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Article
Method Validation for Determination of Thallium by
Inductively Coupled Plasma Mass Spectrometry and
Monitoring of Various Foods in South Korea
Yeon-hee Kim 1 , Wook-jin Ra 1 , Solyi Cho 2 , Shinai Choi 3 , Bokyung Soh 1 , Yongsung Joo 4
and Kwang-Won Lee 1, *
1
2
3
4
*
Citation: Kim, Y.-h.; Ra, W.-j.; Cho,
S.; Choi, S.; Soh, B.; Joo, Y.; Lee, K.-W.
Method Validation for Determination
of Thallium by Inductively Coupled
Plasma Mass Spectrometry and
Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841,
Korea; helly27@korea.ac.kr (Y.-h.K.); ra228@korea.ac.kr (W.-j.R.); bokyung12345@korea.ac.kr (B.S.)
Advanced Food Safety Research Group, School of Food Science and Technology, Chung-Ang University,
Anseong-si 17546, Korea; syjo@knacon.co.kr
KnA Consulting, Yongin-si 16942, Korea; shchoi@knacon.co.kr
Department of Statistics, Dongguk University-Seoul, Seoul 04620, Korea; yongsungjoo@dongguk.edu
Correspondence: kwangwon@korea.ac.kr; Tel.: +82-2-3290-3027
Abstract: Thallium (Tl) is a rare element and one of the most harmful metals. This study validated
an analytical method for determining Tl in foods by inductively coupled plasma mass spectrometry (ICP-MS) based on food matrices and calories. For six representative foods, the method’s
correlation coefficient (R2 ) was above 0.999, and the method limit of detection (MLOD) was 0.0070–
0.0498 µg kg−1 , with accuracy ranging from 82.06% to 119.81% and precision within 10%. We
investigated 304 various foods in the South Korean market, including agricultural, fishery, livestock,
and processed foods. Tl above the MLOD level was detected in 148 samples and was less than
10 µg kg−1 in 98% of the samples. Comparing the Tl concentrations among food groups revealed
that fisheries and animal products had higher Tl contents than cereals and vegetables. Tl exposure
via food intake did not exceed the health guidance level.
Monitoring of Various Foods in South
Korea. Molecules 2021, 26, 6729.
Keywords: thallium; trace element; food contamination; risk assessment; ICP-MS
https://doi.org/10.3390/
molecules26216729
Academic Editors: Željka Fiket and
Martina Furdek Turk
Received: 29 September 2021
Accepted: 3 November 2021
Published: 6 November 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affiliations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
1. Introduction
Since the industrial revolution, modern technological development and associated environmental pollution have increased the exposure to trace metals. With novel applications
in microelectronics, chemotherapies, and other emerging technologies, the toxicological
significance of some uncommon or infrequently used metals has risen [1]. In this regard,
thallium (Tl) is rare but is one of the most harmful metals. William Crookes discovered it
in 1861, and the metallic form was first obtained by Lamy in 1862 [2]. The name is derived
from the Greek word “thallos,” which means young green twig [2]. Tl can be discovered in
pure form or in combination with other elements. Pure Tl has no odor or taste. It is utilized
in the production of optic lenses and glasses and the semiconductor and pharmaceutical
industries [3]. Tl was used as a rodenticide and insecticide until 1972 but was prohibited
because of its potential toxicity to humans [4]. Because the Tl ion charge and radius are
similar to those of potassium ion, Tl can interfere with the biological functions of potassium [5]. Gastroenteritis, polyneuropathy, and alopecia are three significant symptoms of
Tl poisoning in humans [6]. Alopecia is a specific symptom of Tl poisoning. Studies of Tl
toxicity in humans include case studies, clinical reports, and medical investigations. Epidemiological studies associated with long-term exposure to Tl are restricted by small scales
and insufficient information. The average lethal oral dose in adults has been estimated
to be 10–15 mg kg−1 [7]. If not treated, it generally takes 10–12 days to die, but there are
reports of death within 8–10 h [8].
4.0/).
Molecules 2021, 26, 6729. https://doi.org/10.3390/molecules26216729
https://www.mdpi.com/journal/molecules
Molecules 2021, 26, 6729
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The regulation for Tl sets a maximum contaminant level (MCL) of 0.2 µg L−1 in
drinking water in the United States [9], and 0.8 µg L−1 in fresh water and 1 mg kg−1 dry
weight in soil in Canada [10,11]. In South Korea, food regulations for most common toxic
metal(loid)s such as cadmium, lead, mercury, and arsenic are established and managed
to ensure safety throughout food consumption [12]. In contrast, preventive research and
analytical method verification are necessary to avoid food accidents and respond to possible
hazards from excess Tl.
Heavy metal analysis can be performed using such analytical techniques as inductively coupled plasma atomic emission spectrometry (ICP-AES), graphite furnace atomic
absorption spectrometry (GF-AAS), and inductively coupled plasma mass spectrometry
(ICP-MS). The latter is suitable for trace element analysis because of its high resolution and
two to three times lower detection limit than ICP-AES [13]. In recent years, the number
of studies on food analysis using ICP-MS has increased [14–18]. Given that there were no
detailed studies on Tl content in various food products in the South Korean market, we
validated a method for Tl determination by ICP-MS, monitored the commonly consumed
foods in South Korea, and assessed the risk based on the results to provide foundational
information.
2. Results and Discussion
2.1. Method Validation and Quality Control
We validated the external standard calibration curve for Tl. Regression of the correlation (R2 ) showed good linearity: above 0.999. Trueness was represented by certified
reference material (CRM) (BCR-679, white cabbage). The measured average concentration
was 3.03 ± 0.36 µg kg−1, and the mean recovery was 101%. The relative standard deviation
(SD) was 2.94%. Six representative foods were tested for accuracy and precision by adding
standard solutions at low (0.1 µg kg−1 ), medium (0.5 µg kg−1 ), and high (1.0 µg kg−1 )
concentrations (Table S1). The intraday accuracy ranged from 82.06% to 119.81%, interday
accuracy—from 92.05% to 110.44%; the intraday precision—from 0.88% to 9.08%, interday
precision—from 1.09% to 9.79%.
We observed a recovery reduction in the salted solid matrix representative food (sea
salt) to less than 60% during the method validation. Salts (i.e., sodium, calcium, chloride,
and potassium) cause interference in ICP-MS [19]. Higher matrix salt concentrations reduce
the signal and lower the ionization potential, resulting in higher matrix effect [20]. Sample
dilution can reduce the severity of the matrix effect and instrument drift [20,21]. It is
recommended to keep the total dissolved solid level of samples less than 0.2% when using
ICP-MS [21]. The original sample weight was 0.5 g, and the sample decomposition solution
weight was 20 g. To reduce the salt interference effect, we adjusted the sample weight
from 0.5 g to 0.1 g, and the decomposition solution was diluted to 80 g with distilled
water. As a result, the accuracy of sea salt determination was restored from 92.05% to
101.42%. The traditional South Korean diet includes high amounts of sodium [22]. The salt
interference effect can be reduced in evaluating foods with high salt content by diluting
sample solutions to prevent low recovery.
The method limit of detection (MLOD) for representative foods ranged from 0.0070
to 0.0498 µg kg−1 , and the method limit of quantification (MLOQ) ranged from 0.0222 to
0.1585 µg kg−1 (Table 1). We calculated the measurement uncertainty to a non-fatty matrix
(rice) that spiked the Tl standard solution’s intermediate concentration (0.5 µg kg− 1 ). The
expanded uncertainty for rice was 0.51 ± 0.03 µg kg−1 . The expanded uncertainty of the
measurement is stated at an approximately 95% confidence level using coverage of k = 2. If
the target uncertainty is not established in a regulation, an additional 20–30% tolerance can
be considered to allow for variability in the uncertainty calculation process [23].
Molecules 2021, 26, 6729
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Table 1. Selection of representative foods and validation parameters of Tl analysis.
Classification
Representative Food
Regression Equation
R2
MLOD a
(µg kg−1 )
MLOQ b
(µg kg−1 )
Non-fatty Solid
Hydrated Solid
Fatty Solid
Salty Solid
Non-Fatty Liquid
Fatty Liquid
Rice
Apple
Beef
Sea Salt
Orange Juice
Sesame Oil
y = 117366x − 208.52
y = 38252x + 774.86
y = 85346x − 6.9005
y = 67663x + 165.49
y = 88399x − 441.29
y = 111833x − 120.50
1
0.9999
0.9997
0.9999
1
1
0.0092
0.0498
0.0070
0.0207
0.0126
0.0225
0.0293
0.1585
0.0222
0.0661
0.0401
0.0715
a
MLOD refers to the method limit of detection, S/σ = 3.14; b MLOQ refers to the method limit of quantification, S/σ = 10.
2.2. Concentration of Tl in Foods
2.2.1. Concentration of Tl in Agricultural, Fishery, and Livestock Products
We measured the Tl concentration in agricultural, fishery, and livestock products
commonly consumed in the South Korean market and summarized the results into 13 subgroups considering the Korea Food Code (Table 2, Tables S2–S4). Tl above the MLOD was
detected in 120 out of 175 samples. The total number of cereal types tested was 32, with Tl
exceeding MLOD in 16 of them. The mean value was 0.83 µg kg−1, and the highest value
was 5.27 µg kg−1 for sorghum, whereas Tl in rice was reported to be 26.8 µg kg−1 in a Polish
study [24]. Tl contents in rice, barley, and oats were below MLOD in the current study. Tl
levels in tuber crops, such as potatoes and sweet potatoes, exceeded the MLOD. The mean
Tl content in tuber crops was 6.67 µg kg−1 , and the highest of 13.21 µg kg−1 was detected
in sweet potatoes. Tl content in potatoes, 4.04 µg kg−1 , was similar to that in an Italian
study (6.90 µg kg−1 ) and higher than that in a South African one (1.03 µg g−1 ) [25,26]. In
the bean category, three types of beans—white, black, and kidney beans—were examined.
In kidney beans, Tl content was below the detection limit. The average Tl content in beans
was 0.49 µg kg−1 , lower than the Italian study result (13.4 µg kg−1 ) [25].
Apple, mandarin, and pear were the three fruits examined. Tl levels in all mandarins
were below MLOD, whereas Tl was detected in all pears. The mean concentration in the
fruit group was 1.48 µg kg−1 (Table 2). In Italy, the contents in five fruits varied from 0.7 to
3.1 µg kg−1 [25], and we obtained similar values. Fifty vegetables were categorized as root,
leafy, and other vegetables. In the case of root vegetables including carrot, white radish,
garlic, onion, burdock root, and ginseng, Tl was detected in 14 out of 19 samples, with
concentrations ranging from 0.02 to 7.11 µg kg−1 , and the highest detected content was in
white radish. The mean content reduced in the following order: white radish > ginseng >
garlic > carrot > burdock root > onion. Tl was found in 14 out of 16 leafy vegetable samples,
ranging from 0.02 to 15.93 µg kg−1 , with most Tl detected in cabbage. The mean contents
in lettuce and cabbage were relatively high in this group. A previous study discovered that
the Brassicaceae family (cabbage, rape, turnip, and mustard) contains high levels of Tl [27].
Cabbage showed the highest concentration of Tl (4.13 ± 6.82 µg kg−1 ) in our category,
although the SD across samples was large, indicating the differential Tl content in the
soils where the samples were grown. Tl concentrations were below MLOD in zucchini
and cucumber and were 0.60 and 0.49 µg kg−1 in one sample of red pepper and eggplant,
respectively. Tl concentrations in native plants were approximately 0.05 mg kg−1 [28], and
in Chinese crops, they ranged from 0.01 to 0.06 mg kg−1 [29].
In the case of livestock products, Tl in chicken, beef, and pork ranged from 0.51 to
5.72 µg kg−1 with the mean value of 2.46 µg kg−1 . The highest Tl level among animal
products was in eggs, with the mean value of 2.46 µg kg−1 , more than that in a Belgian
study [30]. Tl in eggs came endogenously from chickens, and the natural environment
in which they grew up was conceivable to contribute [30,31]. The mean concentration in
meats was 0.79 µg kg−1 , similar to that reported in a Swiss study [32].
Molecules 2021, 26, 6729
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Table 2. Concentration (fresh weight) and detection frequency of Tl in agricultural, fishery, and livestock products.
Category
Agricultural
Products
Livestock
Products
Fishery
Products
Subcategory
(n) a
Cereals
(32)
Beans
(10)
Tuber crops
(6)
Vegetables
(50)
Mushrooms
(6)
Fruits
(9)
Eggs
(3)
Meats
(9)
Fishes
(21)
Cephalopoda
(6)
Shellfishes
(10)
Crustaceans
(4)
Sea Algae
(9)
Frequency of Detection b
(%)
Min–Max c
(µg kg−1 )
Median
(µg kg−1 )
Mean ± SD c
(µg kg−1 )
50
0.00–5.27
0.20
0.83 ± 1.47
50
0.00–2.31
0.00
0.49 ± 0.80
100
3.78–13.21
5.13
6.67 ± 3.70
66
0.02–15.93
0.34
1.59 ± 3.21
0
0.02–0.02
0.02
0.02 ± 0.00
56
0.02–7.00
0.34
1.48 ± 2.39
100
0.65–5.72
1.03
2.46 ± 2.31
100
0.51–1.63
1.00
0.79 ± 0.38
100
0.41–1.87
1.03
1.06 ± 0.37
100
1.04–1.22
1.07
1.10 ± 0.06
100
1.20–6.02
1.88
2.55 ± 1.46
100
1.07–1.79
1.14
1.28 ± 0.29
22
0.02–12.72
0.02
1.21 ± 1.40
a
n is the number of samples; b frequency of detection is the percentage of samples above the method detection of limit (MLOD) in each
category; c results < MLOD were set equal to 1/2 MLOD values.
Fishery products were classified into fish, cephalopodan, shellfish, crustacean, and sea
alga products. Tl content was higher than the detection limit in 44 out of the 50 samples
tested in this category (Table 2). Tl contents in fishery products ranged from 0.02 to
12.72 µg kg−1 , with a total average concentration of 1.55 µg kg−1 . Tl levels in fishes varied
from 0.41 to 1.87 µg kg−1 , with the mean value of 1.06 µg kg−1 . These values were higher
than those in a Vietnamese study (0.012–0.050 µg g−1 ), but similar to those in the Canadian
(0.01–19.26 ng g−1 ) and Iranian (0.02–0.30 µg kg−1 ) ones [33–35]. As for cephalopods, we
analyzed squid and octopus, and their average Tl content was 1.10 µg kg−1 . Crustaceans
examined were four types of crabs, with an average Tl concentration of 1.28 µg kg−1 .
The average Tl content in shellfish was 2.55 µg kg−1 , with the highest concentration
(6.02 µg kg−1 ) observed in the manila clam. Sea algae were investigated in three types:
laver, sea mustard, and hijiki (Table S4). The hijiki contained the least quantity of Tl, with Tl
concentrations of 5.21 and 12.72 µg kg−1 in one sample of laver and sea mustard samples,
respectively. The SD of Tl levels among sea algae and shellfish was higher than in other
species. Since each sample originated from a different origin site, such a large SD might
represent habitat variations related to the marine environment. The Tl content in aquatic
creatures is influenced by seawater along the coast they inhabit. The Tl concentration
in seawater is about 0.2–20 µg kg−1 , affected by volcanic events, airborne particles, and
deposit inflows from rivers and soil [36]. As a result, if polluted river water or sediment
reaches a habitable area, it may affect the Tl concentration of aquatic species. The presence
of Tl in sediment or soil deposits enables the metal to move into benthic species such
as shellfish (zoobenthos) and sea algae (phytobenthos) rather than fish and cephalopods
due to their habiting nature. In recent years, numerous and broad investigations on Tl
contamination have been undertaken in China [37–41]. There are Tl deposits in India,
Japan, and Uzbekistan in Asia, but China has the most Tl deposits (i.e., Lanmuchang,
Molecules 2021, 26, 6729
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Nanhua, Yunfu, and Chengmenshan deposits) [42]. The presence of Tl in crops is related to
its level in the soil. Tl levels in the crust range from 0.1 to 1.7 mg kg−1 , with sulfide ores
being the most common source [43]. Lee. et al. conducted a study on soil in South Korean
locations and reported the estimated Tl content of 1.20–12.91 mg kg−1 in the soil near a
cement industry facility, and 0.18–1.09 mg kg−1 near a smelter and mine [44]. In addition,
2.28 ± 1.39 mg kg−1 of Tl was found in rhizospheric soils near cement plants in China [45].
Although the Tl content of commonly consumed foods in South Korea is low, additional studies on areas with potential Tl contamination are required, considering the
concentration of Tl in contamination-prone parts of the country.
2.2.2. Concentration of Tl in Processed Foods
The processed foods were monitored on 129 samples of commercial products consumed by the general population (Table 3). Canned foods were excluded because of the
possibility of heavy metal cross-contamination from packaging. Tl was detected in 28 out
of 129 samples of beverages and other processed foods, with considerable supplementary
data (Tables S5 and S6).
Table 3. Concentration (fresh weight) and detection frequency of Tl in processed foods.
Category
Beverages
Other Processed
Foods
Subcategory
(n) a
Infusion Teas
(18)
Other Beverages
(21)
Snacks
(6)
Salted Seafood
processing Products
(12)
Pickled Foods
(12)
Sauces
(18)
Oil Products
(27)
Dairy Products
(6)
Cereal Products (3)
Livestock Processing
Products (3)
Noodles (3)
Frequency of Detection b
(%)
Min–Max c
(µg kg −1 )
Median
(µg kg −1 )
Mean ± SD c
(µg kg −1 )
39
0.01–1.99
0.52
0.61 ± 0.63
33
0.01–4.12
0.01
0.49 ± 0.97
100
0.01–0.01
1.10
0.01 ± 0.00
25
0.01–1.41
0.01
0.30 ± 0.52
8
0.01–19.46
0.01
1.63 ± 5.38
0
0.01–0.01
0.01
0.01 ± 0.00
0
0.01–0.01
0.01
0.01 ± 0.00
0
0.01–0.01
0.01
0.01 ± 0.00
0
0.00–0.00
0.00
0.00 ± 0.00
0
0.01–0.01
0.01
0.01 ± 0.00
0
0.01–0.01
0.01
0.01 ± 0.00
a
n is the number of samples; b frequency of detection is the percentage of samples above the method detection of limit (MLOD) in each
category; c results < MLOD were set equal to 1/2 MLOD values.
Beverages were subdivided into infusion tea and other beverages (e.g., soda, coffee,
and sports drinks). Other processed foods included various processed products, such as
oils, snacks, and salted foods (e.g., sauces, pickled foods, salted seafood, and ham). In
the beverage category, Tl was detected in 14 out of 39 samples. Tl concentration in tea
infusions ranged from 0.01 to 1.99 µg kg−1 . Matte tea had the highest average concentration
(1.50 µg kg−1 ), followed by Solomon-seal (Polygonatum odoratum var. pluriflorum) and black
tea (Table S5). Our results were lower than the Tl concentrations in herbal tea infusions
reported in Brazil [46]. Regarding other beverages, the average Tl concentration in red
ginseng drink was 2.49 µg kg−1 , followed by fruit juice (0.68 µg kg−1 ) and soy milk
(0.24 µg kg−1 ). Samples with mainly undetected Tl and limited sample size were classified
as other processed foods (Table S6). Potato crisps and fruit jelly were categorized as snacks.
The average Tl concentration in potato crisps was 3.49 µg kg−1 , much lower than in a New
Zealand study (0.17 mg kg−1 ) [47]. The average Tl level in fruit jelly was 0.51 µg kg−1 .
In the processed salty seafood category, Tl was only detected in fish cakes (1.19 µg kg−1 ).
In the pickled food category, one of the three radish kimchi samples had a Tl content of
Molecules 2021, 26, 6729
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19.46 µg kg−1 , indicating that the Tl found in the sample might have come from the raw
material. Tl was not detected in sauces, oil and dairy products, cereals, livestock processed
foods, and noodles.
In our study, processed foods showed a lower Tl detection frequency than raw materials. Processed foods have lower raw material contents and are supplemented with water
and various food additives. Certain processed foods require the use of food extracts rather
than raw foods. As a result, the metal concentration in the final product was somewhat
diluted. Furthermore, cooking processes, such as blanching, boiling, and frying, can lower
the amount of metal in food. Several previous studies have reported this trend [48,49].
For these reasons, heavy metal levels in processed foods are often low. Toxic metal(loid)s
(including nickel, chromium, and manganese) can contaminate cooking utensils or packaging processes [50]. Contrastingly, Tl in foods can come from raw materials cultivated in a
polluted environment (i.e., soil and river).
2.3. Comparing the Concentrations of Tl among Food Groups
We divided the food groups based on biological similarities and compared the intragroup and intergroup differences in Tl concentration (Figure 1). The classification is as
follows: cereals and beans (CB), fruits and vegetables (VF), livestock products (LP), and
fishery products (FP). The processed foods were excluded from the comparison because Tl
was not detected in most of their samples.
When cereals and beans were compared, the median Tl value of beans was higher
than that of cereals, but the difference was not statistically significant (Figure 1a). Legumes
are the fruits of plants in the family Fabaceae, while cereals are fruits (caryopsis) with
a seed coat (testa). The fact that there was no significant difference between the groups
might be due to their comparable morphological forms. In the VF group, vegetables were
subdivided into root vegetables, leafy vegetables, tuber crops, and other vegetables. The Tl
concentration reduced in the following order: tuber crops > root vegetables > leafy vegetables > fruits > other vegetables (Figure 1b). A tuber is a nutrient storage organ that grows
underground and belongs to the root type, but we separated it based on the classification
standard of the Korea Food Code [12] and monitored potato (Solanum tuberosum L. ) and
sweet potato (Ipomoea batatas) in the group (Table S2). The Tl concentration in sweet
potato (9.29 µg kg−1 ) was higher than that of potato (4.04 µg kg−1 ), given that potato is
a stem tuber crop and sweet potato is a root tuber crop. In a recent Chinese study, the
bio-concentration factor (BCF) and transfer factor (TF) of Tl in sweet potato were more than
one, indicating that the plant can accumulate metals [51]. The median Tl concentration in
root vegetables was negligibly higher than that in leafy vegetables and fruits. In the other
vegetable group, which included eggplant, red pepper, zucchini, and cucumber, Tl content
was significantly (p < 0.05) lower than in the other VF groups. Based on the biological
classification, fishery products were divided into fishes, cephalopods, shellfish, crustaceans,
and sea algae. Tl levels in aquatic animals were significantly (p < 0.05) higher than those in
aquatic plants among the FP groups (Figure 1c). While there was no significant difference
between the aquatic animals, the shellfish group had the highest median Tl value. Livestock
products were classified as eggs and meats. The median value of eggs was insignificantly
higher than that of meats (Figure 1d).
Examining the significant differences among the CB, VF, LP, and FP groups revealed
that Tl content decreased in the following order: LP and FP > VF > CB (p < 0.05). Tl
concentrations in animal foods (LP and FP) were significantly (p < 0.05) higher than those
in the CB and VF groups (Figure 1e). Fisheries and livestock products belong to the upper
trophic level of the food chain. Although sea algae are in the marine product group, Tl
concentration in animals is apparently higher than that in plants. In other words, it seems
that Tl accumulation can occur as it goes up the food chain. Toxic metal(loid)s accumulate
in the food chain in natural environments [52,53]. Tl is enriched in the natural environment
through anthropogenic sources (e.g., cement production and coal combination) and natural
Molecules 2021, 26, x FOR PEER REVIEW
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Molecules 2021, 26, 6729
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Tl value. Livestock products were classified as eggs and meats. The median value of eggs
was insignificantly higher than that of meats (Figure 1d).
sources, such as volcanic activity and mineral ore mining [36]. Tl accumulates in plants
and animals through the food chain, eventually causing harm to humans.
(a)
(b)
(c)
(d)
(e)
Figure
1. Box
plots
thallium
(Tl)
foodgroups
groupsbybycategories.
categories.(a)
(a)cereals
cerealsand
andbeans,
beans, (b)
(b) fruits,
fruits, vegetables, and tubers,
Figure
1. Box
plots
of of
thallium
(Tl)
inin
food
tubers,
livestock
products,
fishery
products,
cereals
beans;
vegetable
fruits;
livestock
products;
(c) (c)
livestock
products,
(d)(d)
fishery
products,
(e) (e)
CB,CB,
cereals
andand
beans;
VF,VF,
vegetable
andand
fruits;
LP,LP,
livestock
products;
FP,
FP,
fishery
products
(line
=
median,
box
=
25th
and
75th
percentiles,
whiskers
=
minimum
and
maximum
observations
fishery products (line = median, box = 25th and 75th percentiles, whiskers = minimum and maximum observations below
below upper fence); IQR (Interquartile Range) 75th–25th percentile. Means within box plots with different letters are
upper fence); IQR (Interquartile Range) 75th–25th percentile. Means within box plots with different letters are significantly
different at p < 0.05 by testing between mean using t-test or one-way ANOVA with Tukey’s multiple comparison.
Molecules 2021, 26, 6729
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2.4. Health Risk Assessment for Thallium through Food Consumption
We evaluated Tl exposure through food intake based on the monitoring results. Food
intake and the average weight by age were calculated from the National Health and
Nutrition Survey in Korea (KNHANES) data [54]. The information on chronic exposure
to Tl in the human impact case study is limited because it is mainly associated with
unintentional intake or suicide attempts. A recent study by Andrew et al. used a healthbased guidance value (HBGV) approach based on the tolerable daily intake (TDI) of the
Netherlands National Institute for Public Health and the Environment (RIVM) while
considering human toxicological uncertainty [47,55]. In the present study, the same method
was applied to evaluate the dietary exposure and risk assessment for Tl.
Table 4 displays the exposure and risk assessment results based on the Tl concentrations in foods. The exposure through food consumed was very low, below 0.0032 µg kg−1
bw day−1 for all food groups. Among them, the most exposed food group was vegetables
(0.0000–0.0032 µg kg−1 bw d−1 ), followed by tubers (0.0012–0.0029 µg kg−1 bw d−1 ), eggs
(0.0003–0.0028 µg kg−1 bw d−1 ), and fruits (0.0000–0.0011 µg kg−1 bw d−1 ). We estimated
the dietary exposure level specifically by age in both lower bound (LB) and upper bound
(UB) (Tables S7 and S8). At 0–2 years, Tl exposure through vegetables was the highest,
with 0.0000–0.0055 µg kg−1 bw d−1 . The exposure through eggs reached a maximum level
at 3–6 years (0.0010–0.0086 µg kg−1 bw d−1 ), 7–12 years (0.0006–0.0052 µg kg−1 bw d−1 ),
and 13–19 years (0.0003–0.0028 µg kg−1 bw d−1 ), respectively. In adults aged 20–64 years,
exposure through vegetables ranged from 0.0000 to 0.0031 µg kg−1 bw d−1 , and in the
people aged 65 years and older, tubers were the highest at 0.0009–0.0043 µg kg−1 bw d−1 .
Because our detection limits and Tl concentrations were low, the difference between LB
and UB was minimal. Thus, we reported the exposure dose and % HBGV based on the
UB (Table 4).
Table 4. Exposure assessment and risk characterization for Tl.
Food Group
Agricultural Products
Livestock Products
Fishery Products
Processed Products
Cereals
Fruits
Beans
Mushrooms
Tuber Crops
Vegetables
Eggs
Meats
Crustaceans
Fishes
Cephalopoda
Shellfishes
Seaweeds
Beverages
Other Processes
Products
Concentration
(µg kg−1 )
Exposure Dose
(µg kg−1 bw day−1 )
% HBGV
0.01–5.27
0.01–7.00
0.01–2.32
0.01
3.78–13.21
0.01–15.93
0.65–5.72
0.51–1.63
1.07–1.79
0.41–1.87
1.04–1.22
1.20–6.02
0.01–12.72
0.01–4.12
0.0000–0.0001
0.0000–0.0011
0.0000–0.0001
0.0000–0.0000
0.0012–0.0029
0.0000–0.0032
0.0003–0.0028
0.0001–0.0009
0.0000–0.0000
0.0000–0.0001
0.0000–0.0001
0.0000–0.0001
0.0000–0.0002
0.0000–0.0003
0.0000–0.0345
0.0000–0.5625
0.0000–0.0549
0.0001–0.0002
0.6050–1.4682
0.0001–1.5807
0.1604-1.4109
0.0502-0.4326
0.0130-0.0216
0.0027-0.0440
0.0143-0.0445
0.0106-0.0606
0.0000-0.0900
0.0001-0.1549
0.00–19.46
0.0000–0.0014
0.0000-0.6881
HBGV is the health-based guidance value: % HBGV = (estimated daily intake/HBGV) × 100% HBGV was calculated based on HBGV of
Tl = 0.2 µg kg−1 bw day−1 .
In all age and dietary groups, no exposure estimation surpassed the health-based
guidance value. Our results indicate that the risk of exposure through Tl-contaminated
food ingestion is at a minimum level.
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3. Materials and Methods
3.1. Instrumentation
Tl was determined using an Agilent 7700x (Agilent Technologies, Santa Clara, CA, USA)
ICP-MS with ion monitoring at mass-to-charge ratios (m/z) 203 and 205 to gather the data.
The specific instrument parameters and analytical conditions are described in Table S9.
3.2. Reagents and Solutions
The reagents used for this study were of analytical grade, and the water was deion-ized
and purified below 18.2 MΩ using a YL WPS System (Young Lin Instrument Co., Anyang-si,
Kyunggi-do, Korea). As a standard solution for Tl, the periodic table mix 1 for the ICP solution (Sigma-Aldrich, St. Louis, MO, USA) was diluted with 2.5% nitric acid to 100 µg kg−1
of stock solution. Analytical grade nitric acid (Chemitop, Jin-cheon, Cheongbuk-do, Korea)
and hydrogen peroxide (Chemitop, Jincheon, Cheongbuk-do, Korea) were used. BCR-679
(white cabbage) certified reference materials (CRM) for method validation and quality
assurance were purchased from the European Commission (European Com-mission, Joint
Research Centre, Institute for Reference Materials and Measurements, Geel, Belgium).
3.3. Sample Collection and Processing
According to KNHANES, monitored samples were chosen to represent commonly consumed foods. Additionally, we referred to a report on the reassessment of toxic metal(loid)s
criteria in South Korean food published by the Korea Ministry of Food and Drug Safety
(MFDS) [54,56]. For representativeness, agricultural (113 samples), animal (12 samples),
and fishery products (50 samples) were purchased from more than three distinct locations.
For processed foods (129 samples), more than three products were purchased per manufacturer. Before homogenizing, the samples were washed with deionized water, and the
surface moisture and non-edible components (e.g., peels, seeds, and bones) were removed.
Fruits and vegetables were dried at 65 ◦ C for 18 h before homogenization with a lab blender
for 60 s and sealed at −18 ◦ C. For infusion teas, 1.2 g of tea sachet was immersed in 100 mL
of distilled water at 80 ◦ C for 2 min. We used the infused water as the samples.
3.4. Sample Preparation
The current study’s sample preparation procedure was based on the elemental analysis
manual of the US Food and Drug Administration (US FDA) [57] and guidelines for the
analysis of toxic metal(loid)s in food by MFDS [58]. We classified foods into six categories
based on their energy (i.e., calories) and matrix. The calorific value of each food was
determined using the MFDS food nutrient database [59]. The classification included nonfatty solids, hydrated solids, fatty solids, salty solids, non-fatty liquids, and fatty liquids.
Table 5 lists the sample weights for various sample classifications. The samples were
pre-decomposed on a hot plate with 4 mL of nitric acid (70 vol%) and 1 mL of hydrogen
peroxide (30 vol%) in a polytetrafluoroethylene (PTFE) vessel. After cooling at 25 ◦ C, 3 mL
of nitric acid (70 vol%) was added. PTFE vessels were sealed and placed in a microwave
digestion system (ETHOS Easy, Milestone, Italy). After digestion, the samples were cooled
at 25 ◦ C and diluted to 20.0 g (in the case of salty foods, they were diluted to 80.0 g) with
distilled water. All samples were analyzed in triplicates. The hydrated solid foods were
converted to wet-based concentrations by considering the moisture content before drying.
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Table 5. Classification into food matrix groups and sample weights for preparation.
Classification
Sample Weight (g)
Food Group
Non-Fatty Solid
Hydrated Solid
Fatty Solid
Salty Solid
Non-Fatty Liquid
Fatty Liquid
0.5
0.5
0.5
0.1
0.5
0.3
Cereals, Tubers, Beans, Snacks, Cereal Products
Fruits, Vegetables, Mushrooms, Sea Algae
Meats, Eggs, Fishes, Cephalopods, Shellfishes, Crustaceans
Sauces, Pickled Foods, Salt-Added Products, Noodles
Infusion Teas, Other Beverages
Oil Products, Dairy Products
3.5. Single-Lab Validation and Quality Control
Food comprises various organic components. In the method validation for determining toxic metal(loid)s, the matrix effect is one of the critical factors in ICP-MS analysis. We
selected six representative foods for each category, taking into account the effects of matrices, such as salts and fats, and validated the method for determining Tl. We confirmed the
factors for method validation in terms of linearity, trueness, accuracy, and precision. The
linearity for external standard calibration curves was measured in stock solution diluted to
0.025, 0.05, 0.1, 0.25, 0.5, 1.0, and 2.0 µg kg−1 . The trueness of Tl determination was verified
using CRM Tl mass fractions. The measured values were compared with the certification
values seven times. The accuracy and precision were calculated intraday and interday.
The standard solution was spiked into each representative sample, resulting in the final
concentrations of 0.1 µg kg−1 (low concentration), 0.5 µg kg−1 (medium concentration),
and 1.0 µg kg−1 (high concentration) in seven replicates, respectively. The measurement
uncertainty was calculated for the rice matrix spiked with a medium concentration of Tl
standard solution. The sources of measurement uncertainty were expressed in the form of
a fishbone diagram (Figure S1).
The MLOD and MLOQ were determined using the standard addition method, which
involved adding five different Tl concentrations (0.05, 0.1, 0.25, 0.5, and 1.0 µg kg−1 )
to representative foods for calibration curve with seven repetitions. The equations are
as follows:
MLOD = 3.14× S/σ and MLOQ = 10 × S/σ
(1)
where 3.14 = student t factor for seven replicates, S = slope mean, and σ = standard
deviation of intercept. For quality control, CRM was analyzed for every batch of samples,
and recovery was checked within 80–120%.
3.6. Statistical Analysis
The analysis data were processed using SPSS 21 (IBM Corporation, New York, NY, USA)
to compare the levels among the food groups. Significant differences were defined using
t-tests or one-way ANOVA. Statistical significance was set at p < 0.05. For statistical calculation, values below MLOD were replaced with half the MLOD values, and its natural
logarithm was used.
3.7. Risk Assessment and Exposure to Thallium
Risk assessment was conducted based on monitoring data, daily consumption value,
and the average body weight derived from KNHANES. To evaluate the risk of potential
exposure, we used the HBGV approach. In the present study, % HBGV was calculated
using 0.2 µg kg−1 bw day−1 as the HBGV of Tl [46]. Estimated daily intake (EDI) and
health risk (% HBGV) were determined using the following equations:
EDI (µg/kg bw/day) =
Daily consumption (g/day) × Tl concentration (µg/kg)
Average body weight (kg)
(2)
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where average body weights (kg) by age are: 0 to 2 years (11.52 kg), 3 to 6 years (18.75 kg),
7 to 12 years (35.76 kg), 13 to 19 years (59.72 kg), 20 to 64 years (64.93 kg), over 65 years
(60.20 kg).
EDI
% HBGV =
× 100
(3)
HBGV
4. Conclusions
ICP-MS was used to validate analytical methods for six typical foods selected based
on food matrices and calories. Tl levels were monitored in 304 commonly consumed food
items. As a result, Tl was detected in 148 samples, with 97% of the measured samples
containing less than 10 µg kg−1 Tl. We examined Tl levels in agricultural, fishery, and
livestock products and found that Tl concentrations in animal foods are higher than in
plant foods. The results confirmed that the level of Tl exposure through food intake was
within the health-based guidance value. Further research and regular monitoring of Tl in
foods are necessary to protect human health and avoid food-borne contamination.
Supplementary Materials: The following are available online: Table S1: Evaluation of intraday and
interday accuracy and precision at low, medium, and high concentrations of thallium (Tl) in representative foods; Table S2: Tl content (fresh weight) in specific items of agricultural products; Table S3: Tl
content (fresh weight) in specific items of livestock products; Table S4: Tl content (fresh weight) in
specific items of fishery products; Table S5: Tl content (fresh weight) in specific items of beverages;
Table S6: Tl content (fresh weight) in specific items of other processed foods; Table S7: Estimated
dietary exposure to Tl by age (Lower Bound); Table S8: Estimated dietary exposure to Tl by age
(Upper Bound); Table S9: Instrumental conditions of ICP-MS for the determination of Tl; Figure S1:
Fishbone diagram for Tl analysis uncertainty for rice.
Author Contributions: Conceptualization, methodology, data curation, software, formal analysis,
and writing—original draft preparation, Y.-h.K. and K.-W.L.; validation, Y.-h.K. and W.-j.R.; formal
analysis, Y.-h.K., W.-j.R., and B.S.; risk assessment, S.C.(Solyi Cho) and S.C.(Shinai Choi); data curation
and supervision, Y.J.; supervision, writing—review and editing, K.-W.L.; project administration
and funding acquisition, K.-W.L. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Ministry of Food and Drug Safety, Republic of Korea
(grant number 20162MFDS037) in 2020.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: This work was supported by the School of Life Sciences and Biotechnology for
BK21 PLUS, Korea University. The authors thank the Korea University–CJ Food Safety Center (Seoul,
Korea) for providing the equipment and facilities.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Not available.
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| 7,946 | 52,155 |
W4383067948.txt | Клинический случай І Case report
DOI: 10.33925/1683-3031-2023-642
Эстетическая и функциональная реабилитация
детей раннего возраста с использованием
искусственных коронок из диоксида циркония
С.В. Каменева, Д.М. Омехина, Н.В. Кущенко, А.В. Голоднюк
Первый Санкт-Петербургский государственный медицинский университет имени академика И.П. Павлова,
Санкт-Петербург, Российская Федерация
АННОТАЦИЯ
Актуальность. Необходимость в функциональной реабилитации детей раннего возраста – достаточно распространенная проблема в современной детской стоматологии. Это связано с тем, что ребенок в возрасте
до трех лет имеет поведенческие особенности, которые затрудняют оказание стоматологической помощи.
Относительно недавно появились стандартные детские коронки из диоксида циркония, обладающие высокими эстетическими и механическими характеристиками
Цель исследования. Проанализировать на примере клинических случаев применение коронок из диоксида циркония для функциональной реабилитации и профилактики заболеваний пародонта у детей раннего возраста.
Описание клинического случая. В исследование представлен клинический случай функциональной и эстетической реабилитации детей в возрасте до трех лет при помощи искусственных коронок из диоксида циркония.
Заключение. Коронки из диоксида циркония являются перспективной альтернативой другим способам восстановления центральных зубов у детей раннего возраста. На основании клинического случая показано, что циркониевые коронки выглядят естественно, в полной мере восполняют дефект, восстанавливают нарушенную функцию без отдаленных осложнений, а также не оказывают отрицательного воздействия на состояние пародонта.
Ключевые слова: детская стоматология, острый детский кариес, коронки из диоксида циркония, профилактика заболеваний пародонта у детей.
Для цитирования: Каменева СВ, Омехина ДМ, Кущенко НВ, Голоднюк АВ. Эстетическая и функциональная реабилитация детей раннего возраста с использованием искусственных коронок из диоксида циркония.
Стоматология детского возраста и профилактика. 2023;23(2):184-190. DOI: 10.33925/1683-3031-2023-642.
Aesthetic and functional rehabilitation
of young children using zirconia crowns
S.V. Kameneva, D.M. Omekhina, N.V. Kushchenko, A.V. Golodniuk
Pavlov First Saint Petersburg State Medical University, Saint Petersburg, Russian Federation
ABSTRACT
Relevance. The need for functional restoration in young children is a sufficiently common problem in modern pediatric dentistry due to the behaviour characteristics of a child under three years old, which hinder dental treatment. Prefabricated zirconia crowns for children appeared relatively recently and have high aesthetic and functional properties.
Purpose. To analyze the application of zirconia crowns for functional rehabilitation and periodontal disease prevention in young children, exemplified by clinical cases.
Clinical case description. The study presents a clinical case of functional and aesthetic restoration with zirconia
crowns in a child under three years old.
Conclusion. Zirconia crowns are a promising alternative to other methods of restoring anterior teeth in a toddler.
The clinical case shows that zirconia crowns look natural, fully compensate for the defect, restore the lost function
without long-term complications and do not adversely affect the periodontium.
Key words: pediatric dentistry, severe early childhood caries, zirconia crowns, prevention of periodontal disease in children.
For citation: Kameneva SV, Omekhina DM, Kushchenko NV, Golodniuk AV. Aesthetic and functional rehabilitation
of young children using zirconia crowns. Pediatric dentistry and dental prophylaxis. 2023;23(1):184-190. (In Russ.).
DOI: 10.33925/1683-3031-2023-642.
184
2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis
Клинический случай І Case report
АКТУАЛЬНОСТЬ
В современной детской стоматологии наблюдается
тенденция снижения возраста пациентов, обращающихся к стоматологу для лечения. Наиболее распространенным заболеванием зубов в детском возрасте
является кариес, 60-90% детей имеют хотя бы одну
кариозную полость. Наиболее агрессивным видом
кариеса считается острейший кариес временных зубов, который встречается у детей в возрасте до трех
лет [1]. Он имеет свои особенности, среди которых
можно выделить: типичную локализацию (резцы
верхней челюсти), наличие трех-четырех кариозных
полостей на одном зубе, плоскостное и циркулярное
распространение кариеса, симметричность поражения, высокий риск вовлечения пульпы и периодонта
с последующим отколом коронки. Все это в совокупности часто влечет за собой эстетические и функциональные нарушения. В раннем детском возрасте
также высока вероятность травмы зубов, обусловленная пиком любознательности и развития детей при
отсутствии «охранительного» рефлекса. Исходя из
клинической практики и проведенных исследований,
вывихи и переломы коронок верхних центральных
резцов встречаются наиболее часто [2].
Обширное разрушение передней группы зубов
вследствие различных причин ведет как к функциональному, так и к эстетическому нарушению.
С точки зрения функционального нарушения важно
рассмотреть два аспекта. Во-первых, при разрушении передней группы зубов происходит нарушение
функции откусывания и отрывания пищи, что влечет
за собой перекладывание данной функции на боковой отдел зубов, который не приспособлен к ней за
счет отсутствия режущих краев. Во-вторых, передние зубы играют важную роль в звукообразовании.
Так, при разрушении или утере резцов формируется
передний «открытый» прикус, который характеризуется наличием вертикальной щели в переднем отделе зубного ряда, которая, в свою очередь, влияет
на ухудшение произношения таких звуков, как Л, Т,
Д, Н, а также ведет к межзубному произношению
свистящих и шипящих звуков [3]. Проблема эстетической неполноценности вследствие разрушения зубов носит очень серьезный характер. Это обосновано
тем, что внешний вид ребенка влияет на его социализацию. Как правило, путь социализации ребенка
начинается с двух лет в дошкольном учреждении.
К этому возрасту дети способны сравнивать предметы, а также проявлять интерес к собственному отражению в зеркале. Безусловно, в раннем возрасте
дети не будут подвержены насмешкам со стороны
сверстников, но видеть отличие своих разрушенных
зубов от здоровых они будут.
При восстановлении разрушенных зубов необходимо помнить и о риске возникновения локальных
заболеваний пародонта, обусловленных неправильно восстановленным контактным пунктом [4].
Необходимость в функциональной реабилитации детей раннего возраста – достаточно распространенная
проблема в современной детской стоматологии. Это
связано с тем, что ребенок в возрасте до трех лет имеет
поведенческие особенности, которые затрудняют оказание стоматологической помощи. Применение стандартных коронок из нержавеющей стали у детей является
рекомендованным методом протезирования детей при
поражении кариесом двух и более поверхностей зуба.
Этот клинически эффективный и безопасный вариант
восстановления зубов часто отклоняется родителями
по причине неудовлетворительной эстетики. Родители
предпочитают обычные реставрации с использованием
эстетических материалов – композитов или СИЦ. На сегодняшний день все большее распространение для восстановления зубов в детской стоматологии получают
стандартные коронки из диоксида циркония. Высокий
спрос на использование искусственных коронок из диоксида циркония объясняется их более высокими эстетическими характеристиками. Стоит также отметить,
что циркониевые коронки не уступают по механическим
характеристикам коронкам из нержавеющей стали и могут применяться у детей любого возраста как в переднем
отделе, так и в боковом. Но несмотря на повышенный
спрос, искусственные коронки из диоксида циркония
не применяют повсеместно, причина тому – отсутствие
литературы с доказательствами эффективности применения данного вида коронок у детей раннего возраста.
Поэму целью данного исследования стал анализ
клинического случая применения коронок из диоксида циркония у детей раннего возраста.
ОПИСАНИЕ КЛИНИЧЕСКОГО СЛУЧАЯ
Пациент А. Пол: женский. Дата рождения: 10.10.2021 г.
(на момент обращения возраст 1 год 7 месяцев).
Жалобы: дефект резцов верхней челюсти. Нарушение
приема пищи, отказ ребенка от кислой и сладкой пищи.
Анамнез жизни:
1. Перенесенные заболевания: аллергоанамнез не
отягощен, заболевания со стороны сердечно-сосудистой
системы, эндокринной системы и ЖКТ отсутствуют.
2. Инфекционные заболевания родители отрицают.
3. Непереносимость лекарственных препаратов:
отсутствует.
4. Наследственность: не отягощена.
5. Беременность у матери первая, токсикоз I триместра беременности, родоразрешение путем кесарева сечения, гипоксия плода. Искусственное
вскармливание с 1,5 месяцев.
6. Вредные привычки: ночное кормление молочными продуктами из бутылочки.
7. Гигиена полости рта: нерегулярная чистка зубов
один раз в день при помощи силиконовой зубной
щетки и зубной пасты «Дракоша».
Развитие настоящего заболевания: родители обратились в клинику с целью реставрации разрушенных
центральных резцов верхней челюсти.
2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis
185
Клинический случай І Case report
Внешний осмотр: общее состояние удовлетворительное, сознание ясное, положение активное, тип
телосложения нормостенический, рост 83 см, вес 11
кг. Температура тела 36.6, кожа нормального цвета
без высыпных элементов, конфигурация лица не изменена, лимфатические узлы овальные, пальпация
безболезненна, симметричная.
Осмотр полости рта: слизистая оболочка бледнорозовая, умеренно увлажнена, высыпных элементов
нет. В полости рта присутствует 10 зубов (5.4, 5.2, 5.1,
6.1, 6.2, 6.4, 7.2, 7.1, 8.1, 8.2). На вестибулярной поверхности 5.2, 5.1, 6.1, 6.2 отмечаются симметричные
пятна. При зондировании на 5.1 и 6.1 зубе под слоем
пораженной эмали обнаружен размягченный дентин.
5.2 и 6.2 зуб разрушены, пульпарная камера вскрыта.
Индекс Loe & Silness: 5.2, 5.1, 6.1, 6.2 – 2 балла (умеренное воспаление – покраснение, отечность, кровоточивость при зондировании); 5.4, 6.4, 7.2, 7.1, 8.1,
8.2 – 1 балл (незначительное воспаление – легкая отечность, отсутствие кровоточивости при зондировании);
общая оценка: 1.4 балл (гингивит средней тяжести).
Основной диагноз: К00.40 Гипоплазия эмали 5.1,
5.2, 6.1, 6.2 зубов.
Осложнение: К.02.1 Кариес дентина 5.1, 6.1 зубов.
К.04.0 Обратимый пульпит 5.2, 6.2 зубов.
Ход лечения
Лечение проведено в условиях общего обезболивания (ингаляционный газ Севоран). В условиях изоляции при помощи системы коффердам проведена механическая и медикаментозная обработка кариозных
полостей 5.1 и 6.1 зуба. 5.2 и 6.2 зуб – пульпотомия с
применением препарата «Биодентин», изолирующая
подкладка IRM. Произведена фиксация искусственных коронок из диоксида циркония на 5.1, 5.2, 6.1, 6.2
зубы при помощи цемента «Фуджи 1».
Рекомендации:
1. Посещение стоматолога 1 раз в 3 месяца.
2. Гигиена полости рта 2 раза в день под контролем родителей при помощи мягкой зубной щетки и
пастой с содержанием фтора 1000 ppm.
ОБСУЖДЕНИЕ
Восстановление разрушенных передних молочных зубов у детей требуется для профилактики нарушения прикуса, нормализации функции откусывания пищи и звукообразования, предотвращения
развития заболеваний пародонта. Формирование
нейтрального прикуса зависит от соотношения челюстей и положения зубов. В свою очередь, челюсти
Рис. 1. Клиническая картина
до начала лечения.
Гипоплазия эмали 5.1, 5.2, 6.1, 6.2
зубов, осложненная кариозным
процессом:
а)
вестибулярная
поверхность;
а/a
б) небная поверхность
Fig. 1. Clinical pictures before
the treatment. Enamel hypoplasia
of teeth 5.1, 5.2, 6.1, 6.2
complicated by caries:
a) labial surface
b) palatal surface
б/b
а/a
б/b
186
Рис. 3. Клиническая картина
после окончания лечения.
Искусственные коронки
из диоксида циркония
на 5.1, 5.2:
а) вестибулярная поверхность;
б) небная поверхность
Fig. 3. Clinical pictures before
the treatment. Enamel hypoplasia
of teeth 5.1, 5.2, 6.1, 6.2
complicated by caries:
a) labial surface
b) palatal surface
Рис. 2. Прицельная рентгенограмма
5.1, 5.2, 6.1, 6.2 зубов
Fig. 2. Periapical X-rays of teeth
5.1, 5.2, 6.1, 6.2
Рис. 4. Контрольная прицельная
рентгенограмма после фиксации
коронок на 5.1, 5.2, 6.1, 6.2 зубы
Fig. 4. Control X-ray after crown
cementation to teeth 5.1, 5.2, 6.1, 6.2
2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis
Клинический случай І Case report
развиваются и растут за счет жевательной нагрузки
как на кость, так и на зубы во время жевания. Выпадение из окклюзии хотя бы одного зуба ведет к
перераспределению жевательного давления и, как
следствие, к нарушению роста челюстей и формированию патологического прикуса [5]. Кроме скелетной
формы патологического прикуса, вызванной неправильным ростом челюстей, существует зубная форма, обусловленная неправильным положением зубов
и их ретенцией. В большинстве случаев (61,8%) ретенция постоянных резцов обусловлена ранней потерей и разрушением временных резцов [6]. В свою
очередь, неправильно расположенные центральные
резцы влияют на формирование постоянного прикуса. Торк центральных резцов верхней и нижней
челюсти имеет важное значение в становлении прикуса у ребенка. Во-первых, торк влияет на глубину
резцового перекрытия – может сформироваться открытый, глубокий или прямой прикус во фронтальной плоскости. Во-вторых, значение угла наклона
центральных резцов влияет на положение больших
и малых коренных зубов. В свою очередь, положение
жевательной группы зубов влияет на межальвеолярную высоту и формирование нейтрального прикуса.
Особую роль играет оральная поверхность верхних
центральных резцов, так как она является направляющей при движении нижней челюсти вперед. При
нарушении длины и угла наклона верхних центральных резцов происходит изменение суставной и зубной траектории нижней челюсти [7].
Применение стандартных коронок из нержавеющей стали (SSC) у детей является рекомендованным
методом протезирования детей при поражении кариесом двух и более поверхностей зуба. Этот клинически эффективный и безопасный вариант восстановления зубов часто отклоняется родителями по
причине неудовлетворительной эстетики.
Прямая реставрация молочного зуба композитом – одна из наиболее классических методик восстановления разрушенного зуба. К ее преимуществам
следует отнести: эстетичность (возможность подбора цвета в стоматологическом кресле), доступность
и дешевизну. Среди недостатков прямых реставраций композитным материалом следует выделить:
прямую зависимость результата реставрации от мануальных навыков врача, большое количество времени, необходимое для восстановления анатомической формы зуба, низкую износостойкость, высокую
степень усадки, необходимость полной изоляции
зуба от ротовой жидкости, высокий риск сколов при
объемном восстановлении, небольшой срок службы,
нарушение краевого прилегания пломб (и, как следствие, рецидив кариозного процесса), нуждаемость
в регулярной полировке пломбировочного материала с целью профилактики возникновения ретенционных пунктов зубного налета, способных привести
к воспалительным заболеваниям пародонта (например, локальному гингивиту).
Прямые реставрации молочных зубов можно выполнить также стеклоиономерными цементами и
их производными – компомерами. Главным преимуществом данных пломбировочных материалов
является выделение в окружающую среду ионов
фтора, за счет которого на границе твердых тканей
зубов образуются фторапатиты, что обеспечивает
дополнительную профилактику кариеса [8]. Однако
они обладают весомым недостатком в виде высокого водопоглощения, что ведет к гигроскопическому
расширению материала. Стоит отметить, что эстетичность и цветостойкость у них ниже, чем у композиционных материалов.
Альтернативной методикой восстановления разрушенных молочных зубов являются искусственные
коронки. В современной детской стоматологии для
восстановления передних молочных зубов применяются металлические коронки с композитной облицовкой, стрип-коронки и циркониевые коронки.
Металлические коронки с композитной облицовкой – это детские стандартные штампованные
коронки, покрытые слоем композиционного материала, имеющие различную форму и размер. К преимуществам коронок данного вида стоит отнести:
эстетичность, отсутствие рецидива кариеса, щадящее препарирование зуба без создания уступа, низкую стоимость.
Однако данные коронки имеют большое количество недостатков. Среди них можно выделить:
низкую цветостойкость и износостойкость, слабую
адгезию композита с металлической основой, неудовлетворительную прочность композита на сжатие (ведет к появлению сколов), высокий риск расцементировки.
Данный вид искусственных коронок является
приоритетным при восстановлении зубов с гипоминерализованной эмалью [9].
Альтернативным эстетическим вариантом для реставрации зубов переднего отдела являются стрипкоронки. Они представляют собой целлулоидные
колпачки, которые имеют различную форму. Колпачки после припасовки заполняются композиционным материалом (преимущественно микронаполненным или гибридным) [10]. Данная методика
имеет ряд преимуществ: эстетичность, щадящее
препарирование, простота методики, низкая стоимость и времязатратность. Несмотря на доступность
данной методики, повсеместное ее применение
ограничено из-за ряда негативных свойств: необходимость полной изоляции, высокая степень усадки
из-за объема композиционного материала, недостаточная прочность материала на сжатие, низкая износостойкость, образование ретенционных пунктов
из-за несовершенства полировки, высокий риск осложнений со стороны пародонта [11].
Согласно данным литературы искусственные коронки из диоксида циркония на передние зубы – это один
из способов реабилитации детей раннего возраста.
2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis
187
Клинический случай І Case report
Стандартные коронки из диоксида циркония получают все большее распространение для восстановления фронтальной группы зубов у детей. Среди положительных сторон данных коронок, кроме эстетики,
можно выделить: биосовместимость, предупреждение рецидива кариеса, высокую прочность на изгиб,
стабильность цвета, низкую степень износа. Кроме
этого, при сравнении уровня гигиены у детей спустя
год после протезирования было выявлено, что у детей, зубы которых покрыты коронками из диоксида
циркония, зубной налет скапливается в меньшей степени, чем у детей, чьи зубы покрыты иными видами
искусственных коронок. В результате снижается риск
возникновения заболеваний пародонта (в частности
локального гингивита), отсутствует негативное влияние на пародонт зуба. При протезировании зубов
коронками из диоксида циркония кровоточивость
отмечается в меньшем количестве случаев, чем при
протезировании композитными коронками и коронками из нержавеющей стали [11]. К недостаткам циркониевых коронок можно отнести большую редукцию твердых тканей по сравнению с коронками из
нержавеющей стали, а также сложность регулировки
для механической ретенции [12]. При фиксации искусственных коронок из диоксида циркония лишь
на одну из челюстей происходит стирание твердых
тканей зубов-антагонистов. Было проведено исследование, во время которого отслеживали степень износа зубов-антагонистов по индексу Смита и Найта
спустя 3, 6, и 12 месяцев после фиксации циркониевых коронок. Спустя 3 и 6 месяцев состояние эмали
не изменилось. Спустя 12 месяцев у 11,7% исследуемых была отмечена минимальная потеря контуров
и минимальные изменения поверхности эмали (р <
0,02). Данная динамика свидетельствует о том, что со
временем стираемость противоположных зубов увеличивается и данные коронки лучше не использовать
у пациентов с низкой степенью минерализации эмали, либо необходимо фиксировать искусственные коронки и на зубы-антагонисты с целью профилактики
износа [11]. В то же время Murad Alrashdi и соавторы
(2022) на основании обзора литературы утверждают,
что достоверно значимого стирания зубов-антагонистов не было выявлено. Фиксация края искусственной
коронки в области биологической ширины зуба при
тонкой альвеолярной кости может привести к атрофии костной ткани и рецессии десны. Расположение
края искусственной коронки глубже биологической
ширины зуба может повлечь за собой рецидивирующий гингивит, при этом уровень костной ткани остается неизменным [14].
Стоит также отметить, что в качестве осложнений
встречаются редкие случаи расцементировки искусственной коронки, которая происходит за счет того,
что несоответствие формы культи зуба и стандартной коронки компенсируется большим количеством
композиционного цемента. Циркониевые коронки,
как и другие конструкции, имеют свои показания, к
ним следует отнести: мультиповерхностный кариес,
кариес с вовлечением режущего края, травму зуба,
покрытие зубов после проведенного эндодонтического лечения по поводу пульпита и периодонтита,
дисколорит передних зубов, «бутылочный» кариес.
Однако коронки из диоксида циркония имеют относительные противопоказания: повышенная стираемость зубов, скученное положение зубов, воспаление пародонта [14].
ЗАКЛЮЧЕНИЕ
Коронки из диоксида циркония являются перспективной альтернативой другим способам восстановления центральных зубов у детей раннего
возраста. Эстетико-функциональная реабилитация
детей раннего возраста с использованием искусственных коронок из диоксида циркония может быть
предпочтительным вариантом лечения маленьких
детей с зубами, имеющими обширные (более 50%)
разрушения коронковой части зуба. Данный вид
конструкций не только восполняет эстетический
и функциональный дефекты и нарушенную функцию, но и препятствует дальнейшему разрушению
зубного ряда, способствует правильному формированию прикуса и полноценному звукообразованию.
Преимуществом циркониевых коронок является
минимизация рисков возможных осложнений, что
важно при лечении детей до трех лет. Лечение детей раннего детского возраста чаще всего проходит
в условиях общего обезболивания, что осложняет
возможности проведения повторного лечения для
устранения осложнений. На основании клинических
случаев показано, что циркониевые коронки выглядят естественно, в полной мере восполняют дефект,
восстанавливают нарушенную функцию без отдаленных осложнений, а также не оказывают отрицательного воздействия на состояние пародонта.
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Teeth. Int J Pedod Rehabil. 2017.1:75-78.
doi: 10.4103/2468-8932.196491
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in primary anterior teeth in children: a randomized
clinical trial. BMC Oral Health. 2020;20(1):313.
doi: 10.1186/s12903-020-01305-1
12. Alzanbaqi SD, Alogaiel RM, Alasmari MA, Al Essa AM,
Khogeer LN, Alanazi BS, et al. Zirconia Crowns for Primary
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Actual problems of pediatric dentistry. Materials of
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Journal of Positive School Psychology. 2022;6(8):1718-1724.
Available from:
https://journalppw.com/index.php/jpsp/article/
view/10035
СВЕДЕНИЯ ОБ АВТОРАХ
Каменева Саида Вячеславовна, кандидат
медицинских наук, доцент кафедры стоматологии терапевтической и пародонтологии Первого
Санкт-Петербургского государственного медицинского университета имени академика И. П. Павлова,
Санкт-Петербург, Российская Федерация
Для переписки: sannysvk87@gmail.com
ORCID: https://orcid.org/0000-0003-2929-6565
Автор, ответственный за связь с редакцией:
Кущенко Николай Викторович, кандидат
медицинских наук, доцент кафедры стоматологии терапевтической и пародонтологии Первого
Санкт-Петербургского государственного медицин-
ского университета имени академика И. П. Павлова,
Санкт-Петербург, Российская Федерация
Для переписки: nikolaiknv@mail.ru
ORCID: https://orcid.org/0000-0003-1154-249X?lang=ru
Омехина Дарья Михайловна, врач-стоматолог,
Санкт-Петербург, Российская Федерация
Для переписки: d.kuratch52@gmail.com
ORCID: https://orcid.org/0000-0001-7937-0212
Голоднюк Анна Вячеславовна, врач-стоматолог
детский, Санкт-Петербург, Российская Федерация
Для переписки: d.kuratch52@gmail.com
ORCID: https://orcid.org/0009-0009-5991-1603
INFORMATION ABOUT THE AUTHORS
Saida V. Kameneva, DMD, PhD, Associate Professor,
Department of Restorative Dentistry and Periodontology, Pavlov First Saint Petersburg State Medical University, Saint Petersburg, Russian Federation
For correspondence: sannysvk87@gmail.com
ORCID: https://orcid.org/0000-0003-2929-6565
Anna V. Golodniuk, DMD, Pediatric Dentist, Saint
Petersburg, Russian Federation
For correspondence: d.kuratch52@gmail.com
ORCID: https://orcid.org/0009-0009-5991-1603
Corresponding author:
Nikolai V. Kushchenko, DMD, PhD, Associate Professor, Department of Restorative Dentistry and Periodontology, Pavlov First Saint Petersburg State Medical
University, Saint Petersburg, Russian Federation
For correspondence: nikolaiknv@mail.ru
ORCID: https://orcid.org/0000-0003-1154-249X?lang=ru
Конфликт интересов:
Авторы декларируют отсутствие
конфликта интересов/
Сonflict of interests:
The authors declare no conflict of interests
Поступила / Article received 06.05.2023
Поступила после рецензирования / Revised 25.05.2023
Принята к публикации / Accepted 29.05.2023
Daria M. Omekhina, DMD, Saint Petersburg, Russian Federation
For correspondence: d.kuratch52@gmail.com
ORCID: https://orcid.org/0000-0001-7937-0212
190
2023; 23(2) Стоматология детского возраста и профилактика / Pediatric dentistry and dental prophylaxis
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ISSN 2222-1735 (Paper) ISSN 2222-288X (Online)
Vol.14, No.15, 2023
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Effectiveness of the Matheladder as a Review Material on the
Achievement and Motivation Levels of Grade 9 Learners in
Trigonometry
Ferrylou L. Guadiario
Mindanao State University – Iligan Institute of Technology,
Andres Bonifaco Ave., Tibanga, Iligan City
ferrylou.guadiario@g.msuiit.edu.ph
Douglas A. Salazar
Mindanao State University – Iligan Institute of Technology,
Andres Bonifaco Ave., Tibanga, Iligan City
douglas.salazar@g.msuiit.edu.ph
Abstract
This study aimed to look into the effectiveness of the Matheladder as a review material for grade 9 learners’
achievement and motivation level in learning trigonometry. The study was administered to grade 9 learners on
the fourth quarter from Acelo C. Badelles Sr. Memorial High School who were selected through purposive
sampling. This study employed mixed method of research which used quasi-experimental design as the
quantitative method. Quantitative and qualitative data was utilized from the achievement test, motivation
questionnaire and interview guide questions. Findings revealed that the experimental groups have significantly
higher achievement level compared to the control group. It also reveals that all experimental groups have
significantly different motivation levels than the control group. The experimental groups, in particular, had a
higher level of motivation than the control group. The drawn insights of the learners revealed that some learners
demonstrated an interest in trigonometry and appreciated the game’s value in helping them comprehend the
teachings in an enjoyable way. Furthermore, the game’s Question-and-Answer component, as well as the
concept of winning the game, is one of the most captivating parts of the game. The participants had difficulty in
answering the test due to the lack of sufficient knowledge, of how hard it is to understand the topics,
unfamiliarity with the problem-solving process, and the negative notion of trigonometry. Following are some
recommendations based on the foregoing conclusions: (1) Conduct the study in a longer span of time and with
larger sample. (2) Encourage the use of the Matheladder game as a formative assessment instead of using it as a
review material. (3) Contextualize and incorporate Matheladder in mathematics activities. (4) Replicate the study
in the Grade 9 level in order to know the reasons behind the low achievement level in trigonometry. (5) Students
and teachers need to focus on conceptual learning rather than rote memorization for meaningful understanding in
trigonometry.
Keywords: achievement level, educational board-game, Matheladder, motivation level, trigonometry
DOI: 10.7176/JEP/14-15-05
Publication date:May 31st 2023
1. Introduction
Mathematics is an integral part of education and is a required subject across all levels of the basic education
curriculum. At the secondary level, Mathematics requires more than just performing the four fundamental
operations in mathematics or using a formula to find an answer to a given problem. One of its main goals is to
make students become effective problem solvers (SEI-DOST & MATHTED, 2011). Mathematics is an essential
part of the educational curriculum, as it is used in both daily life and the study of other disciplines. However, it is
commonly believed that the majority of students despise mathematics due to a variety of factors including
instruction, learners' cognitive, emotional, and psychomotor characteristics, subject matter, and learning
environment (Gafoor & Kurukkan, 2015).
Moreover, trigonometry is one branch of mathematics that high school students still find difficult, despite
the fact that it has been taught since elementary school (Gur, 2009). Students frequently make mistakes when
solving trigonometric questions, such as using incorrect equations, sequences of operations, inappropriate use
of sines and cosines, misinterpretations of languages, illogical conclusions, distorted definitions, and technical
error computations (Tuna, 2013).
The COVID-19 pandemic has wreaked havoc on education systems around the world, affecting almost
1.6 billion students across 190 countries and continents. The distance learning method is thought to be the
most applicable to the COVID-19 pandemic condition. It is surely not simple to implement because it
involves the preparation of facilities that support instructors, institutions, and students (Cortez, 2020). This
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crisis, on the other hand, has sparked innovation in the education sector. We've seen some innovative methods
to ensure the continuity of education and training (UNESCO, 2020). Remote learning at home is becoming
more than a trend; it is becoming a necessity to ensure that education continues. According to recent research
conducted by Mak (2021), the pandemic has affected children's motivation to learn. It is as frustrating as it is
for teachers who are working incredibly hard to engage their students via new platforms in this new normal.
For many students, playing games and discussing about games was clearly more essential than remotely
completing homework (Miller, 2021).
As a result, every concerned educator is looking for solutions to the problem. One method is to improve
the teaching process by including instructional aids just like mathematical games. Mathematical games have
been used to teach mathematics and science in many countries, including here in the Philippines. This is a
result of the usefulness of games in the educational process. A study conducted by Veray (2016), which aimed
to determine the effectiveness of engaging students through board games, revealed that the experimental
group who was exposed to the board games has higher academic performance. The study also recommends
engaging students in board games as part of improving their academic performance. Games can facilitate the
mathematical environment as they release boredom, tension and establish a friendly atmosphere which allows
for the growth of skills and knowledge.
It has long been known that board games can be used as instructional materials for math-related
disciplines. This study is a continuation of the undergraduate thesis conducted by Guadiario & Dela Cruz
(2019) entitled “Development and Validation of Matheladder as an Enrichment Activity on Basics of
Trigonometry”. Matheladder is a developed and validated board game that was patterned from the Snakes and
Ladders game but with the infusion of Trigonometry on it. Furthermore, the board game was tried out on
Grade 9 learners in Mindanao State University - Iligan Institute of Technology Integrated Developmental
School (MSU-IIT IDS) on the fourth quarter of S.Y. 2018-2019. Results showed that Matheladder is
creatively made and is well designed. It has no construction errors and sharp edges that can cause injury and it
is very easy to use. The rules are also understandable and clear enough that all could easily participate.
However, its effectiveness has not been explored. One of the recommendations of the study is to implement
the board game. Hence, in this present study, the researcher looked into the effectiveness of the Matheladder
as a review material on the learners’ achievement level and motivational level.
2. Methods
2.1 Research Design
The researcher used the Mixed method of research which is an intellectual and practical synthesis based on the
combination of quantitative and qualitative research. The experimental design was used under the quantitative
research design. The quasi-experimental research approach was used for the quantitative data. Specifically, the
researcher used the posttest-only group design where the learners are grouped into the experimental group and
the control group. The control group had their traditional review session while the experimental groups received
the intervention by utilizing the Matheladder board game as a review material through a series of trials. The
achievement test was then given to both groups that shows the difference between the achievement levels of the
students from the control and experimental groups. Furthermore, the qualitative data was obtained from the
insights of the learners through the interview guide questions.
2.2 Research Setting and Participants
This study was conducted at Acelo C. Badelles, Sr. Memorial High School. It is a government owned school of
the Department of Education that provides junior and senior high school education. It is located at Purok 6,
Tipanoy, Iligan City.
2.3 Participants of the Study
The participants of the study are the Grade 9 learners in Acelo C. Badelles, Sr. Memorial High School which
will be selected through purposive sampling. The selection for the control and experimental group was also
done purposively. The chosen section in Grade 9 was divided into two groups as per direction of the school and
as one of the protocols in implementing the limited face-to-face classes. There are 42 learners in the section but
only 40 students were considered. The mean grades of the learners were used to show the comparability of the
participants.
2.4 Research Instruments
The study will use the following research instruments:
2.4.1 Achievement Test
This is a 40-item multiple choice test questionnaire made by the researcher following the Most Essential
Learning Competencies stipulated by Deped, and Table of Specification. The coverage of the test includes all the
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topics discussed for the whole duration of the study which are: illustrates the six trigonometric ratios, finds the
trigonometric ratios of special angles, illustrates angles of elevation and angles of depression, uses trigonometric
ratios to solve real-life problems involving right triangles, illustrates laws of sines and cosines, and solves
problems involving oblique triangles. The achievement test was given to both experimental and control groups.
The test questionnaire was pre-evaluated by two in-service teachers and the section adviser/resource teacher. A
dry-run was conducted with 30 learners in the Grade 10 level at Iligan Capitol College due to the availability and
considering the online modality of the school. In addition, an item analysis was done where eleven of the
questions out of forty items were revised and rest is to be retained. The researcher used the suggestions received
during the pre-evaluation to finalize the instrument and make it ready for use.
2.4.2 Motivation Questionnaire
The researcher adapted the motivation questionnaire which was developed by Fiorella et al., (2021) to assess
learners' motivation toward trigonometry in both the control and experimental groups. It is modified accordingly
for it to be suited to the study. This questionnaire contains several statements aim at investigating the level of
learners’ motivation towards Trigonometry. This is composed of 19 Likert-scale questions that are indicated into
five latent factors: intrinsic value, self-regulation, self-efficacy, utility value, and test anxiety. Intrinsic Value
pertains to the student’s feeling that learning trigonometry is personally valuable. Self-regulation refers to a
student’s tendency to engage in behavioral learning strategies. Self-efficacy is a student’s belief that they have
the necessary competence and capability to perform well in trigonometry. Utility value is a student’s perception
that trigonometry is personally relevant to their lives or future careers. Lastly, test anxiety refers to a student’s
feeling of anxiety toward being assessed in trigonometry or compared with others. All items were on a fivepoint Likert-type instrument where students will assess their level of agreement in every statement. A dry-run
was conducted to the students of Iligan Capitol College in Roxas Avenue, Iligan City. To identify the internal
consistency of the motivation questionnaire, the Cronbach’s alpha was computed. It resulted with an overall
Cronbach’s alpha of 0.808 which implies that the internal consistency of the motivation questionnaire exceeds
the acceptable reliability. Therefore, the instrument is reliably good.
2.4.3 Interview Guide Questions
The interview guide questionnaire is made by the researcher that consists of open-ended questions relevant to the
study. This interview questionnaire serves as a reference for assessing the learners' experiences with
Matheladder and to get their insights after playing the Matheladder board game. These guide questions were
evaluated by two in-service teachers and the resource teacher.
2.5 Data Gathering Procedure
The following procedure was undertaken by the researcher to conduct the study.
a.) Asking for Permission
To guarantee the credibility of the research process before collecting data, a letter had been prepared addressed
to the Department of Education asking for permission to conduct the study in the chosen school. A formal letter
addressed to the School Principal of Acelo C. Badelles, Sr. Memorial High School was also presented along with
the letter from the DepEd in order to gain permission in entering and utilizing some school facilities during the
conduct of the study. Consent letters addressed to the parents of the learners will also be given. Upon the
approval of the school, a section from the Grade 9 level was selected as the participants of the study. The
learners were grouped accordingly so as to form the desired groups.
b.) Orientation
The researcher introduced herself and explained the purpose of the visit. All participants were asked to join the
orientation to be held at the school, bringing with them the consent letter signed by their parents or guardian.
The concept of the Matheladder board game was introduced only to the experimental group to make the learners
aware of what the game is all about, thus preparing them for the actual game. Prior to the conduct of the
orientation and/or the study, certain protocols are observed including protective measures, hygiene practices,
safety procedures, and necessary documents for the school and other stakeholders.
c.) Implementation
The experimental groups were able to utilize the Matheladder board game as review material before taking the
achievement test while the control group was given the traditional way of review. This was conducted by batch
due to the current pandemic situation and a series of trials was made. Groups 1, 2, and 3 was considered the trials
1, 2, and 3 respectively. This is to see if there is a consistency of the results from all the trials conducted. Upon
completion of the activity, the achievement test and the motivation questionnaire were given which concludes
the gathering of quantitative data. For the qualitative data, the researcher conducted an interview based on an
interview guide question and provided a feedback slip for the insights of the learners.
d.) Intervention
Given the pandemic situation, the resource school has implemented a progressive limited face-to-face class.
Following the Operational Guidelines on the Implementation of Face-to-Face Learning Modality, the chosen
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section in Grade 9 was divided into two. The researcher is required to follow the guidelines so as the original
schedule of the Grade 9 learners that is, the other half is following the MWF schedule while the other half is
following the TTh schedule. The selected section has 42 learners but only 40 of them was considered as the
participants of the study due to some absences. There are 20 learners for the MWF schedule and another 20
learners for the TTh schedule, both utilizing the 7:30am-9:30am classes.
d.) Conducting the Posttest
Upon completion of the review, the achievement test and the motivation questionnaire were given to both the
experimental and control groups. The conduct of the posttest was done on Friday for the learners following the
MWF schedule. On the other hand, the conduct of the posttest for the learners following the TTh schedule was
on Friday afternoon, this is with regards to the special agreement between the researcher, resource teacher and
the assistant principal.
2.6 Data Analysis
The study's participants' achievement and motivation levels were determined using both quantitative and
qualitative methods. The achievement test and Motivation Questionnaire provided quantitative data, while
interview results provided qualitative data. The tables below are used for the interpretation of data. These serve
as basis to classify and determine the description of the intervals they belong to.
Table 2
Achievement Level Classification
Score Range
Rating
Classification
36-40
90% above
Outstanding
34-35
85% - 89%
Proficient
32-33
80% - 84%
Approaching Proficient
30-31
75% - 79%
Developing
29-0
Below 74%
Beginning
Note: From https://www.ciit.edu.ph/k-to-12-grading-system/
With a passing average of 75 percent, the table indicates the achievement test score ranges, percentages,
and achievement level classification. This is based on the K-12 curriculum grading system approved by the
Department of Education. The score ranges are set based on the number of items on the achievement test
given with the lowest interval 0-29 classified as ‘Beginning’ and the highest interval of 36-40 classified as
‘Outstanding’. This table is used to determine the classification of the learners ‘achievement level from the
result on their achievement test scores.
Table 3
Description and Interpretation of the Different Levels of Motivation
Score
Interpretation
Description
4.51 – 5.0
Very Highly Motivated
Always feel motivated towards trigonometry.
3.51 – 4.50
Highly Motivated
Often feel motivated towards trigonometry.
2.51 – 3.50
Moderately Motivated
Sometimes feel motivated towards trigonometry.
1.51 – 2.50
Lowly Motivated
Rarely feel motivated towards trigonometry.
1.00 – 1.50
Very Lowly Motivated
Never feel motivated towards trigonometry.
Note: Adapted from the study “Online Learning and Students’ Mathematics Motivation, Self-Efficacy, and
Anxiety in the “New Normal” by L. Mamolo, 2022. From https://doi.org/10.1155/2022/949634
Table 3 shows the description and interpretation of the different levels of motivation from the study of
Mamolo (2021). The researcher made modifications that suited the present study. This is used to determine
the classification of the learner’s motivation level based from their responses to the motivation questionnaire.
2.7 Statistical Tools
The researcher will use the following statistical tools to analyze the gathered data.
a. Mean and Standard Deviation
This is used to measure the average achievement level of the respondents and to determine the index of
variability or the dispersion of the students’ scores in the motivation questionnaire and achievement test.
b. ANOVA
This is used to compare the collected data from the experimental group and the control group and to
determine if there is a difference between the two. It was computed using a licensed SPSS and tested at
0.05 level of significance.
c. Thematic Analysis
Thematic analysis was used in the analysis of qualitative data which came from the interview and
insights of the students after the implementation of the study.
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2.8 Coding of Data
The data gathered during the implementation of the study was coded accordingly. Since the study is comprised
with two groups namely, experimental group and control group, it was coded as EG and CG respectively.
Following the coding of each group, “EG1” was used to represent ‘Experimental Group 1’, and “CG1” for
‘Control Group learner 1’.
3. Results and Discussions
Objective 1: Determine the achievement level of the Grade 9 learners in trigonometry.
Table 4
Learner’s Achievement Level in Trigonometry (n=40)
Group
Mean
Std. Dev.
Interpretation
1 (Experimental Group 1)
29.6 (~30)
1.26
Developing
2 (Experimental Group 2)
29.9 (~30)
1.79
Developing
3 (Experimental Group 3)
29.4
1.84
Beginning
4 (Control Group)
19.1
2.77
Beginning
Total
27.0
5.00
Beginning
Table 4 presents the achievement level of the Grade 9 learners in trigonometry. Based on the calculated
mean score of 19.1 for control group, it shows that the learners under this group exhibit “beginning” level of
achievement in trigonometry. This is much lower when compared to the learners under the experimental
groups. Two out of three in the experimental group obtained an average rating of 75%-79% with a mean score
of approximately 29.6 and 29.9 which is interpreted as “Developing”. Moreover, the third group obtained a
mean score of 29.4 which has the interpretation as “Beginning” but it doesn’t show much difference on the
other experimental groups. It can be observed that the calculated mean of the experimental groups is on the
range of 29.4 – 29.6 which shows a consistent result among the three trials conducted that is higher than the
control group. On average, learners regardless of the intervention, still has low achievement level since it is
only in the developing level. This indicates that the groups don’t have much difference in terms of their
learning ability. However, it is still worthy to note that though they do have generally low achievement levels,
it still shows that the experimental groups have higher achievement levels than the control group.
As such, there was an improvement from the control and experimental group. It is clear enough that the
learners in the experimental group have higher achievement levels compared to the control group. With this,
it appears that the matheladder is an effective review material in learning trigonometry for the Grade 9
learners.
Objective 2: Determine the learners’ motivational level towards Trigonometry.
The researcher adapted a motivation questionnaire that was given to the participants as part of the posttest after
playing Matheladder.
Table 5
Learners’ Motivational Level toward Trigonometry (n=40)
Group
Mean Std. Dev. Description
Interpretation
1 (Experimental Group 1)
3.71
0.24
Often feel motivated towards trigonometry.
Highly Motivated
2 (Experimental Group 2)
3.90
0.15
Often feel motivated towards trigonometry.
Highly Motivated
3 (Experimental Group 3)
4.08
0.21
Often feel motivated towards trigonometry.
Highly Motivated
3.15
0.15
Sometimes
feel
motivated
towards Moderately
4 (Control Group)
trigonometry.
Motivated
Total
3.71
0.40
Table 5 presents the motivational level of the Grade 9 learners toward trigonometry for both control and
experimental groups. The three experimental groups got a “High” remark while the control group has
“Moderate” remark. Again, it can be observed that the control group obtain a lower mean score level of
motivation when compared to the experimental groups. Clearly, Grade 9 learners who experienced the
matheladder exhibit much higher motivational level compared to those learners who are not exposed to
matheladder. That is, learners under experimental groups often feel motivated toward trigonometry while
those learners from control group only sometimes feel motivated towards trigonometry. It shows that the
matheladder is an effective review material for improving the motivational level of the Grade 9 learners
towards trigonometry.
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Objective 3: Compare the mean scores in the achievement level of the learners between the experimental and
control group.
Table 6
ANOVA Table on the Mean Score Difference in Learner’s Achievement Level
Source
SS
df
MS
F
Sig. (P-value)
Between Groups
833.4
3
277.8
70.13
0.0001*
Error Variance
142.6
36
3.96
Total
976
39
25.03
Table 6 presents the result of one-way analysis of variance (ANOVA) in testing the significant mean
score difference of learner’s achievement level among groups. As observe, the computed p-value is lesser than
the alpha level (0.0001<0.05). Therefore, there is sufficient evidence to believe that the mean score of the
learner’s achievement level is significantly different among groups. This means that the achievement level of
the learners under for experimental groups is higher compared to control group. This shows that the
matheladder is an effective review material for improving the achievement level of the Grade 9 learners
toward trigonometry.
Table 7
Post Hoc Analysis: Bonferroni Comparison Test
Groups
Mean
Mean Dif. (Control-Trial)
Sig.
1(Experimental Group 1)
29.6
-10.5
0.0001*
2(Experimental Group 2)
29.9
-10.8
0.0001*
3(Experimental Group 3)
29.4
-10.3
0.0001*
4 (Control Group)
19.1
* Significant at 5% level (if p<0.05)
Now, to see which among the groups differ significantly, the Bonferroni comparison test is used. Based
on the result of Bonferroni multiple comparison test, it reveals that all experimental groups have significantly
higher achievement level compared to the control group.
Objective 4: Compare the mean scores in the motivational level of the learners between the two groups.
Table 8
ANOVA Table on the Mean Score Difference in Learner’s Motivation Level
Source
SS
df
MS
F
Sig. (P-value)
Between Groups
4.87
3
1.62
44.99
0.0001*
Error Variance
1.30
36
0.04
Total
6.17
39
0.16
Table 8 presents the result of one-way analysis of variance (ANOVA) in testing the significant mean
score difference of learner’s motivation level among groups. As presented in the table above, there is enough
data to conclude that the mean score of motivation level for both control and experimental groups varies
significantly. This means that matheladder is also an effective review material in improving the motivation of
the Grade 9 learners toward trigonometry.
Table 9
Post Hoc Analysis: Bonferroni Comparison Test
Groups
Mean
Mean Dif. (Control-Trial)
Sig.
1(Experimental Grp1)
4.21
-0.978
0.0001*
2(Experimental Grp2)
4.24
-1.002
0.0001*
3 (Experimental Grp 3)
4.30
-1.062
0.0001*
4 (Control Group)
3.24
* Significant at 5% level (if p<0.05)
Moreover, the result of Bonferroni comparison test shows that all groups under experimental have
significantly different motivation level compared to control group. Specifically, the experimental groups have
statistically higher motivation level than the control group.
Objective 5: Draw insights from the learner’s experience after playing Matheladder.
After the conduct of the activity, a semi-structured interview was conducted to the learners under the
experimental group. Here are some of the responses of the learners on the given questions.
1.) The use of Matheladder affected the participants’ feelings towards trigonometry.
From the responses of the participants, seven out of thirty participants find Matheladder as fun and
exciting. Positive feelings are also evident as it allows them to be comfortable with the lessons and
have relived their boredom and anxiety. Furthermore, five participants responded that Matheladder
helps in understanding the lessons in trigonometry more and in recalling the basics of trigonometric
values. Though one participant is not much affected, there are some participants who showed some
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liking towards trigonometry.
2.) Performance on the Matheladder game.
The performance of the participants in playing the Matheladder turned out to be good. Nine participants
claimed to have done better, some had fun and find it an exciting and amazing experience amidst the
nervousness. Some were also delighted for winning the game. Moreover, EG18 also find it nice and worthy
to be shared to others if given a chance.
3.) The participants’ experiences with their classmates when playing Matheladder.
Ten out of thirty participants described their experience while playing the Matheladder with their classmates
as happy and enjoyable. Twelve participants also find their experience fun and exciting. It showed how
they enjoyed their time playing the Matheladder board game and also how challenging it was. Many have
responded about how they had fun and learn at the same time.
4.) Comparing their enjoyment of trigonometry before the use of matheladder?
Most of the participants have responded that they find trigonometry challenging before, but with
Matheladder, they have realized that trigonometry can be fun and interesting while still challenging. In
addition, four participants have mentioned again the helpfulness of the game in understanding the lessons in
a fun way.
5.) The participants’ favorite aspect of the game.
The Question-and-Answer portion of the game is one of the most likeable parts based on the responses of
the participants. They find it more challenging on their end since the player can’t move to his/her next move
if the question will be answered correctly. EG7 also pointed it out to be a good thing so as to know if he/she
really learned something. Additionally, EG20 also mentioned that even though he/she didn’t get the correct
answer, he/she still learned from it also because of the answers of the other players. It shows that sharing of
ideas is present even if this game is not necessarily a group effort type of game. Three participants also like
the part of winning which is a vital result of the game. Throwing and rolling the dice also sparked an
interest in the other five participants. The overall game experience is also of the liking of the other six
participants which highlighted their enjoyment in playing and meaningful company with their classmates.
4. Conclusions
The main purpose of this study is to delve into the effectiveness of the Matheladder as a review material for
Grade 9 learners in trigonometry. Findings reveal that the Matheladder is an effective review material. The
learners in the control group are said to have a “beginning” level of achievement in trigonometry. This is
considerably lower than the "developing” achievement level of the experimental group. There is enough
information to conclude that the mean score of the learners’ achievement level differs significantly between
groups. This suggests that the experimental groups’ learners’ achievement levels are higher than the control
group. It also reveals that all experimental groups have significantly higher achievement level compared to the
control group.
Furthermore, the experimental group’s participants provided more positive answers to the motivation
questionnaire. As can be seen, the control group had a lower mean motivation score than the experimental
group. The learners under the control group only sometimes feel motivated towards trigonometry, whereas
learners in the experimental groups often feel motivated towards trigonometry. There is enough evidence to
establish that the mean motivation level score for both the control and experimental groups varies
significantly. It also reveals that all experimental groups have significantly different motivation levels than the
control group. The experimental groups, in particular, had a statistically higher level of motivation than the
control group.
Lastly, the drawn insights of the learners revealed that the Matheladder is fun, enjoyable, exciting,
challenging, as well as helping in a better understanding of the lessons and recalling trigonometric values.
Some learners also demonstrated an interest in trigonometry and appreciated the game’s value in helping them
comprehend the teachings in an enjoyable way. The majority of the participants also recognized that with
Matheladder, trigonometry can be both fun and challenging. Additionally, the game’s Question-and-Answer
component, as well as the concept of winning the game, is one of the most captivating parts of the game.
Furthermore, the participants had difficulty in answering the test due to the lack of sufficient knowledge, of
how hard it is to understand the topics, unfamiliarity with the problem-solving process, and the negative
notion of trigonometry.
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W2346288794.txt | Hindawi Publishing Corporation
Journal of Diabetes Research
Volume 2016, Article ID 1540267, 8 pages
http://dx.doi.org/10.1155/2016/1540267
Review Article
Physiological and Pharmacological Roles of FGF21 in
Cardiovascular Diseases
Peng Cheng,1,2,3 Fangfang Zhang,1,2,3 Lechu Yu,1 Xiufei Lin,1,2,3 Luqing He,1,2,3
Xiaokun Li,2,3 Xuemian Lu,1 Xiaoqing Yan,1,2,3 Yi Tan,1,2,4 and Chi Zhang1,2,3
1
The Chinese-American Research Institute for Diabetic Complications, Wenzhou Medical University, Wenzhou 325035, China
Ruian Center of the Chinese-American Research Institute for Diabetic Complications, The Third Affiliated Hospital,
Wenzhou Medical University, Wenzhou 325200, China
3
School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325035, China
4
Kosair Children Hospital Research Institute, Department of Pediatrics, University of Louisville School of Medicine,
Louisville, KY 40202, USA
2
Correspondence should be addressed to Yi Tan; yi.tan@louisville.edu and Chi Zhang; zhangchi515@126.com
Received 26 October 2015; Revised 26 February 2016; Accepted 18 April 2016
Academic Editor: Raffaella Mastrocola
Copyright © 2016 Peng Cheng 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.
Cardiovascular disease (CVD) is one of the most severe diseases in clinics. Fibroblast growth factor 21 (FGF21) is regarded as an
important metabolic regulator playing a therapeutic role in diabetes and its complications. The heart is a key target as well as a
source of FGF21 which is involved in heart development and also induces beneficial effects in CVDs. Our review is to clarify the
roles of FGF21 in CVDs. Strong evidence showed that the development of CVDs including atherosclerosis, coronary heart disease,
myocardial ischemia, cardiac hypertrophy, and diabetic cardiomyopathy is associated with serum FGF21 levels increase which
was regarded as a compensatory response to induced cardiac protection. Furthermore, administration of FGF21 suppressed the
above CVDs. Mechanistic studies revealed that FGF21 induced cardiac protection likely by preventing cardiac lipotoxicity and the
associated oxidative stress, inflammation, and apoptosis. Normally, FGF21 induced therapeutic effects against CVDs via activation
of the above kinases-mediated pathways by directly binding to the FGF receptors of the heart in the presence of 𝛽-klotho. However,
recently, growing evidence showed that FGF21 induced beneficial effects on peripheral organs through an indirect way mediated by
adiponectin. Therefore whether adiponectin is also involved in FGF21-induced cardiac protection still needs further investigation.
1. Introduction
Cardiovascular diseases (CVDs) are the leading cause of
death worldwide composed of heart and blood vessel diseases. In the recent years, the incidence of CVDs has been
increasing at a sharp rate globally. According to the World
Health Report 2010, CVDs contributed to 17.5 million deaths
and these numbers are estimated to increase to 23.3 million by
2030 [1, 2].
Fibroblast growth factor (FGF) is a cytokine superfamily
with pleiotropic biological functions including regulating cell
growth, differentiation, development, and metabolism [3–7].
Human FGFs contain 22 members which can be divided into
7 subfamilies based on phylogeny and sequence [8–10]. Due
to the lack of a heparin binding domain, FGF19 subfamily
members (FGF19, FGF21, and FGF23) function in an
endocrine manner rather than an autocrine manner as other
subfamily members of FGFs [9]. Among them, FGF21 is
a polypeptide with 209/210 (human/rodent) amino acid
residues that is primarily produced and secreted by the liver,
adipose tissue, and thymus [11]. FGF21 expression is mainly
regulated by peroxisome proliferator-activated receptor 𝛼
(PPAR𝛼) in the liver [12] and PPAR𝛾 in adipocytes [13,
14]. FGF21 was firstly cloned in 2000 [11] and received
global attention in recent years due to its outstanding ability
on regulating carbohydrate and lipid metabolism including
improving insulin sensitivity, lowering blood glucose, reducing hepatic/plasma triglycerides, inducing weight loss by
increasing energy expenditure, and reducing fat mass [15–18].
Further studies indicated that FGF21 functions by binding
2
to (FGFR)1c and (FGFR)2c in the presence of coreceptor
𝛽-klotho and activation of downstream signaling pathway
[19, 20]. Although FGF21 and other members of FGFs share
the same FGF receptors, the coreceptors are different (𝛽klotho for FGF21 and heparin for others) which determined
that they have different bioactivity due to activation of
various pathways [21, 22]. Unlike traditional insulin therapy
in clinics, FGF21 did not cause hypoglycemia [16]. The possible explanation is that FGF21 induces physiological role in
healthy condition and pharmacological role under unhealthy
condition [23, 24]. Additionally, FGF21 does not lead to
carcinogenic event due to lack of mitogenic function which
makes it possible to be administrated in vivo in clinics [16].
Therefore, FGF21 may hold promise as a clinically therapeutic
option due to the abovementioned characters and advantages.
In recent clinical and preclinical studies, CVDs have
been closely associated with serum FGF21 which increased
in the patients with atherosclerosis, coronary heart disease,
myocardial ischemia, cardiac hypertrophy, and diabetic cardiomyopathy [25–27]. Therefore FGF21 has the potential to
be considered as a biomarker for the above CVDs.
Whether the increased serum FGF21 level is the basis for
CVD pathogenesis or is induced to protect the heart form
CVDs is still under discussion. However, growing evidence
indicated that administration of exogenous FGF21 induces
preventive effects on most of the above CVDs, suggesting
that FGF21 not only is a simple marker of cardiovascular
risk but also induces a protective effect on the cardiovascular
system contributing to a reduction in risk (Table 1). In clinics,
serum FGF21 levels were increased in patients with obesity or
type 2 diabetes which was associated with high risk of CVDs.
The paradoxical phenomenon was supposed to be explained
by a compensatory response to induce cardiac protection or
resistance to FGF21 which impaired its bioactivity [28, 29].
In animal study, we found that at the early-stage of diabetes
serum FGF21 level of mice was sharply increased compared
with nondiabetic mice (C57BL/6J), while it was dramatically
decreased at the late-stage of diabetes which further confirmed that early-stage increase of serum FGF21 was a compensatory response and induced beneficial effect on the heart;
late-stage decrease may be the cause of diabetes-induced cardiac damage [30], since the above CVDs are always attributed
to lipid metabolic disorder. Mechanistic studies indicated
that FGF21-induced cardiac protection in CVDs is possibly
attributed to the suppression of lipotoxicity since the above
CVDs are always the consequences of lipotoxicity. This review
tries to illuminate the underlying relationship between FGF21
and CVDs and the possible mechanisms.
2. FGF21 and Atherosclerosis and
Coronary Heart Disease
Atherosclerosis is a chronic, inflammatory disorder characterized by the deposition of excess lipids in the arterial
intima [31]. The accrued evidence indicated that lipidlowering therapy limits the progression of atherosclerosis
and reduces CAD events [32]. Since FGF21 plays an important
role in the regulation of lipid metabolism, the effect of FGF21
in atherosclerosis is of interest. Clinical studies showed that
Journal of Diabetes Research
increased circulating FGF21 levels were discovered in
atherosclerotic patients or the individuals with high risk of
developing atherosclerosis [33, 34]. Additionally, an in vivo
study demonstrated that increased serum FGF21 was
observed in aortas of apoE−/− mice (C57BL/6J background)
[35]. Strong evidence identified that administration of
exogenous FGF21 significantly improved lipid metabolic
disorders and reduced atherosclerotic plaque areas in these
animals [36]. Moreover, Lin et al. also reported that FGF21
deficiency enhanced atherosclerotic deterioration and
mortality in apoE−/− mice (C57BL/6J background) [35],
implying that increased serum FGF21 in patients with
atherosclerosis described previously induces beneficial
effect rather than the basic for atherosclerotic pathogenesis.
Mechanistic study indicated that FGF21-induced prevention
of atherosclerosis was associated with suppression of
endoplasmic reticulum stress-mediated apoptosis in apoE−/−
mice (C57BL/6J background) [37]. Further mechanistic
studies revealed that prevention of atherosclerosis by
FGF21 was attributed to the fine-tuning of multiorgan cross
talk among the liver, adipose tissue, and blood vessels,
characterized by suppression of hepatic sterol regulatory
element-binding protein-2 and induction of adiponectin in
mice with atherosclerosis [35]. Although FGF21 functions in
an endocrine manner, whether FGF21 can also induce a direct
protection to the blood vessels remains unclear. For decades,
lowering levels of low-density lipoprotein (LDL) cholesterol
and increasing level of high-density lipoprotein (HDL)
have formed the cornerstone of management of patients
with atherosclerotic cardiovascular disease. Strong evidence
demonstrated that FGF-21 dramatically improved the
condition of atherosclerosis in Wistar rats by decreasing
serum LDL levels and increasing serum HDL levels.
Moreover, FGF-21-induced antioxidative function is also
involved in its therapeutic effect in atherosclerotic Wistar rat
characterized by increased levels of superoxide dismutase,
reduced glutathione, and reduced malondialdehyde [38].
Along with the development of atherosclerosis, the
artery’s lining becomes hardened, stiffened, and swollen with
all sorts of “gunge,” including fatty deposits and abnormal
inflammatory cells, to form a plaque and then eventually
deteriorate into coronary heart disease [39–41]. Strong evidence indicated that cardiac endothelial cell dysfunction
may be an early initiating factor for atherosclerosis which
facilitates the development of coronary heart disease [42].
Oxidized LDL (ox-LDL) is a proatherogenic lipoprotein that
accumulates in the vascular wall and contributes to vascular
dysfunction at the early-stage of atherosclerosis development
[43–53]. Enhanced serum ox-LDL and antibodies against its
epitopes are predictive for endothelial dysfunction and subsequent coronary heart disease [43]. Previous in vitro study
indicated that both FGF21 mRNA and protein expressions
were increased in response to ox-LDL treatment in cardiac
endothelial cells and this was protective against apoptosis
caused by ox-LDL [54]. Also, FGF21 has been reported to
prevent high glucose induced cell damage and endothelial
nitric oxide synthase dysfunction through an AMP-activated
protein kinase- (AMPK-) dependent pathway in endothelial
Apolipoprotein E(−/−) mice
Atherosclerosis
Coronary artery ligation (ischemia/reperfusion)
Isoproterenol infusion-induced cardiac
hypertrophy/LPS-induced cardiac hypertrophy
Multiple low-dose STZ-induced type 1 diabetes
Myocardial ischemia
Cardiac hypertrophy
Diabetic cardiomyopathy
Coronary heart disease
Model
Heart disease
Outcomes
Atherosclerotic lesion area collagen composition ↓
Recombinant murine FGF21 was given daily
Total cholesterol ↓
intraperitoneally for 16 weeks
Hypertriglyceridemia ↓
Circulating adiponectin ↑
Cell apoptosis ↓
Mouse FGF21 full length protein was given for 24 or Oxidative stress ↓
48 hours
NO production ↑
eNOS phosphorylation ↑
Recombinant mouse FGF21 was administered
Activity of caspase-3 ↓
intravenously immediately after myocardial injury Degree of myocardial infarction ↓
every 12 hrs for 3 days
Left ventricular function ↑
Cardiomyocyte size ↓
FGF21 was injected intraperitoneally for 7 days or
Δ heart weight/body weight ↓
given for 24 hours in neonatal cardiomyocytes
Inflammation ↓
Cardiac oxidative stress ↓
Oxidative stress ↑
Knockout FGF21 in type 1 diabetic mouse model
Lipid accumulation ↑
Cardiac dysfunction and remodeling ↑
Methods
Table 1: Summary of major pharmacological studies of FGF21 in heart disease.
[66]
[64, 65]
[59]
[55]
[63]
Ref.
Journal of Diabetes Research
3
4
cells [55]. Therefore the relationship between FGF21 and
coronary heart disease is of interest. Shen et al. reported that
serum FGF21 level was positively associated with coronary
heart disease in clinics [56, 57]. Our previous work confirmed
that serum levels of FGF-21 are increased in patients with
coronary heart disease independently associated with adverse
lipid profiles [33]. In contrast, another study indicated that
serum FGF21 has been associated with hypertriglyceridemia,
hyperinsulinemia, and pericardial fat accumulation but not
associated with coronary heart disease [58]. This paradox
may be explained by decreased body mass index of healthy
controls compared to patients with coronary heart diseases.
3. FGF21 and Myocardial Ischemia
Myocardial ischemia, a disorder causing cardiomyocytes
injury and myocardial infarction and malfunction, activates adaptive responses enhancing myocardial tolerance to
ischemia. Liu et al. indicated that, in response to myocardial
ischemia in the C57BL/6J mouse, liver- and adipocytesderived FGF21 was upregulated and secreted into the circulation. After interacting with FGFR1 in cardiomyocytes in
the presence of 𝛽-klotho, FGF21 activates its downstream
kinases and proteins including phosphatidylinositol 3-kinase
(PI3K), protein kinase B (PKB/AKT), and Bcl2 antagonist
of cell death (BAD), thereby reducing myocardial ischemiainduced apoptosis characterized by reduction of caspase-3
activity [59]. However, the adaptive response was not found
in FGF21-deficient mice. Reversely, myocardial ischemic size
was significantly smaller in FGF21 transgenic mice than
that in wild type mice [59], suggesting that upregulated
endogenous FGF21 derived from the liver and adipose tissue
in response to myocardial injury induced cardiac protection
mediated by activation of FGFR1/𝛽-klotho-PI3K-Akt1-BAD
signaling pathway. Although various growth factors and
cytokines were upregulated during myocardial ischemia, the
expression and secretion of cardiac FGF21 had no alteration, implying FGF21 induces cardiac protection against
myocardial ischemia in an endocrine rather than an autocrine
manner [59, 60]. To date, a question of whether administration of exogenous FGF21 can also induce cardiac protection during myocardial ischemia and if so whether the
protection of exogenous FGF21 against myocardial ischemia
can be direct to the heart or cardiomyocytes appears. This
question was answered by Patel group [61]. They found that
administration of exogenous FGF21 induced significant cardioprotection and restored cardiac function following global
ischemia in Langendorff perfused rat hearts. Further study
revealed that inhibition of AKT, extracellular signal-regulated
kinase (ERK1/2), and AMPK impaired FGF21-induced
antimyocardial ischemia effect in the hearts of obese Wistar
rats, suggesting that the above kinases are involved in this cardioprotection of FGF21 [61]. Our previous in vitro study also
confirmed that administration of exogenous FGF-21 attenuated ischemia-reperfusion induced damage in H9c2 cells
characterized by inhibition of oxidative stress and apoptosis
[62]. The mechanistic study revealed that FGF21-induced
protection against ischemia-reperfusion injury in cardiac
Journal of Diabetes Research
cells mainly depended on the activation of Akt-GSK-3𝛽caspase-3 signaling pathway by preventing oxidative stress
and recovery of the energy supply [62].
4. FGF21 and Cardiac Hypertrophy
Hypertrophic remodeling characterized by enlarged cardiomyocytes is an adaptive response of the heart to certain
stresses. And it is also the leading cause of multiple cardiovascular problems including hypertension, myocardial ischemia,
valvular disease, and cardiomyopathy [67–69]. Mature cardiomyocytes are considered to be terminally differentiated
cells with no regenerative ability [70–72]. Under stresses,
cardiac hypertrophy is characterized by cardiomyocytes
enlargement, rather than cells division [73, 74], and this
phenomenon is accompanied by the increase of extracellular
matrix and fibroblasts inside the heart [75, 76].
Recently, cardiac hypertrophy was reported to induce
FGF21 gene expression in the cardiomyocytes of mouse,
and this was subjected to transcriptional regulation of the
hepatic silent mating type information regulation 2 homolog
1/PPAR𝛼 pathway [64]. In turn, FGF21 knockout mice had
greater heart weights and more severe cardiac dysfunction
in response to isoproterenol infusion along with induction of hypertrophic inflammatory markers [64]. However,
administration of recombinant FGF21 significantly prevented
isoproterenol-induced cardiac hypertrophy damage in mice
[64]. Mechanistic studies indicated that FGF21 prevented
cardiac hypertrophy by activating mitogen-activated protein
kinase (MAPK) signaling via activation of FGFR1c/𝛽-klotho
[64, 77]. Additionally, FGF21 prevented cardiac hypertrophy
by promoting multiple antioxidant genes expressions (e.g.,
uncoupling proteins 2 and 3, also superoxide dismutase-2)
and inhibiting the formation of reactive oxygen species in an
autocrine manner [65].
5. FGF21 and Diabetic Cardiomyopathy
Diabetic patients develop the diabetic cardiomyopathy independent of coronary artery disease and hypertension [78, 79].
Diabetic cardiomyopathy is attributed to multiple pathogenic
factors, including hyperglycemia, hyperlipidemia, and
inflammation [80–82]. Cardiomyopathy is a late consequence
of diabetes-induced early cardiac responses especially the
myocardial apoptosis [83, 84]. Thus, treatments to reduce
cardiac apoptosis may help control diabetic cardiomyopathy.
Recently, we reported that cardiac FGF21 mRNA expression was positively associated with the development of
diabetes in the type 1 diabetic mice, suggesting that the
increased cardiac FGF21 expression may be beneficial to the
heart in this regard [30]. In the study we also observed cardiac
apoptosis in early diabetic mice, which was remarkably prevented by administration of recombinant FGF21 [30]. Similar
protection by FGF21 was observed in mice with cardiac
lipotoxicity induced by fatty-acid [30]. Mechanistic studies
indicated that FGF21-induced antiapoptotic effects in vitro
and in vivo were mediated by ERK1/2-p38-MAPK-AMPK
signaling pathway [30]. Thus, FGF21-induced cardioprotection in diabetic mice is mainly attributed to prevention of
Journal of Diabetes Research
5
FGF21
FGFR1c
Cell membrane
𝛽-klotho
𝛼
AR
PP
AMPK
PI3K
p85
Erk1/2
Sirt1
P38
MAPK
Cell damage
Endothelial dysfunction
Atherosclerosis
Akt
BAD
Inflammatory
oxidative stress
Cardiac hypertension
Coronary heart disease
AMPK
Lipotoxicity
Oxidative stress
Apoptosis
Myocardial ischemia
Diabetic cardiomyopathy
Figure 1: FGF21 induces preventive effect on CVDs through multiple signaling pathways. As a classical cytokine, FGF21 functions as a
metabolic regulator by binding with its receptor FGFR1 or FGFR2 in the presence of 𝛽-klotho. Growing studies demonstrated that FGF21
also induced beneficial effects on CVDs probably due to inhibition of glucose or lipid metabolic disorders. For instance, FGF21 prevented
atherosclerosis and the subsequent CHD by inhibition of lipogenesis which was also the possible mechanism of FGF21-induced preventive
effect on CH. Additionally, FGF21 also prevented MI and DC by activation of Akt- and AMPK-mediated signaling pathway which were
usually involved in maintaining glucose and lipid homeostasis.
lipotoxicity by FGF21. Also, long-term treatment of FGF21
prevented diabetic-induced cardiac dysfunction and fibrosis
mediated by the same signaling pathway as above [30]. Our
work also revealed that FGF21 deletion-aggravated cardiac
lipid accumulation is likely mediated by cardiac Nrf2-driven
CD36 upregulation in type 1 diabetic mice, which contributes
to increased cardiac oxidative stress and remodeling, and
eventual development of diabetic cardiomyopathy [66].
cardiomyopathy via Akt- or AMPK-mediated signaling pathways which regulate lipid and glucose metabolisms (Figure 1).
Since serum FGF21 increases in several kinds of CVDs, serum
FGF21 levels might be regarded as a potential biomarker not
only for diagnosis of metabolic disorders but also for diagnosis of CVD in clinics. And supplementation of exogenous
FGF21 might also induce beneficial effect in patients with
CVD based on the conclusion of preclinical studies.
6. Summary
Disclosure
CVD includes atherosclerosis, coronary heart disease, myocardial ischemia, cardiac hypertrophy, and diabetic cardiomyopathy which are all closely associated with severe lipid
metabolic disorders [85–87]. FGF21, a metabolic regulator of
carbohydrates and lipids, has been shown to improve insulin
sensitivity and glucose uptake and suppress lipogenesis and
lipid oxidation [15–18]. Clinical studies indicated that serum
FGF21 changes were positively associated with the development of atherosclerosis, coronary heart disease, myocardial
ischemia, cardiac hypertrophy, and diabetic cardiomyopathy,
which implies that upregulated endogenous FGF21 may
improve CVDs. Specifically, FGF21 prevented atherosclerosis
and subsequent coronary heart disease was attributed to multiorgan cross talk among the liver, adipose tissue, and blood
vessels and was characterized by suppression of lipid accumulation and increased lipid oxidation [63]. Similarly, FGF21
prevented stress-induced CH via enhancing lipid oxidation
mediated by the ERK1/2-CREB-PGC-1𝛼 signaling pathway
[64]. FGF21 also prevented myocardial ischemia and diabetic
The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the paper.
Competing Interests
The authors declare that there are no competing interests
regarding the publication of this paper.
Authors’ Contributions
Peng Cheng, Fangfang Zhang, and Lechu Yu are equal
contributors to the review.
Acknowledgments
This study was supported in part by grants from the National
Science Foundation of China (81370917, to Chi Zhang;
81471045, to Xuemian Lu; 81573435, 81273509, and 30971209
to Yi Tan), the Research Development Fund of Wenzhou
6
Medical University (QTJ13005, to Chi Zhang; QTJ13007 to
Yi Tan), the Medical and Healthy Technological Grant of
Zhejiang Province (201472233, to Chi Zhang), the Project
of Public Welfare of Wenzhou (2014Y0416, to Chi Zhang),
and the Project for Selected Overseas Chinese Supported by
Zhejiang Technology Foundation (to Chi Zhang) and the
Key New Drug Development Grant 2012ZX09103-301-016 (to
Xiaokun Li and Yi Tan) and the grants from the American
Diabetes Association and Juvenile Diabetes Research Foundation (1-13-JF-53 and 1-INO-2014-122-A-N to Yi Tan).
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MACNAIR—LIMESTONES IN THE GLASGOW DISTRICT.
No.
37
V.—THE
STRATIGRAPHY OF THE LIMESTONES LYING
IMMEDIATELY ABOVE THE CALCIFEROUS LAVAS IN THE GLASGOW
DISTRICT.
By PETER MACNAIR, F.R.S.E.,
F.G.S., and
HARRY R. J. CONACHER.
[Read 13th March, 1913.]
THE group of sediments under discussion is that commonly
known as the Lower Limestones, with the addition of those
which occupy the space between the base of the Hurlet Seam
and the top of the lavas, that is, from the Hosie Limestones
at the top to below the Hollybush. These limestones crop
out practically all around the basin in which Glasgow lies,
and are seen in burn sections, quarries, and railway cuttings
along the base of the hills of Calciferous lava which form its
rim.
The strata below the Hurlet Seam are grouped by the officers
of the Geological Survey with the Calciferous series, but as
there is no essential break at this horizon in our district such
as exists in the Lothians, we propose to deal with all these
beds as one series.
The district dealt with may be defined as including Hurlet,
Howood, Paisley, Bridge of Weir, Duntocher, Bearsden,
Campsie, Kilsyth.
The literature of the subject is meagre. There are references
in the Geological Survey's publications dealing with Sheet No.
31 of their 1-inch Geological Map.
In the Transactions of
our Society there is a paper by Mr. Hugh M'Phail dealing
with the Barrhead-Paisley District; there is Dr. Young's
paper of 1860 on Campsie and the Glasgow District; and there
is the Memoir of the Geological Survey, published in October,
1911.4
1
2
3
1
Memoirs of t h e Geological Survey, Scotland. Explanation of Sheet 3 1
(1879).
On t h e Carboniferous Sections of the Levern V a l l e y , Renfrewshire.
Glas. Geol. Soc. Transactions, vol. iii., p. 254.
On t h e Geology of the Campsie District.
Trans. Geol. Soc. Glas.
vol. i., p. 1, 1863.
Memoirs of t h e Geological Survey of Scotland. T h e Geology of t h e
-Glasgow District, 1911.
2
3
9
4
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38
TRANSACTIONS
GEOLOGICAL
SOCIETY
OP
GLASGOW.
Prior to the issue of the last-named publication, no definite
co-relation of the beds under discussion had been published..
It was while going over the ground with that book in hand
that we came on the points which we now bring before you.
Hurlet District.—In the Memoir, the following horizons are
recognised and described in the district, and may be examined
at the localities named: —
Hosie Limestone—River Levern at Barrhead.
Blackhall Limestone—Jenny's Well, Paisley, and Levernside,.
Crookston.
Hurlet Limestone—Not now seen, except at Arkleston Cutting,.
north-east of Paisley.
Blackbyre Limestone—Railway Cutting at Barrhead.
Hollybush Limestone—Hollybush Quarries, and Hawkhead
Dam.
With regard to the Hosie Limestone at Barrhead, we are
unable to offer any opinion or criticism of this identification.
The Blackhall Limestone, as seen at Jenny's Well (first
described by Mr. John Smith in vol. xii. of our Transactions)
shows definite and constant characters which enable it to be
reoognised with certainty throughout the district. It is in
several beds, the top one marine, the lower fresh water, and
contains a parting of entomostracan oil shale.
It can be seen
to overlie a series of clay-band ironstones, which, again, are
above the Hurlet Seam, as shown by old workings.
The Blackbyre Limestone, as seen in the railway cutting
at Blackbyre Farm, is also in several beds, the upper composed
of small encrinite fragments, and the lower slabby and crowded
with small producti and containing occasional plant remains.
The Hollybush Seam is accessible with difficulty in the old
quarries at Hollybush and below high-water level in the Hawkhead Dam. In both localities its most striking characteristic
is the abundance of large producti (giganteus, latissimus, and
semi-reticulatus), and branching and solitary corals.
The stratigraphical relations of the Blackbyre and Hollybush
Limestones cannot be certainly seen in the type district, but
the positions assigned to them in the Memoir are confirmed
by neighbouring sections, which are now described for the
first time.
r
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MACNAIR—LIMESTONES
I N THE GLASGOW DISTRICT.
39"
Nethercraigs.—At Nethercraigs, south of Paisley, there is a
small syncline, truncated to the south by the fault which
brings up the lavas of the Ferneze Hills, and in this basin—
no more than half a mile across—we see the Hurlet Lime stone
the Blackbyre, and the Hollybush.
The Hurlet Limestone,
which at Hurlet is stated to be no more than 4 to 5 feet thick,
has here increased, by the addition of limey shales, to about
30 feet. This is a surprising phenomenon, but it is also seen
to occur at Howood, in the G-ryfe, and at Campsie.
The
y
Fig. 1.—Plan a n d Section across Limestones a t Nethercraigs, Paisley.
(a) S t r a t a below Hollybush L i m e s t o n e ; (b) Hollybush L i m e s t o n e ;
(c) Hollybush Sandstone ; (d) Blackbyre L i m e s t o n e ; (e) A s h y m u d s
w i t h limey n o d u l e s ; ( / ) Hurlet Limestone.
alum shale and coal, which usually underlie the encrinital
Hurlet Limestone, are not seen here, but a very characteristic
brecciated limestone bed with ashy mud, recognised by us in
the same position at Howood, Bridge of Weir, and Campsie,
is well displayed in old quarry section and in the neighbouring
burn. Below it comes a thickness of sandstone whose lower
boundary is a line of unconformity, which is also seen in the
Campsie district.
The Blackbyre Limestone comes in at this
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40
TRANSACTIONS
GEOLOGICAL
SOCIETT
OP
GLASGOW.
point and is, as elsewhere, a typically marine deposit.
The
beds underlying it are seen in the burn section to consist
mainly of sandstone with thin coal (the "Lady Anne"), and
then comes the Hollybush Limestone. This seam is not well
seen m section, but it contains the typical large producti and
corals.
At Nethercraigs the burn section goes only a short
distance below the Hollybush, showing nothing but sandstone,
but at Hollybush we get a glimpse in burn and railway
sections of volcanic muds and oolitic beds which seem every
where to occupy a position immediately above the lavas.
There are also some ironstones containing plant remains.
Howood.—In old quarries here, and in a burn section at
Meikle Corseford, we find the Hurlet Limestone exhibiting the
Nethercraigs characteristics and immediately underlain by a
shale and coal. Some parts of the shale contain a marine
fauna (producti, &c), while others are matted with a lamellibranch which appears to be Cypricardia
(Sanguinolites)
tricostata?
The peculiar brecciated limestone is seen in the
burn, and it here contains a few marine fossils, and is overlain
by an ashy bed with nodules, like that of Nethercraigs. The
equivalent of the Blackbyre Seam is also exposed, but differing
somewhat in character from that of the type. Below it sandstone
appears, overlying limey beds which are crowded witE giganteid
producti and lithostrotion, which we regard as the equivalent
of the Hollybush. Unfortunately the section gives no certain
information beyond this point, and the boundary fault comes
in. The modification of the characters of the Blackbyre Lime
stone seen in this section is interesting as confirming the
identification of the small, isolated exposure of limestone seen
in the railway cutting at Auchinback, east of Barrhead,
providing a link in the transition of this bed from the characters
seen in the type section to those prevailng at Thorntonhall
and in north Ayrshire.
Bridge of Weir.—In the Gryfe, to the north-east of Bridge
of Weir, there is an excellent section which, with but one
important gap, is complete from the top of the lavas to the
top Hosie Limestone.
Beginning at Bridge of Weir and
working down stream, there is an interesting section of muds
and conglomerate of lava pebbles lying in hollows on the
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on July 11, 2015
M A C N A I R — L I M E S T O N E S I N THE GLASGOW DISTRICT.
41
eroded l a v a surface.
Above this comes ashy fireclay, with
rootlets, irregular coal, and greenish sandstone.
A horizon
of fossiliferous nodules and shales then comes in, which is found
t o develop into a fairly compact limestone in places, crowded
with l a r g e producti and branching corals.
I t can b e
examined at intervals along the b a n k s of the Gryfe from the
c o t t a g e on the west b a n k to the ford. A t the latter place the
Fig. 2 — P l a n a n d Section along the Gryfe, from Bridge of Weir t o Crosslee.
Lettering s a m e a s tables in t e x t (see page 42).
nodular p h a s e is well seen.
This limey horizon overlies a thin
coal. A sill penetrates the s t r a t a , and the sequence is obscured
b y small disturbances which run parallel to the line of section.
F r o m the ford to the turn in the stream a t the old limestone
workings, there are no exposures, but below water level a t the
sharp bend, the nodular green limestone can be seen, contain
i n g corals and encrinites.
The coal and a l u m shale a r e not
now exposed, b u t fragments can b e picked u p in the b i n g s .
T h e evidence for identifying this seam as the Hurlet is fairly
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on July 11, 2015
42
TRANSACTIONS—GEOLOGICAL
SOCIETY
OF
GLASGOW.
complete, and as already mentioned, this section also displays
the augmentation of its thickness by the addition of limey
shales, while the more typical encrinital part, 4 to 5 feet
thick, has been worked out.
The gap in this section occurs
below the nodular green bed, just described, and we believe
that at this point the Blackbyre Limestone exists, which
opinion is confirmed by the evidence of the following bore put
down at Goldilee and quoted in the Transactions of the High
land and Agricultural Society : —
BORE AT GOLDILEE, BRIDGE OF WEIR, RENFREWSHIRE.
CORRELATED WITH THE HURLET SECTION.
Feet. Inches.
Clay,
Flag,
Limestone,
Till,
Coal,
Fire-clay,
Limestone,
Fire-clay,
Black Till,
Coal,
Grey Till,
Greenstone,
Grey Till,
Freestone,
Coal Till,
Coal,
Fire-clay,
Whinstone,
-
-
12
20
7
1
2
5
4
2
19
13
5
5
5
1
5
-
0
0
0
6
^
10 }
J
g Hurlet Limestone.
/ A l u m Shale.
e Hurlet Coal.
d A s h y Muds w i t h Limey Nodules.
c Blackbyre Limestone.
b Equivalent of Hollybush Sandstone.
Intrusive Sill.
a Hollybush Limestone.
9
3
Lavas.
The limey beds, containing large producti and corals, we
correlate with the Hollybush Seam.
Proceeding with the stream, a small fault is crossed, which
brings down the top of the Hurlet, and then the shales and
ironstones, with a thin coal, are met. These are overlain by
a very interesting series of strata, of which we give a detailed
section—
18" encrinital limestone.
14" strong shale.
12" shale or fireclay w i t h stigmaria and entomostracan shale.
12" entomostracan limestone.
12" shale.
4" coal.
Shale w i t h ironstones (containing Lithostrotion in places) and coal.
Downloaded from http://trngl.lyellcollection.org/ at West Virginia University
on July 11, 2015
Trans. Geol. Soc. of Glasgow.
Vol. XV., Plate V
F i g 3.
Plan and section across the South Hill of Campsie from Craigenglen
to Baldow Glen,
Lettering same as Table in text, see page 45.
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on July 11, 2015
MACNAIR—LIMESTONES
I N THE GLASGOW DISTRICT.
43
There can be no doubt that this represents the Blackhall
Limestone with some interesting variations.
Above these strata comes a fresh series of shales with iron
stones, some of which contain entomostraca and almost develop
into limestone, and then we reach the two encrinital bands of
the Hosie, with shale containing Posidonomya
corrugatcfi
(marked i in section).
We consider that the Nethercraigs, Howood, and Gryfe
sections together afford important evidence bearing upon the
co-relation of the Hurlet section with those in the north of
Ayrshire. Thus it seems to us that the lower division of the
main limestone, as seen at Broadstone, Beith, and elsewhere,
with its underlying coal is the equivalent of the Hollybush
seen in the three Renfrewshire localities.
The fragmental
deposits lying between the lower and upper divisions of the
main limestone of Ayrshire, as seen at Broadstone, is the
representative of the Hollybush Sandstone; while the upper
division of the main limestone, as seen at Dockra, is the
equivalent of the Blackbyre Limestone, the position of this
limestone being fixed over a large part of the north of Ayrshire
by the capping of ashy muds with limey nodules, which, we
have shown, is so characteristic of this horizon in the Renfrew
shire sections.
The Hurlet in the north of Ayrshire has
dwindled to less than its thickness in the type section, and
the top of the Lower Limestone Series is marked by beds of
limey shale with Posidonomya corrugata, which have a very
striking resemblance to the shales and limestones of Calderwood Glen. We hope, however, to deal with this matter in
more detail next session.
Crossing now to the north side of the Clyde, we take the
Campsie district first, because there we find the clearest
sections. There are no sections so complete as that on the
Gryfe, but, in the numerous small burns, exposures occur which
can be pieced together. In the Memoir it is stated that the
Hollybush Limestone is not seen north of the Clyde, but we
6
See Trans. Geol. Soc. Glas. vol. xiv., part iii., page 309, " On t h e Dis
tribution of Posidonomya
corrugata, Ether. J u n . , in the Carboniferous
Limestone of t h e Glasgow District," by Peter Macnair, F . R . S . E . , F . G . S . ,
and Harry R. J. Conacher.
f
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on July 11, 2015
44
TRANSACTIONS—GEOLOGICAL
SOCIETY
OF
GLASGOW.
believe its exposures there are more plentiful than those of
any other beds.
South Brae of Campsie.—In Craigen Glen there is a section
of a series of shales which are famous for the variety of their
fossils.
These beds are correlated by the Survey with the
Blackbyre Seam (Productus Shales), but we are of opinion that
they represent the Hollybush horizon. The section, beginning
at the base, shows an oolite in fireclay-like beds overlain by
sandstone containing a thin coal.
The limey shales contain
several bands of calcareous ironstone nodules, which were
formerly mined; these nodules, as well as the shales, contain
producti and corals which, in some cases, have been completely
removed by percolating water, leaving very striking casts of
the calcareous parts of the organism. The section passes up
into sandstone and fireclay apparently underlying the Hurlet,
whose position is traceable, but the section at this point is
obscure.
In Glen Wynd, immediately to the west, the
Craigen Glen shales can be identified passing under sandstone
which is overlain by a limestone, consisting of beds of small
producti, with plant remains, and an encrinital upper part.
These characters at once suggest the Blackbyre Limestone of
Barrhead, and it also has an unconformity above it, as noted
at Nethercraigs. Above this unconformity is a peculiar deposit,
weathering with a characteristic red crust; if sampled near its
base it is found to be a hard sandstone with rootlets and
spirorbis, but the upper portions are distinctly limey, and in
other sections it is found to be a calmy limestone with many
entomostraca. It can be examined to advantage at the Linn
of Baldernock, where thin shale bands subdivide it, as is also
the case elsewhere.
In Glen Wynd this limestone is not well
seen, and the succeeding strata are also either worked out or
hidden from sight, with the exception of a black shale, baked
by a sill of white trap, whose identity is betrayed by its fauna
as the alum shale of Lennoxtown district. North-west of Glen
Wynd a small pine wood is fronted by extensive signs of old
workings, and at one point, where a small burn falls into the
old waste, a few feet of an encrinital limestone, with a few
small producti, is seen overlain by black shales with small
ironstones.
These old workings, with occasional exposures of
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on July 11, 2015
MACNAIR—LIMESTONES
I N THE GLASGOW DISTRICT.
45
the limestone, can be traced practically right round the hill,
and at Boyd's Burn and Culloch Slap work is still going on.
For a short distance to the south of the wood just mentioned
the tirring of old opencasts encumbers the ground, but above
an old mine mouth in the former bed of the stream we see
again the sandy base of the Baldernock Limestone, with its
line of unconformity, and it is possible to recognise below it
the same beds as seen in Glen Wynd. The structure in this
glen (Borraston) is complicated by a number of small faults,
which repeat part of the sequence.
At Baldernock Linn, or higher in the burn which forms it,
the following section is exposed: —
Black shale with small ironstone nodules.
Thin limestone, with small encrinites, &c, and pebbles of
dolomite. (Shields bed).
Black shales with small ironstone nodules.
Encrinital
t \ -i
• » • here,
i
~
- v bedj
• m t EN. i Limestone. 1 Dolerite
intrusion
seen in
Black Shale.
V
•> . *.
,of burn and in old quarry.
Entomostracan Limestone.
Sandy Limestone.
Unconformity.
Shales with plant remains and Lingula.
Encrinital Limestone.
c
u
A
This section is much distorted by the dolerite intrusion near
the linn, and a small dyke and sill of white trap also appear
higher up.
West of here, and near to the Auld Wives' Lifts, the sill
is again seen, with fragments of limestone, &c, sufficient to
identify it, and its outcrop across the slope to the south-east
is marked by the depression left by old workings. No doubt
this is the locality referred to in "The Geology of Campsie"
where the author speaks of an encrinital limestone passing
under the Craigmaddie Sandstones; in reality it is thrown
against them by a fault.
Passing now to the north face of the South Brae, we get
several glimpses of our encrinital limestone—sometimes accom
panied by the sill—in old workings. At Culloch Slap (above
Lennoxtown), where work is still going on, we see that this
limestone, 4 to 5 feet thick, overlies alum shale, coal, and
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on July 11, 2015
46
TRANSACTIONS—GEOLOGICAL
SOCIETY OP
GLASGOW.
entomostracan limestone, and in Baldow Glen we see the
position of these strata in the sequence.
Beginning at the
road to Lennox Castle, we see, in ascending order,
a Sandstones.
b Fossiliferous limey shales, w i t h nodules and some more solid b e d s
Productus giganteus (Craigen Glen beds). (Hollybush.)
c Sandstones and fire-clays.
d L i m e y shales crowded w i t h small Producti and t w o p o s t s of
limestone composed of small encrinite fragments (Glen W y n d
beds). (Blackbyre.)
e Shale w i t h Lingula.
f Unconformity.
g Sandy Limestone.
h Entomostracan Limestone (Baldernock).
i Fireclay.
j Gap. (Campsie Main Limestone worked out.) (Hurlet.)
Jc Black shales w i t h small ironstone nodules.
I Limestone band w i t h dolomitic pebbles, encrinites, &c. (Shields
bed.)
m Black shales w i t h large C.B. ironstones at top.
n Limestone, in five beds, top one encrinital, others entomostracan
and red crusted, partly oolitic, shale partings, one an oilshale (Baldow). (Blackhall.)
o Shale w i t h nodules (Posidonomya becheri bed at base).
The rest of this section shows only black shale and ironstone
nodules.
It is stated by the miners that in some parts of this district
there is a 14-inch coal below the entomostracan limestone
{Baldernock Limestone), but we have seen no trace of this.
In the new Memoir, reference is made to a blue limestone
cropping out in this glen, now no longer visible.
This we
suppose to be the limey shales, &c, which have yielded
Productus giganteus. The exposure is very small and obscured
by drift, but we identify it as the representative of the
Craigen Glen shales, and we feel no doubt also that the
productus shales with encrinital beds are the same as those
seen in Glen Wynd.
The upper part of the Baldernock Limestone here contains
a nodular brecciated band with rootlets, whose appearance at
once suggests that already described from Thorntonhall,
Gryfe, and Nethercraigs.
In Baldow, the Hurlet Coal and
Limestone are not seen, and the alum shale is barely visible,
but all can be examined in neighbouring workings.
In
€ulloch Slap and Boyd's Burn the alum shale contains numerous
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MACNAIR—LIMESTONES
I N THE GLASGOW DISTRICT.
47
limestone nodules which closely resemble, when seen lying
broken and weathered, the Baldernock Limestone, but they
contain no entomostraca, but frequently a marine fauna, and
are intricately brecciated like septarian nodules, which indeed
they are. The alum shale contains a varied fauna; at Culloch
Slap, Boyd's Burn, and Glen Wynd beds occur which are
crowded with Leppodomus costellatus, while others bear only
Lingula, small Producti, or Bellerophcm, and an occasional plant
print (Sph&nopteris?), and in parts fish remains and ento
mostraca are abundant.
At Boyd's Burn and Glen Wynd,
and in old bings east of Craigen Glen, we have found an
occasional Posidonomya corrugata (one specimen from each
locality).
In Baldow Glen, at the sharp bend above the Hurlet outcrop,
there is well exposed the striking bed which we refer to as the
" Shields Bed."
This bed is seen with approximately the
same features in the burn above Baldernock Linn, in diminished
size in the burn at Flutcher, in the wood between Langshot
and Newlands (in small burn at east side of wood), and in Glen
Whapple (north-west of Upper Carlston).
In the last-named
locality it has become partly oolitic and sandy enough to
contain annelid burrows. In the type locality (Shields Burn,
Milton of Campsie), it is a 3- to 4-feet bed of sandy encrinital
limestone; while it is constant in some form throughout this
district, the distance above the Hurlet Limestone varies.
The section of the Baldow Limestone in Baldow Glen is the
best in the district, but it is also seen in Glen Whapple, where
it has been wrought, and its position is marked by old workings
and numerous fine fragments at the mouth of Craigen Glen,
to which position it is brought by a fault.
On the South Hill, no higher strata than the Hosie Sandstone
•occur, and this is to be seen in one or two small, old quarries
south of Clochcore and at Newlands.
7
7
I n the alum shale which comes between the Cobbinshaw limestone and
coal at Cobbinshaw and a t W e s t Mains there occurs an exactly similar
fauna t o t h a t j u s t described, and i t is certainly very striking t h a t in t w o
places so widely separated there should be found such a close resemblance
i n t h e strata and their contents ; in both districts the descending sequence
i s encrinital limestone, alum shale, pyritous coal, fire-clay, and entomostracan limestone.
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on July 11, 2015
48
TRANSACTIONS
GEOLOGICAL
SOCIETY OF
GLASGOW.
North
Brae of Campsie,
&c.—Leaving
the South
Hill and crossing to Balgrochan, north of Lennoxtown,
we find 4 to 5 feet of encrinital limestone capped by
many feet of flaggy limestone, with pecten, productus,
&c, and limey shales, a state of affairs similar to that
at Nethercraigs, Gryfe, and Howood. Below this seam occurs
the alum shale and coal, then the Baldernock Limestone, and
a long section of the Glen Wynd Limestone is found in burns
at Balgrochan and east of Bencloich Mains.
In the burn at
the latter place the Shields bed can be seen in its more typical
form, and higher up the Baldow Limestone comes in. Above
it at this point the shales contain an interesting fauna, not
yet fully examined, including Posidonomya becheri (first noticed
in this position by Mr. Reoch at Corrie Burn), orthoceras, and
nautilus. This fauna we have also found in the same position
in Baldow Glen and at Shields Cottage, but where the lime
stone crops in Glen Whapple the shales are too much crushed
by an adjacent fault to yield fossils. The Baldow Limestone
is also exposed in the Bught Glen at Maiden Castle, where it
is curious to note that the burn has sunk its ravine many feet
right through that structure.
In the burn east of Shields Farm we get our first sight of
the Hosie Limestone, where it is of a cement-like nature in
two bands about 8 feet apart, accompanied by black shales
and one or two ironstone and kingle ribs.
A few feet above
the upper limestone the Posidonomya corrugafebeds
occur in
good form.
The group is not again seen .early until we
reach the Corrie district, but there the burn west of Burnhead
gives an excellent section.
Above the Hosie Limestone along the south face of the
Campsie Hills between Corrie and Spouthead there occurs a.
mass of sandstone alternating with shales and fakes, the alter*nation being repeated several times with remarkable regularity.
Near the top of the series there occurs a sandstone with annelid
burrows and Spirophytan
caudagalli and small encrinities,
and the latter bed is also to be seen near Burnhead.
At Corrieburn there is a very interesting section, which
passes from the zone of the volcanic detritus, with rough
conglomerates and fine muds to the strata above the Upper
Downloaded from http://trngl.lyellcollection.org/ at West Virginia University
on July 11, 2015
Vol. XV., Plate VI.
Trans. Geol. Soc. of Glasgow.
Fig. 4.
Plan and section across Limestones at Corrieburn, K i l s y t h .
Lettering same as Table in t e x t , see page 49.
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MACNAIR—LIMESTONES
I N THE GLASGOW DISTRICT.
49
Hosie Limestone. The details of the section as observed by
us differ from those given in the Survey Memoir, and we set
them out side by side below—
Survey
Memoir.
Black oily shale w i t h Lingula.
Limestone w i t h Bellerophon (Hosie
Limestone).
Strata w i t h thick yellow sandstone
(Hosie Sandstone).
Black shale w i t h septarian ironstone
nodules. Marine fossils near base.
Limestone, partly marine w i t h encrinites, partly dolomitic and
oolitic w i t h entomostraca (Black
hall).
Strata—black shales and sandstones
w i t h I . S . band.
Hurlet
j Upper hard band,
-r«
,
i Calmy band.
Limestone. |
/
L
o
w
r
h
a
r
d
b
a
n
d
Large growth forms of brachiopods
and mollusca.
Strata w i t h thin coal.
Coral limestone.
Black shales, sandstone bands, iron
stones and calcareous n o d u l e s ;
w i t h alternations of purplish
volcanic
detritus and
rolled
fragments.
Lingula
in shales
and volcanic muds.
Thick volcanic muds and con
glomerates resting on lavas (pp.
15 and 24 of Memoir).
Present
Authors.
z Posidonomya
corrugata beds and
Lingula shale.
y Black limestone w i t h Bellerophon.
x Black shales w i t h
Leptodomus
costellatus, Pinna, &c.
w Limey shale.
v Black limestone w i t h Bellerophon
and Orthoceras.
u Shale.
t Sandstone.
s Shale with nodules ( P . becheri
bed at base).
r Encrinital limestone w i t h small
solitary corals.
q Brown - crusted
entomostracan
limestone.
p Sandstone, flaggy and ripplemarked, limey and oolitic in
parts.
0 Limey shales w i t h large encrinites
and Polyzoa.
n Encrinital limestone (Hurlet).
m Black shale w i t h
Leptodomus
costellatuSy and small ironstone
nodules.
1 Coal.
k Fire-clay.
j Nodular green limestone w i t h
corals.
i Coral limestone, eroded in places
(Hollybush).
h Limey shales w i t h
Rhynconella
and nodules containing corals
and brachiopods.
g Shale with ironstone.
/Naiadites
crassa bed.
e Sphenopteris (?) bed.
d Beds containing young of Naia
dites crassa.
c Shales grading down into volcanic
mud w i t h oolitic ironstone
nodules.
b Sandstone.
a Muds and conglomerate.
The Hurlet Limestone appears to be correctly identified
here, and the overlying beds at once betray their identity,
but below the coal there is difficulty. We are inclined to think
that here the line of erosion, elsewhere noted above the Glen
VOL. XV., PT. I.
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50
TRANSACTIONS—GEOLOGICAL
SOCIETY
OF
GLASGOW.
Wynd Limestone, has cut out that seam, and that the coral
limestone and the accompanying very fossiliferous beds are
equivalent to the Craigen Glen shales.
East of Corrie there are only one or two unimportant,
isolated exposures of the Baldow Limestone at Berryhill, hints
of it at Linns, while the Hosie is seen at Drumnessie.
At North Bardowie, the Hurlet group can be traced by old
workings, at Flutcher the Shields bed is seen, and at Dowans
there is an excellent section of the Craigen Glen beds involved
in a dolerite intrusion, the intervening strata being hidden.
West of Milngavie, the Hurlet group is not seen, but old
workings mark its outcrop.
At Hardgate Quarry, in the burn at Duntocher Mill and at
Auchentoshan and on and near the golf course at Lawmuir,
are exposures of what is evidently the same calcareous series,
containing corals and large producti.
The character of these
beds, their relation to the Hurlet outcrop, and the nature of
the adjacent strata (sandstone with thin coal above and below),
cause us to regard them as the equivalent of the Craigen Glen
beds.
From the foregoing, it will be seen that throughout the
Glasgow district the limestones are remarkably constant in
occurrence, and that those modifications which take place can
be traced in their development from one locality to another,
showing that the Blackhall, Hurlet, Blackbyre, and Hollybush
Limestones of the type locality have their counterparts north
of the Clyde in the district stretching from Kilsyth to Dun
tocher in the Baldow, Campsie Main, Glen Wynd, and Craigen
Glen beds respectively.
| 5,214 | 30,334 |
W4294190135.txt | Journal of Educational
and Psychological Sciences
مجلة العلوم
التربوية والنفسية
Volume (6), Issue (36): 30 Aug 2022
P: 57 - 73
م2022 أغسطس30 :)36( العدد،)6( املجلد
73 - 57 :ص
ISSN: 2522- 3399
The obstacles which are facing mathematics teachers in activating
professional learning communities from their point of view
Fahad Ibrahim Mokli
Ahmed Abdullah Zaylaee
Sabya Education Department || Ministry of Education || KSA
Abstract: The study aimed to identify the obstacles facing mathematics teachers in activating professional learning
communities in Sabya's Educational directorate. The questionnaire was used in the study. which was consisting of three
dimensions. (administrative obstacles, physical obstacles, and personal obstacles), the number of study sample members
reached (716) teachers. the study found a set of obstacles, most notably: high number of classes given to the teacher, lack of
training programs on professional learning communities, and teachers' low conviction in the process of change and
development in their performance. The study also concluded that there were no statistically significant differences at the
level (0.05) between the opinions of the sample members about the three dimensions of the study due to years of
experience. The study recommended a number of recommendations.
Keywords: Obstacles, professional learning communities, mathematics teachers.
ً
املعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات التعلم املهنية وفقا آلرائهم
فهد بن إبراهيم موكلي
أحمد بن عبد هللا زيلعي
إدارة التعليم بمحافظة صبيا || وزارة التعليم || اململكة العربية السعودية
هدفت الدراسة إلى التعرف على املعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات التعلم املهنية بإدارة تعليم:املستخلص
املعيقات الشخصية) وبلغت عينة، املعيقات املادية، وقد تم استخدام استبانة مكونه من ثالثة محاور وهي (املعيقات اإلدارية،صبيا
ً )716( الدراسة
قلة البرامج التدريبية حول، ارتفاع أنصبة املعلمين: وقد توصلت الدراسة إلى مجموعة من املعوقات من أبرزها،معلما
كما توصلت الدراسة إلى أنه ال توجد فروق ذات. ضعف قناعة املعلمين بعملية التغيير والتطوير في أدائهم،مجتمعات التعلم املهنية
وقد أوصت الدراسة،) بين آراء أفراد العينة حول محاور الدراسة الثالثة تعزى إلى سنوات الخبرة0.05( داللة إحصائية عند مستوى
.بمجموعة من التوصيات
معلمي الرياضيات- مجتمعات التعلم املهنية- معيقات:الكلمات املفتاحية
. املقدمة-1
من،فرضت التغيرات املتسارعة في شتى مناحي الحياة إحداث مدخالت تطويرية حديثة في العملية التعليمية
ً
ً عامال
واالهتمام بتحقيق الجودة في أداء،مؤثرا في تحسين مخرجات التعليم
شأنها تطوير األداء املدرس ي الذي يعد
. وبما يمكن من مواكبة التغيرات،املعلمين
DOI: https://doi.org/10.26389/AJSRP.H030122
)57(
Available at: https://www.ajsrp.com
المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس 2022م
ويستلزم تطوير التعليم االهتمام بالتنمية املهنية املستدامة للمعلمين وتحسين ثقافتهم املهنية واألكاديمية
من خالل تدريبيهم على سد الفجوات بين واقع ممارساتهم التعليمية وبين متطلبات النظم الحديثة لإلصالح املدرس ي،
والتجديدات التربوية (السليمي والزلفي.)2019 ،
وتعد مجتمعات التعلم املهنية إحدى املدخالت التطويرية الحديثة في التعليم واإلصالح املدرس ي التي تتبنى
التنمية املهنية املستدامة للمعلمين من خالل تزويدهم باملعارف واملهارات ،وتحسين ممارساتهم التدريسية ،وزيادة
ً
إيجابيا من خالل استخدام عدة استراتيجيات فعالة ،كما تحد من العمل الفردي بين
فاعليتهم ،والتأثير عليهم
املعلمين ،وتتغلب على ثقافة االنعزال ،األمر الذي يؤدي إلى تطوير األداء املدرس ي (حسن بت2016 ،؛ إبراهيم
وحسان2018 ،؛ البرعمي وعبد الرشيد2020 ،؛ .)Herrington, 2011
وتقوم فكرة مجتمعات التعلم املهنية على تشكيل مجموعات تخصصية تجتمع باستمرار لتبادل األفكار،
وتحسين التدريس (الزايد وعمر .)2016 ،كما جاءت متمحورة حول زيادة املعرفة املهنية للمعلمين من خالل
اندماجهم وتفاعلهم في هذه املجتمعات وتأملهم للخبرات واملمارسات اليومية التي يتشاركونها فيما بينهم (شركة تطوير
للخدمات التعليمية.)2014 ،
وتدعم مجتمعات التعلم املهنية املناخ املدرس ي والثقافة التنظيمية ،باإلضافة إلى أنها تدمج املعلمين في
املشروعات واألنشطة التعاونية ،وتزيد من قدراتهم وإمكاناتهم على العمل ،مما يسهم في وجود بيئة مدرسية منتجة،
وتحسين عملية التعليم والتعلم (.)Kalkan, 2016
وتهدف مجتمعات التعلم املهنية إلى مساعدة املعلمين ليكونوا أكثر فاعلية في عملهم مع الطالب ،وبالتالي
التركيز على التعلم والنتائج ،وكذلك دعم زمالئهم واالهتمام باملمارسات التدريسية وتأملها ونقدها وذلك من خالل بناء
ثقافة تعزيز التعاون بين العاملين في املدرسة (.)Battersby & Verdi, 2015
ً
وتعرف مجتمعات التعلم املهنية على أنها مجاال لتطوير األداء املدرس ي ،يركز على تمكين املدرسة من تحديد
املشكالت والتحديات التي تقابلها والقدرة على مواجهتها بفاعلية ،وذلك من خالل تحويل املدرسة إلى مجتمعات تعلم
مهنية لها أنشطة تستهدف تنمية قدرات املعلمين وتحسين مستويات التعلم لدى طالبهم (إبراهيم واملرزوقي.)2018 ،
إن االفتراض الذي تقوم عليه مجتمعات التعلم املهنية هو ارتباط تحسين تعلم الطالب بالتنمية املهنية
املستدامة ملعلميهم) . (Dufour et. al, 2016وقد أكدت دراسة الصالحية والهاشم ( )2018على دور مجتمعات التعلم
املهنية في رفع املستوى التحصيلي للطالب.
وتناولت األدبيات مجتمعات التعلم املهنية ،وأكدت على االهتمام بها ،والتركيز عليها ،وأشارت إلى أهميتها،
ً
اتجاها ع ً
امليا إلصالح التعليم في كثير من الدول :كأمريكا ،وبريطانيا ،وفرنسا،
حيث أصبحت مجتمعات التعلم املهنية
وأملانيا ،والصين (ناصف.)2012 ،
كما تؤكد دراسة دوقان ( )Dogan et al, 2017أن مجتمعات التعلم املهنية من الركائز الرئيسية في
مشروعات التطوير املنهي للمعلمين في كثير من دول العالم حيث تؤثر بصورة إيجابية في تنمية قدرات املعلمين
ومهاراتهم وتركز على جهود التعاون والرؤية املشتركة بينهم.
وقد وقفت اململكة العربية السعودية على عدد من التجارب الناجحة ،واالستعانة بالعديد من الدراسات
العاملية التي تناولت األنظمة التربوية والتعليمية (املطيري .)2018 ،واحتلت مجتمعات التعلم املهنية ً
حيزا ً
كبيرا ضمن
ً
ً
رئيسيا في نموذج تطوير
ومنطلقا
تطوير التعليم في اململكة العربية من خالل مشروع امللك عبد هللا لتطوير التعليم،
املدارس ،وداعمة لعمليتي التعليم والتعلم (أنموذج تطوير املدارس.)1436 ،
موكلي ،زيلعي
()58
المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات
التعلم المهنية وفقًا آلرائهم
المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس 2022م
وقد أجريت العديد من الدراسات العربية واألجنبية حول مجتمعات التعلم املهنية ،ومنها دراسة سونق
( )Song,2012التي توصلت إلى أن تطبيق مجتمعات التعلم املهنية يساعد املدارس على تحسين التعلم ،وعلى جعل
املعلمين أكثر تقبال لإلصالحات التربوية ،كما توصلت دراسة فولر ( )Fuller, 2014إلى األثر اإليجابي ملجتمعات التعلم
املهنية على ممارسات املعلمين ،وكذلك على التحصيل الدراس ي للطالب ،وتوصلت دراسة األخناوي ( )2016إلى ضعف
تطبيق مجتمعات التعلم املهنية ،وأن هناك معوقات تحول دون تفعيلها ،كما توصلت دراسة محمد ( )2019إلى دور
مجتمعات التعلم املهنية في تحسين األداء األكاديمي في مدارس التعليم العام ،وقد أوصت هذه الدراسات بضرورة
تفعيل مجتمعات التعلم املهنية ،وتذليل الصعوبات التي تعيق تفعيلها.
وقد اقتصر الباحثان في هذه الدراسة على معلمي الرياضيات؛ كون الرياضيات مجال تخصص الباحثين.
ويمكن تحقيق مجتمعات التعلم املهنية ملعلمي الرياضيات من خالل تشكيل مجتمع التعلم املنهي ملعلمي
الرياضيات ،وتحديد املعلم املشرف ،وإعطاء الوقت الجتماع املعلمين فيما بينهم ،وبناء الخطة التطويرية لتنمية
معرفتهم بمحتوى الرياضيات وطرق تدريسه ،حيث إن إتاحة الفرصة لتطوير معرفة معلمي الرياضيات وممارساتهم
التدريسية وفق احتياجاتهم ،ومعالجة القصور لديهم يؤثر في تعلم الطالب ،وبالتالي في تحصيلهم (موكلي.)2019 ،
قصورا واضحاً
ً
وبالرغم من أهمية مجتمعات التعلم املهنية في تحسين تعليم وتعلم الرياضيات إال أن هناك
في تفعيلها من قبل معلمي الرياضيات في الوقت الذي يؤكد املشروع الوطني لتطوير التعليم في اململكة العربية
السعودية على ضرورة تطبيق مجتمعات التعلم املهنية لنجاح عملية تطوير األداء املدرس ي ،وتحسين نتائج الطالب،
لذلك كان من األهمية بمكان القيام بدراسة للتعرف على املعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات
التعلم املهنية.
مشكلة الدراسة:
تسهم مجتمعات التعلم املهنية في تطوير كفايات املعلمين ورفع أدائهم التدريس ي وتحسين عمليات التعليم
والتعلم ،كما تعد ر ً
افدا للمدرسة في التنبؤ بمشكالتها املستقبلية ،وقدرتها على التحسين والتطوير.
وقد أكدت نتائج دراسة (هلل ،)2013 ،ودراسة بيرتش ) (Bertsch,2013أن مجتمعات التعلم املهنية تسهم
في التقليل من عزلة املعلم ،وتزيد من التزامه بمهامه وتحقيق أهداف املدرسة .كما أكدت دراسة (الزايد )2018 ،على
ً
إيجابيا باملمارسات التأملية في مجتمعات التعلم
أن الجانب املعرفي ،واملمارسات الصفية ،واملعتقدات التربوية تأثرت
املهنية .وتؤكد دراسة محروس ( )2015أن مجتمعات التعلم املهنية استراتيجية فعالة تغيير وتطوير املدارس ،ومدخل
قوي لإلصالح املدرس ي املنشود.
وقد أكدت دراسة كل من :الحازمي ( ،)2016وناصف ( ،)2012على أهمية تفعيل مجتمعات التعلم املهنية
داخل املدرسة ،وأوصت دراسة (العجالن )2016 ،بتعميم تجربة مجتمعات التعلم املهنية على جميع املدارس باململكة
العربية السعودية ،وأوصت دراسة (الزايدي )2013 ،بنشر ثقافة تحول املدارس إلى مجتمعات تعلم مهنية من خالل
تصميم برامج تدريبية لجميع منسوبي املدارس في السعودية .وتؤكد دراسة توفيق ( )2017على أهمية تفعيل
مجتمعات التعلم املهنية لتحقيق اإلصالح والتطوير التعليمي.
وبالرغم من أهمية مجتمعات التعلم املهنية ودورها في عمليتي التعليم والتعلم إال أن الباحثين من خالل
عملهما مشرفين تربويين في وحدة تطوير املدارس ،ومتابعتهما لتطبيق مجتمعات التعلم املهنية الحظا أن هناك ً
ضعفا
في تفعيل مجتمعات التعلم املهنية لدى املعلمين ،وكذلك ما توصلت له الدراسات السابقة من وجود ضعف في
موكلي ،زيلعي
()59
المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات
التعلم المهنية وفقًا آلرائهم
المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس 2022م
تفعيل مجتمعات التعلم املهنية ،ووجود معيقات تحول دون تفعيلها ،وبذلك يمكن تحديد مشكلة الدراسة في محاولة
التعرف على املعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات التعلم املهنية ً
وفقا آلرائهم.
أسئلة الدراسة:
بناء على ما سبق؛ تتحدد مشكلة هذه الدراسة في السؤال الرئيس اآلتي:
ً
ما املعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات التعلم املهنية وفقا آلراء املعلمين؟
ويتفرع منه األسئلة التالية:
ً
-1ما املعيقات اإلدارية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية وفقا آلرائهم؟
-2ما املعيقات املادية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية ً
وفقا آلرائهم؟
-3ما املعيقات الشخصية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية ً
وفقا آلرائهم؟
-4هل توجد فروق ذات داللة احصائية عند مستوى ( )α≤0.05بين آراء معلمي الرياضيات حول املعيقات التي
تواجه معلمي الرياضيات في تفعيل مجتمعات التعلم املهنية تعزى للخبرة التدريسية؟
أهداف الدراسة
سعت الدراسة لتحقيق األهداف التالية:
ً
.1التعرف على املعيقات اإلدارية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية وفقا آلراء
املعلمين.
.2التعرف على املعيقات املادية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية ً
وفقا آلراء
املعلمين.
ً
.3التعرف على املعيقات الشخصية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية وفقا آلراء
املعلمين.
.4الكشف عن وجود فروق ذات داللة احصائية عند مستوى ( (α≤0.05بين آراء معلمي الرياضيات حول
املعيقات التي تواجههم في تفعيل مجتمعات التعلم املهنية تعزى للخبرة التدريسية.
أهمية الدراسة
تكمن أهمية الدراسة في أنها قد تفيد في:
تقديم تغذية راجعة للمسئولين في وزارة التعليم باململكة العربية السعودية للتعرف على املعيقات التي تواجهمعلمي الرياضيات في تفعيل مجتمعات التعلم املهنية.
االسهام في تقديم الحلول لتالفي املعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات التعلم املهنية.حدود الدراسة.
اقتصرت الدراسة على الحدود اآلتية:
ً
الحدود املوضوعية :املعيقات التي تحول دون تفعيل معلمي الرياضيات ملجتمعات التعلم املهنية وفقا آلرائهم.
الحدود املكانية :املدارس الحكومية للبنين في إدارة تعليم صبيا.
الحدود الزمانية :طبقت الدراسة في الفصل الدراس ي الثاني من العام الدراس ي 1442 -1441هـ
موكلي ،زيلعي
()60
المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات
التعلم المهنية وفقًا آلرائهم
المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس 2022م
مصطلحات الدراسة
مجتمعات التعلم املهنية :هي مجموعات تخصصية يشكلها مدير املدرسة ويقوم بدعمها ،ويتم فيها بناء الخطةالتطويرية للمجموعة ،وتبادل التجارب بين أفرادها ،وتشارك الخبرات املهنية ،وتأمل املمارسات التدريسية
ونقدها ،ومناقشة املحتوى الرياض ي ،وتقويم الطالب.
-2منهجية الدراسة وإجراءاتها.
منهج الدراسة:
ً
نظرا لهدف الدراسة وهو التعرف على املعيقات التي تحول دون تفعيل معلمي الرياضيات ملجتمعات التعلم
املهنية .فقد تم استخدام املنهج الوصفي املسحي منهجا للدراسة ملالءمته لهدفها.
مجتمع الدراسة:
ً
تألف مجتمع الدراسة من جميع معلمي الرياضيات في مدارس إدارة تعليم صبيا والبالغ عددهم ( )716معلما
للعام الدراس ي 1440 -1439هـ.
عينة الدراسة:
ً
شملت عينة الدراسة ( )279معلما ،أي ما يمثل ( )%39من مجتمع الدراسة ،حيث تم االختيار بالطريقة
العشوائية.
توزيع أفراد العينة:
أ -توزيع أفراد العينة ملتغير سنوات الخبرة التدريسية
جدول ( )1عدد أفراد العينة وفقا ملتغير سنوات الخبرة التدريسية
النسبة املئوية
التكرار
سنوات الخبرة التدريسية
%7.53
21
أقل 5سنوات
%8.60
24
من 10 -6سنوات
%83.87
234
أكثر من 10سنوات
%100
279
املجموع
ً
يتضح من الجدول ( )1توزيع أفراد العينة وفقا ملتغير عدد سنوات الخبرة التدريسية ،حيث يظهر أن عدد
أفراد العينة التي تتراوح خبرتهم أقل من 5سنوات ( )21ويمثلون نسبة ( )%7.53من إجمالي أفراد العينة ،ويبلغ عدد
أفراد العينة الذين تتراوح خبرتهم من 10 -6سنوات ( )24ويمثلون نسبة ( )%8.60من إجمالي أفراد العينة ،بينما يبلغ
عدد أفراد العينة التي تزيد خبرتهم التدريسية عن 10سنوات ( )234ويمثلون نسبة ( )%83.87من إجمالي أفراد
العينة.
ب -توزيع أفراد العينة وفقا ملتغير املؤهل
جدول ( )2عدد أفراد العينة وفقا ملتغير املؤهل
النسبة املئوية
التكرار
املؤهل
%96.77
270
بكالوريوس
موكلي ،زيلعي
()61
المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات
التعلم المهنية وفقًا آلرائهم
المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس 2022م
النسبة املئوية
التكرار
املؤهل
%3.23
9
الشهادات العليا
%100
279
املجموع
يتضح من جدول ( )2توزيع أفراد عينة الدراسة وفقا ملتغير عدد املؤهل العلمي ،حيث يتبين أن ( )270من
العينة الذين يمثلون ما نسبته ( )96.77من إجمالي عينة الدراسة كان مؤهلهم بكالوريوس ،في حين أن ( )9من العينة
الذين يمثلون ما نسبته ( )3.23من إجمالي العينة كان مؤهلهم دراسات عليا.
أداة الدراسة:
استبانة
الهدف :التعرف على املعيقات التي تحول دون تفعيل معلمي الرياضيات ملجتمعات التعلم املهنية.
إجراءات بناء االستبانة:
-1قراءة الدراسات السابقة التي تناولت مجتمعات التعلم املهنية.
-2تحديد محاور االستبانة والتي تمثل معوقات مجتمعات التعلم املهنية .وهي (إدارية ،مادية ،شخصية).
-3كتابة مجموعة من العبارات املناسبة لكل محور.
-4عرض االستبانة على مجموعة من الخبراء في مجال تعليم الرياضيات.
-5كتابة األداة بالصورة النهائية .حيث جاءت بالشكل التالي:
املحور األول :املعوقات اإلدارية وتضمن ( )10عبارة. املحور الثاني :املعوقات املادية وتضمن ( )11عبارات. املحور الثالث :املعوقات الشخصية وتضمن ( )9عبارة.صدق االستبانة وثباتها
ً
أوال :صدق االستبانة
للتعرف على صدق أداة الدراسة ،فقد تم عرضها على مجموعة من أعضاء هيئة التدريس املتخصصين في
املناهج وطرق تدريس الرياضيات ومجموعة من املشرفين التربويين ومجموعة من الباحثين في موضوع مجتمعات
ً
التعلم ،وفي ضوء آراء املحكمين أعيدت صياغة بعض الفقرات لغويا ،وعدلت بعض الفقرات ،وتم اعتماد الفقرات
التي كان معدل اتفاق املحكمين حولها ( )%90فما فوق.
ً
ثانيا -ثبات االستبانة
للتأكد من ثبات االستبانة ،قام الباحثان بحساب معامالت ارتباط درجة كل عبارة باملحور الذي تنتمي له،
وذلك باستخدام معامل ارتباط (بيرسون) كما يوضحه الجدول (:)3
جدول ( :)3درجة ارتباط كل عبارة باملحور الذي تنتمي له
معامل االرتباط
معامل االرتباط
معامل االرتباط باملحور
املفردة
املفردة
املفردة
باملحور
باملحور
0.580
22
0.705
11
0.561
1
0.803
23
0.795
12
0.792
2
موكلي ،زيلعي
()62
المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات
التعلم المهنية وفقًا آلرائهم
المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس 2022م
معامل االرتباط
معامل االرتباط
املفردة
املفردة
باملحور
باملحور
0.796
24
0.822
13
0.826
3
0.679
25
0.858
14
0.810
4
0.814
26
0.804
15
0.811
5
0.848
27
0.711
16
0.859
6
0.795
28
0.897
17
0.866
7
0.563
29
0.852
18
0.832
8
0.718
30
0.781
19
0.715
9
0.719
20
0.493
10
0.766
21
يتضح من الجدول ( )3أن جميع معامالت ارتباط العبارات مع محاورها دالة إحصائيا عند مستوى داللة
( ،)0.01حيث تراوحت ملحور املعوقات اإلدارية بين ( ،)0.866 -0.493بينما تراوحت للمعوقات املادية بين (-0.705
،)0.897وتراوحت للمعوقات الشخصية بين ( ،)0.848 -0.563مما يدل على ارتباط كل عبارة من عبارات االستبانة
باملحور الذي تنتمي له ،مما يعني توافر درجة عالية من ثبات عبارات لالستبانة.
كما يوضح جدول ( )4حساب معامل ألفا كرونباخ للثبات:
جدول ( :)4قيم ألفا كرونباخ لثبات محاور االستبانة
قيمة ثبات املحاور
عدد العبارات
محاور االستبانة
املحور األول
0.94
10
0.95
11
املحور الثاني
0.92
9
املحور الثالث
0.96
30
الثبات العام ملحاور الدراسة
يتضح من جدول ( )4أن معامل الثبات ملحاور األداة (االستبانة) مرتفع ،حيث يتراوح بين ( )0.95~0.92تم
حساب معامل ألفا كرونباخ ،حيث بلغت نسبة ثبات األداة ( .)0.96وهذا يدل على أن األداة تتمتع بدرجة عالية من
الثبات ،ويمكن االعتماد عليها والوثوق بنتائج الدراسة.
املفردة
معامل االرتباط باملحور
-3عرض النتائج ومناقشتها.
النتائج املتعلقة بالسؤال الفرعي األول" :ما املعيقات اإلدارية التي تحد معلمي الرياضيات من تفعيل مجتمعات
التعلم املهنية ً
وفقا آلرائهم؟
ولإلجابة تم حساب التكرارات والنسب املئوية واملتوسطات الحسابية واالنحرافات املعيارية لكل مفردة من
مفردات األداة والجدول ( )5التالي يوضح تلك النتائج.
موكلي ،زيلعي
()63
المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات
التعلم المهنية وفقًا آلرائهم
المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس 2022م
جدول ( :)5املعيقات اإلدارية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية
أوافق بشدة
%
ك
م
العبارة
1
يبني املدير خطة
املدرسة دون إشراك
املعلمين
2
3
4
5
6
7
8
9
1
0
غياب دعم مدير
املدرسة إلتاحة الوقت
املناسب الجتماع
املعلمين
غياب دعم مدير
املدرسة إلتاحة املكان
املناسب الجتماع
املعلمين
غياب العمل من قبل
مدير املدرسة على
تشكيل فرق مجتمعات
التعلم املهنية
ضعف تهيئة مدير
املدرسة للمناخ املالئم
لعقد الحوارات
واملناقشات الهادفة
ضعف متابعة مدير
املدرسة لعمل فرق
مجتمعات التعلم املهنية
ضعف قناعة مدير
املدرسة بأهمية
مجتمعات التعلم املهنية
ودورها في تحسين
عملية التعلم
ضعف العالقة بين
مدير املدرسة واملعلمين
في املدرسة
ضعف العالقة بين
املعلمين داخل املدرسة
قلة البرامج التدريبية
حول مجتمعات التعلم
املهنية
72
أوافق
%
ك
54
25.81
19.35
إلى حد ما
%
ك
60
21.51
ال أوافق
%
ك
66
23.66
ال أوافق أبدا
%
ك
27
9.68
املتوسط
الحسابي
2.72
االنحراف
املعياري
0.791
الترتيب
10
45
16.13
39
13.98
54
19.35
87
31.18
54
19.35
3.24
0.835
6
30
10.75
54
19.35
69
24.73
75
26.88
51
18.28
3.23
0.590
7
42
15.05
45
16.13
39
13.98
102
36.56
51
18.28
3.27
0.807
5
45
16.13
36
12.90
54
19.35
84
30.11
60
21.51
3.28
0.818
4
48
17.20
33
11.83
66
23.66
81
29.03
51
18.28
3.19
0.810
8
30
10.75
57
20.43
48
17.20
81
29.03
63
22.58
3.32
0.743
3
39
13.98
36
12.90
48
17.20
81
29.03
75
26.88
3.42
0.838
2
33
11.83
57
20.43
78
27.96
66
23.66
45
16.13
3.12
0.562
9
66
23.66
81
29.03
72
25.81
39
13.98
21
7.53
3.53
0.769
1
3.23
0.48
املتوسط الحسابي العام
يتضح من جدول ( )5ما يلي:
موكلي ،زيلعي
()64
المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات
التعلم المهنية وفقًا آلرائهم
المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس 2022م
-1
-2
-
-
-3
-4
هناك تقارب في استجابات عينة الدراسة ملحور املعيقات اإلدارية التي تحد معلمي الرياضيات من تفعيل
مجتمعات التعليم املهنية ،حيث تراوحت متوسطات استجاباتهم على املحور األول ما بين (2.72إلى )3.53وهي
متوسطات تقع في الفئتين :الثالثة (متوسط) والرابعة (مرتفع) من درجات املقياس املتدرج الخماس ي املشار
ً
إليه سابقا (في أداة الدراسة) ،ويالحظ أن متوسط املوافقة العام على فقرات املحور بلغ ( 3.23درجة من )5
مما يعني أن هناك (موافقة متوسطة) من أفراد العينة على عبارات هذا املحور.
ً
ً
ترتيب عبارات هذا املحور حسب درجة املوافقة عليها ترتيبا تنازليا كما يلي:
كانت استجابات أفراد العينة على الفقرة رقم ( )10وهي( :قلة البرامج التدريبية حول مجتمعات التعلم
املهنية) باملرتبة األولى بين الفقرات بمتوسط الحسابي ( ،)3.53وهذا يعني أن هناك موافقة بين أفراد العينة
أن من أهم املعيقات اإلدارية هي( :قلة البرامج التدريبية حول مجتمعات التعلم املهنية).
جاءت استجابات أفراد العينة على الفقرات املتبقية )8( :و ( )7و ( )5و ( )4و ( )2و ( )3و ( )6و ( )9و ()1
بمتوسطات حسابية على التوالي،)3.12( ،)3.19( ،)3.23( ،)3.24( ،)3.27( ،)3.28( ،)3.32( ،)3.42( :
()2.72؛ وهذا يعني أن هناك موافقة بدرجة (مرتفعة) للفقرة ( )8وموافقة متوسطة للفقرات (،2 ،4 ،5 ،7
)1 ،9 ،6 ،3بين أفراد العينة على تلك املعيقات؛ ومن ثم تعد تلك الفقرات معيقات بدرجة متوسطة.
ً
ترتيب املعيقات بحسب درجة تحققها وفقا لآلتي:
قلة البرامج التدريبية حول مجتمعات التعلم املهنية.
ضعف العالقة بين مدير املدرسة واملعلمين في املدرسة.
ضعف قناعة مدير املدرسة بأهمية مجتمعات التعلم املهنية ودورها في تحسين عملية التعلم.
ضعف تهيئة مدير املدرسة للمناخ املالئم لعقد الحوارات واملناقشات الهادفة.
غياب العمل من قبل مدير املدرسة على تشكيل فرق مجتمعات التعلم املهنية.
غياب دعم مدير املدرسة إلتاحة الوقت املناسب الجتماع املعلمين.
غياب دعم مدير املدرسة إلتاحة املكان املناسب الجتماع املعلمين.
ضعف متابعة مدير املدرسة لعمل فرق مجتمعات التعلم املهنية.
ضعف العالقة بين املعلمين داخل املدرسة.
يبني املدير خطة املدرسة دون إشراك املعلمين.
يالحظ أن هناك اتفاقا بين أفراد العينة في أن قلة البرامج التدريبية حول مجتمعات التعلم املهنية للمعلمين
يعد معيقا بدرجة مرتفعة؛ وهذا معيق كبير وال شك ،فقلة البرامج التدريبية حول مجتمعات التعلم املهنية ال
يتيح تفعيل مجتمعات التعلم املهنية بالصورة املطلوبة.
النتائج املتعلقة بالسؤال الفرعي الثاني" :ما املعيقات املادية التي تحد معلمي الرياضيات من تفعيل مجتمعات
التعلم املهنية ً
وفقا آلرائهم؟
ولإلجابة تم حساب التكرارات والنسب املئوية واملتوسطات الحسابية واالنحرافات املعيارية لكل مفردة من
مفردات األداة والجدول ( )6التالي يوضح تلك النتائج.
موكلي ،زيلعي
()65
المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات
التعلم المهنية وفقًا آلرائهم
المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس 2022م
جدول ( :)6املعيقات املادية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية
م
1
2
3
4
العبارة
أوافق بشدة
ك
نقص الكوادر املؤهلة
علميا ومهنيا لقيادة
84
أنشطة مجتمعات التعلم
املهنية داخل املدرسة
ارتفاع أنصبة املعلمين
183
التدريسية
زيادة األعباء غير
التدريسية على املعلم
159
غياب التجهيزات واملرافق
141
في املدرسة لتفعيل
مجتمعات التعلم املهنية
ضعف الحوافز املادية
168
واملعنوية لتفعيل
مجتمعات التعلم املهنية
ضعف الدعم املالي
املخصص ملجتمعات
156
التعلم املهنية في امليزانية
التشغيلية
أوافق
ك
%
%
إلى حد ما
%
ك
ال أوافق
ك
%
ال أوافق أبدا
ك
%
املتوسط االنحراف
الحسابي املعياري
الترتيب
30.11
81
29.03
66
23.66
30
10.75
18
6.45
2.83
0.646
9
65.59
51
18.28
27
9.68
9
3.23
9
3.23
4.11
0.525
1
56.99
69
24.73
36
12.90
15
5.38
0
0.00
4.04
0.812
4
50.54
75
26.88
42
15.05
9
3.23
12
4.30
3.91
0.68
7
60.22
63
22.58
24
8.60
18
6.45
6
2.15
4.05
0.57
3
55.91
75
26.88
30
10.75
12
4.30
6
2.15
3.98
0.842
6
7
كثافة مقررات
الرياضيات ومتطلباتها
162
58.06
66
23.66
33
11.83
6
2.15
12
4.30
3.99
0.670
5
8
كثافة أعداد الطالب في
الفصول
135
48.39
78
27.96
33
11.83
24
8.60
9
3.23
3.84
0.641
8
9
قلة أعداد معلمي
الرياضيات في املرحلة
الدراسية
168
5
6
66
60.22
23.66
30
10.75
املتوسط الحسابي العام
9
3.23
6
2.15
4.09
0.808
3.87
0.39
يتضح من جدول ( )6ما يلي:
-1هناك تقارب في استجابات عينة الدراسة ملحور املعيقات املادية التي تحد معلمي الرياضيات من تفعيل
مجتمعات التعليم املهنية وفقا آلراء املعلمين ،حيث تراوحت متوسطات استجاباتهم على املحور األول ما بين
( 2.83إلى )4.11وهي متوسطات يقع في الفئتين الثالثة (متوسط) والرابعة (مرتفع) من درجات املقياس،
ويالحظ أن متوسط املوافقة العام على فقرات املحور بلغ ( )3,87مما يعني أن هناك (مرتفعة) من أفراد العينة
على عبارات هذا املحور.
ً
ً
-2ترتيب عبارات هذا املحور حسب درجة املوافقة عليها ترتيبا تنازليا كما يلي:
موكلي ،زيلعي
()66
المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات
التعلم المهنية وفقًا آلرائهم
2
المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس 2022م
-
-
-3
-4
كانت استجابات أفراد العينة على الفقرة رقم ( )2وهي( :ارتفاع أنصبة املعلمين التدريسية) باملرتبة األولى بين
الفقرات بمتوسط الحسابي ( ،)4.11وهذا يعني أن هناك موافقة بين أفراد العينة أن من أهم املعيقات
املادية هي( :ارتفاع أنصبة املعلمين التدريسية).
جاءت استجابات أفراد العينة على الفقرات املتبقية )9( :و ( )5و ( )3و ( )7و ( )6و ( )4و ( )8و ()1
بمتوسطات حسابية على التوالي)2.83( ،)3.84( ،)3.91( ،)3.98( ،)3.99( ،)4.04( ،)4.05( ،)4.09( :؛ وهذا
يعني أن هناك موافقة بدرجة (مرتفعة) بين أفراد العينة على تلك املعيقات؛ ومن ثم تعد تلك الفقرات
معيقات بدرجة مرتفعة.
ً
ترتيب املعيقات بحسب درجة تحققها وفقا لآلتي:
ارتفاع أنصبة املعلمين التدريسية.
قلة أعداد معلمي الرياضيات في املرحلة الدراسية باملدرسة.
ضعف الحوافز املادية واملعنوية لتفعيل مجتمعات التعلم املهنية.
زيادة األعباء غير التدريسية على املعلم.
كثافة مقررات الرياضيات ومتطلباتها.
ضعف الدعم املالي املخصص ملجتمعات التعلم املهنية في امليزانية التشغيلية.
غياب التجهيزات واملرافق في املدرسة لتفعيل مجتمعات التعلم املهنية.
كثافة أعداد الطالب في الفصول.
نقص الكوادر املؤهلة علميا ومهنيا لقيادة أنشطة مجتمعات التعلم املهنية داخل املدرسة.
يالحظ أن هناك اتفاقا بين أفراد العينة في أن ارتفاع أنصبة املعلمين التدريسية يعد معيقا بدرجة مرتفعة؛
وهذا معيق كبير وال شك ،فارتفاع أنصبة املعلمين التدريسية ال يتيح تفعيل مجتمعات التعلم املهنية بالصورة
املطلوبة.
النتائج املتعلقة بالسؤال الفرعي الثالث" :ما املعيقات الشخصية التي تحد معلمي الرياضيات من تفعيل
مجتمعات التعلم املهنية ً
وفقا آلرائهم؟
ولإلجابة تم حساب التكرارات والنسب املئوية واملتوسطات الحسابية واالنحرافات املعيارية لكل مفردة من
مفردات األداة والجدول التالي يوضح تلك النتائج.
جدول ( :)7املعيقات الشخصية التي تحد معلمي الرياضيات من تفعيل مجتمعات التعلم املهنية
أوافق بشدة
م
العبارة
1
ضعف الثقة املتبادلة
بين أعضاء مجتمع
التعلم املنهي
48
2
غياب وعي املعلمين
بمفهوم مجتمعات
التعلم املهنية
60
ك
%
17.20
21.51
موكلي ،زيلعي
أوافق
ك
66
93
%
23.66
33.33
أوافق إلى حد
ما
%
ك
78
84
27.96
30.11
()67
ال أوافق
ك
69
30
%
24.73
10.75
ال أوافق أبدا املتوسط االنحراف
الحسابي املعياري
%
ك
18
12
6.45
4.30
2.45
2.43
0.603
0.761
المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات
التعلم المهنية وفقًا آلرائهم
الترتيب
6
7
المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس 2022م
م
العبارة
أوافق بشدة
ك
3
4
5
6
7
8
9
10
11
ضعف قناعة املعلمين
بأهمية مجتمعات التعلم
املهنية في تحسين
العملية التعليمية
ضعف قناعة املعلمين
بعملية التغيير والتطوير
في أدائهم
قلة رغبة املعلمين في
ممارسة العمل الجماعي
غياب تقبل املعلمين
للمقترحات والنقد بينهم
بروح ايجابية
ضعف رغبة املعلمين في
تفعيل مجتمعات التعلم
املهنية
ضعف معرفة املعلمين
بأدوارهم في مجتمعات
التعلم املهنية
نقص الوعي بمتطلبات
تطبيق مجتمعات التعلم
املهنية
غياب التعاون بين
معلمي املدرسة لتفعيل
مجتمعات التعلم املهنية
ضعف القدرة على
تخطيط مجتمعات
التعلم املهنية
أوافق إلى حد
ما
%
ك
أوافق
ك
%
%
ال أوافق
ك
%
ال أوافق أبدا املتوسط االنحراف
الحسابي املعياري
%
ك
الترتيب
57
20.43
87
31.18
78
27.96
45
16.13
12
4.30
2.53
0.828
3
54
19.35
78
27.96
78
27.96
51
18.28
18
6.45
2.57
0.684
1
78
27.96
78
27.96
81
29.03
24
8.60
18
6.45
2.38
0.768
11
63
22.58
72
25.81
87
31.18
39
13.98
18
6.45
2.56
0.580
2
60
21.51
84
30.11
75
26.88
48
17.20
12
4.30
2.41
0.695
9
63
22.58
87
31.18
87
31.18
33
11.83
9
3.23
2.42
0.737
8
66
23.66
75
26.88
108
38.71
15
5.38
15
5.38
2.40
0.759
10
66
23.66
69
24.73
96
34.41
39
13.98
9
3.23
2.48
0.809
5
57
20.43
75
26.88
102
36.56
36
12.90
9
3.23
2.52
0.722
4
2.47
0.48
املتوسط الحسابي العام
يتضح من جدول ( )7ما يلي:
-1هناك تقارب في استجابات عينة الدراسة ملحور املعيقات الشخصية التي تحد معلمي الرياضيات من تفعيل
مجتمعات التعليم املهنية وفقا آلراء املعلمين ،حيث تراوحت متوسطات استجاباتهم على املحور األول ما بين
( 2.38إلى )2.57وهي متوسطات يقع في الفئتين الثانية (متدنية) من درجات املقياس ،ويالحظ أن متوسط
املوافقة العام على فقرات املحور بلغ ( )2.48مما يعني أن هناك (متدنية) من أفراد العينة على عبارات هذا
املحور.
ً
ً
-2ترتيب عبارات هذا املحور حسب درجة املوافقة عليها ترتيبا تنازليا كما يلي:
موكلي ،زيلعي
()68
المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات
التعلم المهنية وفقًا آلرائهم
المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس 2022م
-
-
-3
-4
كانت استجابات أفراد العينة على الفقرة رقم ( )4وهي( :ضعف قناعة املعلمين بعملية التغيير والتطوير في
أدائهم) باملرتبة األولى بين الفقرات بمتوسط الحسابي ( ،)2.57وهذا يعني أن هناك موافقة بين أفراد العينة
أن من أهم املعيقات الشخصية هي( :ضعف قناعة املعلمين بعملية التغيير والتطوير في أدائهم).
جاءت استجابات أفراد العينة على الفقرات املتبقية )6( :و ( )3و ( )11و ( )10و ( )1و ( )2و ( )8و ( )7و
( )9و ( )5بمتوسطات حسابية على التوالي،)2.42( ،)2.43( ،)2.45( ،)2.48( ،)2.52( ،)2.53( ،)2.56( :
()2.38( ،)2.40( ،)2.41؛ وهذا يعني أن هناك موافقة بدرجة (متدنية) بين أفراد العينة على تلك املعيقات؛
ومن ثم تعد تلك الفقرات معيقات بدرجة متدنية.
ً
ترتيب املعيقات بحسب درجة تحققها وفقا لآلتي:
ضعف قناعة املعلمين بعملية التغيير والتطوير في أدائهم.
غياب تقبل املعلمين املقترحات والنقد بينهم بروح ايجابية.
ضعف قناعة املعلمين بأهمية مجتمعات التعلم املهنية في تحسين العملية التعليمية.
ضعف القدرة على تخطيط مجتمعات التعلم املهنية.
غياب التعاون بين معلمي املدرسة لتفعيل مجتمعات التعلم املهنية.
ضعف الثقة املتبادلة بين أعضاء املجتمع التعلم املنهي.
غياب وعي املعلمين بمفهوم مجتمعات التعلم املهنية.
ضعف معرفة املعلمين بأدوارهم في مجتمعات التعلم املهنية.
ضعف رغبة املعلمين في تفعيل مجتمعات التعلم املهنية.
نقص الوعي بمتطلبات تطبيق مجتمعات التعلم املهنية.
قلة رغبة املعلمين في ممارسة العمل الجماعي.
يالحظ أن هناك اتفاقا بين أفراد العينة في أن ضعف قناعة املعلمين بعملية التغيير والتطوير في أدائهم يعد
املعيق األعلى من بين املعيقات الشخصية؛ وهذا معيق وال شك ،فضعف قناعة املعلمين بعملية التغيير
والتطوير في أدائهم ال يتيح تفعيل مجتمعات التعلم املهنية بالصورة املطلوبة.
النتائج املتعلقة بالسؤال الفرعي الرابع" :هل توجد فروق ذات داللة احصائية عند مستوى ( )α≤0.05بين آراء
معلمي الرياضيات حول املعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات التعلم املهنية تعزى للخبرة
التدريسية"؟
جدول ( )8حساب تحليل التباين األحادي لداللة الفروق في متوسطات إجابات العينة على املحاور الثالثة تعزى
لسنوات الخبرة
املحور
األول
الثاني
مصدر التباين
بين املجموعات
داخل املجموعات
املجموع
بين املجموعات
داخل املجموعات
املجموع
موكلي ،زيلعي
درجات
الحرية
2
276
278
2
276
278
مجموع
املربعات
57.227
9322.516
9379.743
61.543
9754.783
9816.326
()69
قيمة ف
()F
متوسط
املربعات
28.614
33.777
0.847
30.772
35.343
0.871
الداللة
اإلحصائية
0.624
0.661
المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات
التعلم المهنية وفقًا آلرائهم
المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس 2022م
املحور
مصدر التباين
الثالث
بين املجموعات
داخل املجموعات
املجموع
درجات
الحرية
2
276
278
مجموع
املربعات
49.768
7592.743
7642.511
متوسط
املربعات
24.884
27.507
قيمة ف
()F
0.905
الداللة
اإلحصائية
0.543
يتضح من جدول ( )8عدم وجود فروق ذات داللة إحصائية عند مستوى ( )0.05فأقل في آراء أفراد العينة
حول جميع محاور الدراسة الثالثة ترجع إلى سنوات الخبرة؛ وهذا يعني أن جميع أفراد العينة بمختلف خبراتهم
التدريسية يرون الرأي نفسه في املعيقات وال فرق بينهم في ذلك ،وهذا يؤكد وجود املعيقات التي اتضحت أثناء
مناقشة كل محور على حدة ،وأن وجودها بالدرجة نفسها.
مناقشة النتائج:
بناء على ما تقدم يمكن الخلوص إلى النتائج اآلتية:
-1املعيقات وفقا آلراء املعلمين عالية الدرجة وعددها ( )10من ( )30معيقا أي ما نسبته ( )%33.33من املعيقات
التي تحد من تفعيل مجتمعات التعلم املهنية في تعلم وتعليم الرياضيات هي:
ارتفاع أنصبة املعلمين التدريسية. قلة أعداد معلمي الرياضيات في املرحلة الدراسية باملدرسة. ضعف الحوافز املادية واملعنوية لتفعيل مجتمعات التعلم املهنية. زيادة األعباء غير التدريسية على املعلم. كثافة مقررات الرياضيات ومتطلباتها. ضعف دعم مالي مخصص ملجتمعات التعلم املهنية في امليزانية التشغيلية. غياب التجهيزات واملرافق في املدرسة لتفعيل مجتمعات التعلم املهنية. كثافة أعداد الطالب في الفصول. قلة البرامج التدريبية حول مجتمعات التعلم املهنية. ضعف العالقة بين مدير املدرسة واملعلمين في املدرسة. -2بقية املعيقات وفقا آلراء املعلمين متوسطة الدرجة وعددها ( )9من ( )30معيقا أي ما نسبته ( )%30من
املعيقات ،ومتدنية الدرجة وعددها ( )11من ( )30معيقا أي ما نسبته ( )%36.67من املعيقات.
التوصيات واملقترحات.
-1
-2
-3
-4
-5
-6
-7
بالنظر إلى نتائج الدراسة يوص ي الباحثان ويقترحان بما يلي:
ضرورة تدريب وتأهيل املعلمين لتطبيق مجتمعات التعلم املهنية
تخفيض أنصبة املعلمين بما يتناسب مع تفعيل مجتمعات التعلم املهنية بالصورة املطلوبة.
تحفيز املعلمين معنويا وماديا على تفعيل مجتمعات التعلم املهنية
تخفيف األعباء غير التدريسية على املعلمين من أجل تفعيل مجتمعات التعلم املهنية
تأمين تجهيزات ومرافق املدرسة لتطبيق مجتمعات التعلم املهنية
تعاون مدير املدرسة ومعلميها وتقوية العالقة بينهم لتفعيل مجتمعات التعلم املهنية
كما يقترح الباحثان إجراء الدراسات اآلتية:
موكلي ،زيلعي
()70
المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات
التعلم المهنية وفقًا آلرائهم
المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس 2022م
.1دراسة معيقات تفعيل مجتمعات التعلم املهنية في التخصصات األخرى.
.2دراسة أثر مجتمعات التعلم املهنية على تحسن نتائج الطالب في مادة الرياضيات.
.3دراسة أثر مجتمعات التعلم املهنية على تحسن أداء املعلمين تدريسيا.
قائمة املراجع.
ً
أوال -املراجع بالعربية:
إبراهيم ،حسام الدين؛ املرزوقي ،أحمد سعيد ( .)2018املشكالت التي تواجه مجتمعات التعلم املهنية في املدارسفي سلطنة عمان .مجلة الفنون واألدب وعلوم اإلنسانيات واالجتماع ،ع (.337 -306 ،)29
إبراهيم ،محمد عبد الناصر؛ حسان ،حسن محمد ( .)2018مجتمعات التعلم املهنية مدخل ملواجهة بعضمشكالت مدارس التعليم الثانوي الصناعي في مصر .دراسة ميدانية ،رسالة ماجستير غير منشورة ،كلية التربية،
جامعة املنصورة ،مصر.
األخناوي ،محمد السيد ( )2016متطلبات تجويد األداء املدرس ي بالتعليم الثانوي الصناعي في مصر في ضوءمدخل مجتمعات التعلم املهنية .مجلة كلية التربية.162 -82 ،)64( 4 ،
البرعمي ،يوسف أحمد؛ عبد الرشيد ،ناصر سيد ( .)2020تصور مقترح لتفعيل اإلشراف التربوي املعتمد علىمجتمعات التعلم املهنية .مجلة البحوث التربوية والنفسية.247 -212 ،)65( 17 ،
البرنامج الوطني لتطوير املدارس باململكة العربية السعودية ( .)2016مجتمعات التعلم املهنية .الرياض ،مشروعامللك عبد هللا بن عبد العزيز لتطوير التعليم العام.
توفيق ،فيفي أحمد ( .)2017سيناريو مستقبلي لتفعيل مجتمعات التعلم بمدراس التعليم العام بمحافظةسوهاج .املجلة التربوية ،كلية التربية بسوهاج.260 -113 ،)47( 47 ،
الحازمي ،زكي رزيق ( .)2016املجتمعات املهنية للمعلمين "دراسة تطبيقية" .بحث مقدم للمؤتمر الخامس إلعداداملعلم (إعداد وتدريب املعلم في ضوء مطالب التنمية ومستجدات العصر) 4 -2 ،فبراير ،كلية التربية جامعة أم
القرى بمكة املكرمة.
حسن بت ،حسين بن قاسم ( .)2016الكفايات املهنية املنشودة لدى القيادات املدرسية لتطبيق معاييرمجتمعات التعلم بمؤسسات التعليم العام السعودية :نموذج مقترح ،الثقافة والتنمية -مصر-1 ،)103( 16 ،
.16
الزايد ،زينب بنت عبد هللا ( .)2018تأثير املمارسة التأملية في مجتمعات التعلم املهنية في تعلم معلمات املرحلةالثانوية .مجلة رسالة التربية وعلم النفس ،جامعة امللك سعود.79 -55 ،)62( ،
الزائد ،زينب عبد هللا؛ عمر ،سوزان حسين ( .)2016تأثير مجتمعات التعلم املهنية على االنترنت في فهم معلماتالعلوم لطبيعة العلم ومواسا تدريسها .املجلة األردنية في العلوم التربوية .362 -349 ،)3( 12
الزايدي ،أحمد محمد ( .)2013متطلبات التنمية املهنية في مجتمعات التعلم مدخل إلصالح املدارس الثانويةنموذج مقترح .مجلة كلية التربية بجامعة األزهر ،مصر.406 -333 ،)152( 3 ،
السليمي ،خالد بن سعد؛ الزلفي ،وافي بن صالح ( .)2019التنمية املهنية املستدامة للمعلمين أثناء الخدمة.املؤتمر االول للجمعية السعودية للمعلم (جسم) ،في الفترة بين 5 -4ديسمبر 2019جامعة امللك خالد ،أبها،
.446 -436
موكلي ،زيلعي
()71
المعيقات التي تواجه معلمي الرياضيات في تفعيل مجتمعات
التعلم المهنية وفقًا آلرائهم
المجلة العربية للعلوم ونشر األبحاث ــ مجلة العلوم التربوية والنفسية ــ المجلد السادس ــ العدد السادس والثالثون ــ أغسطس 2022م
-
-
-
-
الصالحية ،فاطمة محمد؛ الهاشم ،نور حياتي ( .)2018تطبيق مجتمعات التعلم املهنية ودورها في رفع مستوى
التحصيل الدراس ي للطالب .مجلة دراسات عربية في التربية وعلم النفس ،رابطة التربويين العرب-447 ،)97( ،
.472
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| 6,148 | 43,901 |
W3166822786.txt | ADMJ (Ahmad Dahlan Medical Journal)
VOL 2, No. 1, 10-18
http:// http://journal2.uad.ac.id/index.php/admj
Albumin Description of Continuous Ambulatory
Peritoneal Dialysis Patients in Sardjito Hospital
Yogyakarta
Barkah Purwanto1*, Dewi Yuniasih2, Metalia Puspitasari3
* Email
(Corresponding Author): barkahdj@gmail.com
of Internal Medicine, Ahmad Dahlan University
2Department of Pharmacology, Ahmad Dahlan University
3Department of Internal Medicine, Universitas Gadjah Mada
1Department
ARTICLE INFO
Article history
Received 5 May 2021
Revised 17 May 2021
Accepted 29 May 2021
Keywords
Albumin profile, quality of life,
CAPD, Cross sectional
ABSTRACT
Albumin profile is an important sign of nutritional status as a qualityof life parameter, whereas hypoalbuminemia has been shown to be
associated with a high mortality rate for patients with End Stage
Renal Disease (ESRD) undergoing dialysis including CAPD. The study
was conducted to determine serum albumin levels in patients with
ESRD undergoing CAPD therapy. The study was conducted in a crosssectional manner on ESRD patients who were still actively
undergoing CAPD in the outpatient Installation of Dr. Sardjito
Hospital Yogyakarta until November 2018. Subjects were examined
for serum albumin levels. There were 57 research subjects with the
category of albumin levels <3.5 g / dL as many as 19 (33.3%),
between 3.5-4.0 g / dL as many as 27 (47.3%) and albumin levels>
4.0 g / dL of 11 (19.3%). There were no significant differences in all
age groups, BMI and duration of CAPD. Albumin profile can be an
independent parameter of quality of life and even a predictor of
mortality in CAPD patients because it does not depend on age group,
BMI and length of CAPD.
This is an open access article under the CC–BY-SA license.
Pendahuluan
Penyakit Ginjal Kronik (PGK) terjadi akibat penurunan fungsi ginjal progresif sehingga ginjal
tidak dapat mengendalikan hemostasis dalam tubuh (El Nahas and Bello, 2005; Polonsky and
Bakris, 2019). Peningkatan prevalensi PGK menjadi kekhawatiran global dengan berbasis data
semakin meningkatnya jumlah pasien End Stage Renal Disease (ESRD) atau stadium Gagal Ginjal
Terminal (GGT) yang ditandai dengan peningkatan jumlah pasien yang melakukan transplantasi
ginjal (Mencarelli, Busutti and Montini, 2015; Anand et al., 2017; Dhondup et al., 2018; Muhammed,
Kibria and Crispen, 2020; Pu et al., 2020). Dengan rata-rata peningkatan jumlah global pasien GGT
hingga dua kali lipatnya maka fenomena GGT akan menjadi beban sistem layanan kesehatan di
semua negara dunia (Mencarelli, Busutti and Montini, 2015; Polonsky and Bakris, 2019). Kebijakan
yang lebih baik pada tatalaksana terapi pasien GGT dengan segala komplikasinya menjadi
pertimbangan yang urgen bagi tiap negara agar menurunkan beban layanan kesehatan.
10
ADMJ Vol. 2. No 1, May 2021 p. 10-18
Modalitas terapi pengganti ginjal (TPG) pada pasien GGT yang terbanyak adalah hemodialisis
(HD) selanjutnya peritoneal dialisis (PD) dan transplantasi ginjal. Di Indonesia, pasien PD
umumnya dengan continuous ambulatory peritoneal dialysis (CAPD) atau Dialisis Peritoneal
Mandiri Berkesinambungan (DPMB). Pasien GGT yang menjalani CAPD mempunyai kebebasan
yang lebih baik, rutinitas hidup tanpa mesin, perasaan lebih nyaman dan biaya yang relatif lebih
murah dibanding melalui modalitas hemodialisis (Widyatmoko, A., 2009).
Modalitas CAPD dinilai semakin baik dan pengalaman secara kolektif semakin banyak, tetapi
angka mortalitas pasien GGT yang menjalani dialisis di Indonesia tetap tinggi (Bandiiara et al.,
2012). Berbagai penelitian mengaitkan tingginya prevalensi mortalitas pasien dialisis ini akibat
kondisi malnutrisi pasien GGT yang menjalani terapi dialisis (Tortorelli, Fanelli and Luciani, 2005;
Al-othman et al., 2016; Naini et al., 2016).
Penentuan tingkat malnutrisi pasien secara biokimia tetap menjadi tantangan dunia medis
karena penentuan indikator malnutrisi sulit ditentukan. Salah satu indikator malnutrisi pasien GGT
yang menjalani TPG adalah dengan penentuan kadar albumin serum (Gama-axelsson et al., 2012;
Tortorici et al., 2020). Kajian tentang pentingnya profil albumin serum perlu diperhatikan karena
penurunan kadar albumin serum dari standar adalah satu tanda progres inflamasi (Sridhar and
Josyula, 2013). Konsekuensinya, rendahnya serum albumin berefek negatif pada pasien hingga
potensi terjadinya mortalitas (Amaral et al., 2015; Sun et al., 2020; Tortorici et al., 2020). Gambaran
tingkat keparahan (severity level) dari pasien dibandingkan dengan nilai normal albumin serum
standar dengan kriteria sangat parah (Q1) < 3,5 g/ dl, parah (Q2) 3,5 g/dL – 4,0 g/dL dan normal
(Q3) > 4,0 g /dL. (Amaral et al., 2015).
Profil albumin serum pasien CAPD diperlukan sebagai informasi awal agar terapi terhadap
pasien terkontrol secara efektif dan lebih menyeluruh. Profil albumin serum juga diperlukan
sebelum pasien menjalani TPG untuk mencegah efek negatif setelah pasien menjalani CAPD dengan
cara meningkatkan nutrisi pasien untuk mendapatkan manfaat optimal yang diharapkan (Szeto
Cheuk et al., 2000; Levin and Kotanko, 2006).
Berdasar latar belakang tersebut, peneliti bermaksud melakukan penelitian untuk mengetahui
kadar albumin serum sebagai parameter status nutrisi dan kualitas hidup pada pasien GGT yang
menjalani terapi CAPD.
Metode Penelitian
Penelitian dilakukan di Instalasi Rawat Jalan CAPD Unit Penyakit Dalam RSUP Dr Sardjito
Yogyakarta pada bulan Oktober 2018 sampai dengan bulan November 2018. Populasi target adalah
pasien GGT dengan CAPD sebagai terapi pengganti ginjal. Populasi terjangkau adalah pasien GGT
dengan CAPD yang menjalani pemeriksaan rutin selama periode penelitian dan bersedia mengikuti
Albumin Description of Continuous Ambulatory Peritoneal Dialysis Patients in Sardjito Hospital Yogyakarta
(Barkah Purwanto, et.al.)
11
ADMJ Vol 2. No 1, May 2021 p. 10-18
penelitian ini dengan menandatangani informed consent. Sampel penelitian adalah pasien GGT
dengan CAPD yang memenuhi kriteria inklusi dan ekslusi. Kriteria inklusi adalah pasien GGT yang
menjalani CAPD, berusia antara 18 sampai 65 tahun, kontrol rawat jalan di RSUP Dr Sardjito,
menjalani pemeriksaan kadar albumin, kurun waktu dari pemasangan tenckhof lebih dari 1 bulan,
dan bersedia mengikuti penelitian dengan menandatangani informed consent. Kriteria eksklusi
meliputi pasien yang sedang mengalami sepsis, peritonitis, infeksi, overload cairan dan hasil
pemeriksaan tidak lengkap. Gambaran tingkat keparahan (severity level) kadar albumin berdasar
kategori sangat parah (Q1) < 3,5 g/ dl, parah (Q2) antara 3,5 – 4,0 g/dL dan normal (Q3) > 4,0 g
/dL.
Hasil Penelitian
Selama periode penelitian didapatkan 82 pasien CAPD yang kontrol rutin. Sebanyak 25 subyek
tidak diikutkan dalam penelitian terdiri dari 11 subyek usia kurang dari 18 tahun, 3 subyek
mengalami overload cairan, 3 subyek tidak bersedia ikut penelitian, 5 subyek tidak dapat dihubungi
dan 3 subyek hasil pemeriksaan tidak lengkap.
Tabel 1. Karakteristik data dasar 57 subyek CAPD
Variabel
Jenis kelamin
Laki-laki
Perempuan
Usia (tahun)
Diabetes Melitus
Ya
tidak
Hipertensi
Ya
Tidak
Lama CAPD
BMI (kg/m2)
Under weight
Normal
Overweight
Obese
Kadar Albumin
< 3,5
3,5 – 4,0
> 4,0
n (%)
35 (61,4)
22 (38,6)
Rerata ±SB
37,84±16,16
4 (7,0)
53 (93,0)
14 (24,6)
43 (75,4)
40.44 + 28.63
22.19 + 3.33
11 (19,3)
35 (61,4)
9 (15,8)
2 (3,51)
19 (33,3)
27 (47,3)
11 (19,3)
Analisis dilakukan pada 57 subyek, didapatkan kadar albumin
3,62±0,56
< 3,5 sebanyak 19 (33,3%),
antara 3,5-4,0 sebanyak 27 (47,3%) dan kadar albumin > 4,0 sebanyak 11 (19,3%) (Gambar 2).
Subyek terdiri dari 35 (61,4 %) laki-laki dan 22 (38,6 %) perempuan. Rerata usia 37,84±16,16
tahun dengan rerata lama CAPD 40.44 ± 28.63 bulan. Subyek dengan diabetes melitus sebanyak
7% dan hipertensi 24,6%. Rerata BMI 22,19±3,33 dengan BMI normal sebanyak 61,4%,
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ADMJ Vol. 2. No 1, May 2021 p. 10-18
underweight 19,3%, overweight 15,8% dan obese 3,5%. (Tabel 1).
Gambar 1. Gambaran tingkat keparahan pasien CAPD berdasarkan kadar albumin
Sementara itu, gambaran tingkat keparahan pasien CAPD berdasarkan kadar albumin (gambar 1),
didapatkan hasil 47,3% berada di level Q2 atau parah, diikuti 33,3% berada pada tingkatan sangat
parah (Q1) dan terakhir 19,3% pasien CAPD pada level normal.
Tabel 2. Perbedaan variabel berdasarkan kadar Albumin serum
Kadar Albumin Serum
Karakteristik
Usia
BMI
Lama CAPD
Total (n=57)
Q1 (< 3.5)
N = 19
rerata + SD
37.84 + 16.16
37.58 + 15.353
22.19 + 3.33
40.44 + 28.63
21.52 + 2.87
45.11 + 36.52
Q2 (3.5 - 4,0)
N = 27
rerata + SD
37.41 +
16.768
22.32 + 3.19
38.30 + 26.42
Q3 (>4.0)
N = 11
rerata + SD
39.36 + 17.449
23.03 + 4.37
37.64 + 17.64
P
value
0.943
0.476
0.691
Pada table 2, tampak secara statistik tidak ada perbedaan yang bermakna antara rata-rata usia,
BMI dan lama CAPD terhadap albumin (p > 0.05).
Pembahasan
Profil Albumin Pasien CAPD
Jumlah pasien GGT yang menjalani terapi CAPD di RSUP Dr. Sardjito lebih banyak berjenis
kelamin laki-laki. Selain itu, pasien pada kategori Q1 dan Q2 dengan kadar serum albumin di bawah
standar juga didominasi pasien laki-laki, sementara pada kategori Q3 dengan kadar albumin
normal jumlahnya berimbang. Fenomena pasien CAPD dengan gender laki-laki lebih banyak dari
pada perempuan umumnya karena pola hidup lelaki yang lebih tinggi faktor risikonya, misalkan
pekerja keras, perokok dan faktor lingkungan lainnya (Wu, Wu and Tarng, 2014; Napierala et al.,
2019; Muhammed, Kibria and Crispen, 2020).
Albumin Description of Continuous Ambulatory Peritoneal Dialysis Patients in Sardjito Hospital Yogyakarta
(Barkah Purwanto, et.al.)
13
ADMJ Vol 2. No 1, May 2021 p. 10-18
Profil Albumin sebagai penanda status nutrisi pasien CAPD
Profil albumin relevan sebagai penanda penting tingkat nutrisi pasien dialisis, termasuk
CAPD (Joseph et al., 1996; Kim et al., 1999; Swartzendruber, 2020). Selain itu, profil kadar albumin
pada pasien gagal ginjal kronik sangat penting diketahui, terutama pasien yang menderita
kekurangan albumin atau hipoalbuminemia karena komplikasi penyakit dan terapi yang dijalani.
Pada kajian ini, pasien lebih banyak pada kondisi hipoalbuminemia sebanyak 80,7 %. Fenomena ini
merupakan prevalensi komplikasi umum yang ditemui pada penyakit ginjal kronik (Sridhar and
Josyula, 2013). Karenanya, tingkat relevansi mendapatkan profil serum albumin pada pasien CAPD
juga sangat penting karena akan berpengaruh juga terhadap optimalisasi adekuasi CAPD (Joseph et
al., 1996; Cueto-manzano et al., 1997; Yang et al., 1997; Kim et al., 1999; Szeto Cheuk et al., 2000;
Sezer, Tutal and Haberal, 2005).
Risiko malnutrisi pasien CAPD di RSUP Dr Sardjito
Secara umum pasien CAPD berpotensi mengalami risiko malnutrisi. Penyebab malnutrisi
pada pasien dialisis antara lain karena minimnya asupan energi akibat anoreksia, kehilangan
nutrien selama terapi dialisis, pembatasan makanan/diet sebelum dialisis, peningkatan
katabolisme protein dan asidosis (Tortorelli, Fanelli and Luciani, 2005; Al-othman et al., 2016; Naini
et al., 2016). Berdasarkan profil serum albumin pada penelitian ini, kekurangan serum albumin
pada pasien CAPD RSUP Sardjito dapat berdampak progres negatif pasien yang sedang melakukan
terapi gagal ginjal tersebut. Selain adanya konsekuensi malnutrisi, memungkinkan juga terjadinya
inflamasi progresif (Gelder et al., 2018). Albumin menentukan tekanan osmotik plasma darah,
karenanya penurunan albumin dalam peredaran darah akan menggeser cairan dalam ruang
intravaskuler (Sridhar and Josyula, 2013).
Pada penelitian ini diperoleh gambaran bahwa hanya 19,3% pasien CAPD memiliki kadar
albumin serum normal (>4 g/ dL), sementara sebanyak 80,7% pasien lainnya memiliki kadar
albumin kurang dari kondisi normal. Dari pasien yang mengalami hipoalbuminemia, terindikasi
33,3 % pasien pada kriteria Q1 (sangat parah) dan 47,3 % pada level Q2 (parah). Bila
memperhatikan kajian retrospektif dari Sun et al, 2020, profil albumin serum pasien CAPD di RSUP
Dr. Sardjito ini perlu mendapatkan perhatian serius karena pada pasien yang memiliki rentang
kadar albumin serum tingkat Q1 dan Q2 memiliki faktor risiko all-cause mortality (ACM) relatif
tinggi, masing-masing dengan probabilitas 28% pada tingkat Q1, 20% pada tingkat Q2 dan 8% pada
level Q3.
Pasien GGT dengan terapi CAPD pada mayoritas penelitian mengalami hipoalbuminemia
yang berpotensi memperburuk kelangsungan hidupnya pasca terapi (Cueto-manzano et al., 1997;
Sezer, Tutal and Haberal, 2005; Al-othman et al., 2016) seperti pada pasien dengan hemodialisis,
14
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ADMJ Vol. 2. No 1, May 2021 p. 10-18
kadar albumin kurang dari 3,5 gram/dl termasuk faktor risiko utama mortalitas (Pifer, Cullough,
Port and Dadvid A G Goodkin, 2002; Pifer, Cullough, Port and David A G Goodkin, 2002; Sridhar and
Josyula, 2013; Gelder et al., 2018).
Kajian-kajian lain juga menekankan perhatian pada terapi pasien gagal ginjal kronik yang
mengalami penurunan kadar serum albumin. Penelitian-penelitian tersebut memaparkan relevansi
rendahnya serum albumin dengan peningkatan prevalensi mortalitas (Ustace et al., 2004b; Levin
and Kotanko, 2006; Herselman et al., 2010; Sridhar and Josyula, 2013; Amaral et al., 2015; Gelder
et al., 2018). Penurunan albumin pada pasien GGT yang menerima terapi CAPD di RSUP Sardjito
tersebut harus mendapatkan terapi peningkatan albumin (Of et al., 2004; Gelder et al., 2018). Di
masa yang akan datang, perlu pengkajian tatalaksana yang lebih optimal terkait terapi albumin bagi
pasien CAPD.
Pada penelitian ini didapatkan bahwa kadar albumin serum dapat menjadi prediktor
independen terhadap kualitas hidup bahkan mortalitas karena tidak didapatkan perbedaan
bermakna pada kelompok usia, Body-Mass-Index (BMI) dan lama CAPD. Selanjutnya, tantangan
penelitian berikutnya di bidang ini adalah untuk menggali teori lebih detail tentang mekanisme
terjadinya penurunan level albumin pasca CAPD. Kehilangan albumin pada pasien CAPD sudah
dikaji sebelumnya, yang menguatkan fakta bahwa pasien CAPD rentan malnutrisi karena potensi
kehilangan albumin selama terapi (Iktzler et al., 1994; Ustace et al., 2004a; Herselman et al., 2010;
Gelder et al., 2018; Tanaka et al., 2020). Perlu dilakukan penelitian lebih detail tentang mekanisme
dan tahapan terjadinya kehilangan albumin pada pasien CAPD selama mengikuti terapi sehingga
potensi hipoalbuminemia dikurangi dan menghasilkan penanganan pasien lebih baik selama terapi
(Cueto-manzano et al., 1997; Sezer, Tutal and Haberal, 2005; Guest, 2013; Sridhar and Josyula,
2013).
Kesimpulan
Penelitian telah mengkaji profil albumin pada responden pasien CAPD di RSUP Sardjito.
Albumin merupakan indikator dari kondisi nutrisi pasien. Hasil penelitian menunjukkan bahwa
sebanyak 80,7% pasien CAPD yang ditangani di RSUP Dr. Sardjito memiliki kadar albumin lebih
rendah dari standar normal. Secara gender, pasien laki-laki lebih banyak dari pasien wanita. Kadar
albumin rendah /hipoalbuminemia seperti pada responden penelitian ini adalah indikasi dari
malnutrisi. Dari hasil kajian penelitian sebelumnya menguatkan korelasi positif hipoalbuminemia
dengan peningkatan prevalence mortalitas pasien GGT. Karenanya, terapi peningkatan kadar
albumin pasien CAPD RSUP Dr. Sardjito yang mengalami hipoalbuminemia perlu dilakukan. Fokus
penelitian ini hanya pada pemaparan profil albumi pasien CAPD, karenanya perlu dilakukan kajian
berikutnya untuk memberikan kontribusi lebih banyak lagi pada kajian klinis dan akademis di
Albumin Description of Continuous Ambulatory Peritoneal Dialysis Patients in Sardjito Hospital Yogyakarta
(Barkah Purwanto, et.al.)
15
ADMJ Vol 2. No 1, May 2021 p. 10-18
bidang ini. Beberapa topik penelitian berikutnya yang menjadi perhatian pada kajian ini diuraikan
diakhir pembahasan dengan besar harapan menjadi kontribusi untuk penelitian-penelitian
berikutnya di bidang ini yang lebih detail sehingga membantu terapi pasien CAPD yang lebih
optimal.
Terimakasih
Penulis mengucapkan banyak terimakasih kepada RS Sarjito Yogyakarta dan seluruh
pasien yang telah berkontribusi dalam penelitian ini.
Daftar Pustaka
Al-othman, A. M. et al. (2016) ‘Association of malnutrition in peritoneal dialysis patients of Saudi
Arabia’, Arabian Journal of Chemistry. King Saud University, 9, pp. S1059–S1062. doi:
10.1016/j.arabjc.2011.11.015.
Amaral, S. et al. (2015) ‘Serum Albumin Level and Risk for Mortality and Hospitalization in
Adolescents on Hemodialysis’. doi: 10.2215/CJN.02720707.
Anand, S. et al. (2017) ‘Prevalence of chronic kidney disease and risk factors for its progression: A
cross-sectional comparison of Indians living in Indian versus U.S. cities’, PLoS ONE, 12(3),
pp. 1–14. doi: 10.1371/journal.pone.0173554.
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Influence of some anthropometric and motor parameters on triple jump
Ramadani L.1, Rashiti N.2, Shkodra M.2, Heta G.3
University of Prishtina, Faculty of Physical Education and Sport, Prishtina, Kosova
Abstract
Athletics disciplines are characterized by motor movements that can be successfully applied
during the educational process or through other forms of exercise, which significantly affect the
development of general psychophysical abilities of individuals. Each of the disciplines of
athletics acts on the development of individual abilities, but also on the general psychophysical
status of man and for this reason athletics is called the "queen of sports". Athletic activities have
a characteristic of participant inclusion. The values of the paper will be based on the purpose of
anthropometric, basic motor and specific motor characteristics of students aged 21 years ± 6
months. A total of 70 male students, 7 anthropometric variables, 7 motor variables and 1 specific
motor variable that were tested during the period of May, of the academic year, 2020/2021 were
included in the research. In the basic parameters students, based on the results achieved in this
anthropometric variable, are presented as a moderately homogeneous group. Although it is seen
that the acquired flexibility and convexity (Skewness and Kurtosis) have a pronounced
asymmetry, in the vast majority of anthropometric variables. Although it is seen that the acquired
flexibility and convexity (Skewness and Kurtosis) have a pronounced asymmetry, in the vast
majority of motor and criterion variables. Thus the statistically significant coefficients of the
variables with the highest degree of statistical inference (p <0.01) are denoted by two asterisks.
With easier statistical conclusion criterion (p <0.05) correlation coefficients. All variables have
statistically significant correlation to all anthropometric, basic and criterion motor variables.
Keywords: anthropometric variables, motor, specific, student, regression.
INTRODUCTION
Athletic jumps are exercises, characterized by an extended phase without support, running over
obstacles as a result of momentum and energetic beating on the ground with one foot. [1] These
types of exercises are associated with great neuromuscular strain, within a short time [2] As such
they develop with priority explosive force and speed as well as sensations of orientation in space
38
and time. Long and triple jumps are natural forms of movement, in general, and athletics in
particular. These types constitute one of the basic tools of all-round training: physical, functional
and psychological. This is the main reason why they are constantly appreciated, especially in the
context of peak exercise for sporting achievement. [3] The data show that the triple jump is the
discipline of the old Olympic Games and the first result found is 16 m, but this result is assumed
to be the sum of the results of three separate jumps, to jump in a row. The triple jump is part of
the group of monostructural movements and is quite complex. Momentum, as the first phase, is
cyclic motion, while the other phases belong to acyclic motions. [1,2,4] During the jump, large
loads (300-500 kg) are exerted on the moving system of the jumpers, so the three-step jump is
approached after the students have reached a proper quantum of basic motor properties, such as
speed, strength, mobility and proper condition. Speed and strength mainly dominate in this
discipline. The sporting result depends on the size of the initial flight speed, the flight angle as
well as the height of the overall center of gravity. that the jumper makes in space on every flight.
[5,6] High ump is a complex movement. Its structure consists of: momentum as cyclic motion,
thrust, flight and fall belong to the acyclic group of motions. High jump belongs to the
monostructural group of movements where the determining element is strength and speed. [7,9,10]
The complexity of this discipline increases with the increase of the sports result which is
characterized by the high degree of connectivity of these elements as well as by the high degree
of harmony and coordination of movements in space.
THE PURPOSE OF THE STUDY
The problem which is addressed in this paper is as important as it is current, because all students
have different characteristics, so even in the sport of athletics there is an increasing need for
objectification of indicators of the impact of physical exercise and especially the various jumps,
can safely and efficiently diagnose, program, directly manage and control transformational
processes in the subject of athletics and sports in general at Faculty of Physical Education and
Sports. The main purpose of this research is to determine the impact of some anthropometric,
motor and criterion variables in the triple jump to the students of the Faculty of Physical
Education and Sports at the University of Prishtina "Hasan Prishtina" - Prishtina, male students in
the first year of study of the academic year 2020/2021, aged 19 years ± 6 months. It also aims to
verify the anthropometric, motor and motor specific characteristics of the Faculty of Physical
Education and Sports students.
The basic criteria for participation in the research was: a) that during the year they were
always present in athletics classes, b) they were present in the teaching process, c) they were not
sick during the test.
VARIABLES SAMPLE
1. Anthropometric variables
The following anthropometric parameters have been applied to identify anthropometric variables:
Body height (BH), Arm length (AL), Leg length (LL), Body weight (BW), Subcutaneous
abdominal fat tissue (SAFT), adipose tissue of the arm (ATA), adipose tissue of the thigh
(ATTH).
39
2. Specific-motor variables
Standing long jump with two legs (SLJTL), Standing high jump (SHJ), Standing long jump with
the left foot (SLJLF), Standing long jump with the right foot. (SLJRF), Hand tapping (HT), Foot
tapping (FT), Flexibility - body bending forward (BBF) and running 30 meters high start (RHS
30 m.).
3. Specific motor variables
Triple jump with momentum
METHODS FOR PROCESSING THE RESULTS
Taking into account all these facts, for the purposes of this research, procedures have been chosen
that are considered to correspond to the nature of the problems being researched. For each
variable, the following values will be calculated:
1. Central basic and Distribution Parameters: Arithmetic Mean (AM), Standard Deviation
(SD), Minimum Value (Min), Maximum Value (Max).
2. The distribution curve is tested by the coefficient of asymmetry (Skewness), and the
degree of curvature of the vertex of the distributed results curve (distribution height)
through the flat coefficient (Kurtosis).
3. Interconnections reports between variables in the manifest space, as well as correlations
between the systems of variables.
4. To determine the relationship between predictor variables (anthropometric and motor
characteristics) and criterion variables (triple jump with momentum), regression analysis
was applied in the manifest space.
DESCRIPTIVE ANALYSIS OF ANTHROPOMETRIC AND MOTOR SPECIFIC VARIABLES
In Table 1 descriptive analysis of anthropometric and motor specific variables is presented to the
19-year-old students of the Faculty of Physical Education and Sports.
The sample included a group of 60 male students, where the arithmetic mean values, minimum
value, maximum value, standard deviation, distribution or asymmetry parameters (Skewness - tilt,
asymmetry) and the degree of extension of the curvature peak of the distribution of results
(Kurtosis - convexity).
40
Table 1. Basic statistical parameters of anthropometric and motor specific variables in students
of the Faculty of Physical Education and Sports.
The arithmetic mean of the anthropometric variable, body height (BH), (Table 1 and Histogram 1)
is (178.67 cm). The minimum result (165.50 cm) and the maximum result (197.60 cm) of the
variable, body height shows that there is a significant difference between students in this
anthropometric variable, and a slight tilt of the variables has the lowest results, because in the
asymmetry test dominates the results in between, the asymmetry test is mesocortical
(mesokurtical).
Students based on the results achieved in this anthropometric variable are presented as a
moderately homogeneous group with a larger number of values towards the lower ones. Although
there is a marked difference between the minimum and maximum result, it is seen that the
acquired flexibility and convexity (Skewness and Kurtosis) have pronounced asymmetry. All
variables presented in table 1 are homogeneous; no variable presented in this table is
heterogeneous.
INTERCORRELATION MATRIX BETWEEN BASIC AND SPECIFIC
ANTHROPOMETRIC AND MOTOR VARIABLES
Intercorrelation coefficients of basic and specific anthropometric and motor variables in
students of year (I) of the Faculty of Physical Education and Sports
Table 2 presents the intercorrelation matrix of anthropometric and motor-specific variables in
Faculty of Physical Education and Sports students. Due to the better reflection of the table and to
more clearly observe the intercorrelation coefficients, the significant coefficients of the different
connection levels are identified with the asterisk. Thus the statistically significant coefficients of
the variables with the highest statistical conclusion (p <0.01) are marked with two asterisks. With
the easiest statistical conclusion criterion (p <0.05) the correlation coefficients are marked with
41
an asterisk. The interpretation of simple linear correlation coefficients as it is known in most
cases depends on the number of tested, respectively more precise on the degree of freedom. The
sample of 70 students tested, the degree of freedom number is 48. With the inspection of the table
of limited values, with Pearson coefficients it is noticed that the value of the limit for 48 degrees
of freedom (df (N-2) is r = 0.250 with the easiest criterion (p = 0.05) respectively r = 0.325 with
stricter statistical inference criteria (p = 0.01) (Bala, 1990).
Table 2. Intercorrelation coefficients of basic and specific anthropometric and motor
variables in students of year (I) of the Faculty of Physical Education and Sports
By inspecting the intercorrelation coefficients of anthropometric variables in students we
notice that all anthropometric variables among themselves have a high correlation and that that
coefficient or all correlation coefficients are statistically significant at the level of p <0.001.
Especially the correlation coefficients that belong to the longitudinal dimension (arm lengths) and
those that belong to the dimension of body mass and body volume are higher. r = 0.989; p <0.01.
leg length. r = 0.758; p <0.01., body lengths r = 0.501; p <0.01, arm adipose tissue r = 0.481; p
<0.01, adipose tissue of the thigh r = 0.440; p <0.01 etc. By inspecting the intercorrelation
coefficients of the basic motor variables in basketball players (table 3) are grouped among
themselves. In the first group we have the correlation coefficients belonging to the explosive
forces of the lower extremities, standing long jump and standing high jump r = 0.278; p <0.05. In
the second group we have the correlation coefficients belonging to the running speed 30 meters r
= 0.780; p <0.01. triple jump r = 0.345; p <0.01, etc.
42
Table 3. Regression analysis - correlation and influence of anthropometric and motor
variables as independent (predictor) variables on the dependent (criterion) variable - triple
jump with momentum (TJM).
Model
R
1
.607a
Model
Regression
1
Model Summary
R Square
Adjusted R
Std. Error of the
Square
Estimate
.369
.193
.71194
ANOVAa
Sum of Squares
df
Mean Square
15.983
15
1.066
Residual
27.370
54
Total
43.354
69
F
2.102
Sig.
.024b
.507
a. Dependent Variable: K3HAP
b. Predictors: (Constant), MV30M, AGJKEM, MPPARA, IDHKRA, MKMAJ, MTKEM, APTRU,
MTDOR, AIDHKOF, KLVEN, AIDHBA, MKGJVE, AGJKRA, MKDJA, ALATR
Coefficientsa
Model
1
Standardized
Coefficients
Beta
Unstandardized Coefficients
B
Std. Error
(Constant)
15.386
7.572
ALATR
AGJKRA
AGJKEM
APTRU
AIDHBA
IDHKRA
AIDHKOF
MKGJVE
KLVEN
MKMAJ
MKDJA
MTDOR
MTKEM
MPPARA
MV30M
.099
-.137
-.056
.001
-.109
.127
.025
-.785
-.063
.027
.107
.015
.012
-.010
-1.196
.117
.747
.244
-.487
.031
-.341
.021
.011
.056
-.351
.063
.298
.032
.117
.749
-.194
.032
-.347
.057
.091
.048
.487
.031
.065
.035
.045
.017
-.074
1.273
-.180
a. Dependent Variable: K3HAP
t
Sig.
2.032
.047
.847
-.563
-1.839
.057
-1.948
2.027
.779
-1.047
-1.965
.482
2.221
.486
.337
-.600
-.939
.401
.576
.071
.955
.057
.048
.439
.300
.055
.632
.031
.629
.737
.551
.352
The regression analysis in students shows that the value of the correlation between the
group of independent predictor variables (basic anthropometric and motor variables with the
criterion dependent variable - Triple jump with momentum (TJM) has been verified. Connectivity
of the whole system of independent predictOR variables (basic anthropometric and motor
variables) - Triple jump with momentum (TJM). Connectivity of the whole system of
independent predictor variables (basic anthropometric and motor variables): Body height (BH),
Arm length (AL), Leg length (LL), Body weight (BW), Abdominal subcutaneous adipose tissue
(ASAT), Adipose tissue of the arm (ATA), Adipose Tissue of the Thigh (ATT), Standing Long
43
Jump (SLJ), Standing High Jump (SHJ), Standing Long Jump with the Left Foot (SLJLF),
Standing Long Jump with the Right Foot (SLJRF), Hand Tapping (HT) , Foot Tapping (FT),
Forward Body Bend Flexibility (FBBF) with the criterion variable - Triple Jump with Momentum
(TJM), these have been proven with multiple correlations. The multiple correlation coefficient
has the value R = 0.607, which explains the common variability between the system of predictor
variables and the criterion variable about 40% (R Square = 0.369). The distribution (F) is
obtained as the quotient distribution of the two variances, and in these cases it is always
necessary to assign the two degrees of freedom. The first degree of freedom is equal to the
number of predictor variables (df = n) respectively (df = 15), while the second is performed so
that the number of subjects (70) is reduced by the number of predictive variables minus 1 (df = N
- n - 1) respectively (df = 70 - 15– 1). We can emphasize that the test (F-test) is always more
valuable if the multiple correlation is greater, in the concrete case of this paper is significant (Sig
= 0.024), because the value of the F-test is 3.926. In this research, after obtaining a statistically
significant multiple correlation (R = 0.642), it is necessary to look for the coefficient in the
column (Beta) as well as the value of (t-test), which show the impact of each predictor variable
( independent) in the dependent or criterion variable (Table 3).
CONCLUSION
Athletic jumps are exercises, characterized by an extended phase without support, as a
result of momentum and energetic beating on the ground with one foot. These types of exercises
are associated with great neuromuscular strain, within a short time. As such they develop with
priority explosive force and speed as well as sensations of orientation in space and time. In the
formation of anthropological features of a population, the genetic factor has a primary influence,
but the influence of external factors, thus ecological factors should not be left aside. This paper
aimed to ascertain the impact of anthropometric, motor - specific parameters on Faculty of
Physical Education and Sports students aged 19 years. A total of 70 students were included in this
research. The measurements were executed during the month of May in the physical education
hall of the Faculty of Physical Education and Sports. Based on the scope and problem of the
research and in accordance with the purpose and statistical methods for processing the results,
four hypotheses are presented. A total of 15 motor variables and 1 specific motor variable were
applied. All these applied variables have been subjected to basic statistical processing, on the
basis of which it was proved that all these variables met the application conditions and have had
satisfactory metric characteristics. Descriptive analysis were applied in order to determine the
arithmetic mean, standard deviation and to determine the asymmetry and curve. In order to
determine the factors in psychomotor variables regression analysis has been applied. Regression
analysis shows that student results are presented as homogeneous results. The intercorrelation
coefficients of the basic motor and specific motor parameters of some variables, the obtained
values have a significant statistical correlation, which has been proven based on the research so
far according to many authors.
The arguments obtained show that the most realistic picture regarding the selection of
students for the sport of athletics based on psychomotor skills are processed through regression
analysis.
44
The application of scientific research methods, in a good way creates the information base
in order to valorize the values and program contents during the holding of classes in athletics and
other sports as well as training processes. The results of the research for pedagogues and trainers
will present important information on the impact of anthropometric and basic motor
characteristics on the performance of specific motor tasks in the sport of athletics in order to dose
the volume and intensity of loads during regular hours of training processes, special hours and
various recreational activities. Based on what was said above, it can be concluded that the results
and conclusions of the research will be in function of the identification, monitoring, programming
and control of students of this age group who regularly attend the subject of athletics in the
Faculty of Physical Education and Sports. The whole research is presented in tabular form of
results. I think that this paper will be a small window in this field of science and sports in the
subject of athletics.
Based on what was said above, it can be concluded that the results and conclusions of the
research will be in function of the identification, monitoring, programming and control of
students of this age group who regularly attend the subject of athletics at the Faculty of Physical
Education and Sports.
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Correspondence to:
Prof.Dr. Rashiti Naser, Ph.D.
University Prishtina
Faculty of Physical Education and Sport
Kosova
Phone number: +383 49 666 619
E-mail: naser.rashiti@uni-pr.edu
46
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W4383551190.txt | BMC Cancer
Xu et al. BMC Cancer
(2023) 23:636
https://doi.org/10.1186/s12885-023-11135-3
Open Access
R E T R AC T I O N N OT E
Retraction Note: A synthetic dsRNA, as a TLR3
pathwaysynergist, combined with sorafenib
suppresses HCC in vitro and in vivo
Yu-Yin Xu1,2†, Li Chen1*†, Gui-Lan Wang1, Jia-Ming Zhou1, Yi-Xin Zhang3, Yin-Ze Wei3, Yuan-Yuan Zhu4 and Jing Qin1
Retraction Note: BMC Cancer 13, 527 (2013)
https://doi.org/10.1186/1471-2407-13-527
The Editor has retracted this article because it appears
that some of the text and figures have been previously
published by some of the same authors. Specifically:
• there is substantial text overlap with results section
in [1]
• Figure 2B appears to present the same data as Fig. 1C
in [1]
• It appears the same FACS plots have been presented
in Fig. 3B (for srfn, poly (I:C) and control) as in
Fig. 4A in [1]
• Figure 3C (sifn and Poly(I:C) ) and 3D appear to
show the same images as those published previously
in [1] in Fig. 3A
Author Yu-Yin Xu does not agree to this retraction. None
of the other authors have responded to any correspondence from the editor or publisher about this retraction
References
1. Chen, L., Xu, Y., Zhou, J., Wu, Y., E, Q., Zhu, Y. “TLR3 dsRNA agonist inhibits
growth and invasion of HepG2.2.15 HCC cells”. Oncology Reports 28.1 (2012):
200–206.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
†
Equal contributors.
The online version of the original article can be found at https://doi.
org/10.1186/1471-2407-13-527
*Correspondence:
Li Chen
bl1@ntu.edu.cn
1
Department of Pathological Anatomy, Nantong University, Nantong,
China
2
Department of Nephrology, Affiliated Hospital of Nantong University,
Nantong, Jiangsu, China
3
Department of Pathology, Affiliated Tumor Hospital, Nantong University,
Nantong, China
4
Biomics (Nantong) Co., Ltd, Nantong, China
© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,
sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this
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in this article, unless otherwise stated in a credit line to the data.
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W4377970269.txt | Scientific and practical journal EJCRP&P № 1(2) 2023
5 Психолого-педагогические исследования
МНРТИ 15.01.79; 14.35.01
УДК 159.99; 37.082
https://doi.org/10.46914/2959-3999-2023-1-1-59-65
Г.О. Абдуллаева,*¹
к.пед.н., ассоц. профессор.
*e-mail: g.abdullayeva@turan-edu.kz
ORCID ID: 0000-0002-8359-3117
И. Сулейменов,¹
магистрант.
e-mail: 22232256@turan-edu.kz
ORCID ID: 0009-0006-8299-4070
1
Университет «Туран»,
г. Алматы, Казахстан
Карьерные ориентации студентов: подходы к исследованию
Аннотация
В статье раскрывается актуальность и существующие подходы к изучению карьерных ориентаций студентов, определены перспективы и стратегия дальнейшего исследования. Отражены методологические основания исследования, среди которых философские положения о диалектическом методе познания, сущности
деятельности, участие в которой преобразует личность; теории системно-комплексного, личностно-деятельностного подхода; идеи формирования личности, становление профессионала; связи теории с практикой.
Представлен анализ подходов к пониманию карьеры и карьерных ориентаций в психологии, а также показана
авторская позиция, которая выражается в том, что карьерные ориентации включены в систему профессиональной направленности личности и закладывают основу построения карьеры.
Ключевые слова: карьера, карьерные ориентации, профессиональное самоопределение, профессиональная направленность, студенты вуза.
Введение
Постоянно усиливающийся темп жизни, инновации и преобразования в социально-экономической сфере, бурное развитие информационных технологий, ускоряющиеся процессы
глобализации и интернационализации, неспокойная геополитическая обстановка в мире оказывают серьезное влияние на жизнь молодежи и заставляют ее адекватно реагировать на проявляющиеся вызовы. Прежде всего это отражается на профессиональном самоопределении и
построении карьеры молодых людей.
Сегодня, чтобы быть конкурентоспособным на рынке труда, важны не только hard skills,
но особую значимость приобретают soft skills. Благодаря сформированности так называемых
«мягких навыков» современные специалисты могут успешно решать сквозные профессиональные задачи в русле непосредственной профессиональной деятельности: эффективно коммуницировать с разными людьми, гибко реагировать на изменения в деятельности, уметь работать в
ситуации многозадачности, выстраивать профессиональную деятельность в ситуациях неопределенности и т.п. Все это способствует успешному построению карьеры современного специа
листа. Чем раньше человек начнет осознанно задумываться о выборе профессионального и
карьерного пути, тем большей ответственностью и качеством будет отличаться его учебно-профессиональная деятельность, например, в ходе обучения в вузе.
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В период обучения по выбранному профессиональному направлению студент пытается
осознать, соответствует ли выбор его желаниям, личностным ценностям, жизненным целям и
смыслам. Если выбор профессии сделан правильно, то индивидуально-личностный потенциал
раскрывается подобно расцветающему цветку и открываются реальные перспективы для планирования и построения карьеры. Таким образом, складываются все предпосылки для профессионального развития и самореализации личности.
К сожалению, практика показывает, что современная молодежь испытывает серьезные затруднения в профессиональной социализации. Связано это с размытостью представлений о
выбранной профессии и построении карьеры в ней [1, с.61]. Чаще всего молодые люди ассоциируют карьеру с должностью и статусом в обществе, что на самом деле не всегда отражает
уровень профессионального самоопределения и самореализации их личности.
Вышеизложенное определяет необходимость эффективной профориентации и формирования карьерных ориентаций молодых людей на этапе их обучения в вузе. В этом и заключается
актуальность выбранной проблемы исследования.
Материалы и методы
Для решения задач исследования использован комплекс теоретических методов, адекватных природе и сущности построения карьеры и формирования карьерных ориентаций студентов на разных этапах обучения в вузе:
1) определение теоретико-методологической базы исследования карьерных ориентаций
студентов предполагает обращение к методам теоретического анализа (феноменологический,
логический, сравнительно-сопоставительный) и обобщения философских, социологических,
психолого-педагогических исследований;
2) научное обоснование изучаемой проблемы основано на методах теоретического анализа,
обобщения практического опыта, системно-структурном методе.
Методологическую основу исследования составляют философские положения о диалектическом методе познания, сущности деятельности, участие в которой преобразует личность;
теории системно-комплексного, личностно-деятельностного подхода; идеи формирования личности, становление профессионала; связи теории с практикой.
Основные положения
Важно понимать, что карьерные ориентации в структуре профессиональной направленности представляют собой динамическое образование. Они развиваются, трансформируются
вместе с развитием личности студента. Возможность изучения этого процесса позволит прежде
всего подобрать релевантные методы диагностики карьерных ориентаций студентов на разных
этапах обучения в вузе. С другой стороны, исследование теории вопроса, этиологии ключевых
понятий позволит нам выстроить четкую концепцию исследования и найти эффективные способы, позволяющие развивать карьерные ориентации студентов на разных этапах обучения в
вузе.
Таким образом, следуя логике организации научного исследования, целесообразно выделить существующие подходы к изучению карьерных ориентаций студентов, что позволит нам
выстроить стратегию собственного исследования.
По нашему мнению, формирование карьерных ориентаций напрямую коррелирует с профессиональной направленностью. Иначе говоря, они выступают в качестве значимого элемента
в системе профессиональной направленности личности, процесса построения карьеры.
Литературный обзор
Ю.П. Хяюринен выделяет две базовые модели исследования карьеры:
В первой модели изменения, которые происходят в обществе, и карьера взаимосвязаны.
В ней психологические и социальные факторы взаимодействуют между собой.
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Вторая модель акцентирует внимание на неизменности человеческой природы. Успех в
карьере связывают с возрастными особенностями и различиями между индивидами.
Исходя из данного подхода карьера, с одной стороны, рассматривается в качестве структурного компонента жизненного пути человека. С другой – выступает структурным элементом
рынка труда [2].
С позиции рынка труда довольно популярны исследования карьеры в сфере психологии
управления. Для работодателя важно создать благоприятные условия сотрудникам в продвижении их карьеры. Решение этих задач зачастую определяет необходимость разработки программ
развития карьеры в компаниях. Такой подход довольно популярен в управленческой деятельности западных компаний [3].
Термин «карьера» в советское время воспринимался зачастую негативно, поскольку ассоциировался у многих с погоней за должностями [4]. Это объясняется существующей в то
время общественной идеологией, ценностями общественно значимых целей в выполнении любой деятельности. Карьера связывалась с индивидуальностью, персонализированностью, что
противоречило установленным канонам в обществе.
Перемены в социально-политической и экономической жизни в конце ХХ столетия, распад
СССР привели к пересмотру отношения и взгляда на термин «карьера» на территории постсоветского пространства.
В российской науке основательно начал исследовать проблему карьеры Е.А. Климов, который рассматривал ее как часть профессионального самоопределения [5, c. 20–35].
Именно с началом появления его работ в российской и отечественной психологии сфор
мировался подход, в русле которого карьера понимается как процесс, направленный на поиск
путей реализации профессионального и личностного потенциала человека. Существенной
особенностью процесса построения карьеры является приложение человеком собственных
усилий, раскрытие своих способностей, умений, талантов, направленных на достижение
определенного статуса в обществе, успеха в профессиональной сфере. Так, по мнению
Е.А. Могилевкина, Л.В. Куликова и др. карьера – это последовательность этапов развития
человека в разных (основных) сферах жизни, которая помимо продвижения в профессиональ
ной сфере способствует получению значимости для других и признания своей неповтори
мости [6, 7].
Исходя из этого можно заключить, что карьера определяется не только успехом в профессио
нальной деятельности, но и успехом в жизни вообще. Подтверждение этой мысли мы находим
в трудах современных психологов [8].
Результаты и обсуждение
В построении нашего исследования мы разделяем точку зрения таких ученых, как
Д. Сьюпер [9] и Н.С. Пряжников, которые считают, что построение карьеры начинается и
зависит от успешности профессионального самоопределения. Действительно, человеку в ходе
его жизни приходится сталкиваться с выбором профессии после окончания школы, опреде
ляться после окончания вуза, при переходе в другую сферу деятельности и т.д.
Таким образом, карьеру мы понимаем, как процесс профессионального самоопределения
личности, основанный на развитии Я-концепции и ценностного отношения к определенной
профессиональной сфере.
Проведенный анализ исследований карьеры показал, что они актуализированы в сфере
управления и направлены на изучение связи между индивидуально-личностными характеристиками работника и построением карьеры; различных аспектов мотивации в построении карь
еры; особенностях карьерного роста в различных профессиональных сферах и т.п. [10].
Если обратиться к анализу психологических исследований карьеры и карьерных ориентаций в Казахстане, то необходимо отметить их малочисленность. Существующие исследования
в основном посвящены вопросам взаимосвязи карьерных ориентаций с различными аспектами
трудовой деятельности в разных профессиональных сферах [11, 12]. Хотя необходимо отметить, что проблема профессионального самоопределения, вопросы профориентации набирают
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актуальность и вызывают интерес у казахстанских исследователей, при раскрытии которых
авторы обращаются и к вопросам развития карьеры [13].
Однако исследований, посвященных изучению развития карьеры и карьерных ориентаций,
у студентов недостаточно.
Среди наиболее ярких и значимых исследований по указанной проблематике можно выделить работы американского ученого Э. Шейна [14].
Он проводил много эмпирических исследований, результаты которых показали важность
карьерных ориентаций как элемента профессиональной идентичности личности. В ходе эмпирических исследований им была установлена значимая взаимосвязь карьерных ориентаций с
развитием карьеры, что позволило определить карьерные ориентации в качестве определяющего фактора оригинальности, индивидуальности процесса управления карьерой. По мнению
Э. Шейна, карьерные ориентации тождественны карьерным установкам, в связи с чем начинают проявляться с началом приобретения профессионального опыта или профессиональной
социализации [15].
По мнению Э. Шейна, в любой профессиональной деятельности существует несколько
направлений продвижения личности, соответствующих ее карьерным ориентациям («якоря
карьеры»), которые, в свою очередь, связаны с представлением человека о себе. Исследование,
проведенное ученым, показало, что личностные характеристики человека, демонстрируемые
во время профессионального обучения, не являются определяющими факторами его будущей
карьеры. Это связано с тем, что в начале карьеры человек открывает для себя неожиданные
соответствия и расхождения собственных ценностей, потребностей, способностей с требованиями организационной среды, в которой он находится при выполнении своей работы. Постепенное прояснение образа «я», сопряженного с собственными мотивами, потребностями, ценностями и талантами в профессиональной сфере, было названо процессом нахождения «якоря
карьеры», который, стабилизируясь, начинает действовать в карьере как ведущая сила. «Якорь
карьеры» – это «группа потребностей, ценностей и талантов, реализация которых в наибольшей степени значима для человека при осуществлении профессиональных выборов» [15, с. 23].
В начале Э. Шейн выделил «пять якорей» карьеры: менеджмент, профессиональная компетентность, стабильность, автономия/независимость, предпринимательство. В ходе последующих исследований, проведенных в 80-х гг. прошлого века, ученым выделено еще три карьерные ориентации: вызов, служение, интеграция стилей жизни.
Карьерные ориентации, по мнению Н.И. Юртаевой [16], можно рассматривать в качестве
регулятора поведения личности в социальной среде, благодаря которому выбирается оптимальный способ достижения успеха в карьере. Иначе говоря, карьерные ориентации выражают способ достижения успеха в построении карьеры и ее ценности.
Заключение
Таким образом, проведенный анализ исследований указанной проблемы показал, что чаще
всего они сосредоточены на изучении различных аспектов развития карьеры в ходе трудовой
профессиональной деятельности. Как за рубежом, так и в нашей стране интерес исследователей привлекают аспекты взаимосвязи карьеры с личностными характеристиками, мотивацией
и др.
Актуализируются исследования, посвященные различным аспектам профессионального
самоопределения и его влияния на формирование успешной карьеры. Иначе говоря, большинство исследований реализованы в русле психологии менеджмента, организационной психологии и ориентированы на человека, который уже трудится (является сотрудником какой-либо
компании).
Однако мы считаем, что основы успешной карьеры закладываются еще на этапе обучения
в вузе. Основу карьеры закладывают именно карьерные ориентации, которые включены в сис
тему профессиональной направленности личности. В этой связи необходимо изучить особенности развития карьерных ориентаций на этапе обучения в вузе. Значимость этого, по нашему
мнению, определяется тем, что период обучения в вузе важен не только для формирования
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Scientific and practical journal EJCRP&P № 1(2) 2023
профессиональных компетенций, но и для определения целей, планов для успешного карьернопрофессионального развития.
Конкретизация представлений о своем профессионально-личностном будущем в русле осваиваемой профессии происходит благодаря карьерным ориентациям. Следовательно, изучение личностных факторов, определяющих карьерные ориентации студентов на разных этапах
обучения, и составляет перспективное направление нашего исследования.
ЛИТЕРАТУРА
1 Харланова Т.Н. Развитие карьерных ориентаций студенческой молодежи в процессе профессиональной подготовки. Дисс. ... канд. психолог. н. – М., 2015. – 261 c.
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6 Могилевкин Е.А. Карьерный рост: диагностика, технологии, тренинг / Е.А. Могилевкин. – СПб.:
Речь, 2007. – 336 с.
7 Куликов Л.В. Психология личности в трудах отечественных психологов: хрестоматия /
Л.В. Куликов. – СПб.: Питер, 2008. – 460 с.
8 Пряжников Н.С. Мотивация трудовой деятельности / Н.С. Пряжников. – М.: Академия, 2012. –
368 с.
9 Super D.E. The psychology of career / D.E. Super. New York: Evanston, Harper & Row, 1957. 362 р.
10 Соловьева Е.М. Подходы к определению понятия «карьерные ориентации» / Е.М. Соловьева //
Психология карьеры: материалы Всерос. научно-практ. конф. (заоч.), 2012. – С. 24–27.
11 Тажина Г.О., Таирова К.Т. Карьерные ориентации как предикторы организационной лояльности
преподавателей университетов // Bullletin-psysoc.kaznu.kz/ URL: http://doi.org/10.26577/JPsS.2020.v73.
i2.01 (дата обращения: 18.02.2023)
12 Лазаренко Д.В. Особенности мотивации и карьерных ориентаций государственных служащих //
«Public administration and civil service» International scientific-analytical journal. URL: http:repository.apa.kz
(дата обращения: 18.02.2023)
13 Сыздыкова К. Развитие системы профориентационной работы в школы как фактор расширения
доступа к высшему техническому и профессиональному образованию // Программа для молодых исследователей в области публичной политики Фонда Сорос-Казахстан. – Алматы, 2018. URL: http:soros.kz
(дата обращения: 20.02.2023)
14 Шейн Э. Организационная культура и лидерство: построение, эволюция, совершенствование /
Э. Шейн – СПб.: Питер, 2002. – 336 с.
15 Schein E.H. Career anchors: the changing nature of work and careers / E.H. Schein, J. Maanen. San
Francisco: Wiley, 2013. 88 p.
16 Юртаева Н.И. Управление карьерными ориентациями в профессиональном развитии студентов
инженерного вуза. Вестник Казанского технологического университета. – 2012. – Т. 15. – 1: 300–306. –
С. 37.
REFERENCES
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podgotovki. Diss. ... kand. psiholog. n. M., 261 p. (In Russian).
2 Zeer Je.F. (2003) Psihologicheskie osobennosti upravlenija kar'eroj / Je.F. Zeer, O.V. Taranova //
Ezhegodnik Rossijskogo psihologicheskogo obshhestva. Materialy 3-go Vserossijskogo s#ezda psihologov.
SPb. V. 3. (In Russian).
3 Moll E.G. (2012) Upravlenie kar'eroj menedzhera / E.G. Moll. SPb.: Piter, 198 p. (In Russian).
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4. Kirt N.L. (2000) Dinamika predstavlenij o professional'noj kar'ere psihologov obrazovanija v processe
professional'nogo samoopredelenija: dis. ... kand. psihologicheskih nauk: 19.00.07 / N.L. Kirt. Moskva, 158 p.
(In Russian).
5 Klimov E.A. (1998) Vvedenie v psihologiju truda / E.A. Klimov. M.: Juniti. P. 8. (In Russian).
6 Mogilevkin E.A. (2007) Kar'ernyj rost: diagnostika, tehnologii, trening / E.A. Mogilevkin. SPb.: Rech',
336 p. (In Russian).
7 Kulikov L.V. (2008) Psihologija lichnosti v trudah otechestvennyh psihologov: hrestomatija /
L.V. Kulikov. SPb.: Piter, 460 p. (In Russian).
8 Prjazhnikov N.S. (2012) Motivacija trudovoj dejatel'nosti / N.S. Prjazhnikov. M.: Akademija, 368 p. (In
Russian).
9 Super D.E. (1957) The psychology of career / D.E. Super. New York: Evanston, Harper & Row, 362 p.
(In English).
10 Solov'eva E.M. (2012) Podhody k opredeleniju ponjatija «kar'ernye orientacii» / E.M. Solov'eva //
Psihologija kar'ery: materialy Vseros. nauchno-prakt. konf. (zaoch.) P. 24–27. (In Russian).
11 Tazhina G.O., Tairova K.T. Kar'ernye orientacii kak prediktory organizacionnoj lojal'nosti
prepodavatelej universitetov // Bullletin-psysoc.kaznu.kz/ URL: http://doi.org/10.26577/JPsS.2020.v73.i2.01
(data obrashhenija: 18.02.2023). (In Russian).
12 Lazarenko D.V. Osobennosti motivacii i kar'ernyh orientacij gosudarstvennyh sluzhashhih // «Public
administration and civil service» International scientific-analytical journal. URL: http:repository.apa.kz (data
obrashhenija: 18.02.2023). (In Russian).
13 Syzdykova K. (2018) Razvitie sistemy proforientacionnoj raboty v shkoly kak faktor rasshirenija dostupa
k vysshemu tehnicheskomu i professional'nomu obrazovaniju // Programma dlja molodyh issledovatelej v oblasti
publichnoj politiki Fonda Soros-Kazahstan. Almaty. URL: http:soros.kz (data obrashhenija: 20.02.2023). (In
Russian).
14 Shejn Je. (2002) Organizacionnaja kul'tura i liderstvo: postroenie, jevoljucija, sovershenstvovanie / Je.
Shejn – SPb.: Piter, 336 p. (In Russian).
15 Schein E.H. (2013) Career anchors: the changing nature of work and careers / E.H. Schein, J. Maanen.
San Francisco: Wiley, 88 p. (In English).
16 Jurtaeva N.I. (2012) Upravlenie kar'ernymi orientacijami v professional'nom razvitii studentov
inzhenernogo vuza. Vestnik Kazanskogo tehnologicheskogo universiteta. V. 15. 1: 300–306. P. 37. (In Russian).
Г.О. Абдуллаева,*¹
пед.ғ.к., қауымд.профессор.
*e-mail: g.abdullayeva@turan-edu.kz
ORCID ID: 0000-0002-8359-3117
И. Сүлейменов,¹
магистрант.
e-mail: 22232256@turan-edu.kz
ORCID ID: 0009-0006-8299-4070
1
«Тұран» университеті, Алматы қ., Қазақстан
Студенттердің мансаптық бағдарлары: зерттеу тәсілдері
Андатпа
Мақалада студенттердің мансаптық бағдарларын зерттеудің өзектілігі мен қолданыстағы тәсілдері ашы
лып, зерттеудің алдағы болашағы мен стратегиясы анықталады. Зерттеудің әдіснамалық негіздері көрсетілген,
олардың арасында танымның диалектикалық әдісі туралы философиялық ережелер, қатысуы жеке тұлғаны
өзгертетін іс-әрекеттің мәні; жүйелік-кешенді, тұлғалық-белсенділік тәсілінің теориялары; жеке тұлғаны қа
лыптастыру идеялары, кәсіби маманның қалыптасуы; теорияның практикамен байланысы. Психологиядағы
мансап пен мансаптық бағдарларды түсіну тәсілдеріне талдау жасалады, сонымен қатар мансаптық бағдарлар
жеке тұлғаның кәсіби бағдарлау жүйесіне еніп, мансаптық құрылыстың негізін қалайтын авторлық ұстаным
көрсетілген.
Тірек сөздер: мансап, мансаптық бағдар, кәсіби өзін-өзі анықтау, кәсіби бағдар, ЖОО студенттері.
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Scientific and practical journal EJCRP&P № 1(2) 2023
G.O. Abdullaeva,*¹
c.ped.sc., ass. professor
*e-mail: g.abdullayeva@turan-edu.kz
ORCID ID: 0000-0002-8359-3117
I. Suleimenov,¹
master student.
e-mail: 22232256@turan-edu.kz
ORCID ID: 0009-0006-8299-4070
1
Turan University, Almaty, Kazakhstan
Career orientations of students: approaches to research
Abstract
The article reveals the relevance and existing approaches to the study of students orientations, identifies the
prospects and strategy for further research. The methodological bases of research, among which philosophical
positions about a dialectical method of knowledge, essence of activity, participation in which transforms a personality;
theories of system-complex, personality-activity approach; ideas of formation of a personality, formation of the
professional; connection of the theory with practice are reflected. The article presents the analysis of approaches
to the understanding of career and career orientations in psychology, and also shows the author's position, which is
expressed in the fact that career orientations are included in the system of professional orientation of the person and
lay the foundation for career building.
Key words: career, career orientations, professional self-determination, professional orientation, university
students.
65
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W4389558636.txt | http://doi.org/10.3126/njr.v13i2.59964
REVIEW ARTICLE
Intracardiac KCI Injection in Cesarean Scar Pregnancy: A Review Article
Gupta A1, Thapa M2
1
Department of Intervention Radiology, Sri Ganga Ram Hospital, New Delhi, India
Department of Intervention Radiology, Nepal Army Institute of Health Sciences, Kathmandu, Nepal
2
Received: September 25, 2023
Accepted: November 10, 2023
Published: November 24, 2023
Cite this paper:
Gupta A, Thapa M. Intracardiac KCl injection in cesarean scar pregnancy: A Review Article. Nepalese Journal of Radiology
2023;13(2):3-8. http://doi.org/10.3126/njr.v13i2.59964
ABSTRACT
Embryo implantation in a previous caesarean scar (CS) resulting in a caesarean scar pregnancy (CSP) is
another rare but potentially catastrophic complication of a previous caesarean birth. CSP can be detected
between days 33 and 94 of pregnancy. Methotrexate and potassium chloride (KCl) are the most common
feticidal agents used. intracardiac KCl causes feticide and reduces the uteroplacental blood flow, thereby
decreasing the rate of hemorrhage due to placenta previa, caesarian scar pregnancy and in cases of fibroids;
it is easy to evacuate the macerated fetus. Intracardiac KCl causes feticide and reduces the uteroplacental
blood flow, thereby decreasing the rate of hemorrhage and abortion time before termination of pregnancy
(TOP) leading to an easy evacuation of the macerated fetus. In conclusion, feticide with intracardiac KCl
is a safe procedure.
Keywords: Cicatrix; Leiomyoma; Methotrexate; Potassium Chloride; Pregnancy
INTRODUCTION
many factors including clinical presentation, β-hCG
levels, imaging features, and patient choice. In IR,
the local treatment is widely used for termination of
pregnancy using MTX as the most common
feticidal agent or other agents such as potassium
chloride, ethanol and hyperosmolar glucose.1,2,3,4,5,6,7
Cesarean scar pregnancy is defined as an ectopic
pregnancy embedded in the myometrium of a
previous Cesarean scar. The first case of CSP was
reported in 1978 by Larsen and Solomon. The first
detection of a 7-week cesarean scar pregnancy
made in transvaginal US occurred in 1990. Studies
indicate that CSP can be detected between days 33
and 94 of pregnancy. The incidence of CSP has been
estimated to range from 1/1800-1/2500 of all CD
performed and 1/531 for women who had at least
one cesarean delivery. To date there have been only
a few randomized studies on CSP and evidencebased management remains unclear. Until then,
treatment should be individualized according to
The technique of intracardiac KCl to induce fetal
demise is well established.27 Intracardiac KCl is
frequently used for fetal reduction in multiple
pregnancies. Its use to induce fetal demise to reduce
hemorrhage in medical termination of pregnancy is
not very frequent, however, intracardiac KCl causes
feticide and reduces the uteroplacental bl
Correspondence to: Dr. Manish Thapa
Department of Intervention Radiology
Nepal Army Institute of Health Sciences
Kathmandu, Nepal
Email: emaildrmanish@gmail.com
Licensed under CC BY 4.0 International
License which permits use, distribution and
reproduction in any medium, provided the
original work is properly cited
NJR VOL 13 No. 2 ISSUE 22 November, 2023
3
Gupta A et al. KCl
flow, thereby decreasing the rate of hemorrhage due
to placenta previa, caesarian scar pregnancy and in
cases of fibroids; it is easy to evacuate the
macerated fetus.
Types of CSP
There are two types of cesarean scar pregnancy
proposed by Vial et al. CSP directed towards
cervicoisthmic space or uterine cavity (type I,
endogenic type) and CSP with a deep invasion of
scar defect with progression towards the bladder
and abdominal cavity (type II, exogenic type).
The endogenic type of CSP could result in a viable
pregnancy; yet with a high risk of bleeding at the
placental site.13,14
METHODS
We searched MEDLINE/PubMed electronic
database using medical subheading search words
such as ‘caesarean scar pregnancy’; ‘caesarean
scar’; ‘caesarean scar local management’;
‘caesarean scar minimally invasive management’;
‘ectopic pregnancy’; ‘caesarean scar ectopic
pregnancy’; ‘pregnancy, previous caesarean scar’;
and ‘unusual ectopic pregnancies’ to obtain a
comprehensive list of articles concerning this
condition from January 1978 to March 2021. We
also searched using the ‘Scholar Google’ search
engine on the internet (www.scholar.google.com)
for articles on this topic since 1987. The citations
were cross-checked via related articles and links in
PubMed. Additional articles were obtained from the
cross-references of the relevant publications for
bibliographical purposes.
Presentation
The symptoms can be seen from 5-6 weeks and
as late as 16 weeks. Mild vaginal bleeding is the
presenting symptom in 39% and approximately
16% complain of associated mild to moderate pain
and 9% complain of only abdominal pain.15,16,17
Imaging
Ultrasound
It is the primary diagnostic tool for cesarean scar
and pregnancy The advent of TVUS has made it
possible to diagnose ectopic pregnancies at earlier
gestational ages, reducing the risk of significant
morbidity and mortality. The early diagnosis of CSP
at approximately 6 weeks of gestation and as late as
16 weeks of gestation enables non-surgical
treatment, resulting in short-term hospitalisation
and preserving fertility. Early correct CSP diagnosis
increases confidence and implementation of
minimally invasive surgery.18
Predisposing
factors,
mechanism
and
pathophysiology
The exact cause and mechanism is not well
understood but it is possible that the conceptus
penetrates the myometrium through a microscopic
dehiscent tract of the cesarean scar or the gestational
sac implantation occurs in a poorly healed cesarean
section scar. Such a tract can also develop from the
trauma of other uterine surgery, e.g. curettage,
myomectomy, metroplasty, hysteroscopy and even
manual removal of placenta. In CSP, the gestation
sac is surrounded by myometrium and the fibrous
tissue of the scar, quite separate from the
endometrial cavity. Cesarean delivery is associated
with increased risks of placenta previa, uterine
rupture and placenta accreta. Among women with
placenta previa and a previous cesarean section, the
risk of placenta accreta was significantly higher
when the placenta was implanted over the scar,
supporting the theory that trophoblast adherence or
invasion is enhanced when the scant decidualization
of the lower uterine segment is further impaired by
previous myometrial disruption.8,9,10,11,12
The following criteria have been followed for
diagnosis of CSP:5,19,20,21
An empty uterine cavity and endocervical canal.
Gestational sac located within the anterior
myometrium of previous cesarean scar site in LUS.
Color Doppler flow around the sac is characterized
by a high velocity (peak velocity 0.20 cm/s) and low
impedance (pulsatility index,1) blood flow;
Negative ‘sliding organs sign’, that is inability to
displace the gestational sac from its position at the
level of the internal os using gentle pressure applied
by the transvaginal probe.
less than 5mm of myometrium or scar tissue
NJR VOL 13 No. 2 ISSUE 22 November, 2023
4
Gupta A et al. KCl
separating the sac from the bladder
contamination through the needle tract during
insertion, the initial 1 ml of aspirated heart blood
was discarded. Thereafter, 2ml of 15% KCl was
injected into the fetal heart under direct vision. A
further 2ml was injected every 30 seconds until
permanent fetal asystole was achieved, and the time
was recorded. The fetal heart was observed on scan
for a further 5 minutes of asystole with the needle in
situ. To minimise any risk of maternal
contamination with KCl, the needle was flushed
with 5ml sterile water or saline before removal.
Provided there were no contraindications to vaginal
delivery, the women were given the choice to have
labour induced or to await spontaneous onset of
labour. Labour was induced if women did not go
into spontaneous labour within 3 weeks.26
In most of the cases, less than 5mm thickness of
myometrium between the gestational sac and
bladder has been reported.22
It can also help to detect placenta accreta spectrum
i.e. placenta or gestational sac embedded in the
hysterotomy scar.
Non-visualisation of myometrium between the
urinary bladder and sac can also help in
differentiation from cervicoisthmic pregnancy.8
In spontaneous abortion, the avascular gestational
sac should be seen in the cervical canal on TVS,
indicating detachment of sac from its implantation
site, whereas in caesarean scar pregnancies, the
gestational sac would appear well-perfused and
would be located in the anterior uterine wall at the
isthmus.23
Treatment
Because of the rarity of the condition, the majority
of CSPs are case reports or small case series
reported in the literature, with no consensus on the
preferred mode of treatment. Generally, termination
of pregnancy (TOP) in the first trimester is strongly
recommended, as there is a high risk of subsequent
uterine rupture, massive bleeding and lifethreatening complications. This, in turn, may
warrant life-saving hysterectomy and loss of future
fertility.
Magnetic resonance imaging
MRI imaging can be an added advantage in
equivocal cases for gestational sac location
confirmation and surrounding organs. MRI and
TVS are equally accurate in the diagnosis of CSP
but scar implantation can be better evaluated with it.
MRI T2-weighed sagittal section was used to
identify the cesarean scar defect, trophoblastic
layer, and myometrium separately. The MRI
findings of cesarean scar pregnancy are the same as
above mentioned sonographic findings concepts
implantation on cesarean section scar, myometrial
defect in association with empty endometrial cavity
and placenta accreta spectrum.14,24,25
There are following types of CSP intervention
Technique
At least half an hour before the feticide procedure,
all the patients were given pethidine 50 mg
intravenously for sedation and an anti-emetic if
necessary. After excluding allergies, a secondgeneration cephalosporin (mefoxin) 2gm was given
intravenously as a prophylactic antibiotic.
Lignocaine 2% was used to infiltrate the maternal
abdominal skin at the site of entry into the fetal
heart. A 20-gauge 15cm spinal needle was traversed
through the predetermined tract, targeting the left
ventricle or the most accessible chamber of the fetal
heart. To reduce the risk of feto-maternal
KCl only
KCl + MTX
MTX only
Expectant
Intracardiac potassium chloride (KCl) was first
described as a method for inducing fetal demise
in 1988 and has since grown in use to induce feticide
before an abortion, by either intracardiac or
umbilical
vein
administration.
Post-KCl
administration causes fetal asystole by disrupting the
balance of intra- and extracellular potassium, which
subsequently decreases myocyte action potential.
As a result of this in-induced demise, the fetal tissue
becomes macerated and softened, mimicking a
spontaneous fetal demise, and thereby theoretically
resulting in an easier surgical procedure.28,29
NJR VOL 13 No. 2 ISSUE 22 November, 2023
5
Gupta A et al. KCl
There are very few studies and case reports where
fetal demise is induced with intracardiac KCl before
medical TOP to reduce the risk of hemorrhage either
because of placenta previa or ectopic pregnancy at
the scar site or due to large fibroids distorting the
uterine anatomy.
birth. The exponential rise in its incidence during
the past 5-6 years may be a true increase in
incidence because of the rising caesarean section
rate worldwide or an apparent increase as a result of
a more liberal use of TVS in early pregnancy. Little
is known about its exact mechanism and natural
history.
According to Kaur et al., an average of 1.8cc of
intracardiac KCl was required to achieve cardiac
asystole which is less as compared to other studies.
This is because the gestational age was less (10+4
weeks to 19 weeks) in our study as compared to a
study by Sfakianaki et al. (range, 15.4-24.9 weeks)
where on an average of 10 mL of strong KCl was
required to reliably achieve fetal cardiac asystole.30,31
In conclusion, feticide with intracardiac KCl is a
safe procedure and reduces the risk of hemorrhage
and abortion time before TOP in scar pregnancy.
CONFLICT OF INTEREST
None
SOURCES OF FUNDING
Another study showed the average amount of
potassium chloride required in their study was
4.7ml (range 2–10 ml), much less than the amounts
reported by other authors. If the needle is correctly
positioned in the left ventricle, a minimal amount of
potassium chloride is required to achieve asystole as
the potassium chloride is injected rapidly into the
coronary circulation inducing immediate asystole.32
None
REFERENCES
The data shows that it is safe to use up to 20 mL of
15% KCl by the intracardiac route, but that on
average only 10 mL of strong KCl is required to
reliably achieve fetal cardiac asystole.33
The role of intracardiac KCl is to reduce the blood
flow and thus the risk of hemorrhage or to reduce
abortion time either in terms of conservative
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Tufa et al., and Lohr et al., Inducing fetal demise
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Uterine artery embolization has also been used in
previous studies to manage scar pregnancy but
uterine artery embolization is a more invasive
procedure with a longer duration than intracardiac
KCl.36
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W2995547367.txt | RESEARCH ARTICLE
Motion prediction enables simulated MRimaging of freely moving model organisms
Markus Reischl1☯, Mazin Jouda ID2☯, Neil MacKinnon2, Erwin Fuhrer ID2,
Natalia Bakhtina ID2¤, Andreas Bartschat1, Ralf Mikut ID1, Jan G. Korvink ID2*
1 Institute for Automation and Applied Informatics, Karlsruhe Institute of Technology, Hermann-vonHelmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany, 2 Institute of Microstructure Technology,
Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen,
Germany
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
☯ These authors contributed equally to this work.
