks black scaling across the pale bluish band along vein 8,
the basal part of vein 7 and the radius. Upperside pale discal forewing band wide. No bluish-white spot
present in space 3 at the cell-end on the hindwing upperside. Graphium bathycles bathycloides (male) bathycles bathycloides (Honrath, [1884]). Fig. 13. Upperside (left) and underside (right) of the two taxa. a. Graphium chironides malayanum
Eliot, 1982. b. G. bathycles bathycloides (Honrath, [1884]). Fig. 13. Upperside (left) and underside (right) of the two taxa. a. Graphium chironides malayanum
Eliot, 1982. b. G. bathycles bathycloides (Honrath, [1884]). 114 PHON C.-.K. et al., Graphium chironides (Lepidoptera) in Peninsular Malaysia Where there is a need, dissection can be used to confirm identity. However, difficulties in identification
based on wing markings should rarely arise if locality information is correct since, as far as is known,
G. bathycles does not occur on the continent, its northernmost limits being the southern half of the
Isthmus of Kra, and G. chironides does not occur in Java. Where there is a need, dissection can be used to confirm identity. However, difficulties in identification
based on wing markings should rarely arise if locality information is correct since, as far as is known,
G. bathycles does not occur on the continent, its northernmost limits being the southern half of the
Isthmus of Kra, and G. chironides does not occur in Java. Synonymies and corrections to literature Only historical name changes and current corrections are shown in the brief synonymic lists below. For explanatory notes on the nomenclatural history of G. chironides and the stability of its name, see
Supp. file 4 (Part A). Our rationale for rejecting the synonymy of malayanum with chironides suggested
by Ek-Amnuay (2012) is explained further in Supp. file 4 (Part B). The errors in Saigusa et al. (1977)
corrected below are a mix-up in the plates that do not appear to have affected the analyses and conclusions
of the authors. Graphium chironides, syn. chironicum Eliot, 1982 – Eliot 1983: 283–284 (chironicum was the name
proposed for the Indian subspecies in combination with Graphium clanis). raphium clanis Jordan, 1909 – Eliot 1982: 180; replacement name. Papilio chiron var. chironides Honrath, 1884: 397, pl. 10 fig. 4; type locality Darjeeling, Sikkim Graphium chironides (Honrath, [1884]) Graphium chironides (Honrath, [1884]) p
(
[
])
Papilio chiron Wallace, 1865: 66; type locality Assam, Sylhet; preoccupied by Papilio chiron Fabricius,
1775.i p
(
[
])
Papilio chiron Wallace, 1865: 66; type locality Assam, Sylhet; preoccupied by Papilio chiron Fabricius,
1775.i Papilio chiron var. chironides Honrath, 1884: 397, pl. 10 fig. 4; type locality Darjeeling, Sikkim. Graphium clanis Jordan, 1909 – Eliot 1982: 180; replacement name. 115 European Journal of Taxonomy 917: 94–121 (2024) Graphium bathycles bathycloides (Honrath, [1884])i Graphium chiron – Saigusa et al. 1977: figs 39 and 41 mislabelled, recte G. bathycles bathycloides;
fig. 39 (showing upperside) likely same specimen shown on underside in fig. 38 as “bathycles”; see
also correction to Saigusa et al. (1977) above. Graphium chiron – Saigusa et al. 1977: figs 39 and 41 mislabelled, recte G. bathycles bathycloides;
fig. 39 (showing upperside) likely same specimen shown on underside in fig. 38 as “bathycles”; see
also correction to Saigusa et al. (1977) above. Habitat differences of taxa in Peninsular Malaysia Graphium c. malayanum inhabits the foothills and mountains of Peninsular Malaysia and is more
frequently encountered in the highlands than G. b. bathycloides. The latter occurs mainly in the lowlands
and foothills and is not rare, but it is also occasionally encountered in the highlands. Relatively few
specimens of G. c. malayanum are found in collections or in photographs taken in the wild in comparison
to G. b. bathycloides, which suggests it is relatively scarce in the Peninsula. The recognition of the
existence of this species in the Peninsula and its occurrence as a distinct subspecies has conservation
importance. Graphium chironides malayanum Eliot, 1982 Graphium chironides malayanum Eliot, 1982
Graphium clanis malayanum Eliot, 1982: 180–181; genitalia illustrated, p. 181; type locality Selangor
(Peninsular Malaysia). Graphium clanis malayanum Eliot, 1982: 180–181; genitalia illustrated, p. 181; type locality Selangor
(Peninsular Malaysia). Graphium bathycles bathycloides – Saigusa et al. 1977: fig. 37 mislabelled, recte G. chironides
malayanum (upperside), likely the same specimen shown on underside in fig. 40 as “chiron” (i.e.,
chironides); see also correction to Saigusa et al. (1977) below. Graphium chironides malayanum – Eliot 1983: 283–284. — Corbet & Pendlebury 1992: 75–76, 594,
pl. 6 no. 2; 2020: 69, 492, pl. 10 no. 5, text and related couplets in identification key quote Corbet &
Pendlebury (1992) verbatim.i Graphium chironides malayanum – Eliot 1983: 283–284. — Corbet & Pendlebury 1992: 75–76, 594,
pl. 6 no. 2; 2020: 69, 492, pl. 10 no. 5, text and related couplets in identification key quote Corbet &
Pendlebury (1992) verbatim. Graphium chironides chironides – Ek-Amnuay 2012: 120; unjustified synonymy. Graphium bathycles bathycloides (Honrath, [1884])i Discussion malayanum in their sample
contributed to the conclusion that the status of G. chironides malayanum is in question, implying that
G. b. bathycloides and G. c. malayanum could be a single taxon as stated in the original dissertation
(Karen-Chia 2014). However, this view is untenable and is a result of confusion over the morphology of
the two taxa. As shown in the foregoing analysis of specimens occurring in the Peninsula, the two taxa
are both morphologically and genetically distinct. The wider width of the pale blue forewing discal band was the first and most diagnostic character of G. c. malayanum pointed out by Eliot (1982). In particular, the band’s terminal spot in space 5 was emphasised,
and its size range was stated. The pale blue discal spots that make up the band are the largest markings
on the forewing except in space 5, where it is small (Fig. 11). However, Wilson et al. (2014) mistook a
very small streak joined to the inner margin of the discal spot in space 1a as the band referred to by Eliot
(1982), while in space 5 they appear to have used two spots instead of just the discal spot. In our data,
the width ranges of the spots in spaces 4 and 5 that form part of the discal band were mutually exclusive
for the two taxa. In fact, the differences in the width of these distal spots on the forewing of the two taxa
are sufficiently large and reliable to enable the taxa to be differentiated by simple comparison without
the need for measurement. Thus, we do not consider the two taxa “particularly hard to distinguish” or all
the characters used to distinguish them “subtle” or “obscure”. The magnitude of the differences in these
diagnostic characters also makes it extremely unlikely that overlap in the ranges would occur had our
sample size of G. c. malayanum been larger. Other characters that we analysed were less diagnostic. Some were generally reliable, some were helpful
to a limited extent, and others were not useful. It is common for a new taxon to be described from a
limited number of samples. As more samples become available and the extent of variation within the
taxon becomes better understood, character differences described by the original author are re-evaluated
and refined by subsequent authors. Discussion Based on our analysis of wing and genitalia morphology as well as genetic sequences, we affirm that
G. chironides occurs in the Peninsula, and that the Peninsular Malaysian subspecies G. chironides
malayanum is a good subspecies. Ordination of the width of the forewing band against forewing length
and forewing angle showed the existence of two very distinct phenotypic clusters in specimens that we
measured against a scale. Ordination of the width ratio of the forewing bluish spot in space 5 against
forewing angle enabled inclusion of specimens from published figures, and similarly produced two
clusters, with the holotype of G. c. malayanum and syntype of G. bathycles bathycloides clustering
with the wide- and narrow-spotted phenotypes, respectively. Genitalia morphologies of representative
specimens of each cluster were also distinctly different and clearly diagnostic. The cluster with broader
forewing bands and a less falcate forewing was identifiable from published genitalia illustrations of
specimens from other regions as G. chironides, and the other cluster was identifiable as G. bathycles. Although some variation in genitalia morphology occurred within the two taxa, they were readily
distinguishable by major differences, especially in the location of the middle arm of the harpe. Genetic
sequencing also confirmed the existence of two species in Peninsular Malaysia by virtue of two well-
separated clades. The Peninsular Malaysian specimens that were identified as G. chironides formed a
monophyletic clade with specimens of G. chironides from China, with which they formed a closely
related sister group. We determined from our analysis that all the specimens examined by Wilson et al. (2014) in the MZUM
were G. b. bathycloides. Wilson et al. (2014) did also assign all their specimens as G. b. bathycloides
in their paper despite some of the specimens they examined in the MZUM being misidentified as G. c. malayanum on their original specimen labels. They also rightly stated that the genitalia were similar
to those of G. b. bathycloides and that there was a lack of specimens and sequence data. This should 116 PHON C.-.K. et al., Graphium chironides (Lepidoptera) in Peninsular Malaysia have led to the conclusion that G. c. malayanum was absent in their sample. However, based on a
morphological analysis, the taxa were deemed to have ambiguous and overlapping wing characters. The
very similar DNA barcodes that were obtained due to the absence of G. c. Discussion This involves narrowing down the most diagnostic characters and
sometimes identifying additional characters that might have been overlooked. However, the conclusion
reached by Wilson et al. (2014) that G. c. malayanum has ambiguous and overlapping wing characters
was not so much due to a lack of usefulness of some characters as it was to their misjudgement of most
of the characters. In some cases, they misinterpreted the characters. In other cases, character states were
misassigned. Occasionally, characters were not understood, or two characters that should have been
evaluated separately were combined. However, we agree with Wilson et al. (2014) that colour characters
can be subjective to apply, and that colours may fade in older specimens. Our observations were that
these colour characters were not reliable, and therefore we did not attempt to use them. There were
many specimens of G. b. bathycloides that had pale orange postdiscal markings in spaces 1b to 4 on the
hindwing underside, and even more that have yellow-tinged basal and subbasal pale blue spots in space
8 on the hindwing underside. In addition to the diagnostic characters mentioned by Eliot (1982), the less falcate forewing in
G. chironides that was implied by Wallace (1865) is a good character, as the ranges for the angle that
we measured to quantify this character in the two species were mutually exclusive. The less falcate
forewing is also seen in a shorter forewing length and longer length of vein 2 and vein 5 on average in
comparison to G. b. bathycloides. In other words, the forewing is shorter and wider in its proportions in
G. c. malayanum, in addition to being less curved outwards at the apex. Reliance on incorrectly identified specimens for gene analysis was the first of a sequence of problems
in the work of Wilson et al. (2014). The misidentifications should have become apparent from the
absence of clear genetic and genitalic differences. However, their inability to understand some of the 117 European Journal of Taxonomy 917: 94–121 (2024) differentiating characters on the wings, including the most diagnostic characters of malayanum, and
their misjudgements in the assigning of character states to their specimens in a few other characters,
led them to the wrong conclusion that malayanum has ambiguous and overlapping wing characters and
is therefore of doubtful status. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper. Author responsibilities The joint first authors were responsible for the morphological and taxonomic work in this article. DNA extraction
and sequencing was the work of the third author, and its analysis was the joint work of the first-named and third
authors. Discussion Their problems with this taxon were further compounded by inherent
variation in some of the characters originally proposed for its diagnosis. The confusion caused by
their conclusions shows the importance of a good understanding of morphology as a basis for accurate
identification, and the importance of both morphology and identification in the interpretation of gene
data. The utility of DNA barcoding for the identification of taxa is lost if the reference specimens on
which it is based are not correctly identified in the first place on the basis of their morphology. Mistakes
in the recognition of morphological characters inevitably lead to mistakes in taxon identification and can
subsequently lead to wrong inferences in the molecular phylogenies of taxa, or even cast doubt on the
validity of genuine taxa like G. c. malayanum. Acknowledgements The authors express their gratitude to the following museums and their curators and staff for access to
physical specimens or digital images: H. Omar, A.A. Azidah, N. Mamat and S. Amni Bazilah of the
University of Malaya Museum of Zoology, Kuala Lumpur; S. Farizawati and J. Mohd. Khairill of the
Natural History Museum of the Malaysian National Museum, Kuala Lumpur; W.S. Hwang of the Lee
Kong Chian Natural History Museum, Singapore. We are grateful to C.Y. Chong and J. Arshad, and to
N.L. Liew, for the use of specimens from their personal collections. We are also thankful to C.Y. Chong
for providing valuable advice, especially on the choice of characters, to A.M. Cotton for useful
discussions and comments on the manuscript, and to K. Willmott and F.L. Condamine for reviewing an
earlier version of the manuscript and suggesting improvements. We also thank C.Y. Chong, A.M. Cotton
and S. Takagi for providing useful reference material, J. Hao of Anhui Normal University, Wuhu for
locality information of his G. chironides sequence, and M. Soh for locality information of his field
photograph of G. c. malayanum. This study was funded by the Economic Planning Unit of the Prime
Minister’s Department, Malaysia under the Twelfth Malaysia Plan project entitled “Documentation and
Conservation of Biodiversity for the Well-Being of Forests and Sustainability of Natural Resources
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3 Jan. 2023]. Manuscript received: 7 January 2022 Manuscript accepted: 4 August 2023 120 PHON C.-.K. et al., Graphium chironides (Lepidoptera) in Peninsular Malaysia Published on: 9 January 2024
Topic editor: Tony Robillard
Section editor: Jurate de Prins
Desk editor: Pepe Fernández Published on: 9 January 2024
Topic editor: Tony Robillard
Section editor: Jurate de Prins
Desk editor: Pepe Fernández Printed versions of all papers are also deposited in the libraries of the institutes that are members of the
EJT consortium: Muséum national d’histoire naturelle, Paris, France; Meise Botanic Garden, Belgium;
Royal Museum for Central Africa, Tervuren, Belgium; Royal Belgian Institute of Natural Sciences,
Brussels, Belgium; Natural History Museum of Denmark, Copenhagen, Denmark; Naturalis Biodiversity
Center, Leiden, the Netherlands; Museo Nacional de Ciencias Naturales-CSIC, Madrid, Spain; Leibniz
Institute for the Analysis of Biodiversity Change, Bonn – Hamburg, Germany; National Museum of the
Czech Republic, Prague, Czech Republic. Supp. file 1. A. References Methods used in the dissection and illustration of genitalia. B. General structure of the
genitalia of Graphium bathycles bathycloides (Honrath, [1884]) and Graphium chironides malayanum
Eliot, 1982. C. Additional differences between the genitalia of G. b. bathycloides and G. c. malayanum. https://doi.org/10.5852/ejt.2024.917.2391.10541 Supp. file 2. A. Specimen data and measurements. B. Predicted size limits of forewing spot in space 5
for specimens figured by Wilson et al. (2014) that we were unable to trace and measure physically. https://doi.org/10.5852/ejt.2024.917.2391.10543 Supp. file 3. Reanalysis of phylogenies in the genus Graphium Scopoli, 1777 based primarily on the
data of Wilson et al. (2014). https://doi.org/10.5852/ejt.2024.917.2391.10545 Supp. file 3. Reanalysis of phylogenies in the genus Graphium Scopoli, 1777 based primarily on the
data of Wilson et al. (2014). https://doi.org/10.5852/ejt.2024.917.2391.10545 Supp. file 4. A. Further notes on the nomenclatural history of Graphium chironides (Honrath, [1884]). B. Unjustified synonymy of Graphium chironides malayanum Eliot, 1982. https://doi.org/10.5852/ejt.2024.917.2391.10547 Supp. file 4. A. Further notes on the nomenclatural history of Graphium chironides (Honrath, [1884]). B. Unjustified synonymy of Graphium chironides malayanum Eliot, 1982. https://doi.org/10.5852/ejt.2024.917.2391.10547 Supp. file 4. A. Further notes on the nomenclatural history of Graphium chironides (Honrath, [1884]). B. Unjustified synonymy of Graphium chironides malayanum Eliot, 1982. https://doi.org/10.5852/ejt.2024.917.2391.10547 121 121
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Need for redefining needs.
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Health and quality of life outcomes
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cc-by
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Abstract Defining needs is difficult due to the inherent complexity of the concept of 'need', so it is not
surprising that numerous definitions have been proposed. 'Health' consists of a wide range of
characteristics so 'health needs' ought to include personal and social care, health care,
accommodation, finance, education, employment and leisure, transport and access. Target-driven standards in areas of health care with a high political profile appear to be replacing
the concept of universal provision and clinical need; this major change in clinical care warrants a re-
evaluation of health care outcomes. Identifying who might benefit from this new approach to health
care is equally important if scarce resources are to be fully and appropriately utilised. If the goal of
care is 'optimal health', the key marker of success ought to be to ascertain individual patients' health
care needs (HCN) and tailor services accordingly. Wide variation in the description of 'needs'
directly affects policies and services intended to meet a population's health care needs. Consequently, the definition of 'needs' has important implications for healthcare provision- the
more constrained the definition, the less healthcare will be made available and vice versa. This
paper describes some common definitions of needs and discusses their respective benefits and
disadvantages in terms of health care provision and their potential impact on health policy. y
Need for redefining needs
Mohsen Asadi-Lari1, Chris Packham2 and David Gray*1 Address: 1Division of Cardiovascular Medicine, University Hospital, Nottingham, UK, NG7 2UH and 2Division of Epidemiology and Public
Health, University of Nottingham, Nottingham, UK Email: Mohsen Asadi-Lari - msxma@nottingham.ac.uk; Chris Packham - chris.packham@nottingham.ac.uk;
David Gray* - d.gray@nottingham.ac.uk * Corresponding author Published: 21 August 2003
Health and Quality of Life Outcomes 2003, 1:34 Received: 03 June 2003
Accepted: 21 August 2003 Health and Quality of Life Outcomes 2003, 1:34 Health and Quality of Life Outcomes 2003, 1:34
This article is available from: http://www.hqlo.com/content/1/1/34 This article is available from: http://www.hqlo.com/content/1/1/34 © 2003 Asadi-Lari et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL. Commentary
Need for redefining needs
Mohsen Asadi-Lari1, Chris Packham2 and David Gray*1 Open Access Societal view from health care provision' [14,15]; in other words, 'need'
exists only if there is a 'capacity to benefit' from a particu-
lar healthcare service.[10,16] Need may be assumed to
exist, therefore, when there is an effective treatment [17]
or 'health gain'.[18] Ability to benefit from health care can
be influenced by several factors including epidemiological
aspects such as incidence and prevalence of disease and
the effectiveness of interventions. Applying this defini-
tion, the outcomes of health interventions assume greater
importance. In a sociological environment, Bradshaw defined need as:
normative (distinguished by professionals, such as vacci-
nation), felt (wants, wishes and desires), expressed (vocal-
ised needs or how people use services) and comparative
needs, which indicates that needs arising in one location
may be similar for people with similar socio-demographic
characteristics living in another location [6]. Bradshaw's typology of need creates a definition which is
more practical for health service research workers,
although it does not include the concept of cost contain-
ment. He recently argued that his taxonomy of need was
constrained because of inherent problems with the con-
cept of need.[7] First of all, Culyer [10] proposes that 'capacity to benefit'
(as an outcome measure) differs from needs (as a resource
input), so these two concepts are measurable in different
ways which do not necessarily match. Physical, physiolog-
ical, and social benefits may be identified in individuals as
well as groups or communities. In addition, the benefits
of health care can be determined as improvement in clin-
ical status, reassurance, supportive care, and relief of car-
ers rather than a narrow medical definition in which
merely objective, measurable clinical improvements are
recorded. The rational result of this definition is that ben-
efit from healthcare may be affected inversely by the sever-
ity of disease. For instance someone who suffers from
mild symptoms of coronary heart disease may have a
greater chance of being offered coronary bypass surgery
than an older patient with severe 3-vessel coronary dis-
ease, whose life expectancy may not be extended greatly
by surgery, on the grounds of having less capacity to benefit. Moreover, equity in access to healthcare is fundamental to
the economists' definition, otherwise it might not be equi-
table. Also, this definition minimises the influence of lay
people; focuses on "health care" rather than "health" con-
trary to Bradshaw's model [6]; and is concentrated on a
causal model. Societal view This can be problematic when studying
human behaviour based on complex interactions
between: individual behaviour, social circumstances, cul-
tural beliefs and genetic construction.[19] Furthermore it
often leads to a belief that current services are the basis for
healthcare needs assessment. [20] Even the supporters of
the definition concede this, arguing that measured needs
are only based on existing services.[15] On the other
hand, this terminology is innovation-disoriented, that is it
limits population healthcare needs to readily available
services, ignoring potential needs arising from emerging
health technologies. One example is the increased 'need'
that followed the introduction of automatic implantable
cardioverter defibrillators in late 1990s. Even so, individ-
uals who have more 'capacity' to improve their health sta-
tus or prevent deterioration might benefit more from
healthcare provision -for example health professionals
have more knowledge about their health/ill-health condi-
tions, therefore may benefit from health services at higher
levels. Introduction
h
l h g
A wide variety of definitions of 'need' has been developed. Although each was intended to improve service delivery to
the population, ambiguity increased to such an extent that
"it may be an illusion to suppose that there might ever be
a consensus about the meaning of needs" [4]. It is impor-
tant to recognise the different perspectives illuminating
the relationship between the concepts of need, and health-
care needs. Davis proposed a relatively simple definition of
need as 'a subjective feeling state that initiates the process
of choosing among medical resources' [5]. In health care, need has a variety of meanings which may
change over time so it is not surprising that different
groups of health professionals refer to 'needs assessment'
in very different ways [1]. Stevens et al [2] considered that
interest in a needs-driven health system passed through
several stages. A sociological approach in the 1960s was
followed by 'rational planning' and resource allocation
based on deprivation and epidemiology (RAWP [3]) in
the 1970s; in the 1990s, National Health Service reform
introduced need-target resource allocation and by the year
2000 the focus was on 'collaborative action" where the
need for health care was to be collectively identified by
interested 'stakeholders'. Page 1 of 5
(page number not for citation purposes) Health and Quality of Life Outcomes 2003, 1 Health and Quality of Life Outcomes 2003, 1 Health and Quality of Life Outcomes 2003, 1 Health and Quality of Life Outcomes 2003, 1 http://www.hqlo.com/content/1/1/34 Philosophical points Some experts describe needs as 'instrumental' or funda-
mental to the achievement of a desirable goal [8] while
others highlight a non-instrumental (or absolute) sense of
needs[9,10]. Baldwin [11] proposed a rather theoretical
definition of need, that is a 'tension need' which implies a
desire to compensate for some dis-equilibrium such as
thirst due to fluid loss. He also proposed a 'teleological
need' reflecting the gap between actual and desired status,
such as a desire for coronary bypass surgery to improve
both quality and longevity of life. This approach to need
implies 'necessity to be explicit about whether it is effec-
tive, how effective it is and for whom' [4]. Baldwin consid-
ered teleological need to arise 'when the goal is not realised
and there is a need of a certain thing when this is necessary for
realising the goal' [11], which seems to be a characteristic
attributable to any kind of need. While this definition use-
fully expounds the concept of need, a significant improve-
ment in health services is unlikely without specific efforts
to develop needs-oriented services. Pragmatic view Green and Kreuter considered need as 'whatever is required
for health or comfort' [12], covering personal, social and
environmental conditions, including family planning
information, smoke-free zones, seat belt rules, and health
'hot lines' but appears ineffective in terms of 'life creativ-
ity' and cost-effectiveness. Doyal and Gough suggested
'objective needs', asserting that 'health needs' and 'auton-
omy' are not only two universal human needs, but also
basic human rights [13], as some have previously claimed
[9]. Page 2 of 5
(page number not for citation purposes) Geographical variations A distinction needs to be made between individual and
population-based health. Several approaches have been
adopted as a proxy for assessing population's healthcare
needs: mortality rates, [25–28] socio-economic sta-
tus[29], service utilisation,[30] or prevalence rates,[31]
which are all at macro level. However, needs can be
defined at micro level too, as demonstrated by the doctor-
patient relationship, consultation with health profession-
als, or patients' healthcare needs at a local surgery or
health centre. Both macro- and micro-health needs are
important in different settings of health decision mak-
ing.[10] Nevertheless, in routine clinical management,
health professionals deal with rather wider aspects of
healthcare needs than 'capacity to benefit', such as social
support, informational needs and equipment for daily
activities. Demand for healthcare may also be affected by geograph-
ical variation [35,36] and medical charges.[37] Healthcare
providers too may constrain patients' ability to benefit
from healthcare; for example, low-referring General Prac-
titioners may fail to refer patients who need special
care.[35] Hospital utilisation data cannot be assumed to
be a valid proxy for need since hospital use is a product of
many variables including service supply and clinical deci-
sion-making rather than population need[38]. These data
more likely reflect patients' propensity to consult, the will-
ingness of family doctors to refer, access to hospital beds
and the availability of alternative facilities provided by the
private sector.[39] The Economists' approach Cost containment is the focus of policy-makers' attention,
therefore combining satisfactory services with cost-effec-
tiveness could provide a solution to health care rationing
issues. The most widely presented definition of need
favoured by economists is 'the ability of people to benefit Page 2 of 5
(page number not for citation purposes) Page 2 of 5
(page number not for citation purposes) Health and Quality of Life Outcomes 2003, 1 http://www.hqlo.com/content/1/1/34 Literature review reveals that cost effectiveness is already
receiving greater emphasis, although there is no evidence
that direct questioning of individuals to establish their
health care requirements is being overlooked. attitude of the population can all influence demand for
health care, while medical guidelines and effectiveness of
interventions may affect the provision and availability of
health care. Ideally, the provision of health care services
should meet most of the populations' needs but the latter
may not be constant. Consequently health needs assess-
ment surveys are necessary both locally and nation-wide
to establish what services are required to meet these needs. A health service approach? The Medical Research Council considers need to exist
when a patient's functioning falls below -or threatens to
fall below- some minimum specified level and there is a
remediable cause. This definition takes into account the
effectiveness of the care process and implies that a need is
met 'when it has attracted some at least partly effective inter-
vention'.[21] In a similar vein, Buchan et al defined health
service needs as 'those for whom an intervention produces a
benefit at reasonable risk and acceptable cost' [22]. This defi-
nition does incorporate effectiveness and cost-effective-
ness. Some health economists define demand as a measure for
desire, wherein willingness to pay or spending time reflects
the extent of demand. If health care services become more
accessible (for economic, physical and cultural reasons),
the demand for healthcare based on need will increase. In
the past, demand for health care such as attendance at
clinic has often been used as a proxy for need [32], but this
approach generates various problems. Converting felt need
to demand requires numerous factors- individuals' beliefs
and the imposed costs (as well as time off work) are
involved. A more reasonable definition of needs is 'the requirement
of individuals to enable them to achieve, maintain or restore an
acceptable level of social independence or quality of life, as
defined by particular care agency or authority' [23]. Taking
this definition into account, health authorities and other
health-related organisations at local, regional, and
national level set out to provide appropriate services to
meet its population needs, targeting an acceptable level of
social independence and improved quality of life. If
assessing needs is being considered to change current
healthcare services, [24] definitions that focus on 'maxi-
mum health' seem preferable. Need, demand and supply do overlap in Venn-like fash-
ion to some extent, although each has its own distinctive
characteristics. There is no standard model. In the NHS,
service provision or supply has almost always been less
than demand or need. Individual needs usually exceed
their expressed needs or apparent demands, although this
hypothesis remains to be fully evaluated.[33] Interven-
tions may become more effective when they are targeted
to fulfil need [34]. Do existing definitions satisfy clinically relevant
health care needs? Coronary heart disease is increasingly common with
advancing age and has a significant impact on daily life. It
constitutes a large proportion of the clinical workload for
UK general and hospital practitioners, but a range of phar-
macological and surgical interventions are available. Our
clinical experience led us to suspect that this patient group
had specific needs that existing definitions failed to cover. Demand and supply in relation to need Demand and supply in relation to need
'Demand' is defined as what people ask for, and the
media, advances in medical technology and social and
educational background can have a profound influence
on patients' and society's expectations. Geographic varia-
tion, socio-economic status, knowledge about health and Page 3 of 5
(page number not for citation purposes) Page 3 of 5
(page number not for citation purposes) Health and Quality of Life Outcomes 2003, 1 Health and Quality of Life Outcomes 2003, 1 http://www.hqlo.com/content/1/1/34 We developed a comprehensive, self-administered needs
tool In order to identify cardiac patients' specific health
care needs through patient interviews, expert opinions
and literature review and administered this to 240 consec-
utive patients admitted to an acute cardiac unit. The meth-
odology has been described extensively elsewhere [40]
but briefly the needs assessment questionnaire consists of
46 questions in 5-score Likert scale (1 indicates more
needs versus 5 with no needs) in five domains of 'physical
needs, 'satisfaction', 'informational needs', 'social needs,
and 'concerns', with satisfactory internal consistency
(Cronbach's alpha ranged between 0.83–0.89). This was
administered with a specific (Seattle Angina Question-
naire) and generic instruments (SF-12 and EQ-5D).[40] approach to health care and towards a needs driven sys-
tem. In addition, it is important to ensure that patients
express their needs to a suitable agency, which can provide
the sort of specialist information required. Providing
patients with a forum in which to express their needs to
access health professionals might be productive. Politicians keen to propose how they intend to meet the
needs of the voting public may find that it is easy to be
seduced by definitions of 'need' which lead to a situation
where limited resources appear sufficient. While some
genuine needs will be met, others, perhaps of greater value
if met, will be denied. The comprehensiveness of 'health'
deserves a definition of health needs which over-rides
political considerations, or providers' limitations, and
embraces current political strategy to conceptualise and
meet health need in the widest sense.[41] If assessing
needs is being proposed as a trigger to change current
healthcare services, definitions that address optimum lev-
els of health are preferable and must be clinically appro-
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potential for improvement in caring services. Health Quality Life
Outcomes 2003, 1: 26:. Publish with BioMed Central and every
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Clinical pharmacist interventions in elderly patients with mental disorders in primary care focused on psychotropics: a retrospective pre–post observational study
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11007 TPP0010.1177/20451253211011007Therapeutic Advances in PsychopharmacologyM Stuhec and L Lah 11007 TPP0010.1177/20451253211011007Therapeutic Advances in PsychopharmacologyM Stuhec and L Lah 11007 TPP0010.1177/20451253211011007Therapeutic Advances in PsychopharmacologyM Stuhec and L Lah Therapeutic Advances in Psychopharmacology Original Research Original Research https://doi.org/10.1177/20451253211011007
https://doi.org/10.1177/20451253211011007
Ther Adv Psychopharmacol
2021, Vol. 11: 1–8
DOI: 10.1177/
20451253211011007
© The Author(s), 2021. Article reuse guidelines:
sagepub.com/journals-
permissions Keywords: clinical pharmacist, long-term interventions, mental disorders, primary care
setting, psychotropics Keywords: clinical pharmacist, long-term interventions, mental disorders, primary care
setting, psychotropics Received: 20 August 2020; revised manuscript accepted: 27 March 2021. Received: 20 August 2020; revised manuscript accepted: 27 March 2021. prevalence of mental health disorders in older
adults to be 19.1%. The prevalence was associ-
ated positively with age and increased from 14.8%
in the 55–59 age group to 28.9% in the 80–84 age
group. The most common mental disorders were Clinical pharmacist interventions in
elderly patients with mental disorders in
primary care focused on psychotropics:
a retrospective pre–post observational study Matej Stuhec
and Lea Lah Abstract Background: Mental disorders pose a significant clinical burden and affect approximately
one-third of older adults. Although studies have shown positive impacts of clinical pharmacist
(CP) interventions within the general population, the long-term effects of such cooperation
on geropsychiatric patients in primary care settings are not yet known. This study evaluated
whether CP interventions have a long-term impact on the quality of medication prescribing in
geropsychiatric patients. Methods: We conducted a retrospective non-interventional observational pre–post study
for the 2015–2017 period, involving patients aged 65 or above for whom a medication review
was provided by a CP. The study included participants with mental disorders treated with
polypharmacy, including at least one psychotropic. Potentially inappropriate medications
(PIMs) in elderly patients were determined with the Priscus list, and potential type X drug–
drug interactions (pXDDIs) with Lexicomp®. Up-to-date treatment guidelines were used to
evaluate patient pharmacotherapy, and patient medication was evaluated before the initial
medication review and again 6 months later. Results: The study included 48 patients (79.4 years, SD = 8.13) receiving a total of 558
medications (155 for the treatment of mental disorders). The number of medications
decreased by 9.5% after the medication review. The CP proposed 198 interventions related to
psychotropics, of which 108 (55%) were accepted by the general practitioners. All accepted
(99.1%) interventions except one were still maintained 6 months after the interventions had
been proposed. They led to a significant decrease in the total number of medications, PIMs,
and pXDDIs (p < 0.05), and improved treatment guidelines adherence. Conclusions: CP interventions decreased the number of medications, PIMs, and pXDDIs, and
almost all interventions were maintained 6 months later. These results provide evidence for
the positive effects of CP interventions in a primary care setting. Additional research with a
larger sample size and a randomized study design is needed. Correspondence to:
Matej Stuhec
Faculty of Pharmacy,
University of Ljubljana,
Askerceva Cesta 7,
Ljubljana, SI-1000,
Slovenia
matejstuhec@gmail.com
Department of Clinical
Pharmacy, Ormoz
Psychiatric Hospital,
Ormoz, Slovenia
& Department for
Pharmacology, Maribor,
Slovenia & Faculty of
Pharmacy Ljubljana,
University of Ljubljana
Lea Lah
University of Ljubljana,
Ljubljana, Slovenia Introduction Mental disorders impose a significant clinical and
economic burden globally and affect about one-
third of older adults. A cross-sectional study of
35 general practices in Ireland estimated the Correspondence to:
Matej Stuhec
Faculty of Pharmacy,
University of Ljubljana,
Askerceva Cesta 7,
Ljubljana, SI-1000,
Slovenia
matejstuhec@gmail.com
Department of Clinical
Pharmacy, Ormoz
Psychiatric Hospital,
Ormoz, Slovenia
& Department for
Pharmacology, Maribor,
Slovenia & Faculty of
Pharmacy Ljubljana,
University of Ljubljana
Lea Lah
University of Ljubljana,
Ljubljana, Slovenia urnals.sagepub.com/home/tpp
1
Creative Commons CC BY: This article is distributed under the terms of the Creative Commons Attribution 4.0 License (https://creativecommons.org/licenses/by/4.0/)
which permits any use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open
Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). Creative Commons CC BY: This article is distributed under the terms of the Creative Commons Attribution 4.0 License (https://creativecommons.org/licenses/by/4.0/)
which permits any use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open
Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). journals.sagepub.com/home/tpp Lea Lah A mul-
tiple regression analysis showed an inverse asso-
ciation of PIMs with cognitive impairment and
significant positive associations with permanent
restlessness and permanent negative attitude.5
Furthermore, many geropsychiatric patients are
treated with irrational polypharmacy: unneces-
sary polypharmacy used while there are alterna-
tive treatments with fewer medications that are
safer and/or more effective.6,7 Both PIMs and
irrational polypharmacy can result in harm, treat-
ment failure, and increased treatment costs.8,9 In
Slovenia, most patients with mental disorders are
treated in primary care, which accounts for two-
thirds of antidepressants and the majority of anxi-
olytics prescriptions, so further research on
psychotropics in primary care is needed.10
One approach to minimizing PIMs and irrational depression (17.1%), panic/anxiety (11.3%), cog-
nitive disorders (5.6%), and alcohol (3.8%) and
substance misuse (3.8%).1 Mental disorders in
this population are most often treated with psy-
chotropics, due primarily to difficulties with other
interventions and a lack of resources (e.g., for
psychotherapy). Although effective, psychotrop-
ics are often over-prescribed in this population,
and more prudent prescribing strategies are
needed to minimize the risks of over-prescribing. Psychotropics, especially antidepressants, anxio-
lytics, and antipsychotics, have several important
adverse effects and drug–drug interactions
(DDIs), which can lead to treatment failure and
serious harm. Potentially inappropriate medica-
tions (PIMs) in elderly patients can be avoided
using several PIM lists (e.g., Priscus, STOPP/
START, Beers), which also list many psycho-
tropics.2–4 PIMs, in particular antipsychotics and
anxiolytics, are commonly used in older adults. An Austrian study of most nursing homes in
Voralberg (n = 1844) found that 70.3% of all resi-
dents had at least one PIM 70.3% [95% confi-
dence interval (CI) 67.2–73.4] and that 1014
(55%) residents were using at least one psycho-
tropic PIM. The most frequently prescribed
PIMs were prothipendyl (25.9% of residents),
lorazepam (14%), and diclofenac (6.1%). Lea Lah A mul-
tiple regression analysis showed an inverse asso-
ciation of PIMs with cognitive impairment and
significant positive associations with permanent
restlessness and permanent negative attitude.5
Furthermore, many geropsychiatric patients are
treated with irrational polypharmacy: unneces-
sary polypharmacy used while there are alterna-
tive treatments with fewer medications that are
safer and/or more effective.6,7 Both PIMs and
irrational polypharmacy can result in harm, treat-
ment failure, and increased treatment costs.8,9 In
Slovenia, most patients with mental disorders are
treated in primary care, which accounts for two-
thirds of antidepressants and the majority of anxi-
olytics prescriptions, so further research on
psychotropics in primary care is needed.10 Lea Lah 1 journals.sagepub.com/home/tpp Creative Commons CC BY: This article is distributed under the terms of the Creative Commons Attribution 4.0 License (https://creativecommons.org/licenses/by/4.0/)
which permits any use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open
Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). Therapeutic Advances in Psychopharmacology 11 This study evaluates the long-term impact of CP
interventions in a primary care setting on the
quality of medication prescription, as measured
by the number of overall medications, PIMs, and
DDIs in geropsychiatric patients treated with
polypharmacy. We hypothesized a negative asso-
ciation between clinical pharmacist interventions
and the number of PIMs, medications, and
potential type X drug-drug interactions (pXD-
DIs, contraindicated potential DDIs as defined
by Lexicomp®) as well a positive association
between CP interventions and treatment guide-
lines adherence. depression (17.1%), panic/anxiety (11.3%), cog-
nitive disorders (5.6%), and alcohol (3.8%) and
substance misuse (3.8%).1 Mental disorders in
this population are most often treated with psy-
chotropics, due primarily to difficulties with other
interventions and a lack of resources (e.g., for
psychotherapy). Although effective, psychotrop-
ics are often over-prescribed in this population,
and more prudent prescribing strategies are
needed to minimize the risks of over-prescribing. Psychotropics, especially antidepressants, anxio-
lytics, and antipsychotics, have several important
adverse effects and drug–drug interactions
(DDIs), which can lead to treatment failure and
serious harm. Potentially inappropriate medica-
tions (PIMs) in elderly patients can be avoided
using several PIM lists (e.g., Priscus, STOPP/
START, Beers), which also list many psycho-
tropics.2–4 PIMs, in particular antipsychotics and
anxiolytics, are commonly used in older adults. An Austrian study of most nursing homes in
Voralberg (n = 1844) found that 70.3% of all resi-
dents had at least one PIM 70.3% [95% confi-
dence interval (CI) 67.2–73.4] and that 1014
(55%) residents were using at least one psycho-
tropic PIM. The most frequently prescribed
PIMs were prothipendyl (25.9% of residents),
lorazepam (14%), and diclofenac (6.1%). Methods Study design and inclusion/exclusion criteria
We conducted a non-interventional retrospective
observational pre–post study. It included patients
serviced by the Ljutomer primary health center in
southeast Slovenia who had been referred to a CP
between 1 February 2015 and 1 July 2017. Patients were selected according to GP medical
referral forms (no impact on selection criteria),
which are used by GPs in Slovenia to refer patients
to various medical specialists. The CP performed
medication reviews for patients with a medical
referral from their GP. A medication review with
recommendations was sent back to the respective
GP, who accepted or rejected the recommenda-
tions at the patient’s next visit. This service is
paid for by the Health Insurance Institute of
Slovenia. Each patient was considered a separate
observational episode. For each patient, the data
were collected at their first visit to their GP after
the CP’s medication review and again 6 months
later (long-term acceptance). The study included patients aged 65 years or
above at the time of their initial examination, who
were concurrently treated with five or more medi-
cations (i.e., polypharmacy), including at least
one psychotropic (ATC code N Nervous system
including psychotropics) and were diagnosed
with at least one mental disorder as defined by the
10th revision of the International Statistical
Classification of Diseases and Related Health
Problems (ICD-10).12 Only patients without
missing data were included. The STROBE
Statement checklist was used to insure the inclu-
sion of all items required in reports of observa-
tional studies.13 The patients’ health data,
including diagnoses, were obtained from their One approach to minimizing PIMs and irrational
polypharmacy is the inclusion of clinical pharma-
cists (CPs) in primary care settings where general
practitioners (GPs) work, which has been the
subject of research, particularly in the United
States (US).11 Despite some recent research
examining large populations, there are no data on
the long-term acceptance of CP recommenda-
tions in primary care in Central Europe.6,7 2 journals.sagepub.com/home/tpp M Stuhec and L Lah contact between the researchers and GPs or
patients. LL was an MPharm student and MS is
an experienced psychiatric CP with over 10 years
of work experience in a psychiatric hospital, includ-
ing daily rounds and ward activities, and ambula-
tory clinical pharmacy service. The latest guidelines
for the treatment of individual diseases were con-
sidered for this study. Methods The PRISCUS list was used
to determine PIMs in the elderly.2 Only pXDDIs
as defined by Lexicomp® 3.0.2, were included in
the study, as in our previous study.15 The impact
on treatment guidelines adherence was also evalu-
ated with various treatment guidelines. medical documentation (patient charts and the
CP medication reviews). The CP in this study was an experienced CP
(PharmD/PhD) with over 10 years of work expe-
rience in a psychiatric hospital (ward rounds,
medication reviews, meetings with patients and
psychiatrists). The CP did not communicate with
the GPs and patients after providing a medication
review. The GPs also did not provide feedback on
why they accepted or rejected the CP’s recom-
mendations. The CP did not have any conversa-
tion with GP and patients after the visit, and,
therefore, pharmacotherapy after 6 months was
also checked (long-term acceptance). Outcomes
Th
i The primary outcomes (number of medications,
PIMs, pXDDIs) were noted after the medication
review and 6 months later. The long-term accept-
ance was calculated as the difference between
6 months and immediate GP acceptance). In
addition, the authors examined several treatment
guidelines (secondary outcome) to evaluate the
impact of CP recommendations on treatment
guidelines adherence.2–4,16–18,19–20 For this pur-
pose, all patients with all various diagnoses (e.g.,
schizophrenia,
insomnia,
dementia)
were
included and guidelines adherence was assessed
case by case. When treatment guidelines provided
insufficient data, various studies and summaries
of product characteristics were used to determine
if the medication use was appropriate. Interventions and data collection
The selection criteria were applied to all patients
referred to the CP by GPs. The CP provided a
medication review, which was given to the GPs
who made the final decision on whether to accept
or reject the proposed changes. The Slovenian
Pharmacy Act allows the pharmacist to conduct
medication reviews in primary care settings and
hospitals. In primary care, all GPs can refer
patients to CPs. In hospitals, CPs are part of the
multidisciplinary teams on the wards and provide
medication reviews for inpatients.14 For details on
the medication review service, see our other stud-
ies and reports.7,14 Medication changes were
retrieved from patients’ medical charts. This
study examined only three main intervention
types: individual drug discontinuation (ineffec-
tive medication, no indication), drug initiation
(untreated indication, re-initiation), and drug
dosage adjustment (dose change, dose fre-
quency, titration, or renal function adjustment). The results of the CPs’ interventions were deter-
mined by examining the patients’ medical
records after the medication review was pro-
duced. This study only included prescription
medication, so over-the-counter medications,
dermal preparations, and medications on an
as-needed basis were excluded. Statistical analysis
h
i
h Statistical analysis
h
i
h y
The main characteristics of the sample were
described using descriptive statistics. Patient
medical records were retrieved immediately after
the first conversation with the CP when the medi-
cation review was performed and 6 months later. The Shapiro–Wilk test was used to test for data
normality. A t test for the dependent samples
(paired t test) was used for normally distributed
variables, and a non-parametric Wilcoxon signed-
rank test was used for non-normally distributed
variables. Patients with missing data were
excluded from the study, which also addressed
loss to follow up. Bias was assessed as descriptive
bias (see Discussion). The p value used was 0.05
and the required sample size was not calculated. The analyses were performed with the Statistical
Package for Social Science 22.0 for Windows®
(SPSS, Chicago, IL, USA). Researchers (LL and MS) examined all medica-
tion reviews and patient charts, classified the
changes into intervention types as well as noted if
the accepted recommendations were maintained
6 months after the GPs received the medication
review (long-term acceptance). All data were
retrieved retrospectively, so there was no direct 3 journals.sagepub.com/home/tpp Therapeutic Advances in Psychopharmacology 11 Figure 1. A flow chart of the main study outcomes. DDI, drug–drug interaction; PIM, potentially inappropriate medication. Figure 1. A flow chart of the main study outcomes. DDI, drug–drug interaction; PIM, potentially inappropriate medication. Primary outcomes The CP proposed 198 interventions (134 drug
discontinuations, 45 drug initiations, and 19 drug
adjustments), which amounted to 4.1 interven-
tions per patient (median = 4), of which the GPs
accepted 108 (55%). The mean of accepted inter-
ventions per patient was 2.25 (median = 2)
(p < 0.05). The highest number of accepted inter-
ventions was eight (in one patient). In 36 patients
(75%), the GPs accepted all proposed recom-
mendations. The interventions were maintained
6 months after introduction in all patients. General results General results The sample included 48 patients [79.4 years, stand-
ard deviation (SD) = 8.13] (Figure 1, Flowchart). The mean number of diagnoses per patient was
4.96 (median = 5). Dementia (50%), schizophrenia
(29%), and depression (19%) were the most preva-
lent. In total, the participants were treated with 558
different medications (mean = 12.6 medications,
median = 11), of which 155 (28%) were psycho-
tropics and used to treat mental disorders (3.2 psy-
chotropics per patient); 38 patients (79.2%) were
treated with at least one antipsychotic and 30
patients (62.5%) with an antidepressant. Before the medication review, 61 PIMs (as
defined by the PRISCUS list) were prescribed, 4 journals.sagepub.com/home/tpp journals.sagepub.com/home/tpp M Stuhec and L Lah Figure 2. Number of patients with PIMs before medication review and after 6 months according to the
PRISCUS list. Figure 2. Number of patients with PIMs before medication review and after 6 months according to the
PRISCUS list Figure 2. Number of patients with PIMs before medication review and after 6 months according to the
PRISCUS list. Table 1. Comparison of treatment guidelines adherence in patient groups before and after the medical review. Patients group
Depression
Anxiety
Insomnia
Dementia
Schizophrenia
No of diagnosis
30
26
24
19
38
No of proposed
interventions
19
11
12
3
15
No of accepted
interventions
11
5
4
3
9
Treatment guidelines
adherence (before) %
patients
33.3% (n = 10)
61.5% (n = 16)
29.2% (n = 7)
89.4% (n = 17)
71.1% (n = 27)
Treatment guidelines
adherence (after) %
patients
73.3% (n = 22)
80.8% (n = 21)
54.2% (n = 13)
100.0% (n = 19)
89.5% (n = 34)
Difference
+40.0% (p < 0.05)
+19.3% (p < 0.05)
+25.0% (p < 0.05)
+11.6% (p = 0.157)
+18.4% (p < 0.05) mparison of treatment guidelines adherence in patient groups before and after the medical review. Table 1. Comparison of treatment guidelines adherence in patient groups before and after the m diazepam (13% PIMs). The number of pXDDIs
also decreased significantly after the interventions
from 8 to 1 (p < 0.05). which represented 10.3% of all prescribed medi-
cations. On average, patients received 1.05 PIMs
(median = 1). Detailed results are presented in
Figure 2. After GPs received and accepted or
rejected the proposals in the medication review,
the total number of PIMs decreased significantly
from 61 to 31, which is a 49% reduction
(p < 0.05). General results The average number of PIMs per
patient also decreased from 1.05 to 0.7 (post-
review median = 1). Psychotropics represented
91.8% of all PIMs and 39.3% of all PIMs were
benzodiazepines. The most common PIM was
the hypnotic zolpidem (23% PIMs) followed by Discussion This study is the first retrospective pre–post study
assessing the impact of a CPs’ medication review
service in geropsychiatric patients in Central
Europe. Our results provide three key findings
and suggest the service may be beneficial in a pri-
mary care setting. Firstly, the results suggest that the proposed medi-
cation changes were relatively well accepted, as
over 90% of the recommendations were still main-
tained 6 months after introduction, although the
patients’ clinical outcomes were not measured. Acceptance rates may vary between different work
environments, as the rate in this study was higher
than in our previous study of a nursing home, but
lower than in a study of a Slovenian psychiatric
hospital (88.0%).6,15,21 A CP in a psychiatric hos-
pital is in daily contact with psychiatrists and
patients (e.g., during ward rounds), whereas pri-
mary care patients do no receive follow up with a
CP, unless referred by their GP anew. Future
research could evaluate the effects of additional
appointments with CPs and the effects of inde-
pendent prescribing by CPs, which is currently
not possible in Slovenia, but is elsewhere (e.g.,
collaborative plan agreements in the US).11 The third finding is that the service improved treat-
ment guidelines adherence. Most commonly, the
interventions were related to depression treatment. Improved depression treatment adherence was
also reported in a US study, where this service is
available in some states.11 However, the CP in our
study did not have prescribing rights, which could
be explored in future studies. A study with a large
sample size reported that depression is not ade-
quately recognized and treated in primary care:
although 51.6% (95% CI, 46.1–57.2) of 12-month
cases received treatment for depression (n = 9,090),
the treatment was adequate in only 41.9% of them
(95% CI, 35.9–47.9), resulting in 21.7% (95% CI,
18.1–25.2) of 12-month depression cases being
treated adequately.22 The most commonly pro-
posed interventions regarding antidepressants in
our study were sertraline initiation (often instead
of escilatopram, which featured in pXDDIs), and
mirtazapine and trazodone initiations (often
instead of benzodiazepines). Secondary outcomes Patients were grouped according to their diagno-
ses and medications. Table 1 shows the full review
of treatment groups according to the proportion
of accepted interventions and treatment guide-
lines adherence. In the three largest groups of
patients, the CP interventions improved treat-
ment guidelines adherence (p < 0.05). journals.sagepub.com/home/tpp 5 Therapeutic Advances in Psychopharmacology 11 important pXDDIs involving antipsychotics and
antidepressants, which is a larger reduction than in
our previous studies.6,15,21 Furthermore, the total
number of PIMs (PRISCUS list) also decreased. The largest PIM reduction (32%) occurred with
hypnotics and sedatives (e.g., zolpidem, bromaze-
pam, alprazolam, and diazepam) (Figure 1), which
is in line with recommendations,2–4 and reflects our
previous studies, as benzodiazepines should be
avoided in elderly patients due to their negative
effect on falls and cognitive decline.5–7 important pXDDIs involving antipsychotics and
antidepressants, which is a larger reduction than in
our previous studies.6,15,21 Furthermore, the total
number of PIMs (PRISCUS list) also decreased. The largest PIM reduction (32%) occurred with
hypnotics and sedatives (e.g., zolpidem, bromaze-
pam, alprazolam, and diazepam) (Figure 1), which
is in line with recommendations,2–4 and reflects our
previous studies, as benzodiazepines should be
avoided in elderly patients due to their negative
effect on falls and cognitive decline.5–7 Discussion This is also in line
with the guidelines.18 Insomnia guidelines adher-
ence improved, as benzodiazepines were often dis-
continued in line with the guidelines and the
PRISCUS and Beers lists.2,3 For schizophrenia
treatment, discontinuation of small doses of que-
tiapine was recommended in some cases, as such
use is common in clinical practice despite poor evi-
dence for it.18 Our results reflect the findings of our
2019 study examining antipsychotic treatment
guidelines, in which 9 out of 21 different CP inter-
ventions (42.8%) were accepted by GPs. The
acceptance rate of the recommendations, but not
patient age, improved treatment guidelines adher-
ence for antipsychotics (p = 0.041) and quetiapine
was found to be the most frequently used antipsy-
chotic, prescribed to 30 out of 49 patients included
in the study (61.2%).7,10 Some important dose
adjustment interventions were also provided in
dementia treatment. Secondly, CP recommendations may curb irra-
tional polypharmacy. In this study, the service
reduced the total number of medications, which
corroborates our previous studies in a primary
community setting and a nursing home.6,15 This
service can thus strongly reduce polypharmacy and
associated negative effects in line with the goals of
health insurance bodies, such as the Health
Insurance Institute of Slovenia (Slovene: Zavod za
zdravstveno zavarovanje Slovenije – a funding body
in Slovenia), which funds the service described in
this paper.15 We found psychotropics were often
involved with several PIMs and pXDDIs, which we
also observed in our previous studies.6,15,21
Additionally, nearly all pXDDIs were removed by
the medication review service, which is a larger
reduction than in our previous studies.6,15 The
remaining pXDDI (between rivastigmine and pro-
pranolol), was due to the patient needing both
beta-blockers for heart failure and rivastigmine for
dementia. Other pXDDIs were combinations con-
taining quetiapine (three times: metoclopramide,
trospium and amiodarone), olanzapine (once: tro-
spium), clozapine (once: carbamazepine) and riv-
astigmine (twice: metoclopramide, propranolol). Our results show that antipsychotics were often
part of pXDDIs and that the CP removed all Despite positive results, this study has several
important limitations. The researchers did not 6 journals.sagepub.com/home/tpp journals.sagepub.com/home/tpp M Stuhec and L Lah contact the study participants directly to gather
data on their response to the service. Discussion The method-
ology used in this study has limitations that may
affect the results and introduce high bias (i.e., no
control group, no randomization, selection bias,
no outcomes measuring, polypharmacy, heteroge-
neous population, small sample size, monocentric
study, risk of type II error). These could all be
addressed in future studies. Despite these limita-
tions, we provide valuable data on the effects of
CP interventions in a primary care facility in
Slovenia. The study adds to the research on
healthcare provision for elderly patients with men-
tal disorders in Central Europe and demonstrates
what effects can be expected from implementing
the service in countries that do not yet have it. References 1. McCombe G, Fogarty F, Swan D, et al. Identified mental disorders in older adults in
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https://orcid.org/0000-0001-
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https://orcid.org/0000-0001-
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Different functional networks
underlying human walking
with pulling force fields acting
in forward or backward directions
OPEN Tetsuya Ogawa 1,2*, Hiroki Obata 2,3, Hikaru Yokoyama 2, Noritaka Kawashima 4 &
Kimitaka Nakazawa 2 Walking with pulling force fields acting at the body center of mass (in the forward or backward
directions) is compatible with inclined walking and is used in clinical practice for gait training. From
the perspective of known differences in the motor strategies that underlie walking with the respective
force fields, the present study elucidated whether the adaptation acquired by walking on a split-
belt treadmill with either one of the force fields affects subsequent walking in a force field in the
opposite directions. Walking with the force field induced an adaptive and de-adaptive behavior of the
subjects, with the aspect evident in the braking and propulsive impulses of the ground reaction force
(difference in the peak value between the left and right sides for each stride cycle) as parameters. In
the parameters, the adaptation acquired during walking with a force field acting in one direction was
transferred to that in the opposite direction only partially. Furthermore, the adaptation that occurred
while walking in a force field in one direction was rarely washed out by subsequent walking in a force
field in the opposite direction and thus was maintained independently of the other. These results
demonstrated possible independence in the neural functional networks capable of controlling walking
in each movement task with an opposing force field. Despite its stereotypical features that exhibit stable rhythmicity and reproducibility, human locomotion is flexible
enough to meet changing task demands. From the perspective of its kinematics, the lower limb joints exhibit simi-
larity to repeat flexion and extension over gait cycles, regardless of demand. However, recent studies have shown
the possibility of different neural mechanisms (or motor strategies) underlying locomotion that is dependent on
detailed tasks1–6. Based on the locomotor adaptation that occurs in a particular locomotive task, the occurrence
of aftereffects in other locomotive tasks or contexts was less evident1–6. These studies particularly focused on
slow component of locomotor adaptation that occurs over minutes as a consequence of comparison between
predicted and actual limb movement and the recalibration of motor output through cerebellar function7 upon
exposure to a novel mechanical environment. These studies suggest the possibility of different neural networks
responsible for each locomotive task (i.e., direction, speed, gait mode [walk and run], and use of hand-held poles). www.nature.com/scientificreports www.nature.com/scientificreports Materials and methods Participant. Sixteen volunteers (15 males and 1 female; mean ± SD age, 28.6 ± 6.6 years; height, 172.0 ± 7.7 cm;
body weight (BW), 66.0 ± 12.0 kg) without a history of neurological or orthopedic disorders were included in
this study. Each participant was tested using two of the four experimental protocols (Fig. 1B). Eight of them
participated in Experiments 1 and 3, while the other eight participated in Experiments 2 and 4, with the order of
participation randomly distributed among subjects to overcome any ordering effects. All participants provided
written informed consent before participation. All experimental procedures were approved by the local Ethics
Committee of the School of Arts and Sciences of the University of Tokyo and were conducted following the
Declaration of Helsinki. Experiment. The experiments consisted of walking under one of two physical conditions (either with an
“aiding” the force field or an “impeding” force field, see Fig. 1A). Force fields were applied to the participants
via a belt stranded around the torso near the COM, which was then attached to the counterweight (2 kg, cor-
responding for 3.12 ± 0.54% BW of the subjects) through two carabiners, a rigid cable, and two low friction
pulleys. Subjects were pulled horizontally forward in the aiding force field, while a backward pull was applied in
the impeding force field.h i
The participants were instructed to walk on a split-belt treadmill (Bertec, Columbus, OH, USA) with two
separate belts, and the speed of each belt was controlled independently. In the present experiment, the treadmill
was operated under one of two conditions, tied (two belts moving together at the same speed) or split (separately
at different speeds), using a custom-written computer program written in Lab-VIEW (National Instruments,
Austin, Texas, USA). The speeds were set at 0.75 ms−1 for both belts under the tied, and while in the split, the
belt on the left side was 0.5 ms−1 and another on the right was 1.0 ms−1 (ratio, 1:2). The limb on the slower (left)
side speed of the treadmill under the split was defined as the “slow limb” and the limb on the faster (right) side
speed as the “fast limb”.h The experimental protocols consisted of baseline, adaptation, re-adaptation, and three washout periods, as
dictated by the protocol (Fig. 1B). Participants always accompanied one of the two force fields (impeding or
aiding) throughout the experiments. www.nature.com/scientificreports/ and increases 6.13% per 1% body weight of horizontal impeding force12. Along with the changing mechanical
demands, the application of these force fields reduced (aiding force field) and increased (impeding force field)
the effort to walk and the activity of the plantar flexor muscle (medial gastrocnemius), which is necessary for
propulsion11,13. Furthermore, a recent study demonstrated detailed characteristics in the adjustment of walking
with these force fields, including different COM dynamics and spinal motor output14. In this study, the analysis
of EMG activity in 16 muscles demonstrated not only a general increase or decrease in activity, but also specific
changes dependent on muscles and the direction of the force fields, particularly in terms of activity in the lumbar
and sacral motor pools of the spinal cord. Importantly, the results of the force fields acting on the COM resemble
those obtained when walking on a slope15 resulting in modifications of positive and negative COM mechanical
power production14 and the strategies of neuromuscular control16,17. To add, similarity in the mechanical adjust-
ments of locomotion with the application of force fields to those performed on slope have been performed in
running, recently18. Following its compatibility with inclined walking in our daily lives along with its possible
use in the clinical practice of gait training19, a detailed analysis of walking with these force fields is expected to
play a crucial role in further understanding gait control.h The present study utilized locomotor adaptation on a split-belt treadmill, a novel environment in which two
belts (one underneath each foot) were driven at different velocities to another. Known that the aspect of adapta-
tion thorough walking on the split-belt treadmill is evident in the interlimb adjustment of gait, particularly in
the kinetic adjustments between that limb20, rather than intralimb21, the present study focused on the asymmetry
of the ground reaction force between the two limbs3–6 and addressed whether the adaptation was transferred
between walking with different force fields. With the known task-dependent specificity in the neural mechanisms
underlying human locomotion as demonstrated in a series of motor adaptation studies1–6, along with difference
in COM dynamics and spinal motor output14 it was possible that further specificity underlies between walking
in the specific mechanical environment with the pulling force field into opposite directions. www.nature.com/scientificreports/ It was hypothesized
that adaptation transfers only limitedly and is maintained independently between walking conditions with
pulling force filed acting into opposite directions as a reflection of specific neural mechanisms being capable of
walking in the given environment. That is, after adaptation to walk on an asymmetrically-driven split-blet tread-
mill under pulling force filed in one direction, aftereffect is evident as asymmetry of the ground reaction force
between the two limbs when subsequently walking on a symmetrically-driven treadmill under force filed into
the same direction, but less evident under force field into opposite direction. Further, aftereffect (asymmetry of
the ground reaction force between the two limbs) acquired through walking on an asymmetrically-driven split-
blet treadmill under pulling force filed in one direction is washed out (or decays) through subsequent walking
on a symmetrically-driven treadmill under force filed into same direction, but less affected by walking under
force field into opposite direction. https://doi.org/10.1038/s41598-023-29231-6 Different functional networks
underlying human walking
with pulling force fields acting
in forward or backward directions
OPEN Contrary, several studies suggest the use of common neural network among different locomotor tasks on the
basis of analysis of limb kinematics8–10 and electromyographic (EMG) activities in locomotor-related lower limb
muscles8,10. Provided that humans locomote in a variety of environments (such as leveled ground, slopes, slippery
floor) by utilizing different tasks (such as walking and running, or those under different speed) depending on
demand, it is particularly important to understand the responsible control mechanisms. p
y
p
p
To further investigate the basic features of the neural mechanisms underlying human locomotion and particu-
larly from a perspective of motor adaptation, the present study focused on walking with force fields acting at the
body center of mass (COM), which pull subjects forward (aiding force field) or backward (impeding force field). Applying these force fields to normal unperturbed walking has been shown to alter the mechanical demands
associated with braking and propulsion. Aiding force fields are known to increase braking and reduce propulsive
impulses, while the impeding force field increases propulsive but reduces braking impulses11. Recently, it has
been demonstrated that an application of horizontal impeding force affects metabolic power in running gait 1Department of Clothing, Faculty of Human Sciences and Design, Japan Women’s University, Tokyo 112‑8681,
Japan. 2Graduate School of Arts and Sciences, The University of Tokyo, Tokyo 153‑8902, Japan. 3School of
Engineering, Kyushu Institute of Technology, Kita‑Kyushu 804‑8550, Japan. 4National Rehabilitation Center for
Persons with Disabilities, Saitama 359‑8555, Japan. *email: ogawat@fc.jwu.ac.jp Scientific Reports | (2023) 13:1909 | https://doi.org/10.1038/s41598-023-29231-6 www.nature.com/scientificreports/ Materials and methods Given that the emergence of the aftereffect is not
stable but can decay throughout the experiments4,22, the order of exposure to the washout periods with different
force fields was alternated depending on the experiments (between experiments 1 and 2, 3, and 4, respectively)
to overcome possible ordering effects. 1, for example, the degree of adaptation was tested by assessing the magnitude of the aftereffect while walking
with the aiding force field on the tied belt during washout 1 (catch trial) after adapting to walk on the split-belt
with the force field in the same (aiding) direction. The transfer of adaptation, on the other hand, was tested by
walking with the impeding force field in washout 2 (post-adaptation) after adapting to walking (re-adaptation
period) with the force field in the opposite (aiding) direction. Given that the emergence of the aftereffect is not
stable but can decay throughout the experiments4,22, the order of exposure to the washout periods with different
force fields was alternated depending on the experiments (between experiments 1 and 2, 3, and 4, respectively)
to overcome possible ordering effects. p
gf
Subjects underwent an extra washout period (washout 3, ➂ and ➇ in the experiments 1 and 3, respectively
and those in the experiments 2 and 4 not subject for further analysis) to walk on a tied belt with force fields in the
same direction as the adaptation period, but were different from those during the washout 2 period. The purpose
of this additional washout period was to evaluate the degree to which the adaptation acquired through walking
on the split-belt could be maintained (or washed out) after walking with a force field in a different direction. Between each testing period, upon changing the belt speeds and/or direction of the force field, there was a 15-s
time interval in which subjects stepped on platforms on both sides of the treadmill. They were then allowed to
step on the treadmill with the left leg when a sufficient belt speed was reached and the appropriate force fields
were mounted. During the experiments, subjects were instructed to walk while watching a wall approximately
3 m in front of them and not to look down at the belts. They were allowed to hold onto the handrails mounted
on either side of the treadmill in case of risk of falling. All subjects completed the test sessions without holding
on. Materials and methods During the baseline, the treadmill was tied and participants walked with
the impeding and aiding force fields for 1 min each. The treadmill was then operated in a split and the partici-
pants underwent a 10-min adaptation, followed by a 1-min catch trial (washout 1) to walk on the treadmill in
tied. The treadmill was then returned to split, and the participants again underwent adaptation to walking on
a split-belt (re-adaptation) for 5 min, which was again followed by a 1-min catch trial (washout 2) to walk on a
tied belt. The force fields in the two catch trial periods (washouts 1 and 2) were different in direction (imped-
ing washout 1 and aiding in washout 2 or vice versa) to address both the degree of adaptation by evaluating the
magnitude of the aftereffect and how it could transfer to walking with the opposite force field. In Experiment Scientific Reports | (2023) 13:1909 | https://doi.org/10.1038/s41598-023-29231-6 www.nature.com/scientificreports/ Figure 1. (A) Experimental apparatus used to impose aiding (left) and impeding (right) force fields on subjects. The belt stranded around the torso near the center of mass (COM) was attached to the weight (2 kg) by a cable
and two low friction pulleys. (B) Experimental protocols used in the present study. In Experiments 1 and 2,
subjects underwent a split-belt adaptation with aiding force field and in Experiments 3 and 4, with impeding
force field. Figure 1. (A) Experimental apparatus used to impose aiding (left) and impeding (right) force fields on subjects. The belt stranded around the torso near the center of mass (COM) was attached to the weight (2 kg) by a cable
and two low friction pulleys. (B) Experimental protocols used in the present study. In Experiments 1 and 2,
subjects underwent a split-belt adaptation with aiding force field and in Experiments 3 and 4, with impeding
force field. 1, for example, the degree of adaptation was tested by assessing the magnitude of the aftereffect while walking
with the aiding force field on the tied belt during washout 1 (catch trial) after adapting to walk on the split-belt
with the force field in the same (aiding) direction. The transfer of adaptation, on the other hand, was tested by
walking with the impeding force field in washout 2 (post-adaptation) after adapting to walking (re-adaptation
period) with the force field in the opposite (aiding) direction. Materials and methods To ensure safety, one experimenter stood by the treadmill. Data recording and analysis. Force sensors mounted underneath each treadmill belt were used to deter-
mine the dimensional ground reaction force (GRF) components: mediolateral (Fx), anteroposterior (Fy), and
vertical (Fz). Force signals were sampled at 1 kHz, stored on a computer via an analog-to-digital converter, and
low-pass filtered at a cut-off frequency of 8 Hz (Power Lab; AD Instruments, Sydney, Australia). The anteropos-
terior (Fy) and vertical (Fz) components were used in the later off-line analysis. The magnitude of the anteropos-
terior (Fy) GRF component was evaluated for each stride cycle. The timing of foot contact and toe-off for each
stride cycle was determined based on the vertical Fz component of the GRF for both fast and slow sides using
custom-written software (VEE Pro 9.3, Agilent Technologies, Santa Clara, CA, USA).i t
g
g
To address the degree of adaptation and transfer of motor patterns across walking with force fields in opposite
directions, the degree of asymmetry in the anteroposterior (Fy) component of the GRF was calculated for each
stride cycle of walking. As depicted in Fig. 2A, this GRF component includes braking and propulsive components
that appear at different phases during the gait cycle. For each component, the peak amplitude during each gait https://doi.org/10.1038/s41598-023-29231-6 Scientific Reports | (2023) 13:1909 | www.nature.com/scientificreports/ Figure 2. (A) Representative examples of the anteroposterior GRF (N) and the vertical GRF (N) during
baseline with exposure to different force fields. Each set of waveforms represents the time-series changes of
the force for ten consecutive stride cycles (from heel contact to subsequent heel contact and including both
left and right sides, superimposed) in a single subject. The blue and red lines represent walking with an aiding
force field and an impeding force field, respectively. The calibration bars indicate 100 N (Fy), and 200 N (Fz) for
the vertical axis and 500 ms for the horizontal axis, respectively. (B) Mean amplitude (absolute) of each GRF
component tested during the baseline with different force fields. The error bars represent the SEM. Statistically
significant differences: **P < 0.01, ***P < 0.001, n = 16. Figure 2. (A) Representative examples of the anteroposterior GRF (N) and the vertical GRF (N) during
baseline with exposure to different force fields. Materials and methods Each set of waveforms represents the time-series changes of
the force for ten consecutive stride cycles (from heel contact to subsequent heel contact and including both
left and right sides, superimposed) in a single subject. The blue and red lines represent walking with an aiding
force field and an impeding force field, respectively. The calibration bars indicate 100 N (Fy), and 200 N (Fz) for
the vertical axis and 500 ms for the horizontal axis, respectively. (B) Mean amplitude (absolute) of each GRF
component tested during the baseline with different force fields. The error bars represent the SEM. Statistically
significant differences: **P < 0.01, ***P < 0.001, n = 16. cycle was calculated as the absolute value for both the fast and slow sides (upper panels of Figs. 3, 4). The degree
of asymmetry, which represents the difference in the absolute values, was then calculated by subtracting the value
of the slow limb from that of the fast limb on a stride-by-stride basis (lower panels of Figs. 3, 4).fil y
(
p
g
)
As exposure to different force fields (aiding and impeding) influences the magnitude of the GRF components
during walking, it does not allow for direct comparisons of the degree of asymmetry between walking with dif-
ferent force fields. In addition, to consider the influence of the natural walking movement of the subjects, which
is not perfectly symmetrical and allows for comparisons between different force fields, the obtained degree of
asymmetry underwent a normalization process. For both the braking and propulsive components of the GRF,
the degree of asymmetry obtained in the washout periods (1, 2 and 3) was subtracted by the mean values of those
under the respective baseline with different force fields. The normalized values were then divided into bins of
5 s and averaged for each bin. Statistics. Two-way analysis of variance (ANOVA) with repeated measures was used to test for statisti-
cally significant differences in the degree of asymmetry (in terms of both acquisition and transfer of adapta-
tion) between walking with different force fields (either aiding or impeding) and different time periods of the
experiment (initial or final phase of washout periods). When ANOVA revealed significant results, Bonferroni’s
post hoc comparisons were performed to identify significant differences between variables. Materials and methods In addition, to test
whether the adaptation acquired during walking with one force field was washed out (or maintained) by walking
with the other force field, a paired Student’s t-test was performed to compare the degree of asymmetry between
the final phase of washout 2 and the initial phase of washout 3 periods. A paired Student’s t-test was used to com-
pare the magnitude of the GRF components and the cadence between walking under the two force conditions. Data are presented as mean ± standard error of the mean (SEM) values. Statistical significance was set at P < 0.05. Resultsh The addition of both aiding and impeding force fields during walking resulted in a systematic modification of
the magnitude of the GRF components. Figure 2A,B shows the changes in each GRF component depending on
the force field while walking normally on the tied belt during the baseline. Figure 2A shows the typical GRF
waveforms for 10 consecutive stride cycles (heel contact to heel contact, superimposed) under each force field
in a single subject, and Fig. 2B shows the group means of the peak magnitude of each GRF component for each https://doi.org/10.1038/s41598-023-29231-6 Scientific Reports | (2023) 13:1909 | www.nature.com/scientificreports/ Figure 3. (Upper panels) Example of typical time-series changes in the peak amplitude (absolute values) of the
braking (A) and the propulsive (B) component of the GRF on a stride-by-stride basis in a single subject (same
subject as in Fig. 4) from Experiment 2 (split-belt adaptation with aiding force field) for both the fast (filled
circle) and slow (open circle) sides. (Lower panels) The differences in the peak forces between the fast and slow
sides (degree of asymmetry) for each stride cycle. Figure 3. (Upper panels) Example of typical time-series changes in the peak amplitude (absolute values) of the
braking (A) and the propulsive (B) component of the GRF on a stride-by-stride basis in a single subject (same
subject as in Fig. 4) from Experiment 2 (split-belt adaptation with aiding force field) for both the fast (filled
circle) and slow (open circle) sides. (Lower panels) The differences in the peak forces between the fast and slow
sides (degree of asymmetry) for each stride cycle. Figure 4. (Upper panels) Example of typical time-series changes in the peak amplitude (absolute values) of the
braking (A) and the propulsive (B) component of the GRF on a stride-by-stride basis in a single subject (same
subject as in Fig. 3) from Experiment 4 (split-belt adaptation with impeding force field) for both the fast (filled
circle) and slow (open circle) sides. (Lower panels) The differences in the peak forces between the fast and slow
sides (degree of asymmetry) for each stride cycle. Figure 4. (Upper panels) Example of typical time-series changes in the peak amplitude (absolute values) of the
braking (A) and the propulsive (B) component of the GRF on a stride-by-stride basis in a single subject (same
subject as in Fig. Resultsh 3) from Experiment 4 (split-belt adaptation with impeding force field) for both the fast (filled
circle) and slow (open circle) sides. (Lower panels) The differences in the peak forces between the fast and slow
sides (degree of asymmetry) for each stride cycle. stride cycle as well as the cadence. The anteroposterior Fy component includes the braking component and the
propulsive component. With the aiding force field, the braking component was significantly greater than with
the impeding force field (P < 0.001). In contrast, the propulsive component was significantly larger with the
impeding force field than with the aiding force field (P < 0.001). There was also a statistically significant differ-
ence in cadence (P < 0.01), where it was slightly higher with aid (102.2 ± 1.2 steps/min) than with the impeding
force field (98.3 ± 1.3 steps/min). stride cycle as well as the cadence. The anteroposterior Fy component includes the braking component and the
propulsive component. With the aiding force field, the braking component was significantly greater than with
the impeding force field (P < 0.001). In contrast, the propulsive component was significantly larger with the
impeding force field than with the aiding force field (P < 0.001). There was also a statistically significant differ-
ence in cadence (P < 0.01), where it was slightly higher with aid (102.2 ± 1.2 steps/min) than with the impeding
force field (98.3 ± 1.3 steps/min). https://doi.org/10.1038/s41598-023-29231-6 Scientific Reports | (2023) 13:1909 | www.nature.com/scientificreports/ Figures 3 and 4 show representative examples of time-series changes in the braking (A) and propulsive
component (B) of the GRF throughout the experiments on a single subject. In both figures, the upper panels
show the peak amplitude of the GRF components on a step-by-step basis for each limb (left to right), whereas
the lower panels show the difference in the peak amplitude between the limbs (degree of asymmetry) for each
stride cycle. During the baseline period, both the braking and propulsive components showed similar ampli-
tudes between the sides with certain variability between each stride cycle, regardless of whether the force field
was aiding or impeding (upper panels of Figs. 3, 4). Consequently, the values of the degree of asymmetry (lower
panels of Figs. 3, 4) for each stride cycle are scattered around zero (horizontal lines indicate perfect symmetry). Resultsh 6), differences dependent on
the type of force field were only evident in the propulsive component (Fig. 6B) and not in the braking component
(Fig. 6A). There was a difference in the braking component depending on the time (F(1,15) = 16.24, P < 0.001),
but not on the type of force field (F(1,15) = 0.14, P = 0.72). This interaction was not significant (F(1,15) = 0.22,
P = 0.65). In contrast, in the propulsive component (Fig. 6B), there was a significant difference in the type of
force field (F(1,15) = 11.07, P < 0.01) and time (F(1,15) = 10.44, P < 0.01). This interaction was not significant
(F(1,15) = 1.16, P = 0.30). The size of the aftereffect was greater while walking with an impeding force field than
with aiding in the initial phase of the catch trial/washout period (P < 0.05). g
p
p
(
)
Provided that the transfer of adaptation occurred only partially in the particular combination of the force
field and the GRF component (Figs. 5A, 6B), the extent of washout in the adaptation was further investigated. Figures 7 and 8 show the group means of the degree of asymmetry for washout period 2 and subsequent washout
period 3 among subjects who underwent Experiments 1 and 3, respectively. As shown in Fig. 5A, the motor
pattern acquired through walking with an aiding force field was partially transferred to subsequent walking with
an impeding force field, as observed in the braking GRF component. A similar tendency is shown in Fig. 7 (red
line) as a group means of eight subjects, where the degree of asymmetry deviates in the positive direction and
decays in the subsequent 60 s washout period. Once the direction of the force field was switched from impeding
to aiding, the degree of asymmetry increased significantly (blue line). There was a significant difference in the
degree of asymmetry between the final epoch of the second washout period and the initial epoch of the third
washout period (bar graph, P < 0.05). In the result of the propulsive GRF component after adaptation to the
impeding force field (Fig. 8), the degree of asymmetry showed a large increase upon a change in the direction
of the force field from aiding to impeding between washout periods 2 and 3. Resultsh With exposure to the split-belt, subjects exhibited a pronounced limp in their walking pattern in the early
phases, followed by slower changes to walk more stably. These modifications in walking patterns are quantified
as changes in the GRF in both the braking and propulsive components, in which the braking component gener-
ally showed more prominent changes than the propulsive component. With a return to the tied belt in the catch
trial and post-adaptation periods, significant “aftereffects” (hereafter, used to show fast (right) limb–slow (left)
limb asymmetry of the GRF) with the degree of asymmetry deviating in the opposite direction to those during
the early phases of adaptation and re-adaptation periods were observed. In particular, a significant after effect
was observed in the braking and propulsive components, which showed only minor effects in previous studies
of split-belt adaptation without additional force fields19,21.fi p
pi
In Figs. 5 and 6, the extent of the transfer of adaptation between walking with different force fields is portrayed
by comparing the degree of asymmetry between walking with an aiding force field and that with an impeding
force field during the catch trial and washout periods. A significant aftereffect was evident, regardless of the
GRF component (braking or propulsive). However, the magnitude of the aftereffect was largely dependent on
the component and the combination of the force field between the adaptation and catch trial/washout periods. In the braking component after adaptation to the aiding force field (Fig. 5A), ANOVA revealed that there were
significant main effects for the type of force field (aiding or impeding) (F(1,15) = 57.91, P < 0.001) and time (ini-
tial or final epoch of the catch trial and washout periods) (F(1,15) = 27.20, P < 0.001) and significant interaction
(F(1,15) = 5.98, P < 0.05). The aftereffect was greater when walking with an aiding force field than with an imped-
ing one in the initial (P < 0.01) and final (P < 0.01) epochs of the catch trial/washout periods. However, there were
significant differences in the propulsive component (Fig. 5B) depending on the time (F(1,15) = 33.23, P < 0.001)
but not on the type of force field (F(1,15) = 1.08, P = 0.31). This interaction was also significant (F(1,15) = 5.93,
P<0.05). )
In comparison of aftereffects after adaptation to the impeding force field (Fig. Resultsh There was a significant difference
in the degree of asymmetry between the final epoch of the second washout period and the initial epoch of the
third washout period (bar graph, P < 0.05). Discussionh The present study investigated whether the adaptation of walking with force fields acting on the body COM of
subjects in one direction (either forward or backward by aiding and impeding force fields, respectively) transfers
to another in the opposite direction. Subjects walked on a split-belt treadmill where two belts were driven at
different speed to each other (one belt at 0.5 ms−1 and another at 1.0 ms−1 (therefore, 1:2 in ratio). Known that
the number of stride cycle needed for adaptation is dependent on the speed ratio of the two belts21, subjects
underwent adaptation to store novel motor pattern to walk in the constrained environment. With the subjects
fully-adapted to walk in the constraints, subsequent change of the belt speeds to normal condition (two belts
driven in the same speed) resulted in the emergence of after effect (fast (right) limb − slow (left) limb asymmetry
of the GRF). With the braking and propulsive components of the GRF as a parameter, the transfer occurred only
limitedly between walking with the opposite force field, with the results dependent on the parameter. When
adaptation occurred by walking with an aiding force field, the aftereffect size was greater in walking with an aid-
ing force field than in walking with impeding only in the braking component. The aftereffect was smaller with
an impeding force field; however, it became larger when the force field was switched from impeding to aiding, https://doi.org/10.1038/s41598-023-29231-6 Scientific Reports | (2023) 13:1909 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. Comparison of the after effect sizes in the braking component (A) and the propulsive component
(B) of the GRF between walking with aiding (blue lines) and impeding (red lines) force fields after adaptation
with the aiding force field. Over the 1 min washout period, data were averaged for every 5-s bin. Data of eight
subjects were obtained during washout 1 (corresponding to ➀ or ➃ in Fig. 1B), while those of the other eight
subjects were from washout 2 (➁ or ➄) to overcome the ordering effects (n = 16). Lines show the mean (solid)
and standard error of the mean (dotted), respectively. The bar graphs highlight the initial and final 5 s of the
1 min washout period. The error bars are the standard error of the mean. **P < 0.01. Figure 5. Figure 6. Comparison of the after effect sizes in the braking component (A) and the propulsive component (B)
of the GRF between walking with aiding (blue lines) and impeding (red lines) force fields after adaptation to the
impeding force field. Over the 1 min washout period, data were averaged for every 5-s bin. Data of eight subjects
were obtained during washout 1 (corresponding to ➅ or ➈ in Fig. 1B), while those of the other eight subjects
were from washout 2 (➆ or ➉) to overcome the ordering effects (n = 16). Lines show the mean (solid) and
standard error of the mean (dotted), respectively. The bar graphs highlight the initial and final 5 s of the 1 min
washout period. The error bars are the standard error of the mean. **P < 0.01. Figure 6. Comparison of the after effect sizes in the braking component (A) and the propulsive component (B)
of the GRF between walking with aiding (blue lines) and impeding (red lines) force fields after adaptation to the
impeding force field. Over the 1 min washout period, data were averaged for every 5-s bin. Data of eight subjects
were obtained during washout 1 (corresponding to ➅ or ➈ in Fig. 1B), while those of the other eight subjects
were from washout 2 (➆ or ➉) to overcome the ordering effects (n = 16). Lines show the mean (solid) and
standard error of the mean (dotted), respectively. The bar graphs highlight the initial and final 5 s of the 1 min
washout period. The error bars are the standard error of the mean. **P < 0.01. mechanisms dependent on the direction of force fields but also changed the strategies used by the subjects to
adapt to walking on a split-belt treadmill (i.e., adaptive and de-adaptive aspects were evident in parameters dif-
ferent from walking without force fields). h
f h
ft
ff
h
d d
d
l
h In the emergence of the aftereffect, the present study demonstrated results contrasting with our previous
ones, in which adaptive processes and subsequent aftereffects were less evident in the propulsive component of
the GRF20,22. The results showed clear adaptive and de-adaptive aspects only in the braking component associ-
ated with activity in the tibialis anterior muscle during the early stance phase, suggesting that predictive feed-
forward control was required to set the optimal ankle stiffness upon ground contact20. Discussionh Comparison of the after effect sizes in the braking component (A) and the propulsive component
(B) of the GRF between walking with aiding (blue lines) and impeding (red lines) force fields after adaptation
with the aiding force field. Over the 1 min washout period, data were averaged for every 5-s bin. Data of eight
subjects were obtained during washout 1 (corresponding to ➀ or ➃ in Fig. 1B), while those of the other eight
subjects were from washout 2 (➁ or ➄) to overcome the ordering effects (n = 16). Lines show the mean (solid)
and standard error of the mean (dotted), respectively. The bar graphs highlight the initial and final 5 s of the
1 min washout period. The error bars are the standard error of the mean. **P < 0.01. showing limited washout with the opposite force field. Meanwhile, when adaptation was acquired by walking
with an impeding force field, the aftereffect was greater in walking with an impeding force field than in walking
with aiding force field only in the propulsive component. At this time, the smaller aftereffect of walking with the
aiding force field became larger as the direction of the force field switched to impeding. Together, these results
support the hypothesis of the present study and suggest that human locomotion with force fields acting in dif-
ferent directions is controlled by different neural circuits in the central nervous system.hi f
The aiding and impeding force field used in the present study was smaller in size (2 kg counterweight accounts
for approximately 3% mean body weight of the subjects) compared to those used in previous studies using up
to 15% body weight of the subjects11,14. However, the application of this relatively smaller force field resulted in
constant changes in both the braking and propulsive impulses, along with other GRF components and cadence
(Fig. 2). The application of the force field with different directions not only revealed the use of different neural https://doi.org/10.1038/s41598-023-29231-6 Scientific Reports | (2023) 13:1909 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ 5, 6) in addi-
tion to the braking component. Given that the predictive feedforward process of the nervous system underlies
both adaptive and de-adaptive processes, the present results showed possible changes in the adaptive strategy
with the additional involvement of predictive feedforward control to recalibrate the motor output necessary for
propulsion in the given environment. The application of the force field while walking reduces (aiding force field)
and increases (impeding force field) the effort to walk and muscle activity in the plantar flexor muscle necessary
for propulsion11. Therefore, emphasizing the propulsion phase with the application of the impeding force field
is possible to enhance the use of the predictive feedforward process at the end of the stance; therefore, adaptive
and de-adaptive (aftereffect) processes emerged in the propulsive GRF component. Meanwhile, adaptive and
de-adaptive processes were also evident in the propulsive GRF component, which was also evident with the
application of the aiding force field and is contradictory to the perspective of propulsion effort. It is possible
that the adaptation strategy of the subjects to walk on the split-belt may have been affected by the application of
the force fields regardless of the direction (aiding or impeding). In the present study focusing on transfer and
washout of adaptation between walking with different task demand (application of pulling force field of differ-
ent direction), it is of particular interest that both transfer and washout took place only limitedly in the specific
parameters. The result was evident in the GRF component that were emphasized during the adaptation periods
(in the braking component after adaptation with aiding force and in the propulsive component after adaptation
with impeding force) but not in others.fi p
g
What factors were responsible for the partial transfer of washout between walking with different force fields
acting on the subjects? Given the possibility of different functional networks depending on detailed locomo-
tion tasks, studies have demonstrated differences in the combination of muscle or neural sites used in different
locomotion tasks. For example, in forward and backward walking, the organization of muscle synergies in the
activity of lower extremity muscles has been demonstrated to be different23. Among the different speeds of walk-
ing and running, spinal mapping reconstructed from the activity of 14 lower extremity muscles demonstrated
that a different site of the spinal cord was used depending on gait and speed24. This was in contrast to
the propulsive component that showed only constant (not adaptive) changes following the fast/slow speed of
the split-belt along with activity in the gastrocnemius muscle during the stance phase, showing the use of pas-
sive feedback control for the production of reflexively induced propulsive force at the end of the stance phase20. Scientific Reports | (2023) 13:1909 | https://doi.org/10.1038/s41598-023-29231-6 www.nature.com/scientificreports/ Figure 7. Degree of washout in the acquired asymmetrical movement pattern in the braking GRF component
with the aiding force field by subsequent walking with the impeding force field in washout 2 (➁) and the aiding
force field in washout 3 (➂) periods (n = 8). The lines represent the mean (solid) and the standard error of the
mean (dotted). The bar graph compares the mean values between the last 5 s bin of washout 2 (➁) and the first
5 s bin of washout 3 (➂). The error bars represent the standard error of the mean. *P < 0.05. Figure 7. Degree of washout in the acquired asymmetrical movement pattern in the braking GRF component
with the aiding force field by subsequent walking with the impeding force field in washout 2 (➁) and the aiding
force field in washout 3 (➂) periods (n = 8). The lines represent the mean (solid) and the standard error of the
mean (dotted). The bar graph compares the mean values between the last 5 s bin of washout 2 (➁) and the first
5 s bin of washout 3 (➂). The error bars represent the standard error of the mean. *P < 0.05. Figure 7. Degree of washout in the acquired asymmetrical movement pattern in the braking GRF component
with the aiding force field by subsequent walking with the impeding force field in washout 2 (➁) and the aiding
force field in washout 3 (➂) periods (n = 8). The lines represent the mean (solid) and the standard error of the
mean (dotted). The bar graph compares the mean values between the last 5 s bin of washout 2 (➁) and the first
5 s bin of washout 3 (➂). The error bars represent the standard error of the mean. *P < 0.05. Meanwhile, in this study, clear adaptive processes and aftereffects in the propulsive component were evident
(as portrayed in the representative example, especially in Fig. 4, and in the mean values in Figs. Differences in the activity of the
spinal cord based on spinal mapping were also demonstrated in walking with the application of force fields in
the trunk, as in the present study. When subjects walked on a treadmill at speeds of 0.83, 1.39, 1.94 ms−1, either
one aiding or impeding force, each corresponding to 15% of the body weight of the subjects, was applied at the
COM14. The results showed that the activity of the sacral motor pool increased with the impeding force and
decreased with the aiding force, while in the lumbar motor pool, the activity increased with both aiding and
impeding forces. Interestingly, the results were similar to those obtained when walking on a slope. While the Scientific Reports | (2023) 13:1909 | https://doi.org/10.1038/s41598-023-29231-6 www.nature.com/scientificreports/ Figure 8. Degree of washout in the acquired asymmetrical movement pattern in the propulsive GRF
component with the impeding force field by subsequent walking with the aiding force field in washout 2 (➆)
and impeding force field in washout 3 (➇) periods (n = 8). The lines represent the mean (solid) and the standard
error of the mean (dotted). The bar graph compares the mean values between the last 5 s bin of washout 2 (➆)
and the first 5 s bin of washout 3 (➇). The error bars represent the standard error of the mean. *P < 0.05. Figure 8. Degree of washout in the acquired asymmetrical movement pattern in the propulsive GRF
component with the impeding force field by subsequent walking with the aiding force field in washout 2 (➆)
and impeding force field in washout 3 (➇) periods (n = 8). The lines represent the mean (solid) and the standard
error of the mean (dotted). The bar graph compares the mean values between the last 5 s bin of washout 2 (➆)
and the first 5 s bin of washout 3 (➇). The error bars represent the standard error of the mean. *P < 0.05. ctivity of the sacral motor pool increased on a positive slope and decreased on a negative slope, in the lumbar
motor pool, the activity increased on both positive and negative slopes15.f Underlying the differences in the combination of muscles and neural sites used depending on the detailed
task of locomotion, differences in cadence cannot be ruled out. In experiments investigating the locomotion of
nonhuman animals, the emergence of locomotor behavior concerning the underlying neural mechanisms was
dependent on cadence (movement frequency). In the swimming behavior of larval zebrafish, McLean et al.25
demonstrated that particular groups of spinal interneurons were active in a particular swimming frequency
range and were inhibited and kept silent at other swimming frequencies. In the spinal interneurons responsible
for the alternate movement from left to right of the stepping movement in cats, the alternate movement of the
limbs was diminished with a specific frequency of movement when a particular set of interneurons underwent
lesions26. The present results demonstrated a minor but constant difference in cadence between walking with
aiding (102.2 ± 1.2 steps min−1) and impeding force fields (98.3 ± 1.3 steps min−1). These differences could influ-
ence the limited transfer and washout of adaptation between walking with different force fields. p
gfi
As a limitation of the present study, it was possible that the traction force imposed on the subject was not
constant through each stride cycle. This is because the present experiment used a rigid cable and it was there-
fore an issue with inertia. A use of rubber tubing or spring placed in series with the rigid cable along with force
transducer was more appropriate for the purpose of the present study.i To summarize, the present results revealed that the adaptation that occurs while walking with a force field
acting on the COM of the subjects in one direction does not transfer to walking with a force field in the opposite
direction and is rarely washed out by each other. These results demonstrate the independence of neural control
of these locomotor tasks and provide basic knowledge to better understand the specificity of the tasks underlying
human locomotion. With its compatibility with walking on slopes, the results can provide helpful information
to develop intervention strategies for gait training in clinical practice. Given that the training effect was more
evident within the specific task or environment of walking and less under others, clinical practice of gait directed
towards everyday life, conducted in a variety of environments and by utilizing different tasks should take account
for the specificity of adaptation (training effect) dependent on the environment and the task of walking. References Ogawa, T., Kawashima, N., Obata, H., Kanosue, K. & Nakazawa, K. Distinct motor strategies underlying split-belt adaptation in
human walking and running. PLoS ONE 10, e0121951 (2015). 3. Grasso, R., Bianchi, L. & Lacquaniti, F. Motor patterns for human gait: Backward versus forward locomotion. J. Neurophysiol. 80
1868–1885 (1998). 4. Yokoyama, H., Ogawa, T., Shinya, M., Kawashima, N. & Nakazawa, K. Speed dependency in α-motoneuron activity and locomotor
modules in human locomotion: Indirect evidence for phylogenetically conserved spinal circuits. Proc. R. Soc. B 284, 20170290
(2017).t 25. McLean, D. L., Masino, M. A., Koh, I. Y. Y., Lindquist, W. B. & Fetcho, J. R. Continuous shifts in the active set of spinal interneurons
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26. Talpalar, A. E. et al. Dual-mode operation of neuronal networks involved in left-right alternation. Nature 500, 85–88 (2013) www.nature.com/scientificreports/ www.nature.com/scientificreports/ Received: 11 May 2022; Accepted: 31 January 2023 Author contributions T.O. performed experiments; T.O. analyzed data; T.O., H.O., H.Y., N.K., K.N. interpreted results of experiments;
T.O. prepared figures; T.O. drafted manuscript; T.O., H.O., H.Y., N.K., K.N. approved final version of manuscript. T.O. performed experiments; T.O. analyzed data; T.O., H.O., H.Y., N.K., K.N. interpreted results of experiments;
T.O. prepared figures; T.O. drafted manuscript; T.O., H.O., H.Y., N.K., K.N. approved final version of manuscript. F
di T.O. performed experiments; T.O. analyzed data; T.O., H.O., H.Y., N.K., K.N. interpreted results of experiments;
d fi
d ft d
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5. Dewolf, A. H., Ivanenko, Y. P., Zelik, K. E., Lacquaniti, F. & Willems, P. A. Differential activation of lumbar and sacral motor pools
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6. Rozumalski, A., Steele, K. M. & Schwartz, M. H. Muscle synergies are similar when typically developing children walk on a treadmil
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at different speeds and slopes. J. Biomech. 64, 112–119 (2017). f
17. Saito, A., Tomita, A., Ando, R., Watanabe, K. & Akima, H. Similarity of muscle synergies extracted from the lower limb including
the deep muscles between level and uphill treadmill walking. Gait Posture 59, 134–139 (2017).h 8. Mesquita, R. M. et al. The bouncing mechanism of running against hindering, or with aiding traction forces: A comparison with
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9. Na, K.-P., Kim, Y. L. & Lee, S. M. Effects of gait training with horizontal impeding force on gait and balance of stroke patients. J
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revealed by split-belt locomotor adaptation. J. Exp. Biol. 218, 3192–3198 (2015). vealed by split-belt locomotor adaptation. J. Exp. Biol. 218, 3192–3 y p
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5. Ogawa, T., Obata, H., Yokoyama, H., Kawashima, N. & Nakazawa, K. Velocity-dependent transfer of adaptation in human running
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645–649 (2006).f 8. Lamb, T. & Yang, J. F. Could different directions of infant stepping be controlled by the same locomotor central pattern generator? J. Neurophysiol. 83(5), 2814–2824 (2000). p y
9. Ivanenko, Y. P., Cappellini, G., Dominici, N., Poppele, R. E. & Lacquaniti, F. Modular control of limb movements during human
locomotion. J. Neurosci. 27(41), 11149–11161 (2007). locomotion. J. Neurosci. 27(41), 11149–11161 (2007). 10. Dewolf, A. H., Sylos-Labini, F., Cappellini, G., Ivanenko, Y. & Lacquaniti, F. Age-related changes in the neuromuscular control of
forward and backward locomotion. PLoS ONE 16(2), e0246372 (2021). 11. Gottschall, J. S. & Kram, R. Energy cost and muscular activity required for propulsion during walking. J. Appl. Physiol. 94,
1766–1772 (2003).h 2. da Silva, E. S., Kram, R. & Hoogkamer, W. Data availabilityh y
The datasets used and/or analysed during the current study available from the corresponding author on reason-
ble request. https://doi.org/10.1038/s41598-023-29231-6 Scientific Reports | (2023) 13:1909 | Fundingh g
This work was supported by the Grant-in-Aid for Scientific Research (C) (#18K10847) from Japan Society for
he Promotion of Science (JSPS) to T.O. g
This work was supported by the Grant-in-Aid for Scientific Research (C) (#18K10847) from Japan Society for
the Promotion of Science (JSPS) to T.O. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
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English
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Coupled quintessence and the impossibility of an interaction: a dynamical analysis study
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European physical journal. C, Particles and fields
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cc-by
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a e-mail: bernardiff@gmail.com
b e-mail: rlandim@if.usp.br Coupled quintessence and the impossibility of an interaction:
a dynamical analysis study Fabrízio F. Bernardia, Ricardo G. Landim
b
Instituto de Física, Universidade de São Paulo, Caixa Postal 66318, São Paulo, SP 05314-970, Brazil Received: 16 March 2017 / Accepted: 25 April 2017 / Published online: 6 May 2017
© The Author(s) 2017. This article is an open access publication Abstract
We analyze the coupled quintessence in the light
of the linear dynamical systems theory, with two different
interactions: (1) proportional to the energy density of the dark
energy and (2) proportional to the sum of the energy densities
of the dark matter and dark energy. The results presented here
enlarge the previous analyses in the literature, wherein the
interaction has been only proportional to the energy density
of the dark matter. In the first case it is possible to get the
well-known sequence of cosmological eras. For the second
interaction only the radiation and the dark-energy era can be
described by the fixed points. Therefore, from the point of
viewofdynamicalsystemtheory,theinteractionproportional
to the sum of the energy densities of the dark matter and dark
energy does not describe the universe we live in. When a scalar field is in the presence of a barotropic fluid
(with equation of state wm = pm/ρm, where pm is the pres-
sure and ρm is the energy density of the fluid) the relevant
evolution equations can be converted into an autonomous
system. Such approach is a good tool to analyze asymptotic
states of cosmological models and it has been done for uncou-
pled dark energy (quintessence, tachyon field, phantom field
and vector dark energy, for instance [63–70]) and coupled
dark energy [48,54,71–76]. The coupling assumed for the
quintessence field has been proportional to the energy density
of the dark matter ρm. However, there are other possibilities,
as for instance the coupling proportional to the energy density
of the dark energy ρφ or the sum of the two energy densities
ρm + ρφ. Similar kernels have been widely studied in the
literature [77–84]. In particular, the dark-energy evolution
at high redshifts measured by the BOSS-SDSS Collabora-
tion [85] shows a deviation from the cosmological constant
which can be explained assuming interacting dark-energy
models [86]. Eur. Phys. J. C (2017) 77:290
DOI 10.1140/epjc/s10052-017-4858-x Regular Article - Theoretical Physics b e-mail: rlandim@if.usp.br 1 We could be more economic if we had written the matter and radiation
equations in a joint form, as a general barotropic fluid with equation of
state wb. The results would be, of course, unchanged. 2 Interacting dark energy and dynamical analysis In addition, if any eigenvalue is a complex number, the fixed
point can be stable (Re μ < 0) or unstable (Re μ > 0) spiral,
due to the oscillatory behavior of its imaginary part. We consider that dark energy is described by a scalar field
with energy density ρφ and pressure pφ, and with an equa-
tion of state given by wφ = pφ/ρφ. We assume that the
scalar field is coupled with dark matter, in such a way
that total energy-momentum tensor is still conserved. In the
flat Friedmann–Lemaître–Robertson–Walker (FLRW) back-
ground with a scale factor a, the continuity equations for both
components and for radiation are 3 Quintessence dynamics where the prime denotes the derivative with respect to φ. The interaction between the quintessence field with DM
enters in the right-hand side of Eq. (7). In the presence of matter and radiation, the Friedmann
equations for the scalar field are H2 = 1
3
˙φ2
2 + V (φ) + ρm + ρr
,
(8)
˙H = −1
2
˙φ2 + ρm + 4
3ρr
,
(9) (8) √
6H
To deal with the dynamics of the system, we will define
dimensionless variables. The new variables are going to char-
acterize a system of differential equations in the form (9) X′ = f [X],
(4) (4) X′ = f [X], and the equation of state becomes wφ = pφ
ρφ
=
˙φ2 −2V (φ)
˙φ2 + 2V (φ). (10) where X is a column vector of dimensionless variables and
the prime is the derivative with respect to log a, where we set
the present scale factor a0 to be one. The critical points Xc
are those ones that satisfy X′ = 0. In order to study stability
of the fixed points we consider linear perturbations U around
them, thus X = Xc+U. At the critical point the perturbations
U satisfy the following equation: where X is a column vector of dimensionless variables and
the prime is the derivative with respect to log a, where we set
the present scale factor a0 to be one. The critical points Xc
are those ones that satisfy X′ = 0. In order to study stability
of the fixed points we consider linear perturbations U around
them, thus X = Xc+U. At the critical point the perturbations
U satisfy the following equation: (10) We are now ready to proceed to the dynamical analysis of
the system. 1 Introduction Sixty-eight percent of our universe [1] consists of a still
mysterious component called “dark energy” (DE), which is
believed to be responsible for the present acceleration of the
universe [2,3]. In addition to ordinary matter, the remain-
ing 27% of the energy content of the universe is a form of
matter that interacts in principle only gravitationally, known
as dark matter (DM). Among a wide range of alternatives
for the dark energy, which includes the cosmological con-
stant, scalar or vector fields [4–16], holographic dark energy
[17–29], metastable dark energy [30–34], modifications of
gravity and different kinds of cosmological fluids [35–41],
the usage of a canonical scalar field, called “quintessence”,
is a viable candidate [42–46]. A dynamical analysis remained to be done for these two
kernels. In this paper we use the linear dynamical systems
theory to investigate the critical points that come from the
evolution equations for the quintessence, assuming the inter-
action between DE and DM proportional to (i) ρφ and (ii)
ρφ + ρm. We found that in the case (i) there are fixed points
that can describe the sequence of three cosmological eras. In
the second case either radiation era or dark-energy era can be
described by fixed points, but the matter-dominated universe
is absent. The remainder of this paper is structured as follows. In
Sect. 2 we present the basics of the interacting dark energy
and the dynamical analysis theory. The quintessence dynam-
ics is presented in Sect. 3 and the dynamical system theory
is used to study the coupled quintessence in Sect. 4, wherein
the critical points are shown. We summarize our results in
Sect. 5. We use Planck units (¯h = c = Mpl = 1) throughout
the text. In addition, the two components of the dark sector may
interact with each other [21–24,39,47–59] (see [60] for a
review) and the interaction can eventually alleviate the coin-
cidence problem [61,62]. 12 3 3 290
Page 2 of 7 Eur. Phys. J. C (2017) 77 :290 3 Quintessence dynamics The scalar field φ is described by the Lagrangian L = −√−g
1
2∂μφ∂μφ + V (φ)
,
(6) (6) ˙ρφ + 3H(ρφ + pφ) = −Q,
(1)
˙ρm + 3Hρm = Q,
(2)
˙ρr + 4Hρr = 0,
(3) where V (φ) is the scalar potential given by V (φ) = V0e−λφ
and V0 and λ are constants. This choice is motivated by the
autonomoussystem,asweshallseesoon.Forahomogeneous
field φ ≡φ(t) in an expanding universe with FLRW metric
with scale factor a ≡a(t), the equation of motion is ˙ρr + 4Hρr = 0, respectively, where H = ˙a/a is the Hubble rate, Q is the
coupling and the dot is a derivative with respect to the cos-
mic time t. The indices m and r stand for matter and radi-
ation, respectively.1 The case of Q > 0 corresponds to
dark-energy transformation into dark matter, while Q < 0
is the transformation in the opposite direction. In princi-
ple, the coupling can depend on several variables Q =
Q(ρm, ρφ, ˙φ, H, t, . . . ), so we assume for the quintessence
the coupling is (i) Q = Qρφ ˙φ and (ii) Q = Q(ρφ + ρm) ˙φ,
where Q is a positive constant. The case with negative Q is
the same as the case with Q > 0 but with negative fixed point
x, described in the next section by
˙φ
√
6H (11). respectively, where H = ˙a/a is the Hubble rate, Q is the
coupling and the dot is a derivative with respect to the cos-
mic time t. The indices m and r stand for matter and radi-
ation, respectively.1 The case of Q > 0 corresponds to
dark-energy transformation into dark matter, while Q < 0
is the transformation in the opposite direction. In princi-
ple, the coupling can depend on several variables Q =
Q(ρm, ρφ, ˙φ, H, t, . . . ), so we assume for the quintessence
the coupling is (i) Q = Qρφ ˙φ and (ii) Q = Q(ρφ + ρm) ˙φ,
where Q is a positive constant. The case with negative Q is
the same as the case with Q > 0 but with negative fixed point
x, described in the next section by
˙φ
√
6H (11). ˙φ( ¨φ + 3H ˙φ + V ′(φ)) = 0,
(7) (7) where the prime denotes the derivative with respect to φ. 4 Autonomous system Notice that y cannot be negative and recall that
r = z2. The fixed points of the system are obtained by setting
dx/dN = 0, dy/dN = 0, dz/dN = 0 and dλ/dN = 0
in Eqs. (17)–(20). When = 1, λ is constant; the poten-
tial is V (φ) = V0e−λφ [63,64].2 The fixed points are shown
in Table 1. Notice that y cannot be negative and recall that
r = z2. 0 ≤x2 + y2 ≤1,
(14) (14) due to 0 ≤φ ≤1. The equation of state wφ becomes due to 0 ≤φ ≤1. The equation of state wφ becomes The point x f is The point x f is wφ = x2 −y2
x2 + y2 ,
(15)
The point x f is
x f = −3+Qλ−λ2+√
Q2(λ2−12)−2Qλ(λ2−9)+(λ2−3
2
√
6(Q−λ) wφ = x2 −y2
x2 + y2 ,
(15 wφ = x2 −y2
x2 + y2 , x f = −3+Qλ−λ2+√
Q2(λ2−12)−2Qλ(λ2−9)+(λ2−3)2
2
√
6(Q−λ)
. (22) (15) (22) The eigenvalues of the Jacobian matrix were found for each
fixed point in Table 1. The results are shown in Table 2, where
the eigenvalues μ f ± are and the total effective equation of state is and the total effective equation of state is weff =
pφ + pr
ρφ + ρm + ρr
= x2 −y2 + z2
3 ,
(16)
the eigenvalues μ f ± are
μ f ± = 1
4{−12 +
√
6x f (−2Q + 3λ)
± [48(Q
λ)λ + 96
√
6 3(
Q + λ) weff =
pφ + pr
ρφ + ρm + ρr
= x2 −y2 + z2
3 ,
(16)
with an accelerated expansion for weff < −1/3. 4 1
i
the eigenvalues μ f ± are
μ f ± = 1
4{−12 +
√
6x f (−2Q + 3λ)
± [48(Q −λ)λ + 96
√
6x3
f (−Q + λ)
+ 6x2
f (−24 + 4Q2 + 28Qλ −31λ2) μ f ± = 1
4{−12 +
√
6x f (−2Q + 3λ)
± [48(Q −λ)λ + 96
√
6x3
f (−Q + λ)
+ 6x2
f (−24 + 4Q2 + 28Qλ −31λ2)
+ 8
√
6x f (9λ + 2λ3 −2Q(3 + λ2))]1/2}. (23) (16) with an accelerated expansion for weff < −1/3. 4 Autonomous system U ′ = J U,
(5) (5) U ′ = J U, The dimensionless variables are defined as The dimensionless variables are defined as where J is the Jacobian matrix. The stability around the
fixed points depends on the nature of the eigenvalues (μ)
of J , in such a way that they are stable points if they all
have negative values, unstable points if they all have positive
values and saddle points if at least one eigenvalue has positive
(or negative) value, while the other ones have opposite sign. x ≡
˙φ
√
6H
,
y ≡
√V (φ)
√
3H
,
z ≡
√ρr
√
3H
,
λ ≡−V ′
V ,
≡V V ′′
V ′2 . x ≡
˙φ
√
6H
,
y ≡
√V (φ)
√
3H
,
z ≡
√ρr
√
3H
,
λ ≡−V ′
V ,
≡V V ′′
V ′2 . (11) (11) The dark-energy density parameter is written in terms of
these new variables as The dark-energy density parameter is written in terms of
these new variables as φ ≡ρφ
3H2 = x2 + y2,
(12) (12) 12 3 Eur. Phys. J. C (2017) 77 :290 Page 3 of 7
290 Page 3 of 7
290 Table 1 Critical points (x, y and z) of Eqs. (17)–(19) for the quintessence field with interaction Qρφ. The table shows the correspondent equation
of state for the dark energy (15), the effective equation of state (16) and the density parameter for dark energy (12) Table 1 Critical points (x, y and z) of Eqs. (17)–(19) for the quintessence field with interaction Qρφ. The table shows the correspondent equation
of state for the dark energy (15), the effective equation of state (16) and the density parameter for dark energy (12) Table 1 Critical points (x, y and z) of Eqs. (17)–(19) for the quintessence field with interaction Qρφ. 4 Autonomous system The table shows the correspondent equation
of state for the dark energy (15), the effective equation of state (16) and the density parameter for dark energy (12)
Point
x
y
z
wφ
φ
weff
(a)
2
√
6
3λ
2√2Q+λ
√
3λ2(λ−Q)
1 −4(λ−4Q)
λ2(λ−Q)
1
3
1 −4Q
λ
4
λ(λ−Q)
1
3
(b)
0
0
1
−
0
1
3
(c)
−
√
6
3Q
0
1 −
2
Q2
1
2
3Q2
1
3
(d)
0
0
0
−
0
0
(e)
Q±√
Q2+6
√
6
0
0
1
x2
e
x2
e
(f)
x f
1 + x2
f −
√
2x f λ
0
−3+
√
6x f λ
3+6x2
f −
√
6x f λ
1 + 2x2
f −
2
3 x f λ
−1 +
2
3 x f λ (f) so that Eq. (8) can be written as
where so that Eq. (8) can be written as
φ + m + r = 1,
(13)
where
H−1 dH
dN = −3
2
1 + x2 −y2 + z2
3
. (21) (13)
H−1 dH
dN = −3
2
1 + x2 −y2 + z2
3
. (21) (21) where the matter and radiation density parameter are defined
by i = ρi/(3H2), with i = m,r. From Eqs. (12) and (13)
we see that x and y are restricted in the phase plane by the
relation where the matter and radiation density parameter are defined
by i = ρi/(3H2), with i = m,r. From Eqs. (12) and (13)
we see that x and y are restricted in the phase plane by the
relation 4.1.1 Critical points The fixed points of the system are obtained by setting
dx/dN = 0, dy/dN = 0, dz/dN = 0 and dλ/dN = 0
in Eqs. (17)–(20). When = 1, λ is constant; the poten-
tial is V (φ) = V0e−λφ [63,64].2 The fixed points are shown
in Table 1. Notice that y cannot be negative and recall that
r = z2. The fixed points of the system are obtained by setting
dx/dN = 0, dy/dN = 0, dz/dN = 0 and dλ/dN = 0
in Eqs. (17)–(20). When = 1, λ is constant; the poten-
tial is V (φ) = V0e−λφ [63,64].2 The fixed points are shown
in Table 1. 2 The equation for λ is also equal zero when x = 0 or λ = 0, so that λ
should not necessarily be constant, for the fixed points with this value of
x. However, for the case of a dynamical λ, the corresponding eigenvalue
is equal zero, indicating that the fixed points is not hyperbolic. 4 Autonomous system Table 2 Eigenvalues and stability of the fixed points for the quintessence field with interaction Qρφ
Point
μ1
μ2
μ3
Stability
(a)
See the main text
Saddle
(b)
1
3 −
√
6Q
3 −
√
6
2 λ
Saddle or unstable
(c)
1 −
√
2
Q
1 +
√
2
Q
2 −λ
Q
Saddle or unstable
(d)
−3
2
3
2
−1
2
Saddle
(e)
1
2
2 + Q
Q ±
6 + Q2
1
2
6 + Q
Q ±
6 + Q2
1
2
6 + (Q −λ)
Q ±
6 + Q2
Saddle or unstable
(f)
−2 +
3
2 x f λ,
μ f +
μ f −
Stable Table 2 Eigenvalues and stability of the fixed points for the quintessence field with interaction Qρφ Fig. 2 Allowed regions of Q, for the fixed points (a) (yellow), (c) (blue)
and (f) (red) Fig. 2 Allowed regions of Q, for the fixed points (a) (yellow), (c) (blue)
and (f) (red) x
y Fig. 1 Phase plane for the fixed point (f) with λ = 1 and Q = 1/4 Fig. 2 Allowed regions of Q, for the fixed points (a) (yellow), (c) (blue)
and (f) (red) saddle point. Similar results are found for other values of λ
and Q. Both points (b) and (c) also describe the radiation era and
are unstable or saddle points. The eigenvalues μ2 and μ3 of
the point (b) can be either positive or negative, depending on
the values of λ and Q. On the other hand, the first eigenvalue
μ1 is always positive. The same happens with the eigenval-
ues of the point (c) and in this case the interaction must be
Q >
√
2 for the fixed points be real and Q > 20/(3
√
3) for
BBN
φ
< 0.045. Therefore, the sequence of cosmological eras (radiation
→matter →dark energy) is reached considering the transi-
tion: (b) or (c) →(d) or (e) →(f). 4.2 Interaction Q(ρφ + ρm) The dynamical system for the variables x, y, z and λ with
the interaction proportional to ρφ + ρm is φ
The matter-dominated universe is described by the saddle
point (d) and also by the point (e), provided that Q is suf-
ficiently large for the latter case. 4 Autonomous system 4.1 Interaction Qρφ 4.1 Interaction Qρφ (23) The fixed point (a) describes a radiation-dominated uni-
verse and in order to the fixed points be real and φ satisfy the
nucleosynthesis bound BBN
φ
< 0.045 [87] we should have The dynamical system for the variables x, y, z and λ with
the interaction proportional to ρφ is dx
dN = −3x +
√
6
2 y2λ −
√
6
2 Q(x2 + y2) −x H−1 dH
dN ,
(17)
dy
dN = −
√
6
2 xyλ −yH−1 dH
dN ,
(18)
dz
dN = −2z −zH−1 dH
dN ,
(19)
dλ
dN = −
√
6λ2x ( −1) ,
(20) dx
dN = −3x +
√
6
2 y2λ −
√
6
2 Q(x2 + y2) −x H−1 dH
dN ,
(17)
dy
dN = −
√
6
2 xyλ −yH−1 dH
dN ,
(18)
dz
dN = −2z −zH−1 dH
dN ,
(19)
dλ
dN = −
√
6λ2x ( −1) ,
(20) φ
λ > 20
√
2
3
and Q ≤9λ2−800
9λ
. The eigenvalues were found
numerically. For λ = 10 and the upper limit for the interac-
tion (Q = 10/9) we get the eigenvalues μ1 = −0.7 + 2.1i,
μ2 = −0.7 −2.1i and μ3 = 1, so this critical point is a φ
λ > 20
√
2
3
and Q ≤9λ2−800
9λ
. The eigenvalues were found
numerically. For λ = 10 and the upper limit for the interac-
tion (Q = 10/9) we get the eigenvalues μ1 = −0.7 + 2.1i,
μ2 = −0.7 −2.1i and μ3 = 1, so this critical point is a 2 The equation for λ is also equal zero when x = 0 or λ = 0, so that λ
should not necessarily be constant, for the fixed points with this value of
x. However, for the case of a dynamical λ, the corresponding eigenvalue
is equal zero, indicating that the fixed points is not hyperbolic. 12 3 3 Eur. Phys. J. 4 Autonomous system C (2017) 77 :290 290
Page 4 of 7 Table 2 Eigenvalues and stability of the fixed points for the quintessence field with interaction Qρφ
Point
μ1
μ2
μ3
Stability
(a)
See the main text
Saddle
(b)
1
3 −
√
6Q
3 −
√
6
2 λ
Saddle or unstable
(c)
1 −
√
2
Q
1 +
√
2
Q
2 −λ
Q
Saddle or unstable
(d)
−3
2
3
2
−1
2
Saddle
(e)
1
2
2 + Q
Q ±
6 + Q2
1
2
6 + Q
Q ±
6 + Q2
1
2
6 + (Q −λ)
Q ±
6 + Q2
Saddle or unstable
(f)
−2 +
3
2 x f λ,
μ f +
μ f −
Stable
x
y
Fig. 1 Phase plane for the fixed point (f) with λ = 1 and Q = 1/4
saddle point. Similar results are found for other values of λ
and Q. Both points (b) and (c) also describe the radiation era and
are unstable or saddle points. The eigenvalues μ2 and μ3 of
the point (b) can be either positive or negative, depending on
the values of λ and Q. On the other hand, the first eigenvalue
i
l
iti
Th
h
ith th
i
l
Fig. 2 Allowed regions of Q, for the fixed points (a) (yellow), (c) (blue)
and (f) (red)
Therefore, the sequence of cosmological eras (radiation
→matter →dark energy) is reached considering the transi-
tion: (b) or (c) →(d) or (e) →(f). 4 Autonomous system Whatever the value of the
interaction all eigenvalues of the point (e) cannot be simul-
taneously negative. dx
dN = −3x +
√
6
2 y2λ −
√
6
2 Q(1 −z2) −x H−1 dH
dN ,
(24)
dy
dN = −
√
6
2 xyλ −yH−1 dH
dN ,
(25)
dz
dN = −2z −zH−1 dH
dN ,
(26)
dλ
dN = −
√
6λ2x ( −1) ,
(27) The last fixed point (f) is an attractor and it describes the
dark-energy dominated universe if either λ ≤
√
2 and Q < λ
or λ >
√
2 and −2λ+λ3
4+λ2
≤Q < λ. The real parts of the
eigenvalues are negative for this range of values, thus the
fixed point is stable or stable spiral. Its behavior is illustrated
in Fig. 1, where we plot the phase plane with λ = 1 and
Q = 1/4. (27) where where The allowed values of λ and Q, for the fixed points
(a), (c) and (f), are shown in Fig. 2. From the figure we
see that the fixed points (a) and (f) do not have common
regions. H−1 dH
dN = −3
2
1 + x2 −y2 + z2
3
. (28) (28) 123 4.2.1 Critical points As before fixed points of the system are obtained by setting
dx/dN = 0, dy/dN = 0, dz/dN = 0 and dλ/dN = 0 in
Eqs. (24)–(27). The fixed points are shown in Table 3, where The radiation era is also described by the points (b) and
(c). They are saddle points and for (c) the interaction must be
Q ≥20
√
3
27
in order not to conflict the nucleosynthesis bound. The radiation era is also described by the points (b) and
(c). They are saddle points and for (c) the interaction must be
Q ≥20
√
3
27
in order not to conflict the nucleosynthesis bound. The matter-dominated universe can be described by the
point (d) but only if the interaction is zero, which in turn is
known in the literature [35]. The matter-dominated universe can be described by the
point (d) but only if the interaction is zero, which in turn is
known in the literature [35]. wφe =
λ2
λ2 −3 −
12Qλ + (λ2 −3)2
3
λ(λ + 2Q) + 3 −
12Qλ + (λ2 −3)2
. (29) (29) The fixed point (e) can describe the current stage of accel-
erated expansion of the universe for some values of Q and
λ. The critical points are real with 0 ≤φ ≤1 and
weff < −1/3 for 0 < λ ≤
√
2 and 0 < Q ≤λ or for
λ >
√
2 and λ2−2
3λ
< Q ≤λ. For these ranges of λ and Q
the real part of the eigenvalues are negative, so the point is
stable or stable spiral. The attractor point has φ = 1 and
wφ = weff = −1 for Q = λ. The eigenvalues of the Jacobian matrix were found for
each fixed point of Table 3. The results are shown in Table
4, where μe1 = 1
4
λ2 −5 −√μ
,
(30)
μe2,e3 =
1
8λ2 (3λ4 + 3λ2(5 −√μ)
±
√
2(λ2(−72(−3 + √μ))
−6Qλ(7λ2 −48 + 8√μ)
+ λ2(λ4 −63 −(3 + λ2)√μ))1/2)
(31)
and μe1 = 1
4
λ2 −5 −√μ
,
(30) (30) μe2,e3 =
1
8λ2 (3λ4 + 3λ2(5 −√μ)
±
√
2(λ2(−72(−3 + √μ))
−6Qλ(7λ2 −48 + 8√μ)
+ λ2(λ4 −63 −(3 + λ2)√μ))1/2)
(31)
and
μ = 12Qλ + (λ2 −3)2. 4.2.1 Critical points (32) Therefore, both radiation and dark-energy-dominated uni-
verse can be described by the fixed points, however, none of
them represent the matter era. (31) and 123 The
equation of state for the dark energy (15), the effective equation of state (16) and the density parameter for dark en Table 3 Critical points (x, y and z) of Eqs. (24)–(26) for the quintessence field with interaction Q(ρφ + ρm). The table shows the correspondent
equation of state for the dark energy (15), the effective equation of state (16) and the density parameter for dark energy (12)
Point
x
y
z
wφ
φ
weff
(a)
2
√
6
3λ
2√6Q+λ
√
3λ2(λ+3Q)
λ(λ+3Q)−4
λ(λ+3Q)
λ
3λ+12Q
4((λ+4Q)
λ2(λ+3Q)
1
3
(b)
0
0
1
−
0
1
3
(c)
−
√
6
9Q
0
1 −
2
9Q2
1
2
27Q2
1
3
(d)
3x3
d −3xd −
√
6Q = 0
0
0
1
x2
d
x2
d
(e)
3+λ2±√
12Qλ+(λ2−3)2
2
√
6λ
x2e −(
√
6/3)xeλ + 1
0
wφe
λ2+2Qλ+3−√
12Qλ+(λ2−3)2
2λ2
λ2−3−√
12Qλ+(λ2−3)2
6 See the main text
−1
2
9Q2 −3
See the main text
μe2 μe3 All equations above but the first one are identical to the pre-
vious case. nucleosynthesis bound BBN
φ
< 0.045 [87] we should have
20
√
2
3
< λ < 40
√
6
9
and Q ≤9λ3−800λ
27λ2−3200 or λ ≥40
√
6
9
for any
value of positive Q. The eigenvalues were found numerically
and similarly to the case of the previous section, the fixed
point is a saddle point for the allowed values of λ. 123 Page 5 of 7
290 Eur. Phys. J. C (2017) 77 :290 Page 5 of 7
290 Table 3 Critical points (x, y and z) of Eqs. (24)–(26) for the quintessence field with interaction Q(ρφ + ρm). The table shows the correspondent
equation of state for the dark energy (15), the effective equation of state (16) and the density parameter for dark energy (12)
Point
x
y
z
wφ
φ
weff
(a)
2
√
6
3λ
2√6Q+λ
√
3λ2(λ+3Q)
λ(λ+3Q)−4
λ(λ+3Q)
λ
3λ+12Q
4((λ+4Q)
λ2(λ+3Q)
1
3
(b)
0
0
1
−
0
1
3
(c)
−
√
6
9Q
0
1 −
2
9Q2
1
2
27Q2
1
3
(d)
3x3
d −3xd −
√
6Q = 0
0
0
1
x2
d
x2
d
(e)
3+λ2±√
12Qλ+(λ2−3)2
2
√
6λ
x2e −(
√
6/3)xeλ + 1
0
wφe
λ2+2Qλ+3−√
12Qλ+(λ2−3)2
2λ2
λ2−3−√
12Qλ+(λ2−3)2
6
Table 4 Eigenvalues and
stability of the fixed points for
the quintessence field with
Q(ρφ + ρm)
Point
μ1
μ2
μ3
Stability
(a)
See the main text
Saddle
(b)
2
−1
1
Saddle
(c)
−
2
9Q2 −3
2
9Q2 −3
2 +
λ
3Q
Saddle
(d)
See the main text
Saddle or unstable
(e)
μe1
μe2
μe3
Stable Table 3 Critical points (x, y and z) of Eqs. (24)–(26) for the quintessence field with interaction Q(ρφ + ρm). The table shows the correspondent
equation of state for the dark energy (15), the effective equation of state (16) and the density parameter for dark energy (12) Table 3 Critical points (x, y and z) of Eqs. (24)–(26) for the quintessence field with interaction Q(ρφ + ρm). The table shows the correspondent
equation of state for the dark energy (15), the effective equation of state (16) and the density parameter for dark energy (12) Table 3 Critical points (x, y and z) of Eqs. (24)–(26) for the quintessence field with interaction Q(ρφ + ρm). The table shows the correspondent
equation of state for the dark energy (15), the effective equation of state (16) and the density parameter for dark energy (12) Table 3 Critical points (x, y and z) of Eqs. (24)–(26) for the quintessence field with interaction Q(ρφ + ρm). References 28. M. Li, X.-D. Li, S. Wang, Y. Wang, Dark energy. Commun. Theor. Phys. 56, 525–604 (2011) 1. P.A.R. Ade et al., Planck 2015 results. XIII. Cosmological param-
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https://hal.parisnanterre.fr//hal-03148895/document
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Visual Attentional Training Improves Reading Capabilities in Children with Dyslexia: An Eye Tracker Study During a Reading Task
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To cite this version: Simona Caldani, Christophe-Loïc Gerard, Hugo Peyre, Maria Pia Bucci. Visual Attentional Training
Improves Reading Capabilities in Children with Dyslexia: An Eye Tracker Study During a Reading
Task. Brain Sciences, 2020, 10 (8), pp.558. 10.3390/brainsci10080558. hal-03148895 Visual Attentional Training Improves Reading
Capabilities in Children with Dyslexia: An Eye Tracker
Study During a Reading Task
Simona Caldani, Christophe-Loïc Gerard, Hugo Peyre, Maria Pia Bucci Visual Attentional Training Improves Reading
Capabilities in Children with Dyslexia: An Eye Tracker
Study During a Reading Task
Simona Caldani, Christophe-Loïc Gerard, Hugo Peyre, Maria Pia Bucci Received: 24 July 2020; Accepted: 12 August 2020; Published: 15 August 2020 Abstract: Dyslexia is a specific disorder in reading abilities. The aim of this study was to explore
whether a short visual attentional training could improve reading capabilities in children with reading
disorders by changing their oculomotor characteristics. Two groups (G1 and G2) of 25 children with
reading disabilities and who are matched in IQ (intelligence quotient), sex, and age participated in
the study. The allocation of a subject to a specific group (G1 = experimental group; G2 = control
group) was generated in an unpredictable random sequence. The reading task was recorded twice
for G1, i.e., before (T1) and after (T2) 10 min of visual attentional training. Training consisted of
oculomotor tasks (saccades and pursuits movements) and searching tasks (three different exercises). For G2, the two reading tasks at T1 and T2 were done at an interval of 10 min instead. We found that
at T1, oculomotor performances during reading were statistically similar for both groups of children
with reading disabilities (G1 and G2). At T2, the group G1 only improved oculomotor capabilities
significantly during reading; in particular, children read faster, and their fixation time was shortest. We conclude that short visual attentional training could improve the cortical mechanisms responsible
for attention and reading capabilities. Further studies on a larger number of dyslexic children will
be necessary in order to explore the effects of different training types on the visual attentional span
given its important role on the orienting and focusing visuospatial attention and on the oculomotor
performance in children with dyslexia. Keywords: eye movements; dyslexia; children; reading disabilities; visual attentional training Visual Attentional Training Improves Reading
Capabilities in Children with Dyslexia: An Eye
Tracker Study During a Reading Task Simona Caldani 1,2,*, Christophe-Loïc Gerard 3, Hugo Peyre 3,4 and Maria Pia Bucci 1,2
1
UMR 7114 MoDyCo, CNRS-Université Paris Nanterre, 92000 Nanterre, France; mariapia.bucci@gmail.com
2
EFEE—Centre D’Exploration Fonctionnelle de L’Équilibre Chez L’Enfant, Robert Debré Hospital,
75019 Paris, France
3
Child and Adolescent Psychiatry Department Robert Debré Hospital 75019 Paris France; Simona Caldani 1,2,*, Christophe-Loïc Gerard 3, Hugo Peyre 3,4 and Maria Pia Bucci 1,2
1
UMR 7114 MoDyCo, CNRS-Université Paris Nanterre, 92000 Nanterre, France; mariapia.bucci@gm
2
EFEE—Centre D’Exploration Fonctionnelle de L’Équilibre Chez L’Enfant, Robert Debré Hospital,
75019 Paris, France
3
Child and Adolescent Psychiatry Department, Robert Debré Hospital, 75019 Paris, France;
christophe.loic@gmail.com (C.-L.G.); peyrehugo@yahoo.fr (H.P.)
4
Université de Paris, 75000 Paris, France
*
Correspondence: simona.caldani@gmail.com Simona Caldani 1,2,*, Christophe-Loïc Gerard 3, Hugo Peyre 3,4 and Maria Pia Bucci 1,2
1
UMR 7114 MoDyCo, CNRS-Université Paris Nanterre, 92000 Nanterre, France; mariapia.bucci@gmail.com
2
EFEE—Centre D’Exploration Fonctionnelle de L’Équilibre Chez L’Enfant, Robert Debré Hospital,
75019 Paris, France
3
Child and Adolescent Psychiatry Department, Robert Debré Hospital, 75019 Paris, France;
christophe.loic@gmail.com (C.-L.G.); peyrehugo@yahoo.fr (H.P.)
4
Université de Paris, 75000 Paris, France
*
Correspondence: simona.caldani@gmail.com Simona Caldani
, ,*, Christophe-Loïc Gerard , Hugo Peyre
, and Maria Pia Bucci
,
1
UMR 7114 MoDyCo, CNRS-Université Paris Nanterre, 92000 Nanterre, France; mariapia.bucci@gmail.com
2
EFEE—Centre D’Exploration Fonctionnelle de L’Équilibre Chez L’Enfant, Robert Debré Hospital, *
Correspondence: simona.caldani@gmail.com *
Correspondence: simona.caldani@gmail.com HAL Id: hal-03148895
https://hal.parisnanterre.fr/hal-03148895v1
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teaching and research institutions in France or
abroad, or from public or private research centers. brain
sciences brain
sciences 1. Introduction Dyslexia is a disorder reported in 5–10% of school-aged children [1]. Given the heterogeneity of
this disorder in the literature there are still different theories that have tried to explain its etiology [2]. Actually, it is known that genetical investigations indicate a family risk, and the presence of a
phonological impairment in dyslexia could be considered the most robust hypothesis suggesting that
dyslexic children showed difficulties to read because they fail to acquire the skill of separating the sounds
in a word in order to match them to their visual letter counterparts [2]. However, other hypotheses have
been put forward. For instance, several studies reported deficits in auditory capabilities [3], as well
as in visual perception and oculomotor performances [4,5]. Nicolson and Fawcett [6] advanced the
hypothesis of a cerebellar impairment that leads to poor automaticity and motor control in children with
dyslexia. Facoetti et al. [7] reported also in children with dyslexia a specific disability in orienting as well www.mdpi.com/journal/brainsci Brain Sci. 2020, 10, 558; doi:10.3390/brainsci10080558 www.mdpi.com/journal/brainsci 2 of 13 Brain Sci. 2020, 10, 558 as in sustained focusing visuospatial attention. Our group further explored oculomotor capabilities in
dyslexic children and suggested a deficit in visual attentional processing in relation to the immaturity of
cortical structures responsible for saccades triggering [8–10]. Reading, in fact, corresponds to a complex
cognitive process during which several mechanisms are involved: visual perception, eye movements
such as saccades and fixations, and semantic and linguistic abilities; consequently, a deficit in one of
these different components could cause impairment in reading acquisition. Previous studies from our
group [11] and other groups [12,13] reported a longer duration of fixation in children starting to read,
explained by the immaturity of cortical structures responsible for eye fixation [14]. During the normal
development of reading skills, children learn to read rapidly by having shorter durations of fixations,
larger prosaccades, and fewer regressions; in other words, eye movement patterns become similar to
those observed in adults. In contrast, in children with dyslexia this oculomotor pattern is not observed; indeed, it is
well documented that children with dyslexia have abnormal oculomotor patterns during reading,
e.g., a slower reading speed, longer duration of fixations, frequent and smaller prosaccades, and several
regressions [15]. Interestingly, such characteristics have been reported in children with dyslexia
of different countries speaking different languages (Greek speaking [16]; English speaking [17];
Italian speaking [18], and German speaking [19]). 1. Introduction However, the hypothesis of an abnormal oculomotor
pattern in dyslexia and related visual deficits is not shared by other researchers; for instance, the authors
in [20] showed that visual deficit impairments could be causal in dyslexia, and other researchers
advanced the hypothesis that poor oculomotor behavior is a consequence and not the cause of
dyslexia [21]. This question is still under debate. One of the most important objectives of research conducted on children with dyslexia is to develop
training types able to improve reading skills, particularly reading speed—even if it is influenced by
the type of language (more or less transparent, see previously cited studies)—as it is an important
parameter for dyslexic children during school activities, given that if children need a longer time to
read they cannot do the other school exercises as nondyslexic children do. Although linguistic training
is the most studied [22], research has also been carried out on nonlinguistic training types. Firstly, Solan et al. [23], in order to study some of the visual processes of reading disability,
compared two types of training (eye movement and comprehension training for 12 one-hour sessions)
in two groups of 15 children of 11 years old with dyslexia. For the eye movement training, the perceptual
accuracy/visual efficiency method was used, while for the comprehension training the child must correct
missing words in a sentence. They found that the number of fixations as well as the number of regressive
saccades and comprehension were both improved by eye movement training; similar results were
obtained by comprehension training, supporting the notion of a cognitive link between visual attention,
eye movement’s performance, and reading comprehension. These findings support the existence of a
cognitive relationship among visual attention, eye movements, and reading comprehension. Meng et al. [24] reported reading enhancement in Chinese-speaking children with dyslexia after
ten sessions within four weeks of visual perceptual training (texture discrimination tasks lasting about
50 min/session); interestingly, such improvement was still observed two months later. These authors
advanced the hypothesis that temporal processing and spatial attentional capabilities leading to
enlarging the visual span could be the possible cause of the training effect. This study suggested
that, at least in Chinese readers, visual perceptual processing and reading ability could share a
similar network. Several studies conducted in children with dyslexia have reported an improvement of reading
performance by decreasing the crowding phenomenon (i.e., increasing letter spacing [25–27]). 1. Introduction It should
be noted, however, that for most of these studies the number of children with dyslexia was small,
and no eye movement recording during reading was performed. Our group recorded eye movements
during reading and confirmed in French children with dyslexia the beneficial effect of large letter
spacing between words for reading [28]. Recently, we showed also that a computer-based oculomotor
training (15 min per day for five/seven days per week for 8 weeks) in a small group of 16 Italian 3 of 13 Brain Sci. 2020, 10, 558 children with dyslexia could improve reading abilities by shortening the reading time and the duration
of fixation objectively measured (by an eye tracker), perhaps via visual attentional capabilities [29]. More recently, Cancer et al. [30] compared in a small group of dyslexic children (n = 12) the effect
of rhythmic reading training (RRT) on reading speed and accuracy with respect to those of two training
types already validated (i.e., Bakker’s visual hemisphere-specific stimulation and the action video
game training). Both training types were administrated for 13 h over nine days. The authors reported
that RRT improved more significantly the pseudoword reading speed, and the other training increased
general reading accuracy. Authors suggested that these different results could be due to different
cognitive mechanisms trained; in other words, the RTT could affect phonological awareness while the
other training could have an effect on rapid automatized naming. Based on these results we could
make the hypothesis that specific training types could be developed for different types of dyslexia. This hypothesis, however, needs to be tested on a large population with dyslexia. Peters and colleagues [31] made a systematic review on the effect of visual attentional training on
reading abilities in children with dyslexia aged 5–15 years old. These authors selected eighteen studies
and analyzed three types of visual attentional interventions: action video games, reading acceleration,
and visual perceptual training. Their findings showed that action video games improved speed reading
and fluency, while reading acceleration programs increased accuracy, and speed reading and visual
perceptual training benefited reading fluency and comprehension. Interestingly, these interventions
can improve reading capabilities for at least two months after training. It should be noted, however,
that other researchers [32] did not confirm these results. 1. Introduction It is important also to point out that no study
recorded eye movement behavior to confirm or not the eventual benefit of interventions, with the
exception of one study in which a small group of children were tested (nine children only) [33]. Based on these findings, the aim of the present study is to measure by an eye tracker the effect
of a short visual attentional training on the reading skills in French children with reading disorders. Eye movement recording is an objective tool that allow us to better understand the eventual changes
in oculomotor patterns in children with dyslexia after training. Our final purpose is to develop a new
functional training program for this kind of children. 2.1. Subjects Fifty children with reading disorders (from 7.8 to 12 years old) were recruited from the Robert
Debré Pediatric Hospital. The children in the study underwent a complete evaluation (see our previous
studies [8–15]). For each child, the L2MA (Langage Oral Écrit Mémoire Attention) [34] was used to measure text
comprehension and the ability to read words and pseudowords. The child was included in the study if
(i) their L2MA score was more than two standard deviations from the mean (all children included had
dyslexia); (ii) the intelligent quotient (IQ, evaluated using the) was in normal range (between 85 and
115); (iii) they had normal visual acuity at near vision (both eyes ≥10/10); (iv) any hyperactivity deficit
was excluded, using the ADHD (Attention-deficit/hyperactivity disorder) Rating Scale (ADHD-RS)
parental report. It should be noted also that no treatment was given to the children, and all of them
were naive of therapeutic intervention. For each child we also evaluated the reading age by using the ELFE test (Évaluation de la Lecture
en FluencE) (www.cognisciences.com, Grenoble, France) and the visual attentional (VA) span test [35]. For more details, the ELFE test is used to measure the reading age of children, and it is widely used in
French laboratories/clinicians to evaluate the reading age of children. Each child was allocated to a specific group (G1 = experimental group; G2 = control group)
using an unpredictable random sequence; the group G1 has only benefited from visual attentional
rehabilitation. It should be noted that the children were unaware of the experimental manipulations,
and only parents were informed about the goal of the study. The investigation adhered to the 4 of 13 Brain Sci. 2020, 10, 558 principles of the Declaration of Helsinki and was accepted by the Institutional Human Experimentation
Committee of CPP (Comité de protection des personnes) Ile de France I (INSERM CEEI-IRB, N◦16-290,
Hotel-Dieu Hospital, Paris). p
Below are details for the ELFE test and the VA (visual attention) span test. The ELFE test consisted
of two texts, i.e., “Le Geant Egoiste” and “Monsieur Petit”. The child was invited to read aloud one text
during 1 min, and the examiner counted the number of words read. 2.1. Subjects It should be noted that the two
texts are similar and comparable for their difficulty and orthography; the number of words with high
and low frequency as well as the number of longer and shorter words were similar, and consequently
the two texts can be used for measuring reading age before and after training without any risk of
learning effect. The VA span test consisted of 20 trials, in which the child was required to orally report a string of
5 letters that was briefly presented at the center of the monitor screen. At the beginning of each trial,
a central fixation point was presented for 1000 ms followed by a blank screen for 500 ms. A letter string
was then presented at the center of the display for 200 ms. The child had to report verbally all the
letters immediately after they disappeared. After having written their response, the examiner pressed a
button to start the next trial. At the end the examiner counted the number of letters accurately reported
across the trials. Clinicians in France frequently use this test. 2.2. Reading Task The text was presented on a 22-inch LCD (liquid-crystal display) screen with “full HD
(High-Definition) resolution” (image size of 1920 × 1080 pixels). The child was asked to read
aloud a text of four lines from a children’s book (extract from Jojo Lapin Fait des Farces, Gnid Bulton,
Hachette). The paragraph contained 40 words and 174 characters. The text was 29◦wide and 6.4◦high;
mean character width was 0.5◦, and the text was written in black Courier font on a white background. This reading task is showed in Appendix A (Texts 1 and 2, see Figures A1 and A2); it should be noted
that only the length, subject, and writing style were balanced. Eye movements during the reading task
were recorded two times at T1 and T2, i.e., before and after, respectively, 10 min of visual attentional
training for the group G1 (experimental group) and also before and after 10 min for the group G2
(control group). The order of the text (Texts 1 and 2) presented in T1 and T2 was counterbalanced. It should be noted that the child was instructed to read the text without asking for comprehension. 2.2.1. Visual Attentional Training Visual attentional training consisted of both oculomotor exercises without recording eye
movements (pursuits, saccades) and three searching tasks by using Metrisquare© Lebe Business
Centers Sittard, Sittard, Nederland. It should be noted that the child received specific instructions for
each test (see below in the “Oculomotor Task” and “Searching Task” sections). 2.2.3. Searching Task
2.2.3. Searching Task
Th
diff Three different types of searching exercises were used. The child had to search for some small
objects and to remove them with a pencil following the instructions of the experimenter. The objects
were presented on a tablet that was connected to a PC (Metrisquare©), and the pencil was also
connected with this PC, allowing for an objective measurement of the time needed to execute the
exercise, the errors, and the omission of each exercise (for more details, see Chatard et al. [36]). The three
exercises are referred to as “house”, “cat”, and “space rockets”. Three different types of searching exercises were used. The child had to search for some small
objects and to remove them with a pencil following the instructions of the experimenter. The objects
were presented on a tablet that was connected to a PC (Metrisquare©), and the pencil was also
connected with this PC, allowing for an objective measurement of the time needed to execute the
exercise, the errors, and the omission of each exercise (for more details, see Chatard et al. [36]). The
three exercises are referred to as “house”, “cat”, and “space rockets”. In the house exercise 36 colored houses on a white background of (2 × 2 cm) were presented In the house exercise, 36 colored houses on a white background of (2 × 2 cm) were presented,
among which 24 houses are on fire. The child was asked to remove the houses that are not on fire
(see Figure 1A). In the cat exercise, 24 black heads of cats on a white background were presented
together with 24 black stars and 24 black trees (0.5 × 1 cm). The child was asked to remove all the
heads of the cats (see Figure 1B). In the space rockets exercise, 104 black space rockets having different
characteristics were presented on a white background (2 × 1 cm). The child was requested to remove
16 rockets that match the model (see Figure 1C). In the house exercise, 36 colored houses on a white background of (2 × 2 cm) were presented,
among which 24 houses are on fire. The child was asked to remove the houses that are not on fire
(see Figure 1A). In the cat exercise, 24 black heads of cats on a white background were presented
together with 24 black stars and 24 black trees (0.5 × 1 cm). 2.2.2. Oculomotor Task Two oculomotor tasks were used: horizontal visually-guided saccades and horizontal pursuits
presented on the PC (personal computer) that was used for the reading task. For horizontal visually-guided saccades, the stimulus (both the central and eccentric targets) was
a white filled circle subtending a visual angle of 0.5◦. After a variable fixation period ranging between
2000 and 3500 ms, the central target disappeared, and a target at the left or at the right side of the
screen simultaneously appeared for 1000 ms. The central fixation target then reappeared, signaling the
beginning of the next trial. Each child performed two blocks of 30 visually-guided saccades randomly
presented to the left and to the right side of different amplitudes (5◦, 10◦, 15◦, and 20◦). The child was
instructed to look at the target as accurately and as rapidly as possible. The pursuit task requires the child to follow a slowly moving visual target displayed on the PC. The target velocity was of 15◦/s. The target (a white circle of 0.5◦) was initially placed in the central
position (0◦) and then moved horizontally to one side until it reached a ±20◦location, where it reversed 5 of 13
5 of 14 Brain Sci. 2020, 10, 558
Brain Sci. 2020, 10, x FO abruptly and moved to the opposite side. A total of nine cycles were run. Children were instructed to
keep their eyes on the target, wherever it moved. Children had to perform the same test four times. The duration of the pursuit task was 2 min. reversed abruptly and moved to the opposite side. A total of nine cycles were run. Children were
instructed to keep their eyes on the target, wherever it moved. Children had to perform the same test
four times. The duration of the pursuit task was 2 min. 2.2.3. Searching Task
2.2.3. Searching Task
Th
diff The child was asked to remove all the
heads of the cats (see Figure 1B). In the space rockets exercise, 104 black space rockets having different
characteristics were presented on a white background (2 × 1 cm). The child was requested to remove
16 rockets that match the model (see Figure 1C). Figure 1. Visual attentional exercises by Metrisquare©: house (A), cats (B), and space rockets (C). h d
f h
h
bl
f
d
d
h
Figure 1. Visual attentional exercises by Metrisquare©: house (A), cats (B), and space rockets (C). The duration of three searching exercises was variable (from 5 to 7 min), depending on the tim Figure 1. Visual attentional exercises by Metrisquare©: house (A), cats (B), and space rockets (C). Figure 1. Visual attentional exercises by Metrisquare©: house (A), cats (B), and space rockets (C). Figure 1. Visual attentional exercises by Metrisquare©: house (A), cats (B), and space rockets (C). Figure 1. Visual attentional exercises by Metrisquare©: house (A), cats (B), and space rockets (C). The duration of three searching exercises was variable (from 5 to 7 min), depending on the time
needed for the child to perform the tasks. Any feedback on the performance was given to the child
after doing the Metrisquare task
The duration of three searching exercises was variable (from 5 to 7 min), depending on the time
needed for the child to perform the tasks. Any feedback on the performance was given to the child
after doing the Metrisquare task. Eye Movement Recording During the execution of reading task, eye movements were recorded using an Eye Brain
T2® (SuriCog, Paris, France) head-mounted eye tracker. This eye tracker is a medical EC
(European Commission) certified device. The accuracy of this system is 0.25◦, and its recording
frequency reaches 300 Hz. This device can record the position of the eyes horizontally and vertically,
independently and simultaneously for each eye. Calibration is done before eye movement recording. The calibration consists of a succession of red dots (diameter of 0.5◦), presented on a flat PC screen
of dimensions 512 × 288 mm, corresponding to the nominal diagonal size of 22 inches. During this
procedure, we asked the children to look a grid of 13 points mapping the screen at a distance of 60 cm. Calibration is calculated for a 250 ms fixation period for each point. There is no obstruction of the
visual field during registration with the recording system, and the calibrated zone covers a visual angle
of ±22◦[35]. After the calibration procedure, the reading task was explained to the child. Duration of
each task varied accordingly to the specific child’s reading speed. 2.5. Statistical Analysis Statistical analysis using the GLM (general linear models) in STATISTICA®(12.0, Palo Alto,
California, United States) was performed. Univariate one-way ANOVA was performed in order to
compare the age, the IQ, and the number of words read in 1 min in the ELFE test and the visual
attentional span test (the number of letters accurately reported across the trials) in two groups of
children (G1 and G2). Repeated measurements of ANOVA were also run between the two children
groups (G1 and G2) on the oculomotor parameters recorded at T1 and T2. An analysis of covariance
(ANCOVA) on the duration of fixation with the total reading time as a covariable was also performed,
in order to eliminate variance due to reading time differences. Post hoc comparisons were made with
the Bonferroni test. The effect of a factor was considered significant when the p-value was below 0.05. 2.4. Data Analysis We used the software MeyeAnalysis (provided with the eye tracker, SuriCog, Paris, France) in
order to calibrate and to extract saccadic eye movements from the data. The onset and the end of each
saccade was automatically determined through a built-in saccade detection algorithm [37]. The number
and the amplitude of saccades or prosaccades, the number of regressions, the duration of fixation,
and the total duration of reading were calculated in a computerized manner for each reading task. Accuracy was not measured, given that the present study focuses on eye movement pattern changes
before and after visual attentional training. 2 3 Procedure
2.3. Procedure 2.3. Procedure
After the screening for dyslexia (done by the two coauthors, Dr. C.-L.G. and H.P.), the child was
invited to participate in our experiment. Firstly, the examiners (two coauthors S.C. and M.P.B.)
After the screening for dyslexia (done by the two coauthors, Dr. C.-L.G. and H.P.), the child
was invited to participate in our experiment. Firstly, the examiners (two coauthors S.C. and M.P.B.) 6 of 13 Brain Sci. 2020, 10, 558 measured their reading age by the ELFE test; children were assigned randomly to each group (G1 or
G2) in accordance to their performances in the VA and ELFE tests. Afterwards, the child underwent
oculomotor recording (T1), and then only children of G1 (experimental group) underwent visual
attentional training (i.e., both oculomotor types—without eye movements recording—and searching
task by using the Metrisquare apparatus). In contrast, children of G2 (control group) did not perform
the training, but they spoke for about 10 min with the two examiners. Then, T2 oculomotor recording
was run for both groups of children (G1 and G2). The duration of all these tests was about 45 min
because some breaks were done to avoid fatigue. 3. Results Table 1 shows clinical characteristics of the two groups of children with reading disorders (G1 and
G2). The one-way ANOVA failed to report any statistical difference between G1 (experimental group)
and G2 (control group) (F(1,48) = 0.15, p = 0.7; F(1,48) = 0.10, p = 0.8; F(1,48) = 0.02, p = 1.0; F(1,48) = 0.01,
p = 0.9; F(1,48) = 0.03, p = 1.0; F(1,48) = 0.04, p = 0.8; and F(1,48) = 0.84, p = 0.4; respectively for the age, IQ,
L2MA (oral and written languages and memory), ELFE test, and VA span test). 7 of 13
G2
E Brain Sci. 2020, 10, 558
Table 1. Clinica
o t ol
ou ) Table 1. Clinical characteristics of the two groups of dyslexic children (G1 = experimental group;
G2 = control group) with mean and standard deviations of age (years), IQ, of words/min read in the
ELFE test and visual attentional span test (the number of letters accurately reported across the trials). test and visual attentional span test (the number of letters accurately reported across the trials). G1 (n = 25)
G2 (n = 25) G1 (n = 25)
G2 (n = 25)
Age (years)
9.56 ± 0.29
9.74 ± 0.38
IQ (WISC-IV)
100 ± 6
102 ± 5.1
ADHD-RS score
5.2 ± 1
4.9 ± 1.2
L2MA standard deviation from
the mean
Oral Language
2.8
2.9
Written Language
2.6
2.7
Memory
2.7
2.8
ELFE test
48 ± 5.3
50 ± 5.8
VA span
63 ± 3.0
60 ± 2.1
Note: For the L2MA test done in both group of children the standard deviation from normal mean is reported. Age (years)
9.56 ± 0.29
9.74 ± 0.38
IQ (WISC-IV)
100 ± 6
102 ± 5.1
ADHD-RS score
5.2 ± 1
4.9 ± 1.2
L2MA standard deviation from the mean
Oral Language
2.8
2.9
Written Language
2.6
2.7
Memory
2.7
2.8 The ANOVA reported a significant training effect for the total reading time (F(1,48) = 4.78, β = 0.09
p < 0.03) and also a significant interaction T × G effect (F(1,48) = 39.56, β = 0.45 p < 0.0001). The Bonferroni
post hoc test showed only that the time of reading for G1 (experimental group) at T2 decreased with
respect to T1 (p < 0.0001) (see Figure 2). 3. Results ELFE test
48 ± 5.3
50 ± 5.8
VA span
63 ± 3.0
60 ± 2.1
Note: For the L2MA test done in both group of children the standard deviation from normal mean is reported. Figure 2. Means and standard deviations of the total time (s) of reading the text at T1 and T2 for both
groups of children (G1 = experimental group; G2 = control group). Figure 2. Means and standard deviations of the total time (s) of reading the text at T1 and T2 for both
groups of children (G1 = experimental group; G2 = control group). Figure 2. Means and standard deviations of the total time (s) of reading the text at T1 and T2 for both
groups of children (G1 = experimental group; G2 = control group). Figure 2. Means and standard deviations of the total time (s) of reading the text at T1 and T2 for both
groups of children (G1 = experimental group; G2 = control group). The ANOVA showed a significant training effect for the duration of fixation (F(1,48) = 6.43, β =
0.11 p < 0.01) and a significant interaction T × G effect (F(1,48) = 17.82, β = 0.27 p < 0.0001). The Bonferroni
post hoc test showed that for G1 (experimental group) only, the duration of fixations at T2 was shorter
in contrast to T1 (p < 0.0001). ANCOVA showed a significant group effect in T2 (F(1,48) = 10.70 p <
0.001), suggesting that all significant findings survived the removal of variance due to reading time
differences (see Figure 3). The ANOVA showed a significant training effect for the duration of fixation (F(1,48) = 6.43, β = 0.11
p < 0.01) and a significant interaction T × G effect (F(1,48) = 17.82, β = 0.27 p < 0.0001). The Bonferroni
post hoc test showed that for G1 (experimental group) only, the duration of fixations at T2 was shorter in
contrast to T1 (p < 0.0001). ANCOVA showed a significant group effect in T2 (F(1,48) = 10.70 p < 0.001),
suggesting that all significant findings survived the removal of variance due to reading time differences
(see Figure 3). differences (see Figure 3). Table 2 shows the values of amplitude of prosaccades and regressions at T1 and T2 for both
groups of children (G1 and G2). 3. Results ANOVA failed to show any significant group or time effect for either
interaction
g
Table 2 shows the values of amplitude of prosaccades and regressions at T1 and T2 for both
groups of children (G1 and G2). ANOVA failed to show any significant group or time effect for
either interaction. interaction. Therefore, in order to explore the eventual effect of the text in Table 3, the total time of reading
and the duration of fixation are reported for children of G1 (experimental group) who read Text 1 and
Text 2 before and after training (at T1 and T2, respectively). ANOVA failed to show any significant text
effect, suggesting no effect of the type of the text on reading performance. 8 of 13 Brain Sci. 2020, 10, 558
59 ± Figure 3. Means and standard deviations of the duration of fixations (ms) at T1 and T2 for both groups
of children (G1 = experimental group; G2 = control group). Figure 3. Means and standard deviations of the duration of fixations (ms) at T1 and T2 for both groups
of children (G1 = experimental group; G2 = control group). Figure 3. Means and standard deviations of the duration of fixations (ms) at T1 and T2 for both groups
of children (G1 = experimental group; G2 = control group). Figure 3. Means and standard deviations of the duration of fixations (ms) at T1 and T2 for both groups
of children (G1 = experimental group; G2 = control group). Finally, ANOVA reported only a significant interaction T × G effect for the number of regressions
(F(1,48) = 6.02, β = 0.11 p < 0.02), as seen in Figure 4. The Bonferroni test failed to show any statistical
difference between the groups and the time of testing (T1 and T2). Finally, ANOVA reported only a significant interaction T × G effect for the number of regressions
(F(1,48) = 6.02, β = 0.11 p < 0.02), as seen in Figure 4. The Bonferroni test failed to show any statistical
difference between the groups and the time of testing (T1 and T2). Brain Sci. 2020, 10, x FOR PEER REVIEW
9 of 1 Figure 4. Means and standard deviations of the number of prosaccades (A) and number of regressions
Figure 4. 4. Discussion The aim of this study was to evaluate the effect of a short visual attentional training on the reading
performance in French children with reading disabilities. Our results indicate that these children could
benefit from a short visual attentional training for reading faster with a decrease in the duration of
fixations. It should be noted, however, that such short visual attentional training did not lead to any
change concerning the number and the amplitude of prosaccades and regressions. These findings are
discussed below. In the literature, the beneficial effects of visual perceptual training on the reading comprehension
and fluency in children with dyslexia were recently reviewed (see the review of Peters et al. [31]
described in the Introduction). However, that in this review, only one study [33], recorded eye
movements during reading of the text. In more details, Judica and collaborators evaluated reading
skills after a reading training program (1 h, two times for week for 5 months) in a small group of
nine children with dyslexia (age mean of 11 years old). Training consisted of both reading a word
aloud or silently and writing it on the keyboard. Eye movements were recorded using an infrared
pupil reflection system with recording frequency at 200 Hz, and the duration of fixation, the number,
and the amplitude of pro saccades and regressions were measured. The authors reported after
training a significant shortening of fixations, suggesting that training could facilitate the process of
extracting visual information on the words. In other terms, dyslexic children became more efficient in
extrapolating information from the unit of letters composing the word. Indeed, during the reading
task the most important eye movement parameter is the duration of fixation, given that it is during
this time that the reading process takes place. These findings suggested that visual attentional training could act on the neural network
responsible of duration of fixations during reading task. In other words, the capability of orienting and
focusing visuospatial attention could be improved by a visual attentional training type even if it is
short in time. We recall that neuroimaging studies reported that a complex neural network is activated
during reading, predominantly the left-hemisphere area of inferior frontal, temporoparietal,
and occipitotemporal cortical regions [38]. Concerning attentional system, research found that
it is associated to the cingulo-frontoparietal network, linked with frontostriatal and frontoparietal
pathways [39]. 3. Results Mean and standard deviations of the total time of reading and of duration of fixation for
children of G1 = experimental group, who read Texts 1 and 2 before and after training (in T1 and T2). Total Time of Reading (s)
Duration of Fixation (ms)
Before Training (T1)
After Training (T2)
Before Training (T1)
After Training (T2)
Text 1
Text 2
Text 1
Text 2
Text 1
Text 2
Text 1
Text 2
59 ± 5
57 ± 6
45 ± 6
47 ± 5
465 ± 25
475 ± 29
411 ± 21
415 ± 20 3. Results Means and standard deviations of the number of prosaccades (A) and number of regressions
(B) at T1 and T2 for both groups of children (G1 = experimental group; G2 = control group). Figure 4. Means and standard deviations of the number of prosaccades (A) and number of regression
Figure 4. Means and standard deviations of the number of prosaccades (A) and number of regressions
(B) at T1 and T2 for both groups of children (G1 = experimental group; G2 = control group). 9 of 13 Brain Sci. 2020, 10, 558 Table 2. Means and standard deviations of the amplitude of prosaccades and regressions at T1 and T2
for both groups of children (G1 = experimental group; G2 = control group). Table 2. Means and standard deviations of the amplitude of prosaccades and regressions at T1 and T2
for both groups of children (G1 = experimental group; G2 = control group). Amplitude of Prosaccades (◦)
Amplitude of Regressions (◦)
T1
T2
T1
T2
G1 (experimental group)
2.7 ± 1.1
2.5 ± 0.1
2.6 ± 0.1
2.6 ± 0.1
G2 (control group)
2.5 ± 0.1
2.4 ± 0.1
2.7 ± 0.1
2.6 ± 0.1
Table 3. Mean and standard deviations of the total time of reading and of duration of fixation for
children of G1 = experimental group, who read Texts 1 and 2 before and after training (in T1 and T2). Total Time of Reading (s)
Duration of Fixation (ms)
Before Training (T1)
After Training (T2)
Before Training (T1)
After Training (T2)
Text 1
Text 2
Text 1
Text 2
Text 1
Text 2
Text 1
Text 2
59 ± 5
57 ± 6
45 ± 6
47 ± 5
465 ± 25
475 ± 29
411 ± 21
415 ± 20 for both groups of children (G1 = experimental group; G2 = control group). Amplitude of Prosaccades (◦)
Amplitude of Regressions (◦)
T1
T2
T1
T2
G1 (experimental group)
2.7 ± 1.1
2.5 ± 0.1
2.6 ± 0.1
2.6 ± 0.1
G2 (control group)
2.5 ± 0.1
2.4 ± 0.1
2.7 ± 0.1
2.6 ± 0.1
Table 3. Mean and standard deviations of the total time of reading and of duration of fixation for
children of G1 = experimental group, who read Texts 1 and 2 before and after training (in T1 and T2). Amplitude of Prosaccades (◦)
Amplitude of Regressions (◦) Table 3. 4. Discussion In the literature, dyslexia has been associated to a dysfunctional connectivity of
attentional networks as well as frontal and parietal regions [40]. These authors compared the resting
state using fMRI in a group of 33 children with dyslexia (age range = 7.7–15.7 years) and in a group of
control children (n = 11), and they found in children with dyslexia a reduced activity of left intraparietal Brain Sci. 2020, 10, 558 10 of 13 sulcus and left middle frontal gyrus; interestingly, they reported that the lower activity of these circuits
was also associated with higher ratings of inattention. We could suggest that a visual attentional training could play an important role in the cerebral
networks responsible of both eye movement control and attentional mechanisms, given that attention
and eye movements are linked [41]. However, attentional tests could be introduced to further explore
the role of attention abilities in dyslexic children to improve their reading capabilities. It should be noted, however, that such short visual attentional training did not lead to any change
concerning the number and the amplitude of prosaccades and regressions. Judica et al. [33] reported
similar findings, and they suggested that the lack of change in the number and the amplitude of saccades
could be explained by the fact that the dyslexic child was not able to acquired information from the
word as a single unit, and they still need to split the words. As discussed by Reichle and Sheridan [42],
reading skills develop with age, and fixation duration is an important parameter for the reading
capabilities. Interestingly, other studies in adult nondyslexic subjects showed improvement in reading
capabilities by decreasing fixation durations and the number of saccades, in cases of a manipulation
of text difficulty or text repetition [43] or shorter fixation durations when adult nondyslexic subjects
had a text with larger spaces between the letters, but they failed to report any change in the number
of fixations, in the total reading time, and in the comprehension scores [44]. Further studies on such
issues will be necessary in order to better understand the relationship between the fixation and the
saccade systems and their role in reading skills. We suggested that such an oculomotor pattern of children with dyslexia that consists of smaller
and more frequent prosaccades and regressions could be explained by the children’s reduced visual
attentional span. 4. Discussion The visual attentional span corresponded to the number of distinct visual elements that
can be processed [35], and in the literature, it was reported that the orienting and focusing visuospatial
attention is impaired in dyslexia [45]. More recently, Chen and colleagues [46], using a pathway analysis,
studied the impact of visual attentional span in a group of 105 subjects with dyslexia (mean age 17
years old), and they observed that visual attentional span have an important impact on the reading
comprehension, underlining the pivotal role of visuospatial pathways for reading processes. A reduced
visual attentional span, in fact, did not permit the subjects to skip small words during reading as
found in expert readers; after visual attentional training, dyslexic children are still reading as inexpert
readers, and the pattern of the number and amplitude of saccades is not affected by our training type. Actually, in our coming studies we try to perform tests using visual exercises to increase the visual
attention span in dyslexic children in order to explore possible improvement also in the number and
amplitude of saccades during reading, as it has been recently showed by Zhao et al. [47]. 5. Limitations The present study has several limitations that are worth noting. First, we did not examine the
psycholinguistic effect of the texts used, and the type of font is known to be quite complex for dyslexic
children. Secondly, the paragraph that the child has to read was quite short given the technical set up of
eye movement recordings, particularly due to the calibration of the eye tracker system. Indeed, to avoid
head movements of the child during the reading task, a short paragraph was presented in the center of
the screen, and reading accuracy was not recorded. Another important factor needing to be taken in account is the motivation of children; children of
the control group may have been less motivated with respect to children of the experimental group. Further studies taking in account these arguments need to be done on a large group of dyslexic children
by using a more ecological experimental setup. 11 of 13
ren
up. Brain Sci. 2020, 10, 558
Another im
of the control gr 11 of 13
ren
up. 6. Conclusions
children by Conflicts of Interest: The authors have no financial and personal relationships with other people or Conflicts of Interest: The authors have no financial and personal relationships with other people or organizat
hat could inappropriately influence or bias their work in this study. for lending the Metrisquare© system, and Emilie Lacroix for leading the visual searching tests. Conflicts of Interest: The authors have no financial and personal relationships with other people or Availability of Supporting Data: The datasets analyzed during the current study are available from
orresponding author on reasonable request. organizations that could inappropriately influence or bias their work in this study. Availability of Supporting Data: The datasets analyzed during the current study are available from the Availability of Supporting Data: The datasets analyzed during the current study are available from the
corresponding author on reasonable request. organizations that could inappropriately influence or bias their work in this study. Availability of Supporting Data: The datasets analyzed during the current study are available from the Appendix A. Texts Used in T1 and T2 for Eye Movement Recording
corresponding author on reasonable request.
A
di A Texts Used in T1 and T2 for Eye Mo ement Re ording Brain Sci. 2020, 10, x FOR PEER REVIEW
12 of 14 1.
Peterson, R.L.; Pennington, B.F. Developmental dyslexia. Lancet 2012, 379, 1997–2007, doi:10.1016/s014
6736(12)60198-6
1.
Peterson, R.L.; Pennington, B.F. Developmental dyslexia. Lancet 2012, 379, 1997–2007. [CrossRef] 6. Conclusions
children by A short visual attentional training could improve the cortical mechanism responsible for focus
attention and reading capabilities in children with dyslexia. Further studies will be necessary in order
to test different training types for improving both visual attentional span and eye movements during
reading in a dyslexic population. 6. Conclusions
A short visual attentional training could improve the cortical mechanism responsible for focus
attention and reading capabilities in children with dyslexia. Further studies will be necessary in order
to test different training types for improving both visual attentional span and eye movements during Author Contributions: Conceptualization: M.P.B. and S.C.; selection of patients: C.-L.G. and H.P.; oculomotor
measure and data analysis: S.C. and M.P.B.; writing original draft: S.C. and M.P.B.; review and editing: S.C.,
M.P.B., H.P., and C.-L.G. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization: M.P.B. and S.C.; selection of patients: C.-L.G. and H.P.; oculomotor
measure and data analysis: S.C. and M.P.B.; writing original draft: S.C. and M.P.B.; review and editing: S.C.,
M P B H P and C L G All authors have read and agreed to the published version of the manuscript Acknowledgments: The authors would like to thank the children who participated in the study, Ben Vaessen for
lending the Metrisquare© system, and Emilie Lacroix for leading the visual searching tests. Funding: This research received no external funding. Acknowledgments: The authors would like to thank the children who participated in the study, Ben Vaessen
f
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t Acknowledgments: The authors would like to thank the children who participated in the study, Ben Vaessen
ending the Metrisquare© system, and Emilie Lacroix for leading the visual searching tests. Funding: This research received no external funding. Acknowledgments: The authors would like to thank the children who participated in the study, Ben Vaessen
f
l
di
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i
©
d E
ili L
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l
di
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hi Conflicts of Interest: The authors have no financial and personal relationships with other people or organizat
hat could inappropriately influence or bias their work in this study. for lending the Metrisquare© system, and Emilie Lacroix for leading the visual searching tests. 6736(12)60198-6.
2.
Stein, J. What is Developmental Dyslexia? Brain Sci. 2018, 8, 26.
2.