¤ Current address: ETH Zurich, Zurich, Switzerland
* jan.korvink@kit.edu
Abstract
OPEN ACCESS
Citation: Reischl M, Jouda M, MacKinnon N,
Fuhrer E, Bakhtina N, Bartschat A, et al. (2019)
Motion prediction enables simulated MR-imaging
of freely moving model organisms. PLoS Comput
Biol 15(12): e1006997. https://doi.org/10.1371/
journal.pcbi.1006997
Editor: Rahul Singh, San Francisco State
University, UNITED STATES
Received: March 30, 2019
Accepted: November 8, 2019
Published: December 19, 2019
Copyright: © 2019 Reischl et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files.
Funding: Support for the C. elegans Archive and
for WormAtlas comes from NIH OD 010943 to D.
H. Hall. MJ, NM, EF, NB and JGK acknowledge
financial support from the European Research
Council (ERC) under grant 290586 NMCEL. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
Magnetic resonance tomography typically applies the Fourier transform to k-space signals
repeatedly acquired from a frequency encoded spatial region of interest, therefore requiring
a stationary object during scanning. Any movement of the object results in phase errors in
the recorded signal, leading to deformed images, phantoms, and artifacts, since the
encoded information does not originate from the intended region of the object. However, if
the type and magnitude of movement is known instantaneously, the scanner or the reconstruction algorithm could be adjusted to compensate for the movement, directly allowing
high quality imaging with non-stationary objects. This would be an enormous boon to studies
that tie cell metabolomics to spontaneous organism behaviour, eliminating the stress otherwise necessitated by restraining measures such as anesthesia or clamping. In the present
theoretical study, we use a phantom of the animal model C. elegans to examine the feasibility to automatically predict its movement and position, and to evaluate the impact of movement prediction, within a sufficiently long time horizon, on image reconstruction. For this
purpose, we use automated image processing to annotate body parts in freely moving
C. elegans, and predict their path of movement. We further introduce an MRI simulation
platform based on bright field videos of the moving worm, combined with a stack of high resolution transmission electron microscope (TEM) slice images as virtual high resolution
phantoms. A phantom provides an indication of the spatial distribution of signal-generating
nuclei on a particular imaging slice. We show that adjustment of the scanning to the predicted movements strongly reduces distortions in the resulting image, opening the door for
implementation in a high-resolution NMR scanner.
Author summary
Magnetic resonance imaging (MRI) requires its subjects not to move, since movement
will cause image artifacts. This is hard to achieve for adult humans, whom we can ask to
PLOS Computational Biology | https://doi.org/10.1371/journal.pcbi.1006997 December 19, 2019
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Motion prediction enables simulated MR-imaging of freely moving model organisms
Competing interests: The authors have declared
that no competing interests exist.
comply, but can currently only be achieved by sedation for other freely moving biological
specimens. Because of the importance of non-invasive MRI as a technique to also capture
metabolic information during activity, this is a huge deficiency of the methodology that is
hampering progress. In our paper we ask the question whether it is possible to computationally combine optical information on specimen movement with MRI. Our approach is
to predict the future movement and position of the specimen and thereby anticipate
where it will be so as to specify correct MRI parameters. Our computer simulations show,
for a freely moving worm, that a reasonable prediction is already possible for a short time
window, and that we can control the amount of error of the resulting MRI image. Importantly, with the continuous speedup of computation, our simulations suggest that it is
opportune now to implement such a system in hardware.
Introduction
A major challenge in biological science is to relate molecular regulation at the cellular level to
response and behaviour at the organism level. Knowing this relationship lies at the foundation
of every disease, and indeed also in understanding the healthy organism. An experiment establishing this relationship, as schematically shown in Fig 1, requires i) in vivo cellular-level
detection of regulation-relevant molecules, such as metabolites and their production rates,
ii) precise access to and application of biological perturbation mechanisms, and iii) an unburdened (naturally responding) organism. Clearly, even taken individually, these items are very
hard to achieve in general, and correspond to major research areas in their own right.
Fig 1. A freely moving organism is subject to a stimulus (mechanical, chemical, light, etc.) causing a response at
all levels of detail (metabolomic, behaviour, etc.). The experiment designer relates the correlated output to a
biological hypothesis and may adapt the stimulus.
https://doi.org/10.1371/journal.pcbi.1006997.g001
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Motion prediction enables simulated MR-imaging of freely moving model organisms
Small organisms such as C. elegans are biological model organisms useful for studying
many human disorders, including neurodegenerative diseases [1, 2]. Model organisms have
been the mainstay of biological sciences for decades, and thus a broad knowledge base already
exists starting from the genome level, through developmental cycles, and up to behavioural
response to applied stress. These platforms offer the opportunity to address the connection
between molecular phenotype, which can be conveniently implemented due to the very rapid
yet standardized life cycle of C. elegans, to behaviour. What remains is the technological challenge of satisfying the three requirements for robustly linking phenotype to behaviour.
Nuclear magnetic resonance (NMR), which is a noninvasive and non-destructive technique, together with its imaging modality (MRI), is a strong candidate as the analytical method
of choice, towards the ultimate goal of in vivo measurement of the molecular response. NMR
is based on exciting the spin-active nuclei of the magnetized organism with radio frequency
(RF) signals, and detecting their response via induced RF signals. Many nuclei are NMR sensitive, but in vivo molecular concentrations of metabolites are typically below millimolar levels,
requiring high sensitivity to detect. NMR is a non-ionizing technique, making it superior to
computed tomography (CT) based on X-rays.
Although MRI microscopy currently only achieves spatial resolutions down to 4 μm, new
techniques are partially overcoming these limits, such as the use of nitrogen vacancy centers in
diamond, or the use of hyperpolarisation techniques. MRI is a Fourier imaging technique that
uses magnetic field gradients to selectively excite parts of the object, and perform consecutive
phase and frequency encoding of the excited spins. This so-called spatial encoding makes MRI
a relatively slow imaging technique, as a single MRI image requires multiple consecutive signal
acquisitions from the object that is being imaged. In conventional MRI, it is assumed that the
organism (or object) being imaged is fixed in space, so that spatio-temporally varying magnetic
fields are only due to the technical system. Any movement of the organism results in measurements from shifted spatio-temporal positions resulting in image artifacts and ensuing difficulty
in data interpretation (see Fig 2). Motion-induced artifacts are the challenge since spatially
localized spectroscopy (required for molecular profiling) necessarily requires repeated measurements to bring the signal level above the noise.
In clinical applications of MRI, free body motion remains a challenge and there is a concerted effort underway to i) collect the MRI data faster than the motion; ii) collect the MRI
data at moments when motion is minimal (triggering); iii) track patient motion and correct
the MRI data during post processing; iv) track the motion and guide the spatial encoding to
reflect the instantaneous geometrical configuration [3–6]; v) predict the motion and adjust the
spatial encoding system in real-time, e.g., the prospective motion correction based on respiratory motion prediction [7]. Whilst these methods have been highly successful to control artefacts due to breathing, heartbeat, and low amplitude head movement, their assumptions for
the kinematics of the underlying movement or the periodicity of motion are limiting. For
example, the head is motion-captured in a model that assumes rigid six-degree-of-freedom
body movement involving translations and rotations (x, y, z, θx, θy, θz) along the three
orthogonal Cartesian axes. The direct translation of these techniques to MR measurements of
small samples is not straightforward primarily because of the reduced size, more complex
organism motion including writhing and wiggling, and rapid displacement across the detector’s sensitive region. Methods used to immobilise an organism can be considered to avoid
these motion artifacts, such as clamping or freezing; however, such drastic measures typically
introduce an undesired stress response into the molecular profile. The challenge of molecular
measurement of non-stressed, small model organisms therefore still remains open. Given the
advances in image processing, we believe there is an opportunity to address this challenge
computationally.
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Motion prediction enables simulated MR-imaging of freely moving model organisms
Fig 2. Concept of moving frame imaging: An object of interest is placed close to an NMR sensor (coil). For conventional
cartesian acquisition of an MR image, N repetitions are required to fill the k-space matrix. The signal of one repetition is used to fill
one k-space line. This acquisition scheme results in a total acquisition time of TR � N. The time scales of worm motion and repetition
time TR are of the same magnitude, and with a standard Eulerian or fixed frame MR procedure, strong motion blurring occurs. To
enable imaging free of blurring of a freely moving worm, the concept of a Lagrangian moving frame is required.
https://doi.org/10.1371/journal.pcbi.1006997.g002
Advances in computer vision have revolutionized the speed and accuracy with which image
analyses can be performed, and aim to reduce the need for expert knowledge e.g. in medical
image analysis [8] and automated experiment handling [9]. These technologies are entering
public awareness through the automation of highly complex processes, such as trajectory generation for self-driving vehicles (road, aerial) and surveillance for public safety. This is accomplished by real-time processing of dynamic images, in which accuracy and speed are of
paramount importance. To name an example, advances in image and data processing algorithms are expected to make real-time dynamic spectrum imaging (achieving a hyperspectral
imaging cube or hypercube) possible at all electromagnetic wavelengths. For instance, recording image and spectral data over 500 × 500 pixels, with a spectral resolution of 5nm over the
visible spectrum, at 5 f s−1 has already been demonstrated [10]. Enormous data storage and
processing power is required to perform such operations with sufficient spectral, spatial, and
temporal resolution, and has motivated an increased effort in sparse imaging modalities.
MRI also suffers from limitations in acquisition speed, which in this case are due to physical
constraints such as the relaxation times. This renders MRI a relatively slow imaging technique
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Motion prediction enables simulated MR-imaging of freely moving model organisms
compared to techniques that are based on the visual range of wavelengths. In MRI, data is
acquired in ð~
k; tÞ-space (~
k being the spatial frequency), and the observer requires the transformed data in ð~
x ; tÞ space. Hence, a large number of acquired data is associated with each
final image pixel or voxel, and motion or geometrical warping during imaging will introduce
errors into the reconstruction algorithms. To obtain sufficient acquired signal power per
voxel volume, object tracking must be implemented in order to ensure correct spatial and
spectral co-localization over time. This is especially important in MRI microscopy, which
depends on accumulated sampling for sufficient image resolution. With the novel capabilities
of image and data processing algorithms, real-time image processing becomes feasible. This
enables the extraction of information and motion prediction based on a conventional video
stream which then can be used to steer the MR sequence in real-time. The prediction horizon
will therefore be dependent on the signal acquisition time at a given frame rate and voxel spatial resolution.
In this contribution we consider the preconditions for performing MRI experiments on
unburdened small organisms. We will focus our attention on the nematode C. elegans, mainly
because it fits in best with our own efforts towards in vivo metabolomic profiling, but we
will address the underlying problem in more generality so that it is relevant also for other
organisms.
In this contribution we explore the possibility to completely remove the requirement of
organism immobilisation, by providing the host observing technical system (e.g., a spectrometer or microscope) with a real time co-evolving Lagrangian coordinate system centered in the
organism that provides the organism’s current center-of-gravity position and shape, paired
with a robust prediction of the organism’s future position and deformed shape.
There is a multitude of publications of so-called motion-trackers, which mainly quantify
rigid body motions and ignore strain fields. Important ones are [11] which categorises behavior and morphology features out of C. elegans-videos by applying segmentation and tracking
algorithms and [12], which applies methods of machine vision, data processing and tracking
to evaluate drug assays and [13] to follow multiple worm within the same environment. Furthermore, motion decomposition methods using so-called Eigenworms apply methods from
oscillation analysis to divide the worm motion into so called eigenmodes [14]. There are automated software-packages being able to quantitatively assess movement parameters (e.g. TrackA-Worm [15], Parallel Worm Tracker [16] etc.). However, there is a lack of an easy-to-use and
sufficiently fast prediction algorithm for individually chosen positions within a single worm to
be used to adjust the gradient system following a body part to be imaged. The prediction horizon available for the host system depends on the speed of image processing and latency of the
capturing hardware (essentially the frame rate), as well as the smoothness of the organism’s
motion. To gain insight into how movement prediction can enhance MRI signal detection, the
paper introduces:
• Data of a virtual phantom, combining high resolution electron microscope slice images [17]
with conventional video-recordings of C. elegans moving in a Petri dish (Section Phantom
Generation);
• A new concept for location prediction in C. elegans, and detailed movement prediction,
exploiting characteristic worm movements (Section MRI Simulation);
• A computational platform, being able to evaluate the outcome of MR imaging with and without adapting the imaging parameters based on the movement prediction (Section MRI
Simulation).
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Motion prediction enables simulated MR-imaging of freely moving model organisms
• A measure for simulation success that compares prediction to the ‘true’ image through a
similarity measure sxy, a technique first introduced in [18] and described in detail in the supporting information.
Using this simulation model, we demonstrate the capabilities of the motion-prediction
algorithm in MRI.
Materials and methods
Phantom generation
The study is based on eight 10 s duration AVI-videos (sampling frequency 12 Hz) of C. elegans.
The recording was done in a controlled environment with a fixed camera and constant illumination over time. The worm was enclosed in a technical setup including a microfluidic channel
and a Petri dish of the host system, suitable for optical recording and other real-time measurements. Typically, if natural state studies are relevant, the organism can also be provided with
an optically transparent nutritional substrate such as a gel containing E. coli bacteria.
To simulate MR imaging of the moving worm, we artificially linked transmission electron
microscope (TEM) images to the video of the worm, such that virtually scanning each location
within the worm would deliver a simulated high-resolution MR-image. At each time-frame,
the 1.2mm worm was segmented into 50 slices perpendicular to its center line. This resulted in
a reasonable slice thickness of roughly 24μm. Subsequently, we assigned voxel MR signals
adapted from the TEM images to each slice as follows: first, we removed the background from
the TEM images and scaled them such that the dimensions of the body part in each slice were
realistic. Second, we inverted the color map of the images, since the white regions of the TEM
images corresponded to low density material, and therefore would appear dark in MR imaging
due to the low proton signal intensity. This color inversion is just to make the sample images
look like MR images. The reader should bear in mind that TEM and MRI use completely different imaging mechanisms, and therefore converting one to the other is not a straightforward
task. In fact, having true MRI sample images is not essential to prove the efficiency of motion
prediction in imaging enhancement, and indeed any arbitrary set of morphologically correct
sample images would work. Finally, we reduced the number of pixels of the TEM images to
64 × 64, which corresponded to an MRI in-plane resolution of approximately 1.6 μm, and then
assigned the voxel signals to the virtual MR slices. Although such a high volumetric resolution
lies beyond the capabilities of currently available MRI scanners, it was chosen on purpose to
allow more accurate assessment of the prediction algorithm. It is thus a good compromise
between being close to a realistically achievable resolution, and being high enough for the
structural similarity measure to work more accurately. Furthermore, considering the rapid
advances in the field of MRI including higher fields, stronger gradients, and hyperpolarization
techniques, the mentioned resolution of 1.6μm is believed to be achievable in the near future.
Motion prediction
New concept. In order to adjust the gradient system of the MRI, the worm needs to be
detected and its future positions need to be predicted. Therefore, we introduced a new concept
of real-time image processing of the worm, suitable for any video format, regardless of color
model, and also independent of background structures.
Fig 3a shows a block diagram of the basic steps of the concept. Starting with a raw image
matrix Xraw, a robust estimation of the worm and its position Xworm within the video were
determined. Based on the segmented worm, characteristic points such as center of gravity
(COG) and position of the head region were determined. To introduce a coordinate system
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Motion prediction enables simulated MR-imaging of freely moving model organisms
Fig 3. a: Concept for the prediction of defined locations within a worm, b: Parametrization of the worm, c-j: Snapshot of a sample
video. c: grayscale image/video Xraw[k], d: Image with subtracted background, e: difference image, f: overlay with worm, g:
skeletonization, h-j: Point of interest (POI, gray marker) and predictions for Δk = 30 (= 2.5 s) time samples (white marker). h-j: Time
samples k = 50, k = 80, k = 110. Figures show the position every 30 samples and the prediction of the POI 30 samples ahead. Thus,
the similarity of POI(80) vs. POIpred(80) and POI(110) vs. POIpred(110) shows the quality of the prediction.
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along the center line of the worm, a skeletonization was applied, see Fig 3b. Assuming that
body regions excluding the head would move along the center line, the worm velocity was
used to predict positions xc,p of arbitrary selected points xc within the worm. A detailed
description is given in the supporting information.
Worm detection. This section briefly covers the worm detection and movement
prediction. The preprocessing for the worm detection aims to reduce the computational complexity and consists of a grayscale conversion (standard Matlab conversion) of the videos as
well as a decrease of the resolution by a factor of 9 (Fig 3c) (Decreasing the resolution proves
real time processing to be possible / usage of low-end imaging hardware to be accurate (see
Discussion)). For the segmentation of the worm, the background of the video is estimated and
removed from the images (Fig 3d).
The direction of motion is determined by relying on the position of the head and the COG.
The COG is calculated based on the foreground pixels of the segmentation and is smoothed to
obtain variations for future predictions (1st order low-pass filtering over time). The position of
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Motion prediction enables simulated MR-imaging of freely moving model organisms
Fig 4. Evaluation (mean values and standard deviation) of the dataset given in section phantom generation. a: fixed location in
the worm (s = 0.9), varying prediction horizons, b: fixed prediction horizon (Δk = 12, 1 second, varying locations, prediction horizon
too high for s < 0.3).
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the head is computed utilizing difference-images (Fig 3e) of the last 10 frames in the video. Fig
3f shows the detected head.
Coordinate prediction. The position of the COG moves linearly and is predicted by linear
extrapolation based on its past five positions. To predict the position of an arbitrary point of
interest (POI) within the worm (which can easily be identified e.g. by a mouse click in the an
NMR-simulation), we use the skeleton line as the center line of the segmentation and introduce a normalized coordinate s along it (head: s = 0, tail: s = 1, see Fig 3b and 3g).
Assuming that each worm segment moves according to the current shape of the worm, following its predecessor segment with the velocity of the worm (The assumption is not perfectly
valid, but it significantly simplifies processing and delivers reasonable results (see also Fig 4)),
the velocity and the shape is used to predict the location sc of the POI after Δk time steps using
sc = s − vΔk, (Fig 3h–3j) (If the prediction horizon is chosen too high, negative sc are avoided
by setting the prediction to the topmost point (s = 0)).
The prediction quality is evaluated using an Euclidean distance. Given the prediction horizon Δk, the predicted position based at time point k + Δk is calculated (x1,c,p[k + Δk], x2,c,p[k +
Δk]) and the distance to the true position (x1,c[k + Δk], x2,c[k + Δk]) is measured. The average
for all time samples is termed the mean prediction error. The dependency of the prediction
accuracy to the prediction horizon, as well as to the predicted position of the worm, is given in
Fig 4: The prediction error increases if the prediction horizon becomes larger. Regions near
the head cannot be reliably predicted, since the head moves at a much higher frequency and in
random patterns while scanning the surroundings and deciding on the moving direction. Furthermore, if the prediction horizon is set too high the predicted point lies outside the current
shape of the worm (e.g. for Δk = 12 for all s < 0.3) (depending on the speed of the worm, however wild type worms mostly have the same velocity of approximately 0.13 mm s−1) and the
topmost point (s = 0) is chosen as prediction, resulting in deviations).
For all other regions, the uncertainty of the prediction stays roughly constant.
Results
MRI simulation
Simulation-platform. The magnetic resonance imaging process is simulated using
Matlab. The worm is assumed to reside within a strong and constant magnetic field B = (0, 0,
Bz) of the MRI scanner. The software mimics a simplified yet acceptably accurate MR image
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Motion prediction enables simulated MR-imaging of freely moving model organisms
acquisition process via a standard gradient-echo sequence [19] that takes place during worm
motion as follows:
• An image slice at a parametrised position s along the worm axis is defined as the image
plane perpendicular to the worm axis. As previously mentioned, the worm is segmented
into 50 slices whose voxels’ intensities are obtained from the TEM images. Thus s is the
center of one of those slices. Ideally, to do MRI of a slice s, the center of the gradient system
(the field of view FoV), GC, should be exactly at s over the entire signal acquisition process.
This is unfortunately not the case as the prediction imposes some error resulting in GC
being at another slice, at the desired slice but off-center, or both. In this case the MR
excitation is done by calculating the signal intensity of each voxel in the FoV depending
on the position of GC. If, for instance, GC is off-center, then some voxels of the true slice
will be out of the FoV and the corresponding ones in the FoV will then be filled with zeros.
The excitation step is followed by a phase encoding step, in which each voxel of the FoV
is given a phase proportional to its position along one cross-sectional axis ξ. After a time
delay TE/2, where TE (echo time) is the time from excitation to the center of the MR
signal (echo), a frequency encoding gradient is applied, whereby each voxel of the FoV
is assigned a frequency proportional to its position along an axis η perpendicular to
the phase encoding axis (so that ξ � η = 0). The superposition of all voxel RF signals are
simultaneously detected by a virtual coil. This so-called echo is recorded at instant TE,
resulting in one line of k-space. The detection system is assumed to have a uniform spatial
sensitivity.
• After a time delay TR (repetition time), which is the time it takes to repeat the sequence in
order to acquire a new line of the k-space, the algorithm calculates the anticipated new position and orientation of the selected slice (as the worm would have already moved to a new
position). Now the slice at this new position is excited, phase encoded, and frequency
encoded, resulting in a new line of k-space being filled.
• The entire process is repeated until all the lines of k-space (in our case 64 lines) are complete.
Once the imaging procedure is complete, the program reconstructs the anticipated MR
image from the k-space data via Fourier transform.
Algorithm 1 Gradient-echo MR imaging
1: procedure GRE(TE, TR)
⊳ Echo time and repetition time.
2:
s = 0
⊳ s parametrises slice position along worm axis.
3:
while s 6¼ 1 do
⊳ cycle through the slices.
4:
s
s + δs
⊳ Next slice.
5:
t = 0
⊳ Initialize time counter.
6:
N = 64
⊳ Number of k-space lines.
7:
k = −π
⊳ Set initial phase.
8:
J = 1
⊳ Set index.
9:
while t 6¼ N � TR do
10:
Pos. Pred.
⊳ Predict next position.
11:
Mov. Grad.
⊳ Update imaging gradient.
12:
Exc.
⊳ Excite slice.
13:
t
t + TE/2
⊳ Advance time to half echo time.
14:
Ph. Enc. (k + J � 2π/N)
⊳ Phase encode.
15:
t
t + TE/2
⊳ Advance time to echo time.
16:
Freq. Enc.
⊳ Frequency encode.
17:
Echo
⊳ Signal acquisition.
18:
t
t + TR − TE
⊳ Advance time to next line.
19:
J
J + 1
20:
end while
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Motion prediction enables simulated MR-imaging of freely moving model organisms
21:
end while
22: end procedure
The simulation is integrated into an interactive graphical user interface (GUI) to enable the
user to set and change the imaging parameters easily and execute the processing without any
programming skills (Fig 5).
The simulated MRI signals can be chosen by the user and are either MR-like signals translated from real transmission electron microscopy (TEM) images, or any virtual MR-like
images provided by the user.
To start the simulation, an arbitrary slice of interest (with coordinate s) within the worm’s
body is selected. For this point, the simulated MR image of a slice without movement is
shown. In the display, the slice position s is denoted by a red line perpendicular to the center
line of the worm, while the predicted position to which the virtual imaging gradient coordinate
is set is denoted by a green line.
Furthermore, the software allows the user to set the TR and TE imaging parameters, and to
choose the prediction horizon (a value between 1 and 10 frames), which refers to the number
of frames ahead for which the predicted worm position will be calculated. Our algorithm does
not correct for the worm motion occurring during recording of a line of k-space. This is equivalent to the assumption that the MRI pulse sequence is based on very short echo times, which
is possible, but taken at the expense of increased acquisition bandwidth and thus reduced signal-to-noise ratio (SNR).
Simulation paradigms. We performed three MRI simulations using the optical video
dataset from Section Phantom Generation to:
Fig 5. Graphical user interface of a Matlab program that simulates the MR imaging experiment of a moving worm. The
microscope movie of the worm is shown to the bottom left, and emulates real-time observation. The slice position is also shown. The
top right shows the phantom MR image of the worm at the position of the slice.
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Motion prediction enables simulated MR-imaging of freely moving model organisms
1. confirm that the prediction enhances the MR imaging of the moving worm in general (Simulation 1);
2. measure the efficiency of the prediction algorithm as the desired resolution of the MR
images increases in comparison to the resolution of the optical video used for the prediction
(Simulation 2); and
3. measure the effect of an increased prediction horizon (the number of frames ahead for
which the position is predicted, Simulation 3).
In all the simulations, the repetition time (TR) was set to 83 ms, which corresponds to the
frame rate of the optical video on which the prediction was based.
We evaluate the simulation by quantifying the structural similarity sxy 2 f0; 1g 2 R between
the true image and simulated image as introduced in [18]. Identical images return sxy = 1,
whereas structural inequality delivers sxy = 0.
Body position (Simulation 1). In Simulation 1, three slices (slice definition from Section
Phantom Generation, first slice from the head (s = 0.02), second from the middle body
(s = 0.5), third from the tail (s = 1) of the worm) are selected and an MR imaging simulation is
performed (prediction horizon Δk = 1 (83 ms)).
The results are illustrated in Fig 6a, each row shows the true slice on the left, the simulated
MR image based on the prediction algorithm in the middle, and the simulated image when no
position prediction is involved on the right.
Clearly, the prediction algorithm significantly reduces the motion artifacts that would otherwise occur if the gradient system did not follow the worm as it moves. Moreover, we observe
that prediction quality varies along the worm’s body. More specifically, the prediction performs better for the slices from the body and tail when compared with prediction of the head.
This is axiomatic, since the worm rapidly moves its head laterally whilst scavenging for nutrition, thus the motional entropy of the head is higher (see also Fig 4). Fig 6b shows the simulation results of eight videos of different worms. The abscissa represents the slice position
starting from the head (s = 0) to the tail (s = 1), while the ordinate shows the structural
Fig 6. a: effect of position prediction on the quality of the MR images in the head (s = 0.02), body (s = 0.5) and tail (s = 1) regions.
The prediction horizon in this case is 83 msec. b: dependence of the MR imaging quality (structural similarity) on the position of the
slice. The figure displays the simulation results from eight videos of different worms.
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Motion prediction enables simulated MR-imaging of freely moving model organisms
Fig 7. Effect of decreasing the ratio of the MR image resolution to the optical video resolution on the imaging
quality for a prediction horizon of 1. The rows show the results for MR image:video resolution ratios of 4:1 and 2:1
respectively. Each row displays, from left to right, the reference slice, the MR image with position prediction, and the
MR image without position prediction.
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similarity between the prediction-based MR images and the original slices. To very good agreement with Figs 4 and 6b shows that the quality of the prediction-based MR images are higher
for slices from the body and tail than the images taken near the head where the prediction
uncertainty is usually higher.
Image resolution (Simulation 2). In Simulation 2, a slice from the middle body section of
the worm (s = 0.72) was selected and an MRI simulation was performed for different resolution ratios (= MR image: optical video). The idea here is to explore the effect of changing the
MRI resolution (voxel size) on the quality of the prediction-based imaging. Imagine, for example, that one pixel of the optical video is 10 μm × 10 μm and the desired MRI isotropic resolution is also 10 μm × μm × 10 μm × 10 μm, then one pixel error in prediction results in one
voxel error in the MRI k-space. On the other hand, if higher MRI resolution is wished, then
one pixel error in prediction results in more than one voxel error in the MRI k-space. Decreasing the optical resolution can be used to speed up image processing, and thus decreasing the
prediction horizon, if needed. The ratios of 4:1 and 2:1 are heuristically chosen, for which each
pixel in the optical video respectively corresponds to 4 and 2 pixels in the MR image. Fig 7
demonstrates the results of this simulation—the rows correspond to the different resolution
ratios while the columns depict the original slice, the image with prediction, and the image
without prediction. The figure shows that the efficiency of the prediction declines as the
desired resolution of the MR image increases (pixel size decreases), or alternatively, the optical
resolution should be higher or at least equal to the desired MR resolution for a high quality
prediction-based MR image. Indeed, the commercial optical imaging solutions can easily meet
such demands of high resolution; however, upon implementation, any increase in number of
pixels will be at the expense of the prolonged processing time needed for prediction. Nevertheless, this can be overcome by utilizing more powerful processors and by employing techniques
such as parallel computing.
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Fig 8. Effect of enlarging the prediction horizon on the quality of the imaging (s = 0.52, see Fig 6). The images (1a-10a) show the
reconstructed MR images based on the prediction algorithm for prediction horizons from 1 to 10 frames. The images (1b-10b) show
the reconstructed MR images when no prediction is involved and when the initial position of the imaging gradient is set to the
position of the slice after 1 (83 ms) to 10 (830 ms) frames respectively. (c) The similarity measure of the simulated image versus the
prediction horizon.
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Prediction horizon (Simulation 3). Simulation 3 varies prediction horizons for one slice
(s = 0.52). Fig 8 illustrates the results of this simulation: Fig. (1a–10a) are the prediction-based
simulated images for prediction horizons of Δk 1 to 10, respectively. In contrast, Fig. (1b–10b)
display the results when prediction is not in action. Because only a few lines of the k-space are
collected from the correct slice (depending on TR and the speed of the worm), the images in
this figure exhibit a noticeable decrease in quality as the prediction horizon increases, leading
to the conclusion that one should, whenever possible, minimize the prediction horizon. Of
course, in an actual hardware realization of the system, the choice of the prediction horizon
will be bounded by the speed of the image acquisition and processing units.
The effect of increasing the prediction horizon is described quantitatively in Fig 8c by measuring the structural similarity between the original slice and the simulated image for both
cases with and without prediction. The results confirm the variation in predictability along the
length of the worm. The prediction horizon degradation is linear for the rear two-thirds of the
worm, but falls off more rapidly for the head section, as expected from Fig 4.
Moreover, a statistical assessment of the effect of the prediction horizon on the quality of
the MR imaging was done: Regarding simulation results of four slices along the worm using
the given eight videos it can be shown that the accuracy of prediction and thus the MRI quality
decays with the increased prediction horizon but is in mean for all parameter combinations
roughly three times better than without prediction.
Discussion
The results demonstrate that our prediction algorithm can markedly improve MR image quality of arbitrarily moving and deforming objects. The complete processing pipeline (including
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Motion prediction enables simulated MR-imaging of freely moving model organisms
functions which will not be used in real-time processing, e.g. rotation, prediction of COG,
bounding boxes etc.) takes 76 msec per frame on an average Laptop PC (core I7, fifth generation, 16 GB RAM) and has various possibilities for optimization (modern hardware, software
parallelisation, hardware-filtering etc.). Decreasing the resolution of the optical video offers an
additional option to reduce processing time up to a factor of 8 without significant loss of quality. Based on the current implementation, real-time processing is already possible for sample
frequencies smaller than 13 Hz and application of the algorithm to a real MRI system is
feasible.
For the predictive information to be useful, the characteristic time (velocity) of sample
motion tmotion and predictive accuracy needs to be determined. This can be done by recording
a freely moving sample with the desired optical setup followed by an image analysis routine as
described in this report. Models of the motion kinematics can be tested in order to maximize
the prediction horizon given a user-specified predictive accuracy threshold. The threshold can
be selected based on the expected error introduced within a single voxel with pre-selected
dimensions. The predictive horizon together with the calculation time tpred then can be used to
determine if the sample is ‘MR imageable’, i.e. by comparing the timescales to those required
for MRI. For example, in this report it was observed that a prediction horizon of 83 ms requiring a calculation time of 76 ms yielded a predictive accuracy of approximately 57% as indicated
by the structural similarity measure of the prediction-based MRI simulation, Fig 8c. On the
other hand, a prediction horizon of 830 ms requiring a calculation time of 76 ms resulted in a
prediction accuracy of approximately 30%.
With the timescales of motion and predictive calculation defined, one must evaluate
whether MRI is possible by comparing to the instrumental timescale (Only a standard cartesian MRI sequence is considered. The authors acknowledge the existence of more advanced
sampling schemes, but these are outside the scope of the present discussion). The shortest relevant timescale is the time between spatial encoding steps, which in the case exemplified here is
the repetition time TR (TR includes TE and the data acquisition time). It is during TR that prediction and hardware adjustments must be done prior to the subsequent spatial encoding step.
In the MRI simulations described here, TR was 83 ms (including a TE of 4 ms) while tpred was
76 ms. Given the organism motion and hardware/experiment timescale regimes, one can estimate the potential for sample imaging with correction, summarized as follows:
• tmotion < TR: the object is not MR imageable without motion artifacts. Conditions to slow
the natural motion of the sample should be identified and implemented.
• TR < tmotion, tpred: the object is MR imageable. Careful choice of TE and TR must be done so
that the prediction calculation is complete before the next spatial encoding period. This
places a restriction on the types of contrast that can be implemented.
• tpred < TR < tmotion: the object is MR imageable. There is no restriction on the contrast
weightings that can be implemented.
To further improve the predictive quality, it is important to have kinematic models appropriate to the organism to be imaged. C. elegans is a convenient model for this reason given the
advanced studies about behavioural phenotypes [11, 12, 20] and motion decomposition using
so-called Eigenworms [14]. Extension of these models to organisms featuring similar motion
characteristics should be straightforward (i.e. oscillation/undulation along the long axis—
worms, snakes, swimming fish). As the kinematics becomes more complicated and/or sporadic, it will become necessary to introduce a method to guide the organism in order to introduce a predictive nature to its motion (food source, temperature gradient, etc.). Alternatively,
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Motion prediction enables simulated MR-imaging of freely moving model organisms
shorter prediction horizons can be targeted together with faster MR imaging sequences such
as the echo planar imaging (EPI), most likely at the expense of spatial resolution.
To bring the presented method of in-situ real-time video capturing and motion prediction
experiments for MR microscopy into daily routine, we so far lack available MR-compatible
optical microscopes for high-field vertical bore magnets. However, there is strong progress to
reach this technical integration in the near future. Initial results have already been presented in
the early 2010s and a patent describing the adaptive objective to be integrated into an optical
system has been granted in 2019 under the title “MR-compatible microscope, EP 2824471B1
(see e.g. https://worldwide.espacenet.com)”. Once the remaining hardware challenges are
overcome the presented procedure for a tracking control loop will give the possibility to fully
exploit and expedite these novel techniques for conducting MR microscopy of free-moving,
undisturbed microscopic organisms.
Supporting information
S1 Text. This file describes the principal structure of the motion prediction algorithm.
(PDF)
S1 Fig. Structural similarity versus prediction horizon for slices (s = 0.1, s = 0.3, s = 0.5,
and s = 0.9) for eight videos.
(TIF)
Acknowledgments
We sincerely thank Dr. David H. Hall of Albert Einstein College of Medicine in New York for
providing the high resolution images of C. elegans that we used as phantoms for high resolution NMR images. These lent our images more realism. The original images are from the
MRC/LMB C. elegans Archive now curated by the Hall lab. These were generously donated by
John White and Jonathan Hodgkin to the Hall lab.
Author Contributions
Conceptualization: Ralf Mikut, Jan G. Korvink.
Data curation: Neil MacKinnon, Jan G. Korvink.
Formal analysis: Mazin Jouda, Neil MacKinnon.
Funding acquisition: Jan G. Korvink.
Investigation: Markus Reischl, Mazin Jouda, Neil MacKinnon, Natalia Bakhtina, Andreas
Bartschat.
Methodology: Markus Reischl, Mazin Jouda, Neil MacKinnon, Erwin Fuhrer.
Project administration: Neil MacKinnon, Ralf Mikut, Jan G. Korvink.
Resources: Ralf Mikut.
Software: Markus Reischl, Mazin Jouda, Natalia Bakhtina, Andreas Bartschat.
Supervision: Ralf Mikut, Jan G. Korvink.
Validation: Mazin Jouda, Natalia Bakhtina.
Visualization: Markus Reischl, Mazin Jouda, Erwin Fuhrer, Andreas Bartschat.
Writing – original draft: Markus Reischl, Ralf Mikut, Jan G. Korvink.
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Motion prediction enables simulated MR-imaging of freely moving model organisms
Writing – review & editing: Markus Reischl, Mazin Jouda, Neil MacKinnon, Erwin Fuhrer,
Ralf Mikut, Jan G. Korvink.
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W1499177907.txt | British Journal of Cancer (2000) 82(12), 1984–1990
© 2000 Cancer Research Campaign
doi: 10.1054/ bjoc.2000.1163, available online at http://www.idealibrary.com on
Enhancement of chemotherapy and radiotherapy of
murine tumours by AQ4N, a bioreductively activated
anti-tumour agent
LH Patterson1, SR McKeown2, K Ruparelia1, JA Double3, MC Bibby3, S Cole4 and IJ Stratford4,5
1
School of Pharmacy and Pharmaceutical Sciences, De Montfort University, Leicester LE1 9BH, UK; 2School of Biomedical Sciences, University of Ulster,
Jordanstown, Newtownabbey BT37 OQ, Northern Ireland; 3Clinical Oncology Unit, University of Bradford, Bradford BD7 1DP, UK; 4MRC Radiation and Genome
Stability Unit, Harwell, Oxon OX11 ORD, UK; 5School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, UK
Summary AQ4 (1,4-Bis-{[2-(dimethylamino-N-oxide)ethyl]amino}5,8-dihydroxyanthracene-9, 10-dione) is a prodrug designed to be excluded
from cell nuclei until bioreduced in hypoxic cells to AQ4, a DNA intercalator and topoisomerase II poison. Thus, AQ4N is a highly selective
bioreductive drug that is activated in, and is preferentially toxic to, hypoxic cells in tumours. Five murine tumours (MAC16, MAC26, NT,
SCCVII and RIF-1) have been used to investigate the anti-tumour effects of AQ4N. In only one tumour (MAC16) was AQ4N shown to be
active as a single agent. However, when combined with methods to increase the hypoxic tumour fraction in RIF-1 (by physical clamping) and
MAC26 tumours (using hydralazine) there was a substantial enhancement in anti-tumour effect. Notably, RIF-1 tumours treated with AQ4N
(250 mg kg–1) followed 15 min later by physically occluding the blood supply to the tumour for 90 min, resulted in a 13-fold increase in growth
delay. When combined with radiation or chemotherapy, AQ4N substantially increased the effectiveness of these modalities in a range of in
vivo model systems. AQ4N potentiates the action of radiation in both a drug and radiation dose-dependent manner. Further the enhancement
observed is schedule-independent with AQ4N giving similar effects when given at any time within 16 h before or after the radiation treatment.
In combination with chemotherapy it is shown that AQ4N potentiates the activity of cyclophosphamide, cisplatin and thiotepa. Both the
chemotherapeutic drugs and AQ4N are given at doses which individually are close to their estimated maximum tolerated dose (data not
included) which provides indirect evidence that in the combination chemotherapy experiments there is some tumour selectivity in the
enhanced action of the drugs. © 2000 Cancer Research Campaign
Keywords: AQ4N; radiotherapy; bioreduction; cyclophosphamide; cisplatin; thiotepa
Hypoxia occurs in regions of solid tumours as a result of poorly
formed micro-vasculature coupled with the metabolic demand of
tumour cells close to the available blood supply; this compromises
the oxygen availability to more distant tumour cells (Brown and
Giaccia, 1994). Hypoxic regions are known to be resistant to
radiotherapy and conventional chemotherapy and this has led to
much interest in the discovery of cytotoxins that kill hypoxic
tumour cells (reviewed in Stratford and Workman, 1998). These
agents are designed to be bioreduced selectively in cells low in
oxygen to produce potent cytotoxins. Synthetic compounds with
chemotherapeutic potential as bioreductive agents are usually
based on nitroaromatic or quinone structures, e.g. nitroimidazoles
and mitosenes (indoloquinones) (Workman and Stratford, 1993;
Denny et al, 1996; Patterson and Raleigh, 1998). These agents are
cytotoxic, on reduction, due to their ability to covalently bind to
DNA that will not occur in oxygenated cells. A benzotriazene diN-oxide, tirapazamine, is currently the most promising bioreductive drug and is in phase II/III clinical trials. This agent can act as
Received 1 October 1999
Revised 11 February 2000
Accepted 17 February 2000
Correspondence to: LH Patterson, The School of Pharmacy, University of
London, 29/39 Brunswick Square, London WC1N 1AX, UK
1984
a chemosensitizer and radiation enhancer due primarily to toxicity
of the free radical species formed transiently during its reduction
to the non-toxic reduction product SR4317. The toxic effect is
probably caused by direct local damage to DNA that results in
DNA strand-breaks and chromosome breaks that are difficult to
repair (Brown and Wang, 1998).
To date, therefore, bioreductive agents generate transient reactive species that are entirely dependent on hypoxia to generate
the toxic species. In contrast, we report here preclinical data on
a new class of bioreductive agent exemplified by AQ4N (1,4-bis{[2-(dimethylamino-N-oxide)ethyl]amino}5,8-dihydroxyanthracene-9, 10-dione). This agent is a prodrug designed to be
excluded from cell nuclei (Patterson, 1993; Smith et al, 1997b)
until metabolized in hypoxic cells to give AQ4, a stable, oxygeninsensitive metabolite (Smith et al, 1997a) (Figure 1). AQ4 is a
DNA intercalator and potent inhibitor of DNA type II topoisomerase (Patterson, 1993; Patterson et al, 1994; Smith et al, 1997a).
Previously we have shown that AQ4N is an effective enhancer of
the anti-tumour effect of radiation in one murine tumour model,
i.e. the T50/80 mammary carcinoma grown in BDF mice
(McKeown et al, 1995, 1996). We now report on the efficacy of
this drug in several murine models. These models were selected as
they have previously been used successfully to show the efficacy
of other bioreductive drugs when used in combination with
radiation or chemotherapeutic agents (Stratford et al, 1989;
Bremner et al, 1990; Bibby et al, 1989).
Enhancement of chemotherapy and radiotherapy by AQ4N 1985
O
OH
O
NH(CH2)2N(CH3)2
OH
O
NH(CH2)2N(CH3)2
+2e
OH
O
NH(CH2)2N(CH3)2
AQ4N
O
OH
O
NH(CH2)2N(CH3)2
OH
O
NH(CH2)2N(CH3)2
+2e
O
OH
NH(CH2)2N(CH3)2
AQM
O
AQ4
Figure 1 AQ4N and its reduction metabolities
MATERIALS AND METHODS
Materials
AQ4N and 1,4-bis-{[2-(dimethylamino)ethyl]amino}5,8-dihydroxy-anthracene-9,10-dione (AQ4) were synthesized as
described previously (Patterson et al, 1994). Cisplatin, cyclophosphamide, thiotepa and hydralazine were obtained from commercial sources. Materials for cell culture were from Flow Labs
(Irvine, UK) and all other chemicals were used as purchased from
Sigma (Poole, Dorset, UK).
Murine tumour models
MAC 16, MAC26, RIF-1, NT and SCCV11 tumour-bearing
animals
Pure strain NMRI male mice, aged 6–8 weeks were used for experiments with MAC16 and MAC26 tumours (Double and Ball,
1975; Bibby et al, 1987). Male C3H mice (8–12 weeks) were used
for experiments using the RIF-1 and SCCVII tumours. NT
tumours were grown in male CBA mice (Denekamp and Harris,
1975). RIF-1 and SCCVII tumours were maintained in mice by in
vivo–in vitro passage as described previously by Twentyman et
al (1980). Tumours were passaged using tumour brei from a donor
mouse or by implantation of fragments. Tumours for experiments
were implanted subcutaneously in the mid dorsal region of the
back. All animal procedures were carried out under project
licences issued by the Home Office, London, and UKCCCR
guidelines (Workman et al, 1988) for the use of animals in experiments were adhered to throughout.
Tumour systems and treatments
Radiation treatments To allow local irradiation
of tumours, unanaesthetized mice were gently restrained in
polyvinyl jigs with lead shielding and a cut-away section over the
tumour. Irradiations were done with 250 kV X-rays as described
by Stratford et al (1989). In most experiments, single high doses of
radiation were used (20 Gy for the SCCVII tumour; 25 Gy for the
RIF-1 tumour, 12 Gy for the NT tumour). These doses were
chosen to kill most of the oxic cells in these tumours, thereby
creating tumours which contained predominantly radio-resistant
hypoxic cells. This mode of treatment should reveal the
cytotoxicity of bioreductive drugs selectively toxic to hypoxic
cells when injected into mice after local irradiation of the tumours.
Drug treatments AQ4N was usually given at its maximum tolerated single dose (MTD) which was 200–250 mg kg–1
(data not shown). However, in drug combination experiments,
toxicity was observed with some chemotherapy agents, most
notably cisplatin. The doses used were therefore at or close to the
© 2000 Cancer Research Campaign
MTD of the combination as determined from pilot experiments
(data not shown). All drugs were dissolved in phosphate-buffered
saline (PBS) and administered to mice by the intraperitoneal (i.p.)
route in a 0.1 ml injection volume.
Induction of hypoxia in tumours
Two methods were employed to temporarily reduce the oxygen
status of the tumours. The first of these was physically to occlude
the blood supply to the tumours by applying a hinged, metal Dshaped clamp around the base of the tumour to occlude the vasculature for 90 min (Denekemp et al, 1983). The second was to treat
mice with 10 mg kg–1 hydralazine. Both these methods can induce
100% radiobiological hypoxia in experimental tumours (Acker
and Chaplin, 1987). In these experiments, mice were treated with
AQ4N, then 15 min later, when it is presumed that tumour levels
of drug will be high, the tumours were clamped or mice treated
with hydralazine.
Measurement of tumour response to treatment
Tumour growth delay
For NT tumours, treatment was initiated when the tumour reached
6.5–7.5 mm GMD (geometric mean of three orthogonal diameters).
Tumours were measured three times weekly and the time taken to
reach double its treatment volume was used as a measure of antitumour efficacy. For RIF-1 tumours, treatment was initiated when
the tumours reached 100–200 mm3 and their growth followed until
they had reached 4 × the treatment volume. MAC16 and MAC26
tumours were treated when the two longest orthogonal diameters
were at least 4 and 5 mm. Tumour response was measured twice
weekly by two-dimensional caliper measurements. For RIF-1,
MAC16 and MAC26, tumour growth delay was calculated by
subtracting the mean time to grow to 4 × treatment volume for
control tumours from that obtained after drug exposure.
In vivo–in vitro clonogenic assay
C3H mice bearing SCCVII or RIF-1 tumours were treated upon
reaching a volume of 100–200 mm3. After 24 h tumours were
excised, minced and digested with an enzyme cocktail (6 mg
DNase, 2 mg collagenase and 2 mg pronase per 10 ml PBS) to
produce a single cell suspension. The cells were counted, diluted
as necessary, plated and incubated at 37°C in 95% air/5% carbon
dioxide for 12–14 days. Colonies were fixed, stained with 0.4%
crystal violet in methanol and scored by eye. Surviving fraction
(SF) was calculated as the number of colonies counted divided by
the number of cells plated for a given treatment, divided by the
same fraction determined for untreated control tumours.
RESULTS
Activity of AQ4N when combined with methods to
induce hypoxia
To investigate the effect of tumour hypoxia on AQ4N activity a
physical clamp and a vascular ‘steal’ agent, hydralazine were used.
Table 1 shows that AQ4N, clamping or hydralazine alone does not
significantly delay tumour growth in RIF-1 or MAC26 tumours.
However, making the tumours completely hypoxic for
90 min, using a physical clamp, after AQ4N administration does
significantly delay tumour growth by 13-fold. Hydralazine, which
British Journal of Cancer (2000) 82(12), 1984–1990
1986 LH Patterson et al
Table 1 Activity of AQ4N in combination with hypoxia inducing protocols
Treatment
Tumour model
Hypoxia
AQ4N
Clamp alone
AQ4N + clamp
AQ4N
AQ4N + hydralazine
Hydralazine
Radation
Radiation (25 Gy)
Radiation (25 Gy) + AQ4N
(AQ4N immediately after radiation)
AQ4N
Radiation (12Gy)
AQ4N
(AQ4N 30 min before radiation)
+ Radation (12 Gy)
Growth delaya (days)
n
RIF-1
RIF-1
RIF-1
MAC26
MAC26
MAC26
0.6±0.5
1.1±0.9
14.1±1.7
0
5.5*
0
5
14
8
5
5
5
RIF-1
RIF-1
31.1±2.0
59.7±4.8
19
5
0.05±0.6
3.76±2.1
13.76±3.8**
8
8
8
NT
NT
NT
Growth delay = time to reach 4 × tumour volume at treatment (treated group minus untreated group) for RIF -1
and MAC26; NT tumours were followed to 2 × treatment volume. AQ4N dosed i.p. MAC tumours: 50 mg kg–1;
Hydralazine (10 mg kg–1) was given 10 min after AQ4N, RIF-1 and NT tumours: 200 mg kg–1; clamping for
90 min was carried out 15 min after AQ4N administration. See Materials and Methods for experimental details.
Mann–Whitney U-test: * P < 0.05, **P < 0.01.
Table 2 Activity of AQ4N in combination chemotherapy against murine tumours in vivo
Treatment and
Time schedule
in minutes
AQ4N
Thiotepa
AQ4N 1 min + thiotepa
AQ4N
CPM
AQ4N 60 min + CPM
AQ4N
CPM
AQ4N 60 min + CPM
AQ4N
Cisplatin
AQ4N 30 min + cisplatin
AQ4N
Cisplatin
AQ4N 30 min + cisplatin
Drug dose
(mg kg–1)
50
10
50/10
200
50
200/50
250
100
250/100
100
8
100/8
100
8
100/8
Tumour
MAC16
MAC16
MAC1615.2**
RIF-1
RIF-1
RIF-1
SCCVII
SCCVII
SCCVII
RIF-1
RIF-1
RIf-1
SCCVII
SCCVII
SCCVII
Growth delaya
(days)
4.6
3.7
–
0.5
0.1
11.4***
–
–
–
–
–
–
–
–
–
Surviving
fractionsb
–
–
–
–
–
1.4
0.0052
<0.00001
0.7
0.00023
<0.000001
0.77
0.0004
<0.000001
a
Time to 4 × initial tumour volume (treated group minus untreated group). bSurviving fraction of clonogenic cells following treatment.
All drugs were dosed i.p. See Materials and Methods for experimental details. CPM = cyclophosphamide. All results (n ≥ 5 per treatment
group). Mann-Whitney U-test: *P < 0.05, **P < 0.01, ***P < 0.001.
acts as a partial pharmacological clamp also, increases the antitumour effect of AQ4N in MAC26 tumours.
Activity of AQ4N in combination chemotherapy
Table 2 shows that when AQ4N (50 mg kg–1) or thiotepa
(10 mg kg–1) alone were tested for anti-tumour efficacy against the
MAC16 tumour only a small effect (approx. 4 days delay) on
tumour growth was seen; combination of AQ4N and thiotepa at
these concentrations significantly delayed tumour growth by over
15 days (P < 0.01). Table 2 also shows that against RIF-1 tumours,
a combination of AQ4N (200 mg kg–1) and cyclophosphamide
British Journal of Cancer (2000) 82(12), 1984–1990
(50 mg kg–1) significantly delays tumour growth whereas, as single
agents, neither drug has a measurable effect. Similar results are
seen with SCCVII where clonogenic cell survival was below the
limit of detection following a combination of AQ4N (250 mg kg–1)
and cyclophosphamide (100 mg kg–1). Scheduling of drug administration is important since tumour regression (in RIF-1) and clonogenic cell kill (in SCCVII) is greater when cyclophosphamide is
administered 1 h prior to AQ4N compared to 1 h following AQ4N
(results not shown). In both the RIF-1 and SCCVII models the antitumour effect of 8 mg kg–1 cisplatin was significantly enhanced by
combination with 100 mg kg–1 AQ4N (Table 2).
© 2000 Cancer Research Campaign
Enhancement of chemotherapy and radiotherapy by AQ4N 1987
0.1
20 Gy only
0.01
Surviving fraction (SCCVII)
Surviving fraction (SCCVII)
0.1
0.001
0.0001
0.00001
Limit of detection
0.000001
0
50
100
150
200
250
Dose of AQ4N (mg kg–1) after X-rays (20 Gy)
Figure 2 The effect on surviving fraction of SCCVII tumours of various
doses of AQ4N after 20 Gy of X-radiation. C3H mice bearing the SCCVII
tumour were treated (five per group) with a range of AQ4N doses 30 min
after exposure to 20 Gy X-radiation. Mice were sacrificed 24 h later, the
tumour excised and the number of clonogenic cells determined as described
in Materials and Methods. Results are given for individual tumours. Those
which showed no clonogenic cells within the limits of detection of the assay
are indicated with downward pointing arrows
Surviving fraction (SCCVII)
10
0.01
20 Gy only
0.001
0.0001
0.00001
0.000001
–25 –20 –15 –10 –5 0
5 10 15 20 25
Time interval (h) between
AQ4N (250 mg kg–1) and X-rays (20 Gy)
Figure 4 The effect on surviving fraction of SCCVII tumours of dosing
AQ4N at different times before or after X-radiation. C3H mice bearing the
SCCVII tumour (at least three animals per treatment group) were irradiated
with 20 Gy X-radiation. They were also administered with a single i.p. dose of
AQ4N (250 mg kg–1) at known times intervals up to 24 h before (o) or after
radiation (▼ ). Mice were sacrificed 24 h after each dosing interval, the tumour
excised and the number of clonogenic cells determined as described in
Materials and Methods. Results are given for individual tumours
AQ4N only
1
0.1
0.01
0.001
0.0001
0.00001
0.000001
0
5
10
15
20
25
30
X-rays dose (Gy)
Figure 3 The effect on surviving fraction of SCCVII tumours of 250 mg kg–1
of AQ4N after various doses of X-radiation. C3H mice bearing the SCCVII
tumour were treated with a range of X-radiation doses with (open circles) or
without (closed squares) 250 mg kg–1 of AQ4N. Drug was administered
30 min after exposure to radiation. Mice were sacrificed 24 h later, the tumour
excised and the number of clonogenic cells determined as described in
Materials and Methods. Results are the means ± s.e.m. n = 5. Tumours
which showed no clongenic cells within the limits of detection of the assay
are indicated with downward pointing arrows
AQ4N in combination with ionizing irradiation
Previously we have shown (McKeown et al, 1995, 1996) that in
the T50/80 tumour AQ4N enhances the effect of radiation. This
was confirmed in the RIF-1 and NT tumour models (Table 1) and
led to a more detailed study in the SCCVII model (Figures 2, 3 and
4). Figure 2 shows that AQ4N considerably enhances the clonogenic cell kill produced by a single dose of ionizing radiation
(20 Gy) over a range of AQ4N concentrations (100–250 mg kg–1).
In fact, AQ4N doses greater than 200 mg kg–1 in combination with
ionizing radiation were so effective that the number of surviving
clonogenic cells was below the limit of detection. At this dose of
drug alone there was little, if any, measurable killing of tumour
cells.
We also investigated the effect of a range of radiation doses
(0–30 Gy) immediately followed by a single dose of AQ4N
(250 mg kg–1) in C3H mice bearing SCCVII tumours (Figure 3).
There was a marked decrease in clonogenic cell survival as
compared to irradiation alone especially at radiation doses above
© 2000 Cancer Research Campaign
10 Gy. This will be because, at doses of 10 Gy and below, the
response of the tumour will be dominated by the aerobic population of tumour cells, i.e. killing of the radiation-resistant hypoxic
cells will only be revealed with single doses of radiation > 10 Gy.
Since the level of tumour cell killing following 15 Gy plus AQ4N
is only seen when radiation alone is given at 30 Gy, we can calculate a dose modification of 2.0 at this level of cell kill. The effect of
administering AQ4N at various times before or after irradiation of
SCCVII tumour-bearing mice was subsequently investigated (see
Figure 4). This shows that i.p. injection of AQ4N (250 mg kg–1) up
to 16 h before irradiation or up to 16 h following irradiation (20
Gy) produced a substantial increase in cell kill when these intervals were increased to 24 h the potentiating effect is lost.
DISCUSSION
The rationale underlying the development of AQ4N was to design
a drug with potential for targeting DNA and DNA associated
enzymes, but which was not active until metabolized in hypoxic
tumour cells. The DNA affinity of several clinically useful agents,
including the anthracyclines and mitoxantrone, is associated with a
planar, electron-deficient chromophore bearing cationic amino
side arm(s). It was proposed that an N-oxide of a tertiary aliphatic
amine is electrically neutral and thus will exhibit a markedly
decreased DNA binding affinity compared to the reduced cationic
tertiary amine (Patterson, 1993). In fact previous studies have
shown that AQ4 has excellent DNA binding affinity (Patterson et
al, 1994) and is a DNA type II topoisomerase inhibitor (Smith et
al, 1997a). In contrast AQ4N had a equilibrium binding to DNA
which was too low to measure and AQ4N exhibited a 50-fold
decrease in topoisomerase II inhibitory activity as compared to
AQ4. AQ4N, in combination with hypoxia was a very poor cytotoxin against rodent and human cell lines whilst its reduction
product, AQ4 is a potent cytotoxic agent (Smith et al, 1997a). This
was confirmed in several other cell lines (Wilson et al, 1996).
Previously using the T50/80 murine tumour model in vivo we
have observed potent anti-tumour activity of AQ4N when used in
British Journal of Cancer (2000) 82(12), 1984–1990
1988 LH Patterson et al
combination with radiation (McKeown et al, 1995, 1996). For all
bioreductive drugs to be useful clinically, tumour expression in
vivo of appropriate reductases will be an important determinant of
efficacy. Although not addressed in this study AQ4N is known to
be reductively metabolized by haem containing enzymes including
cytochrome P450s (CYPs). Specifically, AQ4N is a substrate for
the CYP3A subfamily under hypoxic conditions (Raleigh et al,
1998) and CYP3A has been measured in RIF-1 and SCCVII
tumours (Murray et al, 1998). CYP3A isoforms have been
detected in a broad spectrum of human cancers including colon
(Massaad et al, 1993; McKay et al, 1993) breast (Murray et al,
1993) and lung (Kivisto et al, 1995). Unlike tirapazamine it has
not proved possible to metabolize AQ4N in cultured tumour cells,
this is almost certainly due to the fact that cytochome P450s are
known to be down-regulated ex vivo (Patterson et al, 1998). We
have previously shown that the potential to metabolize AQ4N
disappears within 24 h of excision in the T50/80 tumour (Hejmadi
et al, 1996). The present studies were undertaken to further
examine the potential of AQ4N to enhance radiation and
chemotherapy in a number of in vivo murine tumours and hence
support its use as a hypoxia activated bioreductive agent in the
clinic.
enhancement effects of other bioreductive agents, including tirapazamine (Kim and Brown, 1994; Siim et al, 1997) and E09
(Bibby et al, 1993) in these tumour types. MAC16 is an unusual
model since it is poorly responsive to most conventional anticancer drugs (Bibby et al, 1988). This may be in part because
MAC16 is a relatively slow growing tumour becoming necrotic as
it grows suggesting the presence of a significant level of hypoxia.
The significantly enhanced effect of thiotepa in combination with
AQ4N suggests that this approach might have particular value in
control of colon tumours.
Standard chemotherapy drugs typically target the well-perfused
and hence oxygenated tumour regions. The enhancement by
AQ4N on the anti-tumour effects of these drugs may be explained
by the existence in the hypoxic regions of AQ4 which would exert
its cytotoxicity on cells as they attempt to re-enter the cell cycle, in
response to the damage accrued by the well oxygenated cells.
Recently, AQ4N over a range of concentrations has been shown
to enhance the anti-tumour effects of cyclophosphamide in
T50/80 implants. In particular, the anti-tumour effect of AQ4N
(100 mg kg–1) combined with cyclophosphamide (100 mg kg–1)
was shown to be equivalent to that produced by a single 200 mg
kg–1 dose of cyclophosphamide alone, i.e. a 50% reduction in
cyclophosphamide dose (McKeown et al, 1998).
Enhancement of AQ4N metabolism in vivo by hypoxia
Evidence that AQ4N is acting as a hypoxia-activated cytotoxin
comes from the combination of this agent with clamping or
hydralazine (Table 1). Clearly, the production of 100% hypoxia
with a tumour clamp enhances the anti-tumour effect of AQ4N
administered as a single agent and supports the contention that
AQ4N can be bioreductively activated in vivo. Enhancement of
the anti-tumour activity of bioreductive drugs with the vasoactive
agent, hydralazine, has previously been shown with RSU 1069
(Acker and Chaplin, 1987; Stratford et al, 1997; Brown et al,
1987) and mitomycin-C (Adams et al, 1989; Quinn et al, 1992). It
is known that the treatment of mice bearing the MAC26 colon
tumour with hydralazine produces 80% shut down in functional
tumour vasculature and greater than 60% reduction in tumour
blood perfusion (Quinn et al, 1992). The present results show that
hydralazine significantly enhances the anti-tumour efficacy of
AQ4N again supporting the hypothesis that it is bioreductively
activated in vivo despite poor activation in vitro. Our interpretation of the present data supports other published studies. Notably,
AQ4N was active against the mouse mammary tumour MDAHMCa-4 when combined with the tumour blood flow inhibitor 5, 6dimethylxanthenone-4-acetic acid (DMXAA) (Wilson et al,
1996). In addition, the anti-tumour effect of AQ4N in vivo was
potentiated in T50/80 tumours by combination with hypobaric
hypoxia with a dose enhancement ratio of 5.1 (McKeown et al,
1995).
Enhancement of chemotherapy by AQ4N
From the results presented in Table 2 it can clearly be seen that
AQ4N significantly enhances the effect of three standard
chemotherapy agents (cisplatin, cyclophosphamide and thiotepa).
This supports previous results that showed that AQ4N enhances
the anti-tumour effect of cyclophosphamide and cisplatin in the
T50/80 tumour (McKeown et al, 1998). The results with the RIF-1
and SCCVII model systems are consistent with the chemotherapeutic
British Journal of Cancer (2000) 82(12), 1984–1990
Enhancement of radiation therapy by AQ4N
Combination of AQ4N (200 mg kg–1) with ionizing radiation
(25 Gy) slowed RIF-1 tumour growth by over 40% compared to
radiation alone (Table 1). This encouraging result was explored
more thoroughly using SCCVII implants, which showed that
AQ4N, over a range of concentrations, greatly enhanced the positive effects of radiation (20 Gy). When AQ4N and radiation are
administered at approximately the same time, a positive interaction can be explained on the basis that the two modalities are cytotoxic to different (i.e. oxic or hypoxic) cell populations. However,
with AQ4N the observed enhancement is maintained even when
drug is administered up to 16 h before radiation in the SCCVII
tumour model (Figure 4); the stability and DNA affinity of AQ4 is
likely to explain this phenomenon (Smith et al, 1997a; 1997b).
Once produced, AQ4 will persist in the hypoxic cell fraction and
kill any cell which attempts to re-enter the cell cycle following
destruction of oxic cells by irradiation. When AQ4N was administered up to 16 h after irradiation, an enhancement was also
observed. This suggests that formation of AQ4 in hypoxic cells
that have not yet re-oxygenated, can still prevent them from repopulating the tumour. In T50/80 tumours a similar, although even
longer, positive interaction time was observed with a 50% dosesparing effect reported for up to 4 days before and 6 h after irradiation (Hejmadi et al, 1996; McKeown et al, 1996). An enhanced
anti-tumour activity post radiation was also observed in MDAHMca-4 tumours (Wilson et al, 1996). Overall this and previous
studies are consistent with AQ4N acting as a bioreductive radiation enhancer. Differences in the time scales of the radiation
enhancement by AQ4N in SCCVII compared to T50/80 tumours
may be related to tumour clearance and/or host animal clearance
of compound. The ability of cells to survive in the hypoxic
compartment of tumours may also be a factor. Moore (1988) has
reported that T50/80 tumours have a particularly high hypoxic
fraction (60%) which suggests that T50/80 cells are capable of
remaining viable, when hypoxic, for some considerable time; a
© 2000 Cancer Research Campaign
Enhancement of chemotherapy and radiotherapy by AQ4N 1989
factor which might also account for the very long times of interaction in this model previously observed (McKeown et al, 1995). In
contrast, SCCVII tumours are significantly better oxygenated and
have a faster doubling time than T50/80. This may explain the
shorter time of maximal interaction, i.e. 16 h versus 96 h in the
T50/80 tumor. The stable DNA-affinic agent AQ4, which is
produced in the hypoxic cells, will compromise the cells in which
it is generated for as long as they are viable. Once these cells die
and become necrotic the cells, and the associated AQ4, are no
longer capable of influencing tumour responses. This is likely to
occur in fast growing tumours (e.g. SCCVII) more quickly than
slow growing tumours especially those with a high proportion of
hypoxic cells (e.g. T50/80).
In conclusion AQ4N acts like a bioreductive drug when
combined with hypoxia, chemotherapy or radiation treatment in
vivo. The efficacy of the drug has now been shown against
MAC16, MAC 26, NT, RIF-1 and SCCVII, T50/80 (McKeown et
al, 1995, 1996) and MDAH-Mca-4 (Wilson et al, 1996). They all
support the use of AQ4N as a bioreductive agent that has little or
no intrinsic activity per se but is a very efficient enhancer of radiation as well as standard chemotherapy agents such as cyclophosphamide, cisplatin and thiotepa. An additional benefit of this
bioreductively activated cytotoxin is the stable nature of the reduction product, AQ4, which may be capable of eliciting a bystander
cell killing effect on proximate non-hypoxic tumour regions. In
support of this confocal microscopy shows that viable cells
preloaded with AQ4, the authentic reduction product of AQ4N
liberate drug within 2 h despite the high DNA affinity of the latter
(Smith et al, 1997b).
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W4281676468.txt | Close kin influence COVID-19 precautionary
behaviors and vaccine acceptance of older
individuals
Bruno Arpino ( bruno.arpino@unifi.it )
University of Florence https://orcid.org/0000-0002-8374-3066
Valeria Bordone
University of Vienna
Giorgio Di Gessa
University College London https://orcid.org/0000-0001-6154-1845
Article
Keywords:
Posted Date: June 2nd, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1699988/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
Page 1/26
Abstract
The family plays a central role in shaping health behaviors of its members through social control and
support mechanisms. We investigate whether and to what extent close kin (i.e., partner and children) have
mattered for older people in taking on precautionary behaviors (e.g., physical distancing) and vaccination
during the COVID-19 pandemic in Europe. Drawing on data from the Survey of Health, Ageing and
Retirement in Europe (SHARE), we combine its Corona Surveys (June-August 2020 and June-August
2021) with pre-COVID information (October 2019-March2020). We find that having close kin (especially a
partner) is associated with a higher probability of both adopting precautionary behaviors and accepting a
COVID-19 vaccine. Results are robust to controlling for other potential drivers of precautionary behaviors
and vaccine acceptance, as well as to accounting for co-residence with kin. Our findings suggest that
policy makers and practitioners may differently address kinless individuals when promoting public policy
measures.
Full Text
In the early phases of the COVID-19 pandemic, individual precautionary behaviors were the only weapon
to protect people from infection and reduce the spread of the virus in the community. Due to herd
immunity remaining a distant target1 and given that COVID-19 vaccines neither permanently nor
completely protect against infection2-4, precautionary health behaviors have remained crucial also after
the launch of COVID-19 vaccination campaigns. Thus, governments across the globe have imposed or
recommended behaviors such as physical distancing, mask-wearing, and frequent hand-washing.
Although these have been presented as general guidelines for everyone, individuals at greater risk of
developing a more severe case of COVID-19 when infected by the Coronavirus, such as older individuals,
have been particularly encouraged to adopt precautionary health behaviors during all phases of the
pandemic in order to limit admissions to Intensive Care Units and deaths from COVID-19-related
conditions5-10. Monitoring and understanding compliance with COVID-19-preventive behaviors have thus
become a prime target for research since the beginning of the pandemic11-18.
More recently, the fight against the pandemic has entered a new stage with the approval by health
authorities of effective COVID-19 vaccines. Although it is widely recognized that effective and equitable
distribution of COVID-19 vaccines is a key policy priority19-20, ensuring their acceptance by the population
is just as important. Thus, several studies have aimed at understanding the determinants of vaccine
acceptance (i.e., actual vaccine intake and intention to be vaccinated)21-30.
Despite the acknowledged importance of precautionary health behaviors and vaccines to limit the spread
of the virus, compliance with guidelines and vaccine acceptance are anything but universal. Even among
older adults, that are at the highest risk of COVID-19 complications, studies have shown that a large share
of individuals does not follow the recommended precautionary behaviors11-12 and/or is unvaccinated
and not willing to be vaccinated23-26. Socio-demographic characteristics of individuals such as gender
Page 2/26
and education, and health conditions have been shown to be associated with both the adoption of
precautionary health behaviors14-18 and vaccine acceptance27-30. For example, highly educated
individuals and those in poorer health conditions were more likely to follow the guidelines and get
vaccinated, while a gender “paradox” emerged31: women are more likely to adopt precautionary behaviors
but less likely to accept COVID-19 vaccines. Other studies have considered the role of antiintellectualisms32, science skepticism33, information trust34 and misinformation35. We extend the
existing work by analyzing whether having close kin (a partner and/or children) influences the adoption
of precautionary health behaviors and vaccine acceptance.
Indeed, social ties are known to influence health behaviors throughout the lifecourse36-38. In particular,
partner and children tend to represent the most important social ties for older adults in terms of
emotional closeness and intensity of support38-41. Therefore, numerous studies have investigated the role
of having close kin (i.e., a partner and/or children) on health behaviors (see the reviews in [42-44]). The
theoretical social-behavioral explanations of the importance of the family for health behaviors focus on
the instrumental and emotional support that family members provide to each other complying with social
norms of family obligations45-46. Family members complement thus the role of the health care system by
providing material support, information, and motivation to prevent diseases and help adhering to medical
treatments or recommendations47.
The power of close kin to improve health is also explained by the social control function of family
members, which exert pressures and control to inhibit unhealthy behaviors and to promote positive habits
and lifestyles38,48,49. Social control affects health behaviors directly (through sanctions for deviant
behaviors, regulation, and physical interventions) and indirectly (through internalization of norms of
healthful behavior and facilitation of positive health behaviors)48. Partnership and parenthood, in
particular, enhance a sense of obligation and greater self-regulation that discourage harmful behaviors
and boost healthy ones48,50. Although under certain circumstances (e.g., family conflicts or multiple roles
overload) kin may have a negative effect on health behaviors47,51, most studies have found positive
effects38,43. Moreover, partnership tends to be more consistently beneficial for health and health
behaviors as compared to parenthood48,52,53. The influence of family members on health behaviors is
particularly strong when they live together48 and later in life54.
In the context of the COVID-19 pandemic, the social control function of partners and children might have
been particularly relevant to vehiculate information about the importance of adopting precautionary
behaviors (e.g., wearing masks) and of vaccination. Similarly, children might have provided instrumental
support to their older parents with (online and in-person) shopping in order to limit their in-person
contacts. Thus, we should expect individuals with a partner and children to be more likely to adopt
precautionary behaviors and (to be willing) to get vaccinated compared to their counterparts who lack
these kin ties. Based on findings from the literature on family and health behaviors mentioned above, the
effect should also be stronger for partnership than parenthood status.
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We test the role of close kin in precautionary health behaviors and COVID-19 vaccine acceptance using
large-scale representative data from the Survey of Health, Ageing and Retirement in Europe (SHARE), a
survey on individuals aged 50 or more implemented in several European countries55. We combine data
from the two SHARE Corona Surveys, administered in June-August 2020 and June-August 2021, with
information from the latest pre-COVID wave (regular wave 8; October 2019-March 2020) from 27
countries. Our findings suggest that partnership and parenthood are positively associated with the
likelihood of adopting precautionary health behaviors and to accept COVID-19 vaccine. The effect of
having a partner is found to be stronger than that of having children. Our results urge policy makers and
practitioners in the health sector to pay special attention to kinless individuals when designing
interventions and recommendations related to precautionary health behaviors and vaccination. This
research provides important insights to be better prepared for the next phases of the COVID-19 pandemic
and in case of future pandemics.
Results
Having close kin and precautionary behaviors
We first present the results based on the SHARE Corona Survey 1 (SCS1) which collected information on
nine precautionary behaviors in June-August 2020. To ease interpretation of results, we present them
graphically in Fig. 1 in terms of Average Marginal Effects (AMEs) with 95% confidence intervals obtained
from fully-adjusted logistic regression models (see Methods for the socio-demographic and health
variables we controlled for). The full table of regression estimates (log-odds) is reported in the
Supplementary Materials (Table S.1).
Results in Fig. 1 show that, overall, respondents who have close kin (partner or children) are more likely to
adopt the suggested precautionary health behaviors against the spread of the virus compared to kinless
older adults. As an example, compared to older people who do not have a partner, partnered older adults
(independently of whether they have children or not) are about 6 percentage points more likely to use
hand sanitizer or disinfection fluids more frequently than before the outbreak of the pandemic. The
positive effect of kin is particularly evident for partnership: for most outcomes, having a partner and no
children is more often associated with a higher probability of adopting precautionary behaviors than
having children and no partner. In addition, the AMEs for those who have a partner and children are
usually very similar and not statistically different to the AMEs for those who have a partner and no
children. The only precautionary behavior where the combined effect of partnership and parenthood is
both significantly and substantially higher than the effect of partnership alone is for reporting less
shopping: partnered parents are about 5 percentage points more likely to report having left home for
shopping less often or not at all since the outbreak of the pandemic than partnered respondents without
children.
Having close kin and vaccine acceptance
Page 4/26
Next, we present results about vaccine acceptance based on SHARE Corona Survey 2 (SCS2; June-August
2021), the only SHARE survey where this information is available. Figure 2 presents estimated AMEs
(with 95% confidence intervals) obtained from a fully-adjusted multinomial logistic regression (full
regression estimates are available in Table S.2 of the Supplementary Materials). Figure 2 shows that the
probability of being already vaccinated or planning to do so is about 5 percentage points higher for
respondents who have a partner (independently of whether they have children or not). Correspondingly,
older adults in a partnership are less likely of both being undecided about vaccination and of not
intending to get vaccinated. Parenthood, instead, does not seem to play a role in vaccine acceptance. In
fact, the AMEs of having children and no partner are very close to zero, and not statistically significant. In
addition, the effect of partnership is neither substantially nor statistically modified by its combination
with parenthood.
Heterogeneity analyses
Figures 3–8 present results obtained when interactions with gender, age groups, and country groups were
considered to rule out the specificity of results for certain demographic or country groups (full regression
estimates are available in Tables S.3-S.8 of the Supplementary Materials). Overall, results are very similar
across gender (Figs. 3 and 4), age groups (Figs. 5 and 6) and country groups (Figs. 7 and 8), with
statistically significant differences only observed in a bunch of cases, therefore confirming the
importance of kinship (partnership, in particular) for precautionary behaviors and COVID-19 vaccine
acceptance.
Accounting for co-residence with kin
The stronger effect on precautionary behaviors and vaccine acceptance found for partnership as
compared to parenthood might be driven by typical living arrangements with different kin at older ages. In
our sample, the vast majority (96.2%) of partnered older adults live with their partner. Instead, only 16.3%
of older parents co-reside with at least one of their children. Thus, partners might be more likely to provide
support and to exert control as compared to children simply because of the higher amount of time (and
resources) shared. However, even analyses that account for living arrangements show that (co-residing)
partners more clearly influence precautionary behaviors and vaccine acceptance compared to (coresiding) children (see Figure S.1 for precautionary behaviors and Figure S.2 for vaccine acceptance in
the Supplementary Materials).
Additional analyses
As explained in the Methods section (see below), information on precautionary behaviors has been
collected very differently in the second SHARE Corona Survey (SCS2) as compared to the first one.
Nonetheless, analyses based on items in SCS2 yielded similar results to those based on items in SCS1:
having close kin, and especially a partner, is associated with a higher probability of adopting
precautionary behaviors (see Figures S.3 and S.4 in the Supplementary Material).
Page 5/26
Finally, in additional preliminary analyses (available upon request) we also distinguished parents by their
number of children but did not find this to matter. Also, given the slightly different sample size available
for each outcome, we run the regression models selecting only observations available for all outcomes
but results were barely affected.
Discussion
Precautionary behaviors have demonstrated efficacy at containing the spread of COVID-1956–58.
Similarly, COVID-19 vaccines have been found to reduce the risk of infection, hospitalization, and
death59–61. Thus, to slow the spread of the Coronavirus and limit its negative health consequences it is
crucial to understand the factors associated with individuals’ adoption of precautionary behaviors and
acceptance of COVID-19 vaccines. Our study focuses on the role of kin ties among older people, which
the general (pre-COVID) literature on health behaviors found to be crucial for the adoption of healthy
behaviors42–44,47−49,51−52.
Our results show that having close kin is overall positively associated with older individuals’ likelihood of
adopting precautionary behaviors and of being vaccinated or willing to get a COVID-19 vaccine. In
particular, we find individuals in a partnership to be more likely to accept vaccine and to adopt (almost
all) precautionary behaviors considered in this analysis. Results are robust to controlling for several other
drivers of precautionary behaviors and vaccine acceptance (such as health and education), as well as to
accounting for co-residence with kin. In addition, results are not specific for age, gender, or country
groups. Most statistically significant associations are also substantially important. We find an adjusted
difference in the probability of adoption of certain precautionary behavior (washing hand, using hand
sanitizer, covering coughs and sneezes, reduced shopping) and of accepting COVID-19 vaccines of about
5 percentage points between partnered and unpartnered older adults, which is similar to the effect found
in previous research for gender, health perception and chronic conditions11,15,18,30−31.
Although our data do not include direct measures of social control, the positive effect of kin on older
people’s adoption of precautionary behaviors and vaccine acceptance in the context of the COVID-19
pandemic is in line with predictions from the pre-COVID literature which finds ample evidence of positive
effects of family social control on health behaviors, such as avoidance of alcohol and cigarette
consumption38,62−64. Thus, it can be speculated that during a pandemic partners and children have an
important role in encouraging and controlling the respect of public health measures and
recommendations to reduce the risk of contagion and its negative health effects. Evidence in our study is
also consistent with social support mechanisms identified in pre-COVID studies, showing that
motivational and practical help from close kin may positively influence health behaviors38,54,65. In the
context of a pandemic, partners and children may provide assistance and useful information to
understand the importance of precautionary behaviors and vaccination. Practical help may also be a
mechanism at work. Indeed, among the health behaviors analyzed, we find that having children is
especially important for a specific outcome, i.e., limiting in-person shopping. Children, in this case, might
Page 6/26
take the burden to go shop or order groceries online for their parents in order to reduce their risk of
meeting strangers in a crowded indoor space, and therefore their risk of infection66–68.
The generally stronger role that we find for partners compared to children in influencing precautionary
behaviors and vaccine acceptance also fits with the predominant evidence in the general literature on
family and health behaviors that reports larger associations with health behaviors of being in a
partnership than of having children48,52−53. This is in part explained by the stronger and more effective
social control received by partners38,63, and by their usually greater provision of emotional and practical
support41,52,69. In addition, partners have been found to bilaterally influence each other’s behaviors, thus
reinforcing the social support and control function of being in a partnership70. In addition, previous
studies found that concerns about the possible consequences of COVID-19 for family members
influences precautionary behavior and vaccine acceptance30. This mechanism might also contribute
explaining the stronger effect we find for partnership than for parenthood: older individuals might be
more concerned about reducing the risk of infecting their partner than their children, because partners are
more likely to be older individuals with health pre-conditions.
Our findings should be considered in light of some limitations. Our data could not account for the quality
of relationships with partner and children for those individuals who have these ties. Previous research
found that in case of conflicting relationships, family ties may also lead to health-compromising
behaviors as coping mechanisms to deal with stress71. Also, the effectiveness of social control may vary
with the type of behavior of the agent of the control72. Future research could examine more in detail
possible heterogeneity in the role of kin ties in the context of the COVID-19 pandemic related to these and
other factors (e.g., geographical distance to children and availability of friends). Also, an interesting
avenue for future research is to examine the role of kin’s characteristics such as education and health.
Finally, our results might be affected by differential response rates by family status during the pandemic.
However, data quality controls showed that response rates remained satisfactory73.
Despite these limitations, our findings shed some light on the complex role of kin ties during the COVID19 pandemic. It has been argued that family relationships (measured, for example, in terms of coresidence, frequent face-to-face contacts, etc.) may increase the chances of getting in contact with an
infected person, thus constituting a risk to contract the virus. While it has been shown that conditional on
having a (co-resident) family member infected the risk of getting the Coronavirus substantially
increases74–75, the evidence on the (unconditional) risk of Coronavirus infection due to family ties per se
is still scarce and, with few exceptions, is based on macro-level data. Also such macro-level analyses
show mixed results76–82. A recent study83 based on part of the same individual-level data we used
(SCS1) found that living with children was associated with a lower risk of Coronavirus infection for older
women. Although it was not the focus on their study, the authors also found a similar effect for living
with a partner for both men and women. These results are consistent with our findings of a positive
association of close kin ties with precautionary behaviors and vaccine acceptance.
Page 7/26
As Ross and colleagues52 wrote well before the onset of the COVID-19 pandemic, “a family is more than
just a collection of people who might expose each other to infections and pollutants.” Thus, on the one
hand, as all types of in-person contact, family contact can constitute a risk factor for Coronavirus
infection. On the other hand, our study shows that partners, and to a lesser extent, children can also
positively influence precautionary behaviors and vaccination. The overall effect of kin on risk of
contagion and death is not easy to predict and it may vary with several factors, including extra-family
(horizontal) relationships84 working status85 and age86 of family members. Our findings point to a
potential positive role of kin in helping public health institutions to fight the pandemic and suggest that
when analyzing the role of social relationships on COVID-19 outcomes rather than social network size per
se one should account for (precautionary) behaviors and all types of contact (not limited to a specific
type of ties, e.g., family) a person has. Understanding under which conditions social relationships may
play a positive role in the context of a pandemic is of paramount importance and our study offers a new
perspective and empirical evidence on this matter. Our findings that kin can have a positive influence on
precautionary behaviors and vaccine acceptance suggest that policy makers and practitioners should
focus especially on kinless individuals, especially those who are unpartnered, when designing measures
to encourage the uptake and adherence to public health measures for COVID-19 prevention or in future
pandemics.
Methods
Data
The present study used data from the Survey of Health, Ageing and Retirement in Europe (SHARE)55.
SHARE is a longitudinal survey on non-institutionalized individuals aged 50 + and their partners in 27
European countries and Israel. It is conducted biannually since 2004 and 9 waves of data have been
collected till date. We use data from wave 8, which started in October 2019 but was suspended in all
countries in March 2020 due to the COVID-19 outbreak. Regular data collection is based on computerassisted personal interviewing (CAPI), which provides pre-COVID information87. A special dataset, SHARE
Corona Survey 188, was added to wave 8. This survey has been administered with CATI (computer
assisted telephone interviewing) between June and August 2020 to collect information on individuals’
behaviors and conditions during the pandemic (SHARE Corona Survey 1; SCS1). We excluded
observations from Portugal (because Portugal started the fieldwork of the regular wave 8 only a few
weeks before the start of the first lockdown due to the pandemic), thus restricting the analyses to
individuals from the 27 countries included in both regular and SCS1 data. We also use data from wave 9,
i.e. SHARE Corona Survey 2 (SCS2)89 collected between June and August 2021. Our outcome variables
(precautionary behaviors and vaccine acceptance) come from the two SHARE Corona Surveys;
independent variables, instead, are measured from the pre-COVID wave 8 of SHARE because these
variables not available in the Corona Surveys. We dropped individuals older than 85 because they
constituted a small share of the sample (about 4%) with almost no variation for certain outcomes.
Results were however not affected by this selection. After discarding observations with missing values on
Page 8/26
the independent variables (640), our analytic samples comprise between 27,432 and 33,097 individuals
depending on the outcome (the sample sizes differ because some outcomes do not apply to all
respondents – those who declared who never left their home since the beginning of the pandemic – and
because of missing values).
Measures
Using data from SCS1 we built nine outcome variables corresponding to nine different precautionary
health behaviors. The questionnaire of the SCS1 is available at: http://www.share-project.org/datadocumentation/questionnaires/corona-questionnaire-1.html. All outcome variables are binary and coded
so that 1 represents a precautionary behavior. More specifically the variables are constructed as follows
(note that all of them refer to activities done or not since the outbreak of the pandemic and that italicized
words refer to the names of the variables used in the models and reported in figures and tables). Washing
hands equals one for respondents who report washing hands more than usual. Sanitizing hands equals
one for respondents who report using special hand sanitizer or disinfection fluids more frequently than
usual. Covering coughs and sneezes equals one for respondents who report paying special attention to
covering cough and sneeze. Wearing masks equals one for respondents who report always wearing a
face mask when in a public space. Keeping distance equals one for respondents who report always
keeping distance from others in public. Less shopping equals one for respondents who report to have left
their home for shopping less often or not at all since the outbreak of the pandemic. Less walks equals
one for respondents who report to have left their home for going out for a walk less often or not at all
since the outbreak of the pandemic. Less meetings equals one for respondents who report to have left
their home for meeting with more than 5 people from outside their household less often or not at all since
the outbreak of the pandemic. Less visits equals one for respondents who report to have left their home
for visiting other family members less often or not at all since the outbreak of the pandemic.
The SCS2 used a different questionnaire (available at: http://www.share-project.org/datadocumentation/questionnaires/corona-questionnaire-2.html), with questions which are not directly
comparable with those in SCS1. Some of the questions about precautionary behaviors investigated in
SCS1 were not kept in SCS2 (Washing hands; Sanitizing hands; Wearing masks; Less walks; Less visits);
others changed the time reference (no longer ‘since the outbreak of the pandemic’ but either ‘in the three
months preceding the survey’ or ‘compared to the first wave of the pandemic’); and there were some
additional behaviors not included in SCS1 (such as going out to a restaurant). Therefore, these questions
asked in SCS2 were only analyzed as robustness checks (and presented in Supplementary Files). Based
on SCS2 items we built the following variables. Infrequent shopping equals one for respondents who
report going out for shopping less often than once a week during the three months preceding the survey.
Infrequent meetings equals one for respondents who report to have left their home for meeting with more
than 5 people from outside their household less often than once a week during the three months
preceding the survey. More covering of cough/sneeze equals one for respondents who report paying
special attention to covering cough and sneeze more frequently. Keeping distance equals one for
respondents who report to always pay special attention to keep distance from others in public during the
Page 9/26
three months preceding the survey. Infrequent restaurants equals one for respondents who report going
out to a restaurant less often than once a week during the three months preceding the survey.
SCS2 additionally collected information in two consecutive steps on vaccination status and intent to get
vaccinated. First, respondents were asked whether they had been vaccinated against COVID-19 at least
once. Second, those who had not yet been vaccinated, were asked about their intention to do so,
distinguishing whether they already had scheduled an appointment for vaccination, wanted to get
vaccinated, did not want to get vaccinated, or were still undecided. We combined the information from
these two questions and built a three-level categorical outcome variable: vaccinated or willing to get the
vaccine (including vaccinated individuals and those who intend to get vaccinated); undecided; not willing
to get the vaccine.
The explanatory variable combined information on partnership and parenthood status: has a partner and
children (respondents who are in a partnership and have at least one child); has a partner, no children
(childless respondents with a partner); no partner, has children (unpartnered respondents, including
widowed or divorced respondents, with at least one child); no partner, no children (respondents with no
close kin – reference category in the models). In the main analyses we do not distinguish our main
independent variable of interest according to living arrangements, i.e. we only account for having kin
independently of whether respondents live with their partner or child(ren). In a robustness check we built a
similar variable but considering kin availability only in case of co-residence (i.e. we dropped respondents
who did not live with their partner or at least one child). The resulting variable had the following
categories: has co-residing partner and children (respondents who live with their partner and with at least
one child); has cores. partner, no children (childless respondents with a co-resident partner); no partner,
has co-res. children (unpartnered respondents with at least one co-resident child); no partner, no children
(respondents with no close kin – reference category).
Control variables included the following: Age (in 5-year categories: 50–54 – reference category; 55–59;
60–64; 65–69; 70–74; 75–79; 80–85); Female (gender of respondent; male – reference category);
Education (low – reference category; medium; high); Working status (retired – reference category;
working; other); (Equivalized) Household income (continuous); Self-rated health (= 1 for respondents who
rate their health as fair or poor; = 0 if health is rated as good, very good or excellent); Diagnosed illness (=
1 for respondents who self-reports of at least one doctor-diagnosed conditions including hypertension,
diabetes, cancer, lung disease, heart disease, stroke and arthritis; = 0 otherwise); Gali (global activity
limitations; = 1 for respondents whose activities are limited or severely limited because of health
problems; = 0 otherwise); Respondent or close relatives tested positive (= 1 if the respondent or a close
relative (partner, children, parents) has been tested for the Coronavirus and the result was positive; = 0
otherwise); Country of residence (reference category: Austria); Week of interview. As for education, the
three groups are defined based on the International Standard Classification of Education (ISCED;
http://www.uis.unesco.org/): low (ISCED 0–1, no or primary education, and ISCED 2, lower secondary
education), medium (ISCED 3–4, higher secondary education), and high (ISCED 5–6, tertiary education).
The independent variables have been measured using information from the regular SHARE wave 8 either
Page 10/26
because they are time-invariant characteristics of because of unavailability of the information in the two
SHARE Corona Surveys.
Analyses
Each of the precautionary health behavior described above represented a different binary outcome that
we modeled using logistic regression. Thus, based on data from the SCS1 we estimated nine logistic
regression models, one for each outcome. For vaccine acceptance, we use a multinomial logistic
regression model. Although an ordering of the three categories of the outcome can be established (in
terms of vaccine acceptance), a multinomial model allowed a higher degree of flexibility compared to an
ordered logistic regression (i.e., it was possible to estimate separate effects of the independent variables
for each category of the outcome).
To ease interpretation of results, the main findings are reported in the main text graphically as Average
Marginal Effects (AMEs) for the explanatory variable with 95% confidence intervals. Due to the
categorical nature of our outcomes and explanatory variables, the AMEs are to be interpreted as the
discrete effect of the independent variable (compared to the reference category – no partner, no children),
i.e. as the difference between the predicted probabilities (in percentage points) across the groups being
compared (e.g., has a partner and children vs no partner, no children). Full tables of regression estimates
(estimated coefficients; log-odds) are reported in the Supplementary Materials. All control variables listed
above have been included in all regression models.
Among the additional analyses implemented, we considered heterogeneity analyses to rule out that the
main findings only applied to certain demographic (gender and age) or country groups. More specifically,
we have re-estimated the models that generated the main results by adding interactions between the
explanatory variable and, in turn, gender, age (two groups: 50–64; 65+) and country groups. Countries
have been grouped geographically: Northern/Central Europe (Denmark, Finland, France, Germany,
Luxembourg, Netherlands, Sweden, Switzerland) and Southern/Eastern Europe (Bulgaria, Croatia, Cyprus,
Czech Republic, Estonia, Greece, Hungary, Israel, Italy, Latvia, Lithuania, Malta, Poland, Romania, Slovakia,
Slovenia, Spain). Although simple, this classification allows to capture considerable variation in family
norms and similar grouping have been used in a previous study about the role of families on health and
mortality90. Similarly to what we did for the main analyses, results are presented graphically, showing the
estimated AMEs for the three categories of the explanatory variable corresponding to having close kin.
However, this time we estimated separate AMEs for the two groups defined, in turn, by gender, age or
country. In the few cases of a statistically significant difference (at the 5% level) between the AMEs this is
indicated by an “x” used as a marker (the “x” is used for the highest AME among the two compared). Full
tables of regression estimates (estimated coefficients; log-odds) are reported in the Supplementary
Materials.
Declarations
Acknowledgments
Page 11/26
This paper uses data from SHARE Wave 8 and 9 (DOIs: 10.6103/SHARE.w8.800,
10.6103/SHARE.w8ca.800, 10.6103/SHARE.w9ca800)), see Börsch-Supan et al. (2013) for
methodological details. The SHARE data collection has been funded by the European Commission, DG
RTD through FP5 (QLK6-CT-2001-00360), FP6 (SHARE-I3: RII-CT-2006-062193, COMPARE: CIT5-CT-2005028857, SHARELIFE: CIT4-CT-2006-028812), FP7 (SHARE-PREP: GA N°211909, SHARE-LEAP: GA
N°227822, SHARE M4: GA N°261982, DASISH: GA N°283646) and Horizon 2020 (SHARE-DEV3: GA
N°676536, SHARE-COHESION: GA N°870628, SERISS: GA N°654221, SSHOC: GA N°823782, SHARECOVID19: GA N°101015924) and by DG Employment, Social Affairs & Inclusion through VS 2015/0195,
VS 2016/0135, VS 2018/0285, VS 2019/0332, and VS 2020/0313. Additional funding from the German
Ministry of Education and Research, the Max Planck Society for the Advancement of Science, the U.S.
National Institute on Aging (U01_AG09740-13S2, P01_AG005842, P01_AG08291, P30_AG12815,
R21_AG025169, Y1-AG-4553-01, IAG_BSR06-11, OGHA_04–064, HHSN271201300071C, RAG052527A)
and from various national funding sources is gratefully acknowledged (see www.share-project.org).
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90. Zueras, P., Rutigliano, R., & Trias-Llimós, S. (2020). Marital status, living arrangements, and mortality
in middle and older age in Europe. International journal of public health, 65(5), 627–636.
Figures
Figure 1
Having close kin (partner and children) and COVID-19 precautionary behaviors
Page 18/26
Notes: The graph shows the effect of the explanatory variable (having kin) in the form of Average
Marginal Effects (AMEs) with 95% confidence intervals from nine separate logistic regression models
(one for each of the considered precautionary behaviors). Each AME compares the predicted probability
of adopting a precautionary behavior for one of the three groups of older adults who have kin available
(e.g., those who have both a partner and children) with the predicted probability of the outcome for the
reference group (kinless, i.e. older adults who lack both partner and children). All control variables are
included in the models. Full estimates are available in Table S.1 in the Supplementary Materials. Data are
from SHARE Corona Survey 1 (June-August 2020).
Figure 2
Having close kin (partner and children) and COVID-19 vaccine acceptance
Notes: The graph shows results for the effect of the explanatory variable (having kin) in the form of
Average Marginal Effects (AMEs) with 95% confidence intervals from a multinomial logistic regression
model for the three-level categorical outcome vaccine acceptance. Each AME compares the predicted
probability of a certain outcome category (e.g., being vaccinated or willing to get the vaccine) for one of
Page 19/26
the three groups of older adults who have kin available (e.g., those who have both a partner and children)
with the predicted probability for the reference group (kinless, i.e. older adults who lack both partner and
children). All control variables are included in the models. Full estimates are available in Table S.2 in the
Supplementary Materials. Data are from SHARE Corona Survey 2 (June-August 2021).
Figure 3
Having close kin (partner and children) and COVID-19 precautionary behaviors by age group (65+ in blue;
50-64 in red)
Notes: The graph shows the effect of the explanatory variable (having kin) in the form of Average
Marginal Effects (AMEs) with 95% confidence intervals from nine separate logistic regression models
(one for each of the considered precautionary behaviors). Each model includes an interaction between
the explanatory variable and a dummy variable for age that distinguishes two groups. Thus, separate
AMEs by age are obtained (65+ in blue; 50-64 in red). Each AME compares the predicted probability of
adopting a precautionary behavior for each one of the three groups of older adults who have kin available
(e.g., those who have both a partner and children) with that for the reference group (kinless, i.e. older
Page 20/26
adults who lack both partner and children). Statistically significant differences (p<0.05) between the
AMEs of the two considered age groups are indicated by an “x” in correspondence of the bigger AME. All
control variables are included in the models. Full estimates are available in Table S.3 in the
Supplementary Materials. Data are from SHARE Corona Survey 1 (June-August 2020).
Figure 4
Having close kin (partner and children) and COVID-19 vaccine acceptance by age group (65+ in blue; 5064 in red)
Notes: The graph shows results for the effect of the explanatory variable (having kin) in the form of
Average Marginal Effects (AMEs) with 95% confidence intervals from a multinomial logistic regression
model for the three-level categorical outcome vaccine acceptance. Each model includes an interaction
between the explanatory variable and a dummy variable for age that distinguishes two groups. Thus,
separate AMEs by age are obtained (65+ in blue; 50-64 in red). Each AME compares the predicted
probability of a certain outcome category (e.g., being vaccinated or willing to get the vaccine) for each
one of the three groups of older adults who have kin available (e.g., those who have both a partner and
Page 21/26
children) with that for the reference group (kinless, i.e. older adults who lack both partner and children).
Statistically significant differences (p<0.05) between the AMEs of the two considered age groups are
indicated by an “x” in correspondence of the bigger AME. All control variables are included in the models.
Full estimates are available in Table S.4 in the Supplementary Materials. Data are from SHARE Corona
Survey 2 (June-August 2021).
Figure 5
Having close kin (partner and children) and COVID-19 precautionary behaviors by gender (women in blue;
men in red)
Notes: The graph shows the effect of the explanatory variable (having kin) in the form of Average
Marginal Effects (AMEs) with 95% confidence intervals from nine separate logistic regression models
(one for each of the considered precautionary behaviors). Each model includes an interaction between
the explanatory variable and a dummy variable for gender. Thus, separate AMEs by gender are obtained
(women in blue; men in red). Each AME compares the predicted probability of adopting a precautionary
behavior for each one of the three groups of older adults who have kin available (e.g., those who have
Page 22/26
both a partner and children) with that for the reference group (kinless, i.e. older adults who lack both
partner and children). Statistically significant differences (p<0.05) between the AMEs of the two genders
are indicated by an “x” in correspondence of the bigger AME. All control variables are included in the
models. Full estimates are available in Table S.5 in the Supplementary Materials. Data are from SHARE
Corona Survey 1 (June-August 2020).
Figure 6
Having close kin (partner and children) and COVID-19 vaccine acceptance by gender (women in blue; men
in red)
Notes: The graph shows results for the effect of the explanatory variable (having kin) in the form of
Average Marginal Effects (AMEs) with 95% confidence intervals from a multinomial logistic regression
model for the three-level categorical outcome vaccine acceptance. Each model includes an interaction
between the explanatory variable and a dummy variable for gender. Thus, separate AMEs by gender are
obtained (women in blue; men in red). Each AME compares the predicted probability of a certain outcome
category (e.g., being vaccinated or willing to get the vaccine) for each one of the three groups of older
Page 23/26
adults who have kin available (e.g., those who have both a partner and children) with that for the
reference group (kinless, i.e. older adults who lack both partner and children). Statistically significant
differences (p<0.05) between the AMEs of the two genders are indicated by an “x” in correspondence of
the bigger AME. All control variables are included in the models. Full estimates are available in Table S.6
in the Supplementary Materials. Data are from SHARE Corona Survey 2 (June-August 2021).
Figure 7
Having close kin (partner and children) and COVID-19 precautionary behaviors by country groups (SouthEast Europe in blue; North-West Europe in red)
Notes: The graph shows the effect of the explanatory variable (having kin) in the form of Average
Marginal Effects (AMEs) with 95% confidence intervals from nine separate logistic regression models
(one for each of the considered precautionary behaviors). Each model includes an interaction between
the explanatory variable and a dummy variable for country that distinguishes two groups (South-East
Europe in blue; North-West Europe in red). Each AME compares the predicted probability of adopting a
precautionary behavior for each one of the three groups of older adults who have kin available (e.g., those
Page 24/26
who have both a partner and children) with that for the reference group (kinless, i.e. older adults who lack
both partner and children). Statistically significant differences (p<0.05) between the AMEs of the two
considered country groups are indicated by an “x” in correspondence of the bigger AME. All control
variables are included in the models. Full estimates are available in Table S.7 in the Supplementary
Materials. Data are from SHARE Corona Survey 1 (June-August 2020).
Figure 8
Having close kin (partner and children) and COVID-19 vaccine acceptance by country groups (South-East
Europe in blue; North-West Europe in red)
Notes: The graph shows results for the effect of the explanatory variable (having kin) in the form of
Average Marginal Effects (AMEs) with 95% confidence intervals from a multinomial logistic regression
model for the three-level categorical outcome vaccine acceptance. Each model includes an interaction
between the explanatory variable and a dummy variable for country that distinguishes two groups
(South-East Europe in blue; North-West Europe in red). Each AME compares the predicted probability of a
certain outcome category (e.g., being vaccinated or willing to get the vaccine) for each one of the three
Page 25/26
groups of older adults who have kin available (e.g., those who have both a partner and children) with that
for the reference group (kinless, i.e. older adults who lack both partner and children). Statistically
significant differences (p<0.05) between the AMEs of the two considered country groups are indicated by
an “x” in correspondence of the bigger AME. All control variables are included in the models. Full
estimates are available in Table S.8 in the Supplementary Materials. Data are from SHARE Corona Survey
2 (June-August 2021).
Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.
SupplementaryMaterials.docx
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W2266414995.txt | Communicatio Socialis 34 (2001), Nr. 1: 99–106
Quelle: www.communicatio-socialis.de
Frank
Marcinkowski
Autopoietische Systemvorstellungen in der Theorie der
Massenmedien
Vorschläge und Einwände
Im Gefolge einiger Pionierarbeiten der frühen 90er Jahre (Spangenberg
1993, Marcinkowski 1993, Blöbaum 1994), in denen versucht worden
war, Begriffe und Konzepte der neueren soziologischen Systemtheorie für
die Theorie der Massenmedien fruchtbar zu machen, hat sich bis zum
Ende des Jahrhunderts ein beachtlicher Fundus einschlägiger Diskussionsbeiträge angesammelt, unter denen die Reaktionen von Niklas
Luhmann (1996; 1997, 1096-1109) besondere Aufmerksamkeit erregt
haben. Ist bei Luhmann selbst durchaus Sympathie für die Idee der Konzeptualisierung von Massenmedien als autopoietischem Funktionssystem der modernen Gesellschaft erkennbar geworden, hat die - je nach
Standort - gefeierte oder gefürchtete "Autopoietisierung" der Medien
gerade innerhalb der Kommunikationswissenschaft nicht nur Epigonen,
sondern auch vehemente Kritiker hervorgebracht und nicht zuletzt einer
(Wieder-)Belebung handlungstheoretischer Konzepte der Journalismusforschung (Baum 1994, Neuherger 1996) Vorschub geleistet. Aber auch
unter denen, die innerhalb des autopoietischen Paradigmas argumentieren, herrscht keineswegs Einigkeit in der Frage, wie das Mediensystem
unter systemtheoretischen Vorzeichen im Detail beobachtet und beschrieben werden muss.l Die nachfolgenden Bemerkungen beziehen
1
Dem gewissermaßen vorgelagert ist die Frage, ob man das Konzept der Autopoiesis
im fachfremdem - nämlich sozialwissenschaftliehen - Kontext überhaupt verwenden
soll. Hierzu hat sich namentlich Humberto R. Maturana, der Erfinder des Begriffs,
dezidiert geäußert. Maturana verwendet den Begriff der Autopoiesis ausschließlich
zur Definition und Abgrenzung des Lebendigen. Soziologische Analogieschlüsse, wie
sie Nildas Luhmann entwickelt hat, überzeugen ihn demgegenüber nicht. Maturana
beharrt (als Nicht-Soziologe) vielmehr darauf, dass sich das Soziale durch andere
Merkmale treffender charakterisieren lässt, als durch die Behauptung, alles Soziale
ist im wesentlichen ein selbsterschaffendes Kommunikationsnetzwerk. Letztlich
scheint ihm der Mensch, seine Einstellungen, Wünsche, Interessen und das daraus
resultierende Handeln zur Charakterisierung des Sozialen unverzichtbar. Die
Vorstellung einer Gemeinschaft (auch einer Theorie von Gemeinschaft). die das
Individuum mundtot macht, ausschließt, negiert, scheint ihm vor dem Hintergrund
autobiographischer Erfahrungen mit der Diktatur in seinem Heimatland unerträglich.
Nüchtern betrachtet zielen diese Argumente allerdings auf den Entdeckungs- bzw.
(potenziellen) Verwendungszusammenhang des Konzepts und scheinen mir insoweit
im Begründungszusammenhang soziologischer Theorie von untergeordneter Bedeutung.
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MARCINKOWSKI
Stellung zu einigen der dabei sichtbar gewordenen Streitpunkte und sollen insoweit auch zur Präzisierung von Positionen beitragen, die der
Verfasser in den vergangeneu Jahren wiederholt vorgetragen hat.
Funktionsproblem
Von grundlegender Bedeutung ist zunächst der Hinweis, das gesellschaftliche Funktionsproblem, mit dem auf die Ausdifferenzierung des
Systems der Massenmedien reagiert wird, sei bisher noch nicht klar
benannt. Träfe die Kritik zu, geriete die ganze Konstruktion ins Wanken.
Tatsächlich lässt sich der Vorwurf bei genauem Hinsehen kaum halten.
Sowohl Luhmann als auch Marcinkowski gehen bei ihren Überlegungen
von der Riskanz und Anfälligkeit einer primär unter funktionalen Gesichtspunkten differenzierten Gesellschaft aus. Eine solche Formation,
die ihren inneren Zusammenhalt nicht von einer hierarchischen Spitze
aus beziehen kann, muss ständig um das Auseinanderdriften ihrer egoistischen Sonderperspektiven und Sondersprachen fürchten. Daran lässt
sich die Überlegung knüpfen, dass gleichwohl in jedem Gesellschaftssystem Vorsorge dafür getroffen werden muss, dass sich gesellschaftliche
Kommunikation auch thematisch auf sich selbst beziehen lässt, Gesellschaften also "imaginäre Konstruktionen der Einheit des Systems" (Luhmann 1997, 866) als Rahmenbedingungen der eigenen Kommunikation
erzeugen und verwenden. Für diese Aufgabe, so lautet die Annahme, wird
ein eigenständiges Funktionssystem ausdifferenziert, nämlich das System der Massenmedien. Sein Funktionsproblem ist die permanente
Selbstbeobachtung und Anfertigung einer Selbstbeschreibung als (thematischer) Rahmen gesellschaftsintern möglicher Kommunikation. Sie
macht unwahrscheinliche Kommunikation (und damit die Reproduktion
der Gesellschaft) generell wahrscheinlicher, weil sie einen Entwurf von
Welt allgemein zugänglich macht, der von jeder Kommunikation vorausgesetzt werden kann und nicht mitproduziert werden muss. Nun könnte
man darüber streiten, ob eine solche Weltbeschreibung nicht auch in
anderen Funktionsbereichen der Gesellschaft angefertigt wird und auf
dieser Basis die Exklusivität des medialen Funktionsproblems bestreiten. Als Konkurrenten kämen in dem Falle etwa die Kunst oder die
Wissenschaft in Frage und tatsächlich ist gar nicht auszuschließen, dass
jede Gesellschaft eine Mehrzahl von Selbstbeschreibungen verfügbar
hält. Die Besonderheit massenmedialer Konstruktion läge neben ihrer
sachlichen Beliebigkeit und der Besonderheit der Themenbehandlung
vor allem im ungeheuren Inklusionspotential der Massenmedien. Denn
auf der Ebene der Beteiligung von Menschen an der Funktionserbringung
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AUTOPOIETISCHE SYSTEMVORSTELLUNGEN IN DER THEORIE DER MASSENMEDIEN
ist das Mediensystem das bei weitem inklusivste Sozialsystem moderner
Gesellschaften und zweifellos inklusiver als Wissenschaft oder Kunst.
Und schließlich kann die Argumentationskette noch einen Schritt
weitergeführt werden: denn die Funktion der Massenmedien besteht
nicht nur darin, dass sie eine Beschreibung der Gesellschaft verfügbar
macht, sie besteht vor allem darin, dass sie diese Beschreibung beobachtbar macht. In den Medien kann man beobachten, wie man von anderen
beobachtet wird. Medienrealität eröffnet damit allen Beobachtern in der
Gesellschaft einen Zugang zum Modus der Beobachtung zweiter Ordnung und zur Reflexion. Reflexiv sind Systeme insoweit, als sie die Folgen ihres Operierens in der Umwelt und den Systemen ihrer Umwelt
durch die Rückwirkungen auf sich selbst kontrollieren und als Konsequenz dieser Beobachtung die eigenen Möglichkeiten beschränken. Die
Medienfunktion besteht folglich nicht darin, dass sie teilsystemische Abstimmung aktiv herstellt, wie die ältere Theorie dies noch fordert (,,Integrationsfunktion"), sondern darin, dass sie als Bedingung ihrer Möglichkeit fungiert, in dem sie den Aufbau von Reflexionskapazität bei den
Teilen ermöglicht (vgl. dazu ausführlich Marcinkowski 2001).
Systemgrenzen
Ein zweiter Kritikpunkt knüpft unmittelbar an die Frage des Funktionsproblems an: die mangelnde Erkennbarkeit der Systemabgrenzung.
Wenn man ein Funktionssystem als den Zusammenhang aller Kommunikationen bezeichnet, die sinnhaftauf ein spezifisches Bestandserfordernis der Gesellschaft bezogen sind, dann ist die Klärung der Funktion
unabdingbare Voraussetzung der Grenzziehung. In dem Zusammenhang
wird vermutet, gerade der Luhmannsche Entwurf würde zur näheren
Bestimmung des Mediensystems zu stark auf die technischen Verbreitungsmedien zurückgreifen, die in ihm verwendet werden (vgl. etwa:
Kohring 2000).
Eine alternative Lesart besteht darin, die Medientechnik als eine Bedingung der Besonderung publizistischer Kommunikation zu verstehen,
unter anderem, weil sie es erlauben, Themen nach eigenen Routinen
auszuwählen und zu bearbeiten um sie anschließend einer spezifischen
Karriere auszusetzen. Gerade weil technisch verbreitete Kommunikation
nicht unmittelbar beantwortbar und insoweit keine Interaktion ist, 2 kann
sie sich im Gegenteil aus den Interaktionskontexten des täglichen Le2
Wie in dem Zusammenhang die digitalen, "interaktiven" Online·Medien einzuschätzen sind, ist eine der vielen offenen Fragen, über die zu diskutieren wäre.
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MARCINKOWSK/
bens lösen und eine besondere (und abgesonderte) Art der Codierung und
Programmierung von Kommunikation praktizieren. Außerdem zwingt die
Technik zu programmierter Selektion, denn durch die Entwicklung technischer Kommunikationsmittel potenzieren sich Kommunikationsmöglichkeiten und das führt zu einer noch schärferen Selektion dessen, was
dann tatsächlich mitgeteilt werden kann, also zu eigenen Selektionskriterien. Und es ist diese Art der Codierung, die die Identität des Systems
ausmacht, nicht etwa die Technik.
Der Sinngehalt, den diese Form der Kommunikation mitführt, ist nicht
der technische Akt der Publikation, sondern die Untersteilbarkeit des
Bekanntseins bzw. das Bekanntsein des Bekanntseins von Themen und
Beiträgen. Rein technisch gesehen erhöhen Verbreitungsmedien lediglich die soziale Reichweite von Kommunikation und steigern insoweit das
Inklusionspotential des Systems. Zugleich wird aber Kommunikation anonymisiert. 3 Im Falle von Printmedien kann man nicht genau wissen, wer
welche Texte wann gelesen hat, und im Hinblick auf elektronische Medien, wer wann was gesehen oder gehört hat. Das heißt aber, man kann
zunächst gar nicht anders, als die Bekanntheit publizierter Sachverhalte
zu unterstellen und dann beobachten, ob Kommunikation mit dieser Erwartung fortgesetzt werden kann. Und auf der anderen Seite kann man
sich dem Druck kaum entziehen, über Themen zu reden, die allgemein als
bekannt unterstellt werden. Man wird das auch dann versuchen, wenn
man nur vage ahnt, wovon die Rede ist, und man wird versuchen, beim
nächsten Mal besser vorbereitet zu sein. Um das auszudrücken, wurde
auch vom symbolisch generalisierten Erfolgsmedium "Publizität" gesprochen. Exakt in diesem Sinne sind publizistische Systemgrenzen als Sinngrenzen etabliert.
Autonomie vs. Abhängigkeit
Schwer vermittelbar ist schließlich auch der Gedanke autonomer Verfügung über die eigenen Operationsgrundlagen in einem autopoietisch
gedachten Mediensystem. Die klassische Theorie war es gewohnt, die
Medien im Hinblick auf ihr Verhältnis zu den übergeordneten Bezugssytemen zu definieren, für das im Falle des (öffentlich-rechtlichen) Rundfunks an erster Stelle die Politik, im Falle des privatwirtschaftlich organisierten Printsektors zuvorderst die Ökonomie in Frage kamen. Auf der
anderen Seite wurden dem gesamten Mediensystem, also auch seinen
3
Dies wiederum gilt für Online-Kommunikation in gleichem, wenn nicht in gesteigertem Maße.
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AUTOPOIETISCHE SYSTEMVORSTELLUNGEN IN DER THEORIE DER MASSENMEDIEN
kommerziellen Teilen, politische Funktionen zugeschrieben und mit der
Zulassung privater Veranstalter wurden auch im Rundfunksystem Kriterien der Wirtschaftlichkeit implementiert. Das führte allerdings nicht zur
Verunsicherung von derlei Dependenztheorien, sondern zu der heute
weithin geteilten Annahme, der Rundfunk habe lediglich sein Referenzsystem gewechselt und werde nunmehr, genau wie die Printmedien auch,
nicht mehr von der Politik, sondern von der Wirtschaft dominiert. Mit der
Annahme einer "Kommerzialisierung" des gesamten Mediensystems
wird diese Beobachtung auf den theoretischen Begriff gebracht. Systemisch gesprochen hätten wir es bei den Massenmedien insoweit lediglich
mit einem Anwendungsfall von Wirtschaft zu tun.
Demgegenüber hat eine autopoietische Medientheorie die These zu
verteidigen, dass operativ geschlossene Kommunikationssysteme allenfalls wechselseitig resonanzfähig sind, aber keinesfalls extern beherrschbar. Unbefriedigend bleibt dabei zweifellos, dass mit Konzepten wie Irritation und Resonanz bisher lediglich hoch abstrakte Thesen über
tatsächliche Wechselbeziehungen von publizistischem System und Umweltsystemen wie Wirtschaft oder Politik formulierbar sind, was die
Erklärungs- und Prognosekraft des Ansatzes im Einzelfall erheblich
einschränkt. Die relativ groben Analysen der Resonanzfähigkeit der Systeme füreinander verleiten dabei tendenziell zu reduktionistischen Annahmen über ihre wechselseitigen Einflusschancen, die dann nicht selten
von drastischen Beispielen für Intersystemeffekte überrascht werden.
Darüber hinaus wird die These von der operativen Schließung immer
wieder in der Weise missverstanden, als verneine sie jegliche
Kontaktmöglichkeit zwischen den Systemen. Dazu trägt auch der Umstand bei, dass das Konzept der strukturellen Kopplung von Systemen,
mit dem an dieser Stelle gearbeitet werden müsste, nach wie vor als nicht
hinreichend ausgearbeitet gilt, ein Mangel, der freilich nicht allein der
systemischen Medientheorie anzulasten ist.
Am intensivsten wurde bisher das Verhältnis von Mediensystem und
Politik behandelt. Eine differenzierte Betrachtung der Publizistik/Politik-Beziehungen auf den Referenzebenen Interaktion, Organisation und
Gesellschaft steht allerdings auch für diesen Fall aus. Erste Versuche
bezogen sich vornehmlich auf die Makroebenen und nahmen insoweit die
Frage einer spezifisch publizistischen Codierung in den Blick. Auch
wenn hierüber keine Einigkeit unter den Beteiligten besteht, liegen doch
immerhin diskutable Entwürfe vor. Danach orientieren die Massenmedien, wie alle anderen Funktionssysteme moderner Gesellschaft auch, ihre
Umweltbeobachtung an einer exklusiven Sonderperspektive, nämlich an
dem Gesichtspunkt ihrer unbedingten Präferenz für Information (Luhmann 1996, ebenso Blöbaum 1994) bzw. an der Akzeptanz gesellschaftshttps://doi.org/10.5771/0010-3497-2001-1-99, am 20.05.2024, 16:46:48
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MARCINKOWSK/
weit unterstellbarer Bekanntheit von ausgewählten Sachverhalten als
Themen publizistischer Kommunikation (Marcinkowski 1993). In beiden
Fällen hat man es im Hinblick auf die Machtkommunikation von Politik
mit deutlich unterscheidbaren Leitwerten zu tun.
Auf der Ebene publizistischer Organisationen führt die Beobachtung
systeminterner Differenzierung der Kriterienstruktur in Form dauerhaft
verfestigter Programmbereiche zur Erklärung relativ enger Kopplungen
von Massenmedien und Umweltsystemen, wobei Publizistik und Politik
offensichtlich über den politischen Journalismus (bei Luhmann der Programmhereich "Nachrichten und Berichte") gekoppelt sind (vgl. Marcinkowski/Bruns 2000; Luhmann 1996, 53-82).
Interaktionsbeziehungen zwischen Politikern und Journalisten wurden bisher nicht als Beispiel struktureller Kopplung angeführt. Offenbar
sind Interaktionssysteme zu flüchtig, um dauerhafte, strukturell angelegte Beziehungen zu erklären. Der gelegentliche, meist buchstäblich
"vorübergehende" Kontakt zwischen Journalisten und Politikern, so
wichtig er im Einzelfall auch sein mag, scheint jedenfalls zu unbeständig,
um Intersystembeziehungen darauf aufzubauen. Das hängt mit der hohen
Störanfälligkeit des Anwesenheitsprinzips zusammen. Wenn Politiker
verhindert sind oder Vertraulichkeit vereinbaren, können die Journalisten nur noch miteinander fachsimpeln. Die Bedeutung solcher Interaktionen, soweit sie denn zustande kommen, liegt darin, dass die Teilnehmer sich in der Interaktion aus den Rollenerwartungen lösen mögen, an
die ihre Kommunikation innerhalb der Funktionssysteme gebunden
wäre: Wenn der Journalist zusagt, nicht gleich zu veröffentlichen, mag er
Mitteilungen erhalten, die der Position (Macht!) seines Gesprächspartners nicht unbedingt zuträglich sind. Das heißt, jenseits funktionaler
Kommunikationsbezüge kann über andere Themen anders gesprochen
werden. Damit baut die Interaktion freilich hohe Erwartungshaltungen
auf, nämlich die, das genau das auch getan wird. Werden solche Erwartungen wiederholt enttäuscht, bricht die Interaktion auseinander.
Insgesamt ist die Ablösung des Mediensystems von politischen Imperativen gerade vor dem Hintergrund medialer Expansion, Differenzierung
und Komplexitätssteigerung auch empirisch relativ leichter vermittelbar.
Schwieriger liegt der Fall mit der sogenannten "Kommerzialisierung", die
einer medialen Verselbständigung so offensichtlich zu widersprechen
scheint. Hier verführt die Bedeutung des Mediums Geld für Ausbildung
und Bestand von Organisationen und die Bedeutung der Organisation für
die Spezialisierung der Funktionssystems viele Beobachter dazu, alle
zentralen Gesellschaftsbereiche (nach der CD U-Affäre unter Einschluss
der Politik) als wirtschaftlich dominiert anzusehen. Unter systemtheoretischen Vorzeichen muss freilich zwischen dem "Erfolgsmedium" des
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AUTOPOIETISCHE SYSTEMVORSTELLUNGEN IN DER THEORIE DER MASSENMEDIEN
Funktionssystems und dem Medium Geld als Voraussetzung der Organisationsbildung im System unterschieden werden, um nicht weite Bereiche, darunter das System der Massenmedien umstandslos als Wirtschaft
zu beschreiben. Das man eine Zeitung verkaufen muss, wenn man sie
unterhalten will, ist selbstverständlich richtig und gilt im übrigen so
lange, wie es überhaupt publizistische Medien gibt. Aber dieser Verkauf
ist keine publizistische Operation, sondern eine ökonomische. So viel
Differenzierungsvermögen muss man unterstellen können. Wer am Kiosk
eine Zeitung aus dem Ständer nimmt und das passende Geldstück auf die
Theke legt, der wird verstanden, auch wenn kein einziges Wort gefallen
ist. Man kann insoweit von gelungener Kommunikation sprechen, aber
einer Kommunikation, die ausschließlich der Autopoiesis der Wirtschaft
nutzt und eben keine publizistische Kommunikation ist. Was die Zeitung
mitteilt, was der Nutzer liest und welche Information er aus dem Gelesenen generiert, kann dem Kioskbesitzer im Prinzip egal sein, ebenso wie
dem Verleger. Für den Leistungsbereich der Publizistik (Journalismus)
ist die Information freilich wichtig, aber nicht unter ökonomischen Gesichtspunkten, sondern um zu sehen, woran man mit der nächsten Ausgabe erfolgreich anschließen kann. Das Medium Geld und der Zahlungsvorgang selbst liefert zu diesem Prozess jedenfalls keinen operativen
Zugang. Natürlich mag es für Gründung und Unterhalt eines Mediums
ökonomische Motive geben. "Neue Medien" entstehen, weil und solange
man damit Geld verdienen kann. Aber dieses Motiv allein reicht keineswegs aus. Um gelungene oder gar erfolgreiche (folgenreiche) Kommunikation zu initiieren, sind journalistische Orientierungen unverzichtbar.
Denn der erste Blick am Kiosk gilt nicht dem Preis einer Druckschrift.
Man liest bestimmte Zeitungen und nicht andere aus Gründen, die die
Kommunikation selbst mitliefert, nicht der Geld- bzw. Kostenmechanismus. Weder auf der Sender- noch auf der Empfängerseite dirigieren
ökonomische Kriterien den Erfolg publizistischer Kommunikation.
Ist der Journalismus insoweit autopoietisch? In der Form scheint mir
die Frage falsch gestellt. Welche Einsichten resultieren, wenn man sich
entschließt, Journalismus und Medien als autopoietisches Sozialsystem
zu beschreiben? Die Frage gilt es zu beantworten. Und es ist dabei bewusst zu halten, dass und wie die Verhältnisse sich ändern, wenn man
sie mit anderen Mitteln beobachtet. Es kann auch in der Medien- und
Journalismustheorie nicht darum gehen, nicht-autopoietische Sichtweisen zu ersetzen oder gar zu eliminieren. Es geht um das Angebot einer
"Sehhilfe", das nur dann angenommen zu werden verspricht, wenn es an
undurchsichtigen Stellen mehr Klarheit zu schaffen vermag. Die bisherigen Erträge des systemtheoretischen Diskurses - so unvollständig sie
noch lange Zeit bleiben werden - stimmen diesbezüglich optimistisch.
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Literatur
Baum, Achim, 1994: Journalistisches Handeln. Eine kommunikationstheoretisch begründete Kritik des Journalismus, Opladen.
Blöbaum, Bemd, 1994: Journalismus als soziales System. Geschichte,
Ausdifferenzierung und Verselbständigung. Opladen.
Kohring, Matthias, 2000: Komplexität ernst nehmen. Grundlagen einer
systemtheoretischen Joumalismustheorie, in: Löffelholz, Martin
(Hrsg.), Theorien des Journalismus. Ein diskursives Handbuch,
Opladen, S. 153-168.
Luhmann, Niklas, 1996: Die Realität der Massenmedien. Opladen.
Luhmann, Niklas, 1997: Die Gesellschaft der Gesellschaft, 2 Bde.
Frankfurt/M.
Marcinkowski, Frank 1993: Publizistik als autopoietisches System.
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Marcinkowski, Frank 2001: Massenmedien und die Integration differenzierter Gesellschaften: Was leistet Reflexion? In: Imhof, Kurt/Blum,
Roger/Jarren, Otfried (Hrsg.): Medien und Integration. Mediensymposium Luzem, Bd. 7, Opladen (in Vorb.).
Marcinkowski, Frank/Bruns, Thomas, 2000: Autopoiesis und strukturelle Kopplung. Inter-Relationen von Journalismus und Politik, in:
Löffelholz, Martin (Hrsg.), Theorien des Journalismus. Ein diskursives Handbuch, Opladen, S. 209-223.
Neuberger, Christoph, 1996: Journalismus als Problembearbeitung.
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Spangenberg, Peter, 1993: Stabilität und Entgrenzung von Wirklichkeiten. Systemtheoretische Überlegungen zu Funktion und Leistung der
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W2587924389.txt | www.impactjournals.com/oncotarget/
Oncotarget, 2017, Vol. 8, (No. 14), pp: 22825-22834
Research Paper
Rottlerin as a novel chemotherapy agent for adrenocortical
carcinoma
Yi Zhu1,2, Minjie Wang3, Xu Zhao2,4, Lei Zhang2, Yigao Wu2, Bangqi Wang2,
Weilie Hu2
1
Third Military Medical University, Chongqing, P.R. China
2
Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, Guangdong, P.R. China
3
No. 422 Hospital of PLA, Zhanjiang, Guangdong, P.R. China
4
Southern Medical University, Guangzhou, Guangdong, P.R. China
Correspondence to: Weilie Hu, email: huwl-mr@vip.sina.com
Keywords: adrenocortical carcinoma, rottlerin, Wnt/β-catenin, anticancer agent
Received: October 07, 2016 Accepted: January 24, 2017 Published: February 09, 2017
ABSTRACT
Adrenocortical carcinoma (ACC) is a rare, but aggressive endocrine malignancy
with a generally poor clinical outcome. There is no effective therapy for advanced
and metastatic ACC. In our study, we found that an existing drug (rottlerin) exerted
its tumour-suppressive function in ACC. Specifically, rottlerin inhibited cellular
proliferation of ACC cell lines (NCI-H295R and SW-13) in a dose- and time-dependent
manner. We also found that rottlerin induced cell apoptosis and promoted G0/G1
cell cycle arrest in ACC cell lines. The cellular migration and invasion of ACC cell lines
were decreased after treatment with rottlerin. Further, the molecular expression of
lipoprotein receptor related protein 6 (LRP6) and β-catenin were down-regulated in
rottlerin-treated ACC cells, which indicated that Wnt/β-catenin signaling was involved
in the tumour-suppressive function of rottlerin. To further confirm the anti-tumour
function of rottlerin, a nude mouse ACC xenograft model was used. The xenograft
growth curves and TUNEL assays demonstrated that rottlerin inhibited proliferation
and induced apoptosis in the ACC xenograft model. Furthermore, we verified that
rottlerin down-regulated the expression of LRP6 and β-catenin in vivo. The ACC cell
line and xenograft mouse model data indicated that rottlerin significantly inhibited
proliferation and induced apoptosis of ACC cells, likely via suppression of the Wnt/βcatenin signaling pathway. Our study indicated the potential therapeutic utility of
rottlerin as a novel and potential chemotherapeutic agent for ACC.
INTRODUCTION
ACC. The combination of a cytotoxic drug and mitotane is
recommended as first-line therapy in advanced ACCs, but
the poor therapeutic efficiency (< 40%) and severe side
effects limits clinical utility [4]. Therefore, new agents for
ACC treatment are urgently required.
Rottlerin(1-[6-[(3-acetyl-2,4,6-trihydroxy-5methylphenyl)methyl]-5,7-dihydroxy-2,2dimethyl-2H1-benzopyran-8yl]3-phenyl-2-Propen-1-one),also known
as mallotoxin, is a natural plant polyphenol.Rottlerin
is a traditional Indian medicine previously used as an
antagonist of fertilization and treatment for cestode and
trematode infections [5]. In recent years, rottlerin has
been shown to regulate several cell processes, including
cell circle regulation, cellular proliferation, apoptosis,
Adrenocortical carcinoma (ACC) is a rare, but
typically aggressive malignancy with an estimated
annual incidence of 0.7–2.0 cases per million population.
ACCs are highly aggressive with a poor prognosis and
an overall survival rate at 5 years of only 25%–50% in
most series [1, 2]. Until recently, radical surgical resection
has been the only potentially curative option for ACC;
however, approximately 40% of patients initially present
with distant metastases. Even after seemingly complete
removal of the tumor, recurrences occur in approximately
60%–80% of ACC patients [3]. Unfortunately, there is no
effective therapy for patients with advanced or recurrent
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autophagy, and migration [6]. Numerous studies have
demonstrated that rottlerin plays an important role in antitumor activity in several cancers like prostate cancers
and pancreatic cancer,and has great potential as a novel
chemotherapeutic agent. Other studies have shown that
rottlerin suppresses tumor cell survival in breast cancer,
likely via suppression of the Wnt/β-catenin signal pathway
[7]. Within the adrenal cortex, the Wnt/β-catenin cascade
is involved in adrenal development and differentiation [8].
Indeed, abnormal functioning of the Wnt/β-catenin signal
pathway may lead to adrenal tumourigenesis [9, 10]. In
patients with ACC, β-catenin activation is associated with
decreased overall survival. The Wnt/β-catenin pathway
is expected to be an efficient targeting strategy for the
treatment of ACC [11, 12]. Therefore, we sought to
investigate the potential role and preliminary mechanisms
of rottlerin in tumor cell proliferation, migration, and
invasion in ACC cell lines and an ACC xenograft
mouse model. In addition, we explored the potential
therapeutic utility of rottlerin as a novel and potential
chemotherapeutic agent for ACC.
the rottlerin-treated group compared with the control and
DMSO groups. After treatment with 2.5 μM rottlerin for
48 h, the percentage of apoptotic cells reached 13.0% in
the NCI-H295R cells and 10.9% in the SW-13 cells. After
treatment with 5 μM rottlerin for 48 h, the percentage of
apoptotic cells reached 43.3% in the NCI-H295R cells and
38.3% in the SW-13 cells. The results suggest that rottlerin
induces apoptosis in a dose-dependent manner. The results
of AO/EB staining support these conclusions regarding
morphology changes. The apoptosis rate increased in
the rottlerin-treated group compared with the control
and DMSO groups with AO/EB staining (Figure 2B,
*p < 0.05). The apoptosis rate in the 5μM rottlerin-treated
group was higher compared to the 2.5μM rottlerin-treated
group (Figure 2B, *p < 0.05).
Rottlerin caused cell cycle arrest of ACC cells
in vitro
Rottlerin inhibited cellular proliferation and may
also be involved with cell cycle arrest. Therefore, we
investigated the effect of rottlerin on the cell cycle of ACC
cells using flow cytometry analyses. After treatment with
5 μM rottlerin for 48 h, G0/G1 cell cycle arrest occurred
in ACC cells (Figure 1C). The percentage of cells in the
G1 phase was 85.14% in NCI-H295R cells and 81.88% in
SW-13 cells, which was significantly increased compared
with the control group (Figure 1D, *p < 0.05).
RESULTS
Rottlerin inhibits ACC cell proliferation in vitro
To investigate the effects of rottlerin on ACC, we
first evaluated whether or not rottlerin decreases viability
in ACC cell lines. NCI-H295R and SW-13 cells were
treated with rottlerin at the indicated concentration for 24,
48, 72, 96, and 120 h. It was shown that rottlerin inhibited
proliferation of ACC cells in a time- and dose-dependent
manner (Figure 1A). At a concentration of 4 μM, rottlerin
inhibited approximately 44.0% and 43.2% of NCI-H295R
and SW-13 cells, respectively, after treatment for 48 h.
In contrast, when exposed to 8μM rottlerin for 72 h, the
inhibition ratio (1-viable cell%) was up to 70% in SW13 cells and 80% in NCI-H295R cells. The 5-day growth
curves showed that the growth of rottlerin-treated cells
was relatively slower than cells not treated with rottlerin in
a dose-dependent manner (Figure 1B). The 48-h IC50 was
5.02 μM in NCI-H295R cells and 4.87 μM in SW-13 cells.
Rottlerin inhibited cell invasion and migration of
ACC cells
Invasion and migration are related to the malignant
features of ACCs [13]. To determine if rottlerin inhibits
cell invasion and migration of ACC cells, we performed
transwell invasion and migration assays. In the rottlerin
treatment group, the invasion of ACC cells was impaired
by rottlerin (5 μM); significantly fewer cells migrated to
the lower side of the transwell chamber membrane than
the control group (Figure 2C, *p < 0.05).
The results of wound healing assay demonstrated that
the migration rate of SW-13 cells was significantly reduced
by rottlerin (5 μM) compared with the control group
(Figure 3A). The 24 h migration rate was significantly
decreased in SW-13 cells (Figure 3C, *p < 0.05).
Rottlerin induced ACC cell apoptosis in vitro
To further elucidate the mechanism by which
rottlerin inhibits cellular growth in ACC cells, we
determined whether or not rottlerin induces apoptosis in
ACC cells. Flow cytometry was performed to show the
apoptosis rate of ACC cells after treatment with rottlerin
at the indicated concentration (5 μM [approximately the
48 h IC50] and 2.5 μM [approximately half of the 48 h
IC50]). The percentage of apoptosis (early apoptosis plus
late apoptosis) was identified in NCI-H295R and SW13 cells after rottlerin treatment (Figure 2A). The results
showed an increase in the percentage of apoptotic cells in
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Rottlerin inhibited tumor growth in vivo
To investigate the effect of rottlerin on anti-tumor
activities in vivo, we established a SW-13 xenograft tumor
model in nude mice. With the administration of rottlerin
for 4 weeks, tumor sizes and body weight were measured
every 4 days. Assessment of tumor volume showed that the
rottlerin-treated groups had delayed tumor growth compared
to the control group and the group treated with DSMO
(Figure 4A). There was no significant weight differences
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the control and DMSO-treated groups in vivo (Figure 5,
*p < 0.05). Moreover, in the higher dose-treated group
(rottlerin 4 mg/kg), the expression of β-catenin, LRP6,
and p-LRP was lower than the lower dose-treated group
(rottlerin 2 mg/kg).
among the groups and there was no toxicity or side effects
in the mice after 4 weeks of treatment (Figure 4B). Then,
mice were sacrificed and the xenografts were extirpated. As
shown in Figure 5, tumors were smaller in the experimental
groups than the control and DSMO groups, and tumors
were smaller in the rottlerin 4 mg/kg group than the rottlerin
2 mg/kg group. The mean volume of the tumors decreased
from 3.23 cm3 (control) and 3.35 cm3 (DMSO) to 1.56 cm3
(rottlerin 4 mg/kg) and 1.94 cm3 (rottlerin 2 mg/kg); the
difference was statistically significant (p < 0.05). TUNEL
assays were performed to assess the anti-tumor activity of
rottlerin in vivo (Figure 4C). The TUNEL assays indicated
that the rottlerin-treated groups had higher apoptotic rates
compared with the control and DMSO-treated groups.
DISCUSSION
There is no effective treatment for patients with
locally advanced and metastatic ACC. Development of
effective treatment or novel agents for patients with locally
advanced and metastatic ACC is significant [14]. Rottlerin,
which is extracted from Mallotus philippinensis, is a
polyphenol that has been used as an ant-helminthic or a
fertilization antagonist. Rottlerin has been used in humans
for many years, has a good safety profile, and exhibits
little toxicity, even after long-term exposure [5]. Recently,
rottlerin has been shown to exhibit anti-neoplastic activity
in several cancers, including breast, prostate, colorectal,
and pancreatic cancers [7, 15, 16] by inhibiting various
cellular pathways known to play important roles in
carcinogenesis,like Wnt/β-catenin signaling pathway and
mammalian target of rapamycin complex 1 (mTORC1)
signaling.however, its antineoplastic activity in ACC has
rarely been studied.
Wnt/β-catenin signaling plays an important role in
embryonic development and can lead to tumor formation
when aberrantly activated [14, 17]. The Wnt pathway
has been examined in relation to many cancers, such as
leukemia, melanoma, lung cancer, and colorectal cancer
Rottlerin suppressed the Wnt/β-catenin signaling
pathway
To determine if rottlerin suppresses the Wnt/βcatenin signaling pathway in ACC, and to elucidate
the mechanism by which rottlerin mediates ACC cell
apoptosis, some signaling molecules in the Wnt/β-catenin
pathway were examined. Based on result of Western
blotting, We can conclude that rottlerin reduced the
expression of β-catenin, LRP6, and p-LRP in ACC cells
in a dose-dependent manner (Figure 3B).
Furthermore, we performed H&E (Figure 4D) and
immunohistochemistry staining in xenograft tissues, and
the results showed that rottlerin also down-regulated the
expression of β-catenin, LRP6, and p-LRP compared with
Figure 1: (A) Rottlerin both inhibited proliferation of ACC cells in a dose- and time-dependent manner. (B) Growth curve of NCI-H295R
and SW-13 cells treated with rottlerin (0, 0.5, 1, 2, 4, and 8 μM) for 5 days. The growth of rottlerin–treated cells was relatively slower than
untreated cells in a dose-dependent manner. (C) Rottlerin caused cell cycle arrest of ACC cells at the G0/G1 phase after treatment with
5 μM rottlerin for 48 h based on flow cytometric analysis. (D) Rottlerin caused cell cycle arrest of ACC cells at the G0/G1 phase compared
with control (*p < 0.05).
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[18]. Activation of the Wnt/β-catenin pathway occurs in
ACC and is frequently caused by activating mutations of
the gene encoding Wnt/β-catenin [18]. Therefore, blocking
the Wnt/β-catenin pathway is expected to be an efficient
targeting strategy for the treatment of ACCs [4]. Recent
studies have reported that rottlerin blocks the Wnt/βcatenin signaling pathway in some cancers, including
colorectal, prostate, and breast cancers [7].
In this study, we examined the effect of rottlerin
on ACC cells. The results showed that rottlerin inhibited
proliferation, induced apoptosis and G0/G1 cell cycle
arrest, and decreased invasion and mobility of ACC cell
lines. Furthermore, the results of animal experiments
showed that rottlerin inhibited tumor growth in vivo, and
there was no obvious toxic effect in nude mice. The results
showed that rottlerin has anti-neoplastic activity in ACC,
and could serve as a novel agent for the treatment of ACC.
We then turned to study the mechanism underlying
the antineoplastic activity of rotterlin against ACC. We
detected the expression of several key components of
the Wnt/β-catenin signaling pathway in ACC cell lines
and xenografts of nude mice treated with rottlerin. Based
on Western blot and immunohistochemistry staining, the
expression of β-catenin, LRP6, and p-LRP6 was downregulated after treatment with rottlerin. We deduced that
rottlerin plays a role as an anti-tumor agent in ACC related
to Wnt/β-catenin signaling pathway,likely via suppression
of the Wnt/β-catenin signaling pathway.
β-catenin, which is encoded by the CTNNB1
gene, is a key component of this signaling pathway
and has multiple functions, including mediation of cell
adhesion and signal transduction [19, 20]. β-catenin
combines with a variety of proteins to regulate cell
proliferation and differentiation, which is critical for
embryonic development and tumorigenesis [21]. In ACC,
accumulation of β-catenin has frequently been noted,
indicating activation of the Wnt signaling pathway.
Mutations in β-catenin have been associated with a poor
prognosis in patients with ACC, and higher-grade ACC is
associated with higher β-catenin expression [22]. In our
study,β-catenin was down-regulated by rottlerin in ACC
cells, both in vitro and in vivo.
LRP6, an indispensable co-receptor for the Wnt
signaling pathway, is a member of the low-density
lipoprotein receptor family, which interacts with the
seven transmembrane receptor of the Frizzled (Fzd)
Figure 2: (A) Flow cytometric analysis of annexin-V/PI staining of NCI-H295R and SW-13 cells. Cells were treated with rottlerin at
the indicated concentration (2.5 μM or 5 μM) for 48 h. The dual parameter dot plots combining annexin-V-fluorescein isothiocyanate
(FITC) and PI fluorescence show the viable cell population in the lower left quadrant (annexin-V2, PI2), apoptotic cells in the lower right
quadrant (annexin-V+, PI2) and upper right quadrant (annexin V+, PI+), and necrotic cells in the upper left quadrant (annexin-V2, PI+).
(B) Morphologic changes of ACC cells based on AO/EB staining. After treatment with rottlerin (2.5 μM or 5 μM) for 48 h, apoptosis was
observed using a fluorescence microscope.In order to indicate ,the blue arrow point to a normal cell, the red arrow point to an early apoptotic
cell, and the yellow arrow point to a late apoptotic cell. The rate of apoptosis in the rotterlin-treated group was higher compared with the
control and DMSO groups (*p < 0.05). The apoptotic rate of cells in the higher concentration groups after treatment with 5 μM rottlerin
for 48h was higher compared with the lower concentration groups (*p < 0.05). (C) The transwell invasion and migration assay showed that
rottlerin inhibited cell invasion and migration of ACC cells (*p < 0.05).
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family to activate the Wnt/β-catenin signaling pathway
[23]. LRP6 is expressed in human cancer cell lines and
up-regulated in human malignant tissues [24], and LRP6
silencing weakens Wnt/β-catenin signaling and inhibits
cell proliferation and tumor growth in breast and prostate
cancers [25]. LRP6 is readily expressed in ACC cells
[26], but there has been limited research involving LRP6
in ACC. In our study, LRP6 and its phosphorylated form
(p-LRP6) were down-regulated by rottlerin in ACC cells,
both in vitro and in vivo.
Blocking the Wnt signaling pathway is not the
only mechanism underlying rottlerin anti-tumor activity.
Some studies have indicated that rottlerin decrease
carcinogenesis by impacting the mammalian target
of rapamycin complex 1 (mTORC1) signaling [27].
Furthermore, rottlerin inhibits the markers of angiogenesis
(COX-2, VEGF, VEGFR, and IL-8), and metastasis
(MMP-2 and MMP-9), thus blocking production of
tumorigenic mediators in the tumor microenvironment
[28]. Rottlerin also inhibits epithelial-mesenchymal
transition by up-regulating E-cadherin [29, 30]. According
to these studies, we know that the anti-neoplastic activity
of rottlerin is a multiple-signal pathway. The anti-tumor
activity of rotterlin against ACC warrants further study.
The recommended chemotherapy regimens for
ACC are etoposide +doxorubicin+cisplatin+mitotane
(EDP/M) or streptozotocin+mitotane (Sz/M). Whatever the
chemotherapy regimen, mitotane is a key agent; however,
the effective rate of treatment is only approximately 40%.
Moreover, mitotane has intense side effects, thus limiting
its clinical use [31, 32]. In this study, we attempted to
identify a novel agent which suppresses ACC more
efficiently and safer than traditional therapy. Based on our
results, rottlerin is a novel and potential chemotherapeutic
agent for ACC based on multiple signal pathway-related
anti-neoplastic activity.
Further studies should involve clinical trials better
defining the anti-neoplastic activity of combination
rottlerin and traditional chemotherapeutics or cytotoxic
drugs.
MATERIALS AND METHODS
Cell lines and culture
The ACC cell lines, NCI-H295R and SW-13,
were purchased from the Cell Repository of the Chinese
Academy of Science and originated from the American
Type Culture Collection (ATCC; Manassas, VA, USA).
The NCI-H295R cell line was cultured in DMEM
(GIBCO, Grand Island, NY,USA), supplemented with
10% fetal bovine serum (MPBIO, Carlsbad, CA, USA)
in a standard humidified incubator at 37°C in a 5% CO2
atmosphere. The SW-13 cell line was cultured in L15
media (GENOM, Shanghai, China) supplemented with
10% fetal bovine serum in a standard humidified incubator
at 37°C in an atmosphere without CO2.
Drugs and antibodies
R00ottlerin was purchased from Sigma Aldrich (St.
Louis, MO, USA). Rottlerin was dissolved in dimethyl
Figure 3: (A) The wound healing assay showed that rottlerin reduced the cell migration rate of SW-13 cells compared with the control
and DMSO groups. (B) WB assays of β-catenin, LRP6, and p-LRP were performed in NCI-H295R and SW-13 cells. The results showed
that rottlerin down-regulated the expression of β-catenin, LRP6, and p-LRP in a dose-dependent manner. (C) The 24 h migration of SW-13
decreased after rottlerin treatment compared with the control and DMSO-treated groups (*p < 0.05).
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Apoptosis analyses by flow cytometry
sulfoxide (DMSO; GIBCO) at 10mM and stored in
dark at 4°C. The final concentrations of rottlerin used
for different experiments were prepared by diluting the
stock solution with media solution. The antibodies used
for Western blotting and immunohistochemistry staining
were as follows: anti-LRP6 and anti-p-LRP6 antibodies
were purchased from Affinity Bioscience (Cincinnati, OH,
USA); and anti-β-catenin antibody was purchased from
ABCAM (San Francisco, CA, USA).
NCI-H295R and SW-13 cells were cultured
in 6-well plates and treated with rottlerin in the dark
(concentration : 5 μM and 2.5 μM ). The control group was
not treated with rottlerin and a DMSO treated-group was
established as well. After incubation for 48 h, cells were
harvested, washed with PBS twice, resuspended in binding
buffer, stained with an annexin-V/PI solution (Dojindo) at
room temperature, then analyzed using a FACScan system
(BD Biosciences, San Jose, CA, USA).
Cell viability
Acridine orange/ethidium bromide (AO/EB)
staining
NCI-H295R and SW-13 cells were plated at a
density of 2000 cells/well in flat bottom 96-well plates
(100μl media per well). After 24 h, cells were treated
with rottlerin at various concentrations (0, 0.5, 1, 2, 4, and
8 μM) in the dark. Cell viability was measured after
24, 48, 72, 96, and 120 h using the Cell Counting Kit
8 (Dojindo, Osaka, Japan) with a microplate reader at
450 nm. All experimental concentrations were assessed
in triplicate.The half maximal inhibitory concentration
(IC50) was calculated with GraphPad Prism 5.
Morphologic assessment of apoptotic cells was
performed using the AO/EB staining method. NCIH295R and SW-13 cells were cultured in 6-well plates
(5 × 104 cells/ml). After a 24-h incubation, the cells were
treated with rottlerin in the dark (concentration = 5 μM
and 2.5 μM) for 48 h. After washing twice with PBS, the
cells were stained with 1ml of AO/EB (Sigma Aldrich)
Figure 4: (A) Tumors in the rottlerin-treated groups were smaller than the control and DMSO-treated groups. (B) There was no significant
difference in weight among the groups (*p > 0.05), and the rottlerin-treated groups showed delayed tumor growth compared to the control
and DSMO-treated groups (*p < 0.05); there was no significant difference in tumor volume between the control and DSMO-treated groups
(**p > 0.05). Tumors in the rottlerin 4 mg/kg group grow more slowly compared with the rottlerin 2 mg/kg group (*p < 0.05). (C) Tunnel
assays indicated that rottlerin induced apoptosis in vivo. The tumor sections were observed under a light microscope. The apoptotic cells
appeared brown in color, when the normal cells appeared blue. The apoptotic rates of the groups showed that the rottlerin-treated groups had
higher apoptotic rates than the control and DMSO-treated groups (*p < 0.05). The apoptotic rate in the rottlerin 4mg/kg group was higher
than the rottlerin 2 mg/kg group (*p < 0.05). (D) Hematoxylin and eosin (H&E) staining of tumor tissue.
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for 3min and imaged under an inverted fluorescence
microscope.Five hundred cells were counted under a
microscope (magnification = 200×). The apoptosis rate
was calculated using the following formula: apoptosis
rate = (early apoptotic cells + late apoptotic cells)/the total
number of cells.The nuclei of early apoptotic cells had a
bright green fluorescence, the late apoptotic cell nuclei
had a bright orange fluorescence, and the nuclei of normal
cells had a ground glass-like dim green fluorescence.
pre-treated with the respective concentrations of rottlerin
(5 μM) for 24 h. The cells (1 × 105) in 0.2 ml of culture
medium without fetal bovine serum were added to the
upper chamber. The lower chamber was filled with 0.6 ml
of complete medium containing 30% fetal bovine serum.
After a 24 h incubation,cells remaining on the upper side
of the transwell membrane were removed using a cotton
swab.The membrane was washed with ice cold PBS
twice.Cells that invaded the underside were fixed with
4% formaldehyde, stained with crystal violet, and counted
in five randomly selected fields under a microscope
(magnification of 200×).
We also measured the migration rate using a wound
healing assay.SW-13 cells were plated in 6-well plates
(1 × 106cells/ml); after attachment the cells were scratched
using a sterile pipette tip.The experimental group was
treated with 5μM rottlerin. The cells were photographed
at various time points (0, 12, and 24 h; magnification =
100×). The migration rate at 24 h was calculated using the
following formula:migration rate = (1-the width of the cell
wound at 24 h/the width of the cell wound at 0 h)%.
Cell cycle analyses by flow cytometry
NCI-H295R and SW-13 cells were plated in 6-well
plates. After a 24 h incubation, the cells were treated with
rottlerin in the dark (concentration = 5 μM and 2.5 μM)
for 48 h. The cells were collected, washed twice in cold
PBS, mixed in 300 ml of 1×binding buffer, and incubated
at room temperature for 15 min with propidium iodide
(PI), NP-40, and RNaseA (Beyotime, Shanghai, China).
The cell cycle was analyzed by flow cytometry and the
percentage of cells in the different phases was calculated
using ModFit LT software (Verity Software House).
Animal experiments
Invasion and migration assay
Animal experiments were performed using 4-weekold male nude mice (athymic, BALB/C nu/nu), which
were purchased from the Provincial Animal Center
(Guangdong, China). The animal experiments were
performed according to the Guide for the Care and Use
of Laboratory Animals and approved by the Animal
Cell invasion and migration were assessed using a
Transwell chamber (GreinerBio, Frickenhausen, Germany)
containing a polycarbonated filter with 8lM pores coated
with Matrigel (BD Biosciences, BD Biosciences,San
Jose, CA, USA).The cells in the experimental group were
Figure 5: The expression of β-catenin, LRP6, and p-LRP by immunohistochemistry staining in xenograft tissues. The
positive expressing proteins appeared brown in color.The results showed that rottlerin down-regulated the expression of β-catenin, LRP6,
and p-LRP compared with the control and DMSO-treated groups (*p < 0.05) in vivo. The suppression was stronger in the higher dosetreated group (rottlerin 4 mg/kg) than the lower dose-treated group (rottlerin 2 mg/kg; **p < 0.05).
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Investigation Committee of the General Hospital of
Guangzhou Military Command. Mice were housed in a
standard animal laboratory with free access to water and
food. The mice were kept under constant environmental
conditions with a 12 h light-dark cycle. SW-13 cells
were (3 × 106 cells) suspended in 200 μl of serum-free
culture medium and inoculated subcutaneously in the flank
region of nude mice. Tumors were permitted to grow to
approximately 4mm, and mice were randomized into 4
treatment groups (control, DMSO, rottlerin 4 mg/kg, and
rottlerin 2 mg/kg) with 5 mice per group. All the mice were
injected intraperitoneally daily for 4 weeks. Rottlerin was
dissolved in DMSO, and diluted with normal saline (NS).
The control group was injected with 0.5 ml of NS and
the DMSO group was injected with 0.45 ml of NS mixed
with 50 μl of DSMO. The rottlerin groups were injected
with rottlerin (4 mg or 2 mg per kilogram). The mouse
weights and tumor volumes were monitored every 4 days.
Using Vernier calipers, the tumor volume was calculated
according to the following formula: A × B × B/2, where
A is the length of the tumor and B is the width. After
4 weeks, the mice were euthanized and the xenografts
were removed from the animals. Tumor tissues were
fixed in 10% formalin solution and paraffin-embedded.
The sample sections were prepared for hematoxylin and
eosin (HE) staining, immunohistochemistry staining, and
terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL) assays.
and ethanol. The slides were rinsed twice with PBS and
treated for 15 min at 37°C with proteinase K (15 mg/ml in
10 mM Tris/HCl [pH 7.4–8.0]). Endogenous peroxidases
were blocked using 3% hydrogen peroxide in methanol
at room temperature for 10 min. The tissue sections were
then analyzed with an in situ Cell Death Detection KitPOD (Roche, Basel, Switzerland) in accordance with the
manufacturer’s instructions. The reaction was visualized
using microscopy. Cell numbers of five randomly selected
fields(400×) were counted under a microscope and
analyzed with Imaga-ProPlus 6.0 software.
Western blotting
NCI-H295R and SW-13 cells were treated with rottlerin
in the dark (concentrations = 0, 2.5, 5, and 10 μM) for 48 h.
The cells were washed twice with PBS solution, then lysed
with RIPA Lysis Buffer (Beyotime, Shanghai, China) and a
protease inhibitor (Thermo Scientific, Waltham, MA, USA).
Protein concentrations were determined with a Pierce BCA
Protein Assay Kit (Beyotime). Equivalent amounts of total
protein (60 μg) were boiled and electrophoretically separated
on a 10% polyacrylamide gel at 80 volts. The proteins were
transferred to a polyvinylidene difluoride membrane. The
membranes were blocked for 60 min with a 5% milk solution
prepared in PBS. The membranes were incubated overnight at
4°C with 1:500 (anti-p-LRP6 and anti-LRP6) or 1:3000 (antiβ-catenin) dilutions of the primary antibodies. The membranes
were washed three times for 5 min each with Tween 20
(1:1000 dilution in PBS) and incubated for 45 min with
the appropriate peroxidase-conjugated secondary antibody
(1:5000 dilution). The membranes were washed with Tween
20-PBS three times for 10 min each and were developed using
an Odyssey two-color infrared laser imaging system. The
signal generated by β-actin was used as an internal control.
Immunohistochemistry staining
Tumor tissues sections from paraffin-embedded
tumors were de-paraffinized and rehydrated using xylene
and ethanol, and immersed in 3% hydrogen peroxide
solution for 10 min to block endogenous peroxidase.
Sections were boiled for 30 min in 10 mM citrate buffer
solution (pH 6.0) for antigen retrieval. Slides were
incubated for 45 min with 5% bovine serum albumin
and incubated overnight at 4°C with anti-LRP6, antip-LRP6, and anti-β-catenin antibodies. The specimens
were incubated for 45 min at 37°C with the appropriate
peroxidase-conjugated secondary antibody and visualized
using the DAB Detection Kit (BOSTER, Shanghai, China)
following the manufacturer’s instructions. All sections
with immunohistochemical staining were observed and
the pictures of 6 randomly selected but homogeneous in
staining and cell numbers fields (400×) were photographed
by an Olympus microscope (IX-70 OLYMPUS, Japan)
under high power view. The integrated optical density
(IOD) in each image was measured with the same setting
for all the slides, and the results were analyzed with
Image-ProPlus 6.0 software[33].
Statistics
Statistical analyses were performed using SPSS17.0
software and all the data from at least three experiments
are presented as the mean ± S.D. The statistical difference
between the means was analyzed with a Student’s t-test or
one-way ANOVA. Compared with the control, a p < 0.05
was considered to be statistically significant.
ACKNOWLEDGMENTS
We thank Dr. WANG Jie and Dr. XIA Bin for
providing instructions for experiments, and Dr. WANG
Wei for providing advise in experimental design.
CONFLICTS OF INTEREST
TUNEL assays
The authors declare that there are no conflicts
of interest that could be perceived as prejudicing the
impartiality of the research reported.
Tumor tissue sections from paraffin-embedded
tumors were de-paraffinized and rehydrated using xylene
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FUNDING
This research did not receive any specific grant from
any funding agency in the public, commercial, or not-forprofit sectors.
10.
Authors’ contributions
Conceived and designed the experiments: Weilie Hu
and Yi Zhu. Performed the experiments: Yi Zhu, Minjie
Wang,Xu Zhao, Lei Zhang.Analyzed the data: Yi Zhu ,
Minjie Wang,Yigao Wu and Bangqi Wang. Contributed
reagents/materials/analysis tools: Yi Zhu, Minjie Wang,
and Xu Zhao. Wrote the paper: Yi Zhu.
11.
12.
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| 6,006 | 40,137 |
W4386339713.txt | ISSN: 2597-8012 JURNAL MEDIKA UDAYANA, VOL. 10 NO.9,SEPTEMBER, 2021
JMU
Jurnal medika udayana
Diterima: 2021-01-20 Revisi: 2021-02-02 Accepted: 15-09-2021
HUBUNGAN KARAKTERISTIK, KEPUASAN KERJA, MOTIVASI
PERAWAT DAN DOKTER DENGAN KOMITMEN PENCAPAIAN STANDAR
PELAYANAN MINIMAL DI INSTALASI GAWAT DARURAT RUMAH
SAKIT UMUM DAERAH KABUPATEN BADUNG MANGUSADA
Ni Made Widya Juliati, Dyah Pradnyaparamita Duarsa, Putu Cintya Denny Yuliyatni
Departemen Ilmu Kesehatan Masyarakat, Fakultas Kedokteran Universitas Udayana
Email : madejuliatiwidya@gmail.com
ABSTRAK
Latar Belakang : Rendahnya angka pencapaian standar pelayanan minimal di RSUD Badung
dan tidak tercapainya target standard pelayanan minimal (SPM) yang telah ditetapkan
menunjukkan beberapa permasalahan. Motivasi, kinerja dan kepuasan kerja yang rendah dapat
mempengaruhi target tercapainya komitmen dalam pencapaian standar pelayanan minimal yang
ditetapkan di IGD.
Tujuan : Tujuan penelitian ini untuk mengetahui hubungan karakteristik, kepuasan kerja,
motivasi perawat dan dokter dengan komitmen pencapaian standard pelayanan minimal di IGD.
Metode: Penelitian ini menggunakan metode cross-sectional dengan 59 sampel yaitu seluruh
tenaga kesehatan di IGD, dengan instrument penelitian berupa kuesioner dengan metode angket.
Selanjutnya dilakukan analisis univariat, bivariat dengan chi-square menggunakan nilai P
dengan tingkat kesalahan 5% atau sebesar 0,05, analisis multivariat dengan regresi logistik yang
disertakan dalam uji multivariat dengan nilai p<0,25.
Hasil: Analisis univariat pada variabel komitmen pencapaian SPM ditemukan 61% responden
tidak mempunyai komitmen Pada analisis bivariat tenaga kesehatan yang mempunyai
komitmen lebih banyak dijumpai pada tenaga kesehatan umur 36-45 tahun, jenis kelamin laki
– laki, pendidikan sarjana, masa kerja lebih dari 5 tahun, status perkawinan menikah, dan
penghasilan > 2,5 juta. Variabel yang paling dominan adalah Jenis kelamin dengan nilai OR
0,187 (95%IK = 0,043 - 0,805).
Simpulan: Secara umum, dengan rendahnya komitmen pencapaian SPM khususnya pada
tenaga kesehatan berjenis kelamin perempuan, perlu menjadi perhatian pihak rumah sakit.
Manajemen rumah sakit diharapkan dapat menyeimbangkan antara jenis kelamin laki-laki dan
perempuan dalam rekrutmen tenaga kesehatan serta memeberikan kompensasi rutin apabila
target tercapai, memperjelas aturan dan kebijakan serta mensosialisaikannya.
Kata kunci: Kepuasan Kerja, Motivasi, Komitmen, Komitmen Standar Pelayanan Minimal
ABSTRACT
Background : The low level of achievement of minimum service standards at Badung Regional
Hospital and not achieving the minimum service standard targets that have been set indicate
several problems. Motivation, performance and low job satisfaction can affect the target of
achieving commitment in achieving the minimum service standards set in the IGD.
https://ojs.unud.ac.id/index.php/eum
doi:10.24843.MU.2020.V10.i9.P01
1
HUBUNGAN KARAKTERISTIK, KEPUASAN KERJA, MOTIVASI PERAWAT…, Ni Made Widya Juliati, Dyah
Pradnyaparamita Duarsa, Putu Cintya Denny Yuliyatni
Objectives: The purpose of this study was to determine the relationship between characteristics,
job satisfaction, motivation of nurses and doctors with a commitment to achieving minimum
service standards in the emergency department
Methods: This study used a cross-sectional method with 59 samples, namely all health workers
in the ER, with a questionnaire as the research instrument. After the data was collected, editing,
coding, processing, and cleaning were carried out, then univariate, bivariate analysis with chisquare analysis was carried out and multivariate analysis along with other control variables
using logistic regression.
Results: Univariate analysis on the variable commitment to achieving MSS found that 61% of
respondents had no commitment. In the bivariate analysis there was a significant relationship
between age, sex, education, years of service, income, job satisfaction, compensation, work
load, attitude to work procedures and supervision with a commitment to achieving minimum
service standards. The most dominant variable was gender with an OR value of 0.187 (95% CI
= 0.043 - 0.805).
Conclusion: Most respondents fall into the category of not having a commitment to achieving
minimum service standards. The dominant variable is gender, female gender is less committed
than male gender. Hospitals are expected to balance the sexes of men and women in the
recruitment of health workers. Build a regular communication platform, provide regular
compensation when targets are achieved, clarify rules and policies and disseminate them.
Keywords: Job Satisfaction, Motivation, Commitment, Commitment to Minimum Service
Standar
PENDAHULUAN
Pelayanan kesehatan yang baik akan
meningkatkan efektifitas mutu pelayanan
kesehatan. Penerapan pelayanan kesehatan
dengan penerapan keselamatan pasien dapat
meminimalkan tingkat kesalahan dan berdampak
pada penurunan biaya yang harus dibayar oleh
pasien akibat masa rawat yang lama¹.
Instalasi gawat darurat merupakan salah
satu jenis pelayanan rumah sakit yang ditujukan
untuk
memberikan
pelayanan
kesehatan
secepatnya pada kasus gawat darurat untuk
mengurangi risiko kematian atau cacat. Instalasi
Gawat Darurat adalah ujung tombak pelayanan
kesehatan di rumah sakit³.
Hasil laporan monitoring pelayanan IGD
dari sembilan indikator standar pelayanan
minimal yang telah ditetapkan oleh rumah sakit,
terdapat tiga indikator yang belum tercapai.
Jumlah kematian pasien selama tahun 2014 (satu
tahun) ≥ 24 jam sebanyak 26 orang dengan nilai
0,026 pada tahun 2015 sebanyak 65 orang dengan
nilai 0,065, sedangkan standar yang ditetapkan
oleh rumah sakit sebesar 0,003. Jumlah pasien
dirujuk pada tahun 2014 sebanyak 450 orang
(4,5%), sedangkan tahun 2015 sebanyak 686
orang (19%), jumlah tersebut tidak mencapai
target karena standar yang ditetapkan ≤ 3% per
tahun. Jumlah tenaga perawat yang bersertifikat
https://ojs.unud.ac.id/index.php/eum
doi:10.24843.MU.2020.V10.i9.P01
ATLS/BTLS/ACLS/PPGD yang masih berlaku
pada tahun 2014 untuk tenaga dokter dan perawat
16 orang (36%) sedangkan tahun 2015
sebanyak18 orang (37%), sedangkan jumlah
tenaga dokter dan perawat yang belum
mempunyai
atau
setifikat
ATLS/BTLS/ACLS/PPGD yang tidak berlaku
tahun 2014 sebanyak 29 (64%), sedangkan tahun
2015 sebanyak 31 orang (63%), indikator ini tidak
mencapai target karena standar nilai yang
ditetapkan rumah sakit sebesar 100% untuk
tenaga
Penelitian
ini
dilakukan
untuk
mengetahui hubungan karakteristik, kepuasan
kerja, dan motivasi dengan komitmen dalam
pencapaian standar pelayanan minimal di IGD
RSUD Kabupaten Badung Mangusada.
METODE
Penelitian ini merupakan penelitian
kuantitatif analitik, menggunakan rancangan
potong-lintang. Penelitian dilakukan pada bulan
Januari 2020 di IGD RSUD Kabupaten Badung
Mangusada. Jumlah sampel sebanyak 59 orang,
dengan kiteria insklusi semua dokter dan perawat
yang bekerja di IGD dengan status pegawai
Negeri Sipil dan Tenaga Kontrak dan kriteria
eksklusi Dokter dan perawat magang dari instansi
pendidikan ataupun instansi lain. Penelitian ini
2
HUBUNGAN KARAKTERISTIK, KEPUASAN KERJA, MOTIVASI
PERAWAT…,
menggunakan kuesioner yang sebelumnya telah
dilakukan uji validitas dan reliabelitas.
Penelitian ini telah mendapatkan
kelaikan etik dari Komite Etik Penelitian Fakultas
Kedokteran Universitas Udayana/Rumah Sakit
Umum Pusat Sanglah Denpasar dengan Nomor:
2954/UN/14.2.2.VII.14/LP/ 2019 tertanggal 6
Desember.
HASIL
Hasil Analisis Univariat
Diketahui dari tabel 1 sebagian besar
responden dengan kelompok umur 36-45 tahun
(86,4%), jenis kelamin perempuan (72,9%),
pendidikan tingkat sarjana (89,8%), masa kerja >
5 tahun (86,4%), dengan status menikah (96,6%),
penghasilan lebih dari 2,5 juta (83,1%)
mempunyai komitmen.
Tabel 1. Karakteristik Tenaga Kesehatan di IGD
RSUD Mangusada
Variabel
Frekuen Persentase
(n=59)
si (f)
(%)
Umur
20-35 tahun
36-45 tahun
Jenis kelamin
Perempuan
Laki-laki
Pendidikan
Diploma
Sarjana
Masa kerja
< 5 Tahun
≥ 5 Tahun
Status
BlmMenikah
Menikah
Penghasilan
< 2,5 Juta
>2,5 Juta
8
51
13,6
86,4
43
16
72,9
27,1
6
53
10,2
89,8
8
51
13,6
86,4
2
57
3,4
96,06
10
49
16,9
83,1
Tabel 2. Komitmen, Kepuasan Kerja, Motivasi
di IGD RSUD Mangusada
Variabel
(n=59)
Komitmen
Punya
Tidak
Kep. Kerja
Puas
Tidak puas
Motivasi
Rendah
Tinggi
Kompensasi
Rendah
Tinggi
Kondisi kerja
Tdk nyaman
Nyaman
Beban kerja
Rendah
Tinggi
P.prosedur
kerja
Tidak sesuai
Sesuai
Supervisi
Tidak
Ada
Frekuensi
(f)
Persentase
(%)
36
23
61
39
53
6
89,8
10,2
24
35
40,7
59,3
8
51
13,6
86,4
16
43
27,1
72,9
15
44
25,4
74,6
9
50
15,3
84,7
6
53
10,2
89,8
Hasil Analisis Bivariat
Tenaga kesehatan yang mempunyai
komitmen lebih banyak dijumpai pada tenaga
kesehatan umur 36-45 tahun (45,1%), jenis
kelamin laki – laki (56,33%), pendidikan sarjana
( 56,6%), masa kerja lebih dari 5 tahun (45,1%),
status perkawinan menikah (40,4%), dan
penghasilan > 2,5 juta (44,9%), disajikan pada
tabel 3.
Secara umum tenaga kesehatan di RSUD
Mangusada
Kabupaten
Badung
kurang
mempunyai komitmen (61%). Hasil penilaian
kepuasan kerja, sebagian besar merasa sangat
puas dalam bekerja (89,8%). Responden memiliki
motivasi kerja yang tinggi (59,3%).
https://ojs.unud.ac.id/index.php/eum
doi:10.24843.MU.2020.V10.i9.P01
3
Ni Made Widya Juliati, Dyah Pradnyaparamita Duarsa,
Putu Cintya Denny Yuliyatni
Tabel
3. Hubungan Karakteristik dengan
Komitmen Pencapaian SPM di IGD
RSUD Mangusada
Var.
Bebas
Umur
20-35
36-45
JK
P
L
Pddkn
D1/3
S1
Masa
kerja
< 5 Th
≥ 5 Th
Status
Blm
Mnikah
Mnikah
Penghasi
lan
<2,5 Jt
≥2,5 Jt
Komitmen
Tidak
Mempun
Nila
Mempun
yai
ip
yai
n(%)
n(%)
Variabel
Bebas
PR
8(100)
28(54,9)
0
23(45,1)
0,0
15
1,8
2
29(67,4)
7(43,8)
14(32,6)
9(56,33)
0,09
7
1,5
4
6(100)
30(56,6)
0(0)
23(43,4)
0,03
9
1,7
6
8(100)
28(54,9)
0(0)
23(45,1)
0,01
5
1,8
2
2(100)
34(59,6)
0(0)
23(40,4)
0,25
0
1,6
7
9(90)
27(55,1)
\1(10)
22(44,9)
0,03
9
1,6
3
Tenaga kesehatan yang mempunyai
komitmen lebih banyak ditemukan pada tenaga
kesehatan yang mempunyai kepuasan kerja yang
baik
(41,5%), Motivasi tinggi (97,1%),
kompensasi baik (43,1%), kondisi kerja nyaman
(41,9%), beban kerja rendah (53,3%), penyikapan
prosedur kerja sesuai (44%), dilakukannya
supervise (43,4%).
https://ojs.unud.ac.id/index.php/eum
doi:10.24843.MU.2020.V10.i9.P01
Tabel 4. Hubungan Kepuasan Kerja dan Motivasi
dengan Komitmen Pencapaian SPM
di IGD RSUD Mangusada
Kep.
kerja
Tidak
puas
Puas
Motivasi
Rendah
Tinggi
Kompen
sasi
Rendah
Baik
Kondisi
kerja
Tidak nyaman
Nyaman
Beban
kerja
Rendah
Tinggi
P.prosed
ur kerja
Tidak
sesuai
Sesuai
Supervis
i
Tidak
Ada
Komitmen
Tidak
Mempu
Mempu
nyai
nyai
n(%)
n(%)
Nil
ai p
P
R
5(83,3)
31(58,6)
1(16,7)
22(41,5)
0,2
37
1,4
2
4(16,7)
1(2,9)
20(83,3)
34(97,1)
0,0
61
7,2
9
7(87,5)
29(56,9)
1(12,5)
22(43,1)
0,0
99
1,5
3
11(68,8)
25(58,1)
5(31,3)
18(41,9)
0,4
58
1,1
8
7(46,7)
29(65,9)
8(53,3)
15(34,1)
0,1
87
1,4
1
8(88,9)
28(56)
1(11,1)
22(44)
0,0
63
1,5
8
6(100)
30(56,6)
0(0)
23(43,4)
0,0
39
1,7
6
4
HUBUNGAN KARAKTERISTIK, KEPUASAN KERJA,
MOTIVASI PERAWAT…,
Hasil Analisis Multivariat
Tabel 5 menunjukkan hanya jenis
kelamin yang berhubungan dengan komitmen
dengan nilai p kurang dari 0,05. Jenis kelamin
memiliki nilai p =0,017, dengan nilai OR 7,833
dan 95% IK sebesar 1,440 – 42,603.
Tabel 5. Analisis Uji Multivariat Hubungan
Karakteristik,
Kepuasan
Kerja,
Motivasi Perawat dan Dokter dengan
Komitmen
Pencapaian
Standar
Pelayanan Minimal di IGD RSUD
Mangusada
Variabel
OR
Nilai p
95% IK
(n =59)
Umur
1512788 0,999
0,000 - (-)
272,803
JK
7,833
0,017
1,440 –
42,603
Tk.pendid
-ikan
27173,4 1,000
0,000 – (-)
47
Status
P.kawinan 13,432
1,000
0,000 – (-)
Penghasilan
1,843
0,676
0,105 –
32,412
Kepuasan
kerja
0,000
0,999
0,000 – (-)
Kompens
asi
1,843
0,676
0,105 –
P.
32,412
prosedur
kerja
5,147
0,266
0,286 – 92,
528
supervisi
7987233 1,000
0,000 – (-)
001115,
020
PEMBAHASAN
Hasil analisis komitmen pencapaian
standar pelayanan minimal diperoleh (61%)
responden tidak mempunyai komitmen.
Menurut Sopiah, komitmen merupakan dimana
orang-orang dalam organisasi tetap tinggal oleh
karena membutuhkan gaji atau karyawan
tersebut tidak diterima kerja di tempat lain atau
bertahannya karyawan di dalam suatu
organisasi disebabkan karena organisasi
tersebut memberikan banyak keuntungan yang
tidak didapatkan di organisasi yang lain⁸.
https://ojs.unud.ac.id/index.php/eum
doi:10.24843.MU.2020.V10.i9.P01
Jenis kelamin memberikan pengaruh
tidak langsung kepada kinerja individu, karena
ada perbedaan bawaan antara pria dan wanita⁷.
Perubahan peran pada perempuan di masa kini
dimana perempuan juga memiliki kebebasan
dan keinginan untuk pengembangan peran,
tetapi faktor status perkawianan dimana wanita
menikah mempunyai tanggung jawab lebih
pada rumah tangga, sedangkan laki – laki
cenderung lebih agresif dan lebih besar
kemungkinan dalam memiliki harapan untuk
sukses¹º.
SIMPULAN
Sebagian besar responden masuk
katagori tidak mempunyai komitmen pencapaian
standar pelayanan minimal. Variabel yang
berhubungan dengan komitmen pencapaian
standard pelayanan minimal adalah jenis kelamin.
Variabel yang tidak berhubungan dengan
komitmen pencapaian standard pelayanan
minimal antara lain: karakteristik (umur,
pendidikan, masa kerja, status perkawinan,
penghasilan), kepuasan kerja, motivasi terdiri dari
lima sub variabel diantaranya: kompensasi,
kondisi kerja, beban kerja, penyikapan prosedur
kerja dan supervisi.
SARAN
1. Bagi Manajemen Rumah Sakit
a. Rumah
sakit
diharapkan
dapat
menyeimbangkan tenaga kesehatan antara
jenis kelamin laki-laki dan perempuan
dalam rekrutmen karyawan.
b. Target indikator yang ditentukan rumah
sakit dalam pencapaian SPM perlu dikaji
lagi. Apabila target indikator tercapai
tenaga kesehatan perlu diberikan reward.
2. Untuk Peneliti Selanjutnya
Mengingat beberapa kelemahan dari
penelitian ini, diharapkan dijadikan sebagai
data dasar untuk mengadakan penelitian yang
lebih kompleks dan spesifik menggunakan
desain
penelitian
kualitatif
agar
mengeksplorasi lebih dalam komitmen
pencapaian standard pelayanan minimal
dengan jenis kelamin.
5
Ni Made Widya Juliati, Dyah Pradnyaparamita Duarsa,
Putu Cintya Denny Yuliyatni
DAFTAR PUSTAKA
1.
2.
3.
4.
5.
6.
7.
8.
Gerri, Kaufan & Dorothy, M.C. The Effect of
Organisational Culture on Patient SafetyResearch Databese, The university of York.
The
Univercity
of
York.
2013.
https://doi.org/10.7748/ns2013.06.27.43.50.
e7280
Departemen Kesehatan RI. Pedoman Insiden
Keselamatan
Pasien
Rumah
Sakit
(Patient safety). Jakarta. 2008.
Scharein, P &Trendelenburg., M. Critical
Incidents in a Tertiary Care Clinic for
Internal Medicine. BMC Research Notes. 6
(1). 2013. 1 . https://doi.org/10.1186/17560500-6-276
RSUD Kabupaten Mangusada. Laporan
Standar Pelayananminimal Rumah Sakit
Umum Daerah Kabupatrn Mangusada. 2015.
Didik Widiyanti. Pengaruh Disiplin Kerja,
Motivasi Kerja, Gaya Kepemimpinan Kerja
Terhadap Kualitas Kinerja Karyawan Dinas
Kesehatan Kabupaten Sragen, Tesis.
Surakarta: Fakultas Ekonomi & Bisnis
Program Study Ekonomi Manajemen
Universitas Muhamadiyah Surakarta. 2015.
Angraeni, S. Analisis Faktor-Faktor yang
Berhubungan dengan Kepuasan Kerja
Perawat Pelaksana di Ruang Rawat Inap RS.
MH. Thamrin Jakarta Pusat. Tesis Program
Pasca sarjana FIK-UI Jakarta. 2011.
Chan,
Sow
Hup.
Organizational
identification and commitment of members
of human development organization, Jurnal
of Management Development. 2006.
Sopiah. Prilaku Organisasi. Yogyakarta.
ANDI. 2008.
https://ojs.unud.ac.id/index.php/eum
doi:10.24843.MU.2020.V10.i9.P01
9.
10.
11.
12.
13.
14.
15.
16.
Wijono, S. Psikologi Industri & Organisasi
dalam suatu Bidang Gerak Psikologi
Sumber Daya Manusia. Jakarta.
Kencana. 2010.
Chan, Sow Hup. Organizationalidentification
and commitment of members of human
development organization, Jurnal of
Management Development. 2006.
Wibowo. Manajemen Kinerja. Jakarta:
Rajawali Pers. 2011.
Siangian, Sondang. Manajemen Sumber
Daya Manusia. Jakarta. Bumi Aksara. 2006.
Robbins, S.P. Alih Bahasa Pujaatmaka, H
&Molan, B. Perilaku Organisasi: Konsep
Kontroversi, Aplikasi. Jakarta. PT
Prelindo. 2006.
Smith,S.
Reducing
Medical
Errors
andIncreasing Patient Safety in Health Care.
Proreques Dissertasions Publishing. Utica
Collage. 2016. https://doi.org/10183250.
Luthans. Alih Bahasa Yuwono, dkk. Perilaku
Organisasi. Yogyakarta. ANDI. 2006.
Ilyas Y. Perencanaan SDM Rumah Sakit.
Teori. Metoda dan Formula. Jawa Barat:
Fakultas Kesehatan Masyarakat UI. 2004.
6
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W4220929057.txt | MATCHING FUND SEKTOR PARIWISATA DESA MINGGIRSARI
DALAM PERSPEKTIF HUKUM PARIWISATA
Ongky Ramadhan Baseno
Fakultas Hukum Universitas 17 Agustus 1945 Surabaya
Jl. Semolowaru 45, Surabaya, 60118
E-mail: ongkyramadhan26@gmail.com
Abstrak
Indonesia adalah negara yang berpotensi untuk menjadi tujuan pariwisata di dunia.
Pariwisata adalah suatu kegiatan yang menyediakan jasa akomodasi, transportasi, makanan,
rekreasi, serta jasa-jasa lainnya. Perdagangan jasa pariwisata melibatkan beberapa aspek, yaitu
aspek ekonomi, budaya, sosial, agama, lingkungan, keamanan dan aspek lainnya. Dari berbagai
aspek tersebut, aspek ekonomilah yang mendapat perhatian paling besar dalam pembangunan
pariwisata sehingga pariwisata dikatakan sebagai suatu industri. Berdasarkan pada ketentuan
latar belakang masalah tersebut di atas maka dapat ditentukan rumusan masalah, Bagaimana
pengelolaan desa wisata Minggirsari yang sesuai dengan prinsip hukum kepariwisataan. Jenis
penelitian yang digunakan dalam penulisan paper ini adalah metode penelitian hukum
normatifPariwisata adalah suatu kegiatan yang secara langsung menyentuh dan melibatkan
masyarakat, sehingga membawa berbagai dampak terhadap masyarakat setempat. Bahkan
pariwisata dikatakan mempunyai energi dobrak yang luar biasa, yang mampu membuat
masyarakat setempat mengalami metamorphose dalam berbagai aspeknya.
Pendahuluan
1. Latar Belakang Masalah
Indonesia adalah negara yang berpotensi untuk menjadi tujuan pariwisata di dunia.
Pariwisata adalah suatu kegiatan yang menyediakan jasa akomodasi, transportasi, makanan,
rekreasi, serta jasa-jasa lainnya. Perdagangan jasa pariwisata melibatkan beberapa aspek, yaitu
aspek ekonomi, budaya, sosial, agama, lingkungan, keamanan dan aspek lainnya. Dari berbagai
aspek tersebut, aspek ekonomilah yang mendapat perhatian paling besar dalam pembangunan
pariwisata sehingga pariwisata dikatakan sebagai suatu industry.1
Peranan sektor pariwisata nasional semakin penting sejalan dengan perkembangan dan
kontribusi yang diberikan sektor pariwisata melalui penerimaan devisa, pendapatan daerah,
1
I Putu Gelgel, Industri Pariwisata Indonesia Dalam Globalisasi Perdagangan Jasa (GATS-WTO)
ImplikasiHukum dan Antisipasinya, Refika Aditama, Bandung, 2009, h. 22.
pengembangan wilayah, maupun dalam penyerapan investasi dan tenaga kerja serta
pengembangan usaha yang tersebar di berbagai pelosok wilayah di Indonesia. Menurut Buku
Saku Kementerian Pariwisata (2016), kontribusi sektor pariwisata terhadap Produk Domestik
Bruto (PDB) nasional pada tahun 2014 telah mencapai 9 % atau sebesar Rp 946,09 triliun.
Sementara devisa dari sektor pariwisata pada tahun 2014 telah mencapai Rp 120 triliun dan
kontribusi terhadap kesempatan kerja sebesar 11 juta orang. Melalui mekanisme tarikan dan
dorongan terhadap sektor ekonomi lain yang terkait dengan sektor pariwisata, seperti hotel dan
restoran, angkutan, industri kerajinan dan lainlain. Melalui multiplier effect-nya, pariwisata
dapat dan mampu mempercepat pertumbuhan ekonomi dan penciptaan lapangan kerja. Itulah
mengapa, percepatan pertumbuhan ekonomi dan penciptaan lapangan kerja yang lebih luas
dapat dilakukan dengan mempromosikan pengembangan pariwisata.2
Matching Fund adalah bentuk nyata dukungan dari Kementerian Pendidikan dan
Kebudayaan (Kemendikbud) Republik Indonesia untuk penciptaan kolaborasi dan sinergi
strategis antara Insan Dikti (lembaga perguruan tinggi) dengan pihak Industri. Lulusan
pendidikan tinggi mendapat pekerjaan yang layak. Matching Fund menjadi salah satu nilai
tambah terbentuknya kolaborasi antara dua pihak melalui platform Kedaireka. Dukungan
Matching Fund ini diprioritaskan bagi kolaborasi yang berkontribusi terhadap pencapaian 8
(delapan) Indikator Kinerja Utama Perguruan Tinggi yang telah ditetapkan oleh Kemendikbud:
•
Lulusan pendidikan tinggi mendapat pekerjaan yang layak
•
Mahasiswa mendapat pengalaman di luar kampus
•
Dosen berkegiatan di luar kampus
•
Praktisi mengajar di dalam kampus
•
Hasil kerja dosen berguna bagi masyarakat dan diakui internasional
•
Program studi kampus bekerja sama dengan mitra kelas dunia
•
Kelas bersifat kolaboratif dan partisipatif
•
Program studi berstandar internasional
Kaitannya di dalam hal ini ialah mahasiswa diberi tugas atau program kerja di dalam
pelaksanaan Kuliah Kerja Nyata (KKN) untuk meningkatkan sektor ekonomi yang berada pada
Desa Minggirsari. Mengingat adanya Pandemi Covid-19 juga tentunya berdampak pada sektor
ekonomi khususnya di bidang pariwisata Desa Minggirsari.
2. Rumusan Masalah
2
Isharyanto dkk., Hukum Kepariwisataan Dan Negara Kesejahteraan (Antara Kebijakan Dan
Pluralisme Lokal), Halaman Moeka Publishing, Bogor, 2019, h. 3
Berdasarkan pada ketentuan latar belakang masalah tersebut di atas maka dapat ditentukan
rumusan masalah sebagaimana berikut:
Bagaimana pengelolaan desa wisata Minggirsari yang sesuai dengan prinsip hukum
kepariwisataan?
3. Metode Penelitian
Jenis penelitian yang digunakan dalam penulisan paper ini adalah metode penelitian
hukum normatif (normative legal research), dimana untuk menemukan peraturan-peraturan
yang berlaku, prinsip-prinsip, dan disertai pula dengan doktrin-doktrin, guna menjawab isu
hukum yang dihadapi.3
Pembahasan
Alasan sektor pariwisata dipacu untuk dijadikan komoditi andalan di samping migas
sebagai komoditi pendukung kelangsungan pembangunan nasional , antara lain adalah:4
a. Pola perjalanan wisata dunia yang terus menerus meningkat dari tahun ketahun;
b. Pariwisata tidak begitu terpengaruh gejolak ekonomi dunia, di samping
pertumbuhannya yang lebih cepat daripada pertumbuhan ekonomi dunia;
c. Meningkatkan kegiatan ekonomi daerah dan pengaruh ganda dari pengembangan
pariwisata tampak lebih nyata;
d. Komoditi pariwisata tidak mengenal proteksi atau quota seperti komoditi lainnya;
e. Potensi pariwisata Indonesia yang tersebar diseluruh wilayah Indonesia yang
beranekaragam macamnya tak akan habis terjual;
f. Pariwisata sudah menjadi kebutuhan hidup manusia pada umumnya.
Dalam living culture, unsur-unsur yang bisa dijadikan sebagai daya tarik antara lain tradisi
suatu suku bangsa tertentu, upacara dan ritual keagamaan, seni pertunjukan, dan sebagainya.
Sedangkan dalam cultural heritage, daya tarik yang ditawarkan dapat berupa benda-benda
peninggalan sejarah dan purbakala, lansekap budaya, dan sebagainya. Wisata berbasis budaya
adalah salah satu jenis kegiatan pariwisata yang menggunakan kebudayaan sebagai objeknya.
Pariwisata jenis ini dibedakan dari minat-minat khusus lain, seperti wisata alam, dan wisata
petualangan. Pariwisata budaya sebagai salah satu produk pariwisata merupakan jenis
pariwisata yang disebabkan adanya daya tarik dari seni budaya suatu daerah. Pariwisata budaya
pada intinya merupakan jenis pariwisata yang menawarkan kebudayaan yang berupa atraksi
budaya baik yang bersifat tangibel atau konkret maupun intangibel atau abstrak, juga yang
3
4
Peter Mahmud Marzuki, Penelitian Hukum, Jakarta, Kencana Pernada Media Group, 2010, h. 35.
Hari Karyana, Kepariwisataan, Penerbit PT. Gramedia Widiasarana Indonesia, Jakarta, 1997, h. 7
bersifat living culture (budaya yang masih berlanjut) dan cultural heritage (warisan budaya
masa lalu), sebagai daya tarik utama untuk menarik kunjungan wisatawan.
Pariwisata secara teori dapat ditinjau dari berbagai fenomena yang mempengaruhi
berkembangnya kegiatan pariwisata. Menurut Hari Karyana ada 5 (lima) fenomena yang
berpengaruh terhadap penyelenggaraan pariwisata yaitu fenomena sosial, fenomena ekonomi,
fenomena psikologi, fenomena geografi dan fenomena budaya.5 Keinginan pemerintah untuk
mengembangkan pariwisata di Indonesia pada dasarnya disebabkan oleh beberapa faktor.
Pertama adalah semakin berkurangnya peranan minyak sebagai penghasil devisa jika
dibandingkan dengan waktu yang lalu; kedua merosotnya nilai ekspor kita dari sektor non
minyak; ketiga prospek pariwisata yang tetap memperlihatkan kecenderungan meningkat
secara konsisten; dan keempat besarnya potensi yang kita miliki bagi pengembangan
pariwisata di Indonesia, khususnya pada Desa Minggirsari.
Pariwisata adalah suatu kegiatan yang secara langsung menyentuh dan melibatkan
masyarakat, sehingga membawa berbagai dampak terhadap masyarakat setempat. Bahkan
pariwisata dikatakan mempunyai energi dobrak yang luar biasa, yang mampu membuat
masyarakat setempat mengalami metamorphose dalam berbagai aspeknya.6 Pengembangan
pariwisata di Indonesia sebenarnya sangat potensial yang terkait dengan daya tarik wisata
berbasis budaya. Terkait dengan hal itu salah satu sistem yang dikehendaki oleh Program
Pembangunan Nasional (Propenas) dibidang pariwisata adalah pembangunan pariwisata yang
berbasis komunitas budaya karena disadari bahwa memang keanekaragaman budaya bangsa
Indonesia merupakan kekayaan yang tidak ternilai harganya dan jumlahnya sangat melimpah
dari Sabang sampai Merauke.
Dalam ketentuan Undang-Undang Nomor 10 Tahun 2009 dapat disimpulkan pada bagian
pasal-pasal undang-undang sebagai berikut :
a. Pasal 2 , asas-asas yang menggambarkan bahwa UU No. 10 Tahun 2009 melibatkan
masyarakat dalam pengembangan pariwisata yaitu asas manfaat, asas kekeluargaan,
asas adil dan merata, asas partisipatif, asas demokrasi.
b. Pasal 3 yang mengatur fungsi kepariwisataan.
c. Pasal 4 yang mengatur mengenai tujuan penyelenggaraan kepariwisataan.
d. Pasal 5, prinsip penyelenggaraan kepariwisataan huruf c, d, e.
5
6
Ibid.
I Gde Pitana dan Putu G. Gayatri , Sosiologi Pariwisata, Penerbit Andi, Yogyakarta, 2005, h. 109.
e. Pasal 19 yang secara tegas menyebutkan peran serta masyarakat dalam
penyelenggaraan pariwisata: Ayat (1) Setiap orang berhak memperoleh kesempatan
memenuhi kebutuhan wisata; melakukan usaha pariwisata;, menjadi pekerja/buruh
pariwisata;. Berperan dalam proses pembangunan kepariwisataan. Ayat (2)Setiap
orang dan/atau masyarakat di dalam dan disekitar destinasi pariwisata mempunyai hak
prioritas untuk menjadi pekerja/buruh; hak konsinyasi ; dan hak pengelolaan.
Pengembangan pariwisata berbasis masyarakat merupakan sebuah pendekatan yang
melibatkan dan meletakkan masyarakat sebagai pelaku penting dalam konteks paradigma baru
pembangunan yaitu pembangunan berkelanjutan (suistainable development paradigm).
Pariwisata berbasis masyarakat merupakan peluang untuk menggerakkan segenap potensi dan
dinamika masyarakat guna mengimbangi peran pelaku usaha pariwisata berskala besar.
Pariwisata berbasis masyarakat tidak berarti merupakan upaya kecil dan local semata , tetapi
perlu diletakkan dalam konteks kerjasama masyarakat secara global. Dalam konsep pariwisata
berbasis masyarakat terkandung di dalamnya konsep pemberdayaan masyarakat. Upaya
pemberdayaan masyarakat pada hakekatnya selalu dihubungkan dengan karakteristik
seseorang sebagai suatu komunitas yang mempunyai ciri, latar belakang dan pemberdayaan
masyarakat , yang terpenting adalah dimulai dengan bagaimana menciptakan kondisi suasana
atau iklim yang meningkatkan potensi masyarakat untuk berkembang.
Di dalam pelaksanaan KKN pada Desa Minggirsari terdapat beberapa kendala antara lain
yakni:
•
Bantuan marketing dalam pembuatan sosial media agar wisatawan lebih mudah
mengetahui wisata alam yg berada di desa minggir sari.
•
Memperbaiki rute jalan ke wisata alam agar lebih baik dan mudah untuk di akses.
•
Pendampingan dan pelatihan untuk menjaga situs bersejarah di Desa Plunturan
•
Tidak terdapat tempat pemandian atau toilet di daerah sumber air
•
Tidak ada tempat parkir yang luas untuk wisatawan
•
Kurang banyaknya tempat sampah
•
Kurang petunjuk arah untuk menuju ke tempat wisata
Hal tersebut yang memuat program kerja sebagaimana KKN yang dilaksanakan pada Desa
Minggirsari Blitar. Namun dengan bantuan para warga sekitar pelaksanaan Kuliah Kerja Nyata
(KKN) Tematik Matching Fund pada Desa Minggirsari Blitar dapat terselenggara dengan baik
dan memberikan dampak yang positif bagi masyarakat sekitar
Penutup
1. Kesimpulan
Berdasarkan pada ketentuan tersebut di atas maka, pelaksanaan Kuliah Kerja Nyata (KKN)
Tematik Matching Fund pada Desa Minggirsari Blitar dapat terselenggara dengan baik dan
memberikan dampak yang positif bagi masyarakat sekitar. Disamping itu diharapkan program
kerja yang dilakukan mahasiswa ini akan menimbulkan dampak yang bagus baik dilingkup
sektor pariwisata, ekonomi, maupun hukum. Diharapkan Desa Minggirsari kedepannya dapat
mengembangkan beberapa sektor khusunya pada sektor pariwisata, mengingat para
masyarakat warga Desa Minggirsari sangat antusias terhadap hal seperti ini.
2. Ucapan Terimakasih
Penulis mengucapkan terimakasih yang sebesar-besarnya kepada beberapa pihak yang
membantu untuk kelancaran pelaksanaan Kuliah Kerja Nyata (KKN) ini. Lebih lanjut saya
ucapkan terimakasih yang sebesar-besarnya kepada dosen pembimbing serta seluruh perangkat
dan warga Desa Minggirsari.
DAFTAR PUSTAKA
Hari Karyana, Kepariwisataan, Penerbit PT. Gramedia Widiasarana Indonesia, Jakarta, 1997
I Gde Pitana dan Putu G. Gayatri , Sosiologi Pariwisata, Penerbit Andi, Yogyakarta, 2005
I Putu Gelgel, Industri Pariwisata Indonesia Dalam Globalisasi Perdagangan Jasa (GATSWTO) ImplikasiHukum dan Antisipasinya, Refika Aditama, Bandung, 2009
Isharyanto dkk., Hukum Kepariwisataan Dan Negara Kesejahteraan (Antara Kebijakan Dan
Pluralisme Lokal), Halaman Moeka Publishing, Bogor, 2019
Peter Mahmud Marzuki, Penelitian Hukum, Jakarta, Kencana Pernada Media Group, 2010
| 1,717 | 13,396 |
W4289262792.txt | “Talqin va tadqiqotlar” Respublika ilmiy-uslubiy jurnali №7
FIZIKA FANI RIVOJLANISH TARIXINI O’RGANISHNING DIDAKTIK
JIHATLARI
Muydinova Madina Alisherovna
Andijon davlat pedagogika instituti katta o’qituvchisi
Abdunabiyeva Maftunaxon Solijon qizi
Andijon davlat pedagogika instituti
Ta’limda axborot texnologiyalari mutaxassisligi magistranti
Qosimjonova Xamidaxon Botirjon qizi
Andijon davlat universiteti Fizika-matematika fakulteti talabasi
Annotatsiya: Ushbu maqolada fizika fanini rivojlanish tarixi o’rganish
bosqichlari, vazifalari, rivojlanishidagi fanlar bilan bog’liqligi va rivojlanish
tarixining davrlari haqida keng yoritilgan. Bundan tashqari, o’quv darslarida fizika
fani tarixini o’qitishning ahamiyati va samaradorligi tahlil etilgan.
Kalit so’zlar: rivojlanish tarixi, analitik ma’lumot, tarixiy fakt, tabiatshunoslik,
jamiyat qonunlari, astornomiya, ishlab chiqarish, ilmiy kashfiyot.
Insoniyat o’zi yashayotgan olam to’g’risidagi bilimlarni to’satdan va tayyor
tugallangan holda ololmaydi. Oddiy, misol: Nyutonning qonunlari degan nom bilan
ataluvchi hozirgi maktab o’quvchisiga ham tanish bo’lgan harakat qonunlarini
tushunish minglab yillarni talab qilgan. Yana ko’p yuz yillar davomida bu
qonunlarning har doim ham har qanday holatlar uchun ham qo’llab bo’lmasliginii
tushunildi[1]. Ya’ni juda katta tezlikdagi va juda kichik o’lchamdagi zarrachalar
harakatiga bu qonunlarni qo’llab bo’lmasligi aniqlandi.
Insoniyat bilmaslikdan bilishga bo’lgan uzoq va mashaqqatli masofani bosib
o’tdi. Bunda u unchalik aniq va to’liq bo’lmagan bilimlarni aniq va to’liq
tushunchalar bilan almashtirib bordi.
Hech bir ish yo’q joydan boshlanmaydi, balki har bir yangi avlod o’zidan
avvalgi o’tganlar to’xtagan joydan ishni boshlaydi va o’zi amalga oshirgan ishlarni
keyingi keluvchilarga topshiradi.
Bu yerda I. Nyutonning quyidagi so’zlarini eslash muxum ahamiyat kasb etadi.
“Agar men boshqalarga nisbatan uzoqni ko’ra olgan bo’lsam, buning sababi, men
buyuk kishilarning yelkalarida turganligim uchundir”[2]. Yevklid va Arximedlarsiz
Nyuton, Nyutonsiz Eynshteyn yoki Bor, Xorazmiy va Forobiysiz Beruniy yoki Ibn
Sino, Umar Xayyom va Umar Chag’moniysiz Ulug’bek, bularsiz Qozizoda Rumiy
(zamonasining Aflotuni) yoki Ali Qushchi bo’lmas edi. Ya’ni, har bir avlod o’zidan
avval o’tganlar merosini o’rganib, ular qoldirgan ishlar zaminida fan va texnikaning
kelgusi rivojini amalga oshiradi.
172
“Talqin va tadqiqotlar” Respublika ilmiy-uslubiy jurnali №7
Har qanday bilimning rivojlanish jarayonini o’rganish alohida bir fan - tarix
fanining asosiy masalasini belgilaydi. Shu jumladan fizika fani rivojlanish tarixi, fan
tarixining bir bo’lagini tashkil qilib, u fizika fanining rivojlanishi jarayonini
o’rganadi. Har qanday fanning asosiy vazifasi shu fan shug’ullanayotgan sohada
amalda bo’lgan qonunlarni ochishdan iborat bo’lganligi kabi fizika tarixi fanining
ham vazifasi fan rivojlanishini boshqaruvchi qonunlarni yaratishdan iborat. Yuzaki
qaraganda bunday qonunlarning bo’lishi mumkin emasdek ko’rinadi. Oldindan
Farobiy, Beruniy, Ibn Sino, Arximed, Nyutonlarning dunyoga kelishini bilib
bo’lmaydi, olimlar fikri va tafakkurini boshqarib bo’lmaydi. Tashqaridan bir
ko’rishda fan tarixi buyuk daholarning nazorat qilib bo’lmaydigan faoliyatlari
natijasiga o’xshab ketadi. Albatta ular faoliyatini tabiatda yuz beruvchi biror harakat
qonunlariga o’xshash qolipdagi qonunlarga o’xshatib bo’lmaydi[3].
Mubolag’asiz aytish mumkinki, fan bu insoning murakkab tafakurri va ijodiy
faloiyat mahsulidir. Biroq fan rivoji muhum o’rin tutuvchi ma’lum bir tarixiy
sharoitlarda amalga oshadi. Bu narsani esa, albatta, ilmiy tahlil qilish mumkin.
Tarixan ma’lumki ilm-fan rivoji iqtisodiy rivojlanish bosqichlariga bog’liq. Qaysi
joyda ishlab chiqarish, sanoat, madaniyat rivojlanishi yuqori bo’lsa o’sha joyda fan
rivojlanishi ham yuqori darajada bo’ladi. Hozirgi kunda bu yanada yaqqol namayon
bo’lib bormoqda.Chunki, ilm fan hozirgi davr rivojida juda katta ijtimoiy mablag’ni
talab qiladi.
Fizika fanini rivojlanish tarixi o’rganish bir qator bosqichlarda amalga
oshiriladi:
–ma’lumotlar yig’ish
–analitik ma’lumotlar orasidagi bog’lanish va sabablar
- analitik natijalarni umumlashtirish va fandagi asosiy qonunlarni ochish.
Fanning rivojlanish tarixi bu umumiy fizikaviy bilimlarning ajralmas bir
qismdir, chunki fizika fanning o’rganish bilan uning tarixi ham o’rganib boriladi, bu
fanning qonun qoidalarning ochilishi, ushbu qonun ochishda olimlarning tutgan
o’rnidir[4]. Fizika tarixini bilish uchun asosan shu qonunlarning yozilishini, asl
nusxasini o’rganish uchun asil ma’nosini o’rganishda muhim o’rin egallaydi.
Har qanday fanning tarixini o’rganishda, bu fan aslida qanaqangi fan, bu fan o’z
taraqqiyotida umumbashariy rivojlanishda jamiyat taraqqiyotida qanaqangi o’rin
tutadi va qanday metodlar bilan o’rganilishi kerak. Shu nuqtai nazardan qaragan
fizika tarixi o’rganish, jamiyat taqqiyotining bosqichini o’rganishda tabiatshunoslik
fanning yetakchilari bilan bir qatorda tutgan o’rni muhimdir. Chunki fizika
atrofimizni o’rab olgan tabiat qonunlarni o’rganish bilan bog’liqlikligi tufayli, uni
o’rganish tarixi ham juda qadimiy bo’lib, hozirgi kungacha 2000 yildan ortiq vaqtni
o’z ichiga oladi[5].
173
“Talqin va tadqiqotlar” Respublika ilmiy-uslubiy jurnali №7
Fizika fani o’tgan XX asr davomida shunchalik o’zgarib ketdiki, fizika fani
o’rganishda uning oddiy fizik xodisalardan boshlab uning hozirga kundagi murakkab
taraqqiyotda davom etayogan taraqqiyotini inson ongiga singdirib borish uchun ham
fizikani fan tarixi bilan biog’lab o’rganish muximdir. Fizika tarixi fani fizikaviy
bilimlarning ajralmas bir qismdir.
Shu sababli fizika fanini o’rganish fizika o’qituvchilaridan fizikani chuqur
o’rganish, uni analiz qilish, qonunlarining bir biriga bog’liqligini ochib berish,
o’quvchilarga uni bir-biri bilan uzviy bog’liqligini ko’rsatib berish, qonunlarni
moxiyatini tushuntirish, fanning jamiyat taraqqiyotida tutgan o’rnini ochib
berilishidan iboratdir.
Fizikaning rivojlanish tarixini o’rganishda quyidagi vazifalarni amalga
oshiriladi:
- Tarixiy faktlarni topish va oydinlashtirish;
- Tarixiy faktik materiallarni analiz qilish, uning moxiyatini ochish;
- Fizikaviy umumiy qonunlarni o’zaro bog’liqligini ochish.
Dunyo taraqqiyotidagi barcha fanlar tarixi asosiy imkoniyat yo’nalishidagi
vazifani hal qiladi:
1.
Tabiat qonunlari tarixi;
2.
Jamiyat qonunlari tarixi.
Tarixiy fanlar shuni ko’rsatadiki, fizika fani o’z rivojlanishida boshqa fanlar
bilan birinchi navbatda tabiatshunoslik fani tarixi bilan uzviy bog’liqdir, fizika fani
esa bu fanlarning rivojlanishida o’zining munosib xissasini qo’shadi. Shuning uchun
ham ko’pgina fanlar rivojlantirgan olimlarni ham ko’p qirrali deb qarashimiz
mumkin. Bular fizika, astronomiya, ximiya, biologiya, matematika va h.k. fanlarning
rivojlanishiga kattagina hissa qo’shgan olimlar Abu Rayxon Beruniy, Mirzo
Ulug’bek, Ibn Sino, R. Boel, E. Mariott, G.Kavendesh va boshqalar shular
jumlasidandir. Fanlar tarixini o’rganar ekanmiz qadimda bu fanlarning barchasi
falsafiy qarashlardan kelib chiqqanini ko’rishimiz mumkin. Qadimda olimlarning
“Donolik ishqibozlari” deyilgan[6]. So’ngra fanlar o’rganilib qonuniyatlarning
ochilishi natijasida barchasi alohida fan sifatida shakllana bordi.
Fizika fani ham o’z rivojlanishida ko’pgina fanlar bilan bog’liqdir.
Masalan fizika fani o’z rivojlanish tarixida matematika fani bilan uzviy
bog’liqdir, chunki matematikasiz fizik qonunlari tushuntirish mumkin emas.
Matematikani ilmiy metod sifatidagi ahamiyati fizikani o’qitishda juda keng va
yaxshi o’z aksini topdi. Ular matematika formulalarida ifodalanadi, bular esa fizik
qonunlardan xulosaalar chiqarishda ayrim holatlarni isbotlashda va fizik masalalarni
echishda ishlatiladi. Fan tarixida minglab yillar davomida astronomlar osmon
xodisalari to’g’risida faqat ko’zga ko’rinadigan yorug’lik vostasida olinadigan
informastiya bilan cheklanib keladilar. Ular bu xodisalarni elektromagnit
174
“Talqin va tadqiqotlar” Respublika ilmiy-uslubiy jurnali №7
nurlanishlarning keng spektridagi torgina tirqishi orqali o’rganar edilar deb aytish
mumkin. 1965- yillar radiofizikasining rivojlanishi tufayli radioastronomiya vujudga
keldi va bizning koinot haqidagi tasavvurlarimiz xaddan tashqari kengayib ketdi.
So’ngi yillarda muxim o’zgarishlar bo’ldi. Astronomiyadagi astrofizika ko’proq
ahamiyat kasb eta boshladi va ishlab chiqarishni rivojlantirishda, ishlab chiqarish
jamiyatning moddiy farovonligining o’sishiga olib keladi[7]. Demak, fanning
rivojlanishi ishlab chiqarishni boshqarar ekan, ishshlab chiqarish fizikaning yangi
qonunlar ochishiga har doim ta’zyiq o’tkazib turadi. Masalan, Gyugensning mayatnik
nazariyasi – mexanik soatning ixtiro qilishga olib keldi. Termodinamikadan S.Karno
stikli bug’ mashinsining ixtiro etinlishiga asos bo’ldi. A.S.Popov tomonidan
radioning kashf etilishi radiofizikaning rivojlanishiga olib keldi va hakozolar.
Shuningdek fizika fanining rivojlanish tarixi nazar solsak, fanning taraqqiyoti
bevosita ishlab chiqarishni rivojlantirish tarixi bilan bog’liqligini ko’ramiz. Chunki
fizik fanining yutuqlari bevosita texnika va texnalogiyalarda qo’llanib, u esa o’z
navbatida ishlab chiqarish kularini rivojlantirishda muxim o’rni egallaydi. Fizika
fanining rivojlanishi jamiyatning ijtimoiy tuzilishiga ham bog’liqdir. Chunki fanning
rivojlanishida jamiyatning iqtisodiy siyosiy tuzilishi ham muhim rol o’ynaydi.
Masalan: Quldorlik tuzumi davridan keyingi ilk feodalizm davrida fan taassurotlar
bilan rivojlanmadi, chunki o’sha davrdagi quldor ham, feodal ham ishlab chiqarishni
rivojlantirishi bilan qiziqmas edi.
Fizika faqat ishlab chiqarish bilan bog’liq bo’libgina qolmay, u barcha tabiiy
fanlar bilan ham bog’liqdir. Buni qanning tarixi o’rganishimizda ko’rib boramiz.
Fizika fani rivojlanish tarixini quyidagi davrlari uning rivojlanishiga qarab
o’rganib boriladi.
1. Qadimgi zamon fizikasi(fizik tushunchalarning shakillanish davri)
a)
antik davr fizikasi (eramizdan oldingi III asrga qadar bo’lgan davr)
b)
O’rta asr fizikasi (III-X asr)
v) Geliostentrik sistema uchun kurash(X-XVI asr )
2. Klassik fizikaning asosiy yo’nalishlar shakllanisi davri
a)
Ilmiy inqilobning tugallanishi (XVIII asr).
b)
Fizikaning asosiy yo’nalishlarining rivojlanishi (XIX asr).
3. Fizikada ilmiy inqilobning asosiy yo’nalishlari (zamonaviy fizika shakllanisl
davri)
a)
Elektron nazariya va elektrodinamikaning rivojlanishi.
b)
Eynshteyning nisbiylik nazariyasi
v) Atam va yadro fizikasining rivojlanishi.
g) Fizikaviy inqilobning birinchi bosqichi Rezerford-Bor nazariyalar, kvant
mexanikaning rivojlanishi.
d) yadro fizikasining rivojlanishi 1918 – 1938 yillar.
175
“Talqin va tadqiqotlar” Respublika ilmiy-uslubiy jurnali №7
Fizika fani mazmuni fizikaning rivojlanish bosqichlari bo’yicha tanlansa,
maqsadga muvofiq bo’ladi[8]. Chunki fizika fanining rivojlanishi jamiyatning
iqtisodiy va siyosiy rivojlanishi bilan birgalikda qaralishi lozim. Ma’lumki, har
qanday fanning rivojlanishi jamiyat ehtiyojlarini qondirish bilan uzviy bog’liqdir.
Shunday qilib fizika fani rivojlanish tarixini o’rganish o’quv jarayoni va
tarbiyaning ko’pgina muammolarini hal etish bilan birgalikda, fizika fanini o’qitish
uslubining ham rivojlanishida muhim omil hisoblanadi.
Tarixiy hujjatlardan dars jarayonida quyidagi shakllarda foydalanish tavsiya
etiladi:
- tarixiy voqyea va hujjatlar asosida kirish so’zi bilan muammoli
vaziyatni yuzaga keltirish orqali darsning samaradorligini oshirish;
- o’tilgan mavzuni yakunlashda o’quvchilar bilimini bir tizimga
keltirish maqsadida tarixiy hujjatlardan foydalanish;
- o’quvchilar bilimining ishonarli bo’lishiga erishish uchun buyuk kashfiyotlar,
fundamental tajribalar tarixini bayon etish;
- o’quvchilar shaxsini takomillashtirish, milliy mafkura va tarbiyani amalga
oshirish maqsadida buyuk olimlar hayoti va ilmiy faoliyatidan ayrim lavhalarni
keltirish;
- tarixiy mazmundagi masalalardan dars jaryonida foydalanish. Tarixiy
hujjatlarni ushbu shaklda bayon etish shartli bo’lib, ba’zan, bir necha usullar
birgalikda qo’llanilishi mumkin[9]. O’tilayotgan mavzu ishonarli bo’lishi uchun
tarixiy tajribalar, kashfiyotlar, chizmalar, grafiklar, rasmlar, modellar orqali
tushuntiriladi. Xuddi shuningdek, moddiy va abstrakt modellar, kinofilmlardan
foydalanish ham mumkin.
O’rta maktab fizika kursida 100 dan ortiq fizik olimlar nomi qayd etiladi. Ushbu
olimlarning hayoti va ilmiy kashfiyotlari qanchalik darajada va qanday tartibda
berilishi kerak. Albatta, o’quv rejasida keltirilgan vaqt davomida barcha olimlarning
hayoti va ilmiy faoliyatini to’la yoritishning imkoniyati yo’q. Shu sababli ba’zi
olimlar hayotidan ayrim lavhalarni, jumladan, dars mavzusiga mos kelganlarini
tanlab olish maqsadga muvofiqdir. Ba’zan olimlar tomonidan aytilgan hikmatli
so’zlar, masalan,T.Yung aytganidek, «Odam qilgan ishni boshqa odam ham bajara
oladi» yoki olimlar tomonidan aytilgan hazil-mutoyibalar darsning qiziqarli o’tishiga
sabab bo’ladi.
Buyuk olimlar hayotidan lavhalar keltirish orqali yoshlarda mehnatsevarlik,
halollik, vatanparvarlik kabi tuyg’ularni shakllantirishga erishiladi. Masalan:
alXorazmiy, Ibn Sino, Beruniy, Forobiy, Ulug’bek, Qori Niyoziy, R.Bekjonov, A.K.
Otaxo’jayev kabi olimlarimizning hayoti va ilmiy faoliyatini yoritish orqali
milliy g’urur, iftixor, mustaqillik, ozodlik g’oyalarini o’quvchilar ongiga singdirish
imkoniyati yaratiladi. Olimlar hayoti bilan tanishish jarayonida ularning faqat ijobiy
176
“Talqin va tadqiqotlar” Respublika ilmiy-uslubiy jurnali №7
tomonlari olinmasdan, balki, turmushda va ilmiy izlanishda yo’l qo’ygan xato va
kamchiliklari to’g’risida ham to’xtash maqsadga muvofiqdir. Masalan V. Tomson va
Klauziuslarning «Olamning issiqlik halokati» to’g’risidagi fikri yoki "Efir",
"teplorod" nazariyasi tarafdorlarining noilmiy fikrlari darsning qizikarli bo’lishiga
xizmat qiladi.
Xulosa qilib shuni aytish mumkinki, dars jarayonida buyuk fizik olimlarning
dunyoqarashi va ilmiy kashfiyotlari haqida ma’lumotlar berish orqali darsni qiziqarli
o’tish va uning samaradorligini oshirish kabi bir qator didaktik vazifalarni amalga
oshirish mumkin.
Foydalanilgan adabiyotlar:
1.
Kudryavsev P.S. Kurs istorii fiziki. M. 1989;
2.
Ziyaxanov R.F., Mirzayev A.T., Astronomiya va fizika tarixi, I qism,
Toshkent, 2008;
3.
Pannekuk A, Istoriya astronomii, M., Nauka, 1966;
4.
Mirzayev A.T. Astronomiya tarixi. (ma’ruzalar matni). T. О‘zMU. 2000;
5.
Mamadazimov M.M. Astronomiya va fizika tarixi. (ma’ruzalar matni),
T. TDPU, 2001;
6.
Mamadazimov M.M. Ulug‘bek va uning rasadxonasi, T. О‘zbekiston,
1994;
7.
Gorbatskiy V.G., Leksii po istorii astronomii. SPgu, 2002;
8.
Astronomicheskiye dati i otkritiya, http://astro.websib.ru/istor/istor.htm;
9.
http://naturalhistory.narod.ru/Page_1.htm.
177
| 1,766 | 14,777 |
W4390711231.txt | Bangabandhu Sheikh Mujib Medical University Journal 2023;16(4):198-204
bsmmuj.org
ORIGINAL ARTICLE
Quality of life among adolescents with substance use
disorders
Rubaiya Khan1, Mohammad Shamsul Ahsan2, Md. Tanvir Rahman Shah2, Bijoy Kumar Dutta3, Mukul Chandra Nath4
1
Department of Psychiatry, Khwaja Yunus Ali Medical College and Hospital, Sirajganj, Bangladesh
of Psychiatry, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh
3Department of Psychiatry, Chottogram Medical College Hospital, Chottogram, Bangladesh
4Department of Psychiatry, Marine City Medical College and Hospital, Chottogram, Bangladesh
2Department
Correspondence to: Dr. Rubaiya Khan, Email: rubaiyakhan1201@yahoo.com
ABSTRACT
Background: Adolescents are vulnerable to substance use, which profoundly affects their func on, feelings and quality of life
(QoL). This study aimed to assess the physical health, psychological, social rela onships, and environmental domains of QoL
among adolescents with substance use disorders (SUDs).
Methods: A cross-sec onal study was done from October 2020 to September 2021 among 44 adolescents selected purposively
from Central Drug Addic on Treatment Center (CDC), Dhaka and Ashok Punorbashon Nibash (APON), Manikganj. Clinical
diagnosis was assigned using The Diagnos c and Sta s cal Manual of Mental Disorders, Fi h Edi on (DSM-5) by psychiatrists.
Then, they were interviewed with a ques onnaire and a Bengali Version of the World Health Organiza on quality of life scale
brief version (WHOQOL-BREF).
Results: Among the 44 adolescents with SUD, 18 (40.9%) rated their QoL as poor, 14 (31.8%) as very poor, 8 (18.2%) as neither
poor nor good, and only 4 (9.1%) as good. Nearly half of them (47.7%) reported that they were dissa sfied, one third (29.5%)
were very dissa sfied with their health. Among the four domains, the psychological domain (9.8±2.3) had the lowest mean
score, followed by physical health (10.5±2.7), social rela onships (10.6±2.6), and environmental domain (11.5±2.2) of the
WHOQOL-BREF. Mul ple drug users had poorer scores of their QoL than single drug user.
Conclusion: Our study demonstrated that all domains of QoL (physical, psychological, social, and environmental) are impaired in
adolescents with SUDs.
Keywords: quality of life, substance use disorders, adolescents
National Institute of Mental Health, Dhaka during the
INTRODUCTION
Substance use disorders (SUDs) are one of the alarming
mental health issues in adolescents throughout the
world, which lead to impairment and distress. There
periods of 2017 to 2018 showed a 2.9% prevalence of
substance use among seven years and above children
and adolescents.3
are substantial geographical variations in the prevalence
In several studies, it was found that quality of life (QoL)
of SUD among adolescents. A study done in the United
is poorer among substance-dependent individuals and
States using data from the National Household Survey
substance use disorders treatment seekers than among
on Drug Use and Health (NSDUH) shows that 7-9% of
cohorts without substance use disorders.4, 5 SUDs are
adolescents in the US between the ages of 12-17 years
associated with a wide range of serious health, social,
disorders.1
and economic complications. The health status of
In a community-based cross-sectional study carried out
alcohol and drug abusers is generally affected by their
among 3564 children of Bangladesh aged 5 to 17 years
abuse. Consequently, their life expectancy is often much
in 2009, substance-related disorders was found in 0.8%
lower
of respondents. In the case of adolescents aged 12 years
relational, and judicial problems are also well-
and above, substance abuse was 2.1%.2 A nationwide
documented among substance abusers. Drug and
met the criteria for an alcohol or illicit drug
household-based cross-sectional study conducted by the
than
the
general
population.6-9 Housing,
alcohol abuse further causes high costs due to frequent
Received: 18 June 2022; Revised version received: 16 Oct 2023; Accepted: 4 Nov 2023; Published online: 23 Nov 2023
DOI: https://doi.org/10.3329/bsmmuj.v16i4.60341; ISSN 2074-2908, eISSN 2224-7750; © CC-BY-NC-4.0
This work encompasses the MD thesis of Dr Rubaiya Khan
Quality of life among adolescents with substance use disorders
199
Research instruments
HIGHLIGHTS
1. Quality of life is lower among adolescents with substance use
disorders.
2. Among the four domains of the WHOQOL-BREF scale, the psychological domain is affected most by adolescent substance
usage.
3. Multiple substance use affect QoL more adversely than single
substance use.
a. Questionnaire for socio-demographic and related
variables for the study of QoL
A semi-structured questionnaire in Bangla was designed
by the researcher to collect information regarding sociodemographic variables such as age, sex, residence,
religion, level of education, family type, family history of
substance use, age of onset of taking the drug, the
and multiple hospitalizations and treatment episodes.10
reason for initiation of substance use, name of
substance currently using, route of administration,
A study revealed that adolescents who met the criteria
monthly expenditure, history of receiving treatment and
for SUDs had reduced scores across all domains of QoL,
admission in hospital for substance use, etc.
with
the
most
observable
effects
on
academic
achievement and school adjustment.11 Stevanovic et al.
b. DSM-5 criteria for SUD
found that adolescents who reported using substances
The DSM-5 criteria for substance use disorders were
had significantly lower scores in total, physical,
used to diagnose SUDs in adolescents. The DSM-5
emotional, social, and school functioning domains of
recognizes SUDs resulting from the use of ten separate
the Pediatric Quality of Life Inventory (PedsQL) than
classes
those who did not report using any substance.12 On the
hallucinogens, inhalants, opioids, sedatives-hypnotic-or
other hand, a moderate reduction in the frequency of
anxiolytics, stimulants, tobacco, and other unknown
substance use among adolescents was associated with
substances.14
improvements in
of
drugs:
alcohol,
caffeine,
cannabis,
QoL.13
c. World Health Organization quality of life scale, brief
The primary objective of our study was to assess the
version (WHOQOL-BREF)
quality of life in relation to sociodemographic factors
among adolescents with SUDs. Our secondary objective
This study used WHOQOL-BREF to assess the quality
was to compare the quality of life among adolescents
of life of adolescents with SUD. The scale had 26 items,
using single and multiple substances.
among which the first two items are used to measure an
METHODS
Study design and participants
This cross-sectional study was done from October 2020
to September 2021 in the Central Drug Addiction
Treatment Centre (CDC), Tejgaon, Dhaka, and Ashokti
Punorbashon Nibash (APON), Singair, Manikganj. A
total of 44 adolescents within the range of 11 to 19 years
of either sex with SUD who attended the inpatient and
outpatient department of the CDC and APON were
purposively included in the study. However, adolescents
in an intoxication or withdrawal state who were mute
and those with non-communicable were excluded from
the study. We obtained ethical approval from the
Institutional Review Board of Bangabandhu Sheikh
Mujib Medical University.
individual’s overall perception of QoL and overall
perception of their health. The remaining 24 items
measured the following four domains: physical health,
psychological, social relationships, and environment.15
The WHOQOL-BREF has been translated into Bangla
and validated for Bangladeshi adolescents.16
Data collection
Prior permission was taken from the authority of two
institutions: CDC and APON. Participants and their
parents were informed about the study's purpose,
method, and outcome. Informed written assent/consent
was taken from the patients and one of their parents
before data collection, as appropriate, by face-to-face
interviews. Qualified psychiatrists made clinical
diagnoses by applying DSM-5 criteria of SUD. Then,
socio-demographic information was documented using
the semi-structured questionnaire. The Bangla version
Khan R et al. Bangabandhu Sheikh Mujib Medical University Journal 2023; https://doi.org/10.3329/bsmmuj.v16i4.60341
Quality of life among adolescents with substance use disorders
of the WHOQOL-BREF was applied to evaluate the
QoL. One of the investigators (RK) read out the
questions and recorded the answers given by the
participants.
Data analysis
TABLE 1 Socio-demographic characteristics and substance use
among the study participants with substance use disorder (n=44)
Variables
Age, years
Age of onset of substance use, years
Monthly expenditure due to substance use (BDT)
Descriptive statistics such as frequency (percent) or
mean (standard deviation) were used as appropriate.
Student’s t test was used to compare the mean score of
four domains (physical health, psychological health,
social relationship, and environment) of the WHOQOLBREF categories. The analysis of variance was used to
compare more than two categories (e.g., education).
P<0.05 was considered statistically significant.
RESULTS
Forty-four adolescents with SUDs were included in this
study, among which 24 were from APON and 20 from
CDC. The mean age of the study participants was 16.5
(2.3), 88.6% being male. The majority (79.6%) of the
respondents were from urban areas. About half (43.2)
of the parents were divorced, widows, widowers, or
separated. About 80% were from nuclear families
(TABLE 1).
About 47.7% of the respondents had a family history of
substance use. The mean age of onset of substance use
was 12.2 years. The mean monthly expenditure due to
substance use was BDT 11,579.
A little less than half (40.9%) of them rated their QoL as
poor, and 31.8% as very poor. About 48.0% of the
respondents were dissatisfied with their health, but
9.1% were satisfied (FIGURE 1). The most commonly
used substance was tobacco (75.0%), followed by
cannabis (65.9%), stimulant (45.5%), alcohol (38.6%),
inhalant (18.2%), sedative (15.9%), and opioid (9.1%)
(FIGURE 2).
The mean standard deviation scores of physical health,
200
Sex
Male
Female
Residence
Urban
Rural
Education
Primary or below
Secondary school
Higher secondary or above
Type of family
Nuclear
Joint
Marital status of parents
Married
Separated/ divorced/ widow/ widower
Family history of substance use
Yes
No
Reason for initiation of substance use*
Peer pressure
Curiosity
Broken family
Family negligence
Failure in study
Failure in a love affair
Number of substances currently used
Single
Multiple
Route of administration*
Smoking
Swallowing
Inhalation
Injection
History of treatment for substance use
Yes
No
History of hospital admission for substance use
Yes
No
Findings
Mean
16.5
12.2
SD
2.3
3.5
11,579
Number
11,117
Percent
39
5
88.6
11.4
35
9
79.5
20.5
16
13
15
36.4
29.5
34.1
35
9
79.5
20.5
25
19
56.8
43.2
21
23
47.7
52.3
18
19
9
11
2
6
40.9
43.2
20.5
25.0
4.5
13.6
10
34
22.7
77.3
40
25
17
1
90.9
56.8
38.6
2.3
24
20
54.5
45.5
17
27
38.6
61.4
SD indicates standard deviation
*Multiple responses
DISCUSSION
psychological, social relationships, and environmental
domains were 10.5 (2.7), 9.8 (2.3), 10.6 (2.6), and 11.5
SUDs
(2.2), respectively. According to the WHOQOL-BREF
uncontrolled use of a particular substance despite
are
complex
condition
characterized
by
scale, a higher score indicates a better quality of life.
harmful consequences. Adolescents are at greater risk of
Therefore, the psychological domain was most impaired
substance use. During adolescence, development occurs
among the four domains, followed by the physical
in multiple domains like-emotional, social, cognitive,
health, social relationships, and environmental domain
and biological, and conditions like SUDs may affect
(TABLE 2).
adolescents’ behavior, lifestyle, and future life. Nowadays,
SUD has increased significantly among adolescents and
Khan R et al. Bangabandhu Sheikh Mujib Medical University Journal 2023; https://doi.org/10.3329/bsmmuj.v16i4.60341
Quality of life among adolescents with substance use disorders
201
young adults.17 SUD impairs individuals’ physical and
psychological health and also jeopardizes general safety
and social performance and, thereby, overall quality of
TABLE 2 Relationship of WHOQOL-BREF scores and sociodemographic variables of the respondents (n=44)
Variables
Mean (standard deviation) scores of Quality of Life
Physical
Health
Psychological
Social
Relationships
Environmental
Overall
10.5 (2.7)
Gender
Male
10.5 (2.7)
Female
10.6 (2.9)
P*
0.97
Residence
Urban
10.7 (2.5)
Rural
10.6 (2.9)
P*
0.74
Education
Primary or below
9.6 (2.5)
Secondary school
11.1 (2.4)
Higher secondary or
11.1 (2.6)
above
P*
0.18
Type of family
Nuclear
10.5 (2.5)
Joint
10.6 (3.5)
P*
0.90
Marital Status of Parents
Married
10.9 (3.0)
Separated/divorced/
9.3 (2.7)
widow/widower
P*
0.27
History of receiving treatment
Yes
9.4 (2.3)
No
12.0 (2.5)
P*
0.001
History of admission to the hospital
Yes
9.4 (2.3)
No
11.3 (2.7)
P*
0.02
Number of substances
Single
13.0 (1.9)
Multiple
9.8 (2.5)
P*
0.001
9.8 (2.3)
10.6 (2.6)
11.5 (2.2)
9.8 (2.3)
9.6 (2.3)
0.85
10.6 (2.6)
10.2 (2.3)
0.74
11.4 (2.2)
11.4 (2.0)
0.91
9.8 (2.3)
10.1 (2.3)
0.57
11.0 (2.4)
11.0 (3.1)
0.61
11.8 (2.3)
11.6 (2.0)
0.32
9.2 (2.3)
10.0 (2.1)
10.3 (2.5)
11.0 (2.6)
11.5 (1.8)
11.1 (2.8)
12.3 (2.8)
11.5 (2.4)
12.5 (2.5)
0.41
0.88
0.64
9.9 (2.4)
9.2 (1.8)
0.35
10.9 (2.6)
9.1 (1.1)
0.03
11.6 (2.2)
10.5 (1.5)
0.09
10.2 (2.7)
9.2 (1.7)
10.2 (2.2)
9.8 (2.1)
12.0 (2.3)
11.4 (2.8)
0.03
0.37
0.98
8.8 (2.3)
10.1 (2.2)
0.002
10.5 (2.1)
10.7 (3.1)
0.90
11.2 (2.1)
11.5 (2.3)
0.90
9.1 (2.2)
10.3 (2.4)
0.10
10.7 (2.1)
10.6 (2.9)
0.81
11.4 (2.3)
11.5 (2.1)
0.81
11.8 (2.2)
9.2 (2.1)
0.005
12.1 (2.3)
10.1 (2.5)
0.03
12.5 (2.2)
11.1 (2.1)
0.11
life. Thus, the present study aimed to evaluate the
quality of life among adolescents with SUD.
FIGURE 1 Perception of quality of life and satisfaction about health
among the participants of substance use disorder (n=44).
Our study's mean scores for four domains were from 9.8
to 11.5 According to the WHOQOL-BREF scale, the
higher the mean scores, the better the quality of life. We
found that the mean scores of physical health,
psychological, social relationships, and environmental
domains of WHOQOL-BREF were higher in the healthy
male adolescent population of both slum and residential
areas of Dhaka city than the mean scores of these four
domains of our respondents who were substance
users.18 This indicates that all four domains of the QoL
of adolescents with SUD were low compared to the
healthy adolescent male population.
*Student t-test or ANOVA
relationships, and environmental domains were low.19
An international study involving 2393 adolescents from
A study done in Etawah, India, on 145 street children 13
seven countries using the Pediatric Quality of Life
-18 years of age abusing substances revealed that mean
Inventory
scores
substances have a significantly lower score in PedsQL
of
physical
health,
psychological,
social
(PedQL)
showed
adolescents
using
physical, emotional, social, and school functioning
domains.12
Most adolescents rated their QoL as poor or very poor.
Most of the substance-dependent adolescents were
dissatisfied with their health, and only a few were
satisfied. Though the QoL among adolescents with SUD
is a major public health concern, QOL assessment using
the WHOQOL-BREF among adolescents was minimal.
Therefore, comparing this study’s results concerning the
perception of quality of life and satisfaction with health
FIGURE 2 Proportion of type of substance use among the respondents with substance use disorders (n=44).
with other studies was impossible.
Khan R et al. Bangabandhu Sheikh Mujib Medical University Journal 2023; https://doi.org/10.3329/bsmmuj.v16i4.60341
Quality of life among adolescents with substance use disorders
202
Among the four domains, the psychological domain was
educational background is almost identical to the
mostly affected in adolescents with SUD. Psychological
national data. Moreover, it is similar to other studies.25
distress is a common experience for patients with SUD,
Broken family and history of substance use in the family
partly due to substance use and its consequences and
increase the risk of adolescent substance use.26
partly due to psychiatric disorders that often coexist
with SUDs, resulting in a double burden of symptoms
and problems.20 Approximately 70-80 percent of youths
presenting to treatment for SUD are duly diagnosed.21 A
study in Bangladesh revealed that among the patients
In the present study, the average age of onset of
substance use was 12 years, and the reasons for
initiation were peer pressure, curiosity, broken family,
family negligence, academic failure, and relationship
with first-episode psychosis, lifetime SUD was double
failure. Sawhney and Kaur also reported similar age of
that of control.22 The temporal relationship between
onset, and the reasons were enjoyment, showing
substance use and psychological symptoms, such as
manhood and fun, frustration, curiosity, peer pressure,
depression, anxiety, or psychosis, should be considered.
and imitating their father and siblings.25
Substance use may be a perpetuating factor for
The finding of this research suggests that adolescents
deteriorating mental health. On the other hand,
with SUD have reduced QoL across all domains.
impaired mental health may also have preexisted before
the substance use, precipitated the substance use, and
may have deteriorated further.23 Therefore, assessment
of the psychological domain and providing appropriate
interventions toward treating both SUD and psychiatric
disorders simultaneously is mandatory for a better QoL.
Therefore, a biopsychosocial approach through a
multidisciplinary team should be implemented while
managing such patients. Management should include
treatment of co-morbid physical and psychiatric illness,
psychological intervention, and social rehabilitation to
improve and maintain QoL. Proper psychological
The impairment in physical health may be due to
assessment by a psychiatrist and a more integrated
increased risk for injury and violence associated with
approach are needed for a better QoL. Awareness
substance use, respiratory problems due to smoking, or
programs for society about the disease, treatment, and
inhalation of substances. Co-occurring psychiatric
outcome of SUD should be implemented. Psychiatrists,
disorders may have contributed to lower scores in the
governmental organizations, and non-governmental
psychological
social
working on drug use should deal with this condition
relationships, adolescents with multiple substance use
holistically. The QoL assessment, therefore, should be
may be more neglected by the family or society due to
considered in health care. QoL measures are needed to
social stigma. Those who go to hospital might have
be routinely included in the evaluation of treatments.
domain.
In
an
aspect
of
severe problems of SUD as indicated by our data.
Clear
male
dominance
was
(88.6%)
This study has several limitations too. First, SUDs were
observed.
diagnosed by psychiatrists using DSM-5 criteria, where
Throughout the world, the prevalence of substance use
a structured interview could give a more accurate
is higher in males than in females.24 Due to social
diagnosis. Second, small number of participants from
restrictions and the society's attitude, male dominance
selected drug addiction treatment centres, lack the
is also observed in our country. The majority (79.6%) of
representativeness, and therefore, findings cannot be
the patients hailed from urban areas. As the sample was
generalized. Third, a cross-sectional study design
collected from two centres near Dhaka city, most
cannot provide a causal relationship.
attendants belonged to the metropolitan area. It may
also refer that adolescents from urban areas have better
The quality of life is impaired in adolescents with SUD.
access to services.
Education might have some link to adolescents'
addiction
behaviour.
Conclusion
However,
our
participant's
The psychological domain is the most impaired among
the four domains of the WHOQOL-BREF scale. In
addition, physical health, psychological, and social
Khan R et al. Bangabandhu Sheikh Mujib Medical University Journal 2023; https://doi.org/10.3329/bsmmuj.v16i4.60341
Quality of life among adolescents with substance use disorders
203
relationships domains are significantly impaired in
4.
Donovan D, Mattson ME, Cisler RA, Longabaugh R, Zweben
A. Quality of life as an outcome measure in alcoholism
treatment research. Journal of Studies on Alcohol,
Supplement. 2005 Jul(15):119-39. DOI: https://
doi.org/10.15288/jsas.2005.s15.119.
5.
Smith KW, Larson MJ. Quality of life assessments by adult
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Stevanovic D, Atilola O, Balhara YP, Avicenna M, Kandemir
H, Vostanis P, Knez R, Petrov P. The relationships between
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Izutsu T, Tsutsumi A, Islam A, Matsuo Y, Yamada HS, Kurita
H, Wakai S. Validity and reliability of the Bangla version of
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multiple substance users. The physical health domain is
significantly lower among those who seek services. A
comprehensive management plan is necessary to
improve
the
health
of
adolescents
with
SUDs.
Longitudinal studies using a more representative
sample are warranted.
Acknowledgments
The authors are grateful to all the adolescents who participated in
this study.
Author Contributions
Conception and design: RK, MSA. Acquisition, analysis and
interpretation of data: RK, MSA, MTRS, BKD, MCN. Manuscript
drafting and revising it critically: RK, MSA. Approval of the final
version of the manuscript: RK, MSA, MTRS,BKD, MCN. Guarantor
accuracy and integrity of the work: RK, MSA.
Funding
The study received financial support from Bangabandhu Sheikh
Mujib Medical University.
Conflict of Interest
Authors declare no conflict of interest.
Ethical Approval
The study was approved by the Institutional Review Board of
Bangabandhu Sheikh Mujib Medical University (Memo No:
BSMMU/2021/126 issued on 13 February 2021).
ORCID iD
Rubaiya Khan https://orcid.org/0009-0007-4193-6090
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| 4,125 | 29,408 |
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Registered charity number: 207890
| 68 | 499 |
W4298355692.txt | Adaptive divergence despite small population sizes and
strong genetic drift in English yew (Taxus baccata L.)
M. Mayol, M. Riba, D. Grivet, Giovanni G Vendramin, L. Vincenot, Santiago
C. González-Martínez
To cite this version:
M. Mayol, M. Riba, D. Grivet, Giovanni G Vendramin, L. Vincenot, et al.. Adaptive divergence despite
small population sizes and strong genetic drift in English yew (Taxus baccata L.). 14. MEDECOS
& 13. AEET meeting. Human driven scenarios for evolutionary and ecological changes, Jan 2017,
Séville, Spain. 268 p., 2017, XIV MEDECOS & XIII AEET meeting. Human driven scenarios for
evolutionary and ecological changes. �hal-02735247�
HAL Id: hal-02735247
https://hal.inrae.fr/hal-02735247
Submitted on 2 Jun 2020
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Distributed under a Creative Commons Attribution 4.0 International License
XIV MEDECOS
&
XIII AEET meeting
Human driven scenarios for evolutionary and ecological changes
Abstract book
31st January - 4th February 2017
Seville, Spain
Abstract book of the XIV MEDECOS & XIII AEET meeting,
Seville, Spain, 31st January - 4th February 2017
Issued in Open Access, under the terms of Commons attribution Non Commercial License
Doi.: 10.7818/MEDECOSandAEETCongress.2017
Editorial deadline: 20th December, 2016.
Publisher: Asociación Española de Ecología Terrestre (AEET)
C/ Tulipán s/n, 28399, Móstoles, Madrid, Spain
Layout: Imprenta Sand, S.L. Seville. Spain.
Symposium 09
XIV MEDECOS & XIII AEET meeting. Human driven scenarios for evolutionary and ecological changes
Seville, Spain, 31st January - 4th February 2017
Sp.09: Local adaptation in the Mediterranean
Sp.09-1-Main talk
Why do we need to incorporate phenotypic variation into species distribution models?
Benito Garzon, M.1
(1) INRA, France
Correspondence e-mail: [marta.benito@gmail.com]
Here I will focus on trees and explore i) how considering phenotypic variation can change species distributions ii) the difficulties
arising for accounting for phenotypic variation due to the experimental designs and iii) how species and populations models based
on the phenotypic variation can be used in management decisions. Extensive experiments with translocations of several tree species
across Europe do exist and can be used to estimate the amount of the variation due to plasticity and to local adaptation with statistical
approaches that account for the origins of phenotypic variation. However, in some cases, these experiments do not cover the entire
range of the species because they have not been designed to understand the ecophysiological limits of the species. . Including
phenotypic variation into species distribution models gives a more realistic output than classical species distribution models and
allows us to visualize the phenotypic variation at species and population levels, a useful tool to design assisted migration programs for
the future.
Sp.09-2-Oral
Ecological mechanisms underlying hybridization and its constraint in genus Erysimum
Abdelaziz, M.1, Muñoz-Pajares, A.J.2, Perfectti, F.3, Gómez Reyes, J.M.4
(1) University of Granada. (2) CIBIO-InBIO, Universidade do Porto. (3) University of Granada. (4) CSIC
Correspondence e-mail: [mabdelazizm@ugr.es]
Hybrid zones have the potential to shed light on evolutionary processes driving local adaptation and speciation. Secondary contact
hybrid zones are particularly powerful natural systems where study the interaction between divergent genomes to understand the
mode and rate at which reproductive isolation accumulates during speciation. Using 10 microsatellites markers we studied five
populations from two Erysimum (Brassicaceae) species presenting a secondary contact zone in Sierra Nevada Mountains (SE Spain).
We have estimated the genetic differentiation, the genetic structure, and the amount of recent gene flow between populations. We
found a narrow unimodal hybrid zone where the hybrid genotypes were more frequent than the parental genotypes. Afterwards, we
carried out two experiments to explore the pre-zygotic and post-zygotic reproductive barriers underlying the origin and maintenance
of this narrow hybrid zone. Post-zygotic barriers were evaluated performing over 1000 hand crosses on 44 plants from both species
till the second generation. While to evaluate the pre-zygotic barriers, a plot of parental and hybrid flowering plants were presented to
the pollinators in the natural populations to evaluate their preferences. We did not identify any significant reproductive barrier at the
generation of F1 hybrids nor the pre-zygotic barriers. However, we identify a strong and significant postzygotic reproductive barrier in
the F2 hybrid production. Our results suggest that the pollinators are the main promoters of the hybrid zone, but a constant migration
of plants from the parental population would be necessary for the long-term maintenance of that narrow hybrid zone.
173
Symposium 09
XIV MEDECOS & XIII AEET meeting. Human driven scenarios for evolutionary and ecological changes
Seville, Spain, 31st January - 4th February 2017
Sp.09-3-Poster
Spatio-temporal flowering patterns in Mediterranean Poaceae. A community study in SW Spain
Cebrino Cruz, J.1, García-Castaño, J.L.2, Domínguez-Vilches, E.3, Galán, C.4
(1) University of Córdoba. (2) University of Seville. (3) University of Córdoba. (4) University of Córdoba
Correspondence e-mail: [jcebrinocruz@gmail.com]
Grasses are one of the largest plant families and they are worldwide distributed. Numerous studies have focused on the phenology
of different plants. However, lower attention has been given to grass species and to the effects of different land covers. The goals of
this work were (i) to study the grass community composition in different land covers; and (ii) to assess the importance of the species
and the land cover in the grass flowering phenology start and duration in two study years. Twenty-nine sampling points were randomly
selected in three different land covers. We found differences among plants covers in grass species composition, showing the scrub the
highest number of species. In addition, we detected differences on the flowering start date for some species –i.e. Hyparrhenia hirta
(L.) Stapf was the earliest species whereas Melica magnolii Gren. & Godr. was the latest one-. Moreover, we detected differences for
the land cover type and the study year. Species in the scrub flowered the latest whereas they flowered the earliest in the pasture in
2014 and in the riverbank in 2015. In general, the flowering start occurred later in 2014 than in 2015. In relation to the phenological
length, differences for some study species were detected, being the shortest season for Aegilops triuncialis L. and the longest one for
Hyparrhenia hirta. This study provides information for species and, especially, a spatially dependent answer, which might be important
in the scenario of the predicted climate change.
Sp.09-4-Oral
Clinal variation in phenological behavior, size at flowering and biomass allocation reveals adaptation to climatic divergence in
annual Thrincia hispida across its range
Pedro, M. de1, Mayol, M.2, González-Martínez, S.C.3, Regalado, I.4, Riba, M.5
(1) CREAF (Centre for Ecological Research and Forestry). (2) CREAF (Centre for Ecological Research and Forestry). (3) INRA. (4) CREAF (Centre for Ecological Research and
Forestry). (5) CREAF (Centre for Ecological Research and Forestry)
Correspondence e-mail: [manueldepedrorodriguez@gmail.com]
Understanding and predicting the consequences of global change on species persistence and distribution requires considering the
extent and potential for local adaptations. It is generally accepted that local adaptation in plants is a widespread phenomenon. However,
recent theoretical and empirical work suggests that adaptation could be constrained in short-lived plants during range expansions due
to demographic processes and phenotypic and genetic correlations among traits. Our aim is to produce new experimental evidence
of evolutionary mechanisms during plant range expansions. Here we present preliminary data on population differentiation in several
life history traits: time to germination and flowering, size at flowering and biomass allocation in 42 populations covering the whole
range of the short-lived colonizing plant Thrincia hispida. We found significant phenotypic variation among population in all measured
traits in a common garden experiment. The timing of germination and flowering followed environmental clines with temperature
and inter-annual variation of precipitation, indicating potential drivers of selection. Size at flowering was strongly correlated with
phenology. Compared with plants from northern populations, plants from southern populations germinated later and flowered earlier,
were smaller and allocated more biomass to reproduction. We conclude that southern populations have evolved to face short growing
periods by evolving traits favoring short life cycles. This study provides clear evidence for local adaptation in phenological traits in
response to environmental varying selection, and highlights the role of selection on phenological traits in explaining the variability in
other correlated traits (e.g. size) closely linked to fitness.
174
Symposium 09
XIV MEDECOS & XIII AEET meeting. Human driven scenarios for evolutionary and ecological changes
Seville, Spain, 31st January - 4th February 2017
Sp.09-5-Oral
Reproductive isolation of colour morphs in Lysimachia arvensis through a combination of incomplete pre- and postzygotic barriers
Jiménez-López, F.J.1, Pannell, J.R.2, Ortiz, P.L.3, Arista, M.4
(1) Universidad de Sevilla. (2) Université de Lausanne. (3) Universidad de Sevila. (4) Universidad de Sevilla
Correspondence e-mail: [fjimenez16@us.es]
The boundaries that define plant species have been much debated. Although absolute reproductive isolation is accepted as an
unequivocal sign of speciation, partial isolation between divergent populations can also lead to speciation. Evolution of reproductive
isolation depends on a considerable number of barriers. Tto understand the speciation process, it is necessary to identify all reproductive
barriers that limit hybridization. In this study, we estimated the strength of prezygotic and postzygotic barriers between the two flower
colour phenotypes, red and blue, of the herb Lysimachia arvensis. Our study points to a temporal separation between morphs at
two stages: the blue morph begins to flower 10-20 days earlier than the red morph; and blue flowers open about 1 hour earlier in
the morning and close about 1 hour later in the evening that red flowers. Pollen flow between morphs carried out by pollinators was
always less frequent than expected, based on morph frequencies. Pollen of each morph showed the same fertilization capacity on the
ovules of the other morph. F1 and F2 hybrids had lower seed production than parental plants. Our results indicate that none of these
barriers to hybridization are complete, but it seems that a sum of incomplete barriers may cause effective isolation of each morph,
reducing the chance of F1 hybridisation and contributing to the speciation process.
Sp.09-6-Poster
Adaptive divergence despite small population sizes and strong genetic drift in English yew (Taxus baccata L.)
Mayol, M.1, Riba, M.2, Grivet, D.3, Vendramin, G.G.4, Vincenot, L.5, González-Martínez, S.C.6
(1) Centre de Recerca Ecològica i Aplicacions Forestals (CREAF-UAB). (2) CREAF-UAB. (3) INIA-CIFOR. (4) National Research Council of Italy. (5) Université de Rouen.
(6) INRA
Correspondence e-mail: [maria.mayol@uab.es]
English yew (Taxus baccata L.) is a dioecious long-lived gymnosperm occurring in a great diversity of habitats in most of the European
continent. However, populations are often small and isolated, show irregular size/age structures and biased sex ratios, and neutral
genetic diversity is highly structured, so that the ability of this species to cope with local changing conditions in the face of impending
climate change may be severely reduced. Here we provide an overview of our research on adaptive patterns of variation in this conifer,
both at the phenotypic and molecular levels. Results from a clonal common garden experiment revealed the existence of two basic
adaptive phenological patterns according to the regional origin (“mild-type” and “continental-type”) associated to temperature and
precipitation clines. At the molecular level, new-generation outlier detection and environmental association analysis of 25,726 SNPs
(1,210 candidate genes) in 120 individuals distributed along an environmental gradient in Europe identified several outliers associated
with temperature and precipitation. In particular, 4 SNPs located on an early-responsive to dehydration stress protein (ERD4) were
identified as a target of selection by all the different approaches used. Our results suggest that, despite small population sizes and
strong genetic drift, divergent selection have contributed to population divergence in this conifer. However, we found that both current
and past climatic data could explain the environmental associations detected, so adaptive patterns might be likely reflecting processes
occurred in the distant past.
175
| 1,978 | 13,810 |
W3135425860.txt | life
Communication
Ferlins and TgDOC2 in Toxoplasma Microneme, Rhoptry and
Dense Granule Secretion
Daniel N. A. Tagoe 1,† , Allison A. Drozda 1,† , Julia A. Falco 2 , Tyler J. Bechtel 2,‡ , Eranthie Weerapana 2 and
Marc-Jan Gubbels 1, *
1
2
*
†
‡
Citation: Tagoe, D.N.A.; Drozda,
A.A.; Falco, J.A.; Bechtel, T.J.;
Weerapana, E.; Gubbels, M.-J. Ferlins
and TgDOC2 in Toxoplasma
Microneme, Rhoptry and Dense
Granule Secretion. Life 2021, 11, 217.
Department of Biology, Boston College, Chestnut Hill, MA 02467, USA; dnatagoe@gmail.com (D.N.A.T.);
drozda@bc.edu (A.A.D.)
Department of Chemistry, Boston College, Chestnut Hill, MA 02467, USA; falcoju@bc.edu (J.A.F.);
tylerbechtel@gmail.com (T.J.B.); eranthie@bc.edu (E.W.)
Correspondence: gubbelsj@bc.edu; Tel.: +1-617-552-8722
These authors contributed equally.
Present Address: Merck & Co., Inc., Cambridge, MA 02141, USA.
Abstract: The host cell invasion process of apicomplexan parasites like Toxoplasma gondii is facilitated
by sequential exocytosis of the microneme, rhoptry and dense granule organelles. Exocytosis is
facilitated by a double C2 domain (DOC2) protein family. This class of C2 domains is derived
from an ancestral calcium (Ca2+ ) binding archetype, although this feature is optional in extant
C2 domains. DOC2 domains provide combinatorial power to the C2 domain, which is further
enhanced in ferlins that harbor 5–7 C2 domains. Ca2+ conditionally engages the C2 domain with
lipids, membranes, and/or proteins to facilitating vesicular trafficking and membrane fusion. The
widely conserved T. gondii ferlins 1 (FER1) and 2 (FER2) are responsible for microneme and rhoptry
exocytosis, respectively, whereas an unconventional TgDOC2 is essential for microneme exocytosis.
The general role of ferlins in endolysosmal pathways is consistent with the repurposed apicomplexan
endosomal pathways in lineage specific secretory organelles. Ferlins can facilitate membrane fusion
without SNAREs, again pertinent to the Apicomplexa. How temporal raises in Ca2+ combined with
spatiotemporally available membrane lipids and post-translational modifications mesh to facilitate
sequential exocytosis events is discussed. In addition, new data on cross-talk between secretion
events together with the identification of a new microneme protein, MIC21, is presented.
https://doi.org/10.3390/life11030217
Keywords: Apicomplexa; Toxoplasma; microneme; rhoptry; dense granule; exocytosis; DOC2
Academic Editor: Stanislas Tomavo
Received: 14 February 2021
Accepted: 5 March 2021
1. Introduction
Published: 9 March 2021
The opportunistic apicomplexan parasite Toxoplasma gondii causes significant disease
in people with immature or compromised immune systems [1]. Central to the pathogenesis
of all apicomplexan parasites, including malaria causing Plasmodium falciparum, is the rapid
serial completion of lytic replication rounds causing tissue damage and inflammation.
Completion of the lytic cycle requires host cell invasion and egress, which are mediated by
sequential exocytosis of, respectively, the apically located micronemes and rhoptries and
the more scattered dense granules organelles [2,3] (Figure 1A,B).
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Life 2021, 11, 217. https://doi.org/10.3390/life11030217
https://www.mdpi.com/journal/life
Life 2021, 11, 217
2 of 22
Figure 1. Schematic representation of the function and timing of secretion events in the T. gondii lytic cycle. (A). Schematic
representation of T. gondii and its three secretory organelles, serving as legend for the other panels. (B). Schematic
representation of secretion events throughout the lytic cycle. (C). Schematic representation of the levels of secretion of
each organelle and the levels of intracellular calcium [Ca2+ ]c corresponding with the secretion events throughout the lytic
cycle. Timeline on the X-axis corresponds with the events in panel B. Note that the invasion process completes in less than
a minute and is shown in a stretched-out time window. The two spikes in [Ca2+ ]c at the time of egress correspond with
the release of intracellular stores followed by the influx of extracellular Ca2+ , while the [Ca2+ ]c oscillates during gliding in
concert with bursts of motility [4]. The activity of DOC2 and FER1 and FER2 is also indicated.
Microneme protein function ranges from pore-forming protein (PLP1) facilitating
egress, to a spectrum of adhesion molecules enabling the gliding motility and invasion
machinery that mature through the action of microneme secreted-serine proteases (subtilisins) [5,6]. Microneme secretion is dosed as long as parasites are between host cells,
a necessity to support sustained gliding motility, which also powers host cell invasion.
Upon contact with an appropriate host cell the parasite injects the contents of its rhoptry
organelles directly into the host cell cytosol. Rhoptry neck proteins (RONs) residing closest
to the site of ejection, are released first to establish the moving junction, which is critical to
efficient invasion. The rhoptry bulb proteins (ROPs) are released in the same secretion event
(Figure 1B,C), and subvert a variety of host cell pathways to secure parasite replication
and survival [7–9]. In addition, the rhoptries contain cholesterol and phosphatidylcholine
presumably required to establish the parasitophorous vacuole membrane [10,11]. Upon
abortion of an invasion event, rhoptries are still secreted as ‘evacuoles’ into noninvaded
host cells and enable manipulation of these host cells similar to invaded host cells [12,13].
Dense granule proteins (GRA) are lastly secreted after PVM formation into the PV lumen
from where they traffic to the PVM, intravacuolar network (IVN), host cell cytoplasm
and even to the nucleus [14]. Dense granules with non-overlapping contents have been
described, although it is not known whether these have different functions and/or different secretion kinetics [15]. GRA proteins have been implicated in several functions from
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interference with host cell signaling [16], transfer of small molecules between host cells
and PV [17], manipulation of host cell immune response [18] and even parasite egress [19].
C2 domains are composed of 80–160 amino-acids that fold into β-sheets with specific
binding characteristics that are found in a large and diverse set of eukaryotic proteins that
can be grouped into several families [20]. Most C2 domains have lipid binding characteristics that are family specific [21], but they can also interact with other proteins [22–24]. The
most diverse C2 domain protein family is derived from a protein kinase C (PKC) archetype
that is unique among C2 domains in that it can stabilize Ca2+ ions (up to 3 per C2 domain)
in a pocket composed of three loops emanating from the β-sheets. This family almost
exclusively functions in signal transduction, membrane trafficking and fusion through
conditionally engaging lipids and/or membranes upon changes in [Ca2+ ]c . However, not
all members have retained the Ca2+ -binding capacity and their membrane association
could be conditional upon the presence of lipids in certain membranes. In principle, these
C2 domain have two different lipid or membrane binding sites, which are not variably
conserved: one in the Ca2+ -binding loop region, and one in a lateral concavity in the β
sheets [20].
The versatility of the PKC-C2 domain family is further enhanced by domains of
tandemly positioned C2 domains creating a double C2 or DOC2 domain. This provides
more flexibility in the interaction repertoire by creating cooperatively functioning C2
domains. Importantly this effect can modulate the response to varying [Ca2+ ]c . DOC2
proteins control numerous Ca2+ -dependent exocytic processes, but have been studied in
most detail for the Ca2+ -dependent release of neurotransmitter from neurons [22,25–27]
(Figure 2). For Ca2+ mediated fusion of vesicles to the plasma membrane to occur in general,
a DOC2 Ca2+ -sensor with a transmembrane (TM) domain anchored in the secretory vesicle
is required, represented by the synaptotagmin (Syt) family [22,28–30]. These typically
respond to fast, high Ca2+ fluxes and are required for fast-synchronous neurotransmitter
release [23,31–33]. Soluble sensors like mammalian DOC2 proteins (Figure 2B: note that
their domain structure is very different from TgDOC2) are sensitive to lower [Ca2+ ]c
concentration and regulate constitutive and spontaneous asynchronous neurotransmitter
release [23,34–37]. Upon the correct Ca2+ concentration, DOC2 proteins act on congregating
and clamping on SNARE proteins residing in the secretory vesicle (v-SNARE) and target
membrane (t-SNARE) and result in fusion of the two membranes, thereby releasing the
vesicle contents.
The ferlin family comprises a distinct family of DOC2 proteins absent from neurons
that facilitate secretory processes in other cell types. Ferlins are defined by a C-terminal
TM domain and 5–7 C2 domains, typically organized in three DOC2 domains (Figure 2).
Vertebrate ferlins function in the secretory, endocytic and lysosomal pathways with structural and functional importance in health and disease [38]. Secretion of synaptic vesicles
from the inner ear cells upon mechanical stimulation by sound, only a single Ca2+ -sensor,
otoferlin, is required for exocytosis, at the site of secretion, thereby deviating significantly
from the standard model of Ca2+ -dependent exocytosis established in neurons [39–41].
Humans encode six ferlins that cluster closely together compared to the three different
apicomplexan ferlins (Figure 2C). This suggests that the apicomplexan diversity represents
unique clades that originated independently of the mammalian ferlins, which appear to
have radiated out of a single ancestral mammalian ferlin.
Here, we discuss the apicomplexan ferlins in light of newly emerging insights regarding the triggers and machinery facilitating microneme and rhoptry exocytosis. In addition.
T. gondii ferlin and TgDOC2 mutants were used to explore their respective contributions
to the various secretion events, which fortuitously led to the identification of a novel
microneme protein family, MIC21, conserved in the Coccidia sub-class of the Apicomplexa.
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Figure 2. Comparative topologies of Ca2+ -mediated exocytosis machinery in Toxoplasma and humans and ferlin phylogeny.
(A). Toxoplasma encodes four DOC2 domain proteins: TgDOC2 and three ferlin proteins. Ferlins are defined by 5 to 7 C2
domains. TgFER3 contains an N-terminal signal peptide (SP), which, in combination with the C-terminal TM domain,
could signal GPI-anchor addition at the C-terminus. Yellow shades in TgFER3 represent coiled-coil domains. Light blue C2
domains are degenerate (defined as having a p-value below the cut-off in PFam database searches); light blue shaded Ca2+
binding to C2 is based on modeling. The C2 domains labeled A–F by established conventions [28,30,42]. TM: C-terminal
transmembrane domain; FerI: conserved ferlin domain of unknown function. Modified from [43]. (B). The spectrum of
types of C2 domain containing proteins in humans with roles in Ca2+ -dependent secretion. Calcium sensors are present in
the Syt-x, DOC-x, and ferlin (otoferlin shown as representative) families, though not all family members are Ca2+ sensors.
MID = MUN Interaction Domain. MUN = Mammalian Uncoordinated domain. ZF = Zn Finger; PxxP = Pro-rich. (C).
Phylogenetic analysis of apicomplexan, chromerid and human ferlins. The following abbreviations are used: human
ferlins “L1-L5” FR1L1-5 (FR1L1 (dysferlin; O75923.1), FR1L2 (otoferlin; Q9HC10.3), FR1L3 (myoferlin; Q9NZM1.1), FR1L4
(A9Z1Z3.1), FR1L5 (A0AVI2.2), FR1L6 (Q2WGJ9.2)), Ot: green algae Ostreococcus tauri (Q01FJ7); Chromerids: “Vb” Vitrella
brassicaformis (VbFER1 (Vbre_12074 + Vbra_12075), VbFER2 (Vbra_9198)) and “Cv” Chromera velia (CvFER1 (Cvel_17519.2)
and CvFER2 (Cvel_9223)); Apicomplexa: “Tg” Toxoplasma gondii (TgFER1 (TGME49_309420), TgFER2 (TGME49_260470),
TgFER3 (TGME49_295472 + TGME49_295468)) “Nc”, Neospora caninum (NcFER1 (NCLIV_053770), NcFER2 (NCLIV_026570),
NcFER3 (NCLIV_002280)), “Em” Eimeria maxima (EmFER1 (EMWEY_00002120), EmFER2 (EMWEY_00009280), EmFER3
(EMWEY_00017650)), “Pf” Plasmodium falciparum (PfFER1 (PF3D7_0806300), PfFER2 (PF3D7_1455600)), “Pb” Plasmodium
berghei (PbFER1 (PBANKA_122440), PbFER2 (PBANKA_131930)), “Cp” Cryptosporidium parvum (CpFER1 (cgd8_2910),
CpFER2 (cgd2_2320)), “Gn” Gregarina niphandrodes (GnFER1 (GNI_063830), and GnFER2 (GNI_073830)). Alignment and
unrooted Jukes-Cantor phylogenetic tree were generated in Geneious (v.6.1.6) [44]) from a MUSCLE alignment using
neighbor-joining. Note that the FER1 and FER2 nodes for Tg and Nc are barely discernable at this scale. From [43].
2. Materials and Methods
2.1. Parasites and Host Cells
Transgenic derivatives of the RH strain were maintained in human foreskin fibroblasts
(HFF) or hTERT immortalized HFF cells as previously described [45]. The following
previously reported parasite lines were used: line ts-DOC2 is the recapitulated temperature
sensitive (ts) mutation [46] in a RH∆HX background with an addition of a C-terminal 5xTY
tag on DOC2 [47], parasite line DN-FER1 is conditional overexpression of an exogenous
FER1 gene controlled by the destabilization DD domain and the ligand Shield-1 with an
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additional Myc-tag [46], and in parasite line FER2-cKD the FER2 gene is placed under a
tetracycline regulatable promoter with an additional N-terminal Myc-tag [47].
2.2. Cloning
Plasmid pmic21-MIC21-YFP(MCS)/sagCAT was cloned by PCR amplification of genomic DNA containing the ORF and the 1400 bp promoter upstream of the newly annotated
start codon using primers #5085 gPro234380YFP-F GCCGGTGTTAGGAGATGACGAGACGTTTAAACCGCTGCCAGTGGATATATCTACGG and #5086 gPro234380YFP-R CTCCTCGCCCTTGCTCACCATCCTAGGAAGGTATTTCTTCAAAAGGTGTCAAGGG (restriction
sites underlined) which was cloned into PmeI/AvrII digested ptub-YFP-YFP(MCS)-30 dhfr/
sagCATsag [48] to replace the α-tubulin promoter and the first YFP ORF by Gibson assembly (NEB).
2.3. SILAC and ESA Collection
We adapted published methods to differentially label T. gondii cultures with isotopes [49]. Parasites grown at the permissive condition (35 ◦ C) were metabolically labeled
with heavy (C13, N15) Lys and Arg (L-Lysine-2-HCl (Thermo/Life Cat # 89987) and LArginine-HCl (Thermo/Life Cat # 88210;) for two lysis rounds using established T. gondii
SILAC protocols [49]. Medium was prepared in phenol free DMEM Media for SILAC
(Thermo/Life Cat # 89985). Specifically, parasites were grown in 5 mL medium (containing
1% FBS) in a T25 flask that was pre-equilibrated with isotope containing medium 24 hours
(hrs) prior to parasite inoculation. Following lysis 48 hrs later, parasites were passed in one
T25 flask under similar conditions. 5 mL freshly lysed parasites were passed into two T175
flasks per condition, pre-equilibrated for 24 hrs in isotope containing medium. Following
growth for 48 hrs at the restrictive condition (40 ◦ C, 1 µg/mL anhydrous tetracycline (ATc),
or 1 µM Shield-1) in isotope labeled medium, or 48 hrs at the permissive condition (35 ◦ C
or no ligands) in non-isotope labeled medium, monolayers were scraped with a rubber
policeman and parasites released by passage through a 21 gauge needle. Parasites were
collected by centrifugation at 1000× g for 20 min at RT, resuspended in 1 mL of serum free
SILAC medium containing 10 mM Hepes pH 7.3 and centrifuged again. Following one
additional wash step, parasites were resuspended to 1.2 × 108 parasites/mL in serum free
SILAC medium. 200 µL aliquots were placed in round bottom 96 well plates ((CELLTREAT
Scientific Products)). Secretion was triggered by 2% ethanol incubation at 37 ◦ C for 15 min
as described [50,51]. The plate was centrifuged at 1000× g at 4 ◦ C for 10 min to pellet cells
and 100 µL of the supernatant was transferred to an Eppendorf tube followed by addition
of 10 µL protease inhibitor (100× stock; Sigma # P88490). Parasites and supernatant remaining in the plate were resuspended and transferred into a separate set of tubes, centrifuged
at 1000× g at 4 ◦ C for 10 min, supernatant discarded and pellet resuspended in 180 µL PBS
before addition of 20 µL 10% SDS as control for parasite number. Protein concentrations
were determined using the BCA plate assay (Pierce #23227).
2.4. Silver Staining
Silver staining of SDS-PAGE gels was performed using a Silver Stain Kit (Pierce,
Thermo Scientific) following the manufacturer’s protocol. Briefly, 25 µg each of Heavy
Labelled (HL) and Light Labeled (LL) ESA proteins were run on 8–12% SDS-PAGE MOPS
gel. The gel was washed twice 5 min in water and fixed in 30% ethanol/10% acetic acid for
2 × 15 min. The gel was washed twice in 10% ethanol for 10 min followed by 2 washes for
1 min in water. The gel was sensitized for 1 min in Sensitizer working solution followed by
2 × 1 min washes with water. Proteins were stained for 30 min in Stain working solution
followed by 2 × 20 sec washes with water. Gels were developed in Developer Working
Solution for 3 min and the reaction stopped with 5% acetic acid for 10 min.
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2.5. LC-MS/MS Analysis
LC–MS/MS analysis was performed on an LTQ-Orbitrap XL mass spectrometer
(Thermo Fisher) coupled to an EASY-nLC 1000 nanoLC (Thermo Fisher). Samples were
pressure loaded onto a 250 µm fused silica desalting column packed with 4 cm of Aqua
C18 reverse-phase resin (Phenomenex). The peptides were then pushed onto a column
(100 µm fused silica with a 5 µm tip, packed with 10 cm C18) and eluted with a gradient
of 0–55% Buffer B in Buffer A (Buffer A: 95% water, 5% acetonitrile, 0.1% formic acid;
Buffer B: 20% water, 80% acetonitrile, 0.1% formic acid). The flow rate through the column
was set to 400 nl/min and the spray voltage was set to 3.5 kV. One full MS1 scan (FTMS;
400–1800 MW) was followed by seven data dependent scans (ITMS) of the nth most intense
ions with dynamic exclusion enabled.
The generated tandem MS data were searched using the SEQUEST algorithm [52]
using a concatenated target/decoy variant of the T. gondii GT1 ToxoDB-V28 database
combined with a target/decoy non-redundant variant of the human UniProt database. Data
sets were searched independently with the following parameter files; for the light search, all
amino acids were left at default masses; for the heavy search, static modifications on lysine
(+6.02013) and arginine (+10.00826) were specified. A static modification of +57.02146 on
cysteine was specified in all searches to account for iodoacetamide alkylation. SEQUEST
output files were filtered using DTASelect 2.0 [53]. Reported peptides were required to be
unique to the assigned protein (cannot be attributed to both human and T. gondii derived
protein), with a minimum of two unique peptides per protein. Discriminant analyses were
performed using the CIMAGE quantification package as previously described [54]. The
H/L ratios generated for unique peptides were grouped by protein with the median H/L
ratio chosen as the representative ratio for that protein. Human proteins were removed
from the dataset. Ratios from each sample were normalized to the median ratio of T. gondii
derived proteins within that sample in order to correct for variations in isotope labeling.
2.6. Fluorescence Microscopy
RH∆HXGPRT parasites were co-transfected with 15 µg each of plasmid pmic21MIC21-YFP(MCS)/sagCAT and PG53 encoding MIC8-mCherry ([55] a kind gift from
Markus Meissner) or pMIC2-mCherry-Myc (Luc in plasmid pMIC2-Luc-Myc, kindly provided by David Sibley [56], was replaced with mCherry). 18 hrs post transfection, parasites
were imaged with a Zeiss Axiovert microscope and a ORCA-FLASH 4.0 camera was used
in capturing images using 100x objective with oil and 0.55 Numerical Aperture (NA).
2.7. Western Blots
SDS-PAGE (8–12%, MOPS) separated ESA samples and corresponding total parasite
lysates were blotted onto PVDF membrane and probed with mouse α-MIC2 (1:8000) [57],
mouse α-TY (MAb BB2; 1:1000; a kind gift from Sebastian Lourido), mouse α-tubulin
(12G10) (1:2000; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), rabbit
α-GRA7 (1:2000; a gift from John Boothroyd) [58]. Procedures and signal intensity quantification as previously described [43,47]. A two-tailed, two sample equal variance Student’s
t-test was performed on protein levels normalized to α-tubulin.
2.8. MIC21 Phylogeny
The reannotated TGGT1_234380 with signal peptide was used for a BLASTP search
of select representative Apicomplexa against EuPathDB [59]. Manually curated orthoand para-logs were aligned using ClustalO 3.0 (CLC Main Genomic Workbench 20, Qiagen). The alignment was used to generate an unrooted phylogenetic tree using the
Neighbor joining Algorithm, Jukes-Cantor protein distance and 100x bootstrap analysis
(CLC Main Genomic Workbench 20, Qiagen). The following sequences were included in
the analysis: MIC21: TGGT1_234380, HHA_234380, NCLIV_049520, CSUI_003785; MIC21L1: TGGT1_222188, HHA_222188, NCLIV_005580, SN3_01200570, CSUI_000781 (2a),
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CSUI_009221 (2b); MIC21-L2: EBH_0032420, ETH_00028875, EAH_00066010, LOC34621018;
MIC21-L3: ETH_00004565, LOC34624209.
3. Results
3.1. Apicomplexan Protein Trafficking to the Secretory Organelles
The secretory pathway that subsequently passes through the endoplasmic reticulum
(ER) and Golgi apparatus mediates protein trafficking to the three secretory organelles.
Targeting proteins to the dense granules does not require any additional signals and
comprises the default pathway secretory organelle. Specifics on the protein trafficking
pathways to the micronemes [60] and rhoptries [61] and their biogenesis were recently
reviewed. Although some rhoptry specific targeting signals have been identified [62,63],
specific sorting signals for microneme proteins have remained elusive. Following the
Golgi compartment, microneme and rhoptry proteins traffic through an endosome like
compartment (ELC) (reviewed in [64,65]), where they undergo maturation by proteolytic
removal of a pro-peptide [66]. Specific Rab GTPases have been associated with the first
leg of the trafficking through the endosomal pathway, but are absent from the mature
organelles or their sites of secretion [67]. Peculiarly, a subset of microneme proteins
(MIC3, MIC5, MIC8 and MIC11) traffic, in Rab5A/C-dependent fashion while the others
are Rab5A/C-independent [67]. Sustained microneme secretion in gliding extracellular
parasites is balanced with active endocytosis [68]. During cell division, micronemes of the
mother are re-directed into the newly forming daughters, which hints at a mechanism that
supports trafficking of micronemes in more than one direction [69].
3.2. Organization of Organelle Secretion: Small Molecules and Machinery
Microneme release is turned off during intracellular replication and only occurs during egress, gliding and host cell invasion (Figure 1C). Pharmacological studies allowed for
dissection of the controls of microneme exocytosis and established a role for three signaling
molecules: cytoplasmic Ca2+ , phosphatidic acid (PA) and cGMP [70]. The levels of cytoplasmic calcium ([Ca2+ ]c ) correlate with the level of microneme secretion [71] (Figure 1C), and
the micronemes are considered a bona fide Ca2+ -dependent secretory organelle [72]. Ca2+
mediators are involved at several levels. Firstly, the exocytosis event has been associated
with Centrin2 [73], which has four EF-hand domains putatively binding Ca2+ , and two
double C2 domain (DOC2) proteins, TgDOC2 [46] and FER1 [47]. An indirect role is also
found in the requirement of a Ca2+ -dependent protein kinase, CDPK1, for microneme
exocytosis, but the key phosphorylation events are still unknown [74,75]. A parallel set of
controls is mediated by phosphatidic acid (PA) produced upon activation of egress which
is deposited in the plasma membrane. PA is recognized by an essential acylated pleckstrin
homology (PH) domain-containing protein (APH) on the surface of the micronemes [76].
Micronemes are exocytosed at the very apical tip of the parasite, where the rhoptries are
discharged as well.
The triggers for rhoptry exocytosis are not well understood besides that this requires
microneme secretion first, and that it only occurs upon contact with a host cell. The
connection to the micronemes resides in critical roles for microneme proteins MIC8 [55],
AMA1 [77] and CLAMP [78], but the detailed mechanisms have not been resolved. In
addition, rhoptry secretion is mediated by PA and phosphatidylinositol 4,5 bisphosphate
(PIP2) [79], which accumulate at the plasma membrane following induction of the microneme secretion [77]. C2 and PH domains in Rhoptry Associated Surface Proteins
(RASPs) engage with PA and PIP2, bringing the rhoptry in close contact with the plasma
membrane and/or apical vesicle residing at the very apical between the rhoptry and the
plasma membrane. [70]. In addition, a machinery shared with the release of trichocysts by
ciliates comprises several Nd proteins, names for their ‘not discharged’ genetic phenotype
in the ciliate Paramecium [80]. The Nd proteins are critical for the apical rosette, a structure
in the plasma membrane through which the rhoptries and trichocysts are secreted. The
apical vesicle is positioned on the cytoplasmic side of the rosette opposed to the rhoptry tip.
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It is of note that microneme secretion is not mediated by the rosette as they are positioned at
the plasma membrane next to the apical vesicle and, moreover, microneme secretion is also
not affected by disruption of the rosette structure in TgNdP1 and TgNdP2 mutants [80].
Some Nd proteins reside in the cytoplasm and others at the apical vesicle, but the Nd
proteins are either directly critical for the rosette structure, or for the exocytosis event [80].
Furthermore, the Nd proteins interact with another DOC2 domain protein, FER2, which
is essential for rhoptry secretion [43]. Two models have been proposed for the role of
Nd proteins in controlling the fusion step: 1. the correct assembly of Nd6, Nd9, NdP1
and NdP2 as a complex together with a GTPase (TGME49_277840) is mediated by the
Nd6 guanine exchange factor (GEF) domain and Ca2+ -dependent action of FER2, or; 2.
cytosolic Nd9 and/or NdP1 interact transiently with the apical vesicle associated Nd6
to trigger the assembly of the rosette [80]. Although no pharmacological compounds are
known to trigger rhoptry secretion, an inhibitor, 4-bromophenacyl bromide (4-BPB), is
available [81]. This compound typically inhibits A2 phospholipases, but its target in T.
gondii is unknown [81]. Overall, the structural architecture of the site of rhoptry secretion
comprises a critical role for several C2 domain proteins engaging with various lipids, while
providing a putative role for Ca2+ through FER2 [79].
Dense granule secretion is the least understood. Not even the site of secretion has
been conclusively established. Although there is evidence that the dense granules are
secreted somewhere at the apical end of the parasite [82,83], their secretion has also been
inferred from a specialized invagination at the posterior end of the parasite where the basal
complex resides [84]. However, an alternative interpretation is that dense granule proteins
are secreted at an apical site from the parasite and subsequently transition to the posterior
end [85]. In either scenario dense granule proteins facilitate the assembly of multilamellar
vesicles in the vacuole at the posterior end of the parasite, which subsequently transition
into tubulated structures making up an intravacuolar tubulovesicular network [84]. Since
dense granules with different contents are present, it is also possible these might have
distinct secretion sites and/or kinetics.
3.3. Ferlins in Mammals
The mammalian ferlins come in two basic flavors differentiated by their sub-cellular
localization at either the plasma membrane or on intracellular compartments. Localization
relates to their function in either late endosomal transit or trans-Golgi recycling [86]. Dysferlin and otoferlin have been the most studied. Dysferlin is critical for muscle development
and functions in vesicular trafficking and lysosome exocytosis during muscle plasma membrane damage repair [38,87]. Dysferlin does not localize to lysosomes in intact myotubes,
but dysferlin-containing vesicles fuse with lysosomes upon sarcolemma damage, possibly
in response to a raise in [Ca2+ ]c [88]. This two-step process might mimic the role of the apical vesicle wedged between the rhoptry tip and the plasma membrane, which also required
a two-step fusion event [80]. A further parallel with the Apicomplexa is the indication
that rhoptry targeting tyrosine motifs are shared with mammalian lysosomal trafficking
signals [63,89]. This suggests a kinship at several levels between mammalian lysosomes
and rhoptries, with a possibly comparable role for dysferlin and FER2, respectively.
Otoferlin functions as both a scaffolding protein in the secretory pathway as well as
in the actual membrane fusion during exocytosis [39,41,90,91]. Otoferlin is expressed in
many tissues but in Cochlear Hair Cells (CHCs) in the inner ear auditory systems where is
localizes to synaptic vesicles and the plasma membrane and is essential in neurotransmitter
release upon mechanical triggers: a variety of mutations across otoferlin lead to hearing
loss [90,92]. Under low [Ca2+ ]c interactions among C2C, C2D, C2E, and C2F is promoted,
whereas high [Ca2+ ]c leads to interaction with phospholipids, the prelude to membrane
fusion [90].
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3.4. DOC2 Protein Repertoire in T. gondii
The Apicomplexa encode two widely conserved ferlin proteins, FER1 and FER2 ([43]
and one quite unconventional alveolate-wide conserved DOC2 protein without a TM
domain [46] (Figure 2A,C). Some parasites, including T. gondii, encode a degenerate third
ferlin, TgFER3 [43]. We were able to knock out TgFER3 without any effect on the lytic cycle
and concluded that it is not involved in organelle secretion, at least not in the tachyzoite
stage (Tagoe and Gubbels, unpublished). TgFER1 and TgFER2 mediate exocytosis of the
micronemes and rhoptries, respectively [43,47]. Besides T. gondii, ferlins have only been
studied in one other apicomplexan parasite, Plasmodium berghei. The ferlin-like protein,
PbFLP, the TgFER1 ortholog, is essential for male gametocyte egress, and its knock out
phenotype was consistent with a role in exocytosis [93].
The conserved DOC2 protein without TM domain, TgDOC2, is unconventional in
its relatively long length without other recognizable domains (Figure 2). TgDOC2 is
essential for microneme protein secretion, a function that is conserved in Plasmodium
falciparum [46]. PfDOC2 has been shown to bind Ca2+ and is present in the membrane
fraction of replicating parasites [94]. In conclusion, all conserved DOC2 domain proteins
function in the exocytosis of the specialized apicomplexan secretory organelles defining
the obligate intracellular lifestyle of these parasites. Comparing the function and behavior
of mammalian ferlins to the T. gondii repertoire points at otoferlin as relevant to understand
FER1 and dysferlin as a role model for FER2.
Regarding the function of TgDOC2, comparison with the mammalian repertoire points
to the DOC2 family: cytoplasmic Ca2+ sensors like mammalian DOC2 proteins (Figure 2B:
note that their domain structure is very different from TgDOC2) which are sensitive to
lower [Ca2+ ]c concentration and regulate constitutive and spontaneous, asynchronous
neurotransmitter release [34–37]. They act in concert with a membrane bound, TM domain
containing Ca2+ sensor like the synaptotagmins or otoferlin that typically respond to
fast, high Ca2+ fluxes required for fast-synchronous neurotransmitter release [23,31–33].
Tentatively, this dual player DOC2 system seems to fit the micronemes with their dynamic
secretory nature (Figure 1C), whereas as single DOC2 protein seems sufficient for the single
burst in rhoptry release. However, based on the unconventional structure of TgDOC2 we
cannot exclude other potential roles fulfilled by different mammalian multiple C2 domain
proteins such as MUNC13 or RIMs outlined in Figure 2B. Munc13 (mammalian ortholog
of C. elegans uncoordinated protein) stabilizes the SNARE and Ca2+ -sensor complex and
is absolutely essential for any type of Ca2+ -mediated exocytosis [95,96]. MUNC13 is not
a Ca2+ sensor but a CAPS (Calcium-dependent Activator Protein for Secretion), which
prime vesicles for Ca2+ -triggered exocytosis. MUNC13s harbor 2 or 3 C2 domains and
a diacylglycerol-binding C1 domain [97] (Figure 2B). The MUN domain stabilizes the
SNARE complex [97,98]. However, the T. gondii genome does not encode a protein with
a domain reminiscent of a MUN domain, which indicates a mechanism distinct from the
well-studied systems. RIMs are involved in vesicle priming, docking and organization at
the site of secretion. RIM stands for Rab3 Interacting Molecules. They harbor multiple C2
domains (Figure 2B) and bind multiple presynaptic proteins, e.g., SNAREs, Rab3, Munc13’s,
mediated by the C2A, C2B and Pro-rich domains [99]. RIMs receive the secretory vesicle
from the Rab-mediated trafficking machinery and relay it to the fusion proteins. Knock-out
of RIMs reduces neurotransmitter release to 60% [100]. The relevance to T. gondii goes
off the rails by the absence of Rabs and SNAREs at the sites of microneme and rhoptry
secretion [67]. However, the unconventional nature might bypass the need for these
players, as ferlins equally have the capacity to function without. Moreover, otoferlin is even
considered to be the one and only Ca2+ -sensor for secretory vesicle exocytosis in CHCs,
since stimulus–secretion coupling is eliminated in otoferlin knock-out mice [39–41].
In conclusion, the role of apicomplexan ferlins in microneme and rhoptry exocytosis
is consistent with mammalian ferlins acting on the endosomal pathway, wherein the micronemes and rhoptries reside. However, given the specialized nature of the apicomplexan
secretory organelles combined with the ancient divergence between FER1, FER2 and the
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mammalian ferlin families (Figure 2C), this comparison is only at a high level. The ferlins
act in the context of lineage specific proteins like TgDOC2 [46] and APH [101] for the
micronemes, and RASP [79] and Nd [80] proteins in rhoptry exocytosis.
3.5. FER1 and FER2 in Microneme and Rhoptry Fusion with the Plasma Membrane
The fine details on how FER1, FER2 and TgDOC2 orchestrate the exocytosis of micronemes and rhoptries is associated with several questions. In particular, T. gondii Rabs are
absent from the microneme and rhoptry secretion site in the apical conoid [67]. Although
Rab11A has been shown to be critical in constitutive secretion of dense granules, this is not
a triggered event [102]. In addition, no Toxoplasma’s SNARE proteins have been detected at
the site of microneme and rhoptry secretion site in the very apical plasma membrane [103].
The most forward localizing SNARE in the secretory pathway identified is a syntaxin,
TgStx12, which is critical in trafficking of proteins from ELC to the micronemes and rhoptries, but not for their secretion. Thus, this is different from neurotransmitter release where
SNAREs play a key role in the membrane fusion event facilitating exocytosis.
In general, the absolute requirement for SNAREs in exocytosis, certainly regarding
ferlin-mediated exocytosis, has come under debate. It has been postulated that fast, Ca2+ dependent exocytosis is inconsistent with the role of SNAREs [104] and some exocytosis
in absence of SNAREs is possible [21,105]. Furthermore, some neurotransmitter is still
released when all relevant SNAREs are depleted [105], and alternative models for SNARE
independent neurotransmitter release have been postulated [104]. Most germane to the
situation in parasites is the absence of SNAREs from the site of otoferlin-mediated neurotransmitter release in the auditory hair cells [106]. Furthermore, at the sites of membrane
lesions in cultured myotubes SNAP23, syntaxin4 and VAMP4 do not accumulate, but
myoferlin does and is required for membrane repair [107,108]. Thus, these ferlins are
essential for specialized membrane fusion events, but are not dependent on SNARE proteins. This leaves the door open for SNARE-independent fusion, or alternatively, an as yet
uncharacterized class of SNAREs [90,106].
Regarding the modular nature of the DOC2 domains in ferlins, the multiple C2
domains have been proposed to be able to support membrane binding integrating the Ca2+ sensing and membrane fusion events [31,42]. Indeed, pairs of C2 domain can interact cooperatively to take on a closed or open conformation [109] or coordinate a driver-facilitator
relationship [110,111]. In this manner C2-C2 interactions facilitate protein scaffolding,
determine Ca2+ sensitivity and direct lipid binding affinity. In this division of labor, a single
C2 domain may bind a recruited protein, while others within the ferlin are responsible for
binding Ca2+ or lipid. In this way all domains of the protein coordinate to allow fusion of
membranes [112,113]. The combination of several C2 domains in a single protein allows
for dynamic conformational changes that reveal or hide different C2 domains and thus
change its function and as such ferlins might facilitate Toxoplasma exocytosis in absence of
SNAREs. Our working model is therefore that TgDOC2 and FER1 and FER2 can fulfill the
missing function of SNAREs and Rabs at the plasma membrane.
3.6. Ferlins in Organellar Trafficking
FER1 does not only function in microneme exocytosis but also in microneme organelle
trafficking [47]. Two different phenotypes in different mutants were observed: conditional overexpression of dominant negative FER1 allele without its TM domain results
in a reversible, retrograde microneme transport away from the apical cortex to a pile up
of micronemes in an apical cytoplasmic location, whereas conditional overexpression of
the full-length FER1 resulted in an anterograde transport of the micronemes into the very
apical tip of the parasite and even premature exocytosis [47]. The retrograde transport was
interpreted as being germane to the process of microneme recycling from the mother into
the emerging daughters during the second half of cell division [69]. The actin-dependent
trafficking also provides a tentative connection between FER1 and a myosin. The anterograde trafficking supports a role in replenishment of secretory vesicles at the site of
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secretion. It is assumed that micronemes travel along the sub-pellicular microtubules as
they follow this organization pattern in their cortical localization pattern [67]. However,
there is no direct experimental data supporting this assessment. The microtubule based
motor, dynein TgDLC8a, has been associated with replenishment, but since it is only observed in the very apical tip, is unlikely to transport the organelles along the sub-pellicular
microtubules, but more likely through the conoid [73]. Either way, dual roles in membrane
fusion and vesicle trafficking are also seen in the vertebrate ferlins [86], such as otoferlin in replenishment of synaptic vesicles [40,41] through myosin VI [114,115]. The role
of dysferlin in vesicular trafficking in quite diverse [38], but is also dependent on actin
myosin for plasma membrane repair [116]. Overall, these observations and similarities
with vertebrate ferlins indicate that the role of FER1 changes under different conditions
organellar trafficking during intracellular replication, and microneme exocytosis upon
activation of egress, during gliding and host cell invasion. Currently, we have no indication
that FER2 has such a dual role. The physical feature of the rhoptries are in alignment with
this model: they are constitutively docked at the apical end and do not traffic [80] whereas
their relatively large size would also not be very amendable to an organelle recycling
pathway [61].
3.7. T. gondii DOC2 Proteins Act Differentially on the Various Secretory Organelles
We performed knock-out or dominant negative allele studies on FER1 [47], FER2 [6],
and TgDOC2 [46] to reveal the organelles these factors are acting on. However, to obtain
more subtle, quantitative data on the exact nature of secretion events and to refine the
mechanistic models we performed Stable Isotope Labeling with Amino acids in Cell
culture (SILAC) proteomics on the proteins released in our set of secretion mutants. We
induced all conditional mutant phenotypes overnight followed by mechanical release of
the parasites from their host cells and then stimulated exocytosis with 2% ethanol for
15 min. We collected the excreted and secreted antigens (ESA) under both induced and
non-induced control conditions, which were differentially labelled with light- and heavyisotope containing amino acids, respectively, before mixing in equal ratios and analysis by
mass spectrometry (Figure S1). Changes in the heavy:light ratio reveal which proteins are
differentially secreted in each mutant.
For the temperature sensitive (ts)-TgDOC2 lines, the results confirmed the dominant
role of TgDOC2 on micronemes (Figure 3A). We also confirmed that the ts allele results in
protein instability and loss of TgDOC2 from the parasites at restrictive, high temperature
(Figure S2). However, we also identified significantly less secretion of both the dense granules, and, to a lesser but still significant extent, the rhoptries. Induction of DN-FER1, which
redirects the vast majority of micronemes away from the cortex thereby inhibiting their secretion, results in a significant reduction in secretion of many microneme proteins, whereas
the ones below the significance cut-off trend strongly to reduced secretion (Figure 3B).
There is no effect on the rhoptries. However, we observed a secretion trend for the dense
granules, mimicking the micronemes, albeit largely below the statical relevance limit.
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Figure 3. SILAC data for T. gondii DOC2 and Ferlin mutants. (A). ts-DOC2 is a recreated temperature sensitive allele of
DOC2 conditionally treated at 35 ◦ C and 40 ◦ C [43,47]; (B). DN-FER1 is a DD-Myc conditional overexpression allele induced
with 1 µM Shield-1 [47]; (C). cKD-FER2 is tetracycline regulated promoter replacement induced with 1 µM ATc [59]; (D). the
wild type control consisted of RH∆Ku80 parasites treated at 35 ◦ C and 40 ◦ C. All inductions were performed for 48 hrs on
intracellularly replicating parasites. For ESA collection, parasites were mechanically lysed from the host cell, and stimulated
with 2% ethanol for 15 min. Parasites grown under permissive conditions were grown in light-isotope (L) labeled amino
acids; parasites at restrictive conditions in heavy-isotope (H) labeled amino acids. Equal amounts of ESA collected under
permissive and restrictive conditions were mixed and subjected to mass spectrometry. Averages of two biological repeats
are shown, except for cKD-FER2 which was a single experiment. Marked in pink, the cKD-FER2 experiment is the only
one in which some ER proteins (ER-2 pool defined in [117]) were detected (TGGT1_229480: putative calcium binding
protein precursor; TGGT1_221210: cyclophilin; TGGT1_211680; protein disulfide isomerase). Dotted lines mark the −0.5
and +0.5 Log2 arbitrary cut-offs for decreased or increased significant changes, respectively, in excretion/secretion above
background.
3.8. Do DOC2 Proteins Act on Multiple Secretory Organelles?
The suggestive role of TgDOC2 in microneme and rhoptry discharge and that of FER1
in dense granule exocytosis can be interpreted in two different ways: 1. a direct role for
TgDOC2 in rhoptry and dense granule exocytosis and for FER1 in dense granule exocytosis;
2. induced dense granule and rhoptry release requires secretion of the micronemes first.
Discharge of rhoptries indeed requires microneme secretion first [54], which would explain
the strong effect seen in the ts-DOC2 line. However, the dependence of dense granules
on either TgDOC2 or FER1 is less clear. In fact, an opposite correlation between induced
microneme and dense granule exocytosis has been reported [118]. However, dense granule
exocytosis was not completely inhibited, and the differences in protein secretion we detect
are of course relative to the non-induced condition. The secretion drop we see therefore
drops even below the reported reduced level [118].
To validate the reduction in dense granule exocytosis we performed Western blots
and quantified the differential secretion of the microneme and dense granules protein
reporters MIC2 and GRA7, respectively (Figure 4). This revealed that like MIC2, GRA7
secretion is completely inhibited. The other notable new insight is that the expression
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levels of both MIC2 and GRA7 are trending upward upon TgDOC2 depletion. Although
this upregulation does not reach a statistically significant difference, it trends contrary
to the complete disruption of secretion, thereby indicating the potential presence of a
feedback loop to increase protein expression as a compensatory mechanism for the block
in exocytosis.
Figure 4. Dense granule secretion is TgDOC2 dependent. (A). Representative Western blots of ts-DOC2 secretion assays as
performed for the ESA-SILAC experiment. “total” represents the pellet of the ESA assay. Antiserum against α-tubulin was
used as loading control. Relative to the first lane for each condition, only half the protein amount was loaded in the second
lane. (B). Quantification of secreted and total MIC2 normalized against α-tubulin shows that MIC2 expression is 3-fold
upregulated in ts-DOC2 at the restrictive condition (40 ◦ C), yet none is secreted. (C). Quantification of secreted and total
GRA7 normalized against α-tubulin shows that GRA7 is not upregulated, but its stimulated secretion is dependent on DOC2.
All blots used for quantification are provided in Supplementary Figure S2 and the quantification data in Supplementary
Table S2. Statistics was performed with a two-tailed, two sample equal variance Student’s t-test; n.s.: non significant.
3.9. Identification of a Novel Microneme Protein: MIC21
Besides the annotated microneme, rhoptry and dense granule proteins, we also identified differentially secreted proteins outside these categories. These were mostly typical
contaminants such as SAG surface antigen, ribosomal proteins, as well as mitochondrial or
cytoplasmic proteins (although we cannot exclude the possibility that their secretion is truly
dependent on the DOC2 domain proteins; Figure 5 and Table S1). Either way, one protein
in the ts-DOC2 dataset stood out for its extremely strong differential secretion putting it
right between the other microneme proteins: TGGT1_234380 (named MIC21; Figure 3A).
This protein lacked a signal peptide prediction in ToxoDB [119], but was identified in
the microneme fraction of the recent subcellular compartment proteome hyperLOPIT
study of T. gondii [117]. Furthermore, we detected orthologs and a paralog across the
Coccidia, which in contrast to TGGT1_234380 all contained a signal peptide. We therefore
scrutinized the TGGT1_234380 annotation and noted a continuous open reading frame
omitting the single intron that results in inclusion of a signal peptide comparable to the
para- and orthologs (Figure 5A). We further identified two conserved domains (CD1 and
CD2), without significant homology to known domains, which therefore does not direct
further functional roles.
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Figure 5. Characterization of putative microneme protein TGGT1_234380. TGGT1_234380 was the top hit in the ts-DOC2
SILAC experiment and annotated as a hypothetical protein on ToxoDB. Recently, the protein was detected in the microneme
fraction by HyperLOPIT [117]. Querying select, representative apicomplexan and chromerid annotated genomes in
EuPathDB identified only orthologs in the Coccidia. All except the T. gondii annotation of the predicted gene contained an
N-terminal signal peptide (SP), which upon closer examination, was also present in the T. gondii gene if the gene was not
spliced. This re-annotated version of the protein, which we named microneme protein 21 as this is the next available number
in the microneme protein family (MIC21), was used to assemble a protein alignment that is the basis of data presented in
panels A-C. (A). Schematic representation of MIC21. Two highly conserved domains (CD1 and CD2) were identified whose
conservation across orthologs and paralogs is shown in the magnified panel as % conservation and amino acid logo plots. A
PFam search identified domain defined in the peroxisomal acyl-CoA oxidase-II domains 3 and 4 (SCOP domain d1is2a3)
with a non-significant e-value of 1, and furthermore this domain is not conserved across ortho- and para-logs. (B). Unrooted
(neighbor-joining, Jukes-Cantor, CLC Workbench) phylogenetic tree of representative MIC21 related sequences identified
in the Coccidia resolves into 4 branches of ortho- and paralogs (MIC21 cluster next to MIC21-like proteins MIC21-L1,
MIC21-L2, and MIC21-L3 clusters). Bootstrap analysis (100x) supported all branches with over 70%, except for the two
branch points as indicated. Hh: Hammondia hammondi; Nc: Neospora caninum; Cs: Cystoisospora suis; Sn: Sarcocystis neurona;
Ea, Eb, Et: Eimeria acervulina, brunetti and tenella, respectively; Cc: Cyclospora cayetanensis. (C). Heatmap representing the
percentage of sequence similarity across the DSP1 protein family. Percentage similarity based on ClustalOmega alignment.
(D). RNAseq data available on ToxoDB reveals MIC21-L1 is not expressed in tachyzoite but only in the enteric stages. MIC21
is robustly expressed tachyzoites, bradyzoites and the enteric stages [120]. Neither MIC21 nor MIC21-L1 is expressed in
sporulating oocysts [121]. Error bars represent standard error [120]. (E,F). Exogenous, transient expression of genomic DNA
spanning the TgMIC21 promoter (1.4 kb) and coding sequence N-terminally fused to the YFP reporter co-transfected with a
MIC8-mCherry (E) or MIC2-mCherry (F) reporter indicates MIC21 resided in the micronemes though does not perfectly
co-localize with either MIC8 or MIC2.
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Phylogenic analysis revealed that the ortho- and paralogs formed four subgroups,
two of which span the cyst-forming coccidia (Hammondia hammondi, Neospora caninum,
Sarcocystis neurona, Cystoisospora suis) whereas the other two are present in the non-cyst
forming coccidia (Eimeria spp. and Cyclospora cayetanensis) (Figure 5B,C). Analysis of T.
gondii stage specific gene expression data revealed that TGGT1_234380 is expressed in tachyzoites, bradyzoites and merozoites but not sporozoites, whereas the paralog TGGT1_222188
(MIC21-L1) is restricted to the merozoites (Figure 5D). Finally, we experimentally validated
localization of MIC21 to the micronemes by exogenous expression of a C-terminal YFP
fusion construct under control of the endogenous MIC21 promoter. MIC21 localized to the
apical region reminiscent of the micronemes. Co-transfections with either MIC2-mCherry
or MIC8-mCherry reporters showed partial co-localization, whereas MIC21 always was
present in larger apical puncta (Figure 5E,F). These puncta likely reflect some mis-trafficking
induced by the fusion protein, which fits with the inability to obtain parasites stably expressing the MIC21-YFP protein. Altogether, we conclude that TGGT1_234380 is a bona fide
microneme protein and we named it MIC21; the as yet unvalidated paralogs were named
MIC21-like proteins MIC21-L1, MIC21-L2, and MIC21-L3.
4. Discussion
Pharmacological triggering of microneme release facilitated studies on the proteins
discharged by the parasite from this organelle [50,51]. Our quantitative proteomics using
the DOC2 and ferlin secretion mutants confirmed the previously assigned roles for TgDOC2
and FER1 in microneme exocytosis, but in addition revealed cross-organelle impacts on the
discharge dynamics of the various organelles. Notably, TgDOC2 depletion also resulted
in decreased rhoptry and dense granule protein levels in the ESA (Figures 3A and 4).
The former might not be surprising given that microneme exocytosis is a requirement
for rhoptry expulsion, but the connection to reduced dense granules release was not
immediately obvious. This hints at a potential role for TgDOC2 as a general factor in
secretory organelle fusion with the plasma membrane. We envision a dual function for
TgDOC2, with a Ca2+ -dependent role on microneme membrane fusion with the plasma
membrane, whereas it might be a general factor, less dependent on Ca2+ , in the membrane
fusion event of rhoptries and dense granules, e.g., as a MUNC13-like clamp to stabilize the
complex of molecules required for membrane fusion.
The dynamic of the overexpression of a dominant negative FER1 allele indicated
this was acting equally on micronemes and dense granules, but not on the rhoptries
at all. The microneme phenotype in the DN-FER1 line originates in mistrafficking of
micronemes, while the presence of a wild type FER1 allele still facilitates a low level of
microneme protein secretion [47]. We did not assess dense granule trafficking in the DNFER1 line and therefore we can currently not differentiate a role for FER1 in dense granule
trafficking effect from a function in actual dense granule membrane fusion with the plasma
membrane. A putative model should consider the two modes of dense granule exocytosis:
a steady, constitutive level of dense granule release, and a specifically regulated burst upon
completion of host cell invasion (Figure 1). It is possible that FER1 plays a differential role
in these different events.
TgDOC2 and the ferlins are derived from the ancestral PKC family of C2 domains
that binds Ca2+ . However, not all extant C2 domains retained the Ca2+ -binding capacity,
and this is an unsettled question for the apicomplexan family members. We analyzed
the canonical C2 domains and determined that at least one C2 domain has a minimal set
of negatively charged aspartic acid or glutamic acid residues in the key position in the
C2 domain loops (FER1-C2D, FER2-C2F and TgDOC2-C2B: Supplementary Figure S3).
Overexpression of FER1 allele with a C2D domain wherein the Ca2+ -binding residues were
mutated resulted in distinct microneme trafficking phenotype, suggesting physiological
relevance of this in sillico analysis [47]. However, these assignments need experimental
validation. It is of note that dysferlin and otoferlin each only have three predicted Ca2+ binding sites, but experimentally, all seven dysferlin C2 domains and five of the six otoferlin
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C2 domains were able to bind Ca2+ , albeit, to variable degrees [122,123]. Hence, the
presented prediction could underestimate the Ca2+ -biding capacity in the T. gondii proteins.
Details on lipid and membrane binding capacities are another open question key to
understand the role of the ferlins and TgDOC2 in membrane fusion. In particular the possibility to ‘sculpt’ the membrane structure of phosphatidylserine membranes as a prelude
to membrane fusion [124] is of interest for FER1 since this is a membrane lipid preferentially flipped by the guanyl-cyclase-flippase required for microneme discharge [125].
Furthermore, PIP2 and PIP4 are common lipids bound by ferlins [123,126], which again
are lipids with critical roles in apicomplexan exocytosis [127]. Furthermore, (conditional)
lipid binding of the key RASP and Nd proteins associated with rhoptry release suggest that
lipid binding by FER2 is quite likely an essential event in this process [79,80]. Furthermore,
rhoptry secretion requires recruitment of all Nd proteins to the apical vesicle or rosette,
suggestion protein-protein interactions are another critical aspect wherein FER2 could
participate. Thus, the spatiotemporal conditions that could change ferlin conformation
range from the local availability of lipids in the plasma membrane (e.g., phosphatidylserine [125] and phosphatidic acid [101] for the micronemes) to the increase in [Ca2+ ]c as
well the potential contribution of Ca2+ -dependent phosphorylation by, e.g., CDPK1 [74],
PKA [128] or PKG [129,130]. For example, phosphorylation of otoferlin regulates the
Ca2+ -sensitivity of C2C and C2F domains [131]. Currently there is no data supporting
phosphorylation of FER1 or FER2, but three phosphorylated residues of TgDOC2 are
contained in the tachyzoite phosphoproteome [132]. Furthermore, a vacuolar H+ -ATPase
(VP1) in a compartment at the very apical tip of the parasite has been related to a critical
role in microneme secretion [133,134]. The identity of this compartment is not known, but
it is unlikely the apical vesicle that is not essential for microneme secretion.
In summary, the DOC2 proteins of T. gondii are woven deeply throughout the fabric of
the exocytic organelles and events. The endosomal ancestry of these organelles fits well
with endolysosomal context wherein the vertebrate ferlins facilitate vesicular trafficking
and Ca2+ -dependent exocytosis. Open questions regarding the mechanism of how the
DOC2 proteins exert their function pertain to their Ca2+ , lipid and protein binding capacities
together with conditional conformational changes that might integrate environmental
conditions and post-translational modifications.
Supplementary Materials: The following are available online at https://www.mdpi.com/2075-1
729/11/3/217/s1, Figure S1: Silver stained SDS-PAGE gels of ESAs, Figure S2: Western blots of
ESAs, Figure S3: In sillico analysis of Ca2+ -binding potential of C2 domains, Table S1: ESA mass
spectrometry SILAC data, Table S2: Western blot quantification data.
Author Contributions: Conceptualization, M.-J.G. and E.W.; methodology, D.N.A.T., A.A.D., T.J.B.,
J.A.F., M.-J.G. and E.W.; validation, D.N.A.T. and M.-J.G.; data curation, T.J.B., J.A.F. and. E.W.;
writing, A.A.D. and M.-J.G.; visualization, D.N.A.T., A.A.D. and M.-J.G.; supervision, E.W. and
M.-J.G.; project administration, M.-J.G.; funding acquisition, M.-J.G. All authors have read and agreed
to the published version of the manuscript.
Funding: This study was supported by National Institutes of Health grants AI122042, AI099658, and
AI122923, and American Cancer Society grant RSG-12-175-01-MPC.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The mass spectrometry proteomics data have been deposited to the
ProteomeXchange Consortium via the PRIDE [135] partner repository with the dataset identifier
PXD024210.
Acknowledgments: We thank John Boothroyd, Sebastian Lourido, Markus Meissner, and David
Sibley for sharing reagents and Gubbels lab members for discussing this project.
Conflicts of Interest: The authors declare no conflict of interest.
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W3121785009.txt | Article citation info:
45
Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D. Analysis of the neutral grounding modes influence on the reliability
characteristics of local systems with renewable energy sources. Diagnostyka 2021; 22(1): 45-56. https://doi.org/10.29354/diag/132834
DIAGNOSTYKA, 2021, Vol. 22, No. 1
ISSN 1641-6414
e-ISSN 2449-5220
DOI: 10.29354/diag/132834
ANALYSIS OF THE NEUTRAL GROUNDING MODES INFLUENCE ON THE
RELIABILITY CHARACTERISTICS OF LOCAL SYSTEMS WITH RENEWABLE
ENERGY SOURCES
Oleksandr OSTAPCHUK 1, Valeriy KUZNETSOV 2, Włodzimierz KRUCZEK 3,
Vitaliy KUZNETSOV 4, Dmytro TSYPLENKOV 5
1
Department of Renewable Energy, Igor Sikorsky Kyiv Polytechnic Institute, Prosp. Peremohy, 37, Kyiv,
Ukraine, 03056, e-mail: O.Ostapchuk@kpi.ua
2
Electric Power Department, Railway Research Institute, Chlopickiego str., 50, Warsaw, Poland,
e-mail: vkuznetsov@ikolej.pl
3
Electric Power Department, Railway Research Institute, Chlopickiego str., 50, Warsaw, Poland,
e-mail: wkruczek@ikolej.pl
4
Department of the Electrical Engineering and Electromechanic, National Metallurgical Academy of Ukraine,
Gagarina ave., Dnipro, Ukraine, 49600, e-mail: wit1975@i.ua
5
Department of Electrical Engineering, Dnipro University of Technology, av. Dmytra Yavornytskoho, 19,
Dnipro, Ukraine, 49005, e-mail: tsyplenkov.dv@nmu.one
Abstract
When comparing the performance indicators of electrical networks with different types of neutral
grounding, along with the fulfilment of the requirement to ensure reliability of power supply to consumers,
serious attention is drawn to the main network parameters influencing the performance of power supply
systems. Analysis of research and its results, reported above, on the influence of the neutral ground of power
networks on the reliability and electrical safety conditions of the power supply systems as a whole, on the
damage of distribution networks and electrical equipment as well as on the working capacity of relay
protection devices, provides an opportunity to estimate each specific operation mode of the neutral and to
develop recommendations aimed at strengthening the positive indicators of the corresponding modes. Other
things being equal, reliability of power supply to electrical receivers or reliability of distribution networks is
mainly determined by the damage to network elements and the performance of relay protection devices. The
degree of influence of these factors on the reliability of distribution networks depends on the neutral mode,
which in turn determines the level of internal overvoltages and the nature of transient processes at earth fault.
Keywords: electric networks, protection, reliability, ground failure current, electrical safety, system with isolated neutral,
system with resonant grounding, system with low-impedance neutral grounding.
1. RELEVANCE
The rapid development of renewable energy
systems in Europe and the world creates new
directions for their application. One of these areas
in recent years has been the use of a distributed
generation system to provide power to non-traction
consumers of railway transport [1-4]. Such systems
are called local systems and in their specificity of
work, structure and execution: they significantly
differ from general-purpose systems [5-9]. A
feature of the work of these systems is the provision
of power to consumers of the first and second
categories, namely: circuits of automatic and semiautomatic blocking, signalling, communications,
outdoor track lighting, automatic heating control
systems for buildings etc. [10-12]. Accordingly, for
such networks it is necessary to determine the
indicators on which the reliability of providing
electric energy depends [13-15]. The reliability and
safety of maintenance of these systems is largely
determined by the neutral grounding method, which
is established according to the instructions of
normative and technical documents and by
technical and economic comparison according to
established criteria. In many countries of the world,
these conditions have developed historically, under
the influence of various factors that changed during
the development of networks [16-19]. Today,
prerequisites have arisen to analyse these factors of
influence on the reliability, efficiency and safety of
servicing local networks.
In accordance with [16] medium voltage (MV)
networks can have system with isolated neutral or
system with resonant grounding or system with
low-Impedance neutral grounding (resistance) [20].
When comparing the performance indicators of
electrical networks with different types of neutral
46
DIAGNOSTYKA, Vol. 22, No. 1 (2021)
Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ...
grounding, along with the fulfilment of the
requirement to ensure reliability of power supply to
consumers, serious attention is drawn to the main
network parameters influencing the performance of
power supply systems, these include:
- insulation levels and overvoltage resistibility
(overvoltage protection) [21, 22];
- provision of single-phase-to-ground failure
protection and its performance;
- selectivity of protection actions against ground
failures;
- safety protection from the effect of touch and step
voltages at earth failure.
Other things being equal, reliability of power
supply to electrical receivers or reliability of
distribution networks is mainly determined by the
damage to network elements and the performance
of relay protection devices.
At the same time, the main indicators of failure
will be following:
- the probability of failure Q (t);
- MTTF T 0 ;
- the average recovery time T R.
The degree of influence of these factors on the
reliability of distribution networks depends on the
neutral mode, which in turn determines the level of
internal overvoltages and the nature of transient
processes at earth failure. The level of overvoltage
has a decisive influence on the first and second
indicators, and the nature of transient processes - on
the third [23].
Distribution networks MV with system with
isolated neutral are most common, but such
operation mode of the network neutral is not always
optimal in terms of the specified earlier criteria.
Power supply systems with a fully insulated neutral,
as compared to networks with other neutral modes,
do not require additional capital expenditures.
However, the operational costs of such networks
due to their greater damage rate as well as due to
the power outages, are significantly greater than
those in the networks operating with other neutral
modes [20, 24].
The efficiency of the capacitive current
compensation and the performance system with
resonant grounding largely depends on the tuning
mode of the compensating device. In this context,
most researchers give preference to the resonant
inductance tuning (it is extremely difficult to
provide it in dynamic networks) of the
compensating device with a network capacity
relative to the earth. From an economic point of
view, distribution networks with the compensation
of the capacitive ground failure current require
additional capital expenditures on the arc
suppression reactors and devices for their
connection. With regard to operating costs, they are
significantly less than those in the networks with an
isolated neutral due to a lower level of damage to
the elements of the system. With resonance tuning
of the compensating device and minor disorders of
compensation in electric networks, the value of
dielectric strength in relation to the operating
overvoltages increases up to 30% [25, 26].
Electrical networks with a neutral resistor have,
in comparison with system isolated neutral and
resonant grounding, higher reliability because of
the
suppression
of
transient
processes
accompanying single-phase ground failures, the
reduction of damage to the elements of the power
supply system (the latter is caused by a significant
decrease of internal overvoltages) and the
elimination
of
ferro-resonance
processes.
Considerable additional capital costs for the
implementation of networks with a neutral resistor,
as compared to networks with a fully isolated
neutral, at the earth failure currents will be greater
than 5 ... 10 A. In this case, it is required to include
a high-voltage resistor and devices for its
connection into the neutral network, which in
addition to switching devices may also include
special transformers needed for the connection of
resistors. In networks with ground-failure currents
up to 5 A, additional capital costs decrease
practically to zero, since in this case it is possible to
manage available in the network measuring
transformers and low-voltage resistors [21].
2. EFFECTS OF THE NEUTRAL MODE ON
THE PHASE CHARACTERISTICS OF
EMERGENCY CURRENTS
In local systems, the earth failures are the most
common damages (up to 70-85% of the total
number of accidents) [27, 23]. In such networks,
possible overvoltages can reach 3.95 the phase
voltage value. Hence greater attention and higher
requirements to the operational performance of
protection devices, otherwise the unreasonable
interruptions in power supply to responsible
consumers (under the mistaken a protective actions)
or further development of the failure is probable (in
case of protection failure, transition of a singlephase earth failure into a double one is possible).
To study phase characteristics of voltage and
zero sequence currents, we use the equivalent
circuit (Fig. 1), which is presented in the form of
two attachments connected to one power
transformer [28].
Given that, we believe that conductance of
appropriate phase-to-ground insulation of the
controlled connection ( Y A1 = Y B1 = Y C1 = Y 1 ) and
the
rest
of
the
distribution
network
( Y 'A = Y 'B = Y C' = Y '
formula Y 1' + Y '
)
are
related
by
= Y ...
1
1
Then Y = + jC; Y 1 = + jC1;
R1
R
1
1
1
1
YK =
−j
; Y = 1 ; y = ; yi = .
RK
L N RN
ri
r
the
DIAGNOSTYKA, Vol. 22, No. 1 (2021)
47
Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ...
The Figure 1 shows the zero sequence current
filter TA installed on the damaged connection and
intended to isolate the zero sequence current I 0 in
the monitored line; currents flowing through the
conductance of phase-to-ground insulation of the
monitored line ( I A1 , I B1 , I C1 ), and currents flowing
through the appropriate phase conductance of the
external network ( I 'A , I 'B , I C' ) ; I g 1 , I g
- single-
phase ground failure currents in the monitored line
and in the external network, respectively; I N current flowing through the conductance of the
network neutral point with respect to ground. We
proceed from the fact that a single-phase earth
failure can occur in the supervised line ( y1 0 ) or
in the external network ( y 0 ).
L1 L2 L3
I0
Q
ТA
IN
Q1
I A1
I B1
I C1
YH
I A I B IC Q2
I g1
I g
y1
YA1
YB1
YC1
y
Y A
Y B
YC
Fig. 1. The equivalent circuit diagram of the
distribution network for study of phase
characteristics of voltage and zero-sequence
currents
2.1 System with isolated neutral ( YN = 0 )
By using the basic laws of electrical engineering
for the presented equivalent circuit diagram, we
have done necessary transformations and obtained a
mathematical equation for the zero-sequence
current in the case of an error in the monitored line:
I 0C = −3U 0 Y ' = −3U 0 (Y − Y 1 )
or
( R − R)+ j(C −C1 ) R R1
(1)
I 0 = −3U 0 1
,
R R1
Where R and C are, respectively, the insulation
resistance and the capacity of the whole electrically
connected network with respect to ground; R1 and
C1 are, respectively, the active insulation resistance
and the capacity of the supervised connection with
respect to ground.
Equations for the intrinsic current of the
monitored line (zero-sequence current in the
monitored line with an external earth failure) are as
follows:
I 0C = 3 U 0 Y 1
or
1 + j C1 R1
R1
The analysis of the equations (1) and (2) shows
that in the network with the fully isolated neutral,
the zero-sequence current in the failure line is
determined by the zero-sequence voltage and
insulation parameters of the external network with
respect to ground, that is, by the isolation
parameters of the entire network with respect to
ground, excluding insulation of the faulted
connection, whereas the intrinsic current of the
monitored connection (zero-sequence current in the
supervised line with external faults) is determined
by the zero-sequence voltage and insulation
parameters with respect to ground, only in relation
to the supervised connection. From the obtained
expressions, it can be concluded that on the basis of
the appearance of voltage and current of the zero
sequence it is possible to establish the fact of
asymmetric insulation damage, and by their
magnitude to assess the degree of this failure. The
presence of these indicators can be used to build
diagnostic models.
Using these equations, with account of their
imaginary and real parts, we obtain formulas that
determine the phase dependence of the zerosequence current vector:
I 0C = 3 U 0
01 = 1800 + arctg
(C −C1 ) R1 R
R1 − R
(3)
,
and the phase dependence of the intrinsic current
vector of the monitored line (the current of the zero
sequence in the monitored line with the external
single-phase-to-ground fault)
02 = arctg ( C1 R1 ),
(4)
in relation to the zero sequence voltage vector on
the insulation parameters with respect to ground of
the whole network and of the controlled connection.
The analysis of the obtained equations (3) and
(4) shows that taking into account the real values of
the insulation parameters, relative to the earth, of
the whole network and of a separate connection,
and also taking into account the actual interrelation
of the capacitive and active insulation resistance,
the following conclusions can be drawn:
- the angle between the zero-sequence current
vector and the zero-sequence voltage vector does
not depend on the completeness of the closure (the
transient resistance at the closing point) and it is
almost 270 el. degrees, or minus 90 el. degrees;
- the angle between the intrinsic vector of the
monitored line (zero-sequence current in the
monitored line with the external single-phase-toground fault) and the zero-sequence voltage vector
is determined by the isolation parameters relative to
the ground only of the controlled connection and it
is almost 90 el. degrees.
48
DIAGNOSTYKA, Vol. 22, No. 1 (2021)
Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ...
2.2. Compensated neutral network upon
1
1
condition ( Y N = Y K =
)
−j
RK
L
For this network, we similarly obtain the
equations [6, 18] for the zero-sequence current in
case of damage in the controlled line:
L (3R1 RK − 3R RK + R R1 )
−
I 0 = −U 0 [
R R1 RK L
−j
R R1 RK ( − 3 2 C1L)
]
R R1 RK L
,
(5)
for the intrinsic current of the monitored line (zero
sequence current in the monitored line with the
external earth fault):
1 + j C1 R1
I 0C = −3 U 0
,
(6)
R1
and also the formulas determining the phase
dependence of the zero sequence current vector:
01=180 −
− arctg
(
R Rk R1 − 3 2 C1 L
)
,
(7)
L ( 3Rk R1 − 3Rk R + R R1 )
and the phase of the intrinsic current vector of the
monitored line with respect to the zero-sequence
voltage vector on the insulation parameters
02 = arctg ( C1 R1 )
(8)
with respect to the zero sequence voltage vector
from the insulation parameters relative to the earth
of the whole network, the controlled connection and
the degree of the compensating device
detuning = 1 − 3 2 C L from the resonance
mode with the network capacity.
The analysis of the equations (5) - (8) shows
that at the single-phase earth fault in the network
with a compensated neutral, taking into account the
actual values of insulation parameters relative to the
ground of the whole network and of the separate
connection as well as taking into account the actual
values of the arc suppression reactor parameters, we
can state:
- the zero sequence current in the faulty line is
determined by the zero sequence voltage and the
insulation parameters of the external network
relative to the ground, i.e. the insulation
parameters of the entire network relative to the
ground (including the compensating device
parameters), minus the insulation parameters of
the damaged connection;
- the angle between the zero-sequence current
vector and the zero-sequence voltage vector
does not depend on the completeness of the
closure (transient resistance at a closing point)
and is determined to a large extent by the value
of detuning of the compensating device from the
resonant mode and for resonance tuning it is
approximately 180 el. degrees; when the
compensating device is detuned from the
resonance mode both in the direction of
overcompensation and under compensation, the
current vector deviates by an angle of
respectively up to plus 90 el. degrees and minus
90 el. degrees, so the range of angle variation
between the zero-sequence current vector and
the zero-sequence voltage vector is theoretically
180 el. degrees;
- the intrinsic current of the controlled connection is
determined by the zero-sequence voltage and
the insulation
parameters with respect to ground
(5)
only of the controlled connection;
- the angle between the intrinsic current vector of
the monitored line and the zero-sequence
voltage vector is determined by the insulation
parameters with respect to ground only of the
controlled connection and it is almost 90 el.
degrees.
2.3 Network with a neutral resistor ( Y N = Y R = 1
/ R N ).
For such a network, in accordance with the
equivalent circuit diagram (Fig. 1), the formulas [3,
4, 7] for the studied currents and their phase
characteristics are as follows:
I 0 = −U 0
[
L (3RN R1 − 3R RN + R R1 )
R R1 RN
R R1 RN (C − C1 )
];
R R1 RN
1 + j C1 R1
I 0C = −3 U 0
;
R1
−
(9)
−j
01=180 + arctg
(10)
R RN R1 ( C − C1 )
; (11)
3RN R1 − 3R RN + R R1
02 = arctg ( C1 R1 ),
(12)
and their analysis shows that in case of a singlephase earth fault in the network with a neutral
resistor:
− the zero-sequence current in the faulty line is
determined by the zero sequence voltage and the
insulation parameters of the external network
with respect to ground as well as by the
resistance value of the neutral resistor, and the
phase of this current in relation to the zero
sequence voltage vector does not depend on the
completeness of the closure and in contrast to
the networks with a fully insulated neutral it is
not fixed and depends on the resistance value of
the resistor, besides it can theoretically take
values in the range from 180 to 270 el. degrees
in relation to the zero-sequence voltage vector;
for the real network insulation parameters with
respect to ground and the recommended
resistance value of the resistor, this angle is
approximately 225…240 el. degrees, i.e. in
contrast to the network with a fully isolated
neutral, a shift of the emergency current vector
is possible by an angle from 30 to 45 el.
degrees;
DIAGNOSTYKA, Vol. 22, No. 1 (2021)
49
Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ...
− the intrinsic current of the controlled connection
is determined by the zero-sequence voltage and
insulation parameters with respect to ground
only of the controlled connection, and the angle
between the vector of this current and the zerosequence voltage vector is determined in the
same way as in the networks with a fully
isolated and compensated neutral − by the
insulation parameters relative to the ground only
of the controlled connection and it is almost 90
el. degrees.
When constructing diagnostic models, it’s must
be taken into account the fact that the position of
the zero-sequence current vector with respect to the
zero-sequence voltage vector is generally
determined by the nature and mode of the network
neutral ground, and the phase of the intrinsic
current of the controlled connection (zero-sequence
current in the monitored line with the external
single phase-to-ground fault does not depend on the
neutral operation mode − it is determined only by
the parameters of the directly controlled connection
and practically it is rigidly linked to the zerosequence voltage.
3. EFFECTS OF THE NEUTRAL MODE
ON THE PERFORMANCE OF GROUNDFAILURE PROTECTION
At present, in the distribution networks, the
ground failure protection devices, responsive to the
steady-state parameters of the emergency mode,
which can conditionally be divided into three types:
current (maximum) protection - devices that react
to the zero sequence current; voltage protection devices that respond to the zero sequence voltage;
directional protection - devices that respond to the
zero sequence power [17, 24, 29].
Studies have shown that the use of simple
current protection in networks with a fully isolated
neutral is justified by the sensitivity conditions
under the capacitance of the protected line 5…7.5
times less than the capacity of the entire electrically
connected network. If the specified condition is not
observed, selectivity of its operation is disrupted.
The necessity of fulfilling this condition
significantly limits the application area of current
protection devices, particularly if to take into
consideration the fact that, while in operation, the
capacity of network, as well as of individual lines,
varies greatly. The ratio of the zero sequence
currents in case of external earth fault and in the
protection zone, as well as the conditions ensuring
selectivity performance of the current protection
devices, make it difficult to use them in networks
with a compensated neutral. In networks with the
capacitance current compensation in the steadystate ground-fault mode, when adjusting the
compensating device in resonance with the network
capacity relative to the earth, as well as with
insignificant disorders from the resonance mode,
the zero sequence current of the protected
connection (intrinsic current) is nearly always
greater than the closing current and the zero
sequence current in the case of an error in the
protected connection. This circumstance practically
excludes the possibility of applying current
protection devices in networks with a compensated
neutral. The most favourable conditions for the
application of simple residual-current protective
devices are created in networks with a neutral
resistor due to the suppression of transient
processes, however it is difficult to achieve the
operational performance of current protection
devices in case of faults through the transition
resistances.
Protective devices responding to the zerosequence voltage are characterized by the simplicity
of their circuits and elements; in local networks
they are mainly used either in the form of backup
units for networks with high security requirements
The principal drawback of such protective devices
is an inability to ensure the performance selectivity.
In system with resonant grounding, application of
such protectors is also practically impossible
because of the high value of the neutral bias voltage
at resonance (or close to resonance) tuning of the
arc suppression reactor.
Devices of directional protection against singlephase ground failure, responding to the parameters
of the steady-state closing mode, ensure the
operation selectivity due to the phase comparison of
the zero sequence current and voltage. Such devices
are largely used only for networks with a fully
isolated neutral, since in such networks the angles
between zero sequence currents and voltages
practically do not depend on the parameters of the
network insulation and the transition resistance at
the fault point in case of one- phase ground failure
and they are practically fixed.
The
presence
of
transient
processes
accompanying the occurrence of a phase-to-ground
failure and primarily the shutdown of the damaged
connection should be considered as the determining
reasons for the unsatisfactory operation of the
directional protection devices in networks with a
fully isolated neutral. The reason for this lies in the
transient process which accompanies the process of
recovery of phase-to-ground voltage and which is
oscillating, damping and rather lengthy, with a
frequency close to the industrial one.
The reasons, causing false operation of existing
directional protection devices, should also include
the imperfection of circuitries. From this group of
reasons special attention should go to the following
ones [30, 31]:
- insufficient detuning of devices through the zero
sequence current and voltage channels from
higher harmonic components, the level of which
can be significantly high, especially at closures
through the alternating arc;
− a wide angular operating area that is
approximately 180−210°, which leads to a time
coincidence of the compared signals due to their
50
DIAGNOSTYKA, Vol. 22, No. 1 (2021)
Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ...
phase distortions caused by angular errors of
current and voltage transformers as well as
phase shifts of signals directly in the device
circuit, etc.
In networks with a compensated neutral, the
specified principle of protection implementation is
impossible, as in these networks the angles between
zero sequence currents and voltages are determined
mainly by the tuning mode of the compensating
device and the value of the transition resistance at a
point of damage and it may vary within 180 ° (from
minus 90° to plus 90°) which practically excludes
the selectivity principle of the protection operation
[29].
In networks with a neutral resistor, perspectives
of using the directional protection devices from
single-phase earth faults, operating on the basis of
phase comparison of the zero sequence current and
voltage can be considered good for the following
reasons:
- in such networks, the false operation of devices
from transient processes is practically excluded,
because the presence of a neutral resistor greatly
suppresses the duration and amplitude of the
transient processes;
- the angular error for the values, recommended for
quarry
networks
and
introduced
by
superimposing an active component of the earth
fault current on the network, is in the range of
30 ° ... 40 ° and it refers exclusively to the zero
sequence current in the faulty line.
The current ground-failure protection devices,
which received their application in networks with a
compensated neutral, are devices responding to the
higher harmonic components of the zero sequence
current.
Reliable functioning of the devices under
consideration is possible only at a sufficiently high
level and stability of the natural harmonics used for
the protection operation. The absence of a
compensating device (network with a fully
insulated neutral) and the inclusion of the resistor in
the neutral network contribute to a decrease in the
level and spectrum of natural harmonics.
A general lack of the devices responsive to
higher harmonics in the zero sequence current is a
complex dependence of the structure and level of
natural harmonics on: the number of lines, the
tuning mode of the compensating reactor, the
resistance at the point of fault, as well as the
presence of interferences.
Investigations have shown that for the singlephase ground failure through the transition
resistance of several tens of ohms, the level of the
higher harmonics components sharply decreases.
So, with a change in the coefficient of the closure
completeness from 1 to 0.4, the zero sequence
current for the 5th harmonic decreases 11.5 times,
for the 7th harmonic − 16 times, for the 11th
harmonic − 25 times [27,32].
Protection devices responding to the transient
process parameters are used in system with
resonant grounding, since for the protection
implementation of the ground fault capacitive
current compensation doesn't allow, as a rule, the
usage of simpler and more reliable principles based
on the parameter checkout of the zero sequence
currents and voltages.
To the shortcomings of the earth-fault
protection devices, responding to the amplitude and
wave characteristics of the transient process, which
largely limit their spread in the quarry distribution
networks, it is necessary to include the following
ones:
- the inrushes of the initial capacitive current as
well as the currents and voltages of waves of the
transient discharge process are determined, to a
significant extent, by the values of voltage and
its phase at the moment the earth fault occurs.
At the same time, the aerial quarry electrical
networks are characterized by a high probability
of a mechanical insulation damage, which can
occur at the moments when the voltage of the
damaged phase hasn't reached its highest value
yet and when the transient process hasn't
actually arisen;
- the presence of a transition resistance at the point
of the phase-to-ground fault leads to a decrease
in the amplitude and time characteristics of the
transient process. Operational experience with
distribution networks shows that when the value
of the transient resistance is of the order of
hundreds of ohms, the transient process
practically does not arise;
- the absence of repeated actions of the protective
devices when the signal is acknowledged in the
conditions of a steady earth fault.
The ground-failure protection devices that react
to the network-imposed currents of non-industrial
frequency can be used in networks with any neutral
mode. The disadvantage of such devices is the
absence of operation selectivity (the transformer is
usually switched off).
For the devices reacting to the superimposed
alternating current of non-industrial frequency, the
value of the superimposed current is composed of
the components determined, in addition to the
voltage level of the source Us, by the value of the
transition resistance r at the fault location and the
sum of the phase network capacities with respect to
ground:
I v.s = I v.s + I v.s =
U v .s
U v.s
+
,
z + r z + 3 xc
(13)
where z - is the value of the total resistance of
transformer windings, line resistance and ground
resistance for the current of the superimposed
frequency; xc - is the capacitive resistance of one
phase of the entire network with respect to ground
(for the superimposed frequency).
The second component of the superimposed
current is apart from the transition resistance value
and rises with an increase of the network capacity
and of the control voltage frequency. This
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Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ...
component is distributed across all transmission
lines of the network in proportion to their
capacities.
A general lack of the protection reacting to the
superimposed current of both low and high
frequency is the impossibility of creating highly
sensitive devices, since it is necessary to avoid the
leakage of operating current through the
capacitance of the protected line.
In addition, one must note the possibility of
false operation of protection devices during the
transient processes, since the spectrum of currents
in the transient process can contain components of
operating frequency [33].
The principle of superimposing the operational
voltage of non-industrial frequency on the protected
network is most preferable for system with resonant
grounding both by the simplicity implementation of
the protection device itself and operational source.
4. INFLUENCE OF THE NETWORK
NEUTRAL MODE ON ELECTRICAL
SAFETY CONDITIONS.
In accordance with the electrical safety
conditions, when a person directly touches the
current-carrying parts of the distribution networks,
none of the proposed neutral operation modes can
be considered as an advantageous one. Regardless
of the neutral mode with account of the real
insulation parameters of the distribution networks,
as well as the action time of protection devices and
used switching equipment, the current value
passing through the human body will significantly
exceed the safe levels. None of the protective
devices can guarantee safe outcomes when a person
touches current-carrying parts in networks with any
neutral mode at a voltage of 6 kV and higher. Even
in the networks with the compensation of the
capacitive fault current, the presence of a transient
process arising at the moment of touching one of
the phases, the benefits of such networks, in respect
to electrical safety, are practically reduced to zero.
At the moment of touching, the so-called free
current of the compensating device will pass
through the human body, the frequency of which is
determined by the physical parameters of the
electrical network and the arc-suppression device,
and its value is approximately 25% of the
capacitive fault current [28].
Therefore, with regard to the distribution
networks MV, one should speak about the indirect
electrical safety of people from the effect of touch
and step voltage, when operating electrical
networks.
The degree of danger of an electrical network
for a person who touches the energized electrical
equipment and installations housings because of the
damage of one of the phases depends, to a large
extent, on the neutral mode.
Especially this study is of interest for the
widespread mode of unstable closure (through an
alternating arc). In this mode, which in networks
with an isolated neutral at certain network
parameters poses danger to humans, in networks
with a neutral resistor the danger of touching the
frames of damaged equipment significantly
decreases due to a sharp reduction in the duration of
the transient process as well as to a decrease in the
amplitude of the earth fault current inrushes.
When superimposing an additional active
current equal to at least 50% of the capacitive
current at the earth fault location, it is interesting to
analyse the touch and step voltages arising on the
earthed enclosures of electrical equipment with the
alternating arc faults in the network. Since the
touch and step voltages are determined by the earth
fault current (flowing from the grounding
electrode), it makes sense to consider the effect of
the active neutral resistance on the value of this
current in the transient mode.
For the air-cable distribution network, which
can be represented in the form of L, C, r of the
contour (without taking into account the
longitudinal active resistance of the network),
connected to the instantaneous phase voltage
U = U m cos(t ) ; at the beginning of the closure as
well as throughout the entire process of burning of
the alternating (or intermittent) arc free and steadystate components of the emergency current arise.
The maximum inrush of a free capacitive current
with sufficient accuracy for practical calculations is
determined by the formula [21]:
U
i fr = m e− t sin ( t ) ,
L
or
i fr = 2 I C K e− t
2fr
sin ( t ) , (14)
where Um – is the maximum value of the voltage on
the damaged phase; K – is the voltage multiplicity
of the damaged phase at the moment of breakdown;
δ = r/(2L) – is the damping coefficient of the free
r2
1
– is
−
4 L2 3L C
the angular frequency of natural oscillations;
1
; L – is the zero-sequence equivalent
fr =
3L C
inductance of one phase; r – is the earth fault
resistance, keeping in mind that the minimum
possible resistance for the arc combustion is taken
equal to the resistance of the quarry grounding
network − 4 Ohm.
Calculations performed by the expression (14)
show that initial values of the free circuit current
can be tens of times greater than the amplitude of
the steady-state capacitive current IC in the event of
a ground failure. At several repeated one after
another extinctions and ignitions of the arc, free
current surges are also repeated. The effective value
of the current for one period of the industrial
circuit current; = 2 − fr =
52
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Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ...
frequency (assuming that the arc goes out and
ignites once per period) with the accuracy sufficient
for practical purposes can be determined by the
formula [21]:
0.013
i fr =
K IC ,
(15)
rC
So, for example, in the network voltage of 6 kV
with IC = 10 А, r = 4 Ω and K=1 the effective value
of free current will be Ifr = 38 А.
When grounding the network neutral through
the resistor that provides creation of an active
component of the failure current at the level of 50 –
100 % of the capacitive one, in case of an arc
closure to grounded electrical equipment, the
effective value of the resulting failure current will
decrease 1.4…2.8 times compared to the possible
value in the same network with a fully isolated
network neutral [22]. This reduction occurs owing
to the effective limitation of overvoltages in the
network after each break in the arc of the earth
failure.
|In view of the overvoltage reduction on the
undamaged phases (up to 2.2…2.4 U), as a result of
the superimposition of the active current, the
probability of developing single-phase failures in
the closures of two phases at different points of the
network also significantly decreases.
5. COMPARATIVE ESTIMATION OF THE
NEUTRAL MODE EFFICIENCY IN
TERMS OF LOCAL DISTRIBUTION
NETWORKS
An analysis of the above results of studies of the
degree of influence of the neutral grounding
method on the operational characteristics of
autonomous power supply systems, as well as the
performance of relay protection devices, allows us
to evaluate each method and develop practical
recommendations
aimed
at
their
further
development.
5.1. System with isolated neutral
An insulated neutral system is widespread and
has extensive operating experience. However, such
a grounding method network neutral mode is not
always optimal in terms reviewed of the criteria
specified earlier.
Taking into account possible changes in the
lowering coefficients, for the actual parameters of
distribution networks, the maximum voltage
between the healthy phases and the ground is at a
level of 4.5 of phase voltage. For these same
networks, the theoretical maximum of the neutral
point displacement voltage is a threefold phase
voltage.
Besides, the single-phase earth failures in
networks with an insulated neutral are
accompanied by transient processes arising at the
moment of failure occurrence and at the moment of
disconnection of the damaged section (the process
of voltage recovery in the network), which support
a significant part of false responses of the groundfailure protective devices.
The disadvantages of networks with an
insulated neutral may include: the instability of the
neutral voltage, favourable conditions for the
occurrence of arc closures, ferro-resonance
phenomena, increased touch and step voltages in
case of arc ground failures, increased multiplicities
of internal overvoltages, etc.
5.2. System with resonant grounding
When grounding the neutral through the arc
suppression reactor (tuned inductance), the
overvoltages of arc closures is smaller and, what is
especially important for insulation, they last for a
relatively short time interval (usually about half a
period). However, this advantage is minimized
when the compensation mode is detuned by more
than 5% of the resonance value. Also, the
disadvantage of the neutral grounding system
through the arc suppression reactor is the
complexity of providing resonant tuning.
Therefore, for small currents, the use of this type
of grounding is not economically feasible.
If to assess the reliability of power supply to
electric consumers by the damage of network
elements and by the operating quality of the relay
protection, it should be noted that application of
compensated networks, where the protective
shutdown action is required, is constrained by the
second condition. As for the damage rate of the
elements of distribution networks, one must note
that this indicator directly relates to the tuning
mode of the compensating device, since it is the
degree of the compensating device detuning from a
resonant mode that determines the level of
overvoltages in the network under single-phase
earth failures.
Figure 2 shows that with the resonant tuning of
the compensating device, as well as with its
detuning within 5%, even theoretically, the
overvoltages on the undamaged phases cannot
exceed more than 2.75Uf. The decrease in the level
of overvoltage is due to the creation of a
convenient path for the drainage of static charges
in phases due to the inclusion of the inductance of
the arc suppression reactor in the neutral of the
network [27].
An increase in the degree of compensation
detuning from 5 to 30...40% leads to a rapid
increase in the level of overvoltages. It should be
noted that with a detuning of the compensating
device by 20% of the resonant one, the
effectiveness of the compensating devices in terms
of limiting overvoltages during earth faults is
practically not felt in comparison with networks
with a completely isolated neutral.
The overvoltage in the neutral of the network is
approximately 1.5...2 times less than the frequency
of overvoltages in the undamaged phases, that also
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Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ...
helps to reduce the damage to the elements of the
power supply system.
From an economic point of view, distribution
networks with capacitive earth fault current
compensation require additional capital costs for
arc suppression reactors and devices for their
connection. With regard to operating costs, they
are significantly less than in networks with a
completely isolated neutral due to the less damage
to the system elements. With a resonant adjustment
of the compensating device and with insignificant
compensation mismatches in electrical networks,
the margin of dielectric strength of insulation in
relation to the impacting overvoltages increases to
30%.
Fig. 2. Dependences of the maximum
overvoltage multiplicity on the degree of
compensation detuning in the mode:
1 - overcompensation; 2 - undercompensation
5.3. System with low-Impedance neutral
grounding (active resistance)
The system with low-impedance neutral
grounding (active resistance), as compared to other
neutral grounding systems, have higher reliability
due to the improved performance of the protection
devices against single-phase earth failures
(suppression of post-accidental transient processes),
the elimination of ferro-resonance processes and the
reduction of the damage to the elements of the
power supply system. The latter is conditioned by a
significant decrease in internal overvoltages. As the
active component of the failure current increases
with respect to the capacitive component, the
overvoltage multiplicity decreases to 2.4 provided
that active and capacitive failure currents are equal.
The magnitude of overvoltage in a three-phase
network with an active resistance in the neutral is
determined by the expression:
0
U ov = 3 U f sin ( ig + 30 ) +
+U f sin
ig − (sin arc − 0.2 )e− k
a ( ig − arc )
(16)
( − )
C 1 d ,
C +CM
where
ig
– phase of voltage of the damaged phase
at the moment of ignition, rad.;
arc – phase of the
voltage of the damaged phase at the moment of arc
extinction, at which the bias voltage reaches its
maximum; ka is the ratio of active and capacitive
fault current components.
Figure 3 shows the dependence of the
maximum multiplicity of internal overvoltages in
the network with a resistor in the neutral on the
ratio of the active and capacitive components of
the single-phase earth fault current, obtained taking
into account the most unfavorable conditions
according to expression (16).
Fig. 3. Dependence of the maximum multiplicity of
overvoltage in a network with a resistor in neutral from
the ratio of the active and capacitive components of the
fault current
As the active component of the fault current
grows in relation to the capacitive component, the
overvoltage ratio decreases to 2.4, with the active
and capacitive fault current being equal. Figure 3
shows that a further increase in the active
component practically does not lead to a significant
decrease in the overvoltages ratio.
From the point of view of providing the singlephase ground failure protection, as well as the
quality of its performance (which is directly related
to the electrical safety and continuity of power
supply to electrical receivers), the power networks
with a neutral resistor, in other words, with the
overlay of the additional active component on the
ground failure current, can be considered as the
most favourable ones. In such networks, currents
and directional devices used in the insulated neutral
networks show more reliable and efficient operation
by suppressing the transient processes. On
condition of providing the single-phase ground
failure protection, the electrical networks with a
compensated neutral are in the most unfavourable
position.
54
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Ostapchuk O, Kuznetsov V, Kruczek W, Kuznetsov V, Tsyplenkov D.: Analysis of the neutral modes ...
According to the conditions of electrical safety
of power networks, when a person directly touches
the current-carrying parts, none of the possible
neutral operation modes can be considered as a
favourable one. According to the conditions of
indirect danger, preference should be given to
electrical networks, then the electrical network with
the neutral resistor should be considered as the most
favourable one.
With the development of autonomous networks
and an increase in the total capacity of the earth
failure, the combined neutral mode of the network
is advisable. It takes place when the resistor and the
arcing reactor are connected in parallel to the
neutral of the network. In this case, in addition to
creating the inductive component of the current of a
single-phase earth failure, the active component is
simultaneously superimposed on the network. The
value of the active component imposed on the
compensated network should be at the level of 30 50% of the capacitive component, that is, be
selected from the condition:
I a = (0.3...0.5) IC .
(17)
The recommended values of the emergency
current active component, ensure the suppression of
transient processes in the event of ground failures,
the increased efficiency of ground-failure protective
devices (signalling), the exclusion of the
ferroresonance phenomena, which leads to an
increase in the level of electrical safety and
reliability, as well as to the improvement of
performance characteristics, adequate to the power
supply systems only with the neutral resistor, even
at the detuning of an arc suppression reactor to 50%
of the resonance compensation mode [34-35].
Fig. 4 shows a comparison between the zone of
maximum multiplicity of overvoltages on the
degree of detuning compensation from the resonant
mode in a network with the compensated neutral
(zone 1) and in a network with the combined
neutral operation mode (zone 2).
4.4
Uov/U
1
4.0
3.6
3.2
2
2.8
2.4
2.0
0
0.2
0.4
0.6
0.8
Fig. 4. Dependence of the multiplicity of internal
overvoltages at earth failures on the degree of
detuning compensation mode: 1 - in a network
with the compensated neutral; 2 - in a network
with the combined neutral grounding
The upper and the lower boundaries of the
zones correspond to the values of the coefficient
overvoltage reduction, equal respectively to 1 and
0.8, which takes into account network physical
characteristics, relative damage location, etc.
6. CONCLUSIONS
1.The carried out studies allowed to establish the
fact that the reliability of distribution networks
in local systems is determined by the failure rate
of network elements and the effectiveness of
relay protection devices. The degree of
influence of these factors on the reliability of
distribution networks depends on the neutral
mode, which in turn determines the level of
internal overvoltages and the nature of transient
processes at single-phase-to-ground failures.
2. As regards the provision of earth failure
protection as well as of its performance (that is
directly related to the electrical safety and the
power supply continuity to electrical
consumers), electrical networks with the resistor
in a neutral can be considered as most
advantageous ones, where the current and
directional protection devices applied in the
networks with the insulated neutral, due to the
suppression of transient processes, show more
reliable and effective operation. In terms of
provision of the single-phase-to-ground failure
protection, electric grids with the compensated
neutral are in the most unfavourable position.
3. The lowest level of operational reliability
corresponds to the networks with the fully
isolated neutral, as well as to the ones with the
compensated neutral in case of compensation
detuning to 20% and higher from the resonant
value. This is because of the high level of
damage to the elements of power supply
systems by the effect of internal overvoltages
and of ferroresonance phenomena.
4. In the case of compensated networks in which it
is difficult to provide a resonant tuning mode, it
is recommended that the combined neutral
mode of operation, with the creation of an active
component, be at the level of 30 … 50% of the
capacitive one, that provides performance
indicators adequate to power supply systems
with only a resistor in the neutral, even during
malfunctions arc suppression reactor up to 50%
of the resonant compensation mode.
5. None of the considered methods of neutral
grounding allows to achieve the required level
of reliability in autonomous systems and
requires the use of additional devices that
continuously monitor the above indicators.
ACKNOWLEDGMENTS
This paper is elaborated in the framework of the
project co-financed by the Polish National Agency
for Academic Exchange
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28. Stojanovic, ZN, & Djuric, MB An algorithm for
directional earth-failure relay with no voltage inputs.
Electric Power Systems Research,. 2013;96: 144-149.
https://doi.org/10.1016/j.epsr.2012.11.004
29. Guo MF, Yang GJ, Huang SY. Adaptive faulty line
selection based on transient characteristics for
resonant
groundingsystem.
Electric
Power
Automation Equipment. 2012; 32(12):25-31.
30. Ayuurzana E, Mambetzhanov AD, Takyrbashev BK,
Dzhamagidze D, Petrov MI, Kuzmin AA. Evaluation
of the components of the current of a single-phase
earth failure during field experiments in mediumvoltage networks for various purposes. Bulletin of the
Chuvash University. 2016;3:21-29.
31. Wang Q, Liu X, Liu Z. Research on Diagnosis and
Positioning of Single-Phase Arc Grounding Failure in
the Distribution Network. 2018 Chinese Automation
Congress (Cac), 2018:2141-2146.
32. Kolesnikova KV, Gusev YP, Smotrov NN,
Lkhamdondog, AD. The Impact of the Sequence of
Measures for the Transition to a Resistive-Grounded
Neutral in 6-35kV Cable Networks on Investment
Profitability. In 2019 IEEE Conference of Russian
Young Researchers in Electrical and Electronic
Engineering (EIConRus). 2019; 992-995
33. Ravlić S, Marušić A, Havelka J. Neutral grounding in
middle voltage power systems: Simulation model. In
2015 38th International Convention on Information
and Communication Technology, Electronics and
Microelectronics (MIPRO). 2015 1124-1130.
34. Guldbrand A. System earthing. Report, Sweden.
2006.
35. Vinokurova TY, Dobryagina OA, Shagurina ES,
Shuin VA. Selective complex single-phase earth
failure protection for distribution medium-voltage
networks. Universitätsbibliothek Dortmund. New
Method for Calculation of the Harmonics in the
Residual Earth Failure Current in Isolated and
Compensated Networks. 2015.
https://doi.org/10.17877/DE290R-7398
Received 2020-06-30
Accepted 2021-01-25
Available online 2021-01-27
Oleksandr OSTAPCHUK
Department of Renewable
Energy, Igor Sikorsky Kyiv
Polytechnic Institute. Research
interests:
Power supply systems of
industrial enterprises, problems
of integration of distributed
generation sources into the
power system, relay protection,
electricity metering systems,
intelligent power supply systems, electrical part of
stations and substations.
E-mail: O.Ostapchuk@kpi.ua
Valeriy KUZNETSOV. prof.
dr. hab. Electric Power
Department, Railway Research
Institute, Poland. Obtained
dr.hab. degree in electric
transport in 2012. His research
interests concern: Energy
saving
methods,
energy
management, reliability of
traction power supply systems.
E-mail: vkuznetsov@ikolej.pl
Włodzimierz
KRUCZEK,
M.Sc. Eng: Railway Research
Institute,
Electric
Power
Engineering Department. His
research interests concern:
electric shock protection in
traction
systems,
power
equipment
of
traction
substations,
traction
converters, overhead contact
line and traction return system.
PhD student at the Warsaw
University of Technology
Email: wkruczek@ikolej.pl
Vitaliy KUZNETSOV, PhD,
Department of the electrical
engineering and electromechanic,
National metallurgical academy of
Ukraine. Received PhD in
electrical complexes and systems
in 2013. His research interests
concern:
Induction
motors,
Modelling, Simulation, and Power
quality monitoring systems.
E-mail: wit1975@i.ua
Dmytro TSYPLENKOV
Department
of
Electrical
Engineering, Dnipro University
of Technology
PhD, Associate Professor, Head
of the Department of Electrical
Engineering. He graduated from
the State Mining Academy of
Ukraine in 1997. Research
interests:
Electric machines, wind energy, renewable energy
E-mail: tsyplenkov.dv@nmu.one
| 9,462 | 60,088 |
W4380085519.txt | PHYSICAL REVIEW D 107, 115010 (2023)
Deconstructing squark contributions to di-Higgs production at the LHC
Stefano Moretti,1,2,* Luca Panizzi,2,† Jörgen Sjölin ,3,‡ and Harri Waltari
2,§
1
School of Physics and Astronomy, University of Southampton,
Highfield, Southampton SO17 1BJ, United Kingdom
2
Department of Physics and Astronomy, Uppsala University,
Box 516, SE-751 20 Uppsala, Sweden
3
Department of Physics, Stockholm University, 10691, Stockholm, Sweden
(Received 14 February 2023; accepted 17 May 2023; published 9 June 2023)
We present a novel approach to the study of di-Higgs production via gluon-gluon fusion at the LHC. The
relevant Feynman diagrams involving two Standard Model-like Higgs bosons hh are computed within a
simplified model approach that enables one to interpret possible signals of new physics in a modelindependent way as well as to map these onto specific theories. This is possible thanks to a decomposition
of such a signal process into all its squared amplitudes and their relative interferences, each of which has a
well-defined coupling structure. We illustrate the power of this procedure for the case of both a minimal and
next-to-minimal representation of supersymmetry, for which the new physics effects are due to top squarks
entering the loops of gg → hh. The squarks yield both a change of the integrated cross section and peculiar
kinematic features in its differential distributions with respect to the Standard Model. These effects can in
turn be traced back to the relevant diagrammatic and coupling structures and allow for a detailed analysis of
the process. In order to do so, we perform systematic scans of the parameter spaces of such new physics
scenarios and identify benchmark points which exhibit potentially observable features during the current
and upcoming runs of the LHC.
DOI: 10.1103/PhysRevD.107.115010
I. INTRODUCTION
The past few years have seen full data taking at Run 1 &
2 of the Large Hadron Collider (LHC), with Run 3 ongoing,
and this has been heralding a new era of precision Higgs
physics. The importance of its detailed understanding
cannot be overstated since the Higgs properties, originating
from the mass generation mechanism, play a key role in
searches for new physics (NP) which interacts with the
electroweak (EW) sector. The measured Higgs couplings so
far still allow for a minimal version of the Higgs mechanism, but one should bear in mind that several of the most
important properties directly related to the Higgs potential
(i.e., the Higgs self-couplings and Higgs-top couplings)
are still very weakly constrained. For this reason, Higgs
*
s.moretti@soton.ac.uk
stefano.moretti@physics.uu.se
†
luca.panizzi@physics.uu.se
‡
sjolin@fysik.su.se
§
harri.waltari@physics.uu.se
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.
2470-0010=2023=107(11)=115010(17)
physics remains one of the highest priorities in the global
high energy physics program.
Of all Higgs channels where beyond the Standard Model
(BSM) effects can be searched for, one which carries
particular relevance is di-Higgs production, as it can give
direct access to the structure of the scalar potential
triggering EW symmetry breaking (EWSB). At the LHC,
this process primarily takes place via gg-fusion [1,2], i.e.,
via the loop subprocess gg → hh involving triangle and box
graphs. BSM physics can therefore enter here via loops of
new particles at the same perturbative order as those of SM
ones, chiefly, the top-quark. In fact, another circumstance
which renders these virtual effects from BSM physics
potentially accessible specifically in di-Higgs production
is that there exists a strong cancellation between the
aforementioned triangle and box diagrams involving the
top quark [3] at (or near) the SM limit.
This is the context of our present study, which therefore
dispenses of the case involving “resonant” production of
heavy Higgs states decaying into (pairs of) the SM one, as
we are concerned here with “nonresonant” di-Higgs production. This has been studied extensively at the LHC to
date [4–12] and the standard approach is to parametrize
BSM effects via modifications of the Yukawa coupling of
the top quark (entering both the triangle and box diagrams)
and/or the Higgs self-coupling (entering solely the triangle
115010-1
Published by the American Physical Society
MORETTI, PANIZZI, SJÖLIN, and WALTARI
PHYS. REV. D 107, 115010 (2023)
ones). In turn, these can be interpreted in terms of an
effective field theory (EFT), wherein any new particle
entering gg → hh production via loops is essentially
integrated out.
Our approach is different. We allow for a BSM spectrum
in which the presence of new states is accounted for exactly,
at one-loop level, so that they can have any masses,
including those comparable to the dynamical scale of the
LHC (of order TeV), for which an EFT approach cannot
be adopted. We do so by exploiting a simplified model
approach that can easily be translated into any fundamental
theory responsible for the EWSB dynamics chosen by
Nature. This comes in the form of a numerical toolbox
enabling Monte Carlo (MC) studies at a level of sophistication comparable to actual experimental analyses (albeit
the version used in the present analysis is one-loop only).
Borrowing as reference BSM framework the one of
supersymmetry (SUSY), we will prove that the very same
particles (i.e., the top quark companions in SUSY, so-called
squarks) responsible for enabling extensions to the SM
without hierarchy problems can give sizeable effects in diHiggs production, when their typical masses are indeed of
order TeV.
The plan of the paper is as follows. In the next section we
will introduce the realizations of SUSY that we will be
adopting for exemplifying our approach, including both a
minimal and nonminimal version [13]. We will then
describe our toolbox. Numerical results for gg → hh
will follow, for both Run 3 and the High-Luminosity
LHC (HL-LHC) [14], in turn preceding our conclusions.
II. STOPS AND THE HIGGS MASS
We shall first consider the minimal supersymmetric
standard model (MSSM), which is based on the superpotential
W MSSM ¼ yu QHu U c þ yd QH d Dc þ yl LH d Ec þ μHu Hd :
ð1Þ
The Higgs couples strongly to the top-stop sector, so the
(s)tops will have a large impact on both the Higgs mass and
its production cross sections. The stop mass matrix at treelevel is
1 2 2
2
2
2
2
Mt̃L t̃L ¼ mQ̃ þ mt þ mZ cos 2β − sin θW ; ð2Þ
33
2 3
M2t̃L t̃R ¼ mt ðμ cot β − At Þ;
M 2t̃R t̃R ¼ m2Ũ
33
2
þ m2t þ m2Z cos 2β sin2 θW :
3
SUSY breaking coupling H0u t̃L t̃R . There are loop corrections to this mass matrix [15], which are potentially large.
In the MSSM there is a tree-level bound on the (lightest)
SM-like Higgs mass,
m2h ≤ m2Z cos2 2β;
so large loop corrections are needed to produce the
observed Higgs mass of 125 GeV. Since the top Yukawa
coupling is the largest coupling in the MSSM, the most
economical way to achieve this is to take tan β to be large
(so that the tree-level mass is maximal and the SM-like
Higgs boson couples maximally to the top/stop), make the
stops heavy (so that the top-stop Supersymmetric cancellation is incomplete) and to introduce large stop mixing
(which maximizes their loop corrections).
Large mixing in the stop sector creates a large mass
splitting between the stops. If one of the soft masses m2Q̃ or
33
m2Ũ is somewhat smaller than the other, we might end up
33
with a relatively light stop together with a heavy one.
Currently, stop masses down to 600 GeV are allowed if
the mass splitting between the stop and the Lightest
Supersymmetric Particle (LSP) is small [16,17], since
searches based on missing transverse momentum lose their
sensitivity in the compressed case.
In the MSSM, a large stop mass splitting is a necessity to
achieve a 125 GeV Higgs mass, so searches based on
missing transverse momentum are sensitive to the heavier
stop. Hence, it must be heavy, the lower bound being
around 1250 GeV [17,18] Since the stop mixing parameter
At needs to be large in the MSSM, the bubble, triangle and
box diagrams with trilinear stop couplings (see following
section) give a large contribution to Higgs pair production,
as known from literature [19–21] and which we will
elaborate upon.
It is well known that in the MSSM the Higgs trilinear
self-coupling is always close to its SM value [22,23].
Numerical scans show that deviations can be at most at the
3% level [24]. Hence, in the MSSM, the modifications of
the Higgs self-coupling will not lead to observable effects
in Higgs pair production at the LHC as the predicted
precision of the di-Higgs cross section measurement is
around 40% [25].
In the next-to-MSSM (NMSSM), the superpotential is
ð3Þ
κ
W ¼ W MSSM ðμ ¼ 0Þ þ λSHu Hd þ S3 :
3
ð4Þ
Here m2Q̃ and m2Ũ are the soft SUSY breaking squark
masses, tan β ¼ hH0u i=hH 0d i and At is the trilinear soft
ð5Þ
ð6Þ
In this case there are additional tree-level contributions to
the lightest Higgs boson mass so that the bound reads
as [26]
115010-2
DECONSTRUCTING SQUARK CONTRIBUTIONS TO
m2h
≤
m2Z
cos2 2β þ
2λ2
2
sin 2β :
g2 þ g02
…
PHYS. REV. D 107, 115010 (2023)
ð7Þ
If 1 ≲ tan β ≲ 3 and λ is large, the Higgs mass can be
close to 125 GeV without loop corrections. Hence in the
NMSSM one can have two light stops [27] but, due to the
experimental constraints [17], they need to be nearly
degenerate with each other and the LSP. In such a case
we need to require stop mixing to be minimal, μ cot β ≃ At
[see (3)] and hence the contribution from the trilinear
bubble and triangle diagrams will be small. Furthermore,
due to the extended Higgs sector, it is possible to have a
large deviation from the SM prediction to the trilinear
Higgs self-coupling even if other Higgs couplings are
SM-like [24]. An enhancement can occur when tan β is
close to 1 and λ is large while a suppression requires a
second, singlet-dominated scalar to be light and λ to be
small. This second option is incompatible with light
squarks. We give a qualitative argument for this behavior
in Appendix B.
In our analysis, we perform a comprehensive scan in the
MSSM and then select some benchmark points from the
NMSSM to represent cases not present in the MSSM, but
relevant for the di-Higgs process. The parameter space of
the MSSM is chosen as follows. We choose the mostly
right-handed stop to be the light one and to have a small
mass splitting,
mt̃1 − mχ̃ 01 < 10 GeV;
DI-HIGGS
ð8Þ
with the lightest neutralino, which we take to be
Higgsino-like. This mass splitting can always be
arranged by choosing the value of the μ-parameter.
The mostly left-handed squark doublet will then be heavy
(mt̃2 > 1250 GeV). We scan over the values of m2Q̃ , m2Ũ ,
33
33
tan β and At , which are the ones that determine the squark
contribution to Higgs pair production. The other soft
masses are fixed such that the rest of the sparticle spectrum
is heavier than the stops and the Higgsinos.
We also show a few NMSSM benchmarks representing
cases that cannot be realized in the MSSM. One is when we
make both stops as light as possible and adjust the Higgs
mass to 125 GeV by tuning tan β and λ. For this benchmark
the Higgs trilinear self-coupling is about 50% larger than in
the SM, but the trilinear Higgs-stop couplings are much
smaller than in the MSSM case. A second benchmark point
represents a case where the Higgs trilinear self-coupling is
about 60% larger than in the SM and the stop masses and
mixing are similar to the MSSM points.
III. NUMERICAL ANALYSIS
The contributions to the di-Higgs final state from the
SM are represented by the well-known, destructivelyinterfering topologies shown in Fig. 1, where the only
FIG. 1.
SM topologies for di-Higgs production at the LHC.
coupling parameters entering the amplitudes are proportional to the Higgs trilinear self-coupling λv and the
Yukawas yt;b .
The SUSY-inspired parameters that are relevant for the
di-Higgs process (squark masses and couplings) will be
included in a simplified Lagrangian which assumes that
every other SUSY particle does not participate significantly
in the process: this means all other scalars are too heavy or
their couplings are too small and all other squarks are also
decoupled.
First of all we notice, trivially, that the electric charge of
the squarks propagating in the loops does not play any
role in the process. What actually matters for a modelindependent treatment of the process is that, besides
introducing NP modifications of the SM couplings, there
are new colored particles propagating in the loops. Then,
the key properties of such particles are their representation
under SUð3Þ of QCD, their spin, how many they are and the
size of their couplings with the Higgs boson. Only if the
new particles are fermions, their electric charge plays a role,
due to the fact that heavy quarks with charge 2=3 or −1=3
can mix with the SM quarks.
Limiting our treatment to only two scalars in the
fundamental representation of SUð3Þ, the simplified squark
Lagrangian we consider in this analysis only contains
interactions between those two squarks q̃i (with i ¼ 1, 2
labeling the mass eigenstates), h, t, and NP contributions to
the couplings h3 and htt̄ (we will neglect modifications to
the bottom Yukawa coupling in the following.). The model
contains two colored scalars in the loop so it can be adapted
to the SUSY scenarios described in the previous section,
and also considering that it is realistically very hard (if not
plainly inconsistent) to build scenarios in which one of the
two scalar top partners is light enough to produce visible
effects at the LHC and the other is too heavy and decoupled
from low-energy physics. The simplified interaction
Lagrangian therefore generically reads as
TABLE I. Parameter ranges for the scan in the MSSM. All
other soft scalar masses are larger than those mentioned. The
μ-parameter is adjusted so that the Higgsino-like LSP is slightly
lighter than the lightest stop.
Parameter
Minimum
Maximum
tan β
At (GeV)
m2Ũ (GeV2 )
7
1500
1.35 × 106
50
3500
2 × 106
2.2 × 106
3.5 × 106
33
m2Q̃ (GeV2 )
115010-3
33
MORETTI, PANIZZI, SJÖLIN, and WALTARI
PHYS. REV. D 107, 115010 (2023)
FIG. 2. Representative examples of LO di-Higgs cross sections (shown as a color gradient) as function of pairs of MSSM input
parameters (left), stop masses (center), or the parameters of the simplified Lagrangian equation (9) (right), highlighting their
correlations.
1
Lq̃NP ¼ −ðλSM þ κ hhh Þvh3 − pffiffiffi ðySM
t þ κ htt Þht̄t
2
!
κ11
κ 12
q̃1
h
q̃
q̃
h
q̃
q̃
þ vhðq̃1 q̃2 Þ
q̃2
·
κ 22
hq̃q̃
!
11
12
κ hhq̃q̃ κ hhq̃q̃
q̃1
þ hhðq̃1 q̃2 Þ
;
22
q̃2
·
κ hhq̃q̃
ð9Þ
where the trilinear and quartic couplings are kept independent to account in a model-independent way for further NP
effects which may alter the relation between the two. In the
following, we identify q̃1;2 with t̃1;2 , and all the couplings
are assumed to be real.
This Lagrangian has been implemented in FeynRules [28]
to obtain a UFO [29] output suitable for simulations at nextto-leading order (NLO) in QCD with the MG5_aMC [30]
Monte Carlo (MC) generator.1 For di-Higgs production this
is obviously necessary as the process is at one-loop at
leading order (LO). In all our results we used the
NNPDF3.0 LO PDF set [32].
The following analysis is aimed at finding which values
of the parameters of NP can alter significantly the signal
cross section and at the same time exhibit peculiar
kinematic features which could lead to its observation
during Run 3 or the HL-LHC. Crucially, our implementation of the gg → hh process starting from the aforementioned simplified Lagrangian is such that we are able to
identify each component entering the total cross section,
i.e., the contributions of the square of the relevant diagrams
as well as the interferences between all of these, thereby
1
The model is publicly available on HEPMDB [31] at this link:
https://hepmdb.soton.ac.uk/hepmdb:0223.0337 and it actually
contains four new colored scalars in the fundamental of SUð3Þ
(recall that we only consider two of these for this analysis). The
additional squarks have been included to account for possible
extensions.
affording us with significant diagnostic power of the
emerging signal. While this approach, as emphasized, is
model independent, we will be applying it to both the
MSSM and NMSSM described in the previous section, in
order to show its effectiveness. Studies of squark effects of
the kind looked for here have been performed in literature
before (e.g., see [21]), but what this approach adds is the
ability to directly reverse engineer the dynamics involved
and interpret it already at the experimental analysis stage.
A. Signal cross section and parametric dependence
We first have performed a parametric scan within the
MSSM: the scan parameters and their ranges are given in
Table I. The spectra were calculated with SPheno v4.0.4
[33,34]. We required the lighter stop to be heavier than
600 GeV and the heavier stop to be heavier than 1250 GeV
with the Higgs mass being in the interval 125.2 0.3 GeV
(∼2σ around the central value). We set the μ parameter so
that the Higgsino-like neutralino LSP is slightly lighter than
the lighter stop, hence, we may use 600 GeV as the lower
bound for the lighter stop mass. Other parameters are kept
fixed such that the rest of the BSM spectrum is decoupled,
as previously mentioned.
The resulting MSSM couplings and masses have been
translated into parameters of the simplified Lagrangian
equation (9). With the obtained set of benchmark points we
have performed MC simulations and determined correlations between parameters which maximize the cross section, as shown in Fig. 2.
We found preferred directions in the parameter space for
which the cross section increases, around specific combinations of the stop masses and for specific values of the
trilinear parameters, especially the modifiers of SM couplings κ hhh and κ htt .
In Fig. 2 we show some of the parametric dependencies.
Obviously light squark masses lead to larger deviations
from the SM, so the maximal cross sections are seen close
115010-4
DECONSTRUCTING SQUARK CONTRIBUTIONS TO
DI-HIGGS
to mðt̃1 Þ ¼ 600 GeV, mðt̃2 Þ ¼ 1250 GeV, the lighter stop
mass being the more important one in increasing the
cross section. Large tan β ≳ 20 is preferred as the top-stop
correction to the Higgs mass is maximal and the 125 GeV
Higgs mass can then be reached with lighter stops than is
the case for low tan β.
In the MSSM with fixed squark masses and tan β you
either have an interval of possible At values leading to an
acceptable Higgs mass (at low tan β) or two intervals of At ,
where between these intervals the Higgs is too heavy. In the
lower of these intervals the squark bubbles and triangles
give a smaller contribution and the cross section is SM-like.
In the higher interval significant enhancements are possible, if the squarks are light. The region of allowed values
for At depends on the average stop mass2: the heavier the
stops, the larger the maximum value for At. Therefore, the
maximal cross sections are obtained with values of At
which are intermediate within the scan range, but which are
maximal for the given stop masses and tan β.
The point which produces the highest cross section,
defined in Table II, will be considered in the following as
benchmark point for the kinematic analysis.
…
PHYS. REV. D 107, 115010 (2023)
TABLE II. Benchmark point for the MSSM. Left: tree-level
input parameters for SPheno. The loop corrections to the stop
mass matrix are large, so the soft masses plus the loop corrections
are approximately m2Ũ ¼ 5.3 × 105 GeV2 and m2Q̃ ¼ 1.43 ×
33
33
106 GeV2 and the effective value for At ¼ 2750 GeV after loop
corrections. Right: mass spectrum and couplings, as defined in
the simplified model Lagrangian of Eq. (9). For di-Higgs
production the κ12
hht̃ t̃ coupling is not relevant.
MSSM benchmark point
Input parameter
Value
tan β
At (GeV)
m2Ũ (GeV2 )
45.4
2595
1.547 × 106
33
m2Q̃ (GeV2 )
2.447 × 106
33
MSSM benchmark point
Masses and couplings
mt̃1 (GeV)
mt̃2 (GeV)
κ hhh
κ htt
11 12
B. Kinematic distributions
1. Deconstruction of the signal
To perform a differential analysis of the signal associated
with the propagation of stops in the loops, we deconstruct
the signal into basic independent components. The total
signal is then obtained as a weighted sum of such
components. This procedure allows us to analyze separately their peculiar kinematic features, assess their relevance for the final result and understand semianalytically
which range of parameters maximizes the potential observability of the signal.
Limiting our treatment to only two stops we can build a
limited number of elements. Labeling with a black dot (•)
the NP contributions to SM couplings, with a red dot ( ) the
purely NP couplings and with red Feynman propagators the
new particles (t̃1;2 ), the signal amplitudes can be deconstructed in a small number of elements, listed in Table III.
κ ht̃ t̃ κ ht̃ t̃
· κ 22
ht̃ t̃
κ 12
κ 11
hht̃ t̃ hht̃ t̃
· κ 22
hht̃ t̃
Value
600.6
1301.0
3.34 × 10−3
−1.68
× 10−3
−6.690 7.228
·
8.519
−0.6702 −0.0174
·
−0.6374
The signal contributions and their interferences can be
parametrized in terms of a sum of different terms, proportional to unique functions of the couplings and to reduced
cross sections σ̂ depending exclusively on the stop masses.
Labeling as “B” the SM irreducible background, as “M” the
contribution of the topologies containing only modified SM
couplings (1 to 4 in Table III) and as “S” the contribution of
topologies associated with the propagation of squarks (5 to
8) the complete set of contributions is:
σ M ¼ κ 2hhh σ̂ 1 þ ðκ hhh κ htt Þ2 σ̂ 3 þ κ 4htt σ̂ 4 ;
ð10aÞ
X X jj
X jj
ii 2
ii
2
2
κ iiht̃ t̃ κht̃ t̃ σ̂ int
κ ht̃ t̃ σ̂ int
σS ¼
6i ðmt̃i;j Þ
5i ðmt̃i;j Þ þ κ hhh ðκ ht̃ t̃ Þ σ̂ 6d ðmt̃i Þ þ κ hhh κ ht̃ t̃
i¼1;2
þ
j>i
ðκ iiht̃ t̃ Þ4 σ̂ 7d ðmt̃i Þ
j>i
X ij
X
X ij
ii 2
2 int
þ
ðκht̃ t̃ Þ4 σ̂ 7o ðmt̃i;j Þ þ ðκ iiht̃ t̃ Þ2 ðκjj
ðκ ht̃ t̃ Þ2 σ̂ int
7ido ðmt̃i;j Þ
ht̃ t̃ Þ σ̂ 7idd ðmt̃i;j Þ þ ðκ ht̃ t̃ Þ
j>i
X jj
ii
2
ii
int
þ ðκ hht̃ t̃ Þ σ̂ 8d ðmt̃i Þ þ κ hht̃ t̃ κ hht̃ t̃ σ̂ 8i ðmt̃i;j Þ ;
j>i
j≠i
ð10bÞ
j>i
This is essentially to avoid a color-breaking vacuum. In general a large ratio of X t =MS , where X t ¼ At − μ cot β and M S is the
average tree-level stop mass, can lead to a color-breaking vacuum, though the relation is not so simple if loop corrections are taken into
account [35,36]. Our scan ranges have X t =M s < 2.5, which is considered to lead to a stable enough color-conserving vacuum.
2
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MORETTI, PANIZZI, SJÖLIN, and WALTARI
PHYS. REV. D 107, 115010 (2023)
σ int
MB ¼ κ hhh σ̂ 1B þ κ htt σ̂ 2B ;
σ int
SB
¼
X
κiiht̃ t̃ σ̂ int
5B ðmt̃i Þ
ð10cÞ
X ij
ii
2
int
int
þ
ðκht̃ t̃ Þ σ̂ 7oB ðmt̃i;j Þ þ κhht̃ t̃ σ̂ 8B ðmt̃i Þ ;
3
3
2
int
2
int
int
int
σ int
MM ¼ κ hhh κ htt σ̂ 1;3 þ κ hhh κ htt σ̂ 1;4−2;3 þ κ htt σ̂ 2;4 þ κ hhh κ htt σ̂ 3;4 ;
σ int
SS
ð10dÞ
j>i
i¼1;2
ð10eÞ
X
X
X jj
X ij
ii 3 int
ii
ii
int
κ hhh κiiht̃ t̃ κ jj
¼
ðκ ht̃ t̃ Þ2 σ̂ int
ðκht̃ t̃ Þ2 σ̂ int
5;7i ðmt̃i;j Þ þ κ ht̃ t̃
5;7o ðmt̃i;j Þ
ht̃ t̃ σ̂ 5;6i ðmt̃i;j Þ þ ðκ ht̃ t̃ Þ σ̂ 5;7d ðmt̃i Þ þ κ ht̃ t̃
j>i
i¼1;2
j≠i
j≠i
X jj
X jj
ii
int
ii 3 int
ii
þ κiiht̃ t̃ κ iihht̃ t̃ σ̂ int
ðm
Þ
þ
κ
κ
σ̂
ðm
Þ
þ
κ
ðκ
Þ
σ̂
ðm
Þ
þ
κ
κ
ðκ ht̃ t̃ Þ2 σ̂ int
hhh
hhh
t̃
t̃
t̃
6;7d
6;7i ðmt̃i;j Þ
5;8d
hht̃ t̃ 5;8i
ht̃ t̃
ht̃ t̃
ht̃ t̃
i
i;j
i
j≠i
þ
κhhh κ iiht̃ t̃
j≠i
X ij
X jj
ii 2 int
ii
ðκht̃ t̃ Þ2 σ̂ int
κ hht̃ t̃ σ̂ int
6;7o ðmt̃i;j Þ þ κ hhh ðκ ht̃ t̃ Þ σ̂ 6;8d ðmt̃i Þ þ κ hhh κ ht̃ t̃
6;8i ðmt̃i;j Þ
j≠i
j≠i
X ij
X jj
ii 2 ii
int
ii 2
int
2 ii
int
κ hht̃ t̃ σ̂ 7i;8 ðmt̃i;j Þ þ
ðκ ht̃ t̃ Þ κhht̃ t̃ σ̂ 7o;8 ðmt̃i;j Þ ;
þ ðκht̃ t̃ Þ κ hht̃ t̃ σ̂ 7d;8 ðmt̃i Þ þ ðκ ht̃ t̃ Þ
j≠i
σ int
MS
ð10fÞ
j≠i
X
X ij
ii
int
ii
int
κ2hhh κ iiht̃ t̃ σ̂ int
¼
ðκht̃ t̃ Þ2 σ̂ int
1;7o ðmt̃i;j Þ þ κ hhh κ hht̃ t̃ σ̂ 1;8 ðmt̃i Þ þ κ htt κ ht̃ t̃ σ̂ 2;5 ðmt̃i Þ
1;6 ðmt̃i Þ þ κ hhh
j>i
i¼1;2
þ
κ hhh κ htt κ iiht̃ t̃ σ̂ int
2;6−3;5 ðmt̃i Þ
þ
κ 2hhh κ htt κ iiht̃ t̃ σ̂ int
3;6 ðmt̃i Þ
þ
κ hhh κ htt κ iihht̃ t̃ σ̂ int
3;8 ðmt̃i Þ
þ κhtt ðκ iiht̃ t̃ Þ2 σ̂ int
2;7d ðmt̃i Þ þ κ htt
X ij
ii
int
ðκ ht̃ t̃ Þ2 σ̂ int
2;7o ðmt̃i;j Þ þ κ htt κ hht̃ t̃ σ̂ 2;8 ðmt̃i Þ
j>i
þ
κhhh κ htt ðκiiht̃ t̃ Þ2 σ̂ int
3;7d ðmt̃i Þ
þ κ hhh κhtt
X ij
ðκht̃ t̃ Þ2 σ̂ int
3;7o ðmt̃i;j Þ
j>i
þ
κ2htt κiiht̃ t̃ σ̂ int
4;5 ðmt̃i Þ
þ
κhhh κ 2htt κ iiht̃ t̃ σ̂ int
4;6 ðmt̃i Þ
X ij
ii
2
2
int
2
int
þ κ htt ðκ ht̃ t̃ Þ σ̂ 4;7o ðmt̃i;j Þ þ κ htt κ hht̃ t̃ σ̂ 4;8 ðmt̃i Þ ;
þ κ 2htt ðκ iiht̃ t̃ Þ2 σ̂ int
4;7d ðmt̃i Þ
ð10gÞ
j>i
int
2
int
σ int
MSB ¼ κ hhh κ htt σ̂ 1;2−3B þ κ htt σ̂ 2−4B þ
X
ii 2 int
ii 2 int
½κhhh κ iiht̃ t̃ σ̂ int
1;5−6B ðmt̃i Þ þ κ hhh ðκ ht̃ t̃ Þ σ̂ 1;7d−5;6 ðmt̃i Þ þ ðκ ht̃ t̃ Þ σ̂ 5d−7dB ðmt̃i Þ:
ð10hÞ
i¼1;2
The reduced cross sections have been labeled according
int
to the numbering scheme of Table III: σ̂ int
x;y and σ̂ xB
represent, respectively, the interference terms between
the x and y classes of topologies and between x and the
int
SM background, while σ̂ int
x;y−j;k or σ̂ x;y−jB signal combinations proportional to the same function of couplings.
Further, σ̂ xd means that the topology class x contains
couplings between same quark flavor, σ̂ xo between different
ones and finally, σ̂ xi or σ̂ int
x;yi mean interference between
topologies where two different squarks circulate in the
loops. Factors of 2 from interference terms have been
included into the σ̂’s.
The total cross section induced by NP is given by the
sum of all the terms of Eq. (10). This parametrization
allows for a model-independent analysis of the process: the
kinematics of each individual term of Eq. (10) is determined exclusively by the masses of the particles circulating
in the loops, while the couplings only affect the relative
proportion between the various terms. Total and differential
quantities can be thus determined for multiple benchmarks
characterized by the same stop masses and different
couplings by performing a unique set of numerical simulations [one for each term of Eq. (10)] for those masses. The
simulation syntax is explained in Appendix A.3
In the following sections, the analysis of a specific
distribution—the invariant mass of the di-Higgs system—is
3
Notice that, if more colored scalars are present, other terms
depending on more than two masses would have to be considP
P
2
ered, such as i;k ðκiiht̃ t̃ Þ2 j;k≠i ðκkj
ht̃ t̃ Þ σ̂ 7idi ojk ðmt̃i;j;k Þ, and further
simulated samples would have to be included in the database.
Considering that the topologies of Table III contain at most two
different squark propagators, a complete set of σ̂ elements can be
obtained considering four colored scalars. The particle content of
our UFO model indeed allows one to explore such possibilities.
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…
PHYS. REV. D 107, 115010 (2023)
TABLE III. Complete list of topologies describing di-Higgs production with modified SM couplings hhh and htt, and loop
propagation of any number of stop squarks t̃i . The topologies are classified according to the different products of new couplings [defined
in Eq. (9)] to which the amplitudes are proportional.
Topology type
Feynman diagrams
Amplitude
1
Modified Higgs trilinear coupling
Ai ∝ κ hhh
2
One modified Yukawa coupling
Ai ∝ κhtt
3
Modified Higgs trilinear coupling
and modified Yukawa coupling
Ai ∝ κhhh κhtt
4
Two modified Yukawa couplings
Ai ∝ κ2htt
5
Bubble and triangle with ht̃t̃ couplings
Ai ∝ κiiht̃ t̃
This class of topologies involves only diagonal couplings between the Higgs and the squarks, due to the absence of FCNCs
in strong interactions and the presence of one ht̃t̃ coupling.
6
Modified Higgs trilinear coupling þ
Ai ∝ κhhh κ iiht̃ t̃
Bubble and triangle with ht̃t̃ coupling
7
8
Only diagonal couplings between the Higgs and the squarks due to the strong interaction.
Triangle and box with two ht̃t̃ couplings
2
Ai ∝ jκ ij
ht̃ t̃ j
Ai ∝ κ iihht̃ t̃
Bubble and triangle with hht̃t̃ coupling
Only diagonal couplings between the Higgs and the squarks due to the strong interaction.
discussed in detail following the aforementioned procedure, both before and after the decays of the Higgs
bosons.
2. Invariant mass of the di-Higgs system
before Higgs decay
One of the key observables to explore the contributions
of NP in di-Higgs production at the LHC is the invariant
mass of the di-Higgs system. Its shape is affected by the
modification of SM couplings and by the presence of new
particles in the loop. In this section we will consider the
MSSM benchmark point in Table II to describe the analysis
strategy.
The invariant mass distribution is built from the individual components of Eq. (10), evaluated for mðt̃1;2 Þ ¼
f600; 1300g GeV, where the masses are approximated to
the values on the simulation grid.
Considering Eq. (10b) as an example, one can evaluate
each differential cross section corresponding to the various
σ̂ terms using the corresponding MC samples, as shown in
the left panel of Fig. 3. From this, the differential
distribution of σ S can be evaluated by weighting each
component with the numerical values of the coupling
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PHYS. REV. D 107, 115010 (2023)
FIG. 3. Left panel: dσ̂=dMhh distributions of all components of σ S . Right panel: each component of the left panel is weighted with the
associated coupling products corresponding to the MSSM benchmark point of Table II and converted into the number of expected events
with at the end of Run 3 with nominal luminosity of 300 fb−1 . The black curve corresponds to the sum of all components and represents
the M hh distribution of Eq. (10b).
product factor and sum all terms, as shown in the right
panel of Fig. 3, where the distributions are also multiplied
by the nominal luminosity at the end of Run 3 of the LHC
to evaluate the number of expected physical events. From
the distributions evaluated before their weighting, it is
possible to notice the threshold effects around mhh ¼
1200 GeV corresponding to the propagation of the
600 GeV stop, and the presence of regions with negative
interference. The importance of each term is however
reshuffled by the couplings: for example, the negative
value of the σ̂ int
5i ðmt̃1;2 Þ contribution is entirely due to the
22
11
negative coupling product (κ 11
ht̃ t̃ κ ht̃ t̃ with κ ht̃ t̃ < 0) in front
of it. However, when summed with the other contributions,
the overall net effect is a positive cross section in the whole
mhh range, with a relevant enhancement of the 1200 GeV
threshold peak.
The very same procedure is applied to each term in
Eq. (10) and the final result for the MSSM benchmark point
is shown in Fig. 4. In general there are three types of
contributions. Those that involve only modifications to the
SM couplings (σ M , σ MM and σ MB ) have a peak at low Mhh
and then decay exponentially. Such contributions are small
in the MSSM, but can be more pronounced in, e.g.,
NMSSM, where the trilinear Higgs coupling can deviate
from its SM value. Second, we have squark contributions
squared (σ S and σ SS ), which are relatively flat over a large
range and peak at M hh ¼ 2mt̃ . Finally we have interference
contributions between squarks and the SM-type contributions (σ SB , σ MS and σ MSB ), which are positive at low M hh,
negative between mt̃ < Mhh < 2mt̃ and turn positive again
at high Mhh . This leads to an almost complete cancellation
of BSM effects in the range mt̃ < M hh < 2mt̃ .
Looking at the MSSM benchmark of Fig. 4 multiple
effects are in place: the threshold peak generated by the
terms in Eq. (10b) is clearly standing out with respect to the
SM irreducible background, even if negative interference
contributions from Eq. (10f) slightly reduce its impact, but
the increase in the total cross section (SM þ Signal≃
24.5 fb) is mostly due to the excess of events on the global
peak of the distribution, generated by the interference terms
of Eq. (10h). Inspecting the elements of Eq. (10h), analogously to what was done in Fig. 3, the largely dominant
contribution (in the whole Mhh range) is given by the term
2 int
ðκ 11
ht̃ t̃ Þ σ̂ 5d−7dB ðmt̃1 Þ, which sums the pure signal contribution from topology 5 of Table III and the interference
FIG. 4. Invariant mass distribution of the di-Higgs system at
parton level and before Higgs decay for the MSSM benchmark
point of Table II, displaying the number of expected events at the
end of Run 3 with nominal luminosity of 300 fb−1 . Curves for the
SM intrinsic background (black), pure signal (red) and their sum
(blue) are shown as solid lines. For the signal, the individual
contributions of its components are also shown as dashed lines, to
assess their relative role in the construction of its shape.
115010-8
DECONSTRUCTING SQUARK CONTRIBUTIONS TO
FIG. 5. Same as Fig. 4, but showing only the SM and Signal þ
SM curves with 1σ uncertainty bands representing the combined
scale and PDF systematics. In the bottom panel, the ratio between
central values, ðSM þ SignalÞ=SM, is displayed.
DI-HIGGS
…
PHYS. REV. D 107, 115010 (2023)
uncertainties are obtained by considering the largest deviations from the central value, while PDF uncertainties are
obtained using the standard deviation on the whole PDF
set, as described in [38]. The scale and PDF systematics are
then linearly summed. In Fig. 5 the distributions for
the SM background and the sum of signal and background
are shown: it is possible to see that while the excess
on the global peak is completely hidden within the
uncertainties, the threshold peak can indeed be potentially
discriminated.
So far, only distributions before Higgs decays have
been considered. If no shape discrimination was possible
already at this stage, any further investigation would have
been pointless. Given the optimistic results, however,
the next step of the analysis involves letting the Higgs
decay and identify promising final states for experimental
detection.
3. Invariant mass of the di-Higgs system after
Higgs decay
between topology 7 and the SM background, both proportional to the same product of new couplings. Given the
small modifications to the SM couplings, the topologies
proportional to κ hhh and κ htt have an almost negligible
impact on the determination of the final shape of
dσ̂ S =dMhh .
Despite the excess in the global peak and the presence of
a relatively sizeable threshold peak with respect to the
background distribution, an estimation of the systematic
uncertainties is in order to assess if the different shapes can
be potentially discernible using real data. A dedicated MC
simulation has been performed for this estimation, validating the results obtained with the deconstruction method
as a by-product. The combination of scale and parton
distribution function (PDF) systematics is done using
the same method of [37]: bin-by-bin, the asymmetric scale
FIG. 6.
In this part of the analysis we consider three final states,
motivated either by large branching ratios or by cleanliness
of detection. In all cases we select final states where at least
one of the Higgs boson decays to 2 bottom quarks, and the
other to either 2 photons, 2τ leptons or other 2 bottom
quarks. The reconstruction of final state objects is done
using FastJet [39] through MadAnalysis 5 [40–42], using the
anti-kT jet clustering algorithm [43] with jet radius parameter R ¼ 0.4.
The corresponding distributions of physical events as
function of the invariant mass of the di-Higgs decay
products are shown in Figs. 6–8: in all figures, the left
panels show results at reconstruction level without any cut,
while the right panels correspond to the basic selection cuts
reported in Table IV. The preselection cuts are inspired by
current experimental prospects [44]. For the bbγγ final state
Invariant mass distribution of the bbγγ system at reconstruction level (left) and after the cuts of Table IV (right).
115010-9
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FIG. 7. Same as Fig. 6 but for the bbττ final state.
FIG. 8. Same as Fig. 6 but for the 4b final state.
a diphoton invariant mass cut around the Higgs boson mass
has been applied to reduce the contribution from radiated
photons and enhance the contribution of photons from
Higgs decay.
The depletion in number of events in the bbγγ and bbττ
case makes it impossible to discriminate any NP contribution induced by the scenarios we are considering during
the Run 3 of the LHC. While the bbbb final state looks
more promising, a careful evaluation of all sources of
TABLE IV.
backgrounds (especially QCD-induced ones) must be
performed in order to establish potential discrimination
possibilities.
IV. DEVIATIONS BEYOND THE MSSM
After the first two runs of the LHC, the parameter space of
the MSSM has become constrained, especially when it
comes to parameters related to Higgs pair production. We
Selection and kinematic cuts for the three final states considered in the analysis.
bbγγ
NðbÞ > 1
NðγÞ > 1
pT ðbÞ > 45ð20Þ GeV
jηðbÞj < 2.5
jηðγÞj < 2.5
120 GeV < MðγγÞ < 130 GeV
bbττ
bbbb
NðbÞ > 1
NðτÞ > 1
pT ðbÞ > 45ð20Þ GeV
jηðbÞj < 2.5
jηðτÞj < 2.5
NðbÞ > 3
115010-10
pT ðbÞ > 40 GeV
jηðbÞj < 2.5
DECONSTRUCTING SQUARK CONTRIBUTIONS TO
know that both the top Yukawa coupling and the Higgs
trilinear coupling must be close to the SM values, so
deviations can mainly arise from a light stop around the
threshold of 2mt̃ and the interference effects between squark
and SM diagrams. This is not the case in nonminimal
Supersymmetric models, as an extended Higgs sector can
lead to deviations from the SM in the Higgs trilinear
coupling. The top Yukawa is more constrained experimentally as the tt̄h production rate depends on it [45].
The squark contribution arises mainly from the diagrams
involving trilinear Higgs-stop-stop couplings. The trilinear
coupling at tree-level is proportional to Xt ¼ At − μ cot β.
Large stop mixing is needed to get a 125 GeV mass for the
SM-like Higgs in the MSSM, so the stop contribution is
always relatively large in the MSSM. Large stop mixing
also enhances the mass splitting between the stops, so one
of the stops needs to be heavy so it will not contribute to the
threshold excess.
In the NMSSM one can achieve the 125 GeV Higgs mass
already at tree-level, so one may try a setup with Xt ≃ 0,
which would allow for two light stops, both being insensitive to searches targeting missing transverse momentum.
The requirement of mh ¼ 125 GeV requires low tan β and
large λ so the term jλj2 jH u Hd j2 leads to an enhancement of
the triple Higgs coupling. A larger Higgs trilinear coupling
can lead to a first order EW phase transition. In the EFT
limit, where all BSM particles are decoupled, a 50%
enhancement is needed for a first order phase transition
[46]. Additional light degrees of freedom, such as light
neutralinos [47], could change the needed enhancement.
Nevertheless, an enhancement up to 100% is possible in the
NMSSM [24] so it is natural to ask how well could we
distinguish such a case.
In the left panels of Fig. 9 we show a case with two stops
having masses of approximately 600 GeV, tan β ¼ 1.35,
λ ¼ 0.64, the trilinear Higgs coupling being 50% larger
than in the SM, trilinear Higgs-stop couplings being
significantly smaller than those of the MSSM and the
top Yukawa being close to the SM value, κ t ≃ 0.985. We
see that there is a deficit of events at low M hh : this is due to
the enhanced trilinear Higgs coupling. In addition, the
excess at the squark threshold has become smaller than the
uncertainties4 (while still being around twice the SM rate),
which emphasizes the significance of the bubble and
triangle diagrams.
In the right panel of Fig. 9 we have a “MSSM-like” case
in the NMSSM. The stop masses and mixings are similar to
those of the MSSM, but we have tan β ¼ 1.38 and
λ ¼ 0.69, which again lead to a significant deviation from
the SM in the trilinear Higgs coupling, being about 1.6
times the SM value. This results in a deficit of events at low
4
We remark, however, that the uncertainties in the plots are at
LO and therefore bound to significantly reduce once higherorders corrections are taken into account.
DI-HIGGS
…
PHYS. REV. D 107, 115010 (2023)
Mhh and an excess of events at high M hh , the intermediate
range being SM-like due to the cancellation of the various
BSM amplitudes. As the bbγγ channel is sensitive to low
invariant masses and the 4b channel is sensitive to high
invariant masses, we should see a deficit in the former and
an excess in the latter.
If such a case were interpreted just through the modification of SM couplings, κt and κλ , the results between
different channels would show an inconsistency, which
implies that the model is not sufficient to describe the
physics. Such an effective description can be somewhat
misleading, unless a complete EFT basis is used, even in the
low Mhh region as the interference between the squark
diagrams with the SM ones can lead to a 30% deviation in the
event rate as our MSSM benchmark (with essentially
κt ¼ κ λ ¼ 1) shows. In the case of a large coupling deviation
the κ framework leads to a better estimate, though even there
we would have some uncertainty from the nondecoupling
squark effects (difference between green (MB) and red
(signal) curves in the upper right panel of Fig. 9).
In the lowest panels of Fig. 9 the ratios of the event rate
compared to the SM are shown. The enhanced trilinear
Higgs couplings lead to a deficit of about 60%–70% in the
lowest mass bins. Such a precision would be needed to
show that a first order EW phase transition is possible
without additional light degrees of freedom.
V. PROSPECTS AT THE HL-LHC
To estimate the prospects for model exclusion/discovery
around the 1 TeV region one can, e.g., use information from
recent searches for resonant di-Higgs production at the
LHC. One such example is an ATLAS search
pffiffiin
ffi the bb̄bb̄
final state using 139 fb−1 of data collected at s ¼ 13 TeV
[48]. In the Mhh mass range 1–1.2 TeV ATLAS expects
Oð10Þ signal events over a background of O(100) events
resulting in an excluded cross section around 10 pb. The
signal acceptance times efficiency is around 10% given a
fiducial preselection. Comparing to the particle level
preselected yields in Fig. 8 it seems not possible to make
any exclusion of the benchmark point using only current
LHC Run 2 data. However, with 20 times the data expected
from HL-LHC as shown in Fig. 11, compared to the
ATLAS search example, the situation for exclusion looks
much more promising.
This is of course true for the MSSM benchmark point of
Table II, which has been selected due to its high cross
section. It is important to stress that the cross section
strongly depends on the stop masses, as can be seen in the
central panel of Fig. 2, and that when the mass of the
lightest stop approaches 1 TeV, the MSSM cross section
collapses to values similar to the SM case. The threshold
effects corresponding to 2mt̃1 in the di-Higgs invariant mass
distribution, however, can be seen also when the stop mass
approaches 1 TeV: the excess on the stop threshold is in fact
be compensated by a smooth and continuous depletion at
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FIG. 9. The invariant mass distributions for the two NMSSM benchmarks described in the text. The integrated luminosity is in this
case 3000 fb−1 , corresponding to the nominal reach of the HL-LHC phase. The left panels correspond to a case with two light stops with
minimal mixing, while the right panels have a light and a heavy stop. Both have a trilinear Higgs coupling that is larger than that
of the SM.
FIG. 10. Invariant mass distributions for 3000 fb−1 and for
different MSSM benchmark points characterized by different t̃1
masses, from 600 GeV to 1 TeV, and mt̃2 ¼ 1600 GeV.
smaller invariant mass values, with higher event counts
than the peak, leading to a total cross section which is
similar to the SM one. This can be seen in Fig. 10 for
different representative MSSM benchmark points characterized by the same value of mt̃2 and different values of mt̃1 ,
from 600 GeV to 1000 TeV (couplings are not reported as
not relevant in this context). For mt̃1 ≳ 800 GeV the
number of expected events is however already small (some
units) at parton level, and therefore the actual observability
of higher masses at HL-LHC is rather challenging, if just
not possible.
The NMSSM benchmarks also introduce large effects at
low invariant Mhh mass. If one focuses only on the MðhhÞ
region below the stop mass where an EFT descriptions is
valid, the NP contributions are dominated by the modified
Higgs triple gauge coupling interfering with the rest of the
SM background which is visible in Fig. 9 (labeled MB).
This effect is measured in the experiments using coupling
modifiers (κ-framework) or more consistently with an EFT,
115010-12
DECONSTRUCTING SQUARK CONTRIBUTIONS TO
DI-HIGGS
…
PHYS. REV. D 107, 115010 (2023)
FIG. 11. Invariant mass distributions for the MSSM benchmark point and for 3000 fb−1. The top left panel shows the distribution
before Higgs decays, the other panels show the distributions for the 3 final states described in Sec. III B 3 and after application of the cuts
of Table IV.
and it can, as previously mentioned, have important
cosmological implications. The current estimated HLLHC 95% exclusion limits for κ λ using e.g. the ATLAS
experiment is [0, 2.5] [44], while the NMSSM benchmarks
have κ λ ¼ 1.6. This indicates that the low mass effects
might be detectable during HL-LHC with more accurate
theory modeling such as an EFT or techniques outlined in
this paper, reduced experimental systematic uncertainties
and combinations across different experiments.
Note that the sensitivity estimates made in this section do
not include any di-Higgs signal corrections to account for
missing higher order contributions (K-factors). For NP
processes these are estimated to be in the range K ≃
1.6–2.4 [49].
VI. CONCLUSIONS
In summary, we have provided a proof-of-concept that
nonresonant di-Higgs production at the LHC (involving the
SM-like Higgs boson already discovered) can be used as a
proxy to viable models of the EW scale, such as SUSY, via
loop effects induced by low-mass (i.e., nondecoupled) stops
that can appear through both an increase (or decrease) of the
inclusive (integrated) rates and significant shape changes in
exclusive (differential) distributions of the gg → hh process.
Chiefly, among the latter, the invariant mass of the Higgs
boson pair may display a large enhancement or depletion just
above the 2mh threshold (where the production rates are
maximal) and a local maximum when mhh ≈ 2mt̃1 (in
correspondence to a loop threshold involving the lightest
stop state), both of which can be used for diagnostic purposes
of the underlying EWSB scenario, separately or simultaneously depending on the benchmark point being targeted.
While near the 2mt threshold the effects are more spread out,
the event rate for gg → hh can be enhanced by up to an order
of magnitude compared to that of the SM at the 2mt̃ threshold.
Quite remarkably, this can happen in the absence of stop
cascade signals at the LHC, owing to compressed sparticle
mass spectra which can easily be obtained, in compliance
with current exclusion limits, in two popular SUSY
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PHYS. REV. D 107, 115010 (2023)
realizations, minimal and nonminimal, like the MSSM and
NMSSM, respectively, which we have adopted here. So
that, in the end, the effects investigated in this paper could
well be the first evidence of SUSY (at least of the squark
sector of it) at the LHC during the HL-LHC run.
(Unfortunately, Runs 2 and 3 cannot afford one with
significant sensitivity in this respect.)
In order to prove this, we have resorted to an exact
computational framework implementing a simplified
model approach that, on the one hand, can easily be
translated into any fundamental theory responsible for
EWSB dynamics and, on the other hand, dispenses with
the necessity of invoking any EFT limit of it, as it can
compute (event level) integrated and differential rates for
essentially any BSM spectrum of masses and couplings. In
fact, the framework relies on a deconstruction method
which makes it flexible to study a wide range of scenarios
beyond those described here.
In this paper, we have shown all this being true while
maintaining the hh pair strictly on-shell. However, we have
also argued that such stop loop effects might persist after
backgrounds (both irreducible and reducible) are accounted
for in the most popular channels leading to di-Higgs
detection, following a reconstruction-level MC analysis
that we have performed. This might be the case even
considering both statistic and systematic uncertainties on
the signal, which in our analysis are relatively large due to
its LO nature. Among these, upon an initial assessment
based on existing literature, the 4b channel is likely to be
the one affording an actual experimental study with the best
sensitivity (above and beyond bb̄γγ and bb̄τþ τ− ), so long
that the QCD-induced background can be brought under
control. We postpone to a future paper the detailed detector
level MC analysis of these final states, including dedicated
selections targeting the described kinematic features.
ACKNOWLEDGMENTS
This work would not have been possible without
financing from the Knut and Alice Wallenberg
Foundation under the Grant No. KAW 2017.0100, which
supported L. P., J. S. and (partially) S. M. within the SHIFT
project. S. M. is also supported in part through the NExT
Institute and the STFC Consolidated Grant No. ST/
L000296/1. H. W. is supported by Carl Trygger
Foundation under Grant No. CTS18:164. L. P. acknowledges the use of the Fysikum HPC Cluster at Stockholm
University. All authors thank Arnaud Ferrari, Venugopal
Ellajosyula, José Eliel Camargo-Molina and Rikard Enberg
for discussions.
APPENDIX A: SIMULATION SYNTAX IN
MG5_AMC FOR DI-HIGGS WITH THE
DECONSTRUCTION METHOD
In this appendix we describe how to obtain the various
terms of Table III using the UFO model in https://hepmdb
.soton.ac.uk/hepmdb:0223.0337. All our simulations have
been made using MG5_AMC version 3.4.1.
The UFO model contains 4 colored scalars, named sq1,
sq2, sq3 and sq4. Their trilinear couplings with the
Higgs boson are proportional to the dimensionless parameters KHSQ1SQ1, KHSQ1SQ2..., which multiply a
VEV factor, according to Eq. (9). Each coupling is
associated to a specific coupling order HSQ1SQ1,
HSQ1SQ2... which can be invoked when performing
simulations in MG5_aMC to restrict the number of possible
topologies. The quadrilinear couplings between the squarks
and the Higgs boson are proportional to the dimensionless
parameters KHHSQ1SQ1, KHHSQ1SQ2..., according to
Eq. (9). Also for the quadrilinears, specific coupling orders
have been defined, HHSQ1SQ1, HHSQ1SQ2..., which
allow to select or remove these couplings. Finally, the
modifications to the SM hhh, htt and hbb couplings are
labeled as KHHH, KHTT and KHBB respectively, with
coupling orders HHH, HTTMOD and HBBMOD respectively.
The convention for these couplings is again the same
of Eq. (9).
In order to reproduce the results of this paper, the model
has to be imported in MG5_AMC with a restriction which
removes sq3, sq4 and their corresponding interactions.
While not strictly necessary, the restriction greatly speeds up the calculations by ignoring irrelevant interactions and
propagators. Alternatively it would be enough to set all coupling orders associated to sq3 and sq4 to zero in the topologygeneration phase, or set all the numerical values of their couplings to zero when generating events. The baseline syntax to
generate di-Higgs is completely analogous to what one does in the SM:
However, this syntax alone is not sufficient (and MG5_aMC would raise an error message), as the other couplings orders of
the model have to be specified. In our analysis we want to isolate specific topologies to apply the deconstruction method: a
careful tuning of the coupling orders allow us to do it in an efficient and unambiguous way.
115010-14
DECONSTRUCTING SQUARK CONTRIBUTIONS TO
DI-HIGGS
…
PHYS. REV. D 107, 115010 (2023)
The SM irreducible di-Higgs background can be obtained in different ways, either by adding to the baseline syntax all
coupling orders associated to NP set to 0:
or by removing all squarks from propagation and setting only the SM modified coupling orders to 0:
The signal and interferences can then be simulated using different combinations of the coupling orders. One example for
each component of Eq. (10) should be sufficient to describe the whole procedure.
M: κ2hhh σ̂ 1
22 int
S: κ11
ht̃ t̃ κ ht̃ t̃ σ̂ 5i ðmt̃1;2 Þ
MB: κ htt σ̂ int
2B
int
SB: κ 11
ht̃ t̃ σ̂ 5B ðmt̃1 Þ
MM: κ2hhh κ htt σ̂ int
1;3
12 2 int
SS: κhhh κ 11
ht̃ t̃ ðκ ht̃ t̃ Þ σ̂ 6;7o ðmt̃1;2 Þ
int
MS: κhhh κ htt κ 11
hht̃ t̃ σ̂ 3;8 ðmt1 Þ
2 int
MSB: κ hhh ðκ 11
ht̃ t̃ Þ σ̂ 1;7d−5;6 ðmt̃1 Þ
APPENDIX B: TRILINEAR HIGGS COUPLING IN
THE MSSM AND THE NMSSM
In the SM the Higgs potential is particularly simple. The
Higgs self-interactions arise solely from the term λjHj4 . Since
λ also determines the Higgs mass, we get a unique prediction
λSM ¼
g2 m2h
:
8m2W
ðB1Þ
In the MSSM it is easier to achieve the 125 GeV mass in
the large tan β regime. In such a case the SM-like Higgs in
the alignment limit is practically the neutral component of
the Hu doublet. Thus the only relevant term of the loopcorrected scalar potential that is relevant for the SM-like
Higgs mass generation is of the form λeff jHu j4 and hence
necessarily λeff ≃ λSM in the MSSM. This is why you never
get large deviations in the quartic or trilinear Higgs
coupling in the MSSM.
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MORETTI, PANIZZI, SJÖLIN, and WALTARI
PHYS. REV. D 107, 115010 (2023)
When the mass matrix is computed, the contribution of
the bilinear term comes in a form that leaves no contribution to the SM-like Higgs mass, just like the soft SUSY
breaking Bμ Hu Hd term does not contribute to the Higgs
mass in the MSSM. Terms of the form of jH0u H0d j2 do not
contribute to the diagonal elements of the mass matrix as
they get eliminated by the tadpole equations. Hence we get
two types of contributions to the mass matrix that affect the
SM-like Higgs mass: diagonal elements from jH 0u;d j4 ,
which come with a factor (from differentiating the potential) of 4 × 3 and off-diagonal elements from jH 0u H 0d j2 ,
which come with a factor of 2 × 2, in addition there are
factors depending on β. As we typically have tan β > 1, the
most important contributions are those from jH 0u j4 and
jH0u H0d j2 , which have relative weights of sin2 β and cos2 β,
respectively. Hence any effect from the jλj2 term gets
suppressed as tan β increases.
In the trilinear Higgs coupling the relative factors are
different, one gets a factor of 4 for the jH0u j4 term and two
contributions with factors of 2 from jH 0u H 0d j2 , again the
former having a factor of sin2 β and the latter having a factor
cos2 β. Therefore the relative contribution of the jH0u H0d j2
term to the trilinear Higgs self interaction is larger than to its
mass. Hence if λ is larger than the gauge couplings, it will
enhance the Higgs trilinear from its SM value and if it is
smaller, it will reduce it. However, this effect is suppressed
by cos2 β, so you can only have a significant deviation in the
region 1 ≲ tan β ≲ 3. Obviously, the precise computation of
the trilinear Higgs coupling will need the loop corrections to
the scalar potential (B2), but this tree-level discussion should
allow us to understand the results.
The enhancement is relatively easy to combine with any
stop masses, as the tree-level contribution reduces the need
of large loop corrections, so you can always find a suitable
level of stop mixing. The parameter space, where the Higgs
trilinear is smaller than in the SM is very narrow. The best
chances are when the singlet-dominated state is lighter than
the SM-like Higgs so that the mixing can push the SM-like
Higgs mass up [50]. Even in that case large loop corrections
are typically needed, so heavy stops and sbottoms are
preferred. In addition, as the singlet is not decoupled, you
cannot neglect the trilinear soft interaction term Aλ Hu Hd S,
so that scenario is more complex than the case where the
trilinear coupling is enhanced.
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In the NMSSM the situation is different. In addition to
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previous argument it is obvious that in the large tan β region
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deviations from the SM value of the Higgs self-coupling.
With tan β close to one the situation is different.
We now assume that the singlet state is decoupled, so
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the Higgs self-coupling. At tree-level the quartic selfcoupling and the Higgs masses arise from the following
terms of the scalar potential
VðH0u ; H0d Þ ¼
g2 þ g02
1
jjH0u j2 − jH0d j2 j2 þ jλj2 jH0u H 0d j2
4
8
2
v
v
þ Aλ psffiffiffi þ κλ s H0u H0d …
ðB2Þ
2
2
115010-16
DECONSTRUCTING SQUARK CONTRIBUTIONS TO
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115010-17
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W3107447193.txt | Lingu@ Nostr@ - Revista Virtual de Estudos de Gramática e Linguística
ISSN 2317-2320
PROCESSO DE ALFABETIZAÇÃO:
AS RELAÇÕES ENTRE POLÍTICAS E PRÁTICAS DO/NO MUNICÍPIO
DE VITÓRIA DA CONQUISTA/BA (2002-2012)
LITERACY PROCESS:
THE RELATIONSHIP BETWEEN POLICIES AND PRACTICES IN / IN THE
MUNICIPALITY OF VITÓRIA DA CONQUISTA / BA (2002-2012)
Claudionor Alves da Silva1
Resumo: O início das reflexões sobre o processo de alfabetização se dá a partir das inquietações que surgiram
com o constante contato com as professoras alfabetizadoras da rede municipal de ensino de Vitória da Conquista,
desde o ano de 2004. Esse contato se deu por meio do desenvolvimento de oficinas de formação continuada e
das atividades curriculares desenvolvidas nas disciplinas de Metodologia da Alfabetização e de Prática de ensino
do curso de Pedagogia da Universidade Estadual do Sudoeste da Bahia. O constante contato com essas
professoras, a escuta atenta de seus relatos sobre as práticas pedagógicas foi motivador para o desenvolvimento
de uma investigação intitulada “Processo de alfabetização escolar: concepções de alfabetização e práticas
alfabetizadoras de professoras da rede municipal de Vitória da Conquista, Bahia, Brasil”, na qual se insere este
trabalho. Entre as perguntas que deram origem a esta investigação, destaca-se: Quais são as concepções de
alfabetização das professoras alfabetizadoras e sua relação com os referenciais teóricos-metodológicos que
fundamentam suas práticas pedagógicas? Daí, o objetivo deste trabalho é apresentar uma análise das relações
entre as políticas e as práticas de alfabetização desenvolvidas na rede municipal de ensino de Vitória da
Conquista, Bahia, entre os anos de 2002 a 2012. Para a construção dos dados, realizou-se entrevistas com vinte
professoras do Ciclo de alfabetização e análise de documentos. Segundo o que se constatou, as políticas e ou
programas implementados entre os anos de 2002 a 2012 pelo sistema municipal de educação de Vitória da
Conquista, embora o tenha feito com a justificativa de melhorar os índices educacionais, em especial na
alfabetização, as preocupações estavam muito mais voltadas para as questões político-burocráticas do que
político-pedagógicas. Como consequência, não houve avanço nas práticas pedagógicas, que permaneceram, até
esse período, mecânicas, cujas atividades de leitura e escrita eram destituídas de sentido.
Palavras-chave: Alfabetização; Políticas de alfabetização; Práticas de alfabetização.
Resumen: El inicio de las reflexiones sobre el proceso de alfabetización se basa en las preocupaciones que
surgieron con el contacto constante con los profesores de alfabetización de la red de educación municipal de
Vitória da Conquista, desde 2004. Este contacto se produjo a través del desarrollo de talleres de educación
continua y las actividades curriculares desarrolladas en las disciplinas de Metodología de Alfabetización y
Práctica Docente del curso de Pedagogía de la Universidad Estatal de Sudoeste de la Bahía. El contacto
constante con estos profesores, la escucha atenta de sus informes sobre prácticas pedagógicas fue motivador para
el desarrollo de una investigación titulada "Proceso de alfabetización escolar: concepciones de alfabetización y
prácticas de alfabetización de profesores de la red municipal de Vitória da Conquista, Bahía, Brasil", en la que se
inserta este trabajo. Entre las preguntas que dieron origen a esta investigación, destacan las siguientes: ¿Cuáles
son las concepciones de alfabetización de los profesores de alfabetización y su relación con las referencias
teórico-metodológicas que apoyan sus prácticas pedagógicas? El objetivo de este trabajo es presentar un análisis
de las relaciones entre las políticas y prácticas de alfabetización desarrolladas en la red de educación municipal
de Vitória da Conquista, Bahía, entre 2002 y 2012. Para la construcción de los datos, se realizaron entrevistas
con veinte profesores del Ciclo de alfabetización y análisis de documentos. De acuerdo con lo encontrado, las
1
Mestre em Ciências da Linguagem; Especialista em Psicopedagogia e professor de Alfabetização da
Universidade Estadual do Sudoeste da Bahia. Endereço eletrônico: profclaudionor@yahoo.com.br.
Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020.
405
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ISSN 2317-2320
políticas y/o programas implementados entre 2002 y 2012 por el sistema educativo municipal de Vitória da
Conquista, aunque lo hizo con la justificación de mejorar los índices educativos, especialmente en la
alfabetización, las preocupaciones se centraron mucho más en cuestiones político-burocráticas que políticopedagógicas. Como consecuencia, no hubo ningún avance en las prácticas pedagógicas, que permanecieron,
hasta este período, mecánicas, cuyas actividades de lectura y escritura no tenían sentido.
Palabras-claves: Alfabetización; Políticas de alfabetización; Prácticas de alfabetización.
1. Introdução
Presencia-se, no Brasil, desde os anos de 1990, uma expansão quantitativa no sistema
educacional brasileiro. Em nome da redemocratização da sociedade, as políticas públicas,
entre elas, as educacionais, têm reconhecido a dívida histórica, principalmente com os
segmentos sociais menos favorecidos. Em nome do pagamento dessa dívida ou parte dela,
ampliou-se o número de vagas e de escolas para o atendimento à população que estava
excluída desse cenário. Nesse movimento, conforme percebe Gatti (2008), houve mudanças
nos segmentos sociais, que passaram a exigir da formação de professores mudanças radicais.
A formação de professores, nessa perspectiva, é vista como uma das variáveis capazes de
incidir na melhoria da qualidade do ensino brasileiro.
Tal cenário foi propício para a implementação de políticas educacionais, motivadas
pela Lei de Diretrizes e Bases da Educação Nacional, no ano de 1996. Daí, surgem novas e ou
se ampliam abordagens teórico-metodológicas, sobretudo para o processo de alfabetização.
Ampliam-se também, a partir desse momento, os estudos e pesquisas voltados para a
alfabetização em função de dois aspectos: primeiro, a ampla divulgação dos estudos acerca da
psicogênese da língua escrita de Ferreiro e Tebersoky, a partir da segunda metade dos anos de
1980, que exerceram influências para a implementação dos Parâmetros Curriculares
Nacionais de Língua Portuguesa e o segundo refere-se aos sucessivos resultados de exames
nacionais e internacionais sobre o desempenho dos estudantes, em leitura e escrita.
Esses aspectos são aqui vistos como contraditórios uma vez que não se verifica que os
avanços dos estudos, pesquisas e implementação de políticas educacionais específicas
contribuíram para a melhoria do desempenho dos estudantes, pois os índices referentes à
leitura e escrita permanecem abaixo do desejado. Conforme os resultados das avaliações, a
média brasileira ainda está abaixo do esperado; a média do Estado da Bahia está abaixo da
média nacional e alguns municípios baianos têm sua média abaixo da média do Estado,
inclusive o de Vitória da Conquista, o terceiro maior do Estado.
Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020.
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ISSN 2317-2320
O início dessas reflexões sobre o processo de alfabetização se dá a partir das
inquietações que surgiram com o constante contato com as professoras alfabetizadoras da rede
municipal de ensino de Vitória da Conquista, desde o ano de 2004. Esse contato começou
com o desenvolvimento de oficinas de formação continuada e das atividades curriculares
desenvolvidas nas disciplinas de Metodologia da Alfabetização e de Prática de ensino do
curso de Pedagogia da Universidade Estadual do Sudoeste da Bahia. Como exigência da
disciplina de Prática de ensino, o professor deveria supervisionar a atividades de regência
desenvolvidas pelos discentes do curso de Pedagogia.
Esse contato possibilitou, sobretudo, o diálogo com as professoras alfabetizadoras,
momento no qual elas tinham a oportunidade de desabafar sobre suas angústias e frustrações
em relação ao trabalho pedagógico que desenvolvem. Nessas oportunidades, elas reclamavam
de um problema crescente nos últimos anos, relacionado às dificuldades enfrentadas no
processo de ensino e aprendizagem da leitura e da escrita. Segundo as professoras, está cada
vez mais difícil alfabetizar seus alunos que, segundo elas, enfrentam muitas dificuldades para
se alfabetizarem.
Isso foi motivador para o desenvolvimento de uma investigação intitulada “Processo
de alfabetização escolar: concepções de alfabetização e práticas alfabetizadoras de professoras
da rede municipal de Vitória da Conquista, Bahia, Brasil”, na qual se insere este trabalho.
Entre as perguntas que deram origem a esta investigação, destaca-se: Quais são as concepções
de alfabetização das professoras alfabetizadoras e sua relação com os referenciais teóricosmetodológicos que fundamentam suas práticas pedagógicas? O objetivo deste trabalho é
apresentar uma análise das relações entre as políticas e as práticas de alfabetização
desenvolvidas na rede municipal de ensino de Vitória da Conquista, Bahia, entre os anos de
2002 a 2012.
A construção dos dados aqui analisados se constituiu pela aplicação de entrevistas
sobre o processo de alfabetização com professoras do Ciclo de alfabetização e análise de
documentos relacionados com a alfabetização proposta pelo sistema municipal de educação.
Foi realizada uma reunião em duas escolas, com todas as professoras das classes de
alfabetização, com o intuito de apresentar a proposta de investigação para que, por adesão, as
professoras participassem, como sujeitos desta investigação. Assim, participaram 20
professoras, sendo 09 de uma escola e 11 de outra.
As entrevistas com as professoras foram realizadas em horários e datas previamente
agendados, conforme a disponibilidade de cada uma delas. As entrevistas foram gravadas em
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áudio, com a permissão de cada uma delas e, em seguida foram transcritas. Para qualificar
ainda mais as informações, elaborou-se um roteiro com perguntas sobre a concepção de
alfabetização, o método que se utiliza para alfabetizar, os marcos teóricos e sua relação com
as práticas de ensino, o trabalho com os gêneros textuais etc.
Segundo o que se constatou, as políticas e ou programas implementados entre os anos
de 2002 a 2012 pelo sistema municipal de educação de Vitória da Conquista, embora o tenha
feito com a justificativa de melhorar os índices educacionais, em especial na alfabetização, as
preocupações estavam muito mais voltadas para as questões político-burocráticas do que
político-pedagógicas. Evidencia-se daí que não houve preocupação por parte do sistema
municipal de ensino em formar os professores, no sentido de muni-los dos referenciais
teórico-metodológicos de cada uma das propostas. Como consequência, não houve avanço
nas práticas pedagógicas.
2. Antecedentes
As políticas públicas educacionais implementadas nas últimas décadas, considerando o
marco institucional da LDB 9394/96, também não foram ou não têm sido suficientes para
garantir o sucesso de todos os alunos, segundo os resultados das avaliações desenvolvidas por
organismos governamentais e não-governamentais. Tais avalições revelam que a maioria dos
estudantes brasileiros tem tido desempenho muito inferior ao esperado. Entre essas políticas
incluem as leis 11.114/05, que tornou o ensino obrigatório a partir dos seis anos de idade e
11.274/06, que estendeu a duração do Ensino Fundamental para nove anos, mantendo o início
aos seis anos.
A implantação do Ensino Fundamental de nove anos também significou, para os
sistemas educacionais, a expansão do Ciclo de alfabetização para três anos, ou seja, os três
primeiros anos do Ensino Fundamental. No entanto, os dados indicam que um número
significativo de crianças não é alfabetizada nesse período. Das crianças matriculadas no
terceiro ano da rede pública brasileira, em 2013, 11,9% foram reprovadas e 19,9% tinham
distorção idade/série (BRASIL, 2014).
A redução da taxa de reprovação no Ciclo de alfabetização, a partir do ano de 2010,
ocorre em função de as Diretrizes Curriculares Nacionais para o Ensino Fundamental de nove
anos não permitirem a retenção de alunos do primeiro e segundo anos. As taxas do fracasso
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registradas nesses anos são provenientes da evasão escolar. Em 2013, a taxa de evasão no
primeiro ano foi de 1,4% e de 3,6% no terceiro ano. (BRASIL, 2013).
Para verificar a qualidade e a eficiência do Ciclo de alfabetização das escolas públicas,
o Ministério da Educação (MEC) criou a Avaliação Nacional de Alfabetização (ANA) pela
Portaria MEC nº 482, de 7 de junho de 2013. De acordo com os termos desta Portaria, em seu
Artigo 4, os objetivos da ANA são: I - avaliar a qualidade, a equidade e a eficiência
(incluindo condições de oferta) do Ciclo de alfabetização das redes públicas; e II - produzir
informações sistemáticas sobre as unidades escolares, para que cada unidade receba o
resultado global.
Os resultados da primeira avaliação, publicada em 2014, denunciam que 54% dos
estudantes do terceiro ano têm desempenho insuficiente em leitura, interpretação de textos e
operações matemáticas básicas. Nessa avaliação, os estudantes tinham de localizar
informação específica, explícita em textos simples. Apenas 11% deles conseguiram oferecer
respostas de forma satisfatória. Entre os 46% dos estudantes que leem e interpretam um texto
de forma adequada se encontram aqueles que estão no nível apropriado e aqueles que estão no
nível desejado.
Os dados apresentados aqui sobre a alfabetização no cenário educacional brasileiro
revelam que o país ainda tem que enfrentar vários desafios para que seus estudantes alcancem
um nível satisfatório em relação à leitura e à escrita. Para Soares (2007) os baixos índices de
desempenho dos alunos no processo de alfabetização resultam de diferentes perspectivas do
processo de alfabetização, de diferentes áreas do conhecimento (psicologia, linguística e
pedagogia), cada uma das quais trata o tema de forma independente. Em segundo lugar, são
dados que, exclusivamente, buscam a explicação do problema ora no aluno, ora no contexto
cultural do aluno, ora no professor, ora no método, ora no material didático, ora finalmente no
próprio meio, o código escrito (a questão das relações entre o sistema fonológico e o sistema
ortográfico da língua portuguesa).
Garantir a aprendizagem da leitura e da escrita a todos os que se matriculam na escola
pública brasileira é um grande desafio. A falta de professores com capacitação técnica é um
dos fatores que impedem essa realização, segundo a posição de Cagliari (2006). Para ele, o
professor alfabetizador, que tem uma boa preparação para o exercício de sua função, sabe
avaliar se um método é bom ou não. Além das escolas de formação professores formarem mal
os professores alfabetizadores, Cagliari (2006), atribui a culpa também aos pacotes
educacionais e à atitude conformista do próprio professor. Como resultado, o professor
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permanece com suas práticas pedagógicas medíocres, que não têm contribuído para a
aprendizagem de forma mais significativa dos alunos.
A discussão sobre o fracasso escolar no Brasil, referente ao aprendizado da leitura e da
escrita, ocorreu em dois momentos: no primeiro momento, até 1996, o discurso girou em
torno do alto índice de reprovação e evasão dos alunos e, no segundo momento, com a
instituição da nova LDB 9394/1996, o discurso gira em torno da não aprendizagem dos
alunos. A partir de então, amplia-se o acesso das crianças brasileiras ao processo de
escolarização, embora carecem de condições de permanência e sucesso escolar. (MORTATTI,
2004).
O município de Vitória da Conquista ficou nacionalmente conhecido, de forma
negativa, por ter tido o pior desempenho educacional, segundo o Índice de Desenvolvimento
da Educação Básica (IDEB), no ano de 2009, quando atingiu apenas 2,8. Esse resultado
denunciou o quanto é baixo o nível de desempenho dos alunos com o mínimo de cinco anos
de escolaridade. Cabe lembrar, no entanto, que os resultados anteriores, a contar de 2005 e
dos posteriores, 2011 e 2013, o município ficou abaixo da meta projetada, embora tenha tido
um resultado mais significativo. Conforme os trabalhos de Soares (1985), Morais (2010),
entre outros, o problema do fracasso escolar tem atingido a maioria das crianças pertencentes
a setores socialmente marginalizados a ponto de muitos não aprenderem a ler e escrever.
A seguir, serão abordadas as políticas e programas educacionais implementados pelo
município de Vitória da Conquista entre os anos de 2002 a 2012 e suas relações com as
práticas pedagógicas. Nessa relação serão discutidos os resultados ou efeitos dessas políticas
para a melhoria ou não dos índices educacionais no município, considerando que todas elas
justificaram o baixo desempenho educacional dos estudantes.
3. Políticas e práticas de alfabetização do/no município de Vitória da Conquista
Os resultados negativos dos alunos, em relação ao seu desempenho em leitura e
escrita, apontados acima, motivaram o município de Vitória da Conquista a organizar, no ano
de 2002, o Ensino Fundamental (1ª a 4ª série) em Ciclos de Aprendizagem: Ciclo I
(correspondendo ao 1ª e 2ª série do Ensino Fundamental e Ciclo II (3ª e 4ª série), mediante a
Resolução Nº 003/02. O ponto básico dessa organização pelo município foi desenvolver o
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currículo com base em competências e habilidades. Por isso, passou-se a exigir uma série de
mudanças no sistema de ensino, especialmente na prática pedagógica dos professores.
O debate acerca do conceito de competência amplia-se, na década de 1990, na área
pedagógica, destinado ao ensino de crianças nos primeiros anos de escolaridade. O
desenvolvimento de habilidades por parte de um estudante, segundo Perrenoud (1999), deve
ser em torno de três anos em cada ciclo escolar. Em 2006, é instituído o Ensino Fundamental
de 9 anos, por meio da Lei N° 11.274, implementando, assim, o Ciclo da alfabetização (do 1º
ao 3º ano).
A organização do ensino em ciclos sofreu diversas críticas por parte dos professores
da rede municipal de ensino, devido à adoção do regime de progressão continuada,
denominada também no município de aprovação automática, proposta por essa organização.
Os fragmentos de fala a seguir, denunciam isso:
“Desde a implantação do Ciclo, a SMED2 não sabe o que fazer com a educação do município. Isso
bagunçou a cabeça do professor e com o aprendizado dos alunos. Ainda hoje sofremos com a
imposição de propostas que, de fato, não conseguimos entender. A avaliação dos alunos foi a pior
parte do Ciclo porque exigia que fosse toda qualitativa. Não tínhamos como ter um conhecimento
assim dos alunos, pois o tempo era curto e tínhamos de ensinar os conteúdos. Antes dessa proposta
de Ciclo, eu sabia alfabetizar meus alunos, pois era muito mais fácil. [...]. O resultado de tudo isso
foi o fracasso dos alunos” (Professora Júlia. Entrevista de 9 de abril de 2014).
“A aprovação automática significou o fim da educação no município. Foi a pior medida tomada pela
Secretaria de Educação. [...] Nunca tínhamos visto nada parecido e não funcionou de forma
significativa. A aprovação automática foi a condição que o município encontrou para resolver o
problema do fracasso. Não houve, a meu ver, uma preocupação com a aprendizagem dos alunos [...]
(Professora Beatriz. Entrevista de 11 de março de 2014).
“A proposta é que os alunos sejam avaliados de forma contínua e cumulativa, com prevalência de
aspectos qualitativos sobre aspectos quantitativos. [...] O problema é que não nos foi orientado a
fazer isso. Tínhamos que preecher um parecer descritivo que, na verdade não condizia com a
realidade do aluno. [...] tive que inventar muito, porque não sabia o que escrever sobre os alunos.
Era uma opinião individual”. (Professora Ana. Entrevista de 11 de março de 2014).
“[...] A avaliação era exigida de uma maneira que os professores não sabiam como fazer e não
fomos orientados para tal. [...]. Todos os alunos do 1º e 2º anos eram aprovados, sabendo ou não”
(Professora Fábia. Entrevista de 05 de abril de 2014).
Evidencia-se, por meio desses fragmentos de fala, que não houve participação dos
professores nesse processo de mudança de abordagem, ou seja, a proposta foi imposta pelo
sistema municipal de ensino. Evidencia-se ainda que a preparação dos professores não tenha
acorrido para a implementação dessa proposta, o que pode justificar a carência dos
professores em relação à adoção de novas metodologias, pois não tinham conhecimento dos
2 Secretaria Municipal de Educação.
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fundamentos teóricos da proposta instituída. A denúncia apresentada por meio do fragmento
de fala da professora Beatriz, a seguir, parece demonstrar o objetivo da gestão municipal:
“A aprovação automática foi a condição que o município encontrou para resolver o problema do
fracasso. Não houve, a meu ver, uma preocupação com a aprendizagem dos alunos [...]”.
(Professora Beatriz. Entrevista de 11 de março de 2014).
Nessa perspectiva, concorda-se com Jacomini (2004), ao analisar a institucionalização
do Ciclo na rede de ensino do Estado de São Paulo. Ela aponta três problemas que foram
evidenciados pelos professores participantes desta investigação: 1) elaboração de novas
propostas sem avaliar os resultados das políticas que as antecederam; 2) ausência de discussão
com os professores; 3) e, o não oferecimento das condições materiais e organizacionais para
que as propostas sejam implementadas a contento.
As reformas escolares fracassam, novos programas não são aplicados, belas ideias são
pregadas como métodos ativos, construtivismo, avaliação formativa ou pedagogia
diferenciada, mas nunca são praticadas. Isso porque, na área da educação, o desvio
astronômico entre o que é prescrito e o que é praticado nas condições efetivas do trabalho
docente não é medido suficientemente. (PERRENOUD, 2007). Isso também foi evidenciado
por meio dos fragmentos de fala das professoras, como os seguintes:
“Nos últimos anos, a SMED tem exigido muito de nós. O maior problema é que ela não tem dado as
condições adequadas. Ela quer que ensinamos de outra maneira, mas nós não sabemos como [...]”.
(Professora Carla, Entrevista de 19 de março de 2014).
“[...] do que adianta estar tudo perfeito no papel, mas na prática não funciona. Baixa as portarias,
mas nós professores não temos esse conhecimento, não sabemos como se operacionaliza isso. Nós
reclamamos toda reunião, mas não nos escuta, não nos orienta, mas as cobranças vêm. [...] Parece
até uma perseguição a nós professores. (Professora Ana. Entrevista de 11 de março de 2014).
A resistência por parte dos professores da rede municipal aumentava a cada ano em
relação à organização do currículo em Ciclos de Aprendizagem. Tal resistência levou a
Secretaria Municipal de Educação a declinar da ideia da expansão da organização do currículo
em Ciclos de Aprendizagem para a segunda etapa do Ensino Fundamental. No entanto, por
meio da Resolução nº 009/06, a rede municipal de ensino institui a organização do currículo
em Ciclos de Formação Humana, revogando, assim, a Resolução nº 003/2002. Por meio dessa
Resolução, o Ensino Fundamental foi instituído em três cilos, cada um com duração de três
anos.
Dentre os objetivos do Ciclo de Formação Humana, destacam-se: o estabelecimento
do processo de formação humana, focado no pleno desenvolvimento do aluno, sua preparação
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para o exercício da cidadania; o desenvolvimento da capacidade de aprender, tendo como
meio básico o pleno domínio da leitura, escrita e cálculo; o desenvolvimento da capacidade de
aprendizagem, levando em consideração a aquisição de competências e habilidades, bem
como a formação de atitudes e valores; a continuidade do processo de aprendizagem de
crianças, pré-adolescentes e adolescentes que ingressam no sistema escolar. (SMED, 2008). A
organização do Ensino Fundamental em Ciclos de Formação Humana deu continuidade ao
regime de Progressão Continuada nos mesmos moldes do Ciclo de Aprendizagem.
O Ciclo I corresponde ao período da infância, que inclui alunos de seis a oito anos.
Nas classes desse Ciclo deve-se incluir a matrícula de novos alunos, transferidos de outro
regime e daqueles que não tiverem comprovação de escolaridade anterior. Com essa medida,
a rede municipal de ensino registra a presença de um número significativo de crianças com
distorção idade/ciclo, além de evasão e reprovação. Esses casos geralmente estão intimamente
relacionados à situação socioeconômica dos alunos, uma vez que o fracasso escolar tem
atingido principalmente os alunos pertencentes aos contextos desfavoráveis, economicamente.
A avaliação, de acordo com a organização do Ciclo de Formação Humana, é
diagnóstica, contínua, processual, participativa e emancipatória, e sua concepção deve ser
construída de forma a caracterizar as observações das competências propostas. Nesse sentido,
são desenvolvidas ações de reorientação para alunos com dificuldades de aprendizagem e
apoio processual em relação ao desenvolvimento do aluno. Predomina, portanto, nesse
processo de avaliação os aspectos qualitativos sobre os quantitativos. (SMED, 2008). No
entanto, os professores não aprovaram a nova proposta implementada pelo sistema municipal
de ensino, conforme os fragmentos de fala apresentados a seguir:
“No Ciclo não tem sentido avaliar os alunos, pois não pode haver reprovação. Passa os alunos de
qualquer jeito [...]. Avaliar acaba sendo perda de tempo”. (Professora Beatriz. Entrevista de 11 de
março de 2014).
“[...] Os alunos, sabendo que não serão reprovados, nem demonstram interesse pelos conteúdos
porque serão aprovados de todo modo”. (Professora Elisa. Entrevista de 17 de março de 2014).
De acordo com esses fragmentos de fala, evidencia-se que a avaliação seja reduzida à
verificação da aprendizagem. Evidenciou-se ainda que as dificuldades apresentadas pelas
professoras, em relação ao trabalho precário em sala de aula, estava relacionado à não
existência de um projeto consolidado de formação continuada dos professores. As professoras
continuam reivindicando a sua participação na elaboração e implementação das propostas
pedagógicas. Segundo Arroyo (2011), é necessário avaliar o que os professores consideram
mais importante em suas práticas para que uma proposta seja apresentada como inovadora. Na
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mesma direção, Masetto (2011) acredita que um projeto, que pretende ser inovador, deve se
concentrar na prática pedagógica do professor.
Em 2009, como já afirmado, o município teve o seu pior Ideb e ficou entre os piores
do Brasil, reafirmando, assim, que os investimentos feitos de 2004 até esse momento, não
trouxeram resultados positivos. A solução apresentada pelo municío foi revogar a Resolução
nº 009/06, ao reorganizar o Ensino Fundamental de nove anos, por meio da Resolução nº
002/09, em Ciclos de Aprendizagem, assim divididos: o Ciclo I, do primeiro ao terceiro ano,
denominado ciclo de alfabetização; Ciclo II, quarto e quinto ano; Ciclo III, sexto e sétimo ano
e Ciclo IV, oitavo e nono anos.
Identificou-se que as diretrizes propostas por essa Resolução, em sua essência, são
textualmente as mesmas da Resolução nº 009/06. A diferença identificada entre essas
resoluções é em relação à progressão continuada: a Resolução de 002/2009 prevê a aprovação
automática apenas no ciclo I, enquanto a Resolução de 009/2006 previa a aprovação
automática em todos os anos de cada Ciclo. Cabe lembrar que a aprovação automática, nos
dois primeiros anos de escolaridade, está prevista nas Diretrizes Nacionais do Ensino
Fundamental, não sendo, portanto, prerrogativa da rede municipal de ensino de Vitória da
Conquista.
A Resolução nº 002/09, semelhante à Resolução nº 009/2006, parece apresentar
equívocos quanto à concepção de um sistema de ensino organizado em Ciclos de
aprendizagem. A questão da avaliação é um dos equívocos mais evidentes: embora o conceito
e critérios de avaliação das duas resoluções sejam os mesmos, a 002/09 exclui a emissão de
parecer descritivo sobre o desempenho acadêmico de cada aluno e não aborda sobre aspectos
qualitativos, o que prevalecem os aspectos quantitativos, exatamente como era no sistema de
seriação. Sobre essa questão, os professores assim afirmaram:
“Pelo menos a avaliação voltou ao normal. Não somos obrigadas a arovar de qualquer jeito os
alunos [...]”. (Professora Julia. Entrevista de 09 de abril de 2014).
“Com essa nova proposta, a escola fica livre para decidir como avaliar seus alunos. Não ter que
fazer parecer para cada aluno já é uma avanço. [...]”. (Professora Fábia. Entrevista de 05 de abril de
2014).
“Os pareceres descritivos de cada aluno era uma grande mentira. a gente copiava [...] Aquilo não
representava a verdade sobre os alunos. Era uma perda de tempo fazer aquilo e nao servia para nada
pois ninguém lia”. (Professora Elisa. Entrevista de 17 de março de 2014).
A adoção dessa postura em relação à avaliação parece ter sido agradável aos
professores, conforme os fragmentos de fala acima. Pelo menos em relação à avaliação, podese afirmar que essa proposta, que utiliza uma terminologia de Ciclos de Aprendizagem, mas
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não assumem seus fundamentos teórico-práticos. Segundo Mainardes (2009), a organização
do currículo escolar em ciclos é uma política complexa que possui justificativas filosóficas,
políticas, psicológicas, antropológicas e sociológicas.
No caso do município de Vitória da Conquista, evidencia-se, conforme suas
justificativas, que a organização do currículo em Ciclos teve a intenção apenas de reduzir as
altas taxas de reprovação e evasão escolar, para amenizar o fracasso escolar. Conforme os
depoimentos das professoras, constata-se que a rede municipal de ensino não tenha assumido
os fundamentos teórico-práticos, de nenhuma das propostas adotadas, o que permite dizer que
o sistema de ensino municipal não tenha desenvolvido práticas pedagógicas fundamentadas
no modelo teórico adotado.
Os resultados das avaliações divulgados no ano de 2011, apontam que o Ideb
observado, da primeira etapa do Ensino Fundamental salta de 2,8 para 3,4. Embora o
município tenha registrado esse avanço, continua abaixo da meta projetada (4,0). Nesse ano, o
município adere ao Programa Pacto de Alfabetização com o Município, lançado pelo Governo
do Estado da Bahia. Entre as ações desse Programa destacam-se a formação contínua dos
professores alfabetizadores; monitoramento e avaliação; distribuição de materiais didáticos
para professores e alunos; oferta de reforço escolar e organização de espaços de leitura nas
escolas, preferencialmente em cada sala de aula. Embora a adesão a esse Programa tenha sido
em 2011, a sua operacionalização só ocorreu a partir do ano seguinte, quando o município
também adere ao Programa Nacional de Alfabetização na Idade Certa [PNAIC ], lançado pelo
governo federal.
O PNAIC se configura como um compromisso formal assumido pelo governo federal,
pelo Distrito Federal, pelos estados e municípios, com a intenção de garantir que todas as
crianças sejam alfabetizadas até os oito anos de idade. A adesão ao PNAIC implica
compromisso com a alfabetização de todas as crianças em língua portuguesa e em
matemática; realização das avaliações anuais universais, aplicadas pelo INEP, entre elas a
ANA, com os alunos 3º ano do Ciclo da alfabetização.
De acordo com o PNAIC, ser alfabetizado aos oito anos de idade significa
compreender como o sistema de escrita funciona; o domínio das correspondências grafemasfonemas; a fluência da leitura e domínio das estratégias de compreensão e produção de textos
escritos. Para tal, as ações do PNAIC se baseiam em quatro linhas de ação: 1. Formação
presencial para professores de alfabetização e seus orientadores de estudos; 2. Material
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didático, obras literárias, trabalhos de apoio educacional, jogos e tecnologias educacionais; 3.
Avaliações sistemáticas e 4. Gestão, mobilização e controle social. (BRASIL, 2012).
A formação continuada dos professores é a principal atividade do PNAIC que,
segundo Klein e Guizzo (2017), se converteu em um instrumento importante para a busca de
melhorias da qualidade da educação básica, afim de satisfazer as novas demandas por meio de
cursos de capacitação para que os professores atualizem suas práticas pedagógicas.
Intensificar a formação continuada dos professores fortalece os vínculos entre a teoria e a
prática.
As professoras consideram fundamental a formação continuada e concordam que é por
meio dela que pode ocorrer o domínio dos referenciais teórico-metodológicos para se
apropriarem dos novos conceitos e, consequentemente, desenvolver práticas mais
significativas. No entanto, elas reclamam que é insuficiente a formação que o sistema de
ensino tem oferecido.
“Para nós, professoras, é muito difícil mudar a nossa prática. Temos uma prática cristalizada,
baseada em nossas crenças. Não podemos inovar só com esses cursos de capacitação que a SMED
oferece. Tinha de ter uma formação continuada mais sólida”. (Professora Beatriz. Entrevista de 19
de março de 2014).
“Me esforço para fazer o melhor que posso, porém […]a SMED não ajuda muito com a formação
que oferece. Muitas vezes, considero perda de tempo porque é sempre a mesma coisa e não ensina
nada de novo. Nem pode exigir mudanças de nossa prática porque o que ensina nessa formação já é
antigo”. (Professora Iris. Entrevista de 08 de abril de 2014).
A partir das narrativas das professoras, pode-se constatar que o sistema municipal de
educação instituiu propostas pedagógicas com fins mais políticos que pedagógicos,
acreditando resolver o problema do fracasso escolar. Por meio das narrativas se verificou
também que as professoras foram resistentes às propostas, primeiro por não terem participado
da construção de nenhumas delas e, segundo por não terem tido conhecimento dos seus
referenciais teóricos, o que as impede, de algum modo, de desenvolver novas práticas ou
práticas orientadas por tais propostas. Fica evidente, desse modo, que a descontinuidade das
propostas pedagógicas não foi favorável para a melhoria dos índices educacionais dos alunos.
A análise realizada a partir de documentos e entrevistas sobre as relações entre as
políticas e práticas pedagógicas desenvolvidas pelas professoras das classes de alfabetização
na rede municipal de ensino de Vitória da Conquista e os referenciais teórico-metodológicos
aponta para: a) uma preocupação mais com aspectos políticos do que com questões
pedagógicas por parte do sistema de educação; b) o desconhecimento ou não domínio dos
referenciais teórico-metodológicos das propostas pedagógicas implementadas pelo sistema
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municipal de educação e c) as práticas pedagógicas de alfabetização contraditórias à proposta
vigente, ou seja, a permanência de práticas mecânicas de ensino da leitura e da escrita.
De 2002 a 2012, o sistema de ensino municipal foi marcado, conforme o que se
verificou no contexto desta investigação, por propostas pedagógicas descontínuas cuja
preocupação por parte da gestão municipal era mais política que pedagógica. Cada uma
dessas propostas foi implementada com o objetivo de melhorar os índices educacionais,
principalmente no ciclo de alfabetização. No entanto, essas propostas não promoveram as
melhorias esperadas, como evidenciado no contexto dessa análise.
Conclusão
Neste trabalho de investigação buscou analisar as relações entre os referenciais
teórico-metodológicos que fundamentam as políticas educacionais (e as propostas
pedagógicas) implementadas pela rede municipal de ensino de Vitória da Conquista e as
práticas pedagógicas das professoras alfabetizadoras. A alfabetização é aqui vista como um
processo que vai além da simples capacidade mínima de decodificar e codificar a escrita; é
entender e conceber usar os sociais da leitura e da escrita na vida cotidiana e pressupõe que
haja entendimento mínimo para fazer uso dessas capacidades. Assumir essa posição é dar-se
conta de que o processo de construção da linguagem escrita, devido a sua complexidade,
requer muito mais que técnicas ou treino mecânico; e que a capacidade de ler e escrever
depende da compreensão de como funciona a linguagem e como se usa no contexto social.
Segundo Borzone, Silva y Rosemberg (2004), Morais (2012) y Soares (2015), para
que a criança aprenda a linguagem escrita, é necessário o desenvolvimento de habilidades
cognitivas e linguísticas. Esta posição também parte do princípio legal de que as crianças
tenham direito de aprender a ler e escrever, dado que este direito tenha sido negado às
crianças pobres durante muitos anos, no Brasil. É um direito de as crianças não só
ingressarem na escola, como terem acesso a uma educação de qualidade que lhes proporcione
situações mais autônomas, de modo que ao final do chamado ciclo de alfabetização, tenham
desenvolvido habilidades de leitura e produção de textos para continuar p processo
educacional de forma exitosa.
Os dados construídos e analisados nesta investigação revelam aspectos importantes
sobre as relações entre os referenciais teórico-metodológicas e as práticas pedagógicas das
Língu@ Nostr@, Vitória da Conquista, v. 8, n. 1, p. 405-419, jan-julho. 2020.
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professoras alfabetizadoras. Ao abordar esses aspectos, no processo de alfabetização, permitiu
optar por referências teóricas da Linguística, Psicolinguística e Educação, a fim de manter um
diálogo entre elas. Neste sentido, apresenta em seguida algumas conclusões: a) permanece
mecânico o ensino da leitura e da escrita e desvinculado do círculo cultural dos alunos; b) as
atividades de ensino não têm em conta o conhecimento dos alunos nem seu grau de
dificuldade; c) a linguagem utilizada nas atividades de ensino privilegia apenas a padrão,
desenvolvidas como se todos os alunos dominassem a leitura e a escrita e d) carência de
materiais didático-pedagógicos adequados.
Esta investigação, pelo menos no que se refere ao ensino da leitura e da escrita,
denuncia a fragilidade das práticas de alfabetização no interior da escola pública e constata
que as professoras têm muito o que aprender, todavia em relação ao trabalho com o texto, a
leitura, a escrita e a análise linguística. Isto reafirma o quanto tem sido precária a formação
continuada dos professores na rede municipal de ensino de Vitória da Conquista. Espera-se
que a formação continuada de forma mais sólida possa contribuir com a superação do
mecanicismo no processo de alfabetização; oferecer aos alunos, no que se refere à linguagem,
as devidas condições de expressão de si mesmos, de possibilitar a leitura de mundo, de modo
que eles organizem os conhecimentos construídos; promover a interação entre as funções
cognitivas superiores dos alunos como leitores e os conteúdos expressos nos textos entre
outras.
Enfim, cabe ressaltar que a elaboração e implementação de uma proposta pedagógica
somente fará sentido se o objetivo for discutir e repensar as referências teóricas e
metodológicas e o conteúdo das áreas de conhecimento para atualizá-las, levando em
consideração os avanços científicos. O não cumprimento desse objetivo promove a
continuidade das práticas cristalizadas, ou seja, que valorizam o mecanismo do ensino da
leitura e da escrita, apesar de, no Brasil, as teorias construtivistas apoiarem as propostas de
alfabetização.
Referências
ARROYO, M. G. Currículo, território de disputa. Petrópolis, RJ: Vozes, 2011.
BRASIL.
Pacto Nacional
pela Alfabetização na Idade
Certa. Disponível
pacto.mec.gov.br. 2012. Acesso em 05 de jan. de 2015.
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em:
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_____. Saeb - Sistema De Avaliação da Educação Básica. Disponível em: http://www.adurrj.org.br/4poli/gruposadur/gtpe/portaria_482_7_6_13.htm, 2013. Acesso em set. de 2014.
_____. Inep - Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira.
Indicadores Educacionais. Brasília: INEP. Disponível em: http://portal.inep.gov.br, 2014.
Acesso em 10 nov. 2018.
BORZONE, A. M.; SILVA, M. L. & C. ROSEMBERG. Programa de promoción del
desarrollo lingüístico y cognitivo para los jardines de infantes de la provincia de Entre Ríos.
Consejo
General
de
Educación:
gobierno
Entre
Ríos,
2004.
Disponível
em:
https://www.researchgate.net/profile. Acesso em 20 de nov. 2018.
CAGLIARI, L. C. Alfabetização sem ba-be-bi-bo-bu. São Paulo: Scipione, 2006.
JACOMINI, Márcia A.
A escola e os educadores em tempo de ciclos e progressão
continuada: uma análise das experiências no estado de São Paulo Educação e Pesquisa, São
Paulo, v.30, n.3, set./dez. 2004.
KLEIN, J. M & GUIZZO, B. S. Problematizando representações docentes nos Cadernos de
formação do Programa Pacto Nacional pela Alfabetização na Idade Certa (PNAIC).
Disponível em: http://dx.doi.org/10.24109/2176-6681.rbep.98i249.2763. 2017. Acesso em 12
de out. 2018.
MAINARDES, J. A escola em ciclos: fundamentos e debates. São Paulo: Cortez. 2009.
MASETTO, M. T. Competência pedagógica do docente universitário. São Paulo: Summus
Editorial. 2011.
MORAIS, A. G. Psicologia, Educação Escolar e Didáticas Específicas: refletindo sobre o
ensino e a aprendizagem na alfabetização. Trabalho escrito apresentado em concurso para
docente titular no Depto. de Psicologia e Orientação Educacionais da UFPE, Recife. 2010.
_____. Sistema de escrita alfabética. São Paulo: Melhoramentos. 2012.
MORTATTI, M. do R. L. Educação e Letramento. São Paulo: UNESP. 2004.
PERRENOUD, P. Construir as competências desde a escola. Porto Alegre: Artmed. 1999.
_____. A formação dos docentes no século XXI. In: PERRENOUD, P. et al (2007). As
competências para ensinar no século XXI: a formação dos docentes e o desafio da avaliação.
Porto Alegre: Artmed. 2007.
SMED. Plano Municipal de Educação. Vitória da Conquista, Bahia. 2008.
SOARES, M. As muitas facetas da alfabetização. Cad. Pesq., São Paulo. 1985.
_____ . Alfabetização e Letramento: 5ª ed. São Paulo: Contexto. 2007.
_____. Alfabetização no Brasil: a questão dos métodos. São Paulo: Contexto. 2015.
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W2799771798.txt | Revista Brasileira de Zootecnia
Full-length research article
Brazilian Journal of Animal Science
© 2018 Sociedade Brasileira de Zootecnia
ISSN 1806-9290
www.sbz.org.br
R. Bras. Zootec., 47:e20170159, 2018
https://doi.org/10.1590/rbz4720170159
Non-ruminants
The role of yeast culture (Saccharomyces cerevisiae) on performance, egg
yolk fatty acid composition, and fecal microflora of laying hens
Bülent Özsoy1*, Özlem Karadağoğlu2, Akın Yakan3, Kadir Önk4, Elif Çelik5, Tarkan Şahin6
1
Mustafa Kemal University, Veterinary Medicine Faculty, Department of Animal Nutrition and Nutritional Disease, Hatay, Turkey.
Kafkas University, Kars Vocationel High School, Department of Agriculture and Animal Production, Kars, Turkey.
3
Mustafa Kemal University, Veterinary Medicine Faculty, Department of Genetic, Hatay, Turkey.
4
Kafkas University, Veterinary Medicine Faculty, Department of Animal Science, Kars, Turkey.
5
Kafkas University, Veterinary Medicine Faculty, Department of Microbiology, Kars, Turkey.
6
Kafkas University, Veterinary Medicine Faculty, Department of Animal Nutrition and Nutritional Disease, Kars, Turkey.
2
ABSTRACT - This study investigated the effects of different levels (0.05, 0.1, and 0.2%) of yeast culture supplementation
on body weight, feed intake, feed conversion ratio, egg production, egg weight, egg quality traits, egg yolk fatty acid composition,
and microbiological flora in feces. A total of 240 laying hens at 18-19 weeks of age were divided into four groups and fed a
basal diet containing 2750 kcal/kg metabolizable energy and 16% crude protein for 16 weeks. The basal diet was supplemented
with 0.05, 0.1, and 0.2% commercial yeast culture product obtained from Saccharomyces cerevisiae. The different levels of
yeast culture supplementation to the diets did not statistically affect body weight change among the treatments. However, feed
intake was lowest in the group fed 0.2% of yeast culture. The highest egg weights were obtained from the groups fed 0.1 and
0.2% yeast culture, when compared with control group. Regarding fatty acid composition, linolenic acid (C18:2 n6) was lowest
in the group fed 0.2% yeast culture. However, yeast culture supplementation to the diet did not alter the microbial flora. Yeast
culture (S. cerevisiae) supplementation to the diet of laying hens is beneficial for increasing feed intake and egg weight of
laying hens without affecting the microbial flora in their digestive system.
Key Words: laying hen, fatty acid, microbiological flora, yeast culture
Introduction
Feed additives, which are defined as substances, have
long been added to animal diets to increase the utilization of
feeds as well as the quantity and quality of animal products.
Yeast cultures are complex fermented products containing
metabolic agents produced by yeast during fermentation.
Live yeast cultures contain digestive enzymes such as
amylase, maltase, sucrose, lactate dehydrogenase, proteinase,
polypeptides, dipeptidase, deaminase, transaminase, lipase,
phospholipase, phosphatase, and phytase and provide
higher digestibility (Stanley et al., 1993). Saccharomyces
cerevisiae, which produces these properties, has been
proven to have beneficial effects on poultry production,
e.g., in reproduction, growth rate, egg production, and feed
efficiency (Dawson, 1993). In addition, supplementation
of yeast, yeast extracts, and yeast cultures to feed has
Received: June 29, 2017
Accepted: December 5, 2017
*Corresponding author: bulent58@gmail.com
Copyright © 2018 Sociedade Brasileira de Zootecnia. This is an Open Access
article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is
properly cited.
provided economic and environmental benefits in poultry
diets for the past 40 years (Stone, 2007). Saccharomyces
cerevisiae is known to increase the biological value of
nitrogenous compounds in the digestive tract and to reduce
stress-producing factors in animals. Yeast culture also
helps digestion by increasing the availability of nutrients
(Stanley et al., 1993).
The positive effects of supplementation of yeast culture,
which might also be called probiotics on the performance
values of animals, have been attributed to many factors
including (but not limited to) microbial flora, dose added
to the feed, environmental conditions, and physical
condition of the animal (Ceylan and Çiftçi, 2003). It has
been shown that probiotic microorganisms reach the villi of
the intestinal lumen earlier than pathogenic bacteria, thus
preventing these pathogens from becoming trapped in the
digestive tract (Hassanein and Soliman, 2010).
In other words, probiotic microorganisms compete
with pathogenic microorganisms for binding to receptors
(competitor exclusion). The positive effect may also be due
to the strengthening of the natural defense system and the
biological regulation of intestinal microflora of the host, as
well as to the direct effect of the probiotic on health or the
2
Özsoy et al.
nutritional form of the probiotic (Shareef and Al-Dabbagh,
2009; Hassanein and Soliman, 2010).
This study aimed to investigate the effects of the
different levels (0.05, 0.1, and 0.2%) of yeast culture
supplementation on feed intake (FI), feed conversion ratio
(FCR), egg production, egg quality traits, egg yolk fatty
acid composition, and microbiological flora in the feces of
laying hens.
Material and Methods
A total of 240 laying hens at 18-19 weeks of age were
divided into four groups (control and three trial groups) and
fed for 16 weeks. Each group comprised 10 sub-groups,
with each sub-group having six animals. The animals were
fed a basal diet containing 2750 kcal/kg metabolizable
energy and 16% crude protein (Table 1). The yeast culture
(S. cerevisiae) was added to the diet at different levels
(0.05, 0.1, and 0.2% body weight – BW). The BW was
determined at the beginning and at the end of the study.
The daily egg production of the groups was measured. The
FI and FCR of animals were recorded weekly (Table 2).
In the 4th, 8th, 12th, and 16th weeks of the study, the
egg internal and external quality traits were determined in 10
randomly selected eggs from each group, and measurements
were repeated four times. Eggs were weighed twice a week
for two successive days, and average weights were recorded
(Table 3).
Egg yolk oil extraction was conducted using nine
egg samples from each group as reported by AOAC
(2000). Afterwards, methyl esters were formed with
boron trifluoride. The fatty acid methyl esters were
condensed under nitrogen gas and then analyzed by gas
Table 1 - Composition and nutrient level of basal diet
Item
Ingredient (g kg−1)
Corn
Soybean meal
Sunflower seed meal
Meat and bone meal
DL-methionine
Limestone
Dicalcium phosphate
Salt
Vitamin and mineral premix1
645
150
78
30
0.5
82
10
2
2.5
Calculated nutrient level
Metabolizable energy (kcal kg−1)
Crude protein (g kg−1)
Ca (g kg−1)
P (g kg−1)
2753
160
34.5
6.2
1
Provides per kg: 15,372.00 mg vitamin A; 6.28 mg vitamin E; 0.64 mg vitamin K3;
27.36 mg Mn; 89 mg Fe; 25 mg Zn; 8.76 mg Cu; 0.03 mg Co; 0.05 mg I;
0.91 mg Se.
chromatography-mass spectroscopy (HP 6890/5972). The
Agilent HP88 100 × 250 × 250 µm column was used in
the analysis. The column starting temperature was 120 °C,
the final temperature was 230 °C, and the injector and
detector temperature was 250 °C. The injection speed was
set to 50:1. Helium was used as a carrier gas.
Fecal samples, which were collected from each
group, were placed into sterile plastic tubes in the last
week of the experiment. Stool samples were diluted as
10:1 in physiological saline (FTS) containing 0.9% NaCl.
Subsequently, the samples were diluted in a ten-fold (log10)
dilution series by using six tubes. Inoculation began from
the lowest dilution rate. Plate count agar was used for
general live (total mesophilic aerobic microorganism),
MacConkey agar for coliform bacteria, and Sabouraud
dextrose agar for yeast. Plate count agar was incubated in
aerobic conditions for 48 h at 30 °C, MacConkey agar was
incubated for 24 h at 37 °C for coliform bacteria count,
and Sabouraud dextrose agar was incubated at 45 °C for
72 h (Arda, 1985; Arda et al., 1997). After incubation,
the media were counted and the averages were taken. The
mean numbers for all dilution steps were then determined,
and microorganism counts were recorded for each sample
according to these results.
One-way ANOVA was used to determine the
significance of the differences between the statistical
calculations and mean values of the groups. The Duncan
test was employed to determine the significance among the
groups (Dawson and Trapp, 2001).
Results
During the experimental period, there was no side
effect on the health of animals due to supplementation of
yeast culture at the test concentrations added to the feed.
The different levels of yeast culture supplementation to
diets did not affect BW. The lowest average FI (101.96
g/day) was obtained from the group fed 0.2% yeast culture
supplementation, when compared with control group
(106.43 g/day) (Table 2).
The highest egg production was obtained from the
groups fed 0.2 (91.54%) and 0.1% supplemention (91.93%),
while control group had an average egg production of
90.30% (P>0.05).
Yolk diameter was lowest in the group fed 0.1%
supplementation, followed by the 0.05% supplementation,
control, and 0.2% supplementation, in that order. In
comparison with the control and 0.05% supplementation
groups, the highest Haugh units were obtained from the 0.2%and 0.1%-supplementation groups, respectively (Table 4).
R. Bras. Zootec., 47:e20170159, 2018
The role of yeast culture (Saccharomyces cerevisiae) on performance, egg yolk fatty acid composition, and fecal microflora of laying...
In terms of fatty acid composition (Table 5),
hexadecanoic (C16:0) and oleic acids (C18:1) were the
main fatty acids in the egg yolk. Myristoleic (C14:1) and
hexadecanoic acid (C16:0) values were higher in the 0.1
and 0.2% yeast culture-supplementaation groups, when
compared with control group (P>0.05). Polyunsaturated
fatty acids (PUFA) values were numerically lower in the
0.2% yeast culture-supplementation group than in the other
3
groups. However, PUFA:saturated fatty acids (SFA) and
C18:2 n6 values were significantly different between the
control and experimental groups (P<0.05).
The microbiological analysis of feces (Table 6) revealed
that dietary supplementation of yeast culture did not
influence the total bacterial number (P = 0.50). The number
of coliform bacteria was lowest (1.35±0.59 log10 cfu/g) in
the 0.2% yeast culture-supplementation group (P = 0.89).
Table 2 - Effects of yeast culture on laying hen performance
Control group
Initial body weight (g)
Final body weight (g)
Egg production/hen/day (%)
Feed intake (g/day per hen)
Feed conversion ratio (g feed/g egg)
Experimental group 1
1757±19.12
1720±17.86
90.30±0.60
106.43±1.17a
2.03±0.03
1740±18.20
1707±16.90
90.93±0.52
105.23±0.85a
1.99±0.03
Experimental group 2
1758±14.34
1750±19.36
91.54±0.53
104.82±1.05ab
1.97±0.02
Experimental group 3
1761±8.53
1756±21.23
91.35±0.43
101.96±1.15b
1.94±0.07
P-value
0.812
0.211
0.35
0.03
0.07
Control group: basal diet; Experiemental group 1: basal diet + 0.05% yeast culture; Experimental group 2: basal diet + 0.1% yeast culture; Experimental group 3: basal diet + 0.2%
yeast culture.
a-b - Means within a row with different letters are different at P<0.05.
Table 3 - Weekly egg weights (g)
Week
Control group
Experimental group 1
Experimental group 2
Experimental group 3
P-value
1-2
56.97±0.51
n = 55
57.70±0.46
n = 55
58.78±0.51
n = 58
58.37±0.59
n = 56
57.67±0.71b
n = 51
57.97±0.62b
n = 53
58.84±0.61b
n = 54
58.85±0.59b
n = 51
58.15±0.20b
n = 433
56.83±0.44
n = 55
57.69±0.46
n = 58
58.52±0.44
n = 60
57.86±0.51
n = 58
58.28±0.64ab
n = 53
58.77±0.57ab
n = 52
59.89±0.57ab
n = 55
58.16±0.55b
n = 54
58.24±0.19b
n = 445
57.38±0.51
n = 53
58.49±0.54
n = 53
58.79±0.56
n = 57
60.19±0.65a
n = 58
58.55±0.54ab
n = 56
59.80±0.65a
n = 56
60.96±0.53a
n = 56
60.47±0.66a
n = 57
59.35±0.21a
n = 446
56.34±0.53
n = 51
58.58±0.62
n = 53
59.17±0.53
n = 50
60.36±0.54a
n = 57
59.97±0.55a
n = 58
60.44±0.53a
n = 56
60.51±0.46a
n = 56
60.96±0.47a
n = 56
59.59±0.19a
n = 437
0.54
3-4
5-6
7-8
9-10
11-12
13-14
15-16
All periods
0.44
0.85
0.003
0.052
0.019
0.041
0.002
<0.00
Control group: basal diet; Experiemental group 1: basal diet + 0.05% yeast culture; Experimental group 2: basal diet + 0.1% yeast culture; Experimental group 3: basal diet + 0.2%
yeast culture.
a-b - Means within a row with different letters are different at P<0.05.
Table 4 - Effects of yeast culture on egg quality traits of laying hens
Egg weight (g)
Shape index (%)
Shell weight (g)
Albumen weight (g)
Yolk weight (g)
Shell weight percentage (%)
Albumen weight percentage (%)
Yolk weight percentage (%)
Yolk diameter (mm)
Yolk height (mm)
Haugh unit
Control group
Experimental group 1
Experimental group 2
Experimental group 3
P-value
60.07±0.69
78.46±0.58
5.94±0.09
39.72±0.62
14.41±0.17
9.90±0.13
66.04±0.36
24.06±0.35
39.23±0.31ab
21.49±0.28
79.86±0.30b
58.85±0.66
77.28±0.43
5.81±0.13
38.79±0.61
14.25±0.24
9.87±0.19
65.85±0.49
24.28±0.44
38.86±0.43ab
21.31±0.28
79.71±0.31b
59.66±0.73
77.81±0.36
6.05±0.11
39.18±0.67
14.44±0.21
10.14±0.16
65.58±0.45
24.28±0.41
38.08±0.41b
21.25±0.26
80.49±0.37ab
59.78±0.63
78.02±0.59
6.08±0.09
37.62±1.12
16.08±1.04
10.19±0.18
62.91±1.69
26.90±1.67
40.01±0.43a
21.48±0.35
81.18±0.29a
0.62
0.42
0.62
0.29
0.07
0.38
0.06
0.08
0.01
0.92
0.005
Control group: basal diet; Experiemental group 1: basal diet + 0.05% yeast culture; Experimental group 2: basal diet + 0.1% yeast culture; Experimental group 3: basal diet + 0.2%
yeast culture.
a-b - Means within a row with different letters are different at P<0.05.
R. Bras. Zootec., 47:e20170159, 2018
4
Özsoy et al.
Table 5 - Effects of dietary supplementation of yeast culture on yolk fatty acids (% of total methyl esters of fatty acids) of laying hens
Fatty acid (%)
Control group
Experimental group 1
C14:0
C14:1
C:16:0
C16:1
C17:0
C17:1
C18:0
C18:1
C18:2 n6
C18:3 n6
C18:3 n3
C20:0
C20:1
C20:2 n6
C20:3 n3
C20:4 n6
C22:6 n3
C24:0
SFA
MUFA
PUFA
UFA
PUFA:SFA
n6
n3
n6:n3
Nutritive value1
Atherogenic index2
Thrombogenic index3
0.31±0.015
0.014±0.004
23.96±0.44
2.97±0.15
0.18±0.04
0.22±0.046
8.87±0.23
41.07±0.69
18.09±.54ab
0.51±0.044
0.88±0.040
0.075±0.017
0.34±0.045
0.21±0.014
0.19±0.018
1.44±0.083
0.54±0.042
0.12±0.013
33.54±0.39
44.61±0.64
21.86±0.66
66.46±0.39
0.65±0.02ab
20.24±0.59
1.61±0.08
12.65±0.37
2.09±0.06
0.15±0.005
0.57±0.01
0.31±0.016
0.015±0.005
24.14±0.35
3.07±0.16
0.20±0.015
0.15±0.018
8.24±0.23
40.72±0.42
19.09±0.37a
0.43±0.043
0.97±0.035
0.061±0.006
0.27±0.016
0.25±0.024
0.19±0.011
1.28±0.10
0.46±0.057
0.11±0.005
33.10±0.39
44.22±0.46
22.67±0.49
66.89±0.39
0.69±0.02a
21.06±0.43
1.61±0.07
13.16±0.47
2.03±0.04
0.14±0.003
0.57±0.009
Experimental group 2
0.32±0.022
0.023±0.005
24.07±0.34
3.15±0.12
0.17±0.018
0.13±0.008
8.30±0.20
41.00±0.58
18.81±0.37a
0.48±0.039
0.94±0.048
0.091±0.019
0.26±0.019
0.21±0.021
0.17±0.011
1.24±0.09
0.47±0.041
0.14±0.018
33.12±0.40
44.56±0.66
22.32±0.48
66.88±0.40
0.67±0.02a
20.74±0.43
1.59±0.06
13.14±0.38
2.05±0.04
0.14±0.004
0.56±0.01
Experimental group 3
P-value
0.30±0.020
0.029±0.002
24.79±0.32
3.27±0.11
0.26±0.062
0.18±0.030
8.47±0.16
41.98±0.82
16.80±0.79b
0.45±0.028
0.84±0.058
0.12±0.025
0.29±0.04
0.21±0.030
0.19±0.01
1.27±0.03
0.47±0.021
0.10±0.007
34.04±0.27
45.73±0.89
20.23±0.87
65.96±0.27
0.59±0.03b
18.73±0.81
1.50±0.06
12.50±0.17
20.04±0.05
0.15±0.003
0.58±0.01
0.96
0.09
0.41
0.48
0.41
0.19
0.15
0.55
0.03
0.56
0.23
0.18
0.3
0.45
0.77
0.33
0.59
0.10
0.25
0.44
0.06
0.25
0.03
0.07
0.58
0.47
0.83
0.23
0.50
Control group: basal diet; Experiemental group 1: basal diet + 0.05% yeast culture; Experimental group 2: basal diet + 0.1% yeast culture; Experimental group 3: basal diet + 0.2%
yeast culture.
MUFA - monounsaturated fatty acids; PUFA - polyunsaturated fatty acids; UFA - unsaturated fatty acids; SFA - saturated fatty acids.
1
(C18:0 + C18:1)/C16:0.
2
(4*C14:0) + C18:0)/UFA.
3
(C14:0 + C16:0 + C18:0)/(0.5*C18:1) + (0.5*MUFA) + (0.5*n6) + (3*n3) + (n3/n6).
a-b - Means within a row with different letters are different at P<0.05.
Table 6 - Effects of dietary supplementation of yeast culture on fecal microflora (log10 cfu/g) of laying hens
Total bacteria
Coliform bacteria
Yeast
Control group
Experimental group 1
Experimental group 2
Experimental group 3
P-value
6.32±0.57
1.71±0.58
0.15±0.043
6.13±0.99
1.79±0.29
0.36±0.15
4.85±0.99
1.80±0.39
0.23±0.09
4.78±0.97
1.35±0.59
0.29±0.05
0.50
0.89
0.46
Control group: basal diet; Experiemental group 1: basal diet + 0.05% yeast culture; Experimental group 2: basal diet + 0.1% yeast culture; Experimental group 3: basal diet + 0.2%
yeast culture.
Non-significant differences P>0.05.
Discussion
Studies on laying hens reported that addition of
yeast culture or yeast metabolites did not affect BW
(Yousefi and Karkoodi, 2007; Yalçın et al., 2012; Yalçın
et al., 2014; Hassanein and Soliman, 2010). Similarly,
our results also did not show any difference in the BW of
laying hens. On the other hand, some studies indicated that
yeast culture supplementation to broiler diets positively
affected their BW (Shareef and Al-Dabbagh, 2009; Fasina
and Thanissery, 2011; Fathi et al., 2012), which could
be attributed to the strains of yeast and animal species,
among other factors.
Although yeast culture supplementation had no
statistical effect on egg production, a numerical increase
was observed. Similar results were recorded for yeast or
yeast metabolite supplementation to laying hen diets in
many studies (Ayanvale et al., 2006; Asli et al., 2007;
Yousefi and Karkoodi, 2007; Yalçin et al., 2008; Yalçın et al.,
2012; Gül et al., 2013; Sacakli et al., 2013). On the other
hand, Hassanein and Soliman (2010) observed an increase
in egg production in laying hens by using yeast culture
supplementation at much higher concentrations (0.4, 0.8,
1.2, and 1.6%).
The different levels of yeast culture supplementation to
diets significantly affected FI (P<0.05). Consistent with our
R. Bras. Zootec., 47:e20170159, 2018
The role of yeast culture (Saccharomyces cerevisiae) on performance, egg yolk fatty acid composition, and fecal microflora of laying...
findings, yeast culture supplementation positively affected
FCR of broilers (Onifade and Babatunde 1996; Miazzo et
al., 2005; Shareef and Al-Dabbagh, 2009) and laying hens
(El-Ella et al., 1996; Katoch et al., 2003; Yalçın et al., 2012;
Gül et al., 2013; Yalçın et al., 2015). Conversely, FI and
FCR was not improved by yeast culture supplementation in
laying hens (Nursoy et al., 2004; Asli et al., 2007; Yousefi
and Karkoodi, 2007; Hassanein and Soliman, 2010; Sacakli
et al., 2013), in laying quail (Önol et al., 2003), and in broiler
turkeys (Özsoy and Yalçın, 2011).
The highest egg weight was observed in hens fed 0.1
and 0.2% yeast culture during the experimental period,
and this difference was statistically significant (P<0.05).
Our findings corroborate previous studies (Ayanvale et al.,
2006; Yalçin et al., 2008; Yalçın et al., 2015; Zhong et al.,
2016), but disagree with others (Nursoy et al., 2004; Asli
et al., 2007; Hassanein and Soliman, 2010; Gül et al., 2013;
Sacakli et al., 2013).
No statistical difference was observed for shape index,
shell weight, albumen weight, or yolk height. These results
are consistent with those of other studies (Asli et al., 2007;
Hassanein and Soliman, 2010; Yalçın et al., 2012; Yalçın
et al., 2014; Yalçın et al., 2015). Yolk weight (P<0.07), yolk
weight percentage (P<0.08), and yolk diameter (P<0.01)
were the highest in the group fed 0.2% yeast culture
supplementation. The lowest albumen weight percentage
was also found in the third trial group (P<0.06). Haugh
unit was calculated as 79.86, 79.71, 80.49, and 81.18 in
the control and the three trial groups, respectively. The best
Haugh unit was found in the third trial group (P<0.005).
Similarly, previous studies (Ayanwale et al., 2006; Yousefi
and Karkoodi, 2007; Asli et al., 2007; Zhong et al., 2016)
reported that egg yolk weight was increased by the
supplementation of yeast culture.
At the end of the experiment, egg yolk fatty acids
C14-C24 were determined. The C18:2 n6 values were
statistically different between the control and experimental
groups (P<0.05). In addition, PUFA (P = 0.06), PUFA/SFA
(P<0.03), and n6 (P = 0.06) values were different between
control and experimental groups. Yalçın et al. (2010)
similarly reported that yeast autolysate supplementation
decreased monounsaturated fatty acids (MUFA) and
C18:1, but increased C14:0, C14:1, C20:0, C20:3n-3,
and C20:5n-3. Yalçın et al. (2012) also reported that
only C18:1 and MUFA levels increased, and the other
fatty acid parameters were not affected by yeast culture
supplementation.
The positive effects of yeast culture supplementation
on the natural defense system of the host animal and on
the biological regulation of its intestinal microflora and the
5
direct effect of the probiotic on health or the nutritional form
of the probiotic have been previously reported (Shareef
and Al-Dabbagh, 2009; Hassanein and Soliman, 2010). In
the current study, total microbial counts, coliform bacteria,
and yeast values were not altered in stool samples during
the last week of the study. The lowest coliform bacteria
value was found in the group fed 0.2% yeast culture
supplementation. Fecal yeast count was higher in the yeast
culture groups than in the control group. Gül et al. (2013)
reported that the number of total bacteria was decreased, but
the number of total yeast was increased in the intestine by
yeast supplementation to laying hen diets. Another study
(Hassanein and Soliman, 2010) reported that different
levels (0.4, 0.8, 1.2, and 1.6%) of yeast supplementation
to laying hen diets significantly reduced the number of
total bacteria, especially in the 1.6% supplementation
group. However, Wang et al. (2015) found that yeast
supplementation increased the total number of bacteria in
the intestine of laying hens.
Conclusions
Yeast culture supplementation, as a probiotic to laying
hen diets, does not cause any health problems. It is thus
suggested that the different levels of yeast supplementation
(S. cerevisiae) in laying hen diets could increase the
productivity of laying hens in terms of feed intake, egg
weight, and egg Haugh unit.
Acknowledgments
The present study was supported by the Scientific
Research Project Unit of Kafkas University, Turkey (Project
Number: BAP 2016 TS 19).
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W4220812676.txt | ACADEMIC RESEARCH IN MODERN SCIENCE
International scientific-online conference
MODERN PEDAGOGICAL TECHNOLOGIES ENSURING THE QUALITY
OF TEACHING IN ENGLISH LESSONS IN ELEMENTARY GRADES
Mirkamilova Naima Sadiliyevna
English teacher school 328
mirkamilovakamila5@gmail.com
https://doi.org/10.5281/zenodo.5889345
Annotation: Today, the focus is on the student, his personality, and his
unique inner world. Therefore, the main goal of a modern teacher is to choose
methods and forms of organizing students' educational activities that optimally
correspond to the set goal of personal development. In recent years, the issue of
the use of new information technologies in secondary schools has been
increasingly raised. These are not only new technical means, but also new forms
and methods of teaching, a new approach to the learning process.
Keywords: psychological development, story-role-playing game, selforganization, intensive methodology
Based on the peculiarities of the psychological development of younger
schoolchildren, one of which is the predominance of the emotional sphere over
the intellectual one, I chose game technology as the leading pedagogical
technology in early English language teaching. In particular, as a leading
educational activity – a story-role-playing game. In the story-role-playing game,
the impressions of a junior schoolboy are constantly replenished, clarified, and
qualitatively changed. The knowledge he has, thanks to their practically effective
reproduction in the plot-role-playing game, becomes more durable, more
conscious. Freedom, independence, self-organization and creativity of children in
the story-role-playing game are manifested with special completeness. The
creation of a unified system of scheduled and extracurricular work on the subject
is the main task of the educational process. Only under the condition of an organic
combination of regular and extracurricular work, a foreign language can make a
significant contribution to the formation and development of a child's personality.
The initial training course is divided into three stages:
I. "Let's play English". Students in a playful way master the main types of
speech activity - speaking, listening, get acquainted with the letters and sounds of
the English alphabet, learn to read the first words.
II. "Let's read and write English". At this stage, all types of speech activity are
being developed, but special attention is paid to reading and writing.
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ACADEMIC RESEARCH IN MODERN SCIENCE
International scientific-online conference
III. "Let's enjoy your English". At this stage, there is a further development of
students' communicative competencies, as well as systematization and selfimprovement of the acquired knowledge and skills.
In an effort to update the content and introduce new approaches in teaching
a foreign language, focused on the development of pupils' communicative
competence based on game technologies. Elements of intensive methodology,
various forms of play activity, dramatization, dramatization contribute to the
intensification of the learning process and ensure a high level of student learning.
In my classes, I rely on the methods which, from my point of view, successfully
combine all learning goals, take into account the psychological characteristics of
children of early age. The program allows you to make learning understandable
and joyful. The child learns by playing. To do this, interesting tasks are set for
students in the course of training. The obvious advantage of this program is the
presentation of lexical and grammatical material in a playful way, which is very
important for children. Tired of serious educational material in other subjects, the
school children easily and actively perceive problematic stories interesting to
them in English lessons, plunge into the world of fairy tales. All this contributes to
the emergence of a positive motivation for the subject English, and also
encourages him to participate in activities in the classroom, at the same time
develops the child himself. When school children have already mastered the basic
skills of reading and writing in their native language, he has formed basic general
educational skills, so he is ready for a more serious assimilation of more complex,
not always corresponding to the equivalents of their native language, material in
English. To combine programs, grades 2, 3, 4, I use such techniques and tools that
allow me to build a lesson as an integral communication system: game situations
like "fairy tales", interesting stories, the same actors familiar from other grades.
The pupil must understand the meaning of everything that he has to reproduce,
including grammatical constructions. Already from the first stage of learning
English, I try to explain grammatical phenomena, but I do it with the help of game
techniques, where dolls are also assistants. This is the formation of the plural of
nouns, the negative structure with the verb can; the use of have (has, etc.). A young
child loves to play very much, so one of the effective teaching methods is the use
of various plots (for example, "Journey to the land of the Four Fairies", "Magic
City"), stories (secrets, secrets of letters, the eternal adventures of Simon), game
situations (the appearance of Karabas, Master Lomaster, Santa Claus). The main
characters of my game plots in the lessons are dolls and toys. Dolls are my best
assistants in English lessons: Alice, Simon, Pinocchio, Malvina, Dunno, etc. These
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ACADEMIC RESEARCH IN MODERN SCIENCE
International scientific-online conference
dolls are present from the first lessons, they talk about the countries of the
language being studied, their families, friends, hobbies, teach children, enter into
an argument with them, travel, arrange holidays. These dolls have their own
biographies, a certain character, their own life stories. So, Alice and Simon are
present at almost all lessons, Pinocchio sells us goods in the magic store, and is
also a guide to school to Malvina. Malvina teaches us the letters and sounds of the
English alphabet, tells us about her school. Dunno appears in those moments
when we do not know something, we are experiencing difficulties, but together
we find a way out of a difficult situation.
References
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Publishing. New York. 2011
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W2516315368.txt | Typifying Mechanisms for Gender Digital Equity in
Latin America
Gabriela Marín-Raventós, Marta Calderón-Campos
To cite this version:
Gabriela Marín-Raventós, Marta Calderón-Campos. Typifying Mechanisms for Gender Digital Equity
in Latin America. 6th IFIP World Information Technology Forum (WITFOR), Sep 2016, San José,
Costa Rica. pp.159-170, �10.1007/978-3-319-44447-5_15�. �hal-01429744�
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Typifying Mechanisms for Gender Digital Equity
in Latin America
Gabriela Marín-Raventós1, Marta Calderón-Campos2
1
CITIC-ECCI, Universidad de Costa Rica
gabriela.marin@ucr.ac.cr
2
ECCI, Universidad de Costa Rica
marta.calderon@ucr.ac.cr
Abstract. Gender digital equity in Latin America and the Caribbean is our goal.
We analyze gender specific structural inequalities that constitute barriers for
women´s access to ICT: such as education, traditional cultural beliefs and practices, and economic inequality. We argue also that male-dominated ICT design
and implementation (functionality, content, and human-computer interaction)
can inhibit gender digital equity. We present a model to typify the existing mechanisms for reaching gender digital equity and propose a new mechanism:
creating gender consciousness in ICT design and construction, and attracting
women to the ICT field. The proposed model reflects the four mechanisms and
depicts the possible means through which they can be implemented. Moreover,
the model appoints actors capable of and responsible for such means. Recommendations for the ICT community regarding gender digital equity are also included.
Keywords: ICT and Gender · Inequity · Digital Divide · Digital Equity
1
Introduction
Latin America and the Caribbean is a middle-income region, with the majority of its
42 countries and territories belonging to that category. It is a heterogeneous region,
ranging from low income countries, such as Honduras, Nicaragua, Guatemala, Paraguay, Bolivia, and Dominican Republic to higher income, and regarded as more developed countries, such as Chile, Mexico, Argentina, and Brazil [1]. According to the
Socio-Economic Database for Latin America and the Caribbean (SEDLAC), 34.3% of
the Latin American population is middle class, with income between $10 a day and
$50 a day; 25.3% is still under the poverty line of $4 a day [1].
In last decades, many countries in Latin America and the Caribbean have made a
big effort to close their gender gaps [2]: poverty has been reduced to lower levels,
more girls are in school, child mortality has dropped, and diseases are being fought.
However, gender issues remain and reduction in gender gaps does not mean that
women´s empowerment has been reached [2]. In particular gender digital divide is
still prevalent in the Region.
Digital divide is “an inequality in the power to communicate and to process information digitally” [3]. In most existing published research about digital divide, the
word gender refers to sex, that is, to biological women and men, since information
about gender identity is not available [3]. In our research, we adopt this position.
In the next section we analyze general gender gaps in Latin America and the Caribbean. In section 3, numbers and aspects of the Region´s digital divide are presented. Causes of gender digital divide are presented in section 4, with the objective
of trying to identify means for obtaining gender digital equity. Understanding ICTs
and their potential for the empowerment of women is necessary in order to overcome
the gender digital divide. Section 5 describes some different types of projects that
have been implemented throughout the region to overcome gender digital divide. Our
main contribution is in Section 6 where we present a model to typify the existing three
mechanisms for reaching gender digital equity and propose a new mechanism: creating gender consciousness in ICT design and construction, and attracting women to the
ICT field. Finally, Section 7 includes the conclusions and recommendations for the
ICT community, which includes government, academia, non-profit organizations, and
industry, as a guide for them to design and implement solutions for each of the four
mechanism categories mentioned above, in order to contribute to reach gender digital
equality and equity.
2
General Gender Gaps in the Region
Figure 1 shows that women are still underrepresented in the best-paid professions and
receive significantly lower salaries than men. The average gender earning gap between men and women is 58% in the region. Engineering, including computer-related
professions, is the field in which female representation reaches the lowest percentage
(17%), and the second highest gender earning gap (76%).
The World Economic Forum introduced the Global Gender Gap Index (GGGI) in
2006 for capturing the magnitude of gender-based differences [2]. Three concepts are
important for understanding the GGGI. The first is that it measures gaps rather that
levels of attainment. The second is that it measures outcome variables instead of input
variables. This means that variables such as rights, culture or customs, considered
inputs, are not reflected in the GGGI. Output variables are related to basic rights, such
as education and health. The third concept is that the index ranks countries by gender
equity rather than by women´s empowerment. The GGGI considers four categories:
Economic Participation and Opportunity, Educational Attainment, Health and Survival, and Political Empowerment.
According to the World Economic Forum, Latin America and the Caribbean have
closed the gender gap by 70%. Since 2006, this region “has shown the most improvement on the overall GGGI and second-most improvement on both the Economic
Participation and Opportunity and Political Empowerment sub-indexes” [2].
Fig. 1. 10 TOP-Paying Occupations in Latin America, CIRCA 2007. (Source: [4])
However, according to the Inter-American Development Bank [5], even with more
education than men, women are still concentrated in lower-paid occupations. When
comparing men with women of the same age and educational level, men earn 17 percent more than women. Changing household roles and stereotypes is essential to attaining gender equity in the labor market.
Women´s reality is still difficult. Too many women still die in childbirth. Employment, salary and decision making gaps still exist. More needs to be done in order to
enhance access to education, job opportunities, and reproductive health services.
3
Digital Divide in Latin America and the Caribbean
The digital divide is rooted in the very issues that constrain Latin America’s overall
economic development: income inequality, lack of infrastructure, and still-nascent
technological knowledge base [6]. Note that income inequality is not necessarily
related to a country´s wealth. Figure 2 depicts some statistics that highlight the heterogeneity in Latin America´s income inequality. Uruguay ranks as the most equitable
country in Latin America, with the wealthiest 20 percent receiving 8 times more than
the poorest 20 percent. Venezuela comes in at number two. The United States takes
eleventh place -- behind Costa Rica and ahead of Colombia, and several spots below
its southern neighbor Mexico. Brasil, Guatemala, and Honduras are regarded as the
countries with highest inequality, and therefore, more susceptible to digital divide.
Information and communication technologies (ICT) face constraints in the region,
such as a poor legal framework for the development of the ICT sector, heavy administrative burdens, almost non-existing government prioritization for ICT development,
low Internet penetration, rates and pervasive brain drain which undermines the potential for faster growth of the economies’ ICT sectors [8].
Uruguay
Venezuela
El Salvador
Peru
Ecuador
Mexico
Nicaragua
Chile
Argentina
Costa Rica
United States
Colombia
Panama
Dominican Republic
Bolivia
Paraguay
Brasil
Guatemala
Honduras
Inequality index: the annual
income of the richest 20%
divided by the annual income of
the poorest 20%
0
5
10
15
20
25
30
Fig. 2. Inequality in the Americas (Source: [7])
Despite the digital divide has been closing in Latin America and the Caribbean, improvement has been lower amongst women, youth, indigenous peoples, afrodescendants, and rural populations [9].
In order to close this gap, it is necessary to understand the problem of the digital
divide. The most difficult barrier to overcome is not related to access (telecommunication infrastructure and dissemination of artifacts), but to use [10]. Opportunities
created by technological innovations depend on the use made of them and how they
affect the professional development and life of people. The Economic Commission
for Latin America and the Caribbean (ECLAC) [11] points out that there is a lag in
the ability to adopt and disseminate information technology and systems innovation in
Latin America and the Caribbean. They point out that this panorama is even more
discouraging given the fact that in recent years, the region´s connectivity growth has
been the world´s fastest. Unequal access of different social sectors to this new technology is their concern. ECLAC warns that the risk of widening the domestic digital
divide is greater within the region, than the threat of increasing the gap between the
region and the developed world [11].
In particular we are interested in examining how to diminish digital divide existing
amongst Latin American and the Caribbean women.
4
Gender Digital Divide
Gender digital divide is a reality in many developing countries. Hilbert found that
women used Internet less than men, both in Latin America and Africa [3]. Other two
differences between men and women, related to having or not a job and attending an
educational institution, were found. In both cases, men reach higher employment and
educational attainment percentages. However, when eliminating these two facts, that
is, when considering only educated and working women and men, women become
more active Internet users than men. Technophobic arguments, such as thinking that
women have a negative attitude toward ICTs, are no longer valid [3] [12]. However,
there are gender specific structural inequalities that constitute barriers for women´s
access to ICTs. According to [10], they are education, traditional cultural beliefs and
practices, as well as economic inequality.
The gender digital divide can only be overcome reaching gender equality and
gender equity. According to the International Fund for Agricultural Development,
gender equality “means that women and men have equal opportunities, or life
chances, to access and control socially valued goods and resources. This does not
mean that the goal is that women and men become the same, but it does mean that we
will work towards women's and men's equal life chances [13].” On the other hand,
gender equity “means fairness of treatment for women and men, according to their
respective needs. This may include equal treatment or treatment that is different but
which is considered equivalent in terms of rights, benefits, obligations and opportunities [13].” We believe gender equality is the basis upon which gender equity can be
sought.
5
Towards Gender Equity: Types of Projects
First, we should be aware that ICTs uses are not gender neutral, even when producers´
goal is to sell technology with the least variation in order to lower costs. For example,
in a study of How Women and Men Use the Internet [14], Deborah Fallows identified
several gender differences: (1) Men are more interested than women en technology
and are more tech savvy. (2) Moreover, significantly more men than women: maintain and fix their own computers, are more likely to try new gadgets and applications
and software, and are more confident in themselves as searchers and geeks, and pursue and consume information online more aggressively. (3) Women are more enthusiastic online communicators, use email in a more robust way, and are more concerned about general criminal use of the internet than men.
Understanding ICTs and their potential for the empowerment of women is necessary in order to overcome the gender digital divide. Empowering women will give
them the capacity to cope with life requirements more efficiently and to transform life
conditions. Initiatives for building these two capacities exist and have to be encouraged [10].
In order to build the capacity of coping with life requirements, it is necessary to
provide women with the skills that will give them self confidence to use technology.
Projects such as TIC-as (ICT-as), under the leadership of the cooperative Sulá Batsú
[14], can help to achieve this goal, integrating more women in rural areas to ICTs.
Women need to get basic knowledge for using word processors and spreadsheets and
navigating in the web. They can also use technology in order to learn about other
fields, such as agriculture, legislation, negotiation, marketing, and finance. This can
help them to find a job, or a better job, or even, start their own business.
Attracting women to ICT studies is also important because it can help women
transform life conditions. ICT is not gender neutral [15]. There are differences be-
tween men and women on hardware and software requirements, desired interaction
modes, use contexts, and acquisition capacity. Therefore, increasing the participation
of women in ICT design and development can help to promote gender digital equity,
create awareness of the importance of including gender issues when designing and
developing technology, destroy the public perception that ICT is a male domain, ensure a competitive workforce, and enhance the quality of women in general [16].
Initiatives such as Meninas Digitais (digital girls) in Brazil [17] and Work Intel
Network (WIN) [18] aim primarily at students of high school and technological education, to motivate them to follow careers in ICT. The robotic program of the Fundación Omar Dengo in Costa Rica is designed to develop creativity and innovation skills
in children creating and programming robots [19].
It is interesting to note that most initiatives to empower women and to reduce
gender digital divide are women driven, and do not correspond to structured public
policies.
6
Typifying Mechanisms for Reaching Gender Digital Equity
Mechanisms for reaching digital equity must attack several dimensions, since it is not
only an economical issue. Several mechanisms have been proposed and implemented.
We present a characterization of mechanisms based on the degree of ICT appropriation that women can reach.
The first category is providing women with access to technology. This seems
simple, but the effect is not necessarily the expected. Women who have not received
training on computers can feel frustrated. If the computer is at home and available for
all the members of the family, there is no warranty that women will use it. Women
could think that the computer is for her husband and kids, and that it is not worth
using it [20]. Access can also be provided through public computer centers, but some
women may not visit these places by themselves. If there is a charge for using a computer, women with low income can hardly afford it, and if they can, probably they
will find other priorities to invest the money, forexample, to enhance the quality of
living of her children. Moreover, moving to a public computer center may not be
possible for some women who have to take care of other members of the family.
Access to mobile telephones plays an important role, not only because they are low
cost devices, but because they are for personal use (not for family use), and can help
overcome the impossibility of moving to a public computer center.
The second category is allowing women to learn how to use standard software
tools (for example, word processing and spreadsheets). This can be a social activity,
so that a group of women can learn together and support amongst themselves. However, these software tools may not be useful enough to make women enthusiastic TIC
users. Additionally, as stated before, women may not be able to leave their house and
move to the training center, because of distance, cost, or obligation of taking care of
someone else.
The third category is empowering women through enabling women to learn how
to use ICTs to take control of their lives. TICs can be tools for improving life con-
ditions. Women must be empowered such that they can find out how technology can
help them improve their own quality of life: for example, help them control domestic
expenses, sell products they produce, increase their income, find employment opportunities, or learn about topics they need to know, such as health, child care and nutrition, among others [3]. Even more importantly, ICTs can help women increase their
self esteem. ICTs can become channels to reunite women groups to fight for their
own rights.
Evaluation methodologies are required in order to determine whether an ICT
project really improves women´s social conditions and promotes a positive change at
personal, institutional, community, and social levels. For example, the Gender Evaluation Methodology (GEM), developed by the Association for Progressive Communications (APC), has been used in many initiatives since 2002 [21]. GEM, created
with the participation of experts on ICT development in 25 countries from Latin
America, Asia, Africa, and Central and East Europe, is useful for determining what
role ICTs play in changes that empower women and how this changes shift gender
relations between women and men. Methodologies like GEM provide policy makers
and the ICT community with feedback that can help them improve the social effects
of ICT projects [21].
Moreover, when men and women share the learning experience and work together,
in an environment without discrimination in which both have the same participation
opportunities, men can change their opinion about women´s ability to use computers.
This is very important because respect and power relations can become more favorable for women. In Latin America, more than 300 community telecenters have been
installed [12]. At these places, people can learn about ICTs and how to use them to
promote community development. People, from very different backgrounds, work at
these telecenters. The Women´s Networking Support Programme (WNSP), of the
APC, is “a network of women throughout the world committed to using technology
for women’s empowerment” [22]. WNSP experiences in telecenters show that the
diversity of technological solutions and innovation are promoted when men and
women participate in an equitable environment [12].
Previous three categories to pursue gender digital equity have been studied and
promoted in most countries. We want to highlight the importance of higher female
representation in ICT related jobs as a means to enhance gender digital equity. It constitutes our forth category of mechanisms: creating gender consciousness in ICT
design and construction, and attracting women to the ICT field. Women have
special gender needs that will hardly be considered if they do not participate in ICT´s
design and implementation. An interesting example of gender consciousness is that
Hewlett-Packard (HP) discovered that quilters used HP ink-jet printers to transfer
patterns onto cloth [23]. This is a female need that HP seriously considered and developed custom quilting software. This decision was taken due to the presence of a
significant number of women at HP. Women in the ICT field should become aware of
situations such like the shown in this example. Many women in the world are owners
of small businesses which could take advantage of ICTs. Paying more attention to
their needs could help ICT developers, or women themselves,find new business opportunities which would improve female living and working conditions.
Women on ICT jobs would be the voice of many unheard women and would help
achieve gender digital equity. Attracting women to ICT college careers and empowering them to play a proactive role in the professional field is a challenge. Numbers
show that the percentage of women studying ICT careers is very low and it is even
decreasing not only in our Region [24]. Consciousness on this fact has been obtained
but researchers and university department authorities had not succeeded in attracting
more women.
Figure 3 evidence the actors, and the means they normally use, to implement the
four mechanisms identified to enhance gender digital equity. Most proposals to diminish gender digital divide concentrate on the first three mechanisms. However, we
believe the fourth mechanism, creating gender consciousness in ICT design and construction, can facilitate the previous three, not only in Latin America and the Caribbean, but globally. Women can design and develop ICT technologies better suited for
female needs, can develop more female friendly applications and e-learning tools to
promote ICT use amongst the female community, and can help empower women.
Fig. 3. Mechanisms for Reaching Gender Digital Equity
7
Recommendations and Conclusions
The ICT community, which includes government, academia, non-profit organizations,
and industry, can contribute to design and implement solutions for each of the four
mechanism categories mentioned above, in order to contribute to reach gender digital
equality and equity.
For the first category, the ICT community should continue developing cost efficient devices, protocols and communication technologies, in order to warranty low
cost access to women. Community access centers are a source to provide access, if
schedules are designed to incorporate women´s needs, and daycare facilities for children and elderly are provided. Moreover, they must foster an environment in which
women can interact with someone who can solve her doubts. Mobile technology may
play an important role since they avoid the need to attend community centers. They
are easy to use and low cost.
Government policies can provide incentives and financial sources to supply mobile
technology access to women. They can impose conditions on companies that want to
operate in a country, such as obliging an operator to donate telephone connections to
the lowest income population.
For the second category, it is important to be aware of the limitations women can
face. Developing on-line training programs may be very convenient, because women
would not have to leave home, and could choose when to connect and interact with
the applications. E-learning is a challenging option requiring the participation of professionals from different backgrounds. Content creation is a major issue, because
training applications should be easy to use and should offer options to solve doubts.
Usability is a very important feature, since women value it more than men, and may
not have too much time to experiment with the application [15].
For the third category, the ICT community should understand women´s needs.
These are not necessarily standard, since needs could depend on the natural and social
environment. An important issue is promoting innovation and entrepreneurship
among women. Introducing women to electronic commerce and social networks to
sell their products is an example of how this could be achieved.
Another possibility is thinking about how to introduce women in the service sector,
that, in general, offers better income conditions that the manufacturing sector. Telework may become helpful to overcome limitations women face when looking for a
job. Women could receive necessary job training through on-line applications.
Creating online content that can help women improve their living conditions must
be a cooperative activity result of the interaction of the ICT community and women.
Examples of key areas of content are education, health, nutrition, and child and elder
care. Language used has to be understandable and adapted to the natural and social
environment.
Women should not be only content consumers. Introducing women to peer production in any of its two modalities, commons-based and corporate-based, could help
them to introduce them in the creation of information (e.g., financial services, accounting) and culture (e.g., music) [25]. This would allow women to get an income
and would help balance gender-power relations.
Giving women the possibility of participating in community decisions related to
ICT projects makes sense when they have understood the opportunities technology
offers. Government policies can enforce female participation in decision making, but
cultural aspects could neutralize the possible effects of these policies. Teaching women how they can assume an active role in the Internet governance, through commonsbased peer production, would help them to become more autonomous, because they
could create services and information by and for themselves and could freely organize
in non-hierarchical organizations [25].
The ICT community should also take advantage of international volunteer programs, in which experts in ICT volunteer as tutors in education programs. It would
help to lower program costs. For example, from 2001 to 2013, Korea Internet Volunteers (KIT) has contributed with more than 900 volunteer teams, mostly formed by
college professors and students, who have supported ICT education programs in 70
countries in Latin America, Africa and other continents [26]. Governments and nonprofit organizations should promote local volunteer programs, in which active and
retired ICT experts could participate. ICT enterprises could establish social programs
in which both the company and the employees contribute according to their capabilities. Companies could provide materials and ICT tools helpful to women, and employees could volunteer to work as instructors.
However, the ICT community should understand that introducing ICT in a community is not sufficient to achieve positive social changes. Reviewing existing evaluating methodologies and promoting its use is a pending task.
For the fourth category, creating gender consciousness in ICT design and construction, especially attracting women to the ICT field, is a pending task worldwide.
Moreover, retaining them not only while they study, but also during their professional
life, requires also our attention. Their presence in classrooms and working places can
contribute to get higher levels of innovation and competitiveness.
Women conceptualize a profession mostly based on the opinion of others and the
messages they receive from the society [24] [27]. On the other hand, men make their
career decision based on their personal experiences with technology, starting from
toys they have played with. Therefore, attracting young women to computer-based
careers requires of several changes. Young girls should have the same opportunity as
boys of playing with toys that let them solve problems and design solutions. Girls
should receive support from their parents and professors when they decide to study a
technological college career. The ICT community can develop promoting programs
showing what ICT professionals do, highlighting the opportunities that ICT offer to
improve people´s lives and promoting professional development. Media can highlight
the achievements of women in technological fields, which would help to change stereotypes that keep young girls away from ICT careers and would provide them with
models to follow.
Universities can play a more proactive role in recruiting ICT students. Both female
and male professors can visit primary and high schools to talk about possible professional development paths and to resolve the doubts students could have. Some
changes on Computer Science curricula can also be beneficial. Changing the program
of the career introductory course from a technological approach to an ICT potential
impact approach can help women to be sure they made the right decision when choosing career. A mentoring system during the first college year, in which women are
empowered and motivated to express their needs and opinions and their self esteem is
increased, could help retain them in the ICT field. Promoting a non-discrimination
study environment and working on teams in which at least a woman is always present
might help male classmates understand that they get better technological solutions
when both sexes cooperate.
The ICT community must become aware of the importance of designing technology considering gender differences. The field of gender human-computer interaction
has contributed to understand that women and men use computers for different purposes and goals [15], due to differences in their personal interests and in the roles
both sexes play within their families and communities. Physical differences (e.g., in
weight, height, and strength) should also be considered, in order to avoid personal
damage and fatigue. However, there are no general rules that can be applied when
identifying special needs of men and women, because human beings are complex and
their motivations and interests sometimes can seem contrary to what is expected. If
the aforementioned mechanisms for empowering women and helping them empower
themselves are effective, more Latin American women could become business owners, new needs may emerge, and ICT women may think about them more seriously
than men would. It is difficult to do research to prove the possible positive impacts of
(1) gender conscious technology design, and (2) greater presence of women in the
ICT field, on gender digital equity. Cause effect factors on long term societal phenomena are very hard to measure. We invite the international community to try to propose mechanisms to address this issue, and help us demonstrate the impact of our
proposed mechanism.
Finally, to foster gender digital equity, women in ICT related jobs should not limit
their activities to designing and building technology. They should also have the opportunity of participating in the definition of related policies, making sure that regulations and incentives necessary to motivate and facilitate the creation and use of applications favoring women, are created and implemented.
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26. Yochai Benkler, La Riqueza de las Redes. Icaria Editorial, Barcelona, España (2015)
27. Red de Redes Telecentros LAC, Proyectos,
http://www.telecentros.org/proyectos.php?id=2
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Springer
Received: November 24, 2023
Revised: January 5, 2024
Accepted: January 10, 2024
Published: January 23, 2024
Elliptic genera from classical error-correcting codes
a,b
and Shinichiro Yahagia
a
Department of Physics, Faculty of Science, The University of Tokyo,
Bunkyo-Ku, Tokyo 113-0033, Japan
b
Department of Physics, Osaka University,
Machikaneyama-Cho 1-1, Toyonaka 560-0043, Japan
E-mail: kawabata-koki382@g.ecc.u-tokyo.ac.jp,
yahagi@hep-th.phys.s.u-tokyo.ac.jp
Abstract: We consider chiral fermionic conformal field theories constructed from classical
error-correcting codes and provide a systematic way of computing their elliptic genera. We
exploit the U(1) current of the N = 2 superconformal algebra to obtain the U(1)-graded
partition function that is invariant under the modular transformation and the spectral flow.
We demonstrate our method by constructing extremal N = 2 elliptic genera from classical
codes for relatively small central charges. Also, we give near-extremal elliptic genera and
decompose them into N = 2 superconformal characters.
Keywords: Field Theories in Lower Dimensions, Superstrings and Heterotic Strings, Scale
and Conformal Symmetries
ArXiv ePrint: 2308.12592
Open Access, © The Authors.
Article funded by SCOAP3 .
https://doi.org/10.1007/JHEP01(2024)130
JHEP01(2024)130
Kohki Kawabata
Contents
1
2 Elliptic genera and weak Jacobi forms
2.1 N = 2 superconformal symmetry
2.2 Elliptic genera as weak Jacobi forms
2.3 Extremal elliptic genera
3
4
6
9
3 Elliptic genera from classical codes
3.1 Fermionic code CFTs
3.2 Constraints on U(1) current
3.3 Elliptic genera of fermionic code CFTs
3.4 Spectral flow and chiral ring
10
10
14
16
22
4 Extremal N = 2 elliptic genera
4.1 Central charge 12
4.2 Central charge 24
25
25
27
5 Other examples
5.1 n = 12 , p = 2
5.2 n = 24 , p = 3
5.3 n = 36 , p = 3
28
28
29
31
6 Discussion
33
A N = 2 superconformal characters
35
1
Introduction
Recently, the construction of conformal field theories (CFTs) from error-correcting codes
has been revisited and extended to fermionic CFTs with supersymmetry. It has been widely
known that error-correcting codes can be exploited to construct CFTs for a long time. The
program initially began with chiral bosonic CFTs constructed from classical error-correcting
codes [1–3], driven by insights from the understanding of the Monstrous moonshine [4].
Afterward, via fermionization, some of their fermionic counterparts have been shown to have
supersymmetry [5–8]. These pioneering developments have inspired the direct construction
of supersymmetric chiral CFTs from classical ternary codes [9] and its generalization to
classical p-ary codes [10].1 The chiral fermionic CFTs, termed fermionic code CFTs, have
Although this paper focuses on chiral CFTs constructed using classical error-correcting codes, there is
another approach that builds nonchiral CFTs from quantum error-correcting codes [11–13]. Recently, the
direction has taken significant advancement in various aspects, including the modular bootstrap program [14–16]
and the relation to holographic duality [17–19]. See [20–27] for other related progress.
1
–1–
JHEP01(2024)130
1 Introduction
n
h
i
TrHR (−1)F q L0 − 24 ,
(1.1)
where q = e2πiτ , τ is the torus modulus, and L0 is the zero mode of the Virasoro generators.
To compute the elliptic genus, we extend the Witten index to the partition function graded
by U(1) charge J0
n
h
i
TrHR (−1)F q L0 − 24 y J0 ,
(1.2)
where y = e2πiz and z ∈ C.
Then, we obtain the following main theorem (Theorem 3.1): if a classical code C satisfies
• The Construction A lattice Λ(C) is an odd self-dual lattice of rank n ∈ 12Z.
• The minimum norm of characteristic vectors in Λ(C) is n/3.
the graded torus partition function (1.2) of a fermionic code CFT becomes a weak Jacobi
form by taking an appropriate U(1) charge J0 .
To extend torus partition functions to elliptic genera, we identify the U(1) current
of the N = 2 superconformal algebra in fermionic code CFTs. Since elliptic genera are
torus partition functions with the insertion of U(1) current describing the R-symmetry, we
need to identify the R-symmetry in fermionic code CFTs. Fermionic code CFTs consist of
free chiral bosons and have U(1) currents generated by the free bosons. We take a linear
combination of them and impose some constraints to describe the R-symmetry. We see that
the constraints are satisfied if one determines a linear combination by a lattice vector called
a characteristic vector [29, 33, 34]. The corresponding U(1)-graded partition functions show
the modular invariance and spectral flow invariance for the central charge being a multiple of
12 (Proposition 3.3). Furthermore, when satisfying an additional condition, the U(1)-graded
partition functions turn out to be weak Jacobi forms (Theorem 3.1). With these observations
in hand, we investigate the N = 2 structure of fermionic code CFTs. One characteristic
–2–
JHEP01(2024)130
demonstrated their utilities in verifying conjectured constraints on superconformal indexes [9]
and searching for theories with large spectral gaps [10].
The construction of fermionic code CFTs exploits the relationship between classical
error-correcting codes and Euclidean lattices. Concretely, a code can specify a lattice by
the method called “Construction A” [28]. Then the lattice can be regarded as a momentum
lattice in a CFT and provides a set of vertex operators in the Neveu-Schwarz (NS) sector.
The Ramond (R) sector can be obtained from the shadow of the lattice, which is a set of
points shifted by half of a certain vector, called the characteristic vector [29].
This paper aims to compute the elliptic genera of fermionic code CFTs. In supersymmetric quantum field theories, the Witten index [30, 31] plays an essential role in characterizing
a theory through a topological invariant under continuous deformations preserving supersymmetry. For N = 2 superconformal theories, one can refine the Witten index to the elliptic
genus [32] by inserting the U(1) charge. The elliptic genera are invariant under the modular
transformation and the spectral flow and are described as weak Jacobi forms in number theory.
For a fermionic code CFT with central charge n, the Witten index can be computed by the
partition function twisted by the fermion parity (−1)F in the Ramond sector HR [9, 10]
2
Elliptic genera and weak Jacobi forms
In this section, we review N = 2 supersymmetric CFTs with special attention to their
elliptic genera following [36, 40, 42–47]. Section 2.1 is devoted to introducing the N = 2
superconformal algebra and its spectral flow. We also review N = 2 structures such as the
Ramond ground states and chiral primary fields. Noting that the elliptic genera are invariant
–3–
JHEP01(2024)130
of N = 2 superconformal algebra is the spectral flow connecting a periodicity condition to
a different one (e.g., the anti-periodic one to the periodic one) [35]. In the Hilbert space
of a fermionic code CFT, we show that the spectral flow can be regarded as a shift of a
momentum lattice by a characteristic vector, and an operator acting as the spectral flow
arises as a vertex operator whose momentum is a characteristic vector. Also, we consistently
identify the BPS states known as chiral primary states and show that they obey a strong
constraint between their conformal weights and U(1) charges [36] in fermionic code CFTs.
We demonstrate our computation of elliptic genera through the construction of extremal
N = 2 elliptic genera from fermionic code CFTs. The notion of extremal CFTs was introduced
to explore potential holographic duals to pure AdS3 gravity [37] (see also [38, 39]). It was
generalized to theories with N = 2 supersymmetry by defining extremal elliptic genera [40],
with some of them realized using actual CFTs with relatively small central charges [6, 41].
However, to our best knowledge, these extremal elliptic genera have been found independently,
and there has not been a unified construction yet. In this paper, we uniformly reproduce
the extremal N = 2 elliptic genera with the central charges 12, 24 in [6, 41] by employing
our computation with classical codes. We also construct other elliptic genera from classical
codes and interpret them as near-extremal elliptic genera, which obey a weaker condition on
extremality [40]. Moreover, we decompose these elliptic genera into N = 2 superconformal
characters and discuss their implications.
The organization of this paper is as follows. In section 2, we review N = 2 superconformal
field theories and their elliptic genera. Taking the spectral flow of importance, we explain
the Ramond ground states, chiral primary states, and their bounds on conformal weights
and U(1) charges. Also, we introduce elliptic genera as weak Jacobi forms and define their
polar regions and extremal elliptic genera, which we will use in later sections. Section 3 is the
central part of the present paper. Starting with a brief review of fermionic code CFTs, we
develop the computation of their elliptic genera. We obtain partition functions that return
the elliptic genera for N = 2 theories. To compute elliptic genera concretely, we explain how
to choose an appropriate U(1) current in fermionic CFTs constructed from p-ary codes. Also,
we identify the spectral flow operator and chiral primary states in fermionic code CFTs, and
we see that, especially in the ternary case (p = 3), those expressions become much simple.
In section 4, we exemplify the calculation of elliptic genera by reproducing extremal elliptic
genera. We deduce classical p-ary codes that yield extremal elliptic genera for small prime
p and obtain fermionic code CFTs with extremal elliptic genera for central charges 12, 24.
Section 5 deals with other examples of elliptic genera. Considering some classical codes, we
give their elliptic genera that admit an N = 2 character decomposition and discuss their
near-extremality. In section 6, we summarize the results and their implications. Appendix A
aligns representations of the N = 2 algebra and their characters.
under the modular transformation and spectral flow, we describe them with characterization
as weak Jacobi forms in section 2.2. In section 2.3, we introduce extremal and near-extremal
elliptic genera defined in [40], which we will reproduce from classical error-correcting codes
in section 4 and section 5, respectively.
2.1
N = 2 superconformal symmetry
Then, the supercurrents G± (z) have charge Q = ±1 under the U(1) current J(z). For
an N = 2 theory with the central charge n, these operators have the following operator
product expansions (OPEs):
T (z)T (w) ∼
n
2
(z −
T (z)G± (w) ∼
w)4
3
2
+
2
1
T (w) +
∂T (w) ,
2
(z − w)
z−w
G± (w) +
1
∂G± (w) ,
z−w
(z −
J(w)
∂J(w)
T (z)J(w) ∼
+
,
(z − w)2
z−w
J(z)J(w) ∼
w)2
n
3
G± (z)G± (w) ∼ 0 ,
J(z)G± (w) ∼ ±
,
(z − w)2
2
2J(w)
2T (w) + ∂J(w)
3n
G+ (z)G− (w) ∼
+
+
.
3
2
(z − w)
(z − w)
z−w
G± (w)
,
z−w
(2.2)
The mode expansions of the operators are
T (z) =
Lm
,
z m+2
m∈Z
X
G± (z) =
X G±
r
r
z
r+ 32
,
J(z) =
X
m∈Z
Jm
z m+1
,
(2.3)
where r ∈ Z + 1/2 in the Neveu-Schwarz (NS) sector and r ∈ Z in the Ramond (R) sector.
Accordingly, the commutation relations of the N = 2 algebra are
n
[Lm , Lk ] = (m − k) Lm+k + (m3 − m) δm+k,0 ,
12
±
±
Lm , Gr = (m/2 − r) Gm+r ,
[Lm , Jk ] = −kJm+k ,
(2.4)
±
[Jm , Jk ] = (n/3) m δm+k,0 ,
Jm , G ±
r = ±Gm+r ,
n
o
n 2 1
+
−
Gr , Gs = 2 Lr+s + (r − s)Jr+s +
r −
δr+s,0 ,
3
4
+
−
−
and {G+
r , Gs } = {Gr , Gs } = 0.
The N = 2 superconformal algebra has the spectral flow isomorphism [35]. This connects
different sectors (e.g., the NS sector and the R sector) by twisting the boundary condition
of the operators by the U(1) current J(z). Using a twist parameter θ, the spectral flow
operator acting on the Hilbert space H0 is denoted by
Uθ : H0 → Hθ ,
–4–
(2.5)
JHEP01(2024)130
An N = 2 superconformal theory has stress tensor T (z), two supercurrents G1 (z), G2 (z),
and SO(2) ∼
= U(1) current J(z) describing the R-symmetry. It is convenient to define
√
G± (z) = G1 (z) ± i G2 (z) / 2 .
(2.1)
where Hθ is the Hilbert space quantized on the twisted boundary condition with the parameter
θ. Under the spectral flow, the Virasoro generators and the modes of the U(1) current and
the supercurrents transform by ([36])
Uθ Lm Uθ−1 = Lm + θJm +
n 2
θ δm,0 ,
6
n
θδm,0 ,
3
= G−
r−θ .
Uθ Jm Uθ−1 = Jm +
−1
Uθ G+
= G+
r Uθ
r+θ ,
−1
Uθ G−
r Uθ
(2.6)
Accordingly, a state |h, Q⟩ ∈ H0 with conformal weight h and U(1) charge Q transforms
into the twisted state
(2.7)
For θ ∈ Z + 1/2, the spectral flow connects the NS and R sectors. Then, the NS sector
and the R sector are isomorphic in N = 2 superconformal theories [35]. For θ ∈ Z, the
spectral flow takes NS to NS and R to R.
A supersymmetric theory has a unitarity bound on the conformal weight in the R sector.
From the N = 2 algebra, we have the relation
n
o
+
G−
= 2L0 − n/12 .
0 , G0
(2.8)
Since the left-hand side is a positive operator, any state in the R sector must satisfy the
bound h ≥ n/24. One can show that the bound is saturated (h = n/24) if and only if a
state satisfies the condition
G±
m |ϕ̃⟩ = 0 ,
(m ≥ 0) .
(2.9)
To see this, suppose |ϕ̃⟩ satisfies h = n/24, which implies that
n
2
o
−
+
+
⟨ϕ̃| G−
0 , G0 |ϕ̃⟩ = 0 = G0 |ϕ̃⟩ + G0 |ϕ̃⟩
2
.
(2.10)
−
Hence we have G+
0 |ϕ̃⟩ = G0 |ϕ̃⟩ = 0. Note that the operators Jm (m > 0), which lower the
L0 eigenvalue, must annihilate a state |ϕ̃⟩ saturating h = n/24. Otherwise, the unitarity
bound does not hold. Therefore, using the N = 2 commutation relation
h
i
±
Jm , G±
0 = ±Gm ,
(2.11)
we obtain G±
m |ϕ̃⟩ = 0 for m ≥ 0. These states have the lowest energy in the R sector, so
they are called the Ramond ground states.
Since the NS and R sectors are isomorphic, there exist states in the NS sector, which are
connected to the Ramond ground states by the spectral flow. Let us consider the spectral
flow with θ = −1/2. Then, the supercurrents transform into
−1
+
U−1/2 G+
r U−1/2 = Gr−1/2 ,
−1
−
U−1/2 G−
r U−1/2 = Gr+1/2 .
(2.12)
Using these relations, the condition (2.9) for the Ramond ground states becomes
−1
±
U−1/2 G±
m U−1/2 U−1/2 |ϕ̃⟩ = Gm∓1/2 |ϕ⟩ = 0
–5–
(m ≥ 0) ,
(2.13)
JHEP01(2024)130
n
n
Uθ |h, Q⟩ = h − θQ + θ2 , Q − θ ∈ Hθ .
6
3
where |ϕ⟩ = U−1/2 |ϕ̃⟩ is in the NS sector. Hence, the resulting state |ϕ⟩ satisfies the conditions
G+
−1/2 |ϕ⟩ = 0 ,
+
G−
m+1/2 |ϕ⟩ = Gm+1/2 |ϕ⟩ = 0
(m ≥ 0) .
(2.14)
n
o
+
G−
1/2 , G−1/2 |ϕ⟩ = (2L0 − J0 ) |ϕ⟩ .
(2.15)
As the left-hand side is a positive operator, any state in the NS sector satisfies the bound
h≥
Q
.
2
(2.16)
Chiral primary states saturate the bound and have the conformal weight h = Q/2. Similarly,
an anti-chiral primary state with charge Q has the conformal weight h = −Q/2. Additionally,
we can show that any state with h = Q/2 is both chiral and primary. We can also obtain
other bounds on chiral primary states from the N = 2 algebra
n
o
+
G−
3/2 , G−3/2 = 2L0 − 3J0 + 2n/3 .
(2.17)
Since the left-hand side is a positive operator and any chiral primary state satisfies h = Q/2,
we have the inequality
(2.18)
h ≤ n/6 .
Via the state-operator isomorphism, the operators corresponding to chiral primary states
are called chiral primary fields. Using the inequalities (2.16) and (2.18), it is straightforward
to see that the OPEs between chiral primary fields do not have singular terms and are welldefined without regularizations [36]. Such OPEs are closed in chiral primary fields. Hence,
the space of chiral primary fields is called a chiral ring with the product structure by the
operator product. In section 3.4, we identify the spectral flow operator, the Ramond grounds
states, and the chiral ring in fermionic CFTs constructed from classical error-correcting codes.
2.2
Elliptic genera as weak Jacobi forms
Let us consider a supersymmetric CFT with the central charge n = 6m (m ∈ Z) on the torus
with the modulus τ = τ1 + iτ2 . The Witten index is defined by the Ramond sector partition
function with the insertion of the fermion parity (−1)F
n
h
n
i
TrHR (−1)F q L0 − 24 q̄ L̄0 − 24 ,
–6–
(2.19)
JHEP01(2024)130
These states are called chiral primary states [36], which can be obtained from the Ramond
ground states by the spectral flow with θ = −1/2. In the opposite direction, the spectral
flow with θ = 1/2 transforms chiral primary states into the Ramond ground states. Similarly,
anti-chiral primary states are defined by only replacing G+ with G− in (2.14) and they are
mapped to the Ramond ground states by the spectral flow θ = −1/2.
Chiral primary states are subject to a strong constraint on the conformal weight and
the U(1) charge. From the N = 2 algebra, we have the relation
where q = e2πiτ . For an N = 2 superconformal theory, the Witten index can be refined
to an elliptic genus using the U(1) current J(z) of the N = 2 algebra.2 Following [49], we
define the elliptic genus by
n
h
n
i
ZEG (τ, z) = TrHR (−1)F q L0 − 24 q̄ L̄0 − 24 y J0 ,
(2.20)
where J0 is the zero mode of J(z) and y = e2πiz with z ∈ C.
If the left- and right-moving R charges (Q, Q̄) satisfy Q − Q̄ ∈ Z for any state, we can
define the fermion parity F = FL + FR in terms of the U(1) current [36, 49, 50]
(2.21)
In this paper, we restrict our attention to theories with integral U(1) charges. Then, the
modularity properties together with the spectral flow invariance and the unitarity bound
in the R sector can be summarized that the elliptic genus ZEG (τ, z) is a weak Jacobi form
of weight 0 and index m [43].
Definition ([51])
A weak Jacobi form ϕ(τ, z) of weight w and index m ∈ Z satisfies the transformation laws
aτ + b
z
ϕ
,
cτ + d cτ + d
2
cz
w 2πim cτ
+d
= (cτ + d) e
ϕ(τ, z + lτ + l′ ) = e−2πim(l
2 τ +2lz)
a b
c d
ϕ(τ, z) ,
!
∈ SL(2, Z) ,
l, l′ ∈ Z .
ϕ(τ, z) ,
(2.22)
(2.23)
where q = e2πiτ , y = e2πiz and has a Fourier expansion in terms of the integer coefficients
c(n, l)
ϕ(τ, z) =
X
(2.24)
c(n, l) q n y l ,
n≥0,l∈Z
with c(n, l) = (−1)w c(n, −l).
The modular transformation (2.22) is generated by the T and S transformations:
T : (τ, z) → (τ + 1, z) ,
ϕ(τ + 1, z) = ϕ(τ, z) ,
1 z
S : (τ, z) → − ,
τ τ
1 z
ϕ − ,
τ τ
,
11
01
2
w 2πim zτ
=τ e
ϕ(τ, z) ,
!
0 −1
1 0
(2.25)
,
!
,
(2.26)
where the matrices are in SL(2, Z). The shift of z (2.23) is generated by
(τ, z) → (τ, z + 1) ,
(τ, z) → (τ, z + τ ) ,
ϕ(τ, z + 1) = ϕ(τ, z) ,
ϕ(τ, z + τ ) = e
−2πim(τ +2z)
(2.27)
ϕ(τ, z) .
(2.28)
These generators are not independent since the shift by τ (2.28) can be derived by combining
the S transformation (2.26) and the shift by integer (2.27). In addition, if ϕ(τ, z) can be
2
For another refinement called a mod-2 elliptic genus, see e.g. [48].
–7–
JHEP01(2024)130
(−1)F = exp [iπ(J0 − J¯0 )]
written as (2.24), then the T transformation (2.25) and (2.27) are automatically satisfied.
The coefficients condition c(n, l) = (−1)w c(n, −l) can be derived from S 2 , i.e., (τ, z) →
(τ, −z). Thus, to prove that ϕ(τ, z) is a weak Jacobi form, it is enough to check the S
transformation (2.26) and the Fourier expansion (2.24).
A weak Jacobi form ϕ(τ, z) can be decomposed into a set of vector-valued modular forms
hµ (τ ) and theta functions θm,µ (τ, z) with µ ∈ Z/2mZ:
ϕ(τ, z) =
hµ (τ ) θm,µ (τ, z) .
X
(2.29)
µ ∈ Z/2mZ
hµ (τ ) =
cµ (n) q n/4m ,
X
θm,µ (τ, z) =
X
ql
2 /4m
yl ,
(2.30)
l∈Z
l = µ mod 2m
n = −µ2 mod 4m
2
with cµ (n) = (−1)2ml c( n+l
4m , l) and l = µ mod 2m. There has been an attempt to construct a
Jacobi form from a prescribed set of coefficients [46] (one can refer to [52] as math literature).
For our purposes, it is more convenient to use the following expression. A weak Jacobi form
of even weight can also be expressed as a polynomial consisting of four generators ([40, 51])
E4 (τ ) , E6 (τ ) , ϕ0,1 (τ, z) , ϕ−2,1 (τ, z)
(2.31)
(w, m) = (4, 0) , (6, 0) , (0, 1) , (−2, 1) .
(2.32)
of weights and indices
Here, E4 and E6 are the Eisenstein series
E4 (τ ) = 1 + 240
E6 (τ ) = 1 − 504
∞
X
n3 q n
1 − qn
n=1
∞
X
n5 q n
n=1
1 − qn
= 1 + 240q + 2160q 2 + O(q 3 ) ,
(2.33)
= 1 − 504q − 16632q + O(q ) ,
2
3
and ϕ0,1 and ϕ−2,1 are given by
ϕ0,1 (τ, z) = 4
θ2 (τ, z)2 θ3 (τ, z)2 θ4 (τ, z)2
+
+
θ2 (τ, 0)2 θ3 (τ, 0)2 θ4 (τ, 0)2
!
= y + 10 + y −1 + (10y 2 − 64y + 108 − 64y −1 + 10y −2 )q + O(q 2 ) ,
θ1 (τ, z)2
ϕ−2,1 (τ, z) =
η(τ )6
(2.34)
= y − 2 + y −1 + (−2y 2 + 8y − 12 + 8y −1 − 2y −2 )q + O(q 2 ) .
More explicitly, a weak Jacobi form ϕ(τ, z) of weight w ∈ 2Z and index m ∈ Z can be written as
ϕ(τ, z) =
X
cq,r,s,t (E4 )q (E6 )r (ϕ0,1 )s (ϕ−2,1 )t ,
cq,r,s,t ∈ R
(2.35)
where the sum is taken for q, r, s, t ∈ Z≥0 such that 4q + 6r − 2t = w and s + t = m. When
w = 0, the number of terms is round((m + 3)2 /12) where round(x) = ⌊x + 12 ⌋.
–8–
JHEP01(2024)130
where
According to [40], the polar region of index m is defined by
n
o
P (m) = (n, l) ∈ Z2 | 1 ≤ l ≤ m , 0 ≤ n , p := 4mn − l2 < 0 .
(2.36)
The term q n y l such that (n, l) ∈ P (m) is called the polar term. For example, the polar
terms of small even m are
m = 2 : y2, y
m = 4 : y ,y ,y ,y
4
3
2
5
4
3
2
6
5
(2.37)
· · · 4 terms,
(2.38)
· · · 8 terms.
(2.39)
A weak Jacobi form ϕ(τ, z) of weight 0 and index m is uniquely determined by the coefficients
of the polar terms since the number of the polar terms of index m is greater than or equal
to the number of the independent terms in (2.35) [40, 53].
2.3
Extremal elliptic genera
The NS vacuum irreducible character of the N = 2 algebra at the central charge n = 6m is
n
h
L0 − 24 2πizJ0
ch(m)
e
vac (τ, z) = TrV0 q
i
1
=q
−m
4
1
∞
Y
(1 + yq k+ 2 )(1 + y −1 q k+ 2 )
(1 − q)
(1 − q k )2
k=1
,
(2.40)
where V0 denotes the vacuum Hilbert space. Using spectral flow, we get the corresponding
terms for the R sector as
SFθ ch(m)
vac
X
θ∈ 12 +Z
:=
X
1
τ, z +
2
n
h
1
TrV0 Uθ q L0 − 24 e2πi(z+ 2 )J0 Uθ−1
i
(2.41)
θ∈ 12 +Z
= (−1)m (1 − q) y m
∞
Y
(1 − yq k+1 )(1 − y −1 q k )
(1 − q k )2
k=1
+ (Non-polar terms) .
Note that only SF1/2 ch(m)
vac is explicitly written since all terms from θ ̸= 1/2 are non-polar.
According to [40], we define the extremal elliptic genus as the “closest” weak Jacobi form to
SF1/2 ch(m)
vac . More precisely, we call a weak Jacobi form ϕ(τ, z) of weight 0 and index m as
the extremal elliptic genus when the coefficients of the polar terms are equal to SF1/2 ch(m)
vac .