Stein, J. What is Developmental Dyslexia? Brain Sci. 2018, 8, 26. [CrossRef] [PubMed] Referenc
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PL S ONE 2012
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Marine caves of the Southern Tyrrhenian Sea: a First Census of Benthic Biodiversity
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Journal of marine science: research & development
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Marine caves of the Southern Tyrrhenian Sea: a First Census of Benthic
Biodiversity Gianfranco Scotti1, Pierpaolo Consoli1, Valentina Esposito1,2*, Renato Chemello3, Teresa Romeo1 and Franco Andaloro1
1ISPRA (Institute for Environmental Protection and Research), Department of environmental monitoring and protection and biodiversity conservation, Italy
2Istituto Nazionale di Oceanografia e di Geofisica Sperimentale (OGS), Sezione Oceanografia, Italy
3Department of Earth and Marine Sciences, University of Palermo, Italy *Corresponding author: Valentina Esposito, ISPRA, Department of environmental monitoring and protection and biodiversity co
Milazzo, ME, Italy, Tel: +39 0650074063; Fax: +39 0909241832; E-mail: valentina.esposito@isprambiente.it tina Esposito, ISPRA, Department of environmental monitoring and protection and biodiversity conservation, Via dei Mille 46, 98057
074063; Fax: +39 0909241832; E-mail: valentina.esposito@isprambiente.it Received date: June 28, 2017; Accepted date: September 13, 2017; Published date: September 18, 2017 Received date: June 28, 2017; Accepted date: September 13, 2017; Published date: September 18, 2017 Copyright: © 2017 Scotti G, 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: © 2017 Scotti G, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution
use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract This is the first paper documenting research on a selection of marine caves located along the coast of Capo
Milazzo in the southern Tyrrhenian Sea. Three submarine and one semi-submerged caves were surveyed and
sampled using underwater photo sampling. Surveys have only taken into account the sessile species belonging to
the main taxa: Porifera, Anthozoa, Bryozoa and Polychaeta. Diversity indices and abundances were calculated for
three sections within each explored cave: the Entrance Zone, Intermediate Zone and Bottom Zone. The richest
group was Porifera with 21 taxa, followed by cnidarians, (Anthozoa), with 8 taxa, Polychaeta (5 taxa), and Bryozoa
(5 taxa). Among Porifera, the presence of Petrobiona massiliana, a protected species according to SPA/BIO
Protocol and the Bern Convention, must be highlighted. The encrusting forms were dominant in the Bottom Zone,
the massive forms in the Intermediate Zone and the arborescent forms in the Entrance Zone. Generally, the
percentage coverage of each morphological group showed a decline in the Intermediate Zone and a general
increase in the Dark Zone within each cave. The S, H’ and J values showed different trends in the five caves. These
differences, also evidenced by Permanova analysis, depend on the topographic specificity of each cave which, in
turn, affects the gradients of the biotic and abiotic parameters. Finally, no horizontal gradient of rarefaction of the
benthic sessile fauna has been detected. This study represents an important step for the management and
conservation practices of these fragile ecosystems, especially in view of the forthcoming establishment of the Marine
Protected Area. Keywords Marine caves; Benthic biodiversity; Photographic census;
Mediterranean Sea; Conservation and/or ecological islands. Moreover, the marine caves [l] are
considered a link between closed habitats, (hard and soft substrata,
seagrass bed, coralligenous assemblages) [6,7]. The caves also play an
important role economically for a local diving centre due to the high
frequency of requests from divers to explore them (pers. comm.). Management action will need to be evaluated in order to reduce and
prevent the impact of recreational divers on the benthic community
[8,9]. J Marine Sci Res Dev, an open access journal
ISSN:2155-9910 Journal of Marine Science: Research
& Development Journal of Marine Science: Research
& Development Scotti et al., J Marine Sci Res Dev 2017, 7:7
DOI: 10.4172/2155-9910.1000238 OMICS International Marine caves of the Southern Tyrrhenian Sea: a First Census of Benthic
Biodiversity Introduction doi:10.4172/2155-9910.1000238 Scotti G, Consoli P, Esposito V, Chemello R, RomeoT, et al. (2017) Marine caves of the Southern Tyrrhenian Sea: a First Census of
Benthic Biodiversity. J Marine Sci Res Dev 7: 238. doi:10.4172/2155-9910.1000238 Citation: Page 2 of 9 Figure 1: Study area with caves location. Figure 2: 2D plans of surveyed caves. Mediterranean caves surveyed versus only 738 in the eastern
Mediterranean. Mediterranean caves surveyed versus only 738 in the eastern
Mediterranean. Figure 1: Study area with caves location. In Italy, several studies have been conducted on the benthic
populations or single species of the underwater caves [6,11,16,29-33]
and
have
often
been
limited
to
Marine
Protected
Areas
[8,9,13,19,21,22,34-47]. There are still many caves to survey and further intensive studies on
the biology and ecology of these areas are needed. The description of the caves considered in this paper represents the
first contribution to the knowledge of these environments along the
north-eastern coast of Sicily. Although the seabeds along the promontory of Capo Milazzo have
been designated as Marine Protected Areas of forthcoming
establishment and the terrestrial part of the promontory represents a
Site of Community Interest (SCI), no scientific information is available
on the faunal composition of local submerged and semi-submerged
caves. The purpose of this work is therefore: a) to provide the first data on
the 2-D morphology of three submerged and one semi-submerged
caves at Capo Milazzo; b) to provide further data on the biodiversity of
these environments, paying special attention to the presence of
protected species included in the lists of international conventions. Figure 1: Study area with caves location. Material and Methods Figure 2: 2D plans of surveyed caves. J Marine Sci Res Dev, an open access journal
ISSN:2155-9910 Citation:
Scotti G, Consoli P, Esposito V, Chemello R, RomeoT, et al. (2017) Marine caves of the Southern Tyrrhenian Sea: a First Census of
Benthic Biodiversity. J Marine Sci Res Dev 7: 238. doi:10.4172/2155-9910.1000238 Introduction According to Annex 1 of the ‘Habitats’ EC Directive 92/43, the
submerged and semi-submerged caves, (code 8330), are natural
habitats of community interest. Habitat and associated species, (semi-
dark cave habitats), have been included in the Action Plan, (AP),
regarding the conservation of habitats and species associated with
seamounts, underwater caves and canyons, aphotic hard beds and
chemo-synthetic
phenomena
in
the
Mediterranean
Sea
[1]. Management procedures involve enacting laws aimed at regulating
human activities likely to affect dark populations and permit their
long-term conservation. Over the past 15 years, several studies focused on the biodiversity of
marine caves [4,10-15], their conservation [9,16-19] and the
occurrence of non indigenous species [20] and references therein] have
been carried out worldwide. In Sicily, several studies were carried out
on Bryozoa, Brachiopoda, Serpuloidea and Floristic macroalgal
diversity of some submerged caves [21-25]. The presence of submerged and semi-submerged caves has also
helped to strengthen the decision making criteria in support of the
establishment of marine protected areas in the Mediterranean Sea [26];
about 66% of these protected areas include submerged caves [27]. The AP asserts (B.2–Setting up MPAs) that “the institution of a
Marine Protected Area intended to permit more efficacious
conservation of these dark assemblages must be based on the
identification of emblem sites on the basis of the criteria: uniqueness or
rarity, particular importance for species biological stages, importance
for threatened, endangered or declining habitats or species,
vulnerability and reduced recuperative capacity after disturbance,
biological productivity, biological diversity and naturality” [1]. About 4000 caves have been surveyed in the Mediterranean in 14
European countries [28]. The biodiversity, also linked to the presence
of alien species [20], and the community structure of these particular
habitats have been investigated. But there is also a large gap between
the studies on biodiversity and animal community structure between
the western and eastern Mediterranean caves [13], with 3262 western Mediterranean marine caves play a valuable role as ‘reservoirs’ for
the restocking of valuable species [2-5] and show a significant
ecological [6] value in relation to their function of habitat/refuge Volume 7 • Issue 7 • 1000238 J Marine Sci Res Dev, an open access journal
ISSN:2155-9910 ation:
Scotti G, Consoli P, Esposito V, Chemello R, RomeoT, et al. (2017) Marine caves of the Southern Tyrrhenian Sea: a First Census of
Benthic Biodiversity. J Marine Sci Res Dev 7: 238. Description and plans of surveyed caves Gamba di donna cave (CGD): This is a small semi-submerged cave
located on the westernmost tip of Capo Milazzo. It develops
horizontally within a carbonatic rock layer running N-S. The entrance
is located at a depth of -1.5 m. The cave extends for about 14 m inside
the promontory without further links with the outside. It is only accessible from the sea. The entrance, about 1.5 meters
wide and 2 meters high, 1 m of which is outside the water, is visible
from the surface, but develops largely below the surface level. Del Cristo cave (CCR): This cave is located inside a small rocky
outcrop about 50 m off the west coast of the cape. It is part of a small
system of cavities and caves from -5 to -20 m depth. The cave is 27 m
long; its entrance is located at a depth of -18 m. The Intermediate Zone
is linked to the outside through a cracking of the roof It has a modestly
sized side tunnel of about 2 meters. The floor of the cave is covered by
a thin, sandy sediment and is littered with rocks and boulders. This
cave is often visited by divers, also due to the presence of a statue of
Christ placed there years ago. Four morphological categories have been identified [50]: Encrusting
(EN), Massive (MA), Arborescent (AR), and Tubular (TU). For each
category and for each sector, coverage in cm2 and percentage were
calculated and the differences in morphological categories were
observed through the horizontal development of the caves, (Entrance-
Bottom Cave). Delle Corvine cave (CCO): The cave is located on the south-western
side of the promontory. It has a predominantly horizontal development
within the metamorphic rock. The entrance is at a depth of -12 m; the
cave extends for about 50 m and consists of 4 main cavities connected
to each other by narrow passages. At about 16 m from the entrance
there is a small secondary branch that connects the cave with the
external environment at a depth of -5 m. The last room ends with two
small tunnels that are not accessible to divers. The semi-dark area is
limited to the first 10 m from the entrance, while the remaining part is
dark. The floor of the cave is covered by fine sediments. Inside there
are numerous individuals of Sciaena umbra. Description and plans of surveyed caves Secca di Levante cave (CLE): The cave, formed by marine erosion on
the calcareous rock, is located about 400 m off the coast of Capo
Milazzo. The entrance is at -30 m depth and the cave extends
horizontally for about 40 m in a NW-SE direction, having an average
width of 2.60 meters, and consisting of 3 connected rooms. The final
part is completely dark, while the intermediate part has cracks in the
roof that allow in light filtering from outside and that connect it with
the top of the shoal at a depth of 18 m. Coarse sand and organic
detritus originate from the disintegration of organisms that colonise
the walls and the vault covering the bottom up to 15 m from the
entrance. This cave is subject to strong currents. Muddy sand covers
the back part of the cave bottom. This cave, as it is easy to explore, and
for the presence of numerous encrusting taxa, (Porifera, Bryozoa), and
numerous cavities inhabited with Plesionika sp., Conger conger and
Phycis phycis, has been visited by the diving centre as well as
independent divers. A metal pole was attached to the camera to indicate the height the
camera needed to be held above the benthos to ensure that the width
of the photograph was 400 cm2. Each photograph was high quality and
between 6 and 9 MB in size. Then, the sampling led to the collection of 10 spatial replicates per
sector, (S1-5-S6-10 Figure 2), for a total of 30 samples for each cave. The images were transferred to a PC for the taxonomic recognition at
the lowest taxonomic level, (whenever possible), while the coverage
percentages for each species or colony were calculated using
photoQuad software [49]. Additionally, during the survey, some
specimens of each taxon were collected in order to identify at specific
level the benthic fauna present in the photoquadrats. Surveys have only
taken into account the sessile fauna in the caves belonging to the
following taxa: Porifera, Anthozoa, Bryozoa and Polychaeta. Photographic sampling was chosen as it provides a good compromise
between optimal resolution and conservation of the habitat, although it
could probably lead to the underestimation of small and cryptic
species. Furthermore, the non-destructive methodological approach
obtaining biodiversity data through the analysis of images is suitable
for studying marine benthic communities in Marine Protected Areas
and vulnerable habitats. Study Site The promontory of Capo Milazzo is a small peninsula that stretches
northwards for about 6 km from the northern coast of Sicily, (Figure
1), with a maximum width of about 1.3 km. The coastal profile appears
steep and rugged. The exposed area is classified as a Site of Community Importance,
(code ITA030032 ‘Capo Milazzo’), according to the EC ‘Habitats’
Directive 92/43, (ordinary supplement n. 167 to the Official Gazette no. 170 of 24 July 2007). In addition, since January 2014, the submerged
part of the promontory of Capo Milazzo was included in the list of the
Marine Protected Areas of Gathering, (Law 27 December 2013, n. 147
ordinary supplement n. 87 to the Official Gazette n. 302 of 12.27.2013),
and then, from August 2014, following the economic, social and
environmental investigations commissioned by the Italian Ministry of
the Environment and Protection of Land and Sea to ISPRA, Capo
Milazzo was proposed as an MPA. The bedrock of the peninsula is formed by metamorphic rocks
covered by Upper Miocene reef limestones and Upper Pliocene- Lower
Pleistocene marls and marly limestone [48]. The studied caves are located at different depths, (from 0 to -30 m
u.s.l), and distances from the coast and show different morphogenesis. Three submerged caves (“Delle Corvine” (CCO) “Secca di Levante”
(CLE) and “Del Cristo” (CCR) and one semi-submerged, “Gamba di
Donna” (CGD)), have been described (Figure 2). Figure 2: 2D plans of surveyed caves. Volume 7 • Issue 7 • 1000238 J Marine Sci Res Dev, an open access journal
ISSN:2155-9910 Citation: Page 3 of 9 Description and plans of surveyed caves It is the only cave, among
those documented in this work, where there is the presence of
individuals of Grammonus ater (Osteichthyes-Bythitidae). Results Sampling was conducted during summer 2010. Photographic
samples were taken on vertical rocky walls using a Canon G12
PowerShot with an underwater case and two electronic strobes. Within
each site five 20 × 20 cm square frames were positioned, randomly, on
both vertical walls of the cave and at three levels of distance, (sectors),
from the entrance: Entrance Sector, Intermediate Sector and Bottom
Sector. Each sampling level was chosen relying on cave morphology:
total length, presence of chambers and/or niches. Data analysis The macrobenthic assemblages were characterized by calculating
the following diversity indices for each cave and cave sectors,
(Entrance Zone - Intermediate Zone - Bottom Zone): species richness,
(S), expressed as the total number of species observed in the images
collected in each sampling sector; the Shannon-Wiener diversity index,
(H’) [51], calculated as where pi is the proportion of specimens in the
ith species, and the Pielou’s evenness index, (J), expressed as J=H’/lnS;
where H’ is the Shannon-Wiener diversity index value and S is the
species richness. Statistical analyses were performed using the PRIMER6 &
PERMANOVA+ software packages [52,53]. To evaluate potential
variations in the values of each of the three diversity indices in relation
to the factors “cave” and “sector”, a two-way crossed non-parametric
univariate analysis of variance, (PERMANOVA), was performed. Data
was analyzed on the basis of Euclidean distance, (4999 permutations). Pairwise comparisons were computed when significant differences,
(p<0.05), among factor levels were detected. Citation:
Scotti G, Consoli P, Esposito V, Chemello R, RomeoT, et al. (2017) Marine caves of the Southern Tyrrhenian Sea: a First Census of
Benthic Biodiversity. J Marine Sci Res Dev 7: 238. doi:10.4172/2155-9910.1000238 J Marine Sci Res Dev, an open access journal
ISSN:2155-9910 J Marine Sci Res Dev, an open access journal
ISSN:2155-9910
Volume 7 • Issue 7 • 1000238 J Marine Sci Res Dev, an open access journal Taxonomic composition 2.4
9.7
16
IZ
MA
Demospongiae
Ircinia variabilis
4.8
63
IZ,BZ
MA
Demospongiae
Petrosia ficiformis
17.7
5.9
EZ,IZ,BZ
EN
Demospongiae
Phorbas sp
19.88
EZ,IZ
EN
Demospongiae
Spirastrella cunctatrix
28.86
7.6
EZ,IZ,BZ
MA
Anthozoa
Astroides calycularis
15.6
7.62
21.04
EZ,IZ,
AR
Anthozoa
Cornularia cornucopiae
62.5
EZ
TU
Anthozoa
Lepsosammia pruvoti
15.43
4.8
3.4
5.3
EZ,IZ,BZ
AR
Anthozoa
Pachyceriantus solitarius
1
IZ
TU
Anthozoa
Paracyathus pulchellus
31.25
EZ
AR
Anthozoa
Parazoanthus axinellae
25
EZ
MA
Anthozoa
Phyllangia
americana
mouchezii
13
IZ
EN
Anthozoa
Polycyathus muellerae
25
45
EZ,IZ,BZ
EN
Polychaeta
Filograna implexa
16
BZ
EN
Polychaeta
Josephella marenzelleri
39.25
41
IZ,BZ
EN
Polychaeta
Protula tubularia
12.5
2.6
1.9
EZ,IZ,BZ
EN
Polychaeta
Serpula vermicularis
12.5
1.3
EZ,BZ Volume 7 • Issue 7 • 1000238 EN
Polychaeta
Vermiliopsis
infundibulum
18.56
EZ, BZ
AR
Gymnolaemata
Margaretta cereoides
27.5
EZ
AR
Gymnolaemata
Myriapora truncata
26.75
14.3
EZ,IZ
MA
Gymnolaemata
Reteporella grimaldii
4.6
EZ,IZ,BZ
Table 1: Taxonomic list of species with relative morphological groups, and % coverage assessed in each of the studied cave. MA: massive; EN:
encrusting; AR: arborescent; TU: tubular. EZ: entrance zone; IZ: Intermediate zone; BZ: bottom zone; CCO: Corvine cave; CLE: Secca di Levante
cave; CCR: Cristo cave; CGD: Gamba di donna cave. Citation:
Scotti G, Consoli P, Esposito V, Chemello R, RomeoT, et al. (2017) Marine caves of the Southern Tyrrhenian Sea: a First Census of
Benthic Biodiversity. J Marine Sci Res Dev 7: 238. doi:10.4172/2155-9910.1000238
Page 5 of 9 Citation:
Scotti G, Consoli P, Esposito V, Chemello R, RomeoT, et al. (2017) Marine caves of the Southern Tyrrhenian Sea: a First Census of
Benthic Biodiversity. J Marine Sci Res Dev 7: 238. doi:10.4172/2155-9910.1000238 Citation: Page 5 of 9 EN
Polychaeta
Vermiliopsis
infundibulum
18.56
EZ, BZ
AR
Gymnolaemata
Margaretta cereoides
27.5
EZ
AR
Gymnolaemata
Myriapora truncata
26.75
14.3
EZ,IZ
MA
Gymnolaemata
Reteporella grimaldii
4.6
EZ,IZ,BZ
Table 1: Taxonomic list of species with relative morphological groups, and % coverage assessed in each of the studied cave. MA: massive; EN:
encrusting; AR: arborescent; TU: tubular. EZ: entrance zone; IZ: Intermediate zone; BZ: bottom zone; CCO: Corvine cave; CLE: Secca di Levante
cave; CCR: Cristo cave; CGD: Gamba di donna cave. Table 1: Taxonomic list of species with relative morphological groups, and % coverage assessed in each of the studied cave. MA: massive; EN:
encrusting; AR: arborescent; TU: tubular. Taxonomic composition EZ: entrance zone; IZ: Intermediate zone; BZ: bottom zone; CCO: Corvine cave; CLE: Secca di Levante
cave; CCR: Cristo cave; CGD: Gamba di donna cave. Figure 3: % coverage of each morphological groups in each cave
sector: entrance, intermediate and bottom zone. EN: encrusting,
MA: massive, TU: tubular, AR: arborescent. CCO: Corvine Cave,
CLE: Levante Cave, CCR: Cristo cave, CGD: Gamba di Donna cave. Anthozoans, mostly belonging to the coralligenous assemblages,
such as Parazoanthus axinellae and Leptopsammia pruvoti, are also
represented by taxa belonging to the community of dark and semi-
dark caves, such as Cornularia cornucopiae, Paracyathus pulchellus
and Polycyathus muellerae [54-56]. Among the bryozoans, shade-tolerant species and coralligenous
taxa, (Reteporella grimaldii), and representatives of photophilic
communities under strong currents, (Margaretta cereoides), were
found only in the first sectors, (Entrance Zone), of the CLE caves. With regards to polychaetes, species found inside the caves of Capo
Milazzo are exclusively serpulids typical of shady and coralligenous
environments,
(Vermiliopsis
infundibulum,
Filograna
implexa,
Josephella marenzelleri, Protula tubularia), and are widely distributed
in the Italian submarine caves [57]. Considering the three sectors of the 4 investigated caves and the 4
morphological categories, (Encrusting - EN, Massive - MA,
Arborescent - AR, Tubular - TU), the encrusting forms showed a
percentage of coverage higher in the Bottom Zone in all explored
caves, whereas the massive and tubular forms were more abundant in
the Intermediate Zone. The Entrance zone presents all morphological
categories with a dominance of arborescent and massive forms (Figure
3). Percentage of coverage of each taxa show highest values at the
Entrance Zone, (71.44%), a decrease in the Intermediate Zone,
(42.15%) and an increase in the Bottom Zone, (49.96%) within each
cave. Anthozoans, (Cornularia cornucopiae, Parazoanthus axinellae),
and encrusting forms of some species of sponges, (Dendroxea sp., S. cunctatrix, Ircinia variabilis), covered the walls at the entrance of the
caves. In the Intermediate Zone of each cave, sponges grow
significantly, reaching considerable sizes (A. oroides, P. ficiformis),
while the percent coverage of Anthozoa and Polychaeta decreases. Reteporella grimaldii and Myriapora truncata represent the only
Bryozoa species located in the Intermediate Zone. In the Bottom Zone,
percent coverage of encrusting forms of serpulids, (Vermiliopsis
infundibulum, Serpula vermicularis, Josephella marenzeller) and
porifera (Dysidea fragilis and I. variabilis) (Figure 4), increases and
showed the highest coverage values. Figure 3: % coverage of each morphological groups in each cave
sector: entrance, intermediate and bottom zone. J Marine Sci Res Dev, an open access journal
ISSN:2155-9910 Taxonomic composition Overall, 37 taxa belonging to 4 phyla were observed (Table 1). The
most numerous group was Porifera, with 21 taxa, followed by Cnidaria
(Anthozoa) with 8 taxa, Annelida, (5 taxa of Polychaeta), and Bryozoa
(3 taxa). Porifera were represented by species frequently found in cave
environments such as Petrosia ficiformis, Spirastrella cunctatrix, Volume 7 • Issue 7 • 1000238 J Marine Sci Res Dev, an open access journal
ISSN:2155-9910 Citation:
Scotti G, Consoli P, Esposito V, Chemello R, RomeoT, et al. (2017) Marine caves of the Southern Tyrrhenian Sea: a First Census of
Benthic Biodiversity. J Marine Sci Res Dev 7: 238. doi:10.4172/2155-9910.1000238 Citation: Page 4 of 9 Clathrina clathrus and Agelas oroides [10,13,16,41]. Of remarkable
importance is the presence of protected species Petrobiona massiliana,
(Annex II of the SPA/BIO Protocol of the Barcelona Convention and Annex II of the Bern Convention), inside the Gamba di Donna (CGD)
cave. Morphological groups
Classes
Species
CCO
CLE
CCR
CGD
Cave sector
MA
Calcarea
Clathrina clathrus
4.4
EZ,IZ,BZ
EN
Calcarea
Petrobiona massiliana
16.79
EZ,IZ,BZ
MA
Homoscleromorpha
Corticium sp. 1.5
IZ,BZ
MA
Demospongiae
Aaptos sp. 2.5
5.7
EZ,IZ,BZ
MA
Demospongiae
Agelas oroides
1.6
5.8
18.44
EZ,IZ,BZ
MA
Demospongiae
Axinella cf. verrucosa
2.8
EZ
MA
Demospongiae
Axinella cf. damicornis
3.2
EZ
MA
Demospongiae
Axinella sp
2.5
1.8
7.75
EZ,IZ,BZ
MA
Demospongiae
Chondrosia reniformis
18.6
50
EZ,IZ,BZ
EN
Demospongiae
Crambe sp. 30.65
EZ,IZ,BZ
EN
Demospongiae
Dendroxea sp. 11.42
EZ,IZ,BZ
EN
Demospongiae
Dysidea fragilis
40.83
EZ, BZ
EN
Demospongiae
Dysidea sp. 21.31
EZ,IZ,BZ
MA
Demospongiae
Haliclona sp. 3.5
25.25
IZ,BZ
EN
Demospongiae
Haliclona (Halichoclona)
fulva
8.4
IZ
MA
Demospongiae
Ircinia cf. oros
10.2
70
EZ,IZ,
MA
Demospongiae
Ircinia sp. Taxonomic composition EN: encrusting,
MA: massive, TU: tubular, AR: arborescent. CCO: Corvine Cave,
CLE: Levante Cave, CCR: Cristo cave, CGD: Gamba di Donna cave. Volume 7 • Issue 7 • 1000238 J Marine Sci Res Dev, an open access journal
ISSN:2155-9910 Citation:
Scotti G, Consoli P, Esposito V, Chemello R, RomeoT, et al. (2017) Marine caves of the Southern Tyrrhenian Sea: a First Census of
Benthic Biodiversity. J Marine Sci Res Dev 7: 238. doi:10.4172/2155-9910.1000238 Citation: Page 6 of 9 lowest average values of the diversity indices were recorded in CGD,
(S=2.3 ± 1.2; H’=0.5 ± 0.4). Regarding the sectors, the highest average
values of species richness, (S=7.0 ± 1.9), and the Shannon diversity
index, (H’=1.6 ± 0.2), were recorded in the Intermediate Zone of CLE. Figure 4: % coverage of each morphological groups in each cave
sector: entrance, intermediate and bottom zone. EN: encrusting;
MA: massive; TU: tubular; AR: arborescent; CCO: Corvine Cave;
CLE: Levante Cave; CCR: Cristo cave; CGD: Gamba di Donna cave. S, H' and J’ values showed different trends in each of the five caves. S, H and J values showed different trends in each of the five caves. Cave
Sector
S
H'
J
Corvine (CCO)
entrance
2
0.2
0.5
intermediate
3.6
1
0.8
bottom
2.8
0.6
0.6
total
2.8
0.6
0.6
Cristo (CCR)
entrance
5.2
0.4
0.8
intermediate
3.6
1
0.9
bottom
3.4
0.7
0.6
total
4.1
0.7
0.8
Gamba donna (CGD)
entrance
3
0.7
0.7
intermediate
1.8
0.3
0.7
bottom
2
0.7
1
total
2.3
0.5
0.8
Levante (CLE)
entrance
4.2
0.7
0.8
intermediate
7
1.6
0.9
bottom
4.8
1.4
0.9
total
5.3
1.2
0.9
Table 2: Average values of the diversity indices calculated for each
sector of the explored caves Table 2: Average values of the diversity indices calculated for each
sector of the explored caves Table 2: Average values of the diversity indices calculated for each
sector of the explored caves The Intermediate Zones showed the highest H' values except in
Gamba di Donna, (CGD), (Figure 5). Diversity values decrease in the
Bottom Zones of the 4 examined caves, (Table 2). This aspect has
already been found in other Mediterranean [12-14] and Black Sea [57]
caves. Figure 4: % coverage of each morphological groups in each cave
sector: entrance, intermediate and bottom zone. Taxonomic composition EN: encrusting;
MA: massive; TU: tubular; AR: arborescent; CCO: Corvine Cave;
CLE: Levante Cave; CCR: Cristo cave; CGD: Gamba di Donna cave. Species richness (S), follows the trend of diversity (H’), in CCO,
CLE and CGD. Reverse trends of S are found in CCR. Results of the two-way crossed PERMANOVA performed on the
Shannon diversity index, (H’), indicated significant differences,
(p<0.01), for all the considered factors. The two-way crossed
PERMANOVA performed on the species richness index, (S), revealed
significant differences between caves, (F=13.393; p<0.01), and between
sectors across all caves, (F=3.2587; p<0.05), but no significant
differences between cave sectors. Pairwise comparisons performed on
the factor “Cave” showed significant differences among all caves except
between CCO and CGD (Table 3). Biodiversity patterns of the benthic assemblages The average values of the diversity indices for each sector of the
caves are reported in Table 2. On the whole, the richest and most diverse macrobenthic
community resulted in CLE, (S=5.3 ± 2.1; H’=1.2 ± 0.5), while the
lowest average values of the diversity indices were recorded in CGD,
(S=2.3 ± 1.2; H’=0.5 ± 0.4). Regarding the sectors, the highest average
values of species richness, (S=7.0 ± 1.9), and the Shannon diversity
index, (H’=1.6 ± 0.2), were recorded in the Intermediate Zone of CLE. The pairwise test conducted between levels of the “Cave” factor
pointed out Levante, (CLE), as significantly different from all the other
caves, (p<0.01), (Table 3). On the whole, the richest and most diverse macrobenthic
community resulted in CLE, (S=5.3 ± 2.1; H’=1.2 ± 0.5), while the Volume 7 • Issue 7 • 1000238 J Marine Sci Res Dev, an open access journal
ISSN:2155-9910 Citation:
Scotti G, Consoli P, Esposito V, Chemello R, RomeoT, et al. (2017) Marine caves of the Southern Tyrrhenian Sea: a First Census of
Benthic Biodiversity. J Marine Sci Res Dev 7: 238. doi:10.4172/2155-9910.1000238 Citation: Page 7 of 9 Figure 5: Trend of biodiversity indices calculated for the benth
communities of each of the surveyed cave and in each cave secto
entrance, intermediate and bottom zone. CCO: Corvine Cave; CLE
Levante Cave; CCR: Cristo cave; CGD: Gamba di Donna cave; H
Shannon-Wiener diversity; S: Species richness; J: Pielou’s evenne
index. The same test conducted on the “Cave sector” factor indicated
significant differences among all the levels considered, (p<0.01), except
for the comparison between the Bottom Zone and Intermediate Zone. Examining the Pielou Evenness Index, (J), the PERMANOVA
analysis revealed significant differences for the Cave factor, (F=3.0864;
p<0.05), and the interaction factor Cave x Sector (F=2.3545; p<0.05). Pairwise comparisons performed on the Cave factor, showed
significant differences between CCO Vs CCR and CCO Vs CLE, (Table
3). Species
Richness
Shannon diversity
Pielou Evenness
Groups
T
p
Groups
T
p
Groups
T
p
CCO
vs
CCR
3.042
**
CCO
vs
CCR
1.17
2
n.s
CCO
vs
CCR
2.277
*
CCO
vs
CGD
1.254
n.s. Biodiversity patterns of the benthic assemblages CCO
vs
CGD
0.02
9
n.s
CCO
vs
CGD
1.028
n.s
CCO
vs
CLE
4.642
***
CCO
vs
CLE
4.76
5
***
CCO
vs
CLE
2.659
*
CCR
vs
CGD
3.689
***
CCR
vs
CGD
1.18
2
n.s
CCR
vs
CGD
1.038
n.s
CCR
vs
CLE
2.179
*
CCR
vs
CLE
4.46
2
***
CCR
vs
CLE
0.455
n.s
CGD
vs
CLE
4.755
***
CGD
vs
CLE
4.51
7
***
CGD
vs
CLE
1.417
n.s
Table 3: Results of the pairwise comparisons among caves performed
for each index. CCO: Corvine Cave, CLE: Levante Cave, CCR: Cristo
cave, CGD: Gamba di Donna cave.*P<0.05; **P<0.01; ***P<0.001; n.s. = not significant. Table 3: Results of the pairwise comparisons among caves performed
for each index. CCO: Corvine Cave, CLE: Levante Cave, CCR: Cristo
cave, CGD: Gamba di Donna cave.*P<0.05; **P<0.01; ***P<0.001; n.s. = not significant. Citation:
Scotti G, Consoli P, Esposito V, Chemello R, RomeoT, et al. (2017) Marine caves of the Southern Tyrrhenian Sea: a First Census of
Benthic Biodiversity. J Marine Sci Res Dev 7: 238. doi:10.4172/2155-9910.1000238 Discussion A case
study from a Mediterranean deep cave. ICES Journal of Marine
Science 67: 871-874. In conclusion, it is possible to recognize a pool of species,
distributed along a horizontal axis, Entrance-Bottom Zone, whose
specific composition includes: coral species located mostly in the
Entrance Zone, (Parazoanthus axinellae, Paracyathus pulchellus,
Cornularia cornucopiae), species of the "semi-obscure" biocenosis,
(shaded areas), and "dark", (sensu Pérès & Picard), [61] located in the
darkest parts of the caves, (Bottom Zone), (Petrobiona massiliana,
Spiraserpula massiliensis, Haliclona fulva). 10. Gerovasileiou V, Voultsiadou E (2012) Marine caves of the Mediterranean
Sea: A sponge biodiversity reservoir within a biodiversity hotspot. PLoS
One 7: e39873. 11. Gerovasileiou V, Voultsiadou E (2014) Mediterranean marine caves as
biodiversity reservoirs: a preliminary overview. Symposia on the
conservation of Mediterranean marine key habitats, Tunis. 12. Martí R, Uriz MJ, Ballesteros E, Turon X (2004). Benthic assemblages in
two Mediterranean caves: Species diversity and coverage as a function of
abiotic parameters and geographic distance. J Mar Biol Assoc UK 84:
557-572. In some cases these species aggregate together generating different
patterns of abundance and coverage which probably depend on abiotic
factors such as light, (that may filter through cracks even in the
intermedian areas of the caves; i.e. CCR, CLE), hydrodynamism,
(strong currents), and cave morphology, (presence of branches and
niches). 13. Madonna A, Alwany MA, Rabbito D, Trocchia S, Labar S, et al. (2015)
Caves biodiversity in the Marine Area of Riviera d’Ulisse Regional Park,
Italy: Grotta del Maresciallo overview. J Biodivers Endanger Species. 14. Bell JJ (2002) The sponge community in a semi-submerged temperate sea
cave: Density, diversity and richness. Mar Ecol 23(4): 297-311. Sponges and the cnidarians anthozoans represent the most
representative taxa at the entrance and middle areas of each cave. The
sea worms serpulids, with encrusting sponges, have a significant role in
the internal sector, (Bottom Zone). 15. Uiblein F, Ott J, Stachowitsch M (editors) (1996) Deep sea sponges in
Mediterranean cave. In Deep-sea and extreme shallow-water habitats:
affinities and adaptations. Biosystematics and Ecology Series 11: 299-312. 16. Bussotti S, Terlizzi A, Fraschetti S, Belmonte G, Boero F (2006) Spatial
and temporal variability of sessile benthos in shallow Mediterranean
marine caves. Mar Ecol Prog Ser 325: 109-119. Improving the available knowledge on these particular marine
habitats and their species assemblages is extremely important
considering that recreational activities such as diving may negatively
affect them. Discussion This study aims to improve the knowledge of the macrobenthic
diversity of some submerged and semi-submerged caves around Capo
Milazzo, an area selected as an MPA. The sampling method, not
invasive and ethically acceptable for a forthcoming MPA, surely
contributed to the knowledge of these particular environments. According to our results, the diversity index showed a clear pattern,
generally increasing from the entrance to the intermediate zone in
most of the explored caves, (CCO, CLE and CCR). The high values of H 'and S observed in the Intermediate Zones may
be due to openings and cracks in the cave roofs. A small number of
species were found in the Intermediate Zone of Gamba di Donna,
(CGD), cave, which doesn’t have external links. In addition, being a
semi-submersible cave exposed to strong northwestern winds, it is
subject to strong hydrodynamism generated by N-NW winds and this
condition may be responsible for the lower values of S, H’ and J
observed in its Intermediate Zone. The Intermediate Zone, in all the examined caves, appears to
represent a discontinuity area in terms of total coverage percentages,
distribution of animal populations and presence of rare species,
(Haliclona fulva, Ircinia sp., Phyllangia americana mouchezii). In
particular, the different morphology of the four examined caves could
be responsible for the benthic fauna variability. In fact, differences in
the assemblage of the caves probably depend on the topographic Figure 5: Trend of biodiversity indices calculated for the benthic
communities of each of the surveyed cave and in each cave sector:
entrance, intermediate and bottom zone. CCO: Corvine Cave; CLE:
Levante Cave; CCR: Cristo cave; CGD: Gamba di Donna cave; H’:
Shannon-Wiener diversity; S: Species richness; J: Pielou’s evenness
index. J Marine Sci Res Dev, an open access journal
ISSN:2155-9910 Volume 7 • Issue 7 • 1000238 Citation: Page 8 of 9 specificity of each cave which, in turn, affects the gradients of biotic
and abiotic parameters [14,16,55,58-60]. Strong currents in the CLE
cave, for example, may be responsible for the presence of Margaretta
cereoides in the Entrance Zone. 8. Di Franco A, Milazzo M, Baiata P, Tomasello A, Chemello R (2009) Scuba
diver behaviour and its effects on the biota of a Mediterranean marine
protected area. Environmental Conservation 36: 32-40. 9. Di Franco A, Ferruzza G, Baiata P, Chemello R, Milazzo M (2010). Can
recreational scuba divers alter natural gross sedimentation rate? Discussion Monitoring studies of these caves and their biodiversity
are required for the management and conservation practices of these
fragile ecosystems, especially in view of the new MPA establishment. 17. Milazzo M, Chemello R, Badalamenti F, Camarda R, Riggio S (2002) The
impact of human recreational activities in marine protected areas: What
lessons should be learnt in the Mediterranean Sea?. Mar Ecol 23: 280-290. 18. Lloret J, Zaragoza N, Caballero D, Riera V (2008) Impacts of recreational
boating on the marine environment of Cap de Creus (Mediterranean
Sea). Ocean Coast Manag 51: 749-754. Acknowledgements 19. Guarnieri G, Terlizzi A, Bevilacqua S, Fraschetti S (2012) Increasing
heterogeneity of sensitive assemblages as a consequence of human impact
in submarine caves. Marine BiologyMar Biol 159: 1155-1164. We thank Marzia Bo for her valuable assistance in the
determination of anthozoa and sponges and Salvatore Pasta for the
critical revision of the text. We are grateful to Gemma Bevan for the
English revision of the manuscript. 20. Gerovasileiou V, Voultsiadou E, Issaris Y, Zenetos A (2016a) Alien
biodiversity in Mediterranean marine caves. Mar Ecol 37: 239-256. 21. Rosso A, Sanfilippo R, Taddei Ruggiero E, Di Martino E (2013) Faunas
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ISSN:2155-9910
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English
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Visualizing locus-specific sister chromatid exchange reveals differential patterns of replication stress-induced fragile site breakage
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Oncogene
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cc-by
| 10,615
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* Jacqueline H. Barlow
jhbarlow@ucdavis.edu Abstract Chromosomal fragile sites are genomic loci sensitive to replication stress which accumulate high levels of DNA damage, and
are frequently mutated in cancers. Fragile site damage is thought to arise from the aberrant repair of spontaneous replication
stress, however successful fragile site repair cannot be calculated using existing techniques. Here, we report a new assay
measuring recombination-mediated repair at endogenous genomic loci by combining a sister chromatid exchange (SCE)
assay with fluorescent in situ hybridization (SCE-FISH). Using SCE-FISH, we find that endogenous and exogenous
replication stress generated unrepaired breaks and SCEs at fragile sites. We also find that distinct sources of replication stress
induce distinct patterns of breakage: ATR inhibition induces more breaks at early replicating fragile sites (ERFS), while
ERFS and late-replicating common fragile sites (CFS) are equally fragile in response to aphidicolin. Furthermore, SCEs
were suppressed at fragile sites near centromeres in response to replication stress, suggesting that genomic location
influences DNA repair pathway choice. SCE-FISH also measured successful recombination in human primary lymphocytes,
and identificed the proto-oncogene BCL2 as a replication stress-induced fragile site. These findings demonstrate that SCE-
FISH frequency at fragile sites is a sensitive indicator of replication stress, and that large-scale genome organization
influences DNA repair pathway choice. Visualizing locus-specific sister chromatid exchange reveals
differential patterns of replication stress-induced fragile
site breakage a Waisertreiger1
●Katherine Popovich1
●Maya Block1
●Krista R. Anderson1
●Jacqueline H. B Received: 16 November 2018 / Revised: 26 September 2019 / Accepted: 2 October 2019 / Published online: 21 October 2019
© The Author(s) 2019. This article is published with open access 1
Department of Microbiology and Molecular Genetics, University
of California, Davis, CA 95616, USA Oncogene (2020) 39:1260–1272
https://doi.org/10.1038/s41388-019-1054-5 Oncogene (2020) 39:1260–1272
https://doi.org/10.1038/s41388-019-1054-5 ARTICLE ARTICLE ARTICLE 2
Genome Center, University of California, Davis, CA 95616, USA SCE-FISH reveals spontaneous DNA repair at
endogenous fragile sites Unrepaired fragile site breaks are readily detected fol-
lowing induced replication stress. However, it is not known
if fragile sites experience spontaneous replication stress that
is normally repaired, as no prior studies measured suc-
cessful DNA repair at fragile sites on the single cell level. DNA blotting and PCR amplification-based measurements
of successful recombination have limited utility at fragile
sites [15]. Both techniques require mapping break sites
within 2–20 kilobases (kb), while fragile sites span >100 kb
[16, 17]. In addition, only ~10% of cells contain damage at
an individual fragile site, further hampering detection by
these methods. To measure DNA damage and repair at individual fragile
sites, we performed SCE-FISH in antigen-stimulated WT
and XRCC2-deficient mouse primary B cells undergoing
rapid proliferation [18]. We measured breaks and SCEs at
two ERFSs (GIMAP and BCL2), two CFSs (IMMP2L and
FHIT), and two control loci termed cold sites (64O1 and
164J15)—chosen for their distance from mapped fragile
sites (>15 MB). Xrcc2f/f cells act as a positive control, as
~10% of metaphases contain DNA breaks compared with
0–2% in wild type cells [8], and damage at GIMAP is a
frequent event (Fig. 1d, Supplementary Fig. 1b, c). Here we combine sister chromatid exchange (SCE) with
FISH—SCE-FISH—to measure successful HR-mediated
repair at endogenous fragile sites in mouse and human
primary lymphocytes. We found that replication stress from
inhibition of either ATR or DNA polymerase induced DNA
breaks and SCEs at ERFSs and CFSs in WT and HR-
deficient
Xrcc2f/f mouse B cells. Further, SCE-FISH
revealed that Xrcc2 is not required for replication stress-
induced SCE formation. We also observed distinct differ-
ences in SCE frequency at ERFSs and CFSs in response to
ATRi and APH, indicating that exogenous sources of
replication
stress
differentially
affect
early
and
late-
replicating fragile sites. We also investigate the effects of
genomic location on fragile site stability and repair pathway
choice. In the absence of exogneous replication stress, WT cells
contained virtually no DSBs (<0.01 breaks/metaphase), and
no breaks at fragile or cold sites (Fig. 2a, c). Xrcc2f/f cells
harbored ~0.25 breaks/metaphase with ~4% of breaks at the
ERFS GIMAP; these were the only spontaneous fragile site
or cold site breaks observed (Fig. 2a, b). In contrast to
DSBs, we observed extensive spontaneous SCE formation
(Fig. 2c). Similar to previous reports, we observed 15%
fewer spontaneous SCEs in Xrcc2f/f cells than WT cells [19]
(Fig. 2c). Introduction Common fragile sites (CFS) were identified as sites of
recurrent DNA breaks in cells exposed to the DNA poly-
merase inhibitor aphidicolin (APH). CFSs primarily occur
in gene-poor, late replicating regions enriched for AT
repeats prone to forming secondary structures [5–7]. We
recently identified a new class of fragile sites occurring in
gene-rich regions with a high density of replication origins
termed early replicating fragile sites (ERFS) [8]. ERFS are
transcriptionally active, and are enriched for CpG islands—
a common feature of mammalian promoters. Studies of
fragile site stability directly measure unsuccessful DNA
repair using fluorescent in situ hybridization (FISH) to
visualize DNA breaks in metaphase chromosome spreads. Though CFSs and ERFSs have distinct genetic and epige-
netic features, FISH studies revealed that oncogene over-
expression and ATR inhibition induce frequent DNA breaks
at both sites in primary B cells [6, 8, 9]. Replication stress is a potent source of DNA breaks in
proliferating cells and is frequently elevated in cancer cells
[1, 2]. Disruptions in replication fork stability generate
replication stress, leading to increased fork stalling or col-
lapse. Specific genomic regions called fragile sites are
exquisitely sensitive to replication stress, accumulating high
levels of DNA breaks in response to chemical or genetic
perturbations of DNA replication [3, 4]. Supplementary information The online version of this article (https://
doi.org/10.1038/s41388-019-1054-5) contains supplementary
material, which is available to authorized users. Collapsed replication forks contain a double-strand break
(DSB) intermediate, and homologous recombination (HR)
plays a critical role in fork recovery. Cells lacking the HR
factors Brca1, Rad51, Xrcc2, or Mus81 exhibit increased
DNA breaks at fragile sites, suggesting that HR suppresses 1
Department of Microbiology and Molecular Genetics, University
of California, Davis, CA 95616, USA Visualizing locus-specific sister chromatid exchange reveals differential patterns of replication. . . 1261 FISH procedures (Fig. 1b, c). In addition, SCE-FISH helped
visualize mitotic chromosome damage; BrdU staining
helped
differentiate
between
chromosomes
harboring
chromatid breaks from twisted but intact sister chromatids
(Fig. 1c, Supplementary Fig. 1a). spontaneous
replication
stress-associated
damage
[8, 10–12]. Xrcc2 is a Rad51 paralog, forming a hetero-
tetrameric complex with its family members Rad51B,
Rad51C, and Rad51D (BCDX2) [13]. This complex sti-
mulates HR and influences the choice between short and
long-tract gene conversion [14]. SCE-FISH reveals spontaneous DNA repair at
endogenous fragile sites Both WT and Xrcc2f/f cells harbored 2.5-fold
more SCEs at the ERFSs GIMAP and BCL2 than cold sites,
and 1.8-fold more SCEs at the CFS IMMP2L (Fig. 2d). Intriguingly, the SCE frequency at FHIT was similar to cold
sites. These results suggest that GIMAP, BCL2, and
IMMP2L experience more spontaneous DNA damage and
recombination than cold sites. ATR inhibition induces DNA damage and
recombination at ERFSs and CFSs d Example of
a spontaneous DNA break at GIMAP in Xrcc2f/f B cells. GIMAP is in
green, DAPI in greyscale. Probe for fragile sites in green, telomeres in
red, BrdU in cyan, and DAPI in greyscale. Images in c and d taken
from cells exposed to 1 μM ATRi C
telomeres
merge
BCL2
BrdU
FISH with
telomere
probe
e 2
DSB
HR
3
D
DAPI
DAPI
GIMAP
ends facilitates cytogenetic analysis of DNA damage. FISH probes are
shown in green, telomere-specific probe is in red, and BrdU shown in
cyan. b SCE-FISH validation showing a spontaneous SCE at the ERFS
locus BCL2. c Chromatid break at the fragile site BCL2. d Example of
a spontaneous DNA break at GIMAP in Xrcc2f/f B cells. GIMAP is in
green, DAPI in greyscale. Probe for fragile sites in green, telomeres in
red, BrdU in cyan, and DAPI in greyscale. Images in c and d taken
from cells exposed to 1 μM ATRi BrdU
BrdU detection
using anti-BrdU
antibodies
FISH with
locus-specific
probe
FISH with
telomere
probe
Cell Cycle 1
Cell Cycle 2
BrdU
no DSB
DSB
HR
or
A
1
2
3 A D BrdU detection
using anti-BrdU
antibodies
FISH with
locus-specific
probe
or
1
2
B
telomeres
merge
BCL2
BrdU
Fig. 1 SCE-FISH measures successful recombination-mediated repair
at endogenous genomic loci. a SCE-FISH assay scheme. SCE is an
event where the two strands of DNA exchange after repair of a DSB,
resulting in a crossover event. SCEs can be visualized by differentially
labeling the two sister chromatids with the nucleotide analog BrdU. Combining single locus FISH with BrdU staining to measure SCE
events allows the measurement of successful DSB repair at a specific
locus on a single cell level. Telomere probe to visualize chromosome B
merge
BrdU C
merge
BrdU B C telomeres
BCL2 Fig. 1 SCE-FISH measures successful recombination-mediated repair
at endogenous genomic loci. a SCE-FISH assay scheme. SCE is an
event where the two strands of DNA exchange after repair of a DSB,
resulting in a crossover event. SCEs can be visualized by differentially
labeling the two sister chromatids with the nucleotide analog BrdU. Combining single locus FISH with BrdU staining to measure SCE
events allows the measurement of successful DSB repair at a specific
locus on a single cell level. Telomere probe to visualize chromosome ends facilitates cytogenetic analysis of DNA damage. ATR inhibition induces DNA damage and
recombination at ERFSs and CFSs FISH probes are
shown in green, telomere-specific probe is in red, and BrdU shown in
cyan. b SCE-FISH validation showing a spontaneous SCE at the ERFS
locus BCL2. c Chromatid break at the fragile site BCL2. d Example of
a spontaneous DNA break at GIMAP in Xrcc2f/f B cells. GIMAP is in
green, DAPI in greyscale. Probe for fragile sites in green, telomeres in
red, BrdU in cyan, and DAPI in greyscale. Images in c and d taken
from cells exposed to 1 μM ATRi WT cells have an SCE at IMMP2L while fewer than 5%
have an SCE at FHIT (Supplementary Fig. 2d). We
observed similar levels of damage at FHIT and IMMP2L
(Fig. 2b, Supplementary Table 1, Supplementary Fig. 2b),
therefore these results raise the possibility that SCE for-
mation is suppressed at FHIT. DNA damage in 2.5–3% of Xrcc2f/f cells, compared with no
breaks at cold sites [8, 23] (Fig. 2b, Supplementary Fig. 2b). WT cells have an SCE at IMMP2L while fewer than 5%
have an SCE at FHIT (Supplementary Fig. 2d). We
observed similar levels of damage at FHIT and IMMP2L
(Fig. 2b, Supplementary Table 1, Supplementary Fig. 2b),
therefore these results raise the possibility that SCE for-
mation is suppressed at FHIT. To calculate the rate of successful repair, we next ana-
lyzed SCE formation. In response to 1 μM ATRi, total SCEs
increased 1.5-fold in WT and Xrcc2f/f cells (Fig. 2c). The
number of SCEs also increased at GIMAP, BCL2, and
IMMP2L—however the relative frequency of SCE forma-
tion at fragile sites was comparable between ATRi-treated
and untreated cells (Fig. 2d, Supplementary Table 1). These
results support the hypothesis that ATR inhibition induces
genome instability by impeding the cellular response to
spontaneous replication stress. In addition, the SCE fre-
quency at individual fragile sites was similar in WT and
Xrcc2f/f cells in the presence or absence of ATRi (Fig. 2d). Together, these results indicate that Xrcc2 is not required
for spontaneous and ATRi-induced SCE formation at
fragile sites. ATR inhibition induces DNA damage and
recombination at ERFSs and CFSs HR-mediated repair involves invasion of the adjacent sister
chromatid to prime new DNA synthesis. The resulting
cruciform
structure—the
Holliday
junction—can
be
resolved as noncrossover or crossover events, the latter
generating SCEs. SCEs are visualized through the differ-
ential labeling of sister chromatids by incorporating the
nucleoside analog bromodeoxyuridine (BrdU) into DNA for
two rounds of replication (Fig. 1a). To simultaneously
visualize SCEs and single-locus FISH, we detected BrdU by
immunofluorescent staining (Fig. 1a). Unlike immuno-FISH
involving protein detection, the bromine-modified thymi-
dine analog recognized by the BrdU antibody is heat, pro-
tease, and formamide-insensitive, yielding robust and
repeatable fluorescent signal when combined with standard To measure replication stress-induced fragile site breakage,
we analyzed DNA aberrations in WT and Xrcc2f/f cells
exposed to a small molecule inhibitor of ATR, ETP-46464
(ATRi). The DNA damage checkpoint kinase ATR is a
central player in the replication stress response, and loss of
ATR
activity
leads
to
replication-associated
genome
instability and cell death [20–22]. Approximately 2% of
WT cells contained breaks at GIMAP, IMMP2L, and FHIT
in response to 1 μM ATRi, however total damage (0.4
breaks/cell) was too low to calculate break frequency
accurately at individual loci. Therefore, we measured ATRi-
induced damage in Xrcc2f/f cells where breaks were 3.5-fold
higher (Fig. 2a). All four fragile sites co-localized with 1262 I. Waisertreiger et al. C
telomeres
merge
BCL2
BrdU
BrdU
BrdU detection
using anti-BrdU
antibodies
FISH with
locus-specific
probe
FISH with
telomere
probe
Cell Cycle 1
Cell Cycle 2
BrdU
no DSB
DSB
HR
or
A
1
2
3
D
DAPI
DAPI
GIMAP
B
telomeres
merge
BCL2
BrdU
Fig. 1 SCE-FISH measures successful recombination-mediated repair
at endogenous genomic loci. a SCE-FISH assay scheme. SCE is an
event where the two strands of DNA exchange after repair of a DSB,
resulting in a crossover event. SCEs can be visualized by differentially
labeling the two sister chromatids with the nucleotide analog BrdU. Combining single locus FISH with BrdU staining to measure SCE
events allows the measurement of successful DSB repair at a specific
locus on a single cell level. Telomere probe to visualize chromosome
ends facilitates cytogenetic analysis of DNA damage. FISH probes are
shown in green, telomere-specific probe is in red, and BrdU shown in
cyan. b SCE-FISH validation showing a spontaneous SCE at the ERFS
locus BCL2. c Chromatid break at the fragile site BCL2. Increasing ATRi concentration enhances fragile site
damage One micromolar ATRi induces modest levels of damage in
WT cells; therefore, we increased the drug concentration to
confirm that ERFSs and CFSs are sensitive to ATRi. Compared with 1 μM ATRi, 5 μM ATRi increased total
DNA damage 5-fold in WT cells and 2.5-fold in Xrcc2f/f
cells (Fig. 3a). Exposure to 5 μM ATRi led to distinct dif-
ferences in the frequency of fragile site breaks in WT and
Xrcc2f/f cells. ERFS harbored extensive damage in WT and
XRCC2-deficient cells—breaks at GIMAP or BCL2 com-
prised of ~5% of total aberrations (Fig. 3b, Supplementary
Table 2). In contrast, CFS breaks occurred more frequently Similar to untreated cells, SCEs at FHIT were fourfold
lower than IMMP2L in ATRi-treated cells (Fig. 2d, Sup-
plementary Table 1). This difference is highlighted by SCE
frequency within the cell population: 15% of ATRi-treated Visualizing locus-specific sister chromatid exchange reveals differential patterns of replication. . . 1263 C
A
B
D
0
0.5
1
1.5
2
# of breaks
per metaphase
WT
WT_ATRi
Xrcc2
Xrcc2_ATRi
0
WT
1
2
3
4
5
6
7
8
frequency of breaks (%)
WT_ATRi
Xrcc2
Xrcc2_ATRi
#
#
*
*
0
1
2
3
4
5
6
7
8
9
# of SCEs
per metaphase
WT
WT_ATRi
Xrcc2
Xrcc2_ATRi
0
1
2
3
4
5
6
7
#
#
*
*
frequency of SCEs (%)
WT
WT_ATRi
Xrcc2
Xrcc2_ATRi
GIMAP
BCL2
IMMP2L
FHIT
64O1
164J15
CFS
ERFS
cold sites
Fig. 2 Exposure to 1 μM ATR inhibitor induces DNA breaks and SCE
events at ERFSs and CFSs. a Number of DNA aberrations per
metaphase in response to 1 μM ATRi in WT and Xrcc2f/f cells. 1 μM
ATRi induces an average of 0.4 breaks/cell in WT and 1.7 breaks/cell
in Xrcc2f/f cells. b Frequency of DNA aberrations at individual ERFSs,
CFSs, and cold sites. c Number of SCEs per metaphase in response to
1 μM ATRi in WT and Xrcc2f/f cells. Untreated WT cells harbor an
average of 4.6 SCE/cell, and Xrcc2f/f cells contain 3.8 SCE/cell. d
Frequency of SCEs at individual ERFSs, CFSs, and cold sites. Error
bars show the standard error of mean (SEM) from three independent
experiments. Statistics: *p < 0.05 comparing untreated and ATRi-
treated cells for each genotype; #p < 0.05 comparing WT and Xrcc2f/f
cells treated with ATRi. Increasing ATRi concentration enhances fragile site
damage All break frequencies with standard error and
pairwise P values for individual loci are provided in Supplementary
Table 1. For each independent FISH experiment, B cells were isolated
and cultured from a separate mouse. A minimum of 50 metaphase
spreads were analyzed for each experiment, resulting in a minimum of
150 metaphases analyzed A
0
0.5
1
1.5
2
# of breaks
per metaphase
WT
WT_ATRi
Xrcc2
Xrcc2_ATRi
#
#
*
* C
0
1
2
3
4
5
6
7
8
9
# of SCEs
per metaphase
WT
WT_ATRi
Xrcc2
Xrcc2_ATRi
#
#
*
* C B
0
WT
1
2
3
4
5
6
7
8
frequency of breaks (%)
WT_ATRi
Xrcc2
Xrcc2_ATRi D
0
1
2
3
4
5
6
7
frequency of SCEs (%)
WT
WT_ATRi
Xrcc2
Xrcc2_ATRi
GIMAP
BCL2
IMMP2L
FHIT
64O1
164J15
CFS
ERFS
cold sites B D Fig. 2 Exposure to 1 μM ATR inhibitor induces DNA breaks and SCE
events at ERFSs and CFSs. a Number of DNA aberrations per
metaphase in response to 1 μM ATRi in WT and Xrcc2f/f cells. 1 μM
ATRi induces an average of 0.4 breaks/cell in WT and 1.7 breaks/cell
in Xrcc2f/f cells. b Frequency of DNA aberrations at individual ERFSs,
CFSs, and cold sites. c Number of SCEs per metaphase in response to
1 μM ATRi in WT and Xrcc2f/f cells. Untreated WT cells harbor an
average of 4.6 SCE/cell, and Xrcc2f/f cells contain 3.8 SCE/cell. d
Frequency of SCEs at individual ERFSs, CFSs, and cold sites. Error bars show the standard error of mean (SEM) from three independent
experiments. Statistics: *p < 0.05 comparing untreated and ATRi-
treated cells for each genotype; #p < 0.05 comparing WT and Xrcc2f/f
cells treated with ATRi. All break frequencies with standard error and
pairwise P values for individual loci are provided in Supplementary
Table 1. For each independent FISH experiment, B cells were isolated
and cultured from a separate mouse. A minimum of 50 metaphase
spreads were analyzed for each experiment, resulting in a minimum of
150 metaphases analyzed 0
1
5
6
A
# of breaks
per metaphase
ATRi doses (uM)
4
2
3
0
1
5
WT
Xrcc2f/f
#
*
* than cold sites only in Xrcc2f/f cells (~3% vs. ~1% of total
damage). Thus, ERFSs are more sensitive to ATRi-induced
replication stress than CFSs. Increasing ATRi concentration enhances fragile site
damage Intriguingly, 5 μM ATRi exposure increased the number
of cells with DNA breaks at both fragile site alleles,
revealing that replication fork stress occurred at the same
0
1
5
6
A
# of breaks
per metaphase
ATRi doses (uM)
4
2
3
0
1
5
WT
Xrcc2f/f
#
*
*
B
0
WT
1
2
3
4
5
6
frequency of breaks (%)
Xrcc2f/f
GIMAP
BCL2
IMMP2L
FHIT
64O1
164J15
CFS
ERFS
cold sites
Fig. 3 5 μM ATR inhibitor induces high level of DNA breaks at ERFS. a Number of DNA aberrations per metaphase in response to 5 μM
ATRi treatment. We observed an average of 2 breaks/cell in WT cells
and 4.4 breaks/cell Xrcc2f/f cells. b Frequency of DNA aberrations at
ERFSs, CFSs, and cold sites. Error bars show SEM from three
independent experiments. Statistics: *p < 0.05 comparing untreated
and ATRi treated cells for each genotype; #p < 0.05 comparing WT
and Xrcc2f/f cells exposed to ATRi. All break frequencies with stan-
dard error and pairwise P values for individual loci are provided in
Supplementary Table 2 B
0
WT
1
2
3
4
5
6
frequency of breaks (%)
Xrcc2f/f
GIMAP
BCL2
IMMP2L
FHIT
64O1
164J15
CFS
ERFS
cold sites
S. M
lls
at
ee
independent experiments. Statistics: *p < 0.05 comparing untreated
and ATRi treated cells for each genotype; #p < 0.05 comparing WT
and Xrcc2f/f cells exposed to ATRi. All break frequencies with stan-
dard error and pairwise P values for individual loci are provided in
Supplementary Table 2 B
0
WT
1
2
3
4
5
6
frequency of breaks (%)
Xrcc2f/f
GIMAP
BCL2
IMMP2L
FHIT
64O1
164J15
CFS
ERFS
cold sites
S. M
s
at
e
independent experiments. Statistics: *p < 0.05 comparing untreated
and ATRi treated cells for each genotype; #p < 0.05 comparing WT
and Xrcc2f/f cells exposed to ATRi. All break frequencies with stan-
dard error and pairwise P values for individual loci are provided in
Supplementary Table 2 B than cold sites only in Xrcc2f/f cells (~3% vs. ~1% of total
damage). Thus, ERFSs are more sensitive to ATRi-induced
replication stress than CFSs. Fig. 3 5 μM ATR inhibitor induces high level of DNA breaks at ERFS. a Number of DNA aberrations per metaphase in response to 5 μM
ATRi treatment. We observed an average of 2 breaks/cell in WT cells
and 4.4 breaks/cell Xrcc2f/f cells. Increasing ATRi concentration enhances fragile site
damage All break frequencies with standard error and pair-
wise P values for individual loci are provided in Supplementary
Table 3 D
C
0
5
10
15
20
# of SCE
per metaphase
WT
WT_APH
Xrcc2
Xrcc2_APH
#
#
*
*
0
WT
1
2
3
4
5
6
7
WT_APH
Xrcc2
Xrcc2_APH
frequency of SCE (%)
GIMAP
BCL2
IMMP2L
FHIT
64O1
164J15
CFS
ERFS
cold sites
show the standard error of mean (SEM) from three independent
experiments. Statistics: *p < 0.05 comparing untreated and APH trea-
ted cells of each genotype; #p < 0.05 comparing WT and Xrcc2f/f cells
exposed to APH. All break frequencies with standard error and pair-
wise P values for individual loci are provided in Supplementary
Table 3 #
*
*
A
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
# of breaks
per metaphase
WT
WT_APH
Xrcc2
Xrcc2_APH C
0
5
10
15
20
# of SCE
per metaphase
WT
WT_APH
Xrcc2
Xrcc2_APH
#
#
*
* C A B
0
WT
2
4
6
8
10
12
WT_APH
Xrcc2
Xrcc2_APH
frequency of breaks (%) D
0
WT
1
2
3
4
5
6
7
WT_APH
Xrcc2
Xrcc2_APH
frequency of SCE (%)
GIMAP
BCL2
IMMP2L
FHIT
64O1
164J15
CFS
ERFS
cold sites B D Fig. 4 Exposure to aphidicolin induces DNA breaks and SCE events at
ERFSs and CFSs. a Number of DNA aberrations per metaphase in
response to APH in WT and Xrcc2f/f cells. b Frequency of DNA
damage at ERFSs, CFSs, and cold sites. c Number of SCEs per
metaphase in response to APH in WT and Xrcc2f/f cells. APH induces
18 SCE/cell in WT cells, and 14.2 SCE/cell in Xrcc2f/f cells. d Fre-
quency of SCE formation at ERFSs, CFSs and cold sites. Error bars show the standard error of mean (SEM) from three independent
experiments. Statistics: *p < 0.05 comparing untreated and APH trea-
ted cells of each genotype; #p < 0.05 comparing WT and Xrcc2f/f cells
exposed to APH. All break frequencies with standard error and pair-
wise P values for individual loci are provided in Supplementary
Table 3 locus on both chromosomes (Supplementary Table 2). This
finding raises the possibility that the lack of an intact repair
template prevents inter-homolog recombination, resulting in
unrepaired breaks in mitosis. hypersensitive to APH, we re-expressed wild-type murine
XRCC2 (MIGR1-X2) in Xrcc2f/f cells by retroviral infection
and measured fragile site DSBs. Increasing ATRi concentration enhances fragile site
damage b Frequency of DNA aberrations at
ERFSs, CFSs, and cold sites. Error bars show SEM from three Fig. 3 5 μM ATR inhibitor induces high level of DNA breaks at ERFS. a Number of DNA aberrations per metaphase in response to 5 μM
ATRi treatment. We observed an average of 2 breaks/cell in WT cells
and 4.4 breaks/cell Xrcc2f/f cells. b Frequency of DNA aberrations at
ERFSs, CFSs, and cold sites. Error bars show SEM from three independent experiments. Statistics: *p < 0.05 comparing untreated
and ATRi treated cells for each genotype; #p < 0.05 comparing WT
and Xrcc2f/f cells exposed to ATRi. All break frequencies with stan-
dard error and pairwise P values for individual loci are provided in
Supplementary Table 2 than cold sites only in Xrcc2f/f cells (~3% vs. ~1% of total
damage). Thus, ERFSs are more sensitive to ATRi-induced
replication stress than CFSs. Intriguingly, 5 μM ATRi exposure increased the number
of cells with DNA breaks at both fragile site alleles,
revealing that replication fork stress occurred at the same 1264 I. Waisertreiger et al. B
D
C
0
5
10
15
20
# of SCE
per metaphase
WT
WT_APH
Xrcc2
Xrcc2_APH
#
#
*
*
0
WT
2
4
6
8
10
12
WT_APH
Xrcc2
Xrcc2_APH
frequency of breaks (%)
0
WT
1
2
3
4
5
6
7
WT_APH
Xrcc2
Xrcc2_APH
frequency of SCE (%)
GIMAP
BCL2
IMMP2L
FHIT
64O1
164J15
CFS
ERFS
cold sites
#
*
*
A
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
# of breaks
per metaphase
WT
WT_APH
Xrcc2
Xrcc2_APH
Fig. 4 Exposure to aphidicolin induces DNA breaks and SCE events at
ERFSs and CFSs. a Number of DNA aberrations per metaphase in
response to APH in WT and Xrcc2f/f cells. b Frequency of DNA
damage at ERFSs, CFSs, and cold sites. c Number of SCEs per
metaphase in response to APH in WT and Xrcc2f/f cells. APH induces
18 SCE/cell in WT cells, and 14.2 SCE/cell in Xrcc2f/f cells. d Fre-
quency of SCE formation at ERFSs, CFSs and cold sites. Error bars
show the standard error of mean (SEM) from three independent
experiments. Statistics: *p < 0.05 comparing untreated and APH trea-
ted cells of each genotype; #p < 0.05 comparing WT and Xrcc2f/f cells
exposed to APH. Increasing ATRi concentration enhances fragile site
damage APH-induced DNA
damage decreased over 50% in Xrcc2f/f cells complemented
with XRCC2 than cells infected with empty vector
(MIGR1-EV) (Supplementary Fig. 3e). However Xrcc2 re-
expression did not affect fragile site breakage; GIMAP and
IMMP2L breaks occurred at the same frequency in MIGR1-
EV
and
MIGR1-X2-expressing
cells
(Supplementary
Fig. 3f). ERFSs are hotspots of aphidicolin-associated DNA
damage ERFSs and CFSs have contrasting epigenetic and genetic
features [24], therefore distinct sources of replication stress
may differentially impact ERFS and CFS stability. To test
this, we exposed cells to the B-family DNA polymerase
inhibitor aphidicolin (APH) which hinders replication
initiation and progression [25]. Exposure to 0.4 μM APH
induced extensive DNA damage in WT and Xrcc2f/f cells
(Fig. 4a). In WT cells, ~6% of aberrations occurred at the
CFSs FHIT and IMMP2L compared with ~1% of aberra-
tions co-localizing with cold site probes (Fig. 4b, Supple-
mentary Fig. 3a, b). We observed a similar break frequency
for the ERFS GIMAP and BCL2 in WT cells, contrary to
previous experiments [8]. APH also induced more aberra-
tions in Xrcc2-deficient cells, however this result was not
statistically significant. To confirm Xrcc2f/f cells were To measure APH-associated repair, we next analyzed
SCEs. APH induced a 3.5-fold increase in total SCEs in WT
and Xrcc2f/f cells (Fig. 4c). SCEs also occurred more fre-
quently at GIMAP, BCL2, and IMMP2L than cold sites in
WT and XRCC2-deficient cells (Fig. 4d). Unlike ATRi, we
found no difference in SCE frequency between ERFSs and
CFSs—SCEs at all three sites were twofold higher than cold
sites (Fig. 4d, Supplementary Table 3). However the SCE
frequency at fragile sites was significantly lower than in
untreated or ATRi-treated cells (Fig. 4d). WT and Xrcc2f/f
cells had a similar SCE frequency, indicating that XRCC2 is
not required for replication stress-induced SCE formation. We propose that APH provokes replication fork stalling Visualizing locus-specific sister chromatid exchange reveals differential patterns of replication. . . 1265 throughout S phase, affecting both early and late-replicating
fragile sites. allelic repair [28]. If both repair templates are damaged,
then DSBs may persist into mitosis. The majority of HR-
mediated repair events result in noncrossover products,
therefore we are likely underestimating this phenomenon. We propose that damage at both alleles—and the absence of
a viable repair template—accounts for a significant portion
of persistent fragile site breaks observed in mitosis. Similar to ATRi, we observed few SCEs at FHIT in
response to APH—while 25–30% of cells harbor one or
more SCEs at IMMP2L, <15% had an SCE at FHIT (Fig. 4d, Supplementary Fig. 3d). However ~15% of cells con-
tained damage at FHIT—similar to other fragile sites
(Supplementary Fig. 3b, Supplementary Tables 1, 3). ATR activity promotes APH-induced radial fusion
formation ATRi and APH both induce replication stress, however
radial chromosomes only form in response to APH. To
define the impact of ATR inhibition on APH-induced radial
chromosome formation, we characterized DNA aberrations
and SCE formation in cells exposed to both 1 μM ATRi and
0.4 μM APH (ATRi + APH). As expected, total DNA
aberrations were higher in ATRi + APH-treated cells than
single treatments (Fig. 5f). ATRi + APH induced a lower
rate of total SCEs per cell than APH alone (Fig. 5g), sug-
gesting that ATR activity is required for a subset of SCE
events. Neither SCE nor break frequency were significantly
different at fragile sites in ATRi + APH-treated cells com-
pared with single treatments (data not shown). Intriguingly,
radial chromosomes were greatly reduced in response to
ATRi + APH
treatment
compared
with
APH
alone
(Fig. 5f, g). SCEs are suppressed at centromere-proximal fragile
sites From yeast to humans, centromeres experience meiotic
crossover suppression that can affect adjacent genes
[26, 27]. FHIT is located 7 Mb from the centromere, raising
the possibility that it experiences centromere-associated
crossover suppression. To test this, we performed SCE-
FISH at IKZF1, an ERFS located ~7 Mb from the chro-
mosome 11 centromere. In response to 0.4 μM APH, the
break frequency at IKZF1 was twofold higher than cold
sites (Supplementary Fig. 4a, Supplementary Table 3). Similar to FHIT, IKZF1 harbored APH-induced SCEs at
cold site levels (Supplementary Fig. 4b, Supplementary
Tables 1, 3). In addition, SCEs at IKZF1 occurred at cold
site levels in response to 1 μM ATRi (Supplementary Table
3, Supplementary Fig. 4b). The break frequency of IKZF1
in response to 1 μM ATRi was too infrequent to measure
accurately in WT cells, therefore we exposed cells to 5 μM
ATRi. Here, IKZF1 harbored extensive DNA damage—the
break frequency was similar to the ERFS GIMAP and BCL2
(Supplementary Table 2, Supplementary Fig. 4b). We were
unable to measure fragile site SCEs in 5 μM ATRi-treated
cells due to the low mitotic index; BrdU-labeled metaphases
were insufficient. Taken together, this data suggests that
SCE formation is suppressed at fragile sites proximal to
centromeres. ATRi and APH induce distinct rearrangement types Both ATRi and APH induce dicentric chromosomes, chro-
mosome breaks, and chromatid breaks—the last comprises
over 70% of observed damage (Fig. 5a–c). APH also induces
the formation of radial chromosome fusions while ATRi
does not (Fig. 5b–e). APH induces more unrepaired breaks
than1 μM ATRi; therefore it is possible that having multiple
exposed DNA ends in a single cell promotes radial formation
(Figs. 2c and 4c). However exposure to 5 μM ATRi dra-
matically increases the number of unrepaired breaks without
inducing radials (Fig. 3a, Fig. 5d, e), therefore increasing
DNA breaks is insufficient to drive radial formation. Further,
Xrcc2f/f cells harbor high levels of DNA damage yet contain
no radial chromosomes in response to either 1 μM or 5 μM
ATRi (Fig. 3a, Fig. 5d, e). Thus, a high level of DNA
damage is not sufficient to induce radial formation. ERFSs are hotspots of aphidicolin-associated DNA
damage These
results further support the hypothesis that replication stress-
induced SCEs are suppressed at FHIT. Replication stress induces fragile site breaks on
both alleles d Frequency of
l chromosomes (% of total damage) in response to different
treatments. e Percent of cells containing radial chrom
response to different treatments. f Number of DNA aber
metaphase in response to combination of 1 μM ATRi and 0. in WT cells. g Number of SCEs per metaphase in respon
bination of 1 μM ATRi and 0.4 μM APH in WT cells. Error
the SEM from three independent experiments. Statistics:
comparing differentially treated cells
I. Waisertr 1266 I. Waisertreiger et al. A A 0
20
40
60
80
100
untreated
1 µM ATRi
5 µM ATRi
0.4 µM APH
types of damage (%)
C B
0
20
40
60
80
100
untreated
1 µM ATRi
5 µM ATRi
0.4 µM APH
1 µM ATRi
+ 0.4 µM APH
types of damage (%) C B D
0
2
4
6
8
10
12
untreated
1 µM ATRi
5 µM ATRi 0.4 µM APH 1 µM ATRi
+ 0.4 µM APH
radial chromosome frequency (%)
* 0
5
10
15
20
25
% of cells containing
radial chromosomes
untreated
1 µM ATRi
5 µM ATRi 0.4 µM APH
1 µM ATRi
+ 0.4 µM APH
E
* D E E G
0
5
10
15
20
# of SCEs
per metaphase
untreated
1 µM ATRi
0.4 µM APH
1 µM ATRi
+ 0.4 µM APH
*
* F
0
1
2
3
4
5
6
7
8
# of breaks
per metaphase
untreated
1 µM ATRi
0.4 µM APH
1 µM ATRi
+ 0.4 µM APH
* G treatments. e Percent of cells containing radial chromosomes in
response to different treatments. f Number of DNA aberrations per
metaphase in response to combination of 1 μM ATRi and 0.4 μM APH
in WT cells. g Number of SCEs per metaphase in response to com-
bination of 1 μM ATRi and 0.4 μM APH in WT cells. Error bars show
the SEM from three independent experiments. Statistics: *p < 0.05
comparing differentially treated cells Fig. 5 ATRi and APH exposure induce distinct types of DNA aber-
rations. a Representative images of the types of rearrangements pro-
duced. Telomere-specific probe visualized in red, DAPI is in
greyscale. b Frequency of DNA damage types caused by different
replication stress treatments in WT cells. c Frequency of DNA damage
types caused by different treatments in Xrcc2f/f cells. Replication stress induces fragile site breaks on
both alleles ERFSs replicate early, suggesting that damaged forks per-
sist many hours to be observed in mitosis. One possible
explanation for this persistence is both chromosomes
experience damage leaving no intact template for repair. We
found evidence for such events: in WT cells exposed to
5 μM ATRi, 4/21 cells with GIMAP damage contained
breaks at both alleles (Supplementary Table 2c). Further,
10.3% of cells contained an SCE on both BCL2 alleles, and
3.7% at GIMAP (Supplementary Table 1e). HR shows a
strong preference to use the sister chromatid in mammals,
however the homologous chromosome is also utilized in Radials are potentially cytotoxic DNA rearrangements;
chromosome fusions containing more than one centromere
promote mitotic errors [29, 30]. Therefore it is possible
combined treatment with APH and ATRi leads to increased
apoptosis, complicating the analysis of radial formation. To
test this, we measured cell viability in ATRi- and APH-
treated cells. No treatment increased cell death more than
10% by propidium iodide staining (Fig. 6a). Similarly, the B
0
20
40
60
80
100
untreated
1 µM ATRi
5 µM ATRi
0.4 µM APH
1 µM ATRi
+ 0.4 µM APH
types of damage (%)
0
20
40
60
80
100
untreated
1 µM ATRi
5 µM ATRi
0.4 µM APH
types of damage (%)
chromosome breaks
chromatid breaks
dicentrics
radials
fusions
C
A
G
F
0
5
10
15
20
# of SCEs
per metaphase
untreated
1 µM ATRi
0.4 µM APH
1 µM ATRi
+ 0.4 µM APH
*
*
0
1
2
3
4
5
6
7
8
# of breaks
per metaphase
untreated
1 µM ATRi
0.4 µM APH
1 µM ATRi
+ 0.4 µM APH
*
D
0
2
4
6
8
10
12
untreated
1 µM ATRi
5 µM ATRi 0.4 µM APH 1 µM ATRi
+ 0.4 µM APH
radial chromosome frequency (%)
0
5
10
15
20
25
% of cells containing
radial chromosomes
untreated
1 µM ATRi
5 µM ATRi 0.4 µM APH
1 µM ATRi
+ 0.4 µM APH
E
*
*
5 ATRi and APH exposure induce distinct types of DNA aber-
ns. a Representative images of the types of rearrangements pro-
d. Telomere-specific probe visualized in red, DAPI is in
cale. b Frequency of DNA damage types caused by different
cation stress treatments in WT cells. c Frequency of DNA damage
caused by different treatments in Xrcc2f/f cells. Replication stress induces fragile site breaks on
both alleles d Frequency of
radial chromosomes (% of total damage) in response to different Visualizing locus-specific sister chromatid exchange reveals differential patterns of replication. . . 1267 0
0.5
1
1.5
2
2.5
3
proliferation index
untreated
1 uM ATRi
5 uM ATRi
0.4 uM APH
WT
Xrcc2f/f
0
0.5
2.5
3.5
4
mitotic index (%)
3
2
1
1.5
untreated
1 uM ATRi
5 uM ATRi
0.4 uM APH
WT
Xrcc2f/f
D
C
WT untreated
WT 1 uM ATRi
WT 5 uM ATRi
WT 0.4 uM APH
Xrcc2f/f untreated
Xrcc2f/f 1 uM ATRi
Xrcc2f/f 5 uM ATRi
Xrcc2f/f 04.uM APH
85
95
100
A
% of living cells
90
48 h
72 h
untreated
1 uM
ATRi
5 uM
ATRi
0.4 uM
APH
1 uM
ATRi + 0.4
uM APH
0
10
50
70
80
60
40
20
30
% TUNEL-positive cells
WT
Xrcc2f/f
B
Fig. 6 WT and Xrcc2f/f cells exhibit elevated cell death in response to
replication stress. a Viability of WT and Xrcc2f/f cells in response to
ATRi and APH treatment measuring cell fragmentation by flow
cytometry. b Percent of TUNEL-positive WT and Xrcc2f/f cells in
response to ATRi, APH, and combination of ATRi and APH treat-
ment. Error bars show the SEM from three independent experiments. c
Proliferation index of of WT and Xrcc2f/f cells in response to ATRi and
APH. d Mitotic index of WT and Xrcc2f/f cells in response to ATRi
and APH WT untreated
WT 1 uM ATRi
WT 5 uM ATRi
WT 0.4 uM APH
Xrcc2f/f untreated
Xrcc2f/f 1 uM ATRi
Xrcc2f/f 5 uM ATRi
Xrcc2f/f 04.uM APH
85
95
100
A
% of living cells
90
48 h
72 h
B ATR kinase activity is required for radial chromosome
formation. ATR kinase activity is required for radial chromosome
formation. A SCE-FISH is elevated at fragile sites in human cells CFSs were first identified in primary human lymphocytes
exposed to APH [3, 4]. To measure successful fragile site
repair in primary human cells, we exposed stimulated per-
ipheral blood mononuclear cells (PBMC) from whole blood
to 0.4 μM APH for 20 h then performed SCE-FISH. Similar
to previous reports, APH treatment induced breaks and
SCEs in human PBMCs [31] (Fig. 7a, c). APH induced a
high level of DNA aberrations, particularly at CFSs (Fig. 7b, Supplementary Fig. 5b). We also observed breaks at the
ERFS BCL2, albeit at lower levels than either CFS (Fig. 7b;
Supplementary Fig. 5b; Supplementary Table 4). APH also
increased SCEs at CFSs and ERFSs relative to cold sites
(Fig. 7d, Supplementary Table 4). SCEs were strongly
elevated at CFSs, correlating with DNA breakage. Nearly
70% of cells harbored an SCE at FHIT, compared with
~14% in mouse (Supplementary Fig. 5d). The FHIT region
shares high sequence homology between mouse and human,
however it is located ~30 Mb from the centromere in
humans [32]. We hypothesize that chromosomal location
drives the difference in SCE rate between human and mouse
rather than sequence variation. ERFS also exhibited ele-
vated SCE formation in response to APH; SCEs at BCL2
were threefold higher than cold sites (Fig. 7d). Together,
these results demonstrate that replication stress generates
DNA damage at ERFSs and CFSs in human peripheral
lymphocytes, and CFSs are significantly more prone to
APH-induced damage than ERFSs. B C
untreated
1 uM
ATRi
5 uM
ATRi
0.4 uM
APH
1 uM
ATRi + 0.4
uM APH
0
10
50
70
80
60
40
20
30
% TUNEL-positive cells
WT
Xrcc2f/f
B 0
0.5
1
1.5
2
2.5
3
proliferation index
untreated
1 uM ATRi
5 uM ATRi
0.4 uM APH
WT
Xrcc2f/f
C
uM C D 0
0.5
2.5
3.5
4
mitotic index (%)
3
2
1
1.5
untreated
1 uM ATRi
5 uM ATRi
0.4 uM APH
WT
Xrcc2f/f
D Discussion a Number of DNA breaks per meta-
se in response to 0.4 μM APH. b Frequency of DNA aberrations at
FSs, CFSs and cold sites. c Number of SCEs per metaphase in
ponse to 0.4 μM APH. d Frequency of SCEs at ERFSs, CFSs, and
d sites. Error bars show the SEM from three independent
experiments. *p < 0.05 comparing untreated and 0.4 μM APH-treated
cells. For each independent experiment, human peripheral blood spe-
cimens obtained from separate donors were used. A minimum of 50
metaphase spreads were analyzed for each experiment, resulting in a
minimum of 150 metaphases analyzed A
B
# of breaks per metaphase
0
0.5
1
1.5
2
2.5
3
untreated
0.4 uM APH
* C
# of SCEs per metaphase
0
5
10
15
20
25
untreated
0.4 uM APH
* C A D
0
untreated
1
2
3
4
5
frequency of SCE (%)
0.4 uM APH
GIMAP
BCL2
IMMP2L
FHIT
TUBB
PBMS2
CFS
ERFS
cold sites
experiments. *p < 0.05 comparing untreated and 0.4 μM APH-treated
cells. For each independent experiment, human peripheral blood spe-
cimens obtained from separate donors were used. A minimum of 50
metaphase spreads were analyzed for each experiment, resulting in a
minimum of 150 metaphases analyzed D D
5 B
0
untreated
2
4
8
6
10
12
frequency of breaks (%)
0.4 uM APH B B D
0
untreated
1
2
3
4
5
frequency of SCE (%)
0.4 uM APH
GIMAP
BCL2
IMMP2L
FHIT
TUBB
PBMS2
CFS
ERFS
cold sites Fig. 7 SCE-FISH reveals CFS breakage and repair in primary human
peripheral blood lymphocytes. a Number of DNA breaks per meta-
phase in response to 0.4 μM APH. b Frequency of DNA aberrations at
ERFSs, CFSs and cold sites. c Number of SCEs per metaphase in
response to 0.4 μM APH. d Frequency of SCEs at ERFSs, CFSs, and
cold sites. Error bars show the SEM from three independent experiments. *p < 0.05 comparing untreated and 0.4 μM APH-treated
cells. For each independent experiment, human peripheral blood spe-
cimens obtained from separate donors were used. A minimum of 50
metaphase spreads were analyzed for each experiment, resulting in a
minimum of 150 metaphases analyzed BrdU staining at centromeric heterochromatin (Fig. 1b, c);
therefore it is possible that SCEs at FHIT and IKZF1 were
not detected. Discussion 5 uM ATRi
0.4 uM APH 1 uM ATRi Fragile sites were discovered over 30 years ago, leading to
the hypothesis that fragile site instability promotes cancer
initiation and development. Indeed, fragile site instability is
observed in many human cancers [8, 33–35]. While mul-
tiple studies revealed that fragile sites are hypersensitive to
exogenous replication stress, assessing their instability in
unperturbed cells has remained elusive. Fig. 6 WT and Xrcc2f/f cells exhibit elevated cell death in response to
replication stress. a Viability of WT and Xrcc2f/f cells in response to
ATRi and APH treatment measuring cell fragmentation by flow
cytometry. b Percent of TUNEL-positive WT and Xrcc2f/f cells in
response to ATRi, APH, and combination of ATRi and APH treat-
ment. Error bars show the SEM from three independent experiments. c
Proliferation index of of WT and Xrcc2f/f cells in response to ATRi and
APH. d Mitotic index of WT and Xrcc2f/f cells in response to ATRi
and APH Using SCE-FISH to measure successful fragile DNA
repair, we found that spontaneous DNA damage and
repair at fragile sites frequently occurs in proliferating
cells. Furthermore, exposure to ATRi and APH elicit
distinct responses in ERFS and CFS breakage rates. ERFSs harbored more SCEs than CFSs in untreated and
ATR inhibitor-treated cells, suggesting that early repli-
cating fragile sites experience more HR-repaired sponta-
neous
damage
than
late-replicating
counterparts. However, ERFSs and CFSs experience elevated damage fraction of TUNEL-positive cells modestly increased in
response to ATRi + APH (40% vs. 25–30%, Fig. 6b). In
contrast, we found that 60% of cells were TUNEL-positive
in 5 μM ATRi-treated cells—a twofold increase from 1 μM
ATRi (Fig. 6b). Thus, it is unlikely that the lack of radial
chromosomes observed in ATRi + APH-treated cells is due
to increased cell death (Fig. 6b). These results indicate that 1268 I. Waisertreiger et al. A
C
B
D
# of SCEs per metaphase
0
untreated
1
2
3
4
5
frequency of SCE (%)
0.4 uM APH
0
5
10
15
20
25
untreated
0.4 uM APH
*
# of breaks per metaphase
0
untreated
2
4
8
6
10
12
frequency of breaks (%)
0.4 uM APH
0
0.5
1
1.5
2
2.5
3
untreated
0.4 uM APH
*
GIMAP
BCL2
IMMP2L
FHIT
TUBB
PBMS2
CFS
ERFS
cold sites
7 SCE-FISH reveals CFS breakage and repair in primary human
ipheral blood lymphocytes. Discussion However this explanation is unlikely: SCEs
were visible at both FHIT and IKZF1 even in highly
compacted chromosomes (Supplementary Fig. 4d). We
hypothesize that SCEs are suppressed at FHIT and IKZF1
due to their proximity to the centromere, similar to cross-
over suppression in meiosis [26, 39]. In yeast, the Ctf19
complex promotes cohesion enrichment in the pericen-
tromeric region, suppressing break formation and crossover
formation [40]. In mouse, loss of the histone methyl-
transferase DNMT1 or DNMT3A and DNMT3B leads to
increased SCE within centromeres—demonstrating that
epigenetic modifications also regulate crossover formation
in pericentromeric repeats [41]. Further studies employing
SCE-FISH will define whether mitotic SCE suppression is
governed by similar or distinct mechanisms. in response to APH, indicating that perturbation of pol α
primase
disturbs
fork
stability
in
early-
and
late-
replicating regions equally. It will be interesting to
determine if these differences arise solely from replication
timing, or are governed by additional factors such as
transcriptional activity. HR
preferentially
repairs
transcriptionally
active
euchromatin
while
condensed
heterochromatin
favors
NHEJ, suggesting that defective HR would preferentially
increase ERFS breaks. However, ERFS and CFS break
frequency was similar in Xrcc2f/f and WT cells. Xrcc2f/f cells
exhibited a modest but reproducible reduction in sponta-
neous and replication stress-induced SCEs, however SCE
frequency at fragile sites is similar to WT cells. These
results show that XRCC2 suppresses replication stress-
associated instability, however it is largely dispensable for
replication stress-induced SCE formation. XRCC2 pro-
motes HR and replication fork protection [36–38], therefore
the increase in unrepaired damage in response to ATRi is
likely a combination of increased fork collapse and reduced
noncrossover repair. Different sources of replication stress also induced dis-
tinct types of DNA damage. ATRi and APH both generated
chromosome and chromatid breaks, however APH also
induced radial chromosome fusions. Both ERFS and CFS
probes frequently localized at radial fusion junction sites,
indicating they are both rearrangement hotspots. We
hypothesize that APH exposure generates specific DNA SCE formation at the two fragile sites located near cen-
tromeres—CFS FHIT and ERFS IKZF1— was similar to
cold sites in mouse B cells. We did not observe differential Metaphase chromosome preparation structures that promote radial formation. Radial chromo-
somes contain multiple centromeres, and have profound
effects on genome stability. Multiple centromeres promote
severe chromosome segregation defects, mitotic defects,
and entry into “breakage-fusion-bridge” (BFB) cycle. Importantly, BFB cycles are implicated in driving oncogene
amplification and tumorigenesis in multiple cancers [42–
44]. Intriguingly, we found that ATR kinase activity pro-
moted APH-induced radial chromosome formation. It will
be interesting to determine if ATR inhibition impacts the
viability of cells experiencing BFB. To visualize SCE, 1 μM BrdU (Sigma, B5002) was added
to medium for 20–40 h, depending on cell cycle length. Cells were arrested in metaphase by a 1-h treatment with
0.1 μg/ml
demecolcine
(Sigma,
D1925),
treated
with
0.075 M KCl, fixed in methanol:acetic acid (3:1), spread
onto glass slides and air-dried. Visualizing locus-specific sister chromatid exchange reveals differential patterns of replication. . 1269 FISH and FISH-SCE FISH and FISH-SCE studies were performed on metaphase
cells using probes described (Supplementary Table 5). A
total of 200 ng of each probe were hybridized to target DNA
and blocked with ~15-fold excess of human COT DNA
(Roche, 11581074001) and salmon sperm DNA (Ambion,
AM9680). Prior to hybridization, slides were briefly heated
over an open flame, denaturing DNA for BrdU detection. Slides were pretreated at 72 °C in 2 × SSC for 2 min,
washed in 1 × PBS at room temperature (RT) for 5 min,
post-fixed in 1% formaldehyde at RT for 5 min, and washed
in 1 × PBS at RT for 5 min. Slides were dehydrated in
ethanol (75, 85, and 100%) at RT for 2 min each and air-
dried. Cells and probes were co-denatured at 75 °C for
3 min and incubated overnight at 37 °C in a humid chamber. Slides were washed post-hybridization in 0.4 × SSC/0.3%
NP-40 at 72 °C (2 min), then 2 × SSC/0.1% NP-40 at RT
(2 min). Slides were probed with 0.25 μM telomere probe
(PNA Bio, F1002) for 2 h at RT. Slides were then washed in
1 × PBST (1X PBS, 0.5% Triton-X-100) three times for
5 min at 37 °C. BrdU detection: the primary mouse-anti-
BrdU (BD, 347580; 1:200) and secondary Cy5 goat-anti-
mouse antibodies (Invitrogen, A10524; 1:200) were used,
then washed in 1 × PBST (1X PBS, 0.5% Triton-X-100)
three times for 5 min at 37 °C. Slides were counterstained Drug treatments ATRi
(mTOR
Inhibitor
XIII,
ETP-46464,
Millipore,
5.00508.0001) or APH (Fisher Scientific, BP615-1) were
added to the cell culture medium 20 h prior to harvest at the
designated concentration. ATRi
(mTOR
Inhibitor
XIII,
ETP-46464,
Millipore,
5.00508.0001) or APH (Fisher Scientific, BP615-1) were
added to the cell culture medium 20 h prior to harvest at the
designated concentration. Fragile sites have emerged as replication stress-specific
sites of DNA damage and are exquisitely sensitive to a
range of genotoxic agents. HR efficiently repairs sponta-
neous fragile site damage, therefore why does replication
stress induce such a profound increase in unrepaired
damage at these sites? Two possibilities likely contribute:
(1) fragile sites experience more fork collapse, and (2) the
resulting DNA breaks are difficult to repair. Fragile sites are
enriched for repetitive DNA, prone to forming secondary
structures, and associate with RNA:DNA hybrid (R loop)
formation [4, 45, 46]. All three can perturb replication fork
progression, and often require additional enzymes for break
resolution [47–49]. Bacterial artificial chromosome probes All Bacterial Artificial Chromosomes (BACs) used for
custom-designed probes were purchased from Children’s
Hospital Oakland
Research
Institute Resource Center
(BACPAC). Probes were direct-labeled using a nick trans-
lation kit (Abbott Molecular, Inc., 07J00-001) with DY-
495-dUTP (Dyomics, 495-34) and hybridized to metaphase
cell preparations of a karyotypically normal donor to con-
firm correct mapping prior to experimentation. Here we show that SCE-FISH reveals the ongoing
spontaneous DNA damage and successful repair occurring
at ERFSs and CFSs in mouse and human B cells. For non-
centromeric loci, enhanced SCE formation directly cor-
relates with increases in DNA breaks observed in mitosis
—whether they are induced by genetic defects in DNA
repair or the application of chemical agents. We predict
that high levels of crossovers measured by SCE-FISH at
fragile sites can act as a biomarker for patients with high
risk
for
developing
second
cancers
or
proliferative
syndromes. Viability assay 2. Zeman MK, Cimprich KA. Causes and consequences of replica-
tion stress. Nat Cell Biol. 2014;16:2–9. Live cells were rinsed twice in 1× Hanks’ Balanced Salt
Solution (HBSS) (Gibco, 14065-056), then incubated in
1xHBSS supplemented with 2 mg/ml propidium iodide
(Invitrogen, P1304MP) for 10 min at RT. Fluorescence-
activated cell sorting (FACS) analysis was carried out on a
Becton Dickinson CantoII flow cytometer (BD Bios-
ciences). Up to 20,000 live cells were analyzed for each
condition, and data analysis was performed using FlowJo
8.8.32 software. 3. Glover TW, Berger C, Coyle J, Echo B. DNA polymerase alpha
inhibition by aphidicolin induces gaps and breaks at common
fragile
sites
in
human
chromosomes. Hum
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Struct Mol Biol. 2016;23:103–9. TUNEL TUNEL assay was performed using the In Situ Cell Death
Detection Kit (Roche, 11684795910). At least 100 nuclei
per experiment were analyzed by microscopy. At least three
independent experiments were performed for each data set. The statistical significance of differences was estimated by
Student’s-criterion. Microscopy and analysis B cells were isolated and cultured from a separate mouse for
each experiment. A minimum of 50 metaphases were ana-
lyzed for each experiment. Metaphases images were
acquired using an epifluorescent Nikon microscope with
NIS Elements AR4.40.00 software (Nikon). Downstream
analysis used ImageJ32 software (NIH). Acknowledgements We thank Dr. Neil Hunter, Jack McTiernan, and
all members of the Barlow and Hunter labs for helpful discussions and
suggestions. This work was supported by a NIHK22 grant to JHB
from the NCI (K22CA188106) and NIH S100D018223. Mice and cells All experiments were performed in accordance with proto-
cols approved by the UC Davis Institutional Animal Care
and Use Committee (IACUC protocol #20042). Mice used
in this study include CD19cre and Xrcc2f/f [50, 51]. Splenic
B cells were isolated using the Dynabeads untouched CD43
mouse B cell isolation kit (Thermo Fisher, 11422D) and
cultured as previously described [8]. Human lymphocytes
were obtained from peripheral blood of three unrelated
volunteers and cultured for 72 h in MF-Chang medium
(Irvine Scientific, 91005), supplemented with 10% heat
inactivated fetal bovine serum and 3 μg/mL phytohe-
magglutinin (Remel, Inc., 30852701). 1270 I. Waisertreiger et al. with Vectashield mounting medium containing DAPI
(Vector laboratories Inc., H-1200). with Vectashield mounting medium containing DAPI
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spinoculated at 2500 RPM for 90 minutes. After 4 h at 37 C,
viral supernatant was replaced with B cell media with
RP105/LPS/IL-4. At 96 h, GFP + cells were collected by
flow cytometry, then harvested for FISH. Statistics Conflict of interest The authors declare that they have no conflict of
interest. Statistical significance of differences was estimated by
Student’s-criterion. To determine if distinct sites have sig-
nificantly different SCE or break frequencies, we compared
individual loci using pairwise analysis. To estimate the
correlation between co-occurrence of ERFS and CFS in
individual
cells,
the
Cochran–Mantel–Haenszel
test
was used. Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as
long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons license, and indicate if
changes were made. The images or other third party material in this
article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not
included in the article’s Creative Commons license and your intended
use is not permitted by statutory regulation or exceeds the permitted
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repair by interchromosomal recombination: suppression of chro-
mosomal translocations. Genes Dev. 1998;12:3831–42. 9. Jiang Y, Lucas I, Young DJ, Davis EM, Karrison T, Rest JS. et al. Common fragile sites are characterized by histone hypoacetyla-
tion. Hum Mol Genet. 2009;18:4501–12. (Research Support, N.I. H., Extramural). 29. Gascoigne KE, Cheeseman IM. Induced dicentric chromosome
formation promotes genomic rearrangements and tumorigenesis. Chromosome Res. 2013;21:407–18. 10. Ying S, Minocherhomji S, Chan KL, Palmai-Pallag T, Chu WK,
Wass T, et al. MUS81 promotes common fragile site expression. Nat Cell Biol. 2013;15:1001–7. 30. McClintock B. The stability of broken ends of chromosomes in
Zea Mays. Genetics. 1941;26:234–82. 11. Arlt MF, Xu B, Durkin SG, Casper AM, Kastan MB, Glover TW. BRCA1 is required for common-fragile-site stability via its G2/M
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Res. 1985;144:257–63. 32. Shiraishi T, Druck T, Mimori K, Flomenberg J, Berk L, Alder H,
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Sage C, et al. Homologous recombination and nonhomologous
end-joining repair pathways regulate fragile site stability. Genes
Dev. 2005;19:2715–26. 13. Yonetani Y, Hochegger H, Sonoda E, Shinya S, Yoshikawa H,
Takeda S, et al. Differential and collaborative actions of Rad51
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Acids Res. 2005;33:4544–52. 33. Retroviral preparation and B cell infection ATR: a master conductor of cellular responses
to
DNA
replication
stress. Trends
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Sci. 2011;36:133–40. 42. Gisselsson D, Pettersson L, Hoglund M, Heidenblad M, Gorunova
L, Wiegant J, et al. Chromosomal breakage-fusion-bridge events
cause genetic intratumor heterogeneity. Proc Natl Acad Sci USA. 2000;97:5357–62. 21. Gaillard H, Garcia-Muse T, Aguilera A. Replication stress and
cancer. Nat Rev Cancer. 2015;15:276–89. 43. Garsed DW, Marshall OJ, Corbin VD, Hsu A, Di Stefano L,
Schroder J, et al. The architecture and evolution of cancer neo-
chromosomes. Cancer Cell. 2014;26:653–67. 22. Cimprich KA, Cortez D. ATR: an essential regulator of genome
integrity. Nat Rev Mol Cell Biol. 2008;9:616–27. 23. Lopez-Contreras AJ, Specks J, Barlow JH, Ambrogio C, Desler C,
Vikingsson S, et al. Increased Rrm2 gene dosage reduces fragile
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et al. Palindromic amplification of the ERBB2 oncogene in pri-
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ondary structures in fragile site breakage along human chromo-
some 10. Hum Mol Genet. 2013;22:1443–56. (Research Support,
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where replication-transcription collisions cause genetic instability. EMBO J. 2013;32:493–5. (Review). 25. Baranovskiy AG, Babayeva ND, Suwa Y, Gu J, Pavlov YI,
Tahirov TH. Structural basis for inhibition of DNA replication by
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mediated mutagenesis in mice. Nucleic Acids Res. 1997;25:1317–8. 49. Leon-Ortiz AM, Svendsen J, Boulton SJ. ;
51. Rickert RC, Roes J, Rajewsky K. B lymphocyte-specific, cre-
mediated mutagenesis in mice. Nucleic Acids Res. 1997;25:1317–8. 52. Robbiani DF, Bothmer A, Callen E, Reina-San-Martin B, Dorsett
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2008;135:1028–38. DNA repair pathways during nervous system development. Proc
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secondary structures at the eukaryotic replication fork. DNA
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end-joining
and
homologous
recombination
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A large nabothian cyst causing chronic urinary retention
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Medicine
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cc-by
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Abstract Abstract
Rationale: Nabothian cysts are mucus-filled cervical cysts that are usually asymptomatic unless they become very large. Chronic
urinary retention is the persistent inability to empty the bladder despite maintaining an ability to urinate. Chronic urinary retention
caused by a large, deep nabothian cyst has not been reported previously. Patient concerns: A 46-year-old woman presented with chronic urinary retention and a cervical cys
size. Diagnosis: Based on histopathological evidence, our patient was diagnosed with a nabothian cyst stopathological evidence, our patient was diagnosed with a nabothian cyst. Diagnosis: Based on histopathological evidence, our patient was diagnosed with a nabothian cyst. Interventions: A hysterectomy was performed. Diagnosis: Based on histopathological evidence, our patient was diagnosed with a nabothian cyst. Interventions: A hysterectomy was performed. Interventions: A hysterectomy was performed. Outcomes: The urinary symptoms of the patient resolved after she performed clean, intermittent self-catheterizations for 5 days
after the operation. She was discharged on postoperative day 6. an cysts are rare but may account for some unusual symptoms including unexplained urinary difficulties in
treating symptomatic nabothian cysts with local cystectomies or hysterectomies. Lessons: Large nabothian cysts are rare but may account for some unusual symptoms including une
women. We recommend treating symptomatic nabothian cysts with local cystectomies or hysterecto Abbreviations: CUR = chronic urinary retention, MDA = minimal-deviation adenocarcinoma, PV
volume. = chronic urinary retention, MDA = minimal-deviation adenocarcinoma, PVR = postvoid residual urine Keywords: cervix, chronic urinary retention, nabothian cyst Keywords: cervix, chronic urinary retention, nabothian cyst 1. Introduction and nabothian are asymptomatic unless they become very
large.[1,2] Nabothian cysts are mucus-filled cysts that occur on the surface
of the cervix. They are usually 0.2 to 0.3cm in diameter, but they
can exceed 1cm in diameter. In most cases, nabothian cysts reflect
the physiological changes in the cervix, but they are sometimes
related to chronic cervicitis. Treatment is usually unnecessary, Urinary retentionisthe inabilitytocompletely empty the bladder
of urine, and chronic urinary retention (CUR) is the persistent
inability to completely empty the bladder despite maintaining an
ability to urinate. CUR results in elevated postvoid residual urine
volumes (PVRs). Two common causes of CUR are bladder muscle
dysfunction and urinary tract obstructions.[3] Herein, we report the case of a 46-year-old woman who
presented with a large, deep nabothian cyst that contributed to
CUR. This study was supported by the Sichuan Science and Technology Program of
China (grant nos. 2018SZ0248 and 2019YFS0404). This study was supported by the Sichuan Science and Technology Program of
China (grant nos. 2018SZ0248 and 2019YFS0404). The authors have no conflicts of interest to disclose. Medicine
®
OPEN Clinical Case Report A large nabothian cyst causing chronic urinary
retention Zhao Wu, MD, PhDa, Bingyu Zou, MDa, Xun Zhang, MDa, Xue Peng, MD, PhDb,c,∗ 2. Case report a Department of Obstetrics and Gynecology, Sichuan Academy of Medical
Sciences & Sichuan Provincial People’s Hospital, Chengdu 610072,
b Department of Obstetrics and Gynecology, West China Second University
Hospital, Sichuan University, Chengdu 610041, c Key Laboratory of Birth Defects
and Related Diseases of Women and Children, Sichuan University, Ministry of
Education, Chengdu 610041, China. a Department of Obstetrics and Gynecology, Sichuan Academy of Medical
Sciences & Sichuan Provincial People’s Hospital, Chengdu 610072,
b Department of Obstetrics and Gynecology, West China Second University
Hospital, Sichuan University, Chengdu 610041, c Key Laboratory of Birth Defects
and Related Diseases of Women and Children, Sichuan University, Ministry of
Education, Chengdu 610041, China. A 46-year-old woman (gravida 3, para 2) was admitted to our
gynecology
clinic. She
reported
that
an
ultrasonography
assessment performed 5 years earlier revealed a cervical cyst
with a 3-cm diameter. However, she did not report experiencing
any specific symptoms at the time. Follow-up revealed that the
cyst had gradually increased in size. During the previous 2 years,
she experienced gradually worsening urinary difficulties in
addition to dysmenorrhea and menorrhagia but did not seek
any further medical attention. ∗Correspondence: Xue Peng, Department of Obstetrics & Gynecology, West
China Second University Hospital, Sichuan University, No. 20 3rd Section, South
Renmin Road, Chengdu 610041, Sichuan, China (e-mail:
@ li
) Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc. This is an open access article distributed under the Creative Commons
Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Pelvic examinations revealed a uterus with normal bilateral
adnexa, but the size was equivalent to that of a uterus during the
12th week of pregnancy. The enlarged cervix was palpable
during a rectovaginal examination. Transvaginal ultrasonogra-
phy confirmed that the uterus was abnormally large and showed
that it had a heterogeneous myometrial echotexture and an
unremarkable endometrium with a 5cm4.5cm anechoic cyst in How to cite this article: Wu Z, Zou B, Zhang X, Peng X. A large nabothian cyst
causing chronic urinary retention: A case report. Medicine 2020;99:6(e19035). How to cite this article: Wu Z, Zou B, Zhang X, Peng X. A large nabothian cyst
causing chronic urinary retention: A case report. Medicine 2020;99:6(e19035). ∗Correspondence: Xue Peng, Department of Obstetrics & Gynecology, West
China Second University Hospital, Sichuan University, No. 20 3rd Section, South
Renmin Road, Chengdu 610041, Sichuan, China (e-mail:
pengxuesnow@aliyun.com). Medicine Medicine Wu et al. Medicine (2020) 99:6 An indwelling Foley catheter was inserted, and 900ml of urine
was drained. Subsequent laboratory tests showed that our
patient’s hemoglobin level was 52g/l (normal range, 110–160g/l)
and that her tumor biomarker carcinoembryonic antigen 125
level was 74.5U/ml (normal range, <35U/ml). She underwent a
blood transfusion to treat her anemia. Cervical and endometrial
biopsy samples tested negative for atypia. Figure 1. Pelvic computed tomography scan showing an enlarged and
distended bladder and a cervical cyst (arrow). Because she had adenomyosis and severe anemia, a laparo-
scopic hysterectomy was selected as the best treatment option. As
shown in an intraoperative image (Fig. 2), after dissection of the
posterior leaf of the broad ligaments and uterosacral ligaments,
we identified the ureters; the cyst was visible, and the cervix was
pushed to the right lateral anterior side by the cyst. The cyst was
filled with mucous fluid and was located in the posterior left
lateral wall of the cervix (Fig. 3). Additionally, we noted diffuse
uterine enlargement and thickened myometrium with scattered
nodules and small hemorrhagic foci. Histopathological
examinations of recovered cyst tissue
showed that the cyst wall was lined with columnar epithelium,
and endometrial glands and stroma were present deep within the
myometrium (Fig. 4). We, therefore, diagnosed our patient with
nabothian cyst and adenomyosis. She performed clean intermit-
tent self-catheterizations for 5 days after the operation. By
postoperative day 6, her urinary symptoms were completely
resolved, and she was discharged from the hospital. No evidence
of recurrence was observed during the 12 months of follow-up. Our patient gave written informed consent for the publication
of her medical history and images. Figure 1. Pelvic computed tomography scan showing an enlarged and
distended bladder and a cervical cyst (arrow). 2. Case report Received: 5 June 2019 / Received in final form: 4 December 2019 / Accepted: 7
January 2020 Received: 5 June 2019 / Received in final form: 4 December 2019 / Accepted: 7
January 2020 http://dx.doi.org/10.1097/MD.0000000000019035 http://dx.doi.org/10.1097/MD.0000000000019035 1 3. Discussion To the best of our knowledge, this is the first case report to
describe a large, deep nabothian cyst contributing to CUR. Few
existing case reports describe the treatment of large, symptomatic
nabothian cysts, although transvaginal or laparoscopic cystec-
tomies are generally performed to avoid unnecessary hysterecto-
mies.[4,5] In our patient’s case, due to the concurrent presence of the posterior cervical wall. Pelvic computed tomography revealed
an enlarged and distended bladder and bilaterally dilated ureters
and confirmed the presence of a cervical cyst with a 5-cm
diameter (Fig. 1). Figure 2. Intraoperative image showing the cyst (star) and the left ureter (arrow). Figure 2. Intraoperative image showing the cyst (star) and the left ureter (arrow). Figure 2. Intraoperative image showing the cyst (star) and the left ureter (arrow). 2 Wu et al. Medicine (2020) 99:6 www.md-journal.com Figure 3. Image of the uterus showing the dissected cervical nabothian cyst
(star). can serve as a universal definition of CUR, the American
Urological Association defines CUR as a PVR of >300ml.[6]
There are many potential causes of urinary retention in women,
and they can be subdivided into anatomic and functional
subgroups. Anatomic causes can include obstructions resulting
from pelvic organ prolapse, gynecological tumors, and intrinsic
urethral lesions. Moreover, anatomic obstructions may arise
from
iatrogenic
procedures
such
as
anti-incontinence
or
gynecological surgeries. Functional causes include abnormal
contraction of the periurethral muscle and failure to relax the
muscles surrounding the urethra or bladder neck prior to
urination.[7] Nabothian cysts are caused by squamous epithelium obstruct-
ing the cervical crypt orifices.[8] Nabothian cysts can occur in any
part of the cervix, but it is generally large, deep nabothian cysts
that produce cervical swelling, as observed in our patient. Nabothian cysts are usually asymptomatic and mostly benign. Treatment is usually unnecessary. However, as nabothian cysts
increase in size, symptoms may gradually emerge due to
compression of the surrounding organs. For example, compres-
sion of the rectum can result in abnormal defecation and
tenesmus.[9] Nabothian cysts that originate from the anterior lip
of the cervix can cause hematometra and recurrent lower
abdominal pain.[10] Large nabothian cysts and nabothian cysts occurring in
clusters, especially those located deep in the cervix, and they
should be distinguished from minimal-deviation adenocarcinoma
(MDA), which is a rare mucin-producing cervical adenocarcino-
ma. Patients with MDA usually present with abnormal watery
discharges from the vagina. 3. Discussion Magnetic resonance imaging is used
to distinguish MDA from typical nabothian cysts.[11] Figure 3. Image of the uterus showing the dissected cervical nabothian cyst
(star). Figure 3. Image of the uterus showing the dissected cervical nabothian cyst
(star). In adult women, uterine fibroids are the most common benign
tumors that compress the urethra, impair urine flow, and cause
CUR. Several case reports claimed that the surgical removal of
uterine fibroids can alleviate urinary retention problems.[12,13]
The mechanism by which a nabothian cyst caused CUR in our
patient may be similar to the mechanism by which uterine fibroids
cause CUR. A nabothian cyst may obstruct the bladder’s outlet
by displacing the cervix and thereby compressing the urethra or
the bladder neck.[13] Another potential pathophysiological
mechanism involves the mass causing radiculopathy by com-
pressing the pudendal and sacral nerves and thus, interrupting the
innervation of the detrusor muscle.[14] adenomyosis and severe anemia, hysterectomy was considered as
the best treatment option after discussion with the patient. This
treatment successfully resolved her CUR. CUR results in urinary accumulation that leads to adverse
clinical outcomes if left untreated. The PVR is the metric used to
quantify urinary retention levels. Although no single PVR value Figure 4. Histopathological images of the nabothian cyst showing the
columnar epithelium without atypia. Scale bar 100mm (hematoxylin and eosin
staining; magnification, 200). In conclusion, this case report shows that large, deep nabothian
cysts can cause CUR in women. Therefore, although large
nabothian cysts are rare, they should be considered as potential
causes of pelvic masses and urinary symptoms in women. Nabothian cysts can be distinguished from other mucus-
producing cervical malignancies on the basis of symptomatology,
preoperative examination results, and imaging techniques such as
magnetic resonance imaging. Author contributions Conceptualization: Zhao Wu, Xue Peng. Data curation: Bingyu Zou, Xue Peng. Formal analysis: Zhao Wu, Bingyu Zou, Xue Peng. Funding acquisition: Zhao Wu, Xue Peng. Investigation: Zhao Wu, Xun Zhang. Methodology: Bingyu Zou, Xue Peng. Project administration: Zhao Wu. Resources: Zhao Wu. Bingyu Zou, Xun Zhang. Conceptualization: Zhao Wu, Xue Peng. Figure 4. Histopathological images of the nabothian cyst showing the
columnar epithelium without atypia. Scale bar 100mm (hematoxylin and eosin
staining; magnification, 200). 3 Wu et al. Medicine (2020) 99:6 Medicine [7] Jung J, Ahn HK, Huh Y. Clinical and functional anatomy of the urethral
sphincter. Int Neurourol J 2012;16:102–6. Supervision: Bingyu Zou, Xue Peng. Writing – original draft: Zhao Wu. [8] Pelosi MAIII, Pelosi MA, Rudelli RD. Symptomatic cervical macrocyst as
a late complication of subtotal hysterectomy. A case report. J Reprod
Med 1999;44:567–70. Writing – review & editing: Zhao Wu, Xue Peng. [9] Temur I, Ulker K, Sulu B, et al. A giant cervical nabothian cyst
compressing the rectum, differential diagnosis and literature review. Clin
Exp Obstet Gynecol 2011;38:276–9. References [1] Lobo
RA,
Gershenson
DM,
Lentz
GM,
et
al. Comprehensive
Gynecology. 7th ed.Philadelphia: Elsevier; 2017. [10] Torky HA. Huge Nabothian cyst causing hematometra (case report). Eur
J Obstet Gynecol Reprod Biol 2016;207:238–40. [2] Hoffman BL, Williams JW. Williams Gynecology. 2nd ed.New York:
McGraw-Hill Medical; 2012. [11] Oguri H, Maeda N, Izumiya C, et al. MRI of endocervical glandular
disorders:threecasesof a deep nabothian cyst andthree cases of a minimal-
deviation adenocarcinoma. Magn Reson Imaging 2004;22:1333–7. [3] Juma S. Urinary retention in women. Curr Opin Urol 2014;24:375–9. [4] Nassif J, Nahouli H, Mourad A, et al. Laparoscopic excision of an
unusual presentation of a nabothian cyst: case report and review of the
literature. Surg Technol Int 2017;31:140–3. [12] Derbent A, Turhan NO. Acute urinary retention caused by a large
impacted leiomyoma. Arch Gynecol Obstet 2009;280:1045–7. [5] Nigam A, Choudhary D, Raghunandan C. Large nabothian cyst: a rare
cause of nulliparous prolapse. Case Rep Obstet Gynecol 2012;2012:
192526. [13] Wu CQ, Lefebvre G, Frecker H, et al. Urinary retention and uterine
leiomyomas: a case series and systematic review of the literature. Int
Urogynecol J 2015;26:1277–84. [14] Andrada AO, De Vicente JM, Cidre MA. Pelvic plexus compression due
to a uterine leiomyoma in a woman with acute urinary retention: a new
hypothesis. Int Urogynecol J 2014;25:429–31. [6] Stoffel JT, Peterson AC, Sandhu JS, et al. AUA white paper on
nonneurogenic chronic urinary retention: consensus definition, treatment
algorithm, and outcome endpoints. J Urol 2017;198:153–60. 4
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English
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Prunus persica var. platycarpa (Tabacchiera Peach): Bioactive Compounds and Antioxidant Activity of Pulp, Peel and Seed Ethanolic Extracts
|
Plant foods for human nutrition
| 2,015
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public-domain
| 7,584
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TITLE RUNNING HEAD: Tabacchiera peach chemical composition and antioxidant
1
activity
2
ORIGINAL PAPER
3
Prunus persica var. platycarpa (Tabacchiera Peach): bioactive compounds and
4
antioxidant activity of pulp, peel and seed ethanolic extracts
5
6
Monica R. Loizzo,† Deborah Pacetti,§ Paolo Lucci,# Oscar Núñez°,‡, Francesco
7
Menichini†, Natale Giuseppe Frega§ & Rosa Tundis,†,*
8
9
†Department of Pharmacy, Health and Nutritional Sciences, University of Calabria,
10
87036 Rende (CS), Italy.
11
§Department of Food Science, Politecnica delle Marche University, via Brecce Bianche,
12
60131 Ancona, Italy.
13
#Department of Nutrition and Biochemistry, Faculty of Sciences, Pontificia Universidad
14
Javeriana, Bogotà D.C., Colombia.
15
°Department of Analytical Chemistry, University of Barcelona, Martíi Franquès 1-11,
16
08028 Barcelona, Spain.
17
‡Serra Hunter Fellow, Generalitat de Catalunya, Spain.
18
19
CORRESPONDING AUTHOR FOOTNOTE: ROSA TUNDIS, Department of
20
Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende (CS),
21
It l
T l
39 984 493246 F
39 984 493107 E
il
t
di @
i
l it
22 TITLE RUNNING HEAD: Tabacchiera peach chemical composition and antioxidant
1
activity
2
ORIGINAL PAPER
3
Prunus persica var. platycarpa (Tabacchiera Peach): bioactive compounds and
4
antioxidant activity of pulp, peel and seed ethanolic extracts
5
6
Monica R. Loizzo,† Deborah Pacetti,§ Paolo Lucci,# Oscar Núñez°,‡, Francesco
7
Menichini†, Natale Giuseppe Frega§ & Rosa Tundis,†,*
8
9
†Department of Pharmacy, Health and Nutritional Sciences, University of Calabria,
10
87036 Rende (CS), Italy. 11
§Department of Food Science, Politecnica delle Marche University, via Brecce Bianche,
12
60131 Ancona, Italy. 13
#Department of Nutrition and Biochemistry, Faculty of Sciences, Pontificia Universidad
14
Javeriana, Bogotà D.C., Colombia. 15
°Department of Analytical Chemistry, University of Barcelona, Martíi Franquès 1-11,
16
08028 Barcelona, Spain. 17
‡Serra Hunter Fellow, Generalitat de Catalunya, Spain. 18
19
CORRESPONDING AUTHOR FOOTNOTE: ROSA TUNDIS, Department of
20
Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende (CS),
21
It l
T l
39 984 493246 F
39 984 493107 E
il
t
di @
i
l it
22 TITLE RUNNING HEAD: Tabacchiera peach chemical composition and antioxidant
1
activity
2 °Department of Analytical Chemistry, University of Barcelona, Martíi Franquès 1-11,
16
08028 Barcelona, Spain. 17 CORRESPONDING AUTHOR FOOTNOTE: ROSA TUNDIS, Department of
20
Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende (CS),
21
Italy. Tel. +39 984 493246, Fax: +39 984 493107, E-mail: rosa.tundis@unical.it
22 CORRESPONDING AUTHOR FOOTNOTE: ROSA TUNDIS, Department of
20
Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende (CS),
21
Italy. Tel. +39 984 493246, Fax: +39 984 493107, E-mail: rosa.tundis@unical.it
22 Abstract A comparative analysis of ethanol extracts from peel, pulp and seed of Prunus
23
persica var. platycarpa (Tabacchiera peach) was done. The total phenol, flavonoid and
24
carotenoid content as well as the antioxidant properties by using different in vitro assays
25
(DPPH, ABTS, FRAP, Fe-chelating, β-carotene bleaching test) were evaluated. Pulp
26
extract was subjected to liquid chromatography-electrospray-tandem mass spectrometry
27
(HPLC-ESI-MS/MS). Gallic
acid,
protocatechuic
acid,
protocatechualdehyde,
28
chlorogenic acid, p-coumaric acid, and ferulic acid were identified as main constituents. 29
Pulp extract was characterized by the highest total phytonutrients content and exhibited
30
the highest antioxidant activity in all in vitro assays (IC50 values of 2.2 µg/mL after 60
31
minutes of incubation by using β-carotene bleaching test and 2.9 µg/mL by using Fe-
32
chelating assay). Overall, the obtained results suggest that P. persica var. TITLE RUNNING HEAD: Tabacchiera peach chemical composition and antioxidant
1
activity
2
ORIGINAL PAPER
3
Prunus persica var. platycarpa (Tabacchiera Peach): bioactive compounds and
4
antioxidant activity of pulp, peel and seed ethanolic extracts
5
6
Monica R. Loizzo,† Deborah Pacetti,§ Paolo Lucci,# Oscar Núñez°,‡, Francesco
7
Menichini†, Natale Giuseppe Frega§ & Rosa Tundis,†,*
8
9
†Department of Pharmacy, Health and Nutritional Sciences, University of Calabria,
10
87036 Rende (CS), Italy.
11
§Department of Food Science, Politecnica delle Marche University, via Brecce Bianche,
12
60131 Ancona, Italy.
13
#Department of Nutrition and Biochemistry, Faculty of Sciences, Pontificia Universidad
14
Javeriana, Bogotà D.C., Colombia.
15
°Department of Analytical Chemistry, University of Barcelona, Martíi Franquès 1-11,
16
08028 Barcelona, Spain.
17
‡Serra Hunter Fellow, Generalitat de Catalunya, Spain.
18
19
CORRESPONDING AUTHOR FOOTNOTE: ROSA TUNDIS, Department of
20
Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende (CS),
21
It l
T l
39 984 493246 F
39 984 493107 E
il
t
di @
i
l it
22 platycarpa
33
displays a good antioxidant activity and its consumption could be promoted. 34 Abstract A comparative analysis of ethanol extracts from peel, pulp and seed of Prunus
23
persica var. platycarpa (Tabacchiera peach) was done. The total phenol, flavonoid and
24
carotenoid content as well as the antioxidant properties by using different in vitro assays
25
(DPPH, ABTS, FRAP, Fe-chelating, β-carotene bleaching test) were evaluated. Pulp
26
extract was subjected to liquid chromatography-electrospray-tandem mass spectrometry
27
(HPLC-ESI-MS/MS). Gallic
acid,
protocatechuic
acid,
protocatechualdehyde,
28
chlorogenic acid, p-coumaric acid, and ferulic acid were identified as main constituents. 29
Pulp extract was characterized by the highest total phytonutrients content and exhibited
30
the highest antioxidant activity in all in vitro assays (IC50 values of 2.2 µg/mL after 60
31
minutes of incubation by using β-carotene bleaching test and 2.9 µg/mL by using Fe-
32
chelating assay). Overall, the obtained results suggest that P. persica var. platycarpa
33
displays a good antioxidant activity and its consumption could be promoted. 34 Keywords Prunus persica L. Stokes ex Batsch var. platycarpa • Phenols • HPLC-ESI-
35
MS/MS• Dietary antioxidants
36 Keywords Prunus persica L. Stokes ex Batsch var. platycarpa • Phenols • HPLC-ESI-
35
MS/MS• Dietary antioxidants
36 Keywords Prunus persica L. Stokes ex Batsch var. platycarpa • Phenols • HPLC-ESI-
35
MS/MS• Dietary antioxidants
36 Abbreviations
37
ABTS 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
38
DPPH 2,2-Diphenyl-1-picrylhydrazyl
39
FRAP Ferric Reducing Ability Power
40
ROS Reactive oxygen species
41
TEAC Trolox Equivalent Antioxidant Capacity
42 2 Introduction
46 Among them methanol was the most widely used [7-14] since
72
it offers a high recovery of antioxidant compounds. Differently, the ethanol proves to be
73
least effective solvent for extracting vegetable phenolic compounds. However, due the
74
safety and acceptability for human use of the ethanol, it is very important to encourage
75
the evaluation of the potentiality of ethanol extract as valuable sources of bioactive
76
compounds. The scientific knowledge generated could be used by pharmaceutical and
77
food industries to identify alternative value-added ingredients for the development of
78
new functional intermediates or products with health-benefits. 79 bioactivity and functionality present in different peach varieties. Several research papers
70
reported the analysis of bioactive constituents of P. persica using different solvents in
71
the extraction procedure. Among them methanol was the most widely used [7-14] since
72
it offers a high recovery of antioxidant compounds. Differently, the ethanol proves to be
73
least effective solvent for extracting vegetable phenolic compounds. However, due the
74
safety and acceptability for human use of the ethanol, it is very important to encourage
75
the evaluation of the potentiality of ethanol extract as valuable sources of bioactive
76
compounds. The scientific knowledge generated could be used by pharmaceutical and
77
food industries to identify alternative value-added ingredients for the development of
78
new functional intermediates or products with health-benefits. 79 new functional intermediates or products with health-benefits. 79
The aim of this study is to evaluate the compositional profile of Prunus persica var. 80
platycarpa var. “Tabacchiera” or “Saturnina” ethanolic extracts and to teste the
81
antioxidant activities using different in vitro methods. The phenol composition of peach
82
pulp was elucidated by HPLC- ESI-MS/MS. 83
84
Materials and Methods
85
Chemicals and Reagents
86
All chemicals and reagents used in this study were purchased from Sigma-Aldrich
87
Chemical Co. Ltd (Milan, Italy) and VWR International (Milan, Italy) and, unless
88
specified otherwise, were analytical grade or higher. 89
90
Plant Material and Extraction Procedure
91
Prunus persica var. platycarpa fruits were purchased at commercial maturity as
92
assessed by peel fruit colour and pulp firmness maturity in the local market in Catania
93 The aim of this study is to evaluate the compositional profile of Prunus persica var. 80
platycarpa var. “Tabacchiera” or “Saturnina” ethanolic extracts and to teste the
81
antioxidant activities using different in vitro methods. Introduction
46 Several epidemiological studies have confirmed the relationships between diet and
47
diseases [1]. The health-promoting properties of fruits are due to the presence of some
48
secondary metabolites such as phenols that have aroused substantial attention due to
49
their protective potential against diseases [2]. Oxidative stress is considered to play a
50
very important role in the pathogenesis of several health problems [3]. A diet rich in
51
antioxidants can reduce the risk of several diseases. Adequate level of antioxidants
52
provided with diet induces immunological processes and increases defensive abilities of
53
cell in proper way [4]. Several tests were developed to analyse the food antioxidant
54
properties. These tests differ in the generation of radicals, target compounds, and in the
55
mode of assessment the end points. Antioxidants may act in vivo through different
56
mechanisms, so the measure of antioxidant activity required a multifactorial approach
57
based on the use of different analytical techniques. Therefore, choosing an adequate
58
assay is critical and in practice several test procedures are carried out for evaluating
59
antioxidant activities with the samples of interest [5, 6]. The growing interest in the
60
substitution of synthetic antioxidants by natural ones has promoted research on
61
vegetable sources for new antioxidants. Oxidation reactions are not an exclusive
62
concern for the food industry, and antioxidants are widely needed to prevent
63
deterioration of other oxidisable products, such as cosmetics and pharmaceuticals
64
products. 65 P. persica (Rosaceae) is the second most widely cultivated fruit tree [7]. Peach fruit
66
is known for its nutritional value and therapeutic properties as consequence of its
67
bioactive constituents (phenols and carotenoids) [7-13]. Thus, in order to evaluate the
68
potential of peach fruits, significance efforts have been directed on the estimation of
69 P. persica (Rosaceae) is the second most widely cultivated fruit tree [7]. Peach fruit
66
is known for its nutritional value and therapeutic properties as consequence of its
67
bioactive constituents (phenols and carotenoids) [7-13]. Thus, in order to evaluate the
68
potential of peach fruits, significance efforts have been directed on the estimation of
69 3 3 bioactivity and functionality present in different peach varieties. Several research papers
70
reported the analysis of bioactive constituents of P. persica using different solvents in
71
the extraction procedure. Introduction
46 The phenol composition of peach
82
pulp was elucidated by HPLC- ESI-MS/MS. 83 All chemicals and reagents used in this study were purchased from Sigma-Aldrich
87
Chemical Co. Ltd (Milan, Italy) and VWR International (Milan, Italy) and, unless
88
specified otherwise, were analytical grade or higher. 89 4 (Sicily, Italy) in June 2012. P. persica var. platycarpa “Tabacchiera” or “Saturnina” is a
94
deciduous tree cultivated in Etna volcanic area (Sicily, Italy). Fruits were examined for
95
integrity and absence of dust and insect contamination, were cleaned by using distilled
96
water and were peeled. Pulp was separated by seeds. Peel (60.80 g), pulp (483.31 g),
97
and seed (33.05 g) were exhaustively extracted at 25 °C by ethanol (absolute, ≥ 99.5%)
98
(400, 1500 and 250 mL, respectively) (3 × 72 h). 99 High Performance Liquid Chromatography (HPLC) – Tandem Mass Spectrometry (MS)
105
Condition and Analysis of Phenolic Compounds in Pulp
106 High Performance Liquid Chromatography (HPLC) – Tandem Mass Spectrometry (MS)
105
Condition and Analysis of Phenolic Compounds in Pulp
106 The determination of the phenolic profile of pulp peach was performed by means of
107
liquid chromatography-electrospray-tandem mass spectrometry, as already described
108
[17]. Twenty-six selected compounds belonging to different phenolic classes (gallic
109
acid, (+)-catechin hydrate, p-coumaric acid, p-salicylic acid, caffeic acid, chlorogenic
110
acid, (−)-epicatechin, (−)-epigallocatechin ethyl gallate, gallate, ferulic acid, fisetin,
111
gentisic acid, homogentisic acid, polydatin, protocatechuic acid, protocatechualdehyde,
112
quercetin dehydrate quercitrin hydrate, resveratrol, syringic acid, syringaldehyde,
113
taxifolin, umbelliferon, sinapic acid, kaempferol, and vanillic acid) were quantified. 114
115 The determination of the phenolic profile of pulp peach was performed by means of
107
liquid chromatography-electrospray-tandem mass spectrometry, as already described
108
[17]. Twenty-six selected compounds belonging to different phenolic classes (gallic
109
acid, (+)-catechin hydrate, p-coumaric acid, p-salicylic acid, caffeic acid, chlorogenic
110
acid, (−)-epicatechin, (−)-epigallocatechin ethyl gallate, gallate, ferulic acid, fisetin,
111
gentisic acid, homogentisic acid, polydatin, protocatechuic acid, protocatechualdehyde,
112
quercetin dehydrate quercitrin hydrate, resveratrol, syringic acid, syringaldehyde,
113
taxifolin, umbelliferon, sinapic acid, kaempferol, and vanillic acid) were quantified. 114
115 ABTS and DPPH Radical Scavenging Activity Assay Radical scavenging activity was evalòuated by ABTS and DPPH assays [5, 18]. 117 5 5 β-Carotene Bleaching Test
118
Antioxidant activity was determined as previously described [19]. Briefly, β-carotene
119
solution was added to linoleic acid and 100% Tween 20. Introduction
46 persica
155
var. platycarpa cultivar “Sweet-cap” and “ASF-06-83” as a rich source of phenols
156
(35.97 and 41.37 mg gallic acid equivalent/100 g FW, respectively). Our results
157
demonstrated that pulp extract from Tabacchiera is richer in phenols and flavonoids
158
than peel extract. These results are in contrast with previous reports in a wide range of
159
peach cultivars including the flat peach varieties [11-14, 24]. 160
The observed differences might be related to the kind of solvent used for the
161 Dunnett’s test (p< 0.01).The concentration-response curve was obtained by plotting the
141
percentage of inhibition versus the concentrations. 142 Tabacchiera peach peel, pulp and seed are exhaustively extracted with ethanol
146
(yield of 12.09, 7.83 and 3.69%, respectively). The values of total phenols, flavonoids
147
and carotenoids content for peach pulp, peel and seed ethanol extracts were reported in
148
Table 1. The total phenols content of Tabacchiera peach varied significantly among the
149
tree fruit part tested. The pulp exhibited the highest phenols content (921.8 mg
150
chlorogenic acid equivalent/100 g FW), followed by the peel (448.6 FW) and the seed
151
(111.3 FW). This trend was observed with total flavonoid content. Correlation analysis
152
revealed that phenols are correlated with flavonoids (r= 0.89, p< 0.01), implying that
153
this group of phytochemicals an important group of phenols in peach fruit [7, 13]. 154 Previously, Legua et al. [14] reported the methanol extract of the pulp of P. persica
155
var. platycarpa cultivar “Sweet-cap” and “ASF-06-83” as a rich source of phenols
156
(35.97 and 41.37 mg gallic acid equivalent/100 g FW, respectively). Our results
157
demonstrated that pulp extract from Tabacchiera is richer in phenols and flavonoids
158
than peel extract. These results are in contrast with previous reports in a wide range of
159
peach cultivars including the flat peach varieties [11-14, 24]. 160 Previously, Legua et al. [14] reported the methanol extract of the pulp of P. persica
155
var. platycarpa cultivar “Sweet-cap” and “ASF-06-83” as a rich source of phenols
156
(35.97 and 41.37 mg gallic acid equivalent/100 g FW, respectively). Our results
157
demonstrated that pulp extract from Tabacchiera is richer in phenols and flavonoids
158
than peel extract. These results are in contrast with previous reports in a wide range of
159
peach cultivars including the flat peach varieties [11-14, 24]. Introduction
46 The absorbance of the
120
samples, standard and control was measured at 470 nm against a blank at t= 0 and
121
successively at 30 and 60 minutes. 122
123
FRAP (Ferric Reducing Ability Power) Assay
124
The FRAP test is based on the redox reaction that involves TPTZ (2,4,6-tripyridyl-s-
125
triazine)-Fe3+ complex [20]. 126
127
Fe2+Chelating Activity Assay
128
The chelating activity was measured according to the previously reported method [21]. 129
Briefly, extract was mixed with water, 2 mM FeCl2 and 5 mM ferrozine. After 10 min at
130
room temperature, the absorbance was measured at 562 nm. 131
132
Pulp Peach Nutritional Analysis
133
The total nitrogen content, moisture content, ash content, fat content, crude fiber
134
content, total carbohydrates, minerals, and energy values were evaluated [22, 23]. 135
136
Statistical Analysis
137
The inhibitory concentration 50% (IC50) was calculated by non-linear with the use of
138
Prism Graphpad Prism version 4.0 for Windows, GraphPad Software, San Diego, CA,
139
USA. Differences were evaluated by ANOVA test followed by multicomparison
140 β-Carotene Bleaching Test
118
Antioxidant activity was determined as previously described [19]. Briefly, β-carotene
119
solution was added to linoleic acid and 100% Tween 20. The absorbance of the
120
samples, standard and control was measured at 470 nm against a blank at t= 0 and
121
successively at 30 and 60 minutes. 122 6 Dunnett’s test (p< 0.01).The concentration-response curve was obtained by plotting the
141
percentage of inhibition versus the concentrations. 142
143
Results and Discussion
144
Extraction Yield and Phytonutrients Content
145
Tabacchiera peach peel, pulp and seed are exhaustively extracted with ethanol
146
(yield of 12.09, 7.83 and 3.69%, respectively). The values of total phenols, flavonoids
147
and carotenoids content for peach pulp, peel and seed ethanol extracts were reported in
148
Table 1. The total phenols content of Tabacchiera peach varied significantly among the
149
tree fruit part tested. The pulp exhibited the highest phenols content (921.8 mg
150
chlorogenic acid equivalent/100 g FW), followed by the peel (448.6 FW) and the seed
151
(111.3 FW). This trend was observed with total flavonoid content. Correlation analysis
152
revealed that phenols are correlated with flavonoids (r= 0.89, p< 0.01), implying that
153
this group of phytochemicals an important group of phenols in peach fruit [7, 13]. 154
Previously, Legua et al. [14] reported the methanol extract of the pulp of P. Introduction
46 160 The observed differences might be related to the kind of solvent used for the
161
extraction. It is well documented that the quali-quantitative profile of phytonutrient
162
fraction is markedly affected by the polarity of extracting solvent and the solubility of
163
the compounds in the solvent used for the extraction process [25]. Generally, extraction
164 7 7 with aqueous methanol results in a higher recovery of total extractable compounds,
165
whereas ethanol gave the lowest recovery of antioxidant compounds [26]. Nevertheless,
166
due the largely use of ethanol as solvent in human, it is very important to investigate the
167
healthy potentiality of ethanolic extract. Unlike to phenol and flavonoid contents, the
168
peel had significantly higher concentration of carotenoids than the pulp (about 6-fold). 169
This content was similar to those reported for other white-fleshed peaches and
170
nectarines, in which the major constituent was β-carotene followed by β-criptosanthin
171
and lutein [12, 13, 24, 27]. Carotenoids are effective against free radicals, singlet
172
oxygen suppressors and work as chain-breaking antioxidants, protecting cells against
173
attack from radicals [28]. 174 with aqueous methanol results in a higher recovery of total extractable compounds,
165
whereas ethanol gave the lowest recovery of antioxidant compounds [26]. Nevertheless,
166
due the largely use of ethanol as solvent in human, it is very important to investigate the
167
healthy potentiality of ethanolic extract. Unlike to phenol and flavonoid contents, the
168
peel had significantly higher concentration of carotenoids than the pulp (about 6-fold). 169
This content was similar to those reported for other white-fleshed peaches and
170
nectarines, in which the major constituent was β-carotene followed by β-criptosanthin
171
and lutein [12, 13, 24, 27]. Carotenoids are effective against free radicals, singlet
172
oxygen suppressors and work as chain-breaking antioxidants, protecting cells against
173
attack from radicals [28]. 174 175
HPLC-ESI-MS/MS Phenolic profile of Pulp Extract
176
The phenolic profile of pulp extract, that showed the most interesting antioxidant
177
activity, was outlined by means HPLC-ESI-MS/MS analysis. The used methodology
178
was able to provide a comprensive evaluation of twenty-six selected phenols belonging
179
to different phenolic classes, such benzoic and cinnamic acids, flavanols and flavones. 180
Among them, the Tabacchiera pulp extract contained benzoic acids (i.e. gallic and
181
protocatechuic acids), cinnamic acids (i.e. p-coumaric and ferulic acids), chlorogenic
182
acid and protocatechualdehyde (Figure 1, Table 3). Introduction
46 The present study confirmed that
183
chlorogenic acid is the main phenolic compound in peach pulp. In fact, according to
184
previously reported [9] in round peaches and nectarines, our results showed that
185
chlorogenic acid represent the most abundant compound in Tabacchiera pulp extract,
186
followed by gallic acid, protocatechuic acid, protocatechualdehyde, p-coumaric acid and
187
ferulic acid. Singularly, the Tabacchiera pulp extract resulted devoid of catechin,
188 HPLC-ESI-MS/MS Phenolic profile of Pulp Extract
176
The phenolic profile of pulp extract, that showed the most interesting antioxidant
177
activity, was outlined by means HPLC-ESI-MS/MS analysis. The used methodology
178
was able to provide a comprensive evaluation of twenty-six selected phenols belonging
179
to different phenolic classes, such benzoic and cinnamic acids, flavanols and flavones. 180
Among them, the Tabacchiera pulp extract contained benzoic acids (i.e. gallic and
181
protocatechuic acids), cinnamic acids (i.e. p-coumaric and ferulic acids), chlorogenic
182
acid and protocatechualdehyde (Figure 1, Table 3). The present study confirmed that
183
chlorogenic acid is the main phenolic compound in peach pulp. In fact, according to
184
previously reported [9] in round peaches and nectarines, our results showed that
185
chlorogenic acid represent the most abundant compound in Tabacchiera pulp extract,
186
followed by gallic acid, protocatechuic acid, protocatechualdehyde, p-coumaric acid and
187
ferulic acid. Singularly, the Tabacchiera pulp extract resulted devoid of catechin,
188 8 8 epicatechin, epigallocatechin and quercitin whom were previously reported in aqueous
189
methanol extract of pulp of round peaches and nectarines [29]. This difference could be
190
related to the peach variety as well as to the kind of the solvent used for the phenolic
191
extraction from the pulp. 192 epicatechin, epigallocatechin and quercitin whom were previously reported in aqueous
189
methanol extract of pulp of round peaches and nectarines [29]. This difference could be
190
related to the peach variety as well as to the kind of the solvent used for the phenolic
191
extraction from the pulp. 192 Antioxidant Activity of P. persica var. platycarpa Extracts
193 The antioxidant capacity of fruits varies in relation to antioxidant moieties present in the
194
different species, although variations can occur among cultivars within a single species
195
[9, 12, 24]. A concentration-effect relationship was found for all tested samples (Table
196
3). The radical scavenging activity of peach extracts was evaluated using DPPH and
197
ABTS systems. Introduction
46 Pulp extract exhibited the highest DPPH scavenging ability with an IC50
198
value of 12.0 µg/mL followed by peel (IC50 45.3 µg/mL). This trend was observed also
199
against ABTS radical with value of 6.8 and 8.4, respectively. The Ferric Reducing
200
Ability Power of Tabacchiera peach was screened by using FRAP assay. Both pulp and
201
peel extracts resulted the most active (values of 30.2 and 78.9 µM Fe(II)/g,
202
respectively). The reducing ability of both extract was strongly correlated with the
203
phenolic levels (p<0.01, r= 0.99). A strong correlation between high level of phenol and
204
FRAP value was previously reported also by Hong et al. [30]. Phenols exhibited redox
205
properties acting as reducing agents, hydrogen donators and singlet oxygen quenchers. 206
The redox potential of phenolic phytochemicals plays a crucial role in determining the
207
antioxidant properties. Pulp extract chelates ferrous ions with an IC50 value of 2.9
208
µg/mL (value 2.2-time higher than the positive control BHT). Carotenoid content was
209
the only value that correlated significantly (p< 0.01) with antioxidant activity (r= 0.98,
210
0.99, 0.98, 0.98, 0.98, and 0.99 for DPPH, ABTS, β-carotene bleaching test after 30 and
211
60 minutes of incubation, FRAP and Fe-Chelating activity assay, respectively). 212 9 9 Tabacchiera peach is a rich source of phytochemicals with known antioxidant
213
activity. Phenols and carotenoids are able to inhibit oxidation process by acting as free
214
radical scavengers, metal chelators and also trough their effects on cell signaling
215
pathways and on gene expression. The antioxidant activity results from a complex
216
interaction between phytochemicals, which produce synergistic responses. 217
218
Pulp Nutritional Analysis
219
Tabacchiera peach pulp nutritional analysis is reported in Table 4. Moisture content and
220
ash content of pulp were 84.76 and 0.43%, respectively. The fat content was determined
221
by using petroleum ether founding a percentage of 0.21% that revealed that Tabacchiera
222
is a low fat fruit that can be eaten to prevent weight gain and obesity. Protein content
223
was found to be 0.68%. Gravimetric method was used for the determination of dietary
224
fiber founding a value of 1.78 %. Carbohydrates present in the sample were calculated
225
to be 12.14%. The energy value was calculated to be 53.17 Kcal per 100 g of pulp. Introduction
46 Further in vivo
242
studies are warranted to confirm these findings and support the use of this matrix. 243 fruits, especially for the purposes of juice, and/or processed sauces and slice production,
237
result in the generation of large quantity of seeds and peel as agro-wastes. The results
238
could be useful to provide a solid base for the development and utilization of peach
239
ethanolic extracts, and also for the agro-wastes containing peel and seed as ingredient in
240
formulations of functional or nutraceutical products useful in diseases prevention such
241
as neurodegenerative diseases, cardiovascular diseases, cancer etc. Further in vivo
242
studies are warranted to confirm these findings and support the use of this matrix. 243
244 Acknowledgment
245
The authors are grateful to Dr. D. Sturino, Department of Pharmacy, Health and
246
Nutritional Science, University of Calabria, for English revision of the manuscript. The
247
authors are also grateful to the Spanish Ministry of Economy and Competitiveness
248
(project CTQ2012-30836), and to the Agency for Administration of University and
249
Research Grants (Generalitat de Catalunya, Spain) (Project 2014 SGR-539). 250
251
Conflict of Interest The authors declare that they have no conflict of interest. 252
253
References
254
1. Schreiner M, Korn M, Stenger M, Holzgreve L, Altmann M (2013) Current
255
understanding and use of quality characteristics of horticulture products. Scientia
256
Horticul 163:63-69
257
2. Pandey KB, Rizvi SI (2009) Plant polyphenols as dietary antioxidants in human
258
health and disease. Oxid Med Cell Longev 2:270-278
259 Introduction
46 The
226
mineral content showed the presence of high level of sodium and potassium, a moderate
227
iron content which can be utilized to metabolize proteins and helps red blood cells in the
228
production of haemoglobin. 229
230 Tabacchiera peach is a rich source of phytochemicals with known antioxidant
213
activity. Phenols and carotenoids are able to inhibit oxidation process by acting as free
214
radical scavengers, metal chelators and also trough their effects on cell signaling
215
pathways and on gene expression. The antioxidant activity results from a complex
216
interaction between phytochemicals, which produce synergistic responses. 217 Tabacchiera peach pulp nutritional analysis is reported in Table 4. Moisture content and
220
ash content of pulp were 84.76 and 0.43%, respectively. The fat content was determined
221
by using petroleum ether founding a percentage of 0.21% that revealed that Tabacchiera
222
is a low fat fruit that can be eaten to prevent weight gain and obesity. Protein content
223
was found to be 0.68%. Gravimetric method was used for the determination of dietary
224
fiber founding a value of 1.78 %. Carbohydrates present in the sample were calculated
225
to be 12.14%. The energy value was calculated to be 53.17 Kcal per 100 g of pulp. The
226
mineral content showed the presence of high level of sodium and potassium, a moderate
227
iron content which can be utilized to metabolize proteins and helps red blood cells in the
228
production of haemoglobin. 229 Prunus persica var. platycarpa ethanolic extract was analysed for its potential as a
232
functional food. Our results highlight that pulp is a rich source of bioactive compounds
233
particularly phenols. Due the safety and acceptability for human use of this solvent it is
234
very important verify the potentiality of ethanolic extract as valuable sources of
235
bioactive compounds. Moreover, the commercial and domestic uses of large quantity of
236 10 fruits, especially for the purposes of juice, and/or processed sauces and slice production,
237
result in the generation of large quantity of seeds and peel as agro-wastes. The results
238
could be useful to provide a solid base for the development and utilization of peach
239
ethanolic extracts, and also for the agro-wastes containing peel and seed as ingredient in
240
formulations of functional or nutraceutical products useful in diseases prevention such
241
as neurodegenerative diseases, cardiovascular diseases, cancer etc. Acknowledgment
245 The authors are grateful to Dr. D. Sturino, Department of Pharmacy, Health and
246
Nutritional Science, University of Calabria, for English revision of the manuscript. The
247
authors are also grateful to the Spanish Ministry of Economy and Competitiveness
248
(project CTQ2012-30836), and to the Agency for Administration of University and
249
Research Grants (Generalitat de Catalunya, Spain) (Project 2014 SGR-539). 250 The authors are grateful to Dr. D. Sturino, Department of Pharmacy, Health and
246
Nutritional Science, University of Calabria, for English revision of the manuscript. The
247
authors are also grateful to the Spanish Ministry of Economy and Competitiveness
248
(project CTQ2012-30836), and to the Agency for Administration of University and
249
Research Grants (Generalitat de Catalunya, Spain) (Project 2014 SGR-539). 250 The authors are grateful to Dr. D. Sturino, Department of Pharmacy, Health and
246
Nutritional Science, University of Calabria, for English revision of the manuscript. The
247
authors are also grateful to the Spanish Ministry of Economy and Competitiveness
248
(project CTQ2012-30836), and to the Agency for Administration of University and
249
Research Grants (Generalitat de Catalunya, Spain) (Project 2014 SGR-539). 250 2. Pandey KB, Rizvi SI (2009) Plant polyphenols as dietary antioxidants in human
258
health and disease. Oxid Med Cell Longev 2:270-278
259 11 11 3. Dröge W (2002) Free radicals in the physiological control of cell function. Physiol
260
Rev. 82:47-95
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4. Bendary E, Francis RR, Ali HMG, Sarwat MI, El Hady S (2013) Antioxidant and
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structure-activity relationships (SARs) of some phenolic and anilines compounds. 263
Ann AgrSci 58:173-181
264
5. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C (1996)
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Antioxidant activity applying an improved ABTS radical cation decolorization
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6. Antolovich M, Prenzler PD, Patsalides E, McDonald S, Robards K (2002) Methods
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7. Abidi W, Jiménez S, Moreno MÁ, Gogorcena Y (2011) Evaluation of antioxidant
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compounds and total sugar content in a nectarine [Prunus persica (L.) Batsch]
271
progeny. Int J Mol Sci 12:6919-6935
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8. Abidi W, Cantín CM, Jiménez S, Giménez R, Moreno MÁ, Gogorcena Y (2015)
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Influence of antioxidant compounds, total sugars and genetic background on the
274
chilling injury susceptibility of a non-melting peach (Prunus persica (L.) Batsch)
275
progeny. J Sci Food Agric 95:351-358
276
9. Acknowledgment
245 Tomás-Barberán FA, Gil MI, Cremin P, Waterhouse AL, Hess-Pierce B, Kader
277
AA.(2001) HPLC-DAD-ESIMS analysis of phenolic compounds in nectarines,
278
peaches, and plums. J Agric Food Chem 49:4748-4760. 279
10. Gil MI, Tomás-Barberan FA, Hess-Pierce B, Kader AA (2002) Antioxidant
280
capacities, phenolic compounds, carotenoids, and vitamin C contents of nectarine,
281
peach, and plum cultivars from California. J Agric Food Chem 50:4976-4982. 282
11. Vizzotto M, Cisneros-Zevallos L, Byrne DH (2007) Large variation found in the
283 5. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C (1996)
265
Antioxidant activity applying an improved ABTS radical cation decolorization
266
assay. Free Radical Biology Medicine 26:1231-1237
267 7. Abidi W, Jiménez S, Moreno MÁ, Gogorcena Y (2011) Evaluation of antioxidant
270
compounds and total sugar content in a nectarine [Prunus persica (L.) Batsch]
271
progeny. Int J Mol Sci 12:6919-6935
272 8. Abidi W, Cantín CM, Jiménez S, Giménez R, Moreno MÁ, Gogorcena Y (2015)
273
Influence of antioxidant compounds, total sugars and genetic background on the
274
chilling injury susceptibility of a non-melting peach (Prunus persica (L.) Batsch)
275
progeny. J Sci Food Agric 95:351-358
276 9. Tomás-Barberán FA, Gil MI, Cremin P, Waterhouse AL, Hess-Pierce B, Kader
277
AA.(2001) HPLC-DAD-ESIMS analysis of phenolic compounds in nectarines,
278
peaches, and plums. J Agric Food Chem 49:4748-4760. 279 10. Gil MI, Tomás-Barberan FA, Hess-Pierce B, Kader AA (2002) Antioxidant
280
capacities, phenolic compounds, carotenoids, and vitamin C contents of nectarine,
281
peach, and plum cultivars from California. J Agric Food Chem 50:4976-4982. 282 11. Vizzotto M, Cisneros-Zevallos L, Byrne DH (2007) Large variation found in the
283 12 phytochemical and antioxidant activity of peach and plum germplasm. J Am Soc
284
Hort Sci 132:334-340. 285
12. Cevallos-Casals BA, Byrne D, Okie WR, Cisneros-Zevallos L (2006) Selecting
286
new peach and plum genotypes rich in phenolic compounds and enhanced
287
functional properties. Food Chem 96:273-280
288
13. Cantín CM, Moreno MA, Gogorcena Y (2009) Evaluation of the antioxidant
289
capacity, phenolic compounds, and vitamin C content of different peach and
290
nectarine (Prunus persica L. Batsch) breeding progenies. J Agric Food Chem
291
57:4586-4592
292
14. Legua P, Hernández F, Díaz-Mula HM, Valero D, Serrano M (2011) Quality,
293
bioactive compounds, and antioxidant activity of new flat-type peach and nectarine
294
cultivars: a comparative study. J Food Sci 76:C729-C735
295
15. Acknowledgment
245 Gao X, Ohlander M, Jeppsson N, Björk L, Trajkovski V. (2000) Changes in
296
antioxidant effects and their relationship to phytonutrients in fruits of Sea
297
buckthorn (Hippophae rhamnoides L.) during maturation. J Agric Food Chem
298
48:1485-1490
299
16. Loizzo MR, Tundis R, Bonesi M, Menichini F, Mastellone V, Avallone L,
300
Menichini F (2012) Radical scavenging, antioxidant and metal chelating activities
301
of Annona cherimola Mill. (cherimoya) peel and pulp in relation to their total
302
phenolic and total flavonoid contents. J Food Comp Anal 25:179-184
303
17. Puigventós L, Navarro M, Alechaga E, Núñez O, Saurina J, Hernández-Cassou S,
304
Puignou
L
(2015)
Determination
of
polyphenolic
profiles
by
liquid
305
chromatography-electrospray-tandem mass spectrometry for the authentication of
306
fruit extracts. Anal Bioanal Chem 407:597-608
307 12. Cevallos-Casals BA, Byrne D, Okie WR, Cisneros-Zevallos L (2006) Selecting
286
new peach and plum genotypes rich in phenolic compounds and enhanced
287
functional properties. Food Chem 96:273-280
288 13. Cantín CM, Moreno MA, Gogorcena Y (2009) Evaluation of the antioxidant
289
capacity, phenolic compounds, and vitamin C content of different peach and
290
nectarine (Prunus persica L. Batsch) breeding progenies. J Agric Food Chem
291
57:4586-4592
292 14. Legua P, Hernández F, Díaz-Mula HM, Valero D, Serrano M (2011) Quality,
293
bioactive compounds, and antioxidant activity of new flat-type peach and nectarine
294
cultivars: a comparative study. J Food Sci 76:C729-C735
295 15. Gao X, Ohlander M, Jeppsson N, Björk L, Trajkovski V. (2000) Changes in
296
antioxidant effects and their relationship to phytonutrients in fruits of Sea
297
buckthorn (Hippophae rhamnoides L.) during maturation. J Agric Food Chem
298
48:1485-1490
299 16. Loizzo MR, Tundis R, Bonesi M, Menichini F, Mastellone V, Avallone L,
300
Menichini F (2012) Radical scavenging, antioxidant and metal chelating activities
301
of Annona cherimola Mill. (cherimoya) peel and pulp in relation to their total
302
phenolic and total flavonoid contents. J Food Comp Anal 25:179-184
303 17. Puigventós L, Navarro M, Alechaga E, Núñez O, Saurina J, Hernández-Cassou S,
304
Puignou
L
(2015)
Determination
of
polyphenolic
profiles
by
liquid
305
chromatography-electrospray-tandem mass spectrometry for the authentication of
306
fruit extracts. Anal Bioanal Chem 407:597-608
307 13 18. Molyneux P (2004) The use of the stable free radical diphenylpicryl-hydrazyl
308
(DPPH) for estimating antioxidant activity. Songklanakarin J Sci Technol 26: 211–
309
219
310
19. Acknowledgment
245 Amin I, Zamaliah MM, Chin WF (2004) Total antioxidant activity and phenolic
311
content in selected vegetables. Food Chem 87:581–586. 312
20. Benzie IFF, Strain JJ (1996) The Ferric Reducing Ability of Plasma (FRAP) as a
313
Measure of ‘‘Antioxidant Power’’: The FRAP Assay. Analytical biochemistry
314
239:70-76. 315
21. Dinis TCP, Madeira VMC, Almeida LM (1994) Action of phenolic derivatives
316
(acetaminophen, salicylate and 5-aminosalicylate) as inhibitors of membrane lipid
317
peroxidation and peroxyl radical scavengers, Archives of Biochemistry and
318
Biophysics 315:161-169
319
22. Official methods of analysis, AOAC, Association of Official Analytical Chemists. 320
Edition 18: AOAC, 2005
321
23. Official methods of analysis, AOAC, Association of Official Analytical Chemists,
322
Edition 16, Arlighton VA, USA: AOAC, 1995
323
24. Tavarini S, Degl’Innocent E, Remorini D, Massai R, Guidi L (2008) Preliminary
324
characterisation of peach cultivars for their antioxidant capacity. Int J Food Sci
325
Technol 43:810-815
326
25. Alothman M, Bhat R, Karim AA (2009) Antioxidant capacity and phenolic content
327
of selected tropical fruits from Malaysia, extracted with different solvents. Food
328
Chem 115:785-788
329
26. Kchaou W, Abbès F, Blecker C, Attia H, Besbes S (2013) Effects of extraction
330
solvents on phenolic contents and antioxidant activities of Tunisian date varieties
331 18. Molyneux P (2004) The use of the stable free radical diphenylpicryl-hydrazyl
308 19. Amin I, Zamaliah MM, Chin WF (2004) Total antioxidant activity and phenolic
311
content in selected vegetables. Food Chem 87:581–586. 312 20. Benzie IFF, Strain JJ (1996) The Ferric Reducing Ability of Plasma (FRAP) as a
313
Measure of ‘‘Antioxidant Power’’: The FRAP Assay. Analytical biochemistry
314
239:70-76. 315 21. Dinis TCP, Madeira VMC, Almeida LM (1994) Action of phenolic derivatives
316
(acetaminophen, salicylate and 5-aminosalicylate) as inhibitors of membrane lipid
317
peroxidation and peroxyl radical scavengers, Archives of Biochemistry and
318
Biophysics 315:161-169
319 22. Official methods of analysis, AOAC, Association of Official Analytical Chemists. 320
Edition 18: AOAC, 2005
321 22. Official methods of analysis, AOAC, Association of Official Analytical Chemists. 320
Edi i
18 AOAC 2005
321 23. Official methods of analysis, AOAC, Association of Official Analytical Chemists,
322
Edition 16, Arlighton VA, USA: AOAC, 1995
323 23. Official methods of analysis, AOAC, Association of Official Analytical Chemists,
322
Edition 16, Arlighton VA, USA: AOAC, 1995
323 24. Acknowledgment
245 Tavarini S, Degl’Innocent E, Remorini D, Massai R, Guidi L (2008) Preliminary
324
characterisation of peach cultivars for their antioxidant capacity. Int J Food Sci
325
Technol 43:810-815
326 25. Alothman M, Bhat R, Karim AA (2009) Antioxidant capacity and phenolic content
327
of selected tropical fruits from Malaysia, extracted with different solvents. Food
328
Chem 115:785-788
329 25. Alothman M, Bhat R, Karim AA (2009) Antioxidant capacity and phenolic content
327
of selected tropical fruits from Malaysia, extracted with different solvents. Food
328
Chem 115:785-788
329 26. Kchaou W, Abbès F, Blecker C, Attia H, Besbes S (2013) Effects of extraction
330
solvents on phenolic contents and antioxidant activities of Tunisian date varieties
331 26. Kchaou W, Abbès F, Blecker C, Attia H, Besbes S (2013) Effects of extraction
330
solvents on phenolic contents and antioxidant activities of Tunisian date varieties
331 14 (Phoenix dactylifera L.). Ind Crops Produ 45:262-269
332
27. Di Vaio C, Graziani G, Marra L, Cascone A, Ritieni A (2008) Antioxidant
333
capacities, carotenoids and polyphenols evaluation of fresh and refrigerated peach
334
and nectarine cultivars from Italy. Eur Food Res Technol 227:1225–1231
335
28. Edge R, McGarvey DJ, Truscott TG (1997) The carotenoids as anti-oxidants--a
336
review. J Photochem Photobiol B 41:189-200
337
29. Campbell OE, Padilla-Zakour OI (2013) Phenolic and carotenoid composition of
338
canned peaches (Prunus persica) and apricots (Prunus armeniaca) as affected by
339
variety and peeling, Food Res Int 54:448-455
340
30. Hong Y, Lin S, Jiang Y, Ashraf M (2008) Variation in contents of total phenolics
341
and flavonoids and antioxidant activities in the leaves of 11 Eriobotrya species. 342
Plant Foods Hum Nutr 63:200-204
343
344
Table 1 Total phenols, flavonoids and carotenoids content in Prunus persica var. platycarpa fruits. 345
Phytochemicals
Pulp
Peel
Seed
Phenols a
921.8 ± 2.5
448.6 ± 3.5
111.3 ± 1.5
Flavonoids b
726.5 ± 8.2
231.9 ± 2.0
76.8 ± 1.1
Carotenoids c
61.9 ± 1.8
344.7 ± 1.6
109.3 ± 1.7
Values represent means (n= 3) ± S.D. amg chlorogenic acid equivalents/100 g FW; bmg quercetin equivalents/100 g
346
FW; cmg β-carotene equivalents/100 g FW. Acknowledgment
245 347
348
349
Table 2 Phenolic compounds present in ethanolic extract obtained from fresh pulp peach samples
350
Compound
Mean (mg/kg extract)
Gallic acid
1.654 ± 0.9
Protocatechuic acid
0.190 ± 0.06
Protocatechualdehyde
0.020 ± 0.04
Chlorogenic acid
15.029 ± 1.3
p-Coumaric acid
0.153 ± 0.09
Ferulic acid
0.226 ± 0.08
Values represent means (n= 3) ± S.D. 351
352
353
354 (Phoenix dactylifera L.). Ind Crops Produ 45:262-269
332 27. Di Vaio C, Graziani G, Marra L, Cascone A, Ritieni A (2008) Antioxidant
333
capacities, carotenoids and polyphenols evaluation of fresh and refrigerated peach
334
and nectarine cultivars from Italy. Eur Food Res Technol 227:1225–1231
335 variety and peeling, Food Res Int 54:448-455
340
30. Hong Y, Lin S, Jiang Y, Ashraf M (2008) Variation in contents of total phenolics
341
and flavonoids and antioxidant activities in the leaves of 11 Eriobotrya species. 342
Plant Foods Hum Nutr 63:200-204
343
344
Table 1 Total phenols, flavonoids and carotenoids content in Prunus persica var. platycarpa fruits. 345
Phytochemicals
Pulp
Peel
Seed
Phenols a
921.8 ± 2.5
448.6 ± 3.5
111.3 ± 1.5
Flavonoids b
726.5 ± 8.2
231.9 ± 2.0
76.8 ± 1.1
Carotenoids c
61.9 ± 1.8
344.7 ± 1.6
109.3 ± 1.7
Values represent means (n= 3) ± S.D. amg chlorogenic acid equivalents/100 g FW; bmg quercetin equivalents/100 g
346
FW; cmg β-carotene equivalents/100 g FW. 347
348
349
Table 2 Phenolic compounds present in ethanolic extract obtained from fresh pulp peach samples
350 15 Table 3 Radical scavenging and antioxidant capacities of Prunus persica var. platycarpa extracts
356
Sample
DPPH
(IC50µg/mL)
ABTS
(TEAC value)
β-Carotene bleaching test
(IC50µg/mL)
FRAP
(µM Fe(II)/g)
Fe-Chelating activity
(IC50µg/mL)
30 min
60 min
Pulp
12.0 ± 1.9**
6.8 ± 0.6**
2.7 ± 0.4**
2.2 ± 0.2**
30.2 ± 1.5**
2.9 ± 0.5**
Peel
45.3 ± 2.7**
8.4 ± 1.0**
9.2 ± 0.8**
5.2 ± 0.8**
78.9 ± 2.9**
8.7 ± 0.9**
Seed
85.3 ± 3.4**
11.7 ± 1.4**
15.7 ± 1.0**
12.3 ± 1.0**
130.5 ± 3.9**
18.8 ± 1.7**
Propyl gallatea
-
-
1.0 ± 0.01
1.0 ± 0.01
Ascorbic acida
2.0 ± 0.01
0.96 ± 0.03
-
-
BHTa
63.2 ± 4.5
1.3 ± 0.05
Data are given as media ± S.D. Acknowledgment
245 (n = 3); DPPH Radical Scavenging Activity Assay; Antioxidant Capacity Determined by Radical
357
Cation (ABTS+), β-Carotene bleaching test, FRAP Ferric ion reducing antioxidant power, ; a Propyl gallate, ascorbic acid and
358
BHTwere used as positive control. Differences within and between groups were evaluated by one-way analysis of variance test ***
359
P< 0.0001 followed by a multicomparison Dunnett’s test: ** P< 0.01 compared with the positive controls. 360
361
362
363
Table 4 Nutritional analysis and minerals in pulp from Tabacchiera peach
364
Nutritional constituents
Content (%)
Moisture
84.76
Ash
0.43
Fat
0.21
Protein
0.68
Fiber
1.78
Carbohydrates
12.14
Energy
53 Kcal/100g
Minerals
Content (mg/100 g)
Sodium
18
Potassium
37
Zinc
0.64
Copper
0.14
Iron
1.40
365
366
367
368
Figure 1. LC-ESI-MS/MS chromatogram of polyphenols found in Prunus persica var. platycarpa pulp
369 Table 3 Radical scavenging and antioxidant capacities of Prunus persica var. platycarpa extracts
356
Sample
DPPH
(IC50µg/mL)
ABTS
(TEAC value)
β-Carotene bleaching test
(IC50µg/mL)
FRAP
(µM Fe(II)/g)
Fe-Chelating activity
(IC50µg/mL)
30 min
60 min
Pulp
12.0 ± 1.9**
6.8 ± 0.6**
2.7 ± 0.4**
2.2 ± 0.2**
30.2 ± 1.5**
2.9 ± 0.5**
Peel
45.3 ± 2.7**
8.4 ± 1.0**
9.2 ± 0.8**
5.2 ± 0.8**
78.9 ± 2.9**
8.7 ± 0.9**
Seed
85.3 ± 3.4**
11.7 ± 1.4**
15.7 ± 1.0**
12.3 ± 1.0**
130.5 ± 3.9**
18.8 ± 1.7**
Propyl gallatea
-
-
1.0 ± 0.01
1.0 ± 0.01
Ascorbic acida
2.0 ± 0.01
0.96 ± 0.03
-
-
BHTa
63.2 ± 4.5
1.3 ± 0.05
Data are given as media ± S.D. (n = 3); DPPH Radical Scavenging Activity Assay; Antioxidant Capacity Determined by Radical
357
Cation (ABTS+), β-Carotene bleaching test, FRAP Ferric ion reducing antioxidant power, ; a Propyl gallate, ascorbic acid and
358
BHTwere used as positive control. Differences within and between groups were evaluated by one-way analysis of variance test ***
359
P< 0.0001 followed by a multicomparison Dunnett’s test: ** P< 0.01 compared with the positive controls. 360
361
362
363
Table 4 Nutritional analysis and minerals in pulp from Tabacchiera peach
364
Nutritional constituents
Content (%)
Moisture
84.76
Ash
0.43
Fat
0.21
Protein
0.68
Fiber
1.78
Carbohydrates
12.14
Energy
53 Kcal/100g
Minerals
Content (mg/100 g)
Sodium
18
Potassium
37
Zinc
0.64
Copper
0.14
Iron
1.40
365
366 356 Table 3 Radical scavenging and antioxidant capacities of Prunus persica var. Acknowledgment
245 platycarpa extracts 363
Table 4 Nutritional analysis and minerals in pulp from Tabacchiera peach
364
Nutritional constituents
Content (%)
Moisture
84.76
Ash
0.43
Fat
0.21
Protein
0.68
Fiber
1.78
Carbohydrates
12.14
Energy
53 Kcal/100g
Minerals
Content (mg/100 g)
Sodium
18
Potassium
37
Zinc
0.64
Copper
0.14
Iron
1.40
365 367
368
Figure 1. LC-ESI-MS/MS chromatogram of polyphenols found in Prunus persica var. platycarpa pulp
369 Figure 1. LC-ESI-MS/MS chromatogram of polyphenols found in Prunus persica var. platycarpa pulp
369 Figure 1. LC-ESI-MS/MS chromatogram of polyphenols found in Prunus persica var. platycarpa pulp 16
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https://openalex.org/W4367394483
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https://link.springer.com/content/pdf/10.1007/s11831-023-09930-z.pdf
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English
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Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis Review
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Archives of computational methods in engineering
| 2,023
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cc-by
| 24,839
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Abstract The machine learning (ML) paradigm has gained much popularity today. Its algorithmic models are employed in every field,
such as natural language processing, pattern recognition, object detection, image recognition, earth observation and many
other research areas. In fact, machine learning technologies and their inevitable impact suffice in many technological trans-
formation agendas currently being propagated by many nations, for which the already yielded benefits are outstanding. From
a regional perspective, several studies have shown that machine learning technology can help address some of Africa’s most
pervasive problems, such as poverty alleviation, improving education, delivering quality healthcare services, and addressing
sustainability challenges like food security and climate change. In this state-of-the-art paper, a critical bibliometric analy-
sis study is conducted, coupled with an extensive literature survey on recent developments and associated applications in
machine learning research with a perspective on Africa. The presented bibliometric analysis study consists of 2761 machine
learning-related documents, of which 89% were articles with at least 482 citations published in 903 journals during the past
three decades. Furthermore, the collated documents were retrieved from the Science Citation Index EXPANDED, compris-
ing research publications from 54 African countries between 1993 and 2021. The bibliometric study shows the visualization
of the current landscape and future trends in machine learning research and its application to facilitate future collaborative
research and knowledge exchange among authors from different research institutions scattered across the African continent. Abbreviations
CU, Egypt
Cairo University, Egypt
UKZN, South Africa
University of KwaZulu-Natal,
South Africa
UCT, South Africa
University of Cape Town, South
Africa
MansU, Egypt
Mansoura University, Egypt
ZU, Egypt
Zagazig University, Egypt
UW, South Africa
University of Witwatersrand,
South Africa
BU, Egypt
Benha University, Egypt
MU, Egypt
Menoufia University, Egypt
UP, South Africa
University of Pretoria, South
Africa
ASU, Egypt
Ain Shams University, Egypt
UJ, South Africa
University of Johannesburg, South
Africa
UWC, South Africa
University of Western Cape, South
Africa
SU, South Africa
Stellenbosch University, South
Africa
HU, Egypt
Helwan University, Egypt
TU, Egypt
Tanta University, Egypt
UTEM, Tunisia
University of Tunis El Manar,
Tunisia
AU, Egypt
Alexandria University, Egypt
UT, Tunisia
University of Tunis, Tunisia
* Absalom E. Ezugwu
absalom.ezugwu@nwu.ac.za
* Yuh‑Shan Ho
ysho@asia.edu.tw
Olaide N. Oyelade
olaide_oyelade@yahoo.com
Abiodun M. Ikotun
biodunikotun@gmail.com
Jeffery O. Machine Learning Research Trends in Africa: A 30 Years Overview
with Bibliometric Analysis Review Received: 18 February 2023 / Accepted: 19 April 2023 / Published online: 29 April 2023
© The Author(s) 2023 Abstract Agushaka
jefshak@gmail.com
1
Unit for Data Science and Computing, North-West
University, 11 Hoffman Street, Potchefstroom 2520,
South Africa
2
Department of Computer Science, Faculty of Physical
Sciences, Ahmadu Bello University, Zaria, Nigeria
3
Trend Research Centre, Asia University, No. 500, Lioufeng
RoadWufeng, Taichung 41354, Taiwan Abbreviations
CU, Egypt
Cairo University, Egypt
UKZN, South Africa
University of KwaZulu-Natal,
South Africa Archives of Computational Methods in Engineering (2023) 30:4177–4207
https://doi.org/10.1007/s11831-023-09930-z Archives of Computational Methods in Engineering (2023) 30:4177–4207
https://doi.org/10.1007/s11831-023-09930-z Archives of Computational Methods in Engineering (2023) 30:4177–4207
https://doi.org/10.1007/s11831-023-09930-z REVIEW ARTICLE Abbreviations Abbreviations
CU, Egypt
UKZN, South Africa Cairo University, Egypt
University of KwaZulu-Natal,
South Africa 1
Unit for Data Science and Computing, North-West
University, 11 Hoffman Street, Potchefstroom 2520,
South Africa 2
Department of Computer Science, Faculty of Physical
Sciences, Ahmadu Bello University, Zaria, Nigeria 3
Trend Research Centre, Asia University, No. 500, Lioufeng
RoadWufeng, Taichung 41354, Taiwan (0121 3456789)
3 4178 A. E. Ezugwu et al. Suez Canal University, Egypt
University of Carthage, Tunisia SCU, Egypt
UC, Tunisia Suez Canal University, Egypt
University of Carthage, Tunisia Suez Canal University, Egypt
University of Carthage, Tunisia SCU, Egypt
UC, Tunisia 1.1 A Brief Background of ML and Its Evolution
from AI‑ML‑DL The drive to replace human capability with machine
intelligence led to the evolvement of various methods of
AI, which is now defined as the science and engineering
of achieving machine intelligence as often exhibited in
the form of computer programs and often in controlling
and receiving signals from hardware [1]. An upsurge of
research in AI has resulted in the outstanding performance
of machines that now perform complex tasks intelligibly. Several AI paradigms have now evolved, including natu-
ral language processing (NLP), constraint satisfaction,
machine learning, distributed AI, machine reasoning,
data mining, expert systems, case-based reasoning (CBR),
knowledge-representation, programming, robotics, belief
revision, neural network, theorem proving, theory com-
putation, logic, and genetic algorithm. This evolvement
follows a historical trend, as shown in Fig. 1, which dem-
onstrates a continuous improvement of methods and algo-
rithms to increase accuracy in the exhibition of machine
intelligence. This timeline shows that research in AI
advanced through some challenging exploits until around
the 1970s, when ML conceptualization began to mani-
fest interesting results and performances. Interestingly,
with these advances came the challenge of addressing
ethical issues so that AI-driven systems are not allowed
to infringe on human rights, nor will the moral status of Fig. 1 Evolvement of AI from dream to reality 1 Introduction The evolvement of the development and use of computers
in intelligently solving problems predates the creation and
testing of the Turing machine in 1950. Such systems aim
to demonstrate their suitability in interfacing with human
beings in a manner that shows a high level of intelligence
compared to humans. However, this new set of systems
was earlier motivated by the design of 1940s systems such
as ENIAC, which aimed to emulate humans in promoting
learning and thinking. The outcome of this led to computer
game applications competitively gaming with humans. Furthermore, this motivated the design of perceptron,
which accumulated into a broader design of machine learn-
ing used for classification purposes. Further research and
applications in statistics have promoted machine learning
so that the intersection of statistics and computer science
has advanced studies on artificial intelligence (AI). In this
section, we organize the discussion to provide background
knowledge on AI, ML and deep learning (DL). We provide
a summary of a multi-disciplinary approach to research on
ML to show recent methods and major application areas of
ML in addressing real-problems. We conclude this section
by providing a motivation for the bibliometric analysis and
highlighting the study's contribution. Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… 4179 such systems be compromised [2]. That notwithstanding,
the evolvement peaked from the basic Turing’s concept to
the current Industry 4.0 by connecting multi-disciplinary
approaches, including those from computer science but
also psychology, philosophy, neuroscience, biology, math-
ematics, sociology, linguistics, and other areas [3].i computational learning theory, neural networks, stochas-
tic modeling, and pattern recognition. The resulting algo-
rithms have demonstrated state-of-the-art performances
in email filters, NLP, pattern recognition, computer vision
and autonomous vehicle design. Deep learning (DL) belongs to the broader family of ML
and can analyse data intelligently through transformations,
graph technologies and representation patterns. Derived
from the simulation of the human brain from the basic Artifi-
cial Neural Networks (ANN), convolutional neural networks
are designed in a manner that outperforms traditional ML
algorithms. The approach leverages increasingly available
training data from sensors, the Internet of Things (IoT),
surveillance systems, intrusion detection system, cyber-
security, mobile, business, social media, health, and other
devices. These data, often in an unstructured format, are
analyzed and automated for identification of features leading
to either classification or regression analysis [4]. 1.2 A Brief Background of Multi‑disciplinary ML
Research Contribution from Different Scientists
Across Major African Universities In related work, performance enhance-
ment techniques such as data augmentation in improving DL
models have been researched by Oyelade & Ezugwu [20]
using the CNN model to detect architectural distortion in
breast images. Concerning the challenge of deploying ML
methods to address COVID-19, studies have been conducted
using DL architectures to detect and classify the disease in
chest x-ray samples [21]. Similarly, the need to harness the
deployment of Internet of Things (IoT) devices to curb the
spread of COVID-19 using ML algorithms has been advo-
cated [22]. On the issue of security, an investigative study
has been carried out assessing the level of deployment of
AI and its associated ML methods in curbing terrorism and
insurgency in Nigeria [23]. The use of artificial neural net-
work (ANN) and logistic regression (LR) models have also
been used to predict floods in susceptible areas in Nigeria
[24]. Regarding finance and the digital economy, AI-based
methods have been recommended for innovation and policy-
making [25]. Studies in the ML application from Morocco cut across
medicine, solar power and climate. In particular, deep
learning models, CNN, have been proposed for detecting
and classifying breast cancer cases using histopathology
samples [8]. The RNN variant of a DL model has been
adapted to address the problem of daily streamflow over the
Ait Ouchene watershed (AIO). The study used the Short-
Term Long Memory (LSTM) network, a type of RNN, to
achieve this simulation [9]. Research applying ML methods
in the remote sensing field using a popular algorithm such
as support vector machines (SVM) in mapping Souk Arbaa
Sahel in a lithological manner has been reported by Bachri
et al. [10]. In the financial sector, researchers have investi-
gated the use of ML in revolutionizing the banking ecosys-
tem for precise credit scoring, regulation and operational
approaches [11]. In another study, the country's location
motivates research on using ML to harness solar power in
grid management at power plants. Both ML algorithms and
DL have been drafted for predicting solar radiation using
models such as ANN, multi-layer perceptron (MLP), back
propagation neural network (BPNN), deep neural network
(DNN), and LSTM [12]. Researchers in Uganda have also employed AI in health-
care management by observing the performance of an AI
algorithm called Skin Image Search, applied to dermatologi-
cal tasks. 1.2 A Brief Background of Multi‑disciplinary ML
Research Contribution from Different Scientists
Across Major African Universities can identify students at-risk of dropping out of school and
isolate the causative of this challenge [13]. A novel hybrid
DL model capable of detecting features supportive of face
recognition has been proposed to apply the trained model to
build a face clustering system based on density-based spa-
tial clustering of applications with noise (DBSCAN) [14]. Similarly, generative adversarial networks (GANs), a com-
position of DL models adversarial positioned for generative
purposes, have been investigated for kinship face synthe-
sis [15]. Also, identification systems have been built using
CNN by extracting input from video files to apply vision
surveillance [16]. The contextualization of optical character
recognition (OCR) systems to solve local problems has been
researched using CNN, DNN and the SVM classifier to rec-
ognise different classes accurately [17]. Another interesting
application of DL is in the task of Automatic License Plate
Detection and Recognition (ALPR) for Egyptian license
plates (ELP) [18]. There is widespread research using ML to address contextual
problems across African countries. In most cases, this is
promoted by a local conference called Indaba, which pro-
motes the application of DL and ML to help ensure that
knowledge, capacity, recognizing excellence in ML research,
and application are well harnessed to develop the conti-
nent. In this section, a summary of studies on ML and DL
in Africa is reviewed to demonstrate the level of involve-
ment of the researchers in research on ML. It is reported
that AI-based research is improving communities across the
Sub-Saharan Africa (SSA) regions. In Kenya, it is being
applied to aid health worker–patient interaction to detect
blinding eye disorders, and in Egypt, in aiding automated
decision-making systems for health-care support. In South
Africa, it is aiding drug prescription, and with a multinomial
logistic classifier-based application, it is being applied to
human resource planning. ML-trained models are primarily
deployed in medicine in Nigeria, and an example is their use
in the diagnosis of birth asphyxia and identification of fake
drugs. Other cases are the use of ML to diagnose diabetic
retinopathy in Zambia and the diagnosis of pulmonary tuber-
culosis in Tanzania [7]. The ML and DL models have been used primarily in
Nigeria's medicine, security and climate issues. For instance,
the use of CNN in investigating a solution to the classifica-
tion problem of breast cancer using digital mammograms has
been reported [19]. 1 Introduction The DL
has been widely adapted to address application problems,
including audio and speech, visual data, and NLP. Design
patterns for DL have appeared as Convolutional neural net-
work (CNN)—the most popular and widely used of DL
networks—recursive neural networks (RvNNs), recurrent
neural networks (RNNs), Boltzmann machine (BM), and
auto-encoders (AE). While the RNN is often applied to text
or signal processing, RvNN, which uses a hierarchical struc-
ture, can classify outputs utilizing compositional vectors [5]. Results obtained from different studies showed that DL had
obtained good outstanding performance across a variety
of applications [6]. This has now motivated its integration
into reinforcement learning to achieve Deep Reinforcement
Learning (DRL). Considering this evolvement and perfor-
mance of ML and DL, we focus the next sub-section on
presenting brief research and application of methods in this
field among African researchers. ,
gy,
g
,
[ ]
The field of machine learning (ML) branched out of
AI and is focused on evolving computational methods
and algorithms learning and building learning machines
to leverage an object's natural pattern of learning fea-
tures. ML has been reputed to advance AI dramatically
because of its problem-solving approach of recognizing
patterns in domain-specific datasets to gather artificial
experience from the observed data. This follows a data
extraction pipeline through training and prediction using
new data. This learning, shown in Fig. 2, is approached
from and has evolved into different perspectives, includ-
ing popular supervised, unsupervised, semi-supervised,
and reinforcement learning. Over the years, algorithms
have been designed and further evolved in each aspect
of learning. These algorithms address real-life problems
involving classification and regression problems using
supervised learning methods, clustering and association
using unsupervised learning methods, and the problem
of understanding and manoeuvring an environment using
reinforcement learning. The learning process in ML uses
both symbolic and numeric methods as incorporated into
some of its popular algorithms such as linear regression,
nearest neighbor, Gaussian Naive Bayes, decision trees,
support vector machine (SVM), random forest, K-Means,
density-based spatial clustering of applications with noise
(DBSCAN), balanced iterative reducing and clustering
(BIRCH), temporal difference (TD), Q-Learning, and
deep adversarial networks. The design of these algorithms
includes a broad domain of statistics, genetic algorithms, Fig. 2 Evolvement of machine learning evolved from supervised learning to reinforcement Learning Fig. 2 Evolvement of machine learning evolved from supervised learning to reinforcement Learning 1 3 3 4180 A. E. Ezugwu et al. 1.2 A Brief Background of Multi‑disciplinary ML
Research Contribution from Different Scientists
Across Major African Universities The algorithm was trained using a local dataset
from The Medical Concierge Group (TMCG) to diagnosti-
cally analyze and extract the gender, age and dermatological
diagnosis [26]. A researcher from Kenya confirmed that an
investment of US$74.5 million is being made to support the
use of ML models in healthcare [27]. In the same country,
DL architecture, namely the LSTM network, has been inves-
tigated for drought management by forecasting vegetation's
health [28]. In Egypt, research in ML has enjoyed application to
learner-ship, face recognition, visual surveillance, and
optical character recognition (OCR). In a study, the rate of
school-dropout has been investigated and predicted using
ML algorithms, specifically a Logistic classifier. The model 3 1 3 4181 Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… Research on the application of ML is widespread in
South Africa, with more consideration given to language
processing, medical image analysis, and astronomy. In
addition to using ML algorithms, DL and NLP have been
well-researched to aid development [29]. Generative model
GAN has been applied to enable automatic speech recog-
nition (ASR), improving the features of mismatched data
prior to decoding [30]. In another related work, the ASR
system has been researched by combining multi-style train-
ing (MTR) with deep neural network hidden Markov model
(DNN-HMM) [31]. The use of CNN in exploring classifi-
cation accuracy on SNR data has been reported by Andrew
et al. [32]. A study has been channeled to investigate the
role of loss functions in aiding the behavior of deep neural
network optimization purposes [33]. Feedforward neural net-
works have been used to study the space physics problem in
storm forecasting [34]. Optimizing hyperparameter issues
in embedding algorithms has been considered for improving
training word embeddings with speech-recognized data [35]. science. NLP methods have received wider consideration
and study for mainstreaming the use of local languages
across the continent. This includes translating the Yoruba
language to French, automation regarding the use of Swa-
hili, and automatic Arabic Diacritization. Other interesting
areas generating the application of ML algorithms on the
continent are optical communications and networking [36],
deployment of AI to software engineering problems [37],
and advancing medical research and appropriating clinical
artificial intelligence in check-listing research [38]. 1.4 Strong Motivation and Need for the Current
Employment of Bibliometric Analysis Study This study is motivated by the availability of large research
databases providing a considerable number of publications
and research outputs suitable for aiding the search required
for the study. This data availability has helped to guide the
decision on the need to use bibliometric techniques in draw-
ing out important findings from the data collected from the
scientific databases. Bibliometrics is used to facilitate the
examination of large bodies of knowledge within and across
disciplines. The use of bibliometric techniques in this study
will support the aim of the study in identifying hidden but
useful patterns capable of illustrating the research trend on
ML and DL by researchers in African universities. This
study intends to leverage the presentational nature of bib-
liometric analysis to allow policymakers to easily discover
interesting research works in ML on the continent to aid
their decision-making process. All these clear indications show that there is now a strong
increase in research in ML, including its associated sub-
fields of DL and NLP in African universities, with most
applications aimed at healthcare, climate, and security. In
the following sub-section, we summarize the major appli-
cation areas of ML in the continent. This is necessary to
give perspective to the current state of research on ML in
the domain and to serve as a motivation for enabling future
research on ML. 2 Methodology To do bibliometric analyses, data were extracted from the
online databases of the Science Citation Index Expanded
(SCI-EXPANDED) (data extracted on 10 October 2022). Quotation marks (“”) and Boolean operator “or” were used,
which ensured the appearance of at least one search keyword
in terms of TOPIC (title, abstract, author keywords, and Key-
words Plus) from 1991 to 2021 [73]. The search keywords:
“machine learning”, “machining learning”, “machine learn-
able”, “machine learn”, “machine learns”, “machine learn-
ers”, “machine learner”, “machine learnings”, “machines
learning”, “machine learnt”, “machine learned”, and
“machines learn” that were found in SCI-EXPANDED
were considered. To have accurate analysis results, some
terms missed spaces were found and employed including
“machine learningmethods”, “machine learningmetrics”,
“machine learningbased”, “machine learningalgorithm”,
and “machine learningclassifiers”. Furthermore, related
keywords which were misspelt such as “machine learnig”,
“machine learnin”, “maching learning”, and “machin learn-
ing” were also used as search keywords. African countries
including “Algeria”, “Angola”, “Benin”, “Botswana”, “Bur-
kina Faso”, “Burundi”, “Cameroon”, “Cape Verde”, “Cent
Afr Republ”, “Chad”, “Comoros”, “Dem Rep Congo”,
“Rep Congo”, “Cote Ivoire”, “Djibouti”, “Egypt”, “Equat
Guinea”, “Eritrea”, “Eswatini”, “Ethiopia”, “Gabon”,
“Gambia”, “Ghana”, “Guinea”, “Guinea Bissau”, “Kenya”,
“Lesotho”, “Liberia”, “Libya”, “Madagascar”, “Malawi”,
“Mali”, “Mauritania”, “Mauritius”, “Morocco”, “Mozam-
bique”, “Namibia”, “Niger”, “Nigeria”, “Rwanda”, “Sao
Tome & Prin”, “Senegal”, “Seychelles”, “Sierra Leone”,
“Somalia”, “South Africa”, “South Sudan”, “Sudan”, “Tan-
zania”, “Togo”, “Tunisia”, “Uganda”, “Zambia”, and “Zim-
babwe” were also searched in terms of the country (CU). A
total of 2770 documents, including 2477 articles, were found
in SCI-EXPANDED from 1993 to 2021. In summary, a The entire record and the annual number of citations for
each document were checked and placed into Excel Micro-
soft 365, and additional coding was manually executed. The
functions in Excel Microsoft 365, for example, Concatenate,
Counta, Freeze Panes, Len, Match, Proper, Rank, Replace,
Sort, Sum, and Vlookup, were applied. The journal impact
factors (IF2021) were based on the Journal Citation Reports
(JCR) issued in 2021. In the SCI-EXPANDED database, the corresponding
author is designated as the “reprint author”; “corresponding
author” will continue to be the primary term rather than the
reprinted author [48]. In single-author articles where author-
ship is not specified, the single author is considered the first
and corresponding author [49]. Likewise, in single-institu-
tional articles, institutions are classified as first-author and
corresponding author institutions [50]. All corresponding
authors, institutions, and countries were considered in multi-
ple corresponding author articles. 1.3 A Brief Highlight on the Significance of ML
Application Within the Continent Interestingly, we found that the proposed method will
allow for discovering leading contributors to ML research. This method will undoubtedly enable this study to uncover
new directions and themes for future research in ML. As
observed in subsequent sections, bibliometric analysis ena-
bled us to evaluate the impact of publications by regions,
research institutions and authors and obtain relevant sci-
entific information on a topic. The quantitative, scalable
and transparent approach of bibliometric analysis fits them
closely as informetrics and scientometrics. In the next sub-
section, the approach to applying the bibliometric techniques
in this study to achieve the aim of the study is outlined. Findings from the reviewed process detailed in the study
showed that the fields of medicine and healthcare delivery
management, agricultural studies, security and surveillance,
natural language modelling and process and many others
had benefited immensely from the application of ML on
the continent. These ML applications include research on
DL in cyber security intrusion detection and, likewise, the
detection of DDoS in cloud computing. Disease detection
in plants and crops has also been investigated using ML
algorithms with an example of tomato disease detection. The
sugarcane leaf nitrogen concentration estimation has been
reported to map irrigated areas using Google Earth Engine. Several sub-fields of medicine have received research atten-
tion in promoting healthcare delivery and improving dis-
ease detection and management. Examples of ML methods
in this aspect are automatic sleep stage classification, face
mask detection in the era of the COVID-19 pandemic, pro-
tein sequence classification, and temporal gene expression
data. Several studies have also been applied to study the
design of optimization and clustering methods to solve dif-
ficult optimization problems in engineering, medicine and The following highlights are the major contributions of
this study: • We first apply the analysis of research publications to
uncover the developments with ML in African universi-
ties.i • The study identifies core research in ML and DL and
authors and their relationship by covering all the publica-
tions from African researchers. • We analyze the research status and frontier directions and
predict the future of ML research in Africa. 1 3 4182 A. E. Ezugwu et al. • An analysis of entities such as authors, institutions or
countries in African universities is compared to their
research outputs. PRISMA flow diagram is shown in Fig. 3. 1.3 A Brief Highlight on the Significance of ML
Application Within the Continent It visually depicts
the review process of finding published data on the topic
and the authors decisions’ on whether to include it in the
review. This study selected only articles from Science Cita-
tion Index Expanded (SCI-EXPANDED) with keywords as
explained earlier. Quotation marks (“”) and Boolean opera-
tor “or” were used, which ensured the appearance of at least
one search keyword in terms of TOPIC (title, abstract, author
keywords, and Keywords Plus) from 1991 to 2021. The remaining part of the paper is organized as follows:
Sect. 2 describes the data collection process and the method-
ology used in this paper. Extensive bibliometric analysis is
performed in Sect. 3, and this section covers the presentation
of significant narratives and a detailed discussion of findings
from the conducted study analysis. We provide a detailed
literature review of the last few years in Sect. 4. Section 5
concludes the paper by summarizing the study’s findings of
30 years of ML-dedicated research efforts in several univer-
sities across the African continent. Keywords Plus provides additional search terms extracted
from the titles of articles cited by authors in their bibliogra-
phies and footnotes in the Institute of Science Information
(ISI) (now Clarivate Analytics) database. It substantially
augments title-word and author-keyword indexing [39]. It
was noticed that documents only searched out by Keywords
Plus are irrelevant to the search topic [40]. Ho’s group first
proposed the “front page” as a filter to improve bias by
using the data from SCI-EXPANDED directly, including
the article title, abstract, and author keywords [41]. It has
been pointed out that a significant difference was found by
using the ‘front page’ as a filter in bibliometric research in
wide journals classified in SCI-EXPANDED, for example,
Frontiers in Pharmacology [42], Chinese Medical Journal
[43], Environmental Science and Pollution Research [44],
Water [45], Science of the Total Environment [46], and Jour-
nal of Foot and Ankle Surgery [47]. The ‘front page’ filter
can avoid introducing unrelated publications for bibliometric
analysis. Six publication indicators are used to assess the publica-
tion performance of countries and institutions [52, 53]: TP:
total number of articles; IP: number of single-country (IPC)
or single-institution articles (IPI); CP: number of interna-
tionally collaborative articles (CPC) or inter-institutionally
collaborative articles (CPC); FP: number of first-author
articles; RP: number of corresponding-author articles; and
SP: number of single-author articles. Moreover, publications
were assessed using the following citation indicators: Cyear:
the number of citations from Web of Science Core Collec-
tion in a year (e.g. C2021 describes citation count in 2021)
[48]; and TCyear: the total citations from Web of Science
Core Collection received since publication year till the end
of the most recent year (2021 in this study, TC2021) [53, 54]. Six citation indicators (CPP2021) related to the six publi-
cation indicators were also applied to evaluate the publica-
tion's impact on countries and institutions [55]: TP-CPP2021:
the total TC2021 of all articles per the total number of articles
(TP); IP-CPP2021: the total TC2021 of all single-country arti-
cles per the number of single-country articles (IPC-CPP2021)
or single-institutions articles per the number of single-insti-
tutions articles (IPI-CPP2021); CP-CPP2021: the total TC2021 of all internationally per the number of internationally col-
laborative articles (CPC-CPP2021) or inter-institutionally
collaborative articles per inter-institutionally collaborative
articles (CPI-CPP2021); FP-CPP2021: the total TC2021 of all
first-author articles per the number of first-author articles
(FP); RP-CPP2021: the total TC2021 of all corresponding-
author articles per the number of corresponding-author
articles (RP); and SP-CPP2021: the total TC2021 of all single-
author articles per the number of single-author articles (SP). 2 Methodology For more accurate analysis
results, affiliations were checked and reclassified. Author
affiliations in England, Scotland, North Ireland (Northern
Ireland), and Wales were regrouped under the heading of the
United Kingdom (UK) [51]. Furthermore, SCI-EXPANDED
has the article of the corresponding author. Only the address
without the name of the affiliations is found, and the address
is changed to the name of the affiliations. 1 3 3 4183 Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… g
y
Fig. 3 A summary of the data
extraction and screening process
from SCI-EXPANDED Fig. 3 A summary of the data
extraction and screening process
from SCI-EXPANDED 3.1 Document Type and Language of Publication Six citation indicators (CPP2021) related to the six publi-
cation indicators were also applied to evaluate the publica-
tion's impact on countries and institutions [55]: TP-CPP2021:
the total TC2021 of all articles per the total number of articles
(TP); IP-CPP2021: the total TC2021 of all single-country arti-
cles per the number of single-country articles (IPC-CPP2021)
or single-institutions articles per the number of single-insti-
tutions articles (IPI-CPP2021); CP-CPP2021: the total TC2021 The characteristics of document type based on their CPPyear
and the average number of authors per publication (APP)
as basic document type information in a research topic
were proposed [56]. Recently, the median of the number
of authors was also applied to a research topic with a large
number of authors in a document [57]. Using the citation
indicators TCyear and CPPyear has advantages compared 1 3 4184 A. E. Ezugwu et al. and Eissa from Cairo University in Egypt first mentioned
“machine learning” as the authors’ keywords in Case-based
reasoning algorithms applied in a medical acquisition tool
[69]. The number of articles increased slightly from 14 in
2010 to 98 in 2017 (Fig. 4). After that, a sharply rising trend
reached 1035 articles in 2021. The highest CPP2021 was 54
in 2013, which can be attributed to the article entitled Mul-
tiobjective intelligent energy management for a microgrid
[70], ranking at the top in TC2021 with 402 (rank 3rd). to citation counts directly from the Web of Science Core
Collection because of their invariance and ensuring repro-
ducibility [58]. A total of 2761 machine learning-related
documents by authors affiliated with several institutions in
Africa published in the SCI-EXPANDED from 1993 to 2021
were found among 11 document types which are detailed in
Table 1. The majority were articles (89% of 2761 articles)
with an APP of 15 and a median of 4.0. The largest number of authors in an article is “Y Machine
learning risk prediction of mortality for patients undergo-
ing surgery with perioperative SARS-CoV-2: the COVID-
Surg mortality score” [59] published by 4,819 authors from
784 institutions in 71 countries including African coun-
tries: Egypt, Ethiopia, Gabon, Libya, Morocco, Nigeria,
South Africa, Sudan, and Zimbabwe. The document type
of reviews with 235 documents had the greatest CPP2021
value of 18, which was 1.6 times of articles. Five of the
top 12 most frequently cited documents were reviews by
Carleo et al. 3.3 Web of Science Categories and Journals African published machine learning-related articles in 903
journals were classified in 159 of the 178 Web of Science
categories in SCI-EXPANDED. Recently, the characteris-
tics of the Web of Science categories based on TP, APP,
CPP2021, and the number of journals in each category were
proposed [71]. Table 2 shows the top 12 productive Web
of Science categories with over 100 articles. A total of 906
articles (37% of 2468 articles) were published in the top
four productive categories: electrical and electronic engi-
neering containing 278 journals (385 articles; 20% of 2468
articles), information systems computer science containing
164 journals (439 articles; 18%), artificial intelligence com-
puter science containing 145 journals (334 articles; 14%),
and telecommunications containing 94 journals (308 arti-
cles; 12%). Comparing the top 12 productive categories,
articles published in the ‘interdisciplinary applications
computer science’ and ‘remote sensing’ categories had the
greatest CPP2021 of 15, respectively. Articles published in
the ‘information systems computer science category’ had a
lower CPP2021 of 8.9. Articles published in the category of
‘environmental sciences’ had the greatest APP of 6.6, while
articles in the category of ‘artificial intelligence computer
science’ had an APP of 3.5. The interaction of publication Web of Science document type of articles were further
analyzed as they included the entire research hypothesis,
methods and results. Only three non-English articles were
published by the French in Traitement du Signal [65, 66] and
Annales Des Télécommunications [67]. 3.1 Document Type and Language of Publication [60] (TC2021 = 426; rank 3rd), Merow et al. [61]
(TC2021 = 268; rank 6th), Nathan et al. [62] (TC2021 = 231;
rank 9th), Oussous et al. [63] (TC2021 = 206; rank 11th), and
Ben Taieb et al. [64] (TC2021 = 204; rank 12th). Table 1 Citations and authors
based on the document types TP total number of publications, AU number of authors, APP average number of authors per publication,
TC2021 total number of citations from Web of Science Core Collection since publication year to the end of
2021, CPP2021 average number of citations per publication (TC2021/TP) Table 2 Top 12 most productive
Web of Science categories with
TP > 100 3.2 Characteristics of Publication Outputs 4 Number of articles and
the average number of citations
per publication by year
1.0
0
25
6.0
18
7.0
0
0
11
12
0
6.7
20
11
10
2.0
23
40
10
29
54
40
37
32
37
22
16
10
2.1
0
10
20
30
40
50
60
0
200
400
600
800
1000
1200
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
2015
2016
2017
2018
2019
2020
2021
citations per publication
number of articles
Number of articles
Citations per publication
Year
Table 2 Top 12 most productive
Web of Science categories with
TP > 100
TP total number of publications, No. J number of journals in a category in 2021, APP average number of
authors per publication, CPP2021 average number of citations per publication (TC2021/TP)
Web of Science category
TP (%)
No. J
APP
CPP2021
Electrical and electronic engineering
485 (20)
278
4.3
13
Information systems computer science
439 (18)
164
4.4
8.9
Artificial intelligence computer science
334 (14)
145
3.5
14
Telecommunications
308 (12)
94
4.4
9.3
Environmental sciences
192 (7.8)
279
6.6
11
Multidisciplinary sciences
163 (6.6)
73
6.2
13
Interdisciplinary applications computer science
154 (6.2)
113
5.7
15
Multidisciplinary geosciences
142 (5.8)
202
5.9
12
Theory and methods computer science
138 (5.6)
110
4.3
11
Remote sensing
120 (4.9)
34
5.6
15
Imaging science and photographic technology
108 (4.4)
28
5.3
13
Energy and fuels
101 (4.1)
119
4.5
11 development among Web of Science categories is discussed
using Fig. 4, comprising the number of publications versus
the year of publication [72]. Figure 5 shows the development
trends of the top four Web of Science categories with more
than 300 articles. The first articles were published in 1993
d 1997 i th ‘i f
ti
t
t
i
’
d
‘electrical and electronic engineering’ category since 2014. The first article in the category of ‘telecommunications’
was found in 2015. It had a sharp increase since 2018 and
reached 139 articles in 2021, much higher than the 93 arti-
cles in the ‘artificial intelligence computer science category’. R
tl
th
h
t i ti
f th j
l b
d
th i
Fig. 3.2 Characteristics of Publication Outputs 4 Number of articles and
the average number of citations
per publication by year
1.0
0
25
6.0
18
7.0
0
0
11
12
0
6.7
20
11
10
2.0
23
40
10
29
54
40
37
32
37
22
16
10
2.1
0
10
20
30
40
50
60
0
200
400
600
800
1000
1200
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
2015
2016
2017
2018
2019
2020
2021
citations per publication
number of articles
Number of articles
Citations per publication
Year
Table 2 Top 12 most productive
Web of Science categories with
TP > 100
TP total number of publications, No. J number of journals in a category in 2021, APP average number of
authors per publication, CPP2021 average number of citations per publication (TC2021/TP)
Web of Science category
TP (%)
No. J
APP
CPP2021
Electrical and electronic engineering
485 (20)
278
4.3
13
Information systems computer science
439 (18)
164
4.4
8.9
Artificial intelligence computer science
334 (14)
145
3.5
14
Telecommunications
308 (12)
94
4.4
9.3
Environmental sciences
192 (7.8)
279
6.6
11
Multidisciplinary sciences
163 (6.6)
73
6.2
13
Interdisciplinary applications computer science
154 (6.2)
113
5.7
15
Multidisciplinary geosciences
142 (5.8)
202
5.9
12
Theory and methods computer science
138 (5.6)
110
4.3
11
Remote sensing
120 (4.9)
34
5.6
15
Imaging science and photographic technology
108 (4.4)
28
5.3
13
Energy and fuels
101 (4.1)
119
4.5
11 Fig. 4 Number of articles and
the average number of citations
per publication by year
1.0
0
25
6.0
18
7.0
0
0
11
12
0
6.7
20
11
10
2.0
23
40
10
29
54
40
37
32
37
22
16
10
2.1
0
10
20
30
40
50
60
0
200
400
600
800
1000
1200
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
2015
2016
2017
2018
2019
2020
2021
citations per publication
number of articles
Number of articles
Citations per publication
Year Fig. 4 Number of articles and
the average number of citations
per publication by year TP total number of publications, No. 3.2 Characteristics of Publication Outputs A relationship between the annual number of articles (TP)
and their CPPyear by the years in a research field has been
applied as a unique indicator [68]. Machine learning research
was not considered in Africa before 2010, with an annual
number of articles of less than 10. In Africa, Elgamal, Rafeh, TP total number of publications, AU number of authors, APP average number of authors per publication,
TC2021 total number of citations from Web of Science Core Collection since publication year to the end of
2021, CPP2021 average number of citations per publication (TC2021/TP)
Document type
TP
%
AU
APP
Median
TC2021
CPP2021
Article
2468
89
37,770
15
4.0
28,350
11
Review
235
8.5
1391
5.9
4.0
4287
18
Proceedings paper
32
1.2
213
6.7
3.0
365
11
Meeting abstract
28
1.0
359
13
7.5
15
0.54
Editorial material
22
0.80
113
5.1
3.0
88
4.0
Correction
4
0.14
24
6.0
6.0
0
0
Letter
3
0.11
24
8.0
4.0
49
16
Data paper
2
0.072
29
15
15
16
8.0
Book chapter
1
0.036
1
1.0
1.0
0
0
Retraction
1
0.036
4
4.0
4.0
0
0
Withdrawn publication
1
0.036
1
1.0
1.0
4
4.0 TP total number of publications, AU number of authors, APP average number of authors per publication,
TC2021 total number of citations from Web of Science Core Collection since publication year to the end of
2021, CPP2021 average number of citations per publication (TC2021/TP) 1 3 Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… 4185 4185
Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis…
development among Web of Science categories is discussed
‘electrical and electronic engineering’ category since 2014. Fig. 3.2 Characteristics of Publication Outputs Among the 44 African countries that
published machine learning-related articles, 28 countries
(64% of 44 African countries) had no single-country arti-
cles, while only Niger had no internationally collaborative 3.2 Characteristics of Publication Outputs J number of journals in a category in 2021, APP average number of
authors per publication, CPP2021 average number of citations per publication (TC2021/TP)
Web of Science category
TP (%)
No. J
APP
CPP2021
Electrical and electronic engineering
485 (20)
278
4.3
13
Information systems computer science
439 (18)
164
4.4
8.9
Artificial intelligence computer science
334 (14)
145
3.5
14
Telecommunications
308 (12)
94
4.4
9.3
Environmental sciences
192 (7.8)
279
6.6
11
Multidisciplinary sciences
163 (6.6)
73
6.2
13
Interdisciplinary applications computer science
154 (6.2)
113
5.7
15
Multidisciplinary geosciences
142 (5.8)
202
5.9
12
Theory and methods computer science
138 (5.6)
110
4.3
11
Remote sensing
120 (4.9)
34
5.6
15
Imaging science and photographic technology
108 (4.4)
28
5.3
13
Energy and fuels
101 (4.1)
119
4.5
11 TP total number of publications, No. J number of journals in a category in 2021, APP average number of
authors per publication, CPP2021 average number of citations per publication (TC2021/TP) ‘electrical and electronic engineering’ category since 2014. The first article in the category of ‘telecommunications’
was found in 2015. It had a sharp increase since 2018 and
reached 139 articles in 2021, much higher than the 93 arti-
cles in the ‘artificial intelligence computer science category’. development among Web of Science categories is discussed
using Fig. 4, comprising the number of publications versus
the year of publication [72]. Figure 5 shows the development
trends of the top four Web of Science categories with more
than 300 articles. The first articles were published in 1993
and 1997 in the ‘information systems computer science’ and
‘electrical and electronic engineering’ categories, respec-
tively. However, more articles have been published in the i
Recently, the characteristics of the journals based on their
CPPyear and APP as basic information of the journals in a
research topic were proposed [73, 74]. Table 3 shows the 1 4186 A. E. Ezugwu et al. Fig. 5 Development of the top
four productive Web of Science
categories, TP > 300
0
50
100
150
200
250
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
2014
2016
2018
2020
2022
2024
electrical and electronicengineering, TP = 485
information systems computer science, TP = 439
artificial intelligence computer science, TP = 334
telecommunications, TP = 308
Number of articles
Year Fig. 3.2 Characteristics of Publication Outputs 5 Development of the top
four productive Web of Science
categories, TP > 300 top 12 most productive journals with journal impact fac-
tors, CPP2021, and APP. The IEEE Access (IF2021 = 3.476)
published the most, 192 articles, representing 7.8% of
2,468. Compared to the top 12 productive journals, arti-
cles published in the Expert Systems with Applications
(IF2021 = 8.665) had the greatest CPP2021 of 30. In con-
trast, articles in the CMC-Computers Materials & Con-
tinua (IF2021 = 3.860) had only 2.2. The APP ranged from
16 in the Monthly Notices of the Royal Astronomical Soci-
ety to 2.8 in the Journal of Big Data. According to IF2021,
the top five journals which have an IF2021 of more than 60
were World Psychiatry (IF2021 = 79.683) with two articles,
Nature (IF2021 = 69.504) with one article, Nature Energy
(IF2021 = 67.439) with one article, Nature Reviews Disease
Primers (IF2021 = 65.038) with one article, and Science with
one article (IF2021 = 63.714). indicators and six related citation indicators (CPP2021)
[55] were applied to compare the 44 African countries
(Table 4). Egypt dominated in all the six publication indi-
cators with a TP of 777 articles (31% of 2468 articles), an
IPC of 186 articles (29% of 649 single-country articles),
a CPC of 591 articles (32% of 1819 internationally col-
laborative articles), an FP of 345 articles (14% of 2468
first-author articles), an RP of 449 articles (18% of 2467
corresponding-author articles), and an SP of 21 articles
(32% of 66 single-author articles). Compared to the top 17
productive countries with 20 articles or more, Sudan had
a TP of 33 articles, an IP of 3 articles, a CP of 30 articles,
an FP of 6 articles, and an SP of 3 articles, with the great-
est TP-CPP2021 of 20, IPC-CPP2021 of 23, CPC-CPP2021
of 20, FP-CPP2021 of 14, and SP-CPP2021 of 23 respec-
tively. Libya had an FP of 3 articles and an RP of 4, with
the greatest FP-CPP2021 of 14 and RP-CPP2021 of 23. Ten
of the 54 African countries such as Angola, Cape Verde,
Central African Republic (Cent Afr Republ), Comoros,
Djibouti, Equatorial Guinea (Equat Guinea), Eritrea, Sao
Tome and Principe (Sao Tome & Prin), Seychelles, and
South Sudan had no machine learning-related articles in
SCI-EXPANDED. Energies TP total number of articles, % percentage of articles in all articles, IF2021 journal impact factor in 2021, APP average number of authors per pub-
lication, CPP2021 average number of citations per paper (TC2021/TP) 3.4 Publication Performances: Countries Altogether, 649 articles (26% of 2468 articles) were sin-
gle-country articles from 16 African countries with an
IPC-CPP2021 of 10 and 1819 (74%) were internationally
collaborative articles from 146 countries, including 43
African countries and 103 non-African countries with a
CPC-CPP2021 of 12. The results show citations by inter-
national collaborations increased slightly. Six publication 1 3 4187 Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… Table 3 Top 12 most productive journals with TP > 20
TP total number of articles, % percentage of articles in all articles, IF2021 journal impact factor in 2021, APP average number of authors per pub-
lication, CPP2021 average number of citations per paper (TC2021/TP)
Journal
TP (%)
IF2021
APP
CPP2021
Web of science category
IEEE Access
192 (7.8)
3.476
4.5
11
Information systems computer science
Electrical and electronic engineering
Telecommunications
Remote sensing
59 (2.4)
5.349
5.6
8.6
Environmental sciences
Multidisciplinary geosciences
Remote sensing
Imaging science and photographic technology
PLoS One
46 (1.9)
3.752
5.7
18
Multidisciplinary sciences
Sensors
45 (1.8)
3.847
5.3
11
Analytical chemistry
Electrical and electronic engineering
Instruments and instrumentation
Scientific reports
38 (1.5)
4.996
7.6
12
Multidisciplinary sciences
Expert systems with applications
37 (1.5)
8.665
3.9
30
Artificial intelligence computer science
Electrical and electronic engineering
Operations research and management science
Applied sciences-basel
34 (1.4)
2.838
4.7
3.2
Multidisciplinary chemistry
Multidisciplinary engineering
Multidisciplinary materials science
Applied physics
Monthly notices of the royal astronomical society
24 (1.0)
5.235
16
18
Astronomy and astrophysics
CMC-Computers materials & continua
22 (0.89)
3.860
4.8
2.2
Information systems computer science
Multidisciplinary materials science
Journal of big data
22 (0.89)
10.835
2.8
5.0
Theory and methods computer science
Sustainability
22 (0.89)
3.889
5.1
3.5
Green and sustainable science and technology
Environmental sciences
Environmental studies
Energies
21 (0.85)
3.252
4.4
6.7
Energy and fuels 3.5 Publication Performances: Institutions articles. Similarly, 14 (32%), 10 (23%), and 35 (80%)
countries had no first-author, corresponding-author, and
single-author articles, respectively. Concerning institutions, 382 African articles (15% of 2468
articles) originated from single institutions with an IPI-
CPP2021 of 9.9, while 2086 articles (85%) were institutional
collaborations with a CPI-CPP2021 of 12. The institutional col-
laborations slightly increased the citations. The top 20 produc-
tive African institutions and their characteristics are presented
in Table 5. Cairo University in Egypt ranked top with a TP
of 142 articles (5.8% of 2468 articles) and a CPI of 127 arti-
cles (6.1% of 2086 inter-institutionally collaborative articles). However, the University of KwaZulu-Natal in South Africa
ranked top in three of the six publication indicators with an IP
of 19 articles (5.0% of 382 single-institution articles), an FP
of 48 articles (1.9% of 2468 first-author articles), and an RP
of 64 articles (2.6% of 2467 corresponding-author articles). In addition, the University of Johannesburg in South Africa
and the Council of Scientific and Industrial Research (CSIR)
in South Africa ranked top with an SP of four articles (6.1%
of 66 single-author articles), respectively. Compared to the
top 20 African countries, the University of KwaZulu-Natal in
South Africa had a TP of 104 articles, a CPI of 85 articles, an Development trends in the publication of the top six
productive countries with more than 100 articles are
presented in Fig. 6. From the results obtained, the first
machine learning-related article in Africa (by Egypt) dates
back to 1993. In 1995, 1998, 2001, 2004, and 2009, the
first articles were published by South Africa, Tunisia,
Morocco, Algeria, and Nigeria, respectively. Egypt and
South Africa had similar development trends. However,
Egypt sharply increased in the last three years to reach
324 articles in 2021. Algeria and Tunisia also had similar
development trends. Ten of the 103 non-African countries had 100 internation-
ally collaborative articles or more with Africa, as shown
in Fig. 7. The USA had a CPC of 431 articles with CPC-
CPP2021 of 15, followed by Saudi Arabia (CPC of 338 arti-
cles; CPC-CPP2021 of 9.3), the UK (295 articles; 14), China
(252; 14), France (211; 10), India (174; 11), Germany (156;
20), Canada (154; 14), Australia (146; 13), and Spain (124; 1 A. E. Ezugwu et al. TP total number of articles, TPR (%) rank of total number of articles and percentage in 2,468 articles, IPCR (%) rank of single-country articles
and percentage in 649 single-country articles, CPCR (%) rank of internationally collaborative articles and percentage in 1819 internationally
collaborative articles, FPR (%) rank of first-author articles and percentage in 2468 first-author articles, RPR (%) rank of corresponding-author
articles and percentage in 2467 corresponding-author articles, SPR (%) rank of single-author articles and percentage in 66 single-author articles,
CPP21 average number of citations per publication (TC2021/TP), N/A not available 3.5 Publication Performances: Institutions 4188 Table 4 African countries published machine learning articles
TP total number of articles, TPR (%) rank of total number of articles and percentage in 2,468 articles, IPCR (%) rank of single-country articles
and percentage in 649 single-country articles, CPCR (%) rank of internationally collaborative articles and percentage in 1819 internationally
collaborative articles, FPR (%) rank of first-author articles and percentage in 2468 first-author articles, RPR (%) rank of corresponding-author
articles and percentage in 2467 corresponding-author articles, SPR (%) rank of single-author articles and percentage in 66 single-author articles,
CPP21 average number of citations per publication (TC2021/TP), N/A not available
Country
TP
TP
IPC
CPC
FP
RP
SP
TPR (%)
CPP21
IPCR (%)
CPP21
CPCR (%)
CPP21
FPR (%)
CPP21
RPR (%)
CPP21
SPR (%)
CPP21
Egypt
777
1 (31)
13
1 (29)
10
1 (32)
14
1 (14)
11
1 (18)
11
1 (32)
8.6
South Africa
562
2 (23)
14
2 (28)
12
2 (21)
14
2 (11)
12
2 (13)
14
2 (21)
18
Morocco
215
3 (8.7)
9.1
3 (15)
13
6 (6.4)
5.8
4 (6.2)
10
3 (5.9)
10
8 (3.0)
2.0
Algeria
209
4 (8.5)
12
5 (10)
9.2
3 (8.0)
13
3 (6.5)
10
4 (5.3)
8.6
3 (17)
5.5
Tunisia
202
5 (8.2)
7.6
4 (10)
7.8
4 (7.5)
7.5
5 (5.1)
8.4
5 (4.9)
7.1
5 (6.1)
4.0
Nigeria
143
6 (5.8)
9.0
6 (2.8)
3.3
5 (6.9)
10
6 (2.1)
5.9
6 (2.6)
5.8
4 (9.1)
1.3
Ethiopia
86
7 (3.5)
5.5
8 (1.2)
3.8
7 (4.3)
5.7
8 (0.93)
6.1
7 (2.0)
4.0
6 (4.5)
2.0
Kenya
78
8 (3.2)
13
11 (0.46)
0.67
8 (4.1)
14
9 (0.85)
5.8
9 (0.89)
5.3
N/A
N/A
Ghana
63
9 (2.6)
8.8
7 (1.7)
4.2
9 (2.9)
10
7 (1.1)
4.1
8 (1.3)
7.8
8 (3)
0
Tanzania
44
10 (1.8)
12
N/A
N/A
10 (2.4)
12
11 (0.28)
4.9
12 (0.36)
4.4
N/A
N/A
Sudan
33
11 (1.3)
20
11 (0.46)
23
11 (1.6)
20
12 (0.24)
14
11 (0.45)
16
6 (4.5)
23
Uganda
33
11 (1.3)
5.3
9 (0.92)
10
12 (1.5)
4.3
10 (0.45)
8.3
10 (0.53)
7.2
N/A
N/A
Cameroon
21
13 (0.85)
10
N/A
N/A
13 (1.2)
10
14 (0.2)
7.4
13 (0.32)
7.1
N/A
N/A
Libya
21
13 (0.85)
11
N/A
N/A
13 (1.2)
11
19 (0.12)
14
18 (0.16)
23
N/A
N/A
Rwanda
21
13 (0.85)
4.2
N/A
N/A
13 (1.2)
4.2
12 (0.24)
3.5
13 (0.32)
3.0
N/A
N/A
Zambia
21
13 (0.85)
6.3
N/A
N/A
13 (1.2)
6.3
16 (0.16)
3.3
16 (0.20)
4.4
N/A
N/A
Zimbabwe
20
17 (0.81)
7.1
14 (0.15)
5.0
17 (1)
7.2
16 (0.16)
4.8
13 (0.32)
12
N/A
N/A
Botswana
19
18 (0.77)
15
13 (0.31)
6.0
18 (0.93)
16
21 (0.081)
6.0
21 (0.081)
6.0
N/A
N/A
Senegal
15
19 (0.61)
10
N/A
N/A
19 (0.82)
10
16 (0.16)
12
21 (0.081)
15
N/A
N/A
Cote Ivoire
13
20 (0.53)
6.4
N/A
N/A
20 (0.71)
6.4
21 (0.081)
25
21 (0.081)
25
N/A
N/A
Burkina Faso
11
21 (0.45)
39
14 (0.15)
114
22 (0.55)
32
19 (0.12)
91
19 (0.12)
38
N/A
N/A
Dem Rep Congo
11
21 (0.45)
4.1
N/A
N/A
21 (0.60)
4.1
N/A
N/A
N/A
N/A
N/A
N/A
Malawi
10
23 (0.41)
7.9
N/A
N/A
22 (0.55)
7.9
N/A
N/A
21 (0.081)
23
N/A
N/A
Mozambique
10
23 (0.41)
8.0
N/A
N/A
22 (0.55)
8.0
25 (0.041)
14
21 (0.081)
24
N/A
N/A
Madagascar
9
25 (0.36)
21
N/A
N/A
25 (0.49)
21
21 (0.081)
3.5
19 (0.12)
2.7
N/A
N/A
Mauritius
8
26 (0.32)
5.0
10 (0.62)
2.0
29 (0.22)
8.0
14 (0.2)
2.0
16 (0.2)
2.0
N/A
N/A
Benin
6
27 (0.24)
4.7
N/A
N/A
26 (0.33)
4.7
21 (0.081)
2.5
21 (0.081)
2.5
N/A
N/A
Gambia
6
27 (0.24)
14
N/A
N/A
26 (0.33)
14
N/A
N/A
28 (0.041)
2.0
N/A
N/A
Sierra Leone
6
27 (0.24)
9.3
N/A
N/A
26 (0.33)
9.3
25 (0.041)
3.0
21 (0.081)
3.0
N/A
N/A
Gabon
4
30 (0.16)
12
N/A
N/A
29 (0.22)
12
N/A
N/A
28 (0.041)
5.0
N/A
N/A
Mali
4
30 (0.16)
30
N/A
N/A
29 (0.22)
30
N/A
N/A
28 (0.041)
8.0
N/A
N/A
Namibia
4
30 (0.16)
21
N/A
N/A
29 (0.22)
21
N/A
N/A
N/A
N/A
N/A
N/A
Guinea
3
33 (0.12)
5.0
N/A
N/A
33 (0.16)
5.0
25 (0.041)
4.0
28 (0.041)
4.0
N/A
N/A
Togo
3
33 (0.12)
5.0
N/A
N/A
33 (0.16)
5.0
25 (0.041)
11
28 (0.041)
11
N/A
N/A
Burundi
2
35 (0.081) 1.5
N/A
N/A
35 (0.11)
1.5
N/A
N/A
N/A
N/A
N/A
N/A
Chad
2
35 (0.081) 4.5
N/A
N/A
35 (0.11)
4.5
N/A
N/A
N/A
N/A
N/A
N/A
Somalia
2
35 (0.081) 10
N/A
N/A
35 (0.11)
10
25 (0.041)
8.0
28 (0.041)
11
N/A
N/A
Eswatini
1
38 (0.041) 0
N/A
N/A
38 (0.055)
0
N/A
N/A
N/A
N/A
N/A
N/A
Guinea Bissau
1
38 (0.041) 28
N/A
N/A
38 (0.055)
28
N/A
N/A
N/A
N/A
N/A
N/A
Lesotho
1
38 (0.041) 5.0
N/A
N/A
38 (0.055)
5.0
N/A
N/A
N/A
N/A
N/A
N/A
Liberia
1
38 (0.041) 5.0
N/A
N/A
38 (0.055)
5.0
N/A
N/A
N/A
N/A
N/A
N/A
Mauritania
1
38 (0.041) 1.0
N/A
N/A
38 (0.055)
1.0
N/A
N/A
N/A
N/A
N/A
N/A
Niger
1
38 (0.041) 1.0
14 (0.15)
1.0
N/A
N/A
25 (0.041)
1.0
28 (0.041)
1.0
N/A
N/A
Rep Congo
1
38 (0.041) 18
N/A
N/A
38 (0.055)
18
N/A
N/A
N/A
N/A
N/A
N/A Table 4 African countries published machine learning articles TP total number of articles, TPR (%) rank of total number of articles and percentage in 2,468 articles, IPCR (%) rank of single-country articles
and percentage in 649 single-country articles, CPCR (%) rank of internationally collaborative articles and percentage in 1819 internationally
collaborative articles, FPR (%) rank of first-author articles and percentage in 2468 first-author articles, RPR (%) rank of corresponding-author
articles and percentage in 2467 corresponding-author articles, SPR (%) rank of single-author articles and percentage in 66 single-author articles,
CPP21 average number of citations per publication (TC2021/TP), N/A not available 1 3 3 4189
Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis…
1 3
Fig. Fig. 7 Development of the top
five most collaborative coun-
tries with Africa, TP > 200 3.5 Publication Performances: Institutions 6 Development of the top
six productive countries with
TP > 100
0
50
100
150
200
250
300
350
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
2014
2016
2018
2020
2022
2024
Egypt, TP = 777
South Africa, TP = 562
Morocco, TP = 215
Algeria, TP = 209
Tunisia, TP = 202
Nigeria, TP = 143
Number of articles
Year
Fig. 7 Development of the top
five most collaborative coun-
tries with Africa, TP > 200
0
50
100
150
200
250
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
2014
2016
2018
2020
2022
2024
USA, TP = 431
Saudi Arabia, TP = 338
UK, TP = 290
China, TP = 252
France, TP = 211
Number of articles
Year Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… 4189 Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… 4189
Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis…
Fig. 6 Development of the top
six productive countries with
TP > 100
0
50
100
150
200
250
300
350
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
2014
2016
2018
2020
2022
2024
Egypt, TP = 777
South Africa, TP = 562
Morocco, TP = 215
Algeria, TP = 209
Tunisia, TP = 202
Nigeria, TP = 143
Number of articles
Year Fig. 3.5 Publication Performances: Institutions 6 Development of the top
six productive countries with
TP > 100
0
50
100
150
200
250
300
350
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
2014
2016
2018
2020
2022
2024
Egypt, TP = 777
South Africa, TP = 562
Morocco, TP = 215
Algeria, TP = 209
Tunisia, TP = 202
Nigeria, TP = 143
Number of articles
Year 0
50
100
150
200
250
300
350
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
2014
2016
2018
2020
2022
2024
Egypt, TP = 777
South Africa, TP = 562
Morocco, TP = 215
Algeria, TP = 209
Tunisia, TP = 202
Nigeria, TP = 143
Number of articles
Year 0
50
100
150
200
250
300
350
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
2014
2016
2018
2020
2022
2024
Egypt, TP = 777
South Africa, TP = 562
Morocco, TP = 215
Algeria, TP = 209
Tunisia, TP = 202
Nigeria, TP = 143
Number of articles
Year Fig. 7 Development of the top
five most collaborative coun-
tries with Africa, TP > 200 1
0
50
100
150
200
250
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
2014
2016
2018
2020
2022
2024
USA, TP = 431
Saudi Arabia, TP = 338
UK, TP = 290
China, TP = 252
France, TP = 211
Number of articles
Year 3 A. E. Ezugwu et al. 3.5 Publication Performances: Institutions The University of Pretoria in
South Africa had an IPI of 15 articles with the greatest IPI-
CPP2021 of 20, while the Mansoura University in Egypt had an
SP of two articles with the greatest SP-CPP2021 of 29. directly using data from the database, total citations from the
Web of Science Core Collection from the year of publica-
tion to the end of the most recent year of 2021 (TC2021) were
applied [74]. The citation history of the most frequently
cited articles assessed by TCyear in a research topic was pre-
sented to understand the impact history of the articles [48,
53, 74]. Highly cited articles may not always significantly
impact a research field [49, 50, 53]. Table 6 shows the top
ten most frequently cited machine learning-related articles in
Africa. Five of the top ten articles were published by Egypt,
followed by South Africa with two articles and one each by
Nigeria, Kenya, and Morocco. Five non-Africa institutions had 30 inter-institutionally
collaborative articles or more with Africa. King Saud
University in Saudi Arabia had a CPI of 62 articles with
CPI-CPP2021 of 10, followed by Taif University in Saudi
Arabia (CPI of 39 articles; CPI-CPP2021 of 2.3), Univer-
sity of Oxford in the UK (38 articles; 15), King Abdulaziz
University in Saudi Arabia (34; 4.1), and Prince Sattam Bin
Abdulaziz University in Saudi Arabia (31; 7.6). The most cited article was entitled Ranger: a fast imple-
mentation of random forests for high dimensional data in
C + + and R [74] by Wright and Ziegler from the University
of Lubeck in Germany and the University of KwaZulu-Natal
in South Africa and had a TC2021 of 683 (rank 1st) and a
C2021 of 330 (rank 2nd). An article entitled Peeking inside
the black-box: A survey on explainable artificial intelligence
(XAI) [75] by Adadi and Berrada from the Sidi Mohammed 3.5 Publication Performances: Institutions 4190 Table 5 Top 20 most productive African institutions Table 5 Top 20 most productive African institutions
TP total number of articles, TP R (%) total number of articles and percentage of total articles, IPI R (%) rank and percentage of single-institution
articles in all single-institution articles, CPI R (%) rank and percentage of inter-institutionally collaborative articles in all inter-institutionally col-
laborative articles, FP R (%) rank and percentage of first-author articles in all first-author articles, RP R (%) rank and percentage of correspond-
ing-author articles in all corresponding-author articles, SPI R (%) rank and percentage of single-author articles in all single-author articles, TP-
CPP the total TC2021 of all articles per the total number of articles (TP), IPI-CPP the total TC2021 of all single-institution articles per the number
of single-institution articles (IPI), CPI-CPP the total TC2021 of all inter-institutionally collaborative articles per the number of inter-institutionally
collaborative articles (CPI), FP-CPP the total TC2021 of all first-author per the number of first-author articles (FP), RP-CPP the total TC2021 of
all corresponding-author articles per the number of corresponding-author articles (RP), N/A not available
Institution
TP
TP
IPI
CPI
FP
RP
SP
R (%)
CPP
R (%)
CPP
R (%)
CPP
R (%)
CPP
R (%)
CPP
R (%)
CPP
CU, Egypt
142
1 (5.8)
17
3 (3.9)
18
1 (6.1)
17
4 (1.3)
23
2 (2.1)
14
5 (3.0)
7.0
UKZN, South Africa
104
2 (4.2)
24
1 (5.0)
8.8
3 (4.1)
27
1 (1.9)
27
1 (2.6)
34
3 (4.5)
5.0
UCT, South Africa
101
3 (4.1)
14
10 (1.8)
5.9
2 (4.5)
15
6 (0.93)
10
4 (1.3)
6.6
12 (1.5)
5.0
MansU, Egypt
92
4 (3.7)
12
5 (3.4)
10
4 (3.8)
12
2 (1.5)
8.1
3 (1.9)
10
5 (3.0)
29
ZU, Egypt
69
5 (2.8)
16
27 (0.79)
5.0
5 (3.2)
17
8 (0.81)
12
7 (1.1)
10
N/A
N/A
UW, South Africa
66
6 (2.7)
11
19 (1.0)
10
6 (3.0)
11
8 (0.81)
4.8
12 (0.89)
5.5
N/A
N/A
BU, Egypt
64
7 (2.6)
13
36 (0.52)
0.50
6 (3.0)
13
14 (0.57)
24
9 (1.0)
18
12 (1.5)
1.0
MU, Egypt
60
8 (2.4)
10
27 (0.79)
1.3
8 (2.7)
11
20 (0.41)
2.8
14 (0.81)
10
N/A
N/A
UP, South Africa
59
9 (2.4)
12
3 (3.9)
20
10 (2.1)
9.4
5 (1.3)
13
4 (1.3)
12
N/A
N/A
ASU, Egypt
55
10 (2.2)
14
9 (2.1)
3.3
9 (2.3)
15
7 (0.89)
19
7 (1.1)
16
5 (3.0)
2.0
UJ, South Africa
54
11 (2.2)
5.1
2 (4.7)
8.1
12 (1.7)
3.6
3 (1.4)
7.1
6 (1.3)
7.4
1 (6.1)
7.8
UWC, South Africa
48
12 (1.9)
11
14 (1.3)
13
11 (2.1)
11
17 (0.53)
7.8
15 (0.69)
8.9
12 (1.5)
20
SU, South Africa
42
13 (1.7)
7.8
8 (2.4)
7.9
13 (1.6)
7.7
12 (0.77)
9.2
9 (1)
7.2
12 (1.5)
0
HU, Egypt
36
14 (1.5)
10
19 (1.0)
19
14 (1.5)
8.6
18 (0.49)
15
19 (0.61)
12
12 (1.5)
1.0
TU, Egypt
33
15 (1.3)
12
66 (0.26)
9.0
14 (1.5)
12
36 (0.28)
6.0
27 (0.45)
11
N/A
N/A
UTEM, Tunisia
33
15 (1.3)
3.1
14 (1.3)
1.2
16 (1.3)
3.5
8 (0.81)
3.8
11 (1.0)
1.7
N/A
N/A
AU, Egypt
30
17 (1.2)
9.0
36 (0.52)
10
16 (1.3)
8.9
55 (0.16)
7.3
24 (0.53)
6.9
12 (1.5)
7.0
UT, Tunisia
28
18 (1.1)
10
13 (1.6)
5.3
23 (1.1)
11
8 (0.81)
11
13 (0.85)
11
12 (1.5)
5.0
SCU, Egypt
27
19 (1.1)
23
36 (0.52)
3.0
19 (1.2)
24
37 (0.24)
10
19 (0.61)
21
N/A
N/A
UC, Tunisia
27
19 (1.1)
8.6
10 (1.8)
6.3
26 (1.0)
9.4
23 (0.36)
5.1
22 (0.57)
14
3 (4.5)
3.7 TP total number of articles, TP R (%) total number of articles and percentage of total articles, IPI R (%) rank and percentage of single-institution
articles in all single-institution articles, CPI R (%) rank and percentage of inter-institutionally collaborative articles in all inter-institutionally col-
laborative articles, FP R (%) rank and percentage of first-author articles in all first-author articles, RP R (%) rank and percentage of correspond-
ing-author articles in all corresponding-author articles, SPI R (%) rank and percentage of single-author articles in all single-author articles, TP-
CPP the total TC2021 of all articles per the total number of articles (TP), IPI-CPP the total TC2021 of all single-institution articles per the number
of single-institution articles (IPI), CPI-CPP the total TC2021 of all inter-institutionally collaborative articles per the number of inter-institutionally
collaborative articles (CPI), FP-CPP the total TC2021 of all first-author per the number of first-author articles (FP), RP-CPP the total TC2021 of
all corresponding-author articles per the number of corresponding-author articles (RP), N/A not available TP total number of articles, TP R (%) total number of articles and percentage of total articles, IPI R (%) rank and percentage of single-institution
articles in all single-institution articles, CPI R (%) rank and percentage of inter-institutionally collaborative articles in all inter-institutionally col-
laborative articles, FP R (%) rank and percentage of first-author articles in all first-author articles, RP R (%) rank and percentage of correspond-
ing-author articles in all corresponding-author articles, SPI R (%) rank and percentage of single-author articles in all single-author articles, TP-
CPP the total TC2021 of all articles per the total number of articles (TP), IPI-CPP the total TC2021 of all single-institution articles per the number
of single-institution articles (IPI), CPI-CPP the total TC2021 of all inter-institutionally collaborative articles per the number of inter-institutionally
collaborative articles (CPI), FP-CPP the total TC2021 of all first-author per the number of first-author articles (FP), RP-CPP the total TC2021 of
all corresponding-author articles per the number of corresponding-author articles (RP), N/A not available FP of 48 articles, and an RP of 64 articles, with the greatest
TP-CPP2021 of 24, CPI-CPP2021 of 27, FP-CPP2021 of 27, and
RP-CPP2021 of 34 respectively. 3.7 Research Foci In the last decade, Ho’s research group proposed distribu-
tions of words in article titles and abstracts, author key-
words, and Keywords Plus of different periods to determine
research foci and trends [83, 84]. Among 2468 articles,
2,464 articles (99.8% of 2468 articles) had record informa-
tion of article abstracts; 2,103 (85.2%) articles had author
keywords; and 2069 (83.8%) articles had Keywords Plus. The 20 most frequent keywords are listed in Table 7. The
classification was ranked in the top 20 in article titles and
abstracts, author keywords, and Keywords Plus, respectively. The development of the top four topics in machine learn-
ing in Africa, such as deep learning, classification, feature
extraction, and random forest, is shown in Fig. 8. 3.6 Citation Histories of the Ten Most Frequently
Cited Articles These two articles keep
increasing in citations. or author keywords were classified as classification-related
articles. In 1996, Gouws and Aldrich from the University of
Stellenbosch in South Africa reported that using machine
learning techniques and the classification rules on a super-
visory expert system shell or decision support system for
plant operators could consequently make a significant impact
on the way notation plants [85]. Highly cited articles with
TC2021 of 100 or more [50], such as Deep learning for
tomato diseases: Classification and symptoms visualization
[86] and Learning machines and sleeping brains: Automatic
sleep stage classification using decision-tree multi-class
support vector machines [87] were published by African
authors from Algeria and Tunisia respectively. An article
entitled A predictive machine learning application in agri-
culture: Cassava disease detection and classification with
imbalanced dataset using convolutional neural networks
[88] was published in the most recent year 2021 by Samba-
sivam and Opiyo from International Business, Science And
Technology University (ISBAT) in Uganda. 3.6 Citation Histories of the Ten Most Frequently
Cited Articles The total citations in the Web of Science Core Collection are
updated from time to time. To improve bibliometric studies 1 3 Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… 4191 Table 6 The top ten most frequently cited articles by African countries
TC2021 number of citations from Web of Science Core Collection since its publication to the end of 2021, C2021 number of citations from Web of
Science Core Collection in 2021
Rank (TC2021) Rank (C2021) Title
Country
Reference
1 (683)
2 (330)
Ranger: A fast implementation of random
forests for high dimensional data in
C + + and R
Germany, South Africa
Wright and Ziegler [74]
2 (675)
1 (435)
Peeking inside the black-box: A survey on
explainable artificial intelligence (XAI)
Morocco
Adadi and Berrada [75]
3 (402)
22 (56)
Multiobjective intelligent energy manage-
ment for a microgrid
Japan, Egypt, Saudi Arabia
Chaouachi et al. [70]
4 (313)
31 (47)
Computer-aided diagnosis of human brain
tumor through MRI: A survey and a new
algorithm
Egypt, UK
El-Dahshan et al. [76]
5 (255)
8 (87)
Predicting carbon dioxide and energy fluxes
across global FLUXNET sites with regres-
sion algorithms
Italy, Germany, USA, Japan, Spain,
Romania, Canada, Ireland, Switzer-
land, Kenya
Tramontana et al. [77]
6 (251)
15 (64)
An introduction to quantum machine learn-
ing
South Africa
Schuld et al. [78]
7 (228)
20 (59)
An empirical comparison of machine learn-
ing models for time series forecasting
Egypt, USA
Ahmed et al. [79]
8 (200)
48 (35)
A support vector machine: Firefly algorithm-
based model for global solar radiation
prediction
Malaysia, Nigeria, Iran, Serbia, India
Olatomiwa et al. [80]
9 (199)
13 (73)
Machine learning with big data: Challenges
and approaches
Canada, Egypt
L'Heureux et al. [81]
10 (185)
6 (94)
Linear discriminant analysis: A detailed
tutorial
Germany, Egypt
Tharwat et al. [82] m Web of Science Core Collection since its publication to the end of 2021, C2021 number of citations from Web of Ben Abdellah University in Morocco had the most impact
on the most recent year of 2021 with a C2021 of 435 (rank
1st) and a TC2021 of 675 (rank 2nd). These two articles keep
increasing in citations. Ben Abdellah University in Morocco had the most impact
on the most recent year of 2021 with a C2021 of 435 (rank
1st) and a TC2021 of 675 (rank 2nd). 3.7.1 Classification Supporting words for deep learning were deep learning, deep
neural network, deep neural networks, deep transfer learn-
ing, deep reinforcement learning, deep convolutional neural Articles containing supporting words such as classification,
classifications, and misclassification in their title, abstract, 1 4192 A. E. Ezugwu et al. 3.7.1 Classification Table 7 The 20 most frequently used keywords
Words in title
TP
R (%)
Words in Abstract
TP
R (%)
Author keywords
TP
R (%)
Keywords Plus
TP
R (%)
Learning
886
1 (36)
Learning
1,930
1 (78)
Machine learning
918
1 (44)
Classification
279
1 (13)
Machine
739
2 (30)
Machine
1,925
2 (78)
Deep learning
189
2 (9.0)
Prediction
207
2 (10)
Detection
246
3 (10)
Model
1,104
3 (45)
Classification
101
3 (4.8)
Model
164
3 (7.9)
Classification
235
4 (10)
Accuracy
1,067
4 (43)
Feature extraction
101
3 (4.8)
Algorithm
116
4 (5.6)
Prediction
232
5 (9.4)
Proposed
1,013
5 (41)
Random forest
95
5 (4.5)
System
89
5 (4.3)
Approach
196
6 (7.9)
Paper
928
6 (38)
Feature selection
80
6 (3.8)
Diagnosis
88
6 (4.3)
Deep
192
7 (7.8)
Methods
868
7 (35)
Artificial intelligence
75
7 (3.6)
Selection
85
7 (4.1)
Model
166
8 (6.7)
Models
865
8 (35)
Support vector machine
59
8 (2.8)
Regression
82
8 (4)
Analysis
155
9 (6.3)
Approach
812
9 (33)
Support vector machines
55
9 (2.6)
Neural-networks
80
9 (3.9)
Neural
143
10 (5.8)
Analysis
780
10 (32)
Optimization
54
10 (2.6)
Performance
79
10 (3.8)
System
128
11 (5.2)
Classification
745
11 (30)
Covid-19
53
11 (2.5)
Neural-network
75
11 (3.6)
Algorithms
123
12 (5)
Algorithm
731
12 (30)
Artificial neural network
52
12 (2.5)
Identification
64
12 (3.1)
Network
122
13 (4.9)
Techniques
714
13 (29)
Data mining
45
13 (2.1)
Models
63
13 (3)
Networks
117
14 (4.7)
Algorithms
705
14 (29)
Neural networks
44
14 (2.1)
Optimization
61
14 (2.9)
Algorithm
115
15 (4.7)
Method
703
15 (29)
Prediction
44
14 (2.1)
Design
60
15 (2.9)
Techniques
115
15 (4.7)
Compared
691
16 (28)
Remote sensing
44
14 (2.1)
Feature-selection
57
16 (2.8)
Feature
105
17 (4.3)
Neural
622
17 (25)
Convolutional neural network
41
17 (1.9)
Systems
49
17 (2.4)
Hybrid
103
18 (4.2)
Network
621
18 (25)
Machine learning algorithms
41
17 (1.9)
Features
46
18 (2.2)
Selection
98
19 (4)
Features
616
19 (25)
Big data
37
19 (1.8)
Support vector machine
46
18 (2.2)
Models
96
20 (3.9)
Support
604
20 (25)
Internet of things
37
19 (1.8)
Random forest
43
20 (2.1) q
y
y
n title
TP
R (%)
Words in Abstract
TP
R (%)
Author keywords
TP
R (%)
Keywords Plus
TP
R (%)
g
886
1 (36)
Learning
1,930
1 (78)
Machine learning
918
1 (44)
Classification
279
1 (13)
739
2 (30)
Machine
1,925
2 (78)
Deep learning
189
2 (9.0)
Prediction
207
2 (10)
n
246
3 (10)
Model
1,104
3 (45)
Classification
101
3 (4.8)
Model
164
3 (7.9)i
ation
235
4 (10)
Accuracy
1,067
4 (43)
Feature extraction
101
3 (4.8)
Algorithm
116
4 (5.6)
on
232
5 (9.4)
Proposed
1,013
5 (41)
Random forest
95
5 (4.5)
System
89
5 (4.3)
h
196
6 (7.9)
Paper
928
6 (38)
Feature selection
80
6 (3.8)
Diagnosis
88
6 (4.3)
192
7 (7.8)
Methods
868
7 (35)
Artificial intelligence
75
7 (3.6)
Selection
85
7 (4.1)
166
8 (6.7)
Models
865
8 (35)
Support vector machine
59
8 (2.8)
Regression
82
8 (4)
155
9 (6.3)
Approach
812
9 (33)
Support vector machines
55
9 (2.6)
Neural-networks
80
9 (3.9)
143
10 (5.8)
Analysis
780
10 (32)
Optimization
54
10 (2.6)
Performance
79
10 (3.8)
128
11 (5.2)
Classification
745
11 (30)
Covid-19
53
11 (2.5)
Neural-network
75
11 (3.6)
ms
123
12 (5)
Algorithm
731
12 (30)
Artificial neural network
52
12 (2.5)
Identification
64
12 (3.1)
122
13 (4.9)
Techniques
714
13 (29)
Data mining
45
13 (2.1)
Models
63
13 (3)
s
117
14 (4.7)
Algorithms
705
14 (29)
Neural networks
44
14 (2.1)
Optimization
61
14 (2.9)
m
115
15 (4.7)
Method
703
15 (29)
Prediction
44
14 (2.1)
Design
60
15 (2.9)
ues
115
15 (4.7)
Compared
691
16 (28)
Remote sensing
44
14 (2.1)
Feature-selection
57
16 (2.8)
105
17 (4.3)
Neural
622
17 (25)
Convolutional neural network
41
17 (1.9)
Systems
49
17 (2.4)
103
18 (4.2)
Network
621
18 (25)
Machine learning algorithms
41
17 (1.9)
Features
46
18 (2.2)
n
98
19 (4)
Features
616
19 (25)
Big data
37
19 (1.8)
Support vector machine
46
18 (2.2)
96
20 (3.9)
Support
604
20 (25)
Internet of things
37
19 (1.8)
Random forest
43
20 (2.1) 1 3 Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… 4193 Fig. 3.7.1 Classification 8 Development trends of
the four most popular topics in
Africa
0
50
100
150
200
250
300
350
400
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
2014
2016
2018
2020
2022
2024
classification, TP = 841
deep learning, TP = 430
feature extraction, TP = 375
random forest, TP = 190
Number of articles
Year network, and deep convolutional neural networks. Deep
learning was first mentioned in an article on Deep learning
framework with confused sub-set resolution architecture for
automatic Arabic Diacritization [89] by authors from Egypt
and Kuwait. Highly cited machine learning article was pub-
lished by African authors, for example, Deep learning for
tomato diseases: Classification and symptoms visualization
[86] by authors from Algeria and Deep learning for cyber
security intrusion detection: Approaches, datasets, and com-
parative study [90] by authors from Algeria and the UK. The
most impactful article about deep learning in 2021 was A
hybrid deep transfer learning model with machine learning
methods for face mask detection in the era of the COVID-19
pandemic [91] by authors from Egypt, USA, and Taiwan. Minimum redundancy maximum relevance feature selection
approach for temporal gene expression data [94] by authors
from the USA, Serbia, and Egypt. In 2021, Metaheuristic
algorithms on feature selection: A survey of one decade of
research (2009–2019) [95] was published by authors from
India, Saudi Arabia, and Egypt. Minimum redundancy maximum relevance feature selection
approach for temporal gene expression data [94] by authors
from the USA, Serbia, and Egypt. In 2021, Metaheuristic
algorithms on feature selection: A survey of one decade of
research (2009–2019) [95] was published by authors from
India, Saudi Arabia, and Egypt. 3.7.4 Random Forest Supporting words for the random forest were random for-
est, random forests, and random decision forest. In 2010,
Auret and Aldrich [96] from the University of Stellenbosch
in South Africa published the first article about the random
forest in machine learning. Highly cited random forest-
related articles were published in the last decade in Africa,
for example, Ranger: A fast implementation of random for-
ests for high dimensional data in C + + and R [74] by Wright
and Ziegler from Germany and South Africa and Random
forest regression and spectral band selection for estimating
sugarcane leaf nitrogen concentration using EO-1 Hyperion
hyperspectral data [97] by authors from South Africa and
Sudan. In 2021, The application of the random forest classi-
fier to map irrigated areas using Google Earth Engine [98]
was presented by authors from South Africa. 4.2 Research Trends in Africa ML algorithms have found significant application in bioin-
formatics, especially in genetic testing of microscopic spots
stored in DNA microarrays, genomics, and proteomics. Also, the medical or biological fields have been receiving
significant attention from ML researchers, particularly in
areas of medical engineering, epidemiology, and the study
and early detection of genetic diseases and disorders such as
Alzheimer's disease, diabetes, cancer, arthritis, high blood
pressure, hemochromatosis, cystic fibrosis, Huntington's dis-
ease, sickle cell anemia, and Marfan syndrome [99–101]. Focusing on diseases prevalent in Africa, machine learning
has been used to improve the genetic resistance to malaria,
early detection and eradication of diabetes, classification
of sickle cell anemia, improvement of genetic resistance
to HIV/AIDS, and detection of uterine fibroids in women
[102]. In entrepreneurship, ML has been leveraged to deliver
innovative research and products. Hepta Analytics developed
a product called Najua, which uses ML to present web con-
tent in local languages [121]. A start-up company in Nigeria
developed a mobile app called Ubenwa, which is used to
detect early prenatal asphyxia in newborn babies by analyz-
ing acoustic signatures [122]. ML has also found tremendous application in the econ-
omy and is argued to be the bedrock of the fourth industrial
revolution [103]. The developed countries have keyed into
this to avoid missing out on the revolution [104, 105]. Actors
in government and private sectors have developed strategies
that key into the revolution. Africa is lagging in this regard
with little or no efforts towards actualizing the fourth indus-
trial revolution. Some agencies from the West have tried
to assist developing countries [106, 107]. Countries like
Rwanda and others have taken the initiative of developing
plans driven by AI to achieve economic sustainability [108]. 4.1 Preliminary Overview Western technologies like Farmbeats
have been applied in Africa using low-cost, sparsely distrib-
uted sensors and aerial imagery to generate precision maps. 3.7.3 Feature Extraction Supporting words for the feature extraction were feature
extraction, feature selection, and feature evaluation. Saidi
et al. from France and Tunisia published the first feature
extraction-related article entitled Protein sequences clas-
sification by means of feature extraction with substitution
matrices [92] in Africa. Highly cited articles about feature
extraction were Ensemble-based multi-filter feature selection
methods for DDoS detection in cloud computing [93] by
authors from South Africa, Australia, China, and the UK and The yearly development trends of the four most popu-
lar topics in Africa, shown in Fig. 8, illustrated that the 1 3 4194 A. E. Ezugwu et al. A. E. Ezugwu et al. classification (TP = 841 articles) was the most concerned
with machine learning in Africa. Research about deep learn-
ing was more popular than feature extraction. However, they
have shown the same development trends in recent years. Different AI techniques, such as ML and the Internet
of Things (IoT), drive the energy sector. Africa is not left
behind in this aspect. ML is used in pay-as-you-go energy
products to predict demand, score users' activities, and
develop models that make products available, affordable
and adaptable [109]. For example, an energy company can
use the predictive analysis aspect of ML to make available
energy services or products to areas without access to energy
products and services [110, 111].f 3 4.1 Preliminary Overview The agricultural sector offers a fertile ground for ML to
display the ability to improve productivity and efficiency
all along the value chain. It provides solutions for subsist-
ence and mechanized farmers to improve yield and increase
profits through developing models for the detection and
precision treatment of pests and diseases, optimal fertilizer
application, soil monitoring, and many more. Solutions like
Gro intelligence in Kenya deploy AI techniques such as ML
to achieve food security [112]. Climatic conditions for pre-
cision agriculture have been achieved through the use of
drone technology with the capability of knowing the optimal
interventions needed for optimal yield [113]. Machine learning (ML) is a subfield of artificial intelligence. The central idea is that the machine learns by interacting
with the input data and develops a corresponding model
capable of classifying a new input or predicting an outcome
based on new inputs. The input data is usually divided into
two: the training data used to teach the machine and the
classification data used for testing the accuracy of the trained
model. Different ML algorithms have been used to solve
problems such as early disease detection and classification in
medicine and agriculture, plants or crops disease detection,
data mining, clustering, quantum computing technology,
engineering optimization, earth observation, food security,
climate change, pollution, and many more. ML has also been used to develop systems that could
identify in real time the appropriate agronomic interventions
that should be made using sensor data such as pH level,
soil moisture level, temperature, and more. In Kenya and
Mozambique, projects like Third Eye drive this process for
better yield [114, 115]. Western technologies like Farmbeats
have been applied in Africa using low-cost, sparsely distrib-
uted sensors and aerial imagery to generate precision maps. The system is attached to a smartphone carrying helium bal-
loons, which is a low-cost drone system [116, 117]. Intel-
ligent drones with high ML capabilities have been deployed
to survey elephants in Burkina Faso, anti-poaching rhinos
in South Africa, and analysis of flood risks in Tanzania
[118–120]. ML has also been used to develop systems that could
identify in real time the appropriate agronomic interventions
that should be made using sensor data such as pH level,
soil moisture level, temperature, and more. In Kenya and
Mozambique, projects like Third Eye drive this process for
better yield [114, 115]. 4.3.3 Internet of Things (IoT) and Smart Cities The Internet of Things (IoT) is another vital area of the
fourth Industrial revolution. The goal is to make objects
smart by allowing them to transmit data and automate
tasks without human interaction. Therefore, IoT is a fron-
tier in enhancing human activities, such as smart homes,
cities, agriculture, governance, healthcare, and more. Adenugba et al. [129] proposed a machine learning-based
Internet of Everything for a smart irrigation system for
environmental sustainability in Africa. Their solar-pow-
ered smart irrigation system uses a machine learning radial
basis function network to predict the environmental condi-
tion that controls the irrigation system. 4.3 Major Application Areas ML is a major driver of the Fourth Industrial Revolution
(4IR). It has improved outcomes in various application areas
by utilizing its learning and prediction abilities. This section
summarizes and discusses major popular application areas
of machine learning. Figure 9 gives the main branches of
machine learning and the offshoot disciplines of each. It also
depicts how different researchers have used the major ML
algorithms to solve problems in the respective domains. The 4195 Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… used to design security models that can be used on large-
scale security datasets [125]. Mbona and Eloff [126]
designed a semi-supervised machine learning approach
to detect zero-day (new unknown) intrusion attacks based
on the law of anomalous numbers to identify significant
network features that effectively show anomalous behav-
iour. Similarly, Benlamine et al. [127], used a machine
learning model to evaluate emotional reactions in virtual
reality environments where the face is hidden in a virtual
reality headset, making facial expression detection using a
webcam impossible. Several machine learning techniques
have been used to identify and classify spam e-mails [128]. application areas of ML are vast, as seen by the depiction in
Fig. 9. Therefore, this study summarizes the application area
into ten elaborate areas which are discussed below. 4.3.1 Predictive and Decision‑Making Most ML research has been carried out in this domain,
where ML drives the intelligent decision-making process
through data-driven predictive analytics, for instance, sus-
pect identification, fraud detection [123], and many more. ML is also helpful in identifying customer preferences and
behavior, production line management, scheduling optimiza-
tion, and inventory management. As seen from Table 7, the
keywords “prediction” and “detection” represent the third
and fourth most frequently used keywords for research in
ML. Nwaila et al. [124] designed a machine learning algo-
rithm for point-wise grade prediction and automatic facies
identification based on gold assay and sedimentological data
for the South African Witwatersrand Gold ores. 4.3.9 Sustainable Agriculture Sustainable agricultural practices help improve agricultural
productivity while reducing negative environmental impacts
[150, 151]. Sustainable agriculture is knowledge-intensive
and information-driven, where farmers make decisions
based on available information and technology such as the
Internet of Things (IoT), mobile technologies, and devices. Machine learning techniques are applied to predict crop
yield, soil properties, irrigation requirements, weather, dis-
ease detection, weed detection, soil nutrient management,
livestock management, demand estimation, production
planning, inventory management, consumer analysis, and
more. Machine learning techniques have been used to predict
the level of insect infestation with its associated damage in
maize farms [152]. In Hengl et al. [153], spatial predictions
of soil micro and macro nutrients were carried out using 4.3.7 Natural Language Processing (NLP) NLP and sentiment analysis involve processes that could
enable computer reading, understanding, and processing of
spoken or written language [145]. Some examples of NLP-
related tasks include virtual personal assistants, chatbots,
speech recognition, document description, and language or
machine translation. Sentiment Analysis or Opinion Min-
ing uses the result of NLP to mine information or trends
that could translate to moods, views, and opinions from
huge data collected from different social media platforms
[146]. For instance, politicians can use sentiment analysis
to ascertain the perceived views of the electorate about their
candidate. 4.3.5 Healthcare Machine learning techniques have been applied in healthcare
for diagnosing and prognostic diseases, omics data analy-
sis, patient management, and more [132]. The Coronavirus
disease (COVID-19) outbreak elicited the use of machine-
learning techniques to help combat the pandemic [133]. Deep learning also provides exciting solutions to medical
image processing problems and is a crucial technique for
potential applications, particularly for the COVID-19 pan-
demic [134]. Machine learning technique has also been
used in Malaria incidence prediction to address the seri-
ous challenge it poses to socio-economic development in
Africa [135]. Heart failure phenotypes were clustered based
on multiple clinical parameters using unsupervised machine
learning techniques by Mpanya et al. [136] to assist in diag-
nosing, managing, risk stratification and prognosis of heart
failure. Machine learning has been deployed in predicting
the present or future status of a disease or a disease's future
course using machine learning and regression models [137]. Patients can be classified based on disease risk or disease
probability estimation through machine learning approaches
[138]. Brain MRIs can be classified for detecting brain
tumors using a machine learning-based deep neural network
classifier [139]. Other medical diagnoses that use machine
learning include electrocardiograms [140] and cancer dis-
ease diagnosis [141]. 4.3.4 Traffic Prediction creditworthiness can be determined through customers'
credit scoring based on machine learning classification
methods [143]. In retail market operations, a machine learn-
ing tool has been designed to assist retailers in increasing
access to essential products by improving essential product
distribution in uncertain times due to the problem of panic
buying [144]. The economy of a city or country thrives when an efficient
transport system exists. A community's economic growth
comes with challenges such as high traffic volume, acci-
dents, emergencies, high pollution, and more. Therefore,
ML-driven smart city models can help predict traffic anoma-
lies [130]. Also, ML techniques can analyze travel history
data to predict possible hitches or recommend alternative
routes to commuters [131]. 4.3.8 Image, Speech, and Pattern Recognition Machine learning has significant application in this domain,
where different ML techniques have been used to identify
or classify real-world digital images [147]. A typical exam-
ple of image recognition includes labeling digital images
from an X-ray as cancerous. Like image recognition, speech
recognition deals with sound and linguistic models [148]. Finally, pattern recognition aims to identify patterns and
expressions in data [149]. Several machine-learning tech-
niques, such as classification, feature selection, clustering,
or sequence labeling, have been used in this area. 4.3.2 Cybersecurity and Threat Intelligence Cybersecurity is a cardinal area of intervention in Indus-
try 4.0, typically protecting networks, systems, hardware,
and data from digital attacks. Machine learning techniques
have been used to detect security breaches through data
analysis to identify patterns and detect malware or threats. The common ML technique for identifying cyber breaches
is the clustering technique. Also, deep learning has been Machine
learning
Supervised
learning
Unsupervised
learning
Reinforced
learning
Dimensionality
reduction
Clustering
Classification
Clustering
Customer Retention
Diagnostics
Identity Fraud Detection
Image Classification
Advertising Popularity Prediction
Weather Forecasting
Market Forecasting
Estimating Life Expectancy
Population Growth Prediction
Real-Time Decision
Robot Navigation
Learning Task
Skills Acquisition
Game AI
Customer Segmentation
Targeted Marketing
Recommender System
Big Data Visualization
Meaningful Compression
Structure Discovery
Feature Elicitation
Quantum Computing
Fig. 9 The main branches of machine learning and the offshoot disciplines of each Machine
learning Identity Fraud Detection Fig. 9 The main branches of machine learning and the offshoot disciplines of each 1 3 A. E. Ezugwu et al. 4196 3 4.3.6 E‑commerce ML techniques have been used to build systems that help
businesses understand customers' preferences by analyzing
their purchasing histories. These systems can recommend
products to potential customers. Companies would use
these systems to know where to position product adverts or
offers. Many online retailers can better manage inventory
and optimize logistics, such as warehousing, using predic-
tive modeling based on machine learning techniques [142]. Furthermore, machine learning techniques enable compa-
nies to maximize profits by creating packages and content
tailored to their customer's needs, allowing them to maintain
existing customers while attracting new ones. Customers' 3 Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… 4197 life informed the need for its study and prediction. Machine
learning models have been employed to study the relation-
ship between greenhouses gases emissions and climate
variable change rhythm. Ibrahim, Ziedan & Ahmed [161]
explored the application of ML techniques to climate data
for building an ML models for predicting climate variable
states for the long and short term in North-East Africa. This
is employed in climate mitigation and adaptation as well as
in determining the acceptable level of greenhouse gases with
their corresponding concentration to avoid climate crises and
events. Sobol, Scott & Finkelstein [162] utilized supervised
machine learning to modern pollen assemblages in South-
ern Africa to understand biome responses to global climate
change and determine specific biomes or bioregions repre-
sentations. Probabilistic classification for fossil assemblages
was generated for the reconstruction of past vegetation. machine learning techniques to support agricultural develop-
ment, monitoring and intensifying soil resources. Identifying
and mapping ecosystems are important in supporting food
security and other important environmental indicators for
biotic diversity. Tchuenté et al. [154] developed two machine
learning approaches to ecosystem mapping in the African
continent-scale to classify the African ecosystem based on
the Normalized Difference Vegetation Index (NDVI) dataset. Andraud et al. [155] applied machine learning for Benthic
habitat mapping to characterise seafloor substrate using
geophysical data at Table Bay, southwestern South Africa. Computer vision and machine learning techniques have been
used in the evaluation of food quality and the grading of
crops. Semary et al. [156] designed machine learning tech-
niques using feature fusion and support vector machines for
classifying infected or uninfected tomato fruits based on the
external surface of the tomato fruits. 4.3.10 Pollution Control Air pollution is regarded as one of the world's most immense
public and environmental health challenges, with its adverse
effects on the ecosystem, human health, and climate. Gaps
in air quality data in the middle- and lower-income coun-
tries limit the development of policies relating to air pollu-
tion control with its resultant negative health impacts due to
exposure to ambient air pollution. Long-term exposure to
ambient air pollution is associated with an increase in mor-
tality rates in these countries. There is a need for accurate
and reliable estimates of air pollution prediction for land use
regression. Coker et al. [157] proposed a land use regres-
sion model based on low-cost particulate matter sensors
and machine learning to accurately estimate the exposure to
air pollution in eastern and central Uganda—a sub-Saharan
African country. The goal is to use low-cost air quality sen-
sors in land use regression modelling to accurately predict
the fine ambient particulates matter air pollution in the urban
areas which will be estimated monthly. Amegah [158] also
used machine learning techniques with low-cost air quality
sensors for air pollution assessment and prediction in urban
Ghana. Zhang et al. [159] developed a machine learning
model using the random forest for estimating the daily fine
particulate matter concentration in the industrialized Gaut-
eng province in South Africa based on socioeconomic, sat-
ellite aerosol optical depth, meteorology and land use data. 4.3.6 E‑commerce g
p
g
The continual negative effect of climate change and
human-induced ecological degradation worsens the envi-
ronmental pressures on human livelihoods in many regions,
resulting in an increased risk of violent conflict. With refer-
ence to the African continent, Hoch et al. [163] projected
sub-national armed conflict risk along three representative
concentration pathways and three shared socioeconomic
pathways using machine learning methods. The role of
hydro-climatic indicators in driving armed conflict was
assessed. According to their report, climate change increases
the projection for armed conflict risk in Northern Africa and
substantial parts of Eastern Africa. The role of ML in armed
conflict risk projection is to assist the policy-making process
in handling climate security. To combat the adverse effect of
deforestation and climate change on accurate weather infor-
mation, Nyetanyane & Masinde [164] proposed a machine
learning model that uses climate data, vegetation index and
indigenous knowledge to predict the onset of favourable
weather seasons for crop cultivation, monitoring and pre-
diction of crop health. 4.3.12 Soil Analysis The need for detailed soil information to assist in agricul-
tural productivity modelling as well as to aid global estima-
tion of the organic carbon in the soil has grown over time. Moreover, in areas affected by climate change, the need
arises for spatial information about the parameters of soil
waters. According to Folberth et al. [165], obtaining accurate
information about soil may be important in the prediction of
the effect of climate change on food production. Hengl et al. [166] presented an improved version of the SoilGrids system
for global predictions for standard numeric soil properties,
including the organic carbon, Cation Exchange Capacity,
bulk density, soil texture fractions, coarse fragments and
pH, as well as predicting the distribution of soil classes and 4.4 Quantum‑Based Machine Learning Research Another prominent research area in machine learning that
has been actively engaged in Africa is the deployment of
quantum computing to improve classical machine learning
algorithms. Quantum computing manipulates the quan-
tum system for information processing for a substantial
computational speed. In quantum computing, the classical
two states 0 and 1 of conventional computing are replaced
with the superposition of qubit (quantum bit) of the two
states ∣0⟩ and ∣1⟩, which allows many different computation
paths simultaneously. Quantum machine learning involves
the development of quantum algorithms for solving typical
machine learning problems to harness the efficiency of quan-
tum computing. The classical machine learning algorithms
are adapted to run on a quantum computer. In the current
era of the explosive growth of information, the adoption
of quantum machine learning for various machine learn-
ing applications has been an active area of research as it is
a promising area of an innovative approach to improving
machine learning. The development of the quantum kernel method and
quantum similarity-based binary classifier exploiting feature
quantum Hilbert space and quantum interference brought a
great opportunity for enhancing classical machine learning
through quantum computing. In Park, Blank and Petruc-
cione's [171] work, the general theory of the quantum ker-
nel-based classifier was extended to lay the foundation for
advancing quantum-enhanced machine learning. The authors
focused on using squared overlap between quantum states as
the similarity measure to examine the minimal and essential
ingredients for quantum binary classification. Their work
also considered other extensions relating to measurement,
ensemble learning and data type. Schuld, Sinayskiy, & Petruccione [78] presented a sys-
tematic overview of the emerging field of quantum machine
learning, describing the approaches, technical details, and
future quantum learning theory. The presentation included
discussions on the various approaches for relating seven
standard methods of the classical machine learning algo-
rithms: support vector machine, k-nearest neighbour, neural
network, k-means clustering, hidden Markov model, deci-
sion trees and Bayesian theory to quantum physics. The dis-
cussion focused mainly on the quantum machine learning
approach for pattern classification and clustering.i Schuld, Sinayskiy and Petruccione [172] designed an
algorithm for pattern classification with linear regression
on a quantum computer. Their approach focused on solving
linear regression problems from the perspective of machine
learning, where new inputs are predicted based on the data-
set. 4.3.11 Climate System In estimating global gridded net radiation and sensible and
latent heat alongside their uncertainties, machine learning
has been deployed to merge energy flux measurements with
meteorological and remote sensing data for accurate estima-
tion [160]. The negative impact of climate change on human 1 4198 A. E. Ezugwu et al. depth to bedrock based on the USDA and World Reference
Base classification system. depth to bedrock based on the USDA and World Reference
Base classification system. pattern classification examples to briefly introduce quan-
tum machine learning. Their work presented an algorithm
for quantum pattern classification using Trugenberge's
proposal to measure Hamming distance on the quantum
computer. Schuld, Fingerhuth and Petruccione [168]
implemented a distance-based classifier using a quantum
interference circuit. In their approach, a new perspective
was proposed where the distance measure of a distance-
based classifier was evaluated using quantum interference
in quantum parallel instead of the usual approach of the
quantum machine merely mimicking the classical machine
learning methods. Their approach was demonstrated on a
simplified supervised pattern recognition task based on
binary pattern classification. i
In the following paragraph, we critically discuss one of
the research niche areas in which Africa has led after the
United States, Canada and China, specifically in Quantum
Computing machine learning research. The South Africa
Quantum Technology Initiative (SA QuTI) was established
in 2021 as a national undertaking that seeks to create con-
ducive conditions for a globally competitive research envi-
ronment in quantum computing technologies. Moreover, the
University of KwaZulu-Natal has been leading in producing
significant research output in the quantum machine learning
research domain, championed by Professor Petruccione. A
more detailed discussion of the quantum computing research
in presented next. i
The kernel-based machine learning method is another
aspect of machine learning where quantum computing has
been applied for data analysis application areas. The ability
of quantum computing to efficiently manipulate exponen-
tially large quantum space enables the fast evaluation of the
kernel function more efficiently than classical computers. Blank et al. [169] presented a compact quantum circuit for
constructing a kernel-based binary classifier. Their model
incorporated compact amplitude encoding of real-valued
data, which reduced the number of qubits by two and lin-
early reduced the number of training steps. Another kernel-
based quantum binary classifier was presented by Blank
et al. [170]. 4.3.11 Climate System Their distance-based quantum classifier has its
kernel designed using the quantum state fidelity between the
training and the test data so that the quantum kernel can be
systematically tailored with a quantum circuit. The training
data can be assigned arbitrary weight, and the kernel can be
raised to arbitrary power. 4.5 Renewable Energy In renewable energy and bioprocess modelling, Kana et al. [175] reported on the modelling and optimization of biogas
production on mixed substrates of sawdust, cow dung,
banana stem, rice bran and paper waste using a hybrid learn-
ing model that combines ANN and Genetic Algorithm. In
another study, Whiteman and Kana [176] investigated the
relevance of ANN in modelling the relationships between
several process inputs for fermentative biohydrogen pro-
duction and, after that, they suggested that the ANN model
is more reliable for navigating the optimization space rela-
tive to the different parameters at play for the biohydrogen
production system. The authors Sewsynker et al. [177] also
reported the use of ensembles of ANNs in the modelling
of biohydrogen yield in microbial electrolysis cells. The
study showed that the employed ANNs model could accu-
rately model the non-linear relationship between the phys-
icochemical parameters of microbial electrolysis cells and
hydrogen yield due to the ANNS capability to successfully
navigate the optimization window in microbial electroly-
sis cell scale-up processes. ML has been used for multi-
objective intelligent energy management for the microgrid
to improve efficiency in microgrid operation [178]. A hybrid
ML technique has been used for predicting solar radiation
based on meteorological data [80] with an analysis of the
influence of weather conditions in different regions of Nige-
ria. A machine learning model for predicting the daily global The diverse applicability and techniques promoting the
use of AI systems have received more research efforts from
ML. The increasing use of ML algorithms and their sub-
sidiary methods, such as DL, has further shown the com-
putational power of CNN, RNN, LSTM and hybrid models. These models have demonstrated outstanding performance
in pattern recognition, classification, feature extraction,
segmentation and other learning approaches. Interestingly,
while current studies and state-of-the-art are majoring
in hybridizing sequence models such as RNN with pixel
models such as CNN for multimodal computation, little is
mentioned on machine reasoning. The descent of machine
reasoning from the aspect of knowledge representation and
reasoning may not be directly associated with machine learn-
ing. Still, the successful integration of these two branches
of AI holds the possibility for achieving high-performing
systems in the near future. Machine learning, on the one
hand, allows for fine-tuning models and their parameters in
a manner that sets those parameters to enable the machine
to behave in a manner simulated by a human. 4.4 Quantum‑Based Machine Learning Research Their algorithm produced the same result as the least
square optimisation method for classical linear regression in
a logarithmic time dependent on the feature vector's number i
Pattern classification is one of the major tasks under
supervised machine learning. Most quantum machine
learning algorithms are built to address this area of
machine learning to extend or improve the classical ver-
sion. Schuld, Sinayskiy and Petruccione [167] used the 3 4199 Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… solar radiation was designed in Morocco by Chaibi et al. [179]. solar radiation was designed in Morocco by Chaibi et al. [179]. N and independent of the training dataset size if presented
as quantum information. as quantum information. In Schuld and Petruccione [173], the authors introduced
the quantum ensembles of quantum classifiers with parallel
execution of each quantum classifier and the resulting com-
bined decision accessed using a single qubit measurement. An exponentially large machine learning ensemble increases
the performance of individual classifiers in terms of their
predictive power and the ability to bypass the need for the
training session. The ensemble was designed in the form
of a state preparation scheme to evaluate each classifier's
weight. Their proposed framework permits the exponential
combination of many individual classifiers that require no
training, like the classical Bayesian learning, and is credited
with a quantum computing learning that is optimization-free. In most kernel-based quantum binary classifiers, the algo-
rithms require an expensive, repetitive procedure of quantum
data encoding to estimate an expectation value for reliable
operation resulting in high computational cost. Park, Blank
and Petruccione [174] proposed a robust quantum classifier
that explicitly calculates the number of repetitions necessary
for classification score estimation with a fixed precision to
minimize the program resource overhead. In Schuld and Petruccione [173], the authors introduced
the quantum ensembles of quantum classifiers with parallel
execution of each quantum classifier and the resulting com-
bined decision accessed using a single qubit measurement. An exponentially large machine learning ensemble increases
the performance of individual classifiers in terms of their
predictive power and the ability to bypass the need for the
training session. The ensemble was designed in the form
of a state preparation scheme to evaluate each classifier's
weight. 4.4 Quantum‑Based Machine Learning Research Their proposed framework permits the exponential
combination of many individual classifiers that require no
training, like the classical Bayesian learning, and is credited
with a quantum computing learning that is optimization-free.i 4.6 Prospects, Challenges, and Recommendations The prospects of ML research in Africa are enormous. It also
has challenges, such as bioinformatics research in Africa
being limited by the availability of diverse and high-vol-
ume biomedical data for accurate analysis [101]. As data
is central to ML, the Human Heredity & Health in Africa
(H3Africa) consortium is championing efforts at generating
and publicly publishing large genomics datasets of Africans
[180]. Another obstacle is the lack of a computing backbone
which includes internet connectivity and cloud computing,
which leads to data outsourcing to the developed world
[181]. In most kernel-based quantum binary classifiers, the algo-
rithms require an expensive, repetitive procedure of quantum
data encoding to estimate an expectation value for reliable
operation resulting in high computational cost. Park, Blank
and Petruccione [174] proposed a robust quantum classifier
that explicitly calculates the number of repetitions necessary
for classification score estimation with a fixed precision to
minimize the program resource overhead. Similarly, the prospects of ML will be inactive if appro-
priate investments in this direction are not made. Also,
teaching AI techniques, including ML, must be improved
and sustained. An adequate legal framework must be in place
to ensure ethical research and innovative development [182]. A framework for support and collaboration with foreign
agencies must be encouraged. For instance, the strategic
partnership between the Smart Africa alliance and the Ger-
man Ministry for Economic Cooperation and Development
aims to support Africa's development through digital innova-
tions [106, 183]. 4.5 Renewable Energy On the other hand, machine reasoning provides means for
formalising the existing body of knowledge siloed away in
legacy systems for achieving reasoning and inference. Com-
bining these two aspects of machine automation will pro-
mote what is termed neuro-symbolic systems, which allows
for neural networks and rules with formalized knowledge to 1 1 3 4200 A. E. Ezugwu et al. is in the area of crime monitoring and surveillance. For the
latter, the progress made in Computer Vision combined with
the Internet of Things (IoTs) has already provided for the
deployment of facilities to aid the state's surveillance system
and the law enforcement commissions. The former crime
detection and monitoring concept will benefit from recent
deep learning-driven natural language processing (NLP)
methods to analyze a pool of data floating on different social
media platforms and other text-driven systems for effective
crime detection. Motivated by the increasing hosting of deep
learning indaba conferences in Nigeria, Tunisia and South
Africa, with most of them promoting DL-NLP, there is now
a greater prospect of the application of these methods to
crime detection and monitoring. In addition to this, this DL-
NLP method showed that the rich multi-lingual formation
across all tribes and peoples in Africa could interact more
effectively and develop information-sharing mechanisms
through the use of machine translation. For instance, it is
well known that peoples speak languages like Hausa, Swa-
hili, Yoruba, Arabic, and isiZulu in different countries. The
adoption of machine translation will therefore help to build
on this communication skill and close gaps. Lastly, with the
plethora of research outcomes in medical image analysis and
AI-driven computer-aided diagnosis (CAD) systems, health-
care delivery and medical sciences will receive a boost in
health centres across Africa. interface in a manner to drive new state-of-the-art AI appli-
cations. We motivate for redirection of study in AI, ML,
and DL among African researchers to consider this aspect
of learning and reasoning. g
g
Another prospective integration of branches of AI
which promises to promote the discovery of super intelli-
gent systems is the application of clustering and optimiza-
tion methods to the models of DL and deep reinforcement
learning (DRL). Research in the design of DRL models is
now yielding and controlling self-driving cars, fully auto-
mated systems, robotics and other aspects of autonomous
systems. 4.5 Renewable Energy Although DRL draws from the concept of DL, we
consider that identifying some features in DL models (e.g. CNN, RNN, LSTM, GRU and their hybrids) and effectively
integrating them with DRL will uncover some outstanding
high-level performance with regards to machine intelligence. Researchers in Africa are likely to develop an interesting
outcome in this aspect, considering their progress in using
these models in their current isolated form of use. Moreover,
clustering and metaheuristic methods promise to provide
relevant and hardcore optimization solutions to improve the
integration of the hybrids mentioned earlier in this para-
graph. Of course, we have seen several usages of metaheuris-
tic methods in DL models and with the increasing use of
clustering methods. This study motivates a way forward for
an in-depth look into the possible interfacing of DRL, DL
and some clustering methods with the use of optimization
techniques for bolstering performance and computational
cost. A current challenge which needs to be addressed to pro-
mote research in ML in Africa is an intensive and inten-
tional investment in computational infrastructure. ML and
DL experiments demand high computational power with
the requirement for memory and graphical processing units
(GPU), and reliable power grids. Stakeholders and govern-
ment must integrate their thinking and resources to build
a cohesive and robust computational infrastructure to help
support researchers' efforts during experimentation and
deployment. This is necessary to allow for rigorous testing
and experimentation of new models capable of becoming
new state-of-the-art globally. Moreover, the sustenance of
startup hubs, as seen in Morocco, Nigeria, Ghana, Kenya
and South Africa, needs to be promoted to allow for the
convergence of test hubs for AI solutions being developed
by African youths. The applicability of the resulting intelligent systems from
the current and future state-of-the-art in AI, ML and DL is
still in its infancy stage in Africa. The COVID-19 pandemic
demonstrated that Africa still lags behind in adopting some
of the research outcomes from its researchers. Although the
effect of the pandemic is considered not to be very desta-
bilizing when compared with other continents, the lesson
that must be learnt is that Africa must prepare for a future
pandemic by leveraging on the research outcome coming
from research centres in Africa. Therefore, this holds pros-
pects and challenges that can spur on or open up new inter-
esting research areas. 4.5 Renewable Energy For instance, consider applying ML
methods to building smart cities across Africa. This will
draw from significant AI methods and systems successfully
designed and developed for smarting out all infrastructures
and facilities in such cities. Consider also the application
of research efforts in Computer Vision to the challenge of
aiding Africa's transport and communication (T&C) system. Firstly, the pedestrian system must be automated and inte-
grated with the T&C system for an effective AI-driven com-
puting network. We advocate for state-sponsored research
in this direction as it holds the prospect of improving road
connectivity and trade across the continent. Another inter-
esting aspect of AI's applicability to Africa's peculiarities 5 Conclusions Machine learning evolved as a branch in AI, focusing on
designing computational methods and learning algorithms
that model humans' natural learning patterns to address
real-life problems where human capability is limited or
restricted. This paper presents a background study of ML
and its evolution from AI through ML to DL, elaborating on
the various categories of learning techniques (supervised, 3 3 Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis… 4201 articles are classification, deep learning, feature extraction
and random forest. unsupervised, semi-supervised and reinforcement learning)
that have evolved over the years. It also presents the con-
tribution of different ML researchers across major African
universities from niche areas or multi-disciplinary domains. Furthermore, a review of machine learning techniques
and their applications in Africa in recent years was pre-
sented, identifying the main branches of ML and their off-
shoot disciplines. The nine most significant machine-learn-
ing application areas in Africa were identified and discussed. Research on quantum implementations of machine learning
algorithms in Africa for performance improvement of the
classical machine learning techniques was also reviewed. Moreover, quantum machine learning is one area of interest
in ML research which has positively projected the image
of African research scholars from the University of Kwa-
Zulu-Natal and has equally attracted global attention from
quantum computing enthusiasts. Finally, the prospects and
challenges with recommendations regarding ML research in
Africa were discussed in detail. Moreover, a bibliometric study of machine learning
research in Africa is presented. In total, 2761 machine learn-
ing-related documents, of which 89% were articles with at
least 482 citations, were published in 903 journals in the Sci-
ence Citation Index EXPANDED from 54 African countries
between 1993 and 2021. There are 12 topmost frequently
cited documents, of which five were review articles. Signifi-
cant interest in machine learning research in Africa began
in 2010, with the number of articles increasing slightly
from 14 to 98 in 2017 and which then increased with a huge
leap to 1035 articles by 2021. The highest article citation
was recorded in 2013. The top four productive categories
in the Web of Science, where more than 100 articles were
published, include “electrical and electronic engineering”,
“information systems computer science”, “artificial intel-
ligence computer science”, and “telecommunication”, each
recording 20%, 18%, 14% and 12% of the total number of
articles respectively. Acknowledgements NA. Funding Open access funding provided by North-West University. NA. Data Availability All data generated or analyzed during this study are
included in this article. The top five journals with IF2021 of more than 60 pub-
lished six of the articles: World Psychiatry (2), Nature (1),
Nature Energy (1), Nature reviews (1) and Science (1). Inter-
national collaborative articles recorded the highest number
of articles, 74% involving 43 African countries and 103
non-African countries, while the remaining single-country
articles were from 16 African countries. Egypt dominated
with 31% of the total article publication, 29% being single-
country articles and 32% being internationally collabora-
tively published. Ten African countries had no publica-
tion in machine learning-related articles, while 64% of the
remaining countries had no single-country articles. Egypt
and South Africa had similar development trends, but Egypt
recorded a noticeably sharp increase in the last three years. Cairo University in Egypt ranked top among the most pro-
ductive African institutions, with the University of Kwazulu-
Natal in South Africa ranking top in three of the six publica-
tion indicators. King Saud University in Saudi Arabia tops
the list of the five non-African institutions with 30 or more
inter-institutionally collaborative articles with Africa. Ethical Approval NA. Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 5 Conclusions The most productive journal is IEEE
Access, with 192 articles (7.8%). Declarations Competing interests The authors declare that there is no conflict of
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Recommendations for the management of sarcoptic mange in free–ranging Iberian ibex populations
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Animal biodiversity and conservation
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Recommendations
for the management of sarcoptic mange
in free–ranging Iberian ibex populations
J. Espinosa, J. M. Pérez, A. Raéz–Bravo, J. Espinosa, J. M. Pérez, A. Raéz–Bravo,
P. Fandos, F. J. Cano–Manuel, R. C. Soriguer,
J. R. López–Olvera, J. E. Granados Espinosa, J., Pérez, J. M., Baéz–Bravo, A., Fandos, P., Cano–Manuel, F. J., Soriguer, R. C., López–Olvera, J. R., Granados, J. E., 2020. Recommendations for the management of sarcoptic mange in free–ranging Iberian
ibex populations. Animal Biodiversity and Conservation, 43: 137–149, https://doi.org/10.32800/abc.2020.43.0137 Abstract Recommendations for the management of sarcoptic mange in free–ranging Iberian ibex populations. In recent
decades, sarcoptic mange has become the main driver of demographic changes in Iberian ibex (Capra pyrenaica)
populations in the Iberian Peninsula. Given this species' economic and ecological importance, priority must be
given to management measures aimed at limiting the effects of this disease. However, despite the wealth of
research on sarcoptic mange in ibex, no common patterns of action are yet available to manage this disease
under field conditions. The lack of national and international protocols aimed at controlling sarcoptic mange has
favoured the spontaneous emergence of various disease management initiatives in Spain. However, very little
information is available concerning this trend and what there is tends to be available only as 'grey literature' or is
consigned to the memory of local observers. Traditional strategies designed to combat this disease include the
administration of medicated feed and the non–selective culling of mangy ibex. Here, we propose a management
approach that takes into account aspects relating to the ecology and conservation of ibex populations, as well
as public–health–related factors. Our recommendations are based on knowledge of the disease and host–para
site interaction, and aim to promote long–term advances in its control. Moreover, we discuss the efficacy of the
measures traditionally used in mange management. The overall aim is to encourage debate between wildlife
managers and motivate the development of alternative management strategies. Key words: Capra pyrenaica, Conservation, Management strategies, Sarcoptes scabiei, Wild population 137 137 Animal Biodiversity and Conservation 43.1 (2020) Resumen Recomendaciones para el manejo de la sarna sarcóptica en poblaciones silvestres de cabra montés. En las últimas
décadas, la sarna sarcóptica se ha convertido en la principal causa de los cambios demográficos en las poblaciones
silvestres de cabra montés (Capra pyrenaica) de la península ibérica. Dada la importancia ecológica y económica de
esta especie, se debe dar prioridad a las medidas de gestión destinadas a limitar los efectos de esta enfermedad. Sin
embargo, a pesar del gran número de estudios que existen sobre la sarna sarcóptica en la cabra montés, actualmente
no hay ningún protocolo de actuación común para el manejo de esta enfermedad sobre el terreno. La ausencia de
protocolos nacionales e internacionales destinados a controlar la sarna sarcóptica ha favorecido la aparición espontánea
de diversas iniciativas de gestión en España. Sin embargo, existe muy poca información sobre esta tendencia y la
que hay solo suele estar disponible en la literatura gris o en la memoria de los observadores locales. Algunas de las
estrategias tradicionales diseñadas para combatir esta enfermedad son la administración de piensos medicados y el
sacrificio generalizado de los animales afectados. En este trabajo, proponemos un enfoque de gestión que tenga en
cuenta aspectos relacionados con la ecología y la conservación de la cabra montés, además de factores relacionados
con la salud pública. Nuestras recomendaciones se basan en el conocimiento de la enfermedad y la interacción entre
el parásito y el hospedador y tienen por objeto impulsar progresos a largo plazo en su control. Además, analizamos
la eficacia de las medidas utilizadas tradicionalmente en el manejo de la enfermedad. El objetivo general es fomentar
el debate entre los gestores de fauna silvestre y motivar la elaboración de estrategias de gestión alternativas. alabras claves: Capra pyrenaica, Conservación, Estrategias de gestión, Sarcoptes scabiei, Poblacione
vestres © [2020] Copyright belongs to the authors, who license the
journal Animal Biodiversity and Conservation to publish the
paper under a Creative Commons Attribution 4.0 License. ISSN: 1578–665 X
eISSN: 2014–928 X ISSN: 1578–665 X
eISSN: 2014–928 X Espinosa et al. ORCID ID: J. Espinosa: 0000-0002-9036-1402; J.M. Pérez: 0000-0001-9159-0365; A. Ráez-Bravo: 0000-
0002-3190-1659; P. Fandos: 0000-0002-9607-8931; R. C. Soriguer: 0000-0002-9165-7766; J. López-Olvera:
0000-0002-2999-3451; J. E. Granados: 0000-0002-9787-9896 Introduction sarcoptic mange has led to the emergence in Spain of
spontaneous disease management initiatives that lack
any consensus regarding which strategies are the most
appropriate. Traditional strategies include the adminis
tration of medicated feed or the non–selective culling of
mangy ibex. However, many management techniques
generate serious social conflicts between animal rights
activists, hunters and the local environmental agencies
in charge of hunting activities. Furthermore, very little
information is available on this question and what is
available is generally either 'grey literature', that is,
unpublished reports and conference proceedings, or
it simply resides in the memories of local observers
(Sánchez–Isarria et al., 2007a, 2007b). For this reason,
we believe that it is essential to draw up a series of
proposals for improved management and control of
the spread of mange in the Iberian ibex. Awareness of the importance of actively managing
infectious diseases in wild animals is a relatively novel
phenomenon. Until recently, the general attitude was
that 'nature can manage on its own'. However, the
presence of humans and the enormous pressure they
exert on the environment as a means of satisfying their
requirements distorts this natural balance and artificial
control measures are needed (Lyles and Dobson,
1993). Two good examples of such distortions are the
elimination of large predators and the loss of biodiver
sity (Packer et al., 2003; Keesing et al., 2006). In many
zones, this has led to an unsustainable overabundance
of wild animals in their chosen habitats, which creates
ideal conditions for the flare–up of disease (Rossi et
al., 2005; Gortázar et al., 2006; Vicente et al., 2007). If these diseases are zoonotic in character or imply
a threat to human economic activities, human action
becomes inevitable as, for example, in the cases of
rabies in wild carnivores (Pastoret and Brochier, 1999),
classical swine fever in wild boar (Kaden et al., 2000)
and tuberculosis in badgers (Woodroffe et al., 2005). Furthermore, the emergence of virulent forms of in
fectious agents or highly susceptible hosts may also
jeopardize the structure of wild populations (Woodroffe,
1999), as occurred in the case of sarcoptic mange
caused by the Sarcoptes scabiei mite. Introduction p
g
We believe that the selection of the most appropriate
management techniques requires a clear understan
ding of the cause and ecology of this disease, as
well as full knowledge of the course of the disease
in individual ibex and the population biology of the
parasite–host interaction. In light of the four catego
ries used for the management of wildlife diseases
(prevention, control, eradication and doing nothing
i.e. laissez–faire) (Wobeser, 1994, 2002; Artois et al.,
2001, Artois, 2003), here we propose action that lies
halfway between laissez–faire and control, given that
prevention and eradication under field conditions is an
extremely complex task. We use published scientific
evidence on sarcoptic mange in the ibex to develop
a more 'ecological' approach to the management of
this disease, which we believe is the best strategy
for both the conservation of the species and future
prevention. Additionally, we discuss the effectiveness
of the measures traditionally used in mange manage
ment. We hope that this work will stimulate a debate
among wildlife managers and motivate the development
of alternative management strategies. Our aim is to
promote a consensus regarding the best measures
to adopt when confronted with sarcoptic mange, while
ensuring optimal conservation of ibex. y
In the Iberian ibex (Capra pyrenaica), sarcoptic
mange is at the root of the most serious demographic
changes affecting this mountain ungulate in the Iberian
Peninsula (Fandos, 1991; Pérez et al., 1997), to the
extent that representative populations have become se
verely depleted and others are currently threatened (e.g. ibex populations in the Ports of Tortosa and Beceite and
in the Game Reserve of Muela de Cortes) (unpublished
data). Although sarcoptic mange is a constant threat to
all populations of this mountain ungulate, currently this
disease has not curbed the demographic trend of this
species. However, the ecological, economic and social
importance of the ibex (Granados, 2001), together with
the sanitary risks that sarcoptic mange poses, obliges
authorities and wildlife managers to instigate man
agement control measures. One of the great inherent
difficulties is that, like other infectious diseases (Kock
et al., 2018), mange epidemics break out unexpectedly,
often for obscure reasons (Pence and Ueckermann,
2002). Introduction As well, the pathogenesis of sarcoptic mange
(in terms of morbidity, mortality and population effects)
generally varies greatly from one affected area to an
other (Fandos, 1991; Mörner, 1992; Pérez et al., 1997;
Fernández–Morán et al., 1997) and its effects are diffi
cult to predict in affected populations for the first time. Given that the eradication of this disease is practically
impossible (Wobeser, 2002), attempts are made to
reduce its impact to 'tolerable' levels using a variety of
control strategies areas; as such, management tasks
designed to combat this disease are extremely complex. D
it
th
t
f
h th t h
b Resumen 138 Received: 3 IX 19; Conditional acceptance: 10 XII 19; Final acceptance: 10 I 20 Received: 3 IX 19; Conditional acceptance: 10 XII 19; Final acceptance: 10 I 20 José Espinosa, Department of Animal Health, Instituto de Ganadería de Montaña (IGM), CSIC–ULe, Facultad
de Veterinaria, Campus de Vegazana s/n., 24071 León, Spain.– Jesús María Pérez, Departamento de Biología
Animal, Biología Vegetal y Ecología, Universidad de Jaén, Campus Las Lagunillas s/n., 23071 Jaén, Spain. –Arián
Ráez–Bravo, Jorge Ramón López–Olvera, Wildlife Ecology & Health group (Wild&EH) and Servei d’Ecopatologia
de Fauna Salvatge (SEFaS), Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat
Autònoma de Barcelona (UAB), 08193, Bellaterra, Barcelona, Spain.– Paulino Fandos, Agencia de Medio Ambiente
y Agua, Isla de la Cartuja, E–41092 Sevilla, Spain.– Francisco Javier Cano–Manuel, Departamento Actuaciones
Forestales, Delegación Territorial, Consejería de Agricultura, Pesca, Ganadería y Desarrollo Sostenible, Granada,
Spain.– Ramón Casimiro Soriguer, Estación Biológica de Doñana (CSIC), Av. Américo Vespucio, s/n., E–41092
Sevilla, Spain.– José Enrique Granados, Espacio Natural Sierra Nevada, Carretera Antigua de Sierra Nevada
km 7, E–18071 Pinos Genil, Granada, Spain. Corresponding author: José Espinosa. E–mail: jespic@unileon.es Animal Biodiversity and Conservation 43.1 (2020) 139 Selective removal of infested ibexes From a clinical point of view, under natural conditions
the multi–systemic clinical picture is severe and entails
a very marked reduction in body condition, disorders
of haematological and biochemical parameters (Pérez
et al., 2015), septicaemic processes (Espinosa et al.,
2017d), oxidative stress phenomena (Espinosa et al.,
2017c), and an increase in inflammation biomarkers
causing tissue damage in dermal and non–dermal tis
sues (Raéz–Bravo et al., 2015; Espinosa et al., 2017d),
all of which greatly reduce survival possibilities and/
or hamper the recovery of ibexes in chronic phases
of disease. For ethical and humanitarian reasons, the
ending of the suffering of infected animals is necessary. In addition, ibex that have reached these stages of the
disease can be a direct or indirect potential source of
infestation for the rest of the population (Arlian et al.,
1984; Pérez et al., 2011). Thus, unlike a non–inter
vention (laissez–faire) strategy (Wobeser, 2002), our
low–level intervention approach will help reduce the
risks of mange transmission within a population.i Pro–active management measures include actions
that aim to prevent future outbreaks of the disease
and/or reverse the effects of mange epidemics. This group of techniques includes research, habitat
improvement and the setting up of infrastructure. Furthering knowledge of pathological, immunological
and epidemiological aspects of this disease improves
our understanding of its development and helps de
termine in a coherent fashion the best measures for
reducing its impact (Espinosa, 2018). Other important
preventive measures in mange management in ibex
include the restoration of pastures, the sowing of fo
rage in time of scarcity or the modification of drinking
fountains, aimed at strengthening the health status
of the population; the veterinary control of livestock
and safe translocations of ibex, to reduce the risk of
mange transmission between domestic herbivores and
ibex and/or disease–free ibex populations (Fandos
1991; Pérez et al., 1996); or the establishment of a
stock reservoir of ibex in order to combat massive
mortality outbreaks or for the strengthening some of
ibex populations (Espinosa et al., 2017b). Sarcoptic mange has side–effects that, in final
phases of the disease, negatively affect the reproduc
tive physiology of both male and female ibex (Sarasa
et al., 2011; Espinosa et al., 2017a) and hinders their
reproductive success. This makes them ineffective in
prolonging the species and therefore unable to trans
mit to their offspring any type of response developed
against the disease. Preliminary considerations Initially, it is important to highlight certain aspects of
the disease that will serve as premises in control stra
tegies: (1) As a parasitic disease whose main mode of
transmission is direct contact, sarcoptic mange can be
categorized as a density–dependent process (Pence
and Windberg, 1994; León–Vizcaino et al., 1999). (2)
The clinical course in affected individuals (and the
refore its effect on populations) is variable (Fandos,
1991; Górtazar et al., 1998; González–Candela et al.,
2004). Effects will be conditioned by intrinsic factors
relating to each individual and/or population (sex, age,
genetics, health status, previous contact with the mite,
etc.) and/or extrinsic factors (time of year, population
density, infective dose, availability of trophic resources,
etc.) (Rossi et al., 2007; Sarasa et al., 2010; López–
Olvera et al., 2015; Pérez et al., 2017). (3) Except in
residual or highly fragmented populations, in which the Despite the amount of research that has been
carried out on sarcoptic mange in the ibex, no common
evidence–based management strategy designed to
combat this disease under field conditions exists. This
absence of any national or international protocol for 140 Espinosa et al. provide more long–term benefits and better protection
against future mange outbreaks. capacity to respond to changes is low (Skerratt, 2005),
although mortality may at first be high (in many cases
favoured by elevated densities), initial appearances
can be deceptive and have no significant long–term
effects on population dynamics (Pérez et al., 1997;
Little et al., 1998). In the interaction between sarcoptic mange and the
Iberian ibex, four pathological phases or periods have
been used to characterize the severity of infestations,
according to the percentage of skin surface affected:
0, ibexes without skin lesions; 1, skin surface affected
< 25 %; 2, skin surface affected > 25 and < 50 %; 3,
skin surface affected > 50 and < 75 %; and 4, skin
surface affected > 75 %) (León–Vizcaíno et al., 1999;
Pérez et al., 2011). Given this, our management pro
posals focus exclusively on the selective culling of ibex
in the chronic or final phases of the disease (phases
3 and 4). We rule out massive culling in mangy ibex
populations and attempts to control the disease in the
wild using pharmacological treatments. Selective removal of infested ibexes In addition, given that mange is
transmitted mainly by direct contact, recently born
young are likely to be infected and to increase the
affected population. This assumption is based on the
observation of very young ibex with sarcoptic mange
in herds with mangy adult specimens (Espinosa et al.,
2017a), as well as the finding of mangy carcasses
of juvenile ibex (J. E. Granados, pers. comm.).Thus,
severely ill ibexes, with a low reproductive capacity
and with a high risk of spreading the infestation to
the rest of ibex population are determining factors to
selectively remove these individuals and thus con
tribute to the control of the disease. Preliminary considerations As we argue
below, we believe that selective culling of ibexes in
the final phases of the disease is the most appropriate
and most reasonable measure for tackling sarcoptic
mange in the ibex. Given these considerations, the management
measures taken to limit the effects of sarcoptic mange
can be either active or pro–active. Active management refers to actions applied to
the affected ibex population and/or the environment if
sarcoptic mange is present in the ibex population and
there is a desire to reduce its impact. Given its speed
and effectiveness, host density reduction by culling is
the most commonly used method (Sánchez–Isarria et
al., 2007a). In the case of directly transmitted infections,
population reduction is based on the epidemiological
theory that –regardless of the severity of lesions–
the per capita rate of disease transmission and the
prevalence of the disease will grow as the population
increases (Wobeser, 2002; Maxwell et al., 2013). The
use of medicated feed containing Ivermectin or other
macrocyclic lactones placed at different points in the
wild has also been used as a mange management
strategy in free–ranging ibex populations (Sánchez–
Isarria et al., 2007b). Management proposals against sarcoptic
mange and discussion Here we suggest a series of alternative strategies
for managing sarcoptic mange in free–ranging ibex
populations. The selection of the most appropriate ma
nagement technique requires a clear understanding of
the ecology of the disease (including how the disease
affects the individual), as well as intimate knowledge of
the population biology of the parasite–host interaction. In the case of mange in ibex, we propose selective,
less invasive action based on scientific evidence that,
compared to traditional management measures, will Animal Biodiversity and Conservation 43.1 (2020) 141 Fig. 1. Iberian ibex with sarcoptic mange: A, female ibex in early phases of the disease; B, male ibex
with severe sarcoptic mange. Unlike the previous animal, according to our management proposals, this
ibex should be removed from the ibex population. A B Fig. 1. Iberian ibex with sarcoptic mange: A, female ibex in early phases of the disease; B, male ibex
with severe sarcoptic mange. Unlike the previous animal, according to our management proposals, this
ibex should be removed from the ibex population. Fig. 1. Cabras montesas afectadas por sarna sarcóptica. A, hembra de cabra montés en las primeras fases
de la enfermedad; B, macho de cabra montés con sarna sarcóptica intensa. A diferencia del animal ante
rior, de acuerdo con nuestras propuestas de manejo, este ejemplar debería ser eliminado de la población. In all cases, the selective culling of these ibex
must be carried out whenever possible by specialized
staff using firearms. Capture using anaesthetic darts
or systems of physical restraint that involves the
pharmacological administration of a lethal drug –and
therefore further manipulation– is a far more laborious
and costly process (e.g. additional staff, the transfer of
carcasses for incineration, etc.) with greater chances
of failure on the welfare ground (due to additional
capture–related stress; López–Olvera et al., 2009). The thorough disposal of the mangy skin of culled
ibex reduces the possibilities of disease contamination
and transmission and, unlike laissez–faire strategies
(Wobeser, 2002) that advocate leaving dead ibex in
the wild, eliminates a risk factor for the rest of the
ibex population and for sympatric species. ibex in regression stages and those that are resistant
to the disease. We believe that ibex in the initial and
intermediate stages of the disease (phases 1 and 2)
(León–Vizcaíno et al., 1999; Pérez et al., 2011) should
never be removed from the population (fig.1). Management proposals against sarcoptic
mange and discussion In the initial stages of the disease (Phases 1 and 2),
the identification of sarcoptic mange with binoculars or
telescopes can be confused in ibexes that, although
without being parasitized by S. scabiei, show altera
tions of the coat due the seasonally heavy shedder
(Valldeperes et al., 2019). Experimental infestations carried out on ibex from
the Sierra Nevada Natural Space showed a wide
variety of clinical responses varying from animals that
progressed to severe or chronic phases to others that
developed self–limiting clinic processes with mange
lesions covering less than 50 % of the body surface,
spontaneous regression lesions (move from phases
3–4 to 2–1) and even full recovery (Espinosa et al.,
2017c) (fig. 2). Furthermore, scientific evidence show
that a considerable proportion of mangy ibex recover
naturally (Alasaad et al., 2013). Similar results were
obtained in experimentally infested Northern chamois
(R. rupicapra) in the Alps (Menzano et al., 2002) Non–lethal control: drug treatment of infested animals Therefore, it
may never be possible to successfully implement
long–term sarcoptic mange control in free–ranging
ibex populations since dispensing drugs in this fas
hion implies no control of doses, no guarantee of
repeated individual treatment, and very little acquired
knowledge of effective therapeutic doses. It is also
important to know whether other sympatric species
act as reservoirs of the disease and contribute to its
maintenance in the target host population, as shown
in other multi–species models (Gakuya et al., 2012). We also believe that other ecological, ethical
and public health issues must be addressed when
contemplating the free dispensing of drugs in the
natural environment (Artois et al., 2011; Crozier and
Schulte–Hostedde, 2014). For example, the distribution
of medicated feed at specific points can involve the
aggregation of animals and therefore greater contact
and an increase in infestation rates (Anderson and
May, 1978). Environmental pollution via excreta with
antiparasitic residues can cause a loss of biological
diversity amongst invertebrate, vertebrate and microbial
fauna, which in turn can be competitors in exogenous
phases of other parasites (Verdú et al., 2018). Another
important aspect is the development of acquired
resistance by parasites to the chemicals present in
the medication, which, due to the selective pressure
in resistant organisms, is a clear risk in any program
dependent upon the continued and widespread use of
chemotherapy (Wobeser, 2002). This is significant not
only for the treatment of mange in the ibex but also
for the management of disease in domestic livestock
and even in humans. Other less well–known effects,
such as the teratogenic effects of Ivermectin intake
during pregnancy or lactation, must also be taken into
account (Bialek and Knobloch, 1999). When attempting to control sarcoptic mange
epizootics in ibex by administrating drugs orally, it
must be taken into account that many areas of the
natural habitat of the ibex are large and inaccessi
ble (Acevedo and Cassinello, 2009). Therefore, it
may never be possible to successfully implement
long–term sarcoptic mange control in free–ranging
ibex populations since dispensing drugs in this fas
hion implies no control of doses, no guarantee of
repeated individual treatment, and very little acquired
knowledge of effective therapeutic doses. It is also
important to know whether other sympatric species
act as reservoirs of the disease and contribute to its
maintenance in the target host population, as shown
in other multi–species models (Gakuya et al., 2012). Effects of massive lethal control of sarcoptic mange Social struc
ture disruption with increased movement and therefore
increased contact rate (Donnelly et al., 2006) may lead
to depopulation. This has been reported in the case
of bovine tuberculosis in cattle and European badgers
(Meles meles) (Woodroffe et al., 2006). Non–lethal control: drug treatment of infested animals The clinical management of wildlife is becoming incre
asingly frequent but is usually conducted only at the
population level since individual treatment is largely
impractical. Nonetheless, in tiny populations facing high
extinction risks, vaccination and individual treatment may
help manage a variety of infections (Wodroffe, 1999;
Wobeser, 2002). For example, successful treatment
against sarcoptic mange in a small population of Arctic
foxes (Alopex lagopus) and individual treatment with
anthelmintics in red grouse (Lagopus lagopus scoticus)
have been reported (Dobson and Hudson 1992; Borns
tein et al., 2001). Usually, it is impossible to capture and
treat an entire population and so not all management
programs have obtained conclusive results. Despite
showing positive effects at an individual level in some
cases, data on long–term population effects or re–infec
tion rates of treatment against mange in Mednyi arctic
foxes (Alopex lagopus semenovi), cheetahs (Acinonyx
jubatus) and mountain gorillas (Gorilla beringei beringei)
are inconclusive (Mwanzia et al., 1995; Goltsman et al.,
1996; Kalema–Zikusoka et al., 2002). In the same way,
following field trials the use of Ivermectin ‘bio–bullets’
to treat bighorn sheep (Ovis canadensis nelsoni) for
psoroptic mange was rejected as a realistic manage
ment tactic (Jessup et al., 1991). If, in tiny populations,
pharmacological treatment as a management strategy
proves not to be totally efficient, in larger populations
or over larger areas, such intervention may be inappro
priate, unsustainable or simply impractical. Part of the
problem in this regard is that the drugs that are used
have often not been tested extensively in free–ranging
wild animals, so that their actual efficacy is unknown. In addition, there is no consensus between the dose of
drug administered and the severity of the infestation of
the treated animals. Most of the studies consulted on
attempts to control mange in free–ranging populations
based their outcomes upon a few recaptured or remo
tely observed individuals and the long–term effects at
the population level were in most cases inconclusive
(see table 1). mortality (López Olvera et al., 2009). When attempting to control sarcoptic mange
epizootics in ibex by administrating drugs orally, it
must be taken into account that many areas of the
natural habitat of the ibex are large and inaccessi
ble (Acevedo and Cassinello, 2009). Effects of massive lethal control of sarcoptic mange Attempts to reduce or eliminate sarcoptic mange from
the population by culling all mangy ibex– regardless
of the severity of lesions– may also have unintended
consequences on the population. No effective results
for this type of management measure have ever
been reported. For example, the spread of mange in
Northern chamois (Rupicapra rupicapra) in the Eas
tern Alps (Italy, Austria and Slovenia) and Southern
chamois in the Cantabrian Mountains (Spain) could
not be controlled by culling visibly infected individuals
(Meneguz et al., 1996; Fernández–Morán et al., 1997). One of the problems of this technique is that, in the
event of epizootic outbreaks of disease, most of the
large–scale culls take place before epidemiological
studies of the response of the population to the disea
se are performed and so do not discriminate between As well, the loss of genetic diversity from the po
pulation through the removal of resistant ibex or those
in a recovery stage can have long–term negative con
sequences and even give rise to future, more severe
epidemics due to a loss of herd immunity (Ebinger et
al., 2011).The culling of diseased animals can even
select for increased virulence as it means that there
will be a greater number of relatively more susceptible
hosts available for pathogens; this, in turn, stimulates
pathogens to transmit more quickly to susceptible hosts
to avoid being culled along with their hosts (Choo et 142 Espinosa et al. Captive mangy ibex treated with Ivermectin via sub
cutaneous injection needed four weeks of treatment
before complete parasitological and clinical recovery. However, no positive results were obtained in chroni
cally ill ibex (León–Vizcaíno et al., 2001), similarly as
in other species (Skerratt, 2003; Kido et al., 2014). We
consider that in ibex with severe sarcoptic mange the
multi–systemic complications derived from the action
of the mites (Espinosa et al., 2017d, 2017c) require
additional treatment if the animal is to completely
recover, which include the provision of intravenous
fluids, antimicrobials and high–caloric nutrition. In
free–ranging ibex, parenteral antiparasitic treatment
is considered impractical for economic reasons, while
the capture and handling of severely affected ibex for
treatment purposes may often result in short–term
mortality (López–Olvera et al., 2009). al., 2003). Another counterproductive consequence is
that a reduction in population size by culling is often
offset by the effects of compensatory reproduction and
immigration (Caughley and Sinclair, 1994). Non–lethal control: drug treatment of infested animals p
(
y
,
)
We also believe that other ecological, ethical
and public health issues must be addressed when
contemplating the free dispensing of drugs in the
natural environment (Artois et al., 2011; Crozier and
Schulte–Hostedde, 2014). For example, the distribution
of medicated feed at specific points can involve the
aggregation of animals and therefore greater contact
and an increase in infestation rates (Anderson and
May, 1978). Environmental pollution via excreta with
antiparasitic residues can cause a loss of biological
diversity amongst invertebrate, vertebrate and microbial
fauna, which in turn can be competitors in exogenous
phases of other parasites (Verdú et al., 2018). Another
important aspect is the development of acquired
resistance by parasites to the chemicals present in
the medication, which, due to the selective pressure
in resistant organisms, is a clear risk in any program
dependent upon the continued and widespread use of
chemotherapy (Wobeser, 2002). This is significant not
only for the treatment of mange in the ibex but also
for the management of disease in domestic livestock
and even in humans. Other less well–known effects,
such as the teratogenic effects of Ivermectin intake
during pregnancy or lactation, must also be taken into
account (Bialek and Knobloch, 1999). In the Iberian Peninsula, attempts to control
sarcoptic mange outbreaks in ibex and chamois
(Rupicapra pyrenaica parva) (Fernández–Morán et
al., 1997) by dispersing feed treated with Ivermectin
is a commonly used management strategy. Taking
into account results in other species (Rowe et al.,
2019), we believe that this strategy is impractical as
to date there is no proof as to its effectiveness. In
fact, treatment with Ivermectin is likely not warranted
until designed studies have demonstrated its efficacy
in free–ranging Iberian ibex populations. The abuse of antiparasitic drugs can also affect the
nature of the parasite–host interaction. For example,
it is well known that previous contact with the mite
induces a more intense and effective immune response
in re–infestations (Sarasa et al., 2010). Thus, host–
parasite relationships can be modified by continuous Animal Biodiversity and Conservation 43.1 (2020) 143 Fig. 2. Iberian ibex experimentally infested with Sarcoptes scabiei showing a self–limiting process. Non–lethal control: drug treatment of infested animals A, development of the first lesions in the inoculation area (inter–scapular region); B, extension of the
lesions over 50 % of the body surface; C, regression of the lesions, leaving small lesion centres on the
rump; D, complete recovery of the animal with the disappearance of mangy lesions. To date this animal
has not developed any further lesions or clinical signs of disease (Espinosa et al., 2017c). A B
C D B A B B A D C D C Fig. 2. Iberian ibex experimentally infested with Sarcoptes scabiei showing a self–limiting process. A, development of the first lesions in the inoculation area (inter–scapular region); B, extension of the
lesions over 50 % of the body surface; C, regression of the lesions, leaving small lesion centres on the
rump; D, complete recovery of the animal with the disappearance of mangy lesions. To date this animal
has not developed any further lesions or clinical signs of disease (Espinosa et al., 2017c). Fig. 2. Cabra montés infectada experimentalmente con Sarcoptes scabiei que muestra un proceso de
infección autolimitante. A, aparición de las primeras lesiones en la zona de inoculación (región interesca
pular); B, extensión de las lesiones en el 50 % de la superficie corporal; C, regresión de las lesiones, que
dejan pequeños centros de lesión en la grupa; D, recuperación completa del animal con la desaparición
de las lesiones sarnosas. En la actualidad, este animal no ha vuelto a manifestar lesiones ni signos
clínicos de la enfermedad (Espinosa et al., 2017c). interference to the immune system as a result of the
lack of sufficient parasitic antigens able to produce
efficient and protective stimulation (Pedersen and
Fenton, 2015). Therefore, the development of a correct
immune response and the appearance of resilient
ibex may be delayed or interrupted. Even if treatment
does manage to eliminate mites from mangy ibex,
recovered animals will not acquire long–lasting immu
nity and re–infestation may occur (Pederson, 1984;
Wobeser, 2002). Another unintended consequence of
the non–selective use of antiparasitic drugs in the wild
is the possible modification of the balance with other
parasites in both healthy and mangy ibex (Pérez et
al., 2003; Thomas et al., 2005). Based on the above,
we believe that in free–ranging species the application
of uncontrolled antiparasitic treatment is inappropriate
since evidence of proven efficacy is lacking and the
re is no solid scientific base to support its use as a management measure. Non–lethal control: drug treatment of infested animals Until such scientific support is
obtained, this type of management measure should be
limited exclusively to the control of sarcoptic mange in
captive wildlife (Rowe et al., 2019) or domestic livestock
sharing territory with the ibex, thereby ensuring the
safe and efficient control of a significant risk factor for
the ibex population (Granados, 2001). On the other
hand, it will be interesting to evaluate the efficacy of
the new generation of isoxazoline parasiticides as an
alternative to the use of macrocyclic lactones in the
treatment of sarcoptic mange in ibex (Van Wick and
Hashem, 2019). For more information on the success
of such treatment in wild species, treated animals will
have to be fitted with radio–collars to guarantee better
individual monitoring. Finally, we believe that, bearing in mind the
considerations outlined above, the culling of mangy
ibex in chronic phases is justified. The public can be Espinosa et al. 144 Table 1. Summary of attempts to treat mange in free–ranging wildlife and reported results. Treatments
in captive wildlife are excluded: I, infestation (OM, Otodectic mange; PM, Psoroptic mange; SM,
sarcoptic mange). Table 1. Summary of attempts to treat mange in free–ranging wildlife and reported results. Treatments
in captive wildlife are excluded: I, infestation (OM, Otodectic mange; PM, Psoroptic mange; SM,
sarcoptic mange). Host species I Severity Treatment and doses
Mednyi artic fox
OM
Unreported
Alugan spray and ivermectin
(Alopex lagopus semenovi)
Unknow doses
Cheetah
SM
Mild to severe
Ivermectin: one doses
(Acinonyx jubatus) and others
(200 μg/kg SC)
Bighorn sheep
PM
Mild or severe
Ivermectin 'bio–bullet'. Non–lethal control: drug treatment of infested animals (Ovis canadensis)
Doses unknown strategy
Red fox
SM
Unreported
Ivermectin;
(Vulpes vulpes)
one doses (300 μg/kg SC)
Southern hairy nosed wombat SM
Mild or severe
Ivermectin:
(Lasiorhinus latrifrons)
one doses (300 μg/kg SC)
Bare–nosed wombat
SM
Mild to moderate
Ivermectin: two doses
(Vombatus ursinus)
(400–800 μg/kg SC) +
Amitraz: one doses
(0.02 5% topical wash)
Mountain gorilla
SM
Mild to severe
Ivermectin: one doses
(Gorilla beringei beringei)
(170–670 μg/kg IM) +
Antibiotic and
vitamin supplements
Hanuman langur
SM
Moderate
Tebrub: 30 doses (250 mg PO)
(Semnophitecus entellus)
Mebhydrolin: 30 doses (25 mg PO)
Ivermectin: one doses (1 mg/kg SC)
Chlorpheniramine maleate:
one dose (10 mg SC)
Vicuña
SM
Mild to severe
Ivermectin (unknown doses
(Vicugna vicugna)
Giraffe
SM
Moderate to severe Unreported
(Giraffa camelopardis)
Agile wallaby
SM
Moderate
Ivermectin:
(Macropus agilis)
two doses (300 μg/kg SC)
Gorilla
SM
Severe
Ivermectín: two doses
(Gorilla beringei beringei)
(200 mg/Kg SC) +
Antibiotic
Iberian ibex
SM
Mild to severe
Foxim: two doses
(Capra pyrenaica)
(500 mg/l topical wash) +
Ivermectin: two doses
(02 mg/kg SC)
Koala
SM
Unreported
Amitraz: two doses
(Phascolarcos cinereus)
(0.025 % topical wash)
p
g ) Animal Biodiversity and Conservation 43.1 (2020) 145 Tabla 1. Resumen de los intentos de tratar la sarna en especies de fauna silvestre y resultados
obtenidos. Quedan excluidos los tratamientos en cautividad: I, infestación (OM, sarna otodéctica; PM,
sarna psoróptica; SM, sarna sarcóptica). Effects on population Reference
Non–significant increase in cub survival; effect
Goltsman et al. (1996)
on population viability unknown
Recovery of some treated individual; inconclusive
Gayuka et al. (2012)
long–term population effects
Not reported, but dismissed as a management
Jessup et al. (1991)
Ineffective results both in the individual
Newman et al. (2002)
and in the population
Effective treatment only in animals with mild
Ruykys et al. (2013)
mange; population effects unknown
Recovery of some treated individual; population
Skerratt et al. (2004)
effects unknown
Recovery of all affected animals and mange
Kalema–Zikusoka et al. (2002)
control in the gorilla population
Recovery only of animals with parenteral
Chhangani et al. (2001)
treatment; population effects untested
Inconclusive treatment results
Gómez–Puerta et al. (2013)
Treated animals recovered. Certain success
Alasaad et al. (2012)
at the population level
Successful on an individual level; population
McLelland and Youl (2005)
effects not evaluated
Successful on an individual level and mange control
Graczyk et al. (2001)
in the gorilla population
Recovery of some treated mild mangy individual. Conclusions Parasites & Vectors, 6(1): 242. Parasites are natural parts of ecosystems and for this
reason the presence of parasites in hosts does not
necessarily imply that these wild populations are in
danger of disappearing. Wildlife is a societal resource
and provides ecological services that are vital for sus
taining economies and human health. However, some
times (as in the case of sarcoptic mange in Spanish
ibex) a parasitic disease causes the death of a part
of the population, thereby endangering the economic
activities (e.g. ECO–tourism, hunting) that are derived
from it. When attempting to control the disease and
limit its effects, a lack of information and the need to
make decisions quickly in a 'crisis' situation lead to the
application of strategies that are not appropriate for
the management of the disease. We believe that the
management of diseases in wild animals generally re
quires solid scientific evidence grounded on corrective
measures with potentially irremediable consequences
for the future of the wild population. Short–term specific
measures including pharmacological treatment and
mass culls are too costly, too limited in duration and
have little effect on overall population health. Given
that it is difficult to predict where and when the next
outbreak of sarcoptic mange will occur, further research
is needed on the real effectiveness of the different
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financed by the projects CGL2012–40043–C02–01,
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(MEC, Spanish Government) and by the PAIDI Re
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Novel Rht-1 dwarfing genes: tools for wheat breeding and dissecting the function of DELLA proteins
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Journal of experimental botany
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© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 354 | 354 | the number of seminal roots in maize co-map with the root developmental
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systems in sorghum and maize. Plant and Soil 333, 287–299. Insight Novel Rht-1 dwarfing genes: tools for wheat breeding and
dissecting the function of DELLA proteins The most widely | 355 believed that the effect of these mutations is to produce an
N-terminally truncated protein which cannot be bound by the
GID1–GA receptor, therefore resisting GA-mediated degra-
dation and acting to constitutively repress GA-responsive
growth and development. The severe dwarfing allele, Rht-
B1c, also contains a lesion in the N-terminal coding region,
which is predicted to have the same effect on perturbing
GA signalling. However, in this case, the increased stability
of RHT-B1C is due to a 30-amino acid insertion within the
GID1–GA binding domain (Pearce et al., 2011; Wu et al.,
2011). Although conclusive biochemical evidence is lack-
ing, the current consensus of opinion regarding the milder
GA-insensitive phenotype observed in Rht-B1b and Rht-D1b is caused by mutations in the homoeologous DELLA genes
Rht-B1 and Rht-D1 (Peng et al., 1999; Pearce et al., 2011). DELLAs are members of the GRAS family of transcrip-
tional regulators (Thomas et al., 2016), containing two distinct
domains: an N-terminal regulatory domain and a C-terminal
functional GRAS domain (Box 1). The N-terminal domain
is required for binding the GID1–GA receptor complex, a
process which ultimately triggers DELLA degradation and
promotes GA-responsive growth. The agronomically impor-
tant Rht-B1b and Rht-D1b semi-dwarfing alleles contain
mutations that introduce premature stop codons in the region
of these genes encoding the N-terminal GID1–GA binding
domain (Peng et al., 1999; Pearce et al., 2011; Box 1). It is Novel Rht-1 dwarfing genes: tools for wheat breeding and
dissecting the function of DELLA proteins Stephen G. Thomas utilized Rht dwarfing genes in worldwide wheat breeding
programmes are those containing lesions at the Rht-1 locus. These include the Rht-B1b and Rht-D1b semi-dwarfing alleles
which, at around the turn of the last century, were estimated
to be present in over 70% of wheat cultivated worldwide
(Evans, 1998). Remarkably, both of these Rht-1 homoeo-
alleles originated from the same Japanese variety, Norin-10,
and were successfully exploited in US wheat breeding pro-
grammes in the 1950s (Hedden, 2003; Wilhelm et al., 2013). Reduced height-1 (Rht-1) dwarfing alleles have provided
an essential breeding tool for increasing wheat grain
yields and providing lodging resistance under high inputs. In this issue (pages 443–455), Van De Velde et al. dem-
onstrate the potential of novel Rht-1 alleles for allowing
more flexible control of stature and preharvest sprouting
resistance in wheat breeding programmes. On a funda-
mental level, these alleles provide an important opportu-
nity to uncover the signalling events that are responsible
for improving these traits. Gibberellins (GAs) are plant hormones that promote
many aspects of vegetative and reproductive development,
including stem elongation and germination (Sponsel, 2016). The central regulators of the GA signalling pathway are
nuclear-localized DELLA proteins (DELLAs) that act to
repress GA-responsive growth through their physical asso-
ciation with transcription factors and other down-stream
components (Thomas et al., 2016). Bioactive GAs relieve the
DELLA growth repression by targeting their rapid degrada-
tion through a GID1–GA receptor-mediated signalling path-
way (Nelson and Steber, 2016). The Rht-1 dwarfing alleles are
known to reduce stem extension by causing partial insensitiv-
ity to GAs (Gale and Marshall, 1973). This altered response During the Green Revolution, the benefits of intensive agro-
nomic practices to increase wheat grain yields could only
be fully achieved when combined with varieties containing
Reduced height (Rht) dwarfing genes (Hedden, 2003). The
beneficial effects of these dwarfing alleles on grain yields
are twofold: first, they prevent excessive stem elongation in
response to high nitrogen fertilizer regimes that are prone to
make the crop susceptible to environmental damage through
lodging. Second, they allow a higher proportion of photosyn-
thate to be partitioned to the grain by increasing the number
of grains within the spikelets of the spike. Novel Rht-1 dwarfing alleles Despite the success of Rht-1 dwarfing alleles in controlling
wheat stature and grain yields, the current range of genetic
and phenotypic diversity is limited. An elegant screen per-
formed by Chandler and Harding (2013) has greatly extended
this diversity (Box 2). In this study they identified taller sup-
pressor lines, designated overgrowth (ovg) alleles, following
mutagenesis of a severely dwarfed line containing Rht-B1c. A total of 35 intragenic Rht-B1c suppressor alleles were iden-
tified, the majority of mutations resulting in amino acid sub-
stitutions within conserved regions of the functional GRAS
domain (Chandler and Harding, 2013; Van De Velde et al.,
2017; Box 1). As well as providing alleles that have the poten-
tial to allow breeders to precisely control stature over a wider
range, the nature of these mutations provides us with the tools
for understanding how DELLAs act to control diverse devel-
opmental processes. These studies demonstrate the exciting
potential of these alleles on both of these levels. Rht-1 alleles with differential effects on
GA-responsive traits During the past decade there have been many studies dem-
onstrating that DELLAs interact with a multitude of differ-
ent classes of proteins to repress aspects of GA-responsive
growth (Thomas et al., 2016). The regulatory mechanisms
that these associations affect also differ depending on the
interaction partner. Despite the large variety of DELLA
partners and the diverse regulatory mechanisms involved,
the majority of these studies have identified the GRAS
domain as responsible for mediating these associations and
exerting control. This is in agreement with the position of
ovg mutations causing amino acid substitutions, which To establish whether the ovg alleles represented useful alter-
natives to the classical dwarfing gene, Rht-B1b, Van De Velde
et al. (2017) selected fourteen for further phenotypic charac-
terization. Based on stem height these were classified as semi-
dwarfing or tall depending on whether they were shorter or Box 1. Rht-1 dwarfing mutations The upper panel shows an amino acid alignment of a region of the N-terminal regulatory
domains of the wheat, rice and Arabidopsis DELLAs. The blue bars and asterisks below the
alignment indicate conserved residues required for binding to the GID1–GA receptor (Murase
et al., 2008), targeting them for degradation and relieving growth repression. The positions of
mutations are shown in Rht-B1c (purple triangle indicating the site of a 30 amino acid inser-
tion) and Rht-B1b (green triangle indicating the introduction of a premature stop codon; Q64*). Translational reinitiation (potential sites are indicated by a green bar) is thought to produce an
N-terminally truncated RHT-B1B protein. The current model suggests that RHT-B1C and RHT-
B1B constitutively repress GA-responsive growth because they do not bind to the GID1–GA
complex and are therefore not degraded in response to the GA signal. The lower panel shows
a schematic diagram illustrating conserved domains within RHT-B1 and the predicted effect of
Rht-1 dwarfing mutations on the encoded proteins. The positions of amino acid substitutions
caused by the ovg missense mutations are indicated within the GRAS domain (vertical blue
lines) (Chandler and Harding, 2013; Van de Velde et al. 2017). 356 | compared to Rht-B1c is due to a lower level of accumulation
of the N-terminally truncated proteins produced by a process
of translational reinitiation (Peng et al., 1999). taller than Rht-B1b, respectively (Box 2). This allowed the
selection of three alleles (two semi-dwarfing and one tall)
whose agronomic potential was established following intro-
gression into several elite spring wheat cultivars that have
markedly different continental growing environments. Highly
consistent effects on stem length and other architectural char-
acteristics were observed in the presence of the ovg alleles. Importantly, no obvious detrimental effects on grain yields
were observed under field conditions. The stability that they
exert on these crop traits in different genetic backgrounds and
under diverse environments clearly demonstrates their poten-
tial in wheat breeding programmes. Box 2. Wheat overgrowth mutants The wheat overgrowth mutants (var. Maringa) were identified by mutagenizing the severe dwarf
line Rht-B1c and screening for M2-suppressor lines that displayed increased leaf elongation
or final height (Chandler and Harding, 2013). The screen resulted in the identification of many
new Rht-B1 dwarfing alleles that can be used for more precise control of wheat stem height. Illustrating this, the image shows two ovg mutants (Rht-B1c.32 and Rht-B1c.3) compared to
wild-type Rht-B1a (tall control), and the classical Rht-1 dwarfing lines Rht-B1b (semi dwarf)
and Rht-B1c (extreme dwarf). Picture courtesy of Karel Van De Velde. | 357 genome is that the presence of functionally redundant
homoeologues impedes the identification of recessive loss-
of-function alleles, probably obscuring genetic diversity
that can be exploited by wheat breeders. The recent devel-
opment of robust reverse genetics platforms for functional
genomics in wheat, including TILLInG populations (King
et al., 2015) and CRISPR/Cas-based mutagenesis (Wang
et al., 2014), raises the exciting prospect of unlocking this
genetic diversity. An advantage of polyploid genomes is
that mutagenesis events can be chemically induced at much
higher frequencies than those achieved in diploid species,
allowing the generation of increased genetic diversity (Parry
et al., 2009). The unparalleled collection of DELLA muta-
tions identified by Chandler and Harding (2013) clearly
emphasizes these benefits. It also illustrates the power of
novel screening strategies for generating improved genetic
diversity that is unavailable with conventional breeding
approaches. were identified solely within the region encoding the GRAS
domain of RHT-B1C (Chandler and Harding, 2013; Van
De Velde et al., 2017). Van De Velde et al. (2017) have used
the recently resolved crystal structure of the rice GRAS
protein OsSCL7 to model the effect of ovg mutations (Li
et al., 2016). Many of these substitutions are suggested to
occur within the interior of the DELLA protein, potentially
affecting interacting residues (Van De Velde et al., 2017). It
is tempting to speculate that the impact of these is to alter
the 3D structure and perturb interactions with down-stream
GA signalling components. To establish whether this is the
case, it will first be necessary to identify RHT-1-interacting
partners and then determine the impact of the ovg mutations
on these interactions. Based on our current understanding of how Rht-1
dwarfing genes cause GA insensitivity it might be reason-
able to expect that the impact of a particular allele has an
equivalent effect across different developmental processes
controlled by GAs. References Borrill P, Adamski N, Uauy C. 2015. Genomics as the key to unlocking
the polyploid potential of wheat. New Phytologist 208, 1008–1022. Chandler PM, Harding CA. 2013. ‘Overgrowth’ mutants in barley and
wheat: new alleles and phenotypes of the ‘Green Revolution’ DELLA gene. Journal of Experimental Botany 64, 1603–1613. Evans LT. 1998. Feeding the ten billion: plants and population growth. Cambridge University Press. Gale MD, Marshall GA. 1973. Insensitivity to gibberellin in dwarf wheats. Annals of Botany 37, 729–735. Gooding MJ, Uppal RK, Addisu M, Harris KD, Uauy C, Simmonds
JR, Murdoch AJ. 2012. Reduced height alleles (Rht) and Hagberg falling
number of wheat. Journal of Cereal Science 55, 305–311. Hedden P. 2003. The genes of the Green Revolution. Trends in Genetics
19, 5–9. Hirano K, Kouketu E, Katoh H, Aya K, Ueguchi-Tanaka M, Matsuoka
M. 2012. The suppressive function of the rice DELLA protein SLR1 is
dependent on its transcriptional activation activity. The Plant Journal 71,
443–453. King R, Bird N, Ramirez-Gonzalez R, Coghill JA, Patil A, Hassani-
Pak K, Uauy C, Phillips AL. 2015. Mutation scanning in wheat by exon
capture and next-generation sequencing. PLoS ONE 10, e0137549. Li S, Zhao Y, Zhao Z, Wu X, Sun L, Liu Q, Wu Y. 2016. Crystal structure
of the GRAS domain of SCARECROW-LIKE7 in Oryza sativa. The Plant
Cell 28, 1025–1034. Murase K, Hirano Y, Sun TP, Hakoshima T. 2008. Gibberellin-induced
DELLA recognition by the gibberellin receptor GID1. Nature 456, 459–463. Nelson SK, Steber CM. 2016. Gibberellin hormone perception: down-
regulating DELLA repressors of plant growth and development. In: Hedden
P, Thomas SG, eds. The gibberellins. Annual Plant Reviews, Volume 49. Chichester, UK: John Wiley and Sons, 153–187. Box 2. Wheat overgrowth mutants However, there is evidence that this is
not the case: a recent study of Rht-1 near-isogenic lines
has indicated that those alleles containing premature stop
codons, including the severe dwarf Rht-D1c, do not dra-
matically affect grain dormancy whereas Rht-B1c enhances
it strongly (Gooding et al., 2012). The characterization of
germination responses in the ovg mutants derived from Rht-
B1c clearly confirms these differences between alleles, with
the majority of them producing a level of dormancy that is
consistent with their effect on stature (Van De Velde et al.,
2017). In contrast, this correlation was not observed in
the Rht-B1b semi-dwarf controls, in which dormancy was
unaffected. This suggests an uncoupling of GA-regulated
developmental responses that are controlled by the RHT-1
repressors lacking 70 amino acids of the N-terminus (Van
De Velde et al., 2017). It is interesting to note that there is
evidence supporting a functional role for the N-terminus of
the rice DELLA SLR1 (Hirano et al., 2012). If the DELLA
N-terminus does have a functional role, what is perhaps
surprising is the absence of ovg missense mutations in this
region. A plausible explanation for this could be the design
of the original screen, which involved identifying enhanced
leaf or stem elongation (Chandler and Harding, 2013). If
feasible, a similar screen focusing on germination responses
may deliver alternative intragenic suppressor alleles. Nevertheless, from the perspective of crop improvement, it
is clear that the enhanced dormancy observed in the ovg
mutants provides the potential to increase resistance to pre-
harvest sprouting beyond that obtained with the currently
deployed Rht-1 alleles. Key words: DELLA proteins, dormancy, gibberellins, Green Revolution,
preharvest sprouting, Rht-1 dwarfing mutations, stem height, wheat
(Triticum aestivum). Journal of Experimental Botany, Vol. 68 No. 3 pp. 354–358, 2017
doi:10.1093/jxb/erw509 Journal of Experimental Botany, Vol. 68 No. 3 pp. 354–358, 2017
doi:10.1093/jxb/erw509 Key words: DELLA proteins, dormancy, gibberellins, Green Revolution,
preharvest sprouting, Rht-1 dwarfing mutations, stem height, wheat
(Triticum aestivum). Journal of Experimental Botany, Vol. 68 No. 3 pp. 354–358, 2017
doi:10.1093/jxb/erw509 Wheat: a new model plant Attempts aimed at manipulating specific signalling path-
ways to improve traits in bread wheat have been severely
hindered by its hexaploid nature and the lack of a reference
genome sequence. The recent availability of a near-com-
plete genome sequence now raises exciting possibilities for
quickly identifying genetic elements underlying these traits
(Borrill et al., 2015). An obvious problem with the hexaploid Parry MA, Madgwick PJ, Bayon C, et al. 2009. Mutation discovery for
crop improvement. Journal of Experimental Botany 60, 2817–2825. Pearce S, Saville R, Vaughan SP, et al. 2011. Molecular
characterization of Rht-1 dwarfing genes in hexaploid wheat. Plant
Physiology 157, 1820–1831. Peng J, Richards DE, Hartley NM, et al. 1999. ‘Green revolution’ genes
encode mutant gibberellin response modulators. Nature 400, 256–261. 358 | Sponsel VM. 2016. Signal achievements in gibberellin research: the second
half-century. In: Hedden P, Thomas SG, eds. The gibberellins. Annual Plant
Reviews, Volume 49. Chichester, UK: John Wiley and Sons, 1–36. Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu JL. 2014. Simultaneous editing of three homoeoalleles in hexaploid bread wheat
confers heritable resistance to powdery mildew. Nature Biotechnology 32,
947–951. Thomas SG, Blazquez MA, Alabadi D. 2016. DELLA proteins: Master
regulators of gibberellin responsive growth and development. In: Hedden
P, Thomas SG, eds. The gibberellins. Annual Plant Reviews, Volume 49. Chichester, UK: John Wiley and Sons, 189–228. Wilhelm EP, Boulton MI, Barber TES, Greenland AJ, Powell W. 2013. Genotype analysis of the wheat semidwarf Rht-B1b and Rht-D1b ancestral
lineage. Plant Breeding 132, 539–545. Wu J, Kong X, Wan J, et al. 2011. Dominant and pleiotropic effects of a
GAI gene in wheat results from a lack of interaction between DELLA and
GID1. Plant Physiology 157, 2120–2130. Van De Velde K, Chandler PM, Van Der Straeten D, Rohde A. 2017. Differential coupling of gibberellin responses by Rht-B1c suppressor alleles
and Rht-B1b in wheat highlight a unique role for the DELLA N-terminus in
dormancy. Journal of Experimental Botany 68, 443–455.
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A aplicação foliar de molibdênio na fase de enchimento de vagens do feijão-comum pode reduzir a qualidade da semente
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Revista Ceres
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cc-by
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Submetido em 23/04/2014 e aprovado em 24/06/2015.
1Epamig, Viçosa, Minas Gerais, Brasil. rfvieira@epamig.br; trazilbo@epamig.br; roberto.araujo@epamig.br
2Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brasil. adalgisa.prado@ufv.br
3Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brasil. miller.lehner@ufv.br
4Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brasil. rhaphael.silva@ufv.br
*Autor para correspondência: rfvieira@epamig.br RESUMO Com o objetivo de aprimorar a tecnologia de produção de sementes ricas em molibdênio (Mo), foram avaliados
os efeitos da aplicação foliar de 600 g ha-1 de Mo na qualidade fisiológica da semente de feijão. Em campo, essa
dose de Mo foi aplicada na fase V4 (terceira folha trifoliolada), ou parcelada de duas a quatro vezes e aplicada em
V4, R6 (floração), R7 e, ou, R8 (enchimento de vagens). A aplicação de 300 g ha-1 de Mo em R6 não influenciou a
qualidade da semente, em comparação com a do controle (90 g ha-1 de Mo aplicado em V4). Dos três tratamentos
em que 200, 255 ou 300 g ha-1 de Mo foram aplicados em R7, apenas com 255 g ha-1 o Mo reduziu a germinação e
vigor das sementes, em comparação com as do controle. Quando 200, 255 ou 300 g ha 1 de Mo foram aplicados em
R8 (cinco tratamentos), geralmente o Mo reduziu a germinação e, em dois casos, também o vigor das sementes, em
comparação com os do controle. Concluiu-se que 255 g ha-1 de Mo aplicado em R7 e, sobretudo, doses iguais ou
mais altas que 200 g ha-1 de Mo aplicadas em R8 podem reduzir a qualidade fisiológica da semente de feijão. alavras-chave: Phaseolus vulgaris, molibdênio na semente, germinação, vigor de semente. Palavras-chave: Phaseolus vulgaris, molibdênio na semente, germinação, vigor de semente. Rogério Faria Vieira*1, Trazilbo José de Paula Júnior1, Adalgisa Leles do Prado2, Roberto Fontes Araújo1,
Miller da Silva Lehner3, Rhaphael Alves Silva4 o Faria Vieira*1, Trazilbo José de Paula Júnior1, Adalgisa Leles do Prado2, Roberto Fontes Araújo1,
Miller da Silva Lehner3, Rhaphael Alves Silva4 Rogério Faria Vieira*1, Trazilbo José de Paula Júnior1, Adalgisa Leles do Prado2, Roberto Fontes Araújo1,
Miller da Silva Lehner3, Rhaphael Alves Silva4 http://dx.doi.org/10.1590/0034-737X201562040012 Rev. Ceres, Viçosa, v. 62, n.4, p. 415-419, jul-ago, 2015 MATERIAL E MÉTODOS Instalou-se ensaio, em 14 de março de 2010, em
Coimbra (20º 49’ S, 42º 45’ W, altitude de 716 m), mu-
nicípio da Zona da Mata de Minas Gerais, em Argissolo
Vermelho-Amarelo com 560 g kg 1 de argila, 190 g kg 1
de silte e 250 g kg 1 de areia e deficiente em Mo (Vieira
et al., 2005). Na instalação do ensaio, a análise do solo
na camada de 0-20 cm indicou: pH em água (relação
1:2,5) de 4,8; 11 e 96 mg dm-3 de P e K disponíveis,
respectivamente (extrator Mehlich); 0,5, 1,7 e 0,8 cmolc
dm-3 de Al, Ca e Mg, respectivamente, extraídos com KCl
mol L-1; 34% de saturação por bases e 1,4 dag kg-1 de
matéria orgânica (Walkley-Black). O solo foi prepara-
do com grade aradora, seguida de grade niveladora. Em feijão-comum (Phaseolus vulgaris), a preven-
ção ou correção da deficiência de Mo geralmente é fei-
ta pelo tratamento de sementes ou, sobretudo, pela pul-
verização de plantas com solução de Mo. Esses méto-
dos são usados por agricultores bem informados e em
regiões onde há disponibilidade de adubo molíbdico no
comércio. Recentemente, com o aprimoramento da téc-
nica para enriquecer sementes com Mo (Vieira et al.,
2005, Vieira et al., 2011; Pacheco et al., 2012), é possí-
vel fornecer aos agricultores, a baixo custo (Vieira et
al., 2011), esse micronutriente, por meio da semente. Nesse caso, a disponibilização de Mo para a planta
independe de a tecnologia ser conhecida, ou não, pelo
agricultor, e de o adubo molíbdico estar disponível, ou
não, no comércio local. Para produzir sementes ricas
em Mo, as plantas são pulverizadas, geralmente mais de
uma vez, com doses de Mo mais altas que as usadas para
prevenir ou corrigir deficiência de Mo no solo. Os tratamentos constaram de formas de parcela-
mento de 600 g ha-1 de Mo, nestas fases: V4, R6
(floração), R7 (formação de vagens) e,ou, R8 (enchi-
mento de vagens) (Tabela 1). Aplicada em V4, a dose de
90 g ha-1 de Mo, recomendada para prevenir ou corrigir
deficiência de Mo na Zona da Mata (Berger et al., 1996),
foi usada como controle. Na Zona da Mata de Minas Gerais, para se prevenir
ou corrigir deficiência de Mo do solo, aplicam-se de 70
a 100 g ha-1de Mo, entre 14 e 28 dias após a emergência
dos feijoeiros (Berger et al., 1996). Foliar molybdenum applied to common bean pod filling may reduce seed quality Aiming to improve the technology of producing seed with high Mo content, the effects of 600 g ha-1 of
foliar-applied Mo on common bean seed quality were evaluated. In the field, this Mo rate was applied at the V4
growth stage (third trifoliolate leave) or divided into two or four rates applied at V4, R6 (flowering), R7 and/or R8
(pod filling). Mo (300 g ha-1) applied at R6 did not affect seed quality relative to control (90 g ha-1 of Mo applied at
V4). In the three treatments in which 200, 255 or 300 g ha-1 of Mo were applied at R7, only in one (255 g ha-1) Mo
decreased seed germination and vigor relative to control. When 200, 255 or 300 g ha-1 of Mo were applied at R8
(five treatments), in general, Mo decreased seed germination and, in two cases, also seed vigor relative to control. It was concluded that 255 g ha-1 of Mo applied at R7 and, especially, doses of 200 g ha-1 of Mo or higher applied at
R8 can reduce common bean seed quality. Key words: Phaseolus vulgaris, molybdenum in seed, germination, seed vigor. Rogério Faria Vieira et al. 416 INTRODUÇÃO ha-1 de Mo aplicados durante a fase de enchimento de
vagens não influenciou a qualidade fisiológica das se-
mentes (Milani et al., 2010). No entanto, em relação ao
feijão-comum, não há estudo a esse respeito. O objeti-
vo deste trabalho foi avaliar os efeitos de parcelamentos
e de épocas de aplicação do adubo molíbdico em fases
reprodutivas do feijão-comum sobre a qualidade fisio-
lógica das sementes colhidas. O molibdênio (Mo) é exigido em pequena quantida-
de pela planta e, nela, suas funções estão associadas a
mudanças de valência, ao participar de enzimas. Oxida-
do, ele existe como Mo (VI), reduzido, como Mo (V) e
Mo (IV). O Mo participa das enzimas nitrogenase e ni-
trato redutase. Esta última é chave na assimilação do ni-
trato. Logo, quando há deficiência dessa enzima, a plan-
ta depende do amônio como fonte de nitrogênio (N). A
nitrogenase é necessária para a fixação biológica do N
(Mendel, 2011). MATERIAL E MÉTODOS Percentagem de germinação de sementes (PGS), P
função de parcelamentos de 600 g ha-1 de Mo em até quatro fas
Dose de molibdênio em g ha-1
(fase de desenvolvimento do feijão)(1)
90 (V4)
600 (V4)
300 (V4) + 300 (R6)
300 (V4) + 300 (R7)
300 (V4) + 300 (R8)
90 (V4) + 255 (R6) + 255 (R7)
90 (V4) + 255 (R6) + 255 (R8)
90 (V4) + 255 (R7) + 255 (R8)
200 (V4) + 200 (R6) + 200 (R7)
200 (V4) + 200 (R6) + 200 (R8)
200 (V4) + 200 (R7) + 200 (R8)
90 (V4) + 170 (R6) + 170 (R7) + 170 (R8)
Média
CV(%)
(1) V4 = terceira folha trifoliolada, R6 = floração, R7 = formação de vag
(2) São apresentados os dados originais, os quais foram previamente t
* Médias, na coluna, seguidas de asterisco diferem significativamente
pelo teste de Dunnett. da floração e dez dias depois, para o controle preventivo
do mofo-branco (Sclerotinia sclerotiorum). Irrigações
semanais foram feitas com aproximadamente 40 (fase
vegetativa) ou 50 mm (fase reprodutiva) de água. 2009). Nos testes de germinação e vigor, usou-se o
delineamento inteiramente casualizado, com sete re-
petições. Avaliaram-se, ademais, o teor de molibdênio das se-
mentes provenientes de três tratamentos: 90 (V4), 600
(V4) e 90 (V4) + 170 (R6) + 170 (R7) + 170 (R8). Fez-
se essa avaliação com as sete repetições de campo, com
o objetivo de confirmar se o Mo aplicado na folhagem
aumentou o teor de Mo da semente. A metodologia usa-
da para avaliar o teor de Mo das sementes é descrita por
Vieira et al. (2005). Foi usado o delineamento em blocos ao acaso, com
sete repetições. As parcelas constaram de quatro filei-
ras de 2 m, espaçadas de 0,6 m. Foram distribuídas 15
sementes por metro. A parcela útil constou das duas fi-
leiras centrais. Avaliaram-se estande final, produtividade, massa
de 100 sementes, germinação e vigor das sementes
colhidas. A produtividade e a massa de 100 sementes
foram obtidas com sementes com aproximadamente
13% de água (base úmida). Utilizaram-se os seguin-
tes testes de vigor: envelhecimento acelerado,
condutividade elétrica e frio. Nos testes de germina-
ção e de vigor, realizados 35 dias após a colheita, usa-
ram-se quatro replicatas de 50 sementes de cada tra-
tamento de campo. MATERIAL E MÉTODOS Os testes de germinação, envelhe-
cimento acelerado e condutividade elétrica foram re-
alizados conforme descrito por Leite et al. (2009). No teste de frio, as sementes foram distribuídas em
papel germitest previamente umedecido, como feito
no teste de germinação. Os rolos foram colocados no
interior de sacos plásticos, vedados com fita adesiva
e mantidos em câmara incubadora BOD, a 10°C, du-
rante sete dias (Loeffler et al., 1985). Em seguida, os
rolos foram transferidos para germinador, regulado à
temperatura de 25 °C, onde permaneceram por cinco
dias antes da contagem de plântulas normais (Brasil, Os dados, previamente transformados por arco seno
de
, foram submetidos à análise de variância. Uti-
lizou-se o teste de Dunnett para comparar os tratamen-
tos de parcelamento de 600 g ha-1 de Mo com o controle
(90 g ha-1 de Mo aplicado em V4). MATERIAL E MÉTODOS Sementes colhidas
de feijoeiros pulverizados com doses nessa faixa e nes-
sa época de aplicação têm melhor qualidade fisiológica
que as colhidas de plantas que não receberam Mo
(Meireles et al., 2003; Leite et al., 2009). Usou-se o cultivar BRSMG Majestoso, do grupo
comercial carioca e de hábito de crescimento indeter-
minado (tipo III), que é bom acumulador de Mo na se-
mente (Vieira et al., 2014). No sulco de plantio, distri-
buíram-se 350 kg ha-1 do formulado 8-28-16 (N – P2O5
– K2O). Em V4, os feijoeiros de todas as parcelas rece-
beram, além das respectivas adubações com Mo (Tabela
1), adubação em cobertura, com 100 kg ha-1 de ureia,
distribuídos em filete e a 10 cm da fileira. Com pulveri-
zador de CO2, pressão de 245 kPa, equipado com bicos
XR 11002 espaçados de 0,5 m, distribuiu-se o molibdato
de sódio (Na2MoO4.2H2O) sobre a folhagem. Adicionou-
se à solução de Mo um espalhante adesivo. Lona plásti-
ca foi estendida entre as parcelas vizinhas às tratadas para
interceptar a deriva. Fez-se o controle de plantas dani-
nhas com os herbicidas fomesafen (0,25 kg ha-1) e
fluazifop-p-butil (0,20 kg ha-1) e, quando necessário, tam-
bém com capinas manuais. Controlaram-se pragas com
os inseticidas metamidofós (500 mL ha-1) ou clorpirifós
(400 mL ha-1) e doenças foliares com o fungicida
epoxiconazol (12,5 mL ha-1) ou azoxystrobin (50 g ha-1). O fungicida fluazinam (0,5 L ha-1) foi aplicado no início Para produzir sementes ricas em Mo, a dose a ser
usada depende da disponibilidade de Mo no solo, pH do
solo, do cultivar, etc. Na Zona da Mata, 600 g ha-1 de Mo
geralmente elevam o conteúdo de Mo para pelo menos
3,6 µg por semente (Vieira et al., 2014), teor com que
os feijoeiros não mais respondem à aplicação de Mo na
folhagem (Vieira et al., 2011). Nessa região, o conteú-
do de Mo da semente de feijão quase dobrou quando a
dose de 1440 g ha-1 de Mo foi dividida em quatro
subdoses e aplicada entre V4 (terceira folha trifoliolada)
e R5 (pré-floração), em comparação com o efeito da
aplicação única em V4 (Vieira et al. 2005). Há indica-
ções de que a aplicação do Mo na fase reprodutiva do
feijão (Kubota et al., 2008) seja boa estratégia para pro-
duzir sementes ricas nesse nutriente. Em soja, 1000 g Rev. Ceres, Viçosa, v. 62, n.4, p. (1) V4 = terceira folha trifoliolada, R6 = floração, R7 = formação de vagens, R8 = enchimento de vagens. Rev. Ceres, Viçosa, v. 62, n.4, p. 415-419, jul-ago, 2015 * Médias, na coluna, seguidas de asterisco diferem significativamente do tratamento controle (90 g ha-1 de Mo aplicado em V4) a 5% de probabilidade,
pelo teste de Dunnett. ados originais, os quais foram previamente transformados por arco seno de
para a análise de variância. MATERIAL E MÉTODOS 415-419, jul-ago, 2015 A aplicação foliar de molibdênio na fase de enchimento de vagens do feijão-comum pode... 417 da floração e dez dias depois, para o controle preventiv
do mofo-branco (Sclerotinia sclerotiorum). Irrigaçõe
semanais foram feitas com aproximadamente 40 (fa
vegetativa) ou 50 mm (fase reprodutiva) de água. Foi usado o delineamento em blocos ao acaso, co
sete repetições. As parcelas constaram de quatro file
ras de 2 m, espaçadas de 0,6 m. Foram distribuídas 1
sementes por metro. A parcela útil constou das duas f
leiras centrais. Avaliaram-se estande final, produtividade, mass
de 100 sementes, germinação e vigor das semente
colhidas. A produtividade e a massa de 100 semente
foram obtidas com sementes com aproximadamen
13% de água (base úmida). Utilizaram-se os seguin
tes testes de vigor: envelhecimento acelerado
condutividade elétrica e frio. Nos testes de germin
ção e de vigor, realizados 35 dias após a colheita, us
ram-se quatro replicatas de 50 sementes de cada tr
tamento de campo. Os testes de germinação, envelh
cimento acelerado e condutividade elétrica foram r
alizados conforme descrito por Leite et al. (2009
No teste de frio, as sementes foram distribuídas e
papel germitest previamente umedecido, como feit
no teste de germinação. Os rolos foram colocados n
interior de sacos plásticos, vedados com fita adesiv
e mantidos em câmara incubadora BOD, a 10°C, du
rante sete dias (Loeffler et al., 1985). Em seguida, o
rolos foram transferidos para germinador, regulado
temperatura de 25 °C, onde permaneceram por cinc
dias antes da contagem de plântulas normais (Brasi
Tabela 1. RESULTADOS E DISCUSSÃO Os tratamentos não influenciaram o estande final (F
= 0,778, p = 0,660, CV = 21,8%, média de 177 mil plan-
tas ha-1), a produtividade de grãos (F = 0,804, p = 0,636,
CV = 10,5%, média = 4148 kg ha-1) e a massa de 100
sementes (F = 0,440, p = 0,949, CV = 6,1%, média =
27,3 g). O efeito não significativo dos tratamentos na
produtividade era esperado, pois os feijoeiros de todos
os tratamentos receberam Mo e N em cobertura. As sementes originadas de plantas adubadas com 90,
600 (ambas em V4) e 600 g ha-1 de Mo parcelados em
V4, R6, R7 e R8, apresentaram teor médio de Mo de
0,59; 11,83 e 15,2 µg g-1, respectivamente. Portanto, os Tabela 1. Percentagem de germinação de sementes (PGS), PGS após envelhecimento acelerado (EA) e estresse de frio (EF) em
função de parcelamentos de 600 g ha-1 de Mo em até quatro fases de desenvolvimento do feijão, em Coimbra, MG
Dose de molibdênio em g ha-1
(fase de desenvolvimento do feijão)(1)
90 (V4)
81,8
86,4
92,7
600 (V4)
79,4
83,3
91,1
300 (V4) + 300 (R6)
77,7
83,1
88,9
300 (V4) + 300 (R7)
76,7
85,8
89,3
300 (V4) + 300 (R8)
72,1*
77,3*
83,1*
90 (V4) + 255 (R6) + 255 (R7)
74,0*
78,7*
87,4
90 (V4) + 255 (R6) + 255 (R8)
72,4*
79,0*
86,7
90 (V4) + 255 (R7) + 255 (R8)
75,3*
85,0
89,7
200 (V4) + 200 (R6) + 200 (R7)
76,9
83,1
91,1
200 (V4) + 200 (R6) + 200 (R8)
73,8*
81,3
89,0
200 (V4) + 200 (R7) + 200 (R8)
76,9
83,6
88,7
90 (V4) + 170 (R6) + 170 (R7) + 170 (R8)
77,2
86,8
90,9
Média
76,2
82,8
89,1
CV(%)
5,1
5,7
5,0
(1) V4 = terceira folha trifoliolada, R6 = floração, R7 = formação de vagens, R8 = enchimento de vagens. (2) São apresentados os dados originais, os quais foram previamente transformados por arco seno de
para a análise de variância. * Médias, na coluna, seguidas de asterisco diferem significativamente do tratamento controle (90 g ha-1 de Mo aplicado em V4) a 5% de probabilidade,
pelo teste de Dunnett. PGS(2) (%)
EA(2) (%)
EF(2) (%) Tabela 1. (2) São apresentados os dados originais, os quais foram previamente transformados por arco seno de
para a análise de variân ada, R6 = floração, R7 = formação de vagens, R8 = enchimento de vagens. CONCLUSÕES Dose de 300 g ha-1 de Mo aplicada na floração (R6)
do feijão-comum não influencia a qualidade fisiológica
das sementes. Dose de 255 g ha-1 de Mo aplicada na fase de forma-
ção de vagens (R7), antecedida da aplicação de 255 g ha-1
de Mo em R6, pode reduzir a qualidade fisiológica das
sementes. Os três tratamentos com aplicação de 255 g ha-1 de
Mo em R7 ou R8 (após aplicação de 90 g ha-1 de Mo em
V4 e 255 g ha-1 em R6 ou R7), reduziram a germinação
e, em dois casos, o vigor das sementes (teste de enve-
lhecimento acelerado). Um dos três tratamentos com
aplicação de 200 g ha-1 de Mo em R7 ou R8 (após 200 g
ha-1 em V4 e 200 g ha-1 em R6 ou R7), causou redução da
germinação, em comparação com a do controle. Os re-
sultados desses seis tratamentos, em que a dose de Mo
foi parcelada três vezes, podem estar indicando que o
aumento da dose usada em V4, de 90 para 200 g ha-1 de
Mo, com consequente redução das doses usadas na fase
reprodutiva do feijão, pode minimizar os efeitos dano-
sos do Mo na qualidade das sementes. Desconhece-se o
mecanismo que explica os efeitos de doses relativamente
altas de Mo, aplicadas na fase reprodutiva do feijão, na
qualidade fisiológica das sementes colhidas. Dose de 200 g ha-1 de Mo e, sobretudo, doses mais
altas que essa, aplicadas na fase de enchimento de va-
gens (R8) podem reduzir a qualidade das sementes, mas
dose de 170 g ha-1 de Mo, mesmo aplicada em R8, não
influencia a qualidade fisiológica das sementes. AGRADECIMENTOS À Fapemig, pelo financiamento deste estudo; ao
CNPq, belas bolsas concedidas. RESULTADOS E DISCUSSÃO Percentagem de germinação de sementes (PGS), PGS após envelhecimento acelerado (EA) e estress
função de parcelamentos de 600 g ha-1 de Mo em até quatro fases de desenvolvimento do feijão, em Coimbra, MG agem de germinação de sementes (PGS), PGS após envelhecimento acelerado (EA) e estresse de frio (EF) em
mentos de 600 g ha-1 de Mo em até quatro fases de desenvolvimento do feijão, em Coimbra, MG Rogério Faria Vieira et al. 418 feijoeiros que receberam 600 g ha-1 de Mo chegaram a
produzir sementes com teor de Mo 26 vezes mais alto
que os que receberam 90 g ha-1 de Mo. O teor de Mo
mais alto neste estudo ficou um pouco abaixo do obtido
em Coimbra, com o cultivar Majestoso, no estudo de
Vieira et al. (2014). características diferentes (especialmente quanto a óleo
e proteína) das do feijão-comum, eles não usaram con-
trole com dose baixa de Mo para comparação e, tam-
bém, não empregaram o teste de envelhecimento acele-
rado. Este teste de vigor foi o que mais diferenciou os
tratamentos, neste estudo. O teste de condutividade elétrica não detectou dife-
rença significativa entre tratamentos (F = 0,686, p =
0,747, CV = 10,3%). A dose de 600 g ha-1 de Mo aplica-
dos em V4 não influenciou significativamente a germi-
nação e o vigor das sementes, em comparação com os
do controle, com 90 g ha-1 de Mo (Tabela 1). A aplicação
de 300 g ha-1 de Mo em V4 e, em seguida, de 300 g ha-1
em R6 ou R7 também não influenciou a germinação e o
vigor das sementes, mas a diferença entre as médias das
variáveis dependentes do tratamento 300(V4) + 300
(R8), em comparação com os do controle, foi significa-
tiva. A diferença não significativa entre as médias de ger-
minação e de vigor do tratamento 90 (V4) + 170 (R6) +
170 (R7) + 170 (R8) e as do controle sugerem que o
uso de dose de 170 g ha-1 de Mo, ou mais baixas, mesmo
se aplicadas em R8, não reduzem a qualidade fisiológica
das sementes. Milani GL, Oliveira JA, Pereira E de M, Carvalho BO, Oliveira GE &
Costa RR (2010) Aplicação foliar de molibdênio durante a maturação
de sementes de soja. Ciência e Agrotecnologia, 34:810-816. Vieira RF, Salgado LT & Ferreira ACC (2005) Performance of common
bean using seeds harvested from plants fertilized with high rates of
molybdenum. Journal of Plant Nutrition, 28:393-377. Rev. Ceres, Viçosa, v. 62, n.4, p. 415-419, jul-ago, 2015 REFERÊNCIAS Berger PG, Vieira C & Araújo GAA (1996) Efeitos de doses e épocas de
aplicação de molibdênio sobre a cultura do feijão. Pesquisa
Agropecuária Brasileira, 31:473-480. Considerados em conjunto, os resultados do parce-
lamento da dose de Mo em duas ou três aplicações (nove
tratamentos) sugerem que 200 e, sobretudo, 255 ou 300
g ha-1 de Mo aplicados em R8, podem reduzir a qualida-
de fisiológica das sementes. Também indica que 255 g
ha-1 de Mo aplicados em R7 (depois de 255 g ha-1 em
R6) podem reduzir a qualidade fisiológica das semen-
tes. Em outro estudo, conduzido na Zona da Mata, a apli-
cação de 1000 g ha-1 de Mo, aos 71 DAE (fase R7), não
influenciou significativamente a germinação das semen-
tes (Vieira et al., 2010). Logo, mais estudos com com-
binações de doses de Mo aplicadas em R7 são necessá-
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fase de enchimento de vagens não mostraram efeito sig-
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semente (Milani et al., 2010). No entanto, além de es-
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via foliar, na qualidade fisiológica das sementes de feijão. Revista
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Report, 30:1787-1797. Rev. Ceres, Viçosa, v. 62, n.4, p. 415-419, jul-ago, 2015 Pacheco RS, Brito LF, Staliotto R, Pérez DV & Araújo AP (2012) Seeds
enriched with phosphorus and molybdenum as a strategy for
improving grain yield of common bean crop. Field Crops Research,
136:97-106. A aplicação foliar de molibdênio na fase de enchimento de vagens do feijão-comum pode... 419 Vieira RF, Salgado LT, Pires AA & Rocha GS (2010) Conteúdo de
molibdênio das sementes de feijoeiro em resposta a doses do
micronutriente pulverizado sobre as plantas. Ciência Rural, 40:666-
669. Vieira RF, Salgado LT, Pires AA & Rocha GS (2010) Conteúdo de
molibdênio das sementes de feijoeiro em resposta a doses do
micronutriente pulverizado sobre as plantas. Ciência Rural, 40:666-
669. Pacheco RS, Brito LF, Staliotto R, Pérez DV & Araújo AP (2012) Seeds
enriched with phosphorus and molybdenum as a strategy for
improving grain yield of common bean crop. Field Crops Research,
136:97-106. Vieira RF, Paula Júnior TJ, Pires AA, Carneiro JES & Rocha GS (2011)
Common bean seed complements molybdenum uptake by plants from
soil. Agronomy Journal, 103:1843-1848. Vieira RF, Paula Júnior TJ, Carneiro JES & Queiroz MV (2014) Genotypic
variability in seed accumulation of foliar-applied molybdenum to
common bean. Revista Brasileira de Ciência do Solo, 38:205-213.
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Predictors of positive response to cardiac resynchronization therapy
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BMC cardiovascular disorders
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RESEARCH ARTICLE Open Access * Correspondence: diana.rinkuniene@gmail.com
1Lithuanian University of Health Sciences, Kaunas, Lithuania
2Hospital of Lithuanian University of Health Sciences Kaunas Clinics, Kaunas,
Lithuania
Full list of author information is available at the end of the article Abstract Background: Approximately 30% of patients treated with cardiac resynchronization therapy (CRT) do not achieve
favourable response. The purpose of the present study was to identify echocardiographic and clinical predictors of
a positive response to CRT. Methods: The study included 82 consecutive heart failure (HF) patients in New York Heart Association (NYHA)
functional class III or IV with left bundle branch block (LBBB), QRS duration ≥120 ms and left ventricular ejection
fraction (LVEF) ≤35%. Statistical analysis was performed using IBM SPSS statistical software (SPSS v.21.0 for Mac OS
X). A p value < 0.05 was considered statistically significant. Results: Echocardiographic response was established in 81.6% and clinical response was achieved in 82.9% of
patients. Significant univariate predictors of favourable echocardiographic response after 12 months were smaller left
ventricular end-diastolic diameter (LVEDD) (odds ratio [OR] 0.89; 95% confidence interval [CI] 0.82 - 0.97, p = 0.01), and
smaller left ventricular end-systolic diameter (LVESD) (OR 0.91; 95% CI 0.85 - 0.98, p = 0.01). Lower uric acid concentration
was associated with better echocardiographic response (OR 0.99; 95% CI 0.99 - 1.0, p = 0.01). Non-ischemic HF etiology
(OR 4.89; 95% CI 1.39 - 17.15, p = 0.01) independently predicted positive clinical response. Multiple stepwise regression
analysis demonstrated that LVEDD lower than 75 mm (OR 5.60; 95% confidence interval [CI] 1.36 - 18.61, p = 0.01) was
the strongest independent predictor of favourable echocardiographic response. Conclusions: Smaller left ventricular end-diastolic and end-systolic diameters and lower serum uric acid concentration
were associated with better response to CRT. Left ventricular end-diastolic diameter and non-ischemic heart failure
etiology were the strongest independent predictors of positive response to CRT. Keywords: Cardiac resynchronization therapy, Heart failure, Response discharge diagnosis in elderly patients. Development
of HF is characterised by progressive left ventricular
(LV) remodelling, that further impedes LVEF and is
responsible for progression of clinical symptoms. Results
from mechanistic studies, observational evaluations and
randomised controlled trials consistently demonstrated
significant improvement in quality of life, functional
status, and exercise capacity in HF patients in NYHA
class III and IV who were assigned to active CRT. However,
CRT does not provide any benefit to approximately
30% of patients [3-5]. Lack of response to CRT in
these studies may be in part attributed to inappropriate
echocardiographic
and/or electrocardiographic criteria
used to select patients for CRT. The purpose of our study Predictors of positive response to cardiac
resynchronization therapy Diana Rinkuniene1,2,3*, Silvija Bucyte1,2, Kristina Ceseviciute1, Silvijus Abramavicius1, Kristina Baronaite-Dudoniene1,2,
Jolanta Laukaitiene1,2, Tomas Kazakevicius1,2, Vytautas Zabiela1,2, Vytautas Sileikis2, Aras Puodziukynas1,2
and Renaldas Jurkevicius1,2 Rinkuniene et al. BMC Cardiovascular Disorders 2014, 14:55
http://www.biomedcentral.com/1471-2261/14/55 Rinkuniene et al. BMC Cardiovascular Disorders 2014, 14:55
http://www.biomedcentral.com/1471-2261/14/55 © 2014 Rinkuniene et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated. Two-dimensional echocardiography Echocardiography was performed in all patients at rest
in the lateral decubitus position, at baseline before
device implantation and was repeated at 12-months
follow-up. Transthoracic Doppler echocardiography was
performed using a GE Vivid 7 system (GE Vingmed
Ultrasound AS N-3190, Horten, Norway) with an M4S
transducer. Standard transthoracic echocardiographic mea-
surements were performed according to the Guidelines of
the American Society of Echocardiography [6]. A standard
evaluation of LV volumes was performed, and LVEF
was calculated according to the Simpson’s equation. To Background Approximately 1 – 2% of the adult population in developed
countries have HF, and its prevalence rises to ≥10% in
individuals 70 years of age or older [1]. Coronary artery
disease (CAD) is the cause of approximately two-thirds of
cases of systolic HF, although in many cases hypertension
and diabetes are likely contributing factors [2]. HF is associated with substantial mortality and
morbidity, and remains the most common hospital * Correspondence: diana.rinkuniene@gmail.com
1Lithuanian University of Health Sciences, Kaunas, Lithuania
2Hospital of Lithuanian University of Health Sciences Kaunas Clinics, Kaunas,
Lithuania
Full list of author information is available at the end of the article Page 2 of 8 Rinkuniene et al. BMC Cardiovascular Disorders 2014, 14:55
http://www.biomedcentral.com/1471-2261/14/55 Rinkuniene et al. BMC Cardiovascular Disorders 2014, 14:55
http://www.biomedcentral.com/1471-2261/14/55 was to identify initial echocardiographic and clinical
parameters that predict positive response to CRT. minimize variability of measurements, all echo/Doppler
evaluations were performed and analysed by the same
physician, also the same transducer position and sample
volume location were maintained throughout the record-
ings. All images were digitally stored for off-line analysis
(EchoPac V.6.0.0; GE Vingmed). Statistical analysis Statistical analysis was performed using IBM SPSS statistical
software (SPSS v.21.0 for Mac OS X). Normally distributed
continuous variables were presented as mean ± SD and
were compared using Student t-test for paired and unpaired
data. Statistical significance of differences between groups
was analysed by Mann–Whitney U test for non-parametric
continuous variables and categorical variables were
compared using the maximum likelihood (ML) Chi-square
test. Correlation between continuous variables was analysed
by Spearman rank correlation test. Receiver operating
characteristic (ROC) curve was used to determine a cut-off
point of categorical predictors. Variables significant in uni-
variate analysis were added to logistic regression to deter-
mine independent predictors of response to CRT. Stepwise
variable selection with forward selection and backward
elimination demonstrated identical results. Precision of the
model was verified with the Hosmer-Lemeshow test of
goodness of fit test. A p value < 0.05 was considered statisti-
cally significant. Before the study all patients signed informed consent
approved by Local Ethics Committee (Kaunas Regional
Biomedical Research Ethics Committee, ref. n. BE-2-54). Blood samples for uric acid concentration were taken
one to two days before CRT implantation and were
analysed in the laboratory of Lithuanian University of
Health Sciences Hospital Kaunas Clinics. Before the study all patients signed informed consent
approved by Local Ethics Committee (Kaunas Regional
Biomedical Research Ethics Committee, ref. n. BE-2-54). Device implantation
C T
l CRT implantation was performed through the subclavian
or axillary vein access. Coronary sinus venogram was
obtained using balloon catheter. LV lead was inserted into
the posterolateral vein, which was selected according to
anatomical characteristics of the vessels enter the appropri-
ate electrode. The optimal position of the LV lead was
defined according to LV lead impedance, threshold and no
nervus phrenicus stimulation. The right atrial lead was
conventionally positioned in the right atrium appendage
and right ventricular lead in to the ventricular septum or
right ventricular apex. Finally, all leads were connected to a
dual chamber biventricular CRT device. The optimal atrio-
ventricular (AV) delay was determined during simultaneous
biventricular pacing by the simplified mitral inflow method:
a long AV interval (200 ms) was programmed and gradually
reduced by 20 ms, until A-wave truncation was observed. Ischemic cardiomyopathy (ICMP) was diagnosed in
patients with previous myocardial infarction, or coronary
artery bypass graft surgery, or percutaneous coronary
intervention (balloon and/or stent angioplasty), or angio-
grafically documented significant coronary artery disease
and history of angina pectoris. All patients were in sinus rhythm, electrocardiogram at
rest was recorded (measured on surface electrocardiogram
leads, at a paper speed of 25 mm/s) and QRS duration
was measured at baseline and after 12 months post CRT
implantation. Clinical evaluation and two-dimensional echocardiog-
raphy were performed before CRT device implantation
and repeated at 12 months of follow-up. Clinical evaluation
included assessment of NYHA class and performance of
6 minute walk test (6-MWT). At 12 months of follow-up,
patients, who achieved improvement in NYHA of at least
1 class and a ≥15% increase in 6-MWT, were classified as
clinical responders. Patient population The study included 82 consecutive HF patients, who
underwent CRT implantation at Cardiology Department
at Hospital of Lithuanian University of Health Sciences
Kaunas Clinics, between January 2009 and December
2011. All patients met the following inclusion criteria:
NYHA class III or IV despite optimal medical therapy,
LBBB, QRS width ≥120 ms and LVEF ≤35%. Patients
with previously implanted pacemaker or defibrillator, re-
cent myocardial infarction or coronary artery bypass graft
surgery (≤6 months), or decompensated HF were excluded. Echocardiographic response was defined as an increase in
LVEF of ≥5% and decrease of left ventricular end-systolic
volume (LVESV) and left ventricular end-diastolic volume
(LVEDV) by ≥15%. Results Baseline characteristics of the subjects are summarized
in Table 1. A total of 82 consecutive patients were
included in the study. The study population consisted Rinkuniene et al. BMC Cardiovascular Disorders 2014, 14:55
http://www.biomedcentral.com/1471-2261/14/55 Rinkuniene et al. BMC Cardiovascular Disorders 2014, 14:55
http://www.biomedcentral.com/1471-2261/14/55 Page 3 of 8 Rinkuniene et al. BMC Cardiovascular Disorders 2014, 14:55
http://www.biomedcentral.com/1471-2261/14/55 Table 1 Baseline characteristics
Patient characteristics
Baseline (n = 82)
Age (yrs.)