(m)
The polar terms of SF1/2 chvac at small even m are
m = 2 : y 2 + 0y ,
(2.42)
m = 4 : y 4 + 0(y 3 + y 2 + y) ,
(2.43)
m = 6 : y + 0(y + y + y + y + y) + q(y − y )
6
5
4
3
2
6
5
(2.44)
where the polar terms with coefficients 0 are also shown. As already mentioned, we can
uniquely determine the weak Jacobi form from these polar terms. For example, at m = 2, a
–9–
JHEP01(2024)130
m = 6 : y , y , y , y , y , y, qy , qy
6
· · · 2 terms,
weak Jacobi form of weight 0 can be decomposed into ϕ20,1 and ϕ2−2,1 E4 as (2.35), and (2.42)
is satisfied when
1
5
(m=2)
Zext (τ, z) = ϕ20,1 + ϕ2−2,1 E4
6
6
(2.45)
= y 2 + 22 + y −2 + O(q) .
Similarly, at m = 4, (2.43) is satisfied when
(m=4)
Zext
1 4
1
11
67 4
ϕ + ϕ2 ϕ2 E4 + ϕ0,1 ϕ3−2,1 E6 +
ϕ
E2
432 0,1 8 0,1 −2,1
27
144 −2,1 4
= y 4 + 46 + y −4 + O(q) .
(τ, z) =
(2.46)
n
o
P (m) = (n, l) ∈ Z2 | 1 ≤ l ≤ m , 0 ≤ n , 4mn − l2 ≤ −β .
(2.47)
Following [40], we call a weak Jacobi form ϕ(τ, z) of weight 0 and index m as a β-extremal
elliptic genus when the coefficients of the terms in the β-truncated polar region are equal to
SF1/2 ch(m)
vac . Note that a β-extremal elliptic genus is not unique for general β.
3
Elliptic genera from classical codes
In this section, we provide a way of computing the elliptic genera of fermionic code CFTs.
Section 3.1 briefly reviews the construction of fermionic CFTs from classical p-ary codes.
Choosing the U(1) current consistent with the N = 2 algebra from fermionic code CFTs
in section 3.2, we obtain the U(1)-graded partition function that is invariant under the
modular transformation and the spectral flow in section 3.3. Also, we identify the spectral
flow operator and chiral ring of fermionic code CFTs in section 3.4.
3.1
Fermionic code CFTs
In this subsection, we review the construction of fermionic CFTs from classical codes over
finite fields Fp = {0, 1, · · · , p − 1} of prime order p [10] (see [9] for the ternary case (p = 3)).
A p-ary linear code of length n can be defined as a subspace C of Fnp and the dimension of
C is denoted by k ≤ n (see e.g. [29, 54–60] for more details). It is convenient to introduce
a generator matrix G to define a linear code. A generator matrix G is a k × n matrix of
rank k and generates a linear code
C=
n
c ∈ Fnp c = x G , x ∈ Fkp
o
(3.1)
.
The inner product between elements v = (v1 , · · · , vn ), w = (w1 , · · · , wn ) ∈ Fnp is given by
P
the standard Euclidean product v · w = ni=1 vi wi ∈ R. Associated with the inner product,
we define the dual code of C by
C⊥ =
n
v ∈ Fnp c · v = 0
– 10 –
mod p , c ∈ C
o
.
(3.2)
JHEP01(2024)130
Note that such a weak Jacobi form may not exist for general m, and even if it does, there
may be no actual CFT that realizes the extremal elliptic genus. In fact, at m = 6, we
cannot get (2.44) from (2.35) with any coefficients, which means the non-existence of the
extremal elliptic genus.
Therefore, a relaxed definition of near-extremal elliptic genus is also useful. The βtruncated polar region for β > 0 is defined by
We call C self-orthogonal if C ⊂ C ⊥ . Then, the dimension of C must be k ≤ n/2. Also,
we call C self-dual if C = C ⊥ . In this case, C must satisfy k = n/2, then n ∈ 2Z. For the
binary case (p = 2), we can further classify self-orthogonal codes into two classes. A binary
linear code C is doubly-even if any c ∈ C satisfies c · c ∈ 4Z. On the other hand, C is called
singly-even if there exists an element c ∈ C such that c · c ∈ 4Z + 2.
A linear code C ⊂ Fnp can construct a Euclidean lattice via Construction A [28, 29, 60]
(
Λ(C) =
c + pm
∈ Rn c ∈ C, m ∈ Zn
√
p
)
,
(3.3)
Λ ∗ = λ ′ ∈ Rn | λ · λ ′ ∈ Z , λ ∈ Λ .
(3.4)
A lattice is called integral if Λ ⊂ Λ∗ and self-dual if Λ = Λ∗ . Additionally, for an integral
lattice Λ, we call Λ even if any element λ ∈ Λ has an even norm λ · λ ∈ 2Z, and odd if there
exists an element λ ∈ Λ such that λ · λ ∈ 2Z + 1.
The Construction A lattice Λ(C) inherits the property of a linear code C. The following
propositions are essential to constructing fermionic code CFTs.
Proposition 3.1 ([10])
For an odd prime p, the Construction A lattice Λ(C) is odd self-dual if and only if a linear
code C ⊂ Fnp is self-dual.
Proposition 3.2 ([10])
For p = 2, the Construction A lattice Λ(C) is odd self-dual if and only if a linear code C ⊂ Fn2
is singly-even self-dual.
These propositions guarantee that odd self-dual lattices can be constructed from self-dual
codes for an odd prime p and singly-even self-dual codes for the binary case (p = 2). In what
follows, we focus on such classes of linear codes and the corresponding odd self-dual lattices
to yield fermionic CFTs. Specifically, we define the NS sector of a fermionic code CFT by
using the Construction A lattice Λ(C), and the R sector by the shadow of Λ(C).
To introduce the shadow of a lattice, consider an element χ ∈ Λ(C) ⊂ Rn such that
χ·λ=λ·λ
mod 2 ,
(3.5)
for any λ ∈ Λ(C). Such an element χ ∈ Λ(C) is called characteristic [29, 33, 34]. A
characteristic vector always exists for an odd self-dual lattice and the choice of a characteristic
vector is not unique to a lattice. It is known that all the characteristic vectors have norm
χ · χ = n mod 8 [33, 61]. Since self-dual codes C ⊂ Fnp exist only for n ∈ 2Z, characteristic
vectors of the Construction A lattice Λ(C) have even norms:
χ · χ ∈ 2Z ,
χ ∈ Λ(C) .
(3.6)
Using a characteristic vector χ ∈ Λ(C), we can define the shadow of Λ(C) by
S(Λ(C)) = Λ(C) +
χ
χ
= λ+
λ ∈ Λ(C) .
2
2
– 11 –
(3.7)
JHEP01(2024)130
which has the standard Euclidean inner product denoted by ·. As in classical codes, the
dual lattice of Λ ⊂ Rn is
The shadow of a lattice is not a lattice since it is not closed under addition. While there
are several choices for a characteristic vector, the shadow itself does not depend on the
choice of a characteristic vector χ ∈ Λ(C).
We construct the NS sector of a fermionic CFT from the Construction A lattice using the
lattice construction of chiral CFTs (see e.g. [62, 63]).3 Let X(z) be an n-dimensional chiral
boson. We fix the normalization by the OPE X(z)X(w) ∼ − log(z − w). To define the NS
sector, we associate an element λ ∈ Λ(C) of the Construction A lattice to the vertex operator
Vλ (z) = : eiλ·X(z) : ,
λ ∈ Λ(C) ,
(3.8)
Li (z) = i ∂X i (z) ,
T (z) = −
1
: ∂X(z) · ∂X(z) : ,
2
Vλ (z) = : eiλ·X(z) : .
(3.9)
Via the state-operator isomorphism, vertex operators Vλ (z) are mapped to momentum states
|λ⟩, which are eigenstates of the Virasoro generator L0 with eigenvalue h = λ2 /2. Combining
with the excitation by the bosonic oscillators αki , the NS sector of a fermionic code CFT is
n
i1
ir
HNS (C) = α−k
· · · α−k
|λ⟩
r
1
o
λ ∈ Λ(C) , r ∈ Z≥0 ,
(3.10)
where the oscillators are in negative modes (k1 , · · · , kr > 0). The conformal weight of
P
i1
ir
α−k
· · · α−k
|λ⟩ is h = λ2 /2 + rj=1 kj , which is an integer if λ2 ∈ 2Z and a half-integer
r
1
if λ2 ∈ 2Z + 1. From the spin-statics theorem, such a state is bosonic when λ2 ∈ 2Z and
fermionic when λ2 ∈ 2Z + 1. Thus, we can define the fermion parity in the NS sector by
(−1)F = (−1)χ·λ ,
(3.11)
where χ ∈ Λ(C) is a characteristic vector satisfying (3.5). This motivates us to divide the
Construction A lattice into two parts: Λ(C) = Λ0 ∪ Λ2 where
Λ0 = {λ ∈ Λ(C) | χ · λ = 0
mod 2} ,
Λ2 = {λ ∈ Λ(C) | χ · λ = 1
mod 2} .
(3.12)
On the other hand, the R sector can be defined by associating each element in the
shadow S(Λ(C)) to the vertex operator
χ
χ
Vλ+ χ (z) = : ei(λ+ 2 )·X(z) : ,
λ + ∈ S(Λ(C)) .
(3.13)
2
2
The R sector of a fermionic code CFT is
χ
χ
i1
ir
HR (C) = α−k
·
·
·
α
λ
+
λ
+
∈
S(Λ(C))
,
r
∈
Z
(3.14)
≥0 ,
−kr
1
2
2
where the bosonic oscillators are in negative modes (k1 , · · · , kr > 0). In the R sector, the
fermion parity is well-defined only up to an overall factor. In analogy with the NS sector (3.11),
i1
ir
we give the fermion parity of α−k
· · · α−k
λ + χ2 ∈ HR (C) by
r
1
χ
(−1)F = (−1)χ·(λ+ 2 ) .
(3.15)
Recently, the classification program of chiral fermionic CFTs has developed. Up to now, the classification
has been completed for central charges less than or equal to 24. See [64–66].
3
– 12 –
JHEP01(2024)130
where we omit the cocycle factor because it does not matter for our purpose. The fundamental
operators in a fermionic code CFT can be given by
Note that χ · (λ + χ2 ) is an integer from (3.6). Thus, we can naturally divide the shadow
S(Λ(C)) into two parts: S(Λ(C)) = Λ1 ∪ Λ3 where
χ
Λ1 = λ + ∈ S(Λ(C)) χ · λ +
2
χ
Λ3 = λ + ∈ S(Λ(C)) χ · λ +
2
χ
=0
2
χ
=1
2
mod 2
mod 2
,
(3.16)
.
Let us place a fermionic code CFT on the torus with the modulus τ = τ1 + iτ2 . Then,
the torus has the spacial and timelike cycles
timelike : w ∼ w + 2πτ ,
(3.17)
where w is the cylindrical coordinate. Depending on the periodicity (correspondingly, the
choice of spin structures), we have the four partition functions
n
h
i
ZNS (τ ; Λ(C)) = TrHNS q L0 − 24 =
h
F
ZN
fS (τ ; Λ(C)) = TrHNS (−1) q
h
ZR (τ ; Λ(C)) = TrHR q
n
L0 − 24
i
1
η(τ )n
n
L0 − 24
1
η(τ )n
2
λ ∈ Λ(C)
q
1
(λ+ χ
)2
2
2
(3.18)
,
λ ∈ Λ(C)
i
1
η(τ )n
1
Here, η(τ ) is the Dedekind eta function η(τ ) = q 24
can be collectively denoted by
1
η(τ )n
1
(−1)χ·λ q 2 λ ,
X
X
F L0 − 24
ZR
=
e (τ ; Λ(C)) = TrHR (−1) q
Z α,β (τ ; Λ(C)) =
2
q 2λ ,
λ ∈ Λ(C)
=
1
=
η(τ )n
n
h
i
1
X
X
χ
1
χ 2
(−1)χ·(λ+ 2 ) q 2 (λ+ 2 ) .
λ ∈ Λ(C)
Q∞
m=1 (1 − q
χ
m ).
1
These partition functions
χ 2
(−1)βχ·(λ+α 2 ) q 2 (λ+α 2 )
(3.19)
R : (1, 0) ,
(3.20)
X
λ ∈ Λ(C)
where (α, β) corresponds to each sector by
NS : (0, 0) ,
′
f : (0, 1) ,
NS
e : (1, 1) .
R
′
Note that Z α,β (τ ; Λ(C)) = Z α ,β (τ ; Λ(C)) for integers α ≡ α′ , β ≡ β ′ mod 2.
On the torus, any modular transformation is generated by the modular S transformation: τ → −1/τ and the modular T transformation: τ → τ + 1. Under these modular
transformations, the partition functions behave as
Z α,β (τ + 1; Λ(C)) = eiπ(3α
2 −1) n
12
n
Z α,α+β+1 (τ ; Λ(C)) ,
Z α,β (−1/τ ; Λ(C)) = (−1)αβ 2 Z β,α (τ ; Λ(C)) .
(3.21)
The phases caused by the modular transformations can be canceled by coupling with 2n
right-moving Majorana-Weyl fermions. Then, the partition functions of a fermionic code
CFT show the expected modular transformation laws.
– 13 –
JHEP01(2024)130
spacial : w ∼ w + 2π ,
3.2
Constraints on U(1) current
Li (z) = i ∂X i (z) ,
(i = 1, 2, · · · , n) .
(3.22)
Let us consider a linear combination of these U(1) currents and denote it by
J(z) =
n
X
ai Li (z) ,
(3.23)
i=1
where a = (a1 , a2 , · · · , an ) ∈ Rn . Although J(z) gives a U(1) current in a fermionic code
CFT, such a choice of the coefficients does not always realize the R-symmetry. We figure out
some conditions for the U(1) current to describe the R-symmetry and explicitly construct
the U(1) current J(z) satisfying those constraints.
The first constraint comes from the consistency with the N = 2 superconformal algebra (2.2). Since we have the OPE between a pair of the current J(z)
J(z)J(w) ∼
a·a
,
(z − w)2
(3.24)
we require J(z) to satisfy the norm condition
a·a=
n
,
3
(3.25)
where n is the central charge.
Another condition comes from the relationship (2.21) between the U(1) charge Q and
the fermion parity (−1)F . As shown in the previous section, both the NS and R sectors of
a fermionic code CFT are built out of the vertex operators
(
Vκ (z) = : eiκ·X(z) : ,
κ∈
Λ(C)
(NS sector)
S(Λ(C))
(R sector) .
(3.26)
Taking the OPE between the U(1) current J(z) and a vertex operator Vκ (w), we obtain
J(z) Vκ (w) ∼
– 14 –
a·κ
.
z−w
(3.27)
JHEP01(2024)130
We have introduced chiral fermionic CFTs constructed from classical codes using Euclidean
lattices. It has been known that all the fermionic code CFTs have N = 2 supersymmetry in
the ternary case (p = 3) [9]. Also, there is strong evidence for the presence of supersymmetry
in a class of fermionic code CFTs in other cases (p ̸= 3) [10]. For an N = 2 supersymmetric
theory, the elliptic genus can be computed by the partition function with the U(1) charge
inserted. Here, the corresponding U(1) current must be consistent with the N = 2 algebra
and describes the R-symmetry. We need to identify the U(1) current in fermionic code
CFTs to compute their elliptic genera. This section aims to find the U(1) current by solving
constraints for a U(1) current to realize the R-symmetry.
By construction, a fermionic code CFT with the central charge n always has the following
U(1) currents:
Thus, each vertex operator Vκ (z) has the charge Q = a · κ with respect to the U(1) current
J(z). On the other hand, combining (3.11) and (3.15), the fermion number of Vκ (z) is
F =χ·κ
mod 2 ,
(3.28)
eπi Q = (−1)F ,
(3.29)
which results in Q = F mod 2. From (3.27) and (3.28), the U(1) charge of Vκ (z) is given by
Q = a · κ and the fermion number is F = χ · κ mod 2. Therefore, we obtain an additional
constraint on the coefficients a
a·κ=χ·κ
mod 2 ,
(3.30)
where κ ∈ Λ(C) for the NS sector and κ ∈ S(Λ(C)) for the R sector.
In the NS sector, the constraint requires the coefficients a ∈ Rn to satisfy
a·λ=χ·λ=λ·λ
mod 2
λ ∈ Λ(C) ,
(3.31)
where we used the definition of a characteristic vector χ ∈ Λ(C). This requires a ∈ Rn to
be a characteristic vector of Λ(C). Since characteristic vectors constitute a coset of 2Λ(C)
in Λ(C): χ + 2Λ(C) = 2S(Λ(C)) [61], the vector a can be written as
a = χ + 2λ′ ,
λ′ ∈ Λ(C) .
(3.32)
On the other hand, a vertex operator in the R sector takes the form Vλ+ χ (z) where
2
λ ∈ Λ(C). In this case, the constraint becomes
χ
a· λ+
2
χ
=χ· λ+
2
mod 2 ,
λ ∈ Λ(C) .
(3.33)
Assuming the constraint (3.32) in the NS sector, the constraint coming from the R sector
returns χ · λ′ = 0 mod 2, from which we conclude
a = χ + 2λ′ ,
λ ′ ∈ Λ0 .
(3.34)
Note that χ is a characteristic vector that fixes the fermion parity.
From the above discussion, we essentially have two constraints (3.25) and (3.34) on
the choice of the U(1) current. Now we propose to define the U(1) current J(z) by taking
λ′ = 0 ∈ Λ0 . Accordingly, we fix the coefficients a ∈ Rn by taking the same vector as χ:
a = χ.
– 15 –
(3.35)
JHEP01(2024)130
where χ ∈ Λ(C) is a characteristic vector of Λ(C).
Let us assume that the U(1) charge Q of each state is always integral (we check the
consistency soon in this subsection). From (2.21), the fermion parity (−1)F is the Z2 subgroup
of the R-symmetry in N = 2 theories. Then, the fermion number F and the U(1) charge
Q must be related by
Then, to meet the constraints, we only need to choose a characteristic vector χ ∈ Λ(C)
satisfying χ · χ = n/3. The corresponding U(1) current is
J(z) =
n
X
χi Li (z) .
(3.36)
i=1
The Construction A lattice Λ(C) ⊂ Rn does not necessarily have a characteristic vector
χ ∈ Λ(C) with χ · χ = n/3. Even if present, such a characteristic vector is not unique to Λ(C)
in general. For later convenience, we define a set of characteristic vectors with norm n/3 by
(3.37)
Note that this set becomes empty unless n ∈ 12Z since χ · χ = n mod 8. Hence, we only
have to specify an element χ ∈ Xn/3 to define the U(1) current J(z). We will see that, for the
choice of the U(1) current (3.36), the U(1)-graded partition function exhibits the expected
modular invariance together with the spectral flow invariance.
Once we pick up a characteristic vector χ ∈ Xn/3 for defining the fermion parity, the
conditions on the U(1) current are satisfied by choosing the coefficients a ∈ Rn according
to (3.35). For the choice of J(z), a vertex operator Vλ (z) in the NS sector has the U(1)
charge Q = χ · λ and Vλ+ χ (z) in the R sector has the U(1) charge Q = χ · λ + χ2 . These
2
U(1) charges are integral because all the characteristic vectors of the Construction A lattice
have even norms χ · χ ∈ 2Z as in (3.6). This is consistent with the assumption of (2.21)
that we have used to determine the U(1) current J(z).
Using the fact that the bosonic oscillators are neutral with respect to the U(1) current
i1
ir
J(z), the state-operator isomorphism tells us that an NS state α−k
· · · α−k
|λ⟩ ∈ HNS (C)
r
1
has the following conformal weight and U(1) charge:
h=
r
λ2 X
+
kj ,
2
j=1
Q = χ · λ,
(3.38)
i1
ir
On the other hand, a Ramond state α−k
· · · α−k
|λ + χ2 ⟩ ∈ HR (C) has
r
1
h=
1
χ
λ+
2
2
2
+
r
X
kj ,
j=1
Q=χ· λ+
χ
2
.
(3.39)
Here, kj (j = 1, 2, · · · , r) are positive integers. The next section reveals that the Ramond
partition function with the corresponding U(1) charge inserted shows the same modular
transformation law as weak Jacobi forms.
3.3
Elliptic genera of fermionic code CFTs
In this section, based on the choice of U(1) current (3.36), we compute the Ramond-Ramond
partition function with the corresponding U(1) charge inserted, which can be expected to
return elliptic genera for N = 2 superconformal theories. Furthermore, we show that, under
the modular transformation and the spectral flow, the partition functions behave in the
same manner as weak Jacobi forms.
– 16 –
JHEP01(2024)130
Xn/3 = { χ ∈ 2S(Λ(C)) | χ · χ = n/3 } .
Consider a fermionic CFT constructed from a self-dual code C ⊂ Fnp (singly-even self-dual
code for p = 2) and pick a characteristic vector χ ∈ Xn/3 . Using the U(1) current given
by (3.36), we define the U(1)-graded partition function
n
1
h
i
ZEG (τ, z ; Λ(C), χ) = TrHR e2πi (z+ 2 ) J0 q L0 − 24 ,
(3.40)
where Jn (n ∈ Z) is the mode expansion of the U(1) current J(z) and q = e2πiτ . The
conformal weight and U(1) charge of each state in the Ramond sector are given by (3.39).
Then, the partition function can be written as
1
η(τ )n
X
χ
1
χ 2
1
e2πi (z+ 2 ) χ·(λ+ 2 ) q 2 (λ+ 2 ) ,
(3.41)
λ ∈ Λ(C)
where η(τ ) is the Dedekind eta function. For an N = 2 superconformal theory, one can
expect that the graded partition function gives the elliptic genus and becomes a weak Jacobi
form of weight 0 and index n/6. The following proposition guarantees that the partition
function exhibits the expected modular transformation for n ∈ 12Z.
Proposition 3.3
Let Λ(C) be an odd self-dual lattice of rank n ∈ 12Z and χ ∈ Xn/3 . Then, the partition
function ZEG (τ, z ; Λ(C), χ) satisfies
ZEG
cz 2
aτ + b
z
,
; Λ(C), χ = e2πim cτ +d ZEG (τ, z ; Λ(C), χ) ,
cτ + d cτ + d
ZEG (τ, z + lτ + l′ ; Λ(C), χ) = e−2πim(l
where
a b
c d
2 τ +2lz)
(3.42)
ZEG (τ, z ; Λ(C), χ) ,
(3.43)
∈ SL(2, Z) and l, l′ ∈ Z, and m = n/6.
Proof. The modular transformation by SL(2, Z) is generated by the modular T transformation
τ → τ + 1 and S transformation τ → −1/τ . First, we consider the T transformation
ZEG (τ + 1, z ; Λ(C), χ) =
1
η(τ + 1)n
χ 2
X
χ
1
1
χ 2
eπi(λ+ 2 ) e2πi (z+ 2 ) χ·(λ+ 2 ) q 2 (λ+ 2 ) ,
λ ∈ Λ(C)
(3.44)
where we used q → e2πi q under the T transformation. By definition of a characteristic vector
χ 2
χ, the phase factor eπi(λ+ 2 ) becomes
χ 2
πi
πin
eπi(λ+ 2 ) = eπi(λ·λ+λ·χ) · e 4 χ·χ = e 12 .
(3.45)
This exactly cancels an overall phase coming from the T transformation of η(τ ). Then, we
conclude the invariance of ZEG (τ, z ; Λ(C), χ) under τ → τ + 1, which agrees with (3.42).
Second, we consider the modular S transformation by fixing the parameters a = d = 0,
b = −1, c = 1. Then it implements τ → −1/τ and z → z/τ :
ZEG (−1/τ, z/τ ; Λ(C), χ) =
1
η(−1/τ )n
X
λ ∈ Λ(C)
– 17 –
z
1
χ
πi
χ 2
e2πi ( τ + 2 ) χ·(λ+ 2 ) e− τ (λ+ 2 ) .
(3.46)
JHEP01(2024)130
ZEG (τ, z ; Λ(C), χ) =
Since the Fourier transform is given by
fˆ(ω) =
Z
z
Rn
χ
1
χ 2
πi
dn λ e−2πiλ·ω e2πi ( τ + 2 ) χ·(λ+ 2 ) e− τ (λ+ 2 ) ,
z2
n
χ 2
1
(3.47)
χ
1
= (−iτ ) 2 e2πim τ q 2 (ω− 2 ) e2πi (z+ 2 ) χ·(−ω+ 2 ) ,
the Poisson summation formula tells us
ZEG (−1/τ, z/τ ; Λ(C), χ) =
1
η(−1/τ )n
2
2πim zτ
ω ∈ Λ(C)∗
X
q
1
(ω+ χ
)2
2
2
(3.48)
e
2πi (z+ 12 ) χ·(ω+ χ
)
2
,
ω ∈ Λ(C)
√
where we used χ · χ = n/3 ∈ 4Z, the modular S transformation η(−1/τ ) = −iτ η(τ ) and
self-duality Λ(C)∗ = Λ(C), and we changed the variable ω → −ω. Hence, we conclude the
modular S transformation (3.42).
Finally, we consider the transformation (3.43) under the spectral flow:
e2πim(l
2 τ +2lz)
ZEG (τ, z + lτ + l′ ) =
1
η(τ )n
1
=
η(τ )n
X
χ
1
χ 2
1
e2πi (z+ 2 ) χ·{(λ+lχ)+ 2 } q 2 {(λ+lχ)+ 2 } ,
λ ∈ Λ(C)
X
e
2πi(z+ 12 )χ·(λ+ χ
)
2
q
1
(λ+ χ
)2
2
2
(3.49)
,
λ ∈ Λ(C)
where we used χ · χ = n/3 ∈ 2Z and changed the variable λ → λ − lχ ∈ Λ(C) (l ∈ Z). (The
above transformation suggests that the shift z → z + lτ can be understood as the shift of
Λ(C) by lχ in terms of a lattice.)
Since the above proposition only ensures the modular transformation of
ZEG (τ, z ; Λ(C), χ), we need to require an additional condition to ensure that it is a weak Jacobi form. In the presence of supersymmetry, any operator in the Ramond sector must satisfy
h ≥ n/24, which is a unitarity bound from the superconformal algebra. Then, from (3.40),
the U(1)-graded partition function can be expanded as
ZEG (τ, z ; Λ(C), χ) =
X
c(n, l) q n y l .
(3.50)
n≥0,l∈Z
In this case, by combining the transformation law, we can conclude that ZEG (τ, z ; Λ(C), χ)
is a weak Jacobi form.
For fermionic code CFTs, the unitarity bound h ≥ n/24 in the Ramond sector can be
rephrased as in the following proposition.
Proposition 3.4
Let Λ(C) be an odd self-dual lattice. Then, the Ramond sector satisfies h ≥ n/24 if and only
if any characteristic vector χ ∈ 2S(Λ(C)) satisfies χ · χ ≥ n/3.
Proof. Let us rewrite the bound h ≥ n/24 in the Ramond sector. We choose an element
χ ∈ 2S(Λ(C)). Then, from (3.39), the unitarity bound becomes
1
χ
λ+
2
2
2
– 18 –
≥
n
.
24
(3.51)
JHEP01(2024)130
e
=
η(τ )n
fˆ(ω) ,
X
Using a characteristic vector χ ∈ 2S(Λ(C)), any characteristic vector can be represented by
χ′ = χ + 2λ (λ ∈ Λ(C)). Therefore, the above inequality can be written as
1
2
′ 2
χ
2
≥
n
24
⇔
χ′ 2 ≥
n
.
3
(3.52)
Therefore, the unitarity bound in the R sector is equivalent to the bound χ · χ ≥ n/3 for any
characteristic vector.
Theorem 3.1
Let Λ(C) be an odd self-dual lattice of rank n ∈ 12Z and the minimum norm of characteristic
vectors in Λ(C) is n/3. For an element χ ∈ Xn/3 , ZEG (τ, z ; Λ(C), χ) is a weak Jacobi form
of weight 0 and index m = n/6.
Under the assumption in Theorem 3.1, the partition function ZEG (τ, z ; Λ(C), χ) becomes
a weak Jacobi form of weight 0 and index n/6. This is the characterization of the elliptic
genus, so it is expected to return the elliptic genus. In section 4, we exploit the method
described above to reproduce extremal elliptic genera, which gives further evidence of the
validity of our computation of elliptic genera.
Note that the U(1)-graded partition function depends on the choice of a characteristic
vector χ ∈ Xn/3 . Generally, there can be several inequivalent choices for χ ∈ Xn/3 , which
give different weak Jacobi forms ZEG (τ, z ; Λ(C), χ). This can reflect that there can exist
several ways of realizing the N = 2 superconformal symmetry depending on the choice of the
U(1) current. In what follows, we separately discuss how to choose characteristic vectors in
fermionic code CFTs for an odd prime p and the binary case (p = 2).
Characteristic vector for odd prime p. For an odd prime p, the Construction A lattice
is obtained from a self-dual code C ⊂ Fnp . Then, a characteristic vector of Λ(C) is given by
the following proposition, which is an improved statement of Proposition 3.7 in [10].
Proposition 3.5
Let p be an odd prime and C ⊂ Fnp a self-dual code. Then χ ∈ Rn is a characteristic vector
√
of the Construction A lattice Λ(C) if and only if χ = m/ p ∈ Λ(C) where each component
mi is an odd integer for i ∈ {1, 2, · · · , n}.
Proof. We explicitly prove the necessary condition (“only if”). From the construction of
Λ(C), λ ∈ Λ(C) can be written as λ = √1p s, s ∈ Zn . Since Λ(C) is self-dual and p is odd,
λ · λ is an integer and
λ · λ ≡ pλ · λ = s · s ≡
n
X
si
mod 2 .
(3.53)
i=1
On the other hand, if χ is a characteristic vector, χ · λ must be an integer, which means that
√
χ ∈ Λ(C). Then χ can be written as χ = m/ p , m ∈ Zn and
χ · λ ≡ pχ · λ =
n
X
mi s i
i=1
– 19 –
mod 2 .
(3.54)
JHEP01(2024)130
Therefore, if one assumes that the minimum norm of characteristic vectors is n/3, then the
U(1)-graded partition function ZEG (τ, z ; Λ(C), χ) has a Fourier expansion (3.50). Combining
Proposition 3.3, we arrive at the following theorem.
Since any Λ(C) contains vectors with one non-zero element such as λ =
definition of characteristic vector:
∀λ ∈ Λ(C) , λ · λ = χ · λ
√1 (p, 0, · · ·
p
mod 2
, 0), the
(3.55)
is satisfied if and only if all mi are odd.
ZEG (τ, z; Λ(C), χ) =
1
η(τ )n
χ
1
X
1
χ 2
e2πi(z+ 2 )χ·(λ+ 2 ) q 2 (λ+ 2 ) ,
λ ∈ Λ(C)
(3.56)
1
=
WC ({fa (τ, z)}) ,
η(τ )n
Here, the complete weight enumerator of C ⊂ Fn3 is defined by
WC ({xa }) =
X
Y
(3.57)
a (c)
xwt
,
a
c ∈ C a ∈ F3
where wta (c) = |{i | ci = a}|, and
fa (τ, z) =
X
1
a
1
3
a
1 2
e2πi(z+ 2 )(m+ 3 + 6 ) q 2 (m+ 3 + 6 ) ,
a ∈ F3 .
(3.58)
m∈Z
Characteristic vector for p = 2. For a binary singly-even self-dual code C ⊂ Fn2 , one
can define the shadow of the code, from which we can obtain the shadow of the lattice Λ(C).
We divide C into two subsets C0 and C2 consisting of doubly-even and singly-even
codewords of C, respectively:
C0 = {c ∈ C | c · c ∈ 4Z} ,
C2 = {c ∈ C | c · c ∈ 4Z + 2} ,
(3.59)
where · is the Euclidean inner product: c · c′ = i ci c′i ∈ R. Note that C0 is a doubly-even
self-orthogonal code and C0 ⊂ C ⊂ C0⊥ . The shadow S(C) of the code C is defined by ([67])
P
S(C) = C0⊥ \ C .
(3.60)
We can relate the shadow of the code to characteristic vectors of the lattice by the following
proposition.
Proposition 3.6 ([61])
Let C ⊂ Fn2 be a singly-even self-dual code. Then χ ∈ Rn is a characteristic vector of Λ(C) if
√
and only if χ = 2 (s + 2m) where s ∈ S(C) and m ∈ Zn .
– 20 –
JHEP01(2024)130
Table 1 shows characteristic vectors χ = √1p (c + pm) with χ · χ = n/3. For p > 3 (and
p = 2 as we will see shortly), the number of types increases as n becomes larger. For p = 3,
there is only one type of vector at any n. In particular, if C contains the codeword 1n ∈ C,
√
we can take the characterstic vector χ = 1n / 3. Then, the U(1)-graded partition function
can be written in terms of the complete weight enumerator of C as
(a) p = 3
χ ∈ Xn/3
n
12
√1 ([±1
3
1
√ ([±1
3
1
√ ([±1
3
24
36
c∈C
× 12])
([(1 or 2) × 12])
× 24])
([(1 or 2) × 24])
× 36])
([(1 or 2) × 36])
χ ∈ Xn/3
n
√1 ([±1
5
√1 ([±1
5
√1 ([±1
5
√1 ([±1
5
12
24
36
c∈C
× 11, ±3 × 1])
([(1 or 4) × 11, (2 or 3) × 1])
× 22, ±3 × 2])
([(1 or 4) × 22, (2 or 3) × 2])
× 33, ±3 × 3])
([(1 or 4) × 33, (2 or 3) × 3])
× 35, ±5 × 1])
([0 × 1, (1 or 4) × 35])
Table 1. The characteristic vectors χ ∈ 2S(Λ(C)) ⊂ Rn with χ · χ = n/3 and the corresponding
vectors in Fnp for odd prime p = 3, 5. The meaning of “corresponding” is that there exists m ∈ Zn such
that χ = √1p (c + pm). For example, ([±1 × 2, 0 × 10]) refers to a vector such that two components
are 1 or −1 and ten components are 0.
χ ∈ Xn/3
n
√
12
24
36
2([±1 × 2, 0 × 10])
([1 × 2, 0 × 10])
2([±1 × 4, 0 × 20])
([1 × 4, 0 × 20])
2([±2 × 1, ±1 × 2, 0 × 33])
√
2([±1 × 6, 0 × 30])
([1 × 2, 0 × 34])
√
√
s ∈ S(C)
([1 × 6, 0 × 30])
Table 2. The characteristic vectors χ ∈ 2S(Λ(C)) ⊂ Rn with χ · χ = n/3 and the corresponding
√
vectors in Fn2 . The meaning of “corresponding” is that there exists m ∈ Zn such that χ = 2(s + 2m).
√
Table 2 shows characteristic vectors χ = 2(s + 2m) with χ · χ = n/3. Note that
√
([1 × 2, 0 × 34]) ∈ C at n = 36 also corresponds to the characteristic vector χ′ = 2([±1 ×
2, 0 × 34]), which does not satisfy the unitarity bound from Proposition 3.4.
In the rest of this subsection, we show a practical way to find shadows for specific codes.
Here, we use the inclusion map ι : F2 → {0, 1} ⊂ Z to avoid some confusion.
Proposition 3.7
Let C ⊂ Fn2 be a singly-even self-dual code that has an
the left side is the identity matrix In/2 . Then,
n
2
× n generator matrix G such that
s = P (G1 ), . . . , P (Gn/2 ), 0, . . . , 0 ∈ Fn2
– 21 –
(3.61)
JHEP01(2024)130
(b) p = 5
is an element of the shadow S(C) where
Gi ∈ C : the i-th row of G ,
(
P (c) =
(3.62)
0
(c · c ∈ 4Z) ,
1
(c · c ∈ 4Z + 2) .
(3.63)
n/2
Proof. For any codeword c = x G ∈ C, x ∈ F2 , there exists m ∈ Zn such that ι(c) =
ι(x)ι(G) + 2m. Then, via mod 4 computation, we obtain
≡ (ι(x)ι(G))2
=
X
ι(xi )2 ι(Gi ) · ι(Gi ) + 2
i
≡2
X
ι(xi )ι(xj ) ι(Gi ) · ι(Gj )
(3.64)
i̸=j
X
ι(xi P (Gi )) .
i
In the last line, we used c · c ≡ 2P (c) and c · c′ ∈ 2Z for c, c′ ∈ C from the self-duality of C.
On the other hand, since the left part of G is the identity matrix In/2 ,
c · s = x G · P (G1 ), . . . , P (Gn/2 ), 0, . . . , 0 =
X
ι(xi P (Gi )) ,
mod 2 .
(3.65)
i
Thus, the equivalences
c · s = 0 mod 2 ⇔ c ∈ C : doubly-even
c · s = 1 mod 2 ⇔ c ∈ C : singly-even
hold, which means that s ∈ S(C).
From the proposition, we can read off an element of the shadow S(C) from the generator
matrix G. Since the shadow of C is a coset of C in Fn2 : s + C = S(C), the whole set of S(C)
can be easily obtained once we have an element s ∈ S(C).
3.4
Spectral flow and chiral ring
We have shown that the U(1)-graded partition function becomes a weak Jacobi form under
some assumptions. This strongly supports the existence of N = 2 supersymmetry in the
corresponding fermionic code CFT. In this subsection, we consider the N = 2 structure and,
more concretely, aim to identify the spectral flow and chiral rings in fermionic code CFTs.
Let us consider a fermionic CFT constructed from an odd self-dual lattice Λ(C) where the
minimum norm of characteristic vectors is n/3 and specify the U(1) current by (3.36) where
χ ∈ Xn/3 . For the fermionic code CFT, a state with charge Q = χ · κ can be represented by
(
Vκ (z) = : e
iκ·X(z)
:,
κ∈
– 22 –
Λ(C)
(NS sector) ,
S(Λ(C))
(R sector) ,
(3.66)
JHEP01(2024)130
c · c = (ι(x)ι(G) + 2m)2
up to neutral operators. The spectral flow by θ can be obtained by shifting the momentum κ
by θχ, i.e. κ → κ − θχ. In fact, under the flow, a state Vκ (z) with h = κ2 /2 and Q = κ · χ
transforms into Vκ−θχ (z) = : exp[i (κ − θχ) · X(z)] : with
1
n
h′ = (κ − θχ)2 = h − θQ + θ2 ,
2
6
n
′
Q = χ · (κ − θχ) = Q − θ ,
3
(3.67)
where we used χ · χ = n/3. This reproduces the transformation rule (2.7) of quantum numbers
under the spectral flow. Therefore, we identify the spectral flow operator as
(3.68)
This is also consistent with the observation in the proof of Proposition 3.3: the spectral flow
can be understood as the shift of Λ(C) by χ in terms of a lattice.
From Proposition 3.4, the assumption that the minimum norm of characteristic vectors
is n/3 implies that any operator in the R sector satisfies h ≥ n/24. In this case, we may
identify the Ramond ground states as
Vχ′ /2 (z) = : exp(i χ′ · X(z)/2) : ,
(3.69)
where χ′ ∈ Xn/3 . The vertex operators have the expected conformal weight h = n/24.
Furthermore, there is another unitarity bound (2.16) in the NS sector. Interestingly,
the fermionic code CFT also satisfies the unitarity bound h ≥ Q/2 in the NS sector. To
see this, suppose a characteristic vector χ ∈ 2S(Λ(C)) with the minimum norm. Then, any
characteristic vector χ′ ∈ 2S(Λ(C)) is written in the form χ′ = χ − 2λ (λ ∈ Λ(C)). Since
χ ∈ 2S(Λ(C)) is minimum: χ′ · χ′ ≥ χ · χ, we obtain
λ · λ ≥ λ · χ,
(3.70)
which implies h ≥ Q/2 for a vertex operator Vλ (z). The bosonic oscillators increase the L0
eigenvalue and do not change the U(1) charge, so any operator in the NS sector satisfies
the bound.
As explained in section 2.1, the spectral flow by θ = −1/2 flows the Ramond ground
states into chiral primary fields. Now we obtain
V(χ+χ′ )/2 (z) = : exp[i (χ + χ′ ) · X(z)/2] : .
(3.71)
The conformal weight h and U(1) charge Q of the states are
1 χ + χ′ 2
h=
= χ · (χ + χ′ )/4 ,
2
2
Q = χ · (χ + χ′ )/2 ,
(3.72)
which implies h = Q/2, reproducing the known relation between quantum numbers for chiral
primary fields. Also, the conformal weight h satisfies
h = χ · (χ + χ′ )/4 ≤ χ · (χ + χ)/4 = n/6 .
– 23 –
(3.73)
JHEP01(2024)130
Uθ = : exp (−i θ χ · X(z)) : .
This also agrees with the property of chiral primary fields as shown in (2.18).
Let us consider the operator product expansion of two chiral primary fields
χ+χ′ χ+χ′′
· 2
2
V(χ+χ′ )/2 (z) V(χ+χ′′ )/2 (w) ∼ (z − w)
Vχ+(χ′ +χ′′ )/2 (w) .
(3.74)
Using the fact that any operator satisfies h ≥ Q/2 and chiral primary fields saturate it,
in this case, we obtain
χ + χ′ χ + χ′′
·
≥ 0.
2
2
(3.75)
R = ra a =
χ + χ′
, χ′ ∈ Xn/3
2
.
(3.76)
Here, the product is ra · rb = ra+b subject to ra · rb = 0 if a · b > 0 where a = (χ + χ′ )/2
and b = (χ + χ′′ )/2.
In the ternary case. For a ternary code (p = 3), the above discussion can be further
simplified. In the rest of this section, we move on to the convenient frame F3 = {−1, 0, 1}
using the isomorphism φ : {0, 1, 2} → {−1, 0, 1} where
φ : 0 7→ 0 ,
1 7→ 1 ,
2 7→ −1 .
(3.77)
Then, a characteristic vector χ ∈ Xn/3 takes the form χ = √13 ([(±1) × n]), and the corresponding codeword is c = ([(±1) × n]) ∈ C. This type of codeword is called maximal and we denote
the set of maximal codewords by Cmax . Note that a self-dual ternary code contains maximal
codewords only when n ∈ 12Z. Then, a characteristic vector χ ∈ Xn/3 can be written as
√
χ = c/ 3 ,
(3.78)
c ∈ Cmax .
√
Choosing a characteristic vector by χ = c/ 3 (c ∈ Cmax ), the spectral flow operator is
√
Uθ = : exp(−i θ c · X(z)/ 3) : ,
(3.79)
where θ is a twist parameter. The Ramond ground states can be identified as
Vχ′ /2 (z) = : e 2
i
√
3
c′ ·X(z)
:,
c′ ∈ Cmax ,
(3.80)
√
where we used χ′ = c′ / 3. Correspondingly, the chiral primary fields in the NS sector are
V(χ+χ′ )/2 (z) = : e 2
i
√
3
(c+c′ )·X(z)
:,
(3.81)
which gives the chiral ring
n
o
R = r(c+c′ )/2 c′ ∈ Cmax .
– 24 –
(3.82)
JHEP01(2024)130
Therefore, if one takes the limit z → w, the operator product does not vanish if and only
if (χ + χ′ ) · (χ + χ′′ ) = 0. Hence the chiral ring can be written as
The U(1) character valued degeneracy of the Ramond ground states is
h
TrR tJ0
i
G±
0 =0
=
′
tχ·χ /2 =
X
χ′ ∈X
c′ ∈C
n/3
′
X
(3.83)
tc·c /6 ,
max
√
where we used χ = c/ 3. By the spectral flow, we obtain the counterpart in the NS sector
called the Poincaré polynomial [36]
h
i
P (t) := TrNS tJ0 |R =
X
′
tc·(c+c )/6 = tn/6
c′ ∈Cmax
X
′
tc·c /6 ,
(3.84)
c′ ∈Cmax
Extremal N = 2 elliptic genera
4
In this section, we construct extremal N = 2 superconformal theories with the central charge
n = 12 and 24. Originally, bosonic and N = 1 extremal CFTs are introduced to understand
the holographic duality of the pure AdS3 gravity [37].4 For N = 2 theories, extremal CFTs
are defined using extremal elliptic genera [40]. The extremal N = 2 SCFTs with the central
charge n = 12 and 24 have been explicitly constructed in [6, 41]. We will give an alternative
systematic construction of the extremal elliptic genera from linear codes. In this and the
following sections, we use the graded character defined in appendix A. Since we only consider
(R)
the partition function in the R sector, we abbreviate Chl;h,Q as Chl;h,Q .
4.1
Central charge 12
In this subsection, we employ our method to obtain the extremal elliptic genus with the
central charge n = 12 (correspondingly, m = 2) [41]. As in (2.45), the q 0 term in the
extremal elliptic genus at m = 2 is
y 2 + 22 + y −2 .
(4.1)
From the discussion around (3.69), the corresponding lattice Λ(C) must have 24 orthogonal
characteristic vectors in Xn/3 , i.e., |Xn/3 | = 24 and χ · χ′ = 0 for χ, χ′ ∈ Xn/3 , χ ̸= ±χ′ .
In what follows, we search for classical codes that give the extremal elliptic genus by
imposing constraints from the orthogonality of characteristic vectors. Here, we consider
classical codes over Fp where p = 2, 5.
When p = 2, from table 2, the shadow S(C) of the code C ⊂ F12
2 must have 6 vectors
with two 1 and ten 0. In addition, from the orthogonality, all ‘1’s must be in different
positions such as
(1, 1, 010 ) , (02 , 1, 1, 08 ) , (04 , 1, 1, 06 ) , (06 , 1, 1, 04 ) , (08 , 1, 1, 02 ) , (010 , 1, 1) .
(4.2)
There have been numerous works on the relation between extremal CFTs and pure gravity. See, e.g., [68–79]
for the developments.
4
– 25 –
JHEP01(2024)130
where we used c · c = n. These polynomials are completely determined by the set of maximal
codewords. The Poincaré polynomial is known to be closely related to the cohomology of
the target manifold [36].
In this case, the code C must include the codewords as (1, 1, 1, 1, 08 ) since s − s′ ∈ C for
any s, s′ ∈ S(C) and t ∈ C2 ⊂ C must be in the form like (1, 0, 1, 0, 1, 0, 1, 0, 1, 0, 1, 0) since
t · s = 1 for any s ∈ S(C). In summary, the code must be generated by
1
1
1
1
1
1
1
1
1
1
1
0
1
0
0
0
0
1
1
0
0
0
0
0
0
1
0
0
0
1
0
1
0
0
0
0
0
0
1
0
0
1
0
0
1
0
0
0
0
0
0
1
0
1
0
0
0
1
0
0
0
0
0
0
1
1
0
0
0
,
0
1
0
(4.3)
3
1
1
1
1
1
1
3
4
4
4
4
1
4
3
4
4
4
1
4
4
3
4
4
1
4
4
4
3
4
1
4
4
4
4
3
1
4
1
4
4
4
1
4
4
1
4
4
1
4
4
4
1
4
1
4
4
4
4
1
1
1
1
1
1
1
1
1
4
.
4
4
4
(4.4)
The Construction A lattice from either code is D+
12 (see [29, 81] for the names of lattices).
We can explicitly check that for any characteristic vector χ ∈ Λ(C) with norm n/3 = 4,
the elliptic genus is
ZEG (τ, z; Λ(C), χ) = y 2 + 22 + y −2 + O(q)
1
5
= ϕ20,1 + ϕ2−2,1 E4
6
6
= 23 Ch 3 ; 1 ,0 + Ch 3 ; 1 ,2
2 2
+
∞
X
(4.5)
2 2
At,1 (Ch 3 ; 1 +t,1 + Ch 3 ; 1 +t,−1 ) + At,2 Ch 3 ; 1 +t,2
2 2
t=1
2 2
2 2
where At,1 = {770, 13915, . . . } and At,2 = {231, 5796, . . . } for t = {1, 2, . . . } [7]. This means
that both codes with p = 2, 5 correspond to the CFT with the extremal elliptic genus.
In the ternary case, we can perform further analysis using the complete weight enumerator.
We consider the ternary Golay code C ⊂ F12
3 generated by
1
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
2
2
2
2
2
1
0
1
2
2
1
1
1
0
1
2
2
1
2
1
0
1
2
1
2
2
1
0
1
1
1
2
,
2
1
0
(4.6)
whose corresponding lattice Λ(C) is D+
12 . The complete weight enumerator is
12
12
6 6
6 6
6 6
3 3 6
3 6 3
6 3 3
WC (x0 , x1 , x2 ) = x12
0 + x1 + x2 + 22(x0 x1 + x0 x2 + x1 x2 ) + 220(x0 x1 x2 + x0 x1 x2 + x0 x1 x2 ) .
(4.7)
– 26 –
JHEP01(2024)130
up to permutations of the columns.
When p = 5, from table 1, the code C ⊂ F12
5 must have 24 codewords with eleven 1 or 4
and one 2 or 3. All self-dual codes over F5 with length 12 are known [80] and there are 3
such codes up to permutations, all satisfying the orthogonality. One of them is generated by
The code contains the codeword 1 = (1, . . . , 1) ∈ C and thus we can take the characteristic
√
vector χ = 1/ 3. From (3.56), it can be verified that the q 0 term of the elliptic genus
6 6
12
ZEG (τ, z; Λ(C), χ) is y 2 + 22 + y −2 , which comes from x12
0 + 22x0 x2 + x2 .
4.2
Central charge 24
In this subsection, we obtain the extremal elliptic genus with the central charge n = 24
(correspondingly, m = 4) [6]. As in (2.46), the q 0 term in the extremal elliptic genus at m = 4 is
y 4 + 46 + y −4 .
(4.8)
(m=4)
SF1/2 Zext
1
τ, z −
2
1
= q −1 + 24 + q 2 (2048y + 2048y −1 ) + O(q) .
(4.9)
i |0⟩, thus the
The terms q −1 and 24 come from the vacuum |0⟩ and its descendants α−1
1
minimum norm of the lattice Λ(C) must be 3, which corresponds to q 2 . There is only one
such odd self-dual lattice: the odd Leech lattice [82]. In the ternary case, the odd Leech
lattice can be constructed from the code with the minimum Hamming weight 9. There are
two such self-dual codes and one is generated by
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
1
2
0
1
2
2
0
2
2
2
1
0
1
2
0
0
2
0
2
1
2
0
1
2
0
2
1
0
2
2
1
0
2
1
1
0
0
0
2
1
1
2
1
0
1
1
1
2
2
1
2
1
0
1
2
2
1
1
2
1
2
0
2
2
2
1
1
1
1
2
2
1
0
0
0
2
1
2
0
2
1
0
0
1
1
1
1
2
2
1
1
0
0
2
1
1
1
1
2
0
2
2
1
2
1
1
0
0
1
1
0
1
1
1
2
0
2
0
2
0
2
0
2
1
1
1
0
2
2
1
0
2
0
1
2
,
0
1
2
0
2
2
which is modified from the top matrix for F3 , n = 24, d = 9 in [80].
– 27 –
(4.10)
JHEP01(2024)130
From the discussion around (3.69), the corresponding lattice Λ(C) must have 48 orthogonal
characteristic vectors in Xn/3 .
As in the previous case, we would like to find classical codes that return the extremal
elliptic genus. Below we consider classical codes over Fp where p = 2, 3.
When p = 2, from table 2, we have to choose the U(1) current such as J(z) = χ ·
√
L(z), χ = 2 (1, 1, 1, 1, 020 ). However, in this case, s = √12 (−1, 1, 1, 1, 020 ) is an element of
S(Λ(C)) and χ · s = 2, thus Vs (z) corresponds to q 0 y 2 in the elliptic genus. The existence of
this term is inconsistent with y 4 + 46 + y −4 , which means that the extremal elliptic genus
cannot be constructed from classical codes over F2 .
When p = 3, from table 1, the code C ⊂ F24
3 must have 48 codewords consisting of only
1 and 2. In addition, from the orthogonality, if we choose χ ≡ √13 c, c ∈ C, then 46 codewords
except for ±c must have 12 components with the same value as c (both 1 or both 2) and
12 components with different values (one is 1 and the other is 2).
In this case, it is also useful to consider the NS sector. The corresponding partition
function to the extremal elliptic genus is
√
For any characteristic vector χ ∈ Λ(C) with norm n/3 = 8 such as χ = 1/ 3, the
elliptic genus is
ZEG (τ, z; Λ(C), χ) = y 4 + 46 + y −4 + O(q)
1 4
1
11
67 4
=
ϕ + ϕ2 ϕ2 E4 + ϕ0,1 ϕ3−2,1 E6 +
ϕ
E2
432 0,1 8 0,1 −2,1
27
144 −2,1 4
= 47 Ch 7 ;1,0 + Ch 7 ;1,4
2
+
(4.11)
2
∞
X
3
X
t=1
k=1
!
At,k (Ch 7 ;1+t,k + Ch 7 ;1+t,−k ) + At,4 Ch 7 ;1+t,4
2
2
2
5
Other examples
In the previous section, we constructed the extremal elliptic genera. In this section, we
give other examples of the elliptic genera, especially near-extremal elliptic genera, which are
slightly modified in the polar regions from extremal ones as in (2.47).
By Theorem 3.1, for the partition function ZEG (τ, z ; Λ(C), χ) to be a weak Jacobi form,
the minimum norm of characteristic vectors in Λ(C) ⊂ Rn must be n/3. From the fact that
χ · χ = n mod 8 for any characteristic vector χ, if n = 12, 24, it is sufficient to verify the
existence of a characteristic vector with norm n/3, i.e., Xn/3 ̸= ∅. In terms of codes, this is
equivalent to the existence of the codewords listed in table 1 and 2. If n = 36 or greater,
there may be characteristic vectors with a norm less than n/3. Therefore, we should choose
a code C whose corresponding lattice Λ(C) does not contain such vectors. In the ternary
case, the minimum norm of the characteristic vectors is always greater than or equal to n/3
from Proposition 3.5, thus at any n ∈ 12Z the conditions are summarized as the existence
of the maximal codewords (consisting of only 1 and 2), i.e., Cmax ̸= ∅.
5.1
n = 12 , p = 2
4
12
In dimension n = 12, there are only 3 odd self-dual lattices: D+
12 , E8 ⊕ Z , and Z . As
+
we have seen in section 4.1, D12 corresponds to the CFT with the extremal elliptic genus.
Since Z12 is trivial, we consider the case E8 ⊕ Z4 .
For a code C ⊂ F12
2 generated by
1
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
0
0
0
0
0
1
– 28 –
0
0
0
1
1
1
0
0
1
0
1
1
0
0
1
1
0
1
0
0
1
,
1
1
0
(5.1)
JHEP01(2024)130
where At,1 = {32890, 2969208, . . . }, At,2 = {14168, 1659174, . . . }, At,3 = {2024, 485001, . . . },
and At,4 = {23, 61984, . . . } for t = {1, 2, . . . }. Therefore, the CFT constructed from this
code certainly has the extremal elliptic genus.
√
For χ = 1/ 3, from (3.56), the terms y 4 + 46 + y −4 in the elliptic genus come from
12 12
24
x24
0 + 46 x0 x2 + x2 in the complete weight enumerator.
the Construction A lattice Λ(C) is E8 ⊕ Z4 . We can easily verify that the first four columns
correspond to Z4 and the rest to E8 .
For any characteristic vector χ ∈ Λ(C) with norm n/3 = 4 such as
χ=
√
2 (1, 0, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0) ,
(5.2)
the elliptic genus is
= ϕ2−2,1 E4
(5.3)
= 7 Ch 3 ; 1 ,0 + 4(Ch 3 ; 1 ,1 + Ch 3 ; 1 ,−1 ) + Ch 3 ; 1 ,2
2 2
+
∞
X
2 2
2 2
2 2
At,1 (Ch 3 ; 1 +t,1 + Ch 3 ; 1 +t,−1 ) + At,2 Ch 3 ; 1 +t,2
2 2
t=1
2 2
2 2
where At,1 = {750, 13875, . . . } and At,2 = {255, 5868, . . . } for t = {1, 2, . . . }. This is not
extremal but a β-extremal elliptic genus with β > 1 since it includes the term q 0 y.
5.2
n = 24 , p = 3
We consider a code C ⊂ F24
3 generated by
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
2
2
0
2
1
1
1
2
2
1
1
1
1
2
2
0
2
2
2
1
2
1
1
0
0
1
1
1
0
2
2
0
2
1
2
0
0
0
0
2
2
2
1
1
2
2
1
1
2
2
2
2
1
2
0
2
1
0
0
2
0
2
0
0
2
0
0
1
2
2
0
2
0
1
1
1
2
0
2
2
0
0
2
2
1
1
2
0
1
0
2
1
0
0
1
0
2
2
0
1
0
0
0
0
1
1
0
1
1
0
1
1
2
2
1
0
0
2
2
1
2
0
2
0
0
2
1
1
0
2
1
1
1
0
2
,
1
0
1
0
0
2
(5.4)
which is the matrix for F3 , n = 24, d = 6 in [80].
In this case, the elliptic genus ZEG depends on the choice of U(1) current, i.e., characteristic vector χ ∈ Xn/3 . There are 72 characteristic vectors in Xn/3 , which are divided
into two types.
For 24 characteristic vectors, e.g.,
1
χ1 = √ (1, 1, 1, 1, 1, −1, 1, 1, −1, 1, 1, −1, 1, −1, 1, −1, −1, −1, −1, −1, −1, 1, −1, 1) ,
3
– 29 –
(5.5)
JHEP01(2024)130
ZEG (τ, z; Λ(C), χ) = y 2 − 4y + 6 − 4y −1 + y −2 + O(q)
the elliptic genus is
ZEG (τ, z; Λ(C), χ1 ) = y 4 − 24y + 22 − 24y −1 + y −4 + O(q)
=−
1 4
7
41
137 4
ϕ0,1 + ϕ20,1 ϕ2−2,1 E4 +
ϕ0,1 ϕ3−2,1 E6 +
ϕ
E2
864
48
108
288 −2,1 4
= 23 Ch 7 ;1,0 + 24(Ch 7 ;1,1 + Ch 7 ;1,−1 ) + Ch 7 ;1,4
2
+
2
∞
X
3
X
t=1
k=1
2
(5.6)
2
!
At,k (Ch 7 ;1+t,k + Ch 7 ;1+t,−k ) + At,4 Ch 7 ;1+t,4
2
2
2
1
χ2 = √ (1, 1, 1, 1, −1, 1, 1, 1, 1, 1, −1, −1, 1, 1, −1, 1, −1, −1, 1, −1, 1, 1, −1, 1) ,
3
(5.7)
the elliptic genus is
ZEG (τ, z; Λ(C), χ2 ) = y 4 + y 2 − 12y + 44 − 12y −1 + y −2 + y −4 + O(q)
=
1 4
5
83
137 4
ϕ0,1 + ϕ20,1 ϕ2−2,1 E4 +
ϕ0,1 ϕ3−2,1 E6 +
ϕ
E2
864
36
216
288 −2,1 4
= 45 Ch 7 ;1,0 + 12(Ch 7 ;1,1 + Ch 7 ;1,−1 ) + (Ch 7 ;1,2 + Ch 7 ;1,−2 ) + Ch 7 ;1,4
2
+
2
∞
X
3
X
t=1
k=1
2
2
2
2
!
At,k (Ch 7 ;1+t,k + Ch 7 ;1+t,−k ) + At,4 Ch 7 ;1+t,4
2
2
2
(5.8)
where At,1 = {32922, 2969505, . . . }, At,2 = {14125, 1658880, . . . }, At,3 = {1989, 484920, . . . },
and At,4 = {45, 62120, . . . } for t = {1, 2, . . . }. The difference with the extremal one in the
polar region is the terms q 0 y 2 and q 0 y, thus this is a β-extremal elliptic genus with β > 4.
This means that the earlier case is nearer to the extremal elliptic genus.
From the generator expansion, it can be seen that the absolute values of the coefficients
4
of ϕ0,1 are equal in both cases. It can be understood as follows.
Let ϕ(τ, z) be a weak Jacobi form of weight w = 0 and index m ∈ Z. Since ϕ0,1 (τ, 0) = 12
and ϕ−2,1 (τ, 0) = 0, ϕ(τ, 0) is a constant determined by the coefficient of ϕm
0,1 . On the other
hand, ZEG (τ, 0 ; Λ(C), χ) = ZR
e (τ ; Λ(C), χ) and the dependence of ZR
e on χ is only the overall
m
sign. Thus, the absolute value of the coefficient of ϕ0,1 in ZEG (τ, z ; Λ(C), χ) does not depend
on the choice of characteristic vector χ.
We can also observe that the coefficients of ϕ4−2,1 E42 are equal in both cases. This is not
n/6
n/12
a coincidence. In what follows, we show that the coefficient of ϕ−2,1 E4
in the U(1)-graded
partition function (3.41) does not depend on the choice of characteristic vectors χ ∈ Xn/3 .
Let ϕ(τ, z) be a weak Jacobi form of weight w and index m ∈ Z. Substituting a = d =
0, b = −1, c = 1 and τ = i in (2.22), we get
2
ϕ(i, −iz) = iw e2πmz ϕ(i, z) .
– 30 –
(5.9)
JHEP01(2024)130
where At,1 = {32866, 2969208, . . . }, At,2 = {14192, 1659246, . . . }, At,3 = {1976, 484929, . . . },
and At,4 = {47, 62056, . . . } for t = {1, 2, . . . }. The difference with the extremal one in the
polar region is the term q 0 y, thus this is a β-extremal elliptic genus with β > 1.
For 48 characteristic vectors, e.g.,
When z =
1
2 (1
− i), from −iz = z − 1 and (2.23),
1
1
ϕ i, (1 − i) = iw e−iπm ϕ i, (1 − i)
2
2
(5.10)
.
1
1
ZEG (i, (1 − i) ; Λ(C), χ) =
2
η(i)n
1
=
η(i)n
X
χ
χ 2
eπχ·(λ+ 2 ) e−π(λ+ 2 )
λ ∈ Λ(C)
X
(5.11)
2
e
−π(λ2 − χ4 )
.
λ ∈ Λ(C)
If we fix χ2 = n/3, this does not depend on the choice of χ. Combining them, we can conclude
n/12 n/6
that the coefficient of E4 ϕ−2,1 in ZEG (τ, z ; Λ(C), χ) does not depend on χ.
5.3
n = 36 , p = 3
For the central charge 36, the polar terms of the extremal elliptic genus are
y 6 + 0(y 5 + y 4 + y 3 + y 2 + y) + q(y 6 − y 5 ) .
(5.12)
However, as shown in section 2.3, the extremal elliptic genus cannot be realized as a weak
Jacobi form. Moreover, it is not possible to construct the elliptic genus such that the coefficient
of qy 6 is 1 from codes over any Fp since if we choose the U(1) current as J(z) = χ · L(z),
χ ∈ Xn/3 , then the state |χ/2⟩ in the R sector corresponds to q 0 y 6 and its descendants
i |χ/2⟩ , i = 1, . . . , 36 to 36qy 6 in the elliptic genus.
α−1
From the discussion at n = 24, it is natural to expect that a code with a large minimum
Hamming weight gives a near-extremal elliptic genus. In the ternary case, the largest minimum
Hamming weight that a self-dual code with length n = 36 can have is known to be 12. Thus,
– 31 –
JHEP01(2024)130
Thus, ϕ(i, 12 (1 − i)) can be nonzero only if w + 2m ∈ 4Z. For the four generators, it follows
that E6 (i) = 0 and ϕ0,1 (i, 12 (1 − i)) = 0. Then, when a weak Jacobi form ϕ(τ, z) such that
w + 2m ∈ 4Z is decomposed by generators, only the term consisting of E4 (τ ) and ϕ−2,1 (τ, z),
i.e. (E4 )(w+2m)/4 (ϕ−2,1 )m , can have nonzero contribution at τ = i, z = 12 (1 − i). In other
words, the coefficient of such a term is determined solely by the value ϕ(i, 12 (1 − i)). On the
other hand, from (3.41), the elliptic genus at that values is
we consider a code C ⊂ F36
3 generated by ([n, k, d] = [36, 18, 12] code in [83])
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
2
2
1
2
1
1
1
2
2
1
1
1
2
1
2
2
1
2
0
2
2
1
2
1
1
1
2
2
1
1
1
2
1
2
1
2
2
0
2
2
1
2
1
1
1
2
2
1
1
1
2
1
1
1
2
2
0
2
2
1
2
1
1
1
2
2
1
1
1
2
1
2
1
2
2
0
2
2
1
2
1
1
1
2
2
1
1
1
1
1
2
1
2
2
0
2
2
1
2
1
1
1
2
2
1
1
1
1
1
2
1
2
2
0
2
2
1
2
1
1
1
2
2
1
1
1
1
1
2
1
2
2
0
2
2
1
2
1
1
1
2
2
1
2
1
1
1
2
1
2
2
0
2
2
1
2
1
1
1
2
1
2
2
1
1
1
2
1
2
2
0
2
2
1
2
1
1
1
1
1
2
2
1
1
1
2
1
2
2
0
2
2
1
2
1
1
1
1
1
2
2
1
1
1
2
1
2
2
0
2
2
1
2
1
1
1
1
1
2
2
1
1
1
2
1
2
2
0
2
2
1
2
1
2
1
1
1
2
2
1
1
1
2
1
2
2
0
2
2
1
1
1
2
1
1
1
2
2
1
1
1
2
1
2
2
0
2
2
1
2
1
2
1
1
1
2
2
1
1
1
2
1
2
2
0
2
1
2
2
1
2
1
1
1
2
2
1
1
1
2
1
2
2
0
1
1
1
1
1
1
1
1
1
1
.
1
1
1
1
1
1
1
1
0
(5.13)
As in the previous example, the elliptic genus ZEG depends on the choice of U(1) current,
i.e., characteristic vector χ ∈ Xn/3 . There are 888 characteristic vectors in Xn/3 , which
are divided into two types.
For 72 characteristic vectors, the elliptic genus is
ZEG (τ, z; Λ(C), χ)
= y 6 − 408y + 70 − 408y −1 + y −6 + (36y 6 − 36y 5 + . . . )q + O(q 2 )
=−
+
31
365 4 2
269 3 3
3071 2 4
ϕ60,1 +
ϕ0,1 ϕ−2,1 E4 +
ϕ0,1 ϕ−2,1 E6 +
ϕ ϕ
E2
124416
41472
7776
13824 0,1 −2,1 4
271
3281 6
1231 6
ϕ0,1 ϕ5−2,1 E4 E6 +
ϕ−2,1 E43 +
ϕ
E2
648
13824
15552 −2,1 6
(5.14)
= 71 Ch 11 ; 3 ,0 + 408(Ch 11 ; 3 ,1 + Ch 11 ; 3 ,−1 ) + Ch 11 ; 3 ,6
2
+
2
∞
X
5
X
t=1
k=1
2
2
2
2
2
2
!
At,k (Ch 11 ; 3 +t,k + Ch 11 ; 3 +t,−k ) + At,6 Ch 11 ; 3 +t,6
2
2
2
2
2
2
where At,1 = { 580550 , 167398422 , . . . } , At,2 = { 298452 , 104775760 , . . . } , At,3 = { 71400 ,
39702684 , . . . }, At,4 = {7140, 8410920, . . . }, At,5 = {0, 843115, . . . }, and At,6 =
{35, 29154, . . . } for t = {1, 2, . . . }.
– 32 –
JHEP01(2024)130
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
For 816 characteristic vectors, the elliptic genus is
ZEG (τ, z; Λ(C), χ)
= y 6 + 93y 2 − 36y + 628 − 36y −1 + 93y −2 + y −6 + (36y 6 − 36y 5 + . . . )q + O(q 2 )
=
241 4 2
569 3 3
3133 2 4
31
ϕ60,1 +
ϕ0,1 ϕ−2,1 E4 +
ϕ0,1 ϕ−2,1 E6 +
ϕ ϕ
E2
124416
41472
15552
13824 0,1 −2,1 4
+
2137
3281 6
631 6
ϕ0,1 ϕ5−2,1 E4 E6 +
ϕ−2,1 E43 +
ϕ
E2
5184
13824
7776 −2,1 6
(5.15)
2
+
2
∞
X
5
X
t=1
k=1
2
2
2
2
2
2
2
2
2
2
!
At,k (Ch 11 ; 3 +t,k + Ch 11 ; 3 +t,−k ) + At,6 Ch 11 ; 3 +t,6
2
2
2
2
2
2
where At,1 = { 581480 , 167402049 , . . . } , At,2 = { 296499 , 104768320 , . . . } , At,3 = { 72795 ,
39708264, . . . }, At,4 = {6768, 8409525, . . . }, At,5 = {0, 842185, . . . } and At,6 =
{35, 29712, . . . }, for t = {1, 2, . . . }.
They are both β-extremal elliptic genera with β > 12 since they include the term 36qy 6 .
In addition, the coefficients of ϕ6−2,1 E43 and the absolute values of the coefficients of ϕ60,1 are
equal respectively, as shown in the previous example.
6
Discussion
In this paper, we have considered fermionic CFTs constructed from classical code over Fp [10]
and given a systematic way of computing the U(1)-graded partition function that returns
elliptic genera for N = 2 theories. Taking the U(1) current to be consistent with the N = 2
algebra, we have shown that the U(1)-graded partition function exhibits modular invariance
and spectral flow invariance. Using some examples of classical codes, we demonstrated the
unified construction of extremal N = 2 elliptic genera for central charges n = 12, 24. Also,
we discussed other examples that give near-extremal elliptic genera.
Except for the ternary case, the existence of N = 2 supersymmetry is not manifest
in fermionic code CFTs. However, Theorem 3.1 dictates that, under some assumptions,
the U(1)-graded partition function becomes a weak Jacobi form, which is invariant under
the modular transformation and spectral flow. Additionally, we can consistently identify
the spectral flow operator and chiral primary fields. These observations naturally lead us
to the following conjecture.
Conjecture 6.1
If Λ is an odd self-dual lattice of rank n ∈ 12Z and the minimum of characteristic vectors
in Λ is n/3, then the fermionic CFT whose set of vertex operators is specified by Λ admits
N = 2 supersymmetry.
If an odd self-dual lattice can be constructed from a ternary self-dual code up to rotation,
then the resulting CFT admits N = 2 supersymmetry [9]. However, as of now, we are not
certain whether any lattice satisfying the assumption in Conjecture 6.1 accepts the construction
from ternary codes. It would be interesting to pursue the validity of the conjecture.
– 33 –
JHEP01(2024)130
= 629 Ch 11 ; 3 ,0 + 36(Ch 11 ; 3 ,1 + Ch 11 ; 3 ,−1 ) + 93(Ch 11 ; 3 ,2 + Ch 11 ; 3 ,−2 ) + Ch 11 ; 3 ,6
Whereas this paper focuses on N = 2 supersymmetry in fermionic code CFTs, there
is a possibility that N = 4 supersymmetry emerges in fermionic code CFTs. In this case,
the R-symmetry becomes SU(2) symmetry rather than U(1). Note that, in the binary case,
any fermionic code CFT contains affine SU(2) symmetry as discussed in the bosonic case [3].
Hence, the binary construction might be helpful to provide N = 4 superconformal theories.
In section 4, we constructed extremal N = 2 elliptic genera for central charges n = 12
and n = 24. To construct extremal CFTs with larger central charges, we have to care about
the descendants of the bosonic oscillators. Those descendants always appear in CFTs based
on lattices and are obstacles to constructing extremal CFTs. One possible candidate to
resolve the problem is to take the orbifold by an appropriate symmetry as in the construction
of the Monster CFT [1], which is an extremal bosonic CFT. As discussed in the ternary
case [9], it would be interesting to study the fermionic orbifold of code CFTs.
In section 4 and 5, we computed the elliptic genera and decompose them into N = 2
superconformal characters. The Mathieu moonshine was discovered by noting the relationship
between the coefficients of the characters in the elliptic genus and the dimensions of the
irreducible representations [84] (see, e.g., [85] and references therein). Our construction of
elliptic genera uses classical error-correcting codes, and their automorphisms (symmetries)
have been studied extensively [29]. There may be an intriguing relationship between automorphisms of classical codes and coefficients of characters in elliptic genera. It would help
acquire a better understanding of the moonshine phenomena.
In the present paper, we computed examples of the U(1)-graded partition function with
the Fourier expansion (3.50). In other words, the unitarity bound (h ≥ n/24) in the R sector
holds. However, one can consider the U(1)-graded partition function whose Fourier expansion
has singular terms. For example, if we take the last example in appendix B of [10], the
(supersymmetric) unitarity bound (h ≥ n/24) in the R sector does not hold. However, the
– 34 –
JHEP01(2024)130
Although this paper deals with the construction of extremal N = 2 CFTs from classical
codes, it would be interesting if one could construct extremal N = 4 CFTs, some of which
have been realized in actual CFTs at small central charges [7, 40, 41]. For the central charge
n = 12, it is known that extremal N = 2 and N = 4 elliptic genera are the same functions,
so the fermionic code CFT constructed in section 4.1 realizes both the extremal N = 2
and N = 4 theories. For the central charge n = 24, the extremal N = 2 elliptic genus is
different from the N = 4 one. To construct N = 4 extremal CFT, we attempted to find
binary codes (p = 2) that return the Ramond-Ramond partition function consistent with
the N = 4 extremal elliptic genus [7]. However, we could not find such binary codes from
the database [80]. Also, in the ternary case (p = 3), it is straightforward to deduce from
Theorem 1.3 in [9] that any ternary code cannot yield the N = 4 extremal elliptic genus. If
one considers classical codes over p ≥ 5, there would be some chance to obtain the N = 4
extremal CFT. Additionally, there would be some possibilities that N = 4 extremal CFTs
arise if one can generalize the construction of fermionic code CFTs to the finite rings Zk for
an integer k. Recently, the construction of Narain CFTs from quantum codes over the rings
has been given [13]. Analogously, we may construct fermionic CFTs from classical codes over
rings. We can expect that such an extension widely broadens the scope of fermionic code
CFTs and yields theories inaccessible in the present paper.
Construction A lattice contains a characteristic vector with norm n/3 = 12. Therefore, we
can construct the U(1)-graded partition function that shows modular invariance and spectral
flow invariance while it does not admit the Fourier expansion (3.50). It would be interesting
to figure out its property with the help of number theory [51, 86, 87].
Acknowledgments
A
N = 2 superconformal characters
We list the graded characters of the representations of the N = 2 superconformal algebra. The
following notation is consistent with [41] up to overall signs. For more details, see e.g. [88–91].
The elliptic genus of an N = 2 SCFT is invariant under integer spectral flows. Therefore,
it should be decomposed into graded characters Ch(R) (τ, z) in the R sector defined by
Ch(R) (τ, z) =
X
SFθ ch(NS) τ, z +
θ∈ 12 +Z
1
2
(A.1)
where SFθ is defined in (2.41) and ch(NS) (τ, z) is an irreducible character in the NS sector.
The characters are classified into three types: massive, massless, and graviton. Let
the central charge n ∈ 6Z, l = n6 − 12 , q = e2πiτ , y = e2πiz , with h and Q representing the
conformal weight and the U(1) charge, i.e., the eigenvalues of L0 and J0 .
Massive. For the NS sector, the irreducible character of the massive representation with
the highest weight h, Q is
1
(NS)
chl;h,Q (τ, z)
=q
n
h− 24
Q
y
1
∞
Y
(1 + yq k− 2 )(1 + y −1 q k− 2 )
(1 − q k )2
k=1
.
(A.2)
The contribution (1 − q k )−1 = 1 + q k + q 2k + . . . comes from L−k and J−k , 1 + yq r from
−
−1 r
G+
−r , and 1 + y q from G−r .
For the R sector, the graded character of the massive representation with the highest
weight h′ , Q′ is given by
(R)
Chl;h′ ,Q′ (τ, z)
=
X
(NS)
SFθ chl;h,Q
θ∈ 12 +Z
where
Q
n
h′ = h + +
,
2
24
Q′ =
1
τ, z +
2
Q + n
Q +
– 35 –
6
n
6
(Q′ > 0)
−1
(Q′ < 0)
(A.3)
.
(A.4)
JHEP01(2024)130
We are grateful to Hee-Cheol Kim, Minsung Kim, and Yutaka Matsuo for their valuable
discussions. The work of K. K. and S. Y. was supported by FoPM, WINGS Program, the
University of Tokyo. The work of K. K. was supported by JSPS KAKENHI Grant-in-Aid
for JSPS fellows Grant No. 23KJ0436.
From (2.6), we obtain a more explicit form as
(R)
Chl;h′ ,Q′ (τ, z)
1
=
X
q
n
h− 24
+Qθ+ n
θ2
6
(−y)
Q+ n
θ
3
θ∈ 12 +Z
=
X
(1 − q k )2
k=1
n
n
1
1
∞
Y
(1 − yq k− 2 +θ )(1 − y −1 q k− 2 −θ )
1 2 1
− 2 m(m+1)
q h− 24 +Q(m+ 2 )+ 6 (m+ 2 )
n
1
(−y)Q+ 3 (m+ 2 )−m
m∈Z
∞
Y
(1 − yq k )(1 − y −1 q k−1 ) (A.5)
(1 − q k )2
k=1
′
′
n
′
∞
Y
(1 − yq k )(1 − y −1 q k−1 ) X
(1 − q k )2
k=1
(−1)m q lm
2 +(Q′ +δ− 1 )m
2
y 2lm
m∈Z
where δ is 1 if Q′ < 0 and 0 if Q′ > 0.
Massless. For the NS sector, the irreducible character of the massless representation with
the highest weight h = − Q2 , Q is
(NS)
ch
(τ, z)
l;h=− Q
,Q
2
=q
n
h− 24
Q
y
1
1
∞
Y
(1 + yq k− 2 )(1 + y −1 q k− 2 )
1
(A.6)
(1 − q k )2
1
1 + y −1 q 2
k=1
1
Q
where 1/(1 + y −1 q 2 ) comes from G−
−1/2 |h = − 2 , Q⟩ = 0.
For the R sector, the graded character of the massless representation with the highest
n
weight h′ = 24
, Q′ is given by
(R)
Chl;h′ = n ,Q′ (τ, z)
24
=
X
θ∈ 12 +Z
(NS)
SFθ ch
l;h=− Q
,Q
2
1
τ, z +
2
(A.7)
where Q′ = Q + n6 . From a similar discussion as the massive representation,
′
(R)
Chl;h′ = n ,Q′ (τ, z) = (−1)Q y Q
24
′
∞
Y
(1 − yq k )(1 − y −1 q k−1 )
(1 − q k )2
k=1
×
1
X
m∈Z
1 − y −1 q
(−1)m q lm
−m
2 +(Q′ − 1 )m
2
(A.8)
y 2lm .
Graviton. According to the equation (7.5) in [41], the graded character of the graviton
n
representation with the highest weight h′ = 24
, Q′ = n6 in the R sector is given by
n
−1
6
(R)
(R)
Chl;h′ = n ,Q′ = n (τ, z) = q −s Chl; n +s, n +
24
6
24
n
6
6
X
(R)
(R)
(−1)k Chl; n +s, n −k + Chl; n +s,k− n
24
k=1
6
24
6
(A.9)
(R)
+ (−1) Chl; n ,0
24
where s is an arbitrary positive integer.
Using these graded characters, we can decompose the elliptic genus of an N = 2 SCFT as
ZEG (τ, z) =
(R)
A0,0 Chl; n ,0
24
+
∞
X
t=0
n
−1
6
X
(R)
At,k Ch n
k=1
l; 24 +t,k
+
(R)
Chl; n +t,−k
24
+ At, n6
(R)
Chl; n +t, n
24
6
(A.10)
where At,k ∈ Z.
– 36 –
JHEP01(2024)130
= (−1)Q +δ q h − 24 y Q +δ
Open Access. This article is distributed under the terms of the Creative Commons
Attribution License (CC-BY4.0), which permits any use, distribution and reproduction in
any medium, provided the original author(s) and source are credited.
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W4200096228.txt | PLOS ONE
RESEARCH ARTICLE
Genetic risk factors associated with
gestational diabetes in a multi-ethnic
population
Paula Benny1, Hyeong Jun Ahn2, Janet Burlingame1, Men-Jean Lee ID1, Corrie Miller1,
John Chen2, Johann Urschitz ID3*
1 Department of Obstetrics, Gynecology, and Women’s Health, John A. Burns School of Medicine, University
of Hawaii, Honolulu, HI, United States of America, 2 Department of Quantitative Health Sciences, John A.
Burns School of Medicine, University of Hawaii, Honolulu, HI, United States of America, 3 Department of
Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI,
United States of America
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
* johann@hawaii.edu
Abstract
Aims
OPEN ACCESS
Citation: Benny P, Ahn HJ, Burlingame J, Lee M-J,
Miller C, Chen J, et al. (2021) Genetic risk factors
associated with gestational diabetes in a multiethnic population. PLoS ONE 16(12): e0261137.
https://doi.org/10.1371/journal.pone.0261137
Editor: Carla Pegoraro, PLOS, UNITED KINGDOM
Received: May 18, 2021
Accepted: November 24, 2021
Genome-wide association studies have shown an increased risk of type-2-diabetes (T2DM)
in patients who carry single nucleotide polymorphisms in several genes. We investigated
whether the same gene loci confer a risk for gestational diabetes mellitus (GDM) in women
from Hawaii, and in particular, Pacific Islander and Filipino populations.
Methods
Blood was collected from 291 women with GDM and 734 matched non-diabetic controls
(Pacific Islanders: 71 GDM, 197 non-diabetic controls; Filipinos: 162 GDM, 395 controls;
Japanese: 58 GDM, 142 controls). Maternal DNA was used to genotype and show allele frequencies of 25 different SNPs mapped to 18 different loci.
Published: December 20, 2021
Copyright: © 2021 Benny et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All raw data files have
been made freely accessible and have been
uploaded to a public data repository; doi:https://
data.world/pbenny/gdm-snp-analysis.
Funding: Funding for this project was partially
supported by NIH Awards U54MD00760131,
U54GM104944, 1R21HD092716 and
P30GM131944.
Competing interests: The authors have declared
that no competing interests exist.
Results
After adjusting for age, BMI, parity and gravidity by multivariable logistic regression, several
SNPs showed significant associations with GDM and were ethnicity specific. In particular,
SNPs rs1113132 (EXT2), rs1111875 (HHEX), rs2237892 (KCNQ1), rs2237895 (KCNQ1),
rs10830963 (MTNR1B) and rs13266634 (SLC30A8) showed significant associations with
GDM in Filipinos. For Japanese, SNPs rs4402960 (IGFBP2) and rs2237892 (KCNQ1) were
significantly associated with GDM. For Pacific Islanders, SNPs rs10830963 (MTNR1B) and
rs13266634 (SLC30A8) showed significant associations with GDM. Individually, none of the
SNPs showed a consistent association with GDM across all three investigated ethnicities.
Conclusion
Several SNPs associated with T2DM are found to confer increased risk for GDM in a multiethnic cohort in Hawaii.
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Genetic risk factors for type II diabetes and gestational diabetes
Introduction
Normal pregnancy is associated with a period of relative insulin resistance that progressively
leads to maternal hyperglycemia and transplacental passage of glucose to promote fetal growth.
This is similar to the type of insulin resistance found in patients with type 2 diabetes (T2DM)
[1–5]. Maternal hyperglycemia usually results in an increase in insulin secretion by maternal
pancreatic beta-cells [6]. However, in approximately 1 out of 15 pregnant women in the USA
[7], the diagnosis of gestational diabetes (GDM) is made when this compensatory mechanism
is impaired. It has been postulated that there may be an underlying baseline level of insulin
resistance that predates the pregnancy and that changes of glycemic regulation associated with
the secretion of human placental lactogen, also known as human chorionic somatomammotropin, from the placenta, leads to a compromise in glycemic control and the diagnosis of
GDM.
Maternal age, overweight/obese status and a family history of diabetes are risk factors for
GDM [8, 9]. Recent studies have also described combinatorial effects of risk factors in the
manifestation of GDM, together with the integration of first trimester fasting glycemia measurements [10, 11]. Fetuses of pregnant women with poorly controlled GDM are at an
increased risk for macrosomia, polyhydramnios, birth injury, neonatal hypoglycemia, and
other neonatal metabolic disturbances. In addition to these perinatal complications, the likelihood of long-term consequences for the health and wellbeing of both mothers and their offspring is significantly higher compared to normoglycemic pregnancies. GDM is not only
associated with a higher incidence of adult obesity and the development of T2DM later in life,
but also with a predisposition for cardiovascular diseases [12, 13] and metabolic disorders [14–
16]. While GDM usually resolves after delivery, the increased probability of future deterioration in insulin resistance and beta-cell insufficiency puts women who have had GDM at a
greater risk of developing T2DM than those who had a normal pregnancy [17]. Women with a
previous history of GDM have a 35% to 60% chance of developing diabetes in the next 10–20
years [18].
GDM prevalence rates have been reported to vary in direct proportion to the prevalence of
T2DM in several ethnic populations [8]. Along with high rates of T2DM, GDM is particularly
prevalent in certain minority populations including Asian, Native American, Hispanic and
African-American women, who are at higher risk for GDM than non-Hispanic Caucasian
women [19]. The local prevalence of T2DM in Hawaii is increasing, especially among Asian
and Pacific Islander parturients. These groups have demonstrated significantly higher rates of
impaired glucose tolerance (15.5%) and T2DM (20.4%) compared to the overall U.S. population [20]. This increase in T2DM in Asian and Pacific Islanders is likely to correlate with
increasing number of pregnancies complicated by GDM [20, 21]. A recent report on the prevalence of GDM in Hawaii found that GDM rates were highest in Filipino women, followed by
Chinese, Japanese and Native Hawaiian/Pacific Islander women. Caucasians in Hawaii had
the lowest GDM rate of all ethnic groups [22]. Such differences in GDM incidences are likely
to be multi-factorial and are expected to include genetic factors [23].
Several pioneering genome-wide association studies (GWAS) have identified over 60
genetic loci in genes responsible for insulin secretion, insulin resistance, lipid and glucose
metabolism, and other pathways that are associated with type 2 diabetes [24–29]. While most
of these studies were from European populations, more recent GWAS have identified novel
loci specific to other populations such as South Asians [30], Japanese [31, 32], Mexicans [33],
Chinese [34, 35], African Americans [36, 37], and Asian Indians [38]. Since women with
GDM are at increased risk of T2DM and both T2DM and GDM are polygenic, multifactorial
diseases, it has been suggested that both diseases are manifestations of similar
PLOS ONE | https://doi.org/10.1371/journal.pone.0261137 December 20, 2021
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PLOS ONE
Genetic risk factors for type II diabetes and gestational diabetes
pathophysiological processes [39]. Several studies have validated these loci to also infer an
increased risk of gestational diabetes [40–43]. While these studies have examined several different ethnic populations, few prior studies have investigated the specific associations of
T2DM in the multiethnic Hawaiian populations [44]. One such study to focus on the Hawaiian
population is the MEC-PAGE (Multi-Ethnic Cohort Population Architecture using Genomics
and Epidemiology). While this large study provided data on risk assessments of chronic diseases such as diabetes [45], colorectal [46] and prostate cancer [47] in Native Hawaiians, this
was predominantly in an older population (age 45–75 years old). Another recent study focused
on disease occurrence in Native Hawaiians and identified a locus on Chr6 associated with
T2DM [48]. However, these loci could not be validated as the replication study size was a
small, predominantly Samoan cohort. As Native Hawaiians comprise approximately 10% of
the State’s population, a more accurate representation of the different populations of Hawaii is
currently lacking.
Therefore, in this study, we investigated whether a select panel of 25 significantly reported
single nucleotide polymorphisms (SNPs) known to be associated with T2DM conferred a risk
for GDM in Pacific Islander, Filipino and Japanese women in a unique cohort of pregnant
women living in Hawaii. The results of this cross-sectional study would provide new knowledge on the genetic risks of GDM susceptibility occurring in the distinct populations of
Hawaii.
Materials and methods
Subjects
A case–control study was performed comparing genotype frequencies of 25 different SNPs
(Table 1) in healthy pregnant women and pregnant women with GDM. Ethical approval for
the study was obtained from the Institutional Review Board (IRB) of the University of Hawaii.
All participants provided written informed consent. Samples were obtained from the RMATRIX Biorepository of the John A. Burns School of Medicine, University of Hawaii following
the study’s inclusion/exclusion criteria. The Hawaiian Biorepository houses over 9,000 biospecimens collected from pregnant women from 2006–2012. All pregnant women had been routinely screened with a 1-hour 50-g oral glucose challenge test (GCT) at 24–28 weeks of
gestation. Gestational age was determined by ultrasound examination. Individuals with blood
Table 1. Clinical characteristics of participants according to ethnic group.
Variable
N
Mean
Std Dev
Min
Max
N
Mean
Std Dev
Min
Max
Filipino
Maternal Age
395
30.5
5.7
18.0
44.0
162
31.7
5.5
19.0
42.0
0.029
BMI
395
24.2
4.4
15.4
43.5
155
25.9
5.2
15.9
44.6
<0.001
Gravida
395
2.8
1.7
1.0
13.0
162
2.8
1.6
1.0
8.0
0.930
Parity
395
1.2
1.1
0.0
6.0
162
1.4
1.3
0.0
5.0
0.031
Japanese
Pacific Islander
Control
p
Ethnicity
GDM
Maternal Age
142
35.0
4.8
19.0
45.0
58
35.8
5.0
27.0
50.0
0.300
BMI
141
22.4
3.6
15.7
43.5
51
24.9
3.8
17.7
34.5
<0.001
Gravida
142
2.5
1.5
1.0
7.0
58
3.2
1.9
1.0
7.0
0.020
Parity
142
0.9
0.9
0.0
4.0
58
0.9
1.1
0.0
4.0
0.950
Maternal Age
197
28.0
5.9
18.0
43.0
71
30.0
6.1
19.0
46.0
0.016
BMI
192
29.4
6.2
16.5
50.0
56
32.7
7.1
18.3
47.8
<0.001
Gravida
197
3.4
2.2
1.0
12.0
71
3.3
2.3
1.0
12.0
0.870
Parity
197
2.0
1.9
0.0
8.0
71
1.8
1.8
0.0
8.0
0.410
https://doi.org/10.1371/journal.pone.0261137.t001
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Genetic risk factors for type II diabetes and gestational diabetes
glucose levels greater than 130 mg/dL underwent a 3-hour 100-g oral glucose tolerance test
(OGTT) in the morning after an overnight hour fast.
Pregnant women were diagnosed with GDM when 2 or more glucose values were at or
above the specified Coustan-Carpenter thresholds for diagnosing gestational diabetes; fasting
95 mg/dL, 1-hour 180 mg/dL, 2-hour 155 mg/dL, and 3-hour 140 mg/dL [49]. Blood was collected from 291 women who met Coustan-Carpenter criteria for GDM and 734 non-diabetic
controls (normal glucose tolerance). Only subjects, where self-reported ethnicity was concordant with that of all four biological grandparents’ ethnicity were included in this study.
According to the US Census Bureau, race and ethnicity are defined as a person’s self-identification with one or more social groups [50]. Subjects may report multiple races and ethnicities
based on geographical, social, and cultural affiliations associated with human civilization. Selfidentified population stratifications and geographic ancestry have been demonstrated to be
highly correlated with self-identified race/ethnicity as well as a determinant of genetic structure [51]. The GDM cohort consisted of 71 Pacific Islander, 162 Filipino, and 58 Japanese
women from Hawaii. The control cohort consisted of 197 Pacific Islander, 395 Filipino, and
142 Japanese women from Hawaii with normal glucose tolerance (NGT). Information regarding Height (m), weight (kg), age, BMI, ethnicity (self-reported) and GCT, OGTT results were
obtained from prenatal records.
Genotyping
The 25 single nucleotide polymorphisms (SNPs) were chosen due to their strong association
with T2DM in other ethnic groups including Caucasians/United States [25], Europeans [26]
and Caucasians/Canada [27]. While these SNPs were associated with T2DM in other ethnic
groups, this is the first study to evaluate the association in Native Hawaiian, Pacific Islander,
Filipino and Japanese populations. Genomic DNA was extracted from whole blood using the
Autopure DNA isolation system (Gentra Systems, Minneapolis, MN) following the manufacturer’s protocol. All SNPs selected for genotyping (Table 2) were candidate or GWAS-identified variants shown to be associated with T2DM or GDM. Genotyping was performed on the
TaqMan OpenArray platform (Life Technologies, Foster City, CA) using 125ng of genomic
DNA. TaqMan OpenArray Genotyping Mastermix was added to the DNA and this solution
was then distributed across the OpenArray plates pre-loaded with TaqMan genotyping assays
for 32 SNPs. Polymerase chain reaction (PCR) was performed on a Bio-Rad Slide Chambers
Dual-Block Alpha unit using PCR conditions set by the manufacturer. Allelic discrimination
was done on a Biotrove OpenArray System using the OpenArray SNP Genotyping Analysis
Software Version 1.0.3. All SNPs were analyzed using TaqMan Genotyper Software Version
1.0.1. Quality control criteria included fingerprint blanks, which are used to identify the plate
and verify cross-contamination and or sample error and no template controls (NTC), which
serve as a background detector and review of any reagent problems. DNA samples that failed
in more than 25% of genotyping assays were excluded, assuming poor DNA quality and uncertain measurements. A genotyping error of 0.3% was determined by blind duplicate genotyping. Automated call rates for all 25 SNPs exceeded 97%.
Statistical analysis
Demographic and clinical variables (age, BMI, gravidity, and parity) were summarized by
descriptive statistics; means, standard deviations, minimums and maximums. Differences in
patient characteristics between case and control groups for each ethnicity were examined
using independent two-sample t-test and differences in patient characteristics among the three
race/ethnicity groups were also examined using one-way Analysis of Variance (ANOVA).
PLOS ONE | https://doi.org/10.1371/journal.pone.0261137 December 20, 2021
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Genetic risk factors for type II diabetes and gestational diabetes
Table 2. Association of 25 SNPs with GDM risk.
Filipino
Gene
SNP
ADCY5
Japanese
Pacific Islanders
Alleles
MAF
HWE
Alleles
MAF
HWE
Alleles
MAF
HWE
rs2877716
C/T
0.7
0.028
C/T
0.3
1.000
C/T
4.3
1.000
ARAP 1
rs11603334
G/A
5.5
1.000
G/A
3.4
1.000
G/A
5.5
0.415
CDKAL1
rs7754840
G/C
49.6
0.258
G/C
39.4
0.718
G/C
45.6
0.877
CDKN2A/2B
rs10811661
T/C
30.8
0.285
T/C
42.4
0.300
T/C
36.4
0.754
DGKB
rs2191349
T/G
24.3
0.476
T/G
31.9
0.565
T/G
15.1
0.030
�
EXT2
rs1113132
C/G
27.1
0.082
G/C
34.3
0.856
C/G
14.2
0.546
FTO
rs8050136
C/A
15.5
0.843
C/A
16
1.000
C/A
11.7
0.737
G6PC2
rs3755157
C/T
35.0
0.571
C/T
38.2
0.258
T/C�
48.5
0.748
G6PC2
rs16856187
A/C
37.3
0.330
A/C
33.9
0.573
A/C
43.8
1.000
<0.010
G6PC2
rs560887
C/T
2.0
0.015
C/T
1.8
1.000
C/T
1.2
GCK
rs4607517
G/A
13.9
<0.010
G/A
22.7
1.000
G/A
18.9
0.815
GCKR
rs780094
C/T
41.7
0.210
T/C�
49.2
0.735
C/T
27.2
0.207
HHEX
rs1111875
T/C
17.0
0.032
T/C
30.9
1.000
T/C
35.7
0.02
HHEX
rs7923837
A/G
9.5
0.325
A/G
18.7
0.567
A/G
27.1
0.077
IGFBP2
rs4402960
G/T
26.5
0.358
G/T
29.4
0.551
G/T
22.4
0.399
KCNQ1
rs2237892
C/T
38.7
0.462
C/T
40.9
0.601
C/T
28.7
1.000
KCNQ1
rs2237895
A/C
33.1
0.406
A/C
32.3
0.848
A/C
24.1
1.000
MADD
rs10501320
G/C
0.5
1.000
G/C
0.3
1.000
G/C
1.2
1.000
MTNR 1B
rs10830963
G/C
47.8
0.606
C/G�
43.2
0.286
G/C
48.9
0.281
PKN2
rs6698181
C/T
18.8
0.623
C/T
40.2
0.476
C/T
8.7
1.000
SLC30A8
rs13266634
T/C
49.9
0.749
C/T�
38.8
0.858
C/T�
41.9
0.084
TCF7L2
rs7903146
C/T
1.8
1.000
C/T
5.2
1.000
C/T
11.5
0.041
TCF7L2
rs7901695
T/C
1.7
1.000
T/C
5.2
1.000
T/C
9.7
0.399
TCF7L2
rs12255372
G/T
1.7
1.000
G/T
3.6
1.000
G/T
9.2
0.688
TCF7L2
rs11196205
G/C
4.1
0.134
G/C
6.7
1.000
G/C
11.2
0.720
Alleles: major/minor, MAF = Major Allele Frequency, HWE = Hardy-Weinburg Equilibrium. SNPs not at Hardy–Weinberg equilibrium (p<0.05) are shown in blue.
Reversed minor allele compared to Filipino Controls are in red with an asterisk. Risk alleles are bold and underlined.
https://doi.org/10.1371/journal.pone.0261137.t002
Genotype minor allele frequencies (MAF) were summarized by percentages. The BreslowDay test [52] was used to assess the homogeneity of MAFs and odds ratios (OR) among the
three ethnic groups. We used chi-square tests to determine whether an individual polymorphism was in Hardy–Weinberg equilibrium. For the dominant and recessive model of each
polymorphism, unadjusted odds ratios were calculated with p values using chi-square tests
within each ethnic group. The dominant/recessive model was chosen over other models such
as the additive or multiplicative model as it increased the resolution of assessing disease risk,
similar to other studies of diabetes [53, 54]. We evaluated the homogeneity of the unadjusted
OR among the three ethnic groups by Breslow-Day test and used Mantel-Haenszel formula
[55] to calculate summary unadjusted OR with 95% confidence interval (CI). We then selected
SNPs which showed that 1) unadjusted OR were significantly different from 1.0; 2) p values of
Breslow-Day test were less than 0.05; 3) the summary of unadjusted OR was significantly different from 1.0 based on Mantel-Haenszel formula. For the selected SNPs, multivariable logistic regression models were used to further adjust and control for confounding factors, such as
age, BMI, parity, and gravidity. All data analyses were performed in R-package and SAS 9.3
and a two-tailed p<0.05 was regarded as statistically significant.
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Genetic risk factors for type II diabetes and gestational diabetes
Results
We compared the genotype frequencies in 291 women from Hawaii who met Coustan-Carpenter criteria for GDM and 734 matched non-diabetic controls (Pacific Islander: 71 subjects
with GDM and 197 non-diabetic controls; Filipinos: 162 GDM patients and 395 controls; Japanese: 58 GDM patients and 142 controls).
The clinical baseline characteristics are listed in Table 1. The mean BMI was significantly
higher in all three GDM groups (Filipino and Japanese p<0.001, Pacific Islander p = 0.016)
when compared to the ethnically matched controls. Additionally, mean maternal age
(p = 0.029) and mean parity (p = 0.031) were significantly higher in Filipino women with
GDM than in their control (p = 0.001), whereas parity (p = 0.020) and maternal age differed in
the Japanese GDM cohort and Pacific Islander women respectively.
25 SNPs reported to be associated with T2DM were genotyped. Table 2 reports the SNP
alleles, as well as minor allele frequency (MAF) and the results from the Hardy–Weinberg
equilibrium analysis in the control group for all 25 SNPs studied. All SNPs were in Hardy–
Weinberg equilibrium at the significance level of p>0.05 except for (i) rs4607517 (GCK) and
rs2877716 (ADCY5) in Filipinos, (ii) rs2191349 (DGKB) and rs7903146 (TCF7L2) in Pacific
Islanders, (iii) rs1111875 (HHEX) and rs560887 (G6PC2) in both Filipino and Pacific Islanders
(Table 2, blue values). For certain SNPs, there was also a reversal of major/minor alleles among
the subpopulations (as in an allele that is major in one race becomes minor in another race
and vice versa) (Table 2, red alleles).
Unadjusted odds ratios (OR) and associated p-values were subsequently calculated for the
association of genotypes with GDM in the dominant and recessive model of each polymorphism (Tables 3 and 4 respectively). Interestingly, for each race, several SNPs showed significant associations with GDM (Tables 3 and 4, blue values). However, none of the SNPs
appeared to show a consistent association with GDM across all three investigated ethnicities.
To evaluate this observation further, we assessed the homogeneity of the unadjusted OR
among the three ethnic groups using the Breslow-Day (BD) test as well as the Mantel-Haenszel
(MH) formula that calculates a summary of unadjusted OR with 95% confidence interval. In
the dominant model, the association of rs2237892 (KCNQ1, Table 3) with GDM differed significantly between our populations (BD p = 0.024, green value, Table 3). Indeed, while highly
significant for Filipinos (p<0.001), we detected only a moderate association of rs2237892
(KCNQ1) with GDM in Japanese subjects (p = 0.03) and none in Pacific Islanders (p = 0.49)
(Table 3). Similarly, for the recessive model, rs13266634 (SLC30A8, Table 4) showed a significant association with GDM in Pacific Islanders (p = 0.005), but not in Filipinos (p = 0.301) or
Japanese (p = 0.230) (BD p = 0.004, green value, Table 4). SNPs with values from MantelHaenszel formula that were significantly different from 1.0 in the dominant and recessive
models are highlighted in red in Tables 3 and 4, respectively.
Next, we used multivariable logistic regression models to adjust for age, BMI, gravidity, and
parity. Such regression analyses were applied only to selected SNPs that originally showed
either significant unadjusted OR p values (p<0.05; blue) or significant Breslow-Day test p values (p<0.05; green) or values from Mantel-Haenszel formula significantly different from 1.0
(red) in the dominant and recessive models shown in Tables 3 and 4. The results of the regression analyses for the SNPs that originally showed significant unadjusted OR p values (p<0.05)
(blue values in Tables 3 and 4) are shown in Table 5. The results of the regression analyses for
the SNPs that originally showed significant Breslow-Day test p values (p<0.05) (green values
in Tables 3 and 4) are shown in Table 6. The results of the regression analyses for the SNPs
that showed values from Mantel-Haenszel formula significantly different from 1.0 (red values
in Tables 3 and 4) are shown in Table 7.
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Genetic risk factors for type II diabetes and gestational diabetes
Table 3. Association of SNPs with GDM risk using a dominant model (unadjusted odds ratios; OR, Breslow-Day test; BD, Mantel-Haenszel formula; MH).
Filipino
Japanese
P
OR
PI
Gene
SNP
OR
P
OR
BD_FJPI
OR.mh
Low. B
Up. B
ADCY5
rs2877716
0.39
(
0.337
)
0.61
(
0.176
)
0.27
(
0.046
P
)
0.889
0.34
0.11
1.04
ARAP 1
rs11603334
1.11
(
0.725
)
1.08
(
0.906
)
0.76
(
0.601
)
0.821
1.02
0.64
1.63
CDKAL1
rs7754840
1.5
(
0.071
)
0.66
(
0.195
)
1.82
(
0.089
)
0.057
1.27
0.92
1.75
CDKN2A/2B
rs10811661
0.88
(
0.499
)
1.17
(
0.641
)
0.57
(
0.054
)
0.248
0.83
0.63
1.11
DGKB
rs2191349
1.13
(
0.507
)
0.78
(
0.433
)
0.74
(
0.365
)
0.406
0.96
0.72
1.29
EXT2
rs1113132
1.52
(
0.028
)
4.00
(
0.032
)
1.04
(
0.907
)
0.218
1.48
1.09
2.02
FTO
rs8050136
1.11
(
0.604
)
0.9
(
0.767
)
0.7
(
0.335
)
0.539
0.97
0.71
1.34
G6PC2
rs16856187
0.74
(
0.12
)
0.68
(
0.217
)
1.17
(
0.626
)
0.393
0.80
0.60
1.07
G6PC2
rs3755157
0.83
(
0.346
)
0.52
(
0.047
)
1.18
(
0.676
)
0.252
0.80
0.59
1.07
G6PC2
rs560887
0.44
(
0.156
)
0.4
(
0.35
)
1.43
(
0.293
)
0.745
0.41
0.15
1.12
GCK
rs4607517
1.45
(
0.088
)
1.17
(
0.632
)
0.91
(
0.753
)
0.456
1.22
0.90
1.65
GCKR
rs780094
0.89
(
0.56
)
1.41
(
0.367
)
0.67
(
0.17
)
0.307
0.89
0.66
1.19
HHEX
rs1111875
1.5
(
0.047
)
0.61
(
0.13
)
1.43
(
0.229
)
0.053
1.22
0.91
1.63
HHEX
rs7923837
1.51
(
0.085
)
1.19
(
0.605
)
1.84
(
0.036
)
0.607
1.52
1.11
2.08
IGFBP2
rs4402960
0.97
(
0.888
)
0.5
(
0.031
)
0.83
(
0.534
)
0.205
0.82
0.62
1.09
KCNQ1
rs2237892
0.49
(
< .001
)
0.5
(
0.033
)
1.22
(
0.488
)
0.024
KCNQ1
rs2237895
1.1
(
0.624
)
0.96
(
0.907
)
1.79
(
0.047
)
0.28
1.20
0.91
1.59
MADD
rs10501320
0.59
(
0.627
)
0.61
(
0.176
)
1.43
(
0.293
)
0.556
0.43
0.10
1.95
0.83
MTNR 1B
rs10830963
0.63
(
0.025
)
0.71
(
0.368
)
0.52
(
0.037
)
0.799
0.61
0.45
PKN2
rs6698181
0.89
(
0.551
)
0.82
(
0.555
)
1.02
(
0.95
)
0.91
0.89
0.66
1.22
SLC30A8
rs13266634
0.58
(
0.011
)
0.74
(
0.387
)
1.09
(
0.781
)
0.239
0.72
0.53
0.98
TCF7L2
rs11196205
0.95
(
0.89
)
0.7
(
0.465
)
0.82
(
0.583
)
0.878
0.84
0.54
1.31
TCF7L2
rs12255372
0.92
(
0.876
)
0.64
(
0.496
)
0.72
(
0.411
)
0.902
0.76
0.43
1.34
TCF7L2
rs7901695
0.91
(
0.866
)
0.8
(
0.683
)
0.92
(
0.817
)
0.979
0.89
0.52
1.50
TCF7L2
rs7903146
1.2
(
0.724
)
0.8
(
0.683
)
0.67
(
0.288
)
0.655
0.82
0.48
1.38
BD-FJPI = Breslow Day Test for Filipino, Japanese and Pacific Islander. SNPs with significant unadjusted OR p values (p<0.05) are highlighted in blue. SNPs with
significant Breslow-Day test p values (p<0.05) are highlighted in green. SNPs with values from Mantel-Haenszel formula significantly different from 1.0 are highlighted
in red.
https://doi.org/10.1371/journal.pone.0261137.t003
Overall, after adjusting for age, BMI, parity and gravidity, SNPs rs1113132 (EXT2),
rs1111875 (HHEX), rs2237892 (KCNQ1), rs10830963 (MTNR1B) and rs13266634 (SLC30A8)
showed significant associations with GDM in the dominant model for Filipinos, while
rs2237892 (KCNQ1), rs2237895 (KCNQ1) and rs10830963 (MTNR1B) were significantly
associated with GDM in the recessive model (Table 8). For Japanese women, the SNPs
rs4402960 (IGFBP2) and rs2237892 (KCNQ1) showed significant associations with GDM in
the dominant model, but no SNP was significantly associated with GDM in the recessive
model (Table 8). For Pacific Islander women, the SNP rs10830963 (MTNR1B) showed significant association with GDM in the dominant model, while rs13266634 (SLC30A8) was significantly associated with GDM in the recessive model (Table 8).
Discussion
GDM and T2DM are both multifactorial diseases and are believed to share similar epidemiologic risk factors such as obesity, hypertension, polycystic ovarian syndrome, racial-ethnic
background, and family history. Several studies have demonstrated a shared genetic susceptibility for both T2DM and GDM among various populations. Cho et al. investigated various
PLOS ONE | https://doi.org/10.1371/journal.pone.0261137 December 20, 2021
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Genetic risk factors for type II diabetes and gestational diabetes
Table 4. Association of SNPs with GDM risk using a recessive model (unadjusted odds ratios; OR, Breslow-Day Test; BD, Mantel-Haenszel formula; MH).
Filipino
Japanese
P
OR
PI
Gene
SNP
OR
P
OR
BD_FJPI
OR. mh
Low. B
ADCY5
rs2877716
1.13
(
0.99
)
NA
(
NA
)
NA
(
NA
P
)
NA
0
0
Up. B
0
ARAP 1
rs11603334
2.39
(
0.544
)
NA
(
NA
)
2.85
(
0.471
)
0.931
2.605
0.364
18.643
CDKAL1
rs7754840
0.82
(
0.375
)
1.38
(
0.441
)
1.33
(
0.436
)
0.366
0.997
0.71
1.4
CDKN2A/2B
rs10811661
0.63
(
0.167
)
0.36
(
0.031
)
1.17
(
0.701
)
0.18
0.667
0.425
1.047
DGKB
rs2191349
1.48
(
0.327
)
0.94
(
0.903
)
0.31
(
0.204
)
0.32
1.057
0.587
1.903
EXT2
rs1113132
0.73
(
0.392
)
1.52
(
0.201
)
1.14
(
0.332
)
0.131
1.03
0.652
1.627
FTO
rs8050136
1.06
(
0.923
)
2.56
(
0.267
)
1.95
(
0.484
)
0.662
1.514
0.655
3.499
G6PC2
rs3755157
1.04
(
0.898
)
1.49
(
0.374
)
1.19
(
0.621
)
0.792
1.168
0.788
1.732
G6PC2
rs560887
1.13
(
0.99
)
NA
(
NA
)
1.14
(
1
)
0.372
0.518
0.06
4.461
G6PC2
rs16856187
1.04
(
0.898
)
1.44
(
0.406
)
1.36
(
0.387
)
0.745
1.198
0.821
1.749
GCK
rs4607517
1.02
(
0.969
)
1.06
(
0.935
)
0.48
(
0.46
)
0.794
0.919
0.438
1.929
GCKR
rs780094
0.76
(
0.268
)
1.10
(
0.785
)
1.35
(
0.6
)
0.521
0.9
0.616
1.316
HHEX
rs1111875
1.13
(
0.798
)
1.1
(
0.857
)
1.15
(
0.729
)
0.998
1.128
0.679
1.873
HHEX
rs7923837
1.81
(
0.448
)
1.65
(
0.595
)
1.3
(
0.592
)
0.923
1.456
0.703
3.013
IGFBP2
rs4402960
0.75
(
0.435
)
0.37
(
0.156
)
0.84
(
0.826
)
0.679
0.665
0.361
1.224
KCNQ1
rs2237892
0.55
(
0.03
)
0.78
(
0.565
)
0.82
(
0.736
)
0.726
0.634
0.412
0.977
KCNQ1
rs2237895
2
(
0.015
)
0.69
(
0.517
)
1.64
(
0.404
)
0.25
1.604
1.025
2.508
MADD
rs10501320
NA
(
NA
)
NA
(
NA
)
NA
(
NA
)
NA
0
0
0
MTNR 1B
rs10830963
0.39
(
< .001
)
0.61
(
0.15
)
0.7
(
0.296
)
0.339
0.506
0.356
0.72
PKN2
rs6698181
0.59
(
0.325
)
1.89
(
0.117
)
1.14
(
1
)
0.185
1.2
0.653
2.205
SLC30A8
rs13266634
0.79
(
0.301
)
0.55
(
0.23
)
2.77
(
0.005
)
0.004
TCF7L2
rs11196205
1.13
(
0.99
)
NA
(
NA
)
1.14
(
0.57
)
0.363
0.432
0.052
3.622
TCF7L2
rs12255372
NA
(
NA
)
NA
(
NA
)
1.14
(
1
)
NA
0
0
0
TCF7L2
rs7903146
1.13
(
0.296
)
NA
(
NA
)
1.14
(
0.345
)
0.028
TCF7L2
rs7901695
NA
(
NA
)
NA
(
NA
)
1.14
(
0.571
)
NA
0
0
0
BD-FJPI = Breslow Day Test for Filipino, Japanese and Pacific Islander. SNPs with significant unadjusted OR p values (p<0.05) are shown in blue. SNPs with significant
Breslow-Day test p values (p<0.05) are shown in green. SNPs with values from Mantel-Haenszel formula significantly different from 1.0 are shown in red.
https://doi.org/10.1371/journal.pone.0261137.t004
SNPs known to be associated with an increased risk of T2DM in women with and without
GDM in a Korean population [40]. Allelic differences in CDKAL1 (Cdk5 regulatory associated
protein 1-like 1), CDKN2A-2B (cyclin-dependent kinase inhibitor 2A), HHEX (hematopoietically expressed homeobox), IGFT2BP2 (Insulin Like Growth Factor Binding Protein 2),
SLC30A8 (Solute Carrier Family 30 Member 8), and TCF7L2 (Transcription Factor 7 Like 2)
conferred an increased risk of GDM [40]. More recently, MTNR1B (rs10830962) was also
found to have an excess association with T2DM and GDM in a Korean population [56].
rs2237895 on the KCNQ1 gene has been confirmed to be associated with both GDM and
T2DM in Pakistani and Chinese cohorts [57, 58]. Gene variants of HMG20A (rs7178572) and
HNF4A (rs4812829) showed significant association with GDM in Asian Indians [59].
The unique multiethnic population of Hawaii is comprised of both immigrants and multigenerational descendants of Japanese, Chinese, and Filipino heritage, in addition to those of
Native Hawaiian ancestry. While many patients presenting for care identify as multiethnic, the
inclusion criteria for this study population was strictly limited to those self-reporting one ethnicity according to all 4 grandparents. This allowed us to compare across multiple ethnicities
at once without disproportionate heterogeneity. Identifying genotypic differences may account
for the variation in disease phenotypes that are seen in various populations of pregnant
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Genetic risk factors for type II diabetes and gestational diabetes
Table 5. Multivariable logistic regression analysis (OR).
Filipino
Dominant
Recessive
Gene
SNP
ADCY5
rs2877716
EXT2
rs1113132
G6PC2
rs3755157
HHEX
rs1111875
HHEX
rs7923837
IGFBP2
rs4402960
KCNQ1
rs2237892
KCNQ1
rs2237895
MTNR 1B
OR
Japanese
P_value
OR
1.53 (
0.034
)
1.60 (
0.027
)
0.5 (
< .001
)
rs10830963
0.62 (
0.028
)
SLC30A8
rs13266634
0.49 (
0.002
)
KCNQ1
rs2237892
0.51 (
0.019
)
KCNQ1
rs2237895
1.84 (
0.046
)
CDKN2A/2B
rs10811661
MTNR 1B
rs10830963
0.34 (
< .001
)
SLC30A8
rs13266634
PI
P_value
OR
7.40 (
0.063
)
0.57 (
0.133
)
0.32 (
0.003
)
0.45 (
0.035
)
0.35 (
0.053
P_value
0.37 (
0.153
)
1.55 (
0.196
)
1.72 (
0.108
)
0.42 (
0.021
)
2.43 (
0.03
)
)
List of SNPs (adjusted for age, BMI, parity, gravidity) which showed significant unadjusted OR p values (p<0.05) in the Dominant (Table 3) and Recessive (Table 4).
https://doi.org/10.1371/journal.pone.0261137.t005
women. For example, an East Asian cohort of women living in New York City with GDM
were found to have a normal BMI, have a low risk for fetal macrosomia and GDM phenotype
that was relatively easy to control with only diet modification [60]. However, a cohort of Arabian women with GDM living in Scandinavia were found to be much more insulin-resistant
than Caucasian Scandinavian pregnant women diagnosed with GDM using a homeostasis
model assessment [61]. Of note, multiple testing adjustments such as Bonferroni or Benjamini-Hochberg corrections were not included in this study owing to the modest sample size.
This was to maintain the capacity to discover any potential associations between the selected
SNPs, ethnicity and diabetes in this unique population.
Looking at allelic variations in our population demonstrates the importance of identifying
phenotypic factors to screen for gestational diabetes as well as investigating drug treatments
that target ethnic-specific mechanisms that can be used to develop personalized medicine. For
instance, KCNQ1 gene encodes for potassium inwardly rectifying channel subfamily J, member 11 [62], and polymorphisms associated with potassium channels are likely to control insulin secretion in the pancreatic islet cells. The dominant model demonstrated SNPs at the
CDKN2A loci, a gene coding for a cyclin-dependent kinase inhibitor 2A, were highly associated with an increased risk of GDM in Japanese participants. The results from our candidate
Table 6. Multivariable logistic regression analysis (BD).
Filipino
Gene
Dominant
Recessive
SNP
OR
Japanese
P_value
OR
PI
P_value
OR
P_value
KCNQ1
rs2237892
0.5 (
< .001
)
0.45 (
0.035
)
1.18 (
0.622
)
TCF7L2
rs7903146
NA (
0.99
)
NA (
NA
)
NA (
0.98
)
SLC30A8
rs13266634
0.73 (
0.17
)
0.62 (
0.41
)
2.43 (
0.03
)
List of SNPs (adjusted for age, BMI, parity, gravidity) which showed significant Breslow-Day test p values (p<0.05) in the Dominant (Table 3) and Recessive models
(Table 4).
https://doi.org/10.1371/journal.pone.0261137.t006
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Genetic risk factors for type II diabetes and gestational diabetes
Table 7. Multivariable logistic regression analysis.
Filipino
Dominant
Recessive
OR
Japanese
P_value
OR
PI
Gene
SNP
P_value
OR
EXT2
rs1113132
1.33 (
0.13
)
6.05 (
0.085
)
1.16 (
P_value
0.69
)
HHEX
rs7923837
1.48 (
0.095
)
1.01 (
0.97
)
1.56 (
0.17
)
MTNR 1B
rs10830963
0.63 (
0.025
)
0.81 (
0.59
)
0.39 (
0.006
)
SLC30A8
rs13266634
0.56 (
0.006
)
0.73 (
0.38
)
1.02 (
0.96
)
KCNQ1
rs2237895
1.5 (
0.14
)
0.61 (
0.46
)
1.19 (
0.8
)
MTNR 1B
rs10830963
0.37 (
< .001
)
0.52 (
0.092
)
0.56 (
0.14
)
List of SNPs (adjusted for age, BMI, parity, gravidity) which showed values from Mantel-Haenszel formula significantly different from 1.0 in the Dominant (Table 3)
and Recessive (Table 4) models.
https://doi.org/10.1371/journal.pone.0261137.t007
gene approach are also consistent with prior studies showing B-cell dysfunction and impaired
insulin secretion in Japanese populations [63]. Upstream of this locus are sequences that
encode for p15INK4b and p16 INK4a, which both inhibit CDK4 and decrease pancreatic beta
cell replication [64].
MTNR1B polymorphisms also have associations with GDM in Filipino and Pacific Islander
populations. rs10830963 has been reported to have a strong correlation in a meta-analysis of
GDM GWAS studies [65], and this finding has been seen among Korean, Greek [66], Russian
[67] and Chinese women [42]. MTNR1A and MTNR1B encode for melatonin receptors and
high-risk allele carriers at rs10830963 also have higher expression of MTNR1B in pancreatic
beta cells, which leads to impaired insulin secretion [68]. Interestingly, the presence of this
SNP was associated with a lower frequency of GDM in Pacific Islander women in our cohort.
We observe that no single T2DM associated SNP is replicated across all Hawaiian populations
who develop GDM, and this could be attributed to our modest sample size. However, we have
refined the resolution of SNPs associated specifically with Japanese, Filipino and Pacific
Islander populations in Hawaii. Future studies could validate these observations in a larger
cohort. In addition, further studies could build on our findings and investigate a larger panel
of candidate genes such as BACE2 and HKDC1 which are associated with maternal metabolic
traits but were not included in this study [69].
Table 8. Summary table illustrating SNPs with significant associations with GDM, according to race.
Filipino
Gene
Dominant
Recessive
SNP
�
OR
Japanese
P_value
OR
EXT2( )
rs1113132
1.53 (
0.034
)
HHEX(� )
rs1111875
1.60 (
0.027
)
0.5 (
< .001
)
IGFBP2(� )
rs4402960
KCNQ1(� )(�� )
rs2237892
MTNR 1B (� )(��� )
rs10830963
0.62 (
0.028
)
SLC30A8(� )(��� )
rs13266634
0.56 (
0.006
)
KCNQ1(� )
rs2237892
0.51 (
0.019
)
KCNQ1(� )
rs2237895
1.84 (
0.046
)
rs10830963
0.37 (
< .001
)
�
MTNR 1B( )(
���
SLC30A8(� )(�� )
)
rs13266634
PI
P_value
OR
0.32 (
0.003
)
0.45 (
0.035
)
P_value
0.42 (
0.021
)
2.43 (
0.03
)
The table lists SNPs that showed significant unadjusted OR p values (p<0.05; � ), significant Breslow-Day test p values (p<0.05; �� ) or values from Mantel-Haenszel
formula significantly different from 1.0 (��� ) in the Dominant (Table 3) and Recessive (Table 4) models. Significance was determined after multivariable logistic
regression analysis was performed to control for confounding factors.
https://doi.org/10.1371/journal.pone.0261137.t008
PLOS ONE | https://doi.org/10.1371/journal.pone.0261137 December 20, 2021
10 / 15
PLOS ONE
Genetic risk factors for type II diabetes and gestational diabetes
Conclusion
These findings elucidate the pathophysiology of increased risk of T2DM later in life for
women affected by GDM and highlight the importance of lifestyle modifications to decrease
this risk. Such associations need to be validated in larger Asian and Pacific Islander cohorts
and further characterized into clinically meaningful phenotypes that can be designed to guide
personalized medicine. While deleterious genetic mutations may provide increased susceptibility for gestational diabetes and impaired glucose tolerance postnatally, the environmental
risk factors for this condition should also be considered for treatment.
Author Contributions
Conceptualization: Janet Burlingame, Johann Urschitz.
Data curation: Hyeong Jun Ahn, John Chen, Johann Urschitz.
Formal analysis: Paula Benny, Hyeong Jun Ahn, Men-Jean Lee, Corrie Miller, John Chen,
Johann Urschitz.
Funding acquisition: Johann Urschitz.
Investigation: Johann Urschitz.
Methodology: Hyeong Jun Ahn, Janet Burlingame, Men-Jean Lee, Corrie Miller, John Chen,
Johann Urschitz.
Project administration: Johann Urschitz.
Resources: Johann Urschitz.
Software: Hyeong Jun Ahn, John Chen.
Supervision: Johann Urschitz.
Visualization: Johann Urschitz.
Writing – original draft: Paula Benny, Men-Jean Lee, Corrie Miller, Johann Urschitz.
Writing – review & editing: Paula Benny, Men-Jean Lee, Corrie Miller, Johann Urschitz.
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REVIEW
Rare lung diseases II: Pulmonary alveolar proteinosis
Stephen C Juvet MD1, David Hwang MD PhD2, Thomas K Waddell MD PhD3, Gregory P Downey MD1,4
SC Juvet, D Hwang, TK Waddell, GP Downey. Rare lung
diseases II: Pulmonary alveolar proteinosis. Can Respir J
2008;15(4):203-210.
Les maladies pulmonaires rares II :
La protéinose alvéolaire pulmonaire
The present article is the second in a series on rare lung diseases. It
focuses on pulmonary alveolar proteinosis (PAP), a disorder in which
lipoproteinaceous material accumulates in the alveolar space. PAP
was first described in 1958, and for many years the nature of the
material accumulating in the lungs was unknown. Major insights into
PAP have been made in the past decade, and these have led to the
notion that PAP is an autoimmume disorder in which autoantibodies
interfere with signalling through the granulocyte-macrophage
colony-stimulating factor receptor, leading to macrophage and neutrophil dysfunction. This has spurred new therapeutic approaches to
this disorder. The discussion of PAP will begin with a case report,
then will highlight the classification of PAP and review recent
insights into the pathogenesis of PAP. The approach to therapy and
the prognosis of PAP will also be discussed.
Le présent article est le deuxième d’une série sur les maladies pulmonaires
rares. Il porte sur la protéinose alvéolaire pulmonaire (PAP), un trouble
qui provoque l’accumulation de substances lipoprotéinacées dans les
espaces alvéolaires. La PAP a été décrite pour la première fois en 1958, et
pendant de nombreuses années, la nature de la substance s’accumulant
dans les poumons est demeurée inconnue. On a fait d’importantes observations sur la maladie depuis dix ans, lesquelles ont permis d’établir que la
PAP est une maladie auto-immune qui provoque l’interférence des autoanticorps avec la signalisation par le récepteur des facteurs stimulant les
colonies de granulocytes-macrophages, provoquant un dysfonctionnement des macrophages et des neutrophiles. Ce constat a suscité de nouvelles démarches thérapeutiques pour cette maladie. L’exposé sur la PAP
commence par une étude de cas, puis porte sur la classification de la PAP
et l’analyse des récentes observations sur la pathogénèse de la maladie. La
démarche à l’égard du traitement et du pronostic de la PAP sera également abordée.
Key Words: Autoimmunity; GM-CSF; Growth factor; Respiratory
failure; Restrictive lung disease
CASE PRESENTATION
A 46-year-old woman presented with a six-month history of
progressive exertional dyspnea and fatigue. There was no history of fever, cough, weight loss, chest pain or peripheral edema.
Her medical history included iron deficiency anemia that
required parenteral iron replacement. She took no medications, except for iron replacements, and was a lifelong nonsmoker. She worked as a cashier, drank alcohol socially, had no
known allergies, no pets and no history of recent travel.
On physical examination she appeared well, with no respiratory distress. The following parameters were recorded: blood
pressure 110/65 mmHg, heart rate 78 beats/min, respiratory
rate 20 breaths/min and body temperature 37.2°C. Her arterial
O2 saturation on room air was 91%. Her jugular venous pressure was not elevated. Cardiac examination revealed normal
first and second heart sounds, with no murmurs or extra sounds.
Auscultation of the chest revealed scattered inspiratory crackles
bilaterally. Her abdominal examination was unremarkable,
without organomegaly, and there was no peripheral cyanosis,
clubbing or edema.
Laboratory findings revealed a normal complete blood count
and differential. Serum electrolytes, liver and renal function
tests were all normal. Arterial blood gas analysis on room air
revealed pH 7.37, partial pressure (P) of CO2 37 mmHg, PO2
62 mmHg, and bicarbonate 24 mmol/L. A chest radiograph
demonstrated bilateral diffuse airspace opacities affecting both
lungs (Figure 1). A computed tomography scan of the chest
(Figure 2) revealed diffuse, multifocal, patchy ground glass
opacities, with superimposed reticulations resulting in a ‘crazy
paving’ appearance.
The patient underwent bronchoscopy with bronchoalveolar
lavage from the right middle lobe. The lavage fluid was sent for
routine cytology, cell count and differential, Gram stain and
bacterial culture, acid-fast stain and mycobacterial culture, and
fungal stain and culture; all returned negative. A video-assisted
thoracoscopic lung biopsy revealed the presence of eosinophilic
foamy material in the alveolar spaces (Figure 3). A diagnosis of
primary alveolar proteinosis (PAP) was made, and she underwent bilateral whole lung lavage (WLL), which resulted in significant improvement in clinical, radiological and pulmonary
function parameters. However, her symptoms recurred after
six months. She underwent repeat bilateral WLL, with temporary improvement, followed by a recurrence of symptoms and
radiographic abnormalities within a few months. A third WLL
resulted in only minor improvement. Therapy with inhaled
granulocyte-macrophage colony-stimulating factor (GM-CSF)
was initiated and continued for 12 weeks. This resulted in
improved clinical and radiological parameters, and she has
remained stable over the past two years.
PAP
Clinical features and classification of PAP
PAP is a rare lung disease characterized by the accumulation
of excess lipoproteinaceous (surfactant-like) material in the
of Respirology, Department of Medicine; 2Department of Laboratory Medicine and Pathobiology; 3Department of Surgery, University of
Toronto and the Toronto General Hospital of the University Health Network, Toronto, Ontario; 4Departments of Medicine, Pediatrics and
Immunology, National Jewish Medical and Research Center and University of Colorado Denver, Denver, Colorado, USA
Correspondence: Dr Gregory P Downey, National Jewish Medical and Research Center, K701b, 1400 Jackson Street, Denver, Colorado 80206,
USA. Telephone 303-398-1436, fax 303-270-2243, e-mail downeyg@njc.org
1Division
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Figure 1) Posterior-anterior chest x-ray of a 46-year-old woman with
alveolar proteinosis, characterized by bilateral perihilar alveolar infiltrates
Figure 3) Open lung biopsy stained by hematoxylin and eosin, illustrating the eosinophilic foamy material present within the alveolar
spaces. A The scale bar represents 1.0 mm. B The scale bar represents
0.05 mm
Figure 2) Chest computed tomography scan of the patient with alveolar proteinosis, illustrating bilateral ground glass alveolar infiltrates and
interlobular septal thickening consistent with a ‘crazy paving’ pattern
alveolar compartment. It was first recognized as a distinct
clinical entity in 1958 (1). There are three clinically recognized categories of PAP (2): idiopathic PAP, with onset occuring in adulthood; secondary PAP, also typically occurring in
204
adulthood and resulting from another disorder or exposure;
and congenital PAP, which occurs in the neonatal period, and
causes severe respiratory distress.
Idiopathic PAP, the most common form of the disorder, is
generally diagnosed in young to middle-aged adults, with a
median age at diagnosis of 39 years (2). A recently published
analysis (2) of 410 cases of PAP revealed that the median delay
between symptom onset and diagnosis is seven months. The
disorder is two to three times more common in men, and
approximately 72% of patients are smokers (2). More than
90% of cases of PAP are classified as idiopathic (1,3,4), with no
identifiable familial or secondary etiology.
Patients with PAP report progressive exertional dyspnea,
often associated with malaise and low-grade fever. However,
prominent fevers should prompt an aggressive search for an
underlying infection, as discussed in more detail below. Weight
loss may be a clinical feature. A nonproductive cough and,
occasionally, hemoptysis may occur.
On examination, patients with PAP usually do not exhibit
any specific findings (2). Digital clubbing is uncommon; auscultation of the chest may reveal fine crackles, but is more
frequently unrevealing. With these nonspecific symptoms, it is
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Pulmonary alveolar proteinosis
not surprising that many patients are misdiagnosed with
chronic bronchitis. When chest radiography is performed, the
radiographic findings are often strikingly more severe than
would be expected based on the patient’s history and physical
examination.
When evaluating a patient with suspected PAP, care must
be taken to consider possible exposure to environmental hazards that have been associated with secondary PAP. It should
be noted, however, that many of these putatively causal exposures are only described in single case reports or small case
series, and hence, direct evidence of causation is lacking.
Examples of occupational exposures that have been associated
with PAP include a variety of organic and inorganic dusts
(flour, cement, wood) and fumes (chlorine gas, gasoline, plastics). Exposure to a heavy inhalational burden of silica has
long been recognized to trigger acute ‘silicoproteinosis’ (5,6),
a condition that has many similarities to alveolar proteinosis,
but this disorder is now uncommon in the era of workplace
safety standards that mandate the use of personal protective
equipment.
A variety of systemic disorders have been reported to be
associated with PAP, and these cases are classified as secondary
PAP. Most frequently, secondary PAP is reported in association
with hematological disorders such as chronic myelogenous
leukemia and myelodysplastic syndromes (7,8). Altered immunity resulting from HIV infection (9), hypogammaglobulinemia (8), thymic alymphoplasia (10) and immunoglobulin (Ig) A
deficiency (11) have all been associated with secondary PAP.
The rare genetic disorder known as lysinuric protein intolerance, caused by a mutation in the y+L amino acid transporter-1
gene (12,13), leads to impaired membrane transport of dibasic
amino acids. Its consequences include hematological abnormalities and secondary PAP (14,15).
Congenital PAP, first described in 1981 (16), is a rare cause
of neonatal respiratory distress syndrome. Clinically, such
infants present with respiratory distress that is not responsive
to surfactant or corticosteroid therapy. In such infants, lung
transplantation is the only available life-saving therapy. As discussed further below, the pathogenesis of congenital PAP is
quite distinct from that of PAP developing in adulthood.
Biology and pathogenesis of PAP
As described above, PAP of all types is characterized by the
accumulation of surfactant-like material in the alveolar spaces.
That the accumulated material in PAP may be a functional
surfactant was first proposed in 1965 (17). In an elegant series
of in vivo and in vitro experiments, Ramirez and Harlan (18)
demonstrated in 1968 that the accumulation of phospholipids
in the alveolar space in PAP was the result of decreased clearance, rather than of increased synthesis, of this material. They
further demonstrated that the nature of the phospholipid in
PAP is chemically similar (but not identical) to that of a naturally occurring surfactant. Although the latter assertion was initially challenged on the basis that the material isolated from the
lungs of PAP patients lacked surface-active properties, subsequent studies have demonstrated that the material has bona
fide surfactant properties (interested readers are referred to reference 2 for a review).
Alveolar macrophage dysfunction: The finding that surfactant
clearance is impaired in PAP led to the hypothesis that PAP is
the result of a defect in alveolar macrophage function. In the
1970s, several studies demonstrated that alveolar macrophages
Can Respir J Vol 15 No 4 May/June 2008
obtained from PAP patients by bronchoalveolar lavage (BAL)
were phenotypically immature (19) and exhibited reduced ability to phagocytose Candida pseudotropicalis (19) and
Staphylococcus aureus (20). These findings also supported the
clinical observation that patients with PAP are predisposed to
pulmonary infections. An early case series (21) appeared to support this latter hypothesis, describing an increased frequency of
opportunistic infections in PAP patients. Many of the patients
included in the studies in the present review, however, likely
had secondary PAP related to an underlying hematological
malignancy; many had also received immunosuppressive therapies, and it is therefore difficult to conclude from this evidence alone that PAP confers a predisposition to infection.
Unexpected insights from knockout mice – the role of GMCSF: In the 1970s and 1980s, intense interest in GM-CSF had
developed in the field of experimental hematology. GM-CSF is
a potent stimulator of myeloid hematopoiesis, and was cloned
in 1984 (22). GM-CSF binds to a cell surface receptor that is
comprised of a distinct alpha chain and a beta (β) chain. The
latter is also a component of the receptors for interleukin (IL)-3
and IL-5. Knockout mice lacking either the gene for GM-CSF
itself (23,24), or for the β chain of the receptor (25,26) were
generated in the 1990s. To the surprise of the investigators,
these mice did not have abnormal hematopoiesis, but instead
reliably developed a pulmonary disorder indistinguishable from
PAP. Furthermore, they also exhibited defective clearance of
radiolabelled surfactant components, rather than increased
surfactant synthesis, in keeping with the earlier observations
made by Golde et al (19).
The GM-CSF receptor is expressed on type II pneumocytes
and on alveolar macrophages (27,28); pulmonary epithelial
cells are a source of GM-CSF (27). Local intrapulmonary
delivery of exogenous GM-CSF (29) or alveolar epithelial
overexpression of GM-CSF (29) can correct the pulmonary
pathology observed in GM-CSF-deficient mice. Notably, GMCSF-deficient mice also have other abnormalities, including a
predisposition to infections (30,31) and impaired macrophage
function (32,33). Together, this series of investigations suggested that a GM-CSF deficiency in mice is a model that very
closely resembles human PAP.
The link between GM-CSF and human idiopathic PAP was
revealed when anti-GM-CSF antibodies were detected in the
serum and BAL fluid of patients with the disease (34-36). This
key finding explained the attenuated hematopoietic response
observed in studies of GM-CSF used as a therapeutic agent for
idiopathic PAP (37-39). Furthermore, the presence of antibodies that neutralize GM-CSF in patients with idiopathic PAP
provides a pathogenetic explanation for the development of
the disease in the absence of genetic abnormalities of GM-CSF
or its receptor. Idiopathic PAP can therefore now be classified
as an autoimmune disorder (2).
Why does the lack of GM-CSF, either due to its complete
absence (in the knockout mouse) or to its binding by autoantibodies (in idiopathic PAP), lead to surfactant accumulation?
The precise mechanism by which this occurs is not yet
known, but it has been demonstrated that alveolar
macrophages from GM-CSF-deficient mice exhibit decreased
expression of PU.1, a transcription factor required for the
functional maturation of these cells (40). These cells were
unable to metabolize surfactant; however, when provided with
GM-CSF in vitro, they expressed PU.1 and cell surface markers characteristic of mature macrophages, and acquired the
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ability to metabolize surfactant. PAP patients treated with
GM-CSF had higher levels of PU.1 expression than healthy
controls or PAP patients before treatment (41).
Additional signalling events downstream of the GM-CSF
receptor also appear to be important for surfactant catabolism.
Peroxisome proliferator-activated receptor-gamma (PPAR-γ)
is activated by GM-CSF stimulation, which in turn activates
the transcription of genes required to metabolize lipids and glucose. PPAR-γ messenger RNA transcripts are absent in the
alveolar macrophages of PAP patients, but are present in the
alveolar macrophages of healthy control subjects (42).
Moreover, GM-CSF therapy restores PPAR-γ expression to
control levels in patients with PAP (42).
Together, these animal and human observations illustrate
the critical role of anti-GM-CSF autoantibodies in the
pathogenesis of idiopathic PAP. They reveal the importance
of GM-CSF in maintaining surfactant homeostasis in the
alveolar microenvironment. Furthermore, they demonstrate
that GM-CSF acts by driving the functional maturation of
alveolar macrophages.
Infections associated with PAP: Although micro-organisms
can be isolated from the lungs of PAP patients by BAL, many
of these potential pathogens are rarely associated with clinical
evidence of infection. For example, while the lung lavages of
eight of 19 sequential patients treated between 1984 and 1992
grew nontuberculous mycobacteria, none of these patients
manifest clinical evidence of disease related to infection from
these organisms (43). In contrast, other investigators have
observed an increased prevalence of disseminated opportunistic infections in patients with PAP. In particular, infection of
the central nervous system by Aspergillus species (44) and
Nocardia species (45-47) has been described. In Seymour and
Presneill’s 2002 review (2) of 410 cases of PAP, Nocardia
species were isolated in 34 cases. They hypothesized that this
observed increase in the frequency of disseminated and opportunistic infections may indicate a systemic defect in immunity
in patients with PAP, although they did not detect any difference in neutrophil counts, corticosteroid use or cigarette
smoking among patients with and without these infections (2).
A recent publication by Uchida et al (48) provided additional insight into the increased prevalence of infections
observed in patients with PAP. The authors hypothesized that
the presence of GM-CSF antibodies may also contribute to
neutrophil dysfunction in patients with PAP, and in the GMCSF knockout mouse. Although neutrophil ultrastructure was
normal, neutrophil phagocytic capacity was reduced, as were
markers of neutrophil activation in response to GM-CSF stimulation. These deficiencies were observed both in mouse neutrophils and in neutrophils from patients with PAP, but not in
those from healthy control subjects. Bacterial killing by neutrophils was also moderately diminished in both the mouse
model and in patients with PAP, as compared with unaffected
controls. Therefore, anti-GM-CSF antibodies appear to mediate functional impairment in both neutrophils and
macrophages in PAP, which translates into a global but subtle
defect in the innate immune response to bacterial infection.
As previously mentioned, secondary PAP has been described
in the setting of HIV (9,49-52). However, this association is
still restricted to case reports, and a direct pathogenetic relationship between HIV and secondary PAP has not been established. Some authors have suggested that Pneumocystis jirovecii
pneumonia is responsible for these cases of PAP (50) and not
206
HIV infection per se. Moreover, the propensity for patients
with either PAP (of any type) or HIV to present with opportunistic pulmonary infections such as those due to P jirovecii
and Nocardia asteroides could conceivably lead to a diagnostic
dilemma. In the event of uncertainty, clinicians should consider
ordering HIV serology to exclude this diagnosis.
Pathogenesis of congenital PAP: Deficiency of surfactant protein (SP)-B was reported in a patient with congenital PAP in
1993 (53). In fact, an inherited autosomal recessive mutation
in the SP-B gene is the cause of this disorder in many cases
(54). However, abnormalities in other proteins have been
reported, such as the common β chain of the IL-3, IL-5 and
GM-CSF receptors (55), and in SP-C (56). It appears that
congenital PAP can result from diverse genetic abnormalities,
with many cases as yet unexplained (57).
Physiological testing
On pulmonary function testing, a restrictive ventilatory defect
is usually apparent in PAP patients, with reductions in total
lung capacity and vital capacity (2,3). The diffusing capacity
for carbon monoxide is often reduced out of proportion to the
degree of the restrictive defect. Hypoxemia with an elevated
alveolar-arterial PO2 gradient is the result. Arterial blood gas
analysis often reveals compensated respiratory alkalosis (8). A
markedly elevated shunt fraction while breathing 100% O2 has
been reported in PAP patients (58).
Radiographic abnormalities
In PAP, posteroanterior and lateral chest radiographs typically
demonstrate bilateral and symmetrical airspace opacities that
seem out of proportion to the relatively modest and indolent
symptoms and clinical presentation. The opacities are generally
most dense in the perihilar regions, and may exhibit a ‘butterfly’ configuration, similar to pulmonary edema or P jirovecii
pneumonia (1,59). Other patterns may be seen, including focal
infiltrates, reticular markings, nodules and asymmetrical infiltrates. Pleural effusions and lymphadenopathy are absent but,
generally, the plain radiographic findings are nonspecific.
Computed tomography provides better visualization of the
pulmonary parenchyma and illustrates the nature of the infiltrates. Again, however, opacities may be peripheral or central,
focal or diffuse (60). Typically, ground glass opacities and
increased interstitial markings are seen. Airspace opacities may
be sharply demarcated from adjacent normal parenchyma in a
nonanatomical fashion, creating a geographic appearance
(60). The combination of interlobular septal thickening and
ground glass opacification has been termed the ‘crazy paving’
appearance, due to its resemblance to cobblestones laid in an
irregular, unpredictable fashion.
The crazy paving appearance was once believed to be pathognomonic for PAP, but has since been described in other conditions. Examples include bronchioloalveolar cell carcinoma,
P jirovecii pneumonia, alveolar hemorrhage, sarcoidosis, cryptogenic organizing pneumonia and nonspecific interstitial pneumonia (61). Exogenous lipoid pneumonia and drug-induced
lung diseases may also produce the same appearance (61). Given
this broad differential diagnosis, a further careful diagnostic evaluation is required.
Diagnosis of PAP
Laboratory studies: Most routine laboratory studies are normal in patients with PAP. Serum lactate dehydrogenase may be
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elevated (58,62), but this is a nonspecific finding. Similarly, elevated levels of SP-A, SP-B and SP-D have been reported in PAP
(63-66), but again, this finding is present in other disorders as
well. Serum and BAL levels of the mucin-like protein KL-6 (67)
are markedly elevated in PAP, and appear to correlate with disease activity. Overall, however, these laboratory indicators have
not proven useful in making the diagnosis of PAP.
Anti-GM-CSF antibodies, as described above, are central
to the pathogenesis of idiopathic PAP, and have shown promise as a diagnostic test. In the initial report by Kitamura et al
(34), 11 idiopathic PAP patients, two secondary PAP patients,
53 healthy individuals and 14 patients with other lung diseases
were examined. PAP was diagnosed by BAL and lung biopsy.
BAL was performed in all of the patients and in 30 of the
healthy subjects. Blood samples were obtained from 30 of the
healthy subjects, five of the idiopathic PAP patients and one of
the secondary PAP patients. The authors found that polyclonal
anti-GM-CSF IgG antibodies were present in all serum and
BAL fluid samples from idiopathic PAP patients, but were not
present in the serum or BAL fluid of any of the other subjects,
including those with secondary PAP. Furthermore, serum from
the three patients with idiopathic PAP in remission (ie, no
symptoms, clear chest radiograph) still contained the antibodies (34). Therefore, although this was a small study, it provides
powerful evidence that IgG anti-GM-CSF antibodies drive the
development of idiopathic PAP and may represent a useful
diagnostic tool.
Bronchoalveolar lavage and lung biopsy: The diagnosis of
PAP can be established definitively with BAL and/or lung
biopsy. BAL fluid from patients with PAP is opaque and milky
in appearance. When allowed to stand, the fluid spontaneously
separates into a pale yellow, almost translucent supernatant
and a thick sediment (8). A cytospin preparation demonstrates
the presence of amorphous, granular, eosinophilic material.
The enlarged alveolar macrophages obtained in this manner
are characteristically vacuolated and foamy in appearance.
Electron microscopy demonstrated the presence of tubular
myelin, lamellar bodies and fused membrane structures within
the amorphous extracellular material (3). These electron
microscopical characteristics are morphologically the same as
those of surfactant.
Open lung biopsy, traditionally considered to be the gold
standard diagnostic test, is now largely unnecessary in the
majority of cases of PAP; the combination of the clinical
presentation, imaging findings and BAL results are generally
sufficient to make the diagnosis (2). Biopsy specimens reveal
multiple nodular areas of consolidation, with periodic acidSchiff stain-positive acellular material filling the alveoli and
terminal bronchioles. There may be a mild associated lymphocytic infiltrate in the interstitium (1), but this feature is
minor in comparison with the findings in the alveolar compartment. Importantly, the underlying architecture of the
lung is usually preserved. In advanced cases, fibrotic changes
may develop, which can lead to architectural distortion,
although this is uncommon (68-70). As is the case with BAL
samples, electron microscopy can be employed to examine
the acellular material for the presence of tubular myelin in
difficult cases.
Therapy
Treatment for idiopathic PAP has evolved from the use of a
variety of nonspecific and largely ineffective agents, to the
Can Respir J Vol 15 No 4 May/June 2008
physical removal of the lipoproteinaceous material from the
lungs, to the development of specific therapy targeted at the
underlying pathogenesis of the disorder.
The first publication to describe PAP (1) also discussed its
unsuccessful treatment with postural drainage, antibiotics and
corticosteroids. Inhalation of aerosolized trypsin (71) and
lavage with heparin and saline (72) have been reported to be
helpful in isolated cases, as has the mucolytic agent ambroxol
(73,74). Overall, however, these therapies have not shown
success in large numbers of patients (2), unlike WLL.
WLL: WLL has long been considered the definitive therapy
for PAP. The idea that the accumulated material could be
physically removed from the lungs of PAP patients was first
advanced in the early 1960s, when selective segmental lavage
was reported by Ramirez et al (18,75). This was a trying experience for patients and physicians because it involved the
transtracheal blind administration of warmed saline with only
minimal analgesia, several times daily, for several weeks (76).
However, in addition to providing the conceptual basis for the
development of WLL, it represented the advent of the use of
BAL as a therapeutic tool in modern pulmonary medicine.
WLL is now performed under general anesthesia, with a
double-lumen endotracheal tube in place. The lungs are ventilated for 20 min with 100% O2 to wash out nitrogen. The lung
to be treated is then isolated at end-expiration, and the volume
of that lung is estimated from the preoperative functional
residual capacity. Warmed 0.9% saline is then instilled at a rate
of 100 mL/min, which approximates the rate of absorption of
the 100% O2 from the lung. Once the functional residual
capacity has been replaced with saline, tidal volume-sized
aliquots of saline are instilled under gravitational force and
then allowed to drain out. The procedure is repeated, often
using 10 L to 12 L of saline per lung, until the effluent, which
is typically initially milky and turbid, becomes clear (77).
Once this occurs, residual fluid is aspirated and the lung is ventilated with 100% O2 again. A decision can then be made
whether to proceed to WLL of the contralateral lung (77).
Chest percussion is commonly included in the procedure to
promote clearance of the lipoproteinaceous material (78). The
WLL procedure takes 2 h to 4 h and should be performed in a
centre with anesthesiologists skilled in the placement and
management of double-lumen endotracheal tubes. Care must
be taken to avoid barotrauma. Saline thorax is another potential complication; insertion of a chest tube is occasionally
required. Diuresis is forced during the procedure and for several
days afterward to ensure resorption of all excess extravascular
lung water. In severely hypoxic patients with PAP, WLL has
been performed with extracorporeal membrane oxygenation
and in hyperbaric chambers (79,80).
Because no prospective studies of WLL are available, it is
difficult to determine the effect of this procedure on prognosis.
However, the vast majority of cases of idiopathic PAP have
been reported to improve on clinical, radiographic and physiological grounds following WLL (2). Improvement in PO2,
forced vital capacity, total lung capacity and diffusing capacity
for carbon monoxide is expected for several days following the
procedure (81). The goal of therapy is a return to normal functioning, with freedom from symptoms such as exertional dyspnea (77). The median duration of freedom from recurrent
symptoms appears to be approximately 15 months, but is highly
variable, with many patients needing only a single lavage (2).
A small number of patients do not respond to WLL, and
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younger age appears to be the major predictor of a poor
response. Nonresponders had a similar long-term prognosis to
responders, save for slightly decreased survival (87%±9% versus 97%±2%) in these patients in the first six months after
WLL (2). It should be noted that congenital PAP appears to be
particularly unlikely to respond to WLL (2).
GM-CSF therapy for idiopathic PAP: The discovery that
GM-CSF-deficient mice develop a disorder resembling PAP,
which resolves when GM-CSF is replaced, led to the hypothesis that GM-CSF therapy could be effective in humans with
the disorder. The first prospective study of GM-CSF therapy in
PAP patients began in 1995. Fourteen patients with idiopathic
PAP were treated with 5 μg/kg/day of GM-CSF given subcutaneously. Five patients (36%) responded, as judged by improvements in the alveolar-arterial gradient for O2; a sixth patient
responded following an escalation of the dose to 20 μg/kg/day
(38). The remaining eight patients did not respond to GMCSF. All patients had anti-GM-CSF antibodies in serum
obtained before the initiation of therapy. A second study,
using a dosage of 5 μg/kg/day to 9 μg/kg/day, reported significant clinical, radiographic and physiological improvements
in five of seven patients, including improvements in the
alveolar-arterial gradient (82,83). Benefits are generally not
seen for eight to 12 weeks (83). Interestingly, the hematopoietic response to GM-CSF is blunted, which appears to result
from the presence of anti-GM-CSF antibodies (38,83). In summary, although GM-CSF is generally well tolerated, the overall
response rate to this therapy in these two small, nonrandomized
studies appears to be less than 50%, which is in stark contrast to
the ability of WLL to produce rapid improvements in symptoms
and pulmonary function in the majority of patients. There are
reports of the successful use of inhaled GM-CSF for the treatment of PAP. Further studies of GM-CSF therapy in idiopathic
PAP are clearly warranted to better define its role.
Treatment of secondary PAP: If secondary PAP is diagnosed, the underlying disorder must be treated, or the patient
should be removed from exposure to the suspected environmental agent. Additional treatment with WLL may still be
necessary, and has been performed successfully, in secondary
PAP (84-86). Because secondary PAP is not associated with
anti-GM-CSF antibodies, there is no rationale for GM-CSF
in its treatment.
Treatment for congenital PAP: As with secondary PAP,
GM-CSF therapy should not be used for congenital PAP,
which is the result of SP-B deficiency or a mutation in the
β chain of the GM-CSF receptor (and perhaps other unrecognized mutations). Unfortunately, congenital PAP does not
respond well to WLL (2). At the present time, lung transplantation appears to be the only therapeutic option in cases
of congenital PAP.
Prognosis
The prognosis in idiopathic PAP is considerably better than
might be expected, based on the severity of physiological
derangements that can arise in this condition. Actuarial fiveand 10-year overall survival rates are approximately 75% and
68%, respectively (2). Disease-specific survival is higher;
approximately 88% at five years (2). The only specific adverse
prognostic factor identified is age, with patients younger than
five years (many of whom likely had congenital PAP) having an
actuarial survival of only 14% at five years after diagnosis.
Patients older than the median age at diagnosis (39 years) were
208
at higher risk of death than younger patients, but not for diseasespecific mortality (2). Although smokers are at increased risk for
the development of PAP, continued smoking did not appear to
influence survival (2). Overall, the risk of death from respiratory
failure appears to be approximately 10% to 15% (2).
Spontaneous improvement, or rarely, complete resolution,
occurs in a minority of idiopathic PAP patients. In their review
of all reported cases of PAP to 2002 (2), Seymour and Presneill
reported that 7.9% exhibited some spontaneous improvement.
That such improvement may occur has been recognized since
PAP was first described, with five of the original 26 patients
reported by Rosen and colleagues (1) demonstrating “definite
improvement”. It should be noted that radiographic or physiological improvements are often incomplete (2).
Pulmonary fibrosis has occurred rarely in PAP, typically late
in the course of the disease (69,70). Its clinical and prognostic
significance is unclear, but its presence would be expected to
result in incompletely reversible physiological abnormalities.
Whether the pulmonary fibrosis resulting from PAP is progressive is unclear.
CONCLUSIONS
Like lymphangioleiomyomatosis, PAP is a disease in which significant conceptual and therapeutic advances have been made
as a result of investigations into the fundamental biological
aberrations that characterize the disorder. The serendipitous
discovery of PAP-like disease in the GM-CSF-deficient mouse,
and the subsequent finding of anti-GM-CSF antibodies in idiopathic PAP patients, has recast it as an autoimmune disorder.
Despite these advances, numerous challenges remain.
Much about the biology of idiopathic PAP is still incompletely understood. For example, what is the role of cigarette
smoking in the pathogenesis of PAP? What is the significance
of the interstitial lymphocytic infiltrate that accompanies
PAP? Although the evidence strongly implicates anti-GMCSF antibodies in the pathogenesis of the disease, what triggers their synthesis in individual patients and what factors are
responsible for the waxing and waning course in some
patients?
Therapy for PAP is also an area that merits further investigation. What is the effect of treatment on the natural history
of the disease? WLL effectively treats PAP in most instances,
but it is cumbersome and associated with both morbidity and
mortality. A treatment that can be administered by the patient
is conceptually much more appealing. However, although GMCSF can be used, its 50% success rate and expense makes it
suboptimal. Clearly, more research needs to be done to understand why some patients respond and others do not.
Alternative therapeutic approaches are also worth considering. Given that idiopathic PAP results from the presence of an autoantibody, B cell-specific treatments may be
useful. Rituximab, a monoclonal antibody directed at the
CD20 molecule expressed on the surface of mature B cells,
was originally employed in the treatment of non-Hodgkin
B cell lymphomas, but has shown efficacy in a variety of
autoimmune diseases (87). Conceivably, this agent, or a similar one, may be useful in the treatment of PAP. Similarly, it
may be possible to deliver high concentrations of GM-CSF
to the alveolar space to overwhelm anti-GM-CSF antibodies
at their site of action. Aerosolized GM-CSF has been used
successfully in PAP, and it may be possible to induce alveolar
epithelial overexpression of GM-CSF using gene therapy, as
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has been achieved in animal models (29,88). Congenital
PAP, while rare, is a devastating illness that has a completely
different pathogenesis. Deficiency of SP-B or the β chain of
the GM-CSF receptor could also theoretically be replaced
using genetic or cellular therapies directed at the alveolar
compartment.
With our improved understanding of the various forms of
PAP, it is hoped that further advances in therapy are forthcoming. Increased understanding of PAP with the use of careful and well-designed in vitro, animal and human studies will
hopefully also stimulate further research into the mechanisms
of other unusual and more common lung diseases.
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W2086634749.txt | Respawn: A Distributed Multi-Resolution
Time-Series Datastore
Maxim Buevich †
Anne Wright ‡
Randy Sargent ‡
Anthony Rowe †
Dept. of Electrical & Computer Engineering
‡ Robotics Institute
Carnegie Mellon University, U.S.A.
{mbuevich,arwright,rsargent,agr}@andrew.cmu.edu
†
Abstract—As sensor networks gain traction and begin to scale,
we will be increasingly faced with challenges associated with
managing large-scale time-series data. In this paper, we present
a cloud-to-edge partitioned architecture called Respawn that is
capable of serving large amounts of time-series data from a
continuously updating datastore with access latencies low enough
to support interactive real-time visualization. Respawn targets
sensing systems where resource-constrained edge node devices
may only have limited or intermittent network connections
linking them to a cloud-backend. The cloud-backend provides
aggregate storage and transparent dispatching of data queries
to edge node devices. Data is downsampled as it enters the
system creating a multi-resolution representation capable of lowlatency range-base queries. Lower-resolution aggregate data is
automatically migrated from edge nodes to the cloud-backend
both for improved consistency and caching. In order to further
mask latency from users, edge nodes automatically identify
and migrate blocks of data that contain statistically interesting
features. We show through simulation and micro-benchmarking
that Respawn is able to run on ARM-based edge node devices
connected to a cloud-backend with the ability to serve thousands
of clients and terabytes of data with sub-second latencies.
I.
I NTRODUCTION
Technological advances in low-power processors, communication and sensors are rapidly accelerating our ability to
record information about the physical world. This data has
the potential to revolutionize application domains including
critical infrastructure monitoring, health care, transportation,
defense systems, manufacturing, smart buildings and city-wide
energy optimization. However, for this data to be actionable,
we need scalable and efficient solutions designed to handle the
increasingly enormous amount of data being generated.
Current large-scale sensing systems are typically composed
of a tiered architecture where resource constrained sensor
nodes transmit data to a server-backend. Limited networking
capabilities (for example in wireless networks) often force
designers to significantly filter data at the source. In many
cases, this data filtering removes information that could be used
to support new functionality beyond the original design scope.
Given the current trend in disk and flash memory technologies,
it is now possible to store large amounts of data locally on an
embedded gateway device.
Unlike most of the approaches used in current cloud computing systems, embedded gateways are CPU limited, memory
constrained and exist as field devices on networks with high
latencies. Many distributed sensing systems also suffer network
outages which without coordinated local storage lead to data
loss. To support interactive visualization and querying of data,
these systems need to intelligently organize, pre-process and
pre-fetch data to provide timely access to data. Ideally, a sensor
networking datastore should also have provisions to support
streaming communication that might be required by control
applications. The availability of fresh (recent) data often conflicts with the throughput gains achievable by batching data.
In this paper, we present Respawn, a distributed time-series
datastore designed to manage hundreds of thousands of sensor feeds while providing range-based queries at sub-second
latencies from resource-constrained devices. To achieve this,
Respawn leverages two concepts: cloud-to-edge partitioning
and multi-resolution storage. In Respawn’s distributed architecture, communication is load balanced between a few serverclass machines and many inexpensive, low-end embedded edge
devices. This is achieved by partitioning the data between
the cloud and the edge, storing the low-resolution aggregate
data in cloud nodes and the high-resolution data at fielddeployed edge nodes. A dispatcher front-end is responsible for
directing queries between the cloud and the edge and, in effect,
maintaining low request latencies. The dispatcher is designed
to operate predominantly out of memory to support tens of
thousands of concurrent connections. A bloom filter-based
caching layer is used to determine the location of requested
data and to avoid costly network accesses.
For multi-resolution storage, Respawn leverages the opensource Bodytrack Datastore (BTDS) [1], a light-weight multiresolution datastore for time-series data. On edge node gateway
devices, BTDS is responsible for down-sampling incoming
data and storing hierarchically-organized copies of the data
at differing resolutions. This pre-processing upon ingest of
the data significantly improves range-based queries where a
subset of data is requested from an arbitrary timespan. In the
evaluation section, we show that BTDS can perform rangebased queries running on an embedded platform at nearly the
same speed as range-based requests for standard relational
databases running on server-class hardware. We also extend
BTDS to support lossless compression, where different levels
of compression are applied to different levels in the hierarchy,
allowing for more commonly accessed data to be served faster.
Since edge nodes typically have latencies of almost an
order of magnitude greater than the cloud, selective data
migration can be used to further improve latency. A Qualityof-Service (QoS) parameter at each gateway node is used to
determine how much bandwidth is available for data and hence
how aggressively tiles can be migrated. Top-level aggregate
tiles are migrated first since these are typically used as starting
points for range-based queries. Low-level (higher resolution)
tiles are migrated based on both client access patterns and
based on data metrics like standard deviation. Standard deviation is one of many metrics that can be used to pinpoint
tiles that would be of more interest to clients. We evaluate
these different migration schemes through a small user study
in which participants are required to search through data.
In order to evaluate the performance of Respawn, we
benchmark ingest and query times on a single server machine
comparing against MySQL, SQLite and OpenTSDB. This includes an evaluation of the impact of using LZ77 compression.
We also benchmark BTDS on memory-contrained 450Mhz
ARM9 embedded hardware which cannot support MySQL
or OpenTSDB. Finally, we evaluate our latency masking
techniques using user traces from a network consisting of
an embedded gateway board, dispatcher, and cloud datastore.
Using trace-driven simulation, we evaluate how the system
scales across large networks with thousands of edge nodes.
In summary, this paper makes the following three main
contributions. First, it provides the design of a highly-scalable
time-series datastore that leverages local multi-resolution storage, along with compression, and is able to operate on embedded hardware. Second, we present a cloud-to-edge handoff mechanism with intelligent caching to reduce latencies for
interactive queries and visualization. Finally, we evaluate the
system in terms of single-server performance and horizontal
scalability through the addition of edge nodes.
II.
R ELATED W ORK
It is becoming increasingly apparent that no single approach can solve all data storage problems [2]. For sensor
networking applications, it is important to be able to store
and process many streams of data simultaneously. There have
been several efforts to build upon databases like MySQL to
support stream processing functionality like pattern correlation
queries [3]. Other similar work has tried to incorporate stream
processing engines and historical storage [4]. These efforts
illustrate many of the challenges involved with large-scale
time series data, even in the context of highly-connected and
resource abundant datacenters. In this section, we highlight a
few of the key distinctions between traditional databases and
those designed for sensing applications.
A. Time Series Datastores
There have been many examples of time series databases
for infrastructure monitoring (for example in data centers)
and financial analysis, such as DataGarage[5], RRDtool [6],
tsdb [7], TSDS [8], OpenTSDB [9], Dremel [10], Vertica [11]
and DataSeries [12]. Some of these databases already support
multi-resolution queries [5], [6], [9], [11]. However, none of
them natively support sub-second time stamping. RRDtool
is one of the few options that can operate on an embedded
target, but it is designed to run locally and does not provide
any distributed management features. Many of these databases
do not natively support access control [5], [6], [7], [9], [10],
[12]. Even the ones that do have access control [11] lack perstream permissions and do not operate in a distributed manner.
Respawn in contrast embraces a cloud-to-edge distributed
operation with per-sensor stream security through use of the
a Publish-Subscribe access control model. The OceanStore
[13] project has very similar goals and features to Respawn.
OceanStore is a global persistent data store designed to be
highly scalable. One of its primary goals is to provide consistent and highly-available service to an infrastructure running
on top of untrusted devices. It also strives to reduce latency
by capitalizing on data locality whenever possible. However,
OceanStore is not primarily a time series database and hence
does not provide multi-resolution access. Because the data
could be significantly more general, it utilizes access-based
caching schemes rather than proactive data migration. It is
also not optimized to run on embedded targets.
B. Streaming and Aggregation
Another approach to managing continuous data is to perform on-the-fly aggregation using stream processing techniques [14] [15]. This approach works well for networks that
have enough provisioning to transfer data in real-time, but suffer in that they potentially lose information that could be processed later. In Respawn, we leverage an XMPP communication layer to deliver real-time transducer information and then
provide a componentized storage facility that operates in the
background. Decoupling the design of the stream-processing
functionality from the historical data allows the system to more
flexibly batch entries to increase ingest throughput.
C. Sensor Network Databases
Many databases have been proposed that operate within
sensor networks, such as Diffusion[16], Tag [17], TinyDB
[18], Cougar [19] and DIMENSIONS [20]. Diffusion provides
a routing and in-networking processing approach that propagates queries using flooded messages. Cougar uses a similar
approach only it uses a static configuration of nodes that is
selected using a proxy front-end. This proxy front-end is still
responsible for dispatching requests out to the leaf-nodes of
the sensor network for data collection. There is no notion of
storage or caching at the proxy. TinyDB focuses on running the
database within resource constrained sensor nodes on microcontroller hardware. Queries are processed directly on endpoint sensor devices. Respawn is more geared towards data
management that runs on sensor gateway collector devices
rather than on the leaf nodes themselves. In many cases,
these gateway collectors could be connected to proprietary
networks where it would be difficult to run custom software.
For example, one could connect a Respawn edge node to a
buildings BACnet data feed and it would act as a consolidator
of potentially hundreds of sensor streams. Dimension further
improves upon the idea of in-network storage by using wavelet
transforms to deal with data aging and multi-resolution.
D. Middleware
Certain sensor networking middleware frameworks also
provide storage capabilities. sMAP [21], for example, focuses
on with data delivery, but also provides querying and retrieval
of sensor data. sMAP is compatible with MySQL for storage,
but can also use a custom binary time-series datastore called
readingDB to help cope with the performance challenges. readingDBs performance is significantly improved over MySQL,
latency/bandwidth
barrier
edge
data
request
WSN
client
edge
nodes
address
request
mobile
device
dispatcher
cloud
data
request
cloud
node
migra;on
Fig. 1: Respawn High-Level Architecture
but it is still primarily a centralized datastore without builtin load balancing. readingDB does however provide delta
encoding and zlib compression and can be manually distributed
based on how sMAP is configured. In contrast, Respawns design is centered around the notion of edge-to-cloud partitioning
and is highly distributed by default. Its primary goal is latency
masking which it achieves through intelligent data migration.
E. Why Respawn?
Data centers have become increasingly effective at providing low-latency access to vast amounts of data. Services
like Google Maps allows people to interactively scroll through
databases that are hundreds of Terabytes in size with hundreds
of Gigabytes of indexing information. Sensor data and the
way in which it is collected differ significantly from the
type of information that current data center practices are so
efficient at handling. Typical operations performed on time
series data include plotting, zooming, correlation, clustering,
prediction, pattern matching queries and summarization. Time
series processing systems often have both a historical and
real-time component. In order to optimize ingest throughput,
databases often batch large segments of data. Unfortunately,
this introduces significant delay. Most traditional databases
deal with read and write transactions that are for the most
part independent. In time series databases, there is a constant stream of appends with intermittent queries. Specific
to sensing systems, data sources are often distributed and
must be collected on field devices. Unlike data centers with
high-speed network interconnects, field devices are frequently
connected with expensive links like cellular or satellite where
customers are charged per kilobyte. Few open-source time
series databases provide the timing granularity required for
capturing physical events. The majority of these systems were
designed for data center monitoring or financial applications
that require integer second timing granularity. Sensing systems
should be able to distinguish sub-second events. As sensor
networks are finding their way into a wider range of large-scale
complex applications, there is a growing need for a scalable
storage platform customized for these particular needs.
III.
D ESIGN AND I MPLEMENTATION
The Respawn architecture enables interactive exploration
and responsive querying of time-series sensor data, despite
the data originating from embedded nodes behind a latencybandwidth barrier. A high-level representation of the Respawn
system architecture is displayed in Figure 1. Sensor readings
are generated at the edge, in most cases from mobile devices
and wireless sensor network deployments. The sensor data
streams are transmitted through a series of adapters to Respawn
edge nodes, low-cost embedded gateways outfitted with large
amounts of flash storage. Edge nodes process and store the
data locally and periodically notify a Respawn cloud node
of new data received. Cloud nodes are server-class machines
which handle much of the load of and serve as low-latency
data caches for the Respawn system. The cloud nodes also
facilitate a continuous migration of a subset of the total data
being stored by the edge nodes. When data is migrated from
the edge, the cloud node notifies the Respawn dispatcher.
The dispatcher serves as the system entry point for almost
all data requests initiated by clients. It is designed to handle
large volumes of small requests and to maintain thousands of
open connections at once. The dispatcher’s primary function
is to redirect requests from clients to end-nodes. Redirections
are decided based on client-perceived latency, as well as load
on individual edge nodes and load on the entire network. In
Section IV, we show that despite the centralized nature of the
dispatcher, it scales to handle thousands of simulataneous users
and does not represent a significant performance bottleneck in
the architecture given our target network sizes.
A. Multi-Resolution Datastore
For the purpose of local data storage, Respawn leverages
Bodytrack Datastore (BTDS), an open-source time-series datastore which targets multi-resolution queries and interactive
visualization. BTDS stores raw data streams in a lossless
format and additionally generates lossy aggregate versions of
the streams for query acceleration. In the current implementation, aggregates are calculated by finding a mean average of
datapoint values over a set window, however, it is possible
to incorporate other aggregation functions. BTDS supports
a command line interface for local client requests and an
HTTP/JSON interface for web requests.
1) Data Model: A single instance of the datastore is a
collection of data streams, with each stream separated into a
single raw version and many aggregate version (Figure 2). An
aggregate is a representation of the raw data down-sampled
by a power of two, and therefore corresponds to a distinct
level of resolution. A single aggregate is composed of many
tiles, tiles in turn are composed of one-point buckets. Formally,
a tile represents a contiguous time-range and a collection of
m equally-sized buckets. A bucket is a ”smaller” contiguous
time-range containing either a single datapoint or no datapoint.
In a single instance of the datastore, all tiles contain exactly
m buckets (regardless of resolution level) and thus contain
a maximum of m points. Buckets at level L are half the
size temporally of buckets at level L+1, thus tiles at level L
represent a time-range half the size of tiles at L+1 (please refer
to Figure 2). This also means that, for any arbitrarily-chosen
range in time, the level L view of the data contains twice the
buckets and is up to twice the resolution of the level L+1 view
of the data. By convention, the bucket ”width” (time duration
represented by a bucket) at an arbitrary level is equal to 2level
seconds; thus at level 0 a bucket has a ”width” of 1 second.
A tile is uniquely addressed by its resolution level and
offset. A tile’s offset at level L corresponds to its distance in
time from the Unix epoch as measured in tiles of level L.
…
(le
2
offset
1
0
resolu&on
level
bucket
1
1
0
2
3
0
0
2
1
3
4
6
5
each time-series datapoint will contain a timestamp, a mean
value, a standard deviation, and a count. Floating point values
are stored with 64-bit precision. Count and standard deviation
are meaningful for low-resolution aggregate tiles because they
represent the number of raw datapoints used for calculating
the mean value and the standard deviation of the datapoints.
The format for a JSON tile response is given below:
7
-‐1
(response example:)
{
…
"fields": [
"time", "mean", "stddev", "count"
],
"data": [
[1367968303.677585, 486.696, 12.053,
[1367969280.41341, 494.2760, 4.347,
[1367970305.185479, 498.36,
3.0824,
...
[1368391167.9347,
476.883, 5.690,
],
"level": 10,
"offset": 2609,
0
4
8
Unix
&me
(seconds)
12
raw
data
stream
Fig. 2: BTDS Multi-Resolution Storage
Figure 2 shows an example of how aggregate data tiles are
organized within BTDS. The raw data stream and three of
the aggregate versions are displayed (other aggregates are not
shown). Each aggregate is represented by tiles of m=4 buckets
(a small m value was chosen for presentation purposes). The
stream in this example begins at the Unix epoch, thus the
leftmost tiles have an offset of 0. At each level of resolution,
tile offsets are incremented as time progresses.
BTDS stores data streams as uncompressed binary files
on disk (In Section IV we explore the impact of adding
compression to BTDS). Sets of contiguous tiles at a single
resolution are organized into files in much the same way that
buckets are organized into tiles. Files are named according
to their corresponding range of offsets and are organized into
two directory levels. Top-level directories separate individual
data streams while bottom-level directories separate resolution
levels within data streams. The structure of BTDS maps well
to the organization of many common filesystems and enables
performance benefits associated with filesystem-level caching
and optimized lookup.
2) Making Requests: Respawn queries are implemented on
top of independent BTDS tile requests. In Respawn, BTDS
tile requests are made via HTTP and return JSON (Javascript
Object Notation) representations of tiles. A tile request must
specify a data stream, a tile resolution level, and a tile offset.
Data stream names are split into two parts: a device name
and a channel name. Channels which are grouped into devices
can share timestamp values, resulting in accelerated ingest and
saved disk space. The level and offset specified in a request
maps directly to a tile position in the data stream. Because all
tiles are precomputed at the time of ingest, requests are made
in constant time, regardless of the resolution level. The format
for an HTTP tile request is given below:
(request format:)
GET /tiles/DEVICE.CHANNEL/LEVEL.OFFSET.json
(example:)
GET /tiles/sensor.temperature/10.2609.json
Each tile request yields JSON data for a single tile. A
response contains an array of datapoints, an array of fields describing each datapoint, and the tile level and offset. Typically,
1125],
1507],
1520],
1531]
}
B. Distribution Layer Components
Time-series sensor data destined for storage enters through
the edge nodes. It is first buffered in memory for a predefined
amount of time so as not to incur the overhead of constant
writes to the BTDS datastore. Periodically, the buffered datapoints are written to the datastore and committed to disk.
The BTDS datastore parses the incoming points and, based
on timestamp, collects them into tiles. Raw timestamp and
value information is stored in the highest-resolution raw tiles;
all other tiles are aggregate tiles generated from raw tiles. In
addition, edge nodes run security key generators that publish
keys to XMPP, which maintains network-wide access control.
A primary function of the cloud node is to serve data
summaries collected from the edge nodes in the network.
To improve latency, the cloud node maintains a collection of
persistent and non-persistent data structures. The first of these
is the Summary datastore, an instance of the BTDS datastore
containing migrated edge tiles which are likely to be requested
in the future. The migrated tiles represent an approximation
of the total data in the distributed datastore. The storageconstrained nature of the edge nodes necessitates data aging,
but this is not required of the cloud node. Additional modules
are maintained to optimize accesses to the Summary datastore
and to enforce access control policies; these are discussed
further in the section on data migration.
The dispatcher acts as the first point of entry for almost
all client-initiated data requests. It redirects requests for data
tiles of particular channels to the appropriate end-nodes, either
edge or cloud. The dispatcher does not serve time-series data;
requests and response payloads are kept small (i.e. smaller than
100 bytes). This, coupled with the fact that most dispatcher
data structures are kept in memory, enables the dispatcher
to serve a steady-state stream of over ten thousand requests
per second (as described in Section IV). The dispatcher’s
data structures are primarily copies of the structures generated
by the cloud node. For most requests, the dispatcher decides
where clients are redirected to and, as a result, how load in
the system is distributed.
(B)
Cloud
Hit
(A)
Unmigrated
(C)
Cloud
Miss
client
client
l/bw
barrier
edge
node
A2
B1
l/bw
barrier
A1
data
store
DS
dispatcher
edge
node
B2
data
store
DS
dispatcher
cloud
node
cloud
node
A1=AddrRequest
A2=EdgeRequest
client
B1=AddrRequest
B2=CloudRequest
C4
l/bw
barrier
edge
node
C3
DS
dispatcher
C1
C2
data
store
cloud
node
C1=AddrRequest
C2=CloudRequest
C3=EdgeAddrRequest
C4=EdgeRequest
Fig. 3: Request Transaction Types
An XMPP back-end hosts a publish-subscribe network and
is used by the three primary components for the sharing of
event notifications and other lightweight metadata. XMPP also
provides a delivery system for streaming data, as well as access
control and security.
D. Data Migration
C. Request Redirection
1) Predictive Caching: As an alternative to the classic
caching scheme we have developed methods for Predictive
Caching in order to minimize the effect of the latencybandwidth barrier. Predictive caching leverages the structure
of time-series sensor data by analyzing and isolating certain
characteristics of the data. The data characteristics are used in
the construction of a prediction model which decides which
portions of data are likely to be requested and should be
preemptively cached. We have defined and implemented two
predictive caching schemes in Respawn, Periodic Migration
and Proactive migration.
In this section, we discuss the details of the Respawn
dispatcher and the movement of a client’s data fetch through
the Respawn system as shown in Figure 3. All of the requests
described in this section are standard BTDS-format HTTP
requests extended with a security key.
A data retrieval is initiated by an AddrRequest arriving at
the dispatcher. The dispatcher uses an access control list to
map the requested channel to the security key currently in use
by the channel’s edge node. A key mismatch causes an error
response to be sent. Upon a key match, a list of base resolutions
and a summary bloom filter are checked. If either match, a
redirection to the cloud node is returned. The Bloom filters are
sized to keep the probability of false-positives low, implying a
reasonable assurance that in the case of a match the requested
tile exists at the cloud node. If the previous check resulted in
false outputs, the requested tile has not been migrated and a
redirection to the edge is returned, causing the client to make
a subsequent EdgeRequest.
The probabilistic nature of the CTR Bloom filters allows
for false-positive cloud redirections to occur. This results in
three main types of request traces in the Respawn system:
”Unmigrated”, ”Cloud Hits,” and ”Cloud Misses.” The ”Unmigrated” traces are the simplest of the three. When a request
processed by the dispatcher fails to positively match, the tile
has not been migrated and the client is redirected to edge.
When a match on the bloom filters occurs, it is only likely
that the tile has been migrated. Hence, a small percentage
(i.e. 1%) of CTR Bloom filter matches result in ”Cloud
Miss” traces. In such cases, the client is redirected to the
cloud node, causing it to send a CloudRequest. The request
is checked against the full Cloud Tile Registry (CTR), which
returns a negative result, signifying that the tile has not been
migrated. The negative result is returned to the client, causing
the client to issue an EdgeAddrRequest. An EdgeAddrRequest
is functionally identical to an AddrRequest, with the exception
that it is guaranteed to redirect the client to the tile’s edge node.
Once the client receives a response to the EdgeAddrRequest, it
uses the response to generate an EdgeRequest, which it sends
to the appropriate edge node. Upon receiving the request, the
edge node verifies the client’s access, retrieves the tile from
the local datastore, and serves the client’s request.
For distributed storage systems which leverage storage at
the edge, the quality of network links to the edge devices can
be a substantially limiting factor for performance, especially in
terms of latency. In this section we will describe the mechanics
of data migration used to reduce access latencies.
Periodic Migration: When browsing, clients will frequently
request tiles with the lowest resolution first, in order to
see a ”big-picture” view of a dataset. From there they will
”drill down” to explore particular sections of the data at a
higher resolution. The effect of this phenomenon is that lowresolution tiles are on average requested more often than highresolution tiles. Thus, it is often practical to bias tile migration
toward lower-resolutions. The Respawn cloud node periodically migrates all tiles at or below a predefined resolution,
which can be assigned on a per-edge-node or per-device basis.
Periodic Migration is most effective for periodic data feeds
from time-triggered sensing systems.
Proactive Migration: Whereas Periodic Migration leverages resolution information about data tiles for migration decisions, Proactive Migration involves extracting characteristics
from the data itself for the same purpose. In Respawn, each
tile generated at the edge is assigned a value corresponding to
the standard deviation of the sensor values contained within
the tile. For many datasets, standard deviation was found to
be a good scalar approximation of the statistical importance of
a time-series data-range. Furthermore, standard deviation over
tiles is simple enough that it can be computed upon initial
ingest of the data, even on a low-end embedded platform. We
found Proactive Migration based on standard deviation to be
especially effective at accelerating access times for sparse data
feeds containing isolated events of interest. This suggests that
Proactive Migration is especially suited for handling data feeds
from event-triggered sensing systems.
2) Mechanics of Migration: Respawn maintains and migrates a collection of data structures to facilitate request redirection. The first of these is the Summary datastore, a subset
edge
node
*
cloud
node
Edge
Node
dispatcher
IP
address,
security
key
Edge
Node
List
Edge
Node
List
XMPP
Cloud
Node
Dispatcher
IP/Key
Event
Base
Resolu7ons
Summary
Datastore
Summary
Tiles
*
BTDS
Mul7-‐Res
Data
*
latency/bandwidth
barrier
*
Summary
Bloom
Filters
Base
Resolu7ons
fetch
Cloud
Tile
Registry
(CTR)
HTTP
Summary
Bloom
Filters
New
Tile
Event
XMPP
Summary
Update
Event
fetch
*locally
generated
data
structure
Fig. 4: Data Structure Migration within Respawn
of the tiles in the system chosen for migration (Figure 4).
Two additional modules are maintained to optimize accesses
to the Summary datastore: The Base Resolutions and the Cloud
Tile Registry (CTR). The Base Resolutions are a mapping of
time-series data channels to their base migration resolution,
the resolution R for which the cloud node is guaranteed to
migrate all tiles belonging to that channel whose resolution is
R or lower. The CTR is a registry containing the addresses of
all migrated tiles not represented by the Base Resolutions. Like
the rest of the data structures maintained by the cloud Node,
the CTR is segregated by channel and is thus implemented as
a map from channel to list of unique tile addresses.
The cloud node makes migration decisions on a per-datachannel basis and thus a set of migrated tiles for one channel
is independent from the set of another channel. Access to CTR
addresses are further optimized by the addition of Summary
Bloom filters. This enables faster, no-false-negative lookups
on the existence of addresses. The last high-level structure
maintained by the cloud node is the Edge Node List, which
maps channels to their associated IPs, security keys, and other
access control metadata. This listing is checked on every
request in order to verify that a client requesting cached data
originating at an edge node is authorized to access the edge
node.
3) HTTP vs. XMPP: Respawn separates two common
modes of communication, event notifications and bulk transfers, and implements them with two distinct layer-7 protocols.
This choice has the benefit of increasing responsiveness to
client requests, horizontal scaling potential, and reliability
in overload cases. Event notifications, the first mode, are
always sent as the direct or indirect result of an asynchronous
sensor event entering the system through an edge node. These
notifications are lightweight data transfers whose destination
is a single node or a subset of all nodes in the system; global
broadcasts are very rare. In many cases, event notifications cannot be predicted and thus it is difficult for a receiver to control
data flow without severely hindering communication. Respawn
uses the XMPP publish-subscribe protocol for these types of
transactions. XMPP provides an event node abstraction for
which each event node has one or more publishers and one
or more subscribers, in addition to tunable quality-of-service
parameters to place limits on data flow. Each event node
is also associated with an access control list. Respawn uses
these access control features as a basic building block in its
security model. XMPP was explicitly designed for lightweight,
asynchronous massaging and is thus a good fit for Respawn
event messages.
Data fetches represent bulk transfers of structured data
facilitated by client-server interactions. They can occur both
periodically and as a result of an asynchronous event being
received. Respawn uses HTTP for transferring time-series tiles
and large metadata structures. HTTP serves these purposes
well as a result of its simplicity, its bulk transfer efficiency, and
its request-driven model. One common limitation of HTTPbased systems is the need to periodically ”poll” for data, which
can result in wasted bandwidth in cases where data is not
yet available or does not exist. This effect is mitigated in
Respawn because most fetches are triggered by the receipt
of a lightweight XMPP event. The use of HTTP’s requestdriven model is also key in preventing overload for the most
important components of Respawn. In terms of bandwidth,
most back-end data transferred by the dispatcher and cloud
node are initiated by the dispatcher and cloud node. Thus, it
would be difficult to overload the dispatcher and cloud node
through the addition of edge nodes.
4) Bloom Filter: A bloom filter is a probabilistic data
structure used for quickly and space-efficiently testing an element’s membership in a set. Bloom filters are most commonly
implemented as bit arrays in which each element is mapped
to multiple bit positions via multiple unique hash functions.
A set is queried for an element by hashing the element with
the same set of hash functions and checking whether all of
the bit positions are set to 1. Querying a bloom filter does not
return an exact result; false positives are possible while false
negatives are not.
Respawn maintains a bloom filter per data stream in the
Summary Bloom Filters data structure. As tiles are added to
a Bloom filter, the filter becomes less accurate and produces
a larger percentage of false positives. A Bloom filter which
produces a false positive ratio of 1% contains 9.57 bits of
space per element in the set. When the number of elements in
a Bloom filter becomes too great to maintain a false positive
ratio of 1%, the cloud node will ”resize” the bloom filter by
generating an empty one of double size, rehashing the values,
and reinserting them.
1K-point range query time (ms)
100000
Fig. 5: ARM9 Embedded Gateway
IV.
E VALUATION
In this section we evaluate the primary components of
the Respawn system. By micro-benchmarking BTDS against
MySQL, SQLite, and OpenTSDB, we profile the ingest and
query performance of a multi-resolution architecture against
standard relational and time-series architectures. We also
show initial compression ratio and compute the overhead
for performing gzip compression on a large environmental
sensor dataset. We then perform a design-based simulation
of various dispatcher architectures and then benchmark the
actual throughput of our implementation. Finally, we use traces
collected from users browsing a typical time-series dataset to
evaluate various Respawn migration schemes.
A. Multi-Resolution Storage
Each database was profiled on an 8-core E5320 Intel Xeon
CPU clocked at 1.86GHz with 4GB of RAM. Since BTDS
fits within a small enough footprint to operate on edge-node
routers, we also benchmark it on a 450MHz ARM9 processor
with 32MB of RAM operating from a 64GB flash micro-SD
card (shown in Figure 5). Unless otherwise specified, each
point on the following graphs represents the average of at least
one thousand runs.
Table I shows the average request time required to fetch a
1024-point (1K) block of data from each database at a random
location within a one million point dataset. We see that SQlite
performs best and BTDS runs slightly faster than MySQL and
significantly faster than OpenTSDB. Enabling compression
adds a factor of six slowdown in BTDS performance. The
embedded platform runs approximately 13 times slower on
the embedded target. Given that each embedded edge node
will receive a small fraction of the requests as compared to the
main server, the overall response time of 310 ms is promising.
Database
BTDS
BTDS Compressed
BTDS Embedded
SQlite
MySQL
OpenTSDB
1K-Point Random
Query Time (ms)
23.86
159.51
310.98
11.98
25.49
1206.38
TABLE I: Full resolution query performance
BT datastore
SQLite
MySQL
OpenTSDB
10000
1000
100
10
100000
1x106
1x107
Dataset Size (# of datapoints)
Fig. 6: Range-based query performance (log-log scale)
Next we evaluate the performance of range-based queries
that are defined by a starting timestamp, an end timestamp and
a resolution. On standard relational databases like MySQL, this
requires fetching raw values and then computing aggregates.
BTDS performs its processing on ingest. Figure 6 shows the
difference in performance of each database with respect to 1Kpoint range-based query. The x-axis shows the performance as
the size of the database increases. For standard databases, as
the size of the dataset increases this incurs a larger penalty
when computing aggregates. Note the log-scale on both the
x and y axis. On small datasets, SQLite outperforms BTDS.
However as the dataset grows, BTDS’s ability to efficiently
traverse pre-computed aggregates allows it to provide a constant query response time independent of the dataset size. Both
SQLite, MySQL and OpenTSDB show drastically increasing
delay as the data scales.
Next, we evaluate the ingest penalty required for fast
range-based queries. Table II shows the total time required to
ingest a 1M-point dataset. BTDS is approximately 1.7 times
slower than SQLite and over 9 times slower than MySQL.
The embedded BTDS target is more than 70 times slower
than its server counter-part due to poor disk I/O performance.
Since the embedded nodes are closest to the streaming data, it
would be rare for the system to need to ingest a large (1 year)
batch of data. The compression overhead on write ends up
being approximately a 400% decrease in performance. As the
database increases in size, BTDS must aggregate data across
increasingly larger datasets. Figure 7 shows that the BTDS
ingest performance scales as the dataset increases but rapidly
levels off. MySQL, SQLite and OpenTSDB remain nearly
constant since they are typically just seeking to the end of
a structure and appending data. With a sensor input rate of
1Hz, the average ingest performance stabilizes at about half a
year worth of data and still remains competitive with MySQL.
Database
BTDS
BTDS Compressed
BTDS Embedded
SQlite
MySQL
OpenTSDB
1M-Point Ingest Time (seconds)
373.03
1503.32
26404.00
217.18
41.12
6072.56
TABLE II: Ingest performance with 1K batches
ingest time for 1K pts per stream (s)
1K-point Ingest Time (seconds)
BT Datastore (raw)
BT Datastore (smoothed)
mySQL (smoothed)
Sqlite (smoothed)
OpenTSDB (smoothed)
10
1
0.1
0
5x106
1x107
1.5x107
2x107
2.5x107
400
200
0
0
2000
Stored Data Size (number of raw datapoints)
4000
6000
8000
10000
Number of streams
Fig. 9: Ingest Performance vs Number of Streams
BT datastore time
SQLite time
MySQL time
OpenTSDB
10000
600
500
1000
# of occurrences
1M-point ingest time (seconds)
600
3x107
Fig. 7: Ingest Performance vs Datastore Size (log scale)
100
10
1
G=1, P=1
G=10, P=1
G=100, P=1
G=1, P=10
G=10, P=10
G=100, P=10
400
300
200
100
0
0
5000
10000
15000
batch size (number of raw datapoints)
20000
0
0.5
1
1.5
2
Compression Ratio
Fig. 8: Ingest Performance vs Data Batch Size (log scale)
Fig. 10: Compression Ratio for Example Dataset
The distributed nature of Respawn also horizontally scales to
alleviate central ingest bottlenecks.
motion, pressure, humidity and audio as well as a wide variety
of HVAC sensors that monitor blower speeds and water intake
temperatures. We see that in general, the data compresses to
25% of its original size, however the runtime performance
overheads are non-trivial. As future work, we intend to investigate utilizing the tile structure of our database to compress only
infrequently access tiles or ones with low-standard-deviation
numbers that would achieve higher compression ratios.
One mechanism used to optimize reads and writes in
streaming storage is batching of data. Figure 8 shows how
each database scales in terms of ingest time as the batches
of points increase in size. They all follow a similar trend and
level off with approximately 1K data points. 1K data points on
a 1Hz sensor feed corresponds to 15 minutes of data. There is
a trade-off with how much a sensor should be willing to batch
in memory and how soon data becomes available to incoming
queries. Approaches like [22] have proposed making queries
on both disk and in-memory to reduce those overheads.
Another technique for improving ingest performance is
to group sensors that have identical timestamps. In BTDS,
grouping can be used to save on the bookkeeping overhead
normally required to setup a unique timestamp entry. Figure 9
shows the time required to ingest 1K-points per stream as the
number of total streams is increased. Each line represents a
different grouping size G and a different number of processor
cores P used by the server. We see that grouping sensor
improves efficiency up to a point, but saturates at about 10
inputs. We also see that BTDS becomes CPU limited since
increasing the number of cores is able to double performance.
Figure 10 shows a histogram of the compression ratios
achieved when running gzip compression on a 21GB repository
containing 6 months of mixed time series sensor data. The
data consisted of environmental sensors like light, temperature,
B. Dispatcher
Next, we compare against the architectures shown in Figure 11. The first is a centralized architecture where all nodes
stream data to the cloud for storage and there is no database
running on the edge nodes. The second architecture, proxy, is
one where storage exists on the edge nodes and requests are
routed through the cloud. The final redirection architecture is
the most distributed option, where a a lightweight dispatcher
redirects requests either to the cloud or, in this case, only to
edge nodes that are running a local copy of the database.
Figure 12 shows the limit on number of requests per
second in each architecture. These lines were generated using
a simulation based on hardware profiles. Networking overhead
was determined by running requests from a separate machine
against the database or dispatcher until its performance saturated. Real requests were generated and rerouted to closely
mimic the real system. Since we do not have access to
thousands of edge nodes, we assume that these requests can
Centralized
Proxy
Client
Redirec2on
Fig. 11: Dispatcher Architectures
Request limit (requests/second)
18K
Client
Client
Central Storage
Classic Cache
Redirection/Hand-off
16K
14K
12K
10K
8K
6K
4K
2K
0
0
be serviced outside of the local network independently (i.e.
requests are directly handed off to edge nodes do not interfere
with each other). We also assume that the requests for each
edge node arrive in an equally independent fashion.
The centralized storage architecture is limited by the number of simultaneous requests that can be made to BTDS (in
this case 80 per second). As the number of nodes in the
system scales, the performance gradually worsens as additional
edge nodes consume the server’s bandwidth. This approach,
which is most common in simple sensor collection systems,
scales very poorly. The proxy architecture shows improved
scalability due to its ability to offload the majority of the
database workload to the edge. The system becomes network
bandwidth constrained at about 2000 requests per second.
For many applications, this approach is simple and able
to handle medium workloads. The Respawn components are
flexible enough that they can be easily configured to support
this type of access pattern if so desired. The redirection and
hand-off approach which best represents the standard Respawn
configuration scales quite well supporting up to 16,000 requests per second. Eventually, the limiting bottleneck is the
performance of the dispatcher. These operating points can be
used as general rules of thumb to determine if a particular
scheme meets the expected application demands.
C. Migration
In this section, we evaluate three different tile migration
schemes. The first is a simple periodic migration technique
where top-level aggregate tiles are periodically migrated based
on available bandwidth of the edge node. Since each Respawn
edge node is configured with a QoS limit by the user or is
limited by the network connection itself, tiles are transmitted
at a fixed rate so as to keep average throughput below the
configured setting. The second scheme is a statistical pre-fetch
mechanism that prioritizes which tiles get migrated based on
standard deviation of the data. The intuition being that clients
are more likely to investigate areas where the data is exhibiting
significant changes. Again, these tiles are migrated within the
specified QoS limits. The final scheme is a combination of
the first two approaches. In this case, tiles are selected in a
round-robin fashion from the periodic and the stddev-based
transmit queues. The cloud caches data periodically at a lower
granularity and also maintains a few regions of highly variable
data ready to serve.
In order to evaluate these migration approaches, we performed a small user study where participants were asked to
browse around a web-based plot of a week worth of time-series
2000
4000
6000
8000
10000
Number of edge nodes
Fig. 12: Architecture Request Limit vs Number of Edge Nodes
data from environmental sensors placed in our lab. Users were
asked to determine approximately how many hours in the last
week a lab was occupied based on sensor data. This forced
users to navigate and zoom into each region to determine the
length of each section and if it was continuous. Each click and
zoom selected on the plotting tool logged the corresponding
range-based query to the database. These traces could then be
played back against a version of the system executing each of
the migration schemes.
Figure 13 shows the latency per time of a characteristic
trace performed on the data. In (a), there was no migration.
Each request shown by a small circle was redirected to an
embedded cloud node that was running on the campus wireless
network. These requests took on average 340 ms. The grey
background shading shows a sliding window average of the
latency to help visualize the impact of clusters of high and
low latency requests. In trace (b), the system utilized periodic
tile migration. Accesses that went to the cloud instead of the
edge are shown with much lower latencies that correspond
with connecting to a wired server machine on campus. In trace
(c), the variance-based prefetching approach is used to migrate
data with higher levels of variation, a scheme which has a
slight performance increase over periodic migration. Finally,
trace (d) shows both periodic and variance-based prefetching,
which achieves the best performance at an average latency of
85 ms. In all of these cases, the migration bandwidth was kept
constant, and 5% of edge tiles were migrated. We see that
combining both approaches is most effective in our tests. This
can be attributed to users making a reasonable number of lowresolution tile accesses to get an overview and subsequently
”drilling down” into regions containing interesting features.
V.
C ONCLUSIONS AND F UTURE W ORK
In conclusion, in this paper we present a highly-scalable
distributed time series datastore called Respawn. Respawn is
able to run a small multi-resolution datastore on embedded
leaf nodes that pre-processes incoming data in order to accelerate range-based queries. A high-speed dispatcher server
is responsible for routing client requests to either a cloud
storage backend or directly to the edge node. We propose
and evaluate various data migration techniques that define how
edge nodes should push tiles to the cloud backend to help
mask latency for client request. This architecture is ideal for
supporting interactive visualization tasks, allowing scientists to
Latency (secs)
Latency (secs)
0.8
0.6
0.4
0.2
0
0
20
40
60
80
100
0.8
0.6
0.4
0.2
0
120
0
20
40
Trace Time (secs)
0.6
0.4
0.2
0
20
40
60
80
100
120
80
100
120
(b) periodic
Latency (secs)
Latency (secs)
(a) no migration
0.8
0
60
Trace Time (secs)
80
100
0.8
0.6
0.4
0.2
0
120
0
Trace Time (secs)
20
40
60
Trace Time (secs)
(c) prefetch
(d) periodic and prefetch
Fig. 13: Trace latency given different tile migration schemes
easily browse large repositories of sensor data. Respawn allows
large-scale sensing systems to horizontally scale in a manner
that still provides responsive access to raw or aggregate data.
Through micro-benchmarking, we show that Respawn is able
to support range-based queries on large datasets with nearly
constant access time while other commonly used databases
scale exponentially. We also show that the data ingest penalty
can be kept at levels similar to that of MySQL databases by
batching and grouping data. We show that in terms of network
scalability, that Respawn can support thousands of edge nodes
handling tens of thousands of requests. Finally, we demonstrate
how intelligent data migration can be used in cloud-to-edge
systems to automatically mask latency. As future work, we
intend to investigate different aggregation functions as well as
more sophisticated data compression and aging techniques.
VI.
[9]
[10]
[11]
[12]
[13]
ACKNOWLEDGMENTS
This research was funded in part by the Intel Science and
Technology Center on Embedded Computing, the Bosch Research and Technology Center in Pittsburgh and TerraSwarm,
one of six centers of STARnet, a Semiconductor Research
Corporation program sponsored by MARCO and DARPA.
We would like to thank Ilari Shafer for the many interesting
discussions about time-series databases, and David O’Hallaron
for his insightful advice and feedback.
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Daniel J. Abadi, Don Carney, Ugur Çetintemel, Mitch Cherniack,
Christian Convey, Sangdon Lee, Michael Stonebraker, Nesime Tatbul,
and Stan Zdonik. Aurora: a new model and architecture for data stream
management. The VLDB Journal, 12(2):120–139, August 2003.
Jianjun Chen, David J. Dewitt, Feng Tian, and Yuan Wang. Niagaracq:
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John Heidemann, Fabio Silva, Chalermek Intanagonwiwat, Ramesh
Govindan, Deborah Estrin, and Deepak Ganesan. Building efficient
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Hong. Tag: a tiny aggregation service for ad-hoc sensor networks.
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| 8,533 | 56,407 |
W4226126446.txt | Qeios, CC-BY 4.0 · Review, December 8, 2021
Review of: "Everybody Nose: Molecular and Clinical
Characteristics of Nasal Colonization During Active
Methicillin-Resistant Staphylococcus Aureus
Bloodstream Infection"
Wolfdgang Witte
Potential competing interests: The author(s) declared that no potential competing interests exist.
The manuscript reports a study on nasal MRSA colonization in patients with MRSA blood stream infection
(BSI). It was performed from July 2018 until May 2019 at the Mount Sinai Hospital, NYC. 63 patients with
BSI were enrolled, finally data on demographic and clinical characteristics as well as results from
bacteriology were available for 53 of them. Primary cultures were carefully examined by colony
morphology for probably heterogeneous colonization. Representative isolates were subjected to molecular
typing by means of spa-typing.
Background of this study is the widely accepted concept that nasal colonization with Staphylococcus
aureus is a substantial reservoir for infections with this pathogens in these carriers. This had also been
demonstrated for methicillin resistant S.aureus (MRSA) by studies performed nearly 20 years ago.
The study is aimed to analyse the diversity of colonizing MRSA within hosts and of the demographic and
clinical features associated with concomitant nasal MRSA colonization in patients suffering from MRSA BSI.
The reported data on heterogeneous nasal colonization, on the frequency of concomitant nasal MRSA
colonization (70%) and the congruence of results from typing nasal and blood stream isolates (95%)
largely confirm results from studies in Europe where at that time MRSA epidemiology was different from
that in the USA at present. Furthermore, these previous studies did not include such a broad analysis of
clinical patient characteristics.
From my side there are following comments/suggestions:
1. Abstract; conclusions a little bit more concrete explanations on the consequences of the findings for
prevention/treatment would be desirable.
2. Introduction: For those who are not active in the field of MRSA infections a short description of the
changing MRSA epidemiology including the discrimination of HA-MRSA, CA-MRSA and discrimination of
widely disseminated epidemic strains of both categories would be helpful.
3. Methods:
1. Either here or at the beginning of the results section more details on the study setting with respect to
prevalence of MRSA infections, and, if performed, on results from MRSA screening at admission
would be of interest.
Qeios ID: AAHFZA · https://doi.org/10.32388/AAHFZA
1/2
Qeios, CC-BY 4.0 · Review, December 8, 2021
2. 3.2.The study is based on 53 patients (37 colonized and 16 noncolonized), for which at least different
12 clinical variables had been recorded. I am concerned about the statistical power. Was a power
calculation carried out when designing the study ?
4. Results. Starting point is the widely accepted concept that nasal colonization with Staphylococcus
aureus is a substantial reservoir for infections with this pathogens in these carriers. This had also been
demonstrated for methicillin resistant S.aureus (MRSA) by studies performed nearly 20 years ago. We
should, however, keep in mind, that nosocomial infections (e.g. surgical wound infections) can result
from direct transmission to the site of infection and must not necessarily start from nasal colonization.
With respect to the comparatively low prevalence of MRSA BSI it would have been a tremendous effort
to screen each patient before medical procedures which may result in an infection for MRSA
colonization. One of the results is the observation that 38% of the colonized and 2 from 14 of the
noncolonized patients had a history of previous MRSA colonization (Table 2 ). Were the corresponding
isolates typed or are they still available for typing ?
5. The authors assume that spa-type t002 is indicative for MRSA CC5 “USA100” which is widely
disseminated as HA-MRSA in the USA and t008 for the “notorious” CA-MRSA “USA300”. There are
however, nosocomial MRSA that also exhibit t008. Therefor it would be highly advisable to perform at
least PCR for luk-PV, better in addition also for arcA. (We have to keep in mind that high resolution
typing based next generation sequencing [core genome MLST represents the state of the art).
6. Discussion. It would not be revealing to discuss the differences observed for MRSA CC8 and CC5
against the background of HA-MRSA and CA-MRSA epidemiology.
Qeios ID: AAHFZA · https://doi.org/10.32388/AAHFZA
2/2
| 688 | 4,555 |
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4
Pulsed Laser Welding
Hana Chmelíčková and Hana Šebestová
Institute of Physics of the Academy of Sciences of the Czech Republic,
Joint Laboratory of Optics of Palacký University and Institute of
Physics of the Academy of Sciences of the Czech Republic
Czech Republic
1. Introduction
Metal joining by means of components heating to the melting temperatures was known
thousands years ago in old age Greece. Heat sources have been developed from forging
furnace to modern methods of plasma arc welding, electric resistance welding, oxy-fuel
welding or laser welding. Laser, as a source of intensive light beam, starts to be implemented
into industrial welding systems due to its advantages in comparison with classic methods, for
example narrow heat affected zone, deep penetration, flexibility and many others. Besides the
welding of compatible metals it is also possible to weld plastics.
The principle of “light” welding is the same for all suitable laser wavelengths. The
absorption of laser radiation in thin work piece surface layer leads to the temperature rise to
the melting or vaporization point. Due to the conduction of generated heat to the
surrounding material volume sufficient weld pool is melted. However heat conduction also
causes essential energy losses.
Laser welding important processing parameters are laser beam properties (power, beam
quality and diameter, wavelength, focusing lens length), weld conditions (focus position
towards the material surface, relative motion of the work piece towards the laser spot, weld
type, processing gas) and physical properties of welded material and work piece
dimensions. Typical power densities applied in laser welding lie between 105 – 107 W.cm-2.
Lasers with active media formed by CO2, semiconductors, Yb:YAG, Nd:YAG crystals or
Ytterbium doped fibres can be used in industry for various components welding. High power
gaseous continual CO2 laser with wavelength 10 600 nm and wall-plug efficiency about 10 %
has excellent beam quality, near Gaussian mode and high depth of focus, thus it is suitable for
deep penetration welding. Far infrared radiation of this laser type cannot be transferred by
means of optical fibre. Therefore typical CO2 laser welding system is equipped with fixed
processing head and work piece positioning mechanism. A mixture of helium and nitrogen is
recommended as a processing gas to suppress plasma shielding effect.
Solid state pulsed Nd:YAG laser’s wavelength 1 064 nm is suitable for fibre guiding from
resonator to the processing head, fixed on a robot arm. This allows welding few meters far
from the laser source which is especially suitable for large or in shape complicated
components. Laser beam has a multimode profile with quality 20 – 30 mm.mrad, wall – plug
efficiency of the flash-lamp pumped Nd:YAG lasers reaches only about 3%. In recent years
www.intechopen.com
42
Nd YAG Laser
new and much more efficient laser types were developed – diode lasers with beam quality
up to 30 mm.mrad, diode pumped disc Yb:YAG lasers and Ytterbium doped fibre lasers
with excellent beam quality, both in near infrared region. With more compact design, higher
efficiency 30 % (fibre laser) to 50 % (diode laser) and lower running costs these systems are
going to replace above mentioned Nd:YAG and CO2 lasers (Němeček & Mužík, 2009).
Nevertheless many pulsed laser welders are already installed in small and middle
enterprises and various materials are being processed. Processing parameters optimization
is a goal of many research projects to improve productivity and decrease occurrence of
defects that results in lower production costs.
2. Processing parameters optimization
To achieve fully penetrated high quality weld it is necessary to set optimal combination of
processing parameters. They involved three different groups: laser beam properties
(wavelength, power, diameter, divergence) material properties (density, thermal
conductivity, specific heat, latent heat of melting and vaporization, thickness, joint
configuration) and very important interaction parameters (welding speed, focusing element
length, focus plane position towards the material surface, shielding gas direction and flow,
absorptivity of material surface) (Duley, 1998). At room temperature almost all metals have
absorptivity about 10 % - 20 %. It increases during material heating and leaps to 80 % - 90%
when metal melting point is reached.
Heat Q necessary to melt material mass m is given by the well known equation
Q m[c(Tm T0 ) Lm ]
(1)
where c is specific heat, Tm melting temperature, T0 initial temperature and Lm latent heat of
melting. In the case of continual laser welding this equation can be transformed to the
following form
Q
Dhv[c(Tm T0 ) Lm ]
t
(2)
where Q/t represents power required for melting, ρ volume density, D spot diameter,
h penetration depth and v welding speed. Then, penetration depth can be expressed as
follows
Q
t
.
h
Dv[c(Tm T0 ) Lm ]
(3)
Including surface absorption and heat conduction losses, penetration depth can be roughly
estimated
hK
P
Dv
(4)
where P is laser power and K constant resulting from material physical characteristics
including surface reflectivity and other energy losses. Thus, penetration depth is
www.intechopen.com
43
Pulsed Laser Welding
proportional to the applied laser power and inversely proportional to the spot diameter and
welding speed.
P/v ratio defines the heat input to a unit length. Melted area cross section linearly rises with
the heat input (Ghaini et al., 2007). In the case of continual laser welding processing
parameters like laser power and welding speed are simply entered into the welding device
control system pursuant to the material thickness and physical properties. Much more
complicated setting of pulsed laser parameters is outlined in paragraph 2.1.
Surface power density or laser beam intensity is defined as a portion of laser power and
laser spot area on material surface. Three welding modes are used in praxis, heat conduction
mode, penetration mode and deep penetration (keyhole) mode.
Heat conduction welding is characterized by power density in the interval 104 – 106 W.cm-2
that causes only surface melting up to 1 mm. The weld is wide and shallow with aspects
ratio about 2:1. Only laser beam without sharp intensity peak can be used, or the focal plane
must be shifted some millimetres above the material surface. In the case of pulsed laser,
pulse length 1 ms – 10 ms is used. When power density balances around the critical point
106 W.cm-2, produced welds are deeper than in the case of the conduction welding. Aspect
ratio is about 1:1 that indicates penetration mode welding (Lapšanská et al., 2010).
Keyhole welding mode starts when energy density exceeds 106 W.cm-2. Laser beam is
focused on the material surface and the fusion zone rapidly heats up to the boiling point.
Melted material begins to vaporize at the centre of the weld spot and creates a blind hole
(keyhole) in the centre of the weld line (Fig. 1). The pressure of hot metal vapour keeps the
hole open during the welding. Presence of the keyhole allows the laser energy to reach
deeper into the fusion zone and consequently to achieve deeper weld with lower aspect
ratio (Kannatey-Asibu Jr., 2009). Keyhole mode welding is a typical application of high
power continuous lasers or high energy pulsed lasers.
During the deep penetration laser welding plasma can be generated above the keyhole.
Ionised metal vapour and shielding gas absorb laser light, change its direction and cause
lower process efficiency. Inert gases such as argon, nitrogen, helium and their special
mixtures are used for plasma reduction. On the other hand, thanks to the plasma plume
presence, welding process can be controlled by means of plasma intensity measurement
(Aalderink et al., 2005).
a)
b)
c)
d)
Fig. 1. Keyhole formation, a) surface irradiation, b) surface melting, c) vaporisation and
cavity formation and d) light absorption inside the keyhole.
www.intechopen.com
44
Nd YAG Laser
2.1 Pulsed laser welding parameters
In the case of pulsed laser, more parameters are involved. Three basic parameters that must
be set at laser source control panel are frequency f (Hz), pulse length t (ms) and flash lamp
charging voltage U (V). These parameters define actual pulse energy E (J). Peak power Ppeak
(kW) is defined as a portion of energy and pulse length
Ppeak
E
.
t
(5)
Peak power determines interaction intensity of laser beam with material for given spot size.
According to the material thickness and welding mode peak power values 0.2 kW to 5 kW
are recommended in the operation manuals of laser welders.
Laser average power P (W) is given as a product of actual energy and pulse frequency
P Ef
(6)
and determines the welding speed.
There are two possible welding methods using pulsed laser. The first and simpler one is a
spot welding which often replaces resistance welding nowadays. One or more pulses land
material surface to reach required penetration depth in spot welding. No mutual motion
between the processing head and material is applied. Spot welding is also often used for
rough fastening of components to be subsequently seam welded using either pulsed or
continual laser. This procedure reduces final distortions resulting from high thermal
gradients corresponding to high value of applied power densities.
To achieve continuous tight welds using a pulsed laser, pulse overlap must be applied. This
is realized using suitable combination of processing parameters. Pulse overlap PO is defined
as follows
PO 1
v
Df
(7)
To achieve hermetic tight joints pulse overlap is recommended to be 80 %.
In comparison with conventional welding methods and continual laser welding, higher
peak power densities in laser pulse mode causes higher heating and cooling rates which can
result in weld defects and inhomogeneous microstructure. Many experimental works have
been realised to optimise pulsed laser welding parameters for different kind of metals with
goal to eliminate defects. For instance (Ghaini and al., 2006) studied overlap bead on plate
welding of low carbon steel, (Tzeng, 1999) made successful welds without gas formed
porosity in lap joints of zinc-coated steel. Another important parameter was introduced in
these studies which is effective peak power density.
Effective peak power density EPPD is defined as a product of peak power density PPD and
pulse overlapping index F
EPPD F PPD
www.intechopen.com
(8)
45
Pulsed Laser Welding
where
F
1
.
1 PO
(9)
This parameter was introduced to better formulate the real power reaching material surface.
When seem welding is required more pulses land material surface (Fig. 2) and their
contribution adds. Thus the effective peak power density can be used to compare energy
requirements of welds accomplished with different pulse overlap.
a)
b)
Fig. 2. Top view and longitudinal weld cross section.
2.2 Experimental work
Pulsed Nd:YAG laser system LASAG KLS 246 – 102 with maximal average power 150 W
and beam parameter product 22 mm.mrad was used to carry out experiments focused on
the study of the effect of processing parameters on weld dimensions and its surface
character. Material to be welded was 0.6 mm thick stainless steel AISI 304.
So as the results were not affected by an accidental misalignment of components to be butt
joined, by possible contamination or presence of surface defects of contact areas, or other
unsuitable initial conditions influencing weld properties, it was decided not to join two sheets
but to make a deep remelting of one sheet, which is, in fact, bead-on-plate welding. This
strategy ensures that only the effect of energy changes will be studied. Laser welding is very
demanding on pieces to be welded preparation, especially when narrow laser beam is used in
near focal position. Therefore, highly precise prepared edges and minimal gap between the
components to be joined are always supposed, which are conditions that need to be fulfilled in
every precise butt joint laser welding application to prepare a high-quality weld. Then, the
results of bead-on-plate experiments can be applied to the real sheet welding at conditions
suitable for a high-quality weld joint preparation. This simplification can be used for the effect
of processing parameter changes study in different laser applications.
Welding itself was realized 4 mm under the focal plane to ensure the sufficient beam
diameter on the specimen which was 0.85 mm. Focussing lens with 100 mm focal length was
www.intechopen.com
46
Nd YAG Laser
used. Cleaned degreased weld pieces were clamped in a mounting jig. Pure argon gas at
coaxial 8 l.min-1 flow rate was used to protect the weld pool against its oxidation. In each set
of experiments, only one parameter was changed keeping all the other parameters constant
to be able to identify the effect of the one examined parameter. Table 1 presents processing
parameters of each set of experiments.
series
1
2
3
4
5
E (J)
3.5
3.5 – 6.5
5.0
5.0
1.8 – 6.2
P (W)
45.5
45.5 – 80.6
45.5
15.0 – 35.0
23.4 – 80.6
Ppeak (kW)
1.59 – 0.8
1.03 – 1.82
1.47
1.47
1.2
t (ms)
2.2 – 4.4
3.4
3.4
3.4
1.5 – 5.1
v (mm.s-1)
4.0
4.0
1.5 – 6.0
4.0
4.0
f (Hz)
13.0
13.0
13.0
3.0 – 7.0
13.0
Table 1. An overview of processing parameters of realised experiments
2.2.1 Pulse length effect
The first set of experiments was focused on the effect of pulse length which was changed in
the interval from 2.2 ms to 4.4 ms. To keep constant beam energy 3.5 J charging voltage had
to be decreased when pulse length was increased (Fig. 3). Thus the average power remained
constant and peak power decreased (Fig. 4).
charging voltage (V)
220
200
180
160
140
120
100
2
2.4
2.8
3.2
3.6
4
4.4
4.8
pulse length (ms)
Fig. 3. Charging voltage vs. pulse length keeping constant pulse energy.
1800
peak power
power (W)
1500
average power
1200
900
600
300
0
2
2.4
2.8
3.2
3.6
4
4.4
4.8
pulse length (ms)
Fig. 4. Average and peak power vs. pulse length keeping constant pulse energy.
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Pulsed Laser Welding
Pulse length increase led to the decrease of penetration depth (Fig. 5) that corresponds to the
decrease of peak power which seems to be a critical parameter.
penetration depth (mm)
0.3
penetration depth
0.25
linear fit
0.2
0.15
0.1
0.05
0
2
2.4
2.8
3.2
3.6
4
4.4
4.8
pulse length (ms)
Fig. 5. Penetration depth vs. pulse length.
These results also showed that applied parameters were not sufficient for the full
penetration of 0.6 mm metal sheet (Fig. 6). However the effect of pulse length is evident.
b)
a)
c)
Fig. 6. Perpendicular cross sections of welds at a) 2.8 ms, b) 3.2 ms and c) 3.8 ms.
Laser scanning confocal microscope LEXT OLS 3100 was used to image and analyse laser
weld surfaces. Spot diameter varied in the interval from 0.704 mm to 0.759 mm and its
slightly decreasing tendency with increasing pulse length was detected (Fig. 7). Average
value of spot diameter reached 0.73 mm. According to the 0.025 mm deviations measured
within the each sample, no definite relationship between spot diameter and pulse length can
be determined in the investigated region.
spot diameter (mm)
0.88
spot diameter
0.83
linear fit
0.78
0.73
0.68
0.63
0.58
2
2.4
2.8
3.2
3.6
pulse length (ms)
Fig. 7. Spot diameter vs. pulse length.
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4
4.4
4.8
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Nd YAG Laser
Fig. 8 presents an example of surface longitudinal central profile. In technical practise,
strict claims on weld surface quality are often posed. Suitable combination of welding
parameters can minimise post processing mechanical treatment. Fig. 9 presents surface
images of selected samples. Pulse overlap 63 % remained constant during all the
experiments of this set.
Fig. 8. 3D surface reconstruction and central profile of the weld realised at 3.8 ms.
a)
b)
c)
Fig. 9. Weld surface in case of pulse length a) 2.6 ms, b) 3.4 ms and c) 4.4 ms.
2.2.2 Pulse energy effect
The second series of experiments studied the effect of pulse energy which was set in the
interval from 3.5 J to 6.5 J via charging flash lamp voltage changes (Fig. 10). Increasing
energy naturally increases average as well as peak power (Fig. 11).
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Pulsed Laser Welding
7
pulse energy (J)
6
5
4
3
2
1
0
160
170
180
190
200
210
220
charging voltage (V)
Fig. 10. Pulse energy vs. charging voltage.
1800
power (W)
1500
1200
peak power
900
average power
600
300
0
3
3.4
3.8
4.2
4.6
5
5.4
5.8
6.2
6.6
pulse energy (J)
Fig. 11. Average and peak power vs. pulse energy.
Higher laser power leads to the deeper penetration which corresponds to the higher applied
power resulting in higher heat input. Nevertheless penetration depth evolution is not linear
(Fig. 12).
penetration depth (mm)
0.7
0.6
0.5
0.4
0.3
0.2
penetration depth
0.1
polynomic fit
0
3
3.4
3.8
4.2
4.6
5
pulse energy (J)
Fig. 12. Penetration depth vs. pulse energy.
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5.4
5.8
6.2
6.6
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Nd YAG Laser
Fig. 13 presents reached aspect ratios (penetration depth to weld width) as a function of
effective peak power density. In this case pulse overlap 63 % was applied. Thus pulse
overlapping index was 2.7. It means that power really touching a unit of material surface is
2.7 times higher in comparison with laser output power assuming no energy losses between
laser output and material surface.
0.9
aspect ratio
0.75
0.6
0.45
0.3
PPD
0.15
EPPD
0
0
2000
4000
6000
8000
10000
(effective) peak power density (W.mm-2)
Fig. 13. Aspect ratio vs. effective peak power density.
Penetration depth slightly increases with pulse energy until about one half of the sheet
thickness is penetrated. Then, when the formation of the keyhole starts (4.7 J), it increases
steeply until the full penetration is reached at 5.9 J (1.7 kW). Keyhole formed at lower peak
powers is not stable enough to establish true keyhole welding, and penetration welding
mode can be observed (Fig. 14).
a)
b)
c)
Fig. 14. Perpendicular cross sections of welds at a) 3.8 J, b) 4.7 J and c) 6.2 J.
Aspect ratio increases with increasing effective peak power density until the full
penetration is reached at about 8.6 kW.mm-2. At the moment of full penetration spot
diameter is maximal, and aspect ratio decreases. Once the full penetration has been
achieved, increasing effective peak power density does not lead to another weld width
grow. Rather, conversely, the width decreases because the beam can escape from the melt
pool because of the full penetration and because less energy is absorbed. That is why the
aspect ratio increases again. These data corresponds to the weld cross-section fusion area
measurement (Fig. 15).
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cross-section fusion area (mm2)
Pulsed Laser Welding
0.35
0.3
0.25
0.2
0.15
0.1
fusion area
0.05
polynomic fit
0
3
3.5
4
4.5
5
5.5
6
6.5
pulse energy (J)
Fig. 15. Cross-section fusion area vs. pulse energy.
Fig. 16 presents the effect of beam energy on spot diameter. Beam diameter on the specimen
was 0.84 mm for all carried out experiments. Spot diameter does not reach this value for
almost all spots. This fact corresponds to the relative low energy application, non-uniform
heat distribution, resulting from characteristic profile of beam intensity, and is supported by
relatively low thermal conductivity of welded material.
spot diameter (mm)
0.88
0.83
0.78
0.73
0.68
0.63
0.58
3
3.4
3.8
4.2
4.6
5
5.4
5.8
6.2
6.6
pulse energy (J)
Fig. 16. Spot diameter vs. pulse energy.
Spot shape is also very demanding on pulse energy. The volume of melted material is
higher at higher energies which leads to the higher deformation of the spot shape (Fig. 17).
a)
b)
Fig. 17. Weld surface in case of pulse energy a) 3.5 J, b) 5 J and c) 6.2 J.
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52
Nd YAG Laser
2.2.3 Welding speed effect
The third experiment was focused on the effect of welding speed. Welding speed was
changed from 1.5 mm.s-1 to 6 mm.s-1.
pulse overlap (%)
100
80
60
40
20
0
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
welding speed (m.s-1)
Fig. 18. Pulse overlap vs. welding speed.
Speed changes correspond to the pulse overlap change from 86 % to 40 % (Fig. 18, Fig. 19).
b)
a)
c)
Fig. 19. Weld surface in case of welding speed a) 1.5 mm.s-1, b) 3.5 mm.s-1 and c) 6 mm.s-1.
These experiments did not prove any significant change in penetration depth (Fig. 20). On
the other hand, weld width decreased with increasing welding speed (Fig. 21). These give us
positive information that slight required changes of pulse overlap via welding speed do not
significantly influence penetration depth.
penetration depth (mm)
0.7
0.6
0.5
0.4
0.3
0.2
penetration depth
0.1
linear fit
0
0
0.001
0.002
0.003
0.004
welding speed (m.s-1)
Fig. 20. Penetration depth vs. welding speed.
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0.005
0.006
0.007
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Pulsed Laser Welding
spot diameter (mm)
0.88
0.83
0.78
0.73
0.68
spot diameter
0.63
polynomic fit
0.58
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
welding speed (m.s-1)
Fig. 21. Spot diameter vs. welding speed.
2.2.4 Pulse frequency effect
Another experiment concerned with pulse frequency effect. Pulse frequency was changed
from 9 Hz to 17 Hz. In this case peak power remained constant, while the average power
increased (Fig. 22).
average power (W)
100
80
60
40
20
0
6
9
12
15
18
pulse frequency (Hz)
Fig. 22. Average power vs. pulse frequency.
Frequency increase naturally increases pulse overlap (Fig. 23, Fig. 24). Pulse overlap must be
high enough so as also bottom side of the sheet is continuously penetrated (Fig. 25).
80
pulse overlap (%)
70
60
50
40
30
20
10
0
6
9
12
pulse frequency (Hz)
Fig. 23. Pulse overlap vs. pulse frequency.
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18
54
Nd YAG Laser
a)
b)
c)
Fig. 24. Weld surface in case of frequency a) 9 Hz, b) 13 Hz and c) 17 Hz.
a)
b)
c)
Fig. 25. Weld bottom side in case of frequency a) 9 Hz, b) 13 Hz and c) 17 Hz.
Power increase led to the increase of penetration depth (Fig. 26) as well as the weld width.
Frequency 11 Hz was sufficient for the full penetration. Spot diameter slowly increases with
increasing frequency (Fig. 27).
penetration depth (mm)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
6
9
12
pulse frequency (Hz)
Fig. 26. Penetration depth vs. pulse frequency.
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15
18
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Pulsed Laser Welding
spot diameter (mm)
1
0.8
0.6
0.4
spot diameter
0.2
polynomic fit
0
6
9
12
15
18
pulse frequency (Hz)
Fig. 27. Spot diameter vs. pulse frequency.
2.2.5 Combination of pulse energy and pulse length effect
In the last experiment beam energy and pulse length were changed simultaneously to keep
constant peak power (Fig. 28). Although the peak power is still the same average power
increases (Fig. 29).
pulse length (ms)
6
5
4
3
2
1
constant peak power
0
0
1
2
3
4
5
6
7
pulse energy (J)
keeping peak power constant
pulse energy and pulse overlap
Fig. 28. Pulse length vs. pulse energy keeping constant peak power.
7
6
5
4
3
2
pulse energy (J)
1
pulse length (ms)
0
0
20
40
60
80
100
average power (W)
Fig. 29. Combination of pulse energy and pulse length (keeping constant peak power) vs.
average power.
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Nd YAG Laser
The higher energy and pulse length the deeper penetration depth (Fig. 30). This result points
out the fact that even peak power can not be used as a definite indicator of penetration
depth in pulsed laser welding.
Fig. 31 presents a comparison of these results with results of the effect of pulse energy
(chapter 2.2.2). It is obvious that pulse energy is a critical parameter since it defines the
volume of melted material.
penetration depth (mm)
0.6
0.5
0.4
0.3
0.2
0.1
constant peak power
0
0
20
40
60
80
100
average power (W)
Fig. 30. Penetration depth vs. average power keeping constant peak power.
weld dimensions (mm)
1
0.8
0.6
0.4
h (const. t)
d ( const. t)
h (const. Ppeak)
d (const. Ppeak)
0.2
0
0
1
2
3
4
5
6
7
pulse energy (J)
Fig. 31. Weld width d and penetration depth h vs. pulse energy for constant and variable
pulse length.
3. Conclusion
High power Nd:YAG lasers with millisecond pulses are used in industry for spot or
overlap welding. Many research papers reporting on pulsed laser welding parameters
optimisation leading to the production of sufficiently deep welds without defects have
been published. Usually more parameters were simultaneously changed in such
optimisation processes. The aim of our research work was to identify the effect of each
parameter separately.
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Pulsed Laser Welding
57
Pulsed overlap bead-on-plate welding of 0.6 mm thick AISI 304 stainless steel was realised
in our laboratory. Flash lamp pumped pulsed Nd:YAG laser KLS 246-102 with multimode
beam profile was used for five series of welding experiments. The influence of different
process parameters – pulse length, pulse energy, welding speed, pulse frequency and
combination of pulse energy and pulse length on weld dimensions was examined in five
separate experiments. Weld cross sections and laser spots overlap on samples surfaces were
observed and measured by means of laser scanning confocal microscope LEXT OLS 3100.
The effect of penetration depth and surface spot diameter on applied parameters was
outlined.
Following from the above mentioned results, each processing parameter more or less
influences weld characteristics. It is obvious that the knowledge of only one parameter, for
example beam energy or average power, is not sufficient for the prediction of weld
dimensions in pulsed laser seam welding. Peak power, pulse length and frequency in
combination with processing speed are also very important. Suitable combination of
processing parameters must be always found.
Experiments with flash lamp pumped solid state laser with very low efficiency will be
followed by new studies on modern laser systems – diode and fibre lasers.
4. Acknowledgment
The Academy of Sciences of the Czech Republic supports this work under the project no.
KAN301370701.
5. References
Aaldering, B. J.; Aarts, R. G. K. M.; Jonker, J. B. & Meijer, J. (2005). Weld Plume Emission
During Nd:YAG Laser Welding, Proceedings of the third International WLT Conference on Lasers in Manufacturing, Munich, Germany, June 13-16, 2005,
Available from http://doc.utwente.nl/52666/2/weld_plume.pdf
Duley, W. W. (1998). Laser welding, A Wiley-Interscience publication, ISBN 978-0-471-246794, Hoboken, New Jersey, USA
Ghaini, F.M.; Hamedi, M. J.; Torkamany & Sabbaghzadeh, J. (December 2006). Weld metal
microstructural characteristics in pulsed Nd:YAG welding, In: Scipta Materiala 56
(2007), 955 – 958, Elsvier Ltd., Available from
http://www.sciencedirect.com/science/article/pii/S1359646207001194
Kannatey-Asibu Jr., E. (2009). Principles of Laser Material Processing, A John Wiley & Sons,
Inc., Publication, ISBN 978-0-470-17798-3, Hoboken, New Jersey, USA
Lapšanská, H.; Chmelíčková H. & Hrabovský, M. (2010). Effect of Beam Energy on Weld
Geometric Characteristics in Nd:YAG Laser Overlapping Spot Welding of Thin
AISI 304 Stainless Steel Sheets, Metallurgical and Materials Transactions B, Vol. 41,
No. 5 (2010), pp. 1108-1115. ISSN 1073-5615
Němeček, S. & Mužík T. (2009). Laser Material Processing – Hardening and Welding,
Proceedings of METAL 2009 18th International Conference on Metallurgy and Materials,
Hradec nad Moravicí, Czech Republic, May 19-21, 2010, Available from
http://www.nanocon.cz/data/metal2009/sbornik/List/Papers/014.pdf
www.intechopen.com
58
Nd YAG Laser
Tzeng, Y. F.(1999). Pulsed Nd:YAG Laser Seam Welding of Zinc-Coated Steel Welding
research supplement, Vol.6, No.4, (July 1999), pp. 238-s -211-s, ISSN 1729-8806
www.intechopen.com
Nd YAG Laser
Edited by Dr. Dan C. Dumitras
ISBN 978-953-51-0105-5
Hard cover, 318 pages
Publisher InTech
Published online 09, March, 2012
Published in print edition March, 2012
Discovered almost fifty years ago at Bell Labs (1964), the Nd:YAG laser has undergone an enormous
evolution in the years, being now widely used in both basic research and technological applications. Nd:YAG
Laser covers a wide range of topics, from new systems (diode pumping, short pulse generation) and
components (a new semiorganic nonlinear crystal) to applications in material processing (coating, welding,
polishing, drilling, processing of metallic thin films), medicine (treatment, drug administration) and other various
fields (semiconductor nanotechnology, plasma spectroscopy, laser induced breakdown spectroscopy).
How to reference
In order to correctly reference this scholarly work, feel free to copy and paste the following:
Hana Chmelíčková and Hana Šebestová (2012). Pulsed Laser Welding, Nd YAG Laser, Dr. Dan C. Dumitras
(Ed.), ISBN: 978-953-51-0105-5, InTech, Available from: http://www.intechopen.com/books/nd-yaglaser/pulsed-laser-welding
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W4366705116.txt | Vol. 8 No. 1
Language and Semiotic Studies
Spring 2022
Exploring Saussure’s Analogy between
Linguistic and Monetary Signs
Kristian Bankov
New Bulgarian University, Bulgaria
Abstract
Beginning with a few of Saussure’s ideas from Course of General Linguistics1 the author
tracks the development of money as signs fulfilling their pecuniary function. The principle
of the arbitrariness of linguistic signs, postulated in Course, is the leading concept under
which all forms of money are grouped for study. In its evolution from the primitive
consumable forms such as salt, leather and grain to the last abstract forms of accountable
digital writing, the money sign is increasing its level of arbitrariness between signifier and
signified value and thus becomes “better for thinking” (Levy-Strauss) and communicating.
The second part of the paper considers the main contributions to the field of semiotics
pertaining to money and economic value, and thus highlights the originality of the
model, in which the arbitrariness of monetary signs and the scarcity of money tokens
in circulation form the model’s foundation. The author’s analysis, applying the dyadic
sign, reveals the relationship between the value or meaning of a monetary sign and the
temporal dimension of our experience. The article concludes with some hypotheses about
the homology between the primordial finitude of human temporality (Heidegger) and the
principle of scarcity/finitude of money in circulation as a condition of their value.
Keywords: arbitrariness, money sign, fungibility, scarcity, sign production
To determine what a five-franc piece is worth one must therefore know: (1) that it can
be exchanged for a fixed quantity of a different thing, e.g., bread; and (2) that it can be
compared with a similar value of the same system, e.g., a one-franc piece, or with coins of
another system (a dollar, etc.). In the same way a word can be exchanged for something
dissimilar, an idea; besides, it can be compared with something of the same nature, another
word. (Course, 1959, p. 115)
1. An Analogy for the Arbitrariness of Linguistic and Monetary Signs
One of the characteristics of Saussure’s Course is its essentialness. Its exact, short
and clear formulations have inspired innumerable researchers and form the basis of
Structuralism, one of the most popular research paradigms. The short passage, which
postulates the necessity of semiology, is a good example of Saussure’s style (and that
of his students who published the Course). Several references to the analogy between
linguistics and economics (and between linguistic and monetary signs in particular),
114
Kristian Bankov
which have great potential for further development, are also set out in the text. It is
these that I intend to examine here, and though I am not the first to undertake such a
study, I argue that the approach proposed here is the first of its kind.
At the beginning of the third chapter of Course, the various sciences are reviewed
in order to separate those with a distinct synchronic and diachronic approach from
those in which there is no reason for such a division. It is there that Saussure separates
economics and linguistics from the rest (Course, 1959, p. 119), and compares
political economy to the innovative “synchronic” linguistics he offers. Old-fashioned
comparative linguistics is compared to economic history. In a second passage, quoted
above (but see also p. 149), he develops the analogy specifically at a sign level.
Until now, however, there has been no analysis of the analogy between the two
types of sign in terms of the linguistic sign’s fundamental characteristic, arbitrariness.
According to many scholars of Saussure, and particularly in De Mauro’s classical
reading, the discovery of linguistic signs’ arbitrariness is the most important and
mature step in the establishment of Saussure’s research paradigm (De Mauro, 2005,
pp. 325-334). However, Saussure captures the language and the market in a so to
speak “synchronic parallel” that would function differently in a different moment
of the evolution of the money sign. He uses the value of a 5-franc silver coin as an
analogy for the semantic value of the linguistic sign but does not compare it with a
paper or commodity monetary sign.
This is where my contribution comes in. A brief overview of the different
historical money forms shows that they follow a semiotic development, going from
a very low degree of arbitrariness between the signifier (e.g., the mental image of the
currency token) and its value, as in the primitive forms, to a degree of arbitrariness
and formal perfection beyond that of the linguistic sign (as in the case of electronic
money). However, a small misunderstanding may come from the way the study is set
up. For Saussure the study of the origin and development of language “is not even
worth asking” (Course, 1959, p. 72), when it comes to the object of linguistics and
where the notion of arbitrariness belongs. On the contrary here I am using this notion
for a diachronic comparative analysis. For Saussure anything in language that is
“prehistory”, e.g., before written records, was not suitable for scientific analysis for
obvious reasons. The money sign, however, evolves after the linguistic written records
were available and what follows is a series of synchronic comparisons, made with
the same logic as Saussure’s comparison with the silver coin. In philosophy Simmel
gives quite concrete examples of the evolution of the material substance of money in
Chapter II of Philosophy of Money (see Simmel, 1990), but his perspective is different
and I would say complementary to the one proposed here.
Also, we know not very much on the origin of language, but still there has been a
consistent scientific research on the matter since the early nineties of the XX century and
it will not be too speculative if we imagine that it evolved in a similar way as the money
sign—from contextually dependent forms of social interaction with zero degree of
arbitrariness for prehistoric man, to the strongly formalized “arbitrary” languages of the
present day.2 What would have changed Saussure’s mind on the study of the prehistoric
linguistic practices is the scientific evidence which today is far more available.
2. The Different Forms of Money Signs
Bartering is the first and most primitive form of economic exchange. In a barter
system, commodities are exchanged according to their utilitarian value. A minimum
115
Exploring Saussure’s Analogy between Linguistic and Monetary Signs
degree of conventionality is required to determine what quantity of one commodity
corresponds to another. Here, hypothetically, we can assume that any good possessed
in excess functions as a sign for a future need that could be satisfied as the result of
a future exchange. But the conventions are too numerous, and above all, the whole
system is contextually dependent—there must be a coincidence of excess and need in
time to motivate the two exchanges. Barter systems easily evolved into the commodity
money system. Some of the merchandise typically exchanged under the barter system
acquire a privileged function in the commodity money system by combining a higher
utilitarian value with a higher durability. This includes grain, livestock, salt, leather,
etc.—consumable goods, which become a convertible object as they provide a greater
guarantee for future exchange. These are also sought after by those who do not have
the need (for salt, leather, wheat, etc.), but are acquired with a view to their future
conversion to goods available in excess. In this sense, they have a clearer semiotic
function (and a higher degree of arbitrariness), since their possession is independent
of both the possessor’s needs and his productive abilities. However, this is still reliant
on the persistence of the market as a social convention.
Economists often neglect the difference between this kind of commodity money and
commodity money made from precious metals, cowries, ritual objects, stones, beads,
etc. According to a semiotic analysis, the latter has a higher degree of arbitrariness, since
their value comes not from the utilitarian function but from their symbolic value. They
are not consumed, but just owned and exchanged. A hybrid option, and an important
step to the next phase of the evolution of money, are instruments as monetary signs
(mostly in China). The most well-known are spade money and knife money.
A real turning point in currency development is the invention of the coin, as
evinced by both a semiotic analysis and the historical facts that followed the spread of
the use of coins. In the official histories of money,3 however, there is again no explicit
emphasis placed on this link. The qualitative difference that the coin introduces into
market-based economic relations is expressed by the English term fungibility. Unlike
commodity money, which was consumable or bearers of other functions and had to
be judged separately for each deal, the coins in their conception were identical and
countable. The coin introduces structural discretion into the thinking of economic
value, just as phonetically distinct words emerged from the amorphous sound and
visual matter of the gesture and shouting phase. Saussure quite accurately compares
the word as an acoustic image pattern with a coin that, regardless of the particular
token in our hands, always carries the same value.
The coin turns money signs into “economic” writing. Much has been written about the
changes in our civilization since the discovery of the printing machine, but only few note
that coin minting is the first form of printed writing. Accordingly, its effects are remarkable:
the coin provides a much more complete tool for political governance and a completely
new stage in the organization of military campaigns and, above all, a qualitatively new
level of entrepreneurship and market exchange. There are convincing studies about the
role of coins in the Hellenic world, the Roman Empire, and ancient China.4
All this, according to the analysis suggested herein, is due to the fact that the
monetary sign, before all else, becomes good for thinking with its increased level of
arbitrariness. As the hypothesis that thinking is linguistic in nature dominated the
20th century, and as Saussure’s insights reflect the mechanism of this dependence,
civilization’s progress as a result of the invention of a monetary sign with the
properties of a linguistic sign becomes easier to explain.
But money does not stop developing with the invention and refinement of the
116
Kristian Bankov
coin, though it has dominated economic exchange for nearly two millennia. Financial
institutions began to develop intensively particularly from the fourteenth century
onwards, with the beginning of the great geographic discoveries and the Trade
revolution. Meanwhile we witness the emergence of bank institutions, credit, shared
financial risk in investments, etc. All this multiplies the money turnover and so-called
“representative money”, which, unlike the coins, do not have their own material value
but only guarantee that they can be exchanged for gold or other valuables by a bank.
From the name itself, it is clear that the new monetary form is a next step towards the
arbitrariness of the monetary signs. Being much easier and safely portable than gold,
they open a new level in trade and financial manipulation. If a coin makes economic
value better for thinking, representative money makes it better for communication. The
banknote is the most perfect form of representative money. It is no coincidence that the
most complex work about the profound social function of money—Simmel’s Philosophy
of Money (1900)—is inspired by the historical period dominated by the banknote.
Banknotes still exist today, but after the phase of representative money there is
another structural change in the monetary system, which brings the symbolic function
of money (defined by its level of arbitrariness) to that of the linguistic sign, on the
side of the signified. For a long time, national banks have complied with banknote
coverage. Banknotes were guaranteed with deposits of gold. Accordingly, the
monetary sign had a triadic structure: 1) a banknote - 2) an economic value - 3) a gold
equivalent deposited in the bank.
But the dynamics of socio-economic processes, expressed in monetary relations,
have surpassed the capacity of the “gold standard”. Its crisis began after the World
War I; one by one countries started to abandon it; and the USA was the last in 1971.
The next money form was the so-called fiat money (or legal tender). The value of
this money form does not depend on an external reference, but on state law. This
law serves as the social convention in a linguistic sign, defining the relation between
signifier and signified. The sign structure of fiat money is identical from many
perspectives to Saussure’s linguistic sign. In contrast to natural language, however,
the level of arbitrariness also gains an ethical dimension in monetary signs. The value
of money depends on the amount of money put into circulation. Some see the lagging
gold standard as an emanation of neoliberalism and financial-corporate order, wherein
the arbitrariness of a currency is a prerequisite for arbitrary financial manipulations,
for example, with which a government can impoverish the savers through a single
decision. But this topic exceeds the objectives of the article.
The last form of money to date is e-money. Within this category we can
distinguish two main types—electronic banking of ordinary national currencies,
and new forms of digital currency such as Bitcoin. In the first case, we have a direct
continuation along the axis of a currency’s rising arbitrariness. I get my salary by bank
transfer, meaning that the sensory indicator of the occurrence of this important event
for me is an electronic record that I could reproduce on a screen. For conventional
currency, the same “signified”, e.g., the same monetary value, I receive as a bundle
of banknotes that, although extremely comfortable for daily purchases, carry a large
number of constraints. By contrast, electronic payments make it possible for me to pay
for a good or service at any point on the planet, and that does not require the recipient
to be present to receive the sum. E-money is perfect from a communication point of
view, and this is a qualitatively different shift in monetary sign forms. Regarding the
independence of the signified from the signifier, e-money is of a different nature than
the classic, arbitrary linguistic signs described by Saussure.
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Exploring Saussure’s Analogy between Linguistic and Monetary Signs
Even more interesting is the case of digital currencies. The main difference from
electronic banking of national currencies is that digital currencies are not issued by
central banks; there is no legal government protection behind them. As an analogy with
language, digital currencies are like jargon, whose importance depends on the number of
people who use them, naturally in parallel with the literary language / official currency.
In fact, my overall interest in the semiotic study of money was inspired by digital
currencies, and from the lack of satisfactory socio-cultural models for their nature. This
study is part of the theoretical basis for a larger research project, and so will not discuss
digital currencies analysis, which would, however, keep us from linking with Saussure’s
fundamental work. Here the main focus was to track the evolution of the sign-value
relation’s arbitrariness to the type of currency, in the course of the emerging different
forms of money. Accordingly, as a central difference between the two sign systems
on which a reliable model of economic value can be built, “scarcity” is a necessary
condition, on which the value of a currency in circulation depends. This has long been
a central problem for central banks after the penetration of fully “arbitrary” monetary
value, independent from any third-party valuables like the Gold standard.
In the second part of the article, I will try to position the above-mentioned
theoretical hypothesis among the most significant existing contributions to sociosemiotic analyses of money and economic value.
3. Dynamic, Triadic, and Holistic Approaches
As with many other topics, as well as that of economic value and money, semiotics is
divided into two major paradigms. The two authors who devoted most of their work
to this problem belong to different paradigms and although chronologically there is
a complete coincidence in their contributions (both debuted in 1968 and during the
next 10-12 years published several books), they entirely ignore each other. These
are Ferruccio Rossi-Landi and Jean Baudrillard. Both authors are fierce critics of
capitalism and bourgeois ideology, and they both rely on the theories of Karl Marx, in
particular the theme of alienation (although at the end Baudrillard turns even against
Marx), but their hypotheses are totally mutually incompatible. Rossi-Landi’s critique
is based on a broadly developed analogy (or homology, as he prefers to call it) between
physical and linguistic work (Rossi-Landi, 1975, pp. 1862-1888). In his model of the
sign, the third member is always present, and in most cases, it constitutes the embodied
work of “linguistic workers”. Not only does he disregard the above-mentioned
difference in language and currency, but he insists that it is ideologically condemnable
to assume that systems of natural language spontaneously
arise beyond the individual
wishes of community members (pp. 1932 ff.). It is precisely such an assumption that
conceals the fact that they, as the material artefacts, are the product of human labor
that the masses have produced, but that elites control the channels of distribution and
accordingly take advantage of something that does not rightfully belong to them.
Augusto Ponzio, a student and principal follower of Rossi-Landi, goes even
further in this doctrine by developing a direct analogy between the model of the
Saussurian sign and the marginalist (liberal) school in economics. His main criticism
is that both emphasize the moment of exchange, the market in one case, and the
exchange of messages in the other, completely ignoring production and subjectivity,
which are the real cause and source of exchange, the latter only constituting the
surface of these processes (Ponzio, 1990, pp. 185 ff.).
Rossi-Landi’s influence extends beyond the Bari circle. Umberto Eco himself
118
Kristian Bankov
is strongly influenced by Rossi-Landi’s doctrine, to such an extent that in Theory of
Semiotics the typology of signs is replaced by a typology of the various forms of sign
production (Eco, 1975, Chapter 3.6). However, it should be noted that in one of the
most important chapters, which clarifies the characteristics of culture as a semiotic
phenomenon, Eco pays special attention to prove that economic exchange can be
considered as a semiotic interaction (together with the production of tools and the
exchange of women). There he uses an entirely Marxist model of economic value, and
at the same time he is the only one of the authors cited in this tradition who explicitly
formulates the principle of currency’s scarcity:
The only difference between a coin (as sign-vehicle) and a word is that the word can be
reproduced without economic effort while a coin is an irreproducible item (which shares
some of the characters of its commodity-object). This simply means that there are different
kinds of signs which must also be differentiated according to the economic value of their
expression-matter. (Eco’s emphasis, ibid., pp. 25-26)
Eco, however, never returns to this typology of signs according to the economic value of
their expressive matter—something that I have tried to do in the first part of this article.
Baudrillard, opposite to Rossi-Landi, not only uses the dyadic model of the sign to
criticize the capitalist economy, but also directs all his inquiries toward consumption
rather than production. That is why both discourses are so distant, although their
purpose and disciplinary membership are the same. More a visionary than a researcher,
Baudrillard opens a discursive perspective on a semiological reading of the consumer
society. His main diagnosis is that in new post-war capitalism, the leading instrument
for class exploitation is no longer labor but consumption. Thus, the system deforms
its logic. From a material process based on the real needs of people, consumption
develops into an autonomous network of signifieds that (typical of the overall logic of
postmodernism) loses touch with material reality and begins to refer to its components
in a meaningless carousel of signifiers. In such a system media, advertising, and fashion
accelerate the course of the carousel, which distracts the individuals rotated by it more
and more from their authentic existence, thus increasing their alienation. Baudrillard
exclusively uses Saussure’s conceptual apparatus in this analysis, though he does not
pay attention to monetary signs’ analysis anywhere. Instead, he makes a direct parallel
between the post-modern situation in culture and the transition from the financial logic
of the gold standard (currency) to the wholly de-facto (arbitrary) monetary system:
[...] this process culminates in the virtual international autonomy of finance capital, in the
uncontrollable play of floating capital. Once currencies are extracted from all productive
cautions, and even from all reference to the gold standard, general equivalence becomes
the strategic place of the manipulation. (Baudrillard, 1975, p. 129 n. 9)
He also offers some valuable insights into the function of money and credit in the
following direction: “The illusionism is truly remarkable: society appears to extend
credit to you in exchange for a formal freedom, but in reality, it is you who are giving
credit to society, alienating your future in the process.” (Baudrillard, 1968, p. 160)
Thus, he points out the relationship of finance to the temporal dimension of human
existence, although his reading is entirely negative.
In confirming that the dyadic tradition going from the Course more accurately captures the dynamics of monetary signs, we turn to Derrida’s remarkable 1991 work Given
119
Exploring Saussure’s Analogy between Linguistic and Monetary Signs
Time (Derrida, 1992). This is his main treatise on economics, money and gifts. Derrida begins a long deconstruction of the metaphysical idea of giving
or donating time,
the link between economic value and temporality being direct. In the chapter entitled
“The Madness of the Economic Reason”, he deconstructs the paradoxical situation described in one of Baudelaire’s short stories where one of the characters gives a beggar a
fake coin, and at the end of the book expresses the analogy that credit is différance. Of
course, Derrida’s aphoristic and blatant way of expression does not allow for identifying
any model directly from his work, but the genius of some of his insights into money
and finance is attested to by the detailed comment that Michel Tratner makes in his vast
article entitled “Derrida’s Debt to Milton Friedman” (Tratner, 2003). It is here that the
paradoxes of the “deconstructed” linguistic sign projected on the financial history of the
nineteenth and twentieth centuries have quite logically led to the financial relativism of
fiat money after 1971. Deconstruction in culture and monetarism in financial politics are
similar in their deep structure, their apogee being in the seventies and eighties of XX
century, and the socio-economic effect of the latter is probably part of the conditions that
contributed to the enormous success of the former (Derrida’s debt, namely, p. 798).
Baudrillard and Derrida also direct us to a third type of approach where money
and economic value are at the heart of the study, and the semiotic paradigm is
pragmatically chosen in terms of its adequacy for their research goals. In these
approaches, which I call “holistic”, it is common that, thanks to semiotics, the greater
importance of money for economic reality is emphasized above the economic and
financial thought accepted in the mainstream.
In his article from 1989 “Making Semiotic Sense of Money as a Medium of
Exchange” Alan Dyer confronts the deep semiotic understanding of money as a
medium of thinking with the major economic theories of money. In his words “By
re-presenting objects and experiences as comparable containers of exchange value,
money symbolically transforms life and, thus, mediates our understanding of it”
(Dyer, 1989, p. 505). This also means that market transactions are not just culturally
neutral economic exchanges, but a deeply human interaction of sharing a common
understanding and a worldview. Steven Horwitz departs from these premises in his
article “Monetary Exchange as an Extra-Linguistic Social Communication Process”
(1992) and elaborates on the affinity between the subjectivist theory of the economic
value (the famous Austrian school) and the hermeneutic ontology of Gadamer. His
most important insights are about the parallel between the two systems where money
does not correspond to messages as most of the researchers think, but they correspond
to the language itself, within which prices have the function of word or message (p.
208). In his model also the notion of text is projected on the economic reality and it
corresponds to the notion of market (pp. 210 ff.)
A detailed presentation of these methods as well as his own contribution is proposed
by Wennerlind (see Wennerlind, 2001), and in any case the social dimension lies at the
center of the hypotheses. So, he divided the institutional, liberal, and Marxist approaches.
4. Instead of a Conclusion: Difference vs Scarcity
Saussure’s insight into the analogy between political economy and linguistics (as
well as between linguistic and monetary signs) opens a phenomenological research
phenotype for the typology of the different monetary signs in terms of how “good”
they are for thinking. From the review of existing contributions, those inspired by the
dyadic model of the sign go further in that direction, comprising even the temporal
120
Kristian Bankov
dimension of the phenomenon and bringing our attention to a radical difference in
the basic principle of semiosis between the linguistic and the monetary systems. If
in the Saussurean langue system is the difference between the signifiers to determine
the value of the sign, in the monetary system, on the contrary, the more identical the
signifiers the better for thinking they are. Therefore, in our analysis the invention of
the coin is a cornerstone in the evolution of the money sign. But if it is not from the
difference then where does the value of the money sign come from? As we have seen,
the value of each money sign comes from the principle of scarcity, from its position in
a system of quantity rather than in a system of differences.
Now, can we identify a phenomenological ground for the perception of the
economic value/scarcity, 5 comparable to the “condemnation” to semantic meaning,
postulated by Merleau-Ponty – Greimas, as an actualization of the Saussurean heritage?
Derrida often includes Heidegger in his arguments, precisely in relation to the
paradoxical status of the given time and the metaphysical implications that the
German philosopher exposes, interpreting everyday expressions close to giving and
receiving time. Derrida, however, does not come to the central question from the
point of view of my hypothesis, though his analysis often revolves around it. And this
hypothesis is related to what Heidegger calls “the thesis of the primordial finitude
of temporality” (Heidegger, 1996 [1927], p. 330). This means that the fundamental
characteristic of human existence is its finitude in time, the ontological inevitability
of death to which each life project is oriented. According to Heidegger, this situation
precedes and is the condition for the everyday conceptualization of time, for the
common meaning of temporality (p. 331). This means that despite any cultural
differences in the perception of time, religious beliefs and poetic fantasies about the
immortality of the human soul, the inevitable finitude is grounding the primordial
comprehension of the surrounding world. On a more superficial level, where this topic
meets economic issues, we can note that the finitude of human life and its phases from
birth to death are at the heart of all public institutions. They would lose sense, just
like any normal subject of conversation, at the moment when the finitude of human
existence is suspended and life becomes eternal.
I believe that the principle of scarcity of the money sign corresponds to the
primordial finitude of human temporality, the being of the being there as a thrown
project. “Time is money” is a cliché, behind which lies the deep homology between
two dimensions, whose meaning and value depend on the limitations of their
availability. It is this homology that makes money good for thinking, and gives
authors such as Goetzmann the insistence that finance is the foundation of “the ability
of humans to imagine and calculate the future” (Goetzmann, 2016, p. 2).
Notes
1
2
3
4
5
Saussure, F. de (1959 [1916]). A course in general linguistics (Trans., W. Baskin). New
York: Philosophical Library (quoted as Course in the text).
[…] it is only in the context of inherently meaningful collaborative activities, coordinated by
“natural” forms of communication such as pointing and pantomiming, that totally arbitrary
linguistic conventions could have come into existence. (Tomasello, 2008, pp. 327-328)
Davies, 1997; Ferguson, 2008; Goetzmann, 2016.
Seaford, 2004; Goetzmann, 2016.
“Scarcity is the fundamental economic problem of having seemingly unlimited human
wants in a world of limited resources” (Sexton, 2017, p. 65).
121
Exploring Saussure’s Analogy between Linguistic and Monetary Signs
References
Baudrillard, J. (1968). Le Système des objectes. Paris: Gallimard.
Baudrillard, J. (1975). Mirror of production (Trans., M. Poster). St. Louis: Telos Press.
Davies, G. (1997). A history of money from ancient times to the present day. Cardiff:
University of Wales Press.
Derrida, J. (1992). Given time: I. Counterfeit money (Trans., P. Kamuf). Chicago: University
of Chicago Press.
Dyer, A. W. (1989). Making semiotic sense of money as a medium of exchange. Journal of
Economic Issues, 23(2), 503-510.
Eco, U. (1975). A theory of semiotics. Bloomington: Indiana University Press.
Ferguson, N. (2008). The ascent of money: A financial history of the world. New York:
Penguin.
Goetzmann, W. N. (2016). Money changes everything: How finance made civilization possible.
Princeton and Woodstock: Princeton University Press.
Heidegger, M. (1996 [1927]). Being and time (Trans., J. Stambaugh). Albany: State University
of New York Press.
Horwitz, S. (1992). Monetary exchange as an extra-linguistic social communication process.
Review of Social Economy, 50(2), 196-214.
Mauro, T. de (2005). Notizie biografiche e critiche su F. De. Saussure. In Saussure, F. de
2005/1916 Corso di linguistica generale. Bari: Laterza.
Ponzio, A. (1990). Man as a sign. Essays on the philosophy of language (Edited and translated
from Italian and with an introduction by S. Petrilli). Berlin and New York: Mouton de
Gruyter.
Rossi-Landi, F. (1975). Linguistics and economics (= Janua Linguarum, Series Maior 81).
The Hague: Mouton, 2nd ed. 1977 [first publ. in: Current Trends in Linguistics. Vol. XII:
Linguistics and Adjacent Arts and Sciences. The Hague: Mouton 1974, Part 8, 1787-2017]
Saussure, F. de (1959 [1916]). A course in general linguistics (Trans., W. Baskin). New York:
Philosophical Library. (quoted as Course in the text)
Seaford, R. (2004). Money and the early greek mind. Cambridge: Cambridge University Press.
Sexton, R. L. (2017). Exploring economics. CTI Reviews, Content Technologies.
Simmel, G. (1990). The philosophy of money (Ed., D. Frisby; Trans.,T. Bottomore & D.
Frisby). London and New York: Routledge.
Tomasello, M. (2008). Origins of human communication. Cambridge, MA: MIT Press.
Tratner, M. (2004). Derrida’s Debt to Milton Friedman. New Literary History, 34, 791-806.
Wennerlind, C. (2001). Money talks, but what is it saying? Semiotics of money and social
control. Journal of Economic Issues, 35, 557-574.
About the author
Kristian Bankov (kbankov@nbu.bg) is Professor in Semiotics at New Bulgarian
University (NBU), visiting professor at Sichuan University and Director of the
Southeast European Center for Semiotic Studies at NBU. His interests in semiotics
started during the early nineties when he was studying in Bologna, following the courses
of Ugo Volli and Umberto Eco. He graduated in 1995 and since then teaches semiotic
courses at New Bulgarian University. In 2000 he defended his PhD in Helsinki. In 2006
he became Associate Professor in Semiotics, and he became Professor in 2011. He has
written five books and numerous articles in Bulgarian, English, and Italian. Since 2006
he has directed the organization of the international Early-Fall School of Semiotics
(EFSS); in 2014 he was elected as the Secretary General of the International Association
for Semiotic Studies.
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W4213436629.txt | Jurnal Diversita, 7 (2) Desember (2021) ISSN 2461-1263 (Print) ISSN 2580-6793 (Online)
DOI: https://doi.org/10.31289/diversita.v7i2.5038
Jurnal Diversita
Available online http://ojs.uma.ac.id/index.php/diversita
Studi Meta-Analisis : Efektivitas Cognitive Behavioural Therapy
untuk Meningkatkan Self-Esteem
Meta-Analysis Study : The Effectiveness Cognitive Behavioural Therapy
for Increase Self-Esteem
Rizki Sakinah Dewi(1*) & Ananta Yudiarso(2)
Fakultas Psikologi, Universitas Surabaya, Indonesia
Disubmit: 23 Maret 2021; Diproses: 02 Agustus 2021; Diaccept: 02 September 2021; Dipublish: 02 Desember 2021
*Corresponding author: E-mail: rizkisakinahdewi@gmail.com
Abstrak
Teknik Cognitive Behavioural Therapy adalah sebuah teknik intervensi yang cukup efektif untuk dapat meningkatkan
self-esteem individu. Self-esteem yang dimiliki oleh individu akan memengaruhi tingkat kesejahteraannya dan akan
memengaruhi bagaimana ia dapat memandang dirinya sendiri secara positif atau negatif. Oleh sebab itu, penelitian
ini dilakukan untuk dapat mengetahui apakah teknik intervensi Cognitive Behavioural Therapy dapat membantu
meningkatkan self-esteem pada individu. Metode yang digunakan untuk melihat tingkat efektivitas penggunaan
teknik Cognitive Behavioural Therapy tersebut ialah metode meta-analysis, yang dilakukan dengan melihat effect size
dari 20 jurnal eksperimen penelitian terdahulu. Effect size didapatkan dengan melihat jumlah partisipan (N), mean
(M), dan standard deviasi (SD) pada setiap kelompok eksperimen dan kontrol. Hasil penelitian ini menunjukkan
bahwa teknik Cognitive Behavioural Therapy tidak cukup efektif untuk meningkatkan self-esteem pada individu
(Hedges’g = 0.34), (95%CI = 0.11 hingga 0.3; dan I2 = 88.0% ), hal ini dikarenakan keberhasilan seorang individu
untuk meningkatkan self-esteem juga dapat dipengaruhi oleh dukungan sosial yang diterimanya dari lingkungan,
baik keluarga maupun sosial.
Kata Kunci: Cognitive Behavioural Therapy; Meta-Analisis; Self-Esteem
Abstract
Cognitive Behavioral Therapy technique is an intervention technique that is quite effective to increase individual selfesteem. The possessed self-esteem will affects individual level of well-being and how to evaluate theirself positively or
negatively. Therefore, this study was conducted to determine whether Cognitive Behavioral Therapy intervention
techniques can help increase self-esteem in individuals. Method: The method used to see the level of effectiveness of the
Cognitive Behavioral Therapy technique is the meta-analysis method, which was carried out with effect sizes from 20
previous research experimental journals. Effect size was obtained by looking at the number of participants (N), mean
(M), and standard deviation (SD) in each experimental and control group. Result: The results of this study indicates that
the Cognitive Behavioral Therapy technique is not effective enough to increase self-esteem in individuals (Hedges'g =
0.34), (95%CI = 0.11 to 0.3; and I2 = 88.0%), this is due to the success of an individual to improve self-esteem. Self-esteem
can also be influenced by social support received from the environment, both family and social.
Keyword: Cognitive Behavioural Therapy; Meta-Analysis; Self-Esteem
How to Cite: Dewi, R.S. & Yudiarso, A. (2021). Studi Meta-Analisis : Efektivitas Cognitive Behavioural
Therapy untuk Meningkatkan Self-Esteem. Jurnal Diversita, 7 (2): 231-239.
231
Dewi, R.S. & Yudiarso, A. Studi Meta-Analisis : Efektivitas Cognitive Behavioural Therapy
PENDAHULUAN
Self-esteem menjadi topik yang
menarik dalam penelitian ini dikarenakan
tingkat self-esteem pada seorang individu
akan
sangat
berpengaruh
pada
kesejahteraan individu tersebut. Terlebih
self-esteem merupakan bagian penting
dalam konsep diri seseorang, bagaimana
seseorang tersebut akan menanamkan
orientasi secara positif maupun negatif
terhadap dirinya (Ismi Isnani Kamila &
Mukhlis, 2013). Self-esteem pada setiap
individu akan menetapkan apakah individu
tersebut dapat tampil dengan maksimal
dan kompeten pada bidang-bidang yang
penting bagi mereka, baik dalam hal
akademik, sosial, keterampilan maupun
keadaan atau kondisi fisiknya (Santrock,
2012). Self-esteem sendiri merupakan
cara yang digunakan seseorang untuk
dapat mengevaluasi dirinya sendiri
(Santrock, 2012). Menurut Branden (1969)
self-esteem diartikan sebagai keyakinan
seseorang
untuk
kompeten
dalam
menjalani hidupnya dan layak hidup
(Waite et al., 2012).