63.5 ± 10.5
Gender (male,%)
65 (79.3)
QRS duration (ms)
174.8 ± 17.0
NYHA class III n (%)
68 (82.9)
Six minute walk test (m)
300.8 ± 70.4
Diabetes n (%)
9 (10.8)
Hypertension n (%)
66 (80.5)
Paroxysmal AF n (%)
28 (34.5)
VT n (%)
25 (30.5)
CRT-D n (%)
36 (43.9)
LVEF (%)
20.3 ± 6.5
LVEDD (mm)
68.5 ± 9.7
LVESD (mm)
61.9 ± 10.0
LVEDV (ml)
220.6 ± 76.3
LVESV (ml)
175.3 ± 69.8
LAV (ml)
93.3 ± 28.5
Beta blockers n (%)
77 (93.9)
ACE-I n (%)
61 (74.4)
ARB n (%)
16 (19.5)
MRA n (%)
70 (85.4)
Diuretics n (%)
68 (82.9)
Amiodarone n (%)
24 (29.3)
Statin n (%)
40 (48.8)
Aspirin n (%)
29 (35.4)
Warfarin n (%)
27 (32.9)
Uric acid (μmol/l)
426.3 ± 133.2
NYHA - New York Heart Association, AF – atrial fibrillation, VT- ventricular
tachycardia, CRT-D – cardiac resynchronization therapy and defibrillator,
LVEF – left ventricular ejection fraction, LVEDD – left ventricular end-diastolic
diameter, LVESD - left ventricular end-systolic diameter, LVEDV - left ventricular
end-diastolic volume, LVESV - left ventricular end-systolic volume, LAV – left
atrial volume; ACE-I – angiotensin converting enzyme inhibitors; ARB -
angiotensin receptor blockers; MRA - mineralocorticoid receptor antagonists. At 12 months of follow-up, a significant increase in
LVEF (mean 10.4 ± 7.6%, p < 0.001), significant reduc-
tion in LV diameters (LVEDD −10.7 ± 16.5 mm, p <
0.001,
LVESD
−6.7 ± 7.0,
p < 0.001),
LV
volumes
(LVEDV −47.4 ± 53.7 ml, p < 0.001; LVESV −48.1 ± 50.2 ml,
p < 0.001) and left atrial volume (LAV) (−14.0 ml ± 19.0,
p < 0.001) were attained (Figure 1). In addition, a sig-
nificant increase in 6-MWT (from 300.8 ± 70.4 m to
405.5 ± 65.7 m; p < 0.001) and decrease in QRS duration
(from 174.8 ± 17.0 ms to 137.2 ± 15.0 ms; p = 0.001) were
observed. Six patients died within 12 months of CRT implantation. Clinical response At 12 months follow-up 54 (71.1%) of patients had a
significant improvement in NYHA class (p < 0.001). Distribution of NYHA class at baseline and after 12 months
post CRT implantation is provided in Figure 2. An increase
in 6-MWT by ≥15% was observed in 57 (75%) patients
(p = 0.001). Mean increase in 6-MWT post CRT was
121.2 ± 66.1 m in clinical responders and 11.3 ± 27 m
in non-responders (p = 0.001). Combined clinical response
(improvement in NYHA class ≥1 class and/or ≥15%
increase in the 6-MWT) was achieved in 63 (82.9%)
patients (Table 2). Compared to responders, non-responders were more
likely to have ischemic cardiomyopathy (63.5% vs 36.5%,
p = 0.01). Echocardiographic response was observed in
87.3% combined clinical responders (p = 0.03). NYHA - New York Heart Association, AF – atrial fibrillation, VT- ventricular
tachycardia, CRT-D – cardiac resynchronization therapy and defibrillator,
LVEF – left ventricular ejection fraction, LVEDD – left ventricular end-diastolic
diameter, LVESD - left ventricular end-systolic diameter, LVEDV - left ventricular
end-diastolic volume, LVESV - left ventricular end-systolic volume, LAV – left
atrial volume; ACE-I – angiotensin converting enzyme inhibitors; ARB -
angiotensin receptor blockers; MRA - mineralocorticoid receptor antagonists. Results Due to the lack of full 12 month follow-up assessment,
data of these patients were not included into the analyses
of CRT response. Echocardiographic response g
p
p
At 12 months of follow-up, LVEF increase of ≥5%
was observed in 81.6% patients. Increase in LVEF was
higher in patients with non-ICMP (11.2 ± 8.0 vs 7.7 ± 7.1;
p = 0.04). Combined echocardiographic response (LVEF
increase ≥5%,
and/or
LVESV
decrease ≥15%
and/or
LVEDV decrease ≥15%) was established in 81.6% of the
overall study population (Table 2). Compared to responders,
non-responders were more likely to have lower LVEF, larger
LVEDD, LVESD diameters and LV and LA volumes, AF
and VT episodes at baseline, although only LV diameters,
LAV, AF, VT achieved statistical significance (Table 3). Also, a negative association of warfarin use and echo-
cardiographic response was found (p = 0.01). No signifi-
cant differences in QRS duration, gender, age and
CRT type between echocardiographic responders and
non-responders were found. of 65 men (79.3%) and 17 women (20.7%), mean age
63.5 ± 10.5 years. ICMP related HF was diagnosed in
37 (45.1%) patients. Most of the patients (82.9%) were
in NYHA class III. Mean 6-MWT was 300.8 ± 70.4 m. CRT and defibrillator (CRT-D) were implanted in 36
(43.9%) patients, twenty-five of them had developed
paroxysmal monomorphic ventricular tachycardia (VT)
before implantation. According to inclusion criteria all
patients had a wide QRS complex (174.8 ± 17.0 ms), sinus
rhythm, LBBB configuration and were treated accord-
ing
to
HF guidelines
[7],
including
beta-blockers,
angiotensin converting enzyme inhibitors (ACE-I) or
angiotensin receptor blockers (ARB), mineralocorticoid
receptor antagonists (MRA), and diuretics at maximum
tolerated doses. of 65 men (79.3%) and 17 women (20.7%), mean age
63.5 ± 10.5 years. ICMP related HF was diagnosed in
37 (45.1%) patients. Most of the patients (82.9%) were
in NYHA class III. Mean 6-MWT was 300.8 ± 70.4 m. CRT and defibrillator (CRT-D) were implanted in 36
(43.9%) patients, twenty-five of them had developed
paroxysmal monomorphic ventricular tachycardia (VT)
before implantation. According to inclusion criteria all
patients had a wide QRS complex (174.8 ± 17.0 ms), sinus
rhythm, LBBB configuration and were treated accord-
ing
to
HF guidelines
[7],
including
beta-blockers,
angiotensin converting enzyme inhibitors (ACE-I) or
angiotensin receptor blockers (ARB), mineralocorticoid
receptor antagonists (MRA), and diuretics at maximum
tolerated doses. Significant univariate predictors of favourable echocar-
diographic response after 12 months included smaller
LVEDD (OR 0.89, 95% CI 0.82 - 0.97; p = 0.01) and
LVESD (OR 0.91, 95% CI 0.85 - 0.98; p = 0.01). Lower Rinkuniene et al. Echocardiographic response BMC Cardiovascular Disorders 2014, 14:55
http://www.biomedcentral.com/1471-2261/14/55 Rinkuniene et al. BMC Cardiovascular Disorders 2014, 14:55
http://www.biomedcentral.com/1471-2261/14/55 Page 4 of 8 78.6 ± 23.7*
173 ± 66.7*
124.4 ± 59.8*
54.6 ± 10.5*
61.4 ± 10.2*
31 ± 9.1*
93.3 ± 28.5
220.6 ± 76.3*
175.3 ± 69.8
61.9 ± 9.9
68.5 ± 9.7
20.3 ± 6.5
LAV (ml)
LVEDV (ml)
LVESV (ml)
LVESD (mm)
LVEDD (mm)
LVEF (%)
Baseline
After 12months
Figure 1 Change in echocardiographic parameters during 12 months post CRT implantation (n = 76). *p < 0.001 between the same initial and
12 months follow up echocardiographic parameter. LVEF – left ventricular ejection fraction, LVEDD – left ventricular end-diastolic diameter, LVESD - left
ventricular end-systolic diameter, LVEDV - left ventricular end-diastolic volume, LVESV - left ventricular end-systolic volume, LAV – left atrial volume. Baseline
After 12months 54.6 ± 10.5*
61.9 ± 9.9 uric acid concentration was associated with better
echocardiographic response (OR 0.99, 95% CI 0.99 - 1.0;
p = 0.01). The precision of the model was verified
with the Hosmer-Lemeshow test of goodness of fit
t
t (
0 1)
Non-ischemic HF etiology was an independent pre-
dictor of a positive clinical response (OR 4.89, 95%
CI 1.39 - 17.15; p = 0.01). The precision of the model was
verified with the Hosmer-Lemeshow test of goodness o
fit t
t (
0 49)
Figure 1 Change in echocardiographic parameters during 12 months post CRT implantation (n = 76). *p < 0.001 between the same initial and
12 months follow up echocardiographic parameter. LVEF – left ventricular ejection fraction, LVEDD – left ventricular end-diastolic diameter, LVESD - left
ventricular end-systolic diameter, LVEDV - left ventricular end-diastolic volume, LVESV - left ventricular end-systolic volume, LAV – left atrial volume. Figure 1 Change in echocardiographic parameters during 12 months post CRT implantation (n = 76). *p < 0.001 between the same initial and
12 months follow up echocardiographic parameter. LVEF – left ventricular ejection fraction, LVEDD – left ventricular end-diastolic diameter, LVESD - left
ventricular end-systolic diameter, LVEDV - left ventricular end-diastolic volume, LVESV - left ventricular end-systolic volume, LAV – left atrial volume. uric acid concentration was associated with better
echocardiographic response (OR 0.99, 95% CI 0.99 - 1.0;
p = 0.01). The precision of the model was verified
with the Hosmer-Lemeshow test of goodness of fit
test (p = 0.1). Discussion Prognosis of an HF patient depends on demographic,
echocardiographic, haemodynamic, neurohormonal, and
functional factors [8]. Each of these factors provided
powerful independent prognostic information, yet they
were poorly interrelated [9]. The present
study
confirmed
that
most
patients
treated with CRT demonstrated favourable clinical and
echocardiographic responses during 12 months follow-up
period. The baseline LV diameters, uric acid concentration
and ischemic etiology were the main predictors of
response to CRT. Only four parameters (Table 4) selected by regression
analysis reached statistically significant cut-off values in
ROC analyses, with sensitivity ranging from 59% to 81%
and specificity from 61% to 77% (Figure 3). Echocardiographic response was related to outcomes,
but it was not associated with symptoms or quality of life. Dominique Auger et al. LVEF – left ventricular ejection fraction, LVEDD – left ventricular end-diastolic diameter, LVESD - left ventricular end-systolic diameter, LVEDV - left ventricular
end-diastolic volume, LVESV - left ventricular end-systolic volume, LAV – left atrial volume, AF – atrial fibrillation, VT- ventricular tachycardia, ACE-I – angiotensin
converting enzyme inhibitors, ARB - angiotensin receptor blockers, MRA - mineralocorticoid receptor antagonists.
p value for the comparison between responders and non-responders. Echocardiographic response uric acid concentration was associated with better
echocardiographic response (OR 0.99, 95% CI 0.99 - 1.0;
p = 0.01). The precision of the model was verified
with the Hosmer-Lemeshow test of goodness of fit
test (p = 0.1). Non-ischemic HF etiology was an independent pre-
dictor of a positive clinical response (OR 4.89, 95%
CI 1.39 - 17.15; p = 0.01). The precision of the model was
verified with the Hosmer-Lemeshow test of goodness of
fit test (p = 0.49). Figure 2 Distribution of NYHA functional classes at baseline and after 12 months of CRT implantation (p
0 001) NYHA
New York Figure 2 Distribution of NYHA functional classes at baseline and after 12 months of CRT implantation (p = 0.001). NYHA - New York
Heart Association. Figure 2 Distribution of NYHA functional classes at baseline and after 12 months of CRT implantation (p = 0.001). NYHA - New York
Heart Association. Figure 2 Distribution of NYHA functional classes at baseline and after 12 months of CRT implantation (p = 0.001). NYHA - New York
Heart Association. n of NYHA functional classes at baseline and after 12 months of CRT implantation (p = 0.001). NYHA - New York Rinkuniene et al. BMC Cardiovascular Disorders 2014, 14:55
http://www.biomedcentral.com/1471-2261/14/55 Page 5 of 8 Table 2 Clinical and echocardiographic response to CRT
at 12 months of follow-up
After 12 months (n = 76)
Combined echocardiographic response
62 (81.6%)
LVEF increase ≥5%
62 (81.6%)
LVESV decrease ≥15%
56 (73.7%)
LVEDV decrease ≥15%
55 (72.4%)
Combined clinical response
63 (82.9%)
NYHA improvement ≥1 functional class
54 (71.1%)
Six minute walk test ≥15%
57 (75%)
NYHA - New York Heart Association, LVEF – left ventricular ejection fraction, LVEDV -
left ventricular end-diastolic volume, LVESV - left ventricular end-systolic volume. Table 2 Clinical and echocardiographic response to CRT
at 12 months of follow-up Table 2 Clinical and echocardiographic response to CRT as the strongest independent predictor of favourable
echocardiographic response, and non-ischemic HF etiology
as the independent predictor of positive clinical response
(OR 4.88, 95% CI 1.39 - 17.15; p = 0.01). Discussion [10] reported NYHA improvement y
g
Multiple stepwise regression analysis identified LVEDD
of less than 75 mm (OR 5.60, 95% CI 1.36 - 18.61; p = 0.01) Table 3 Comparison of echocardiographic and clinical parameters between combined echocardiographic responders
and non-responders at baseline
Responders (n = 62)
Non-responders (n = 14)
p value
LVEF (%)
21.2 ± 6.5
17.9 ± 5.4
0.09
LVEDD (mm)
66.8 ± 9.8
75.3 ± 6.5
0.01
LVESD (mm)
60.0 ± 10.0
68.4 ± 7.8
0.01
LVEDV (ml)
211.0 ± 77.6
255.4 ± 50.6
0.05
LVESV (ml)
166.4 ± 69.3
201 ± 53.5
0.09
LAV (ml)
89.1 ± 30.2
104.5 ± 17.6
0.02
QRS duration (ms)
174.5 ± 16.6
175.7 ± 19.6
0.89
Six minute walk test (m)
305.6 ± 71.5
307.7 ± 57.3
0.72
Age (yrs.)
63.2 ± 10.1
60.2 ± 11.4
0.32
Gender (male,%)
51 (81)
9 (69.2)
0.34
Paroxysmal AF n (%)
18 (28.6)
8 (61.5)
0.02
VT n (%)
16 (25.4)
7 (53.9)
0.04
CRT-D n (%)
25 (39.7)
8 (61.5)
0.15
Beta blockers n (%)
61 (96.8)
12 (92.3)
0.45
ACE-I n (%)
48 (76.2)
9 (69.2)
0.59
ARB n (%)
13 (20.6)
2 (15.4)
0.66
MRA n (%)
52 (82.5)
12 (92.3)
0.38
Diuretics n (%)
51 (81)
13 (100)
0.08
Amiodarone n (%)
16 (25.4)
5 (38.5)
0.33
Statin n (%)
33 (52.4)
6 (46.2)
0.68
Aspirin n (%)
23 (36.5)
4 (30.8)
0.69
Warfarin n (%)
16 (25.4)
8 (61.5)
0.01
Uric acid (μmol/l)
397.7 ± 111.5
506.3 ± 159.0
0.03
LVEF – left ventricular ejection fraction, LVEDD – left ventricular end-diastolic diameter, LVESD - left ventricular end-systolic diameter, LVEDV - left ventricular
end-diastolic volume, LVESV - left ventricular end-systolic volume, LAV – left atrial volume, AF – atrial fibrillation, VT- ventricular tachycardia, ACE-I – angiotensin
converting enzyme inhibitors, ARB - angiotensin receptor blockers, MRA - mineralocorticoid receptor antagonists. l
f
h
i
b
d
d
d Table 3 Comparison of echocardiographic and clinical parameters between combined echocard
and non-responders at baseline cardiographic and clinical parameters between combined echocardiographic responders Rinkuniene et al. Discussion BMC Cardiovascular Disorders 2014, 14:55
http://www.biomedcentral.com/1471-2261/14/55 Page 6 of 8 Table 4 Echocardiographic and clinical parameters in the prediction of response to CRT
Area under curve
(AUC)
95% CI
Sensitivity (%)
Specificity (%)
P value
LVEDD < 75mm
0.77
0.64 - 0.89
81
62
0.03
LVESD < 64mm
0.74
0.6 - 0.88
70
69
0.01
LAV< 90ml
0.70
0.57 - 0.83
59
77
0.03
Uric acid < 440μmol/l
0.69
0.53 - 0.86
71
61
0.03
CI – confidence interval, LVEDD – left ventricular end-diastolic diameter, LVESD - left ventricular end-systolic diameter, LAV – left atrial volume. Table 4 Echocardiographic and clinical parameters in the prediction of response to CRT Previous studies [13,14] demonstrated that patients
with ischemic heart disease had a lower likelihood of
response to CRT. Gasparini et al. [15] reported that
patients in the non-coronary artery disease (CAD) group
had a significantly greater increase in LVEF (p = 0.007)
and decrease in NYHA class (p < 0.05). Non-CAD
patients had a greater increase in LVEF and decrease in
NYHA functional class compared to patients with CAD. Sylvain Reuter et al. [16] explained differences of response
according to etiology of HF, suggesting that left ventricular
pacing lead was not placed at the optimal site with
regard to ischemic areas. In contrast to our data and
previously discussed studies, Molhoek et al. [17] reported
no differences in CRT response in ischemic HF vs
idiopathic dilated cardiomyopathy groups. However, in this
study response to CRT was defined only by improvement in ≥1 functional class in 80%, and combined clinical
response in 84% of patients. Similar results were obtained
in a study by Viviane Tiemi et al. [11], demonstrating
improvement in NYHA functional class in 79% of patients
after six months of CRT implantation. The 6-MWT was
used as a measure of response to CRT in a number of
studies, and was more sensitive compared to NYHA
functional class [12]. Our data did not contradict with the
findings of previous studies, as almost 82% of patients
achieved significant improvement in clinical and echocar-
diographic parameters. The need for complicated assess-
ment of CRT response is debatable, because from a
patient’s perspective improvement in clinical condition
matters most, hence changes in NYHA functional class
and six-minute walk distance may be unsophisticated and
important criteria for evaluation of response to CRT. 1.
Mosterd A, Hoes AW: Clinical epidemiology of heart failure. Heart 2007,
93:1137–1146. 1. Mosterd A, Hoes AW: Clinical epidemiology of heart failure. Heart 2007,
93:1137–1146. 1. Mosterd A, Hoes AW: Clinical epidemiology of heart failure. Heart 2007,
93:1137–1146. 2. McMurray JJ, Adamopoulos S, Anker SD, Auricchio A, Böhm M, Dickstein K,
Falk V, Filippatos G, Fonseca C, Gomez-Sanchez MA, Jaarsma T, Køber L,
Lip GY, Maggioni AP, Parkhomenko A, Pieske BM, Popescu BA, Rønnevik PK,
Rutten FH, Schwitter J, Seferovic P, Stepinska J, Trindade PT, Voors AA,
Zannad F, Zeiher A, European Society of Cardiology. ESC Guidelines for
the diagnosis and treatment of acute and chronic heart failure 2012: The
Task Force for the Diagnosis and Treatment of Acute and Chronic
Heart Failure 2012 of the European Society of Cardiology. Developed
in collaboration with the Heart Failure Association (HFA) of the ESC. Eur Heart J 2012, 33:1787–1847. Higher uric acid concentration is associated with
increased risk of mortality and morbidity in patients
with HF [25,26]. Importance of uric acid in risk stratifi-
cation is recognized by the Seattle Heart Failure Model. In our study, uric acid concentration was associated with
response to CRT, with possible multifactorial mecha-
nisms, i.e. increased xanthine oxidase activity induced
oxidative stress and inflammation, and renal dysfunction
related to hypoperfusion and diuretic therapy [25]. Further detailed studies are needed to define the exact
mechanism of the association of increased uric acid and
response to CRT. 3. Abraham WT, Fisher WG, Smith AL, Delurgio DB, Leon AR, Loh E, Kocovic
DZ, Packer M, Clavell AL, Hayes DL, Ellestad M, Trupp RJ, Underwood J,
Pickering F, Truex C, McAtee P, Messenger J: Cardiac resynchronization in
chronic heart failure. N Engl J Med 2002, 346:1845–1853. 4. Cazeau S, Leclercq C, Lavergne T, Walker S, Varma C, Linde C, Garrigue S,
Kappenberger L, Haywood GA, Santini M, Bailleul C, Daubert JC,
Multisite Stimulation in Cardiomyopathies (MUSTIC) Study Investigators:
Effects of multisite biventricular pacing in patients with heart failure
and intraventricular conduction delay. N Engl J Med 2001, 344:873–880. 5. Auricchio A, Stellbrink C, Sack S, Block M, Vogt J, Bakker P, Huth C,
Schöndube F, Wolfhard U, Böcker D, Krahnefeld O, Kirkels H, Pacing
Therapies in Congestive Heart Failure (PATH-CHF) Study Group: Long-term
clinical effect of hemodynamically optimized cardiac resynchronization
therapy in patients with heart failure and ventricular conduction delay. Acknowledgements The authors are grateful to Indre Ceponiene for reviewing the English
manuscript. LA dilatation is a sensitive marker of chronic left heart
disease. Pressure and/or volume overload associated with
left ventricular involvement lead to gradual LA enlarge-
ment, electrical remodelling, and fibrosis. Considerable
evidence has been collected, demonstrating relationship
between increased LA size and cardiovascular morbidity
and mortality [19-21] in general population [22] and in
patients with left ventricular dysfunction [23,24]. In our
study, baseline LA volumes were substantially lower in
responders than in non-responders (89.1 ± 30.2 ml/m2 vs
104.5 ± 17.6 ml/m2, p = 0.02), and added to the prediction
of response to CRT. More research is needed to analyse
association of LA dilatation and response to CRT. Competing interests Competing interests
The authors declare that they have no competing interest. Competing interests 1.
Mosterd A, Hoes AW: Clinical epidemiology of heart failure. Heart 2007,
93:1137–1146. In our study, there were no statistically significant
differences in medication use at baseline between
echocardiographic
responders
and
non-responders,
except in warfarin use (p = 0.01). Indirect influence of
warfarin to the response to CRT might be explained by
the AF incidence (non-responders 61.5% vs responders
28.5%), or by larger left atrium and ventricular diameters
in non-responders. ,
Lang RM, Bierig M, Devereux RB, Flachskampf FA, Foster E, Pellikka PA,
Picard MH, Roman MJ, Seward J, Shanewise JS, Solomon SD, Spencer KT,
Sutton MS, Stewart WJ, Chamber Quantification Writing Group; American
Society of Echocardiography’s Guidelines and Standards Committee;
European Association of Echocardiography: Recommendations for chamber
quantification: A report from the American Society of Echocardiography’s
Guidelines and Standards Committee and the Chamber Quantification
Writing Group developed in conjunction with the European Association of
Echocardiography, a branch of the European Society of Cardiology. J Am
Soc Echocardiogr 2005, 18:1440–1463. Limitations of our study included a relatively small
sample
size,
single
centre
participation
and
short
study duration. Also, only patients with sinus rhythm
were enrolled, the proportions of genders in the study
were not equal. Dickstein K, Cohen-Solal A, Filippatos G, McMurray JJ, Ponikowski P, Poole-Wilson
PA, Strömberg A, Van Veldhuisen DJ, Atar D, Hoes AW, Keren A, Mebazaa A,
Nieminen M, Priori SG, Swedberg K, ESC Committee for Practice Guidelines
(CPG): ESC Guidelines for the diagnosis and treatment of acute and chronic
heart failure 2008. The Task Force for the Diagnosis and Treatment of Acute
and Chronic Heart Failure 2008 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association of the ESC
(HFA) and endorsed by the European Society of Intensive Care Medicine
(ESICM). Eur Heart J 2008, 29:2388–2442. Authors’ contributions In this study, receiver-operating characteristic curve
analysis defined the optimal cut-off value of 64 mm for
LVESD and 75 mm for LVEDD to predict the response
to CRT, confirming previous analyses, [13,18] suggesting
the larger cardiac diameters are associated with poorer
response to CRT. Study design: DR, SB, RJ. Data collection: DR, JL, KBD, KC, SA. Writing the first
draft: DR. Data interpretation, discussion and preparation of the final
manuscript: DR, SB, RJ, VZ, TK, VS, AP. All authors read and approved the final
manuscript. Dickstein K, Cohen-Solal A, Filippatos G, McMurray JJ, Ponikowski P, Poole-Wilson
PA, Strömberg A, Van Veldhuisen DJ, Atar D, Hoes AW, Keren A, Mebazaa A,
Nieminen M, Priori SG, Swedberg K, ESC Committee for Practice Guidelines
(CPG): ESC Guidelines for the diagnosis and treatment of acute and chronic
heart failure 2008. The Task Force for the Diagnosis and Treatment of Acute
and Chronic Heart Failure 2008 of the European Society of Cardiology.
Developed in collaboration with the Heart Failure Association of the ESC
(HFA) and endorsed by the European Society of Intensive Care Medicine
(ESICM). Eur Heart J 2008, 29:2388–2442. Received: 2 March 2014 Accepted: 23 April 2014
Published: 29 April 2014 Received: 2 March 2014 Accepted: 23 April 2014
Published: 29 April 2014 J Am Coll Cardiol 2002, 39:2026–2033. Author details
1
h 1Lithuanian University of Health Sciences, Kaunas, Lithuania. 2Hospital of
Lithuanian University of Health Sciences Kaunas Clinics, Kaunas, Lithuania. 3Department of Cardiology, Lithuanian University of Health Sciences, Kaunas
Clinics, Eiveniu 2, LT-50009 Kaunas, Lithuania. Rinkuniene et al. BMC Cardiovascular Disorders 2014, 14:55
http://www.biomedcentral.com/1471-2261/14/55 in NYHA functional class. In our study patients with
non-ICMP achieved a better clinical response compared
to ischemic patients (63.9% vs 36.1%, p = 0.009). Castel MA, Méndez F, Tamborero D, Mont L, Magnani S, Tolosana JM,
Berruezo A, Godoy M, Sitges M, Vidal B, Roig E, Brugada J: Six-minute Cazeau S, Leclercq C, Lavergne T, Walker S, Varma C, Linde C, Garrigue S,
Kappenberger L, Haywood GA, Santini M, Bailleul C, Daubert JC,
Multisite Stimulation in Cardiomyopathies (MUSTIC) Study Investigators:
Effects of multisite biventricular pacing in patients with heart failure
and intraventricular conduction delay. N Engl J Med 2001, 344:873–880.
Auricchio A, Stellbrink C, Sack S, Block M, Vogt J, Bakker P, Huth C,
Schöndube F, Wolfhard U, Böcker D, Krahnefeld O, Kirkels H, Pacing
Therapies in Congestive Heart Failure (PATH-CHF) Study Group: Long-term
clinical effect of hemodynamically optimized cardiac resynchronization
therapy in patients with heart failure and ventricular conduction delay.
J Am Coll Cardiol 2002, 39:2026–2033.
Lang RM, Bierig M, Devereux RB, Flachskampf FA, Foster E, Pellikka PA,
Picard MH, Roman MJ, Seward J, Shanewise JS, Solomon SD, Spencer KT,
Sutton MS, Stewart WJ, Chamber Quantification Writing Group; American
Society of Echocardiography’s Guidelines and Standards Committee;
European Association of Echocardiography: Recommendations for chamber
quantification: A report from the American Society of Echocardiography’s
Guidelines and Standards Committee and the Chamber Quantification
Writing Group developed in conjunction with the European Association of
Echocardiography, a branch of the European Society of Cardiology. J Am
Soc Echocardiogr 2005, 18:1440–1463.
Dickstein K, Cohen-Solal A, Filippatos G, McMurray JJ, Ponikowski P, Poole-Wilson
PA, Strömberg A, Van Veldhuisen DJ, Atar D, Hoes AW, Keren A, Mebazaa A,
Nieminen M, Priori SG, Swedberg K, ESC Committee for Practice Guidelines
(CPG): ESC Guidelines for the diagnosis and treatment of acute and chronic
heart failure 2008. The Task Force for the Diagnosis and Treatment of Acute
and Chronic Heart Failure 2008 of the European Society of Cardiology.
Developed in collaboration with the Heart Failure Association of the ESC
(HFA) and endorsed by the European Society of Intensive Care Medicine
(ESICM). Eur Heart J 2008, 29:2388–2442.
Castel MA, Méndez F, Tamborero D, Mont L, Magnani S, Tolosana JM,
Berruezo A, Godoy M, Sitges M, Vidal B, Roig E, Brugada J: Six-minute Discussion in ≥1 functional class in 80%, and combined clinical
response in 84% of patients. Similar results were obtained
in a study by Viviane Tiemi et al. [11], demonstrating
improvement in NYHA functional class in 79% of patients
after six months of CRT implantation. The 6-MWT was
used as a measure of response to CRT in a number of
studies, and was more sensitive compared to NYHA
functional class [12]. Our data did not contradict with the
findings of previous studies, as almost 82% of patients
achieved significant improvement in clinical and echocar-
diographic parameters. The need for complicated assess-
ment of CRT response is debatable, because from a
patient’s perspective improvement in clinical condition
matters most, hence changes in NYHA functional class
and six-minute walk distance may be unsophisticated and
important criteria for evaluation of response to CRT. 1 - Specificity
1.0
0.8
0.6
0.4
0.2
0.0
Sensitivity
1.0
0.8
0.6
0.4
0.2
0.0
ROC Curve
Figure 3 ROC curve for the association of echocardiographic response and uric acid concentration. (AUC 0.69, p = 0.03). ROC Curve Figure 3 ROC curve for the association of echocardiographic response and uric acid concentration. (AUC 0.69, p = 0.0 Page 7 of 8 Page 7 of 8 Rinkuniene et al. BMC Cardiovascular Disorders 2014, 14:55
http://www.biomedcentral.com/1471-2261/14/55 Rinkuniene et al. BMC Cardiovascular Disorders 2014, 14:55
http://www.biomedcentral.com/1471-2261/14/55 Rinkuniene et al. BMC Cardiovascular Disorders 2014, 14:55
http://www.biomedcentral.com/1471-2261/14/55 Rinkuniene et al. BMC Cardiovascular Disorders 2014, 14:55
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g p y
12. Foley PW, Leyva F, Frenneaux MP: What is treatment success in cardiac
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Madrid A, Pérez-Castellano N, Sitges M, Pavón-Jiménez R, Barba J, Cavero MA,
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Madrid A, Pérez-Castellano N, Sitges M, Pavón-Jiménez R, Barba J, Cavero MA,
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Comparison of characteristics in responders versus nonresponders with
biventricular pacing for drug-resistant congestive heart failure. Conclusions Smaller left ventricular end diastolic and end systolic
diameters and lower serum uric acid concentration
were associated with better response to CRT. LVEDD and
non-ischemic HF etiology were the strongest independent
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25. Wu AH, Ghali JK, Neuberg GW, O’Connor CM, Carson PE, Levy WC: Uric acid
level and allopurinol use as risk markers of mortality and morbidity in
systolic heart failure. Am Heart J 2010, 160(5):928–933. 26. Huang H, Huang B, Li Y, Huang Y, Li J, Yao H, Jing X, Chen J, Wang J: Uric
acid and risk of heart failure: a systematic review and meta-analysis. Eur J Heart Fail 2014, 16:15–24. • Convenient online submission doi:10.1186/1471-2261-14-55
Cite this article as: Rinkuniene et al.: Predictors of positive response to
cardiac resynchronization therapy. BMC Cardiovascular Disorders
2014 14:55.
|
https://openalex.org/W2999910794
|
http://ira.lib.polyu.edu.hk/bitstream/10397/82139/1/Tam_JNK_pathway_mediates.pdf
|
English
| null |
JNK Pathway Mediates Low Oxygen Level Induced Epithelial–Mesenchymal Transition and Stemness Maintenance in Colorectal Cancer Cells
|
Cancers
| 2,020
|
cc-by
| 12,850
|
Received: 23 December 2019; Accepted: 14 January 2020; Published: 16 January 2020 Abstract:(1)Background: Epithelial–mesenchymaltransition(EMT)andcancercellstemnessmaintenance
(SM) are important factors for cancer metastasis. Although hypoxia has been considered as a possible
factor for EMT induction and promotion of SM, studies in this area, apart from hypoxia-inducible factor
(HIF) pathways and severe hypoxia, are scant. This study aimed to evaluate the effects of different oxygen
levels on EMT induction and SM and elucidate the signaling pathways involved in colorectal cancer cells. (2) Methods: Cell morphological analysis, migration assay, immunofluorescence staining of cytoskeleton
and Western blotting were performed on human colorectal cancer cells HT-29, DLD-1, and SW-480
cultured at 1%, 10%, and normal (21%) O2 levels. The role played by c-Jun N-terminal kinase (JNK) was
evaluated through the use of the specific JNK inhibitor SP600125. (3) Results: This study evaluated 1%
and 10% O2 are possible conditions for EMT induction and SM. This study also demonstrated the partial
relieve of EMT induction and SM by SP600125, showing the importance of the JNK pathway in these
processes. Furthermore, this study proposed a novel pathway on the regulation of Akt by JNK-c-Jun. (4) Conclusions: This study suggests 10% O2 as another possible condition for EMT induction, and SM
and JNK pathways play important roles in these processes through multiple factors. Inhibition of JNK
could be explored as treatment for inhibiting metastasis in colorectal cancer cells. Keywords: Akt; colorectal cancer; epithelial–mesenchymal transition; hypoxia; JNK; oxygen level;
stemness maintenance cancers cancers cancers Cancers 2020, 12, 224; doi:10.3390/cancers12010224
Received: 23 December 2019; Accepted: 14 January 2020; Published: 16 January 2020 www.mdpi.com/journal/cancers Article Shing Yau Tam
, Vincent W.C. Wu *
and Helen K.W. Law *
Department of Health Technology and Informatics, Faculty of Health and Social Sciences, The Hong Kong
Polytechnic University, Hong Kong, China; shing-yau.tam@connect.polyu.hk
* Correspondence: vincent.wu@polyu.edu.hk (V.W.C.W.); hthelen@polyu.edu.hk (H.K.W.L.) Shing Yau Tam
, Vincent W.C. Wu *
and Helen K.W. Law *
Department of Health Technology and Informatics, Faculty of Health and Social Sciences, The Hong Kong
Polytechnic University, Hong Kong, China; shing-yau.tam@connect.polyu.hk
* Correspondence: vincent.wu@polyu.edu.hk (V.W.C.W.); hthelen@polyu.edu.hk (H.K.W.L.) Shing Yau Tam
, Vincent W.C. Wu *
and Helen K.W. Law *
Department of Health Technology and Informatics, Faculty of Health and Social Sciences, The Hong Kong
Polytechnic University, Hong Kong, China; shing-yau.tam@connect.polyu.hk
* Correspondence: vincent.wu@polyu.edu.hk (V.W.C.W.); hthelen@polyu.edu.hk (H.K.W.L.) 1. Introduction Metastasis is an important event in cancer progression and accounts for majority of cancer related
deaths including colorectal cancer (CRC) [1]. Although much of the exact mechanism remains unknown,
epithelial–mesenchymal transition (EMT) has been regarded as an important event for metastasis as it
allows cancer cells to transform from the closely packed epithelial cell type to mesenchymal cell type
with migratory and invasive properties [2]. The loss of adherent junctions between cells alters the cytoskeletal composition and cell polarity to
form spindle-shaped cells. In cancer cells undergoing EMT, the actin cytoskeleton is reorganized from
cortical thin bundles into thick contractile stress fibers at the ventral cell surface [3]. The monomers
of actin polymerize to form filamentous-actin (F-actin) and start the formation of various migratory
protrusions including podosomes, invadopodia, filopodia, and lamellipodia. This process, known as
dynamic actin reorganization, is a prerequisite for the migration and invasion of cancer cells [4,5]. Hypoxia induces EMT mainly by hypoxia-inducible factor (HIF) activation [6]. HIF-1α could bind
directly to twist-related protein 1 (TWIST1), matrix metalloproteinase-9 (MMP-9), and histone deacetylase 3
(HDAC3), which eventually promote transcription of Snail [7]. Meanwhile, HDAC3 regulates the formation
of histone methyltransferase complexes to induce vimentin and N-cadherin. For CRC, ubiquitin-specific www.mdpi.com/journal/cancers Cancers 2020, 12, 224 2 of 16 protease 47 (USP47) reduced E-cadherin expression by Snail regulation under hypoxic conditions [8], and
the overexpression of TWIST1 and Snail is associated with poor prognosis [9]. protease 47 (USP47) reduced E-cadherin expression by Snail regulation under hypoxic conditions [8], and
the overexpression of TWIST1 and Snail is associated with poor prognosis [9]. Apart from EMT of the primary tumor, cancer stem cells (CSCs) have been widely accepted
as precursors of metastases [10]. CSCs are pluripotent for multilineage differentiation, capable for
self-renewal, and have long longevity [11]. They vary in number among different types of cancer cell lines
and human xenografts, affecting tumor differentiation and aggressiveness [12]. CSC proliferation and
maintenance of stemness properties are controlled by similar pathways as EMT-inducing pathways such as
Wnt, transforming growth factor-β (TGF-β), and Notch [13]. The pathways exert their influences on
stemness maintenance (SM) by regulating pluripotency markers including octamer-binding transcription
factor 4 (Oct-4), sex determining region Y-box 2 (SOX2), and Nanog. In CRC cells, these pluripotency
markers are regarded as poor prognosis indicators of CRC. 1. Introduction In CRC stem cells, aberrant Wnt/β-catenin
signaling is observed, and this is suggested to cause treatment resistance and disease relapse [13]. Although HIFs have been suggested to influence EMT and stemness though multiple pathways
under hypoxic conditions, the influence of other hypoxia-related molecules such as c-Jun N-terminal
kinase (JNK) has received little attention [6,13]. JNK is activated by various stresses and was initially
regarded as an inducer of inflammation and apoptosis. However, JNK could also be activated by chronic
hypoxia and influence autophagy [14]. JNK may influence EMT though TGF-β SMAD-independent
pathway [6] and SM through Wnt/bone morphogenetic protein (BMP)/T cell factor (TCF)/Notch
or interleukin 6 (IL-6)/Janus kinase (JAK)/signal transducer and activator of transcription 3 (Stat3)
pathways [15]. Cancer cells constantly face different oxygen level during development from hypoxia to about 10%
oxygen level during metastasis in the bloodstream. In our previous autophagy study, we have reported
that 10% oxygen level could exert prominent effects on oxygen level driven regulators including HIFs
and JNK [14]. Therefore, the impact of various oxygen levels on EMT and SM was further investigated
in this study. 2. Results 2.1. Low Oxygen Levels Induced Morphological Signs of EMT, Which Was Reversed by JNK Inhibition 2.1. Low Oxygen Levels Induced Morphological Signs of EMT, Which Was Reversed by JNK Inhibition From the photomicrographs taken after 48 h incubation, the three CRC cell lines (HT-29, DLD-1, and
SW-480) showed different responses to different oxygen levels. For HT-29, which had more epithelial cell
features among the three cell lines, cells became more loosely packed colonies under 1% O2 (Figure 1A),
showing signs of EMT induction in the loss of cell–cell interactions [16]. Also, cell colonies incubated
under 1% and 10% O2 became more irregularly shaped from the dominantly round shape in 21% O2. In the presence of JNK inhibitor, the cell colony shape in all three oxygen levels became rounder, similar to
the control, when compared with those without the addition of JNK inhibitor. For DLD-1, which had intermediate epithelial and mesenchymal cell features among the three
studied cell lines, there was no prominent changes of morphological features under different oxygen
levels (Figure 1B). Whereas, in the presence of JNK inhibitor, cell colonies of DLD-1 cultured in 10% O2
became more closely packed. SW-480 normally had the most mesenchymal cell features among the three cell lines. In the 1%
and 10% O2 conditions, they acquired larger cell colonies of spindle-shaped cells when compared with
cells in 21% O2, showing signs of EMT induction (Figure 1C). In the presence of JNK inhibitor, the EMT
induction in 1% and 10% O2 were partially reversed by having more epithelial cell colonies and less
spindled-shaped mesenchymal-like cells. 3 of 16 Cancers 2020, 12, 224 Cancers 2020, 12, x
3 of 16 Figure 1. Cell morphological changes under different oxygen levels. HT-29 acquired more loosely
packed colonies (red arrows) under 1% O2 conditions, while the presence of SP600125 (10 µM)
changed the cell colony to be more rounded and more densely packed (yellow arrows) under all
oxygen levels investigated (A) DLD-1 did not show prominent changes in cell morphology under
different oxygen level incubation. Cell colonies became more closely packed in 10% O2 conditions
(yellow arrows) in the presence of SP600125 (B) SW-480 showed more mesenchymal features at 1%
and 10% O2 by having more spindle-shaped cells (red arrows). More epithelial cell colonies (yellow
arrows) formed under different oxygen levels with SP600125 addition, especially in 1% and 10% O2
conditions (C). Sample images from 5 independent experiments performed (200× magnification). 2.2. Cell Migration Was Reduced by JNK Inhibition
Cell migration was investigated by wound healing assay. Preliminary experiments using HT-29
Figure 1. showed an extremely slow migration rate, probab
Figure S1); hence, they have been excluded in th
2.2. Cell Migration Was Reduced by JNK Inhibition
)
yg
conditions (C). Sample images from 5 independe g
y
y
that there was no significant difference in migratory rate when cells were cultured in different oxygen
levels (Figure 2). Whereas, the addition of JNK inhibitor generally mildly reduced the migratory rate
among different oxygen levels. A significant reduction of migratory rate at 10% O2 in DLD-1 was found. Cell migration was investigated by wound healing assay. Preliminary experiments using HT-29
showed an extremely slow migration rate, probably due to their epithelial properties (Supplementary
Figure S1); hence, they have been excluded in the analysis. Results for DLD-1 and SW-480 showed that
there was no significant difference in migratory rate when cells were cultured in different oxygen levels
(Figure 2). Whereas, the addition of JNK inhibitor generally mildly reduced the migratory rate among
different oxygen levels. A significant reduction of migratory rate at 10% O2 in DLD-1 was found. 2.2. Cell Migration Was Reduced by JNK Inhibition
Cell migration was investigated by wound healing assay. Preliminary experiments using HT-29
showed an extremely slow migration rate, probably due to their epithelial properties (Supplementary
Figure S1); hence, they have been excluded in the analysis. Results for DLD-1 and SW-480 showed
that there was no significant difference in migratory rate when cells were cultured in different oxygen
levels (Figure 2). Whereas, the addition of JNK inhibitor generally mildly reduced the migratory rate
among different oxygen levels A significant reduction of migratory rate at 10% O2 in DLD 1 was found Figure 2. Wound healing assay under different oxygen levels. Wound healing assay showed that there
were no significant differences of migration rate between different oxygen level incubation in DLD-1
(A,C) and SW-480 (B,D) cells. While the presence of JNK inhibitor SP600125 (10 µM) generally
Figure 2. Wound healing assay under different oxygen levels. Wound healing assay showed that there
were no significant differences of migration rate between different oxygen level incubation in DLD-1
(A,C) and SW-480 (B,D) cells. While the presence of JNK inhibitor SP600125 (10 µM) generally
Figure 2. Wound healing assay under different oxygen levels. Wound healing assay showed that there
were no significant differences of migration rate between different oxygen level incubation in DLD-1
(A,C) and SW-480 (B,D) cells. While the presence of JNK inhibitor SP600125 (10 µM) generally reduced
the migration rate among different oxygen levels in both cell lines, with 10% O2 having significant
reduction in DLD-1. 2.1. Low Oxygen Levels Induced Morphological Signs of EMT, Which Was Reversed by JNK Inhibition Cell morphological changes under different oxygen levels. HT-29 acquired more loosely
packed colonies (red arrows) under 1% O2 conditions, while the presence of SP600125 (10 µM) changed
the cell colony to be more rounded and more densely packed (yellow arrows) under all oxygen levels
investigated (A) DLD-1 did not show prominent changes in cell morphology under different oxygen
level incubation. Cell colonies became more closely packed in 10% O2 conditions (yellow arrows) in
the presence of SP600125 (B) SW-480 showed more mesenchymal features at 1% and 10% O2 by having
more spindle-shaped cells (red arrows). More epithelial cell colonies (yellow arrows) formed under
different oxygen levels with SP600125 addition, especially in 1% and 10% O2 conditions (C). Sample
images from 5 independent experiments performed (200× magnification). Cancers 2020, 12, x
3 of 16
Figure 1. Cell morphological changes under different oxygen levels. HT-29 acquired more loosely
packed colonies (red arrows) under 1% O2 conditions, while the presence of SP600125 (10 µM)
changed the cell colony to be more rounded and more densely packed (yellow arrows) under all
oxygen levels investigated (A) DLD-1 did not show prominent changes in cell morphology under
different oxygen level incubation. Cell colonies became more closely packed in 10% O2 conditions
(yellow arrows) in the presence of SP600125 (B) SW-480 showed more mesenchymal features at 1%
and 10% O2 by having more spindle-shaped cells (red arrows). More epithelial cell colonies (yellow
arrows) formed under different oxygen levels with SP600125 addition especially in 1% and 10% O2 Figure 1. Cell morphological changes under different oxygen levels. HT-29 acquired more loosely
packed colonies (red arrows) under 1% O2 conditions, while the presence of SP600125 (10 µM)
changed the cell colony to be more rounded and more densely packed (yellow arrows) under all
oxygen levels investigated (A) DLD-1 did not show prominent changes in cell morphology under
different oxygen level incubation. Cell colonies became more closely packed in 10% O2 conditions
(yellow arrows) in the presence of SP600125 (B) SW-480 showed more mesenchymal features at 1%
and 10% O2 by having more spindle-shaped cells (red arrows). More epithelial cell colonies (yellow
arrows) formed under different oxygen levels with SP600125 addition, especially in 1% and 10% O2
conditions (C). Sample images from 5 independent experiments performed (200× magnification). 2.2. Cell Migration Was Reduced by JNK Inhibition
Cell migration was investigated by wound healing assay. Preliminary experiments using HT-29
Figure 1. 2.1. Low Oxygen Levels Induced Morphological Signs of EMT, Which Was Reversed by JNK Inhibition Cell morphological changes under different oxygen levels. HT-29 acquired more loosely
packed colonies (red arrows) under 1% O2 conditions, while the presence of SP600125 (10 µM) changed
the cell colony to be more rounded and more densely packed (yellow arrows) under all oxygen levels
investigated (A) DLD-1 did not show prominent changes in cell morphology under different oxygen
level incubation. Cell colonies became more closely packed in 10% O2 conditions (yellow arrows) in
the presence of SP600125 (B) SW-480 showed more mesenchymal features at 1% and 10% O2 by having
more spindle-shaped cells (red arrows). More epithelial cell colonies (yellow arrows) formed under
different oxygen levels with SP600125 addition, especially in 1% and 10% O2 conditions (C). Sample
images from 5 independent experiments performed (200× magnification). Figure 1. Cell morphological changes under different oxygen levels. HT-29 acquired more loosely
packed colonies (red arrows) under 1% O2 conditions, while the presence of SP600125 (10 µM)
changed the cell colony to be more rounded and more densely packed (yellow arrows) under all
oxygen levels investigated (A) DLD-1 did not show prominent changes in cell morphology under
different oxygen level incubation. Cell colonies became more closely packed in 10% O2 conditions
(yellow arrows) in the presence of SP600125 (B) SW-480 showed more mesenchymal features at 1%
and 10% O2 by having more spindle-shaped cells (red arrows). More epithelial cell colonies (yellow
arrows) formed under different oxygen levels with SP600125 addition especially in 1% and 10% O2 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation
g
(
)
p
4 (DLD-1). 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation
4 (DLD-1). 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation
4 (DLD-1). Immunofluorescence staining of F-actin by phalloidin was conducted to evaluate the actin
cytoskeleton. For HT-29, majority of cells had lamellipodia formation (Figure 3A). There was a trend of
increased filopodia formation at lower oxygen levels. Ventral stress fibers are described as thick and
directed contractile fibers [3]. There was a decreasing percentage of cells with ventral stress fibers with
lowering oxygen levels. JNK inhibition reduced the formation of lamellipodia, filopodia, and ventral
stress fibers in different oxygen levels (Figure 3A). 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation
Immunofluorescence staining of F-actin by phalloidin was conducted to evaluate the actin
cytoskeleton. For HT-29, majority of cells had lamellipodia formation (Figure 3A). There was a trend
of increased filopodia formation at lower oxygen levels. Ventral stress fibers are described as thick
and directed contractile fibers [3]. There was a decreasing percentage of cells with ventral stress fibers
with lowering oxygen levels. JNK inhibition reduced the formation of lamellipodia, filopodia, and
t
l t
fib
i
diff
t
l
l (Fi
3A) Fi
3 I
fl
i i
f F
i
d
diff
l
l
I
fl Figure 3. Immunofluorescence staining of F-actin under different oxygen levels. Immunofluorescence
staining of F-actin (red) and DAPI (blue) showed an increase in filopodia formation (yellow arrows)
and a decrease in ventral stress fiber formation (green arrows) in 1% and 10% O2 conditions, while
lamellipodia formation (white arrows) was similar among different oxygen levels in HT-29 (A) DLD-
1 also showed an increase in filopodia formation in 1% and 10% O2 with similar lamellipodia
formation among different oxygen levels (B) SW-480 showed similar amounts of lamellipodia and
Figure 3. Immunofluorescence staining of F-actin under different oxygen levels. Immunofluorescence
staining of F-actin (red) and DAPI (blue) showed an increase in filopodia formation (yellow arrows)
and a decrease in ventral stress fiber formation (green arrows) in 1% and 10% O2 conditions, while
lamellipodia formation (white arrows) was similar among different oxygen levels in HT-29 (A) DLD-1
also showed an increase in filopodia formation in 1% and 10% O2 with similar lamellipodia formation
among different oxygen levels (B) SW-480 showed similar amounts of lamellipodia and filopodia
formation among different oxygen levels, and ventral stress fibers were not observed in SW-480. showed an extremely slow migration rate, probab
Figure S1); hence, they have been excluded in th
2.2. Cell Migration Was Reduced by JNK Inhibition
)
yg
conditions (C). Sample images from 5 independe The data (means ± SEM) were expressed as the relative migration rate against the
rate under 21% O2 + DMSO (C,D). ** p < 0.01, DMSO versus SP600125. N = 3 (SW-480) and 4 (DLD-1). Figure 2. Wound healing assay under different oxygen levels. Wound healing assay showed that there
were no significant differences of migration rate between different oxygen level incubation in DLD-1
(A,C) and SW-480 (B,D) cells. While the presence of JNK inhibitor SP600125 (10 µM) generally Figure 2. Wound healing assay under different oxygen levels. Wound healing assay showed that there
were no significant differences of migration rate between different oxygen level incubation in DLD-1
(A,C) and SW-480 (B,D) cells. While the presence of JNK inhibitor SP600125 (10 µM) generally
Figure 2. Wound healing assay under different oxygen levels. Wound healing assay showed that there
were no significant differences of migration rate between different oxygen level incubation in DLD-1
(A,C) and SW-480 (B,D) cells. While the presence of JNK inhibitor SP600125 (10 µM) generally reduced
the migration rate among different oxygen levels in both cell lines, with 10% O2 having significant
reduction in DLD-1. The data (means ± SEM) were expressed as the relative migration rate against the
rate under 21% O2 + DMSO (C,D). ** p < 0.01, DMSO versus SP600125. N = 3 (SW-480) and 4 (DLD-1). Cancers 2020, 12, 224
reduced t
i
ifi a 4 of 16 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation
g
(
)
p
4 (DLD-1). (C) The
presence of JNK inhibitor SP600125 slightly reduced filopodia and lamellipodia formation among the
three cell lines, while ventral stress fiber formation was promoted in DLD-1. Sample images were taken
from 3 independent experiments. The counting results were shown as means ± SEM in percentage
among all evaluated cells. At least 200 cells were evaluated for each sample. * p < 0.05 DMSO versus
SP600125. N = 3. Figure 3. Immunofluorescence staining of F-actin under different oxygen levels. Immunofluorescence
staining of F-actin (red) and DAPI (blue) showed an increase in filopodia formation (yellow arrows)
and a decrease in ventral stress fiber formation (green arrows) in 1% and 10% O2 conditions, while
lamellipodia formation (white arrows) was similar among different oxygen levels in HT-29 (A) DLD-
1 also showed an increase in filopodia formation in 1% and 10% O2 with similar lamellipodia
formation among different oxygen levels (B) SW-480 showed similar amounts of lamellipodia and
Figure 3. Immunofluorescence staining of F-actin under different oxygen levels. Immunofluorescence
staining of F-actin (red) and DAPI (blue) showed an increase in filopodia formation (yellow arrows)
and a decrease in ventral stress fiber formation (green arrows) in 1% and 10% O2 conditions, while
lamellipodia formation (white arrows) was similar among different oxygen levels in HT-29 (A) DLD-1
also showed an increase in filopodia formation in 1% and 10% O2 with similar lamellipodia formation
among different oxygen levels (B) SW-480 showed similar amounts of lamellipodia and filopodia
formation among different oxygen levels, and ventral stress fibers were not observed in SW-480. (C) The
presence of JNK inhibitor SP600125 slightly reduced filopodia and lamellipodia formation among the
three cell lines, while ventral stress fiber formation was promoted in DLD-1. Sample images were taken
from 3 independent experiments. The counting results were shown as means ± SEM in percentage
among all evaluated cells. At least 200 cells were evaluated for each sample. * p < 0.05 DMSO versus
SP600125. N = 3. Cancers 2020, 12, 224 5 of 16 For DLD-1, a smaller population of cells had lamellipodia formation when compared with HT-29,
while a similar increase of filopodia formation in lower oxygen levels was observed (Figure 3B). JNK
inhibition could slightly reduce lamellipodia and filopodia formation in 1% and 10% O2 but generally
promoted ventral stress fiber formation in DLD-1. 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation
g
(
)
p
4 (DLD-1). In SW-480, similar percentages of cells with lamellipodia and filopodia formations were recorded
among different oxygen levels (Figure 3C). The presence of JNK inhibitor reduced formations of lamellipodia
and filopodia. There was no observable ventral stress fiber formation in SW-480. 2.4. EMT Induced by Low Oxygen Levels via JNK Pathway Was Confirmed by EMT Markers and Transcription
Factors To further investigate the underlying signaling pathways in leading to these morphological and
cytoskeleton changes and the role of JNK in EMT and stemness pathways, the expression of related
proteins including EMT markers, EMT transcription factors, JNK pathway markers, other EMT related
pathway markers, and SM markers were evaluated by Western blotting. The key EMT marker, E-cadherin, was significantly down-regulated in 1% and 10% O2 in all three
studied cell lines (Figure 4A). JNK inhibitor SP600125 (10 µM) could generally promote E-cadherin in
different oxygen levels, especially for 10% O2 in HT-29 (Figure 4B). The mesenchymal marker fibronectin was generally promoted in 1% and 10% oxygen levels among
the three cell lines with significant promotion in 1% O2 on HT-29 (Figure 4C). The impact of SP600125
on fibronectin had diverged responses among the three cell lines (Figure 4D). Down-regulation of
fibronectin in HT-29 was found in lower oxygen levels, whereas DLD-1 and SW-480 experienced
up-regulation trends in 10% and 21% O2. Another mesenchymal marker, vimentin, was only detectable in SW-480. It was mildly promoted
in 10% O2 but not in 1% O2 (Figure 4E). JNK inhibitor reduced vimentin expression under different
oxygen levels (Figure 4F). EMT transcription factors Snail and TWIST1 were also investigated in DLD-1 and SW-480 cells. Both Snail (Figure 4G) and TWIST1 (Figure 4I) were up-regulated in 1% and 10% O2 among both
cell lines, with SW-480 having significant promotions in both Snail and TWIST1 at lower oxygen
levels, while DLD-1 could attain significant increase in TWIST1. Both Snail (Figure 4H) and TWIST1
(Figure 4J) were generally inhibited by JNK inhibitor in DLD-1 and SW-480, especially for DLD-1 in
lower oxygen levels. 6 of 16 Cancers 2020, 12, 224 Cancers 2020, 12, x
6 of 16 Ca ce s 0 0,
,
6 o
6
Figure 4. Epithelial–mesenchymal transition (EMT) marker and transcription factor expressions
under different oxygen levels. 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation
g
(
)
p
4 (DLD-1). Epithelial–mesenchymal transition (EMT) marker and transcription factor expressions under
different oxygen levels. EMT markers and transcription factors were evaluated by Western blotting
in different oxygen levels on HT-29, DLD-1, and SW-480 cell lines. The key epithelial EMT marker
E-cadherin was significantly down-regulated in all three studied cell lines in 1% and 10% O2 (A)
JNK inhibitor SP600125 (10 µM) could generally up-regulate E-cadherin in different oxygen levels,
especially for 10% O2 in HT-29 (p < 0.05) (B) The mesenchymal EMT marker fibronectin was generally
up-regulated in lower oxygen levels with 1% O2 in HT-29 having significant up-regulation (C) HT-29
experienced down-regulation of fibronectin in the presence of SP600125, while DLD-1 and SW-480 had
up-regulation of fibronectin in 10% and 21% O2 by SP600125 (D) Another mesenchymal EMT marker,
vimentin, was only detectable in SW-480; it was mildly promoted in 10% O2 but not in 1% O2 (E)
SP600125 could substantially reduce vimentin expression under different oxygen levels, especially for
10% and 21% O2 (F) EMT transcription factors Snail and TWIST1 expression levels in different oxygen
levels were evaluated in DLD-1 and SW-480 (G–J) Substantial promotions of Snail in lower oxygen
levels were found with SW-480 having significant increase in 1% and 10% O2 (G) Snail was generally
suppressed by SP600125 with more prominent effects in DLD-1 (H) TWIST1 also had significant
promotion in lower oxygen levels among SW-480 and DLD-1 (I) and it experienced a suppression
effect by SP600125 in both 1% and 10% O2 (J) The data (means ± SEM) were expressed as the relative
expression compared with 21% O2 group, N = 9 (HT-29 and DLD-1) or 10 (SW-480) (A,C,E,G,I) The
data (means ± SEM) were expressed as the relative expression compared with 21% O2 + DMSO group. N = 9 (HT-29 and DLD-1) or 10 (SW-480) for DMSO group, N = 4 (HT-29) or 5 (DLD-1 and SW-480) for
SP600125 group (B,D,F,H,J). * p < 0.05, ** p < 0.01, *** p < 0.001. D: DMSO (0.1%), S: SP600125 (10 µM). Figure 4. Epithelial–mesenchymal transition (EMT) marker and transcription factor expressions
under different oxygen levels. EMT markers and transcription factors were evaluated by Western
blotting in different oxygen levels on HT-29, DLD-1, and SW-480 cell lines. 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation
g
(
)
p
4 (DLD-1). The key epithelial EMT
marker E-cadherin was significantly down-regulated in all three studied cell lines in 1% and 10% O2
(A) JNK inhibitor SP600125 (10 µM) could generally up-regulate E-cadherin in different oxygen levels,
especially for 10% O2 in HT-29 (p < 0.05) (B) The mesenchymal EMT marker fibronectin was generally
up-regulated in lower oxygen levels with 1% O2 in HT-29 having significant up-regulation (C) HT-29
experienced down-regulation of fibronectin in the presence of SP600125, while DLD-1 and SW-480
had up-regulation of fibronectin in 10% and 21% O2 by SP600125 (D) Another mesenchymal EMT
marker, vimentin, was only detectable in SW-480; it was mildly promoted in 10% O2 but not in 1% O2
(E) SP600125 could substantially reduce vimentin expression under different oxygen levels, especially
for 10% and 21% O2 (F) EMT transcription factors Snail and TWIST1 expression levels in different
oxygen levels were evaluated in DLD-1 and SW-480 (G–J) Substantial promotions of Snail in lower
oxygen levels were found with SW-480 having significant increase in 1% and 10% O2 (G) Snail was
generally suppressed by SP600125 with more prominent effects in DLD-1 (H) TWIST1 also had
significant promotion in lower oxygen levels among SW-480 and DLD-1 (I) and it experienced a
suppression effect by SP600125 in both 1% and 10% O2 (J) The data (means ± SEM) were expressed as
the relative expression compared with 21% O2 group, N = 9 (HT-29 and DLD-1) or 10 (SW-480)
(A,C,E,G,I) The data (means ± SEM) were expressed as the relative expression compared with 21% O2
+ DMSO group. N = 9 (HT-29 and DLD-1) or 10 (SW-480) for DMSO group, N = 4 (HT-29) or 5 (DLD-
1 and SW-480) for SP600125 group (B,D,F,H,J). * p < 0.05, ** p < 0.01, *** p < 0.001. D: DMSO (0.1%), S:
SP600125 (10 µM). Figure 4. Epithelial–mesenchymal transition (EMT) marker and transcription factor expressions under
different oxygen levels. EMT markers and transcription factors were evaluated by Western blotting
in different oxygen levels on HT-29, DLD-1, and SW-480 cell lines. 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation
g
(
)
p
4 (DLD-1). EMT markers and transcription factors were evaluated by Western
bl
i
i
diff
l
l
HT 29 DLD 1
d SW 480
ll li
Th k
i h li l EMT
Figure 4. Epithelial–mesenchymal transition (EMT) marker and transcription factor expressions under
different oxygen levels. EMT markers and transcription factors were evaluated by Western blotting Figure 4. Epithelial–mesenchymal transition (EMT) marker and transcription factor expressions
under different oxygen levels. EMT markers and transcription factors were evaluated by Western
blotting in different oxygen levels on HT-29, DLD-1, and SW-480 cell lines. The key epithelial EMT
marker E-cadherin was significantly down-regulated in all three studied cell lines in 1% and 10% O2
(A) JNK inhibitor SP600125 (10 µM) could generally up-regulate E-cadherin in different oxygen levels,
especially for 10% O2 in HT-29 (p < 0.05) (B) The mesenchymal EMT marker fibronectin was generally
up-regulated in lower oxygen levels with 1% O2 in HT-29 having significant up-regulation (C) HT-29
experienced down-regulation of fibronectin in the presence of SP600125, while DLD-1 and SW-480
had up-regulation of fibronectin in 10% and 21% O2 by SP600125 (D) Another mesenchymal EMT
marker, vimentin, was only detectable in SW-480; it was mildly promoted in 10% O2 but not in 1% O2
(E) SP600125 could substantially reduce vimentin expression under different oxygen levels, especially
for 10% and 21% O2 (F) EMT transcription factors Snail and TWIST1 expression levels in different
oxygen levels were evaluated in DLD-1 and SW-480 (G–J) Substantial promotions of Snail in lower
oxygen levels were found with SW-480 having significant increase in 1% and 10% O2 (G) Snail was
generally suppressed by SP600125 with more prominent effects in DLD-1 (H) TWIST1 also had
significant promotion in lower oxygen levels among SW-480 and DLD-1 (I) and it experienced a
suppression effect by SP600125 in both 1% and 10% O2 (J) The data (means ± SEM) were expressed as
the relative expression compared with 21% O2 group, N = 9 (HT-29 and DLD-1) or 10 (SW-480)
(A,C,E,G,I) The data (means ± SEM) were expressed as the relative expression compared with 21% O2
+ DMSO group. N = 9 (HT-29 and DLD-1) or 10 (SW-480) for DMSO group, N = 4 (HT-29) or 5 (DLD-
1 and SW-480) for SP600125 group (B,D,F,H,J). * p < 0.05, ** p < 0.01, *** p < 0.001. D: DMSO (0.1%), S:
SP600125 (10 µM). Figure 4. 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation
g
(
)
p
4 (DLD-1). The key epithelial EMT marker
E-cadherin was significantly down-regulated in all three studied cell lines in 1% and 10% O2 (A)
JNK inhibitor SP600125 (10 µM) could generally up-regulate E-cadherin in different oxygen levels,
especially for 10% O2 in HT-29 (p < 0.05) (B) The mesenchymal EMT marker fibronectin was generally
up-regulated in lower oxygen levels with 1% O2 in HT-29 having significant up-regulation (C) HT-29
experienced down-regulation of fibronectin in the presence of SP600125, while DLD-1 and SW-480 had
up-regulation of fibronectin in 10% and 21% O2 by SP600125 (D) Another mesenchymal EMT marker,
vimentin, was only detectable in SW-480; it was mildly promoted in 10% O2 but not in 1% O2 (E)
SP600125 could substantially reduce vimentin expression under different oxygen levels, especially for
10% and 21% O2 (F) EMT transcription factors Snail and TWIST1 expression levels in different oxygen
levels were evaluated in DLD-1 and SW-480 (G–J) Substantial promotions of Snail in lower oxygen
levels were found with SW-480 having significant increase in 1% and 10% O2 (G) Snail was generally
suppressed by SP600125 with more prominent effects in DLD-1 (H) TWIST1 also had significant
promotion in lower oxygen levels among SW-480 and DLD-1 (I) and it experienced a suppression
effect by SP600125 in both 1% and 10% O2 (J) The data (means ± SEM) were expressed as the relative
expression compared with 21% O2 group, N = 9 (HT-29 and DLD-1) or 10 (SW-480) (A,C,E,G,I) The
data (means ± SEM) were expressed as the relative expression compared with 21% O2 + DMSO group. N = 9 (HT-29 and DLD-1) or 10 (SW-480) for DMSO group, N = 4 (HT-29) or 5 (DLD-1 and SW-480) for
SP600125 group (B,D,F,H,J). * p < 0.05, ** p < 0.01, *** p < 0.001. D: DMSO (0.1%), S: SP600125 (10 µM). Cancers 2020, 12, 224 7 of 16 7 of 16 2.5. Confirmation of JNK and Akt Pathway Activation in Low Oxygen Levels
Cancers 2020, 12, x 2.5. Confirmation of JNK and Akt Pathway Activation in Low Oxygen Levels
Cancers 2020, 12, x 2.5. Confirmation of JNK and Akt Pathway Activation in Low Oxygen Levels
Cancers 2020,
, JNK and Akt pathways are possible regulators of EMT transcription factors. 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation
g
(
)
p
4 (DLD-1). It was confirmed that
JNK phosphorylation in CRC was detected in both 1% and 10% O2 conditions for the three studied cell
lines, with significant activations in HT-29 (1% and 10% O2) and DLD-1 (10% O2) (Figure 5A). JNK
inhibitor did not directly affect the phosphorylation status of JNK (Figure 5B). However, it suppressed
the direct target of JNK c-Jun by inhibiting the phosphorylation status of p-c-Jun (Figure 5C) in different
oxygen levels among all three studied cell lines. 2.5. Confirmation of JNK and Akt Pathway Activation in Low Oxygen Levels
JNK and Akt pathways are possible regulators of EMT transcription factors. It was confirmed
that JNK phosphorylation in CRC was detected in both 1% and 10% O2 conditions for the three
studied cell lines, with significant activations in HT-29 (1% and 10% O2) and DLD-1 (10% O2) (Figure 5A). JNK inhibitor did not directly affect the phosphorylation status of JNK (Figure 5B). However, it
suppressed the direct target of JNK c-Jun by inhibiting the phosphorylation status of p-c-Jun (Figure 5C)
in different oxygen levels among all three studied cell lines he direct target of JNK c-Jun by inhibiting the phosphorylation status of p-c-Jun (Figure 5C) in different
xygen levels among all three studied cell lines. JNK inhibitor did not directly affect the phosphorylation status of JNK (Figure 5B). However, it
suppressed the direct target of JNK c-Jun by inhibiting the phosphorylation status of p-c-Jun (Figure 5C)
in different oxygen levels among all three studied cell lines. Figure 5. JNK and Akt pathway activation under different oxygen levels. The key markers of JNK
pathways were evaluated by Western blotting. JNK was activated under lower oxygen levels in HT-
29, DLD-1, and SW-480 with significant promotion in HT-29 and DLD-1 (A) There were generally
mild reductions in JNK phosphorylation status by SP600125 (10 µM) in 1% and 10% O2 among the
three cell lines, with greater effects on SW-480, while JNK phosphorylation was slightly promoted in
21% O2 (B) For the JNK direct downstream target c-Jun, its phosphorylation status p-c-Jun has been
largely suppressed by SP600125 in lower oxygen levels, with significant reductions found in HT-29
and DLD-1 (C) The indirect downstream effector of JNK pathway p62 showed mild up-regulation in
10% O and down regulation in 1% O (D) In the presence of SP600125 significant down regulations
Figure 5. JNK and Akt pathway activation under different oxygen levels. 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation
g
(
)
p
4 (DLD-1). JNK was activated under lower oxygen levels in HT-29,
DLD-1, and SW-480 with significant promotion in HT-29 and DLD-1 (A) There were generally mild
reductions in JNK phosphorylation status by SP600125 (10 µM) in 1% and 10% O2 among the three cell
lines, with greater effects on SW-480, while JNK phosphorylation was slightly promoted in 21% O2
(B) For the JNK direct downstream target c-Jun, its phosphorylation status p-c-Jun has been largely
suppressed by SP600125 in lower oxygen levels, with significant reductions found in HT-29 and DLD-1 8 of 16 Cancers 2020, 12, 224 (C) The indirect downstream effector of JNK pathway p62 showed mild up-regulation in 10% O2 and
down-regulation in 1% O2 (D) In the presence of SP600125, significant down-regulations were found in
different oxygen levels among DLD-1 and SW-480 (E) However, for HT-29, significant up-regulation of
p62 was evaluated in 10% O2. Akt, which is another important EMT promoter, also demonstrated an
increase in activation under lower oxygen levels in the three cell lines, with SW-480 having significant
activation (F) The 1% and 10% O2 activated Akt was abruptly inhibited by SP600125, particularly in
HT-29 and SW-480 (G) The data (means ± SEM) were expressed as the relative expression compared
with 21% O2 group, N = 9 (HT-29), 8–9 (DLD-1), or 10 (SW-480) (A,D,F). The data (means ± SEM) were
expressed as the relative expression compared with 21% O2 + DMSO group. N = 4–9 (HT-29), 5–9
(DLD-1), or 5–10 (SW-480) for DMSO group; N = 4 (HT-29) or 5 (DLD-1 and SW-480) for SP600125
group (B,C,E,G). * p < 0.05, ** p < 0.01, *** p < 0.001. D: DMSO (0.1%), S: SP600125 (10 µM). Cancers 2020, 12, x
8 of 16
were found in different oxygen levels among DLD-1 and SW-480 (E) However, for HT-29, significant
up-regulation of p62 was evaluated in 10% O2. Akt, which is another important EMT promoter, also
demonstrated an increase in activation under lower oxygen levels in the three cell lines, with SW-480
having significant activation (F) The 1% and 10% O2 activated Akt was abruptly inhibited by
SP600125, particularly in HT-29 and SW-480 (G) The data (means ± SEM) were expressed as the For the indirect downstream effector of JNK pathway, p62 had diverse changes under lower
oxygen levels with general promotion at 10% O2 and general down-regulation at 1% O2 (Figure 5D). 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation
g
(
)
p
4 (DLD-1). The key markers of JNK
pathways were evaluated by Western blotting. JNK was activated under lower oxygen levels in HT-29,
DLD-1, and SW-480 with significant promotion in HT-29 and DLD-1 (A) There were generally mild
reductions in JNK phosphorylation status by SP600125 (10 µM) in 1% and 10% O2 among the three cell
lines, with greater effects on SW-480, while JNK phosphorylation was slightly promoted in 21% O2
(B) For the JNK direct downstream target c-Jun, its phosphorylation status p-c-Jun has been largely
suppressed by SP600125 in lower oxygen levels, with significant reductions found in HT-29 and DLD-1 r different oxygen levels The key markers of JNK r different oxygen levels. The key markers of JNK
K
i
d
d
l
l
l i
HT
r different oxygen levels. The key markers of JNK Figure 5. JNK and Akt pathway activation under different oxygen levels. The key markers of JNK
pathways were evaluated by Western blotting. JNK was activated under lower oxygen levels in HT-
29, DLD-1, and SW-480 with significant promotion in HT-29 and DLD-1 (A) There were generally
mild reductions in JNK phosphorylation status by SP600125 (10 µM) in 1% and 10% O2 among the
three cell lines, with greater effects on SW-480, while JNK phosphorylation was slightly promoted in
21% O2 (B) For the JNK direct downstream target c-Jun, its phosphorylation status p-c-Jun has been
largely suppressed by SP600125 in lower oxygen levels, with significant reductions found in HT-29
and DLD-1 (C) The indirect downstream effector of JNK pathway p62 showed mild up-regulation in
Figure 5. JNK and Akt pathway activation under different oxygen levels. The key markers of JNK
pathways were evaluated by Western blotting. 2.3. JNK Inhibition Reduced Lamellipodia and Filopodia Formation
g
(
)
p
4 (DLD-1). JNK inhibitor suppressed p62 in DLD-1 and SW-480 and activated p62 in 10% O2 of HT-29 (Figure 5E). This evidence demonstrated the successful general JNK inhibition. relative expression compared with 21% O2 group, N = 9 (HT-29), 8–9 (DLD-1), or 10 (SW-480) (A,D,F). The data (means ± SEM) were expressed as the relative expression compared with 21% O2 + DMSO group. N = 4–9 (HT-29), 5–9 (DLD-1), or 5–10 (SW-480) for DMSO group; N = 4 (HT-29) or 5 (DLD-1 and SW-480)
for SP600125 group (B,C,E,G). * p < 0.05, ** p < 0.01, *** p < 0.001. D: DMSO (0.1%), S: SP600125 (10 µM). For the indirect downstream effector of JNK pathway, p62 had diverse changes under lower For Akt phosphorylation, all three cell lines showed general activation under lower oxygen levels
with statistically significant up-regulation in SW-480 (Figure 5F). For the parallel EMT-inducing Akt
pathway, JNK inhibition could abruptly inhibit low oxygen level activated Akt, with statistically
significant suppression found among HT-29 and SW-480 (Figure 5G). For the indirect downstream effector of JNK pathway, p62 had diverse changes under lower
oxygen levels with general promotion at 10% O2 and general down-regulation at 1% O2 (Figure 5D). JNK inhibitor suppressed p62 in DLD-1 and SW-480 and activated p62 in 10% O2 of HT-29 (Figure
5E). This evidence demonstrated the successful general JNK inhibition. For Akt phosphorylation, all three cell lines showed general activation under lower oxygen
levels with statistically significant up-regulation in SW-480 (Figure 5F) For the parallel EMT- 2.6. Promotion of SM under Low Oxygen Levels Was Partially Reversed by JNK Inhibition
inducing Akt pathway, JNK inhibition could abruptly inhibit low oxygen level activated
statistically significant suppression found among HT 29 and SW 480 (Figure 5G) 2.6. Promotion of SM under Low Oxygen Levels Was Partially Reversed by JNK Inhibition
inducing Akt pathway, JNK inhibition could abruptly inhibit low oxygen level activated A
statistically significant suppression found among HT-29 and SW-480 (Figure 5G) Changes of SM markers of CSC, including Oct-4 and Nanog, in different oxygen levels were also
studied. Results revealed general promotion of both markers in lower oxygen levels with greater
promotion of Oct-4, especially for HT-29 and DLD-1 (Figure 6A,B). Significant up-regulations were
found at Oct-4 in HT-29 (1% and 10% O2) and DLD-1 (10% O2) and Nanog in SW-480 (10% O2). For the
study of the effect of JNK inhibition on Oct-4 and Nanog (Figure 6C), diverged results had been
discovered in Oct-4 among the three cell lines. Oct-4 was generally suppressed by JNK inhibition
among HT-29 and SW-480, while DLD-1 experienced slight up-regulation instead. In the meantime,
Nanog was generally down-regulated by JNK inhibition among all three cell lines in 1% and 10% O2. 2.6. Promotion of SM under Low Oxygen Levels Was Partially Reversed by JNK Inhibition
Changes of SM markers of CSC, including Oct-4 and Nanog, in different oxygen levels were also
studied. Results revealed general promotion of both markers in lower oxygen levels with greater
promotion of Oct-4, especially for HT-29 and DLD-1 (Figure 6A,B). Significant up-regulations were
found at Oct-4 in HT-29 (1% and 10% O2) and DLD-1 (10% O2) and Nanog in SW-480 (10% O2). For
the study of the effect of JNK inhibition on Oct-4 and Nanog (Figure 6C), diverged results had been
discovered in Oct-4 among the three cell lines. Oct-4 was generally suppressed by JNK inhibition
among HT-29 and SW-480, while DLD-1 experienced slight up-regulation instead. In the meantime,
Nanog was generally down-regulated by JNK inhibition among all three cell lines in 1% and 10% O2 Figure 6. Cont. 9 of 16 Cancers 2020, 12, 224 Figure 6. Oct-4 and Nanog expressions under different oxygen levels. The key stemness maintenance
markers Oct-4 (A) and Nanog (B) changes in lower oxygen levels were evaluated by Western blotting. Results showed that both stemness maintenance markers were generally up-regulated under lower
oxygen levels with significant up-regulation of Oct-4 in HT-29 (1% and 10% O2) and DLD-1 (10% O2)
and Nanog in SW-480 (10% O2). For the impact of JNK inhibitor SP600125 (10 µM) on Oct-4 (C),
diverse impacts with general down-regulation in lower oxygen levels among HT-29 and SW-480 and
slight up-regulation in DLD-1 by SP600125 were found. 2.6. Promotion of SM under Low Oxygen Levels Was Partially Reversed by JNK Inhibition
inducing Akt pathway, JNK inhibition could abruptly inhibit low oxygen level activated A
statistically significant suppression found among HT-29 and SW-480 (Figure 5G) While for Nanog, general inhibition in 1%
and 10% O2 was discovered among all three studied cell lines with greater impact on DLD-1 and SW-
480 (C). The data (means ± SEM) were expressed as the relative expression compared with 21% O2
group, N = 9 (HT-29 and DLD-1) or 10 (SW-480) (A,B) The data (means ± SEM) were expressed as the
relative expression compared with DMSO 21% O2 group. N = 9 (HT-29 and DLD-1) or 10 (SW-480) for
DMSO group; N = 4 (HT-29) or 5 (DLD-1 and SW-480) for SP600125 group (C) * p < 0.05, ** p < 0.01. D:
DMSO (0.1%), S: SP600125 (10 µM). 3. Discussion
Figure 6. Oct-4 and Nanog expressions under different oxygen levels. The key stemness maintenance
markers Oct-4 (A) and Nanog (B) changes in lower oxygen levels were evaluated by Western blotting. Results showed that both stemness maintenance markers were generally up-regulated under lower
oxygen levels with significant up-regulation of Oct-4 in HT-29 (1% and 10% O2) and DLD-1 (10% O2)
and Nanog in SW-480 (10% O2). For the impact of JNK inhibitor SP600125 (10 µM) on Oct-4 (C), diverse
impacts with general down-regulation in lower oxygen levels among HT-29 and SW-480 and slight
up-regulation in DLD-1 by SP600125 were found. While for Nanog, general inhibition in 1% and 10%
O2 was discovered among all three studied cell lines with greater impact on DLD-1 and SW-480 (C). The data (means ± SEM) were expressed as the relative expression compared with 21% O2 group,
N = 9 (HT-29 and DLD-1) or 10 (SW-480) (A,B) The data (means ± SEM) were expressed as the relative
expression compared with DMSO 21% O2 group. N = 9 (HT-29 and DLD-1) or 10 (SW-480) for DMSO
group; N = 4 (HT-29) or 5 (DLD-1 and SW-480) for SP600125 group (C) * p < 0.05, ** p < 0.01. D: DMSO
(0.1%), S: SP600125 (10 µM). Figure 6. Oct-4 and Nanog expressions under different oxygen levels. The key stemness maintenance
markers Oct-4 (A) and Nanog (B) changes in lower oxygen levels were evaluated by Western blotting. Results showed that both stemness maintenance markers were generally up-regulated under lower
oxygen levels with significant up-regulation of Oct-4 in HT-29 (1% and 10% O2) and DLD-1 (10% O2)
and Nanog in SW-480 (10% O2). EMT h
3. Discussion the relatively stationary epithelial cell type to invasive mesenchymal cell type [2]. From the results of
48 h of incubation, lower oxygen levels (1% and 10% O2) induced morphological changes in CRC
cells, and the cells became less organized as the cell–cell separation increased, especially for 1% O2. This demonstrated the loss of cell–cell contact, which is an initial step for EMT. In the presence of
JNK inhibitor, dramatic changes of cell morphology were observed, and JNK inhibition could reverse
the situation by inducing pro-epithelial cell morphology changes. Cancer cells undergoing EMT may have increased invasiveness and migratory power. The
wound healing assay evaluated the impact of lower oxygen levels and JNK inhibition on the
i
t
t
f CRC
ll
B
f th
i
t
t
f HT 29
l
DLD 1
d SW 480
EMT has been regarded as an important event for metastasis as it transforms cancer cells from
the relatively stationary epithelial cell type to invasive mesenchymal cell type [2]. From the results
of 48 h of incubation, lower oxygen levels (1% and 10% O2) induced morphological changes in CRC
cells, and the cells became less organized as the cell–cell separation increased, especially for 1% O2. This demonstrated the loss of cell–cell contact, which is an initial step for EMT. In the presence of JNK
inhibitor, dramatic changes of cell morphology were observed, and JNK inhibition could reverse the
situation by inducing pro-epithelial cell morphology changes. migratory rate of CRC cells. Because of the poor migratory rate of HT-29, only DLD-1 and SW-480
were analyzed. We did not detect a significant difference of relative migratory rate in different oxygen
levels, probably due to the time limitation of this simple wound healing assay, which could only
provide relatively accurate data within 24 h as cell division may affect the assay reliability, and EMT
is a relatively slow process. Moreover, the high confluence of cell culture required in a wound healing
Cancer cells undergoing EMT may have increased invasiveness and migratory power. The wound
healing assay evaluated the impact of lower oxygen levels and JNK inhibition on the migratory rate
of CRC cells. Because of the poor migratory rate of HT-29, only DLD-1 and SW-480 were analyzed. 2.6. Promotion of SM under Low Oxygen Levels Was Partially Reversed by JNK Inhibition
inducing Akt pathway, JNK inhibition could abruptly inhibit low oxygen level activated A
statistically significant suppression found among HT-29 and SW-480 (Figure 5G) For the impact of JNK inhibitor SP600125 (10 µM) on Oct-4 (C),
diverse impacts with general down-regulation in lower oxygen levels among HT-29 and SW-480 and
slight up-regulation in DLD-1 by SP600125 were found. While for Nanog, general inhibition in 1%
and 10% O2 was discovered among all three studied cell lines with greater impact on DLD-1 and SW-
480 (C). The data (means ± SEM) were expressed as the relative expression compared with 21% O2
group, N = 9 (HT-29 and DLD-1) or 10 (SW-480) (A,B) The data (means ± SEM) were expressed as the
relative expression compared with DMSO 21% O2 group. N = 9 (HT-29 and DLD-1) or 10 (SW-480) for
DMSO group; N = 4 (HT-29) or 5 (DLD-1 and SW-480) for SP600125 group (C) * p < 0.05, ** p < 0.01. D:
DMSO (0.1%), S: SP600125 (10 µM). 3. Discussion
Figure 6. Oct-4 and Nanog expressions under different oxygen levels. The key stemness maintenance
markers Oct-4 (A) and Nanog (B) changes in lower oxygen levels were evaluated by Western blotting. Results showed that both stemness maintenance markers were generally up-regulated under lower
oxygen levels with significant up-regulation of Oct-4 in HT-29 (1% and 10% O2) and DLD-1 (10% O2)
and Nanog in SW-480 (10% O2). For the impact of JNK inhibitor SP600125 (10 µM) on Oct-4 (C), diverse
impacts with general down-regulation in lower oxygen levels among HT-29 and SW-480 and slight
up-regulation in DLD-1 by SP600125 were found. While for Nanog, general inhibition in 1% and 10%
O2 was discovered among all three studied cell lines with greater impact on DLD-1 and SW-480 (C). The data (means ± SEM) were expressed as the relative expression compared with 21% O2 group,
N = 9 (HT-29 and DLD-1) or 10 (SW-480) (A,B) The data (means ± SEM) were expressed as the relative
expression compared with DMSO 21% O2 group. N = 9 (HT-29 and DLD-1) or 10 (SW-480) for DMSO
group; N = 4 (HT-29) or 5 (DLD-1 and SW-480) for SP600125 group (C) * p < 0.05, ** p < 0.01. D: DMSO
(0.1%), S: SP600125 (10 µM). EMT h
3. Discussion We did not detect a significant difference of relative migratory rate in different oxygen levels, probably
due to the time limitation of this simple wound healing assay, which could only provide relatively
accurate data within 24 h as cell division may affect the assay reliability, and EMT is a relatively
slow process. Moreover, the high confluence of cell culture required in a wound healing assay 10 of 16 Cancers 2020, 12, 224 could reduce the effectiveness of EMT induction [17]. Nevertheless, the presence of JNK inhibitor
reduced the migratory rate in different oxygen levels, further supporting the notion that JNK promotes
CRC migration. Apart from the macroscopic changes in cell morphology and migration, we also
demonstrated EMT induction based on the cytoskeleton changes due to dynamic actin reorganization. Actin polymerization could occur by increasing F-actin formation to promote the formation of various
migratory structures including lamellipodia, filopodia, and stress fibers and the inhibition of migratory
structure formation by JNK inhibitor. y
Epithelial marker E-cadherin and mesenchymal markers fibronectin and vimentin are important
EMT markers. The loss of E-cadherin was confirmed in all three CRC cell lines in 1% and 10% O2. Although hypoxia (1% O2) has been described in previous research as a factor of EMT induction,
the significant loss of E-cadherin in 10% O2 found in this study is a novel finding. E-cadherin expression
was enhanced in the presence of JNK inhibitor with greater enhancement in 10% O2 and in HT-29. This suggests the general relieve of EMT induction by JNK inhibition. Moreover, the ineffective relieve
in 1% O2 suggests the activation of other EMT-inducing pathways, such as the HIF pathway, while the
predominant EMT-inducing pathway in 10% O2 is JNK-mediated. In line with the observation of EMT-related morphological changes, our results showed that
fibronectin was generally up-regulated under lower oxygen levels, especially in HT-29 and SW-480. While, for another mesenchymal marker, vimentin, its expression level was observable only in SW-480. This suggests that SW-480 had the highest mesenchymal status among the three cell lines studied. Results found that vimentin was generally up-regulated in 10% O2 but not for 1% O2. Vimentin is
usually promoted by HIF-1α-HDAC3 in hypoxia [6], but this promotion was not seen in 1% O2 samples
in this study. EMT h
3. Discussion It may be due to the short-lived nature of HIF-1α, while the promotion trend in 10%
O2 may be due to another pathway that was activated at a low oxygen level. In the presence of JNK
inhibitor, vimentin was significantly suppressed. The trends of the results in mesenchymal markers
coincided with E-cadherin results, as HT-29 achieved better relieve of EMT by JNK inhibitor. Moreover,
the results suggested JNK mediation of vimentin. Various EMT transcription factors have been proposed among different cancer types, including
Snail, Slug, TWIST1/2, and zinc finger E-box-binding homeobox 1/2 (ZEB1/2), as they may affect
transcription of E-cadherin and other EMT markers [6]. Among the three cell lines studied, only Snail
and TWIST1 could be detected in DLD-1 and SW-480 by Western blotting. This indicates the higher
mesenchymal statuses of DLD-1 and SW-480 than that of HT-29. In HT-29, the low expression of
Snail may be due to low expression of HDAC3 [18]. Snail binds to the promoter of CDH1 to repress
E-cadherin transcription and therefore promote EMT [6]. The highest expression level of Snail in
SW-480 among the three cell lines probably is due to the highest expression of HDAC3 [18,19], in which
HDAC3 could promote Snail by HIF-1α-HDAC3-Snail pathway. From the results of this study, SW-480
had significant up-regulation of Snail in both 1% and 10% O2, while DLD-1 only had substantial
activation in 10% O2 but not for 1% O2. The difference of activation in 1% O2 mainly is due to the
difference of activation between SW-480 and DLD-1 by HIF-1α-HDAC3-Snail pathway. Although
HIF-1α-HDAC3-Snail may account for the activation of Snail in 1% O2, HIF-mediated EMT induction
could not account for Snail up-regulation in 10% O2 and there should be another pathway leading to
the up-regulation of Snail [20]. TWIST1 belongs to the basic helix-loop-helix (bHLH) transcription family, which is involved in cancer
metastasis and flanks the CDH1 gene to repress E-cadherin [6]. TWIST1 is found to be a direct target of
HIF-1α, rather than as an indirect target like Snail [6]. Our results demonstrated significant up-regulation of
TWIST1 in both the HDAC3-deficient DLD-1 and HDAC3-activated SW-480. Similar to Snail, TWIST1 was
also significantly up-regulated in 10% O2 for both DLD-1 and SW-480, demonstrating activation by another
EMT-inducing pathway other than HIF-1α under low oxygen level conditions, as HIF-1α does not activate
in 10% O2 [20]. EMT h
3. Discussion Both Snail and TWIST1 confirmed EMT induction in lower oxygen levels. Moreover, both
Snail and TWIST1 were inhibited by JNK inhibition in both DLD-1 and SW-480. Cancers 2020, 12, 224 11 of 16 In this study, we have evaluated JNK pathway related proteins including JNK, its indirect
down-stream regulator p62, and its possible up-stream regulator Akt. The JNK pathway is a possible
delayed pathway that is activated in 10% O2 in light of our previous study [14]. Also, there was research
showing that JNK signaling may contribute to Snail and TWIST1 activation by DNA methyltransferase 1
(DNMT1) or TGF-β1-induced EMT pathway by promoting fibronectin and vimentin [21–27]. Our results
demonstrated the general activation through hyperphosphorylation under low oxygen levels among
all three cell lines, and this confirmed the activation of JNK pathway among CRCs. p62 is an autophagy adaptor protein that may bind to various EMT regulators including TWIST1,
mothers against decapentaplegic-4 (SMAD4), and vimentin [28–30]. Our results showed general
down-regulation of p62 in 1% O2 while opposite results were found in 10% O2. Down-regulation
of p62 in 1% O2 may be due to the promotion of autophagy under severe oxidative stress for cell
survival [31], and this correlates with the lower expression of vimentin in SW-480 under 1% O2 versus
10% O2. While the up-regulation of p62 in 10% O2 corresponds to the activation of JNK-c-Jun-p62 and
could contribute to the up-regulation of TWIST1 and vimentin in 10% O2. Consistent with the past study on the effect of SP600125 on a variety of kinases and enzymes [32],
SP600125 did not inhibit the phosphorylation of JNK but rather by inhibiting its downstream targets
such as c-Jun. Results of p-c-Jun expression in the three cell lines demonstrated the effective inhibition
by SP600125, especially at low oxygen levels. The results of p-c-Jun and EMT transcription factors
suggest the possible JNK-c-Jun-Snail/TWIST1 pathway for EMT induction under low oxygen levels. Moreover, fibronectin and vimentin down-regulation by SP600125 found in HT-29 suggests possible
activation of JNK-c-Jun-Fibronectin/vimentin pathway under low oxygen levels. While for the indirect
JNK target p62, SP600125 could achieve significant inhibitions for DLD-1 and SW-480 in different oxygen
levels. However, SP600125 could up-regulate p62 expression in HT-29 instead. The down-regulations
of p62 and Snail/TWIST1 in DLD-1 and SW-480 suggest another possible JNK-mediated EMT-inducing
pathway by JNK-c-Jun-p62-Snail/TWIST1 under low oxygen levels. EMT h
3. Discussion Akt, which is usually seen as an activator of EMT and an inhibitor of autophagy through
phosphoinositide 3-kinase (PI3K)-Akt-Snail/Slug and PI3K-Akt-mechanistic target of rapamycin
(mTOR) pathways, respectively, has also been regarded as a possible up-stream regulator of the JNK
pathway in gastric cancer cells [33,34]. Our results showed that SW-480 had the most prominent
up-regulation of Akt phosphorylation under lower oxygen levels, while HT-29 was less activated. This could be due to the difference in phosphatase and tensin homolog (PTEN) status among the three
cell lines. PTEN is a tumor suppressor and can inhibit Akt activation [35]. As PTEN has the highest
expression in HT-29 [36], this causes the weakened Akt activation in HT-29 as shown in the result
of this study. SP600125 surprisingly caused hypophosphorylation of Akt, especially for HT-29 and
SW-480, in which Akt was previously considered as a possible upstream regulator of JNK in previous
research [33]. This suggests a novel feedback mechanism of JNK-c-Jun-Akt for further promotion of
EMT as a positive feedback mechanism and inhibition of autophagy through Akt-mTOR pathway
as negative feedback mechanism by preventing simultaneous stimulation of autophagy through
Akt-mTOR and JNK pathways. There are numerous cancer stemness markers proposed in previous studies including pluripotency
markers Oct-4, Nanog, and SOX2, which are considered as poor prognosis indicators of CRC [37,38]. Also, JNK may influence these markers through Wnt/BMP-TCF/Notch or IL-6-JAK-Stat3 pathways [15]. Oct-4 is the primary transcription factor required for stemness properties in CSCs. Overexpression of
Oct-4, together or separately with other pluripotency markers such as Nanog, leads to tumor metastasis
and recurrence in different cancer types [39]. From the results, both Oct-4 and Nanog were generally
stimulated under lower oxygen levels, with HT-29 and DLD-1 having more prominent up-regulation. This verified both 1% and 10% O2 as possible stimulants of SM of CRC cells. In the presence of JNK
inhibitor, the low oxygen level driven general up-regulations of Oct-4 and Nanog among the three cells
lines were partially inhibited, except for Oct-4 in DLD-1. This suggests the importance of JNK in SM
under low-oxygen environments, which could sequentially affect CSCs and the related tumorigenesis Cancers 2020, 12, 224 12 of 16 12 of 16 processes such as metastasis and radiosensitivity. The possible pathway could be Wnt/BMP-TCF/Notch
or IL-6-JAK-Stat3 pathways as suggested in previous studies [15], which could be further confirmed in
future studies. EMT h
3. Discussion Combining the results of this study, we showed the hypoxia-driven EMT induction/SM and
proposed 10% O2 as another possible condition for EMT induction and SM among CRC cells. The blood
oxygen level is about 10% in general. This study suggests that the regulation of EMT/SM and its related
tumor progression events such as metastasis could be triggered by the oxygen level in blood. Moreover,
the partial relieve of EMT induction and SM promotion by JNK inhibitor was demonstrated, and this
suggests the role of JNK pathway mediation of EMT under low oxygen levels. Additionally, as the
majority of CRC cells usually have null SMAD4 expressions in microsatellite instability (MSI)-negative
CRC cell lines, including HT-29 and SW-480, but not for MSI-positive cell lines such as DLD-1, and
this depends on SMAD-independent pathways such as JNK pathway [40,41], the JNK pathway has
unique importance in EMT induction of CRC. Furthermore, this study proposes a novel pathway on
the regulation of Akt by JNK-c-Jun, which could further control EMT and autophagy in CRC cells
under low oxygen conditions. Inhibition of JNK is a potentially rewarding strategy for inhibiting
EMT progression and SM in CRC cells. Further investigations with in vivo and clinical studies are
recommended to establish JNK inhibition as a possible strategy to limit metastasis in CRC. 4.3. Cell Morphological Analysis HT-29, DLD-1, or SW-480 cells (0.5 M) were seeded in T25 flasks. After overnight settling and 24 h
serum starvation, cells were incubated in different O2 levels for 48 h with or without JNK inhibitor
SP600125 (10 µM). The effect of oxygen level in EMT induction was evaluated by photomicrographs
taken by light microscopy with 200× magnification. The cell morphology was visually compared and
analyzed in 5 independent experiments. 4.1. Cell Lines and Culture Conditions Human colorectal adenocarcinoma cell line HT-29 was purchased from PerkinElmer, Inc. (Waltham,
MA, USA). Human colorectal adenocarcinoma cell lines DLD-1 and SW-480 were obtained from
Professor Jun Yu, Department of Medicine and Therapeutics, Faculty of Medicine, The Chinese
University of Hong Kong (Hong Kong). All cells were cultured in DMEM medium with GlutaMAX
supplement and HEPES and supplemented with 10% fetal bovine serum (FBS). 4.2. Hypoxic Condition and JNK Inhibition The hypoxic and blood oxygen condition was maintained by the SCI-tive Dual chamber hypoxia
workstation (Baker Ruskinn, Sanford, ME, USA) at 1% and 10% O2 with 5% CO2, 37 ◦C, and 100%
humidity respectively. Cells cultured in normal incubator at an atmospheric oxygen level of 21% were
used as the control. For JNK inhibition, cells were treated for 48 h with 0.1% DMSO (control) or 10 µM of JNK inhibitor
SP600125 (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 0.1% DMSO as titrated in a preliminary
experiment [32]. 4.6. Western Blotting The cell density, incubation condition, and JNK inhibition were the same as that of the cell
morphological analysis. Protein extraction and gel electrophoresis were performed as described in our
previous paper [14]. The primary antibodies used are listed in Supplementary Table S1. The relative
protein expression of each sample was evaluated by ImageJ software with β-Actin as loading controls. At least 4 independent sets were performed for each set of protein expression analyses. (All original
western blot figures can be found in Supplementary Figures S2–S4) 4.5. Immunofluorescence Staining Fifty thousand HT-29, DLD-1, or SW-480 cells were seeded in 24-well plates with coverslips inserted. The serum starvation and incubation under different oxygen levels with or without JNK inhibitor
SP600125 was the same as that of the cell morphological analysis. After incubation, cells were first gently
washed by phosphate-buffered saline (PBS) for three times, then fixed with 4% paraformaldehyde (PFA)
in PBS for 30 min. After washing by PBS for another three times, the cells were blocked with 2% bovine
serum albumin (BSA) in PBS for 30 min. Alexa Fluor 594 Phalloidin (Thermo Fisher Scientific, Waltham,
MA, USA) was added to the cells for staining F-actin in 1 h. Cells were mounted with ProLong Gold
Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) after PBS
washing. Cells and their cytoskeletons were visualized by confocal microscopy (Leica TCS SPE, Leica
Microsystems, Wetzlar, Germany). The effect of oxygen level on cytoskeleton was quantified in terms
of lamellipodia, filopodia, and ventral stress fibers. At least 200 cells were scored in each of the three
independent experiments, and the percentages of cells having the three cytoskeleton structures of
different conditions were plotted and compared. 4.4. Cell Migration Assay Wound healing assay was employed to assess cell migration properties. Thirty-five thousand HT-29,
DLD-1, or SW-480 cells were seeded in each compartment of a Culture-Insert 2 Well in µ-Dish 35 mm
(Ibidi LLC, Munich, Germany). After overnight settling and 24 h serum starvation, the wound gap was
made by removing the silicone insert. After that, fresh culture medium was added with or without JNK
inhibitor SP600125 (10 µM), and cells were allowed to migrate under different oxygen levels for 24 h. Photomicrographs were taken before and after incubation. The gap area was measured using the MRI Cancers 2020, 12, 224 13 of 16 13 of 16 Wound Healing Tool macro for ImageJ software (NIH) (http://dev.mri.cnrs.fr/projects/imagejmacros/wiki/
Wound_Healing_Tool). The invasion rate was calculated as the relative gap area difference between
0 and 24 h against the control among at least 3 independent experiments. 4.7. Data Analysis Different statistical tests have been used in analyzing the data. In general, values were plotted
as mean ± SEM. Comparisons of means between independent groups were conducted by Student’s
t test (2 groups) or Kruskal–Wallis one-way ANOVA (3 or more groups) with pairwise comparison. Comparison of mean between paired groups was conducted by paired t tests. Statistical analysis was
conducted by Statistical Product and Service Solutions (SPSS) version 22 (IBM Corp, Armonk, NY,
USA), and significance level of p < 0.05 was considered as statically significant. Author Contributions: Conceptualization, S.Y.T. and H.K.W.L.; Data curation, S.Y.T.; Formal analysis, S.Y.T.;
Investigation, S.Y.T.; Methodology, S.Y.T. and H.K.W.L.; Resources, H.K.W.L.; Supervision, V.W.C.W. and H.K.W.L.;
Validation, S.Y.T. and H.K.W.L.; Visualization, S.Y.T. and H.K.W.L.; Writing—original draft, S.Y.T.; Writing—review
& editing, V.W.C.W. and H.K.W.L. All authors have read and agreed to the published version of the manuscript. 5. Conclusions It is clear that JNK, JNK pathway proteins, and transcription factors play important roles in EMT
induction and SM of CRC cells under low-oxygen levels through multiple factors. Moreover, this study
proposes a novel pathway of JNK-Akt serving as further control of EMT and other tumor progression
processes such as autophagy. Thus, we suggest inhibition of JNK as a promising way for inhibiting
EMT progression and SM in CRC cells. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/1/224/s1,
Figure S1: Preliminary experiment of HT-29 wound healing assay; Figure S2: Original Western blots of Figure 4;
Figure S3: Original Western blots of Figure 5; Figure S4: Original Western blots of Figure 6. Table S1: List of
primary antibodies used in Western blotting. Author Contributions: Conceptualization, S.Y.T. and H.K.W.L.; Data curation, S.Y.T.; Formal analysis, S.Y.T.;
Investigation, S.Y.T.; Methodology, S.Y.T. and H.K.W.L.; Resources, H.K.W.L.; Supervision, V.W.C.W. and H.K.W.L.;
Validation, S.Y.T. and H.K.W.L.; Visualization, S.Y.T. and H.K.W.L.; Writing—original draft, S.Y.T.; Writing—review
& editing, V.W.C.W. and H.K.W.L. All authors have read and agreed to the published version of the manuscript. 14 of 16 14 of 16 Cancers 2020, 12, 224 Funding: This research was supported by a Postgraduate Studentship to S.Y.T. and the Departmental Seeding
Fund for H.K.W.L. from The Hong Kong Polytechnic University. Acknowledgments: We would like to acknowledge Jun Yu, Department of Medicine and Therapeutics, Faculty of
Medicine, The Chinese University of Hong Kong, for the provision of DLD-1 and SW-480 cell lines. We would also
like to acknowledge the Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic
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Factors that influence health literacy in patients with coronary artery disease
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* Paper extracted from master’s thesis “Self-care, health
literacy and knowledge about the disease in patients with
coronary artery disease”, presented to Universidade de
São Paulo, Escola de Enfermagem, São Paulo, SP, Brazil.
This study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) -
Finance Code 001, Brazil.
1 Faculdade Wenceslau Braz, Departamento de Enfermagem,
Itajubá, MG, Brazil.
2 Universidade de São Paulo, Escola de Enfermagem, São
Paulo, SP, Brazil.
3 Instituto Dante Pazzanese de Cardiologia, Departamento
de Enfermagem, São Paulo, SP, Brazil.
4 Florida Atlantic University, Christine E. Lynn College of
Nursing, Boca Raton, Florida, United States of America. * Paper extracted from master’s thesis “Self-care, health
literacy and knowledge about the disease in patients with
coronary artery disease”, presented to Universidade de
São Paulo, Escola de Enfermagem, São Paulo, SP, Brazil.
This study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) -
Finance Code 001, Brazil. Rev. Latino-Am. Enfermagem
2023;31:e3879
DOI: 10.1590/1518-8345.6211.3879
www.eerp.usp.br/rlae Original Article Factors that influence health literacy in patients with
coronary artery disease* Ana Caroline da Costa1,2
https://orcid.org/0000-0002-6936-6137
Ana Paula da Conceição2,3
https://orcid.org/0000-0002-1598-807X
Howard Karl Butcher4
https://orcid.org/0000-0002-8394-516X
Rita de Cassia Gengo e Silva Butcher2,4
https://orcid.org/0000-0002-7307-2203 Highlights: (1) Specific knowledge about the disease does
not exert any influence on HL in patients with CAD. (2) HL
is positively influenced by higher schooling levels and better
work status. (3) HL is negatively influenced by age and
arterial hypertension. (4) The factors that exert an influence
on HL should be included in educational interventions. Highlights: (1) Specific knowledge about the disease does
not exert any influence on HL in patients with CAD. (2) HL
is positively influenced by higher schooling levels and better
work status. (3) HL is negatively influenced by age and
arterial hypertension. (4) The factors that exert an influence
on HL should be included in educational interventions. Objective: to investigate the factors that exert an influence on health
literacy in patients with coronary artery disease. Method: a cross-
sectional study, including 122 patients with coronary diseases (60.7%
male; 62.07 ± 8.8 years old). Health literacy and specific knowledge
about the disease were evaluated through interviews with the
participants by means of the Short Test of Functional Health Literacy in
Adults and the Short version of the coronary artery disease education
questionnaire. The data were described by means of central tendency
measures and frequencies. The factors that exert an influence on
health literacy were determined by means of a linear regression model. The significance level adopted was 5%. The study was approved by the
Research Ethics Committee. Results: age and arterial hypertension
presented an inverse and significant relationship with health literacy. On the other hand, higher schooling levels and having a job were
associated with better scores in the health literacy instrument. Specific
knowledge about the disease did not exert any influence on health
literacy. The variables included in the regression model explained
55.3% of inadequate literacy. Conclusion: in this study, knowledge
about the disease exerts no influence on health literacy: however,
the professionals should consider the sociodemographic and clinical
factors to plan the interventions. * Paper extracted from master’s thesis “Self-care, health
literacy and knowledge about the disease in patients with
coronary artery disease”, presented to Universidade de
São Paulo, Escola de Enfermagem, São Paulo, SP, Brazil. This study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) -
Finance Code 001, Brazil. Introduction that it has been associated with unfavorable outcomes
for health, such as less knowledge about the disease
and non-adherence to the treatment(9-10). A systematic
review that synthesized the literature in relation to HL in
people with CAD showed knowledge about the disease,
age, schooling, socioeconomic disadvantaged status,
non-white ethnicity and comorbidities are associated
with low HL(9). Complexity of the varied information and
treatment options is acknowledged as a barrier for
a successful implementation of health interventions. Using unnecessarily complicated language or inaccurate
translations in educational materials, or other education
sources for patients generally leads to poorly informed
decision-making and decreased participation of the
patients in interventions with potential benefits for health. Such complexity exerts a negative impact on health
results, especially among patients with low health literacy
(HL)(1-2) levels. According to the conceptual model on HL, widely
discussed in the literature, text comprehension,
numbering, vocabulary and prior knowledge are necessary
resources to effectively deal with health information(4). However, the influence of specific knowledge about CAD
on HL is not fully understood. Although some studies have
shown the relationship between HL and specific knowledge
about the disease in different groups of patients(11,16-17),
other surveys that included patients with chronic and
infectious diseases failed to demonstrate that specific
knowledge about the disease influences HL(18-21). A meta-
analysis that included more than 18,000 participants with
diabetes showed that higher HL levels were associated
with higher levels of knowledge about the disease
(r=0.308, p<0.001) and with better clinical outcomes,
such as lower levels of glycated hemoglobin (HbA1C) (r
=-0.048, p = 0.027)(18). Health literacy is defined as a person’s ability to
acquire, process and understand all the health-related
information required to make suitable decisions and
achieve positive health results. It involves acquiring
specific skills to perform activities of daily living related to
health and tasks, as well as making decisions to improve
health outcomes(3-4). The prevalence of low HL is relatively high at
the global level, particularly among people with low
socioeconomic status(5). A number of research studies
have evidenced that low HL is an independent health
determinant and that it is associated with worse
health results, such as increased hospitalizations, use
of emergency services, low adherence to medication
regimes and higher mortality rates. Factors that influence health literacy in patients with
coronary artery disease* 1 Faculdade Wenceslau Braz, Departamento de Enfermagem,
Itajubá, MG, Brazil. 2 Universidade de São Paulo, Escola de Enfermagem, São
Paulo, SP, Brazil. 3 Instituto Dante Pazzanese de Cardiologia, Departamento
de Enfermagem, São Paulo, SP, Brazil. 4 Florida Atlantic University, Christine E. Lynn College of
Nursing, Boca Raton, Florida, United States of America. Descriptors: Health Literacy; Coronary Artery Disease; Nursing;
Health Education; Nursing Care; Socioeconomic Factors. How to cite this article
Costa AC, Conceição AP, Butcher HK, Butcher RCGS. Factors that influence health
literacy in patients with coronary artery disease. Rev. Latino-Am. Enfermagem. 2023;31:e3879. [Access
day
month
year
]; Available in:
URL
. https://doi.org/10.1590/1518-8345.6211.3879 How to cite this article
Costa AC, Conceição AP, Butcher HK, Butcher RCGS. Factors that influence health
literacy in patients with coronary artery disease. Rev. Latino-Am. Enfermagem. 2023;31:e3879. [Access
day
month
year
]; Available in:
URL
. https://doi.org/10.1590/1518-8345.6211.3879 2 Rev. Latino-Am. Enfermagem 2023;31:e3879. Type of study This is an observational and cross-sectional study
with a quantitative approach that is part of a main project
aimed at analyzing the association between self-care and
HL in patients with CAD. Inadequate HL is acknowledged as a barrier for health
maintenance and prevention of coronary artery disease
(CAD) and is associated with non-adoption of self-care
behaviors for management of the disease(9-11). In Brazil,
a developing country with profound social inequalities and
weaknesses in its health and education systems, studies
on HL are still scarce, specifically in patients with CAD. To the present date, no national studies analyzing HL in
this group of patients have been found, however, data
from other groups of patients show that the HL level in
the country is low or limited(12-15). This study followed The Strengthening the Reporting
of Observational Studies in Epidemiology (STROBE)
guideline(23). Introduction In addition, patients
with low HL have more difficulty understanding health
information, less knowledge about their disease and
less support to discuss health problems, in addition
to feeling less comfortable communicating with health
professionals and discouraged or even embarrassed, to
ask questions in order to clarify the information they
received(5-6). On the other hand, the literature shows
higher HL levels are associated with better health self-
management indicators such as adherence to healthy
lifestyles and lower rates of obesity, smoking and
hospital readmissions(7-8). Genuine patient-centered care, especially in patients
with CAD, will only be attained if high HL levels are taken
into account(22). Knowing the factors associated with HL
will increase success: in the implementation of health
interventions in CAD management, directing and adapting
the communication between health professionals and
patients and planning of educational interventions. Thus,
the current study investigated the factors that exert an
influence on health literacy in patients with CAD. www.eerp.usp.br/rlae Population and sample The population consisted of patients with CAD
registered in the tertiary-level Coronary diseases
outpatient service, a reference in Cardiology from the
city of São Paulo, Brazil. For the main study that aimed at “Analyzing the
association between self-care ability, health literacy and
knowledge about the disease in patients with CAD”, the
minimum sample size was 84 participants, calculated by
means of the G Power statistical program, version 3.1(24),
based on an infinite population, moderate correlation
(r = 0.30), 80% test power and 5% type I error. The
correlation value adopted for sample size calculation
allows analyzing the existence of a correlation between the
variables of interest, without excessively increasing the
sample size to the point of rendering the study unfeasible. In turn, the numbering assessment consists in
presenting four cards for the participant to interpret the
information. The first card is a medication label. The
second refers to how to interpret a glycaemia value. Card
three deals with the date of next appointment, considering
the one printed in the card. Finally, in card four, the
participant has to calculate the time to take a medication. Two points are assigned to each correct answer, so that
the numbering component has a total score of 28 points. The individuals included in the study were those aged
at least 18 years old, with a medical diagnosis of CAD
documented in their medical charts and who stated being
able to read. The Mini-Mental State Examination (MMSE)
was used as pre-screening tool to determine presence of
cognitive impairment, when the score obtained by the
participants was below 20. This pre-screening is necessary
because impairment in cognitive functions might result
in poor performance in the other assessments due to
difficulties understanding the tasks. This procedure is well-
adopted in the literature(15). The participants excluded from
the study were those with deficits in visual, auditory and/
or verbal communication skills that precluded application
of the data collection instruments. It is recommended that the reading comprehension
and numbering components should be finished in seven
and five minutes, respectively, therefore, application of
the instrument should be timed. The total S-TOFHLA score varies from 0 to 100 and
comprises the sum of the reading comprehension (from
0 to 72) and numbering (from 0 to 28) scores. Period The data collection period was from August to
October 2019. The HL level was assessed by means of the Short
Test of Functional Health Literacy in Adults (S-TOFHLA),
validated for use in Brazil(12). Choice of this instrument
was due to the fact that it assesses both reading
comprehension and numbering. S-TOFHLA contains
36 reading comprehension items organized into two
passages: A and B. Passage A includes information related
to a gastrointestinal examination and Passage B refers to
a term of rights and responsibilities of a patient admitted
to a hospital. Each passage has a space (each space
corresponds to an item) and, below each space, there are
five or six word options, from which the participant has
to select one to complete the sentence and make sense
of the it. Two points are assigned to each word that is
properly selected, so that the total score is 72 points. Population and sample Based on
the score obtained, the participants’ HL level is classified
as inadequate (from 0 to 53), marginal or borderline (from
54 to 66) or adequate (from 67 to 100). The original
instrument’s internal consistency was 0.68 for the four
numbering items and 0.97 for the 36 items from both
passages of the reading comprehension test(12,25). Data collection setting Data collection was performed in the outpatient
service for coronary diseases of a tertiary-level hospital
in the city of São Paulo, Brazil. Choice of hospital was due
to the fact that it is a reference in care and research in the
Cardiology area in Brazil, in addition to being renowned Specifically in patients with CAD, the literature
evidences that the prevalence of inadequate HL is
considered high, with a frequency of up to 74.5% and Costa AC, Conceição AP, Butcher HK, Butcher RCGS. 3 Costa AC, Conceição AP, Butcher HK, Butcher RCGS. Costa AC, Conceição AP, Butcher HK, Butcher RCGS. www.eerp.usp.br/rlae Instruments for data collection worldwide for its excellence. In 2018, the hospital had
376 beds and served nearly 178,927 outpatients, among
which 13,973 were registered in the Coronary diseases
outpatient service. The sociodemographic and clinical data were collected
from the medical charts or through the participants’ self-
reports. The data of interest for this study were sex, age,
race, schooling, marital status, professional status, per
capita income of the patients, number of medications,
smoking habit, comorbidities (hypertension, diabetes and
dyslipidemia) and previously-received health education. Data treatment and analysis The data were analyzed with the aid of the R software,
version 3.5.380. Absolute and relative frequencies were
calculated for the categorical variables; and mean,
standard deviation, median and maximum and minimum
values were calculated for the quantitative ones. The
difference in the participants’ distribution in relation to the
HL categories was determined by means of the chi-square
test. A linear regression model was used to determine
the influence of the patients’ previous knowledge and
sociodemographic characteristics on HL. The researchers
selected the independent variables based on the theoretical
framework after a comprehensive reading of studies about
the factors that influence HL levels. Brazil, 2019
Variables
Mean age (SD*)
62.1 ± 8.8
Schooling, mean (SD*)
7.8 ± 4.2
Male sex, n (%)
74 (60.7%)
Skin color, white, n (%)
71 (58.2%)
Marital status, married, n (%)
90 (73.8%)
Work status, active, n (%)
32 (26.2%)
Per capita income > 1 minimum wage†, yes, n (%)
86 (70.5%)
Smoker, yes, n (%)
9 (7.4%)
Hypertension, n (%)
119 (97.5%)
Dyslipidemia, n (%)
114 (93.4%)
Diabetes, n (%)
59 (48.4%)
*SD = Standard deviation; †Minimum wage in force = R$ 998.00, Brazil, 2019 Normality of the regression analysis residuals was
evaluated by means of Q-Q plots. Multicollinearity of the
independent variables was assessed by means of the
variance inflation factor. The significance level adopted
in all the tests was 5%, with 95% confidence intervals. Internal consistency of the instruments used to
measure HL and knowledge about CAD in the sample of
this study was calculated by means of Cronbach’s alpha(27). *SD = Standard deviation; †Minimum wage in force = R$ 998.00, Brazil, 2019 Procedures for data collection The potential study participants, that is, those aged
at least 18 years old and with a medical diagnosis of CAD,
were identified based on the coronary diseases outpatient
service schedule of appointments. Subsequently, the main
researcher herself invited the potential participants to the
study. Those who agreed signed the informed consent
form and answered MMSE. For the participants included
in the study, individual meetings were held to apply the
other data collection instruments, as described below. The patients’ knowledge about CAD was evaluated
through the Short Version of the Coronary Artery Disease
Education Questionnaire (CADE-Q SV), also validated for
use in Brazil(26) and because it is a specific instrument
for assessing knowledge about CAD. The questionnaire
consists of 20 items and is organized into four knowledge
areas: clinical condition, risk factors, exercise, diet and 4 Rev. Latino-Am. Enfermagem 2023;31:e3879. The participants’ mean age was 62.1 ± 8.8 years old
and the mean schooling level reported corresponded
to 7.8 ± 4.2 years. Most of the patients were male,
Caucasian, married, and earned incomes of less than
one minimum wage. Only 26.2% were employed,
63.9% were retired and 9.8% were unemployed. The vast majority (>90%) had comorbidities such as
hypertension and dyslipidemia. psychosocial risk. Each of the four areas has four items
that are randomly arranged. The answer options are as
follows: “True”, “False” and “I don’t know”. Each correct
answer equals one point; therefore, the total score
varies from 0 to 20. The higher the score, the better the
patient’s knowledge about CAD. The Brazilian version of
the instrument presented adequate evidence in terms of
reproducibility and its intraclass correlation coefficient
was greater than 0.70 for all the items(26). Table 1 – Sociodemographic characteristics of participants
(n=122) of the study in the assessments of health literacy
and knowledge about coronary artery disease. São Paulo,
Brazil, 2019 Ethical aspects The mean total S-TOFHLA score was 60.5 ± 23.1,
whereas the mean reading comprehension score was
33.6 ± 21.7 and the numbering score was 26.7 ± 2.3;
41.8% (n = 51) of the patients had inadequate HL,
35.2% (n = 43) had adequate HL and 23.0% (n = 28)
had borderline HL (p = 0.035). Internal consistency
of S-TOFHLA was 0.96 for reading comprehension and
0.12 for numbering. The mean CADE-Q SV score was
12.3 ± 2.5 and its internal consistency was 0.47. The study was approved by the Research Ethics
Committee at the hospital and the Nursing School of the
University of São Paulo, with Certificate of Presentation
for Ethical Appreciation (Certificado de Apresentação para
Apreciação Ética, CAAE) number 12805619.5.3001.5462
and 12805619.5.0000.5392, respectively. All the
participants freely signed an Informed Consent Form. Results Figure 1 shows that there was no statistically
significant correlation between the CADE-Q SV and
S-TOFHLA scores. The sociodemographic data and clinical
characteristics of the sample are detailed in Table 1. www.eerp.usp.br/rlae 5 Costa AC, Conceição AP, Butcher HK, Butcher RCGS. r* = 0.175; p† = 0.054; 95% CI‡ 0.003-0.342
*r = Pearson’s correlation coefficient; †p = Significance level; ‡CI = Confidence Interval; CADE-Q SV = Coronary Artery Disease Education Questionnaire –
Short Version; S-TOFHLA = Short Test of Functional Health Literacy in Adults
Figure 1 - Correlation of the scores obtained by the study participants (n = 122) in the health literacy and knowledge
about coronary artery disease assessments. São Paulo, Brazil, 2019 r* = 0.175; p† = 0.054; 95% CI‡ 0.003-0.342
*r = Pearson’s correlation coefficient; †p = Significance level; ‡CI = Confidence Interval; CADE-Q SV = Coronary Artery Disease Education Questionnaire –
Short Version; S-TOFHLA = Short Test of Functional Health Literacy in Adults
Figure 1 - Correlation of the scores obtained by the study participants (n = 122) in the health literacy and knowledge
about coronary artery disease assessments. São Paulo, Brazil, 2019 *r = Pearson’s correlation coefficient; †p = Significance level; ‡CI = Confidence Interval; CADE-Q SV = Coronary Artery Disease Education Questionnaire –
Short Version; S-TOFHLA = Short Test of Functional Health Literacy in Adults
Figure 1 - Correlation of the scores obtained by the study participants (n = 122) in the health literacy and knowledge
about coronary artery disease assessments. São Paulo, Brazil, 2019 year of schooling increased the S-TOFHLA score by
2.14 points (95% CI = 1.230; 3.045, p < 0.001) and
being employed increased the S-TOFHLA score by 8.56
points with respect to those who were not (95% CI =
0.422; 16.695, p = 0.039). The variables included in
the regression model explain 55.3% of inadequate HL
in the sample under study. Table 2 shows the linear regression model
corresponding to the factors that exert an influence on
HL. Each additional year of age decreased the S-TOFHLA
score by 0.55 points (95% CI = -0.966; -0.125) and
having hypertension decreased such score by 29.9
points in relation to those without hypertension (95%
CI = -54.124; -5.696, p = 0.016). Each additional Table 2 – Factors that exert an influence on health literacy (n = 122), adjusted for confusion variables (arterial
hypertension, diabetes mellitus and dyslipidemia). *CI = Confidence Interval: Min. - Minimum Max.- Maximum; †p = Significance level; ‡Health guidance = It refers to any guideline or information received
about health, examinations and treatments in general and not specifically about coronary artery disease; §CADE-Q SV = Coronary Artery Disease Education
Questionnaire – Short Version Results São Paulo, Brazil, 2019
Variables
Regression coefficient
Standard error
CI* 95% min
CI* 95% max
p value†
Male sex
4,104
3,275
-2,388
10,596
0,213
Age
-0,546
0,212
-0,966
-0,125
0,012
Skin color, non-white
-6,381
3,231
-12,785
0,024
0,051
Schooling
2,137
0,458
1,230
3,045
<0,001
Marital status, not married
5,774
3,844
-1,846
13,393
0,136
Work status (employed)
8,559
4,104
0,422
16,695
0,039
Per capita income
1,943
1,593
-1,215
5,102
0,225
Number of medications
-0,120
1,071
-2,243
2,003
0,911
Health guidance‡
3,788
3,552
-3,253
10,829
0,289
Hypertension
-29,910
12,215
-54,124
-5,696
0,016
Diabetes Mellitus
-5,686
3,707
-13,034
1,663
0,128
Dyslipidemia
8,536
7,727
-6,781
23,854
0,272
CADE-Q SV§
0,698
0,661
-0,612
2,008
0,293
*CI = Confidence Interval: Min. - Minimum Max.- Maximum; †p = Significance level; ‡Health guidance = It refers to any guideline or information received
about health, examinations and treatments in general and not specifically about coronary artery disease; §CADE-Q SV = Coronary Artery Disease Education
Questionnaire – Short Version Table 2 – Factors that exert an influence on health literacy (n = 122), adjusted for confusion variab
hypertension, diabetes mellitus and dyslipidemia). São Paulo, Brazil, 2019 Rev. Latino-Am. Enfermagem 2023;31:e3879. Rev. Latino-Am. Enfermagem 2023;31:e3879. 6 In addition, normal distribution was observed for the residuals of the variables included in the regression model
by means of Q-Q graph, as shown in Figure 2. Figure 2 – Analysis of the normality of the residuals of the variables included in the regression model (n = 122). São
Paulo, Brazil, 2019 Figure 2 – Analysis of the normality of the residuals of the variables included in the regression model (n = 122). São
Paulo, Brazil, 2019 The a posteriori analysis showed that 122 subjects
was an adequate sample size to demonstrate that, at
least one variable (predictor), significantly altered the
dependent variable (HL assessed by means of the Short
Test of Functional Health Literacy in Adults). Assuming
a 5% type I error and 20% type II error, with 80% test
power and considering that the regression model consisted
of 14 predictors with a coefficient of determination (R²)
of 0.553, the required sample would have been 30
participants. Table 3 shows that there was no evidence of
multicollinearity among the variables included in the
regression model. The highest value obtained was 1.78,
which does not indicate presence of multicollinearity. Table 3 – Multicollinearity analysis of the variables included
in the regression model (n = 122). Results São Paulo, Brazil, 2019
Variables
VIF*
Sex
1.17
Age
1.58
Skin color
1.16
Schooling
1.69
Marital status
1.31
Work status
1.49
Per capita income
1.32
Number of medications
1.78
Health guidance‡
1.14
Hypertension
1.64
Diabetes Mellitus
1.57
Dyslipidemia
1.67
CADE-Q SV§
1.23
*VIF = Variance Inflation Factor; ‡Health guidance = It refers to any guideline
or information received on health, examinations and treatments in general
and not specifically on coronary artery disease; §CADE-Q SV = Coronary
Artery Disease Education Questionnaire – Short Version Table 3 – Multicollinearity analysis of the variables included
in the regression model (n = 122). São Paulo, Brazil, 2019 www.eerp.usp.br/rlae Discussion They also consider that the social factors
tend to exert a strong influence not only on development
of the skills required for HL, but also on the way in which
patients use health information(4,28-29). The literature shows that low HL is associated
with poor blood pressure control(30). Although there is
evidence that hypertensive people with adequate HL
have better adherence to the treatment(29,31), a study
showed that the contribution of HL to explain such
adherence was minimal(30). In relation to age, the older adults’ low performance
in tests that evaluate HL can be attributed to decline in
cognition, hearing and vision, as well as to less access
to educational information in the past(31). A study showed
that participants with lower S-TOFHLA scores, classified
as inadequate or marginal, were significantly older, prone
to be dependent on daily activities and to perform worse
in cognitive domains tests, such as MMSE(32). Regarding knowledge about the disease, CADE-Q SV
has been used in few studies, which hinders comparisons. In the sample under study, the mean CADE-Q SV score
was 12.3 ± 2.5 (possible range from 0 to 20). In the
validation study of this instrument in Brazil, the authors
found a mean CADE-Q SV score of 13.1(26). The findings of
another research conducted with Brazilian and Canadian
patients suggested that the subjects had good knowledge
about the disease(39). It is possible that socioeconomic and
cultural differences may influence these findings. There
is diverse evidence in the literature that lower levels of
knowledge related to the disease are associated with lower
schooling levels, lower incomes and advanced age(26). In
fact, among patients with CAD, although knowledge about
the disease seems to be a predictor of decision-making, it
is not sufficient to change health behaviors(40). A number
of authors found even though CAD patients had moderate
knowledge about the disease, less than one-third of the
sample was consistently involved in physical activities
or underwent regular follow-up with health providers(40). In another study that included a sample of 575
patients with heart failure, the authors showed that older
patients were more likely to have low HL and that the
health outcomes (readmissions and mortality) were worse
in those aged over 65 years and with low HL. In addition
to that, the authors were able to show that HL mediates
the relationship between age and health results(33). Discussion This study analyzed the influence of specific
knowledge about the disease, sociodemographic and
clinical factors, including comorbidities (hypertension,
diabetes and dyslipidemia and number of medications
taken daily) on HL in patients with CAD. The mean
S-TOFHLA score was 60.5 ± 23.1, with a statistically
significant predominance of patients with inadequate HL. The frequency of inadequate HL levels in patients with CAD
varies widely in the literature (from 14.3% to 74.5%)(9-10). However, several studies show that inadequate HL is
common in patients with cardiovascular diseases in
general(12-15), particularly in those with CAD(9-10). In this study, the factors that influenced HL were
age, schooling, employment and hypertension. There
was a reduction in HL with increasing age and diagnosis 7 7 Costa AC, Conceição AP, Butcher HK, Butcher RCGS. Costa AC, Conceição AP, Butcher HK, Butcher RCGS. being, in addition to having other positive attitudes and
behaviors(25,34). It is possible that individuals with high
schooling levels feel safer and have more clarity when
communicating their needs to health professionals(34-35). of hypertension, while individuals with some professional
occupation and with more years of study presented higher
HL levels. A systematic review also showed that the HL
level was worse in unemployed people, of advanced age,
with low schooling levels and multiple comorbidities(9). In relation to employment, work status is considered
as the economic inclusion degree that grants or restricts
people’s access to the necessary resources(36). Thus, being
employed can be seen as an indicator of socioeconomic
status. Actually, socioeconomic status does not directly
affect health status. However, it was recognized as an
important determinant of health-related results(37). Those
who are unemployed, retired or on sick leave and who earn
low incomes and are devoid of additional health resources
such as health insurance, tend to have low adherence to
preventive practices and less contact with health-related
information(37). In addition, as they are not part of the
workforce, they may feel disabled and vulnerable. As a
result, they delegate some activities to family members,
such as scheduling appointments and exams or controlling
drug therapy and end up distancing themselves from
the health system, thus limiting communication with
professionals and, in some cases, not making decisions
related to their own health(38). Recently, a study also
showed that HL is a mediator of the relationship between
socioeconomic level and health status(37). Other authors agree that HL is a multidimensional
concept influenced by personal, family, environmental and
social factors. www.eerp.usp.br/rlae Discussion With regard to schooling, the findings of this study
are consistent with the literature, where it is observed
that individuals with lower schooling levels have lower
HL scores, worse reading skills, less autonomy to seek
health information in different sources and difficulty
understanding and judging what is most appropriate for
their own well-being(25,34). A cross-sectional study that
included 252 patients with cardiovascular diseases showed
that the lower the schooling level, the lower the HL level. The authors showed that patients without schooling had
lower scores in all nine dimensions of the HL instrument
used in the study (Health Literacy Questionnaire - HLQ)
(34). In a systematic review, education was considered a
predictive factor for HL in patients with heart failure. The
studies included in that review showed that the patients
who had less than High School were more likely to have
low HL levels(35). Our findings showed, although considered relevant
by professionals, previous knowledge about the disease
was not related to HL. The assumption that knowledge
is necessary for HL, not confirmed by the results of
this study, was based on the HL model adopted in this
research and widely disseminated in the literature, where
knowledge is an individual ability subdomain. According
to this model, familiarity with specific vocabulary and Individuals with Higher Education tend to have better
reading skills, more autonomy to seek health information
in different sources and better ability to understand
and judge what is most appropriate for their own well- 8 Rev. Latino-Am. Enfermagem 2023;31:e3879. with specific issues related to body functioning and the
disease would contribute to HL(4). the sample and, therefore, limited generalization of the
results. The study did not investigate whether the patients
had participated in previous programs that contributed to
specific knowledge about the disease or improved their HL-
related skills. As this is a cross-sectional research, it was
not possible to establish causal relationships. Therefore,
our results should be interpreted with caution and further
studies should be conducted to confirm our findings. Corroborating the findings of this study, the results of
a recent survey aimed at characterizing the impact exerted
by HL on knowledge and attitudes towards preventive
strategies against COVID-19 also did not evidence any
association between HL and knowledge (OR = 1.141;
95% CI: 0.981; 1.326, p = 0.086)(41). Discussion Similarly, a cross-
sectional study that evaluated HL and knowledge about
diabetes in a sample of 2,895 participants showed no
significant associations between HL and knowledge about
diabetes (p=0.67)(42). The findings in this study have relevant implications
for the clinical practice. It is important to recognize that
hypertensive older adults, with lower schooling levels
or unemployed, are more likely to have inadequate HL
levels. Therefore, they need to be better supported in their
clinical path by someone who can develop and better use
the skills in communicating, searching, and processing
health information in order to apply them in the daily
practice for their own benefit. In this sense, it becomes
necessary that health information be transmitted in a clear
and objective way, considering the social characteristics
that influence HL, as the patients may have difficulty
understanding the health information conveyed to them
by the team and may often feel uncomfortable to ask
for clarifications leading to communication failures and
discontinuity of the bond for the care to be provided. However, other studies support the association
between knowledge about the disease and HL(16,18,43). A
cross-sectional study including 48 patients with atrial
fibrillation showed that those with inadequate HL levels
had significantly less knowledge about anticoagulant
treatment than those with adequate HL levels (55.8 ±
15.9 vs. 66.1 ± 14.4, p = 0.02). In addition to that, a
lower percentage of patients with inadequate HL levels
were aware of the indication for the anticoagulant therapy
(57.1% vs 85.2%, p = 0.04), the mechanism of action
of the medication (42.9% vs 88.9%, p = 0.001) and its
adverse effects (28.6% vs 70.4%, p=0.03)(43). A meta-analysis of more than 18,000 patients with
diabetes showed that higher HL levels were significantly
associated with better knowledge about diabetes and,
in particular, the performance-based HL measures were
the best predictors for knowledge about diabetes(18). Interestingly, tools for assessing HL with a numbering
section led to a significantly smaller effect size than those
that did not include it(18). In addition, the health system as a whole should be
reorganized to assist patients according to their HL levels
to reduce the complexity inherent in navigating across
the health services, whether for simple clarification of
doubts about their health condition, for scheduling, and
for referral to services and other specialized professionals. Discussion The interventions need to be focused on helping patients
organize the information in a meaningful and simple way
so they can put interventions into practice to improve
decision-making about their health management. In this study, most of the participants answered the
numbering questions correctly, which may suggest that
the content or the random answer were easy for the
sample under study. As a result, the HL score may have
been overestimated, affecting the Cronbach’s alpha value. This is not to say that the instrument is not suitable for
HL assessments but, rather, that the size of our sample
was not sufficient to calculate psychometric parameters. In this sense, it is not possible to draw conclusions
regarding validity of the instrument based on our results. Although the sample size was adequate for this purpose,
it would be expected that performance of the instrument
would not be as good when testing it in people with CAD
when compared to the general population, in which it
was validated. Studies evaluating the performance of
S-TOFHLA in specific groups of people, such as those with
CAD, are still required. There is a need for studies on HL levels in different
scenarios, as well as for HL assessments to be routinely
performed by the health professionals during the first
appointment with the patients so that interventions
can be designed according to the individual and
socioeconomic characteristics and to each patient’s degree
of understanding. www.eerp.usp.br/rlae Conclusion In the scope of this study, HL was influenced by
age, hypertension diagnosis, schooling, and professional
status but not by specific knowledge about the disease
in patients with CAD, suggesting that age, clinical, and
social factors may play an important role in the way in
which health information is obtained, processed and
understood by patients for appropriate decision-making. Health professionals should be aware of the factors that
exert an influence on HL, especially in the planning of This study has limitations that need to be taken
into account. The use of convenience sampling from a
single center predominantly consisting of white-skinned
participants may have compromised heterogeneity of 9 Costa AC, Conceição AP, Butcher HK, Butcher RCGS. 11- Jin K, Neubeck L, Koo F, Ding D, Gullick J. Understanding Prevention and Management of
Coronary Heart Disease Among Chinese Immigrants
and Their Family Carers: A Socioecological Approach. J Transcult Nurs. 2020;31(3):257-66. https://doi. org/10.1177/1043659619859059
12- Carthery-Goulart MT, Anghinah R, Areza-Fegyveres
R, Bahia VS, Brucki SM, Damin A, et al. Performance of a
Brazilian population on the test of functional health literacy
in adults. Rev Saude Publica. 2009;43(4):631-8. https://
doi.org/10.1590/S0034-89102009005000031
13- Paes RG, Mantovani MF, Silva ATM, Boller C, Nazário
SS, Cruz EDA. Letramento em saúde, conhecimento da
doença e risco para pé diabético em adultos: estudo
transversal. Rev Baiana Enferm. 2022;36:e45868. https://
doi.org/10.18471/rbe.v36.45868 health interventions, which are more challenging for
patients with inadequate HL. 11- Jin K, Neubeck L, Koo F, Ding D, Gullick J. Understanding Prevention and Management of
Coronary Heart Disease Among Chinese Immigrants
and Their Family Carers: A Socioecological Approach. J Transcult Nurs. 2020;31(3):257-66. https://doi. org/10.1177/1043659619859059
12- Carthery-Goulart MT, Anghinah R, Areza-Fegyveres
R, Bahia VS, Brucki SM, Damin A, et al. Performance of a
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in adults. Rev Saude Publica. 2009;43(4):631-8. https://
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doi.org/10.18471/rbe.v36.45868 11- Jin K, Neubeck L, Koo F, Ding D, Gullick J. Understanding Prevention and Management of
Coronary Heart Disease Among Chinese Immigrants
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J Environ Res Public Health. 2021;18(17). https://doi. org/10.3390/ijerph18179316 43- Rolls CA, Obamiro KO, Chalmers L, Bereznicki LRE. Copyright © 2023 Revista Latino-Americana de Enfermagem
This is an Open Access article distributed under the terms of the
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This license lets others distribute, remix, tweak, and build upon
your work, even commercially, as long as they credit you for the
original creation. This is the most accommodating of licenses
offered. Recommended for maximum dissemination and use of
licensed materials. Authors’ contribution Study concept and design: Ana Caroline da Costa,
Ana Paula da Conceição, Rita de Cassia Gengo e Silva
Butcher. Obtaining data: Ana Caroline da Costa. Data
analysis and interpretation: Ana Caroline da Costa, Ana
Paula da Conceição, Howard Karl Butcher, Rita de Cassia
Gengo e Silva Butcher. Statistical analysis: Ana Caroline
da Costa, Howard Karl Butcher, Rita de Cassia Gengo e
Silva Butcher. Drafting the manuscript: Ana Caroline da
Costa, Ana Paula da Conceição, Howard Karl Butcher, Rita
de Cassia Gengo e Silva Butcher. Critical review of the
manuscript as to its relevant intellectual content:
Ana Caroline da Costa, Ana Paula da Conceição, Howard
Karl Butcher, Rita de Cassia Gengo e Silva Butcher. All authors approved the final version of the text. All authors approved the final version of the text. Conflict of interest: the authors have declared that
there is no conflict of interest. Conflict of interest: the authors have declared that
there is no conflict of interest. Received: May 2nd 2022
Accepted: Nov 6th 2022 Associate Editor:
Maria Lúcia do Carmo Cruz Robazzi Copyright © 2023 Revista Latino-Americana de Enfermagem
This is an Open Access article distributed under the terms of the
Creative Commons (CC BY). This license lets others distribute, remix, tweak, and build upon
your work, even commercially, as long as they credit you for the
original creation. This is the most accommodating of licenses
offered. Recommended for maximum dissemination and use of
licensed materials. Copyright © 2023 Revista Latino-Americana de Enfermagem
This is an Open Access article distributed under the terms of the
Creative Commons (CC BY). This license lets others distribute, remix, tweak, and build upon
your work, even commercially, as long as they credit you for the
original creation. This is the most accommodating of licenses
offered. Recommended for maximum dissemination and use of
licensed materials. www.eerp.usp.br/rlae
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INDICADORES MORFOMÉTRICOS EN NUEVAS VARIEDADES MEGATÉRMICAS DE Cenchrus purpureus TOLERANTES AL ESTRÉS HÍDRICO
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Tropical and subtropical agroecosystems
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R. C. Arias1, J. J. Reyes2, 3, J. V. Ray1, D. G. Benítez1, L. G. Hernández4 and J.
L. Ledea4,* 1Instituto de Investigaciones Agropecuarias “Jorge Dimitrov”. Estación
Experimental de Pastos y Forrajes, km 10½, Carretera Bayamo – Tunas. Bayamo,
Granma, Cuba
2Universidad Técnica de Cotopaxi. Extensión La Maná. Av. Los Almendros y Pujilí,
Edificio Universitario, La Maná, Ecuador. 3Universidad Técnica Estatal de Quevedo. Av. Walter Andrade. Km 1 ½ vía a Santo
Domingo. Quevedo, Los Ríos, Ecuador. 4Centro de Investigaciones Biológicas del Noroeste (CIBNOR). Instituto Politécnico
Nacional. N° 195. Col. Playa Palo de Santa Rita Sur, La Paz, Baja California Sur,
México. Email: ledea1017@gmail.com
*Corresponding author 2Universidad Técnica de Cotopaxi. Extensión La Maná. Av. Los Almendros y Pujilí,
Edificio Universitario, La Maná, Ecuador. 3Universidad Técnica Estatal de Quevedo. Av. Walter Andrade. Km 1 ½ vía a Santo
Domingo. Quevedo, Los Ríos, Ecuador. 4Centro de Investigaciones Biológicas del Noroeste (CIBNOR). Instituto Politécnico
Nacional. N° 195. Col. Playa Palo de Santa Rita Sur, La Paz, Baja California Sur, RESUMEN El objetivo del presente estudio fue caracterizar mediante índices de crecimiento nuevas variedades megatérmicas de
Cenchrus purpureus con tolerancia al estrés hídrico en las condiciones del Valle del Cauto. Para ello se trabajó con
nuevas variedades de C. purpureus (CT-601, CT-603, CT-605, CT-608, CT-609 y CT-115 como control), en diferentes
edades de rebrote (60, 90, 120 y 150 días), las cuales fueron dispuestas en un diseño de bloques al azar con arreglo
factorial con cuatro réplicas. Dentro de la época de pocas lluvias el CT-601 se destacó en la tasa de crecimiento absoluto
(106,6 g día-1), pero sin presentar diferencias significativas (p≥0,05) con el resto de las variedades en esta edad. Mientras que la variedad CT-115 prersentó los mayores valores para tasa de crecimiento relativo a los 60 días (114,4
g día-1), área foliar en las edades de 90 y 120 (285,8 y 211,6 cm2, respectivamente) e índice de área foliar (41,4) entre
90 y 150 días el area foliar a los 120 días de edad (48,5 cm2 días-1) (p≤0,001). Para la época lluviosa solo la TCA se
afectó por la interacción variedad y edad, y la representó el CT-608 a los 60 días (p≤0,01) con 347,7 g día-1 de
acumulación de materia seca. Se concluye que el régimen de pocas lluvias, característico del Valle del Cauto
condicionó la respuesta morfo fisiológica de forma marcada cuando se combinó la utilizaron de las nuevas variedades
en diferentes edades de rebrote, mientras que, en las lluvias, solo la tasa de crecimiento absoluto se afecta por este
efecto. Palabras claves: Pennisetum purpureum; ecosistemas adversos; tolerancia a la sequía. † Submitted December 24, 2018 – Accepted March 11, 2019. This work is licensed under a CC-BY 4.0 International License.