Menurut Rosenberg (1965) selfesteem adalah sebuah evaluasi secara
global yang dilakukan individu terhadap
dirinya sendiri. Self-esteem sendiri
merupakan komponen dari skema kognitif
yang difokuskan pada dirinya sendiri
(Neac, 2013). Menurut skala pengukuran
self-esteem
milik
Rosenberg
1965
(Nurhidayati, 2014) terdapat dua aspek
dalam
pengukuran
self-esteem,
diantaranya ialah: 1) Self-worth 2) Selfacceptance. Menurut Harter (dalam
Brooks, 1999) terdapat dua faktor yang
berhubungan dengan tinggi dan rendahnya
self-esteem pada individu, yaitu: 1)
Perasaan dan pikiran
individu akan
kemampuannya serta bahwa dirinya
232
berharga; 2) Individu mendapatkan
dukungan sosial, baik dari lingkungan
keluarga, sosial, maupun teman sebaya
(Nurhidayati, 2014)
Individu dengan rendahnya selfesteem ini sebagian besar berasal dari latar
belakang keluarga yang bermasalah atau
terdapat konflik, individu akan merasa
ditolak oleh keluarga dan sosial, pernah
mengalami
kekerasan,
serta
tidak
mendapatkan dukungan sosial (Santrock,
2012).
Individu
yang
mengalami
rendahnya self-esteem cenderung lebih
rentan
terhadap
berbagai
macam
gangguan klinis, diantaranya ialah
kecemasan, kesepian, dan mengalami
gangguan makan. Selain itu, ketika berada
dalam kondisi yang kurang baik, mereka
akan memandang semuanya secara negatif
dan hanya mengingat dan memikirkan
perilaku-perilaku yang buruk saja (Myers,
2014).
Menurut McClure., et. al. (2010)
rendahnya self-esteem akan memengaruhi
kondisi psikologis, fisik dan sosial
seseorang, selain itu bagi perkembangan
anak juga akan memengaruhi prestasi
akademiknya
(Wibowo, 2016). Dalam
sebuah studi menyatakan bahwa dari
326.641 individu dengan rentang usia 9 –
90 tahun, self-esteem pada masa remaja
cenderung menurun dan mulai meningkat
di usia 20 hingga 50 tahun, lalu Kembali
menurun diusia 70 hingga 80 tahun. Pada
anak remaja perempuan self-esteem biasa
menurun pada usia 12 hingga 14 tahun,
sedangkan pada remaja laki-laki biasa
menurun di usia 16 tahun (Ismi Isnani
Kamila & Mukhlis, 2013).
Cognitive Behaviour Therapy (CBT)
merupakan sebuah teknik intervensi yang
dapat meningkatkan self-esteem pada
seseorang. Teknik CBT sendiri memiliki 2
Jurnal Diversita, 7 (2) Desember 2021: 231-239.
model dasar yang dilakukan, yaitu
Cognitive restructuring therapy, dimana
pada model ini klien akan diajak untuk
merubah
distorsi
kognitif
yang
mempertahankan perilaku menyimpang
mereka dan merubahnya menjadi kognitif
yang lebih adaptif, serta Cognitivebehavioural
coping
skills
therapy
mengajarkan klien untuk memberikan
respon yang adaptif dan efektif dalam
menghadapi
situasi-situasi
yang
bermasalah (Spiegler & Guevremont,
2013). Dalam penelitian yang dilakukan
(Handley
et
al.,
2015)
terdapat
peningkatan yang signifikan pada selfesteem setelah diberikan teknik CBT. Selain
itu, penelitian lain yang dilakukan oleh
(Salehyan
&
Aghabeiki,
2011)
menunjukkan bahwa hasil rata-rata
posttest self-esteem dalam kelompok
eksperimen yang mendapatkan intervensi
CBT
meningkat
secara
signifikan
dibandingkan kelompok kontrol yang tidak
menerima intervensi. Hal ini menunjukkan
bahwa teknik CBT cukup efektif dalam
meningkatkan self-esteem.
Selain itu dijelaskan pula oleh
(Spiegler & Guevremont, 2013) apabila
penerapan teknik cognitive behavior
therapy ini dilakukan dengan secara
konsisten maka akan meningkatkan
kepercayaan dan harga diri (self-esteem)
mereka terkait kondisi atau keadaan
dirinya. Berdasarkan penjelasan diatas,
oleh karena itu peneliti akan memberikan
fokus penelitian untuk melihat apakah
penggunaan teknik Cognitive Behaviour
Therapy (CBT) efektif untuk meningkatkan
self-esteem pada individu. Jenis penelitian
yang dilakukan ialah meta-analysis, hal ini
dikarenakan peneliti melihat adanya
perbedaan penemuan dari literaturliteratur sebelumnya. Sehingga penelitian
233
meta-analysis ini perlu dilakukan untuk
melihat effect size dari penelitian terdahulu
yang serupa.
METODE PENELITIAN
Jenis
penelitian
yang
akan
digunakan ialah meta-analysis, yang
mengikuti pedoman Prisma 2012 dan
Meta-analysis Review Standart (MARS).
Pengumpulan data penelitian terdahulu
dilakukan dengan menggunakan Science
Direct dan Google Scholar, dengan
menggunakan kata kunci (1) Cognitive
Behaviour Therapy for Self-esteem (2)
Cognitive Behavioural Therapy Group for
Self-esteem.
Pencarian
jurnal
penelitian
terdahulu ini dibatasi hanya untuk jurnal
internasional dengan rentang waktu 20112020 (10 tahun terakhir). Hasil dari
pencarian yang dilakukan, ditemukan 20
jurnal penelitian terdahulu yang dapat
diolah. Namun penelitian ini hanya akan
dibatasi
untuk
melihat
efektivitas
penerapan teknik Cognitive Behavioral
Therapy (CBT) untuk meningkatkan selfesteem pada individu.
Hasil penelitian terdahulu akan
diolah dengan menggunakan Free Online
Meta-Analysis
Service
(www.metamar.com) yang menggunakan indeks
Hedges’g untuk mengetahui effect size.
Pengolahan data dilakukan dengan
melihat hasil mean (M), standard deviation
(SD), dan jumlah sampel (N) pada posttest
setelah diberikan intervensi. Inconsistensy
(𝐼 2 ) akan digunakan untuk melihat
keberagaman
data
penelitian
2
(heterogenitas), 𝐼 > 75% maka hasil
penelitian semakin beragam. Menurut
standar Hedges’g (Ellis, 2010) terdapat 3
kategori nilai effect size, yaitu (1) Small
Dewi, R.S. & Yudiarso, A. Studi Meta-Analisis : Efektivitas Cognitive Behavioural Therapy
effect size apabila nilai hedges’g 0,2; (2)
Medium effect size apabila nilai hedges’g
0,5; (3) Large effect size apabila nilai
hedges’g 0,8.
15.927 jurnal untuk hasil pencarian
Cognitive Behavioral Therapy Group
for self-esteem pada Science Direct
7.023 jurnal hasil untuk pencarian
Cognitive Behavioral Therapy Group for
self-esteem pada Science Direct setelah
difokuskan tahun 2011 - 2020
Jurnal tidak menggunakan
teknik intervensi CBT atau tidak
menggunakan variable selfesteem
91 jurnal hasil penerapan Cognitive
Behavioral Therapy pada self esteem
Beberapa jurnal menggunakan
korelasi dan one group (pretest
& posttest)
20 jurnal eksperimen penerapan teknik
CBT untuk meningkatkan self-esteem
Gambar 1. Skema Alur Pencarian Jurnal Terdahulu Penerapan CBT
Untuk Meningkatkan Self-Esteem
No
Peneliti
1
(Tylee
et
al.,
2014)
(Kothari
et
al.,
2019)
(Roghan
chi et al.,
2013)
(Egan et
al.,
2014)
(Premk
umar et
al.,
2011)
2
3
4
5
6
Partisipan
Eksperim
en
N = 171
M = 14.6
SD = 5.0
N = 31
M = 14.59
SD = 2.78
N = 24
M = 9.68
SD = 3.78
N = 16
M = 29.38
SD = 5.21
N = 25
M = -1.04
SD = 6.15
(Waite
N = 11
et
al., M = 122.73
2012)
SD = 18.69
Tabel 1. Data 20 Jurnal Penelitian Terdahulu
Metode
Alat
Effect
Ukur
Size (g)
Kontrol
N = 193
M = 12.9
SD = 4.8
N = 42
M = 13.15
SD = 1.49
N = 24
M = 6.78
SD = 3.84
N = 18
M = 27.87
SD = 4.60
N = 17
M = 2.25
SD = 3.84
N = 11
M = 84.81
SD = 18.86
SEg
g_lower
g_upper
CBT fennell’s RSES
model
0,346577
0,105
587
0,139627
0,553528
Internet
RSES
based (ICBT)
0,668106
0,240
712
0,19631
1,139902
REBT + art SEI
therapy
0,756398
0,294
25
0,179668
1,333129
CBT face to RSES
face
0,301166
0,337
458
-0,36025
0,962583
CBT
for RSES
psychosis
(CBTp)
+
standard
care
CBT
RSCQ
-0,60367
0,315
385
-1,22183
0,01448
1,942989
0,504
055
0,955041
2,930937
234
Jurnal Diversita, 7 (2) Desember 2021: 231-239.
7
(Shu et
al.,
2019)
N = 94
M = 16.71
SD = 3.80
N = 94
M = 16.73
SD = 5.08
8
(Meirong et
al.,
2019)
(Sönme
z et al.,
2020)
(Salehya
n
&
Aghabei
ki,
2011)
(Shimot
su et al.,
2018)
(Hazell
et
al.,
2018)
(Charkh
andeh et
al.,
2016)
(Handle
y et al.,
2015)
(Joseph
et
al.,
2016)
(Tanoue
et
al.,
2018)
(Neac,
2013)
N = 67
M = 28.43
SD = 4.91
N = 57
M = 27.78
SD = 5.14
N = 32
M = 27.1
SD = 5.9
N = 15
M = -5.6
SD = 1.55
N = 40
M = 30.60
SD = 8.80
N = 14
M = 15.07
SD = 5.37
N = 65
M = 3.09
SD = 1.44
9
10
11
12
13
14
15
16
17
18
19
20
N = 21
M = 28.44
SD = 0.36
N = 48
M = 18.65
SD = 4.93
N = 20
M = 32.06
SD = 10.46
N = 40
M = 19.22
SD = 6.23
(Hullu
N = 46
et
al., M = 37.57
2017)
SD = 10.36
(Freema N = 15
n et al., M = 99.9
2014)
SD = 21.6
(Tarrier N = 17
et
al., M = 88.0
2014)
SD = 27.0
Internet
RSES
based CBT
for
perfectionis
m (ICBT-P)
CBT
SES
0,075487
0,145
328
-0,20936
0,36033
0,128764
0,179
269
-0,2226
0,480131
N = 31
M = 26.8
SD = 7.6
N = 15
M = -1.7
SD = 1.55
CBT
+ RSES
Treatment
as usual
CBT
RSES
0,043641
0,248
927
-0,44426
0,531538
-2,66957
0,494
975
-3,63972
-1,69942
N = 46
M = 29.26
SD = 9.10
N = 14
M = 10.71
SD = 5.37
N = 63
M = 3.97
SD = 1.70
CBT
RSES
0,148182
0,214
554
-0,27234
0,568708
CBT
+ RSES
Treatment
as usual
CBT
CDI
0,78827
0,381
775
0,03999
1,53655
-0,55599
0,179
146
-0,90712
-0,20487
N = 21
M = 25.73
SD = 0.30
N = 52
M = 18.00
SD = 4.65
N = 23
M = 31.36
SD = 10.20
N = 40
M = 16.76
SD = 4.61
N = 35
M = 34.74
SD = 9.93
N = 14
M = 82.9
SD = 25.6
N = 19
M = 77.3
SD = 24.7
CBT
for RSES
perfectionis
m (CBT-P)
CBT
RSES
8,024086
0,926
379
6,208384
9,839789
0,134761
0,198
853
-0,25499
0,524513
CBT
RSES
0,066573
0,300
201
-0,52182
0,654966
Cignitive
Behavioural
Intervention
CBT
RSES
0,444559
0,224
221
0,005085
0,884033
RSES
0,275425
0,223
215
-0,16208
0,712927
CBT
RSQ
0,699826
0,372
699
-0,03066
1,430315
0,405388
0,329
908
-0,24123
1,052007
Cognitive
SERS
Behavioural
Prevention
of Suicide in
Psychosis
protocol
(CBSPp)
(Sumber : www.meta-mar.com)
235
Dewi, R.S. & Yudiarso, A. Studi Meta-Analisis : Efektivitas Cognitive Behavioural Therapy
kurang signifikan dalam meningkatkan
self-esteem.
Hasil dari pengolahan data 20
jurnal internasional menggunakan metamar online, dengan nilai random effect
model 0,34 (small to medium), (95%CI =
0.11 hingga 0.3) serta nilai inkonsistensi
(𝐼 2 = 88.0%) yang tergolong tinggi. Hasil
tersebut menunjukkan bahwa CBT tidak
dapat secara signifikan meningkatkan selfesteem. Selain itu juga menunjukkan
adanya
keberagaman
data
yang
digunakan, baik dalam hal perbedaan alat
ukur yang digunakan, jumlah partisipan
penelitian, perbedaan jenis kelamin
partisipan, perbedaan cara penerapan
teknik CBT.
HASIL DAN PEMBAHASAN
Pada 20 jurnal internasional telah
dipaparkan hasil effect size dengan cara
mengolah nilai jumlah partisipan (N), mean
(M), dan standar deviasi (SD), dari setiap
kelompok eksperimen dan kelompok
kontrol. Hasil 20 jurnal internasional
menunjukkan bahwa penerapan teknik
Cognitive Behaviour Therapy ini adalah
sebuah teknik intervensi yang cukup dapat
meningkatkan self-esteem yang dimiliki
individu, meskipun tidak terlalu efektif. Hal
tersebut nampak berbeda dari hasil
perhitungan effect size pada penelitian ini,
yang menunjukkan bahwa penerapan
teknik Cognitive Behavioural Therapy
Tabel . Summary Of Result - Fixed And Random Effect Models
Hedges’g
(SMD)
0.21
SEg
95%CI
Fixed Effect Model
0.05
[0.11,0.3]
Random Effect
0.34
0.156 [0.031,0.643]
Model
(Sumber Tabel: www.meta-mar.com)
z score
p value
Heterogenity
4.078
4.5e-05
𝐼 =88.0%, 𝐶ℎ𝑖 2 = 158.08, 𝑑𝑓 = 19
2.156
0.03105
𝐼 2 =88.0%, 𝑇𝑎𝑢2 = 0.39
2
Gambar 2. Forestplot Random Effect Model
(Sumber : www.meta-mar.com)
Gambar 3. Funnel Plot Random Effect Model
(Sumber : www.meta-mar.com)
Self-esteem yang dimiliki setiap
individu akan memengaruhi bagaimana
seorang individu dapat tampil dengan
maksimal dan kompeten pada berbagai
macam
bidang
penting
dalam
kehidupannya, baik dalam bidang akademik
atau pendidikan, keterampilan sosial
maupun keadaan atau kondisi fisiknya
(Santrock, 2012). Ketika seorang individu
memiliki self-esteem yang rendah, maka ia
236
Jurnal Diversita, 7 (2) Desember 2021: 231-239.
akan cenderung rentan mengalami
berbagai macam gangguan klinis, seperti
kecemasan, kesepian hingga mengalami
gangguan makan. Selain itu, ketika berada
dalam kondisi yang kurang baik, mereka
akan memandang semuanya secara negatif
dan hanya mengingat dan memikirkan
perilaku-perilaku yang buruk saja (Myers,
2014). Terdapat dua hal yang dapat
memengaruhi self-esteem pada individu,
yaitu perasaan dan pikiran individu akan
kemampuannya serta bahwa dirinya
berharga serta dukungan sosial yang
dimiliki individu, baik dari lingkungan
keluarga, sosial, maupun teman sebaya
(Nurhidayati, 2014).
Berdasarkan pembahasan diatas,
menunjukkan bahwa self-esteem sangatlah
penting untuk kesejahteraan individu, oleh
sebab itulah berbagai macam teknik
intervensi
dilakukan
untuk
dapat
meningkatkan self-esteem seorang individu,
diantaranya ialah cognitive behavioural
therapy (CBT), group therapy, art therapy,
rational emotive behavior therapy (REBT),
dan berbagai macam alternatif lain yang
diharapkan dapat meningkatkan selfesteem seorang individu. Namun, beberapa
penelitian menyatakan bahwa Cognitive
Behavioural Therapy (CBT) ialah salah satu
teknik yang seringkali digunakan serta
dapat meningkatkan self-esteem pada
individu.
Pada hasil penelitian ini menunjukkan
bahwa CBT kurang efektif untuk dapat
meningkatkan
self-esteem.
Hal
ini
ditunjukkan pada nilai hedges’g 0,34,
dimana menurut standar Hedges’g nilai
effect size tersebut tegolong dalam small
effect size. Hasil tersebut dapat dipengaruhi
oleh beberapa faktor, diantaranya ialah
dukungan sosial yang diterima individu,
baik dari lingkungan sosial maupun
keluarga. Hal tersebut menunjukkan bahwa
individu yang telah mendapatkan teknik
Cognitive Behavioural Therapy (CBT),
namun tidak mendapatkan dukungan baik
dari keluarga, teman maupun lingkungan
sosialnya, akan cenderung tetap memiliki
self-esteem yang rendah. Selain itu
ditemukannya keberagaman data pada
hasil penelitian yang cukup besar,
ditunjukkan pada nilai inkonsistensi (𝐼 2 =
88.0%). Nilai inkonsistensi yang tergolong
tinggi tersebut dapat dipengaruhi oleh
beberapa faktor, diantaranya ialah jumlah
partisipan yang berbeda-beda pada tiap
jurnal penelitian, perbedaan metode
intervensi CBT yang digunakan pada setiap
jurnal, perbedaan jenis kelamin partisipan,
serta perbedaan alat ukur self-esteem yang
digunakan pada setiap jurnal penelitian
terdahulu.
SIMPULAN
Penelitian
meta-analysis
ini
dilakukan untuk melihat efektivitas
penerapan teknik Cognitive Behavioural
Therapy untuk meningkatkan self-esteem.
Teknik CBT sendiri merupakan teknik yang
menggabungkan 2 model dasar, yaitu
pikiran dan perilaku. Penerapan CBT ini
diharapkan dapat merubah distorsi
kognitif atau pemikiran yang tidak tepat
pada individu dan merubahnya menjadi
pemikiran yang lebih adaptif sehingga
dapat memunculkan perilaku yang lebih
adaptif dan efektif pula (Spiegler &
Guevremont, 2013). Sehingga penerapan
teknik CBT ini diharapkan dapat merubah
pemikiran negatif individu terhadap
dirinya sendiri serta dapat lebih
menghargai dirinya sendiri, agar individu
tersebut dapat lebih kompeten dan positif
dalam menjalani kehidupannya, baik dalam
lingkungan sosial maupun akademik.
237
Dewi, R.S. & Yudiarso, A. Studi Meta-Analisis : Efektivitas Cognitive Behavioural Therapy
Namun hasil dalam penelitian metaanalysis ini ditemukan bahwa penerapan
teknik CBT kurang efektif atau signifikan
untuk meningkatkan self-esteem individu,
hal ini dikarenakan selain mendapatkan
terapi, individu juga seharusnya mendapat
dukungan sosial yang cukup, baik dari
keluarga,
teman
sebaya,
maupun
lingkungan sosialnya, agar self-esteem
dapat meningkat (Nurhidayati, 2014). Hal
ini juga mendapat dukungan dari penelitian
lain yang menjelaskan bahwa belum dapat
dipastikan apakah penerapan teknik CBT
ini dapat merubah proses kognitif individu
yang mendasari rendahnya harga diri
(Kolubinski et al., 2018).
Selain itu, hal ini juga dapat
dipengaruhi oleh hasil perhitungan yang
heterogen atau beragam, baik dari
perbedaan alat ukur yang digunakan,
perbedaan penerapan teknik CBT, jumlah
partisipan, dan perbedaan jenis kelamin
partisipan. Sehingga diharapkan untuk
penelitian selanjutnya terkait topik ini
dapat lebih memperhatikan metode dan
partisipan yang digunakan, serta lebih
dapat menggali literatur atau teknik
intervensi lain yang mungkin dapat
menjadi treatment tambahan untuk
membantu meningkatkan
self-esteem
dalam diri seseorang.
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Levinson, Meira, and Sanford Levinson. 2003. "Getting Religion": Religion, Diversity, and
Community in Public and Private Schools. In Wrestling with Diversity, ed. Sanford Levinson,
90-123. Durham, NC: Duke University Press.
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W4243708183.txt | Hindawi
BioMed Research International
Volume 2018, Article ID 3782512, 1 page
https://doi.org/10.1155/2018/3782512
Corrigendum
Corrigendum to (Evaluation of Plasma MicroRNAs as
Diagnostic and Prognostic Biomarkers in Pancreatic
Adenocarcinoma: miR-196a and miR-210 Could Be
Negative and Positive Prognostic Markers, Respectively)
Qi Yu,1 Changqing Xu,2 Wei Yuan,3 Chengfeng Wang ,4
Ping Zhao ,4 Lianzhen Chen ,5 and Jie Ma 6
1
Gastrointestinal Rehabilitation Center, Beijing Rehabilitation Hospital, Capital Medical University, Beijing 100144, China
Department of Digestive Medicine, Qianfoshan Hospital, Shandong University, 16766 Jingshi Road, Jinan 250014, China
3
State Key Laboratory of Molecular Oncology, National Cancer Center/Cancer Hospital,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China
4
Department of Abdominal Surgery, Cancer Hospital of Chinese Academy of Medical Sciences,
Peking Union Medical College, Beijing, China
5
Department of Pharmacy, National Cancer Center/Cancer Hospital,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China
6
Department of Biotherapy, Beijing Hospital, National Center of Gerontology, Beijing 100730, China
2
Correspondence should be addressed to Ping Zhao; dr.zhaoping@263.net and Lianzhen Chen; yjkclz@163.com
Received 1 August 2018; Accepted 2 August 2018; Published 6 September 2018
Copyright © 2018 Qi Yu 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.
In the article titled “Evaluation of Plasma MicroRNAs as
Diagnostic and Prognostic Biomarkers in Pancreatic Adenocarcinoma: miR-196a and miR-210 Could Be Negative and
Positive Prognostic Markers, Respectively” [1], there were
errors in the authors’ affiliations. The correct affiliation list is
shown above.
References
[1] Qi Yu, Changqing Xu, Wei Yuan et al., “Evaluation of Plasma
MicroRNAs as Diagnostic and Prognostic Biomarkers in Pancreatic Adenocarcinoma: miR-196a and miR-210 Could Be Negative and Positive Prognostic Markers, Respectively,” BioMed
Research International, vol. 2017, Article ID 6495867, 10 pages,
2017.
| 309 | 2,298 |
W1935588882.txt | Oncotarget, Vol. 6, No. 26
www.impactjournals.com/oncotarget/
MYC amplifications in myeloma cell lines: correlation with
MYC-inhibitor efficacy
Toril Holien1, Kristine Misund1, Oddrun Elise Olsen1, Katarzyna Anna Baranowska1,
Glenn Buene1, Magne Børset1,2, Anders Waage1,3, Anders Sundan1,4
1
.G. Jebsen Center for Myeloma Research, Department of Cancer Research and Molecular Medicine, Norwegian University
K
of Science and Technology, Trondheim, Norway
2
Department of Immunology and Transfusion Medicine, St. Olav’s University Hospital, Trondheim, Norway
3
Department of Hematology, St. Olav’s University Hospital, Trondheim, Norway
4
EMIR (Centre of Molecular Inflammation Research), Department of Cancer Research and Molecular Medicine, Norwegian
C
University of Science and Technology, Trondheim, Norway
Correspondence to:
Toril Holien, e-mail: toril.holien@ntnu.no
Keywords: oncology, carcinogenesis, molecular and cellular biology, signal transduction, cell cycle
Received: March 09, 2015
Accepted: May 20, 2015
Published: June 02, 2015
ABSTRACT
In multiple myeloma, elevated MYC expression is related to disease initiation
and progression. We found that in myeloma cell lines, MYC gene amplifications were
common and correlated with MYC mRNA and protein. In primary cell samples MYC
mRNA levels were also relatively high; however gene copy number alterations were
uncommon. Elevated levels of MYC in primary myeloma cells have been reported
to arise from complex genetic aberrations and are more common than previously
thought. Thus, elevated MYC expression is achieved differently in myeloma cell lines
and primary cells. Sensitivity of myeloma cell lines to the MYC inhibitor 10058-F4
correlated with MYC expression, supporting that the activity of 10058-F4 was through
specific inhibition of MYC.
MYC activity has been shown to be associated with many
features of cancer cells including cell metabolism and
proliferation. [7] In multiple myeloma it was commonly
thought that activation of MYC was a late-stage event. [8]
The importance of MYC in myeloma disease progression
has lately become clearer, and increased MYC activity
has been implicated in progression from MGUS to fullblown myeloma. [9–12] Recently, two papers showed
that complex rearrangements positioning MYC in the
proximity of super-enhancers caused elevated MYC
expression in primary myeloma cells. [13, 14] Moreover,
MYC was shown to be the most frequent translocation
partner in aberrations involving the immunoglobulin light
chains. [15] Altogether, MYC rearrangements were found
in nearly half of the myeloma patients leaving MYC the
most commonly mutated gene in multiple myeloma. [13]
We have earlier shown that MYC expression
was important for in vitro survival of myeloma cells
using different approaches for targeting MYC. [16–18]
The question we wanted to address in this study was
whether the vulnerability of multiple myeloma cells
INTRODUCTION
Multiple myeloma is the second most common
hematological malignancy and accounts for about 2% of
cancer-related deaths. The cancer cells arise from postgerminal center plasma cells and usually reside in the
bone marrow. Myeloma is preceded by a benign condition
termed monoclonal gammopathy of undetermined
significance (MGUS), [1] and the annual risk of
progression to myeloma is about 1%. [2] Treatment of
multiple myeloma has improved during the last decades
by the introduction of proteasome inhibitors such as
bortezomib and carfilzomib as well as immunomodulatory
drugs (IMIDs) such as thalidomide and lenalidomide.
However; the median overall survival time from diagnosis
is still no more than 5–7 years. [3, 4]
The transcription factor c-MYC (hereafter termed
MYC) and the related N-MYC and L-MYC oncogenes
are involved in the development of up to 70% of all
cancers. [5] Under normal conditions MYC increases
cell proliferation and halts differentiation. [6] Abnormal
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for MYC inhibition correlated to cellular levels of
MYC. Pharmacological targeting of MYC activity has
been challenging. One option is to use small molecule
inhibitors that target MYC-MAX heterodimerization
thereby preventing transactivation of MYC target genes.
[19, 20] We found that the small molecule inhibitor of
MYC, 10058-F4, suppressed proliferation and survival
of myeloma cells, arguing for a distinct role of MYC in
multiple myeloma. The importance of MYC was further
supported by an inverse correlation between IC50 of the
inhibitor and the level of MYC in myeloma cell lines.
indicate that the main determinant of elevated MYC
expression in myeloma cell lines is amplification of the
MYC gene.
We went on to investigate the variation in MYC
gene copy numbers in myeloma patient samples by the
same method as applied for cell lines. Interestingly, most
of the primary samples (n = 21) had two copies of the
MYC gene and the samples deviating from this (n = 7)
had MYC gene copies varying from 1 to 4 (data not
shown). The levels of MYC mRNA, on the other hand,
showed remarkable variation (Figure 3A). Thus, in
contrast to myeloma cell lines, MYC levels in primary
cells apparently are not determined by the number of gene
copies as measured here, but by other mechanisms.
Interestingly, we originally compared MYC mRNA
levels in cell lines and primary cells applying GAPDH
mRNA as the only reference, getting higher MYC levels in
primary cells than in myeloma cell lines. However, when
comparing GAPDH mRNA levels in cell lines with primary
cells using the Nanostring nCounter technology and using
several genes as reference; it turned out that the difference
in GAPDH mRNA was even greater than the difference in
MYC mRNA. Thus, when comparing MYC mRNA levels
in primary cells and cell lines, the patient cells as a group
had lower MYC expression levels than cell lines (Figure 3B).
Patients (n = 28) had a median MYC level of 2345, range
660–15969, whereas the cell lines (N = 11) had a median
level of 8411, range 85–18859. Furthermore, the cell lines
had a median GAPDH level of 33347, range 23653–59757,
whereas the primary cells had a median GAPDH level of
8345, range 2297–15769 (Figure 3C). We also treated the
primary myeloma samples with the MYC inhibitor to obtain
IC50 values. However, the cell viability varied and only 18
out of 28 samples had a viability > 50% without treatment.
Those samples were included in analysis (data not shown).
When comparing IC50 values for 10058-F4 in the samples
with baseline viability > 50% with MYC mRNA expression,
we were not able to see the same correlation as in cell lines.
However, analysis of only 18 samples may not be sufficient
to see such correlations. There was no correlation between
cell viability in the patient samples and 10058-F4 IC50
values (n = 28, data not shown).
The primary cells studied were both from newly
diagnosed, untreated patients (n = 13), as well as from
treated patients (n = 15), i.e. patients at a later stage of
disease. Clinical characteristics of the patient samples
are found in Supplementary Table I. Interestingly,
the MYC mRNA levels were not different between
previously treated and untreated patients (Figure 3D),
indicating that untreated patients had myeloma cells with
as high MYC expression as patients in later stages of
disease. These observations suggest that both untreated
and treated patients may have plasma cells with high
MYC expression that possibly could be targeted by
MYC inhibition.
RESULTS
We have earlier shown that the small molecule
MYC inhibitor 10058-F4 induces apoptosis in myeloma
cell lines and primary cells. [17, 20] The inhibitor
downregulated MYC protein and mRNA in a dosedependent manner in myeloma cells (Supplemenatry
Figure 1A–1C). We wanted to find out if the baseline
MYC expression could determine myeloma cell
sensitivity to 10058-F4. A panel (n = 11) of myeloma cell
lines were treated with increasing amounts of inhibitor
for three days. The combined effects on cell proliferation
and viability were determined using CellTiter Glo which
measures the ATP content in cells (Supplementary
Figure 2). IC50 values were determined from doseresponse curves and related to MYC transcript numbers
measured by the nCounter Nanostring technology
(Figure 1A, Supplementary Figure 3A) and protein
levels using immunoblotting (Figure 1B, Supplementary
Figure 3B, 3C). There was a negative correlation
between IC50 values and mRNA (R2 = 0, 548) or protein
(R2 = 0, 585) levels. Taken together, the correlation
between MYC expression and sensitivity to the 10058F4 compound, supports that 10058-F4 is a relatively
specific inhibitor of MYC activity. Secondly, the finding
that the cell lines with the highest MYC concentration
were the most sensitive suggests that cell lines expressing
high levels of MYC are more dependent on the MYC
expression for proliferation or survival than cell lines
expressing lower amounts of MYC.
Next, we measured MYC gene copy numbers
in all 11 myeloma cell lines using PCR with primers
for exon 3 (Supplementary Figure 3D) and correlated
the copy numbers with MYC mRNA, as well as with
protein levels (Supplementary Figure 3A, 3B and 3C).
In cell lines, the MYC gene copy numbers varied from
two to nine. The measured copy numbers were almost
identical using primers that were specific for exon 1
and exon 2 (Supplementary Figure 3D), indicating the
presence of the whole gene rather than fragments of
the gene. Interestingly, the MYC gene copy numbers
correlated well with both mRNA (R2 = 0.847) and protein
(R2 = 0.607) levels (Figure 2A and 2B). The results thus
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Figure 1: MYC gene copy numbers determine expression of MYC mRNA and protein in myeloma cell lines. In a panel
of myeloma cell lines the levels of MYC gene copy numbers as measured by PCR was related to A. MYC mRNA measured using nCounter,
and B. MYC protein levels measured using immunoblotting and normalized to GAPDH. The slope and R2-values are shown in the plots.
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Figure 2: Expression of MYC in myeloma cell lines correlated positively with sensitivity to MYC inhibition. The IC50-
values of the MYC inhibitor 10058-F4 calculated from the results shown in Supplementary Figure 2 was compared with A. MYC mRNA
values or B. MYC/GAPDH relative protein levels. The slope and R2-values are shown in the plots.
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Figure 3: MYC and GAPDH mRNA levels in primary myeloma cells. A. The levels of MYC mRNA in myeloma patient
samples (n = 28), P01-P28, were measured using nCounter and the calculated mRNA copy numbers were plotted. Box plots comparing the
levels of MYC B. and GAPDH C. mRNA in 28 isolated plasma cells from myeloma patients with 11 myeloma cell lines. D. The levels of
MYC mRNA measured as in A were plotted for treated (n = 15) versus untreated (n = 13) patients in a box plot. The upper and lower borders
of the box indicate upper and lower quartile, whereas the line inside the box indicates the median value. Whiskers indicate upper and lower
adjacent values, whereas outside values are indicated by (o) and far out values are indicated by asterisks (*).
DISCUSSION
A major concern when applying small molecule
inhibitors is the specificity of the inhibitor. The 10058-F4
drug has been proposed to be a specific inhibitor of MYCMAX heterodimerization at concentrations < 100 μM.
[20, 21] However, even if 10058-F4 clearly inhibits the
MYC-MAX interaction, the compound may also affect
other molecules within a cell. The correlation between
MYC expression and sensitivity for the 10058-F4 drug
found in cell lines indicated that the activity of the drug
was specifically directed towards MYC activity. As MYC
is a factor that is functionally non-redundant it may be
particularly attractive as a therapeutic target. [22] MYC
could already indirectly be targeted in the clinic since
many drugs, such as dexamethasone and lenalidomide,
have been shown to kill myeloma cells concomitantly with
partial suppression of MYC, [23, 24] albeit, these drugs
clearly also have other effects.
We found higher MYC mRNA levels in myeloma
cell lines than in primary cells; although the range was
approximately the same in these two groups. Nevertheless,
gene copy number variations in MYC were only common
in cell lines, indicating that primary cells had other
ways of increasing MYC levels than cell lines. Indeed,
recent publications indicate that different rearrangements
involving MYC cause elevated MYC expression in nearly
half of newly diagnosed patients. [13–15] MYC expression
has been reported to affect cell proliferation and energy
metabolism in rapidly proliferating cancer cells. [7]
Amplification of the MYC gene seen in myeloma cell
lines may be a consequence of the selection pressure for
proliferation and survival in tissue culture flasks. Cell lines
The most important data presented here is that
there was a positive correlation between MYC expression
and sensitivity to the MYC activity inhibitor, 10058-F4,
in myeloma cell lines. That finding supports that MYC
is important for myeloma cell survival and that the
inhibitor specifically targets MYC. Secondly, although
both myeloma cell lines and primary cells expressed high
amounts of MYC, there are differences in the underlying
mechanisms behind the expression. And thirdly, MYC
expression was in the same range in plasma cells from
untreated compared to treated patients indicating that
MYC activation is an early oncogenic event in multiple
myeloma.
Cell lines expressing the highest levels of MYC
were the most sensitive to MYC-inhibition, indicating that
these cells were more dependent on MYC for survival and
proliferation than low-expressors. Thus, we hypothesize
that patients with myeloma cells expressing high levels
of MYC could benefit from MYC inhibition. MYC
protein and mRNA are very unstable and may be rapidly
degraded. When isolating primary myeloma cells from
bone marrow samples the procedure usually takes a few
hours and there is a risk that MYC protein and mRNA
levels are not maintained at the same levels as when the
cells are situated in the bone marrow. Thus, the most
reliable way to measure MYC levels in patients could be
by quantitative in situ immunohistochemistry using bone
marrow biopsies. Unfortunately, it was not possible for us
to perform such experiments in this study.
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are highly proliferating, whereas primary myeloma cells in
the bone marrow in general proliferate very slowly, with a
Ki-67 labelling index less than 5% for most patients. [25]
Amplification of the MYC gene may be a simple way of
achieving high MYC levels in cell lines that over years
have been selected for cell proliferation in vitro. Another
difference is that, unlike myeloma cell lines, primary
cells depend on the bone marrow microenvironment and
are unable to survive in tissue culture. There was also a
difference in GAPDH expression levels between cell
lines and primary cells that could reflect differences in
utilization of glycolysis for ATP production.
In patients, a negative relationship between MYC
expression and progression-free and overall survival has
been described, suggesting an important role for MYC in
the regulation of tumor mass. [14, 26–28] We compared
the MYC mRNA expression with clinical information
on progression-free and overall survival in our patient
samples, but could not find any relationship (data not
shown). However, the number of informative patients
was too low to draw any conclusions. Nevertheless, our
findings that MYC mRNA is as high in untreated patients
as in patients at later stages of disease suggest that MYC
may be important during the whole course of disease.
To summarize, our results suggest that many
myeloma patients, but first of all patients with cells
expressing high MYC levels, might benefit from MYC
inhibition. If a specific and clinically applicable MYCinhibitor became available, it would be important to
characterize which patients might benefit from inhibition
of MYC.
Norway) whereas KJON was maintained in 5% HS,
all in RPMI and IL-6 (2 ng/mL). Cells were cultured at
37oC in a humidified atmosphere containing 5% CO2. For
experiments 2% HS in RPMI was used as medium, with
IL-6 (1 ng/mL) added for all IL-6 dependent cell lines.
MATERIALS AND METHODS
Real-time quantitative reverse transcription
PCR (qRT-PCR)
Primary cells
Primary CD138+ myeloma cells were isolated
from bone marrow specimens included in the Norwegian
Myeloma Biobank using RoboSep automated cell
separator and Human CD138 Positive Selection Kit
(StemCell Technologies, Grenoble, France). The study
was approved by the Regional Ethics Committee (approval
# 2011/2029) and all patients had given informed consent.
Peripheral blood mononuclear cells (PBMC) were
obtained from EDTA-blood from healthy controls by
density gradient centrifugation using Lymphoprep (AxisShield, Oslo, Norway).
Nucleic acid extraction
Genomic DNA and total RNA were extracted from
frozen cell pellets using AllPrep DNA/RNA Micro Kit and
Qiacube (Qiagen, Venlo, the Netherlands) following the
manufacturer’s instructions. The concentration and quality
of DNA and RNA was determined using NanoDrop
spectrophotometer (Thermo Fisher Scientific, Waltham,
MA, USA), and samples were stored at −80°C until
further use.
Cell lines and reagents
Complementary DNA (cDNA) was synthesized
from total RNA using the High Capacity RNA-to-cDNA
kit (Applied Biosystems, Carlsbad, CA, USA). PCR was
performed using StepOne Real-Time PCR System and
Taqman Gene Expression Assays (Applied Biosystems).
The comparative Ct method was used to estimate relative
changes in gene expression using MYC Taqman assay
(Hs00153408_m1) and GAPDH (Hs99999905_m1) as
housekeeping gene.
A panel of 11 myeloma cell lines was used. Four
of the cell lines were in-house: OH-2, IH-1, URVIN and
KJON, whereas 7 were from other sources: INA-6, CAG,
JJN3 and ANBL-6 were kind gifts from Dr M. Gramatzki
(University of Erlangen-Nurnberg, Erlangen, Germany),
Dr J. Epstein (University of Arkansas for Medical
Sciences, Little Rock, AR, USA), Dr J. Ball (University
of Birmingham, UK), and Dr D. Jelinek (Mayo Clinic,
Rochester, MN, USA), respectively, KMS-12-BM was
obtained from DSMZ (Braunschweig Germany), and
RPMI-8226 and U266 were from ATCC (Rockville, MD,
USA). URVIN, INA-6 and ANBL-6 cells were grown in
10% heat inactivated fetal calf serum (FCS) in RPMI-1640
(RPMI) supplemented with interleukin (IL)-6 (1 ng/mL)
(Biosource, Camarillo, CA, USA). CAG, JJN3, KMS12-BM, RPMI-8226 and U266 were grown in RPMI
with 10, 10, 20, 20 or 15% FCS, respectively. OH-2 and
IH-1 were maintained in 10% heat-inactivated human
serum (HS) (Department of Immunology and Transfusion
Medicine, St. Olav’s University Hospital, Trondheim,
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Copy number variation (CNV)
Real-time reverse transcriptase quantitative
polymerase chain reaction (real-time qPCR) was
performed using the TaqMan Copy Number Assay
Hs01764918_cn (MYC, exon 3), Hs00834648_cn
(MYC, exon 2), Hs02758348_cn (MYC, exon 1), and
TaqMan Copy Number Reference Assay (RNAse P) for
internal control (Applied Biosystems). All PCR reactions
were performed in triplets with genomic DNA using a
StepOnePlus PCR system (Applied Biosystems). CNV was
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Statistical analysis
analyzed using CopyCaller Software (Applied Biosystems)
and DNA isolated from PBMC was used for calibration.
Statistical analysis and box plots were made in IBM
SPSS Statistics 20 (IBM Corp., Armonk, NY, USA).
Gene expression analysis
ACKNOWLEDGMENTS
For mRNA transcript counting the nCounter
Human Cancer Reference Kit (cat.no GXA-CR1–12)
and nCounter Technology (Nanostring Technologies,
Seattle, WA, USA) was used. The standard mRNA Geneexpression experiment protocol provided by Nanostring
was used, the only exception being that kit probes were
diluted 1:2. Briefly, 100 ng total RNA from myeloma
cell lines or patient samples was hybridized with reporter
probes overnight at 65°C. The nSolver Analysis Software
(Nanostring) was used for calculations of transcript
numbers. Sample data was normalized against internal kit
positive controls and the following housekeeping genes:
CLTC, GAPDH, GUSB, HPRT1, PGK1, and TUBB.
The authors are grateful for technical help provided
by Lill-Anny Gunnes Grøseth, Solveig Kvam, Berit
Størdal and Hanne Hella.
FUNDING
This work was funded by grants from the
Norwegian Cancer Society, the Liaison Committee
between the Central Norway Regional Health Authority
and the Norwegian University of Science and Technology,
and the KG Jebsen Foundation for Medical Research.
The funding sources did not participate in study design,
collection, analysis or interpretation of the data, writing of
the paper or deciding to submit the paper for publication.
Immunoblotting
Cells were treated as indicated, washed with icecold phosphate-buffered saline (PBS) and lysed for
30′ on ice. The lysis buffer contained 1% NP40 (SigmaAldrich, St Louis, MO, USA), 150 mM NaCl, 50 mM
Tris-HCl (pH 7.5), a protease inhibitor cocktail (Roche,
Basel, Switzerland), 1 mM Na3VO4 and 50 mM NaF.
Samples were electrophoresed on pre-cast agarose gels
and blotted onto nitrocellulose membranes using the
NuPAGE system (Life Technologies, Carlsbad, CA,
USA). The membranes were blocked with 5% nonfat
dry milk in Tris-buffered saline with 0.01% Tween
20 (TBS-T). The primary antibodies used were c-MYC
(RRID: AB_2148606, Cat# 551102, BD Biosciences,
Trondheim, Norway), and GAPDH (RRID:AB_2107448,
Cat# Ab8245, Abcam, Cambridge, UK). The secondary
antibody was horseradish peroxidase-conjugated goatanti-mouse (Dako Cytomation, Glostrup, Denmark).
Positive bands were detected using the luminescence
substrate SuperSignal West Femto (Thermo Fisher
Scientific) and Odyssey Fc imager (LI-COR Biosciences,
Ltd., Cambridge, UK).
CONFLICTS OF INTEREST
The authors state no conflicts of interest.
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www.impactjournals.com/oncotarget
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W4225879952.txt | bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
1
Asymmetric effects of acute stress on cost and benefit learning
2
Stella Voulgaropoulou1*, Fasya Fauzani1, Janine Pfirrmann1, Claudia Vingerhoets1, 2, Thérèse
3
van Amelsvoort1, Dennis Hernaus1
4
5
1
6
4-6, 6211 LK Maastricht, The Netherlands
7
2
8
Location AMC, Meibergdreef 5, 1105 AZ Amsterdam, The Netherlands.
Department of Psychiatry & Neuropsychology, Maastricht University, Minderbroedersberg
Department of Radiology & Nuclear Medicine, Amsterdam University Medical Centre,
9
10
Abstract
11
Stressful events trigger a complex physiological reaction – the fight-or-flight response – that
12
can hamper flexible decision-making. Inspired by key neural and peripheral characteristics of
13
the fight-or-flight response, here we ask whether acute stress changes how humans learn
14
about costs and benefits. Participants were randomly exposed to an acute stress or no-stress
15
control condition after which they completed a cost-benefit reinforcement learning task.
16
Acute stress improved learning to maximize benefits (monetary rewards) relative to
17
minimising energy expenditure (grip force). Using computational modelling, we demonstrate
18
that costs and benefits can exert asymmetric effects on decisions when prediction errors that
19
convey information about the reward value and cost of actions receive inappropriate
20
importance; a process associated with distinct alterations in pupil size fluctuations. These
21
results provide new insights into learning strategies under acute stress – which, depending on
22
the context, may be maladaptive or beneficial - and candidate neuromodulatory mechanisms
23
that could underlie such behaviour.
1
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
24
Introduction
25
Stress is ubiquitous in everyday life. From recurrent, brief, events (a work meeting, moving
26
to a new house) to major life events (armed combat, pandemic, financial crisis), humans are
27
continuously exposed to challenges in their daily environment. The immediate central and
28
peripheral physiological cascade triggered by such events, collectively termed the fight-or-
29
flight (or acute stress) response (Cannon, 1915), serves an allostatic role that enables
30
organisms to adequately respond to environmental demands (de Kloet, Joëls, & Holsboer,
31
2005). Although beneficial for survival, this allostatic process comes at a cost: stress-induced
32
redistributions of neural resources - e.g., towards vigilance or threat detection - may hamper
33
the deployment of strategies that support adaptive and optimal decision-making (Hermans,
34
Henckens, Joëls, & Fernández, 2014).
35
Optimal decisions essentially depend on the ability to rapidly learn from the positive
36
and negative outcomes of previous actions, also known as reinforcement learning (Niv,
37
2009). Considerable evidence now suggests that acute stress impairs aspects of reinforcement
38
learning (Carvalheiro, Conceição, Mesquita, & Seara-Cardoso, 2020; de Berker et al., 2016;
39
Raio, Hartley, Orederu, Li, & Phelps, 2017). Acute stress, among others, modulates the
40
impact of positive outcomes on future decisions - both positively and negatively - (Berghorst,
41
Bogdan, Frank, & Pizzagalli, 2013; Carvalheiro et al., 2020; Lighthall, Gorlick, Schoeke,
42
Frank, & Mather, 2013; Petzold, Plessow, Goschke, & Kirschbaum, 2010), likely driven by
43
changes in reward sensitivity and the signalling of reward prediction errors (RPEs)
44
(Berghorst et al., 2013; Carvalheiro et al., 2020; Huys, Pizzagalli, Bogdan, & Dayan, 2013);
45
putatively dopaminergic teaching signals that represent the mismatch between actual and
46
expected outcomes, which are used to flexibly adjust behaviour (Niv, 2009; Rescorla, 1972).
47
Alterations in the influence of RPEs on future decisions play a key role in the development of
2
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
48
motivational impairments, which are frequently observed in behavioural disorders associated
49
with repeated and/or prolonged stress exposure (Huys et al., 2013).
50
Intuitive as it is, the notion that the impact of acute stress on (potentially maladaptive)
51
decisions primarily involves changes in how reward value influences action may be
52
oversimplified. Decisions are not only motivated by appetitive properties; they equally
53
depend on the – cognitive (e.g., mental effort) or physical (e.g., energy) – cost associated
54
with actions (Hauser, Eldar, & Dolan, 2017; Pessiglione, Vinckier, Bouret, Daunizeau, & Le
55
Bouc, 2017; Schmidt, Lebreton, Cléry-Melin, Daunizeau, & Pessiglione, 2012). Expectations
56
about action costs are also updated according to a prediction error rule (Skvortsova, Degos,
57
Welter, Vidailhet, & Pessiglione, 2017; Skvortsova, Palminteri, & Pessiglione, 2014)
58
(henceforth “effort” prediction errors; EPEs), which due to the aversive and resource-
59
consuming nature of effort, optimal learners should utilize to minimize effort expenditure.
60
When decisions involve a potential cost and benefit, the former is subtracted from the latter
61
to compute a “net” or subjective decision value (i.e., effort-discounted reward value) (Klein-
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Flügge, Kennerley, Friston, & Bestmann, 2016; Skvortsova et al., 2017; Skvortsova et al.,
63
2014). Notably, stress exposure impairs cost-benefit decisions in rodents when learning is not
64
explicitly required (Friedman et al., 2017; Shafiei, Gray, Viau, & Floresco, 2012). Moreover,
65
in a reinforcement learning context, acute stress blocks the flexible updating of aversive
66
value (Raio et al., 2017), an inherent property of costly actions. These results suggest that
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decisions during acute stress may involve a complex shift in reinforcement learning strategies
68
that serve to balance the cost versus benefits of decisions; a hypothesis that hitherto has
69
remained unexplored.
70
Although computationally similar in nature, distinct neural correlates of RPEs (e.g.,
71
striatal subdivisions, ventromedial prefrontal cortex [vmPFC]) and EPEs (e.g., parietal
72
cortex, insula, dorsomedial PFC) can be observed in cost-benefit reinforcement learning
3
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
73
paradigms (Hauser et al., 2017; Skvortsova et al., 2014). The ascending dopaminergic (e.g.,
74
RPEs, action cost, reward value) (Schultz, Dayan, & Montague, 1997; Skvortsova et al.,
75
2017; Yohn et al., 2016), noradrenergic (e.g., mobilizing energy) (Pessiglione et al., 2017;
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Varazzani, San-Galli, Gilardeau, & Bouret, 2015) and serotonergic (e.g., aversive value,
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overcoming action costs) (H. E. den Ouden et al., 2015; Meyniel et al., 2016)
78
neuromodulatory systems, moreover, encode partly dissociable aspects of goal-directed
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actions that involve learning about costs and benefits, which together support optimal
80
decision-making. These observations are noteworthy because the initial fight-or-flight
81
response triggers a large-scale reorganization of brain networks that is driven by alterations in
82
the firing mode of midbrain dopaminergic ventral tegmental area and noradrenergic locus
83
coeruleus neurons (Arnsten, 2015; Hermans et al., 2014); neurons that signal prediction
84
errors (Steinberg et al., 2013) and that are also responsive to reward value, action cost and
85
energy expenditure (Del Arco, Park, & Moghaddam, 2020; Varazzani et al., 2015). Thus,
86
catecholaminergic mechanisms that are recruited by the fight-or-flight response may
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differentially impact cost and benefit reinforcement learning, resulting in a potential scenario
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in which costs and benefits exert asymmetric influences on decisions.
89
As mentioned above, the central (i.e., neural) effects of acute stress trigger a shift in
90
cognitive strategies, including reinforcement learning. The peripheral counterpart of the acute
91
stress response, however, mobilizes the energy (i.e., adrenaline-mediated glucose release (de
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Kloet et al., 2005; Russell & Lightman, 2019)) that is required to exert effortful actions
93
aimed at preserving homeostasis (Cannon, 1915). Therefore, decision-making and learning
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policies regarding physical costs may be especially susceptible to stress: both via
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computational (neural) mechanisms that support learning about and representation of action
96
cost, as well as peripheral mechanisms that co-determine the amount of available energy that
97
can be directed towards effortful actions. Indeed, preliminary evidence suggests that acute
4
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
98
stress alters the willingness to exert physical effort for rewards (Bryce & Floresco, 2016) and
99
reward-associated cues in a Pavlovian-instrumental transfer context (Pool, Brosch,
100
Delplanque, & Sander, 2015).
101
How acute stress impacts reinforcement learning involving costs and benefits has not
102
been investigated to date in humans. Based on the above considerations, we expect that
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computationally frugal learning strategies, in concert with increased energy availability,
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during acute stress should asymmetrically impact cost versus benefit learning. Using an acute
105
stress-induction paradigm, a cost-benefit learning paradigm and computational model of cost-
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benefit reinforcement learning (Skvortsova et al., 2017; Skvortsova et al., 2014), we
107
demonstrate that acute stress asymmetrically prioritizes reward (maximization) learning over
108
physical effort (minimization) learning. Better benefit versus cost learning results from a
109
stress-induced change in the influence of RPEs versus EPEs on future decisions, and is
110
associated with altered pupil encoding of RPEs, EPEs, and subjective decision value. These
111
results reveal how neural and peripheral mechanisms that support the fight-or-flight response
112
may facilitate a shift in reinforcement learning strategies that confers strategic benefits during
113
acutely stressful situations (e.g., ignoring high action costs to achieve a desirable outcome),
114
yet might also give rise to maladaptive behaviour (e.g., stress-induced relapse in substance
115
users).
5
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
116
Results
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Experiment design
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Healthy human participants were randomly assigned to the acute stress (19 males/21 females;
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age M=23.48, SD=3.94) or no-stress control condition (18 males/22 females; age M=23.80,
120
SD=4.23) of the Maastricht Acute Stress Task (MAST) (Smeets et al., 2012), a validated
121
psychological and physical stress-induction paradigm (see Materials and Methods).
122
Immediately post-MAST and within the confines of the acute stress response (Hermans et al.,
123
2014), all participants completed a ~40 minute probabilistic cost-benefit reinforcement
124
learning paradigm, adapted from Skvortsova et al. (Skvortsova et al., 2017; Skvortsova et al.,
125
2014), in which they learned to select stimuli with high reward value (20 Eurocents) and
126
avoid stimuli with high action cost (exerting grip force above a pre-calibrated individual
127
threshold of 50% maximum voluntary contraction for 3000ms), followed by a surprise test
128
phase. A detailed overview of the paradigm is provided in Figure 1 and the Materials and
129
Methods. Pupil size was continuously recorded while participants performed the task (see
130
Materials and Methods).
6
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
131
Figure 1
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133
Reward maximisation/action cost minimization reinforcement learning task.
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Visual depiction of the learning phase. Participants were presented with four distinct stimulus
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pairs, and all stimuli were associated with a predetermined chance of a €0.20 monetary
136
reward (versus no reward) and a chance of having to exert physical effort (grip force) using a
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dynamometer (versus no grip force required). Stimulus-outcome probabilities were yoked in
138
such a way that, for a given pair, two stimuli only differed in the probability of earning a
139
reward (“reward learning”, RL, left) or the probability of having to exert effort (“effort
140
learning”, EL, right). That is, for RL (left)/EL (right) pairs, reward/effort outcomes were
141
choice-dependent, respectively (see “Reward feedback” for RL and “Effort feedback” for EL
142
for outcome contingencies). For RL pairs, effort outcomes were independent of choice and
143
fixed (see “Effort feedback” for RL), while for EL pairs, reward outcomes were independent
7
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
144
of choice and fixed (see “Reward feedback” for EL). Percentages in blue and red refer to
145
outcomes for the Easy RL/EL and Hard RL/EL pair, respectively.
146
147
Acute stress manipulation
148
We first ascertained whether the acute stress manipulation was successful. Subjective stress,
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physiological and neuroendocrine measurements are displayed in Figure 2. Acute stress and
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no-stress control groups did not differ on physiological, subjective stress, or neuroendocrine
151
measurements pre-MAST (all p-values>0.05). We observed significant Condition-by-Time
152
interactions for subjective stress ratings [PANAS negative: F(1,78)=52.66, p<0.001,
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n2G=0.10; PANAS positive: F(1,78)=9.82 p=0.002, n2G=0.02] and physiological measures
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[systolic blood pressure (SBP): F(1,78)=15.50, p<0.001, n2G= 0.04; heart rate: F(1,78)=6.83,
155
p=0.011, n2G= 0.02]. Simple main effect analyses revealed that only the acute stress group
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exhibited pre-to-post increases in negative affect [control pre-post: t(39)=4.21, p<0.001;
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stress pre-post: t(39)=-6.17, p<0.001; control-stress post-MAST: t(55.1)=-5.78, p<0.001],
158
and greater pre-to-post decreases in positive affect [control pre-post: t(39)=4.09, p<0.001;
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stress pre-post: t(39)=6.45, p<0.001; control-stress post-MAST: t(72.8)=2.53, p=0.014]
160
(Figure 2). Similarly, only the acute stress group exhibited stress-induced increases in SBP
161
[control pre-post: t(39)=1.60, p<0.117; stress pre-post: t(39)=-3.66, p<0.001; control-stress
162
post-MAST: t(69.1)=-3.27, p=0.002] and heart rate [control pre-post: t(39)=1.21, p=0.234;
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stress pre-post: t(39)=-2.78, p=0.008; control-stress post-MAST: t(76.9)=-3.14, p=0.002]
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(Figure 2a-d).
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An expected Condition-by-Time interaction was found for salivary cortisol (sCORT)
166
responses [F(5,390)=18.05, p<0.001, n2G= 0.04], with the acute stress group displaying
167
greater sCORT levels 10 min post-MAST and onwards (all p-values<0.01). We additionally
168
observed a main effect of Condition on sCORT area-under-the-curve with respect to increase:
8
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
169
(AUCi) (Pruessner, Kirschbaum, Meinlschmid, & Hellhammer, 2003) (t(56.32)=-5.28,
170
p<0.001) and salivary alpha-amylase (sAA) AUCi (t(67.45)=-2.50, p=0.015; after excluding
171
one extreme outlier from the control group), suggesting greater sCORT and sAA levels in
172
response to acute stress (Figure 2e-g). These results confirm that the MAST robustly induced
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stress on all levels of inquiry.
174
175
Figure 2
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177
Neuroendocrine, physiological and subjective stress ratings.
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PANAS negative (a) and positive (b) subscale sum scores, systolic blood pressure (mmHg:
179
millimetres of mercury; c) and heart rate (bpm: beats per minute; d) are displayed for no-
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stress control (blue) and acute stress (red) groups separately for pre (light blue/red) and post
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(dark blue/red) MAST time points. SCORT responses for both conditions across the 6
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timepoints are displayed in panel e (“t+00” represents the first post-MAST measurement, and
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the start of the reward maximization/action cost reinforcement learning paradigm; “t-10”
184
represent a baseline sample). Panel f and g show AUCi for sCORT (nmol/l: nanomoles per
185
litre) and sAA (U/mL: Units per millilitre) responses for both MAST conditions. Significant
9
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
186
differences are denoted by asterisks (*: p < 0.05, **: p < 0.01, ***: p < 0.001). In the upper
187
panel, the top line denotes a significant Condition-by-Time interaction; lower lines represent
188
simple main effects of Condition or Time.
189
190
Participants use reinforcement learning to optimize decisions
191
Next, we investigated whether participants in both conditions exhibited evidence of
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reinforcement learning to optimize actions, which in this paradigm should be reflected by an
193
increased tendency to select stimuli with high reward value and avoid stimuli with high
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action cost as a function of increasing number of stimulus pair presentations (i.e., “time”).
195
This intuition was confirmed by a main effect of Time on two distinct trial types: trials on
196
which participants could learn to accumulate frequent rewards while the probability of effort
197
(action cost) was kept constant (RL; selecting the stimulus more frequently associated with
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€0.20) [control: F(2,78)=10.16, p<0.001, n2G= 0.06; stress: F(2,78)=20.44, p<0.001, n2G=
199
0.17] and trials on which participants could learn to frequently avoid effort while the
200
probability of reward was kept constant (EL; selecting the stimulus more frequently
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associated with avoidance of physical energy expenditure) [control: F(2,78)=12.35, p<0.001,
202
n2G= 0.07; stress: F(2,78)=9.76, p<0.001, n2G= 0.05]. We additionally observed greater than
203
chance-level performance (≥0.5) on both trial types during the final part of the task
204
(presentation 21-30; all p-values<0.001 Figure Supplement 1).
205
206
Asymmetric cost-benefit reinforcement learning during acute stress
207
After having observed evidence for reward (maximization) learning and action cost
208
(minimization) learning, we tested our key assumption; that acute stress would induce a
209
reprioritization in learning to maximize reward value versus learning to minimize action cost.
210
Crucially, we observed a significant Condition-by-Trial Type interaction [F(1,78)=6.53,
10
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
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p=0.013, n2G= 0.039] (Figure 3a) with pairwise comparisons indicating that the acute stress
212
group performed significantly better on RL than EL trials [t(39)=5.40, p<0.001], while the
213
no-stress control group performed similarly on both trial types [t(39)=1.01, p=0.320]. A main
214
effect of Condition on RL-EL accuracy difference scores [t(74.02)=-2.55, p=0.013] (Figure
215
3b) and one-sample t-tests revealed that RL-EL accuracy difference scores were significantly
216
greater than zero in the acute stress group but not in the no-stress controls. Simple main
217
effects of Condition on RL [t(65.9)=-1.75, p=0.085] and EL [t(77.5)=1.80, p=0.076]
218
performance showed numerical trends for group differences that failed to reach significance.
219
When we included participants that still performed at chance level at the end of the
220
learning phase (see Participants) in the Condition-by-Trial Type interaction analysis, the
221
interaction remained significant [F(1,91)=7.30, p=0.035, n2G= 0.04], with the acute stress
222
group displaying better RL vs. EL performance (t(46)=5.83, p<0.001), while no-stress
223
controls performed similarly on both trial types (t(45)=1.24, p=0.22). Participants in the acute
224
stress group outperformed participants in the no-stress control group on RL (t(91)=2.04,
225
p=0.04), but no simple main effect of Condition was observed for EL (t(91)=-1.67, p=0.1).
226
These participants were excluded for all other analyses reported below.
227
The use of different reinforcement probabilities for each RL and EL pair (Figure 1)
228
allowed us to discern whether the observed pattern of results reflected a specific change in
229
cost (EL) versus benefit (RL) reinforcement learning, or a more general impairment in
230
reinforcement learning for more difficult stimulus-response associations. We found no
231
evidence for the latter scenario in Condition-by-Trial Type-by-Difficulty [F(1,78)=1.05,
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p=0.310] or Condition-by-Difficulty [F(1,78)=0.36, p=0.549; Figure Supplement 2]
233
interaction analyses.
234
235
When we investigated the use of win-stay (resampling a stimulus following a positive
outcome) and lose-shift (switching to the other stimulus following a negative outcome)
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made available under aCC-BY 4.0 International license.
236
strategies (Hanneke E. M. den Ouden et al., 2013), we observed no significant Condition-by-
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Strategy interaction [F(1,78)=2.99, p=0.087, n2G= 0.03]. However, post hoc comparisons
238
revealed that participants in the acute stress condition [t(39)=-3.73, p<0.001] but not those in
239
the no-stress control condition [t(39)=-1.30, p=0.200], exhibited different win-stay compared
240
to lose-shift (difference) rates. Separate Condition (main effect) analyses indicated that acute
241
stress participants compared to no-stress controls were more likely to win-stay for rewards
242
(RL trials) than for avoidance of action cost (EL trials) [t(78)=-2.28, p=0.025], but not for
243
lose-shifting for reward omissions (RL trials) compared to exerting effort (EL trials)
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[t(77.7)=-0.23, p=0.820]. Differences in win-stay rates for RL compared to EL trials (one-
245
sample t-test) were greater than zero for the acute stress [t(39)=4.91, p<0.001] but not the no-
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stress control group [t(39)=1.68, p=0.101] (Figure 3c).
247
Taken together, our model-free results indicate that acute stress leads to a
248
reinforcement learning strategy that favours learning to maximise reward value over
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minimisation of action cost, which based on analyses of win-stay/lose-shift rates, could be
250
attributed to increased sensitivity to positive reinforcement (i.e., reward delivery) compared
251
to negative reinforcement (i.e., avoidance of physical effort).
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made available under aCC-BY 4.0 International license.
252
Figure 3
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254
Acute stress leads to improved benefit versus cost learning.
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Panel a: Average accuracy (choices of the optimal stimulus) for RL and EL trials, for each
256
condition separately. Panel b: RL-EL average accuracy difference scores. Panel c:
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Win-stay (WSRL-WSEL) and lose-shift (LSRL-LSEL) difference scores for each condition
258
separately. Means ± SD, individual data points, distribution and frequency of the data are
259
displayed. In panel a, the top line indicates a significant Condition-by-Trial type interaction.
260
Significant differences are denoted by asterisks (*: p < 0.05, **: p < 0.01, ***: p < 0.001).
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Source files of task performance data used for the analyses are available in the Figure 3 –
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Source Data 1.
263
264
Asymmetric cost-benefit reinforcement learning biases actions in acute stress subjects
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During a surprise 64-trial test phase (D. Hernaus, Gold, Waltz, & Frank, 2018), we asked
266
participants to discriminate original and novel combinations of stimuli on the basis of reward
267
value or action cost without receiving feedback (n=16 trials for original combinations; n=48
268
for novel combinations; see Materials and Methods). The surprise test phase allowed us to
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
269
assess learned choice tendencies without having to arbitrarily choose a given number of final
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learning phase trials, during which participants may still learn. This approach also allowed us
271
to assess the degree to which learned tendencies would carry over to novel contexts.
272
First, both groups chose the optimal (most rewarding/effort avoiding) stimulus on
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surprise test phase trials involving the original four pairs [one-sample t-test against chance;
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controlRL: t(39)=8.73, p<0.001; controlEL: t(39)=3.72, p=0.002; stressRL: t(39)=13.54,
275
p<0.001; stressEL: t(39)=4.47, p<0.001], confirming that both groups had developed a
276
preference for the optimal stimulus.
277
Although we observed no Condition-by-Trial type (reward value, action cost
278
discrimination) interaction or main effects of Condition for novel stimulus combinations
279
[F(1,78)=1.10, p=0.298, n2G= 0.01; stress vs. controls reward value discrimination: t(75.9)=
280
0.15, p=0.878; stress vs. controls action cost discrimination: t(78)= 1.77, p=0.080], pairwise
281
comparisons revealed that the acute stress group performed better on reward discrimination
282
compared to action cost discrimination trials [t(39)=-2.23, p=0.032], while no-stress controls
283
performed similarly on both trial types [t(39)=-0.87, p=0.387]. These results provide some
284
evidence that a reward maximisation-over-action cost minimisation reinforcement learning
285
policy might bias future actions in novel contexts (Figure Supplement 3).
286
287
Computational cost-benefit reinforcement learning model: Model Fitting, Selection,
288
Demonstrations, and Simulations
289
To uncover latent mechanisms by which acute stress affects cost-benefit reinforcement
290
learning, we turned to computational cognitive modelling. Trial-by-trial choices of
291
participants were fit to all candidate models described in the Materials and Methods (see
292
Model Space). To calculate model fit, log likelihood was updated trial-wise by the log of the
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probability of the observed choice, calculated via a softmax rule (see Materials and Methods,
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
294
equation 6), and best-fitting parameters were identified using fmincon in MATLAB v.2019B
295
(Mathworks, Natick, MA, USA).
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Bayesian Model Selection (BMS; spm_BMS function in SPM12,
297
http://www.fil.ion.ucl.ac.uk/spm/software/spm12/) using the Akaike Information Criterion
298
(AIC) as a fit statistic that penalizes for the number of model parameters (Myung, Tang, &
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Pitt, 2009), suggested that the 2LR_γ model was the most likely model, as indicated by the
300
protected exceedance probability (pxp, φ = 0.99) (Rigoux, Stephan, Friston, & Daunizeau,
301
2014) and expectation of the posterior [p(r|y), 0.70] (Figure 4a for p(r|y) of all candidate
302
models). We note that 2LR_γ remained the most likely model when we considered additional
303
models with greater redundancy and/or lesser biological plausibility (e.g., models with all
304
combinations of reward value/action cost discounting and weight parameters).
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The 2LR_γ model contains separate learning rates that weight the importance of RPEs
306
and EPEs (αR, αE), an action cost discounting parameter (γ), and an inverse temperature
307
parameter (β), which in previous work could account for performance on a conceptually
308
similar cost-benefit learning task (Skvortsova et al., 2014). To demonstrate the effect of
309
changes in parameters values on choice preferences within the 2LR_γ architecture, we first
310
simulated choices from 50 artificial agents (averaged across 10 repetitions) performing the
311
reward maximization/action cost minimization reinforcement learning task using a range of
312
parameter values. As expected, greater values of αR and αE primarily impacted the speed of
313
RL and EL choice preferences, while low values of γ lead to asymmetric choice preferences
314
through discounting of action cost, and lower values of β lead to non-selective increases in
315
random sampling (Figure 4b).
316
In post hoc simulations, i.e., generating participant choices using the obtained
317
parameters, we additionally observed moderate-to-high correlations between simulated and
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empirical RL/EL for the acute stress and no-stress control group [ρRL_control = 0.55, p < 0.01;
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made available under aCC-BY 4.0 International license.
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ρRL_stress = 0.84, p < 0.01; ρEL_control = 0.56, p < 0.01; ρEL_stress = 0.77, p < 0.01; see Figure
320
Supplement 4], although the canonical performance difference in RL versus EL accuracy was
321
not selective to the acute stress group [tcontrol(39)=-6.72, p<0.001; tstress(39)=-6.01, p<0.001].
322
However, after we fixed β and γ to group-level averages, to better demonstrate the effect of
323
group differences in the learning rate parameters, we recovered a small but significant
324
simulated difference in RL versus EL performance for the acute stress group [t(39)=2.27,
325
p=0.029], which was not predicted in the no-stress control group [t(39)=0.91, p=0.367]
326
(Condition-by-Trial Type interaction: [F(1,78)= 0.77, p=0.38, n2G= 0.006]) (Figure 4c for
327
empirical versus simulated data, averaged across 100 repetitions per subject).
328
Importantly, even if a given model is the most likely one based on model fitting and
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post hoc simulation results from the entire sample, there is still the possibility that different
330
models can better explain task performance in the no-stress control and acute stress
331
condition. When repeating BMS for each condition separately, 2LR_γ was the most likely
332
model in the no-stress control group [φ=0.99, p(r|y)=0.83], while for acute stress subjects
333
2LR_γ was not convincingly the most likely model [φ=0.47 p(r|y)=0.46]. Here, the 2LR
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model (containing αR, αE, and β parameters) was equally likely to be the optimal model
335
[φ=0.53 p(r|y)=0.47]. Post-hoc simulations from the 2LR model also correlated with actual
336
data, both for no-stress control [ρRL = 0.65, p < 0.001; ρEL = 0.60, p < 0.001] and acute stress
337
participants [ρRL = 0.81, p < 0.001; ρEL = 0.75, p < 0.001].
338
Similar to the 2LR_ γ model (results discussed in next section), the 2LR model
339
seemingly also explained stress-induced changes in cost-benefit reinforcement learning via
340
changes in learning rates; in the 2LR model, the acute stress group exhibited greater values of
341
αR versus αE (t(39)=2.65, p=0.01), while no-stress control subjects did not (t(39)=0.69,
342
p=0.50) (Condition-by-Learning Rate interaction: [F(1,78)=2.88, p=0.094, n2G= 0.01]. The
343
difference in learning rates between 2LR_γ (where αR and αE are similar for the acute stress
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
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group, see next section) and 2LR (where αR > αE for the acute stress group) can be explained
345
by the absence of discounting parameter γ: 2LR is a special case of 2LR_γ, where γ=1, and
346
thus asymmetric effects of acute stress on reward value maximization and action cost
347
minimization can only be explained by dissimilarity in learning rates.
348
Although the effects of acute stress on reward value and action cost learning rates are
349
opposite in 2LR_ γ versus 2LR architectures, these results bolster our confidence in the
350
overall model space, as well as the interpretation that acute stress primarily impacts reward
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value and action cost learning rates, and not discounting. The observations that I) 2LR_ γ fit
352
better in the entire group of participants, II) 2LR is fully contained within the 2LR_ γ model,
353
and III) 2LR_ γ displayed good recoverability (see below) motivated our choice to focus on
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the 2LR_γ model.
355
In model recoverability analyses i.e., re-fitting the simulated data from the model to
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all candidate models (Wilson & Collins, 2019), BMS confirmed that the simulated 2LR_γ
357
data (that is, simulations without fixed parameters) were most likely to be generated from
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2LR_γ [φ=0.99, p(r|y)=0.71].
359
To assess the stability of 2LR_γ parameters, we repeated model fitting using a
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Bayesian hierarchical model fitting approach consisting of two steps, as described previously
361
(Daw, 2011; Frey, Frank, & McCabe, 2019). In the first step we fit the 2LR_γ model to trial-
362
wise choices to obtain subject-specific parameters; in a second step we again fit the model to
363
trial-wise choices, but this time we used the group-level average and covariance matrix of
364
every parameter as priors, thereby shrinking the parameter search space. Motivated by recent
365
work showing that group-specific priors, compared to a single prior for the entire sample, can
366
better account for between-group differences in task performance, as well as improve
367
parameter robustness and recoverability (Valton, Wise, & Robinson, 2020), we used separate
368
mean and covariance matrices for the acute stress and no-stress control groups.
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
369
Highly similar parameter estimates were obtained after hierarchical fitting (for
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parameter estimates after Bayesian hierarchical model fitting see Figure Supplement 5).
371
Similar to post hoc simulations using parameters from the non-hierarchically fit 2LR_γ
372
model, we observed moderate-to-high correlations between empirical and simulated data
373
using parameters obtained from the hierarchically fit model [ρRL_control = 0.65, p < 0.01;
374
ρRL_stress = 0.84, p < 0.01; ρEL_control = 0.37, p =0.02; ρEL_stress = 0.78, p < 0.01; see Figure
375
Supplement 6]. All in all, these results confirm parameter stability within the 2LR_γ
376
architecture.
377
In light of model fitting results, post-hoc simulations, model and parameter
378
recoverability analyses, we used parameters and trial-by-trial predictions of the non-
379
hierarchically fit 2LR_ γ model in all analyses reported below.
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made available under aCC-BY 4.0 International license.
380
Figure 4
381
382
Model selection, demonstrations, and post hoc simulations of the winning model.
383
Panel a: Expectation of the posterior for all candidate models. Panel b: Model
384
demonstrations. To demonstrate how different parameter values within the 2LR_γ
385
architecture impact choice preferences for the optimal stimulus (“accuracy”), αR, αE, and γ
386
were set to 0.01/0.33/0.66/0.99, while β, a non-linear parameter, was set to 0.1/5/10/20.
387
Parameter effects were always demonstrated for a single parameter (columns), while all other
388
parameter values were kept constant (αR and αE=0.25, γ=1, β=25). Greater values of αR and
389
αΕ selectively increase the speed with which the agent develops a preference for the optimal
390
RL and EL stimulus, respectively. Lower values of γ produce an asymmetric decision-
391
making policy that emphasises reward value over action cost, leading to better performance
392
on RL versus EL trials, while greater values of γ correct this asymmetric choice bias. Finally,
393
greater β values lead to more deterministic sampling of optimal stimuli. Panel c: Post-hoc
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
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simulations after fixing β and γ to group-level averages. Coloured lines represent mean ± SD.
395
Dashed lines denote chance level (0.5). *: p < 0.05, **:p < 0.01, ***: p < 0.001.
396
397
Acute stress selectively reduces the difference between reward and action cost learning
398
rates
399
Comparing 2LR_γ parameters between conditions, we observed a significant Condition-by-
400
Learning Rate (αR, αE) interaction [F(1,78)= 6.42, p=0.01, n2G= 0.03; 95% highest density
401
interval (HDI) for Bayesian mixed ANOVA = -0.405 to -0.023, mean= -0.219]. with greater
402
EPE relative to RPE learning rates in no-stress control participants [t(39)=-4.75, p<0.001],
403
while learning rates in the acute stress group did not significantly differ [t(39)=-1.61,
404
p=0.116]. No between-group differences in αR and αE or in the other parameters (γ, β) were
405
observed (all p-values>0.05) (Figure 5).
406
Paradoxically, symmetric reward value and action cost learning rates in the presence
407
of lower values of γ will lead to more efficient RL compared to EL. This is because lower
408
values of γ bias decisions towards reward value (via greater discounting of action cost) and
409
similar absolute values of αR/αE will not counteract this bias. Asymmetric learning rates
410
(αE>αR) in combination with lower values of γ, however, will lead to more symmetric
411
performance on RL and EL trials via more efficient updating of action cost versus reward
412
expectations. This interpretation is supported by our demonstration of model parameters
413
(Figure 4b) and post hoc simulations (Figure 4c), as well as the observation that lower values
414
of γ (i.e., greater action cost discounting) were associated with greater learning rate
415
asymmetry (αE>αR; more efficient EL) in no-stress controls (ρ=-0.40, p=0.040), who
416
displayed similar RL and EL performance. These results demonstrate that, in a context where
417
all decisions involve a potential cost and benefit, acute stress selectively reduces the
418
difference between EPE and RPE learning rates, while leaving action cost discounting and
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
419
choice stochasticity unaffected. The direction of the change in learning rates (i.e., greater
420
similarity) implies a stress-induced failure to modulate learning rates in the service of
421
overcoming an asymmetric choice bias that emphasises reward value.
422
In analyses using posterior parameters obtained from the hierarchically fit model, we
423
recovered the key Condition-by-Learning Rate interaction (95% HDI for Bayesian mixed
424
ANOVA = -0.406 to -0.128, mean=-0.269) [and acute stress and no-stress control subjects
425
differed from each other on αE (95% HDI = 0.0841 to 0.281, mean=0.183) but not αR (95%
426
HDI = -0.186 to 0.0102, mean=-0.0872)] (Figure Supplement 7). Similar to the non-
427
hierarchically fit parameters, acute stress and control subjects did not differ on posterior
428
estimates of γ (95% HDI = -0.0914 to 0.139, mean=0.0182) and β (95% HDI = -0.0331 to
429
0.213, mean=0.0895) .
430
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
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Figure 5
432
433
Acute stress reduces the difference between reward and effort prediction error learning
434
rates.
435
Free parameters (αR, αE, γ, β) of the winning 2LR_ γ model for both groups. Black lines
436
denote means ± SD, dots represent individual data points, and the violin-like shape denotes
437
distribution and frequency of the data. *: p < 0.05, **: p < 0.01, ***: p < 0.001.
438
439
Pupil size fluctuations track asymmetric cost-benefit reinforcement learning during
440
acute stress
441
We employed pupillometry to better understand whether task-relevant computational
442
processes may be encoded by fluctuations in pupil dilation, which are thought to be
443
controlled by ascending midbrain modulatory systems that play a role in value-based
444
decision-making and the acute stress response (Arnsten, 2015; Hermans et al., 2011; Joshi,
445
Li, Kalwani, & Gold, 2016).
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
446
In model-free analyses – that is, comparing bins of pupillometry data between
447
conditions - we observed no main effect of Condition on pupil size fluctuations during
448
choice, effort outcome, and reward outcome epochs, suggesting that acute stress was not
449
associated with more general changes in pupil size (all bin-level p>0.05; Figure 6, a. Model-
450
free; Figure Supplement 8 for all effort trials).
451
Next, we conducted model-based pupillometry analyses (Lawson, Bisby, Nord,
452
Burgess, & Rees, 2020) to understand how trial-wise estimates of computational processes of
453
interest were encoded by fluctuations in pupil size. These analyses revealed effects of
454
Condition on pupil encoding of subjective decision value, EPEs and RPEs (Figure 6, b.
455
Model-based) in a manner commensurate with task performance results. First, immediately
456
prior to the stimulus choice, acute stress reduced pupil encoding of subjective decision value,
457
as evidenced by the absence of an association between pupil size and subjective decision
458
value (control>stress; stress n.s., control>0). Second, briefly after the presentation of effort
459
avoidance outcomes, both groups exhibited different pupil size-EPE associations, with no-
460
stress controls showing a non-significant numerically positive association between pupil size
461
and action cost prediction errors (control>stress, both groups n.s. different from 0). In model-
462
based analyses using all effort outcome trials, we were not able to uncover group differences
463
in pupil encoding of EPEs, which were likely eclipsed by prominent grip force-related effects
464
on pupil size (Figure Supplement 8). Third, during the reward outcome phase, acute stress
465
participants exhibited greater positive associations between pupil size and RPEs compared to
466
no-stress controls (stress>control, stress>0, control n.s.). The average pupil size-RPE slope
467
for bins in which no-stress control and acute stress participants differed (Figure 6b) correlated
468
significantly with stress-induced changes in SBP [ρstress(38)= -0.41, p(permutation)=0.019]
469
and PANAS negative affect changes [ρstress= -0.46, p(permutation)=0.005] in the stress group.
23
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
470
Crucially, group differences in pupil encoding of subjective decision value, EPEs, and
471
RPEs imply that the ascending neuromodulatory systems may have facilitated a stress-
472
induced shift in asymmetric cost versus benefit learning.
473
474
Figure 6
475
476
Model-based analysis reveals altered pupil encoding of prediction errors and decision
477
value during acute stress .
478
a. Model-free analyses of pupil size during choice, effort outcome, and reward outcome
479
phase revealed no main effect of Condition (no-stress control, acute stress). b. Model-based
480
analyses revealed a stress-induced shift in pupil encoding of subjective decision value (left),
481
action cost prediction errors (middle) and reward prediction errors (right). Black line
482
indicates significant main effect of Condition; blue and red line indicate significance against
483
zero for no-stress control and acute stress groups, respectively (cluster and bin level
484
apermute<0.05, 2000 permutations). Group differences in pupil encoding of action cost and
485
reward prediction errors were observed at similar times (note the x-axis differences for effort
24
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
486
outcome and reward outcome epochs). Source files of pupillometry data used for the analyses
487
are available in Figure 6 – Source Data 2.
488
489
Discussion
490
Stress-induced alterations in adaptive decision-making are commonly studied using
491
paradigms that isolate positive and negative reinforcement, such as the receipt of a reward or
492
avoidance of a loss. However, it remains poorly understood how acute stress affects the
493
complex process that entails learning about costs and benefits, a critical and pervasive feature
494
of everyday decisions. Participants completed a paradigm in which all actions (stimulus
495
choices) contained a potential cost (exerting physical effort) and a financial benefit (€0.20).
496
Crucially, acute stress induced a shift in reinforcement learning strategies that improved
497
maximization of monetary rewards relative to minimisation of energy expenditure. When
498
presented with novel stimulus arrangements and in the absence of feedback, individuals in
499
the acute stress condition, moreover, exhibited better discrimination of stimulus reward value
500
compared to action cost.
501
Relative improvements in reward versus action cost learning align well with previous
502
reports of enhanced reward learning during acute stress (Byrne, Cornwall, & Worthy, 2019;
503
Lighthall et al., 2013; Petzold et al., 2010), although such effects may depend on stressor
504
timing (Joëls, Pu, Wiegert, Oitzl, & Krugers, 2006), stressor type (Carvalheiro et al., 2020),
505
and/or sample characteristics (Evans & Hampson, 2015; Morris & Rottenberg, 2015). While
506
reports on action cost learning during acute stress are scarce, acute exposure to stress in
507
rodents impairs cost-benefit decisions via a selective change in sensitivity to physical effort, a
508
process mediated by corticotropin-releasing factor and dopamine (Bryce & Floresco, 2016).
509
Our analyses of win-stay/lose-shift rates indicate that asymmetric cost-benefit learning can be
25
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
510
driven by a relative increase in the sensitivity to monetary gains compared to the avoidance
511
of costly deterrents.
512
How might maximization of reward value take precedence over minimisation of
513
action cost? Acute stress leads to a redistribution of finite cognitive resources (Hermans et
514
al., 2014): this process limits the availability of computationally intensive strategies,
515
including working memory (Otto, Raio, Chiang, Phelps, & Daw, 2013; Qin, Hermans, van
516
Marle, Luo, & Fernández, 2009) and goal-directed instrumental actions (Lars Schwabe &
517
Wolf, 2011). Assuming that acute stress does not merely increase random responding - which
518
we verified via the choice stochasticity model parameter - a computationally cheap heuristic
519
in our task should present itself as better learning for one modality over the other. Increased
520
energy availability (Hermans et al., 2014), insensitivity to aversive stimuli (Timmers et al.,
521
2018), and impaired aversive value updating (Raio et al., 2017) under stress may have
522
reduced the ability – or urgency – to dedicate cognitive resources to strategies that minimize
523
action cost. Importantly, effort expenditure increases the perceived value of rewards
524
(Hernandez Lallement et al., 2014; Inzlicht, Shenhav, & Olivola, 2018). Thus, frequent
525
expenditure of physical effort, due to suboptimal action cost learning, may increase the
526
perceived value of rewards, and thus tilt learning towards the maximization of reward value.
527
Using a computational model of reinforcement learning (Skvortsova et al., 2017;
528
Skvortsova et al., 2014), we confirmed that biased cost-benefit learning can arise when
529
inappropriate (i.e. more similar) importance is afforded to teaching signals that convey
530
information about reward value (RPEs) and action cost (EPEs). Humans display presumably
531
instinctive biases, such as more efficient learning from better-than-expected outcomes
532
(Lefebvre, Lebreton, Meyniel, Bourgeois-Gironde, & Palminteri, 2017) (compared to worse-
533
than-expected outcomes) and asymmetric “Go”/approach learning (Guitart-Masip et al.,
534
2012) (compared to “No-Go”/avoidance learning), the latter being a bias that is also
26
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
535
modulated by acute stress (de Berker et al., 2016). From this perspective, no-stress controls,
536
who assigned greater importance to EPEs than RPEs, may have used a computationally
537
costly learning strategy that provides counterweight to a decision-making policy that is
538
biased towards the reward value of actions (captured by action cost discounting parameter γ).
539
Paradoxically, when decisions are by default tilted towards reward value, similar reward and
540
action cost learning rates will facilitate reward learning but hamper action cost learning.
541
Reduced learning rate asymmetry in the presence of action cost discounting may therefore
542
represent a computational reformulation of a heuristic that is employed when cognitively
543
demanding learning strategies are unavailable and the policy towards energy expenditure is
544
more liberal, such as during acute stress.
545
Importantly, stress-induced changes in task performance may crucially depend on the
546
release of catecholamines in neural circuits that support motivation and learning. Dopamine’s
547
actions at D1 and D2 receptors in the basal ganglia mediate approach and avoidance learning
548
(Frank, Seeberger, & Reilly, 2004), and acute stress can improve associative learning by
549
augmenting reward-evoked DA bursts in selective striatal subdivisions (Stelly, Tritley,
550
Rafati, & Wanat, 2020). Dopamine’s enhancement via L-DOPA administration, moreover ,
551
improves reward but not action cost learning (Skvortsova et al., 2017). To the degree that
552
pupillometry can be considered a proxy measure of activity of ascending neuromodulatory
553
systems, these findings are consilient with greater encoding of RPEs by pupil size
554
fluctuations during acute stress. Negative correlations between SBP and PANAS negative
555
affect and RPE-pupil size slopes suggest that primarily moderately stressed participants
556
displayed a preference for maximizing reward value, which might be consistent with an
557
inverted U-shape relationship between cognitive performance and DA transmission which is
558
modulated by stress (Arnsten & Goldman-Rakic, 1998; Baik, 2020). Noradrenaline, however,
559
mobilizes available energy to complete effortful actions and locus coeruleus neurons track
27
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
560
energy expenditure (Varazzani et al., 2015). Stress-induced sAA concentrations, increased
561
heart rate, and group differences in the association between pupil size fluctuations and EPEs
562
all point to the involvement of the noradrenaline system. Thus, our model-based pupillometry
563
and stress-induction results hint at stress-sensitive dopaminergic and noradrenergic
564
mechanisms that may regulate cost and benefit learning, which could be explored in future
565
work using targeted pharmacological approaches.
566
The results presented here may improve understanding of stress-related
567
psychopathology. While asymmetric cost-benefit learning during acute stress may be
568
beneficial to reach a desired goal state (e.g., safety) despite high action cost, such strategies
569
could also be maladaptive. For example, stress exposure can lead to drug or smoking relapse
570
(L. Schwabe, Dickinson, & Wolf, 2011), a context in which reward value and action cost
571
may be misaligned. Cost-benefit reinforcement learning may provide a useful framework to
572
test hypotheses regarding stress-related impairments in learning and decision-making.
573
Some study limitations need to be acknowledged. First, pupil dilation associated with
574
effort expenditure greatly reduced our power to detect robust associations between EPE
575
encoding and pupil size fluctuations. Future studies should, therefore, consider a temporal
576
delay between effort outcome and effort expenditure phases. Second, while our
577
computational model was able to recover overall task performance patterns in both groups,
578
such effects were subtle and dependent on the contribution of other (non-learning)
579
parameters, which may highlight the importance of interindividual differences in model
580
parameters.
581
To summarize, we present evidence of asymmetric effects of acute stress on cost
582
versus benefit reinforcement learning during acute stress, which computational analyses of
583
task behaviour explain as a failure to assign appropriate importance to RPEs versus EPEs,
584
and our model-based pupillometry tentatively link to activity of ascending midbrain
28
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
585
neuromodulatory systems. These results highlight for the first time how learning under acute
586
stress can be tilted in favour of acquiring good things and away from the avoidance of costly
587
things.
588
589
Materials and Methods
590
Participants
591
Adult participants were recruited via paper and online advertisements. All participants were
592
screened for a DSM-5 psychiatric and/or neurological disorder, substance use, endocrine
593
and/or vascular disorder, abnormal BMI (>40 or <18), smoking and drinking (>10
594
cigarettes/units per week), psychotropic medication use (lifetime) and hormonal
595
contraceptive use (current; female participants only). All participants completed the ~2-hour
596
experiment between 12:00h and 18:00h to minimize diurnal cortisol fluctuations (Bailey &
597
Heitkemper, 2001). Participants were instructed to refrain from alcohol (starting the evening
598
before the day of the experiment), smoking, food, caffeine intake, strenuous physical activity
599
and brushing their teeth (all >2 hr prior to experiment), which was verified verbally at the
600
start of the session. Four participants were excluded due to an equipment failure (n=4). Three
601
participants quit during stress-induction (n=2) or task procedures (n=1). Because chance-
602
level performance on reinforcement learning tasks might indicate a successful manipulation,
603
a lack of motivation, or a failure to comprehend the task instructions, participants that
604
performed at or below chance level (0.5) on both RL and/or both EL pairs near the end of the
605
experiment (final 10 presentations) were excluded (n=13; 6 acute stress, 7 no-stress control).
606
Including these participants did not alter our key finding that acute stress was associated with
607
asymmetric cost versus benefit learning (see Results). Pupillometry and neuroendocrine data
608
were not processed further for these participants. The study was approved by the ethics
609
committee of the Faculty of Psychology and Neuroscience, Maastricht University (ERCPN-
29
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
610
197_03_08_2018) and carried out in accordance with the Declaration of Helsinki.
611
Participants were remunerated in gift vouchers or research participation credits. Task
612
earnings were paid out in gift vouchers.
613
614
Acute stress induction
615
The MAST is a validated stress-induction paradigm combining both psychological and
616
physiological stressors, and robustly increases neuroendocrine, physiological, and subjective
617
indices of acute stress (Smeets et al., 2012). During a 5-min preparation phase, participants
618
are instructed about the upcoming task via oral and visually displayed instructions, followed
619
by a 10-min stress-induction phase consisting of alternating blocks of cold-water immersion
620
(non-dominant hand; 2oC) and backward counting in steps of 17 (while receiving negative
621
evaluative feedback from an experimenter), with a (non-recording) camera continuously
622
directed at the participant’s face, which was displayed to the participant on a second display.
623
During the MAST no-stress control condition, participants immerse their hand in lukewarm
624
water (36oC) and perform simple mental arithmetic, e.g., counting from 1 to 25, without
625
receiving feedback or fake camera recordings.
626
627
Neuroendocrine, physiological and subjective stress measurements
628
sCORT and sAA were collected to measure stress-induced increases in hypothalamic-
629
pituitary-adrenal (HPA) axis and sympathetic-adrenal-medullary (SAM) axis activity,
630
respectively (Dickerson & Kemeny, 2004; Koh, Ng, & Naing, 2014). Saliva samples were
631
obtained using synthetic Salivette® devices (Sarstedt, Etten-Leur, the Netherlands) during 3-
632
min sampling periods at 6 time points. A baseline sample was collected 10 min prior to the
633
MAST (baseline: t1 = t-10) and five samples post-MAST (t2= t+00, t3= t+10, t4= t+20, t5= t+30, t6=
634
t+40). SAA assessments were obtained only for t1-t4, due to the rapid decay of sAA post-stress
30
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
635
induction (Dennis Hernaus, Quaedflieg, Offermann, Casales Santa, & van Amelsvoort, 2018;
636
Nater et al., 2005). For all participants, t2 marked the starting point for the reward value
637
maximisation/action cost minimisation task. Samples were stored at -20oC immediately after
638
completion of each session. SCORT and sAA levels were determined using a commercially
639
available luminescence immune assay kit (IBL, Hamburg, Germany) and kinetic reaction
640
assay (Salimetrics, Penn State, PA, USA), respectively.
641
Systolic blood pressure (SBP) and heart rate (HR) as an index of autonomic nervous
642
system (ANS) arousal (Schubert et al., 2009; Wright, O'Brien, Hazi, & Kent, 2014) were
643
assessed at t1 and t2 using an OMRON M4-I blood pressure monitor (OMRON Healthcare
644
Europe B.V., Hoofddorp, The Netherlands). Subjective affect ratings were assessed at t1 and
645
t2 using the 20-item Positive and Negative Affect Scale (PANAS) (Watson, Clark, &
646
Tellegen, 1988).
647
648
Reward maximization versus action cost minimization reinforcement learning task
649
All participants completed a probabilistic stimulus selection paradigm during which they
650
learned to select stimuli with high reward value (20 Eurocents) and avoid stimuli with high
651
action cost (exerting force above a pre-calibrated individual threshold for a duration of
652
3000ms). This reinforcement learning task is conceptually similar to a previously-validated
653
probabilistic action selection task that has been employed to study the neural signatures of
654
reward and effort prediction errors and dopaminergic drug effects on reward-effort
655
computations (Skvortsova et al., 2017; Skvortsova et al., 2014). The paradigm was designed
656
in PsychoPy v3.0.0b11 (Peirce et al., 2019) and presented on a 24″ monitor (iiyama ProLite
657
b2483HSU). Physical effort (in mV/kgf) was registered using a hand-held dynamometer in
658
combination with a transducer amplifier (DA100C) and data acquisition system (MP160; all
659
manufactured by BIOPAC Systems, Inc). Individual effort thresholds used throughout the
31
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
660
task were obtained by calculating 50% of each participant’s maximal voluntary contraction
661
(MVC) (Le Heron et al., 2018) reached over three calibration trials by squeezing the
662
dynamometer with the dominant hand.
663
On each of 120 trials, participants chose between two paired distinct black-and-white
664
images (“stimuli”) that were probabilistically associated with both the receipt of a monetary
665
reward and exertion of physical effort (see Figure 1 for a graphical overview). At trial onset,
666
a fixation cross flanked by two images was presented; participants chose one image by
667
pressing the V/B button for the left/right option, respectively. A 440Hz/600Hz tone for
668
left/right choice (200ms) was presented to confirm the participant’s choice. Next, a
669
thermometer with the command “SQUEEZE” or “DON’T SQUEEZE” was displayed. If
670
participants were required to exert effort, they were instructed to squeeze the dynamometer
671
until the mercury level reached the top. The mercury bar only moved if participants exerted
672
above-threshold levels of force and stopped moving if exerted force fell below. The
673
cumulative above-threshold time was 3000ms. If no effort production was required, an
674
animation of a rising mercury bar was displayed (3000ms). Finally, a screen was presented
675
showing either a €0.20 coin or a crossed-out coin, indicating no reward (3000ms).
676
Participants learned to choose the optimal (most reward or most effort avoiding)
677
stimulus for four distinct image pairs, 30 presentations each, with yoked reward and action
678
cost contingencies. For 2/4 pairs, participants could regularly acquire rewards by selecting
679
one (optimal) stimulus over the (suboptimal) other (henceforth, “reward learning”/RL pairs),
680
while the probability of having to exert effort was identical for both stimuli. For the other two
681
pairs, choices of one stimulus were more frequently followed by the avoidance of effort
682
(“effort learning”/EL pairs), while the probability of reward was kept constant between both.
683
For all pairs, the probability of the stimulus property that was kept constant (reward/effort)
32
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
684
was set to a 33.3% chance of positive outcome upon selection (reward/ effort avoidance) and
685
66.6% chance of negative outcome (no reward/effort).
686
To assess whether any acute stress effects on reward maximization (measured using
687
RL pairs) and effort cost minimization (measured using EL pairs) learning were potentially
688
mediated by task difficulty, we employed different difficulty levels for each RL and EL pair.
689
That is, for one RL and one EL (“easy”) pair, a choice for the optimal stimulus was followed
690
by a positive outcome in 83% (vs. 17% negative outcome) of all trials (83% negative/17%
691
positive outcome for suboptimal stimulus); for the other RL and EL (“hard”) pair a choice for
692
the optimal stimulus was followed by a positive outcome in 70% (vs. 30% negative outcome)
693
of all trials (and 70% negative/30% positive outcome) for the suboptimal stimulus. This
694
approach allowed us to disentangle whether acute stress primarily impacted domain-specific
695
(RL vs. EL) or general (easy vs. hard) reinforcement learning (the latter which also might
696
involve other cognitive skills that might be beneficial to performance and sensitive to change
697
under stress, such as working memory (Schoofs, Wolf, & Smeets, 2009). The task
698
contingencies described above were based on extensive pilot tests to identify a reinforcement
699
schedule that would enable us to detect stress-induced improvements and decreases in task
700
performance. We selected task contingencies based on pilot sessions involving a no-stress
701
control condition and chose a reinforcement schedule associated with non-ceiling/floor
702
performance on RL and EL trials.
703
Following the learning phase, participants completed a surprise test phase, similar to
704
previous work (D. Hernaus et al., 2019; D. Hernaus et al., 2018). This phase consisted of 64
705
trials in which participants were presented with the original four, as well as six novel,
706
stimulus combinations. Participants were asked to choose the stimulus with the highest
707
reward value or the lowest action cost - depending on a coin or thermometer image presented
708
in the middle of the screen - and received no choice feedback. This allowed us to assess
33
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
709
acquired choice tendencies, as well as generalizability of this information to novel situations.
710
The four original pairs were presented four times (total n=16) during which we only asked
711
participants to discriminate on the basis on the reward value (for RL) or action cost (for EL).
712
For novel stimulus combinations, we only presented stimuli that differed in reward
713
value/action cost if reward value discrimination/action cost discrimination was assessed (total
714
n=48: n=4 presentations for the 6 combinations).
715
For every participant, stimuli were randomly assigned to pairs, optimal/suboptimal
716
stimulus orientation was balanced (50% of all optimal stimulus presentations occurred on the
717
left-hand side) and misleading outcomes (e.g., negative outcomes for optimal stimuli) were
718
equally spaced out across the thirty presentations (and balanced for left/right side). Trial
719
presentation order was pseudo-randomized such that I) a given pair would never be presented
720
more than twice in a row and II) the gap between two presentations of a given pair was never
721
greater than four trials.
722
Prior to performing the actual task and prior before acute stress/no-stress control
723
procedures, participants received standard verbal instructions and completed a 16-trial
724
practice round of the learning phase. Participants were not informed about stimulus-outcome
725
contingencies; they were only advised to accrue as much money as possible and avoid
726
exerting unnecessary effort. A 60% accuracy performance threshold was used to confirm that
727
participants understood the general task procedure. The practice round was repeated if
728
participants failed to reach 60% accuracy. To prevent learning, we used deterministic
729
stimulus-outcome probabilities and different stimuli.
730
731
Computational cost-benefit reinforcement learning model: model space
732
In an attempt to uncover latent mechanisms by which acute stress affects reward
733
maximization and/or action cost minimization, we turned to cognitive computational
34
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
734
modelling. We employed a modified reinforcement learning framework based on Rescorla
735
and Wagner (Rescorla, 1972), and used in Skvortsova et al. (Skvortsova et al., 2017;
736
Skvortsova et al., 2014) to investigate whether acute stress impacted learning about
737
sensitivity to, and/or discounting of reward value and action cost. We first describe the model
738
space.
739
Various reinforcement learning models assume that choice preferences of an agent are
740
updated via the prediction error, i.e., the mismatch between outcome and expectation
741
(equation 1A, 1B) and the critical quantity that drives learning (Rescorla, 1972):
742
743
RPE(t) = r(t) – QR(t)(s, a) (1A)
744
EPE(t) = e(t) – QE(t)(s, a) (1B)
745
746
Here, QR(t)(s, a) and QE(t)(s, a) represent the expected reward value and action cost
747
(i.e., effort), where s reflects the given pair and a refers to the more abstract action of
748
selecting a stimulus (not to be confused with action selection), r(t) and e(t) represent the reward
749
and effort outcome for the chosen stimulus at trial t. RPE(t) and EPE(t), thus, represent the RPE
750
and EPE at trial t, respectively.
751
In order to allow for the possibility that humans do not calculate the prediction error
752
against the actual outcome but, rather, what the outcome “feels” like (Huys et al., 2013) , we
753
considered a scenario in which reward and effort outcomes are first multiplied by a free
754
parameter that captures the weight that reward and effort outcomes receive (“WR” and WE” in
755
equation 2A and 2B). As the value of these parameters approaches 1, rewards are
756
increasingly valued more positively, and effort more negatively. These parameters, therefore,
757
control the maximum size of the prediction error.
758
35
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
759
RPE(t) = (r(t)*WR) – QR(t)(s, a) (2A)
760
EPE(t) = (e(t)*WE) – QE(t)(s, a) (2B)
761
762
In various formulations of reinforcement learning, such as Q-learning (Watkins &
763
Dayan, 1992) and the actor-critic framework (Niv, 2009; Rescorla, 1972), the degree to
764
which prediction errors update choice preferences is represented by α, the learning rate
765
(equation 3A), which determines how current prediction errors update choice preferences on
766
the subsequent trial. High values of α allow for rapid updating of choice preferences, while a
767
low α implies that choice preferences are updated at a slower pace and are thus co-
768
determined by outcomes further into the past.
769
770
QR(t)(s, a) = QR(t-1)(s, a) + αR*RPE(t-1)(s, a) (3A)
771
QE(t)(s, a) = QE(t-1)(s, a) + αE*RPE(t-1)(s, a) (3B)
772
773
Extensive evidence suggests that organisms use different learning systems for different types
774
of information, including reward value and action cost (Palminteri & Pessiglione, 2017;
775
Skvortsova et al., 2017; Skvortsova et al., 2014) (equation 3A/B). Thus, the use of separate
776
learning rates for RPEs and EPEs allows for asymmetrical learning about these types of
777
information.
778
While the learning rate controls the speed at which choice preferences are updated,
779
learning rate (nor reward/effort weight) alone does not explain how learned estimates of
780
reward value and action cost may compete at the decision stage (i.e., when participants
781
choose between two stimuli). Agents weight costs against benefits to calculate a subjective
782
decision value (Pessiglione et al., 2017; Skvortsova et al., 2017), which is used to guide
783
choices (equation 4).
36
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
784
785
Q(t)(s, a) = QR(t)(s, a) - QE(t)(s, a) (4)
786
787
In its simplest form, Q, the subjective decision value of a stimulus is represented by the
788
difference between the expected reward and action cost value at trial t (equation 4)
789
(Skvortsova et al., 2014). However, this particular operationalization of subjective value does
790
not take into account the observation that humans tend to discount or prioritize certain types
791
of information in their decisions (Apps, Grima, Manohar, & Husain, 2015; Inzlicht et al.,
792
2018). We, therefore, allowed for variation in the calculation of subjective decision value via
793
action cost discounting (equation 5). While discounting rates can be linear or hyperbolic
794
(Hartmann, Hager, Tobler, & Kaiser, 2013), here we only considered linear discounting in
795
light of previous work using a similar task design (Skvortsova et al., 2017; Skvortsova et al.,
796
2014). As the value of γ approaches zero, action cost discounted increases leading the agent
797
to ignore action cost/only utilize reward value to make a decision.
798
799
Q(t)(s, a) = QR(t)(s, a) - γ*QE(t)(s, a) (5)
800
801
Once the subjective decision value has been computed, the degree to which
802
participants deterministically sample the optimal stimulus is captured by a softmax decision
803
function (equation 6).
804
805
pr(s, a) = exp(Q(t)(s, a)) / sum(exp(β*Q(t)(s))) (6)
806
807
808
Here, pr is the probability of selecting an action, β is the inverse temperature parameter
that among others captures the balance between exploration and exploitation (Nassar &
37
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
809
Frank, 2016), Q(t)(s, a) is the net value of the chosen option and Q(t)(s) represents the net
810
values of both stimuli in the pair.
811
Within the above-described model space our predictions of acute stress effects on reward
812
maximization and action cost minimization could, thus, be explained by changes in
813
sensitivity to reward value and/or action cost (WR, WE), changes in how much weight RPEs
814
and EPEs are afforded (i.e., learning rates, αR, αE), and/or changes in the discounting of
815
reward value by action cost (γ). If acute stress leads to more random responses, such effects
816
should be captured by β.
817
Based on our predictions and the obtained pattern of results (most notably asymmetrical
818
RL/EL performance in the acute stress condition), we considered six candidate models that
819
could capture these various scenarios: I) a model with 2 distinct learning rates for reward and
820
effort (αR, αE) [2LR]; II) a model with 2 learning rates (αR, αE) and a discounting parameter
821
(γ) (2LR_ γ); III) a model with 2 learning rates (αR, αE), a reward weight (WR) and an effort
822
weight parameter (WE) (2LR_WR_WE), IV) a model with a single learning rate (α), reward
823
weight (WR), effort weight (WE), and a discounting (γ) parameter (LR_ WR__WE_ γ); V) a
824
model with 2 learning rates (αR, αE), a reward weight (WR), and a discounting (γ) parameter
825
(2LR_WR_γ); VI) a model with 2 learning rates (αR, αE), a reward weight (WR), effort weight
826
(WE) and discounting (γ) parameter (2LR_ WR_ WE_ γ).
827
Lower/upper bounds for all parameters were set to [0,1] and all models contained a β
828
parameter. Consistent with previous work (Skvortsova et al., 2017; Skvortsova et al., 2014),
829
reward and action cost outcomes were set to [0,1 for no/yes reward] and [-1,0 for no/yes
830
effort avoidance], respectively.
38
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
831
Pupillometry
832
Fluctuations in pupil diameter were continuously measured using an SR-Research Eyelink
833
1000 Tower Mount infrared eye tracker while participants performed the reward
834
maximization/action cost minimization reinforcement learning task (1000Hz sampling rate,
835
except for three participants, whose data were obtained at 500Hz). Participants placed their
836
head on an adjustable chin rest and against a forehead bar to minimize motion. Eye-tracker
837
calibration was performed at the start of the paradigm, and subsequently every 10 min.
838
Stimulus luminance was matched using the SHINE toolbox (Willenbockel et al., 2010) in
839
MATLAB (v. 2014B; The MathWorks, Inc., Natick, Massachusetts, United States). Due to
840
the COVID-19 pandemic, pupillometry data were not collected for the final eight
841
participants. Three participants, moreover, failed the quality control for eye-tracking data (2
842
no-stress control/1 acute stress) leaving a final sample of 69 participants with eye-tracking
843
data (34 no-stress control/35 acute stress).
844
Eye-tracking data were pre-processed using an open source pre-processing toolbox
845
(Kret & Sjak-Shie, 2019) and in accordance with previous work (Jackson & Sirois, 2009).
846
Blinks and other invalid samples, due to dilation speed, deviation from the trend line, and
847
extreme values (Kret & Sjak-Shie, 2019) were removed, interpolated, smoothed (4Hz low-
848
pass filter, fourth-order Butterworth filter) (Jackson & Sirois, 2009), z-scored and down-
849
sampled to 50hz (i.e., 20ms). Bins with fewer than 80% valid samples were removed
850
(Lawson et al., 2020). For analyses, we considered three epochs of interest: choice (-1500ms
851
pre-choice - 15000ms post-choice), effort outcome (0-1000ms post-outcome), and reward
852
outcome (0-2000ms post-outcome). We reduced the duration of the effort outcome epoch to
853
1000ms to minimize force exertion-related effects on pupil size (see below). Recent work has
854
shown that expectation violations (prediction errors) are encoded by pupil size fluctuations
855
within this timeframe (Lawson et al., 2020). Given that we observed large grip force-
39
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
856
associated effects on the pupillometry signal (see Figure Supplement 8 middle row, for a
857
comparison between effort and effort avoidance trials), we limited effort outcome analyses in
858
the main text to effort avoidance trials, although we also report analyses involving all effort
859
outcome trials in Figure Supplement 8.
860
861
Statistical analyses
862
Statistical analyses were conducted using R, version 3.6.2 (Team, 2020) and, where
863
applicable, results were visualised using Raincloud Plots (Allen, Poggiali, Whitaker,
864
Marshall, & Kievit, 2019). Acute stress measurements were analysed using mixed ANOVAs
865
involving Condition (between-factor condition: no-stress control, acute stress induction) and
866
Time (within-factor: 2 pre/post-MAST or 6 levels for sCORT).
867
For the reward maximisation/action cost minimisation reinforcement learning task, an
868
accuracy score was calculated by dividing the number of optimal stimulus choices by the
869
total trial amount (n=30 per pair). Mixed ANOVAs involving Condition, Trial Type (RL, EL)
870
and Difficulty (Easy, Hard pairs) were carried out. For analyses involving Time effects (i.e.,
871
repeated presentations of stimulus pairs), accuracy scores were averaged per bin of ten
872
presentations (presentation 1-10, 11-20, and 21-30). To better understand whether acute
873
stress effects on task performance were primarily driven by changes in sensitivity to positive
874
or negative outcomes, win-stay (repeating a choice following a positive outcome) and lose-
875
shift (choosing the other stimulus following a loss) rates were calculated for RL and EL trials
876
(Hanneke E. M. den Ouden et al., 2013). For RL trials, we calculated win-stay/lose-shift rates
877
using reward outcomes (yes/no reward); for EL trials we used effort outcomes (yes/no effort).
878
We refer to the 2-level factor representing win-stay/lose-shift rates as “Strategy”. For surprise
879
test trials involving the original four pairs (n=4 presentations per pair), we investigated final
880
choice tendences using a one-sample t-test against chance level (0.5). Participants’ ability to
40
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
881
discriminate stimuli based on reward value and action cost in novel stimulus arrangements
882
(n=48, 24 reward value and 24 action cost discrimination trials) were investigated using
883
mixed ANOVAs involving Condition and Trial Type.
884
Group differences in model parameters from the non-hierarchically fit model were
885
investigated using Condition-by learning rate (αR, αΕ) mixed ANOVAs and independent
886
samples t-tests. Given that we used separate priors for the two groups, we report the Bayesian
887
analogue of a t-test and mixed-ANOVA (Kruschke, 2014) - a more robust test of group
888
differences - for posterior parameters obtained from the hierarchically fit model (for
889
reference, we also report these analyses for the non-hierarchical data).
890
Post hoc (simple) main effect analyses for all ANOVAs were conducted using
891
independent sample (Condition), paired-samples (Time, Trial Type, Strategy), and one-
892
sample t-tests (≠ 0 or 0.5). Greenhouse–Geisser-corrected statistics were reported when
893
sphericity assumptions were violated. We report statistical significance as p<0.05 (two-
894
sided), but we note that most main and interaction effects involving Condition survived at a
895
more stringent threshold (p<.01), except for some strategy and surprise test phase effects,
896
which should be interpreted with caution. In case of statistically significant results,
897
generalized eta square (ges; n2G) was reported, with n2G values of 0.02, 0.06, and 0.14
898
representing a small, medium, and large effect size, respectively (Lakens, 2013).
899
With respect to pupillometry, we conducted model-free and model-based analyses. In
900
model-free analyses, we investigated group differences in pupil size during the choice, effort
901
outcome, and reward outcome stage, for every bin of interest. To better understand how
902
putative activity of ascending neuromodulatory systems may drive stress-induced changes in
903
computational strategies that support reward maximisation/action cost minimisation learning,
904
we conducted model-based pupillometry analyses using computational parameters from the
905
winning (2LR_ γ) model (Lawson et al., 2020). First, we used linear regression to estimate
41
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
906
beta weights for the association between pupil size and computational estimates of task-
907
related behaviour for every participant, for every epoch, for every bin. For the choice phase,
908
we regressed trial-wise measures of pupil size against trial-wise estimates of the subjective
909
decision value (i.e., effort-discounted reward value) of the chosen stimulus. For the effort and
910
reward outcome phase, trial-wise EPEs and RPEs were the primary predictors of interest,
911
respectively. Trial number (1-120) and presented images/pair (RL_easy, RL_hard, EL_easy,
912
EL hard) served as additional predictors of interest for all models. Additional epoch-specific
913
variables of interest were included for the choice (optimal choice yes/no), effort (action cost
914
of chosen stimulus, effort avoidance yes/no), and reward (reward value of chosen stimulus,
915
reward yes/no, effort avoidance yes/no) outcome phase. Similar results were obtained when
916
repeating the analyses with more elaborate GLMs (e.g., the addition of yes/no most likely
917
outcome based on reward/effort outcome probabilities [“surprise”] and reward/action cost for
918
EL/RL trials). Secondly, in group-level GLMs, we compared the resulting beta weights I)
919
against zero (for the no-stress control/acute stress condition separately), to investigate when
920
the pupil encoded the computational process of interest, and II) between groups, to assess
921
stress-induced changes in associations between pupil size and computational processes. To
922
control the false positive rate, we conducted permutation tests at the bin- and cluster-level
923
(2000 permutations, apermute=0.05). All correlations were performed using Spearman’s ρ
924
correlations. Permutation tests were also conducted for correlation analyses involving acute
925
stress measures and pupil encoding of predictions errors.
42
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
926
Figure 3 – Source Data 1
927
Source files for task performance data.
928
This link contains all task performance data used for the analyses shown in Figure 3. Raw
929
data can be found under “task_performance”.
930
https://osf.io/ydv2q/
931
932
Figure 6 – Source Data 2
933
Source files for pupillometry data.
934
This link contains pupillometry data used for the analyses shown in Figure 6. Raw data can
935
be found under “pupillometry”.
936
https://osf.io/ydv2q/
937
938
Acknowledgements
939
We are indebted to Drs. Conny Quaedflieg, Edwin S. Dalmaijer. and Ross D. Markello for
940
advice on stress-induction procedures and paradigm development. We thank Dr. Michael
941
Frank for advice on hierarchical computational modelling procedures. We thank Katya Brat-
942
Matchett for involvement in participant recruitment and Truda Driessen for administrative
943
support.
944
945
Competing interests
946
D.H. has received financial compensation as a consultant for P1vital Products Ltd. These
947
activities were unrelated to the work presented in this manuscript. The authors declare no
948
competing interests.
43
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
949
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Figure Supplement 1
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1211
Evidence of reward and action cost reinforcement learning.
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Optimal stimulus choices (“accuracy”) on reward learning (RL) and effort learning (EL)
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(rows) trials for both conditions (columns). Trials were binned into groups of 10
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0.001).
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1217
Figure Supplement 2
1218
1219
Acute stress does not affect difficulty learning.
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Easy and hard pairs collapsed across RL/EL trials depicted for each condition separately.
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While all participants sampled the optimal choices more frequently for Easy vs. Hard pairs,
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displayed. Significant differences are denoted by asterisks (*: p < 0.05, **: p < 0.01, ***: p <
1225
0.001).
56
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
1226
Figure Supplement 3
1227
1228
Surprise test phase performance.
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The acute stress group performed better on reward than action cost discrimination trials.
1230
Means ± SD, individual data points, distribution and density of the data are displayed.
1231
Significant differences are depicted with asterisks (*: p < 0.05, **: p < 0.01, ***: p < 0.001).
57
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
1232
Figure Supplement 4
1233
1234
Correlations between empirical and simulated 2LR_γ choices.
1235
Actual and post hoc simulated choices for RL and EL (rows) were highly correlated both for
1236
no-stress control and acute stress subjects (columns). Simulations were averaged across 10
1237
repetitions per subject. Solid and shaded lines represent mean ± CI95%. Dots represent
1238
individual data points. Horizontal dashed lines indicate chance level (0.5).
58
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
1239
Figure Supplement 5
1240
1241
Parameter estimates after Bayesian hierarchical model fitting.
1242
Hierarchical model fitting reproduced the overall pattern of parameter estimates (Figure 5 for
1243
comparison) .
59
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
1244
Figure Supplement 6
1245
1246
Correlations between empirical and simulated 2LR_γ choices after Bayesian
1247
hierarchical model fitting.
1248
Correlations between actual and post hoc simulated choices for RL and EL (rows) for no-
1249
stress control and acute stress subjects (columns). Simulations were averaged across 10
1250
repetitions per subject. Solid and shaded lines represent mean ± CI95%. Dots represent
1251
individual data points. Horizontal dashed lines indicate chance level (0.5).
60
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
1252
Figure Supplement 7
1253
1254
Bayesian estimation analysis to evaluate group differences in posterior parameter
1255
distributions
1256
Panel A. Bayesian estimation (mixed-ANOVA) using posterior parameters (following
1257
hierarchical fitting) revealed evidence for a credible Condition-by-Learning Rate interaction.
1258
The observed mean difference from zero that falls outside the 95% HDI suggests that the
61
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
1259
difference between αE and αR was greater in no-stress controls compared to acute stress
1260
subjects. Panel B. Both groups did not differ in the magnitude of αR, as indicated by a 95%
1261
HDI that included 0. Panel C. Acute stress compared to no-stress control subjects exhibited a
1262
lower value of αE, as indicated by a 95% HDI that falls well above zero.
62
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.25.441347; this version posted April 27, 2021. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
made available under aCC-BY 4.0 International license.
1263
Figure Supplement 8
1264
1265
Pupillometry analyses using all effort outcome trials.
1266
Left: Model-free analyses of pupil size using all effort outcome trials. Middle: Pupil size
1267
differences during effort/effort avoidance outcomes in the entire sample; force exertion was
1268
associated with large effects on pupil size and these trials were therefore excluded from
1269
analysis. Right: Model-based action cost prediction error analyses using all effort outcome
1270
trials.
63
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8
Appendicitis in Children
Ngozi Joy Nwokoma
Addenbrooke’s Hospital, Cambridge University Hospitals, Cambridge
United Kingdom
1. Introduction
Abdominal pain is a common clinical problem in children. The challenge is to determine
which could be secondary to serious pathology. For the paediatric surgeon, the evaluation of
a child with abdominal pain is often to ascertain if there is a surgically amenable pathology.
The first clinical report of appendicitis in 1711 is credited to a German surgeon called Lorenz
Heister (Ramsted et al., 1993). Appendicitis is the commonest acute childhood surgical
abdominal emergency in developed countries. The peak incidence of acute appendicitis in
children is in the second decade of life, at about 12years of age (Pearl et al., 1995; Tsze et al.,
2011). It is uncommon in children less than 5years old, rare in infants and neonates, slightly
more frequent in males than females with an incidence ratio of 1:1.5. The overall lifetime risk
of appendicitis is 7%, slightly higher in females.
2. Embryology
The appendix develops as a true diverticulum of the caecum and becomes visible at the
eighth week of gestation. It becomes more distinct as the inferior border of the caecum fails
to enlarge as rapidly as the rest of it (Swain, 2005). As the proximal colon enlarges the
caecum undergoes a downwards displacement into the right iliac fossa region of the
abdomen. In certain congenital anomalies the final position of the appendix is outside the
right lower quadrant. In situs inversus, the orientation of the intra-abdominal organs is
reversed so that left sided organs are on the right and vice versa. The thoracic organs may
also be involved in situs inversus totalis. In this condition, the appendix ends up in the left
lower quadrant. In developmental arrest of the normal rotation of the midgut, the appendix
may lie in the subhepatic region or towards the left side of the abdomen.
3. Anatomy
3.1 Position
The base of the appendix is located in the posteromedial aspect of the caecum; below and
within 3cm of the ileocaecal junction. Though the base of the appendix assumes a relatively
fixed position the final position of the appendix body and tip is variable (Figure 1). It
commonly lies behind the caecum (retrocaecal: 64%) or crossing the pelvic brim into the
pelvic cavity (pelvic: 32%). It could also lie posterior to the proximal colon (retrocolic),
posterior to the terminal ileum (retroileal), anterior to the terminal ileum (preileal), just
below the caecum (subcolic), along the lateral border of the caecum and colon
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134
Appendicitis – A Collection of Essays from Around the World
(paracolic/precaecal) or it may be an obturator appendix crossing over the obturator
internus muscle (Moore & Dalley, 2006; Standring et al., 2005). Rarely, the appendix may lie
on the right kidney or duodenum with a retroperitoneal tip and has been reported to
ulcerate into the duodenum (Ellis & Mahadevan, 2010).
Fig. 1. Positional variation of appendicular body and tip: A. retrocaecal; B. Pelvic;
C. retrocolic; D. retroileal; E. preileal; F. subcolic; G. paracolic/precaecal; H. obturator.
The superficial landmark of the base of the appendix corresponds to the level of the first
segment of the sacral vertebrae (S1) at the McBurney’s point. The McBurney’s point is the
junction of the outer and middle thirds of an imaginary line running from the right anterior
superior iliac spine to the centre of the umbilicus. However, the appendix is located within
5cm of the McBurney’s point less than 50% of the time (Karim et al., 1990).
3.2 Innervation
The midline development of the intra-abdominal viscera and associated innervation results
in visceral pain being perceived in the midline. The level of pain may also be different from
the level of the organ from which the pain stimulus arises due to the cranial migration of the
nervous system. In line with the foregoing, epigastric pain is typically associated with
pathology or irritation of the organs that originate from the foregut, periumbilical pain
relates to midgut organs while infraumbilical or suprapubic pain relates to disease in the
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Appendicitis in Children
135
hindgut. The midgut stretches from the second part of the duodenum to the midpoint of the
transverse colon. Being a midgut-originated structure, the initial pain sensation from the
appendix is felt in the periumbilical region. Perception of abdominal pain occurs when the
nociceptors in the respective organ or region of the abdomen have been stimulated by
appropriate agents. Appendicitis represents inflammation of a magnitude great enough to
stimulate these nociceptors.
The nerve supply to the appendix is derived from the autonomic nervous system and has
fibres that respond to stretch rather than pain which explains the poorly localised symptoms
until the parietal peritoneum becomes involved. The sympathetic nerve supply is from the
superior mesenteric plexus while the parasympathetic nerve supply is from the Vagus
nerve.
3.3 Structure
The appendix is commonly referred to as the vermiform appendix because of its worm-like
tubular structure. The length of the appendix is variable ranging from 2 – 25cm but can be
up to 31cm. It is longer in children, than in adults probably due to age-related atrophy. The
external diameter could range from 3 – 8mm and the luminal diameter between 1 – 3mm
(Williams & Myers, 1994; Petras & Goldblum, 1996). The maximum transverse diameter of
the appendix is attained by the age of 4 years. It progressively narrows with age with
increasing fibrosis after 40 years.
The three taeni coli of the proximal colon converge at the base of the appendix. The anterior
taenia colon is commonly used as a landmark to identify the base of the appendix. In the
neonate, the characteristic haustration of the large bowel are absent appearing within the
first 6months and the taenia coli are thin (Standring et al., 2005).
The appendicular wall consists of four main layers: mucosa, sub-mucosa, muscularis
propria and the serosa. The mucosa is similar to the colonic mucosa and consists of the
epithelial lining, the lamina propria and the muscularis mucosa. The epithelial lining is a
single layer of surface epithelial cells including columnar cells with basally located nuclei,
goblet cells, apical mucin and absorptive cells as well as scattered paneth and endocrine
cells. The lamina propria contains crypts of Lieberkühn. The muscularis mucosa of the
appendix is poorly developed unlike the rest of the gastrointestinal tract. The sub-mucosa
contains a rich network of arterioles, venules, capillaries and lymphatics in a connective
tissue framework. It also contains a plexus of nerves, the Meissner’s plexus. The
neurosecretory cells in the submucosa are few till the age of 9years. The age-related increase
in the number of these cells is thought to explain the increase in number of carcinoid
tumours in older patients.
The muscularis propria contains muscles which are arranged in a similar pattern as those of
the small intestine. The outer longitudinal muscle fibres aggregate into the taenia coli to
become continuous with them at the base of the appendix. The inner circular muscles are
thicker. Between these muscle layers is the myenteric or Auerbach’s plexus of nerves which
is morphologically similar to the Meissner’s plexus in the submucosa, unlike the rest of the
gastrointestinal tract where the Meissner’s plexus is thinner.
3.4 Lymphatics
The appendix belongs to the group of lymphatic organs called the Mucosa Associated
Lymphatic Tissue which also includes the intestinal Peyer’s patches, the tonsils and the
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lymphoid follicles in the walls of the bronchi. They are thought to protect the
gastrointestinal tract and the respiratory tract from recurrent infections from foreign matter
and organisms entering these body cavities (Snell, 2004b). However, its role in immune
protection in the gastrointestinal tract is unclear. The submucosa of the appendix contains
prominent lymphoid tissue similar to that in the terminal ileum; this feature differentiates
the appendix from the colon. These may become hypertrophic in the presence of
inflammation and may obstruct the lumen in acute appendicitis. Lymphoid hyperplasia is at
its peak during the second decade of life. This has been postulated to be the reason behind
the high incidence of appendicitis in this age group. Lymphoid hyperplasia is thought to be
responsible for 60% of acute appendicitis and occurs mainly in children.
The appendicular lymphatic vessels drain into the lymph nodes in the mesoappendix, the
anterior ileocolic lymph nodes which often become enlarged during acute appendicitis and
then into the right para-aortic lymph nodes.
3.5 Vasculature
The appendicular artery arises from the inferior branch of the ileocaecal artery and the vein
drains through the ileocaecal vein into the portal venous system. The meso-appendix
connects the appendix to the ileal mesentry. The artery enters the mesoappendix a short
distance from the appendicular base where it gives off the recurrent branch which
anastomosis with a branch of the posterior caecal artery. It is common to find accessory
arteries associated with the appendix (Standring et al., 2005). These must be handled
carefully to limit blood loss during appendicectomy. The appendicular artery runs through
the meso-appendix along its free edge and lies on the appendix wall in its distal aspect. The
anastomosis at the base gives rise to a good blood supply but it is an end artery from the
midpoint to distal appendix where its close proximity to the appendix makes it susceptible
to thrombosis as the appendix enlarges during acute inflammation.
4. Aetiology of appendicitis
The aetiology is multi-factorial and may involve interplay of factors including obstruction,
infections, ischaemia and hereditary factors. Obstruction from lymphoid hyperplasia is a
common causal factor and this has been addressed in detail elsewhere in this chapter. A
faecolith is a small stone-like mass of stool. Its formation starts with entrapment of vegetable
fibre. Like the colonic mucosa, the appendix mucosa is well equipped for water absorption
resulting in concentration of its contents with mucous entrapment. Several layers of deposits
eventually result in increase in diameter and a faecolith diameter of 1cm leads to
appendicular obstruction. Faecoliths are less common in children than in adults; 7.7% versus
42% (Gillick et al., 2001). A primary neoplasm of the appendix is found in 0.5-1.0% of
specimens removed for appendicitis. The neoplasm could be mucinous adenoma, mucinous
adenocarcinoma, colonic type adenocarcinoma, non-Hodgkins lymphoma, classical
carcinoid tumour, or goblet cell carcinoid tumour. 30-50% of patients with carcinoid present
with acute appendicitis, being associated with obstruction of the appendix in 25% of cases.
An appendicular diameter greater than 15mm should raise suspicion as to the presence of
an appendicular tumour (Pickhardt et al., 2002). Carcinoid tumours mostly are located in the
distal tip of the appendix, taking the form of a bulbous solid tumour of about 2-3cm
diameter. In children it is usually of a diameter of less than 2cm. 75% is at the tip; 20% mid-
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appendix and 5% at the base. The incidence of carcinoid tumours in surgical specimens is
about 0.08-0.7%; 0.2-0.5% in children. It is the most frequent tumour of the gastrointestinal
tract in childhood and adolescence. It occurs more in white females. A mucocele is a dilated
appendix filled with mucinous substance. It may present as an obstructed appendix
containing insipissated mucin or be a consequence of mucinous cystadenoma or mucinous
cystadenocarcinoma.
Bacterial and fungal infections can also lead to appendicitis. The bacteria involved are usually
of a mixed aerobic and anaerobic population; most commonly Bacteroides fragilis and
Escherichia coli. Others include Streptococcus milleri (associated with a seven-fold increased
risk of abscess formation) and Campylobacter jejuni (Feneglio-Preser et al., 2008). Infections
may further lead to fibrin thrombi which can block the small appendicular vessels leading to
secondary ischaemia. The appendix is particularly prone to ischaemic insult because the
appendicular artery is an end artery beyond the base of the appendix. Torsion of the appendix
may occur resulting in ischaemic appendicitis; but, this condition is rare (Fenoglio-Preiser et
al., 2008). Familial aggregation of appendicitis suggests polygenic inheritance and the
appendicitis usually manifests before the age of 10years. The hypothesis of appendicitis being
associated with low fibre diet is weakened by the finding in Africa that populations on high
fibre diet did not have a lower appendicitis rate (Naaeder & Archampong, 1998).
5. Pathophysiology
The human appendix secretes up to 2ml of clear fluid containing mucin, amylase and
proteolytic enzymes, which may be produced by bacteria each day. The appendicular
aperture is guarded by semilunar mucosal folds which give it a valve effect. The basal
intraluminal caecal pressure is approximately 5cm of water while the appendicular
intraluminal pressure ranges from 15 – 25cm of water creating a pressure gradient of about
10cm of water. This is believed to keep gut contents from entering the appendicular lumen.
Experimental studies have shown that the obstruction of exteriorised human appendices can
raise the intra-luminal pressures to an extent that exceeds the perfusion pressure in the
vascular plexus within the wall of the appendix. The distal end of the appendix is most
vulnerable to this reduction in blood flow. Electrical stimulation of the appendix has been
demonstrated to cause closure of the ileocaecal valve (Williams and Myers, 1994). This may
be a contributing factor to the nausea and vomiting associated with acute appendicitis.
The peritoneum consists of a continuous visceral and parietal layer. Both layers are of
mesodermal origin, but develop separately with independent nerve supplies. The visceral
layer covers the intra-abdominal organs and is supplied by autonomic nerves. The parietal
peritoneum lines the under surface of the abdominal wall and is supplied by somatic nerves.
Pathways for pain differ in each layer and so also the quality of pain. Visceral pain has a
dull aching character, often crampy and may be associated with nausea and sweating.
Parietal pain on the other hand is mostly sharp, severe and persistent in nature. Visceral
organs have limited response to pain stimulus but the stretching of the mesentry and
irritation of the parietal peritoneum produces severe pain.
Visceral afferent fibres carrying sensation of distension and pressure are responsible for the
initial pain of appendicitis, poorly localised initially and referred to the periumbilical region.
Afferent nerve fibres from viscera enter the dorsal horn of the spinal cord along with
afferent nerve fibres from cutaneous structures of the corresponding dermatome. These two
groups of nerve fibres overlap at the synaptic junctions in the dorsal horn leading to the
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phenomenon of referred pain whereby pain is perceived by the brain as arising from the
corresponding cutaneous structures. Nerve fibres decussate and travel up to the thalamus
along the lateral spinothalamic tract and then onwards to the cerebral cortex. Increased
intravisceral pressure by stretch, distension or contraction of the viscus especially against an
obstruction leads to visceral pain. The dermatomal distribution associated with the midgut
relates to the umbilical region, with nerves entering the spinal column at the tenth thoracic
spinal segment (T10). The midgut extends from the second part of the duodenum to the
midpoint of the transverse colon. Therefore, pain arising from the midgut is felt initially in
the umbilicus before the parietal peritoneum becomes involved (Klish, 2006).
In 1886, the American pathologist - Reginald Fitz became the first person to describe the
pattern of the pathophysiological basis of appendicitis in literature. He noted that the
condition started with onset of inflammation, followed by perforation, abscess formation
and peritonitis (Morrow & Newman, 2005). Appendicitis is commonly secondary to luminal
obstruction which is demonstrable in 50-80% of cases (Turner, 2010). As stated previously,
the commonest cause of luminal obstruction in children is lymphoid hyperplasia or
hypertrophy which mostly results from dehydration and viral infection. Faecoliths take
several years to form. They are commoner in older children and may cause direct focal or
diffuse mucosal ulceration. The stasis that results creates an environment which favours
bacterial proliferation and also causes ischaemic injury.
The fore-going results in inflammatory changes including oedema, neutrophilic infiltration
of the lumen, muscular wall and periappendicular soft tissue. In early appendicitis,
subserosal vessels become congested and perivascular neutrophilic infiltrate develops
within all the layers of the wall leading to loss of lustre which gives the appendix a dull
granular erythematous appearance. Therefore, the histological diagnosis of acute
appendicitis must demonstrate neutrophilic infiltration of the muscularis propria not just
within the lumen (Turner, 2010). Figure 2 illustrates the sequence of events that follow
appendicular obstruction.
Luminal obstruction Increased mucous production
and bacterial overgrowth
│
↓
Dilatation/swelling of the appendix
│
↓
Impaired venous drainage worsening congestion and oedema
Impaired lymphatic drainage
Increasing oedema and turgidity
Loss of the appendicular lustre
│
↓
Further appendicular swelling
Reduction in arterial blood flow
Thrombosis of appendicular vessels
Necrosis of the appendicular wall.
Fig. 2. Sequence of events that follow appendicular obstruction
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More severe inflammation results in prominent neutrophilic exudate which generates
serosal fibrino-purulent reaction that gives the appendix the creamy yellow appearance
associated with this stage of the inflammatory process. If the inflammatory process is not
curbed at this stage, it progresses to formation of focal abscesses within the appendicular
wall; this is acute suppurative appendicitis. Progressive increase in the intraluminal
pressure leads to venous flow compromise. Laplace law suggests that the wall tension of a
tubular structure is directly proportionate to the thickness of the wall divided by the square
of the radius. Further increase in the wall tension culminates in necrosis of the appendix.
Further inflammation leads to the formation of large areas of haemorrhagic ulceration with
gangrenous necrosis that extends to the serosal layer; this is acute gangrenous appendicitis.
Rupture of the appendix follows with suppurative peritonitis (Turner, 2010). The risk of
perforation of the appendix rises with the duration of symptoms being about 30% for
<24hours and greater than 70% in >48hours (Swain, 2005). The perforation rate also varies
with the age of the child. The average rate is 30-45% which may be as high as 80% in those
under 5years and nearly 100% in those under 2years (Morrow & Newman, 2005; Stevenson,
2003).
The normally glistening serosal and peritoneal surface becomes dull and lustreless; serous
or slightly turbid fluid begins to accumulate within 2-4hours of the onset of inflammation.
With progression of the inflammatory process creamy suppurative material with
increasing viscosity accumulates. At this point, the process can take the form of
localisation by the omentum and viscera to be controlled in a small area of the abdominal
cavity, or become widespread filling the entire abdomen. The cellular response results in
the formation of dense collections of neutrophils and fibrinopurulent debris that coat the
viscera and abdominal wall at the site of the inflammation (Turner, 2010). The greater
omentum is smaller in children and only at the level of the umbilicus in the neonate
containing small amount of fat and therefore providing limited omental protection
(Standring et al., 2005).
Inflammation of the peritoneum and surrounding intra-abdominal organs follows with
peritonitis. Bowel obstruction may result from the adhesive inflammatory process. Irritation
of the rectosigmoid may lead to enteritis manifesting with frequent loose stools. Irritation of
the bladder by the inflammatory process may cause dysuria, increased frequency of
micturition and urgency simulating urinary tract infection. Severe inflammation may lead to
haemorrhagic cystitis.
The inflammatory process may also be accompanied by increased tissue porosity or
permeability with bacterial translocation. Peritonitis from bacterial translocation across the
porous inflamed wall of the appendix may still occur in the absence of obvious perforation
or faecal contamination.
6. Histopathological features
In the acute phase, serosal injection leads to loss of the normal appendiceal lustre; if
inflammation progresses purulent exudate forms on the surface of the appendix followed by
perforation. There may be fecolith within the appendix lumen; or the lumen may be
distended with pus or mucous. Enterobius vermicularis may be present in the lumen and
sometimes within the mucosa where they may induce a granulomatous reaction. They can
be identified on microscopy by their lateral spines evident on the cross section of the
transected worms (Sebire et al, 2010).
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Histological features of acute appendicitis include
Acute transmural inflammatory aggregation of neutrophils and eosinophils
Hyperplasia of mucosal lymphoid tissue
Haemorrhagic changes in the mucosa
Pus within the appendicular lumen
Mucosal ulceration
Acute serosal inflammation
Haemorrhagic necrosis of the appendicular wall
Adenovirus inclusions may be seen in the epithelial cell nuclei
There may be vasculitic changes with or without thrombi within the vessels in the wall
Following interval appendicectomy, there may be chronic inflammatory changes with
fibrosis of the wall with or without occlusion of the appendicular lumen (Sebire et al,
2010).
Inflammation without mucosal ulceration is of uncertain significance. In acute intraluminal
appendicitis, there is increased neutrophil presence in the appendicular lumen with no
evidence of mucosal infiltration. Similar findings have been documented in incidentally
removed appendix specimens (Feneglio-Preser et al., 2008).
The issue of sending normal appearing appendix for histological analysis is supported by
the fact that certain conditions may present in the appendix with macroscopically normal
appearance. These include polyarteritis nodosa, tuberculosis, amoebiasis, parasitic
infestations including bilharzisis, schistosomiasis, trichuriasis, ascariasis and clonorchiasis,
actinomycosis as well as epithelial tumours. 2 – 5% of macroscopically normal appendices
may have significant unsuspected pathological condition (Williams & Myers, 1994).
Furthermore, neurogenic appendicopathy may appear macroscopically normal and can only
be diagnosed with certainty on histological analysis (Zaupa et al., 2011).
7. Microbiological perspective
Peri-appendicular abscesses may occur from bacteria usually present in the bowel including
Escherichia coli, Proteus species, other enterobacteriaceae, Bacteroides species, anaerobic cocci
and other anaerobes. Infections are therefore commonly polymicrobial. The resultant
secondary peritonitis commonly yields Escherichia coli and other enterobacteriaceae and
anaerobes from intra-operative peritoneal pus swabs. Some authors argue that the precise
value of peritoneal swabs in many cases of secondary peritonitis is difficult to assess because
the bacteriology seldom influences antibiotic treatment which is given empirically on
clinical grounds for short duration, often ending within 2-3days before the full bacteriology
results become available (Baker et al, 2004).
The gastrointestinal tract like other portals of entry into the human body, has a normal flora
that helps protect it against pathogenic micro-organisms. The flow rate reduces from the
small intestine to the large intestine giving the bacteria more time to colonise and reproduce
leading to higher concentration of the organisms. The amount of flora increases in number
and varies in type as the gastrointestinal tract progresses from the oral cavity to the
anorectum. About one thousand species of micro-organisms are present in the large
intestine. Approximately 20% of the volume of faecal matter in the healthy person consists
of bacteria, most of which come from the colon. The terminal ileum flora is similar to colonic
flora. More than 90% of these are anaerobes, mostly Bacteroides, Fusobacterium,
Eubacterium and Clostridium. Others include E. coli, enterococci, yeasts and numerous
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others (VanMeter et al, 2010). Organisms commonly isolated from peritoneal
microbiological tests in secondary peritonitis are mostly anaerobes which outnumber
aerobes in the bowel by a thousand fold (Forbes et al, 2007).
Enterobius vermicularis is the most common nematode infection in humans and can be
found in up to 3% of appendices in the USA. Schistosomiasis of the appendix is rare.
Strongiloides stercoralis infection results in eosinophilic appendicitis. Viral appendicitis has
been associated with measles in the prodromal phase. Other viruses that may cause
appendicitis are adenovirus and gastrointestinal cytomegalovirus infection. Acute infectious
mononucleosis and Epstein Barr virus infection rarely may give rise to abdominal pain
(Petras & Goldblum, 1996).
8. Diagnosis
8.1 Clinical presentation
Making a diagnosis of acute appendicitis in children can be a difficult task even for the
experienced paediatric surgeon. Negative appendicectomy rate was found to be higher
among children operated upon in district general hospital than in a specialist paediatric
centre, 20% versus 4% (Whisker et al., 2009). Chang et al., (2010) found that approximately
12-15% of paediatric appendicitis were missed at the first visit to the emergency department
with the rate of perforation of 73.1% versus 49% in those diagnosed at first presentation. The
duration of symptoms was longer in the former group and the rate of perforation higher the
longer the duration of symptoms.
Generally, clinical symptoms are the patient’s report of the manifestation(s) of dysfunction
in the normal body physiology. Thus, the younger the child, the less accurate the report of
symptoms can be expected to be. Neurodevelopmental immaturity precludes accurate
understanding, interpretation and description of symptoms by children particularly those
younger than eight years of age. Not surprisingly, this is the age group that commonly
presents late with advanced appendicitis. Furthermore, parents of infants often ascribe
febrile illness and vomiting to “teething” and do not seek medical evaluation early. The
clinical symptoms of appendicitis are often secondary to luminal obstruction leading to
colicky abdominal pain at onset which progresses to constant pain with progression of the
inflammatory process. Nausea and vomiting are commonly present. Clinical signs are
discussed in detail later in this chapter and often include tenderness in the right lower
quadrant of the abdomen.
Advanced appendicitis is often associated with delayed presentation especially in children
below the age of five years and also with retroileal, retrocolic or pelvic appendicitis.
Irritation of pelvic structures may produce symptoms and signs suggestive of urinary tract
infection or enteritis.
8.2 History
Rigorous pursuit of a detailed history is invaluable in the diagnosis of appendicitis. In
children, patience is an indispensable virtue and a rushed history increases the risk of
misdiagnosis. Possession of the clinical skills required for eliciting appropriately focused
and chronologically accurate history from the child and parent is key to early diagnosis. The
surgeon therefore has to make the most of open-ended and direct questioning at appropriate
key moments of the history taking applying sensitivity to the emotional climate.
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Background information of the child’s usual status of health should be obtained. The onset
of the current symptoms should be carefully ascertained. Site of onset of abdominal pain
and its present location may suggest migration of pain which may be associated with acute
appendicitis; usually starting in the peri-umbilical region, the pain later localises to the
region of the right iliac fossa. In addition it is often preceded by nausea or vomiting.
Characterising the abdominal pain is key to accurate diagnosis of its source. The onset of the
pain associated with acute appendicitis is often gradual, progressively worsening. It may be
intermittent initially then sharp and constant within a few hours. Children may not give this
typical presentation; even the older ones may become very quiet and distracted by other
issues including pain, fear, strange environment with unfamiliar people or even
psychosocial circumstances in the family.
The usual duration of symptoms at presentation is 24 – 36hours. There may be a history of
pain being made worse by road bumps on the way to the hospital. This suggests the
presence of rebound tenderness. Enquiry into the presence of associated factors should be
made. Nausea and vomiting may be present in up to 90% of patients. Diminished appetite
may be absent in children with appendicitis. Diarrhoea may suggest irritation of the
anorectum by inflamed tissue in the rectovesical pouch. The sigmoid colon is often
redundant in children, with a tendency to loop into the pelvis. Consequently, it may come in
contact with an inflamed appendix manifesting as diarrhoea. Care must be taken not to
mistake this for gastroenteritis. Dysuria may be associated with appendicitis from irritation
of the urinary bladder by an inflamed pelvic appendix.
The history should also explore other possible causes of abdominal pain. Symptoms of
upper respiratory tract infection may suggest mesenteric adenitis. Cough may suggest
pneumonia with referred abdominal pain as a diagnosis. Vulvovaginal irritation with or
without vaginal discharge may suggest pelvic inflammatory disease. Abdominal pain may
also be referred from an acute scrotal condition and older boys in particular do not offer this
important information without direct questioning. Constipation may produce symptoms in
children that may imply the presence of pathology and should be considered. In addition,
enquiry should be made about any previous history of abdominal pain, previous abdominal
surgery, recent foreign travel, current or recent medications as well as the presence of
similar condition in other family members or pupils in the same school.
8.3 Differential diagnosis of acute abdominal pain in children
The cause of acute abdomen in children may vary according to sex and age. Possible causes
are presented below. The list is by no means exhaustive and not in order of frequency. In
addition, some conditions may co-exist.
8.3.1 Infants
Viral enteritis
Intussusception
Pyelonephritis/ other urinary tract infection (UTI)
Gastro-oesophageal reflux
Bacterial enterocolitis
Chest infection
Appendicitis
Pyloric stenosis
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Strangulated hernia of the anterior abdominal wall
Testicular torsion
Mesenteric cysts
Ruptured abdominal tumour
Pancreatitis
Meckel’s diverticulitis
Hirschsprung’s disease with or without enterocolitis
Poisoning
Trauma
Non-accidental injury
8.3.2 Children aged between 2-10years old
Meckel’s diverticulitis
Cystitis
Pyelonephritis
Viral enteritis
Bacterial enterocolitis
Appendicitis
Non-specific abdominal pain
Crohn’s disease
Abdominal trauma, including non-accidental injury
Chest infection
Mesenteric adenitis
Neutropenic enterocolitis
Pancreatitis
Ruptured intra-abdominal tumours
Poisoning
8.3.3 Children above 11years old
Viral enteritis
Bacterial enterocolitis
Appendicitis
Non-specific abdominal pain
Mesenteric adenitis
Pelvic inflammatory disease
Tubo-ovarian cysts
Tubo-ovarian abscess with or without rupture
Torsion of an ovarian cyst
Haemorrhage in an ovarian cyst
Endometriosis
Mittleschmerz
Crohn’s disease
Pancreatitis
Neutropenic enterocolitis
Chest infection
Haematocolpos
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Peptic ulcer disease
Psychosomatic condition
Trauma
Ectopic pregnany
Dysmenorrhoea
Gall stone disease including cholecystitis, biliary colic, cholangitis
Urinary tract infections
Neuronal abdominal wall pain including shingles, spinal or nerve root problem,
iatrogenic peripheral nerve injury
Spontaneous rectus sheath haematoma
8.4 Physical examination
A thorough physical examination would compliment the clinical suspicion formed from the
reported symptoms. A general examination of the child with abdominal pain is imperative
and requires experience in identifying the sick child. The child’s appearance should be
noted – body habitus, facial expression, position, willingness or reluctance to move,
alertness, pallor and whether the child is flushed or sweaty. Assess the child’s pulse for
volume and rate. The temperature in early appendicitis may be normal or mildly raised. A
temperature above 38◦C should prompt further investigation or evaluation to exclude other
causes. Ears, throat and lymph nodes should be evaluated. Tachypnoea, recessions, shallow
breathing and flaring of the alar nasi may suggest a respiratory tract problem or be
secondary to circulatory system contraction. An antalgic gait, leaning to right side, limping
on the right leg and slow motion are all cues to the presence of abdominal pathology.
Flexion at the hip suggests abdominal wall discomfort with or without peritoneal irritation.
Younger children typically poorly localise pain. Most of the under five-year-olds point to
their umbilical region as the site of all pain; perhaps because the umbilicus is a central
feature with a unique appearance that sets it out as a point of focus which captures the
child’s attention. The demonstration of certain clinical signs may further qualify the pain but
atypical abdominal pain is seen in about 40 – 45% of patients. One should beware of the
child who is on antibiotic therapy for other presumed infection who presents with
attenuated features of appendicitis.
The child’s anxiety should be taken into consideration and reducing the number of people in
the room or creating a distraction may help. Distraction may be accomplished with the help
of a paediatric play therapist. Building a rapport with the parents gains the child’s trust and
allays anxiety. A warm child-friendly environment is desirable and is common practice in
many paediatric specialist centres. Focused examination of the abdomen should commence
with inspection for distension, abdominal wall excursions with respiratory activity, hernia
orifices, external signs of trauma, scars or visible peristalsis. Percussion of the abdomen may
reveal the presence of rebound tenderness suggesting peritonism. Palpation of the abdomen
should in the first instance be superficial and general, starting away from the site of pain.
This gives the examiner the opportunity to explore all the quadrants of the abdomen and
improves the chance of identifying non-appendicular pathology. This gentle approach
reassures the child and allows him or her to trust the examiner, and also to relax the
abdominal wall. Guarding may be present as well as tenderness. Depending on the child’s
level of development and co-operation, he or she may be encouraged to cough, laugh,
distend the abdomen or retract the abdominal wall. Rebound tenderness may be present if
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these activities elicit pain. This is followed by deep palpation to explore the presence of an
intra–abdominal mass. The character of any palpable mass should be evaluated – soft, firm,
mobile or fixed to surrounding structures, regularity of its palpable surface and possible
organ of origin. Due to the variability of position of the appendicular tip as previously
discussed, the parietal pain may be related to the right upper quadrant, right loin or pelvis.
The practice of gently rocking the pelvis from side to side is still practised by some surgeons
and may elicit rebound tenderness.
Auscultation should evaluate bowel sounds but is generally not very useful. Bowel sounds
may be absent or diminished in advanced inflammation. However, the presence of normal
bowel sounds does not exclude advanced appendicitis. For the tense anxious child, using
the stethoscope as a palpation tool can help with the evaluation of the abdomen. Also
palpating over the child’s hand can play the same role. An auscultation of the chest is part of
the evaluation for probable appendicitis to rule out or confirm chest pathology. In the
presence of positive chest signs, the abdomen should still be carefully evaluated for the
presence of possible co-existing intra-abdominal pathology.
Children are not good at responding to the question – “Does this hurt?” The young child is
very likely to respond in the affirmative when asked such questions. Conversely, beware of
the older child who denies any pain for fear of being admitted into the hospital. The child
with acute appendicitis would often be reluctant to move and may express discomfort by
facial grimace or tears rather than verbally. Therefore careful observation of the child’s facial
expression and non-verbal responses is paramount to the interpretation of clinical signs.
Right lower quadrant pain, tenderness and rebound tenderness should be elicited. The
traditional method of eliciting rebound tenderness by suddenly withdrawing the hand
following a deep palpation, is not advisable in children. It results in sudden severe pain
which may make the child loose confidence in the doctor. Rather, rebound tenderness is
usually tested for by asking the child to increase the intra-abdominal pressure by coughing
(Dunphy sign). This brings the inflamed appendix or surrounding tissues to the anterior
abdominal wall manifesting as rebound tenderness. Similarly, the abdominal pain may also
be exaggerated by attempting to move the abdominal wall outwards – “blowing out the
abdomen” or moving the abdominal wall inwards – “sucking the abdomen inwards”.
McBurney’s sign is the presence of maximum tenderness over the McBurney’s point. This
was first described by McBurney who was the first to recommend appendicectomy for
treatment of appendicitis (Morrow, 2005).
Rovsing sign is positive if there is perception of pain in the right lower quadrant on
palpation of the left lower quadrant. Obturator sign may be positive. To elicit this sign,
the patient lies supine with the right knee and hip flexed to 90degrees. The examiner,
holding the patient’s right ankle in the right hand, places the left hand on the knee.
Outward rotation of the flexed right knee causes internal rotation of the right hip which
causes the obturator internus to become tense. The test is positive if pain in the right
lower quadrant is elicited; usually in appendicitis in the pelvic or obturator positions
where the appendicular tip lies over the obturator fascia covering the obturator internus
muscle. The iliopsoas comprising of the powerful hip flexors – iliacus and psoas major,
can become inflamed in appendicitis which is retrocaecal and therefore retroperitoneal
giving a positive psoas or iliopsoas sign. This can be evaluated by two approaches. With
the patient lying supine, the examiner’s hand is placed just above the right knee and the
patient asked to flex the right hip against resistance. Eliciting pain means positive psoas
sign. An alternative method is to have the patient lie on the left side, if hyper-extension on
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the right hip elicits pain, the sign is positive. A psoas abscess from a different cause would
elicit similar pain (Liu & McFadden, 2003).
The introduction of the algometry for the diagnosis of acute abdominal pain in children has
been welcomed by many paediatric surgeons (Vajcner et al., 2011). This device is used to
predict acute appendicitis by observing the abdominal tenderness threshold which is the
minimum pressure applied to the anterior abdominal wall to produce discomfort. With
regards to diagnosing acute appendicitis, when combined with other clinical findings, it was
found to have a sensitivity of 82% specificity of 73% and positive likelihood ratio of 3.03.
This new innovation may become popular in the future but it needs evaluation through
appropriately designed clinical trials.
9. Investigations
In cases where the clinical history or physical signs are atypical and inconclusive for the
diagnosis of acute appendicitis, various investigations may be used to complement the
clinical findings, strengthen the diagnosis and exclude the presence of alternative pathology.
They may also aid the peri-operatively management of the patient. These may be bed-side,
laboratory, radiological or laparoscopic investigations.
There is no one investigation that can accurately diagnose appendicitis every time. The
clinical value and economic benefit of laboratory investigations for the diagnosis of
appendicitis has been the cause of much debate (Liu and McFadden, 2003).
The general rule to the selection of an investigation is that it would:
Complement the history and examination
Determine what other clinical tests may be required
Alter treatment approach.
An ideal diagnostic test should offer the following benefits:
High level of accuracy: high sensitivity and specificity
Capable of assessing the extent of disease
Cost effective: cost of investigation should be less than the consequences of treatment
without the benefit of the information derived from the examination
Short length of study
Quick and easy access to result or diagnostic information
Non-invasive
Suited to local needs, resources and available expertise (Hernanz-Schulman, 2010)
9.1 Bedside investigations
Bedside investigations can be done alongside the initial evaluation. Urinalysis with urine
dipstix may suggest urinary tract irritation or infection, diabetes or pregnancy-related
conditions. A bedside blood sugar test is a quick check for possible diabetic ketoacidosis.
9.2 Laboratory investigations
Laboratory tests commonly used to evaluate acute abdominal pain include full blood count,
electrolyte studies, C-reactive protein (CRP), urine microscopy and culture, liver function
tests and serum amylase level. Approximately two-thirds of the patients would have
elevated white blood cell (WBC) count with neutrophilia but this is not specific to
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appendicitis. The relative neutrophil count may be above normal range even in the presence
of a normal total white blood count. Serum levels of inflammatory markers may not be
raised in early appendicitis. Repeating the investigations at least 6hours after the initial test
may increase the diagnostic yield (Wu & Fu, 2010).
Laboratory urine microscopy would assess for presence of pyuria and micro-organisms.
Urinalysis may be abnormal in up to 48% of patients with acute appendicitis. This may
show microscopic haematuria, pyuria or proteinuria (Rothrock & Pagane, 2000). Pyuria may
arise as a consequence of irritation of the urinary bladder or the ureter by the inflamed
appendix or surrounding inflamed tissue. Serum or urine βhCG tests should be performed
in young women of child-bearing age and if positive, an ectopic pregnancy should be
excluded by further evaluation involving the gynaecologist. Moreover, pregnancy and
appendicitis can co-exist.
Serum electrolytes and creatinine levels should be requested and any abnormalities corrected
appropriately. Blood glucose should be obtained and any abnormality appropriately
managed. It must be borne in mind that diabetic ketoacidosis may present as acute abdomen.
The CRP is an acute phase reactant synthesized in the liver which is often raised within
12hours of an acute inflammatory process. It may be raised in 50-90% of patients with acute
appendicitis but again it is non-specific. Serum levels of inflammatory markers including CRP
and WBC count cannot be reliably used to distinguish between acute appendicitis and other
causes of abdominal pain (Dalal et al., 2005). They are more effective in supporting a clinical
diagnosis of appendicitis than excluding the diagnosis (Birchley, 2006).
9.3 Radiological investigations
Radiological investigations should be tailored to the specific presentation and possible
differential diagnoses. A chest radiograph may be useful in the presence of clinical suspicion
of lower respiratory tract infection or complications there from.
9.3.1 Plain abdominal radiograph
Plain abdominal radiographs are not commonly used in the evaluation of abdominal pain in
children particularly when appendicitis is felt to be a likely cause. There are several reasons
for this stance, one being that children present with abdominal pain commonly and
obtaining an abdominal radiograph each time may lead to a significant amount of radiation.
Extremes of age are more sensitive to radiation and it should be avoided as much as
possible. The average plain abdominal radiograph exposes the patient to a typical effective
radiation dose of 0.7millisieverts (mSv), equivalent to 4months of natural background
radiation which is equal to 35 chest radiographs (Hampson and Shaw, 2010). It is of limited
use in the evaluation of abdominal pain in children but it may be useful in atypical
presentation where no obvious diagnosis can be made after adequate history, examination
and laboratory investigations. It is noteworthy that only 10% of patients with an acute
abdomen have abnormalities on plain radiographs. A study in the adult population
demonstrated that the specificity of abdominal radiograph for acute appendicitis can be as
low as 0% (Hampson & Shaw 2010).
An adequate abdominal radiograph should include the diaphragm and pelvis; anteroposterior and lateral shoot through views may be required if the patient is unable to sit up.
The preperitoneal fat often gives rise to a fine line of fat on a plain abdominal radiograph.
Inflammation of a retrocaecal appendix may be associated with infiltration of the
preperitoneal fat and lead to a focal absence of this fine line of fat. In addition, a mass
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between the preperitoneal fat and ascending colon, gas in the appendix lumen, a faecolith
above the anterior superior iliac spine combined with haustral irregularity of the ascending
colon can raise the suspicion of appendicitis on plain abdominal radiograph. As stated
earlier faecoliths are uncommon in children. Retrocaecal extraperitoneal gas suggests
perforation. Extraluminal gas on radiograph from a perforated appendicitis may be
demonstrable in 1% of perforated cases. Loss of shadow of the right psoas suggests
advanced appendicitis with retroperitoneal inflammation.
An abdominal radiograph may also demonstrate dilated loops of bowel suggesting
obstruction or extraluminal gas in perforation of abdominal viscus. Bowel obstruction in the
absence of features of peritonism may be secondary to adhesive obstruction. It has a
significant role in the evaluation of the neonate with suspected intra-abdominal pathology
where it may demonstrate radiological features of necrotising enterocolitis as clinical signs
would not conclusively demonstrate perforations. In addition, it may demonstrate the renal
outline with a huge outline being suggestive of obstructive uropathy.
B
A
Fig. 3. Plain abdominal radiograph of a 2year old showing: A. Faecolith; B. Focal absence of
fine line of preperitoneal fat (uninterrupted on the left side). Note also, the absence of bowel
gas in the same region.
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9.3.2 Ultrasonography
Where clinical observation by an experienced paediatric surgeon over a 48hr period still
reveals equivocal diagnosis and suspicion of appendicitis persists, imaging is
recommended, mainly by way of abdominal ultrasonography (Lander, 2007).
Ultrasonography for the evaluation of appendicitis was introduced by Puylaert in 1986. It
is a useful investigation in the further evaluation of abdominal pain with atypical and
inconclusive findings. Some authors suggest that its specificity and sensitivity may be
higher in children than in adults (Rothrock & Pagane, 2000). This is particularly relevant
to peripubertal and older girls where ovarian pathology may mimick appendicitis. Even a
left pedunculated ovarian cyst could present with right-sided symptoms if it assumes a
right lower quadrant position. Abdominal ultrasonography can usually be performed
without any sedation and the sonographer can communicate with the child and ask where
the pain or tenderness is maximal. However, this may be distracting in children who
localise pain poorly. The closeness is reassuring to the child and also allows the
sonographer to observe the child’s facial expression or reaction to contact with the
examination probe. Appropriate application of the probe relies heavily on co-operation
from the patient and the graded compression can be limited by the presence of guarding.
In addition, ultrasound is operator dependent and has reduced efficacy in obese patients.
It can achieve up to 98.5% sensitivity, 98.2% specificity, 98.0% positive predictive value
and 98.7% negative predictive value in experienced hands (Strouse, 2010). A repeat
ultrasound in case of persisting clinical borderline suspicion may increase diagnostic yield
(Schuh et al., 2011).
Ultrasonographic features suggestive of appendicitis include:
1. Rigid non-compressible appendix
2. Tenderness on attempted compression
3. Non-peristalsing appendix
4. Appendicular wall thickness of > 6mm
5. Distension of the appendicular lumen
6. Presence of abscess in the peri-appendicular region
7. Increased amount of intraperitoneal fluid
8. Inflammatory changes in surrounding tissues
9. Discontinuity of the appendicular wall
10. Extruded faecolith
11. Thickening of ileum or caecum which may represent part of the inflammatory mass
around the inflamed appendix but may also suggest a diagnosis of Crohn’s disease.
9.3.3 Computed Tomography (CT)
CT has been demonstrated to be more effective than ultrasonography in the diagnosis of
appendicitis and evaluation of abdominal pathology in general. The radiation load from an
abdominal CT remains a hindrance to its widespread application in children. The typical
effective radiation dose from a CT of abdomen/pelvis is 10 mSv (Hampson and Shaw,
2010). For a single abdominal CT study in a 5 year old child, the life time risk of radiation
induced malignancy would be 26.1/100 000 in girls and 20.4/100 000 in boys.
Reported CT sensitivity is 79-98%, increased with intravenous contrast. Luminal contrast
may further improve its sensitivity (Theoni and Thornton, 2007). Kaiser et al., (2002)
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demonstrated that compared to graded compression ultrasound in acute childhood
appendicitis, CT sensitivity is 97%, with accuracy of 95%, negative predictive value of 92%
while ultrasound sensitivity was found to be 80%, accuracy of 89% and negative predictive
value of 88%. CT is also preferable in obese patients and those with significant ileus or
bowel gas. It was found to lead to a reduction in negative appendicectomy rates in children.
The negative appendicectomy rate without imaging was found to be 14%, 17% with
ultrasound but reduced to 2% with CT. No difference was observed in perforation rate
(Theoni and Thornton, 2007). Lower abdominal CT should be performed with intravenous
contrast where possible. Features suggesting appendicitis include (Theoni and Thornton,
2007):
Appendicular diameter of more than 6mm
Presence of inflammatory changes in the peri-appendicular area combined with a
dilated or thickened appendix
Inflammatory changes extending to the psoas muscle
A calcified faecolith may be seen
There may be free fluid with or without an enhancing rim suggesting abscess
Thickened caecum and terminal ileum with inflamed appendix
Periappendicular fat stranding
Air in the appendix wall, retroperitoneum or abdomen associated with inflammatory
changes in the area around the appendix
Advanced appendicitis may give CT findings of pericaecal phlegmon or abscess
The right lower quadrant may demonstrate free air which suggests perforation.
Early appendicitis may not be distinguishable from normal appendix because the features
mentioned above would be absent. Consequently, failure to visualise the appendix
radiologically does not rule out acute appendicitis. It is noteworthy that air within the
appendix lumen may be normal in the absence of other features of periappendicular
inflammation and the appendix may not be demonstrable in the presence of focal
inflammatory changes of the appendix. Thickening of the wall of the appendix observed on
axial images as three concentric rings or as single thick ring of enhancement with or without
periappendicular soft tissue stranding may be the only feature present. Disadvantages of CT
include the following:
Risk of radiation.
CT costs more to perform
Patients are at risk of allergic reaction to the contrast agent
It takes longer to perform
It may have a lower sensitivity in patients with low body fat (Rothrock & Pagane, 2000).
9.3.4 Radionuclide scanning
Radionuclide scanning using 99mTc-hexamethylpropyleneamine oxime (HMPAO) labelling
of patient’s leucocyte or technetium-99m-labelled antigranulocyte antibodies can be used to
evaluate abdominal pain in children presenting with equivocal clinical and laboratory
findings. Accumulation of the radionuclide material in the right lower quadrant of the
abdomen indicates positivity for appendicitis. The sensitivity is between 91-94% and
specificity is 82-94%. The disadvantages to its use include the issue that it is not universally
available, takes long to perform and interpretation of the scan is operator dependent (Sarosi
& Turnage, 2002 ).
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9.3.5 Contrast studies
A contrast enema is not usually employed in children for the diagnosis of acute appendicitis
because it is unpleasant to the child, may require sedation, involves contrast going through
probably inflamed bowel and may not contribute much to the evaluation following the use
of other radiological investigations. If it is done, it may show failure of the appendix to fill
with contrast. However, 10-20% of normal appendixes do not fill during contrast study.
False negative result may be caused by distal appendicitis at the tip without proximal
obstruction or partial obstruction in early appendicitis. It may demonstrate right colonic or
terminal ileal mucosal changes secondary to infective enterocolitis e.g from Yersinia
enterocolitica, Salmonella spp. Shigella spp. Campylobacter spp. Bacteroides spp.
Escherichia coli, as well as changes due to Crohn’s disease or non-specific inflammatory
bowel disease. It may compliment CT and US in equivocal cases, particularly in recurrent
abdominal pain. An upper gastrointestinal contrast study may be used to evaluate the
rotational status of the midgut in such cases.
Contrast studies offer advantages of being
1. Simple
2. Safe
3. Readily available where ultrasound and CT are not available
Disadvantages include:
Up to 40% of barium studies may be equivocal where CT and US have been equivocal
(Liu and McFadden, 2003)
In the presence of perforation, contrast may extravasate into the peritoneal cavity
It takes time to set up
It may require sedation.
9.3.6 Laparoscopy
Up to 59% of patients with right lower quadrant pain may have appendicitis confirmed at
laparoscopy for suspected appendicitis. 35% of the females with suspected appendicitis may
be found to have gynaecological pathology at laparoscopy (Liu and McFadden, 2003).
Laparoscopy also offers the advantages of direct inspection of all the intra-abdominal
organs as well as the opportunity to treat the identified pathology where appropriate.
9.4 Clinical scoring systems
Several scoring systems have been put forward to facilitate the diagnosis of appendicitis.
Unfortunately, paucity of validation studies limits their clinical application. It should be
borne in mind however that achieving a maximum score in any of the scoring systems may
still lead to a negative appendicectomy. Two of these are discussed.
The Paediatric Appendicitis Score (PAS) for the evaluation of children aged between 415years with probable appendicitis is based on scores assigned to the clinical history,
presenting signs and laboratory results. A score of ≤5 implies the diagnosis is unlikely to be
appendicitis; ≥ 6 is compatible and 7-10 indicates a high probability of appendicitis. PAS has
been advocated and shown to reduce the rate of normal appendicectomy to less than 5%
giving a mean score of 3.1 ± 1.1 in non-appendicitis cases and 9.1 ± 0.1 in appendicitis
(Samuel, 2002). Samuel (2002) also demonstrated that the PAS had a sensitivity of 100%,
specificity of 87%, positive predictive value of 90% and negative predictive value of 100%.
Table 1 shows the details of the scoring system.
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Diagnostic indicator
Tenderness with cough or percussion or hopping
Anorexia
Pyrexia
Nausea/ vomiting
Tenderness in right lower quadrant
Leucocytosis ≥ 10,000 (109/L)
Neutrophilia
Migration of pain
PAS (maximum 10)
2
1
1
1
2
1
1
1
Table 1. Paediatric Appendicitis Score
Similarly, the Alvarado score (Table 2) employs clinical and laboratory values in predicting
the possibility that the cause of abdominal pain is acute appendicitis. Shreef et al., (2010) in
their review of 350 children demonstrated that with an Alvarado score of ≥6, the sensitivity
of the scoring system could be as high as 100%, specificity 84.4%, positive predictive value
of 83% and accuracy of 91.1%.
Diagnostic indicator
Tenderness in right iliac fossa
Rebound tenderness in right iliac fossa
Anorexia
Pyrexia >37.3
Nausea/Vomiting
Leucocytosis
Neutrophilia (>75%)
Migration of pain
Alvarado score(maximum 10)
2
1
1
1
1
2
1
1
Table 2. Alvarado Score
10. Treatment
10.1 Suspected appendicitis
Where a definite diagnosis is not reached following history taking and examination in a
child with significant symptoms, admission for observation should be undertaken. The
child should be managed according to symptoms with analgesia and rehydration therapy
where appropriate. The gastric emptying in children with inflammatory intestinal
problems is delayed, therefore, these patients should be kept on clear liquid diet to avoid
aggravating the condition and also to minimise the risk of aspiration during induction of
anaesthesia should this subsequently becomes necessary. Surana et al., (1995)
demonstrated that active observation of children with suspicion of appendicitis was not
associated with a significant increase in complication rate; 5.5% vs. 4.2% in those
diagnosed at presentation. Moreover, after the inflammation reaches the submucosa, it
progresses quickly to involve the rest of the appendix (Fenglio-Preiser et al., 2008).
Therefore, hospital admission and active observation is recommended with regular
review of the patient at intervals of 4-6hours.
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10.2 Immediate treatment
The immediate management of a child with presumed acute appendicitis should include
resuscitation, analgesia +/- abdominal decompression with a nasogastric tube. The child’s
clinical status should be evaluated to determine the appropriate level of care most suitable
for the individual child. Some children would require level 2 intensive care nursing, or
higher, before and/or after surgical treatment. Fluid resuscitation should be commenced
and the child should be well-hydrated to ensure safe surgery. Broad spectrum antibiotics
should be administered once the diagnosis of acute appendicitis has been made and surgery
planned. There is evidence that commencing antibiotics at least 4hours before surgery
reduces the risk of post-operative wound infection particularly when the duration of
symptoms is longer than 48hours (Krukowski et al., 1987; Lander et al., 1992). Using a
protocol involving adequate fluid resuscitation and a minimum of two pre-operative doses
of antibiotics (Coamoxiclav +/- Gentamicin), Cleeve et al., (2011) demonstrated a
complication rate of 6% in children with advanced appendicitis. The choice of antibiotics
should cover the micro-organisms expected at the site of infection as described in the
microbiology section of this chapter. Commonly, a third or fourth generation cephalosporin
is used with or without a penicillin. An aminoglycoside, often Gentamicin, should be added
where there are features suggesting advanced appendicitis. In the supine position, the
lowermost levels of the peritoneal cavity are the right subphrenic space and the pelvic
cavity. In peritonitic patients the rate of absorption of toxins from the intraperitoneal
infection can be reduced by keeping them in the 45˚ position to encourage gravitation into
the pelvis where the rate of toxin absorption is slow (Snell, 2004).
10.3 Conservative treatment
Delayed diagnosis is associated with higher rate of perforation, pelvic abscess, longer
duration of hospital stay, delayed return to normal activities and greater risk of adhesive
bowel obstruction. Up to 30% of children under 3years of age present with appendix mass
with a duration of symptoms usually longer than 4-5days (Stevenson, 2003). In cases with
long duration of symptoms, ultrasound should be performed before planning surgery if the
clinical status of the abdomen precludes adequate palpation, or if the presence of a mass
cannot be excluded. In the presence of a clinically palpable or radiologically identified
appendicular mass and absence of gross peritonitis, conservative management with broad
spectrum intravenous antibiotics can be safely undertaken. Hoffman et al., (1984)
demonstrated that up to 80% of patients successfully managed with antibiotics for an
appendix mass required no further treatment, 14% of these presented with recurrent
abdominal pain not related to appendicitis; 20% had recurrent appendicitis and 66% of these
occurred within 2 years of initial treatment. Swain et al., (2005) also demonstrated that an
appendix-related abscess of ≤ 2cm can be successfully treated conservatively. Larger
abscesses should be drained whenever this can be safely undertaken either by radiologyguided approach or surgically using laparoscopy or into the rectum.
Careful monitoring of physical signs, both local and systemic should be undertaken at
regular intervals. The temperature, heart rate, respiratory rate, abdominal tenderness and
size of inflammatory mass should be observed. Laboratory investigations should be used to
compliment clinical findings. Repeat radiological investigations may also be required. The
resolution may take a few days to become evident though generally a definite improvement
should be noticed after 48 hours. If the acute appendicitis settles, interval appendicectomy
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should be performed within 6 weeks to 3 months. For those who show persistent or
worsening clinical signs, early appendicectomy should be undertaken to avoid more serious
complications.
10.4 Definitive surgery
Complications of appendicitis include pyelophlebitis, portal venous thrombosis, cholangitis,
liver abscesses and bacteraemia. Also, fistula formation may result from appendicitis
including enteroenteric, enterovaginal, enterocutaneous and enterovesical fistulae.
Therefore, in the presence of strong suspicion of appendicitis, it is less of a clinical risk to
undertake the removal of a normal appendix than expose the patient to the significant
morbidity associated with advanced appendicitis. A negative appendicectomy rate of 5-10%
can be expected (Stevenson, 2003). Oyetunji et al., (2011) observed a reduction in the
negative appendicectomy rate over the years from 8.1 % in 2000 to 5.2% in 2006, being
higher in rural areas, younger children, and girls. Of patients with negative
appendicectomy, 12% may have a different surgical condition, 18-20% may have nonsurgical pathology and 60% may have no identifiable pathology. Complication rate for
negative appendicectomy may range from 5-15% including wound related problems,
pulmonary complications, urinary tract infection and small bowel obstruction (Sarosi &
Turnage, 2002).
Following induction of anaesthesia, palpation of the abdomen should be undertaken. In the
presence of a clearly defined mass which was not identified earlier, further management
would involve two main secondary options: to continue with the planned surgery, or, to
defer the operation and further evaluate the child with treatment using intravenous
antibiotics. The latter view was strongly expressed by Surana and Puri (1995). Gillick et al.,
(2001) found that children who had a palpable mass under anaesthesia, which was not
diagnosed clinically earlier, had a shorter duration of symptoms (mean 2days) than those
with clinically palpable or ultrasound diagnosed mass (mean 4days). In their series, half of
the children aged ≤ 2years and one-third of those ≤ 3years had an appendix mass present at
the time of first evaluation. 15.8% of their patients failed to settle with conservative
management, 41.5% of whom had abscess drainage followed by appendicectomy, while 26%
required early appendicectomy; 50% of these had post-operative complications. 10% of
those who settled with conservative management had recurrent appendicitis. Considering
the short duration of symptoms associated with a mass that was not palpable before
anaesthesia, the author recommends that surgical treatment should proceed in these cases;
having commenced antibiotic therapy at least 4hours before surgery where the duration of
symptoms was longer than 48hours as suggested above. This recommendation is also given
by Stevenson, (2003) and adopted by many paediatric surgeons in the United Kingdom.
10.4.1 Anaesthetic considerations
Appendicitis is usually an acute illness in otherwise healthy persons. It is often associated
with gastroparesis and a patient who is admitted for observation for a probable diagnosis of
appendicitis should be given fluid diet if not nil per oral as the stomach may not empty as
well as in other conditions. Intraoperative precautions should be observed as for patients
with a full stomach with rapid sequence intravenous induction of anaesthesia (Oberhelman
& Malott, 2004). Once anaesthetised, the stomach should be promptly emptied with a
nasogastric tube. The presence of associated peritonitis and abdominal distension may lead
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to splinting of the diaphragm which in turn reduces the functional lung volume. Respiratory
impairment may be present especially in very young children. Tachypnoea may be a
manifestation of respiratory embarrassment, pain, dehydration or sepsis. The circulatory
system may be affected by hypoperfusion from associated fever, vomiting, diarrhoea or
nausea with resultant reduced oral intake. This may manifest as increased heart rate and
end organ signs including increased capillary refill time, reduced peripheral temperature,
dry mucous membranes and reduced urine output. Preoperative correction of any
hypovolaemia is mandatory for safe anaesthesia. There may be coexistent electrolyte
imbalance and this also needs to be appropriately corrected preoperatively (Oberhelman &
Malott, 2004).
Muscle relaxation is required to facilitate surgery whether open approach where muscle
splitting is applied or laparoscopy which requires adequate exposure by
pneumoperitoneum using the lowest possible intra-abdominal pressure. The physiological
challenges posed by the pneumoperitoneum required for laparoscopic surgery needs careful
attention from the anaesthetists (Nwokoma & Tsang, 2011).
10.4.2 Laparoscopic approach
Since the description of laparoscopic appendicectomy by the German gynaecologist Kurt
Semm in 1983 (Semm, 1983), this approach to appendicectomy has continued to gain wide
acceptance. With the advances in laparoscopic surgery in recent years, it has become
common practice in many centres to have laparoscopic approach to appendicectomy in the
absence of contraindications (Table 3).
Patient unsuitable for open surgery
Uncontrolled bleeding or coagulation problems
Multiple previous abdominal surgery
Table 3. Contraindications to paediatric laparoscopy
Where the child presents with features of advanced appendicitis with bowel obstruction,
this may constitute a relative contraindication to the use of laparoscopy due to increased
risk of injury to the dilated bowel loops. Previous abdominal surgery predisposes the
patient to intra-abdominal adhesions which increase the risk of bowel injury and bleeding
but this risk is less if the previous surgery was performed laparoscopically (Nwokoma et al.,
2009b).
Laparoscopic approach has been safely used to treat advanced appendicitis in children with
results similar to that in open approach. We demonstrated that laparoscopic approach
offered significant advantages with better outcomes than open approach in paediatric
advanced appendicitis with less wound-related complications: 8.6% versus 17.6%
(Nwokoma et al., 2009a), and a conversion rate of 0%. Brügger et al., (2011) and Garg et al.,
(2009) drew similar conclusions from their studies. Brügger et al., (2011) further
demonstrated the rate of wound infections (0.50% vs. 6.98-7.97%), post-operative ileus
(0.15% vs. 0.33%), urinary complications (0.13% vs. 0.66%) and pulmonary complications
(0.18 vs. 1.19%) to be lower in their group of laparoscopically treated appendicitis than data
from large studies using the open approach.
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The age-long principles of safe surgery include quick and adequate access, adequate target
organ visualisation and minimal tissue trauma. In children, access can be quite a challenge
because of the smaller height/width ratio of the abdomen particularly observed in those
under 8years of age. In many cases, however long the incision, gaining access to the target
organ or indeed to the four quadrants of the abdomen and pelvis, can be very difficult.
Laparoscopy offers the paediatric surgeon the advantage of been able to visualise these
areas while reducing the trauma usually consequent upon use of large abdominal wall
incisions (Nwokoma & Tsang, 2011).
There is growing evidence that laparoscopy has more advantages and benefits to offer
children than was earlier presumed to be the case. These benefits have been widely reported
(Table 4) and significantly outweigh any concerns regarding the technical difficulties (Table
5) which are largely overcome with increasing experience and further developments in the
laparoscopic equipment.
Reduced wound size
Reduced wound trauma
Less wound infection
Less incisional hernia
Less wound dehiscence
Less wound pain
Early mobilisation
Less bleeding
Less heat loss from tissue
Wider field of vision
Less postoperative adhesions
Less postoperative ileus
Earlier return to usual activities
Earlier commencement of chemotherapy
Less respiratory complications
Less risk of thromboembolism
Reduction in nerve entrapment
Table 4. Advantages of laparoscopy
Loss of tactile sensation
Loss of spatial and depth orientation
Two-dimensional imaging
Difficulty with control of bleeding
Difficulty with extraction of resected tissue or organ
Table 5. Technical difficulties of laparoscopy
A 10mm primary port should be inserted using the Hasson’s open technique either in the
suprapubic region, half way between the symphysis pubis and the umbilicus making sure
that the urinary bladder is not in the path of entry or in the umbilical region – centrally or
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infraumbilically. Two secondary 5mm ports should be inserted under laparoscopic guidance
in the left lower quadrant for instruments. Alternatively, with an umbilical primary port,
each of the two secondary ports can be placed on either side in the left and right lower
quadrants. Single port transumbilical laparoscopy-assisted appendicectomy is gaining
popularity and has been demonstrated to give results comparable to standard laparoscopic
appendicectomy for uncomplicated appendicitis (Guanà et al., 2010). It has been successfully
used to treat uncomplicated appendicitis as day case procedures (Alkhoury et al., 2011).
Local anaesthetic injection into the port sites is advisable. Safe pneumoperitoneum should
be established with 5-8mmHg in the newborn, 10-12mmHg in infants and <15mmHg in
older children (Nwokoma & Tsang, 2011). Pus can be obtained with the suction device for
microbiological analysis. The appendix is dissected free, the appendicular vessels divided
by diathermy cauterisation or between endoclips. Ligation of the appendix should be
carried out with three endosurgical loops; two proximally and one distally, as close to the
base as possible to avoid the complications of stump appendicitis and enterocutaneous
fistula (Lintula et al., 2002). Stump appendicitis which can occur following open or
laparoscopic appendicectomy may occur in residual appendix as small as 6mm (Waseem &
Devas, 2008) and is associated with significant morbidity. Cauterisation of the appendicular
stump may prevent later formation of a mucocele. All incisions ≥ 5mm should be closed
with absorbable sutures to the deep fascia and subcutaneous tissue to avoid port site hernia.
Advanced appendicitis poses a significant challenge for the paediatric surgeon and many
opt for the open approach if this is suspected preoperatively. This is because the abdomen in
children is shorter in height and relatively wider than in adults, especially children younger
than eight years of age which is the group that commonly present with advanced
appendicitis. However, as we demonstrated above, advanced appendicitis can be safely
managed laparoscopically in children with outcome comparable to those of open approach.
An inflammatory mass may be present during surgery and this can be drained
laparoscopically with good vision of all four quadrants of the abdomen. Following
laparoscopic drainage of the abscess, liberal peritoneal lavage should be performed as
appropriate and the inflammatory mass should be assessed with regards to safety of
continuing with the operation. Where the tissues are very friable, it is preferable to postpone
the appendicectomy and treat with intravenous antibiotics with a view of performing
interval appendicectomy safely at a later date. It is preferable to place the patient in a
reverse Trendelenburg position and drain the purulent material from the pelvic cavity
before putting the patient in the Trendelenburg position required for good access for the
appendicectomy. This practice should reduce the risk of post-operative subphrenic,
subhepatic and parasplenic abscesses.
Following laparoscopic appendicectomy in 7446 cases (age range between 12 and 100years),
Brügger et al. (2011) observed an overall complication rate of 8.63% with individual
complications detailed in Table 6.
10.4.3 Open appendicectomy
The open approach to appendicectomy has been established for over a century. The first
recorded appendicectomy was performed by Claudius Amyand in 1735 at St. George’s
Hospital in London (United Kingdom) where he removed an appendix containing a calcified
mass around a pin in a patient presenting with inguinal hernia. Lawson Tait performed the
first successful appendicectomy for appendicitis in 1880 (Williams and Myers 1994).
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Complication
Intraoperative complications
% frequency
1.88
Haematoma/intra-abdominal bleeding
Haematoma/ abdominal wall bleeding
Injury to intra-abdominal organ
Injury to stomach/ bowel
Vascular injury
Inadvertent bowel puncture by trocar
Inadvertent puncture by Veress needle
Other intraoperative complications
0.6
0.28
0.13
0.08
0.07
0.07
0.01
0.63
Postoperative Complications
6.75
Surgical postoperative complications
Abscess
Peritonitis
Paralytic ileus
Haematoma/intra-abdominal bleeding
Haematoma/abdominal wall bleeding
Haematoma/bleeding requiring transfusion
Wound infection
Obstructive ileus
Intestinal perforation
Stricture
Other surgical complications
4.24
0.98
0.59
0.56
0.50
0.34
0.13
0.50
0.15
0.04
0.01
0.44
General postoperative complications
Cardiac complication
Pulmonary embolism
Urinary tract infection
Jaundice
Pneumonia
Deep vein thrombosis
Stroke
Nerve compression
Other general postoperative complications
2.51
0.36
0.15
0.13
0.05
0.03
0.01
0.01
0.01
1.75
Table 6. Complication rates following laparoscopic appendicectomy
The commonly applied incision in children is the Lanz incision. It is an almost transverse
incision in the right lower quadrant, about 2cm above and medial to the anterior superior
iliac spine with its centre in the McBurney’s point. The Lanz incision is more popular in
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children than the gridiron incision which also has its centre as the McBurney’s point but
runs perpendicular to an imaginary line between the anterior superior iliac spine and the
pubic tubercle. This is because the Lanz incision has better cosmesis and healing, being
along the Langerhan’s lines. It also offers the surgeon the qualities of a good incision
including easy and quick access to the abdominal cavity, extendable if required and easy to
close. It crosses less dermatomal regions making the post-operative pain less and easier to
control. As mentioned earlier, the abdomen of young children is relatively wider than its
height on the longitudinal axis. Therefore access to the abdominal organs during surgery is
best achieved by an incision that can go across the abdomen; the Lanz incision offers this
advantage. Some authors advocate palpating the abdomen just before induction of
anaesthesia and placing the incision just below the point of maximum tenderness. The
problem with this is that the point of maximum tenderness usually marks the appendicular
tip and may be far from the base. This may result in a longer than necessary incision. For
example a pelvic appendix tip may give maximum tenderness suprapubically and a
retrocaecal appendix may give maximum tenderness in the right upper quadrant. An
incision over the region of the base of the appendix works best with various positions of the
appendix body and tip. In certain situations, lengthening of the incision is necessary to
perform a four-quadrant examination and drainage of associated pus. The Lanz incision
allows such an extension of the incision to be undertaken safely and effectively.
Muscle splitting is preferable to the muscle cutting approach because the reduced tissue
trauma is associated with reduced risk of bleeding, infection and post-operative pain. The
peritoneum should be entered between clips, avoiding damage to the underlying bowels by
ensuring bowel clearance from the edges of the clips. A microbiological swab of the
peritoneal fluid should be taken, preferably from the appendix itself to increase microorganism yield. Pus is the creamy-yellow viscid fluid present in infected tissues which
consists of bacteria – living and dead, dead polymorphonuclear leukocytes, extravasated
plasma and damaged host cells or tissue debris (Eykyn, 1998). If purulent material is
present, a sample of it should be sent for analysis as well as a swab sample. The caecum
should be identified and the anterior taenia followed inferomedially to the appendix base.
Any inflammatory adhesion should be carefully released by blunt digital dissection. The
peritoneal folds along the lateral and inferior borders of the caecum may need to be divided
to adequately mobilize the caecum and deliver the appendix into the wound, especially so
when it lies retroperitoneally. The mesoappendix is narrowest at the tip and widest at the
base with the appendicular vessels within its edge. Ligation of the vessels usually
commences from the tip towards the base. This is the antegrade dissection. In some cases
retrograde dissection from the base may be required for safe appendicectomy. The appendix
base should be crushed with a straight clamp as close to the caecum as can be safely
achieved. Reapplying the clamp just above the crushed portion, the appendix should be
transfixed and ligated with strong absorbable suture material, then cut above this.
Cauterisation of the appendicular stump reduces risk of formation of a mucocele. Inversion
of the appendicular stump with a purse string suture or a Z- stitch anchored within the
taenia coli on the caecum adjacent to the base is still common practice. Taking too much
caecum into the purse string suture or Z-stitch may lead to the development of a mucocele
or become a lead point for intussusception (Swain, 2005). With local purulent peritonitis,
local irrigation is preferable to wide spread lavage in other to minimise any dissemination of
infective agents. On the other hand, if free pus is present, liberal peritoneal lavage is
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recommended. The addition of Betadine (Povidone iodine) or antibiotic agent(s) to the
lavage fluid is widely practiced. However, it is noteworthy that Schneider et al., (2005)
reported no significant advantage from the use of adjuvant peritoneal Taurolidine lavage in
children with appendicitis associated with localised peritonitis. Local anaesthetic injection
into the wound at this point compliments immediate post-operative analgesia. The
abdominal wall should be closed carefully with absorbable suture material in layers or as
mass closure. Subcuticular absorbable sutures should be used to close the skin. Pauniaho et
al., (2010) demonstrated a reduced incidence of wound-related complications in acute
appendicitis using subcuticular absorbable sutures than with the use of non-absorbable
sutures.
The complication rate following open appendicectomy in children varies with age and
severity of the appendicitis. Intra-abdominal abscesses may complicate up to 20% of
perforated appendicitis; wound abscess <5%; faecal fistula <1% and wound haematoma
<0.5%. Other complications include intestinal obstruction, missed bowel injury and
bleeding. Mortality for non-perforated appendicitis is <0.1% and for perforated appendicitis
this rises to up to 2% (Oberhelman & Malott, 2004).
10.5 Post-operative management
Careful monitoring of the patient in the post-operative period should follow the principles
of management of the critically ill surgical patient. As stated earlier, level 2 (or higher)
nursing care may be required. Careful management of respiratory and cardiovascular
system should be continued. In the very young patient a urinary catheter may be a useful
adjunct to fluid management and opiate analgesia may make the child prone to urinary
retention. A nasogastric tube may be required if features of bowel obstruction are present. A
peripherally inserted central line may be inserted intra-operatively if prolonged antibiotics
or significant delay to return of bowel function is anticipated.
Post-operative analgesia may initially be administered as a patient or nurse-controlled
intravenous opiate analgesia. Where advanced appendicitis has necessitated a wide incision
and laparotomy, an epidural analgesia may be preferable. If epidural analgesia is used, a
urethral catheter should be placed. Oral analgesia should be introduced when
gastrointestinal function returns.
The administration of antibiotics for any reason can potentially upset the balance of the
normal gastrointestinal flora. This may create an environment that is favourable for the
multiplication of exogenous pathogens as well as the overgrowth of select pathogenic
strains. Antibiotic-related complications are common with use beyond 5 days (Mui et al.,
2005).
Principles for the selection of antibiotic therapy (Raftery, 2002) are as follows;
There should be clinical evidence of infection
Best guess antibiotics to cover known likely infective micro-organism(s)
Where possible, remove infected tissue or foreign body
Appropriate specimen collection from the site of infection for microbiology examination
Cheapest and most effective drug or drug combination with known effectiveness over
known likely organisms
Monitoring of clinical response to treatment
Appropriate route to achieve therapeutic levels of drug at site of infection
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Duration of administration should cover acute infection period, avoiding prolonged
antibiotic treatment
Re-evaluate clinical response with microbiology result and change antibiotics if
clinically indicated.
Appendicectomy creates a contaminated wound with an infection risk of 12%. In the
presence of pus or a perforation, a dirty wound results, with infection risk of 25% (Raftery,
2002). Perioperative antibiotics administration should follow local sensitivities, usually –
Amoxicillin/ Gentamicin/ Metronidazole or a cephalosporin given instead of Amoxicillin.
The former combination is used in our institution. Antibiotics should be given at least one
hour before the skin incision is made to ensure adequate therapeutic plasma levels. Further
antibiotic therapy should be based on intraoperative findings. In the presence of normal
looking appendix, no further antibiotic is required. Single dose combined antibiotic therapy
has been demonstrated to be adequate surgical prophylaxis in non-perforated appendicitis
(Mui et al., 2005). In addition, Lee et al., (2010) observed that single or double agent
antibiotics were effective and of lower cost than triple therapy. The author’s
recommendation in the case of an inflamed non-perforated appendix with no pus present is
that a 24hr antibiotic therapy of single or double agents be given. This is because inflamed
bowel is known to be associated with some micro-organism translocation. If heart rate and
temperature remain within normal limits at 24 hours, antibiotics can be discontinued. A
perforation may not be evident on resected specimen due to the extensive inflammation
(Fenoglio-Preiser et al., 2008). In a cutaneous abscess, it is possible to clear out the pus. In the
abdominal cavity, this is not possible and one must assume that infective agents remain free
in the peritoneal cavity even after extensive peritoneal lavage. Five days of intravenous
antibiotics is recommended in the presence of pus or an obvious perforation, preferably a
triple agent therapy. The results of the microbiology analysis of any pus sample should be
ascertained before the end of the five-day antibiotic therapy There is evidence that
intraperitoneal abscess formation is commoner with Streptococcus milleri (Feneglio-Preser
et al., 2008) and a longer duration of antibiotics, about 7days, is recommended in these
situations.
Thromboprophylaxis should be administered by mechanical and/or chemical means as
appropriate to each patient. In particular, older children above average weight or on
contraceptive medication or who smoke should have peri-operative thromboprophylaxis.
Some children show signs of gastric irritation following appendicectomy more with
advanced appendicitis. If features of gastric irritation are observed including new onset
epigastric pain and coffee-ground appearance of the vomitus, H-2 antagonists or proton
pump inhibitors should be given to cover the period of acute illness till symptoms resolve.
11. Special considerations
11.1 The normal appendix: Remove or not remove?
Since the introduction of the Antegrade Colonic Enema procedure to aid the management of
functional problems of the large bowel, the need to preserve the normal appendix
particularly in children has been the subject of much discussion. Children without
functional bowel problem or spina bifida at the time of presentation are unlikely to require
the ACE procedure in the future. The appendix is also used for urinary diversion or vesicocutaneous channel in the Mitrofanoff procedure and for biliary drainage (Swain, 2005).
Arguably, while this may not be required at the time of surgery, the child’s condition might
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change in the future. The likelihood that a child would need an appendix-related
reconstructive surgery in the absence of a previous health problem is less than the likelihood
of having appendicitis (Morrow, 2005). On this premise, it would appear that incidental
appendicectomy has more benefits to offer by avoiding a future appendicitis.
Contraindications of incidental appendicectomy include impaired immunity, presence of
surgical implants, presence of Crohn’s disease in the adjacent caecum, intra-operative instability,
history of recent abdominal radiation and an inaccessible appendix (Stevenson, 2003).
As discussed earlier, a normal-appearing appendix may be pathological. It is arguable that
the presence of a Lanz incision may imply that appendicectomy had previously been
undertaken which could be misleading with needless delay to the diagnosis of appendicitis
where the appendix had actually not been removed. On the other hand, removing a normal
appendix converts a clean operation into a dirty operation with increased risk of
complications. The author recommends appropriate pre-operative evaluation and the
removal of the normal-appearing appendix discovered intra-operatively.
11.2 Neurogenic appendicopathy
This condition is caused by the proliferation of nerve fibres in the appendix and can only be
diagnosed with certainty on histological analysis. It may be present in up to 4.2% of
specimens removed for presumed appendicitis. It is commoner in girls and older children,
with up to 80% of specimens showing no histological features of inflammation. The use of
antiserotonin or antihistamine therapy is advocated in suspicious cases (Zaupa et al., 2011).
11.3 Inflammatory bowel disease in appendicitis
Crohns disease: The appendix is involved in 25% of Crohn’s disease leading to surgical
treatment. However, Crohn’s disease manifesting as appendicitis at the time of diagnosis is
rare with less than 100 cases reported in literature. 7-10% of these are thought to progress to
Crohn’s disease at other sites. Ulcerative colitis: The appendix is involved in up to 50% of
resected ulcerative colitis specimens. Some of these manifest as skip lesions without caecal
involvement or in continuity with caecal disease (Petras & Goldblum, 1996).
11.4 Acute appendicitis in the neonate
Neonatal acute appendicitis is rare but associated with high morbidity and mortality of
about 50-80%. Diagnosis is often late or missed and found at post-mortem (Swain, 2005). It
may result from the presence of necrotising enterocolitis, cystic fibrosis, Hirschsprung’s
disease or bacteraemia associated with maternal chorioamnionitis (Pressman et al., 2001).
The neonatal anatomy presents special challenge due to its difference from the rest of the
paediatric population. The abdomen in these children is often protuberant due to the flat
diaphragm, shallow pelvis and reduced sacral curvature. Consequently, the organs that
would have been within the rib cage and pelvis are intra-abdominal (Standring et al., 2005).
11.5 Chronic appendicitis
An organising phase of acute appendicitis occurs with the finding of granulation tissue and
a mixture of acute and chronic inflammatory changes as well as recently laid down
connective tissue. However, true chronic appendicitis with lymphocyte and plasma cells
present in the muscularis propria and serosa without significant acute inflammation is rare
(Petras and Goldblum, 1996).
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11.6 Tuberculosis of the appendix
Appendicular tuberculosis occurs in 0.1-3% of patients with tuberculosis but isolated
tuberculosis of the appendix is rare. Appendicectomy followed by antituberculous
chemotherapy is the treatment of choice. Abdominal tuberculosis in children affects the
immunocompromised and those who have not received the BCG vaccine. It manifests with
weight loss, malaise, abdominal distension, abdominal pain, anaemia raised white cell count
and altered albumin: globulin ratio (Rangabashyam et al., 2000)
11.7 Neoplasm of the appendix
Neoplasm of the appendix is found in 1.08 to 1.3% of appendicectomy specimens. The
carcinoid tumour is very rare but it is the most common neoplasm of the gastrointestinal
tract in children. It may be found in 0.3% of paediatric appendicectomies. Mean peak age of
incidence in children is 15years though children as young as 6years old may be affected
(Stevenson, 2003). Carcinoid syndrome comprises of flushing, diarrhoea and cardiac disease.
It is usually associated with liver or retroperitoneal metastasis; with increased urinary 5hydroxyindoleacetic acid. Lymph node metastasis is seen in 4-5% of paediatric patients with
carcinoid tumour but distant metastasis of appendicular carcinoid is very rare in children. It
may also be associated with multiple endocrine neoplasia type 2. (Christianakis et al., 2008).
Adenocarcinoma of the appendix is exceedingly rare. More a problem of older patients, it
develops in the appendicular base with appendicitis from luminal occlusion being the
commonest mode of presentation. 50% are metastasized at diagnosis. It commonly spreads
to the peritoneum directly. Adenocarcinoids are also rare. The histological features are of
combined carcinoid and adenocarcinomas. The treatment of neoplasm of the appendix is
largely limited to appendicectomy. Extension beyond the appendix requires treatment by
right hemicolectomy (Liu & McFadden, 2003). Cystadenocarcinomas are mucin-filled.
Perforation results in mucin-secreting peritoneal deposits manifesting as pseudomyxoma
peritonei which is treated by repeated debulking and eventually fatal. In advanced cases,
the abdomen is filled with tenacious semisolid mucin.
11.8 Recurrent appendicitis
Recurrent appendicitis is becoming increasingly accepted as a diagnosis. Appendicitis like
any other inflammation in the human body may become arrested and not progress to fullblown process. To lend support to this, approximately a quarter of patients with
histologically proven acute appendicitis report a history of prior episodes of abdominal pain
of similar character as that which culminated in appendicectomy. Furthermore, sometimes
histopathological analysis of acute appendicitis specimen shows both acute and chronic
inflammatory characteristics. Also, about 60% of patients who respond well to treatment of
advanced appendicitis report abdominal pain suggestive of recurrent appendicitis prior to
interval appendicectomy (Swain, 2005).
11.9 Antibiotic-associated Clostridium difficile infection
Clostridium difficile (C. diff.) is a spore-forming gram positive rod. It produces its
pathogenic features by the production of toxins. Toxin A is an enterotoxin while toxin B is a
cytotoxin. Diagnosis is by detecting these toxins in stool. C. diff. toxins can be detected in
the stool of 2-5% of the general population and up to 50% of infants. Its clinical significance
is anchored on being the causative agent of antibiotics associated diarrhoea included in this
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chapter as a likely complication of prolonged perioperative antibiotic administration. It has
been found to be responsible for approximately 30% of cases of the simple uncomplicated
diarrhoea that often follows antibiotic administration. It is associated with 90% of cases
where pseudomembranous colitis is present. Alteration of colonic flora especially by
Ampicillin or Cephalosporins and Clindamycin favours the proliferation and virulence of C.
diff. The clinical manifestation of C diff. colitis depends on which toxin is predominant in
the colon. In situation of toxin A predominance, watery diarrhoea manifests; with toxin B
predominance pseudomembranous colitis results. C. diff. diarrhoea onset is usually 5-10
days after starting antibiotics but this could range from day 1 to weeks after cessation of the
therapy. Clinical effects may be mild and watery or bloody with or without severe crampy
abdominal pain, raised levels of white blood cells and raised temperature. Treatment of C.
diff colitis consists of discontinuation of implicated antibiotics which would usually lead to
complete resolution of symptoms. However, if there is no response to antibiotic withdrawal
or the patient is severely ill, Metronidazole or Vancomycin given orally is recommended.
The risk of relapse or re-infection requiring repeat treatment may be up to 20% (Ryan & Ray,
2010).
11.10 Appendicitis in cancer patients on chemotherapy
Appendicitis in the neutropaenic child on anticancer chemotherapy is a great challenge. As
much as possible, surgery should be avoided and conservative management with antibiotics
instituted with a plan to perform interval appendicectomy when the child is better. A CT
scan is often required to differentiate this from typhlitis. Joint care should be undertaken
with specialist paediatric oncology staff. Granulocyte Colony Stimulating agents are often
used to improve the neutrophil count. Some authors recommend elective appendicectomy
in patients diagnosed with malignancy who are about to commence chemotherapy if
surgery for other reason was to be performed before chemotherapy with a view of
preventing appendicitis that may occur in neutropenic patients while on chemotherapy.
This practice was not found to be associated with increased complications rate but only 0.2%
of patients who did not have incidental appendicectomy went on to have appendicitis
during a median follow up period of 5years (Morrow, 2005).
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Appendicitis – A Collection of Essays from Around the World
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www.intechopen.com
Appendicitis - A Collection of Essays from Around the World
Edited by Dr. Anthony Lander
ISBN 978-953-307-814-4
Hard cover, 226 pages
Publisher InTech
Published online 11, January, 2012
Published in print edition January, 2012
This book is a collection of essays and papers from around the world, written by surgeons who look after
patients of all ages with abdominal pain, many of whom have appendicitis. All general surgeons maintain a
fascination with this important condition because it is so common and yet so easy to miss. All surgeons have a
view on the literature and any gathering of surgeons embraces a spectrum of opinion on management options.
Many aspects of the disease and its presentation and management remain controversial. This book does not
answer those controversies, but should prove food for thought. The reflections of these surgeons are
presented in many cases with novel data. The chapters encourage us to consider new epidemiological views
and explore clinical scoring systems and the literature on imaging. Appendicitis is discussed in patients of all
ages and in all manner of presentations.
How to reference
In order to correctly reference this scholarly work, feel free to copy and paste the following:
Ngozi Joy Nwokoma (2012). Appendicitis in Children, Appendicitis - A Collection of Essays from Around the
World, Dr. Anthony Lander (Ed.), ISBN: 978-953-307-814-4, InTech, Available from:
http://www.intechopen.com/books/appendicitis-a-collection-of-essays-from-around-the-world/appendicitis-inchildren
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| 17,227 | 116,217 |
W2342988727.txt | DOI: 10.1501/Tarimbil_0000000982
TARIM BILIMLERI DERGISI 2000, 6 (3), 132-134
Yaş l ı Broiler Dam ı zl ı k Sürüsünden Elde Edilen Yumurtalar ı Plastik
Torba İ çinde K ısa Süre Bekletmenin Kuluçka Özelliklerine Etkisi
Okan ELIBOL'
Geli ş Tarihi: 10.04.2000
Özet : Bu araşt ı rma, yaşl ı broiler dam ızl ı klar ından elde edilen yumurtalar ı plastik torba içinde k ısa süreli bekletmenin
kuluçka özelliklerine etkisini belirlemek amac ı yla yürütülmü ştür. Çal ışman ı n yumurta materyali, 57 haftal ı k yaş da olan Ross
Pm3 broiler dam ızl ı kları ndan elde edilmi ştir. Ara şt ı rmada toplam 4500 adet kuluçkal ı k yumurta kullan ı lm ışt ı r. Depolama
süresi olarak 4 gün ve bekletilmeyen (0 gün) olmak üzere 2 grup olu şturulmuştur. Bunun yan ı nda depolama süresi 4 gün olan
yumurtalar ı n yarıs ı plastik torba içinde di ğer yarı sı ise ayn ı koşullarda plastik torbaya konulmadan tutulmu ştur.
Depolama süresi 4 gün olan plastik torbas ı z ve plastik torba içinde bekletilen gruplar ile depolanmayan(depo süresi 0
gün ) grubun ç ı k ış gücü de ğerleri s ı ras ı yla % 86.7, % 90.9 ve % 90.7 olarak tespit edilmi ştir. Gruplar ı n mukayesesi, plastik
torbas ı z olarak 4 gün bekletilen grupta ç ı kış gücünün, di ğer 2 gruptan dü şük olduğ unu ortaya koy ınuştur.(P < 0.05).
Sonuç olarak, ya şl ı broiler dam ızl ı klardan elde edilen yumurtalar ı n ovipozisyon sonial en k ı sa sürede geli şim
makinesine yüklenmesi eğ er bu sağ lanam ıyorsa k ı sa süreli de olsa plastik torba içinde bekletilmesinin ç ı k ış gücünü art ı rd ığı
tespit edilmiştir.
Anahtar Kelimeler: Broiler dam ızl ı k, yumurta depolama süresi, plastik torba, ç ı k ış gücü
The Effect of Short Term Storage in Plastic Bags on Hatchability of Eggs
from OId Broiler Breeders
Abstract : The experiment was conducted to study the effect of storing eggs in plastic bags during short-term
storage(0 and 4 days). The trial involved a total of 4500 hatching eggs from broiler breeder flock aged 57th wks. The eggs
kept for 4 days were divided into 2 groups, one of them was stored in plastic bags(Group 1), other was stored without
plastic(Group 2). Hatchability of fertile eggs, group 1 and group 2 were 90.9% and 86.7% respectively whilst this fıgure was
90.7% for stored 0 days (group 3). Group 2 gaye signif ıcantly lower hatchability than the other two groups(P< 0.05)
This data suggests that for optimum hatchability, eggs from older hens should be set as soon as possible, if it is not
available, even short-term stroge periods, eggs might be stored in plastic bags
Key Words:Broiler breeder, egg stroge period, plastic bag, hatchability
Giri ş
Genellikle broiler civciv üreten kuluçkahanelerde
depolama süresi 1 haftay ı geçmemektedir. Bu sürenin
ç ı k ış gücü üzerine olumsuz etkisinin olmad ığı
bildirilmektedir ( Funk,1950; Oluyemi ve George1972;
Mayes ve Takeballi,1984). Ancak Wilson(1991) ve
Walsh(1993) kuluçkal ı k yumurtalarda maksimum ç ı k ış
gücü de ğ erinin elde edilmesi için yumurtalar ı n
ovipozisyon sonras ı hemen makineye yüklenmesi
gerekti ğ ini belirtmi ş lerdir. Ayr ı ca depolama süresi ile sürü
ya şı aras ı nda önemli seviyede interaksiyon oldu ğ unu,
ya ş l ı broiler anaçlardan elde edilen yumurtalar ı n en k ı sa
sürede geli ş im makinesine konulmas ı gerekti ğ ini, aksi
takdirde ç ı k ış gücünde önemli seviyelerde dü ş ü ş ün
olaca ğı n ı belirten ara şt ı r ı c ı lar da vard ı r(Reis ve ark.
1997, Lapao ve ark 1999).
Ç ı k ış gücüne önemli etkisi olan albümin kalitesinin
depolama ko ş ullar ı ve süresi(Kosin ve Konishi 1973;
Hurnik ve ark. 1978) yan ı nda sürü ya şı ndan da etkilendi ğ i
ve ya ş l ı sürülerin yumurtalar ı nda albümin kalitesinin daha
dü ş ük oldu ğ u belirtilmi ştir ( Burley ve Vadehra 1989;
Walsh 1993; Brake ve ark. 1993, Lapao ve ark 1999 ).
Ya ş l ı sürü yumurtalar ı nda depolama süresinin artmas ı
ile ç ı kış gücünde meydana gelen kayb ı n, albümin
kalitesinin dü ş mesinden kaynaklanabilece ğ i bildirilmi ştir
(Walsh 1993, Brake 1996, Brake ve ark. 1997).
1
Ankara Univ. Ziraat Fak. Zootekni Bölümü - Ankara
Depolama ko ş ullar ı ndan kaynaklanan sorunlar ı en
aza indirebilmek için yap ı lan uygulamalardan birisi de,
yumurtalar ı n depolama süresince plastik torba içinde
bekletilmesidir. Bu uygulama, yumurtada CO2 kayb ı n ı n
azalmas ı ile albümin pH seviyesinin yükselmesini
engelleyerek albümin kalitesindeki dü ş ü ş ün yava ş lamas ı
ve yumurtada a ğı rl ı k kayb ı n ı n azalmas ı sonucu ç ı k ış
gücünü olumlu yönde etkilemektedir(Davis ve Beeckler
1962; Becker ve ark. 1964; Proudfoot ve Hulan 1976).
Plastik torban ı n olumlu etkisinin depolama süresi
uzad ı kça daha belirgin olarak görülebilece ğ ini ifade eden
ara şt ı r ı c ı lar ço ğ unluktad ı r (Davis ve Beeckler,1962;
Becker,1964; Warren ve ark.1965; Proudfoot ,1966; Kirk
ve ark. 1980; Elibol, 1997).
Bu ara şt ı rmada üretimin do ğ as ı gere ğ i bekletme
süresi uzun olmayan ya ş l ı broiler anaçlardan elde edilen
yumurtalar ı plastik torba içinde k ı sa süreli bekletmenin
kuluçka özelliklerine etkisini ortaya koymak
amaçlanm ışt ı r.
Materyal ve Yöntem
Ara şt ı rma bir özel broiler entegre i ş letmesinin
kuluçkahanesinde yürütülmü ştür. Çal ış mada, Petersime
ELiBOL, O. "Yaşl ı broiler dam ızl ı k sürüsünden elde edilen yumurtalar ı plastik torba içinde k ı sa süre bekletmenin
kuluçka özelliklerine etkisi"
(57600 yumurta kapasiteli geli şim, 19200 yumurta
kapasiteli ç ı k ı m makinesi) marka tam otomatik kuluçka
makineleri kullan ı lm ışt ı r.
Ara şt ı rman ı n yumurta materyali, ayn ı entegrasyona
ait dam ızl ı k i ş letmesinde 57 haftal ı k ya şta bulunan broiler
anaçlardan (Ross-Pm3) elde edilmi ştir.
i ş letmede yumurtalar, kümesten kuluçkahaneye
ö ğ le ve akşam olmak üzere her gün 2 parti halinde
getirilmi ştir. Bütün yumurtalar kuluçkahane giri ş inde
fümigasyon i ş lemine tabii tutulmu ş lard ı r. Denemede, 1.
partideki (ö ğ le) yumurtalardan tesadüfi olarak seçilmi ş ve
tasnif i ş lemi sonras ı 20 °C ve °k 75 nispi nem içeren
depoda 4 gün muhafaza edilmi ş yumurtalar ile yükleme
günü ayn ı şekilde toplanan yumurtalar, depolanmaks ız ı n
kullan ı lm ışt ı r. Dört gün depolanacak yumurtalar kendi
içinde rasgele e ş it 2 gruba ayr ı lm ış ve depolama
süresince bir gruptaki yumurtalar plastik torba içinde
muhafaza edilirken, di ğ er grup ayn ı depoda normal
ko ş ullarda bekletilmi ştir. Depolama süresi 4 gün olan
yumurtalar, yükleme günü elde edilen
yumurtalarla(depolanmayan) birlikte önce ikinci kez
fümigasyon i ş lemine ve daha sonrada 25 °C de yakla şı k
8 saat ön ı s ıtmaya tabii tutulduktan sonra ayn ı geli ş im
makinesine yüklenmi ş lerdir.
Çal ış mada, ç ı k ış zaman ı nda ı skarta ve ölü civcivler
ile ç ı kışı olmayan yumurtalar ayr ı lm ışt ı r. Ç ı k ışı olmayan
yumurtalar k ı rı larak döllü olup olmad ı klar ı tespit edilmi ş ,
döllü yumurtalarda ise embriyo ölüm ya şı ( erken
dönem,0-5 gün;orta dönem, 6-17 gün; son dönem
ölümleri, 18-21 gün ve kabu ğ u k ı rı p ölen embriyo) ile
kontamine yumurtalar belirlenmi ştir. Bu veriler
kullan ı larak ba şta ç ı k ış gücü olmak üzere kuluçka
özelliklerine ait de ğ erler hesaplanm ışt ı r.
Ara şt ı rmada toplam 4500 adet kuluçkal ı k yumurta,
her biri 150 adet yumurta alabilen tepsilere dizilmi ş ve her
bir tepsi bir tekerrür olarak de ğ erlendirilmi ştir. Gruplar
aras ı ndaki farkl ı l ığı belirlemek için varyans analizinden,
farkl ı l ığı n hangi gruptan ileri geldi ğ inin tespiti için de
Duncan testinden yararlan ı lm ışt ı r (Düzgüne ş ve ark.
1983).
Bulgular ve Tart ış ma
Sürü ya şı 57 hafta olan broiler anaçlar ı ndan elde
edilen, yükleme öncesi farkl ı süre ve ş ekillerde bekletilen
yumurtalar ı n kuluçka özelliklerine ait ortalama de ğ erler
Çizelge 1 de sunulmu ştur.
Depolama süresi 4 gün (plastik torbas ı z) ve O gün
(depolanmaks ız ı n yüklenen) olan yumurtalar aras ı nda,
erken ve son dönem embriyo dlümleri, ı skarta oran ı ile
ç ı k ış gücü bak ı m ı ndan farkl ı l ığı n önemli (P<0.05) oldu ğ u
belirlenmi ştir. Bu sonuçlar, ya ş l ı sürülerden elde edilen
yumurtalarda optimum ç ı k ış gücü için yumurtalar ı n,
ovipozisyon sonras ı en kı sa sürede makineye
yüklenmesini tavsiye eden Reis ve ark. (1997), Lapao ve
ark: (1999yn ı n bulgular. ile uyum halindedir. Fakat
depolama süresinin 7 güne kadar artmas ı n ı n ç ı k ış
gücünü dü ş ürmedi ğ ini belirten Mayes ve Takeballi
133
Bunun yan ı nda
(1984)'nin bulgular ı ndan farkl ı d ı r.
depolama süresi 4 gün olan yumurtalar ı n plastik torba
içinde bekletilmesi, erken ve son dönem embriyo
dlümleri, ı skarta oran ı ve ç ı kış gücü üzerine olumlu etkide
bulunmu ştur. Nitekim, 4 gün plastik torba içinde
bekletilen grupta ç ı k ış gücü % 90.90 olurken plastik
torbas ı z 4 gün depolanan ve depolanmayan gruplarda
ç ı k ış gücü s ı ras ı yla % 86.70 ve % 90.70 olarak tespit
edilmi ştir(Çizelge 1). Yap ı lan istatistik kontrolde plastik
torbas ız olarak 4 gün bekletilen grubun ç ı kış gücünün
di ğ er 2 gruba göre dü ş ük oldu ğ u saptanm ıştı r(P<0.05).
Bu sonuçlar, depolama süresi k ı sa da olsa, ya ş l ı sürü
yumurtalann ı n plastik torba içinde bekletilmesinin ç ı kış-gücü üzerinde pozitif etkiye neden olabilece ğ ini belirten.,
Brake (1996)'in bulgulan ile de uyum içindedir. Ovipozisyon s ı ras ı nda vizkositesi yüksek ve pH's ı
7.6 civar ı nda olan albüminin, pH değ eri depolama
süresinin artmas ı yla 9.0-9.5' a kadar yükseli ı-(Kosin ve
Konishi 1973; Goodrum ve ark. 1989) ve yo ğ unlu ğ u
azal ı r. Bu değ i ş iklikler albümin kalitesinin dü ş mesi
anlam ı na gelir. Albümin kalitesinin dü ş mesine ba ğ l ı
olarak yumurtan ı n merkezinde bulunan blastodermin
konumu değ i ş ir, kabu ğ a doğ ru hareket eder (Brake 1996)
ve sonucunda erken dönem embriyo ölümleri
artar(Romanoff 1960). Ancak bu de ğ i ş likler, genç ve ya ş l ı
sürü yumurtalar ı nda ayn ı değ ildir. Genç sürülerde,
ya ş l ı lara göre albümin kalitesi daha yüksektir. Ayr ı ca
albümin kalitesinin bozulma ve pH s ı n ı n yükselme h ız ı
daha dü ş üktür( Sauveur 1988;Brake ve ark. 1993). Bir
ba ş ka ifadeyle genç sürü yumurtalar ı nda albümin kalitesi
daha uzun süre muhafaza edilebilmektedir. Bunun
yan ı nda Sauveur ve ark. (1967) ve Walsh(1993) albürnin
kalitesinin belirli bir seviyenin üstünde ve alt ı nda olmas ı
durumunda embriyo ölümlerinin artaca ğı n ı ileri sürerek
optimurn ç ı kış gücü için inkubasyon ba ş lang ı c ı nda uygun
pH seviyesinin 8.2-8.8 aras ı nda olabileceğ ini
bildirmi ş lerdir.
Genç sürülerden elde edilen yumurtalarda albümin
yo ğ unlu ğ u blastoderme oksijen ve besin maddeleri
aktarı m ı n ı s ı n ı rlayacak kadar yüksektir(Meuer ve
Baumann 1988; Brake 1996). Bu durum genç sürü
yumurtalann ı n depolanmadan makineye konulmas ı nda
sorunlar yaratmakta, özellikle erken dönem embriyo
ölümlerini art ı rmaktad ı r(Brake ve ark. 1993; Walsh ve
ark.1995; Brake ve ark. 1997).
elde edilen
sürülerden
karşı l ı k ya ş l ı
Buna
yumurtalarda gençlere göre zaten dü ş ük olan albümin
kalitesi h ı zla azalmakta, pH seviyesi de yükselmektedir.
Albümin kalitesindeki bu h ızl ı dü ş ü ş ün embriyo geli ş imini
engelleyecek seviyeye ula ş mamas ı için de ya ş l ı sürü
yumurtalar ı n ı , genç sürülerden elde edilenlerin aksine, en
k ı sa sürede makineye koymak gerekrnektedir(Brake
(1996; Reis ve ark.1997; Brake ve ark. 1997; Lapao ve
ark.1999). Bu sa ğ lanamad ığı takdirde CO2 ç ı kışı
engellenerek albümin kalitesindeki kayb ı en az seviyede
tutacak yollar aranmal ı d ı r. Bu yollardan birisi de
yumurtan ı n plastik torbada bekletilmesidir. Nitekim,
Brake (1996) de ayn ı yolu önermekte yaln ı z yumurtalar ı
plastik torba içinde bekletmenin sadece; ya ş l ı sürü
yumurtalar ı nda, albürnin kalitesi dü ş ük olan -hatlarda ve
uzun süre depolanacak yumurtalarda olumlu etki
gösterebilece ğ ini belirtmektedir.
134
TARIM BILIMLERI DERGISI 2000, Cilt 6, Say ı 3
Çizelge 1. K ısa süreli depolama ve plastik torba kullan ım ı n ya şlı broiler dam ızl ı kları ndan elde edilen yumurtalarda kuluçka özellikleri (%)
Depolama
süresi
Özellikler
4 gün
Depolama şekli
0 gün
Plastik torbas ız
5.38±0.43a
4.03± 0.43b
Plastik torba
3.84±0.43b
Orta dönem
0.75±0.21
0.97±0.21
0.45±0.21
Son dönem
3.85±0 56
5.78±0.56a
Iskarta oran ı
4.04t0.56b
w
0.53±0.35
b
1.72±0.35 a
Ç ı kış gücü
90.7±0.89b
90.9±0.89b
86.7±0.89b
Embriyo
blüm leri
Erken dönem
0.45±0.35
a-b: P<0.05
Sonuç
Sonuç olarak, ya ş l ı broiler dam ızl ı klardan elde
edilen yumurtalar ı n ovipozisyon sonras ı en k ı sa sürede
geli ş im rnakinesine yüklenmesi, e ğ er bu sa ğ lanam ı yorsa
bunlar ı n, k ı sa süreli de olsa, basit ve kolay bir uygulama
olan plastik torba içinde bekletilmesi tavsiye edilebilir
Kosin, I. L. and T. Konishi, 1973. Pre-incubation storage conditions
and the effect on the subsequent livability of chicken embryos:
Exogenous CO2, plastic bags and extended holding periods
as factors. Poultry Sci. 52: 296-302.
Lapao. C., L. T. Gama. and M. C. Soares, 1999. Effects of broiler
breeder age and lenght of egg storage on albumen
characteristics and hatchability. Poultry Sci 78:640-645.
.
Kaynaklar
Mayes, F. J. and M. A. Takeballi, 1984.Storage of the eggs of the
fowl before incubation. World Poultry Sci. Journal, 40:131140.
Becker, W. A. 1964. The storage of White Leghorn hatching eggs
in plastic bags. Poultry Sci.43: 1109-1112.
Meuer, H. J., R. Baumann, 1988. Oxygen pressure in intra and
extraembryonic blood vessels of early chick embryo.Resp.
Physiol. 71:331-342.
Becker,W. A., J. V. Spencer and J. L.Swartwood, 1964. The preincubation storage of turkey eggs in closed environments.
Poultry Sci.43: 1526-1534.
Oluyemi, J. A. and O. George, 1972. Some factors affecting
hatchability of chicken eggs. Poultry Sci. 51: 1762-1763.
Brake, J. T., J. Walsh, and S. V. Vick, 1993. Relationship of egg
storage time,storage conditions,flock age,eggshell and albumen
characteristics, incubation conditions and machine capacity to
broiler hatchability-Review and model synthesis. Zootech Int.
16(1):30-41.
Brake, J. T., J. Walsh, C. E. Benton., J. N. Petittle, R. Meijerhof., G.
Penalva, 1997. Egg handling and stroge. Poultry Sci. 76: 144151.
Brake. J. T. 1996. Optimization of egg handling and storage.World
Poultry-Misset, vol 12, no 9:33-39.
Burley. R. W. and D. V. Vadehra., 1989. Pages 68-71,372 in: The
avian egg. John Wiley and sons New York.
Davis. G. T. and A. F. Beeckler., 1962. Plastic packaging of eggs.1.
Methods of packaging. Poultry Sci. 41:453-458.
Düzgüneş , O., T. Kesici, F. Gürbüz, 1983. Istatistik Metotlar ı . A.Ü.
Bas ı mevi,Ankara.
Elibol, O. 1997. Kuluçka sonuçlar ı n ı etkileyen etmenler ve kuluçka
aksakl ı klar ı n ı n giderilmesi, belirlenmesi üzerinde ara şt ı rmalar.
Doktora tezi. A.Ü. Fen Bilimleri Enstitüsü(bas ı lmam ış).
Funk, E. M., J. Forward and H. C. Kempster, 1950. Effect of holding
temperature on hatchability of eggs. Missouri Agric. Exp.Stn.
Bull 539,Columbia.
Goodrum, J. W., W. M. Britton and J. B. Davis, 1989. Effect of
storage conditions on albumen pH and subsequent hardcooked eggs peelability and albumen shear strength. Poultry
Sci. 68:1226-1231.
Hurnik, G. I, .B. S. Reinhart and J. F. Hurnik ,1978. Relationship
between albumen quality and hatchability in fresh and stored
eggs.Poultry Sci. 57:854-857.
Kirk., S, G. C. Emmans, R. McDonald and D.Arnot, 1980. Factors
affecting the hatchability of eggs from broiler breeders.British
Poultry Sci. 21:37.
Proudfoot , F .G. 1966. Hatchability of stored chicken eggs as
affected by daily turning during storage and prewarming and
vacuuming eggs enclosed in plastic with nitrogen.Canadian
J.Animal Sci .46:47-50.
Proudfoot ,F. G. and H. W. Hulan, 1976. Care of hatching eggs
before incubation. Pages 1-17 in: Agriculture Canada
Pub.1573/E,Research Station, Kentville,NS, Canada.
Reis, L. H., T. Gama, M. C.Soares, 1997. Effects of short storage
conditions and broiler breeder age on hatchability,hatching
time and chick weights. Poultry Sci 76: 1459-1466.
Romanoff, A. L., 1960. The avian embryo;structural and functional
development.MacMillan Co. New York.
Sauveur, B., R. Ferre, and L. Lacassagne, 1967. Conservation
d'oeufs de poule sous atmosphere enrichie en gaz
carbonique.Ann.Zootech 16:351.
Sauveur, B. R. 1988. Reproduction des volailles et production
d'Oeufs. Institut national da la recherche agronomique,Paris.
Walsh, T. J. 1993. The effects of flock age, storage humidity,carbon
dioxide and lenght of storage on albumen characteristics,
weight loss and embriyonic development of broiler eggs.
Master's thesis. North Caroline State University.NC.
Walsh, T. J., R. E. Rizk and J. Brake, 1995. Effects of storage for 7
and 14 days at two temperatures in the presence or absence
of carbon dioxide on albumen characteristics, weight loss and
early embriyonic mortality of broiler hatching eggs. Poultry Sci.
74: 1403-1410.
Warren, D. C., H. A. Roff and E. Long, 1965. Hatchability of eggs
stored in plastic lined egg cases.Poultry Sci. 44:1278-1280.
Wilson, H. R. 1991. Interrelationships of egg size, chick size,
posthatching growth and hatchability . World's Poultry Sci.J.
47:5-20.
TARIM BILIMLERI DERGISi 2000, 6 (3) 135-140
Türkiyede Üretilen Konik Hüzmeli Baz ı Meme Plakalar ı nda Delik Çap ı ve
Düzgünlü ğ ünün Belirlenmesi
Ergin DURSUN'
Yurdaer KARAHAN
2
Ibrahim ÇILINGIR'
Geli ş Tarihi :28.04.2000
Özet : Konik hüzmeli memeler gövde, süzgeç, girdap plakas ı , meme plakas ı ve başl ı k olmak üzere be ş parçadan
ibarettir. Meme plakas ı ; ilaç da ğıl ı m düzgünlüğ ü, verdi, damla büyüklü ğ ü ve dağı l ı mı nı etkileyen en önemli parçad ı r. Bu
çal ış mada, ülkemizde dört farkl ı üretici firma taraf ı ndan üretilen pülverizatör meme plakalar ı nda delik çaplar ı ve delik
kenar düzgünlükleri belirlenmi ştir. Bundan ba ş ka incelenen plakalarla verdi ve hacimsel s ı vı dağı lı m ı ölçümleri
yap ı lm ışt ır. Araşt ı rma sonuçları na göre plaka delikleri düzgün olmay ı p tam bir dairesellik göstermemektedir. Ayn ı grup
plakalar ı n delik çaplar ı nda farkl ı l ı klar bulunmuştur. ölçülen ortalama delik çaplar ı ile firman ı n çap de ğerleri aras ında
önemli farkl ı lı klar belirlenmi ştir. Plaka delikierinin pürüzlülü ğ ü ve delik çap ı farkl ıl ı kları , verdi ve hacimsel s ı v ı
da ğı l ı mlar ı nda de ğiş imlere neden olmu ştur.
Anahtar Kelimeler : Meme plakas ı , delik çap ı , delik düzgünlü ğ ü
The Determination of the Orifice Diameter and Correctness of Some Cone Nozzle
Orifice Plates Produced ir ı Turkey.
Abstract : Cone nozzles have five parts including body, fı lter, swirl plate, orifice plate and cap. Orifice plate is the
most important nozzle part effecting spray distribution, flow rate and droplet size. In this study, orifice diameters and
correctness of orifice edges of the sprayer orifice plates were determined produced by four different company in Turkey.
Moreover, flow rate and volumetric spray distribution measurements were performed for examined orifice plates.
According to the research results, orifıces of the plates were inccorrect and not perfect circle. Office diameters of the
same group plates were different. The important differences were determined between measured orifice diameters and
company's diameter values. The roughness of orifices and the differences of orifice diameter resulted in variations at
flow rate and volumetric spray distributions.
Key Words : Orifıce plate, orifice diameter, orifıce correctness
Giriş
Memeler pülverizatörlerin en ucuz parçalar ı ndan biri
olmalar ı na karşı n hastal ı k, zararl ı ve yabanc ı otlarla
mücadelede arzulanan biyolojik kontrolün sa ğ lanmas ı nda
oldukça önemli bir etkiye sahiptirler. Pülverizatörün
ba ş ar ı s ı , büyük ölçüde ilac ı n son ç ı k ış noktas ı olan
memelerin, görevlerini uygun bir ş ekilde yapmalar ı na ba ğ l ı
olmaktad ı r. Memelerin verdilerine, damla çaplar ı na ve ilaç
da ğı l ı m paternlerine ba ğ l ı olarak ilaç uygulama etkinli ğ i
de ğ i şmektedir. Memelerin görevi; s ı vı ilac ı olabildi ğ ince
ayn ı büyüklükteki damlalar halinde ilaçlama süresince
verdilerinde de ğ i ş me olmaks ız ı n ve hedef yüzeylerde
düzgün bir ilaç de ğ ilim] olacak şekilde uygulamakt ı r.
Pülverizatörlerde
en
fazla
kullan ı lan
meme
tiplerinden birisi konik hüzmeli memelerdir. Bu memelerde
s ı vı , bir girdap plakas ı veya yivli govdeden geçerek dönü
hareketi kazanmakta ve meme plakas ı deli ğ inden ç ı kan
ince s ı v ı şeridi, eksenel ve te ğ etsel h ız bile ş enlerinin
etkisiyle içi bo ş konik hüzme şeklini almaktad ı r. Içi dolu bir
konik hüzme elde edilmesi için di ğ er bir s ıvı jetinin girdap
odas ı na eksenel yönde girmesi ve meme ç ı kış deli ğ i
içinde olu ş an hava çekirde ğ ini ortadan kald ı rmas ı
gerekmektedir ( Matthews 1992 ). Içi bo ş konik hüzmeli
2
memeler, yüzey kaplaman ı n ve penetrasyonun gerekli
oldu ğ u insektisit, fungusit ve büyüme düzenleyici
hormonlar ı n uygulanmas ı nda, içi dolu konik hüzmeli
memeleler ise ekim öncesi herbisit uygulamalar ı nda
yayg ı n bir ş ekilde kullan ı lmaktad ı rlar (Ozkan 1995).
Konik hüzmeli mernelerde ilaç da ğı t ı mı n ı etkileyen
çok say ı da faktör vard ı r. Meme plakas ı delik ça' p ı , girdap
plakas ı ndaki yar ı klar ı n say ı s ı , girdap odas ı 'yüksekli ğ i ve
s ıvı bas ı nc ı na ba ğ l ı olarak memelerin verdileri , hüzme
aç ı lar ı , damla büyüklükleri ve ilaç da ğı l ı m düzgünlükleri
de ğ i ş mektedir (Matthews 1992). Bu faktörlerin d ışı nda
meme imalat ı ve imalatta kullan ı lan teknoloji de oldukça
önemlidir. Zeren (1974), pülverizatörlerde kullan ı lan konik
ve yelpaze hüzmeli memelerde da ğı l ı m ve pülverizasyon
karakteristikleri üzerinde yapt ığı çal ış mada, yerli yap ı
memelerde süzgeç, meme deli ğ i ve helezon gövde
yap ı lar ı n ı n yeterli düzgünlükte olmad ığı n ı belirtmi ştir.
Meme deli ğ inin pürüzlü olmas ı n ı n ve deli ğ in eksantrik
aç ı lmas ı n ı n, meme plakas ı n ı n meme ba ş l ığı içinde sa ğ a
sola kaymas ı n ı n ve helisel kanallar ı n yeterli düzgünlükte
i ş lenememesinin pülverizasyon karakteristiklerini önemli
ölçüde etkiledi ğ ini vurgulam ışt ı r. Lefebre (1989), düzgün
Ankara Üniv. Ziraat Fak. Tanm Makinalan Bölümü - Ankara
Harran Only. Meslek Yüksekokulu Tar ı m Makirıaları Program ı - Ş anl ı urfa
136
TARIM BILIMLERI DERG İ S İ 2000, Cilt 6, say ı 3
bir ilaç da ğı l ı m ı ve tekdüze bir damla da ğı l ı m ı n ı n hassas
olarak i ş lenmi ş memelerle elde edilebildi ğ ini aç ı klam ıştı r.
Dursun ve Çilingir (1991), meme deli ğ inin tam bir daire
şeklinde ve pürüzsüz olmas ı gerekti ğ ini, çapak, kanalc ı k
ve düzgün aç ı lmam ış deli ğ in ilaç da ğı l ı m ı n ı bozdu ğ unu
aç ı klam ış lard ı r. Naman ve Nordby (1965) yapt ı kları
ara şt ı rmalarda meme ba ş l ığı n ı n meme gövdesine
ba ğ lanmas ı s ı ras ı nda, meme plakas ı ekseninin meme
eksenine göre sapmas ı n ı n ilaç dağı l ı m düzgünlü ğ ü ve
damla büyüklü ğ ünü önemli derecede etkiledi ğ ini
belirtmi ş lerdir. Balc ı ve Ya ğ c ı o ğ lu (1994), s ı rt
pülverizatorlerinde kullan ı lan yerli tip hidrolik memelerin
verdilerini ve da ğı l ı m düzgünlüklerini belirlemi ş lerdir. Verdi
ölçümlerinden elde edilen sonuçlara göre ortalama
verdideki sapman ı n, ele al ı nan 40 memede % 0-5,5,
5 memede % 6,3-10,4, 6 memede ise % 14-21,1 aras ı nda
değ i şti ğ ini belirtmi ş lerdir. Delik çaplar ı n ı n düzgün
olu şturulamamas ı n ı n dairesel da ğı l ı m ı olumsuz yönde
etkiledi ğ ini ortaya koymu ş lard ı r.
Bu çal ış man ı n amac ı , ülkemizde imal edilen konik
hüzmeli meme plakalar ı nda hem delik kenar düzgünlükleri
ve delik çap ı de ğ i ş imleri, hem de bunlar ı n meme verdisi ve
hacimsel s ıv ı da ğı l ı m ı na etkilerini belirlemektir.
Materyal ve Yöntem
Bu ara ştı rmada, deneme materyali olarak ülkemizin
değ i ş ik bölgelerinde faaliyet gösteren 4 farkl ı üretici firma
taraf ı ndan imal edilen konik hüzmeli meme plakalar ı ele
al ı nm ışt ı r. Her meme plakas ı bir anma ad ı ile belirtilmi ştir.
Anma adlar ı s ı ras ı yla Al, A2, B, G1, G2 ve T'dir. Bu
plakalar ı n üretici fı rmalarca belirtilen delik çaplar ı Al ve T
plakalar ı nda 1 mm, A2 ve G2 plakalar ı nda 1,5 mm, B ve
G1 plakalar ı nda 1,2 mm'dir. Bütün plakalar paslanmaz
çelik malzemeden imal edilmi ş lerdir. Her meme plakas ı
grubundan 5'er adet tesadüfi olarak seçilmi ş ve gerekli
incelemeler bu plakalar üzerinde yap ı lm ışt ı r. Meme
plakas ı delik çaplar ı n ı n ölçülmesi için öncelikle plakalar ı n
görüntüleri bir taray ı c ı (scanner) yard ı m ı yla bilgisayar
ortam ı na aktar ı lm ışt ı r. Delik çaplar ı n ı n hassas bir ş ekilde
ölçülmesi amac ı yla görüntüler 15 kat büyütülmü ştür. Daha
sonra bir görüntü i ş leme program ı yla delik çaplar ı her bir
plaka için delik çevresi boyunca 5 farkl ı noktadan
ölçülmü ştür. Bu ölçüm de ğ erlerinden ortalama delik
çaplar ı hesaplanm ışt ı r.
Ayr ı ca incelenen meme plakalann ı n hepsi için verdi
ve hacimsel da ğı l ı m ölçümleri yap ı lm ışt ı r. Verdi
ölçümlerinde püskürtme süresi 60 saniye olup ölçümler 6
ve 8 bar bas ı nçlarda yap ı lm ışt ı r. Püskürtülen s ı v ı bir
dereceli silindirde toplanarak verdi de ğ erleri Umin olarak
belirlenmi ştir. Verdi ölçümleri, her meme plakas ı için 5
tekerrürlü yap ı lm ış ve ölçülen de ğ erlerin ortalamas ı
al ı nm ışt ı r. Hacimsel ilaç da ğı l ı m ı , Tar ı m Bakanl ığı Tar ı m
Alet ve Makinalar ı Test Merkezi Müdürlü ğ ünde bulunan
tek meme denemesine uygun bir paternatör yard ı m ı yla
belirlenmi ştir. ölçümler her meme plakas ı için 3 tekerrürlü
olarak yap ı lm ış ve elde edilen de ğ erlerin ortalamas ı
al ı narak hacimsel da ğı l ı mlar ve bu da ğı l ı mlara ili ş kin
varyasyon katsay ı ları (% CV) bulunmu ştur. Hacimsel
da ğı l ı m ölçümlerinde bas ı nç 8 bar, meme yüksekli ğ i ise 50
cm olarak sabit tutulmu ştur. Bu ölçümler s ı ras ı nda
özellikle meme ekseni ile meme plakas ı ekseninin ayn ı
olmas ı na dikkat edilmi ştir.
Bulgular ve Tart ışma
Deney materyali olarak ele al ı nan meme plakalar ı
deliklerinin 5 farkl ı noktadan ölçülen çap de ğ erleri,
ortalama çap de ğ erleri ve bu çap de ğ erlerine ili ş kin
standart sapma (S) ile varyasyon katsay ı s ı (% CV)
değ erleri Çizelge l'de verilmi ştir Çizelge 1'de
görülebilece ğ i gibi incelenen meme plakalann ı n herbiri için
5 farkl ı noktadan ölçülen delik çaplar ı ve bu çaplardan
hesaplanan ortalama delik çaplar ı birbirinden farkl ı l ı klar
göstermektedir. Al plaka grubunda ortalama delik çap ı
0,989-1,047 mm aras ı nda, A2 grubunda 1,504-1,603 mm
aras ı nda, B grubunda 1,193-1,238 mm aras ı nda, G1
grubunda 1,133-1,180 mm aras ı nda, G2 grubunda 1,4441,605 mm aras ı nda ve T grubunda 0,904-0,980 mm
aras ı nda bulunmu ştur. Plaka deliklerinin 5 farkl ı noktadan
ölçülen çap de ğ erlerine ili ş kin CV değ erleri Al grubundaki
plakalarda % 1,547-5,190, A2 grubunda % 1,696-4,478, B
grubunda % 0,992-3,772, G1 grubunda % 2,128-5,286,
G2 grubunda % 2,743-3,024 ve T grubunda ise % 2,1824,741 aras ı nda de ğ i şmi ştir. Bu sonuçlardan
anla şı labilece ğ i gibi ; ayn ı gruptaki meme plakalar ı n ı n
ortalama delik çaplar ı nda farkl ı l ı klar oldu ğ u gibi, her
plakan ı n delik çevresi boyunca farkl ı noktalardan ölçülen
delik çaplar ı nda da farkl ı l ı klar olmaktad ı r.
Meme plakas ı delik çaplar ı ndaki düzgün ş üzlük ve
buna ba ğ li olarak deliklerin daireselliklerindeki de ğ i şimler,
deliklerin i ş lenme durumuyia ilgilidir. Ş ekil l'de her plaka
grubundan üçer adet meme plakas ı n ı n , ,delik görünümleri
verilmi ştir. Plaka deliklerinin genel görünümleri
incelendi ğ inde, delik çaplar ı ndaki düzgünsüzlük çok net bir
şekilde görülebilir. Bütün plakalarda delik kenarlar ı girintili
ç ı kı nt ı l ı ve delik ş ekli tam bir dairesellik göstermemektedir.
Deliklerin düzgünsüzlü ğ ünden ba ş ka deliklerin delinmesi
s ı ras ı nda oluş an havş alar da Şekil l'de görülebilir. Bu
havş alar özellikle Al, G1 ve T grubu plakalarda oldukça
belirgindir. Ayrı ca gözle ve mikroskop alt ı nda yap ı lan
incelemelerde ço ğ u meme plakas ı n ı n delik çevresi
boyunca çapaklar oldu ğ u saptanm ışt ı r.
firmalar
üretici
meme plakalar ı n ı n
Incelenen
tarafı ndan belirtilen delik çap ı değ erleri ile ölçülen
ortalama delik çaplar ı Çizelge 2'de kar şı la şt ı rmal ı olarak
verilmi ştir. Çizelge 2'deki % fark de ğ erlerinden (-) ile
gösterilenler, ölçülen delik çap ı n ı n firman ı n bildirdi ğ i delik
çap ı ndan küçük oldu ğ unu ifade etmektedir. Çizelge 2
incelendi ğ inde, üretici firmalar ı n bildirdikleri plaka delik
çaplar ı ile ölçülen delik çaplar ı n ı n birbirinden farkl ı oldu ğu
görülebilir. Al, A2 ve B grubu plakalarda ölçülen ortalama
hariç
delik çaplar ı , Al ve B gruplar ı nda birer plaka
değ erlerinden daha büyük
firmalar ı n bildirdi ğ i çap
plakalar ı n
bulunmu ştur. G1, G2 ve T gruplar ı ndaki
ölçülen ortalama delik çaplar ı ise G2 grubundaki . 2 plaka
hariç firma de ğ erlerinden daha küçük bulunmu ştur. En
küçük farkl ı l ı klar % -0,58 ile % 3,17 aras ı nda B grubu, en
büyük farkl ı l ı klar % -2,00 ile % -9,60 aras ı nda T grubu
plakalar ı nda elde edilmi ştir.
DURSUN, E. ve ark."Türkiye'de üretilen konik hüzmeli baz ı meme plakalar ı nda delik çap ı ve düzgünlüg' ünün belirlenmesi"
Çizelge 1.Meme plakalarm ın delik çaplar ına ilişkin sonuçlar
Plaka
tipi
Al
A2
G1
G2
No
di
1
2
3
4
5
1
1,106
1,015
1,038
O 987
1 067
1,521
1 535
1 535
1 459
1 570
1 264
1 219
1 254
1 238
1 228
1,196
1,219
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
T
3
4
Standart sapma
Delik çap ı (mm)
d2
0,970
1,015
1,033
1 040
O 975
1,468
1 467
1 584
1 543
1 488
1 223
1146
1 241
1167
1 270
1,053
1,103
d3
1,015
,z0,945
0,999
1 022
1 055
1,532
1 625
1,625
1,582
1 496
1 264
1 219
1 241
1189
1 190
1,151
1,151
1,061
0,989
1,020
1 072
0993
1,509
1 502
1 555
1 490
1 567
1 229
1196
1 233
1 282
1 230
1,188
1,072
d5
1,083
0,980
1,011
1 030
1 017
1,490
1 557
1 716
1 603
1 612
1 181
1187
1 220
1,195
1 208
1,102
1,120
don
1,047
0,989
1,021
1 030
1,021
1, 04
1 537
1 603
1 535
1-546
1,232
1193
1 238
1 214
1 225
1,138
1,133
1,169
1,180
dı
Varyasyon katsay ısı
(S)
. , 0,054
0,029
,' O .015
O 030
O 039
0,025
O 059
0 071
O 060
. O 053
O 034
0 029
O 012
O 045
O 029
0,060
(% CV)
5,190
2,954
1,547
2 982
3,850
1,696
3 882
4,478
3 947
3 426
2 786
2 510
O 992
3 772
2 439
5,286
0,055
0,025
0,025
0,057
0,444
0,043
4,915
2,173
2,128
4,879
0,044
0,026
3,009
1 675
1,196
1,205
1,188
1,670
1,142
1,172
1,151
1,188
1,196
1,193
1,161
1,140
1,063
1,177
1,205
1,180
1,163
1,548
1,593
1,596
1,615
1,605
1,467
1,535
1 548
1,472
0 880
0,880
O 993
O 890
0,938
1,500
1,523
1 560
1 433
O 926
0,925
1,399
, 1,444
1 572
1 495
O 903
0,948
1,453
1,436
1 508
1 422
O 890
0,921
1,401
1,495
1 569
1 517
O 921
0,910
1,444
1,487
1 551
468
O 040
2 743
O 904
0,917
0 903
O 970
1 029
O 932
0,970
O 917
0,993
O 895
0,971
0,940
0,988
O 973
O 915
0,980
O 019
0,024
0 046
O 022
2 182
2,699
4,741
2,409
0,028
2,849
1,012
A2
Şekil 1. Meme plakası deliklerinin genel görünümleri
2,749
3,024
137
138
TARIM BIL İ MLER İ DERG İ S İ 2000, Cilt 6, Sayı 3
B
G1
Şekil 1'in devam ı
DURSUN, E. ve ark."Türkiye'de üretilen konik hüzmeli baz ı meme plakalar ı nda delik çap ı ve düzgünlü ğ ünün belirlenmesi"
139
Çizelge 2. Meme plakası deliklerinin ölçülen ve üretici fırmalarca bildirilen delik çaplar ı nı n karşı laştırılmas ı
Plaka
tipi
Delik çap ı (mm)
ölçülen
1
1,047
1
0,989
1
1,021
1
1,030
1
1,021
1,5
1,504
1,5
1,537
1,5
1,603
1,5
1,5
1 546
1,2
1,232
1,2
1,2
1,238
1,2
1,214
1,2
1,225
Firma değ eri
Al
A2
B
1,53
Fark
(96)
4,70
-1,10
2,10
3,00
2,10
0,27
2,47
6,87
2,33
3,07
Basınç
(Bar)
6
8
6
8
Al
A2
B
8
6
8
6
8
6
8
G1
G2
T
Ortalama
verdi
(Umin)
0,710
0,815
1,210
1,361
0,947
1 070
0,800
0,921
1,119
1,405
0,690
O 804
Standart
sa ma
(S)
14,5
19,0
16,3
30,4
36,1
28,3
10,9
10,1
28,4
48,8
20,7
25,8
-0,58
3,17
1,17
2,08
Varyas. kat.
(% CV)
2,0
2,3
1,3
2,2
3,8
2,7
1,3
1,1
2,5
3,5
3,0
3,2
Çizelge 4. Hacimsel da ğılı mlara ait varyasyon katsay ı ları
Plaka
tipi
Al
A2
B
G1
G2
T
1
29 , 4
47,0
25,7
37,2
32,4
37,7
G1
G2
2,67
1,93
Çizelge 3 Incelenen meme plakaların ı n kullan ı lmas yla ölçülen
ortalama verdiler
Plaka
tipi
Plaka
tipi
Varyasyon katsay ısı (% CV)
2
3
4
38,3
39,6
45 , 3
33,3
36,5
27,0
35,2
28,6
42,5
38,4
33,2
33,3
40,4
35,4
35,2
35,5
31,9
44,1
5
41,5
40,6
44,4
37,0
31,3
39,7
ilaç da ğı l ı m düzgünlü ğ ünü etkileyen
önemli
faktörlerden biri meme verdilerindeki de ğ iş imdir. Verdiyi
etkileyen önemli faktörlerden biri ise meme plakas ı delik
çap ı d ı r. Diğ er ko ş ullar sabit iken delik çap ı ndaki de ğ i şime
ba ğ l ı olarak meme verdisi de ğ i şmektedir. Incelenen meme
plakalar ı n ı n kullan ı lmas ı yla ölçülen ortalama verdiler ile bu
verdilere ili şkin standart sapma ve varyasyon katsay ı ları
Çizelge 3'te verilmi ştir. 6 bar bas ı nçta en dü ş ük CV değ eri
% 1,3 ile A2 ve G1 grubu plakalar ı nda, en yüksek CV
değ eri % 3,8 ile B grubu plakalar ı nda elde edilmi ştir. 8 bar
bas ı nçta ise en dü ş ük CV % 1,1 ile G1, en yüksek CV
3,5 ile G2 grubu plakalar ı nda bulunmuştur.
ilac ı n son çı kış noktas ı olan meme plakas ı
deliklerinin düzgün olmamas ı ve delik kenarlar ı nda çapak
ve havş aları n bulunmas ı , ilaç dağı l ı m düzgünlü ğ ünün
bozulmas ı na neden olmaktad ı r. incelenen meme
T
Delik çap (mm)
Firma de ğ eri
ölçülen
1,2
1,138
1,133
1,2
1,169
1,2
1,180
1,2
1,163
1,2
1 605
1,5
1,5
1 444
1,487
1,5
1,5
1,551
1,468
15
0,904
1
0,917
1
1
0,973
0,915
1
0,980
1
Fark
(96 )
-5,17
-5,58
-2,58
-1,67
-3,08
7,00
-3,73
-0,87
3,40
-2,13
-9 60
-8 30
-2 70
-8,50
-2,00
plakalar ı n ı n kullan ı lmas ı yla elde edilen hacimsel s ı vı
da ğı l ı mlanna ait varyasyon katsay ı ları Çizelge 4'te
verilmi ştir. Varyasyon katsay ı ları örtme (overlapping)
yap ı lmaks ız ı n tek meme için hesaplanm ışt ı r. Çizelge 4
incelendi ğ inde, ayn ı gruptaki plakaları n % CV değerlerinde
önemli farkl ı l ı klar olduğ u görülebilir. Ayn ı gruptaki
plakalar ı n % CV değ erleri aras ı nda en büyük farkl ı l ı k % 20
ile A2 grubunda, en küçük farkl ı l ı k ise % 5,2 ile G1
grubunda bulunmu ştur. Ayn ı gruptaki farkl ı plakalar ı n
kullan ı lması yla elde edilen hacimsel s ıvı da ğı l ı mları na
ilişkin varyasyon katsay ı lar ı n ı n % 20'lere varan bir farkl ı l ı k
göstermesi, ilaçlama s ı ras ı nda dü ş ük veya a şı rı dozlu
alanlar ı n olu şmas ı na neden olacakt ı r.
Sonuç
Bu ara ştı rmadan elde edilen sonuçlar a şa ğı daki
şekilde özetlenebilir;
• Bütün plaka gruplar ı nda plaka delik çevresi
boyunca 5 farkl ı noktadan ölçülen çap de ğ erleri ayn ı plaka
için farkl ı l ı klar göstermektedir. Çap farkl ı l ı kları n' ifade eden
CV değ erleri, meme plakas ı gruplar ı na ba ğ l ı olarak %
0,992 ile % 5,286 aras ı nda değ i ş mektedir.
• Ayn ı gruptaki meme plakalar ı n ı n ortalama delik
çaplar ı nda farkl ı l ı klar bulunmaktad ı r.
• Üretici firmalar taraf ı ndan bildirilen plaka delik
çaplar ı ile ölçülen ortalama delik çaplar ı aras ı nda önemli
farkl ı l ı klar bulunmaktad ı r. Baz ı plakalar ı n ölçülen ortalama
delik çaplar ı firmalar ı n bildirdikleri çap de ğ erlerinden daha
küçük, baz ı ları daha büyüktür.
• Meme plakas ı delikleri yeterli düzgünlükte olmay ı p,
delik kenarlar ı nda çapak ve hav şalar bulunmaktad ı r.
kullan ı lmas ı yla
• incelenen meme plakaları n ı n
ölçülen meme verdileri de belirli s ı n ırlar aras ı nda
değ i şmi ştir. Verdi değ erlerine ili şkin CV'ler plaka gruplar ı
ve bas ı nca ba ğ l ı olarak % 1,1 ile % 3,8 aras ı nda
de ğ i şmektedir.
• Hacimsel da ğı l ı mlara ili ş kin % CV de ğerleri her
plaka grubundaki plakalar aras ı nda önemli farkl ı l ı klar
göstermi ştir. % CV de ğ erlerindeki bu farkl ı l ı klar ayn ı
gruptaki plakalar için en az % 5,2 ve en fazla % 20 olarak
bulunmu ştur.
140
TARIM B İ L İ MLER İ DERG İ S İ 2000, Cilt 6, Say ı 3
Kaynaklar
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Karakteristikleri. Tar ı msal Mekanizasyon 15. Ulusal
Kongresi, s.231-240, Antalya.
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West Lafayette, Indiana, 421 p, USA.
Matthews, G. A. 1992. Pesticide Application Methods. 2.Edition,
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Derlemeler 49, 34 s., Ankara.
Ozkan, H. E. 1995. Herbicide Application Equipment. Chapter
6.1n; Handbook on Weed Management Systems, Ed; A.E.
Smith, Marcel Dekker Inc., USA.
Naman, J. and A. Nordby, 1965. Intluence of the Eccentric Nozzle
Orifıce Position on the Spray Pattern and Droplet Size.
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Yelpaze Hüzmeli Memelerde Da ğı l ı m ve Pülverizasyon
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| 6,944 | 36,724 |
W2785710465.txt | Turnbull et al. BMC Family Practice (2018) 19:25
DOI 10.1186/s12875-018-0716-7
RESEARCH ARTICLE
Open Access
What gives rise to clinician gut feeling, its
influence on management decisions and its
prognostic value for children with RTI in
primary care: a prospective cohort study
Sophie Turnbull1,10*, Patricia J. Lucas2, Niamh M. Redmond3, Hannah Christensen4, Hannah Thornton1,
Christie Cabral1, Peter S. Blair5, Brendan C. Delaney6, Matthew Thompson7, Paul Little8, Tim J. Peters9
and Alastair D. Hay1
Abstract
Background: The objectives were to identify 1) the clinician and child characteristics associated with; 2) clinical
management decisions following from, and; 3) the prognostic value of; a clinician’s ‘gut feeling something is wrong’
for children presenting to primary care with acute cough and respiratory tract infection (RTI).
Methods: Multicentre prospective cohort study where 518 primary care clinicians across 244 general practices in
England assessed 8394 children aged ≥3 months and < 16 years for acute cough and RTI. The main outcome
measures were: Self-reported clinician ‘gut feeling’; clinician management decisions (antibiotic prescribing, referral
for acute admission); and child’s prognosis (reconsultation with evidence of illness deterioration, hospital admission
in the 30 days following recruitment).
Results: Clinician years since qualification, parent reported symptoms (illness severity score ≥ 7/10, severe fever < 24 h,
low energy, shortness of breath) and clinical examination findings (crackles/ crepitations on chest auscultation, recession,
pallor, bronchial breathing, wheeze, temperature ≥ 37.8 °C, tachypnoea and inflamed pharynx) independently contributed
towards a clinician ‘gut feeling that something was wrong’. ‘Gut feeling’ was independently associated with increased
antibiotic prescribing and referral for secondary care assessment. After adjustment for other associated factors, gut feeling
was not associated with reconsultations or hospital admissions.
Conclusions: Clinicians were more likely to report a gut feeling something is wrong, when they were more experienced
or when children were more unwell. Gut feeling is independently and strongly associated with antibiotic prescribing and
referral to secondary care, but not with two indicators of poor child health.
Keywords: Child, Respiratory tract infections, Primary health care, Prognosis, Emotions, Decision making,
Paediatric, Cough
* Correspondence: Sophie.turnbull@bristol.ac.uk
1
Centre for Academic Primary Care, Population Health Sciences, Bristol
Medical School, University of Bristol, Canynge Hall, 39 Whatley Road, Bristol
BS8 2PS, UK
10
Centre for Academic Primary Care, School of Social and Community
Medicine, University of Bristol, Bristol BS8 2PS, UK
Full list of author information is available at the end of the article
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Turnbull et al. BMC Family Practice (2018) 19:25
Background
Acute cough with respiratory tract infection (RTI) in
children is the most common problem managed by
health services internationally [1, 2]. While the majority
of childhood RTIs are self-limiting, a small number
result in serious illness and hospitalisation [3]. Clinicians
report that uncertainty regarding both the diagnosis and
prognosis of some children with RTIs are important
drivers of antibiotic prescribing, [4–6] contributing to
over-prescribing [7] and antimicrobial resistance [8].
Knowing the prognosis of children presenting to primary
care with acute RTIs can be challenging as clinicians
cannot always be certain where in the illness trajectory
the child has presented, and whether red flag symptoms
and signs are absent or are yet to develop. In cases
where there is uncertainty, clinical intuition or ‘gut feeling’ is thought to play a part in management decisions.
The current literature offers two definitions of
clinician gut feeling. Both propose it arises when the
clinician feels uncertain or has a low ‘feeling of rightness’,
[9, 10] and both have been shown to influence management decisions [11]. In some studies, a comparison has
been made between a sense of reassurance versus a feeling that ‘something is wrong’ which defines a broad set
of cases where clinicians are worried [11]. In others,
authors define a gut feeling as a sense of dissonance
between intuitive and analytic reasoning; a gut feeling
something is wrong despite a lack of clinical markers [9].
The latter definition draws on theories of clinical reasoning distinguishing between “intuitive” processing, which
is rapid and unconscious and “analytic” processing that
involves slow deliberative reasoning [12–14].
In primary care, gut feeling has been described as an
incorporation of clinician knowledge, experience and
information about the patient [9]. In the case of childhood RTI, clinicians report using immediately apparent
symptoms and signs (the child’s energy, pallor and
breathing) to distinguish severe from non-severe cases,
using pattern recognition and drawing on past experience [5]. A recent study in children with any acute
illness, found that: general appearance, breathing pattern, weight loss, history of convulsions and parental
concern that the illness was different from any previously experienced predicted the feeling something was
wrong when clinical impression was of non-serious
illness [15]. This evidence goes some way to characterising the factors influencing clinical gut feeling, but does
not identify the specific clinical features associated with
particular illnesses, or provide objective assessments of
illness severity.
Despite qualitative studies indicating the importance
of clinician intuition for primary care doctors and in
nursing, [16] there is a paucity of quantitative evidence
regarding the prognostic value of gut feelings and
Page 2 of 12
whether primary care clinicians utilise them in their
decision making. A systematic review suggested that gut
feeling in itself should be viewed as a ‘diagnostic red flag’
and that it had greater diagnostic value than the majority
of illness specific symptoms and signs [17]. Another
study indicated that in situations where there was uncertainty after clinical assessment, gut feeling was highly
predictive of serious infective illness [15].
We used a large prospective cohort study of children
presenting to primary care with acute cough and RTI to
address three objectives regarding ‘gut feeling that something is wrong’ (from here on ‘gut feeling’): (i) to
describe the clinician and child characteristics that drive
clinician to have a gut feeling that something is wrong;
(ii) to investigate if gut feeling influences management
decisions; and (iii) to evaluate the prognostic value of
gut feeling in relation to primary care reconsultations
with evidence of illness deterioration, and hospital
admissions.
Methods
Design and setting
The ‘TARGET’ study [18] was a multicentre, prospective
cohort study that recruited children presenting to general practices with acute cough and RTI, between July
2011 and May 2013. Practices were recruited and trained
by four University hubs (Bristol, London, Oxford and
Southampton). The primary aim of the study was to
develop a clinical rule that could help clinicians improve
their use of antibiotics by using baseline clinical characteristics to predict the children that would be hospitalised in the next 30 days [3]. Here we present findings
from secondary analysis of this dataset, which are
reported following the Strengthening the Reporting of
Observational Studies in Epidemiology (STROBE)
recommendations [19].
Participants
Clinicians (General Practitioners: GPs and ‘prescribing’
Nurse Practitioners: NPs), were eligible to recruit to the
study if they were working in participating primary care
practices and reported that they prescribed antibiotics in
≤30% children with RTIs (to reduce the possibility of
confounding by indication). Clinicians were asked to
specify a priori their preferred recruitment strategy (for
example, consecutive or first eligible on the day).
Children were eligible if aged ≥3 months to < 16 years
and presenting with an acute (≤28 days) cough and other
symptoms of an RTI. Children with an infected exacerbation of asthma and those severely unwell (for example,
requiring same day hospital assessment or admission)
were included. Children were excluded if: presenting
with a non-infective exacerbation of asthma; immunocompromised; previously recruited or recently
Turnbull et al. BMC Family Practice (2018) 19:25
participated in other research; or temporarily registered
at the GP practice.
Data collection
Clinician characteristics
Clinician characteristics included the clinician type (GP
or NP), number of years qualified and evidence of further medical/ health qualifications (Diploma in Child
Health (DCH) or Membership of the Royal College of
General Practitioners (MRCGP)).
Child characteristics
At the baseline consultation the recruiting clinician used a
structured case report form (CRF) to record sociodemographic information, parent-reported symptoms
(including severity of symptoms in the past 24 h) and
physical examination findings. A parent and clinician reported illness severity score was collected, scored 0-10
(see Fig. 1. Case report form). Information about the
child’s past medical history was collected from the primary
care medical record. The 2010 Indices of Multiple
Deprivation (IMD) provided a measure of neighbourhood
deprivation linked to the child’s home postcode [20].