ISSN: 1870-0462 Tropical and Subtropical Agroecosystems, 22 (2019): 115-125 Tropical and Subtropical Agroecosystems, 22 (2019): 115-125 Arias et al., 2019 INTRODUCCIÓN uno de los resultados más relevantes que, la mayor
TCC ocurrió con el intervalo de corte de siete semanas
en el período lluvioso en condiciones de intensa sequía
estacional, donde concluyeron que la edad de corte o
de utilización de la planta intervenía de forma activa
en el logro satisfactorio del desarrollo morfo
fisiológico del pasto. Los índices de crecimiento, constituyen indicadores
básicos para comprender la fisiología del crecimiento
y producción de los cultivos, así como para definir y
comprender los mecanismos a través de los cuales se
explica la acción limitante o estimulante de los factores
del crecimiento (Fortes, 2012). El desbalance estacional del Valle del Cauto, que está
regido por el régimen pluviométrico define el
comportamiento del crecimiento de los cultivos y las
tasas de maduración, así como la eficiencia del
desarrollo (Ledea et al.,2018). Como resultado, en el
período lluvioso abunda y se desaprovecha alimento, y
en el período de pocas lluvias, es escaso y en ocasiones
inexistente para la alimentación de la masa ganadera
de la región (Benítez et al., 2010). Por lo tanto, es hace
necesario el empleo de variedades que, en primer orden
toleren las condiciones de intensa sequía que
identifican a este ecosistema, y en segundo orden, se
pueda definir un sistema de manejo que sugiera los
momentos óptimos de utilización de los cultivos a
partir de criterios fisiológicos. La clasificación de los índices de crecimiento,
establecida por Hunt (1990) se divide en cinco grupos:
a) Tasa de Crecimiento Absoluto (TCA); b) Tasa de
Crecimiento Relativo (TCR); c) relaciones simples,
que incluyen la Relación de Área Foliar (RAF), el Área
Foliar Específica (AFE), la relación de peso foliar y el
Índice de Área Foliar (IAF); d) componentes de las
tasas de crecimiento, denominadas también tasas de
crecimiento compuesto, como la Tasa de Asimilación
Neta (TAN) y la Tasa de Crecimiento del Cultivo
(TCC) y e) duraciones integrales, como la Duración de
Área Foliar (DAF) y de la biomasa (DBM). El análisis del crecimiento a través de los índices
mencionados posibilita identificar las adaptaciones en
las plantas a diferentes condiciones edafoclimáticas, y
dentro de estas respuestas seleccionar las más
promisorias. También permite obtener información
acerca de los efectos del ambiente, el manejo, entre
otros factores en las especies de pastos que se estudien,
de ahí la importancia que tiene su uso en los estudios
morfofisiológicos. INTRODUCCIÓN Sin embargo, son esporádicos los
resultados referentes con este enfoque en la variedad
en estudio, Herrera (2010) ya había señalado esta
limitante y Fortes (2012) lo corroboró, este último
autor tuvo que recurrir para desarrollar la discusión de
su estudio a autores externos, que contemplaban
diferentes condiciones de manejo y escenarios
experimentales sin similitudes con respecto al que
desarrolló
su
trabajo,
debido
a
esto,
tomó
alternativamente los patrones de comportamiento de
las
diferentes
variables
que
describían
el
comportamiento morfo fisiológico y no los valores
absolutos, consideración que se tendrá en cuenta en el
presente estudio. Actualmente se cuentan con cultivos megatérmicos
que
fueron
obtenidos
a
partir
de
técnicas
biotecnológicas con tolerancia al estrés hídrico a partir
del CUBA vc CT-115. Estas nuevas variedades, han
sido evaluadas en el occidente (Álvarez et al.,2016), y
caracterizadas en el oriente, atendiendo criterios
agronómicos y morfológico (Ledea et al.,2018), (Arias
et al., 2018). Sin embargo, aún no se cuentan con los
criterios de los índices de crecimiento, información
que puede permitir nuevas inferencias de la
productividad, además de constituir un nuevo aporte al
conocimiento de estos nuevos cultivares en las
condiciones del Valle del Cauto. Es objetivo de este
estudio,
caracterizar
mediante
los
índices
de
crecimiento nuevas variedades megatérmicas de
Cenchrus purpureus con tolerancia al estrés hídrico en
las condiciones del Valle del Cauto. SUMMARY The objective of the present study was to characterize growth rates of new megatérmicas varieties of Cenchrus
purpureus with tolerance to water stress in Valle del Cauto conditions. To this end, new varieties of C. purpureus were
used (CT-601, CT-603, CT-605, CT-608, CT-609 and CT-115 as control), at different regrowth ages (60, 90, 120 and
150 days), which were arranged in a random block design with factorial arrangement with four replicas. Within the
low rainfall season, CT-601 stood out in the absolute growth rate (106.6 g day-1), but did not present significant
differences (p≥0.05) with the rest of the varieties in this age group. . While the CT-115 variety presented the highest
values for the relative growth rate at 60 days (114.4 g day-1), foliar area at the ages of 90 and 120 (285, 8 and 211.6
cm2, respectively ) and leaf area index (41.4) between 90 and 150 days the leaf area at 120 days of age (48.5 cm2 days-
1) (p≤0.001). For the rainy season only TCA was affected by the interaction variety and age, and was represented by
CT-608 at 60 days (p≤0.01) with 347.7 g day-1 of dry matter accumulation. It is concluded that the low rainfall regime,
characteristic of Valle del Cauto, conditioned the morphological response in a marked way when combined the use of
the new varieties in different ages of regrowth, while, in the rains, only the absolute growth rate is affected by this
effect. Keywords: Pennisetum purpureum; adverse ecosystems; tolerance to drought. 115 Arias et al., 2019 Tropical and Subtropical Agroecosystems, 22 (2019): 115-125 Arias et al., 2019 Arias et al., 2019 MATERIAL Y MÉTODOS (Época lluviosa, R2 ajustada:0,98 EE: 7,26)
𝐴𝐹= 2,269(±0,639815) + 83,540(±0,534048)𝐿∗𝐴
(Época poco lluviosa, R2 ajustada: 0,98 EE: 8,19)
𝐴𝐹= 1,874(±0,519636) + 73,426(±0,586023)𝐿∗𝐴 (Época lluviosa, R2 ajustada:0,98 EE: 7,26)
𝐴𝐹= 2,269(±0,639815) + 83,540(±0,534048)𝐿∗𝐴 Figura 1. Comportamiento de las precipitaciones
durante el período experimental y media histórica por
meses. (Época poco lluviosa, R2 ajustada: 0,98 EE: 8,19)
𝐴𝐹= 1,874(±0,519636) + 73,426(±0,586023)𝐿∗𝐴 Donde: AF: Área foliar, L: largo de la hoja, cm (desde
la lígula hasta el ápice) A: ancho de la hoja (promedio
de los puntos; base, medio y apical respectivamente) y
se ajustó usando el método gravimétrico en papel de
peso homogéneo según Mielke et al. (1995). El suelo del área experimental es de tipo Fluvisol poco
diferenciado, según la nueva versión de clasificación
genética de los suelos de Cuba (Hernández et
al.,2015). Presenta buen drenaje general, la topografía
es llana y generalmente su fertilidad natural se
encuentra entre valores medios y bajos con un pH
medianamente ácido en KCl (4.7) y ligeramente ácido
en H2O (6.3). Presenta niveles de salinidad muy bajos
hasta los 60cm de profundidad, los tenores de
nutrientes en general se encuentran bajos (≤ 2.10%
MO) solo la capa superior manifiesta niveles medios
(Ledea et al., 2018a). La humedad del suelo en los
primeros 60 cm en este período osciló entre 21,6
(agosto) hasta 46,4% (septiembre) Ledea op. cit. Se calcularon las variables fisiológicas: Tasa de
Crecimiento Absoluta (TCA), Tasa de Crecimiento
Relativa (TCR), a partir del control de la biomasa aérea
de las plantas, utilizando los procedimientos descritos
por De Armas et al. (1988). 𝑇𝐶𝐴 (𝑔 𝑑í𝑎 −1) = 𝑃 𝑓𝑖𝑛𝑎𝑙−𝑃 𝑖𝑛𝑖𝑐𝑖𝑎𝑙
𝑇𝑓𝑖𝑛𝑎𝑙−𝑇𝑖𝑛𝑖𝑐𝑖𝑎𝑙
𝑇𝐶𝑅 (𝑔 𝑔−1 𝑑í𝑎−1) = (𝑙𝑛𝑀𝑆𝑓𝑖𝑛𝑎𝑙−𝑙𝑛𝑀𝑆𝑖𝑛𝑖𝑐𝑖𝑎𝑙)𝑥 (𝑇𝑓𝑖𝑛𝑎𝑙
−𝑇𝑖𝑛𝑖𝑐𝑖𝑎𝑙) 𝑇𝐶𝐴 (𝑔 𝑑í𝑎 −1) = 𝑃 𝑓𝑖𝑛𝑎𝑙−𝑃 𝑖𝑛𝑖𝑐𝑖𝑎𝑙
𝑇𝑓𝑖𝑛𝑎𝑙−𝑇𝑖𝑛𝑖𝑐𝑖𝑎𝑙 𝑇𝐶𝑅 (𝑔 𝑔−1 𝑑í𝑎−1) = (𝑙𝑛𝑀𝑆𝑓𝑖𝑛𝑎𝑙−𝑙𝑛𝑀𝑆𝑖𝑛𝑖𝑐𝑖𝑎𝑙)𝑥 (𝑇𝑓𝑖𝑛𝑎𝑙
−𝑇𝑖𝑛𝑖𝑐𝑖𝑎𝑙) 𝑇𝐶𝑅 (𝑔 𝑔−1 𝑑í𝑎−1) = (𝑙𝑛𝑀𝑆𝑓𝑖𝑛𝑎𝑙−𝑙𝑛𝑀𝑆𝑖𝑛𝑖𝑐𝑖𝑎𝑙)𝑥 (𝑇𝑓𝑖𝑛𝑎𝑙
−𝑇𝑖𝑛𝑖𝑐𝑖𝑎𝑙) 𝑇𝐶𝑅 (𝑔 𝑔−1 𝑑í𝑎−1) = (𝑙𝑛𝑀𝑆𝑓𝑖𝑛𝑎𝑙−𝑙𝑛𝑀𝑆𝑖𝑛𝑖𝑐𝑖𝑎𝑙)𝑥 (𝑇𝑓𝑖𝑛𝑎𝑙
−𝑇𝑖𝑛𝑖𝑐𝑖𝑎𝑙) Material vegetal utilizado: Los cultivares sometidos a
estudio fueron, el CT-601, CT-603, CT-605, CT-608,
CT-609 y CT-115. Estos nuevos cultivares se
obtuvieron en el Instituto de Ciencia Animal, a través
de mutaciones inducidas en cultivo de tejidos, a partir
de la variedad CUBA vc. CT-115, la cual se utilizó
como control por su amplia utilización en Cuba para la
producción de biomasa forrajera, debido, a elevados
rendimientos, aceptable calidad y que se adapta, crece
y desarrolla en amplia variedad de suelos y
características climáticas (Herrera y Ramos, 2006). MATERIAL Y MÉTODOS Donde: P: peso; T: tiempo Donde: P: peso; T: tiempo A partir del AF, se calculó el Índice de área foliar
(IAF), Duración de la biomasa (DBM) y Duración del
área foliar (DAF) (Fortes, 2012). 𝐼𝐴𝐹=
(Á𝑟𝑒𝑎 𝑓𝑜𝑙𝑖𝑎𝑟𝑓𝑖𝑛𝑎𝑙+ Á𝑟𝑒𝑎 𝑓𝑜𝑙𝑖𝑎𝑟𝑖𝑛𝑖𝑐𝑖𝑎𝑙)
2(1
𝑆)
Para: S: Área del suelo
𝐷𝐵𝑀 (𝑘𝑔 𝑑í𝑎𝑠−1) = [(𝑃𝑓𝑖𝑛𝑎𝑙+ 𝑃𝑖𝑛𝑖𝑐𝑖𝑎𝑙)
2
](𝑇𝑓𝑖𝑛𝑎𝑙−𝑇𝑖𝑛𝑖𝑐𝑖𝑎𝑙)
𝐷𝐴𝐹 (𝑐𝑚2𝑑í𝑎𝑠−1) = (𝐴𝐹𝑓𝑖𝑛𝑎𝑙+ 𝐴𝐹𝑖𝑛𝑖𝑐𝑖𝑎𝑙) −(𝑇𝑓𝑖𝑛𝑎𝑙−𝑇𝑖𝑛𝑖𝑐𝑖𝑎𝑙)
2
Donde: AF: Área foliar; P: peso; T: tiempo 𝐼𝐴𝐹=
(Á𝑟𝑒𝑎 𝑓𝑜𝑙𝑖𝑎𝑟𝑓𝑖𝑛𝑎𝑙+ Á𝑟𝑒𝑎 𝑓𝑜𝑙𝑖𝑎𝑟𝑖𝑛𝑖𝑐𝑖𝑎𝑙)
2(1
𝑆) MATERIAL Y MÉTODOS Localidad, clima y suelo: El estudio se llevó a cabo
durante las estaciones lluviosa y poco lluviosa, de abril
de 2010 a abril de 2012, en la Estación Experimental
de Pastos y Forrajes perteneciente al Instituto de
Investigaciones Agropecuarias “Jorge Dimitrov” en la
provincia Granma. Situada en los 20º 18´13” de latitud
norte y los 76º 39´ 48” de longitud oeste. En investigaciones relacionadas con las tasa e índices
de crecimiento en Cuba, se encuentra la desarrollada
Ramírez de la Ribera et al. (2008) en el pasto
Mombaza (Megathyrzus maximus Jacq) manejado con
diferentes intervalos de corte en las condiciones del
Valle del Cauto, ecosistema que se caracteriza por
regímenes pluviométricos con distribución poco
uniforme, y escasos milímetros precipitados con
respecto a la media nacional de Cuba (1335mm), la
distribución de las precipitaciones dicta que, cerca del
80% precipita en la estación de lluvias, enmarcada en
el período mayo-octubre (ONEI, 2008) y el resto de las
precipitaciones en el período de lluvias (noviembre-
abril). Ramírez de la Ribera op. cit. señalaron como El clima donde está ubicada la estación, se clasifica
como tropical relativamente húmedo (Barranco y Días,
1989). Durante el período experimental, en el área de
estudio
las
precipitaciones
totales
fueron
de
3317,6mm, que representó el 19,06% del acumulado
de 17 años. Durante el período se constataron
fluctuaciones entre cero y 296 mm anuales (Figura 1). 116 Tropical and Subtropical Agroecosystems, 22 (2019): 115-125 Arias et al., 2019 Arias et al., 2019 Las precipitaciones de la época lluviosa representaron
el 86,9% en el período evaluado, y en período poco
lluvioso (noviembre-abril) solo se acumuló el 13,06%,
con períodos de intensa sequía, en la Figura 1 los
marcadores
en
blanco
representan
meses
sin
precipitaciones. riego (250 m3 ha-1) a intervalos de 21 días en la época
de pocas precipitaciones, y estratégicamente cuando se
identificaron síntomas de estrés hídrico dentro del
período lluvioso. Evaluaciones en la planta: Se contabilizó el número de
hojas por plantas en diferentes estados fenológicos por
período climático, 1289 hojas en la época lluviosa y
1324 hojas en la época poco lluviosa se calculó el área
de las mismas (cm2) utilizando las longitudes de la
porción
media
y
longitudinal
siguiendo
las
recomendaciones de Fortes (2012), y se determinó el
Área Foliar (AF) a partir de las siguientes expresiones
de regresión múltiple: Figura 1. Comportamiento de las precipitaciones
durante el período experimental y media histórica por
meses. El El modelo
matemático empleado fue el siguiente: 𝑌𝑖𝑗𝑘= 𝜇+ 𝑅𝑖+ 𝐸𝑅𝑗+ 𝑉𝑘+ (𝐸𝑅× 𝑉)𝑗𝑘+ 𝜀𝑖𝑗𝑘 Para el AF (Tabla 2), la respuesta fue errática, algunas
variedades manifestaron la expansión del AF a lo largo
de las edades de rebrote evaluadas (CT 601, 603 y
605), el resto (CT 609 y 115) lo revelaron en la edad
de 90 días. La variedad CT-115 en las edades de 90 y
120 días presentó valores similares al obtenido por las
variedades CT-605 y CT-609 a los 150 y 90 días de
rebrote, respectivamente. Las variedades CT 601, 603
y 608 no mostraron diferencias con los valores propios
dentro de las edades (p≥0,05). Las variedades CT 605,
609 y 115 que tuvieron valores diferentes a los 60 días
con respecto a los 150 días de rebrote dentro de cada
variedad (p≤0,001). Dónde:Yijkl = variable respuesta, µ= constante común
a todas las observaciones, Ri = efecto de la i-ésima
réplica (i=1, ….,4), ERJ = efecto de la j-ésima edad de
rebrote (j=1,…., 4), Vk = efecto del k-ésima variedad
(k= 1, …,5), ER x Vjk = efecto combinado de la j-ésima
edad de rebrote en el k-ésima variedad, eijk= error
aleatorio ~ N (0, σ2e). El Procedimiento
experimental:
El
área
de
experimentación ya estaba establecida en parcelas de 5
x 4 m con área cosechable de 12 m2, y cinco surcos en
cada parcela con marcos de plantación de 1 x 0.75. Se
realizó un corte para lograr uniformidad en el material
experimental en el mes de noviembre del 2009, y
seguidamente, se aplicó fertilización orgánica de
estiércol bovino, con 1.38 % de nitrógeno, en dosis de
20 t ha-1 fraccionada en dos aplicaciones, una en cada
período climático. El estudio comenzó en 2010 hasta
2012, durante todo el período experimental se aplicó Para: S: Área del suelo Para: S: Área del suelo
𝐷𝐵𝑀 (𝑘𝑔 𝑑í𝑎𝑠−1) = [(𝑃𝑓𝑖𝑛𝑎𝑙+ 𝑃𝑖𝑛𝑖𝑐𝑖𝑎𝑙)
2
](𝑇𝑓𝑖𝑛𝑎𝑙−𝑇𝑖𝑛𝑖𝑐𝑖𝑎𝑙)
𝐷𝐴𝐹 (𝑐𝑚2𝑑í𝑎𝑠−1) = (𝐴𝐹𝑓𝑖𝑛𝑎𝑙+ 𝐴𝐹𝑖𝑛𝑖𝑐𝑖𝑎𝑙) −(𝑇𝑓𝑖𝑛𝑎𝑙−𝑇𝑖𝑛𝑖𝑐𝑖𝑎𝑙)
2 Diseño, tratamiento y análisis estadístico: Se utilizó un
diseño de bloques al azar en arreglo factorial con 117 Tropical and Subtropical Agroecosystems, 22 (2019): 115-125 Arias et al., 2019 La TCR como representante de la eficiencia de la
planta en la formación de tejidos nuevos, coincidió
para los 60 días en todas las variedades destacándose
la variedad CT-115 con el mayor valor (p≤0,001), este
fue común con el resto de las variedades en esta edad,
excepto para el CT-608. El valor del CT-115 en
comentario difirió con sus propios promedios y con los
del resto de las variedades en estudio en las edades
posteriores a 60 días. cuatro réplicas, se evaluaron 24 tratamientos
resultantes de la combinación edad de rebrote (60, 90,
120 y 150 días), y nuevos cultivares de Cenchrus
purpureus (CT-601, CT-603, CT-605, CT-608, CT-
609 y CT-115 testigo). Para la distribución normal de
los datos se empleó la prueba de Kolmogorov-Smirnov
(Massey, 1951) y para la homogeneidad de varianzas
la prueba de Bartlett (1937), las medias resultantes de
las interacciones se compararon mediante la dócima de
Keuls (1952). Los valores significativamente inferiores se obtuvieron
en las variedades CT-605 y CT-601 a los 90 y 120 días
de rebrote, respectivamente. El resto de las variedades
manifestaron promedios intermedios en las diferentes
edades y que no se diferenciaron de los valores que
aportó el CT-115, solo el CT-601 a los 90 días se
diferenció (p≤ 0,001) de la variedad CT-608 a los 150
días. Para los análisis estadísticos se empleó el paquete
Statistica sobre Windows, versión 12.0 (StatSoft
2011). Se realizaron análisis de varianza según las
exigencias del diseño experimental. a-g Al menos con una letra en común no presentan diferencias significativas según Keuls (1952)
Números entre paréntesis corresponden a valores transformados para log(x)1 y log(x+3)2 Al menos con una letra en común no presentan diferencias significativas según Keuls (1952)
Números entre paréntesis corresponden a valores transformados para log(x)1 y log(x+3)2 g Al menos con una letra en común no presentan diferencias significativas según Keuls (1952)
Números entre paréntesis corresponden a valores transformados para log(x)1 y log(x+3)2 RESULTADOS Para TCA se observaron diferencias en la variedad CT-
601 a la edad de 150 días y CT-609 a los 60, con los
obtenidos por la variedad CT-605 a los 60, 90 y 120
días de edad, y con los del CT-608 a los 90 y 120 días,
también con el CT-601 a los 60 días (p≤0,001). Con
respecto al testigo no se apreciaron diferencias
significativas en ninguna de las edades evaluadas
(Tabla 1). Para el IAF los mayores valores fueron aportados en
las edades más prolongadas. ndicadores fisiológicos de crecimiento en nuevas variedades de Cenchrus purpureus en
l é
ll
i (
)
Tabla 1. Comportamiento de indicadores fisiológicos de crecimiento en nuevas variedades de Cenchrus purpureus en
diferentes edades de rebrote en la época poco lluviosa. Variedades
(CT)
1TCA (g día-1)
2TCR (g día-1)*10-3
60
90
120
150
60
90
120
150
Edades de rebrote
Edades de rebrote
601
40,83c-g
(3,6)
72,58abc
(4,28)
80,10abc
(4,37)
106,6a
(4,6)
89,5ab
(4,48)
10,8ef
(2,22)
4,7g
(1,55)
6,6fg
(1,66)
603
71,50abc
(4,24)
52,22abc
(3,81)
60a-d
(4,05)
64,6a-d
(4,15)
51,1abc
(3,85)
30,7bcde
(3,3)
11,5def
(2,37)
15,1cdef
(2,70)
605
27,08efg
(3,28)
28,11g
(3,09)
22,91fg
(3,23)
76abc
(4,29)
85,8ab
(4,44)
4,56g
(0,69)
15,9cdef
(2,61)
9,6ef
(2,19)
608
71,50abc
(4,24)
33,05d-g
(3,48)
48,95b-e
(3,85)
58,50a-d
(4)
28,2bcde
(3,28)
14,5def
(2,41)
22,4cde
(3,10)
42,4abcd
(3,7)
609
113,12a
(4,7)
58,88a-d
(4,03)
80,41a-c
(4,37)
93,33ab
(4,5)
86ab
(4,44)
32,4bcde
(3,6)
19,4cdef
(2,9)
11,3def
(2,4)
115
75,50abc
(4,30)
73,33a-c
(4,26)
58,02a-d
(4,04)
57,83a-d
(4,03)
114,4a
(4,73)
9,1ef
(2,21)
18,9cdef
(2,86)
11def
(2,37)
EE
0,066
0,019
P
0,00001
0,00001
a-g Al menos con una letra en común no presentan diferencias significativas según Keuls (1952) (
)
Tabla 1. Comportamiento de indicadores fisiológicos de crecimiento en nuevas variedades de Cenchrus purpureus en
dif
t
d d
d
b
t
l é
ll
i Tabla 1. Comportamiento de indicadores fisiológicos de crecimiento en nuevas variedades de Cenchrus purpureus en
diferentes edades de rebrote en la época poco lluviosa. 118 Tropical and Subtropical Agroecosystems, 22 (2019): 115-125 Arias et al., 2019 Tabla 2. Comportamiento del área foliar e índice de área foliar en nuevas variedades de Cenchrus purpureus en
diferentes edades de rebrote en la época poco lluviosa. Á
Í
Á Tabla 2. Comportamiento del área foliar e índice de área foliar en nuevas variedades de Cenchrus purpureus en
diferentes edades de rebrote en la época poco lluviosa. Tabla 2. RESULTADOS Comportamiento del área foliar e índice de área foliar en nuevas variedades de Cenchrus purpureus en
diferentes edades de rebrote en la época poco lluviosa. Variedades
(CT)
1Área Foliar (cm2)
2Índice de Área Foliar
60
90
120
150
60
90
120
150
Edades de rebrote (días)
Edades de rebrote (días)
601
84,9fgh
(4,43)
102,7e-h
(4,56)
131,3c-g
(4,84)
147,4b-g
(4,92)
7,07e
(2,08)
15,62d
(2,79)
19,5bd
(2,98)
23,2bd
(3,14)
603
94,4e-h
(4,53)
117,2d-g
(4,75)
120,3c-g
(4,78)
132,9c-g
(4,88)
7,87e
(2,17)
19,35bd
(2,98)
19,8bd
(3,02)
21,1bd
(3,09)
605
62,40h
(4,08)
79,4gh
(4,36)
112,8d-g
(4,72)
224,10ab
(5,40)
5,22e
(1,79)
15,5d
(2,72)
16,02d
(2,83)
28,07abc
(3,36)
608
81,1gh
(4,36)
93,5e-h
(4,52)
113,7d-g
(112,8)
131,6c-g
(4,85)
6,75e
(2,02)
14,5d
(2,72)
17,25cd
(2,89)
20,42bd
(3,04)
609
77,12gh
(4,34)
206,1a-d
(5,26)
183,7b-d
(5,17)
161,6b-e
(5,05)
6,42e
(2)
23,6d
(3,17)
17,25ab
(3,41)
28,7abc
(3,36)
115
62,97h
(4,08)
285,8a
(5,65)
211,6abc
(5,65)
144,2b-f
(4,96)
5,25e
(1,83)
29,07abc
(3,41)
41,4a
(3,71)
29,6ab
(3,47)
EE
0,057
0,027
P
0,00001
0,00001
a-h Al menos con una letra en común no presentan diferencias significativas según Keuls (1952)1
a-d Al menos con una letra en común no presentan diferencias significativas según Keuls (1952)2
Números entre paréntesis corresponden a valores transformados para log(x)1 y log(x+1)2 Tabla 3. Duración del área foliar de nuevas variedades
de Cenchrus purpureus en diferentes edades de rebrote
en la época poco lluviosa. Variedad
(CT)
1Duración del área foliar
(cm2 días-1)
Edades de rebrote (días)
60
90
120
150
601
69,9g
(8,34)
172,7ef
(13,06)
219,1cde
(18,44)
263,7cde
(16,04)
603
79,4g
(8,87)
196,7ef
(14,01)
222,5cde
(14,89)
238,2b-e
(15,42)
605
47,4g
(6,72)
126,9f
(11,24)
177,3ef
(14,89)
321,9bcd
(17,9)
608
66,10g
(8,05)
159,6ef
(12,56)
192,2ef
(13,29)
230,3cde
(15,08)
609
69,9g
(7,86)
268,2bcd
(16,24)
374,6b
(13,80)
330,1bcd
(18,03)
115
47,9g
(6,91)
333,8bcd
(18,27)
48,5a
(19,10)
340,7bc
(18,96)
EE
2,14
P
0,00001
a-g Al menos con una letra en común no presentan
diferencias significativas según Keuls (1952)
Números entre paréntesis corresponden a valores
transformados para log(x) Tabla 3. Duración del área foliar de nuevas variedades
de Cenchrus purpureus en diferentes edades de rebrote
en la época poco lluviosa. La variedad CT-115 mostró valores similares a los 90,
120 y 150 días, también fueron similares a los
obtenidos en la variedad CT-609 a los 120 y 150 días,
y en esta última edad con los valores del CT-605. DISCUSIÓN En la época poco lluviosa, los valores de la TCR (Tabla
1), coinciden con el estadío de crecimiento del pasto,
al ser una variable que relaciona la formación de
tejidos nuevos por unidad de tiempo, y que se expresa
en base seca, es lógico que en los primeros momentos
la planta manifieste un incremento positivo en el
desarrollo de nuevos tejidos, y que luego, en respuesta
del propio hábito de crecimiento el proceso se haga
más lento, tal como figura en las edades posteriores a
60 días. Los promedios obtenidos se encuentran
próximos a 0,02 a 0,5 g g día-1, rangos definidos por
Badle (1993) como aceptables para la mayoría de las
especies de plantas C4 en diversas condiciones
ambientales, lo que identifica a las nuevas variedades
como promisorias para el tipo de ambiente en las que
se desarrollan, y sobre todo, en la época de evaluación
que es donde predominan bajas temperaturas, días
cortos, condiciones que atentan con un desarrollo
eficiente de la fotosíntesis, y con ello menor TCR. El
tipo de condiciones climáticas según Fortes (2012),
son de las que determina la TCR, además de especie o
cultivar y estadio de crecimiento. En la época poco lluviosa, los valores de la TCR (Tabla
1), coinciden con el estadío de crecimiento del pasto, Para el período lluvioso, otras variables fueron
afectadas por el efecto principal edad de rebrote (Tabla
5). Para el AF la edad de 90 días fue el mayor valor
para esta variable, las edades 60 y 150 días propiciaron
los menores valores (p≤0,001). Mientras que el IAF y
DAF fue a la edad de 120 días donde se presentó el
valor significativamente superior y a los 60 días el
menor para ambas variables. Sin embargo, la DBM
tuvo dos momentos para aportar biomasa de forma
significativa, a los 120 y 150 días de rebrote. La TCR
tuvo su mejor (p≤0,001) momento en la edad de 60
días, y se fue reduciendo la capacidad de acumulación Tabla 5. Efecto de la edad de rebrote en indicadores fisiológicos de nuevas variedades de C. purpureus en la época
lluviosa. RESULTADOS Los
valores significativamente inferiores se obtuvieron de
forma general para todas las variedades a los 60 días
de rebrote (p≤0,001) (Tabla 2). Las variedades CT-601, 603, 605 y 608 mostraron
similares valores en la DAF en las edades de 90 a 150
días, excepto el valor intermedio del CT-605 a los 90
días. Los promedios del CT-609 y CT-115 fueron
comunes a los obtenidos por el CT 601, 603 en la edad
de 120 días y en la posterior a esta (Tabla 3). Para la época lluviosa la interacción de segundo grado
solo afectó la variable TCA (Tabla 4). La mayor tasa
de crecimiento la alcanzó el CT-608 a los 60 días. Hasta los 120 días mostró diferencias estadísticas con
sus propios promedios (120 y 150 días) y con los de las
variedades CT 601 (90, 120 y 150 días), 603 (120 días)
y 605 (90 días). El valor significativamente inferior se
obtuvo también en la variedad CT-608 a los 150 días,
y este solo difirió del promedio del CT-601 a los 150
días, con el resto de las variedades los superíndices
fueron similares p≥0,01. El CT-115 estuvo favorecido
por el incremento de la TCA hasta los 120 días,
comportamiento similar solo manifestó el CT-609, el
resto de las variedades a partir de los 90 días,
decayeron en valores de TCA. Lo que le resalta al CT-
115 y CT-609 capacidad de acumular materia seca por
unidad de tiempo en comparación al resto de las
variedades. El efecto independiente edad de rebrote en el período
lluvioso modificó la DBM, el mayor valor (p≤0,001),
se obtuvo a los 120 días (606,1 kg días-1), y este fue
común con el promedio a los 150 días (545,1 kg días-
1), pero diferente significativamente con los valores
que se obtuvieron a los 60 y 90 días (484,3 kg días-1,
para ambas edades), con un error estándar de 0,57. 119 Tropical and Subtropical Agroecosystems, 22 (2019): 115-125 Arias et al., 2019 Arias et al., 2019 Tabla 4. Comportamiento de la Tasa de Crecimiento
Absoluto en nuevas variedades de Cenchrus purpureus
en diferentes edades de rebrote en la época lluviosa. Números entre paréntesis corresponden a valores transformados para log(x)1 y log(x+2)2 RESULTADOS Variedad
(CT)
TCA (g día-1)
Edades de rebrote (días)
60
90
120
150
601
201,6abc
(5,27)
176,1bc
(5,13)
156,2bd
(5,10)
125cde
(4,80)
603
286,07ab
(5,62)
222,8abc
(5,37)
174,6bc
(5,12)
90ef
(4,45)
605
244,5abc
(5,50)
150,6bc
(4,93)
285,4abc
(5,55)
89,17def
(4,45)
608
347,7a
(5,87)
239,5abc
(5,42)
175bcd
(5,10)
71,15f
(4,12)
609
288,7ab
(5,65)
213,6abc
(5,35)
184,3abc
(5,28)
78,3ef
(4,32)
115
194,5abc
(5,27)
207,9abc
(5,26)
184,5abc
(5,7)
89,1def
(4,45)
EE
0,248
P
0,00562
a-f Al menos con una letra en común no presentan
diferencias significativas según Keuls (1952)
Números entre paréntesis corresponden a valores
transformados para log(x) Tabla 4. Comportamiento de la Tasa de Crecimiento
Absoluto en nuevas variedades de Cenchrus purpureus
en diferentes edades de rebrote en la época lluviosa. de biomasa por día en función del incremento de la
edad de rebrote (Tabla 5). En la Tabla 6 se muestra el efecto de las avriedades de
C. purpureus sobre los indicadores fisiológicos en la
época lluviosa. Para el AF el CT-608 difirió con el CT-
603. Mientras que para el IAF y DAF fueron en las
variedades CT-603 y CT-605 en las que se obtuvieron
valores diferentes de los promedios del CT 601, 608 y
609 (p≤0,001). p
Variedad
(CT)
TCA (g día-1)
Edades de rebrote (días)
60
90
120
150
601
201,6abc
(5,27)
176,1bc
(5,13)
156,2bd
(5,10)
125cde
(4,80)
603
286,07ab
(5,62)
222,8abc
(5,37)
174,6bc
(5,12)
90ef
(4,45)
605
244,5abc
(5,50)
150,6bc
(4,93)
285,4abc
(5,55)
89,17def
(4,45)
608
347,7a
(5,87)
239,5abc
(5,42)
175bcd
(5,10)
71,15f
(4,12)
609
288,7ab
(5,65)
213,6abc
(5,35)
184,3abc
(5,28)
78,3ef
(4,32)
115
194,5abc
(5,27)
207,9abc
(5,26)
184,5abc
(5,7)
89,1def
(4,45)
EE
0,248
P
0,00562 , b, c Letras diferentes en una misma fila difieren entre sí según Keuls (1952)
1 DISCUSIÓN esta
respuesta ocurre por una compensación del sistema
fotosintético en función del crecimiento, sin embargo,
no tuvieron en cuenta que en las condiciones en que se
desarrolló su estudio, la disponibilidad hídrica no es
una limitante al precipitar 4000 mm en la Amazonia
Ecuatoriana (Uvidia et al., 2013), y que además de la
disponibilidad de humedad que ofrece, la molécula de
agua aporta a partir del higrógeno, parte de los
esqueletos
carbonados
para
la
formación
de
carbohidratos estructurales y no estructurales (Ledea et
al., 2016), efecto que se relaciona con el desarrollo y
crecimiento,
no
solamente
con
la
eficiencia
fotosíntética. El
comportamiento
de
inflexión
observado, se ha descrito en otras especies de
Cenchrus purpureus vc King grass (Leonard et al.,
2014) y en otras gramíneas tropicales como Setaria
splendida y Brachiaria hibrido cv mulato (Leonard et
al., 2017), ambos ejemplos en las condiciones de la
Amazonía Ecuatoriana, también en ecosistemas áridos
con el pasto Bouteloua curtipendula (Mich.) Torr. en
Chiuahua (Álvarez et al., 2017), México. De lo que se
instuye, que este es un mecanismo muy particular de
las gramíneas
tropicales
influenciado por las
características climáticas que imperan en la zona y/o
región donde se desarrollen las mismas. Tabla 6. Efecto de la variedad en algunos indicadores
fisiológicos de nuevas variedades de C. purpureus en
la época lluviosa. a, b Letras diferentes en una misma columna difieren
entre sí según Keuls (1952) Fortes op. cit. obtuvo valores inferiores a los del
presente estudio cuando evaluó a la variedad CT-115
en pastoreo, también fueron inferiores al intervalo
señalado por Beadle op. cit. El primer autor referido
fundamentó que en su estudio, el comportamiento
obedeció a los propios mecanismos fisiológicos de la
planta producto al incremento de la edad en la que se
sometió el pastoreo, ya que no coincidía con edades
tempranas donde prevalecen las hojas jóvenes con gran
capacidad fotosintética y elevada fijación de CO2, sino
que predominaron procesos de senescencia, y con ello
reducción de eficiencia fotosintética de las hojas y
aumento de las pérdidas respiratorias de la planta,
procesos que atentan a una eficiente TCR, y que están
manifiestos en el presente estudio en las edades
posteriores a 60 días. DISCUSIÓN Indicador
Edad de rebrote (días)
EE
P
60
90
120
150
Área foliar, cm2
167,6c
258,8a
231,1b
168,6c
1,69
0,0001
1Índice de área foliar
13,9c
(2,55)
35,4b
(3,54)
41,16a
(3,70)
33,30b
(3,49)
0,17
1Duración del área foliar, cm2
días-1
152,6c
(4,92)
416,5b
(6)
477,9a
(6,15)
384,7b
(5,94)
0,19
1Duración de la Biomasa, kg
días-1
121,6b
(4,7)
370,7a
(5,56)
167,2b
(5,02)
260,8a
(5,51)
0,03
2TCR (g día-1)*10-3
162,7a
(5,09)
25,8b
(2,85)
22,6b
(2,09)
10,3c
(2,97)
0,34
a, b, c Letras diferentes en una misma fila difieren entre sí según Keuls (1952) dad de rebrote en indicadores fisiológicos de nuevas variedades de C. purpureus en la época Tabla 5. Efecto de la edad de rebrote en indicadores fisiológicos de nuevas variedades de C. purp
l
i 120 Tropical and Subtropical Agroecosystems, 22 (2019): 115-125 Arias et al., 2019 Arias et al., 2019 Tabla 6. Efecto de la variedad en algunos indicadores
fisiológicos de nuevas variedades de C. purpureus en
la época lluviosa. Variedades
(CT)
Indicadores
Área
foliar
(cm2)
Índice de
Área foliar
Duración
del área
foliar
(cm2 días-1)
601
218.9ab
27,76b
(3,24)
330,4b
(5,87)
603
194,1b
33,85a
(3,47)
391,1a
(5,70)
605
225,8ab
37,53a
(3,54)
435,4a
(5,91)
608
248,2a
29,40b
(3,15)
337,8b
(5,98)
609
207,9ab
29,87b
(3,27)
342,15b
(5,67)
115
207,8ab
33,01ab
(3,43)
381,21ab
(5,87)
EE
52,5
1,75
24,1
P
0,0357
0,00013
0,00013
a, b Letras diferentes en una misma columna difieren
entre sí según Keuls (1952)
Números entre paréntesis corresponden a valores
transformados para log(x) productivo en la primera edad según Leonard et al.,
(2014) se relaciona con las condiciones climáticas y no
mecanismos intrínsecos de las plantas. Se debe señalar
que en esta primera edad ocurre la llamarada de
crecimiento producto de las reservas acumuladas en las
raíces en caso de las plantas del presente estudio que
ya estaban establecidas, luego se entiende que se
reduce el incremento de la TCA por la utilización de
las mencionadas reservas y de los carbohidratos
sintetizados para la producción de hojas y otras
estructuras como parte intrínseca del desarrollo y no
del crecimiento. En el criterio de Leonard op. cit. DISCUSIÓN Atendiendo
a que en la época climática en cuestión estás variables
se manifiestan de forma inversa a las necesidades para
un buen desarrollo del índice foliar, y lo cual se reflejó
en la expresión tardía de la misma (120-150 días de
forma general), se infiere que fue la humedad residual
del suelo propició un desarrollo favorable en las
plantas, en este sentido se ha planteado que la
elongación celular y en menor grado la división celular
se ve afectada por el déficit hídrico, lo que influye en
desarrollo foliar y por tanto en su índice de expansión. esta variable, con presunción de acumulación de
material muerto producto al efecto de la senescencia. Por lo general este es un comportamiento de cultivos
de ciclos cortos, y le puede atribuir a las nuevas
variedades, en edades no muy distantes de la que se
manifiesta la expansión foliar, un incremento de
producción de biomasa de calidad. Este comportamiento no se corresponde con el
observado por Calsina et al. (2014), sin embargo, se
justifica mediante la eficiencia que manifiestan las
plantas al invertir su crecimiento temprano en la
expansión foliar para un mejor aprovechamiento de la
radiación solar (Gardner et al.,2000). Sin embargo,
debe considerarse que el auto sombreado ejerce un
efecto negativo sobre el aprovechamiento de esta
respuesta, y que puede llegar a deprimirse si transcurre
mucho tiempo luego de la edad en que mostró el mayor
potencial foliar, también debe considerarse que la
prolongada edad se traduciría en una afectación de la
calidad química por el engrosamiento de la pared
celular, y prevalencia de carbohidratos estructurales
(Ledea, 2016) por lo tanto, es necesario no hacer una
utilización tardía del acúmulo de biomasa cuando esta
se manifieste. Las nuevas variedades y sistema de manejo establecido
posibilitaron de forma mayoritaria se manifestara entre
los 120 y 150 días los valores más significativos de
IAF, estos resultados coinciden con los obtenidos por
Rueda et al.,(2016), cuando evaluaron ocho variedades
de C. purpureus con potencialidades para la
producción de energía en Veracruz, México. Está
variable está directamente relacionada con la
producción de biomasa según las experiencias de
Tavares et al.,(2010) e Silva de Aquino y de Conti
(2014) en caña de azúcar. DISCUSIÓN Se debe considerar que un incremento de la TCA
sostenido no siempre debe interpretarse como positivo,
ya que unas de las particularidades de las gramíneas
tropicales es la acumulación de biomasa, y acelerada
maduración de tejidos por la amplia capacidad de
asimilación de radiación que poseen, lo que trae
consigo una afectación química por modificaciones de
la pared celular con la consecuente pérdida de valor
nutritivo (Ledea et al., 2018b). Se observaron comportamientos variables en algunas
variedades (CT 603, 608 y 115) en función de las
edades evaluadas, señalando para este fenómeno
respuestas individuales de cada variedad en función de
eficiencia
fotosintética
y
acumulación
de
fotoasimilados, que se relacionan con la cantidad de
hojas del dosel y la intercepción de la radiación, en este
sentido destacó la importancia de la estructura vertical
de las plantas en la captación de la energía radiante, y
de esta forma la TCR. Se observa en la Tabla 2 que las variedades referidas
recientemente con comportamiento irregular en la
TCR, fueron las variedades que menor área foliar
mostraron y para el CT-115, su abrupto incremento
para los 120 días, se reflejó en la TCR. La respuesta del AF fue la esperada (Tabla 2), con un
incremento continuo en función del aumento de la edad
de rebrote, aunque algunas (CT 609 y 115) tuvieron un
comportamiento prematuro en la expansión del área
foliar, y luego manifestaron un declive progresivo de Para la TCA exepto el CT-601, todas las variedades
tuvieron un incremento ascendente a los 60 días de
edad, con una inflexión a los 90 días. Este incremento 121 Tropical and Subtropical Agroecosystems, 22 (2019): 115-125 Arias et al., 2019 Arias et al., 2019 se identifican como favorables según el criterio de
Fortes (2012), al comentar que esta variable depende
de los niveles de irradianza, expansión de hojas y altas
temperaturas para su favorable expresión. DISCUSIÓN Reportes similares de AF se han registrado en el
período lluvioso (Fortes, 2012) y en transiciones de
estaciones climáticas (verano-invierno) (Colabelli et
al.,1998), donde la particularidad es la prevalencia de
días largos con altas temperaturas, y por tanto, mayor
exposición a niveles de irradianza que justifican la
expansión foliar, todo lo contrario ocurre en períodos
de poca duración de horas luz y bajas temperaturas, sin
embargo, es de considerarse que el sistema de manejo
a partir de las edades establecidas para el corte
redujeron el efecto negativo de la estación climática
sobre la expresión de esta variable fisiológica. Para complementar los índices anteriores se determinó
la DAF (Tabla 3) que expresa la magnitud y
persistencia del área foliar durante el período de
crecimiento de la planta (Hunt, 1990), esta variable
manifestó valores similares a los expresado por el AF
y el IAF. El comportamiento representado en la Tabla
3 no se corresponde con lo observado por Fortes (2012)
al referir que esta variable disminuyó en función del
incremento de la edad de rebrote en dos de los cuatro
ciclos que evalúo en su estudio, según este autor la
DAF guarda una relación inversamente proporcional
con el AF y con la cantidad de hojas verdes, y responde
favorablemente al incremento de la eficiencia
fotosintética y crecimiento de la planta, no obstante,
observó comportamientos erráticos que no se
relacionaban con lo antes mencionado, provocados
seguramente en respuesta al sistema de manejo
impuesto, que consistió en sometimiento a pastoreo. Castro et al. (2013) plantearon que las defoliaciones
poco frecuentes o a edades equis distantes, posibilitan
el desarrollo de hojas largas producto al tiempo de
recuperación foliar que le imprime la edad de rebrote,
en su defecto, se desarrollarán hojas cortas con
predominios de tallos. A su vez, Zepeda et al. (2018)
señalaron que el crecimiento, y por consiguiente el
desarrollo foliar del género Cenchrus, se condiciona
principalmente con la edad en que se utilice la planta,
en las condiciones del Valle del Cauto y ecosistemas
similares, es preciso mantener frecuencias de
utilización de la planta similares o cercanas a las que
se dictan en el presente trabajo cuando se desarrollen
estudios agronómicos con intereses productivos. REFERENCIAS En la Tabla 5 se observa otro grupo de variables fueron
condicionadas por la edad de rebrote. Se observó que
no hubo relación del momento de expresión del
potencial entre las variables en discusión, de esta
forma la mayor área foliar se alcanzó a los 90 días,
mientras que el IAF fue los 120, coincidiendo con la
DAF. La DBM manifestó dos picos, uno a los 90 y otro
a los 150 días, este comportamiento se justifica a partir
del desarrollo de la planta lo cual define la edad de la
misma. Las superposiciones de las hojas van a generar
un incremento del área foliar, y con ello el paulatino
incremento del IAF para la recepción de luz y
acumulación de MS, este mismo efecto va a generar
que las hojas más viejas y desarrolladas se vean
afectadas por el auto sombreado y se afecte la DAF. Mello y Pedreira (2004), observaron un decrecimiento
en el desarrollo del dosel cuando se llegó a interceptar
hasta el 95% de la energía radiante, efecto que pudo
manifestarse en el presente estudio y que condicionó el
comportamiento de la DBM con dos picos de esta
variable. En este sentido Ledea et al. (2018a), observó
que a la edad de 120 días, las dimensiones de las hojas
se redujeron con respecto al resto de las edades que
evalúo, lo cual se corresponde con la distribución del
área foliar con hojas más pequeñas, que condiciona
menor área foliar, pero mayor DBM al no verse
afectadas las mismas por el auto sombreado, lo cual
posibilita intercepción de luz sin interrupciones
incrementando por tanto la tasa fotosintética de las
mismas, con una acumulación discreta de biomasa. Álvarez, A., Febles, G., Fernández, J. M. 2016. Space
distribution
of
Pennisetum
purpureum,
according to projections for climate change in
Cuba. Cuban Journal of Agricultural Science
50(2): 291-303. ISSN: 2079-3480 Álvarez, A., Morales, C.R., Corrales, R., Sierra, J.S.,
Villarreal, F. 2017. Análisis del crecimiento
de cinco genotipos de pasto banderita
[Bouteloua curtipendula (Mich.) Torr.], bajo
condiciones
de
invernadero. Revista
Tecnociencia Chihuahua. 11(1): 25-32. ISSN:
1870-6606 Arias, R.C., Ledea, J.L., Benítez, D.G., Ray, J.V.,
Ramírez, J.L. 2018. Performance of new
varieties of Cenchrus purpureus, tolerant to
drought, during dry period. Cuban Journal of
Agricultural Science 52(2). 1-12. ISSN:
2079-3480 Barranco, G. and Díaz, L.R. 1989. Clima. Nuevo Atlas
Nacional de Cuba. Cuba: Instituto de
Geografía de la ACC, ICGC, MINFAR. Bartlett, M. DISCUSIÓN Desde los 90 hasta los 150 días existe un incremento
paulatino de la eficiencia fotosintética y crecimiento de
la planta, con excepción del CT-115 que a los 120 días
presentó un pico de la DAF, mientras que el CT-605 lo
manifestó a los 150 días, lo que sugiere que hasta los
120 días todas las variedades se mantienen
acumulando biomasa. Sin embargo, es importante
resaltar que esta variable no presenta un patrón por el
cual se pueda interpretar, sino que responde a
estímulos como lo constituyen el sistema de manejo y
período climático. El IAF, variable que se mantuvo en consonancia con el
desarrollo del área foliar como variable respuesta, pero
expresando el área foliar que esta fotosintéticamente
activa a partir de la relación del área foliar de un cultivo
por área de suelo, esta respuesta y los valores obtenidos 122 Arias et al., 2019 Tropical and Subtropical Agroecosystems, 22 (2019): 115-125 En la época lluviosa la única variable que se modificó
por el efecto de la interacción de segundo grado
(Variedad x Edad) fue la TCA (Tabla 4), se puede
observar claramente que las nuevas variedades están
acumulando biomasa hasta los 120 días de rebrote, y
que el clima de este período favoreció esta respuesta al
presentarse
las
condiciones
idóneas
para
el
crecimiento, desarrollo y acumulación de biomasa. A
los 150 días ocurrió un descenso y se debió a un
balance negativo que se establece entre la fotosíntesis
y la respiración que resulta en una ineficiencia
biológica en la acumulación de biomasa en la referida
edad (Fortes et al., 2015). Esto se debe a la relación
que existe entre la vía fotosintética que utilizan las
gramíneas tropicales con la respiración de estas, sin
obviar las interacciones entre los atributos genéticos,
ambientales,
fisiológicos
y
las
características
morfológicas que determinan la productividad del
pastizal (Pedreira, 2006), que este período no marcó las
diferencias según el objetivo del presente estudio. mecanismo respuesta a la exposición de altas
temperaturas y ciclo de crecimiento más acelerado, que
si bien, incrementa la biomasa por el aporte de este
órgano, la hace inaprovechable por las características
químicas que las caracteriza (Ledea et al., 2018b). CONCLUSIÓN El Valle del Cauto condicionó la respuesta morfo
fisiológica de forma marcada cuando se utilizaron las
plantas en diferentes edades de rebrote en el período de
pocas lluvias, con excepción de la DAF que la
determinó las características intrínsecas de cada
variedad. Mientras que en el período lluvias solo la
TCA se afecta por la combinación de cortes dentro de
esta estación climática. Sin embargo, las variables que
responden de forma mayoritaria a la permanencia y
acumulación de biomasa se afectaron por la edad de
rebrote y variedad, pero de forma separada. REFERENCIAS 1937. Properties of suffiiency and
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producción de biomasa no es comparable con la
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animal.com.ar/produccion_y_manejo_pastur
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CT-115 utilizado como banco de biomasa. Indicadores morfofisiológicos. Cuban Journal
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morfofisiológicas
do
capim-tanzânia
(Panicum maximun cv. Tanzânia) irrigado à
intensidade
de
desfolha
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lotação
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35982004000200003 Hernández, A., Pérez, J.M., Bosch. D., Rivero, L.,
Camacho, I. 2015. Nueva versión de la
clasificación genética de los suelos de Cuba. Instituto de Suelos. Ministerio de la
Agricultura. AGRINFOR. Ciudad de La
Habana, Cuba. p 64. Herrera, R.S. and Ramos, N. 2006. Factores que
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ganadería tropical. R. S. Herrera, G. J. Febles
y G. J. Crespo (Eds.). EDICA, Instituto de
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e de campo para estimativa da área foliar em
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90161995000100015 124 Tropical and Subtropical Agroecosystems, 22 (2019): 115-125 Arias et al., 2019 ONEI. 2008. Medio Ambiente (1958-2008). Medio
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.007 Silva de Aquino, M and de Conti, C. 2014. Produtividade e índices biométricos e
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204X2014000300003 Pedreira, B.C. 2006. Interceptação de luz, arquitetura
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Ok, USA: StatSoft, Inc. Tavares, O.C.H., Lima, E., Zonta, E. 2010. REFERENCIAS Crescimento e produtividade da cana‑planta
cultivada em diferentes sistemas de preparo
do solo e de colheita. Acta Scientiarum
Agronomy. 32. 61‑68. DOI:
10.4025/actasciagron.v32i1.2051. Ramírez de la Ribera, J.L., Leonard, I., López, Y.,
Álvarez, Y., López, B. 2005. Efecto de la
edad de rebrote en el valor nutritivo de dos
especies de pastos tropicales. Disponible en:
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Cisneros, M., Tamayo, W. 2008. Effect of age
and growing season on DM yield and leaf to
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Research for Rural Develoment. 20(9): 23-27. Disponible
en
http://www.lrrd.org/lrrd20/9/rami20148.htm Uvidia, H., Leonard, I., Benítez, D., Buestan, D. 2013. Dinámica del crecimiento de la Maralfalfa
(Pennisetum sp.), en condiciones de la
Amazonia Ecuatoriana. Revista Amazónica
Ciencia y Tecnología, 2(1), 14-18. ISSN
1390-8049 en Zepeda, R.G., Lazo, J.A., Sánchez, E.M.O., García, B. H., Sánchez, Y.J.J., Hernández, J.O.A.,
Martínez, R.T. 2018. Evaluación de cultivares
de Cenchrus purpureus para la producción de
forraje. Livestock
Research
for
Rural
Development. 30 (2): 1-8. ISSN 0121-3784 Rueda, J.A., Ortega, E., Hernández, A., Enríquez, J. F.,
Guerrero, J.D, Requero, A. 2016. Growth,
yield, fi ber content and lodging resistanc e in
eight varieties of Cenchrus purpureus
(Schumach.) Morrone intended as energy
crop. Biomass and Bioenergy, 88. 59-65. 125
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https://e-journal.uingusdur.ac.id/iqtida/article/download/iqtida030106/734
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Indonesian
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Pesan Dakwah dalam Media Sosial
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IQTIDA
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cc-by-sa
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Abstract This research was conducted to find out the meaning of denotation, connotation, and myths of
da'wah messages about the three great sins on Ustadz Abdul Somad's Official Youtube account. The part studied is the contents of Ustadz Abdul Somad Official's YouTube da'wah content. The
data was taken from content entitled "3 Great Sins" during the recitation after dawn at the Sultan
Mahmud Badaruddin Mosque on the link https://www.youtube.com/watch?v=qLHIBaIVSuQ
which was uploaded on May 1, 2021 from Ustadz Abdul Somad's Official Youtube account . This
research is a qualitative research model of Roland Barthes's semiotic analysis. The purpose of
this research is to see how the contents of da'wah messages on Ustadz Abdul Somad Official's
Youtube account, especially content with a discussion of the three great sins. The results of the
study, there is a discussion that is included as a grave sin, namely, associating partners with
Allah, disobedience to parents, and false witnesses. p
f
Keywords: da'wah messages, social media, the three great sins Submitted: 16 Februari 2023
Revised: 15 Mei 2023 Submitted: 16 Februari 2023
Revised: 15 Mei 2023
Accepted: 6 Juli 2023 Journal of Da’wah and Communication Journal of Da’wah and Communication Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 Abstrak Penelitian ini dilakukan untuk mengetahui makna denotasi, konotasi, dan mitos pesan dakwah
tentang tiga dosa besar dalam akun Youtube Ustadz Abdul Somad Official. Bagian yang diteliti
adalah isi konten dakwah Youtube Ustadz Abdul Somad Official. Data diambil pada konten
dengan judul “3 Dosa Besar” Cawisan Ba’da Shubuh, Masjid Sultan Mahmud Badaruddin pada
ling https://www.youtube.com/watch?v=qLHIBaIVSuQ yang diunggah pada 1 Mei 2021 dari
akun Youtube Ustadz Abdul Somad Official. Penelitian ini merupakan penelitian kualitatif
model analisis semiotik Roland Barthes. Tujuan dari penelitian ini adalah untuk melihat
bagaimana isi pesan dakwah pada akun Youtube Ustadz Abdul Somad Official terutama konten
dengan pembahasan tiga dosa besar. Hasil penelitian, terdapat pembahasan yang masuk sebagai
dosa besar yaitu, mempersekutukan Alloh, durhaka kepada orang tua, dan saksi palsu. Kata kunci: pesan dakwah media sosial tiga dosa besar Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 107 Journal of Da’wah and Communication Journal of Da’wah and Communication para da’i untuk menyampaikan pesan dakwah seperti di media sosial sehingga semua
masyarakat dapat mengetahui perkembangan Islam secara beragam dan menyeluruh. para da’i untuk menyampaikan pesan dakwah seperti di media sosial sehingga semua
masyarakat dapat mengetahui perkembangan Islam secara beragam dan menyeluruh. Dengan kecanggihan teknologi, menonton video sudah bisa dilakukan dengan
menggunakan handphone. Salah satu media yang menyediakan berbagai macam video
dan sangat digemari oleh berbagai kalangan mulai dari anak-anak hingga orang dewasa
yaitu Youtube. Youtube merupakan sebuah media yang menfasilitasi penggunanya untuk
berbagi video atau menonton video. Hingga saat ini Youtube telah diunduh lebih dari 5
juta kali (Ainul Haq, 2020). YouTube adalah media paling strategis, yang memuat konten
video untuk menyebarkan informasi sebagai seni berkomunikasi dengan melibatkan para
pengguna ( Guntur dan Nibros, 2019). Di Indonesia sendiri, menurut data ComScore per Maret 2021 pengguna Youtube
diperkirakan mencapai 100 juta pengguna. Terdiri dari kalangan millenials, tokoh publik,
dosen, ekonom, peneliti, pengamat, politikus, publik speaker, vlogger, artis hingga para
ustadz. Para Ustadz di Indonesia, menggunakan youtube sebagai syiar dakwah. Menyebarkan ide-ide Islam kepada para netizen dari berbagai kalangan! Berbuah manis,
antusias para subscriber juga positif. Jumlah kanal YouTube asal Indonesia yang
memiliki lebih dari 1 juta pelanggan (subscriber) kini telah berjumlah 9.500 kanal,
termasuk kanal youtube yang digawangi oleh para ustadz saat ini (idemuslim.com). YouTube sebagai media komunikasi yang baru juga menjadi tempat dakwah baru
bagi beberapa ustadz dan ulama. Beberapa nama seperti Ustadz Abdul Somad (UAS),
Ustadz Adi Hidayat (UAH), Gus Muwafiq (GM), Ustadzah Mumpuni Handayekti
(UMH), dan Felix Siauw (FS) menggunakan YouTube sebagai media penyebaran konten
dakwah. Dengan menggunakan berbagai strategi penyebaran konten, hal ini memperkuat
alasan digunakannya YouTube oleh mereka sebagai media komunikasi baru PENDAHULUAN Dakwah merupakan upaya untuk mengajak, mendorong, atau memanggil manusia
untuk menyebarluaskan Agama Islam dan merealisasikan ajaran agama di tengah
masyarakat serta kehidupan agar memeluk Agama Islam dan mengamalkannya. Hal ini
seperti yang dijelaskan dalam QS. Al- Imran/3: 104. yang berbunyi: ُاُوﻟٰۤﯨﻚَ ھُﻢ َوَﻟْﺘَﻜُﻦْ ﻣِّﻨْﻜُﻢْ اُﻣﱠﺔٌ ﯾﱠﺪْﻋُﻮْ نَ اِﻟَﻰ اﻟْﺨَﯿْﺮِ وَﯾَﺄْﻣُﺮُوْ نَ ﺑِﺎﻟْﻤَﻌْﺮُوْ فِ وَﯾَﻨْﮭَﻮْ نَ ﻋَﻦِ اﻟْﻤُﻨْﻜَﺮِ ۗ و
اﻟْﻤُﻔْﻠِﺤُﻮْ ن “Dan hendaklah ada diantara kamu segolongan ummat yang menyeru kepada
yang ma‟ruf dan mencegah dari yang munkar, merekalah orang-orang yang
beruntung” (Departemen Agama RI, 2004). Menurut Saputra, dakwah didefinisikan sebagai penyiaran, propaganda, seruan
untuk mempelajari dan mengamalkan ajaran agama Islam (2012:1). Dakwah Islam
dilakukan untuk mengajak manusia kepada jalan yang benar sesuai dengan perintah
Tuhan, untuk kemaslahatan dan kebahagiaan di dunia dan akhirat. Dakwah dapat
dipandang sebagai proses penyampaian pesan dari da’i kepada mad’u untuk selalu berada
dijalan Allah, menjauhi larangan-Nya dan mengikuti perintah-Nya. Dalam melaksanakan
dakwah tidak ada batas ruang dan waktu. Dalam proses penyampaianya tidak semua bisa
berdiri di depan mimbar karena tidak semua mempunyai kemampuan tersebut. Proses
dakwah dapat dilaksanakan melalui beberapa kegiatan dengan lisan atau tulisan. Di
zaman sekarang ini pergerakan dakwah sangat beragam, ada yang secara personal, ada
juga yang bergerak secara berkelompok dengan sarana-sarana dan prasaran yang serta
media berbeda-beda. Menurut Aziz (2004:404) media merupakan sarana yang dapat
mempermudah tercapainya tujuan dakwah, contohnya media elektronik, televisi, dan juga
internet. Munculnya teknologi yang semakin maju, bermunculan juga media sosial dengan
tokoh para da’i yang memanfaatkan media sosial sebagai media dakwah. Kehadiran
media tersebut dapat dimanfaatkan di tengah masyarakat untuk menyampaikan pesan
dakwah khususnya melalui internet. Perkembangan internet menjadikan kegiatan yang
awalnya terasa sulit menjadi mudah. Melalui internet, internet sebagai jaringan
komunikasi yang bisa menghubungkan seluruh orang di dunia termasuk memudahkan Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 108 Journal of Da’wah and Communication Journal of Da’wah and Communication isi pesan dakwah dengan pembahasan tiga dosa besar pada konten dengan judul “3 Dosa
Besar” Cawisan Ba’da Shubuh, Masjid Sultan Mahmud Badaruddin pada ling
https://www.youtube.com/watch?v=qLHIBaIVSuQ yang diunggah pada 1 Mei 2021
dengan 2,8 juta kali tonton dan 23 ribu like. Data penelitian diambil dengan cara
mengidentifikasi isi pesan dakwah akun Youtube Ustadz Abdul Somad Official
terutama konten dengan pembahasan tiga dosa besar. Dalam penelitian ini, data disajikan
berdasarkan analisis menggunakan model analisis semiotik Roland Barthes. isi pesan dakwah dengan pembahasan tiga dosa besar pada konten dengan judul “3 Dosa
Besar” Cawisan Ba’da Shubuh, Masjid Sultan Mahmud Badaruddin pada ling
https://www.youtube.com/watch?v=qLHIBaIVSuQ yang diunggah pada 1 Mei 2021
dengan 2,8 juta kali tonton dan 23 ribu like. Data penelitian diambil dengan cara
mengidentifikasi isi pesan dakwah akun Youtube Ustadz Abdul Somad Official
terutama konten dengan pembahasan tiga dosa besar. Dalam penelitian ini, data disajikan
berdasarkan analisis menggunakan model analisis semiotik Roland Barthes. Di Youtube dapat ditemukan da’i sebagai content creator (komunikan) dengan
beragam materi dakwah. Salah satu content creator yang terdapat di Youtube adalah akun
Ustadz Abdul Somad Official. Akun tersebut mempublikasi konten-konten dakwah
dengan beragam tema pembahasan. Akun Ustadz Abdul Somad Official memiliki 3,05
juta subscriber dan 1,7 ribu video (12/10/2022). Dari 1,7 ribu postingan tentang dakwah
di akun Ustadz Abdul Somad Official, peneliti memilih konten “3 Dosa Besar” Cawisan
Ba’da Shubuh, Masjid Sultan Mahmud Badaruddin yang diunggah pada 1 Mei 2021
dengan 2,8 juta kali tonton dan 23 ribu like (12/10/2022). Dari jumlah jam tonton konten dakwah yang mencapai 2,8 juta menjadi
fenomena yang menarik untuk diteliti. Dalam konten video tersebut membahas tentang
tiga dosa besar. Pesan dakwah yang disampaikan berkaitan dengan dosa-dosa dengan
kategori dosa besar yang dilakukan oleh manusia. Pesan yang akan dileliti dalam Youtube
ini adalah pesan-pesan tentang dosa besar yang berbentuk quote, dalam semiotika quote
dibahas dalam tanda. Semiotika yang digunakan pada penelitian ini adalah semiotika
Roland Barthes. Barthes menganalisis makna dari tanda, yaitu konotasi, denotasi, dan
mitos. Masalah tentang quote pada konten pembahasan tiga dosa besar di akun Ustadz
Abdul Somad Official menarik untuk dikaji dalam bentuk artikel dengan judul Pesan
Dakwah dalam Media Sosial Youtube Ustadz Abdul Somad Official: Tiga Dosa Besar. Hal yang dijadikan permasalahan dalam penelitian ini adalah makna denotasi pesan
dakwah, makna konotasi pesan dakwah dan mitos pesan dakwah tentang tiga dosa besar
dalam akun Youtube Ustadz Abdul Somad Official. Vol. 3 No. 1 Juni 2023. Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 METODE PENELITIAN Penelitian ini merupakan penelitian kualitatif. Peneliti akan melihat bagaimana
isi pesan dakwah pada akun Youtube Ustadz Abdul Somad Official terutama konten
dengan pembahasan tiga dosa besar. Bagian yang dianalisis dalam penelitian ini adalah Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 109 Journal of Da’wah and Communication HASIL DAN PEMBAHASAN Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 Journal of Da’wah and Communication P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 110 Journal of Da’wah and Communication diantaranya karena faktor psikologis mad’u, situasi, kemampuan da’i, dan waktu
penyampaian (Basit, 2017: 140-141). diantaranya karena faktor psikologis mad’u, situasi, kemampuan da’i, dan waktu
penyampaian (Basit, 2017: 140-141). Dan agar pesan yang disampaikan mengena pada sasarannya, maka pesan harus
memenuhi syarat-syarat: 1) Pesan harus direncanakan secara baik-baik, serta sesuai
dengan kebutuhan kita. 2) Pesan tersebut dapat menggunakan bahasa yang dapat
dimengerti kedua belah pihak. 3) Pesan harus menarik minat dan kebutuhan pribadi
penerima serta menimbulkan kepuasan (Uchyana, 1992: 35). Dakwah Dakwah adalah massage, yaitu simbol-simbol. Dalam literatur berbahasa Arab,
pesan dakwah disebut maudu’ ad- da’wah. Istilah pesan dakwah dipandang lebih tepat
untuk menjelaskan, isi dakwah berupa kata, gambar, lukisan dan sebagainya yang
diharapkan dapat memberikan pemahaman bahkan perubahan sikap dan perilaku mitra
dakwah (Moh. Ali Aziz, 2004: 318). Dakwah merupakan upaya mewujudkan masyarakat
yang menjunjung tinggi kehidupan beragama dengan merealisasikan ajaran Islam secara
penuh dan menyeluruh (Ilahi, 210: 101). Dakwah merupakan kewajiban yang tidak bisa ditawar-tawar lagi. Kewajiban
dakwah merupakan suatu yang tidak mungkin dihindarkan dari kehidupannya, karena
melekat erat bersamaan dengan pengakuan diri sebagai penganut Islam (muslim) (Muria,
2000: 6). Dengan kata lain setiap muslim secara otomatis mengemban misi dakwah. Dengan demikian dakwah merupakan bagian yang sangat esensial dalam kehidupan
orang muslim, dimana esensinya berada pada ajakan dorongan (motivasi), rangsangan
serta bimbingan terhadap orang lain untuk menerima ajaran Islam dengan penuh
kesadaran. Dakwah berkaitan dengan penyampaian pesan. Pesan dakwah merupakan salah satu unsur dalam dakwah. Tanpa adanya pesan,
kegiatan dakwah tidak ada artinya. Ada tiga dimensi yang saling terkait dengan istilah
pesan dakwah. Pertama, pesan dakwah menggambarkan sejumlah kata-kata atau
imajinasi yang diekspresikan dalam bentuk kata-kata. Maksudnya, pesan dakwah
mengandung dua aspek penting yaitu isi pesan dan lambang. Isi pesan meliputi pikiran,
sedangkan lambang meliputi kata-kata atau bahasa. Kedua, makna dari pesan dakwah
sangat berkaitan dengan persepsi atau pemahaman dari penerima dakwah. Makna
merupakan proses yang diciptakan atas kerjasama antara komunikator (da’i) dan
komunikan (mad’u). Ketiga, mad’u sebagai penerima pesan dakwah. Setiap pesan
dakwah dapat dimaknai dan dipahami secara berbeda oleh mad’u yang berbeda. Dapat
dipahami bahwa proses penerimaan dakwah tidak dapat mencapai 100%. Banyak faktor
yang menyebabkan pesan dakwah tidak dapat dipahami sepenuhnya oleh mad’u. Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 111 Journal of Da’wah and Communication
diantaranya karena faktor psikologis mad’u, situasi, kemampuan da’i, dan waktu
penyampaian (Basit, 2017: 140-141). 3. Berbayar 4. Sistem offline
Youtube mempunyai fitur baru bagi para pengguna untuk menonton videonya yaitu
sistem offline. Sistem ini memudahkan para pengguna untuk memonton videonya pada
saat offline tetapi sebelumnya video tersebut harus didownload terlebih dahulu. 5. Tersedia editor sederhana
Pada menu awal mengunggah video, pengguna akan ditawarkan untuk mengedit
videonya terlebih dahulu. Menu yang ditawarkan adalah memotong video, memfilter
warna, atau menambah efek perpindahan video (Faiq dan Amir, 2016). Journal of Da’wah and Communication 3. Berbayar
Saat ini seperti yang sedang menjadi viral dimana-dimana, youtube memberikan
penawaran bagi siapapun yang mengunggah videonya ke youtube dan mendapatkan
minimal 1000 viewers atau penonton maka akan diberikan honorarium. 3. Berbayar
Saat ini seperti yang sedang menjadi viral dimana-dimana, youtube memberikan
penawaran bagi siapapun yang mengunggah videonya ke youtube dan mendapatkan
minimal 1000 viewers atau penonton maka akan diberikan honorarium. Youtube Youtube adalah video online dan yang utama dari kegunaan situs ini adalah
sebagai media untuk mencari, melihat, dan berbagi video yang asli dari segala penjuru
melalui suatu web (Budiargo, 2015, 47). Kehadiran Youtube membawa pengaruh besar
kepada masyarakat, khususnya masyarakat yang memiliki minat di bidang pembuatan
video mulai dari film pendek, dokumenter hingga video blog yang tidak memiliki lahan
atau tempat untuk mempublikasikan karyanya. Selain mudah untuk digunakan dengan
mendownload aplikasi kemudian membuat akun, Youtube juga tidak memerlukan biaya
tinggi. Dan dapat diakses dimanapun dengan gadge yang terhubung jaringan internet. Youtube dapat menginspirasi banyak orang di seluruh dunia dan dapat bertindak sebagai
platform distribusi bagi pembuat konten asli dan pengiklan, baik yang besar maupun
kecil. Selain itu, terdapat forum bagi pengguna Youtube untuk saling berhubungan dan
memberikan informasi (Ainul Haq, 2020). Youtube memiliki karakteristik yang membuat banyak dari sebagian pengguna
betah menggunakannya, yaitu: 1. Tidak ada batasan durasi untuk mengunggah video
Hal ini yang membedakan youtube dengan beberapa aplikasi lain yang mempunyai
batasan durasi minimal waktu semisal instagram, snapchat, dan sebagainya. Hal ini yang membedakan youtube dengan beberapa aplikasi lain yang mempunyai
batasan durasi minimal waktu semisal instagram, snapchat, dan sebagainya. 2. Sistem pengamanan yang mulai akurat
Youtube membatasi pengamanannya dengan tidak mengizinkan video yang
mengandung sara, illegal, dan akan memberikan pertanyaan konfirmasi sebelum
menggunggah video. Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 112 Journal of Da’wah and Communication Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 Journal of Da’wah and Communication Journal of Da’wah and Communication dia juga sering menghadiri sejumlah undangan, mulai dari khotbah Jumat, acara
peringatan hari besar Islam, diskusi keislaman, seminar,sampai acara tabligh akbar. dia juga sering menghadiri sejumlah undangan, mulai dari khotbah Jumat, acara
peringatan hari besar Islam, diskusi keislaman, seminar,sampai acara tabligh akbar. Belakangan, pada akhir 2011-an, dia aktif mengunggah video ceramahnya ke
Youtube, hingga menjadi ulama populer seperti saat ini. Semua yang ditampilkannya di
ruang daring adalah hasil pengambilan gambar dan video ceramahnya di ruang luring. Namun, yang menjadi perbedaan adalah media yang mengemasnya. Ceramahnya di ruang
luring hanya dapat dinikmati oleh audiensnya yang hadir secara langsung dalam acara
dakwah, sedangkan ceramahnya di ruang daring dapat menyebar secara luas damelintasi
batas-batas geografis, agama, budaya dan usia. Berkat keterlibatannya di ruang luring
maupun daring, ceramah dari Ustaz Abdul Somad begitu populer di tengah masyarakat. Selain itu, popularitasnya sebagai ulama juga diperolehnya berkat perpaduan antara
kultur tradisional dan selebriti, yang didapatkannya dari media (Rohman, 2022). Ustadz Abdul Somad Ustaz Prof. H. Abdul Somad Batubara, Lc., D.E.S.A., Ph.D., Datuk Seri Ulama
Setia Negara atau lebih dikenal dengan Ustaz Abdul Somad. Ustadz Abdul Somad
merupakan seorang pendakwah dan ulama Indonesia yang sering menjelaskan kajian
agama Islam, khususnya kajian terkait ilmu hadis dan ilmu fikih. Dia dikenal oleh
berbagai kalangan masyarakat karena dakwah yang disampaikannya lugas dan mudah
dicerna. Ustaz yang sering dipanggil dengan UAS ini merupakan keturunan suku Batak
dan Melayu. Ayahnya memiliki darah Batak, sedangkan ibunya berdarah Melayu. Ustaz
Abdul Somad lahir di kampung yang bernama Silo Lama, Asahan, Sumatra Utara pada
18 Mei 1977. Dia lahir di lingkungan yang agamis, yang membentuknya menjadi seorang
taat sejak masih belia. Sebagai seorang ulama yang memiliki latar belakang pendidikan Islam tradisional
dan akses ke sumber kitab-kitab klasik,Ustaz Abdul Somad mampu bertahan dan
beradaptasi dengan kehadiran media baru. Dia memperoleh popularitas sebagai ulama
berkat keterlibatannya dengan media baru. Seperti halnya ulama-ulama pada umumnya, Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 113 Semiotika Roland Barthes Aanalisis menggunakan semiotika perlu memperhatikan hal-hal mendasar
sebagai instrumen. Hal ini dijelaskan oleh Bronwen Martin dan Felizitas Ringham
(Basit, 2017). First, the meaning is not inherent in the object and the object does not have the
meaning by itself. Second, the semiotics views the text as a unit that delivers their
own requirements (autonomous). Therefore, the study of semiotics is started by
the studies of structure and the existing language in the text. Third, semiotics
shows that the structure of the story or narration is built based on the
comprehensive discourse, not only based on explicit knowledge. Fourth,
semiotics also shows a level of meaning or ideas. Penggunaan semiotika dalam instrumen analisis, terdapat empat hal yang harus
diperhatikan, yaitu: 1) Artinya tidak melekat pada objek dan objek tidak memiliki arti
dengan sendirinya. 2) Semiotika memandang teks sebagai unit yang memberikan
persyaratan mereka sendiri (otonom), oleh karena itu, studi semiotika dimulai oleh studi
struktur ahasa yang ada dalam teks. 3) Semiotika menunjukkan bahwa struktur cerita
atau narasi dibangun berdasarkan wacana komprehensif, bukan hanya pengetahuan
eksplisit. 4) Semiotika menunjukkan tingkat makna atau ide. Sobur (2002: 123) mengemukakan bahwa, semiotika menjadi pendekatan
penting dalam teori media. Semiotika karya Roland Barthes menyatakan bahwa semua Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 114 Journal of Da’wah and Communication Journal of Da’wah and Communication objek kultural dapat diolah secara tekstual. Semiotika adalah ilmu mengenai bentuk
(form). Studi semiotika mengkaji signifikasi yang terpisah dari isinya (content). Semiotika tidak hanya meneliti mengenai signifier dan signified, tetapi juga hubungan
yang mengikat mereka, tanda yang berhubungan secara keseluruhan. Teks yang
dimaksud Roland Barthes adalah dalam arti luas. Teks tidak hanya berkaitan dengan
aspek linguistik saja. semiotik dapat meneliti teks dimana tanda-tanda terkodifikasi
dalam sebuah sistem. Dengan demikian, semiotik dapat meneliti bermacam-macam teks
seperti berita, film, iklan, fashion, fiksi, puisi, dan drama. Roland merupakan tokoh besar dalam sejarah semiotika. Menurutnya semiotika
adalah ilmu yang digunakan untuk memaknai suatu tanda. Bahasa merupakan susunan
dari tanda yang memiliki pesan – pesan tertentu dari masyarakat. Selain bahasa tanda
dapat berupa lagu, not musik, benda, dialog, gambar, logo, gerak tubuh, dan mimik
wajah. (Sobur, 2017:15) Barthes mencetuskan model analisis tanda signifikasi dua tahap, yaitu denotasi
dan konotasi. Signifikasi tahap pertama merupakan hubungan antara petanda dan
penanda dalam bentuk nyata. Barthes menyebutnya sebagai denotasi, yaitu makna asli
atau makna umum yang mutlak dipahami oleh kebanyakan orang. Contohnya, kata kursi
memiliki makna denotasi yaitu, tempat duduk, benda padat dan bisa diduduki. Penjelasan tersebut merupakan makna umum yang tentunya semua orang akan paham
maksudnya. (Sobur, 2017:70) Penelitian ini memaparkan pesan dakwah kepada manusia tentang dosa besar
yang dilakukan dan perlu untuk dihindari. Dengan menggunkan konsep analisis
semiotika model Roland Barthes, bagaimana pesan disampaikan. Tabel 1 Tabel 1
Analisis Pesan Dakwah Model Roland Barthes
Isi Pesan Dakwah Tiga Dosa Besar
Menit ke- 13:15
Mempersekutukan Alloh: menjadikan ada yang lain disembah selain Alloh
Signifier (Penanda)
Signified (Petanda) Tabel 1
Analisis Pesan Dakwah Model Roland Barthes
Isi Pesan Dakwah Tiga Dosa Besar
Menit ke- 13:15
Mempersekutukan Alloh: menjadikan ada yang lain disembah selain Alloh
Signifier (Penanda)
Signified (Petanda) Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 115 Journal of Da’wah and Communication Journal of Da’wah and Communic
Pada pesan dakwah tentang tiga dosa
besar yang pertama, penanda terdiri dari
kata
mempersekutukan,
Alloh,
menjadikan yang lain, selain Alloh, dan
disembah. Journal of Da’wah and Communication Pada pesan dakwah tentang tiga dosa
besar yang pertama, penanda terdiri dari
kata
mempersekutukan,
Alloh,
menjadikan yang lain, selain Alloh, dan
disembah. Pada pesan dakwah tentang tiga dosa
besar yang pertama, penanda terdiri dari
kata
mempersekutukan,
Alloh, a. Kata mempersekutukan
merupakan kata berimbuhan dari
kata dasar sekutu. Mempersekutukan dapat
didefinisikan membuat jadi
bersekutu (tentang Allah) (KBBI
daring). b. Alloh (Allah dalam KBBI daring),
nama Tuhan dalam bahasa Arab;
pencipta
alam
semesta
Yang
Mahasempurna; Tuhan Yang Maha
Esa yang disembah oleh orang
yang beriman. c. “Menjadikan”,
diartikan
mengangkat (memilih) sebagai. Kata “yang “ menggambarkan kata
yang menyatakan bahwa bagian
kalimat
yang
berikutnya
menjelaskan kata yang di depan. Sedangkan
kata
“lain”
dapat
diartikan kecuali; tidak termasuk
(dalam hitungan, golongan, dan
sebagainya)(KBBI daring). Jadi,
frasa “menjadikan yang lain”
maksudnya adalah mengangkat
atau memilih, yang diangkat atau
dipilih adalah tidak termasuk
dalam golongan dalam hal ini tidak
memilih masuk dalam golongan
alloh. d. d. Frasa
selain
Alloh,
dapat
dijabarkan
dijabarkan
suatu
pengecualian, dari Tuhan Yang
Maha Sempurna, Tuhan Yang
Maha Esa. Hal ini disimpulkan dari
uraian kata “selain” yaitu
kecuali; lain daripada sedangkan
kata Alloh (Allah dalam KBBI
daring), nama Tuhan dalam bahasa
Arab; pencipta alam semesta Yang
Mahasempurna; Tuhan Yang Maha
Esa yang disembah oleh orang
yang beriman. e. Disembah, dari kata sembah yang
diartikan pernyataan hormat dan
khidmat (dinyatakan dengan cara Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 116 Journal of Da’wah and Communication Journal of Da’wah and Communication
menangkupkan kedua belah tangan
atau menyusun jari sepuluh, lalu
mengangkatnya hingga ke bawah
dagu atau dengan menyentuhkan
ibu jari ke hidung)1). kata atau
perkataan yang ditujukan kepada
orang yang dimuliakan 2) (KBBI
daring). Denotative Sign (Tanda Denotatif)
Mempersekutukan Alloh: menjadikan ada yang lain disembah selain Alloh
Conotative Signiier (Penanda
Konotatif)
Conotative Signified ( Petanda
Konotatif)
Mempersekutukan Alloh: menjadikan
ada yang lain disembah selain Alloh
Kalimat
pernyataan
ini
memberikan
gambaran
bahwa
manusia
sebagai
makhluk
ciptaan-Nya
maka
harus
menyembah
Alloh,
tetapi
pada
kenyatannya
ada
manusia
yang
mempersekutukan
Alloh,
manusia
menjadikan ada hal lain selain Alloh yang
disembah
Conotative Sign (Tanda Konotatif)
Kondisi yang menggambarkan bahwa ada kondisi manusia yang mempersekutukan
Alloh, mereka ada yang menjadikan hal lain selain Alloh untuk disembah
Mitos
Manusia adalah ciptaan Alloh, maka sudah seharusnya manusia menyembah Alloh. Ketika manusia menyembah selain Alloh akan mendapatkan dosa menangkupkan kedua belah tangan
atau menyusun jari sepuluh, lalu
mengangkatnya hingga ke bawah
dagu atau dengan menyentuhkan
ibu jari ke hidung)1). kata atau
perkataan yang ditujukan kepada
orang yang dimuliakan 2) (KBBI
daring). Denotative Sign (Tanda Denotatif)
Mempersekutukan Alloh: menjadikan ada yang lain disembah selain Alloh
Conotative Signiier (Penanda
Konotatif)
Conotative Signified ( Petanda
Konotatif)
Mempersekutukan Alloh: menjadikan
ada yang lain disembah selain Alloh
Kalimat
pernyataan
ini
memberikan
gambaran
bahwa
manusia
sebagai
makhluk
ciptaan-Nya
maka
harus
menyembah
Alloh,
tetapi
pada
kenyatannya
ada
manusia
yang
mempersekutukan
Alloh,
manusia
menjadikan ada hal lain selain Alloh yang
disembah
Conotative Sign (Tanda Konotatif)
Kondisi yang menggambarkan bahwa ada kondisi manusia yang mempersekutukan
Alloh, mereka ada yang menjadikan hal lain selain Alloh untuk disembah
Mitos
Manusia adalah ciptaan Alloh, maka sudah seharusnya manusia menyembah Alloh. Ketika manusia menyembah selain Alloh akan mendapatkan dosa Denotative Sign (Tanda Denotatif) Denotative Sign (Tanda Denotatif) Conotative Sign (Tanda Konotatif) Dari pesan pada menit ke-13:15 di atas, merupakan suatu pernyataan kondisi
penggambaran manusia yang tidak menyembah Alloh, manusia sebagai seorang hamba
melakukan tindakan menyembah selain Alloh. Manusia yang tidak memuliakan Alloh
tidak sesuai dengan hukum Alloh, sebagaimana firman Alloh SWT: Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 117 Journal of Da’wah and Communication َﯾَٰٓﺄَﯾﱡﮭَﺎ ٱﻟﻨﱠﺎسُ ٱﻋْﺒُﺪُوا۟ رَﺑﱠﻜُﻢُ ٱﻟﱠﺬِى ﺧَﻠَﻘَﻜُﻢْ وَ ٱﻟﱠﺬِﯾﻦَ ﻣِﻦ ﻗَﺒْﻠِﻜُﻢْ ﻟَﻌَﻠﱠﻜُﻢْ ﺗَﺘﱠﻘُﻮن
ۚ ْﺎ ﻟﱠﻜُﻢ ًﻟﱠﺬِيْ ﺟَﻌَﻞَ ﻟَﻜُﻢُ اﻻْ َرْ ضَ ﻓِﺮَ اﺷًﺎ وﱠ اﻟﺴﱠﻤَﺎۤءَ ﺑِﻨَﺎۤءً ۖوﱠ اَﻧْﺰَ لَ ﻣِﻦَ اﻟﺴﱠﻤَﺎۤءِ ﻣَﺎۤءً ﻓَﺎَﺧْﺮَ جَ ﺑِﮫٖ ﻣِﻦَ اﻟﺜﱠﻤَﺮٰ تِ رِ زْﻗ
َﻓَﻼَ ﺗَﺠْﻌَﻠُﻮْ ا ﻟِﻠﱣﮫِ اَﻧْﺪَادًا وﱠ اَﻧْﺘُﻢْ ﺗَﻌْﻠَﻤُﻮْ ن “Hai manusia, sembahlah Tuhanmu Yang telah menciptakanmu dan orang-orang
yang sebelummu, agar kamu bertakwa. (21) Dialah Yang menjadikan bumi sebagai
hamparan bagimu dan langit sebagai atap, dan Dia menurunkan air [hujan] dari
langit, lalu Dia menghasilkan dengan hujan itu segala buah-buahan sebagai rezki
untukmu; karena itu janganlah kamu mengadakan sekutu-sekutu bagi Allah [5]
padahal kamu mengetahui”. (22). (Q.S. Al-Baqarah [2]: 21-22). Ayat ini merupakan seruan kepada seluruh manusia yang telah dapat berpikir,
untuk menyembah Allah Subhanahu wa Ta’ala, yang telah menciptakan manusia. Allah
mengkhususkan nikmat-Nya yang dianugerahkan kepada manusia berupa penciptaan
mereka (ni’matul khalq). Menurut Ibnu ‘Abbas, ayat ini ditujukan kepada dua golongan
besar , yaitu golongan orang-orang kafir dan golongan orang-orang munafik. Namun
walaupun ayat ini ditujukan kepada golongan orang-orang kafir dan golongan orang-
orang munafik, tapi Allah juga bermaksud menyeru dengan seruan yang bersifat umum,
yaitu seruan kepada seluruh umat manusia untuk menyembah atau beribadah kepada
Allah (https://minanews.net/). Seruan dari Allah kepada seluruh umat manusia agar menyembah kepada-Nya
adalah karena memang Allah telah mencurahkan berbagai nikmat-Nya yang tak terhingga
kepada manusia. Allah menciptakan hamba-hamba-Nya dari yang sebelumnya tiada
menjadi ada, dan Allah pula yang menciptakan orang-orang yang terdahulu. Jadi, sangat
wajar jika Allah menyeru kepada manusia untuk menyembah-Nya, Dzat yang pantas
untuk disembah oleh manusia. Inilah tugas para Nabi dan Rasul menyeru daan mengajak manusia untuk
menyembah Allah, seperti juga firman-firman-Nya pada ayat: َو ِﻣَﺎٓ اَرْﺳَﻠْﻨَﺎ ﻣِﻦْ ﻗَﺒْﻠِﻚَ ﻣِﻦْ رﱠﺳُﻮْ لٍ اِﻻﱠ ﻧُﻮْﺣِﻲْٓ اِﻟَﯿْﮫِ اَﻧﱠﮫٗ ﻻَ ٓ اِﻟٰﮫَ اِﻻﱠ ٓ اَﻧَﺎ۠ ﻓَﺎﻋْﺒُﺪُوْ ن “Dan Kami tidak mengutus seorang rasulpun sebelum kamu, melainkan Kami
wahyukan kepadanya: “Bahwasanya tidak ada Tuhan [yang hak] melainkan Aku,
maka sembahlah olehmu sekalian akan Aku”. (Q.S. Al-Anbiya [21]: 25). Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 118 Journal of Da’wah and Communication Pada pesan dakwah tentang tiga dosa
besar yang kedua durhaka kepada
orangtua
a. Durhaka didefinisikan ingkar
terhadap perintah (Tuhan, orang
tua, dan sebagainya) (KBBI
daring). b. Kepada merupakan kata depan
untuk menandai tujuan orang
(KBBI daring). c. Orangtua yaitu, ayah ibu kandung
atau (orang tua) orang yang
dianggap tua (cerdik pandai, ahli,
dan sebagainya); orang-orang yang
dihormati (disegani) di kampung;
tetua (KBBI daring). Denotative Sign (Tanda Denotatif)
Durhaka kepada Orangtua
Conotative Signiier (Penanda
Konotatif)
Conotative Signified ( Petanda
Konotatif)
Durhaka kepada Orangtua
Kalimat pernyataan tersebut menyatakan
bahwa sebagai manusia, atau sebagai
seorang anak maka tidak diperkenankan
durhaka kepada orangtua, tetapi pada
kenyataannya masih ada anak durhaka
kepada orang tua
Conotative Sign (Tanda Konotatif)
Kondisi yang menyatakan ada anak durhaka kepada orangtua, mereka ingkar terhadap
orangtua atau kandung atau oatang yang dianggap tua
Mitos
Sebelum dilahirkan seorang anak ada dalan kandungan ibu selama kurang lebih
sembilan bulan, setelah lahir oleh ibu dan bapaknya diberi kasih sayang serta dipelihara
dengan baik, sudah seharusnya seorang anak patuh kepada orangtua, tetapi pada
kenyataannya masih ada anak durhaka kepada orangtua, dan ini merupakan dosa besar. Pada pesan dakwah tentang tiga dosa
besar yang kedua durhaka kepada
orangtua Pada pesan dakwah tentang tiga dosa
besar yang kedua durhaka kepada
orangtua a. Durhaka didefinisikan ingkar
terhadap perintah (Tuhan, orang
tua, dan sebagainya) (KBBI
daring). dan sebagainya); orang orang yang
dihormati (disegani) di kampung;
tetua (KBBI daring). Denotative Sign (Tanda Denotatif)
Durhaka kepada Orangtua
Conotative Signiier (Penanda
Konotatif)
Conotative Signified ( Petanda
Konotatif)
Durhaka kepada Orangtua
Kalimat pernyataan tersebut menyatakan
bahwa sebagai manusia, atau sebagai
seorang anak maka tidak diperkenankan
durhaka kepada orangtua, tetapi pada
kenyataannya masih ada anak durhaka
kepada orang tua
Conotative Sign (Tanda Konotatif)
Kondisi yang menyatakan ada anak durhaka kepada orangtua, mereka ingkar terhadap
orangtua atau kandung atau oatang yang dianggap tua
Mitos
Sebelum dilahirkan seorang anak ada dalan kandungan ibu selama kurang lebih
sembilan bulan, setelah lahir oleh ibu dan bapaknya diberi kasih sayang serta dipelihara
dengan baik, sudah seharusnya seorang anak patuh kepada orangtua, tetapi pada
kenyataannya masih ada anak durhaka kepada orangtua, dan ini merupakan dosa besar. Dari pesan menit ke-33:47, menggambarkan kondisi adanya anak durhaka kepada
orangtua. (https://dalamislam.com/), menjabarkan anak adalah suatu amanah atau
titipkan dari Allah SWT yang harus dijaga oleh setiap orang tua. Mereka bertanggung
jawab atas segala macam kebutuhan anak-anaknya , mulai dari pemberian sandang Vol. 3 No. 1 Juni 2023. P-ISSN. “Dan sesungguhnya Kami telah mengutus rasul pada tiap-tiap umat [untuk
menyerukan]: “Sembahlah Allah [saja], dan jauhilah Thaghut
itu”, maka di
antara umat itu ada orang-orang yang diberi petunjuk oleh Allah dan ada pula di
antaranya orang-orang yang telah pasti kesesatan baginya. Maka berjalanlah
kamu di muka bumi dan perhatikanlah bagaimana kesudahan orang-orang yang
mendustakan [rasul-rasul]”. (Q.S. An-Nahl [16]: 36). Pengertian thagut seperti pada ayat ini Pengertian thagut seperti pada ayat ini
artinya secara bahasa adalah ‘melampaui batas’. Thaghut secara istilah syar’i yaitu segala
sesuatu yang menyebabkan seorang hamba melebihi batasannya, baik itu sesuatu yang
diibadahi,
diikuti,
atau
dithaati. (Menurut
Ibnul
Qayyim
Al-Jauziyyah
(https://minanews.net/). Pembahasan tentang mempersekutukan Alloh sering disebut syirik, kata syirik
terulang sebanyak 162 kali dalam al-Qur’an. Kemusyrikan adalah suatu perkara yang
tidak dianggap mudah karenanya berkaitan dengan akidah seseorang. Di dalam al-Qur’an
Allah SWT menyatakan semua dosa dapat diampunkan kecuali syirik kepadanya. Pembalasan atau azab bagi mereka yang melakukan syirik kepada Allah SWT penyiksaan
yang amat pedih dan dimasukkan ke dalam neraka Jahannam. Kemusyrikan sering
dikaitkan dengan iktiqad dan perbuatan seseorang dalam melakukan perkara yang
dilarangoleh Allah SWT, perintah dan larangan menjahui syirik jelas dinyatakan di dalam
al-Qur’an. Oleh karena itu sikap berhati-hati agar tidak terpengaruh dengan perkara-
perkara yang boleh membawa kepada kemusyrikan terhadap Allah SWT adalah perlu dan
wajib dilaksanakan demi kemurnian akidah (Mohammad, 2013). Tabel 2 Tabel 2
Analisis Pesan Dakwah Model Roland Barthes
Isi Pesan Dakwah Tiga Dosa Besar
Menit ke- 33:47
Durhaka kepada Orangtua
Signifier (Penanda)
Signified (Petanda) Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 119 Journal of Da’wah and Communication Journal of Da’wah and Communication pangan, kasih sayang dan pendidikan agar kelak si buah hati bisa tumbuh menjadi
generasi yang berkualitas dan berakhlakul karimah. Begitupun sebaliknya, anak juga
diwajibkan untuk menghormati kedua orang tuanya. Sebab perjuangan ibu takala ia
mengandung selama 9 bulan tentu sangatlah berat. Kemudian, ibu bertaruh nyawa untuk
melahirkan, menyusui dan merawat anaknya hingga tumbuh besar. Sedangkan peranan
ayah adalah mencari nafkah demi memenuhi segala kebutuhan keluarga. Sungguh, jasa
kedua orang tua itu tiada bandingnya. Allah SWT berfirman: ﻠٰﺜُﻮْ نَ ﺷَﮭْﺮًا َوَ وَﺻﱠﯿْﻨَﺎ اﻻْ ِﻧْﺴَﺎنَ ﺑِﻮَ اﻟِﺪَﯾْﮫِ اِﺣْﺴَﺎﻧًﺎ ۗﺣَﻤَﻠَﺘْﮫُ اُﻣﱡﮫٗ ﻛُﺮْ ھًﺎ وﱠ وَﺿَﻌَﺘْﮫُ ﻛُﺮْ ھًﺎ ۗوَﺣَﻤْﻠُﮫٗ وَﻓِﺼٰﻠُﮫٗ ﺛ
“Kami perintahkan kepada manusia supaya berbuat baik kepada dua orang ibu
bapaknya, ibunya mengandungnya dengan susah payah, dan melahirkannya
dengan susah payah (pula). Mengandungnya sampai menyapihnya adalah tiga
puluh bulan. (al-Ahqâf:15).” ﻠٰﺜُﻮْ نَ ﺷَﮭْﺮًا َوَ وَﺻﱠﯿْﻨَﺎ اﻻْ ِﻧْﺴَﺎنَ ﺑِﻮَ اﻟِﺪَﯾْﮫِ اِﺣْﺴَﺎﻧًﺎ ۗﺣَﻤَﻠَﺘْﮫُ اُﻣﱡﮫٗ ﻛُﺮْ ھًﺎ وﱠ وَﺿَﻌَﺘْﮫُ ﻛُﺮْ ھًﺎ ۗوَﺣَﻤْﻠُﮫٗ وَﻓِﺼٰﻠُﮫٗ ﺛ
“Kami perintahkan kepada manusia supaya berbuat baik kepada dua orang ibu
bapaknya, ibunya mengandungnya dengan susah payah, dan melahirkannya
dengan susah payah (pula). Mengandungnya sampai menyapihnya adalah tiga
puluh bulan. (al-Ahqâf:15).” “Kami perintahkan kepada manusia supaya berbuat baik kepada dua orang ibu
bapaknya, ibunya mengandungnya dengan susah payah, dan melahirkannya
dengan susah payah (pula). Mengandungnya sampai menyapihnya adalah tiga
puluh bulan. (al-Ahqâf:15).” Ayat diatas menegaskan bahwa islam mengajarkan seorang anak untuk berbuat
baik kepada ibu dan bapaknya. Namun sayang, dewasa kini perlakuan anak kepada orang
tua bisa dikatakan jauh dari kata sopan. Bahkan tak jarang mereka berlaku durhaka tanpa
mengindahkan perintah agama. Ayat diatas menegaskan bahwa islam mengajarkan seorang anak untuk berbuat
baik kepada ibu dan bapaknya. Namun sayang, dewasa kini perlakuan anak kepada orang
tua bisa dikatakan jauh dari kata sopan. Bahkan tak jarang mereka berlaku durhaka tanpa
mengindahkan perintah agama. Hal tersebut menunjukkan bahwa anak memiliki kewajiban penuh terhadap orang
tuanya baik masih ada di dunia maupun ketika orang tua telah wafat. Baik orang tua
sepaham maupun tidak sepaham atau bahkan kafir. Pemenuhan kewajiban-kewajiban
anak terhadap orang tuanya mulai dari taat kepada orang tua, memelihara dan
memberikan nafkah, berbuat baik, berkata lemah lembut dan tidak menyinggung, tidak
sombong dan mendoakan mereka. Terdapat pula larangan bagi anak, antara lain durhaka
kepada orang tua seperti tidak mentaati perintah orang tua, tidak memberikan nafkah aya
menyia-nyiakan mereka, berkata kasar dan menyinggung perasaan orang tua, melupakan
merekan dengan tidak mendoakan. Journal of Da’wah and Communication 2775-5207 E-ISSN: 2808 - 8344 120 Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 Journal of Da’wah and Communication Selain itu, anak memiliki hak untuk memilih dalam
menjalani kehidupannya sendiri, orang tua yang memberikan pengarahan dan ajaran-
ajaran sesuai agama Islam. Namun, orang tua juga hendaknya memperhatikan keinginan
anak sehingga antara anak dan orang tua dapat terjalin hubungan yang baik. Kedekatan
yang terbentuk dengan baik antara orang tua dan anak ini cenderung menetap seumur
hidup (Aulia & Himawanti, 2020). Agar anak dapat menjalankan kewajibannya dan Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 121 Journal of Da’wah and Communication Journal of Da’wah and Communication Journal of Da’wah and Communication Conotative Sign (Tanda Konotatif)
Kondisi yang
menggambarkan seseorang yang memberikan suatu keterangan atas
peristiwa, kejadian atau hal secara palsu, tidak benar
Mitos 122 Journal of Da’wah and Communication
Saksi merupakan seseorang yang mengetahui, melihat sesuatu hal secara langsung,
maka seseorang tersebut yang mengetahui kebenaran akan suatu hal tertentu tentang
hal yang benar dan tentunya harus memberikan kebenaran tersebut kepada orang lain,
tetapi kenyataannya ada seseorang yang memberikan keterangan akan suatu hal
secara tidak benar atau saksi palsu Journal of Da’wah and Communication menjauhi larangan-larangan serta dapat memutuskan pilihan yang tepat harus ada
dukungan dari orang tua melalui pendidikan yang mumpuni sejak kecil (Sholihah, 2012). Tabel 2 menjauhi larangan-larangan serta dapat memutuskan pilihan yang tepat harus ada
dukungan dari orang tua melalui pendidikan yang mumpuni sejak kecil (Sholihah, 2012). Tabel 2 Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344
e-ISSN: 2808 - 8344
menjauhi larangan-larangan serta dapat memutuskan pilihan yang tepat harus ada
dukungan dari orang tua melalui pendidikan yang mumpuni sejak kecil (Sholihah, 2012). Tabel 2
Analisis Pesan Dakwah Model Roland Barthes
Isi Pesan Dakwah Tiga Dosa Besar
Menit ke- 48: 30
Saksi Palsu
Signifier (Penanda)
Signified (Petanda)
Pada pesan dakwah tentang tiga
dosa besar yang ketiga saksi palsu
a. Saksi adalah 1) orang yang melihat atau
mengetahui
sendiri
suatu
peristiwa
(kejadian), 2) orang yang dimintai hadir
pada suatu peristiwa yang dianggap
mengetahui kejadian tersebut agar pada
suatu ketika, apabila diperlukan, dapat
memberikan
keterangan
yang
membenarkan
bahwa
peristiwa
itu
sungguh-sungguh terjadi, 3) orang yang
dapat
memberikan
keterangan
guna
kepentingan penyidikan penuntutan dan
peradilan tentang suatu perkara pidana
yang
didengarnya,
dilihatnya,
atau
dialaminya sendiri (KBBI daring). b. Palsu merupakan 1) curang; tidak jujur
(tentang permainan dan sebagainya), 2)
tidak tulen; tidak sah; lancung (tentang
ijazah, surat keterangan, uang, dan
sebagainya) (KBBI daring). Denotative Sign (Tanda Denotatif)
Saksi Palsu
Conotative Signiier (Penanda
Konotatif)
Conotative Signified ( Petanda Konotatif)
Saksi Palsu
Kalimat pernyataan tersebut menggambarkan
suatu kondisi seseorang sebagai saksi palsu,
menyampaikan sesuatu hal secara tidak jujur. Conotative Sign (Tanda Konotatif)
Kondisi yang
menggambarkan seseorang yang memberikan suatu keterangan atas
peristiwa, kejadian atau hal secara palsu, tidak benar
Mitos Analisis Pesan Dakwah Model Roland Barthes b. Palsu merupakan 1) curang; tidak jujur
(tentang permainan dan sebagainya), 2)
tidak tulen; tidak sah; lancung (tentang
ijazah, surat keterangan, uang, dan
sebagainya) (KBBI daring). Denotative Sign (Tanda Denotatif) Denotative Sign (Tanda Denotatif) Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344
ISSN 2808
8344
Saksi Palsu
Conotative Signiier (Penanda
Konotatif)
Conotative Signified ( Petanda Konotatif)
Saksi Palsu
Kalimat pernyataan tersebut menggambarkan
suatu kondisi seseorang sebagai saksi palsu,
menyampaikan sesuatu hal secara tidak jujur. Journal of Da’wah and Communication Journal of Da’wah and Communication Journal of Da’wah and Communication Journal of Da’wah and Communication Saksi merupakan seseorang yang mengetahui, melihat sesuatu hal secara langsung,
maka seseorang tersebut yang mengetahui kebenaran akan suatu hal tertentu tentang
hal yang benar dan tentunya harus memberikan kebenaran tersebut kepada orang lain,
tetapi kenyataannya ada seseorang yang memberikan keterangan akan suatu hal
secara tidak benar atau saksi palsu Dari pesan menit ke-48: 47 , menggambarkan kondisi adanya seseorang yang
menjadi saksi palsu. (https://muslim.or.id/), seseorang yang memberikan persaksian di
pengadilan, haruslah bersaksi dengan jujur dan tidak memberikan kesaksian bohong. “Memberikan persaksian” yang dimaksud di sini tidak hanya terbatas dengan menjadi
“saksi” saja di muka pengadilan. Akan tetapi, termasuk juga advokat (pengacara) atau
pihak-pihak yang juga ikut berbicara di depan persidangan untuk didengar keterangannya
oleh hakim pengadilan. Ketika saksi atau pengacara itu memberikan kesaksian dengan
memutar fakta dan bersilat lidah, sehingga yang benar tampak salah dan yang salah
tampak menjadi benar, keduanya terancam dengan dalil-dalil di bawah ini. َۚﻮْ ا ﻛُﻮْﻧُﻮْ ا ﻗَﻮﱠ اﻣِﯿْﻦَ ﺑِﺎﻟْﻘِﺴْﻂِ ﺷُﮭَﺪَاۤءَ ﻟِﻠﱣﮫِ وَﻟَﻮْ ﻋَﻠٰٓﻰ اَﻧْﻔُﺴِﻜُﻢْ اَوِ اﻟْﻮَ اﻟِﺪَﯾْﻦِ وَ اﻻْ َﻗْﺮَﺑِﯿْﻦ ُﯾﱡﮭَﺎ اﻟﱠﺬِﯾْﻦَ اٰﻣَﻨ
ﺗُﻌْﺮِﺿُﻮْ ا ْاِنْ ﯾﱠﻜُﻦْ ﻏَﻨِﯿﺎ اَوْ ﻓَﻘِﯿْﺮًا ﻓَﺎﻟﻠﱣﮫُ اَوْﻟٰﻰ ﺑِﮭِﻤَﺎۗ ﻓَﻼَ ﺗَﺘﱠﺒِﻌُﻮا اﻟْﮭَﻮٰٓ ى اَنْ ﺗَﻌْﺪِﻟُﻮْ اۚ وَ اِنْ ﺗَﻠْﻮٗٓ ا اَو
ﻓَﺎِنﱠ اﻟﻠﱣﮫَ ﻛَﺎنَ ﺑِﻤَﺎ ﺗَﻌْﻤَﻠُﻮْ نَ ﺧَﺒِﯿْﺮً ا “Wahai orang-orang yang beriman, jadilah kamu orang yang benar-benar
menjadi penegak keadilan, menjadi saksi karena Allah meskipun merugikan dirimu
sendiri, atau ibu bapak, dan kaum kerabatmu. Jika dia kaya ataupun miskin, maka
Allah lebih tahu kemaslahatannya. Janganlah kamu mengikuti hawa nafsu karena
ingin menyimpang dari kebenaran. Jika kamu memutar-balikkan (kata-kata) atau
enggan menjadi saksi, maka sesungguhnya Allah adalah Maha mengetahui segala
apa yang kamu kerjakan.” (QS. An-Nisa’ [4]: 135). Dalam ayat di atas, Allah Ta’ala memerintahkan kita agar menjadi saksi karena
Allah, meskipun persaksian kita merugikan diri kita atau kerabat kita sendiri karena
memang bersalah dalam kasus yang disidangkan. Hubungan kekerabatan tidaklah
menyebabkan kita bersaksi palsu atau bohong demi menyelamatkan kerabat kita dari
hukuman atas kesalahannya. Disisi lain, secara spiritual ketika seseorang melakukan
kejujuran dan kebaikan maka hatinya akan menjadi tenang dan damai (Himawanti, 2021). Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 123 DAFTAR PUSTAKA Aulia, H., & Himawanti, I. (2020). Tahapan Pendidikan Seks dalam Kajian Psikologi dan
Al-Qur’an. Al Amin: Jurnal Kajian Ilmu dan Budaya Islam, 3(02), 195-212. Aulia, H., & Himawanti, I. (2020). Tahapan Pendidikan Seks dalam Kajian Psikologi dan
Al Q
’
Al A i
J
l K ji
Il
d
B d
I l
3(02) 195 212 Al-Qur’an. Al Amin: Jurnal Kajian Ilmu dan Budaya Islam, 3(02), 195-212. Aziz, M.A. 2004. Ilmu Dakwah. Jakarta: Kencana. ________. 2004. Ilmu Dakwah. Jakarta: Prenadamedia Group. Basit, Abdul. 2017. Filsafat Dakwah. Depok: PT. Raja Grafindo Persada. _________. 2017. Health Communication in the Quran: Charles Saunders Pierce’s
Semiotic Analysis Malaysian Journal of Communication Vol 33 No 4 ________. 2004. Ilmu Dakwah. Jakarta: Prenadamedia Group. Basit, Abdul. 2017. Filsafat Dakwah. Depok: PT. Raja Grafindo Persada. . 2017. Health Communication in the Quran: Charles Sau _________. 2017. Health Communication in the Quran: Charles Saunders Pierce’s
Semiotic Analysis. Malaysian Journal of Communication. Vol.33 No.4. Semiotic Analysis. Malaysian Journal of Communication. Vol.33 No.4. Budiargo, Dian. 2015. Berkomunikasi Ala Net-Generation. Jakarta: PT. Elex Media Semiotic Analysis. Malaysian Journal of Communication. Vol.33 No.4. Budiargo, Dian. 2015. Berkomunikasi Ala Net-Generation. Jakarta: PT. Elex Media Budiargo, Dian. 2015. Berkomunikasi Ala Net-Generation. Jakarta: PT. Elex Media
Komputindo. Komputindo. Departemen Agama RI. 2004. Al-Qurán dan Terjemahnya. Jakarta: CV Naladan. Faiqah. F, Nadjib. M, Amir. A.S. YouTube Sebagai Sarana Komunikasi Bagi Komunitas p
Departemen Agama RI. 2004. Al-Qurán dan Terjemahnya. Jakarta: CV Naladan. Faiqah. F, Nadjib. M, Amir. A.S. YouTube Sebagai Sarana Komunikasi Bagi Komunita p
Departemen Agama RI. 2004. Al-Qurán dan Terjemahnya. Jakarta: CV Naladan. F i
h F N djib M A i
A S Y
T b S b
i S
K
ik
i B
i K
i Faiqah. F, Nadjib. M, Amir. A.S. YouTube Sebagai Sarana Komunikasi Bagi Komunitas
Makassar. Jurnal Komunikasi KAREBA. Vol. 5 No.2 Juli – Desember, 2016. Guntur Cahyono dan Nibros Hassani, Youtube: Seni Komunikasi Dakwah dan Media Makassar. Jurnal Komunikasi KAREBA. Vol. 5 No.2 Juli – Desember, 2016. Guntur Cahyono dan Nibros Hassani, Youtube: Seni Komunikasi Dakwah dan Media Pembelajaran, AL-HIKMAH: Jurnal Dakwah, Volume 13, Nomor 1, Tahun 2019
Himawanti, I. (2021). Analysis of The Effect of Faith on Subjective Well-Being. IQTIDA: Journal of Da'wah and Communication, 1(02), 169-180. https://kbbi.kemdikbud.go.id/entri/ diunduh pada 17 Oktober 2022. https://minanews.net/ diunduh pada 17 Oktober 2022. https://minanews.net/kewajiban-menyembah-allah-dan-larangan-syirik-kajian-al-baqarah- 21-22
https://muslim.or.id/ diunduh pada 17 Oktober 2022. https://muslim.or.id/ diunduh pada 17 Oktober 2022. KESIMPULAN Isi pesan dakwah pada akun Youtube Ustadz Abdul Somad Official terutama
konten dengan pembahasan tiga dosa besar, dapat digris bawahi bahwa perbuatan yang
dapat dikategorika sebagai dosa besar yaitu, mempersekutukan Alloh, durhaka kepada
orangtua dan saksi palsu. Analisis berdasarkan analisis Roland Barthes dihasilkan bahwa
mempersekutukan Alloh, ini merupakan kalimat pernyataan memberikan gambaran
bahwa manusia sebagai makhluk ciptaan-Nya maka harus menyembah Alloh, tetapi pada
kenyatannya ada manusia yang mempersekutukan Alloh, manusia menjadikan ada hal
lain selain Alloh yang disembah. Sedangkan durhaka kepada orangtua, adalah Kalimat
pernyataan tersebut menyatakan bahwa sebagai manusia, atau sebagai seorang anak maka
tidak diperkenankan durhaka kepada orangtua, tetapi pada kenyataannya masih ada anak
durhaka kepada orang tua. Saksi palsu, Kalimat pernyataan tersebut menggambarkan
suatu kondisi seseorang sebagai saksi palsu, menyampaikan sesuatu hal secara tidak jujur. Secara mitos: 1) Manusia adalah ciptaan Alloh, maka sudah seharusnya manusia
menyembah Alloh. Ketika manusia menyembah selain Alloh akan mendapatkan dosa. 2)
Sebelum dilahirkan seorang anak ada dalan kandungan ibu selama kurang lebih sembilan
bulan, setelah lahir oleh ibu dan bapaknya diberi kasih sayang serta dipelihara dengan
baik, sudah seharusnya seorang anak patuh kepada orangtua, tetapi pada kenyataannya
masih ada anak durhaka kepada orangtua, dan ini merupakan dosa besar. 3) Saksi
merupakan seseorang yang mengetahui, melihat sesuatu hal secara langsung, maka
seseorang tersebut yang mengetahui kebenaran akan suatu hal tertentu tentang hal yang
benar dan tentunya harus memberikan kebenaran tersebut kepada orang lain, tetapi
kenyataannya ada seseorang yang memberikan keterangan akan suatu hal secara tidak
benar atau saksi palsu. Penelitian ini masih banyak kekurangannya, meskipun demikian diharapkan
penelitian ini dapat bermanfaat bagi penelitian selanjutnya terutama berkaitan dengan
pembahasan pesan dakwah dengan analisis Roland Barthes. Untuk akun Youtube Ustadz
Abdul Somad Official sudah memberikan pesan dakwah yang bermanfaat bagi
pendengan, untuk konten dakwah berikutnya bisa memberikan isi pesan dakwah yang
lebih memberikan penambahan pengetahuan bagi pendengar. Saran pembaca, supaya Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 124 Journal of Da’wah and Communication Journal of Da’wah and Communication menghindari tiga dosa besar yaitu, mempersekutukan Alloh, durhaka kepada orangtua, dan saksi palsu. DAFTAR PUSTAKA https://www.gramedia.com/best-seller/biografi-ustaz-abdul-somad/ diunduh pada 13 Oktober 2022. Idemuslim.com. 2022,
Inilah 10 Ustadz Indonesia Dengan Subscriber Youtube
Terbanyak!dalam
https://idemuslim.com/inilah-10-ustadz-indonesia-dengan-subscriber- Idemuslim.com. 2022,
Inilah 10 Ustadz Indonesia Dengan Subscriber Youtube
Terbanyak!dalam
https://idemuslim.com/inilah-10-ustadz-indonesia-dengan-subscriber- youtube-terbanyak/ diunduh pada 10 Oktober 2022. Ilahi, Wahyu. 2010. Komunikasi Dakwah. Bandung: PT. Remaja Rosdakarya. Mohammad, Khairul Hadi. 2013. Makna Syirik dalam Al Quran (Kajian Tafsir Tematik y
Q
(
j
dan Kaitannya denagan Fenomena Kehidupan Sekarang). Skripsi. UIN Sultan Syarif Kasim
Riau Fakultas Ushuluddin Riau. Fakultas Ushuluddin. Muria, Siti. 2000. Metode Dakwah Kontemporer. Yogyakarta: Mitra Pustaka. Onong Uchyana, 1992. Dinamika Komunikasi. Bandung: Remaja Rosdakarya. Rohman, Fandy Aprianto. Biografi Ustaz Abdul Somad dan Pola Dakwahnya. Saputra, Wahidin.2012. Pengantar Imu Dakwah. Cet II. Jakarta: Rajawali Pers. Sholihah, Rahmah Farihatus. 2012. Konsep Pendidikan Akhlak Anak terhadap Orangtua Persepektif Al Quran. Thesis. Universitas Muhammadiyah Malang. Sobur, A. (2017). Semiotika komunikasi. Bandung : Remaja Kar
______. 2002. Analisis Teks Media: Suatu Pengantar untuk A Persepektif Al Quran. Thesis. Universitas Muhammadiyah Malang. Sobur, A. (2017). Semiotika komunikasi. Bandung : Remaja Karya
______. 2002. Analisis Teks Media: Suatu Pengantar untuk Analisis Wacana. Analisis Sobur, A. (2017). Semiotika komunikasi. Bandung : Remaja Karya
______. 2002. Analisis Teks Media: Suatu Pengantar untuk Analisis Wacana. Analisis
Semiotik dan Analisis Framing Bandung: PT Remaja Rosdakarya ______. 2002. Analisis Teks Media: Suatu Pengantar untuk Analisis Wacana. Analis Semiotik, dan Analisis Framing. Bandung: PT Remaja Rosdakarya. Vol. 3 No. 1 Juni 2023. P-ISSN. 2775-5207 E-ISSN: 2808 - 8344 125
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Conceptual model for assessing the effectiveness of inter-organizational relations in the tourism network
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* Corresponding author: runatasha21@yandex.ru Conceptual model for assessing the
effectiveness of inter-organizational relations in
the tourism network
Natalia Rubtsova1,*, and Konstantin Solodukhin2
1Baikal State University, Lenin Str., 11, 664003 Irkutsk, Russia
2Vladivostok State University of Economics and Service, Gogol Str., 41, 690014 Vladivostok, Russia Abstract. Tourism industry as an object of management can be represented
as a set of formal relations (interactions) between different organizations. Domestic and foreign researchers are unanimous in the opinion that the
network interaction of companies is an important condition for the tourism
industry effective functioning and for the sustainable development of a
tourist destination as well. In terms of the named vector of scientific
research, the efficiency of the inter-organizational relations (IOR) deserves
special attention. At the initial stage of discussion are both the content of the
IOR efficiency category and its assessment. The article presents a conceptual
model for assessing the IOR efficiency in tourism in the context of a
combination of three approaches: goal-oriented, functional and systemic. The authors identified the functions, goals, and indicators of the efficiency
of inter-organizational relations in the tourism network. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons
Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 208, 05002 (2020)
IFT 2020 E3S Web of Conferences 208, 05002 (2020)
IFT 2020 https://doi.org/10.1051/e3sconf/202020805002 1 Introduction Inter-organizational relations and network interactions in various kinds and profiles of
businesses have been studied by a number of foreign and domestic researchers [1-10]. In the
last decade, the tourism industry has also been in the view of studying the issues of network
interaction [11, 12, 13]. In the literature review on inter-organizational cooperation in the
tourism industry presented by Pechlaner H., Volgger M. [13], the study of integration in
tourism is stated to be currently presented rather fragmentarily. The authors give accent to a
set of ideas which have gained certain development, such as: achievement of common goals,
cooperation importance, successful tourism networks, communications, and joint
innovations. The authors conclude that one of the relevant areas in the aspect of the network
interaction theory, which has been attracting a growing academic interest in recent years, is
network management in the tourism industry. The tourism industry as an object of management can be understood as a set of formal
relations (interactions) between various organizations, which include accommodation
facilities (inns, hotels, hostels, etc.), tour operators and travel agencies, transport companies,
sightseeing tour agencies, public organizations, migration and customs services, as well as E3S Web of Conferences 208, 05002 (2020)
IFT 2020 https://doi.org/10.1051/e3sconf/202020805002 other entities directly and indirectly related to tourism. Inter-organizational relations
(generally abbreviated as IOR in foreign sources) represented by such forms of interaction
as association, alliance and cooperation [14] provide their participants with certain benefits:
sharing resources, staff training, knowledge exchange, cost reduction in marketing and PR
activities, and performance of management functions. In addition, effective IORs in the
tourism network ensure the achievement of common strategic goals and quantitative and/or
qualitative synergistic effects of certain territories where the tourism network operates [3]:
local population employment, sustainable competitive advantage, development of the
territory (tourist destination), and environmental problem solving. other entities directly and indirectly related to tourism. Inter-organizational relations
(generally abbreviated as IOR in foreign sources) represented by such forms of interaction
as association, alliance and cooperation [14] provide their participants with certain benefits:
sharing resources, staff training, knowledge exchange, cost reduction in marketing and PR
activities, and performance of management functions. In addition, effective IORs in the
tourism network ensure the achievement of common strategic goals and quantitative and/or
qualitative synergistic effects of certain territories where the tourism network operates [3]:
local population employment, sustainable competitive advantage, development of the
territory (tourist destination), and environmental problem solving. 1 Introduction According to the unanimous opinion of domestic and foreign researchers, the network
interaction is one of the important conditions for the tourism network effective functioning. Its study in the managerial aspect is becoming an urgent research area. In terms of the named
vector of scientific research, the assessment of the IOR efficiency deserves special attention. The subject of this article is the issue of assessing the IOR efficiency in the tourism network. 2 Materials and Methods Yet many works have been devoted to the study of the IOR efficiency, the very concept of it
still does not have a generally accepted definition. Indicating this notion, researchers use a
whole range of related terms: “effectiveness”, “efficiency”, and “productivity”. The terms
are applied to inter-firm relations without preliminary discussion of when this usage is
excusable. Currently, there are several points of view on the IOR efficiency interpretation. In
particular, Russian authors [15] define it as “satisfaction of the interacting parties with the
perceived values of the result indicators”. Foreign researchers use the term “relationship
quality” [16, 17, 18, 19]. In literature, similar definitions can be named differently:
“strength,” “success,” “value,” and “effectiveness” of relationships. The authors of this
article adhere to the point of view of a goal-oriented approach to the IOR efficiency concept. According to this approach, the IOR efficiency is the degree to which the development goals
of these relations characterizing the contribution to the creation of sustainable competitive
advantages of interacting organizations are achieved. g
g
g
To date, a significant number of models and methods have been proposed to assess the
IOR efficiency. They use both separate indicators and multi-parameter scales and systems
corresponding to a particular configuration or a concept of relationships. The most frequent
measured characteristics are trust and satisfaction [17, p. 355], loyalty [18], intention to
continue the relationships [19]. Summarizing the development results of domestic and
foreign researchers, the IOR efficiency can be stated as a complex category with a significant
number of various characteristics. That statement requires a systematic approach to the IOR
efficiency assessment. A. Walter et al. [20, 21] proposed the functional approach, which also
deserves attention. The approach interprets the IOR efficiency through a number of direct
and indirect functions reflecting various value aspects of relationships. In our opinion, the
functional approach provides a wide range of possibilities for its use. For instance, it is
possible to apply the model of relationship functions to detail the contribution of the
relationships to the financial results of activities of companies belonging to the alliance; it is
also possible to justify the goals of inter-organizational relations development. 3 Results Summarizing the available developments on the issue studied and taking into account the
ideas of the goal-oriented, functional and systemic approaches, the authors have formed a list
of indicators for assessing the performance of certain functions and achieving the goals of 2 E3S Web of Conferences 208, 05002 (2020)
IFT 2020 https://doi.org/10.1051/e3sconf/202020805002 the tourism network relations (Table 1). The presented functions of relations seem to allow
deepening and clarifying the idea of “the efficiency of inter-organizational tourist network
relations”. The named idea is a “complex (integral) characteristic that determines the mutual
purposefulness of parties involved to achieve tourist network development goals of relations
and their contribution to the creation of sustainable competitive advantages”. the tourism network relations (Table 1). The presented functions of relations seem to allow
deepening and clarifying the idea of “the efficiency of inter-organizational tourist network
relations”. The named idea is a “complex (integral) characteristic that determines the mutual
purposefulness of parties involved to achieve tourist network development goals of relations
and their contribution to the creation of sustainable competitive advantages”. Table 1. Relationship functions and examples of performance indicators of inter-organizational
interaction in the tourist network
Function
relations
Purpose
development
relations
Indicators
The nature of
the purpose
and indicator
Profit-
making
Profit
maximization
Net profit
Profitability (return on invested and tied
capital)
Monetary
Market
development
Expanding
market share
Marketing costs
The expansion of the customer base
Monetary
Personnel
development
Reducing the
cost of personnel
management
Search, training and personnel
adaptation costs
Monetary
Development
management
Reduced
management
costs
The cost of performing management
functions (access to information about
customers, competitors, new products,
new needs)
Monetary
Development
innovation
Creation of
innovations
Joint development of tourism products
Reduced development time and launch
of new tourism products
Non-monetary
Quality of
relations
Increased
commitment and
trust in partners
Reliability of partners
Stable relationship
Long-term relationship
Equality of partners
Fairness of relationship
Personalization (flexibility) of relations
Non-monetary
Note: developed by the authors Table 1. Relationship functions and examples of performance indicators of inter-organizational
interaction in the tourist network Note: developed by the authors In the offered list (table 1), the indicators characterizing the IOR efficiency in the tourist
network were selected in accordance with the logic of the implementation of the functions
presented and the achievement of the corresponding development goals of relations. For
instance, the implementation of the “profit” function logically corresponds with the “profit
maximization” goal. 3 Results The use of the “net profit” or “profitability” indicators in this case is
appropriate and expedient. On the ground of the authors’ definition of the tourist network
IOR efficiency idea as well as on the ground of the described functions and development
goals of relations, an appropriate conceptual model can be formed (fig.1). 3 3 https://doi.org/10.1051/e3sconf/202020805002 E3S Web of Conferences 208, 05002 (2020) E3S Web of Conferences 208, 05002 (2020)
IFT 2020 Fig. 1. Conceptual model for assessing the effectiveness of inter-organizational relations in the tourist
network (developed by the authors) Fig. 1. Conceptual model for assessing the effectiveness of inter-organizational relations in the tourist
network (developed by the authors) In the given list of indicators (table 1), a group of non-monetary indicators that reflect the
implementation of the “relationship quality” function deserves special justification. This term
was first introduced in the research of marketing services [16]. Later, there were various
examples of this idea operationalisation. The most common interpretation of the “relationship
quality” contains a certain set of characteristics, such as alignment of interests, joint problem
solving, trust, satisfaction, commitment, perceived value, etc. This paper authors hold to the
view presented in [19, 22, 23], according to which the quality of relations is a derivative of
three key characteristics: trust, commitment, and satisfaction. In accordance with this
approach, the authors define the goal of the relationship quality function implementing as
increasing relationship satisfaction, trust, and commitment to tourism network partners. The
authors believe that network members strive to develop relations with those entities
cooperation with which causes greater satisfaction, trust, and commitment. However, the
concepts of satisfaction, commitment and trust are many-sided and require additional
specification. To demonstrate the creation of relation quality goals, the authors offer a set of
six indicators: 1) stability, 2) continuity, 3) fairness and 4) personification (flexibility) of
relations, 5) reliability and 6) equality of partners. Let us consider them in further detail. We define reliability as a basic condition for trust formation between tourist network
partners. Reliability is “the confidence of firm A in the reliability of firm B and the assurance
of firm A that the actions of firm B will not intentionally lead to negative consequences for
firm A” [24]. Ultimately, reliability forms trust among partners and is one of the key
competitiveness factors of companies. According to Morgan R., Hunt S. 4 Conclusions The paper presents a conceptual model for assessing the efficiency of inter-organizational
relations in the tourism network. The authors have identified the functions, goals and
indicators that characterize the IOR efficiency in the tourism network. The model presented
demonstrates the interrelation between the efficiency and quality of inter-organizational
relations. Special attention is paid to the characteristics of the relation quality components. In addition, the issue of working out the integral indicator of the IOR efficiency (as well
as the indicators included) deserves special attention when assessing the IOR efficiency in
the tourism network with the help of the combination of goal-oriented, functional and
systemic approaches. In particular, the BSC (balanced scorecard) and the SCOR (supply
chain operations-reference model) model, which logic of the formation is widely used by
researchers in supply chain management, can serve as the basis for the integral indicator
formation. There is obvious academic interest in the development of various kinds of systems
for assessing the IOR efficiency. As a result of this interest, varieties of proposals have
appeared recently in this field. Notwithstanding, the issues under consideration are still
insufficiently developed in the tourism industry. Therefore, this fact actualizes their further
deepening and development. E3S Web of Conferences 208, 05002 (2020)
IFT 2020 E3S Web of Conferences 208, 05002 (2020)
IFT 2020 Fairness (profitability) of relations and equality of tourist network partners are also
important conditions for achieving mutually beneficial cooperation, solving emerging
problems and building a constructive dialogue between alliance members. Dependence on a
partner leading to unfairness in relations can be viewed by a company as a fairly significant
risk. Consequently, it can cause unwillingness to maintain and develop any relations. Thus,
fair (mutually beneficial) and equal cooperation reinforce each other and lead to increased
satisfaction with the relations. In our opinion, personification of relations is a condition for the formation of commitment
to a partner. It is predetermined by an increase in the level of heterogeneity and variety of
forms of emerging relations between partners. The level depends on resources, strategy, and
characteristics of companies. It is increased in the process of their adaptation to each other
arising from long-lasting cooperation. 3 Results [25], relationship satisfaction leads to commitment in
interaction with a specific partner. The commitment is defined as an effort to retain mutual
relations or certain activities which are of high account for a company. It is characterized by
continuity and stability (steadiness) in mutual relations and implies both the social
involvement and the resource usage to maintain inter-firm relations. As a result of the
increasing value created together with partners and strengthening personal ties, companies
get economic and social advantages in the market. Therefore, continuity and stability can be
affirmed to be fundamental derivatives of satisfaction with the relations of tourism network
partners and with the resulting commitment. 4 4 4 https://doi.org/10.1051/e3sconf/202020805002 References 6. V. Samarukha, Com Telecom Contr, 3 (2012) 7. L. Sanina, Baik Res J, 3 (2014) 8. T. Svetnik, T. Bubaeva, Baik Res J, 5 (2011) 8. T. Svetnik, T. Bubaeva, Baik Res J, 5 (2011) 9. O. Chepinoga, M. Solodkov, A. Semenova, Baik Res J, 3 (2017) 9. O. Chepinoga, M. Solodkov, A. Semenova, Baik Res J, 3 (2017) 10. O. Chistyakova, Bul Baik St Univ, 3 (2012) 10. O. Chistyakova, Bul Baik St Univ, 3 (2012) 11. M. Augustyn, T. Knowles, Tour. Manag. 21 (2004) 12. A. Morrison, P. Lynch, N. Johns, Int J Contemp Hosp Manage, 16 (2004) 13. H. Pechlaner, M. Volgger, Int J Cont Hosp Manage, 24 (2012) 5 E3S Web of Conferences 208, 05002 (2020)
IFT 2020 https://doi.org/10.1051/e3sconf/202020805002 14. Y. Wang, D. Fesenmaier, Tour Manage, 28 (2007)
15. S. Kushch, A. Afanasiev, Russ J Manage, 1 (2004)
16. L. Crosby, K. Evans, D. Cowles, J Mark, 54 (1990)
17. P. Naude, F. Buttle, Ind Mark Manage, 29 (2000)
18. K. Roberts, R. Bea, Org Dynam, 29 (2001)
19. W. Ulaga, A. Eggert, Eur J Mark, 40 (2006)
20. A. Walter, T. Muller, G. Helfert, T. Ritter, Ind Mark Manage, 32 (2003)
21. A. Walter, T. Ritter, H. Gemiinden, Ind Mark Manage, 30 (2001)
22. K. Hewett, R. Money, S. Sharma, J Acad Mark Sc, 30 (2002)
23. J. Hibbard, N. Kumar, L. Stern, J Mark Res, 38 (2001)
24. U. Andersson, M. Johanson, L. Silver, Department of Business Administration: Uppsala
(1996)
25. R. Morgan, S. Hunt, J Mark, 58 (1994) 14. Y. Wang, D. Fesenmaier, Tour Manage, 28 (2007)
15. S. Kushch, A. Afanasiev, Russ J Manage, 1 (2004)
16. L. Crosby, K. Evans, D. Cowles, J Mark, 54 (1990)
17. P. Naude, F. Buttle, Ind Mark Manage, 29 (2000)
18. K. Roberts, R. Bea, Org Dynam, 29 (2001)
19. W. Ulaga, A. Eggert, Eur J Mark, 40 (2006)
20. A. Walter, T. Muller, G. Helfert, T. Ritter, Ind Mark Manage, 32 (2003)
21. A. Walter, T. Ritter, H. Gemiinden, Ind Mark Manage, 30 (2001)
22. K. Hewett, R. Money, S. Sharma, J Acad Mark Sc, 30 (2002)
23. J. Hibbard, N. Kumar, L. Stern, J Mark Res, 38 (2001)
24. U. Andersson, M. Johanson, L. Silver, Department of Business Administration: Uppsala
(1996)
25. R. Morgan, S. Hunt, J Mark, 58 (1994) 24. U. Andersson, M. Johanson, L. References Silver, Department of Business Administration: Uppsala
(1996) 25. R. Morgan, S. Hunt, J Mark, 58 (1994) 6 6
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Blockade of Hedgehog Signaling Inhibits Pancreatic Cancer Invasion and Metastases: A New Paradigm for Combination Therapy in Solid Cancers
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Figure 1
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Multi-queen breeding is associated with the origin of inquiline social parasitism in ants
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Scientific reports
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OPEN Romain A. Dahan
* & Christian Rabeling
* Romain A. Dahan Social parasites exploit the brood care behavior of their hosts to raise their own offspring. Social
parasites are common among eusocial Hymenoptera and exhibit a wide range of distinct life
history traits in ants, bees, and wasps. In ants, obligate inquiline social parasites are workerless
(or nearly-so) species that engage in lifelong interactions with their hosts, taking advantage of
the existing host worker forces to reproduce and exploit host colonies’ resources. Inquiline social
parasites are phylogenetically diverse with approximately 100 known species that evolved at least
40 times independently in ants. Importantly, ant inquilines tend to be closely related to their hosts,
an observation referred to as ‘Emery’s Rule’. Polygyny, the presence of multiple egg-laying queens,
was repeatedly suggested to be associated with the origin of inquiline social parasitism, either by
providing the opportunity for reproductive cheating, thereby facilitating the origin of social parasite
species, and/or by making polygynous species more vulnerable to social parasitism via the acceptance
of additional egg-laying queens in their colonies. Although the association between host polygyny
and the evolution of social parasitism has been repeatedly discussed in the literature, it has not been
statistically tested in a phylogenetic framework across the ants. Here, we conduct a meta-analysis of
ant social structure and social parasitism, testing for an association between polygyny and inquiline
social parasitism with a phylogenetic correction for independent evolutionary events. We find an
imperfect but significant over-representation of polygynous species among hosts of inquiline social
parasites, suggesting that while polygyny is not required for the maintenance of inquiline social
parasitism, it (or factors associated with it) may favor the origin of socially parasitic behavior. Our
results are consistent with an intra-specific origin model for the evolution of inquiline social parasites
by sympatric speciation but cannot exclude the alternative, inter-specific allopatric speciation model. The diversity of social parasite behaviors and host colony structures further supports the notion that
inquiline social parasites evolved in parallel across unrelated ant genera in the formicoid clade via
independent evolutionary pathways. Both vertebrate and invertebrate species have been the subject of many studies investigating the role of ecology in
the evolution of social structure, and how variation in social systems can lead to major evolutionary transitions1–4. www.nature.com/scientificreports www.nature.com/scientificreports School of Life Sciences, Arizona State University, Tempe, AZ, USA. *email: romain.a.dahan@gmail.com; crabeling@
gmail.com Scientific Reports | (2022) 12:14680 www.nature.com/scientificreports/ On the other hand, the inter-specific model must include a free-living
non-host species as sister to the parasite species, and cannot account for a strict interpretation of Emery’s rule. So far, empirical phylogenetic studies supported the intra-specific route of inquiline social parasite evolution
in Acromyrmex, Ectatomma, Mycocepurus, and Myrmica ants37–42. The inter-specific model has so far garnered
more support in Pseudomyrmex and Temnothorax ants36,43. Secondary speciation events of host and/or parasite
species and host shifts can obscure the original transition to inquiline social parasitism given enough time,
and lead to ambiguous resolutions between models, for example in Pogonomyrmex, Solenopsis, and a clade of
Malagasy Pheidole44–46. To explain the evolutionary transition from a cooperative eusocial lifestyle to a socially parasitic life history,
secondary polygyny has repeatedly been suggested as one of the key factors26,31–33 because: (1) less efficient
nestmate and brood recognition in polygynous colonies may yield a greater non-nestmate tolerance47–49; (2)
the presence of multiple queens in colonies may provide an opportunity for supernumerary queens to cheat,
focusing on the production of sexual offspring without contributing to the colony’s sterile worker force30; (3)
workers and queens of polygynous colonies tolerate supernumerary egg-laying individuals50; and (4) the queen
adoption behavior of established, secondary polygynous colonies may provide a nest-invasion ‘channel’ for social
parasites to exploit32. These predictions have different, yet not mutually exclusive implications regarding the
evolutionary dynamics of inquiline social parasitism. The former two predictions are at least partially required
for intra-specific cheating to evolve, and support a sympatric, within-species origin model of inquiline social
parasitism. On the other hand, the latter two predictions are required for the maintenance of current inquiline
social parasitism, targeting specific host behaviors associated with secondary polygyny, and do not necessarily
favor either speciation model for the origin of inquiline social parasitism. While a strong association between
polygyny and inquiline social parasitism would lend support to any one of these predictions equally, a weak
correlation would fail to support the latter two, as it would show that polygyny is not necessarily required for the
maintenance of inquiline social parasitism. Indeed, secondary evolutionary events, after speciation of a parasite
species, such as diversification or extinction events of either host or parasite species, or host shifts, are expected
to obscure the evolutionary origins of social parasites and weaken an association between polygyny and inqui-
line social parasitism. www.nature.com/scientificreports/ has been observed relatively rarely11,27,28, secondary polygyny has been a particular focus of research studying
the evolutionary consequences of variation in social structure, because it provides avenues for the evolution of
alternative life-history strategies1,29,30, and it is thought to be the prevalent mechanism for polygyny in ants11,26. Secondary polygyny has also repeatedly been associated with the evolution of socially parasitic life-histories in
ants31–33. Social parasites are species that take advantage of the social structure of their eusocial hosts to benefit their
direct fitness. Three main life-histories of social parasitism occur in ants 26,32,34. Temporary social parasites found
colonies by invading host nests, killing the host queen(s), and taking advantage of the remaining host workers
to raise their first brood. Dulotic species similarly found colonies as temporary social parasites by invading host
nests, but subsequently rely on frequent raids on neighboring colonies for new workers. Finally, inquiline social
parasites are predominantly queen-tolerant workerless or nearly-workerless ant species which infiltrate estab-
lished host colonies and take advantage of the present worker force to rear their own sexual brood26. g
p
Inquiline social parasites are of particular interest to evolutionary biology because they are phylogenetically
highly diverse. Inquiline (workerless) social parasitism has evolved independently at least 40 times across 25
genera in the formicoid clade of the ant tree of life34, and there are currently 96 known species of inquiline social
parasites, which includes species arising from secondary speciation events in social parasite clades following the
evolution of the parasitic life history. Empirical studies revealed that many inquiline social parasites are closely
related to their hosts, a pattern known as ‘Emery’s rule’33,35. Thus, models describing the evolutionary origin of
social parasitism must account for the rule and explain why this pattern exists. Two models have been proposed
to explain the origin of inquiline social parasitism in ants. The inter-specific origin model proposes that social
parasites originated as facultatively parasitic lineages of a species closely related to the incipient host(s). In
contrast, the intra-specific origin model proposes that inquiline social parasites originated directly as cheating
lineages from the incipient host species via sympatric speciation26,31–33,36. In all cases, parasites rely on similar
resources, social cues, and environmental conditions as their hosts, requiring them to be fairly closely related
to their hosts33. OPEN Among eusocial insects, ancestral lifetime monogamy provided the key conditions favoring the origin of euso-
ciality across Hymenoptera5–7, and the ants share a single common ancestor suggesting that eusociality evolved
once during the Cretaceous8. However, many extant ant species display social structures deviating from a single,
monandrous queen in a colony3,9,10. While polyandry (multiple mating by females) and polygyny (multi-queen
breeding) are expected to reduce intra-colonial relatedness11, these social structures may be beneficial to colonies. In some cases, however, when nestmate queens are highly related, polygyny can have little to no detectable effect
on colony relatedness12. In eusocial animals, polygyny and polyandry can provide fitness benefits to the colony
as a whole11,13–15. On the other hand, divergences in social structure in ant colonies may also result in the evolu-
tion of alternative life-history strategies, such as alternative dispersal morphs, co-operative colony foundation,
reproductive cheating, and nepotism11,16–19. p
g
p
Multi-queen nesting (polygyny) can evolve as a response to rapidly changing ecological conditions, and can
have profound impacts on the social evolution of ant species11,20–25. In eusocial insects, polygyny may be primary,
resulting from the cooperative foundation of colonies by multiple queens (i.e., pleometrosis), or secondary, result-
ing from the adoption of new queens in established colonies26. While primary polygyny is generally associated
with pleometrotic colony founding under ecological conditions that promote crowding of foundress queens, and Scientific Reports | (2022) 12:14680 | https://doi.org/10.1038/s41598-022-17595-0 www.nature.com/scientificreports/ www.nature.com/scientificreports/ In the intra-specific model, hosts and parasites must necessarily form a monophyletic clade at
the time of speciation, providing a strict explanation for why parasites and hosts tend to be more closely related
than any two species within a genus35,36. On the other hand, the inter-specific model must include a free-living
non-host species as sister to the parasite species, and cannot account for a strict interpretation of Emery’s rule. So far, empirical phylogenetic studies supported the intra-specific route of inquiline social parasite evolution
in Acromyrmex, Ectatomma, Mycocepurus, and Myrmica ants37–42. The inter-specific model has so far garnered
more support in Pseudomyrmex and Temnothorax ants36,43. Secondary speciation events of host and/or parasite
species and host shifts can obscure the original transition to inquiline social parasitism given enough time,
and lead to ambiguous resolutions between models, for example in Pogonomyrmex, Solenopsis, and a clade of
Malagasy Pheidole44–46. g
Inquiline social parasites are of particular interest to evolutionary biology because they are phylogenetically
highly diverse. Inquiline (workerless) social parasitism has evolved independently at least 40 times across 25
genera in the formicoid clade of the ant tree of life34, and there are currently 96 known species of inquiline social
parasites, which includes species arising from secondary speciation events in social parasite clades following the
evolution of the parasitic life history. Empirical studies revealed that many inquiline social parasites are closely
related to their hosts, a pattern known as ‘Emery’s rule’33,35. Thus, models describing the evolutionary origin of
social parasitism must account for the rule and explain why this pattern exists. Two models have been proposed
to explain the origin of inquiline social parasitism in ants. The inter-specific origin model proposes that social
parasites originated as facultatively parasitic lineages of a species closely related to the incipient host(s). In
contrast, the intra-specific origin model proposes that inquiline social parasites originated directly as cheating
lineages from the incipient host species via sympatric speciation26,31–33,36. In all cases, parasites rely on similar resources, social cues, and environmental conditions as their hosts, requiring them to be fairly closely related
to their hosts33. In the intra-specific model, hosts and parasites must necessarily form a monophyletic clade at
the time of speciation, providing a strict explanation for why parasites and hosts tend to be more closely related
than any two species within a genus35,36. Social structure in ants f
There is a possibility that lineages reported as monogynous in the literature might be “false negatives”, i.e.,
lineages were inferred as monogynous because polygynous colonies were not observed. It is impossible to prove
that a species is obligately monogynous, so a certain probability of false negative species can be expected. In
contrast, falsely claiming a species as polygynous appears less likely, as a polygyny inference relies on positive
observations of colonies with multiple queens, and therefore, false positives are much less probable in our dataset. False negatives might bias an analysis by adding erroneous data points in the “monogynous/non-host” group,
simply because of the much larger sampling in the “non-host” category. We attempted to curb this effect by
stringently curating the dataset obtained from previous studies. Thus, we rejected any species where the mating
biology was not directly referenced and addressed in the primary literature, as well as species where the colony
structure was only inferred from lab colonies. Some ant species practice “serial” polygyny, that is the adoption of new queens by colonies to replace an old
queen29,57. While rarely documented, the question arises regarding the categorization of these species as either
functionally monogynous or polygynous. Here, we chose to characterize them as polygynous, because the mecha-
nism of adopting new queens still provides the opportunity for parasites to infiltrate the nest. Furthermore, some
inquiline species have been shown to target old, queenless colonies of their hosts, which might originate from
such a mechanism58,59. Similarly, species with known population-based differences in social structure (where
some populations are strictly monogynous while others are either facultatively or obligately polygynous) were
classified as facultatively polygynous in our dataset. Some authors further differentiate polygyny and oligogyny,
where only a small percentage of the colonies are actually polygynous. We decided against this delimitation, as
it seemed to introduce an arbitrariness to our data with unclear biological meaning. Hosts of inquiline social parasites. Similar to our survey of social structure, we collected information
on hosts of inquiline social parasites from the literature, by searching for the known hosts of inquiline social
parasite species. In these cases, we only retained species for which we had social structure information as well,
resulting in 51 host species in the final dataset (Table 1, Table S2). Phylogenetic inference and correction. Social structure in ants In order to correct for phylogenetic non-independence, we
assembled a cladogram of as many ant species as possible from available published molecular phylogenies
(Table S3). We did so by “grafting” genus-level phylogenies together within subfamilies, and then “transplanting”
these subfamily topologies to the appropriate tip of a subfamily-level phylogeny of Formicidae. Thus, while the
branch length information was lost in the grafting, the topology of the resulting cladogram remained an accurate
representation of the phylogenetic relationships between the species included. If a genus lacked an appropriate
species-level phylogeny in the literature, the genus was treated as representing a hard polytomy. We discarded any
species present in the phylogeny but not in the social structure dataset, and vice versa, resulting in a final dataset
mapped to a cladogram of 294 species, including 48 host species of inquiline social parasites. In some cases, we
had to discard phylogenies which included social parasites, because they did not include information about the
social structure of host or non-host species. We then inferred ancestral states of social structure through this
cladogram using the ‘ace’ function in the ape package for R, with default Maximum Likelihood optimization
method. We inferred the social structure at a node as the one with the highest posterior probability. This proce-
dure allowed for tracking the number of independent evolutionary transitions between social structures along
the cladogram. In contrast, we inferred transitions to becoming a host for inquiline social parasites only if all of
the descendants of a node were hosts, unless a case of host shift could reasonably be inferred, e.g. based on the
paraphyly of a host clade relative to the parasite (see Supplementary Methods). Because this method counts evo-
lutionary transitions toward hosting social parasites, cases in which species are hosts to multiple social parasites
were only counted as a single evolutionary transition. As a result, we recovered a contingency table of independ-
ent evolutionary transitions inferred for both social structure and susceptibility to inquiline social parasites, fol-
lowing methods in Refs.60,61. For example, if a transition to becoming a host happened on a branch which started
from a node inferred as polygynous, such event was counted in the Host × Polygynous cell of the contingency
table. www.nature.com/scientificreports/ The social structure of social parasite species is less of a focus for an intra-specific model
because its main relevance would lie in the phylogenetic signal of either mono- or polygyny of their ancestors. Similarly, while multiple mating by queens (polyandry) can reduce relatedness within a colony, its occurrence
would not account for cheating queen lineages in ancestors of host–parasite pairs in an intra-specific model. Furthermore, polygyny and multiple mating rarely co-occur in ants51. Thus, while multiple mating may play a
role in the emergence of cheating, and/or parasitism in an inter-specific scenario, it is not a part of the sympatric,
intra-specific model for the evolution of inquiline social parasitism per se.h pi
q
p
p
The predicted association between polygyny and inquiline social parasitism has been addressed in numerous
empirical studies and literature reviews29,33,52, however, it was never tested in a statistically rigorous framework. Here, we aim to provide a formal statistical test of the prediction that polygyny and inquiline social parasitism
are associated, by conducting a meta-analysis of the social structure of ants, and performing a phylogenetically
corrected test of independence between social structure and parasitism. The role of both facultative polygyny
and obligate polygyny have been discussed in previous reviews, with contradicting predictions29,33. Thus, we
further test whether obligate or facultative secondary polygyny are associated with social parasitism, in order to
test whether either social structure may favor social parasitism more than the other29,33. Finally, we discuss our
results to assess the plausibility of the four hypotheses linking polygyny and the evolution of social parasitism
(as outlined above). Scientific Reports | (2022) 12:14680 | https://doi.org/10.1038/s41598-022-17595-0 www.nature.com/scientificreports/ Methods Social structure in ants. We compiled a dataset of ants with known social structures using previously
published reviews as a starting point, and complemented the sampling with a thorough literature search adding
species and updating information on taxonomy and social structure of species (Table S1)1,9,13,26,29,53–55. For each
species in the dataset, we confirmed its social structure (monogyny/facultative polygyny/obligate polygyny) by
researching the primary literature reference(s). As in Rissing and Pollock55, we validated a species as monogy-
nous if at least 5 colonies were collected in the field. Records based on colonies that were solely lab-reared were
discarded, as well as records only reported in literature reviews. Consequently, our species list does not include
some of the species that were reported in previous studies, such as Keller and Passera56 or Keller and Reeve53. Every species was checked individually to ensure that the most up-to-date information was recorded. Species
with known primary polygyny were excluded, as the intra-specific model predicts that secondary polygyny is
associated with inquiline social parasitism. We also excluded all social parasites, because we specifically tested
predictions about the social organization of the free-living hosts. In total, our final dataset comprised 331 spe-
cies. In spite of our efforts, we do not claim this to be a comprehensive list of all ants with known social structure.h Social structure in ants Inversely, if a reversal to monogyny was inferred on a branch whose parent node was an inferred host, or
if a branch that included a transition to host had a monogynous parent node, we counted these in the Host ×
Monogyny cell. This method is identical to the procedure described in Schmid-Hempel and Crozier13. https://doi.org/10.1038/s41598-022-17595-0 Scientific Reports | (2022) 12:14680 | www.nature.com/scientificreports/ Table 1. Contingency table of social structure (polygyny or monogyny) in species hosting (or not hosting)
obligate inquiline social parasites, before phylogenetic correction. Percentages represent the proportion of host
and non-host species that are polygynous or monogynous. Host species
Non-host species
Monogyny
9
127
17.6%
45.4%
Polygyny
42
153
82.4%
54.6% Host species
Non-host species
Monogyny
9
127
17.6%
45.4%
Polygyny
42
153
82.4%
54.6% Table 1. Contingency table of social structure (polygyny or monogyny) in species hosting (or not hosting)
obligate inquiline social parasites, before phylogenetic correction. Percentages represent the proportion of h
and non-host species that are polygynous or monogynous. Table 1. Contingency table of social structure (polygyny or monogyny) in species hosting (or not hosting)
obligate inquiline social parasites, before phylogenetic correction. Percentages represent the proportion of host
and non-host species that are polygynous or monogynous. Table 1. Contingency table of social structure (polygyny or monogyny) in species hosting (or not hosting)
obligate inquiline social parasites, before phylogenetic correction. Percentages represent the proportion of host
and non-host species that are polygynous or monogynous. Statistical methods. We analyzed the results to test two hypotheses: (i) that hosts of social parasites are
more likely to be polygynous compared to non-hosts; and (ii) that either facultative or obligate polygyny is
overrepresented among hosts of social parasites. We tested the former by testing for the independence of social
structure (monogyny vs. polygyny) from hosting (or not hosting) an inquiline social parasite. We used a one-
tailed Fisher’s exact test, as the intra-specific model predicts a directional association a priori (i.e., that host and
polygyny should be associated; as opposed to either a lack of association, or a preponderance of host/monogyny
association). In contrast, we tested the second hypothesis that either obligate or facultative polygyny are involved
in the evolution of inquiline social parasitism by testing for overrepresentation of either facultative or obligate
polygynous species among hosts and non-hosts. Results Social structure in ants. In total, our final dataset comprised 331 species, including 51 hosts of inqui-
line social parasites (15.41%). Of the total 331 species, 136 (41.09%) were inferred as monogynous, while 119
(35.95%) were facultative polygynous, 28 (8.46%) were obligate polygynous, and 48 (14.50%) were inferred as
polygynous without specifying whether polygyny was obligate or facultative (Tables 1, 2, Table S1). Among the
51 hosts, 42 (82.35%) were polygynous, compared to 153 polygynous species among the 280 non-host species
(54.64%). Twenty-nine host species (56.86%) were facultatively polygynous, 2 (3.92%) were obligately polygy-
nous, and 11 (21.57%) polygynous species had no reference to any social polymorphism (Table 2). After match-
ing the dataset to our assembled cladogram, the sampling was reduced to 272 species spanning 11 subfamilies,
consisting of 111 monogynous species and 161 polygynous species. Of these 272 species that were represented
in the cladogram and for which reliable information on social organization was available, 47 (17.27%) were hosts
of inquiline social parasites. After estimating the evolutionary history of social structure in ants, we recovered 78
transitions from monogyny to polygyny, as well as 34 reversals to monogyny (Table 3). Association between polygyny and social parasitism. We found a significant association between
polygyny and hosting inquiline social parasites (Fisher’s exact test: Odds ratio = 0.423, p = 0.0443, Table 3), indi-
cating that polygynous species were significantly over-represented among hosts of inquiline social parasites. Specifically, hosts of social parasites were more than twice as likely to be polygynous compared to non-hosts. In
contrast, we did not find a significant association between either obligate or facultative secondary polygyny and
social parasitism (χ2 test of independence: χ2
1 = 3.0115, p = 0.0827, Table 4). Social structure in ants Because this hypothesis lacks an a priori directional prediction,
and the sample size was sufficiently large, we used a χ2 test of independence. For all tests, a significance level of
0.05 was used. All analyses were conducted in R 3.4.3, using the packages ‘ape’ ‘phytools’ and ‘base’62–64. Discussion Contingency table of polygyny type (facultative or obligate) in species hosting (or not hosting)
obligate inquiline social parasites, after phylogenetic correction. Numbers represent independent evolutionary
events. Expected values are given in parentheses. Table 4. Contingency table of polygyny type (facultative or obligate) in species hosting (or not hosting)
obligate inquiline social parasites, after phylogenetic correction. Numbers represent independent evolutionary
events. Expected values are given in parentheses. of additional queens either intra- or inter-specifically. It is important to note that while the association gener-
ally supports an intra-specific model, it does not permit excluding the alternative, inter-specific hypothesis, and
individual, taxon-specific analyses are needed to determine the evolutionary route of inquiline social parasitism
in convergently evolved lineages 34. of additional queens either intra- or inter-specifically. It is important to note that while the association gener-
ally supports an intra-specific model, it does not permit excluding the alternative, inter-specific hypothesis, and
individual, taxon-specific analyses are needed to determine the evolutionary route of inquiline social parasitism
in convergently evolved lineages 34. Monogyny and inquiline social parasitism. The occurrence of monogynous hosts of inquiline social
parasites is at first glance inconsistent with the previously outlined arguments that polygyny promotes the evolu-
tion of inquiline social parasitism33,52. Nonetheless, empirical studies revealed that several social parasites have
monogynous host species including, for example, inquiline species in the genera Acromyrmex, Nylanderia, and
Pogonomyrmex45,67,68. To distinguish between the different evolutionary dynamics of social parasite speciation,
i.e., the origin and the maintenance of social parasitism in a host population, it is important to understand
whether hosts of social parasites were monogynous at the time when the social parasite originated, or whether
host monogyny could be a consequence of a co-evolutionary arms-race between host and parasite69,70. Arms race
dynamics are known to affect the social organization of the host species, and population studies of dulotic Tem-
nothorax ants revealed that the frequency of monogynous colonies increased in highly parasitized host popula-
tions, presumably as a co-evolutionary response to parasitism which allowed for improved parasite detection
and rejection by the host71,72. In general, secondary evolutionary events in either host or parasite, including host
shifts, speciation and extinction events, as well as changes in social structure of either host or parasite colony,
may obscure the original conditions under which social parasitism originated. Discussion We conducted a meta-analysis across the ant tree of life to statistically test for an association between social
structure and being a host of inquiline social parasites, with a phylogenetic correction for independent evolu-
tionary events. We found that polygynous species are over-represented among hosts of inquilines, confirming
a long-standing but hitherto untested observation in the social parasitism literature29,31–33. In contrast to previ-
ous conflicting predictions29,33, we did not find a significant association between either obligate or facultative
polygyny and inquilinism. Polygyny and inquiline social parasitism. The association of polygyny with social parasitism is impor-
tant for both the intra-specific and the inter-specific origin models of social parasite speciation, especially relat-
ing to the tolerance of non-nestmate individuals and the queen adoption mechanism of polygynous hosts. How-
ever, while the role of multi-queen nesting is circumstantial in the inter-specific origin model (as parasites may
target monogynous species as well, see below), it is critical in an intra-specific model, in which parasites evolve
directly from intra-specific lineages. A sympatric origin requires polygyny in the incipient stages of the specia-
tion process, as it would begin as reproductive cheating between nestmate queens. Here, we found a significant
association between polygyny and inquiline social parasitism. While the inferred association is not definitive
evidence of the intra-specific origin model, our results add to the mounting phylogenetic evidence favoring
the sympatric, intra-specific speciation model for the origin of inquiline social parasites in distantly related ant
lineages38–41. Furthermore, these observations are consistent with predictions of mechanistic models for sympa-
tric speciation (see below)33,65,66. Overall, our results emphasize the importance of polygyny across ant species
in the initial emergence of inquiline social parasitism, facilitating the emergence of cheating and the adoption https://doi.org/10.1038/s41598-022-17595-0 Scientific Reports | (2022) 12:14680 | www.nature.com/scientificreports/ Table 2. Contingency table of polygyny type (facultative or obligate) in species hosting (or not hosting)
obligate inquiline social parasites, before phylogenetic correction. Polygynous species for which information
regarding obligate or facultative nature of polygyny was missing were excluded from this table. Percentages
represent the proportion of host and non-host species that are facultatively or obligately polygynous. Host species
Non-host species
Facultative polygyny
29
90
93.6%
77.6%
Obligate polygyny
2
26
6.4%
22.4% Table 2. Discussion Contingency table of polygyny type (facultative or obligate) in species hosting (or not h
Host species
Non-host species
Facultative polygyny
29
90
93.6%
77.6%
Obligate polygyny
2
26
6.4%
22.4% Host species
Non-host species
Facultative polygyny
29
90
93.6%
77.6%
Obligate polygyny
2
26
6.4%
22.4% Table 2. Contingency table of polygyny type (facultative or obligate) in species hosting (or not hosting)
obligate inquiline social parasites, before phylogenetic correction. Polygynous species for which informatio
regarding obligate or facultative nature of polygyny was missing were excluded from this table. Percentages
represent the proportion of host and non-host species that are facultatively or obligately polygynous. Table 2. Contingency table of polygyny type (facultative or obligate) in species hosting (or not hosting)
obligate inquiline social parasites, before phylogenetic correction. Polygynous species for which information
regarding obligate or facultative nature of polygyny was missing were excluded from this table. Percentages
represent the proportion of host and non-host species that are facultatively or obligately polygynous. Table 3. Contingency table of social structure (polygyny or monogyny) in species hosting (or not hosting)
obligate inquiline social parasites, after phylogenetic correction. Numbers represent independent evolutionary
events. Expected values are given in parentheses. Host species
Non-host species
Monogyny background
8
26
(12.45)
(21.55)
Polygyny background
33
45
(28.55)
(49.45) Host species
Non-host species
Monogyny background
8
26
(12.45)
(21.55)
Polygyny background
33
45
(28.55)
(49.45) Table 3. Contingency table of social structure (polygyny or monogyny) in species hosting (or not hosting)
obligate inquiline social parasites, after phylogenetic correction. Numbers represent independent evolutionary
events. Expected values are given in parentheses. Table 3. Contingency table of social structure (polygyny or monogyny) in species hosting (or not hosting)
obligate inquiline social parasites, after phylogenetic correction. Numbers represent independent evolutionary
events. Expected values are given in parentheses. Table 4. Contingency table of polygyny type (facultative or obligate) in species hosting (or not hosting)
obligate inquiline social parasites, after phylogenetic correction. Numbers represent independent evolutionary
events. Expected values are given in parentheses. Host species
Non-host species
Facultative polygyny
24
24
(21.52)
(26.48)
Obligate polygyny
2
8
(4.48)
(5.52) Host species
Non-host species
Facultative polygyny
24
24
(21.52)
(26.48)
Obligate polygyny
2
8
(4.48)
(5.52) Table 4. Contingency table of polygyny type (facultative or obligate) in species hosting (or not hosting)
obligate inquiline social parasites, after phylogenetic correction. Numbers represent independent evolutionary
events. Expected values are given in parentheses. Table 4. Discussion From there, selection might favor the assortative mating of selfish lineages, resulting in genetic
divergence and eventually speciation, resulting from alternative adaptations33,83. Natural history observations
reinforce this second line of evidence, as some inquiline species are known to produce a worker caste, and also
display a less extreme degree of size reduction compared to their hosts (e.g. the leaf-cutting ant parasite Acro-
myrmex insinuator84). It is important to note that the appearance and maintenance of these small queen morphs
may also be adaptive to the colonies producing them, as they may evolve as dispersal or polygynous morphs in
ecologically saturated habitats with limited space for new colony founding and nest establishment17,22,85. Further
investigations of possible sources of reproductive isolation may contribute to a better understanding of how
inquiline species originate from cheating lineages within their hosts38,40,66,86. Queen size polymorphism and inquiline social parasitism. Social structure and reproductive ecol-
ogy are at the core of the evolution of inquiline social parasitism32. Increasingly, however, other traits in the
inquiline syndrome (sensu Wilson73) are believed to play important roles in the emergence of reproductive
cheating. Queen polymorphisms involving differential dispersal strategies, for example, have provided a promis-
ing avenue of research for the investigation of intra-specific reproductive cheating33,74–76. Small queen morphs, Alternative evolutionary trajectories to inquiline social parasitism. In contrast to inquiline spe-
cies that originated as reproductive cheaters, other ant social parasite species likely followed alternative evolu-
tionary routes to inquilinism and workerlessness. In the formicine genus Nylanderia, for example, the hosts of
inquiline species may be strictly monogynous54,67,87, possibly indicating that the inquiline social parasites origi-
nated via a different evolutionary model. Across eusocial Hymenoptera, Emery’s rule is only strictly observed
in a few inquiline social parasites whereas many non-inquiline ant social parasites as well as parasitic bees and
wasps are not the closest relatives of their hosts34,35,88. These diverse phylogenetic relationships between hosts
and parasites suggest that social cheating evolved convergently along independent pathways in convergently
evolved host parasite systems. In at least two different systems, inquiline social parasites are nested in clades
featuring other socially parasitic life histories. Discussion If our results are correct, at least
eight independent origins of inquiline social parasitism occurred in a monogynous background (Table 3), sug-
gesting that this specialized parasitic life history can evolve in a monogynous background. Further studies are
necessary to evaluate whether these eight monogynous events truly represent social parasites that evolved in
monogynous hosts and whether host monogyny reflects the social colony organization at the time of parasite
speciation. Once primarily monogynous hosts can be validated, it will be insightful to study the interactions and
evolutionary dynamics between host and parasite species. Investigating potential natural history traits or com- Scientific Reports | (2022) 12:14680 | https://doi.org/10.1038/s41598-022-17595-0 www.nature.com/scientificreports/ mon ecological niches in these systems may provide insights towards elucidating the evolutionary mechanisms
of inquiline social parasitism in monogynous species, and the convergence of a socially parasitic life history. Queen size polymorphism and inquiline social parasitism. Social structure and reproductive ecol-
ogy are at the core of the evolution of inquiline social parasitism32. Increasingly, however, other traits in the
inquiline syndrome (sensu Wilson73) are believed to play important roles in the emergence of reproductive
cheating. Queen polymorphisms involving differential dispersal strategies, for example, have provided a promis-
ing avenue of research for the investigation of intra-specific reproductive cheating33,74–76. Small queen morphs,
referred to as ‘microgynes’, are in many species associated with alternative dispersal strategies and polygyny
itself, and in some cases microgynes were shown to favor the production of sexual offspring over workers, pro-
viding the basis for reproductive cheating30. In some cases, microgynes are in fact considered intra-specific
inquiline parasites66,77, whereas in other cases microgynous forms were raised to the species level as obligate
inter-specific inquiline parasites78,79. Queen size polymorphism is heavily associated with the evolution of social
parasitism based on two major lines of evidence. First, size reduction is a trait observed in most inquiline social
parasites, and part of the inquiline syndrome32,33,73,80. Second, body size is known to affect developmental trajec-
tory at the larval stages75,81,82. From these observations, it has been hypothesized that a shift of the size threshold
for queen development, which would result in microgyne morphs, may also be associated with reproductive
cheating. In such a case, larvae that were allocated with a certain quantity and quality of resources which would
usually lead them toward worker development would instead develop into queens (the so-called ‘selfish brood
hypothesis’65,75). Discussion For example, molecular phylogenies suggest that the socially para-
sitic Temnothorax corsicus species group, a clade formerly known as Myrmoxenus43,89, which contains primarily
dulotic species, also contains a number of independently evolved so-called ‘murder-parasites’ or ‘degenerate
dulotic’ species, which are workerless or nearly-workerless species that display morphological and behavioral
traits generally associated with inquiline social parasitism90. These origins are thought to likely emerge as a loss
of the worker caste in dulotic species and demonstrate that some inquiline social parasites may evolve from a
dulotic ancestor91. Similarly, Formica talbotae, the only confirmed inquiline species in the wood ant genus For-
mica, likely evolved from a temporary social parasitic ancestor in the F. difficilis group (the former F. microgyna
group)26,92–94. In both cases, an intra-specific origin of social parasitism is unlikely, and these species provide
evidence for inquiline social parasites evolving from either a dulotic or a temporary social parasitic ancestor
instead of a free-living, polygynous ancestor. Furthermore, these Temnothorax and Formica social parasite spe-
cies suggest a potential for evolutionary transitions between socially parasitic life history strategies10,26,32,34,52. Limitations of the study. Considering the current knowledge about the phylogenetic relationships and
the social structure of individual ant species, we chose a conservative approach to test whether polygyny is
statistically associated with being a host of an inquiline social parasite. Nonetheless, we would like to point out
some limitations of our study that arise from uncertainties at different levels of the data collection and analysis,
and therefore, future studies may come to a different conclusion. The results of this study are not invalidated by
these shortcomings, and will hopefully be addressed in the future: Species selection. We assembled a comprehensive and carefully curated dataset of ant species with known social
structure. Nonetheless, the social organization and natural history is only known for a small subset (371 species
in our dataset) of the world’s ant species (14,035 recognized species), representing only 2.4%. Therefore, our
inference may change as new information on the social structure of additional ant species becomes available. Furthermore, we applied a conservative filter to our species selection, only retaining social organization data for
species with documented social structures that were observed in nature. We chose to discard information about
the social structure of species which were solely reported from laboratory-bred colonies. Phylogenetic inference. Ideally, this study would have included a high-quality phylogeny inferred from a com-
prehensive molecular genetic dataset. www.nature.com/scientificreports/ for the topology (i.e., bootstrap values or Bayesian posterior probabilities), as well as branch length information
for a more refined ancestral character state inference. Considering the different datasets (i.e., morphological vs
mitochondrial and nuclear genetic vs genomic and transcriptomic data) that have been utilized over the past
decades to infer phylogenies across diverse ant taxa, assembling a data matrix and inferring a comprehensive
phylogeny spanning the taxa analyzed in this this study was not feasible. Therefore, we decided to construct a
cladogram by grafting trees from previously published phylogenies that were inferred from diverse data matrices
(Table S2). By applying this methodology, we retained only the topological information from these phylogenies
representing the relationships between the species in our dataset, but discarding branch length information
and discarding estimates of confidence regarding the topology. Once comprehensive genetic/genomic data will
become available for the entire ant tree of life, a high-quality phylogeny should be used to quantify how uncer-
tainty at the level of tree inference affects the conclusion of our study. Ancestral character state inference. Because of the method we used to assemble the cladogram, a metric tree
was not available to perform an ancestral character state estimation using Maximum Likelihood or Bayesian
methods. In the absence of branch lengths, and by analyzing discrete characters, our analysis relied on a maxi-
mum parsimony approach, which assumes that transitions from monogyny to polygyny and their reversal (from
polygyny to monogyny) are equally likely on each edge of the tree. Future studies need to evaluate whether
transitions from monogyny to polygyny and vice versa are in fact equally likely. Conclusionh The comparative analysis presented here reveals a significant association between secondary polygyny and being
a host of an inquiline social parasite in ants, and becoming a host of a social parasite appears to be twice as likely
in a polygynous species. However, this association is imperfect, in that it affects species with obligate or facultative
polygyny equally. Our results support the previously formulated hypothesis that polygyny is an important trait
associated with the evolutionary origins of inquiline social parasitism in ants. However, secondary evolution-
ary transitions such as host shifts, speciation and extinction events, as well as changes in colony organization in
either the host or parasite may erode the signal of the original condition under which social parasitism origi-
nated. Furthermore, co-evolutionary arms race dynamics between host and parasite may result in secondarily
monogynous hosts of inquiline social parasites. Considering that obligate inquiline social parasitism has evolved
in parallel many times in distantly related clades across the ant tree of life, it is unrealistic to expect that every
convergent origin of the inquiline phenotype evolved along identical trajectories. We discussed potential avenues
to investigate and contrast different mechanisms through which obligate workerless inquiline parasitism might
have evolved in ants, both in sympatry and in allopatry. Overall, our results emphasize that changes in social
structure can have significant consequences for life history and social evolution in eusocial insects. Data availability
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All data is included in the supplementary material as well as on Dryad data repository, and is available at the
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61. Ridley, M. The Explanation of Organic Diversity: The Comparative Methods and Adaptations for Mating (Oxford Science Publica-
tions, 1983). 62. Paradis, E., Claude, J. & Strimmer, K. APE: Analyses of phylogenetics and evolution in R. Bioinformatics 20, 289–290 (2004 63. Revell, L. J. phytools: An R package for phylogenetic comparative biology (and other things). Methods Ecol. Evol. 3, 217–223 (2
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d 63. Revell, L. J. phytools: An R package for phylogenetic comparative biology (and other things). Methods Ecol. Evol. 3, 217–223 (2012). 64. R Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing (R Foundation for
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65. Wolf, J. I. & Seppä, P. Queen size dimorphism in social insects. Insectes Soc. 63, 25–38 (2015). https://doi.org/10.1038/s41598-022-17595-0 Scientific Reports | (2022) 12:14680 | Author contributions R.A.D. and C.R. designed the study. R.A.D. compiled and analyzed the data, and wrote a first draft of the manu-
script with contributions from C.R. R.A.D. and C.R. edited and revised the manuscript. Competing interests
The authors declare no competing interests. Additional information
Supplementary Information The online version contains supplementary material available at https://doi.org/
10.1038/s41598-022-17595-0. Correspondence and requests for materials should be addressed to R.A.D. or C.R. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
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The authors declare no competing interests. upplementary Information The online version contains supplementary material available at https://doi.org/
0.1038/s41598-022-17595-0. Correspondence and requests for materials should be addressed to R.A.D. or C.R. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. https://doi.org/10.1038/s41598-022-17595-0 Scientific Reports | (2022) 12:14680 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
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
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Formation of Russian magistracy: implementation of the digital economy requests in the educational process
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Воеводина Екатерина Владимировна Воеводина Екатерина Владимировна
Канд. соц. наук, доцент, ФГОБУ ВО «Финансовый университет при Правительстве Российской Федерации», г. Москва,
Российская Федерация
ORCID: 0000-0001-6131-8301
E mail: ekaterinavoevodina@yandex ru оцент, ФГОБУ ВО «Финансовый университет при Правительстве Российской Федерации», г. Москва,
рация Канд. соц. наук, доцент, ФГОБУ ВО «Финансовый университет при Правительстве Российской Федерации», г. Москва,
Российская Федерация
ORCID: 0000-0001-6131-8301
E-mail: ekaterinavoevodina@yandex ru E-mail: ekaterinavoevodina@yandex.ru АННОТАЦИЯ ключевых процессов, в которых задействованы основные
стейкхолдеры (обучающиеся, преподаватели и работодатели). В статье отражены результаты опроса 713 преподавателей
магистратуры регионов России, а также 1 140 магистрантов. В качестве основных результатов приведены сведения об ис-
пользовании современных образовательных технологий в под-
готовке российских магистрантов – сделан вывод о небольшом
распространении онлайн-обучения и сетевых программ в вузах. Приведены данные исследования, характеризующие степень
сформированнности цифровых компетенций студентов
российской магистратуры: поиска и обработки информации,
использования информационных технологий, обеспечения
безопасности информации, умения работать с «большими
данными». Отмечено, что эти навыки хорошо развиты у со-
временных студентов, однако участие вузов в их развитии
невысокое. Сделан вывод, что российская магистратура
характеризуется «переходным» периодом в решении задач
цифровой экономики. Исследовано состояние российской магистратуры в условиях
перехода к цифровой экономике. Затронуты отдельные аспекты
присоединения России к Болонскому процессу – отмечены
проблемы перехода, связанные с недостаточным пониманием
сущности трансформаций основными стейкхолдерами. Проа-
нализированы вопросы социализации обучающихся «цифро-
вого» поколения, наиболее характерные из них – стремление
к постоянным изменениям, нетерпимость однотипных задач,
хорошее владение информационными технологиями. Отмечено,
что российская магистратура сталкивается с необходимостью
адаптации к потребностям цифровой экономики, а также
«поколенческим» особенностям представителей поколения Z. Приведены результаты эмпирического социологического
исследования «Рождение российской магистратуры», выпол-
ненного в рамках проекта Института образования ФГАОУ ВО
«Национальный исследовательский университет «Высшая
школа экономики». Цель исследования – анализ состояния
современной российской магистратуры и определение ключевых процессов, в которых задействованы основные
стейкхолдеры (обучающиеся, преподаватели и работодатели). В статье отражены результаты опроса 713 преподавателей
магистратуры регионов России, а также 1 140 магистрантов. В качестве основных результатов приведены сведения об ис-
пользовании современных образовательных технологий в под-
готовке российских магистрантов – сделан вывод о небольшом
распространении онлайн-обучения и сетевых программ в вузах. Приведены данные исследования, характеризующие степень
сформированнности цифровых компетенций студентов
российской магистратуры: поиска и обработки информации,
использования информационных технологий, обеспечения
безопасности информации, умения работать с «большими
данными». Отмечено, что эти навыки хорошо развиты у со-
временных студентов, однако участие вузов в их развитии
невысокое. Сделан вывод, что российская магистратура
характеризуется «переходным» периодом в решении задач
цифровой экономики. Ключевые слова Высшее образование, студент, магистратура, становление российской магистратуры, Болонский процесс, цифровые ком-
петенции, цифровое общество, современные технологии обучения, онлайн-обучение. Становление российской магистратуры: как
реализуются запросы цифровой экономики
в образовательном процессе УДК 316.4 DOI 10.26425/2658-347X-2019-3-16-24
Получено 05.10.2019 Одобрено 15.11.2019 Опубликовано 31.12.2019 УДК 316.4 DOI 10.26425/2658-347X-2019-3-16-24
Получено 05.10.2019 Одобрено 15.11.2019 Опубликовано 31.12.2019 ABSTRACT The state of Russian magistracy in the transition to a digital
economy has been studied. Some aspects of Russia’s accession
to the Bologna process have been affected – problems of tran-
sition associated with a lack of understanding of the essence
of transformations by the main stakeholders have been noted. The issues of socialization of students of the «digital» generation
have been analyzed, the most characteristic of them – the desire
for constant change, intolerance of the same type of problems,
good knowledge of information technology. It has been noticed,
that Russian magistracy is faced with the need to adapt to the
needs of the digital economy, as well as the «generational»
characteristics of representatives of generation Z. stakeholders (students, teachers and employers) are involved. The results of a survey of teachers of magistracy (713) in Russian
regions, as well as undergraduates (1 140), have been reflect-
ed in the article. As the main results, information on the use
of modern educational technologies in the preparation of Russian
undergraduates has been given – a conclusion has been made
about the small distribution of on-line education and network
programs in universities. The state of Russian magistracy in the transition to a digital
economy has been studied. Some aspects of Russia’s accession
to the Bologna process have been affected – problems of tran-
sition associated with a lack of understanding of the essence
of transformations by the main stakeholders have been noted. The issues of socialization of students of the «digital» generation
have been analyzed, the most characteristic of them – the desire
for constant change, intolerance of the same type of problems,
good knowledge of information technology. It has been noticed,
that Russian magistracy is faced with the need to adapt to the
needs of the digital economy, as well as the «generational»
characteristics of representatives of generation Z. The research data characterizing the degree of digital competencies
of Russian graduate students have been adduced: information
search and processing, use of IT technologies, information security,
ability to work with «big» data. It has been noted, that these skills
are well developed among modern students, but the participation
of universities in their development is low. It has been concluded,
that Russian magistracy is characterized by a «transitional» period
in solving the problems of the digital economy. Voevodina Ekaterina ological Sciences, Associate Professor, Financial University under the Government of the Russian Federation Candidate of Sociological Sciences, Associate Professor, Financial University under the Government of the Russian Federation,
Moscow, Russia
ORCID: 0000-0001-6131-8301
E
il
k t
i
di
@
d E-mail: ekaterinavoevodina@yandex.ru ABSTRACT The results of an empirical sociological study “Birth of a Russian
magistracy”, carried out as part of the project of the Higher
School of Economics Institute of Education, have been present-
ed. The purpose of this study is to analyze the state of modern
Russian magistracy and define key processes in which the main Keywords Higher education, student, magistracy, formation of the Russian magistracy, Bologna process, digital competencies, digital society,
modern educational technologies, online education. Formation of Russian magistracy: implementation
of the digital economy requests in the
educational process DOI 10.26425/2658-347X-2019-3-16-24
Received 05.10.2019 Approved 15.11.2019 Published 31.12.2019 Received 05.10.2019 Approved 15.11.2019 Published 31.12.2019 Цитирование Воеводина Е.В. Становление российской магистратуры: как реализуются запросы цифровой экономики в образовательном
процессе//Цифровая социология. 2019. Т. 2. № 3. С. 16–24. Благодарность. В статье отражены результаты исследования в рамках проекта «Рождение российской магистратуры», реализуемого
Институтом образования НИУ ВШЭ, победителем программы «Стипендиальная программа Владимира Потанина» Благотворительно-
го фонда В. Потанина. © Воеводина Е.В., 2019. Статья доступна по лицензии Creative Commons «Attribution» («Атрибуция») 4.0. всемирная
(http://creativecommons.org/licenses/by/4.0/). 16 16 ЛИТЕРАТУРНЫЙ ОБЗОР Обратимся к анализу работ, позволяющих обо-
значить ключевые особенности российского инсти-
тута образования в совокупности с поколенческими
особенностями современного студенчества. Специ-
фикой высшего образования в России является то,
что наряду с воздействием внешних факторов, об-
условленных процессами цифровизации, оно пре-
терпело ряд внутриинституциональных трансфор-
маций. И хотя переходу на двухуровневую систему
уже более десяти лет, еще не все механизмы в дан-
ной сфере полностью отлажены. Постоянным изме-
нениям подвергаются образовательные стандарты
по ряду направлений подготовки, требования к про-
фессорско-преподавательскому составу, технологии
обучения, формы контроля и др. При этом у основ-
ных стейкхолдеров – абитуриентов, обучающихся,
работодателей, пока еще не укоренилось понимание
произошедших трансформаций. В частности, это ка-
сается осознания отличия уровней вузовской подго-
товки – бакалавриата, магистратуры и аспирантуры. В «Стратегии развития информационного общества
в Российской Федерации на 2017–2030 годы»1, приня-
той в 2017 г., провозглашается формирование нацио-
нальной цифровой экономики, основанной на циф-
ровых данных, обработке больших объемов и исполь-
зовании результатов анализа, позволяющих «суще-
ственно повысить эффективность различных видов
производства, технологий, оборудования, хранения,
продажи, доставки товаров и услуг»2. Достижение по-
ставленных целей трудно представить без качествен-
ной подготовки кадров, которая, требует поиска со-
временных подходов, позволяющих не просто осу-
ществлять транслирование знаний, но и способство-
вать развитию инновационных практик. В исследовании, посвященном анализу Болонско-
го процесса в России, О.М. Карпенко и М.Д. Бершад-
ская [2010] раскрывают сущность каждого уровня выс-
шего образования: выпускник магистратуры при этом
занимает промежуточное положение между бакалав-
ром, имеющим общую специализацию, и аспирантом,
полностью погруженным в исследовательскую деятель-
ность. Магистр, в свою очередь, является «узким» про-
фессионалом, специализирующимся на определенной
области науки и практики, что обеспечивает ему боль-
шую сфокусированность на видах деятельности. Про-
фессиональная значимость бакалавра при этом не умень-
шается, это специалист, решающий общепрофильные
задачи, которые есть в любой сфере. Однако непони-
мание различий между бакалавром и магистром при-
водит к формированию ряда стереотипов среди насе-
ления. Результаты замера Всероссийского центра изу-
чения общественного мнения (ВЦИОМ) под названием
«Студент-2017: рвение к учебе и перспективы трудоу-
стройства»3 показали, что 47 % россиян считают бака-
лавриат недостаточным для успешного трудоустрой-
ства. В других исследованиях отмечено, что работода-
тели неохотно принимают на открытые вакансии мо-
лодых выпускников – невостребованность последних
оценивается на уровне 28–50 % [Соловов, 2016]. В связи с этим приобретает актуальность вопрос
о том, насколько современная система высшего обра-
зования способна отвечать на поставленные цифро-
визацией вызовы. For citation Voevodina E.V. (2019) Formation of Russian magistracy: implementation of the digital economy requests in the educational pro-
cess. Digital sociology. Vol. 2, no 3, pp. 16–24. DOI: 10.26425/2658-347X-2019-3-16-24 Acknowlegements. The article reflects the results of the research in the framework of the project “Birth of the Russian master’s degree”, implemented
by the HSE Institute of education, the winner of the Vladimir Potanin Scholarship program of The V. Potanin Charitable Foundation. © The Author(s), 2019. This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/). 17 Цифровая социология /
/
Т. 2 , № 3
2019 в большей степени связан с генерированием инно-
ваций в научно-исследовательской или прикладной
(проектной) деятельности. 1 Указ Президента РФ от 09.05.2017 № 203 «О Стратегии разви-
тия информационного общества в Российской Федерации на 2017–
2030 годы». Режим доступа: http://www.consultant.ru/document/
cons_doc_LAW_216363/ (дата обращения: 03.10.2019).
2 Там же. ВВЕДЕНИЕ Современное общество характеризуется множеством
изменений и процессов, которые трудно было вообра-
зить простому обывателю еще полвека назад. В ХХI сто-
летии произошел ряд кардинальных изменений в раз-
ных областях науки и практики, что привнесло в нашу
реальность такие связанные между собой категории,
как «цифровизация», «диджитализация», «гаджетиза-
ция». Современная «жизнь в цифре» задает свой стиль,
темп, социальные ценности, которые все больше напо-
минают калейдоскоп из множества ранее несовмести-
мых фрагментов; изобилует огромными потоками со-
циальной информации, складывающейся в «большие
данные». Еще в начале перехода к новому типу обще-
ства, некоторые классики современной социологиче-
ской мысли оценивали будущие трансформации скеп-
тически. В частности, в работах У. Бека [2000], З. Бау-
мана [2008] содержатся утверждения о рискогенности,
неустойчивости социальной структуры, положения
индивида в ней. Постоянные изменения в технологи-
ческой сфере и конкурентная гонка за инновациями
влекут за собой кардинальную перестройку всех со-
циальных институтов, в том числе и высшего образо-
вания как сферы подготовки будущих квалифициро-
ванных кадров для цифровой реальности. 3 «Высшее образование: социальный лифт или потерянное время» /
Инициативный всероссийский опрос «ВЦИОМ-Спутник» (2019) // Сайт
Всероссийского центра изучения общественного мнения. Режим доступа:
https://wciom.ru/index.php?id=236&uid=9808 (дата обращения: 03.10.2019). 2 Там же. ЛИТЕРАТУРНЫЙ ОБЗОР В частности, необходимо оценить
насколько современные студенты владеют информа-
ционно-коммуникативными (цифровыми) компетен-
циями, используются ли в российском образовании
новейшие подходы и технологии. В нашем исследо-
вании мы сосредоточим внимание на магистратуре,
так как данный уровень подготовки, на наш взгляд, 3 «Высшее образование: социальный лифт или потерянное время» /
Инициативный всероссийский опрос «ВЦИОМ-Спутник» (2019) // Сайт
Всероссийского центра изучения общественного мнения. Режим доступа:
https://wciom.ru/index.php?id=236&uid=9808 (дата обращения: 03.10.2019). 18 Цифровая среда Современные студенты – потенциальные предста-
вители класса интеллектуалов, генерирующих инно-
вации. Они являются ключевой группой молодежи,
от которой зависит качество реализации целей циф-
ровой экономики в ближайшем будущем. Поколение
Z, чья активная социализация протекала уже в пери-
од существования сети «Интернет», отличает высокое
владение цифровыми компетенциями, и, одновремен-
но, неустойчивость интересов, низкая способность
к концентрации на однотипных задачах, неприемле-
мость любой «рутины». Это нередко ослабляет инте-
рес к профессии уже на этапе ее освоения. А.В. Бры-
кин отмечает, что 2/3 современных выпускников об-
ладают слабыми представлениями о своей специаль-
ности, они ни разу не были в профильных органи-
зациях и не ориентированы на рынке труда. Почти
половина выпускников после 2-х лет работы, уходит
из организации и меняет свою профессию [Брыкин,
2018]. Также следует привести результаты еще одно-
го опроса ВЦИОМ от 18 июня 2019 г. «Высшее обра-
зование: социальный лифт или потерянное время?»,
которые показывают увеличение доли молодежи, свя-
зывающей сущность высшего образования с традици-
ей и социальным престижем. Так, 74 % опрошенных
в возрасте от 18 до 25 лет заявляют о завышении зна-
чимости высшего образования как обязательного ус-
ловия удачной карьеры; 76% не согласны с утвержде-
нием, что отсутствие высшего образования гаранти-
рует только низкооплачиваемую и непрестижную ра-
боту4. Для сравнения: в старших возрастных группах
45–59 лет доля таких респондентов составляет 63 %;
в группе респондентов старше 60 лет – 52 %. Из этого
можно заключить, что особенности мировосприятия
цифрового поколения выступают в качестве специ-
фичных вызовов, требующих адаптации системы выс-
шего образования (к примеру, внедрения принципов
гибкости и индивидуализации образовательных тре-
ков, перехода на современных технологии и формы
обучения). При этом необходимо оценить, насколь-
ко современное высшее образование готово к форми-
рованию цифровых компетенций, под которыми мы,
прежде всего, подразумеваем, знания, умения и на-
выки в области информационно-коммуникационных
технологий и процессов. Их значимость неоднократ-
но подчеркивалась современными исследователями,
в частности Г.А. Банных отмечает, что без должного
участия университетов в процессе формирования та-
ких компетенций ослабляются возможности профес-
сиональной самореализации будущих специалистов
на рынке труда [Банных, 2016]. 4 Студент-2017: рвение к учебе и перспективы трудоустрой-
ства Инициативный всероссийский опрос ВЦИОМ (2017)//Сайт
Всероссийского центра изучения общественного мнения. Режим
доступа: https://wciom.ru/index.php?id=236&uid=515 (дата обра-
щения: 03.10.2019). ЛИТЕРАТУРНЫЙ ОБЗОР Касаясь вопроса о парадигмальных основах исследо-
вания высшего образования в условиях цифровой эко-
номики отметим, что на сегодняшний день данное на-
правление активно развивается в рамках различных об-
ластей социологии. Поскольку на процессы в высшем
образовании влияет ряд макро- и микро- факторов,
оказывающих взаимовлияние друг на друга, в качестве
теоретической основы исследований может выступать
фрактальный анализ, соединяющий в себе принци-
пы синергетики и рефлексивного подхода [Василен-
ко, 2016]. При этом все чаще такие исследования носят
междисциплинарный характер, формируя целую ко-
горту теорий среднего уровня – в числе которых при-
сутствует и цифровое образование [Василенко, 2014]. Данная исследовательская область объединяет не толь-
ко теории социализации поколения Z, которые влия-
ют на специфику образовательного процесса, но и ме-
тоды исследования digital-социологии. В итоге данный
подход предполагает не только адаптацию эмпириче-
ских методов под специфику изучаемой выборочной
совокупности (например, через использование интер-
нет-опросов), но пополнение знаний о социально-зна-
чимых особенностях индивидов, включенных в про-
странство цифровых технологий. С этой точки зрения
исследование информационно-коммуникативных ком-
петенций современных студентов является важным ша-
гом к понимаю того, как происходит становление бу-
дущих профессионалов в условиях цифровой среды. МЕТОДЫ ИССЛЕДОВАНИЯ Как
видим, большинство преподавателей после присоеди-
нения к Болонскому процессу, отмечает увеличение
«бесполезной бумажной работы» (среднее значение со-
гласия с соответствующим суждением 4,69 из 5), сни-
жение возможностей занятия наукой (3,95 из 5). Не-
высокие показатели и у других пунктов – заработной
платы и возможности совмещать преподавание с ра-
ботой в других организациях. В таблице 1 приведены одномерные частотные
распределения ответов респондентов – преподавате-
лей магистратуры по вопросу «Оцените представлен-
ные ниже утверждения относительно Вас и вуза, в ко-
тором вы работаете» и средние значения по степени
согласия с каждым представленным суждением. Как
видим, большинство преподавателей после присоеди-
нения к Болонскому процессу, отмечает увеличение
«бесполезной бумажной работы» (среднее значение со-
гласия с соответствующим суждением 4,69 из 5), сни-
жение возможностей занятия наукой (3,95 из 5). Не-
высокие показатели и у других пунктов – заработной
платы и возможности совмещать преподавание с ра-
ботой в других организациях. Что касается студентов магистратуры, то их сред-
ний возраст составил 24,73 года при стандартном
отклонении 5,48. Минимальный возраст магистра
в выборке исследования – 20 лет, а максимальный –
64 года. Большинство опрошенных являлись выпуск-
никами бакалавриата 87,4 % (996 респондентов), ре-
же – специалитета (118 респондентов, 10,4 % от об-
щего числа опрошенных) или магистратуры (3 ре-
спондента; 0,3 %). Приведенные данные позволяют
сформировать представление о выборочной совокуп-
ности и приступить к оценке полученных данных. Что касается методов обучения, используемых при
подготовке магистров, то из анализа результатов опроса
преподавателей следует, что пока недостаточно распро-
странены такие современные методы, как онлайн-кур-
сы и совместные сетевые образовательные модули. Чис-
ло преподавателей, отмечающих в своем вузе наличие
высокой доли лабораторных занятий с использованием
качественного оборудования невелико (табл. 2). Также
отметим, что на вопрос «Отличаются ли используемые
Вами методы и методики обучения в программах ба-
калавриата и магистратуры?» большинство препода-
вателей дали положительный ответ (77 %), что свиде-
тельствует о наличии реальной специфики в процес-
се подготовки магистров в целом. МЕТОДЫ ИССЛЕДОВАНИЯ В 2018 г. инициативной группой Института обра-
зования НИУ «Высшая школа экономики» при содей-
ствии региональных исследователей, включая автора
статьи, была начата реализация проекта под назва-
нием «Рождение российской магистратуры». Одной
из приоритетных задач выступил анализ состояния
современной магистратуры и определение тех клю-
чевых процессов, в которых задействованы основные
стейкхолдеры – обучающиеся, преподаватели и рабо-
тодатели. В исследовании использовался метод анке-
тирования по квотным выборкам, география респон-
дентов включала более 23-х регионов Российской Фе-
дерации (включая следующие федеральные округа:
Северо-Западный, Южный, Приволжский, Уральский,
Сибирский, Дальневосточный). Всего были получе-
ны данные опроса 713 преподавателей магистратуры,
а также 3 480 студентов бакалавриата и 1 140 студентов
магистратуры (учитывались вузы, подведомственные
Министерству науки и высшего образования). Опрос
проводился как раздаточным способом, так и через
интернет-платформу Google. Для анализа результа-
тов использовалась программа обработки и анализа
социологических данных SPSS. В свете проблематики
настоящей статьи наибольший интерес представляют 19 Цифровая социология /
/
Т. 2 , № 3
2019 ––
наличие условий для выполнения научно-ис-
следовательской работы НПК; результаты опроса преподавателей, задействованных
в реализации магистерских программ, а также студен-
тов магистратуры, позволяющие охарактеризовать не-
которые аспекты реализации потребностей цифровой
экономики в образовательном процессе. результаты опроса преподавателей, задействованных
в реализации магистерских программ, а также студен-
тов магистратуры, позволяющие охарактеризовать не-
которые аспекты реализации потребностей цифровой
экономики в образовательном процессе. ––
возможности академической мобильности НПК; ––
возможности академической мобильности НПК;
––
возможность участия НПК в практической де-
ятельности в рамках специализации; ––
уровень заработной платы НПК. ––
уровень заработной платы НПК. Обратимся к анализу социально-демографических
переменных, дающих представление о преподавате-
лях и студентах российской магистратуры. Среднее
значение возраста преподавателей магистратуры со-
ставило 45,49 лет при стандартном отклонении 11,032. Минимальный возраст преподавателя магистратуры
в выборке опрошенных составил 24 года, а максималь-
ный – 92. Среднее значение по количеству препода-
вательского стажа насчитывает 19,8 лет при стандарт-
ном отклонении 10,349; при этом минимальный стаж
опрошенных преподавателей – 1 год, максимальный –
59 лет. Наличие ученой степени доктора наук указа-
ли 113 опрошенных, кандидата наук – 511, Phd – 4 че-
ловека; без степени – 76 преподавателей. В таблице 1 приведены одномерные частотные
распределения ответов респондентов – преподавате-
лей магистратуры по вопросу «Оцените представлен-
ные ниже утверждения относительно Вас и вуза, в ко-
тором вы работаете» и средние значения по степени
согласия с каждым представленным суждением. АНАЛИЗ РЕЗУЛЬТАТОВ Assessment of changes in higher education by teachers
Закрытый вопрос: «Оцените представленные ниже
утверждения относительно Вас и вуза, в котором
Вы работаете»
Распределение ответов респондентов по
5-ти балльной шкале, где 5 – полностью
согласен, 1 – полностью не согласен
Средние
значения
по степени
согласия с
утверждением
1
2
3
4
5
В России возможности для академической
мобильности (перехода на работу в другой вуз
в любом регионе) неограниченны
121
300
60
51
74
2,43
Заработная плата на академическом рынке труда
в моем регионе неконкурентоспособна в сравнении
с другими сферами занятости
8
48
139
221
194
3,89
Возможность совмещать преподавание и работу
на предприятии после присоединения к Болонскому
процессу сократилась
99
16
203
158
134
3,34
После перехода к новой системе образования стало
больше бесполезной бумажной работы
4
12
36
64
498
4,69
Заниматься научной работой после перехода
на новую систему образования стало сложнее
8
53
70
290
173
3,95
Составлено автором по материалам исследования / Compiled by the author on the materials of the study
Таблица 2. Распределение ответов преподавателей на вопрос «Что отличает дизайн
вашей магистерской программы?»
Table 2. Distribution of teachers’ answers to the question “What distinguishes the design of your master’s program?”
Закрытый вопрос «Что отличает дизайн вашей магистерской программы?»
Распределение ответов
кол-во ответов,
абс. ед. относ. кол-во
отв., %
Модульный учебный план
34
10,93
Участие работодателей в промежуточной аттестации обучающихся
(комплексные экзамены)
11
3,54
Сетевые модули, реализуемые с российскими вузами
1
0,32
Сетевые модули, реализуемые с зарубежными вузами
1
0,32
Онлайн-курсы на внешних российских платформах
29
9,33
Онлайн-курсы на внешних зарубежных платформах
3
0,96
Междисциплинарность курсов/модулей
18
5,79
Индивидуализация обучения
31
9,98
Интерактивные занятия с приглашенными участниками (практиками,
преподавателями других вузов)
4
1,29
Высокая доля лабораторных занятий с использованием качественного
оборудования
54
17,36
Практико-ориентированный подход
67
21,54
Сочетание онлайн-курсов на зарубежных и российских внешних платформах
1
0,32
Онлайн-курсы на зарубежных платформах в сочетании с высокой долей
лабораторных занятий с использованием качественного оборудования
3
0,96
Д
54
17 36 Таблица 1. Оценка изменений в высшем образовании преподавателями магистратуры
Table 1. АНАЛИЗ РЕЗУЛЬТАТОВ Присоединение России к Болонскому процессу
большинство преподавателей оценивает неоднознач-
но: среди тех, кто дал твердую положительную оценку
трансформациям, оказалось всего 7 % респондентов,
отрицательную – 19 %. В группе «сомневающихся»
мнения распределились следующим образом: скорее
положительными реформы находят 34,5 %, а скорее
отрицательными – 39,5 %. Поскольку присоединение
к Болонскому процессу предполагает стимулирование
конкурентоспособности на международном рынке об-
разовательных услуг, российской системе не только
необходимо адаптироваться под реалии цифровой
экономики, но и способствовать внедрению образо-
вательных инноваций. Показателями того, что выс-
шее образование вовлекается в новые мировые трен-
ды, может служить использование современных техно-
логий – on-line-обучения, высокотехнологичного обо-
рудования, индивидуализации и пр. При этом нельзя
забывать, что качество реализации инноваций зави-
сит от степени развития потенциала научно-педаго-
гических кадров (далее – НПК). На наш взгляд, здесь
следует выделить несколько составляющих: Обратимся к результатам опроса студентов маги-
стратуры. Особый интерес в рамках темы данной ста-
тьи представляют показатели, позволяющие охарак-
теризовать сформированность цифровых компетен-
ций магистрантов. Сюда можно отнести следующие
информационно-коммуникативные навыки: ––
поиск и обработка необходимой информации
из различных источников, в том числе специализи-
рованных баз данных; ––
способность использовать информационные тех-
нологии и обеспечивать безопасность информации; ––
навыки презентации результатов исследований
в виде статей, докладов, отчетов; ––
умение работать с данными, в том числе «большими». С этой целью респондентам было предложено от-
ветить на два вопроса, размещенных в разных блоках
анкеты. Первый касался того, насколько обозначен-
ные выше компетенции сформированы в настоящий
момент, а второй – оценки того, насколько, по мне-
нию респондентов, программа магистратуры, способ-
на обеспечить их формирование в целом. 20 Цифровая среда фр
р
Таблица 1. Оценка изменений в высшем образовании преподавателями магистратуры
Table 1. Цифровая социология /
/
Т. 2 , № 3
2019 Цифровая социология /
/
Т. 2 , № 3
2019 Что касается отношения магистрантов к некото-
рым современным методам обучения, в частности
к онлайн-курсам, то оно пока является неоднознач-
ным. Несмотря на ожидаемую положительную оцен-
ку, обусловленную спецификой поколения центени-
алов, чувствующих себя на просторах интернета как
«рыба в воде», только 590 респондентов из 1 139 отве-
тивших (51,8 %) ответили, что занятия в таком форма-
те должны проводиться часто и очень часто. Приме-
чательно, что 398 респондентов (34,9 %) выбрали ва-
риант «редко», а 151 респондент (13,3 %) считает, что
такие курсы вообще не нужны (для оценки использо-
вался закрытый вопрос «Как интенсивно должны про-
водиться в магистратуре онлайн-курсы?»). По-види-
мому, студенты пока не полностью уверены в эффек-
тивности таких образовательных технологий. Кроме
того, интерактивное общение с преподавателем об-
ладает своими преимуществами, среди которых мож-
но назвать обмен мнениями, возможность дискуссии,
получения комментариев и др. В итоге мнения респондентов по поводу оценки
уже имеющихся компетенций распределились сле-
дующим образом: в числе наиболее сформирован-
ных отмечены навыки поиска и обработки инфор-
мации, а среди наименее сформированных – инфор-
мационные технологии и обеспечение информаци-
онной безопасности (табл. 3). Как видим, респонденты отмечают у себя доста-
точную степень овладения цифровыми компетен-
циями на момент обучения в магистратуре. Однако,
данные навыки могли быть получены вне образова-
тельного процесса, например, путем самостоятельно-
го освоения в профессиональной деятельности или
в рамках дополнительных программ. В связи с этим
один из вопросов анкеты был направлен на то, чтобы
выяснить, как оценивают студенты непосредствен-
но программы магистратуры в части возможностей
формирования обозначенных выше компетенций. Одномерные частотные распределения по данно-
му вопросу представлены в таблице 4. Оценка про-
грамм магистратуры в части возможностей форми-
рования цифровых компетенций. Из представлен-
ных данных мы видим, что максимальный вклад
в формирование цифровых компетенций отмеча-
ет от 40,83 % до 67,69 %. Также в таблице 4 пред-
ставлены оценки обеспеченности формирования
в рамках магистратуры организационно-управлен-
ческих компетенций (системное мышление и уме-
ние решать комплексные задачи) и научно-иссле-
довательских навыков (анализ, способность прове-
дения фундаментальных и прикладных исследова-
ний в профессиональной деятельности). В завершении обзора результатов исследования мож-
но заключить, что обучение в российской магистрату-
ре постепенно перестраивается под реалии Болонско-
го процесса с учетом цифровизации окружающей сре-
ды. Несмотря на некоторые сомнения в эффективно-
сти образовательных трансформаций, высшее образова-
ние, по мнению автора, скорее не вернется к прошлому
опыту, учитывая тенденции глобализации и мировой
конкуренции. АНАЛИЗ РЕЗУЛЬТАТОВ Assessment of changes in higher education by teachers
Закрытый вопрос: «Оцените представленные ниже
утверждения относительно Вас и вуза, в котором
Вы работаете»
Распределение ответов респондентов по
5-ти балльной шкале, где 5 – полностью
согласен, 1 – полностью не согласен
Средние
значения
по степени
согласия с
утверждением
1
2
3
4
5
В России возможности для академической
мобильности (перехода на работу в другой вуз
в любом регионе) неограниченны
121
300
60
51
74
2,43
Заработная плата на академическом рынке труда
в моем регионе неконкурентоспособна в сравнении
с другими сферами занятости
8
48
139
221
194
3,89
Возможность совмещать преподавание и работу
на предприятии после присоединения к Болонскому
процессу сократилась
99
16
203
158
134
3,34
После перехода к новой системе образования стало
больше бесполезной бумажной работы
4
12
36
64
498
4,69
Заниматься научной работой после перехода
на новую систему образования стало сложнее
8
53
70
290
173
3,95
Составлено автором по материалам исследования / Compiled by the author on the materials of the study Таблица 2. Распределение ответов преподавателей на вопрос «Что отличает дизайн
вашей магистерской программы?»
Table 2. Distribution of teachers’ answers to the question “What distinguishes the design of your master’s program?”
Закрытый вопрос «Что отличает дизайн вашей магистерской программы?»
Распределение ответов
кол-во ответов,
абс. ед. относ. кол-во
отв., %
Модульный учебный план
34
10,93
Участие работодателей в промежуточной аттестации обучающихся
(комплексные экзамены)
11
3,54
Сетевые модули, реализуемые с российскими вузами
1
0,32
Сетевые модули, реализуемые с зарубежными вузами
1
0,32
Онлайн-курсы на внешних российских платформах
29
9,33
Онлайн-курсы на внешних зарубежных платформах
3
0,96
Междисциплинарность курсов/модулей
18
5,79
Индивидуализация обучения
31
9,98
Интерактивные занятия с приглашенными участниками (практиками,
преподавателями других вузов)
4
1,29
Высокая доля лабораторных занятий с использованием качественного
оборудования
54
17,36
Практико-ориентированный подход
67
21,54
Сочетание онлайн-курсов на зарубежных и российских внешних платформах
1
0,32
Онлайн-курсы на зарубежных платформах в сочетании с высокой долей
лабораторных занятий с использованием качественного оборудования
3
0,96
Другое
54
17,36
Всего ответов респондентов по данному вопросу
311 (100 %)
Составлено автором по материалам исследования / Compiled by the author on the materials of the study 2. Распределение ответов преподавателей на вопрос «Что отличает дизайн
вашей магистерской программы?» 21 Цифровая социология /
/
Т. 2 , № 3
2019 В целом и преподаватели, и обучающи-
еся осознают новые тренды, что выражается в обраще-
нии к цифровым компетенциям, хотя данные навыки
еще не в полной мере формируются в высшей школе. Таблица 3. Субъективная оценка сформированности цифровых компетенций магистрантов
Table 3. Subjective assessment of the formation of undergraduates’ digital competencies
Закрытый вопрос: «Какие компетенции сформированы
у Вас в большей степени, а каких, на Ваш взгляд,
Вам пока не хватает?»
Степень сформированности компетенций
сформированы
в наибольшей степени
пока не хватает
кол-во
ответов,
абс. ед. относ. кол-во
отв., %
кол-во
ответов,
абс. ед. относ. кол-во
отв., %
Искать и обрабатывать информацию из различных источников,
включая специализированные базы данных
973
85,40
166
14,60
Информационные технологии и обеспечение информационной
безопасности
606
53,20
533
46,70
Презентация результатов исследований в виде статей, докладов,
отчетов
831
72,96
308
27,04
Умение работать с данными, в том числе «большими»
814
71,50
325
28,50
Всего ответов респондентов по данному вопросу
1 139 (100 %)
Составлено автором по материалам исследования / Compiled by the author on the materials of the study 3. Субъективная оценка сформированности цифровых компетенций магистрантов
Table 3. Subjective assessment of the formation of undergraduates’ digital competencies 22 22 Цифровая среда Таблица 4. Оценка программ магистратуры в части возможностей формирования цифровых компетенций
Table 4. Assessment of master’s programs in terms of opportunities for forming digital competencies
Закрытый вопрос: «В какой мере, на Ваш взгляд,
выбранная Вами программа магистратуры, обеспечит
формирование следующих компетенций?»
Степень обеспеченности формирования компетенций
полностью
обеспечивает
в некоторой
степени
не обеспечивает
кол-во
ответов,
абс. ед. относ. кол-во
отв., %
кол-во
ответов,
абс. ед. относ. кол-во
отв., %
кол-во
ответов,
абс. ед. относ. кол-во
отв., %
Искать и обрабатывать информацию из различных
источников, включая специализированные базы данных
771
67,69
324
28,45
44
3,86
Информационные технологии и обеспечение
информационной безопасности
465
40,83
548
48,11
126
11,06
Презентация результатов исследований в виде статей,
докладов, отчетов
720
63,21
367
32,22
52
4,57
Умение работать с данными, в т.ч. «большими данными»
637
55,93
438
38,45
64
5,62
Иные компетенции:
Системное мышление и умение решать комплексные
задачи
552
48,46
533
46,8
54
4,74
Анализ, способность проведения фундаментальных
и прикладных исследований в профессиональной
деятельности
468
41,1
621
54,5
50
4,4
Всего ответов респондентов по данному вопросу
1 139 (100 %)
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политехнического университета. Режим доступа: https://goo.su/0fY0 (дата обращения: 03.10.2019). Соловов А.А. (2016). Подготовка бакалавров: проблема востребованности выпускников на рынке труда// Вестник Самарского университета. История, Педагогика, Филология. № 3-1. С. 58–64. Соловов А.А. (2016). Подготовка бакалавров: проблема востребованности выпускников на рынке труда// Вестник Самарского университета. История, Педагогика, Филология. № 3-1. С. 58–64. ЗАКЛЮЧЕНИЕ магистры, социализированные в цифровой среде,
довольно хорошо ориентируются в поиске нужной
информации, но не столь успешно владеют спосо-
бами обеспечения информационной безопасности,
информационными технологиями, которые сегодня
охватывают многие процессы и сферы профессио-
нальной деятельности. Отдельно следует отметить
и то, что роль университетов в части формирования
данных компетенций у магистрантов должна стать
ведущей – возможные «полюсы роста» можно отне-
сти и к пространству «больших данных». В заверше-
нии отметим, что достижение поставленных задач
цифровой экономики невозможно без развития ре-
сурсной базы университетов – как материальной, так
и социальной ее составляющих, а также комплекс-
ного анализа потребностей всех заинтересованных
сторон – преподавателей, обучающихся, работодате-
лей, администрации вузов. Только посредством про-
дуктивного диалога стейкхолдеров возможно усиле-
ние конкурентоспособности высшего образования
на международном рынке и обеспечение благопо-
лучия государства в целом. Проведенное исследование показывает, что россий-
ская магистратура в целом претерпевает ряд измене-
ний и ей, по-видимому, еще предстоит пересмотр не-
которых ориентиров для успешной реализации целей
цифровой экономики. Проблемные точки находят-
ся в слабой ресурсной базе образовательных учреж-
дений (в частности, отсутствии высокотехнологич-
ного оборудования), перегруженности преподавате-
лей в части работы с учебно-методической докумен-
тацией и, как следствие, снижении возможностей за-
ниматься наукой. На наш взгляд, эти аспекты рано
или поздно должны привести к необходимости вне-
дрения соответствующих управленческих решений –
регулированию соотношения аудиторной и внеауди-
торной работы, усилению научно-исследовательской
составляющей через делегирование некоторых функ-
ций персоналу, осуществляющему методическую ра-
боту (к примеру, оформление учебной документации). Необходимо также обратить внимание на внедре-
ние современных образовательных технологий – со-
вместных программ, сетевого обучения, онлайн-кур-
сов с учетом интересов обучающихся. Современные 23 Цифровая социология /
/
Т. 2 , № 3
2019 3 “Higher education: social Elevator or lost time”, Initiative all-Russian survey VTSIOM-Sputnik” (2019), Website of the all-Russian center for
public opinion research. Available at: https://wciom.ru/index.php?id=236&uid=9808 (accessed 03.10.2019). 4 “Student-2017: zeal for study and employment prospects Initiative all-Russian survey of VTSIOM” (2017), Website of the all-Russian center for
public opinion research. Available at: https://wciom.ru/index.php?id=236&uid=515 (accessed 03.10.2019). TRANSLATION OF FRONT REFERENCES 1 Decree of the President of the Russian Federation dated 09.05.2017 No. 203 “On the strategy for the development of the information society in the
Russian Federation for 2017-2030”. Available at: http://www.consultant.ru/document/cons_doc_LAW_216363/ (accessed 03.10.2019). 2 Ibid. 3 “Higher education: social Elevator or lost time”, Initiative all-Russian survey VTSIOM-Sputnik” (2019), Website of the all-Russian center for
public opinion research. Available at: https://wciom.ru/index.php?id=236&uid=9808 (accessed 03.10.2019). 3 “Higher education: social Elevator or lost time”, Initiative all-Russian survey VTSIOM-Sputnik” (2019), Website of the all-Russian center for
public opinion research. Available at: https://wciom.ru/index.php?id=236&uid=9808 (accessed 03.10.2019). 4 “Student-2017: zeal for study and employment prospects Initiative all-Russian survey of VTSIOM” (2017), Website of the all-Russian center for
public opinion research. Available at: https://wciom.ru/index.php?id=236&uid=515 (accessed 03.10.2019). 4 “Student-2017: zeal for study and employment prospects Initiative all-Russian survey of VTSIOM” (2017), Website of the all-Russian center for
public opinion research. Available at: https://wciom.ru/index.php?id=236&uid=515 (accessed 03.10.2019). 4 “Student-2017: zeal for study and employment prospects Initiative all-Russian survey of VTSIOM” (2017), Website of the all-Russian center for
public opinion research. Available at: https://wciom.ru/index.php?id=236&uid=515 (accessed 03.10.2019). 24
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Work and Speech Interactions among Staff at an Elderly Care Facility
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IFIP advances in information and communication technology
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tetsuro.chino@toshiba.co.jp Abstract. We observed bathing assistance, night shift operations, and hando-
ver tasks at a private elderly care home for 8 days. We collected approximately
400 h of recorded speech, 42,000 transcribed utterances, data from an indoor
location tracking system, and handwritten notes by human observers. We also
analyzed speech interaction in the bathing assistance task. We found that (1)
staff members are almost always speaking during tasks, (2) remote communica-
tion is rare, (3) about 75% of utterances are spoken to the residents, (4) the in-
tended recipient of utterances is frequently switched, and (5) about 17% of ut-
terances contain personal names. We also attempted clustering utterances into
passages, and about 33% of passages contained only one person’s name. These
results should be applicable in semi-automatic long-term care record taking. Keywords: cooperative work, speech interaction, care adfa, p. 1, 2011.