Page 3 of 12
they prescribe antibiotics ‘just in case’ children are subsequently admitted to hospital with their RTI, and we
sought to use an inclusive definition. Second, our previous [21] and more recent [22] research has shown the
symptoms of acute cough and RTI take 25 days for most
(90%) of children to resolve. A hospitalisation outcome
with a ≤ 7 day cut off was also used as a sensitivity analysis to ensure that there was not a temporal association
between the prognostic value of gut feeling and the hospitalisation outcome. We calculated the Kappa statistic
to assess inter-rater reliability of the two reviewers that
independently assessed the primary care medical record
for these two health outcomes.
Statistical analysis
Treatment of variables
At the end of their clinical evaluation, the clinician was
also asked to respond ‘yes’ or ‘no’ to the statement “my
gut feeling is ‘something is wrong’” with the child. A definition was provided in the clinician instruction booklet
that gut feeling in this context “represents clinicians gut
feeling that the child’s illness may be more serious than
is superficially apparent”. This question was located at
the end of the CRF, following the socio-demographic,
symptoms and physical examination findings, but prior
to the reporting of management decisions.
Parent-reported symptom severity (mild, moderate,
severe) in the 24 h prior to consultation was transformed into a binary outcome with a threshold
dependent on overall prevalence: to either ‘severe/not
severe’ (where > 5% of children reported as being severe
or ‘moderate or severe/not moderate or severe’ (where
< 5% of children reported being severe). This pragmatic
cut-off was chosen prior to analysis to avoid severity
variables with very low prevalence. We used accepted
clinical cut offs (temperature > 37.8 °C, [23] capillary
refill time (CRT) ≥3 s, [24, 25] oxygen saturation ≤ 95%
[26]) or 25th percentile or 75th percentile thresholds to
dichotomise continuous data, and we used age-adjusted
thresholds for heart and respiratory rates [27]. Given the
large number of variables being tested, continuous outcomes were initially categorised to help interpret and
contextualise the final model, a sensitivity analysis was
then conducted to assess the distribution of these continuous variables if included in the model.
Management decisions
Determinants of gut feeling
Clinicians were asked to record if the child was referred
for a secondary care assessment and if they prescribed
antibiotics.
We first used univariable multilevel random effects
logistic regression (accounting for between-clinician differences) to assess associations between clinician profile
and children’s baseline characteristics, with gut feeling.
Modelling was conducted using a multivariable multilevel random effects logistic regression model (accounting for clustering at the clinician level) and variable
selection using the backward stepwise method with a 5%
model entry threshold. For the ‘determinant’ analyses,
the outcome variable was the clinician’s gut feeling and
(given the large number of variables investigated) model
retention was set at the 1% level. A more liberal threshold was employed for the univariable stage than for the
multivariable stage because the former was used as a
selection filter for the latter and we did not want to miss
any potential associations of interest, whereas for the
final selection of variables it was necessary to be more
Gut feeling
Prognostic value
Two indicators of poor child health were collected from
the primary care medical record by trained notes
reviewers at least 3 months after study entry (to allow
sufficient time for the hospital discharge information to
reach the medical record): primary care re-consultations
for the same RTI with evidence of symptom deterioration; and hospital admissions for RTI in the 30 days
following recruitment (using hospital discharge summaries). The 30 days threshold for hospitalisation was
selected for two reasons. First, the rationale for the primary aim of the TARGET study [3] (from which the ‘gut
feeling’ paper derives its data) was that clinicians report
Turnbull et al. BMC Family Practice (2018) 19:25
Page 4 of 12
Fig. 1 Case report form
conservative given both the number of variables considered and the large sample size (which could potentially
result in small effects being detected statistically). Global
clinician illness severity score was not included in this
analysis as it was suspected that there was overlap in
what this and gut feeling variables represented to the
clinician. Considering prior literature indicating prognostic value of gut feeling in the absence of clinical signs
of severe illness, we undertook a sensitivity analysis to
explore the contribution of high illness severity score to
the model.
Evidence that clustering by clinician was important in
the multivariable model was explored using the
Likelihood-ratio test (LRT). The accuracy of the resulting model was represented by the Area Under the
Receiver Operating Characteristic (AUROC) curve.
Turnbull et al. BMC Family Practice (2018) 19:25
Multiple imputation was employed as a sensitivity analysis to check whether the model was robust [28].
Management decisions and prognostic value
Univariable and multivariable multilevel logistic regression modelling was employed, using backward stepwise
selection (accounting for between-clinician differences),
as well as variables associated with each of those outcomes retaining variables with a p-value of < 0.05. Two
models were used to investigate the associations between
clinician gut feeling and the decision to: 1) prescribe
antibiotics, and 2) refer the child to hospital for acute
admission. Two further models were used to investigate
the associations between gut feeling and: 1) reconsultation for the same RTI with evidence of deterioration, and; 2) hospitalisation for a RTI in the 30 days
following recruitment. A further multivariable model
was used in the sensitivity analysis exploring the association between gut feeling and the hospitalisation outcome with a ≤ 7 day cut off. To ensure the prognostic
value of gut feeling did not differ with definition of gut
feeling, a univariable analysis was conducted to explore
the association between hospitalisation and the more
focused definition described by a previous study: gut
feeling when clinical impression was of non-serious illness [15]. Due to the exploratory nature of these analyses, and to ensure we did not miss any potential
influences, the more liberal threshold was employed for
the multivariable analysis. LRT was used to explore the
influence of clustering by clinician in the multivariate
models. Data were analysed using STATA version 13.1.
Results
Clinicians and children
Of the 842 clinicians agreeing to participate, 519 (62%)
located at 247 general practices recruited at least one
child (median 5, inter-quartile range (IQR) 2-15
children) into the study. Of the clinicians who recruited
at least one child who was retained in the study, data
were available about clinician type for 502 (97%) clinicians; of these 77 (15%) were NPs and 425 (85%) were
GPs. Data on years since qualification were available for
498/518 (96%) of the clinicians: median of 20 years
(range 1-44, IQR 13-26 years). Of the 519 clinicians, 85
(16%) reported having at least one additional qualification (DCH or MRCGP).
A total of 8613 children were recruited to the study, of
whom 219 (3%) children were excluded: 181 did not meet
the eligibility criteria, 32 did not have baseline data and
six were withdrawn. Of the 8394 children recruited and
retained, clinicians recorded their gut feeling for 8377
(99.8%) - all analyses that follow use these 8377 children.
Participant flow through the study is available in Fig. 2.
Oxygen saturation was removed from all analysis as these
Page 5 of 12
data were missing for 50% of children due to a lack of
available paediatric oxygen saturation monitors. All other
reported variables had < 2% missing data. Children’s median age was 3 years (range 3 months-15 years, IQR, 16 years) and 1390 (17%) were under 1 year. There were
slightly more boys (52%) than girls, and of the 8333
(99.5%) for whom ethnicity data were available the majority (6540, 78%) were white (similar to UK Census 2011
data) [29]. Families’ median deprivation (IMD) score was
16.7 (IQR 8.8-29.5), similar to the English median (17.2,
IQR 9.8-30.2) [20]. Median clinician reported illness severity was 3 (range 0-9, IQR 2-4).
Clinicians reported a gut feeling something was wrong
in 1706 (20%) cases. 3110 (37%) were prescribed an antibiotic, with 2341 (28%) given an immediate script and
769 (9%) given a delayed script. Seventy-four children
were immediately referred for secondary care assessment
at the recruiting consultation, of whom 13 were subsequently admitted. A further 65 children were admitted
to hospital with a RTI in the subsequent 30 days. For
the 78 hospitalised children the median number of days
to hospitalisation was 2 days (IQR 1-12 days). The hospital discharge diagnoses were: lower respiratory tract
infection (19%); bronchiolitis (18%); viral wheeze (15%);
upper respiratory tract infection (13%); croup (8%);
infected exacerbation of asthma (8%); tonsillitis (6%);
viral illness (5%); febrile illness (3%); and pneumonia
(1%). Of the 8193 children for whom data were available
relating to primary care reconsultations in the 30 days
following baseline, 1847 reconsulted for an RTI. Of these
354 (4%) reconsulted for the same illness with evidence
of symptom deterioration, with a median of 5 days (IQR
2-11 days) to this reconsultation. The percentage interrater agreement (kappa) for hospitalisation and reconsultation for the same illness with evidence of deterioration
were 90% (Kappa 0.80) and 84% (Kappa 0.67) respectively. There were 242/8377 (3%) children in whom the
clinician reported a low illness severity score (≤2) whilst
also reporting they a gut feeling something was wrong.
Determinants of gut feeling
Univariable analyses
Fifty-one (84%) of the 61 variables measured were
associated with a gut feeling (with p-values < 0.05
(Additional file 1: Web Appendix, Table S1). Two of the
three clinician characteristics were associated: those with
more years since qualification and NPs reported having
a gut feeling in a higher proportion of the children than
GPs (27% vs. 18%), while additional qualifications were
not associated. Only one of the five sociodemographic
variables collected were associated: being male (55% vs
51%). There was no evidence of a difference in the
children’s ethnicity, age, the mother’s age at the time of
the child’s birth, or home IMD score in the two groups.
Turnbull et al. BMC Family Practice (2018) 19:25
Page 6 of 12
Fig. 2 Flow of participants through the study
All the four variables collected regarding the child’s
medical history were associated with gut feeling. The
child was more likely to: have a chronic condition (21%
vs 18%), a previous or current asthma diagnosis (12% vs
8%), and to have consulted for an RTI on two or more
occasions in the 12 months prior to recruitment (36% vs
35%), than the children for whom the clinician did not
have a gut feeling. There was evidence that 44/49 (90%)
of the symptoms and signs that were recorded at
baseline were associated with gut feeling. The five symptoms and signs that did not reach the 5% threshold were
low illness duration prior to recruitment, barking or
croupy cough, blocked or runny nose, diarrhoea and
stridor (Additional file 1: Web Appendix, Table S1).
Multivariable analysis
There was strong evidence that increasing years since
clinician qualification, five carer-reported symptoms
Turnbull et al. BMC Family Practice (2018) 19:25
Page 7 of 12
and eight clinically-reported signs were independently
associated with gut feeling (Table 1). The strongest
was crackles and crepitations. In the multivariable
model there was no evidence of association with clinician type (NP versus GP), additional clinician qualifications or any of the socio-demographic variables.
There was strong evidence that clustering by clinician
was important in the model (LRT of rho = 0: p <
0.001) when compared with the multivariable model
without clustering. The AUROC curve was 0.82, 95%
confidence interval (CI) 0.80-0.83. Imputing the missing data for the final model made little difference to
the odds ratios, confidence intervals and p-values.
The sensitivity analysis exploring the impact of high
clinician illness score on the model resulted in few
changes: high clinician illness severity score was
retained, while severe fever in the last 24 h and parent reporting that that illness was worse recently
dropped out of the model. There was little change to
the remaining estimates, except for crackles and crepitations, where the OR fell from 14.56 (95% CI 11.71-
18.12) to 9.57 (95% CI 7.64-11.99) and the AUROC
increased from 0.81 (95% CI 0.80-0.83) to 0.82 (95%
CI 0.83-0.85).
Management decisions and gut feeling
There was strong evidence in both the univariable and
multivariable analysis that when clinicians report a gut
feeling, they were more likely to prescribe antibiotics
and refer for same day further secondary care assessment (Table 2, including description of the covariates
retained in the multivariate model at the < 1% level).
These findings were independent of clinician’s impression of illness severity.
Prognostic value of gut feeling
There was no evidence to suggest that the children for
whom the clinician had a gut feeling were more likely
to re-consult for the same illness with evidence of illness deterioration in the 30 days following recruitment
in the univariable or multivariable analysis. They were
over twice as likely to be admitted to hospital for a RTI
Table 1 Determinants: Multivariable associations between parent-reported symptoms, clinician-reported observations, clinician
profile and ‘gut feeling’
Significant predictors
OR
95% CI
p-value
10.3
Ref
Ref
< 0.001 5 d.f.*
423/2213
19.1
0.55
0.30-1.02
80.5
235/1206
19.5
1.44
0.71-2.90
76.9
367/1586
23.1
1.28
0.57-2.87
No gut feeling
Yes gut feeling
n/N
%
n/N
%
1-9 years
646/720
89.7
74/720
10-14 years
1790/2213
80.9
15-19 years
971/1206
20-24 years
1219/1586
Years qualified (Categorical)
25-29 years
875/1159
75.5
284/1159
24.5
1.67
0.76-3.68
30+ years
1170/1493
78.4
323/1493
21.6
1.08
0.47-2.46
High parent illness severity score (≥7/10)
1459/6650
21.9
681/1702
40.0
1.67
1.39-2.01
< 0.001
Fever in the last 24 h (severe)
363/6647
5.5
177/1700
10.4
1.66
1.23-2.26
0.001
Low energy during illness
3404/6669
51.0
1100/1704
65.6
1.65
1.36-1.99
< 0.001
Parent reported symptoms
Shortness of breath during illness
2115/6670
31.7
857/1704
50.3
1.50
1.25-1.81
< 0.001
Illness worse recently
4182/6663
62.8
1339/1704
78.6
1.44
1.18-1.76
< 0.001
695/6661
10.4
901/1704
52.9
14.56
11.71-18.12
< 0.001
Recession
164/6663
2.5
239/1704
14.0
3.31
2.29-4.79
< 0.001
Pallor
6185/6663
7.2
345/1705
20.2
3.10
2.30-4.18
< 0.001
Clinical signs
Crackles and crepitations
Bronchial breath
136/6658
2.0
142/1703
8.3
2.99
1.90-4.70
< 0.001
Wheeze (recorded in consultation)
675/6663
10.1
560/1703
32.9
2.78
2.23-3.46
< 0.001
High temperature (≥37.8 °C recorded in consultation)
629/6653
9.5
415/1702
24.4
2.32
1.84-2.91
< 0.001
High respiratory rate (age related cut offs)
825/6634
12.4
414/1698
24.4
1.64
1.30-2.07
< 0.001
Inflamed pharynx
1873/6647
28.2
521/1702
30.6
1.51
1.23-1.86
< 0.001
N = 8217/8377 (98.1% of the sample) 514 clusters (clinicians)
*
Degrees of freedom
Turnbull et al. BMC Family Practice (2018) 19:25
Page 8 of 12
Table 2 Management decisions: relationship between ‘gut feeling’ and subsequent clinical management
Treatment
No gut feeling
Yes gut feeling
Univariable analysis controlling
for clustering
Multivariable analysis controlling for
clustering and significant covariates
n/N
%
n/N
%
OR
95% CI
P-value
OR
95% CI
P-value
25.6
1403/1706
82.2
20.80
17.42-24.83
< 0.001
5.85
4.67-7.32
< 0.001±
3.4
19.27
9.83-37.79
< 0.001
12.64
6.31-25.32
< 0.001¥
Prescribed antibiotics at baseline consultation
Any antibiotics prescribed
1707/6671
Referred for acute admission (during the recruiting consultation)
Referral for acute admission
16/6671
0.2
58/1706
±
Co-variates of any antibiotic prescription (retained at the < 1% level): illness has got worse recently, child’s age (< 2 years), barking cough, fever, diarrhoea, low
energy and productive cough during the illness, moderate/severe wheeze and severe fever in the last 24 h, recession, crackles and crepitations, wheeze (as
reported by the clinician), bronchial breath, inflamed pharynx, high temperature, high clinician and parent illness severity scores, low illness duration and gut
feeling that something is wrong
¥
Co-variates of referral for acute admission (retained at the < 1% level): Recession, gut feeling that something is wrong and capillary refill time (CRT)
in the 30 days following recruitment. However, after
adjusting for other variables associated with hospital
admission, this relationship was attenuated. This was
also true for the sensitivity analysis examining the 7day hospitalisation outcome. The presence of clinician
reported wheeze and/or recession in the multivariate
model reduced the association with gut feeling below
the < 5% threshold (Table 3, including description of
covariates retained in the multivariate model). There
was evidence of clinician clustering effect for reconsultation (LRT of rho = 0: p < 0.001) but not hospitalisation (30 days LRT of rho = 0: p = 0.30, 7 days LRT
of rho = 0: p = 0.43), indicating that there was a relationship between the clinician who had seen the child
and the likelihood of child reconsulting, but not being
hospitalised. There was also no evidence of an association between hospitalisation and the more focused
definition of gut feeling (described in previous studies)
was explored at univariate level(OR 1.26, 95% CI 0.384.13, p = 0.71).
Discussion
Summary of findings
In a large, rigorously conducted, prospective cohort study,
gut feeling was more commonly reported by more experienced clinicians and in children with parent-reported
symptoms and physical examination signs suggesting more
severe illnesses. Gut feeling was strongly associated with
management decisions – both antibiotic prescribing and
same-day referral for admission. Despite this, we did not
find strong evidence that gut feeling was associated with
indicators of poorer prognosis in the following 30 days,
such as reconsulting in primary care with evidence of illness deterioration or being hospitalised for a RTI, at least
not after controlling for potential confounders.
Strengths and limitations
To our knowledge, ours is the largest, prospective study
to describe the determinants, management decisions and
prognostic value of gut feeling for respiratory tract
infections in children in routine, office-based primary
Table 3 Prognosis: relationship between ‘gut feeling’ and children’s health outcomes
Health outcome
No gut
feeling
Yes gut
feeling
Univariable analysis
controlling for clustering
Multivariable analysis controlling for
clustering and significant covariates
n/N
%
n/N
%
OR
95% CI
P-value
OR
95% CI
P-value
Re-consultation for the same illness with
evidence of illness deterioration (≥1) in the
30 days following recruitment
271/6523
4.2
83/1670
5.0
1.30
0.99 -1.71
0.06
1.08
0.80-1.46
0.60¥
Admitted to hospital in the 30 days following
recruitment
48/6671
0.7
30/1706
1.8
2.47
1.55-3.95
< 0.001
1.11
0.65-1.89
0.71≠
24/6647
0.4
25/1681
1.5
4.20
2.34-7.54
< 0.001
1.62
0.83-3.14
0.16α
Health outcome
Sensitivity analysis
Admitted to hospital in the ≤7 days post
recruitment
¥
Co-variates of re-consultation for the same RTI with evidence of deterioration (retained at the < 5% level): ≥2 consultations for RTI in primary care in the year prior
to baseline, low illness duration prior to baseline, previous asthma diagnosis, current asthma diagnosis, child’s age at baseline (< 2 years), white ethnicity, parent
reported barking cough, wheeze and taking fewer fluids during the illness, moderate/severe vomiting and fewer fluids in the 24 h prior to baseline, severe eating
less in the 24 h prior to baseline, wheeze and recession (as reported by the clinician)
≠
Co-variates of hospitalisation (retained at the < 5% level): child’s age (< 2 years), low illness duration prior to baseline, current asthma diagnosis, moderate/severe
vomiting, recession, wheeze (as reported by the clinician), high temperature (age related cut offs)
α
Co-variates of hospitalisation ≤7 days post recruitment (retained at the < 5% level): child’s age (< 2 years), low illness (< 3 days) duration prior to baseline, current
asthma diagnoses, moderate/severe vomiting, recession, wheeze (as reported by the clinician), high temperature (age related cut offs)
Turnbull et al. BMC Family Practice (2018) 19:25
care [2]. We included a broad range of clinician,
demographic, child and clinical characteristics and the
study focussed on a clinical group in whom clinicians
report diagnostic and prognostic uncertainty [6]. Determinants of gut feeling were measured according to routine clinical practice using standardised reporting forms
on the day of recruitment, blind to the prognostic
outcomes, which were reliably measured from the primary care record. We used rigorous techniques to adjust
for confounding.
We are aware of five main limitations. First, while we
offered some instructions to support completion of the
question “My gut feeling is ‘something is wrong’”, including
a definition, there was no attempt to interpret it and clinicians were simply asked to report its presence/absence.
While the low question refusal suggests clinicians understood the question, it is likely the presence of gut feeling
means different things to different clinicians. Definitions of
gut feeling overlap, and may not be clear to clinicians.
Given our relatively high (20%) prevalence of gut feeling,
the strong associations found with symptoms and signs
(such as crepitations/crackles) and with high clinician
reported illness severity score, it is likely our clinicians were
using a broad definition, such as “the presence of a significant illness”. Second, although focussing on an extremely
common primary care presentation, [2] findings may not
generalise to other illnesses or different age groups. Third,
we adjusted for a wide range of potential confounders but
it is possible that other unmeasured factors could be
important. We are aware of another study that found ‘parent concern that the illness was different to other illnesses’,
weight loss and history of convulsions have been shown to
be associated with gut feeling and these were not collected
in our study [15]. Fourth, while our conservative model
entry and exit thresholds have minimised scope for Type I
error, the relative infrequency of the hospitalisation outcome, and the fact this was a secondary analysis of data collected for another purpose, [18] could have led to Type II
error and us incorrectly concluding that gut feeling does
not have prognostic value. The large study size mitigates
against this for re-consultation. It is plausible some cases of
re-consultation or hospitalisation were avoided where
antibiotics were given and this may mask associations with
gut feeling (so-called confounding by indication). However,
there is limited evidence of the mitigating effects of antibiotics on prognostic outcome for RTI. Two studies, one with
adults [30] and the other with children (in submission [31])
with RTIs found antibiotics did not reduce hospitalisation,
and immediate antibiotics did not reduce reconsultations.
Finally, the outcomes used to explore the prognostic value
of gut feeling are indicators of poor health, but we acknowledge that other factors beyond the child’s illness severity
can influence these, such as parent behaviour and available
services in the area.
Page 9 of 12
Comparison with existing literature
Determinants of gut feeling
Previous studies have described gut feeling in primary care clinicians as being derived from a combination of patient characteristics and clinicians’
knowledge and past experience [32, 33]. Like others
we found that pallor or appearance, energy and
breathing patterns were associated with gut feeling
[5, 15].
Our results contrast with a previous study, which did
not find evidence to support their hypothesis that high
temperature contributed towards gut feeling, but we
found high temperature and carer-reported severe fever
both contributed [15]. The literature is contradictory
regarding the role of clinician experience. For instance,
both we and a previous qualitative study found more
experienced family primary physicians were more likely
to report having a gut feeling something was wrong,
[17] whereas another quantitative study found that
more senior doctors were less likely to report it [15].
These differences may be due to differences in participating children or definition of gut feeling; van den
Bruel’s study included children with any non-serious
acute illness, whereas we studied children with RTI
regardless of clinician-reported illness severity. In their
study they conceptualised gut feeling as “intuitive feeling that something was wrong even if the clinician was
unsure why” and included the 3.1% children where the
clinician reported a gut feeling in addition to the illness
was not severe. Our definition was broader and
included 20% of the children. However, in our sample a
clinician reported a gut feeling combined with a low illness severity score approximates this definition, and we
found the same proportion (3%). The concept that gut
feeling is more commonly reported in more experienced clinicians is supported by the learning perspective of intuition: where intuition is the consequence of
unconscious drawing on an implicit knowledge base
and recognition of patterns of clinical markers that is
developed though experience and may be associated
with previous negative or uncertain outcomes. As demonstrated by our findings, more experienced does not
mean more accurate. The validity of the gut feeling as a
guide to decision-making relies not only on the quantity but also the quality of the feedback. If there is good
feedback during the learning process, this results in
good or accurate gut feeling and associated decisionmaking. Conversely, poor or absent feedback results in
poor intuition [34].
Management decisions and gut feeling
Our findings agree with previous studies [32] that found
gut feeling can play a substantial role in treatment
decisions.
Turnbull et al. BMC Family Practice (2018) 19:25
Prognostic value of gut feeling
A recent systematic review suggested that gut feeling
was of greater diagnostic value than the majority of
symptoms and signs [17]. Two recent prospective studies reported that gut feeling in primary care was also
associated with serious infectious illness in children in
Belgium [15] and serious disease in adults and children
in Denmark [35]. In contrast, we did not find evidence
that gut feeling was associated with subsequent reconsultations with evidence of deterioration. When considered in isolation, we found gut feeling was associated
with hospitalisation, however, when other clinical signs
(wheeze and/or recession) were taken into account, gut
feeling was no longer associated. Indeed, our recent
paper identified seven other characteristics that were
independently associated with hospitalisation – namely
age less than 2 years, current asthma, illness duration of
3 days or less, parent-reported moderate or severe
vomiting in the previous 24 h, severe fever in the previous 24 h or a body temperature of 37·8 °C or more at
presentation, clinician-reported intercostal or subcostal
recession, and wheeze on auscultation [3]. Use of these
may reduce prognostic uncertainty and reduce reliance
on gut feeling.
Clinical, teaching and research implications
Our study suggests that for children presenting to primary care with acute cough and RTI, the gut feeling that
something is wrong, which appears to drive clinical
decision-making in this group, are not associated with
either re-consultation or hospital admission, at least not
after potential confounding effects were taken into
account [30, 31]. Given the high levels of uncertainty
reported by clinicians looking after this group of
patients, [5, 36] it is not surprising they wish to use all
the tools at their disposal, including gut feeling. However, our results suggest some clinicians may over-value
gut feeling in their clinical decision making in this group
of patients and that they are using gut feeling as a proxy
or unconscious marker of other important symptoms
and signs. Gut feeling may have value in the absence of
known, objectively assessed markers of risk, however,
attending to objective assessment of these risks is preferable when this information is available. Future research
may wish to focus on a more specific definition of gut
feeling, including perhaps the sense of dissonance arising
when conscious and unconscious assessment processes
are in conflict.
Conclusions
Clinicians were more likely to report a gut feeling
something is wrong, when they were more experienced
and when children were more unwell. Gut feeling is
independently and strongly associated with antibiotic
Page 10 of 12
prescribing and referral to secondary care, but not with
two indicators of poor child health.
Additional file
Additional file 1: Web Appendix Table S1. Determinants: univariable
associations between sociodemographic, parent-reported symptoms,
clinician-reported observations, clinician profile and gut feeling. This table
presents the univariable associations between child and clinician variables
and gut feeling. (DOCX 21 kb)
Abbreviations
AUROC: Area under the receiver operating Characteristic; CRF: Case report
form; CRT: Capillary refill time; DCH: Diploma in child health; GPs: General
practitioners; IMD: Indices of multiple deprivation; IQR: Inter-quartile range;
LRT: Likelihood-ratio test; MRCGP: Membership of the royal college of
general Practitioners; NPs: ‘Prescribing’ nurse practitioners; RTI: Respiratory
tract infection; STROBE: Strengthening the reporting of observational studies
in epidemiology
Acknowledgements
The National Institute for Health Research funds the Programme Grant for
Applied Research TARGET Programme grant at the University of Bristol and
NHS Bristol Clinical Commissioning Group.
This paper summarises independent research funded by the National
Institute for Health Research (NIHR) under its Programme Grants for Applied
Research Programme (Grant Reference Number RP-PG-0608-10018). HC is
supported by the National Institute for Health Research Health Protection
Research Unit (NIHR HPRU) in Evaluation of Interventions at the University of
Bristol, in partnership with Public Health England (PHE). ADH is funded by
NIHR Research Professorship (NIHR-RP-02-12-012). NMR’s time is supported
by the NIHR Collaboration for Leadership in Applied Health Research and
Care West (CLAHRC West) at University Hospitals Bristol NHS Foundation
Trust. The views expressed are those of the authors and not necessarily those
of the NHS, the NIHR, and the Department of Health of Public Health
England.
The authors are extremely grateful to the children, parents/carers and
families who have participated in the study, all GP practices including
recruiting clinicians, administrative and research contacts and all other staff
whose participation made this study possible. We thank all our colleagues
from the TARGET Programme, the TARGET Programme Management Group
and the TARGET Programme Steering Committee (Sandra Eldridge, Nick
Francis, Joe Kai, Victoria Senior, Anna Thursby-Pelham, Mireille Williams) for
their time, expertise and support. We are grateful to the following individuals
who have helped with the study; James Austin, Denis Baird, Tony Beard,
Stephen Beckett, Issy Bray, Peter Brindle, Kate Brooks, Sue Broomfield, Joanna
Cordell, Judy Cordell, Tania Crabb, Hazel Crabb-Wyke, Mike Crawford, Julie
Cunningham, Christina Currie, Rachel Davies, Elizabeth Derodra, Elena
Domenech, Stevo Durbaba, Lucy Feather, Caroline Footer, Emily Gale, Anna
Gilbertson, Victoria Hardy, Rose Hawkins, Abigail Hay, Lisa Hird, Sandra
Hollinghurst, Julie Hooper, Jonathan Hubb, Catherine Jameson, Grania
Jenkins, Amy Jepps, Mari-Rose Kennedy, Michael Lawton, Mel Lewcock, Lyn
Liddiard, Sandra Mulligan, Sharen O’Keefe, Lucy O’Reilly, Marilyn Peters, Aled
Picton, Ilaria Pinna, Fiona Redmond, Isabel Richards, Kim Roden, Sharon Salt,
Douglas Shedden, Ella Simmonds, Sue Smith, Carol Stanton, Kate Taylor,
Elizabeth Thomas, Nicki Thorne, Sara Tonge, Abby Waterhouse, Eleanor
Woodward. The TARGET study team acknowledges the support of the
National Institute for Health Research, through the Comprehensive Clinical
Research Network. To Robert Ross from the Department of Psychology at
the Royal Holloway who advised on the dual process theory of clinical
reasoning. Also to George Leckie, from the multi-level modelling centre at
Bristol University who advised on statistical methods.
Funding
This paper reports independent research funded by the National Institute for
Health Research (NIHR) under its Programme Grant for Applied Research
(Grant Reference Number RP-PG-0608-10018). The views expressed are those
of the author(s) and not necessarily those of the NHS, the NIHR or the
Department of Health. The funder did not have a role in the design of the
Turnbull et al. BMC Family Practice (2018) 19:25
study, data collection, analysis, interpretation of the data or in writing the
manuscript.
Availability of data and materials
The datasets used and/or analysed during the current study are available
from the corresponding author on reasonable request.
Authors’ contributions
PLu and ST were responsible for conceiving the research question. ADH, PLu,
PLi, PSB, BD, MT, and TJP conceived the original idea for the TARGET cohort
study and were responsible securing funding. ADH, CC, PLu, PLi, PSB, HC, BD,
NMR, MT, ST and TJP were responsible for the study design, data collection
and interpretation. NMR, HC and ST were responsible for study management
and co-ordination. ST, HT, NR, PSB and TJP were responsible for study
analysis; ST led the analysis for this paper. ST drafted the paper. All authors
have read and approved the final manuscript. ST (corresponding and
guarantor author) affirms that the manuscript is an honest, accurate, and
transparent account of the study being reported; that no important aspects
of the study have been omitted; and that any discrepancies from the study
as planned (and, if relevant, registered) have been explained.
Ethics approval and consent to participate
The study was approved by the South West Central Bristol Research Ethics
Committee, UK (reference number: 10/H0102/54) and research governance
approvals were obtained for all areas prior to the start of recruitment. The
study was sponsored by the University of Bristol, which ensured the study
met all regulatory approvals. All participants’ parent or legal carers gave
written, informed consent on behalf of the child. All children aged 11 and
over gave informed assent.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
Centre for Academic Primary Care, Population Health Sciences, Bristol
Medical School, University of Bristol, Canynge Hall, 39 Whatley Road, Bristol
BS8 2PS, UK. 2School for Policy Studies, University of Bristol, 8 Priory Road,
Bristol BS8 1TZ, UK. 3National Institute for Health Research Collaborations for
Leadership in Applied Health Research and Care West (NIHR CLAHRC West),
University Hospitals Bristol NHS Foundation Trust, 9th Floor, Whitefriars,
Lewins Mead, Bristol BS1 2NT, UK. 4Population Health Sciences, Bristol
Medical School, University of Bristol, Oakfield House, Oakfield Grove, Bristol
BS8 2BN, UK. 5Population Health Sciences, Bristol Medical School, University
of Bristol, Level D, St Michael’s Hospital, Southwell St, Bristol BS2 8EG, UK.
6
Department of Surgery and Cancer, Imperial College London, St Mary’s
Hospital, W2 1NY, London, UK. 7Department of Family Medicine, University of
Washington, Seattle, WA 98195-4696, USA. 8Primary Care and Population
Sciences Unit, University of Southampton, Aldermoor Health Centre,
Aldermoor Close, Southampton SO16 5ST, UK. 9School of Clinical Sciences,
University of Bristol, 69 St Michael’s Hill, Bristol BS2 8DZ, UK. 10Centre for
Academic Primary Care, School of Social and Community Medicine,
University of Bristol, Bristol BS8 2PS, UK.
Received: 5 April 2017 Accepted: 24 January 2018
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| 8,167 | 54,970 |
W3198574588.txt | Exosomal MicroRNA-151a-3p Improves the
Sensitivity to Radiotherapy via the Interaction
between p53 and Histone Deacetylase 5
Seung Min Lee
Asan Medical Center
Bo Hyun yoon
Asan Medical Center
Myoung-Hee Kang
Asan Medical Center
Dong Ha Kim
Asan Medical Center
Yong-Hee cho
Asan Medical Center
Min choi
Asan Medical Center
Jin Woo Lee
Asan Medical Center
Tae Keun Kim
Asan Medical Center
Je-Won ryu
Asan Medical Center
Kyunggon Kim
Asan Medical Center
Young Hoon Sung
Asan Medical Center
Chan-Gi Pack
Asan Medical Center
Jin Kyung rho
Asan Medical Center
Sang-wook Lee
Asan Medical Center
Chang Hoon Ha ( chhoonha@amc.seoul.kr )
Asan Medical Center https://orcid.org/0000-0001-7606-4340
Page 1/30
Research
Keywords: Exosome, microRNA, HDAC5, p53, radiotherapy, hepatocellular carcinoma
Posted Date: August 31st, 2021
DOI: https://doi.org/10.21203/rs.3.rs-847360/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read
Full License
Page 2/30
Abstract
Background: Tumor-derived exosomal microRNAs are key elements of the cell-cell communications
response to lots of stimuli. However, various functions of the exosome in tumor suppression by
radiotherapy (RT) are not clearly understood. Our study showed a previously unknown interaction of p53
and histone deacetylase 5 (HDAC5) by radiation exposure in hepatocellular carcinoma (HCC).
Methods: Using serial ultracentrifugation methods, radiation and non-radiation exosomes were purified to
investigate the radioresistance of miRNA151a-3p. Radiation doses were treated in 2gy and 4gy using
radiation equipment X-RAD 320 to observe the expression of HDAC5 and p53 in hepatic cancer cells.
Exosomal miRNA bioinformatics analysis was conducted to find a variation in the miRNA configuration
inside Exosome after radiation exposure.
Results: HDAC5 and p53 interacted by exposure to radiation, which increased exosome release and altered
microRNAs' composition within exosomes. Also, we have described the intercommunication occurring
between irradiated and untreated cells via exosomal microRNAs that affect tumor proliferation. In
particular, the expression of exosomal microR151a-3p was markedly reduced by radiation treatment. We
confirmed that inhibition of exosomal microR151a-3p promotes suppression of non-irradiated cancer cells,
thereby increasing RT sensitivity.
Conclusion: our present findings demonstrated HDAC5 is a key component of the p53-mediated release of
exosomes resulting in tumor suppression through exosomal microRNA-151a-3p in response to radiation.
Finally, we highlight the important role of exosomal microRNA 151a-3p as a biomarker in enhancing RT
sensitivity.
Background
The Exosomes are extracellular vesicles of 30-150 nm in size that are released outside of the cell, which
structurally constitutes a phospholipid bilayer membrane, so there is no possibility of being broken down by
various enzymes in the body. Cancer cells secrete 10 times more exosomes than normal cells, and include
growth factors, chemokines, miRNAs, etc. inside exosomes to regulate the microenvironment around
cancer, and promote the growth and communication of cancer cells [1, 2]. Recent studies on miRNA
contained within exosome have been actively conducted [3, 4]. After release, tumor-derived exosomal
microRNAs (miRNAs) regulate such functions as retaining tumor growth, invasion, metastasis and therapyresistance in tumor cells through cell-cell communication within the tumor derived exosome. Therefore,
exosomal miRNAs have an essential function to mediate tumor progression and can likely be applied as a
promising biomarker in the diagnosis and prediction of cancer [5] [6].
p53 is a tumor suppressor that is very important in cancer and regulates cell-cycle control, aging, apoptosis,
and genomic stability in response to various external stresses [7, 8]. Recent studies have shown that p53
modification is temporarily regulated by histone deacetylase 5 (HDAC5) [9]. It is believed that various posttranslational modifications of p53 play an important role in determining p53 promoter specificity toward its
various target gene sets. During post-translational modifications, Acetylation of p53 at lysine 120 of the
Page 3/30
DNA binding domain has been associated with p53-mediated apoptosis [10]. Further, it is known that the
secretion of exosomes in cells undergoing p53 response by stress is increased by effector TSAP6
downstream of p53. So, exosomes can be regulated by the p53 response [11, 12].
Histone acetylation/deacetylation plays a critical role in the regulation of gene expression [13] [14]. In
particular, HDAC5, one of class II HDACs, has been implicated in modulating cellular signalings by
interactions with various transcriptional cofactors and confers responsiveness to gene expressions.
Previous studies have shown that tumor suppressor p53 is activated by DNA damage that causes
genotoxicity, and as a temporary regulator, HDAC5 interacts with p53 and HDAC5 in the early stages of
stress, thereby deacetylating p53. The pro-apoptotic target gene, modulated by p53, is transactivated by
k120 deacetylation of p53 by HDAC5, which determines the fate of the cell as a regulator of p53
modification [9, 15]. However, little is known about the role of HDAC5 in response to radiation therapy in
tumor.
Radiotherapy (RT) is one of the most commonly used therapeutic methods that can suppress the
proliferation of cancer cells. However, there may be some side effects that reduce sensitivity to RT and
become radiation resistant. In recent years, the goal of RT is to improve the quality of life of patients by
reducing the side effects of radiation treatment. In particular, in hepatocellular carcinoma (HCC), RT has
become a key treatment that prevents the progression of cancer and helps surgical treatment. RT is the
most effective treatment for local tumor metastasis in patients who potentially need liver resection or
transplantation [16, 17].
Here, we showed our findings regarding tumor-derived exosomes, including the miRNA delivering
mechanism. We then described the interactions that occur between irradiated and untreated cells via
exosomal miRNAs that affect tumor proliferation. HDAC5 and p53 interacted by exposure to radiation, the
effect of which increased exosome release and altered the composition of miRNAs within exosomes. In
particular, the expression of Exosomal miRNA 151a-3p was decreased, which is the result of irradiation. As
a result of the experiment, it was confirmed that inhibition of exosomal miRNA 151a-3p promotes
apoptosis of non-irradiated tumor cells, thereby enhancing the RT effect. Finally, we highlight the potential
role of miRNA-151a-3p as biomarkers in tumor therapeutics and sensitivity to RT.
Methods
Cell culture, X-Radiation treatment
HepG2, SK-Hep1 was purchased from the American Type Culture Collection (Rockville, MD) (ATCC HTB177). Hep3B was purchased by Korea Cellular Bank (seoul, Korea). It was cultured in DMEM (Corning) with
10% fetal bovine serum, 4.5 g/L glucose, L-glutamine, sodium pyruvate, and 100 U/mL of penicillin and
streptomycin, and maintained at 37°C in a humidified chamber containing 5% CO2. Radiation was exposed
with 2Gray, 4Gray a using X-RAD320 (1Gray/min at 320KV, 12.5mA, 50cm SSD (HVL≈ 4mm Cu))
equipment.
Page 4/30
Exosome purification
Radiation and non-radiation exosomes purified using ultracentrifugation methods. 1 × 106 cells were
seeded in 100 mm dishes and allowed to recover for overnight. Then, the cells were washed twice with prewarmed PBS, and the culture medium was replaced with exosome-free medium supplemented with 5%
exosome-depleted FBS (SBI). The Radiation exosome is treated 4Gray Radiation using X-RAD 320
(1Gray/min at 320KV, 12.5mA, 50cm SSD (HVL≈ 4mm Cu)) equipment. The medium was gathered and
subjected to gradient centrifugation. Briefly, the medium was first centrifuged at 1000g for 10 min, at 3000g
for 30 min, then centrifuged in a Beckman Coulter Optima™ L-XP Ultracentrifuge System at 100,000 gavg at
4℃ for 90 minutes with a SW 28 Swinging-Bucket Rotor (k-factor: 71) to pellet exosomes. The supernatant
was carefully removed, and crude exosome-containing pellets were resuspended in 1 mL of ice-cold PBS
and pooled. A second round of ultracentrifugation [100,000 gavg at 4℃ for 90 minutes with a Type SW 28
Swinging-Bucket Rotor (k-factor: 71)] was carried out, and the resulting exosome pellet resuspended in 500
μl of PBS.
Western blot analysis
The cells were collected, washed with PBS, and lysed in lysis buffer containing protease inhibitors
(GenDEPOT, USA). After determining the protein concentration with a Bradford Protein Assay Reagent (BioRad), equal amounts of protein were separated on 10% SDS-PAGE, electrically transferred to nitrocellulose
membrane, and blocked with 5% skim milk and 5% BSA. The membranes were incubated with anti-HDAC5
(1:1000; Cell Signaling Technology, USA), anti-p53 (1:2000; Santacruz, USA), anti-p21 (1:2000; Cell
Signaling Technology), anti-Puma (1:2000; Cell Signaling Technology), anti-STEAP3 (1:1000; Proteintech,
USA), anti-Maspin (1:1000; Cell Signaling Technology), anti-CD63 (1:1000; Santacruz), anti- CD81 (1:1000;
Santacruz), anti-HSP90 (1:1000; BD bioscience), anti-BCL2 (1:1000; Cell Signaling Technology), anti-Ecadherin (1:1000; Agilent Dako), anti-Twist1 (1:1000; abcam), N-cadherin (1:1000; Cell Signaling
Technology), Vimentin (1:1000; Calbiochem) or anti-beta-actin (1:1000; Santacruz) primary antibody
overnight shaking at 4 °C. After washing twice, the membranes were then incubated with horseradish
peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibody (Jackson ImmunoResearch
Inc. USA) at room temperature for 1 hour. Finally, the membranes were incubated with WESTSAVE-UP
western blotting substrate (Youngin frontier, Korea), and images were visualized using ChemiDoc imaging
system (Bio-Rad, USA) and recorded.
Duolink proximity ligation assay (PLA)
The DuoLink® In Situ Red Starter Kit Mouse/Rabbit (DUO92101, Sigma-Aldrich, Darmstadt, Germany) was
used to detect interacting target proteins. Cells were seeded in eight-well chamber removable slides (ibidi
GmbH Am Klopferspitz, Germany) and cultured 24hours. Then, HepG2 cells were exposed to 4 g of
radiation, and after 12 hours, the slides were washed with cold 1xPBS and fixed in 4% paraformaldehyde
for 30 minutes. Then slides were blocked with Duolink Blocking Solution in a pre-heated humidified
Page 5/30
chamber for 30 min at 37°C. The primary antibody to detect HDAC5 and p53 was added to the slides and
incubated overnight at 4°C. Then slides were washed with 1×Wash Buffer A and subsequently incubated
with the two PLA probes (1:5 diluted in antibody diluents) for 1 h, then the Ligation-Ligase solution for 30
min, and the Amplification-Polymerase solution for 100 min in a pre-heated humidified chamber at 37°C.
Before imaging, slides were washed with 1×Wash Buffer B and mounted with a cover slip using Duolink In
Situ Mounting Medium with DAPI. Fluorescence images were acquired using a zeiss LSM 780 confocal
microscope. 17
Transmission electron microscopy (TEM)
In order to photograph exosomes, 0.1M of exosome pellets were fixed in 2.5% glutaraldehyde in 0.1 M
cacodylate solution (pH 7.0) for 1 hour and then fixed in 2% osmium tetroxide for another hour. (4°C) After
dehydration using the graded acetone series, embedding was performed through Spurr's medium (Electron
Microscopy Sciences). The resulting section sample was cut at 60 nm with an ultramicrotome (RMC MTXL,
USA), followed by double staining using 2% uranyl acetate for 20 minutes and citrate for 10 minutes.
Prepared sections were photographed at 80 kV using Hitachi H-7600 TEM (Hitachi, Japan) equipment.
Exosome size distribution and concentration measurement.
To determine exosome size distribution and concentration, nanoparticle tracking-based analyses were
performed using a NanoSight (NS500) apparatus (Malvern Instruments Ltd.). Samples were diluted to
provide counts within the linear range of the instrument. The videos of 1-minute duration were recorded for
each sample, with a frame rate of 30 frames per second. Particle movement was analyzed by NTA software
(NTA 2.3; NanoSight Ltd.) according to the manufacturer's protocol. The NTA software was optimized to
first identify and then track each particle on a frame-by-frame basis. 18, 19
Small RNA sequencing
RNA isolation
HepG2-derived Exosomal small RNA was extracted using a miRNeasy Mini Kit (Qiagen Korea Ltd.)
according to the manufacturer’s instructions. RNA quality was assessed by Agilent 2100 bioanalyzer using
the RNA 6000 Pico Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was
performed using a NanoDrop 2000 Spectrophotometer system (Thermo Fisher Scientific, Waltham, MA,
USA).
Library preparation and sequencing
Page 6/30
For control and radiation induced HepG2-drived exosomal small RNAs, the construction of the library was
performed using NEBNext Multiplex Small RNA Library Prep kit (New England BioLabs, Inc., USA) according
to the manufacturer’s instructions. Briefly, for library construction, total RNA from each samples were used
1ug to ligate the adaptors and then cDNA was synthesized using reverse-transcriptase with adaptor-specific
primers. PCR was performed for library amplification and libraries were carried out clean-up using QIAquick
PCR Purification Kit (Quaigen, Inc, German) and AMPure XP beads (Beckmancoulter, Inc., USA). The yield
and size distribution of the small RNA libraries were assessed by the Agilent 2100 Bioanalyzer instrument
for the High-sensitivity DNA Assay (Agilent Technologies, Inc., USA). High-throughput sequences were
produced by NextSeq500 system as way of single-end 75 sequencing (Illumina, SanDiego, CA., USA).
Data analysis
Sequence reads were mapped by bowtie2 software tool in order to obtain bam file. Mature miRNA sequence
is used as a reference for mapping. Read counts mapped on mature miRNA sequence were extracted from
the alignment file using bedtools v2.25.0 (Quinlan AR, 2010) and Bioconductor 20 that uses R statistical
programming language (R development Core Team, 2016). Read counts were used in order to determine the
expression level of miRNAs. Quantile normalization method was used for comparison between samples.
For miRNA target study, miRWalk 2.0 21 was performed. Functional gene classification was performed by
DIANA 22.
Exosomal small RNA sequencing arrays were prepared, hybridized, and scanned at the local authorized
Illumina array service provider (Ebiogen, Seoul, South Korea).
TCGA(The Cancer Genome Atlas) and GEO(Gene Expression
Omnibus) Database analysis
The two gene expression profiling data sets (GSE74618, GSE147889) we analyzed were downloaded from
the NCBI GEO database.23 The GSE74618 database consists of 218 human HCC tumours samples, 10
adjacent cirrhotic non-tumoral tissue samples, and 10 healthy liver samples. We used adjacent cirrhotic
non-tumoral tissue samples and healthy liver samples as controls and compared them with human HCC
tumours samples. The GSE1477889 database consists of 97 samples of HCC tumours and 97 samples of
the same patient's surrounding chronic hepatitis tissue. Surrounding chronic hepatitis tissue was used as a
control and compared with HCC tumour samples. Two data sets were analyzed as volcano plots and mean
difference plots, respectively, using the GEO2R analysis tool. TCGA (The Cancer Genome Atlas) data
analysis was performed using oncomiR (WashU Pan-Cancer miRNome Atlas) online bioinformatics tool.24
Among the TCGA data, the analysis was conducted using the LIHC (Liver Hepatocellular Carcinoma)
database (n=366), and the days' survival rate graph of liver cancer patients was exhibited.
Page 7/30
Kyoto encyclopedia of genes and genomes (KEGG) pathway
enrichment analyses
KEGG is a database that contains genetic information on the human genome, biological cell signalling
pathways, and various diseases and chemicals. The KEGG pathway analysis of exosomal miRNA was
analyzed using the DIANA-miRPath v3.0 analysis tool. DIANA-miRPath v3.0 is a KEGG-based miRNA
pathway prediction tool that actively interacts with the major miRNA analysis tools, DIANA-microT CDS and
TargetScan v6.2 database, to analyze the target pathway.22
miRNA Transfection
Cells were seeded in 6-well plates at 1 × 105 cells/mL/well before the transfection and radiation. On the
following day, After HepG2 cells were exposed 4gy of radiation using X-RAD 320 and transfection was
performed when the cells had reached approximately 80% confluence. miRCURY LNA miR-151a-3p mimic
(miRCURY LNA miRNA Mimic, MIMAT0000757) were purchased from QiAGEN (Qiagen Korea Ltd.). And
scrambled miRNA control were purchased from BIONEER (Seoul, Korea). The final concentrations of miRNA
were 50-100nM Transfections were conducted with Lipofectamine 3000 (Invitrogen) according to the
manufacturer’s instructions.
Quantitative real time reverse transcription polymerase chain
reaction(qRT-PCR)
Total RNA was extracted using a miRNeasy Mini Kit (Qiagen Korea Ltd.) in the manufacturers' protocols.
For the quantification of matured cellular and exosomal miRNAs, the extracted total RNA was
polyadenylated with a poly(A) tailing kit (Ambion, Austin, TX) prior to reverse transcription. And was reverse
transcribed to cDNA using PrimeScript™ Reverse Transcriptase (Takara Bio Inc.). The cDNA samples were
used for quantitative RT-PCR analysis in triplicate to determine the expression levels of miR-151a-3p using
a TB Green® Premix Ex Taq™ (Takara Bio Inc.) and CFX96 Detection System real-time PCR instrument.
Proliferation assay
The cell proliferation assay was performed using the Chromo-CK Cell Viability Assay KIT (monobio seoul,
korea) according to the manufacturer’s instructions. Transfected cells were harvested at the designated
times after seeding. Briefly, the reagent (10 μl/well) was added to 100 μl of medium containing cells in each
well of a 96-well plate and incubated for 1 hours at 37℃ under humidified 5% CO2 in air. For colorimetric
analysis, absorbance at 450 nm was recorded using an ELx800 Absorbance Microplate Reader
(SpectraMax 340PC Microplate Reader, Molecular Devices, USA). Each experiment was repeated at least 3
times.
Page 8/30
Migration invasion assay
Tumor cell migration and invasion were analyzed in 24-well plates with 8-μm pore size polycarbonate
membranes (BD, NJ, USA). For invasion assays, the membranes were coated with diluted Matrigel (BD, NJ,
USA) to form matrix barriers. HepG2 were transfected with 151a-3p or negative control. For the migration
and invasion assays, the cells (5×10^4 for migration, 1×10^5 for invasion) were resuspended in 200μl of
serum-free DMEM at 24 hours post-transfection and added to the upper compartments of the chambers,
and the lower compartments were filled with 600μl of DMEM with 10% FBS (different substrates were
added accordingly). After incubation at 37°C for 10 hours (migration) or 24 hours (invasion), the cells
remaining on the upper surfaces of the membrane were removed. The cells on the lower surfaces of the
membrane were fixed, stained with crystal violet and counted under a light microscope. Each experiment
was repeated at least 3 times.
Analysis of cell cycle
The Transfected cells were collected and resuspended after radiation exposure. Annexin V-fluorescein
isothiocyanate (FITC) and propidium iodide (PI) staining assays were conducted to detect the percentage
of apoptotic (FITC-stained) and necrotic (PI-stained) cells in a given population. Analyses were performed
by a FACS canto flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s
instructions. The procedures were repeated in triplicate. 25
Analysis of cell apoptosis
The Transfected cells were collected and resuspended with cold PBS after radiation exposure. And fixed in
cold 70% ethanol added dropwise to the cell pellet while vortexing. Stored at -20℃ for overnight. Cells were
rehydrated with PBS for 10 min at RT and then cells were stained with propidium iodide (PI) staining
solution contained with 50 μg/ml PI (BD Biosciences, Franklin Lakes, NJ, USA), 100 μg/ml RNase A, DNase
and protease-free (Thermo Scientific) in 1X binding buffer. Analyses were performed by a FACS canto flow
cytometer (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. The
procedures were repeated in triplicate.
Subcutaneous tumor xenograft models
BALB/c nude mice (5-week old male) were purchased from the Charles River Laboratories, Inc (Wilmington,
Massachusetts). The fodder was feed to the normal rodent laboratory animal feed. 5x10^6 HepG2 cells in
total volume 100ul were injected into the right flank of a 5-week-old nude mouse in a 1:1 ratio with Matrigel
(corning 356234). After the average of the transplanted solid cancer size increased by more than 100 mm3,
the invivo-jetPEI/target miRNA complex was treated. invivo-jetPEI was used as an efficient carrier for
delivering miRNA and was mixed with 5% glucose solution at a ratio of miRNA 10ug / invivo-jetPEI 1.2ul
and treated in a total dose of 100 ul. The invivo-jetPEI/target miRNA complex was administered 3 times
Page 9/30
every 3 days by intratumoral injection. Tumor size was measured using a standard ABS digimatic caliper
(CD-15AX) every 3 days after transplantation. Tumor volume was measured using the following formula: ∏
x 4/3 x larger diameter x smaller diameter square. At the time when the volume of the largest tumor of the
implanted nude mice in the experimental group exceeded 1000 mm3, the tumor was separated after
sacrifice, the weight of the tumor was measured, and expression of the target protein in cancer tissue was
analyzed.
Statistical analysis
Each experiment in our study was repeated three times. Mathematical data are expressed as mean ± SD.
Unless indicated, the differences between two groups were analyzed using a Student's t-test (two-tailed).
Unless otherwise noted, significant numerical differences between the two groups were analyzed using
Student's t-test (two-tailed). The overall survival curve was graphed using the Kaplan-Meier method and
compared through the log-rank test. Differences were evaluated statistically significant at P< 0.05. All
statistical analysis was performed using SPSS13.0 software (SPSS, Chicago, IL, USA).
Results
Modulation of p53 and HDAC5 by RT
An experiment was conducted to verify that protein p53 and HDAC5 were modulated by radiation in HCC.
Radiation doses were treated in 2gy and 4gy using radiation equipment X-RAD 320 to observe the
expression of HDAC5 and p53 when radiation was treated in three types of hepatic cancer cells. The
interaction of HDAC5 and p53 by DNA damage has been previously described [9]. However, the interaction
of HDAC5 and p53 in HCC by radiation is not yet known. HepG2 and SK-Hep1 are p53 wild types (p53+/+)
and Hep3B are p53 deletion types (p53-/-). The amount of expression of each protein was measured
through the Western Blot in order of 6 hours, 12 hours, 18 hours, and 24 hours (Fig. 1A-F). In HepG2, the
expressions of HDAC5 and p53 were significantly increased from 6 hours compared to the control group by
radiation 2gy and 4gy. Later, at 2 gy of radiation, HDAC5 showed a time-dependent decrease again.
However, HDAC5 and p53 were significantly increased in radiation 4gy regardless of time. This shows that
the expression of HDAC5 and p53 and its downstream is regulated by irradiation in HepG2 (Fig. 1. A,B). SKHep1 increased the expression of HDAC5 24 hours after 2gy irradiation. However, in 4 gy, it was reduced
compared to the control group. p53 and its downstream showed a significant increase by irradiation, but
HDAC5 did not show a significant expression pattern (Fig. 1. C, D). Finally, Hep3B showed an independent
increase in HDAC5 expression without p53 after irradiation. As a result, it was confirmed that among p53
and HDAC5 increased by irradiation, HDAC5 independently increased protein expression (Fig. 1E, F).
Subsequent experiments were conducted on HepG2, which showed the most significant change by RT
among the three liver cancer cells. To check the relationship between HDAC5 and p53, a HepG2 cell line that
p53-/- was created using the CRISPER CAS9 system. The target site of Human p53:
TGTAACAGTTCCTGCATGGG was investigated by the NGS method. The insertion/deletion frequency was
Page 10/30
99.98% (T insertion:19.9%, CCTG deletion:18.8%, AT insertion:19.5%), and the elimination of the p53 gene
was confirmed to be close to perfection (supplementary Fig. 1). The radiation was exposed for p53+/+,
p53-/-cells and measured the protein expression of HDAC5 and p53, p21, Puma with western blot. The
expression of p53, p21 did not almost appear because p53 was deleted. Regardless of p53 identified an
increase in the expression of HDAC5. As a result, it was confirmed that the expression of HDAC5 was
increased by irradiation regardless of the expression of p53 in cells, which showed the most significant
expression by irradiation, and the relationship between HDAC5 and p53 was identified (Fig. 1. G, H). Using
FACS equipment, it was confirmed that cell cycle arrest and apoptosis are induced in p53+/+, p53-/-cells by
radiation treatment. In p53+/+cell, powerful cell cycle arrest in the G2/M phase was observed from 6 hours
after irradiation and gradually switched to G1 phase cell cycle arrest as time passed. A similar cell cycle
alteration was observed in p53-/-cell, but it was verified that the effect was smaller than that of p53+/+cell.
Also, p53-/-cell had significantly less apoptosis 48 hours after irradiation than p53+/+cell. It showed
stronger cell cycle arrest and apoptosis at p53+/+cell compared to p53-/-cell (supplementary Fig. 2).
Intercommunication between p53 and HDAC5 by RT
resulting in increasing the expression of TSAP6, exosome
modulating protein
To identify the more accurate interaction between p53 and HDAC5, this was investigated using the duolink
PLA assay. When the cells were not exposed to radiation, no red dots were found to interact, and 4 Gy of
radiation revealed multiple red dots, the interaction between p53 and HDAC5. It has been demonstrated that
radiation exposure induces a significant increase in the interaction of p53 and HDAC5 in cells (Fig. 2. A).
Using western blot validated whether TSAP6 (Tumor Suppressor Activation Pathway 6), an exosomemodulated protein in the direct downstream of p53 and controlled by p53 is increased by RT [11, 12]. The
expression of TSAP6 was verified with a western blot at 12 hours and 24 hours after exposure to 4Gy of
radiation. Our results showed that the interaction between HDAC5 and p53 was enhanced by radiation, and
the downstream TSAP6 of p53 was also significantly increased (Fig. 2. B, C).
The characteristics of exosomes derived from p53+/+, p53-/-cells were identified. Exosomes secreted by
cells were observed using an electron microscope. (Fig. 2. D) Western blot analysis showed that
microvesicles released from p53+/+, p53-/-cells expressed exosome markers CD63, CD81, and HSP90. All
exosome markers have been demonstrated [18]. We also identified the discriminating expression of protein
maspin (mammary serine protease inhibitor), which is released by p53 dependently through exosome by
radiation exposure [11]. Inside p53+/+cell derived exosomes, it was found that the expression of maspin
was increased by radiation. However, p53-/-cell derived exosomes demonstrated no expression of maspin
by radiation exposure. (Fig. 2. E, F) The expression of maspin inside the exosome induced by radiation
exposure is regulated by the expression of p53. p53 directly binds to the p53 gene-consensus binding site
present in the maspin promoter, through activating the maspin promoter and inducing maspin expression
[19].
Exosome secretion in a p53-dependent manner by RT
Page 11/30
After exposure to radiation, experiments were conducted to demonstrate that the secretion of exosome
changes over time. The number of cultured p53+/+cells and p53-/-cells was equal to 5x106. Experiments
were conducted at 6, 12, 24, and 48 hour intervals after exposure to 4 Gy of radiation, and exosomes
released at each hour were extracted by ultracentrifugation. The qualitative and quantitative comparison
and analysis of the isolated exosomes were performed using the Nanosight NT300 equipment
(supplementary Fig. 3. A, B). As a result of the analysis, the Exosome showed a heterogeneous exosomal
population with a diameter of 30–300 nm or more. These results suggest that the isolated exosomes had
high purity and satisfy the size, density, structure, and characteristics of vesicles secreted by cells [18].
p53+/+cells manifested a significant increase in the secretion of overall vesicles when exposed to radiation
of 4 Gy. As a result of comparing the total vesicles by each secreted time, it was found that the volume of
secreted vesicles increased more than twice when exposed to radiation. In particular, 24 hours after
exposure to radiation, the total quantity of vesicle secretion was found to be more than three-fold that of
the control, and the most vesicle secretion exhibited at this time (Fig. 3A). Conversely, p53-/-cells exhibited a
decrease in the volume of vesicle secretion after exposure to radiation. From 6 hours to 48 hours, the
quantity of vesicle released in all experimental groups was significantly decreased compared to the control
(Fig. 3B). The secreted extracellular vesicles were analyzed qualitatively by separating them into 30-150nm,
150-300nm, and > 300nm by size. The p53+/+cells secreted Exosome size part (30-150nm) was the most
significant increase of 12 hours after radiation exposure. As time passed after exposure to radiation, the
variation of vesicles was characterized by a change in the direction of increasing their size. It was verified
that exosome-sized vesicles were noticeably reduced in vesicles 48 hours after exposure to radiation (Fig.
3C). In the same way, p53-/-cells derived exosome was analyzed by dividing each size. The exosome-sized
vesicles released after irradiation were also rapidly reduced (Fig. 3D). The exosomes of p53+/+ and p53-/cells released after irradiation were analyzed qualitatively and quantitatively. (Fig. 3E, F) This experiment
proved that the secretion of extracellular vesicles was increased by radiation exposure. Among them, 30150nm-sized exosomes proved to be increased or decreased by p53 dependency. This result suggests that
the expression of p53 induced by RT increases exosome secretion through the TSAP6 pathway.
Alteration of exosomal miRNA’s expressions by RT in HCC
After the release in a cell, exosomes are taken up by neighboring or distant cells, and the miRNAs contained
within modulating such processes as interfering with tumor immunity and the microenvironment, possibly
facilitating tumor growth, invasion, metastasis, angiogenesis, and drug resistance. Therefore, exosomal
miRNAs have a significant function in regulating tumor progression. Exosomal miRNA bioinformatics
analysis was conducted to find a variation in the miRNA configuration inside Exosome after radiation
exposure. The variation of miRNA in exosome secreted from the radiation exposed cell and miRNA in
exosome secreted from without radiation exposure was analyzed through Heatmap utilizing MeV software
(Small RNA sequencing, miRNA-Seq Only Analysis Program developed by Dana-Farber Cancer Institute in
the United States) (supplementary Fig. 4. A). Also, to find out which intracellular function each of the
miRNAs inside the exosome changed after irradiation, it was classified and analyzed into various cell
functional categories using Gene Ontology terms (supplementary Fig. 4. B). Handling the miRNA's fold
change values analyzed, the graph was composed in descending order (Fig. 4. A). It has been confirmed
that 33 miRNAs have significantly reduced due to irradiation among 156 miRNAs that have inside exosome
Page 12/30
(Fig. 4. B). Among the miRNAs with reduced expression, we analyzed using the hepatocellular carcinoma
GEO data set to identify the miRNAs associated with the progression of hepatocellular carcinoma. The GEO
data set was downloaded from NCBI Gene Expression Omnibus, and analyzed by volcano plot and meandifference plot using the GEO2R analysis tool (supplementary Fig. 5. A, B). As a result of analysis utilizing
the HCC miRNA GEO data set (Spain, GSE74618), it was verified that 4 specific miRNAs (miR-151a-3p, miR106b-5p, miR-183-5p, miR-452-5p) were significantly increased in tumor tissues compared to normal (Fig.
4.C) [20]. The HCC miRNA GEO data set (Spain, GSE74618) was re-validated using another HCC miRNA
GEO data set (Japan, GSE147889) to confirm the accuracy of the analysis results (fig S4.B). To ascertain
the relationship between the four miRNAs (miRNA151a-3p, miRNA 106b-5p, miRNA183-5p, miRNA 452-5p)
with increased expression in HCC and the survival rate of liver cancer patients, we analyzed using OncomiR
Cancer miRNome Atlas. As a result of OncomiR analysis, it was found that the higher the expression of
miRNA151a-3p, miRNA 106b-5p, miRNA183-5p, and miRNA 452-5p in the tumour tissues of liver cancer
patients, the significantly decreased the survival rate of liver cancer patients (Fig. 4.D) [21]. We performed
the KEGG pathway analysis to find out which molecular biological processes the four miRNAs, which were
directly related to the prognosis of liver cancer patients, affect cancer (Table 1). Among the analysis results
performed, the cell cycle, a mechanism directly related to radiation therapy, and the p53 signalling pathway,
the central target of our study, was ascertained to be significant signaling mechanisms. In addition,
targetscan (http://www.targetscan.org/vert_72/) analysis tool was used to analyze the target protein of
each miRNA. As a result of the target analysis of miRNA, it was discovered that miRNA151a-3p can bind to
the p53 mRNA target region with a high probability (Fig. 4E). Likewise, in previous studies, it has been
reported that miRNA 151a-3p was bioinformatically analyzed as a target miRNA of p53 and binds to the
p53 mRNA target region, thereby inhibiting the expression of p53 in cells [22, 23]. We considered miRNA
151a-3p interesting and continued the study, and it was confirmed that miRNA 151a-3p is deeply related to
tumorigenesis of various cancers (Table. 2). Furthermore, three cancer types were identified, where patient
survival was significantly associated with miRNA 151a-3p (Table. 3). As expected, miRNA 151a-3p had a
great correlation with the prognosis of liver cancer patients. So, it has been confirmed that miRNA 151a-3p
is a respectable relation to tumorigenesis and survival of Liver Hepatocellular carcinoma (LIHC). We
actually verified that miRNA 151a-3p was reduced by radiation exposure both intracellularly and in
exosomes. The expression of miRNA 151a-3p in cell and exosome after radiation exposure was analyzed
using qRT-PCR. The quantification of miRNA 151a-3p was quantified using miR-Let7a [24]. Cellular miRNA
151a-3p decreased by 1/2 after radiation exposure, whereas exosomal miRNA 151a-3p decreased by more
than 1/4 after radiation exposure. (Fig. 4. F, G) As a result, miRNA 151a-3p had a significant relationship
with the poor survival of HCC. It also after irradiation in hepG2, a HCC cell line, demonstrated a significant
decrease in the expression of miRNA 151a-3p inside the cellular and exosome.
Table 1
Results of KEGG pathway analysis of major miRNAs within exosomes that were reduced after radiation
exposure.
Page 13/30
KEGG pathway
p-value
Genes
Pathways in cancer
6.61E-08
103
Viral carcinogenesis
1.62E-06
56
Cell cycle
1.88E-06
43
Adherens junction
1.88E-06
26
Colorectal cancer
2.49E-06
25
Hepatitis B
2.01E-05
45
Thyroid cancer
2.61E-05
15
TGF-beta signaling pathway
0.000154
27
Bladder cancer
0.000154
19
p53 signaling pathway
0.000226
27
Wnt signaling pathway
0.000238
43
Endometrial cancer
0.000245
19
Renal cell carcinoma
0.000284
24
PI3K-Akt signaling pathway
0.000379
87
RNA transport
0.000401
48
Protein processing in endoplasmic reticulum
0.000401
50
AMPK signaling pathway
0.000401
40
Pancreatic cancer
0.001437
24
Insulin signaling pathway
0.002105
41
mTOR signaling pathway
0.002607
22
Focal adhesion
0.002607
55
Small cell lung cancer
0.002778
27
Non-small cell lung cancer
0.006262
18
Endocytosis
0.006754
50
Sphingolipid signaling pathway
0.008416
32
mRNA surveillance pathway
0.014393
27
Hepatitis C
0.015025
37
Phosphatidylinositol signaling system
0.024036
20
MAPK signaling pathway
0.024051
59
Page 14/30
Table 2
15 cancer types where tumorigenesis is significantly associated with hsa-miR-151a-3p expression.
Table 3
cancer types where survival is significantly associated with hsa-miR-151a-3p.
Cancer
Abbreviation
Log
Rank
Pvalue
Log
Rank
FDR
Zscore
Upregulated
in:
Deceased
Expression
Living
Expression
T-Test
Pvalue
T-Test
FDR
LGG
4.71e02
3.48e02
1.990
Deceased
10.37
10.15
4.53e02
1.98e01
LIHC
5.47e04
1.71e02
3.471
Deceased
11.68
11.34
3.93e03
7.93e02
SARC
8.84e04
5.62e01
3.320
Deceased
11.00
10.65
2.15e02
5.56e01
LGG: brain lower grade glioma; LIHC: liver hepatocellular carcinoma; SARC: sarcoma
Radiotherapy reduced expression of exosomal miRNA-151a3p resulting in tumor suppression
We investigated the radioresistance of miRNA151a-3p. A protein related to cell apoptosis and cell cycle
arrest that can be caused by radiation was verified 24 hours after the transfection of negative control and
miRNA151a-3p in the cell through the western blot. Cancer repression gene p53 significantly reduced. A
Decrease of P21 which inhibits cyclin-dependent phosphorylation to stop cell cycle has been identified. The
BCL-2 controlling the Mitochondrial Outer Membrane Permeabilization Pore, which creates a channel that
increases the permeability of the mitochondrial outer membrane, has been increased. And Puma
associated with apoptosis has been reduced (Fig. 5. A, B). Based on the results of Fig. 5. A-B, the cell cycle
arrest analysis experiment was conducted using FACS to verify the function of miRNA151a-3p in cell cycle
arrest induced by radiation exposure. 24 hours after miRNA151a-3p transfection, 4Gy radiation was
exposed to the cell. After six hours, the cell cycle of each experimental group was analyzed (Fig. 5. C). The
G2/M phase arrest was increased in the N.C group and the 4Gy positive group. In the miR-151a-3p group,
the G2/M phase arrest was reduced relatively (Fig. 5. D). Also, it has been confirmed that miRNA151a-3p
has a function to inhibit cell apoptosis induced by radiation exposure. As a result of the experiment, the
miRNA151a-3p group significantly reduced total cell apoptosis compared to the N.C. and 4Gy positive
groups (Fig. 5. E-F).
Page 15/30
Cancer
Abbreviation
T-Test
P-value
T-Test
FDR
Upregulated
in:
Tumor
Expression
Normal
Expression
BLCA
9.93e05
6.17e04
Tumor
11.82
10.21
BRCA
5.98e05
1.61e04
Tumor
10.98
10.68
COAD
7.53e03
1.78e02
Tumor
10.61
9.57
ESCA
7.78e03
3.89e02
Tumor
10.55
10.14
HNSC
7.69e05
2.63e04
Tumor
11.35
10.96
KICH
1.06e03
3.05e03
Tumor
10.99
10.49
KIRC
8.36e25
2.30e23
Tumor
10.68
9.95
KIRP
1.54e05
7.80e05
Tumor
10.60
10.13
LIHC
1.12e10
2.40e09
Tumor
11.67
10.90
LUAD
1.52e04
7.47e04
Tumor
11.68
11.20
LUSC
1.97e05
7.41e05
Tumor
11.54
11.06
PRAD
9.20e06
4.29e05
Tumor
10.58
10.26
READ
8.54e03
4.23e02
Tumor
11.28
8.51
STAD
3.86e11
2.92e09
Tumor
11.25
10.11
UCEC
9.47e08
7.54e07
Tumor
11.41
10.22
BLCA: bladder urothelial carcinoma; BRCA: breast invasive carcinoma; COAD: colon adenocarcinoma;
ESCA: esophageal carcinoma; HNSC: head and neck squamous cell carcinoma; KICH: kidney
chromophobe; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; LIHC:
liver hepatocellular carcinoma; LUAD: lung adenocarcinoma; LUSC: lung squamous cell carcinoma;
PRAD: prostate adenocarcinoma; READ: rectal adenocarcinoma; STAD: stomach adenocarcinoma;
UCEC: uterine corpus endometrial carcinoma
The expression of EMT marker protein was verified 24 hours after the transfection of negative control and
miRNA151a-3p in the cell through the western blot. E-cadherin, a representative epithelial phenotype marker,
was reduced in the miRNA151a-3p group. There was a significant increase in the mesenchymal phenotype
Page 16/30
marker vimentin and N-cadherin moving inversely to E-cadherin, and a noticeable increase in the expression
of Twist1 associated with p53 metastasis (Fig. 6A, B) [25, 26]. For functional evaluation that induces the
progression of cancer of miR-151a-3p, proliferation assay, Transwell migration, and invasion assay were
implemented. A significant increase in proliferation was identified in the miRNA151a-3p 100nM group,
depending on the concentration of miRNA151a-3p (Fig. 6C). Similarly, the cell movement and the increase
in metastasis were identified depending on the concentration of miRNA151a-3p in cell migration, Invasion
assay using transwell (Fig. 6D). The migrated and invasive cells were dyed and expressed in a graph after
counting (Fig. 6E). So, evidence of the above data has demonstrated that miRNA151a-3p can induce EMTlike phenotype and increase cell proliferation.
In order to confirm the tumor progression ability of miRNA151a-3p in vivo, a nude mouse subcutaneous
xenograft model experiment was conducted. HepG2 cells were injected 5x10^6 subcutaneously in the flank
of nude mice. Thereafter, miRNA-NC and miRNA-151a-3p / in vivo jet-PEI complexes were injected three
times at 3 days intervals using the established in vivo jet-PEI transfection reagent to increase the
expression of miRNA 151a-3p in cells [27]. In the in vivo experiment, the size of the tumor was significantly
increased in miR-151a-3p compared to miRNA-NC, and the growth rate of the tumor was also faster (Fig.
7A-C). In addition, in western blot analysis in tumour tissue, miRNA-151a-3p significantly inhibited the
expression of p53, P21, and Puma α/β (Fig. 7D, E). Comprehensive results of the experiment showed that
overexpression of miR-151a-3p decreased the expression of the target protein in the tumour, and as a result,
promoted tumour progression in the xenograft model.
Discussion
In-depth studies have shown that exosomes, which are widely present in various body fluids, contain many
types of proteins, miRNAs, and various small molecules [3]. After secretion, exosomes are absorbed by
adjacent or distant cells, and the miRNAs in them regulate processes such as promoting tumor growth,
invasion, angiogenesis, and resistance to radiotherapy (RT). Therefore, miRNAs are promising as
biomarkers in clinical targeted therapies in regulating cancer progression. [28]. Exosomes can be
considered as modulators that target and bind underlying genetic molecules in the pathways regulating RT.
RT is one of the important treatments for preventing tumor progression, and through various mechanisms,
it affects target cancer tissue and surrounding cancer cells. Among them, p53 plays a key role in the
suppression of cancer through RT [29],[30].
Our current study demonstrated a previously unknown interaction of p53 and HDAC5 in HCC by radiation
exposure. Moreover, the expression of both proteins was confirmed in a time-dependent manner by
radiation exposure. We confirmed that HDAC5 expression was increased by RT in p53-/-HepG2 cells,
indicating that HDAC5 is an up-regulator of p53. These findings show that HDAC5 plays an important role
in p53-mediated exosome release by radiation.
As previously studied, TSAP6 is known as a protein that regulates the secretion of exosomes [12]. TSAP6,
exosome secretion modulating protein through P53, has increased expression in a radiation exposure
environment. Changes of exosomes after radiation exposure were qualitatively and quantitatively analyzed
Page 17/30
by Nanoparticle Tracking Analysis (NTA). The presence or absence of the p53 gene dramatically changed
exosomes' secretion depending on the time of radiation exposure. In the previous figure, p53+/+ TSAP6
was most expressed after 12 hours of radiation exposure, and secretion of exosome was also most
secreted after 12 hours of radiation exposure. On the contrary, the expression of TSAP6 by radiation
exposure in the p53-/-cells did not change, and the secretion of exosomes after radiation exposure
decreased. This suggests that TSAP6 is clearly involved in exosome secretion and that TSAP6 is regulated
by changes in p53 resulting from radiation exposure. We photographed exosomes separated for exosome
characterization using electron microscopy and analyzed western blot of exosome marker protein. Among
the exosome marker proteins, Mammary Serine Protease Inhibitor (maspin) is a protein regulated by the
p53 gene and functions to suppress cancer, and many studies are underway. Previous studies have
confirmed that maspin inside the exosome is increased by radiation exposure in the lung cancer cell line
[11]. We found that the expression of maspin increased significantly within the exosome secreted from
p53+/+cells after radiation exposure. Besides, we confirmed that the expression of maspin did not increase
in the p53-/-cells. Maspin is mounted on the exosome and released outside the cell, indicating that it
functions to suppress tumor progression in surrounding cancer cells. Maspin is expected to suppress the
cancer progression of the recipient cell.
The exosome is loaded with various substances (ex, protein, mRNA, miRNA., etc.) and plays an important
role in communication between cells. Among them, miRNA is a potential biomarker, and many studies are
underway [31] [32]. As a short RNA strand of the 18-22base pair, it binds to specific mRNAs with the same
sequence and inhibits the gene's protein expression. After radiation exposure, changes in the miRNA
composition balance inside the exosomes were observed. The change of miRNA inside exosome changed
by radiation was thought to be the starting point for communication between cancer cells and changes in
the cancer microenvironment.
Therefore, we generally performed a miRNA microarray to determine the miRNAs' overall expression in
exosomes released in culture and exosomes released from irradiated cells. As a result of the analysis, 156
exosomal miRNAs were expressed out of a total of 2588 miRNAs. We classified exosomal-miRNAs for each
cell function using GO term to determine which cellular function the changed exosomal-miRNAs are
involved in after irradiation. As a result of classifying exosomal-miRNAs with a normalized expression
(log2) of 1.5 or more, the number of exosomal-miRNAs involved in cell proliferation and cell migration
decreased the number of exosomal-miRNAs involved in DNA repair and apoptosis increased. Among them,
we conducted an analysis focusing on 33 miRNAs whose expression almost disappeared after irradiation.
To find out the relationship between the disappeared 33 miRNAs and HCC, each miRNA expression was
analyzed using the miRNA microarray dataset cohort in the cancer tissues of HCC patients. Among the 33
miRNAs that disappeared after irradiation, miR-151a-3p, miR-106b-5p, miR183-5p, and miR452-5p involved
cell proliferation and invasion were significantly higher in expression in HCC cancer tissues.
So, we focused on miRNAs whose expression was reduced in exosomes after irradiation. As a result of
analyzing using the GEO dataset (GSE74618) to find out how exosomal-miRNAs with decreased expression
after irradiation were exhibited in cancer tissues of human liver cancer patients, 4 miRNA candidates with
specifically increased expression were found. Four miRNA candidates showed a statistically significant
Page 18/30
increase in expression in liver cancer tissues. Subsequently, it was analyzed using the TCGA database
(oncomiR) to analyze the association between miRNA candidates and liver cancer patients' survival rate. All
miRNA candidates showed a direct correlation with the poor prognosis of liver cancer patients. These
results are expected to contribute as a part of the mechanism for inhibiting cancer growth caused by
radiation therapy.
MiRNA 151a-3p, one of the four miRNAs that had a significant relationship with the survival rate of liver
cancer patients, was found to bind p53 with a high probability due to target protein analysis through the
targetscan prediction tool. Subsequently, it was confirmed by qRT-PCR that the expression of miRNA151a3p was reduced in cells and exosomes due to radiation exposure. p53 is one of the key genes that maintain
cell homeostasis by increasing expression by DNA damage such as radiation, leading to phenotypes such
as cell cycle and cell death. As expected, miRNA151a-3p significantly reduced the expression of p53. Also,
by inhibiting the expression of p53 downstream molecules P21 and Puma, which play an important role in
the cell cycle and apoptosis in the p53 signaling pathway, it actually prevented cell cycle arrest and
suppressed apoptosis after radiation exposure. Eventually, we confirmed that miRNA 151a-3p induces
radiation resistance by inhibiting p53 signaling.
Subsequent experiments were conducted to determine the effect of miR151a-3p on cells. When
miRNA151a-3p was transfected, Epithelial-mesenchymal transition (EMT)-like phenotype was verified by
expression of related markers, increase of cell growth, migration, and invasion was discovered. The
expression of Twist1, which indirectly inhibits cell cycle arrest and apoptosis functions regulated by p53,
was increased [25, 33]. In our experimental results, in the miRNA151a-3p overexpression group, cell cycle
arrest, and apoptosis were significantly reduced compared to the miRNA NC group. So, we proved that
miRNA151a-3p induces anti-cell cycle arrest and anti-apoptosis effects on cells exposed to radiation.
Conclusions
Here, we confirmed the microRNA delivery mechanism through tumor-derived exosomes in RT. We have
described the intercommunication occurring between irradiated and untreated cells via exosomal miRNAs
that affect tumor proliferation. HDAC5 and p53 interacted by exposure to radiation, which increased
exosome release and altered the composition of miRNAs within exosomes. In particular, the expression of
exosomal miR151a-3p was markedly reduced as a result of the investigation. We confirmed that inhibition
of exosomes miR151a-3p promotes apoptosis of non-irradiated cancer cells, thereby increasing
radiotherapy sensitivity. Understanding the molecular mechanisms of exosome secretion and microRNA
expression will help design novel therapeutics that improve sensitivity to RT in HCC. In conclusion, our
present findings establish HDAC5 as a key component of the p53-mediated release of exosome in response
to radiation treatment resulting in tumor apoptosis through exosomal miR-151a-3p resulting in
improvement of RT.
Abbreviations
Page 19/30
RT, radiotherapy; HDAC5, histone deacetylase 5; HCC, hepatocellular carcinoma; TSAP6, Tumor Suppressor
Activation Pathway 6; miRNA, microRNA; EMT, Epithelial-mesenchymal transition.
Declarations
Ethics approval and consent to participate
Not applicable
Consent for publication
Not applicable
Availability of data and material
Not applicable
Competing interests
The authors declare that they have no competing interests.
Funding
This research was supported by National Research Foundation of Korea (NRF) grants (NRF2021R1A6A1A03040260, NRF-2020R1I1A2072110, NRF-2017M2A2A7A02020212). This work was also
supported by grants (2018IP0600-1, 2019IP0848-1, 2020IL0006-1, 2021IP0048-1) from the Asan Institute
for Life Sciences, Asan Medical Center, Seoul, Korea.
Authors' contributions
SML analyzed the data and developed the study. BHY., MHK, DHK, YHC, MC, JWL, TKK, JWR, KGK, YHS,
CGP, and JKR collected and performed samples in the field. SWL, and CHH planed the study and wrote the
manuscript with input from all of the authors. The authors read and approved the final manuscript. there
are no conflicts where this is the case.
Acknowledgements
We thank the Confocal Microscope core facility and the Comparative Pathology Core at the ConveRgence
mEDIcine research cenTer (CREDIT), Asan Medical Center for support and instrumentation.
Page 20/30
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Figures
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Figure 1
Modulation of protein p53 and HDAC5 expression by radiation exposure in hepatocellular carcinoma.
Radiation was exposed to 2Gy and 4Gy, and the protein expression of p53, P21, and HDAC5 were confirmed
by Western blot analysis in a time-dependent manner up to 24 hours per six hours after exposure. (A-F).
Using the CRISPER CAS9 system, a cell line with p53-/- in HepG2 was produced and the expression of
HDAC5, p53, P21 and PUMA was confirmed through Western blot analysis after radiation exposure. The
expression of each protein was quantified by b-actin. (G, H)
Page 23/30
Figure 2
Interaction of HDAC5 and p53 by radiation exposure in HepG2 cells, and characterization of the exosome
secretion pathway and the released exosomes that are altered by this union. The interaction between
HDAC5 and p53 was confirmed after radiation exposure using Duolink proximity ligation assay in HepG2
(A). The expression of TSAP6 (STEAP3) by radiation exposure was confirmed by Western blot analysis. The
expression of TSAP6 (STEAP3) was quantified by b-actin (B,C). Electron microscopy was used to image
resuspended exosome particles isolated from HepG2 (D). The expression of exosome marker protein
Page 24/30
HSP90, CD63, CD81 and Maspin with each sample 10ug after radiation exposure was verified with western
blot analysis. (E,F).
Figure 3
Time-dependent measurement and identification of the difference in exosome size and release amount
determined by the p53 gene in liver cancer cells. Time-dependent measurements were made to determine
whether the secretion of extracellular vesicles (EV) changes after radiation exposure using Nanosight's
Nanoparticle Tracking Analysis (NTA) (A-B). Based on the analyzed nanosight data, three parts (30-150nm,
Page 25/30
150-300nm, >300nm) were displayed by vesicle size (C,D). The secretion of EVs for the presence or absence
of Tp53 genes after radiation exposure was analyzed qualitatively and quantitatively (E,F).
Figure 4
The expression analysis of exosomal miRNAs altered by irradiation in liver cancer cells and the clinical
meaning exploration and identification of candidates using bioinformatics analysis. Before and after
radiation exposure, exosomal miRNA was arranged into a fold change value and analyzed as a graph and
ven diagram (A,B). Applying the GEO data set, the expression difference between normal and cancer of
each candidate miRNA was analyzed. (C). To investigate the clinical significance of each candidate, it was
analyzed using OncomiR Cancer miRNome Atlas and patient survival was expressed by Kaplan-Meier
analysis (D). The target protein prediction analysis of the major candidate miR-151a-3p was performed by
the Targetscan analysis tool (E). The modulation of a miR-151a-3p the radiation exposure was analyzed by
qRT-PCR in the cell and the exosome. (F,G).
Page 26/30
Figure 5
Cell dysfunction according to radiation exposure is regulated by miR151a-3p. Western blot analysis was
performed to confirm that p53 and its sub-signals P21, PUMA, and BCL-2, which are closely related to
cancer suppression, are modulated by miR151a-3p. (A,B) Cell cycle arrest assay using FACS was performed
by PI staining to confirm that cell cycle arrest induced by radiation exposure was modulated by miR151a3p. The Cell cycle was analyzed by dividing into G1, S, and G2 / M phases. (C,D) To confirm that cell
Page 27/30
apoptosis induced by radiation exposure is modulated by miR151a-3p, it was analyzed using FACS by
Annexin V (FITC), PI staining. (E,F)
Figure 6
The ability of miRNA151a-3p to tumorigenesis. That the representative Epithelial-mesenchymal transition
(EMT) marker of E-cadherin, Twist1, Vimentin, N-cadherin is regulated by the miR151a-3p was confirmed by
Western blot analysis. (A,B) The cell proliferation according to the concentration of the miR-151a-3p was
Page 28/30
analyzed by MTT assay. (C) Cell migration and invasion ability according to the concentration of miR-151a3p were analyzed using transwell assay. (D,E)
Figure 7
Increased tumor progression in a nude mouse subcutaneous tumor transplant model overexpressing
miRNA 151a-3p Acceleration of tumor growth by overexpression of miRNA 151a-3p in a subcutaneous
xenograft model.(n = 5) (A-C) Representative images at day 18 post-transplantation with a subcutaneous
xenograft nude mouse model injected with miR 151a-3p/miR-NC. (A) After the mice were sacrificed on the
18th day, the tumor was separated and the size was compared, and the weight of the tumors of each group
was measured and plotted. (B) In the subcutaneous xenograft model, miR-151a-3p, miR-NC, and invivo jetPEI complex were injected three times every three days from the third day after tumor transplantation, and
the size of the tumors of each group was measured every three days. (C) The expression of the related
target protein and downstream in tumour tissues were analyzed by Western blot. (D,E) A schematic image
Page 29/30
of Radiation-induced exosomal miRNA-151a-3p restrain hepatocellular carcinoma progression: By
radiation, HDAC5 and p53 in HCC cells increase the secretion of exosomes as a result of the interaction.
The exosomal miRNA-151a-3p inside the secreted exosome functions to infiltrate the surrounding recipient
cells and promote the progression of cancer. (F)
Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.
SupplementaryFigures.docx
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W2990859622.txt | Genuino et al. BMC Health Services Research
https://doi.org/10.1186/s12913-019-4715-8
(2019) 19:874
RESEARCH ARTICLE
Open Access
Cost-utility analysis of adjuvant
trastuzumab therapy for HER2-positive
early-stage breast cancer in the Philippines
Anne Julienne Genuino1,2, Usa Chaikledkaew1,3* , Anna Melissa Guerrero2, Thanyanan Reungwetwattana4 and
Ammarin Thakkinstian1,5
Abstract
Background: Breast cancer is the leading malignancy among Filipino women, with about 23.50% of cases
characterized by human epidermal growth factor receptor-2 (HER2) overexpression. Trastuzumab, in addition to
standard chemotherapy, is currently recommended as primary treatment for HER2-positive early-stage breast cancer
(EBC) in the adjuvant settings, and has been listed in the Philippine National Formulary (PNF) since 2008, but with
no current evidence yet on its value for money, to date. Hence, despite several policy enablers, its accessibility
remains to be limited in the Philippines. We performed an economic evaluation to assess the cost-effectiveness and
budget impact of adjuvant trastuzumab therapy for HER2-positive EBC in the Philippines, using healthcare system
and societal perspectives, in aid of guiding coverage decisions.
Methods: A Markov model-based cost-utility and budget impact analyses were conducted to estimate the total
costs incurred and outcomes gained in using 1 year of adjuvant trastuzumab added to standard chemotherapy
versus standard chemotherapy alone, over a lifetime horizon. We discounted both costs and outcomes at 3.5% per
annum. Parameters were estimated using country survival data, systematic review and meta-analysis of the relative
treatment effect, local and international cost data, and published utility data. Univariate and probabilistic sensitivity
analyses were used to account for parameter uncertainty.
Results: Trastuzumab therapy was dominated with an incremental cost-effectiveness ratio (ICER) at PHP 453,505 per
QALY gained from a healthcare system perspective or PHP 458,686 per QALY gained from a societal perspective,
with 10% cost-effectiveness probability at the country cost-effectiveness threshold of PHP 120,000 per QALY gained.
National implementation will cost an additional amount of PHP 13,909 million in year one alone, plus about PHP
2000 to 3000 million annually for the succeeding fiscal years.
Conclusion: At its current cost, 1 year of adjuvant trastuzumab therapy compared to standard chemotherapy alone
for HER2-positive EBC does not represent value for money in the Philippines. Its current cost will have to
significantly lower down by one-half to achieve cost-effectiveness.
Keywords: Adjuvant trastuzumab, Cost-utility analysis, HER2-positive, Breast cancer, Cost-effectiveness, Philippines
* Correspondence: usa.chi@mahidol.ac.th
1
Mahidol University Health Technology Assessment (MUHTA) Graduate
Program, Bangkok, Thailand
3
Social and Administrative Pharmacy Excellence Research (SAPER) Unit,
Department of Pharmacy, Faculty of Pharmacy, Mahidol University, 447
Sri-Ayudhaya Rd., Phayathai, Ratchathewi, Bangkok 10400, Thailand
Full list of author information is available at the end of the article
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Genuino et al. BMC Health Services Research
(2019) 19:874
Background
Breast cancer is currently the globally leading malignancy among women with about 1.7 million diagnosed
cases and 521,907 deaths as estimated by the World
Health Organization (WHO) in 2012 [1]. In the
Philippines, it is recognized as the most prevalent
cancer among women and in both sexes, as well as the
most common cause of cancer deaths among women
[2]. About 15 to 20% of breast cancers overexpresses
human epidermal growth factor receptor-2 (HER2) [3]
– a clinically important subtype of breast cancer that is
associated with an aggressive disease phenotype and
shortened survival outcomes [4], resulting in poorer
prognosis compared to other subtypes. Two large studies of breast cancer data in the United States [5] [6]
reported, however, that women of Asian descent were
more likely to have HER2-positive tumours than
Caucasian women suggesting possible racial differences. In the Philippines, the Department of Health
(DOH) Breast Cancer Control Program reported a
HER2-positivity rate of 23.17%, with an estimated 80%
of them at the early stage [7, 8].
The discovery of revolutionary therapies such as
trastuzumab, the first monoclonal antibody to specifically target HER2, has changed the course of treatment
and improved the prognosis of affected breast cancer
patients. Several key pivotal trials have demonstrated
its relative treatment efficacy versus standard chemotherapy alone, in improving the disease-free and overall survival of HER2-positive early-stage breast cancer
(EBC) patients [9–16]. These same trials though have
reported an associated increased risk for cardiotoxic
effects such as congestive health failure (CHF) and left
ventricular ejection fraction (LVEF) decline [9–16].
Both global [17, 18] and national clinical guidelines
[19] currently recommend the administration of trastuzumab with chemotherapy regimens as primary treatment for HER2-positive EBC in adjuvant settings. It has
been listed in the Philippine National Formulary (PNF)
since 2008, but with no current evidence yet on its value
for money to date. Hence, despite several policy
enablers, its accessibility remains to be limited as the
current government insurance case rate for breast cancer
does not cover the treatment for HER2-positive type;
public hospitals cannot afford to procure and make it
available in their facilities due to its high cost; and, while
it has been recently included in the list of subsidized
medicines under the DOH Breast Cancer Medicine
Access Program (DOH BCMAP) under a negotiated reduced price for national hospitals, the access sites and
medicine stocks are limited.
Assessing cost-effectiveness is critical in establishing
equitable trade-off decisions between the sustainable
access to such effective health technology that can
Page 2 of 12
improve survival and the limited health budget, especially for resource-constrained countries such as the
Philippines. While many published economic evaluation (EE) studies have been previously conducted to
assess its cost-effectiveness, all were conducted in
settings that are not comparable to a lower-middle
income country (LMIC) like the Philippines, as they
were all from upper-middle and high-income countries
[20–35]. Therefore, we conducted this economic evaluation to assess the cost-effectiveness and budget
impact of adjuvant trastuzumab therapy compared to
chemotherapy regimen alone for patients with HER2positive EBC in the Philippines, to guide coverage
decisions.
Methods
We conducted a cost-utility analysis (CUA) using decision analytic Markov model to calculate and compare
the costs and utilities of using 1 year of adjuvant trastuzumab combined with standard chemotherapy (i.e.,
doxorubicin 60 mg/m2 on day 1 plus cyclophosphamide 600 mg/m2 on day 1 every 3 weeks for 4 cycles
followed by docetaxel 100 mg/m2 on day 1 plus trastuzumab 8 mg/kg initial dose then 6 mg/kg on day 1
every 3 weeks for 4 cycles then trastuzumab 6 mg/kg
on Day 1 every 3 weeks for 14 cycles) versus chemotherapy alone (i.e., same chemotherapy minus trastuzumab) for Filipino women with HER2-positive EBC.
The model cohorts were Filipino women with HER2positive EBC who enter the model at the age of 50
years old which is the mean age of patients enrolled in
the Department of Health Breast Cancer Medicines
Access Program (DOH BCMAP) [36]. We modelled
over a lifetime horizon from both publicly-funded
healthcare system and societal perspectives, with a
discounting rate of 3.5% per year applied to both costs
and outcomes. We measured the incremental costeffectiveness ratio (ICER) as Philippine Peso (PHP) per
Life Year (LY) or Quality-adjusted life year (QALY)
gained. We applied the current Philippine costeffectiveness threshold value of PHP 120,000 per
QALY gained that was set by the Formulary Executive
Council in the Philippines based on the value of one
times Gross Domestic Product (GDP) per capita in the
Philippines. In the case that trastuzumab was not costeffective, a threshold analysis was performed to calculate the cost-effective price of trastuzumab therapy.
Further, we estimated the likely budget impact of its
national coverage for five fiscal years.
Model overview
Our model structure as illustrated in Fig. 1 consists of
five health states - disease-free survival (DFS), congestive
heart failure (CHF), local recurrence, distant metastasis,
Genuino et al. BMC Health Services Research
(2019) 19:874
Page 3 of 12
Congestive
Heart Failure
Disease-Free
Survival
Local
Recurrence
Death
Distant
Metastasis
Fig. 1 Schematic diagram of the Markov model
and death. Upon administration of either of the competing interventions, the cohort enters the model at the
DFS state. We applied a cycle length of 1 year which was
run for 49 cycles to represent lifetime horizon. The
model assumed that patients who progressed to CHF
could only experience it once as cardiotoxicity from
trastuzumab is only an asymptomatic decline in LVEF
and is mostly reversible with interruption of trastuzumab or cardiac medication [37]. From CHF state, they
have the option to move to DFS, local recurrence, or
distant metastasis state.
It was also assumed that the cohort does not have
any baseline co-morbidities and the use of hormonal
therapies was not considered. Based on the methodological domains and assumptions, a spread sheet model
was developed using Microsoft Office Excel 2013
(Microsoft Corp., Redmond, WA) to generate the total
lifetime costs incurred and LYs or QALYs gained
through cohort simulation.
distant metastasis, and mortality for the intervention
model cohort when at DFS state. As for the longest
follow-up data available to date showing constant
treatment effect over 11 years [9], it was therefore
assumed that the efficacy of trastuzumab lasts for 11
years. The cardiotoxic effect was incorporated in the
model as increased risk for CHF for the intervention
model cohort.
Utility data
The utility values associated with the model health states
were sourced from a quality of life study among 30 patients each with early and advanced breast cancers at
Hanoi Oncology Hospital and Da Nang Oncology Hospital Vietnam [41], which were derived using the EuroQol
five-dimension with three-level (EQ-5D-3 L) questionnaires. As the only utility weight study derived from an
Asian population in a fellow LMIC setting to date, it was
deemed that Vietnam utilities are comparable and applicable to the Philippine setting.
Model input parameters
Clinical data
Cost data
The primary data source for the baseline TPs between
the health states was obtained from published literatures [31, 38], which were estimated from the fouryear follow-up of joint analysis of data from NCCTG
N9831 and NSABP B-31 trials on the efficacy and
safety trastuzumab plus standard anthracycline-taxanebased CT [39].
We generated the relative treatment effect of trastuzumab by conducting a systematic review and metaanalysis of published trials [9–16] on the efficacy and
safety of adjuvant trastuzumab [40]. The benefit of
trastuzumab was incorporated in the model as a reduction applied in the risk of developing local recurrence,
Direct medical costs (DMC) were comprised of workup, treatment and monitoring costs related to laboratory and diagnostic tests, procedures, admissions and
outpatient visits, and pharmacologic therapy. A preconstructed costing sheet guided by clinical guidelines
[18, 42, 43] was consulted among local experts. We
then valuated the final costing items using the government standard case rate values [44] and data from a
local costing study [45] for the medical services, procedures, and diagnostics; and, the government drug price
reference index [46] and procurement data [47] for all
drug costs. The cost per vial of trastuzumab was based
on the negotiated price under the DOH BCMAP for
Genuino et al. BMC Health Services Research
(2019) 19:874
the 150 mg-IV-vial preparation amounting to PHP 619,
667 for all trastuzumab cycles. The dose calculation
was based on a 65 kg-body weight. All costs were
expressed in PHP 2017 values and inflation adjustments were applied as necessary by using the Consumer Price Indexes (CPI) [48].
Direct non-medical costs (DNMC) were estimated
using the costing values for food and transportation expenses from the Thai Standard Costing Lists for Health
Economic Evaluation [49], in the absence of these standard costing values in the Philippines. These costs (in
2009 Thai Baht values) were converted to 2017 PHP
values by using the 2009 conversion factors 1 USD =
33.129 Thai Baht [50] and 1 USD = PHP 46.421 [51],
then adjusted for inflation using CPI [48]. The utilization
of such costs was assumed to incur for every health facility visit involved relevant for the particular health state.
The list of all input parameters used in the model is
summarized in Table 1.
Sensitivity analysis
Both one-way and probabilistic sensitivity analyses
(PSA) using second order Monte Carlo simulation replicated for 50,000 times were performed to handle
parametric uncertainties. We assigned beta distribution for TPs and utility parameters; log-normal distribution for the relative treatment effect; and, gamma
distribution for cost parameters. The PSA results were
illustrated as incremental cost-effectiveness planes and
cost-effectiveness acceptability curves. A discount rate
of 0 to 6%, and a treatment efficacy duration of 5 years
to 49 years were also applied in the one-way sensitivity
analysis.
Results
Cost-effectiveness analysis
Based on a probabilistic approach, from the healthcare
system perspective, trastuzumab therapy compared to
chemotherapy alone was estimated to incur an additional cost of PHP 452,128 with an expected health gain
of additional 1.20 LY or 1.00 QALY per patient, resulting in an ICER of PHP 377,009 per LY gained, or PHP
453,505 per QALY gained. From a societal perspective,
adopting adjuvant trastuzumab therapy was estimated to
cost an additional PHP 457,131 for a similar additional
health benefit, resulting to an ICER of PHP 381,405per
LY gained, or PHP 458,686 per QALY gained.
The ICERs from both perspectives have consistently
shown that adding 1 year of adjuvant trastuzumab to
chemotherapy for HER2-positive EBC is not costeffective in the Philippines as they exceeded the costeffectiveness threshold by about 3.8 times more. The
lifetime costs, LYs and QALYs gained from using trastuzumab therapy versus chemotherapy alone, and the
Page 4 of 12
resulting ICERs in the probabilistic analysis from the
two perspectives are shown in Table 2.
Threshold sensitivity analysis
Results have shown that the cost-effective price of adjuvant trastuzumab therapy per patient is PHP 596,239
from the healthcare system perspective, or PHP 590,314
from a societal perspective. This implies that the current
cost of adjuvant trastuzumab therapy overall, including
the cost of standard chemotherapy drugs and other supportive medications, chemotherapy administration cost
and cardiac function tests (i.e., PHP 1,076,607), needs to
be decreased further by about one-half in order to
achieve an ICER that will be at least equal to the costeffectiveness threshold.
Probabilistic sensitivity analysis
The results the Monte Carlo simulation with 50,000
replications were plotted in a cost-effectiveness plane,
presented in Fig. 2. Majority of the ICER plots appear
at the upper-right hand quadrant of the plane extending to form an ellipsoid shape which implies the
positive correlation of the incremental cost and the incremental outcomes. The black line represents the
resulting mean ICER while the green line represents
the cost-effectiveness threshold where estimates below
this line are considered cost-effective.
Figure 3 presents the cost-effectiveness acceptability
curve for the healthcare system. Transforming the results of the CE plane to a cost-effectiveness acceptability
curve as illustrated in Fig. 3 demonstrates that at the
cost-effectiveness threshold of PHP 120,000 per QALY
gained (green line), the probability of cost-effectiveness
of adjuvant trastuzumab therapy is 10%, while that for
the chemotherapy alone regimen is 90%, under both perspectives. The current cost-effectiveness threshold has to
increase by about four times more (i.e., PHP 500,000 per
QALY gained) in order for adjuvant trastuzumab to
reach at least 48% probability of cost-effectiveness from
the publicly-funded healthcare system perspective, or
51% probability of cost-effectiveness from the societal
perspective. The trastuzumab curve only started to rise
steeply as the threshold increases at PHP 650,000 per
QALY gained.
Deterministic sensitivity analysis
The results of the one-way sensitivity analyses are presented in Fig. 4. Among all input parameters varied
under both perspectives, the ICER results were most
sensitive to variations in the hazard ratio for DFS, duration of efficacy of trastuzumab, discounting rate for
outcomes, cost of trastuzumab therapy, and TP from
DFS to metastasis state. On the other hand, the model
estimates were negligibly sensitive with respect to
Genuino et al. BMC Health Services Research
(2019) 19:874
Page 5 of 12
Table 1 Input parameters used in the model and their sampling distribution for the probabilistic sensitivity analysis
Parameter
Mean (SE) Distribution Source
Clinical Parameters
Baseline Transitional Probabilities
DFS ➔ CHF
0.0053
(0.0024)
Beta
DFS ➔ Recurrence
0.0294
(0.0029)
Beta
DFS ➔ Metastasis
0.0785
(0.0140)
Beta
DFS ➔ Death
0.0020
(0.0001)
Beta
CHF ➔ Recurrence
0.0294
(0.0029)
Beta
CHF ➔ Metastasis
0.0785
(0.0140)
Beta
CHF ➔ Death
0.1500
(0.0153)
Beta
Dokainish et al., 2017 [52]
Recurrence ➔ Metastasis
0.0785
(0.0140)
Beta
Buendia et al., 2013 [31]
Recurrence ➔ Death
0.2950
(0.2066)
Beta
Metastasis ➔ Death
0. 0.2950
(0.2066)
Beta
Pooled hazard ratio for DFS
0.65
(0.0825)
LogNormal
Pooled hazard ratio for OS
0.67
(0.0493)
LogNormal
Pooled risk ratio for CHF
3.97
(0.2240)
LogNormal
5 – year prevalence of Breast Cancer in the Philippines
64,046
prevalent
cases
–
WHO GLOBOCAN, 2012 [8]
Incidence of Breast Cancer in the Philippines
21,057
new
cases
–
Estimated based on the 2015 new cases (Philippine Cancer
Facts and Estimates, 2015 [2]) adjusted to 2017 values by
applying the incidence rate calculated using the total
population for 2015 [53] and 2017 [54]
Percentage of HER2-positivity of breast cancer in the
Philippines
23.17%
–
DOH Breast Cancer and Control Program, 2013 [7]
Estimated percentage of early-stage HER2-positive
breast cancer cases in the Philippines
80%
–
DOH Breast Cancer and Control Program, 2013 [7]
DMC of adjuvant trastuzumab therapy for the intervention PHP
cohort (per patient per treatment course) – Drugs, CT
1076607
administration, cardiac function assessment
(54929)
Gamma
DOH Philippines -Pharmaceutical Division, 2018 [47]
Philippine Health Insurance Corporation, 2013 [44]
DMC of adjuvant CT for the control cohort (per patient per
treatment course) – Drugs, CT administration, cardiac
function assessment
PHP
194900
(9944)
Gamma
DNMC of adjuvant treatment for intervention cohort
PHP 9432 Gamma
(481)
DNMC adjuvant treatment for control cohort
PHP 3494 Gamma
(178)
DMC at DFS state - imaging, labs, visits/consultation
PHP 9493 Gamma
(484)
Buendia et al., 2013 [31]
Relative treatment efficacy of adjuvant trastuzumab therapy
Genuino et al., 2019 [40]
Epidemiological Data
Cost Parameters
Riewpaiboon, 2014 [49]
Wong, 2018 [45]
Genuino et al. BMC Health Services Research
(2019) 19:874
Page 6 of 12
Table 1 Input parameters used in the model and their sampling distribution for the probabilistic sensitivity analysis (Continued)
Parameter
Mean (SE) Distribution Source
DNMC at DFS state
PHP 1747 Gamma
(80)
Riewpaiboon, 2014 [49]
PHP 3400 Gamma
(1602)
Philippine Health Insurance Corporation, 2013 [44]
DNMC at CHF state
PHP 1049 Gamma
[48]
Riewpaiboon, 2014 [49]
DMC at Local Recurrence state (first year) – work-up,
radiotherapy, CT drugs, CT administration
PHP
567156
(3061)
DNMC at Local Recurrence state (first year)
PHP 8166 Gamma
(1488)
Riewpaiboon, 2014 [49]
DMC at Local Recurrence state (after first year) CT drugs, CT administration
PHP
182437
(15513)
Gamma
DOH Philippines, 2018 [46]
Philippine Health Insurance Corporation, 2013 [44]
DNMC at Local Recurrence state (after first year)
PHP
17817
(178)
Gamma
Riewpaiboon, 2014 [49]
DMC at Local Recurrence state (after first year) –
CT drugs and administration
PHP
516904
(26373)
Gamma
DOH Philippines, 2018 [46]
Philippine Health Insurance Corporation, 2013 [44]
DNMC at Local Recurrence state (after first year)
PHP 5939 Gamma
(273)
Riewpaiboon, 2014 [49]
DMC at Distant Metastasis state (first year) - work-up,
radiotherapy, CT drugs, CT administration, palliative
care drugs
PHP
956172
(48784)
Gamma
DOH Philippines, 2018 [46]
Philippine Health Insurance Corporation, 2013 [44]
DNMC at Distant Metastasis state (first year)
PHP
10131
(465)
Gamma
Riewpaiboon, 2014 [49]
DMC at Distant Metastasis state (after first year) –
CT drugs and administration, palliative care drugs
PHP
1666816
(59531)
Gamma
DOH Philippines, 2018 [46]
Philippine Health Insurance Corporation, 2013 [44]
DNMC at Distant Metastasis state (after first year)
PHP 5939 Gamma
(273)
Riewpaiboon, 2014 [49]
DFS
0.8320
(0.0084)
Beta
Ahn et al., 2014 [41]
CHF
0.6700
(272.71)
Beta
Recurrence
0.8280
(0.0262)
Beta
Metastasis
0.7620
(0.0262)
Beta
DMC at CHF state
-hospital admission, echocardiography, drugs, cardiac
monitoring
Gamma
DOH Philippines, 2018 [46]
Philippine Health Insurance Corporation, 2013 [44]
Utility Parameters
Table 2 Cost-effectiveness results of probabilistic sensitivity analysis
Healthcare system perspective
Trastuzumab-CT regimen
CT only regimen
Difference
Total Lifetime Cost (PHP)
4,462,407
4,010,279
452,128
Total LYs gained
11.07
9.87
1.20
Total QALYs gained
8.99
7.99
1.00
ICER (PHP per LY gained)
377,009
ICER (PHP per QALY gained)
453,505
Genuino et al. BMC Health Services Research
(2019) 19:874
Page 7 of 12
Fig. 2 Cost-effectiveness plane from a publicly-funded healthcare system perspective
variations in the DMC and DNMC for all health states,
and all utilities at the different health states. The yellow bars show the effect on the ICER of applying the
lower limit of the specific parameter, while the green
bars show the effect on the ICER of applying the upper
limit of the specific parameter.
We likewise explored the impact on the costeffectiveness of adjuvant trastuzumab therapy of simultaneous changes in the values of the HR of DFS and the
duration of efficacy. The two-way sensitivity analysis as
illustrated in Fig. 5 shows that over the plausible range
of the said parameters, standard chemotherapy remained
the cost-effective approach.
Budget impact analysis
The estimated number of prevalent cases of HER2positive EBC in the Philippines is 11,872 which was
calculated based on the estimated proportion of earlystage cases (i.e., 80% [7]) among all HER2-positive patients which is about 23.17% (i.e., HER2-positivity
rate in the Philippines [7]) of the total breast cancer
prevalent cases in the country [8]. The same calculation was performed for the generation of the
estimated new cases of 3903 by calculating the proportions of early-stage HER2-positive cases from the
total breast cancer new cases in the Philippines [2]
adjusted to 2017 value.
Fig. 3 Cost-effectiveness acceptability curve – publicly-funded healthcare system perspective
Genuino et al. BMC Health Services Research
(2019) 19:874
Page 8 of 12
Fig. 4 One-way sensitivity analysis from a publicly-funded healthcare system perspective
The total budget for implementing adjuvant trastuzumab therapy to cover all estimated prevalent and new
cases of HER2-positive EBC patients in the Philippines
will demand high health budget as it will incur PHP 16,
983 million per cohort in year one alone, plus more than
PHP 5200 million annually per cohort on the next four
fiscal years. On the contrary, implementing chemotherapy alone to cover the same number of prevalent and
new cases will incur a total cost of PHP 3075 million in
year one, and more than PHP 2300 million annually on
the next four fiscal years. The incremental budget, therefore, is about PHP 13,909 million in year one alone, plus
about PHP 2000 to 3000 million annually on the next
four fiscal years, as presented in Fig. 6.
Discussion
To our knowledge, this is the first published study in
Philippines and among LMICs to evaluate the health
and economic impact of adjuvant trastuzumab therapy
for HER2-positive EBC. The study is intended to guide
coverage decisions on such effective but high cost therapy under a low-resource setting. It is believed that this
economic evaluation faithfully considered and represented all the best available evidence appropriate to a
developing country context by conducting a systematic
review and meta-analysis to estimate the relative treatment effect.
In this model, we project that shifting to 1 year of adjuvant trastuzumab therapy in addition to chemotherapy
Fig. 5 Two-way sensitivity analysis from a publicly-funded healthcare system perspective
Genuino et al. BMC Health Services Research
(2019) 19:874
Page 9 of 12
Fig. 6 Five-year governmental budget impact
for HER2-positive EBC, at its current cost, shall incur
additional cost to the government of PHP 453,505 or
USD 9084, or the society with additional PHP 458,686or
USD 9188 (2017 Exchange Rate: 1 USD = PHP 49.9230
[51],) for every unit of QALY gained. Exceeding the
cost-effectiveness threshold by 3.8 times more, our findings suggest that such therapy is not cost-effective in the
Philippines with 10% of being cost-effective at the
threshold compared to chemotherapy alone at 90%. Its
value for money will improve if its current therapy cost
will reduce by one-half. Apart from cost-effectiveness,
the decision makers will also have to consider the affordability of trastuzumab coverage. The total acquisition
cost for trastuzumab drug alone for a national coverage
implementation will cost about PHP 7.36 billion – an
amount that significantly exceeds the usual annual
budget for the procurement of various breast cancer
medicines covered under the DOH BCMAP (i.e., ranging
from PHP 39 to 92 million from year 2009 to 2017) and
can consume the chunk of the DOH budget allocation
for the procurement of all drugs under the national
health programs (i.e., PHP 9.87 billion for 2018) [55, 56].
Our generalizations are comparable with majority of
previous EEs from upper-middle-income countries
(UMICs) [31–33] which similarly concluded that trastuzumab in their settings is not cost-effective. In Iran [33],
the ICER is about USD 174,901 per QALY gained which
is 17 times more than its cost-effectiveness threshold.
The ICERs range from about USD 63,036 to 153,554 in
Latin American countries [31, 32], similarly exceeding
their cost-effectiveness threshold by 6 to 15 times more.
On the contrary, the CUA studies from high-income
countries (HICs) [20–24, 26–30, 34, 35] concluded that
trastuzumab is cost-effective in their settings with their
ICERs ranging from USD 11,334 to 78,929 per QALY
gained. Trastuzumab though was found to be costeffective in the analyses from two UMICs specifically in
China by Chen et al., 2009 [25] and in Thailand by
Kongsakon et al., 2018 [38], with ICERs at USD 9976
per QALY gained and USD 6527 per QALY gained, respectively. Such relatively lower ICER and opposing conclusion compared to the earlier mentioned findings from
majority of analysis from UMICs [31–33] may be
explained by several factors. Chen et al., 2009 applied a
relatively lower hazard ratio (i.e., 0.54) versus those used
by the studies in Iran (i.e., 0.64) and Latin America (i.e.,
0.59). Second, it applied a 5-year efficacy duration with
decreasing efficacy in a stepwise function, compared to
the studies in Iran and Latin America which applied a 5year duration of efficacy with zero applied benefit onwards. Kongsakon et al., 2018 [38] did not incorporate
cardiac events in their modelling analysis which may
have resulted to an underestimated ICER.
Considering ICER values alone, our ICERs (~ USD
9084 per QALY gained) were in fact lower than the
ICER range from UMICs (USD 63,036 to 174,901 per
QALY gained) which concluded for non-costeffectiveness [31–33], the ICER range from HICs (USD
11,334 to 78,929 per QALY gained) which concluded
for cost-effectiveness [20–24, 26–30, 34, 35]. Two
factors in this analysis may have contributed to our
relatively low ICER: First is the longer duration of trastuzumab efficacy applied in this modelling (i.e., 11
years) versus most of the previous studies (i.e., 5 years)
which was based on the currently available follow-up
data during the time of those EEs; and, second is the
Genuino et al. BMC Health Services Research
(2019) 19:874
lower cost of trastuzumab therapy in our analysis
(USD 12,412) versus other studies (USD 35,349 to 137,
677). Our low ICER, however, did not automatically
translate to favourable cost-effectiveness findings because of the relatively low Philippine cost-effectiveness
threshold at USD 2404 per QALY gained only, compared to the higher thresholds of UMICs (~USD 5000
to 15,000 per QALY gained) and HICs (USD 37,920 to
354,555).
Our results were specifically sensitive to the following parameters i.e., the relative treatment efficacy
which underscores the significance of estimating the
effectiveness from real-world data where a less
favourable treatment effect in the actual clinical practice setting may be possibly observed compared to
follow-up studies under controlled environment; the
duration of clinical efficacy which supports the significance of longer follow-up data on trastuzumab to define its real duration of benefit, and therefore its value
for money; and, the cost of trastuzumab therapy which
emphasizes the need to consider schemes to bring
down its incurred cost to the government in order to
achieve cost-effectiveness. Nevertheless, the model and
its overall findings proved to be relatively robust,
showing minimal sensitivity with variations in the
majority of the parameters.
There were several limitations in this analysis. First,
we referred to the Vietnamese utility data for breast cancer in the absence of such data in the Philippines. While
there may be differences between Filipino and Vietnamese
patients on the valuation of health states because of numerous factors (e.g., cultural differences affecting disease
perception, social support, health system structure), this
approach was deemed acceptable because of a comparable
Asian context and economic status. Second, we used the
Thai cost values for the DNMCs in the societal perspective analysis due to data unavailability. As the only available and accessible standard costing menu from a similar
Asian setting, using the Thai cost values was likewise
deemed acceptable. The recommended study perspective
anyway in the Philippine methods guide for economic
evaluation is publicly-funded healthcare system which, in
our analysis, used purely local costing data. Lastly, the estimation of baseline TPs were obtained from published
data, not local data. Hence, it is recommended in future
analysis to re-run the modelling using local TPs, health
utilities and DNMC values. Exploring the role of subgroups (i.e., hormone-receptor and nodal status) on the
value for money of trastuzumab is likewise proposed.
Conclusion
In conclusion, this CUA suggests that 1 year of adjuvant
trastuzumab therapy, at its current cost, in addition to
the standard chemotherapy for HER2-positive EBC,
Page 10 of 12
might not be cost-effective and unaffordable in the
Philippines. The government should consider schemes
to lower down the cost to improve its value for money
such as price negotiation, facilitation of the entry of
cheaper biosimilar products, or targeted coverage (for
the worst prognosis subtype or high risk subgroups), ultimately towards sustainable access to trastuzumab.
Our findings are designed to aid policy decisions of a
resource-constrained setting on the coverage of such
effective but high cost therapy. As this research is the
first published study among LMICs to evaluate the
health and economic impact of adjuvant trastuzumab
therapy for HER2-positive EBC, our findings may
guide decision-makers from comparable low resource
settings with low capacity to conduct economic evaluations who are also facing a similar policy and research
question.
Abbreviations
CHF: Congestive heart failure; CPI: Consumer price index; CT: Chemotherapy;
CUA: Cost-utility analysis; DMC: Direct medical cost; DNMC: Direct nonmedical cost; DOH BCMAP: Department of Heath Breast Cancer Medicine
Access Program; DOH: Department of Heath; EBC: Early stage breast cancer;
EE: Economic evaluation; HER2: Human epidermal growth factor receptor 2;
HIC: High income country; HR: Hazard ratio; ICER: Incremental costeffectiveness ratio; LMIC: Lower-middle income country; LVEF decline: Left
ventricular ejection fraction decline; LY: Life year; OS: Overall survival;
PCS: Philippine Cancer Society; PHL CE threshold: Philippine costeffectiveness threshold; PHP: Philippine Peso; PNF: Philippine National
Formulary; PSA: Probabilistic sensitivity analysis; QALY: Quality adjusted life
year; RR: Risk ratio; TP: Transition probability; UMIC: Upper-middle income
country; WHO: World Health Organization
Acknowledgements
The authors are grateful to the Pharmaceutical Division and the National
Breast Cancer Control Program of the DOH Philippines, and to Dr. Rachel
Rosario and Mr. Romeo Marcaida of the PCS.
Authors’ contributions
AJG performed the research, analysed data and drafted manuscript. UC
designed the research, validated and interpreted data, as well as drafted
manuscript. AMJ designed the research and drafted manuscript. TR and AT
interpreted clinical data and drafted manuscript. All authors have agreed and
approved the author’s contribution and final manuscript.
Funding
Our study was not supported by a pharmaceutical company. This work is a
part of training in Health Technology Assessment (HTA)'s Master degree,
which scholarship is provided by Mahidol University and the International
Decision Support Initiative (iDSI). This work was produced as part of the
International Decision Support Initiative (www.idsihealth.org), which supports
countries to get the best value for money from health spending. iDSI
receives funding support from the Bill & Melinda Gates Foundation, the UK
Department for International Development, and the Rockefeller Foundation.
The funding agencies of this study have no role in study design, data
collection, data analysis, findings, interpretations, conclusions or writing of
this manuscript.
Availability of data and materials
The patient data that support the findings of this study are available from
the DOH and PCS 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 the DOH and PCS.
Genuino et al. BMC Health Services Research
(2019) 19:874
Ethics approval and consent to participate
The Faculty of Dentistry/ Faculty of Pharmacy, Mahidol University,
Institutional Review Board reviewed the protocol and granted the ethics
approval to access the available aggregated patient data from the PCS,
owned by the DOH Philippines. The DOH Philippines also released a
certification to allow the use of the aggregated survival data for the sole
purpose of this study.
Consent for publication
Not applicable
Competing interests
The authors declare that they have no competing interests.
Author details
1
Mahidol University Health Technology Assessment (MUHTA) Graduate
Program, Bangkok, Thailand. 2Pharmaceutical Division, Department of Health
Philippines, Manila, Philippines. 3Social and Administrative Pharmacy
Excellence Research (SAPER) Unit, Department of Pharmacy, Faculty of
Pharmacy, Mahidol University, 447 Sri-Ayudhaya Rd., Phayathai, Ratchathewi,
Bangkok 10400, Thailand. 4Division of Medical Oncology, Department of
Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University,
Bangkok, Thailand. 5Section for Clinical Epidemiology and Biostatistics,
Faculty of Medicine Ramathibodi Hospital, Bangkok, Thailand.
Received: 9 May 2019 Accepted: 6 November 2019
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| 7,404 | 50,545 |
W4226372876.txt | Postoperative pneumonia in geriatric patients with a
hip fracture: incidence, risk factors and a predictive
nomogram
Xin Zhang
Changhai hospital affiliated to the Second Military Medical University
Xu-zhou Duan
Changhai hospital affiliated to the Second Military Medical University
Da-ke Tong
Changhai hospital affiliated to the Second Military Medical University
Fang Ji ( jifangdirector@sina.com )
Changhai Hospital
Kai-hang Xu
Changhai hospital affiliated to the Second Military Medical University
Rong-zhi He
Changhai hospital affiliated to the Second Military Medical University
Research
Keywords: hip fracture, geriatric, postoperative pneumonia, nomogram
Posted Date: July 16th, 2020
DOI: https://doi.org/10.21203/rs.3.rs-35763/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
Page 1/25
Abstract
Background : To evaluate the incidence and risk factors of postoperative pneumonia (POP) in geriatric
patients with a hip fracture after surgery, to design a predictive nomogram and to validate the accuracy of
the nomogram.
Methods : We retrospectively studied 1285 surgical-treated geriatric patients with a hip fracture from April
2010 to April 2018. Surgical treatment was performed on the patients of this study. The procedure
methods were classified as: total hip arthroplasty, hemiarthroplasty, percutaneous fixation, intramedullary
nail fixation and plate/screw fixation. The primary interest of end point of this study is the development
of POP during the postoperative period. The postoperative period in this study was defined as the time
from 24 hours after surgery to discharge. The diagnostic criteria for pneumonia were set according to the
guidelines built by the Infectious Diseases Society of America and the American Thoracic Society
(Guidelines for the Management of Adults with Hospital-acquired, Ventilator-associated, and Healthcareassociated Pneumonia, 2005). Potential variables for developing POP were identified using logistic
regression analyses initially and were further selected via the method of LASSO. Then the independent
risk factors were identified by multivariable regression analyses. A predictive nomogram was built based
on the multiple regression model, and the calibration abilities of the nomogram was measured by Harrel
C-index, calibration plot and Hosmer-Lemeshow test respectively. Decision curve analysis was carried out
to assess the net benefit due to threshold probability and an on-line questionnaire survey was conducted
among the clinicians to assess the applicability of the nomogram coherently.
Results: Of the 1285 patients, 70 (5.4%) developed POP. COPD, number of comorbidities, ASA
classification > 2, preoperative dependent functional status and cognitive impairment were identified as
independent risk factors of POP. The nomogram built based on the results showed good accordance
between the predicted probabilities and the observed frequency. The decision curve analysis confirmed
the clinical utility of the nomogram when the threshold probabilities were between 5% and 65% due to the
net benefit, while the results of on-line questionnaire among 200 clinicians showed that 91.5% of the
participants had a mental shreshold of intervention between 5-50%.
Conclusion: 1. COPD, number of comorbidities, ASA classification > 2, preoperative dependent functional
status and cognitive impairment were independent risk factors for POP. 2. The nomogram built in this
study has a good accordance between the predictive risk and the observational incidence. The results of
decision curve and questionnaire among clinicians show well applicability of the nomogram.
Background
As a worldwide public health concern, hip fracture has ranked one of the top 10 causes of disability [1]
and contributed to high mortalities and morbidities in the elderly population, which leads to increasing
socioeconomic cost [2, 3]. The one-year mortality rate could be as high as 36% despite progressive
surgical management and accelerated rehabilitation [4]. Many life-threatening complications have been
Page 2/25
investigated in current literature, such as cardiac infarction, stroke, deep vein thrombosis and subsequent
pulmonary embolism [5, 6], while little has been studied regarding pneumonia, a troubling complication
following hip fracture surgery [7]. Little is known for the occurrence of postoperative pneumonia (POP)
during the hospital stay. The purpose of this study is to determine the incidence and independent risk
factors of POP after hip fracture surgeries in geriatric patients, to construct a predictive nomogram for the
development of POP and to evaluate the accuracy and clinical applicability of the nomogram.
Patients And Methods
Patient Population
In this study, we retrospectively studied 1285 patients with hip fractures and treated by surgical
management between April 2010 and April 2018 in a single center. The inclusion criteria of patients are
as follows: 1) age ≥ 65, 2) postoperative diagnosis of unilateral femoral neck fracture, or
intertrochanteric fracture; 3) receiving total hip arthroplasty, or hemiarthroplasty, or percutaneous fixation,
intramedullary nail fixation, or plate/screw fixation. The exclusion criteria included: 1) pathological
fractures or history of diagnostic malignant tumor, 2) multiple injuries, 3) open fractures.
Patients were characterized at baseline according to sex, age, body mass index (BMI), comorbidities,
current smoking status, fracture pattern, risk classification of anesthesia according to American
Association of Anesthesiologists (ASA), type of anesthesia, surgery option, time to surgery, operation
time, blood loss, transfusion, preoperative hemoglobin and serum albumin concentration, intensive care,
and length of hospital stay, etc. (Table 1).. Age was stratified into 65–74, 70–74, 75–79, 80–84, and ≥ 85
years. Hemoglobin was stratified into <120g/L in male and <110g/L in female (anemia) versus ≥120g/L
in male and ≥110g/L in female (normal). Serum albumin was stratified into < 35g/L (hypoalbuminemia)
versus ≥ 35g/L (normal). Transfusion was stratified into > 2 units of RBC or not. Time to surgery, which
was calculated in days, was defined as the time elapsed from hospital admission to surgery [8]. Surgery
procedure was stratified as described above.
Patients were characterized according to whether or not they developed pneumonia in the postoperative
period. The postoperative period in this study was defined as the time from 24 hours after surgery to
discharge. The diagnostic criteria for pneumonia were set according to the guidelines built by the
Infectious Diseases Society of America and the American Thoracic Society (Guidelines for the
Management of Adults with Hospital-acquired, Ventilator-associated, and Healthcare-associated
Pneumonia, 2005) [9]. In the guidelines, pneumonia was defined as the presence of “new lung infiltrate
plus clinical evidence that the infiltrate is of an infectious origin, which includes the new onset of fever,
purulent sputum, leukocytosis, and decline in oxygenation.” Accordingly, in our study, the identification of
development of pneumonia should accord with the following events that could be confirmed in the
patients’ medical/nursing records and radiological database: 1) a radiographic (X-ray or CT scan)
infiltrate that is new or progressive, 2) observable clinical symptoms, which included a new onset of fever
with a axillary temperature over 37.0℃, and/or purulent sputum with a positive sputum culture, 3)
Page 3/25
leukocytosis. Two experienced doctors (the co-first authors of this study) independently identified the
diagnosis of POP for all patients following the diagnostic strategies. Any controversies in diagnosis
between the two doctors were settled by discussion, and a final diagnosis for POP on each patient was
made.
Statistical Analysis
The primary end point of interest in this study was the development of POP. Multivariable regression
analysis was conducted by logistic regression model, and this model was the basis of the POP prediction
model. The potential predictors of POP analyzed in this study included age, sex, body mass index,
smoking status, hypertension, diabetes, COPD, congestive heart failure, preoperative functional status,
fracture pattern, ASA classification, serum albumin, hemoglobin, time to surgery, anaesthesia type,
operation type, operation time, blood loss, transfusion, and ICU stay. An initial univariate logistic
regression model was fitted to evaluate the association between the clinical factors and pneumonia. To
identify independent risk factors, variables achieving a significance level of p < 0.05 were initially selected
out. The method of LASSO was used to select potential strong factors and then independent predictors
were identified by multiple regression analysis. The independent statistically significant predictors in the
final model were determined with the backward method based on the AIC criteria. A predictive nomogram
was established based on results of the multiple regression. A predictive discrimination was evaluated
using Harrel Concordance index (C-index), which measured the probability that, given a pair of randomly
selected patients, the model correctly predicted which patient will experience an event first. The C-index of
the model can range between 0.5, which represents random chance, and 1.0, which represents a perfectly
discriminating model (Harrell et al, 1982). The relatively correctional C-index of the nomogram was
calculated by bootstraps with 1000 resamples. In calibration, the predicted probability of postoperative
pneumonia generated by 10-fold cross validation was compared with the observed cumulative incidence
estimates of pneumonia probability. The consistency of the curves was evaluated by Hosmer-Lemeshow
(H-L) test. All statistical analyses were conducted using R software version 3.5.0 (http://www.rproject.org/), with rms package (Harrell, 2012).In addition, the decision curve analysis was carried out
using the code found at https://www.mskcc.org/departments/epidemiology biostatistics/ healthoutcomes/decision-curve-analysis–01 according to its tutorials, and an on-line questionnaire (Appendix.
1) was conducted on Tencent Questionnaire Platform from February 11, 2019 to February 15, 2019
combined with the decision curve to study the threshold for intervention among clinical practitioners,
which was used to evaluate the clinical applicability of the nomogram comprehensively.
Results
Baseline characteristics
There were 1409 patients who met the inclusion criteria for this study, of which 124 were excluded due to
the exclusion criteria. Finally, 1285 patients were included in this study. The clinical and pathologic
Page 4/25
characteristics of patients were listed in Table 1. There were 389 (30.3%) male and 896 (69.7%) female
patients in total with a median age of 80 (IQR, 75–84 years) at the time of diagnosis. 770 patients
(59.9%) had comorbidities preoperatively. 334 patients (26.0%) had two or more than two kinds of
comorbidities. Hypertension (43.5%) and diabetes (16.4%) were the most common comorbidities. COPD
(3.8%) and congestive heart failure (2.8%) also constituted a small amount of comorbidities. Eventually,
70 (5.4%) patients developed POP, of which 30 (42.9%) were male and 40 (57.1%) were female.
Comparison of the clinicopathological features between
patients with and without POP
Patients were characterized according to whether or not they developed pneumonia in the postoperative
period. The postoperative period in this study was defined as the time from 24 hours after surgery to
discharge. So the observation period of this study was limited to the duration of hospitalization.
Compared to patients without POP, those with POP had more older age individuals (82 vs. 79, p = 0.001),
a higher portion of male sex (42.9% vs. 29.5%, p = 0.026), a higher portion of patients who had an ASA
classification higher than grade 2 (52.6% vs. 25.0%, p = 0.017), more individuals who had at least two
kinds of comorbidities (72.9% vs. 23.3%, p<0.001), more dependent functional status (47.1% vs. 21.3%, p<
0.001) and more preoperative hypoproteinemia (55.7% vs. 36.1%, p = 0.002), a higher rate of diabetes
(28.6% vs. 15.7%, p = 0.008), more COPD (28.6% vs. 2.39%, p < 0.001), more congestive heart failure
(30.0% vs. 1.23%, p < 0.001), postoperative ICU stay (36.8% vs. 15.7%, p<0.001) and more postoperative
delirium (24.3% vs. 13.8%, p = 0.025). (Table 1)..
Multiple regression analysis
The results of the univariate regression analysis were shown in Table 2. In the univariate logistic
regression, male sex (p = 0.022), age (p = 0.002), preoperative cognitive impairment (p<0.001), number of
comorbidities ≥ 2 (p<0.001), diabetes (p = 0.009), COPD (p<0.001), ASA classification > 2 (p<0.001),
preoperative hypoproteinemia (p = 0.001), preoperative dependent functional status (p<0.001) and
postoperative ICU stay (p = 0.006) were found to be significant predictors of POP. These factors were
further filtrated by the method of LASSO. Comparatively weaker factors were eliminated so that the
strength of influence of the factors was enhanced. Finally, in multiple regression analysis, COPD, number
of comorbidities, ASA classification > 2, preoperative dependent functional status and cognitive
impairment were identified as independent risk factors of POP. (Table 3)..
Development of a predictive nomogram and utility of the
nomogram
Page 5/25
To provide the physicians with a quantitative method to predict the risk of postoperative pneumonia, we
constructed a predictive nomogram (Figure 1) according to the strength of influence of each independent
risk factors based on the multivariable logistic regression (Table 3)..
To use the nomogram, a vertical line was drawn up to the top point row to assign points for each variable.
Then, the total number of points was calculated, and a vertical line was drawn downwards from the total
point row to obtain the probability of POP. The risk of POP increased with the increasing total points, due
to the nomogram. The total points of the nomogram ranges from 0 to 35 points, corresponding to the
predicted risk of POP from less than 10% to greater than 85%. (Figure 1).. For example, when the total
points was calculated to be 15, the corresponding risk of postoperative pneumonia was 20%.
Validation of the nomogram
The nomogram was validated by the method of Harrel C-index and a calibration plot using the R software
3.0. H-L test was performed to evaluate the statistical significance of the consistency between the curves
in the calibration plot.
The nomogram displayed a C-index of 0.84 (95% CI 0.78–0.90) which indicated good accuracy of the
predictive model. The calibration plot showed good concordance between predicted probability and
actual probability. (Figure. 2).. The H-L test showed that the curves in the calibration plot had statistical
significance. (p = 0.88)
Based on the threshold probability, the decision analysis curve was leveraged to evaluate the clinical
application of a prediction model. In the decision analysis curve, the nomogram is compared with the null
model for its added value, which turned out that the nomogram was applicable when thresholds were in
the range of 0.05 to 0.65 due to the net benefit (Figure. 3)..
The results of on-line questionnaire among 200 clinicians showed that 89.5% of the participants have
obtained Identification of Physician’s Qualification for more than 5 years. 52.5% had senior professional
qualifications. 88.5% have been working as an orthopedist for more than 5 years. 62% had an experience
of surgical treatment for geriatric hip fractures for more than 100 cases. 97% of the participants declared
that they had encountered POP among geriatric hip fracture patients in their career. 91.5% had a
threshold of intervention when predictive risk of POP ranged from 5% to 50%, which slickly conformed to
the net-benefit threshold interval produced by the decision curve, indicating well clinical applicability of
the nomogram.
Discussion
The increasing number of hip fractures worldwide predicts greater impact to health status in elderly
population. In the United States, it has been estimated that the total number of hip fractures might double
by the year 2040 compared to the 1980s [2]. Greater contributions should be made by Asian countries for
Page 6/25
the increasing number of osteoporotic hip fractures in future decades [10]. The risk of mortality and
morbidity within one year of fracture remains high according to the existing literatures [4, 11~14].
The improvements in surgical and rehabilitation managements these years have improved clinical
outcomes of hip fractures, while complications following hip fracture surgery remain frequent and
troubling and are often complex, which might be caused by various factors, such as general conditions,
comorbidities, traumatic impairment, surgery, anesthesia, and immobilization, etc. Several studies have
focused on the fatal adverse events during the postoperative periods, such as acute myocardial
infarction, stroke, deep vein thrombosis, etc. [16 ~ 20], which were known as the most dangerous events.
Pneumonia is also known to be a devastating complication after hip fracture surgery, which can also be
life-threatening when correlative respiratory and circulatory failure was induced subsequently, and is
especially risky to elderly patients who are more easily affected by the impactions of trauma and
immobilization after fractures and surgeries. The reported incidence of pneumonia in elderly patients with
a hip fracture was 1.2% preoperatively [22] and approximately 4% ~ 5% postoperatively according to
recent literatures [14, 15, 21]. Although the overall incidence was not alarmingly great, it was found that
patients who developed pneumonia would have a higher readmission rate, a higher rate of sepsis, and a
higher mortality rate [15]. In our study, 70 (5.4%) of 1285 patients developed postoperative pneumonia,
which involved the time from 24 hours after surgery to discharge. The latest guidelines built by the
Infectious Diseases Society of America and the American Thoracic Society in 2016 [23] has differed
Ventilator Associated Pneumonia (VAP) from the traditional concept of Hospital Associated Pneumonia
(HAP). However, in developing countries, such as China, situation remains different. Community
mechanical ventilator equipment is far from popularized yet, so ventilators still could only be seen in the
intensive care unit in hospital in most cases. The development of POP will significantly increase the
length of stay, while clinical data would be hard to collect after discharge. Therefore, in this study, we
defined POP as a new-onset pneumonia from 24 hours after surgery to discharge, so that all the clinical
and radiological evidences could be exactly obtained.
Little was known regarding the occurrence of pneumonia in postoperative periods. In a recent
retrospective study of 29, 377 patients, male sex, older age, low body-mass index (BMI), and chronic
obstructive pulmonary disease (COPD) were identified to be the strongest risk factors for pneumonia [15].
Our study has also shown that COPD, ASA classification, number of comorbidities, cognitive impairment
and preoperative dependent functional status were identified to be the independent risk factors for the
development of POP. We considered that the smaller sample and ethnological difference might play a role
in the discrepancy of the results.
The impacts of pneumonia on clinical outcomes and rehabilitations in elderly patients with a hip fracture
has been demonstrated, and it has been suggested that prevention of pneumonia is necessary to improve
the outcome of hip fracture [15]. In a recent research,a nonrandomized, Quasi-experimental study was
designed to evaluate an inpatient pulmonary rehabilitation program by comparing 2 sequential time
periods in a same center [24], and a pulmonary rehabilitation program was demonstrated to be effective
Page 7/25
to reduce the incidence of pneumonia. For elderly patients, prevention for complications is more
important than that for cure. Thus, an effective prediction strategy is urgently needed clinically. However,
previous researches have many limitations regarding the prediction strategies for POP, and prospective
studies were rarely found in current literature.
The main advantages of this study include: first, a nomogram was built for the prediction for the
development of POP, with good accuracy according to the statistical verifications. As a graphical
presentation of predictive model, nomogram has been widely used in the area of oncology [25–28], with
great advantages such as easy operation, convenience and effectiveness. Previously, we have reported
the predictive nomogram for the development of postoperative delirium in elderly patients with a hip
fracture [29]. The nomogram created in this study is supposed to be used clinically as a prediction
method in the preoperative assessment, thus further studies with prospective designs can be conducted
based on the existing preliminary results. Second, we evaluated the clinical applicability of the nomogram
through the method of decision curve analysis combining with the questionnaire of clinical practitioners.
The decision curve showed that the nomogram was applicable when thresholds were in the range of 0.05
to 0.65 due to the net benefit compared with the null model for its added value, while the results of
questionnaire showed 91.5% of the clinicians had a threshold of intervention when predictive risk of POP
ranged from 5% to 50%, which slickly conformed to the net-benefit threshold interval produced by the
decision curve. It gives the presentation of the clinical utility of a predictive model more intuitively, making
up for the deficiency of the traditional receiver operator characteristic curve.
The main limitations of this study include the following: firstly, it was a retrospective study, so the level of
evidence was limited; secondly, it was programmed in a single institution, thus the sample size was small
when compared to multi-center studies, and the selection of medical institution by patients might cause
potential bias. Then, as the definition of the primary end point of this study—POP, the observation period
of this study was limited to the duration of hospitalization, so the follow-up time was short.
Conclusion
This study showed that COPD, number of comorbidities, ASA classification > 2, preoperative dependent
functional status and cognitive impairment were independent risk factors of POP. The predictive
nomogram established in this study showed a good accuracy in predicting the risk of POP and was
applicable for clinical practice.
Abbreviations
POP:postoperative pneumonia, BMI:body mass index, ASA:American Association of Anesthesiologists,
COPD:chronic obstructive pulmonary diseases.
Declarations
Page 8/25
Authors’ contributions
Dr. Da-ke Tong and Dr. Fang Ji conceived and designed this research. Data collection and extraction was
performed by Dr. Xin Zhang and Dr. Xu-zhou Duan.Data was verifed by Dr.Kai-hang Xu and Dr.Rong-zhi
He. Statistical analysis was performed by Dr. Xin Zhang and Dr. Xu-zhou Duan participated in drafting
article. All authors gave fnal approval to the version submitted. All authors approved the fnal manuscript.
Acknowledgements
The authors of this research wish to thank the departments of Orthopedics and Record Room, Changhai
Hospital affiliated to Second Military Medical University.
Funding
No Funding.
Availability of data and materials
Data associated with this study are retained at a central repository at the Orthopaedic Department,
Changhai Hospital affiliated to Second Military Medical University. If there are any questions, please
contact the corresponding author.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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19. Durand, WM; Goodman, AD; Johnson, JP; Daniels, AH. Assessment of 30-day mortality and
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20. Zhang Bin-Fei, Wei Xing, Huang Hai, Wang Peng-Fei, Liu Ping, Qu Shuang-Wei, Li Jia-Hao, Wang
Hu, Cong Yu-Xuan, Zhuang Yan, Zhang Kun. Deep vein thrombosis in bilateral lower extremities after
hip fracture, a retrospective study of 463 patients. Clin Interv Aging, 2018.
21. Lv H, Yin P, Long A, Gao Y, Zhao Z, Li J, Zhang L, Zhang L, Tang P. Clinical characteristics and risk
factors of postoperative pneumonia after hip fracture surgery, a prospective cohort study.
Osteoporos Int.2016, 27(10):3001-9
22. Patterson Joseph T, Bohl Daniel D, Basques Bryce A, Arzeno Alexander H, Grauer Jonathan N. Does
Preoperative Pneumonia Affect Complications of Geriatric Hip Fracture Surgery? Am J Orthop (Belle
Mead NJ).2017, 46(3): E177-E185
23. Kalil AC, Metersky ML, Klompas M, Muscedere J, Sweeney DA, Palmer LB, Napolitano LM, O'Grady
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Cruse P, Knight SL, Brozek JL. Management of Adults With Hospital-acquired and Ventilatorassociated Pneumonia: 2016 Clinical Practice Guidelines by the Infectious Diseases Society of
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Predictive nomogram for postoperative delirium in elderly patients with a hip fracture. Injury, 2019,
50(2): 392-397
Page 11/25
Tables
Table 1. Baseline Characteristics of patients
Page 12/25
ALL
No
N=1285
N=1215
Yes
N=70
Gender:
0.026
female
896 (69.7%)
856 (70.5%)
40 (57.1%)
male
389 (30.3%)
359 (29.5%)
30 (42.9%)
Age, years
80.0 (75.0-84.0)
79.0 (75.0-84.0)
82.0 (79.0-86.8)
Age, years:
0.001
0.014
65-69
130 (10.1%)
127 (10.5%)
3 (4.29%)
70-74
176 (13.7%)
170 (14.0%)
6 (8.57%)
75-79
329 (25.6%)
317 (26.1%)
12 (17.1%)
80-84
330 (25.7%)
308 (25.3%)
22 (31.4%)
≥85
320 (24.9%)
293 (24.1%)
27 (38.6%)
BMI,Kg/m2
p
23.4 (21.1-25.6)
23.4 (21.1-25.6)
23.4 (21.5-26.0)
0.326
0.021
BMI,Kg/m2:
BMI ≤ 18.5
53 (4.12%)
48 (3.95%)
5 (7.14%)
18.5 < BMI ≤ 20.0
147 (11.4%)
143 (11.8%)
4 (5.71%)
20.0 < BMI ≤ 25.0
703 (54.7%)
664 (54.7%)
39 (55.7%)
25.0 < BMI ≤ 30.0
335 (26.1%)
320 (26.3%)
15 (21.4%)
BMI > 30.0
47 (3.66%)
40 (3.29%)
7 (10.0%)
Type of fracture:
0.136
femoral neck fracture
706 (54.9%)
661 (54.4%)
45 (64.3%)
Intertrochanteric fracture
579 (45.1%)
554 (45.6%)
25 (35.7%)
Cognitive impairment:
<0.001
no
1239 (96.4%)
1180 (97.1%)
59 (84.3%)
yes
46 (3.58%)
35 (2.88%)
11 (15.7%)
Smoking:
0.582
never
984 (76.6%)
928 (76.4%)
56 (80.0%)
current/former
301 (23.4%)
287 (23.6%)
14 (20.0%)
Comorbidities:
<0.001
Page 13/25
no
515 (40.1%)
508 (41.8%)
7 (10.0%)
yes
770 (59.9%)
707 (58.2%)
63 (90.0%)
Comorbidities:
<0.001
<2
951 (74.0%)
932 (76.7%)
19 (27.1%)
≥2
334 (26.0%)
283 (23.3%)
51 (72.9%)
Hypertension:
0.99
no
726 (56.5%)
687 (56.5%)
39 (55.7%)
yes
559 (43.5%)
528 (43.5%)
31 (44.3%)
Diabetes mellitus:
0.008
no
1074 (83.6%)
1024 (84.3%)
50 (71.4%)
yes
211 (16.4%)
191 (15.7%)
20 (28.6%)
COPD:
<0.001
no
1236 (96.2%)
1186 (97.6%)
50 (71.4%)
yes
49 (3.81%)
29 (2.39%)
20 (28.6%)
Congestive heart failure:
<0.001
no
1249 (97.2%)
1200 (98.8%)
49 (70.0%)
yes
36 (2.80%)
15 (1.23%)
21 (30.0%)
History of depression:
1
no
1279 (99.5%)
1209 (99.5%)
70 (100%)
yes
6 (0.47%)
6 (0.49%)
0 (0.00%)
hypotensive drugs:
0.99
no
726 (56.5%)
687 (56.5%)
39 (55.7%)
yes
559 (43.5%)
528 (43.5%)
31 (44.3%)
Hypoglycemic drugs:
0.171
no
1184 (92.1%)
1123 (92.4%)
61 (87.1%)
yes
101 (7.86%)
92 (7.57%)
9 (12.9%)
Cardiovascular drugs:
0.001
no
1189 (92.5%)
1132 (93.2%)
57 (81.4%)
yes
96 (7.47%)
83 (6.83%)
13 (18.6%)
Page 14/25
Respiratory drugs:
<0.001
no
1267 (98.6%)
1204 (99.1%)
63 (90.0%)
yes
18 (1.40%)
11 (0.91%)
7 (10.0%)
Other drugs:
0.025
no
1218 (94.8%)
1156 (95.1%)
62 (88.6%)
yes
67 (5.21%)
59 (4.86%)
8 (11.4%)
Traction:
0.648
no
1184 (92.1%)
1121 (92.3%)
63 (90.0%)
yes
101 (7.86%)
94 (7.74%)
7 (10.0%)
ASA classification
2.00 (2.00-3.00)
2.00 (2.00-3.00)
3.00 (2.00-3.00)
ASA(2):
<0.001
<0.001
1-2
915 (71.2%)
888 (73.1%)
27 (38.6%)
3-4
370 (28.8%)
327 (26.9%)
43 (61.4%)
Pre-operative RBC
3.75 (3.33-4.18)
3.76 (3.33-4.17)
3.66 (3.23-4.20)
0.284
Pre-operative WBC
8.20 (6.56-9.60)
8.20 (6.56-9.60)
8.21 (6.49-9.95)
0.955
Pre-operative ALB
36.0 (33.0-38.0)
36.0 (33.0-38.0)
34.0 (30.0-37.0)
0.002
Pre-operative ALB:
0.002
≥35
807 (62.8%)
776 (63.9%)
31 (44.3%)
<35
478 (37.2%)
439 (36.1%)
39 (55.7%)
Pre-operative Cr
67.0 (56.0-82.0)
67.0 (56.0-81.5)
70.0 (61.0-90.5)
0.085
Pre-operative HGB
114 (102-125)
115 (102-125)
112 (98.0-124)
0.291
Pre-operative PLT
173 (140-214)
172 (140-214)
174 (142-214)
0.615
Time to surgery
4.00 (3.00-5.00)
4.00 (3.00-5.00)
4.50 (3.00-6.00)
0.181
Anaesthesia type:
0.582
Intra-spinal/nerve block
1068 (83.1%)
1012 (83.3%)
56 (80.0%)
general
217 (16.9%)
203 (16.7%)
14 (20.0%)
80.0 (60.0-105)
80.0 (60.0-105)
87.5 (70.0-105)
Operation time
Type of operation:
hemiarthroplasty
0.087
0.591
347 (27.0%)
325 (26.7%)
Page 15/25
22 (31.4%)
total hip arthroplasty
298 (23.2%)
280 (23.0%)
18 (25.7%)
intramedullary nail
456 (35.5%)
437 (36.0%)
19 (27.1%)
plate/Screw
153 (11.9%)
144 (11.9%)
9 (12.9%)
multiple screw
31 (2.41%)
29 (2.39%)
2 (2.86%)
200 (100-350)
200 (100-350)
Blood loss
200 (100-300)
Preoperative anemia:
0.407
no
695 (54.1%)
661 (54.4%)
34 (48.6%)
yes
590 (45.9%)
554 (45.6%)
36 (51.4%)
Functional status:
<0.001
independent
993 (77.3%)
956 (78.7%)
37 (52.9%)
dependent
292 (22.7%)
259 (21.3%)
33 (47.1%)
Transfusion > 2iu RBC:
0.983
no
690 (53.7%)
653 (53.7%)
37 (52.9%)
yes
595 (46.3%)
562 (46.3%)
33 (47.1%)
Postoperative ICU:
0.005
no
1066 (83.0%)
1017 (83.7%)
49 (70.0%)
yes
219 (17.0%)
198 (16.3%)
21 (30.0%)
Length of stay
0.264
10.0 (8.00-12.0)
10.0 (8.00-12.0)
Table 2. Univariable logistic regression
Page 16/25
11.5 (9.00-13.0)
0.006
Risk factors
OR
p. ratio
female
Ref.
Ref.
male
1.79 [1.09;2.92]
0.022
Age, years
1.06 [1.02;1.09]
0.002
65~69
Ref.
Ref.
70~74
1.46 [0.36;7.43]
0.604
75~79
1.54 [0.47;7.18]
0.498
80~84
2.89 [0.97;12.87]
0.057
≥85
3.72 [1.28;16.42]
0.013
1.05 [0.98;1.12]
0.196
BMI ≤ 18.5
Ref.
Ref.
18.5 < BMI ≤ 20.0
0.27 [0.06;1.11]
0.069
20.0 < BMI ≤ 25.0
0.55 [0.22;1.68]
0.267
25.0 < BMI ≤ 30.0
0.44 [0.16;1.44]
0.163
BMI > 30.0
1.66 [0.48;6.17]
0.423
intertrochanteric fracture
Ref.
Ref.
femoral neck fracture
1.50 [0.92;2.52]
0.107
no
Ref.
Ref.
yes
6.32 [2.92;12.77]
<0.001
never
Ref.
Ref.
current/former
0.82 [0.43;1.45]
0.499
Ref.
Ref.
Gender:
Age, years:
BMI,Kg/m2
BMI,Kg/m2:
Type of fracture:
Cognitive impairment:
Smoking:
Comorbidities:
no
Page 17/25
yes
6.33 [3.07;15.42]
<0.001
<2
Ref.
Ref.
≥2
8.78 [5.18;15.51]
<0.001
no
Ref.
Ref.
yes
1.04 [0.63;1.68]
0.889
no
Ref.
Ref.
yes
2.15 [1.22;3.65]
0.009
no
Ref.
Ref.
yes
16.28 [8.51;30.77]
<0.001
no
Ref.
Ref.
yes
33.85 [16.50;71.32]
<0.001
no
Ref.
Ref.
yes
1.04 [0.63;1.68]
0.889
no
Ref.
Ref.
yes
1.83 [0.82;3.63]
0.133
no
Ref.
Ref.
yes
3.13 [1.58;5.80]
0.002
no
Ref.
Ref.
yes
12.19 [4.28;32.43]
<0.001
Comorbidities:
Hypertension:
Diabetes mellitus:
COPD:
Congestive heart failure:
Hypotensive drugs:
Hypoglycemic drugs:
Cardiovascular drugs:
Respiratory drugs:
Other drugs:
Page 18/25
no
Ref.
Ref.
yes
2.56 [1.08;5.34]
0.034
no
Ref.
Ref.
yes
1.35 [0.55;2.85]
0.486
2.83 [1.93;4.15]
<0.001
1-2
Ref.
Ref.
3-4
4.31 [2.63;7.18]
<0.001
Pre-operative RBC
0.79 [0.55;1.13]
0.198
Pre-operative WBC
0.99 [0.89;1.09]
0.827
Pre-operative ALB
0.91 [0.87;0.95]
<0.001
≥35
Ref.
Ref.
<35
2.22 [1.37;3.64]
0.001
Pre-operative Cr
1.00 [1.00;1.01]
0.922
Pre-operative HGB
0.99 [0.98;1.01]
0.318
Pre-operative PLT
1.00 [1.00;1.00]
0.645
Time to surgery
1.08 [0.97;1.19]
0.165
intraspinal/nerve block
Ref.
Ref.
general
1.26 [0.66;2.24]
0.471
Operation time
1.00 [1.00;1.01]
0.23
hemiarthroplasty
Ref.
Ref.
multiple screw
1.08 [0.15;3.98]
0.918
plate/screw
0.93 [0.40;2.03]
0.865
total hip arthroplasty
0.95 [0.49;1.81]
0.88
intramedullary nail
0.64 [0.34;1.21]
0.172
Traction:
ASA classification
ASA(2):
Pre-operative ALB:
Anaesthesia type:
Type of operation:
Page 19/25
Blood loss
1.00 [1.00;1.00]
0.553
no
Ref.
Ref.
yes
1.26 [0.78;2.05]
0.345
independent
Ref.
Ref.
dependent
3.29 [2.01;5.37]
<0.001
no
Ref.
Ref.
yes
1.04 [0.64;1.68]
0.884
no
Ref.
Ref.
yes
2.21 [1.27;3.72]
0.006
1.01 [1.00;1.03]
0.118
Preoperative anemia:
Functional status:
Transfusion:
Postoperative ICU:
Length of stay
Table 3. Multiple Logistic Regression for risk factors of POP
Page 20/25
Risk factors
OR
95%CI
COPD
P value
<0.001
No
Ref.
Yes
10.38
4.90-22.00
Number of comorbidities
<0.001
<2
Ref.
≥2
5.50
3.07-9.84
ASA classification
0.001
1-2
Ref.
3-4
2.92
1.68-5.09
Preoperative functional status
<0.001
independent
Ref.
dependent
3.33
1.90-5.84
Cognitive impairment
0.002
no
Ref.
yes
4.26
C-index: 0.8396355 (0.7838565-0.8954145)
HL test: p = 0.8839044
Figures
Page 21/25
1.69-10.70
Figure 1
Predictive nomogram
Page 22/25
Figure 2
Calibration plot
Page 23/25
Figure 3
Decision analysis curve
Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.
Supplementaryinformation.docx
Page 24/25
Appendix1.OnLineQuestionnair.docx
Page 25/25
| 5,715 | 39,152 |
W4391497012.txt | CONHECIMENTO CIENTÍFICO E AS FAKE
NEWS: um obstáculo epistemológico no
ensino de ciências e biologia?
SCIENTIFIC KNOWLEDGE AND FAKE
NEWS: are they an epistemological
obstacle in science and biology teaching?
CONOCIMIENTO CIENTÍFICO Y LAS FAKE
NEWS: ¿una objeción epistemológica en
la enseñanza de ciencias y lo a biología?
Resumo: Este artigo apresenta uma análise
teórica, de natureza qualitativa, por meio de
pesquisa bibliográfica para tentar esclarecer se as
fakes news podem ser caracterizadas como
obstáculo epistemológico na aprendizagem do
conhecimento científico, nas disciplinas de
Ciências e Biologia, no contexto da pandemia da
covid-19 e da disseminação de notícias falsas ou
fake news. Para desenvolver essa proposta
pretende-se identificar na obra de Gaston
Bachelard a noção de obstáculo epistemológico
ao reconstruir, de modo breve, a narrativa do
autor, na tentativa de indicar caminhos para o
conhecimento científico e as implicações das fake
news nesse processo. Sabe-se que o excesso de
informações, a infodemia, apresenta-se como
barreira em função da dificuldade de critérios na
seleção de informações relevantes e seguras
como aliadas no processo educacional. Percebese um intenso movimento anticientífico, que não
é novo, mas é possível observar seu crescimento
mundial nos últimos anos. Será importante que o
educador assuma uma postura investigativa e
reflexiva para que o sentimento de fracasso não
passe despercebido quando o comportamento
instintivo não se apresente em sua prática
pedagógica, por ser o detentor do conhecimento
porque dessa forma todo conselho referente aos
erros
pedagógicos
que
cometem
será
absolutamente inútil. Sendo assim, as fake News,
configuraram-se
em
um
fenômeno
do
pensamento
fraco
ou
em
obstáculo
epistemológico da experiência primeira, que
significa deixar-se levar pelo impulso, pelo
arrebatamento natural, causado pela informação,
sem considerá-la criticamente, porque é na
criticidade que o espírito científico se forma e se
reforma, retificando saberes, quando não
oferecemos resistência ao novo conhecimento.
Palavras-chave: Conhecimento científico. Fake
News. Obstáculo Epistemológico.
Recebido em: 11/03/2023
Aceito em: 26/04/2023
Publicação em: 13/09/2023
Revista Espaço do Currículo
ISSN 1983-1579
Doi: 10.15687/rec.v16i3.66091
http://periodicos.ufpb.br/ojs2/index.php
Edilce Maria Balbinot Borba
Mestra em Ensino de Ciências
Professora da Universidade Tecnológica
Federal do Paraná, Brasil.
E-mail: edilcemb@gmail.com
Orcid: https://orcid.org/0000-0001-61671375
Marcos Antonio Florczak
Doutor em Astronomia
Professor da Universidade Tecnológica
Federal do Paraná, Brasil.
E-mail: marcosflorczak@gmail.com
Orcid: https://orcid.org/0000-0002-38741926
Como citar este artigo
BORBA, E. M. B.; FLORCZAK, M. A.
CONHECIMENTO CIENTÍFICO E AS FAKE
NEWS: um obstáculo epistemológico no
ensino de ciências e biologia? Revista
Espaço do Currículo, v. 16, n. 3, p. 1-7, Ano.
2023.
ISSN
1983-1579.
DOI
https://doi.org/10.15687/rec.v16i3.66091.
REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V. 16, N. 3, P. 1-7, SET/DEZ. 2023.
1
BORBA, E. M. B.; FLORCZAK, M. A
Abstract: This study presents a qualitative and theoretical analysis, based on bibliographical research to
enlighten whether fake news can be characterized as an epistemological obstacle to the learning
process of scientific knowledge, regarding Science and Biology subjects, during Covid-19 pandemic
context and fake news dissemination. Thus, to develop this proposal, it aims at identifying the notion of
epistemological obstacle in Gaston Bachelard's work by briefly reconstructing the author's narrative, in
an attempt to indicate possibilities to scientific knowledge and implications of fake news along this
process. It is known that an overflow of information, the infodemic, has been a barrier due to the
difficulty of criteria to select relevant and safe information as allies in the educational process. There is
an intense anti-scientific movement, which is not new, but it is possible to observe its worldwide
increase in recent years. It will be important for educators to assume an investigative and reflective
attitude so that the feeling of failure does not go unnoticed when instinctive behavior does not appear
in their pedagogical practice, as they hold knowledge. Thus, any advice regarding pedagogical mistakes
will be absolutely useless. At last, fake news was configured as a phenomenon of weak thought or an
epistemological obstacle to the first experience, which means let you be carried away by impulse, by
the natural rapture caused by information, without critically consider it, since criticalness helps on
scientific background, reforms and rectifies knowledge, when we do not offer resistance to new
knowledge.
Keywords: Scientific Knowledge. Fake News. Epistemological Obstacle.
Resumen: Este trabajo presenta un análisis teórico, de naturaleza cualitativa, por intermedio de
pesquisa bibliográfica para intentar aclarar si las fake news pueden ser caracterizadas como objeción
epistemológica en el aprendizaje del conocimiento científico, en las asignaturas de Ciencias y Biología,
en el contexto de la pandemia de la covid-19 y de la diseminación de noticias falsas o fake news. Para
desarrollar esa propuesta se pretende identificar en la obra de Gaston Bachelard la noción de objeción
epistemológica al reconstruir, de pronto modo, la narrativa del autor, en la tentativa de indicar caminos
para el conocimiento científico y las implicaciones de las fake news en ese proceso. Es percibido un
intenso movimiento anticientífico, que no es nuevo, pero es posible observar su crecimiento mundial en
los últimos años. Será importante que el educador haga cargo de una postura investigativa y reflexiva
para que el sentimiento de fracaso no pase desapercibido cuando el comportamiento instintivo no se
presente en su práctica pedagógica, por ser el detentor del conocimiento porque de esa forma todo
consejo referente a los errores pedagógicos que perpetran será absolutamente inútil. Entonces, las fake
News, se configuran en un fenómeno del pensamiento débil o en objeción epistemológica de la
experiencia primera, que significa dejárselo llevar por el impulso, por el arrebatamiento natural, causado
por la información, sin considerarla críticamente, porque es en la criticidad que el espíritu científico se
forma y se reforma, rectificando saberes, cuando no ofrecemos resistencia al nuevo conocimiento.
Palabras clave: Conocimiento científico. Fake News. Objeción Epistemológica.
1 INTRODUÇÃO
A velocidade das informações veiculadas pelas mídias digitais, pelas redes sociais com circulação
em volume global em tempo real é discrepante da velocidade com a qual a ciência produz afirmações
confiáveis. Desta forma, a disseminação de fake news, notícias falsas, que implicam em desserviço à
sociedade, gera confusão, desinformação e preocupações desmedidas em diversos temas, em especial
os temas científicos. E o excesso de informações propagadas por essas redes pode gerar dificuldades
no discernimento sobre o que é verdadeiro e falso (ABIB, 2020).
Neste artigo, será utilizado o termo fake news, quando a referência for notícias falsas.
Segundo Nunes 2020, o movimento anticientífico não é novo, mas é possível observar seu
crescimento mundial nos últimos anos. Na corrida contra o tempo para conter a disseminação da
doença, as fake news constantemente distorcem informações fundamentadas pela ciência e uma das
formas para enfrentar a desinformação é o investimento em educação.
Nessa perspectiva, pretende-se apresentar uma análise teórica, com base na pesquisa
bibliográfica e na obra de Gaston Bachelard, para reconstruir, de modo breve, a proposta
epistemológica do autor, no sentido de compreender o termo obstáculos epistemológicos na tentativa
de indicar caminhos para superar tais obstáculos. Além de buscar a construção do conhecimento
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CONHECIMENTO CIENTÍFICO E AS FAKE NEWS: um obstáculo epistemológico no ensino de ciências e biologia?
científico e as implicações das fake news nesse processo, em época da pandemia da covid-19, na
educação.
Dessa forma, a questão que norteia este texto é: as fake news podem ser caracterizadas como
obstáculos na aprendizagem do conhecimento científico, nas disciplinas de Ciências e Biologia?
Para desenvolver essa proposta, pretende-se, mais especificamente, alcançar o objetivo de
relacionar os obstáculos epistemológicos de Bachelard que em tese foram constituídos pela
disseminação de notícias falsas, as fake news, na construção do conhecimento científico.
A pesquisa se caracteriza como sendo qualitativa, com revisão bibliográfica, segundo Flick (2009).
2 O QUE SÃO E COMO SE CARACTERIZAM AS FAKE NEWS
A desinformação deliberada e a necessidade de inventar e divulgar fatos estão em pleno
andamento, as mentiras e notícias falsas permeiam o debate público, controlando a discussão nos
meios sociais, acadêmicos e políticos, servindo a causa populista. Neste sentido o autor Rolf Arnold em
seu livro: Fake News in Science and Education, 2019, chama esse fenômeno emergente, (ARNOLD, 2019,
tradução nossa).
Segundo Arnold (2019), os cientistas tendem a ser muito mais céticos sobre as "verdades". No
processo de formulação do raciocínio baseado em evidências, fatos incontestáveis não são
deliberadamente ocultados, mas apresentados para debate aberto. A livre expressão de opinião, a
imprensa livre e a liberdade de pesquisa e ensino garantem a discussão e o debate necessários para
argumentar as evidências dos fatos em uma democracia. Uma característica importante da educação
em nosso mundo moderno é orientar os estudantes para lidar com essa mudança, uma espécie de
preparação para a insegurança de não saber o que está por vir e uma abertura que ameaça o
conhecimento validado e comprovado, (ARNOLD, 2019, tradução nossa).
Essa plausibilidade interna nos permite ver o que vemos: os “fatos” revelados em nossa pesquisa
estão longe de ser objetivos, mas sim o resultado de nossa visão metódica, disciplinada, mas individual
do que está acontecendo. A maneira como lidamos com os fatos geralmente mostra que gostamos ou
não – mais sobre nós mesmos e nossos pontos de vista do que sobre o objeto de nosso interesse. A
busca de fatos requer um olhar ampliado por trás dos fatos – uma reflexão metafactual, (ARNOLD,
2019, tradução nossa).
Para Arnold (2019), esse tipo de reflexão nos ajuda a expandir a observação científica das
condições para uma observação de segunda ordem, ou seja, questionar como observamos e
reconhecemos nossa tendência a aceitar achados que não são necessariamente fatos baseados em
evidências apenas porque os geramos e queremos que sejam "certezas". Se esta reflexão metafactual
for omitida ao lidar com fatos, há mais em jogo do que apenas aceitar como fato o que parece ser o
caso. Corre-se o risco de uma reprodução contínua da visão anterior das coisas, porque o “ponto cego”
em sua percepção (e talvez em outras) não é reconhecido, e você considera sua maneira de lidar com as
circunstâncias factualmente indiscutível, (ARNOLD, 2019, tradução nossa). Nesse sentido o autor
também contribui,
John Maynard Keynes disse uma vez: "Quando os fatos mudam, eu mudo de
ideia... e o que você faz?" (citado em Chamberland, 2016, p. 191). Esta é uma
boa pergunta que deve nos confrontar em nosso pensamento e interpretação.
Somos realmente capazes de abandonar nossas opiniões duramente
conquistadas quando a análise racional revela algo melhor? Que emoções nos
perseguem quando temos que perceber que nos enganamos? Corrigimo-nos ou
insistimos, tentando manter uma parte das nossas convicções anteriores? Ou
argumentamos ignorando os fatos - inventando provas que não existem
apenas para ganhar? (ARNOLD, 2019, p.xiii, tradução nossa)
Para muitas pessoas, nem isso basta: não se limitam a inventar fatos, mas também sentem a
necessidade de divulgá-los. Ao fazê-lo, essas proposições ganham significado social, não por sua
veracidade, mas por sua repetição. No ciberespaço, não há mais nenhum controle de qualidade para
garantir que haja evidências reais do que é divulgado. Os temas diante do público não são mais
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3
BORBA, E. M. B.; FLORCZAK, M. A
determinados por fatos comprovados, mas por novidades generalizadas. A desinformação deliberada
tornou-se um meio de controle da política e da opinião pública com consequências devastadoras para o
clima e a cultura do diálogo em nossa sociedade (ARNOLD, 2019, tradução nossa).
O termo fake News tornou-se nos últimos anos uma, uma expressão usada de forma exagerada,
muitas vezes como uma explicação rápida e fácil para os problemas da sociedade atual. Fake news foi
eleita a palavra do ano de 2017 pela editora inglesa Collins (BBC, 2017). O termo tornou-se
extremamente corriqueiro e popular, empregado de forma generalizada e imprecisa, em geral pensada
em associação com a ruptura progressiva das democracias liberais nesse início do século XXI.
(MENESES, 2018; ALVES, MACIEL, 2020).
Uma das característica das fake news é a fonte, sendo na internet que se criam: pela facilidade de
publicação (qualquer pessoa, em qualquer parte do mundo, pode publicar), pela desregulação, não
existindo controle e pela abundância de informações, são milhões de informações disponíveis, de todo
o tipo, pela facilidade de confundir verdadeiro e falso, manipular uma foto ou um vídeo, e pela
facilidade de obter retorno financeiro com publicidade. Certamente, a Internet e o crescimento das
mídias sociais não inventaram o fenômeno da desinformação, mas criaram um ambiente propício para
que houvesse uma difusão em massa de notícias falsas, em velocidade nunca vista na história da
humanidade (MENESES, 2018; ALVES; MACIEL, 2020).
Em suma, o fenômeno contemporâneo das fake news envolve algo mais do que a mera falsidade
da notícia, presente também em formas simples e ingênuas de erros factuais ou equívocos
involuntários. Nesse ponto, contudo, é que o fenômeno contemporâneo das fake news é mais amplo e,
mais do que algo que envolve ações necessariamente insinceras e manipuladoras, pode ser
compreendido de maneira mais adequada como algo que envolve desinformações produzidas em
contextos de embate e disputa ideológica. Via de regra, as fakes news encontram seu motor não no
desejo de negar a verdade, mas sim na vontade de vencer a disputa a qualquer preço, mesmo que para
isso seja preciso falsear a realidade. As pessoas deixam de se perguntar se a notícia é verdadeira ou
falsa.(ALVES; MACIEL, 2020).
3 OBSTÁCULOS EPISTEMOLÓGICOS
A noção de obstáculo epistemológico pode ser estudada no desenvolvimento histórico do
pensamento científico e na prática da educação. Em ambos os casos, esse estudo não é fácil. A história,
por princípio, é hostil a todo juízo normativo. Pois, um obstáculo epistemológico se adere ao
conhecimento não questionado. Hábitos intelectuais que foram úteis e sadios podem, com o tempo,
obstruir a pesquisa (BACHELARD, 1996, p.19).
Na obra ‘A Formação do Espírito Científico: contribuições para uma psicanálise do conhecimento’,
Bachelard busca pelo entendimento de como ocorre o processo formativo do conhecimento e quais são
as principais barreiras ou “obstáculos epistemológicos” que, para serem compreendidos, necessitam de
uma psicanálise do conhecimento (BACHELARD, 1996; FÁVERO; TONIETO, 2017).
É importante compreender tais aspectos para quem ensina e para quem aprende, pois implica
olhar o seu processo formativo para compreender como o outro aprende e como é possível intervir
pedagogicamente. Assim, é importante compreender a proposta de Bachelard sobre desenvolvimento
do espírito científico sistematizada na “lei dos três estados para o espírito científico”, que está
entrelaçada à “lei dos três estados da alma” e ao conceito de “paciência científica”, a partir dos quais o
autor construirá o conceito de “obstáculo epistemológico” e apontará suas implicações para a
epistemologia e para a educação (BACHELARD, 1996; FÁVERO; TONIETO, 2017).
Na visão de Bachelard (1996), é preciso considerar que o dever de todo educador, em qualquer
estágio de formação, deve ser o de criar e manter o interesse pela pesquisa. No entanto, o interesse
pela pesquisa tem uma história e sua força se mostra no decorrer da construção da “paciência
científica”, que não é a mesma paciência da erudição, já que esta trabalha com a acumulação e a
repetição, enquanto aquela trabalha com a retificação.
Segundo Bachelard (1996), “a paciência seria sofrimento, porém, com esse interesse, a paciência
é vida espiritual”, isto é, o interesse pela pesquisa científica desinteressada torna comportamentos
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CONHECIMENTO CIENTÍFICO E AS FAKE NEWS: um obstáculo epistemológico no ensino de ciências e biologia?
como calma, tranquilidade, serenidade, persistência, equilíbrio e constância sinônimos de paciência,
aspectos vitais do espírito científico, já que, do contrário, tornaram-se comportamentos perturbadores
e indesejados, por isso, eliminados pela adesão fácil às verdades primeiras.
Nesse sentido, estabelecer a psicologia da “paciência científica” é, para Bachelard (1996),
adicionar às leis do desenvolvimento do espírito científico a “[...] lei dos três estados da alma [já que] as
forças psíquicas que atuam no conhecimento científico são mais confusas, mais exauridas, mais
hesitantes do que se imagina quando consideradas de fora”, pois “[...] mesmo na mente lúcida, há
zonas obscuras, cavernas onde ainda vivem sombras. Mesmo no novo homem, permanecem vestígios
do homem velho” (BACHELARD, 1996; FÁVERO; TONIETO, 2017).
Desse modo, reconhece que a dúvida é um movimento arriscado, já que trabalha com o
desconhecido, porém não é ingênua, pois sabe que o dado não existe em si mesmo, mas é construído. É
um projeto forjado pela “paciência científica”, já que todo saber científico deve ser constantemente
reconstruído. Aqui a “paciência científica” ganha sua expressão máxima, por reconhecer que tudo o
que é fácil de ensinar é inexato, e como tal, desafia quem ensina e quem aprende, já que inverte a
“dialética prematura”, ou seja, deixa de operar sobre as coisas para operar a partir de axiomas, em que
a experiência deixa de ser a legitimadora de toda e qualquer teoria (BACHELARD, 1979, p. 13-15).
Dessa forma buscar as condições psicológicas do progresso científico é, na visão bachelardiana
(1996, p. 17), reconhecer que “[...] é em termos de obstáculos que o problema do conhecimento
científico deve ser colocado”. E tais obstáculos não são meramente objetivos (complexidade e
fugacidade) ou subjetivos (fragilidade dos sentidos e do espírito humano), mas são intrínsecos ao ato
de conhecer e se mostram como uma “espécie de imperativo funcional, lentidões e conflitos”, isto é,
fazem parte do ato de conhecer como uma condição obrigatória prática, vagarosa e conturbada, na
qual residem as causas da imobilidade e até do retrocesso, as quais são denominadas de “obstáculos
epistemológicos” (BACHELARD, 1996; FÁVERO; TONIETO, 2017).
O primeiro obstáculo a ser superado, em direção à cultura científica, é a opinião, pois ela “[...]
pensa mal; não pensa: traduz a necessidade em conhecimentos. Ao designar os objetos pela utilidade,
ela se impede de conhecê-los”, por isso, o primeiro passo é destruí-la. Não basta remodelá-la, pois
compreendê-la cientificamente significa abster-se de opinar a respeito de questões que não
conseguimos formular claramente (BACHELARD, 1996; FÁVERO; TONIETO, 2017).
A partir deste posicionamento, é possível supor que é preciso saber formular problemas, o que
não acontece de forma espontânea e, por isso, precisa de aprendizagem. Na visão de Bachelard (1996,
p. 18), “[...] para o espírito científico, todo conhecimento é resposta a uma pergunta. Se não há
pergunta, não pode haver conhecimento científico. Nada é evidente. Nada é gratuito. Tudo é
construído.” Assim, a primeira atitude a ser aprendida é formular perguntas científicas. Esta é a primeira
ação do espírito que lhe permite a superação da “alma pueril e mundana” e sair do “estado concreto”,
superar o realismo ingênuo, reino das opiniões.
Contudo, não é qualquer pergunta que provoca tal movimento, pois a “[...] pergunta abstrata e
fraca se desgasta: a resposta concreta fica” (BACHELARD, 1996, p. 18), e causa a inversão da atividade
espiritual e a instalação de um obstáculo epistemológico no conhecimento não questionado.
Diante do exposto, considera-se que o primeiro obstáculo a ser superado nas aulas de Ciências e
Biologia é o da experiência primeira, o aprendiz fica mais apegado à beleza do experimento do que à
sua explicação científica. Nesse obstáculo, dá-se preferência às imagens e não às ideias.
A ausência da explicação, no obstáculo citado anteriormente, faz com que haja uma
generalização. Essa ocorre quando uma lei fica tão clara, completa e fechada, que dificulta o interesse
pelo seu estudo mais aprofundado e pelo seu questionamento.
3 CONSIDERAÇÕES FINAIS: AS FAKE NEWS REPENSADAS NA EPISTEMOLOGIA DE BACHELARD
Na educação, a noção de obstáculo epistemológico deveria ser mais discutida na formação dos
professores, uma vez que esses conceitos ajudariam na elucidação das dificuldades na construção do
conhecimento científico. Neste sentido, as fake news podem agir como instrumentos, pontes para esse
conhecimento.
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BORBA, E. M. B.; FLORCZAK, M. A
Devemos, segundo Bachelard (1996), levar sempre em consideração que os alunos chegam às
aulas de ciências com conhecimentos pré-estabelecidos em suas estruturas de conhecimento, os quais
são referentes aos seus conhecimentos empíricos. Esses podem, em algumas situações, ser obstáculos
para o novo conhecimento que será ensinado pelo professor.
Nesse sentido, para ensinar o aprendiz a “inventar, é bom mostrar-lhe que ele pode descobrir, e
que descobrindo, ele pode perceber que errar não é um mal, mas uma oportunidade de aprendizagem”.
O erro é positivo, normal e útil quando consequência de um interesse, de uma possibilidade de
racionalização, já que a evolução do espírito é a retificação de seus erros pessoais e “[...] não há
verdade sem erro retificado” (BACHELARD, 1996, p. 293).
Para que seja possível construir uma nova compreensão, primeiro é necessário criticar e desfazer
a teia de intuições primeiras, de conhecimentos empíricos, que pela sua segurança e simplicidade,
impede a compreensão racional das leis científicas. Eis o primeiro desafio para a docência universitária,
que busca formar melhor os futuros professores de Ciência e Biologia, ou seja, derrubar os obstáculos
epistemológicos adquiridos e cultivados desde a mais tenra idade. Tal decisão implica levar os sujeitos
da aprendizagem a se atentarem de quão limitadas e ingênuas são suas compreensões de mundo, e
que, se não reconstruídas, serão obstáculos para a compreensão científica (BACHELARD, 1996; FÁVERO;
TONIETO, 2017).
Na educação científica, os obstáculos epistemológicos, as noções de obstáculos se constituem em
obstáculos pedagógicos. Dessa forma, a cultura científica deve começar por uma catarse intelectual e
afetiva. Resta, então, a tarefa mais difícil: colocar a cultura científica em estado de mobilização
permanente, substituir o saber fechado e estático por um conhecimento aberto e dinâmico, dialetizar
todas as variáveis experimentais, oferecer enfim à razão razões para evoluir. Tais observações podem,
aliás, ser generalizadas: são mais visíveis no ensino de Ciências, mas aplicam-se a qualquer esforço
educativo (BACHELARD, 1996; FÁVERO; TONIETO, 2017).
O educador não tem o senso do fracasso justamente porque se acha um mestre. Quem ensina
manda. Daí, a torrente de instintos. Há indivíduos para quem todo conselho referente aos erros
pedagógicos que cometem é absolutamente inútil, porque os ditos erros são a mera expressão de um
comportamento instintivo (BACHELARD, 1996; FÁVERO; TONIETO, 2017).
Dessa forma as fake News, se configuram em obstáculo epistemológico da experiência primeira,
que significa deixar-se levar pelo impulso, pelo arrebatamento natural, causado pela informação, sem
considerá-la criticamente, porque é na criticidade que o espírito científico se forma e se reforma,
retificando saberes, quando não oferecemos resistência ao novo conhecimento. (BACHELARD, 1996).
Segundo Arnold (2019), algumas habilidades podem colaborar no pensamento crítico e ampliar os
pontos de vista: a) refletir sobre suas próprias “construções emocionais da realidade”, b) oferecer
linguagem fundamentada, c) expressar-se efetivamente, d) tolerar complexidade e confusão, e) aceitar
argumentação factual e f) permanecer persuasível. Estas habilidades, requerem uma educação que o
ensine a estar aberto aos outros. E aqueles presos em seus próprios medos estão fechados a pontos de
vista, decisões e posições singulares que lhes proporcionam calorosas garantias de certeza, vagando em
uma realidade particular, buscando impiedosamente realizar apenas sua própria certeza em cada
ambiente social. (ARNOLD, 2019, tradução nossa).
Nesse sentido, para Arnold (2019, tradução nossa) a eficácia da educação é uma questão
importante a ser considerada. " Iluminismo " não designa apenas uma época da história intelectual
(aproximadamente de 1650 a 1800), na qual os esforços para encontrar soluções razoáveis começaram
a substituir a superstição, mas sim o início do exame racional pelo qual a ciência e a tecnologia, bem
como a filosofia, e democracia, puderam assumir a forma que conhecemos hoje.
A maneira tradicional de defender a validade de insights, avaliações ou conclusões foi substituída
pela exigência de “buscar seu próprio entendimento”. Quer dizer com isso, é claro, que não se deve
acreditar em tudo o que se ouve, "mas sim tomar uma postura de ceticismo e pedir evidências,
contradições e provas. No entanto, essa é a única coisa que precisamos para desenvolver nossas visões
de mundo e ações? Nossas respostas também não são criadas a partir de tentativas muito individuais de
alcançar o reconhecimento e a necessidade de ser levadas a sério - mesmo nos casos em que não temos
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CONHECIMENTO CIENTÍFICO E AS FAKE NEWS: um obstáculo epistemológico no ensino de ciências e biologia?
a expertise? séculos XX e XXI? (ARNOLD, 2019, tradução nossa).
Nesse sentido, a superação de tal obstáculo não acontecerá sem a análise das fragilidades das
fake news a luz do conhecimento científico, levando em conta o contexto histórico, político e social no
qual estão inseridas.
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W4385954005.txt | TYPE
Original Research
15 August 2023
10.3389/fsufs.2023.1222455
PUBLISHED
DOI
OPEN ACCESS
EDITED BY
Subhasis Mandal,
National Dairy Research Institute (ICAR), India
REVIEWED BY
László Vasa,
Széchenyi István University, Hungary
Raquel Ajates,
Universidad Nacional de Educación a Distancia
(UNED), Spain
*CORRESPONDENCE
Mingwei Yang
2020651906@email.ctbu.edu.cn
RECEIVED 14
May 2023
August 2023
PUBLISHED 15 August 2023
ACCEPTED 03
CITATION
Liu J, Yang M and Zhang Z (2023) Can rural
cooperatives reduce poverty vulnerability of
smallholder households? Evidence from rural
Western China.
Front. Sustain. Food Syst. 7:1222455.
doi: 10.3389/fsufs.2023.1222455
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Can rural cooperatives reduce
poverty vulnerability of
smallholder households? Evidence
from rural Western China
Jianzhong Liu 1, Mingwei Yang 2* and Zhiyong Zhang 2
School of Marxism Studies, Hunan International Economics University, Changsha, China, 2 Research
Center for Economy of Upper Reaches of the Yangtse River, Chongqing Technology and Business
University, Chongqing, China, 3 Qinghai University Library, Qinghai University, Xining, Qinghai, China
1
Introduction: Poverty eradication is one of the global challenges, and rural
cooperatives provide an effective path to address smallholder households‘ poverty.
However, the effect of poverty reduction can show heterogeneity depending on
the economic capital, human capital, and social capital of households.
Methods: Based on comprehensive research data on the poverty status of 1,622
smallholder households in four provinces in the less developed regions of western
China, using OLS and PSM models, this paper empirically analyzes the impact and
heterogeneous characteristics of rural cooperatives on the poverty vulnerability
of smallholder households.
Results/Discussion: The results show that rural cooperatives have a significant
dampening effect on the poverty vulnerability of smallholder farmers, and the
findings hold true after robustness tests using multiple methods. The impact of
rural cooperatives on the poverty vulnerability of farming households differed
significantly across smallholder households with different characteristics.
Specifically, participation in cooperatives had a more pronounced effect on
reducing poverty vulnerability among non-poor, higher human capital and higher
income farm households compared to poor, lower human capital and lower
income farm households. The results of the study can provide a useful reference
for policy-making on rural mutual assistance and poverty reduction among
farmers.
KEYWORDS
rural cooperatives, poverty vulnerability, smallholder households, rural mutual
assistance, poverty alleviation
1. Introduction
The alleviation and elimination of poverty is a common goal of human development
and a worldwide challenge (Wang et al., 2022). In 2020, the Chinese government announced
that it had achieved the goal of eradicating absolute poverty, which is an important milestone
in the history of the fight against poverty in humanity. However, smallholder households in
rural China, especially in the western region, are still at serious risk of poverty. On the one
hand, China’s poverty standard is only roughly equivalent to the World Bank’s extreme
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poverty line,1 if the low-middle poverty line and the high-middle
poverty line are applied, China still has a large number of
low-income people; On the other hand, many families are at risk
of returning to poverty. Nearly 2 million people who have escaped
poverty are at risk of returning to poverty, and nearly 3 million of
the marginal population are at risk of becoming poor (Xu and Li,
2023; Zhang et al., 2023). Reducing the risk of poverty among
low-income groups is therefore the central task in China’s rural
revitalization strategy.
Between 1979 and 1984, China’s land system underwent a dramatic
shift from collective farming based on production teams (equivalent to
villages) to a family-based system of responsibility under the household
joint production contract. The central feature of this system is the
decentralization of the management of arable land from collective to
family management. Under China’s family contract responsibility policy,
farmers’ motivation to produce gets a boost. However, it also brings with
it the difficulty of agricultural decentralization. Most farming households
have a small landholding, with an average household size of less than
0.67 hectares (Yang et al., 2023). The ‘small scale’ nature of agricultural
production activities makes it necessary for smallholder farmers to
allocate their resources between rural and urban areas in order to obtain
sufficient income to meet household consumption expenditure. This
leads mainly to three distinct vulnerabilities of smallholder households
(Zhang et al., 2016). How effective is education in fighting poverty?
Researchers are still divided on this question. An analysis of Pakistani
households found that educational attainment was negatively associated
with the incidence of poverty among farming households, and that
access to higher levels of education reduced the likelihood of farming
households falling into poverty (Jia and Xu, 2021). However, some
studies have found that some of the educational reform in Uganda
designed for low income groups did not achieve poverty level reductions
(Saz-Gil et al., 2021). Finally, social networks are an important part of
social capital, enhances action by playing a role in instrumental and
expressive action, with resources embedded in social networks. By
embedding social capital in external social networks, cooperatives build
close and strong relationships with other network actors and gain greater
access to knowledge and information exchange to improve the efficiency
of resource acquisition. Social capital plays a role in signaling,
monitoring, steering, reducing inter-organizational transaction costs,
‘collateral’ substitution and risk reduction, and by reducing the level of
mistrust between individuals, it improves collective cohesion and
promotes cooperation (Person et al., 2017; Ajates, 2021). Small farmers,
mainly left-behind farmers, have been integrated into the traditional
social life of the countryside, where social relations are more closed
(Liang et al., 2015; Ma and Abdulai, 2017).
For most smallholder farmers, it is often difficult to effectively
enhance their capacity for autonomous development simply by relying
on their own efforts. Farmers’ cooperatives (hereafter referred to as
cooperatives), as ‘self-organizations’ of farmers, have attracted the
attention of many scholars in terms of increasing farmers’ incomes
and reducing poverty (Deng et al., 2021; Cheng et al., 2022). Some
scholars argue that cooperatives should be regarded as an efficient
organizational innovation in rural poverty governance because of
their ability to fundamentally improve the efficiency of the use of
poverty reduction funds and to improve the income, capacity and
rights poverty of farm households (Ma and Abdulai, 2017).
Cooperatives convey agricultural knowledge while improving the
market competitiveness and social adaptability of poor farmers by
enhancing their individual capacities, and repairing the capacity
deficits of farmers in the new economy (Bacon et al., 2014). The
mechanisms inherent in the participation of poor farmers in
cooperatives to reduce poverty and increase income are partly the
result of the individual empowerment of members through their
business, capital and management participation in the cooperative,
and this capacity-enhancing effect is greater for middle- and highincome farmers (Bernard et al., 2008; Verhofstadt and Maertens, 2014).
In general, most of the available studies confirm the positive role
of cooperatives in reducing poverty and increasing income (Chagwiza
et al., 2016; Cafer and Rikoon, 2018; Kumar et al., 2018). Cooperatives
have an advantage over scattered smallholder farmers in terms of
large-scale farming, use of advanced technologies, coping with market
risks and access to policy subsidies, increasing the added value,
profitability, labor productivity and employment of farmers engaged
in agricultural production (Ito et al., 2012). Co-operatives not only
help farmers reduce transaction costs in the procurement of
agricultural materials and agricultural production services, but also
improve their bargaining power in the sale of agricultural products;
they also provide various types of training and activities to help
farmers improve their ability to obtain information, express their
needs and apply technology, thereby increasing their income (Kumar
et al., 2018).
However, some scholars have also found that farmer group
differences have a key impact on the poverty-reducing effects of
cooperatives. Some cooperatives have evolved into “self-run
enterprises” that do not contribute to the development of their
members or to the income of farmers as a whole (Bernard and
Taffesse, 2012). The natural heterogeneity of smallholder farmers in
terms of their initial resource endowments, such as production and
management capacity, risk tolerance and household livelihood capital,
may lead to “elite capture,” resulting in the diversion of poverty
alleviation resources and misalignment of project implementation,
creating new income inequalities (Beuchelt and Zeller, 2013). Some
scholars also argue that small and medium-sized members of
cooperatives are prone to “free-riding” behavior, unwilling to pay for
the cooperative’s public services and enjoy the benefits without
contributing much, affecting the efficiency of the organization’s
operations and distributional equity (Tadesse et al., 2019; Ishak
et al., 2020).
The above-mentioned studies provide an important theoretical
basis for this paper, but there are still shortcomings: firstly, although
scholars have focused on the impact of cooperatives on poverty
reduction among farmers, they have not yet reached a unanimous
conclusion, and most of them are based on theoretical discussions,
lacking qualitative and quantitative studies on the impact of
cooperatives on farmers’ ability to reduce poverty. Secondly, few
scholars have studied the heterogeneous effects of cooperatives on the
future poverty reduction capacity of smallholder farmers from the
perspective of farmer differentiation, especially the lack of discussion
of groups of farmers with different poverty attributes and different
human capital endowments.
1 In 2018, the World Bank used less than US$1.90, US$3.20 and US$5.50 per
person per day as the extreme, low and medium poverty lines and the upper
secondary poverty line.
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The main contribution of this paper is that it uses the cooperative
empowerment dimension as an entry point to quantify the reduction
effect of cooperatives on farmers’ poverty vulnerability and the
differences in their effects on heterogeneous groups between groups,
enriching the research framework on the “multidimensional
pro-poorness” of cooperatives by using the Predominant Score
Matching (PSM) method. The remainder of the paper is organized as
follows: Section 2 proposes a theoretical analysis and four research
hypotheses. Section 3 introduces the identification strategy, variables,
and data for this study. Section 4 tests four hypotheses and presents
the regression results and covers the heterogeneity analysis and
robustness testing. Section 5 provides the discussions, conclusions and
related policy implications.
and the cost of control or supervision in management, ensures the
smooth implementation of collective decisions, and minimizes
opportunistic behavior on the part of cooperative managers (Bender,
1999; Wollni and Zeller, 2007; Ma and Abdulai, 2016). In general,
cooperatives are effective in reducing the poverty vulnerability of
smallholder farmers through market access, accelerated human
(social) capital formation and empowerment of management. On this
basis, we propose hypothesis 1.
H1: Cooperatives can reduce the poverty vulnerability of
smallholder households.
From the beginning of their development, cooperatives have been
characterized by an external market environment embedded in the
vertical integration of agriculture and supply chain management
(Verhofstadt and Maertens, 2015). Smallholder farmers are at a
distinct disadvantage in terms of enjoying the benefits of global value
chains due to their low sales volume and limited bargaining power, as
well as the fact that smallholder farmers are often severely limited in
their participation in markets by human capital and credit. Especially
with the increasing trend toward globalization of agricultural markets
and the need for higher management skills and logistics techniques to
market agricultural products and meet higher standards of food safety
certification, the problem of smallholder participation in integrating
into global value chains has become more pronounced, and they even
face the risk of being marginalized (An et al., 2015; Fan and Garcia,
2018; Ajates, 2020). As far as the internal environment is concerned,
with socio-economic development and the expansion of cooperatives,
the structure of group membership has stratified and the heterogeneity
of members has increased significantly (Mojo et al., 2017). Therefore,
when studying the impact of cooperatives on the poverty vulnerability
of smallholder farmers, different group characteristic factors should
be included in the examination.
In terms of poverty attributes, on the one hand, poor farmers tend
to have a strong will to escape poverty, but show vulnerability
characteristics such as poor labor skills, sick and disabled members of
the family, heavy child-rearing burden and few risk-averse means
(Deng et al., 2021). The greater the vulnerability to poverty, the more
risk-sensitive and risk-averse they are. They are reluctant to join a
cooperative or even if they do join, they are reluctant to invest in
shares, thus becoming passive or dormant members. The stratification
between poor and non-poor farmers, and the resulting unequal power
patterns, may constrain the accumulation of a virtuous cycle of
poverty vulnerability reduction among poor farmers (Ma and Abdulai,
2016). On the other hand, from the perspective of cooperatives,
although cooperatives are an effective way to reduce poverty through
the organic combination of market and government mechanisms, and
objectively have a poverty-reducing effect, some cooperatives do not
have an obvious motivation to reduce poverty subjectively, let alone a
mature concept of poverty alleviation (Grashuis and Su, 2019). Most
of the leaders of co-operatives have a philosophy of serving their own
economic performance, and their “deliberate care” for the poor tends
to be weakened. Based on economic rationality, co-operatives are
exclusive toward poor farmers who lack resources and have low
development capacity, and tend to favor non-poor groups who are
well endowed with large scale operations and dedicated investments
in poor areas (Deng et al., 2010). Even when cooperatives open their
membership to poor farmers in general, taking care of poor farmers
2. Theoretical analysis and research
hypothesis
The main reason for attracting farmers to join a cooperative is the
economic return it can bring to them. Attached to the economic
function, cooperatives also generate positive externalities by helping
farmers to overcome barriers to market access, improve scientific and
cultural literacy, increase social capital stock and empower
management (Ito et al., 2012; Kumar et al., 2018). They also have
positive externalities in terms of helping farmers overcome barriers to
market access, improving scientific and cultural literacy, increasing
social capital stock and empowering management, which in turn
reduce the poverty vulnerability of smallholder farmers (Chagwiza
et al., 2016; Zhang et al., 2023).
With the development of the market economy, agricultural
markets are becoming more and more mature. For decentralized
smallholders, due to their weakness, asymmetric information, high
transaction costs and low standardization of production, they face
high barriers to market entry and lack sufficient competitiveness and
voice in large markets, and are unable to connect effectively with
markets (mainly high value-added markets) on their own (Loconto
and Simbua, 2012; Richards and Mendez, 2014). Collective action, i.e.,
the formation of cooperatives, is an effective mechanism to help
resolve the conflict between smallholders and large markets and to
increase farmers’ participation in the market (Beuchelt and
Zeller, 2013).
Collective action provides relevant information and services to
smallholders, including technical information and services
(agricultural extension and research and development), educational
services (production skills training, business skills training and
general education), etc. It improves the efficiency and management of
farmers’ access to agricultural technology and also promotes the
sustainable and healthy development of the cooperative (Mavimbela
et al., 2010; Meador et al., 2016; Tray et al., 2021). The human capital
of the farmers is accumulated and the endogenous motivation for
development is further stimulated.
In terms of management participation, democratic management
and control is the foundation and core principle of the cooperative
system. The participation of members in general meetings, councils
and supervisory boards, and the full expression of their views and
demands, not only helps to safeguard their own property rights and
interests and to obtain more residual claims, but also increases trust
within the membership, helps to reduce conflicts in decision-making
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in a unified operation would mean lower returns for non-poor
members (Wollni and Zeller, 2007). The more poor farmers a
co-operative takes on, the greater the risk it may face of a decline in
overall benefits. On this basis, we propose hypothesis 2.
their resource endowments and effectively spill over, thus becoming
the biggest beneficiaries of the development of the co-operative (Shi
et al., 2019; Li et al., 2021). On this basis, we propose hypothesis 4.
H4: Cooperatives are more effective in reducing the poverty
vulnerability of middle- and high-income households than of
low-income households.
H2: The effect of poverty vulnerability reduction due to
cooperative membership is higher on non-poor farming
households than in poor farming households.
Based on the above analysis, the theoretical analysis framework of
this paper is shown in Figure 1.
Poverty is a vague concept, but it has some basic characteristics,
namely that it is mainly marked by “lack,” which appears as “low
income” and “lack of material and services,” but in essence is a lack of
“means,” “capabilities,” “rights” and “opportunities.” Education levels
are closely linked to the ability of smallholder households to escape
poverty (Yang et al., 2023). Better-educated farmers tend to have a
greater ability to accept new knowledge and new things and to
understand and learn, and their rich knowledge base makes them
more likely to accept the organizational system, business philosophy
and production techniques of the cooperative, which makes it easier
for them to join the cooperative (Ito et al., 2012). Moreover, the
stronger the ability to accept new knowledge and technology, the
clearer the perception of the cooperative’s ability to enhance its own
development. On this basis, we propose hypothesis 3.
3. Materials and methods
3.1. Data
The data in this article comes from a comprehensive survey on
the status of rural poverty in Southwest China, July–September 2021.
The region covers four provinces, Yunnan, Guizhou, Sichuan and
Chongqing. Including eight state-defined poor counties (cities) in
Fengjie, Wanzhou, Yunyang, Xishui, Puding, Guang’an, Xuyong, and
Dongchuan, 136 villages with 12 farmers per village, a total of 1,632
households in the sample. The research sample was selected based on
a three-stage sampling: (1) Cluster analysis sampling. The original
592 state-defined poor counties were divided into three categories of
overall poverty status, with experts empirically assessing the worst
category and selecting sample provinces and counties in the worst
category. (2) Probability Proportional Scale Sampling. Sample villages
were selected in proportion to the size of the poor population. (3)
Random sampling. A sample of 12 farmers was randomly selected in
each village to answer the questionnaire. This sample data represents
to a large extent the group of farming households that need the most
attention in the less developed counties of China, and is representative
and typical. Since the focus of this paper is on smallholder
households, farmers with average arable land above 0.67 ha are
excluded. After data cleaning and elimination of the 10 questionnaires
that did not meet the requirements, the actual research population of
this paper is 1,622 households.
H3: Cooperatives are more effective in reducing the vulnerability
to poverty of households with high human capital endowments
than those with low human capital endowments.
Institutional norms of rural cooperatives mainly include formal
institutional arrangements and informal institutional norms. We have
found that rural cooperatives in China tend to be member-based and
rely closely on related enterprises. In terms of management, the
cooperative has adopted the practice of “two brands and one set of
staff ” with the enterprise. The day-to-day management, sales and
technical guidance of the cooperative are all dependent on the relevant
enterprise, with the core members responsible for the management of
the enterprise and the ordinary members only involved in the business
work. The heterogeneity of the membership structure of cooperatives
is shaped by the differentiation of farming households (van Rijsbergen
et al., 2016). This heterogeneity is reflected in the distinction between
core members and general members of the cooperative. These two
types of members have different levels of income and different levels
of participation in the cooperative, resulting in different roles and
division of labor, which leads to differences in their ability to improve
their skills, showing typical asymmetrical characteristics (Valkila and
Nygren, 2010). Compared to core members, general members are
usually low-income, low-capital participation groups, and such groups
often lack the interest and ability to participate in the public affairs of
the cooperative, or even the opportunity to do so (Jitmun et al., 2020).
They rarely participate in the day-to-day management and supervision
of cooperatives, and are mostly limited to basic aspects such as
participation in the purchase of agricultural inputs and materials, the
sale of agricultural products, and access to specialized technical
services and policy concessions. For core members, their material
resource endowments are at an advantage, and they hold the majority
of shares in the co-operative, control most of the residual control and
residual claims, have more say in the daily production and
management activities of the co-operative, and can make full use of
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3.2. Method
3.2.1. Poverty vulnerability measurement
Poverty vulnerability, which connects risk shocks to the degree of
household welfare, is often seen as unobservable, dynamic, and
forward-looking, with a focus on poverty generation expectations
(Wang et al., 2022). Poverty vulnerability is the probability that a
household or individual will fall into poverty or fail to escape from
poverty as a result of exposure to uncertainty risk shocks. Poverty
vulnerability is calculated as follows.
(
ln z − X i βˆFGLS
Φ
Vˆi =
Prob ( ln ci < ln z X i ) =
X i θˆFGLS
04
)
(1)
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FIGURE 1
A theoretical model of the impact of cooperatives on poverty vulnerability of smallholder households.
controls similar to the treatment group to compare their effects,
thus effectively solving the endogeneity problem arising from
sample selection bias (Yang et al., 2023). Since differences in
farmers’ initial endowments can directly cause a “selective bias” in
their willingness or behavior to join a cooperative, and whether or
not to join a cooperative often reflects the ideological tendency of
rational farmers to pursue optimal utility, a simple OLS regression
of Equation (2), which estimates the capacity-enhancing effect of
farmers ignores their own subjective initiative, yields only the
conditional expectation effect of the explanatory variables on the
explanatory variables, and the results obtained may be biased. The
PSM propensity value matching method can effectively solve these
problems by finding a control group (uninvolved farmers) with
similar characteristics that can simulate the counterfactual state of
the treatment group (involved farmers), thus maximizing the
elimination of endogeneity problems due to self-selection bias. The
specific steps are as follows.
Step 1: we used a logistic model to calculate the conditional
probability of a household Participating in rural cooperatives, i.e., the
propensity score.
Step 2: based on the propensity scores obtained through three
methods: nearest neighbor matching, radius matching and kernel
matching, we found a sample of farmers in the control group with
propensity scores as similar as possible to those in the treatment
group, in order to control and eliminate selectivity errors.
Step 3: PSM model requires that the variables used for matching
meet the common support domain assumption and the balance test,
and after the sample has been matched and the matching effect has
been achieved, we calculate the average treatment effect (ATT). The
ATT is calculated as shown below.
Where Vˆi is an estimate of the probability of future poverty for
farmer i, ci is the value of per capita household consumption, z is the
delineated poverty line, Φ is the cumulative distribution function of
the normal distribution, βˆFGLS and θˆFGLS denote the expected
value and variance of future household consumption estimated by the
FLGS method, respectively. Xi is an observable variable, referring to
Wang et al. in their examination of poverty vulnerability by
introducing household characteristics variables (including household
income, household size, land assets, liabilities, agricultural machinery,
etc.) and household head characteristics variables (including age,
gender, education, etc.).
3.2.2. Econometric model
We constructed an OLS model to examine the impact of
cooperatives on poverty vulnerability of smallholder households. The
OLS model is set up as in Equation (2):
yi = α + β Cooperativesi + θ Xi + ε i
(2)
Among them, y i is the poverty vulnerability of smallholder
households. Cooperatives i represents the participation in a rural
cooperative, and X i indicates a series of control variables, mainly
including family characteristics, village characteristics and head of
household Characteristics.
3.2.3. Propensity score matching (PSM) model
The propensity score matching method is a counterfactual
inference method, the basic idea of which is to find a sample of
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PSi = Pr Di = 1|X i = E Di = 0|X i
4. Empirical results
(3)
4.1. Benchmark regression results
ATT =
1
Nt
∑i∈I ∩S {Yi − ∑ j∈I ∩SWijY j }
t
c
(4)
Table 2 reports the results of the benchmark regression of the
impact of rural cooperatives on the poverty vulnerability of
smallholder households. In Model 1, we only control for the
characteristic variables of the household head. In Model 2, we further
controlled for household characteristics variables of smallholder
farmers. In Model 3, we included household head characteristics,
household characteristics and village residence characteristics as
control variables. The results show that, controlling for a range of
variables, participation in cooperatives can significantly reduce the
poverty vulnerability of smallholder farmers. The impact factor is
−0.0162, and is significant at the 5% level, which basically supports
hypothesis 1 that cooperatives can reduce the poverty vulnerability of
smallholder farmers.
The coefficients and signs of the control variables remain
consistent with existing studies. The level of education of the
household head, the net household income, social capital and the
distance from the village to the county have a significant positive
impact on the reduction in poverty vulnerability of smallholder
farmers. Age of head of household and level of household poverty
have a significant negative effect on reduction in poverty vulnerability
of smallholder farmers. In addition, the number of laborers and
migrant workers also show a negative impact on the reduction of
poverty vulnerability, which may be closely related to the demographic
disadvantage of smallholder households.
Among them, N t is the number of samples, I t is the sample set of
the disposal group (Participating in rural cooperatives), I c is the sample
set of the control group (Not Participating in rural cooperatives), Yi is
the observed value of the sample of the disposal group, and Y j is the
sample of the control group. The observations of j, S is the common
support domain set, Wij is the matching weight, and ATT is the average
disposition effect.
3.3. Variables
3.3.1. Dependent variables
To forecast household poverty vulnerability, this article uses
household per capita consumption. One reason for using
consumption to define poverty is that income is easily underestimated
in micro-surveys, whereas consumption can better reflect the level of
family welfare, and the other is that using income as an explanatory
variable can easily lead to strong endogenous problems in the
measurement model. Regarding the choice of the poverty line, there
are primarily two standards of per capita daily consumption of
US$1.9 and US$3.1 proposed by the World Bank in 2015, which
we convert into ¥2,800 and ¥4,570 per capita annual consumption
based on China’s average purchasing power and CPI index (Wang
et al., 2022). In the subsequent analysis, we focus on ¥4,570 as the
poverty standard line.
4.2. Robustness tests of the PSM model
The benchmark regression results show that joining a rural
cooperative can significantly reduce the poverty vulnerability of
smallholder farmers. However, there is also a potential problem that
OLS regression results are susceptible to sample selection bias, and
those factors that are not observed may affect the precision of the
estimates. In order to ensure the credibility and robustness of the
regression results, we further used the come PSM model to verify the
poverty vulnerability reduction effect of cooperatives on farm
households. We have selected control variables that were significant
in the baseline regression model for the propensity score matching, in
order to eliminate the variability of the characteristic variables
between the two sample groups.
After propensity score matching, the question of conditional
independence between the two sample groups needs to be checked,
i.e., there are no significant differences in the characteristics
variables between the matched sample groups, except for differences
in the poverty vulnerability of the farmers. Table 3 reports the
results of the conditional independence hypothesis tests for the
explanatory variables before and after PSM matching. After PSM
matching, the pseudo R2 decreases from 0.013 before matching to
0.001–0.003 after matching. LR chi2, B-values and mean bias-value
have all fallen substantially. All p values are greater than 10%. Thus,
after matching by the PSM model, we significantly eliminate
systematic differences in the distribution of explanatory variables
between the treatment and control groups, minimize sample
selection bias, and propensity score estimation and sample
3.3.2. Independent variables
The core independent variable is whether or not one participates
in a cooperative. The ability of cooperatives to bring significant
capacity enhancement effects to farmers at different stages of
agricultural production depends on whether or not farmers
participate in cooperatives. The core explanatory variable is “whether
or not the farmer participates in a cooperative,” which describes the
impact of cooperatives on the poverty vulnerability of
smallholder households.
3.3.3. Control variables
With reference to existing studies, this paper introduces three
types of control variables, namely, variables on individual household
head characteristics, variables on household characteristics and
variables on village characteristics (Scripcariu et al., 2020; Yin et al.,
2020; Ma and Jin, 2022). Household head characteristics include
gender, age, and education level; Household characteristics include the
number of household labors, net household income per capita, level
of household poverty, whether there are family members working in
the city, total productive assets and annual gift expenses; village
characteristics include village transportation conditions and economic
status. The descriptive statistical characteristics of the specific variables
are shown in Table 1.
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matching are more successful, significantly weakening estimation
bias due to self-selection.
In Table 4, we used five PSM methods to estimate ATT, ATU and
ATE for the impact of cooperatives on the poverty vulnerability of
smallholder farmers. Among them, ATT represents the average
treatment effect of the treatment group; ATU represents the average
treatment effect of the control group; ATE is the average treatment
effect for the overall sample. The results show that the five matching
methods ATT, ATU and ATE all passed the test at the 1% significance
level, which indicates that the results of matching between samples are
relatively robust. The mean value of ATT is −0.0252, which suggests
that cooperatives have a significant dampening effect on the poverty
vulnerability of farm households. In other words, the poverty
vulnerability of farmers who joined cooperatives was reduced by 0.0252
compared to those who did not join cooperatives.
4.3. Results of the heterogeneity analysis
4.3.1. Heterogeneity analysis based on
educational level of household heads
The level of education of the household head is largely
representative of the overall human capital endowment of the
smallholder household. Therefore, we examine the heterogeneity of
the effect of cooperatives on reducing the vulnerability of farm
households to poverty in terms of the educational attainment of the
TABLE 1 Definition and descriptive statistics of the variables involved.
Variables
Definition
Mean
SD
0.1414
0.2089
0.2429
0.4289
48.8199
11.3194
0.6374
0.4808
5.6535
4.3273
8.6364
1.2595
3.3083
1.6904
0.5720
0.3775
0.5758
0.4943
Dependent variables
Vulnerability
Poverty vulnerability of smallholder
households
Independent variables
Cooperative
Whether or not to participate in a
cooperative
Control variables
Age
Gender
Education
Income
Labor
Poverty
Age of the head of household (Years)
Gender of head of household.
Female = 0; male = 1
Years of education of the head of
household
Logarithm of net household income per
capita
Number of members in household aged
15–64
Whether the household is households
registered as living under the poverty
line. Yes = 1; No = 0
Migrant
Whether there are family members
working in the city. Yes = 1; No = 0
Assets
Logarithm of total productive assets
11.9661
1.1318
S-capital
Logarithm of total gift expenses
7.3053
2.0172
51.9343
40.824
4.1003
1.0108
0.3218
0.1509
0.1849
0.2514
0.3817
0.3499
0.1116
0.1724
Distance
Economic
SiChuang
YuNan
ChongQing
GuiZhou
Observation
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Distance between the settlement and the
county (km)
Settled village economic status
assessment
Whether the province is SiChuang.
Yes = 1; No = 0
Whether the province is Yunnan. Yes = 1;
No = 0
Whether the province is ChongQing.
Yes = 1; No = 0
Whether the province is Guizhou.
Yes = 1; No = 0
1,622
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TABLE 2 Baseline regression results of the impact of rural cooperatives on the poverty vulnerability of smallholder households.
Variables
(1)
(2)
Cooperatives
(3)
−0.0443 *** (0.0112)
−0.0182 ** (0.0072)
−0.0162** (0.0076)
Age
0.0032*** (0.0004)
0.0036 *** (0.0002)
0. 0036*** (0.0002)
Gender
0.0424*** (0.0103)
0.0282*** (0.0067)
0.0246*** (0.0060)
−0.0135 *** (0. 0011)
−0.0050 *** (0.0007)
−0.0039*** (0.0006)
Education
−0.0897 *** (0. 0046)
−0.0763*** (0.0041)
Labor
0.0580*** (0.0018)
0.0595 *** (0.0164)
Poverty
0.1332*** (0.0112)
0.0452*** (0.0163)
S-capital
−0.0039*** (0.0007)
−0.0035 *** (0.006)
Migrant
0.0198*** (0.0066)
0.0175*** (0.0059)
Income
Economic
−0.0072 (0.0199)
Distance
−0.0294*** (0.0014)
Province-FE
Yes
Yes
Yes
N
1,622
1,622
1,622
R2
0.1464
0.6433
0.7165
The standard error is shown in parentheses. ** and *** are statistically significant at 5%,1% levels, respectively. Province-FE represents provincial fixed effects.
TABLE 3 Results of conditional independence hypothesis tests for explanatory variables before and after matching.
Matching
method
Ps R2
LR chi2
MeanBias
B-value
Value of p
0.013
24.27
7.8
37.9
0.002
0.001
1.01
2.1
7.2
0.998
Radius matching
0.000
0.54
1.4
5.2
0.997
Kernel matching
0.000
0.51
1.4
5.1
0.999
Mahalas matching
0.001
1.30
1.5
8.1
0.996
0.002
3.81
1.6
7.6
0.874
Unmatched
Nearest neighbor
matching (k = 4)
Partial linear regression
matching
household head. China has a 9-year compulsory education system.
Based on China’s school system, this paper classifies the years of
education for heads of households into two categories, namely lower
education group (0–9 years), and higher education group (more than
9 years).
As shown in Table 5, the effect of cooperatives on reducing poverty
vulnerability is 2.05 times greater in the high quality group (−0.0387)
than in the low quality group (−0.0188) for the education level of the
household heads, which suggests that farmers with high quality
human capital endowments are more likely to improve their poverty
status after joining a cooperative than those with low quality.
Hypothesis 3 was tested.
The reduction effect of cooperatives on poverty vulnerability of
non-poor households (0.0570) is 4.37 times greater than that of
poor households (0.0130). This shows that non-poor households are
more likely to benefit from co-operative seeds than poor
households. Hypothesis 2 is verified.
4.3.3. Heterogeneity analysis based on the
household incomes
This paper classifies farm households into lower-income and
higher-income groups based on their median per capita income levels,
and removes the variable of net household income from the regression.
As shown in Table 7, the reduction effect of cooperatives on poverty
vulnerability of higher income households is greater than that of lower
income households. Overall, the cooperatives had a dampening effect
on poverty vulnerability for both the lower and higher income groups
of farmers, but there were differences in the magnitude of the effect,
with a greater reduction effect for the higher income group than for
the lower income group (−0.0406 > −0.0184), and hypothesis 4
is tested.
4.3.2. Heterogeneity analysis based on the level
of household poverty
We divided the sample into two groups according to whether
the households were registered as living under the poverty line or
not. As shown in Table 6, cooperatives have a negative impact on
the poverty vulnerability of farmers across different poverty
attributes, but there are differences in the magnitude of the effect.
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TABLE 4 Propensity score matching estimates of the impact of cooperatives on the poverty vulnerability of smallholder farmers.
Matching method
ATT
ATU
ATE
Nearest neighbor matching (k = 4)
−0.0239**
−0.0299***
−0.0265***
Radius matching
−0.0235***
−0.0284***
−0.0272***
Kernel matching
−0.0222***
−0.0377***
−0.0340***
Mahalas matching
−0.0238**
−0.0335***
−0.0288***
Partial linear regression matching
−0.0324***
−0.0229***
−0.0276***
−0.0252
−0.0305
−0.0288
Average value
The standard error is shown in parentheses. ** and *** are statistically significant at 5 and 1% levels, respectively. Significance tests for ATT, ATU, and ATE values were obtained using the
bootstrap method of repeated sampling 500 times.
TABLE 5 Results of heterogeneity analysis on the relationship between the cooperatives and educational level of household heads.
Variables
ATT
lower education (0–9 years)
higher education (more than 9 years)
Nearest neighbor matching (k = 4)
−0.0191*
−0.0463***
Radius matching
−0.0179*
−0.0337**
Kernel matching
−0.0180*
−0.0440***
Mahalas matching
−0.0121
−0.0326***
−0.0271**
−0.0369***
−0.0188
−0.0387
Partial linear regression matching
Average value
The standard error is shown in parentheses. *, ** and *** are statistically significant at 10, 5, and 1% levels, respectively. Significance tests for ATT, ATU, and ATE values were obtained using
the bootstrap method of repeated sampling 500 times.
5. Discussion and conclusions
have an advantage over scattered smallholder farmers in terms of
large-scale operation of farmland, use of advanced technology, coping
with market risks and access to policy subsidies, increasing the added
value, profitability, labor productivity and employment rate of farmers
engaged in agricultural production (Sun et al., 2009; Babiarz et al.,
2010). Co-operatives not only help farmers reduce transaction costs
in the procurement of agricultural materials and agricultural
production services, and increase their bargaining power in the
marketing of agricultural products; they also provide a variety of
training and activities to help farmers improve their viable capacity to
access information, express their needs and apply technology, which
helps reduce the poverty vulnerability of smallholder households (Dao
et al., 2023).
In practice, some researchers have focused on the alienation of
cooperatives caused by differences in groups of farmers (Dai et al.,
2023; Zeren et al., 2023). Some co-operatives have evolved into ‘selfrun enterprises’ that do not contribute to the development of their
members, nor do they contribute to the incomes of farming
households as a whole (Wilmsen et al., 2022; Dong et al., 2023). The
natural heterogeneity of smallholder farmers in terms of their initial
resource endowments, such as production and management capacity,
risk tolerance and household livelihood capital, may lead to “elite
capture,” resulting in deviated resources for poverty alleviation and
misplaced project implementation, creating new income inequalities
(Gilcrease et al., 2022). Some scholars also argue that small and
medium-sized members of cooperatives are prone to “free-riding”
behavior, unwilling to pay for the cooperative’s public services and
enjoy the benefits without contributing much, affecting the efficiency
of the organization’s operations and distributional equity (Ito
et al., 2012).
5.1. Discussions
Our explorations of the heterogeneous characteristics of
smallholder farmers leads to a topic worth exploring, namely
whether cooperatives in reality can meet the development
aspirations of a wide range of disadvantaged groups and whether
they really have the desired organizational effectiveness in driving
smallholder farmers. According to classical co-operative theory,
alleviating the inherent tension between smallholders and the
larger market is the purpose of forming a cooperative for ‘weak’
smallholders (Lennard-Jones and Devonshire, 1939; Bleaney, 1963;
Elliott et al., 1971). However, an important prerequisite for the
effective operation of cooperatives is a high degree of homogeneity
in membership. In reality, farmers are highly heterogeneous, and
this is difficult to eliminate in the short term (Cai, 2002). As a
result, the organizational objectives of cooperatives deviate from
the assumptions of classical cooperative theory, and the
organizational performance is biased toward members with
superior resource endowments (Cai, 2002; Wagstaff et al., 2009).
Thus, the organizational objectives of cooperatives deviated from
the assumptions of classical cooperative theory, and organizational
performance was biased in favor of members with superior
resource endowments, so that the development and profitability of
small farmers was reduced.
Cooperatives are an important vehicle for industrial poverty
alleviation (Bernard and Spielman, 2009). The vast majority of studies
have affirmed the positive role of cooperatives in reducing poverty and
increasing income (Sun et al., 2009; Mojo et al., 2017). Cooperatives
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TABLE 6 Results of heterogeneity analysis on the relationship between cooperatives and the poverty levels of farmers.
Variables
ATT
Under the poverty line
Above the poverty line
Nearest neighbor matching (k = 4)
−0.0133**
−0.0407*
Radius matching
−0.0128**
−0.0548**
Kernel matching
−0.0068***
−0.0623***
Mahalas matching
−0.0132***
−0.0655**
Partial linear regression matching
−0.0191**
−0.0617**
−0.0130
−0.0570
Average value
The standard error is shown in parentheses. *, ** and *** are statistically significant at 10, 5 and 1% levels, respectively. Significance tests for ATT, ATU, and ATE values were obtained using
the bootstrap method of repeated sampling 500 times.
TABLE 7 Results of heterogeneity analysis on the relationship between cooperatives and the household incomes.
Variables
ATT
Lower income families
Higher income families
Nearest neighbor matching (k = 4)
−0.0173***
−0.0458 ***
radius matching
−0.0181**
−0.0384**
kernel matching
−0.0168***
−0.0423***
Mahalas matching
−0.0202**
−0.0355**
partial linear regression matching
−0.0195**
−0.0413**
−0.0184
−0.0406
Average value
The standard error is shown in parentheses. ** and *** are statistically significant at 5 and 1% levels, respectively. Significance tests for ATT, ATU, and ATE values were obtained using the
bootstrap method of repeated sampling 500 times.
Heterogeneous characteristics make a difference in both the
motivation of farmers to join and their factor inputs, resulting in
differences in the impact of cooperatives on farmers’ incomes
(Gorczyca et al., 2022). It has been generally agreed that farmers
with better resource endowments and more factor inputs are more
likely to seek more control over their surplus. To accurately detect
the impact of co-operatives on the poverty vulnerability of farm
households, it is necessary to distinguish between the
heterogeneity of farm households and focus on which groups
co-operatives work more significantly for. The findings of this
paper also confirm this phenomenon (Fernandes and Silva, 2022).
Farmers whose heads have higher levels of education, non-poor
families and higher household incomes have gained a more
pronounced reduction in their poverty vulnerability after joining
the cooperative. In research in less developed areas, poverty
alleviation work generally suffers from strong policy input but
weak endogenous motivation enhancement, etc. Most cooperatives
only objectively absorb poor farmers into their societies as a
matter of policy, but subjectively they do not pursue the
effectiveness of bringing poverty, and are not willing to absorb
poor farmers. In addition, the risk-averse nature of poor farmers
with inherent lack of production endowments and social network
resources often makes them reluctant to join the society, or even
if they do, the shares they put in are low due to financial
constraints, which inevitably leads to the problem that the
cooperatives are “pro” the capable rural people and “anti” the
Frontiers in Sustainable Food Systems
disadvantaged (Bernard and Spielman, 2009; Chagwiza et al.,
2016). The problem is that cooperatives are inevitably “pro” rural
people and “pro” disadvantaged groups. However, it is also
important to note that the value of cooperatives in benefiting the
poor should not be dismissed because of their “affinity” to the
rural and disadvantaged groups (Xu and Li, 2023). The empirical
results also show that even if poor farmers find it difficult to
participate in co-operatives because they are ‘excluded’ or less
willing to join them, they can still benefit indirectly through the
spillover effects of co-operatives.
Additionally, Farmers in different income brackets and education
levels have different levels of involvement in the operations and
management of the cooperative, leading to differences in their
poverty vulnerability reduction. Farmers with less physical capital
and less learning capacity have less control and say in the day-to-day
operations of the cooperative, and tend to be in a lower position in
the cooperative than those with more material resources and higher
levels of education (Bouichou et al., 2021). Accordingly, cooperatives
are also less effective in reducing their poverty vulnerability than
members with superior physical capital and high levels of education.
Conversely, farmers with strong economic resource endowments
usually have sufficient accumulation of their own resource factors
and can make full use of and effectively spill over their economic
resource endowments (Alam et al., 2021). As a result, the decision to
join the society is more effective and more rewarding for
these farmers.
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5.2. Conclusion
the cooperatives themselves. The variables in this paper are
selected from the perspective of farmers only, and are based on a
single dimensional characteristic of farmers, without detailed
descriptions and statistics of cooperatives. Second, the paper does
not consider the willingness of cooperatives to take on board.
Whether farmers can become members of cooperatives is not only
based on whether they have a demand for membership, but also
on whether cooperatives have the willingness to open up
membership to the public, which is the result of a combination of
demand and supply factors. However, on the supply side, the
willingness of cooperatives to take up membership varies
depending on the organizational model, governance and other
characteristic factors of cooperatives in less developed western
regions. Thirdly, social capital and the governance model of
cooperatives play an important role in the poverty reduction effect
of rural cooperatives. However, we did not conduct an in-depth
analysis of these two areas due to the availability of data. Finally,
the paper does not include the factors of policy intervention in
cooperatives in its examination. If the policy is to support
excellence and strength, cooperatives will choose to exclude the
rural disadvantaged because they want to improve their
competitiveness; in contrast, if the policy is to regulate the
development of cooperatives and advocate their pro-poor
attributes, then the relevant policy interventions will affect the
exclusion decision of cooperatives as well as the demand of
farmers to join the society. Policy interventions affect both the
willingness of co-operatives to take in and the demand of farmers
to join. The construction of an analytical framework that
incorporates policy interventions, cooperative and farmer
characteristics is an important direction for future research.
Based on micro-survey data from smallholder farmers in eight
counties in four provinces in the underdeveloped regions of western
China, this paper analyses the impact of farmers’ membership in
cooperatives on their poverty vulnerability and further explores the
differences in the poverty reduction effects of cooperatives on groups
with different poverty attributes, different human capital endowments
(education level of the household head), and different income class
heterogeneity. The main conclusions are as follows.
(1) Cooperatives have a significant dampening effect on the
poverty vulnerability of smallholder farmers. Cooperatives
have a positive external impact in terms of helping farmers to
overcome barriers to market access, accelerating the formation
of human (social) capital and empowering management,
which in turn has a combined effect on improving the ability
of farmers to develop themselves, which has a combined effect
on the improvement and enhancement of farmers’ capacity for
autonomous development.
(2) After overcoming the sample selection bias using the PSM
model, the results show that participation in cooperatives still
reduces the poverty vulnerability of smallholder farmers by an
average of −0.0252, and the result remains robust to multiple
tests of the methodology.
(3) The impact of cooperatives on the poverty vulnerability of
smallholder farmers is significantly heterogeneous across
groups. Specifically, participation in cooperatives has a more
pronounced effect on poverty reduction among non-poor,
higher human capital endowment and higher income bracket
households than among poor, lower human capital endowment
and lower income bracket households.
Data availability statement
The validated conclusions outlined above can contribute and
assist in emerging policy enlightenment. Firstly, enhancing the
linkages between the interests of rural ‘elite’ figures and ‘weak’ small
farmers. Policy makers should guide and encourage farmers to join
or start cooperatives and support the development of cooperatives
as an effective initiative to reduce poverty among smallholder
farmers, but they should also see the limitations of the effectiveness
of policy implementation. A top-down push for cooperative
development and the pursuit of incremental growth in order to
achieve an increase in the ability of farmers to reduce poverty will
likely lead to a further widening of the gap in the ability of farmers
to escape poverty in the future. The government should, on the
basis of cultivating the stock of cooperatives, keenly identify the fit
between “elite” figures and “weak” small farmers in terms of
business areas and cooperative relationships, and strengthen the
linkage between the interests of cooperatives and small farmers in
order to minimize the negative effect of “elite capture.” Secondly,
cooperatives are an effective way for smallholder farmers in less
developed areas to escape poverty. Even if poor farmers find it
difficult to participate in cooperatives because they are ‘excluded’ or
have a low willingness to join, they can still benefit indirectly
through the spillover effects of cooperatives.
There are still many limitations in this paper, which can
be seen in the following aspects: Firstly, we have focused more on
the heterogeneity of farmers and neglected the heterogeneity of
Frontiers in Sustainable Food Systems
The original contributions presented in the study are included in
the article/supplementary material, further inquiries can be directed
to the corresponding author.
Ethics statement
Ethical review and approval was not required for the study on
human participants in accordance with the local legislation and
institutional requirements. Written informed consent from the
participants was not required to participate in this study in accordance
with the national legislation and the institutional requirements.
Author contributions
MY: conceptualization, validation, data curation, and supervision.
ZZ: methodology and resources. MY and JL: software, writing—original
draft preparation, writing—review and editing, and visualization. JL, MY,
and JL: formal analysis. JL: investigation. All authors have read and
agreed to the published version of the manuscript.
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10.3389/fsufs.2023.1222455
Funding
Conflict of interest
This research was financially supported by the National Social
Science Fund of China (Grant/Award Number: 17XTQ011); the MOE
Project of Key Research Institute of Humanities and Social Sciences
in Universities of China (Grant/Award Number: 16JJD790063); the
National Social Science Foundation Youth Project of China (Grant/
Award Number: 16JJD79006321CJY044).
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 manufacturer, is not guaranteed or
endorsed by the publisher.
Acknowledgments
The authors acknowledge funding from the National Social
Science Foundation of China and the MOE Project of Key Research
Institute of Humanities and Social Sciences in Universities of China.
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| 10,564 | 73,088 |
W2594599868.txt | Journal of Management Sciences
Vol. 4(1): 27-53, 2017
DOI: 10.20547/jms.2014.1704102
GSP Plus Status and Income Distribution: A CGE Model for
Pakistan
Muhammad Shahzad Iqbal ∗
Sofia Anwar †
Muhammad Aamir Khan
Kakali Mukhopadhay
‡
§
Abstract: The purpose of the study is to investigate the impact of the Generalised System
of Preferences (GSP) plus on a household income of Pakistan. The European Union, the largest
trading partner of Pakistan, granted the status of GSP plus to Pakistan in December 2013. The
study has used MyGTAP, developed by Minor and Walmsley (2013) to calculate the impact at
household level. This MyGTAP model uses the data of the latest available Social Accounting
Matrix (SAM) to make changes in the standard GTAP by including multiple types of household
and labor. The main findings of the simulations, run under MyGTAP model show a positive
change in real GDP, merchandise imports and terms of trade while the first simulation shows a
negative change in merchandise exports. Similarly, EBA status of Pakistan in the EU28 shows
an increase in the household income with maximum gain by the household of rural Sindh with no
agricultural land and a positive change in real wages of most of the factors. However, the large and
medium agricultural household types show a negative change in household income in case of first
simulation. Comparatively low improvement over urban and non-farm household of rural areas.
Keywords: Economic growth, trade, GSP Plus, European Union, CGE model, real GDP,
terms of trade, real investment, household income.
Introduction
For many years EU is the largest importer of Pakistani products. Total exports from
Pakistan to the EU during the year 2014 were US$ 8.13 billion which accounted 29 percent
of the total exports. It was 21.5 percent in 2012 and 24 percent in 2011. Although the
EU is considered to be the dominant importer in Pakistan, but the country always showed
a sluggish export growth, especially in terms of commodity diversification. On the other
hand, penetration into the EU market remained overdue comparing to other competitors.
Pakistan is a member of the preferential trading system of the EU, ever since its evolution. The examination evident that external trade relations of the EU with the developing countries has been conducted with a number of different channels, principally with
the African, Caribbean and Pacific (ACP) states through the Lome Convention, Mediter∗ PhD Scholar, Department of Economics, GC University Faisalabad.
E-mail:shahzad.iqbaal@gmail.com
† Chairman, Department of Economics, GC University Faisalabad.
‡ Senior Researcher, Department of Natural Resource Sciences, McGill University.
§ Assistant Professor, COMSATS Institute of Information Technology Islamabad.
27
Journal of Management Sciences
ranean countries through the Global Mediterranean Policy (GMP) and with the rest of
Latin America and Asian developing countries including Pakistan through the GSP scheme.
This is evident from the EU’s complex network of discriminatory tariff through generalized and country-specific or region-specific trade preferences. While, trade relations with
most industrialized countries have been based on most-favoured-nation treatment (Rashid,
2006).
The common commercial policy uses a spread of instruments to regulate trade among
the EU and its trade partners. It covers not only tariff, but other trade instruments
as well. A complicated system of trade advantages, differentiated according to specific
groups of countries, has drawn up to a hierarchy of trade preferences called as ‘pyramid’
of trade preferences. The examination of EU’s trade regime practice worked out over the
years points out that it uses fairly complicated procedures and a very elaborate panoply
of instruments. Although the system has some of the economic effects hoped for and has
been established for political reasons; it seems advisable to simplify considerably in order to
expand its benefits largely according to ‘trade not aid’ principle (Persson & Wilhelmsson,
2016).
On December 2013, European Union granted GSP plus status and since January 2014,
Pakistan has been enjoying this status. It is expected from the very beginning that the
exports from Pakistan are expected to increase in the EU market under GSP plus status.
This status will substantially increase Pakistan’s exports to the EU-28 especially in textile,
wearing apparel and leather sectors.
Everything But Arms (EBA) is a status that allows almost everything to be exported
to the EU by the beneficiary country. It is considered as a most generous scheme for
developing countries like Pakistan, but Pakistan has not been granted this status yet. It is
expected that the EBA status may bring more benefits for Pakistan than any other status.
The European Union is the largest export destination for Pakistan. Recently, the EU
granted GSP plus status to Pakistan. Our main research problem is to investigate the
likely impact of GSP Plus on Pakistan’s exports to the European Union and hence its
resulting positive effects on economic development. The impact can be underestimated
or overestimated if one tries to capture the impact of the FTA like GSP plus by applying
partial equilibrium analysis. As all the sectors of the economy are interlinked, so any shock
in one sector leads to changes in other sectors. So, the main objective of the study is to
find out the impact of GSP plus status of Pakistan in the EU - on Economic growth of
the country. To capture the impact of these measures, Computable General Equilibrium
models are an ideal tool.
The main objective of the study is to calculate the potential impact of GSP plus on the
household income and real wages by applying MyGTAP. The study also aimed to calculate
the potential economic gains from GSP plus and EBA in terms of GDP and merchandise
exports. The study wants to investigate the possible outcomes from a series of different
policy experiments and suggest some policy recommendations.
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Journal of Management Sciences
Is GSP Plus Different from Normal GSP?
The basic and foremost objective of the preferential system known as the Generalized
System of Preferences (GSP) is to help the economies to reduce the poverty, promote good
governance and sustainable economic growth. These preferences enable the economies to
increase their role in the international trade and especially the exports to the EU that
ultimately help them to reduce the poverty and maintain a sustainable development. The
GSP of EU covers the following regimes (European, 2013).
• The standard/normal GSP that covers more than 6300 tariff lines, transports preferences in 90 developing countries that have been reduced from 177 in 2013.
• GSP Plus that brings special arrangements to promote good governance and sustained development in addition to offering duty free access to more goods from the
vulnerable economies including Pakistan. The list includes 25 countries, adding 9
more to the previous 16. The beneficiary economies have to implement and ratify
certain international conventions.
• The most attractive arrangements for the 50 Least Developed Economies (LDCs)
called Everything But Arms (EBA), provides duty free and quota free access to
nearly all commodities.
In addition to the preferential agreements, EU has established trade relations on the basis of Most Favored Nation (MFN) treatment, allowing all industrialized countries outside
the EU to trade with.
Identification of ‘high potential’ products of Pakistan in EU after the GSP plus status
is first step that should be followed by the identification of potential competitors with same
status or even better. The EU has a range of agreements with different countries, including GSP, GSP plus, Every Thing, but Arms (EBA), Overseas Countries and Territories
(OCT), Economic Partnerships Agreement (EPA) and some more. While considering the
competition among developing economies, EBA is considered to be more attractive than
the GSP plus status (Carbone & Orbie, 2016).
Although, achieving the status of zero tariff on export of all products is a huge opportunity, but the exports from Pakistan may not observe abrupt jump. It is because
Pakistan will continue to face a tough competition from countries enjoying the same or
better treatment in the EU market. The countries with GSP plus status will face an annual
capping mechanism while others with EBA status like Bangladesh will enjoy the tariff free
access throughout the year. In addition to such status in the EU market, the commodity
price, production capabilities and demand for the products will also play key role in such
a competitive environment. Table 1 below summarizes the position of Pakistan and its
competitors with similar status in the EU market.
Literature Review
Many authors have observed the literature on CGE models applied to less developed and
developing countries.
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Journal of Management Sciences
Naqvi (1998) developed a CGE model and used the SAM 1983-84 aiming to analyse the
economy wide impact of energy policy. The results found by the simulation show that a
change in energy tax has varied influences on different commodities, i.e. If distortions are
removed from taxes on petroleum products, it fulfills the objective of social equity while
Table 1
Comparison of Imports by the EU (28) with GSP Plus Beneficiaries (US $ Million)
S.No
1
2
3
4
5
6
7
8
9
10
Source:
Exporters
Peru
Pakistan
Paraguay
Costa Rica
Ecuador
Georgia
Armenia
Bolivia
Mongolia
Cabo Verde
International
2008
2009
2010
5757.8 4606.1 6964.3
6068.4 5295.4 5918.3
732.3
509.9
1313.1
5446.4 4516.8 5310.1
3569.8 3078.3 3047.4
908
604.6
886.9
478.1
241.4
323.2
474.4
420.6
569.5
95.4
75.7
135.9
40.4
39.1
50.6
Trade Centre (ICT)
2011
9044.2
7485.8
1657.4
6077.3
3712.9
923.8
443.1
592.2
114.9
63.9
2012
8245.5
6093.2
1225.1
6790.6
3601
762.9
330.8
603.7
93.3
68.5
2013
7319.7
6868.2
1574.6
6394.6
3897.1
916.3
329.4
761.4
105.6
64.4
2014
6829.2
7220.7
1527.6
6119.1
4003.4
907.5
335.9
879.9
115.8
78.5
removal of distortion in taxes on electricity has no impact on the consumption of rural
household while it showed a negative impact on urban household. On the other hand,
applying a tax on natural gas brought a negative impact on the real consumption of the
household. It was further discovered that removing distortions not only increase the real
GDP, but also bring a positive change in the trade balance. The model was simply a static
model aiming comparative analysis and had nothing to do with the forecasting.
Ghosh (2004) developed a CGE model for Pakistan by using GTAP 4 to investigate
the impact of roads and transport infrastructure on the economy. The study proposed a
multiregional CGE model in order to check the impact of a new road network between
Karachi and Peshawar. The simulation results discovered that new road will bring positive
change in the industrial sector of Punjab and NWFP (North West Frontier Province, Now
KPK (Khyber Pakhtunkhwa) provinces of Pakistan. The road network will increase the
real income of households that ultimately will increase the utility level in both provinces.
The study further concluded that this network will bring positive change of 16% to the
GDP of the economy.
Butt (2006) utilized the CGE model for Pakistan to calculate the impact of tariff cuts
on the regional disparities, output, employment and exports by keeping in view different
regions of the country. The study developed a PAKREG database by utilizing the I-O
table of 1990-91 developed by the Federal Bureau of Statistics (FBS 2001). The study in
this way helped the GTAP to recognize Pakistan as a separate country. The results of
the study revealed a positive impact of trade liberalization on all regions of Pakistan in
terms of improvement in output, exports and employment. The results further discovered
a positive relationship between trade liberalization and regional disparities during the
military regimes and opposite in the case of democratic governments. The cross border
tariff cuts seemed to help the increment in real GDP slightly in the short run but significant
in the long run.
Ahmed and O’Donoghue (2008) described the welfare effect of the external balances on
Pakistan economy. The study used a CGE model to capture economy wide impact of policy
30
Journal of Management Sciences
simulation. Social accounting was used as a database and GAMS software was used to run
the model. The study encompassed12 agriculture sector 16 industrial sector and 6 services
sector. Households have been distributed in rural and urban. The rural households have
been further distributed into 17 categories. The experiment was performed through trade
liberalization simulations. The simulations were concerned with a 50 percent increase in
foreign savings, a 10 percent increase in overall import prices and 10 percent increase in
the import prices of petroleum etc. The results of the study suggested that the external oil
prices possessed the high potential to affect Pakistan socio economic conditions. Increase
in foreign saving decrease poverty in the country. The analysis suggested that poverty is
increasing with the increase in import prices.
Shaikh and Rahpoto (2009) has studied the SAFTA implication on Pakistan economy
using the GTAP model. This study used 10 regions and 10 commodities. The experiments
were based on the unilateral trade liberalization (uniform tariff rate 15 percent), regional
trade liberalization, and unilateral trade liberalization (15 percent) for the rest of the
world. The study used GTAP model to investigate the benefits and costs of granting
MFN (Most Favored Nation) status to India and SAFTA. The results highlighted the
potential industries which need to be expanded or contracted. Pakistan gained highest
welfare in case of SAFTA with 15percent uniform external tariff. There is high demand in
the international trade for Pakistani dates, leather and garments etc. The study identified
a variety of industries in which a high potential existed. The SAFTA role is important
by giving opportunity for member countries to achieve economies of scale, diversify their
exports net and improve competitiveness. The study further explored that if SAFTA is
fully integrated and Pakistan gets a tariff cut of 15%, it would bring highest welfare gain
for the people.
Hussain (2010) applied CGE model using SAM 2002 to investigate the fiscal strictness
and the trade liberalization impact on household welfare and inequality. The study explored
that there are two principal effects of export taxes and tariffs. Firstly, they reduce the
trade volumes on both the import and export sides. Secondly, they impose economic costs
by inducing resource mis-allocation. Therefore, if trade related taxes are eliminated, an
economy can avoid production and consumption distortions. It is an established fact that
free trade leads to enhanced efficiency The case of efficiency for free trade is the converse
process to the tariff’s cost benefit analysis. The study further discovered that for a small
country like Pakistan, imposing a tariff does not allow it to influence world prices. However,
prices for domestic consumers and domestic producers do arise as a result. Consequently,
imports and consumption are reduced and the production of import substitute increased.
Dorosh and Rashid (2013) utilized CGE models to measure the latest terms of trade for
the agriculture sector, comparing it to the industrial sector in Pakistan during the years of
2000-2010 and to study the impact of agriculture income tax on Pakistan economy by using
a social accounting matrix (SAM 2002). The result of the experiment showed a 5% and
10% increase in the government revenue through the imposition of agriculture tax. The
study further elaborated that manufacturing and imports flourished while construction and
exports faced decline. The labor demand in the non - agriculture sector raised, whereas the
demand for labor in agriculture sector reduced due to increase in the agriculture income
tax.
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Journal of Management Sciences
Siddig, Aguiar, Grethe, Minor, and Walmsley (2014) studied the Nigerian socioeconomic policy impacts of reducing fuel subsidies while employing countrywide framework
using the newly developed MyGTAP model. This model incorporated country-specificinformation covering 12 groups of households to examine domestic policies. The study
encompassed 13 regions and 21 commodities. My GTAP incorporated government budget
in two folds; income and expenditure. It is considered that lowering the fuel prices accompanied by a low level of living cost can be the outcome of subsidies incorporated in the
imported goods of the petroleum sector. The study supported a reduction in fuel subsidy
policy because it has non-decreasing effects on the household income-especially the poor
one-hence leading to alleviate poverty in Nigeria.
Kuiper and Shutes (2014) studied the impact of food and nutrition security on multiple
households in Ghana by employing MyGTAP database of Minor and Walmsley (2013).
The study involved a multiple household to study the effects of food policy on the most
vulnerable sector of the society, helping the government to design intervention in order to
provide relief to the poor segment of the society. The study embedded 19 commodities
and 9 households. In the study, the following three approaches were used to incorporate
multiple household data in GTAP data base. Firstly, user weights has been assigned to
household and incorporated in GTAP. Secondly, the study included household data through
national SAM. Thirdly, they directly place household survey in GTAP analysis. The result
suggested that the removal of export subsidy is useful to the poor people of Ghana.
Khan, Saboor, Mohsin, et al. (2015) developed a CGE model to investigate the impact of
agriculture trade liberalization (the elimination of import tariff and the removal of export
subsidies) on income inequality of Pakistan. The study adopted the newly developed
MyGTAP model developed by Minor and Walmsley (2013). The model used a two kind
of data base i.e. GTAP and SAM (2007-08). This study deeply analyzed the impact
of agriculture, trade liberalization on multiple households. The study encompassed 18
households, 12 regions and 37 sectors. The results of agricultural trade liberalization
suggested that income inequality in Pakistan is increased by 0.49% from the baseline.
Medium and large household types are aided, and there is a nominal increase in the real
wages of medium and large agricultural labors. The labor intensive crops are replaced
by capital intensive and cheap imported products that ultimately helped to enhance the
income inequality in Pakistan.
Rationale for a New CGE Study on Pakistan
The majority of the CGE studies conducted in Pakistan includes the following issues,
changes in tax, food policies, government expenditures and investments, policies related
energy crisis, performance of public sector organizations, trade liberalization, Dutch disease
effect, unequal distribution of wealth, and poverty conditions in Pakistan.
All of the previous studies conducted in Pakistan using CGE have some common characteristics that include some behavior related phenomenon like producer behavior, consumer
behavior and international trade are some common features used by many researchers. Secondly, most of these researchers used Neo classical comparative static CGE model in their
studies. These are single country models except (Butt, 2006) who developed PAKREG a
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Journal of Management Sciences
regional CGE model in the study to discuss the tariff cuts, exports and regional disparities,
which aimed to help Global Trade Analysis Project (GTAP) for the trade issues relating
to Pakistan.
Methodology
MyGTAP Model
The CGE model in its global version is supported by the Global Trade Analysis Project
(GTAP) model as it provides the modeling framework as well as the database to the CGE
model. That is; the main source of data for the global CGE model is the GTAP database.
The model of GTAP is the most commonly used and known software for the multi country
trade analysis. It is a multi-region, multi-country and multi- sector CGE model which
assumes perfectly competitive markets and return to scale (Burfisher, 2011). GTAP 09
with reference years 2004, 2007 and 2011 to 140 regions, 57 sectors and 244 countries has
been used to link the Pakistan economy with the rest of the world in general and the
European Union (EU28) in particular.
The study has linked the latest available comprehensive Social Accounting Matrix
(SAM) 2007-08 developed by International Food Policy Research Institute (IFPRI) to
a latest extension of the standard GTAP to make it MyGTAP. The MyGTAP developed
by Minor and Walmsley (2013) is briefly explained here. MyGTAP is developed on the
basis of the standard GTAP model which is commonly used for trade analysis focusing the
tariffs (Hertel & Hertel, 1997). A regional household is represented in the standard GTAP
which combines the income coming from factors of production (land, labor and capital)
and taxes and then distributes it among the private household, savings and government
expenditures on the basis of their shares. The standard GTAP provides the details of the
structure of the expenditures in the economy. It combines the income from all sources and
then determine the expenditures, but it does not require the information that which agent
receives the income, taxes or transfers.
The study under consideration not only requires the income to be split into private
household and government, but also split the household into further categories (poor and
non-poor household). The newly developed MyGTAP explains that government collects
revenue from taxes and foreign aid to spend on government expenditures, funding foreign
governments, providing subsidies and transferring to households. Budget deficit or surplus
is decided on the basis of difference between government spending and income. Income
sources for private households on the other hand, are a function of returns to factor endowments (land, labor and capital), net rent on foreign capital and foreign remittances,
transfer payments made by the government and other households. The household net income is either spent or save. The impact of gain in trade i.e GSP plus status of Pakistan
in the EU28 will be noted upon the change in income of different households, but not on
the basis of change in consumption of the household.
33
Figure 1
Flows of Income and Expenditures in MyGTAP Model
Journal of Management Sciences
34
Journal of Management Sciences
Relationships in MyGTAP Model
The study has already explained the source of data base in MyGTAP. As discussed earlier,
the standard GTAP is modified to make it MyGTAP where the single regional household
is replaced with the multiple private households along with a separate government sector
for one region (Walmsley, Minor, et al., 2013). Just like in case of standard GTAP, the
private households receive income from factors, but in MyGTAP, it also incorporates foreign
remittances and capital, which is further used for consumption and saving purpose. The
new model assumes that the government sector gains income from taxes and foreign aid
in. Government income is consumed on the transfers to the private household and foreign
aid out. Moreover, government entertains the receiving of foreign aid instead of the direct
acceptance by the private households (Minor & Walmsley, 2013).
MyGTAP Model Closure
Model closures are the starting point of this model that assumes perfect competition in all
sectors of the economy. Capital and labor as factors of production are considered to be
fully mobile among different sectors of the economy and land along with natural resources
is immobile. The economy of Pakistan faces the problem of high unemployment rate, so we
assumed that unskilled labor (LASKU) are unemployed. Similarly, it is assumed that the
factors prices influence the foreign income flows in the respective country. Trade balance is
endogenous and expected rate of return determines the investment as in case of standard
GTAP model and total domestic savings by the government budget deficit and sum of the
private household savings.
Any country that gets GSP Plus status in the EU face the annual growth capping
mechanism for products with higher growth rates. The study incorporated the capping
mechanism with quota restrictions.
Data Base and Analysis
Social Accounting Matrix (SAM) for MyGTAP
Pakistan Institute of Development Economics (PIDE) developed the first Social Accounting
Matrix (SAM) for Pakistan in 1985 with base year 1979. Federal bureau of Statistics (FBS)
under Project Improvement of National Accounting System (INAS) with collaboration
with the Netherland government developed the second SAM that was limited to only
single household. Siddiqui and Iqbal (1999) constructed a new SAM for Pakistan with
base year 1989-90 and aggregated the industrial classification of Input-Output (IO) table
into SAM with five production accounts. Dorosh, Niazi, and Nazli (2004) built a broad
SAM with 34 production accounts and 19 household groups with base year 2001-02. These
household groups were disaggregated across provincial basis, hence most suited for policy
analysis that target particular households. Khan et al. (2015) produced a financial SAM
for the year 1999-2000. They disaggregated the workings of the loanable funds market into
disaggregated payments related to physical and financial flows among institutions.
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Journal of Management Sciences
The latest available SAM that depends heavily on concomitant National Accounts
and house-hold data was developed by Debowicz, Dorosh, Haider, and Robinson (2007)
with base year 2007-08, under the Pakistan Strategy Support Program (PSSP) funded by
USAID aiming to support the Government of Pakistan with evidence-based policy reform
for pro-poor economic growth and enhanced food security.
To implement a CGE model with an income distribution component, a consistent data
base is required. MyGTAP in the study pursues the SAM (2007-08) desegregation of activities, commodities, factors and institutions. The model follows the framework developed
by Lofgren et al. (2001). This model is a standard static model rather than dynamic CGE
model. Therefore the second period effects of changes in investment expenditures are not
taken into account. Moreover, the model neither specific about the time horizon of the adjustment nor how the adjustment is sequenced. Otherwise stated, the model cannot resolve
whether adjustment from the base to a new equilibrium takes place over any particular
length of time, or whether a large part of the adjustment occurs in a particular year.
SAM provides actual values for the coefficients in these equations through the calibration process. The model will be solved primarily for equilibrium to make sure that the
base year dataset is reproduced. Afterward, it would be possible to shock the model with
a change in the value of one of the exogenous variables. The model will be resolved for
equilibrium and the changes in the values of the endogenous variables. Moreover these
values will be compared to those of the base-year equilibrium to establish the impact of
the exogenous shock.
To simplify the things, the study aggregated the regions and sectors. The details are
presented in appendix.
Simulations
The study used MyGTAP in order to calculate the impact of two simulations on the real
wage rate and household primarily. The base year used in MyGTAP is 2007, as the latest
available SAM for Pakistan is of year 2007-08. In addition to calculating the impact of
simulations on household income and real wage rate, the study have also discussed some
other areas of the economy, taking into account the overall and regional characteristics of
the production sectors and factors allocation.
Although the evidence is available that trade liberalization is good for the growth,
but how it impact on household income and real wage rate is less clear. Pakistan is a
developing country and is more concerned about the effects of EU’s GSP plus and EBA
status on household income in Pakistan. There are many studies that identified the positive
and negative impact of trade liberalization (Hertel & Hertel, 1997; Winters, McCulloch,
& McKay, 2004; Vanzetti, Huong, et al., 2006). Calculating the impact of GSP plus and
EBA status on poor household and different types of labor is important to formulate the
policy and planning for sustainable growth.
The study have only used the two following simulations for this purpose.
Simulation I: EU-28 GSP Plus status with quota restriction: What would happen
if quota restriction is applied on Pakistan to incorporate the capping mechanism of the
EU-28?
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Journal of Management Sciences
Simulation II: Potential EU28-EBA with Competitors: What would happen if Pakistan gets the Everything But Arms (EBA) status in the EU-28 with no Capping mechanism/Quota restriction, with main competitor Bangladesh that already enjoying the EBA
status?
The results of these simulations are presented below.
Results of the Simulations
GDP and Production
The results of the both simulations are presented in Table 2 which show positive change in
real GDP of Pakistan. In case of first simulation, the GDP of Pakistan increases by US$
21.594 million which is 0.015 percent positive change from the base line value. The results
of second simulation also very encouraging with positive change of US$ 884.047 million in
GDP.
Table 2
GDP Quantity Index EU, Constant 2007 Prices (Percent and Millions US$)
Simulations
GSP Plus status with Quota Restrictions
EBA Status
Base Value (Millions US$)
Post Shock Effects
Change in GDP
Percentage Change
143169.594
143169.594
143191.188
144053.641
21.594
884.047
0.015
0.617
Similarly, Table 3 presents the changes in real output of different sectors of Pakistan
after both simulations. The results of both simulations reveal mixed effects on the real
output of commodities. The results of the first simulation show that there are 13 sectors
out of 38 where output level is increased with maximum increase in services sector (US$
0.383million). The other major winning sectors are construction US$ 0.356 million, sugar
US$ 0.199 million, vegetable, fruit and nuts US$ 0.196 million, livestock and meat products
US$ 0.125 million. While there is decrease in real output in rest of the sectors. The
prominent sectors with decrease in output include machinery and equipment US$ -2.117
million, metals and products US$ -1.875 million, leather products US$ -1.777 million and
oil seeds with US$ -1.722 million.
The results of the second simulation show increase in real output in 25 sectors of
Pakistan economy. The results reveal that paddy rice show maximum gain with US$
2.080 million followed by construction sector (US$ 1.14 million), services sector (US$ 1.26
million), mineral products nec (US$ 0.96 million) and processed food (US$ 0.93 million).
While there is deterioration in the 11 sectors. The prominent sectors with decrease in
output include leather products with US$ -1.99 million, plant based fibers with US$ -1.60
million and textiles with US$ -1.99 million.
The results of second simulation are more encouraging than Simulation 1 which means
that if Pakistan is allowed to export in EU28 without any restriction, the real output level
will increase in most of the sectors of the economy.
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Exports and Imports
The duty free and quota free entry of Pakistan into the EU28 is expected to bring positive
effects on the exports of Pakistan. Similarly, the flow of imports will also increase due to
increased demand of foreign inputs and resultant higher prices of many goods. Figure 2
explains the results of the simulations. The results of both simulations show an increase in
imports and reduction in exports resulting disturbance in the trade balance. The exports
of Pakistan to EU28 reduced by -1.79 percent in case of first simulation while in case of
second simulation, the reduction is -1.282 percent. This reduction is export is due to
Table 3
GDP Quantity Index EU, Constant 2007 Prices (Percent and Millions US$)
Commodity
Base Value (Millions US$)
pdr
wht
gro
vf
osd
Sugar
pfb
ctl
Animalprod
frs
fsh
minerals
oil
Meatfood
vol
mil
sgr
bt
tex
wap
lea
Wood
pc
crp
nmm
Metals
Autoparts
ele
ome
omf
utilities
cns
trd
Transport
cmn
All services
1489.76
2616.37
181.39
7201.54
365.22
5895.7
2953.45
4271.85
20862.25
291.25
879.47
630.01
1468
5029.13
3506.79
4373.73
5333.85
3618.37
23984.3
4404.37
1203
2418.66
8120.48
4289.23
5498.41
2107.05
2733.24
2672.3
420.68
1111.31
22303.91
16914.6
21665.48
18768.37
3009.44
55321.8
GSP Plus with EU Capping (Quota)
Changes in Change in
Value
Percent
-0.205
-0.01
-0.102
0
-0.02
-0.01
0.196
0
-1.722
-0.47
0.087
0
-0.796
-0.03
0.016
0
0.125
0
0.027
0.01
0.076
0.01
-0.396
-0.06
-0.318
-0.02
-0.357
-0.01
-0.528
-0.02
0.073
0
0.199
0
0.117
0
-1.208
-0.01
-1.296
-0.03
-1.777
-0.15
-0.628
-0.03
-0.24
0
-1.07
-0.02
0.119
0
-1.875
-0.09
-0.569
-0.02
-0.795
-0.03
-2.117
-0.5
-1.151
-0.1
-0.216
0
0.356
0
-0.133
0
-0.036
0
0.058
0
0.383
0
Potential EBA Status
Changes in Change in
Value
Percent
2.08
0.14
-0.12
0
0.46
0.26
0.17
0
-0.76
-0.21
-0.11
0
-1.6
-0.05
0.23
0.01
0.25
0
-0.07
-0.02
0.27
0.03
0.2
0.03
-0.23
-0.02
0.93
0.02
-0.21
-0.01
0.62
0.01
0.65
0.01
0.67
0.02
-1.11
0
-0.91
-0.02
-1.99
-0.17
0.28
0.01
0.53
0.01
0.22
0.01
0.96
0.02
0.19
0.01
0.55
0.02
0.58
0.02
-0.94
-0.22
0.28
0.03
0.66
0
1.14
0.01
0.39
0
0.64
0
0.76
0.03
1.26
0
production capacity of Pakistan in 2007 which was adversely affected by load shedding.
The adverse effects of energy crises increased the production cost in Pakistan resulting into
decline in exports. Due to this reason, the results of the simulations produced negative
impacts.
The results of both simulations show a positive increase in the imports of Pakistan.
The increase is 0.558 percent in case of first simulation, while in case of second simulation,
the increase is 1.153 percent. This increase in imports is also a result of increased cost of
production in Pakistan. Similarly, in order to increase the production, Pakistan would also
38
Journal of Management Sciences
require more inputs to import.
Figure 2 shows that the trade balance is highly deteriorated in case of first simulation
while in case of second simulation, increase in imports is slightly less than the decrease
in exports. This is because the quota restriction will restrict exports to EU28, leaving
more products available for the domestic consumer to consume. Hence imports increased
at lower rate. While in case of EBA status, there is no restriction to export resulting
into reduced availability of domestic products. The reduction in availability and increased
demand of imported inputs, increase the overall imports of the country.
Figure 2
Merchandise Exports and Imports of Pakistan (Percent)
Impact on Pakistan’s Terms of Trade
Terms of trade is define as the ratio of prices that a country receives and pays in exchange
of its exports and imports. It is considered important to understand the impact of change
in price on the welfare of public generally. Current study investigated the impact of two
different simulations on the change in price of imports and exports. Pakistan has already
achieved the status of GSP plus in EU28, the restriction free exports from Pakistan in case
of GSP plus and EBA, may increase the export price of Pakistani products. Similarly,
applying quota restriction may increase the price level at lower rate.
Figure 2 explains the effects of different simulations performed on the Pakistan’s termsof-trade. The results of both simulations are positive but very different. In case of first
simulation the export prices that Pakistan receives from EU28 are 0.019 percent higher
than the import prices that Pakistan pays to the EU28.
Highest gain is seen in the results of simulation 2, assuming that if Pakistan gets the
status of EBA in EU28 just like Bangladesh. Due to this status, the exports from Pakistan
may increase rapidly resulting an increase in export prices. Hence, the results of this
simulation show that Pakistan is receiving 1.834 percent higher export price than it is
paying for its imports from EU28.
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Journal of Management Sciences
Changes in Household Income
The study has discussed the impact of two simulations by using MyGTAP focusing on the
issues of trade, GDP, output and prices. A unique feature of the MyGTAP model used
in this study is the capability to disaggregate the regional household into both private
and government entities (Minor & Walmsley, 2013). The study disaggregated the regional
household of standard GTAP model into 18 types to conduct a detailed analysis. The simulations used in the study will calculate the effects on household income distribution and
expenditures. The data and weights required were obtained from the latest comprehensive
Pakistani Social Accounting Matrix (SAM) 2007-08.
Figure 3
Changes in Term of Trade (TOT) of Pakistan, Constant 2007 Prices, (Percent)
Changes in Household Income
The study has discussed the impact of two simulations by using MyGTAP focusing on the
issues of trade, GDP, output and prices. A unique feature of the MyGTAP model used
in this study is the capability to disaggregate the regional household into both private
and government entities (Minor & Walmsley, 2013). The study disaggregated the regional
household of standard GTAP model into 18 types to conduct a detailed analysis. The
simulations used in the study will calculate the effects on household income distribution and
expenditures. The data and weights required were obtained from the latest comprehensive
Pakistani Social Accounting Matrix (SAM) 2007-08.
While conducting the welfare analysis, the studies that employ CGE models, normally
show all household are equally affected due to any change in the trade policy. In case of
MyGTAP, the households are distributed into categories aiming to calculate the impact
on the marginalized population before designing a trade policy. Any change in wage rate
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Journal of Management Sciences
is considered a change in the household income. The household income consist of income
coming from different factors, so any change in income of factors means a change in income
of household.
The results of both simulations are summarized in Figure 4 which show a positive change
in overall household income. There is a change of 0.74 percent in case of first simulation
but in case of second the change is 2.17 percent which means that if Pakistan is allowed
to export duty free and quota free into the EU28, the household income in Pakistan will rise.
Figure 4
Changes in Households Income in Pakistan, Constant 2007 Prices (Percent)
The results of both simulations are summarized in Figure 4 which show a positive
change in overall household income. There is a change of 0.74 percent in case of first
simulation but in case of second the change is 2.17 percent which means that if Pakistan is
allowed to export duty free and quota free into the EU28, the household income in Pakistan
will rise.