© Springer-Verlag Berlin Heidelberg 2011 Work and Speech Interactions among Staff at an Elderly
Care Facility Tetsuro Chino*1, Kentaro Torii*1, Naoshi Uchihira*1, Yuji Hirabayashi*2
*1 Corporate R&D Center, Toshiba Corporation, Kawasaki, Japan. *2 Institute of Technology, Shimizu Corporation, Tokyo, Japan. Work and Speech Interactions among Staff at an Elderly
Care Facility
Tetsuro Chino*1, Kentaro Torii*1, Naoshi Uchihira*1, Yuji Hirabayashi*2
*1 Corporate R&D Center, Toshiba Corporation, Kawasaki, Japan.
*2 Institute of Technology, Shimizu Corporation, Tokyo, Japan. Work and Speech Interactions among Staff at an Elderly
Care Facility
Tetsuro Chino*1, Kentaro Torii*1, Naoshi Uchihira*1, Yuji Hirabayashi*2
*1 Corporate R&D Center, Toshiba Corporation, Kawasaki, Japan. *2 Institute of Technology, Shimizu Corporation, Tokyo, Japan. 1
Introduction Japan’s increasingly aging population is an important issue, one urgent aspect of
which is caring for elderly persons with disabilities. Nowadays, care services are of-
ten provided by the devoted efforts of care staff at long-term care facilities. Care ser-
vices are characterized by what we call “action-oriented intellectual services.” Care
staff makes many decisions regarding medical care which require specialized
knowledge. They also assist elderly persons with disabilities in many activities of
daily living. Care service staff members have many types of professions and special-
ties. Also, care services must operate at all hours, throughout the year, and successful-
ly doing so requires a high degree of information sharing among staff, for example,
keeping records of care performed. [1] pointed out that medical staff spend much time
on indirect care, including record keeping and information sharing. Conventional IT
systems are fundamentally designed for desk work, however, and do not support the
needs for hands-free and eyes-free operations suited to action-oriented intellectual
services. [2] and [3] proposed a “voice tweet system” to overcome these problems. In that
system, “voice tweets” spoken by a staff member are tagged with the staff member’s location and motion, spoken keywords, and associations with background knowledge,
and based on these tags, the tweets are automatically delivered to an appropriate staff-
er. This system provides semi-hands-free and eyes-free communication among medi-
cal and care staff. Such a system requires knowing what kind of speech communica-
tion supports cooperative work in medical and care domains. We therefore performed
field studies at an elderly care home in Japan to analyze cooperative work and speech
interaction among care staff. location and motion, spoken keywords, and associations with background knowledge,
and based on these tags, the tweets are automatically delivered to an appropriate staff-
er. This system provides semi-hands-free and eyes-free communication among medi-
cal and care staff. Such a system requires knowing what kind of speech communica-
tion supports cooperative work in medical and care domains. We therefore performed
field studies at an elderly care home in Japan to analyze cooperative work and speech
interaction among care staff. We report on a series of field studies and the insights extracted from analysis of the
collected data. The remainder of this paper is organized as follows. Section 2 de-
scribes previous works. Section 3 describes the field and the tasks examined. 1
Introduction Section
4 describes the study methodology, and Section 5 presents an analysis of the collected
data. Finally, Section 6 gives our conclusions and proposed areas for future study. 2
Related Works [4] analyzed dynamically formed teams in a hospital emergency room, and identi-
fied three key factors in the design of team communication technology: (1) maintain-
ing awareness within the team, (2) making informative interruptions, and (3) support-
ing role-based calling. However, this study was limited to a few directly connected
rooms on a single floor. Further investigation of more distributed locations, such as in
larger hospitals or care facilities with multiple stories, may therefore be necessary. [5], [6], and [7] reported on the use of a commercial voice communication system
named “Vocera” in actual hospitals. They pointed out (1) that selecting communica-
tion artifacts is necessary, (2) that person-locating features can reduce human move-
ment, and (3) that remote voice communication has positive effects on the prioritiza-
tion and scheduling of tasks. They also described risks and problems associated with
voice communication devices. [8] reported on an analysis of nurse-patient conversations in a Japanese hospital. They found that most utterances effective for risk sharing are spoken by nurses who
are highly skilled at risk recognition and communication. In the “voice tweet system”
described above, sharing this kind of informative utterances as “voice tweets” can
have positive educational effects for the whole team. 3.2
Residents with Disabilities About 30 residents were living in this building during our field study. For conven-
ience, we classify residents into the following groups: ─ Residents with slight disability are independently mobile with the aid of a device
such as a cane or walker. ─ Residents with moderate disability require a wheelchair and staff assistance to
move around. ─ Residents with severe disability are bedridden, and can only be moved by multi-
ple staff using specially designed devices such as a stretcher. 3.1
Care Facility We performed field studies at a Japanese elderly care home over 8 days between
September 2011 and February 2012. Figure 1 shows an overview of the study loca-
tion. The building has four stories, with 40 private resident rooms, common living and
dining rooms, 1 care station, 1 bath area, and other rooms and spaces. Corridors, ele-
vators, and stairways connect the rooms. Fig. 1. Overview of the elderly care facility Fig. 1. Overview of the elderly care facility 3.3
Target Tasks We observed and collected data on 3 tasks. We observed and collected data on 3 tasks. ─ The bathing assistance task involves bathing residents on the first floor of the
building, and is performed by a special day shift team. ─ The nighttime staff performs night shift tasks, which involve monitoring resident
sleep, performing evening and morning care, checking vital signs on a schedule,
responding to nurse and sensor calls, and attending to other needs as they arise. ─ At scheduled shift changes, leaders of the incoming and outgoing shift personnel
perform the handover task. 4.1
Speech Data Collection We collected over 142 h of “subject sounds” via microphones attached to staff
members’ clothing. We furthermore collected another 142 h of “intercom sounds” via
intercom lines, and more than 98 h of “environmental sounds” via microphones set up
at various locations. We used IC recorders to record environmental sounds in
44.1 KHz 16-bit linear PCM stereo, and recorded subject sounds and intercom sounds
in 44.1 KHz 16-bit linear PCM mono. Figure 2 shows the equipment used. (a) Subject sounds
(b) Intercom sounds
(c) Environmental sounds (a) Subject sounds (a) Subject sounds (b) Intercom sounds Fig. 2. Sound data collection equipment 4.2
Extraction and Transcription of Utterances About half of the audio data was selected and automatically split into sound frag-
ments. We set the threshold parameter for length of silence between fragments to 500
ms, and human transcribers flagged fragments that were human utterances. Finally,
the human utterances were transcribed with time stamps and speaker identifiers. This
resulted in more than 42,000 transcribed utterances. 4.3
Other Collected Data We collected more than 64,000 staff location samples using a Bluetooth-based in-
door location tracking system that we developed. Human observers traced staff
movements onto paper, and investigated their actions. The observers recorded the
time, place, participants, media, and a brief memo on the content of observed conver-
sations involving staffers. Observers also videotaped subjects to get precise move-
ment data with timestamps. 5.1
Work Analysis of the Bathing Assistance Task We chose the bathing assistance task for analyzing work and speech interactions,
because this was the most complicated collaborative activity among our target tasks. The following is an overview of the bathing assistance task. 1. An “inner support” staff in the dressing room determines the next resident to
be bathed according to a schedule. Staff performs the bathing task according to the
disability level of the resident as follows: ! For residents with slight disability, the inner-support staff calls the resi-
dent’s room via intercom and asks the resident to come to the bath area. ! For residents with moderate disability, one or two “outer support” staff
members go to the resident’s room, check vital signs, and then ask the resi-
dent to come to the bath area by wheelchair with their assistance. If the resi-
dent agrees, then the staff members assist the resident from the bed to the
wheel chair, and bring the resident to the bath area on the first floor. ! For residents with severe disability, two or three outer support staff mem-
bers go to the resident’s room with a stretcher designed for bathing bedridden
residents, and check vital signs. If the resident agrees and conditions are met,
then the staff members assist the resident from the bed to the stretcher, and
bring the resident to the bath area on the first floor. 2. After arriving at the bath area, the resident undresses with inner or outer staff
assistance, according to need and availability. 3. Assisting staff also check the resident’s body and apply ointments or medicines
if necessary. 4. The inner support staff helps the resident bathe in a specially designed barrier-
free bathroom or a mechanized bathroom, as needed. 5. After bathing, mainly inner support staff helps the resident get dressed. 6. Outer support staff assists the resident from the first-floor dressing room to the
second-floor common living and dining rooms. 7. In the living and dining room, other staff members provide support by drying
the resident’s hair, bringing food, or helping the resident rest in the living room. 8. Finally, staff helps the resident return to his or her room. Since multiple residents bathe at the same time, some procedures are performed
simultaneously. Many factors can force changes in procedure. For example, residents
may not be in their rooms, or their mental or physical condition may preclude bathing. 5.1
Work Analysis of the Bathing Assistance Task It is worth noting that the tasks of getting dressed and undressed provide residents
with valuable opportunities to train and maintain their physical and intellectual abili-
ties, so the staffers provide minimal support and focus on safety. The time needed for
getting dressed and undressed differs from resident to resident and can reach 30 min
in the longest case, but efficiency is not the primary consideration in providing high-
quality services at elderly care facilities. Flexible collaboration is therefore necessary
to ensure these tasks are performed smoothly. 5.2
Utterances Spoken during Bathing Assistance We choose one bathing assistance task involving all female residents for our
speech interaction analysis. The task was performed over one morning, and represents
about 10% of the speech data described in section 4.1. According to records provided by the elderly care home, 6 staff members had bath-
ing assistance duties that morning. Among them, we focused on two outer support
staff members, who we refer to as A and B. We selected them because they moved
throughout the study location and interacted with many residents in many different
situations at multiple times and places. Table 1 shows a summary of utterances by A
and B and other staff and residents. Table 1. Summary of the extracted utterances Table 1. Summary of the extracted utterances Table 1. Summary of the extracted utterances
Speaker
Number
Percentage
Min. Ave. Max. Stdev. A
2,293
22.7%
0.074
1.683
5.697
1.214
B
1,692
16.8%
0.121
1.724
5.059
1.125
Others
6,110
60.5%
0.055
1.635
18.053
1.441
Total
10,095
100.0%
-
-
-
-
Extracted utterances
Length of utterances (s)
Table 1. Number and length of extracted utterances of staffers A and B of the outer support staff and otehers (other
persons, including other staff and residents). From the data, 10,095 utterances were extracted. The outer support staff spoke rel-
atively more than others, perhaps because of numerous stock phrases used with resi-
dents. The lengths of each utterance were similar for all speakers, perhaps due in part
to the influence of our automatic splitting process. Table 2 shows the spatial conditions of each conversation participant. Almost all
utterances (99.4%) were face-to-face communications. The proportion of remote
communication was only 1.0% for staffer A, and 0% for staffer B. Inner support staff
calls slightly disabled residents in this task, so there is little opportunity for remote
communication by outer-support staff. Introducing the voice tweet system at this loca-
tion would have a significant effect, because currently outer support staff does not
have access to remote communication media, which may force unnecessary move-
ment. Table 2. Summary of special conditions of each conversation participant Speaker
Number
Percentage
Number
Percentage
Number
Percentage
A
2,269
99.0%
24
1.0%
2,293
100.0%
B
1,692
100.0%
0
0.0%
1,692
100.0%
Total
3,961
99.4%
24
0.6%
3,985
100.0%
Face-to-face
Total
Table 2. Spatial conditions of conversational participants in utterances of staffers A and B (outer support staff). Remote Speaker
Number
Percentage
Number
Percentage
Number
Percentage
A
2,269
99.0%
24
1.0%
2,293
100.0%
B
1,692
100.0%
0
0.0%
1,692
100.0%
Total
3,961
99.4%
24
0.6%
3,985
100.0%
Face-to-face
Total
Table 2. Spatial conditions of conversational participants in utterances of staffers A and B (outer support staff). Remote 5.3
Frequency and Intended Recipients of Utterances We analyzed utterance frequency and the intended recipients of each utterance to
know how often and to whom the care staff talks during this task. Figure 3 shows a
time plot of utterances spoken by outer support staffer A (the upper half of the graph)
and staffer B (the lower half of the graph) during the 2.5 h of the bathing assistance
task. Dots at the upper, middle, and lower positions denote utterances spoken to resi-
dents, other staff, and oneself (monologues), respectively. Both A and B are almost
always speaking during the task, and the intended recipients quickly changes. In one
instance, the collected data show an outer support staff’s simultaneous conversation
with a resident and other staff in the dressing room. Fig. 3. Time plot and intended recipients of utterances
Time of the day, Hour. to residents
to other staffs
to her/him-self
utterances of
"outer-support" Staff B
to residents
to other staffs
to her/him-self
utterances of
"outer-support" Staff A
AM
AM
(triangle denotes remote communication via phone)
(2.5 hour) Fig. 3. Time plot and intended recipients of utterances Figure 4 shows the distribution of intended recipients for utterances by staffers A
and B. For both, about 75% of utterances are spoken to residents, about 17% to other
staff, and the remaining utterances are monologues. The frequency of utterances to
residents may be evidence of the importance of direct face-to-face verbal communica-
tion between staff and long-term residents. Fig. 4. Distribution of intended recipients of utterances of staffers A and B (outer support staff) Fig. 4. Distribution of intended recipients of utterances of staffers A and B (outer support staff) 5.4
Names in Utterances Accurate long-term care records are important for maintaining and improving the
quality of care services. Meaningful long-term care records require several key types
of information, such as who performed the care task, what kind of care was per-
formed, for whom the care was performed, when the care was performed, and where
it was performed. Mobile device–based support systems like the voice tweet system
can automatically record data on who (staff), when (time), and where (location). But
data about for whom care is performed (resident) may be difficult to acquire, because
devices such as physical tags attached to residents may be considered dehumanizing. We therefore analyzed the appearance of personal names in the utterances spoken by
outer support staffers A and B. Personal names appeared 703 times, 378 times for
staffer A and 325 times for staffer B. Table 3 summarizes the distribution of personal
names in each utterance. About 16.9% of utterances have at least one name that could
be a clue as to who is providing care or to whom care is being provided. Such infor-
mation could be used by an advanced voice tweet system to semi-automate long-term
care record maintenance in future care staff support systems. Table 3. Distribution of personal names in each utterance Speaker
Number
Percentage
Number
Percentage
Number
Percentage
Number
Percentage
A
1,938
84.5%
335
14.6%
20
0.9%
2,293
100.0%
B
1,377
81.4%
305
18.0%
10
0.6%
1,692
100.0%
Total
3,315
83.2%
640
16.1%
30
0.8%
3,985
100.0%
Total
Table 3. Distribution of number of presonal names in utterances of A (outer support staff A) and B (outer support staff B). 2 or more
Only 1
No (0) 5.6
Possible Extensions and Applications Background knowledge on each person who works or lives in the facility (e.g. name,
role, gender, shift/work schedule, profession, behavioral preference or tendency, etc.)
can be utilized to disambiguate the referent of each referring expression (e.g., person-
al names or pronouns) appearing in each utterance or passage. Based on this infor-
mation, together with automatically acquirable information (e.g., who, where, and
when) from a mobile device-based platform, the future stuff support system can gen-
erate and suggest estimated candidates or outlines of long-term care records to the
responsible staff member, and ask for confirmation. This mechanism may contribute
to lower costs and reduced load of care staff for the indirect care, as well as to im-
proved quality of long-term care records. 6
Conclusions and Future Work We reported on a series of field studies at an elderly care facility. For eight days we
observed three tasks: bathing assistance, night shift operations, and shift handover. We collected approximately 400 h of recorded speech, 42,000 transcriptions, data
from a location tracking system, and handwritten notes by human observers. We also
analyzed speech interaction in a bathing assistance task. We found that (1) staff mem-
bers are almost always speaking during the task, (2) remote communication is rare,
(3) about 75% of staff utterances are spoken to residents, (4) utterance targets are
frequently switched, and (5) about 17% of utterances contain at least one personal
name. We also attempted clustering utterances into passages. The rate of passages
with only one personal name can be raised to 33%. These results could be utilized in
semi-automatic long-term care record taking. Future targets for study include (1) an integrated analysis of not only the collected
speech data, but also the location, action, and observed descriptions of staff actions,
and (2) comparison with data from other hospitals to clarify differences and common
aspects, helping to create general models applicable to the broader healthcare domain. 5.5
Clustering Sequences of Utterances into Passages To get more information about who provides care and for whom, from utterances for
a semi-automatic long-term care record system, we attempted to cluster sequences of
utterances into what we call “passages.” A threshold parameter of the time gap be-
tween sequential utterances allows merging series of utterances spoken by each staffer
into passages. Figure 5 shows distributions of passages with specified numbers of
names, for 13 different settings (from 0.1 to 60 s) of the threshold for the time gap
between passages. Given an appropriate parameter value (3 s in this case, at the vertical dotted arrow in
Figure 5.), the percentage of passages with only one person-name (shown as the solid
line in the Figure 5.) can be dramatically improved from 16.1% (the unmerged utter-
ances case in Table 3) to 32.6%, and the average time length of the passage will be
16.7 s. This rate is sufficiently high and this length is sufficiently short to be utilized
for precise semi-automatic long-term care records in future care staff support systems. Fig. 5. Relation between the average length of passages (horizontal axis), and the distribution
of passages with a specified number of personal names (vertical axis), for multiple thresholds Fig. 5. Relation between the average length of passages (horizontal axis), and the distribution
of passages with a specified number of personal names (vertical axis), for multiple thresholds Acknowledgements This project was joint research between the Toshiba Corporation, the Shimizu Cor-
poration, and the Japan Advanced Institute of Science and Technology, and was sup-
ported by the Service Science, Solutions and Foundation Integrated Research Project
Program of the Japan Science and Technology Agency under the Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan. References 1. Lemonidou C., C. Plati, H. Brokalaki, J. Mantas, V. Lanara. Allocation of nursing time. Scand J Caring Sci. 1996; 10(3):131-6, (1996). 2. Uchihira Naoshi., et. al., Innovation for Service Space Communication by Voice Tweets in
Nursing and Caring: Concept and Approach in Japanese National Project, 20th Annual
Frontiers in Service Conference, (2011). 3. Kentaro Torii, Naoshi Uchihira, et. al., Service Space Communication by Voice Tweets in
Nursing, in proc. of AHFE2012, July, (2012). 4. Soyoung Lee, Charlotte Tang, et. al., Loosely Formed Patient Care Teams: Communication
Challenges and Technology Design, in proc. of CSCW2012, pp.867-876, February, (2012). 5. Joshua E. Richardson, The Effects of Hand Free Communication Devices on Clinical Com-
munication: Balancing Communication Access Needs with User Control, in proc. of
AIMA2008, pp.621-625, (2008). 6. Charlotte Tang, Sheelagh Carpendale, A Mobile Voice Communication System in Medical
Setting: Love it or Hate It?, in proc. of CHI2009, pp.2041-2050, April, (2009). 7. Charlotte Tang, Sheelagh Carpendale, et. al., InfoFlow Framework for Evaluating Infor-
mation Flow and New Health Care Technologies, in Intl. Journal of Human-Computer Inter-
action, Vol.26, No.4, pp.477-505, (2010). 8. Misako Nambu, Etsuko T. Harada, et.al. Risk Sharing Communication in Medical Settings:
Analysis of Nurses’ Conversation, Cognitive Studies, Vol.13, No.1, pp.62-79, (in Japanese),
March, (2006). 9. Ole Andreas Alsos, Awareness and Usability Issues in Clinical CSCW Systems, in proc. of
CSCW2010, pp.501-502, (2010).
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Observar las estrellas, llegar hasta las estrellas, ser una estrella. Introducción a un topos astral en la religión, el arte y el poder
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Hispania sacra
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Hispania Sacra, LXXIV
150, julio-diciembre 2022, 385-401, ISSN: 0018-215X, https://doi.org/10.3989/hs.2022.26
OBSERVAR LAS ESTRELLAS, LLEGAR HASTA LAS ESTRELLAS,
SER UNA ESTRELLA. INTRODUCCIÓN A UN TOPOS ASTRAL EN LA
RELIGIÓN, EL ARTE Y EL PODER
POR
Manuel Parada López de Corselas1
Universidad Complutense de Madrid
Y
Diego Chapinal-Heras2
Universidad Complutense de Madrid
Resumen
Este artículo explora el tópico cultural en torno a la conversión del ser humano en estrella (catasterismo) y al privilegio de situarse junto a los astros como recompensa a sus virtudes. Para ello, en primer lugar se expone sucintamente el desarrollo de dicho
topos en la Antigüedad Clásica. A continuación, se proponen varios casos de estudio entre los siglos XIV y XVI en el arte español
para analizar la continuidad y readaptación de este tema de la tradición clásica.
Palabras clave: estrellas; alma; cuerpo astral; apoteosis; Paraíso; memoria póstuma.
GAZING AT THE STARS, REACHING THE STARS, BEING A STAR.
INTRODUCTION TO AN ASTRAL TOPOS IN RELIGION, ART AND POWER
Abstract
This paper analyzes the cultural cliché of turning from human into star (catasterism), as well as the privilege of being placed
next to the stars as a reward for their virtues. In order to do this, the study briefly presents the development of this topos in Classical Antiquity. Afterwards, several cases of study between the 14th to 16th centuries in Spanish art are exposed. Their purpose is
to examine the continuity and readaptation of this topic in Classical tradition.
Key words: stars; soul; astral body; apotheosis; Paradise; posthumous memory.
Cómo citar este artículo / Citation: Parada López de Corselas, Manuel y Diego Chapinal-Heras. 2022. «Observar las estrellas,
llegar hasta las estrellas, ser una estrella. Introducción a un topos astral en la religión, el arte y el poder». Hispania Sacra LXXIV,
150: 385-401. https://doi.org/10.3989/hs.2022.26
Recibido/Received
12-02-2021
Aceptado/Accepted 19-05-2022
1. Introducción
12
La tradición clásica presenta al ser humano como Homo
stellaris. Desde las primeras civilizaciones del Creciente Fértil hasta la icónica frase del astrofísico Carl Edward Sagan, se
1
manparad@ucm.es / ORCID iD: https://orcid.org/0000-00015097-7934
2
chapinalheras@gmail.com / ORCID iD: https://orcid.org/00000002-6992-184X
ha definido al hombre como el único animal que observa las
estrellas, el único que desea llegar a ellas y, en última instancia, convertirse en ellas. Este trabajo propone un recorrido sucinto a través de las conexiones religiosas, morales,
literarias y artísticas de dicho tópico en el mundo antiguo
grecolatino.3 A partir de dicha base teórica, se plantea una
3
Próximo Oriente y Egipto fueron fundamentales en la construcción de las primeras teorías sobre las estrellas. No obstante, el análisis
de dichos contextos excede los objetivos de este trabajo.
Copyright © 2022 CSIC. Este es un artículo de acceso abierto distribuido bajo los términos de la licencia
de uso y distribución Creative Commons Reconocimiento 4.0 Internacional (CC BY 4.0)
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M. PARADA LÓPEZ DE CORSELAS Y D. CHAPINAL-HERAS, OBSERVAR LAS ESTRELLAS, LLEGAR HASTA LAS ESTRELLAS, SER UNA ESTRELLA...
primera aproximación a su continuidad y reinterpretación
en el ámbito hispánico entre los siglos XIV y XVI por medio
de varios casos de estudio.
Las comunidades helénicas, a partir de la época arcaica,
proponen diferentes teorías que buscan explicar el cosmos
por medio de la mitología o bien de métodos científicos.
Dos son las concepciones en las que nos interesa profundizar. Ambas se alejan de las teorías naturalistas que buscaban explicar el porqué de las cosas desde un punto de vista
científico, sin la injerencia de la religión.4 Por un lado, se profundizará en la vinculación de las estrellas con los dioses, ya
como instrumentos de estos, ya interpretando a las estrellas
como las propias deidades.5 Por otro lado, podemos identificar también una serie de razonamientos que asociaban a
los astros con el propio ser humano, al cuerpo humano con
el cuerpo astral. A su vez, dentro de este plano será necesario distinguir entre aquellas teorías que generalizan esta
afirmación para toda la comunidad, de otras en las que se
utilizaron diferentes mecanismos para situar a individuos
concretos en la bóveda celeste. Una suerte de catasterismo
que tenía como objetivo principal la legitimación de la posición de poder de los dirigentes.
2. Cuerpo astral, cuerpo humano
Mientras que Píndaro, en su primera oda, habla del Sol
como «otro astro brillando en el día por el desierto éter»,9
Sófocles sitúa a Zeus bajo las estrellas.10 No se trata de la
única divinidad a la que ya se vinculó en esta época con el
ámbito celeste, pues Eurípides, en su Helena, escrita en torno al 412 a. C., aporta la referencia escrita más antigua sobre Cástor y Pólux como estrellas, es decir, como dioses.11
Esto nos remite al fenómeno conocido como catasterismo,
καταστερισμός, resultado de la unión de κατά y ἀστήρ, es
decir, situado entre las estrellas. El término, que no aparece
con mucha frecuencia en la literatura,12 hace referencia a
la transformación de personajes, normalmente mitológicos,
en constelaciones. Heracles es otra de las figuras más recurrentes en este fenómeno.13
En términos generales, la identificación del dios o diosa con un astro no se generalizaría hasta la llegada de influencias de las religiones orientales y de salvación, como
los cultos isíacos venidos de Egipto. Un buen ejemplo está
en las Metamorfosis de Ovidio o de manera más clara en el
Asno de Oro de Apuleyo. Este autor del siglo II d. C. describe
la aparición de Isis —sincretizada con otras muchas divinidades femeninas que cita el texto, como Deméter, Venus,
Juno, Minerva y Diana— de cuerpo brillante (pellucidum),
con un disco en el pecho que, como la Luna, reflejaba la claridad (candidum lumen emicabat), y cuya túnica multicolor
pasaba del blanco más intenso (nunc albo candore lucida) al
oro del azafrán más florido.14
2.1. Estrellas y dioses
Los versos homéricos, la primera gran obra del mundo
griego, no parecen ligar las estrellas a los dioses. Así se aprecia en la Ilíada, cuando se comparan las fogatas del campamento aqueo, frente a Troya, y el cielo, poblado de estrellas.
Aquí, las estrellas «son visibles», ὡς δ’ ὅτ’ ἐν οὐρανῷ ἄστρα
φαεινὴν ἀμφὶ σελήνην.6 Lo cierto es que hasta el siglo VII
a. C., el mundo de lo divino en la cultura griega parece haber estado más centrado en elementos de nuestra geografía
que en las estrellas.7 Sin embargo, a partir de la época clásica, especialmente del s. V, se definirán varios modelos de
pensamiento relativos a esta idea.8
4
Por ejemplo, Anaximandro (Doxografía 14), Plinio el Viejo (HN
2, 5, 10; 2, 8, 29), Teofrasto (Thphr., CP, 7, 15, 1) y Ptolomeo (Harm. 109,
7-10), que consideraban las estrellas y planetas como entes inertes
cuya presencia se podía explicar de manera razonada sin acudir a la
mitología. Por su parte Aristóteles, siguiendo una línea de análisis cientificista heredada de autores como Eudoxo, se esforzó por explicar la
formación y el movimiento de los astros de acuerdo con leyes físicas
(Arist., Cael. 290a20; Mete. 346a1-6; GA 736b33-737a7). Con todo, él
mismo recoge las creencias, a su entender primitivas, acerca del origen mítico de este fenómeno, con las estrellas como dioses y con lo
divino abarcando la Naturaleza entera. Lo que Aristóteles sí defendía,
por otro lado, era la condición de las estrellas como entes vivos (Arist.,
Cael. 284a y 292b; Arist., Metaph. 1074a-b).
5
Pereda 2001, 70.
6
Hom., Il. 8, 555-561.
7
De un modo sutil, en ocasiones la descripción de los dioses recoge esta connotación estelar, al emanar luz su figura. Esta cualidad
divina está presente en la literatura antigua desde los Himnos homéricos. En las teofanías de Deméter y de Afrodita se puede reconocer a
estas diosas debido a su estatura sobrenatural, su belleza carente de
vejez, su fragancia intensa, su cabello largo y el resplandor de todo su
cuerpo: Himno Homérico II (a Deméter), 184-189; Himno Homérico V
(a Afrodita), 173-174.
8
Campion 2012, 149.
2.2. Estrellas y seres humanos
El gran salto conceptual que implica pasar de contemplar las estrellas como dioses o mensajes divinos descifrables por el ser humano, a considerar que el propio hombre
puede situarse en sus proximidades, o incluso transformarse en estrella, tuvo lugar en el ámbito de la filosofía. En este
sentido, uno de los autores más relevantes fue Platón. La
idea principal del pensador, en lo que respecta a este artículo, radicaba en la asociación del alma con las estrellas. Fue
un razonamiento tomado de la corriente pitagórica pues, en
efecto, la escuela de Pitágoras ya sostenía la existencia de
un vínculo entre almas y estrellas.15
Tal y como explica Platón en su Timeo, las almas habrían
sido creadas por el Demiurgo, el hacedor del universo, en
igualdad numérica con las estrellas; el Demiurgo comenzó
la obra, dejando que la continuaran dioses menores, de ahí
que el ser humano no sea perfecto.16 Dentro de cada astro,
se enseñaba al alma que, una vez en el cuerpo mortal de un
Pi., O., 1, 6 (Ortega).
S., Tr. 1105-1107: ...ὁ τοῦ κατ᾽ ἄστρα Ζηνὸς αὐδηθεὶς γόνος.
11
E., Hel. 137-144. Cf. Platt 2018, 230-234.
12
Sud. s.v. Ἵππαρχος, Νικαεὺς, Φιλόσοφος (Hiparco escribió la
obra Καταστερισμoί); Plin., Ep. 5, 17, 1 (Calpurnio Pisón leía sobre la
astronomía o formación de las constelaciones, περὶ καταστερισμῶν);
Alex.Aphr., in Metaph. 833, 2 (sobre las teorías entre los griegos, caldeos y babilonios).
13
Pàmias 2019, 197-198. Este aspecto queda reflejado de forma
clara en la obra Catasterismos, de Eratóstenes. Este autor de época
helenística describió las diferentes constelaciones, explicando su origen mitológico. Heracles, de un modo u otro, está presente es un gran
número de ellas.
14
Apul., Met. 11, 3, 3-5.
15
Barton 1994, 110; Williams 2003, 14.
16
Wright 1995, 65.
9
10
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hombre, estaba sujeta a las pasiones, tales como el amor,
el placer, el dolor, el temor y la ira; en caso de ser capaz de
dominarlas durante ese periodo, el alma viviría con justicia
y podría regresar a su estrella nativa. Si, por el contrario, en
esa vida se dejara llevar por el vicio, sufriría una metamorfosis hacia una mujer y, de continuar igual en su segunda vida,
pasaría a la naturaleza animal.17
El estoicismo, iniciado por Zenón de Citio, tenía como
uno de sus postulados principales la creencia de que lo divino, el alma y la naturaleza eran idénticos. De acuerdo con
esta corriente, el universo entero estaba vivo y los seres humanos estaban sujetos a un destino inexorable, a la vez que
tenían la capacidad de alterar sus vidas mediante la virtud
y un estilo de vida y educación moderados.18 Así lo vemos
en la epigrafía, pues un epitafio en honor a los caídos en la
batalla de Potidea del 432 a. C. hallado en el Cerámico de
Atenas recoge que el éter recibió sus almas y la tierra sus
cuerpos.19 Esta inscripción, de hecho, es la referencia epigráfica más antigua relativa a la ascensión del alma al éter,
a los cielos.20
Con el paso del tiempo, hubo diferentes momentos de
revitalización de ideas de carácter religioso y escatológico.
Como se ha apuntado antes, el periodo helenístico fue uno
de ellos, a raíz de la interacción de las culturas griega, babilonia y egipcia. Cabe destacar, en el ámbito de la astrología, la recopilación conocida como Corpus Hermeticum, que
entre otros aspectos analiza la relación entre el alma y los
cielos. Este corpus, que contaba con tratados inspirados en
parte en las ideas de Platón, pero combinando elementos
de otras civilizaciones, fue elaborado en Egipto en los siglos
II-I a. C.21
Paulatinamente, Roma fue incorporando muchos elementos de la cultura griega. En el Imperio se profundizará
en los aspectos morales de la relación entre el cuerpo humano y el cuerpo astral, de modo que la virtus será en muchas ocasiones el factor determinante para llegar a las estrellas o convertirse en una de ellas. El neoplatonismo fue una
de las corrientes más prolíficas, manteniendo la creencia de
la conexión entre nuestra entidad espiritual y las estrellas,
aunque con pequeñas variaciones en algunos detalles. Jámblico nos explica que Plotino, Porfirio y Amelio asignaban un
estatus similar a todas las almas, todas ellas procedentes
de una supraalma celestial y destinadas a residir en cuerpos
terrenales.22 También contamos con la aportación de Juan
Lido, quien a su vez nos habla del ensayo de Jámblico titulado Sobre el descenso del alma, el cual se hace eco de la
creencia, igualmente muy antigua, de la importancia del Sol
y la Luna, identificados como Hades y Perséfone, respectivamente, en este proceso de asignación de un espacio a cada
alma.23
Algunas corrientes de pensamiento establecían por tanto un vínculo entre el alma de todo ser humano y su correspondiente estrella en el firmamento. Aunque en Grecia el concepto ya existía, fue el mundo romano, en torno
al cambio de era, el contexto en el que se desarrolló más
profundamente la reflexión sobre las relaciones entre el
cuerpo humano y el cuerpo astral. Ovidio, en el comienzo
de sus Metamorfosis ya muestra la situación predominante
del ser humano sobre el resto de seres vivos del planeta;
al ser creado el hombre, se le ordenó ver el cielo y alzar
erguido el rostro hacia las estrellas (iussit et erectos ad sidera tollere vultus).24 Vitruvio, en su tratado De Architectura,
dirigido a Augusto, retoma la misma idea al considerar que
los seres humanos, «dotados por la naturaleza de un gran
privilegio respecto al resto de animales, como es el que caminaran erectos y no inclinados hacia adelante, observaron
las maravillas del universo y de los astros» (ut non proni sed
erecti ambularent mundique et astrorum magnificentiam
aspicerent).25
Cabe destacar que en la esencia de esta creencia se establece con claridad una norma para que la conversión en
estrella se lleve a cabo, algo que queda por tanto reservado
a una parte muy reducida de la población. Algunos autores
circunscriben este proceso de catasterismo a aquellas personas que hubieran destacado en diferentes ámbitos. Las
artes son uno de estos casos. Aristófanes habla del espíritu
de dos o tres poetas ditirámbicos que planean por el aire
y se refiere explícitamente a Ión de Quíos como una estrella matutina.26 En Roma, Horacio muestra un razonamiento
parecido y cargado de simbolismo, describiéndose a sí mismo —con escasa modestia— como poeta que tocará con
su cabeza los astros.27 También Manilio se jacta de subir a
los cielos para comenzar su poema Astronomica: nec per
iter socios commune regentibus actus,/ sed caelo noscenda
canam, mirantibus astris / et gaudente sui mundo per carmina vatis.28 Frente a dicha tendencia de origen griego, a
través de la cual los autores citados manifiestan su refinado
y erudito filohelenismo, los próceres de la tradición romana
virarán el sentido de dicha concepción del ámbito del virtuosismo hacia el de la virtud moral. Así, para Cicerón, en su
Sueño de Escipión, el cielo está abierto a los que cuidan de
la patria y se rigen siempre de acuerdo con sus costumbres
o mores maiorum; para ellos está reservado un lugar en la
Vía Láctea.29 Más explícito y detallado sobre este tema es
Macrobio, que en su Comentario al Sueño de Escipión (princ.
s. V) subraya que el alma noble, tras separarse del cuerpo,
regresa a las estrellas, de donde había descendido.30
La virtus, por lo tanto, es una condición sine qua non
para aspirar a tener un lugar entre las estrellas.31 De este
modo, en la Eneida Apolo felicita a Julo, hijo de Eneas, diciendo «¡Bravo, muchacho, por tu joven valor! ¡Así se llega a
las estrellas! Tú que has nacido de dioses, engendrarás dioses» (macte nova virtute puer: sic itur ad astra, dis genite et
geniture deos).32 A través de esta fórmula, Virgilio reafirma
el origen divino de la dinastía Julio-Claudia, en la misma tradición del sidus Iulium y retomando la asociación de este
tópico con la legitimación del linaje regio a la manera hele24
25
17
18
19
20
21
22
23
Pl., Ti. 41d-42e.
Campion 2012, 156.
IG I1, 945, l. 6.
Pàmias 2019, 193.
Campion 2012, 150, 158.
Iamb., de An. 6, 26.
Lyd., Mens, 4, 149.
387
26
27
28
29
30
31
32
Ov., Met. 1, 82-86.
Vitr., 2, 1, 2.
Ar., Pax 827-845.
Hor., C. 1, 1, 35-36.
Manilius, Astro. 2, 140-142.
Cic. Resp. 6, 15 y 24.
Macr., Comm. 1, 9, 1-3 y 10.
Wright 1995, 125; Martínez Astorino 2017, 18-19.
Verg., Aen. 9, 641-642.
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M. PARADA LÓPEZ DE CORSELAS Y D. CHAPINAL-HERAS, OBSERVAR LAS ESTRELLAS, LLEGAR HASTA LAS ESTRELLAS, SER UNA ESTRELLA...
nística.33 En la misma obra Virgilio insiste en el significado
virtuoso de esta idea a través de la frase «eligieron las penurias los que siguieron a las estrellas» (opta ardua pennis
astra sequi).34
2.3. Ser una estrella
Ser una estrella no implica necesariamente transformarse en dios. Según el pensamiento platónico el alma al
retornar al cielo no es un dios, sino que está entre los dioses.35 Pero a través de los mecanismos de heroización y de
la apoteosis, el catasterismo se terminaría vinculando a la
divinización de los mortales. Las monarquías helenísticas
desarrollaron esta idea, heredada de las diferentes civilizaciones que abarcó el imperio de Alejandro Magno.36 Berenice II (h. 269-221 a. C.), casada con Ptolomeo III Evergetes,
ofreció su cabellera a Afrodita en el 253 a. C. rogando que
su esposo resultase vencedor en el contexto de la tercera
guerra siria. Este exvoto desapareció misteriosamente; por
ello, el astrónomo Conón de Samos explicó a la reina que
Afrodita había llevado consigo al cielo la cabellera y que con
ella formó la constelación que desde entonces se conoció
como Cabellera de Berenice. Asimismo, el poeta Calímaco
compuso una elegía sobre este asunto. Por supuesto, todo
formaba parte de la propaganda regia ptolemaica, que se
complementó con monedas en las que se incluía el retrato
de la reina y una cornucopia entre dos estrellas.37 Otro caso
similar lo ofrece Ptolomeo V Epífanes (204-181 a. C.). Hazzard vincula a este monarca la acuñación de una tetradracma de plata en cuyo anverso se representa al rey junto con
una estrella de seis puntas o un cometa, mientras que en
el reverso vemos un águila sobre un rayo, ambos símbolos
de Zeus, la inscripción ΠΤΟΛΕΜΑΙΟΥ ΒΑΣΙΛΕΩΣ y, de nuevo,
una estrella de seis puntas o un cometa.38 También de época
helenística datan las tetradracmas acuñadas por Antíoco IV
Epífanes de Siria (175-164 a. C.), donde vemos a veces una
estrella y en otras ocasiones dos.39
En Roma, el concepto de catasterismo se extendió especialmente a partir de la traducción de Ennio de la obra de
Evémero.40 Sin embargo, el gran paso hacia la apoteosis de
individuos específicos llegará en el periodo de la formación
del Imperio. Esta práctica se aplicó a algunos de los emperaPandey 2018, 58-59.
Verg., Aen. 12, 892. Dicho concepto, retomado por Séneca, a
través de su frase «no es cómodo el camino desde la tierra hasta las
estrellas» (non est ad astra mollis e terris via; Sen., Her. F. 437), ha
gozado de gran éxito en la tradición occidental, de modo que ha configurado la máxima ad astra per aspera (hasta las estrellas mediante el
sacrificio), con numerosas variantes.
35
Pl., Ti. 42d.
36
Es posible, de hecho, que el propio Alejandro Magno pusiera
en práctica este mecanismo. Según nos informa Plinio el Viejo, el famoso pintor Apelles elaboró una obra en la que el rey macedonio aparecía
representado junto con los Dioscuros (Plin., HN 35, 36, 93).
37
Gutzwiller 1992; Clayman 2011; Van Oppen de Ruiter 2015,
71-115.
38
Hazzard 1995, 415-430.
39
Head 1911, 762.
40
Seznec 1980, 19. De acuerdo con el evemerismo, los dioses
habían sido humanos de tiempos pasados cuyas hazañas les habían
hecho quedar recordados como seres divinos. Por extensión, esta
creencia facilitaría la apoteosis de un individuo en época posterior gracias a sus hazañas.
dores, incluyendo en varios casos incluso a otros miembros
de la familia imperial. Ovidio conecta el catasterismo de personajes mitológicos, como Rómulo y Hersilia, con la apoteosis de Julio César, a quien Venus «llevó a los celestes astros»
tras ser asesinado.41 Mediante esta versión, el emperador
Octavio Augusto podía consolidar su posición en el trono y
la gens Julia, ya vinculada con Venus, reforzaba esta impronta divina con el ascenso del propio Julio César a los cielos.
No fue necesario crear de la nada este episodio, pues
ya antes había habido un movimiento popular que enlazó
a César con los dioses.42 Cicerón, por ejemplo, criticó en la
primera de sus Filípicas a los que honraban a Julio César
con un altar y lo que parecía un culto emergente en torno
a su figura. Finalmente, en el 42 a. C. se proclamó por ley
que era un dios. Pero incluso desde años antes —a partir
de su muerte, en el 44 a. C.— se había ido normalizando la
representación de Julio César, tanto en esculturas como en
monedas, con atributos divinos de carácter astral como es el
caso de la estrella o sidus y la corona radiada.43 Marco Antonio y Octavio Augusto, en su argumentario como herederos
de Julio César, trataron de apropiarse del sidus Iulium en su
numismática (Fig. 1a-c).44 En este sentido, destaca el denario acuñado por Octavio probablemente en Caesaraugusta
entre el 19 y el 18 a. C. Calígula, a su vez, acuñó monedas
en el año 37 d. C. con su retrato y el de Augusto con corona
radiada y flanqueado por dos estrellas (Fig. 1d).45
Según interpretaciones recientes, en el foro de Augusto se siguieron estos mismos parámetros. En su exedra izquierda se exponía el grupo de Eneas, estatuas de los reyes
de Alba Longa y de miembros de la gens Julia; en la exedra
derecha, esculturas de Rómulo y de los summi viri. Vivó i
Codina y Lamuà Estañol sostienen que los pórticos del foro
de Augusto y ambas exedras, todo ello abovedado, actuarían simbólicamente como la Vía Láctea, cuajada de las citadas estatuas como si se tratasen de cuerpos celestes.46 La
galería izquierda culminaba con el divus Iulius del aula del
Coloso, mientras que la derecha, con una estatua de Agripa
con su propio sidus.47 Ambas figuras estaban enmarcadas
por monumentales serlianas que enfatizaban su carácter
teofánico.48 Asimismo debe recordarse el templo de César,
también conocido como templo del Cometa (véase Fig. 1b),
al cual Ovidio se refiere así por boca de Júpiter:49
33
34
41
279.
Ov., Met. 15, 843-851 (Pérez Vega); cf. Martínez Astorino 2017,
Sobre el sidus Iulium y la política augustea véanse en general
Schmid 2005; Pandey 2013; Pandey 2018, 35-82.
43
Koortbojian 2013, 4-8, 27-28, 48-49, 121-122 y 226. Debemos
poner en relación este fenómeno con la contribución de Virgilio y su
Eneida. Es su exhaustivo estudio, M. F. Williams (2003) demuestra que
el poeta supo utilizar con maestría la estrella de César para legitimar a
la gens Julia, retrocediendo hasta su mismísimo origen.
44
Pandey 2018, 43-50.
45
RIC: 2.
46
Vivó i Codina y Lamuà Estañol 2015, 69-70. Se apoyan en Manilius, Astro. 1, 771-772.
47
Vivó i Codina y Lamuà Estañol 2015, 69. Estos autores sugieren
que la muerte de Agripa (12 a. C.) y la creencia en su apoteosis y sidus
propio (Dión Casio, 53, 30, 2; 53, 29, 7-8) justificaría su inclusión en el
programa del foro de Augusto y la modificación del proyecto de la testa
del pórtico sureste.
48
Parada 2015, 49-50; Pandey 2018, 142-150.
49
Ov., Met. 15, 838-851.
42
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Figura 1
a) Denario en honor de Julio César y Venus Genetrix
acuñado en Roma en el 44 a. C. por P. Sepullius Macer.
b) Denario con retrato de Octaviano y representación
del templo de César con el sidus Iulium en el frontón,
acuñado el 36 a. C. c) Denario con retrato de Augusto y el
sidus Iulium, acuñado en Caesaraugusta el 19-18 a. C. d)
Denario con retratos de Calígula y de Augusto divinizado,
acuñado en Lugdunum el 37 d. C.
389
y de su César arrebató a sus miembros y —sin permitir
que en el aire se disipara— su reciente ánima llevó a
los celestes astros (caelestibus intulit astris), y mientras
la llevaba, que luz cobraba y fogueaba sintió y la soltó
de su seno. Que la luna vuela más alto ella, y llameante
arrastrando de espaciosa senda una crin como estrella
centellea (stella) y de su hijo [Augusto] viendo sus buenas obras confiesa que son que las suyas mayores y de
ser vencido se goza por él.
La literatura en torno a la divinización de Julio César no
está exenta de debate, tanto por la duda acerca de cuándo
empezó a ser considerado un dios, como por los motivos
iconográficos que lo representaban. En lo que concierne a
nuestro tema de estudio, resulta llamativo comprobar que
el proceso de catasterismo de Julio César se simboliza a veces por medio de una estrella, pero también en otras de un
cometa. Supuestamente, cuando Octaviano celebró sus primeros juegos en honor a Venus Genetrix poco después de
la muerte de su padre adoptivo, los espectadores pudieron
observar en el firmamento un cometa, lo que se interpretó
como el alma de Julio César yendo al cielo, en la línea de
lo que más tarde diría Ovidio. Analizando las referencias en
torno a este aspecto, Gurval sugiere interpretar el cometa
como el transporte temporal del alma hacia el mundo celeste, y la estrella como el símbolo eterno de su divinidad.50 A
este respecto, Pandey no ve creíble que Augusto elaborara
este tipo de manipulación en una etapa tan temprana de su
recorrido político.51 Por otro lado, sí parece que circularan
historias que asociaban a un joven Octaviano con el plano
estelar, posiblemente elaboradas cuando aún no era el Princeps Senatus. Se decía por ejemplo que el propio Cicerón
había soñado con un muchacho que había descendido de
los cielos, portando el látigo de Júpiter Capitolino.52
A partir de Augusto fueron varios los emperadores divinizados, en la mayoría de los casos de manera póstuma,
aunque Domiciano llegó al extremo de reivindicar su propia
apoteosis en vida.53 El momento culminante de la consecratio era el ascenso del alma del emperador o emperatriz a
los cielos (relatio ad sidera).54 Este asunto se representa en
el relieve de la apoteosis de Sabina, esposa de Adriano. En
él, un genio alado sostiene una antorcha y lleva sobre sus
espaldas a Sabina, de modo que personifica al sidus divale
de la emperatriz camino de un puesto de honor en los cielos
(Fig. 2).55 Por su parte Septimio Severo plasmó esta creencia
en su propio palacio en el Palatino. En la fachada de este
edificio, el emperador aparecía representado entre los dioses planetarios, a la cabeza de las siete esferas celestiales.
En el interior del palacio, en una bóveda, el propio Septimio
Severo ejercía de juez en un salón cósmico, con el techo decorado con los cielos y horóscopos.56
Fuente: CoinArchives.
Gurval 1997, 39, 70.
Pandey 2013, 445-446.
52
Zanker 2008, 70-71.
53
Stat., Silv. 1, 1, 49-55; Suet., Dom. 13, 2.
54
Arce 1988, 131-140.
55
Liverani 2004.
56
L’Orange 1953, 35. L’Orange subraya además la influencia
oriental en este tipo de representación artística, mostrando el paralelismo con la corte real Arsácida en Babilonia, que contaba con un salón
abovedado en el que se podía ver a los dioses reflejados en la cúpula.
50
Cuando con sus méritos haya igualado sus años,
las etéreas sedes y sus emparentadas constelaciones
tocará (sidera tanget) [César]. Esta ánima, entre tanto,
de su asesinado cuerpo arrebatada, hazla tú luminaria
(iubar), para que siempre los Capitolios nuestros y el
foro, divino, desde excelsa sede vigile Julio». Apenas
ello dicho había [Júpiter] cuando en medio de la sede
del Senado se posó la nutricia Venus, para nadie visible,
51
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Figura 2
Apoteosis de Sabina (detalle), procedente del arco de Portugal en Roma
Fuente: Museos Capitolinos, Roma. Fotografía de Miguel Hermoso Cuesta.
En cuanto a los ciudadanos particulares, la epigrafía latina aporta epitafios que hablan del alma del difunto ascendiendo a las estrellas.57 El relieve de Amiternum (h. 20 a.
C. — h. 20 d. C.) puede interpretarse como un testimonio
temprano en el arte romano de la relación del difunto con
el ámbito sideral durante sus exequias (Fig. 3).58 En él varios porteadores llevan la imago funeraria recostada de un
particular, junto a la cual se dispone un bastidor o baldaquino decorado con ondas marinas y constelado de estrellas
y Luna, quizás para aludir al ascenso del alma del difunto a
los cielos.
En la iconografía funeraria romana desde el siglo II se
registra una identificación entre los difuntos y los dioses o
héroes divinizados por sus virtudes, fenómeno que Matz
denominó Privatapotheose y Wrede consecratio in formam
deorum. Es bien conocido este fenómeno a través del cual
los difuntos se equipararon a Venus y Marte, Selene y Endimión, Hércules y Ónfale y otros.59 En nuestra opinión esta
tendencia, en unión a la heroización familiar —no estatal—,
57
Accesit astros, Tartari fugit domos (CLE 1535B, 2); hic corpus
posuit, sedem dicavit in astris (CLE 1836, 6); corporeos rumpens nexus
qui gaudet in astris (CLE 668, 2); cf. González-Berdús 2018, 256, n. 19
(una investigación centrada más en la esfera cristiana). Véase también
Lattimore 1942, 311-312.
58
Arce 1988, fig. 2.
59
Borg 2019, 192-238.
Figura 3
Relieve con cortejo fúnebre hallado en Amiternum
(detalle)
Fuente: Museo nazionale d’Abruzzo, L’Aquila. Fotografía: Sailko.
podría explicar la iconografía de los techos de las tumbastorre de Jámblico (83 d. C.) —con bustos, águilas y parejas
de figuras aladas— y de Elahbel (103 d. C.) en Palmira. Dichos ejemplos palmirenos recuerdan al techo del peristilo
del templo de Baco en Baalbek (siglo II), en el que una trama
de motivos estrellados y vegetales acoge bustos de dioses,
héroes y otras personificaciones, como si de constelaciones
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Figura 4
Cubierta del peristilo del templo llamado de Baco en
Baalbek
391
bre fondo azul y rodeados de estrellas-flor de seis puntas
(Fig. 5). Los ejemplos palmirenos se han empleado para explicar la tradición que culminaría en el fresco procedente
del techo de la villa romana de época de Constantino en
Tréveris, en el que se representan varios bustos y parejas de
figuras aladas.60
3. Recepción en el ámbito hispánico medieval y
renacentista
Fuente: fotografía de J. E. Bruce.
Figura 5
Vista general de la cámara funeraria de la torre-tumba de
Elhabel, Palmira
El apartado anterior ha esbozado la trayectoria de las
creencias en las estrellas en el mundo clásico como elementos conectados con el plano divino, tratando de ahondar en
la asociación entre los astros y la figura humana. La idea de
la apoteosis celeste se mantuvo, evolucionando de diferentes maneras según cada contexto, en los siguientes siglos.
Con el objetivo de mostrar la recepción de esta tradición en
etapas posteriores y su reflejo iconográfico, a continuación
ofrecemos una serie de casos de estudio en la esfera hispánica medieval y renacentista. Para ello, no obstante, es
esencial recoger al menos de manera sucinta la transmisión
de estas ideas en el ámbito judeocristiano.
En el ámbito judío el topos astral se refleja desde al menos el siglo VI a. C. vinculado a la sanción divina del linaje de
Israel, a través de la promesa de Dios a Abraham: «Y lo llevó
fuera, y le dijo: Mira ahora los cielos, y cuenta las estrellas, si
las puedes contar. Y le dijo: Así será tu descendencia».61 Dicho pacto entre Dios y el pueblo elegido constituye la base
de las tres religiones del Libro. Tras la adoración del becerro
de oro y la rotura de las primeras tablas de la Ley, la alianza
con Dios se renovó a través de la segunda entrega de la Ley
a Moisés: «Acuérdate de Abraham, de Isaac y de Israel tus
siervos, a los cuales has jurado por ti mismo, y les has dicho:
Yo multiplicaré vuestra descendencia como las estrellas del
cielo; y daré a vuestra descendencia toda esta tierra de que
he hablado, y la tomarán por heredad para siempre».62
Como ya había hecho Aristófanes a finales del siglo V a.
C. y como harían Horacio y Manilio en el siglo I a. C., el libro de Daniel (siglo II a. C.)63 aplicó el topos astral al ámbito
de la exaltación de la virtud de los sabios y legisladores. El
profeta declara que «los entendidos resplandecerán como
el resplandor del firmamento; y los que enseñan la justicia
a la multitud, como las estrellas a perpetua eternidad».64 La
identificación de los justos con las estrellas en el más allá
fue constante en los textos judíos como el Libro de la Sabiduría, I-II Henoch, IV Macabeos y otros.65
Por su parte, en el siglo I san Pablo también vinculó el
topos astral con la vida en el más allá, proceso de escatología astrológica que domina en las corrientes herméticas y en
particular en el gnosticismo ya incipiente desde los comienzos del cristianismo. La novedad de san Pablo fue relacionar
las estrellas con la resurrección de la carne: «Una es la gloria
del sol, otra la gloria de la luna, y otra la gloria de las estrellas, pues una estrella es diferente de otra en gloria. Así también es la resurrección de los muertos. Se siembra en co60
61
Fuente: Livius.org.
62
se tratasen (Fig. 4). En el caso de la torre-tumba de Elhabel
observamos a varios difuntos representados en el techo so-
63
64
65
Lavin 1967.
Gen 15,5.
Ex 32,13.
Collins 2000, 2.
Dan 12,3.
Dupont-Sommer 1949.
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rrupción, resucitará en incorrupción. [...] Y así como hemos
traído la imagen del [hombre] terrenal, traeremos también
la imagen del celestial».66 En cuanto a san Mateo, manifiesta
la recepción del topos estelar como señal que indica un hecho extraordinario, concretamente una teofanía, a la manera de las monarquías helenísticas. Así, los magos de Oriente
preguntaron a Herodes «¿dónde está el rey de los judíos,
que ha nacido? Porque su estrella hemos visto en el oriente,
y venimos a adorarle».67 Pero este mismo evangelista aplica la imagen astral también a los bienaventurados: «Entonces los justos resplandecerán como el sol en el reino de su
Padre».68
Una vez constituido en religión oficial del Imperio Romano, el cristianismo asimiló obras fundamentales de la escatología astral clásica como el Sueño de Escipión, única parte
del De re publica de Cicerón que sobrevivió en Occidente
incluso durante la Edad Media —gracias a su carácter soteriológico—, a través del Comentario escrito por Macrobio
a principios del siglo V. Dicho texto se incorporó al gnosticismo cristiano —y a la mística bizantina— y tuvo su reflejo
en las reinterpretaciones medievales de la Escala de Jacob y
en el viaje por el más allá de Dante. Como Cicerón, Macrobio describe al alma descendiendo a la vida terrenal desde
la Vía Láctea y finalmente regresando a ese mismo lugar.69
Para dichos autores, los Campos Elíseos se encuentran en
las alturas de la Vía Láctea, entre las constelaciones de las
estrellas fijas, un reino compuesto de numerosas estrellas,
cada una de las cuales está formada por el resplandor del
alma de un héroe o un espíritu bendito.70 Entre los autores cristianos contemporáneos de Macrobio —a caballo de
los siglos IV y V— destaca el hispano Prudencio, quien en
su poema dedicado a santa Eulalia de Mérida relata el momento cuando ella dejaba los reinos mundanos «y abría un
camino sobre los astros» (et supra astra pararet iter) y a su
alma, en forma de paloma blanca, «se la vio partir, seguir a
los astros» (uisa relinquere et astra sequi).71 Este mismo autor narra la historia de dos hermanos; el primero toma el camino del vicio y el segundo toma el de la virtud y el esfuerzo,
por lo cual solo la cabeza de este último se mezcló con las
estrellas (illum sideribus caput inmiscere propinquis).72
En nuestra opinión, la bóveda anular del Mausoleo de
Constantina, hija de Constantino I —actual iglesia de Santa
Constanza— refleja este tópico de la escatología astral tardoantigua —tanto pagana como cristiana— y constituye un
hito de su transmisión temprana al arte cristiano (Fig. 6).73
En varias de las secciones de dicho mosaico se representan
cabezas, bustos, diversas figuras y genios alados o ángeles
en el interior de una retícula que contiene también motivos
florales y estrellados, junto a otras secciones que representan temas báquicos relacionados con el más allá —presididos a su vez por un busto colocado entre la vegetación— o
delfines atacando a pulpos, iconografía vinculada con la resurrección.74 Tales techos constelados de cabezas y bustos,
66
67
68
69
70
71
72
73
74
1 Cor 15,41-42 y 49.
Mat 2,2.
Mat 13,43.
Latura 2018, 310.
Harris 2012, 277-278.
Prudent., Perist., 3, 60 y 163.
Prud., Ham. 789-801.
Matthiae 1967, 3-53; Johnson 2009, 139-155.
Piazza 2006, 54-65; Arbeiter 2007, 153-230.
recurso que vimos en las torres-tumba de Palmira, también
son habituales en las catacumbas cristianas.75 En estas generalmente se han descrito como elementos «decorativos»,
aunque en el Mausoleo de Constantina se han interpretado como representaciones genéricas de los difuntos en un
Paraíso astral-vegetal o bien —en el caso de los bustos de
mayor tamaño situados frente a los ábsides— retratos de
los ocupantes del mausoleo.76 Subrayamos que en dicha
amalgama astral-vegetal se sitúan genios alados o ángeles,
héroes semidesnudos armados con espada e individuos de
distintas edades, lo cual insiste en la ambientación celeste y
en la idea de asamblea o reunión de virtuosos recogida en el
Sueño de Escipión. Opinamos que el conjunto, realizado hacia 350, guarda relación con la tradición bíblica —en particular con la resurrección según san Pablo— en combinación
con los referentes paganos que pocos años después estimularían el Comentario de Macrobio y los versos de Prudencio.
Asimismo, las figuras aladas podrían guardar relación con
la concepción ambigua que el Antiguo Testamento da a los
astros, los cuales en ocasiones no distingue de los ángeles
de la corte de Dios (Job 38,7; Sal 148,2) o los «ejércitos celestiales» (Gen 2,1), considerados como seres animados al
servicio del Altísimo.77
Figura 6
Mosaico de la bóveda anular del Mausoleo de
Constantina (detalle), actual iglesia de Santa Constanza,
Roma
Fuente: Algargos, Arte e Historia.
En sus Etimologías (princ. S. VII) san Isidoro de Sevilla se
muestra crítico con las interpretaciones mitológicas sobre
los astros, los nombres divinos de los días de la semana y
el zodiaco. Pese a ello, finalmente justifica los saberes astronómicos y su reinterpretación cristiana, recurriendo a
la metáfora del ascenso a las estrellas: «Este orden de las
siete disciplinas seculares, del mismo modo que condujo
desde los filósofos hasta los astros, así también debe levantar los espíritus, entregados al conocimiento humano de las
cosas terrenas, hasta situarlos en la contemplación de las
que son eternas».78 Otro ejemplo de la asimilación del to75
76
77
78
Wilpert 1903, 22-23.
Mackie 1997, 401-403.
Léon-Dufour 1965, 93.
Isid., Etym., III, 71, 41.
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pos que nos ocupa en el siglo VII hispano es la visión del
monje Baldario «el albañil» escrita por su compañero Valerio del Bierzo: «Yacía Baldario en el suelo, paciente de una
penosa enfermedad, cuando al amanecer el alma salió de
su cuerpo. Fue transportada por tres palomas a lo más alto
del cielo, más allá de las estrellas. Allí le condujeron a un
monte de extraordinaria belleza habitado por innumerables
ancianos vestidos de blanco que le llevaron ante la presencia del Señor».79
A partir de la Edad Media encontramos diversos casos a
través de la literatura y el arte en los que se ha amalgamado
el tópico astral escatológico vinculado a las personas virtuosas, particularmente en el ámbito funerario.80 Varios son los
aspectos que confluyen en dicho fenómeno. Por una parte,
la continuidad de la tradición clásica y sus readaptaciones al
cristianismo y al islam.81 Por otro lado, la reinterpretación de
los dioses paganos y los mitos como alegorías moralizadas.82
Finalmente, la relación de las élites, y en particular de las
monarquías europeas, con la astrología83 y con la imagen
solar construida a partir de Augusto como regente del orden
cósmico del Imperio y de Cristo como Sol de Justicia.84
Bajo dicha perspectiva podemos considerar la interpretación de Beato de Liébana en su Comentario al Apocalipsis (VI,
2, 58-61 y 91-107) sobre Cristo como la estrella más luciente
que delega su brillo a las almas beatíficas a través de la luz
interior que transmite el Espíritu Santo, al igual que san Isidoro pensaba que las estrellas no tenían luz propia, sino que
la tomaban del Sol. Dicha luz se transmite a los ángeles, considerados estrellas (Stellas dicit angelos esse ecclesiarum); a
la Iglesia, considerada como Sol, Luna y estrellas (Sol, luna, et
stellae Ecclesiae est, quae lumen veritatis ministrat); alcanza a
las distintas jerarquías de santos o bienaventurados, de nuevo entendidos como estrellas, de quienes finalmente irradia
a toda la comunidad desde el Paraíso: «Stellarum quoque relucentium millia, hoc est patriarcharum, prophetarum, apostolorum, martyrum, sacerdotum, et confessorum, virtutum
suarum luminibus radiantes».85
A partir de la base teórica expuesta líneas arriba, proponemos varios casos de estudio en ámbito hispánico —tanto cristiano como musulmán— entre los siglos XIV y XVI.
Nuestro objetivo se limita a trazar hipótesis de trabajo para
estimular futuros debates, a través de varias obras que habitualmente no se han puesto en relación entre sí, pero que,
bajo nuestro punto de vista, forman parte de la recepción
del topos que analizamos en este trabajo.86
393
de los reyes nazaríes de Granada, reflexiona sobre la memoria póstuma en estos términos:87
Desgraciado el que abandona la buena obra que
puede serle útil, o la bella oración que puede elevarle,
porque vive como una bestia voraz, deja perderse fuera
del buen camino las perlas más delicadas y valiosas
de su vida y despilfarra en los lugares viles y estériles
el depósito que Dios, ensalzado sea, le ha confiado.
Bienaventurado, en cambio el que conoce adónde ha de
ir a parar y aprovecha el breve tiempo de esta vida para
adquirir acciones meritorias, que permanecerán tras
él como estrellas brillantes, y para eternizar virtudes,
que le procurarán alabanza y premio; porque la oración
perseverante atrae y reclama la misericordia y la gracia
divinas y acerca y obtiene el perdón. Trabajen en esto
los hacendosos y tiendan a este fin los anhelosos.
A través de este texto nos encontramos de nuevo ante la
reiteración del topos antiguo que vincula la virtud humana
con la estrella resplandeciente, con la memoria póstuma y,
en última instancia, con el deseo de trascender a través de las
buenas obras. Se trata de un pasaje relacionado con la retórica astral tan común en la oratoria árabe y en las inscripciones
de la Alhambra, en las que existe una relación muy marcada
entre los «tropos astrales, nupciales y de vergel» para exaltar
a los monarcas nazaríes.88 Ibn al-Hatib, fiel servidor de Muhammad V, además de su crónica, también escribió algunas
de las inscripciones que decoran dicha ciudad palatina.89 Debido a tales circunstancias, su crónica se ha utilizado tradicionalmente para explicar quiénes son los personajes representados en posición sedente en una de las pinturas sobre cuero
de la sala de los Reyes (h. 1380) en el palacio de los Leones.90
Independientemente de que dichos personajes se traten de
los sucesivos reyes y/o de sabios vinculados a la corte nazarí,
el aspecto que nos gustaría destacar es el empleo del fondo dorado y la línea de estrellas de ocho puntas que divide
la composición (Fig. 7). Estos recursos, relacionados con la
pintura del gótico internacional para aludir a un ámbito sobrenatural —cielo o Paraíso—, pueden ayudar a completar
la interpretación del conjunto. En nuestra opinión, se trataría
de nuevo de la plasmación simbólica de estos personajes virtuosos de distintas épocas en la Vía Láctea, cuyas «acciones
meritorias» permanecerán como «estrellas brillantes» y que,
desde los cielos, servirán de ejemplo a las generaciones futuras, como nos recordaba Ibn al-Jatib.91 Este imagen astralmoral se completa con las pinturas de las alcobas laterales,
que recogen la segunda connotación del Paraíso en el islam,
de carácter vegetal-hedonista.92
3.1. Ibn al-Jatib y la sala de la Justicia de la Alhambra
Ibn al-Jatib (1313-1374), en su Historia de los Reyes de la
Alhambra, antes de comenzar su análisis sobre los hechos
Merino 2009, 42-43.
Sobre la evolución de la escultura funeraria, recordamos los
estudios clásicos de Panofsky 1964 y Bauch 1976.
81
Grabar 1968, 1973.
82
Seznec 1980.
83
Stierlin 1986; Azzolini 2013; Hankins 2019.
84
Mínguez 2001, 27-52.
85
Merino 2009, 301-303.
86
Las bases para dicha discusión se encuentran en Cumont 1942,
1949, 142-188; S’Jacob 1954, 166-167; Broëns 1972; Camporesi 1988,
20-35; Pereda 2001.
79
80
3.2. La portada del Paraíso de la catedral de Toledo y la
sacristía de las Cabezas de la catedral de Sigüenza
Como es habitual en el arte románico y en el gótico, en
las tres portadas de la fachada occidental o del Perdón de la
Ibn al-Jatib 1998, 4.
Puerta Vílchez 2013, 273-280.
89
Fernández Puertas 2011.
90
Véanse Rallo Gruss 2018; Robinson y Pinet 2008.
91
Este mensaje sería particularmente coherente con las funciones sapienciales, conmemorativas y tal vez funerarias del palacio de
los Leones de la Alhambra, que Ruiz Souza atribuye al conjunto (2001).
92
Parada (en prensa).
87
88
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Figura 7
Reyes nazaríes conversando en los cielos, sala de Reyes del palacio de los Leones, Alhambra de Granada
Fuente: Patronato de la Alhambra y Generalife.
catedral de Toledo (1410-1424)93 se desarrolla un programa
iconográfico que incluye imágenes escatológicas y temas
devocionales vinculados con la titularidad del templo y con
los cultos locales. En el tímpano central se representa la
Descensión de la Virgen para imponer la casulla a san Ildefonso. El Juicio Final, que habitualmente decora el tímpano
central, en la catedral de Toledo se sitúa en el derecho. Por
su parte, el tímpano izquierdo sorprende por su infrecuente
iconografía. Este tímpano, cerrado por una faja doble con
las armas de Castilla y León, está dividido en tres registros
decorados con grandes florones en forma de estrellas de
ocho puntas, rodeadas de otras flores de menor tamaño de
cinco y de ocho pétalos (Fig. 8). Los florones-estrella contienen cabezas o pequeñas figuras en su interior. En el registro
superior observamos una cabeza femenina coronada (reina)
y la de un hombre anciano barbado; en el intermedio, una
pequeña figura masculina, una cabeza masculina imberbe,
una cabeza de león, una cabeza coronada e imberbe (rey)
y una cabeza masculina barbada; en el inferior, una cabeza femenina, una cabeza masculina imberbe, dos cabezas
masculinas barbadas, una figura masculina luchando con un
león y una cabeza femenina. Las cabezas de los extremos
cuyas estrellas están parcialmente cortadas se adaptan al
marco arquitectónico y están escorzadas mirando hacia el
interior, excepto la figurita del registro medio, que se asoma
hacia el exterior del tímpano. Tras la faja heráldica, en la
primera arquivolta se representan profetas, entre ellos dos
figuras simétricamente dispuestas y cada una de la cuales
lleva las tablas de la Ley; podría tratarse de Moisés representado dos veces, en correspondencia a la primera y a la
segunda entrega de la Ley,94 o bien de Moisés y su hermano
Aarón. La segunda arquivolta se decora con sibilas y san93
94
Azcárate 1950, 2.
Ex 31,18-34.
tas con libros y varias mártires con palmas. En la tercera, se
disponen ángeles. En las claves de estas tres arquivoltas se
representan respectivamente las cabezas de un niño, de una
reina y de un hombre.
Figura 8
Portada izquierda o del Paraíso en la fachada occidental
de la catedral de Toledo
Fuente: fotografía de los autores.
Aunque en la tradición popular esta puerta se denomina
del Infierno o de las Palmas, es obvio que su tímpano alude
al Paraíso. La historiografía no ha mostrado gran interés por
ella, como se refleja en algunas síntesis: «La llamada portada del Infierno, a la izquierda, carece de interés artístico.
El tímpano está ornado con decoración vegetal, de donde
penden cabezas humanas relacionadas con el paraíso».95
Su interpretación paradisíaca se ha descrito como «cabezas
95
Franco 1993, 49.
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Figura 9
Descendimiento (detalles del Sol y la Luna) de Benedetto Antelami, catedral de Parma
Fuente: fotografía de los autores.
humanas entre hojas: los bienaventurados como frutos del
Paraíso»96, lectura que actualmente se considera incompleta, pues omite el componente astral de estas «estrellas
vegetales»97. Por nuestra parte, nos gustaría señalar que la
manera de representar a los bienaventurados es muy original y denota influencias italianas, como otras portadas de la
catedral de Toledo. El motivo de la cabeza en el interior de
una forma floral ya se utilizaba en el arte románico italiano
para aludir a un cuerpo astral, como les sucede al Sol y a la
Luna —«luminarias» principales del cielo— representados
en el Descendimiento esculpido por Benedetto Antelami en
1178 que se conserva en la catedral de Parma (Fig. 9). En
Toledo, los motivos vegetales cobran forma de estrella para
acoger en su interior a los bienaventurados, de modo que
nos encontramos ante una reinterpretación de los textos
clásicos que hemos comentado arriba, a través de una solución visual tomada de la iconografía astral del arte románico. Así, la imagen literaria de la apoteosis de aquellas personas virtuosas que recibían un sidus y un puesto de honor en
el firmamento —generalmente en la Vía Láctea— se aplica
en la sede primada toledana al Paraíso cuajado de estrellasflor desde las que brillan las virtudes de los bienaventurados
(Fig. 10).
Personajes como reyes, reinas, jóvenes y ancianos conviven en presencia del león guardián en posición central,
esto es, Cristo, león de la tribu de Judá. A su vez, en la parte
inferior derecha el personaje que vence a un segundo león
podría ser un trasunto de Sansón o de un Hércules moralizado. En suma, el tímpano del Paraíso de la catedral de Toledo
sintetiza la idea pagana y judeocristiana de la conversión del
virtuoso en estrella como premio en la otra vida y subraya
el papel de las jerarquías de bienaventurados como estrellas que guían a la humanidad según la interpretación de
Beato de Liébana. Debido a la potente influencia italiana en
la portada occidental de Toledo, cabe preguntarse si el mo96
97
Pérez Higuera 1988, 50, 52.
Nickson 2015, 190, 192.
Figura 10
Tímpano de la portada del Paraíso de la catedral de
Toledo
Fuente: fotografía de los autores.
saico del Mausoleo de Constantina (Santa Constanza) pudo
servir como referente conceptual interpretado a modo de
síntesis de sus imágenes astrales y vegetales de carácter escatológico.98 De ser así, la sede primada estaría reforzando
su vinculación con Roma y con el prestigioso referente constantiniano.
Asimismo, al igual que en los ejemplos antiguos analizados, sugerimos que el tímpano de Toledo posee un significado funerario y de legitimación dinástica, vinculado con la
capilla de los Trastámara, como veremos después. En este
sentido, resulta importante la relación de la portada con el
mandato divino al pueblo elegido, al que Dios premiará con
98
A este respecto, recordemos la flexibilidad del concepto medieval de copia, cuestión estudiada de modo pionero por Krautheimer,
quien subrayó la importancia del Mausoleo de Santa Constanza en dicho contexto (Krautheimer 1942, 26).
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una descendencia numerosa, como prometió a Abraham.99
Este mensaje se refuerza en la portada de Toledo por la presencia de las dos figuras con las tablas de la Ley en las arquivoltas. A este respecto, no deben olvidarse las palabras de
Dios al renovar la alianza con Moisés y su pueblo a través de
la segunda entrega de la Ley, como expusimos.100
Probablemente la portada del Paraíso de Toledo se tuvo
en cuenta, como referente iconográfico, a la hora de concebir el programa escultórico de la sacristía de las Cabezas en
la catedral de Sigüenza, diseñada por Alonso de Covarrubias
en 1532 y cuya bóveda cerró Juan de Durango en 1552.101 En
las enjutas de los arcos se representan santos y santas, así
como profetas y sibilas que anuncian a Cristo, como sucede
en la capilla Sixtina y en la sacristía del Salvador de Úbeda.102
Esta misma concordatio entre la Antigüedad clásica y el cristianismo afecta a la bóveda de la sacristía de las Cabezas.
Pese a que la sacristía de Sigüenza desarrolla un programa iconográfico sobre la virtud y la Salvación de carácter
humanista, no obstante Herrera advierte en él una «gran
carga de tradición medievalizante»,103 observación que
compartimos. En efecto, la cubierta de la sala se trata de
una verdadera bóveda celeste cuajada de personajes virtuosos de distintos estamentos —entre ellos, varias jerarquías
eclesiásticas—, de ángeles y de florones, como síntesis del
cielo pagano y el Paraíso cristiano (Fig. 11), a la manera del
tímpano del Paraíso de la catedral de Toledo. No creemos
que sean personajes «sufrientes» o condenados —como en
ocasiones se repite— pues están entre flores y en la presencia de ángeles; su gestualidad podría vincularse a la admiración por la contemplación divina. Por su parte los profetas,
sibilas, y santas que en Sigüenza decoran las enjutas, en Toledo se disponían en las arquivoltas. Ambas obras subrayan
la tradicional conexión administrativa, cultural y artística
entre la sede primada toledana y la catedral de Sigüenza,
así como la continuidad de la tradición clásica y medieval en
torno al topos sobre los cuerpos celestes.
El diseño reticular de la bóveda de Sigüenza pudo tomarse, precisamente, de Santa Constanza en Roma a través del
codex Escurialensis, como plantearon Herrera y Marías.104
Quizás la bóveda de Sigüenza sea el testimonio de la recuperación del referente formal del tímpano de Toledo a través
del estudio anticuario. En ella el mensaje forma deriva de la
misma tradición astral escatológica presente en Toledo, pero
las formas se han renovado a través de una recuperación
«arqueológica» de Santa Constanza y de su reinterpretación
manierista. Todo ello gracias al estudio anticuario que permitió realizar empresas artísticas análogas como el Doncel
de Sigüenza105 o las obras promovidas por el cardenal Mendoza.106 A este respecto, contamos con un hito clave que
permite entender esta recuperación cristianizada del sidus
en relación con Pedro González de Mendoza (1428-1495),
cardenal, arzobispo de Toledo y obispo de Sigüenza. El 11
Gen 15,5.
Ex 32,13.
101
Santos y Santos 2003, 136-137, 185-187.
102
Sebastián 1978, 38-45.
103
Herrera 1985, 18.
104
Herrera 1985, 18; Marías 1986, 210-211.
105
Sánchez 2010; Pérez Monzón 2016.
106
Entre la inmensa bibliografía sobre el patrocinio del cardenal
Mendoza destacamos Pereda 2009 por su ámbito de estudio a caballo
de España y la Roma papal.
99
100
Figura 11
Bóveda de la sacristía de las Cabezas (detalle), catedral de
Sigüenza
Fuente: fotografía de los autores.
de enero de 1495, mientras el cardenal Mendoza agonizaba
en Guadalajara y tras su muerte, «en el cielo sobre la casa
veíase una cruz muy blanca de extraordinaria grandeza».107
Este portento repetía el que había anunciado a Constantino
su victoria sobre Majencio, así como el que a su vez anunció a su hijo Constancio II su triunfo sobre Magnencio.108 En
los tres casos, se trataba de una interpretación de la señal
astral —cometa o estrella, transformado en cruz luminosa—
como vía divina de legitimación en clave cristiana. Pero en
el caso específico del cardenal Mendoza, el portento no se
asoció con una victoria militar, sino con el momento de su
muerte en olor de santidad y con la elevación de su alma
al cielo. Así, en la cronística sobre este introductor del renacimiento italianizante en España se llegó a la concordatio
entre el pagano sidus Iulium y el signo constantiniano sancionado por Dios, con miras a la beatificación del cardenal
y reafirmando su relación con la basílica de Santa Croce in
Gerusalemme en Roma.109 Un nuevo sidus se observó en
Madrid a la muerte de la emperatriz María de Austria en
1603: «En el mismo punto que expiraba vieron en el tejado
de su cuarto un maravilloso globo de resplandeciente luz, y
con ser tanta la oscuridad de la noche, parecía claro día».110
3.3. El sepulcro de Juan II e Isabel de Portugal
Como acabamos de exponer, el topos que nos ocupa se
retomó en la España de los siglos XIV-XV, tanto en la Alhambra, sede del poder del reino nazarí de Granada, como en
la catedral de Toledo, sede primada de Castilla. Pereda ha
planteado cómo esta metáfora estelar articula una de las
principales empresas artísticas de Isabel I de Castilla, el sepulcro de sus padres Juan II e Isabel de Portugal, realizado
por Gil de Siloe entre 1489 y 1493 en la cartuja de Miraflores
en Burgos. Su original tipología en forma de estrella (Fig. 12)
se vincula con los cuerpos celestes de los resucitados y pueLayna Serrano 1942, 318.
Parada y Martín 2014, 126.
109
Sobre el cardenal Mendoza y sus políticas artísticas en clave
providencialista véanse Pereda 2009; Parada y Martín 2014.
110
Méndez Silva 1655, f. 49v.
107
108
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de contextualizarse en relación con las palabras de Daniel
(12,3), san Pablo (1 Cor 15,39-49) y la Vita Christi de Ludolfo
de Sajonia, el Cartujano (ca. 1300-1377/78), obra que Isabel
la Católica conoció a través de un manuscrito que le facilitó
fray Hernando de Talavera.111 Completando a san Pablo, el
Cartujano señala que «el cuerpo glorificado será claro y refulgente en estas dos maneras, ca será claro e transparente
a manera de cristal y será luzio e resplandeciente a manera
de oro luzidísimo o de estrella muy relumbrante o de sol en
excesiva manera refulgente».112
Figura 12
Sepulcro de Juan II e Isabel de Portugal, cartuja de
Miraflores, Burgos
Fuente: Cartuja de Miraflores.
Como es sabido, en una obra de arte medieval convergen numerosos niveles de significado. Además de otros
referentes que insisten en el carácter cósmico de este sepulcro, como su posible relación con el esquema de la carta astral,113 en nuestra opinión su imagen estelar también
podría contextualizarse a través de un libro coetáneo, Los
doce trabajos de Hércules de Enrique de Villena (h. 1384 - h.
1434), impreso en Zamora en 1483 y en Burgos en 1499.
En el IX trabajo, Enrique de Villena toma como referente la
interpretación de Daniel 12, 3 por san Jerónimo y compara
a las personas sabias y virtuosas:114
Al firmamento, al sol, a la luna [y] a las estrellas,
como pone Sant Gerónimo trasuntivamente en el prohemio de la Brivia, a[l] mostrar que ellos deven ser firmes como el firmamento y i[n]corruptibles por tales
Pereda 2001, 69-73.
Ibídem, 70.
113
Pérez Monzón 2022, 238-240.
114
BNE, Inc. 2441, f. 20v. http://bdh.bne.es/bnesearch/detalle/
bdh0000179799
397
vicios, claros como el sol dando lumbre en exemplar y
doctrinar a todo el mundo, limpios como la luna [de]
corporal infectión, centell[e]antes como estrella por
castimonia, puridad, eloquencia y traditiva.
Pero además del marcado simbolismo astral en el sepulcro de Juan II, Pereda destaca la presencia reiterada de
leones, algunos de ellos andrófagos. Señalamos que, al igual
que sucede en el registro inferior del tímpano del Paraíso de
la catedral de Toledo, en el sepulcro burgalés se incluye una
representación de Sansón matando al león o bien Hércules.
También figuran otras imágenes leoninas que podrían aludir
a los guardianes del Paraíso o al propio Cristo,115 como sucedía en el registro central del tímpano toledano.
Recordemos que, como en el sepulcro de Juan II e Isabel
de Portugal, también en el tímpano del Paraíso de Toledo se
destaca la vinculación de la imagen escatológica estelar con
la realeza castellana, tanto a través de la faja decorativa con
las armas de Castilla y León, como a través de las cabezas
coronadas situadas en el interior de las estrellas-flor. Probablemente en Toledo se buscaba vincular este programa
salvífico con los enterramientos regios de la catedral. No
olvidemos que la portada del Paraíso (1410-1424) da acceso a la zona en la que se había construido a partir de 1374
la capilla Real de Enrique II, que ocupaba los dos primeros
tramos de la nave norte, junto a la actual capilla del Tesoro.
Enrique II había fundado esta capilla por disposición testamentaria en 1374 para sepultar su cuerpo y el de su esposa Juana Manuel, los de Juan I y su primera esposa Leonor
de Aragón, así como los de Enrique III y su esposa Catalina
de Lancaster. Las obras no se terminaron hasta principios
del siglo XV, coincidiendo con el comienzo de la portada occidental.116 La actual capilla del Tesoro, decorada con una
bóveda de mocárabes perfilados en oro, era la sacristía de
la capilla Real y esta debió de tener una decoración mucho
más rica.117 Las bóvedas de mocárabes habrían contribuido
a reforzar el simbolismo astral del conjunto formado por la
capilla funeraria de los Trastámara fundada por Enrique II
y la portada del Paraíso. De ser correcta esta hipótesis, la
decisión de Isabel la Católica en 1489 al acometer el sepulcro de sus padres estaría vinculada no solamente a la renovación del topos clásico y cristiano del cuerpo astral en
torno al Cartujano, sino que además se explicaría gracias a
sus precedentes en los más prestigiosos ámbitos de reafirmación escatológica y sapiencial de los soberanos nazaríes
y castellanos de los siglos XIV y XV.
3.4. Los sepulcros de Rodrigo Sánchez de Mercado y Per
Afán de Ribera
El libro del Cartujano pudo ser también fundamental
para la reiteración en el siglo XVI español del tópico del
cuerpo glorioso semejante a la estrella. La editio princeps
castellana de la Vita Christi fue la de Estanislao Polono en
Alcalá de Henares en 1502-1503, a la que siguieron sucesivas ediciones en Sevilla en 1520, 1530-1531, 1536-1537,
1543-1544 y 1551 y en Salamanca en 1562.118 Junto a la
111
112
115
116
117
118
Pereda 2001, 61, 67-69.
Azcárate 1950, 1-3.
Ruiz Souza 2006, 14-15.
Quiñones Melgoza 1969.
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obra de Kempis, el libro del Cartujano fue uno de los textos
más influyentes en la espiritualidad del siglo XVI hispano, a
través del impulso que le dieron Francisco de Osuna, Teresa
de Ávila y los Ejercicios ignacianos.119 No obstante, en dicha
centuria el topos astral vinculado al ámbito escatológico retomó las fuentes clásicas de una manera mucho más literal,
aunque probablemente con objetivos mucho más retóricos
que teológicos. Ofrecemos dos ejemplos representativos de
dicha tendencia en la epigrafía funeraria española.
El primero de ellos se encuentra en el sepulcro de Rodrigo Sánchez de Mercado Zuazola (último tercio del siglo XV1548) en San Miguel de Oñate, esculpido por Diego de Siloe
en 1528-1529 (Fig. 13).120 El epitafio latino de esta tumba
retoma la elevación del difunto por encima de las estrellas
debido a sus buenas obras —fundamentalmente, su evergetismo— como ocurría en la apoteosis romana:
santuario, permaneciendo como protector en la gran
ciudad. Como se adornan con tranquilas violetas del
monte y por su sagrado halo se levantan los miembros
muertos, así surgen estos templos por obra suprema y
llevan a su autor por encima de los astros (super astra
ferunt), al cual engendró el honorable Ochoa Mercado
con su esposa Zuazola, los cuales habitaban en este
lugar.
Figura 14
Lauda funeraria de Per Afán de Ribera, cartuja de Sevilla
Figura 13
Sepulcro de Rodrigo Sánchez de Mercado Zuazola, iglesia
de San Miguel, Oñate
Fuente: fotografía de Vicente Lleó Cañal.
El segundo ejemplo lo constituye la lauda funeraria en
bronce de Per Afán de Ribera (1509-1571), I duque de Alcalá
de los Gazules, contratada en 1571 a Juan Bautista Vázquez
el Viejo y fundida en 1573 por Bartolomé Morel en Sevilla
(Fig. 14).121 Además del epitafio, esta lauda contiene una segunda inscripción latina con el siguiente elogio en el que se
equipara al difunto con las estrellas, en la mejor tradición
romana:
Fuente: Jl FilpoC.
Rodrigo, doctor en ambos derechos, con este mármol pario mandó edificar un magnífico mausoleo.
Igualmente obispo de Ávila, construyó este claustro y
119
120
García Mateo 2000.
Gómez-Moreno 1950; González de Zárate 1992, 120-121.
Yace en este túmulo aquel que la virtud llevó hasta
los astros (quem virtus vexit ad astra), a quien la debida
fama cantará hasta el último día: diversos tiempos
gobernó dos amplísimos reinos; mozo el de Barcelona,
anciano el de Nápoles. Mientras estuvo en oriente brilló
casi como el astro de oriente (sidus eoum, lucero de la
mañana), mientras estuvo en las regiones de occidente
fue otro Héspero (lucero de la tarde). Injusto es llorar al
121
Redondo Cantera 1987, 72-73.
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que vivió feliz en todas partes, ante los hombres vivo,
muerto ante los dioses.
En 1635, Luis Muñoz aplicó este tópico a dos devotas
seguidoras de san Juan de Ávila que pertenecían al mismo
contexto cultural de la alta nobleza andaluza que Per Afán
de Ribera y que destacaron por sus virtudes cristianas, Catalina Fernández de Córdoba, marquesa de Priego —fallecida a edad temprana en 1569— y su madre Ana Ponce de
León (1527-1601), condesa de Feria:122 «Ocupara ella sola
la admiración y lenguas de sus vasallos, a no haber concurrido con su madre. Diferénciase en la claridad una estrella
de otra estrella; mas fuéronlo ambas lucidísimas en el cielo
de la Iglesia».
Conclusiones
No cabe duda de que la evolución conceptual del ser
humano frente a las estrellas en el mundo grecorromano
bebe de muchas fuentes. Con la influencia de otras culturas,
especialmente del Próximo Oriente, pero al mismo tiempo
añadiendo elementos de su propio cuño, esta visión astral
fue analizada y paulatinamente surgieron diferentes planteamientos teóricos, que en ocasiones se reflejaron en las
artes. Algunas corrientes defendieron estudiar las estrellas
como entes en el firmamento sin conexión alguna con las divinidades. Otras escuelas protagonizaron intensos debates
en torno a la condición de los astros como dioses, a su relación con el alma humana y, en última instancia, a la conversión del ser humano en una estrella. La tradición griega, en
términos generales, optó por una visión filosófica y metafísica, mientras que la romana se vio más influida por aspectos
religiosos de tradición oriental —incluyendo la legitimación
de las dinastías helenísticas— que Roma enlazó con sus propios argumentos de tipo moral en torno a la virtud.
La incertidumbre inherente a nuestra propia existencia
nos lleva a cuestionarnos nuestro lugar en el mundo. Para
ello, analizamos lo que nos rodea, incluyendo lo que está
sobre nosotros. Las estrellas nos han acompañado siempre
y permanecerán incluso cuando hayamos muerto, por ello,
es razonable que establezcamos la conexión astro-deidad y,
seguidamente, astro-alma. Sin embargo, entre observar las
estrellas y llegar a ellas o convertirse en una, especialmente
cuando se trata de la apoteosis de miembros de la clase dirigente, hay un salto conceptual que implica la utilización de
determinados mecanismos ideológicos y de control social a
partir de creencias populares firmemente arraigadas. La figura líder lo es por designio divino. Con el tiempo, este favor
o apoyo de los dioses va a dar paso a la construcción de un
topos que identificará al gobernante, normalmente una vez
fallecido, como un dios, situándole a él y a otros miembros
de su familia en el firmamento, junto a los demás dioses.
Unas luces que siempre estarán presentes para nosotros.
Las tradiciones judeocristiana e islámica, como tantos
otros aspectos, asimilaron este topos y lo adaptaron a su
cosmovisión, insistiendo en los aspectos morales, soteriológicos y jerárquicos. Ello, unido a la recuperación del mundo
clásico entre los siglos XIV y XVI, se refleja en diversos textos
y obras de arte del ámbito hispánico vinculados a la exalta122
Muñoz 1806, 182.
399
ción escatológica de las élites, primero de la monarquía, y
más tarde del clero y de la nobleza. En dicho desarrollo los
textos de Daniel, san Pablo, Macrobio, Prudencio y Beato de
Liébana jugaron un importante papel, así como el mosaico
de la bóveda anular del Mausoleo de Constantina. Por su
parte, al referirse a los cuerpos resucitados en el cielo como
estrellas lucientes, el Cartujano fusionó varias tradiciones.
En primer lugar, retomó la idea platónica de la equiparación
de las almas con las estrellas, la concepción aristotélica de
las estrellas como entes vivos, así como la interpretación religiosa y moralista del catasterismo, perteneciente al ámbito
judeocristiano y romano. Asimismo, las unió al simbolismo
de la luz ampliamente desarrollado en periodo gótico.123 Finalmente, integró todas estas reflexiones en su propia concepción de Cristo como hombre y en la imitatio Christi, de
modo que los cuerpos gloriosos de los resucitados se equiparan al propio cuerpo de Cristo. Según Beato de Liébana,
los cuerpos de los bienaventurados reciben su luz de Cristo;
según el Cartujano, son luminosos como el cuerpo de Cristo.
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Heat Treatment of AZ91 Magnesium Alloy Coated with an Al2O3 Thin Film with Fluidized Bed Technology
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Materials
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Received: 11 December 2018; Accepted: 6 January 2019; Published: 10 January 2019 Abstract:
Fluidized bed technology is a methodology widely known in the manufacturing
environment for surface treatment of metals. Within the field of surface coating, it has already
been exploited for the coating of a magnesium alloy creating a compact layer of Al2O3. The result
was an improvement in mechanical and tribological properties, along with improved corrosion
resistance. In this context, the work proposed is addressed towards the evaluation of the effects of
thermal post-treatment on the alumina coating produced by means of the fluidized-bed technology. To analyse the effects of heat treatments the morphology, composition and hardness of the samples
were investigated along with adhesion and wear resistance of the alumina film. The results obtained
show how the temperature affects the surface morphology and promotes the diffusion of magnesium
towards the alumina superficial layer. The mechanisms triggered by heat treatments increase the
adhesion of the surface film obtained in the deposition process, improving its mechanical and
tribological properties. ywords: fluidized bed (FB); magnesium alloy; alumina coating; heat treatment; functional coating Article
Heat Treatment of AZ91 Magnesium Alloy Coated
with an Al2O3 Thin Film with Fluidized
Bed Technology Gabriele Baiocco 1,*
, Gianluca Rubino 2 and Nadia Ucciardello 3 1
Dipartimento di Ingegneria Industriale e dell’Informazione e di Economia, University of L’Aquila,
Via G. Gronchi 18, 67100 L’Aquila, Italy
2
Università della Tuscia, DEIM, Largo dell’Università, 01100 Viterbo, Italy; gianluca.rubino@unitus.it
3
Dipartimento di Ingegneria dell’Impresa “Mario Lucertini”, University of Rome of Tor Vergata,
Via del Politecnico 1, 00133 Rome, Italy; nadia.ucciardello@uniroma2.it
*
Correspondence: gabriele.baiocco@uniroma2.it; Tel.: +39-0672597591 1
Dipartimento di Ingegneria Industriale e dell’Informazione e di Economia, University of L’Aquila,
Via G. Gronchi 18, 67100 L’Aquila, Italy
2
Università della Tuscia, DEIM, Largo dell’Università, 01100 Viterbo, Italy; gianluca.rubino@unitus.it
3
Dipartimento di Ingegneria dell’Impresa “Mario Lucertini”, University of Rome of Tor Vergata,
Via del Politecnico 1, 00133 Rome, Italy; nadia.ucciardello@uniroma2.it
*
Correspondence: gabriele.baiocco@uniroma2.it; Tel.: +39-0672597591 2
Università della Tuscia, DEIM, Largo dell’Università, 01100 Viterbo, Italy; gianluca.rubino@unitus.it
3
Dipartimento di Ingegneria dell’Impresa “Mario Lucertini”, University of Rome of Tor Vergata,
Via del Politecnico 1, 00133 Rome, Italy; nadia.ucciardello@uniroma2.it
*
Correspondence: gabriele.baiocco@uniroma2.it; Tel.: +39-0672597591 y
*
Correspondence: gabriele.baiocco@uniroma2.it; Tel.: +39-0672597591 Materials 2019, 12, 216; doi:10.3390/ma12020216 materials materials materials 1. Introduction The application of magnesium and magnesium alloys in engineering fields has grown rapidly in
the last years. The low density and the high specific strength [1,2] make magnesium alloys attractive for
several applications. In particular, automotive and aerospace fields exploit these materials as they lead
to the reduction of fuel consumption and CO2 emission [3–5]. Properties such good castability, good
ductility, high dimensional stability, good biodegradability, and biocompatibility make magnesium
exploitable in a wide range of domains [6–8]. The main problems affecting these materials are
represented by the low corrosion resistance, caused by the high chemical reactivity of magnesium [9,10],
and low wear resistance [11–13]. In particular, sliding motion has been proved to be a serious
problem for magnesium alloys [14]. Furthermore, the chemical reactivity represents a limit also for the
realization of treatment aimed at the improvement of its features. Chemical coating methodologies
and conversion coatings require the use of chemicals that can trigger violent reactions in magnesium
when they are brought into contact. The fluidized-bed (FB) is a well-known technique for manufacturing systems. It allows several
environmentally-friendly treatments of specimens with complex geometries using basic equipment. Many processes may be performed by FB. Among them, the powder coating processes of ferrous www.mdpi.com/journal/materials www.mdpi.com/journal/materials Materials 2019, 12, 216 2 of 13 and non-ferrous alloys [15] are of great interest, as they may enhance tribological and mechanical
behaviour improving wear and corrosion resistance [16]. The FB process involves the formation of a surface layer of a material characterized by a high
hardness, such as metals or ceramics. During the deposition process the powder of the coating
material, whose dimensions are macrometric, impacts on the substrate to be covered. These particles
are embedded on the soft surface of the substrate gradually accumulating and generating a layer. During the process, two kind of impacts occur: sliding impacts and the rolling impacts. While the
former is characterized by high impact speed and angle the latter feature low impact speed and
angle. Anyway, both cause the release of alumina powder fragments that get stuck in the sample
surface producing the coating. The thickness of the superficial layer as a function of the processing
time features an asymptote caused by the embedding phenomenon. Additionally, the impacts of
the particles cause both a compressive residual stress in the substrate and an increased hardening of
the external layer of the specimen [17,18]. 1. Introduction Among the most suitable materials for the production of
functional coatings, ceramic materials are one of the most reliable candidates. In particular, Al2O3
has been proved to provide excellent wear and corrosion resistance. [19,20]. In the context of the
magnesium alloys, this material was exploited for the creation of a protective layer by means of the
FB deposition process, obtaining an increase in the tribological and corrosion resistance properties
of the substrate [21]. However, heat treatments carried out following deposition processes could
affect the properties of the coated sample. A thermal treatment was carried out on a thermal-sprayed
coating of Y2O3 stabilized ZrO2 and Al2O3 powder on René 95 super alloy [22]. The experiment was
performed at 1300 ◦C for 5 h in an argon atmosphere and resulted in an enhancement of the thermal
conductivity. A two-coating process was coated (Ni-P and Ni-P-Al2O3) by means of an electroless
process on an Al-Si sample [23]. At a later stage heat treatments were executed at 400–550 ◦C for
1–8 h under an argon protective atmosphere for the wear resistance improvement evaluation. Heat
treatments were produced on 316L stainless steel produced by means of a selective laser melting
process and the addition of different volume fraction of TiB2 powder [24]. Differently from the
previous works, following a heating at 1150 ◦C two different kind of cooling processes were achieved. The aim of the study was to assess the effect of a secondary annealing treatment on the composite
properties. The fluidized bed method was exploited for the production of an Al2O3 on an aluminium
alloy substrate [25]. The following thermal treatment performed was designed in function of heating
temperature, up to 600 ◦C, and process duration. It has been proven to be effective in the enhancement
of mechanical and tribological properties of the substrate. In this paper, heat treatments on samples of
the magnesium alloy AZ91, in which mechanical properties are reported [26], following a deposition
process of Al2O3 by means of the fluid-bed technology are proposed. Differently from the previous
works [22–24] and in similarity with [25], the experiment was executed in an air atmosphere as
well as the cooling at ambient temperature, which was performed by means of a not forced process. Furthermore, the effect of a second annealing process was not considered. 1. Introduction The heating maximum
heating temperature was set to 450 ◦C as magnesium burns when treatments at higher temperature
were performed. This treatment was aimed at promoting the diffusion of the protective alumina
film and therefore its tribological and mechanical resistance characteristics. The effect of different
temperatures applied during thermal post-treatment was assessed by investigating the morphology
and microstructure of the samples, as well as by mechanical and tribological tests. The results of
the characterization tests show how the protection grade that the coatings were able to provide on
magnesium alloy was related to the temperature of the thermal post-treatment. Samples of magnesium alloy AZ91, for which composition is reported in Table 1, were cut in
squared specimens of dimension 25 × 25 mm2 and thickness of 3 mm. 2.1. Materials Samples of magnesium alloy AZ91, for which composition is reported in Table 1, were cut in
squared specimens of dimension 25 × 25 mm2 and thickness of 3 mm. 3 of 13 Materials 2019, 12, 216 Table 1. AZ91 composition. Element
Weight%
Mg
90.17
Al
9.00
Zn
0.70
Mn
0.13
Table 1. AZ91 composition. Element
Weight%
Mg
90.17
Al
9.00
Zn
0.70
Mn
0.13 Table 1. AZ91 composition. Element
Weight%
Mg
90.17
Al
9.00
Zn
0.70
Mn
0.13
Table 1. AZ91 composition. Element
Weight%
Mg
90.17
Al
9.00
Zn
0.70
Mn
0.13 Before the alumina deposition with the fluidized bed technique, a lapping process was performed
on the samples with an abrasive paper up to 2500 grit. Uncoated samples used as benchmark were
polished to highlights the AZ91 alloy features. Before the alumina deposition with the fluidized bed technique, a lapping process was
performed on the samples with an abrasive paper up to 2500 grit. Uncoated samples used as
benchmark were polished to highlights the AZ91 alloy features. The samples exploited for the thermal process were coated with alumina alpha featuring a 16 mesh
and a shape factor of 0.67 (Smyris Abrasivi srl, Pero, Italy) and dimensions between 1.2 and 1.4 mm,
the same as previously reported [21]. Alpha phase alumina is the strongest and stiffest of the oxide
ceramics. Its high hardness, excellent dielectric properties, refractoriness, good thermal properties
and chemical and thermal stability make it the material of choice for a wide range of applications [27]. Indeed, its application for functional coating lead to high wear and corrosion resistance in addition to
thermal and electrical insulation [28]. The coating process was performed with a rotating frequency of
6 Hz for 240 min while the fluidizing gas (air) was at a pressure of 5 bar. The fluidized bed employed
for the coating process is the same exploited in [17], where the detail concerning the FB device are
reported. The FB device is represented in Figure 1. p
g
g
y
The samples exploited for the thermal process were coated with alumina alpha featuring a 16
mesh and a shape factor of 0.67 (Smyris Abrasivi srl, Pero, Italy) and dimensions between 1.2 and 1.4
mm, the same as previously reported [21]. Alpha phase alumina is the strongest and stiffest of the
oxide ceramics. 2.1. Materials Its high hardness, excellent dielectric properties, refractoriness, good thermal
properties and chemical and thermal stability make it the material of choice for a wide range of
applications [27]. Indeed, its application for functional coating lead to high wear and corrosion
resistance in addition to thermal and electrical insulation [28]. The coating process was performed
with a rotating frequency of 6 Hz for 240 min while the fluidizing gas (air) was at a pressure of 5 bar. The fluidized bed employed for the coating process is the same exploited in [17], where the detail
concerning the FB device are reported. The FB device is represented in Figure 1. Figure 1. FB device exploited for the Al2O3 coating on magnesium substrate
Figure 1. FB device exploited for the Al2O3 coating on magnesium substrate. Figure 1. FB device exploited for the Al2O3 coating on magnesium substrate
Figure 1. FB device exploited for the Al2O3 coating on magnesium substrate. The deposition process allowed the deposition of an alumina layer of about 4 μm, measured by
means of SEM images. The thermal treatments were performed with a convection oven Nabertheram
P330 (Lilienthal, Germany) for 60 min. The temperatures considered for the experimental tests were
150, 300, and 450 °C. Before the thermal treatment of the samples, the oven was pre-heated to the
treatment temperature. The deposition process allowed the deposition of an alumina layer of about 4 µm, measured by
means of SEM images. The thermal treatments were performed with a convection oven Nabertheram
P330 (Lilienthal, Germany) for 60 min. The temperatures considered for the experimental tests were
150, 300, and 450 ◦C. Before the thermal treatment of the samples, the oven was pre-heated to the
treatment temperature. 2.2. Characterization
2.2. Characterization The samples produced were analysed by means of several techniques in order to evaluate the
morphology of the surface in addition to the tribological and mechanical features. The
characterization was performed on the coated samples before and after the thermal process and on
the uncoated AZ91 alloy. The samples produced were analysed by means of several techniques in order to evaluate the
morphology of the surface in addition to the tribological and mechanical features. The characterization
was performed on the coated samples before and after the thermal process and on the uncoated
AZ91 alloy. y
Preliminarily, the surface of the samples was investigated by optical microscopy using a
stereoscope (SMZ745T, Nikon, Düsseldorf, Germany). At a later stage, the evaluation of the
superficial features was exploited with a contact gauge surface profiler (Talysurf CLI 2000, Taylor-
Hobson, Leicester, UK). Three-dimensional surface profiles were acquired on areas of 4 × 4 mm2 with
Preliminarily, the surface of the samples was investigated by optical microscopy using a
stereoscope (SMZ745T, Nikon, Düsseldorf, Germany). At a later stage, the evaluation of the superficial
features was exploited with a contact gauge surface profiler (Talysurf CLI 2000, Taylor-Hobson,
Leicester, UK). Three-dimensional surface profiles were acquired on areas of 4 × 4 mm2 with a 4 of 13 Materials 2019, 12, 216 spacing and resolution of 2 µm and a measurement speed of 2 mm/s. The evaluation of the samples
roughness was obtained acquiring 101 profiles of 10 mm length with a resolution of 1 µm, a spacing
of 100 µm and a measurement speed of 1 mm/s. Finally, the composition and surface morphology
of the deposited alumina film was examined in detail using Scanning Electron Microscopy/Energy
Dispersive X-Ray Spectroscopy microscopy (SEM-EDS, FEG-SEM Leo Supra 35, Zeiss, Oberkochen,
Germany). In particular, images of the surface and of the cross-section of the samples were acquired. Furthermore, an X-ray Diffraction (XRD) analysis was performed by Philips X’Pert Pro diffractometer
(Amsterdam, The Netherlands), equipped with a plane mono-chromator using Cu Kα radiation
(λ = 1.5418 Å). This analysis was carried out to evaluate the possible creation of new chemical species
that may influence the behaviour of the samples [26]. ,
,
of 100 μm and a measurement speed of 1 mm/s. 2.2. Characterization
2.2. Characterization Finally, the composition and surface morphology of
the deposited alumina film was examined in detail using Scanning Electron Microscopy/Energy
Dispersive X-Ray Spectroscopy microscopy (SEM-EDS, FEG-SEM Leo Supra 35, Zeiss, Oberkochen,
Germany). In particular, images of the surface and of the cross-section of the samples were acquired. Furthermore, an X-ray Diffraction (XRD) analysis was performed by Philips X’Pert Pro diffractometer
(Amsterdam, The Netherlands), equipped with a plane mono-chromator using Cu Kα radiation (λ =
1.5418 Å). This analysis was carried out to evaluate the possible creation of new chemical species that
may influence the behaviour of the samples [26]. The mechanical characterization consisted of Vickers micro-hardness tests performed with a The mechanical characterization consisted of Vickers micro-hardness tests performed with a
Micro Combi tester produced by CSM Instruments (Micro Indenter MHT, CSM Instruments, Needham,
MA, USA). Two different loads, 0.5 and 1 N, were considered and applied on coatings and substrate
for 65 s. For each sample, 10 tests were performed to calculate the average hardness. To compare the
results of the samples thermally treated with the uncoated magnesium alloy, untreated samples were
subjected to the same thermal treatments as for the samples coated with alumina particles. p
Micro Combi tester produced by CSM Instruments (Micro Indenter MHT, CSM Instruments,
Needham, MA, USA). Two different loads, 0.5 and 1 N, were considered and applied on coatings and
substrate for 65 s. For each sample, 10 tests were performed to calculate the average hardness. To
compare the results of the samples thermally treated with the uncoated magnesium alloy, untreated
samples were subjected to the same thermal treatments as for the samples coated with alumina
particles. The tribological measurements performed were the dry-sliding linear reciprocating and the scratch
test. The dry sliding linear reciprocating test, executed with a standard tribometer produced by CSM
Instruments (Needham, MA, USA), was performed at a sliding speed of 8 cm/s. The counterpart was a
100Cr6 ball 6 mm in diameter with an applied load of 1 N. The wear volume was evaluated after several
sliding distances, and particularly after 2, 4, 8, 20, 40, 80, 120, 240, and 500 m. The wear trails were 3D
mapped with a spacing on the x and y axis respectively of 2 and 5 µm with a measurement speed of
2 mm/s. 2.2. Characterization
2.2. Characterization The scratch test was performed with a scratch tester CSM Instruments (Micro Indenter MHT,
Needham, MA, USA), a Rockwell C diamond tip and a load up to 10 N. The measurement speed was
1 mm/min and the sliding distance 4 mm. The tribological measurements performed were the dry-sliding linear reciprocating and the
scratch test. The dry sliding linear reciprocating test, executed with a standard tribometer produced
by CSM Instruments (Needham, MA, USA), was performed at a sliding speed of 8 cm/s. The
counterpart was a 100Cr6 ball 6 mm in diameter with an applied load of 1 N. The wear volume was
evaluated after several sliding distances, and particularly after 2, 4, 8, 20, 40, 80, 120, 240, and 500 m. The wear trails were 3D mapped with a spacing on the x and y axis respectively of 2 and 5 μm with
a measurement speed of 2 mm/s. The scratch test was performed with a scratch tester CSM
Instruments (Micro Indenter MHT, Needham, MA, USA), a Rockwell C diamond tip and a load up
to 10 N. The measurement speed was 1 mm/min and the sliding distance 4 mm. 3. Results and Discussion
3. Results and Discussion From the images obtained by stereo microscopy and reported in Figure 2, is evident a
homogeneous deposition of alumina for all the samples treated with the FB deposition process. The sample heated at 300 ◦C features a corrugated surface suggesting an influence of the thermal
treatment on the surface morphology. From the images obtained by stereo microscopy and reported in Figure 2, is evident a
homogeneous deposition of alumina for all the samples treated with the FB deposition process. The
sample heated at 300 °C features a corrugated surface suggesting an influence of the thermal
treatment on the surface morphology. Figure 2. Images of the coated and heat-treated samples acquired by stereoscope. Figure 2. Images of the coated and heat-treated samples acquired by stereoscope. Figure 2. Images of the coated and heat-treated samples acquired by stereoscope. Figure 2. Images of the coated and heat-treated samples acquired by stereoscope. 5 of 13 Materials 2019, 12, 216 The 3D maps of the samples, proposed in Figure 3, show how the unbaked sample has a surface
morphology similar to the uncoated sample as both are characterized by the peak-peak height “St”
of about 10 µm. Furthermore, 3D maps highlight how the morphology of the coating is a function of
the treatment temperature. The sample heated to 150 ◦C shows an oscillation of the surface profile
with a high frequency. This oscillation is reduced with the treatment temperature. Indeed, the sample
treated at 300 ◦C assumes a profile with fewer peaks and the sample heated to 450 ◦C highlights a
smooth surface with a morphology comparable with the uncoated sample. The St values reach the
maximum value in correspondence of a heating temperature of 300 ◦C. These variations are due to the
heat treatments exclusively, as preliminary tests demonstrated that the samples shown comparable
superficial morphologies in consequence of the deposition process only. Materials 2018, 11, x FOR PEER REVIEW
5 of 14
the treatment temperature. The sample heated to 150 °C shows an oscillation of the surface profile
with a high frequency. This oscillation is reduced with the treatment temperature. Indeed, the sample
treated at 300 °C assumes a profile with fewer peaks and the sample heated to 450 °C highlights a
smooth surface with a morphology comparable with the uncoated sample. The St values reach the
maximum value in correspondence of a heating temperature of 300 °C. 3. Results and Discussion
3. Results and Discussion These variations are due to
the heat treatments exclusively, as preliminary tests demonstrated that the samples shown
comparable superficial morphologies in consequence of the deposition process only. Figure 3. 3D maps of the samples uncoated, unbaked and coated and baked at 150 °C, 300 °C, and 400 °C
Figure 3. 3D maps of the samples uncoated, unbaked and coated and baked at 150 ◦C, 300 ◦C, and
400 ◦C. Figure 3. 3D maps of the samples uncoated, unbaked and coated and baked at 150 °C, 300 °C, and 400
Figure 3. 3D maps of the samples uncoated, unbaked and coated and baked at 150 ◦C, 300 ◦C, and
400 ◦C. The same is confirmed by the roughness analysis reported in Figure 4, which lead to th
evaluation of the average roughness “Ra”, maximum height “Rz”, spacing “Rsm”, and RMS (root mea
square) slope “RΔq”. The same is confirmed by the roughness analysis reported in Figure 4, which lead to the evaluation
of the average roughness “Ra”, maximum height “Rz”, spacing “Rsm”, and RMS (root mean square)
slope “R∆q”. q
From the analysis of the roughness parameters it is evident how uncoated, unbaked and baked at
450 ◦C samples show similar values, as determined by means of 3D maps and stereoscope investigation. Compared to the unbaked sample, increasing values of Ra, Rz, and Rsm are observed for temperatures
of 150 and 300 ◦C. Instead, there is a substantial settlement of the values of R∆q. The “Rsm” increase
confirms the increment of the profile oscillation frequency observed in the 3D maps. Furthermore,
it can be noted that the growth of the “Rz” value kept a constant “R∆q”. A trend of this type suggests
the loss of surface material due to the thermal treatments undergone. In particular, magnesium has
a coefficient of thermal expansion greater than alumina. Therefore, the heating treatment induces a
tensile stress state on the alumina particles embedded on the surface causing a gradual detaching. With the 300 ◦C heating treatment, the highest value of Rsm was found. At this temperature there is
the greatest difference in expansion between bulk and coating and, therefore, the detachment of larger
alumina fragments. The heating treatment that was performed at 450 ◦C, about 3/4 of the magnesium
melting temperature, involved a reduction in the mechanical properties of the bulk. 3. Results and Discussion
3. Results and Discussion Due to the poor 6 of 13 Materials 2019, 12, 216 mechanical properties of the bulk, the greater thermal expansion of magnesium cannot induce a stress
on the alumina coating. Therefore, the detachment of coating fragments does not occur. Materials 2018, 11, x FOR PEER REVIEW
6 of 14 Figure 4. Roughness analysis of the samples surface. Figure 4. Roughness analysis of the samples surface. Figure 4. Roughness analysis of the samples surface. Figure 4. Roughness analysis of the samples surface. From the analysis of the roughness parameters it is evident how uncoated, unbaked and baked
at 450 °C samples show similar values, as determined by means of 3D maps and stereoscope
investigation. Compared to the unbaked sample, increasing values of Ra, Rz, and Rsm are observed for
temperatures of 150 and 300 °C. Instead, there is a substantial settlement of the values of RΔq. The
The SEM images of the surface of the samples shown in Figure 5 highlight the variations in
morphology as a function of the heat treatment. With a greater magnification of the SEM images
concerning the sample treated at 300 ◦C, shown in Figure 6, it is possible to observe the cracks caused
by the tension state induced by the heating. Materials 2018, 11, x FOR PEER REVIEW
7 of 14 rms the increment of the profile oscillation frequency obser
be noted that the growth of the “Rz” value kept a constant “
oss of surface material due to the thermal treatments unde
coefficient of thermal expansion greater than alumina. Th
tensile stress state on the alumina particles embedded on t
With the 300 °C heating treatment, the highest value of Rsm
s the greatest difference in expansion between bulk and co
arger alumina fragments. The heating treatment that was p
gnesium melting temperature, involved a reduction in the m
the poor mechanical properties of the bulk, the greater th
induce a stress on the alumina coating. Therefore, the de
occur. es of the surface of the samples shown in Figure 5 highlig
nction of the heat treatment. With a greater magnification
ple treated at 300 °C, shown in Figure 6, it is possible to obser
induced by the heating. Figure 5. SEM images of the samples surface. Figure 5. SEM images of the samples surface. by the heating. Figure 5. SEM images of the samples surface. Figure 5. SEM images of the samples surface. 3. Results and Discussion
3. Results and Discussion As a
consequence of this interface, a greater adhesion of the coating is expected and, therefore, a better The mechanical tests were then performed by means of the Vickers micro-indentation, whose
values are shown in Figure 8. tribological behaviour. The mechanical tests were then performed by means of the Vickers micro-indentation, whose
l
h
i
Fi
8 wn in Figure 8. Figure 8. Hardness test results as function of load and heating treatment. Figure 8. Hardness test results as function of load and heating treatment. Considering the same heat treatment, the uncoated samples show the same hardness values
regardless of the applied load. With increasing treatment temperature, slightly decreasing hardness
values are observed. This behaviour is caused by the enlargement of the grain, as well as by the
reduction of the residual stress. The alumina coating leads to an increase in hardness compared to the uncoated sample, regardless
of the heat treatment suffered. The Vickers micro-hardness passes from values of about 60 HV to values
of about 120 HV. Tests carried out with a minor load presented the highest hardness values, if the same
treatment conditions are considered. The variation in hardness is due to the greater penetration of the
indenter which, therefore, is more heavily affected by the properties of the bulk. Indeed, it is noted
that the hardness reduction with increasing heating temperature is more marked for a load of 1 N with
respect to the load of 0.5 N. The adhesion of the film was tested by means of a scratch test. Aim of the test was the examination
of the images of the trails as well as the measurement of the penetration depth (Pd) and residual depth
(Rd). The results are shown in Figures 9 and 10. With increasing treatment temperatures, greater penetration depths are reached due to the greater
ductility of the substrate, especially for the last two temperatures investigated. Residual depth is
similar for all samples and, therefore, there is an increasing difference between Pd and Rd. This
behaviour indicates a lower tendency of the coating to detach. Furthermore, there is a sudden drop
of the residual depth at the end of the trace that increases with the temperature. This behaviour is
attributable to the phenomenon of front pile up and denotes an accumulation of the coating at the end
of the track and a lower tendency to fragile break. 3. Results and Discussion
3. Results and Discussion 7 of 13 Materials 2019, 12, 216 Figure 6. Highlight of the samples heated at 300 °C. Figure 6. Highlight of the samples heated at 300 ◦C. Figure 6. Highlight of the samples heated at 300 °C. Figure 6. Highlight of the samples heated at 300 ◦C. These fractures are characterized by equiaxial geometries. The cross-section of the specimens
was then analysed by means of the SEM-EDS analysis reported in Figure 7. This allowed to highlight
the composition and thickness of the layer as a result of treatments at different temperatures. These fractures are characterized by equiaxial geometries. The cross-section of the specimens was
then analysed by means of the SEM-EDS analysis reported in Figure 7. This allowed to highlight the
composition and thickness of the layer as a result of treatments at different temperatures. Materials 2018, 11, x FOR PEER REVIEW
8 of 14 igure 7. SEM-EDS images of the specimens highlighting the alumina and magnesium content of the coatin
Figure 7. SEM-EDS images of the specimens highlighting the alumina and magnesium content of
the coating. gure 7. SEM-EDS images of the specimens highlighting the alumina and magnesium content of the coati
Figure 7. SEM-EDS images of the specimens highlighting the alumina and magnesium content of
the coating. All samples had an alumina layer of about 4 μm. It can also be noted that, besides an alumina
sedimentation highlighted in the previous analysis, there was a gradual diffusion of magnesium
towards the superficial layers of the sample. This diffusion increases with the temperature of the
thermal process and creates an interface between bulk and coating without a clear distinction. As a
All samples had an alumina layer of about 4 µm. It can also be noted that, besides an alumina
sedimentation highlighted in the previous analysis, there was a gradual diffusion of magnesium
towards the superficial layers of the sample. This diffusion increases with the temperature of the
thermal process and creates an interface between bulk and coating without a clear distinction. As a Materials 2019, 12, 216
All samples h
sedimentation high 8 of 13
umina
esium consequence of this interface, a greater adhesion of the coating is expected and, therefore, a better
tribological behaviour. p
y
p
p
thermal process and creates an interface between bulk and coating without a clear distinction. 3. Results and Discussion
3. Results and Discussion From the pictures of the scratch test trails, shown
in Figure 10, it can be seen that the delamination is lower for heat treatments at higher temperatures. The damages due to the passage of the indentation are shown to increase in load as the treatment
temperature increases. In fact, the damages due to the indenter begin to be evident at higher loads
with the increase of the treatment temperature, as highlighted in Figure 10. The LC1 (load critical) load
for the unbaked sample and the samples treated at 150, 300 and 450 ◦C is, respectively, of 3.9, 4.1, 5,
and 7 N. Furthermore, the uncovered areas decrease with the treatment temperature. 9 of 13
s the
) and
s the
and Materials 2019, 12, 216
The adhesio
examination of th
The adhesion
examination of th l depth (Rd). The results are shown in Figures 9 and 10. Figure 9. Penetration depth and residual depth of the scratch test. Figure 9. Penetration depth and residual depth of the scratch test. l depth (Rd). The results are shown in Figures 9 and 10. Figure 9. Penetration depth and residual depth of the scratch test. s 2018, 11, x FOR PEER REVIEW dual depth (Rd). The results are shown in Figures 9 and 10. Figure 9. Penetration depth and residual depth of the scratch test. Figure 10. Scratch test trails with the coating detachment highlighted in the white circles. Figure 9. Penetration depth and residual depth of the scratch test. ual depth (Rd). The results are shown in Figures 9 and 10. Figure 9. Penetration depth and residual depth of the scratch test. Figure 10. Scratch test trails with the coating detachment highlighted in the white circles. Figure 10. Scratch test trails with the coating detachment highlighted in the white circles. rials 2018, 11, x FOR PEER REVIEW
10
With increasing treatment temperatures, greater penetration depths are reached due to
ater ductility of the substrate, especially for the last two temperatures investigated. Residual d
imilar for all samples and, therefore, there is an increasing difference between Pd and Rd. aviour indicates a lower tendency of the coating to detach. Furthermore, there is a sudden
he residual depth at the end of the trace that increases with the temperature. 3. Results and Discussion
3. Results and Discussion This behavio
ibutable to the phenomenon of front pile up and denotes an accumulation of the coating a
of the track and a lower tendency to fragile break. From the pictures of the scratch test t
wn in Figure 10, it can be seen that the delamination is lower for heat treatments at hi
peratures. The damages due to the passage of the indentation are shown to increase in load a
tment temperature increases. In fact, the damages due to the indenter begin to be evide
her loads with the increase of the treatment temperature, as highlighted in Figure 10. The
d critical) load for the unbaked sample and the samples treated at 150, 300 and 450 °
pectively of 3 9 4 1 5 and 7 N Furthermore the uncovered areas decrease with the treatm p
(
)
g
p
(
)
g Figure 9. Penetration depth and residual depth of the scratch test. Figure 9. Penetration depth and residual depth of the scratch test. Figure 9. Penetration depth and residual depth of the scratch test. OR PEER REVIEW Figure 9. Penetration depth and residual depth of the scratch test. Figure 9. Penetration depth and residual depth of the scratch test. Figure 9. Penetration depth and residual depth of the scratch test. OR PEER REVIEW Figure 10. Scratch test trails with the coating detachment highlighted in the white circles. Figure 10. Scratch test trails with the coating detachment highlighted in the white circles. Figure 10. Scratch test trails with the coating detachment highlighted in the white circles. With increasing treatment temperatures, greater penetration depths are reached due t
er ductility of the substrate, especially for the last two temperatures investigated. Residual d
milar for all samples and, therefore, there is an increasing difference between Pd and Rd
viour indicates a lower tendency of the coating to detach. Furthermore, there is a sudden
e residual depth at the end of the trace that increases with the temperature. This behavio
utable to the phenomenon of front pile up and denotes an accumulation of the coating a
of the track and a lower tendency to fragile break. From the pictures of the scratch test t
n in Figure 10, it can be seen that the delamination is lower for heat treatments at h
eratures. 3. Results and Discussion
3. Results and Discussion The damages due to the passage of the indentation are shown to increase in load a
ment temperature increases. In fact, the damages due to the indenter begin to be evide
r loads with the increase of the treatment temperature, as highlighted in Figure 10. The
critical) load for the unbaked sample and the samples treated at 150, 300 and 450 °
ctively of 3 9 4 1 5 and 7 N Furthermore the uncovered areas decrease with the treat Figure 10. Scratch test trails with the coating detachment highlighted in the white circles. Figure 10. Scratch test trails with the coating detachment highlighted in the white circles. Figure 10. Scratch test trails with the coating detachment highlighted in the white circles. critical) load for the unbaked sample and the samples treated at 150, 300 and 450 °
ti
l
f 3 9 4 1 5
d 7 N F
th
th
d
d
ith th
t
t Finally, the wear behaviour was considered by means of a dry-sliding linear reciprocating test. To evaluate the test result, the samples were photographed and the trails 3D mapped as shown in
Figures 11 and 12. e pe i e y, o
9,
,
, a
N
u
e
o e,
e u
o e e
a ea
e
ea e
i
e
ea
e
temperature. Finally, the wear behaviour was considered by means of a dry-sliding linear reciprocating test. To evaluate the test result, the samples were photographed and the trails 3D mapped as shown in
Figures 11 and 12 g
Figure 11. Images of the linear reciprocating trails. Figure 11. Images of the linear reciprocating trails. Figure 11. Images of the linear reciprocating trails. Figure 11. Images of the linear reciprocating trails. Materials 2019, 12, 216 10 of 13 10 of 13 Figure 12. 3D maps of the linear reciprocating trails. Figure 12. 3D maps of the linear reciprocating trails. i l
1
11
FOR PEER REVIEW
11
f Figure 12. 3D maps of the linear reciprocating trails. Figure 12. 3D maps of the linear reciprocating trails. The volume of material removed during the test is shown in Figure 13 as a function of the sliding
distance. From the pictures of the samples it is clear that the coating improves the wear behaviour of
the sample. 3. Results and Discussion
3. Results and Discussion Materials 2018, 11, x FOR PEER REVIEW
11 of 14
The volume of material removed during the test is shown in Figure 13 as a function of the sliding
distance. From the pictures of the samples it is clear that the coating improves the wear behaviour of le. Figure 13. Wear volume removed during the test, as function of distance. Figure 13. Wear volume removed during the test, as function of distance. Figure 13. Wear volume removed during the test, as function of distance. Figure 13. Wear volume removed during the test, as function of distance. The first signs of wear are evident after sliding distances increase with the treatment
temperature. At 150 °C the first signs appear after 120 m, while at 300 and 450 °C there are signs of
wear for distances of 240 and 500 m. Furthermore, on the heat-treated samples there were evident
deposits of the ball on the coating to highlight the good adhesion of the alumina powder on the
substrate. The wear volume increased linearly with the sliding distance as expected. At equal
distances, the volume decreases with the treatment temperature. After 500 m of sliding the wear
The first signs of wear are evident after sliding distances increase with the treatment temperature. At 150 ◦C the first signs appear after 120 m, while at 300 and 450 ◦C there are signs of wear for distances
of 240 and 500 m. Furthermore, on the heat-treated samples there were evident deposits of the ball on
the coating to highlight the good adhesion of the alumina powder on the substrate. The wear volume
increased linearly with the sliding distance as expected. At equal distances, the volume decreases
with the treatment temperature. After 500 m of sliding the wear volumes of the uncoated samples, Materials 2019, 12, 216 11 of 13 unbaked samples, and the samples heated at 150, 300, and 450 ◦C were, respectively, 1.4, 1.2, 0.7, 0.6,
and 0.3 mm3. unbaked samples, and the samples heated at 150, 300, and 450 ◦C were, respectively, 1.4, 1.2, 0.7, 0.6,
and 0 3 mm3 Considering the 3D maps and the images, an irregular bottom is evident for the traces produced
by the greater sliding distances. At the inversion point of the counterpart motion there was an increase
in the friction coefficients and a phenomenon of adhesion with the substrate. 3. Results and Discussion
3. Results and Discussion This caused the dragging
of the material in the wear track. Its progressive accumulation is responsible for the irregular bottom,
implying the counterpart bouncing. Furthermore, in the wear patterns left on the sample before the
bottom becomes irregular and with respect to the uncoated sample, there are signs parallel to the
sliding axis. This wear morphology is typical of three-body wear. The best behaviour found in thermally treated samples with respect to the uncoated and unbaked
samples was considered to be due to exclusively the diffusion phenomena previously highlighted. Indeed, the XRD analysis shown in Figure 14 shows how heat treatments do not promote the formation
of new chemical species capable of justifying the improved behaviour of the coated magnesium
samples. The peak in correspondence of the 2θ value equal to 43◦, which it is not present in the
uncoated samples, it was due to the formation of highly crystalline periclase MgO [29]. Its formation
is due to the interaction with the oxygen present in Al2O3 and rapidly develop when treated with high
temperature in a non-controlled atmosphere. Materials 2018, 11, x FOR PEER REVIEW
12 of 1 Figure 14. XRD analysis of the magnesium samples after the heating treatment. (°)
Figure 14. XRD analysis of the magnesium samples after the heating treatment. (°) Figure 14. XRD analysis of the magnesium samples after the heating treatmen
Figure 14. XRD analysis of the magnesium samples after the heating treatment. 4. Conclusion
In the work conducted, depositions of Al2O3 powder were carried out on AZ91 magnesium
substrates using fluidized-bed technology. The coated samples were then heat treated at differen
temperatures in order to improve the performance of the coating. The various samples obtained as a
Furthermore, with the heating at 450 ◦C there was a further improvement compared to the
samples treated at lower temperatures. This temperature allows the formation of a smooth coating. The absence of crests on the surface improves the tribological behaviour of the film coated, as they are
a place of concentration of tensions that may promote the breaking of the coating References 1. Liang, J.; Bala Srnivasan, P.; Blawert, C.; Stormer, M.; Dietzel, W. Electrochemical corrosion behaviour of
plasma electrolytic oxidation coatings on AM50 magnesium alloy formed in silicate and phosphate based
electrolytes. Electrochim. Acta 2009, 54, 3842–3850. [CrossRef] 1. Liang, J.; Bala Srnivasan, P.; Blawert, C.; Stormer, M.; Dietzel, W. Electrochemical corrosion behaviour of
plasma electrolytic oxidation coatings on AM50 magnesium alloy formed in silicate and phosphate based
electrolytes. Electrochim. Acta 2009, 54, 3842–3850. [CrossRef] 2. Mordike, B.L.; Ebert, T. Magnesium: Properties-applications-potential. Mater. Sci. Eng. A 2001, 302, 37–45. [CrossRef] 2. Mordike, B.L.; Ebert, T. Magnesium: Properties-applications-potential. Mater. Sci. Eng. A 2001, 302, 37–45. [CrossRef] 3. Cole, G.S. Issues that Influence Magnesium’s Use in the Automotive Industry. Mater. Sci. Forum 2003,
419–422, 43–50. [CrossRef] 3. Cole, G.S. Issues that Influence Magnesium’s Use in the Automotive Industry. Mater. Sci. Forum 2003,
419–422, 43–50. [CrossRef] 4. Kaneko, T.; Suzuki, M. Automotive applications of magnesium alloys. Mater. Sci. Forum 2003, 419–422,
67–74. [CrossRef] 4. Kaneko, T.; Suzuki, M. Automotive applications of magnesium alloys. Mater. Sci. Forum 2003, 419–422,
67–74. [CrossRef] 5. Schubert, E.; Klassen, M.; Zerner, I.; Walz, C.; Sepold, G. Light weight structures produced by laser beam
joining for future application in automobile and aerospace industry. J. Mater. Process. Technol. 2001, 115, 2–8. [CrossRef] 5. Schubert, E.; Klassen, M.; Zerner, I.; Walz, C.; Sepold, G. Light weight structures produced by laser beam
joining for future application in automobile and aerospace industry. J. Mater. Process. Technol. 2001, 115, 2–8. [CrossRef] 6. Kojima, Y. Platform Science and Technology for Advanced Magnesium Alloys. Mater. Sci. Forum 2000,
350–351, 3–18. [CrossRef] 6. Kojima, Y. Platform Science and Technology for Advanced Magnesium Alloys. Mater. Sci. Forum 2000,
350–351, 3–18. [CrossRef] 7. Xina, Y.; Hu, T.; Chu, P.K. In vitro studies of biomedical magnesium alloys in a simulated physiological
environment: A review. Acta Biomater. 2011, 7, 1452–1459. [CrossRef] 8. Wan, P.; Tan, L.; Yang, K. Surface Modification on Biodegradable Magnesium Alloys as Orthopedic Implant
Materials to Improve the Bio-adaptability: A Review. J. Mater. Sci. Technol. 2016, 32, 827–834. [CrossRef] 9. Wang, L.; Chen, L.; Yan, Z.C.; Wang, H.L.; Peng, J.Z. Growth and corrosion characteristics of plasma
electrolytic oxidation ceramic films formed on AZ31 magnesium alloy. Chin. J. Process Eng. 2009, 9, 592–597. 10. Song, G.; Atrens, A. Understanding magnesium corrosion—A framework for improved alloy performance. 9. Wang, L.; Chen, L.; Yan, Z.C.; Wang, H.L.; Peng, J.Z. result of the d
l
t th i
4. Conclusions conceived and designed the experiments; G.B. and G.R. performed the experiments;
N.U. and G.R. analysed the data; N.U. contributed reagents/materials/analysis tools; and G.B. wrote the paper. Funding: This research received no external funding. Funding: This research received no external funding. Funding: This research received no external funding. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. result of the d
l
t th i
4. Conclusions evaluate their morphology, composition, and mechanical performance. Through the fluidized-bed deposition process it was possible to obtain a homogeneous coating
with a thickness of around 4 μm. However, surface morphology is affected by the temperature of the
heat treatment. The heat treatments up to 300 °C induce stress on the alumina particles embedded on
h
f
b
f h
d ff
ff
h
h
b lk
d
h
In the work conducted, depositions of Al2O3 powder were carried out on AZ91 magnesium
substrates using fluidized-bed technology. The coated samples were then heat treated at different
temperatures in order to improve the performance of the coating. The various samples obtained as Materials 2019, 12, 216 12 of 13 a result of the different heat treatments were characterized by different points of view in order to
evaluate their morphology, composition, and mechanical performance. a result of the different heat treatments were characterized by different points of view in order to
evaluate their morphology, composition, and mechanical performance. Through the fluidized-bed deposition process it was possible to obtain a homogeneous coating
with a thickness of around 4 µm. However, surface morphology is affected by the temperature
of the heat treatment. The heat treatments up to 300 ◦C induce stress on the alumina particles
embedded on the surface, because of the different expansion coefficient that characterizes bulk and
coating. This causes alumina fragments detachment and an increased surface roughness. At higher
temperatures the mechanical properties of magnesium are poor and, although a different thermal
expansion, the bulk cannot create a stress on the coating. The result is a smooth surface whose
morphology is comparable to the unbaked sample. Along with this phenomenon, a diffusion of magnesium towards the alumina layer which
increases with treatment temperature was observed. The diffusion process along with the smooth
surface are solely responsible for the improvements observed in adhesion and wear resistance of the
produced coating. Indeed, no new chemical species, except magnesium oxide, was formed during
thermal treatments, as observed by XRD analysis. Author Contributions: G.R. conceived and designed the experiments; G.B. and G.R. performed the experiments;
N.U. and G.R. analysed the data; N.U. contributed reagents/materials/analysis tools; and G.B. wrote the paper. Author Contributions: G.R. conceived and designed the experiments; G.B. and G.R. performed the experim
N.U. and G.R. analysed the data; N.U. contributed reagents/materials/analysis tools; and G.B. wrote the pa Author Contributions: G.R. References Growth and corrosion characteristics of plasma
electrolytic oxidation ceramic films formed on AZ31 magnesium alloy. Chin. J. Process Eng. 2009, 9, 592–597. electrolytic oxidation ceramic films formed on AZ31 magnesium alloy. Chin. J. Process Eng. 2009, 9, 592–597. 10. Song, G.; Atrens, A. Understanding magnesium corrosion—A framework for improved alloy performance. Adv. Eng. Mater. 2003, 5, 837–858. [CrossRef] 10. Song, G.; Atrens, A. Understanding magnesium corrosion—A framework for improved alloy performance. Adv. Eng. Mater. 2003, 5, 837–858. [CrossRef] 11. Blawert, C.; Dietzel, W.; Ghali, E.; Song, G. Anodizing treatments for magnesium alloys and their effect on
corrosion resistance in various environments. Adv. Eng. Mater. 2006, 8, 511–533. [CrossRef] 12. Lee, Y.K.; Lee, K.; Jung, T. Study on microarc oxidation of AZ31B magnesium alloy in alkaline metal silicate
solution. Electrochem. Commun. 2008, 10, 1716–1719. [CrossRef] 13. Yamauchi, N.; Demizu, K.; Ueda, N.; Cuong, N.K.; Sone, T.; Hirose, Y. Friction and wear of DLC films on
magnesium alloy. Surf. Coat. Technol. 2005, 193, 277–282. [CrossRef] 13 of 13 13 of 13 Materials 2019, 12, 216 14. Wang, S.Q.; Yang, Z.R.; Zhao, Y.T.; Wei, M.X. Sliding wear characteristics of AZ91D alloy at ambient
temperatures of 25–200 ◦C. Tribol. Lett. 2010, 38, 39–45. [CrossRef] 15. Tsipas, D.N.; Anthymidis, K.G.; Flitris, Y. Deposition of hard and/or corrosion resistant, single and
multielement coatings on ferrous and nonferrous alloys in a fluidized bed reactor. J. Mater. Process. Technol. 2003, 134, 145–152. [CrossRef] 16. Weiss, K.D. Paint and coatings: A mature industry in transition. Prog. Polym. Sci. 1997, 22, 203–245. [CrossRef] 17. Barletta, M. Al2O3 graded coatings on aluminium alloy deposited by the fluidized bed (FB) technique: Film
formation and mechanical performance. J. Eng. Mater. Technol. 2010, 132, 031003. [CrossRef] 18. Barletta, M.; Costanza, G.; Polini, R. Al2O3 thin coating of AA 6082 T6 components using a fast regime
fluidized bed. Thin Solid Films 2006, 515, 141–151. [CrossRef] 19. Bensalah, W.; Elleuch, K.; Feki, M.; De Petris-Wery, M.; Ayedi, H.F. Comparative study of mechanical and
tribological properties of alumina coatings formed on aluminium in various conditions. Mater. Des. 2009, 30,
3731–3737. [CrossRef] 20. Ding, H.Y.; Dai, Z.D.; Skuiry, S.C.; Hui, D. Corrosion wear behaviors of micro-arc oxidation coating of Al2O3
on 2024Al in different aqueous environments at fretting contact. Tribol. Int. 2010, 43, 868–875. [CrossRef]
21. Baiocco, G.; Rubino, G.; Tagliaferri, V.; Ucciardello, N. References Al2O3 coatings on magnesium alloy deposited by the
fluidized bed (FB) technique Materials 2018 11 94 [CrossRef] [PubMed] 20. Ding, H.Y.; Dai, Z.D.; Skuiry, S.C.; Hui, D. Corrosion wear behaviors of micro-arc oxidation coating of Al2O3
on 2024Al in different aqueous environments at fretting contact. Tribol. Int. 2010, 43, 868–875. [CrossRef] 21. Baiocco, G.; Rubino, G.; Tagliaferri, V.; Ucciardello, N. Al2O3 coatings on magnesium alloy depos
fluidized bed (FB) technique. Materials 2018, 11, 94. [CrossRef] [PubMed] 22. Dutton, R.; Wheeler, R.; Ravichandran, K.S.; An, K. Effect of heat treatment on the thermal conductivity of
plasma-sprayed thermal barrier coatings. J. Therm. Spray Technol. 2000, 9, 204–209. [CrossRef] 23. Novák, M.; Vojtˇech, D.; Vít ˚u, T. Influence of heat treatment on tribological properties of electroless Ni–P and
Ni–P–Al2O3 coatings on Al–Si casting alloy. Appl. Surf. Sci. 2010, 256, 2956–2960. [CrossRef] 24. Al Mangour, B.; Grzesiak, D.; Yang, J.M. Selective laser melting of TiB2/316L stainless steel composites:
The roles of powder preparation and hot isostatic pressing post-treatment. Powder Technol. 2017, 309, 37–48. [CrossRef] 25. Rubino, G.; Trovalusci, F.; Barletta, M.; Fanelli, P. Heat treatment of AA 6082 T6 aluminum alloy coated with
thin Al2O3 layer by fluidized bed. Int. J. Adv. Manuf. Technol. 2018, 96, 2605–2618. [CrossRef] 26. Luo, A. Processing, microstructure, and mechanical behavior of cast magnesium metal matrix com
Metall. Mater. Trans. A 1995, 26, 2445–2455. [CrossRef] Metall. Mater. Trans. A 1995, 26, 2445–2455. [CrossRef]
27. Munro, M. Evaluated Material Properties for a Sintered alpha-Al2O3. J. Am. Ceram. Soc. 2005, 80, 1919–1928. [CrossRef] 7. Munro, M. Evaluated Material Properties for a Sintered alpha-Al2O3. J. Am. Ceram. Soc. 2005, 80, 1919–1
[CrossRef] 28. Balijepalli, S.K.; Colantoni, I.; Donnini, R.; Kaciulis, S.; Lucci, M.; Montanari, R.; Ucciardello, N.; Varone, A. Elastic modulus of S phase in kolsterized 316L stainless steel. Metall. Ital. 2013, 105, 42–47. 29. Aramendia, M.G.; Benitez, J.A.; Borau, V.; Jimenez, C.; Marinas, J.M.; Ruiz, J.R.; Urbano, F. Characterization
of Various Magnesium Oxides by XRD and H-1 MAS NMR Spectroscopy. J. Solid State Chem. 1999, 144,
25–29. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Automated data cleaning of paediatric anthropometric data from longitudinal electronic health records: protocol and application to a large patient cohort
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www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Automated data cleaning of
paediatric anthropometric data
from longitudinal electronic health
records: protocol and application to
a large patient cohort Hang T. T. Phan1,2 ✉, Florina Borca2,3, David Cable3, James Batchelor1,2, Justin H. Davies3,4 &
Sarah Ennis1,2,4 Hang T. T. Phan1,2 ✉, Florina Borca2,3, David Cable3, James Batchelor1,2, Justin H. Davies3,4 &
Sarah Ennis1,2,4 ‘Big data’ in healthcare encompass measurements collated from multiple sources with various
degrees of data quality. These data require quality control assessment to optimise quality for clinical
management and for robust large-scale data analysis in healthcare research. Height and weight data
represent one of the most abundantly recorded health statistics. The shift to electronic recording of
anthropometric measurements in electronic healthcare records, has rapidly inflated the number of
measurements. WHO guidelines inform removal of population-based extreme outliers but an absence
of tools limits cleaning of longitudinal anthropometric measurements. We developed and optimised
a protocol for cleaning paediatric height and weight data that incorporates outlier detection using
robust linear regression methodology using a manually curated set of 6,279 patients’ longitudinal
measurements. The protocol was then applied to a cohort of 200,000 patient records collected from
60,000 paediatric patients attending a regional teaching hospital in South England. WHO guidelines
detected biologically implausible data in <1% of records. Additional error rates of 3% and 0.2%
for height and weight respectively were detected using the protocol. Inflated error rates for height
measurements were largely due to small but physiologically implausible decreases in height. Lowest
error rates were observed when data was measured and digitally recorded by staff routinely required
to do so. The protocol successfully automates the parsing of implausible and poor quality height and
weight data from a voluminous longitudinal dataset and standardises the quality assessment of data for
clinical and research applications. With the availability of digital electronic health systems, ‘big’ clinical data has become more accessible to the
research community1,2. The big data era, which includes using data obtained from heterogeneous digital sources,
has enabled novel opportunities for conducting empirical clinical research. At the same time there are challenges
using such data for research purposes, including the need to adapt existing and develop new methodologies to
cope with the scale and complexity of the data3. However, a more fundamental issue for researchers is the require-
ment to undertake data cleaning, as incorrect clinical measurements entered into an electronic health record
(EHR) will significantly affect the quality of dataset. Scientific Reports | (2020) 10:10164 | https://doi.org/10.1038/s41598-020-66925-7 Automated data cleaning of
paediatric anthropometric data
from longitudinal electronic health
records: protocol and application to
a large patient cohort Data cleaning can be time-consuming and involve multiple
stages including detailed data analysis to identify error types, data inconsistencies, outlier detection and imple-
ment data transformation where required4,5. Thus, developing automated methods for data cleaning is desirable. h
Height and weight are the most commonly recorded anthropometric measures for the assessment of child
health in both clinical practice and research studies. Longitudinal height measurements give an indication of
well-being and perturbations may be an indication of nutritional, endocrine, cardiac or other abnormalities that
should prompt a clinical decision for investigation or intervention. Body mass index (BMI), defined by heights 1NIHR Southampton Biomedical Research Centre, University Hospital Southampton, Southampton, UK. 2University
of Southampton, Southampton, UK. 3University Hospital Southampton NHS Foundation Trust, Southampton, UK. 4These authors contributed equally: Justin H. Davies and Sarah Ennis. ✉e-mail: hang.phan@soton.ac.uk Scientific Reports | (2020) 10:10164 | https://doi.org/10.1038/s41598-020-66925-7 www.nature.com/scientificreports/ and weights, may be used to establish risks of prevalence of diseases6. In children, longitudinal changes of BMI
provide insight into predisposition to health problems such as obesity, hypertension, type 2 diabetes and nutri-
l
ffi and weights, may be used to establish risks of prevalence of diseases6. In children, longitudinal changes of BMI
provide insight into predisposition to health problems such as obesity, hypertension, type 2 diabetes and nutri-
tional insufficiency. fi
y
World Health Organisation (WHO) guidelines7 can be used to exclude biologically implausible values (BIV)
from the EHR for childhood height, weight and BMI data, by converting the measurements to standard deviation
scores (SDS) and using defined parameters to exclude extreme values (e.g. height to age z-score (HAZ) exclusion
if < −6 or >6). However, there are few studies which have evaluated methods for cleaning periodical longitu-
dinal anthropometric data8. For example, some have identified BIVs for annual longitudinal values where the
mean changes of BMI values exceed 3SDS or −3SDS and height decrements greater than 1 inch/year, and mean
increases in height>3SDS9,10. Others10 have suggested removing weight measurements where annual changes
exceed 22.7 kg or 27.2 kg if the individual was severely obese at baseline, any height decrease and any height
increase >15 cm a year. These methods were developed for identifying extreme changes in periodical measure-
ments and do not detect less extreme changes and so are not applicable to children where growth is dynamic. Neither are they applicable to the big-data scenario where anthropometric measurements are non-periodical. Materials and methods Anthropometric data scope and extraction. Electronically recorded height, weight measurements and
date of birth was extracted for all patients admitted to UHS from 1932–2018 where the patient’s age at date of meas-
urement was between 2–20 years. Data prior to 2008 were paper-based archived data transcribed into the elec-
tronic EPR system since its introduction in UHS. Measurements are recorded to an accuracy of 1 decimal place for
weight (kg) and height (cm). The occupation and department of the staff members entering the data was also cap-
tured. Measurements of children of age less than 2 years were not considered in this assessment as the absence of
gestational age data prevented accurate calculation of height for age z-scores (HAZ), weight for age z-scores (WAZ)
and weight for height z-scores (WHZ). From the raw measurements of height (H, metre) and weight (W, kg),
BMI was calculated as W/H2 and HAZ, WAZ and WHZ were calculated using the LMS method15. Data quality indicators. In assessing the quality of the captured anthropometric height and weight meas-
urements, established data quality indicators for children ≥2 years of age were applied: (i) standard deviation
(SD) of HAZ, WAZ and WHZ16 (ii) Myer’s Index (MI) for height and weight where MI is a measurement of digit
preference of recorded data17. Myer’s Index calculates the divergence in the frequency of the ending digit in the
measurements compared with the expected uniform distribution where there is no digit bias. The higher the
value, the more biased the measurement towards a digit or two in all measurements, reflecting rounding effects. Conventional data cleaning. The thresholds for normal ranges of HAZ, WAZ and WHZ specified by the
WHO Child Growth Standards18 were applied for height, weight and BMI measurements. Those satisfying the
condition of HAZ, WAZ or WHZ being within the [−6,6], [−6,5] and [−5,5] ranges respectively were retained
for further analysis. Implausible flagging of sparse data. When longitudinal measurement data were sparse e.g. the number
of entries per individual was less than four, an implausible increment or decrement flag was applied e.g. gain or
loss of >25% of weight within one day; gain or loss of >40% of weight within three months; gain or loss of >50%
of weight within one year; gain of >15% of height within three months; any decrease in height exceeding 1 cm
were flagged for manual checking. Outlier flagging method for longitudinal data. Automated data cleaning of
paediatric anthropometric data
from longitudinal electronic health
records: protocol and application to
a large patient cohort More recently the jack-knife residual method, applicable to paediatric patients with ≥4 datapoints, was suggested
and applied to a paediatric anthropometric dataset for children ≤2 years old11. Although simple to use, it can be
too strict in defining the range of plausible values hence not allowing more pronounced fluctuations in longitudi-
nal data that are typical in the paediatric clinical setting where an individual can reduce or gain significant weight
during or after a treatment period12,13. gt
p
University Hospital Southampton (UHS) is a large teaching and research hospital serving a population of
nearly 3.5 to 4 million people in South Hampshire. The Southampton Children’s Hospital of UHS initiated elec-
tronical recording of anthropometric measurements in 2012 and subsequently developed an Electronic Growth
Chart (EGC) which was rolled out for use across departments in the hospital in 201314. Since then, anthropomet-
ric data on children has been systematically recorded, improving the accuracy of growth data presentation on a
growth chart and enhancing the experience of sharing growth data by clinicians between paediatric specialities. It has also presented an opportunity for research studies to use longitudinal routine patient care anthropomet-
ric data and make correlations between childhood growth and development of disease or efficacy of therapy. However, data recorded for routine clinical care by end-users can be prone to typographical or default value
entry errors often related to time pressure for care delivery. Hence it is necessary that the anthropometric data be
cleaned and processed before it is used for research purposes. p
p
p
In this study, we developed an automated protocol for identifying outliers of longitudinal routine paediatric
height and weight measurements using state-of-the-art outlier detection methods. Concurrently, a subset of UHS
electronic paediatric height and weight data of patients aged 2–20 years old, the gold-standard dataset manual
curated for parameter optimisation, were assessed for data quality. We demonstrate how dataset scrutiny can
identify and target training needs in anthropometric assessment in a teaching hospital. Box 1 Summary of final protocol for outlier flagging for longitudinal height and weight
measurements of a patient Box 1 Summary of final protocol for outlier flagging for longitudinal height and weight
measurements of a patient Box 1 Summary of final protocol for outlier flagging for longitudinal height and weight
measurements of a patient 1. Flag data not satisfying WHO guidelines for heights, weights and BMIs whose SDS values fall beyond
the ranges [−6,6], [−6,5] and [−5,5] respectively, remain n datapoints g
p
y
p
2. If n < 4: assess the implausible increments/decrements of height and weight measurements: p
g
g
i. For weight: for each pair of consecutive measurements, use the following method to flag extreme
changes as below: •
Time span ≤ 1 day: beyond ±25% •
Time span ≤ 3 months: beyond ± 40% •
Time span ≤ 1 year: beyond ± 50% ii. For height •
If time span ≤ 3 months, height increase is ≥15%l •
If time span ≤ 3 months, height increase is ≥15% •
If height measurement at time point is at least 1 cm smaller than time point, flag data at time
point. 3. With the remaining data, where n > =4: 3. With the remaining data, where n > =4: a. Apply the ordinary least square (OLS) linear regression method of the SDS values as a linear
function of age (number of variables k = 1)lfib b. Calculate influence values: Cook’s distance, dffits, dfbeta for age. Retain data that have Cook’s
distance <1, |dffits | <2 and | dfbeta_age | <2/ to re-estimate the regression line and obtain the SD
of the residuals. c. Any patient whose SD of the residuals for height or weight larger than 0.47 or 0.76 respectively
has their whole series of measurements flagged for manual inspection.l d. Where the SD of the residuals for height or weight is ≤1, flag any individual datapoint with resid-
ual error exceeding θ x SD where θ is 2.9 for weight and 2 for height (as informed by parameter
tuning). e. For height data: i. Perform adult height check: for age measurements not flagged in (2c) within the range 18–20
years, calculate median value for that individual Mh, and flag as outlier any height measure-
ment difference exceeding 1 cm.l f
ii. Across all age ranges and for data not already flagged, perform height decrease check. If
height measurement at time point is at least 1 cm smaller than time point, flag data at time
point. Materials and methods For outlier flagging of longitudinal anthropometric
measurements, robust regressions of the linear regression methodology was adopted19. Robust regressions can
handle multiple outliers by introducing residual statistics including influence measurements such as Cook’s dis-
tance, DFFITS, DFBETAS20 (see Supplementary for method details). Datapoints with influence statistics exceeding
suggested thresholds are temporarily removed from the inference and the regression parameters are re-estimated Scientific Reports | (2020) 10:10164 | https://doi.org/10.1038/s41598-020-66925-7 www.nature.com/scientificreports/ from the remaining data. This results in a regression line that best fits the most reliable data. It is this regression
line that is used to discriminate outlying datapoints from the entire set of datapoints using the SD fold threshold θ. Additional checks on height data. In addition to robust regression analysis of the data to detect outli-
ers, height measurements were additionally inspected to flag anomalies such as variation in adult height and/or
height decrease over time as follow. Final adult height is generally reached at approximately 18 years21, therefore,
variation >1 cm from the median height measurements of patients older than 18 years flagged an error in data
recording. Additionally, any decrease in height exceeding 1 cm also prompted a flag to cross check recorded data
manually. This check was applied regardless of the number of datapoints in any set of measurements.l yh
pp
g
p
y
Details of the overall longitudinal height and weight data outlier flagging protocol is summarised in Box 1. Box 1 Summary of final protocol for outlier flagging for longitudinal height and weight
measurements of a patient Outliers were split into three types: height outlier flagging using linear
regression (LR), height entry error with adult height check and height with height decrease check. check, totalling 1,730 flagged height measurements (3.11%). This yielded a gold-standard dataset with a defined
set of ‘true’ errors.i Sensitivity and specificity metrics were evaluated for θ ∈ [1.5,5.5] using the gold standard dataset. Here, a
true positive (TP) was defined as a datapoint identified as an outlier that was deemed clinically implausible by
the clinician, a true negative (TN) was a value that was not flagged as an outlier by our method and identified as
plausible by the clinician, a false positive (FP) was a true plausible value wrongly flagged as an outlier, and a false
negative (FN) was a truly implausible value not flagged as an outlier by the protocol. Therefore, the positive pre-
dictive value (PPV) is an important metric to consider. Ideally, any given protocol should maximise the number
of true outliers as a proportion of all data flagged for manual review while maintaining good sensitivity to detect
all true outliers.h The gold-standard UHS data were used to calculate sensitivity and PPV for θ ∈ [1.5,5.5] (Fig. S4). For both
height and weight, it was desirable to maintain sensitivity above 0.9 while maximising the PPV. Hence for height,
the typical value of θ = 2 was selected but for weight measurements, it was observed that increasing θ to 2.9 main-
tained sensitivity above 0.9 but had a dramatic effect on reducing the manual curation of false positive outliers
(Table 1). These values were used in the final protocol described in Box 1.hi hi
The final selected values of θ were applied to gold standard data sets for height and weight respectively. From
55,688 height measurements, a subset of 4469 measurements (representing 2635 patients) were flagged as out-
liers for manual inspection. Approximately 92% of the data passed checks and could be automatically classified
as plausible. Of the 8% of flagged measurements, the 1237 (2.2%) due to decreases in height may be excluded
without further clinical review and only 5.8% of the data may be subjected to further expert review or excluded
depending on application. Importantly, the protocol failed to flag 36 measurements across 25 patients that the
clinician subsequently flagged as implausible. This represented 0.06% of possible erroneous measurements that
would go undiscovered by automated cleaning. Box 1 Summary of final protocol for outlier flagging for longitudinal height and weight
measurements of a patient 4. If the total number of datapoints flagged (by any step) exceed 40% of the longitudinal data, the whole
series of longitudinal data is flagged for manual inspection. Parameter tuning. Typically, datapoints exceeding 2 times the SD (θ) of any series of measurements are
nominally flagged as outliers, corresponding to an outlier rate of 5%22. However, for voluminous datasets of
growth data in children, this parameter may be unnecessarily stringent. The tuning of θ was facilitated by a
‘gold-standard’ dataset from UHS, manually curated by an endocrinologist (JHD), where each patient had ≥7
datapoints (Supplementary text). This gold-standard dataset consisted of 6,279 patients with 89,258 weight meas-
urements and 4,396 patients with 55,688 height measurements. Of these, 208 (0.23%) weight and 302 (0.54%)
measurements were deemed ‘implausible’ by the endocrinologist. Additional height checks identified a further
191 (0.34%) height measurements failing the adult height check and 1,237 (2.22%) flagged by the height decrease Scientific Reports | (2020) 10:10164 | https://doi.org/10.1038/s41598-020-66925-7 www.nature.com/scientificreports/ (a) Contingency table of weight outlier flagging
(b) Contingency table of height outlier flagging
Weight
θ = 2.9
Manual curation by clinician
Height
θ = 2
Manual curation by
clinician
Impossible
Plausible
Impossible
Plausible
Flagging by
protocol
Outlier
189
2,110
2,299
Flagging by
protocol
Outlier
1,694
2,775
4,469
Plausible
19
86,940
86,959
Plausible
36
51,183
51,219
208
89,050
89,258
1,730
53,958
55,688
Sensitivity = 90.87%
Sensitivity = 97.91%
PPV = 8.22%
PPV = 37.91%
Table 1. Contingency tables for chosen values of θ for weight and height and their sensitivity and PPV#. #PPV
is Positive Predicted Value, defined as the proportion of positive results that are true positive, PPV = TP/
(TP + FP). Table 1. Contingency tables for chosen values of θ for weight and height and their sensitivity and PPV#. #PPV
is Positive Predicted Value, defined as the proportion of positive results that are true positive, PPV = TP/
(TP + FP). Figure 1. Percentage of datapoints identified as true errors in the gold standard dataset stratified by year for
weight and height, weight for height. Outliers were split into three types: height outlier flagging using linear
regression (LR), height entry error with adult height check and height with height decrease check. Figure 1. Percentage of datapoints identified as true errors in the gold standard dataset stratified by year for
weight and height, weight for height. Scientific Reports | (2020) 10:10164 | https://doi.org/10.1038/s41598-020-66925-7 Results Data quality of gold-standard longitudinal data. The ‘gold-standard’ UHS height and weight data-
set enabled assessment of true data quality. Chronologically, both height and weight measurements across the
2008–2018 were stable with an error rate of ~3% for height and 0.2% for weight (Fig. 1). The discrepancy in error
rates between the two measurements was largely attributable to decreases in height which were deemed physio-
logically impossible. g
y
p
Outlier rate by occupation was highest in the Pharmacist group (0.27%) followed by Others (0.20%) and
Dietician (0.16%) for weight. The Pharmacist group recorded the most errors in height as assessed through man-
ual review (2.4%) and using the adult height check (5.7%, Fig. 2a). This likely reflects the pharmacist’s focus on
estimated weight and not height for prescribing purposes. g
g
p
g p
p
By department, the Others group has the highest error rate for weight (0.48%) followed by Dietetics/Speech
and Language Therapy and Paediatric Neurology (0.16%, Fig. 2b). For height data, the highest rate of data deemed
implausible though manual review was observed in Dietetics/Speech and Language Therapy (0.63%) followed by
Paediatric Medicine (0.44%) and Paediatric Oncology (0.40%). Additional height checks saw the highest combined
error rate in Dietetics/Speech and Language Therapy (2.05%) followed by Paediatric Oncology (1.25%, Fig. 2b). Application of automated cleaning protocol to the entire UHS paediatric height and weight
dataset (n = 68,595 patients). UHS data summary and characteristics. The entire cohort contained all
records for patients aged 2–20 years, dating from 1932 to 31/12/2018. A total of 214,983 weight measurements
(68,273 patients) and 146,635 height measurements (47,616 patients) were obtained for 68,595 paediatric patients
in the UHS EPR (Fig. 3a), resulting in 142,643 BMI values (46,479 patients). ( g
),
g
,
(
,
p
)
The number of records was low prior to 2008 (1932–2008) and increased from 2008, reflecting the gradual
introduction of EPR system into UHS departments, with a sharp increase in 2014 when the EGC was introduced
at the end of 2013 (Fig. 3b). The number of weight measurements recorded was about 30% higher than that of
height during 2014–2018 period. Additional description regarding age group at initial measurement, length of
follow-up time is presented in Supplementary (Fig. S4a,b).h p
p
pp
y
g
Patients were grouped by their respective number of longitudinal height and weight measurements. Box 1 Summary of final protocol for outlier flagging for longitudinal height and weight
measurements of a patient Similarly, for weight, 2299 (2.6%) measurements from 1875
patients were flagged as requiring manual expert review while 97.4% of the data passed automated checks. Only
nineteen datapoints (0.02%) that were deemed by the clinician as implausible were missed by the protocol. p
y
p
y
p
All the data processing and protocol implementation was performed using the open-source programming
language Python version 3.723. The ordinary least square method OLS from the Python package statsmodel24
was used to perform LR. The script for calculating SDS values of anthropometric measurements and outlier Scientific Reports | (2020) 10:10164 | https://doi.org/10.1038/s41598-020-66925-7 www.nature.com/scientificreports/ Figure 2. Manual outlier curation results of UHS gold standard paediatric height and weight data: (a)
Percentage of outliers for each of the occupation categories for weight, height using LR, height with adult height
check, and height with height decrease check. (b) Percentage of outliers for each of the department categories
for weight, height using LR, height with adult height check, and height with height decrease check. Figure 2. Manual outlier curation results of UHS gold standard paediatric height and weight data: (a)
Percentage of outliers for each of the occupation categories for weight, height using LR, height with adult height
check, and height with height decrease check. (b) Percentage of outliers for each of the department categories
for weight, height using LR, height with adult height check, and height with height decrease check. detection described by the pipeline is available for use from https://github.com/hangphan/peanof/. This includes
the portable Docker container25 where all dependencies required for running the script were set up and ready to
be executed on any environment where Docker is made available. Ethics and information governance. The study was approved by the IG management team of the
University Hospital of Southampton (UHS). Ethics approval from the Research Ethics Committee and Health
Research Authority, and informed consent was waived by the internal review board at the R&D Department of
UHS as this is a combination of an Audit against WHO guidance and Service Evaluation. The anthropometric
data in UHS were retrospective data and anonymised. All methods used in this study were performed in accord-
ance with the relevant guidelines and regulations. Scientific Reports | (2020) 10:10164 | https://doi.org/10.1038/s41598-020-66925-7 Results There
is an excess of patients with a single measurement entry and these represent approximately half of the cohort,
reflecting paediatric patients with a single hospital visit to departments such as emergency. Patients with ≥7
entries for height and weight represented ~10% of the cohort but contributed almost half of the entire dataset for
both height and weight (Fig. 3d,e). These represent the patient population whose ill health may confer growth and
developmental irregularities requiring frequent monitoring. Scientific Reports | (2020) 10:10164 | https://doi.org/10.1038/s41598-020-66925-7 www.nature.com/scientificreports/ Figure 3. UHS age 2–20 years’ height and weight data (1932–2018) summary: (a) Number of patients and
records of height and weight, broken down by number of datapoints per patients. (b) Total number of height,
weight and BMI measurements over time from prior to 2008 to 2018 (c) Percentage of data flagged by WHO
guidelines over time. (d) Number of patients within groups of patients defined by their number of longitudinal
datapoints for height and weight. (e) Number of height and weight records per group of patients binned by
number of datapoints per patient. Figure 3. UHS age 2–20 years’ height and weight data (1932–2018) summary: (a) Number of patients and
records of height and weight, broken down by number of datapoints per patients. (b) Total number of height,
weight and BMI measurements over time from prior to 2008 to 2018 (c) Percentage of data flagged by WHO
guidelines over time. (d) Number of patients within groups of patients defined by their number of longitudinal
datapoints for height and weight. (e) Number of height and weight records per group of patients binned by
number of datapoints per patient. Figure 4. One decimal place digit distribution for height and weight measurements, demonstrating the bias in
recording height and weight measurements, rounding to the precision of kg for weight and the precision of cm
or 0.5 cm for height. This bias is reflected in the Myers’ index of height and weight measurements. Figure 4. One decimal place digit distribution for height and weight measurements, demonstrating the bias in
recording height and weight measurements, rounding to the precision of kg for weight and the precision of cm
or 0.5 cm for height. This bias is reflected in the Myers’ index of height and weight measurements. Results WAZ
HAZ
WHZ
DHS RANGE OF SD
1.01–1.49
1.08–2.33
1.01–2.02
PRE-WHO PROCESSING SD
5.29
5.90
15.55
POST-WHO PROCESSING SD
1.45
1.32
1.36
Table 2. Standard deviation of WAZ, HAZ and WHZ of the UHS 2–20 anthropometric measurement data. WAZ
HAZ
WHZ
DHS RANGE OF SD
1.01–1.49
1.08–2.33
1.01–2.02
PRE-WHO PROCESSING SD
5.29
5.90
15.55
POST-WHO PROCESSING SD
1.45
1.32
1.36
Table 2. Standard deviation of WAZ, HAZ and WHZ of the UHS 2–20 anthropometric measurement data. Table 2. Standard deviation of WAZ, HAZ and WHZ of the UHS 2–20 anthropometric measurement data. Table 2. Standard deviation of WAZ, HAZ and WHZ of the UHS 2–20 anthropometric measurement data. Scientific Reports | (2020) 10:10164 | https://doi.org/10.1038/s41598-020-66925-7 www.nature.com/scientificreports/ Figure 5. UHS data characterisation by occupation and by department of staff entering the data (a) Weight
records by occupation (b) Height records by occupation (c) Percentage of height and weight data flagged by
WHO rules by occupation (d) Weight records by department (e) Height records by department (f) Percentage
of height and weight data flagged by WHO rules by department. Figure 5. UHS data characterisation by occupation and by department of staff entering the data (a) Weight
records by occupation (b) Height records by occupation (c) Percentage of height and weight data flagged by
WHO rules by occupation (d) Weight records by department (e) Height records by department (f) Percentage
of height and weight data flagged by WHO rules by department. Data quality by conventional quality indicators. The number of records failing WHO child growth standard
guidelines for weight, height and BMI measurements were 1,386 (0.95%) and 814 (0.38%) and 677 (0.47%)
respectively. The percentage of records excluded based on WHO limits was highest in 2013 at 2.37%, 2.64%, and
2.71 for weight, height and BMI respectively (Fig. 3c). This coincides with the gradual introduction of EGC into
various departments across UHS in 2013, reflecting a transient increase in error rate during the transition period
to the electronic recording of data. A comparison of the five years preceding the transition to electronic data
recording and the five years following 2013 identified a significant reduction (pweight = 9.97 × 10−23, pheight = 1.05
× 10−8) in these extreme data recording errors.h g
The SD of HAZ, WAZ and WHZ was calculated and compared against reported ranges of SD observed in the
52-country DHS survey16 (Table 2). Scientific Reports | (2020) 10:10164 | https://doi.org/10.1038/s41598-020-66925-7 Discussionh This study presents a bespoke protocol for automated anthropometric data cleaning that has been tested across a
sizeable dataset captured from a regional teaching hospital in South England. While more than half the patients
represented within the dataset had only a single measurement recorded, approximately half of the collected data
are from a small proportion (~10%) of the patients with multiple longitudinal records. This subset is likely to be
enriched for patient cohorts that are the subject of health research.hi j
The WHO parameters to detect BIVs was a good first pass analysis to detect overtly incorrect data. WHO
outliers represented <1% of recorded measurements. The discrepancy in data quality indicator before and after
WHO guideline BIV detection is a strong indication of a small subset of extreme data entry errors (e.g. records of
1 kg or 100 cm and/or transposition errors in weight and height measurements respectively). It is essential these
conspicuous errors are automatically flagged and excluded from the data before any model fitting approaches
are applied. Additionally, the WHO thresholds should be applied with caution in specific clinical settings where
patients with true clinical data may fall outside these parameters (e.g. growth disorders, morbid obesity) but still
have BIVs longitudinally. g
y
All protocols for cleaning data are limited when data are sparse. We have developed a robust regression frame-
work for automatic identification of erroneous data that performs most reliably across data series with at least
seven measurements. Application of the protocol automatically classifies the majority of records as plausible
values, leaving a small proportion of flagged data that can be either discarded or manually reviewed. The protocol
is computationally cheap to implement and provides assurance of minimal and consistent quality standard for
downstream analysis. The availability of the code in Github and Docker communities allows for the protocol can
be adopted easily in different settings. The protocol is also modifiable, particularly in the arbitrary thresholds
chosen for height and weight checks to reflect less extreme changes in non-clinical settings.l l
Encouragingly, overall error rates in paediatric data were low, with transient fluxes in data quality observed
over periods where new systems were implemented. A higher error rate was noted in measurements of height
compared to weight, largely due to very small decreases in height likely resultant from inconsistencies in measur-
ing techniques, e.g. shoes on or off. Discussionh The error profile by occupation demonstrated that staff routinely required to
measure and enter the data tended to record better quality measurements.h The application of the protocol allows an assessment and rapid feedback regarding data quality in EHR sys-
tems which is valuable for identifying and targeting training needs and data entry practice. This will further
contribute to the overall impact of improving the quality of data available for longitudinal clinical assessment and
patient management as well as enhancing input data quality for large-scale digital healthcare research. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Patient group
Filter
Weight
Height
All
WHO
1,386 (n = 864)
814 (n = 527)
2–3
Extreme change
119 (n = 114)
655 (n = 607)
4–6
OLS robust, few remain
680 (n = 170)
292 (n = 73)
Large SD
114 (n = 24)
296 (n = 61)
LR
3,626
(n = 3,531)
3,029
(n = 2,987)
Adult height
N/A
114 (n = 77)
Height decrease
N/A
357 (n = 365)
≥7
OLS robust, few remain
0
0
Large SD
1,303 (n = 71)
699 (n = 46)
LR
2,573
(n = 2,055)
3,412
(n = 2,581)
Adult height
N/A
222 (n = 121)
Height decrease
N/A
1,395 (n = 674)
Table 3. Number of flagged implausible values by the application of the final outlier flagging protocol to the
UHS 2–20 data set from 1932 to 31/12/2018. Table 3. Number of flagged implausible values by the application of the final outlier flagging protocol to the
UHS 2–20 data set from 1932 to 31/12/2018. Table 3. Number of flagged implausible values by the application of the final outlier flagging protocol to the
UHS 2–20 data set from 1932 to 31/12/2018. increases>15% within 3 months were detected. For those with 2–3 weight measurements, the extreme weight
change check resulted in 119 (0.29%, 114 patients) measurements flagged. For patients with ≥7 datapoints, the
protocol Section 3c flagged 1,303 weight measurements from 71 patients whose weight data had a large residual
SD (>0.76) and protocol Section 3d identified 2,573 weight measurements (2,055 patients) as LR outliers. increases>15% within 3 months were detected. For those with 2–3 weight measurements, the extreme weight
change check resulted in 119 (0.29%, 114 patients) measurements flagged. For patients with ≥7 datapoints, the
protocol Section 3c flagged 1,303 weight measurements from 71 patients whose weight data had a large residual
SD (>0.76) and protocol Section 3d identified 2,573 weight measurements (2,055 patients) as LR outliers.l pi
g
p
Similarly, Section 3c of the protocol flagged 699 height measurements (46 patients) as having large SD and
Section 3d identified 3,412 height measurements (2,581 patients) as LR outliers and the additional adult height
checks (Section 3e) flagged a further 1,617 datapoints (Table 3). Results The SD values prior to exclusion of WHO extreme datapoints fell significantly
outside the expected ranges. However, after exclusions of these extreme values, the observed SD values for height,
weight and BMI z-scores fall within the expected limits.h g
p
The Myer’s Index (MI) for digit preference of height data (excluding WHO extreme values) is consistent with
the average observed across 51 countries in the DHS survey (MIUHS = 17.91, MI51_country_average = 17.8, Fig. 4). The
MI for weight data is higher (MIUHS = 10.69, MI51_country_average = 4.6) suggesting a greater tendency for estimation
in UHS weight data. Data quality indicators by occupation and department of entry staff. The quality of the extracted data was also
scrutinised by staff occupation and department to understand the most likely source of erroneous data and target
the training in anthropometric assessments.f For 75% of the observed data, the occupation and department of the staff member entering the data was
available for evaluation. Ninety-three different staff occupations across 96 different departments were noted and
the ten staff occupations that most frequently entered height and weight measurements are presented in Fig. 5a,b. Healthcare assistants most frequently recorded weight and height data (24% and 30% respectively) followed by
Healthcare support workers, Staff nurses and Consultants.li f
Application of the WHO flags for extreme values identified a low and consistent level of less than 1% of likely
data entry error across occupations (Fig. 5c). The most striking peak in this type of error was 7.5% noted in the
height data entered by pharmacists. However, given pharmacists entered only a very small proportion of the
overall height data (n = 214 records) this higher error rate reflects a very small number (n = 16) extreme values.h l
The Paediatric outpatient department contributed most data for weight and height measurements (47%
and 58% respectively; Fig. 5d,e). The WHO violation rate by department was small and relatively consistent
across departments. The highest rate identified was 1.2% amongst weight values recorded within the Paediatric
Endocrinology department (Fig. 5f). Outlier detection for patients with longitudinal records in UHS dataset. For those with 2–3 height measurements,
the implausible flagging method identified 655 (2.21%, 607 patients) height decreases >1 cm (Table 3). No height Scientific Reports | (2020) 10:10164 | https://doi.org/10.1038/s41598-020-66925-7 References Kato, S., Ashizawa, K. & Satoh, K. An examination of the definition 21. Kato, S., Ashizawa, K. & Satoh, K. An examination of the definition ‘final height’ for practical use. Vol. 25 (1998). 22 Seo S A Review and Comparison of Methods for Detecting Outliers in Univariate Data Sets Master thesis University of Pittsb 21. Kato, S., Ashizawa, K. & Satoh, K. An examination of the definition final height for practical use. Vol. 25 (1998). 22. Seo, S. A Review and Comparison of Methods for Detecting Outliers in Univariate Data Sets Master thesis, Univer
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22. Seo, S. A Review and Comparison of Methods for Detecting Outliers in Univariate Data Sets Master thesis, University of Pitts
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h The data that support the findings of this study are available from UHS, but restrictions apply to the availability
of these data, which were used under license for the current study, and so are not publicly available. Data are
however available from the authors upon reasonable request and with permission of UHS. Received: 5 December 2019; Accepted: 13 May 2020;
Published: xx xx xxxx Received: 5 December 2019; Accepted: 13 May 2020;
Published: xx xx xxxx Received: 5 December 2019; Accepted: 13 May 2020;
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19. Rousseeuw, P. J. & Leroy, A. M. Robust regression and outlier detection. (John Wiley \& Sons, Inc., 1987).l 19. Rousseeuw, P. J. & Leroy, A. M. Robust regression and outlier del 20. Cook, R. D. Detection of Influential Observation in Linear Regression. Technometrics 19, 15–18, https://doi.org
.1977.10489493 (1977). 21. Kato, S., Ashizawa, K. & Satoh, K. An examination of the definition ‘final height’ for practical use. Vol. 25 (1998). . Author contributions F.B. extracted the data from the UHS EPR system. D.C. and J.B. provided the source of reference and the
background work available within UHS to set up the study. H.P. and J.D. designed the protocol. J.D. manually
review the gold standard data. H.P. and S.E. wrote the main manuscript text and HP prepared all the figures, the
github repository and Docker image. All authors reviewed the manuscripts. Competing interestsh Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-66925-7. Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-66925 Correspondence and requests for materials should be addressed to H.T.T.P. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
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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) 2020 Scientific Reports | (2020) 10:10164 | https://doi.org/10.1038/s41598-020-66925-7
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https://pureportal.coventry.ac.uk/files/72717333/Published.pdf
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English
| null |
Friction and Wear in Stages of Galling for Sheet Metal Forming Applications
|
Lubricants
| 2,023
|
cc-by
| 12,983
|
Friction and Wear in Stages of Galling for
Sheet Metal Forming Applications Devenport, T. M., Griffin, J. M., Rolfe, B. F. & Pereira, M. P. Published PDF deposited in Coventry University’s Repository Original citation:
Devenport, TM, Griffin, JM, Rolfe, BF & Pereira, MP 2023, 'Friction and Wear in
Stages of Galling for Sheet Metal Forming Applications', Lubricants, vol. 11, no. 7,
288. g
Devenport, TM, Griffin, JM, Rolfe, BF & Pereira, MP 2023, 'Friction and Wear in
Stages of Galling for Sheet Metal Forming Applications', Lubricants, vol. 11, no. 7,
288. https://dx.doi.org/10.3390/lubricants11070288 DOI 10.3390/lubricants11070288
ISSN 2075-4442 Publisher: MDPI This article is an open access article distributed under the terms and conditions of
the Creative Commons Attribution (CC BY) license
(https://creativecommons.org/licenses/by/4.0/) lubricants lubricants lubricants lubricants Keywords: galling; wear; aluminum; steel; friction; scratch morphology; lump growth Citation: Devenport, T.M.; Griffin,
J.M.; Rolfe, B.F.; Pereira, M.P. Friction
and Wear in Stages of Galling for
Sheet Metal Forming Applications. Lubricants 2023, 11, 288. https://
doi.org/10.3390/lubricants11070288 Article Timothy M. Devenport 1, James M. Griffin 1
, Bernard F. Rolfe 2,*
and Michael P. Pereira 2 1
Institute for Clean Growth and Future Mobility, Coventry University, Coventry CV1 5FB, UK;
tdevenport@deakin.edu.au (T.M.D.)
2
School of Engineering, Deakin University, Geelong, VIC 3216, Australia; michael.pereira@deakin.edu.au
*
Correspondence: bernard.rolfe@deakin.edu.au 1
Institute for Clean Growth and Future Mobility, Coventry University, Coventry CV1 5FB, UK;
tdevenport@deakin.edu.au (T.M.D.)
2
School of Engineering, Deakin University, Geelong, VIC 3216, Australia; michael.pereira@deakin.edu.au
*
Correspondence: bernard.rolfe@deakin.edu.au Abstract: Aluminum is a very commonly used material at present, and roughly half of the produced
aluminum products undergo forming during manufacturing. Galling is a severe form of wear that
occurs during sheet metal forming operations and is a common failure mode of materials in sliding
contact; however, the causes and mechanisms of galling are poorly understood. In this work, sliding
wear experiments were conducted to produce galling wear between a tool steel ball bearing and
aluminum alloy Al5083, to study the relationship between the coefficient of friction, the lump growth
on the tool and the scratch morphology. Whilst the characteristic friction regimes were observed, the
characteristic damage (grooves running parallel to the scratch direction) was not observed. Instead,
when galling was developed on the indenter, the scratch surface morphology displayed a series of
peaks and grooves perpendicular to the scratch direction. It is likely that the difference in scratch
morphology observed once galling was initiated is due to the lower hardness and reduced work
hardening behavior of the Al5083 alloy, compared to the high strength steels previously examined in
sheet metal forming applications. The evolution of the scratch morphology has been characterized in
a novel way by investigating the distribution of the longitudinal cross-section profile height along the
scratch length in relation to the three-stage friction regime observed. This showed that, as the galling
wear progressed, the longitudinal cross-section profile height distribution shifts towards negative
values, with a corresponding shift in the distribution of material transferred to the tool shifting to the
positive. This indicates that, as the amount of material adhered to the indenter increased, the depth
of the grooves on the scratch surface perpendicular to the sliding direction also increased. 1. Introduction The stamping of an aluminum sheet is a common manufacturing technique used for
near net shape production in several industries—such as the automotive, aerospace and
consumer electronics industries and in industrial machinery—because it is cost effective,
offers lightweight construction, produces little scrappage and is well suited to mass produc-
tion. However, stamping along with punching and other sheet metal forming techniques is
often hindered by galling wear [1–4]. Received: 3 June 2023
Revised: 30 June 2023
Accepted: 30 June 2023
Published: 7 July 2023 Galling is a severe form of wear that occurs during sheet metal forming operations
often resulting in scratches on the sheet [5], leading to significant damage to the tooling
and decreased product quality. Galling failure accounts for up to 71% of the cost of die
maintenance [6]. The effects of galling wear are higher energy needs, shorter tool life, lower
part quality and increased cost [7]. In recent years, there has been a growing interest in
understanding and mitigating the effects of galling in the sheet metal forming industry. Whilst galling is a common failure mode of materials in sliding contact, the causes and
mechanisms behind it are poorly understood [8]. Copyright:
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). Galling wear is a combination of adhesive and abrasive wear, whereby material is
transferred from the soft sheet to the hard tool [9], thus damaging the tool and future parts. https://www.mdpi.com/journal/lubricants Lubricants 2023, 11, 288. https://doi.org/10.3390/lubricants11070288 Lubricants 2023, 11, 288 2 of 16
by mat
ool and Hou et al. [6] explained that galling is a stochastic result of adhesive contact between two
surfaces. Galling most frequently occurs between materials that have a protective oxide
surface layer, such as stainless steel and aluminum [10], and so it is important to further
the understanding of galling wear. two surfaces. Galling most frequently occurs between materials that have a protec
ide surface layer, such as stainless steel and aluminum [10], and so it is important
ther the understanding of galling wear. It is widely reported that galling exhibits three distinct regimes, characterized It is widely reported that galling exhibits three distinct regimes, characterized by an
increase in the coefficient of friction (COF) between each regime, as shown in Figure 1. 1. Introduction [14] as shown in Figure 2A–C. However, Sindi et al. [14]
also report on the surface damage after stage 3, as shown in Figure 2D. increased, as shown in Figure 3c. This means an increased unlubricated contact area at the
interface, which would have caused an increase in the coefficient of friction. At the third
stage (point 4), friction fluctuated severely at a value of approximately 1.5, which could
be due to the breakdown of the lubricant leading to direct contact between the steel pin
and warm (300 °C) aluminum blank [16]. As a result of the dry sliding, the strong adhesive
friction force between the steel and hot aluminum with intimate contact could have con-
tributed to the high value of coefficient of friction. Large numbers of aluminum wear par-
ticles were generated and formed wear debris lumps on the wear track, as illustrated in
Figure 3d, resulting in the severe fluctuation of friction values [16]. transform into severe adhesive wear of the entire contact area. This damage is repo
other authors, such as Sindi et al. [14] as shown in Figure 2A–C. However, Sindi et
also report on the surface damage after stage 3, as shown in Figure 2D. Sheets of the 5000-series AlMg alloys are almost exclusively used for comp
inside the car body, e.g., for the structural parts or for inner closure panels [15]. The
studying the galling behavior of this alloy will allow for the bridging of knowled
tween the well-researched tribopairs such as steel on steel and 7000-series alumin
loys and steel wearing against even softer aluminum alloys such as the 1000, 30
6000 series. Yang et al [16] investigated friction evolution due to lubricant breakdown in
Figure 2. Sheet morphology from a slider on flat sheet tests of steel-on-steel under a 125 N load, and
630 mm/min sliding speed, reproduced with permission from Sindi et al. [14]. Figure 2. Sheet morphology from a slider on flat sheet tests of steel-on-steel under a 125 N load, and
630 mm/min sliding speed, reproduced with permission from Sindi et al. [14]. as Si –C. H loys and steel wearing against even softer aluminum alloys such as the 1000, 30
6000 series. Yang et al [16] in estigated friction e olution due to lubricant breakdo
n in
Figure 2. 1. Introduction Initially, in stage 1 (before galling) the friction coefficient is low and reasonably constant,
increasing sharply into stage 2 (galling initiation and transition), before increasing further
and wildly fluctuating in the final stage, stage 3 (severe galling). increase in the coefficient of friction (COF) between each regime, as shown in Fi
Initially, in stage 1 (before galling) the friction coefficient is low and reasonably co
increasing sharply into stage 2 (galling initiation and transition), before increasing
and wildly fluctuating in the final stage, stage 3 (severe galling). Figure 1. Typical friction curve for progressing galling wear, adapted with permission from
son et al. [11]. Figure 1. Typical friction curve for progressing galling wear, adapted with permission from
Eriksson et al. [11]. EVIEW
3 of 17
friction stage, indicating that the boundary lubrication occurred during this stage (Figure
3a). It was stated that the friction force arose from the adhesion force between the asperity
contacts and the ploughing of hard asperities on the steel pin surface (as well as the effects h
h b
d Figure 1. Typical friction curve for progressing galling wear, adapted with permission from
son et al. [11]. Figure 1. Typical friction curve for progressing galling wear, adapted with permission from
Eriksson et al. [11]. friction stage, indicating that the boundary lubrication occurred during this stage (Figure
3a). It was stated that the friction force arose from the adhesion force between the asperity
contacts and the ploughing of hard asperities on the steel pin surface (as well as the effects Figure 1. Typical friction curve for progressing galling wear, adapted with permission from
son et al. [11]. Figure 1. Typical friction curve for progressing galling wear, adapted with permission from
Eriksson et al. [11]. friction stage, indicating that the boundary lubrication occurred during this stage (Figure
3a). It was stated that the friction force arose from the adhesion force between the asperity
contacts and the ploughing of hard asperities on the steel pin surface (as well as the effects Vilhena et al. [10] examined the galling wear of the aluminum and M2 steel tr
and confirmed a sharp rise in the curve of the friction force vs. normal force can b
as a signal for galling initiation. Heli et al. 1. Introduction [16] reported that a significant number of
asperities at the contact interface came into contact, resulting in the detachment of the
aluminum wear particles and the subsequent formation of wear debris lumps on the wear
track on the blank. As sliding proceeded (point 3), the area covered by wear debris lumps
increased, as shown in Figure 3c. This means an increased unlubricated contact area at the
interface, which would have caused an increase in the coefficient of friction. At the third
stage (point 4), friction fluctuated severely at a value of approximately 1.5, which could
be due to the breakdown of the lubricant leading to direct contact between the steel pin
and warm (300 °C) aluminum blank [16]. As a result of the dry sliding, the strong adhesive
friction force between the steel and hot aluminum with intimate contact could have con-
tributed to the high value of coefficient of friction. Large numbers of aluminum wear par-
ticles were generated and formed wear debris lumps on the wear track, as illustrated in
Figure 3d, resulting in the severe fluctuation of friction values [16]. g
g
y
q
tween the generation and ejection of loose wear particles or the third body (as opp
the erratic third stage). Gaard et al. [13] describe the initial surface damage in steels occurring due to
deformation and the flattening of the track, with subsequent sliding and transfor
into the second frictional regime occurring due to the transition in wear mechan
the abrasive scratching of the sheet surface. At this stage, Gaard et al. [13] report th
width was observed to be significantly wider as compared to the initial regime. further sliding leads to the second transition in friction, where scratching was fo
Gaard et al. [13] describe the initial surface damage in steels occurring due to plastic
deformation and the flattening of the track, with subsequent sliding and transformation
into the second frictional regime occurring due to the transition in wear mechanisms to
the abrasive scratching of the sheet surface. At this stage, Gaard et al. [13] report the track
width was observed to be significantly wider as compared to the initial regime. Finally,
further sliding leads to the second transition in friction, where scratching was found to
transform into severe adhesive wear of the entire contact area. This damage is reported by
other authors, such as Sindi et al. 1. Introduction [12] found the rapid increase in the
coefficient, at the running-in stage under dry sliding contact conditions, was due
formation of an aluminum transfer layer between the contact surfaces. The friction
cient stabilized at a high value, indicating that a dynamic equilibrium was achiev
Vilhena et al. [10] examined the galling wear of the aluminum and M2 steel tribopair
and confirmed a sharp rise in the curve of the friction force vs. normal force can be used as a
signal for galling initiation. Heli et al. [12] found the rapid increase in the friction coefficient,
at the running-in stage under dry sliding contact conditions, was due to the formation of an
aluminum transfer layer between the contact surfaces. The friction coefficient stabilized at
a high value, indicating that a dynamic equilibrium was achieved between the generation
and ejection of loose wear particles or the third body (as opposed to the erratic third stage). of lubricant failure). At the beginning of the second stage (point 2), the coefficient of fric-
tion began to increase, which Yang et al. [16] attributes to aluminum wear particles ag-
glomerating and forming wear debris lumps on the wear track, as shown by the dark areas
in Figure 3b. As the lubricant failed, Yang et al. [16] reported that a significant number of
asperities at the contact interface came into contact, resulting in the detachment of the
aluminum wear particles and the subsequent formation of wear debris lumps on the wear
track on the blank. As sliding proceeded (point 3), the area covered by wear debris lumps Vilhena et al. [10] examined the galling wear of the aluminum and M2 steel tr
and confirmed a sharp rise in the curve of the friction force vs. normal force can b
as a signal for galling initiation. Heli et al. [12] found the rapid increase in the
coefficient, at the running-in stage under dry sliding contact conditions, was due
formation of an aluminum transfer layer between the contact surfaces. The friction
cient stabilized at a high value, indicating that a dynamic equilibrium was achiev
tween the generation and ejection of loose wear particles or the third body (as opp
the erratic third stage). Gaard et al. 1. Introduction [13] describe the initial surface damage in steels occurring due to
deformation and the flattening of the track, with subsequent sliding and transfor
into the second frictional regime occurring due to the transition in wear mechan
the abrasive scratching of the sheet surface. At this stage, Gaard et al. [13] report th
width was observed to be significantly wider as compared to the initial regime. F
further sliding leads to the second transition in friction, where scratching was fo
Vilhena et al. [10] examined the galling wear of the aluminum and M2 steel tribopair
and confirmed a sharp rise in the curve of the friction force vs. normal force can be used as a
signal for galling initiation. Heli et al. [12] found the rapid increase in the friction coefficient,
at the running-in stage under dry sliding contact conditions, was due to the formation of an
aluminum transfer layer between the contact surfaces. The friction coefficient stabilized at
a high value, indicating that a dynamic equilibrium was achieved between the generation
and ejection of loose wear particles or the third body (as opposed to the erratic third stage). Gaard et al. [13] describe the initial surface damage in steels occurring due to plastic
deformation and the flattening of the track, with subsequent sliding and transformation
into the second frictional regime occurring due to the transition in wear mechanisms to
the abrasive scratching of the sheet surface. At this stage, Gaard et al. [13] report the track
width was observed to be significantly wider as compared to the initial regime. Finally,
further sliding leads to the second transition in friction, where scratching was found to
transform into severe adhesive wear of the entire contact area. This damage is reported by
other authors, such as Sindi et al. [14] as shown in Figure 2A–C. However, Sindi et al. [14]
also report on the surface damage after stage 3, as shown in Figure 2D. of lubricant failure). At the beginning of the second stage (point 2), the coefficient of fric-
tion began to increase, which Yang et al. [16] attributes to aluminum wear particles ag-
glomerating and forming wear debris lumps on the wear track, as shown by the dark areas
in Figure 3b. As the lubricant failed, Yang et al. 1. Introduction Sheet morphology from a slider on flat sheet tests of steel-on-steel under a 125 N load, and
630 mm/min sliding speed, reproduced with permission from Sindi et al. [14]. Figure 2. Sheet morphology from a slider on flat sheet tests of steel-on-steel under a 125 N load, and
630 mm/min sliding speed, reproduced with permission from Sindi et al. [14]. Lubricants 2023, 11, 288
Lubricants 2023, 11, x FOR 3 of 16
3 of 17 3 of 16
3 of 17 Sheets of the 5000-series AlMg alloys are almost exclusively used for components
inside the car body, e.g., for the structural parts or for inner closure panels [15]. Therefore,
studying the galling behavior of this alloy will allow for the bridging of knowledge between
the well-researched tribopairs such as steel on steel and 7000-series aluminum alloys and
steel wearing against even softer aluminum alloys such as the 1000, 3000 and 6000 series. friction stage, indicating that the boundary lubrication occurred during this stage (Figure
3a). It was stated that the friction force arose from the adhesion force between the asperity
contacts and the ploughing of hard asperities on the steel pin surface (as well as the effects
of lubricant failure). At the beginning of the second stage (point 2), the coefficient of fric-
tion began to increase, which Yang et al. [16] attributes to aluminum wear particles ag-
glomerating and forming wear debris lumps on the wear track as shown by the dark areas g g
y
Yang et al. [16] investigated friction evolution due to lubricant breakdown in warm
aluminum forming processes and showed how the damage accumulates in AA7075 during
pin on disc tests, as shown in Figure 3. Initially, ploughing grooves were observed by
Yang et al. [16] on the aluminum surface and the wear track was clearly visible at the
low friction stage, indicating that the boundary lubrication occurred during this stage
(Figure 3a). It was stated that the friction force arose from the adhesion force between
the asperity contacts and the ploughing of hard asperities on the steel pin surface (as
well as the effects of lubricant failure). At the beginning of the second stage (point 2),
the coefficient of friction began to increase, which Yang et al. [16] attributes to aluminum
wear particles agglomerating and forming wear debris lumps on the wear track, as shown
by the dark areas in Figure 3b. 1. Introduction As the lubricant failed, Yang et al. [16] reported that a
significant number of asperities at the contact interface came into contact, resulting in
the detachment of the aluminum wear particles and the subsequent formation of wear
debris lumps on the wear track on the blank. As sliding proceeded (point 3), the area
covered by wear debris lumps increased, as shown in Figure 3c. This means an increased
unlubricated contact area at the interface, which would have caused an increase in the
coefficient of friction. At the third stage (point 4), friction fluctuated severely at a value of
approximately 1.5, which could be due to the breakdown of the lubricant leading to direct
contact between the steel pin and warm (300 ◦C) aluminum blank [16]. As a result of the
dry sliding, the strong adhesive friction force between the steel and hot aluminum with
intimate contact could have contributed to the high value of coefficient of friction. Large
numbers of aluminum wear particles were generated and formed wear debris lumps on
the wear track, as illustrated in Figure 3d, resulting in the severe fluctuation of friction
values [16]. glomerating and forming wear debris lumps on the wear track, as shown by the dark areas
in Figure 3b. As the lubricant failed, Yang et al. [16] reported that a significant number of
asperities at the contact interface came into contact, resulting in the detachment of the
aluminum wear particles and the subsequent formation of wear debris lumps on the wear
track on the blank. As sliding proceeded (point 3), the area covered by wear debris lumps
increased, as shown in Figure 3c. This means an increased unlubricated contact area at the
interface, which would have caused an increase in the coefficient of friction. At the third
stage (point 4), friction fluctuated severely at a value of approximately 1.5, which could
be due to the breakdown of the lubricant leading to direct contact between the steel pin
and warm (300 °C) aluminum blank [16]. As a result of the dry sliding, the strong adhesive
friction force between the steel and hot aluminum with intimate contact could have con-
tributed to the high value of coefficient of friction. Large numbers of aluminum wear par-
ticles were generated and formed wear debris lumps on the wear track, as illustrated in
Figure 3d, resulting in the severe fluctuation of friction values [16]. Figure 2. 1. Introduction Sheet morphology from a slider on flat sheet tests of steel-on-steel under a 125 N load, and
630 mm/min sliding speed, reproduced with permission from Sindi et al. [14]. Figure 3. Optical microscopy images of the aluminium alloy wear track, showing how the damage
evolves with friction (at elevated temperature of 300 °C), reproduced with permission from Yang et
al. [16]. Figure 3. Optical microscopy images of the aluminium alloy wear track, showing how the dam-
age evolves with friction (at elevated temperature of 300 ◦C), reproduced with permission from
Yang et al. [16]. Figure 3. Optical microscopy images of the aluminium alloy wear track, showing how the damage
evolves with friction (at elevated temperature of 300 °C), reproduced with permission from Yang et
al. [16]. Figure 3. Optical microscopy images of the aluminium alloy wear track, showing how the dam-
age evolves with friction (at elevated temperature of 300 ◦C), reproduced with permission from
Yang et al. [16]. Lubricants 2023, 11, 288 4 of 16 There are several disagreements/differences in the literature regarding the evolution
of the worn surface morphology during galling, possibly due to the stochastic nature of
galling and the fact that the micro-scale galling behavior is complex and can be sensitive to
small changes in the conditions. Whilst it is widely reported that the tribological behavior
of two surfaces depends strongly on the conditions of the tribosystem, conditions such as
tool geometry and materials of the rubbing surfaces, atmosphere and temperature, possible
lubrication and friction between the contacting surfaces, contact pressure, sliding speed all
contribute to this tribosystem. Moreover, it is known that in ambient room temperature
environments, contaminant films are present on metallic surfaces. The main environmental
effect is the adsorption of oxygen and the oxidation of metals. It has been shown that clean
metal surfaces in contact can result in severe adhesion [17,18]. Therefore, in the laboratory
testing environment, it is important to examine the galling behavior for the triboconditions
that are close to those experienced in the real (industrial) wear processes. The goal of this research is to investigate the underlying mechanisms of galling wear in
sheet metal cold forming by studying the galling behavior of a hard tool in sliding against
an aluminum plate, under conditions not conducive to oxide production in the contact. 1. Introduction This is achieved by conducting dry sliding wear tests between a spherical steel indenter
and an Al5083 plate at room temperature, low sliding speed and low load to reduce the
effects of frictional heating. The primary results examined are the evolution of the friction
coefficient, lump growth on the indenter and scratch morphology. New insights into the
galling behavior and methods of characterizing the scratch surface morphology and galling
stages are gained. 2. Materials and Methods Dry sliding of a spherical steel indenter on an Al5083 plate has been employed to
investigate the galling wear behavior. 2.2. Test Setup
2.2. Test Setup
Scratch t Scratch tests were conducted using a Bruker TriboLab UMT machine, Figure 4. The
Bruker UMT TriboLab can precisely measure the position of the indenter (Z height, mm)
and the position of the stage. The load cell in the Bruker UMT TriboLab was used to
measure the frictional forces (N) and applied load (N). Both the positions and loads were
measured at a sampling rate of 1 kHz. The sample plates were attached to the Bruker stage
via the four bolts shown and the indenter in the indenter holder. Scratch tests were conducted using a Bruker TriboLab UMT machine, Figure 4. T
Bruker UMT TriboLab can precisely measure the position of the indenter (Z height, m
and the position of the stage. The load cell in the Bruker UMT TriboLab was used to me
ure the frictional forces (N) and applied load (N). Both the positions and loads were me
ured at a sampling rate of 1 kHz. The sample plates were attached to the Bruker stage
the four bolts shown and the indenter in the indenter holder. Figure 4. Bruker TriboLab UMT as used for this experimental regime. Figure 4. Bruker TriboLab UMT as used for this experimental regime. Figure 4. Bruker TriboLab UMT as used for this experimental regi
Figure 4. Bruker TriboLab UMT as used for this experimental regime. Immediately after each scratch, the indenters were measured using an Alicona
finiteFocus optical profilometer with no additional cleaning. The sample plates w
measured later, after all scratches were conducted on the plate, also with no additio
cleaning. By using optical profilometry, visual observations of the scratch morphology
the sheet material and associated material built-up edge and lump growth on the t
were conducted. The longitudinal cross-section profile of the scratch was measured wit
the scratch width (width of the measurement = 0.1 mm, as shown in Figure 5), and acr
the lump growth on the indenter. For analysis of the lump growth on the indenter, it w
necessary to use the form removal tool in the Alicona software. This “flattening” of
scan surface allowed an easier comparison between the indenter surfaces, measurem
f th
l
f
t
i l dh
d
d th
f
b tt
d
t
di
f th
lli
Immediately after each scratch, the indenters were measured using an Alicona Infinite-
Focus optical profilometer with no additional cleaning. 2.1. Materials The indenter used was a commercially available Chrome–Molybdenum high tensile
steel (AISI 4140) ball bearing, 10 mm in diameter, with a reported hardness of 207 HV [19]. The same ball bearing was reused for each scratch stroke to observe how the damage
accumulated with the sliding distance. However, once galling had occurred (as identified
by the coefficient of friction and microscopy, described below), a fresh indenter was used. The plate material was an aluminum alloy, Al5083 H32 of 3 mm thickness. This is a
commercially available alloy used in the automotive industry for producing lightweight
car body components. y
p
To simulate industry, both the plate and indenters were tested in the “as-received”
condition. There were no additional in-house treatments for either component. The plates
were wiped with acetone and left to air dry prior to testing to remove any surplus dust or
contaminants. The hardness of the plates was measured to be 100 HV (corresponding to
the manufacturers’ reports of 89 HB), using a Struers Microhardness tester using a Vickers
indenter with a 300 g load. The plate roughness measured using an Alicona InfiniteFocus
optical profilometer is given in Table 1, and the chemical composition is given in Table 2. Table 1. Sample plate roughness values (experimentally measured). Table 1. Sample plate roughness values (experimentally measured). Table 1. Sample plate roughness values (experimentally measured). Plate
Ra (in Sliding Direction) (µm)
Ra (across Sliding Direction) (µm)
1
0.027
0.202
2
0.031
0.275
3
0.024
0.231
4
0.023
0.229
5
0.021
0.286
6
0.031
0.243
Average
0.026
0.244 Plate
Ra (in Sliding Direction) (µm)
Ra (across Sliding Direction) (µm) Lubricants 2023, 11, 288 5 of 16 Table 2. Chemical composition [20]. Element
% Present
Si
0.4
Fe
0.4
Cu
0.1
Mn
0.4–1.0
Mg
4.0–4.9
Zn
0.25
Ti
0.15
Cr
0.05–0.25
Al
Balance
Element
% Present
Si
0.4
Fe
0.4
Cu
0.1
Mn
0.4–1.0
Mg
4.0–4.9
Zn
0.25
Ti
0.15
Cr
0.05–0.25
Al
Balance 2.3. Data Processing
2.3. Data Processing
2.3. Data Processing
As described ab As described above, the coefficient of friction was recorded continuously during the
scratch tests. Using the friction coefficient data, the three stages of galling were differenti-
ated using the MATLAB software package, as illustrated in Figure 6. The transition point
between the end of stage 1 and the start of stage 2 was found using MATLAB software’s
“Ischange” function on the smoothed coefficient of the friction data. This function was
used to find the point of greatest positive gradient change in the coefficient of friction,
prior to the coefficient of friction exceeding 0.5, as it was assumed (based on observation
of all the tests) that a coefficient of friction value of 0.5 or higher was in the middle of stage
2. The transition point at the end of stage 2 and start of stage 3 was determined by the first
instance in which the coefficient of friction was greater than 0.9. Again, this rule was de-
veloped based on the analysis of all of the test data and galling observations. If this start
of post galling was very close to the end of the test, i.e., after more than 95% of the test
was completed, this phase was excluded, and the test characterized as only have stage 1
and stage 2. Similarly, if a transition to stage 2 happened near the end of the scratch, this
test was characterized as only exhibiting stage 1. As described above, the coefficient of friction was recorded continuously during the
scratch tests. Using the friction coefficient data, the three stages of galling were differenti-
ated using the MATLAB software package, as illustrated in Figure 6. The transition point
between the end of stage 1 and the start of stage 2 was found using MATLAB software’s
“Ischange” function on the smoothed coefficient of the friction data. This function was
used to find the point of greatest positive gradient change in the coefficient of friction,
prior to the coefficient of friction exceeding 0.5, as it was assumed (based on observation
of all the tests) that a coefficient of friction value of 0.5 or higher was in the middle of
stage 2. The transition point at the end of stage 2 and start of stage 3 was determined by
the first instance in which the coefficient of friction was greater than 0.9. igu e
E a
p e o
2 4 T
C
di i
2.4. Test Conditions
2.4. Test Conditions 2.4. Test Conditions
The scratch tests were conducted using a load control for the normal force. All
scratches applied a constant 10 N normal load, using a load cell with a load range of 0.5
N to 50 N and a load resolution of ±2.5 mN. This load generates a maximum contact pres-
sure of 650 MPa (mean contact pressure of 430 MPa), based on Hertzian contact pressure
calculations assuming elastic deformation. This contact pressure closely corresponds to
the contact pressures in the sheet metal forming and other works reporting on the galling
of aluminum [12,21]. A total of 34 scratches were successfully performed on the sample
plates. Since some tests were the result of the same ball sample continuing after the first
or second 50 mm scratch length increment, these 34 scratch segments corresponded to 22
The scratch tests were conducted using a load control for the normal force. All
scratches applied a constant 10 N normal load, using a load cell with a load range of 0.5
N to 50 N and a load resolution of ±2.5 mN. This load generates a maximum contact pres-
sure of 650 MPa (mean contact pressure of 430 MPa), based on Hertzian contact pressure
calculations assuming elastic deformation. This contact pressure closely corresponds to
the contact pressures in the sheet metal forming and other works reporting on the galling
of aluminum [12,21]. A total of 34 scratches were successfully performed on the sample
plates. Since some tests were the result of the same ball sample continuing after the first
or second 50 mm scratch length increment, these 34 scratch segments corresponded to 22
individual ball samples as shown in Table 3
The scratch tests were conducted using a load control for the normal force. All
scratches applied a constant 10 N normal load, using a load cell with a load range of 0.5 N
to 50 N and a load resolution of ±2.5 mN. This load generates a maximum contact pressure
of 650 MPa (mean contact pressure of 430 MPa), based on Hertzian contact pressure
calculations assuming elastic deformation. This contact pressure closely corresponds to the
contact pressures in the sheet metal forming and other works reporting on the galling of
aluminum [12,21]. A total of 34 scratches were successfully performed on the sample plates. 2.2. Test Setup
2.2. Test Setup
Scratch t The sample plates were measured
later, after all scratches were conducted on the plate, also with no additional cleaning. By
using optical profilometry, visual observations of the scratch morphology on the sheet ma-
terial and associated material built-up edge and lump growth on the tool were conducted. The longitudinal cross-section profile of the scratch was measured within the scratch width
(width of the measurement = 0.1 mm, as shown in Figure 5), and across the lump growth
on the indenter. For analysis of the lump growth on the indenter, it was necessary to use the
form removal tool in the Alicona software. This “flattening” of the scan surface allowed an
easier comparison between the indenter surfaces, measurement of the volume of material
adhered and, therefore, better understanding of the galling evolution. Lubricants 2023, 11, 288
Lubricants 2023, 11, x FO
Lubricants 2023, 11, x FOR 6 of 16
6 of 17
6 of 17 Figure 5. Example of the profile form measurement of a test that exhibited stage 3 galling. The white
arrow indicates the indenter sliding direction. Figure 5. Example of the profile form measurement of a test that exhibited stage 3 galling. The white
arrow indicates the indenter sliding direction. Figure 5. Example of the profile form measurement of a test that exhibited stage 3 galling. The white
arrow indicates the indenter sliding direction. 2 3 Data P o e i Figure 5. Example of the profile form measurement of a test that exhibited stage 3 galling. The white
arrow indicates the indenter sliding direction. Figure 5. Example of the profile form measurement of a test that exhibited stage 3 galling. The white
arrow indicates the indenter sliding direction. g
p
p
g
g
g
arrow indicates the indenter sliding direction. 2.3. Data Processing
2.3. Data Processing
2.3. Data Processing
As described ab Again, this rule
was developed based on the analysis of all of the test data and galling observations. If this
start of post galling was very close to the end of the test, i.e., after more than 95% of the test
was completed, this phase was excluded, and the test characterized as only have stage 1
and stage 2. Similarly, if a transition to stage 2 happened near the end of the scratch, this
test was characterized as only exhibiting stage 1. As described above, the coefficient of friction was recorded continuously during the
scratch tests. Using the friction coefficient data, the three stages of galling were differenti-
ated using the MATLAB software package, as illustrated in Figure 6. The transition point
between the end of stage 1 and the start of stage 2 was found using MATLAB software’s
“Ischange” function on the smoothed coefficient of the friction data. This function was
used to find the point of greatest positive gradient change in the coefficient of friction,
prior to the coefficient of friction exceeding 0.5, as it was assumed (based on observation
of all the tests) that a coefficient of friction value of 0.5 or higher was in the middle of stage
2. The transition point at the end of stage 2 and start of stage 3 was determined by the first
instance in which the coefficient of friction was greater than 0.9. Again, this rule was de-
veloped based on the analysis of all of the test data and galling observations. If this start
of post galling was very close to the end of the test, i.e., after more than 95% of the test
was completed, this phase was excluded, and the test characterized as only have stage 1
and stage 2. Similarly, if a transition to stage 2 happened near the end of the scratch, this
test was characterized as only exhibiting stage 1. Figure 6. Example coefficient of friction curve for progressing galling wear. Figure 6. Example coefficient of friction curve for progressing galling wear. Figure 6. Example coefficient of friction curve for progressing galling wear. Figure 6 Example coefficient of friction curve for progressing galling wear
Figure 6. Example coefficient of friction curve for progressing galling wear. Figure 6. Example coefficient of friction curve for progressing galling wear. igu e
E a
p e o
2 4 T
C
di i
2.4. Test Conditions
2.4. Test Conditions Since some tests were the result of the same ball sample continuing after the first or second
50 mm scratch length increment, these 34 scratch segments corresponded to 22 individual
ball samples as shown in Table 3. Lubricants 2023, 11, 288 7 of 16 Table 3. Summary of scratch length and galling stages observed for all tests. Ball Sample
Number
Galling Stage(s)
Observed for Scratch
Increment A (0–50 mm)
Galling Stage(s)
Observed for Scratch
Increment B (50–100 mm)
Galling Stage(s) Observed
for Scratch Increment C
(100–150 mm)
Total Scratch
Length (mm)
1
1, 2, 3
-
-
50
2
1
-
-
24
3
1
-
-
24
4
1
-
-
36
5
1, 2
-
-
50
6
1, 2, 3
-
-
50
7
1, 2, 3
-
-
50
8
1, 2, 3
-
-
50
9
1, 2, 3
-
-
50
10
1, 2, 3
-
-
50
11
1
2
3
150
12
1
2
3
150
13
1, 2
3
-
100
14
1
1,2
-
100
15
1, 2
-
-
50
16
1, 2
-
-
50
17
1, 2
3
-
100
18
1, 2
3
-
100
19
1, 2
3
-
100
20
1, 2
3
-
100
21
1, 2
3
-
100
22
1, 2
3
-
100 Table 3. Summary of scratch length and galling stages observed for all tests. The sliding speed was kept to 1 mm/s to minimize the effects of frictional heating
during the test (it has been reported that sliding speed affects galling wear [22]). The
majority of tests were stopped after severe galling was detected; however, some tests were
stopped at the moment the coefficient of friction reached 0.9, so the total scratch length for
each sample varied, depending on when the galling occurred. Due to the stochastic nature
of galling initiation and progression, the total sliding distance for each test varied from
24 mm up to 150 mm. The maximum scratch length that could be achieved in one length on
the sample was 50 mm. Therefore, scratches longer than 50 mm consisted of more than one
scratch (using the same indenter). For example, for the case of the tests with 150 mm scratch
lengths, these consisted of three consecutive scratches each of a length of 50 mm. igu e
E a
p e o
2 4 T
C
di i
2.4. Test Conditions
2.4. Test Conditions The
scratch tests were conducted using a load control for the normal force. All scratches applied
a constant 10 N normal load, using a load cell with a load range of 0.5 N to 50 N and a load
resolution of ±2.5 mN. This load generates a maximum contact pressure of 650 MPa (mean
contact pressure of 430 MPa), based on Hertzian contact pressure calculations assuming
elastic deformation. This contact pressure closely corresponds to the contact pressures in
sheet metal forming and other works reporting on the galling of aluminum. A total of
34 scratches were successfully performed on the sample plates. Since some tests were the
result of the same ball sample continuing after the first or second 50 mm scratch length
increment, these 34 scratch segments corresponded to 22 individual ball samples as shown
in Table 3; most scratch tests were not stopped until the end of the 50 mm scratch increment. Lubricant was not used Lubricants 2023, 11, 288 8 of 16 8 of 16 3. Results
coefficien The galling wear in this manuscript has been characterized and investigated via the
coefficient of friction vs. time measurements, the scratch morphology and the lump growth
on the indenter. growth on the indenter. 3.1. Friction 3.1. Friction
The co
ob e
ed i The coefficient of friction from each experiment, categorized by the stages of galling
observed, is given in Figure 7. The rest of the results are correlated against this friction
measurement. It is evident that the friction did not increase when the galling did not occur
(as shown by Figure 7A). Additionally, an increasing coefficient of friction can be seen in
the second stage of galling. Finally, the third stage exhibits the highest coefficient of friction
of approximately 1. observed, is given in Figure 7. The rest of the results are correlated against this
measurement. It is evident that the friction did not increase when the galling did no
(as shown by Figure 7A). Additionally, an increasing coefficient of friction can be
the second stage of galling. Finally, the third stage exhibits the highest coefficient
tion of approximately 1. Figure 7. Coefficient of friction curves for all tests. Refer to Table 3 for reference on t
stages. Figure 7. Coefficient of friction curves for all tests. Refer to Table 3 for reference on the tests and stag
3 2 L
G
th
th T l Figure 7. Coefficient of friction curves for all tests. Refer to Table 3 for reference on the t
stages. Figure 7. Coefficient of friction curves for all tests. Refer to Table 3 for reference on the tests and stages. 3.2. Lump Growth on the Tool Figure 7. Coefficient of friction curves for all tests. Refer to Table 3 for reference on the
Figure 7. Coefficient of friction curves for all tests. Refer to Table 3 for reference on the tests and stages. Figure 7. Coefficient of friction curves for all tests. Refer to Table 3 for reference on the t
stages. Figure 7. Coefficient of friction curves for all tests. Refer to Table 3 for reference on the tests and stages. 3.2. Lump Growth on the Tool stages.
3.2. Lump Growth on the Tool 3.2. Lump Growth on the Tool
Figure 8 shows an example of an unworn/new indenter ball and the lump gro
the indenter after successive 50 mm scratches (up to 150 mm sliding). Figure 9 sho
measured volume of transferred material on all of the balls of the indenters. Fro
Figure 8 shows an example of an unworn/new indenter ball and the lump growth
on the indenter after successive 50 mm scratches (up to 150 mm sliding). Figure 9 shows
the measured volume of transferred material on all of the balls of the indenters. From
both figures, it is evident that there is more material transferred at later stages of the
galling process. Lubricants 2023, 11, 288
Lubricants 2023, 11, x FO 9 of 16
17 Figure 8. Lump growth on the tool. (A) Unworn, (B) 50 mm sliding, (C) 100 mm sliding, (D) 150
mm sliding. The white arrow indicates the sheet sliding direction. Figure 8. Lump growth on the tool. (A) Unworn, (B) 50 mm sliding, (C) 100 mm sliding, (D) 150 mm
sliding. The white arrow indicates the sheet sliding direction. Figure 8. Lump growth on the tool. (A) Unworn, (B) 50 mm sliding, (C) 100 mm sliding, (D) 150
mm sliding. The white arrow indicates the sheet sliding direction. Figure 8. Lump growth on the tool. (A) Unworn, (B) 50 mm sliding, (C) 100 mm sliding, (D) 150
mm sliding. The white arrow indicates the sheet sliding direction. Figure 8. Lump growth on the tool. (A) Unworn, (B) 50 mm sliding, (C) 100 mm sliding, (D) 150 mm
sliding. The white arrow indicates the sheet sliding direction. Figure 8. Lump growth on the tool. (A) Unworn, (B) 50 mm sliding, (C) 100 mm sliding, (D) 150
mm sliding. The white arrow indicates the sheet sliding direction. Figure 9. Lump growth—volume of material transferred to the indenter for each galling stage. Figure 9. Lump growth—volume of material transferred to the indenter for each galling stage. Figure 9. Lump growth—volume of material transferred to the indenter for each galling stage. Figure 9. Lump growth—volume of material transferred to the indenter for each galling stage. Figure 9. Lump growth—volume of material transferred to the indenter for each galling stage. Figure 10 shows the cross-sectional shape of the build-up on the indenter between each
stage. stages.
3.2. Lump Growth on the Tool Figure 10. Cross-sections of lump growth on all of the indenters after each segment of the scratch
tests, separated by the stage of galling. The sheet sliding direction is from left to right. Figure 10 Cross-sections of lump growth on all of the indenters after each segment of the scratch
Figure 10 Cross sections of lump growth on all of the indenters after each segment of the scratch Figure 10. Cross-sections of lump growth on all of the indenters after each segment of the scratch
tests, separated by the stage of galling. The sheet sliding direction is from left to right. Figure 10. Cross-sections of lump growth on all of the indenters after each segment of the scratch
tests, separated by the stage of galling. The sheet sliding direction is from left to right. Figure 11 shows the distribution of the height of the build-up on the indenter, to mir-
ror the analysis of the scratch morphology in the following section. All stages have a high
number of points in and near the 0 mm profile height, as this is close to the reference plane
and represents the profile height of the unworn surface. However, the number of points
further away from 0 mm is indicative of material build up. In stage 1, most of the data are
centered around the 0 mm profile height, with 100% of the profile height data points
within ±0.002 mm. However, stage 2 shows a difference compared with stage 1, where it
is evident that there is material build-up in the positive area on the histogram. Stage 3
shows more of this build up, in line with the 2D profiles shown in Figures 9 and 10. Figure 11 shows the distribution of the height of the build-up on the indenter, to mirror
the analysis of the scratch morphology in the following section. All stages have a high
number of points in and near the 0 mm profile height, as this is close to the reference plane
and represents the profile height of the unworn surface. However, the number of points
further away from 0 mm is indicative of material build up. In stage 1, most of the data
are centered around the 0 mm profile height, with 100% of the profile height data points
within ±0.002 mm. stages.
3.2. Lump Growth on the Tool Note that the baseline surfaces on the balls in these cross-sections are flat, because
the spherical indenter surfaces were “flattened out” using the Alicona software procedure
described in Section 2.2. When examining the cross-sectional profiles, it is notable that there
is little to no evidence of transferred material on the indenter during stage 1. However, a Figure 9. Lump growth—volume of material transferred to the indenter for each galling stage. Figure 10 shows the cross-sectional shape of the build-up on the indenter between each
stage. Note that the baseline surfaces on the balls in these cross-sections are flat, because
the spherical indenter surfaces were “flattened out” using the Alicona software procedure
described in Section 2.2. When examining the cross-sectional profiles, it is notable that there
is little to no evidence of transferred material on the indenter during stage 1. However, a Lubricants 2023, 11, 288 10 of 16
re pro- 10 of 16
re pro- very uniform lump appears for stage 2, which then typically evolves into a larger lump in
stage 3. However, the lump growth on some of the indenters in stage 3 results in a very
steep build up on the leading edge of the indenter. However, a very uniform lump appears for stage 2, which then typically evolves into a
larger lump in stage 3. However, the lump growth on some of the indenters in stage 3
results in a very steep build up on the leading edge of the indenter. very uniform lump appears for stage 2, which then typically evolves into a larger lump in
stage 3. However, the lump growth on some of the indenters in stage 3 results in a very
steep build up on the leading edge of the indenter. However, a very uniform lump appears for stage 2, which then typically evolves into a
larger lump in stage 3. However, the lump growth on some of the indenters in stage 3
results in a very steep build up on the leading edge of the indenter. steep build up on the leading edge of the indenter. e u
i
a e y
eep ui
up o
e ea i g e ge o
e i
e
e
Figure 10. Cross-sections of lump growth on all of the indenters after each segment of the scratch
tests, separated by the stage of galling. The sheet sliding direction is from left to right. 3.3. Scratch Morphology
3.3. Scratch Morphology
h Figure 12 shows the surface damage observed on the plate surfaces after the testing,
as measured via the optical surface profilometer. This figure shows that there are clear
differences between the scratch surface morphology for each of the galling wear stages. In particular, after stage 1, it is evident that the evolution of the surface damage is char-
acterized by the appearance and evolution of peaks and troughs perpendicular to the
scratch direction. Figure 12 shows the surface damage observed on the plate surfaces after the testi
as measured via the optical surface profilometer. This figure shows that there are cl
differences between the scratch surface morphology for each of the galling wear stages
particular, after stage 1, it is evident that the evolution of the surface damage is charact
ized by the appearance and evolution of peaks and troughs perpendicular to the scra
direction. Figure 12 shows the surface damage observed on the plate surfaces after the testing,
as measured via the optical surface profilometer. This figure shows that there are clear
differences between the scratch surface morphology for each of the galling wear stages. Figure 12 shows the surface damage observed on the plate surfaces after the testi
as measured via the optical surface profilometer. This figure shows that there are cl
differences between the scratch surface morphology for each of the galling wear stages gy
g
g
g
In particular, after stage 1, it is evident that the evolution of the surface damage is char-
acterized by the appearance and evolution of peaks and troughs perpendicular to the
scratch direction. particular, after stage 1, it is evident that the evolution of the surface damage is charact
ized by the appearance and evolution of peaks and troughs perpendicular to the scra
direction. Figure 12. Magnified images of the three stages of galling wear for the different galling stages:
stage 1, (B) stage 2, (C) stage 3. Figure 12. Magnified images of the three stages of galling wear for the different galling stages:
(A) stage 1, (B) stage 2, (C) stage 3. Figure 12. Magnified images of the three stages of galling wear for the different galling stages:
stage 1, (B) stage 2, (C) stage 3. Figure 12. Magnified images of the three stages of galling wear for the different galling stages:
(A) stage 1, (B) stage 2, (C) stage 3. stages.
3.2. Lump Growth on the Tool However, stage 2 shows a difference compared with stage 1, where it is
evident that there is material build-up in the positive area on the histogram. Stage 3 shows
more of this build up, in line with the 2D profiles shown in Figures 9 and 10. VIEW
11 of 17 Figure 11. Distribution of the build-up on the indenter. Figure 11. Distribution of the build-up on the indenter. Figure 11. Distribution of the build-up on the indenter. Figure 11. Distribution of the build-up on the indenter. Lubricants 2023, 11, 288 11 of 16 11 of 16 3.3. Scratch Morphology
3.3. Scratch Morphology
Fi
12 h 3.3. Scratch Morphology
3.3. Scratch Morphology
h The scratch morphology for all tests is summarized by the longitudinal scratch p
files shown in Figure 13, as measured by the optical profilometer. These longitudinal p
files were obtained from the center of the scratch (as described in Section 2.2) and sh
the profile height along the scratch length. This figure shows a clear difference in the p
file of the stage 1 scratch surface, in comparison to stage 3 scratch surface after sev
galling wear. The progressive change in scratch morphology can be observed in Figu
13C, for the scratch test segments that showed all three stages of galling wear. To bet
observe the series of peaks and troughs in the scratch seen in the profile form measu
ment, a histogram of these height measurements is shown in Figure 14. In Figure 14 th
The scratch morphology for all tests is summarized by the longitudinal scratch profiles
shown in Figure 13, as measured by the optical profilometer. These longitudinal profiles
were obtained from the center of the scratch (as described in Section 2.2) and show the
profile height along the scratch length. This figure shows a clear difference in the profile
of the stage 1 scratch surface, in comparison to stage 3 scratch surface after severe galling
wear. The progressive change in scratch morphology can be observed in Figure 13C, for the
scratch test segments that showed all three stages of galling wear. To better observe the
series of peaks and troughs in the scratch seen in the profile form measurement, a histogram
of these height measurements is shown in Figure 14. In Figure 14 there is a clear change in
the profile height distribution between each stage of the galling wear, with each successive
stage shifting the distribution further to the negative values of profile height. This indicates
that there are deeper trough-like features in the center of the scratch in stage 3, which
correlates with the visual observations shown in Figure 12C. There are noticeably more counts, an order of magnitude greater, for Figure 14 in
comparison to Figure 11. This is because the length of the scratch (and therefore the
measured length of the cross-sectional height) is much longer than the length of the cross-
sectional height of the indenter, but the resolution of these measurements is the same. Thus,
there is naturally an order of magnitude more counts for Figure 14 than Figure 11. 3.3. Scratch Morphology
3.3. Scratch Morphology
h Lubricants 2023, 11, 288 12 of 16
ec
hus, y
g
g
g
Figure 13. Evolution of the scratch morphology along the scratch length, within the scratch width. Figure 13. Evolution of the scratch morphology along the scratch length, within the scratch width. REVIEW
13 of 17
Figure 14. Distribution of series of peaks and troughs in the scratch after each stage of galling. Figure 14. Distribution of series of peaks and troughs in the scratch after each stage of galling. REVIEW
13 of 17 Figure 13. Evolution of the scratch morphology along the scratch length, within the scratch width. Figure 13. Evolution of the scratch morphology along the scratch length, within the scratch width. Figure 13. Evolution of the scratch morphology along the scratch length, within the scratch width. Figure 13. Evolution of the scratch morphology along the scratch length, within the scratch widt Figure 14. Distribution of series of peaks and troughs in the scratch after each stage of galling. Figure 14. Distribution of series of peaks and troughs in the scratch after each stage of galling. Lubricants 2023, 11, 288 13 of 16 13 of 16 4.1. Friction The stochastic nature of galling has been observed in the friction curves (Figure 7),
nevertheless the scratch test segments can be split into four categories. “Stage 1 only”
is the stage when the coefficient of friction never rises above a certain threshold (0.32). Conversely, “stage 3 only” occurs when the coefficient of friction is above 0.9, very close to
the start of the scratch test segment (e.g., refer to the galling stages summarized in Table 3). There are some scratch test segments that can be categorized as having all three phases of
galling, and conversely again some tests that only have “stage 1” and “stage 2” phases, but
no “stage 3” phase. g
p
In accordance with the other literature [10,11], each successive stage of galling reaches
a higher coefficient of friction, and so these data have been used to separate the three stages
in the scratch morphology in this manuscript. It is reported in the literature that the rise in
the coefficient of friction is due to the formation of an aluminum transfer layer, and that
this stabilizes when a dynamic equilibrium is reached between the generation and ejection
of loose wear particles [12]. However, the coefficient of friction reported in the tests in
this current study generally monotonically increase during stage 2, rather than a constant
intermediate friction coefficient as reported by Eriksson et al. [11]. Additionally, there was
no “sharp rise” in coefficient of friction between stage 1 and stage 2, or between stage 2
and stage 3, which was also reported by Eriksson et al. [11] and Vilhena et al. [10]. As these experiments were conducted at room temperature, the strong adhesion
Yang et al. [16] report (coefficient of friction reaching 1.5) is not seen. The maximum coeffi-
cient of friction observed in this work stabilized around a maximum of 1. However, as seen
in the measurements of the scratch morphology (Section 3.3), the damage mechanisms and
coefficient of friction measurements for the initial galling wear (stage 1) are similar to the
observations by Yang et al. [16]. 4. Discussion The galling wear in this manuscript has been characterized and investigated
via the evolution of the coefficient of friction, the lump growth on the tool and the
scratch morphology. 4.3. Scratch Morphology 4.3. Scratch Morphology The results show that there is an obvious difference in the scratch morphology as
the galling evolves. It is evident from Figure 12 that the evolution of the damage maybe
characterized by the series of peaks and troughs running across the scratch. The surface damage Yang et al. [16] reported is comparable to the damage seen in
this work for the initial galling wear. However, as wear progresses, the results from
Yang et al. [16] showed similar damage to that seen by Gaard et al. [30], with smaller
scratches forming within the contact along the sliding direction. These previous studies
attribute these longitudinal scratches to hard particles becoming trapped in the contact
region. However, this behavior has not been observed in this current work. g
In the literature it is reported that the scratch morphology is affected by adhesion and
ploughing between surface asperities. Authors such as Gaard et al. [30] (working with steel-
on-steel contacts) and Heinrichs et al. [31] (working with steel and aluminum alloys) state
that the transferred material work hardens, or hardens by oxidation [13,23–26,32–37], in the
contact region. Moreover, in some of the literature it is argued that the adhered material work
hardens to an equal or greater hardness than that of the indenter, due to the work hardening
of austenitic steels or the intrinsic hardness of aluminum oxides [4,13,38–40]. However, the
damage observed in this current study suggests that these hardening processes do not occur
in these tests—i.e., that there are no hard asperities being formed in the contact, during the
material transfer from the Al5083 plate to the tool steel, or during the subsequent sliding. The tests were conducted at room temperature (indicative of cold forming) and low sliding
speeds, which reduces the frictional heating. Therefore, it is possible that a lack of oxide
formation may be playing a role here. Despite this possibility, the effect of oxide formation is complex and it is unclear
whether or not oxide formation will inhibit or accelerate galling wear. In some cases, oxide
formation worsens the galling as it introduces hard particles into the contact; however, in
other cases, the oxides themselves can reduce galling by acting as a barrier to metal-on-
metal contact [7,10,18,39–41]. There is a consensus that metal-on-metal contact generates
high levels of adhesion, typically more than metal–oxide contact. 4.3. Scratch Morphology This further suggests
that oxide layers have not been formed in the contacts in this current study, and the wear
scenario has remained as a metal-on-metal contact. Jue et al. [17,18] found that worn surfaces of 7075 aluminum alloy at different tem-
peratures can be divided into two zones (a smooth zone and rough zone), and the relative
areas of these zones varies with temperature. However, the reported smooth/rough zones
are very close to one another, intermixed in some examples shown in the work [17,18]. The results presented in this manuscript do not have this mix of smooth and rough zones,
rather there is a clear “smooth” zone that becomes a rough zone. g
The scratch morphology, as measured by the optical profilometer, is shown in Figure 13,
as a function of the height along the scratch length. To better observe the series of peaks and
troughs in the scratch seen in the profile form measurement, a histogram of these (height)
measurements is shown in Figure 14. In Figure 14 there is a clear change in the profile
height distribution between each stage of galling wear. Each successive stage of galling
results in a shift of the distribution further in the negative direction, with a corresponding
shift in the distribution of material transferred to the indenter shifting to the positive
direction, as shown in Figure 11. This indicates that, as the amount of material adhered to
the indenter increased, the depth of the grooves on the scratch surface perpendicular to the
sliding direction also increased. To the authors’ best knowledge, there are no other studies
on the distribution of damage within the wear scar. However, Moghadam et al. [42] report
on the number of valleys deeper than 1 µm in relation to the drawing length of their tests. 4.2. Lump Growth on the Indenter Owing to the tribosystem being a closed system, the measurements can only be
made post-test—hence only the transfer of material for the final stage observed can be
investigated. That is, for the tests that are otherwise classified as showing two or three
stages of galling, only the volume of transferred material in the final observed stage can be
commented upon. Nevertheless, immediate transfer was seen in some tests (in agreement
with other works), and later stages see more transferred material. Some studies report
that adhered material can be moved, removed and abraded away, which is evidenced here
as the increase in transferred volume between the stages is greatest for stage 2 to stage 3
(when considering the amount of material transferred). However, when considering the
percentage increase, stage 1 to stage 2 see a far greater transfer of material than from stage 2
to stage 3. g
It is widely reported that adhesion occurs on the harder of the two contacting sur-
faces [10,11,16,23–27], which has been seen in this work as shown by the lump growth
on the steel indenter. Often, studies that have investigated the aluminum/steel tribopair,
it is the tool/indenter that is made from an aluminum alloy and, as such, becomes abra-
sively worn by the harder steel sample plate [7,28,29]. Therefore, the galling behavior and
lump growth for this configuration, where the tool/indenter is the harder material in the
aluminum/steel tribopair, is understudied/under-reported. Lubricants 2023, 11, 288 14 of 16 14 of 16 5. Conclusions The coefficient of friction between a tool steel and Al5083 during dry sliding at slow
sliding speed and low loads, was measured and used to categorize the stages of galling
wear, which were correlated to the lump growth and scratch morphology. 15 of 16 15 of 16 Lubricants 2023, 11, 288 The following conclusions have been made: •
The galling evolution is characterized by the coefficient of friction regimes. •
The galling evolution is characterized by the coefficient of friction regimes. g
g
y
g
•
Stage 1 showed little to no lump growth on the indenter. Stage 2 showed uniform
lump growth, while stage 3 showed less uniform lump growth. In some cases, the
lumps in stage 3 showed the development of a steep edge at the beginning of the lump. p
g
p
p
g
g
g
p
•
The scratch morphology from stage 2 and 3 showed a series of peaks and troughs
across the scratch, as opposed to the expected damage mechanism of longitudinal
scratches in the scratch. •
Using a novel investigation of the distribution of the heights of these peaks and troughs
showed that, as the amount of material adhered to the indenter increased, the depth of
the grooves on the scratch surface perpendicular to the sliding direction also increased. Author Contributions: T.M.D.: conceptualization, experiments, data collection, formal analysis and
draft writing, project administration. M.P.P.: conceptualization, experimental design, supervision,
review and edit of writing, project administration. J.M.G.: supervision, review and proof reading. B.F.R.: supervision, review and proof reading. All authors have read and agreed to the published
version of the manuscript. Funding: This research received no external funding. Data Availability Statement: The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to on-going research. Acknowledgments: The authors would like to thank Alireza Vahid and Reza Parvizi for their
technical support of the experimental regime and microscopy. We would also like to thank Matt
Zampatti, Luke Tyrell and Robert Lovett for their technical support. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Shanbhag, V.V.; Rolfe, B.F.; Arunachalam, N.; Pereira, M.P. Investigating galling wear behaviour in sheet metal stamping using
acoustic emissions. Wear 2018, 414–415, 31–42. [CrossRef] 2. Devenport, T.; Rolfe, B.; Pereira, M.; Griffin, J.M. Analysis of Acoustic Emissions for Determination of
Scratch Tests. Appl. Sci. 2022, 12, 6724. [CrossRef] , B.; Pereira, M.; Griffin, J.M. Analysis of Acoustic Emissions for Determination of the Mechanical Effects of
Sci. 2022, 12, 6724. [CrossRef] 3. Gåård, A.; Sarih, R. Influence of Tool Material and Surface Roughness on Galling Resistance in Sliding against Austenitic Stainless
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Shanbhag VV; Rolfe B F ; Pereira M P Investigation of Galling Wear Using Acoustic Emission Frequency Characteristics 4. Heinrichs, J.; Jacobson, S. Mechanisms of Transfer of Aluminium to PVD-Coated Forming Tools. Tribol. Lett. 2012, 46, 299–312. [CrossRef]
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32. Ghiotti, A.; Simonetto, E.; Bruschi, S. Influence of process parameters on tribological behaviour of AA7075 in hot stamping. Wear
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2019, 426–427, 348–356. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content. References [CrossRef] 33. Choi, H.; Kim, S.; Seo, P.; Kim, B.; Cha, B.; Ko, D. Experimental investigation on galling performance of tool steel in stamping of
UHSS sheets. Int. J. Precis. Eng. Manuf. 2014, 15, 1101–1107. [CrossRef] 34. Määttä, A.; Vuoristo, P.; Mäntylä, T. Friction and adhesion of stainless steel strip against tool steels in unlubricated sliding with
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gainst a carbon steel sheet. Wear 2009, 267, 387–393. [CrossRef] 36. Heinrichs, J.; Jacobson, S. The influence from shape and size of tool surface defects on the occurrence of galling in cold forming of
aluminium. Wear 2011, 271, 2517–2524. [CrossRef] ,
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oxidation in 316L stainless steel. Wear 2020, 450–451, 203234. [CrossRef] 38. Karlsson, P.; Gåård, A.; Krakhmalev, P.; Bergström, J. Galling resistance and wear mechanisms for cold-work tool steels in
lubricated sliding against high strength stainless steel sheets. Wear 2012, 286–287, 92–97. [CrossRef] 38. Karlsson, P.; Gåård, A.; Krakhmalev, P.; Bergström, J. Galling resistance and wear mechanisms for cold-work tool steels in
lubricated sliding against high strength stainless steel sheets. Wear 2012, 286–287, 92–97. [CrossRef] 39. Mao, L.J.; He, X.; Cai, M.J.; Qian, L.Q. Influence of Contact Load on the Dry Sliding Wear Performance of 7075 Aluminum Alloy. Exp. Tech. 2023, 47, 357–367. [CrossRef] 39. Mao, L.J.; He, X.; Cai, M.J.; Qian, L.Q. Influence of Contact Load on the Dry Sliding Wear Performance of 7075 Aluminum Alloy. Exp. Tech. 2023, 47, 357–367. [CrossRef] 40. Heinrichs, J.; Jacobson, S. Laboratory test simulation of aluminium cold forming—influence from PVD tool coatings on the
tendency to galling. Surf. Coat. Technol. 2010, 204, 3606–3613. [CrossRef] 40. Heinrichs, J.; Jacobson, S. Laboratory test simulation of aluminium cold forming—influence from PVD tool coatings on the
tendency to galling. Surf. Coat. Technol. 2010, 204, 3606–3613. [CrossRef] 42. Moghadam, M.; Christiansen, P.; Bay, N. Detection of the Onset of Galling in Strip Reduction Testing Using Acoustic Emission. Procedia Eng. 2017, 183, 59–64. [CrossRef] 42. References Moghadam, M.; Christiansen, P.; Bay, N. Detection of the Onset of Galling in Strip Reduction Testing Using Acoustic Emission. Procedia Eng. 2017, 183, 59–64. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.
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https://europepmc.org/articles/pmc3441308?pdf=render
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English
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RV144-like trial in macaques using ALVAC-SIV & gp120 induces innate immunity and increases the frequency of NK22 & NKG2A+ cells in mucosal tissues
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Retrovirology
| 2,012
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cc-by
| 555
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Background that demonstrated an increased frequency of NK22 cells
at mucosal sites. These NK22 cells expressed CCR6 (a
gut homing marker) and are thought to play a role in
mucosal immunity. Similarly, vaccination also increased
the frequency of NKG2A+ cells that were either cyto-
toxic (CD107a+) or cytokine producing (IFNg+). NK cells play a pivotal role in the innate immunity and
patrol various tissues, including mucosal sites, the portal
of entry of HIV. We recently reproduced in the SIV
mac251- rhesus macaque (RM) model the limited protec-
tion reported in the RV144 HIV vaccine trial in humans
(32% protection from HIV acquisition), using similar
vaccines (ALVAC-SIV & gp120). Conclusion Thus, the ALVAC SIV/gp120 vaccine regimen induces a
significant activation of NK cells and other innate
immune responses. Given that this vaccine platform has
conferred some degree of protection from HIV infection
in humans, understanding the role of innate immunity
in protection from SIVmac251 may guide our effort in
the development of novel vaccine strategies against HIV. Liyanage et al. Retrovirology 2012, 9(Suppl 2):O14
http://www.retrovirology.com/content/9/S2/O14 Open Access Author details
1 Author details
1Animal Models & Retroviral Vaccines Section , Bethesda, MD, USA. 2Vaccine
& Gene Therapy Institute -Florida, FL, USA. 3Vaccine Research Center, NIAID,
NIH, Bethesda, MD, USA. Methods In here, we immunized, by the intramuscular route, 6
RM with 108 PFU of ALVAC-SIV at weeks, 0 (V0), 4
(V2), and with both ALVAC SIV and the SIVgp120
envelope proteins, adjuvanted with ALUM at week 12
(V3) and 24 (V4) and analyzed the profile of gene
expression at 24 hours after each vaccination. In paral-
lel, we studied the phenotypical and functional changes
in the NK cell subsets after vaccination by multi para-
metric flow cytometry. RV144-like trial in macaques using ALVAC-SIV &
gp120 induces innate immunity and increases
the frequency of NK22 & NKG2A+ cells in
mucosal tissues From AIDS Vaccine 2012
Boston, MA, USA. 9-12 September 2012 From AIDS Vaccine 2012
Boston, MA, USA. 9-12 September 2012 Author details
1Animal Models & Retroviral Vaccines Section , Bethesda, MD, USA. 2Vaccine
& Gene Therapy Institute -Florida, FL, USA. 3Vaccine Research Center, NIAID,
NIH, Bethesda, MD, USA. 1Animal Models & Retroviral Vaccines Section , Bethesda, MD, USA
Full list of author information is available at the end of the article Results Published: 13 September 2012 Published: 13 September 2012 Microarray analysis in the PBMC after V1 and V2
showed the up regulation of anti viral (MX1, HERC-5)
and IFN responsive genes and a down regulation of pro-
inflammatory genes. Significant alteration in the gene
expression profile was also observed after the gp120/
Alum boost (V3). Interestingly, NK cells associated
genes were up regulated after V3 vaccination. These
finding paralleled our results observed by FACS analysis doi:10.1186/1742-4690-9-S2-O14
Cite this article as: Liyanage et al.: RV144-like trial in macaques using
ALVAC-SIV & gp120 induces innate immunity and increases the
frequency of NK22 & NKG2A+ cells in mucosal tissues. Retrovirology 2012
9(Suppl 2):O14. 1Animal Models & Retroviral Vaccines Section , Bethesda, MD, USA
Full list of author information is available at the end of the article © 2012 Liyanage 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.
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en
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Behaviour and Welfare of Dairy Buffaloes: Pasture or Confinement?
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Journal of buffalo science
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cc-by
| 3,642
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Journal of Buffalo Science, 2018, 7, 43-48
Behaviour and
Confinement?
Welfare
of
Dairy
Buffaloes:
43
Pasture
or
Patricia Mora-Medina1, Jesús Alfredo Berdugo-Gutiérrez2, Daniel Mota-Rojas3,*,
Jhon Didier Ruiz-Buitrago4, José Nava-Adame3 and Isabel Guerrero-Legarreta5
1
Livestock Science Department, Universidad Nacional Autónoma de México (UNAM), FESC, State of Mexico,
Mexico
2
Latin American Center for the Study of Buffalo, Colombia
3
Neurophysiology, Behavior and Assessment of Welfare in Domestic Animals, Department of Animal
Production and Agriculture, Universidad Autónoma Metropolitana (UAM), Mexico City, 04960, Mexico
4
Grupo de Investigación INCACES, Facultad de Medicina Veterinaria y Zootecnia, Universidad CES,
Medellin, Colombia
5
Department of Biotechnology, Food Science, Universidad Autónoma Metropolitana-Iztapalapa, (UAM-I),
México City, Mexico
Abstract: This review seeks to integrate recent scientific findings on the behaviour of buffalo cows in different production
systems. These issues are discussed in relation to the level of welfare that buffalo cows experience under different
production systems. In extensive conditions, the level of welfare is high because the animals are free to express natural
behaviours related to feeding (grazing, ruminating) and rest. In contrast, intensified livestock-raising methods and
techniques (machine-milking, artificial breeding etc.), first developed for dairy cattle are increasingly being used with
water buffaloes in order to increase milk production. Greater knowledge of the biology of dairy buffaloes in aspects linked
to physiology, behaviour, and health, together with needed adjustments to their production systems, will indicate options
for improving the levels of comfort and welfare of these animals and contribute to increasing the efficiency of this type of
dairy production. It is necessary to appreciate the importance of welfare within the entire chain of animal production
since each of the scientific aspects considered in this manuscript reflects that animal welfare is not an absolute term, but
multidisciplinary, with direct consequences on productivity. The welfare of animals in the production systems must be
considered with the aim of ensuring an adequate nutritional, clinical, sanitary and behavioural status of the animals.
When these aspects are achieved, production can be maximized and, for this reason, it is essential to maintain a
balance between welfare and productivity.
Keywords: Buffaloes, behaviour, welfare, extensive system, intensive system.
INTRODUCTION
The increase in the global demand for animal
products in the last decades of the twentieth century
has intensified considerably, displacing the traditional
systems. This intensification allows increasing the
volume of milk and meat production as well as the
efficiency of resource utilisation, productivity and food
security [1]. However, the intensification can negatively
affect the welfare of animals by modifying the
environmental conditions (confinement, feeding, etc.).
More specifically, the intensification can cause welfare
problems if the animals cannot adequately perform
social and innate behaviors [2].
There are three factors that are combined and that
must be in equilibrium in the production systems:
animal, environment, and human; these are, at the
*Address correspondence to these authors at the Neurophysiology, Behavior
and Assessment of Welfare in Domestic Animals, Universidad Autónoma
Metropolitana (UAM), Calzada del hueso 1100, Del. Coyoacan, 04960, Mexico
City, Mexico; Tel: (525) 54837535; E-mails: dmota@correo.xoc.uam.mx,
dmota100@yahoo.com.mx
ORCID ID: https://orcid.org/0000-0003-0562-0367
ISSN: 1927-5196 / E-ISSN: 1927-520X/18
same time, determined by an economic factor [3], since
farm animals are kept with the aim to gain profit.
However, intensive systems applied at their maximum
expression can lead to physiological and behavioral
imbalances [4, 5]. Currently, the welfare of farm
animals such as poultry, cattle, and pigs, among
others, have taken great importance due, mainly, to
some aspects related to the physical environment in
which they live and the different production or housing
systems in which they are produced, which are
responsible for the welfare and performance of the
animals, and quality and costs of the products [6, 7].
Dairy buffaloes have been treated and handled in
the same manner as bovines; that is, without
considering that this is a more rustic species (with a
great capacity to adapt to adverse environments)
usually raised in extensive systems, in contrast to dairy
cattle, which are more specialized and are bred
intensively. What is important is to assess which type
of production system will ensure dairy buffaloes higher
levels of welfare and comfort that are requirements for
© 2018 Lifescience Global
44
Journal of Buffalo Science, 2018, Vol. 7, No. 3
optimizing their milk yield [1]. Against this background,
our review examines key behavioural indicators in
different production systems to offer readers significant
feedback on this issue. One consequence of the
growing economic interest in consuming foods
produced by these animals is that water buffalo-raising
has undergone a process of intensification [8-10], but
this exposes the animals to novel stimuli generated by
the diverse technologies adapted from dairy cattle (e.g.
milking machines, more contact with humans, modified
diet, reduced space, etc.). These changes affect health
(e.g. more lesions), social behaviour (e.g. greater
frequency of agonistic encounters), and heat
dissipation, and have significant repercussions on milk
ejection, product quality and animal welfare [11, 12].
In extensive production systems, buffaloes devote
most of their time to two main behavioural categories:
feeding (grazing, ruminating) and rest. Another typical
behaviour of this species is wallowing and bathing [13].
However, these systems may also have other welfare
problems, mainly related to nutrition, water
supplementation, parasitic diseases, extreme weather
conditions, lameness, predators or lack of supervision
[14].
In contrast, intensive or confinement systems are
thought to affect negatively the welfare of buffaloes
because freedom of choice and movements are
restricted; however, this classification is not that simple
since in both systems (pasture and confinement) there
is an important factor that influences welfare, which is
the stress experienced by animals [2, 7, 15].
The increasing intensification of milk production
means that more and more buffaloes are raised under
conditions of confinement; thus, it is important to pay
more attention to farming conditions because they not
only restrict this species’ natural behaviours but may
also cause physiological alterations associated with
stress [16].
Against this background, the objective of this review
is to integrate recent scientific findings on the
behaviour of buffalo cows in different production
systems.
THE BEHAVIOUR OF DAIRY BUFFALO
Pasture
It is well-known that suitable environmental
stimulation promotes animal welfare and that some
signs of good welfare –e.g. playfulness– occur more
Mora-Medina et al.
frequently under adequately-enriched environmental
conditions [17-19]. In extensive production systems,
buffaloes devote most of their time to two main
behavioural categories: feeding (grazing, ruminating)
and rest. Another typical behaviour of this species –
wallowing and bathing– occurs more often in the hot
season, when they provide thermoregulation and
protection from ectoparasites [13].
According to Fericean [20], on the prairies of
extensive systems, water buffaloes spend over 99% of
their time feeding, drinking water, ruminating and
resting. In fact, on average feeding and ruminating
occupy 60-65% of their time [11]. During the remaining
1%, buffaloes move around and perform various other
activities [20].
De Rosa et al. [12] observed that if there is a pool of
water and the buffaloes are free to move about through
larger spaces, then their preferred posture is wallowing,
perhaps because it is their main means of dissipating
heat, though it might also foster non-agonistic social
interaction, since more of these activities (i.e., sniffing,
nuzzling) were seen under these conditions. Also,
social licking was more apparent when the Buffaloes
2
enjoyed more ample living spaces (36 m /head vs. 10
2
m /head).
With respect to grazing, size, age, gestation status,
and milk production will affect the duration of feeding
time [20], but grazing time also decreases under high
temperatures and if the land has mud pools. Under
these conditions, buffalo cows tend to stay in the
muddy water for 4-6 hours every day to get refreshed
[20]. In addition to providing shade and a variety of
food sources, silvopastoral environments allow
buffaloes to express their natural behaviours. In these
systems, activities like exploring the surroundings and
their co-specifics increase markedly (Figure 1a and
1b). In these conditions, buffalo cows are usually handmilked (Figure 1c) and often calves are re-joined to the
mothers to promote milk ejection.
The natural selection process has led the water
buffalo to acquire morphological traits that allow it to
adapt to areas with hot, humid climates. One such trait
is its melanin-pigmented skin, which provides
protection from ultraviolet rays; another is its low hair
density, which allows heat to be dissipated by
convection and radiation. In hot dry climates, in
contrast, the low humidity promotes intense heat loss
through evaporation, though this is limited in buffaloes
due to their low number of sweat glands. Finally,
Behaviour and Welfare of Dairy Buffaloes
respiratory evaporation is less efficient in buffaloes
than cattle, for it may cause alkalosis by quickly raising
blood pH [13, 21]. Therefore, buffaloes rely on
wallowing and the consequent evaporation for efficient
heat loss and thermoregulation. This behaviour is
usually performed in extensive conditions where
buffaloes purposely create potholes for bathing.
Journal of Buffalo Science, 2018, Vol. 7, No. 3
45
Two findings from that research are that milk
production is greater in outdoor systems (8.12 vs.
7.77kg), and that cleanliness scores are higher (2.80
vs. 2.41) than under conventional indoor conditions. In
addition, it was determined that confinement resulted in
more animals experiencing lameness (0.01 vs. 0.10).
Other observations indicate that cows who are free to
graze spend more time feeding than those that are kept
in confinement and released outdoors later (78 vs.
25%, respectively). Finally, both cattle and buffaloes
tend to walk greater distances in outdoor systems [13,
22].
Another recent research approach consists in
evaluating extensive grazing systems that allow dairy
farms to conserve biodiversity while promoting
landscape ecology [23]. Extensive rearing systems are
convenient for species that are already well-adapted to
the environment, a category that includes buffaloes and
non-productive animals, such as heifers [24]. Extensive
systems can also lower production costs and lessen
environmental impacts [25], while simultaneously
improving animal welfare [10] and promoting product
differentiation by implementing quality assurance
measures [13, 26].
Confinement
Figure 1: Extensive system (Pasture) of dairy buffalo, area
of the Bajo Cauca, towns (municipalities) of Caucasia
(Antioquia department), Planeta Rica (Córdoba deparment)
and Puerto Boyacá (Boyacá Department), Colombia. Animals
are kept on pasture (a, b) and hand-milked (c).
Tripaldi et al. [16] have demonstrated that when
raised in comfortable environments under conditions
similar to traditional ones that promote their welfare,
buffalo cows show a less pronounced response of the
adrenal cortex possibly because they are free to
choose the sites where they wish to eat, wallow [16] or
explore [13], according to variations in the microenvironmental conditions.
In dairy production systems, welfare includes not
only the animals that produce milk but also the
newborns, the young females that will be used as
replacements and the males in breeding units [27].
Raising calves is one of the most complex activities of
dairy production, being very common infectious and
parasitic diseases leading to increased levels of
mortality [28]. The high level of morbidity and mortality
of buffalo calves has important ethical and economic
implications. It is likely that the problems of buffalo calf
morbidity and mortality, which dairy production systems
often face, are directly related to the management of
the first days of life, including early maternal
separation, artificial rearing, insufficient or late
colostrum ingestion, limited space, inadequate
bedding, lack of opportunity of social interaction and
play, in addition to little interaction with humans [29].
The increasing intensification of milk production
means that more and more buffaloes are raised under
conditions of confinement; thus, it is important to pay
more attention to housing conditions because they not
only restrict this species’ natural behaviours but may
also cause physiological alterations associated with
stress due to the restricted space allowance [16].
46
Journal of Buffalo Science, 2018, Vol. 7, No. 3
Indeed, housing conditions are an indirect indicator of
buffalo welfare, for if they do not include environmental
enrichment, they will generate stress in the animals
[16] (Figure 2a and 2b).
Mora-Medina et al.
characterised by the incorporation of methods
designed to intensify production, coupled with modern
livestock-raising techniques implemented initially for
dairy cattle (milking machines, artificial rearing, and
artificial insemination, among others) in order to
generate higher milk yields. But under these conditions
animals are exposed to novel environmental conditions
that can trigger stress, both physical and physiological
[12] (Figure 2c).
In their work, Tripaldi et al. [16], studied the effect of
two rearing systems on the behavioural and
physiological responses of buffalo cows. The systems
assessed were: 1) with, and 2) without a broad, openair yard with varied vegetation and watering holes or
pools where the cows could bath and wallow, similar to
those found in traditional systems. In their work, the
buffalo cows kept under intensive conditions had
extended periods of idling since there was no access to
yards or pools that enriched the environment of the
corresponding outdoor group [16]. The authors
concluded that one enriching element necessary for
buffaloes is the presence of open spaces with mud
pools because they were found to favour milk
production in the hotter, more humid months of the
year [12]. It should be noted that lower activity levels
were also reflected in a higher percentage of time
spent in various “resting” postures including lying down
[13].
In order to address the machine milking issue,
Cavallina et al. [32] applied the focal sampling
technique to determine –through observation– the
behavioural responses of primiparous and multiparous
buffalo cows to the milking routine. Their results
showed that primiparous buffaloes had a higher
frequency of acute, stress-related behaviours while
being milked that included kicking (36.67% vs.
24.36%), defecating (5% vs. 2.56%), pulling the teat
out of the milking cup (11.67% vs. 5.13%), and
urinating (48.33% vs. 11.54%).
Figure 2: Intensive system of dairy buffalo, area of the Bajo
Cauca, towns (municipalities) of Caucasia (Antioquia
department), Planeta Rica (Córdoba department) and Puerto
Boyacá (Boyacá Department), Colombia. Animals are kept
indoorsure (a, b) and machine-milked (c).
Another consequence of higher dairy buffalo
production is an increase in the use of intensive
management facilities, including the mechanisation of
routine activities on farms [30, 31]. In recent years,
water
buffalo-raising
has
been
increasingly
The results of other research show that keeping and
feeding dairy cows in enclosed yards causes an
increase in lameness and mastitis and, possibly,
agonistic behaviours, while also reducing walking time
[33]. Moreover, reduced ruminant motility is seen in
dairy buffaloes that experience problems with their
hooves or legs, and haematological indexes revealed
significant decreases in Hb, PCV and TEC [34]. It has
further been shown that tie-stall conditions, sometimes
used in small familiar buffalo enterprises, may neglect
fundamental biological and behavioural needs of the
Behaviour and Welfare of Dairy Buffaloes
Journal of Buffalo Science, 2018, Vol. 7, No. 3
animals. In: Mota-Rojas D, Velarde-Calvo A, Maris-Huertas
S, Nelly-Cajiao M, editors. Bienestar Animal una visión global
en Iberoamérica. [Animal welfare, a global vision in IberoAmerica]. 3rd ed. Barcelona, España: Elsevier 2016; pp.
137-54.
animals and, therefore, cause serious difficulties for
their welfare [35].
Buffaloes maintained in intensive conditions with no
access to pasture or water for wallowing have longer
periods of inactivity and spend less time exploring.
Confinement also entails space reduction, but no
studies have yet specifically examined this issue in
order to ascertain precise requirements for water
buffaloes. Restricting the space available to these
animals can impact several aspects, including health
(frequent injuries), social behaviour (more agonistic
interactions), fertility, and heat dissipation [13].
CONCLUSIONS
The welfare of animals in different production
systems must be considered with the aim of ensuring
an adequate nutritional, clinical, sanitary and
behavioural state of the animals. The critical points of
intensive and extensive systems are different; however,
when these aspects are satisfied, production can be
improved in qualitative and quantitative terms in both
extensive and intensive systems and, for this reason, it
is essential to maintain a balance between welfare and
productivity. The welfare of buffaloes should be
promoted through the entire chain of animal production
since each phase has an impact on animal welfare and
process quality with potential effects on consumer
acceptance.
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Received on 22-11-2018
DOI: https://doi.org/10.6000/1927-520X.2018.07.03.2
Accepted on 02-12-2018
Published on 31-12-2018
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THE ACTIVITY OF LYSOSOMAL NUCLEASES IN DIFFERENT AGE GROUPS OF SALMON SALMO SALAR L.
|
Trudy Karelʹskogo naučnogo centra Rossijskoj akademii nauk
| 2,015
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cc-by
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Активность лизосомальных нуклеаз у молоди
лосося Salmo salar L. разных возрастных групп К настоящему времени во всем мире из-за ухудшения экологической обстанов-
ки, загрязнения рек промышленными и бытовыми сточными водами, а также не-
рационального промысла произошло снижение запасов атлантического лосося. На Северо-Западе России одной из немногих рек, где сохранилось естественное
воспроизводство этого ценнейшего промыслового вида, является река Варзуга
(Мурманская обл.). Для разработки стратегии устойчивого сохранения популя-
ций лосося следует вести мониторинг с целью поддержания стабильных условий
обитания в экосистеме реки. Известно, что ферменты, участвующие в белковом,
энергетическом и углеводном обмене, являются достаточно информативными по-
казателями процессов роста, развития и адаптации рыб к изменениям окружаю-
щей среды. Наряду с вышеперечисленными параметрами для оценки состояния
водных экосистем используются лизосомальные гидролазы, в том числе кислые
нуклеазы, принимающие участие в компенсаторных перестройках нуклеинового
и тесно связанного с ним белкового обмена. В работе исследовалась активность
лизосомальных нуклеаз (РНКазы, ДНКазы) в мышцах атлантического лосося Salmo
salar L. разных возрастных групп (0+, 1+, 2+) из русла р. Варзуги (Собачий порог). Показано, что активность изученных ферментов и содержание белка в скелетных
мышцах рыб линейно увеличивались при переходе от одной возрастной группы
к другой и были максимальными у двухгодовиков. Абсолютные значения активно-
сти лизосомальной РНКазы были несколько выше, чем ДНКазы. Полученные дан-
ные свидетельствуют об активном участии лизосомальных нуклеаз в адаптивных
реакциях молоди лосося в постэмбриональном развитии, что выражается в первую
очередь в интенсификации процессов биосинтеза белка. Сделан вывод о том, что
условия обитания в р. Варзуге являются благоприятными для роста и развития мо-
лоди атлантического лосося. К л ю ч е в ы е с л о в а: лизосомальные нуклеазы; раннее развитие; лосось Salmo
salar L.; р. Варзуга. Труды Карельского научного центра РАН
№ 11. 2015. С. 92–98
doi: 10.17076/eb239 Труды Карельского научного центра РАН
№ 11. 2015. С. 92–98
doi: 10.17076/eb239 E. A. Vdovichenko, R. U. Vysotskaya, D. A. Efremov, A. E. Veselov. THE
ACTIVITY OF LYSOSOMAL NUCLEASES IN DIFFERENT AGE GROUPS OF
SALMON, SALMO SALAR L. These data indicate that lysosomal nucleases actively
participate in early post-embryonic adaptive reactions in juvenile salmon, which appear,
first of all, in an intensification of protein biosynthesis. The conclusion was drawn that the
living conditions in the Varzuga are favorable for the growth and development of juvenile
Atlantic salmon. K e y w o r d s: lysosomal nucleases; early stages of development; salmon Salmo salar L.;
Varzuga River. Введение из ценнейших промысловых видов. В последние
годы на севере России ухудшение экологичес-
кой обстановки и загрязнение водоемов быто-
выми и промышленными сточными водами, не-
рациональный и бесконтрольный вылов привели
к значительному спаду объемов вылова лосося
в регионе. Считается, что крупнейшая в Европе
популяция атлантического лосося, где естест-
венное воспроизводство этого вида еще сохра-
нилось, обитает в р. Варзуга [Калюжин, 2003]. Известно, что формирование популяций
рыб
является
результирующей
процессов
размножения, роста, полового созревания
и смертности [Залепухин, 2008]. Одним из по-
казателей, отражающих значимость эмбри-
онального и постэмбрионального развития
для численности поколений, является разно-
качественность рыб на ранних этапах онтоге-
неза [Дехник, 1985]. При изучении популяций
рыб в их естественной среде обитания обычно
оцениваются темпы роста, увеличение мас-
сы и длины, которые демонстрируют степень
адаптации к экологическим условиям. На кле-
точном уровне общую тенденцию адаптивных
изменений у молоди рыб отражают такие па-
раметры, как активность ферментов, участвую-
щих в белковом, углеводном и энергетическом
обмене. Достаточно информативными являют-
ся показатели соотношения РНК/ДНК, уровня
экспрессии генов тяжелой цепи миозина и ци-
тохром с оксидазы [Павлов и др., 2007]. Такой
биохимический параметр, как активность ли-
зосомальных нуклеаз, может служить дополни-
тельным критерием процессов роста, развития
и адаптации рыб к изменяющимся факторам
среды обитания, поскольку лизосомы играют
важную роль на протяжении всего онтогенеза
рыб. Они принимают участие в различных ме-
таболических процессах, связанных с гамето-
генезом, оплодотворением, эмбриогенезом,
выклевом личинок и их последующим постэм-
бриональным развитием [Высоцкая, Немова,
2008; Lanes et al., 2009; Биота…, 2012]. Целью данной работы являлось изучение
динамики активности лизосомальных нуклеаз
(РНКазы и ДНКазы) в мышцах атлантического
лосося из р. Варзуги (Собачий порог) в процес-
се развития молоди разных возрастных групп. E. A. Vdovichenko, R. U. Vysotskaya, D. A. Efremov, A. E. Veselov. THE
ACTIVITY OF LYSOSOMAL NUCLEASES IN DIFFERENT AGE GROUPS OF
SALMON, SALMO SALAR L. Deterioration of the environment, pollution of rivers by industrial and domestic waste-
waters, as well as unsustainable fisheries have caused Atlantic salmon, Salmo salar L.,
stocks to decline all over the world. One of the few rivers in Northwest Russia which has
retained the natural reproduction of this highly valuable commercial species is the Varzuga
River (Murmansk Region). Monitoring is required to work out the strategy for sustainable
conservation of salmon populations, which implies maintenance of stable living condi- 92 92 tions in the river ecosystem. Enzymes involved in the protein, energy and carbohydrate
metabolism are quite informative indicators of fish growth, development and adaptation
to environmental changes. In addition to the parameters mentioned above, the status of
aquatic ecosystems is assessed using lysosomal hydrolases, including acid nucleases
involved in compensatory transformations of the nucleic metabolism and the closely re-
lated protein metabolism. The activity of lysosomal nucleases (RNAse, DNAse) in mus-
cles in different age groups (0+, 1+, 2+) of juvenile Atlantic salmon from the Varzuga River
(Sobachiy rapid) was investigated in the present study. The activity of the enzymes and
the protein content in skeletal muscles were shown to increase linearly with age, reaching
a maximum in 2-year-old salmon. The absolute values of lysosomal RNAse activity were
slightly higher than DNAse activity. These data indicate that lysosomal nucleases actively
participate in early post-embryonic adaptive reactions in juvenile salmon, which appear,
first of all, in an intensification of protein biosynthesis. The conclusion was drawn that the
living conditions in the Varzuga are favorable for the growth and development of juvenile
Atlantic salmon. tions in the river ecosystem. Enzymes involved in the protein, energy and carbohydrate
metabolism are quite informative indicators of fish growth, development and adaptation
to environmental changes. In addition to the parameters mentioned above, the status of
aquatic ecosystems is assessed using lysosomal hydrolases, including acid nucleases
involved in compensatory transformations of the nucleic metabolism and the closely re-
lated protein metabolism. The activity of lysosomal nucleases (RNAse, DNAse) in mus-
cles in different age groups (0+, 1+, 2+) of juvenile Atlantic salmon from the Varzuga River
(Sobachiy rapid) was investigated in the present study. The activity of the enzymes and
the protein content in skeletal muscles were shown to increase linearly with age, reaching
a maximum in 2-year-old salmon. The absolute values of lysosomal RNAse activity were
slightly higher than DNAse activity. Обсуждение Лизосомы
обладают
широким
набором
гидролаз, активных при кислых значениях рН
и способных осуществлять гидролитическое
расщепление практически всех сложных ве-
ществ и биополимеров, из которых построена
живая материя [Покровский, Тутельян, 1976]. Одними из важнейших макромолекул являются
нуклеиновые кислоты, отвечающие за передачу
и хранение наследственной информации, а так-
же ее реализацию в ходе процесса трансляции,
т. е. биосинтеза белка в организме [Спирин,
2001; Bashan, Yonath, 2008]. Кислые нуклеазы
(РНКаза и ДНКаза) участвуют в расщеплении
межнуклеотидных
фосфодиэфирных
связей
в молекулах РНК и ДНК [Покровский, Тутельян,
1976; Высоцкая, Немова, 2008; Pizzo et al., 2008]. Результаты исследования показывают, что
изменения в активности изученных фермен-
тов носят сходный характер при переходе от
сеголеток (0+) к пестряткам (1+, 2+), при этом
максимальный уровень ферментативной актив-
ности выявлен у двухгодовиков. Следует пред-
положить, что линейно повышающийся уровень
активности лизосомальной РНКазы и ДНКазы
в мышцах атлантического лосося связан с ин-
тенсификацией процессов биосинтеза белка
в организме. Это можно подтвердить данными
по уровню содержания белка у рыб разных воз-
растных групп. В более ранних работах по изу-
чению активности лизосомальных ферментов
у молоди лосося было установлено, что актив-
ность кислой фосфатазы – фермента-маркера
лизосом, принимающего участие в липидном
и углеводном обмене, а также щелочной фос-
фатазы – одного из показателей интенсивнос-
ти роста рыб, возрастали в процессе взросле-
ния организма [Высоцкая и др., 2005]. Высокая
активность лизосомальной фосфатазы говорит Лизосомы
обладают
широким
набором
гидролаз, активных при кислых значениях рН
и способных осуществлять гидролитическое
расщепление практически всех сложных ве-
ществ и биополимеров, из которых построена
живая материя [Покровский, Тутельян, 1976]. Одними из важнейших макромолекул являются
нуклеиновые кислоты, отвечающие за передачу
и хранение наследственной информации, а так-
же ее реализацию в ходе процесса трансляции,
т. е. биосинтеза белка в организме [Спирин,
2001; Bashan, Yonath, 2008]. Кислые нуклеазы
(РНКаза и ДНКаза) участвуют в расщеплении
межнуклеотидных
фосфодиэфирных
связей
в молекулах РНК и ДНК [Покровский, Тутельян,
1976; Высоцкая, Немова, 2008; Pizzo et al., 2008]. Определение
активности
РНКазы
(КФ
3.1.4.23) и ДНКазы (КФ 3.1.4.6) проводи-
ли по методам А. П. Левицкого и др. [1973]
и А. А. Покровского с соавт. [Покровский, Арча-
ков, 1968]. В качестве субстратов использовали
0,1%-е растворы РНК и ДНК на 0,2 М растворе
ацетатного буфера (pH 5,2 и 5,0 соответствен-
но). Материалы и методы В качестве объекта исследования была вы-
брана молодь лосося Salmo salar L. разных
возрастных групп (0+, 1+, 2+). В каждой груп-
пе исследовано от 5 до 7 экземпляров. Пробы
отбирали осенью 2014 г. из основного рус-
ла р. Варзуга (Собачий порог), относящейся
к бассейну Белого моря. Собачий порог яв-
ляется мелководным, со средней глубиной
0,35–0,65 м. Длина порога составляет око-
ло 600 м, ширина варьирует в пределах 120–
160 м. Скорость течения изменяется в преде-
лах 0,5–1,2 м/с. По гидрохимическим харак-
теристикам вода имеет слабощелочную среду
(рH 8,42) и температуру 6,2 °C. Грунт преиму-
щественно валунный с отдельными галечными
участками, есть глыбы. На пороге ежегодно
происходит нерест производителей атланти-
ческого лосося и обитает молодь разных воз-
растных групп: 0+, 1+, 2+, 3+. Встречаются кар-
ликовые самцы в возрасте 4+ и 5+. Плотность Атлантический лосось обитает в морских
и пресных водоемах северного полушария в бас-
сейне Атлантического океана и является одним 93 Результаты особей всех возрастов варьирует в пределах
22–54 экз./100 м2 (0,7 экз./м2). Осенью при
снижении обилия дрифта пестрятки переходят
на частичное питание донными организмами
с грунта и прикрепленными к водной расти-
тельности личинками ручейников и моллюсков,
доминирующих в это время года в составе бен-
тоса [Шустов и др., 2012]. Результаты исследования показали, что ак-
тивность изученных ферментов существенно
зависела от возраста рыб. Так, активность ли-
зосомальной РНКазы в мышцах молоди лосо-
ся линейно возрастала при переходе от воз-
растной группы 0+ к 2+ (рис. 1). Активность
кислой ДНКазы в мышцах пестряток (1+ и 2+)
также была достоверно выше, чем у сеголеток
(0+) (рис. 2). При этом абсолютные значения
ДНКазной активности были несколько ниже
РНКазной. Содержание белка было практичес-
ки одинаковым у рыб в возрасте 1+ и 2+ и на
35–48 % (р < 0,05) превышало соответствую-
щий показатель у сеголеток (рис. 3). Cбор проб осуществляли методом электро-
лова. Затем мальков выдерживали в течение
суток в русловых садках для снятия эффекта
воздействия электрического поля. Рыбу доставляли в лабораторию в заморо-
женном состоянии в сосудах Дьюара и хранили
при температуре –80 °С. Мышцы для анализа
отбирали непосредственно в день определения
биохимических показателей. Из навески тканей
готовили 10%-е гомогенаты на 0,25 М раство-
ре сахарозы с добавлением 0,001 М раствора
ЭДТА и 0,1%-го раствора неионного детер-
гента тритона Х-100, затем центрифугирова-
ли при 10 000 g и температуре +4 °С в течение
30 мин. В полученном супернатанте определя-
ли активность лизосомальных нуклеаз и содер-
жание белка. Обсуждение Принцип методов основан на способности
нуклеаз расщеплять соответствующие нукле-
иновые кислоты на низкомолекулярные фраг-
менты, количество которых определяли спект-
рофотометрически при 260 нм после осажде-
ния нерасщепленных нуклеиновых кислот и их
крупных фрагментов 0,5 М раствором хлорной
кислоты и 0,25%-м раствором уранилацета-
та в 0,5 М растворе хлорной кислоты (для ДНК
и РНК соответственно). Активность ферментов
выражали в условных единицах ΔD260 в расчете
на 1 мг белка за единицу времени. Содержание
белка в пробах определяли по методу Лоури
[Биохимические методы…, 1969]. ;
,
,
;
,
]
Результаты исследования показывают, что
изменения в активности изученных фермен-
тов носят сходный характер при переходе от
сеголеток (0+) к пестряткам (1+, 2+), при этом
максимальный уровень ферментативной актив-
ности выявлен у двухгодовиков. Следует пред-
положить, что линейно повышающийся уровень
активности лизосомальной РНКазы и ДНКазы
в мышцах атлантического лосося связан с ин-
тенсификацией процессов биосинтеза белка
в организме. Это можно подтвердить данными
по уровню содержания белка у рыб разных воз-
растных групп. В более ранних работах по изу-
чению активности лизосомальных ферментов
у молоди лосося было установлено, что актив-
ность кислой фосфатазы – фермента-маркера
лизосом, принимающего участие в липидном
и углеводном обмене, а также щелочной фос-
фатазы – одного из показателей интенсивнос-
ти роста рыб, возрастали в процессе взросле-
ния организма [Высоцкая и др., 2005]. Высокая
активность лизосомальной фосфатазы говорит Полученные данные обработаны статисти-
чески с помощью Microsoft Office Excel 2007. Результаты представлены в виде средних и их
ошибок (М ± m). Достоверность различий оце-
нивалась по непараметрическому критерию U
Вилкоксона–Манна–Уитни при уровне значи-
мости р ≤ 0,05 [Гублер, Генкин, 1969]. Работа выполнена на приборно-аналитичес-
кой базе центра коллективного пользования
научным оборудованием Института биологии
КарНЦ РАН. 94 Рис. 1. Активность РНКазы в мышцах молоди лосося из Собачьего порога р. Варзуга. Здесь и далее: *стати-
стически достоверные отличия (p ≤ 0,05) Рис. 1. Активность РНКазы в мышцах молоди лосося из Собачьего порога р. Варзуга. Здесь и далее: *стати-
стически достоверные отличия (p ≤ 0,05) Рис. 2. Активность ДНКазы в мышцах молоди лосося из Собачьего порога р. Варзуга о большом количестве лизосом, содержащих
ферменты, необходимые для расщепления
полимерных компонентов в клетке, в том чис-
ле нуклеиновых кислот [Немова, Высоцкая,
2004]. Образующиеся продукты гидролиза ис-
пользуются для пластических и энергетических
успешным перестройкам метаболизма, свя-
занным с переходом мальков от одной стадии
развития к другой. Обсуждение Все это указывает на то, что
условия обитания молоди лосося (темпера-
тура воды, скорость течения, кормовая база)
способствуют ускоренным темпам роста и раз-
Рис. 2. Активность ДНКазы в мышцах молоди лосося из Собачьего порога р. Варзуга
Рис. 3. Содержание белка в мышцах молоди лосося из Собачьего порога р. Варзуга Рис. 2. Активность ДНКазы в мышцах молоди лосося из Собачьего порога р. Варзуга Рис. 3. Содержание белка в мышцах молоди лосося из Собачьего порога р. Варзуга Рис. 3. Содержание белка в мышцах молоди лосося из Собачьего порога р. Варзуга о большом количестве лизосом, содержащих
ферменты, необходимые для расщепления
полимерных компонентов в клетке, в том чис-
ле нуклеиновых кислот [Немова, Высоцкая,
2004]. Образующиеся продукты гидролиза ис-
пользуются для пластических и энергетических
нужд растущего организма, что способствует о большом количестве лизосом, содержащих
ферменты, необходимые для расщепления
полимерных компонентов в клетке, в том чис-
ле нуклеиновых кислот [Немова, Высоцкая,
2004]. Образующиеся продукты гидролиза ис-
пользуются для пластических и энергетических
нужд растущего организма, что способствует успешным перестройкам метаболизма, свя-
занным с переходом мальков от одной стадии
развития к другой. Все это указывает на то, что
условия обитания молоди лосося (темпера-
тура воды, скорость течения, кормовая база)
способствуют ускоренным темпам роста и раз-
вития особей. Стоит отметить, что активность 95 95 Дехник Т. В., Серебряков В. П., Соин С. Г. Зна-
чение ранних стадий развития рыб в формировании
численности поколений // Теория формирования
численности и рационального использования стад
промысловых рыб. М.: Наука, 1985. С. 56–72. лизосомальной ДНКазы была несколько ниже,
чем РНКазы, что, по всей видимости, обуслов-
лено большей интенсивностью метаболизма
РНК и, соответственно, усилением процессов
биосинтеза белковых молекул. лизосомальной ДНКазы была несколько ниже,
чем РНКазы, что, по всей видимости, обуслов-
лено большей интенсивностью метаболизма
РНК и, соответственно, усилением процессов
биосинтеза белковых молекул. Залепухин В. В. Разнокачественность произво-
дителей карповых рыб и ее роль в формировании
биологических ресурсов // Вестник Астраханского
государственного технического университета. Се-
рия Рыбное хозяйство. 2008. № 3. С. 39–42. Поступила в редакцию 11.09.2015 Поступила в редакцию 11.09.2015 Поступила в редакцию 11.09.2015 Поступила в редакцию 11.09.2015 Заключение Таким образом, в результате исследований
обнаружено сходное повышение активности
лизосомальных нуклеаз (РНКазы и ДНКазы)
в мышцах молоди лосося (0+, 1+, 2+), обитаю-
щей в р. Варзуга (Собачий порог) в процессе
взросления особей. Лизосомальные фермен-
ты, в том числе кислые нуклеазы, осуществляют
деградацию выполнивших свои функции био-
полимеров, тем самым обеспечивая организм
компонентами для синтеза новых нуклеиновых
кислот, принимающих участие в биосинтезе не-
обходимых на новом этапе развития белковых
веществ. Синтезированные белки используют-
ся не только для построения структур и тканей
молоди рыб, но и выполняют каталитическую
и регуляторную функции в организме. Получен-
ные данные позволяют сделать вывод о том, что
условия обитания в основном русле р. Варзуги
являются благоприятными и способствуют ус-
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1985. P. 56–72. the origin of life]. Vest. Ros. Akad. Nauk [Herald Russ. Acad. Sci.]. 2001. Vol. 71, No 4. P. 320–328. formirovaniya chislennosti I ratsional’nogo ispol’zovaniya
stad promyslovykh ryb [Population formation theory and
rational use of commercial fish stock]. Moscow: Nauka,
1985. P. 56–72. Shustov Yu. A., Baryshev I. A., Belyakova E. N. Oso-
bennosti pitaniya molodi atlanticheskogo lososya Salmo
salar L. v subarkticheskoy reke Varzuga I ee malykh prito-
kakh (Kol’skiy poluostrov) [Specific features of the feeding
of juvenile Atlantic salmon Salmo salar L. in the subarctic
Varzuga River and its small tributaries (Kola Peninsula)]. Biol. vnut. vod [Inland Water Biol.]. 2012. No 3. P. 66–70. Gubler E. V., Genkin A. A. Primenenie kriteriev ne-
parametricheskoy statistiki dlya otsenki razlichiy dvukh
grupp nablyudeniy v mediki-biologicheskikh issledo-
vaniyakh [Application of criteria of nonparametric statis-
tics for estimating differences between two study groups
in biomedical research]. Moscow: Meditsina, 1969. 29 p. Vysotskaya R. U., Lomaeva T. A., Amelina V. S., Ve-
selov A. E., Morozov D. N. Aktivnost’ lizosomal’nykh
fermentov u molodi lososya, razlichayushcheysya vy-
borom uchastkov obitania [Lysosomal enzymes activity
in juvenile salmon living in different areas]. Sovremen-
nye problemy fiziologii I biokhimii vodnykh organizmov:
mat. mezd. konf. (Petrozavodsk, 6–9 sentyabrya 2004). Petrozavodsk, 2005. P. 32–35. Kalyzhin S. M. Atlanticheskiy losos’ Belogo morya:
problemy vosproizvodstva i ekspluatatsii [Atlantic salm-
on in the White Sea basin: problems of reproduction and
fisheries]. Petrozavodsk: PetroPress, 2003. 264 p. i
Levitskiy A. P., Barabash R. D., Konovets V. M. Se-
zonnye osobennosti aktivnosti ribonukleasy i α-amilazy
slyuny i slyunnykh zhelez u krys linii Vistar [Seasonal
characteristics of ribonuclease and α-amylase activity
of saliva and salivary glands in Wistar rats]. Biokhimiche-
skaya evolyutsiya [Biochemical evolution]. Leningrad:
Nauka, 1973. P. 192–195. Vysotskaya R. U., Nemova N. N. Lizosomy i lizoso-
mal’nye fermenty ryb [Fish lysosomes and lysosomal
enzymes]. Moscow: Nauka, 2008. 284 p. Zalepukhin V. V. CONTRIBUTORS: Вдовиченко Елизавета Андреевна
аспирант, младший научный сотрудник
Институт биологии Карельского научного центра РАН
ул. Пушкинская, 11, Петрозаводск, Республика Карелия,
Россия, 185910
эл. почта: elizaveta.vdovichenko@gmail.com
тел.: (8142) 769810
Высоцкая Римма Ульяновна
главный научный сотрудник, д. б. н., проф. Институт биологии Карельского научного центра РАН
ул. Пушкинская, 11, Петрозаводск, Республика Карелия,
Россия, 185910
эл. почта: rimma@bio.krc.karelia.ru
тел.: (8142) 769810 Vdovichenko, Elizaveta
Institute of Biology, Karelian Research Centre,
Russian Academy of Sciences
11 Pushkinskaya St., 185910 Petrozavodsk, Karelia, Russia
e-mail: elizaveta.vdovichenko@gmail.com
tel.: (8142) 769810 д
др
аспирант, младший научный сотрудник
Институт биологии Карельского научного центра РАН у
р
у
ц
р
ул. Пушкинская, 11, Петрозаводск, Республика Карелия,
Россия, 185910 эл. почта: elizaveta.vdovichenko@gmail.com
тел.: (8142) 769810 Высоцкая Римма Ульяновна
главный научный сотрудник, д. б. н., проф. Институт биологии Карельского научного центра РАН
ул. Пушкинская, 11, Петрозаводск, Республика Карелия,
Россия, 185910
эл. почта: rimma@bio.krc.karelia.ru
тел.: (8142) 769810 Vysotskaya, Rimma
Institute of Biology, Karelian Research Centre,
Russian Academy of Sciences
11 Pushkinskaya St., 185910 Petrozavodsk, Karelia, Russia
e-mail: rimma@bio.krc.karelia.ru
tel.: (8142) 769810 Высоцкая Римма Ульяновна 97 Efremov, Denis
Institute of Biology, Karelian Research Centre,
Russian Academy of Sciences
11 Pushkinskaya St., 185910 Petrozavodsk, Karelia, Russia
e-mail: veselov@krc.karelia.ru
tel.: (8142) 769810 Ефремов Денис Александрович
научный сотрудник, к. б. н. Институт биологии Карельского научного центра РАН
ул. Пушкинская, 11, Петрозаводск, Республика Карелия,
Россия, 185910
эл. почта: veselov@krc.karelia.ru
тел.: (8142) 769810 Ефремов Денис Александрович
научный сотрудник, к. б. н. Институт биологии Карельского научного центра РАН
ул. Пушкинская, 11, Петрозаводск, Республика Карелия,
Россия, 185910
эл. почта: veselov@krc.karelia.ru
тел.: (8142) 769810 Веселов Алексей Елпидифорович Veselov, Alexey
Institute of Biology, Karelian Research Centre,
Russian Academy of Sciences
11 Pushkinskaya St., 185910 Petrozavodsk, Karelia, Russia
e-mail: veselov@krc.karelia.ru
tel.: (8142) 769810 Веселов Алексей Елпидифорович
главный научный сотрудник, д. б. н., проф. Институт биологии Карельского научного центра РАН
ул. Пушкинская, 11, Петрозаводск, Республика Карелия,
Россия, 185910
эл. почта: veselov@krc.karelia.ru
тел.: (8142) 769810 Veselov, Alexey
Institute of Biology, Karelian Research Centre,
Russian Academy of Sciences
11 Pushkinskaya St., 185910 Petrozavodsk, Karelia, Russia
e-mail: veselov@krc.karelia.ru
tel.: (8142) 769810 Веселов Алексей Елпидифорович
главный научный сотрудник, д. б. н., проф. Институт биологии Карельского научного центра РАН
ул. Пушкинская, 11, Петрозаводск, Республика Карелия,
Россия, 185910
эл. почта: veselov@krc.karelia.ru
тел.: (8142) 769810
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Pathology in Africa: back to basics
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Virchows Archiv
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Pathology in Africa: back to basics Pieter J. Slootweg Received: 13 March 2012 /Accepted: 14 March 2012 /Published online: 29 March 2012
# The Author(s) 2012. This article is published with open access at Springerlink.com Received: 13 March 2012 /Accepted: 14 March 2012 /Published online: 29 March 2012
# The Author(s) 2012. This article is published with open access at Springerlink.com In this issue, Berezowski et al. describe their experience as
volunteers in a pathology laboratory in Malawi. It is not a
surprise that the case mix they were confronted with differs
greatly from what is seen in the daily practice of the major-
ity of diagnostic pathology laboratories: almost half of the
cases consisting of nonneoplastic lesions and within the
group of neoplasias are a large proportion of pediatric cases,
which would be rather unusual for a pathology laboratory in
the northern hemisphere. additional stains: the diagnosis has to be made on a few or
even just one single hematoxylin and eosin-stained slide. Di-
agnostic skill and acumen that tend to languish in an environ-
ment where a brown deposit usually is considered to be more
decisive for diagnosis than careful morphological analysis are
brought to new life when one is left with nothing else than a
microscope of moderate quality, a pair of eyes, and experience. An issue only briefly alluded to by Berezowski et al. but
requiring a little bit more reflection is whether in that setting
histopathological examination plays any role in patient man-
agement. Quite often, patients are treated without prior
biopsy or, in case their specimens are submitted for patho-
logical examination, have already left the hospital and
returned to their villages somewhere in a remote area before
the pathology report has arrived on the clinician's desk. This
precludes any guidance of treatment by pathological analy-
sis, either preoperatively on the basis of a biopsy or postop-
eratively through examination of a resection specimen. Not only the nature of cases is different but also the stage
of development and size of the lesions that are submitted for
pathological examination. Tourists on safari trips to Africa
usually want to see the big five: lion, leopard, rhino, ele-
phant, and buffalo. Getting to see them requires some luck
and effort and this wish is not always fulfilled. Virchows Arch (2012) 460:361
DOI 10.1007/s00428-012-1229-8 Virchows Arch (2012) 460:361
DOI 10.1007/s00428-012-1229-8 INVITED EDITORIAL Open Access
This article is distributed under the terms of the Crea-
tive Commons Attribution License which permits any use, distribution,
and reproduction in any medium, provided the original author(s) and
the source are credited. P. J. Slootweg (*)
Department of Pathology,
Radboud University Nijmegen Medical Center,
P.O. Box 9101, 6500 HB, Nijmegen, Netherlands
e-mail: p.slootweg@pathol.umcn.nl Pathology in Africa: back to basics It does not
take much effort for the volunteering pathologist to encoun-
ter her or his own version of the big five: very large uterine
fibroids, goiters, ovarian tumors, breast cancers, and lesions
sometimes weighing several kilograms. Therefore, if pathology is to play any role in guiding daily
patient care under sub-Saharan African conditions and resour-
ces, it should provide a cheap and quick diagnosis—require-
ments that obviously can be met by diagnostic cytology. Any
volunteering pathologist should master this area and should
preferably also be capable of performing FNAs, as this allows
not only rapid diagnosis and valuable clinical correlations but
also offers an opportunity for personal contacts with less
privileged people whose daily lives are much more difficult
than ours. This experience may make us more humble and
lenient in handling the small inconveniences we all regularly
encounter in either our private or professional lives. However, it is not only the difference in the type of lesion
and their size that makes a stay in an African pathology
laboratory such a rewarding experience. Working there in fact
means practicing pathology as it was done where most of us
reside not that long ago: before immunohistochemistry and
molecular pathology entered the scene. It encompasses looking
at slides that quite often are slightly or even quite a bit thicker
than the 3 or 4 μm we are accustomed to. There is no room for
postponing of decisions by ordering time-consuming P. J. Slootweg (*)
Department of Pathology,
Radboud University Nijmegen Medical Center,
P.O. Box 9101, 6500 HB, Nijmegen, Netherlands
e-mail: p.slootweg@pathol.umcn.nl Open Access
This article is distributed under the terms of the Crea-
tive Commons Attribution License which permits any use, distribution,
and reproduction in any medium, provided the original author(s) and
the source are credited. P. J. Slootweg
Department of Pathology, Kilimanjaro Christian Medical Center,
Moshi, Tanzania
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English
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Extractive Spectrophotometric Method for the Determination of lron(III) in Green Leafy Vegetables using Sodium Pentamethylene Dithiocarbamate
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Zenodo (CERN European Organization for Nuclear Research)
| 1,991
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cc-by
| 874
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Experimental Na(Pipdtc) was prepared and recrystallised frolll
benzene2 and 1% of stock solution was prepared
in double-distilled water. Ferric ammonium sulphate (Proanalysis) disso!vedd
in 2M H2S04 was diluted
with
double-distJIIe
water. All other chemicals were of A.R. grade. Absorbance measurements were made using &
Schimadzu PR-1 spectrophotometer and pH adjust·
ments made on an Elico LI-10 pH meter. A 0.25 x w-s M Feii1 solution (I ml), a buffer of
pH .-..~4.2 (l ml), salting-out agent (0.1 MMgS0~
1
l ml), 1% Pipdtc (l ml), water (6 ml} and ciiCs
(lO ml) were mixed in the above order and tbe
organic layer containing the complex was separate~!·
The extrac<ion of the water"insoluble yellowish gr~n
complex into CHCI 3 was stable for 6 h. Quantita~~o
extraction could be acheived in a single extractJO~
and within 2 min of shaking in the presence 0
magnesium sulphate. The complex showed an
absorption maximum at 350 nm. Neither the reagent
nor the metal ion had any absorbance in the ranS0
of study. Extractive Spectrophotometric Method for the
Determination of lron(m) in Green Leafy
Vegetables using Sodium Pentamethylene
Dithlocarbamate
K. SARASWATI-II*, M. DASARATHA RAMAIAH
and
V.V,RAMANA
Department of Chemistry, S. V. University College.
Ti rupati-517 502
Manuscript received 30 APril 1990, revised 14 Nooemberl990·
accepted 26 January 1991 Extractive Spectrophotometric Method for the
Determination of lron(m) in Green Leafy
Vegetables using Sodium Pentamethylene
Dithlocarbamate
K. SARASWATI-II*, M. DASARATHA RAMAIAH
and
V.V,RAMANA
Department of Chemistry, S. V. University College. Ti rupati-517 502
Manuscript received 30 APril 1990, revised 14 Nooemberl990·
accepted 26 January 1991 IN continuation of our earlier work on metal co~·
plexes of sodium pentamethylene dithiocarbamates'
we report herein a simple ar.d sensitive spectr~·
photometric procedure for the determination offe1 •
Since
sodium
pentamethylene
dithiocarbam~te
(Pipdtc) has been widely used for the determinatiOn
of many metal ions a procedure is adopted for tbe
determination of iron in the leaves of AmaranthU5
viridis and A. gangeticus. IN J, INDIAN CHEM, SOC., VOL, 68, FBBRUAltY 1991 J, INDIAN CHEM, SOC., VOL, 68, FBBRUAltY 1991 Results and Discussion The metal chelate showed maximum absorban~e
in the pH range 3-6 in sodium acetate and acetiC
acid buffer. The pH of 4.2 is selected to eliminate
the interference of some metal ions like AIIII an~
Zn11•
At least a six-fold excess of the reagent. iS
necessary for consistency in absorbance. Quanuta·
tive determif!ation of iron is possible in t.he ran~
1-15 ppm of Felli per 10 ml of .the organic laY~1•
The molar absorptivity of the complex and sensitiVItY 116 NOTES are found to be 1.96 x to-s dm3 mol- 1 cm- 1 and
0.02 pg em- 2, respectively. are found to be 1.96 x to-s dm3 mol- 1 cm- 1 and
0.02 pg em- 2, respectively. Job's method, mole-ratio method and
Asmus
method were employed to obtain the composition of
the complex and the ratio of metal-to-ligand was
found to be 1 : 3. The instability constant as calcula-
ted from Edmond and Birnbaum method and Asmus
method was found to be 2.0 X 10- 9 • The ions, Snll, wvt' Srii' Aliii, Zn11' sulphate,
carbonate, nitrate,
fluoride, chloride, bromide,
oxalate, phosphate. tattrate do not interfere. How-
ever, the interference of Co11, Niii, Cull can be
masked with 2% KCN and that of MoVI by 1%
fluoride solution. Nitrite,
iodate, bromate and
periodate ions interfere in the determination. Application : The procedure was applied for the
determination of Fe'11 in the leaves of A. viridis
and A. gangeticus and the results obtaiPed (in mg/
100 g) are comparable to that by the phenanthroline
method8 (shown in the parenthesis) : 18.70 (18.46),
26.64 (26.58). The present method for the estimation of Fe111 in
the leaves is simple, less time-consuming and free
from interference of other metal ions. Acknowledgement One of the authors (M.D.R.)
i~o thankful to the
U.G.C., New Dell,i, for a Fellowship and to the
management of Sri Sarvodaya College, Nellore, for
granting study leave. 3.
'b;, EMANUEL, ''Mineral Nutrition of Plants Principles
And Perspectives'', Wiley, New York, 1972: A I.
VOGEL. "A Text Book of Quantitative Inorganic
Analysis", 3rd. ed., ELBS, London. H?64. ,
,
,
2.
K. GLUE and R. SCHWAB, Angew Chem., 1950, 61,
320. References 1. K. SARASWATHI,
K
VIJAVALAKSHMI
11nr! K. JYO'l'HIRMAYI, J, Blect,.ochem Soc., 1985,34-3, 184;
K SARASWA'l'HI, K. SAN'l'HA and K. jYO'l'HIRM!IYI,
Bull. Blect,.ochem., 1988, 4, 6'l3 , K. SARASWATHI,
K. SAN'l'HA 11nd K. M. KUMAR!, Analyst, 1990, 115,
4,
465 ; V. V. RAMANA,
K. SANTHA,
M. D. RAMAIAH And K. SAR<\SWA'l'HI, J. Indian Chem. Soc., 1991, 68, 000. 1. K. SARASWATHI,
K
VIJAVALAKSHMI
11nr! K. JYO'l'HIRMAYI, J, Blect,.ochem Soc., 1985,34-3, 184;
K SARASWA'l'HI, K. SAN'l'HA and K. jYO'l'HIRM!IYI,
Bull. Blect,.ochem., 1988, 4, 6'l3 , K. SARASWATHI,
K. SAN'l'HA 11nd K. M. KUMAR!, Analyst, 1990, 115,
4,
465 ; V. V. RAMANA,
K. SANTHA,
M. D. RAMAIAH And K. SAR<\SWA'l'HI, J. Indian Chem. Soc., 1991, 68, 000. ,
,
,
2. K. GLUE and R. SCHWAB, Angew Chem., 1950, 61,
320. ,
,
,
2. K. GLUE and R. SCHWAB, Angew Chem., 1950, 61,
320. 3. 'b;, EMANUEL, ''Mineral Nutrition of Plants Principles
And Perspectives'', Wiley, New York, 1972: A I. VOGEL. "A Text Book of Quantitative Inorganic
Analysis", 3rd. ed., ELBS, London. H?64.
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Using particle swarm optimization to improve visibility-aerosol optical depth retrieval method
|
npj climate and atmospheric science
| 2,021
|
cc-by
| 10,561
|
INTRODUCTION distribution is high in summer and low in winter, showing an
overall increasing trend in recent decades18–21. Aerosols originate from a wide variety of sources, and often have
highly complex compositions, which play an important role in
global and regional environments. Understanding the physical,
chemical, and optical characteristics and distribution of aerosols
aids to assess their role in radiation balance, air quality, and
predict
climate
change1,2. The
composition,
concentration,
absorption, and scattering characteristics of the aerosol are
directly or indirectly observed by remote sensing and ground-
based sensors3–7. The aerosol optical depth (AOD), one of the
most important optical parameters of aerosols, is a key physical
quantity characterizing atmospheric turbidity, and an important
factor used to determine the effect of aerosols on the climate. The
space coverage of satellite data is wide; however, the timelapse is
short, whereas ground-based data acquired by a sunphotometer
provide high accuracy, but few stations. Neither can provide AOD
data spanning a wide range, long time, and high consistency8. AOD retrieval methods worldwide mostly use the original
visibility- AOD conversion model17,22,23. The inversion method is
based on the Elterman model and takes into account the influence
of water vapor. A set of empirical parameters are introduced into
each station and developed into the M-Elterman and the KM-
Elterman models20,21,24, whose retrieval parameters are deter-
mined by the nonlinear least square method (NLSM)20. These AOD
retrieval methods using NLSM suffer from inaccurate parameters,
which limit their wider application. Further, Lin25,26 proposed an
AOD retrieval method using chemical transmission model GEOS-
Chem fusion visibility data, and the results were in good
agreement with MODIS data. g
To address the inaccurate parameters in the M-Elterman model,
this study introduces the particle swarm optimization (PSO)
algorithm into the AOD retrieval method, which originates from
a study on the foraging behavior of birds27. Owing to the
simplicity, stability, and efficiency of this algorithm, as well as its
ability to obtain global or approximately global optimal solutions
of the problem28, it has been widely applied across different fields,
and its use has become particularly widespread in the field of
artificial intelligence29–31. The essence of the PSO algorithm is that
one or more functions (usually nonlinear) can solve the problem of
the global optimal solution in a multi-dimensional space. 1Key Laboratory of Atmospheric Environment and Processes in the Boundary Layer over the Low-Latitude Plateau Region, Department of Atmospheric Science, Yunnan
University, Kunming 650091, China. 2CAS Key Laboratory of Regional Climate-Environment for Temperate East Asia, Institute of Atmospheric Physics, Chinese Academy of
Sciences, Beijing 100029, China. 3Gaochun Meteorological Bureau, Nanjing 211300, China. ✉email: wujian@ynu.edu.cn ARTICLE
OPEN
Using particle swarm optimization to improve
visibility-aerosol optical depth retrieval method Jian Wu
1✉, Shuang Zhang1, Qidong Yang1, Deming Zhao2, Wenxuan Fan1, Jingchuan Zhao1 and In view of the lack of long-term AOD (Aerosol Optical Depth) data, PSO (Particle Swarm Optimization) algorithm is introduced and
joint used with NLSM (the nonlinear least square method) to improve visibility-AOD retrieval method, which is referred to as the
PSO-M-Elterman model and significantly increases data available rate by 8% and correlation by about 20% with the true value in
the experimental group. The mean absolute error, the proportion of the smaller absolute error and the root mean square error in
the PSO-M-Elterman model experimental group are 0.0314 and 91.23%, 0.0509 respectively, which significantly outperforms other
groups. The main increase of AOD was found in the eastern region (South China, East China, Central China) and Taklimakan with the
trend coefficients of 2.67, 2.46, 2.13, and 1.45 (×10−3 yr−1) in recent 55 years, which may not be interpreted by the influence of
relative humidity. Long-term change of AOD in east China is mainly caused by human activity, and the AOD is higher in cities with a
larger population and more human activity. The PSO-M-Elterman model can maximize the advantage of visibility sequence length
to obtain long-term AOD inversion results. npj Climate and Atmospheric Science (2021) 4:49 ; https://doi.org/10.1038/s41612-021-00207-5 www.nature.com/npjclimatsci INTRODUCTION In each step of the iteration, the particle updates its
position by tracking two extrema, one being the optimal solution
found by the particle itself, which is called the individual extrema,
while the other is the optimal solution found by the entire
population, which is the global extrema. After finding these two
extrema, the particle repeatedly updates its velocity and position,
eventually reaching the global extreme for all particles. Compared
with the NLSM, the optimization of parameters is considered to be
a similar process, as random parameter combinations are selected
to minimize the deviation between the observed and simulated
values within a certain period of time, and the parameter
combination with the smallest deviation is considered as the
optimal parameter. Both algorithms require iterative calculation;
the difference lies in the randomized initial parameters of the PSO
algorithm. In the actual application, because the calculation time is
controlled (small storage requirements), the value range of the
parameter is given, and the parameter space to be optimized
becomes a closed area. Thus, the global optimal parameters in this
area can be obtained, which represent the approximate optimal
parameters in the entire open area. according to the conversion schemes described above. Finally,
we compared them with the actual observed visibility distance,
whose difference is considered to be the error between the
converted visibility using the conversion scheme and the actual
visibility, and the relative error is the absolute error divided by
the actual visibility. The comparison of the mean error in the
monthly series (Fig. 1a) shows that the errors fluctuated
periodically over a period of 12 months. The absolute error of
scheme Qin fluctuates within the range of –0.4–1 km. In
contrast, the mean absolute error of the conversion proposed
scheme only fluctuates around zero with an amplitude below
0.1 km, and does not change significantly with time. Further-
more, the distribution of the relative error (Fig. 1b) shows that
the Qin scheme exhibits the highest relative error in terms of
visibility, generally above 10%, whereas in the conversion
scheme, except for a few areas in the southwest of Xinjiang, the
relative error is below 1%. Hence, we demonstrate that the conversion scheme sig-
nificantly improves the credibility of visibility conversion owing
to the consideration of differences and seasonal characteristics
of each station. INTRODUCTION The most
significant difference between this and the traditional algorithm,
which uses tools such as derivatives to find extrema, is that the
initial value, the update of particle velocity and position are
generated randomly. Under the constraint of an objective
function, as long as there is a sufficient number of iterations,
these particles will tend to converge, and thus obtain the optimal
solution of the problem. y
Currently, studies on the long-term change of AOD are scarce
due to limited aerosol data. Several studies revealed an
interaction between aerosols and monsoon systems9–12. There-
fore, obtaining long-term aerosol data to analyze the interaction
between aerosols and climate change factors is an urgent issue
in the effort to understand the impact of human activities on the
climate. AOD can be calculated from the extinction coefficient,
wide-band solar radiation, visibility, and other meteorological
elements13–16. Visibility, as an indicator reflecting atmospheric
transparency, has the advantage of long-term observation and
numerous stations, and it is often used to retrieve AOD and
analyze its spatial and temporal characteristics. AOD retrieval
using visibility data shows that the Indian Peninsula, Central
Europe, West Africa, Southeast North America, and Southeast
China are the largest AOD regions in the world17. The spatial
distribution of inversion AOD by visibility in China is high in the
southeast and low
in
the northwest, while the seasonal The PSO algorithm is implemented by initializing the para-
meters to be optimized to the position of a group of random Published in partnership with CECCR at King Abdulaziz University Fig. 1
Comparison of four schemes for visibility grade conversion. a Monthly time series of national mean visibility error from 1980–2013. b Spatial distribution of mean visibility relative error from 1980 to 2013. J. Wu et al. J. Wu et al. J. Wu et al. J. Wu et al. 2 Fig. 1
Comparison of four schemes for visibility grade conversion. a Monthly time series of national mean visibility error from 1980–2013. b Spatial distribution of mean visibility relative error from 1980 to 2013. es for visibility grade conversion. a Monthly time series of national mean visibility error from 1980–2013. bility relative error from 1980 to 2013. particles, and subsequently finding the optimal solution through
iteration. INTRODUCTION The improved converted visibility provides
more reliable data for the subsequent AOD inversion, and
further improves the accuracy of the inversion results. This
scheme can also be applied to other scientific research that
requires long-term visibility data in China, as it can effectively
improve the homogeneity of visibility. Published in partnership with CECCR at King Abdulaziz University npj Climate and Atmospheric Science (2021) 49 Conversion of visibility grade data Visibility was recorded on a scale of ten before 1980, and the
actual visibility distance (unit: km) was directly recorded after
1980. Therefore, it is necessary to convert the visibility grade
data before 1980 into the corresponding distance data. We
designed a conversion scheme based on the scheme of Qin19
(details are in the Methods section), which is commonly used in
visibility conversion, and compared our results. To estimate
alternative distances in different schemes, we converted the
visibility distance data from 1980 to 2013 into their correspond-
ing grade, and subsequently converted them into distances Spatiotemporal contrast of different inversion results (3) Figure 3 shows the mean spatial distribution and temporal
fluctuation of AOD in the experimental and the extrapolation
groups with MODIS and MISR data denoting the true values,
respectively. The spatial distribution of MODIS/MISR AOD shows a
similar distribution pattern in both the experimental and the
extrapolation groups (Fig. 3a, c): southern Xinjiang, and east China
(North China Plain, the middle and lower reaches of the Yangtze
River, and Sichuan Basin) are the regions with high AOD values,
while parts of Southwest China and Northeast China are the
regions with low AOD values. However, MODIS AOD is generally
higher than MISR AOD, except for some areas in the southwest
(the intersecting area of Tibet, Sichuan, and Yunnan). The time
series (Fig. 3b, d) show evident fluctuation characteristics with a
period of one year. The AOD is higher in the summer and autumn
and lowers in the spring and winter. The monthly average AOD of
MODIS varies from 0.25 to 0.65 with a mean value of 0.4472, while
the monthly average AOD of MISR varies from 0.15–0.5 with a
mean value of 0.2928. (4) where x and y denote the true and inverted AOD, respectively. y
y
According to the definitions of KGE and R, their larger values
contribute to a better fitting effect. To evaluate retrieval results,
we divided the values of KGE and R of each site into four different
evaluation intervals- KGE & R > 0.7; KGE & R > 0.5; KGE & R > 0.3
and KGE & R > 0 (Corresponding to “excellent -A”, “good -B”,
“medium -C”, “poor -D”), then evaluate inversion values in each
interval respectively (When KGE or R < 0, the inversion results can
be considered to be completely unavailable). Figure 2a shows the comparison of the time series of inversion
AOD and the true value of four evaluation intervals when MODIS
data are used as the AOD truth value. The values and fluctuation
characteristics of the inversion AOD are similar to the true value in
the first three KGE and R evaluation intervals, while the inversion
AOD is significantly higher than the true value in the fourth
evaluation interval with poor temporal variations. Therefore, the
reliability and accuracy of inversion results are acceptable when
KGE & R > 0.3 (i.e., the inversion results are evaluated as “medium-
C” or above). Joint use of inversion parameters from different algorithms of KGE (Kling-Gupta efficiency)32 and R:
KGE ¼ 1
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
R 1
ð
Þ2þ α 1
ð
Þ2þ β 1
ð
Þ2
q
(1)
R ¼
Pn
i¼1 xi x
ð
Þ yi y
ð
Þ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Pn
i¼1 xi x
ð
Þ2 ´ Pn
i¼1 yi y
ð
Þ2
q
(2)
α ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Pn
i¼1 ðxixÞ2
n
r
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Pn
i¼1 ðyiyÞ2
n
r
(3)
β ¼ x
y
(4) KGE ¼ 1
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
R 1
ð
Þ2þ α 1
ð
Þ2þ β 1
ð
Þ2
q R ¼
Pn
i¼1 xi x
ð
Þ yi y
ð
Þ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Pn
i¼1 xi x
ð
Þ2 ´ Pn
i¼1 yi y
ð
Þ2
q Joint use of inversion parameters from different algorithms Joint use of inversion parameters from different algorithms
To verify the reliability and accuracy of the inversion, we split the
period of satellite observation AOD into an experimental and an
extrapolation verification group, containing data collected over
94 months from January 2007 to October 2014 and over
72 months from January 2001 to December 2006, respectively. For each station, the parameters were first fitted using the PSO
algorithm and then filtered through the combined criterion npj Climate and Atmospheric Science (2021) 49 Published in partnership with CECCR at King Abdulaziz University J. Wu et al. 3 Fig. 2
Selection of parameter simulation algorithm. a Time series of inversion AOD and true value in different evaluation intervals when
using MODIS data. b Distribution of station selection for PSO and NLSM algorithm in PSO-M-Elterman model. Fig. 2
Selection of parameter simulation algorithm. a Time series of inversion AOD and true value in different evaluation intervals when
using MODIS data. b Distribution of station selection for PSO and NLSM algorithm in PSO-M-Elterman model. Fig. 2
Selection of parameter simulation algorithm. a Time series of inversion AOD and true value
using MODIS data. b Distribution of station selection for PSO and NLSM algorithm in PSO-M-Elterman mulation algorithm. a Time series of inversion AOD and true value in different evaluation intervals when
of station selection for PSO and NLSM algorithm in PSO-M-Elterman model. of KGE (Kling-Gupta efficiency)32 and R: of KGE (Kling-Gupta efficiency)32 and R: of KGE (Kling-Gupta efficiency)32 and R:
KGE ¼ 1
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
R 1
ð
Þ2þ α 1
ð
Þ2þ β 1
ð
Þ2
q
(1)
R ¼
Pn
i¼1 xi x
ð
Þ yi y
ð
Þ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Pn
i¼1 xi x
ð
Þ2 ´ Pn
i¼1 yi y
ð
Þ2
q
(2)
α ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Pn
i¼1 ðxixÞ2
n
r
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Pn
i¼1 ðyiyÞ2
n
r
(3)
β ¼ x
y
(4) M-Elterman model is referred to as the PSO-M-Elterman model. In
this model, 467 stations employed the PSO algorithm to
determine retrieval parameters, and 194 stations employed the
NLSM when MODIS data were used as the true value. Further, the
PSO algorithm and NLSM were used in 308 and 353 stations
respectively, when MISR data were used as true value (as shown in
Fig. 2b, the selection method for specific stations is described in
the discussion section of this article). Published in partnership with CECCR at King Abdulaziz University npj Climate and Atmospheric Science (2021) 49 Spatiotemporal contrast of different inversion results The PSO-M-Elterman model evidently captures more low-value
areas of AOD, whereas areas with a true values below 0.1 are not
well presented. The spatial distribution and probability density of the correla-
tion coefficients between the inversion and true value time series
at each station are shown in Fig. 4. When using MODIS data, the
positive correlation coefficients of the M-Elterman model are
mainly distributed in Southwest China and North China, and most
of the values are below 0.5. Almost half of the correlation
coefficients in the region assume negative values. The PSO-M-
Elterman model not only increases the coverage area of the
positive correlation coefficient, but also enhances the correlation
values. The probability peak of the correlation coefficient appears
between 0.4 and 0.5, and is associated with few negative
correlations. The pattern of the extrapolation group is similar,
but slightly worse than that of the experimental group. The
proportion of R > 0.3 stations in the PSO-M-Elterman experimental
and extrapolation groups are 67.17% and 56.58%, respectively,
which is considerably higher than that in the M-Elterman model. For MISR data, the M-Elterman model likewise yields poor
correlation, and the PSO-M-Elterman model presents a slightly
weaker positive correlation pattern than MODIS data. Further-
more, the area of positive correlation in the experimental group
almost covers the entire region of China. The inversion results of
the extrapolation group are slightly worse than that of the
experimental group, and the proportion of the R > 0.3 stations in
the two groups reach 54.16% and 47.05%, respectively, which is
about 26% and 16% higher than that of the M-Elterman model,
respectively. The inversion results of the PSO-M-Elterman model
show significant improvement in terms of the fluctuation trend
and correlation of time series relative to the M-Elterman model. p
The performance of spatial pattern of AOD obtained by the
PSO-M-Elterman model is not significantly better than that of
the M-Elterman model. The spatial distribution of AOD simulated
by the two models is similar, irrespective of whether MODIS or
MISR data are used as AOD truth values, while there are slight
differences in several regions. The performance of the extra-
polation group was slightly exacerbated compared to that of the
experimental group. For the time series (Fig. Spatiotemporal contrast of different inversion results Thus, the stations of KGE & R > 0.3 were selected to
use the parameters fitted by the PSO algorithm, while other
stations used the parameters fitted by NLSM in the retrieval. This
joint use of retrieval parameters from PSO and NLSM in the When MODIS data are employed as the true value (Fig. 3a), both
models perform better to estimate the spatial distribution pattern
of AOD. However, the AOD simulated by the M-Elterman model in
Southwest Xinjiang and Inner Mongolia is approximately 0.1
higher than the true value. Meanwhile, the AOD simulated by the
PSO-M-Elterman model in the boundary area of Qinghai and
Xinjiang and some areas of the North China Plain is higher than
0.8. The true value of the experimental group in the southwest
and northern regions (Inner Mongolia, Heilongjiang, etc.) is less
than 0.1, which is lower than that of the extrapolation group. npj Climate and Atmospheric Science (2021) 49 Published in partnership with CECCR at King Abdulaziz University J. Wu et al. 4 Fig. 3
Evaluation of the mean value of inversion AOD. The spatial distribution of observed AOD, M-Elterman AOD, PSO-M-Elterman AOD (a)
and the monthly time series from January 2001 to October 2014 (b) when using MODIS data. The spatial distribution of observed AOD,
M-Elterman AOD, PSO-M-Elterman AOD (c) and the monthly time series from January 2001 to October 2014 (d) when using MISR data. Fig. 3
Evaluation of the mean value of inversion AOD. The spatial distribution of observed AOD, M-Elterman AOD, PSO-M-Elterman AOD (a)
and the monthly time series from January 2001 to October 2014 (b) when using MODIS data. The spatial distribution of observed AOD,
M-Elterman AOD, PSO-M-Elterman AOD (c) and the monthly time series from January 2001 to October 2014 (d) when using MISR data. For MISR data (Fig. 3c), the true value of the experimental group is
slightly higher than that of the extrapolation group in Southwest
Xinjiang (<0.05). The spatial distribution of AOD simulated by the
M-Elterman and the PSO-M-Elterman models is similar, except for
some areas in Northeast China, and the overall value is below 0.5. The retrieved AOD of the two models in northern China (Inner
Mongolia, Heilongjiang) and southwestern China (the junction of
Yunnan, Sichuan, and Tibet) is slightly higher than the true value. npj Climate and Atmospheric Science (2021) 49 Published in partnership with CECCR at King Abdulaziz University Spatiotemporal contrast of different inversion results 3b, d), true value
presents a one-year fluctuation period that first increases and
then decrease (MODIS AOD fluctuate between 0.2–0.7, MISR
AOD fluctuate between 0.15–0.5), which indicates that AOD is
higher in summer and lower in spring and winter. But the
fluctuation range of AOD inversed by the PSO-M-Elterman
model is about 0.2 larger than that of the M-Elterman model,
which are more consistent with the actual situation. The
performance of the experimental group is likewise slightly
better than that of the extrapolation group, although extreme
values are not well presented in the two groups. Published in partnership with CECCR at King Abdulaziz University J. Wu et al. 5 Fig. 4
Evaluation of the correlation of inversion AOD. The spatial distribution (a) and probability density plot (b) of correlation coefficients
between inversion AOD in M-Elterman/PSO-M-Elterman models and observed AOD when using MODIS data. The spatial distribution (c) and
probability density plot (d) of correlation coefficients between inversion AOD in M-Elterman/PSO-M-Elterman models and observed AOD
when using MISR data. Fig. 4
Evaluation of the correlation of inversion AOD. The spatial distribution (a) and probability density plot (b) of correlation coefficients
between inversion AOD in M-Elterman/PSO-M-Elterman models and observed AOD when using MODIS data. The spatial distribution (c) and
probability density plot (d) of correlation coefficients between inversion AOD in M-Elterman/PSO-M-Elterman models and observed AOD
when using MISR data. Quantitative evaluation of different inversion results algorithm simulates the average AOD state more effectively. Previous studies20,21 focused more on the spatial distribution and
correlation of the whole region average in the discussion of
inversion results. However, in a smaller area or even a single
station, it is difficult to depict the temporal fluctuation character-
istics
of
AOD. Nevertheless,
the
PSO-M-Elterman
algorithm
significantly improved the inversion results, especially the regions
with a small absolute error and high correlation, which indicates
that it is more applicable to the visibility-AOD inversion formula
than the NLSM. Several indices (Table 1) were used to evaluate the performances
of different retrieval models using different satellite data. The PSO-
M-Elterman model evidently outperforms the M-Elterman model,
irrespective of whether MODIS or MISR data are used as the true
value. For the data unavailability rate, the M-Elterman model
achieves more than 20% using MODIS data, and approximately
15% using MISR data. The PSO-M-Elterman model significantly
increases data availability by 8% and 6%, respectively. The mean
absolute error and the proportion of the smaller absolute error in
the PSO-M-Elterman model experimental group are 0.0314 and
91.23%, respectively, and the root means square error was only
0.0509, which significantly outperforms other groups. While in the
M-Elterman model, the absolute error of each group is generally
greater than 0.04 and the proportion of the smaller absolute error
is less than 85%. At the same time, the correlation coefficient of
each group is lower than 0.4, and there are only less than 40% of
sites with a correlation coefficient above 0.3. Unlike other
parameters, for which the experimental group is superior to the
extrapolation group, the correlation coefficients of the two models
showed better results in the latter. The correlation coefficient of
the PSO-M-Elterman model is the highest with MISR data, while
the proportion of the stronger correlation coefficient is slightly
larger with MODIS data. Published in partnership with CECCR at King Abdulaziz University npj Climate and Atmospheric Science (2021) 49 Long-term changes of retrieval AOD in China Combined with
the time coefficient of the first mode, the AOD in these areas
shows an increasing trend after 1960, and the fluctuations tend to
flatten after about 2005, which indicates that the anthropogenic
aerosol content increased rapidly with the rapid development of
the economy and population in eastern China33. Southern
Xinjiang, Qinghai, and other regions with significant negative
abnormal AOD showed a weak declining trend in the past 50
years. The second spatial distribution mode also shows the
alternation between positive and negative anomalies with small
values, with no significant anomaly center. Only the southwest
region of the Xinjiang positive anomaly is slightly stronger, with
the time coefficient first increasing and subsequently decreasing,
indicating that the AOD in Southwest Xinjiang was higher from
1984 to 2000, and lower in other years. Table 1. Comparison of evaluation parameters of inversion results at different satellite data/experimental groups. Evaluation
Parameters
MODIS
MISR
M-Elterman
PSO-M-Elterman
M-Elterman
PSO-M-Elterman
Experimental group Extrapolation group Experimental group Extrapolation group Experimental group Extrapolation group Experimental group Extrapolation grou
Data Unavailable Rate 22.44%
22.62%
8.09%
8.76%
14.76%
15.04%
6.04%
6.17%
Mean Absolute Error
0.0735
0.0985
0.0440
0.0881
0.0468
0.0634
0.0314
0.0532
Proportion of smaller
absolute error (<0.1)
stations
72.16%
60.67%
85.48%
63.99%
83.36%
75.34%
91.23%
83.21%
Root-Mean-
Square Error
0.1140
0.1294
0.0839
0.1147
0.0668
0.0881
0.0509
0.0713
Correlation
Coefficient
0.3224
0.4407
0.4369
0.4726
0.1806
0.3141
0.4824
0.5621
Proportion of
stronger correlation
coefficient (>0 3)
34.65%
33.43%
67.17%
56.08%
28.29%
30.56%
54.16%
47.05%
npj Climate and Atmospheric Science (2021) 49 We employed power spectrum analysis, wavelet analysis, and
EEMD (Ensemble Empirical Mode Decomposition) analysis to
explore the periodic characteristics of AOD in China in recent
decades; however, none passed the confidence test. This indicates
that the increasing trend of aerosol in China may be predomi-
nantly attributed to the local emission of polluting gases and
particulate matter, rather than the long-distance transport of
atmospheric circulation. Long-term changes of retrieval AOD in China MISR AOD data were used to represent the true value, and the
parameters simulated by the PSO-M-Elterman model from 2007 to
2014 were applied to the AOD retrieval before 2000. Thus, the
long-term AOD series from 1960 could be established by inversion
AOD. The spatial pattern and temporal series of inversion AOD
from 1960 to 2014, and the spatial and temporal fields of the first
two modes of EOF (Empirical Orthogonal Function) analysis are
shown in Fig. 5. The general characteristics of the AOD pattern are
similar to that after 2000: the high-value areas are distributed in
southern Xinjiang, the Sichuan Basin, and southeastern China,
while the low-value areas include northern Xinjiang, Southwest
China, and Northeast China. Compared with the spatial distribu-
tion after 2000 (Fig. 3c), the high-value region (AOD > 0.5)
narrowed in Southeast China, while the low-value region (AOD
< 0.2) broadened slightly in northern Xinjiang (Fig. 5a). g
Considering all evaluation indices, the PSO-M-Elterman model
evidently improves the inversion results relative to the M-Elterman
model with regard to all aspects, and the inversion results using
MISR data outperform those using MODIS data. Further, the
performance of the extrapolation group is slightly lower than that
of the experimental group. The above evaluation of inversion
results from different perspectives shows that the M-Elterman The AOD of the whole region average gradually increased in
time, which was associated with the annual fluctuation exhibiting
a growth rate of 0.0011 yr−1. The fluctuation range of annual AOD npj Climate and Atmospheric Science (2021) 49 Published in partnership with CECCR at King Abdulaziz University J. Wu et al. 6 variation gradually increased with time (Fig. 5b). The variance
contribution rates of the first two modes of EOF decomposition in
AOD anomalies are 47.33% and 12.21%, respectively, (both passed
the North test). The eigenvector of the first spatial mode alternates
between positive and negative, which represents two trending
characteristics of the AOD. In addition to the intersecting region of
Sichuan, Qinghai, Tibet, southern Xinjiang, and part of Inner
Mongolia, other regions are basically positive anomalies, especially
in North China, the middle and lower reaches of the Yangtze River,
northwestern Xinjiang, Guangxi, and Guangdong. Published in partnership with CECCR at King Abdulaziz University DISCUSSION NLSM employs the minimum sum of error squares as the criterion
to estimate the parameters of a static model. Because of the
nonlinearity of the M-Elterman model, the parameter estimation
cannot be obtained by calculating the extreme value of multi-
variate function according to the linear least square method. The
M-Elterman model adopts an iterative algorithm, that is, starting
from an initial value of parameters (i.e., the original parameters of
the Qiu model), and subsequently generating a series of iterative
parameter points. If this parameter sequence converges to a
parameter point that minimizes the objective function, it will be
regarded as the final solution. However, as the initial parameters
are fixed, the simulated parameters are usually only optimized
locally, but not applicable globally, which takes the partial
inversion AOD beyond the reasonable value range. During the
experiment, we found that parameters simulated in different
stations are highly sensitive to variations in the PW (pressure of
water vapor) value. This is because in the product term of the
M-Elterman model, PW exists in two terms of the natural power
(Eq. (15)), and the second term of the product term in the power is
likewise an exponential relation. This means that the values of
parameters b and d (especially d) determine the sensitivity of the
inversion result to the PW value. p
AOD in Sichuan Basin maintains high value all the year round
(>0.4) with nearly no monthly fluctuation (Fig. 6c, d) with a
maximum (83.02%) in October. There is a distinct rising phase
followed by a decreasing phase for both AOD and relative
humidity in North China, East China, South China, southwest
China. In the Taklimakan, however, AOD is lower in winter (<0.3)
and higher in spring and summer (around 0.4), which synchro-
nizes the onset of sandstorms in spring and gradual abating in
autumn, with relative humidity low in months with high AOD. Different from other regions, AOD in Northeast China reaches its
peak in April (0.304) and October (0.296) respectively, and shows a
fluctuation characteristic of ascending - descending - ascending -
descending throughout the year, but relative humidity shows
different variations with high in summer low in other seasons. The
monthly characteristic of AOD and relative humidity are almost
consistent in East China, North China, and Southwest China but
varies in other regions, and the reasons need to be further studied. Distribution characteristics of inversion AOD in different
regions According to the average distribution of inversion AOD and EOF
decomposition, China is divided into 9 regions (each region has
similar distribution and trend characteristics) to further discuss
the regional long-term variation characteristics of AOD. In
addition, many researches pointed that as the relative humidity
increases, the hygroscopic growth of aerosols will continue and
become faster34–37. Considering that water vapor is a major
factor affecting physicochemical and optical properties of
aerosols38–40, the variation characteristics of relative humidity
are also discussed. (Please refer to Supplementary Fig. 1 for
details of regional division). In Fig. 6a, the regional average AOD in the Sichuan Basin
reaches the highest (0.4436) in China, followed by central China
(0.404). AOD fluctuates around 0.3 in South China, East China,
and Taklimakan region. The lowest average AOD in northeast
China and Northwest China are 0.234 and 0.235, respectively. Weak upward trends present in all regions, in which South
China, East China, Central China, and the Taklimakan region
passed the significance t-test at 95% confidence level with the
linear trend coefficients of 2.67, 2.46, 2.13, and 1.45 (×10−3 yr−1)
respectively. These features generally confirm the conclusions of
previous research20. p
As shown in Fig. 6b, the relative humidity of all regions do not
show distinct rising or descending characteristics with a small
fluctuation range. Mean relative humidity are higher than 70% in
East China, Central China, South China, and Sichuan Basin, where
the AOD are also relatively high. But the Taklimakan has the
lowest relative humidity 47.19% and the relatively high AOD
0.3381. This may be due to the fact that aerosol composition
varies greatly in different regions. The hygroscopic properties of
atmospheric aerosol particles influence ambient particle size,
density, and mass which in turn control the lifetime and removal Published in partnership with CECCR at King Abdulaziz University J. Wu et al. 7 Fig. 5
Spatiotemporal characteristics of inversion AOD. a Spatial distribution of AOD during1960–2000. b Annual time series of AOD during
1960–2014 (The upper and lower bounds of the shadow are the highest and lowest AOD values of the year, respectively). c EOF analysis of
spatial field and (d) time coefficient series of AOD during 1960–2014. Fig. 5
Spatiotemporal characteristics of inversion AOD. a Spatial distribution of AOD during1960–2000. b Annual time series of AOD during
1960–2014 (The upper and lower bounds of the shadow are the highest and lowest AOD values of the year, respectively). Distribution characteristics of inversion AOD in different
regions c EOF analysis of
spatial field and (d) time coefficient series of AOD during 1960–2014. mechanisms of the particles, and then affect the value of AOD. The
higher AOD in Taklimakan is mainly caused by dust aerosols,
which are hydrophobic. While the eastern region has more sulfate
or nitrate aerosols caused by human emissions, which are
hydrophilic and more affected by relative humidity. Combined with the analysis of AOD in cities with different
populations in the Supplementary Discussion, long-term change
of AOD in China is mainly caused by human activity, and the AOD
is higher in cities with a larger population and more human
activity. Based on the analysis above, we think that the long-term
increasing trend of AOD is not mainly caused by long-term
changes of relative humidity, but does not deny the influence of
relative humidity on aerosols, which may occur more in local
Spatio-temporal scales and varies in different regions. The long-term variation characteristics of relative humidity and
AOD in different types of large cities in east China are in
Supplementary Fig. 3 for further analysis (Almost all large cities are
located in the east of China, where anthropogenic emissions are
higher and have more hydrophilic aerosols). The classification of
city types is based on Supplementary Fig. 2a. The relative humidity
in the four types of large cities all showed a weak decreasing
trend. Although the downward trend is not obvious, there are
strong negative correlation between relative humidity and AOD
with a value of −0.6168, −0.6521, −0.6872 in ILarge City, IILarge
City, and Extra Large City. This means that a decrease in relative
humidity may be more conducive to aerosol accumulation. The
negative correlation between AOD and relative humidity is not
obvious in Super Large City (only 7 cities), which may be due to
the scattered distribution of cities and different regional environ-
mental impacts. Published in partnership with CECCR at King Abdulaziz University npj Climate and Atmospheric Science (2021) 49 DISCUSSION We randomly selected four different stations and used MISR
data as the true value, while keeping the altitude and actual
visibility unchanged to test the sensitivity of retrieval AOD to PW
value between 0 and 20 hPa (Fig. 7a). The inversion AOD in
stations “50968” and “50114” is within 0.4 and increases with the Published in partnership with CECCR at King Abdulaziz University npj Climate and Atmospheric Science (2021) 49 J. Wu et al. Fig. 6
Annual and monthly variations of AOD and relative humidity in different regions. a Annual series of AOD from 1960 to 2014 (The
regions in the red boxes represent trend significance t-test at 95% confidence level). b Annual series of relative humidity from 1960 to 2014. c Monthly distribution of AOD. d Monthly distribution of relative humidity. J. Wu et al. 8 Fig. 6
Annual and monthly variations of AOD and relative humidity in different regions. a Annual series of AOD from 1960 to 2014 (The
regions in the red boxes represent trend significance t-test at 95% confidence level). b Annual series of relative humidity from 1960 to 2014. c Monthly distribution of AOD. d Monthly distribution of relative humidity. rise of PW, which is in line with the actual situation. However, the
inversion AOD in stations “51330” and “54838” appears as an
extremely large value within a normal PW range. When the PW is
larger than a certain critical value, the AOD conforms to the actual
situation (AOD < 1). This is because the parameters simulated by
NLSM for these two stations are not applicable to the global
situation, and similar situations exist in other stations and the
MODIS data. iterative experiment of the PSO algorithm, the optimal solution
can be selected from different experimental results, increasing the
number and credibility of the available station. Taking the simulation results using MODIS data as an example,
we plot the spatial distribution of evaluation parameters KGE and
R for the four PSO repeated experiments (Fig. 8). The results of
the four experiments show that the better and more stably
performing stations are mainly distributed in the North China
Plain,
Southwest
China
(including
Tibet,
western
Sichuan,
Yunnan, Guangxi, and Guangdong), and southern Xinjiang. However, there are still differences between several stations,
and the inversion results of these stations in different batches of
experiments are not in the same evaluation range. npj Climate and Atmospheric Science (2021) 49 Published in partnership with CECCR at King Abdulaziz University DISCUSSION With MISR
data,
the
repeated
PSO
experiment
results
show
similar
distribution characteristics (abridged). The spatial distribution of the unavailability rate of inversion
AOD data using the NLSM to solve the parameters (Fig. 7b) shows
there is a severe lack of AOD data in Inner Mongolia, Northeast
China, and North China, and the unavailability rate in some areas
reaches more than 50%. When using MODIS data, the unavailable
range is larger, which extends south to Hubei, Hunan, and
Guangxi. According to the monthly distribution (Fig. 7c), the
parameters fitted by NLSM are not applicable in spring and winter,
possibly because the PW in these two seasons is relatively low. Further, both the experimental and the extrapolation groups show
that the unavailability rate when using MODIS data is larger than
when using MISR data. According
to
the
relatively
stable,
but
slightly
different
distribution characteristics of the inversion results, we take KGE
and R as the thresholds and select available stations from different
experimental batches based on the results of the first experiment
(i.e., the stations that meet the criteria are further selected from
the stations not selected in the last experiment). The selection of
stations in each experiment is shown in Table 2. With the increase
in the number of experiments, the available stations in each
evaluation range increased but the number of increased stations is
less and less. Taking KGE & R > 0.3 as an example, in the first
experiment, 413 stations could be selected when using MODIS The application of the PSO algorithm in the M-Elterman mode
has the advantage of global convergence; however, the initial
value of each iteration is random, which leads to different
experimental results. This indicates that the optimal parameter
solution of different experimental simulations for the same station
is not the only solution. In theory, if each station repeats the Published in partnership with CECCR at King Abdulaziz University J. Wu et al. 9 Limitations of NLSM. a Variation of AOD with PW in different stations when using MISR data. b Spatial distribution and (c) Monthly
bution of the unavailable rate of inversion AOD. Fig. 7
Limitations of NLSM. a Variation of AOD with PW in different stations when using MISR data. b Spatial distribution and (c) Monthly
distribution of the unavailable rate of inversion AOD. DISCUSSION data, while in the fourth experiment, 467 sites could be selected,
which amounts to an increase of 54 in total. A total of 308 stations
are selected after four experiments using MISR data, an increase of
56 in total. Table 2 shows that for a specific station, although the
parameters fitted by different PSO algorithms are different each
time, the difference between the simulated results and the real
value is still relatively stable. Nevertheless, repeated experiments
can still help increase the number of credible stations with more
reliable simulation results. The final stations selected for the PSO
algorithm in Fig. 2 are based on the scheme above discussed in
this section. data, while in the fourth experiment, 467 sites could be selected,
which amounts to an increase of 54 in total. A total of 308 stations
are selected after four experiments using MISR data, an increase of
56 in total. Table 2 shows that for a specific station, although the
parameters fitted by different PSO algorithms are different each
time, the difference between the simulated results and the real
value is still relatively stable. Nevertheless, repeated experiments
can still help increase the number of credible stations with more
reliable simulation results. The final stations selected for the PSO
algorithm in Fig. 2 are based on the scheme above discussed in
this section. in China is found to mainly occur in areas with a large population,
which proves that the increase in AOD is caused by human activity
rather than natural causes. The PSO-M-Elterman model can be
generally applied to global AOD retrieval using monthly or daily
visibility data to reconstruct long-term AOD data. npj Climate and Atmospheric Science (2021) 49 Published in partnership with CECCR at King Abdulaziz University Grade-distance conversion method of visibility Visibility was recorded on a scale of ten grads before 1980: below 0.05;
0.05–0.2; 0.2–0.5; 0.5–1; 1–2; 2–4; 4–10; 10–20; 20–50 and above 50 (unit:
km), and the actual visibility distance (unit: km) was directly recorded after
1980. Theoretically, each visibility level can correspond to any value within
the corresponding distance interval, such as the median value of the
interval. Since the interval of visibility distance corresponding to each
visibility grade is not equally spaced, the use of median value instead of
each visibility grade may result in unhomogeneity of data. Qin et al. developed a grade-distance conversion method: This study improves the processing method of inconsistent
recording in 661 meteorological stations in China, which improves
the uniformity and reliability of visibility data collected prior to
1980 and subsequently employs them for AOD retrieval. The PSO
algorithm is introduced into the AOD retrieval and applied in
combination with the NLSM as the PSO-M-Elterman model. MODIS
and MISR satellite data are employed as true values to retrieve
AOD using visibility data. Both the scheme of visibility grade
conversion and the PSO-M-Elterman model refine the evaluation
results to the scale of a single station, and achieve good
experimental results. Furthermore, the long-term change of AOD Firstly, the actual observed visibility distance of all stations after 1980 is
converted into the corresponding grade according to the 10 intervals
above mentioned. (The time range is from 1980 to 2005 in Qin et al.’s
research). npj Climate and Atmospheric Science (2021) 49 Published in partnership with CECCR at King Abdulaziz University Fig. 8
Repeat experiment of PSO algorithm. Distribution of KGE and R in four repeated experiments of PSO algorithm. J. Wu et al. 0 J. Wu et al. 10 Fig. 8
Repeat experiment of PSO algorithm. Distribution of KGE and R in four repeated experiments of PSO algorithm. Fig. 8
Repeat experiment of PSO algorithm. Distribution of KGE and R in four repeated exper Assuming aerosols obey a Junge distribution: Table 2. The comparison of the station numbers that can be selected
for each time in PSO repeated experiment at different KGE and R value
intervals. Grade-distance conversion method of visibility Number of stations
KGE &
R > 0.7
KGE &
R > 0.5
KGE &
R > 0.3
KGE &
R > 0
MODIS 1st experiment
77
221
413
587
2nd experiment
18
25
35
30
3rd experiment
5
7
14
8
4th experiment
1
2
5
8
Total
101
255
467
633
MISR
1st experiment
4
55
252
520
2nd experiment
0
20
35
32
3rd experiment
0
8
17
18
4th experiment
0
0
4
4
Total
4
83
308
574 Table 2. The comparison of the station numbers that can be selected
for each time in PSO repeated experiment at different KGE and R value
intervals. n rð Þ ¼ cr vþ1
ð
Þ n rð Þ ¼ cr vþ1
ð
Þ
(6) (6) where c is a constant and r is the particle radius, and v* = 3 does not
change with height. where c is a constant and r is the particle radius, and v* = 3 does not
change with height. g
g
Under the standard surface air temperature (15 °C) and pressure
(1013 hPa) conditions, the air molecular extinction coefficient is a constant
0.0116, then the aerosol extinction coefficient σλ and the atmospheric
molecules σm at the height of z at the wavelength of λ can be expressed as: σλ ¼ NA z
ð Þ
NA 0
ð Þ
3:912
V
0:0116
0:55
λ
(7) (7) σm ¼ 0:0116 0:00099z σm ¼ 0:0116 0:00099z
(8) σm ¼ 0:0116 0:00099z
(8) (8) Where NA(0) and NA(z) are the concentrations of aerosol particles at the
surface and the altitude of z respectively, and the distribution of NA(z) with
altitude proposed by McClatchey42 can be expressed as: NA z
ð Þ ¼
55 exp z5:5
ð
Þ
H1
h
i
ðz ⩽5:5 kmÞ
55ð5:5 km ⩽z ⩽18 kmÞ
55 exp z18
ð
Þ
H2
h
i
ðz > 18 kmÞ
8
>
>
>
<
>
>
>
:
(9) (9) Then the relationship between the visibility Vz actually observed at the
altitude of z and the visibility V revised to sea level can be represented as: Then, calculate the average value of distance at each visibility grades
according to the corresponding relationship in the first step. Grade-distance conversion method of visibility Vz ¼
3:912
0:0116 0:00099z þ
3:912
V
0:0116
e
z
ð0:886þ0:222VÞ
(10) (10) Finally, the average distance of each grade is substituted as an
estimated value for that grade before 1980. Thus, the AOD τλ can be obtained by integrating the extinction
coefficient in the vertical direction: It is worth noting that all stations share the same conversion scheme in
Qin’s method. However, China has a diverse geographical environment
and observation conditions are complex at different stations. In addition,
visibility also shows seasonal differences. The same transformation scheme
obviously does not represent the individual differences of each station. Therefore, we improved it on the basis of Qin’s method by respectively
calculating the average value of visibility distance for 12 months at each
station after 1980 (The actual time range involved in calculating the
average distance of each visibility grade are only from 1980 to1985 to
eliminate the long-time trending signal of visibility). That is, each station
has its own independent grade-distance conversion scheme for each
month, and there are 661(stations) × 12(months) conversion schemes that
are used for visibility grade data before 1980 in total. τλ ¼
3:912
V
0:0116
0:55
λ
2v
H1 e z
H1 e5:5
H1
þ 12:5e z
H1 þ H2e z
H1
h
i
(11) (11) where H1 ¼ 0:886 þ 0:0222V km
ð
Þ. ð
Þ
Formula (11) is the classical Elterman model of visibility inversion AOD. But it is sensitive to the geographical distribution of China, especially in
coastal areas. Qiu24 developed a parameterized model suitable for Chinese
characteristics by comparing it with the optical depth detected through
direct solar radiation data: τλ ¼
3:912
V
0:0116
0:55
λ
2v
H1 e z
H1 e5:5
H1
þ 12:5e z
H1 þ H2e z
H1
h
i
f (12) npj Climate and Atmospheric Science (2021) 49 Published in partnership with CECCR at King Abdulaziz University CODE AVAILABILITY The computer code of Particle Swarm Optimization algorithm in this study are
available from the corresponding author upon reasonable request. The computer code of Particle Swarm Optimization algorithm in this study are
available from the corresponding author upon reasonable request. DATA AVAILABILITY Meteorology data over 661 Chinese meteorological sites used for the present study
was from Daily data set of Surface climatic data in China V3.0 and provided by the
China
Meteorological
Administration(http://data.cma.cn/data/cdcdetail/dataCode/
SURF_CLI_CHN_MUL_DAY_V3.0/). AOD data for 2001–2014 were from NASA
(National Aeronautics and Space Administration) Goddard Earth Sciences Data and
Information Services Center: Multiangle Imaging Spectro Radiometer (MISR) daily
Level 3 Component Global Aerosol Product (https://asdc.larc.nasa.gov/data/MISR/
MIL3DAE.004/) and Moderate Resolution Imaging Spectroradiometer (MODIS) Level-3
aerosol products of the Terra satellite (https://nrt3.modaps.eosdis.nasa.gov/allData/
61/MODAODHD/). The datasets generated during and/or analysed during the current
study are available from the corresponding author on reasonable request. (15) AODi represents the inversion AOD at the ith station; a, b, c, d, e, f, and g
are the parameters corresponding to each station. AODi represents the inversion AOD at the ith station; a, b, c, d, e, f, and g
are the parameters corresponding to each station. p
p
g
The NLSM is employed to calculate each parameter and define DIF: DIF ¼
X
Yearend
Yearstart
AOD AODi
ð
Þ2¼
X
Yearend
Yearstart
AOD F V; a; b; c; d; e; f; g
ð
Þ
ð
Þ2
(16) (16) An independent set of parameters a, b, c, d, e, f, and g for each station
can be obtained through iterative nonlinear equations. Then, the
corresponding parameters can be applied to the year with the missing
satellite data, and the AOD can be retrieved using visibility. AODi ¼ F V; a; b; c; d; e; f; g
ð
Þ AODi ¼ F V; a; b; c; d; e; f; g
ð
Þ F in the formula is: F in the formula is: F ¼
3:912
V
0:0116
0:55
γ
2v
e V þ f
ð
Þ exp
z
eVþf
exp
5:5
eVþf
h
þ12:5 exp
5:5
eVþf
þ g exp
5:5
eVþf
i
exp
a þ b pw þ c Vz
ð
Þ exp
dV2
Pw
(1 Published in partnership with CECCR at King Abdulaziz University REFERENCES 1. Boucher, O. et al. Clouds and Aerosols, in Climate Change 2013. Vol. 7 (Cambridge
University Press, 2013). where Sk and Ok represent the simulated value and observed value
respectively, and O represents the observed average value. NSE is used to
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<
:
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pressure Pw, altitude z, wavelength, and Junge spectral parameter v*. The revised coefficient f depends on the distribution of aerosol particle
number density with height. f < 1 means that the attenuation of aerosol V ¼ 3:912
σ0:55 (5) npj Climate and Atmospheric Science (2021) 49 Published in partnership with CECCR at King Abdulaziz University Published in partnership with CECCR at King Abdulaziz University be respectively expressed as:
pi ¼ ðpi1; pi2; ¼ ; pinÞ
(23)
Gn ¼ ðpg1; pg2; ¼ ; pgnÞ
(24)
g ¼ min
1⩽i⩽n½FðpiÞ
(25)
where g is the lowest value of the target function and F is the target
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function needs to be defined. Nash-Sutcliffe Efficiency (NSE) is usually used
as the objective function: NSE ¼ 1
P
k ðSk OkÞ2
P
k ðOk OÞ2
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P
k ðSk OkÞ2
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in
¼ wvN
in þ c1r1ðpN
in xN
inÞ þ c2r2ðGN
n xN
inÞ
(21) vNþ1
in
¼ wvN
in þ c1r1ðpN
in xN
inÞ þ c2r2ðGN
n xN
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wvin þ c1r1ðpin
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(21)
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(23)
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(24)
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function. AODi ¼ F V; a; b; c; d; e; f; g
ð
Þ
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6373–6397 (2008). npj Climate and Atmospheric Science (2021) 49 Published in partnership with CECCR at King Abdulaziz University
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Piglet innate immune response to Streptococcus suis colonization is modulated by the virulence of the strain
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© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Streptococcus suis is a zoonotic pathogen of swine involved in arthritis, polyserositis, and meningitis. Colonization of
piglets by S. suis is very common and occurs early in life. The clinical outcome of infection is influenced by the viru‑
lence of the S. suis strains and the immunity of the animals. Here, the role of innate immunity was studied in cesarean-
derived colostrum-deprived piglets inoculated intranasally with either virulent S. suis strain 10 (S10) or non-virulent S. suis strain T15. Colonization of the inoculated piglets was confirmed at the end of the study by PCR and immunohis‑
tochemistry. Fever (≥40.5 °C) was more prevalent in piglets inoculated with S10 compared to T15 at 4 h after inocula‑
tion. During the 3 days of monitoring, no other major clinical signs were detected. Accordingly, only small changes
in transcription of genes associated with the antibacterial innate immune response were observed at systemic sites,
with S10 inducing an earlier response than T15 in blood. Local inflammatory response to the inoculation, evaluated
by transcriptional analysis of selected genes in nasal swabs, was more sustained in piglets inoculated with the virulent
S10, as demonstrated by transcription of inflammation-related genes, such as IL1B, IL1A, and IRF7. In contrast, most of
the gene expression changes in trachea, lungs, and associated lymph nodes were observed in response to the non-
virulent T15 strain. Thus, S. suis colonization in the absence of systemic infection induces an innate immune response
in piglets that appears to be related to the virulence potential of the colonizing strain. Keywords: Streptococcus suis, colonization, innate immunity, pig immunity, gene expression, bacterial virulence S. suis is a zoonotic agent that is receiving increased sci-
entific interest due to Chinese outbreaks in humans in
1998 and 2005 [2]. Piglet innate immune response
to Streptococcus suis colonization is modulated
by the virulence of the strain Carlos Neila‑Ibáñez1,2† , Louise Brogaard3,8† , Lola Pailler‑García1,2 , Jorge Martínez2,4,5 ,
Joaquim Segalés2,4,5 , Mariela Segura6 , Peter M. H. Heegaard7 and Virginia Aragon1,2*† Carlos Neila‑Ibáñez1,2† , Louise Brogaard3,8† , Lola Pailler‑García1,2 , Jorge Martínez2,4,5 ,
Joaquim Segalés2,4,5 , Mariela Segura6 , Peter M. H. Heegaard7 and Virginia Aragon1,2*† Neila‑Ibáñez et al. Veterinary Research (2021) 52:145
https://doi.org/10.1186/s13567-021-01013-w Neila‑Ibáñez et al. Veterinary Research (2021) 52:145
https://doi.org/10.1186/s13567-021-01013-w Open Access Introduction Streptococcus suis is a major bacterial pathogen of swine,
involved in meningitis, arthritis, septicemia, and acute
death, among other clinical syndromes. Disease caused
by S. suis is more prevalent in nursery pigs, but sucklers
and young fatteners can also be affected [1]. Additionally, Streptococcus suis is an early colonizer of the swine
upper respiratory tract, mainly found in tonsil and nasal
cavity [3]. Newborn piglets experience the first contact
with the bacterium in the birth canal during parturition,
as S. suis colonizes the sow’s vaginal tract [4]. In addition,
animals housed in the same pen are exposed to horizon-
tal colonization by direct contact or aerosol, especially
during outbreaks when animals may shed bacteria in
high numbers [5]. 1 IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus
de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
Full list of author information is available at the end of the article Streptococcus suis strains are heterogeneous both
with respect to antigenicity and virulence [6]. Presently, Materials and methods
Animal study Animal experimentation was performed in the BSL3
facilities of IRTA-CReSA (Bellaterra, Spain) follow-
ing good veterinary practices, in accordance with
European (Directive 2010/63/EU) and Spanish (Real
Decreto 53/2013) regulation. The experimental study
was approved by the Ethics Commission in Animal
Experimentation of the Generalitat de Catalunya (Pro-
tocol number 10201). Four pregnant sows were trans-
ported to IRTA facilities and housed for two weeks
before delivery, which was performed by cesarean
section. The genetic background of these piglets was
(Duroc × White Large) ♀ × Landrace ♂, a commer-
cial breed. Piglets were fed the milk substitutive Pata-
vie Porc (Oriane-Celtilait) ad libitum during the first
2 days. Afterwards, animals received Neopigg (Provimi
Cargill) mixed with the milk or dry after 10–15 days of
age. Piglets were treated with colistin (Colimicina SP,
SP Veterinaria S.A., Spain) and enrofloxacin (Baytril
0.5%, Bayer Hispania S.L., Spain), both orally, during
the first nine days of life. Twenty piglets, housed in the
same box, were included in the study. At 25 days of age
(3 days before inoculation), blood samples and nasal
swabs were taken from all piglets. Piglets were ran-
domly assigned to 5 groups of 4 piglets each for inocu-
lation and euthanasia, and housed in 3 separated boxes,
depending on the inoculum assigned to them. One
group was inoculated with strain T15 and euthanized
1 day post-inoculation (dpi), while a second group that
was also inoculated with strain T15 was euthanized at
3 dpi. Similarly, two groups were inoculated with strain
S10 and were euthanized at 1 and 3 dpi, respectively. A fifth group was inoculated with PBS (Phosphate
Buffered Saline) and euthanized at 1 dpi, as negative
control. On day 28 of life, inoculation was performed
intranasally with a nasal atomizer (MAD Nasal™, Only few in vivo S. suis challenge studies in pigs have
focused on characterizing the host immune responses. Genes related to bacterial recognition (TLR4, MYD88)
and inflammatory responses (IL6, CXCL8, CCL2) have
been shown to be expressed in lungs of pigs after intra-
nasal S. suis challenge (serotype 2, strain 05ZY), and
these responses were enhanced by co-infection with
influenza A virus (H1N1) [21]. Intravenous challenge
of pigs with S. suis serotype 2 (strain SC19) induced
expression of bacterial pattern recognition receptors
(TLR2, CD14) in the lung, as well as components of the
inflammatory response (IL1B, IL6, TNF, CXCL8) [22]. © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Neila‑Ibáñez et al. Veterinary Research (2021) 52:145 Page 2 of 15 that inflammatory responses can be induced in vivo by
S. suis serotype 2 challenge. 29 confirmed serotypes have been described based on
the antigenicity of capsular polysaccharides [7, 8]. Pigs
are usually colonized by more than one serotype, but
only a few strains can induce disease. Serotype 2 is the
most frequently isolated serotype from affected organs
in diseased individuals (swine and humans) in most
parts of the world [9]. However, different virulence
results have been reported for the same S. suis serotype,
or even the same S. suis strain [10, 11].f The present study, performed in cesarean-derived
colostrum-deprived (CDCD) piglets, describes the host
early immune response in blood, nasal mucosa, and vari-
ous tissues to intranasal inoculation with S. suis T15 and
S10. These strains belong to serotype 2 and have shown
different virulence in pigs, based on the frequency of clin-
ical signs, leukocytosis, and mortality reported in previ-
ous animal experiments [24]. Inoculation of the strains
was performed without pre-treatment of the mucosa in
order to examine the natural response of the host when
encountering S. suis strains of different virulence. How
this early response might affect disease development is
also discussed. Different animal models have been used to study S. suis pathogenesis, including pigs, mice, rabbits, and
zebrafish [12]. Clinical disease has been reproduced
by respiratory, intraperitoneal, and intravenous routes
in pigs, but reproduction of the disease is difficult [12]. After colonization, the development of disease depends
on the virulence potential of the strain and the inter-
play between the host response and the bacteria [13]. Severe disease is caused by excessive inflammation [14],
and in vitro studies have demonstrated strong induc-
tion of pro-inflammatory cytokines and chemokines
by S. suis serotype 2 [15–18]. A major obstacle to the
investigation of S. suis disease in vivo is the fact that
systemic disease is not easily induced by challenge via
the natural oronasal route of infection. In fact, systemic
disease by intranasal challenge is not induced or is
strongly reduced in the absence of acetic acid pretreat-
ment or prior viral infection [19, 20]. Thus, to repro-
duce systemic disease, it seems necessary to inoculate
S. suis either by injection (e.g. intravenous or intraperi-
toneal) or intranasally after irritation of the mucosa by
pre-treatment. Materials and methods
Animal study Nasal challenge (after acetic acid pre-treatment) with
S. suis serotype 2 (strain 05ZY) induced a primarily
TLR2-dependent cytokine response in the spleen [13]. The hepatic response has been investigated in vivo,
showing that clinical and subclinical disease after S. suis
serotype 2 (strain SS02-0119) challenge by subcutane-
ous inoculation was accompanied by an acute phase
response consisting of the acute phase proteins (APPs)
serum amyloid A, C-reactive protein, haptoglobin, pig-
MAP and Apo A-I [23]. These studies have thus shown Neila‑Ibáñez et al. Veterinary Research (2021) 52:145 Page 3 of 15 Neila‑Ibáñez et al. Veterinary Research (2021) 52:145 Neila‑Ibáñez et al. Veterinary Research (2021) 52:145 Teleflex, Athlone, Ireland) with 2 mL of 1.1 × 109 CFU/
mL of S. suis T15 (non-virulent serotype 2 strain) or
with 2 mL of 1.8 × 109 CFU/mL of S. suis S10 (virulent
serotype 2 strain), while the control group was inocu-
lated with 2 mL of PBS. For the three groups, the inocu-
lated volume was split between the two nostrils. Strains
were provided by Dr Astrid de Greeff and Dr Norbert
Stockhofe from Wageningen Bioveterinary Research
(Wageningen University & Research, the Netherlands). After inoculation, piglets were supervised for clinical
signs, including rectal temperature. RNA extraction and quality control
Extraction of total RNA from lymph nodes, lungs,
trachea, spleen, and liver was performed using the
miRNeasy Mini Kit (Qiagen) according to the manu-
facturer’s instructions. Briefly, approximately 30 mg of
RNAlater stabilized tissue was homogenized in 1 mL
QIAzol Lysis Reagent (in kit) using M-tubes (Miltenyi
Biotec) and a gentleMACS Dissociator (Miltenyi Bio-
tec). Total RNA was isolated from the homogenate by
column-based extraction, including on-column DNase
digestion of contaminating genomic DNA using the
RNase-Free DNase Set (Qiagen) according to the man-
ufacturer’s instructions. Total RNA was eluted in 50 µL
RNase-free water and stored at −80 °C. To study the innate immune response to the inoculated
strains, early time points were chosen for sampling. Nasal
swabs and blood were collected 4 h after inoculation, 1,
2 and 3 dpi. After euthanasia, piglets were examined by
necropsy and lesion scores were calculated as a combina-
tion of the severity of the lesions and the number of body
sites affected. In addition, samples from tissues (trachea,
cranial and caudal lobes of the lung, submandibular and
tracheobronchial lymph nodes, spleen, and liver) were
collected. Materials and methods
Animal study To ensure RNA integrity, blood samples were
obtained in PAXgene Blood RNA tubes (Becton Dickin-
son, Spain) which were kept at room temperature 4 h and
subsequently stored at 4 °C for 72 h and ultimately trans-
ferred to −20 °C. Nasal swabs and tissues were imme-
diately submerged in RNAlater (Invitrogen, Spain) and
stored at 4 °C overnight to allow thorough penetration of
the stabilizing solution into the tissue and subsequently
stored at −20 °C until RNA extraction was performed. Extraction of total RNA from whole blood collected
in PAXgene Blood RNA Tubes was performed using
the PAXgene Blood miRNA Kit (Qiagen) according to
the manufacturer’s instructions, including on-column
DNase digestion as above. RNA was eluted in 40 µL
BR5 buffer (in kit) and stored at −80 °C. f
Total RNA from RNAlater stabilized nasal swabs was
extracted using an in-house optimized protocol. First,
the RNAlater containing the swab was mixed with one
volume (1 mL) RNA Lysis Buffer from the Quick-RNA
Microprep Kit (Zymo Research) and vortexed fol-
lowed by 5 min incubation at room temperature. Then
the swab was removed, and the sample transferred
to a 15 mL tube and mixed with 2.5 volumes (5 mL)
cold (< 0 °C) 100% ethanol, followed by vortexing and
30 min incubation at −20 °C. The supernatant was
carefully removed with a pipette and the precipitate
was washed twice with 70% ethanol at room tempera-
ture. The precipitate was dissolved in 1 mL RNase-
free water and 700 µL was transferred to a Zymo-Spin
IC Column (from the Quick-RNA Microprep Kit) and
centrifuged at 10000 × g for 30 s and flow-through was
discarded. This was repeated until the entire sampled
had been passed through the column. From this point
the Quick-RNA Microprep Kit protocol for RNA puri-
fication was followed according to the manufacturer’s
instructions, including on-column DNase digestion of
contaminating genomic DNA. Total RNA was eluted in
15 µL RNase-free water and stored at −80 °C. Transcriptional analysis in low-EDTA TE buffer). The individual assay mixes
consisted of Assay Loading Reagent (Fluidigm) and
primer pairs (20 µM for each primer). After loading all
samples and reagents onto the chips using appropriate
controllers (RX IFC Controller [Fluidigm] for 192.24
Dynamic Array IFC chips and HX IFC Controller [Flui-
digm] for 96.96 Dynamic Array IFC chips), chips were
transferred to the BioMark instrument for 35 cycles
of amplification followed by melting curve analysis to
ensure specific amplification. Two replicates of cDNA were synthesized from each
RNA sample using the QuantiTect Reverse Transcrip-
tion Kit (Qiagen) according to the manufacturer’s
instructions employing 500 ng RNA for each synthe-
sis for all tissue and blood RNA samples. For the nasal
swabs, due to limited amounts of sample and low RNA
yields, cDNA synthesis was performed using as much
RNA as was possible for the individual samples, varying
from 33 to 323 ng per cDNA synthesis. Two no-reverse
transcriptase controls (reaction not containing reverse
transcriptase, negative controls) were made for each
tissue/sample type. ii
Amplification curves, melting curves, and standard
curves (dilution series) were evaluated using the Flui-
digm Real-Time PCR Analysis software (v. 4.1.3). The
GenEx software (v. 6) was used to correct Cq values with
the obtained qPCR efficiencies, to evaluate potential ref-
erence genes for data normalization with the geNorm
[27] and NormFinder [28] algorithms and subsequently
perform normalization, to average technical repeats, and
to convert Cq values to linear scale by computing rela-
tive quantities. Different subsets of reference genes were
found appropriate for data normalization in different tis-
sues based on the abovementioned reference gene evalu-
ation: whole blood: YWHAZ, RPL13A, HPRT1; nasal
swabs: B2M, RPL13A, PPIA; trachea: RPL13A, GAPDH,
HPRT1; lung: GAPDH, HPRT1, B2M, RPL13A; subman-
dibular lymph node: PPIA, YWHAZ, RPL13A; tracheo-
bronchial lymph node: B2M, HPRT1, PPIA, YWHAZ,
RPL13A, ACTB; liver: PPIA, YWHAZ, RPL13A; spleen:
HPRT1, PPIA, YWHAZ. All cDNA samples (diluted 1:10 in low-EDTA TE
buffer) were pre-amplified using the TaqMan PreAmp
Master Mix Kit (Applied Biosystems) in combination
with a primer mix (each primer at 200 nM) containing all
primer pairs to be used in the subsequent qPCR analysis
(see below for details on primer design). All cDNA sam-
ples from tissues and whole blood were pre-amplified
using 18 cycles of amplification, while cDNA samples
from nasal swabs were pre-amplified using 22 cycles of
pre-amplification. Detection of S. suis serotype 2 by PCR
and immunohistochemistry (IHC) Additional nasal swabs were taken at necropsy for detec-
tion of S. suis by PCR. Swabs were resuspended in PBS
and DNA was extracted using the Nucleospin Blood kit
(Macherey–Nagel, Germany). Four µL of DNA (between
42.0 and 867.2 ng) were used in the PCR to detect the
serotype of the challenge strains, serotype 2, as previ-
ously described [25]. For IHC, tissue samples from respiratory tract, includ-
ing nasal turbinates, cribriform plate of ethmoid, trachea,
and caudal lung lobe, as well as submandibular and tra-
cheobronchial lymph nodes, were fixed by immersion in
10% buffered formalin and embedded in paraffin. Bacte-
rial antigen detection in tissues was performed by IHC
using a rabbit monoclonal anti-S. suis serotype 2 antibody
(SSI Diagnostica, DK), followed by BrightVision Alkaline
Phosphatase (AP)-conjugated anti-rabbit immunoglobu-
lin G (IgG; Immunologic) and Vector Red (Vector Labs). Slides were counter stained with hematoxylin [26]. Addi-
tionally, another consecutive slide from each tissue was
stained with hematoxylin–eosin to study the lesions. RNA concentration (ng/µL) and purity (A260/A280 and
A260/A230 ratios) were assessed using a NanoDrop 1000
spectrophotometer (Thermo Scientific). RNA integrity
number (RIN) was measured using an Agilent 2100
Bioanalyzer (Agilent Technologies) and the RNA 6000
Nano Kit (Agilent Technologies) (Additional file 1). Neila‑Ibáñez et al. Veterinary Research (2021) 52:145 Page 4 of 15 Transcriptional analysis Following pre-amplification, residual
primers were digested using Exonuclease I (New England
BioLabs). Pre-amplified, exonuclease treated cDNA was
diluted 1:10 in low-EDTA TE buffer for use in qPCR, and
pools of pre-amplified, exonuclease treated cDNA were
prepared from each of the tissue/sample types to pro-
duce dilution series in order to experimentally determine
qPCR efficiency of all assays (primer pairs) for all inves-
tigated tissue/sample types. In addition, a non-template
control was prepared to check for background fluores-
cence build-up of all primer pairs in the absence of cDNA
template. Clinical signs and lesions after inoculation Few clinical signs were observed, comprising mild
tremors at 2 and 3 dpi in two piglets inoculated with
S10. Differences among the groups were observed
in rectal temperature after the inoculation of the two
strains. Although both groups of S. suis inoculated
piglets had higher temperature than the control group
(PBS inoculated) at 4 h after the challenge, this dif-
ference was statistically significant only in the piglets
inoculated with S10 (ANOVA Tukey’s HSD, P = 0.044;
Figure 1). Furthermore, the number of piglets with rec-
tal temperature higher than 40.5 °C at 4 h post-inocu-
lation was greater in the group inoculated with S10 (5
out of 8) than in the T15 group (1 out of 8); however,
no statistical difference was found between the two
S. suis-inoculated groups. Temperatures at later time
points were also recorded, and although S10 gave rise
to higher temperature than T15 at 2 dpi, no statistical
differences were found (Figure 1).i f
Gross lesions identified at necropsy were in general
mild, affecting animals in all groups, including the
PBS challenged group. None of these lesions could be
ascribed to the S. suis challenge as S. suis was not re-
isolated or detected by PCR from any of the lesions. f
Gross lesions identified at necropsy were in general
mild, affecting animals in all groups, including the
PBS challenged group. None of these lesions could be
ascribed to the S. suis challenge as S. suis was not re-
isolated or detected by PCR from any of the lesions. In the histological evaluation, no apparent lesions
were found in most of the tissues (98/120) and were not
consistent with characteristic S. suis pathology, with no
differences among the three groups. In the histological evaluation, no apparent lesions
were found in most of the tissues (98/120) and were not
consistent with characteristic S. suis pathology, with no
differences among the three groups. Streptococcus suis serotype 2 detection in the respiratory
tract after inoculation Streptococcus suis serotype 2 detection in the respiratory
tract after inoculation S. suis serotype 2 was detected by PCR in nasal swabs
taken postmortem in piglets inoculated with T15 (7/8) or
S10 (7/8). Amplification was more intense in nasal swabs
from piglets inoculated with S10 than with T15, espe-
cially at 3 dpi (three samples from T15 inoculated pig-
lets yielded a weak amplification and one was negative in
the PCR, while two samples from S10 inoculated piglets
yielded a moderate amplification, one a strong amplifica-
tion and one was negative in the PCR). Using IHC, S. suis
serotype 2 was detected in the upper respiratory tract for
both strains, mostly in the mucus but also in the epithe-
lium of the nasal cavity (2/8 for T15 and 4/8 for S10), cri-
briform plates of ethmoid (7/8 for T15 and 8/8 for S10),
and tracheas (3/8 for T15 and 1/8 for S10). Immunolabel-
ling was also found in the alveolar lumen of the lungs (4/8
for T15 and 5/8 for S10) (Figures 2A and B), but there
was no detection in any of the lymph nodes analyzed. In
the cribriform plate of ethmoid, T15 bacteria were found
only in the mucus (Figure 2C), while some S10 bacteria
were detected deep in the tissue, close to the cartilage
(Figure 2D). This latter location was not observed in
any of the animals infected with the T15 strain. Thus, S. suis serotype 2 was detected in all inoculated animals by
either PCR (14/16) or IHC (15/16), but not in the piglets
from the non-infected control group. Local and systemic transcriptional responses to virulent
and non‑virulent S. suis inoculation High quality RNA was obtained from lymph nodes, tra-
chea, lungs, spleen, liver, and whole blood. RNA obtained
from nasal swabs was of sub-optimal quality, and care was
therefore taken when choosing a strategy for transcrip-
tional analysis of these samples. This included limiting
the focus to relatively few genes that could be expected to
be strongly induced during an inflammatory antibacterial
response, as well as assaying the transcription of several
of the investigated genes with two independent assays
(two different primer pairs targeting the same mRNA
transcript at non-overlapping sites). Mean and range of
RNA quality for the different tissues are summarized in
Additional file 1. Gene expression in longitudinal samples
(whole blood and nasal swabs) and in necropsy samples
(all other tissues) were compared to the expression at
3 days before challenge and the PBS group, respectively,
as indicated above (see section “Materials and methods”). Time, days post-inoculation
Rectal temperature
-2
-1
0
1
2
3
39.5
40.0
40.5
41.0
*
Figure 1 Rectal temperature before and after S. suis intranasal
inoculation. Mean and standard deviation of rectal temperatures of
the piglets before and after intranasal inoculation with S. suis strains
T15 (non-virulent, blue circles) or S10 (virulent, red circles) (n = 8
from -2 to 1 day post-inoculation [dpi]; n = 4 at 2 and 3 dpi, for both
strains). A group of piglets was inoculated with PBS as control (white
squares; n = 4 for all time points). * Statistically significant (P < 0.05)
difference between S10 and PBS groups. Rectal temperature
-2
-1
0
1
2
3
39.5
40.0
40.5
41.0
* Time, days post-inoculation Time, days post-inoculation Figure 1 Rectal temperature before and after S. suis intranasal
inoculation. Mean and standard deviation of rectal temperatures of
the piglets before and after intranasal inoculation with S. suis strains
T15 (non-virulent, blue circles) or S10 (virulent, red circles) (n = 8
from -2 to 1 day post-inoculation [dpi]; n = 4 at 2 and 3 dpi, for both
strains). A group of piglets was inoculated with PBS as control (white
squares; n = 4 for all time points). * Statistically significant (P < 0.05)
difference between S10 and PBS groups. Generally, only small changes in gene expression were
observed in S. suis-challenged animals, with the major-
ity of the transcriptional regulation being <2-fold either
up- or down-regulated, for both the S10 and T15 strains. Statistical analysis
l Data analyses were performed with R (v. 4.0.2, [29]). Rectal temperature after inoculation was analyzed using
ANOVA with Tukey’s Honest Significant Difference
(HSD) post-hoc test with interaction between inoculated
groups and time points. In order to compare changes
in gene expression between groups, relative transcript
quantities were calculated; for longitudinal samples
(nasal swabs and blood) gene expression levels at 4 h
post-inoculation and 1, 2, and 3 dpi were normalized
against 3 days before inoculation. Statistical significance
of the gene expression changes in whole blood and nasal
swab samples was assessed by linear mixed effects regres-
sion with interaction between the different time points
and the inoculum groups, taking into account animal ID
as random-effect. For necropsy samples (all other tissue
samples, taken at 1 and 3 dpi), normalization was done
against the values of the PBS inoculated group. Statistical
significance of the gene expression changes in necropsy
tissues was analyzed using ANOVA with Tukey’s HSD
post-hoc test with interaction between inoculated groups qPCR analysis was carried out using the high-
throughput platform BioMark (Fluidigm) using 192.24
Dynamic Array IFC chips (Fluidigm) (192 samples in
combination with 24 assays, used for lung tissues, tra-
chea, nasal swabs) or 96.96 Dynamic Array IFC chips
(Fluidigm) (96 samples in combination with 96 assays,
used for lymph nodes, liver, spleen, blood). All assays
(primer pairs) employed in the present study were
designed in-house and purchased from Sigma-Aldrich. All qPCR primer sequences and amplification efficien-
cies can be found in Additional file 2. Whenever pos-
sible, primers pairs were designed to span intron/exon
borders in order to prevent amplification of potentially
contaminating genomic DNA. qPCR was carried out
using a sample mix comprising TaqMan Gene Expres-
sion Master Mix (Applied Biosystems), DNA Bind-
ing Dye (Fluidigm), EvaGreen Dye (Biotium), and
pre-amplified, exonuclease treated cDNA (diluted 1:10 Neila‑Ibáñez et al. Veterinary Research (2021) 52:145 Page 5 of 15 and time points. A confidence level of 95% was consid-
ered as statistically significant (P < 0.05). and time points. A confidence level of 95% was consid-
ered as statistically significant (P < 0.05). Local and systemic transcriptional responses to virulent
and non‑virulent S. suis inoculation In addition, only some of these changes were statistically p g
T15 (non-virulent, blue circles) or S10 (virulent, red circles) (n = 8
from -2 to 1 day post-inoculation [dpi]; n = 4 at 2 and 3 dpi, for both
strains). A group of piglets was inoculated with PBS as control (white
squares; n = 4 for all time points). * Statistically significant (P < 0.05)
difference between S10 and PBS groups. Neila‑Ibáñez et al. Veterinary Research (2021) 52:145 Page 6 of 15 Figure 2 Detection of S. suis serotype 2 by immunohistochemistry. Piglets were intranasally inoculated with S. suis T15 (non-virulent) or S10
(virulent) strains, and tissues were collected at 1 and 3 days post-inoculation (dpi). A: Bacteria present at 1 dpi in the bronchiole (arrowhead) and
alveolar lumen of a piglet inoculated with T15. B: Bacteria present at 1 dpi in the epithelial surface of bronchioles (arrowhead) and alveoli of a piglet
inoculated with S10. C: Presence of bacteria at 3 dpi in neutrophils in the mucus of the cribriform plate of ethmoid of a piglet inoculated with T15
(arrowhead). D: Bacteria next to the cartilage of the cribriform plate of ethmoid (CT) at 3 dpi, from a piglet inoculated with S10 (arrowhead). Figure 2 Detection of S. suis serotype 2 by immunohistochemistry. Piglets were intranasally inoculated with S. suis T15 (non-virulent) or S10
(virulent) strains, and tissues were collected at 1 and 3 days post-inoculation (dpi). A: Bacteria present at 1 dpi in the bronchiole (arrowhead) and
alveolar lumen of a piglet inoculated with T15. B: Bacteria present at 1 dpi in the epithelial surface of bronchioles (arrowhead) and alveoli of a piglet
inoculated with S10. C: Presence of bacteria at 3 dpi in neutrophils in the mucus of the cribriform plate of ethmoid of a piglet inoculated with T15
(arrowhead). D: Bacteria next to the cartilage of the cribriform plate of ethmoid (CT) at 3 dpi, from a piglet inoculated with S10 (arrowhead). Figure 2 Detection of S. suis serotype 2 by immunohistochemistry. Piglets were intranasally inoculated with S. suis T15 (non-virulent) or S10
(virulent) strains, and tissues were collected at 1 and 3 days post-inoculation (dpi). A: Bacteria present at 1 dpi in the bronchiole (arrowhead) and
alveolar lumen of a piglet inoculated with T15. Local and systemic transcriptional responses to virulent
and non‑virulent S. suis inoculation B: Bacteria present at 1 dpi in the epithelial surface of bronchioles (arrowhead) and alveoli of a piglet
inoculated with S10. C: Presence of bacteria at 3 dpi in neutrophils in the mucus of the cribriform plate of ethmoid of a piglet inoculated with T15
(arrowhead). D: Bacteria next to the cartilage of the cribriform plate of ethmoid (CT) at 3 dpi, from a piglet inoculated with S10 (arrowhead). (Figure 3, Additional file 3). This response seemed to
continue unabated throughout the experiment in the pig-
lets inoculated with the virulent S10 strain whereas the
response to the T15 challenge was shorter and had a ten-
dency to return to baseline levels by day 3 (Figures 3 and
4). Despite the changes observed between strains (Fig-
ures 3 and 4, Additional file 3), specially at 3 dpi, none
of these were statistically significant, probably due to the
low number of piglets. significant, probably due to the considerable individual
variation in gene expression levels observed within the
groups of animals. However, a group of genes showed quite pronounced
transcriptional responses in nasal swab samples and
clearly demonstrated differential host responses after vir-
ulent and non-virulent challenge, with more genes con-
sistently up-regulated by S10 at 4 h post-inoculation than
by T15 (Figures 3 and 4). These genes included pro- and
anti-inflammatory genes IL1A, IL1B, IL1RN, and IRF1, as
well as the chemokine CXCL10, and were induced early
in the nasal mucosa after challenge with both strains In contrast to the observations in the nasal samples,
gene expression in the submandibular lymph node was
generally less affected with fewer and smaller changes Neila‑Ibáñez et al. Veterinary Research (2021) 52:145 Page 7 of 15 Figure 3 Relative gene expression in nasal samples after S. suis intranasal inoculation. Log2 of the individual values and mean (black bars) of
the relative gene expression in nasal samples in S. suis inoculated piglets (gene expression was normalized relative to the 3 days before challenge
mean for each inoculated group). Piglets were intranasally inoculated with S. suis T15 (non-virulent, blue bars) or S10 (virulent, red bars) strains,
and nasal swabs were taken at 4 h and 1, 2 and 3 days after inoculation. Genes showing significant difference (P < 0.05) in pairwise analysis when
comparing different time points in the S. Local and systemic transcriptional responses to virulent
and non‑virulent S. suis inoculation suis inoculated groups or between strains at the same time point, and with a mean greater than 2-fold
change (log2 = 1) are shown. * P < 0.05 when comparing versus their respective 3 days before challenge time point. n = 8 for both strains at 4 h
post-infection and 1 dpi, n = 4 for both strains at 2 and 3 dpi. IL1B 1 and IL1B 2 are both IL1B assays consisting of two different primer pairs targeting
non-overlapping sites in the IL1B transcript. All expression values and significant differences can be found in Additional file 3. Figure 3 Relative gene expression in nasal samples after S. suis intranasal inoculation. Log2 of the individual values and mean (black bars) of
the relative gene expression in nasal samples in S. suis inoculated piglets (gene expression was normalized relative to the 3 days before challenge
mean for each inoculated group). Piglets were intranasally inoculated with S. suis T15 (non-virulent, blue bars) or S10 (virulent, red bars) strains,
and nasal swabs were taken at 4 h and 1, 2 and 3 days after inoculation. Genes showing significant difference (P < 0.05) in pairwise analysis when
comparing different time points in the S. suis inoculated groups or between strains at the same time point, and with a mean greater than 2-fold
change (log2 = 1) are shown. * P < 0.05 when comparing versus their respective 3 days before challenge time point. n = 8 for both strains at 4 h
post-infection and 1 dpi, n = 4 for both strains at 2 and 3 dpi. IL1B 1 and IL1B 2 are both IL1B assays consisting of two different primer pairs targeting
non-overlapping sites in the IL1B transcript. All expression values and significant differences can be found in Additional file 3. and only by the T15 strain (at 3 dpi), with no signifi-
cant modulation of gene expression observed in animals
inoculated with S10 strain at 1 or 3 dpi (Figure 5). At day
3 after the challenge with the non-virulent T15 strain,
genes IL1B and PTGS2 (indicative of inflammation) and
CCL2 and SELP (involved in recruitment of immune
cells) were significantly >2-fold up-regulated vs. PBS con-
trol (Figure 5). In the case of PTGS2 and CCL2, signifi-
cant differences were also found between T15 and S10
inoculated animals, with up-regulation only by the non-
virulent T15 strain. Local and systemic transcriptional responses to virulent
and non‑virulent S. suis inoculation Samples were taken at 3 days before, and 4 h, 1, 2, and 3 days after the
intranasal inoculation of S. suis strains T15 (non-virulent) and S10 (virulent). A group of piglets inoculated with PBS are also shown and served as
control. All the genes found to be quantifiable are shown irrespectively of their statistical significance. Gene expression was normalized relative
to the 3 days before challenge mean for each inoculated group and log2 transformed. Values are presented as a heat map. Numbers in abscissa
axis represent animal ID. Color scale was limited to ± 5 and out of bounds values displayed with the maximum intensity color. Gene functional
groups: Apop.: Apoptosis; Chemo.: Chemokines; M.: Miscellaneous; P.R.R.: Pattern Recognition Receptors. IL1B_1 and IL1B_2 are both IL1B assays
consisting of two different primer pairs targeting non-overlapping sites in the IL1B transcript. Samples marked with a black cross: expression level
not quantifiable. different only at 1 dpi for the following genes: CASP1,
CCL4, IRF7, STAT1, and STAT2, with higher values in
animals challenged with the non-virulent strain T15 (Fig-
ure 7, Additional file 7). Significant differences lower than
2-fold between strains were observed only at 1 dpi for the
genes IL1B, JAK2, TICAM1, TRIF, and TNF, with higher
values for T15 than for the S10, and also for NFKBIA, but
in the opposite direction (Additional file 7). in TLR6 in the S10 inoculated group (Additional file 4). In the caudal lobe, significant regulation with a >2-fold
change was observed for CXCL8 (IL8) (down-regulated
by T15 at 1 dpi), SAA (up-regulated by T15 at 3 dpi), and
TNF (down-regulated by T15 at 3 dpi, and by S10 at 1
and 3 dpi) compared to the PBS group (Figure 5). When
comparing the responses to the two strains, SAA (>2-
fold, Figure 5) and TLR2 (<2-fold, Additional file 4) were
significantly higher for the non-virulent T15 than the
virulent S10 at 3 dpi. A few other significantly different
(P < 0.05) <2-fold changes compared to the PBS group,
between time points for the same strains, or between
strains at the same time point, are shown in Additional
file 4. i
Consistent with the absence of systemic disease and/or
overt systemic reactions to the intranasal S. suis inocu-
lation, very few genes were significantly affected in liver
and spleen (Additional file 8). Local and systemic transcriptional responses to virulent
and non‑virulent S. suis inoculation Despite the small magni-
tude of these changes (all with <2-fold changes), some
statistical differences between strains were observed in
liver at 1 dpi, with lower values in piglets inoculated with
the virulent strain S10 for BCL2, TNFRSF1A, and TP53. Individual values for all the genes analyzed in these tis-
sues are presented as heat maps in Additional file 9. i
Analysis of whole blood indicated changes in gene
expression patterns in response to the challenge with
both S. suis strains, although the majority of changes
were below 2-fold, with subtle differences in the tem-
poral dynamics depending on the challenge strain (Fig-
ures 7 and 8, Additional file 7). The blood response to
strain S10 was rapid with six genes showing >2-fold up-
regulation at 4 h after challenge (CASP1, CD14, IRF7,
STAT1, STAT2, and TLR4), and maintaining this differ-
ence at 2 dpi for STAT1 and STAT2 (Figure 7, Additional
file 7). The response to the non-virulent strain T15 was
more delayed, with a peak in the number of significantly
up-regulated genes with a >2-fold change on 1 dpi (five
genes, CASP1, CCL4, IRF7, STAT1, and STAT2), and
only one gene (TLR4) significantly up-regulated at 4 h
after the challenge (Figure 7, Additional file 7). The dif-
ferent response observed to both strains was statistically Local and systemic transcriptional responses to virulent
and non‑virulent S. suis inoculation In the tracheobronchial lymph node,
significant up-regulation was only observed for CASP1 at
1 dpi in S10 inoculated pigs (Figure 5). Genes with signif-
icant changes lower than 2-fold when compared vs. the
PBS group for both lymph nodes are included in Addi-
tional file 4. In addition, individual expression changes in submandibular and tracheobronchial lymph nodes
are presented in Figure 6. Although some changes were
observed in individual piglets, the response showed high
variation within the groups and no statistical differences
were found in our model (Figure 6 and Additional file 5). As an example, IL1RN in tracheobronchial lymph node:
mean ± standard deviation of 3.92 ± 2.78 and 3.79 ± 1.18
for T15 strain at 1 and 3 dpi respectively; and 5.88 ± 3.49
and 4.06 ± 2.44 for S10 strain at 1 and 3 dpi respectively
(all tissues values are available in Additional file 5). i i
In trachea, IL10 and TLR6 were significantly down-reg-
ulated in response to both strains, which coincided with a
general trend towards down-regulation of the majority of
genes examined in this tissue (Figure 5, Additional file 6). In lungs, more changes were detected in the caudal than
in the cranial lobe, which only showed minor changes Neila‑Ibáñez et al. Veterinary Research (2021) 52:145 Page 8 of 15 Figure 4 Gene expression in nasal samples after S. suis inoculation. Samples were taken at 3 days before, and 4 h, 1, 2, and 3 days after the
intranasal inoculation of S. suis strains T15 (non-virulent) and S10 (virulent). A group of piglets inoculated with PBS are also shown and served as
control. All the genes found to be quantifiable are shown irrespectively of their statistical significance. Gene expression was normalized relative
to the 3 days before challenge mean for each inoculated group and log2 transformed. Values are presented as a heat map. Numbers in abscissa
axis represent animal ID. Color scale was limited to ± 5 and out of bounds values displayed with the maximum intensity color. Gene functional
groups: Apop.: Apoptosis; Chemo.: Chemokines; M.: Miscellaneous; P.R.R.: Pattern Recognition Receptors. IL1B_1 and IL1B_2 are both IL1B assays
consisting of two different primer pairs targeting non-overlapping sites in the IL1B transcript. Samples marked with a black cross: expression level
not quantifiable. Figure 4 Gene expression in nasal samples after S. suis inoculation. Discussion Pathogens use different mechanisms to evade the innate
immune system, the first line of defense against them,
and to colonize the host. In S. suis infection, the host’s
immune response combined with the virulence of the
infecting strain play important roles in achieving coloni-
zation and, subsequently, in the possible development of
the disease [30]. Although it is difficult to reproduce disease with this
bacterium using the intranasal route of inoculation, it has
been used on numerous occasions to study host–patho-
gen interactions [30]. In the present study, despite the Neila‑Ibáñez et al. Veterinary Research (2021) 52:145 Page 9 of 15 Figure 5 Relative gene expression in different tissues after S. suis intranasal inoculation. Log2 of the individual values and mean (black bars)
of the relative gene expression in different tissues in S. suis inoculated piglets (gene expression was normalized relative to the PBS group). Piglets
were intranasally inoculated with S. suis T15 (non-virulent, blue bars) or S10 (virulent, red bars), and necropsies were performed at 1 and 3 days
post-infection. The values and means are shown for the indicated groups (challenge strain and time point) having at least one significant difference
when compared to the PBS group and with a mean higher than 2-fold change (log2 = 1). LN Tracheobr: Tracheobronchial lymph node. * indicates
significant differences (P < 0.05) versus the PBS group. Differences between strains at the same time point are labelled with # and differences
between time points for the same strain are labelled with+, P<0.05, in both cases. n=4 for each group. All expression values and significant Figure 5 Relative gene expression in different tissues after S. suis intranasal inoculation. Log2 of the individual values and mean (black bars)
of the relative gene expression in different tissues in S. suis inoculated piglets (gene expression was normalized relative to the PBS group). Piglets
were intranasally inoculated with S. suis T15 (non-virulent, blue bars) or S10 (virulent, red bars), and necropsies were performed at 1 and 3 days
post-infection. The values and means are shown for the indicated groups (challenge strain and time point) having at least one significant difference
when compared to the PBS group and with a mean higher than 2-fold change (log2 = 1). LN Tracheobr: Tracheobronchial lymph node. * indicates
significant differences (P < 0.05) versus the PBS group. Discussion n = 4 for each group. All expression values and significant
differences can be found in Additional file 5. fact that systemic disease did not develop in the inocu-
lated animals in the short course of the study, we did
observe various host responses (including fever) induced
by strains S10 and T15, with different known virulence
potential, during the first steps of infection. For most
of the sample types examined in this work, the limited
number of animals and high individual variations made
it difficult to correlate a clear gene expression pattern or
inflammatory marker consistently with the virulence of
the strain. However, for the nasal mucosa the transcrip-
tional response did in fact reflect the virulence potential
of the inoculated S. suis strain. Despite these limitations,
this study used an experimental model that reproduces S. suis natural infection of pigs, providing for the first time
a comprehensive overview of the host innate immune
response induced by S. suis during upper respiratory tract fact that systemic disease did not develop in the inocu-
lated animals in the short course of the study, we did
observe various host responses (including fever) induced
by strains S10 and T15, with different known virulence
potential, during the first steps of infection. For most
of the sample types examined in this work, the limited
number of animals and high individual variations made
it difficult to correlate a clear gene expression pattern or
inflammatory marker consistently with the virulence of
the strain. However, for the nasal mucosa the transcrip-
tional response did in fact reflect the virulence potential
of the inoculated S. suis strain. Despite these limitations,
this study used an experimental model that reproduces S. suis natural infection of pigs, providing for the first time
a comprehensive overview of the host innate immune
response induced by S. suis during upper respiratory tract colonization. In addition, the present study paves the way
for more extensive mechanistic studies on modulation of
host immunity by this important swine pathogen. Interestingly, both strains induced an early pro-
inflammatory response locally in the nasal mucosa;
however, the return to baseline gene expression levels
was faster for the non-virulent strain (T15). Among
genes up-regulated by both strains at the nasal mucosa,
IL1B is a cytokine that acts as a master regulator of
inflammation by controlling a variety of innate immune
processes [31]. Several studies have reported the capac-
ity of S. Discussion Differences between strains at the same time point are labelled with # and differences
between time points for the same strain are labelled with +, P < 0.05, in both cases. n = 4 for each group. All expression values and significant
differences can be found in Additional file 5. Figure 5 Relative gene expression in different tissues after S. suis intranasal inoculation. Log2 of the individual values and mean (black bars)
of the relative gene expression in different tissues in S. suis inoculated piglets (gene expression was normalized relative to the PBS group). Piglets
were intranasally inoculated with S. suis T15 (non-virulent, blue bars) or S10 (virulent, red bars), and necropsies were performed at 1 and 3 days
post-infection. The values and means are shown for the indicated groups (challenge strain and time point) having at least one significant difference
when compared to the PBS group and with a mean higher than 2-fold change (log2 = 1). LN Tracheobr: Tracheobronchial lymph node. * indicates
significant differences (P < 0.05) versus the PBS group. Differences between strains at the same time point are labelled with # and differences
between time points for the same strain are labelled with +, P < 0.05, in both cases. n = 4 for each group. All expression values and significant
differences can be found in Additional file 5. Figure 5 Relative gene expression in different tissues after S. suis intranasal inoculation. Log2 of the individual valu Figure 5 Relative gene expression in different tissues after S. suis intranasal inoculation. Log2 of the individual values and mean (black bars)
of the relative gene expression in different tissues in S. suis inoculated piglets (gene expression was normalized relative to the PBS group). Piglets
were intranasally inoculated with S. suis T15 (non-virulent, blue bars) or S10 (virulent, red bars), and necropsies were performed at 1 and 3 days
post-infection. The values and means are shown for the indicated groups (challenge strain and time point) having at least one significant difference
when compared to the PBS group and with a mean higher than 2-fold change (log2 = 1). LN Tracheobr: Tracheobronchial lymph node. * indicates
significant differences (P < 0.05) versus the PBS group. Differences between strains at the same time point are labelled with # and differences
between time points for the same strain are labelled with +, P < 0.05, in both cases. Discussion suis to induce IL-1 cytokine family members
by a variety of cell types [32, 33]. In addition to IL1B,
up-regulation of the interferon-regulatory factor 1
(IRF1) suggests activation of the interferon (IFN) path-
way during S. suis colonization, including expression of Neila‑Ibáñez et al. Veterinary Research (2021) 52:145 Page 10 of 15 Figure 6 Gene expression in submandibular and tracheobronchial lymph nodes after S. suis intranasal inoculation. Samples from Figure 6 Gene expression in submandibular and tracheobronchial lymph nodes after S. suis intranasal inoculation. Samples from
the submandibular (A) and tracheobronchial (B) lymph nodes were collected at 1 and 3 days after the intranasal inoculation of S. suis T15
(non-virulent) and S10 (virulent). Results at 1 day post-inoculation from piglets inoculated with PBS are also included as control. All the genes
found to be quantifiable are shown irrespectively of their statistical significance. Gene expression was normalized relative to the PBS group and
log2 transformed. Values are presented as a heat map. Numbers in abscissa axis represent animal ID. Color scale was limited to ± 4 and out of
bounds values displayed with the maximum intensity color. Gene functional groups: A.P.P.: Acute Phase Proteins; Ad. M.: Adhesion Molecules; Misc.:
Miscellaneous; P.R.R.: Pattern Recognition Receptors. Figure 6 Gene expression in submandibular and tracheobronchial lymph nodes after S. suis intranasal inoculation. Samples from
the submandibular (A) and tracheobronchial (B) lymph nodes were collected at 1 and 3 days after the intranasal inoculation of S. suis T15
(non-virulent) and S10 (virulent). Results at 1 day post-inoculation from piglets inoculated with PBS are also included as control. All the genes
found to be quantifiable are shown irrespectively of their statistical significance. Gene expression was normalized relative to the PBS group and
log2 transformed. Values are presented as a heat map. Numbers in abscissa axis represent animal ID. Color scale was limited to ± 4 and out of
bounds values displayed with the maximum intensity color. Gene functional groups: A.P.P.: Acute Phase Proteins; Ad. M.: Adhesion Molecules; Misc.:
Miscellaneous; P.R.R.: Pattern Recognition Receptors. Figure 6 Gene expression in submandibular and tracheobronchial lymph nodes after S. suis intranasal inoculation. Samples from
the submandibular (A) and tracheobronchial (B) lymph nodes were collected at 1 and 3 days after the intranasal inoculation of S. suis T15
(non-virulent) and S10 (virulent). Results at 1 day post-inoculation from piglets inoculated with PBS are also included as control. Discussion All the genes
found to be quantifiable are shown irrespectively of their statistical significance. Gene expression was normalized relative to the PBS group and
log2 transformed. Values are presented as a heat map. Numbers in abscissa axis represent animal ID. Color scale was limited to ± 4 and out of
bounds values displayed with the maximum intensity color. Gene functional groups: A.P.P.: Acute Phase Proteins; Ad. M.: Adhesion Molecules; Misc.:
Miscellaneous; P.R.R.: Pattern Recognition Receptors. Neila‑Ibáñez et al. Veterinary Research (2021) 52:145 Page 11 of 15 Figure 7 Relative gene expression in blood after S. suis intranasal inoculation. Log2 of the individual values and mean (black bars) of the
relative gene expression in blood in S. suis inoculated piglets (gene expression was normalized relative to the 3 days before challenge mean for
each inoculated group). Piglets were intranasally inoculated with S. suis T15 (non-virulent, blue bars) or S10 (virulent, red bars), and blood samples
were taken at 4 h and 1, 2, and 3 days after inoculation. Genes with at least 1 significant difference in pairwise analysis (P < 0.05) when comparing
different time points in the S. suis inoculated groups or between strains at the same time point, and with a mean greater than 2-fold change
(log2 = 1) are shown. * indicates significant differences (P < 0.05) versus their respective 3 days before challenge time point. Differences between
strains at the same time point are labelled with # (P < 0.05). n = 8 for both strains at 4 h post-infection and 1 dpi, n = 4 for both strains at 2 and 3 dpi. All expression values and significant differences can be found in Additional file 7. Figure 7 Relative gene expression in blood after S. suis intranasal inoculation. Log2 of the individual values and mean (black bars) of the Figure 7 Relative gene expression in blood after S. suis intranasal inoculation. Log2 of the individual values and mean (black bars) of the
relative gene expression in blood in S. suis inoculated piglets (gene expression was normalized relative to the 3 days before challenge mean for
each inoculated group). Piglets were intranasally inoculated with S. suis T15 (non-virulent, blue bars) or S10 (virulent, red bars), and blood samples
were taken at 4 h and 1, 2, and 3 days after inoculation. Discussion Genes with at least 1 significant difference in pairwise analysis (P < 0.05) when comparing
different time points in the S. suis inoculated groups or between strains at the same time point, and with a mean greater than 2-fold change
(log2 = 1) are shown. * indicates significant differences (P < 0.05) versus their respective 3 days before challenge time point. Differences between
strains at the same time point are labelled with # (P < 0.05). n = 8 for both strains at 4 h post-infection and 1 dpi, n = 4 for both strains at 2 and 3 dpi. All expression values and significant differences can be found in Additional file 7. (e.g., the Muramidase-Released Protein [MRP] or the
extracellular factor [EF] protein) that are absent in T15
[24]. CXCL10, a chemokine gene that can be up-regulated
in response to IFN-γ/IRF1 signaling pathway [34]. The
IFN pathways can play either a regulatory or a patho-
logical role depending on the virulence of the S. suis
strain or the specific clinical manifestation of the dis-
ease, as previously suggested [33, 35]. In the absence of clinical manifestations and histo-
pathological lesions, the observed modulation of the
innate immune response by S. suis colonization could be
considered a homeostasis-restoring state of inflamma-
tion [36], which is considered different from pathological
inflammation. It has been suggested that such state may
be maintained by pattern recognition receptors (PRRs)
expressed in stromal and/or immune cells, detecting
endogenous ligands and/or pathogens [36]. In agreement
with this concept, expression of interleukin-1 receptor
antagonist (IL-1RA; encoded by IL1RN) was observed
in nasal samples and that of the enzyme cyclooxyge-
nase-2 (COX-2), encoded by PTGS2, was found in In the submandibular lymph node, gene expression
related to recruitment of immune cells (such as expres-
sion of the chemokine CCL2 and the adhesion molecule
P selectin encoded by SELP) and to inflammation (IL1B)
was mainly observed after colonization with the non-
virulent strain. These seemingly contradictory results
observed between nasal mucosa and the submandibular
lymph node may reflect intrinsic properties of the strains,
with different molecular composition, including the pres-
ence of virulence-associated proteins in the S10 strain Neila‑Ibáñez et al. Veterinary Research (2021) 52:145 Page 12 of 15 Figure 8 Gene expression in blood after S. suis intranasal inoculation. Discussion Figure 8 Gene expression in blood after S. suis intranasal inoculation. Samples were taken at 3 days before, and 4 h, 1, 2, and 3 days after the
intranasal inoculation of S. suis T15 (non-virulent) and S10 (virulent). A group of piglets inoculated with PBS are also shown and served as control. All
genes found to be quantifiable are shown irrespectively of their statistical significance. Gene expression was normalized relative to the 3 days before
challenge mean for each inoculated group and log2 transformed. Values are presented as a heat map. Numbers in abscissa axis represent animal
ID. Color scale was limited to ± 3 and out of bounds values displayed with the maximum intensity color. Gene functional groups: A.P.: Acute Phase
Proteins; Adhesion M.: Adhesion Molecules; Chemo.: Chemokines; Misc.: Miscellaneous; Pattern Recognition R.: Pattern Recognition Receptors. the IL-1 or the IFN pathways (such as CASP1, IRFs, and
STATs), which seem to predominate during the innate
immune response induced by S. suis colonization. However, massive activation of these and other pro-
inflammatory pathways (cytokine storm) are known
to be involved in pathological inflammation during S. suis systemic disease leading to septic shock [41]. Nev-
ertheless, the transcriptional patterns in blood showed
that the host response to the virulent challenge was
rapid, peaking within hours after challenge, which
coincided with elevated body temperature. In contrast,
the circulating response to the non-virulent challenge
was more delayed and did not coincide with the fever
response. This correlation between up-regulation of
pro-inflammatory cytokines and fever agrees with the
initial course of disease in other pig infection models
[42]. Internal organs such as liver, spleen, kidney, or
heart, are invaded after S. suis reaches systemic circu-
lation [43]. However, in the present study, the piglets
did not develop systemic disease and, accordingly, the
splenic and hepatic response to both S. suis strains submandibular lymph node (and mainly induced by the
non-virulent strain). COX-2-derived metabolites are
important regulators of inflammation [37] and IL-1RA
competitively inhibits IL-1 binding to cell-surface recep-
tors. Maintenance of a balance between IL-1 and IL-1RA
is important in preventing the development or progres-
sion of inflammatory disease [38]. It has been suggested
in other models that selective induction of IL-1RA might
facilitate mucosal colonization by bacteria. IL-1RA also
plays a critical role in maintaining a homeostatic and bal-
anced microbiota [39, 40]. Further studies are required
to delineate the link between S. Discussion Samples were taken at 3 days before, and 4 h, 1, 2, and 3 days after the
intranasal inoculation of S. suis T15 (non-virulent) and S10 (virulent). A group of piglets inoculated with PBS are also shown and served as control. All
genes found to be quantifiable are shown irrespectively of their statistical significance. Gene expression was normalized relative to the 3 days before
challenge mean for each inoculated group and log2 transformed. Values are presented as a heat map. Numbers in abscissa axis represent animal
ID. Color scale was limited to ± 3 and out of bounds values displayed with the maximum intensity color. Gene functional groups: A.P.: Acute Phase
Proteins; Adhesion M.: Adhesion Molecules; Chemo.: Chemokines; Misc.: Miscellaneous; Pattern Recognition R.: Pattern Recognition Receptors. Figure 8 Gene expression in blood after S. suis intranasal inoculation. Samples were taken at 3 days before, and 4 h, 1, 2, and 3 days after the
intranasal inoculation of S. suis T15 (non-virulent) and S10 (virulent). A group of piglets inoculated with PBS are also shown and served as control. All
genes found to be quantifiable are shown irrespectively of their statistical significance. Gene expression was normalized relative to the 3 days before
challenge mean for each inoculated group and log2 transformed. Values are presented as a heat map. Numbers in abscissa axis represent animal
ID. Color scale was limited to ± 3 and out of bounds values displayed with the maximum intensity color. Gene functional groups: A.P.: Acute Phase
Proteins; Adhesion M.: Adhesion Molecules; Chemo.: Chemokines; Misc.: Miscellaneous; Pattern Recognition R.: Pattern Recognition Receptors. Figure 8 Gene expression in blood after S. suis intranasal inoculation. Samples were taken at 3 days before, and 4 h, 1, 2, and 3 days after the
intranasal inoculation of S. suis T15 (non-virulent) and S10 (virulent). A group of piglets inoculated with PBS are also shown and served as control. All
genes found to be quantifiable are shown irrespectively of their statistical significance. Gene expression was normalized relative to the 3 days before
challenge mean for each inoculated group and log2 transformed. Values are presented as a heat map. Numbers in abscissa axis represent animal
ID. Color scale was limited to ± 3 and out of bounds values displayed with the maximum intensity color. Gene functional groups: A.P.: Acute Phase
Proteins; Adhesion M.: Adhesion Molecules; Chemo.: Chemokines; Misc.: Miscellaneous; Pattern Recognition R.: Pattern Recognition Receptors. Additional file 4. Significant gene expression with < 2-fold changes
in lymph nodes, trachea and lungs after S. suis intranasal inocula‑ in lymph nodes, trachea and lungs after S. suis intranasal inocula
tion. Log2 of the individual values and mean (black bars) of the relative
gene expression in different tissues of S. suis inoculated piglets. Piglets
were intranasally inoculated with S. suis T15 (non-virulent, blue bars) or
S10 (virulent, red bars), and necropsies were performed at 1 and 3 days
post-infection. Gene expression was normalized relative to the PBS group. The values and means are shown for the indicated groups (challenge
strain and time point) having at least one significant difference when
compared to the PBS group and with a mean lower than 2-fold change
(log2 = 1). LN Trbr: Tracheobronchial lymph node; Lung Cr: Lung Cranial;
Lung Cd: Lung Caudal. * indicates significant differences (P < 0.05) versus
the PBS group. Differences between strains at the same time point are
labelled with # and differences between time points for the same strain
are labelled with +, P < 0.05, in both cases. n = 4 for each group. All expres‑
sion values and significant differences can be found in Additional file 5. y
post-infection. Gene expression was normalized relative to the PBS group. The values and means are shown for the indicated groups (challenge
strain and time point) having at least one significant difference when
compared to the PBS group and with a mean lower than 2-fold change
(log2 = 1). LN Trbr: Tracheobronchial lymph node; Lung Cr: Lung Cranial;
Lung Cd: Lung Caudal. * indicates significant differences (P < 0.05) versus
the PBS group. Differences between strains at the same time point are
labelled with # and differences between time points for the same strain
are labelled with +, P < 0.05, in both cases. n = 4 for each group. All expres‑
sion values and significant differences can be found in Additional file 5. Additional file 5. Gene expression in tissues samples. Gene expression
from tissues samples, including means, standard deviations, and P-values. Additional file 5. Gene expression in tissues samples. Gene expression
from tissues samples, including means, standard deviations, and P-values. Additional file 6. Gene expression in different respiratory tissues
after S. suis intranasal inoculation. Samples from trachea (A), caudal Additional file 6. Gene expression in different respiratory tissues
after S. suis intranasal inoculation. Discussion suis colonization and the
induction of a homeostasis-restoring state of inflamma-
tion, including a potential regulatory role of IL-1RA and/
or COX-2. The systemic response was limited as no clinical inva-
sive disease was observed and S. suis was not found in
blood. The observed minor changes in gene expres-
sion in systemic samples could be a consequence of
the ongoing local response at the upper respiratory
track. Indeed, up-regulated genes were associated to Neila‑Ibáñez et al. Veterinary Research (2021) 52:145 Page 13 of 15 showed a low number of genes significantly affected
and with low magnitude. This lack of systemic disease
may be explained by the route of inoculation, intrana-
sal, unaided by acetic acid or viral co-infection [19, 20],
the short time of the study, or the ability of the host to
control the infection before bacteria could reach the
bloodstream. Additional file 1. RNA quality. Mean values with the minimum and
maximum range for RNA concentration and qualities for each sample
type. Additional file 8. Significant gene expression in spleen and liver after
S. suis intranasal inoculation. Log2 of the individual values and mean Additional file 8. Significant gene expression in spleen and liver after
S. suis intranasal inoculation. Log2 of the individual values and mean
(black bars) of the relative gene expression in spleen and liver in S. suis
inoculated piglets. Piglets were intranasally inoculated with S. suis T15
(non-virulent, blue bars) or S10 (virulent, red bars), and necropsies were
performed at 1 and 3 days post-infection. Gene expression was normal‑
ized relative to the PBS group. The values and means are shown for the
indicated groups (challenge strain and time point) having at least one
significant difference when compared to the PBS group. * indicates sig‑
nificant differences (P < 0.05) versus the PBS group. Differences between
strains at the same time point are labelled with # and differences between
time points for the same strain are labelled with +, P < 0.05, in both cases. n = 4 for each group. All expression values and significant differences can
be found in Additional file 5. Additional file 9. Gene expression in spleen and liver after S. suis
intranasal inoculation. Samples from spleen (A) and liver (B) were col‑
lected at 1 and 3 days after the intranasal inoculation of S. suis T15 (non-
virulent) and S10 (virulent). Results at 1 day post-inoculation from piglets
inoculated with PBS are also included as control. All the genes found to be
quantifiable are shown irrespectively of their statistical significance. Gene
expression was normalized relative to the PBS group and log2 trans‑
formed. Values are presented as a heat map. Numbers in abscissa axis rep‑
resent animal ID. Color scale was limited to ± 3 and out of bounds values
displayed with the maximum intensity color. Gene functional groups: A.P.P. and Acute P.P.: Acute Phase Proteins; Ad. M.: Adhesion Molecules; Chemo.: Additional file 4. Significant gene expression with < 2-fold changes
in lymph nodes, trachea and lungs after S. suis intranasal inocula‑ Samples from trachea (A), caudal
lung (B), and cranial lung (C) were collected at 1 and 3 days after the intra‑
nasal inoculation of S. suis T15 (non-virulent) and S10 (virulent). Results at
1 day post-inoculation from piglets inoculated with PBS are also included
as control. All the genes found to be quantifiable are shown irrespectively
of their statistical significance. Gene expression was normalized relative
to the PBS group and log2 transformed. Values are presented as a heat
map. Numbers in abscissa axis represent animal ID. Color scale was limited
to ± 4 and out of bounds values displayed with the maximum intensity
color. Gene functional groups: Acute P.P.: Acute Phase Proteins; Chemo.:
Chemokines; P.R.: Pattern Recognition Receptors. Additional file 7. Gene expression in blood. Gene expression from
blood samples, including means, standard deviations, and P-values. This study provides information for understanding
the colonization of S. suis (first step of infection) and the
potential mechanisms involved in the early local innate
immune response, which might either favor colonization
without disease development or rather colonization fol-
lowed by systemic invasion. Our results seem to reflect a
host response to this non-virulent S. suis, which is char-
acterized by rapid control at the site of inoculation, prob-
ably mediated by a sustained immune response at the
associated lymph node. In contrast, the virulent strain
used seem to prevent a robust lymph node response,
and, in consequence, they are maintained at the site of
inoculation, where they continue to elicit inflammatory
mediators. Several factors might dictate these outcomes,
including host and environment factors, as well as the
virulence potential of the strain. Additional file 2. Primer sequences and amplification efficiency.