The results below show change in all 18 categories of households. The regional household is divided into three categories, household in Punjab, household in Sindh and household in rest of the Pakistan. The results further reveals that that every household is not
equally affected. There are some household better off and vice versa.
Household Income of Large and Medium Farm
Table 4 represents the results of both simulations in order to check their impact on the
income of large and medium household of Pakistan. The results reveal that in case of
first simulation, the household other than Sindh and Punjab have positive change of 0.029
percent in its income while the income of household in Sindh is reduced by -0.478 percent
and in Punjab by -0.239 percent. On the other hand, the income of all household increases
with maximum 2.347 percent increase in household of rest of the Pakistan (excluding Sindh
and Punjab provinces).
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Table 4
Changes in Household Income of Large and Medium Farm, Constant 2007 Prices (Percent)
Household Types
Large and medium farm Sindh
Large and medium farm Punjab
Large and medium farm other
HH Code
Population (millions)
Income shares (percent)
GSP Plus with Quota
Potential EBA Status
H-MF1
H-MF2
H-MF3
0.8
2.4
0.6
1.5
6.1
0.8
-0.478
-0.239
0.029
1.831
1.613
2.347
Income of Small Farm Household
The results of the both simulations are presented in Table 5 which show the impact on the
small household living in Pakistan. The results reveal that small farmer living anywhere
in Pakistan is befitted in both cases. The results further reveal that the farmer living in
parts other than Sindh and Punjab is benefitting maximum in case of both simulations
while the small farm household in Sindh is getting minimum benefits in both cases. The
maximum benefit that small farm household of other Pakistan is getting is 2.253 percent
which is in case of Simulation 2 (if Pakistan gets the EBA status in EU28) and small farm
household of Sindh is getting minimum benefit of 0.331 percent in case of first simulation
(applying quota restrictions to justify the capping mechanism of EU28 after GSP plus
status of Pakistan).
Table 5
Changes in Household Income of Small Farmers, Constant 2007 Prices (Percent)
Household Types
HH Code
Population (millions)
Income shares (percent)
GSP Plus with Quota
Potential EBA Status
H-SF1
H-SF2
H-SF3
3.1
16
5.6
1.8
11.5
3.3
0.331
0.487
0.885
2.146
2.179
2.253
Small farm Sindh
Small farm Punjab
Small farm other Pakistan
Income of Landless Farmer Household
In this section, we will discuss the rural household that is farmer but do not own a piece
of agriculture land in any area of Pakistan. The results of both simulations are presented
in Table 6 that show a positive change in income of all rural households that are landless
but are farmers. In case of first simulation landless farmer of Sindh is gaining minimum
(0.405 percent) while the landless farmer of rest of the Pakistan is gaining maximum (0.954
percent). The results of second simulation show that landless farmer of Punjab is gaining
maximum (2.452 percent) while the landless farmer of Sindh is gaining minimum (2.08
percent).
Table 6
Changes in Household Income of Landless Farmers, Constant 2007 Prices (Percent)
Household Types
Landless farmers Sindh
Landless farmers Punjab
Landless farmers other Pakistan
HH Code
Population (millions)
Income shares (percent)
GSP Plus with Quota
Potential EBA Status
H-OF1
H-OF2
H-OF3
2.5
3.6
1.7
1.4
1.8
0.7
0.405
0.68
0.954
2.08
2.452
2.323
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Income of Landless Labor
The household living in rural areas of Pakistan working in agriculture farms as a laborer
and having no land is included in landless labor. The results showing the impact of both
simulations on the household income of landless agriculture labor of Pakistan are presented
in Table 7. The results show maximum gain for the landless labor of whole Pakistan in both
simulations as compared to any other household. In case of first simulation maximum gain
is shown in for the landless agriculture labor of Sindh (1.563 percent) while the landless
agriculture labor of Punjab is getting minimum gain (1.432 percent). In case of second
simulation, the results are quite similar with maximum gain again in case of landless
agriculture labor of Sindh (4.136 percent) while the landless agriculture labor of Punjab is
getting minimum gain (3.512 percent).
Table 7
Changes in Household Income of Rural Agricultural Labor, Constant 2007 Prices (Percent)
Household Types
HH Code Population (millions) Income shares (percent) GSP Plus with Quota Potential EBA Status
Landless agri. Lab Sindh
Landless agri. Lab Punjab
Landless agri. Lab other Pakistan
H-AGW1
H-AGW2
H-AGW3
3
3.3
0.4
1.5
1.4
0.2
1.563
1.432
1.498
4.136
3.512
3.944
Income of Rural Non-farm Household
In rural areas of Pakistan, there are households that have no direct connection with agriculture farming. The results shown in Table 8 reveal the impact of both simulations on
the income of non-farm rural households. The rural non-farm household of Sindh gets
minimum gain (0.994 percent) in case of first simulation while rest of the rural non-farm
households in Pakistan gain (0.997 percent). Similarly, in case of second simulation, rural
non-farm households of Sindh get maximum gain (1.569 percent) and minimum gain (1.356
percent) in case of rural non-farm households of rest of the Pakistan.
Table 8
Changes in Household Income of Rural Non-farm Household, Constant 2007 Prices (Percent)
Household Types
HH Code Population (millions) Income shares (percent) GSP Plus with Quota Potential EBA Status
Rural non-farm quintile 1
Rural non-form quintile 2
Rural non-farm quintile other
H-NFQ1
H-NFQ2
H-NFOTH
8.2
8.9
27.7
2.8
3.3
17.3
0.994
0.997
0.997
1.569
1.539
1.356
Income of Urban Household
Table 9 show the impact of both simulations on the urban household of Pakistan. The urban
household of Sindh is showing maximum gain in both simulations (0.935 percent and 1.342
percent respectively) while minimum gain is seen in the income of urban household of rest
of the Pakistan in case of both simulations (0.86 percent and 1.162 percent respectively).
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Table 9
Changes in Household income of Urban Household, Constant 2007 Prices (Percent)
Household Types
Urban quintile 1
Urban quintile 2
Urban other
HH Code
Population (millions)
Income shares (percent)
GSP Plus with Quota
Potential EBA Status
H-UQ1
H-UQ2
H-UOTH
8.6
8.6
25.7
2.6
3.4
38.7
0.935
0.927
0.86
1.342
1.265
1.162
Overall, factor income remains positive for almost all households. If there is no tariff
and quota restriction from EU28, the income of every household type will increase but
that increase. Primarily, it is because, the increased exports of Pakistan will definitely
increase the economic activities in the economy and the backward and forward linkages of
the industry will bring positive change in the income of every household.
Effects on Real Returns to Factors
Increased trade and especially exports increase the rate of return to factors. The HeckscherOhlin model is although workable only in the economies where amount of goods and the
number of production factors is equal. The products are assumed homogenous in the model,
despite many markets in the world are represented in a better way with differentiated
products (Suranovic, 2010). In case of Pakistan, the model suggests that there will be
decreases in the return to capital as country is already capital deficient as compare to its
competitors (Khan et al., 2015).
Labor force of Pakistan is more than 65 million. Unemployment rate in Pakistan is
about 6 percent (Government of Pakistan, 2015). Despite tremendous Government efforts
for ensuring minimum wages in Pakistan like “Minimum Wages Ordinance 1961, the Punjab
Minimum wages for unskilled Workers Ordinance 1969, Minimum Wages Board,” etc ensure
that Government is dedicated to support low income groups.
The model used for the calculation of effects on real returns to factors is the extension
of standard GTAP model and uses the Armington assumption that categories the products
on the basis of country of origin. Furthermore, assimilation could disturb the rate of return
on capital by virtue of the prices of transitional and capital goods.
The results of both simulations show the change in factor prices with respect to the
price index for private consumption expenditure. However, it fails to consider the impact
of changes in government’s revenue, and government’s capacity to redistribute tax income
to individuals, whether it is through transfer payments or provision of public goods (Khan
et al., 2015).
Wages of Large Agriculture Land Owned Labor
The results of both simulations are presented in the Table 10 showing changes in the wages
of household that owns large agricultural land. The results reveal that in case of first
simulation, the wage of labor decrease by -1.45 percent while in case of second simulation,
it increased by 0.283 percent.
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Table 10
Changes in Household income of Urban Household, Constant 2007 Prices (Percent)
Factor
RF Code
Description
Labor
LA-AGL
Labor - agric (own)-large
GSP Plus with Quota
Potential EBA Status
-1.45
0.283
Wages of Medium Agriculture Land Owned Labor
The results in Table 11 show the changes in the wages of labor that own medium size piece
of agriculture land, after performing both simulations. The results show that in case of
first simulation, the wage of labor with medium sized agriculture land decreased by -1.335
percent in the region of Punjab. The results of second simulation show that wage of all
households with medium sized agriculture land increased with maximum increase in the
case of labor living in parts of Pakistan other than Punjab and Sindh and that is 1.487
percent.
Table 11
Change in Real Wages of Medium Agriculture Land Owned Labor (Percent)
Factor
RF Code
Description
Labor
LA-MF1
LA-MF2
LA-MF3
Labor - agric (own)-med Sindh
Labor - agric (own)-med Punjab
Labor - agric (own)-med OPak
GSP Plus with Quota
Potential EBA Status
-1.244
-1.335
-0.956
1.184
0.285
1.487
Wages of Small Agriculture Land Owned Labor
In case of farmers having small area of agriculture land in all areas of Pakistan, the results
seem quite similar to the case of farmers having medium sized agriculture land. The results
of both simulations are shown in Table 12. According to the results, the wages of the labor
with small size agriculture farm decreased everywhere in Pakistan when country faces quota
restrictions in the EU while a positive change can be seen in case of Simulation 2 i.e. if
Pakistan gets the status of EBA in the EU28.
Table 12
Change in Real Wages of Small Agriculture Land Owned Labor (Percent)
Factor
RF Code
Labor
LA-SF1
LA-SF2
LA-SF3
Description
GSP Plus with Quota
Potential EBA Status
-1.227
-1.017
-0.454
1.136
0.851
1.577
Labor - agric (own)-sm Sindh
Labor - agric (own)-sm Punjab
Labor - agric (own)-sm OPak
Wages of Skilled and Unskilled Labor
One additional simulation is added in the study by assuming unskilled labor is unemployed
in the model and then performing both simulations to check what would be the impact
on real wages of other types of labors. The impact of both simulations on the wage of
skilled and unskilled labor is shown in the Table 13. The results show that in case of first
simulation, the wage of agriculture labor in general is increased by 1.355 percent, in case of
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Journal of Management Sciences
unskilled non-agriculture labor, the wage increased by 0.286 percent and in case of skilled
non-agriculture labor, it is increased by 0.304 percent.
The results of the second simulation are quite different where the wage of non-agriculture
unskilled labor is decreased by -1.851 percent while a maximum increase is seen in the agriculture labor (5.366 percent)
Table 13
Change in Real Wages of Skilled and Unskilled Labor (Percent)
Factor
RF Code
Description
Labor
LA-AGW
LA-SKU
LA-SK
Labor - agric (wage)
Labor - non-ag (unsk)
Labor - non-ag (skilled)
GSP Plus with Quota
Potential EBA Status
1.355
0.286
0.304
5.366
-1.851
0.075
It is worthy to note that the supply of labor is fixed in the agriculture sector, so any
decrease in demand may decrease the wage of the agriculture labor. The results of the
simulations show that the increase in wage of skilled labor is greater than the increase
in wage of unskilled labor. Similarly, the supply of production labor is also fixed which
results into increase in wage rate as the production demand increases. The results of the
simulations show that increased exports will increase the wage rate which opposes the
theory that liberalized trade may reduce the wage rate. For further information, please see
(Stiglitz, 1970; Davis, 1996; Feenstra & Hanson, 1997; Topalova, 2005; Harrison, 2006).
Majority of the exports from Pakistan are textile and agricultural products so increased
volume of exports due to GSP plus and EBA status in the EU28, might shift labor from
the agriculture to industry. This is primarily due to the fact that land is sector specific
but labor is inter-sectorally mobile. So this offset effect might lead industrialization in
Pakistan.
Real Return to Land of Large Agriculture Farms
After labor, land is another factor of production, the rent paid to land is also affected by
certain changes in the trade. The source land is fixed, so any change in demand for the
production of goods requiring more land, may result into change in return to land. The
results shown is Table 14 reveal the effects of both simulations on the large land farms.
The results of first simulation show a negative change in the real return to land in case
of large farms everywhere in Pakistan but in case of Simulation 2, the large land farm
of Punjab is losing in return while a positive change is seen in the farms of Sindh (0.464
percent) and rest of the Pakistan (2.053 percent) which is maximum gain.
Table 14
Change in Real Return to Land of Large Farms (Percent)
Factor
RF Code
Description
Land
LN-LG1
LN-LG2
LN-LG3
Land - large- Sindh
Land - large- Punjab
Land - large - OthPak
GSP Plus with Quota
Potential EBA Status
-1.252
-1.639
-1.085
0.464
-0.175
2.053
46
Journal of Management Sciences
Real Return to Land of Medium Agriculture Farms
The case of change in real return to the land of medium farms is a little bit different than
the previous case. The results of both simulations are presented in Table 15 that show a
reduction in return to the land of medium farms in case of first simulation. The maximum
reduction in return is seen in the province of Punjab (-1.338 percent). While in case of
second simulation, the results are quite encouraging. There is gain in return to the land
of medium farms in all areas of Pakistan with maximum gain in return of 1.467 percent in
the region other than Punjab and Sindh.
Table 15
Change in Real Return to Land of Medium Farms (Percent)
Factor
RF Code
Description
Land
LN-MD1
LN-MD2
LN-MD3
Land - irrigated - med Sindh
Land - irrigated - med Punjab
Land - irrigated - med OthPak
GSP Plus with Quota
Potential EBA Status
-1.228
-1.338
-0.951
1
0.359
1.467
Real Return to the Land of Small Agriculture Farms
The results of both simulations relating to change in return to the land of small farms are
presented in Table 16 which are quite similar to the results of medium size farms. The
gain is negative in case of first simulation for all regions of Pakistan with maximum loss in
return in the province of Sindh (-1.215 percent). The results of the second simulation are
opposite to the first simulation that show a gain in return to the land of small farms in all
areas of Pakistan. The maximum gain is seen in the areas other than provinces of Punjab
and Sindh that is 1.613 percent.
Table 16
Change in Real Return to Land of Small Farms (Percent)
Factor
RF Code
Description
Land
LN-SM1
LN-SM2
LN-SM3
Land - irrigated - sm Sindh
Land - irrigated - sm Punjab
Land - irrigated - sm OthPak
GSP Plus with Quota
Potential EBA Status
-1.215
-1.022
-0.399
0.977
0.942
1.613
Real Return to the Land of Non-Irrigated Agriculture Farms
There are certain areas in Pakistan where traditional canal system is not activated to
irrigate the agriculture land. Those farms are called non-irrigated farms. The results of
both simulations are presented in Table 17 to show the return to the land of non-irrigated
farms. Interestingly the results are very similar to the results of previous two cases (land of
medium and small farms). In case of first simulation, there is negative gain in all areas of
Pakistan where non-irrigated farms exist with loss of -1.12 percent in the province of Sindh
while in rest of the Pakistan the non-irrigated land suffered from loss of -1.337 percent in
return. The case of second simulation shows gain in return of the non-irrigated land with
maximum gain in the Sindh province (0.821 percent) whereas in rest of the Pakistan, the
non-irrigated land gain 0.708 percent in return.
47
Journal of Management Sciences
Table 17
Change in Real Return to Land of Non-Irrigated Farms (Percent)
Factor
RF Code
Description
Land
LN-DR1
LN-DR2
LN-DR3
Land non-irrig - sm/m Sindh
Land non-irrig - sm/m Punjab
Land non-irrig - sm/m OthPak
GSP Plus with Quota
Potential EBA Status
-1.12
-1.337
-1.337
0.821
0.708
0.708
It is interesting to note that the results of first simulation suggest a negative gain in
return to land which also support the results of (Bradley & Norman, 1977) while working
on US economy. While these are contradictory with the findings of Hong (1990) which
suggested that protection increases the return as resources shift towards agricultural productivity. The gain is not possible without technonolgy whether economy is facing restrictions or not (Ghosh, 2004). The Stolper Samuelson Theorem that is based on the famous
Heckscher-Ohlin model also supports the results that liberalized trade lead to increase in
return to land (Leamer et al., 1995). Similarly, a large majority of researchers support
the results that return on land increases if there is no restrictions on the exports of an
economy, for example see (Runge & Halbach, 1990; Chang, 1979).
Real Return to the Capital
We have already discussed the impact of both simulations on the return to labor and
land. In this section, we will focus on the factor of capital. The study has divided the
capital into four categories and results along with categories are presented in Table 18.
The results of both simulations show gain in return to capital in most of the types except
capital other than agriculture (-1.106 percent in case of quota restriction on Pakistan) and
capital formation (-0.054 percent in case if Pakistan gets EBA status in EU28). In case of
both simulation, the maximum gain is seen in capital livestock, 1.818 percent when quota
is applied on exports from Pakistan to justify the capping mechanism of EU28 and 6.24
percent gain in return if Pakistan gets the status of EBA in EU28.
Table 17
Change in Real Return to Land of Non-Irrigated Farms (Percent)
Factor
Capital
RF Code
Description
K-LVST
K-AGR
KFORM
KINF
Capital
Capital
Capital
Capital
GSP Plus with Quota
Potential EBA Status
1.818
-1.106
0.105
0.28
6.24
0.798
0.105
-0.054
livestock
other agriculture
formal
informal
The results of both simulation suggest that there is gain in return to capital in most of
the cases which support the work of researchers like (Chang, 1979; Hong, 1990; Thompson,
2016). Walmsley et al. (2013); Khan et al. (2015) also produced the same results while
studying the Mozambique and Pakistan economy respectively. Although Ghosh (2004) also
support this but adds that gain in return to the capital can be maximized with continuous
improvement in the technology.
48
Journal of Management Sciences
Limitations of the Study
Parallel to many empirical studies, this study was constrained by a variety of factors which
could be considered as limitations. The first and the most important limitation is the
database, similar to most other studies which adopt CGE models. Current study used
GTAP v 9.0 and Social Accounting Matrix with base year 2007-08. This extension of
standard GTAP model is known as MyGTAP which provided the parameters related to
trade elasticity but these parameters are not estimated econometrically. It is noted that
household welfare results are sensitive to parameter values assumed in the model. Likewise,
a superior understanding of implications at household level could have been achieved if we
had been able to use more disaggregated data at household level.
Despite the above mentioned limitations, the global CGE model (standard GTAP)
generated reasonable good results at macro level for the country and the MyGTAP model
with the most latest constructed SAM (2007-08) for Pakistan. The results could be more
practical with current data.
Conclusion and Policy Recommendations
The study has attempted to calculate whether the GSP plus status of Pakistan in the
EU28 produce positive change not only at aggregate level but also at household income
and real wages. The study attempted to calculate the effects of different potential and
current opportunities for Pakistan by using standard MyGTAP. The MyGTAP employed
the latest available Social Accounting Matrix (SAM) with base year 2007-08.
The descriptive analysis of the results of different simulations MyGTAP reveal that
there is overall increase in the GDP of Pakistan. The incentive to export in the EU28 will
increase the production level in the Pakistan. Similarly, the improvement in production
also increase the real wages and household income. Similarly, EBA status of Pakistan in
the EU28 show an increase in the household income with maximum gain by the household
of rural Sindh with no agriculture land and a positive change in real wages of most of
the factors. Comparatively low improvement over urban and non-farm household of rural
areas. Therefore, empirical evidence in terms of the household welfare from this study
supports the overall view that Pakistan can gradually gain from GSP plus status.
The policy implications for the study are straightforward and related to trade policy
which is also the main concern of the research. The competition after the GSP plus
implementations have been improved, so the country needs to improve the competitiveness.
For this purpose, it should be acknowledged that it is firm that can bring success for the
country only if it works under right strategies and clear vision about how and why to
produce and for which target market it is producing. Therefore, the firms wishing to enter
into tough competition require very supportive role from the public authorities in the
Pakistan. The public authorities should ensure the provision of safe and healthy working
environment that may enable firms to compete at domestic and finally in the world market.
The production activities of the country are concentrated towards the sectors of textiles,
wearing apparel, beverages and tobacco and leather products that need to be diversified
with cost effective methods. Moreover, Pakistan can focus on those markets where its
49
Journal of Management Sciences
competitors have a small share.
50
Journal of Management Sciences
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W2756376449.txt | EPJ Web of Conferences 146, 02022 (2017)
DOI: 10.1051/epjconf/201714602022
ND2016
Evaluation of excitation function for 186 W(d,2n)186 Re reaction
Jimin Wanga , Xi Tao, Mengxiao Kang, and Xiaolong Huang, and Youxiang Zhuang
China Nuclear Data Center, China Institute of Atomic Energy, PO Box 275(41), 102413 Beijing, China
Abstract. The excitation function of the 186 W(d,2n)186 Re reaction was evaluated and the thick target yield
(TTY) of the therapeutic radioisotope 186 Re was calculated. The available experimental data from the EXFOR
library and literature were analyzed and corrected with the g-ray branching ratio, radioactive decay constant
and standard cross sections. The excitation function of the 186 W(d,2n)186 Re reaction was recommended below
50 MeV on basis of the least-squares fit with polynomial and the theoretical calculation with TALYS code.
The TTY was calculated using the recommended excitation function of the 186 W(d,2n)186 Re reaction.
1. Introduction
The charged particle excitation functions are the important
part of the nuclear data, applied to the activation analysis,
nuclear medicine investigation, nuclear theory, calculation
of integral thick target yield, and so on.
Rhenium-186 is an important medically radionuclide,
and regarded very suitable for radiotherapy and radioimmunotherapy due to its attractive properties which include
emission of high-energy β-rays 1.07 MeV, low-abundance
(9.42%) gamma emission at 137 keV, and 90.64 h half-life.
The 186 Re has been produced using nuclear reactors
through 185 Re(n,γ )186 Re reaction, but the specific activity
is medium, not in no-carrier-added form. In order to
radiolabel antibodies more efficiently, production of nocarrier-added 186 Re with high specific activity is required.
There are two major routes for the production of nocarrier-added 186 Re by cyclotron, namely 186 W(p,n)186 Re
and 186 W(d,2n)186 Re.
In the present work, the excitation function of
186
W(d,2n)186 Re reaction was evaluated. The available
experimental data include the latest reports [1, 2] that are
not considered in previous works [3, 4] were analyzed
and corrected. The nuclear model calculation was done
using TALYS [5]. The TTY of 186 Re was calculated using
the recommended excitation function of 186 W(d,2n)186 Re
reaction, and compared with the TTY from 186 W(p,n)186 Re
reaction [6].
2. Evaluation of experimental data
The activation method and stacked foil technique are
often used in the charged particle excitation functions
measurement. The formula [7] of charged particle
excitation functions is as follows:
eλtd
M
c 1 1.6 × 10−19 ti
·
· · ·
· 1024 (b)
nεpa N0 t χ
Q
1 − e−λti
(1)
Where, M is the target molecular weight, nis the target
nucleus number in a molecule, ε denotes the detection
σ =
a
e-mail: jmwang@ciae.ac.cn
efficiency of detector at full energy peak, p is the g-ray
branching ratio, a denotes the isotope abundance, N0 is
the Avogadro constant, c/tis the accounting g-ray number
at full energy peak in a unit time, χ denotes the target
weight in a unit area, ti denotes the irradiation time, Qis
the total integrated beam current (in Coulomb), td denotes
the cooling time (start from stop irradiation) and λ is the
radioactive decay constant.
From the mentioned-above formula, the cross sections
are proportional to the radioactive decay constant, and
inversely proportional to the g-ray branching ratio.
Moreover, if the experiment is relative measurement,
the cross sections are proportional to the standard cross
sections. According to these relations, σ can be corrected
by the new p, λ and standard cross sections.
Eight experiments [1, 2, 7–12] of the 186 W(d,2n)186 Re
reaction were founded in the EXFOR library and literature,
listed in Table 1 and shown in Fig. 1.
The experimental data were normalized to 100%
enrichment of 186 W, considering the given enrichment of
the target in the literature and the isotopic composition
of natural tungsten, i.e., 180 W (0.12%), 182 W (26.50%),
183
W (14.31%), 184 W (30.64%) and 186 W (28.43%).
In the measurements of F. Tarkanyi et al. [11],
K.Ochiai et al. [12], S. Manenti et al. [1] and
C. Duchemin et al. [2], the samples were thick
natural W-metal foils, so the normalization factor
is 3.517. In the measurement of F.W. Pement et al. [8], the
sample was WO3 powder, enriched to 97.2% in 186 W, so
the normalization factor is 1.0288.
In the measurement of Tao Zhenlan et al. [7], the g-ray
branching ratio of 137.16 keV are 0.092, and the new value
is 0.0942 [13]. The sample was WO3 powder, enriched
to 99% in 186 W, so the corrected factor is 0.9813. The
experimental data after normalization and correction are
shown in Fig. 2.
3. Nuclear model calculations
Several nuclear model code have been developed, in
the evaluation of data are very helpful. In the process
of evaluation, the parameters of the nuclear model
c 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/).
EPJ Web of Conferences 146, 02022 (2017)
DOI: 10.1051/epjconf/201714602022
ND2016
Table 1. Measurements of 186 W(d,2n)186 Re reaction.
Author
F.W. Pement et al. [8]
Sample
WO3 enriched
to 97.2%
S.J. Nassiff et al. [9]
—
Tao Zhenlan et al. [7]
WO3 enriched
to 99%
N.S. Ishioka et al. [10]
natural
F. Tarkanyi et al. [11]
natural
K. Ochiai et al. [12]
natural
S. Manenti et al. [1]
natural
C. Duchemin et al. [2]
natural
Method
STTA
STTA,
ACTIV
STTA,
ACTIV
STTA,
ACTIV
STTA,
ACTIV
STTA,
ACTIV
STTA,
ACTIV
STTA,
BCINT
Figure 3. The results of nuclear model calculation using TALYS.
Figure 4. The results of fitting from threshold to 14 MeV.
Figure 1. Original experimental data.
rvsdjust was set to 1.2. The pairing energy of 186 Re was set
to 0.0. The theoretical results are shown in Fig. 3.
4. Fitting of experimental data
After normalization and correction, the experimental data
are consistent besides the partial data of S.J. Nassiff
et al. [9] and S. Manenti et al. [1]. S.J. Nassiff et al.
reported six cross section values from 7.3 to 16.7 MeV,
but the errors not given. The data are higher than the
calculated values using TALYS and those reported by other
authors between 10 to 20 MeV. The data of S. Manenti
et al. are highter than the results of calculation and other
measurements at 11.76, 12.38, 12.54 and 13.14 MeV. F.W.
Pement et al. [8] reported nine data points but not given
the errors also. So these data were neglected in fitting of
experimental data.
On basis of least-squares fit with polynomial, the
evaluated experimental data were fitted from threshold to
14 MeV and from 14 to 50 MeV. The curves of fitting are
shown in Fig. 4 and Fig. 5.
The function of fitting from threshold to 14 MeV as
follws:
σd,2n = a − bE d + cE d2 − d E d3
(2)
Figure 2. The experimental data after normalization and
correction.
code need to be adjusted to reproduce the experimental
data.
In this work, the cross sections were calculated using
TALYS, a nuclear model code, developed by Koning
et al. [5]. The nuclei were considered non-spherical in
shape. The compound nucleus contribution was considered
in the frame of Moldaner model. The contributions of
direct reactions were taken into account by ECIS. The
back-shifted Fermi gas model (BFM) was used for level
densities (ldmodel 2). The OM parameters were adjusted,
Where, a = 4.2176, b = 1.4378, c = 0.1555, d = 0.0052.
The function of fitting from 14 to 50 MeV as follws:
σd,2n = exp(a − bE d + cE d2 )
Where, a = 1.8248, b = 0.2544, c = 0.0031.
2
(3)
EPJ Web of Conferences 146, 02022 (2017)
DOI: 10.1051/epjconf/201714602022
ND2016
function of 186 W(d,2n)186 Re reaction are agree with the
experimental data, the maximum value is slightly higher
than the value of IAEA TRS 473, and little lower than the
value of M. Hussain et al.
The thick target yield (TTY) of 186 Re was calculated using the recommended excitation function of
186
W(d,2n)186 Re reaction, and compared with the TTY
from 186 W(p,n)186 Re reaction [6], together with the
measurements [1, 14, 15] are shown in Fig. 7. The TTY
of 186 W(d,2n)186 Re reaction are much higher than the
value of 186 W(p,n)186 Re reaction above 15 MeV. So
the 186 W(d,2n)186 Re reaction may be more suitable
for production of no-carrier-added 186 Re for medical
application, if the deuteron beams are available by
cyclotron and the energy is high enough.
Figure 5. The results of fitting from 14 to 50 MeV.
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[11] F. Tarkanyi, S. Takacs, F. Szelecsenyi, F. Ditroi,
A. Hermanne, M. Sonck, Nuclear Instruments and
Methods in Physics Research B 211, 319 (2003)
[12] K. Ochiai, M. Nakao, N. Kubota, S. Sato,
M. Yamauchi, N.H. Ishioka, T. Nishitani, C. Konno,
EDP Sciences, 1011 (2008)
[13] Xiaolong Huang, Zhigang Ge, Zhendong Wu,
Guochang Chen, Jimin Wang, Xi Tao, Jianhui
Li, V.P. Chechev, S.A. Badikov, T.V. Golashvilli,
O.O. Patarakin, V.I. Rachkov, Nuclear Characteristics of Nuclides, (China Atomic Energy Press, 2013)
[14] S. Mukhammedov, A. Vasidov, E. Pardaev, Soviet
Atomic Energy 56, 56 (1984)
[15] M.L. Bonardi, F. Groppi, S. Manenti, E. Persico,
L. Gini, Applied Radiation and Isotopes 68, 1595
(2010)
Figure 6. The recommended excitation function for
186
W(d,2n)186 Re reaction, compared with experimental data
and results published earlier.
Figure 7. The TTY of 186 W(d,2n)186 Re reaction, compared with
the TTY of 186 W(p,n)186 Re reaction.
5. Results and discussion
The excitation function for 186 W(d,2n)186 Re reaction was
recommended from threshold energy to 50 MeV after
comprehensive evaluation, take into account the results of
calculation and fitting. The recommended curve is given in
Fig. 6, together with the results of M. Hussain et al. [3]
and IAEA TRS 473 [4]. The recommended excitation
3
| 1,781 | 10,824 |
W2528671231.txt | 2 - ORIGINAL ARTICLE
WOUND HEALING
Adipose tissue stromal vascular fraction in the treatment of full thickness burns in rats1
Alexandre Lamaro CardosoI, Maria Márcia. BachionII, Júlia de Miranda MoraisIII, Marcelo Silva FantinatiIV, Vera Lúcia Lima
de AlmeidaV, Ruy Souza Lino JúniorVI
DOI: http://dx.doi.org/10.1590/S0102-865020160090000002
Fellow Master degree, Faculty of Medicine, Universidade Federal de Goiás (UFG), Goiania-GO, Brazil. Intellectual and scientific content of the study,
conception and design of the study, manuscript writing.
II
PhD, Full Professor, Faculty of Nursing UFG, Goiania-GO, Brazil. Acquisition, analysis and interpretation of data.
III
PhD, Associate Professor, Faculty of Medicine, UFG, Goiania-GO, Brazil. Acquisition, analysis and interpretation of data.
IV
PhD, Full Professor, ESEFFEGO, Universidade Estadual de Goiás (UEG), Goiania-GO, Brazil. Acquisition, analysis and interpretation of data.
V
Biomedic, Institute of Tropical Pathology and Public Health, Experimental Pathology Laboratory, UFG, Goiania-GO, Brazil. Acquisition of data.
VI
PhD, Associate Professor, Institute of Tropical Pathology and Public Health, Experimental Pathology Laboratory, UFG, Goiania-GO, Brazil. Scientific
and intellectual content of the study, analysis and interpretation of data, statistical analysis, manuscript preparation.
I
ABSTRACT
PURPOSE: To analyze the healing effects of stromal vascular fraction (SVF) application compared to wound dressing with 2% silver
sulfadiazine in full thickness burn wounds in rats.
METHODS: Animals were divided into two groups: 2% silver sulfadiazine group and SVF group. Both groups received occlusive
bandages while the first one was treated with 2% silver sulfadiazine and the latter was treated with injections of SVF prepared from
adipose tissue extracted from an animal donor. The animals were accompanied through 3, 7 and 30 days for evaluation of macroscopic,
microscopic and morphometric aspects.
RESULTS: On day three, a significant increase (p<0.05) of infiltration of polymorphonuclear, fibrin formation and fibroblasts migration
in SVF group was observed. On the 7th day the mononuclear infiltrate, angiogenesis, collagen and fibroblasts were significantly increased
in the SVF group (p<0.05). At 30 days significantly increased collagen deposition was observed in the SVF group (p<0.05).
CONCLUSION: Adipose tissue derived stromal vascular fraction injections promotes better wound repair than 2% silver sulfadiazine
in the treatment of full thickness burn in rats during the evaluated experimental period.
Key words: Burns. Wound Healing. Rats.
578 - Acta Cirúrgica Brasileira - Vol. 31 (9) 2016
Adipose tissue stromal vascular fraction in the treatment of full thickness burns in rats
Introduction
cell cultivation techniques, injured tissue and patient’s immune
response8. Of particular importance is the recognition of abundant
Full thickness burns are characterized by destruction of
sources of cells, with great therapeutic potential9.
all skin layers. The destruction of the free nerve endings in the
The separation of adipose tissue components by
dermis makes these lesions painless, and grafting procedures
enzymatic digestion and subsequent centrifugation of the material
are often needed to optimize tissue repair that usually evolves
raised new possibilities for using this tissue in regenerative
with formation of hypertrophic scars, severe fibrosis and tissue
therapy. While initially the focus was turned to the use of mature
1
adhesion .
adipocytes, located in the upper region of tissue solution after
The global incidence of burns requiring medical
centrifugation, different studies have explored the therapeutic
intervention in 2004 was about 11 million people. Although the
majority of cases throughout the world are not fatal, fire related
burns are among the leading causes of disability in low and middle
income countries2.
The Brazilian Burn Society estimates that millions of
burn cases occur every year in Brazil. Approximately two hundred
thousand are treated at emergency wards and forty thousand
require hospitalization. Estimates presented in a study conducted
in the Federal District of Brazil showed a mortality rate of 6.2%
among hospitalized burns victims in emergency wards. Among
violent causes of death in Brazil burn injuries represent the third
most important cause after road accidents and homicides3.
The techniques conventionally used in the treatment of
burn wounds vary according to the extent of the lesion, affected
body area, age and previous individual health status. Prevention
of infections, removal of necrosis, and grafting procedures are
routinely used to treat such wounds4.
In full thickness burn injuries surgical debridement
followed by occlusive dressing are generally used in most clinical
centers dedicated to the treatment of these lesions. Moisturizing
agents such as glycerin and microbicides as 2% silver sulfadiazine
are incorporated into the occlusive dressing to promote the healing
process and prevent infection .
5
applications of stromal vascular fraction (SVF)10.
Endothelial cells, lymphocytes, myelocytes, pericytes,
pre-adipocytes and adipose derived stem cells (ADSCs) are part
of its composition and, depending on the stimuli in the tissue
microenvironment, present considerable plasticity, and the
capacity to proliferate and differentiate into distinct cell lines11.
Studies suggest that therapeutic effects of SVF are
primarily a result of the activity of ADSCs7,12 such as modulation
of inflammatory response13, increased migration of fibroblasts14,
collagen production15 an angiogenesis16-18.
Considering the need to develop interventions which
address the reduction of fibrosis, restrictions in tissue mobility,
aesthetic deformities and functional limitations in full thickness
burn injuries, this study aimed to compare the microscopic,
macroscopic and morphometric effects between the treatments
with SVF applied intradermally and occlusive bandages with 2%
silver sulfadiazine in experimentally induced full thickness burn
wounds in rats.
Methods
The study was approved by the Ethics Committee on the
use of animals from the Universidade Federal de Goiás (protocol
While the outcomes in burn wound injury treatments
Number 035/13).
increased significantly in the last decades, as a result of
developments in surgical, nursing and rehabilitation procedures,
Stromal vascular fraction preparation and injection
severe functional and cosmetic limitations are still highly prevalent
procedure
in burn patient populations6. The development of interventions
morphological
One day after the burn procedure, euthanasia of the
reconstruction and functional recovery are necessary in order to
donor animal was performed (lethal injection of thiopental sodium
achieve better results .
100mg/kg) followed by the removal of the visceral adipose tissue
to
reduce
hypertrophic
scarring,
promote
7
Cell therapy is one of the most promising approaches to
by surgical dissection. The material was conducted to laminar flux
morphological reconstruction and functional restoration of skin
in transport solution (Sterile Phosphate Buffer Solution at 2%
burn damage. The effective application of cell populations that
(PBS) and 1% antibiotics: 105 units/ml penicillin and 105ug/ml
optimize the repair processes and tissue regeneration in different
streptomycin) washed in PBS to remove debris and red blood cells
clinical conditions depends on several factors such as type of cell,
and minced with fine tissue scissors. The fragmented tissues were
Acta Cirúrgica Brasileira - Vol. 31 (9) 2016 - 579
Cardoso AL et al.
incubated with type I collagenase (1.5 mg/ml) and kept in water
Animals and treatment
bath at 37ºC for 50 minutes. After enzymatic digestion, the tissue
was filtered using a 70µm filter and collagenase was removed by
dilution with phosphate buffer solution (PBS). The cell suspension
was centrifuged at 363g (1800 rpm)/7 minutes and the supernatant,
composed primarily of mature adipocytes, was removed. The
pellet was formed by cells which were re-suspended in PBS. The
counting of viable cells was performed using the Tripan Blue stain
exclusion method. Injections were prepared adding PBS and a
SVF volume equivalent to 5 x105 cells/ml in 1 ml syringes. Four
injections of 0.2 ml were made in each wound edge and dripping
of 0.2 ml covering the wound bed.
Full thickness burn procedure
After local preparation of the dorsal skin, shaving
and antisepsis, the animals were anesthetized with
0.01ml/g of Ketamine 10% and 0.03ml/g of Xilazine 2%
via intraperitoneal. Afterwards, the full thickness thermal
injury was induced by the immersion of this region in
boiling water as described by Morais et al.19. Briefly, each
animal was placed inside a PVC (polyvinyl chloride) plastic
cylinder with a 2x2 cm opening and sealed extremities,
Thirty male adult Wistar rats weighing between 300 and
350g were used in this study. Adipose tissue extraction was made
once in each experimental series with durations of 3, 7 and 30 days,
with a total of three animals used as donors. Each experimental
group was composed by five animals. Each experimental group
received treatment after the debridement procedure, 2 days after
the injury induction. The 2% silver sulfadiazine group (CT)
received occlusive bandages with sterile gauze impregnated
with glycerine and 2% silver sulfadiazine. The stromal vascular
fraction treated group (SVF) received occlusive bandages with
sterile gauze impregnated with glycerine and injections of stromal
vascular fraction at a concentration of 5 x105 cells/ml. The
injection procedure was made only once after debridement in SVF
group and bandages were change in each group daily during the
experimental series.
The animals euthanasia was performed at each
experimental day with a lethal injection of thiopental sodium
100 mg/kg followed by collection of material for microscopic
examination and photographic register of the injury for
morphometric and macroscopic analysis.
Macroscopic evaluation
the uncovered area was immersed in water at 95 for 14
o
seconds. The accomplishment of a full thickness burn by
the cited method was early verified by histological analysis
with hematoxylin-eosin preparation showing complete
destruction of the epidermis, dermis and hypodermis
confirming a full thickness burn wound with necrosis and
disarrangement of collagen fibers. On the day that followed
the burn procedure, animals received surgical debridement
under general anesthesia (0.06 ml/Kg of 10% ketamine,
0.01 ml/Kg distilled water and 0.03 ml/Kg of 2% xylazine
applied intraperitoneally) to remove necrosis. Debridement
procedure initiated by dissection of skin with a Metzembaum
scissor in panniculus carnosum, at the suprafascial level.
The other edges were sectioned with a straight Mayo scissor
always in the internal border of demarcated area. A full
After euthanasia, wounds were photographed and
the component phases of tissue repair such as inflammation,
proliferation and maturation19 were macroscopically examined.
Parameters of hyperemia, necrosis, hemorrhage, crust formation,
tissue granulation and fibrosis were graded as absent (score 0),
discrete (up to 25% of committed area, score 1), moderate (from
26 to 50% of committed area, score 2) and accentuated (more
than 50% of committed area, score 3) in a semi-quantitative form.
Examination was performed by the same researcher, blind to
group allocation. The results were expressed in mean ± standard
deviation.
Morphometric evaluation
pain management was performed by inclusion of 2 mg/ml
Wound photographs were analyzed using the ImageJ
software (National Institute of Health; version 1.3.1). Wound
areas were circulated by the researcher without knowing the group
allocation, and the contraction level was calculated using the
following equation:
CL= (area T0 – area Teuthanasia day)x 100/area T0,were
paracetamol in the rats’ drinking water.
CL=contraction level; T0= day of burn injury induction; Teuthanasia
thickness skin (4 cm2) was excised from burn area by removing the
epidermis and dermis layer with a surgical blade (0.1 mm) until
the subcutaneous fat,avoiding the panniculus carnosum and the
muscle layer. Early post operative and three days post burn
580 - Acta Cirúrgica Brasileira - Vol. 31 (9) 2016
Adipose tissue stromal vascular fraction in the treatment of full thickness burns in rats
= days after injury induction. The morphometric evaluation
day
was performed at days 7 and 30 due to the absence of wound
contraction at the early stage of the healing process (3 days).
Microscopic evaluation
After euthanasia, the wound tissue and adjacent
skin were removed. This material was incubated with 10%
formaldehyde, included in paraffin blocks, prepared with sections
of 4 µm and stained with hematoxylin-eosin (H&E). Tissue
slides were analyzed by optical microscopy of entire wound
area at magnifications of x10 and x40 and parameters of fibrin,
hemorrhage,
polymorphonuclear
infiltration,
mononuclear
infiltration, hyperemia, angiogenesis, necrosis, fibroblasts and
collagen were graded as absent (score 0), discrete (up to 25% of
committed area, score 1), moderate (from 26 to 50% of committed
area, score 2) and accentuated (more than 50% of committed area,
FIGURE 1 - Macroscopic evaluation of third degree burn wounds
experimentally induced in rats treated with 2% silver sulfadiazine and
adipose tissue stromal vascular fraction. (CT - control group) 2% silver
sulfadiazine. (SVF) adipose tissue stromal vascular fraction treated group.
(A) Arrow indicates discrete hyperemia. (B) Arrow indicates accentuated
granulation tissue. (C) Arrow indicates discrete fibrosis. (D) Arrow
indicates accentuated necrosis at wound edges. (E) Arrow indicates
moderate necrosis. (F) Arrow indicates accentuated fibrosis.
score 3) in a semi-quantitative form. Examination was performed
by the same researcher, blind to group allocation. The results were
expressed in mean ± standard deviation.
Statistical analysis
Statistical analysis was performed with the SigmaStat
2.3 software. Variables were tested for normal distribution
and homogeneous variance (Kolmogorov-Smirnov test). The
macroscopic and microscopic statistical analyses were performed
through the Mann-Whitney test. The morphometric analysis was
performed through the Student T test. Differences were considered
significant when p<0.05.
Results
Macroscopic results
General pathologic process associated with inflammation,
proliferation and maturation phases were observed in days 3, 7
and 30 respectively (Figure 1). Statistically significant differences
were found between the CT group and SVF group regarding
necrosis formation, which was significantly increased in the
CT group at day 3 (p<0.05). Other parameters such as crust,
hemorrhage, hyperemia, granulation tissue and fibrosis did not
present a macroscopic difference when comparing both treatments
TABLE 1 - Macroscopic evaluation (mean ± standard
deviation) of general pathological processes in third degree burn
wound in rats treated with 2% silver sulfadiazine and adipose
tissue stromal vascular fraction at days 3, 7 and 30.
Pathological
CT
SVF
DAI
P
Process
(n = 5)
(n = 5)
3
p=0.01*
1.0 ± 0.6
0.3 ± 0.8
Necrosis
7
0.3 ± 0.6
1.3 ± 0.4
p=0.183
30
0.0 ± 0.0
0.0 ± 0.0
p=1.00
3
0.3 ± 0.6
0.0 ± 0.0
p=0.183
Crust
7
0.3 ± 0.6
0.0 ± 0.0
p=0.183
30
0.0 ± 0.0
0.0 ± 0.0
p=1.00
3
0.3 ± 0.6
1.3 ± 0.4
p=0.42
Hemorrhage
7
0.3 ± 0.6
1.3 ± 0.4
p=0.183
30
0.0 ± 0.0
0.0 ± 0.0
p=1.00
Hyperemia
Granulation
tissue
Fibrosis
3
0.3 ± 0.6
2.6 ± 0.4
p=0,.57
7
30
3
7
30
3
7
30
0.3 ± 0.6
0.0 ± 0.0
0.0 ± 0.0
0.3 ± 0.6
0.3 ± 0.6
0.0 ± 0.0
0.0 ± 0.0
3.0 ± 0.0
2.3 ± 0.4
0.0 ± 0.0
0.0 ± 0.0
2.6 ± 0.4
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
3.0 ± 0.0
p=0.42
p=1.00
p=1.00
p=0.057
p=1.00
p=1.00
p=1.00
p=1.00
SVF: stromal vascular fraction group. CT: 2% silver sulfadiazine group. n: number
of animals in each group. DAI: days after injury induction. *Statistically significant
(p<0.05 – Mann-Whitney).
(Table 1).
Acta Cirúrgica Brasileira - Vol. 31 (9) 2016 - 581
Cardoso AL et al.
Microscopic results
Fibrin
formation,
polymorphonuclear
infiltration
and fibroblast content were significantly increased in the SVF
group compared to the CT group at day 3 (p<0.05) (Figure 2).
The mononuclear infiltration, angiogenesis, fibroblast content
and collagen deposition were significantly increased in the SVF
group compared to the CT group at day 7 (p<0.05). 30 days after
burn injury induction hyperemia and collagen deposition were
significantly increased in the SVF group compared to the CT
group (p<0.05) (Table 2). Collagen disruption and hemorrhage
did not present significant differences between the treated groups
during the experimental days.
FIGURE 2 - Microscopic evaluation of third degree burn wounds
experimentally induced in rats treated with 2% silver sulfadiazine and
adipose tissue stromal vascular fraction. (CT - control group) 2% silver
sulfadiazine. (SVF) adipose tissue stromal vascular fraction treated group.
(A) Arrow indicates moderate fibroblastic proliferation. (B) Arrows
indicate accentuated angiogenesis. (C) Arrows indicate accentuated
collagen formation. (D) Arrow indicates discrete polymorphonuclear cells
inflammatory infiltration. (E) Arrow indicates accentuated mononuclear
cells inflammatory infiltration. (F) Arrow indicates moderate mononuclear
cells inflammatory infiltration (x400 hematoxylin & eosin. Scale Bar
indicates 20 µm).
TABLE 2 - Microscopic evaluation (mean ± standard
deviation) of general pathological process in third degree burn
wound in rats treated with 2% silver sulfadiazine and adipose
stromal vascular fraction at days 3, 7 and 30.
Pathological
DAI CT (n = 5) SVF (n =5)
P
Process
3
3.0 ± 0.0
3.0 ± 0.0
p=1.00
Collagen
7
1.0 ± 0.0
0.3 ± 0.6
p=0.183
disruption
30
0.6 ± 0.5
0.0 ± 0.0
p=0.422
3
0.0 ± 0.0
0.0 ± 0.0
p=1.00
Hemorrhage
7
1.8 ± 0.7
1.0 ± 0.0
p=0.183
30
0.2 ± 0.4
0.3 ± 0.4
p=1.00
3
1.6 ± 0.4
1.6 ± 0.9
p=0.741
Hyperemia
7
0.6 ± 0.8
2.0 ± 0.81
p=0.225
30
p=0.03*
0.2 ± 0.4
2.0 ± 0.0
3
p=0.03*
0.3 ± 0.6
3.0 ± 0.0
Fibrin
7
0.0 ± 0.0
0.0 ± 0.0
p=1.00
30
0.0 ± 0.0
0.0 ± 0.0
p=1.00
3
p=0.03*
0.0 ± 0.0
1.6 ± 0.4
PMN infiltra7
0.6 ± 1.2
1.0 ± 0.0
p=1.00
tion
30
0.0 ± 0.0
0.0 ± 0.0
p=1.00
3
0.6 ± 0.48
1.0± 0.0
p=0.183
MN infiltra7
p=0.01*
0.6 ± 0.4
3.0 ± 0.0
tion
30
0.8 ± 0.7
2.0 ± 0.0
p=0.183
3
0.0 ± 0.0
1.3 ± 0.4
p=0.057
Angiogenesis
7
p=0.01*
0.3 ± 0.6
3.0 ± 0.0
30
0.3 ± 0.6
2.0 ± 0.0
p=1.00
3
p=0.03*
0.3 ± 0.6
2.0 ± 0.0
Fibroblast
7
p=0.03*
0.3 ± 0.6
2.0 ± 0.0
30
0.3 ± 0.6
3.0 ± 0.0
p=0.42
3
0.0 ± 0.0
0.0 ± 0.0
p=1.00
Collagen
7
p=0.03*
0.3 ± 0.6
2.0 ± 0.0
30
p=0.03*
0.3 ± 0.6
3.0 ± 0.0
SVF: stromal vascular fraction group. CT: 2% silver sulfadiazine group.
PMN:plymorphonuclear cells; MN: mononuclear cells. DAI: days after injury induction. *Statistically significant (p<0.05 – Mann-Whitney).
Morphometric results
No significant difference was observed between the SVF
group and the CT group regarding the wound contraction levels at
days 7 and 30 (Table 3).
582 - Acta Cirúrgica Brasileira - Vol. 31 (9) 2016
Adipose tissue stromal vascular fraction in the treatment of full thickness burns in rats
TABLE 3 - Morphometric evaluation of wound
contraction area in third degree burn wound in rats treated with
2% silver sulfadiazine and adipose tissue stromal vascular fraction
at days 7 and 30. Results expressed in percentage ± standard
deviation.
Experimental group
7 DAI
30 DAI
CT
14.476 ± 0.4
88.207 ± 2.6
SVF
15.023 ± 0.5
88.203 ±5.8
p
p= 0.676
p= 0.149
CT: 2% silver sulfadiazine group. SVF: stromal vascular fraction group. DAI: days
after injury induction. Statistical analysis: Student T test.
Discussion
This study demonstrated that the stromal vascular fraction
removed from the adipose tissue improved the microscopic and
macroscopic aspects of the healing process of full thickness
burn wounds experimentally induced in rats. These improvements
were observed in all phases of the healing process, inflammatory,
proliferative and maturation.
Stromal vascular fractions are composed of different cell
populations such as mesenchymal stem cells, T regulatory cells,
endothelial precursor cells, preadipocytes and macrophages. All
of them are capable of influencing the wound microenvironment
in many ways as they share similarities with bone marrow stromal
cells, including the capacity of multilineage differentiation20.
In this study, an amplification of the inflammatory
response evidenced by increased migration of polymorphonuclear
and fibrin deposition was observed at day 3. Studies applying
SVF or ADSCs previously expanded in culture on wound
models observed a reduction of the inflammatory response21.
These different observations suggest that SVF could modulate
inflammation in a bi-directional manner.
Studies have shown that controlled amplification
of initial inflammatory episodes has been associated with
a shift in repair processes of different tissues towards more
regeneration and less scar formation22-24.
Trials designed to elucidate how different SVF
preparations influence burn wound repair, regarding the
type of adipose tissue utilized and dose-response curves for
application of different SVF concentrations are needed to
clarify the reasons for variable experimental findings.
A significant increase of fibrin and fibroblasts in the
SVF group compared to the CT group at day 3 was also
observed. Evidence suggests that fibronectin binding to
fibrin25 and ADSCs paracrine secretion of growth factors
such as platelet derived growth factor (PDGF) and basic
fibroblast growth factor (bFGF) could be possible associated
mechanisms leading to the observed effect26.
At day 7, angiogenesis (p=0.01), fibroblasts
(p=0.03), mononuclear infiltrate (p=0.01) and collagen
production (p=0.03) showed a marked increase in the SVF
group when compared to the control group at the same
experimental day. The secretome repertoire of ADSCs
has been reported to be composed by many cytokines
and growth factors which are able to influence these
parameters12. Expression of potent angiogenic agents such
as vascular endothelial growth factor (VEGF), fibroblast
migration and activity stimulators like Transforming
Growth Factor (TGF) and macrophages chemotaxis agents
were expressed by ADSCs in studies performed in different
models8,14. Collectively, this process could accelerate the
proliferative phase and build better conditions for wound
repair as observed in our results.
Regarding mononuclear infiltration, classically
activated macrophages have been known to express high
levels of pro-inflammatory cytokines like IL-1, IL-6 and IL23 and under its influence, Th17 cells have released IL-17
which induces increased recruitment of polymorphonuclear
cells towards the injury site24. It was possible to observe
an increase in the mononuclear infiltration at day 7 with
no increase in polymorphonuclear infiltration. This result
suggested that the cytokines released by SVF cells could
change the macrophage activation profile from classic to
regulatory which improved the wound healing profile.
Corroborating this hypothesis is the observation of marked
collagen formation in the SVF group. Activated regulatory
macrophages have been known to increase the production
of extracellular matrix components and the release of
anti-inflammatory cytokines such as IL-10, controlling
the extension and intensity of inflammation, therefore
promoting better conditions for the occurrence of the
proliferative process24.
Similarities between ADSCs in vivo morphology
and fibroblasts could also contribute to the observation of
increased fibroblast. Histological preparations with H&E
did not allow the differentiation between the two cell types,
nor the detection of fibroblasts that differentiate directly
from ADSCs27.
The initial stimulation of fibroblasts at 3 and 7 DAI
in the SVF treated group resulted in increased collagen
deposition at day 30. These results are in accordance to the
Acta Cirúrgica Brasileira - Vol. 31 (9) 2016 - 583
Cardoso AL et al.
description that ADSCs up-regulate type I and III collagen
and fibronectin and down-regulate metalloproteinase
1(MMP-1), significantly accelerating healing from the
wound edges and increasing the tensile strength of scar
tissue28. Studies approaching scar tissue tensile strength in
burn wounds treated with SVF and isolated ADSCs could
better elucidate the specific contributions of these different cell
populations regarding the functional outcomes in the burn wound
healing process and guidance of the clinical decision-making
process about which procedure would have a better cost-benefit
relationship.
SVF acquisition in humans using procedures such as
lipoaspiration have been reported as a method that does not cause
marked morbidity and have been easy to apply, particularly in
conditions requiring immediate medical interventions such as in
burn injuries18.
Conclusions
Intradermally
applied
SVF
have
3.
4.
5.
6.
7.
8.
11,
12.
13.
14.
15.
improved
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observed in this study showed that therapeutic application of cells
extracted from adipose tissue could have a significant role in burn
wound care.
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Acknowledgement
To Vânia Beatriz Lopes for help in technical analysis.
Correspondence:
AlexandreLamaro Cardoso
Faculdade de Medicina - UFG
Rua 235, 1a s/n, Setor Universitário
Goiânia-GO Brasil
Tel/Fax: (55 62)3209-6165
alexandrelamaro@hotmail.com
Received: May 21, 2016
Review: July 19, 2016
Accepted: Aug 18, 2016
Conflict of interest: none
Financial source: FAPEG (grant number 2013/0267001091)
Research performed at Laboratory of Experimental Pathology, Institute
of Tropical Pathology and Public Health, Universidade Federal de Goiás
(UFG), Goiania-GO, Brazil.
1
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W3126813359.txt | Bangladesh Journal of Medical Science Vol. 20 No. 02 April’21
Original article:
Impact of Socio-demographic factors on Quality of life in Medical Students of Eastern Saudi Arabia
Mohammad Ayoob1, Abdulrehman Alsultan2, Nurjahan Begam3, Saleh Al Sumaih4, Hamad Waleed Albuali5
Abstract
Background: Quality of life of medical students may influenced by different socio-demographic
components and disrupt their capability to study medicine. An in-depth exploration of these
elements may encourage enhancement in learning and retention of medical students. Aim:
This study was designed to identify the role of socio-demographic factors in quality of life in
undergraduate medical students. Material and Method: A cross-sectional study was conducted
among 223 undergraduate medical students (male = 125, females = 98) at King Faisal University,
Saudi Arabia between February and September 2019. An Arabic version of WHOQOL-BREF
was used to explore the QOL of medical students. Regression analysis were employed to evaluate
association between QOL and socio-demographic factors including age, sex, academic year,
family type, area of residence, monthly income, parental education and housing status. Result:
The results showed a significant relationship between various socio-demographic variables and
quality of life. The findings of the study revealed that gender, academic year, marital status,
monthly income and family type were found significant predictors of quality of life among
medical students. Conclusion: The present study has explored the impact of socio-demographic
factors on the quality of life of medical students.
Keywords: socio-demographic; quality of life; medical students; Saudi Arabia
Bangladesh Journal of Medical Science Vol. 20 No. 02 April’21. Page : 250-257
DOI: https://doi.org/10.3329/bjms.v20i2.51531
Introduction
The World Health Organization (WHO) defined
Quality of Life (QoL) as “an individual’s perception
of their position in life, in the context of the culture
and value systems in which they live, and in
relation to their goals, expectations, standards and
concerns”1 . Quality of life focused all aspects of life,
including physical health, family, security, status,
education, beliefs, and the environment. Quality of
life is influenced by many factors such as, biological,
behavioral, psychological and demographical2.
However, the link between these elements and
QOL are varying among different communities and
population3.
Generally, medical students experience more stress
than general population.4-6 The causes of their stress
could be due to the academic burden, less leisure time,
more material to be retained, high parental expectation,
peer pressure, and repeated formative and summative
examinations.7 Previous research reported that poor
quality of life among medical students is linked with
poor attention and concentration, increased incidence
of errors, negligence, absenteeism, self-medication,
and cheating during examinations.8
1. Mohammad Ayoob, Assistant Professor, Department of Clinical Neurosciences, College of Medicine,
King Faisal University, Saudi Arabia, Email: ayooblone08@gmail.com;
2. Abdulrehman Alsultan, Assistant Professor, Department of Biomedical Science, College of Medicine,
King Faisal University, Saudi Arabia.
3. Nurjahan Begam, Assistant Professor, Department of Clinical Neurosciences, College of Medicine, King
Faisal University, Saudi Arabia.
4. Saleh Al Sumaih, Medical Student, College of Medicine, King Faisal University, Saudi Arabia.
5. Hamad Waleed Albuali, Medical Student, College of Medicine, King Faisal University, Saudi Arabia.
Correspondence to: Mohammad Ayoob, Assistant Professor, Department of Clinical Neurosciences, College
of Medicine, King Faisal University, Saudi Arabia, Email: ayooblone08@gmail.com;
250
Impact of Socio-demographic factors on Quality of life in Medical Students of Eastern Saudi Arabia
Several studies have examined quality of life among
medical students. For instance, in a cross-sectional
study conducted among Saudi medical students,
the students experienced environmental domain as
their highest QOL, whereas physical domain and
social relations domain were experiences as the
lowest.9 Mahmoud and Fareed10 reported that final
year medical students score higher in environmental
domain than students in initial years. Another
study reported that first year and final year medical
students experienced better quality of life in terms of
psychological and social domain.11
There is tremendous research works on the quality of
life among medical students. However, few studies
have examined the impact of demographic factors
on quality of life among medical students12. There is
presently dearth of psychological research on impact
of different demographic factors on quality of life
among medical students especially in Saudi Arabia.
Therefore, the current study aimed to assess the effect
of various socio-demographic factors on the quality
of life of medical students, with the intention of
improving learning and retention at medical college.
Materials and Methods
Setting and participants
The present study was conducted in college of
medicine, King Faisal University, Saudi Arabia. King
Faisal University is one of the leading University
in the Kingdom of Saudi Arabia with more than
42227 students. Among the total student body, 1070
students are enrolled at the college of medicine, with
nearly 300 new medical students enrolling each
semester. The bachelor of medicine and surgery
in Saudi Arabia is six-year programme in which
first three years are preclinical, and years 4–6 are
clinical. College of Medicine utilizes an innovative
teaching methodology to achieve their vision and
mission of preparing future physicians. Participants
of the present study were 223 medical students (Male
=125, Females = 98) studying in college of medicine,
King Faisal University, Saudi Arabia. Ages of these
participants were ranging from18 to 25 years.
Study Design
A cross sectional study was performed among
undergraduate medical students attending College of
Medicine, King Faisal University between February
and September 2019. Medical students in their Ist to 5th
year in College of Medicine voluntarily participated
in the study. In the beginning of the third semester of
the academic year, students were asked to complete
a self-reported questionnaire on demographic factors
and quality of life.
Measures
In order to achieve the goals of the present study,
the different measures were used. Quality of life was
assessed by World Health Organization Quality of
Life-Biomedical Research and Education Facility
(WHOQOL-BREF). Demographic questionnaire
prepared by the researcher was also include in the
study.
Demographic Questionnaire: The information about
demographic profile of the participants was collected
with the help of questions related to their age, sex,
marital status, and academic year. In addition,
information about their family were include area of
residence, family type, education level of parents,
family occupation, income, housing status etc.
World Health Organization Quality of Life Assessment
–Abbreviated Version (WHOQOL-BREF): Quality
of life was measured by using WHOQOL-BREF.13
The scale comprises of twenty six items, covering
four domains: physical health, psychological health,
social relationships, and environment. The first two
questions separately assess the overall perception of
quality of life and general health. Participants were
asked to rate their responses to each item using a
5-point Likert scale ranging from 1 (very dissatisfied/
very poor) to 5 (very satisfied/ very good). The
total score is derived by adding the scores of the
items belonging to different areas and mean scores
are obtained. In this scale higher scores indicate
better QOL14. Mahmoud and Fareed10 have reported
internal reliability for the total scale as 0.83. Internal
consistency reliability (Cronbach’s alpha) of this
measure was found as 0.92.
Procedure
After seeking required permission from concerned
college authorities, the participants were personally
contacted. They were briefed about the purpose of
research and questionnaire used in the study. After
seeking consent of the student, a suitable time and date
was fixed for data collection. Before administering
the questionnaire, the purpose of the study was again
explained to the participants and they were assured
that their responses would be kept confidential and
would be used for research and academic purpose
only. A good rapport was built with the participants
for getting correct responses. Necessary instruction
and guidelines were provided to them for properly
filling the questionnaire. After this, the questionnaires
251
Mohammad Ayoob, Abdulrehman Alsultan, Nurjahan Begam, Saleh Al Sumaih, Hamad Waleed Albuali
were provided to them and they were requested to fill
up the questionnaire as per the instructions given in
the questionnaire. It took 20 minutes to complete the
questionnaire. After completion of the questionnaire
participants returned the questionnaire and they were
thanked for their participation and cooperation.
and socio-demographic factors, multiple regression
models were performed. For this, separate analysis
was run for each aspect of WHOQOL-BREF.
Statistical Analysis
Result
The data were first exported to Microsoft Excel
and then to Statistical Package for Social Sciences
(SPSS version, 22) computer software used for
quantitative statistical analyses. Prior to analysis, all
variables were screened for possible code. In order
to obtain an understanding of the socio-demographic
characteristics of the participants, frequency,
percentage, means, standard deviations and other
descriptive statistics were conducted. To investigate
a relationship between WHOQOL-BREF scores
Ethical clearance: The ethical committee of human
research at the College of Medicine approved the
study protocol.
Socio-demographic
population
characteristics
of
study
The present study invited 250 students’ studying in
colleges of medicine at King Faisal University. A
total of 223 students answered the questionnaire. The
response rate was 89.20%. Among the 223 students,
125 were males and 98 were females. The mean age
of these students was 21.36 years. Table 1 shows that
majority (83.4%) of participant were single. Only 30
(13.5%) participants were married. Most of students
Table 1: Quality of life scores (Mean and Standard Deviation) according to Demographic
characteristics
N (%)
(n=223)
Physical Health
(Mean and SD)
Psychological Health
(Mean and SD)
Social Relationships
(Mean and SD)
Environment
(Mean and SD)
Male
125(57.0)
64.09±16.52*
Female
98(43.0)
54.89 ± 15.74
29.31 ± 22.69
48.47 ± 16.33
63.47 ± 18.22
34.17 ± 17.27
52.38 ± 22.90*
56.36 ± 17.55
Gender
Academic Year
Ist
67(30.0)
63.59 ± 18.52
49.17 ± 21.82**
50.70 ± 16.47
56.11 ± 12.98
2nd
40(17.9)
59.45 ± 12.04
34.70 ± 17.72
41.95 ± 12.52
52.57 ± 16.11
3rd
41(18.4)
55.46 ± 13.79
19.75 ± 11.81
39.14 ± 18.80
51.53 ± 21.91
4th
50(22.4)
60.24 ± 14.25
17.16 ± 11.00
54.88 ± 21.33
65.36 ± 16.89
5th
25(11.2)
74.68± 14.65**
27.32 ± 7.35
75.00 ± 17.26**
75.44 ± 19.16**
Marital Status
Married
30(13.5)
64.93 ± 19.58**
32.37 ± 16.79
71.43 ± 19.18**
71.50 ± 13.27**
Engaged
7(3.1)
61.57± 13.16
23.14 ± 7.88
60.17 ±21.76
69.86 ±14.54
Single
186(83.4)
53.33 ± 16.16
28.78 ± 22.23
48.22 ± 23.45
57.84 ± 19.07
Nuclear
90(40.4)
59.47 ±17.99*
29.44 ± 17.87
56.90 ± 24.26*
64.11 ±20.44*
Joint
133(59.6)
51.71 ± 15.33
28.80 ± 23.37
46.26 ± 22.36
57.31 ± 17.31
<10000SAR
76(34.1)
49.03 ± 16.55
28.92 ± 22.01
44.76 ± 22.45
55.09 ± 19.99
10001-25000
107(48.0)
57.82 ± 6.76
28.64 ± 21.10
53.45 ± 24.04
61.83 ± 17.74
25000>
40(17.9)
57.90 ± 5.06**
30.60 ± 20.81***
53.80 ± 23.51***
64.72 ± 18.11
Own
198(88.8)
54.97 ± 17.09
29.12 ± 21.07
50.88 ± 24.12
60.11 ±18.77
Rented
25(11.2)
53.76 ± 15.11
28.84 ± 23.30
47.96 ± 20.05
Family Type
Monthly Income
Housing Status
*p=.01 (t-test), **p=.01 (One-way ANOVA), ***p=.05 (One-way ANOVA),
252
59.64 ± 20.1
Impact of Socio-demographic factors on Quality of life in Medical Students of Eastern Saudi Arabia
(30.0%) were from Ist year and 25(11.2%) were from
5th year. The percentages of participants belonging
to nuclear and joint families were 40.4 and 59.6
respectively.
With regard to different domains of quality of life
presented in Table 1 showed that male participants
scored higher in physical health domain of QoL than
female participants, whereas, female participants
scored better than male participants in social domain
of QoL. In terms of academic year, fifth year student’s
scored higher in physical, social and environmental
domain of QoL, while as first year students score
better in psychological domain of QoL. For marital
status, married participants scored higher in physical,
social and environmental domain of QoL. Participants
belong to nuclear families showed higher score in
physical and environmental domain of QoL. In terms
of monthly income, participants having more income
perceive higher score in physical, psychological and
social domain of QoL.
Quality of life according to socio-demographic
factors
Separate multiple linear regression analyses were
performed to identify predictors variables for the
physical, psychological, social and environmental
quality of life, as measured by the WHOQOL-BREF.
Result of multiple regression analysis presented in
Table 2 revealed that five variables, gender (β= -.20),
academic year (β= .25), marital status (β= -.12)
monthly income (β= .11) and family type (β= -.15)
are the strongest predictors in the physical domain
of QoL. The obtained results clearly revealed that
gender was negatively and significantly related to
physical domain. This means that male participants
showed good physical quality of life than female
participants. Result also revealed that marital status
was found negatively and significantly related to
physical domain of quality of life. This indicates
that married students showed good physical quality
of life than unmarried students. Similarly, family
type was negatively and significantly related to
physical domain. This means that students belong
to nuclear families showed good physical quality
of life compared to students living in joint families.
However, results clearly revealed that academic years
were found positively and significantly related to
physical component of quality of life. This indicates
that students who are final stage of their degree
showed good physical quality of life than students
of initial years of their course. Also, study revealed
that monthly income was found positively and
significantly related to physical domain of quality of
life. This means that students having high monthly
income experience better physical quality of life than
students having low monthly income.
For the psychological domain, academic year (β= -.42)
and monthly income (β= .13) were found strongest
predictors. Results clearly revealed that academic
years were found negatively and significantly related
to psychological aspect of quality of life. This
indicates that students who are initial stage of their
degree showed good psychological quality of life
Table 2: Association between Quality of life and Predictor variables
Variables
Physical QOL
Psychological QOL
β
p
β
p
Environment QOL
Social QOL
β
p
β
p
Gender
-.20
.00
.01
.90
.16
.02
-.10
.14
Age
.20
.17
.05
.77
-.14
.38
.13
.38
Academic Year
.25
.05
.01
.54
.00
.35
.02
.01
-.42
Marital Status
-.12
.05
-.11
.12
-.03
.67
-.18
Area of residence
-.01
.85
-.04
.49
-.02
.80
.00
.96
Monthly income
.11
.05
.13
.04
.12
.05
.10
.10
Family occupation
-.05
.42
-.06
.32
-.03
.57
-.05
.38
Fathers education
.06
.39
-.05
.44
-.07
.31
.02
.80
Mothers education
-.01
.89
-.02
.74
-.03
.62
-.02
.81
House status
-.05
.38
.04
.54
-.03
.66
-.03
.53
Family type
-.15
.01
-.03
.63
-.14
.03
-.12
R2
38%
31%
26%
.02
36%
253
Mohammad Ayoob, Abdulrehman Alsultan, Nurjahan Begam, Saleh Al Sumaih, Hamad Waleed Albuali
than students of final years of their course. However,
monthly income was positively and significantly
related to psychological quality of life. This means
that students having high monthly income experience
better psychological quality of life than students
having low monthly income.
In regard to the social quality of life of participants
results of multiple regression presented in Table 2
revealed that gender (β= .16), academic year (β= .54),
monthly income (β= .12), and family type (β= -.14)
were found significant predictors of social quality of
life. The obtained results clearly revealed that gender
was positively and significantly related to social
domain of quality of life. This means that female
participants showed good social quality of life than
male participants. Similarly, academic years was
positively and significantly related to social domain.
This indicates that students were in final phase of
their course showed good social quality of life as
compared to students who are in initial years of their
courses. Also, monthly income was positively and
significantly related to social quality of life. This
shows that students having high monthly income
experience better social quality of life than students
having low monthly income. However, family status
was negatively and significantly related to social
domain. This means that students belong to nuclear
families showed good social quality of life compared
to students living in joint families.
Result
of
regression
analysis
predicting
environmental domain from different demographic
factors presented in Table 2 revealed academic year
(β= .35) and marital status (β= -.18) as significant
predictors of environmental quality of life. These
results clearly indicate that academic years were
positively and significantly related to environmental
domain. This means that students were in final phase
of their course showed good environmental quality
of life as compared to students who are in initial
years of their courses. However, result revealed that
marital status was found negatively and significantly
related to environmental component of quality of
life. This indicates that married students showed
good environmental quality of life than unmarried
students.
Discussion
The present study was conducted to examine the role
of demographic factors that potentially influence
254
the quality of life among undergraduate medical
students attending college of medicine, King Faisal
University, Saudi Arabia. It was hypothesized
that demographic factors will significantly predict
quality of life of medical students. The results
showed a significant relationship between various
socio-demographic variables and quality of life. The
findings of the study revealed that gender, academic
year, marital status, monthly income and family type
were found significant predictors of quality of life
among medical students.
Gender effects
Findings of the present study regarding
gender differences in quality of life showed significant
differences between male and female participants
on the measure of WHOQOL-BREF. In the domain
of physical health, male participant reported better
quality of life in comparison to their counterpart
female participants. These results regarding gender
differences in quality of life of the participants
extended support to the previous studies showing
differences between males and females in quality
of life.15-19 Better quality of life reported by the male
participants in the present study may be because of a
tendency to perceive their personal health as excellent
and that they always feel energetic, as compared with
females. This is in line with the cultural norms of the
Saudi Arabia where men are generally considered to
be the stronger and dominant sex, thus men perceive
themselves to be so. These factors may be more
responsible for better quality of life among male
medical students. However, these issues could not
appropriately be explained by our study and requires
further investigation. In the present study, female
students scored higher than males in social domain of
quality of life, studies shown that females are better
than men in dealing with interactions.20 Moreover,
female medical students had handful of experiences
and maturity to manage their social relations and the
environment in comparison to male students.
Academic years
Regarding effect of academic years on quality of life
among medical students, the present study observed
significant relationship of academic years to different
domains of quality of life. In the present study, it was
observed that fifth year student’s scored higher in
physical, social and environmental domain of QoL.
A similar kind of research among Chinese medical
Impact of Socio-demographic factors on Quality of life in Medical Students of Eastern Saudi Arabia
students reported higher scores in psychological
domain and social domain of QoL in clinical years
as compared to third year students.21 Another study
conducted on Brazilian medical students indicated
significant differences in the quality of life according
to academic year, particularly with respect to the
psychological and physical domains.22 The reasons
for better quality of life during later years in medical
course are that the students had less theoretical
content, more experience and maturity to improve
their quality of life in comparison to students of
preclinical years.
Marital Status
Several researches have suggested the effect of marital
status on the quality of life in university students.23
In the present study, the married students have better
physical and environmental domain of quality of life
as compare to the unmarried student. The results are
in line with previous studies examined marital status
and quality of life.24 Previous findings reported that
married people have strong mental health than those
who are single due to the social support received
from their spouse.25 Another research examined the
relationship between marital status and mortality. The
results indicated that people who were single showed
higher mortality in particular diseases compared
with those who were married.26 In the present study,
good quality of life among married students may be
because of care and support provided by their spouses
especially during the tough times of their study.
Monthly income
To the best of our knowledge, this is the first study
to examine the relationship between socio-economic
status and quality of life among undergraduate
medical students. The findings of the present study
indicates that students with high monthly income
showed better physical, psychological and social
quality of life as compared to the students with low
monthly income. These results regarding relationship
between monthly income and quality of life of the
participants received extended support from previous
studies.27 Previous findings suggests that low income
badly affects self-esteem, blocked aspirations,
increase frustrations, reduce efficacy, fatalism and
lower mastery and personal control.28
Family type
Regarding the role of family type on quality of life
among medical students, the present study indicated,
the quality of life is better among the students belongs
to nuclear families as compared to joint family
subjects. This result is consistent with the findings
from a study conducted in India, which suggested the
quality of life is higher in nuclear family participants.29
A nuclear family is a very small family having only
a few members, less engagements, more freedom,
more attention to children and less responsibilities.
However, nuclear family faces lot of problems of
lower tolerance among individuals, lower maturity,
more dependency, adjustment issues and egoism.
Other studies also indicates that living in nuclear
family unit is related to better health among males as
compared to females, than other family structures.30
Several limitations of the present study must be
noted. First, the data of the present study were
collected from only one medical college from eastern
region of Saudi Arabia. Data gathered in this context
may therefore be unique, and it is entirely possible
that a replication of this study in a different parts of
the country might yield different results. Second, the
convenience sampling method of medical students
in eastern region is not likely to be representative
of all medical students studying in other regions
of the Saudi Arabia. Therefore, further study needs
representative samples in order to establish the
generalizability of findings on medical students
studying in other parts of the country. Third, the
cross-sectional design used in the present study does
not allow drawing conclusions regarding causality.
Longitudinal research will be needed to support such
conclusions. Lastly, sample size of the present study
was relatively small and homogeneous which also
limits generalization.
The present study demonstrated the differences in
scores of different domains for the quality of life
related to various demographic factors. Gender,
academic year, marital status, monthly income and
family type are found strongest predictors of quality
of life. Betterment in the quality of life of medical
students is very important to ensure the good medical
education system. It is also important to maintain a
well-balanced academic environment for improved
learning experience. A focus on student needs and
problems can help prevent the harmful effects of stress
on quality of life. In addition, It is also suggested
that all required facilities should be provided to
255
Mohammad Ayoob, Abdulrehman Alsultan, Nurjahan Begam, Saleh Al Sumaih, Hamad Waleed Albuali
the medical students and programmes like career
counseling and stress management training should be
executed for the students to reduce their mental stress
and improve quality of life during medical education.
Conflict of Interest Statement
There are no conflicts of interest or any financial or
personal relationships with other people or
organizations.
Role of funding sources and author’s contribution:
The funding sources did not play a role in the study
conceptualization or design; collection, analysis, or
interpretation of data; writing of the manuscript; or
the decision to submit the article for publication.
Acknowledgment
I would like to thank everybody who was important
to the successful realization of this study, especially
the participants who took this work seriously enough
to complete the study and pass the message on. Also,
our special thanks to the college administration for
allowing us to conduct this study.
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POLÍTICAS PÚBLICAS E DIREITO À SAÚDE: A TENSÃO ENTRE OS PODERES – A
NECESSIDADE DE ESTABELECER CRITÉRIOS JUDICIAIS PARA INTERVENÇÃO
Leonardo Grison1
Têmis Limberger2
RESUMO
O direito a saúde é um direito social e deve ser implementado por meio de
políticas públicas. A experiência de Estado Social de outros países é diferente do Brasil.
As políticas públicas são mandatos formulados pelo legislador e realizados pelo
administrador. A discricionariedade administrativa encontra limites constitucionais. Nos
caso de ineficiência ou omissão na execução das políticas públicas, cabe a intervenção
judicial. As ações coletivas são o foro mais adequado para esta discussão. As ações
individuais são um mecanismo de pressão. A cultura democrática implica no
reconhecimento dos limites e possibilidades do controle judicial para efetividade do
direito á saúde.
PalavrasChave: Controle Judicial. Direito à saúde. Estado Social. Políticas
públicas. Resposta adequada.
INTRODUÇÃO
Os direitos não são um dado, mas um construído. Podese formular esta idéia a
partir do pensamento de Hannah Arendt, que concebe os direitos humanos a partir da
construção da igualdade e a cidadania como o direito a ter direitos. O primeiro direito
humano é o direito da ter direitos. Isto significa pertencer pelo vínculo da cidadania, a
algum tipo de comunidade juridicamente organizada e viver numa estrutura onde vige o
princípio da legalidade, evitandose o surgimento de um novo estado totalitário de
natureza3. Dentro desta perspectiva, quando se comemoram 20 anos da promulgação
da Constituição Cidadã, a questão principal não é mais a declaração de novos direitos,
mas a busca pela efetividade dos mesmos. Aí se insere o direito à saúde, que por ser
um direito social, requer a disponibilidade orçamentária para sua implementação. O
direito à saúde teve a sua trajetória de construção nestas duas décadas, em que pese
muito ainda ter de ser feito.
No Brasil, devido à falta de planejamento, são despendidas elevadas quantias
quando se trata da implementação dos direitos sociais. Ao invés de haver uma atitude
de prevenção, gastase muito mais na reparação. Podese pensar na questão da
educação, moradia, segurança, transporte, trabalho, previdência social, lazer, saúde,
etc. Todos estes direitos sociais previstos no art. 6º, “caput”, da CF se prestam para
investigação, mas devido a um corte metodológico necessário, é imprescindível
delimitar a questão. Dentro desta perspectivas, os dois direitos sociais que têm por
parte dos poderes públicos as chamadas “verbas carimbadas”, no sentido de que o
151
percentual de recursos já se encontra previamente definido são: educação (art. 212, da
CF) e saúde (art. 198, § 2º, da CF), daí se constata a importância de investimento para
estes setores tão prioritários para que o Brasil seja no futuro, um país sem tantas
desigualdades sociais. A relação que se pode estabelecer entre estes dois direitos
sociais: educação e saúde, é no sentido de que quanto maior o nível de instrução mais
saudável é a população, via de regra. Estudos4 demonstram que a educação é uma
ferramenta importante, quando se está a mencionar algumas problemáticas, tais como:
mortalidade infantil, gravidez precoce e número de filhos por núcleo familiar.
O trabalho optou por abordar o direito à saúde, especialmente nestes últimos
anos da recente história constitucional democrática brasileira. O desafio que se impõe é
como fazer com que o direito à saúde não seja um comando meramente programático e
se torne efetivo.
Diante desse quadro, impõese a seguinte indagação: quais as possibilidades e
limites do controle judicial, atinente às políticas públicas que visem à implementação do
direito à saúde?
Para responder a esta questão, algumas tomadas de posição são necessárias,
tais como: por que vigora(ou) no direito administrativo brasileiro o dogma da
impossibilidade de revisão dos atos administrativos, que se albergavam sob o manto da
discricionariedade administrativa; qual o conceito de política pública; quais os critérios
para se estabelecer a intervenção do poder judiciário nesta seara.
O trabalho se propõe a uma abordagem das políticas públicas como
determinações constitucionais, ou seja, mandatos a serem realizados pelo
administrador, sujeitos, portanto, ao controle judicial. Assim, tarefas que são previstas
na Constituição e que devem ser realizadas de acordo com a capacidade orçamentária,
mas que não se constituem em uma discricionariedade administrativa, não ficando,
portanto, imunes à questionamento judicial. Pretendese, então, revisar o conceito de
discricionariedade da administração que foi transposto equivocamente do direito
francês ao direito brasileiro e também, a forma de realização dos direitos sociais, aonde
a doutrina alemã, não se adapta totalmente à realidade brasileira. Por isso, a
necessidade de desenvolver uma teoria, voltada às especificidades de nosso país,
desde a ótica de uma hermenêutica comprometida com uma nova crítica do direito e, ao
final, a busca de uma solução adequada constitucionalmente.
O direito administrativo é o direito constitucional concretizado, em uma síntese,
formulada pela doutrina alemã5. Assim, o administrador na realização das políticas
públicas deve observar os preceitos constitucionais. O mandato político não deve ser
desempenhado conforme critérios subjetivos do governante, mas representam políticas
públicas a serem desenvolvida em conformidade com os ditames constitucionais.
Direitos sociais importantes foram consagrados no art. 6º da CF, visando a execução
pelo administrador. Porém, estes preceitos não têm sido cumpridos, operase uma
situação de crise, pois o Estado brasileiro não foi capaz de atender às demandas do
liberalismo clássico, com os direitos de cunho individual e tampouco conseguiu realizar
as demandas do Estado Social. O Estado imiscuiuse em atividades que não eram
152
próprias do poder público e deixou de realizar atividades que eram prioritárias.
Atualmente, não se afigura um projeto político claro com realização às demandas
prestacionais sociais. Como conseqüência, os serviços públicos não são prestados ou o
são de forma deficiente.
Assim, no Brasil os direitos sociais para que sejam efetivos demandam um
caminho diferente dos países europeus, onde estes são uma determinação da
Constituição ao legislador ordinário ou ao administrador. Em nosso país, diante da
particularidade própria em que estes comandos não são atendidos por seus
destinatários, é importante a intervenção do Poder Judiciário. O desafio ocorre, no
sentido de verificar os limites em que ocorre a atuação do Poder Judiciário, que não
pode se substituir em atividades destes outros dois poderes, mas que tem de propiciar
a efetividade dos direitos fundamentais sociais, quando decorrentes de políticas
públicas insuficientes ou inexistentes.
A realização dos direitos sociais é de suma importância, principalmente em
países de modernidade tardia6 como o Brasil. Na época em que foi realizada a
Constituinte, viviase uma euforia como se a partir da colocação das garantias na
Constituição fosse haver uma transformação em nossa realidade. Pairava na sociedade
a idéia de pensamento mágico, como se da previsão na Constituição fosse advir uma
modificação na situação fática. É claro que a garantia representa um compromisso
importante em termos de desenvolvimento de políticas públicas, isto é, que país
queremos ser no terceiro milênio.
Tiveram que se passar quase 20 anos para que se começasse a amadurecer no
sentido de que os direitos sociais fossem relacionados com os dispositivos
orçamentários. É o que Canotilho denomina de “Constituição Orçamental”7. As
medidas de gestão orçamentária são importantes quando se pretende a realização dos
serviços públicos.
Questões vitais como saúde, educação, segurança e moradia, reclamam para
sua implementação dispêndios por parte do poder público, que precisa contar com
disposições orçamentárias. Assim, o administrador quando concretiza uma política
pública encontra no orçamento o limite objetivo da reserva do possível. Da mesma
forma, diante da omissão ou precariedade da implementação da política pública, o
Poder Judiciário fica também adstrito ao orçamento, mas pode pronunciarse quando
provocado, para efetivar os direitos sociais. Durante muitos anos havia a errônea
concepção de discricionariedade administrativa que servia para agasalhar todos os
desmandos ou ineficácia do executivo. Sob o manto do ato discricionário não poderia o
judiciário se imiscuir na atividade da administração. As ações judiciais interpostas e
algumas decisões judiciais indicam que houve uma mudança de posição, é claro que
isto não significa suprimir ou substituir a atividade administrativa, mas buscar a
efetividade dos direitos sociais.
Da análise das decisões jurisprudenciais proferidas pelo Supremo Tribunal
Federal (preponderantemente) em matéria de políticas públicas, especialmente as que
visam à implementação do direito social à saúde é que se terá um perfil dos limites e
153
critérios de atuação do Poder Judiciário, tomandose como referência a promulgação da
atual Constituição Federal. Deste modo, o trabalho a ser desenvolvido, utilizará o
pensamento de Hannah Arendt, partindose da idéia de que os direitos não são um
dado, mas um construído, neste aspecto o direito à saúde representa um marco
importante de desenvolvimento, no sentido de que sua evolução vem sendo construída
pelos operadores do direito, de modo a promover condição de dignidade para o
cidadão.
Com estes balizadores será possível travar uma discussão da eficácia e limites
de implementação das políticas públicas de concretização dos direitos sociais, quando
são submetidas ao crivo do poder judiciário, especialmente no que diz respeito ao
direito à saúde, não no sentido de sentenças formuladas com critérios eminentemente
subjetivos, mas a decisão adequada8, desde o ponto de vista constitucional.
1 O DOGMA DA IMPOSSIBILIDADE DE REVISÃO JUDICIAL DOS ATOS DA
ADMINISTRAÇÃO, SOB O MANTO DA DISCRICIONARIEDADE ADMININISTRATIVA
Durante muitos anos no direito brasileiro vigorava o entendimento da
impossibilidade de revisão judicial da atividade administrativa. O controle judicial dos
atos da administração não era realizado, fundado na justificativa da discricionariedade
administrativa. Esta posição que vigorou em nosso país de forma inconteste, ainda
encontra seguidores, embora se encontre superada, por no mínimo, três motivos: a
impossibilidade de revisão dos atos administrativos por parte do judiciário, que é
doutrina transposta do direito francês, onde existe uma especificidade que é o
contencioso administrativo, que no direito brasileiro não encontra similar. O segundo
motivo, reside na teoria transposta do direito alemão aonde os direitos sociais são
comandos endereçados ao legislativo e administrativo, não cabendo ao judiciário este
controle. Ocorre que naquele país estes poderes realmente levam a sério a
implementação destas políticas públicas, sem que seja necessário a interferência do
judiciário. Por fim, o terceiro aspecto a ser considerado, é que no Estado Democrático
de Direito, somente existe escolha do administrador vinculada aos preceitos
constitucionais, em especial pela pauta dos direitos fundamentais. O Poder Judiciário
quando efetua esta decisão não pode cair na casuística, autorizando ou negando todas
as pretensões que lhe vêm a julgamento, sem considerar os dispositivos orçamentários,
mas deve buscar a solução adequada constitucionalmente, e aí reside a questão.
Com esta perspectiva, pretendese desenvolver o trabalho para investigar os
limites em que são controladas as políticas públicas judicialmente. Assim, o
questionamento judicial destes atos da administração vêm ocorrendo, mas o importante
é apontar os critérios de como este vem se implementando, para que o judiciário não se
substitua à atividade do administrador e tampouco o administrador fique livre dos
controles judiciais. Esta sindicabilidade tem de ocorrer de forma a concretizar os
preceitos estabelecidos pela Constituição Federal. As grandes questões do direito
público neste terceiro milênio dizem respeito à possibilidade de controle judicial dos
atos da administração referente às políticas públicas.
154
Em um país como o Brasil em que os direitos sociais ainda não estão
assegurados para grande parte da população, a pergunta que se faz é a seguinte: fica
ao alvedrio a implementação destas políticas públicas por parte do Executivo ou o
Poder Judiciário pode ser um importante mecanismo de pressão, na implementação
destes direitos e em que limites? Assim, exemplificativamente, referente ao direito à
saúde, o que a Constituição objetiva assegurar: um tratamento caro no exterior para
uma só pessoa ou o tratamento de doença epidemiológica relevante para uma parcela
expressiva da sociedade? Por isso, quando está ausente ou insuficiente uma política
pública, o judiciário deve agir, mas com critérios e limites, sob pena de se cair no
subjetivismo judicial. Daí advém a necessidade desta investigação, pois em casos de
omissão ou atuação precária do administrador, por vezes o judiciário manifestase de
forma tímida ou sem critérios. Visase com este estudo contribuir para esta questão, no
sentido de apontar critérios formulados a partir dos preceitos constitucionais, sem que
se caia em um casuísmo destituído de fundamento legal.
Com relação ao primeiro aspecto, a inadequação do modelo francês transposto à
realidade brasileira, fazse um retrospecto a respeito dos controles da administração.
Podese afirmar que existem dois sistemas: o da unidade e o da dualidade da
jurisdição. O sistema da unidade ou da dualidade da jurisdição decorrem da
interpretação do Princípio da Separação dos Poderes. É importante salientar que a
doutrina a que se convencionou a denominação “Separação dos Poderes”9, não foi
assim conceituada por seu autor Montesquieu. Da leitura de sua obra, extraemse duas
idéias principais, quais sejam: a distinção tripartite das funções do Estado e a idéia de
controle recíproco entre estas atividades. Em momento algum escreveu o autor a
palavra “separação dos poderes”, isto decorreu da interpretação principal de sua obra,
mormente a apropriação da doutrina que se constituiu em um dos paradigmas
apregoados pela Revolução Francesa. A interpretação e crítica de Louis Althusser10,
não teve fortes ecos, no sentido de que Montesquieu era de origem nobre, conhecido
como Barão de La Brède e formulou a teoria apenas para fortalecer o poder
monárquico, na época em que este se encontrava em crise.
Na França, berço do princípio da Separação dos Poderes devido à doutrina de
Montesquieu, este foi interpretado de forma drástica como a impossibilidade de um
poder exercer o controle sobre outro. A origem histórica desta interpretação encontrase
na desconfiança que os legisladores da Revolução Francesa tinham para com o Poder
Judiciário, pois este tinha se mostrado resistente às conquistas populares. Waline11
assevera que a Lei nº 16, de 24 de agosto de 1790, dispunha sobre a organização
judiciária e proclamava a separação das funções administrativas e judiciais, que foi
reafirmada com o período que se seguiu à Revolução Francesa. Houve proibição legal
expressa aos juízes do conhecimento da matéria administrativa. As reclamações com
conteúdo administrativo não poderiam ser, em nenhum dos casos, encaminhadas aos
tribunais. Deveriam ser submetidas ao rei, então chefe da administração geral12 .
Por contencioso administrativo, entendese o conjunto de regras relativas aos
litígios organizados que questionam a atividade dos administradores públicos. O
progresso do direito administrativo francês deveuse à existência de um contencioso.
Essa instituição garantiu o desenvolvimento do direito propriamente dito, por meio das
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garantias de legalidade criadas, pela importância do aspecto moral da conduta dos
administradores, dando respaldo às teorias subjetivas.
O recurso por excesso de poder é uma criação jurisprudencial devido ao próprio
Conselho de Estado, constituindose o principal instrumento de controle da legalidade
administrativa13. Assim, o Conselho de Estado14 é um órgão de fiscalização dos atos da
administração, situado dentro da próprio Executivo e que desempenha suas funções
decisórias com imparcialidade e em consonância com a legalidade. A justificativa é no
sentido de que o legislativo é o poder constituído a partir da vontade popular e não seria
democrático, a substituição da representação da soberania do povo pelo poder
judiciário, que não é eleito democraticamente.
Esta questão é bem enfrentada por Sérgio Cademartori15, que analisa a questão
da legitimidade. Assim, o poder legislativo e poder judiciário possuem intervenção
calcados em pressupostos de legitimidade distintos. A legitimidade do poder legislativo
decorre da idéia de soberania popular. O poder legislativo é o representante da
população, em que pese poderem ser feitas críticas ao modelo representativo16. Com
pressuposto diferente de legitimidade se encontra o poder judiciário, que possui seu
fundamento no Estado de Direito.
Basicamente, são estas as razões de ordem histórica pelo qual não se permite a
revisão dos atos administrativos no direito francês, que consagra a dualidade de
jurisdição. Esta doutrina é inaplicável ao direito brasileiro em virtude do art. 5º , XXXV,
da CF que consagra a inafastabilidade do controle judicial de todos os atos. Logo, os
atos da administração que traduzem opções de políticas públicas não podem ficar à
margem do questionamento judicial, o desafio está em estabelecer estes critérios.
2 DIREITOS SOCIAIS NO BRASIL E EM PAÍSES QUE REALIZARAM O
ESTADO SOCIAL TRAJETÓRIAS DISTINTAS
O segundo motivo de análise, são as especificidades do modelo alemão que
foram equivocadamente transpostas para o Brasil, sem as necessárias adequações.
Assim, no dizer de Hesse17: “ derechos sociales fundamentales como por ejemplo (...) el
derecho a una vivienda adecuada (...) no se hacen ya efectivos por el hecho de que se
respeten y amparen, sino que requieren de antemano, y em cualquier caso más que en
los derechos fundamentales tradicionales, acciones del Estado tendentes a realizar el
programa contenido en ellos. No sólo exige esto regularmente un actuar del legislador,
sino también el de la Administración; y puede afectar a los derechoslibertad ajenos.
Por ello, los derechos fundamentales no alcanzan a justificar pretensiones de los
ciudadanos invocables judicialmente en forma directa, como cuadraría a la doctrina de
los derechos fundamentales”.
É claro que os direitos sociais têm uma estrutura distinta dos direitos individuais,
do liberalismo clássico. São comandos voltados prioritariamente para o administrador e
legislador, e para a sua implementação dependem de possibilidade orçamentária.
Porém, no contexto brasileiro, diante da inoperabilidade destes poderes, por vezes, o
judiciário representa um fator importante para pressionar a realização das políticas
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públicas, visando assegurar a dignidade da pessoa humana, composto pelo mínimo
existencial18. Um exemplo frutífero que pode ser apontado é o tratamento da AIDS em
nosso país. Diante da inicial omissão dos poderes legislativo e executivo, no sentido de
promover políticas públicas, no tocante à prevenção e tratamento do HIV, ações
reiteradas no âmbito judicial pedindo medicamentos, incentivaram a que o Brasil se
tornasse um país referência nesta área.
Deste modo, apropriadas as considerações de Andreas Krell19, conhecedor das
realidades alemã e brasileira: “ Face aos problemas sociais candentes de um país
periférico como o Brasil, o princípio tradicional da Separação dos Poderes deve ser
entendido sob os parâmetros e dimensões novas e diferentes dos das nações centrais.
Ainda não foram aproveitadas as potencialidades dos modernos instrumentos
processuais do direito brasileiro para correição judicial das omissões dos Poderes
Executivo e Legislativo na área das políticas públicas, como a ação civil pública e a
ação de inconstitucionalidade por omissão”.
Alguns países como a França e Alemanha, em que o Estado Providência20
cumpriu seu papel, assegurando os direitos sociais à população é adequado que se
discuta a permanência ou a diminuição de algumas garantias, devido à proporção
robusta que estas alcançaram. Diferentemente, no Brasil, em que a população não
conseguiu atingir níveis homogêneos em termos de acesso aos direitos sociais.
A “reserva do possível”21 tem sido outro conceito mal transposto ao direito
comparado. Esta teoria é adaptação de uma expressão cunhada no direito alemão, que
entende que a construção de direitos subjetivos à prestação material de serviços
públicos pelo Estado está sujeita à condição da disponibilidade dos respectivos
recursos, ou seja, no campo orçamentário.
Assim, é necessário para avaliar em que limites está ocorrendo o provimento
judicial a respeito das políticas públicas, em um país como o Brasil, com especificidades
próprias, principalmente no que diz respeito às grandes desigualdades econômicas e
culturais. É perigoso importar diretamente conceitos cunhados em outros países com
contexto cultural e sócioeconômico diferentes. O Brasil, que é um país em
desenvolvimento não pode transportar diretamente teorias de países ricos. O debate
europeu sobre a redução dos direitos conquistados durante o Estado Social não pode
ser transferido, porque o Estado Providência nunca foi efetivado plenamente.
Ainda, concernente à eficácia, importante a posição de Ingo Sarlet22, que
posiciona os direitos de cunho prestacional como autênticos direitos fundamentais
constituindose, por isso, direito imediatamente aplicável, nos termos do art. 5º, § 1º, da
CF. Por isto, no embate entre o Estado Social intervencionista e altamente regulador e
a nefasta tentativa de implementar um Estado minimalista desenhado a partir da
globalização e da ideologia neoliberal, o correto manejo da proibição de retrocesso na
esfera dos direitos fundamentais sociais, poderá se constituir uma ferramenta útil para a
construção do perfil do Estado brasileiro, com dignidade para cada indivíduo e,
portanto, uma vida saudável para todos os integrantes do corpo social23.
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Deste modo, o trabalho abordará a da importância dos poderes executivo e
legislativo na implementação de políticas públicas e o papel do judiciário na busca de
efetividade do direito social à saúde, fixando os seus limites e possibilidades de
atuação.
3 O CONTROLE JUDICIAL E SUA PERSPECTIVA CONSTITUCIONAL
No Brasil, os poderes executivo e legislativo não têm implementado as políticas
públicas a contento, por isso, a discussão se insere na órbita judicial. Neste contexto,
outro aspecto a ser considerado é a sindicabilidade das políticas públicas como
decorrência do Estado Democrático de Direito. Atualmente não existe um poder
discricionário livre, mas somente um poder discricionário vinculado24. Vinculação esta,
que se pode relacionar aos ditames constitucionais. Desta maneira, o conteúdo das
políticas públicas não pode ficar ao alvedrio do administrador, mas tem de buscar
efetivar os preceitos contidos na Constituição.
O direito administrativo se estruturou no século XIX, a partir do Princípio da
Legalidade, este foi o primeiro grande freio que foi imposto ao administrador, porém
atualmente com a denominada fuga do direito administrativo ou a tendência
neoliberalizante do Estado, ocasionando a chamada eficácia com relação a terceiros
“drittwirkung”25, novos pressupostos devem ser erigidos. Para García de Enterría26, o
aspecto subjetivo da Justiça Administrativa, que rompeu o mito histórico de sua suposta
objetividade, atrás da qual de escondia uma superioridade formal da Administração
sobre o cidadão, considerado ainda como súdito, que deveria ceder ante a suposta
superioridade dos “interesses gerais”, gestionados pela administração, é uma conquista
irrenunciável do nosso tempo.
Por isso, o grande desafio que é posto aos estudiosos do direito da administrativo
na atualidade é esta possibilidade de controle judicial. Afirma, Moreira Neto27, que há
atuações administrativas constitucionalmente mandatórias, como é o caso das políticas
públicas nacionais e não apenas governamentais, da educação, da saúde e da
segurança pública. Assim, o Estado Democrático de Direito reclama a implementação
dos direitos fundamentais, por meio das políticas públicas. Deste modo, a atividade
administrativa no terceiro milênio deve ser pautada dentro dos balizadores que
compõem o Estado Democrático de Direito: os direitos do homem e a democracia
substantiva. A partir destes dois vetores são estatuídos os quatro paradigmas atuais do
direito administrativo28: legitimidade, finalidade, eficiência e resultados. Devem ser
analisados de forma conjunta e não isoladamente, tendo em vista a realização do
serviço público. Estes critérios serão necessários quando são exercidos os controles
com relação à atividade administrativa quer no âmbito preventivo ou repressivo.
Assim legitimidade e finalidade, que eram requisitos intrínsicos dos atos
administrativos, demandam um novo olhar.
No tocante à legitimidade serão
constitucionais as prestação públicas que respeitem os direitos fundamentais. Não
basta mais somente a legitimidade fundada no que se denominava pacto social, mas
devese perquirir a respeito do atendimento dos valores que visam implementar a pauta
dos direitos humanos, principalmente quanto estes completam 60 anos29. Assim a
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legitimação é, sobretudo, um fenômeno antropológico indissociável do consenso
alcançado pelos grupos sociais, que consiste na verificação da concordância com os
fenômenos políticos e valorativos relacionados ao poder, ou seja, à sua concentração, à
sua distribuição, ao seu acesso, ao seu exercício e ao seu controle30. Concernente à
finalidade, estamos sob a época da redescoberta dos valores e das finalidades, devese
buscar identificar a finalidade nos princípios.
A eficiência é um princípio trazido pela EC 19/98, que traduz um aspecto
axiológico, no sentido de que o direito administrativo deve e pode ser um instrumento
para a realização dos direitos fundamentais e facilitador do desenvolvimento econômico
e social.
Após o aspecto valorativo ocorre o último paradigma que é o do resultado. Cuida
se de mensurador prático, em que são buscadas técnicas de controle da gestão31.
Devese atentar para a prestação do serviço público, que possui um espectro distinto
da seara privada. Já foi mencionado o fenômeno da eficácia horizontal dos direitos
fundamentais, onde os conflitos migraram da seara pública à privada. É a denominada
fuga do direito administrativo em direção ao direito privado32. Isto aconteceu com a
questão da saúde, também. Há porém especificidades próprias, pois a iniciativa privada
somente se interessa por setores aonde há a possibilidade de lucro. As áreas
deficitárias são suportadas pelo poder público. Assim, a prestação de serviço pelo SUS
responde por aproximadamente 75% população brasileira33. Isto demonstra que os 25%
restantes incumbem à iniciativa privada porque são setores com a potencialidade de
lucro. Desta forma, o princípio da eficiência deve ser aferido com características
próprias e não com os mesmos balizadores privados.
4 CONCEITO DE POLÍTICA PÚBLICA, DESDE O PRISMA JURÍDICO, COM
ÊNFASE NO DIREITO À SAÚDE
O conceito de política pública é um conceito que partiu da Ciência Política e da
Administração Pública, mas que hoje encontra importante espaço de reflexão no campo
jurídico. Assim, a definição apresentada na ciência política é a dimensão normativa ou
moral do Estado, que perpassa a filosofia política ocidental e objetiva garantir uma vida
feliz ao cidadão34. A questão da política pública apresenta um núcleo com interseccção
na organização do sistema internacional, de organização na sociedade e da
organização do Estado. Não se apresenta como algo isolado, mas que deve dialogar
tendo em conta os três vértices: internacional, estatal e social.
Assim, já se percebe porque o tema das políticas públicas entra na pauta jurídica,
nos últimos tempos. Da passagem do Estado Liberal com sua feição de abstenção35,
chegase ao Estado Social com seu conteúdo de intervenção, a partir das demandas
concretas formuladas pelo cidadão. É o constitucionalismo social inaugurado no início
do século XX, depois da Revolução Industrial e do Socialismo36, que tem sua marca na
Constituição Mexicana de 1917, Constituição de Weimar de 1919 e em nosso país, na
Constituição de 1934, da era Vargas. Passado o movimento pendular no sentido de
constituições antidemocráticas e democráticas, chegase à Constituição de 1988, onde
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a democracia começa a se estabilizar e confere margem para a efetividade dos direitos.
Deste modo, a discussão das políticas públicas ganha espaço no cenário jurídico.
Um delineamento a respeito do tema das políticas públicas é trazido por Ana
Paula Bucci, como programas de ação destinados a realizar, sejam os direitos a
prestações, diretamente, sejam a organizações, normas e procedimentos necessários
para tanto. As políticas públicas não são, portanto, categoria definida e instituída pelo
direito, mas arranjos complexos, típicos da atividade políticoadministrativa, que a
ciência do direito deve estar apta a descrever, compreender e analisar, de modo a
integrar à atividade política os valores e métodos próprios do universo jurídico37.
Uma questão que surge é no sentido de delimitar quais as ações que conduzem à
implementação das políticas públicas, considerandose as de cunho individual e
coletivo. Estas últimas não suscitam dúvidas. A indagação diz respeito às ações
individuais, prestamse estas para estatuir políticas públicas? É sabido que o
art. 5º,
XXXV, CF consagra o acesso à justiça de forma ampla e irrestrita. Assim, não se pode
impedir o acesso, mas a concessão de medicamentos não previstos como essenciais,
demanda uma atenção para a sua decisão. O art. 196 da CF conjuga o direito à saúde
a políticas sociais e econômicas, para que seja possível assegurar a universalidade das
prestações e preservar a autonomia dos cidadãos, independente do seu acesso maior
ou menor do Poder Judiciário. “Presumese que Legislativo e Executivo, ao elaborarem
as listas referidas, avaliaram, em primeiro lugar, as necessidades prioritárias a serem
supridas e os recursos disponíveis, (...)”38.
Este pressuposto, é corolário de um argumento democrático. Os recursos obtidos
para o fornecimento dos medicamentos são obtidos pelos tributos suportados pela
população. Desta forma, os representantes eleitos pelo processo democrático devem
estabelecer quais são as prioridades na atual conjuntura. Pode ser simpático o
argumento de ampla concessão de qualquer medicamento, porém isto é falacioso, pois
os recursos orçamentários são limitados em qualquer país, não é possível pretender
fazer tudo a qualquer gasto. O argumento de que os recursos públicos são mal
utilizados e, por vezes, desviados, não é cabível nesta discussão, embora seja
necessária a devida responsabilização na órbita da improbidade administrativa. Como
os recursos são limitados não se pode pretender o pagamento de um medicamento, por
vezes com valor altíssimo, não testado suficientemente, em prol de uma lista estatuida
a partir dos critérios legais e de implementação do executivo.
Por isso, importante a decisão proferida pela Ministra Ellen Gracie na
SS
39
3073/RN que considerou inadequado fornecer medicamento que não constava da lista
do Programa de Dispensação em Caráter Excepcional do Ministério da Saúde. A
Ministra enfatizou que o Estado do RN, não estava se negando à prestação dos
serviços à saúde e que decisões casuísticas, ao desconsiderarem as políticas públicas
definidas pelo Poder Executivo, tendem a desorganizar a atuação administrativa,
comprometendo as políticas de saúde ainda incipientes.
O caráter de importância nas ações individuais somente se pode vislumbrar,
quando se tornam mecanismo de pressão para implementação das políticas públicas,
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como aconteceu nos casos dos portadores de vírus HIV40, em que o Brasil se constitui
em um país com tratamento que é uma referência. Nas primeiras demandas, a
contestação do Estado era no sentido de isentarse de responsabilidade e dizer que se
o demandante havia contraído a enfermidade era porque tinha dado causa a isso, numa
atitude nitidamente preconceituosa.
O foro adequado é o das ações coletivas, aí se pode discutir o arrolamento de
algum outro remédio ou tratamento na lista dos essenciais, vejase a questão atinente à
mudança de sexo41. Houve ajuizamento de ação civil pública pedindo a inclusão na
tabela do SUS da transgenitalização, que foi julgada procedente. Tal redundou em
alteração da posição administrativa, ocasionando a portaria do Ministério da Saúde de
nº 1.707/2008, que incorpora a alteração de sexo, como procedimento oferecido pela
rede pública. Três ponderações para que a discussão se trave em âmbito coletivo são
apresentadas por Barroso42. Por primeiro, a discussão no âmbito coletivo exigirá a
análise do contexto em que se situam as política públicas; por segundo, evitase a
questão da microjustiça, ou seja, a preocupação do juiz com o deslinde daquela ação
esquecendose da macrojustiça, atendimento com recursos limitados a demandas
ilimitadas; por terceiro, a decisão proferida em decisão coletiva terá efeitos “erga
omnes”, preservando a igualdade e a universalidade do atendimento à população.
Assim, a política pública visa a implementação pelo poder executivo de um
comando constitucional. Especificamente, atinente ao direito à saúde, constatase que
com o constitucionalismo social inaugurado no Brasil em 1934, o direito à saúde
aparece por primeira vez em sede constitucional. Os textos seguintes limitaramse a
atribuir competência à União para planejar sistemas nacionais de saúde, conferindolhe
a exclusividade da legislação sobre normas gerais de proteção e defesa da saúde e
mantiveram a necessidade de obediência ao princípio que garantia aos trabalhadores
assistência médica sanitária.
Na Constituição atual, o direito à saúde (art. 6º, CF) é reconhecido como direito
social e um dever do Estado (art. 196, CF) que a Constituição institui obrigações para
todos o entes federados. Constituise em competência comum (art. 23,II, CF) à União,
aos Estados, ao Distrito Federal e aos Municípios, competência concorrente sobre a
proteção e defesa da saúde, competindo à União o estabelecimento de normas gerais e
aos Municípios as suplementares ( art. 24, §§ 1º e 2º, c/c art. 30,II, CF) e cooperação
técnica e financeira com o auxílio da União e dos Estados (art. 30, VII, CF). Nenhum
dos entes federativos está isento de atribuições, isto apresenta dupla crítica no sentido
de que a todos incumbem tarefas, mas por outro lado, quando a responsabilidade é tão
partilhada entre todos, fica mais difícil cobrar a atribuição de cada um. Porém, o escopo
deve ser no sentido de construir a responsabilidade no âmbito individual, familiar e do
poder público, em todas as áreas de atuação, conforme apregoa Rogério Gesta Leal43.
Este balizador de atuação deve ser um objetivo para os juristas.
O Sistema Único de Saúde (SUS) se propõe a ser uma estrutura descentralizada,
com direção única em cada esfera do governo, oferece atendimento integral e conta
com a participação da comunidade (art. 198, CF). Em atenção aos preceitos
constitucionais foram elaboradas as Leis nºs 8.080/90 e 8.142/90, conhecidas como Lei
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Orgânica da Saúde LOS, que são leis nacionais, com o caráter de norma geral, onde
contém as diretrizes e os limites que devem ser respeitados pela União, pelos Estados
e pelos Municípios ao elaborarem suas próprias normas para garantir o direito à saúde
para a população brasileira.
Deste modo, a LOS colabora na definição do direito à saúde, estrutura o SUS,
tornando claro seus objetivos e suas atribuições, as diretrizes que devem orientar sua
organização, direção e gestão, a forma como estão distribuídas as tarefas entre as três
esferas de poder e a forma de participação na comunidade na gestão do sistema em
cada uma dessas esferas, além de organizar o seu financiamento, tratando
expressamente dos recursos, da gestão financeira, do processo de planejamento e do
orçamento, inclusive das transferências intergovernamentais de recursos financeiros.
A questão dos medicamentos, de responsabilidade do SUS, encontra previsão
legal na Lei Federal nº 8.080/90, art. 6º, VI, sendo um dos pontos de difícil
operacionalização quando se trata da política de Estado para a saúde pública. Desde
197544 a Organização Mundial da Saúde – OMS aponta para a necessidade de os
Estados formularem uma política que permita o acesso e favoreça o uso racional dos
medicamentos por todas as pessoas, pois se verificou que o mercado mundial era
composto, por aproximadamente 70% de substâncias não essenciais, desnecessárias e
até perigosas, e que 1/3 da população não tem acesso a medicamentos essenciais45.
Para a OMS, a formulação de uma política de medicamentos implica a definição de um
conjunto de diretrizes com a finalidade de assegurar para toda a população uma
utilização racional dos medicamentos. Isto significa diversos momentos que vão desde
a produção até a prescrição dos remédios46.
No Brasil, foi editada a Portaria do Ministério da Saúde nº 3.916/9847 contendo a
Política Nacional dos Medicamentos, baseada nas recomendações da OMS, visando
fornecer os medicamentos necessários à população mais desprovida de recursos
econômicos.
Importante passo para a acessibilidade dos genéricos foi conseguido por meio
dos medicamentos genéricos, disciplinados pela Lei nº 9.787/99. A partir de então, a
Agência Nacional de Vigilância Sanitária – ANVISA, tem estabelecido por meio de
resoluções como deve ser feito o registro, a fabricação, o controle de qualidade, a
prescrição dos medicamentos genéricos.
5 A BUSCA DE CRITÉRIOS
CONSTITUCIONALMENTE
DENTRO
DA ESCOLHA
ADEQUADA
Feitas as considerações iniciais, no sentido de que é cabível a intervenção judicial
dentro da perspectiva da busca da escolha adequada, passase à parte mais instigante,
que é a busca dos critérios para nortear a interpretação jurisprudencial, em um estudo
preliminar.
Por primeiro, a formulação de políticas públicas cabe aos poderes legislativo e
executivo, uma vez que estas opções se constituem em uma decorrência da
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democracia representativa e, por vezes, da participativa. Não cabe aí uma manifestação
jurisprudencial, portanto.
Por segundo, nos casos de ineficiência ou omissão na execução das políticas
públicas, cabe a intervenção judicial. Aí podem se apresentar algumas situações.
O Judiciário está autorizado a intervir, quando o poder público não fizer o aporte
exigido constitucionalmente à área da saúde, em conformidade com o art. 198, § 2º, da
CF. O foro mais adequado para discutir a inclusão ou exclusão de algum medicamento
é por via das ações coletivas, com legitimados ativos representativos e que permitem
um amplo debate a respeito das prioridades, que são estabelecidas a partir das
determinações da Organização Mundial da Saúde, levando em conta as peculiaridades
de cada país e implementadas pelo Ministério respectivo, após debate junto ao
Congresso Nacional.
Atinente aos medicamentos em experimentação, que não têm a sua eficácia
comprovada não podem ser objeto da inclusão em lista, pelo Poder Judiciário. Devese,
ainda, optar pelo genérico de menor custo, quando houver esta possibilidade.
Nos casos de demanda individual, compreendese a agonia do cidadão que
requer o remédio, da família que passa por uma situação difícil e do magistrado que
muitas vezes se encontra em uma decisão entre a vida e a morte, iminentemente, mas
se trava um embate entre as deliberações públicas e privadas e situações de risco em
curto e médio prazo.
Por exemplo, às vezes se gasta mais na parte curativa do que preventiva. Vejam
se os números que são investidos em saneamento básico e construção de redes
potável, comparativamente à saúde48. Os primeiros são muito tímidos se comparados
aos segundos. E a ausência de investimento em saneamento básico é um foco para
disseminar doenças, posteriormente. Então, na premência do momento, descuidase de
uma perspectiva de futuro.
Deste modo, a concessão dos medicamentos não deve se pautar por uma
abordagem individual dos problemas sociais, mas pela busca de uma gestão eficiente
dos escassos recursos públicos, analisandose os custos e benefícios, desde o prisma
das políticas públicas. Aí se pode estabelecer mais uma reflexão a partir do
pensamento de Hannah Arendt, quando trata da dicotomia público e privado49.
As atividades humanas são condicionadas pelo fato de que os homens vivem
juntos. O termo público denota dois fenômenos intimamente correlatos, mas não
perfeitamente idênticos. Significa, em primeiro lugar, que tudo o que vem a público
pode ser visto e ouvido por todos e tem a maior divulgação possível. Tornase
aparente, aquilo que é visualizado e ouvido pelos outros e nós mesmos, constituindose
na realidade. Por segundo, público significa o próprio mundo, na medida em que é
comum a todos nós e diferente do lugar que nos cabe dentro dele. Se o mundo deve
conter um espaço público, não pode ser construído apenas para uma geração e
163
planejado somente para os que estão vivos: deve transcender a vida de homens
mortais.
Dentro desta perspectiva, as decisões de saúde não devem se pautar pelo
casuísmo, ou no dizer de STRECK50, de modo solipsista, mas dentro de uma
perspectiva de cumprimento do texto constitucional, em busca da resposta adequada
dentro dos parâmetros preceituados pela Constituição Federal, sob pena de não haver
avanços para as próximas gerações.
Preocupado com a legitimidade social das decisões judiciais, e com a intenção de
se evitar a discricionariedade (vale dizer, a arbitrariedade), Lênio Streck faz importante
construção teórica, tendo como parâmetros a Hermenêutica Filosófica de Hans Georg
Gadamer e a teoria integrativa de Ronald Dworkin. Deste, toma para si a importância
dos princípios no processo interpretativo, que trazem para o Direito a faticidade, e
daquele relembra como o sentido, no processo de interpretação/aplicação se constrói
socialmente, e historicamente, através da tradição.
De Dworkin vem a lição de que por trás de cada regra há um princípio instituidor
da norma. Há na verdade uma cooriginariedade entre princípio e norma. “Não há
primeiro uma formação subjetiva de princípios e, depois, sua aplicação compartilhada
no mundo da convivência, mas essa formação principiológica é formada pelo mundo e,
ao mesmo tempo, forma mundo, na medida em que pode articular um significado novo
que exsurge da interpretação do próprio mundo”.51
De plano impõese a apreciação dos princípios no plano caso concreto, eis que
eles não existem em abstrato. No plano fático é que se verifica como o princípio é a
história institucional do direito. Assim, a legitimidade de uma decisão advém do fato que
um princípio institui aquela regra/norma. Sem um princípio, não há legitimidade para a
aplicação da regra. Não se trata, portanto, de, como no caso do direito à saúde,
simplesmente citar o direito à vida e alçálo a condição de princípio. De plano percebe
se que não se trata de um princípio, como é o caso da igualdade, do devido processo
legal, da presunção de inocência, da publicidade dos atos estatais, etc. Princípios
possuem força normativa, e encontram sua realização em uma regra.
A esse passo, cabe abordar outro aspecto importante: o sentido da tradição na
interpretação/aplicação do direito. Gadamer52 mostrou como a interpretação autêntica,
fundada nos préconceitos autênticos, é aquela fundada na tradição. Assim, os
princípios adquirem sentidos porque ancorados na jurisprudência (tal qual o precedente
na tradição anglosaxã, a decisão institui um princípio, que aponta para a mesma
aplicação da regra aos casos subseqüentes), na doutrina, e na própria opinião pública.
No caso do direito à saúde, em especial, costuma se vislumbrar uma afronta à Lei
8.080/90. Esta legislação estabelece como se dará a aplicação do direito fundamental à
saúde, estabelecendose os medicamentos de maior relevância, e a atribuição da
competência de cada ente federativo. Ora, tal lei se presume constitucional, e inserese
naquilo que se pode chamar de tradição. É produzida democraticamente, e a
elaboração da lista dos medicamentos é acompanhada por uma série de instituições
164
sociais (como já abordado no capítulo anterior). Portanto, possui uma história
institucional que precisa ser levada em conta quando da sua interpretação/aplicação.
Isso é uma questão de princípio, tal qual trata Dworkin. Esse sentido dado pela tradição
é que faz com que não haja um esvaziamento hermenêutico do direito constitucional à
saúde, que como cabe lembrar, se faz por meio de políticas públicas, portanto não se
traduz em um direito subjetivo, de matriz liberalindividualista, que autorize o cidadão a
postular para si um “direito à saúde” (o que importaria na exclusão de outros referente a
esse direito, pela própria escassez).
Dessa maneira, fica evidente a postura adequada pelos tribunais ao exercerem o
controle que lhes é próprio relativo à implementação do direito fundamental à saúde.
Deve sempre buscar a resposta correta (nem a única, nem a melhor, como bem
salienta STRECK), vale dizer, adequada à Constituição, o que se faz recorrendose a
tradição, que é quem confere sentido à norma. Portanto, impossível não aplicar a lei
orgânica da saúde, e suas determinações em termos de políticas públicas, a menos
que, em sede de jurisdição constitucional se faça: a) controle difuso da
constitucionalidade, declarando a norma inconstitucional; b) deixese de aplicála em
função de antinomia de normas; c) façase uma interpretação conforma à Constituição,
o que implica em alteração do sentido da lei; d) aplicação da nulidade parcial sem
redução de texto, por via da qual há a exclusão de determinada hipótese de aplicação;
ou e) a declaração de inconstitucionalidade com redução de texto, hipótese em que
excluindose uma palavra, mantémse a constitucionalidade do dispositivo.53
Lembrandose que para isso há de existir profunda fundamentação, no sentido de
desvelar adequadamente os princípios constitucionais.
Portanto, é possível dizer (com STRECK) que existe um direito fundamental à
obtenção de uma resposta correta, e um dever fundamental de uma adequada
fundamentação das decisões judiciais que não de azo à discricionariedade. Somente
recorrendose a tradição, e buscandose substrato nos princípios que orientam a
resolução do conflito sob judice que se dará efetividade ao controle judicial das políticas
públicas. Assim como o fundamento do controle é a Constituição, não pode ser ele
quem a viole. Em vez de um sujeito solipsista que tem na interpretação um ato de
vontade54, é preciso que o magistrado, em tempos de Estado Democrático de Direito,
esteja atento para a necessidade de legitimação das decisões judiciais. É atentando
para o caso concreto e tudo que dele exsurge que é possível controlar a
discricionariedade. Pelo fortalecimento das instâncias democráticas de controle da
saúde é que se dá a efetividade das políticas públicas de saúde, e não pela mera
judicialização da política.
CONCLUSÕES
Na perspectiva de novos temas referentes à administração, o controle judicial das
políticas públicas, é o tema pautado neste terceiro milênio, que coincide com o
processo de redemocratização e reconstitucionalização do país, que é fruto da
mudança de paradigma: a busca de efetividade dos direitos fundamentais. Assim,
pretendese contribuir ao debate, a fim de traçar os limites e possibilidades de atuação
judicial, no tocante às políticas públicas, e construir um horizonte, com perspectiva
165
favorável, para milhares de brasileiros que necessitam da eficácia dos direitos
fundamentais sociais.
No final do século XX, produziramse importantes mudanças no âmbito
econômico, social e cultural, devidos à revolução tecnológica, à globalização, à
circulação rápida da informação e das comunicações, aos processos de integração
econômica e política, ao término da guerra fria, a dissolução da URSS, a queda do
muro de Berlim, e o ataque de movimentos fundamentalistas. Todos estes fatores
contribuíram para uma mudança no direito administrativo que até então havia se
construído com o aporte iluminista e se estruturado a partir do princípio da legalidade.
Com a mudança do aspecto territorial, como um dos elementos do Estado, devido à
globalização, um novo contexto de Estado vem se definindo, conferindo ao direito
administrativo um novo horizonte como a efetividade das garantias dos cidadãos, que
fazem superar a concepção tradicional de busca de equilíbrio das prerrogativas da
administração e as garantias dos cidadãos, para almejar o cumprimento do preceituado
pela Constituição por parte dos poderes públicos, em especial, no que diz respeito aos
direitos fundamentais.
O Estado Social produziu a superação do conceito de interesse público, calcado
exclusivamente a partir dos objetivos da administração e foi substituído pelo interesse
coletivo, aberto à participação popular.
Voltando ao pensamento de Hannah Arendt, percebese que o direito à saúde
está sendo construído, por meio das políticas públicas de implementação de
tratamentos e de medicamentos ditos essenciais, constituindose o Poder Judiciário um
instrumento de realização dos direitos dos cidadãos, desde que se apliquem critérios
adequados com os preceitos da Constituição, deixandose o casuísmo e o subjetivismo,
nesta seara. Outro importante aspecto a ser considerado quando da implementação
das políticas públicas dos direitos sociais que devem atentar aos dispositivos
orçamentários. Os últimos vinte anos de constitucionalismo democrático brasileiro
demonstram esta busca de efetividade dos direitos fundamentais sociais.
Deste modo, a crise do direito administrativo é uma mutação para adequarse às
transformações e mudanças sociais vividas no início do século XXI. É necessário voltar
se à pauta dos direitos fundamentais, que até hoje não foram efetivados de maneira
universal, considerando, especialmente, o direito à saúde no Brasil. Somente assim,
poderemos acenar com a construção de um país realmente democrático com sua
população mais saudável, nas próximas gerações.Tal tarefa não é fácil, e passa por
uma transformação de imaginário. Somente por meio da produção de uma cultura
democrática (que implica no reconhecimento dos limites e possibilidades do controle
jurisdicional) é que se pode vislumbrar avanços nesta seara.
166
PUBLIC POLICIES AND RIGHT TO HEALTH: the tension between the powers – the
need to establish judicial criteria to intervention
ABSTRACT
The right to health is a constitutional right, and its meant to be implemented by
public policies. The experience of the Social State of other countries is different of the
Brazilian one. The public policies are mandates created by the legislator and achieved
by the administrator. The administrative discretion find constitutional limits. In cases of
inefficiency or omission in execution of public policies, can occur judicial intervention.
The collective actions are the most adequate way for this discussion. The individual
actions are a mechanism of pressing. The democratic culture implies in the recognition
of the limits and possibilities of the judicial control for the effectivity of the right to health.
Keywords: Adequate answer. Judicial control. Public policies. Right to health.
Social state.
NOTAS
1
2
3
4
5
6
7
Bacharel em Direito pela UNISINOS, Mestrando em Direito Público na UNISINOS.
Doutora em Direito pela Universidade Pompeu Fabra de Barcelona/Espanha, Mestra em Direito pela
UFRS, Profa. do PPG em Direito da UNISINOS, Promotora de Justiça/RS.
ARENDT, Hannah. Los orígenes del totalitarismo. Madrid: Taurus, 1998, p. 368 382.
Vide também The rights of man, what are they? In Hannah Arendt’s Centenary: Political na
Philosophie, vol. 74, number 3, Part I (fall 2007), guest editor: Jerome Kohn.
SCLIAR, Moacyr. Do mágico ao social: trajetória da saúde pública, São Paulo: SENAC, 2002, p. 95.
Assim, o médico sanitarista escreve: “(...) Sabese que a morbimortalidade materna, por exemplo,
correlacionase inversamente com o grau de educação formal, independente das condições
socioeconômicas.
Para que o processo educativo em saúde atinja a plenitude de seus objetivos é preciso percorrer
várias etapas: a partir de um diálogo com a população, procurase informar as pessoas, para que,
informadas adotem uma atitude positiva, que deve gerar um comportamento; esse comportamento,
idealmente, deveria ser introjetado sob a forma de hábito, ou seja, comportamento automatizado.
Disseminado, o hábito evolui para o costume, que é um prática generalizada, incorporada à própria
cultura” (grifos do autor).
Vide SZWARCWALD, Célia L. Mortalidade infantil no Brasil: Belíndia ou Bulgária? Cadernos de
Saúde Pública, vol. 13, nº3, Rio de Janeiro, 1997. Disponível também em:
http://www.scielo.br/pdf/csp/v13n3/0175.pdf Acesso em: 9/10/2009.
COUTO e SILVA, Almiro in apresentação do livro Elementos de Direito Administrativo Alemão,
MAURER, Hart, Porto Alegre: Fabris, 2000, p. 14.
STRECK, Lênio. Jurisdição Constitucional e Hermenêutica: uma nova crítica do direito. 2. ed. Rio de
Janeiro: Forense, 2004, p. 122. Para Streck: “o que houve (há) é um simulacro de modernidade. (...)
Ou seja, em nosso país as promessas de modernidade ainda não se realizaram. E, já que tais
promessas não se realizaram, a solução que o establishment apresenta, por paradoxal que possa
parecer, é o retorno ao Estado (neo)liberal. Daí que a pósmodernidade é vista com a visão
neoliberal. Só que existe um imenso deficit social em nosso país, e, por isso, temos que defender as
instituições da modernidade contra esse neoliberalismo pós moderno”.
CANOTILHO, J.J. Gomes e MOREIRA Vital. Fundamentos da Constituição. Coimbra: Coimbra Ed.,
1991, p.173. Na Constituição orçamental estariam os preceitos relativos ao orçamento do Estado,
principalmente a aprovação parlamentar do orçamento, incluindo a autorização parlamentar anual dos
impostos e a autorização e controle parlamentar das despesas públicas.
167
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
STRECK, Lênio. Decisionismo e discricionariedade judicial em tempos póspositivistas: o solipsismo
hermenêutico e os obstáculos à concretização da Constituição no Brasil. Separata: o Direito e o
Futuro do Direito, Almedina: Coimbra, 2008, p. 99.
MOTESQUIEU, Charles Louis de Secondat. Do espírito das leis. 2. ed., São Paulo: Abril Cultural,
1979, pp.148/54.
ALTHUSSER, Louis. Montesquieu – A política e a história. 2. ed., Lisboa: Editorial Presença.
WALINE, Marcel. Traité Élémentaire de Droit Administratif, 6e édition, Recueil Sirey, Paris, 1952, p.
45.
LAUBADÈRE, André. Manuel de Droi Administratif, 15e édition, L.G.D.J., Paris, 1995, p. 107.
LAUBADÈRE, op. cit., pp. 105/7. A respeito da evolução do recurso de excesso de poder, o autor faz
uma análise de sua evolução até o final do século XX.
Da mesma forma, o Conselho Constitucional é o órgão a quem incumbe o controle da
constitucionalidade das leis, que é realizado de uma maneira preventiva, não se permitindo ao
judiciário o controle destas questões.
CADERMATORI, Sergio. Estado de Direito e Legitimidade: uma abordagem garantista. 2. ed.,
Campinas: Millennium, 2006.
A respeito das insuficiências do modelo representativo e o movimento que ocorre em prol da
democracia participativa, não se tratará desta questão, por não ser objeto do presente trabalho.
HESSE, Conrado. Significado de los derechos fundamentales. In Manual de Derecho Constitucional,
BENDA et als. , Madrid: Instituto Vasco de Administración Pública, 1996, p. 98, g.n.
BARROSO, Luis Roberto. Fundamentos teóricos e filosóficos do novo direito constitucional brasileiro
(pósmodernidade, teoria crítica e póspositivismo). Revista do Ministério Público, Porto Alegre, nº 46,
jan/mar,2002, p. 59.
KRELL, Andreas J. Direitos Sociais e controle judicial no Brasil e na Alemanha: os (des)caminhos de
um direito constitucional comparado. Porto Alegre: Fabris, 2002, p. 109, g.n.
EWALD, François. L’Etat Providence. Paris: Bernard Grasset, 1986.
KRELL, Andreas J. op. cit., p. 51/2. Segundo o Tribunal Constitucional Federal da Alemanha, esses
direitos a prestações positivas “estão sujeitos à reserva do possível no sentido daquilo que o
indivíduo, de maneira racional pode esperar da sociedade”. Essa teoria impossibilita exigências acima
de um certo limite básico social; a Corte recusou a tese de que o Estado seria obrigado a criar
quantidade suficiente de vagas nas universidades públicas para atender todos os candidatos.
SARLET, Ingo.W . A eficácia dos direitos fundamentais, 7. ed. Porto Alegre: Livraria do Advogado,
2007, p. 297.
SARLET, Ingo.W . A eficácia do direito fundamental à segurança jurídica: dignidade da pessoa
humana, direitos fundamentais e proibição de retrocesso social no direito constitucional brasileiro, in
Constituição e Segurança Jurídica, ROCHA, Cármen Lúcia Antunes Coord., Belo Horizonte: Fórum,
2004, p.128.
MAURER, Hart. Elementos de Direito Administrativo Alemão. Porto Alegre: Fabris, 2007, p. 50.
Os conflitos não mais se situam na polaridade Estado x cidadão, mas nas relações grupo x indivíduo.
Isso ocorre porque alguns grupos econômicos possuem poder paralelo ao Estado, decorrente, em
alguns casos, das privatizações, em que muitos serviços públicos são prestados por empresas
privadas. O desenvolvimento da dtittwirkung, também conhecida por eficácia diante de terceiros ou
eficácia horizontal, é hoje questão principal nos direitos do indivíduo. Os litígios foram se deslocando
do plano das relações públicas para o das privadas. A propósito vide BILBAO UBILLOS, Juan Maria.
La eficacia de los derechos fundamentales frente a particulares. Madrid: CEC, 1997, p. 256.
GARCÍA DE ENTERRÍA, Eduardo. Las Transformaciones de La Justicia Administrativa: de excepción
singular a La plenitud jurisdiccional. ¿Um cambio de paradigma? Pamplona: Thomson Civitas, 2007,
p. 147.
MOREIRA NETO, Diogo de Figueiredo. Novos horizontes para o direito administrativo: pelo controle
das políticas públicas. Revista de Direito do Estado, Rio de Janeiro, nº 4: 403412, out/dez, 2006, p.
406.
Vide também ALLI ARANGUREN, JuanCruz. Derecho Administrativo y Globalización, Madrid:
Thomson Arandazi, 2004, p. 377.
MOREIRA NETO, Diogo de Figueiredo. Quatro paradigmas do direito administrativo pósmoderno:
legitimidade, finalidade, eficiência, resultados. Belo Horizonte: Fórum, 2008.
MOREIRA NETO, Diogo de Figueiredo. op. cit., p. 33.
168
30
31
32
33
34
35
36
37
38
39
MOREIRA NETO, Diogo de Figueiredo. op. cit., p. 63.
MOREIRA NETO, Diogo de Figueiredo. op. cit., p. 139. São apresentadas técnicas destinadas a
realizar as seguintes funções: a) a definição de objetivos de políticas públicas; b) a identificação das
estruturas administrativas responsáveis por sua consecução; c) definição das normas de
desempenho que traduzam um compromisso quanto ao grau de realização dos objetivos demarcados
em função dos meios a eles destinados; d) os resultados comensurados e sua comunicação; e)
outorga de facilidades de gestão aos responsáveis proporcionalmente a seu desempenho (conforme
convênios de gestão); f) análise dos dados de desempenho integrados com vista a informar as
subseqüentes decisões sobre recursos e orçamento; g) fixação de acompanhamentos físicos e
contábeis necessários para os controles, correntes e posteriores.
FLEINER, Fritz. Institutionem des Verwaltungsrechts, 8. ed., 1928, p. 326, apud MIR PUIGELAT,
Globalización, Estado y Derecho. Las transformaciones recientes del Derecho administrativo. Madrid:
Civitas, 2004, p.158.
De acordo com a pesquisa mundial sobre saúde, desenvolvida pela OMS e coordenada no país pelo
Centro de Informação Científica e Tecnológica (CICT) da Fiocruz, em estudo que avalia os sistemas
de saúde de 71 nações, consigna que os brasileiros dispensam em média 19% da renda familiar com
saúde, da parcela da população de 25,8% que têm acesso aos planos de saúde privados, enquanto a
maioria 74,2% conta com os serviços do Sistema Único de Saúde – SUS. Disponível em: http://www.
ministeriodasaude.gov.br. Acesso em 20/8/2008.
HOWLETT, Michael e RAMESH, M. Come studiare le poliche pubbliche. Bologna: Il Mulino, 1995, p.
5. Tradução do original Studying Public Policy: Policy cycles and policy subsystems, Oxford, Oxford
University Press, 1995.
A propósito a Constituição dos Estados Unidos de 1787 e a Constituição Francesa de 1791.
SIGMANN, Jean. 1848 Las revoluciones románticas y democráticas de Europa. 3. ed., Madrid: Siglo
veintiuno editorial, 1985.
BUCCI. O conceito de política pública em direito, p. 31, in BUCCI, Maria Paula Dallari (org). Política
públicas: reflexões sobre o conceito jurídico. São Paulo: Saraiva, 2006.
BARROSO, Luís Roberto. Da falta de efetividade à judicialização excessiva: direito à saúde,
fornecimento gratuito de medicamentos e parâmetros para a atuação judicial, p. 28
Disponível em: http: www. lrbarroso.com.br/pt/noticias.medicamentos. pdf. Acesso em: 29/8/2008.
STF, DJU 14/2/2007, SS 3073/ RN, Rel. Min. Ellen Gracie. Verifico estar devidamente configurada a
lesão à ordem pública, considerada em termos de ordem administrativa, porquanto a execução de
decisões como a ora impugnada afeta o já abalado sistema público de saúde. Com efeito, a gestão
da política nacional de saúde, que é feita de forma regionalizada, busca uma maior racionalização
entre o custo e o benefício dos tratamentos que devem ser fornecidos gratuitamente, a fim de atingir
o
maior
número
possível
de
beneficiários.
Entendo que a norma do art. 196 da Constituição da República, que assegura o direito à saúde,
referese, em princípio, à efetivação de políticas públicas que alcancem a população como um todo,
assegurandolhe acesso universal e igualitário, e não a situações individualizadas. A
responsabilidade do Estado em fornecer os recursos necessários à reabilitação da saúde de seus
cidadãos não pode vir a inviabilizar o sistema público de saúde. No presente caso, ao se deferir o
custeio do medicamento em questão em prol do impetrante, estáse diminuindo a possibilidade de
serem oferecidos serviços de
saúde
básicos ao restante
da
coletividade.
Ademais, o medicamento solicitado pelo impetrante, além de ser de custo elevado, não consta da
lista do Programa de Dispensação de Medicamentos em Caráter Excepcional do Ministério da Saúde,
certo, ainda, que o mesmo se encontra em fase de estudos e pesquisas.
Constato, também, que o Estado do Rio Grande do Norte não está se recusando a fornecer
tratamento ao impetrante. É que, conforme asseverou em suas razões, “o medicamento requerido é
um plus ao tratamento que a parte impetrante já está recebendo” (fl. 14).
Finalmente, no presente caso, poderá haver o denominado “efeito multiplicador” (SS 1.836AgR/RJ,
rel. Min. Carlos Velloso, Plenário, unânime, DJ 11.10.2001), diante da existência de milhares de
pessoas
em
situação
potencialmente
idêntica
àquela
do
impetrante.
Ante o exposto, defiro o pedido para suspender a execução da liminar concedida nos autos do
Mandado de Segurança nº 2006.0067950 (fls. 3135), em trâmite no Tribunal de Justiça do Estado
do Rio Grande do Norte.
169
40
41
42
43
http://www.ioc.fiocruz.br/aids20anos/linhadotempo.html Acesso em: 9/10/2009. Em 1991, O Ministério
da Saúde dá início à distribuição gratuita de antirretrovirais. A OMS anuncia que 10 milhões de
pessoas estão infectadas pelo HIV no mundo. No Brasil, 11.805 casos são notificados. O
antirretroviral Videx (ddl) é aprovado nos Estados Unidos e a fita vermelha tornase o símbolo
mundial de luta contra a Aids.
A Fiocruz foi convidada pelo Programa Mundial de Aids das Nações Unidas e Organização Mundial
da Saúde (Unaids/OMS) para participar da Rede Internacional de Laboratórios para Isolamento e
Caracterização do HIV1. Em 1992, pesquisadores franceses e norteamericanos estabelecem
consenso sobre a descoberta conjunta do HIV. A Aids passa a integrar o código internacional de
doenças e os procedimentos necessários ao tratamento da infecção são incluídos na tabela do SUS.
Combinação entre AZT e Videx inaugura o coquetel antiaids.
O início foi uma ação civil pública proposta pelo MPF/ RS, que ocasionou a decisão de 2007 do TRF
4 (Tribunal Regional Federal da 4ª Região) em que obrigava o SUS a fazer esse tipo de cirurgia. A
decisão do TRF determinava que o governo federal tomasse todas as medidas que possibilitem aos
transexuais a realização da cirurgia de mudança de sexo pelo SUS.
“Direito Constitucional. Transexualismo. Inclusão na tabela do SUS. Procedimentos médicos de
transgenitalização. Princípio da proibição de discriminação por motivo de sexo. Discriminação de
gênero. Direitos fundamentais de liberdade, livre desenvolvimento da personalidade, privacidade e à
dignidade humana e direito à saúde. Força normativa da Constituição.”
Rel. Roger Raupp Rios, Processo nº 2001.71.00.0262799. D.E. 22/08/2007.
A União recorreu ao STF (Supremo Tribunal Federal), alegando que as operações gratuitas trariam
prejuízo aos cofres públicos.
Em 12 de dezembro de 2007, a então presidente do STF, Ministra Ellen Gracie, suspendeu os efeitos
da decisão do TRF4, em um pedido de suspensão de tutela antecipada. Agora, o próprio Ministério
da Saúde decidiu incluir a cirurgia na tabela do SUS.
A cirurgia para mudança de sexo fará parte da lista de procedimentos do Sistema Único de Saúde
(SUS). A Portaria n.º 1.707 do Ministério da Saúde, publicada na edição desta terçafeira, no Diário
Oficial da União, prevê a realização do processo nos hospitais públicos.
A regulamentação dos procedimentos caberá à Secretaria de Atenção à Saúde do Ministério da
Saúde. Segundo o texto da portaria publicada nesta terçafeira, a secretaria vai adotar as
providências necessárias à estruturação e implantação do processo transexualizador no SUS,
definindo os critérios mínimos para o funcionamento, o monitoramento e a avaliação dos serviços.
O anúncio de que o SUS faria este tipo de cirurgia foi feito no início de junho, durante a Conferência
Nacional de Gays Lésbicas, Bissexuais Travestis e Transexuais, a primeira, no mundo, organizada
por um governo federal. Na ocasião, o ministro da Saúde, José Gomes Temporão, lembrou que essa
cirurgia é um processo extremamente complexo e delicado, e serão selecionados pouquíssimos
centros no Brasil, possivelmente hospitais universitários para o procedimento, que atualmente é feito
em hospitais particulares, custa caro e muitas vezes envolve riscos para o paciente.
O processo cirúrgico transexualizador vai além da cirurgia e envolve uma equipe multidisciplinar
composta por psicólogos, assistente social e médicos endocrinologista, psiquiatras, urologistas e
ginecologistas. O Ministério promete que todo o atendimento será livre de discriminação.
Disponível
em:
http://www.agenciabrasil.gov.br/noticias/2008/08/19/materia.200808
19.1741052107/view. Acesso em: 9/10/2009. Cirurgia para mudança de sexo passa a fazer parte de
procedimentos do SUS.
BARROSO, Luís Roberto. Da falta de efetividade à judicialização excessiva: direito à saúde,
fornecimento gratuito de medicamentos e parâmetros para a atuação judicial, p. 31.
Disponível em: http://www.lrbarroso.com.br/pt/noticias/medicamentos.pdf Acesso em: 9/10/2009.
LEAL, Rogério Gesta. A quem compete o dever de saúde no direito brasileiro? Esgotamento de um
modelo institucional. Revista de Direito Sanitário, v. 9, p. 5069, 2008.
O autor assevera que o Direito Civil pátrio estatui dever de solidariedade entre as pessoas que
compõem a família. A solidariedade impõe um dever de cuidado, que se exprime, dentre outros, nas
prestações alimentícias. Ocorre que tais prestações não englobam apenas a subsistência, devendo
ela ser adequada, do que se depreende que inclusive a assistência à saúde está incluída, não sendo
apenas uma verba para alimentação. Isso é reflexo do dever de cuidado que os familiares tem para
si, posição que os colocam como devedores solidários da promoção à saúde. Prova disso é que
nossos tribunais ao fixar o valor dos alimentos já incluem o valor das despesas médicas, com plano
170
44
45
46
47
48
49
50
51
52
53
54
de saúde inclusive, não estando apartados os valores. A lei 9.908/93RS, que determina o
fornecimento por parte do Estado de medicamentos, desde que se comprove a condição de pobre,
demonstrando a incapacidade da família em arcar com os custos. A construção de Gesta Leal vai no
sentido de preocuparse com a efetividade da Constituição, rejeitandose os discursos que apregoam
que a simples referência à ela promoveria milagres no plano fático. Cabe lembrar que é pressuposto
de eficácia do Estado Social a solidariedade, razão pela qual há que se impedir o avanço das práticas
clientelistas, que mantém os cidadãos infantilizados.
Resolução WHA 28.66, da 28ª Assembléia Nacional da Saúde.
SOBRAVIME, O que é o uso racional de medicamentos, São Paulo: SOBRAVIME, 2001, p.13.
Organização Mundial da Saúde, Pautas para establecer políticas farmacéuticas nacionales, Ginebra:
OMS, 1988.
Publicada no DOU nº 215E, Seção I, de 10/11/98, p. 1822.
No Estado do Rio de Janeiro foram gastos com os programas de assistência farmacêutica
R$ 240.621.568,00, enquanto com o saneamento básico foram investidos R$ 102.960.276,00.
Comparandose as duas quantias, o gasto com saneamento é menos da metade.
Disponível em: http://www.planejamento.rj.gov.br/orcamentoRJ/2007_LOA.pdf. Acesso em:
9/10/2009.
ARENDT, Hannah, A Condição Humana, 10. ed., 2ª reimp., Rio: Forense, 2002, capítulo II – as
esferas pública e privada, pp. 31/88.
STRECK, Lenio Luiz. Decisionismo e discricionariedade judicial em tempos póspositivistas: o
solipsismo hermenêutico e os obstáculos à concretização da Constituição no Brasil. Separata: o
Direito e o Futuro do Direito, Almedina: Coimbra, 2008, p. 99.
STRECK, Lenio Luiz. Verdade e Consenso. Constituição, Hermenêutica e Teorias Discursivas Da
possibilidade à necessidade de respostas corretas em Direito. 3..ed Rio de Janeiro: Lumen Juris.
2009 p. 499
Nesse sentido ver GADAMER, Hans Georg. Verdade e Método: Traços Fundamentais de uma
Hermenêutica Filosófica. 4. ed. Petrópolis: Vozes, 2002.
Nesse sentido ver STRECK, Lenio Luiz. Op cit. p. 561562.
Tal qual Kelsen explicita em seu cap. 8°, no qual trata da interpretação. KELSEN, Hans. Teoria pura
do direito. 7. Ed. São Paulo: Martins Fontes. 2006.
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Recebido para publicação 23/10/2009
Aceito para publicação 12/12/2009
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W3078796666.txt | RESEARCH ARTICLE
Human Tregs at the materno-fetal
interface show site-specific adaptation
reminiscent of tumor Tregs
Judith Wienke,1 Laura Brouwers,2 Leone M. van der Burg,1 Michal Mokry,3,4 Rianne C. Scholman,1
Peter G.J. Nikkels,5 Bas B. van Rijn,2,6 and Femke van Wijk1
Center for Translational Immunology, 2Wilhelmina Children’s Hospital Birth Center, 3Regenerative Medicine Utrecht,
1
Laboratory of Clinical Chemistry and Hematology, and 5Department of Pathology, Wilhelmina Children’s Hospital,
4
University Medical Center Utrecht, Utrecht University, Netherlands. 6Obstetrics and Fetal Medicine, Erasmus MC University
Medical Center Rotterdam, Rotterdam, Netherlands.
Tregs are crucial for maintaining maternal immunotolerance against the semiallogeneic fetus.
We investigated the elusive transcriptional profile and functional adaptation of human uterine
Tregs (uTregs) during pregnancy. Uterine biopsies, from placental bed (materno-fetal interface)
and incision site (control) and blood were obtained from women with uncomplicated pregnancies
undergoing cesarean section. Tregs and CD4+ non-Tregs were isolated for transcriptomic profiling by
Cel-Seq2. Results were validated on protein and single cell levels by flow cytometry. Placental bed
uTregs showed elevated expression of Treg signature markers, including FOXP3, CTLA-4, and TIGIT.
Their transcriptional profile was indicative of late-stage effector Treg differentiation and chronic
activation, with increased expression of immune checkpoints GITR, TNFR2, OX-40, and 4-1BB;
genes associated with suppressive capacity (HAVCR2, IL10, LAYN, and PDCD1); and transcription
factors MAF, PRDM1, BATF, and VDR. uTregs mirrored non-Treg Th1 polarization and tissue
residency. The particular transcriptional signature of placental bed uTregs overlapped strongly with
that of tumor-infiltrating Tregs and was remarkably pronounced at the placental bed compared
with uterine control site. In conclusion, human uTregs acquire a differentiated effector Treg profile
similar to tumor-infiltrating Tregs, specifically at the materno-fetal interface. This introduces the
concept of site-specific transcriptional adaptation of Tregs within 1 organ.
Introduction
Conflict of interest: The authors have
declared that no conflict of interest
exists
Copyright: © 2020, Wienke et al. This
is an open access article published
under the terms of the Creative
Commons Attribution 4.0 International
License.
Submitted: April 1, 2020
Accepted: August 12, 2020
Published: September 17, 2020.
Reference information: JCI Insight.
2020;5(18):e137926.
https://doi.org/10.1172/jci.
insight.137926.
In the past decade, T cells have been identified in various human and murine nonlymphoid tissues (1, 2).
These tissue-resident memory T cells (TRM) do not recirculate, serve as first-line responders to infections,
and are characterized by expression of signature molecules such as CD69, which prevents their tissue egress
(1, 3–8). TRM adapt to tissue environments by acquiring a specialized functional phenotype that depends
on microenvironmental cues (9, 10). Also Tregs, critical gatekeepers of immune homeostasis (11), have been
recently identified in murine and human tissues (12–16). Like TRM, Tregs can become resident and gain a
polarized phenotype, with functional specialization depending on the tissue or organ, which is controlled
on a transcriptional level (14–22). Although increasing evidence in mice supports functional adaptation of
Tregs to nonlymphoid tissue environments (23), studies in humans are still scarce (14, 17). However, transcriptional adaptation of Tregs has gained special interest in the tumor environment, due to the important
therapeutic implications (24). Tumor-infiltrating Tregs (TITR) display a unique and specialized transcriptional signature (25), associated with activation and functional specialization, including increased suppressive capacity (25–27). Tissue and tumor Tregs undergo differentiation reminiscent of effector Tregs, with
potent suppressive capacity, and are characterized by expression of CD45RO and increased CD25, CTLA-4,
and HLA-DR (28–31). Furthermore, effector Tregs (in tissues and tumors) express high levels of immune
checkpoint molecules OX-40, 4-1BB, GITR, TIGIT, and ICOS and transcription factors such as BLIMP-1
(encoded by PRDM1) and BATF (19, 25–27, 31–34). Effector Tregs can mirror effector Th cell polarization by acquiring coexpression of FOXP3 with chemokine receptors and transcription factors associated
with Th1 (CXCR3, T-bet), Th2 (GATA3, IRF4), or Th17 (RORγt, STAT3) differentiation (19, 35–38).
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This specific polarization is associated with an enhanced suppressive efficacy toward the matching T effector
response (31, 36–45). Since most of these insights have been generated in mice, it is still largely unknown
whether these principles also apply to human tissue Tregs.
As recently highlighted (46), one of the most interesting yet elusive tissue sites for Treg function in
humans is the materno-fetal interface. Pregnancy is a mystifying biological process when viewed from an
immunological perspective, posing a unique challenge to the maternal immune system (47, 48). While
peripheral immunity against pathogens needs to remain intact, the semiallogeneic fetus and placenta,
which may harbor foreign paternal antigens, need to be tolerated (49). The maternal immune response is
therefore delicately balanced and requires tight regulation especially locally at the materno-fetal interface,
which is underlined by the fact that human decidual T cells can recognize and actively respond to fetal cord
blood cells (47–51). Maternal Tregs are consequently indispensable for successful embryo implantation and
pregnancy outcome, and they contribute to materno-fetal tolerance on multiple levels (47, 52, 53). Depletion of murine maternal Tregs causes pregnancy loss due to immunological rejection of the fetus (53, 54).
In humans, maternal Tregs are abundantly present in the gravid uterus (55–62), and normal human pregnancy is characterized by increased numbers of Tregs in the periphery and at the materno-fetal interface
(56, 61, 63, 64). In patients with preeclampsia, a severe hypertensive pregnancy disorder, and patients with
recurrent miscarriages, Treg numbers are reduced both at the materno-fetal interface and in the periphery
(57, 65–70), implying that — also in humans — local presence of Tregs in the pregnant uterus is required
for successful pregnancy outcome.
Previous studies investigating the maternal, uterine immune system in humans have been limited by
the practical challenge of acquiring biopsy material of the uterine wall and have made use of the thin superficial decidual layer attached to the delivered placenta, which is heavily contaminated by fetal immune cells
and may not be representative of the maternal Treg status during pregnancy. Moreover, the functional and
transcriptomic profile of human uterine Tregs (uTregs) from the materno-fetal interface and its relation
to Tregs from other human tissues remain to be elucidated. Here, we investigated functional adaptation
and specialization of highly purified human, exclusively maternal, resident uTregs in myometrial biopsies
from the materno-fetal interface. We performed transcriptomic profiling and functional in vitro assays, as
well as flow cytometry, to study their phenotypic heterogeneity on protein level in single cell resolution. To
identify tissue site–specific functional adaptation, we compared these uTregs with uTregs from a distant
uterine control site and maternal peripheral bTregs, in addition to tissue- and site-matched resident CD4+
non-Tregs. Last, we observed the specific profile of functional adaptation of uTregs compared with known
Treg signatures from other human and murine tissue sites, including tumors.
Results
uTregs are bona fide suppressive Tregs. The frequency of CD25hiFOXP3+ Tregs within the CD4+ T cell
population was similar between blood and uterine tissue and ranged from 2.5% to 13.5% (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.
insight.137926DS1). For transcriptional profiling, the CD3+CD4+CD25hiCD127– population (Tregs) and
CD3+CD4+CD25–CD45RA– memory T cells (Tconv) were FACS sorted from peripheral blood and myometrial biopsies from 5 women with uncomplicated pregnancies undergoing cesarean section. In myometrium, Tconv were selected for CD69 positivity. The sorting strategy is shown in Supplemental Figure 1B.
Confirming the maternal origin of the sorted cells, the female-specific gene XIST was highly expressed in
all samples, whereas transcripts of the male-specific gene SRY were undetectable in all samples, including
pregnancies with male offspring (Supplemental Figure 1C). Principal component analysis (PCA) of transcriptomic profiles showed that uTregs from the materno-fetal interface are clearly distinct from blood-derived Tregs (bTregs), and that also uterine T conv (uTconv) and blood-derived Tconv (bTconv) clearly cluster apart (Figure 1A). Notably, PC1, mounting the difference between the cell sources, accounted for > 60%
of the variance, whereas PC2, explaining variance between Treg and Tconv populations, accounted for only
11% of the variance. To assess whether the sorted population of uTregs consisted of bona fide Tregs, we analyzed enrichment of a published core Treg gene signature (71) in uTregs compared with uTconv and bTregs
by gene set enrichment analysis (GSEA). Expression of Treg core signature genes was not only enriched
compared with uTconv, but, remarkably, also more pronounced in uTregs than in bTregs, indicating that
uTregs are bona fide Tregs with enhanced expression of Treg core signature genes (Figure 1, B and C).
Indeed, expression of many of the published Treg markers (71) was higher in uTreg than bTreg (Figure 1D).
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Figure 1. Tregs at the materno-fetal interface are bona fide Tregs. (A) Principal component analysis of bTregs, bTconv, uTregs, and uTconv (all n = 5). (B
and C) GSEA with published Treg signature gene set (71) comparing uTreg and uTconv (B) and uTreg and bTreg (C) (all n = 5). NES, normalized enrichment score; FDR, FDR adjusted P value. (D) Heatmap of genes in leading edge of GSEA comparing enrichment of published Treg signature genes in
uTregs and bTregs. Expression values were mean centered and scaled per gene. (E) Representative gating strategy of bTregs, uTregs, and uTconv out of
5 experiments. (F) Representative expression of CTLA-4 in uTregs out of 6 experiments. (G) Ex vivo protein expression of core Treg molecules FOXP3 (n
= 10) and CTLA4 (n = 6) measured by flow cytometry. (H) Ex vivo protein expression of Treg signature molecule TIGIT (n = 6) measured by flow cytometry. (G and H) Multiplicity adjusted P value of 2-way ANOVA with Tukey’s post hoc test. (I) GSEA of TIGIT+ Treg signature (n = 5) (72).
Higher expression of the Treg-identifying molecules FOXP3 and CTLA-4 in uTregs than bTregs was confirmed on protein level (Figure 1, E–G). Also TIGIT, a key checkpoint molecule associated with specialized suppressive function (72), was highly expressed in uTregs, with the majority of uTregs being positive for TIGIT (Figure 1H). Consistently, GSEA showed significant enrichment of a previously identified
TIGIT+ Treg signature (Figure 1I) (72). Suppression assays, although technically challenging due to low
cell numbers, confirmed the suppressive potential of uTregs on proliferation and cytokine production of
healthy donor peripheral blood–derived CD4+ T cells (Figure 2 and Supplemental Figure 1, D and E). Two
of 4 uTreg donors showed particularly high suppressive capacity of uTregs on cytokine production of IL-2,
IL-10, IFN-γ, and TNF-α, already at a 1:8 (Treg/Tconv) ratio, compared with bTregs. These results confirm
that the sorted uTregs are bona fide functional Tregs, with enhanced expression of Treg signature genes.
The uTreg signature indicates an activated and effector Treg profile. To investigate the functional adaptation of
uTregs to the specific environment of the materno-fetal interface, we determined both their functional differentiation and Th polarization, both of which may be influenced by the tissue environment (12, 13, 20, 33, 73–75).
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To identify the uTreg-specific transcriptional signature, we assessed their differential gene expression with both
bTregs and uTconv. Many genes were differentially expressed between uTregs and bTregs (Figure 3A), with significant upregulation of 1966 genes and downregulation of 1997 genes in uTregs (adjusted P value [Padj] < 0.05
and |log2FC| > 0.5). To isolate the uTreg-specific signature, we also compared gene expression between uTreg
and uTconv, yielding 465 upregulated genes — including the Treg-identifying genes FOXP3, IL2RA, CTLA4,
TIGIT, and IKZF2 — and 103 downregulated genes in uTregs (Padj < 0.05 and |log2FC| > 0.5; Figure 3B); 236
genes were specifically upregulated (225 after removal of duplicate genes) and 23 genes specifically downregulated in uTregs compared with both bTregs and uTconv (Figure 3C, Supplemental Table 4, and Supplemental
Table 5). Among the downregulated genes were ITGA6, IL7R, CCR7, TTC39C, PLAC8, ATF7IP2, ABLIM1,
MGAT4A, PRKCB, and GIMAPs, as well as transcription factors TCF7, LEF1, and SATB1, indicating late-stage
differentiation of Tregs (76, 77). The 225 upregulated genes were involved in cytokine signaling, TNF receptor
signaling, and glycolysis (Figure 3D). Selected genes from the top 5 pathways included those related to Treg
activation and effector differentiation, such as immune checkpoints of the TNF receptor superfamily (TNFRSF13B [TACI], TNFRSF18 [GITR], TNFRSF1B [TNFR2], TNFRSF4 [OX-40], TNFRSF8 [CD30], TNFRSF9
[4-1BB]) and HLA-DR, CD80, and LRRC32 (GARP). Furthermore, genes associated with suppressive capacity
(CTLA4, ENTPD1, HAVCR2, IL10, IL2RA, LAG3, LAYN, LGALS1, PDCD1, and TOX2) were highly expressed
in uTregs (Figure 3E) (19, 31), and cytokine receptors of the IL-1 and IL-2 family (IL1R1, IL1R2, IL1RAP,
IL1RN, IL2RA, and IL2RB) and specific chemokine receptors (CCR1, CXCR6) showed increased and specific
expression in uTregs (Figure 3E). Transcription factors specifically upregulated in uTregs included BATF, CEBPB, ETS2, ETV7, HES1, IKZF4, MAF, NFIL3, PRDM1, VDR, and ZBTB32 among others (Figure 3E). This transcriptomic profile, and especially high expression of BATF, PRDM1, and immune checkpoint molecules, reflects
previously identified crucial signatures of effector Treg differentiation and function, especially in tissues (29, 32,
33, 78–80). We confirmed upregulation of immune checkpoints associated with effector Treg differentiation/
chronic stimulation GITR, OX-40, 4-1BB, PD-1, HLA-DR, and ICOS in uTregs, even compared with uTconv,
on the protein level (Figure 3F). Since increased expression of many of these genes pointed toward an activated
phenotype, we confirmed this by demonstrating significant enrichment of published gene sets of in vitro–activated Tregs in uTregs (Supplemental Figure 2 and Supplemental Table 3) (81–85). Taken together, uTregs at the
materno-fetal interface have a highly differentiated transcriptional signature suggestive of a specialized function
with high suppressive capacity and high responsiveness to environmental cues, which is reflective of late-stage
effector differentiation and chronic activation.
uTregs have a tissue-resident phenotype and share transcriptional specialization with uTconv. To examine whether uTregs at the materno-fetal interface represent a resident population or rather transiently infiltrating cells,
we assessed the expression of tissue residency–related markers and gene signatures. uTregs had a significantly higher gene and protein expression of key residency molecule CD69 than bTregs and bTconv, similar
to uTconv (Figure 4A). Expression analysis and GSEA with published human TRM signatures showed a
pattern of upregulated and downregulated genes as previously described for human TRM in general (Figure 4B) and specifically in CD4+ (and CD8+) TRM from lung and skin (Figure 4C) (4, 14, 86), confirming
the tissue-resident profile in uTregs as compared with bTregs. Next, we identified the shared tissue-specific
adaptation of uTregs and uTconv to the materno-fetal interface. A large proportion of upregulated and
downregulated genes was shared between uTregs and uTconv compared with their counterparts from blood
(Supplemental Figure 3A; 1032 genes up and 1348 down; Padj < 0.05 and |log2FC| > 0.5), which again suggests that the specific tissue environment at the materno-fetal interface accounts for a significant part of their
adapted transcriptional profile. Shared upregulated genes were involved in cytokine signaling (Supplemental
Figure 3B), highlighting the integration of a spectrum of microenvironmental cues, while shared downregulated genes were reflective of ribosomal processes involved in RNA translation (Supplemental Figure 3C).
Taken together, uTregs have a TRM signature that reflects a shared adaptation to the tissue environment of
the materno-fetal interface between uTregs and uTconv.
uTregs mirror uTconv Th1 polarization with a predominance of T-bet+CXCR3+ Tregs. Effector Tregs can
acquire different Th phenotypes with coexpression of FOXP3 and lineage-defining transcription factors T-bet (TBX21, Th1), GATA3 (Th2), and RORγt (RORC, Th17), as well as lineage-associated cytokine and chemokine receptors (35). We investigated whether uTregs and uTconv underwent a, possibly
shared, Th polarization. uTregs showed significantly increased expression of Th1-related TBX21 compared with bTreg, which mirrored the increased expression of TBX21 in uTconv (Figure 5A). Th2-related GATA3 and Th17-related RORC were not significantly differentially expressed between uTreg and
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Figure 2. Tregs at the materno-fetal interface are bona fide Tregs with suppressive capacity. (A–D) Suppression assay
assessing cytokine production of IL-2 (A), IFN-γ (B), TNF-α (C), and IL-10 (D) by anti-CD3–stimulated (or unstimulated)
healthy CD4+ T cells in the supernatant by multiplex immunoassay after 4 days of coculture with healthy donor bTregs,
maternal bTregs, or uTregs at a 1:8, 1:4, and 1:2 ratios. Data represent median with interquartile range. n = 4 donors, but
not every condition could be measured for each donor due to limited availability of material. Therefore, some conditions contain data from 3 donors.
bTreg (and uTconv and bTconv), although RORC showed a trend toward downregulation, which was
confirmed on the protein level (Figure 5, A and B). Increased expression of T-bet was also confirmed
on the protein level, with 6%–87% (median 22%) of uTregs showing positivity for T-bet (Figure 5, C
and D). Also, the Th1-related cytokine receptor IL-18R1 was increased in both uTregs and uTconv compared with blood T cells on the gene and protein levels (Figure 5E). Investigation of chemokine receptor
expression, related to both Th polarization and tissue-specific homing (87, 88), showed that chemokine receptors associated with naive Tregs and lymphoid tissue environments CCR7 and CXCR5 were
downregulated in uTregs compared with bTregs, on the gene and protein levels (Figure 5, F and G).
Chemokine receptors upregulated in uTregs included CCR2, CCR5, CXCR3, CXCR4, CCR1, and CXCR6
(Figure 5, F and H), which largely mirrored expression by uTconv. CCR1 and CXCR6 were specifically
upregulated in uTregs, both previously identified as part of the conserved murine tissue Treg signature
(18). The Th1-associated CXCR3 (36, 89) and Th1/inflammation-associated CCR5 (89–92) had significantly higher gene and protein expression in uTregs and uTconv compared with their counterparts from
blood (Figure 5, F and H). Although the variable percentage of T-bet+ Tregs suggests heterogeneity
in uTreg subspecialization, virtually all uTregs (and uTconv) were positive for CXCR3 (84%–100%,
median 93%), and the majority expressed CCR5 (22%–83%, median 62%) (Figure 5I). Consistent with
these findings, a previously published gene signature of T-bet+CXCR3+ Tregs from the pancreas of
prediabetic mice was highly enriched in uTregs compared with bTregs (Figure 5J) (38). In conclusion,
uTregs at the materno-fetal interface show Th1 polarization mirroring uTconv, with high expression of
Th1-related markers T-bet and CXCR3. Furthermore, uTregs express an array of chemokine receptors
with which they can integrate a variety of locally produced signals. While some of these receptors are
uTreg specific, others are shared with uTconv. uTreg and uTconv cells may therefore rely on both unique
and shared cues to guide their migration to and retention at the uterine materno-fetal interface.
The uTreg signature at the materno-fetal interface overlaps with TITR signatures. We questioned whether the highly differentiated uTregs from the materno-fetal interface would resemble Tregs from other human and murine
tissue sites or would show a uniquely adapted profile. Well-studied murine tissue Treg populations include
Tregs from visceral adipose tissue (VAT), muscle, and intestines (12, 16, 18, 73, 93). Each population displays
a tissue-specific phenotype with expression of certain (a) transcription factors, (b) chemokine receptors, and
(c) preference toward a Th lineage differentiation when compared with spleen Tregs (12, 13, 16, 18, 73).
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Figure 3. The uTreg core signature. (A and B) Volcano plot of differential gene expression between uTregs and bTregs (A) or uTregs and uTconv (B) (all
n = 5). (C) Venn diagrams yielding genes specifically upregulated (Padj < 0.05 and log2FC > 0.5, left panel) or downregulated (Padj < 0.05 and log2FC <
–0.5, right panel) in uTreg compared with bTreg and uTconv. (D) Pathway analysis (ToppGene pathways) of 236 genes specifically upregulated in uTregs.
P < 0.05 after Bonferroni’s correction were considered significant. (E) Heatmaps showing gene expression of genes in top 5 pathways and selected
downregulated genes in the uTreg core signature, related to Treg activation or effector differentiation (left panel), (cytokine) signaling (middle panel;
including downregulated CCR7 and IL7R), and transcription factors (right panel). Expression values were mean centered and scaled per gene. (F) Protein
expression of GITR (TNFRSF18), OX-40 (TNFRSF4), 4-1BB (TNFRSF9), PD-1 (PDCD1), HLA-DR, and ICOS. uTregs were gated as CD3+CD4+CD25hiFOXP3+
cells. Multiplicity adjusted P value of 2-way ANOVA with Tukey’s post hoc test. n = 6 each.
A murine PAN-tissue signature, shared by VAT, muscle, and intestinal Tregs, was also identified (18).
GSEA in Figure 6A shows that the shared murine PAN-tissue Treg signature was also strongly enriched
in uTregs, again highlighting its generalized expression in tissue Tregs, apparently even conserved across
species. Overlaying significantly upregulated genes in uTreg (versus bTreg) with murine tissue–specific or
murine tissue–shared Treg signatures (18), yielded a large amount of shared genes between uTregs and
murine VAT–, colon–, and muscle–derived Tregs (Figure 6B, numbers in each field represent overlap
of the specific field with significantly upregulated genes in uTreg). Fifty-nine genes were shared among
all 3 murine tissues and uTregs, including IL1RL1 (receptor for IL-33, ST2), AREG, IL10, IRF4, GZMB,
TNFRSF9, BHLHE40, NR4A1, NR4A3, and CCR2, many of which have been described as crucial regulators
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Figure 4. Tregs at the materno-fetal interface have a tissue-resident profile. (A) Gene and protein expression of CD69 in sorted T cell populations.
DE, differentially expressed genes with Padj < 0.05. Box plots with median: box indicates 25th to 75th percentiles, whiskers indicate minimum and
maximum values (n = 5). FACS data: representative plot of 5 experiments. uTregs were gated as CD3+CD4+CD25hiCD127–. MFI, median fluorescence
intensity. Two-way ANOVA with Tukey’s post hoc test. (B) Heatmap with a published human core tissue–resident signature (4) in uTreg compared
with bTreg. Expression values were mean centered and scaled per gene. (C) GSEA with published genes identifying human lung CD4+ and CD8+ TRM
compared with blood memory cells; ref. 86) and genes upregulated/downregulated in skin CD4+ TRM compared with blood CD4+ T cells (14), in uTregs
versus bTregs. NES, normalized enrichment score; FDR, FDR adjusted P value.
for effector and/or tissue Treg function (Figure 6C) (12, 32, 40, 78–80, 94). Twelve of the 59 genes were
even part of the uTreg-specific core signature as defined in Figure 3 (CCR1, CXCR6, ELL2, FGL2, GEM,
IL10, LAPTM4B, SNX9, TNFRSF8, NFIL3, NR4A3, and PRDM1). This indicates that uTregs display features of tissue adaptation, which are highly conserved across tissues and species.
The investigation of human tissue–derived Tregs has proven challenging, and only limited data are
available. To assess how uTregs compare with other human tissue Tregs, we analyzed enrichment of 3
previously published gene sets of significantly upregulated genes in healthy skin, colon, and lung Tregs
compared with bTregs (Supplemental Table 3) (14, 17). All 3 signatures were significantly enriched in
uTregs compared with bTregs, indicating that the tissue profile of uTregs shows similarities with human
Tregs from various tissue sites (Supplemental Figure 4).
Human Tregs infiltrating the unique tissue-environment of tumors (TITR) have been studied more extensively. Comparison of genes significantly upregulated in uTregs versus bTregs with 7 recently published gene signatures of TITR infiltrating a variety of human tumors (Supplemental Table 3) (25, 95–100) yielded a remarkable overlap with each of the TITR signatures, with up to 65% of genes shared with uTregs (Supplemental
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Figure 5. uTreg and uTconv polarization at the materno-fetal interface. (A) Gene expression of lineage-defining transcription factors TBX21 (T-bet),
GATA3 (GATA-3), and RORC (RORγt). P values from differential gene expression analysis. DE, differentially expressed Padj < 0.05. Box plots with median;
box indicates 25th to 75th percentiles, and whiskers indicate minimum and maximum values (all n = 5). (B–D) Protein expression of RORγt (B) and T-bet
(C and D). uTregs were gated as CD3+CD4+CD25hiFOXP3+ cells. MFI, median fluorescence intensity. Multiplicity adjusted P values of 2-way ANOVA with
Tukey’s post hoc test.(n = 5) (E) Gene and protein expression of IL18R1 (IL-18R1). Gene expression: box plots with median — box indicates 25th to 75th
percentiles, and whiskers indicate minimum and maximum values (n = 5). DE, differentially expressed Padj < 0.05. Protein expression: uTregs were gated
as CD3+CD4+CD25hiFOXP3+ cells. Multiplicity adjusted P values of 2-way ANOVA with Tukey’s post hoc test (n = 5). (F) Heatmap showing gene expression
of chemokine receptors. Expression values were mean centered and scaled per gene. DE, differentially expressed Padj < 0.05. (G–I) Protein expression of
chemokine receptors downregulated (G) and upregulated (H and I) in uTregs. uTregs were gated as CD3+CD4+CD25hiFOXP3+ cells. P values of 2-way ANOVA
with Tukey’s post hoc test (n = 5). (J) GSEA with published gene set of CXCR3+T-bet+ Tregs from the pancreas of prediabetic mice (38), comparing uTregs
and bTregs. NES, normalized enrichment score; FDR, FDR adjusted P value.
Table 6). Of the 41 genes that were shared among ≥ 4 of the 7 TITR signatures (Supplemental Table 7), 31 were
also part of the 225 genes in the uTreg core signature. Figure 6D shows the number of genes shared between
the uTreg core signature, as well as each of the TITR signatures and healthy tissue–derived Treg signatures.
Remarkably, 93 (41.3%) of the 225 core uTreg genes were overlapping with specifically upregulated genes from
hepatocellular carcinoma–infiltrating (HCC-infiltrating) Tregs (98), 54 with the unique TITR signature identified by De Simone et al. (25), 49 with breast cancer TITR genes (95), and 40 with OX-40+ Treg from cirrhotic/
tumor liver tissue (Figure 6D) (99). Importantly, the 225 uTreg core signature genes showed less overlap with
healthy tissue–derived Treg–specific signatures from human healthy colon, lung, and skin. The genes that were
most often shared between uTregs and TITR were IL1R2 (7 of 7); TNFRSF1B, CTSC, DPYSL2, and LAPTM4B
(6 of 7) and TNFRSF4, TNFRSF18, LAYN, IL2RA, ENTPD1, NCF4, SDC4, and CRADD (5 of 7) (Figure 6E),
whereas — with healthy tissue-Treg signatures — PDGFA was most often shared (3 of 3) (Figure 6F).
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Figure 6. uTregs share their transcriptional signature with tissue- and tumor-infiltrating Tregs. (A) GSEA with a published murine PAN-tissue gene signature (18), comparing uTregs and bTregs. NES, normalized enrichment score; FDR, FDR adjusted P value. (B) Venn diagram showing the numbers of genes
upregulated in uTregs compared with bTregs (Padj < 0.05) (and genes in the uTreg core signature in parentheses), which are represented in tissue-specific
and tissue-shared published murine gene signatures (18). VAT, visceral adipose tissue. (C) Heatmap showing the expression of the 59 genes that were part
of the murine PAN-tissue signature and upregulated in uTregs compared with bTregs (Padj < 0.05) (18). Expression values were mean centered and scaled
per gene. (D) The number of genes shared between the uTreg core signature and published human TITR signatures or healthy tissue Treg signatures (14,
17, 25, 95–100). Numbers behind bars indicate the number of shared genes out of the total number of genes in the specific signature. (E) The genes that
were most often shared between the uTreg core signature and human TITR signatures (shared in ≥ 4 of 7 signatures). (F) The genes that were most often
shared between the uTreg core signature and human healthy tissue Treg signatures (shared in ≥ 2 of 3 signatures).
GSEA also showed that many of the nonoverlapping genes from the published TITR signatures were
significantly enriched in uTregs compared with bTregs (Figure 7A). Genes in the leading edge of ≥ 3 of
7 tumor-specific GSEA that were highly expressed in uTregs compared with bTregs, but not part of the
uTreg core (mostly because their high expression was shared with uTconv), are shown in Figure 7B. These
included CREB3L2 (6 of 7); EBI3, GCNT1, and ICOS (5 of 7); ACTA2, ARHGEF12, BCL2L1, CCND2,
PRDX3, and SLAMF1 (4 of 7); and CXCR3, CD7, CAECAM1, CD79B, and MICAL2 (3 of 7), among others.
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Remarkably, genes specifically upregulated in breast cancer–infiltrating Tregs compared with Tregs from
normal breast parenchyma or significantly upregulated in colon cancer Tregs compared with healthy
colon Tregs showed a particularly high enrichment in uTregs, suggesting that uTregs are not just similar
to Tregs from breast or colon tissue, but specifically to the highly differentiated/activated Tregs from the
tumor environment (Figure 7C) (26, 100). By overlapping these cancer-versus-healthy tissue Treg signatures with significantly upregulated genes in uTregs (versus bTregs), we identified 12 cancer-specific genes
expressed by uTregs (Figure 7D): CD80, IL1R2, LAYN, MYO7A, TNFRSF4, TNS3, TRAF3, VDR, DUSP4,
HSPA1A, HSPA1B, and IFI6. The first 8 of these were also part of the uTreg-specific core signature, again
highlighting the specificity c.q. importance of receptors IL1R2, LAYN, TNFRSF4, and CD80 and transcription factor VDR for human Tregs in a tumor-like microenvironment. Furthermore, tumor-specific downregulated genes were shared with the uTreg core signature: CCR7, PLAC8, and TCF7. In conclusion, these
results indicate that uTregs from the materno-fetal interface have a transcriptional core signature that is
shared specifically with the specialized transcriptional profile of TITR.
uTregs show site-specific adaptation within the uterus. Next, we wondered whether uTregs would be
merely adapted to the microenvironment in uterine tissue or specifically adapted to the tissue site at the
maternal-interface. To investigate this site-specific adaption within 1 organ, we compared uTregs from
the materno-fetal interface (i.e., placental bed; pbuTregs) with uTregs from a distant uterine site (i.e., the
incision site made during cesarean section; incuTregs). Confirmation of Treg identity and TRM signature for incuTregs is shown in Supplemental Figure 5, A–F. The differentially expressed genes between
inc
uTregs and bTregs were similar to those between pbuTregs and bTregs (Figure 8A). Furthermore,
PCA showed that gene expression profiles of pbuTregs and incuTregs were rather similar, compared with
bTregs (Figure 8B). However, direct comparison of pbuTregs and incuTregs revealed a substantial difference between the 2 populations (Figure 8C). First, protein expression of the core Treg transcription
factor FOXP3 was lower in incuTregs than pbuTregs, comparable with bTegs (Figure 8D). This was not
due to incuTreg contamination with bTregs, since expression of CD69 was similar between pbuTregs
and incuTregs (Supplemental Figure 5D). Protein expression of other core Treg markers CTLA-4 and
TIGIT was also lower in incuTregs than pbuTregs (Figure 8D). This indicates that pbuTregs, derived from
the materno-fetal interface, have a more pronounced expression of Treg signature markers, suggesting
enhanced activation/differentiation in comparison with their uterine counterparts from the incision
site. Differential gene expression analysis revealed 558 upregulated and 125 downregulated genes in
pb
uTregs versus incuTregs (Figure 8E).
The heatmap in Figure 9A shows a selection of previously highlighted genes in this manuscript that
proved to be differentially expressed between pbuTregs and incuTregs. These results suggest that Tregs
cannot only adapt to the microenvironment within a certain tissue, but they will specifically adapt to
the environmental cues at a certain tissue site. Pathway analysis showed that upregulated genes in
pb
uTregs versus incuTregs were related to PD-1 signaling, cytokine signaling, TCR signaling, and Th
cell differentiation (Supplemental Figure 5G). Indeed, PD-1 was expressed higher in pbuTregs than
inc
uTregs on gene and protein levels (Figure 9, A and B), and GSEA showed enrichment of a TCR-activated Treg signature in pbuTregs compared with incuTregs (Supplemental Figure 5H). Furthermore,
pb
uTreg-specific core genes associated with effector Treg differentiation including TNFRSF4 (OX-40
protein, Figure 9C) and transcription factors BATF, MAF, PRDM1, and VDR, among others, were
significantly expressed higher in pbuTreg than in incuTregs (Figure 9A), again suggesting that pbuTregs
show more pronounced differentiation toward an effector Treg phenotype. Since pbuTregs appeared to
be especially differentiated at the materno-fetal interface, we assessed whether the TITR-like profile of
pb
uTregs was also more pronounced than in incuTregs. Remarkably, 5 of 7 tested published TITR signatures were significantly enriched in pbuTregs compared with incuTregs (P < 0.05; Figure 9D). More specifically, GSEA with signatures differentiating between TITR and their counterparts from a matched
healthy tissue site showed significant enrichment in pbuTregs compared with incuTregs (Figure 9E).
CCR8 and ICOS, which were present in 6 of 7 TITR signatures, as well as TNFRSF18 (GITR), were
expressed significantly higher in pbuTregs than in incuTregs and bTregs on the protein level (Figure 9F).
CCR8 has been shown to be highly enriched in tumor Tregs and associated with a poor prognosis in
several cancers (25, 26, 74). Thus, pbuTregs at the materno-fetal interface specifically acquire a highly
differentiated effector profile similar to TITR, which is more pronounced even compared with a uterine tissue site distant from the materno-fetal interface.
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Figure 7. uTregs have a functional profile similar to tumor-infiltrating Tregs. (A) GSEA with published TITR-specific signatures in uTregs versus bTregs
(25, 95–100). NES, normalized enrichment score; FDR, FDR adjusted P value. (B) Heatmap showing expression of genes in the leading edge of ≥ 3 of
7 GSEA from Figure 7A, which were not represented in the uTreg core signature. Expression values were mean centered and scaled per gene. (C) GSEA
with published gene signatures specific to Tregs from tumor tissue compared with the healthy tissue counterpart in uTregs versus bTregs(26, 100). NES,
normalized enrichment score; FDR, FDR adjusted P value. (D) Venn diagrams showing shared genes between uTregs and genes specifically upregulated in
Tregs from tumor tissue compared with the healthy tissue counterpart (26, 100).
Discussion
Here, we demonstrate for the first time to our knowledge that, in pregnancy, human uTregs have a highly
differentiated transcriptional profile, which is specifically enriched at the materno-fetal interface and is
reminiscent of the specialized profile of TITR. With these findings, we answer a long-standing question
on how Tregs are functionally specialized at the materno-fetal interface to modulate local effector T cell
responses, preventing an allo-reaction against the fetus. Moreover, we introduce the potentially novel concept of site-specific adaptation of Tregs within 1 organ or tissue. This again substantiates the notion that
Tregs are capable of adapting their transcriptional program driven by microenvironmental cues (15–19).
We have demonstrated that uTregs at the materno-fetal interface display a highly activated and late-stage differentiated effector profile (32, 34, 76, 77, 79), with high BATF and PRDM1, low SATB1, increased expression
of molecules associated with suppressive capacity, and abundant expression of TNFR superfamily members.
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Figure 8. uTregs show site-specific adaptation to the materno-fetal interface. (A) Venn diagrams of genes upregulated (left panel) and downregulated
(right panel) in both incuTregs and pbuTregs compared with bTregs. (B) PCA of bTregs (n = 5), pbuTregs (n = 5), and incuTregs (n = 4). (C) PCA of pbuTregs (n = 5)
and incuTregs (n = 4). (D) Protein expression of FOXP3, CTLA-4, and TIGIT. uTregs were gated as CD3+CD4+CD25hiFOXP3+ cells. Multiplicity adjusted P value
of 2-way ANOVA with Tukey’s post hoc test. Left upper P value, blood versus placental bed; right upper P value, placental bed versus incision site; lower
P value, blood versus incision site. MFI, median fluorescence intensity. (E) Volcano plot of differentially expressed genes between pbuTregs and incuTregs.
****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.
Also, others have reported that nonlymphoid tissue Tregs display an activated phenotype compared with lymphoid organ and circulating Tregs (12, 20, 73). Both BATF and the TNFRSF/NF-κB signaling axis are crucial
in the survival of Tregs and maintenance of a stable effector Treg phenotype, especially in tissues (15, 28, 32,
33, 78, 79, 101). It is now recognized that Tregs adapt to their tissue environments, with, on the one hand, common
adaptations across many tissues — such as increased expression of IL10, IL1RL1 (encoding ST2, an IL-33 receptor
subunit), AREG (encoding amphiregulin), CTLA4, TIGIT, BATF, and IRF4 and decreased expression of LEF1,
TCF7 — but, on the other hand, importantly, tissue-specific signatures (12, 13, 15, 16, 18). These tissue-specific
signatures counter the notion that tissue Tregs merely have a more activated effector or memory state. Rather,
they have a specialized adapted program (93), likely matching the specific requirements of a certain tissue site
(10, 13, 18, 102, 103).
To our knowledge, the concept of site-specific transcriptional adaptation of Tregs within 1 tissue or
organ is novel, taking into account that tumors represent a completely altered tissue and not a different site
within the same organ. We show that uTregs display features suggestive of a high responsiveness to microenvironmental cues, such as a range of TNF receptor superfamily members and chemokine receptors. With
such a matrix of options to detect signals from the microenvironment, Tregs are likely able to adjust not
only to the tissue or organ of their residence, but even to specific sites within that tissue, based on microenvironmental cues. Most likely, implantation of the placenta (i.e., the multitude of signals produced by
myometrium-invading trophoblast; ref. 104) are the primary cues effectuating microenvironmental changes
at the materno-fetal interface. Trophoblast attracts Tregs to the materno-fetal interface by production of
hCG and CXCL16, the ligand for CXCR6.(105, 106). Moreover, in vitro coculture of HLA-G+ extravillous
trophoblast with CD4+ T cells increased Treg numbers and FOXP3 expression level (107, 108), indicating
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Figure 9. uTregs show site-specific adaptation to the materno-fetal interface. (A) Heatmap with previously highlighted genes in this manuscript that were
differentially expressed between pbuTregs and incuTregs. Expression values were mean centered and scaled per gene. (B and C) Protein expression of PD-1 (B)
and OX-40 (C). uTregs were gated as CD3+CD4+CD25hiFOXP3+ cells. Multiplicity adjusted P value of 2-way ANOVA with Tukey’s post hoc test. Left upper P
value, blood versus placental bed; right upper P value, placental bed versus incision site; lower P value, blood versus incision site. MFI, median fluorescence
intensity (n = 6). (D) GSEA with published TITR-specific signatures in pbuTregs versus incuTregs (25, 95–100). NES, normalized enrichment score; FDR, FDR
adjusted P value. (E) GSEA with published gene signatures specific to Tregs from tumor tissue compared with the healthy tissue counterpart in pbuTregs
versus incuTregs (26, 100). (F) Protein expression of CCR8 (n = 5), ICOS (n = 6), and GITR (n = 6). uTregs were gated as CD3+CD4+CD25hiFOXP3+ cells. Multiplicity
adjusted P value of 2-way ANOVA with Tukey’s post hoc test for protein. Left upper P value, blood versus placental bed; right upper P value, placental bed
versus incision site; lower P value, blood versus incision site. MFI, median fluorescence intensity. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.
that Tregs may also be locally induced or expanded by trophoblast. Thus, it is likely that signals produced
by invading trophoblasts at the materno-fetal interface account for at least some of the site-specific transcriptional adaptations in uTregs.
The Th response at the materno-fetal interface was previously suggested to be skewed away from a proinflammatory Th1 response, to prevent a pathogenic allo-reaction against the fetus, resulting in a Th2-dominant
response during the second trimester. However, during the third trimester, a proinflammatory Th1 response
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may be essential for initiation of labor (reviewed in ref. 48). In line with this, our findings indicate that the
Th response in the uterus at term is dominated by Th1 polarization, although well controlled. uTregs at the
materno-fetal interface appear to be specifically equipped to effectively suppress Th1 responses. Although
we observed heterogeneity of T-bet protein expression in uTreg, CXCR3 expression was remarkably homogeneous, with 84%–100% of uTregs being CXCR3+. CXCR3-expressing (and T-bet– expressing) Tregs are
especially adept to suppress Th1 responses (36, 38, 45, 89). Furthermore, the majority of uTregs expressed
TIGIT, OX-40, and/or CCR5. Tregs expressing TIGIT preferentially inhibit Th1 and Th17 responses (72),
a subpopulation of OX-40–expressing Tregs can differentiate into Th1-suppressing Tregs (109), and also
CCR5 expression on Tregs has been associated with more effective suppression of Th1 responses (90). Thus,
the necessary proinflammatory Th1 response at the materno-fetal interface at term appears to be controlled
by specifically differentiated and Th1-polarized Tregs. So far, Th1-like Tregs have been described mainly in
inflammatory environments, such as infections, autoimmune diseases, and transplantation reactions (45,
89, 110–112), whereas tissue-resident Tregs were mostly characterized as being Th2 skewed (VAT, muscle)
(13, 18) or Th17 skewed (intestines) (73). DiSpirito et al., however, recently also identified a subset of T-bet–
expressing Tregs in muscle and colon (18), indicating that they can also be present in steady-state tissues.
We are the first to our knowledge to study exclusively maternal, myometrial tissue–resident Tregs from
the materno-fetal interface. Previous studies of uTregs had to resort to the use of more easily accessible
decidua, due to the difficulty of acquiring human myometrium. Since decidual tissue is of fetal origin, it
may not only be contaminated with fetal immune cells, but it also does not allow for studying the unique,
and specifically maternal, uterine environment underlying the placenta, in which the complex process of
spiral artery remodeling takes place. The only publications that we know of investigating FOXP3 expression in actual human placental bed biopsies demonstrated that the percentage of FOXP3+ T cells was
significantly decreased in patients with preeclampsia, and FOXP3 mRNA expression was reduced in endometrial biopsies of infertile women, highlighting the importance of functional Tregs for a healthy pregnancy (57, 113). From human decidual data, it is known that the frequency of clonally expanded populations
of effector Tregs is increased in decidua of third trimester cases compared with first trimester cases (114).
Decidual Tregs display a more pronounced suppressive phenotype than in blood, with increased expression
of FOXP3, CTLA-4, CD25, HLA-DR, ICOS, GITR, and OX-40, which recapitulates our findings (58,
59, 63, 115). Recently, 3 types of functional Tregs were identified at the human materno-fetal interface,
of which the CD25hiFOXP3+ population matches the population investigated in this study (108). These
Tregs effectively suppressed CD4+ and CD8+ T cell proliferation and IFN-γ and TNF-α production. Transcripts identified by qPCR array as specific for this subset were IL2RA, FOXP3, TIGIT, ENTPD1, LRRC32,
IL1RL1, BATF, and CCR8, and increased expression of CCR5, IL10, and GITR compared with bTregs
was also observed (108), which confirms our findings of an activated Treg phenotype at the materno-fetal
interface. A study investigating chemokine receptor expression of CXCR3, CCR4, and CCR6 in decidual
Tregs by flow cytometry showed that CCR6–CXCR3+ Th1 cells were increased and CCR6+CCR4+ Th17
cells were nearly absent, whereas CCR4+ Th2 frequencies were similar in blood and decidua (58), which is
also in line with our findings. Taken together, this indicates that the here-identified activated phenotype of
myometrial uTregs has overlapping characteristics with decidual Tregs.
We observed that uTregs from the materno-fetal interface display a peculiar differentiated effector phenotype similar to TITR, defined by high expression of IL1R2, LAYN, CD80, VDR, and TNFRSF4, among
others, with specific enrichment of TITR signatures compared with Treg signatures from matched, unaffected tissue sites. This observation may be explained by recent insights on the similarity of the immune environment at the materno-fetal interface and tumors (48). Both the receptivity of the myometrium toward implantation of the blastocyst and invasiveness of the trophoblast show striking similarities with implantation of
tumor metastases in healthy tissues (116, 117). Tumor cells can modulate their immune environment into an
antiinflammatory milieu and can recruit and/or induce suppressor cells, among which are high numbers of
Tregs (118, 119). Just as in tumors, a tolerogenic mode of antigen presentation with indirect allorecognition
of low levels of antigens predominates at the materno-fetal interface (120). Furthermore, others reported
striking similarities between early Treg responses to embryo and tumor implantation (54). These findings
imply that the microenvironment at the materno-fetal interface may be a unique mammalian tissue site that,
under challenged but physiological conditions, resembles a tumor microenvironment: an actively remodeling tissue site distinct from a steady-state tissue, with low-grade inflammation and newly infiltrating/invading cells. These dynamic characteristics may account for the unique transcriptional adaptation of Tregs.
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Although we observed global changes in gene expression patterns in uTregs, flow cytometry revealed
an expression gradient of many markers across the uTreg population, suggesting that uTregs consist of a
heterogenic population with various stages of differentiation and possibly subspecialization. Single cell
sequencing techniques and mass cytometry are indeed starting to reveal the heterogeneity of Treg populations in tissues and tumors (15, 27, 97, 98, 121–123).
A unique strength of our study is that we compared the transcriptomic profile of a highly specific and
highly purified maternal Treg subset from myometrial biopsies, not only to their counterpart in blood, but
also to a tissue-specific and site-specific Treg control population and matched Tconv population. We validated our key findings on the protein level in single cell resolution by flow cytometry. We only studied term
pregnancies, due to the practical limitation of delivery of the infant and placenta. It would therefore be
interesting to investigate term-dependent changes in uTreg profiles in future studies. Although protocols for
tissue digestion may induce transcriptional changes (124), many of the uTreg-specific genes were previously found not to be affected by a tissue digestion protocol similar to but harsher than the one used here (17).
Our findings have important implications. TITR are currently under heavy investigation as targets in cancer immunotherapy. However, we demonstrate that signatures identified in TITR are not as unique as previously assumed and that they may be shared by Tregs with specialized functions in other human tissues that may
still be unknown. On the other hand, our results may lead to new targets for cancer immunotherapy, since profiling of Tregs in a variety of tissues under physiological, but not necessarily steady-state, conditions may help
to identify truly TITR-specific expression patterns. Moreover, increased understanding of immunoregulatory
mechanisms at the materno-fetal interface during healthy pregnancy gives unique insights into human immunobiology of pregnancy and also aids to elucidate the pathological changes in Tregs in pregnancy disorders
such as preeclampsia, fetal growth restriction, or recurrent miscarriage, as many studies have pointed toward
a role for Treg defects or deficiency in these disorders (65–67, 105, 114, 125, 126). Last, functional adaptation
of human Tregs to different tissues and specific tissue sites is still largely unexplored. The receptivity of Tregs
to their environmental stimuli and subsequent subspecialization may be exploited for therapeutic purposes.
In conclusion, we have shown that human Tregs show functional adaptation with tumor-infiltrating–
like features specifically at the materno-fetal interface, which introduces the concept of tissue site-specific
transcriptional adaptation of human Tregs.
Methods
Participants and biopsies. This study is part of the Spiral Artery Remodeling (SPAR) cohort study, which is
an ongoing effort to investigate the adaptation of the uterus to placental development by obtaining site-specific uterine biopsy samples in women undergoing cesarean section. A detailed description of the study
set-up and protocol was previously published (127). For this analysis, we included 20 women who delivered
by elective cesarean section (i.e., without any contractions or other signs of labor such as rupture of membranes) after an uncomplicated pregnancy and without any major underlying pathology, n = 5 of which
were included for transcriptomics of T cell populations, n = 4 for suppression assays, and n = 11 for flow
cytometry. Baseline characteristics are provided in Supplemental Table 1. One tube of sodium-heparin
blood was taken from each donor before cesarean section. After delivery of the neonate and placenta,
the placental bed was manually located, and 2 biopsies of the central placental bed from the inner uterine
myometrial wall were obtained as previously described (127). Additionally, biopsies were taken from the
incision site when the placenta was not situated on this part of the uterine wall.
Lymphocyte isolation. Peripheral blood mononuclear cells (PBMC) were isolated from blood diluted 1:1
with basic medium (RPMI 1640 [Thermo Fisher Scientific] with penicillin/streptomycin [Thermo Fisher
Scientific], L-glutamine [Thermo Fisher Scientific]), by ficoll-density centrifugation (at room temperature,
1100g for 20 minutes; GE Healthcare-Biosciences, AB). PBMC were washed in basic medium with 2%
FCS (Biowest) and PBS or staining buffer consisting of cold PBS supplemented with 2% FCS and 0.1%
sodium-azide (Severn Biotech Ltd.). The biopsy samples were collected in basic medium supplemented
with 10% FCS and minced into pieces of 1 mm3 in PBS (Thermo Fisher Scientific). The biopsies were enzymatically digested with 1 mg/mL collagenase IV (MilliporeSigma) in medium for 60 minutes at 37°C in a
tube shaker under constant agitation at 120 rpm. To dissolve the remaining biopsy pieces after digestion and
remove any remaining lumps, the biopsies were pipetted up and down multiple times and poured over a 100
μm cell strainer (BD Falcon). Cells were subsequently washed in staining buffer and filtered through a 70 μm
cell strainer, and they were prepared for flow cytometry or flow cytometry-assisted cell sorting.
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Flow cytometry. For flow cytometric experiments without restimulation, PBMC and uterine cells were first
incubated in fixable viability dye eFluor506 (eBioscience) in PBS (1:300) for 20 minutes at 4°C and washed
in PBS. For surface staining, cells were incubated with the antibodies shown in Supplemental Table 2 for 20
minutes in staining buffer at 4°C and were subsequently washed in the same buffer. Cells were permeabilized
with 1 part fixation/permeabilization concentrate and 3 parts fixation/permeabilization diluent (eBioscience)
for 30 minutes at 4°C and subsequently incubated overnight with intracellular antibodies (Supplemental Table
2) in 10× diluted Permeabilization buffer (Perm; eBioscience) 4°C. The next day, cells were washed with Perm
and measured on the LSR Fortessa (BD Biosciences). For intracellular cytokine measurement, PBMC and
uterine cells were first incubated with surface staining, washed, and then restimulated with 20 ng/mL phorbol
12-myristate 13-acetate (PMA, MilliporeSigma) and 1 μg/mL ionomycin (MilliporeSigma) for 4 hours with
addition of Monensin (Golgistop, BD Biosciences) during the last 3.5 hours at 37°C. Afterward, cells were
stained with the viability dye, permeabilized, intracellularly stained, and measured as described above.
Flow cytometry–assisted cell sorting. Cells were incubated with surface antibodies (Supplemental Table 2) for
20 minutes in staining buffer at 4°C, washed in the same buffer, and filtered through a 50 μm cell strainer (Filcon, BD Biosciences). For suppression assays, cells of the CD3+CD4+CD25+CD127– cell population (Tregs)
and CD3+CD4+CD25– cell population (Tconv) were directly sorted into tubes with 500 μL FCS on a FACSAria III (BD Biosciences). For RNA sequencing (RNA-seq), 2000 cells of the CD3+CD4+CD25+CD127– cell
population (Tregs) and CD3+CD4+CD25–CD45RA– (CD69+ from biopsies, CD69– from blood) cell population (Tconv) were sorted into Eppendorfs containing 125 μL PBS. After sorting, 375 μL Trizol LS (Thermo
Fisher Scientific) was added to each vial, and vials were stored at –80°C until RNA isolation.
Suppression assays and cytokine measurement. After sorting, peripheral blood, uTregs, and Tconv were
washed in PBS and resuspended in basic medium with 10% human AB serum (Sanquin). Previously isolated and frozen healthy (HC) PBMC were labeled with 2 μM CellTrace Violet (Thermo Fisher Scientific) as
described previously (128). Treg or Tconv populations were added to 15,000 HC PBMC at different ratios,
and cells were coincubated for 4 days at 37°C. Supernatants were collected for cytokine measurement by
multiplex assay before cells were stained with surface antibodies for CD3, CD4, and CD8 as described
above and measured on a FACS Canto (BD Biosciences).
Whole transcriptome sequencing. For RNA isolation, the vials were thawed at room temperature, and 100 μL
chloroform was added to each vial. The vials were shaken well and spun down at 12,000g for 15 minutes at 4°C.
The aqueous phase was transferred into a new tube, and RNA was mixed with 1 μL of GlycoBlue (Invitrogen)
and precipitated with 250 μL isopropanol. Samples were incubated at –20°C for 1 hour and subsequently spun
down at 12,000g for 10 minutes. The supernatant was carefully discarded, and the RNA pellet was washed twice
with 375 μL 75% ethanol. Vials were stored at –80°C until library preparation. Low-input RNA-seq libraries
from biological sorted cell population replicates were prepared using the Cel-Seq2 Sample Preparation Protocol
(129) and sequenced as 2 × 75 bp paired-end on a NextSeq 500 (Utrecht Sequencing Facility). The reads were
demultiplexed and aligned to human cDNA reference using the BWA (0.7.13) (130). Multiple reads mapping to
the same gene with the same unique molecular identifier (UMI, 6bp long) were counted as a single read.
Statistics. RNA-seq data were normalized per million reads per sample. Differentially expressed genes
between the cell populations were identified using the DESeq2 package in R 3.5.1 (CRAN), with correction
for donor batch (design = ~donor + cell population) and input of all genes. Genes with FDR Padj < 0.05
and |log2FC| > 0.5 were considered differentially expressed. PCA was performed in DESeq2 based on the
constructed model, including donor correction. Pathway enrichment analysis was conducted in the Toppgene Suite publicly available online portal (https://toppgene.cchmc.org/enrichment.jsp), and pathways
with Bonferroni-corrected P < 0.05 were considered statistically significant (131). For heatmap analysis, gene
expression was mean centered and scaled per gene, and hierarchical clustering was performed with Ward’s
method and Euclidian distance. GSEA (132) was conducted with Broad Institute software, by 1000 random
permutations of the phenotypic subgroups to establish a null distribution of enrichment score, against which
a normalized enrichment scores and multiple testing FDR-corrected q values were calculated. Gene sets with
an FDR < 0.05 were considered significantly enriched. Gene sets were either obtained from provided data
in publications or by analyzing raw data using GEO2R (NCBI tool) (133). An overview of used signatures
is provided in Supplemental Table 3. For flow cytometric data, median fluorescence intensities (MFI) and
percentages of positive cells were analyzed in FlowJo. For graphic representation, data were analyzed in
GraphPad Prism. To assess significant differences on protein level between groups, 2-way ANOVA with
Tukey’s post hoc test was used, and multiplicity-adjusted P < 0.05 were considered statistically significant.
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The data sets generated for this study have been submitted to a public repository on GitHub ((https://
github.com/JudithWienke/Human-uterine-Tregs; branch name, master; Commit ID, f788ba7f8163d7f892ce4069a2c9c304cf922902) The raw data files could not be submitted due to GDPR constraints, but any additional
required data can be requested with the corresponding author.
Study approval. All patients received study information and signed informed consent before participation. This study was reviewed and approved by the local Institutional Ethical Review Board of the University Medical Center Utrecht (no. 16-198).
Author contributions
LB recruited and included patients, and collected clinical data. JW, LB, RCS, and LMVDB performed all
wet-lab experiments. MM performed the RNA-seq and helped with data analysis. JW performed all data
analyses and wrote the manuscript. PGJN consulted on biopsy preparation, tissue integrity, and uterine T
cell distribution and phenotype. BBVR and FVW supervised JW, LB, LMVDB, and RCS and were closely
involved in setting up the study protocol, collecting data, conducting data analysis, and writing the manuscript. All authors critically revised the manuscript.
Acknowledgments
We thank Michal Mokry, Noortje van Dunen, and Nico Lansu for their help with RNA-seq. We thank
the multiplex core facility, and especially Jeroen van Velzen and Pien van der Burght, for their advice and
(both practical and moral) support during FACS sorting. We thank Tatjana Vogelvang for her help in the
collection of samples at the Diakonessenhuis in Utrecht and Arie Franx for his involvement in establishing the SPAR study initiative at the Wilhelmina Children’s Hospital. FW was supported by a VIDI grant
(91714332) from the Netherlands Organization for Scientific Research (NWO, ZonMW).
Address correspondence to: Femke van Wijk, Suite KC.02.085.0, Postbus 85090, Lundlaan 6, 3508 AB
Utrecht, Netherlands. Phone: 31.0.88.7554275; Email: f.vanwijk@umcutrecht.nl
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| 12,199 | 85,239 |
W1999051990.txt | Document downloaded from:
http://hdl.handle.net/10251/134138
This paper must be cited as:
Chieregato, A.; Soriano Rodríguez, MD.; Basile, F.; Liosi, G.; Zamora Blanco, S.;
Concepción Heydorn, P.; Cavani, F.... (2014). One-pot glycerol oxydehydration on
multifunctional catalysts: influence of acid and redox properties on the catalytic performance.
Applied Catalysis B Environmental. 150-151:37-46.
https://doi.org/10.1016/j.apcatb.2013.11.045
The final publication is available at
https://doi.org/10.1016/j.apcatb.2013.11.045
Copyright Elsevier
Additional Information
One-pot glycerol oxidehydration to acrylic acid on multifunctional
catalysts: Focus on the influence of the reaction parameters in respect
to the catalytic performance
Alessandro Chieregato1, M. Dolores Soriano2, Francesco Basile1, Giuseppe Liosi1, Segundo Zamora2,
Patricia Concepción2, Fabrizio Cavani*, José M. Lopez Nieto*
1. Dipartimento di Chimica Industriale e dei Materiali, ALMA MATER STUDIORUM Università di
Bologna, Viale Risorgimento 4, 40136 Bologna, Italy. Also: INSTM, Research Unit of Bologna.
2. Instituto de Tecnología Química, UPV-CSIC, Campus de la Universidad Politécnica de Valencia,
Avda. Los Naranjos s/n; 46022 Valencia (Spain)
Abstract
We report here about an investigation of the key catalyst features and reaction parameters in
glycerol oxidehydration into acrylic acid over W-V-Nb mixed oxides with the hexagonal tungsten
bronze structure. The incorporation of Nb allowed to considerably improve the catalytic behaviour
not only because of the higher yield to acrylic acid and acrolein compared to W-V bronzes, but also
to increase of more than order of magnitude the oxygenated productivity rate, because of the
greater concentration of glycerol used in the inlet feed and the lower contact time needed.
Experiments were carried out by reacting acrolein, and by oxidation of methanol, as a model
molecule for the determination of surface acid and redox properties. These experiments
demonstrated that with the W-V-Nb catalyst the optimal ratio between the two consecutive steps
of acid-catalysed glycerol dehydration and acrolein oxidation, and between the two parallel
reactions of acrolein oxidation and transformation into by-products (ketals and oligomers), was
achieved in the presence of defined glycerol-to-oxygen inlet ratio. Indeed, oxygen played the
fundamental role of accelerating the oxidation of the intermediately formed acrolein into acrylic
acid, by allowing a greater concentration of the oxidizing V5+ sites.
1
Introduction
With the aim of reducing the carbon footprint of fuels, bio-diesel represents one of the most
important options, theoretically providing neutral CO2 balance and significant reduction of
greenhouse gas emission.1-3 The increasing trend of biodiesel production goes hand-in-hand with
the availability of large volumes of glycerol that is co-produced in the conventional
transesterification reaction exploited to synthesize the bio-fuel.2,4 Since the late 90s, this abundance
of glycerol has significantly impacted the glycerin market resulting in a decline of its price,5,6 and
finally making glycerol a low-cost raw material desirable to be converted into different chemicals
with higher economic value. On the other hand, it must be mentioned that starting from 1997 the
glycerin market has faced very volatile conditions, increasing price and actual shortage of refined
glycerol supply,7 that nowadays renders difficult any accurate economic evaluation of chemical
processes designed for its transformation. However, since the over-supply of crude glycerol from
biodiesel production (Bio-Crude) is actually one of the main reasons for price volatility, the
transformation of glycerol toward more added-value products represents a key-point so as to
develop a new bio-based chemical industry.
Amongst the several options focused on glycerol conversion, a great deal of attention has
been paid to the dehydration of glycerol into acrolein,1,4,7-31 and more recently also to the
oxidehydration of glycerol into acrylic acid.32-42 From an economic and an engineering point of view,
an interesting option to perform the latter reaction is the one-pot (single-step) transformation, by
means of a multifunctional catalyst able to carry out both the dehydration of glycerol into acrolein
and aldehyde oxidation into acrylic acid. Indeed, the use of a single catalyst can simplify the reactor
engineering and level the heat balance between the endothermic dehydration of glycerol and the
exothermic oxidation step.
2
Previously we reported that W-V oxides with hexagonal tungsten bronze (HTB) structure and
V4+ incorporated in WO3 lattice are effective catalysts for the direct synthesis of acrylic acid from
glycerol;38 under the best conditions these systems showed 25% acrylic acid yield plus 11% yield to
residual acrolein. Later, we studied the influence of Nb addition to V-doped HTB structures, with
the aim of improving the acidic features of the catalysts so as to enhance the glycerol dehydration
step. Three-component catalysts (W-V-Nb) further increased the catalytic performance, finally
leading to 33% acrylic acid yield plus 17% yield to residual acrolein;41 moreover, since the optimum
working condition for W-V-Nb catalysts is located at a much lower contact time (from 0.38 s to 0.15
s) than for the W-V system, the catalyst productivity was also consistently increased.
In order to fully investigate the catalyst activity of the best performing tri-component sample
(reported as WVNb-1 sample)41, we report here a complete study of the catalyst behavior as a
function of the inlet feed composition. Moreover, it is here reported about the methanol gas-phase
transformation into formaldehyde and dimethyl ether (DME) on both W-V and W-V-Nb catalysts;
indeed, methanol is known to be a suitable probe to explore the acid and redox properties of metal
oxides.45,46 The results obtained allow to draw important and general conclusions on the catalyst
features needed to convert oxygenated molecules by means of direct processes, using a single
multifunctional catalyst.
Finally, so as to support the conclusions depicted on HTB doped structures, the same
reactions were carried out on Mo-V-(W) oxides with laminar structure. In order to obtain a complete
picture of the catalysts behavior, the acrolein oxidation reaction was also studied on both HTBs and
laminar-type oxides.
2. Experimental
2.1 Synthesis of W-V, W-V-Nb HTBs and Mo-V-(W) laminar oxide
3
W-V and W-V-Nb catalysts were prepared hydrothermally from gels obtained from aqueous
solution of the corresponding salts. The hydrothermal synthesis was carried out at 175°C 48 h. Then
the solid were washed, dried at 100°C overnight. Finally the solids were heat-treated in N2 at 600°C
during 2 h. More details on the preparation have already been reported elsewhere.38,41
Mo-V-(W) catalyst was prepared by evaporation method; an aqueous solution of ammonium
heptamolybdate, vanadyl sulphate and ammonium tungstate was evaporated in rotavapor. The
solid was dried at 100°C overnight and then calcined in air at 350°C. Finally the solid was heattreated in N2 at 500°C for 2 h. (Josè: MORE DETAILS ON THE LATTER CATALYST ?).
2.2 Catalysts characterization
Surface areas were obtained from N2 adsorption isotherms using the BET method; a
Micromeritics ASAP 2000 instrument was used. The samples were degassed in-situ under vacuum
at a temperature of 250°C. Metal atomic composition of both hydrothermal and heated samples
was determined by inductive coupled plasma (ICP). Powder X-ray diffraction patterns (XRD) were
collected using a PANalytical X’Pert PRO diffractometer with CuKα radiation and an X’Celerator
detector in Bragg-Brentano geometry. Unit cell parameters were refined using the program
FullProf.25 Termogravimetric analysis (TG) was performed on a Mettler Toledo (TGA/SDTA 851)
instrument in the temperature range 20-700°C with a 0.02 g sample. The heating rate was 10°C min1
and the flow air 100 ml min-1.
Infrared spectra were recorded at room temperature in the 300-4000 cm-1 region with a
Nicolet 205xB spectrophotometer equipped with a Data station at a spectral resolution of 1 cm-1
and accumulations of 128 scans. Raman spectra were obtained with an “in via” Renishaw
spectrometer, equipped with an Olympus microscope. The exciting wavelength was 785 nm from a
Renishaw HPNIR laser with a power of approximately 15 mW on the sample. Dehydration of
4
catalysts was carried out using a home-designed microreactor for in situ Raman spectroscopy
measurement. The samples were dehydrated under 20 ml min-1 argon flow at 150°C.
Temperature-programmed reduction (TPR) experiments were carried out on 10–20 mg of
catalyst with a N2:H2 flow (10% H2, total flow 50 ml min-1). The temperature range explored was
from room temperature to 650°C. The heating rate was maintained at 10°C min-1. Temperatureprogrammed desorption of ammonia (TPD) experiments were carried out on a TPD/2900 apparatus
from Micromeritics. 0.30 g of sample was pre-treated in a He stream at 450°C for 1 h. Ammonia was
chemisorbed by pulses at 100°C until equilibrium was reached. Then, the sample was fluxed with
He stream for 15 min, prior to increasing the temperature up to 500°C in a helium stream of 100 ml
min-1 and using a heating rate of 10°C min-1. The NH3 desorption was monitored with a thermal
conductivity detector (TCD) and a mass-spectrometer following the characteristic mass of ammonia
at 15 a.m.u.
X-ray photoelectron spectroscopy (XPS) measurements were performed on a SPECS
spectrometer equipped with a Phoibos 150 MCD-9 detector using a monochromatic Al K-alpha
(1486.6 eV) X-ray source. Spectra were recorded using analyzer pass energy of 50 V, an X-ray power
of 200W, and an operating pressure of 10-9 mbar. Spectra treatment was performed using the CASA
software. Binding energies (BE) were referenced to C1s at 284.5 eV.
2.3 Reactivity experiments
Reactivity experiments for glycerol, acrolein and methanol transformation were carried out
using a continuous flow reactor made of glass, operating at atmospheric pressure. An amount of
catalyst ranging from 0.1 to 0.3 g of catalyst was loaded in the form of powder. Overall reactor gas
residence time was varied. Inlet feed molar ratios were also varied according to the desired
compositions. For each condition, all the reaction parameters are always specified in each figure.
5
For both glycerol oxidehydration and acrolein oxidation the effluent stream was bubbled through
two in-series abatement devices, which were filled with water (but in some cases anhydrous
acetone was used, for the identification of compounds which are less soluble in water) and
maintained at a temperature of 0-2°C; a third refrigerated condenser was left unfilled with any
solvent. After this abatement, the gaseous stream, still containing oxygen and carbon oxides, was
fed to an automatic sampling system for gas-chromatography (GC-TCD) analysis. The water solution
containing the unconverted glycerol and reaction products was analyzed by GC, using a HewlettPackard 5890 instrument equipped with a FID detector. A semi-capillary wide-bore OV 351
(polyethylenglycol treated with terephthalic acid) column was used for the separation of condensed
compounds; oven temperature was set from 40°C to 190°C (heating rate 10°/min, isothermal step
at 190°C, 3 min), then from 190°C to 225°C (heating rate 30°/min, final isothermal step at 225°C, 30
min). Two wide-bore columns were used for the separation of uncondensable products: a Molsieve
5A for oxygen and CO, and a Silica Plot for CO2 (oven temperature 80°C). Compounds were identified
by means of both GC-MS and the injection of pure reference standards for the comparison of
retention times in the GC column. A few unknown compounds were eluted in the GC column; we
attributed to these compounds the same response factor of the corresponding known compound
with the closest retention time. In the figures included with this paper, minor identified products
and unknown compounds have been grouped together under the heading “Others”.
Josè: SPECIFIC REACTION PARAMETERS FOR METHANOL REACTION
3. Result and discussion
3.1 Characterization of mixed-oxide catalysts
Josè: you should write this part. The figures and Tables, those which you sent us, are at the end of
the paper.
6
3.2 Reactivity experiments: oxidehydration of glycerol on W-V-Nb with HTB structure
In order to examine the catalyst activity of W-V-Nb sample (WVNb-1 in ref [41]), the inlet
feed composition was varied; for each value of oxygen-to-glycerol ratio, the catalytic performance
was analyzed in the temperature range 260-410°C. Specifically, Figure N+1 reports the catalytic
results obtained at 265°C, where the maximum acrylic acid yield was registered (however,
analogous trends were obtained for the other temperatures examined). In this experiment both the
glycerol molar content in feed (6%) and the water one (40%) were kept constant; oxygen molar and
inert gas (He) molar contents were varied so as to satisfy both the desired feed composition and the
pursued residence time. The latter was always set constant to 0.15 s (measured at room
temperature).
When a stoichiometric amount of oxygen and glycerol was fed (ratio 3:6), the glycerol
conversion was close to 50% and very low selectivity to both acrolein and acrylic acid were
registered. On the other hand, selectivity into heavy compounds was very high. Increasing the
amount of oxygen, the heavy compounds selectivity dropped in favor of selectivity to the other
products; the acrylic acid selectivity trend showed a maximum (39%) with the oxygen-to-glycerol
molar ratio 12:6, finally decreasing in favor of carbon oxides.
Figure N+2 shows the results of a set of experiments where the oxygen to glycerol molar
ratio was kept constant equal to 2, which showed to be the best ratio in Figure N+1, but the inlet
molar fraction of reactants was proportionally decreased compared to the ratio oxygen-to-glycerol
12:6. In spite of the constant optimal ratio between the reactants, the heavy compounds selectivity
increased mainly in favor of acrylic acid when the molar fraction of reactants was decreased, which
is the opposite one might expect based on the fact that higher reactants loading should favour
condensation reaction leading to heavy compounds. This indicates that the most important
7
parameter affecting catalytic behavior is the oxygen partial pressure; higher oxygen contents
enhance the oxidation of the intermediately formed acrolein into acrylic acid, so limiting the
undesired formation of acrolein condensation into by-products. When instead lower oxygen molar
fractions are used, irrespectively of glycerol partial pressure (i.e., at both low and high glycerol
partial pressure), the kinetically preferred reaction involves the transformation of acrolein into byproducts (Scheme 1). This strongly support the hypothesis that the rate-determining step in the
redox mechanism of acrolein transformation into acrylic acid is the oxidation of the reduced V sites
into V5+, the latter being the species involved in the aldehyde oxidation, and that the surface
concentration of the oxidizing species is thus affected by oxygen partial pressure.
OH
O
O
1/2 O 2
- H 2O
H
OH
OH
OH
H+
Oligomers
Scheme 1. The overall reaction mechanism for glycerol oxidehydration.
On the other hand, if the partial pressure of oxygen is too high, the selectivity to acrylic acid
drops, with a concomitant increase of carbon oxides (Figure N+1). These findings allow to draw the
important conclusion that in order to minimize the undesired competitive reaction of heavier
compounds formation, likely catalyzed by the same acid sites which are also required for the glycerol
dehydration step, the crucial point is to develop a steady-state showing a controlled amount of
oxidizing surface sites on catalyst surface.
Figure N+3 compares the productivity into acrolein + acrylic acid for the W-V catalyst41 and
for the W-V-Nb catalyst under non optimized reaction conditions,38 with productivity achieved
8
under the conditions shown in the present work, and with results reported in the literature as well.
It is shown that the productivity achieved with W-V-Nb is by far higher than that previously reported,
and also much better than that reported for other catalytic systems and for the two-catalyst inseries reactor configuration, which the alternative approach proposed in the literature. Indeed, the
best value achieved of ca 2 h-1 is quite of significance for industrial processes aimed at the synthesis
of bulk chemicals or intermediates.
3.2 Reactivity experiments: oxidehydration of glycerol on W-V-Nb catalyst, time-on-stream test
Figure N+4 (top) shows the catalytic behavior of the W-V-Nb catalyst plotted in function of
time-on-stream (tos), using the experimental conditions that allowed to obtain the best acrylic acid
yield (feed molar ratio O2/Gly/H20/He=12/6/40/40, temperature 265°C, residence time 0.15 s). The
yields shown in figs. N+1 and N+2 were obtained after ca 90 min tos, and well fit the trends here
shown; on the other hand, after 6 h tos an activation phenomenon was registered. Indeed, until ca
40 h tos the consecutive selective oxidation of acrolein to acrylic acid was clearly favored, since the
acrolein yield decreased in favor of acrylic acid. At ca 40 h tos, the highest 50% acrylic acid yield was
registered. However, afterwards the catalyst started to deactivate and the selectivity to acrolein
and acrylic acid inverted their trends. Figure N+4 (bottom), plotting the yield to both acrolein and
acrylic acid registered during experiments shown in Figure N+4 (top), demonstrates that a clear
kinetic relationship between acrolein and acrylic acid exists; indeed, the sum of yields to both
compounds was always close to 55%, even if the relative quantity of the two was consistently
different during the period of the experiment.
The XRD analysis on the spent W-V-Nb sample indicates that at the end of the lifetime
experiment the catalyst structure was deeply changed with a loss of the HTB structure…… (Josè:
9
ANY SUGGESTION AND COMMENT IS HERE VERY WELCOMED! ANY COMMENT FROM XPS/RAMAN
ANALYSIS ON SPENT SAMPLE?).
More complex is the explanation for catalyst activation shown during the 6-to-20 h tos; this
might be due to the formation of different crystalline or amorphous surface structures. Indeed, the
formation of more active amorphous structures is well known to occur on Mo-V-(W) catalysts,
typically used to selectively oxidize acrolein into acrylic acid44 and we cannot exclude that a similar
phenomenon may occur on our W-V-Nb systems. Josè: here it should be necessary to characterise
the catalyst downloaded after the first 20 h tos, that is after the activation has occurred; would it
be possible to make TEM analysis of the freshly calcined, of the activated (after 20 h tos) catalyst
and finally of deactivated catalysts ? We shall prepare samples and mail you.
3.3 Reactivity experiments: reaction with methanol on W-V and W-V-Nb catalysts
Figures N+5 and N+6 report the results obtained when methanol was made react in the
presence of oxygen on W-V-Nb and W-V catalysts; this latter sample was investigated in detail for
glycerol oxidehydration in a previous work.38 As a general trend, at low temperature the formation
of dimethyl ether (DME) was favored over its oxidation into formaldehyde (HCHO) (higher ratio
DME/HCHO); when the temperature was increased the DME-to-formaldehyde ratio decreased. At
high temperature the decomposition and total oxidation were also favored, thus increasing the
overall carbon oxides selectivity, mainly carbon monoxide through formaldehyde decomposition.
Minor amounts of dimethoxymethane and methylformiate also formed.
Comparing the catalytic behavior of W-V and W-V-Nb, it is possible to notice that for the
latter sample a much higher selectivity to DME was shown, in accordance with the higher acid
strength of this sample. Increasing the temperature, the reaction of methanol oxidehydrogenation
to formaldehyde kinetically prevailed over the etherification reaction; finally, at high methanol
10
conversion (90%) formaldehyde selectivity was almost identical for the two catalysts. Moreover, it
is interesting to notice the different temperatures needed to reach similar conversions; for the WV-Nb catalyst, 90% methanol conversion was shown at 330-335°C, whereas for the W-V catalyst the
same conversion was achieved at 380°C; this difference is attributable to higher surface specific
surface area of the Nb-containing sample, which is one of the main features of this catalyst
compared to the W-V; moreover, a contribution to the higher activity may also derive from the
faster reaction of methanol etherification. The CO selectivity also was slightly different for the two
catalysts, respectively 10% for W-V-Nb and 16% for W-V (at the same methanol conversion of 90%).
3.4 Reactivity experiments: acrolein conversion on W-V and W-V-Nb catalysts
Figure N+7 compares the catalytic results obtained when acrolein was made react on W-V
and W-V-Nb catalysts. Under the same working conditions, the two catalyst showed important
differences: W-V sample exhibited both lower acrolein conversion than W-V-Nb, which again can be
explained by taking into account the higher specific surface area of this latter sample, and lower
selectivity into acrylic acid, with higher selectivity to heavy compounds. Therefore, It may be
hypothesized that the improved acid features of this latter sample may help the desorption of acrylic
acid,43 so limiting its combustion. On the other hand, the low selectivity to acrolein oligomers
suggests that the aldehyde oxidation into acrylic acid is much faster than the side acid-catalysed
reactions, which may also relate to the high concentration of V sites. The latter in turn is greatly
affected by oxygen partial pressure (vide supra), but a role of Nb ions in facilitating the reoxidation
of reduced V sites cannot be excluded.
3.4 Reactivity experiments: comparison with Mo-V-(W) laminar oxide
11
With the aim of demonstrating the general conclusions drawn on W-V-Nb catalysts, showing
superior performance compared to W-V, we report here the catalytic results obtained on Mo-V-(W)
laminar oxide, which is a catalyst for the selective oxidation of acrolein into acrylic acid [REF].
Figure N+8 reports the results obtained in the oxidehydration of glycerol using the same
conditions found as optimal for W-V-Nb. As highlighted by the catalyst characterization (NH3
adsorption and NH3-TPD tests, fig. N) this mixed oxide shows very poor acid properties; this finding
is very well reflected on the reactivity results of both glycerol (fig. N+8) and methanol (fig. N+9).
Indeed, when glycerol was made react on this catalyst very high selectivity to heavier compounds
was registered since the unconverted glycerol can react with the produced aldehyde so as to form
ketals (cyclic ethers). In fact, if the catalyst does not hold the acid properties needed to quickly and
selectively dehydrate glycerol into acrolein, the formation of other by-products is detected, eg,
cyclic ethers such as 1,3-dioxan-5-ol and 1,3-dioxolan-4-yl-methanol, formed by reaction between
glycerol and formaldehyde or acetaldehyde, the latter compounds being in turn formed by retroaldol condensation of 3-hydroxy-propanal, one product of glycerol mono-dehydration.35 The
intermediately formed acrolein can react in a similar way with glycerol to form other cyclic ethers
as reported in Scheme 2; indeed, the formation of these ethers was confirmed by means of ESI-MS
analysis of the reaction mixture.
12
OH
O
O
GLY
O
O
O
- H2O
HO
OH
- H2O
O
H
OH
O
H
H
OH
GLY
GLY
OH
HO
O
HO
O
O
O
O
O
HO
O
O
Scheme 2. Ketals formation pathways as by-products of the glycerol oxidehydration reaction.
When the temperature was raised the ketals selectivity decreased mainly in favor of COx and
acrolein formation; on the other hand, the acrylic acid selectivity remained limited, even if the
catalyst is specifically designed for acrolein oxidation into acrylic acid. Apparently, this was due to
both the low selectivity to acrolein and the specific residence time used (optimal for glycerol
dehydration on Nb-V-HTB structures), which is too low to efficiently convert acrolein into acrylic
acid on Mo-V-(W) laminar oxides [REF]. This conclusion was confirmed by reacting acrolein: good
acrylic acid selectivity was obtained on Mo-V-(W) oxide, but with low conversion (figure N+10).
As already reported for glycerol oxidehydration, the lack of acid sites in laminar Mo-V-(W) is
also evident from the catalytic behavior shown in methanol oxidation (fig. N+9); indeed the acid
catalyzed route to DME was very low. On the other hand this system showed to be very selective in
the oxidation of methanol to formaldehyde: a maximum aldehyde selectivity equal to 93% was
registered at 87% methanol conversion (T 350°C). At low temperature, we also noticed the
13
formation of dimethoxymethane, which may derive from the acetalization of formaldehyde with
the unconverted methanol. On the other hand, because of the low specific surface area of this
catalyst, in order to obtain methanol conversions close to those obtained by both W-V and W-V-Nb
catalysts, higher contact time (0.12 s) was needed as compared to that used with the latter systems
(0.06 s). Josè: ARE THESE VALUES of contact time CORRECT ?
Conclusions
The studies carried out on both HTBs and laminar oxides have led to draw important conclusions on
the features needed by multifunctional catalysts for the one-pot oxidehydration starting from
oxygenated compounds. Indeed, the consecutive oxidation step must be fast compared to the
dehydration one, in order to avoid the formation of heavy compounds generated by both ketals
formation and oligomerisation reactions that take place from the intermediately formed aldehyde.
Because of this, strongly oxidizing conditions, the latter being achieved by using oxygen partial
pressures well above that needed for the stoichiometric oxidehydration of glycerol into acrylic acid,
are necessary with the best performing W-V-Nb catalyst. The latter is characterized by higher
surface area and higher concentration of stronger acid sites, properties which on one hand are
important for an efficient dehydration of glycerol into acrolein, but on the other hand might favour
the formation of undesired by-products. Under selected reaction conditions, an outstanding
productivity to acrolein + acrylic acid close to 2 h-1 with an overall yield as high as 55% were
achieved.
Acknowledgements
CIRI (Centro per la Ricerca Industriale), Università di Bologna, is acknowledged for the grant to A.C.
The Valencia group also thanks the financial support of Dirección General de Investigación
Científica y Técnica in Spain (Project CTQ2012-37925-C03-01).
14
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17
Fig. N. XRD spectra of the catalysts. A) Fresh samples; B) W-V-Nb sample,
fresh and after 100 hours of time on stream test.
18
Fig. N. Spectra of fresh catalysts, Mo-V-W (a), W-V-Nb (b) and W-V (c).
A) FTIR spectra, B) Raman spectra
Fig. N. A) H2-TPR B) NH3-TPD
19
Figure N+1
100
80
Gly conversion
60
Acrylic Acid
Acrolein
40
COx
Heavy C.
20
0
Others
0
1
3:6
2
6:6
3
9:6
4
12:6
5
18:6
6
Fig. N+1. W-V-Nb catalyst. Variable oxygen to glycerol ratio. Others: mainly acetaldehyde and acetic
acid (in minor amounts, < 4%: allylic alcohol, acetone, propionaldehyde, propionic acid and
hydroxyacetone).
20
Figure N+2
50
100
45
90
40
80
35
70
30
60
25
50
20
40
15
30
10
20
5
10
0
0
0
1
2:1
2
4:2
38:4
4
12:6
5
Acrylic
Acid
Acrolein
COx
Heavy C.
Others
Gly
conversi
on
Fig. N+2. W-V-Nb catalyst. Constant Oxygen:Glycerol molar ratio (equal to 2). Others: mainly
acetaldehyde and acetic acid (in minor amounts, < 4%: allylic alcohol, acetone , propionaldehyde,
propionic acid and hydroxyacetone).
21
Figure N+4
60
100
90
80
70
Yield (%)
40
60
50
30
40
20
30
20
10
0
Acrylic
acid
Acrolein
Glycerol conversion (%)
50
Others
COx
Gly conv
10
0
10
20
30
40
50
60
70
80
90 100
0
Time on stream (h)
60
Acrylic acid yield (%)
55
50
45
40
35
30
25
20
0
5
10
15
20
25
30
Acrolein yield (%)
Fig. N+4. Top: Time on stream test; feed condition Oxygen/Glycerol/H2O=12/6/40, tau 0.15 s.
22
Figure N+5
DO112d600_(W1V0,2Nb0,15)
Selectivity (%)
70
60
50
40
30
20
10
0
20
40
60
80
100
350
340
330
320
310
300
290
280
270
260
250
Conversion (%)
Fig. N+5. Methanol oxidation on W
23
HCHO
temperature (°C)
80
DME
FM
DMM
CO2
CO
Treaction (º C)
| 5,157 | 33,039 |
W4290964525.txt | 153
A.HMED RASIM'İN
İBRAHIM
MÜTEFERRIKA
YE MATBAACtLIK
ÜZERINEY AZOIK.LAIUNININCELENMESİ
'
'Yaşar A. Tonta *
GIRiş
Bu çalı~manınamacı,VCirP\an
bilgive düşüncelerinkjt1eseıı~şmesindc
en önemli
,
,
etmenlerd~nbirisi olandevingen (müteharrik) h,arflimatbaanınTürkiye'ye girişi konu9Jyla ilgili oJaliık, ö~eıdeAhmed Rasim'in ()Şmanlı Tarihi adlı eserindeyazmışol.
duğu "ibrahim Müteferrika ve~atbaacdık"adl i makalesini değerlendinnek, genelde
konuyla ilgili .basth kaynaklardanyararlanarakAhmed Rasim'in yazdıkları çerçeve.
sinde Müteferri~vematbaa.cllıkkQpU$Unu iôc.lemektir.
Bilindi~i gibi)yaıının bulunuşuyla.birlikte yazı malzemesi de hızla değişmiş ve
kağltbulumıuştut.J.s.
1.yüzyJlcfaÇinlilet tarafından bulunduğu sanılan kağıt,ozamana değin elde edilen bilgi biri,kiminikaYda geçinnekte. kullandmıştır. Böylece,
mevcutbilgi defontıasyonauğ'ramakstZınkijşaktan kuşağa aktarıtabUmiştir..
.
Yazıda mekanizasyondevingen harfii ma1baanıhbulunuşuyla sağlarımış ,Müteferrika'nm dadeğindiğigibi,bir C'nty~azahmetiyıe
b.inlerce.ciltdoğru yazılrmş eser
eldeedebilmeolanağı doğmuştur..
. ÇalışmamlZ,da,matbaanın Türkiye'ye giı'işigemil hatlarıylain~lenmektedir.
Ah.
ıned Rasirn.'in ma:kal~sin.de ilk,basılaneserlece
k,ısaca değiniJdiği,nden Müteferrika
Ma1bclas.1arafındanbasllan eserlerkomı~Qdaay"ıntıh,biıgi
verilmemiştir. Ycızıkaleme
alınırken "kesin anlatım" yerine ';de~yimciantatım If. yeğlenmıştir. Bu dunim, Iı>-,
rahim Müteferrika ve Matbaa,lhkkônu9Jndaaydmlatllmasl~eken
birçok hU9JS ol-
maSındMt ,kaYnaklanmakta~ır.Hatta, çok. kesin. bir 'yargıOlmakla. birlikte' konuyla
.
ilgili olarakNiyazi B~esşunlar.
$Öytonektedir; ",..basılı Oımanlıkaynaklarıncfa
ibrah im MÜ1eferrilf.t üzerine, Qrijinal kaynaklı hiçbir bilgi yoktur; arşlvlerdeki bilgile.
rin hiçbiri de incelenmemiştir :,1'
,
,
AJiMEDRAS~f,:
.
YAŞMlIVE
Y APffLARI
ıstanbul'da doğan yazar gazete,i ve1arihçLA.l:imed ~sim (1864-1932),ç~k.IuğUruakraba veyakınlarıom k()I'UyUc;ulJ.ıjualtında geçirim iştir. Darijşş afaka'y ibirinciHkle bitirdikten sonra bir$Üı:e Qevletmemu,",ı.ığu yapan Ahmed Rasiin, Ahmed Mid.
hat Efendit'niı1 teşviklyle basın Yatarmııaatılrjtjşttr.Çeşitli
gazetelerde makale, söyleşi,şiir ve çevlril~ri yaV,ttnlanınış~1927 ydlndula miUe~kili().Jarak Mec::ll$6ginniş-
tır.
(.)
.,
.
'
Araştırma göıevlisi.N.Ü,
KütiiiIDarteÇilikBö!iiinü
(,1;) N.~rkea,Ti1rkiye;deÇ41dtif~tıf~;t~talt):Jtiı.19'NbJ.Sl
AH~,,!ET
RASi M 'tN,! DHP,r!i
t'~'1~.1UTEFE
r~Ri f{..,4 VE i\.lAT81,...,c..CI'L.i
,<
Ahmed ,Rasim 'in toplanı 140 kadar yapltıva,.dır.İstanbuf
yaşam 101anlatan ÖykÜ
ve romanıarı, dtiy!}lsal aşk öyküleri,çeşitli
kondlarda yazdığı fen kit.ıpları ve çevirileri de olan yazarın yapıtlarından bazılarışunlardır: JIh Sevgi (189'1), Güzel Eleııi
(1892), Me;yl-i Ôil (Gönli! Kayması) (1 892);ıJc/~tL'p 1'1rhadaşıııı (1895), N ciJ;am (mera.
mına Ererneyen) (1898), Askeroğlu (1899).
Ahmed' Rasim Meşıutiyetten sonra tarihsel konuları da işleycreko.rtlokullarda
(idadi) okutulmak üzer~ dört ciltlik Resimli ue Ha/ilah Osmanlı Tarihi'ni (19101912) yazdı. Bu yapıtın asıl değeri "faide" başlığı altında verilen dipnotla~dır. Say,.
fanın yarısını kaplayan bu dipnot/arda ~ski devirlerin gelenek.gö.rencklerine, askeri
ve yönetimsel örgütlenmesine vcta.rihscl terimIere jJJşkin b'jlgile( verilmektedi~. Nitekim'çalışmamıza tertı;çl olan "ibrahim Müteferrika veM;ıtbaacllık"
adlımakale de
ikinci cildin 811-819. sayfa hırında yeralan 18~numaralı "faide" de buluıunakt.dır.
Adı geçen makale metinincelemesi yöntemiyle aşağıdaverilmektedir.
iBRAaİM MÜTEFERRiI(A.VEi MATBAACILlK
,
f
i'
"Osmanı) hizmetine dahil olan Mac;ırlard;ın iki zatiki biiyük hizmetle saiderin.
den serfıraz olmuşlardır (diğerleri nden üstiiıı tıı tuhnuş:lardır). Bunlardan biri topçu
Urban, diğeri matbaacı ibrahim,Müteferrika'dır.
'.
"Urban kendi arzU!ıJ ile Türklerin y.anına gelip Fatih Sultan Mehmet zamanında
Osınanlılara ilk bwü~ toplip'1 döktü. istanbul fethinde şehrin duvarlarında rahneler
(büyük delikler) açan cesim toplar Urban Usta'nı~ malıaretiyle meydana gelmiştir.
"ıbrahim! Miiteferrika kendi arzusıı iledeğilvukua'tın ilcasıylaOsl11anlı hizmetine
gin~ıiştir. MiiialUnileyh (adıgeçen) H Iristi.yanlıkmczaJı ibinden Kaluinist mezhebinde
fakir bir Mac~r aileye mensup olan 108S'teMacaristan'ın
şark tarafında kiiin Kolojva.r §ehrinde ~oğmuştur. 1104 senesinde OrtaMacar Kralı Tököl~BeyOs'narılı ordusuyla müttefikan (birlikte) Nemçeaı~hine
vuku bırtan muhareoode ıbrahim Mütefer>rika esir dÜşıüş. FidYe-i necat (kurtulmalık) veremediği için 1l06'da Istanbul/a getirilip burada esir olarak 5.1nldl. \-lin.i esaretinde (köleliği sırasında) kaba, z.'1limbir ada.
mın elinedüşerek köleliğe ademJ tahammülüne binaen (köl'eliğe tıhammül edeme-
yerek)islamolarakıbrahimte5miyeolundu(adlandırıldı).
'
..ibrah~ Efendi Risale,i Jslamiyye nam,l/altındabirkitap' te'lif etmiş.idi. Kitabın
muhteviyatı dinJ Islamı müdafaaya niÜllhasır makalat..-ilmiyyeden
(bilimsel makale-
.
lerden) olmağla bilahare sadrazam olan 'Damat ıbrahim Paşa'nın nazar-ı dikkatine .
çarpmış ve mumalleyhi (a(fıgeçe,ni)himayesinealmıştır." 2
,
'
Ahmed Rasim 'in makalesinın başınctıu'lyaptığ.m1Zbu alıntıdan sonra verilen
(2) Ahmed Rasim, Asma/ılı Tarihi' eili Il, ısta~bul:~eıns
ss. 811-812.
l\1-atbaası,'1326.-1329
YAŞAR
155
A. TONTA
bilgilere kısaca bir göz atalım; Ahmea--Rasim. Müteferrika'nı" kendi ısteği dışında
müslünfdr'i.olduğunu. ~alvinci bir aileycmenSllp olup H. lO~5'te Kolojvar'da d~duğunu; Osmai'ılı ordu!U ile yapılan savaşta esjr düştüğünü ve kurtUlmalık veremediği için Jstanbul'a getirilip köle olarak s3.tl.ldığını. köleliğe tahammüledemediği için
de islam.iyeti kaPu! ettiğini yazmaktadır. Yazar; Müteferrika'nm RisaLe-i lsliimi~'ye
adlı Islam dinini savunan bilim.seı makaleler içeren bir kitap yazdığını ve yazdığı bu
.de eklemektedir,Bu
kitapla da sadrazamm dik!<;ıtini çekerek him.ayesi,oe.girdiğiıı,i
koroda araHırma yapanlarR~ale-i İslamiyye'nin Islam dirıini savunmak için yaz'ılmış
bir kitap olduğu konuSlInda birleşmektcdirler. OYsak:i. söz konu!U kitabın Islamiyeri
savun.anW eser olmadığı Niyazi . ~fkes'in çalışmaJarısornıcu ortıya çıkarılmıştır.
Berkes şunları söylem~ktedir:
.
"'Gerçektebue$er ne,lstanıhğın savunmaşıdır. nede islam biljmleri üzerine yazılmış bir eseidir.(...) Bu eser ı<atol;ildiğe.papahğa ve testis inancına hücüm edenbir
polemiktir. Asıl önemli yanıbize ıbrahim'in aSlında sanıldığı gibi Kalvinist olmadığını
göstermesindedif. Bunu. öğrendiğimiz zaman Ibrahi~ı'~n TÜrklere karş~ ~vusturya
ordu!Undasavaştığına, kölelik yüzündenmüslümanlığa, geçtiğ ine inanmakgüçleşir.~"
Herkes, Risale-i ISl6mıyyehakkirıda dahagen.iş bilgi vcrdiği:20 ıiumarah dipnot.
ta yazmanın kendisinde bile Rısale-i lsldmi)')'eadının geçn1edlğini; bu adınbir katalogcu tarafından rastgele verilmiş bir ad olduğunu söylemektedir .Dahası. "Islamlığı
bilmeyenbir kiş,i olmayan Ibi'ahim'in Katolik Habsburg'larııi yönetimi altında yaşam:ıktansaÖsriıanh!ığa geçerek.miisliJrıan olduğU
apaçıktır"4
sözleri de Betkes'e ;ut~
.
tir.
Berkes'in verdiği bu bilgilere.dayanatak Ahmed R.asim'in değindiği (Mütefer, rika'nın Kalvincibir aileye memubiyeti, kurtulmahkveremediği. içinköle olarak ~.
tl!ıŞI. köleliğe dayanamadığı için mUslümanoluşu. RisaleJ lsldmiyye'nin "din~
.
isiSını müdafaaya münhasır" bir eser olduğu.yc yine aynıkitap d61ayısıyla müteferrika'nın Damat ıbrahim P3$a'nın him~yesinegirmesi vd. )ansikloı'edik bilgilerin
gerçeklerlebağdaşıriadığt görülmektedir. Sait Maden'in deyişiyle. bu ansiklop~dik
bilgiler şimdiye dek bu' konUda araştırma yapanlarınbirazeksik
birazfaZla. küllana
.
geldikleri bir "ortak mal" duıurnundadtr.s
,
'
Tarihsel bilgTaçlsındal1bu d'önernlerdekldinsel gelişmelere
. dekısacadeğinmek
gerekmektedjr..
.
':
. XVIII. yüzyılda Batı Avrupa Hıristıyanh~ı blrkaçm'eZhebe bö~nmliştü. (Katolik.
(3): Berkes, a-.g.)'., s: 52
(4). a.)'. s:53.
(5). Sait M aden , "İbrahim Müteferrika tçinBil'YenidenDeiteriendirme
.
.
"Milliyet Sanat De rgisi Yeni Dizi No. 1, (Şubat 1980),8. 87
Çalışması,
AHMET
156
RASIM'iN
IBRAHIMMOTEFE;:RRiKA
VE MATBAACtLlK
luterd, Anglikan). Kalvincilik de Qu'mezhebierden birisidir.Hıristiyan dininde görülebuklikleşme o zamana'de~in herl<esçekaböledU1m"tek ve mutlak bir gerçekfik.
ri" üzerinde olums.ız etki yaptı. Dinsel savaşımlarda birçok kan aktL Ama zıt inezhepterin biraradayaŞamasl$rektiği'düş~esiningetişmesıne de katkıda bulundu. Bu
arada, tek ve mutlak bir ger'çek fikrinidevam ettirmek isteyenlerUnitarizmi kurdular. Müteferrika Katvinci d~il, terslne Unitariusçudur. Unitarizmin Islam dinine
yakın bir doktrin oluşu, gene\ÇJetektanncılığa dayanması Müteferrika'nın zor yoluy.
la müslümanolmadığına-da birjkariıtolabitir.ÇÜOkü; kili~ebağnazlığına karşı Çıkan,
din ve inanç ayrımını, h~ türfüdüşünce~gürfüğünü $awnan, diğerbilim dallarıYla
yakıml'anilgilenen, kısacasısöıP1ğün tırnan.lamıyla tam.birentelektüel olan Müteferrika'nın ıslamiyet korAls.ındada bilgisi olması beklenir. Ayrıca, Unitariiminkunıcusu olan Michael Servetus'un (1511-155~) Unitariusçuların inançlarını yaymak için
t<Ui"an'ltlnıkgôstermesinin Müteferrika'yı da etkilemişolabileceği
kuşku!IJz, düşü.
~~~
'.
. KaMncilikle'tJnitariusçuluğun birbirlerinin tam ~rşltl d9ktriı:tlerolquğunu
belirtmek için Michael Servetus'un.diridiri yakıldığını da eklemek~kmektedir_
.
Ahmed Rasim'inkonuiiLerine
kaldığımız yerden sürdürelim:
,
.
.
i
yazmış olduğu makaleden yaptığımız alıntıları
"1128'deAvuSÜ!rya ile zuhureden'muharebedeBelırat'ta içtimaedenMacar-
lara tercümanlık vazifesiyle.memur olarıfs"(1129) Orta Macar Kralı meşhur Rakoçi
de Dersaadet'e gelmekle ıbrahimmumiileyhyanında kalmJŞtır:HattaRakoçi'nin Tekfur dağında ikameti esnasındadahi ıbrahim Efendi'yi beraberindebulmuş ise de ek. ser vaktini ıstanbul'da Sadrazamıbrahim PaŞa neztiinde geçinniştir. Macar efazlM
müverrihinden(erdemlitı.tihçilerinden)olatı Doktor Karaçuiı Efendi bunları yudıktarı sonradiyor ki:'
'
, Sadrazam OS1tanlılararasmda ıılOm Yefün~n Jle Avnıpa medeniyetinin taammüm ettirilmesi {genelf!'Ştirilmesi)arzu!IJı-ı~raz .veizhar etmekte idi. Fransa Sefaretindenavdeteden Yinn ise~'Mehmed Çelebi ile ~Iu Said Efendi' ki bilahare sadıazam olanSaid Paşa'Clır- ıbrahim Paşa'yıteşvikederlerdi.
-
'ıbrahim. Efendi bu m~sudaniilolmakiçin
SadQz~a bir matbaa teşkilw tesisi hakkında müracaat etti. Sadrazamtısvipedip icrasını emreyJedive bu ~!lJS için
ıbrahim Efendi'ye kifayet miktırı para ,dahi verdiğinden ıbrahim Efendi Viyana'dan
hikkiklar ile humfat'dökmecUeri ve ma1b..acdarıgetirip işe başlamak istedi; Ama
gayrıme'mulmiinanaatauğradı(beklenmeyenengellerie kaI'Şllaştı),..6
'
Alıntıdan da anlaşılacağı üzere Ahmed Rasim kaynak olarak Karaçun Efendi'den
yararlanmıştır.
(6). Ahmed Rasim, a.g.y. ss. 812-814
,
YAŞAR
157
A. TONTA
Qgy:ı;ınh sınırıaniçindey.aş,,"aY~
,baştathktan
Doğal ol~rak ibrahim Müteferrika
sonra çeşitli kademelerde birçok görev yapmıştlı;.Başarıh hi~metleri; dolayıs1y,!a(jır
ki kendisine "müteferrika"ltk görevi verilmiştir.'
'
Bilinen o ki, ibrahimMüteferrikaOsm;ın1thizmetine girmezdençok öocelerii3asma scuıatıni1atımaktadır. Berkes,Müteferrika'nın hangi nedenlerlematbaayı tanmış.
olabileceğini şöyle anlatmaktadır.
"Erdel ve Macaristan,Orta Avrupa'da matbaac;ılığ1l1çjlbukyerleştiği yer Qbnuştur. Oımanlt tarihçisi Peçevi'nin doğlim yeri olan Peç'şehrinin /;>aşIıca,kilisesi
1570
de Unjtariusakidesinikabuletmişti. Bir,yüzyılsonra o Zil11anll)en tanınmış ,harf
dökümcüsii ve basınıcl$lol;ın, Misztotf~ulu'Mihail Kiss, Kofojvarşehrinde, 1689
dao ~amanınen önemli U nitarius basımevini kurmuştur. Ora~! ibrani, Enneni V~
Gürcü harfleri. döken bu ~.
bastığı kutsal kitapmetni dolay,ısıyla KaMn,istlerio
hücumunauğranııştl; ıbraHimMüteferrikadaha Türk olmazdan önce basmazenaatını
değilse bile basımeviniyakındanbilen birKişi idi. Yirmisekiz Çeleb1 Metımet Efendi
.'
ye basımevi açılması fikriniogetirmiştir.'"
.
.'
Kaldı ki, Selim 'NiizhetGerçek'a.,gôre Müte.ferrika,dah.a1719-17:20 yıllarında,
ıŞ
şimşir üze~nekazllmışo,lan19.x48,5Grn' boyuüındclbir.harita ba!lt1 ~'altına da
şu notu düşmüştür: "Btnim devleduefendimeğerfennanınlZolursa dahi büyüRI~.
yapılır. Sene 1132.,,8 Ki bu tarih 1719'a karşıltk gemektedir. Oysa Said Efendi'nin
.
Paris'tendöomesi 1725 ylllarınararilam~dır'~.Müteferrikabu
'sır~d,a~ı.:Ieniz
,9 MütefefJi~'nın S~d
.
haritasın'ı basmakla meŞlJlldjjr,;OS01aı:1,ErS9Y daeşerinde,
Efendi'den önce matb~aişleri ilej 19ilendiği.nedair bQtduğu ~ betgedensöze~ekte..
.
dW.
.
Bu'dunımda,matbaa açı.ması fikrinin Said- 'Efendi'yeMüteferrika tarafından
açllmfŞolabilec~i 'düşünÜleibiID'.
EğerŞaid Efendi'nin matbaanın kurulrn~inda bir
rolÜ olduysa
-
ki ,oImuş1Ur.".
bu rol: 1) Matbaanın
açılmaSt iÇingerekli
se.",ayenin
Said Efendi ıarafmCJahbulu'nması;2) Said Efendi'niıf(deVıet kademesindeiyi bir yerde görevli olmasından dolayı) bürokratik engellerin aşılmasında 'yardımcı. olma;
'itorpil" göreviniüsdenmesişek,lindegerçekleşmiş olabilir.
'
('7). Berkes, a.g.Y. s." 57
,
.
'
(8). Selim Niizhet ~çek,
Türk MatbaaedılıI;
Müteferrikil Matbfi481,:İataiıbül1939.
s. 34. ~Eaerin vesikalar bölümünde Marmara Dımizi Haritasmm 'klişesifaksimile
olarakvardır.)
"
(9). Osman &soy, 1firldye~yeMatboanmGirişive Ilk BaSıfanEserler, Ankara,1959
s. 31 '
,
AHrv1 ET
,
,~,'\si ~,1'iN.i EJRAr-'lir,1
,,1 üTEFER;:ıj
KA
VE Mp,TBAACJLI
K
Ahmed Rasim Karaçun Efendi'yi kaynak göstererek Müteferrika'nın Sadrazama
matbaa kurulmasıkonu9:indaba-ş vurduğunu; Sadr#amın bu başvuruyu ,onaylayarak
kun.ılmasını'ernrettiğini ve dahası; bu i,ş için Müt~ferrika'ya yeterli miktarda para
bile verdiğini kaydetmektedir. Ancak, neSadrazamınmatbaanın
kuıulmasını emret~,
tiği ve ne de bu husu,s için Müteferrika'ya yeterli miktarda para verdiği belgelencrıri!.
memektedir.
i
Hakkikların ve hurutat dökmedlerinn nerede~getjrildiği konu'sunda, yapılan ,
araştırmalarda çok değişik görüşler ortaya atılmıştır. Ahmed Rasim'in kaynak göster.
diği KaraçunEfendi. hikkikların ve hurufat dökmedlerinin Viyana'dan getirtildiği.
ni; Selim Nüzhet Gerçek Lork'a~dayanarak "istanbL1l" da isaga edild1ğini"(kalıba d,ö.
olduğunu,
küldüğünü), 10 NecipAsım harfleri dökeninArabo~lu adındabirErmeni
i 1 Carleson ise harflerin ehlıyetli Alman LIstalarınc~ döküldüğünü iddia etmektedir,.
ler. ,1Z Osnı an Ersoy da elindeki belgeleredayanarali harflerin isıanbu i'da döküldüğü
'
ve yapılmaSı' mümkün otanbUtiiı materyalin ifstanbul!'da yapıldığı sonucuna varmak ta-
clır. 13
i,
'
Hikkik ve hurufat dökmedlerıtıin Viyana d..n getirtitdiğihu'su~na olumlu
yönden yaklaşılacak olul'5i1şunlar söylenebilir:Avus-ı..ryave italyao devirdematbaa.
cılığın en fazla getiştiği ülkeler durumunda-ydt.EğetViyana'da Arap harfleriyle ba.
slmCllık daha ileride idıyse, belki de bu nedenle hakkaklar ve hurufat döl-,,,'edleri
Viyana'dan getirtilmiş olabilir. Ancak Duolasılık çokzayıf görijnmekte~ir.
Öte. yandan S.N.Gerçek,Müteferrika'nınkitapbasma
izni almak için verdiği
difekçenin çok giizetıolartfesih.ya:zılatı ileilk basılan,eserlerin yazıların," birbirine
benzemesindendolayı harflerinıstanbul'da dökiirnüş olabUeceğini söylemektedir.1,4
Şu da ,bir ,gerçek ki.is~nbul halkı matbaayı çbk önce~itarihlerden beri tanınıaktıdır. Çünkü, o devirde ıstanbul'da ~lnlıklartırafJr1dan kurulmuş vefaaliyette
olan matbaalar bulunmaktadır. 1,5 ErsoY'jigöreilk Türk matbaaSl ku.rulurker:ıazınlık,
lardanyardımgörü1rn~tür. Ersoy bu t~ini şöyle desteklemektedir:
(10) Gerçek, (l.g.y. s. 60
(ll) Ersoy,(l.g.y.s. 35
,
.
(12) Edvard Carleson, ıbrahim Müteferrika Basıme!)i ue Bastığı ilk ESerler, Yayma
.'
Hazırlayan: M.Akbı.ılut, Ankara 1979.
(13) Ersoy, a.g.y.s.35
(14) Gerçek, a ,g.y. s. 60.
(15) İstanbul'cia kı.ırulim Musevi, &nem
ye Rummatbaalım
konusunda S,N, Gerçek
ineserine bakılabilir. (s.26)' Ayrıca, matbaanın yayılmasıyla ilgili olarak bkz.
Ersoy, a.g.)!. Ek i.
yAŞAR
A.TQNTA
"... ibrahim Müteferrika tarafından' verilen istidanın ortalarındaki bir pafagrafta matbaa işlerinden anlayan edavat ve alat vc mühimmat
san'atında mahir yonaadlı
16
bir Yahudinin yardımındanbahse<l'iImektedir."
Ahmed Rasinı daha sonra makalesine şöyle devam etmektedir:
"Bu menılekette tarik~ ilmiyye ricali zinüfuz olmakla (bilim adamları etkin olmakla) bu ihtir.a-ı cedidc(yenr buluşa) birtürlü cevaz (izin)vcrmediler. Matbaanın
müslünıanlar arasındaisti'mal olunmamasını'(kullanılinam;ısın'ı) Sadrazama alettekrar
(tekrar tekrar~arı ettiler. Ve lüzumundanfazla kitapların meyclan-ı tedavüle vaz' edilmesi ile (dolaşıma konulması ile) asayiş~ umumi (geneldü1;cn) vemuamelat-ı diniyyeye (dinselişlemlere) ihtidi-ı mezkurun (adı gççcn buluşun) tehlikeli olacağını ıikı
reylediler."
.
7'
.
. Sait Maden'e göre Ahmed Rasimyukarıdaki bilgileri De Saussure'danalarak ver.
mektedir. Sait Maden, De Saussure'un bir süre Tekirdağ'da Rakoçi'nin yıımnda.
bulunmuşbir Macar soylu9J olduğunu, bu arada M~tcferrika ilc de .tanışmış otabi.
lec~ini, fakat verdiği bilgilerinbelli tarihlerdenve belgelerdenyoksun olduğunu kay:
detmektedir. 1.8 Bu nedenle de De Saussure'a pek güvenmernekgerekmektedir. Dikkati çeken diğer bir nokta da, DeSaussure'uri,irıatb;ıanın bulurıı.şununüzerindenüç
yüzyıla yakın bir süre geçmesinekarşın, matbaadan.hiila "ihtira-ı
cedid" diye söz
.
.
'.
etmesidir.
'.'
Yukarıdak paragTilfta,matbaanın işler halegeçmesiyle birlikte gereğinden fazla
kitapların piyasaya çıkmasıylagenel düzenin ve dinsel işlemlerin b~zulacağından
sözedilmektedir. Ancak gereğinden fazla ki.tıplarınneşekilde zararı dokunacağı
konusunda.ne yazık ki ayrıntılı bilgi verilmemiştir. Dolay.ısıylabasılacakkitapların
.
nasılıararlı olacağı anlaşılmamaktadır.
Ahmed Rasinı,matbaaya
karşı çıkllmasıyJa ilıjli olarak~akate$inde
şu satırlara
.
da yer vermektedir.
"ıstanbul'da ozamanlar yüzlerce adarnl,n maişeti (geçimi). yalnız kitapların
istinsahı(çoğaltılması) ve yazısı olmayanlarınmektuplarınıyazmojehusuSlna nıüıiha~ıridi. aunlar da matbaanın küşadrndan(açılmasından)mutazarrır (zararlı)olacaklarından vemedar-ı maişetlerini (geçinı,yollarını) kaybedeceklerinden haif oldukJarın.
dan (korktUklarından)hoşnutcilmaddar.Bunlar Sadrazaının matbaayı iltizam ettiği.
(ı 6) E~oy ~O.g.)'.s. 35
(17) Ahmed Rasim,
(Ü3)l\Iad.~n,a.g.m,s.87
.
a.g.y. s. 814
.
i
-~
160
AHMET
~-~
RASI'M'iN.iBRAHiMMOTEFERRiKA
~---:-~~-
--, _.-
VE MATBAACtLIK
l1i (gerekli' bulduğunu) görüncegüya din4rrübinitl (islamdininin) muhafazası bahanesi ileavamı ma1baa aleyhineteşviketmeğe:başladılar,
Fiilen biraz patırtı ve çok
dedikodu oldıı. Artık bunlarla taraftarlarını, tes1dnetmek için"müsag-ı şerıiyi mutazammm fetva-yı şerife olmadıkça (dinsel izni içeren fetva olmadıkça) hiçbir kitabın
tab'olunamayacağını vaad ettiler. Ve bLJ. veçhile matbaa 1139 senesinde ıbrahim
.
Efendi'nin kendi evinde işe başladı. "19
/.
.'
. Geçimlerini kitaplann çoğaltılmasından. ve mektupyazmaktan sağlayan birçok
kimseni!) matbaanın faaliyete geçmesi nden hoşnut olmayacaklan açıktır.Nitekim
kitap esnafı da elikolubaği. du rmamıştır. Bu arada bir de gösteri "yapmışlardır. Gösterinin yapıldığı.tar.ihhakkında
kesin bir bilgi olmamakla birlikte, inceleyebildiğimiz
kaynakların tümünde böyle bir olayın m~ydana geldiği dOğrulanmak tadır. Selim Nüzhet Ger~ek'e &öre hattatlar ve esnaflar "ka~)t ve kalemlerialat ve edavat-ı kitabeti
bir tabutı koyarak bir cenazealayı" düzenlemişlerdir. Ancak bu gÖsteriye katılanlar
"birazr>atırtı ve birçok dedikQ(luya
sebebiyet 'verdikleri haldesonraları süktııet"
.
bulmuşlardır.
20
.
.
.
Jale Baysal da Müteferrika'dan I. Me.ş~ tiyete Kadar Osmanlı '.flirlderinin Bastıkları. Kitaplar: adlıeserindePatrona
HaJit Isyan, slraslJida Sadrazam Damat ıbrahim
Paşa'nın öldürülmesinekarşın basımevinin kınJıp dökülmedensağlamkaimış
olduğuna dik.kati çekerek; sözkonusu düşmanlığın geniş halk kitlelerini etkilemediğini
wrgulamaktadır. Bu düşmanlığın halk kitlelerini etkilememesine neden olarak da,
toplumun basılı kitapla fazla ilgilenmediğini; bir yüzyıl boyunca basılan kitaplann
sayısının 180'~ gewı ediğin i ve Müteferrika öldüğünde terekesindebastığı kitapları
dan birçoğunun satılmadan kal(lığIDI göstennektedir. ..~
.
, Toplum bilimciler kültüriiı m~diunsurlarıı:ıın toplumsal yaşamı belirleyici ol.
duğunu sawnmaktadırlar. Nesnelkoşullar gerektirmedikçe dışarıdan alınan kültürel
Yenilikler toplumsal yaşamda benimsenememektedir.Başkabir deyişle,- nezamali ki
,toplum matbaayagerekslmne wymuş ve bu yeniliği benimsemeyehazırhale,gelmiş,
on.daıı.sonra.matbaa kunılabilmiştir.
.
.
.,
.