List of primers used, including primer sequences, amplicon length and
amplification efficiency by tissue. Additional file 2. Primer sequences and amplification efficiency. List of primers used, including primer sequences, amplicon length and
amplification efficiency by tissue. Additional file 3. Gene expression in nasal samples. Gene expression
from nasal swabs samples, including means, standard deviations, and
P-values. Transcriptional results from trachea and lungs indi-
cate that the host response or bacterial spread beyond
the nasal cavity and further down the respiratory tract
for the duration of the experiment was limited. Specific
serotype detection by IHC was achieved in the tissues
in which more mucus remained after the paraffin treat-
ment, like the alveolar sac in lungs or the characteris-
tic sinuous tissue of the cribriform plate of the ethmoid
bone. In other respiratory tissues, bacteria were only
detected in mucus or a few of them attached to the
epithelium, which is consistent with the sub-clinical
infection and the low response observed in the trachea. Colonization thus appears to primarily affect the host
response locally at the site of colonization, with little or
no widely disseminated response beyond the nasal cav-
ity. Regarding the localization of S. suis S10 detected in
the cribriform plate of the ethmoid, it cannot be ruled
out that this site may serve as a non-hematogenous
route to the central nervous system. This route has pre-
viously been suggested for Streptococcus pneumoniae
[44] and demonstrated for others bacteria such as Neis-
seria meningitidis and Burkholderia pseudomallei [45,
46], as well as for ameboflagellates (Naegleria fowleri;
[47]) or viruses (SARS-CoV-2; [48]). This hypothesis
deserves further analysis.h Additional file 4. Significant gene expression with < 2-fold changes
in lymph nodes, trachea and lungs after S. suis intranasal inocula‑ Funding This work was supported by the European Commission through Grant
number 727966 (Program for Innovative Global Prevention of Streptococcus
suis, PIGSs) from program Horizon 2020. CN-I and LB were funded by the same
project. Funding institution had no role in the design of the study; in the col‑
lection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results. 7. Kerdsin A, Akeda Y, Hatrongjit R, Detchawna U, Sekizaki T, Hamada S,
Gottschalk M, Oishi K (2014) Streptococcus suis serotyping by a new mul‑
tiplex PCR. J Med Microbiol 63:824–830. https://doi.org/10.1099/jmm.0.
069757-0 8. Okura M, Osaki M, Nomoto R, Arai S, Osawa R, Sekizaki T, Takamatsu D
(2016) Current taxonomical situation of Streptococcus suis. Pathogens
5:45. https://doi.org/10.3390/pathogens5030045 Availability of data and materials 9. Goyette-Desjardins G, Auger JP, Xu J, Segura M, Gottschalk M (2014)
Streptococcus suis, an important pig pathogen and emerging zoonotic
agent-an update on the worldwide distribution based on serotyping and
sequence typing. Emerg Microbes Infect 3:e45. https://doi.org/10.1038/
emi.2014.45 All data generated or analysed during this study are included in this published
article (and its additional files). Acknowledgements Dr Xavier Moll-Sánchez and Dr Félix García-Arnas from the Departament
de Medicina i Cirurgia Animal and Hospital Clinic Veterinari of the Universitat
Autònoma de Barcelona (Spain) are thanked for performing cesareans sections. BSL3 personnel from IRTA-CReSA is acknowledged for their excellent care
of the animals, and Mónica Pérez from IRTA-CReSA for the histopathology
laboratory work. Karin Tarp and Seyed Hossein Mirbarati from DTU are thanked
for assistance with RNA isolation from tissues. CN-I, LP-G, JM, JS and VA are also
grateful to the Centres de Recerca de Catalunya (CERCA) Programme. 3. O’Dea MA, Laird T, Abraham R, Jordan D, Lugsomya K, Fitt L, Gottschalk
M, Truswell A, Abraham S (2018) Examination of Australian Streptococ-
cus suis isolates from clinically affected pigs in a global context and the
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Diseases in Europe (IRTA-CReSA), Bellaterra, Barcelona, Spain. 3 Section for Pro‑
tein Science and Biotherapeutics, DTU Bioengineering, Technical University
of Denmark, Kongens Lyngby, Denmark. 4 UAB, Centre de Recerca en Sanitat
Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona,
08193 Bellaterra, Spain. 5 Departament de Sanitat I Anatomia Animals, Facultat
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Diseases Research Centre, Faculty of Veterinary Medicine, University of Mon‑
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29. R Core Team (2020) R: A language and environment for statistical com‑
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Exacerbated type II interferon response drives hypervirulence and toxic
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PFO-DBT:MEH-PPV:PC71BM Ternary Blend Assisted Platform as a Photodetector
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Sensors 2015, 15, 965-978; doi:10.3390/s150100965 Sensors 2015, 15, 965-978; doi:10.3390/s150100965 sensors
ISSN 1424-8220
www.mdpi.com/journal/sensors
OPEN ACCESS sensors
ISSN 1424-8220
www.mdpi.com/journal/sensors
OPEN ACCESS Article Keywords: PFO-DBT; MEH-PPV; ternary blend; organic photodetector 1. Introduction Solution-processable organic photodetectors have attracted significant R&D efforts as a promising
alternative to inorganic semiconductor devices [1,2] by virtue of their low-cost, device architectural flexibility
and large-scale fabrication capability [3–5]. To date, the organic bulk heterojunction approach, particularly
intimately blended D/A binary blend is the dominant theme for image sensing application [6,7]. The
operating principle of BHJ OPDs relies on the efficient dissociation of the photoinduced geminate carrier
pairs at D/A intermolecular contacts. Photodetection parameters intensively rely on electrical properties
including HOMO/LUMO energy levels, the charge carrier mobility of the donor (D) and acceptor (A)
materials as well as the structure of the photodiode. Hoppe et al. report that enhanced interfacial area
between D/A phases within BHJ device design enables efficient charge separation as compared to planar
interface of bilayer devices [8]. For bulk heterojunction approach, the photons absorbed throughout the
interpenetrating network of D/A materials contribute to the photogenerated current. Composite of
judiciously chosen pair of conjugated polymer donors and acceptor material thus yield high photoinduced
charge generation and pronounced charge transfer [9]. Several binary blend-based photo sensing platforms have been proposed so far. In our previous studies,
we have explored a variety of D/A combinations for the applications in organic solar cells and OPDs [10–13]. The main challenge of OPDs design engineering is to develop the sensing layer that can harvest a wide
range of incident photon energies. Optoelectronic devices based on BHJ consisting of two components,
exhibit limited light-harvesting range. Usually, the acceptor materials in D/A binary blend contribute very
little to the light harvesting, furthermore, their selection range to make a suitable D/A combination is also
limited [14]. Light is mainly absorbed by donor moiety (i.e., conjugated polymer) in the blend only [15]. Hence in principle, an ideal donor polymer should exhibit high hole mobility coupled with the broad
absorption spectra to match with visible solar terrestrial radiation [16]. Ternary blend bulk heterojunction
(BHJ) provides a distinct platform and an alternate route to enhance the absorption bandwidth of OPD’S
while maintaining the ease and benefits of a single photoactive layer. Summation of the individual
absorption spectra of two distinct donors leads to a noticeably strong and wide absorption spectrum spanning
over the long range. Yet another benefit of incorporating a second donor material within the photoactive
layer is that charge transfer barriers are believed to reduce in the resulting D/A ternary blend as well [17]. Qayyum Zafar, Zubair Ahmad * and Khaulah Sulaiman Low Dimensional Materials Research Centre (LDMRC), Department of Physics, Faculty of Science,
University of Malaya, 50603 Kuala Lumpur, Malaysia; E-Mails: qayyumzafar@siswa.um.edu.my (Q.Z.);
khaulah@um.edu.my (K.S.) * Author to whom correspondence should be addressed; E-Mail: zubairtarar@um.edu.my;
Tel.: +60-3-7967-4054; Fax: +60-3-7967-4146. * Author to whom correspondence should be addressed; E-Mail: zubairtarar@um.edu.my;
Tel.: +60-3-7967-4054; Fax: +60-3-7967-4146. Received: 27 March 2014 / Accepted: 3 December 2014 / Published: 7 January 2015 Received: 27 March 2014 / Accepted: 3 December 2014 / Published: 7 January 2015 Received: 27 March 2014 / Accepted: 3 December 2014 / Published: 7 January 2015 Abstract: We present a ternary blend-based bulk heterojunction ITO/PEDOT:PSS/PFO-DBT:
MEH-PPV:PC71BM/LiF/Al photodetector. Enhanced optical absorption range of the active
film has been achieved by blending two donor components viz. poly[2,7-(9,9-di-octyl-
fluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2,1,3-thiadiazole]
(PFO-DBT)
and
poly(2-
methoxy-5(2'-ethylhexyloxy) phenylenevinylene (MEH-PPV) along with an acceptor
component, i.e., (6,6)-phenyl-C71 hexnoic acid methyl ester. The dependency of the
generation rate of free charge carriers in the organic photodetector (OPD) on varied incident
optical power density was investigated as a function of different reverse biasing voltages. The photocurrent showed significant enhancement as the intensity of light impinging on active
area of OPD is increased. The ratio of Ilight to Idark of fabricated device at −3 V was ~3.5 × 104. The dynamic behaviour of the OPD under on/off switching irradiation revealed that sensor
exhibits quick response and recovery time of <800 ms and 500 ms, respectively. Besides
reliability and repeatability in the photoresponse characteristics, the cost-effective and
eco-friendly fabrication is the added benefit of the fabricated OPD. Keywords: PFO-DBT; MEH-PPV; ternary blend; organic photodetector Keywords: PFO-DBT; MEH-PPV; ternary blend; organic photodetector Sensors 2015, 15 Sensors 2015, 15 966 Sensors 2015, 15 Sensors 2015, 15 967 present study, PC71BM has been utilized by virtue of its enhanced visible light absorption as compared to
PC61BM [22]. The motivation to choose these MEH-PPV, PFO-DBT and PC71BM materials, is
continuation and improvement in our previous studies. Ternary blend seems to be a potential approach
for tailoring the sensing parameters of OPD which is otherwise not possible by using binary BHJ
approach. In our present systematic study, we therefore aim to achieve relative performance increase by
increasing the spectral sensitivity of the photoactive film, which is prerequisite for improvement in
sensing parameters of the OPD. The spectral responses of the PFO-DBT:PCBM and MEH-PPV:PCBM
devices have previously been investigated by Zhou et al. [23]. It is found that the photoresponse of
PFO-DBT:PCBM device is 50–60 nm more toward the higher wavelength as compared to the
MEH-PPV:PCBM device. This gives the clue that if we make the blend of PFO-DBT:MEH-PPV:PCBM
then the improved spectral response towards the higher wavelengths can be obtained. A ternary blend of
PFO-DBT:MEH-PPV:PC71BM is therefore expected to yield improved sensing parameters, i.e., better
photoresponsivity and an enhancement in photo to dark current ratio. 1. Introduction Ternary blend approach has already been utilized to enhance the power conversion efficiency (PCE) of solar
cells [18–20], however much less attention has been paid to utilize it for photodetection application. In our previous studies, we demonstrated MEH-PPV:VOPCPhO [11] and MEH-PPV:Alq3 [12] binary
blend-based photodetectors. MEH-PPV exhibits fairly good hole mobility and environmental stability [21]. However, the photosensitivities and the photo to the dark current ratios of both binary blends-based OPDs
were relatively lower. In the present work, we aim to demonstrate more sensitive visible wavelength
photodetector with two donor components i.e., MEH-PPV and PFO-DBT in order to enhance the
photosensitivity and the photo to the dark current ratio. Conjugated conducting polymer PFO-DBT is used
as an electron donor component in photoactive layer of organic solar cells. However, it is ineffective to
harvest light in shorter wavelength ranges near 450 nm, thereby limiting the overall device performance. MEH-PPV on the other hand harvests light efficiently in this visible wavelength range. Fullerene derivative
PCBM, undoubtedly remains an ubiquitously utilized acceptor material by virtue of its excellent ability to
induce ultrafast electron transfer and transport properties, in contrast to VOPCPhO and Alq3. In the Sensors 2015, 15 Sensors 2015, 15 Sensors 2015, 15 968 aqueous solution coating. The optical absorption study in visible spectral range was carried out using a
UV-Vis-NIR spectrophotometer (Shimadzu UV-3101PC). The photoluminescence (PL) dynamics of
active film in the visible and near-infrared-spectral ranges were studied by using RENISHAW in via a
Raman microscope instrument. The RENISHAW in via Raman microscope instrument uses the 325 nm
laser wavelength and the detector limits of this system are 400–1000 nm. The I-V characteristics of the
OPD were measured under different light intensities (0–150 mW/cm2) using an Oriel 67,005 solar simulator
and were recorded by a Keithley 236 source measuring unit (SMU). (a)
(b)
(c)
Figure 1. The molecular structure of (a) PFO-DBT (b) MEH-PPV and (c) PC71BM. (b) (a)
(b)
(c)
Figure 1. The molecular structure of (a) PFO-DBT (b) MEH-PPV and (c) PC71BM. (a)
(
(c) (a) (b) (c) Figure 1. The molecular structure of (a) PFO-DBT (b) MEH-PPV and (c) PC71BM. 2. Experimental Poly[2,7-(9,9-di-octyl-fluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2,1,3-thiadiazole] (PFO-DBT) and
[6,6]-phenyl-C71-butyric-acid methyl ester (PC71BM) were obtained from a commercial supplier
Luminescence Technology Corp. (Taiwan, China). No further purification was done and they were
used as received. Poly[2-methoxy-5-(2'-ethylhexyloxy)-p-phenylenevinylene] (MEH-PPV) was
synthesized by established Gilch’s polymerization reaction [24]. The aqueous solution of poly(3,4-
ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) with PH ~1000 and conductivity 900–1000
S/cm was commercially obtained from H.C. Starck (Goslar, Germany ). Molecular structures of
PFO-DBT, MEH-PPV and PC71BM are depicted in Figure 1. Commercially available ITO substrates
(25 mm × 25 mm slides, sheet resistance ~12 Ω/sq), were cleaned according to a well-established protocol
i.e., by ultrasonic agitation in acetone, ethanol, isopropyl alcohol and DI water, respectively, and later
substrates were dried clean by blown nitrogen. An anode buffer layer of PEDOT:PSS was then
spun-coated onto ITO substrates at 3000 rpm for 30 s. The resulting transparent (~40 nm thick)
PEDOT:PSS thin film was annealed at 120 °C for half an hour. Thirty mg/mL concentrated solutions for
PFO-DBT, MEH-PPV and PC71BM each, were prepared separately in chloroform by stirring them
overnight at room temperature. The photoactive layer of PFO-DBT:MEH-PPV:PC71BM ternary blend
with optimum ratio was spun cast on top of PEDOT:PSS layer. Spin speed was maintained at 4000 rpm,
yielding a thickness of 120 nm. Photoactive layer deposition was followed by baking at 120 °C for half
an hour. The optimum mixing ratio (by volume) of donor binary blend was selected by the help of
photoluminescence (PL) study. A thin film of LiF (~10 Å) was used between active layer and top
electrode to enhance the performance of the OPD. Two of the several suggested advantages of LiF coating
are: (a) lowering of the effective work function of top Al cathode and (b) protection of photoactive layer
from hot Al atoms during its deposition [25]. Both LiF and aluminium (Al) were thermally deposited via
shadow mask. The fabricated devices were in circular shapes with diameter ~1 mm. The evaporation rates
for LiF and Al cathodes were controlled to be 0.1 and 3 nm/s to achieve a thickness of 0.8 and 100 nm,
respectively. Finally the fabricated devices were annealed at 120 °C for 30 min. The entire fabrication
process of OPD was carried out into a glove-box with inert N2 atmosphere, except for the PEDOT:PSS 3. Results and Discussion The layer stack of the fabricated OPD (ITO/PEDOT:PSS/PFO-DBT:MEH-PPV:PC71BM/LiF/Al)
and energy level diagram of the components have been depicted in Figure 2a,b, respectively. Figure 2b
shows the energy levels of PFO-DBT, MEH-PPV and PC71BM and work functions of ITO and Al. The
HOMO/LUMO energy levels were obtained from material safety data sheets (MSDS) provided by
the manufacturer of the materials. Same values have also been reported by other researchers in the
literature [23,26,27]. Inorganic fluorides such as LiF have good electron extraction ability from active layer,
further they serve as a hole blocking layer as well [28]. PEDOT:PSS on the contrary, serves as a buffer
layer for hole collection at the ITO anode. The energetic position of the frontier levels of the D/A materials
used in the present work ensures better flow of photogenerated charges to the appropriate direction. The
potential offset at the D/A interfaces provide adequate energy to dissociate electron-hole pairs. The
transport and collection of mobile charge carriers at the electrodes is then facilitated by the external bias
and built-in electric field between PEDOT:PSS and Al work functions. 969 Sensors 2015, 15 (a)
(b)
Figure 2. (a) Schematic view of the device geometry, ITO/PEDOT:PSS/PFO-DBT:MEH-
PPV:PC71BM/LiF/Al with ternary blended donor-acceptor structure and (b) the energy bands
of their component materials. (a) (a) (b) Figure 2. (a) Schematic view of the device geometry, ITO/PEDOT:PSS/PFO-DBT:MEH-
PPV:PC71BM/LiF/Al with ternary blended donor-acceptor structure and (b) the energy bands
of their component materials. Figure 3, compares the optical absorption spectra of the photoactive donor polymers: MEH-PPV,
PFO-DBT and acceptor moiety PC71BM. The peak optical absorption of MEH-PPV is at 510 nm, which
indicates that substantial portion of incident visible light is not harvested by MEH-PPV alone. The
motivation here is to extend the absorption profile of the photoactive film of OPD still further toward
longer wavelengths. The approach used to achieve a comprehensive coverage of the solar spectrum is the
addition of other polymeric donor material with a complimentary absorption profile. For instance, PFO-DBT
exhibits a light complementary absorption to that of MEH-PPV. The UV-VIS spectroscopic data confirms
that PFO-DBT dominates the spectral absorbance above 590 nm, where either of MEH-PPV and PC71BM
fails to absorb photons. The absorption spectrum of PFO-DBT exhibits two maxima; the first is located
at 384 and the second at 540 nm. Yong et al. Sensors 2015, 15 Sensors 2015, 15 devices have already been reported [23]. Further, it can be cross-referenced that the spectral response is
in good agreement with the independent material’s absorption spectra. Figure 3. UV-Vis spectra of PFO-DBT, MEH-PPV, PC71BM neat materials and their ternary
blend thin film. 400
500
600
700
0.0
0.2
0.4
0.6
0.8
1.0
Normalized Absorbance (a.u)
Wavelenghth (nm)
PFO-DBT
PC71BM
MEH-PPV
Blend Figure 3. UV-Vis spectra of PFO-DBT, MEH-PPV, PC71BM neat materials and their ternary
blend thin film. The photoluminescence measurement (PL) of the thin films of PFO-DBT:MEH-PPV binary blend
was obtained by an excitation wavelength of 325 nm in the range from 400 to 1000 mm. Figure 4 (inset)
depicts the PL spectra of thin films of MEH-PPV, PFO-DBT and PC71BM independently. Figure 4, presents
the PL spectrum of binary blend for six different volume ratios (1:0.4, 1:0.6, 1:0.8, 1:1, 1:1.2 and 1:1.4). The photoluminescence (PL) of binary blend has been studied in order to obtain optimized volumetric ratio
providing enhanced photoinduced exciton splitting efficiency within the photoactive layer. The binary
blend exhibits strong emission peak at 700 nm wavelength for all volumetric ratios. At 1:0.6 volumetric
ratio of the binary blend, the PL intensity is markedly quenched, indicating a more efficient charge transfer
between the organic materials. Emission occurs when the photogenerated excitons recombine emissively
instead of splitting into free carriers. Quenching in emission spectra on contrary is an indicator of how
well excitons split into free charges instead of undergoing emissive recombination [8]. Subsequently
improvement in photocurrent can thus be realized for 1:0.6 optimized volumetric ratio of binary blend. PC71BM was incorporated as an acceptor material in the binary donor blend. 1:0.8 volumetric fraction of
D:A has been used in the ternary blend. As it is well known, the absorption of incident photon flux in donor
moiety of BHJ blend generates columbically bound electron-hole bound pairs called excitons. These
photogenerated excitons can either relax back to the ground state or dissociate at D/A interface into free
electrons and holes. Since the photoresponse of OPD results from photogeneration of free charge carriers
so exciton must be dissociated to mobile charges. Splitting of photogenerated excitons is caused by the
difference in electron affinities at the D/A interface of BHJ blend. 3. Results and Discussion [29] suggest that absorption band at smaller wavelengths is
attributed to fluorine segment and the additional longer wavelength absorption band is attributed to DBT
unit of the PFO-DBT polymer. By virtue of complimentary absorption profile, the inlay of PFO-DBT to
the MEH-PPV-PC71BM is believed to yield a broader and consistent absorption spectrum. Further,
PC71BM exhibits strong absorption ability at shorter wavelengths (350–500 nm) [30], hence it is potentially
adopted as an acceptor material in BHJ solar cells with relatively higher power conversion efficiency
(PCE) [31–33]. The spectral response of the MEH-PPV:PCBM and PFO-DBT:PCBM based photovoltaic 970 Sensors 2015, 15 Sensors 2015, 15 electrodes to create photocurrent in external circuit. The whole photodetection phenomenon can thus be
summed up as the generation, separation and transport of charge carriers towards their respective electrodes. Figure 4. PL spectra of PFO-DBT: MEH-PPV blends at different volumetric ratio. (Inset)
PFO-DBT, MEH-PPV and PC71BM PL spectra, when studied independently. 400
500
600
700
800
900
-1x10
4
0
1x10
4
2x10
4
3x10
4
4x10
4
5x10
4
6x10
4
7x10
4
8x10
4
500
600
700
800
900
0.0
5.0x10
3
1.0x10
4
1.5x10
4
2.0x10
4
2.5x10
4
PL Intensity (au)
Wavelength (nm)
MEH-PPV
PFO-DBT
PC71BM
PL Intensity (a.u.)
Wavelength (nm)
1:0.4
1:0.6
1:0.8
1:1
1:1.2
1:1.4
PFO-DBT:MEH-PPV Figure 4. PL spectra of PFO-DBT: MEH-PPV blends at different volumetric ratio. (Inset)
PFO-DBT, MEH-PPV and PC71BM PL spectra, when studied independently. Typically, the photodetector functions as a photodiode in photoconductive mode and is usually operated
in reverse biased configuration. The use of two electrodes (ITO and Al) with different work function,
ensures the diodic behaviour as is evident from characteristic IV curves of diode Figure 5 (inset). With the
increase in optical power density, the generation rate of mobile charge carriers increases and resultantly
photodetector outputs an increased photocurrent. This aforementioned phenomenon forms the basis of
operation principle of photo detector. To evaluate the performance of the ternary blend based OPD, its
reversed biased current-voltage (I-V) characteristics were investigated. Figure 5, shows the photoresponse
of the OPD when exposed to simulated light of varied illumination levels. It can be observed that the
reverse biased current in dark condition (Idark) is quite low as compared to the photo induced current
(Ilight) under the influence of simulated light illumination. It is also evident from Figure 5 that with higher
influx of incident photons on OPD; a higher density of separated charges approaches the electrodes,
giving rise to pronounced magnitude of photocurrent. Furthermore, from Figure 6, it can be inferred that
magnitude of photoresponse is increased with the increase in the electric field (sourced by external reverse
bias voltage). In fact, the couple of bottlenecks to better photoresponse are: (1) enhanced photon absorption;
(2) efficient creation and splitting of excitons into independent holes and electrons; (3) efficient transportation
of charges to collection electrodes. The bulk heterojunction blend approach helps only to improve both
photon absorption and exciton dissociation. Sensors 2015, 15 Band diagram of the ternary blend
(depicted in Figure 2b) indicates desirable offsets in frontier orbital energies (HOMO/LUMO levels) that
are sufficient to overcome the columbic forces present between the photoinduced excitons. The same
built-in potential is the reason of the electron drift, towards LUMO of the material with the larger electron
affinity (PC71BM acceptor phase) and hole drift, toward HOMO of the materials with the lower ionization
potential (MEH-PPV and PFO-DBT donor phases). These separated charges are then transported to the 971 Sensors 2015, 15 However the migration of separated charges towards electrodes
is external field assisted. External biasing serves as a driving force for transportation of charge carriers
through interpenetrating D/A networks. The transportation becomes faster and efficient at higher order
of potential bias, resulting in improved photoresponse. However at lower biasing, mobile charges are
more likely to get trapped or encounter recombination rather than being collected at the electrodes. 972 Sensors 2015, 15 Figure 5. The photoresponce characteristics of the OPD (ITO/PEDOT:PSS/PFO-
DBT:MEH-PPV:PC71BM/LiF/Al)
illustrating
photocurrent
dependence
on
light
illumination levels. The measurements were recorded under different light intensities from
0 to 150 mW/cm2, at room temperature. Inset shows the I-V characteristics of the OPD under
forward and reverse biased condition at room temperature. -3.0
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
-0.6
-0.4
-0.2
0.0
-2
-1
0
1
2
3
-0.3
0.0
0.3
0.6
0.9
Current Density (mA/cm
2)
Voltage/V
Illumination ~ 100 mW/cm
2
Current Density (mA/cm
2)
Voltage/V
dark
50 mW/cm
2
75 mW/cm
2
100 mW/cm
2
125 mW/cm
2
150 mW/cm
2 -3.0
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
-0.6
-0.4
-0.2
0.0
-2
-1
0
1
2
3
-0.3
0.0
0.3
0.6
0.9
Current Density (mA/cm
2)
Voltage/V
Illumination ~ 100 mW/cm
2
Current Density (mA/cm
2)
Voltage/V
dark
50 mW/cm
2
75 mW/cm
2
100 mW/cm
2
125 mW/cm
2
150 mW/cm
2 Voltage/V Figure 5. The photoresponce characteristics of the OPD (ITO/PEDOT:PSS/PFO-
DBT:MEH-PPV:PC71BM/LiF/Al)
illustrating
photocurrent
dependence
on
light
illumination levels. The measurements were recorded under different light intensities from
0 to 150 mW/cm2, at room temperature. Inset shows the I-V characteristics of the OPD under
forward and reverse biased condition at room temperature. Figure 6. Light intensity v’s. current density measurements for ITO/PEDOT:PSS/PFO-
DBT:MEH-PPV:PC71BM/LiF/Al photodetector at different bias voltages (0 to −3 V) at
room temperature. 0
20
40
60
80
100
120
140
-0.60
-0.50
-0.40
-0.30
-0.20
-0.10
0.00
Current Density (mA/cm
2)
Light Intensity (mW/cm
2)
0.0 Volts
- 0.5 Volts
- 1.0 Volts
- 1.5 volts
- 2.0 Volts
- 2.5 Volts
- 3.0 Volts Figure 6. Light intensity v’s. current density measurements for ITO/PEDOT:PSS/PFO-
DBT:MEH-PPV:PC71BM/LiF/Al photodetector at different bias voltages (0 to −3 V) at
room temperature. Table 1, compares the sensing parameters of the present ternary blend based OPD and binary blend
based OPDs previously reported by our research group. Sensing parameters have been compared at −3 V
applied bias. Sensors 2015, 15 Current Density (A/cm2)
MEH-PPV:PCBM [34]
Bias = −3.5 V
PFO-DBT:MEH-PPV:PC71BM
Bias = −3.0 V
Dark Current Density (D)
−2.49 E−7
−1.7 E−8
Photo Current Density (P)
−3.79 E−5
−6.0 E−4
Ratio = P/D
152.29
3.5 E4 The temporal response is another important parameter of the photodetector and is determined by the
average exciton lifetime in photoactive film. Reasonably fast response times can be achieved by
incorporating thinner photoactive films in OPDs [38]. The fabricated OPD demonstrates a sharp change in
its magnitude of photocurrent in response to periodic pulsed simulated solar light (irradiance
~100 mW/cm2, pulse width equals 20 s) as shown in Figure 7. The presented sensor has been investigated
as a whole visible light detector, therefore a solar simulator has been used to impinge whole visible
spectrum on the sensing area instead of a monochromator. The photodetector was externally biased at
−3 V, while probing the time dependent photoresponse characteristics as a function of light modulation
ON/OFF. As explained in the photocurrent generation mechanism earlier, when light impinges on the active
film it causes a progressive rise of the photocurrent amplitude by virtue of photogenerated charges. The
resulting time-resolved photocurrent response is gradual, stable and repeatable as evidenced by three
successive cycles of abrupt switching between ON and OFF states of illumination. The initial photocurrent
spike observed during light exposure is due to the momentary surge in illumination level, which is
commonly observed when a halogen bulb is switched on. As the illumination level of the bulb stabilizes
to 100 mW/cm2, photocurrent also tends to stabilize after the momentary initial spike. For our present
OPD, in response to sudden light irradiation, the photocurrent density progressively increased from
~−0.168 nA/cm2 to −0.330 mA/cm2. The normalized transient reverse biased photocurrent behavior has
been depicted in Figure 7. The time required for the photocurrent to increase from 10% to 90% of the
final settled value or vice versa is generally defined as the rise and decay time (tr and td), respectively [39]. Figure 7, reveals the operation speed of OPD, in present case the rise time (tr) is about 800 ms, and fall
time (tf) is 500 ms. For reference, typically the response times for bulk heterojunction based OPDs are in
the order of (10−8 to 10−3) s [40]. Similar fast response time are observed in the case of inorganic
photodiodes, Liu et al. Sensors 2015, 15 The ratio of Ilight to Idark of fabricated device at −3 V is ~3.5 × 104, which is significantly
higher as compared to our previous study [11]. Similarly, the response/recovery times of the sensor have
also showed marked improvement comparatively. The photoresponsive parametrs of ternary blend based
OPD have also been compared with those of MEH-PPV:PCBM binary blend based OPD, reported earlier
by Barai [34]. Table 2, signifies that with the incorporation of PFO-DBT in MEH-PPV:PCBM blend,
the fabricated OPD has shown marked improvement in its photo response characteristics. For reference, 973 Sensors 2015, 15 typically the response times and responsivity for bulk heterojunction based OPDs are in the order of ms
and mA/W respectively [35–37]. typically the response times and responsivity for bulk heterojunction based OPDs are in the order of ms
and mA/W respectively [35–37]. Table 1. Comparison of the sensor’s parameters at −3 V biasing at 100 mW/cm2. Table 1. Comparison of the sensor’s parameters at −3 V biasing at 100 mW/cm2. OPDs
IPh/IDark
Responsivity
Response and Recovery Time
MEH-PPV:VOPCPhO [11]
5.9
5 × 10−4 mA/W
~4 s both
PFO-DBT:MEH-PPV:PC71BM
3.5 × 104
3.9 mA/W
~800 and ~500 ms
Table 2. Comparison of the binary and ternary blend based sensor’s parameters at reverse biasing. Current Density (A/cm2)
MEH-PPV:PCBM [34]
Bias = −3.5 V
PFO-DBT:MEH-PPV:PC71BM
Bias = −3.0 V
Dark Current Density (D)
−2.49 E−7
−1.7 E−8
Photo Current Density (P)
−3.79 E−5
−6.0 E−4
Ratio = P/D
152.29
3.5 E4 Table 1. Comparison of the sensor’s parameters at −3 V biasing at 100 mW/cm2. OPDs
IPh/IDark
Responsivity
Response and Recovery Time
MEH-PPV:VOPCPhO [11]
5.9
5 × 10−4 mA/W
~4 s both
PFO-DBT:MEH-PPV:PC71BM
3.5 × 104
3.9 mA/W
~800 and ~500 ms
Table 2. Comparison of the binary and ternary blend based sensor’s parameters at reverse biasing. Current Density (A/cm2)
MEH-PPV:PCBM [34]
Bias = −3.5 V
PFO-DBT:MEH-PPV:PC71BM
Bias = −3.0 V
Dark Current Density (D)
−2.49 E−7
−1.7 E−8
Photo Current Density (P)
−3.79 E−5
−6.0 E−4
Ratio = P/D
152.29
3.5 E4 OPDs
IPh/IDark
Responsivity
Response and Recovery Time
MEH-PPV:VOPCPhO [11]
5.9
5 × 10−4 mA/W
~4 s both
PFO-DBT:MEH-PPV:PC71BM
3.5 × 104
3.9 mA/W
~800 and ~500 ms mparison of the binary and ternary blend based sensor’s parameters at reverse biasing. Sensors 2015, 15 for instance, reported response time on the order of microseconds for photodiode
based on high quality ZnO epitaxial films on sapphire substrates [41]. It is worth noting that the ternary
blend based photodetector exhibited fast response and recovery times as compared to binary blend based
devices previously reported by our group (refer to Table 1). The repeatability and reproducibility of the 974 4. Conclusions A solution processable ternary blend-based OPD with improved sensing performance has been
successfully demonstrated without sacrificing the attractive simplicity of single photoactive layer
fabrication design. Marked improvement in sensing parameters, i.e., IPh/IDark and switching time between
stable dark and illuminated states have been observed. The reason for the higher sensitivity may be attributed
to judicious component and compositional selection of the ternary blend. The addition of PFO-DBT in
the MEH-PPV:PC71BM blend significantly enhances the absorption profile of photoactive layer, thereby
increasing the magnitude of photocurrent and improvement in sensing parameters of OPD. Time resolved
measurements of photocurrent response as a function of pulsed illumination revealed response and
recovery times as fast as 800 ms and 500 ms, respectively. Sensors 2015, 15 Sensors 2015, 15 response/recovery time measurements were investigated for five devices and similar robust stability and
reproducibility in response has been observed. Figure 7. The response time of the ITO/PEDOT:PSS/PFO-DBT:MEH-PPV:PC71BM/LiF/Al
under pulsed optical illumination with the intensity of 100 mW/cm2 at −3 V biasing. The
maximum value of current density was ~0.4 mA/cm2 under 100 mW/cm2 at −3 V biasing. 0
20
40
60
80
100
0.0
0.2
0.4
0.6
0.8
1.0
Normalized reverse biased
current density
Time/s Figure 7. The response time of the ITO/PEDOT:PSS/PFO-DBT:MEH-PPV:PC71BM/LiF/Al
under pulsed optical illumination with the intensity of 100 mW/cm2 at −3 V biasing. The
maximum value of current density was ~0.4 mA/cm2 under 100 mW/cm2 at −3 V biasing. Figure 8 depicts the external quantum efficiency (EQE) spectrum for the fabricated OPD. The EQE
spectrum was obtained by using a xenon lamp (150 W Oriel), a monochrometer and with the help of a
calibrated Si-photodetector. It can be inferred from the spectrum that the ternary blend helps to enhance
the light utilization efficiency of the active film, which is vital for improved sensing parameters. EQE
spectrum matches well with the visible absorption spectrum. Compared to the absorption spectrum of
the ternary blend, the photocurrent from 450 nm to 650 nm is mainly due to the two donor materials i.e.,
MEH-PPV and PFO-DBT. Light harvesting at shorter wavelength i.e., near 450 nm is mainly contributed
by MEH-PPV phase whereas at higher wavelengths, PFO-DBT phase contributes more towards light
harvesting. The photosensitivity spectra of the binary blend based photodetectors of MEH-PPV-PCBM
and PFO-DBT-PCBM have already been investigated by Zhou et al. [23]. It can be cross-referenced that
the EQE spectrum of the ternary blend covers the detection range from 450 nm to 650 nm which consist
of the sensitivity range of the both photodetectors i.e., MEH-PPV-PCBM and PFO-DBT-PCBM. Figure 8. EQE spectrum of the ITO/PEDOT:PSS/PFO-DBT:MEH-PPV:PC71BM/LiF/Al photodetector. 450
500
550
600
650
700
750
0.0
0.1
0.2
0.3
0.4
0.5
EQE (%)
Wavelength (nm) Figure 8. EQE spectrum of the ITO/PEDOT:PSS/PFO-DBT:MEH-PPV:PC71BM/LiF/Al photodetector. Sensors 2015, 15 Sensors 2015, 15 975 Acknowledgment This research is supported by High Impact Research MoE Grant UM.S/625/3/HIR/MoE/SC/26 from
the Ministry of Education Malaysia. Author Contributions Qayyum Zafar has conducted the experimental work whereas the experimental designing has been
done by Zubair Ahmad. Khaulah Sulaiman has made the significant intellectual contribution to the paper
by supervising this research work. Conflicts of Interest The authors declare no conflict of interest. The authors declare no conflict of interest. References 1. Monroy, E.; Calle, F.; Pau, J.; Munoz, E.; Omnes, F.; Beaumont, B.; Gibart, P. AlGaN-based UV
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https://openalex.org/W2296145362
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https://biotechnologyforbiofuels.biomedcentral.com/track/pdf/10.1186/s13068-016-0469-2
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English
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Hyper-accumulation of starch and oil in a Chlamydomonas mutant affected in a plant-specific DYRK kinase
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Biotechnology for biofuels
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cc-by
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© 2016 Schulz‑Raffelt et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International
License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any
medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons
license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.
org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Because of their high biomass productivity and their ability to accumulate high levels of energy-rich
reserve compounds such as oils or starch, microalgae represent a promising feedstock for the production of biofuel. Accumulation of reserve compounds takes place when microalgae face adverse situations such as nutrient shortage,
conditions which also provoke a stop in cell division, and down-regulation of photosynthesis. Despite growing inter‑
est in microalgal biofuels, little is known about molecular mechanisms controlling carbon reserve formation. In order
to discover new regulatory mechanisms, and identify genes of interest to boost the potential of microalgae for biofuel
production, we developed a forward genetic approach in the model microalga Chlamydomonas reinhardtii. Results: By screening an insertional mutant library on the ability of mutants to accumulate and re-mobilize reserve
compounds, we isolated a Chlamydomonas mutant (starch degradation 1, std1) deficient for a dual-specificity tyrosine-
phosphorylation-regulated kinase (DYRK). The std1 mutant accumulates higher levels of starch and oil than wild-type
and maintains a higher photosynthetic activity under nitrogen starvation. Phylogenetic analysis revealed that this
kinase (named DYRKP) belongs to a plant-specific subgroup of the evolutionarily conserved DYRK kinase family. Fur‑
thermore, hyper-accumulation of storage compounds occurs in std1 mostly under low light in photoautotrophic con‑
dition, suggesting that the kinase normally acts under conditions of low energy status to limit reserve accumulation. Conclusions: The DYRKP kinase is proposed to act as a negative regulator of the sink capacity of photosynthetic
cells that integrates nutrient and energy signals. Inactivation of the kinase strongly boosts accumulation of reserve
compounds under photoautotrophic nitrogen deprivation and allows maintaining high photosynthetic activity. The
DYRKP kinase therefore represents an attractive target for improving the energy density of microalgae or crop plants. Keywords: Chlamydomonas, DYRK, Kinase, Microalgae, Nutrient deprivation, Oil, Photosynthesis, Starch reserve compounds including starch [5] convertible into
bioethanol and oil convertible into biodiesel [1, 6], algal
productivity must be improved in order to implement
economically viable biofuel production [7]. One of the
major limitations is the requirement for stress conditions
such as nutrient deprivation to trigger accumulation of
reserve compounds, as this results in decreased biomass
productivity [1]. In natural environments, photosyn-
thetic organisms have developed sophisticated strate-
gies to optimize growth and survival under constantly
fluctuating conditions of light, temperature, or nutri-
ent availability. Deprivation of essential macronutrients *Correspondence: gilles.peltier@cea.fr
1 CEA, CNRS, Aix‑Marseille Université, Institut de Biosciences et
Biotechnologies Aix Marseille, Laboratoire de Bioénergétique et
Biotechnologie des Bactéries et Microalgues, CEA Cadarache,
13108 Saint‑Paul‑lez‑Durance, France
Full list of author information is available at the end of the article Biotechnology for Biofuels Biotechnology for Biofuels Biotechnology for Biofuels Schulz‑Raffelt et al. Biotechnol Biofuels (2016) 9:55
DOI 10.1186/s13068-016-0469-2 Hyper‑accumulation of starch and oil
in a Chlamydomonas mutant affected
in a plant‑specific DYRK kinase Miriam Schulz‑Raffelt1,2,3,5, Vincent Chochois1,2,3,6, Pascaline Auroy1,2,3, Stéphan Cuiné1,2,3,
Emmanuelle Billon1,2,3, David Dauvillée4, Yonghua Li‑Beisson1,2,3 and Gilles Peltier1,2,3* Miriam Schulz‑Raffelt1,2,3,5, Vincent Chochois1,2,3,6, Pascaline Auroy1,2,3, Stéphan Cuiné1,2,3,
Emmanuelle Billon1,2,3, David Dauvillée4, Yonghua Li‑Beisson1,2,3 and Gilles Peltier1,2,3* Background Microalgae, the primary biomass producers of oceans,
are a promising and renewable feedstock for the pro-
duction of next-generation biofuels [1–4]. Despite
their high biomass productivity and a marked ability to
accumulate high intracellular amounts of energy-rich *Correspondence: gilles.peltier@cea.fr
1 CEA, CNRS, Aix‑Marseille Université, Institut de Biosciences et
Biotechnologies Aix Marseille, Laboratoire de Bioénergétique et
Biotechnologie des Bactéries et Microalgues, CEA Cadarache,
13108 Saint‑Paul‑lez‑Durance, France
Full list of author information is available at the end of the article Page 2 of 12 Schulz‑Raffelt et al. Biotechnol Biofuels (2016) 9:55 cassette within the third exon of a gene, initially annotated
as DYRK2 (C. reinhardtii genome version 4.0) (Additional
file 1: Figure S1A). A new gene structure was confirmed
based on overlapping RT-PCRs (Fig. 1a), and the locus was
renamed DYRKP based on the subsequent phylogenetic
analysis. The std1 mutant was complemented using a con-
struct containing the wild-type DYRKP genomic sequence
driven by the constitutive psaD promoter (Additional
file 1: Figure S1B). Two independent complemented strains (such as nitrogen or sulfur) strongly affects algal growth
and induces drastic changes in the cellular metabolism,
including decreased protein synthesis, arrested cell divi-
sion, a massive accumulation of energy-rich storage com-
pounds [5, 8], and a down-regulation of photosynthesis
[9–11]. Deciphering regulatory mechanisms that control
photosynthesis and reserve accumulation in response to
nutrient supply is therefore a key issue in understanding
survival strategies of photosynthetic organisms in natural
ecosystems, and is thus crucial for optimization of algal
productivity for biotechnological applications. CreDYRKP
1 2
3
4
5
6
7
8 9
200 bp
RT-PCR
RT-PCR
RT-PCR
AphVIII
10 11
12
13
14
a
b
ACTIN
STD1
250
150
STD1
RBCL
c
CreDYRKP
VcaDYRKP
ChlNC-DYRKP
MicpuDYRKP
OstDYRKP
OsDYRKP-1
OsDYRKP-3
OsDYRKP-2
AtDYRKP-4
VivDYRKP-1
AtDYRKP-1/2
PhypaDYRKP
-1/2/3
VivDYRKP-2
Minibrain
DYRK1A
MNB-DAN
DYRK1B
DdDyrk1
DdDyrk2
Pom1
Ppk5p
DYRK4
dDyrk2
DYRK2/3
dDyrk3
CreDYRK2 VcaDYRK2
PhypaDYRK2
Micpu
DYRK2
ChlNC-DYRK2
AtYak1
VivYak1
YakA Yak1p
AspYak1
Ppk15p
VcaYak1
CreYak1
MicpuYak1
OstYak1
OsYak1/2
PhypaYak
1/2/3/4/5
AtDYRKP-3
DYRK2
Yak1
DYRK1
DYRKP
d
Fig. 1 Molecular and phylogenetic characterization of the Chla-
mydomonas std1 mutant. a In std1, the paromomycin-resistance
cassette (AphVIII gene, white box) is inserted within the third exon
of a gene annotated as a DYRK kinase (Cre07.g337300, v5.5). The
gene structure was deduced from three overlapping RT-PCR. b
STD1 transcript levels were analyzed by RT-PCR in WT, std1 and two
complemented lines (std1::STD1-1 and std1::STD1-2) by amplifying a
1421 bp product. Background A 456 bp RT-PCR actin product was amplified as a
loading control. Sequence primers for RT-PCR are given in Additional
file 1: Table S2. c The level of STD1 protein was analyzed by immu‑
nodetection in WT, std1 and two complemented lines (std1::STD1-1
and std1::STD1-2). d Phylogenetic analysis of the DYRK protein family
indicates that STD1 (referred to here as DYRKP) belongs to a new
bf
l
fi
l Until now only a few regulatory elements and pathways
linking the nutrient status to reserve accumulation and
growth have been identified in plants. These include the
conserved TOR and SnRK1/Snf1/AMPK kinases involved
in the control of growth by nutrient availability [12, 13]. In Chlamydomonas, SNRK2 has been identified as a cru-
cial regulatory element of the S deprivation response [14,
15]. A nitrogen response regulator (NRR1) predicted as a
transcription factor and holding a SQUAMOSA promoter-
binding domain was proposed to regulate the algal oil con-
tent [16]. In yeast, other regulatory elements such as the
DYRK kinase (dual-specificity tyrosine-phosphorylation-
regulated kinase) Yak1 are involved in the cellular response
to nutrient stress [17]. Recently, a Chlamydomonas mutant
deficient in a DYRK kinase belonging to the Yak1 subfam-
ily was isolated from a forward genetic screen; this mutant
is showing a decreased capacity to accumulate oil under N
deprivation when compared to the wild-type [18]. CreDYRKP
1 2
3
4
5
6
7
8 9
200 bp
RT-PCR
RT-PCR
RT-PCR
AphVIII
10 11
12
13
14
a
b
ACTIN
STD1
250
150
STD1
RBCL
c a CreDYRKP
VcaDYRKP
ChlNC-DYRKP
MicpuDYRKP
OstDYRKP
OsDYRKP-1
OsDYRKP-3
OsDYRKP-2
AtDYRKP-4
VivDYRKP-1
AtDYRKP-1/2
PhypaDYRKP
-1/2/3
VivDYRKP-2
Minibrain
DYRK1A
MNB-DAN
DYRK1B
DdDyrk1
DdDyrk2
Pom1
Ppk5p
DYRK4
dDyrk2
DYRK2/3
dDyrk3
CreDYRK2 VcaDYRK2
PhypaDYRK2
Micpu
DYRK2
ChlNC-DYRK2
AtYak1
VivYak1
YakA Yak1p
AspYak1
Ppk15p
VcaYak1
CreYak1
MicpuYak1
OstYak1
OsYak1/2
PhypaYak
1/2/3/4/5
AtDYRKP-3
DYRK2
Yak1
DYRK1
DYRKP
d d With the aim to identify new regulatory mecha-
nisms involved in the dynamics of reserve formation in
response to nutrient availability, a genetic screen was
developed in the unicellular green alga Chlamydomonas
reinhardtii [19]. We report here the characterization
of one such mutant, defected in a plant-specific DYRK
kinase (DYRKP), which accumulates higher starch and
oil amounts than WT in response to nutrient deprivation
in photoautotrophic conditions. The std1 mutant hyper‑accumulates starch and oil
under photoautotrophic nutrient deprivationi Biomass productivity was then measured by using pho-
tobioreactors operated as turbidostats (Fig. 4). WT and
std1 mutant cells were first grown under N replete condi-
tions by maintaining biomass at a constant value through
the addition of fresh MM. At t0, MM was replaced by
N-deprived MM as dilution media, resulting in a complete
N depletion in both cultures after 43 h (Fig. 4a). Upon
starvation, the dilution rate of the std1 culture increased
while the dilution rate of the WT culture progressively
decreased. Biomass productivity, determined from dilu-
tion rate and dry weight measurements, was much higher
in the mutant after 48 h and remained at a high value after
72 h, while the productivity of the WT strongly decreased
and reached a null value after 72 h (Fig. 4b). Starch pro-
ductivity was also higher in the std1 mutant than in the
WT, the effect being more marked at 72 h (Fig. 4c). p
p
p
Although std1 was initially identified as a mutant affected
in starch degradation [19] (Additional file 1: Figure S1C),
further characterization identified an even stronger
starch accumulation phenotype in response to depletion
of nitrogen (Fig. 2a, b) or sulfur (Additional file 1: Fig-
ure S3). Interestingly, this phenotype was dependent on
culture conditions. In WT and complemented strains,
starch accumulation strongly depended on the intracel-
lular energy status. Low or transient accumulations were
respectively observed at low (35 µmol photons m−2 s−1)
or medium light (100 µmol photons m−2 s−1) under pho-
toautotrophic N deprivation (Fig. 2a, b), while high and
persistent accumulation was observed in mixotrophic
conditions, in which acetate was added to the culture
medium (Fig. 2c). In the wild-type strain, accumulation
of reserve compounds depends on light intensity or ace-
tate supply, thus reflecting the effect of the intracellular
energy status on reserve formation. This dependency
was absent in std1, in which high and persistent starch
accumulation was observed under all of these conditions, Molecular characterization and genetic complementation
of the std1 mutant Fig. 1 Molecular and phylogenetic characterization of the Chla-
mydomonas std1 mutant. a In std1, the paromomycin-resistance
cassette (AphVIII gene, white box) is inserted within the third exon
of a gene annotated as a DYRK kinase (Cre07.g337300, v5.5). The
gene structure was deduced from three overlapping RT-PCR. b
STD1 transcript levels were analyzed by RT-PCR in WT, std1 and two
complemented lines (std1::STD1-1 and std1::STD1-2) by amplifying a
1421 bp product. A 456 bp RT-PCR actin product was amplified as a
loading control. Sequence primers for RT-PCR are given in Additional
file 1: Table S2. c The level of STD1 protein was analyzed by immu‑
nodetection in WT, std1 and two complemented lines (std1::STD1-1
and std1::STD1-2). d Phylogenetic analysis of the DYRK protein family
indicates that STD1 (referred to here as DYRKP) belongs to a new
DYRK subfamily specific to plants We previously initiated a genetic approach in the unicellu-
lar green alga C. reinhardtii by screening a DNA insertional
mutant library based on the analysis of starch content
dynamics. Insertion lines were submitted to 5 days N or S
deprivation to induce starch accumulation, then to a 48-h
starch degradation period under nutrient replete condi-
tions. Eighteen mutants showing higher intracellular starch
amounts than WT were isolated from the screen of 15,000
paromomycin-resistant transformants [19], among which
one mutant, named std1 for starch degradation 1, contained
a single insertion of the paromomycin (AphVIII)-resistance gene structure was deduced from three overlapping RT-PCR. b
STD1 transcript levels were analyzed by RT-PCR in WT, std1 and two
complemented lines (std1::STD1-1 and std1::STD1-2) by amplifying a
1421 bp product. A 456 bp RT-PCR actin product was amplified as a
loading control. Sequence primers for RT-PCR are given in Additional
file 1: Table S2. c The level of STD1 protein was analyzed by immu‑
nodetection in WT, std1 and two complemented lines (std1::STD1-1
and std1::STD1-2). d Phylogenetic analysis of the DYRK protein family
indicates that STD1 (referred to here as DYRKP) belongs to a new
DYRK subfamily specific to plants Page 3 of 12 Schulz‑Raffelt et al. Biotechnol Biofuels (2016) 9:55 (std1::STD1-1 and std1::STD1-2) were isolated showing
transcript and protein levels close to that of WT progeni-
tor (Fig. 1b, c), and a rescued starch degradation phenotype
(Additional file 1: Figure S1C). the wild-type phenotype being rescued in complemented
strains (Fig. 2a–c). Chlamydomonas DYRKP (STD1) is a member of a novel
plant‑specific group of the DYRK family i
y
DYRKs are a relatively novel subfamily of eukaryotic
kinases belonging to the CMGC group, including CDKs
(cyclin-dependent kinases) and MAPKs (mitogen-
activated protein kinases). In the yeast Saccharomyces
cerevisiae, the DYRK family member Yak1 is a nega-
tive regulator of cell proliferation under nutrition stress
[20, 21]. DYRK kinases exhibit conserved sequence fea-
tures, in particular the DYRK homology-box (DH-box)
that precedes the conserved catalytic domain [22]. Phy-
logenetic analysis showed that the Chlamydomonas
DYRKP (STD1) belongs to a distinct group, different
from DYRK1, DYRK2, and Yak sub-families (Fig. 1d). This novel group contains only plant members and has
been thus named DYRKP (for plant DYRK) [18]. DYRKP
shares conserved sequence features of the other DYRK
sub-families, including a DH-box motif (Additional file 1:
Figure S2A, B). The length of the N- and C-terminal
regions upstream and downstream of the kinase domain
is quite variable among the DYRK family, but DYRKP
members harbor a very short C-terminal extension
(Additional file 1: Figure S2C). Whereas mosses and vas-
cular plants harbor 2–6 DYRKP homologues, microalgal
genomes tend to contain only one group member. Molecular characterization and genetic complementation
of the std1 mutant The intracellular oil content increase
was also greater in the mutant than in control strains
(Fig. 2f), although the difference occurred later during
starvation in photoautotrophy (Fig. 2d) as compared to
mixotrophy (Fig. 2e). While both complemented lines
showed an intermediary oil phenotype on Fig. 2d, full
restoration was observed in an independent experiment
(Additional file 1: Figure S5). The std1 mutant shows increased biomass production
during photoautotrophic N starvation Biomass production (as estimated from cell pellet size)
was much higher in std1 cultures than in control strains
upon 3 and 10 days of N deprivation (Fig. 3a). While con-
trol lines grew as isolated cells, the std1 mutant formed
aggregates (or palmelloids [23]) of 2, 4, 8 or more cells
enclosed by the mother cell wall (Additional file 1: Fig-
ure S6). Nonetheless, the cell number as determined
after autolysin treatment increased in a similar manner
in both std1 and control strains; the large increase in the
cellular volume observed in the mutant resulted from
an increase in the volume of each single cell (Additional
file 1: Figure S6). After 6 days of N depletion, the bio-
mass increase (measured as dry weight) was about twice
higher in the mutant than in control strains, the differ-
ence being essentially due to the increase in intracellular
starch (Fig. 3b). Transcript analysis demonstrates that
the DYRKP gene is strongly expressed after 1 day of N
depletion, and remained high after 3 days of deprivation
(Fig. 3c), thus indicating a functional role of the kinase in
the response to nitrogen stress. The std1 mutant maintains higher photosynthetic activity
under photoautotrophic N starvation N deprivation is known to induce a decline in the photo-
synthetic activity of microalgae, resulting in a drop in the Page 4 of 12 Schulz‑Raffelt et al. Biotechnol Biofuels (2016) 9:55 Fig. 2 Starch and oil hyper-accumulate in std1 mutant following N deprivation. a Starch content in cells grown photoautotrophically under low
light (LL, 35 µmol photons m−2 s−1) supplemented with 2 % CO2. b Starch content in cells grown photoautotrophically under medium light
(100 µmol photons m−2 s−1) supplemented with 2 % CO2. c Starch content in cells grown mixotrophically (i.e., TAP) under medium light (100 µmol
photons m−2 s−1). d TAG content in cells grown photoautotrophically under medium light (100 µmol photons m−2 s−1) supplemented with 2 %
CO2. e TAG content in cells grown mixotrophically (i.e., TAP media) under medium light (100 µmol photons m−2 s−1). f Nile red fluorescence (lower
panel) and bright field microscopy (upper panel) of indicated cells under photoautotrophic N deprivation. Scale bar 10 µm. MM minimal medium. Data are means ± SD (n = 3). Cell counts, cellular volumes, and chlorophyll content measurements related to these experiments are shown on
Additional file 1: Figure S4 Fig. 2 Starch and oil hyper-accumulate in std1 mutant following N deprivation. a Starch content in cells grown photoautotrophically under low
light (LL, 35 µmol photons m−2 s−1) supplemented with 2 % CO2. b Starch content in cells grown photoautotrophically under medium light
(100 µmol photons m−2 s−1) supplemented with 2 % CO2. c Starch content in cells grown mixotrophically (i.e., TAP) under medium light (100 µmol
photons m−2 s−1). d TAG content in cells grown photoautotrophically under medium light (100 µmol photons m−2 s−1) supplemented with 2 %
CO2. e TAG content in cells grown mixotrophically (i.e., TAP media) under medium light (100 µmol photons m−2 s−1). f Nile red fluorescence (lower
panel) and bright field microscopy (upper panel) of indicated cells under photoautotrophic N deprivation. Scale bar 10 µm. MM minimal medium. Data are means ± SD (n = 3). Cell counts, cellular volumes, and chlorophyll content measurements related to these experiments are shown on
Additional file 1: Figure S4 Fig. 2 Starch and oil hyper-accumulate in std1 mutant following N deprivation. a Starch content in cells grown photoautotrophically under low
light (LL, 35 µmol photons m−2 s−1) supplemented with 2 % CO2. The std1 mutant maintains higher photosynthetic activity
under photoautotrophic N starvation b Starch content in cells grown photoautotrophically under medium light
(100 µmol photons m−2 s−1) supplemented with 2 % CO2. c Starch content in cells grown mixotrophically (i.e., TAP) under medium light (100 µmol
photons m−2 s−1). d TAG content in cells grown photoautotrophically under medium light (100 µmol photons m−2 s−1) supplemented with 2 %
CO2. e TAG content in cells grown mixotrophically (i.e., TAP media) under medium light (100 µmol photons m−2 s−1). f Nile red fluorescence (lower
panel) and bright field microscopy (upper panel) of indicated cells under photoautotrophic N deprivation. Scale bar 10 µm. MM minimal medium. Data are means ± SD (n = 3). Cell counts, cellular volumes, and chlorophyll content measurements related to these experiments are shown on
Additional file 1: Figure S4 PSII yield [9]. When N deprivation was performed under
photoautotrophic conditions, the PSII yield decrease was
less pronounced in std1 than in control strains (Fig. 5a). However, parallel decreases in PSII yields were observed
in std1 and control strains when N deprivation was con-
ducted under mixotrophic conditions (Fig. 5b). Immu-
nodetection experiments revealed a decrease in major
photosynthetic components in both std1 and the WT pro-
genitor in response to N deprivation (Additional file 1: Fig-
ure S7), therefore indicating that the higher photosynthetic
activity observed in std1 does not result from higher levels
of photosynthetic complexes but rather from a higher PSII
activity. Consistently, the higher photosynthetic activity of
the mutant was observed in conditions where the synthe-
sis of reserve compounds was increased (Fig. 2). PSII yield [9]. When N deprivation was performed under
photoautotrophic conditions, the PSII yield decrease was
less pronounced in std1 than in control strains (Fig. 5a). However, parallel decreases in PSII yields were observed
in std1 and control strains when N deprivation was con-
ducted under mixotrophic conditions (Fig. 5b). Immu-
nodetection experiments revealed a decrease in major
photosynthetic components in both std1 and the WT pro-
genitor in response to N deprivation (Additional file 1: Fig-
ure S7), therefore indicating that the higher photosynthetic
activity observed in std1 does not result from higher levels
of photosynthetic complexes but rather from a higher PSII
activity. Consistently, the higher photosynthetic activity of
the mutant was observed in conditions where the synthe-
sis of reserve compounds was increased (Fig. 2). Discussion The 100 %
value corresponds to 7.5 mM NH4
+, which is the ammonium con‑
centration of the minimal medium. Shown are mean ± SD (n = 3). b Biomass productivity (g dry weight L−1 d−1) was determined from
dilution rates and biomass measurements at t0, and 48 and 72 h after
N-removal. Shown are means ± SD (n = 3) shows a sustained photosynthetic activity in response to
nutrient starvation. Discussion All strains were grown photoautotrophically in an MM
medium supplemented with 2 % CO2, at a light intensity of 100 µmol
photons m−2 s−1 and then subjected to N deprivation (at day 0). a
Visual observation of cell pellets from 1 ml N-starved cells harvested
at different time points. b Biomass (measured as dry weight) and
intracellular starch were determined in N-starved strains. Data repre‑
sent means ± SD (n = 3) (upper error bar dry weight, lower error bar,
starch). c Northern blot analysis of DYRKP transcripts in response to N
deprivation a b
WT
std1
std1::STD1-1
std1::STD1-2
starch b Fi
4 Bi
d
ti it
f WT
d td1 Chl
d
ll CBLP2
0 1 2 3
WT
0 1 2
3
std1
STD1
c
Northern blot
days c Fig. 3 Biomass and starch production during photoautotrophic N
deprivation. All strains were grown photoautotrophically in an MM
medium supplemented with 2 % CO2, at a light intensity of 100 µmol
photons m−2 s−1 and then subjected to N deprivation (at day 0). a
Visual observation of cell pellets from 1 ml N-starved cells harvested
at different time points. b Biomass (measured as dry weight) and
intracellular starch were determined in N-starved strains. Data repre‑
sent means ± SD (n = 3) (upper error bar dry weight, lower error bar,
starch). c Northern blot analysis of DYRKP transcripts in response to N
deprivation Fig. 4 Biomass productivity of WT and std1 Chlamydomonas cells
measured in photobioreactors operated as turbidostats during
photoautotrophic N deprivation. Cells were grown under constant
illumination (125 µmol photons m−2 s−1) in the presence of 2 % CO2
enriched air. Cell density was measured using an absorption probe
and maintained at a constant level by injection of fresh medium. Due to the aggregation phenotype of std1, OD880nm was regulated at
different values for WT (OD880nm = 0. 4) and std1 (OD880nm = 0. 3) to
reach similar biomass concentrations (0. 15 g dry weight L−1). After a
48-h stabilization period in the presence of MM, the dilution medium
was replaced by MM-N (t0). a Cumulated amounts of fresh medium
were added to maintain the culture at a constant biomass concentra‑
tion. Measurements of ammonium concentration (dotted lines) in the
culture medium showed complete exhaustion after 45 h. Discussion Accumulation of reserve compounds (starch and oil)
by photosynthetic organisms is part of an acclimation
strategy to nutrient shortage. Despite a strong interest
in microalgae as a renewable feedstock for the produc-
tion of next-generation biofuels, little is known about
molecular mechanisms regulating carbon storage. Using
the unicellular green alga C. reinhardtii as a model, we
developed a forward genetic screen to identify novel
genes involved in the regulation of starch dynamics [19]. We report here the characterization of a mutant (std1)
affected in a DYRK kinase homologue belonging to a
subgroup (called DYRKP) specific to plants. The std1
mutant, the first DYRKP mutant reported so far, accu-
mulates high intracellular starch and oil amounts and Page 5 of 12 Schulz‑Raffelt et al. Biotechnol Biofuels (2016) 9:55 Fig. 4 Biomass productivity of WT and std1 Chlamydomonas cells
measured in photobioreactors operated as turbidostats during
photoautotrophic N deprivation. Cells were grown under constant
illumination (125 µmol photons m−2 s−1) in the presence of 2 % CO2
enriched air. Cell density was measured using an absorption probe
and maintained at a constant level by injection of fresh medium. Due to the aggregation phenotype of std1, OD880nm was regulated at
different values for WT (OD880nm = 0. 4) and std1 (OD880nm = 0. 3) to
reach similar biomass concentrations (0. 15 g dry weight L−1). After a
48-h stabilization period in the presence of MM, the dilution medium
was replaced by MM-N (t0). a Cumulated amounts of fresh medium
were added to maintain the culture at a constant biomass concentra‑
tion. Measurements of ammonium concentration (dotted lines) in the
culture medium showed complete exhaustion after 45 h. The 100 %
value corresponds to 7.5 mM NH4
+, which is the ammonium con‑
centration of the minimal medium. Shown are mean ± SD (n = 3). b Biomass productivity (g dry weight L−1 d−1) was determined from
dilution rates and biomass measurements at t0, and 48 and 72 h after
N-removal. Shown are means ± SD (n = 3) a
Day 3
Day 10
Day 0 a
b
Day 3
Day 10
Day 0
WT
std1
std1::STD1-1
std1::STD1-2
starch
CBLP2
0 1 2 3
WT
0 1 2
3
std1
STD1
c
Northern blot
days
Fig. 3 Biomass and starch production during photoautotrophic N
deprivation. DYRKP, a negative regulator of carbon storage Data are means ± SD (n = 6 for a, and n = 4 for b) accumulation is observed as light increases or acetate is
supplied to the culture medium (Fig. 2a–c). This depend-
ency is abolished in the std1 mutant, in which high and
sustained starch accumulation was observed in all condi-
tions (Fig. 2a–c). We therefore suggest that the DYRKP
(STD1) kinase acts as a negative regulator of carbon stor-
age in conditions of low cellular energy status (Fig. 6). Under nutrient deprivation, photosynthesis would be
restricted in the wild-type strain by the sink capacity of
reserve metabolism, resulting in an increase in excita-
tion pressure at PSII. This restriction would be alleviated
in the std1 mutant, thus explaining the lower excitation
pressure and the higher photosynthetic activity observed
in the mutant (Fig. 5a, b). In WT, the function of DYRKP
(STD1) would be to prevent excessive accumulation of
reserve compounds when the cellular energy status is
low, thereby preserving cellular energy for other pur-
poses. External acetate supply, which increases the intra-
cellular energy status [24] has been reported to boost accumulation of reserves under nitrogen depletion [25,
26]. Such a stimulation of reserve accumulation by ace-
tate, which is absent in std1, reflects the tight regulation
of this phenomenon by the intracellular energy status,
which likely occurs through a DYRKP (STD1)-dependent
mechanism. accumulation is observed as light increases or acetate is
supplied to the culture medium (Fig. 2a–c). This depend-
ency is abolished in the std1 mutant, in which high and
sustained starch accumulation was observed in all condi-
tions (Fig. 2a–c). We therefore suggest that the DYRKP
(STD1) kinase acts as a negative regulator of carbon stor-
age in conditions of low cellular energy status (Fig. 6). Under nutrient deprivation, photosynthesis would be
restricted in the wild-type strain by the sink capacity of
reserve metabolism, resulting in an increase in excita-
tion pressure at PSII. This restriction would be alleviated
in the std1 mutant, thus explaining the lower excitation
pressure and the higher photosynthetic activity observed
in the mutant (Fig. 5a, b). In WT, the function of DYRKP
(STD1) would be to prevent excessive accumulation of
reserve compounds when the cellular energy status is
low, thereby preserving cellular energy for other pur-
poses. External acetate supply, which increases the intra-
cellular energy status [24] has been reported to boost DYRKP, a negative regulator of carbon storage In nutrient-deprived WT cells, accumulation of starch is
strongly dependent on the cellular energy status. While
low starch accumulation occurs at low light, higher Page 6 of 12 Schulz‑Raffelt et al. Biotechnol Biofuels (2016) 9:55 Fig. 5 Photosynthetic activity of WT and std1 Chlamydomonas cells during mixotrophic or photoautotrophic N deprivation. PSII yields were deter‑
mined at different light intensities in the std1 mutant, in the WT and in two complemented mutant lines by measuring pulse amplitude-modulated
chlorophyll fluorescence. Cells were grown under a light intensity of 100 µmol photons m−2 s−1 under a Photoautotrophic conditions (i.e., MM sup‑
plemented with 2 % CO2) or b Mixotrophic conditions (i.e., TAP). At t0, cells were resuspended in an N-free medium and fluorescence measurements
were performed at t0 (N-replete) and after 2 and 3 days of N deprivation. Data are means ± SD (n = 6 for a, and n = 4 for b) Fi
Ph
h
f WT
d
d Chl
d
ll d
h
h
h
N d
PSII
ld
d Fig. 5 Photosynthetic activity of WT and std1 Chlamydomonas cells during mixotrophic or photoautotrophic N deprivation. PSII yields were deter‑
mined at different light intensities in the std1 mutant, in the WT and in two complemented mutant lines by measuring pulse amplitude-modulated
chlorophyll fluorescence. Cells were grown under a light intensity of 100 µmol photons m−2 s−1 under a Photoautotrophic conditions (i.e., MM sup‑
plemented with 2 % CO2) or b Mixotrophic conditions (i.e., TAP). At t0, cells were resuspended in an N-free medium and fluorescence measurements
were performed at t0 (N-replete) and after 2 and 3 days of N deprivation. Data are means ± SD (n = 6 for a, and n = 4 for b) Fig. 5 Photosynthetic activity of WT and std1 Chlamydomonas cells during mixotrophic or photoautotrophic N deprivation. PSII yields were deter‑
mined at different light intensities in the std1 mutant, in the WT and in two complemented mutant lines by measuring pulse amplitude-modulated
chlorophyll fluorescence. Cells were grown under a light intensity of 100 µmol photons m−2 s−1 under a Photoautotrophic conditions (i.e., MM sup‑
plemented with 2 % CO2) or b Mixotrophic conditions (i.e., TAP). At t0, cells were resuspended in an N-free medium and fluorescence measurements
were performed at t0 (N-replete) and after 2 and 3 days of N deprivation. DYRKP are plant‑specific DYRK kinases We propose that DYRKP nega‑
tively regulates the sink capacity in response to both nutrient and
energy signals. DYRKP is induced in response to nutrient limitation,
and would be active in conditions of low energy status. Disruption
of DYRKP in std1 allows sustained synthesis of reserve compounds,
thereby increasing electron sink capacity and maintaining a high
photosynthetic rate DYRK functions and mechanisms Yeast and mammalian DYRKs contain a conserved YxY
motif in the so-called activation loop (subdomain VIII),
which is autophosphorylated at the second tyrosine thus
providing full activity of DYRK kinases [31, 32]. The
autophosphorylation was described as a one-off event,
which is mediated by an intramolecular mechanism and a
transitory intermediate structure [33, 34]. The YxY motif
is not conserved in the DYRKP group, the first tyrosine
being replaced by a cysteine in plants and a serine in C. reinhardtii (Additional file 1: Figure S2A). However, for
all DYRKs analyzed so far, only the second tyrosine of
the YxY motif is phosphorylated and essential for the
kinase activity [35]. Moreover, recent phosphoproteome
studies have shown the existence of phospho-tyrosine
peptides in the activation loops of Arabidopsis AtYak1 Relationships with other signaling pathways of nutrient
and energy signals In yeast and mammals, target of rapamycin (TOR) and
RAS/cAMP are the major signaling pathways involved in
the regulation of cell growth in response to nutrient and
energy-derived signals [41]. The mTORC1 complex senses
the energy status of a cell through AMP-activated protein
kinase (AMPK) [42], and controls anabolic processes such
as ribosome biogenesis and translation through the phos-
phorylation of different targets [42]. In photosynthetic
eukaryotes, both SnRK1 (the plant orthologue of AMPK)
and TOR are involved in the control of cellular processes
through the transduction of nutrient and energy signals
[12, 43]. In yeast, TOR negatively regulates the DYRK
kinase Yak1 via PKA (cAMP-dependent protein kinase)
[44]. Whereas the TOR pathway is conserved in all eukary-
otes and controls cell proliferation and growth in response
to nutrient and energy signals, DYRKP kinases are plant
specific. We suggest here that in green algae and most
likely in vascular plants, DYRKP is part of a regulatory cas-
cade controlling the accumulation of reserve compounds,
a key feature for the survival of photoautotrophic eukary-
otes in fluctuating conditions of nutrient availability. two, respectively). In contrast, algal genomes contain only
one member of each DYRK subgroup (Yak, DYRK2, and
DYRKP). Such a lower redundancy of algal genomes was
previously reported for other gene families. For example,
the Chlamydomonas genome harbors a smaller number
of cell cycle genes than vascular plants or vertebrates [27]. Only two genes encoding heat-shock factors are found in
the Chlamydomonas genome, whereas Arabidopsis has
more than 21 [28]. While mutants of DYRK homologs of
Yak1 have been recently isolated in C. reinhardtii [29] and
A. thaliana [30], this is the first report on the function of a
DYRKP kinase in the green lineage. DYRKP are plant‑specific DYRK kinases Phylogenetic analysis revealed that DYRKs can be
divided into four subgroups, the DYRK1, the DYRK2,
the Yak, and the DYRKP subgroup defined in this study
(Fig. 2; Additional file 1: Figure S2, S8, S9). While vascu-
lar plant DYRKs only belong to two subgroups (Yak and
DYRKP), algal and moss genomes also harbor members
of the DYRK2 subgroup. Vascular plants and mosses pos-
sess more than one homologue of the DYRKP subgroup
(Arabidopsis and Populus contain four members and
Physcomitrella three) and of the Yak subgroup (Physcom-
itrella harbors five and Arabidopsis and rice have one and Page 7 of 12 Page 7 of 12 Schulz‑Raffelt et al. Biotechnol Biofuels (2016) 9:55 Photosynthesis
Acetate supply
Starch and oil
synthesis
Nutrient limitation
PSII
PSI
PSII
PSI
DYRKP
PQ
PQH2
PQ
PQH2
Energy status
Metabolism
Fig. 6 A hypothetical model of the DYRKP (STD1) function in
response to nutrient deprivation. We propose that DYRKP nega‑
tively regulates the sink capacity in response to both nutrient and
energy signals. DYRKP is induced in response to nutrient limitation,
and would be active in conditions of low energy status. Disruption
of DYRKP in std1 allows sustained synthesis of reserve compounds,
thereby increasing electron sink capacity and maintaining a high
photosynthetic rate Photosynthesis
Acetate supply
Starch and oil
synthesis
Nutrient limitation
PSII
PSI
PSII
PSI
DYRKP
PQ
PQH2
PQ
PQH2
Energy status
Metabolism and AtDYRKP3 [36] and of C. reinhardtii CreYak1 and
CreDYRKP [37]. Taken together, this strongly suggests
that activation of plant and algal DYRKP kinases also
depends on tyrosine autophosphorylation. In yeast, the
DYRK homologue Yak1 has been reported to be part of
a glucose-sensing system controlling the arrest of the
cell cycle after translocation to the nucleus and by phos-
phorylating Pop2p [21]. Yak1 was also suggested to be
involved in the control of glycogen storage [38] and to
mediate stress responses by phosphorylation of tran-
scription factors such as Msn2 and Hsf1 upon glucose
depletion [39]. More recently, Yak1 was proposed to lie
at the center of a regulatory cascade controlling growth
and stress response by targeting different transcription
factors [40]. Further studies will be needed to identify
DYRKP targets and the nature of the regulatory cascade
controlled by DYRKP in plants and microalgae. Starch and oil
synthesis Fig. 6 A hypothetical model of the DYRKP (STD1) function in
response to nutrient deprivation. Genetic characterization and complementation of the std1
mutant Despite a strong interest for microalgal biofuels, little is
known concerning regulatory mechanisms controlling
cellular accumulation of storage compounds. By isolat-
ing a Chlamydomonas kinase mutant hyper-accumulat-
ing starch and oil in response to nitrogen starvation, we
show here that accumulation of reserve compounds is a
highly regulated process. The newly discovered DYRKP
kinase is proposed to down-regulate reserve accumula-
tion in the wild-type by integrating nutrient and energy
signals, such a regulation being suppressed in the mutant. Future work will aim at identifying molecular targets of
the kinase as well as the full signaling pathway involved
in this regulatory mechanism. The DYRKP kinase there-
fore represents a valuable target for the biotechnological
improvement of microalgae and other crops to enhance
their energy density for the production of biofuel. To check the integration frequency of the inserted DNA,
Southern blot analysis was performed with wild-type
and std1 mutant cells. Genomic DNA was prepared
as described previously [47], and 4, 6 or 8 µg genomic
DNA restricted with NotI were separated in an 0.8 %
agarose gel, blotted on a nylon membrane and hybrid-
ized with a digoxygenin-labeled probe complementary to
part of the AphVIII gene of the inserted resistance cas-
sette. A PCR DIG Probe Synthesis Kit (Roche) was uti-
lized for probe labeling using primers AphORF_For and
Aph_Tail3. The hybridization with the resulting 400 bp-
PCR fragment was performed overnight at 50 °C using
DIG Easy Hyb buffer (Roche). Anti-Digoxigenin-AP and
CSPD as substrate (Roche) were applied to detect sig-
nals using G:BOX Chemin XL (Syngene). The integration
site of the paromomycin resistance cassette was identi-
fied by genome walking (GenomeWalker Kit, Clontech). Genomic DNA of the strain std1 was digested with FspI
and processed according to the manufacturer’s instruc-
tions. The gene-specific primers GSP1 and GSP2 allowed
the determination of the genomic sequence downstream
of the inserted AphVIII cassette. Amplification was
achieved using the Advantage GC Genomic LA Polymer-
ase (Clontech). A ~1900 bp fragment from the ~4800 bp
pSL-X was found inserted into the std1 genome. For
complementation of the std1 mutant, the genomic DNA
encoding DYRKP gene was amplified using the primers
XbaG4forHyg and XbaG4RevHyg and DyNAzyme™ EXT
DNA Polymerase (Finnzymes Oy). Genetic characterization and complementation of the std1
mutant The amplified PCR
product (6913 bp) was restricted by XbaI and cloned into
the XbaI-digested vector pSL-Hyg (derived from pSL18
[48]), which is under control of the psaD promoter and
carries a resistance cassette for hygromycin [49]. Fol-
lowing autolysin treatment, std1 cells were transformed
with KpnI-linearized pSL-Hyg-STD1 by agitation with
glass beads [50]. Transformed cells were selected on
TAP plates containing 10 µg ml−1 hygromycin B and DYRKP as a target for improving carbon storage
in microalgae and plant crops Increasing the accumulation of reserve compounds is a
major issue for domestication of microalgae [1–4]. While
great improvements of oil or starch content have been
obtained in crop plants by means of classical breeding
techniques, successful biotechnological improvements of
microalgae are still scarce. Regulators of the algal oil con-
tent such as NRR1 [16] may represent potential targets
for biotechnological improvements. It has been recently
suggested that CHT7, a repressor of cellular quiescence Page 8 of 12 Schulz‑Raffelt et al. Biotechnol Biofuels (2016) 9:55 different strains were diluted to reach a similar cellular
concentration before starvation experiments. could provide a target to increase biomass and oil pro-
ductivity in algae [45]. Moreover, it has been hypoth-
esized that vascular plants may increase the carbon flow
into starch and other storage compounds by avoiding
feedback inhibition of photosynthesis, for instance by the
manipulation of signaling pathways mediated by SnRK
protein kinases [13]. Such an effect was clearly observed
here in the std1 mutant, inhibition of the DYRKP kinase
increases the storage of reserve compounds and the sink
strength, thus limiting feedback inhibition of photosyn-
thesis (Fig. 6). The discovery of DYRKP thus opens new
perspectives for bioengineering of microalgae. Photobioreactor experiments were performed using
four photobioreactors operated as turbidostats as
described previously [46]. Cells were grown photoau-
totrophically on a minimal medium by bubbling air
supplemented with 2 % CO2 under a light intensity of
125 µmol photons m−2 s−1. The pH was maintained at
7.2 by addition of 0.2 M KOH. At t0, the dilution medium
was replaced by a nitrogen deprived minimal medium. Ammonium concentration was determined in the culture
supernatant at different time points by using an ion selec-
tive electrode (Orion 9512HPBNWP, Thermo Scientific). Strains and growth conditionsh The C. reinhardtii wild-type strain CC124 (mt−nit1 nit2)
was used for mutant generation as described previously
[19]. The mutant strain std1 was isolated from a mutant
library generated by transformation of the strain CC124
with the KpnI-linearized plasmid (pSL-X) harboring the
paromomycin-resistance cassette AphVIII [19]. The starch
content of mutant strains was assayed using coloration
with iodine vapor following 5 days of nitrogen starvation
to trigger starch accumulation and 48 h in the dark on a
nitrogen replete medium of starch to induce starch break-
down [19]. Cells were grown in flasks either mixotrophi-
cally (Tris–acetate–phosphate or TAP medium) [23] or
photoautotrophically (MOPS-buffered minimal medium
[23] supplemented with 2 % CO2 in the air), under contin-
uous illumination (100 µmol photons m−2 s−1 or ~35 µmol
photons m−2 s−1 for low-light experiment) at 25 °C. Pre-
cultures were grown to a density of 2 to 4 × 106 cells ml−1
before starvation experiments. Due to palmelloid forma-
tion in std1, total cellular volume was measured using a
Multisizer 3 Coulter counter (Beckman Coulter) and the Page 9 of 12 Schulz‑Raffelt et al. Biotechnol Biofuels (2016) 9:55 complemented lines were screened by applying the same
protocol used for mutant isolation [19]. for RT-PCR, and the amplified product was restriction
digested by BamHI and HindIII, which was then cloned
into the BamHI–HindIII-restricted pQE-30 vector (Qia-
gen). Radioactive signals were detected using BAS-IP
MS2040 phosphorimager plates (Raytest; http://www. raytest.de), scanned with a Molecular Imager FX phos-
phorimager
(Bio-Rad;
http://www.biorad.com),
and
imaged using the Quantity One-4.5.1 program (Bio-Rad). RNA analyses and RT‑PCR Total RNA was isolated as previously described [55]. For
RT-PCR reactions, 1 µg of DNaseI-treated total RNA
was utilized with the OneStep RT-PCR Kit (Qiagen). To obtain sequence information of the complete tran-
scribed DYRKP (STD1) gene, three overlapping RT-PCRs
were performed using the primer pairs Std1UTR1 and
Std1P3rev, Std1FW2 and G4rev14, and the primer pair
ACG4_FW3 and ACG4_Rev1. For comparison of tran-
script levels in wild-type, mutant and complemented
strains, the Std1FW2–G4rev14 primer pair was used to
amplify part of the DYRKP transcript. Specific primers
were designed for actin (locus name Cre13.g603700, pro-
tein ID 515031) serving as a constitutively expressed con-
trol gene (Actin_FW and Actin_Rev). For Northern blot analysis, RNA extraction was per-
formed by collecting 15 ml of cell cultures at different
time points on ice and centrifuging for 1 min at 1789g. The 500 µl cell suspension was then transferred to a
1.5 ml-tube on ice and mixed with 500 µl of RNA lysis
buffer. RNA extraction, separation on formaldehyde
agarose gels and northern blot probing were performed
as previously described [55]. Membranes were hybrid-
ized with DNA probes containing a fragment of STD1
or CBLP2 as a loading control. A 1.1-kb BamHI-HindIII
fragment coding for the 3′-part of DYRKP (STD1) and
the 1-kb cDNA of CBLP2 were used for hybridization. The ACG4_FW3 and ACG4_Rev1 primers were used Protein extraction and immunoblot analysis y
To detect DYRKP, soluble cell lysates were prepared as fol-
lows: 100 ml of C. reinhardtii cell culture in the exponen-
tial phase (eq. to 5 × 106 cells ml−1 or 0.8 mm3 ml−1) were
harvested by centrifugation for 2 min at 1789g and resus-
pended in 1 ml lysis buffer (20 mM HEPES–KOH pH 7.2,
10 mM KCl, 1 mM MgCl2, 154 mM NaCl, 0.1× protease
inhibitor cocktail; Sigma P9599). Cells were sonicated on
ice for 90 s with an alternating cycle of 1 s pulse/1 s pause. Lysates were loaded onto sucrose cushions and centri-
fuged in a MLA-55 rotor (Beckman Coulter) for 30 min at
151,300g and 4 °C. Soluble proteins were mixed with one
volume of 2 × sample buffer [28] or 2 × LDS sample buffer
(Invitrogen) and heated for 5 min at 95 °C or 10 min at
70 °C prior to loading on an 8 % SDS–polyacrylamide gel. Immunodetection of DYRKP (STD1) was performed using
a purified antibody obtained by immunizing two rabbits
against two synthetic peptides (DGMDDPGYSRKEVPNP-
cys and PAVNHEDVELFRN-cys) conjugated to KLH (key-
hole limpet hemocyanin) as the carrier protein (http://
www.proteogenix-antibody.com/). DYRKP was detected
by ECL (SuperSignal West Pico Chemiluminescent Sub-
strate, Thermo Scientific). For immunodetection of other
proteins, cell pellets equivalent to 1.2 mm3 total cellular
volume were harvested from cell cultures and stored at
−80 °C until use. Cell pellets were resuspended in 70 µl of
a buffer containing 50 mM Tris pH 8, 10 mM EDTA and
2 % SDS, incubated for 30 min at RT, and centrifuged for
2 min at 4 °C. Protein concentrations were quantified from
2 µl samples by colorimetric measurements with bicin-
chonic acid (Pierce BCA Protein Assay kit, Thermo Scien-
tific). For immunoblot analysis, 10–12 µg of total protein
extracts were separated on 10 % SDS–polyacrylamide gels,
transferred to BioTrace™ NT nitrocellulose membrane
(Pall Life Sciences, http://www.pall.com) and decorated
using antibodies raised against AtpB, COXIIb, Cyt f, PsaC,
PsbD (D2), and RbcL (all purchased from Agrisera). Phylogenetic analysis Amino acid sequences were aligned using MAFFT ver-
sion 6 software [51]. The resulting alignment was then
manually refined using SeaView version 4 [52]; regions
displaying dubious homology were removed from fur-
ther analysis. A total of 313 amino acid positions were
retained for the phylogenetic analysis of DYRK proteins. Phylogenetic analyses were conducted using Neigh-
bour-Joining (NJ), Maximum Likelihood (ML) and Par-
simony (Pars) approaches in the Phylogenetic Inference
Package Phylip (version 3.69) (Fig. 1d; Additional file 1:
Figures S8, S9) [53]. The PROTML program was used
for ML analysis and the sequence input order was ran-
domized (20 jumbles). The SEQBOOT and CONSENSE
programs were used for bootstrap value calculations
on 100 replications and consensus tree reconstruc-
tions, respectively. To examine node confidence, NJ and
Pars analyses were done by using the NEIGHBOR and
PROTPARS programs. Distance matrices used for the
NJ analysis were created with the PROTDIST program. The phylogenetic trees were drawn with MEGA5 [54]. Author details 1 CEA, CNRS, Aix‑Marseille Université, Institut de Biosciences et Biotechnolo‑
gies Aix Marseille, Laboratoire de Bioénergétique et Biotechnologie des Bac‑
téries et Microalgues, CEA Cadarache, 13108 Saint‑Paul‑lez‑Durance, France. 2 CNRS, Biologie Végétale et Microbiologie Environnementale, UMR7265,
13108 Saint‑Paul‑lez‑Durance, France. 3 Aix Marseille Université, Biologie
Végétale et Microbiologie Environnementale, UMR7265, 13284 Marseille,
France. 4 UMR8576, CNRS, Université des Sciences et Technologies de Lille,
59655 Villeneuve d’Ascq, France. 5 Present Address: Molecular Biotechnology
and Systems Biology, TU Kaiserslautern, Paul‑Ehrlich‑Straße 23, 67663 Kai‑
serslautern, Germany. 6 Present Address: Research School of Biology College
of Medicine, Biology and Environment, Linneaus Building 134, The Australian
National University, Canberra, ACT 2601, Australia. Biomass, starch, and chlorophyll measurements p y
Biomass was determined by dry weight measurements
from three 5-ml samples upon filtration of the algal cul-
ture on a glass fiber filter (VWR, Ref. 611-0739) dried
overnight at 80 °C. Intracellular starch and chlorophyll
contents were measured as previously described [19]. Abbreviations DYRK: dual-specificity tyrosine-phosphorylation-regulated kinase; DYRKP:
plant-specific dual-specificity tyrosine-phosphorylation-regulated kinase; MM:
minimal medium; PSII: photosystem II; std1: starch degradation 1; TAP: tris–
acetate–phosphate medium. Chlorophyll fluorescencel Chlorophyll fluorescence was measured using a Dual
Pam-100 (Heinz Walz). Samples were placed into a
cuvette under constant stirring at room temperature and
were dark-adapted for 5–10 min before measurements. Light curves were recorded by increasing stepwise (3 min
per step) the light intensity from 15 to 715 µmol photons
m−2 s−1. Saturating flashes (10,000 µmol photons m−2
s−1, 200 ms duration) were applied to determine PSII
yield, 1-qP, and ETRs [57, 58]. Authors’ contributions MSR, YLB, and GP designed the study. MSR, VC, PA, SC, EB, DD, and YLB per‑
formed the experiments. MSR, VC, DD, YLB, and GP analyzed the data. MSR,
YLB, and GP prepared the manuscript. All authors read and approved the final
manuscript. MSR, YLB, and GP designed the study. MSR, VC, PA, SC, EB, DD, and YLB per‑
formed the experiments. MSR, VC, DD, YLB, and GP analyzed the data. MSR,
YLB, and GP prepared the manuscript. All authors read and approved the final
manuscript. Microscopy The authors declare that they have no competing interests. The authors declare that they have no competing interests. A Leica DMRXA microscope (Leica Microsystems, Ger-
many) was used for light microscopy. When necessary,
cells were fixed with 0.25 % glutaraldehyde in the medium. A Neubauer chamber was used to compare cell concentra-
tions. Images were captured with the Spot Insight 4 soft-
ware (Diagnostic Instruments Inc., Sterling Heights, USA). Fluorescence microscopy following Nile red staining was
performed as previously described [6], but with the addi-
tion of DMSO (10 %, v/v) to aid in the penetration of Nile
red through several cell wall layers of mutant cells.i Received: 4 September 2015 Accepted: 19 February 2016 Received: 4 September 2015 Accepted: 19 February 2016 Oil content quantification Chlamydomonas reinhardtii cells (eq. to 2 mm3 total cel-
lular volume) were harvested by centrifugation at 1000g
for 2 min (at 4 °C). The cells were quenched in hot iso-
propanol for immediate lipid extractions. Total cellu-
lar lipids were extracted using a mixture of hexane and Page 10 of 12 Page 10 of 12 Page 10 of 12 Schulz‑Raffelt et al. Biotechnol Biofuels (2016) 9:55 isopropanol [56]. Organic solvent phase-containing lipids
were collected and dried under a stream of nitrogen gas,
then resuspended into 200 µl chloroform:methanol (2:1,
v/v). Triacylglycerols (TAGs, i.e., oils) were first separated
from other lipid classes on a thin layer chromatograph
and charred with 2 % CuSO4 dissolved in 8 % H3PO4 in
water. TAG content was then calculated based on a den-
sitometry method, after comparison to a standard curve
generated with a C17:0 TAG standard [6]. Additional file 1: Figure S1. Southern blot analysis and complementa‑
tion of the std1 mutant. Figure S2. Conserved sequence features of
DYRKP kinases. Figure S3. Persistently high starch levels were observed
in the std1 mutant during photoautotrophic S deprivation conditions.
Figure S4. Cell counts, total cellular volume data and chlorophyll con‑
tents for the kinetic experiments in nitrogen deprivation shown on Fig. 2.
Figure S5. Oil accumulates in std1 mutant following N deprivation.
Figure S6. The std1 mutant forms cell aggregates enclosed by the
mother cell wall. Figure S7. Protein levels in wild-type and std1 mutant
cells during photoautotrophic N deprivation as determined by immu‑
nodetection. Figure S8. Phylogenetic tree of the DYRK protein family.
Figure S9. Phylogenetic tree of the DYRK protein family by using the
Maximum Likelihood (ML) or the Parsimony (Pars) approach. Table S1.
Accession numbers of the sequences used for the phylogenetic tree in
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0014341&type=printable
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The Role of Climate Variability in the Spread of Malaria in Bangladeshi Highlands
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Abstract Background: Malaria is a major public health problem in Bangladesh, frequently occurring as epidemics since the 1990s. Many factors affect increases in malaria cases, including changes in land use, drug resistance, malaria control programs,
socioeconomic issues, and climatic factors. No study has examined the relationship between malaria epidemics and climatic
factors in Bangladesh. Here, we investigate the relationship between climatic parameters [rainfall, temperature, humidity,
sea surface temperature (SST), El Nin˜o-Southern Oscillation (ENSO), the normalized difference vegetation index (NDVI)], and
malaria cases over the last 20 years in the malaria endemic district of Chittagong Hill Tracts (CHT). Methods and Principal Findings: Monthly malaria case data from January 1989 to December 2008, monthly rainfall,
temperature, humidity sea surface temperature in the Bay of Bengal and ENSO index at the Nin˜o Region 3 (NIN˜ O3) were
used. A generalized linear negative binomial regression model was developed using the number of monthly malaria cases
and each of the climatic parameters. After adjusting for potential mutual confounding between climatic factors there was
no evidence for any association between the number of malaria cases and temperature, rainfall and humidity. Only a low
NDVI was associated with an increase in the number of malaria cases. There was no evidence of an association between
malaria cases and SST in the Bay of Bengal and NIN˜ O3. Conclusion and Significance: It seems counterintuitive that a low NDVI, an indicator of low vegetation greenness, is
associated with increases in malaria cases, since the primary vectors in Bangladesh, such as An. dirus, are associated with
forests. This relationship can be explained by the drying up of rivers and streams creating suitable breeding sites for the
vector fauna. Bangladesh has very high vector species diversity and vectors suited to these habitats may be responsible for
the observed results. Citation: Haque U, Hashizume M, Glass GE, Dewan AM, Overgaard HJ, et al. (2010) The Role of Climate Variability in the Spread of Malaria in Bangladeshi
Highlands. PLoS ONE 5(12): e14341. doi:10.1371/journal.pone.0014341 Editor: Erika Martins Braga, Universidade Federal de Minas Gerais, Brazil Received June 28, 2010; Accepted November 4, 2010; Published December 16, 2010 Copyright: 2010 Haque 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. Abstract Funding: The study was supported by the Global Center of Excellence program, Institute of Tropical Medicine, Nagasaki University, Japan. GG was supported by
the Johns Hopkins Malaria Research Institute. 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: hashizum@nagasaki-u.ac.jp * E-mail: hashizum@nagasaki-u.ac.jp Ubydul Haque1, Masahiro Hashizume1*, Gregory E. Glass2, Ashraf M. Dewan3,4, Hans J. Overgaard5, Taro
Yamamoto1 Ubydul Haque1, Masahiro Hashizume1*, Gregory E. Glass2, Ashraf M. Dewan3,4, Hans
Yamamoto1 1 Department of International Health, Institute of Tropical Medicine (NEKKEN) and The Global Center of Excellence Program, Nagasaki University, Nagasaki, Japan,
2 Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America,
3 Department of Geography and Environment, University of Dhaka, Dhaka, Bangladesh, 4 Department of Spatial Sciences, Curtin University of Technology, Perth, Australia,
5 Department of Mathematical Sciences and Technology, Norwegian University of Life Sciences, Aas, Norway PLoS ONE | www.plosone.org December 2010 | Volume 5 | Issue 12 | e14341 Introduction It is well known that malaria is a
poverty related disease and strongly associated with socioeconomic
status [12]. However, trends in climatic factors are also driving
forces which affect malaria transmission [13]. No previous studies
have elucidated the relationship between changes in the incidence
of malaria and fluctuations of climate variables in Bangladesh. 10.7% per month for each 10 millimeter increase in monthly
rainfall (with a 2–3 month lag) in the highlands of West Kenya
[20]. Natural climatic disasters such as floods and cyclones may
also have significant relationship with malaria outbreaks [21]. and directly affects vector habitats. With land use change, malaria
may either increase or decrease [10]. Drug resistance is also a key
factor and responsible for sharp increases in malaria [8]. An
efficient malaria control program can also significantly reduce
malaria transmission [11]. It is well known that malaria is a
poverty related disease and strongly associated with socioeconomic
status [12]. However, trends in climatic factors are also driving
forces which affect malaria transmission [13]. No previous studies
have elucidated the relationship between changes in the incidence
of malaria and fluctuations of climate variables in Bangladesh. also have significant relationship with malaria outbreaks [21]. Temperatures between 15 to 40uC and humidity between 55 to
80% are suitable for the completion of the P. falciparum and P. vivax
malaria parasite life cycles [22]. Such conditions are found
throughout the seasons in India, where a close association between
temperature, rainfall conditions, and malaria has been reported
[22]. The minimum temperature was strongly associated with the
occurrence of malaria cases in Rwanda [23]. Another study in east
African highlands have shown that a 1uC increase in minimum
temperature with a lag time of 1–2 months and a 1uC increase in
maximum temperatures with a lag time of 2–5 months led to an 8–
95% increase in the number of malaria outpatients [24]. Studies throughout the world have linked changes in malaria
incidence with patterns of rainfall, temperature and humidity
[14,15]. Rainfall is considered to be a major factor influencing
malaria cases in Africa [16] and a causal relationship between
rainfall and malaria transmission is well recognized [17,18,19]. In
Sri Lanka, malaria cases were strongly correlated with rainfall with
a time lag of 0–3 months [14]. Malaria cases increased by 1.4% to Satellites from the U.S. Introduction The malaria vector situation in Bangladesh is complex due to
high species diversity and the presence of species complexes with
many sibling species displaying different ecological behaviors [4]. In Bangladesh An. minimus s.l., An. dirus, An. philippinensis, and An. sundaicus are considered primary malaria vectors and An. aconitus,
An. annularis, and An. vagus as secondary vectors [5]. However,
recent studies have incriminated a range of other species, such as
Anopheles nigerrimus, An. subpictus, An. barbirostris, and An. maculatus
[6,7]. The main vectors in the study area are An. baimai (dirus), An. philippinensis, An. vagus, and An. minimus [7]. Malaria is the most important tropical and parasitic disease in
the world. In 2008, there were an estimated 243 million cases of
malaria, the vast majority of cases (85%) occurring in the African
Region. In 2008, malaria accounted for an estimated 863,000
deaths [1]. Bangladesh is one of ten Asian countries where malaria
is endemic [2]. In 2008, malaria morbidity and mortality in
Bangladesh were recorded as 84,690 and 154, respectively [3]. Malaria is endemic in 13 northern and eastern districts of
Bangladesh along the border with India and Myanmar, with 90%
of morbidity and mortality reported from three hill districts
(Rangamati, Bandarban and Khagrachari) (Figure 1). The malaria
prevalence rate in Bangladesh was 3.97% in 2007 [2]. The
majority of infections was P. falciparum (90.2%), with P. vivax and
mixed infections making up 5.3% and 4.5% respectively [2]. In Bangladesh, malaria became epidemic during the 1990s
possibly due to the ban of DDT (dichlorodiphenyltrichloroethane)
in 1985, lack of malaria control efforts, insecticide resistance and
resistance to chloroquine (1st line drug at that time) [8,9] and
many factors may account for the pattern of malaria infection in
the country. Land use change is a part of environmental change December 2010 | Volume 5 | Issue 12 | e14341 1 December 2010 | Volume 5 | Issue 12 | e14341 PLoS ONE | www.plosone.org Climate Malaria Bangladesh Figure 1. Spatial distribution of malaria prevalence in Bangladesh. doi:10.1371/journal.pone.0014341.g001 Figure 1. Spatial distribution of malaria prevalence in Bangladesh. doi:10.1371/journal.pone.0014341.g001 and directly affects vector habitats. With land use change, malaria
may either increase or decrease [10]. Drug resistance is also a key
factor and responsible for sharp increases in malaria [8]. An
efficient malaria control program can also significantly reduce
malaria transmission [11]. PLoS ONE | www.plosone.org December 2010 | Volume 5 | Issue 12 | e14341 Results survey at the climatic scale. Normalized Difference Vegetation
Index (NDVI) is a measure of vegetation conditions. NDVI values
vary between +1.00 and 21.00; the higher the NDVI value, the
denser or healthier the green vegetation. Strong relationship
between vegetation health (VH) (another measure of vegetation
conditions and similar to NDVI), and malaria cases has been
demonstrated in Bangladesh, indicating that VH can be used as an
indicator of climatic and environmental conditions [25]. The time series of the number of malaria cases, rainfall, tem-
perature, humidity and NDVI from January 1989 to December
2008 are shown in Figure 2. The time series of SST of Bay of
Bengal, NINO3 during the study period is shown (Figure S1). There was a distinct seasonality in the number of malaria cases
with a peak during June to August. High temperatures occurred in
April to September in each year. Except for some small
fluctuations, rainfall occurred between May and October. In
some years, high NDVI was observed in all seasons but the peak
occurred between in October and November. SST was lowest in
January and February, started to increase in March, remained
high until October and decreased from November onwards. El Nin˜o and La Nin˜a years coincide with low and high rainfall
years in southern Asia. El Nin˜o Southern Oscillation (ENSO) has
been used as predictor of climatic events and a significant cor-
relation has been reported between sea surface temperature (SST)
and malaria cases [15,17,18,22]. El Nin˜o Southern Oscillation
showed a significant association with malaria case numbers. A 1uC
increase in Nin˜o 3.4 (region in Pacific) SST was associated with
about a 20% increase in malaria cases in Colombia [26]. An
analysis of 37 years of national statistics in India showed, in
general, that if the number of malaria cases in a particular year
was less than the decadal average, that particular year was
influenced by La Nin˜a; and when the number of malaria cases in a
particular year exceeded the decadal average, that particular year
was influenced by El Nin˜o [22]. The number of malaria cases increased significantly with
increased temperature with a lag time of 0–3 months (p = 0.007)
(Figure 3a) but decreased significantly with higher rainfall with a
lag of 0–3 months (p = 0.002) (Figure 3b). The number of malaria
cases also decreased as humidity increased with a lag time of 0–3
months (p,0.001) (Figure 3c). Introduction National Oceanic and Atmospheric
Administration (NOAA) environmental satellites provide a vegetation PLoS ONE | www.plosone.org December 2010 | Volume 5 | Issue 12 | e14341 2 Climate Malaria Bangladesh Climate Malaria Bangladesh Results The number of malaria was
significantly negatively associated with NDVI at a lag of 0–3
months (p,0.001) (Figure 3d). The risk response relationships adjusted for potential mutual
confounding between malaria cases and temperature, rainfall, and
humidity with lag times of 0–3 months showed no significant
associations (Figure 4a–c). However, malaria cases remained
significantly associated with lower NDVI with a lag time of
0–3 months (Figure 4d). Each 0.1 increase in monthly NDVI
was associated with a 30.4% decrease in malaria cases (95% CI:
19.2–40.1). There is a strong interest in investigating the relationship be-
tween climate variability and malaria transmission and with con-
cerns about potential climate change, this interest has increased. A
rise of about 6% in malaria cases during 2000 in middle income
countries was attributed to climate change [21]. In this study,
we investigate the relationship between climate variability and
malaria cases in the endemic area of Bangladesh. Figure 2. Time series of the number of all malaria cases per month and meteorological data in Rangamati, 1989–2008. doi:10.1371/journal.pone.0014341.g002 Figure 2. Time series of the number of all malaria cases per month and meteorological data in Rangamati, 1989–2008. doi:10 1371/journal pone 0014341 g002 Figure 2. Time series of the number of all malaria cases per month and meteorological data in Rangamati, 1989–2008. doi:10.1371/journal.pone.0014341.g002 December 2010 | Volume 5 | Issue 12 | e14341 PLoS ONE | www.plosone.org 3 Climate Malaria Bangladesh Figure 3. Relationship between the number of malaria cases per month and (a) average mean temperature, (b) total rainfall, (c)
average relative humidity and (d) Normalized difference vegetation index (NDVI) over lags of 0–3 months (shown as a 3 d.f. natural
cubic spline) adjusted for seasonal variation and between-year variations. RR represents the relative risk of malaria (scaled against the
mean monthly number of cases). The centre line in each graph shows the estimated spline curve, and the upper and lower lines represent the 95%
confidence limits. doi:10.1371/journal.pone.0014341.g003 Figure 3. Relationship between the number of malaria cases per month and (a) average mean temperature, (b) total rainfall, (c)
average relative humidity and (d) Normalized difference vegetation index (NDVI) over lags of 0–3 months (shown as a 3 d.f. natural
cubic spline) adjusted for seasonal variation and between-year variations. RR represents the relative risk of malaria (scaled against the
mean monthly number of cases). Discussion After adjusting for potential confounders, our study suggests
that the best leading indicator of the number of malaria cases was
NDVI at a lag of 0–3 months, and that NDVI was negatively
associated with malaria cases. We did not find significant rela-
tionships like other studies; however, this may be due to the fact
that other studies used different methodologies in different regions
of the world where the malaria ecology/epidemiology is quite
different. This study draws attention again to the complex nature
of the relationship between malaria and climate. However, it has
also illustrated the potential value of such studies, both for
identifying local factors which may predict the epidemiology of the
disease as well as for providing a deeper understanding of the
biology of the parasite system. In the unadjusted analysis, rainfall showed a clear negative
correlation with the number of malaria cases, but incorporating
other climatic factors eliminated the significant relationship. It is
difficult to explain why the malaria-rainfall associations became
non-significant when other climatic factors were included in the
model. These results differ from studies carried out in Thailand
where malaria cases were positively associated with rainfall [33]. Rainfall is also a major contributing factor for the increase of
malaria cases in other areas [17,18,19,24]. However, rainfall was
negatively correlated with malaria cases in India [22]. Long term
data from Sri Lankan (January 1972 to December 2005) showed
that the region with the highest rainfall had the least malaria, and
that malaria cases increased with lower rainfall [14]. Moderate
correlation (r = 0.48, p = 0.069) with annual rainfall with malaria
incidence confirmed from Indian Rajasthan. The incidence of Temperature and number of malaria cases were positively
associated when not considering the effect of other climatic con-
founding factors. However, after adjusting for all other parameters, Figure 5. Relationship between the number of malaria cases per month and (a) average sea surface temperature (SST) of the Bay of
Bengal over lags of 0–3 months, (b) average NINO3 over lags of 0–3 months, (c) 4–7 months and (d) 8–11 months (shown as a 3 d.f. natural cubic spline) adjusted for potential mutual confounding between the lags of NINO3, seasonal variation and between-year
variations. RR represents the relative risk of malaria (scaled against the mean monthly number of cases). Results The centre line in each graph shows the estimated spline curve, and the upper and lower lines represent the 95%
confidence limits. doi:10 1371/journal pone 0014341 g003 doi:10.1371/journal.pone.0014341.g003 Figure 4. Relationship between the number of malaria cases per month and (a) average mean temperature, (b) total rainfall, (c)
average relative humidity and (d) Normalized difference vegetation index (NDVI) over lags of 0–3 months (shown as a 3 d.f. natural
cubic spline) adjusted for potential mutual confounding between these 4 variables, seasonal variation and between-year
variations. RR represents the relative risk of malaria (scaled against the mean weekly number of cases). The centre line in each graph shows the
estimated spline curve, and the upper and lower lines represent the 95% confidence limits. doi:10.1371/journal.pone.0014341.g004 Figure 4. Relationship between the number of malaria cases per month and (a) average mean temperature, (b) total rainfall, (c)
average relative humidity and (d) Normalized difference vegetation index (NDVI) over lags of 0–3 months (shown as a 3 d.f. natural
cubic spline) adjusted for potential mutual confounding between these 4 variables, seasonal variation and between-year
variations. RR represents the relative risk of malaria (scaled against the mean weekly number of cases). The centre line in each graph shows the
estimated spline curve, and the upper and lower lines represent the 95% confidence limits. doi:10.1371/journal.pone.0014341.g004 December 2010 | Volume 5 | Issue 12 | e14341 PLoS ONE | www.plosone.org 4 Climate Malaria Bangladesh no association was observed. Undoubtedly, temperature is a key
factor in malaria transmission [24,27,28,29,30,31], directly affecting
mosquito development, survival, reproduction, activity, and the
extrinsic incubation rate. For example, minimum temperatures
during the cool months or the previous month have been associated
with malaria transmission in China and Burundi, respectively [29,32]. The reason we were not able to detect a significant temperature-
malaria relationship could be because of the crude average tem-
perature data used here concealing shorter term effects impacting
vector populations. Furthermore, statistical significance alone does
not always address the complex biological dynamics of mosquito
development and temperature. However, temperature ranges in this
region of Bangladesh are always favorable for mosquito development. Further research should consider multiple study areas, including
biological models of mosquito development [e.g. 36] to improve
detection of temperature effects on malaria transmission specific for
Bangladesh. Results There were no significant associations between malaria cases
and SST using lag times of 0–3 months (Figure 5a) and NINO3
with different lag periods (Figure 5b–d). The incorporation of the Fourier terms (up to fifth harmonics
adding 1 harmonic at a time) into the fully adjusted model, in
place of indicator variables for months, had broadly little effect on
the estimates of the effects of NDVI: model with no seasonal
control (24.8% (95%CI: 14.3–34.0) decrease). Although there was
no evidence for the effect of temperatures in the model adjusted
for season with indicator variables of months, there was a
significant positive effect of temperatures when there was no
seasonal control and with the Fourier terms of 1 harmonic (results
not shown). Discussion However, another study showed a positive
correlation between SST in the Bay of Bengal with June rainfall in
south-eastern Bangladesh, but no significant relationship with May,
July, August and September rainfall [50]. Results have shown that
the Indian summer monsoon and ENSO are negatively correlated
[51]. Therefore, it is clear that this parameter exerts different effects
in different places at different times. Other reasons for the observed result may be that high and
intensive rainfall flush out breeding sites. It may also be that people
are more aware of the risk of malaria during high rainfall and so take
preventative measures. During the post monsoon, when rainfall
decreases or stops, malaria cases increase. Better entomological data
would greatly increase our understanding of the influence of rainfall
on the biology of malaria in Bangladesh. Unfortunately, such
entomological data are not yet available in Bangladesh. In the East African highlands, ENSO events may trigger heavy
rainfall and raised temperatures and were associated with increased
malaria in the southwestern highlands of Uganda [48]. The same
ENSO and heavy rainfall reduced malaria in the Tanzanian
highlands [48]. The relationship between SST and rainfall in
Bangladesh has not been extensively investigated. During the ENSO
years of this study, rainfall decreased significantly in Bangladesh [49],
similar to an earlier study using a 43-year data set that established a
negative association
between
ENSO events
and
rainfall
in
Bangladesh [49]. However, another study showed a positive
correlation between SST in the Bay of Bengal with June rainfall in
south-eastern Bangladesh, but no significant relationship with May,
July, August and September rainfall [50]. Results have shown that
the Indian summer monsoon and ENSO are negatively correlated
[51]. Therefore, it is clear that this parameter exerts different effects
in different places at different times. In our study, a low relative humidity was associated with an
increase in malaria cases in the bivariate analysis but after adjusting
for other local climatic parameters, no significant relationship was
observed. This could be explained by the fact that humidity is
directly dependent on temperature and rainfall, thus confounding
the results. Generally, increased humidity is believed to favor vector
survival [39]. Little has been published on the relationship between
humidity and risk of malaria [30], but Bhattacharya and colleagues
reported humidity levels between 55 and 80% were suitable for both
P. falciparum and P. vivax [22]. Discussion The centre line in each graph shows the
estimated spline curve, and the upper and lower lines represent the 95% confidence limits. doi:10.1371/journal.pone.0014341.g005 Figure 5. Relationship between the number of malaria cases per month and (a) average sea surface temperature (SST) of the Bay of
Bengal over lags of 0–3 months, (b) average NINO3 over lags of 0–3 months, (c) 4–7 months and (d) 8–11 months (shown as a 3 d.f. natural cubic spline) adjusted for potential mutual confounding between the lags of NINO3, seasonal variation and between-year
variations. RR represents the relative risk of malaria (scaled against the mean monthly number of cases). The centre line in each graph shows the
estimated spline curve, and the upper and lower lines represent the 95% confidence limits. doi:10.1371/journal.pone.0014341.g005 December 2010 | Volume 5 | Issue 12 | e14341 PLoS ONE | www.plosone.org 5 Climate Malaria Bangladesh P. falciparum malaria showed a significant correlation (r = 0.61,
p = 0.016) with rainfall [34]. At the same time no clear relationship
was observed between rainfall and malaria incidence in Madhya
Pradesh, central India [35]. The interpretation was that drought
caused pools in the river bed, creating suitable conditions for
mosquito breeding. A similar explanation may be true in
Rangamati, where the main malaria vectors have a comparable
ecology to those in Sri Lanka [36]. For example, An. minimus s.l. is
associated with slow-moving streams [37] as is the primary vector
in Sri Lanka, An. culifacies which breeds in river bed pools [36]. An. dirus, on the other hand, breeds in small temporal pools in heavily
shaded forests and seems to be positively associated with rainfall
[38]. Anopheles minimus and An. dirus are also primary vectors in
Thailand and it is thus difficult to explain the contradictory results
obtained here [33]. An important point to consider here is that we
might be witness of a potential shift in vector importance, which
again stresses the need for continuous monitoring of vector
transmission dynamics and detailed studies of vector bionomics in
the region. of vector species and their siblings, changes in vector transmission
dynamics, geographical and socio-economic settings, drug resis-
tance, immunity among people, or control efforts. It can also be
due to differences among climatic parameters between African
countries and Bangladesh. Summer-winter seasonality may have
some affect on the activity of mosquito vectors. Discussion Humidity was found to be related with
the number of malaria cases in China, where a relative humidity
below 60% shortened the life span of the mosquito so that below
60%, there was a decline in the risk of clinical malaria while above
60% relative humidity the infection rate increased significantly
[29,30]. It was also confirmed that the malaria risk at 80% humidity
was twice as high as that of 60% [29,30]. Further studies are needed
to elucidate the relationship between humidity and malaria
epidemiology in Bangladesh. The main climatic variables associated with malaria transmission
on the Indian sub-continent are rainfall, temperature and humidity
[22]. Studies on the interaction of climate and malaria in Bangladesh
are limited because of a paucity of malaria case data, lack of skilled
manpower and meteorological stations in endemic districts. In the
Chittagong Hill Tracts, for example, there is only one meteorological
station situated in Rangamati. However, the potential role of climate
change and its impact on health, particularly malaria, has received
increasing attention in Bangladesh. Normalized Difference Vegetation Index, both unadjusted and
adjusted for other variables, were negatively associated with
number of malaria cases. In Eritrea, NDVI and malaria cases were
significantly (positively) associated with each other [40]. Our
results differ from those of Bruce et al [41], who showed no
association with NDVI and infection rates in Malawi. In another
study in Indochina Peninsula, overlaying maps of the vegetation
index with indices of P. falciparum and P. vivax infection showed that
areas with NDVI values higher than 0.3 or 0.4 coincided with
areas of high malaria incidence [42]. A similar result was found in
Mali where an NDVI between 0.35 and 0.4 was associated with an
increase in malaria cases [43]. It seems counterintuitive that a low NDVI, an indicator of low
vegetation greenness, is associated with increases in malaria cases,
since the primary vectors in Bangladesh, such as An. dirus, are
associated with forests. However, NDVI is a reasonably reliable
indicator of rainfall and the unadjusted analysis both indicate a
relationship between rainfall and malaria cases: i.e. as rainfall (and
NDVI) decreases malaria increases. This relationship can be
explained by the drying up of rivers and streams creating suitable
breeding sites for the vector fauna. Bangladesh has very high
vector species diversity and vectors suited to these habitats may be
responsible for the observed results. PLoS ONE | www.plosone.org December 2010 | Volume 5 | Issue 12 | e14341 Discussion It is also mostly due
to the climatic dependency of vector behavior and different areas
are likely to experience different effects in the rate of malaria
vector growth because of climatic parameters including NDVI. Interestingly, NDVI has already been shown to be a reliable
estimate of vector population and vector species distribution [44]. A relationship between weekly and monthly NDVI and mosquito
abundance has also been demonstrated [45]. There was no relationship between SST, NIN˜ O3 and malaria
cases. In contrast, analyses of the trends of SST over the eastern
equatorial Pacific indicated that SST during March, April and
May were negatively correlated with malaria cases in India from
1980 to 2000 [22]. Positive relationships were observed between
Southern Oscillation Index and monthly incidences of malaria in
China [46]. Historical epidemic malaria in Punjab between 1868
and 1943 correlates significantly with the sea surface temperature
anomalies in the Eastern Equatorial Pacific. At the same time, 9
out of 16 malaria epidemics in the south west part of Sri Lanka
were recorded between 1870 and 1945 during El Nino years [47]. There was no relationship between SST, NIN˜ O3 and malaria
cases. In contrast, analyses of the trends of SST over the eastern
equatorial Pacific indicated that SST during March, April and
May were negatively correlated with malaria cases in India from
1980 to 2000 [22]. Positive relationships were observed between
Southern Oscillation Index and monthly incidences of malaria in
China [46]. Historical epidemic malaria in Punjab between 1868
and 1943 correlates significantly with the sea surface temperature
anomalies in the Eastern Equatorial Pacific. At the same time, 9
out of 16 malaria epidemics in the south west part of Sri Lanka
were recorded between 1870 and 1945 during El Nino years [47]. In the East African highlands, ENSO events may trigger heavy
rainfall and raised temperatures and were associated with increased
malaria in the southwestern highlands of Uganda [48]. The same
ENSO and heavy rainfall reduced malaria in the Tanzanian
highlands [48]. The relationship between SST and rainfall in
Bangladesh has not been extensively investigated. During the ENSO
years of this study, rainfall decreased significantly in Bangladesh [49],
similar to an earlier study using a 43-year data set that established a
negative association
between
ENSO events
and
rainfall
in
Bangladesh [49]. Malaria and Climate Data All malaria cases were collected from the Rangamati district
hospital (22u 409 N, 92u 119 E) from January 1989 to December
2008. This hospital is the reference hospital for all 10 sub-districts
of Rangamati district. There is no other hospital or clinic in the
central town of Rangamati. Not all cases were confirmed by
microscopy. From 1988 to 2004, cases were characterized as
uncomplicated malaria (UM), treatment failure malaria (TFM)
and severe malaria (SM). Uncomplicated malaria was presump-
tively determined while TFM and SM were confirmed by malaria
microscopy. From 2004 to 2009, cases were confirmed as
uncomplicated malaria presumptive (UMP), uncomplicated ma-
laria confirmed (UMC), as well as SM and VM (vivax malaria). Neither
microscopy nor rapid diagnosis tests (RDT)
were
performed for UMP, but for the others either microscopy or
RDT was used for diagnosis. A survey conducted by the authors in
one of the thana (Rajathali sub-district) in Rangamati district
showed that 93.2% of malaria cases were P. falciparum, 1.9% were
P. vivax and 5.0% were mixed infections (unpublished data). Monthly climatic data including rainfall, temperature, and relative
humidity were obtained from the Bangladesh Meteorological
Department. The meteorological station is in the central town of
the district within 5 km of the study hospital. Normalized
difference vegetation index was derived from the data library of
the International Research Institute (IRI) of Lamont Doherty
Earth Observatory (LDEO) at Columbia University, USA. Mean
monthly SSTs in the Bay of Bengal (20–21uN, 90–91uE) were
derived from the NOAA Optimum Interpolation Sea Surface
Temperature dataset [52,53]. The strength of ENSO was
measured using SST in the Nin˜o 3 region (NIN˜ O3) in the Pacific
Ocean, which were extracted from NOAA climate prediction
center datasets [54]. Discussion Satellite-based vegetation health (VH) indices have also been
compared with malaria epidemiology to study whether they could
be used as a proxy for monitoring malaria epidemics in sixty four
districts of Bangladesh. During drought years, when vegetation
was under stress, fewer people had malaria [25]. The fact that our
findings are not consistent with results from these areas could be
due to within-country variations, such as different ecological habits The current study displays several limitations. First, the cases
were based on one hospital’s data. Although the hospital is the
reference hospital for all 10 sub districts of Rangamati district and
the cases are somewhat representative of the entire district, this PLoS ONE | www.plosone.org December 2010 | Volume 5 | Issue 12 | e14341 December 2010 | Volume 5 | Issue 12 | e14341 6 Climate Malaria Bangladesh sample may be underrepresented as only the severe cases are likely
to be referred to the hospital. However, whether or how this would
introduce a temporal trend in the data that would distort the
results is not evident. Second, P. vivax and P. falciparum malaria
cases were pooled together as total malaria cases. This may
confound more detailed interactions of these two parasites, their
vectors and climatic conditions. However, environmental condi-
tions permitting parasite development broadly overlaps with
P. vivax being somewhat more permissive in its temperature
tolerances. Furthermore, as more than 90% of malaria cases are
due to P. falciparum and the proportion of P. vivax is very small in
this area, separate analysis may not be statistically meaningful. Third, the study was based on only one district, the Chittagong
Hill Tracts, so that extrapolating these results to other parts of the
country or south Asia needs to be done with caution. This region,
however, includes areas with some of the highest rates of malaria
transmission and so is of practical importance in itself. Fourth,
there may be concerns about a possible effect of non-climatic
factors such as land use changes, population growth, development
of drug resistance, change in diagnostic criteria, changes in local
health infrastructure, access to care and public health interven-
tions over the duration of the 20 year study. However, as these
factors are not likely to change on a monthly interval, and it seems
unlikely that they would obscure the short-term dependence of
malaria on the factors investigated in this study. Malaria and Climate Data log E Y
ð
Þ
ð
Þ~azNS TEMP0{3 , 3 df
ð
ÞzNS RAIN0{3 , 3 df
ð
Þ
zNS HUM0{3, 3 df
ð
ÞzNS NDVI0{3 , 3 df
ð
Þ
zi:monthzi:yearzAR1
ð1Þ log E Y
ð
Þ
ð
Þ~azNS TEMP0{3 , 3 df
ð
ÞzNS RAIN0{3 , 3 df
ð
Þ
zNS HUM0{3, 3 df
ð
ÞzNS NDVI0{3 , 3 df
ð
Þ
zi:monthzi:yearzAR1
ð1Þ log E Y
ð
Þ
ð
Þ~azNS SST0{3 , 3 df
ð
Þzi:monthzi:yearzAR1ð2Þ log E Y
ð
Þ
ð
Þ~azNS SST0{3 , 3 df
ð
Þzi:monthzi:yearzAR1ð2Þ E(Y) is the expected monthly case count, NS indicates a natural
cubic spline function, TEMP0–3, RAIN0–3, HUM0–3, NDVI0–3 and
SST0–3 represent average temperature, rainfall, relative humidity,
NDVI and SST at lag 0–3 months, respectively, i.month represents
indicator variables for the month, i.year represents indicator
variables for the year, and AR1 represents a first-order auto-
regressive term. For example, NS(TEMP0–3, 3 df) indicates a linear
term (raw data) and two spline terms of temperature at a lag of 0–3
months. Discussion cases as well as different climate parameters in the study period were
observed graphically using scatter plots. Due to over-dispersed data
for monthly malaria cases, a generalized linear negative binomial
regression model was developed using the number of monthly
malaria cases and climatic parameters. Potentially significant
associations were analyzed by comparing patterns of variation in
incidence of malaria over time with the patterns of each climatic
parameter, using time series regression analysis. The unit of analysis
in this study was the month, thus potential confounders that must be
controlled are those that vary over time, possibly coinciding with
each climate variable. Thus the association between the particular
climatic parameter (e.g. temperature) and malaria incidence can be
confounded by the other climatic parameters (e.g. rainfall, humidity
and NDVI). Temporal associations between climate and disease can be con-
founded by trends and seasonal patterns. To account for seasonality
of malaria that was not directly linked with the climate, we included
indicator variables for each month in the model. Indicator variables
for the years of the study were also incorporated into the model to
allow for long-term trends and other variations between years. To
allow for autocorrelations, an autoregressive term at order 1 was
incorporated into the model [55]. Models for temperature, rainfall, humidity, NDVI and SST
From exploratory analyses, we considered lag times, (the delay
in the effect of climate factors on the number of malaria cases) of
up to 3 months for temperature, rainfall, humidity, NDVI and
SST of the Bay of Bengal. In our initial analyses, we fitted a
natural cubic spline (3 df) [56] to the average climatic factors over
lag times of 0–3 months. Natural cubic splines were used to create
graphs, where the number of malaria cases was plotted as
smoothed functions of climatic factors [56], to visually assess the
functional form of the adjusted relationship, thereby identifying
whether the relationship was likely to be linear or not across the
full range of independent variables. Finally, potential mutual
confounding between temperature, rainfall, humidity and NDVI
were adjusted to identify independent associations of monthly
malaria cases and each particular climatic parameter. Since the
SST of the Bay of Bengal was regarded as a more distant factor
(compared with local climatic factors), we did not adjust it for the
effect of the local climatic parameters of temperature, rainfall,
humidity and NDVI. The final model was: December 2010 | Volume 5 | Issue 12 | e14341 References 1. WHO (2009) World malaria report 2008. 23. Loevinsohn ME (1994) Climatic warming and increased malaria incidence in
Rwanda. Lancet 343: 714–718. 1. WHO (2009) World malaria report 2008. (
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resistant malaria in Bangladesh: an in vitro assessment. Am J Trop Med Hyg 68:
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successfully reduced in Brazil, Eritrea, India, and Vietnam. Am J Trop Med Hyg
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transmission, prevention and treatment of malaria among two endemic
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resurgence in the East African highlands: temperature trends revisited. Proc Natl
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13. Sutherst RW (2004) Global change and human vulnerability to vector-borne
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in Thailand. Journal of Infection 52: 227–230. 15. Lindblade KA, Walker ED, Onapa AW, Katungu J, Wilson ML (1999)
Highland malaria in Uganda: prospective analysis of an epidemic associated
with El Nino. Trans R Soc Trop Med Hyg 93: 480–487. 34. Gupta R (1996) Correlation of rainfall with upsurge of malaria in Rajasthan. J Assoc Physicians India 44: 385–389. 35. Singh N, Sharma VP (2002) Patterns of rainfall and malaria in Madhya P
central India. Ann Trop Med Parasitol 96: 349–359. 16. Abeku TA (2007) Response to malaria epidemics in Africa. Emerg Infect Dis 13:
681–686. 36. Kusumawathie PH, Wickremasinghe AR, Karunaweera ND, Wijeyaratne MJ,
Yapabandara AM (2006) Anopheline breeding in river bed pools below major
dams in Sri Lanka. Acta Trop 99: 30–33. 17. Statistical Analysis We then fitted the data to the linear threshold models i.e.,
models that assume a log-linear increase in risk. The increase in
the number of malaria cases associated with 1% decrease in a The climatic data and malaria case time series data were
computerized and cross checked. Seasonality and peaks of malaria PLoS ONE | www.plosone.org December 2010 | Volume 5 | Issue 12 | e14341 December 2010 | Volume 5 | Issue 12 | e14341 7 Climate Malaria Bangladesh given measure of climatic parameters (estimated as coefficients
from the regression model) was reported as a percentage change. zi:monthzi:yearzAR1 To investigate whether the results were sensitive to the levels of
control for seasonal patterns, analyses were repeated using Fourier
terms of the month up to the fifth harmonic per year, adding one
harmonic at a time. Diagnostics for model (1) including plots of
model residuals, predicted and observed time series plots, par-
tial autocorrelation function of the residuals were calculated
(Figure S2). All analyses were performed by STATA 10 (Stata
Corporation, College Station, Texas). Model for NINO3 We considered lag times of up to 11 months for NINO3. We
fitted a natural cubic spline (3 df) [56] to the average NINO3 over
each 4-month period (i.e. lags 0–3, 4–7 and 8–11 months), as
separate splines that were simultaneously included in the model. Figure S2
Diagnostics of malaria-climate (temperature, rainfall,
humidity and NDVI) models: (a) plots of model residuals, (b)
predicted and observed time series plots, (c) partial autocorrelation
function of the residuals. log E Y
ð
Þ
ð
Þ~azNS NINO3 0{3, 3 df
ð
Þ
zNS NINO3 4{7, 3 df
ð
Þ
zNS NINO3 8{11 , 3 df
ð
Þ
zi:monthzi:yearzAR1
ð3Þ log E Y
ð
Þ
ð
Þ~azNS NINO3 0{3, 3 df
ð
Þ
zNS NINO3 4{7, 3 df
ð
Þ
zNS NINO3 8{11 , 3 df
ð
Þ
zi:monthzi:yearzAR1
ð3Þ Found at: doi:10.1371/journal.pone.0014341.s002 (0.12 MB TIF) Found at: doi:10.1371/journal.pone.0014341.s002 (0.12 MB TIF) Supporting Information Figure S1
Time series of the average sea surface temperature
(SST) of the Bay of Bengal and NINO3, 1989–2008. Found at: doi:10.1371/journal.pone.0014341.s001 (0.11 MB TIF) Figure S1
Time series of the average sea surface temperature
(SST) of the Bay of Bengal and NINO3, 1989–2008. Found at: doi:10.1371/journal.pone.0014341.s001 (0.11 MB TIF) Author Contributions Conceived and designed the experiments: UH MH. Analyzed the data:
UH MH. Wrote the paper: UH MH HJO. Critical input and re-appraisal
of the manuscript: GEG AMD TY. Critical input: HJO. Conceived and designed the experiments: UH MH. Analyzed the data:
UH MH. Wrote the paper: UH MH HJO. Critical input and re-appraisal
of the manuscript: GEG AMD TY. Critical input: HJO. Acknowledgments ð3Þ We are grateful to Malaria control program, Bangladesh for providing
malaria case data. We are also grateful to Dr. Md Mushfiqur Rahman,
national consultant (epidemiologist) World Health Organization (WHO),
Bangladesh for providing additional information. We are grateful to Malaria control program, Bangladesh for providing
malaria case data. We are also grateful to Dr. Md Mushfiqur Rahman,
national consultant (epidemiologist) World Health Organization (WHO),
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