Kuşkusuz matbaanınkurulmasındaki gecikmede birtakım sosyo.ekonomik nedenle~ roloynamıştır. Birbaşka neden olarak da Herkes, Osmanlı devlet sis,temine
özgü lonc.asınırlamaliirıni Qmekgöstermektedir.2 2 Oysa Türklerle ya~yana yaşayan,
amazer1aatvetic.aretleu~aşan Rum, Ermeni ve Yahudi ~!nhklar matt>aarıınbulunu-
(19) Ahmed Rasim, a.g.y. 8.815
(20) Gerçek, o.g.y. 8.59
(21) Jale Baysal,
Müteferrika
'da,n 1. Meş~
Kitaplar, İstanbul 1968. 88.13-14.
(22) Berkes, a.g.y. s. 6.0
.
.
_
tiyet Kadar Osmanlı '.flirklerinin Bastıkları
.
,- -"-~---~--f" -. ,
YAŞAR
161
A. TONTA
malliaakunnuşlardır.
Bunda, Osmanlı yönetiminde. yaşayan
şunda,\hemen,sonra
azınlıklara gösterilen hoşgörünün büyük bir payrolsa gerek. Yoksabazı araş~nnacıla~
rın saviJnduğu gibi, Osmanh devletyöı:1etimidinsel
nedenlerle matbaanın girişini geıştır , kanısındayız. Sözün özü, müslüman top,ciktirmek gibi bir dekadansa uğram <it1
lum o devirlerde kitaba değil, {akat basıh kitaba \ gereksinim duymamaktadır.
,
"',
'
Şeyhülislamın fetVası "av~ı matbaa aleyhine te~vik" eden kimseleriyatıştırmak
ve adı geçen gösteriy! Onlemekiçinçıkartılmıştır,
kanısındayız. Fakat fetvada dinsel kitapların basılarnayacağına ilişkin herhangi birhükiin bJ,ıluıvnadığl hU!1Uu,
23 ' ' fetvan ın V~n Kulu sözlüiij1d~kj~r~tinden Yararlanılarak ortaya .çıkarılmlŞ tır'.
~~~d Rasim'd,en yaptı~ımız alıntıda, esnaf tarafından yapılan gösteriyeulema--dan bazı kimselerin de~trldığından
SÖZ edilmek~ir.
Hiçbirt>illm adamınınbllimselaraştırmalara geniş bOyutlar kazandıracak böyle bir ~rişimin(TürkjYe'de Türkçe
yayın yapacak bir matbaa kurulması girişiminin) karşısında olabileceğini düşij1mek
mümkün &.örünmemektedir, Ma1b~, b,aşta da beli,rttiğimiz gibi, düşüncelerin kitlesel~
leşmesinde önemli rol oynamaktadır. Gerçek bilim a~ının
da yaptığı dUşij1sel
üretimi ne kadar çok kimseye yayabilirse kendisin~ o kadar başarılı sayması gerekir.
Ahmed Rasim 'in verdiğ i bilgileri ak tarmaya devam,edc;lim :
"Ulum ve füıunun ve her nev'i efkar.. medeniyyenin sür'at.. intişarına (her türlü
uygar düşüncelerinhızla yayılmaSına) hizmet etın'işolan ihtlraat.. miiıimmeden (önemli ,buluşlardan)biri de matbaacılık dediğimiz ~bt-ı tıbOGt(basmsanatı)dır. Bu
sanat'atı ilk icad eden G.ıttenberg namında Harlem şehri .-halisindenbir zattıi'. Tarih-4icadi 844 sene.j hicriWe ve 1440sene.j iniladiYYedir.Fakatbizdeki tarih.j icadı
pek sonradır.
,
,
,
"OSınanlılardahuruf-u arabiWeyi (arap harflerini)san'at-ı tıbaata tatbikVe. ilk
tesis edenler bilahare mesned-4sadarete irtika (en yüce makama yükselen) ve 1170
tarihinde dar-i bekaolmuş(ölmüş} Said Mehmed'Paşa ile DergaJ1-ıAli kapıcıbaşılarından Müteferrika ıbrahim Ağa'dır. Kibar-ıhacegan-ı divan-ıhümayundan (dvan-ı
hUmayunefendilerinin büyüklerinden)Yirmisekiz Çelebi Mehmed Efendi sefaret1e
Paris'e izarn olunduğu (gönderildiği)zaman mandumu{oğlu) Said Efendi'yi de kethüdalık hizmetiylem.aiyetinde götürmüş idi, Said Mehmed E~ndi'nin bu seyahatte
(23) Hidayet Yavuz Nub~lu, "Müteferrika Matbaasınm Kurultnası Için Verilen
Fetva tjzerine~ "Türk Kiitiiphaneciler Demeği Basım lie Ya)'lncılığıml%lR 250.
Yıiı Bilimşel 'TopIQntfSllQ-l1 Anılık 1979, ,Ankanı 'Bildiriler,ADkara.1980
ss. 119-126.
'
"
'-"
162
'AH~,1ET
RASiM'iN
iBRAHiM
---
~-~
MüTEFERRiKA
VEMATBAACALIK
en evvel naz;ır-ı dikkatini celbeden şeyParis'in matba~iları oldu. istanbUl'a avdctinde(döntişühde} rhüşa!ıedatını (gö&rirJerini) ve bu babdakiefkarmı
(bu konOda:ki
dÜşüncelerini),M(jteftrrikiıibrahimAğa'ya;ınlattl."24
,
,
\
Buradabirazdurahmvc
birJki noktaya deginclfm:Va,ptığırrıızalıntıcla
matbaanın dışarıdaki "tarih .,jkadı" iie bizdeki. "tı:'rih.,j kadl"miıfaf"klftYlduğu
Söylenmektedir. DÜnyanın herhangi bir ülkesinde yapılan yeni bir buluş bilimin evrenselliği
yasasına tcrs dÜşmeden düriyanmher,tarafına yayılır., Bizde yapılan da,matbaacıh.
ğı n rfrcvcÜtteknikleriÖğreniler~k ü'kerniidekendi idt<tplarıimzıri'basımı için hizmete
sokulmasıdir. Demek istCdiğ imii ,'ülkı!rriiidç inatb~ kunılmadan önce diğer ülkelerde matbaa kurulmuştürve faaiiyettediı',:YOk;a'rnatba'aikinci
kez icadedilmemiştir.
Ayrıca, 15. yÜzyıl ronlarınt1aO!irn'anh iinparatorlÜgu coğrafyasında çalişmakta olan
birçok matbaanın olduğu da bilinmektedir.
,
'Öteya'ri~n
,Said Efend,i'ninPiU"ismatbaalarınl
merakla ,görmesi ve istanbul'a
dÖnlişiipde 'gö'rd,iildei'inima1baakonuıııylaı;iddibir
şeki/ge ilgilenenibrahim Müteferrika'ya anlaımaSldikkat
çekicidir~ Bu da~ibrahim Müteferrika'nın matbaayı daha
önceden.tıı)ıdiğını
gösterin'ekted,k.' Nite~l>u
'SjlvımlZldoğrulayan
belgeler de
bu1unmaktadır: Ersoy'un e.serinde'bu husu'-s/ailgili gen'iş bilgi verilmiştir. 2 s Kaldı ki,
bu husu sla Ilişki! i olarak ,şusonı da' akla g~lmekt~ ir: Ac~a Said Efendi Paris dönüş'ü
gözlernierini ne~ren,birb~şkasına değil' ,de Mütcfernka'ya anlatmıştır? Demek ki, Mü'.
c
-
.
<"'c,"-
.
_'.
.'
.
_ ~.
.'.
."
,'.'
,
_
.
'_',
_
. ..
-'
'"
_
teferrika'nın bu sanatı daha öncedentanıdığı devict yönetiminin üst kademelerinde
görevalan bazı kimselercede bHinrrıekted,ir.
,
~
Ahmed Rasim matbaanın kunıluş çalışmalarını da şöyle anlatmaktadır:
"ibrahim Ağa Vesiletii't-Tıbaanamıyı.r'bir risaleyazaralcbunda :Saniat...tıbaatın
fevaidini (basım'sanatının yararlarını) say<ft<İöktü:Bu(Vesiletü't-Tıbaa)sadrazam ot
zamanolan .ibrahimPaşa'ya :takdim.olundu. Müşaruıiileyh(adıgeçefl) Paşa bunun
ebemmiyetini takdir itehemen-Q devrin uk<1l~veflizelasından (akdlive erdemlilerinden) bir meclis~ meşveret(danışmakuıulu) teşkileyledi. Bunun efkar-i ammeee
(kamuoyunca) mazhar-ıhüsn-ükabul olması ve kavaid.,jşFr'i mübine (islamdini ku.
ra!1~rına ).t;atl? ik, ,ve tevfik ,ile tesisi için şeyhülişlamdan ,isti~ya
(fetVıl istenmesine)
meclisçe karar, verildi. _Şeyhwislam Abpu/lah, Efendi de fetva verdi, Bir de takriz
(takdim:yetak~jjry~ısı) y~dı'. U~emaiJaJ:)
j)ek ç~ ~~vatdatakrizler,yazdıl~r.'
.
"
',.
...
- ile beraber
'''\Tesileiii/t-Tıb,QClmeclis.,j meşveretin ,mazb~tası ve fetva- ve, takrizler
SultınAhm~'Salis'e,
(Üı. Ahm,efe).ari'olunarakbkhatt-ıhümayun
He Said Meh.
meçIEfendi ve şetiki'(orıağ I)Miitefe.rrj}Q Jbr<ıhimAğa 'yamcı~aa tesisi, ve kitap tab ',I
için ruhSat ita kılındı (verildi)."26,
'
(24) Ahmetl Rasiın,a.g,y.
FeritBey'in
ss, .815-'-816
N€vSal.iOsmallJadh
(25 )Ersöy', a.g,y., 8,,31
(28) Ahmed Rasinı, a,g.y, 8; 817
Ahmed Rasim yukarıdakibilgileri
e8eril1d~naktarmaktadır.
'
Oıman
,
YAŞAR
A. TONTA
Yukarıya aldığımız ik i paragrafta Ahmed Rasim ,ibrahim Miiteferrika' nın basım
sanatının yararlarını anlatan bir risale yazarak zamanınsadrazam i Damat ibrahim
Paşa'yasunduğunu;
Sadrazamın Vesilet4't"Tıbdaadını
ta~lyanQurisalenin önemine
binaen bir danışma kurulu kurarak şeyhiilislanıdan fetva istenmesine
karar verildiği.
.
ni ve şeyhülislamın da fetva verdjğiniyazmaktadır.'
Vesiletü't-Tıbaa ad1ıil risalenin b ıgüne kadar bir yazma nüshası bulunamamışt~r;
Ayrıca, günümüzde nadir eserler kita piyasasında baş tarafındaadı geçen risale basılı olan V~n Kulu sözlükleri. olmayan ra oranla dahadeğerli sayılmaktadır.
Kanımızca Vesiletü't-Tıbaabasıla ilgili tüm hazırlıklar tamamlandıktan sonra
Damat ibrahim Paşa'ya su~lmuştıı .Matbaa konusunda kamuoyu 01uştıırmakanıacıyla yazılmıştır. Ortada maıbaa Ileilgili hiçbir hazırlık yokken yazılıp Sadrazama
sunulmuş değildir.
Toplandığıiddia edilen danış akurulunun herhangi bir konu Uzerindckar.ar
verme yetkisi buh.ınmamaktadır.Daışma kurulu boyle bir karar çıkarama)'"acağı
gibi şeyhülislamı fetvavermeye de orlayamaz. Şeyhülislamdan devlct ışleriylc ilgili olarak saltpadişah fetva isteyebil r.
Kaldı ki, danışma kUl\1lunun b" yle bir yetkisininolabiıeceğini
kabullensek bile
söz konusu kararla ilgili mazhata rtada bulunmamaktadır. Verilen bilgilerdeise,
danışma kuruhınun şeyhülislamclan aldığı fetvayı kendi karar mazbatasıylabirlikte
Sultan ııı. Ahmct'e sunduğu; padiş ın da verd1ği hatt':' hÜ01ayun ile Said Mehmed
Efendi. ile ortağı Mütefeıtika'nınbas"mevi kurarak kitap basmalarını' onayladığından
söz edilmektedir. Bu bilgiler Osmanlıyönetim erkinin yapısınauy~n gözükmemektedir. Şöyle ki; danışma kuıulu taratnmn fetVa padişaha sunulmaktadır. Padişah da
bu fetvaya boyuneğerek fermanver ektedir. Yani, kararı, fetvayı veren şeytiülisıam
onaylamakta. padişahise uygulam tadır.Böyle bir durumun -Osmanlı yönetimirÜn
hiyerarşik örgütlenişi içerişinde yeri lmayacağı kanısındayız'.
Ahmed Rasim makalesinin
ilgili bilgiler vermektedir
nda., Müteferrika
matbaasında basılan eserlerle
.
~
"Bunun üzerine mÜ01aileyhimaa (adı geçen iki kişiye) edavat-ı tab'iyyeyi (basım araçlarını) ihzar ve ikmal ile ilk Osmanlı matba~sınıSultanSeiim civarında ibra.
him Ağa'nın hanesindeve 1140 tari ~ hicriyyesinde tesis.cttiler (ki tarih~icadından
,üç asır sonda demektir).-aumatbaa
a 1140'ta ilk basılan kitap meşihlr-i ulemadan
(ünlü bilginlerden) meşhur Vani Me ed bin Mustafa Efendi'nin Van Kulu nam lu- "
gatıdırki Sılıah-, Cevhen ismindeki 19at kitabının Türkçeye tercemcsidir.
Bin tane basılmıştır (1141 senc)
ikinci tabi edilen Katip Çeleb 'nin: Tunfetü'l
bahriye tarihidir.
.
'
.
,-- Kibar fı Esfari'l-Bihar
"'"\m
164
AHMET
RASiM'iN
ıBRAHiM
MüTEFERRiKA
Oçüncü:Terceme~ Tarih~ Seyyah
VE MATBAACILIK
.
Dördüncü: Tarih~ Hind~ Gaıbi EI.Müsenıma bi Hadis~ Nev.
Beşinci: Tarih~ Timur,Gürgan.
Altıncı : Tarih ~.M Isru'I.Kad im ve Mısru 'I-Cedid .
Yedinci: Gülşen~ Hülefa.
.
Sekizinci: Usulü'I.Hikem fi Nizamü'I-'Omem(ibrahifn.
Dokuzunaı: Füyuzat-ı Mıknatısiyye (keza). \
Müteferrika'nın).
Ownaı : Cihann~.a (Katip Çelebi~nin).
.
Onbirinci:Tarih~Naima,cild-ieweL
Onikinci: Tarih-i Naima, cild-i sani.
Tarih-i Raşid ,(cild~ievvel ve sani)." 27
Ahmed Rasim'in konumuzia ii~1i olarak Osmanlı Tarihi adlı eserinde yer alan
makalesi yukarıdaki satırlarla son bul~~tadır. Ancak, yazısının sonundaki birkaç
çelişkiye de değinmek gerekmektedir.
-Müteferrikamatbaasında basılan kitaplanh kaynakçası Ersoy'uneseiinde veril.
'mektedir. 28 Ayrıca,adı geçen matbaada basılan ,ve özellikle resimliolan kitapların.
ayrıntılı özellikleri hakkında-geniş bilgi, konuyagrafik sanatının doğuşu açısından
yaklaşan Sait Maden'in Qevre dergisindeyayıml~nandizi yazısında bulunmaktadır.29
Ahmed R~im, matbaanın faaliyete geçiş tarihiyle ilgili üç değişik tarih vermektedir.ilkind e, "... Ve bu veçhile matbaa 1139 senesinde ibrahim Efendi'nin kendi
evinde işe baş~/\demektedir.3o
Verdiği butariht~nbirxaç sayfasonra; "...ilk
Osmanlı matba.;
Sultan, Se6m civannda ibrahim Ağa'nın hanesinde ve 1140
tarih-i hicriy'yesinde tesis ettiler" 3 i cümlesibulunmaktadır. Birkaç satır sonrada ilk
basılan eser olan VanKulu sözlüğününbasım tarihini verirken: "Bintane basılmıştır
(1141 se06)"3 2 .demektedir. 1139, 1140 ve :1141. 1141 tarihii!k basılan eser olan
Van Kulu'nun yayın ti\rihiolarakdoğrudur. 1139 Ve 1140taiihlerindekiçelişki di.
ğer kaynaklardada karşımıza çıkmaktadır. Yukarıda,Vesileffl't-Tıbaaının matbaa
"
konusunda kamuoyu faratmak,dilekçe ire fetvanın da herhangi birtepkiyi.önlemek
amacıyla ele alındığını ifade etmiştik. Aynca, Vesilem't.Tıbaa'nınmevtut nüshalarına
dade~inmiştik. Bu bilgilerimatbaanın kuruluşunu doğru saptamak amacıyla kullana-
(27) Ahmed Rasim,a.g.y.ss.
818~19
(28) Ersôy,a.g.y.ss.
3845
(29) Sait Maden, "Başlangıcindan .BugüneTürk' Grafik Sanatı (2); Müteferrika: Dönemi, "çeure Cilt I, No. 2, ss. 83-87;ve:Ciltl,No.
3, ss. 87~90,
1980.
. .
.
(30) Ahmed. Rasim, a.g.y. s. 815
(31) a.y. 818
(32)0.y.
.
.
.'
YAŞAR
165
A. TONTA
biliriz. Vesiletii 't-Tıbaa 'rlRlbasılı kopyesinin elde bulunmaması ; ayrıca, dilekçede örnek.
. nüshaların takdim edildiğinden söz edilmesi b,izi, fetvaya dayanan .emr~ hümayun
alınmasından önce matbaanın kurulmuş ve faaliyetegeçmiş olabileceği noktas.ınagô,
türmektedir. Matbaanın hartlerdolayısıy.1a Avrupa'dan ithaldeğil,
tamamen yerli
bir tesis olarak' kurulduğu, ve bunun için gerekli olan zama,n da göz'önündealınırsa,
ma1baanın vertlen, tarihlerdenç~k daha önceki bir tarihte kuıulduğu söylenebilir .Hat-
ta bu tarihi, bir olasılık da olsa, saifÇelebi'nin Paris'ten dönüşüolarak da düşünmek
mümkündür.
.
.
,
Ahmed Rasim, Müteferrika matbaasında basılan eserlerin kaynakçasını verirl<en,
dikkat edileceği üzere Gtammaire Turquie'ten hi~ söz etmemektedir. Bilindiği gibi
Müteferrika, Nôima Tarihi'ne değin bastığıkitapların
kolofonunu Naima Tarihi'
\
nin sonundavermiştir.Bu kolofonda Grammaire Turquie gösterilmemiştirP
Bunun üzerine, Ahmed Rasm'inbasllaf1
kitapların listesini Nôima Tarihi'nde bulunan
kolofondan y.a~rlimarak verdiğini bu nedenle de, Grammaira Thrquie'ü listesine
alm4d1ğını düşünmüştük; Fakat hemen 'sonra, S.N.Gcrçek'ineserinin
"vesikalar"
bölümüoü.karJş,tırırkenbuldu,ğumuz.kolofolJun faksimilini inceieyerek,Ahmed Ra- sim'in baSılan esenerinlistesiniverirkenbukolofondan
yarananmitdığını saptamıŞ
i
bulunmaktayız,.
"
Çünkü kolofondaQrammaire
Thrquie verilmediği halde T"kvimü't-Tevôrih adlı eser
Cihannümô,'dan sonra,"Takvimü't-Tcvarih
.Ii Kitip Çelebi cild 500" şeklinde verilmektedir. Oysaki, Ahmed Rasim'in verdiği listede Takvimü't-Tevarih'in de adı geçmemektedir. OolaYISly1a, listenin hangi kaynaklara dayanılarak hazırlandığı saptanamamıştır. ..
Ayrıca verilen.listede, basılan ka,çıncı eser olduğu belirtilmeksizin Tarihi Raşid'
it birinci ve ikinci ciltlerinin vadığından söz edilmektedir. Basılandiğer eserler konusunda ise araştırma yapma gereği duyuımam ıştır.
',,-
Ahmed Rasipı'in rhakal~sihakkında' genelolarak şunlar söylenebilir: Incelenen
kaynaklar eleştiri süzgecinden geçirilmeksiıin olduğ~ gibi kab",i edilmjştir. Bu nedenle dekaynaklarda mevcutbaiıçe6şkilerin farkına varılamamıştır;
Basımevinin kurulUşu ~nu_5Unda eldeki belgelere dayal'Illarak doğrulu kesin
olarak bilinen vefakat AhmedRasim tarafından hatalı olarak aktarılan bilgilere de
,
rastlanmaktadır.Ömeğin,
Ahmed Rasim, De Saussure'di1n aktararak, istanbul halkının çokeskiden
beri ıanıd1ğımatbaayı
"ibtira-ıcedid"
diye netilendirmeldedir.
ıstanbul'da yaşayan Musevi, Ermeni ve Rum azınlıkların 15. yüzyılın sonundan itibaren istanbul;da kitap bastıklarına 'daha önce değinilmişti. Ayrıca, Avrupa'da basılmış
Şark, eserlerinin Istanbul kit;ı.pçdarınd;ı. sattıdığı ve bunların, itibar görmediği de
.
bilinmektedir.
';133) Gerçek, a.g.y .s. 84
,
166
AHMET
RASir,:'iN
iBRAHiM
MÜTEFERRil<A
VE MATBAACtLlK
Keza,.. Ahmed Rasim' in maKalesindebeli rttiğigib i, matbaan ın ku ıt/lmasına Said
EfendiJ1in düşüncesiylehaşlanmadığı hu G;Isuna yukarıda ayr' tl" bir biçimde yer ve-
rilmişti.
.
l
Tarihsel kaynakların iyi değerlendirilememesincJenve yapılan araştırmalarda
bilimsel kuşkuculukla hareket edilmemesindendolayı yan,lgilariıdüşülmüştür.Oysaki, verilen bilgiler o giiJıkübelgelerdensağlanmıştır. KuşkusllZ doğru bilgiler de bu
.
belgelerde bulunmaktadır..
.
SONÜÇ
Vlusal kültür yaşamımızclaç()k önemlibir yer tutanmatbaanın kurulUşu, .ülke.
mizdeki düşünselgelişmeye ~üyük bir hıt kazandırmış ve toplum eskiye oranla daha
fazla ve nispeten daha ucuza kitap okuyabilme olanağ.u\a ka.wşmuştUr.~ önemli bu.
luşun uygulanmasında :ıynı zamandabir bilim adamı olan ibrahim Mütefernka'nın ro.
iü kuşkusuz çok fazladır. Her konuda derin birbilgiyesahip olan Mütefe~rika'oın bel~
leğinde daha öğrencilikyıll:ırındagöi'çlüğübas,rnevidetin
bulunduguridada buSanatı "lt ra'.' etmiştir. ..
.
izler ~lrakırilŞve olanak
ibr;ıhim Mteferrika devlet kademelerinde üstlendiği görevleri ve matbaatılığı
birarada yürütmüştür,.kanısındayız.Buna karşın azımsanamayacak.sayıdavebirbirin.
den değerli kitaplar basmıştır. öyleki, :bastığı eserlerdenbir kısmı bugünbile birinci
el kaynakolmaözelliğinikorumaktadır.
.
Devingertharflimatbaanın ülkeırtiz(lefaaliy~te geçmesiyle birlikte basılan kitiplarda hızı. bir artıŞ olduğu sôylenebilir. bk ylUama çok y.avaşolan bu hız, yazıda
mekanizasyonun yararlan toplum tarafından gÖrüldükçeartmaya başlamıştır. Hele
hele kutsalkitaplarabir zarar gelmediğini ve saygısızlık edilmediğini gören kimseler
basılı kitaba daha fazla ilgigöstermişlerdir. Gii1ünüzderurkiye'de 2500'e yakın sade.
tegazetertin ba-sllmasl,matbaan4okültürel~lişmemizcieoynactığı rolünen açık kanı.
. .
tı olarak gösterilebilir. kanısındayız.
KAYNÇ
ADIVAR,A. ADNAN
OsmanlıTürklerin'de Ilim,geli ştirilmiş 4. bs. istanbul:
Remzi Kitabevi, 1982.s. 166-170.
AHMED RASiM
OsmanlıTarihi, Cılt 1I,<istanbul; Şems Matbaasl,1326_
1329.55.811-819.
BA YSA L, JA LE
BERKES,
NiYAZi
BERKES, NiYAZi
Müteferrika 'dtin Birinci Meşru tiyete 'Kada~
Türiderinin Bastıkları Kitaplar, istanbul, 1968.
Osmanlı
"ilk TütkMatbaa KUıucu~nun Dini w Fikri Kimliği,"
BeIleten Cilt26.No.104, ss. 720-721, 1962.
Türkiye 'de Çaf{claşlaşnıa, istanbul,1978.
.
YASARA'
167
TONTA'
CARLESON, EDV ARD
ERSOY, OSMAN
GERÇEK,
SELiM
.
lbrahiın kliiteferrika Basımevi ve Bastığı/lk Eser/er, Yayına hazırlayan: Mustafa Akbulut. Ankara: Türk Kütüphaneeilel' Derneği, 1979.
Tiliniye'ye Matbaanın Girişi ve 1II~Bası/an
Eser/er, Anka.
ra,1959
NOZHET
TilrkiUatbaacllıjJı
1939.
i; Müteferrika Matbaası,
Istanbul,
.
'
"ibrahim Müteferrika," lş.ldm Aniihlopedisi, cm 5, ss
KUN, HALASi
896-0
MADEN, SAiT
UBaşlangıcındanBugi~e Türk Grafik Sanatı (2); Müteferrika Dönemi, "Çevre Ciltt,No. 2, SS83-87, Mart-Nisan
.
1980:
MADEN, SAiT
"Başlangıcından BugüneTürk Grafik Sanatı (3); Mütefer.,
rika Dönemi 2,u Çevre Cilt i, No. 2, ss. 87 -90, M,W+laZ
1980.
SIVAVUşGIL,SABRI
TVRKKVTVPHANECILER
...
ESAT "Ahmed Rasim," Islam Ansiklopedisi Cilt 1, s.201.
DERNEG/ Basımve YayıncılIRımIZin 250. Yılı Bilimsel ToptanhsJ 10-11' Aralık 1979, Ankara Bildiriler,
Ankara, 1980, ss. 119-126..
| 4,128 | 37,895 |
W3009565379.txt | https://journal.uwgm.ac.id/index.php/KESMAS
P-ISSN: 2477-1880; E-ISSN: 2502-6623
December 2019, Vol. 5 No. 2
Hubungan Antara Pengetahuan, Motivasi Pasien dan Dukungan Keluarga
Terhadap Kepatuhan Minum Obat Anti Tuberkulosis (OAT) Pada Penderita
Penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota Samarinda
Nindi Elvira Fitriani 1, Tumpak Sinaga 2, Abdul Syahran 3
nindielviraf7@gmail.com, Universitas Widya Gama Mahakam, Indonesia1
siganatumpak@yahoo.com2, abd_syah@yahoo.co.id3, Dinas Kesehatan Provinsi Kalimantan Timur, Indonesia 2 3
Abstrak
Latar Belakang:
Data Puskesmas Pasundan Kota Samarinda, angka keberhasilan pengobatan tahun 2017 sebesar 47,60%, yang
masih dibawah target Nasional sebesar 85%.
Tujuan :
Tujuan penelitian ini mengetahui hubungan antara pengetahuan, motivasi pasien dan dukungan keluarga terhadap
kepatuhan minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) di Puskesmas Pasundan
Kota Samarinda tahun 2019.
Metode Penelitian:
Jenis penelitian kuantitatif dengan desain cross-sectional. Populasi adalah seluruh penderita penyakit TB Paru
dengan BTA Positif yang berkunjung ke di Puskesmas Pasundan Kota Samarinda dari bulan Januari sampai
Oktober tahun 2018 dengan jumlah 31 penderita. Teknik pengambilan sampel total sampling. Analisis data
menggunakan uji chi-square.
Hasil :
Hasil penelitian tidak ada hubungan pengetahuan terhadap kepatuhan minum obat anti tuberkulosis (p value :
0,056 > α : 0,05), tidak ada hubungan motivasi pasien terhadap kepatuhan minum obat anti tuberkulosis (OAT)
(p value : 0,057 > α : 0,05) dan ada hubungan dukungan keluarga terhadap kepatuhan minum obat anti tuberkulosis
(OAT) (p value : 0,002 < α : 0,05).
Kesimpulan:
Tidak ada hubungan pengetahuan, motivasi pasien dan Ada hubungan dukungan keluarga terhadap kepatuhan
minum obat anti tuberkulosis (OAT) pada penderita penyakit TB Paru BTA (+) di Puskesmas Pasundan Kota
Samarinda tahun 2019.
Kata kunci: Pengetahuan, Motivasi, Dukungan Keluarga, Kepatuhan Minum Obat Anti Tuberkulosis.
Abstract
Background:
Data from Pasundan Health Center in Samarinda City, the success rate of treatment in 2017 is 47.60%, which is
still below the national target of 85%.
Objectives:
The purpose of this study was to determine the relationship between knowledge, patient motivation and family
support for adherence to taking anti-tuberculosis drugs (OAT) in patients with pulmonary TB disease (+) in
Pasundan Health Center, Samarinda City in 2019.
Research Metodes:
This type of quantitative research with cross-sectional design. The population was all patients with pulmonary
TB with positive smear who visited Pasundan Health Center in Samarinda City from January to October 2018
with a total of 31 patients. Total sampling technique sampling. Data analysis using the chi-square test.
Results:
The results of the study there is no relationship of knowledge of adherence to taking anti-tuberculosis drugs (pvalue: 0.056> α: 0.05), there is no relationship between patient motivation and adherence to taking antituberculosis drugs (OAT) (p-value: 0.057> α: 0.05 ) and there is a relationship of family support for adherence
to taking anti-tuberculosis drugs (OAT) (p-value: 0.002 <α: 0.05).
Conclusion:
There is no relationship of knowledge, patient motivation and there is a relationship of family support for
adherence to taking anti-tuberculosis drugs (OAT) in patients with pulmonary TB disease (+) in Pasundan Health
Center, Samarinda City in 2019.
P-ISSN: 2477-1880 E-ISSN: 2502-6623
Kesmas Uwigama : Jurnal Kesehatan Masyarakat
Kesmas Uwigama : Jurnal Kesehatan Masyarakat
P-ISSN 2460-0350, E-ISSN 2477-5819
December 2019, Vol. 5 No. 2
Kata kunci: Knowledge, Motivation, Family Support, Compliance with Anti-Tuberculosis Medication.
DOI
:
http://dx.doi.org/10.24903/kujkm.v5i1.838
Received
:
October 2019
Accepted
:
November 2019
Published
:
December 2019
Copyright Notice
This work is licensed under Creative Commons Attribution 4.0 International License.
P-ISSN: 2477-1880 E-ISSN: 2502-6623
PENDAHULUAN
Data Kementerian Kesehatan tahun
Paru pada tahun 2013 sebesar 96,12%,
2011 menunjukkan bahwa insiden semua
kembali mengalami penurunan pada tahun
tipe TB adalah 450.000 kasus atau 189 per
2015 menjadi 82,57% dan meningkat
100.000 penduduk, angka prevalesi semua
kembali pada tahun 2016 menjadi 83,86%.
tipe TB 690.000 atau 289 per 100.000
Angka keberhasilan pengobatan TB Paru
penduduk dan angka kematian TB sebesar
(Succes Rate) per Kabupaten/Kota. Kota
64.000 atau 27 per 100.000 penduduk atau
Samarinda berada di urutan ke 2 terendah
175 orang per hari. Jumlah kasus TB di
setelah Kabupaten Paser dengan jumlah
Indonesia menurut Laporan WHO tahun
keberhasilan pengobatan (Succes Rate)
2015, diperkirakan ada 1 juta kasus TB
sebesar 71,55%. (Data Profil Kesehatan
baru pertahun (399 per 100.000 penduduk)
Provinsi Kalimantan Timur Tahun 2016).
menurun pada tahun 2014 menjadi 90,64%,
dengan 100.000 kematian pertahun (41 per
Menurut Data Dinas Kesehatan
100.000 penduduk) angka notifikasi kasus
Kota Samarinda tahun 2016, penemuan
(Case Notification1 Rate/CNR) dari semua
kasus TB BTA (+) di Kota Samarinda
kasus,
sebanyak
dilaporkan
sebanyak
129
per
457
kasus,
menurut
jenis
100.000 penduduk. Jumlah seluruh kasus
kelamin, jumlah kasus pada laki-laki lebih
324.539 kasus, diantaranya 314.965 adalah
tinggi daripada perempuan yaitu 270 kasus
kasus baru. Adapun jumlah kasus TB paru
pada
berdasarkan
microskofik
2016
perempuan. Menurut kelompok umur,
sebanyak
759/100.000
penduduk
kasus TB Paru pada kelompok umur 25-34
tahun
(Kemenkes RI, 2016).
Menurut Data Dinas Kesehatan
Provinsi Kalimantan Timur tahun 2016,
laki-laki
dan
187
kasus
pada
tahun. Sedangkan kasus TB Paru pada
anak-anak 0-14 tahun sekitar 3% dari total
penemuan
kasus.
Sedangkan
angka
presentase keberhasilan pengobatan TB
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keberhasilan pengobatan (Succes Rate)
lanjutan 4-6 bulan berikutnya. Pengobatan
pada tahun 2017 Kota Samarinda sebesar
yang teratur pada pasien TB paru dapat
42,20%, dengan penemuan BTA (+)
sembuh secara total, apabila pasien itu
berjumlah
sendiri mau patuh dengan aturan-aturan
144
kasus
(Data
Dinas
Kesehatan Kota Samarinda Tahun 2017).
tentang pengobatan TB Paru. Sangatlah
Menurut Data Puskesmas Pasundan
penting bagi penderita untuk tidak putus
Samarinda,
keberhasilan
berobat dan jika penderita menghentikan
pengobatan (Succes Rate) pada tahun 2017
pengobatan, kuman TB Paru akan mulai
sebesar 47,60%. Nilai tersebut masih
berkembang
sangat jauh dari target Nasional yaitu
penderita mengulangi pengobatan intensif
sebesar 85%. penemuan kasus TB BTA (+)
selama 2 bulan pertama. Tanpa pengobatan,
pada tahun 2015 sebanyak 24 kasus, dan
setelah lima tahun 50% dari penderita TB
meningkat pada tahun 2016 sebanyak 34
akan meninggal, 25% akan sembuh sendiri
kasus, dan terus meningkat pada tahun
dengan daya tahan tubuh tinggi, dan 25%
2017 sebanyak 57 kasus, dan pada tahun
sebagai kasus kronik yang tetap menular.
2018 dari bulan Januari sampai dengan
Sebaliknya, jika penderita melaksanakan
Bulan Oktober berjumlah 31 kasus (Data
pengobatan dengan baik atau pengobatan
Puskesmas Pasundan Tahun 2018).
dengan pengawasan minum obat secara
Kota
angka
Mengingat tingginya kasus TB paru
langsung
biak
lagi
yang
sehingga
berarti
mampu
dan risiko penularan terhadap orang lain
mempertahankan diri terhadap penyakit,
yang cukup tinggi maka pemerintah
mencegah masuknya kuman dari luar dan
mengeluarkan
kebijakan
dapat menekan angka kematian yang
penanggulangan
TB
dalam
paru
melalui
disebakan oleh TB Paru (Septia, 2017).
pengadaan obat anti tuberkulosis (OAT).
Penelitian terdahulu yang dilakukan
Kebijakan ini sejalan dengan rekomendasi
Ahsan dkk (2012) menyatakan bahwa salah
WHO dimana penggunaan obat anti
satu faktor yang dapat meningkatkan
tuberkulosis
strategi
kepatuhan pengobatan pada penderita
(Directly Observed Treatment Shortcourse)
dengan penyakit kronik ialah adanya
DOTS
mengurangi
dukungan keluarga yang baik. Dukungan
penyebaran penyakit TB paru. TB paru
keluarga sangat diperlukan terutama pada
adalah penyakit yang dapat diobati dan
penderita
disembuhkan. Pengobatan TB paru dapat
penyakit kronik dan mengharuskan ia
diberikan dalam 2 tahap, yaitu tahap
mengkonsumsi obat dengan jangka waktu
intensif 2 bulan pengobatan dan tahap
yang lama, karena keluarga merupakan lini
(OAT)
bertujuan
dalam
untuk
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TB
yang
juga
merupakan
126
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pertama
bagi
mendapatkan
meningkat
penderita
masalah
apabila
kesehatan
kesehatan
itu
atau
sendiri.
Merupakan salah satu fungsi keluarga
untuk mendukung anggota keluarga yang
sakit dengan berbagai cara, seperti memberi
dukungan dalam mengkonsumsi obat.
Berdasarkan uraian tersebut, maka
HASIL PENELITIAN
Hubungan
pengetahuan
terhadap
kepatuhan minum obat anti tuberkulosis
(OAT) pada penderita penyakit TB Paru
BTA (+) di Puskesmas Pasundan Kota
Samarinda tahun 2019.
Tabel 1. Hubungan pengetahuan terhadap kepatuhan
minum obat anti tuberkulosis (OAT) pada
penderita penyakit TB Paru BTA (+)
peneliti tertarik untuk melakukan penelitian
tentang
mengetahui
pengetahuan,
hubungan
motivasi
pasien
antara
Kepatuhan Minum Obat Anti
Tuberkulosis
No
Pengetahuan
dan
minum obat anti tuberkulosis (OAT) pada
penderita penyakit TB Paru BTA (+) di
Tinggi
2
Rendah
Puskesmas Pasundan Kota Samarinda
%
Patuh
%
Tidak
Patuh
%
7
22,6
5
16,1
12
38,7
4
12,9
15
48,4
19
61,3
11
35,5
20
64,5
31
100
dukungan keluarga terhadap kepatuhan
1
Total
Jumlah
p
value
0,056
tahun 2019.
METODE PENELITIAN
Metode penelitian yang digunakan
adalah
dengan
menggunakan
metode
Hubungan motivasi terhadap kepatuhan
minum obat anti tuberkulosis (OAT)
pada penderita penyakit TB Paru BTA
(+) di Puskesmas Pasundan Kota
Samarinda tahun 2019.
Adapun besarnya sampel pada penelitian
Tabel 2. Hubungan motivasi terhadap kepatuhan
minum obat anti tuberkulosis (OAT) pada
penderita penyakit TB Paru BTA (+)
ini adalah sebesar 31 responden. Jumlah
No
Motivasi
1
Tinggi
9
29
8
2
Rendah
2
6,5
11
35,5
kuantitatif dengan desain cross-sectional.
tersebut sama dengan jumlah populasi,
yaitu seluruh pasien penderita penyakit TB
Jumlah
Paru dengan BTA Positif yang berkunjung
Kepatuhan Minum Obat Anti
Tuberkulosis
Tidak
Patuh
%
%
Patuh
Total
%
25,8
17
54,8
12
38,7
14
45,2
20
64,5
31
100
p
value
0,057
ke di Puskesmas Pasundan Kota Samarinda
dari bulan Januari sampai Oktober tahun
2018 dengan jumlah 31 penderita. Analisis
ini digunakan untuk mengetahui hubungan
atau korelasi antara variabel bebas dan
variabel terikat dengan uji Chi Square
(yang
disajikan
dalam
bentuk
kontigensi 2x2.
P-ISSN: 2477-1880 E-ISSN: 2502-6623
Kesmas Uwigama : Jurnal Kesehatan Masyarakat
Hubungan dukungan keluarga terhadap
kepatuhan minum obat anti tuberkulosis
(OAT) pada penderita penyakit TB Paru
BTA (+) di Puskesmas Pasundan Kota
Samarinda tahun 2019
Tabel 3. Hubungan dukungan keluarga terhadap
kepatuhan minum obat anti tuberkulosis
(OAT) pada penderita penyakit TB Paru
BTA (+)
tabel
No
Dukungan
Keluarga
Kepatuhan Minum Obat Anti
Tuberkulosis
Tidak
Patuh
%
%
Patuh
Total
127
%
p
value
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December 2019, Vol. 5 No. 2
1
Tinggi
10
32,3
5
16,1
15
48,4
2
Rendah
1
3,2
15
48,4
16
51,6
11
35,5
20
64,5
31
100
Jumlah
responden pengetahuan tentang TB Paru
0,002
rendah, namun patuh minum obat anti
tuberkulosis
PEMBAHASAN
Hubungan
pengetahuan
terhadap
kepatuhan minum obat anti tuberkulosis
(OAT) pada penderita penyakit TB Paru
BTA (+) di Puskesmas Pasundan Kota
Samarinda tahun 2019
Berdasarkan hasil penelitian terlihat
tinggi,
proporsi
tertinggi
pada
responden yang patuh minum obat anti
tuberkulosis
berjumlah
7
responden
(22,6%), hal ini dikarenakan responden
tahu dan memahami tentang TB Paru
sehingga berupaya agar sembuh dari
penyakit tersebut dengan patuh minum obat
anti
tuberkulosis. Terdapat
responden
pengetahuan tentang TB Paru tinggi,
namun tidak patuh minum obat anti
tuberkulosis
berjumlah
5
responden
(16,1%). Hal ini dikarenakan sering lupa
waktu minum obat, malas dan bosan
dari
19
responden
pengetahuan tentang TB Paru rendah,
proporsi tertinggi pada responden yang
tidak patuh minum obat anti tuberkulosis
berjumlah 15 responden (48,4%), hal ini
dikarenakan responden kurang tahu dan
kurang memahami
responden
jadwal minum obat yang dianjurkan dokter,
mematuhi saran dokter dan tidak pernah
kesal ataupun bosan dengan pengobatan
tersebut.
Hasil
uji
statistik
dengan
menggunakan uji fisher diperoleh hasil p
value : 0,056 > α : 0,05 sehingga Ho
diterima
yaitu
tidak
ada
hubungan
pengetahuan terhadap kepatuhan minum
obat
anti
tuberkulosis
(OAT)
pada
penderita penyakit TB Paru BTA (+) di
Puskesmas Pasundan Kota Samarinda
tahun 2019.
Hasil penelitian ini sesuai dengan
penelitian
terdahulu
yang
dilakukan
Wulandari (2015) menunjukkan bahwa
tidak ada hubungan pengetahuan dengan
kepatuhan pasien Tuberkulosis Paru tahap
lanjutan untuk minum obat di RS Rumah
dengan pengobatan tersebut.
Adapun
4
(12,9%). Hal ini dikarenakan selalu ingat
dari 12 responden pengetahuan tentang TB
Paru
berjumlah
tentang TB
Paru
sehingga kurang berupaya agar sembuh
dari penyakit tersebut dengan tidak patuh
minum obat anti tuberkulosis. Terdapat
Sehat Terpadu. Begitupula penelitian Dewi
(2011) menunjukkan bahwa tidak ada
hubungan antara pengetahuan dan sikap
penderita dengan kepatuhan penderita
minum Obat Anti Tuberkulosis Paru di
Puskesmas Lidah Kulon Surabaya.
Berdasarkan hasil penelitian juga
diketahui adanya kecenderungan bahwa
pada penderita TB yang memiliki tingkat
pengetahuan tinggi cenderung patuh dalam
pengobatan TB. Hal ini terlihat bahwa pada
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penderita TB
yang memiliki tingkat
cenderung
baik.
Penderita
memiliki
pengetahuan tinggi sebanyak 22,6% patuh
pengetahuan yang kurang akan tetapi
dalam menjalani pengobatan TB dan
penderita memahami sedikit informasi
penderita TB
yang memiliki tingkat
yang mereka dapat dengan baik. Sehingga
pengetahuan rendah sebanyak 48,4% yang
saat penderita memiliki pemahaman yang
tidak patuh dalam menjalani pengobatan
baik maka diharapkan aplikasi juga baik
TB.
(Noorhizmah, 2016).
Pengetahuan
mempengaruhi
penderita
pemahaman
dapat
penderita
Tidak adanya hubungan antara
tentang penyakit TB Paru, semakin rendah
pengetahuan dengan kepatuhan minum
tingkat pengetahuan tentang kesehatan
Obat Anti Tuberkulosis. Hal ini bisa terjadi
maka akan kurang cara pemahaman
dikarenakan oleh pengawasan yang kurang
terhadap suatu penyakit, sehingga dengan
atau penderita sudah bosan, sehingga harus
pengetahuan yang tinggi maka akan
ditingkatkan lebih lanjut. Dimana ada
menunjang terhadap kepatuhan minum
kecenderungan bahwa penderita
Obat Anti Tuberkulosis (OAT) (Wulandari,
memiliki pengetahuan tahu, memahami,
2015).
dan aplikasi kurang memiliki tingkat
Salah satu penunjang keberhasilan
pengobatan
TB
Paru
adalah
dengan
mengetahui seberapa jauh pengetahuan
penderita tentang TB Paru. Pengetahuan
penderita mengenai istilah TB Paru, gejala
dan penularannya di Puskesmas Pasundan
tergolong rendah. Padahal kemampuan dan
pengetahuan masyarakat atau penderita
akan
gejala
bagaimana
penyakit
cara
TB
Paru
penularannya
dan
sangat
mutlak dipahami. Karena dalam tahap ini,
dapat dilakukan pencegahan terjangkitnya
penyakit TB Paru atau setidaknya penyakit
penderita
dapat
diketahui
lebih
dini
sehingga pengobatan yang akan dilakukan
tidak terlalu lama dan memiliki resiko
kegagalan lebih kecil. Tingkat memahami
yang
kepatuhan yang tidak patuh.
Hubungan motivasi terhadap kepatuhan
minum obat anti tuberkulosis (OAT)
pada penderita penyakit TB Paru BTA
(+) di Puskesmas Pasundan Kota
Samarinda tahun 2019
Berdasarkan
hasil
penelitian
mengenai hubungan motivasi terhadap
kepatuhan minum obat anti tuberkulosis
(OAT) pada penderita penyakit TB Paru
BTA (+) di Puskesmas Pasundan Kota
Samarinda tahun 2019 terlihat dari 17
responden motivasi kesembuhan TB Paru
tinggi, proporsi tertinggi pada responden
yang patuh minum obat anti tuberkulosis
berjumlah 9 responden (29%), hal ini
dikarenakan responden memiliki tekat yang
kuat dalam diri sendiri untuk sembuh
sehingga responden patuh minum obat anti
penderita berdasarkan hasil penelitian
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tuberkulosis. Terdapat responden yang
penelitian berbanding terbalik, yaitu pasien
motivasi kesembuhan TB Paru tinggi
yang motivasinya tinggi banyak yang tidak
namun tidak patuh minum obat anti
patuh, sebaliknya pasien yang motivasinya
tuberkulosis
rendah banyak yang patuh dalam menjalani
berjumlah
8
responden
(25,8%). Hal ini dikarenakan responden
pengobatan.
mudah lupa waktu minum obat.
Octaswari (2015) tidak ada hubungan
Adapun dari 14 responden motivasi
kesembuhan TB Paru rendah, proporsi
Begitupula
penelitian
antara motivasi diri dengan kepatuhan
minum obat penderita TB paru.
tertinggi pada responden yang tidak patuh
Adapun
faktor-faktor
yang
minum obat anti tuberkulosis berjumlah 12
mempengaruhi ketidakpatuhan antara lain
responden (38,7%), hal ini dikarenakan
pemahaman tentang instruksi dan kualitas
responden kurang memiliki tekat yang kuat
interaksi. Mungkin motivasi yang dimiliki
dalam diri sendiri untuk sembuh sehingga
pasien untuk berobat tinggi, tetapi jika
responden tidak patuh minum obat anti
dalam menyerap instruksi dari tenaga
tuberkulosis.
Terdapat responden yang
kesehatan tentang program pengobatan
motivasi kesembuhan TB Paru rendah
kurang, akan sangat sia-sia motivasi yang
namun patuh minum obat anti tuberkulosis
dimiliki.
berjumlah 2 responden (6,5%). Hal ini
motivasinya tinggi dan ditunjang oleh
dikarenakan responden selalu ingat waktu
kualitas interaksi yang baik antara tenaga
minum obat.
kesehatan dan pasien, maka pemahaman
Hasil
uji
statistik
Tetapi
sebaliknya,
jika
dengan
tentang instruksi yang diberikan akan
menggunakan uji fisher diperoleh hasil p
sangat tinggi. Kualitas interaksi antara
value : 0,057 > α : 0,05 sehingga Ho
professional
diterima yaitu tidak ada hubungan motivasi
merupakan bagian yang penting dalam
terhadap kepatuhan minum obat anti
menentukan derajat kepatuhan (Latifatul
tuberkulosis
Muna, 2014).
(OAT)
pada
penderita
penyakit TB Paru BTA (+) di Puskesmas
Pasundan Kota Samarinda tahun 2019.
Hasil
penelitian
penelitian
terdahulu
kesehatan
dan
pasien
Dalam penelitian ini dijelaskan
bahwa suatu kondisi akan menimbulkan
ini
didukung
suatu keinginan, dorongan perilaku tertentu
yang
dilakukan
yang mengarah pada kondisi tersebut.
Latifatul Muna (2014) menunjukkan bahwa
Perilaku
tidak ada pengaruh motivasi dengan
mengarahkan mengarah pada perilaku.
kepatuhan berobat pada pasien TB Paru di
Dalam penelitian ini didapatkan hasil
Poli Paru BP4 Pamekasan. Dari hasil
bahwa
P-ISSN: 2477-1880 E-ISSN: 2502-6623
Kesmas Uwigama : Jurnal Kesehatan Masyarakat
dan
respon
motivasi
klien
intrinsik
TB
akan
dalam
130
Kesmas Uwigama : Jurnal Kesehatan Masyarakat
P-ISSN 2460-0350, E-ISSN 2477-5819
December 2019, Vol. 5 No. 2
melaksanakan
program
pengobatan
Pasundan Kota Samarinda tahun 2019
dipengaruhi oleh dua faktor yaitu faktor
terlihat dari 15 responden dukungan
internal dan faktor
eksternal. Faktor
keluarga tinggi, proporsi tertinggi pada
internal yaitu meliputi keinginan dalam diri
responden yang patuh minum obat anti
sendiri, pengetahuan individu, tingkat
tuberkulosis
pendidikan, pengelolaan diri dan juga usia.
(32,3%), hal ini dikarenakan keluarga yang
Sedangkan untuk faktor eksternal
tinggal
berjumlah
serumah
selalu
10
responden
mengingatkan
meliputi faktor ekonomi, agama faktor
jadwal minum obat dan memberikan
pendukung keluarga dan perawat. Motivasi
semangat kepada responden agar sembuh.
untuk penderita TB dipengaruhi oleh dua
Terdapat responden responden dukungan
hal tersebut yaitu dari dalam diri penderita
keluarga tinggi namun tidak patuh minum
itu sendiri dengan adanya dorongan,
obat
keinginan untuk berobat, keinginan untuk
responden (16,1%). Hal ini dikarenakan
melakukan hal yang baik guna mencapai
respondne mulai malas dan bosan, sehingga
kesembuhan dan dukungan dari keluarga,
anjuran keluarga kurang ditaati.
masyarakat dan petugas kesehatan dalam
anti
tuberkulosis
Adapun
dari
16
responden
dukungan
seorang penderita TB dikatakan baik
tertinggi pada responden yang tidak patuh
apabila
dapat
minum obat anti tuberkulosis berjumlah 15
mengendalikan dirinya untuk mencapai
responden (48,4%), hal ini dikarenakan
kebaikan dirinya yaitu keembuhan dengan
keluarga yang tinggal serumah tidak
cara
mengingatkan jadwal minum obat dan tidak
rutin
meningkatkan
tersebut
minum
obat.
Untuk
motivasi
penderita
TB
rendah,
5
menangani penyakit tersebut. Motivasi
penderita
keluarga
berjumlah
proporsi
memberikan semangat kepada responden
diperlukan adanya penyuluhan tentang
agar
penyakit dan bahayanya penyakit tersebut.
dukungan keluarga rendah namun patuh
Hubungan dukungan keluarga terhadap
kepatuhan minum obat anti tuberkulosis
(OAT) pada penderita penyakit TB Paru
BTA (+) di Puskesmas Pasundan Kota
Samarinda tahun 2019
Berdasarkan
hasil
penelitian
sembuh.
Terdapat
responden
minum obat anti tuberkulosis berjumlah 1
responden (3,2%). Hal ini dikarenakan
responden memiliki semangat yang tinggi
untuk cepat sembuh.
Hasil
uji
statistik
dengan
mengenai hubungan dukungan keluarga
menggunakan uji fisher diperoleh hasil p
terhadap kepatuhan minum obat anti
value : 0,002 < α : 0,05 sehingga Ho ditolak
tuberkulosis
penderita
yaitu ada hubungan dukungan keluarga
penyakit TB Paru BTA (+) di Puskesmas
terhadap kepatuhan minum obat anti
131
(OAT)
pada
P-ISSN: 2477-1880 E-ISSN: 2502-6623
Kesmas Uwigama : Jurnal Kesehatan Masyarakat
Kesmas Uwigama : Jurnal Kesehatan Masyarakat
P-ISSN 2460-0350, E-ISSN 2477-5819
December 2019, Vol. 5 No. 2
tuberkulosis
(OAT)
penderita
dimana keluarga inti maupun keluarga
penyakit TB Paru BTA (+) di Puskesmas
besar berfungsi sebagai sistem pendukung
Pasundan Kota Samarinda tahun 2019.
bagi anggota keluarganya. Fungsi dasar
Dukungan
pada
keluarga
sangat
keluarga yaitu fungsi perawatan kesehatan.
diperlukan terutama pada penderita TB
Fungsi
yang juga merupakan penyakit kronik dan
kemampuan
mengharuskan ia mengkonsumsi obat
anggota keluarga yang mengalami masalah
dengan jangka waktu yang lama, karena
kesehatan. Keluarga perlu memberikan
keluarga merupakan lini pertama bagi
dukungan yang positif untuk melibatkan
penderita apabila mendapatkan masalah
keluarga sebagai pendukung pengobatan
kesehatan atau meningkat kesehatan itu
sehingga
sendiri. Merupakan salah satu fungsi
pemantauan pengobatan antara petugasdan
keluarga
anggota keluarga yang sakit.
untuk
mendukung
anggota
perawatan
kesehatan
keluarga
adanya
untuk
kerjasama
adalah
merawat
dalam
keluarga yang sakit dengan berbagai cara,
seperti
memberi
dukungan
dalam
mengkonsumsi obat (Septia, 2017).
Dukungan keluarga yang positif
diharapkan baik mengantar langsung untuk
periksa di puskesmas maupun di rumah
sakit, dokter atau petugas kesehatan lainnya
dan berpartisipasi penuh pada pengobatan
penderita. Dukungan keluarga memilki
hubungan dengan kepatuhan minum obat
pasien TB dimana PMO sebaiknya adalah
anggota keluarga sendiri yaitu anak atau
pasanganya dengan alasan lebih bisa
dipercaya. Selain itu adanya keeratan
hubungan emosional sangat mempengaruhi
PMO selain sebagai pengawas minum obat
juga memberikan dukungan emosional
kepada penderita TB (Septia, 2017).
Dukungan
keluarga
merupakan
salah satu faktor yang mempengaruhi
KESIMPULAN
1. Tidak ada hubungan
pengetahuan
terhadap kepatuhan minum obat anti
tuberkulosis (OAT) pada penderita
penyakit
TB
Paru
BTA
(+)
di
Puskesmas Pasundan Kota Samarinda
tahun 2019.
2. Tidak ada hubungan motivasi pasien
terhadap kepatuhan minum obat anti
tuberkulosis (OAT) pada penderita
penyakit
TB
Paru
BTA
(+)
di
Puskesmas Pasundan Kota Samarinda
tahun 2019.
3. Ada hubungan dukungan keluarga
terhadap kepatuhan minum obat anti
tuberkulosis (OAT) pada penderita
penyakit
TB
Paru
BTA
(+)
di
Puskesmas Pasundan Kota Samarinda
tahun 2019.
kepatuhan untuk pengobatan TB Paru,
P-ISSN: 2477-1880 E-ISSN: 2502-6623
Kesmas Uwigama : Jurnal Kesehatan Masyarakat
132
Kesmas Uwigama : Jurnal Kesehatan Masyarakat
P-ISSN 2460-0350, E-ISSN 2477-5819
December 2019, Vol. 5 No. 2
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| 4,118 | 30,617 |
W2884705992.txt | GENOME SEQUENCES
crossm
New Reference Genome Sequences for 17 Bacterial Strains of
the Honey Bee Gut Microbiota
Kirsten M. Ellegaard,a Philipp Engela
a
Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland
We sequenced the genomes of 17 strains isolated from the gut of
honey bees, including strains representing the genera Lactobacillus, Bifidobacterium,
Gilliamella, Snodgrassella, Frischella, and Commensalibacter. These genome sequences
represent an important step forward in the development of a comprehensive reference database to aid future analysis of this emerging gut microbiota model.
ABSTRACT
T
he honey bee gut is colonized by a remarkably simple community dominated by
only 8 to 10 bee-specific phylotypes (1). However, genome-level analyses have
shown that several of the phylotypes comprise highly divergent strains (2–4). As such,
the honey bee is a promising future model for studying strain-level evolution and
function in gut-associated bacterial communities (5). Here, we present 17 new genome
sequences of strains isolated from the gut of honey bees, which were generated to
facilitate the development of a reference genome database for this community. All
strains were isolated from honey bees collected from our apiary in Lausanne, Switzerland, by culturing gut homogenates on agar plates (6) under microaerophilic or
anaerobic conditions (7).
Four strains of the genus Lactobacillus (Table 1) were selected for sequencing with
PacBio 20K (Pacific Biosciences) single-molecule real-time (SMRT) technology. The
strains were grown overnight in MRS broth supplemented with fructose and cysteine
(8) at 35°C under anaerobic conditions, and total genomic DNA was extracted using a
cetyltrimethylammonium bromide-based extraction protocol (7). De novo genome
assembly was done using the Hierarchical Genome Assembly Process (HGAP) version
2.3. Another 13 strains representing the genera Bifidobacterium, Gilliamella, Snodgrassella, Frischella, and Commensalibacter (Table 1) were selected for sequencing with
Illumina technology. The strains were cultured as described previously (7), and total
genomic DNA was extracted with the GenElute bacterial genomic DNA kit according to
the manufacturer’s instructions (Table 1). Sequencing libraries were prepared with the
TruSeq DNA kit and sequenced on the MiSeq platform (Illumina) using the paired-end
2 ⫻ 250-bp protocol. All 13 genomes were sequenced to a minimum depth of 50⫻
(Table 1). The resulting FASTQ files were trimmed with Trimmomatic (9) to remove
eventual adapter sequences and low-quality reads using the following parameters:
LEADING, 20; TRAILING, 20; SLIDINGWINDOW, 4:15; and MINLEN, 50. The reads were
assembled with SPAdes version 3.7.1 (10) using the “-careful” flag and multiple k-mer
sizes (21, 33, 55, 77, 99, 127). Small contigs (less than 500 bp) and contigs with low
k-mer coverage (less than 5⫻) were removed from the assemblies, resulting in 6 to
40 contigs per assembly, with a median N50 of 529,190 bp. For strains with related
complete genome sequences or scaffolds available, the contigs were reordered with
Mauve (11).
Assembly qualities were checked by remapping reads to assemblies with the
Burrows-Wheeler Aligner (12) and by GC-skew visualization with DNAplotter (13). For
strain ESL0184, the main chromosome was cut into three contigs due to assembly
Volume 7 Issue 3 e00834-18
Received 14 June 2018 Accepted 22 June
2018 Published 26 July 2018
Citation Ellegaard KM, Engel P. 2018. New
reference genome sequences for 17 bacterial
strains of the honey bee gut microbiota.
Microbiol Resour Announc 7:e00834-18.
https://doi.org/10.1128/MRA.00834-18.
Editor J. Cameron Thrash, Louisiana State
University
Copyright © 2018 Ellegaard and Engel. This is
an open-access article distributed under the
terms of the Creative Commons Attribution 4.0
International license.
Address correspondence to Philipp Engel,
philipp.engel@unil.ch.
mra.asm.org 1
Ellegaard and Engel
TABLE 1 Genome assembly statistics and strain information
Genus
Lactobacillus
Lactobacillus
Lactobacillus
Lactobacillus
Bifidobacterium
Bifidobacterium
Bifidobacterium
Bifidobacterium
Bifidobacterium
Gillamella
Gillamella
Gillamella
Gillamella
Gillamella
Snodgrassella
Frischella
Commensalibacter
Species
L. apis
L. helsingborgensis
L. melliventris
L. kulllabergensis
B. asteroides
B. asteroides
B. asteroides
B. asteroides
B. indicum/
B. coryneforme
G. apicola
G. apis
G. apis
NAd
NA
S. alvi
F. perrara
Commensalibacter
sp.
Phylotypea
Firm5
Firm5
Firm5
Firm5
Bifido
Bifido
Bifido
Bifido
Bifido
Sublineage
Firm5-1
Firm5-2
Firm5-3
Firm5-4
Bifido-1
Bifido-1
Bifido-1
Bifido-1
Bifido-2
Strain
ESL0185
ESL0183
ESL0184
ESL0186
ESL0170
ESL0198
ESL0199
ESL0200
ESL0197
Extracted
DNA (g)b
17.9
19.3
19.8
16.1
1.1
1.3
5.3
4.7
1.1
No. of
contigs
1
2
4
1
7
12
7
16
6
N50 (bp)
1,683,102
1,856,015
1,505,590
2,018,944
1,162,986
618,428
558,059
500,320
1,389,647
Assembly
size (bp)
1,683,102
1,867,232
2,036,181
2,018,944
2,175,262
2,235,610
2,167,340
1,933,421
1,715,238
Coverage
(ⴛ)
420
300
320
290
200
280
50
300
300
GC content
(%)
37
37
36
36
60
60
59
60
61
No. of
genesc
1,578
1,780
2,015
1,915
1,771
1,820
1,741
1,621
1,408
Gilliamella
Gilliamella
Gilliamella
Gilliamella
Gilliamella
Snodgrasella
Frischella
Commensalibacter
Gilli-1
Gilli-2
Gilli-2
Gilli-3
Gilli-3
NA
NA
NA
ESL0178
ESL0169
ESL0172
ESL0177
ESL0182
ESL0196
ESL0167
ESL0284
0.3
3.9
1.8
2.0
1.2
3.7
3.1
1.3
18
13
17
19
31
15
40
13
364,598
481,163
374,672
953,736
255,373
1,281,809
277,847
471,180
2,885,657
2,430,778
2,685,772
3,086,198
3,537,173
2,446,304
2,558,525
1,948,862
200
270
200
50
160
130
200
50
34
35
34
35
35
41
34
38
2,602
2,227
2,468
2,868
3,257
2,224
2,313
1,767
aBased
on 16S rRNA amplicon sequencing.
amount of extracted DNA.
cGene count based on the JGI Microbial Genome Annotation Pipeline.
dNA, not applicable.
bTotal
uncertainty generated by a duplicated prophage sequence. Strains ESL0183, ESL0185,
and ESL0186 were submitted as complete genomes, with strain ESL0183 having a small
plasmid contig of 11.3 kb.
Core phylogenies were generated for the Lactobacillus, Bifidobacterium, and Gilliamella strains, including previously published isolates derived from honey bees, using
OrthoFinder (14) for ortholog prediction and RAxML (15) for phylogenetic inference.
Based on the phylogenies, the Lactobacillus and Bifidobacterium strains represent
members of previously reported sublineages, whereas two strains of the genus Gilliamella (ESL0177 and ESL0182) represent a new sublineage, with strain ESL182 having the
largest genome size reported for this genus to date (3.5 Mbp) (Table 1).
Data availability. The complete genome sequences for the strains reported here
have been deposited in GenBank under the accession numbers CP029476, CP029544/
CP029545, and CP029477, and the whole-genome shotgun projects have been deposited under the accession numbers QGLH00000000, QGLJ00000000, QGLK00000000,
QGLL00000000, QGLI00000000, QGLG00000000, QGLQ00000000, QGLN00000000,
QGLO00000000, QGLP00000000, QGLR00000000, QGLS00000000, QGLM00000000, and
QGLT00000000. Additionally, the genomes were annotated using the JGI Microbial Genome Annotation Pipeline, where they have been deposited under the genome identification numbers 2684622912, 2684622914, 2684622911, 2684622916, 2684622918,
2684622919, 2684622920, 2684622917, 2684622913, 2684622925, 2684622922,
2684622923, 2684622924, 2684622926, 2684622927, 2684622921, and 2756170209.
ACKNOWLEDGMENTS
This work was funded by Human Frontier Science Program (HFSP) Young Investigator grant RGY0077/2016, the European Research Council Starting Grant (ERC-StG)
“MicroBeeOme,” Swiss National Science Foundation grant 31003A_160345, and the
Fondation Herbette at the University of Lausanne.
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W2061871085.txt | Eur. Phys. J. C (2014) 74:3153
DOI 10.1140/epjc/s10052-014-3153-3
Regular Article - Theoretical Physics
Formalism of gauge-invariant curvatures and constructing
the cubic vertices for massive spin- 23 field in AdS4 space
I. L. Buchbinder1,2,a , T. V. Snegirev1,b , Yu. M. Zinoviev3,c
1
Department of Theoretical Physics, Tomsk State Pedagogical University, Tomsk 634061, Russia
National Research Tomsk State University, Tomsk, Russia
3 Institute for High Energy Physics, Protvino, Moscow Region 142280, Russia
2
Received: 9 June 2014 / Accepted: 24 October 2014 / Published online: 28 November 2014
© The Author(s) 2014. This article is published with open access at Springerlink.com
Abstract We study the interaction of a massive spin-3/2
field with electromagnetic and gravitational fields in the
four dimensional AdS space and construct the corresponding cubic vertices. The construction is based on a generalization of Fradkin–Vasiliev formalism, developed for massless higher spin fields, to massive fermionic higher spin
fields. The main ingredients of this formalism are the gaugeinvariant curvatures. We build such curvatures for the massive
theory under consideration and show how the cubic vertices
are written in their terms.
construct a gauge invariant object that we will call curvature
and denote as R. Moreover, with the help of these curvatures a
free Lagrangian can be rewritten in explicitly gauge-invariant
form as
R ∧ R.
L0 ∼
Using these ingredients one can construct two types of nontrivial1 cubic vertices:
– Abelian vertices that have the form
L ∼ R ∧ R ∧ ,
1 Introduction
– non-Abelian ones that look like
Till now most of the investigations of consistent cubic interaction vertices for higher spin fields were devoted to bosonic
massless fields (see e.g. [1–9]). The results on massive higher
spin interactions are not so numerous, although there exists
a classification of massive and massless cubic vertices in flat
space by Metsaev [10–12] as well as some concrete examples
[13–21]. At the same time there exist just a small number of
papers devoted to fermionic higher spin interactions [11,22–
24]. At least one of the reasons for this is that technically
such investigations appear to be much more involved. In this
paper, using massive spin 23 as a simple but physically interesting and non-trivial example of massive fermionic higher
spin fields, we apply the so-called Fradkin–Vasiliev formalism [25,26] (see also [21,27–32]) to the construction of electromagnetic and gravitational cubic vertices.
Let us briefly recall the basic properties of this formalism.
A higher spin particle is described by a set of fields that
we collectively denote here and for each field one can
a e-mail:
L ∼ R ∧ ∧ .
For the massless bosonic fields Vasiliev [33] has shown that
any such non-Abelian vertex can be obtained as a result of a
deformation procedure that can be described as follows.
– Construct quadratic in fields deformations for the curvatures and linear corrections to the gauge transformations
R ∼ ∧ , δ1 ∼ ξ,
so that the deformed curvatures R̂ = R + R transform
covariantly,
δ R̂ = δ1 R + δ0 R ∼ Rξ.
snegirev@tspu.edu.ru
c e-mail:
yurii.zinoviev@ihep.ru
(2)
At this step most of the arbitrary parameters are fixed,
however, there still remains some ambiguity. The reason
is that covariance of the transformations for deformed
joseph@tspu.edu.ru
b e-mail:
(1)
1
We call a vertex trivial if it can be constructed using gauge-invariant
curvatures only, i.e. L ∼ R ∧ R ∧ R.
123
3153 Page 2 of 9
Eur. Phys. J. C (2014) 74:3153
curvatures guarantees that the equations of motion will
be gauge-invariant but it does not guarantee that they will
be Lagrangian.
– Non-Abelian cubic vertex arises then one put these
deformed curvatures into the free Lagrangian and using
explicit form of the gauge transformations requires it to
be gauge-invariant
L∼
R̂R̂
⇔
δL ∼
RRξ = 0.
Note, finally, that Vasiliev has shown [33] that any non-trivial
cubic vertex for three fields with spins s1 , s2 and s3 having up
to s1 +s2 +s3 −2 derivatives can be constructed as some combination of Abelian and non-Abelian vertices. However, it has
been pointed out that the Vasiliev construction [33] was initially formulated only for massless higher spin field theories.
In this paper we generalize the Vasiliev approach to massive
higher spin theories on the example of cubic electromagnetic
and gravitational coupling for a massive spin- 23 field in the
AdS4 space. Let us stress that in this paper we systematically
restrict ourselves to cubic terms in the Lagrangians and terms
linear in the fields in the gauge transformation laws, so all
the Lagrangians we obtain are gauge-invariant in the linear
(first non-trivial) approximation only. As is well known for
higher spin theories in four or greater dimensions to proceed
beyond this linear approximation one has to introduce an
infinite number of fields with ever increasing spins and the
corresponding gauge algebra becomes infinite dimensional.
Thus in what follows all the higher order terms (quartic terms
in the Lagrangians and quadratic terms in the gauge transformation laws) will be discarded.
The paper is organized as follows. In Sect. 2 we give
all necessary kinematics formulas. Here we introduce two
gauge-invariant objects (for the physical field ψμ and Stueckelberg one φ) and rewrite the free Lagrangian using these
objects. Sect. 3 devoted to the electromagnetic interaction
for such massive spin- 23 field (for previous results see e.g.
[34–36]2 ). In Sect. 3.1, as an independent check for our calculations as well as for instructive comparison, we consider
this task using straightforward constructive approach. Then
in Sect. 3.2 we consider the same task in the Fradkin–Vasiliev
formalism. At last in Sect. 4 we construct gravitational cubic
vertex in the Fradkin–Vasiliev formalism.
normalized so that3
1
a
2
a
a
[Dμ , Dν ]ξ = λ e[μ ξν] + Γμν ξ , λ2 = − . (3)
2
3
In (3) eμ a is a background (non-dynamical) tetrad linking world and local indices and has the standard definition,
eμ a eν b g ab = gμν , where gμν and g ab are, respectively,
curved world AdS metrics and flat local one. Wherever it
is convenient local indices are converted into the world ones
by eμ a and its inverse eμ a , in particular the matrix γμ with
world index is understood as γμ = eμ a γ a . Also we introduce the following notation for antisymmetric combinations
of eμ a :
μν
μνα
[μ ν]
[μ ν α]
ab = e a e b ,
abc = e a e b e c .
In the expression (3) Γ ab = 21 γ [a γ b] is an antisymmetric
combination of two gamma matrices and is a particular case
of the more general definition which we will use,
1 [a1
γ . . . γ an ]
n!
where n = 2, 3, 4 for four-dimensional space. Let us present
the main properties of such Γ -matrices:
Γ a1 a2 ...an =
Γ ab1 ...bn = − g a[b1 Γ b2 ...bn ] + γ a Γ b1 ...bn ,
Γ ab1 ...bn = g a[b1 Γ b2 ...bn ] + (−1)n Γ b1 ...bn γ a ,
γ a Γ b1 ...bn = 2g a[b1 Γ b2 ...bn ] + (−1)n Γ b1 ...bn γ a ,
(4)
γa Γ ab1 ...bn = (d − n)Γ b1 ...bn ,
Γ ab1 ...bn γa = (−1)n (d − n)Γ b1 ...bn .
The identities (4) are used in the paper to verify the gauge
invariance of the Lagrangians.
All fermionic fields are anticommuting and Majorana. We
also use the Majorana representation of the γ -matrices with
hermitian conjugation defined as follows:
(γ a )† = γ 0 γ a γ 0
(Γ a1 a2 ...an )† = γ 0 Γ an ...a2 a1 γ 0 = −γ 0 Γ a1 a2 ...an γ 0 .
In this case γ 0 γ a and γ 0 Γ ab are symmetric in the spinor
indices, while γ 0 , γ 0 Γ abc , and γ 0 Γ abcd are antisymmetric.
2 Kinematic of massive spin-3/2 field
Notations and conventions
We work in four-dimensional Anti-de Sitter space with cosmological constant . The AdS covariant derivatives Dμ is
For a gauge-invariant description of a massive spin-3/2 field
besides the master vector-spinor field ψμ one has also introduced Stueckelberg spinor field φ. The free Lagrangian is
2
3 We use Greek letters μ, ν, . . . for world indices and Latin letters
a, b, . . . for local ones. Summation over any repeated indices is implied.
For indices in (square) round brackets we use the convention of complete
(anti)-symmetrization without normalization factor. Spinor indices of
(tensor)-spinor fields are omitted.
Some aspects of higher spin equations of motion in an external electromagnetic field have been studied in [37]; however, these equations
are non-Lagrangian.
123
Eur. Phys. J. C (2014) 74:3153
Page 3 of 9 3153
known and has the form
Using the Bianchi identities for the curvatures (7) we obtain
μνα
μναβ ¯
abcd
¯ μν Γ abc α
να + 8i M abc
−3m abcd
μν Γ
μν
¯ μ Γ ab ν = 0.
+8m ab
i μνα
i
abc
Dν ψα + eμ a φ̄γ a Dμ φ
abc ψ̄μ Γ
2
2
3M μν
μ
a
ψ̄μ Γ ab ψν − M φ̄φ.
+3ime a ψ̄μ γ φ −
2 ab
L0 = −
(5)
This Lagrangian is invariant under the following gauge transformations:
δ0 ψμ = Dμ ξ +
δ0 φ = 3mξ
3 Electromagnetic interaction
iM
γμ ξ
2
3.1 Constructive approach
where M 2 = m 2 +λ2 = m 2 −/3 and m is a mass parameter.
Note that such a description works in de Sitter space as well,
provided m 2 > /3, m 2 = /3 being the boundary of the
unitarity region.
Using the explicit form of gauge transformations one can
construct two gauge-invariant objects (curvatures):
m
iM
γ[μ ψν] ,
μν = D[μ ψν] + Γμν φ +
6
2
iM
γμ φ,
μ = Dμ φ − 3mψμ +
2
(6)
which satisfy the Bianchi identities
D[μ να]
D[μ ν]
m
iM
= Γ[μν α] −
γ[μ να] ,
6
2
iM
γ[μ ν] .
= −3mμν −
2
i μνα
¯ μ γ a δφ.
¯ μν Γ abc δψα − ieμ a
2 abc
We prefer to work with Majorana fermions; thus for the
description of electromagnetic interactions we will use a pair
of them, ψμ i , φ i , where i = 1, 2 is the SO(2) index. Let
us switch to the minimal electromagnetic interaction by the
standard rule,
Dμ ψν i ⇒ Dμ ψν i + e0 εi j Aμ ψν j ,
and similarly for φ i . The Lagrangian (5) with the above
replacement for the covariant derivative is not invariant under
the free gauge transformations, and the variation of the
Lagrangian has the form
ie0 i j μνα i abc
ε abc ψ̄μ Γ Fνα ξ j
2
= −3ie0 εi j eμ a ψ̄μ i Γ abc F bc ξ j .
δ0 L0 = −
(7)
Note that the curvatures (6) are closely related with the
Lagrangian equations of motion. It can be seen from the total
variation of Lagrangian (5) that can be written as follows:
δL0 = −
As will be seen in the next section sometimes this ambiguity
may be important in order to reproduce the most general
cubic vertex, so we will not fix it here.
(8)
(11)
To compensate for this non-invariance let us introduce nonminimal interactions:
μν
L1 = εi j ab ψ̄μ i (b1 F ab + b2 Γ abcd F cd )ψν j
+ieμ a ψ̄μ i (b3 F ab γ b + b4 Γ abc F bc )φ j
+ b5 φ̄ i (Γ F)φ j ,
(12)
Moreover, using these curvatures the free Lagrangian (5)
can be rewritten in explicitly gauge-invariant form. The most
general ansatz looks like
μνα
μναβ ¯
abcd
¯ μν Γ abc α
L0 = c1 abcd
να + ic2 abc
μν Γ
μν
¯ μ Γ ab ν .
+c3 ab
(9)
where (Γ F) = Γ ab Fab , which produce the following variations:
The requirement to reproduce the original Lagrangian (5)
partially fixes the parameters
−iεi j eμ a Dμ φ̄ i (b3 F ab γ b − b4 Γ abc F bc )η j
M(b3 + 2b4 )
+
+ 6mb5 εi j φ̄ i (Γ F)η j
2
3c3 = −8c1 , 6c2 m =
1
− 16c1 M.
2
(10)
The remaining freedom in the parameters is related with the
identity
μναβ
abcd
¯
β ] = 0.
abcd Dμ [να Γ
δ0 L1 = 4b1 εi j ψ̄μ i η j (D F)μ − ib3 εi j φ̄ i γμ η j (D F)μ
μν
+2εi j ab Dμ ψν i (b1 F ab + b2 Γ abcd F cd )η j
+iεi j eμ a ψ̄μ i ((2Mb1 + 3mb3 )F ab γ b
+(2Mb2 + 3mb4 )Γ abc F bc )η j
where we have (D F)μ = Dν F νμ as well as the corresponding corrections to the gauge transformations:
δ1 ψμ i = iα1 εi j (Γ F)γμ ξ i ,
δφ i = α2 εi j (Γ F)ξ j ,
δ1 Aμ = β1 εi j (ψ̄μ i ξ j ) + iβ2 εi j (φ̄ i γμ ξ j ),
(13)
123
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Eur. Phys. J. C (2014) 74:3153
which in turn give us
δ1 L0 = β1 εi j ψ̄μ i η j (D F)μ + iβ2 εi j φ̄ i γμ η j (D F)μ
μν
−6α1 εi j ab Dμ ψ̄ν i (2F ab + Γ abcd F cd )η j
−3iεi j eμ a ψ̄μ i [(4Mα1 − 2mα2 )F ab γ b
−(2Mα1 + mα2 )Γ abc F bc )]η j
−iα2 εi j eμ a Dμ φ̄ i (2F ab γ b + Γ abc F bc )η j
−2Mα2 εi j φ̄ i (Γ F)η j .
From these explicit expressions one can see that gauge invariance can be restored and that gives the solution for arbitrary
coefficients:
b1 = 6α1 , b2 = 3α1 , b3 = −2α2 , b4 = α2
Mα2
, β1 = −24α1 , β2 = −2α2 ,
b5 =
3m
provided the following important relation holds:
4Mα1 + 2mα2 = e0 .
approximation only. Indeed, the introduction of cubic terms
in the Lagrangian and linear terms in the gauge transformation laws generate quadratic variations of the form δ1 L1 . To
compensate for these variations one has to introduce quartic
terms and/or additional fields and so on. In supergravities (i.e.
theories containing spins not higher than 2 and 3/2) such a
program can indeed be realized leading to models with spontaneous supersymmetry breaking (see e.g. [38,39] and references therein). But for the theories with spins greater than
2 such a program will require the introduction of an infinite
number of fields with ever increasing spins. The basic aim
of our paper is to construct first a nonvanishing term in the
interacting Lagrangian. It means that we consider only those
terms in the Lagrangian and gauge transformations which
are required for the realization of this aim. The higher order
terms in the gauge transformations do not affect the form of
the above cubic interaction vertex, and therefore we do not
study these higher order terms here.
(14)
3.2 Formulation in terms of curvatures
The Lagrangian (12) together with the correction, stipulated by the minimal switch to the interaction in the free
Lagrangian, is the final cubic electromagnetic interaction vertex for the spin-3/2 field. As we see, this vertex contains two
arbitrary parameters.
A few comments are in order.
– Calculating the commutator of two gauge transformations we obtain
[δ(ξ1 ), δ(ξ2 )]Aμ = −8εi j (6α1 2 + α2 2 )(ξ2 i γ ν ξ1 j )Fμν ,
and it means that for non-zero electric charge e0 any such
model must be a component of some (spontaneously broken) supergravity.
– From the supergravity point of view the remaining freedom in the parameters α1 and α2 is clear: in general our
vector field is a superposition of the graviphoton (i.e. a
vector field from the supermultiplet ( 23 , 1)) and the goldstino’s superpartner (i.e. a vector field from the supermultiplet (1, 21 ) (see e.g. [38,39] and references therein).
– From the expression for the parameter b5 one can see that
we have an ambiguity between flat and massless limits.
Indeed, in the flat case b5 = α32 and does not depend on
the mass any more, while for the non-zero cosmological
term this parameter is singular in the massless limit.
– The most simple case—α2 = 0, i.e. the vector field
is just the graviphoton. In this case the electric charge
e0 = 23 Mα1 becomes zero at the boundary of the unitarity region.
Now we reformulate the results of Sect. 3.1 on the base of
Fradkin–Vasiliev formalism. Following the general procedure we begin with a deformation of the curvatures so that
they transform covariantly; see (1), (2). In the case under
consideration R = {μν i , μ i , Fμν }. For the fermionic curvatures the deformations correspond simply to the minimal
substitution:
μν i = e0 εi j A[μ ψν] j ,
μ i = e0 εi j Aμ φ j ,
(15)
while the transformations for the deformed curvatures look
like
ˆ μi = 0
ˆ μν i = e0 εi j Fμν ξ j , δ
δ
(16)
ˆ μν i = μν i + μν i ,
ˆ μ i = μ i + μ i , and
where
i
i
μν , μ are given by (6).
The most general deformation for the electromagnetic
fields strength quadratic in the fermionic fields can be written
as follows:
Fμν = εi j [a1 ψ̄[μ i ψν] j + ia2 ψ̄[μ i γν] φ j + a3 φ̄ i Γμν φ j ].
(17)
The corresponding corrections to the gauge transformation
have the form
δ1 Aμ = εi j [2a1 ψ̄μ i ξ j − ia2 φ̄ i γμ ξ j ].
(18)
The deformed curvatures will transform covariantly
As we have already noted in the Introduction the Lagrangians
obtained in this paper are gauge-invariant in the linear
123
i
¯ μν
¯ [μ i γν] ξ j ],
δ1 F̂μν = εi j [2a1
ξ j − ia2
(19)
Eur. Phys. J. C (2014) 74:3153
Page 5 of 9 3153
provided the following relation holds:
3ma3 = −
ma1
− Ma2 .
6
(20)
Here F̂μν = Fμν + Fμν .
Now let us consider the Lagrangian
1
μναβ ˆ¯
i abcd ˆ i
αβ
L = − F̂ μν F̂μν + c1 abcd
μν Γ
4
μνα ˆ i abc i
ˆα
¯ μν Γ
+ic2 abc
μν ˆ i ab i
ˆ
¯ Γ
+c
3 ab
μ
ν
(21)
where all initial curvatures (including the electromagnetic
field strength) are replaced by the deformed ones, and let us
require that this Lagrangian be invariant. Non-trivial variations arise only with respect to transformations with the
spinor parameter ξ i . Using the explicit form of the covariant
transformations (16), (19) we obtain
1 μν
¯ μν i (a1 F ab − 48e0 c1 Γ abcd F cd )ξ j
δL = − εi j ab
2
¯ μ i (a2 γ b F ab + 6e0 c2 Γ abc F bc )ξ j . (22)
+iεi j eμ a
To compensate these terms one needs to introduce nonminimal corrections (note that they have exactly the same
form as in the previous subsection):
δ1 ψμ i = iα1 εi j (Γ F)γμ ξ j , δ1 φ i = α2 εi j (Γ F)ξ j ,
(23)
Let us extract the cubic vertex. Using the relations (10),
(25) we get
μν
L1 = 3α1 εi j ab ψ̄μ i (2F ab + Γ abcd F cd )ψν j
−iα2 εi j eμ a ψ̄μ i (2γ b F ab − Γ abc F bc )φ j
Mα2 i j i
ε φ̄ (Γ F)φ j
+
3m
ie0 μνα
+ εi j abc Aμ ψ̄ν i Γ abc ψα j
2
ie0 i j μ
+ ε e a Aμ φ̄ i γ a φ j .
(26)
2
Here the last two lines correspond to the minimal interactions,
while the other terms are non-minimal corrections.
The Lagrangian L1 (26) up to the minimal interaction is
the same as obtained in Sect. 3.1 for the electromagnetic
cubic vertex.
4 Gravitational interaction
4.1 Kinematics for gravity
Let us briefly review basic features of gravity in the AdS4
space at free level. In the frame formulation the gravitational
field is described by dynamical frame h μ a and Lorentz connection ωμ ab being antisymmetric in local indices. The free
Lagrangian in AdS space has the form
L0 =
1 μν ac bc 1 μνα ab
ωμ ων −
ωμ Dν h α c
2 ab
2 abc
μν
+λ2 ab h μ a h ν b
(27)
and is invariant under the gauge transformations
δ0 ωμ ab = Dμ η̂ab − λ2 eμ [a ξ̂ b] ,
which in turn produce the following variations:
δ1 L0 =
μν
¯ μν i (6F ab + 3F de Γ abde )ξ j
−α1 εi j ab
¯ μ i (2γ b F ab + Γ abc F bc )ξ j .
−iα2 εi j eμ a
δ0 h μ a = Dμ ξ̂ a + η̂μ a .
(24)
Tμν a = D[μ h ν] a − ω[μ,ν] a .
a1 = −12α1 , a2 = 2α2 ,
(25)
Thus we see that the choice of the parameters α1 and α2 is
related with the choice of parameters c1 and c2 in the free
Lagrangian. Moreover, if we use the relation
6mc2 =
1
− 16Mc1 ,
2
The gauge-invariant objects (linearized curvature and torsion) have the form
Rμν ab = D[μ ων] ab − λ2 e[μ [a h ν] b] ,
Comparing with (22) we conclude
α1 = 8e0 c1 , α2 = 6e0 c2 .
(28)
(29)
They satisfy the Bianchi identities
D[μ Rνα] ab = λ2 e[μ [a Tνα] b] ,
D[μ Tνα] a = −R[μν,α] a .
(30)
Note that on the mass shell for the auxiliary field ωμ ab by
virtue of (30) we have
Tμν a = 0
⇒
R[μν,α] a = 0,
D[μ Rνα] ab = 0.
(31)
we again obtain
Finally, the free Lagrangian can be rewritten as follows:
4Mα1 + 2mα2 = e0 .
L0 = c0
μναβ
abcd
Rμν ab Rαβ cd , c0 =
1
.
32λ2
(32)
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Eur. Phys. J. C (2014) 74:3153
4.2 Gravitational coupling for massive spin-3/2
Following the general scheme (1), (2) we begin with the
deformation of the curvatures that in the case under consideration are R = {μν , μ , Rμν ab , Tμν a }. As in the case of
electromagnetic interactions deformations for spin-3/2 curvatures correspond to the minimal substitution rules, i.e. to
the replacement of the covariant derivative D → D + ω and
background tetrad eμ a → eμ a + h μ a :
μν
= a1 ω[μ ab Γab ψν] + 2Mi h [μ a γa ψν]
2m
a
a
−
h [μ Γν] φ ,
3
μ = a1 (ωμ ab Γab φ + 2Mi h μ a γa φ).
(33)
2m a
Γμ φ ξ̂a
δ1 ψμ = −a1 Γ ab ψμ η̂ab + 2i Mγ a ψμ ξ̂a −
3
−ωμ ab Γab ξ − 2i Mh μ a γa ξ ,
(34)
while the transformations for the deformed curvatures will
have the form
2m
ˆ μν = −a1 Γ ab μν η̂ab +2i Mγ a μν ξ̂a +
Γ[μ a ν] ξ̂a
δ
3
−Rμν ab Γab ξ − 2i M Tμν a γa ξ ,
ˆ μ = −a1 (Γ ab μ ηab + 2i Mγ a μ ξ̂a ).
δ
(35)
ˆ μ = μ + μ and μν i , i
ˆ μν = μν + μν ,
Here
are given by (6).
Now let us consider the most general deformations for
gravitational curvature and torsion quadratic in the spin-3/2
fields:
Rμν ab = b1 ψ̄[μ Γ ab ψν] +ib2 e[μ [a ψ̄ν] γ b] φ +ib3 ψ̄[μ Γν] ab φ
+b4 e[μ a eν] b φ̄φ + b5 φ̄Γμν ab φ,
Tμν a = ib6 ψ̄[μ γ a ψν] + b7 e[μ a ψ̄ν] φ
+b8 ψ̄[μ Γν] a φ + ib9 φ̄Γμν a φ.
(36)
Note that there are three possible field redefinitions
ωμ ab ⇒ ωμ ab + iκ1 φ̄Γμ ab φ,
h μ a ⇒ h μ a + κ2 ψ̄μ γ a φ + κ3 eμ a φ̄φ
that shift parameters b3 , b6 and b7 .
123
δ1 ωμ ab = 2b1 ψ̄μ Γ ab ξ − ib2 eμ [a φ̄γ b] ξ − ib3 φ̄Γμ ab ξ,
δ1 h μ a = 2ib6 ψ̄μ γ a ξ + b7 eμ a φ̄ξ + b8 φ̄Γμ a ξ.
(38)
Then the curvature and torsion will transform as follows:
¯ μν Γ ab ξ +ib2 e[μ [a
¯ ν] γ b] ξ −ib3
¯ [μ Γν] ab ξ,
δ R̂μν ab = 2b1
¯ μν γ a ξ −b7 e[μ a
¯ ν] ξ + b8
¯ [μ Γν] a ξ, (39)
δ T̂μν a = 2ib6
provided the following restrictions on the arbitrary parameters hold:
The corrections to the gauge transformations will look like
δ1 φ = −a1 (Γ ab φ η̂ab + 2i Mγ a φ ξ̂a ),
In order that deformed curvatures transform covariantly
we have to introduce the following corrections to the gauge
transformations:
(37)
3mb2 = b6 (M 2 − m 2 ) − b1 M 3mb8 = −b6 M + b1 ,
mb1
mb6
+ 2b7 (M 2 − m 2 ) 6mb9 = −
− 2b3 ,
6mb4 =
3
3
mb1
− 2b3 M.
(40)
6mb5 = −
3
Here R̂μν ab = Rμν ab + Rμν ab , T̂μν a = Tμν a + Tμν a
and Rμν ab , Tμν a are given by (29).
The general solution to these relations has four free parameters, for example, b1,3,6,7 . But as we have already noted
three of them are related with possible field redefinitions, so
we have one non-trivial parameter b1 only.
Let us consider the following Lagrangian:
μνα ˆ
μναβ ˆ¯
abcd ˆ
ˆα
¯ μν Γ abc
L = c1 abcd
να + ic2 abc
μν Γ
μν ˆ
ˆ ν + c0 μναβ R̂μν ab R̂αβ cd
¯ μ Γ ab
+c3 ab
abcd
μναβ ¯
+ic4 abcd μν Γ abc α h β d
μνα
¯ μ Γ ab ν h α c
+c5 abc
(41)
where the first four terms are just the sum of free Lagrangians
for the massive spin- 23 case and the massless spin-2 case with
the initial curvatures replaced by the deformed ones, while
the last two terms are possible Abelian vertices. Note that
in dimensions d > 4 we would have to introduce one more
Abelian vertex:
μναβγ ¯
μν Γ abcd αβ h γ e .
abcde
Now we require that this Lagrangian be gauge-invariant. To
fix all coefficients it is enough to consider variations that do
not vanish on-shell. For ξ -transformations we have
μναβ ¯
ab cd
δL = (−24c1 a1 + 4b1 c0 ) abcd
αβ Rμν Γ ξ
μνα
+i(6c2 a1 − 8c0 b3 ) abc Rμν ad ξ̄ Γ bcd α
μνα
+i(6c2 a1 − 8c0 b2 ) abc Rμν ab ξ̄ γ c α ;
Eur. Phys. J. C (2014) 74:3153
Page 7 of 9 3153
the last two terms vanish on-shell and for the second term it
can be seen from the identity:
μνα
μναβ
0 = abcd Rμν,α a ξ̄ Γ bcd β = 3 abc Rμν ad ξ̄ Γ bcd α .
The first term does not vanish on-shell so we have to put
b1 c0 = 6c1 a1 .
(42)
Calculating the variations for the η̂ab -transformations we
obtain
μναβ ¯
abce
αβ η̂de
δL = −8c1 a1 abcd
μν Γ
μνα
¯ μν Γ abd α η̂cd
−i(12c2 a1 − 3c4 ) abc
μν
¯ μ Γ ac ν η̂bc .
+(8c3 a1 − 2c5 )
ab
− 2. For this vertex we obtain
μναβ
L = (−48c1 a1 + 8c0 b1 ) abcd Dμ ων ab ψ̄α Γ cd ψβ = 0.
Vertex
3
2
−
3
2
This expression vanishes due to the obtained expressions and
the restrictions for the arbitrary coefficients.
Vertex 21 − 21 − 2. In this case we have
μν
L = (−c3 a1 + 8c0 b5 ) ab Dμ ων cd φ̄Γ abcd φ
μν
+(8c3 a1 − 2c5 ) ab ωμ ac Dν φ̄Γ bc φ
μν
+(2c3 a1 + 16c0 b4 ) ab Dμ ων ab φ̄φ.
The second term drops out due to the relations among the
coefficients, the first term vanishes on-shell for the gravitational field due to the identity
μνα
0 = abc Rμν,α d φ̄Γ abcd φ
μνα
= 2 abc (Dμ ων,α d + λ2 eμ d h ν,α )φ̄Γ abcd φ
μν
= 6 ab Dμ ων cd φ̄Γ abcd φ,
The first term vanishes in d = 4 due to the identity
μναβγ ¯
abcd
αβ η̂γ e
0 = abcde
μν Γ
μναβ ¯
abce
= 4 abcd
αβ η̂de ,
μν Γ
while the third term can be removed by the field redefinition
while the last two terms give the restrictions
h μ a → h μ a + κ3 eμ a φ̄φ.
c4 = 4c2 a1 , c5 = 4c3 a1 .
(43)
Finally we consider variations under the ξ̂ a transformations
3
μναβ ¯
abc
δ L = −i 16c1 a1 M + mc4 abcd
αβ ξ̂ d
μν Γ
2
μνα
¯ μν Γ ab α ξ̂ c
+(32c1 a1 m + 3mc5 ) abc
μνα
¯ μν Γ abcd α ξ̂ d
+(−4Ma1 c2 + Mc4 ) abc
μν
¯ μ Γ abc ν ξ̂ c
−i((4a1 c2 − c4 )m + (4c3 a1 − c5 )M) ab
μν
¯ μ γ a ν ξ̂ b .
+i(2(8a1 c2 + c4 )m − 4(2c3 a1 + c5 )M) ab
Taking into account (10), (40), (42), and (43) we obtain simply
i μναβ
¯ μν Γ abc αβ ξ̂ d +2ia1 μν
¯ μ γ a ν ξ̂ b .
δL = − abcd
ab
2
To compensate these variations one can use the following
corrections:
− 2. For the last possibility we get
μνα
L = i(−24c2 a1 + 6c4 ) abc ωα da Dμ ψ̄ν Γ bcd φ
μνα
+i(16c0 b2 − 12c2 a1 ) abc Dν ωα ab ψ̄μ γ c φ
μνα
−i(16c0 b3 − 12c2 a1 ) abc Dν ωα dc ψ̄μ Γ abd φ.
Vertex
3
2
−
1
2
The first term drops out due to the relations among the coefficients, the last term vanishes on-shell for the gravitational
field, and the second term can be removed by the field redefinition
h μ a → h μ a + κ2 ψ̄μ γ a φ.
As a result, the expression (41) is the final form for
Lagrangian of coupled spin-2 and spin-3/2 fields in AdS4
space including the cubic interaction vertex. The parameters
c1 , c2 , c3 are fixed in the free gravitational field Lagrangian
(27), the parameter c0 is given by (32). The parameters
c4 , c5 , and the parameters of the gauge transformations are
expressed through the single parameter a1 , which is the only
free parameter of the theory.
δ1 ψμ ∼ μν ξ̂ ν , δ1 φ ∼ μ ξ̂ μ .
Thus the requirement that the Lagrangian be gaugeinvariant fixes the coefficients c4,5 for the Abelian vertices
and also relates the coefficients b1 and c1 (which is just a
manifestation of the universality of the gravitational interaction). Note that by the Metsaev classification [11] in general
dimensions d > 4 we would have two non-minimal vertices
with two derivatives as well as the minimal one. But in d = 4
dimensions these two derivative vertices are absent (on-shell
and up to possible field redefinitions) as we will now show.
5 Conclusion
In this paper we have constructed the cubic vertices for a
massive spin-3/2 field coupled to electromagnetic and gravitational fields in AdS4 space. The corresponding vertices are
gauge-invariant due to the presence of Stueckelberg auxiliary
fields and contain a number of free parameters. The results
are given by the expressions (12) or (26) for the electromagnetic interaction and (41) for the gravitational interaction.
123
3153 Page 8 of 9
The construction of the vertices is based on a generalization of the Fradkin–Vasiliev formalism [25,26] where
the main building blocks of the Lagrangians are the gaugeinvariant curvatures. Although this formalism was known
only for massless higher spin theories, we have shown, for
the example of a spin-3/2 field, that the gauge invariant curvatures can in principle be constructed for massive higher
spin fields as well. Certainly the case of massive fermionic
fields appears to be the most technically involved one. Nevertheless it seems worth to apply this formalism to massive
fermionic fields with spins higher than 3/2.
As we pointed out, the constructed vertices contain some
number of arbitrary parameters (two for the electromagnetic
coupling and one for gravitational coupling) which cannot
be fixed only by gauge invariance. In principle one can hope
that the additional constraints for those parameters can appear
from the requirements of causality. The possibility of such
constraints was already demonstrated in the papers [17,20].
Acknowledgments I.L.B and T.V.S are grateful to the grant for LRSS,
project No. 88.2014.2, RFBR grant, project No. 12-02-00121-a and
RFBR-Ukraine grant, project No. 13-02-90430-a for partial support.
Their research was also supported by grant of Russian Ministry of Education and Science, project TSPU-122. T.V.S acknowledges partial support from RFBR grant, project No. 14-02-31254. Work of Yu.M.Z was
supported in part by RFBR grant Nj. 14-02-01172.
Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited.
Funded by SCOAP3 / License Version CC BY 4.0.
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W2283272671.txt | (IJACSA) International Journal of Advanced Computer Science and Applications,
Vol. 6, No. 10, 2015
Design of ANFIS Estimator of Permanent Magnet
Brushless DC Motor Position for PV Pumping
System
TERKI Amel
MOUSSI Ammar
TERKI Nadjiba
Genie Electric department
Biskra University
Biskra, Algeria
Genie Electric department
Biskra University
Biskra, Algeria
Genie electric department
Biskra University
Biskra, Algeria
Abstract—This paper presents a new scheme for PMBLDC
(permanent magnet brushless direct current) rotor position
estimation based an ANFIS (adaptive network fuzzy inference
system) estimator. The operation of such motor requires accurate
rotor position knowledge. However, most of rotor position
sensors produce undesirable effects such as mechanical losses
and have other disadvantages. In order to overcome the
disadvantages, sensorless scheme seems to offer great
advantages. This work present an ANFIS estimator design.
Combining the adaptive capability of the neural network to
gather with the reasoning ability of the fuzzy logic in ANFIS
modeling results in a fast responding and flexible model. This
procedure lends itself perfectly adapted for complex system such
as PV pumping systems.
Keywords—Photovoltaic system; Brushless DC motor; ANFIS
estimator; Speed controller
I.
INTRODUCTION
In early1980s, DC motors are widely used for PV pumping
applications either with or without intermediate converters [1].
Starting, steady state, and transient performances of solar
power fed DC motors for linear and centrifugal pump load
torques were analyzed and showed a perfect matching [2].
However these motors a bulky, and require frequent
maintenance. Nowadays solar power fed permanent magnet
brushless DC (PMBLDC) motors were being used instead [2].
PMBLDC machine is more popular due its simple
structure and low cost [1, 3]. These machines have the
advantages of light weight, small size, simple mechanical
construction, easy maintenance, good reliability, and high
efficiency [1, 3, 4, 5]. In general, the motors are equipped with
mechanical position detecting devices to provide proper
commutation for power devices in the bridge inverter [6].
These mechanical devices, such as Hall Effect sensor, optical
or inductive sensor produce undesirable effects such as
mechanical losses and also have other disadvantages like
change in mechanical design and requirement of maintenance.
In order to overcome these drawback, there is a need to
develop a sensorless scheme for estimating rotor position.
In recent years, many sensorless drive methods have been
proposed. Of these, the most popular one in the back emf
based Filter (EKF) is used [7]. This method provides excellent
speed response but requires heavy online matrix computing.
An offline FEM assisted position and speed observer has also
been studied in the literature [8]. Zero crossing of line to line
PM flux linkage is used for estimating the speed and position
[9]. Flux Linkage Observer (FLO) based on the integration of
back EMF, with a simple start-up method is proposed [10].
In this paper the design of an ANFIS estimator of
permanent magnet brushless DC motor in PV pumping system
is presented. The Adaptive Neuro Fuzzy Inference System
(ANFIS), is a neural network that is functionally the same as a
Takagi–Sugeno type inference model. The ANFIS is a hybrid
intelligent system that takes advantages of both ANN and
fuzzy logic theory in a single system, by using the ANN
technique to update the Takagi–Sugeno type inference model
parameters. In order to explain the concept of ANFIS
structure, five distinct layers are used. The first layer in the
ANFIS structure is the fuzzification layer; the second layer
performs the rule base layer; the third layer performs the
normalisation of membership functions (MFs); the fourth and
fifth layers are the defuzzification and summation layer
respectively [11].
This rest of the paper is organized as follows. In section 2,
the configuration of PV pumping systems is presented, The
Adaptive network fuzzy inference system estimator is
described in section 3. Section 4 shows the experimental
performance of Adaptive network fuzzy inference system
estimator. Finally, Section 5 concludes our contribution and
merits of this work.
II.
GENERAL SYSTEM LAYOUT
The full system mainly consists of the solar cell array
generator, DC/DC converter with MPPT (maximum power
point tracker) command, PMBLDC motor with its bridge
inverter coupled to a centrifugal pump load. The motor is
controlled through a hysteresis current loop and an outer speed
with PI type controller as shown in fig1.
136 | P a g e
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(IJACSA) International Journal of Advanced Computer Science and Applications,
Vol. 6, No. 10, 2015
Photovoltaic
Generator
DC/DC
PMBLDC
Inverter
Pump
ia=-(ib+ic)
MPPT
i a*
⍵ref
+
e
Speed
reference
Current
generator
Tref
controller
-
ib*
fem
hysteresis
Current
controller
3
ia
ib
ic
ic *
⍵
ᶱ
d/dt
ANFIS
Estimator
Fig. 1. Overall system configuration
Vbn Rib pb eb
A. PV generator model
The characteristic of the Photovoltaic generator can be
presented by the following nonlinear equation [4]:
I I sc I o [exp(
(V Rs I )
(V Rs I )
) 1]
Vth
Rsh
(1)
Isc PV array short circuit current,
PV array reverse saturation current,
The flux expressions are given by the following
expressions:
Rs PV array series resistance,
Vth PV array thermal voltage.
The thermal voltage Vth and t reverse saturation current Io
are successively identified by [4]:
I o I sc
(Vop Rs I op )
I oP exp
Vrh
(2)
(
)
(10)
(
)
(11)
(
)
(12)
Where Ls: the self-inductance and M: the mutual
inductance.
And
(13)
(3)
B. Permanent magnet Brush-Less DC motor model
The simplified schematic of PMBLDC motor who has a
trapezoidal electromotive force, the use of Park transform is
not the best approach in modelling the machine. Instead the
natural approach in used where the emf is generated with
respect to rotor position [4]. The operating sequences of the
machine can be subdivided into six cycles with respect to rotor
position as shown in table II.
Therefore by substituting Eq.13 in Eqs 10, 11 and 12:
(
)
(14)
(
)
(15)
(
)
(16)
From the electrical equations 4, 5 and 6, the following
system is obtained
[
]
[
][ ]
[
][ ]
[ ] (17)
With Leq = LS –M
From this system, the decoupled phase equations are
obtained and the explicit current equations are given by:
The electric of the motor can be described by [4]
Van Ri a pa ea
(7)
(8)
Vcn Vc0 Vn0
(9)
Where R: per phase stator resistance. ia,b,c and a,b,c are
respectively phase currents of phases a, b and c and total flux
linkage of a,b and c. p: Laplace operator.
Rsh PV array equivalent shunt resistance,
(Vop R s I op Voc )
I op
log(1
)
I sc
Van Va0 Vn0
Vbn Vb0 Vn0
IPV Array output current,
Vth
(6)
With
Where:
Io
(5)
Vcn Ric pc ec
(4)
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(IJACSA) International Journal of Advanced Computer Science and Applications,
Vol. 6, No. 10, 2015
⁄
⁄
[ ]
[
[[
⁄
]
[
][ ]
[ ]]
(18)
]
The mechanical part is expressed by the following
equation:
(19)
With:
Te: electromagnetic torque.
Tr: Load torque
: speed
J : moment of inertia
B : viscose friction coefficient
Neglecting the frictional coefficient and taking
where P is the pole pairs number, (19) can written as:
⍵
(
)⁄
⍵
( ) ⍵ ( ) ⍵( )
( )
( )
(
)
The value of the torque reference is given by [4]:
( )
(
)
( )
( )
(25)
) the speed error of previous interval is,
Where (
( ) is the speed error of working interval.
and
are
speed controller gains.
D. Current control
Several techniques can be used to control the phase current
of the PMBLDC motor. In this paper a hysteresis current
controller is used. It has the major advantage of not requiring
machine parameters to be known. However the commutation
frequency is not constant [4]. It depends on many factors such
as the applied voltage, the back emf, hysteresis band ...etc.
(20)
) ⁄⍵
(21)
And the angular position is expressed by
⍵
(23)
(24)
Maximum value of commutation frequency is obtained at
starting and is given by [4]:
The developed torque can be expressed by
(
compared with the reference speed ⍵ ( ) and the resulting
error is estimated at the nth sampling instant as:
(22)
(26)
The commutations are obtained by comparing actual
currents ia.b.c to a rectangular reference i*a.b.c and by keeping
them in hysteresis band . The commutation sequences of
switches are summarised in the table I. [5].
TABLE I.
THE CMMUTATION SEQUENCES OF SWITCHES
*
Si ia (ia
I )
T1 on
T4 off
Va= U/2
* I )
Si ia (ia
Si i (i* I )
b
b
Si i (i* I )
b
b
*
Si ic (ic I )
Si ic (ic* I )
T1 off
T4 on
Va= -U/2
T2 on
T5 off
Vb= U/2
T2 off
T5 on
Vb= -U/2
T3 on
T6 off
Vc= U/2
T3 off
T6 on
Vc= -U/2
E. Pump model
A centrifugal type is used. It can be described as an
aerodynamic load which is characterised by the following load
equation [4]:
Fig. 2. Back e.m.f and current waves forms for phase a, b and c
C. Speed control
PI speed controller is widely used in industry due to its
ease in design and simple structure. The rotor speed ⍵( ) is
(27)
Where A is the pump constant
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TABLE II.
III.
OPERATING SEQUENCES WITH RESPECT TO ROTOR POSITION
ANFIS ESTIMATOR
The adaptive network fuzzy inference system play a
significant role in the field of artificial intelligence. It
combines the advantages of a fuzzy controller as well as quick
response and adaptability nature of artificial neural network
[11]. Hence using this scheme one can avoid the use of a
position sensor which produces mechanical losses in the
motor. Different configurations of adaptive network fuzzy
inference system have been tested and the best one that gives
the minimum error value is presented in fig6.
The Algorithm of the model structure was constructed as
shown in Fig.3. The membership functions were obtained
from the data set of the back emfs which were first normalized
for loading Data. Then generate Fis, built on sugeno structure,
which is obtained by creating of input membership function:
number (tree inputs) & type (triangular) and output
type(linear). The choice of optimization method (hybrid) is
obtained by Train Fis. If the results are satisfactory (error is
minimum) after test Fis, denormalized procedure is done and
go to application thereafter. The ANFIS structure consists of 5
layers of neurons, each of which having a very specific
behavior.
Original Data
Normalization
Load Data
(testing, training, checking)
Generate Fis
Input: 1. Number of Mfs
2. type of Mf
Output: Mf type
Train Fis
Optimization Methode
Epoch number: error
Test fis
From these, second, third and fifth layer have constant
behavior, while layers 1 and 4 have varying parameters.
Layer 1: consists of five adaptive neurons in which the
fuzzification is performed, that is: the grade of membership to
the defined membership functions of the input is evaluated.
f i1 µ Ai (x)
No
yes
Denormalization
(28)
Where x is the input to ith neurons and Ai is membership
function correspond to variable x.
Application
Fig. 3. Algorithm of the ANFIS model
The membership function µAi (x) is triangular.
Layer2: consists of 125neurons, each node output
represents the firing weight of a rule gives than:
Wk = µ Ai (x) µ Bj (y)
Minimum
Error
(29)
Layer3: consists of 125 neurons, every node calculates the
ratio of every rule firing weight to the sum of all rule firing
weights.
k : represent the number of rule, i : represent the number of
x partition and j : number of y partition.
Wk
Wk
Wi
(30)
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Layer 4: consists of 125 neurons. Nodes in layer 4 are
adaptive nodes in which the consequent evaluation inference is
calculated, its output is defined as:
f k4 Wk f k Wk pk x qk y rk (31)
Wk is the output of layer3 and pi , qi , ri is called
consequent parameters.
5
Wk f k4
(32)
k
IV.
150
100
Eab(v)
50
0
-50
-100
Layer5: Finally, is single node in layer 5, where sums all
the outputs from 4th layer to compute the overall output of the
network:
f
200
SIMULATION RESULTS
The entire ANFIS network architecture is represented in
the Fig.6. The inputs are the three line to line back emfs of the
motor namely Eab, Ebc and Eca Fig4. And the desired output
is the estimated rotor position angle. For producing the above
relationship with least possible error, a five layer feed forward
adaptive network fuzzy inference system is used. The input
layer consists of five neurons that have their inputs as the three
line to line back emfs, the three hidden layers consists of 125
each and the output layer consists of a single neuron whose
output is the estimated position of the rotor. The relationship
between the back emf and the position of the rotor is shown in
the Fig.5.
Fig.7 shows estimated rotor position by adaptative network
fuzzy inference system and the error, along with real rotor
position it’s a case without regulation. Fig.8 shows the
estimated rotor position by adaptive network fuzzy inference
system and the error, along with real rotor position the case is
with PI speed control. In the comparison of the cases with
control (PI regulator) and at without control, The controlled
system attains the steady state in (0.04s) greater than the case
without regulation the system attains the steady state
in(0.023s), so the PI regulator slows the dynamic responses.
The performance of thThe performance of the trained
adaptive network fuzzy inference system is found to have very
minimal errors witch is about 1.303e-7 as seen in the Fig.7
(without control).
-150
-200
0
1
2
3
4
Angle teta (rd)
5
6
7
Fig. 5. The back emf Eab
For Fig.8 (with PI controller), the error is 7.8386e -9. The
error of output of ANFIS and real rotor position sensor are
compared through Fig.7 and Fig.8 respectively. It is clear that
the error in Fig.8 is too small (7.8386e -9) than these in Fig.7
(1.303e-7). The proposed scheme works efficiently where the
usage of neural network topology together with fuzzy logic in
The adaptive network, ANFIS, not only includes the
characteristics of both methods, but also eliminates some
disadvantages of their lonely-used case.
Input1, MF
Int1 mf1
Eab
Int2 mf2
Aggregated
Output1
Ebc
teta
The back fems (V)
150
100
50
Eac
0
-50
Int3 mf5
-100
Rule 125
-150
0
0.02
0.04
0.06
0.08
Time (sec)
0.1
0.12
0.14
Output MF 125
Fig. 6. Structure of ANFIS model proposed
Fig. 4. The inputs curves
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Vol. 6, No. 10, 2015
la position réelle
erreur
7
0.06
6
0.04
5
erreur(rd)
teta(rd)
0.02
4
3
2
0
-0.02
1
-0.04
0
0
0.01
0.02
0.03
0.04
temp(sec)
0.05
0.06
0.07
0.08
-0.06
0
0.02
0.04
0.06
la position estimée
7
0.08
0.1
0.12
temp(sec)
0.14
0.16
0.18
Fig. 8. Simulation Results with PI controller
6
5
V.
teta(rd)
4
3
2
1
0
-1
0
0.01
0.02
0.03
0.04
temp(sec)
0.05
0.06
0.07
0.08
erreur
0.05
0.04
0.03
0.02
erreur(rd)
0.01
CONCLUSION
The developed ANFIS models were successful in
estimating rotor position of PMBLDC motor used in PV
pumping system. In this paper the design of adaptive network
fuzzy inference system based sensorless scheme was
developed for permanent magnet brushless DC motor. The
proposed scheme works efficiently for both with control (PI
regulator) and at without control. It was found that the error is
very minimal and this is smaller in system with regulation,
than these without regulation. Also adaptive network fuzzy
inference system based estimator is very flexible.
0
-0.01
-0.02
-0.03
-0.04
-0.05
0
0.01
0.02
0.03
0.04
temp(sec)
0.05
0.06
0.07
0.08
Fig. 7. Simulation Results without control
la position réelle
This scheme not only eliminates the position sensor,
thereby cutting the cost but also transforms the drive into a
highly efficient drive by eliminating losses caused by position
sensors. The estimation error is found to be very minimal
proving that the developed system is a very efficient and a
reliable one.
[1]
7
6
[2]
teta(rd)
5
4
[3]
3
2
1
[4]
0
0
0.02
0.04
0.06
0.08
0.1
0.12
temp(sec)
0.14
0.16
0.18
0.2
la position estimée
[5]
7
6
teta(rd)
5
[6]
4
3
[7]
2
1
0
0
0.02
0.04
0.06
0.08
0.1
0.12
temp(sec)
0.14
0.16
0.18
0.2
REFERENCES
A.Moussi, A. Saadi, A. achour, Greg Asher, Photovoltaic pumping
systems technologies trends, Larhyss journal ,2003,pp.127-150.
PackiamPeriasamy, N.K.Jain,I.P.Singh, A review on development of
photovoltaic water pumping system, Renew.and Sustainable.Energ.
Reviews, Vol. 43 ,2015, pp. 918–925.
MetinDemirtas,AslanDenizKaraoglan, Optimization of PI parameters
for DSP-based permanent magnet brushless motor drive using response
surface methodology, Energy Conversion and Management, Vol. 56,
2012, pp. 104–111.
A. Terki, A. Moussi, A. Betka, N. Terki, An improved efficiency of
fuzzy logic control of PMBLDCfor PV pumping system,
Appl.Math.Model , Vol. 2, 2012, pp. 934-944.
A.Moussi, A.Terki, Greg Asher, Hysteresis current controller of
permanent Magnet Brushless DC motor PV pumping system, ASME,
2005, pp. 523-528 [International Solar Energy conference].
T. Hiyama, Neural network based estimation of maximum power
generation from PV modules using environmental information, IEEE
Trans. EC, Vol.12, 199, pp. 241–247.
A. Ungurean, V. Coroban-Schramel and I. Boldea, Sensorless control
of a BLDC PM motor based on I-f starting and Back-EMF zero-crossing
detection, OPTIM ’10, 2012, pp. 377-382 [Optimization of Electrical
and Electronic Equipment Conference,OPTIM ’10.IEEE 12th
International].
141 | P a g e
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0.2
(IJACSA) International Journal of Advanced Computer Science and Applications,
Vol. 6, No. 10, 2015
[8]
[9]
AlinStirban, Ion Boldea, and Gheorghe-DanielAndreescu, MotionSensorless Control of BLDC-PM Motor With Offline FEM-InformationAssisted Position and Speed Observer, IEEE Trans. Ind. Appl, Vol. 48,
2012, pp. 1950-1958.
LiviuIoanIepure, Ion Boldea and FredeBlaabjerg, Hybrid I-f starting and
observer-based sensorless control of single phase BLDC-PM Motor
drives, IEEE Trans. Ind. Electron, Vol.59, 2012,pp. 3436 – 3444.
[10] SreepriyaR&RagamRajagopal, Sensorless Control of Three Phase
BLDC Motor Drive with Improved Flux observer, ICCC, 2013, pp.292297 [IEEE International Conference on Control Communication and
Computing].
[11] Ali M. Abdulshahed, AndrewP. Longstaff, Simon Fletcher, Alan Myers,
Thermal error modelling of machine tools based on ANFIS withfuzzy cmeans clustering using a thermal imaging camera, Appl. Math. Model,
Vol. 39, 2015, pp. 1837–1852.
APPENDIX
The PV generator, motor and pump used in this study have the following
parameters: PV generator Modules AEG-40.
(Temperature T=25°Cand solar insolation E=1000W/m².)
Open circuit voltage
22.40 V
Short circuit current
2.410 A
Series resistance
0.450
Current temperature coefficient
0.06%/ C
Voltage temperature coefficient
0.40%/ C
Centrifugal pump
Rated speed
3000 rev/min
Rated power
521 W
Flowrate
2.597 l/s
Head
14.11 m
Efficiency
69%
Brushless DC motor
Rated power
690 W
Rated speed
3000 rev/min
Rated voltage
200-220V
Rated current
4.8 A
Per phase resistance
1
Per phase inductance
5 mH
Poles number
6
E.m.f constant
0.47
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| 3,291 | 19,730 |
W4378737784.txt | ISSN:2349- 0012
Vol..2, Issue 5
ИССЛЕДОВАНИЯ ПРОБЛЕМ ОХРАНЫ ТРУДА РАБОТНИКОВ
СЕЛЬСКОХОЗЯЙСТВЕННЫХ ОТРАСЛЕЙ
Чориев Ислом Нормуминович
ассистент
Каршинский инженерно-экономический институт
кафедра «Экология и охрана труда»
Республики Узбекистан
Аннотация: В данной статье рассмотрены уровень подготовленности
по охране труда руководителей, фермеров и других работников в сфере
сельского хозяйства. А также проведено исследования несчастные случаи и
травмы, возникающие при выращивании сельскохозяйственной продукции и
в перерабатывающей промышленности, определяются причины травм и
меры по их устранению.
Ключевые слова и фразы: Сельское хозяйство, охрана труда,
несчастные
случаи,
сельскохозяйственной
продукции,
травмы,
повреждения, опасные факторы, вредные факторы, перерабатывающей
промышленности, химические элементы, биологические вещества,
RESEARCH OF PROBLEMS OF LABOR PROTECTION OF
WORKERS IN AGRICULTURAL INDUSTRIES
Choriyev Islom Normuminovich
assistant
Karshi Engineering and Economic Institute
539
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department "Ecology and labor protection"
Republic of Uzbekistan
Annotation: This article discusses the level of preparedness for labor
protection of managers, farmers and other workers in the field of agriculture, a
special assessment of jobs, as well as accidents and injuries that occur when
growing agricultural products and in the processing industry, determines the
causes of injuries and measures to their elimination.
Key words and phrases: Agriculture, labor protection, accidents, injuries,
damages, agricultural products, processing industry, dangerous factors, harmful
factors, chemicals, biological elements, machinery and equipment.
Актуальность
темы:
Сельское хозяйство
является одной
из
важнейших отраслей, определяющих уровень жизни населения страны, его
благосостояние
и
обеспечение
продовольственную
безопасность.
Сельскохозяйственная продукция перерабатывается во многих отраслях
народного хозяйства (производство соков, табака, кофе, шоколада, сушка
фруктов и др.). В то же время многие промышленные объекты
(машиностроение, нефтегазовое, производство удобрений и др.) работают на
сельское хозяйство. Получается, что прямо или косвенно в сельском
хозяйстве заняты сотни тысяч инженеров и рабочих.
Особое
место
в
обеспечении
занятости
населения
занимает
сельскохозяйственная отрасль. В сельскохозяйственном секторе занято 3,7
млн населения республики, 27,2% из всех занятых (рис-1).
540
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Рисунок -1. Доля занятых в сельском Рисунок-2.
хозяйстве, % (2021 г.)
труда
в
Производительность
сельском
хозяйстве
в
долларах США (2021 г.)
Из этой диаграммы видно, что, несмотря на то, что уровень занятости
населения значительно выше, чем в развитых странах, экономические
показатели находятся на очень низком уровне. Это и есть модернизация
сельского хозяйства, охрана труда работников, занятых в отраслей ,
создание для них достаточных условий на рабочем месте, оплата и
поощрение их труда, защита работников от отравления удобрениями и
ядохимикатами.
вопросов
используется
обеспечения
для
средства
сельскохозяйственных
индивидуальной
культур,
защиты
а
требуют
рассмотрения.
Кроме того, в отраслях также задействованы тысячи сложной тяжелой
техники, опасного технологического оборудования и информационнокоммуникационных технологий. Поэтому сельское хозяйство является
основной отраслью экономики страны со сложной системой. Техногенные
аварии в такой ситуации неизбежны. Также не оставляют своего влияния
первичные
541
и
вторичные
факторы
опасных
природных
явлений
MAY
ISSN:2349- 0012
Vol..2, Issue 5
(геологические, гидрометеорологические, зпизотические, эпифитные). В
таких условиях среди работников могут фиксироваться различного рода
несчастный случай, травмы, отравления и смерти. Кроме того, известны
случаи отравления удобрениями, используемыми в сельском хозяйстве, и
химикатами, используемыми для борьбы с вредителями.
Получается, что все вышеизложенное показало, насколько актуален
вопрос
анализа
охраны
труда
и
техники
безопасности
для
агропромышленного комплекса.
Целью данной статьи является анализ законодательства об охране
труда в сфере сельского хозяйства на предмет его актуальности, подготовка
выводов и предложений на основе анализа.
Исходя из цели статьи, рассматриваются следующие задачи:
- анализ проблем применения законодательства об охране труда в
сфере сельского хозяйства;
-анализ несчастных случаев, происшествий, травм, повреждений и
профессиональных
заболеваний,
произошедших
в
сфере
сельского
совершенствованию
методики
хозяйства;
-разработка
предложений
по
мероприятий в области охраны труда в сфере сельского хозяйства и другие
задачи определены.
Основная часть: Сельскохозяйственный сектор обширен и является
одним из основных звеньев экономической сети страны, а также
локомотивом, который тянет за собой промышленность страны. Безусловно,
важно отметить специфические черты, определяющие экономические и
организационно-правовые отношения по управлению столь сложной и
широкий экономической системой.
1) наряду с экономическими законами, который управляет сельским
хозяйствым отраслей, существуют и независимые от человека естественные
542
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(биологические) законы (погодные условия, эпидемиологическая ситуация,
загрязнение окружающей среды и др.)
2) это также существенно в связи с ограниченной возможностью
неукоснительного
соблюдения
трудовых
отношений
и
трудового
законодательства в сельскохозяйственных условиях;
3) земледелие представляет собой среду, в которой основное
производство территориально ограничено, не может быть ничем заменено и
имеет свойство повышать производительность только при правильном его
использовании;
4) использует в сельском хозяйстве такие средства, как радиационнохимические соединения, опасные для живых организмов;
5) производство сельскохозяйственной продукции рассредоточено на
больших территориях с различными природно-климатическими условиями;
6) производственные процессы в сельском хозяйстве и конечные
результаты труда могут не совпадать;
7) созданный продукт часто является промежуточным продуктом и
перерабатывается в сельском хозяйстве;
8) занятость в сельском хозяйстве носит сезонный характер;
9) отсутствие постоянных рабочих мест (рабочих мест) у большинства
занятых в сельском хозяйстве.
10) большинство работников, работающих в сфере сельского
хозяйства, имеют среднее образование и не обладают достаточными
знаниями о мире, что влияет на соблюдение трудового законодательства.
Реализация вышеуказанных мер требует правового регулирования
трудовых отношений по таким признакам в сельском хозяйстве, разработки
и применения правил охраны труда, защиты прав работников в этой сфере.
Известно, что сельское хозяйство является отраслью, которая
поставляет и перерабатывает продукты питания населению. Создание
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кластеров, терминалов, транспортно-логистических систем для обеспечения
населения
продовольственными
товарами,
статистические
данные
последних 10 лет показывают, что среднегодовой показатель составляет
9...10%. Также с каждым годом увеличивается объем экспорта продукции. В
связи с этим в нашем государстве приняты важные указы и решения,
государственные
программы,
последовательно
обеспечивается
их
выполнение. В частности, согласно постановлению Президента Республики
Узбекистан
от
14
марта
2019
года
«О
мерах
по
развитию
сельскохозяйственной кооперации в области плодоовощного производства»
в Ферганской, Андижанской и Наманганской области в 2020 году и 100 в
2021 году по стране за счет средств международных финансовых
институтов.
Сегодня в 55 районах нашей республики эффективно работают
кластеры, специализирующиеся на производстве плодоовощной продукции.
К
этим
кооперативам
и
кластерам
присоединились
фермеры-
сельхозпроизводители, реализующие свою продукцию на внутреннем и
внешнем рынках, эффективно использующие земельные и водные ресурсы,
модернизирующие
техническое
оснащение.
Наряду
с
повышением
урожайности сельскохозяйственных культур находится решение проблем
охраны
труда
работников.
Это
обеспечивает
снижение
динамики
несчастных случаев в сфере сельского хозяйства.
По
данным
Государственной
службы
статистики,
количество
пострадавших в сельскохозяйственных организациях за последние пять лет
уменьшилось на 48,0% (рис- 3). Но этого показателя недостаточно.
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Рисунок -1. Динамика сокращения численности сельскохозяйственных
рабочих и травматизма в результате несчастных случаев
Именно поэтому управление охраной труда в аграрном секторе имеет
особое значение. Однако если действия в этом направлении осуществляются
только в одностороннем порядке, не может быть достигнута достаточная
безопасность работников при исполнении служебных обязанностей: в этом
случае государственные органы, органы местного самоуправления и
негосударственные органы, работники и работодатели должны соблюдать
действующие законы , стандарты, правила и требования.
Высокий уровень травматизма и возникновения профессиональных
заболеваний среди сельскохозяйственных рабочих связан с уровнем
воздействия опасных и вредных факторов. Их можно разделить на
следующие группы:
- факторы, связанные с работой машин и механизмов.
- неблагоприятные природно-климатические факторы.
-необходимость контакта с удобрениями, пестицидами, опасными
химическими и биологическими соединениями, используемыми в сельском
хозяйстве;
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-риск
заражения
опасными
заболеваниями
при
контакте
с
нездоровыми животными;
-высокий риск для здоровья или жизни в результате укусов (укусов)
диких животных (насекомых, грызунов, рептилий) в полевых условиях;
-высокий
потенциальный
риск,
связанный
с
необходимостью
выполнения определенной работы в часто движущихся механизмах;
-соблюдение законодательства об охране труда и технической
безопасности, следовательно, низкий контроль в области внутреннего
порядка и дисциплины;
- специфика сельскохозяйственных работ, в том числе высокая
степень тяжести и напряженности работ, связанная с их сезонностью;
- другие факторы.
В целях минимизации негативного влияния на профилактику такого
травматизма и профессиональных заболеваний в сельском хозяйстве особое
внимание следует уделять требованиям, установленным законодательством.
Конечно, в сельском хозяйстве, как и в других сферах, общие вопросы
организации безопасного труда регулируются нормами Трудового кодекса.
Однако соблюдается и приказ Минтруда от 27 октября 2020 г. № 746 «Об
утверждении правил охраны труда в сельском хозяйстве», учитывающий
специфику труда для сельского хозяйства.
Охрана труда в сельском хозяйстве включает следующие направления:
- организация рабочего процесса. Соблюдение этих требований
должно находиться под контролем работодателя;
- требования к работникам;
- дополнительные требования к отдельным видам работ.
Требования
к
организации
рабочего
процесса
устанавливает
работодатель. В целях минимизации вреда, который может быть нанесен
работникам под воздействием негативных факторов, работодатель обязан
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приложить все усилия для повышения уровня автоматизации и механизации
рабочих процессов, при наличии опасных химических соединений и других
угроз, использовать методы работы, исключающие непосредственный
контакт с такими угрозами, или к работе допускается только специально
обученный персонал.
Работники, вступающие в контакт с машинами, химическими
соединениями и другими опасностями, представляющими опасность для их
здоровья в связи с выполнением служебных обязанностей, должны быть
обеспечены средствами индивидуальной и коллективной защиты за счет
средств работодателя.
Оборудование, предоставляемое работодателем для выполнения
определенного
работодатель
задания,
должен
должно
быть
обеспечивать
в
исправном
работникам
состоянии,
разумное
а
сочетание
режимов труда и отдыха. По собственной инициативе работодатель может
вводить
дополнительные
нормы
в
области
охраны
труда,
не
противоречащие требованиям действующего законодательства.
Работники
обязаны
соблюдать
требования
охраны
труда
в
агропромышленном комплексе. В частности, они несут ответственность за:
- своевременное прохождение учебных занятий по охране труда в
соответствии с графиком, утвержденным работодателем;
- выполнение должностных инструкций, в том числе по организации
труда и использованию оборудования;
- правильное использование средств индивидуальной и коллективной
защиты соотрудниками;
- сообщать непосредственному вышей организацию о нештатных
ситуациях, возникающих в процессе работы.
По данным ряда зарубежных исследователей, работники сельского
хозяйства подвергаются большему риску, чем большинство работников
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других отраслей. (тяжелый физический труд, повышенный уровень шума и
вибрации, неблагоприятные микроклиматические условия, различные
химические и биологические вещества). Кроме того, несчастные случаи с
участием аварии тракторов и прицепов являются причиной большинства
несчастных случаев с участием сельскохозяйственных животных и полевых
работ.[4] Но, в силу ряда объективных и субъективных причин, часто
случающиеся практически не обрашается внимание на присходящехся на
несчастных случаев, а статическая отчетность не находится под строгим
контролем. например других отраслях строго провирается и проводит
анализ,
Среди отраслей сельского хозяйства наиболее опасной отраслью
является животноводство и растениеводство, в которых в 2021 году
зафиксировано в 1,7 раза больше смертельных травм. Факторами,
вызывающими гибель и тяжкие телесные повреждения работников,
являются:
- отсутствие препятствий в подвижных частях оборудования;
- отсутствие средств защиты или они не в рабочем состоянии;
- неисправность машин, механизмов, инструментов и приспособлений;
- создание рабочих мест, не отвечающих требованиям охраны труда;
- низкий уровень обучения и инструктажа работников по безопасным
методам работы, организации и контроля за соблюдением требований
охраны труда и трудовой дисциплины;
При
возделывании
сельскохозяйственных
культур
чаще
всего
получают травмы водители сельскохозяйственных тракторов, 74,0 % от
общего числа пострадавших и 16,5 % подсобных рабочих (рис-4).
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Рисунок- 3. Самые вредные профессии в растениеводстве
Снижение уровня травматизма и профессиональных заболеваний в
сельском хозяйстве является одной из приоритетных задач в области охраны
жизни и здоровья работников сельского хозяйства.
Человечество существует, потому что его основная деятельность
состоит из труда. Однако сохранение жизни, здоровья и трудоспособности
работников в процессе труда не происходит само по себе, это требует
формирования умения работать с соблюдением правил охраны труда и
техники безопасности.
По
результатам
многочисленных
исследований
причин
производственного травматизма и острых профессиональных заболеваний
все виды можно разделить на три основные группы.
Первая группа причин: связанные с неправильными действиями
непосредственного исполнителя в определенном трудовом процессе.
Вторая группа причин: связанные с неправильной организацией
работы;
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Третья группа причин: связана с техническими, аппаратными,
приборами, неисправностями оборудования или внезапным отказом или
нестабильным течением технологических процессов.
Хронические профессиональные заболевания - возникают в результате
отсутствия доступа к безопасному технологическому оборудованию или
того, что это экономически нецелесообразно, а работники не используют
средства индивидуальной защиты и другие меры безопасности. Стоит
отметить, что не бывает техногенных аварий, причиной которых является
человек, в итоге попавший в ту или иную аварию. Потому что он «что-то не
сделал» или «сделал не так». либо является результатом неправильных
действий отдельных сотрудников.
Кроме того, не следует забывать, что безопасный труд включает в себя
физическую и психологическую подготовку работников к работе «по
правилам» и выполнению своих функций в соответствии с требованиями
охраны
труда,
при
достаточной
профессиональной
грамотности
и
осознанной мотивации.
Поскольку полностью исключить риски с помощью технических и
организационных мер абсолютно невозможно, безопасность работника
зачастую определяется только его поведением. При устранении и
предупреждении
причин
травматизма
(психологических
и
психофизиологических) это связано с подбором работников по их
профессиональным качествам, а также обучением и переподготовкой
работников, инструктажем и формированием их безопасного поведения. [10]
Мировой опыт показывает, что особое значение имеют требования
техники безопасности и охраны труда, обучение оказанию первой помощи
пострадавшим, а самое главное – своевременные профилактические
мероприятия.
В сельском хозяйстве анализ случаев травмирования показывает, что
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более 70% пострадавших не обучены вопросам охраны труда, не проходят
инструктаж по охране труда. Это следствие того, что многие производители
не объединены в крупные кластеры, логистические центры и корпорации и
холдинги. Потому что при регистрации НС и ПЗ не проводится проверка и
контроль по охране труда, а контролирующий орган не требует документов.
В результате исследований охрана труда в сельскохозяйственных
субъектах оставлена как есть, и в целом отмечается недостаток учебной
литературы и научно-исследовательских работ в этой области. Отсюда
следует, что в дальнейшем необходимо реформирование всей системы
сельского хозяйства на государственном уровне.
Практика исследований показывает, что многие несчастные случаи,
когда внешне легкие травмы ограничиваются оказанием первой помощи в
начальный период, в дальнейшем травмы становятся серьезными и
переходят в несчастный случай, закончившийся летальным исходом.
Сегодня
в
следственных
процессах
рассматриваются
только
несчастные случаи, повлекшие за собой временную или постоянную утрату
трудоспособности или смерть. Из общего количества несчастных случаев
2,5% могут быть расследованы контролирующими органами в соответствии
с законодательством, а остальные 75%, на практике, ни комиссия, ни акт Н-1
не выдаются, и к виновнику не принимаются меры. ответственные
сотрудники. По этой причине детальный анализ несчастных случаев в
подобных ситуациях не позволяет определить и в полной мере оценить
причинно-следственную связь в системе «профессиональная деятельность профессиональный риск» для ее предупреждения.
Все дела, связанные с гибелью работников, тяжелыми и групповыми
травмами, расследуются следственными органами, хотя они обладают
большим
объемом
исчерпывающей
информации
о
произошедших
несчастных случаях, условиях и причинах их возникновения, по порядку
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расследования квалифицированных специалисты в области охраны труда
несчастных случаев не имеют полномочий на проведение следственных
данных для анализа причин, в результате чего данные в полной мере не
расследуются и не обнародуются.
Действующие формы статистической отчетности дают возможность
вводить и систематизировать только ту информацию, которая необходима
для полного анализа причин и условий производственного травматизма. К
сожалению, он не подходит для сельскохозяйственных условий.
Одним
из
произошедших
способов
оценки
несчастных
случаев.
риска
травм
Конечно,
является
никто
не
оценка
может
гарантировать, что они не произойдут в будущем. Поэтому очень важно,
чтобы работодатели, отвечающие за предотвращение несчастных случаев на
таких производствах и внедрение эффективных профилактических мер и
техники безопасности, имели доступ к источникам информации.
Кроме того, работодатель должен убедиться, что предпринятые ранее
превентивные меры достаточны для снижения рисков. Однако если на
производстве
произошел
проанализировать
несчастный
причины,
по
случай,
которым
работодатель
должен
профилактические
меры
недостаточны для предотвращения несчастных случаев.
На основании вышеизложенных фактов можно сделать следующий
вывод.
В сельском хозяйстве основная работа в хозяйстве носит сезонный
характер, и многие работы выполняются с помощью наемного труда. С этой
точки зрения, фермеры, дехканские хозяйства, кластеры, логистические,
терминальные и другие субъекты, занятые в сельском хозяйстве, не имеют
четкой государственной единицы, что требует обучения работников
управлению
охраной
законодательством
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ознакомления
руководящими
с
установленными
принципами,
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медицинский вызывает ряд проблем, таких как осмотр.
Большинство
работников,
работающих
в
сельском
хозяйстве,
являются сельскими жителями, а руководители также имеют среднее
образование, поскольку до сих пор в сельскохозяйственных отраслях не
было высшей оброзавательных курсов по управлению сельским хозяйством
или по охране труда и технике безопасности. это касается и области охраны
труда в сельском хозяйстве.
Еще одной причиной появления вышеперечисленных объективных и
субъективных причин, влияющих на обеспечение охраны труда и техники
безопасности в сельском хозяйстве, является нехватка молодежи, способной
работать в сельском хозяйстве, и негативные демографические тенденции.
По этой причине задачи нашего правительства по разработке эффективных
механизмов занятости молодежи в сельском хозяйстве и созданию
безопасных условий труда остается одним из приоритетных направлений
социально-экономического развития республики.
Таким
образом,
основными
направлениями
совершенствования
охраны труда в сельском хозяйстве являются:
- непрерывное продолжение научно-исследовательской работы в
области охраны труда работников сельскохозяйственного производства и
перерабатывающей промышленности и совершенствование нормативных
правовых документов;
- активизация оценки труда в сельскохозяйственных секторах;
-формирование знаний работодателей в области охраны труда путем
повышения качества и количества подготовки руководителей хозяйств и
специалистов по вопросам охраны труда;
-контроль условий труда и охраны труда в сельскохозяйственных
организациях, информирование работников о результатах мероприятий по
улучшению условий труда;
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-совершенствование
системы
управления
охраной
труда
в
сельскохозяйственном производстве.
-уделить внимание вопросам охраны труда путем привлечения
научных
работников
образовательных
учреждений,
научно-
исследовательских учреждений к организации научно-исследовательских
работ по безопасности агротехносферы, безопасности жизнедеятельности и
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в
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местного
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возможность создания «Единой базы данных» несчастных случаев,
произошедших в сельском хозяйстве республики методом "передача снизувверх" этой информации, предсказывающей высокую частоту несчастных
случаев.
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W2576740365.txt | ONCOLOGY REPORTS 37: 1477-1486, 2017
Characterization of the microRNA profile in early-stage cervical
squamous cell carcinoma by next-generation sequencing
Wansong Lin1,2, Mei Feng3, Guilin Chen3, Zhifeng Zhou1,2, Jieyu Li1,2 and Yunbin Ye1,2
1
Laboratory of Immuno-Oncology, Fujian Cancer Hospital and Fujian Medical University Cancer Hospital;
2
Fujian Key Laboratory of Translational Cancer Medicine; 3Department of Gynecologic Oncology,
Fujian Cancer Hospital and Fujian Medical University Cancer Hospital, Fuzhou, Fujian 350014, P.R. China
Received July 6, 2016; Accepted September 1, 2016
DOI: 10.3892/or.2017.5372
Abstract. Squamous cell carcinoma (SCC) is histologically the
most prominent type of cervical cancer. There is accumulating
evidence suggesting that microRNAs (miRNAs) play important regulatory roles in the biological processes of cervical
squamous cell carcinoma (CSCC). Deciphering the miRNA
regulatory network in CSCC could deepen our understanding
at the molecular level of CSCC initiation and progression. In
the present study, we performed next‑generation sequencing
(NGS) to profile miRNA expression in 3 pairs of early-stage
CSCC samples. Quantitative real-time polymerase chain
reaction (qRT-PCR) was used to verify primary findings in
another 20 pairs of CSCC samples. We identified 37 known
miRNAs that exhibited significant alterations in expression
(2-fold change or greater), among which 8 miRNAs were
upregulated and 29 miRNAs were downregulated. Nine of
these miRNAs were selected for further qRT-PCR validation.
A novel miRNA candidate was also reported for the first time
in the present study to be upregulated. The Gene Ontology
(GO) analysis and Kyoto Encyclopedia of Genes and Genomes
(KEGG) pathway enrichment analysis revealed that its target
genes were involved in MAPK, calcium and adherent junction signaling pathways. The present study systematically
characterized the miRNA expression variation in early-stage
CSCC and provides novel biomarkers for diagnosis and treatment as well as an opportunity for further investigation of
the molecular mechanisms underlying the pathogenesis and
development of CSCC.
Introduction
In less developed countries, cervical cancer is the second most
common cancer and the third leading cause of cancer-related
Correspondence to: Dr Yunbin Ye, Laboratory of ImmunoOncology, Fujian Cancer Hospital and Fujian Medical University
Cancer Hospital, 420 Fuma Road, Fuzhou, Fujian 350014, P.R. China
E-mail: zjyunbin@sina.com
Key words: microRNA, cervical cancer, squamous cell carcinoma,
early-stage, next-generation sequencing
deaths in females. There were an estimated 444,500 new
cervical cancer cases and 230,200 deaths in these countries
in 2012 (1). Almost 90% of the deaths occurred in developing
countries and for these countries it is a huge economic health
care burden. The highest cervical cancer death rates are in
Asia, with an estimated 144,400 deaths occurring in 2012 (1).
Squamous cell carcinoma (SCC) is the most common histological type of cervical cancer (~80% of cases) and is closely
correlated with human papillomavirus (HPV) infection (2).
Cervical screening can prevent SCC, as it allows for detection
and treatment of precancerous lesions, and application of the
HPV vaccine also reduces the risk of SCC (3). However, the
high cost is a major barrier to making screening and vaccination widely available to all women in developing countries,
including China.
Cervical squamous cell carcinoma (CSCC) cases that
are classified as International Federation of Gynecology and
Obstetrics (FIGO) stages IB and IIA have a greater possibility
of being cured and patients have better prognosis (4). Since early
detection results in a better prognosis and cervical screening
and vaccines are extremely expensive, there is an urgent need to
identify more efficient early diagnostic biomarkers and therapeutic targets for CSCC. Therefore, it is necessary to augment
our understanding of the molecular mechanisms underlying the
pathogenesis and development of CSCC.
Although previous studies have identified chromosomal
variations, abnormal expression of oncogenes or tumorsuppressor genes and aberrant promoter methylation in CSCC,
few are relevant to microRNAs (miRNAs). miRNAs are a class
of small non-coding RNAs (ncRNAs) ~19-25 nucleotides (nts)
in length that can regulate gene expression by known classic
mechanisms such as binding to messenger RNA (mRNA)
at the 3'-untranslated region (3'-UTR) to downregulate
protein‑coding genes by increasing mRNA degradation (5).
They also act through other newly found mechanisms such
as binding to promoters, proteins, and by directly interacting
with other ncRNAs (5,6). miRNAs play important regulatory roles in various biological processes e.g. differentiation,
development, proliferation, apoptosis, cell cycle control and
metabolism (7,8). Changes in miRNA expression have been
identified in many diseases, such as cardiac and autoimmune
disorders, schizophrenia and cancer (9). miRNAs have been
found to be differentially expressed between almost all types of
1478
lin et al: MicroRNA PROFILING OF HUMAN CERVICAL SQUAMOUS CELL CARCINOMA
analyzed benign and malignant tumors and non-tumor tissues,
including CSCC (10). In addition, miRNAs participate in regulating a variety of neoplastic biological capabilities acquired
during the multistep process of tumor development. Using
miRNA microarray, Wilting et al observed that 46 miRNAs
showed significantly differential expression between normal
cervical squamous epithelium and CSCC (11). Both miR-19a
and miR-19b were reported to promote cervical cancer cell
proliferation (12), whereas miR-125b inhibited cervical cancer
cell apoptosis (13); both processes are involved in cervical
carcinogenesis. Other studies reported that miR-9 (14) and
miR-135a (15) contributed to HPV-induced cervical cancer
formation, and that miR-133b (16) and miR-10a (17) promoted
the progression and development of cervical cancer by regulating tumor cell migration, invasion and metastasis. However,
miR-424 (18), miR-124 (19) and miR-218 (20) have opposite
roles. All of these cited studies analyzed the differences in
expression of cervical cancer-associated miRNAs by reverse
transcription-polymerase chain reaction (RT-PCR) and
hybridization-based microarray that only measure known and
relatively abundant miRNAs, but are unable to identify novel
miRNAs. To date, no studies have focused on the miRNA
profile in early-stage CSCC. Thus, investigating the miRNA
profile in early-stage CSCC by high-throughput sequencing
may facilitate the analysis of expression of all annotated
miRNAs, detection of novel miRNAs and identification of
candidate markers for early diagnosis, and treatment of CSCC.
In the present study, we applied next-generation sequencing
(NGS) to globally and systematically characterize miRNA
alterations in early-stage CSCC by a comparison with paired
normal samples. Furthermore, we validated these miRNAs by
qRT-PCR and analyzed miRNA target genes and performed
function annotation and pathway analysis.
Materials and methods
Clinical sample collection. For sequencing, 3 pairs of human
CSCC tumor (T) and paired normal tissue (N) samples in
FIGO stages I-II were obtained from patients undergoing
surgery of the cervix. Additional, 20 pairs of CSCC and
normal tissues were also collected for quantitative real-time
polymerase chain reaction (qRT-PCR) validation. All tumor
and paired normal tissues were examined by hematoxylin
and eosin (H&E) staining, and all pathologic diagnosis were
confirmed by 2 independent pathologists; tumor tissues
containing >90% tumor cells were selected for further
study (Fig. 1). The study protocol was approved by the
Ethics Committees on Human Research of the Fujian Cancer
Hospital. All patients agreed to join the study and signed a
written informed consent. None of the patients had received
preoperative adjuvant radiotherapy or chemotherapy. All
samples were immediately snap-frozen in liquid nitrogen and
then stored at -80˚C until RNA isolation.
RNA isolation. Total RNA was extracted using TRIzol
(Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations, and quantified using a ND-1000
spectrophotometer (NanoDrop, Rockland, DE, USA).
The RNA quality was determined using an Agilent 2100
Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA)
and the 28S/18S ratio was determined on 1% agarose gel. Only
RNA extracts with an RNA integrity number (RIN) value ≥7
and 28S/18S ratio >1.8 were used for further analysis.
Deep sequencing and bioinformatic analysis. RNA
sequencing and bioinformatic analysis were performed at
the Beijing Genomics Institute (BGI; Shenzhen, China). In
brief, total RNA was ligated to the 5'- and 3'-end adaptors,
and sequentially amplified by an RT-PCR reaction. The fragments of ~62-75 bp (small RNA plus adaptors) were isolated
from the agarose gel and then purified. These products were
directly sequenced with Illumina HiSeq 2500 (Illumina, San
Diego, CA, USA) according to the manufacturer's instructions.
After trimming adaptor sequences, removing contaminated
reads and filtering low quality reads, the remaining clean
reads were mapped to the human genome using short oligonucleotide alignment program (SOAP). The other small RNAs
(rRNA, tRNA, snRNA, snoRNA and piRNA) were detected
by screening against Rfam 10.1 and the GenBank database.
The known miRNAs were identified by aligning them to the
miRBase. The expression differences of the known miRNAs
between the CSCCs and paired normal samples were evaluated by comparing the log2 ratio of the 2 groups. The miRNAs
with at least a 2-fold-change of expression and a P-value <0.05
between the 2 groups in all three patients were selected for
further study. The novel miRNAs were predicted using
MIREAP software.
Validation of mature miRNA expression by qRT-PCR. For
validation of the selected miRNAs that showed differential
expression and predicted novel miRNAs, qRT-PCR was
applied using the Applied Biosystems 7500 Sequence Detection
System (Applied Biosystems, Foster City, CA, USA) according
to the manufacturer's instructions. In brief, the miRNAs were
reverse transcribed into cDNA using the miScript II RT kit
(Qiagen, Valencia, CA, USA) with 5X miScript HiSpec buffer.
The mixture was incubated at 37˚C for 60 min, and then at
95˚C for 5 min and then placed on ice. Quantitative PCR
was performed using the miScript SYBR-Green PCR kit and
10X miScript Primer Assay (both from Qiagen). The reaction
consisted of 1 cycle at 95˚C for 15 min, followed by 40 cycles
at 94˚C for 15 sec and 55˚C for 30 sec and 70˚C for 30 sec.
All reactions were repeated 3 times. The relative expression
levels of miRNAs were calculated using the 2-ΔΔCt method with
snRNA U6 as the internal reference. Quantitative PCR data
were analyzed by SPSS statistics software (version 19.0; IBM,
Armonk, NY, USA). A Student's t-test was used to compare the
difference in expression between CSCC and normal tissues.
P<0.05 was considered to indicate a statistically significant
result.
miRNA target prediction, functional annotation and pathway
analysis. The target genes of the selected miRNAs with
differential expression and novel miRNAs were predicted
using RNAhybrid. For determination of the main biological
functions of the candidate target genes, Gene Ontology (GO)
enrichment analysis was applied. We mapped all candidates
to GO terms in the database (http://www.geneontology. org/),
using a hypergeometric test to find significantly enriched
GO terms. The GO terms with corrected P-values with an
ONCOLOGY REPORTS 37: 1477-1486, 2017
1479
Figure 1. H&E staining of CSCC and paired normal tissues. The H&E staining results of CSCC and paired normal tissues at a magnification of x40 under a
microscope.
Table I. Summary of the small RNA sequencing.
Category
Number
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------T-1
T-2
T-3
N-1
N-2
N-3
Total reads
Clean reads
%
Unique sRNAs
miRNAs
Known miRNAs in miRBase
miRNAs
miRNA-5p
miRNA-3p
miRNA precursors
Unannotated sRNAs
Novel miRNA candidates
12,000,000
11,713,704
98.40
884,315
4,196
1,708
200
414
306
788
232,043
28
10,523,729
10,313,021
98.73
673,581
4,545
1,881
225
431
351
874
205,460
24
12,000,000
11,519,252
96.70
867,499
4,346
1,810
229
418
325
838
255,885
31
12,000,000
11,799,688
99.02
615,904
3,634
1,571
171
385
298
717
291,651
20
12,000,000
11,820,021
99.18
466,839
4,035
1,659
182
394
330
753
156,254
22
12,000,000
11,704,777
98.25
658,536
4,173
1,724
200
400
335
789
240,371
26
T, CSCC tumor tissue; N, paired normal tissue.
enrichment ratio ≤0.05 were defined as significantly enriched
target gene candidates. For identification of significantly
enriched metabolic pathways or signal transduction pathways in target gene candidates, we mapped these candidates
to the Kyoto Encyclopedia of Genes and Genomes (KEGG)
databases (21). The calculation method is the same as that
in the GO analysis. Pathways with corrected P-values with
an enrichment ratio ≤0.05 were considered as significantly
enriched target gene candidates.
Statistical analysis. All quantitative data are expressed as
the mean ± standard deviation (mean ± SD) from at least
3 samples or experiments/data point. Significant differences
were analyzed by the Student's t-test to compare 2 groups of
independent samples.
Results
Overview of small RNA sequencing results. To determine
the miRNA profile of early-stage CSCC, we performed
high‑throughput NGS in 3 CSCC and their paired normal
samples using Illumina HiSeq 2500. After removing adaptors, low quality tags and contaminants, an average of
11,478,411 clean reads (ranging from 10,313,021 to 11,820.021)
remained (Table I). The majority of these clean reads were
18-28 nt in length, with 22 nt RNAs the most abundant.
These clean reads were mapped to the Human Genome and
rRNA, tRNA and snRNA were identified by the alignment to
Rfam 10.1 and GenBank databases. Subsequently, an average
of 1,726 mature miRNAs (ranging from 1,571 to 1,881) were
successfully annotated in the miRBase, and thus identified
1480
lin et al: MicroRNA PROFILING OF HUMAN CERVICAL SQUAMOUS CELL CARCINOMA
Figure 2. Scatter diagrams of miRNA expression between CSCC and normal tissues (x-axis, normal and y-axis, tumor). Red points represent miRNAs with
ratio >2, blue points represent miRNAs with ratio >1/2 and ≤2 and green points represent miRNAs with ratio <1/2. Ratio, normalized expression in the
tumor/normalized expression in the control tissue.
Figure 3. Heatmap of miRNA expression varied significantly between CSCC and normal tissues in the 3 pairs of samples.
as known miRNAs (Table I). The sequences which did not
match with any database were considered as unannotated
sRNAs (Table I).
Identification and validation of differentially expressed
known miRNAs between CSCC and normal tissues. To detect
the known miRNAs which were differentially expressed
between the CSCC and normal tissues, the expression
level of each miRNA was first normalized to provide the
expression of transcripts per million (TPM), and then the
fold-change and P-values were calculated from the normalized expression. There were 424 differentially expressed
miRNAs between T1 and N1, 480 between T2 and N2 and
440 between T3 and N3 (Fig. 2). Subsequently, on the basis
of the cut-off criteria (fold-change >2 and p-value <0.05),
186, 149 and 126 miRNAs were selected from the data
from each of the 3 pairs of patient samples. Finally, only
37 miRNAs exhibited consistent changes in all 3 pairs of
samples; among these 37 differentially expressed miRNAs,
8 were significantly upregulated and 29 were significantly
downregulated (Fig. 3; Table II).
To further validate the deep sequencing results, qRT-PCR
was applied to assess the significantly differentially expressed
miRNAs in 3 pairs of samples used for sequencing and the
additional 20 pairs of samples. Nine miRNAs were selected
from the miRNAs with significantly modified expression
and measured by qRT-PCR. All of the 9 miRNAs displayed
the same expression pattern consistent with the sequencing
data (Fig. 4). This indicated that our high through-put
sequencing results were reliable.
ONCOLOGY REPORTS 37: 1477-1486, 2017
1481
Table II. Summary of the significantly upregulated and downregulated miRNAs in CSCC.
miRNA
Fold-change (log2 tumor/normal)
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------T1 vs. N1
T2 vs. N2
T3 vs. N3
hsa-miR-934
hsa-miR-3927-3p
hsa-miR-34b-5p
hsa-miR-34c-5p
hsa-miR-21-3p
hsa-miR-7-5p
hsa-miR-1277-3p
hsa-miR-2355-5p
hsa-miR-211-5p
hsa-miR-204-5p
hsa-miR-218-1-3p
hsa-miR-202-5p
hsa-miR-218-5p
hsa-miR-1298-5p
hsa-miR-204-3p
hsa-miR-6510-3p
hsa-miR-299-5p
hsa-miR-1247-5p
hsa-miR-206
hsa-let-7c-5p
hsa-miR-99a-5p
hsa-miR-487b-3p
hsa-miR-154-5p
hsa-miR-337-3p
hsa-miR-495-3p
hsa-miR-134-5p
hsa-miR-125b-5p
hsa-miR-376c-3p
hsa-miR-195-5p
hsa-miR-654-3p
hsa-miR-127-3p
hsa-miR-369-5p
hsa-miR-139-5p
hsa-miR-433-3p
hsa-miR-329-3p
hsa-miR-379-5p
hsa-miR-887-3p
8.30318711
4.51831602
9.09373446
4.78205816
6.62522824
5.56817204
3.30345745
2.42581593
-7.08321336
-6.85930564
-7.33109609
-10.57502857
-7.941165
-5.92810297
-6.66817587
-3.36793893
-5.52895357
-7.39024513
-3.37648276
-6.26600709
-6.62465962
-5.00445576
-5.43273636
-2.21193294
-4.59233357
-3.89631011
-5.94077162
-3.55711379
-4.33482863
-4.63279944
-4.56838942
-5.41522226
-2.84273417
-4.37189471
-3.95914876
-3.57432673
-2.49199079
4.2842649
3.10372182
4.03811041
4.92246647
3.4080715
2.07123043
2.463592
3.65621776
-6.80271289
-9.83557679
-7.40258575
-4.68366909
-8.60425671
-4.54163652
-4.05060043
-2.73610137
-3.78516379
-4.67666656
-2.61057144
-2.93147207
-2.58200581
-3.79678741
-3.17579365
-4.38841479
-3.30958264
-3.08449386
-2.27910623
-2.91544225
-2.13708218
-2.26265207
-2.30793519
-2.08867671
-2.67661687
-2.16954783
-2.12515371
-2.49126887
-2.07624607
7.20535341
9.11787682
2.54681543
3.3850871
2.26011983
2.11570518
3.34499026
2.39229062
-11.34275801
-5.26254955
-6.9021942
-5.0642676
-2.97717439
-7.7387003
-7.09486942
-8.822762
-5.511115
-2.41593802
-7.18229503
-3.93093655
-3.74953697
-3.01875682
-2.93133666
-4.62100525
-3.28815361
-4.1468506
-2.73045243
-4.19191414
-4.13398935
-3.39215912
-3.28638674
-2.46223868
-4.37453647
-2.89020567
-3.0926029
-2.8998286
-2.66140231
Each miRNA is listed in the Table with P<0.01 in all 3 pairs of samples. CSCC, cervical squamous cell carcinoma.
Target genes of the significantly altered miRNAs. To demonstrate the differences in gene expression associated with the
miRNA profile in CSCC, the putative target genes of the
miRNAs that were expected to be significantly differently
expressed between CSCC and normal tissues were predicted
using RNAhybrid. GO and KEGG pathway analyses were
performed to enrich the main biological processes and
pathways in which the target gene candidates were involved.
However, the P-value of the enrichment ratio failed to achieve
filter conditions and the analysis was not continued.
Prediction of novel miRNAs, their target genes and pathways.
To detect novel miRNAs, unannotated sRNAs were further
processed using MIREAP. The number of novel miRNA
candidates obtained from the 6 small RNA libraries ranged
from 20 to 31 (Table I). There were 5 common novel miRNA
1482
lin et al: MicroRNA PROFILING OF HUMAN CERVICAL SQUAMOUS CELL CARCINOMA
Figure 4. Validation of the significantly different miRNAs between CSCC and normal tissues by qRT-PCR in 20 pairs of samples (*P<0.05, **P<0.01, ***P<0.001).
Figure 5. Prediction of novel miRNAs in CSCC. (A) Expression of novel_mir_7 by NGS. (B) Expression of novel_mir_7 by qRT-PCR validation in 20 cancer
samples. (C) The sequence and the stem loop structure of novel_mir_7 (*P<0.05, **P<0.01).
candidates in the 3 pairs of CSCC and normal samples. Among
the 5 common miRNAs, only 1 miRNA, novel_miR_7,
displayed a significant difference in expression (fold-change >1
and P-value <0.05) in all 3 pairs of samples: novel_miR_7
was selected as a putative novel miRNA and verified by
qRT-PCR (Fig. 5). Its sequence and stem-loop secondary
structure are shown in Fig. 5. To further understand the function
of the novel miRNA, its target genes were predicted using
RNAhybrid and also assessed by GO and KEGG pathway
analyses. The GO analysis revealed the main biological
processes in which the target genes are involved and is shown in
Fig. 6. The KEGG pathway analysis indicated that 5 pathways
were enriched; including the MAPK, calcium and adherent
junction signaling pathways (Table III).
ONCOLOGY REPORTS 37: 1477-1486, 2017
1483
Figure 6. Functional annotation for predicted target genes of the novel miRNA in CSCC.
Table III. KEGG pathway analysis for predicted target genes
of the novel miRNA in CSCC.
KEGG no.
KEGG pathway
KO:04010
KO:04020
KO:04520
KO:05217
KO:04730
MAPK signaling pathway
Calcium signaling pathway
Adherent junction
Basal cell carcinoma
Long-term depression
Significance
a
b
b
b
b
Q-value <0.05 in all 3 pairs of samples; bQ-value <0.01 in all 3 pairs
of samples. Q-value, corrected P-value. KEGG, Kyoto Encyclopedia
of Genes and Genomes.
a
Discussion
Although CSCC caused by HPV infection can be prevented,
it is still a common cancer in women. There is accumulating
evidence to indicate that HPV infection alone is insufficient
to cause the disease and miRNAs may play critical roles in
carcinogenesis (22-24). Therefore, global analysis of miRNA
expression comparing CSCC with normal tissue may aid in the
better understanding of the function of miRNAs in the course
of the disease as well as finding novel biomarkers for diagnosis,
treatment and prognostic evaluation. To the best of our
knowledge, this is the first study to profile miRNA expression
in early-stage CSCC on the NGS platform. We identified
37 significantly differentially regulated known miRNAs in
CSCC compared with normal tissue and confirmed a sample
of 9 by qRT-PCR. Furthermore, a novel miRNA candidate,
novel_miR_7, was identified for the first time and its target
genes are involved in MAPK, calcium and adherent junction
signaling pathways.
miRNAs are rather stable, even in body fluids such as
serum, plasma, urine and saliva (25). The expression of
miRNAs is unique to tissues or organs and they can be easily
detected (26). These characteristics make them ideal cancer
biomarkers. Juan et al (27) performed deep sequencing on the
serum pools of cervical cancer patients and healthy controls
to identify putative novel miRNAs. They found that only 1 of
17 common novel miRNA candidates may distinguish cervical
cancer patients from healthy controls. Wang et al (28) also
constructed expression profiles of miRNAs in the serum of
CSCC patients by microarray. They discovered that the levels
of 291 of 338 detectable circulating miRNAs were changed
>2-fold in serum samples from CSCC patients vs. controls.
However, they did not validate these results through any other
methodologies. Although detection of miRNAs in serum has
been proposed to be a simpler and less invasive diagnostic
method than sampling tissue, the consequential biological
and technical variability, which is mainly assessed by a series
of different normalization processes based on the expression
of reference genes, is difficult to avoid. Currently, there is
no known suitable extracellular reference RNA for a proper
normalization, particularly in cell-free miRNA analysis (26).
Commonly used references, including U6 small nuclear
RNA (RNU6B) and 5S ribosomal RNA, may be degraded
or expressed less stably than others in serum (29). Therefore,
we decided to analyze miRNA expression in CSCC tissues.
In previous studies, a microarray technique, which is biased
1484
lin et al: MicroRNA PROFILING OF HUMAN CERVICAL SQUAMOUS CELL CARCINOMA
towards abundantly expressed known miRNAs, was the major
platform for miRNA expression profiling (11,28). We chose a
more sensitive and specific method, NGS, which enables highthroughput analysis and novel miRNA discovery, to measure
miRNA expression.
In the present study, we identified 9 miRNAs that exhibited
differential expression in CSCC using deep sequencing and
confirmed by qRT-PCR evaluation. Six were downregulated,
including miR-211-5p, miR-204-5p/3p, miR-218-1-3p/5p and
miR-202-5p, and 3 were upregulated, including miR-34b-5p,
miR-34c-5p and miR-21-3p. Among these miRNAs, miR-218,
miR-21 and miR-34b-5p have previously been discovered
and their functions had been verified to be associated with
cervical cancer. In comparison to normal cervical tissues the
expression of miR-218 was reported to be lower in cervical
cancer tissues (20), and that finding was concordant with
our results. Its downregulation was significantly associated
with worse overall survival, worse disease-free survival and
pelvic/aortic lymph node recurrence. miR-218 was found
to target survivin to inhibit cervical cancer progression by
regulating clonogenicity, migration and invasion. Increased
miR-21 expression was found to be correlated with poorer
histological diagnosis in cervical cancer (30). Meanwhile,
miR-21 was found to be mainly expressed in the tumor
stromal microenvironment where it may be involved in
cervical cancer progression. In addition, expression of miR-21
in HPV-positive samples and SCC was significantly higher
than that noted in HPV-negative samples (31). Constitutive
activation of aberrant STAT3 signaling via miR-21, which is
regulated by the HPV oncoprotein E6, plays a pivotal role in
the initiation and progression of HPV-induced cervical carcinogenesis (32,33). Our sequencing results found that miR-21
was upregulated in CSCC and it may act as an oncogene;
which is consistent with previous studies (30,31). miR34b-5p was found to be downregulated in minimal deviation
adenocarcinoma (MDA) compared with normal proliferative
endocervical tissues (34), but in the present study, it was
found to be upregulated in CSCC. The discrepancy suggests
different expression patterns of miRNAs in different histological subtypes of tumors.
The other differentially expressed miRNAs we described
have seldom been reported in previous studies of cervical
cancer, but they have been demonstrated to be correlated with
other malignant tumors. Trends seen in the expression of certain
miRNAs that were determined by sequencing were in accord
with published functional studies. For example, miRNA-211
functions as a tumor-suppressor in melanoma (35,36) and
glioma (37). It could block tumor invasion and migration
by targeting NUAK1 (38), BRN2 (39), KCNMA1 (40)
or MMP-9 (37). Both miR-204-5p/3p and miR-202 have
tumor-suppressive function as well. Downregulation of
miR-204-5p in colorectal cancer tissues was associated with
poor prognosis: It suppresses colorectal cancer cell growth,
migration, and invasion as well as promotes tumor sensitivity
to chemotherapy by inhibiting RAB22A (41). In endometrial
carcinoma, miR-204-5p exhibits a similar function through
the TrkB-STAT3-miR‑204-5p regulatory pathways (42) and
miR-204-3p inhibits the growth of hepatocellular carcinoma
tumor endothelial cells by blocking the adhesion function
of FN1 (43). miR-202 can suppress osteosarcoma cell
proliferation and induce cell apoptosis by downregulating
Gli2 expression (44). While miR-34c is considered to be a
putative tumor suppressor in a majority of tumors (45-48), we
found it to be upregulated in CSCC, which is in agreement
with previous results that determined expression by miRNA
microarray in cervical cancer (11,28). Thus, we hypothesized
that miR-34c may play different roles in cervical cancer and
other types of malignancy.
NGS technology allows the rapid discovery of novel
miRNAs, for some of which the biological function is
unknown. We identified 5 common novel miRNA candidates
with differential expression between CSCC and normal
tissues. Our focus was narrowed to miRNAs that exhibited
a significant change of expression in all 3 pairs of samples.
We identified novel_miR_7 as a candidate novel miRNA and
confirmed its presence. Functional annotation revealed that its
predicted target genes are in the MAPK signaling pathway,
which is closely related to cell proliferation, cell death, and
the balance between them. Early in 2006, Engelbrecht et al
indicated the involvement of MAPK in cervical cancer and
demonstrated the correlation between the activity of MAPK
and apoptosis in the disease process (49). The HPV 16 E2
protein may induce apoptosis by inhibiting MAPK signaling
in CSCC (50). However, playing a role in cervical cancer
carcinogenesis, the expression level of MAPK may serve as a
marker for cervical cancer progression (51,52) and its inhibitors have shown the potential to be useful for cervical cancer
therapy (53). Therefore, our results provide a solid basis for
the characterization of the role of novel_miR_7 in the pathogenesis and development of CSCC and in the treatment of this
disease.
In summary, for the first time we identified known and
novel miRNAs in early-stage CSCC by high-throughput NGS.
Moreover, we validated these miRNAs in other independent
samples by qRT-PCR. Our analysis of differentially expressed
miRNAs not only support existing findings but also uncovered
various differentially expressed miRNAs which had not previously been reported in CSCC, thus providing new molecular
signatures of the disease. The prediction of novel miRNAs
found that novel_miR_7 may be a promising biomarker for
CSCC. These results have implications for exploring the regulatory network of miRNAs, provide new targets for molecular
mechanistic studies of CSCC as well as for early detection,
targeted therapy and prognostic evaluation of CSCC patients
in the future.
Acknowledgements
The present study was supported by grants from the
Fujian Province Science and Technology Plan Key Project
(no. 2013Y0030) and the National Clinical Key Specialty
Construction Program of China.
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W4249279809.txt | FOCUS: ARCHITECTURE IN PHOTOGRAPHIC EXHIBITIONS 1858–1861
AN EXHIBITION PROPOSAL FOR THE MUSEUM OF FINE ARTS, HOUSTON
By
Natalie Banaszak
BAH Honours, University of Guelph, Guelph, 2014
A thesis presented to Ryerson University
In partial fulfillment for the degree of
Master of Arts
In the program of
Film and Photography Preservation and Collections Management.
Toronto, Ontario, Canada, and Houston, Texas, USA 2016
© Natalie Banaszak 2016
AUTHOR'S DECLARATION FOR ELECTRONIC SUBMISSION OF A THESIS
I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including
any required final revisions, as accepted by my examiners.
I authorize Ryerson University to lend this thesis to other institutions or individuals for the
purpose of scholarly research
I further authorize Ryerson University to reproduce this thesis by photocopying or by other
means, in total or in part, at the request of other institutions or individuals for the purpose of
scholarly research.
I understand that my thesis may be made electronically available to the public.
ii
FOCUS: ARCHITECTURE IN PHOTOGRAPHIC EXHIBITIONS 1858–1861 — AN
EXHIBITION PROPOSAL FOR THE MUSEUM OF FINE ARTS, HOUSTON
Master of Arts, 2016, Natalie Banaszak, Film and Photography Preservation and
Collections Management, Ryerson University.
Abstract
This thesis consists of a proposal for an exhibition titled Focus: Architecture in
Photographic Exhibitions 1858–1861. Drawn from the collection of almost three hundred
nineteenth-century architectural photographs at the Museum of Fine Arts, Houston, the
exhibition explores the topic of architectural photographs exhibited between 1858 and 1861. This
two-part thesis first approaches the exhibition from a practical side, outlining the steps in
preparing the photographs for display. An essay follows, investigating the role of the
Architectural Photographic Association in the context of the photographic market of this time.
The thesis also includes appendices relating to various aspects of the exhibition design process,
including an object checklist, matting and framing recommendations, condition reports, wall
labels, wall elevations, and an informational brochure.
iii
Acknowledgements
First and foremost, this project would not be possible without the help of Thierry Gervais,
Malcolm Daniel, and Marta Braun. The guidance I received from all of you was invaluable to
me. Thierry, thank you for your patience and for always pushing me to be better. Malcolm, thank
you for welcoming me into the department, and always entertaining my ideas and helping me
refine them. Marta, I appreciate you always supporting me, and am grateful for your suggestions
towards this thesis as its second reader.
I cannot begin thank those who have read this thesis, edited it, and provided suggestions. Adrian,
thank you for your never-ending support; from listening to long rants and making those into real
thoughts, withstanding last minute editing requests, and cheering me on. You are a true gem.
Jamie, thanks for keeping me sane in the in-between parts. To my Firecrackers, thank you all for
the support, the critical eyes, and your firm grips on the English language. Congratulations to my
fellow classmates; thank you for always being an inspiration and a soundboard for ideas.
Finally, my thanks go out to the staff at the Museum of Fine Arts, Houston, for taking time out of
their busy schedules to accommodate me, particularly: Selina Lamberti for your genuine
friendship, and the countless hours of advice; Jason Dibley, for the frequent hospitality in your
workspace, and for teaching me so much; Toshi Koseki and August DiStefano for indulging my
conservation and preparatory requests; and Allison Pappas and Adrian Alejo for your willingness
to go above and beyond to help me with anything and everything in between.
iv
Table of Contents
Declaration
Abstract
Acknowledgements
List of Figures
List of Appendices
ii
iii
iv
vi
vii
Introduction
Survey of the Available Literature
1
5
Part One: Preparing an Exhibition
Curatorial statement
Exhibition timeline
Checklist and rationale
List of Photographs
Cataloguing and preparing wall labels
Preservation considerations
Matting and framing
Wall elevations and layout
Preparing an exhibition pamphlet
13
13
14
15
17
23
26
27
28
29
Part Two: The Exhibitions of the Architectural Photographic Association (1858-1861)
The Association’s First Exhibition
The 1859 Exhibition
The Association’s Downfall
Conclusion
33
34
38
41
44
Appendices
References
46
71
v
List of Figures
Figure 1: Fratelli Alinari, Lorenzo Ghiberti’s Bronze Door, 1852–1858,
albumen silver print from glass negative, 42.8 x 31.7 cm,
The Museum of Fine Arts, Houston, 2004.211.
17
Figure 2: Fratelli Alinari, Campanile for Duomo of Santa Maria del Fiore, Florence,
1852–1858, albumen silver print from glass negative, 44.4 x 29.5 cm,
The Museum of Fine Arts, Houston, 2004.212.
17
Figure 3: Édouard Baldus, Pavillon Denon, Louvre, 1855–1856,
salted paper print from glass negative, 44 x 33.8 cm,
The Museum of Fine Arts, Houston, 2004.249.
18
Figure 4: Bisson Frères, Moissac Abbey Cloister, 1857,
albumen silver print from glass negative, 35.6 x 45 cm,
The Museum of Fine Arts, Houston, 2004.313.
18
Figure 5: Charles Clifford, [Main Door, Salmanca Cathedral], before 1858,
albumen silver print from glass negative, 43 x 32.4 cm,
The Museum of Fine Arts, Houston, 2004.350.
19
Figure 6: Charles Clifford, Salamanca. Puerta del Convento de las Duenas, 1853,
albumen silver print from glass negative, 39.7 x 27.3 cm,
The Museum of Fine Arts, Houston, 2004.351.
19
Figure 7: Tommaso Cuccioni, View of the Roman Forum towards the Capitoline Hill,
Looking West, 1855, albumen silver print from glass negative, 27.3 x 38.1 cm,
The Museum of Fine Arts, Houston, 2004.369.
20
Figure 8: Roger Fenton, printed by Francis Frith & Co., Lichfield Cathedral,
Central Doorway, West Porch, 1858 printed 1863–1865,
albumen silver print from glass negative, 41.9 x 34.9 cm,
The Museum of Fine Arts, Houston, 2014.89.
20
Figure 9: Francis Frith & Co., Columns, Pharaoh's Bed, 1856–1861,
albumen silver print from glass negative, 20.4 x 16.4 cm,
The Museum of Fine Arts, Houston, 88.233.
21
Figure 10: Robert Macpherson, View of Rome from the French Academy, Monte Pincio, 21
1851–1858, albumen silver print from glass negative, 20.4 x 16.4 cm,
The Museum of Fine Arts, Houston, 2004.222.
Figure 11: Carlo Naya, Piazzetta of San Marco looking towards the Island of St. George, 22
1857–1859, albumen silver print from glass negative, 42.4 x 53.5 cm,
The Museum of Fine Arts, Houston, 2004.619.
vi
Figure 12: Robertson and Beato, Tophane Fountain, 1854–1858,
albumen silver print from glass negative, 26.5 x 31.2 cm,
The Museum of Fine Arts, Houston, 2000.255.
Figure 13: Digital image taken around 2004 for Tommaso Cuccioni,
View of the Roman Forum towards the Capitoline Hill, Looking West, 1855,
albumen silver print from glass negative, 27.3 x 38.1 cm,
The Museum of Fine Arts, Houston, 2004.369.
22
30
List of Appendices
Appendix A: Wall Labels
Appendix B: Condition Reports
Appendix C: Matting and Framing Swatches and Worksheets
Appendix D: Wall Elevation
Appendix E: Exhibition Brochure
vii
46
52
54
68
69
Introduction
The Department of Photographs at the Museum of Fine Arts, Houston has a fixed
exhibition space along a hallway in the lower level of the Audrey Jones Beck Building. Located
there is a permanent display titled The History of Photography: Selections from the Museum’s
Collection, in which the photographs rotate every four months, and the curators alternate in
sharing the responsibility of selecting the group of objects. The wall directly opposite is a
rotating Focus display, which includes prints by one photographer or a theme from the
Museum’s collection of over thirty thousand photographs. For my thesis, I proposed an
exhibition of nineteenth-century architectural photographs for this space. This selection will
present a history of exhibitions of architectural photography with a focus on the Architectural
Photographic Association, in order to expand the public’s understanding of the demand for
architectural photographs in the 1850s to 1860s. The exhibition would accompany any History of
Photography rotation, which “present the medium's history in a slightly different light. In this
way, an increasingly complex picture of photographic history emerges for those who move
slowly, look closely, and return for more.”1 My exhibition reflects the Museum’s collection
mandate by starting a conversation about how photographs are displayed as art, how they were
used by architects, and how they were distributed by photographic societies and studios in the
nineteenth century.
The exhibition at the Crystal Palace in London in 1851 was, for the majority of those in
attendance, a preliminary exposure to photographs.2 By the mid-1850s, photographic exhibitions
became more common throughout Europe, and particularly in Britain and France, intrinsically
1
"A History of Photography: Selections from the Museum’s Collection." The Museum of Fine Arts, Houston. 2016.
Accessed April 21, 2016. https://www.mfah.org/history-photography-selections-museums-collection/.
2
John Hannavy, "Exhibitions of Photography," in Encyclopedia of Nineteenth-century Photography, New York:
Routledge, Taylor & Francis Group, 2008: 508.
1
linked with the rise of photographic societies.3 The Photographic Society of London, now the
Royal Photographic Society, was founded in 1853, and the following year, the Société Française
de Photographie was formed in Paris. These societies helped foster an international community
of photographers, working to validate their work as art, and to make technical advances in the
medium or profit off of the photographs. Members of photographic societies, like the Société
Française de Photographie, or the Photographic Society of London, or the Glasgow Photographic
Society, were photographers and chemists. They would exchange prints with each other for the
purposes of refining their own techniques and building their personal collections. These societies
held annual exhibitions, in which the photographs were selected by juries to ensure that they
showcased only the finest examples of photographic practices and inventions of the time.
The 1850s also saw a rise in the number of photographers travelling abroad, particularly
to Italy and the Near East.4 Photography was seen as a more reliable and practical medium for
the documentation of encounters overseas.5 As a result, the number of commercial photographic
studios across Europe and Asia increased. Scholar Anne McCauley argues that beginning in the
1850s, commercial photographers defined public perception of photography.6 In 1848, there
were thirteen photographic studios in Paris, and by 1878, that number rose to almost four
hundred.7 McCauley argues that the years following 1855 were the period of greatest growth of
the photographic industry.8 In the 1850s and 1860s, many photographers bought existing studios
to avoid the cost of starting up their own businesses. Most of these studios only survived because
3
Anne M. Lyden, A Royal Passion: Queen Victoria and Photography, Los Angeles: J. Paul Getty Museum, 2014.
Sylvie Aubenas and Jacques Lacarrière, Voyage En Orient, Paris: Hazan, 1999.
5
Risto Sarvas and David M. Frohlich, From Snapshots to Social Media: The Changing Picture of Domestic
Photography, New York: Springer, 2011: 31.
6
McCauley 8.
7
Ibid
8
Ibid 31.
4
2
of funding from family members, or external benefactors.9 According to McCauley, the market
of the 1850s and 1860s consisted of art reproductions,10 photographs of new construction, and
portraiture. Advances in photographic technology at the time aided in the success of such
studios, and enabled photographers to travel. Specifically, the invention of wet collodion glass
plate negatives made it easier to print a high quantity of sharp images upon a photographer's
return home from their travels. This allowed for a larger-scaled production and distribution of
photographic prints than ever before.11 The increased success and popularity of photographic
societies, commercial studios, and wet collodion glass plate negatives amalgamated to create an
unprecedented international community of photography.
This was the climate in which the Architectural Photographic Association began, holding
its inaugural meeting in London in 1858. The Association reveled in its immediate popularity
and success. Architects belonging to the Association would travel locally and abroad, purchasing
prints by well-established photographers. These photographs would then be assembled into an
annual exhibition in London, where a subscription fee of one guinea or upwards per annum
would grant access and allow patrons to come and purchase copies of photographs that were on
display.12 For reference, a guinea in 1858 was equivalent to 21 shillings, and in today’s market,
that has a relative purchasing power of 171.93 Canadian Dollars.13 By 1861, the Association
struggled with overwhelming demand and short finances and so was disbanded. Due to its
inability to send out photographs to subscribers fast enough, it was overcome by its own business
model in just four years. My thesis is therefore guided by the following question: how does the
9
Ibid., 60.
Ibid., 266.
11
Sarvas and Frohlich, 31.
12
The Literary Gazette, 43.
13
Relative purchasing power calculated by: https://www.measuringworth.com/calculators/ukcompare/r
elativevalue.php; calculated April 21 2016.
10
3
work of the Association reveal the existence of a demand and a market for architectural
photography? For the exhibition, I chose twelve architectural photographs from the Museum’s
collection, eight of which are copies of prints that were included in the exhibitions of the
Architectural Photographic Association between 1858 and 1861. The remaining four
photographs were exhibited at either the Société Française de Photographie, the Photographic
Society of London, or the Glasgow Photographic Society during that same time.
My thesis consists of an outline for an exhibition proposal titled Focus: Architecture in
Photographic Exhibitions 1858–1861 and is structured into two parts. The first part of my thesis
includes practical considerations from the start to the finish of the exhibition process, all of
which can be done in-house at the Museum. The tentative exhibition date falls out of the timeline
of this thesis, so I will describe everything short of physically mounting the display. The second
part features an essay analyzing the photographs that were exhibited at the Architectural
Photographic Association and investigating how the exhibitions were a catalyst for the market of
architectural photography between 1858 and 1861.
4
Literature Survey
My literature survey consists of three main components: photographic collection
management and exhibition preparation, the history of exhibitions by photographic societies
from 1858 to 1861, and the role of architectural photography in the nineteenth-century. The first
section contains the practical literature which I consulted in creating the exhibition, in order to
ensure the photographs were handled safely and that public access to these photographs would
continue beyond the exhibition. Secondly, scholarship on the exhibitions of photographic
societies from 1858 to 1861 gives necessary context into the history of photographic exhibitions
and insight into the unique model of the Architectural Photographic Society. Finally, a gap exists
between scholarship about architectural photography and that of the photographic market:
establishing the context for the creation and uses of these images, but failing to discuss their
dissemination.
Photographic Care and Collections Management
Resources on photographic care and museum management lay the foundation for all of
the work done in preparation for the exhibition, specifically with regard to cataloguing for wall
labels and writing preservation considerations. Integral to any exhibition, beyond preservation
and management, is visitor interpretation; therefore, the section closes with a discussion of one
comprehensive text on how best to plan exhibitions with the visitor experience in mind.
Rebecca A. Buck and Jean Allman Gilmore run their own artifact and museum
management firm, Buck & Gilmore LLC. Their textbook, MRM5: Museum Registration
5
Methods14 was published in 2010 by the American Library Association, and proposes a thorough
introduction to every aspect of art and artifact registration in a museum environment. Since it is
not focused on photography, I treated the sections on “In House Exhibitions” and “Digital Asset
Management” as reference texts for general best practices.
After this broad approach, I consulted specific texts regarding the care, management, and
exhibition of photographs. The publication by founding curator of the Image Permanence
Institute, James M. Reilly, titled Care and Identification of Nineteenth Century Photographic
Prints15 provides useful information about how best to treat nineteenth-century photographs,
including specific matting and framing suggestions, and how to provide safe, long-term access
through digitization. In order to gain necessary techniques on cataloguing, digitizing, and
exhibiting the photographs, I consulted Bertrand Lavédrine’s 2009 book titled Photographs of
the Past: Process and Preservation.16 Lavédrine draws from his experience as director of the
Centre de Recherche sur la Conservation des Collections (CRCC) and conservation science
professor at the Muséum National d'Histoire Naturelle in Paris to give recommendations on best
practices for matting and framing, exhibiting, digitizing, and cataloguing photographs. Finally, I
cross-referenced the above information with a series of PDF documents published by the Library
of Congress, found online in their “Cataloguing and Digitizing Toolbox.”17 These tip sheets are
constantly being updated, and referencing them ensures that the information found in the
aforementioned, older publications is still relevant.
14
Rebecca A. Buck and Jean Allman Gilmore, MRM5: Museum Registration Methods, 5th ed., American
Association of Museums: AAM Press, 2010.
15
James M. Reilly, Care and Identification of 19th Century Photographic Prints, Rochester: Eastman Kodak
Company, 1986.
16
Bertrand Lavédrine, Photographs of the Past: Process and Preservation, Los Angeles: Getty Conservation
Institute, 2009.
17
The Library of Congress, "Cataloging & Digitizing Toolbox," Prints and Photographs Reading Room, Library of
Congress, October 22, 2010, http://www.loc.gov/rr/print/cataloging.html.
6
I also consulted manuals regarding writing exhibition labels, such as the 2015 republication of renowned museum consultant Beverly Serrell’s Exhibit Labels: An Interpretive
Approach.18 This manual for preparing an exhibition gives guidance on writing and designing
visitor-friendly exhibition spaces, including considerations for different types of learning styles,
and remedies for accessibility concerns. Her advice on word count when writing different types
of labels, including exhibition titles, introductory labels, group labels, and caption labels guided
my editing process.19 Serrell places a large emphasis on logical flow throughout the exhibition
design process, which helps visitors identify the objectives of the exhibition more clearly.
These texts are outstanding resources out of a vast selection available to guide curators
and collections managers to assist with the process of physically preparing exhibitions and
ensuring photographs are safely stored and displayed. The basis of all interpretive text is
thorough, accurate cataloguing, for without proper management, storage, and care, photographs
would not survive the harsh conditions of exhibition.
Exhibitions of Photographic Societies from 1858 to 1861
The foundation of my essay, cataloguing, and wall labels involved diligent research about
the photographic exhibitions of 1858 to 1861, in order to ensure that proper historical context
was maintained. A study about the Architectural Photographic Association is paired with
writings surrounding the work of the Photographic Society of London or the Société Française de
Photographie, in order to provide a cultural context for the exhibitions during this time period.
Most surviving primary sources on the foundation of the Architectural Photographic
Association are managed by the Royal Institute of British Architects (RIBA), held at the British
18
19
Beverly Serrell, Exhibit Labels: An Interpretive Approach, 2nd ed. Maryland: Rowman & Littlefield, 2015.
Serrell, 33–36.
7
Architectural Library in London. Robert Elwall was the founding curator of photographs at
RIBA. His 1985 article titled “The Foe-to-Graphic Art: The Rise and Fall of the Architectural
Photographic Association”20, published in The Photographic Collector, is the only existing
historical analysis of the Architectural Photographic Association. Elwall produced a thorough
account of the rise and subsequent downfall of the Association, discussing the events
surrounding the institution’s creation, exhibitions, and dissolution. It is from this article that I
learned about the methods by which the Association was collecting and exhibiting prints, the fact
that architects had decisive authority, and the specific details regarding each of the four
exhibitions. What was particularly important to me was Elwall’s conclusion, in which he states
that the Association fell apart by 1861 because the demand on the market exceeded the means of
the Association. The Association was a commercial success in the limited time it existed,
proving that there was a demand for architectural photography in the market of the 1850s to
1860s. This led me to investigate whether or not other photographic societies shared the same
mandates or successes.
The work of scholars such as Roger Taylor and Paul-Louis Roubert reveals that
photographic societies were generating income in a much different way from the Architectural
Photographic Association. Roubert, associate professor in the history of photography and the
current president of the Société Française de Photographie, has published several articles about
exhibitions in Études Photographique. In his “1859, Exposer La Photographie”21 published in
2000, Roubert explained that the Société Française de Photographie was run according to a
system akin to that of Parisian Salons: only the work selected by a jury would be presented.
20
Robert Elwall, ""The Foe-to-Graphic Art": The Rise and Fall of the Architectural Photographic Association," The
Photographic Collector 5.2 (1985): 142–56.
21
Paul-Louis Roubert, "1859, Exposer La Photographie," Études Photographiques 8 (November 2000): 4–21.
8
While the exhibitions featured a wide range of subject matter, jurors favoured portraiture and art
reproductions. Taylor’s 2002 publication Photographs Exhibited in Britain, 1839–1865: A
Compendium of Photographers and Their Works22 reveals similar preferences. Taylor’s book is a
compilation of exhibition catalogues from British photographic exhibitions. Unlike Roubert,
Taylor does not analyze the contents or intentions of the exhibitions; he simply lists the
exhibition date, name, and location, and the title and maker of each photograph included.
Taylor’s book is a useful tool for comparing exhibitions to one another, as it lists the maker and
exhibition title for each photograph exhibited in Britain between 1858 and 1861.
Scholars writing about the exhibitions of photographic societies do not discuss that these
exhibitions also served to help photographers sell prints. This scholarship was useful when
contextualizing the Architectural Photographic Association’s commercial, utilitarian approach to
exhibition, and highlighted ways in which it differentiated them from other photographic
societies.
The Role of Architectural Photographs in the Nineteenth-Century
The final section of this literature survey provides context regarding the utilitarian role,
political goals, and commerce of architectural photography in the 1850s and 1860s. Scholars like
Claude Baillargeon and Donald English identify architectural photography as a promotion of the
enterprises of political entities, while scholars like Franca Malservisi, Maria Rosaria Vitale and
Christine M. Boyer discuss architectural photography as a tool for cultural conservators, artists,
and architects. Photography and art historian Anne McCauley addresses and studies the
distribution and sale of photographs at this time.
22
Roger Taylor, Photographs Exhibited in Britain, 1839–1865: A Compendium of Photographers and Their Works,
Ottawa: National Gallery of Canada, Library and Archives, 2002.
9
The first major role of architectural photography to be recognized throughout the 1850s
and onward is in the political undertakings of city and country leaders. Claude Baillargeon,
associate professor of art history at Oakland University, focuses on how photographs of the
construction of French monuments like the Sacré-Cœur Basilica in the nineteenth-century were a
way for the nation to demonstrate its power and prowess. His article “Construction Photography
in the Service of International Public Relations: The French Connections” was published as a
chapter in the 2013 Ashgate publication Nineteenth-Century Photographs and Architecture:
Documenting History, Charting Progress, and Exploring the World, edited by Micheline
Nilsen.23 The book itself is also politically charged, exploring how architectural photography
aided in political ventures of industrialization, colonization, and national hubris. Many other
scholars approach architectural photography from a political standpoint, but most relevant to this
study is American scholar Donald E. English’s Political Uses of Photography in the Third
French Republic, 1871–1914.24 This text focuses on how photography evolved into a medium
capable of reaching the masses, which was then manipulated by the political Right. He identifies
the direction which the photographic market of the 1860s was heading towards, making it an
important tool when contextualizing the distribution of photographs from 1858 to 1861.
Beyond political interpretations, the role of architectural photography has been long
established as utilitarian, as it was valued for its efficiency and detail. Christine M. Boyer,
professor and urban historian at the Princeton University School of Architecture, argues that the
photographs of the Mission Héliographique of 1851 were crucial to establishing a collective
23
Claude Baillargeon, “Construction Photography in the Service of International Public Relations: The French
Connections,” in Nineteenth-Century Photographs and Architecture: Documenting History, Charting Progress, and
Exploring the World, ed. Micheline Nilsen, Surrey, UK: Ashgate, 2013, 139–53.
24
Donald E. English, Political Uses of Photography in the Third French Republic, 1871–1914, Ann Arbor, MI:
UMI Research Press, 1984.
10
memory and building nationhood in France.25 This study presents the motivation behind
architectural photographs, arguing that they were intended for preservation and restoration
projects. The photographs of the Mission were never distributed, and remain in an archive to this
day. French architectural historian Franca Malservisi and Spanish architectural historian Maria
Rosaria Vitale presented a study in 2013 on the restoration of the Gros-Horloge in Rouen,
arguing that commissioning photographers to document the process was instrumental in
establishing the role of architectural photography in cultural restorations and in preserving
cultural heritage.26 Their analysis shows how professionals such as conservators and architects
used photography to aid in restoration, yet does not discuss whether or not these photographs
were disseminated. Both of these cases demonstrate how scholars approach architectural
photography, placing historical significance in the creation of the photographs and their use, but
not their distribution.
The leading resource on the distribution of photography in the 1850s and 1860s was
written by Elizabeth Anne McCauley, professor of the history of photography and modern art at
Princeton University. Industrial Madness: Commercial Photography in Paris, 1848–187127 is a
comprehensive review of the photographic market in France. McCauley argues that the demand
at the time was for portraiture and art reproductions, in the form of cartes-des-visites and cased
images. She contextualizes the rise of the photographic market within the Second Empire,
establishing the role of the commercial photographer as aspiring bourgeois, seller of bohemia, or
25
Christine M. Boyer, "La Mission Héliographique: Architectural Photography, Collective Memory and the
Patrimony of France, 1851," In Picturing Place: Photography and the Geographical Imagination, edited by Joan M.
Schwartz and James R. Ryan, London: I.B. Tauris, 2003: 21–54.
26
Franca Malservisi and Maria Rosaria Vitale, "Photography versus the Historical Record: The Role of Photography
in Rouen's Gros-Horloge Restoration," Future Anterior 10 no.2 (2013): 49–65.
27
Elizabeth Anne McCauley, Industrial Madness: Commercial Photography in Paris, 1848–1871. New Haven:
Yale UP, 1994.
11
art copyist. She intentionally omits architectural photography from her book,28 claiming that
other scholars already focus on the individual studios, businesses, and careers of major
architectural photographers of the time like Édouard Baldus29 or Roger Fenton.30
The literature analyzed in these three sections gave me the necessary background to
address my thesis subject. First, the practical literature lays groundwork for appropriate and safe
treatment of photographs, thorough cataloguing, safe and optimal digitizing, and proper
interpretation. Second, literature concerning the photographic exhibitions of other societies
proves that the Architectural Photographic Association has yet to be situated within the discourse
of the history of photography. Finally, most scholars of architectural photography focus on its
creation, use, or political aspects, yet omit a discussion of how the images were distributed and
sold; meanwhile, scholars studying the market of photography do not include architectural
photography in their discussions. This thesis will demonstrate how the Architectural
Photographic Association balances the creation and distribution of architectural photographs, and
how it differs from other photographic societies of the time.
28
Ibid., 8.
See: Malcolm R. Daniel and Barry Bergdoll, The Photographs of Édouard Baldus, New York: Metropolitan
Museum of Art, 1994.
30
See: Gordon Baldwin, Malcolm R. Daniel, and Sarah Greenough, All the Mighty World: The Photographs of
Roger Fenton, 1852–1860, New Haven: Yale University Press, 2004.
29
12
Part One: Preparing an Exhibition
Curatorial Statement
Focus: Architecture in Photographic Exhibitions 1858–1861 explores the role that
exhibitions of photographic societies played in the distribution and sale of architectural
photography in the late 1850s to early 1860s. The Great Exhibition of 1851 in London, held at
the Crystal Palace, was much of the general public’s first exposure to photographs. By the mid1850s, photographic exhibitions became more common throughout Europe, particularly in
Britain and France, and are intrinsically linked with the rise of photographic societies. The
Photographic Society of London, now the Royal Photographic Society, was founded in 1853, and
the Société Française de Photographie was formed in Paris a year later. The focus of this
exhibition, the Architectural Photographic Society, was founded in 1858.
This exhibition features photographs shown at the major exhibitions of photographic
societies in France and Britain, to illustrate the significance of the unique business model of the
Architectural Photographic Association. Photographs exhibited at societies like the Société
Française de Photographie and the Photographic Society of London are paired with those
exhibited at the Architectural Photographic Association, in order to demonstrate how each of
these societies responded to the demand for architectural subjects in the European photography
market.
This exhibition is the culmination of a Master of Arts thesis in Film and Photography
Preservation and Collections Management by Natalie Banaszak of Ryerson University in
Toronto, Canada. Drawn from the collections of the Museum of Fine Arts, Houston, these
photographs give a glimpse into a time period where the relationship between photography and
architecture was still in its infancy. The photographs vary in subject matter, place depicted, and
13
modes of representation, demonstrating that there was no prescribed way to photograph
architecture. Some works were commissioned by governments and political powers; while some
were made to be sold to everyone from tradesmen, like architects or artists, to tourists, whether
they had the means to actually travel or just dreamed of seeing distant cities.
Preparing an Exhibition: Timeline
I worked with a timeline of approximately six months, specifically from January 11th to
June 30th 2016, coinciding with the dates of my placement at the Museum of Fine Arts,
Houston.
By January 30th:
● Survey architectural photographs, to establish the strengths and weaknesses of the
collection
By February 29th:
● Define exhibition ambition
● Begin background research
● Receive approval from department head (Malcolm Daniel, Gus and Lyndall
Wortham Curator of Photography)
By March 30th:
● Obtain contact information for representatives in the conservation department and
the matting and framing department
● Finalize checklist, inform collections manager and department head of choices
● Verify cataloguing information in the database and cross-check with research
● Write curatorial statement
By mid-April:
● Finish digitizing the photographs
● Complete matting and framing recommendations, including: colour of mat, size of
mat, size of frame, colour of frame
14
By April 30th:
● Design brochure to accompany the exhibition
By mid-May:
● Complete wall labels, including the introductory panel, section texts, and object
labels
● Enter label text into the database
By May 30th:
● Review all curatorial writing
● Complete wall elevations
June 30th:
●
Final day to complete any outstanding work
Checklist and Rationale
As per the aforementioned timeline, the first step was to survey the collection of
nineteenth-century architectural photography in order to establish its strengths and weaknesses.
The Museum of Fine Arts, Houston, has over thirty thousand photographs, making it the
museum’s largest collection. Searching the database for photographs dated before 1861 yields
2401 results. 1,500 are by the Fratelli Alinari, in the form of twenty albums donated by the estate
of Mary Lynch Kurtz in the year 2000. Aside from the Alinari albums, there remain 875
photographs created before the year 1861. 284 of these are architectural photographs, and 181 of
these came into the museum through the acquisition of Manfred Heiting’s personal collection
between 2002 and 2004. The Heiting encyclopedic collection is made up of over 4000
photographs that he collected over a span of thirty years. Most photographs chosen for this
exhibition come from the Manfred Heiting Collection.
Upon completing the survey, I made a preliminary selection of photographs which
included work by photographers that exhibited their work in Britain or France from 1858 to
15
1861. I chose this particular time period as it is contemporaneous with the existence of the
Architectural Photographic Association. Of this list of over one hundred images, I flagged the
photographs that were featured in the exhibitions of the Architectural Photographic Association,
the Société Française de Photographie, the Photographic Society of Glasgow, and the
Photographic Society of London. My goal was to allocate twelve to fifteen prints in total, a
majority of them copies of prints exhibited at the Architectural Photographic Society. I ended up
with a final checklist of twelve photographs, eight of which were displayed at the Architectural
Photographic Association, while the other four were featured at exhibitions of other
photographic societies at the time. This set of photographs was chosen in order to visually
present the scope of the Association’s work, and to contextualize it within the models established
by other photographic exhibitions and societies. The full checklist with images, in alphabetical
order by photographer’s last name, follows.
16
List of Photographs
Figure 1: Fratelli Alinari, Lorenzo Ghiberti’s Bronze Door, 1852–1858, albumen silver print from glass negative,
42.8 x 31.7 cm, The Museum of Fine Arts, Houston, 2004.211.
Figure 2: Fratelli Alinari, Campanile for Duomo of Santa Maria del Fiore, Florence, 1852–1858, albumen silver
print from glass negative, 44.4 x 29.5 cm, The Museum of Fine Arts, Houston, 2004.212.
17
Figure 3: Édouard Baldus, Pavillon Denon, Louvre, 1855–1856, salted paper print from glass negative, 44 x 33.8
cm, The Museum of Fine Arts, Houston, 2004.249.
Figure 4: Bisson Frères, Moissac Abbey Cloister, 1857, albumen silver print from glass negative, 35.6 x 45 cm, The
Museum of Fine Arts, Houston, 2004.313.
18
Figure 5: Charles Clifford, [Main Door, Salmanca Cathedral], before 1858, albumen silver print from glass
negative, 43 x 32.4 cm, The Museum of Fine Arts, Houston, 2004.350.
Figure 6: Charles Clifford, Salamanca. Puerta del Convento de las Duenas, 1853, albumen silver print from glass
negative, 39.7 x 27.3 cm, The Museum of Fine Arts, Houston, 2004.351.
19
Figure 7: Tommaso Cuccioni, View of the Roman Forum towards the Capitoline Hill, Looking West, 1855, albumen
silver print from glass negative, 27.3 x 38.1 cm, The Museum of Fine Arts, Houston, 2004.369.
Figure 8: Roger Fenton, printed by Francis Frith & Co., Lichfield Cathedral, Central Doorway, West Porch, 1858
printed 1863–1865, albumen silver print from glass negative, 41.9 x 34.9 cm, The Museum of Fine Arts, Houston,
2014.89.
20
Figure 9: Francis Frith & Co., Columns, Pharaoh's Bed, 1856–1861, albumen silver print from glass negative, 20.4
x 16.4 cm, The Museum of Fine Arts, Houston, 88.233.
Figure 10: Robert Macpherson, View of Rome from the French Academy, Monte Pincio, 1851–1858, albumen silver
print from glass negative, 20.4 x 16.4 cm, The Museum of Fine Arts, Houston, 2004.222.
21
Figure 11: Carlo Naya, Piazzetta of San Marco looking towards the Island of St. George, 1857–1859, albumen silver
print from glass negative, 42.4 x 53.5 cm, The Museum of Fine Arts, Houston, 2004.619.
Figure 12: Robertson and Beato, Tophane Fountain, 1854–1858, albumen silver print from glass negative, 26.5 x
31.2 cm, The Museum of Fine Arts, Houston, 2000.255.
22
Cataloguing and preparing wall labels
Research was a necessary precursive step to completing wall labels for the exhibition, in
order to ensure cataloguing information was correct. The Fratelli Alinari photographs only
needed a change to the constituent record. Before the change, all Alinari photographs had three
constituents: Giuseppe Alinari, Romualdo Alinari, and Leopoldo Alinari. This approach did not
leave room for their corporate name, Fratelli Alinari, and made searching more difficult. One had
to assume that searching for any Alinari brother would yield all of the Alinari records. After
consulting with data standards specialist Merrianne Timko, collections manager Jason Dibley,
and photography cataloguer Selina Lamberti, we decided to change all of the Alinari records to
have a primary display constituent of Fratelli Alinari, as this more accurately reflects the shared
attribution between all three brothers.
Most of the database records had accurate titles, credit lines, and dates. There were,
however, two records that needed particular attention: 2004.350, or Charles Clifford’s [Main
Door, Salamanca Cathedral]; and 2000.255, and Robertson and Beato’s Tophane Fountain. The
Clifford record was easy to fix, I replaced an unclear title with a concise, descriptive one. The
previous title read “Salamanca, Nord Thor, Gallerie Sud,” which was based on a modern pencil
inscription on the mount. Searching for that title in other collections like the Metropolitan
Museum of Art, the J. Paul Getty Museum, and the Victoria and Albert Museum yielded no
results, and the translation did not correspond to what was depicted in the photograph. Museum
policy dictates that it is better to have an accurate, descriptive title as the primary display than an
inaccurately attributed title, so I moved the old title lower in the hierarchy and removed it from
display.
23
The virtual record for Robertson and Beato’s Tophane Fountain demanded more
attention. First of all, the photograph was incorrectly attributed to the Fratelli Alinari, despite an
inscription in the negative bearing the signature “Robertson & Beato photog.” Previous
cataloguers had omitted this inscription, which I added into the appropriate field. I first deleted
the Fratelli Alinari from the record, and added James Robertson and Felice Beato, after
consulting sources to be certain it was Felice and not Antonio Beato working with James
Robertson at the time. Most scholars believe that James Robertson worked with Felice Beato
under the corporate body name of Robertson & Beato, and that the corporate body Robertson &
Beato & Co. was used when Antonio Beato joined them.31 Besides the constituent problems, this
record also had a date of c.1860 attributed. I can say for certain that it is taken before 1858, as it
was exhibited at the Architectural Photographic Association that year under the name
“Constantinople: Fountain at Tophanne.”32 Robertson only opened his studio in Constantinople
in 1854 or 1855.33 By 1857, Robertson and Beato departed for a Grand Tour of Jerusalem, and
by 1858, Beato was spending the majority of his time in India.34 Therefore, a likely date range
for the photograph falls between 1854 and 1857. Museum policy is to indicate this range as a
display date, with an en dash (–) in between, like so: “1854–1857.” The start and end dates then
have to be accordingly assigned as 1854 and 1857. The final issue to resolve was the title of the
photograph. The inscription in the negative reads “La fontaine de Xophinna”, so this was the
only title on the record before I catalogued it. Instead, I assigned the English language
31
Anne Lacoste, Felice Beato: A Photographer on the Eastern Road, Los Angeles: J. Paul Getty Museum, 2010:
184.
32
Taylor, 240.
33
Richard Pare, Photography and Architecture, 1839–1939, Montreal: Canadian Centre for Architecture, 1982: 245.
34
Lacoste, 184.
24
translation, Tophane Fountain, as the primary title. This record was the most involved
cataloguing I had to do for the exhibition.
The first step in writing label text was to identify my exhibition’s “big idea,”35 to which
all labels would refer back to. Beverly Serrell indicates that big ideas “define or describe the
content of the exhibition,”36 and “provide a thread of meaning, coherence, and weight.”37
Though the big idea can take the form of different sentence structures, mine became the
following question: where could architects obtain photographs in the 1850s and 1860s, and how
did they use them? After I wrote my big idea I shifted focus to the wall labels, for which I
followed Serrell’s “Ten Commandments:”38
“1. Labels should begin with concrete, visual references to the objects they interpret to
bring them to life.
2. Labels should relate to the big idea of the exhibit, not ramble without focus or
objectives or contain sub-sub-subtopics.
3. Labels should emphasize interpretation (offering provocation) over instruction
(presenting information).
4. Labels should know their audience and address visitors’ prior knowledge, interests,
and/or misconceptions.
5. Labels that ask questions should be visitors’ questions
6. Label design should reflect the label’s content or context and have a recognizable
system of organization of label types.
35
Beverly Serrell, Exhibit Labels: An Interpretive Approach, second ed., Lanham: Rowman & Littlefield, 2015: 7.
Serrell, 9.
37
Ibid., 12.
38
Ibid., 2.
36
25
7. Labels should be written with a vocabulary that is within reach of the majority of
visitors.
8. Labels should be short and concise, more like a tweet than a tome.
9. Labels for interactives should have instructions or interpretations integrated in logical,
intuitive ways.
10. The typography (typefaces, sizes, design, colors, lighting, materials, and placement)
should make them legible and easy on the eyes, not busy or distracting.”39
The completed wall labels can be seen in Appendix A.
Preservation considerations
When works are selected for exhibition at the Museum of Fine Arts, Houston, they are
sent to the conservation department before framing. The conservators complete a condition
report on all of the photographs, and, upon establishing that the photographs are safe for
exhibition, execute any necessary treatment. With nineteenth-century material, this usually
involves a surface clean or the painting-over of a blemish. These particular photographs were all
in stable condition for exhibition. The completed condition reports and specific
recommendations for each photograph can be found in Appendix B, while this section will focus
on general guidelines.
Calculating the estimated lux-hours is important with any exhibition in order to ensure
the photographs are not under duress. Bertrand Lavédrine recommends a maximum light dose of
12000 lux-hours per year for nineteenth-century photographs, identifying them as highly
39
Serrell, 2–3.
26
sensitive to light.40 The Conservation Centre for Art and Historical Artifacts in Philadelphia,
however, labels albumen and salted paper prints as only moderately sensitive to light, and
recommends a much higher maximum light dose of 50,000 lux-hours per year.41 The formula
they prescribe to calculate total lux-hours is: the average lux, multiplied by the exhibition hours
per day, multiplied by the number of days in exhibition.42 The Museum is open for an average of
8.33 hours a day.43 I proposed a four-month exhibition for these photographs, or 108 days. This
total excludes every Monday, since the Museum is closed and the exhibition lights stay off.
Therefore, I calculated that the average lux for each photograph should not exceed 55.57, for a
four-month exhibition display, at 8.33 hours per day. This will yield 50,000 lux-hours per year,
falling within the exposure range recommended by the Conservation Centre for Art and
Historical Artifacts in Philadelphia. Finally, dark time for the photographs should ideally be
three times longer than the exhibition, so I recommended a twelve-month rest period for these
objects.
Matting and Framing
After taking necessary precautions to maintain a safe gallery space for the photographs, I
ensured to do the same with each object's microenvironment. This section features matting and
framing suggestions for each photograph, including a description of the current housing
situation. I examined each photograph, and proposed an appropriate mat size and colour for each.
40
Bertrand Lavédrine, Photographs of the Past: Process and Preservation, Los Angeles: Getty Conservation
Institute, 2009: 297.
41
Sarah Wagner, Connie McCabe, and Barbara Lemmen, Guidelines for Exhibition Light Levels Fir Photographic
Materials, Report, May 2007, Accessed April 20, 2016, http://www.ccaha.org/publications/paper: 3.
42
The Conservation Centre for Art and Historical Artifacts, Light Exposure of Artifacts on Exhibition: CCAHA
Guidelines, Web Publication, http://www.ccaha.org/uploads/media_items/light-exposure-of-artifacts-onexhibition.original.pdf
43
Calculated on 29 May 2016, based on the opening hours available at: http://www.mfah.org/visit/hours-andadmissions/.
27
In tandem with this, I selected frame sizes and colour. As with the condition reports, this section
focuses on general recommendations for all of the photographs, while more detailed suggestions
for each photograph can be found in Appendix C.
All of the photographs are currently in a 4-ply mat, save for one: Roger Fenton’s
Lichfield Cathedral, which has a 4-ply back and an 8-ply front. I recommend all of the
photographs be re-matted to have 8-ply mats, as it provides the most support for the photographs.
Though they are all mounted on a secondary support, a mat made completely out of 8-ply board
will help prevent the photographs from warping, and separates the photographs from the glazing
when framed.44 The photographs should all be mounted to the mat using folded photo corners,
made out of conservation-quality, silversafe paper. The folded corners protect the object from
damage due to harmful adhesives or residue,45 and can be easily opened to remove the object
from the mat at any time, should anyone have to inspect the verso, digitize the photograph, or
perform conservation treatments and exams. Frames for the photographs should be made out of
wood, with an acrylic glazing. Glass glazing can cause problems since it is fragile, and heavy.46 I
recommended an ultra-violet (UV) filtering acrylic for these photographs, as it is lighter, and
serves to protect the objects from damaging UV radiation.47
The Museum of Fine Arts, Houston, uses Artique 8-ply, solid colour core, alpha-cellulose
conservation-quality mat boards, available for purchase from Larson-Juhl. I recommended this
mat board for all of the photographs. Depending on what suited the tonality of each object, I
suggested one of two shades of a tan colour for the mat board. The first shade, ‘Thatch,’ is
slightly lighter than the second shade, ‘Birch.’ I chose shades that are not too different from each
44
Jill Snyder and Maria Reidelbach. Caring for Your Art. New York, NY: Allworth Press, 2001: 59.
Lavédrine, 298.
46
Ibid., 299.
47
Ibid.
45
28
other, to ensure that the photographs maintain an even hierarchy. I assigned “Thatch”, the lighter
shade, to the following photographs: Charles Clifford’s [Main Door, Salamanca Cathedral], the
Robertson and Beato, the Édouard Baldus, Fratelli Alinari’s Campanile for Duomo of Santa
Maria del Fiore, Florence, the Tomasso Cuccioni, the Roger Fenton, and the Carlo Naya. I
recommended “Birch” for the remaining photographs: Charles Clifford’s Puerto del Convento de
las Duenas, Fratelli Alinari’s [Ghiberti’s Baptistery Doors, Florence], the Francis Frith, the
Bisson Frères, and the Robert Macpherson.
Wall Elevations and Layout
The exhibition wall is 13.7 meters (45 feet) long and 2.9 meters (9.5 feet) tall. The
photographs are to be hung with their center point at 152.4 centimeters (60 inches) from the
floor, as this matches and complements the History of Photography design. Once I became
familiar with the gallery space, I created a vector drawing set of the space in a complementary
program, Inkscape. The elevation drawings, which include each photograph in its frame and
corresponding wall label, are attached as Appendix D. The drawing allowed me to visually
confirm there was an adequate amount of space between the photographs and permanent wall
features, such as doors, signs, and electrical outlets, so the exhibition could be mounted with
ease.
Preparing an exhibition pamphlet
Much of the Manfred Heiting Collection was photographed when it came into the
Museum’s collection in 2004. However, a large portion of the photography done between 2000
and 2004 has a very skewed white balance. Some appear overwhelmingly orange or yellow in
29
tone, while others have a green or blue hue. Eight out of the twelve photographs in my exhibition
had this problem, and I needed to photograph them again before including them in my thesis and
in the exhibition pamphlet. The following section describes how I digitized these photographs.
Figure 13: Digital image taken around 2004 for Tommaso Cuccioni, View of the Roman Forum towards the
Capitoline Hill, Looking West, 1855, albumen silver print from glass negative, 27.3 x 38.1 cm, The Museum of Fine
Arts, Houston, 2004.369.
First, I transported the photographs from their home vault to an interim art storage with a
large copy stand that is always set up for digitization. Handling the photographs is easier and
safer with two people, so I sought help from Photography Collections Cataloguer Selina
Lamberti. The copy stand consists of a Nikon D100 camera on a “Super Repro” copy stand made
by an Italian manufacturer, Industria Fototecnica Firenze. Attached are two lights, powered by
Hikari Halogen JDD bulbs, at a 45 degree angle towards the centre of the copy stand. Bertrand
Lavédrine identifies the preservation of digital files to be a problem, since technology is
changing so rapidly.48 The Nikon D100 has the capability to shoot in the Tag Image File Format
48
Lavédrine, 310.
30
(TIFF), which are useful to keep in case the edited images, in JPG format, should ever become
obsolete or corrupt in the process of conversion.
To begin, I placed a fresh piece of white mat board onto the copy stand, to guarantee a
clean surface for the photographs. Next, I placed the photographs one by one on the mat board. I
lay a colourchecker underneath all of the photographs, ensuring that the tonality could be
corrected in Adobe Photoshop when processing the digital files. I also included a label
underneath each photograph, with the accession number and title of the photograph. Doing this
made naming the files and distinguishing nondescript versos of the photographs much simpler.
For each photograph, I set the camera settings according to the Museum standards, which is an
aperture of f/9.5 and a focal length of 35mm. Initially, the photographs were either out of focus
or blurry, because the camera was at the top of the copy stand and vibrating when I pressed the
shutter. I set the camera to a ten second self-timer, which reduced the shake.
After the photographs were digitized, I edited them in Photoshop. For the most part, the
white balance was correct and the colours did not need manipulating. In cases where the tonality
had shifted, I used the colourchecker to set the white balance and correct the digital file. I also
straightened and cropped all of the images, but did not make any modifications beyond that, as
per Lavédrine’s recommendations to keep all photographs as unedited as possible.49 Finally, I
saved two versions of each file: one version cropped to the photograph, and one with the
secondary support included. I uploaded three versions to The Museum System (TMS), the
aforementioned two versions of the recto, and one photograph of the verso. I then selected the
two images of the recto as ‘approved for web’, to appear on the Online Collection Module,
which will reduce the need for handling the physical object.
49
Lavédrine, 306.
31
Digitizing the prints was an essential first component in the design of the brochure. I
chose the form of a brochure for a publication to accompany the exhibition due to its short and
concise, yet informative and visually appealing format. The brochure features the curatorial
statement, information on the Architectural Photographic Association, and reproductions of all of
the images included in the exhibition. Just as in the text panels and label text, I followed
Museum standards and chose the EB Garamond font. I selected colours that were complementary
to the photographs themselves. The text for the brochure is a condensed version of Part Two of
this thesis, and a template I drafted for the brochure is attached as Appendix E.
32
Part Two: The Exhibitions of the Architectural Photographic Association (1858–1861)
The Architectural Photographic Association was formed in May of 1857,50 and officially
inaugurated at a meeting on the night of Thursday, January 7th 1858.51 The president of the
Association was Charles Robert Cockerell (1788–1863), British architect and professor of
architecture at the Royal Academy in London from 1839 to 1859.52 The architects involved with
the Association travelled and purchased prints from photographers’ studios at the destinations of
these travels. They assembled these prints into an exhibition in London, where a subscription fee
of one guinea or upwards per annum would grant access for patrons to come and purchase copies
of photographs that were on display. After the exhibitions, the Association would order the
subscribers’ choices of prints from each photographer.53 At the inaugural meeting, Cockerell
announced that the number of subscribers had already reached 750, and that the Association had
managed to assemble 360 photographs from various European cities, with plans to soon expand
to the Middle East and Asia.54 The organization saw immediate success upon its first exhibition,
revealing a high demand for architectural photographs, and the opportunity for the existence of a
market in 1858. The Association held four annual exhibitions from 1858 until its dissolution in
1861. This essay postulates that the growth, critical and public reception, and ultimate downfall
of the Architectural Photographic Association all demonstrate the demand for photographs as
source material for architects in the 1850s and 1860s.
50
“Société Photographique d’Architecture,” Bulletin de Société Français de Photographie (1858): 48.
"Architectural Photographic Association," The Literary Gazette: Journal of Belles Lettres, Science, and Art, no.
2138 (January 9, 1858): 42.
52
See “C.R. Cockerell, R.A.,” Royal Academy of Arts, Collections Online:
http://www.racollection.org.uk/ixbin/indexplus?_IXACTION_=file&_IXFILE_=templates/full/person.html&person
=5580
53
The Literary Gazette, 43.
54
Ibid.
51
33
The Association’s First Exhibition: 1858
The first exhibition of the Architectural Photographic Association took place from
January to February of 1858. To guarantee the sale of a certain number of prints by each
photographer, subscribers could only choose one subject from each of the screens into which the
exhibition was divided. This prevented subscribers from “assembling a portfolio of photographs
documenting one building.”55 Photographers exhibiting in 1858 included Fratelli Alinari,
Édouard Baldus, Francis Bedford, Bisson Frères, Charles Clifford, Roger Fenton, Robertson &
Beato, Captain Inglefield, William Lyndon Smith, Robert Cade, Royal Engineers, John Wheeley
Gough Gutch, and Reverend Joseph Lawson Sisson.56 This relatively small group of
photographers nevertheless represents the broad geographical scope of the Association right
from the outset: Charles Clifford was photographing Spain, Robertson and Beato in
Constantinople, the Fratelli Alinari were in Italy, Baldus and the Bisson Frères were
photographing French buildings and monuments, and Fenton and Bedford were in the English
countryside.
Amongst the 364 photographs featured in this exhibition were Robertson and Beato’s
Tophane Fountain, Fratelli Alinari’s Campanile for Duomo of Santa Maria del Fiore, Florence,
and Lorenzo Ghiberti's Bronze Door, Gates of Paradise, Baptistery of San Giovanni, Florence,
Édouard Baldus’ Pavillon Denon, Louvre, and Charles Clifford’s Salamanca: Puerto del
Convento de las Duenas and [Main Door, Salamanca Cathedral]. These photographs are all
either albumen or salted paper prints from wet collodion glass plate negatives, a medium which
was favoured for its sharpness — a result of the transparency of the glass. They are all large
55
Elwall, 146.
Roger Taylor, Photographs Exhibited in Britain, 1839–1865: A Compendium of Photographers and Their Works.
Ottawa: National Gallery of Canada, Library and Archives, 2002.
56
34
prints for the time, the smallest being Robertson & Beato’s, measuring 26.5 by 31.2 centimeters,
while the rest are approximately 40 by 30 centimeters. The size of the photographs allows them
to either depict an entire building, or enhance specific architectural details. For instance, Baldus’
Pavillon Denon, Louvre, Alinari’s Campanile, and Robertson and Beato’s Tophane Fountain
portrayed entire buildings. Conversely, doors were a specific architectural detail that was the
focus of Clifford’s [Main Door, Salamanca Cathedral], the Fratelli Alinari’s Lorenzo Ghiberti’s
Bronze Door or Clifford’s Salamanca: Puerto del Convento de las Duenas. While the specific
locations and subjects of these prints vary, they demonstrate the Association’s preference for
larger images with sharp detail which were found useful by architects as source material.
The notion of using photography as source material was still fairly new, developed
chiefly by the 1851 Mission Héliographique. Anne de Mondenard argues that the Mission was
the first joint public commission in the history of photography.57 The Mission commissioned five
photographers to travel throughout France, creating photographs of a list of buildings and
monuments. These photographs were instantly archived for use as visual documents for
preservation and restoration, and ironically undiscovered until the late twentieth-century.58
Though the Mission created photographs, architects did not immediately view them as useful
resources. By 1857, the representation of architecture through photography had become a more
established tradition.59 Henry Cole, director of the South Kensington Museum in London, now
the Victoria and Albert Museum, was very active in the 1857 to 1865 construction of the
57
Anne de Mondenard, La Mission Héliographique: Cinq Photographes Parcourent la France en 1851, Paris:
Centre des Monuments Nationaux, 2002: 12.
58
de Mondenard, 12–13.
59
Martin Barnes and Christopher Whitehead, "The 'Suggestiveness' of Roman Architecture: Henry Cole and Pietro
Dovizielli's Photographic Survey of 1859," Architectural History 41 (1998): 197.
35
Museum’s building and grounds.60 While visiting the Vatican Museum in 1858, Cole wrote: “the
first step to be taken, to turn it to profitable account for [South] Kensington, would be to have the
whole photographed: not the separate objects, but views on each side, in as many portions as
necessary.”61 He wanted the Museum’s architect, Captain Fowke, to travel to Rome himself, but
Fowke did not travel to Italy until 1863. Barnes and Whitehead argue that Cole’s failure to get
Fowke to Italy added a sense of urgency to Cole’s collection of photographic records of the
buildings in Rome.62 In 1859, Cole commissioned Italian photographer Pietro Dovizielli to take
49 photographs of buildings he was particularly fascinated with. Dovizielli’s photographs
focused on architectural details with a “didactic and anti-picturesque bias.”63
While the Association’s first exhibition received excellent reviews and was deemed a
success, certain architects complained about the subject matter of the photographs, claiming that
there were not enough photographs of architectural details and sculptures. This was due to the
fact that the Association was not commissioning photographs, but purchasing examples of prints
which photographers had already taken. Unlike Henry Cole, the Association did not have the
power to dictate what was photographed; the architects only chose which photographs to collect
and how to best arrange them in the exhibitions. The photographs in the first exhibition of the
Association demonstrate the varied approaches photographers took when creating visual records
of buildings. There are no photographs of architectural details, save for the few views of doors
and entryways. Though the photographs were executed with “minimal artistic input from the
photographer,”64 as Cole preferred, the exhibition of the Architectural Photographic Association
60
Barnes and Whitehead, 193.
Ibid., 196.
62
Ibid.
63
Ibid., 198.
64
Ibid., 197.
61
36
in 1858 would not have provided him with sufficient visual documentation to create the South
Kensington plans, lacking crucial detail elements such as colonnades, steps, obelisks, and
fountains.65
Despite the lack of specific subject matter, the Civil Engineers and Architect’s Journal
praised the exhibition and photographs, emphasizing how useful this endeavour would be for
architects.66 The utilitarian nature of the exhibitions was immediately recognized amongst
architects and engineers, who constituted the predominant readership of this journal. Architects,
who could not attend the exhibitions of the Association could read the reviews published in
professional journals, such as the Civil Engineers and Architect’s Journal or Builder. This way,
they could stay current with popular architectural sites, and become acquainted with the names
of photographers should they decide to purchase prints of certain buildings for their own
inspiration.
News of the exhibition exceeded the professional realm, and journals which appealed to a
more general public featured news of the Association. The Literary Gazette writes that “wide as
will be the amount of information spread by this Association, and powerful as is the feeling of
interest it inspires, these are nothing compared with the advantages that will be reaped from it by
architectural students and professors.”67 This review was also translated into French and
published in La Lumière a few weeks later,68 indicating that the Association’s first exhibition had
a widespread impact. The review itself confirms public interest in the subject matter, while also
outlining the photographs’ potential as valuable source material for architects. A hopeful
sentiment for the Association’s future success was echoed by the reviews of its first annual
65
Ibid.
“Architectural Photographic Association,” The Civil Engineers and Architect’s Journal, no. 21 (1858): 62.
67
Ibid.
68
"Association Photographique D'Architecture," La Lumière, no. 4 (January 23, 1858): 14.
66
37
exhibition, many of which were included not only in profession-specific journals but in popular
weekly newspapers and journals as well.69 It was not uncommon for photographic journals and
bulletins of the time to review international exhibitions, but the reviews never before placed a
focus on architectural subject matter alone. The publication of the exhibition reviews in popular
weeklies such as The Spectator suggests that the Association reached a wider audience than just
architectural or photographic professionals. In reading reviews of the exhibitions of the
Architectural Photographic Association, the general public could refine their affinity for the
subject matter, and develop a discerning taste for architectural photographs. The reviews served
as an invaluable network, and, even today, allow a glimpse into the production of major
photographers from Europe and Asia.
The 1859 Exhibition
Despite criticisms and shortcomings, the Association decisively maintained the same
model for its second exhibition, which took place from December 1858 to February 1859. It
featured 383 photographs, including works by: Robert Macpherson, Messrs Cimetta, Arthur
James Melhuish, Robert Cade, Robertson and Beato, Reverend Percy Lousada, Archibald Lewis
Cocke, Édouard Baldus, Francis Frith, William Lyndon Smith, John Wheeley Gough Gutch, the
Royal Engineers, Francis Bedford, and Carlo Ponti.70 Robert Macpherson exhibited his entire
oeuvre of one hundred and twenty photographs of Rome, the largest body of work by a single
photographer. In this group was his photograph titled The Garden-Front of the Villa Medici on
the Pincian Hill. The foreground of this photograph is the fountain of the Villa Medici, while a
panoramic view of Rome unfolds in the background. The photograph is neither focusing on the
69
70
See also “Architectural Photographic Association,” The Spectator, no. 1579 (October 2, 1858): 30–31.
Taylor, 333.
38
Villa, which did not even make it into the frame, its garden, which is barely visible, nor the full
panorama of Rome, which is shielded by trees. It is instead a marriage of the three in which
Macpherson plays into the viewers’ imaginations by including architecture in the context of its
surroundings, allowing them to situate themselves within the scene. This particular image marks
a shift from the previous exhibition of the Association, as the previously mentioned photographs
all showcase entire buildings or specific architectural components, including as little of the
surroundings in the frame as possible. This photograph is a different type of source material for
architects; it conveys the general mood and feel of the environment surrounding the Villa, not the
structure itself. Again in this case, Cole would not have been satisfied with this image. He
referenced the Villa in his journal, and wrote: “The garden front of the house is ascribed to M.
Angelo. It has been arranged so as to receive bas reliefs of antiquity and they are very happily
brought in. We should have a photograph of it and also of the Colonnade at the side. This is
about 25 feet high and I think shows that that height would hardly be sufficient for our own
Colonnade at Kensington.”71 Even though Macpherson’s framing leaves out all of these details,
his entire body of work, which accounted for almost one third of the total photographs in the
exhibition, was highly praised and hailed as the highlight of the exhibition.72
Also in 1859, the Société Française de Photographie held its third annual photographic
exhibition in what is now the Grand Palais73 in Paris. There are differences between the two
exhibitions which help provide context for the workings of the Architectural Photographic
Association. The Association was the only organization that had a specific focus on architectural
71
Barnes and Whitehead, 196.
Elwall, 149.
73
The exhibition catalogue refers to the location as the southwest pavilion of the Palais de Champs-Élysees. This
was actually the Palais d’Industrie, an exhibition space built for the 1855 Paris World Fair, and demolished in 1897
to make way for the Grand Palais, erected in 1900.
72
39
photography. Exhibitions of other photographic societies, like the Société Française de
Photographie or the Photographic Society of London, did not place restrictions on subject matter.
However, research shows that they both predominantly featured art reproductions, portraiture,
and landscapes.74 The Association was filling a void in terms of the availability of architectural
photography. Gustave Le Gray only exhibited a single photograph of architecture in 1859 at the
Société Française de Photographie. In contrast, and reverting to the previous example, Robert
Macpherson had one hundred and twenty photographs from Rome in the Architectural
Photographic Association’s 1859 exhibition. Architects looking to draw source material had
much more to choose from at the Association. In addition, the Société Française de Photographie
had a jury system in place in order to decide which photographs would be on display.75 In that
way, it was run akin to Parisian Salons, thereby attributing the photographs with a status of art
objects. Only what were defined as the best examples of photography would be considered. The
jury was made up of a variety of professionals: photographers, including Hippolyte Bayard,
Gustave Le Gray, and Louis-Rémy Robert, who was also the head of painting at the Sèvres
porcelain manufactory (then called the Royal Porcelain Manufactory); theoretician Léon
Foucault, then a physicien (physicist) at the Imperial Observatory; and deputy of the National
Assembly Adolphe Moreau, to name a few. The heterogeneous jury displayed the society’s
diverse interests and offered differing perspectives on photography. In contrast, the Architectural
Photographic Association was run solely by professionals in the field of architecture, and the
74
This can be deduced from perusing the exhibition catalogues, either the SFP catalogue which can be found at
‘Société Française De Photographie, Catalogue De La Troisième Exposition De La Société Française De
Photographie, Paris: Renou Et Maulde, 1859, doi:http://www.e-rara.ch/doi/10.3931/e-rara-15868,’ or the catalogue
for the 1859 exhibition of the Photographic Society of London, found in Roger Taylor, Photographs Exhibited in
Britain, 1839–1865: A Compendium of Photographers and Their Works. Ottawa: National Gallery of Canada,
Library and Archives, 2002.
75
Société Française De Photographie, Catalogue De La Troisième Exposition De La Société Française De
Photographie, Paris: Renou et Maulde, 1859. doi:http://www.e-rara.ch/doi/10.3931/e-rara-15868: 2.
40
chosen photographs were specifically intended to serve architects and please subscribers. As
architects were recognizing the uses for these photographs, they validated the work of the
Association. The priorities of the Association were simultaneously utilitarian and commercial:
the aim was not only to show triumphs of photography as art, but to distribute these architectural
photographs as visual records and profit from the subscription service.
In all of these ways, the Architectural Photographic Association’s exhibition of 1859 was
unprecedented and helped solidify the Association’s reputation amongst architects. The third and
largest exhibition opened in 1860, and was made up of 510 photographs.76 Reviews indicate that
architects were impressed with photography’s ability to capture details that escaped the eyes of
draughtsmen or sketch artists.77 This exhibition is strangely excluded from Roger Taylor’s
compendium of British photographic exhibition catalogues, making it difficult to access
information regarding which photographers were featured and what types of photographs they
included.
The Association’s Downfall
The year 1861 marked the height of the Association’s activities, and that year’s
exhibition was also its last. Besides the exhibition itself, the Association held a series of lectures,
during which the attendees debated the use of photography versus architectural drawings.78 This
final exhibition was the Association’s most diverse moment, including works by: the Fratelli
Alinari, Thomas Annan, Captain Austin, Édouard Baldus, Thomas J. Barnes, Francis Bedford,
Bisson Frères, William Jr. Church, Archibald Lewis Cocke, Cundall & Downes, Captain
76
Taylor, 309.
Elwall, 149.
78
Ibid 153.
77
41
Dawson, Philip Henry Delamotte, Captain Henry Dixon, Dolamore & Bullock, R. Eaton,
Francis Charles Earl, Francis Frith, W. T. Frost, Captain Robert Gordon, John Wheeley Gough
Gutch, Gustave Le Gray, Dr. William Despard Hemphill, Robert Macpherson, Arthur James
Melhuish, W. J. C. Moens, James Mudd, Dr. John Murray, W. H. Nichols, Carlo Ponti, Rev.
Joseph Lawson Sisson, Thomas Sutton, Stephen Thompson, and Thomas Tyley.79 These
photographers represent the broad scope of the Association and its growth since 1858. Included
in this exhibition was Francis Frith’s photograph from Egypt, Columns, Pharaoh’s Bed.80 The
photographer also exhibited his photographs of Egypt at the Association’s second annual
exhibition in 1859. Columns, Pharaoh’s Bed, Philae is smaller than the other photographs
discussed in this essay, measuring at 20.4 centimeters tall by 16.4 centimeters wide. Despite its
size, this photograph of the Greek-style temple’s columns is so sharp that names and years,
carved into the stone columns, are visible to the naked eye. It embodies what both the
Association and architects valued as useful: a sharp image depicting a particular architectural
style or detail.
Trouble began in 1859, after just two exhibitions. The Association could not distribute
the photographs to subscribers fast enough, and the print quality was often inferior to the prints
in the exhibitions. Critics also regarded the elaborate system for purchasing and distributing
photographs as too commercial, saying: “photographs are put down at a price, so that a more
commercial aspect is given to it than is desirable.”81A motion was put forth to dissolve the
organization altogether but the Association was not disbanded, much to the relief of the Building
News who argued that “without the APA… the supply of photographs to the architect would be
79
Taylor 309–310.
Taylor 333.
81
Elwall, 146.
80
42
threatened — it would be unwise to rely on the output of amateurs who would probably forsake
photography when the novelty had worn off.”82 Instead, members called for a change of
leadership and elected Sir William Tite as president.83 Tite was a British architect, Member of
Parliament from 1855 until his death in 1871, and president of the Royal Institute of British
Architects from 1861 to 1863 and 1867 to 1870.84 Despite these changes, the Association was in
deep trouble by 1861: the exhibition costs exceeded any profits. This time, the Association was
dissolved, and the majority of its collection of prints sold in order to pay off debts.85 It was
reconstituted once more, with an altered collection mandate to commission photographers, not
architects, to travel abroad. The photographers captured negatives that the Association collected
and printed from, sending proofs to subscribers each year. In this way, the Association and its
photographers had more control over the subject matter and style. In 1862, photographers
Cundall and Downes took thirty-six views of Wells Cathedral, and each subscriber was sent
eleven of these. In 1863, the Association produced twenty-two photographs of French
architecture, and journeyed again to France and to Germany in 1864, 1866, and 1868. According
to Elwall, by 1868 the need for the Association diminished as photographs became cheaper and
more widely available on the popular market.86
The Association’s ultimate demise was not due to its inability to adapt to the changing
market. The fact that the Association was disbanded after four years indicates that its focus was
not purely commercial, but instead on supplying photographs to architects. If the Association’s
goals were purely commercial, its downfall would have been avoided at all costs. Like many
82
Elwall 149.
Elwall, 148.
84
Campbell Dodgson, Dictionary of National Biography, 1885–1900. Vol. 56. London: Smith, Elder, 1898: 418.
85
Elwall, 154.
86
Ibid.
83
43
private, commercial, photographic studios, it would have sought investors to stay afloat. The
financial troubles of the Association serve as a portrait of the climate of architectural
photography on the commercial market, since it was overwhelmed by demand after just four
years of exhibitions. The Association became a victim of its own success, ultimately thwarted by
its inability to keep up with the popularity of its unique business model.
Conclusion
Many questions regarding the Architectural Photographic Association still remain. Who
were the Association’s subscribers? How many subscriptions were sold to architects, and how
many to members of the general public? Were there recreational enthusiasts of architectural
photography, and did they subscribe? These questions would help identify how the Association
evolved over time, and how it changed the role of architectural photography in society. This
essay concludes that the Association acted as a catalyst for architectural photography, making it
accessible to architects and the general public alike through exhibitions and their reviews. Its
unique business model and initial success demonstrate the demand for architectural photography
in the 1850s, while its brief history and downfall serve as an indication of how that demand
rapidly evolved into a potential market that could not be served. The Association made the
subject more accessible, and hosted discussions and debates on the benefits of photography as a
draughtsman’s tool. The exhibitions of the Architectural Photographic Association validated
architecture as a more popular subject matter in photography than described in the literature,
presenting many of its uses by the end of the 1850s. The fact that the Association was led by
architects, not photographers, distinguishes it from other societies and certainly helped to solidify
its reputation. The widespread reviews of the Association, published in popular weeklies like The
44
Spectator and specialized photographic and architectural journals alike, demonstrate the
immediate impact the Association had on the public. Finally, the downfall of the Association
proves that not only was there a demand for architectural photography, but that it grew to exceed
the means of the Association in just four years. Overall, this essay serves as a call to action for a
shift in scholar’s emphasis from how photographs were created to how they were distributed. In
writing future histories of architectural photography, the Architectural Photographic Association
should not be ignored, deepening the current understanding of the photographic market from
1858 to 1861.
45
Appendix A: Wall Labels
Francis Frith & Co. (British, founded 1859)
Columns, Pharaoh's Bed
1856–1861
Albumen silver print from glass negative
Museum purchase funded by Mr. and Mrs. Alexander McLanahan in honor of Edward B.
Mayo
88.233
A copy of this photograph was on display at the Architectural Photographic Association’s largest
and final exhibition in 1861. Critics of the Association’s exhibitions always commented on the
lack of photographs of architectural details, and Frith certainly caters to this need with this
particular image. The photograph is sharp, bright, and packed with details: even though it is the
smallest in this exhibition, you can read the names and dates travellers carved into the columns
with your naked eye.
Francis Frith made his first of three tours to Egypt and Lebanon in 1856. He worked
systematically to provide pictorial evidence of Biblical sites, publishing his work for a British
Victorian audience upon his return. Frith’s photographs provide a sense of romantic meandering
from one Middle Eastern site to another, without ever having to leave your armchair.
James Robertson (British, 1813–1888)
Felice Beato (British, born Italy, 1832–1909)
Tophane Fountain
1854–1858
Albumen silver print from glass negative
Gift of Mrs. Efrem Kurtz
2000.255
James Robertson opened a commercial photographic studio in Constantinople (now Istanbul) in
1854, and soon after, Felice Beato joined his practice. They signed their negatives with the
corporate name “Robertson & Beato photog.,” as visible in the lower right of this photograph.
Here, they depict the eighteenth-century Tophane Fountain, with a ladder leaning up against the
side of the wall serving as a measuring tool for viewers to grasp the scale of the structure. They
still capture some of the movement of the busy market square on the right side of the fountain,
giving us a sense of the busy city functioning around this building. However, this does not
interfere with the sharpness of the building details, a style which was favoured amongst
46
architects who found source material amongst the photographs exhibited by the Architectural
Photographic Association in 1858.
Fratelli Alinari (Italian, 1852–)
Gates of Paradise, Baptistery of San Giovanni, Florence
1852–1858
Albumen silver print from glass negative
Museum purchase funded by Alice C. Simkins in honor of Isabel B. Wilson, The Manfred
Heiting Collection
2004.211
Fratelli Alinari (Italian, 1852–)
Campanile for the Cathedral of Santa Maria del Fiore, Florence
1852–1858
Albumen silver print from glass negative
Museum purchase funded by Craig Lidji in memory of Lee Hage Jamail, The Manfred
Heiting Collection
2004.212
The Alinari Brothers first established an international reputation for themselves based on their
photographs of Florence when they exhibited at the Paris Exposition of 1855. This image
captures the entire length of Giotto’s bell tower from a high vantage point, likely the roof of a
nearby building. The façade of the Cathedral, partially obstructed by the Baptistry, is bare; it was
redone with the same red, green, and white marble as the campanile between 1867 and 1887.
Though the photographs cannot capture the colour, the intricacies in the construction and
stonework are still visible. The photographs have a flat focus, with universally sharp details from
the foreground to the background. This allowed architects who saw the print at the Architectural
Photographic Association’s 1858 exhibition to study not only the bell tower and baptistry doors,
but to imagine how they all harmoniously make up the Cathedral complex.
Robert Macpherson (English, 1811–1872)
View of Rome from the French Academy, Monte Pincio
c. 1863
Albumen silver print from glass negative
Museum purchase funded by Alice C. Simkins in memory of W. Stewart Simkins, The
Manfred Heiting Collection
2004.222
47
This particular image marks a shift from the previous exhibition of the Association, in which the
photographs all showcase entire buildings or specific architectural components with as little of
the surroundings in the frame as possible. Here, Macpherson is neither focusing on the Villa
Medici (the French Academy), which is not even in the frame, its garden, which is barely visible,
or the full panorama of Rome, framed by trees in the background. By marrying these three,
Macpherson instead engages the viewers’ imaginations. Including architecture in the context of
its surroundings allows anyone to situate themselves within the scene.
It was among the 120 views of Rome exhibited by Macpherson at the Architectural Photographic
Association’s 1859 exhibition. His body of work, which accounted for almost one third of the
total photographs on view, was hailed as the highlight of this exhibition. It is a different type of
source material for architects; it captures the general mood and feel of the environment
surrounding the Villa, not the structure itself.
Édouard Baldus (French, 1813–1889)
Pavillon Denon, Louvre
1855–1856
Salted paper print from glass negative
Gift of Manfred Heiting, The Manfred Heiting Collection
2004.249
In 1855, Baldus received a commission to photograph the construction of the New Louvre. Here,
he subtly captures this state of flux; though scaffolding is visible, the construction itself is not the
focus of his photograph. He instead celebrates the grandeur of the almost-complete building and
its decoration. Even light rakes across the entire façade of the Pavillon, illuminating the
ornamental details.
By the the end of his commission in 1857, Baldus created more than 1200 photographs of the
Louvre. His images, a triumph of nineteenth-century photography, were so popular that he
continued to photograph the Louvre for the government and his own personal business until the
mid-1860s. Only through photography could he compile such a vast archive as an art form while
simultaneously making his work available to architects as source material. He exhibited this and
other work from the commission at the Architectural Photographic Association’s first exhibition
in 1858.
Louis-Auguste Bisson (French, 1814–1876)
Moissac Cloister
1857
Albumen silver print from glass negative
Gift of Manfred Heiting, The Manfred Heiting Collection
2004.313
This photograph is rich in subtle devices which make it very useful as source material for
architects, but outstanding as an artwork as well. Architects tended to prefer close-up detailed
views of single building components, or views of entire building façades as source material. Yet
48
from this vantage point, the Bisson brothers simultaneously present the sculptural intricacies of
the columns, the wooden beams of the roof, and how the constructed building functions and
interacts within the natural landscape in one single image. It pulls us into the dark, mysterious
corner, and leads us back into the light of the perpendicular hallway and garden. This image was
widely exhibited at photographic societies throughout Europe between 1858 and 1860.
Charles Clifford (English, 1819–1863)
[Main Door, Salamanca Cathedral]
1855–1860
Albumen silver print from glass negative
Gift of Manfred Heiting, The Manfred Heiting Collection
2004.350
Charles Clifford (English, 1819–1863)
Salamanca, Puerta del Convento de las Dueñas
1853
Albumen silver print from glass negative
Gift of Manfred Heiting, The Manfred Heiting Collection
2004.351
Though convents and cathedrals were popular subject matter for British and French architectural
photographers, the sculptural ornamentation on either building seen in Clifford’s photographs is
not an architectural style readily found in either Britain or France. Diplomatic relations between
Britain and Spain were not formed until 1850, allowing curious travellers to venture there for the
first time. In keeping with the tradition of the Grand Tour, these photographs embody British
society’s craving for travel and foreign places, but they also merge architectural photography
with nineteenth-century Europe’s fascination with orientalism. These photographs straddle a fine
line between the East and West, the familiar and unfamiliar, and served to bridged the gap
between representations of familiar European architecture and those of Eastern architecture at the
Architectural Photographic Society’s first exhibition.
Tommaso Cuccioni (Italian, c. 1790–1864)
View of the Roman Forum towards the Capitoline Hill, Looking West
1855
Albumen silver print from glass negative
Museum purchase funded by the Brown Foundation Accessions Endowment Fund, The
Manfred Heiting Collection
2004.369
From the mid-eighteenth century, it had become customary for young, British aristocrats to
travel to Italy. This coach trip, called the Grand Tour, was initially considered an essential part of
their education, but turned into a trendy pastime. The ruins of the Roman Forum were a popular
stop for those undertaking their Grand Tour, and the subject of many drawings, paintings, and,
49
after the 1840s, photographs. In fact, The Museum of Fine Arts, Houston, has three almost
identical photographs taken from the same viewpoint between 1853 and 1860. This print, a copy
of which was exhibited at the 1858 exhibition of the Photographic Society of Scotland, is crisp
and large, with rich, eggplant tones and bright highlights. It is very classical but marvelously
executed, and provided architects and members of the public alike with a chance to study the
Roman Forum without having to leave Scotland.
Carlo Naya (Italian, 1816–1882)
Piazzetta of San Marco looking towards the Island of St. George
1857–1859
Albumen silver print from glass negative
Museum purchase funded by Cathy and Giorgio Borlenghi, The Manfred Heiting
Collection
2004.619
Carlo Naya exhibited a copy of this print at the Société Française de Photographie’s 1859
exhibition, just two years after opening his photographic studio in Venice. Unlike most
photographs selected by the Architectural Photographic Association, this image does not focus
on any one particular building, making its usefulness as source material questionable. Instead, it
captures characteristics of Venice: the water surrounding it, the ornamental, almost-Eastern
architecture, and the hot sun. The dramatic use of shadows on the right, within which three
figures take shelter, contrasted with the bright white highlights on the Doge’s Palace on the left
leave viewers with a sense of the heat of the Venetian afternoon.
Roger Fenton (English, 1819–1869)
Printed by Francis Frith & Co. (British, founded 1859)
Lichfield Cathedral, Central Doorway, West Porch
1858
Albumen silver print from glass negative, printed 1863–1865 by Francis Frith & Co.
Museum purchase funded by James Edward Maloney
2014.89
Fenton worked with large-format glass plate negatives, excelling in many genres of photography:
sweeping and soft English landscapes, monumental architectural views, reportage of the Crimean
War, and portraits of the Royal Family. Here, the priest and parishioner in the doorway serve to
give a sense of scale, while the simultaneously helping the composition of the photograph,
inviting viewers in through the open door. The dark shadows contrast the light stonework,
allowing architects and other viewers to study the intricacies of the doorway. With this image,
included in the 1859 exhibition of the Société Française de Photographie, Fenton shows us his
mastery of the medium, marrying perfect lighting, technique, and Romantic spirit.
The Architectural Photographic Association
The 1850s also saw a rise in the number of photographers travelling abroad, particularly
to Italy and the Near East. Photography was seen as a more reliable and practical medium for the
50
documentation of overseas encounters. As a result, the number of commercial photographic
studios across Europe and Asia rose by the mid-1850s. Beginning in the 1850s, commercial
photographers defined public perceptions of photography. The demand in the 1850s and 1860s
was for art reproductions, photographs of new construction, and portraiture. Advances in
photographic technology at the time aided in the success of photographic studios, and enabled
photographers to travel. Wet collodion glass plate negatives made it possible to print a high
quantity of sharp images upon a photographer's return home from their travels, which resulted in
a larger-scaled production and distribution of photographic prints than ever before. The increased
success and popularity of photographic societies, commercial studios, and wet collodion glass
plate negatives converged to create an unprecedented international community of photography.
Architecture from around the world became a popular photographic subject in the 1850s, and
photographers like Édouard Baldus, Charles Clifford, or Francis Frith devoted the majority of
their practice to the subject.
The Architectural Photographic Association was formed in May of 1857, and officially
inaugurated at a meeting on the night of Thursday, January 7th 1858. The architects belonging to
the Association would travel, purchase prints from photographers’ studios at their destinations,
and then assemble these prints into an exhibition in London. Subscribers could come to the
exhibitions and purchase copies of photographs that were on display. After the exhibitions, the
Association would order the chosen prints from each photographer, and send them to the
subscribers. The Association held four annual exhibitions from 1858 until 1861, when it was
dissolved due to its inability to keep up with an overwhelming demand.
Photographic Exhibitions from 1858 to 1861
The exhibition at the Crystal Palace in London in 1851 was, for the majority of those in
attendance, a preliminary exposure to photographs. By the mid-1850s, photographic exhibitions
became more common throughout Europe, and particularly in Britain and France, intrinsically
linked with the rise of photographic societies. The Photographic Society of London, now the
Royal Photographic Society, was founded in 1853, and a year later, the Société Française de
Photographie was formed in Paris. These societies helped foster an international community of
photographers, working to validate their work as art, and to make technical advances in the
medium or profit off of the sale of photographs. Members of photographic societies, like the
Société Française de Photographie, the Photographic Society of London, or the Glasgow
Photographic Society, were mostly photographers and chemists. They would exchange prints
with each other for the purposes of refining their own techniques and building their personal
collections. These societies held annual exhibitions, curated and selected by juries to ensure that
the exhibitions included only the finest examples of photographic practices of the time.
51
Appendix B: Condition Reports
(Arranged by accession number, in ascending order)
Accession
Number
Condition of Primary
Support/Object
Condition of Secondary
Support
Overall Condition,
Suggestions/Concerns
88.233
Francis Frith
& Co.
Columns,
Pharaoh's
Bed
Microcracks throughout the
emulsion. 2 mm diameter
foxing on photograph in
bottom left. Small (3mm) tear
along bottom edge.
Yellowing of highlights and
slight fading
Slight yellowing, slight
warping, but overall good
condition
Good condition
2000.255
Robertson &
Beato
Tophane
Fountain
Small losses along left edge.
Microcracks and abrasions
throughout. Overall very little
yellowing/fading/
Good condition
Great
2004.211
Fratelli
Alinari
Gates of
Paradise,
Baptistery of
San
Giovanni,
Florence
Losses/discoloration (possible
retouching?) along bottom
edge, especially in bottom left
corner. “159” is inscribed in
the negative, bottom right
corner. Surface abrasion in
shape of a semi-circle, open
right, on the bottom left.
Small losses and
microabrasions throughout.
Ink inscription reads: “Firenze
/ Porta principale del
Battistero.” Blind stamp is
embossed below. Foxing
throughout, increases in
frequency closer to the bottom
edge. Surface dirt throughout,
mostly in bottom left corner
and along the bottom of the
mount. Handling creases along
left and right sides.
Good condition. Stable.
Could use a surface
clean.
2004.212
Fratelli
Alinari
Campanile
for the
Cathedral of
Santa Maria
del Fiore,
Florence
Retouching in bottom right
corner in the form of a 2 inch
long curved line. 2 tears along
the bottom edge: one in the
centre (1 cm long, vertical
orientation) and one near the
right corner (5 mm). Small
surface stains and losses
throughout. Slight yellowing
overall. Silvering very lightly
along the bottom right and
bottom left edges in Dmax
areas.
Dirt throughout, but more near
the right, top, and bottom
edges. Foxing throughout.
Graphite “X” in bottom right
of recto.
Good condition.
Eventually the tears
could be mended, but
too small to intervene
right now.
2004.222
Robert
Macpherson
Fading and yellowing along
left, bottom, and top edges.
Small (<1 mm diameter)
Trace of paper manufacturer's
stamp along the right edge,
whole length. Slight surface
Great condition.
Negative is in worse
shape, makes the print
52
View of Rome
from the
French
Academy,
Monte Pincio
losses and micro abrasions
throughout. 3 mm tear along
right edge. Evidence of
retouching: an oblong circle
along right edge, with a
lengthwise diameter of 3mm,
and a small circle near bottom
edge center, with roughly a
1.5 mm diameter.
stains throughout.
Adhesive/linen residue along
top edge, fabric fraying.
look like it has losses
and cracks in places it
doesn’t.
2004.249
Édouard
Baldus
Pavillon
Denon,
Louvre
Evidence of retouching
bottom left and center right.
Some foxing top left and top
right. Microscope inspection
confirms medium as salted
paper print. Slight yellowing
throughout.
Surface dirt near bottom left
near the edge, bottom right
corner, and centre right near
the edge. Small stain in the
bottom right approx. 2 inches
above the bottom edge.
Good condition. Could
benefit from a surface
clean, but not essential.
2004.313
Bisson Frères
Moissac
Cloister
Losses, filled in with ink
along top and right edge, and
more near the top right
corner. Microcracks and
small losses/surface dirt
throughout. Slight silvering in
Dmax areas along top and
right edge, more concentrated
in top right corner.
Foxing very heavy throughout.
Large tear along left edge,
horizontal orientation, just
under 5 cm long. Circular
wear (abrasion) on bottom
edge, less than 1 mm in
diameter. Surface dirt heavy
throughout, more concentrated
near the edges (especially the
bottom). Graphite inscriptions
and artist’s facsimile signature
stamp (ink) in bottom right
corner. Embossed blind stamp
in bottom centre.
Overall fair condition.
Mount shows more wear
than the photograph
itself, which is in great
condition.
2004.350
Charles
Clifford
[Main Door,
Salamanca
Cathedral]
Blind stamp embossed
(upside down) in the bottom
left corner: “CLIFFORD /
PHOTOGRAPHER.” 2 cm
abrasion in bottom right, runs
diagonally. Micro abrasions
throughout. Small losses in
top right (less than 1 mm) and
evidence of retouching. Loss
along left edge, near top left
corner; and in bottom right
corner along right edge.
Adhesive or label residue in
top left corner. Graphite
inscription bottom left corner
reads “48” and “Salamanca /
Nord Thor Gallery / Sud.”
Slight foxing throughout.
Surface dirt throughout, more
buildup along 4 edges.
Slightly warped/buckling. Top
centre there is a graphite (3
mm) mark parallel to and just
above the top edge of the
photograph. Top right corner
missing 1 layer of paper.
Good condition, minor
surface clean could be
useful in future.
Graphite marks can be
erased. Photograph
should not be in humid
environment, as the
paper will continue to
dangerously buckle.
2004.351
Charles
Clifford
Salamanca,
Puerta del
Convento de
Minimal damage.
Microscratches throughout,
mostly visible to naked eye in
bottom left. Slight tear and
loss in bottom left corner, in
the tip of the corner itself.
Dealer inscription in graphite
in bottom right centre and
corner. Slight
warping/buckling throughout.
Yellowing and dirt along all
four edges. Handling damage
Great condition, mostly
stable, if exposed to
excess humidity the
secondary support will
warp more which could
be unfavourable for the
53
las Duenas
There is a “43” inscribed in
red ink in the bottom right
corner, and a “55” in the
bottom left corner of the
negative.
in bottom left corner (small
loss, layers of paper are
separating)
photographic emulsion.
2004.369
Tommaso
Cuccioni
View of the
Roman
Forum
towards the
Capitoline
Hill, Looking
West
Loss/retouching in upper left,
near top left corner. Slight
foxing in highlights of print.
Cracks in the negative
(scratches?) near left edge,
run diagonally towards
centre, were painted in (in the
negative?). Slight yellowing
of highlights.
Paper manufacturing stamp
visible along entire length of
the right edge (similar to the
Macpherson). Surface dirt
throught. Sparse foxing spots
to the left of the image and
surrounding embossed blind
stamp, which is in the lower
centre. Graphite inscription
reads “Rome / Forum” in
bottom right corner. Slight
warping/buckling.
Great. Exposure to
humidity would be
detrimental and cause
the photograph to buckle
more.
2004.619
Carlo Naya
Piazzetta of
San Marco
looking
towards the
Island of St.
George,
“109” is inscribed in the
negative in the bottom right
corner. Small groups of
abrasions and scratches along
the top edge of the image.
Slight yellowing of
highlights. Microcracks
throughout. Surface stain near
top right corner. Loss/stain
near left edge, around 2 mm
in diameter.
Surface stains along the edges
especially. Foxing slightly.
Yellowing on the edges,
especially the bottom, right,
and left.
Great condition.
Microscopic inspection
would reveal the
intricacies of the stains,
and determine whether
or not they can be
removed.
2014.89
Roger
Fenton,
printed by
Francis Frith
& Co.
Lichfield
Cathedral,
Central
Doorway,
West Porch
Yellowing and fading around
all four edges, could be overmatted. Overall in good
condition. Slight yellowing of
highlights throughout. Small
microcracks and abrasions
throughout.
Good condition
The object is in good,
stable condition.
54
Appendix C: Matting and Framing Swatches and Worksheets
Artique Matboard: Thatch (A4937)
Artique Matboard: Birch (A4837)
55
56
57
58
59
60
61
62
63
64
65
66
67
68
Appendix D: Wall Elevation
With Sightlines:
Without Sightlines:
69
Appendix E: Exhibition Brochure
70
71
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W2192560730.txt | Psychiatr Psychol Klin 2015, 15 (1), p. 4–12
Carlo Garofalo , Patrizia Velotti , Giulio Cesare Zavattini ,
Marco Tommasi3, Roberta Romanelli3, Helena Espírito Santo4,
Aristide Saggino3
1
2
1
Received: 27.03.2015
Accepted: 15.04.2015
Published: 30.04.2015
On the factor structure of the Dissociative Experiences Scale: contribution
with an Italian version of the DES-II
Struktura czynnikowa Skali Przeżyć Dysocjacyjnych: włoska wersja skali DES-II
Department of Dynamic and Clinical Psychology, Sapienza University of Rome, Italy
Department of Education Sciences, University of Genoa, Italy
3
Department of Psychological, Humanistic and Territorial Sciences, University of Chieti-Pescara, Italy
4
Department of Psychology, Miguel Torga Institute, Coimbra, Portugal
Correspondence: Carlo Garofalo, Department of Dynamic and Clinical Psychology, Sapienza – University of Rome, Via degli Apuli, 1,
00185, Rome, Italy, tel.: +393283898543, fax: +39 010 20953728, e-mail: cgarofalo24@gmail.com, carlo.garofalo@uniroma1.it
1
2
Abstract
Aim of the study: Notwithstanding its clinical and empirical relevance, there is no consensus on how to conceptualize
dissociation. This may be partly due to the conflicting results yielded on the factor structure of the gold-standard selfreport measure of dissociation (the Dissociative Experiences Scale-Revised; DES-II, Carlson and Putnam, 1993).
In an attempt to advance research on this topic, we sought to explore the factorial structure of an Italian version of
the DES-II. Material and methods: A sample of 320 subjects (122 inmates and 198 community participants) was
administered the Italian version of the DES-II. Results: The Italian version of the DES-II showed good psychometric
properties and replicated a two-factor structure. Items content seemed to support the distinction into two qualitatively
different forms of dissociative experiences, described as detachment and compartmentalization phenomena. In line
with the expectations, participants in the inmate sample reported higher rates of dissociative experiences than
community participants, on both dimensions. Conclusions: This study provides further support for the validity of the
Italian version of the DES-II for use with community and inmate samples. Furthermore, we corroborated previous
evidence on a two-factor structure of the DES-II, which is consistent with theoretical assumptions describing two
distinct, albeit overlapping, dissociative dimensions (i.e., detachment and compartmentalization).
Key words: dissociation, community sample, inmates, detachment, compartmentalization
Streszczenie
Cel badania: Niezależnie od wartości klinicznej i empirycznej pojęcia, jakim jest dysocjacja, nie ma zgody co do
właściwego sposobu jego konceptualizacji. Częściowo może to być spowodowane sprzecznymi wynikami analizy
struktury czynnikowej złotego standardu wśród narzędzi do samooceny zjawisk dysocjacyjnych, jakim jest Skala
Przeżyć Dysocjacyjnych (Dissociative Experiences Scale-Revised; DES-II, Carlson i Putnam, 1993). Mając na celu
pogłębienie badań na ten temat, autorzy podjęli się analizy struktury czynnikowej włoskiej wersji skali DES-II.
Materiał i metody: Badanie obejmowało próbę 320 osób (122 więźniów i 198 osób należących do populacji ogólnej).
Zastosowano w nim włoską wersję skali DES-II. Wyniki: Włoska wersja skali DES-II wykazała dobre właściwości
psychometryczne i została w niej zreplikowana struktura dwuczynnikowa. Treść pozycji na skali wydawała się
potwierdzać istnienie podziału na dwie jakościowo inne formy przeżyć dysocjacyjnych, ujmowane jako oddzielenie
(ang. detachment) i szufladkowanie (ang. compartmentalization). Zgodnie z oczekiwaniami więźniowie częściej
zgłaszali występowanie przeżyć dysocjacyjnych niż osoby należące do populacji ogólnej, na obu wymiarach. Wnioski:
Niniejsze badanie dostarcza kolejnych dowodów na zasadność stosowania włoskiej wersji skali DES-II w badaniach na
populacji ogólnej oraz więziennej. Potwierdzono również poprzednie dane świadczące o dwuczynnikowej strukturze
skali DES-II, co jest zgodne z założeniami teoretycznymi opisującymi dwa odrębne, choć nakładające się na siebie
wymiary dysocjacji (tj. oddzielenie i szufladkowanie).
Słowa kluczowe: dysocjacja, populacja ogólna, więźniowie, oddzielenie, szufladkowanie
4
© Medical Communications Sp. z o.o. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives
License (CC BY-NC-ND). Reproduction is permitted for personal, educational, non-commercial use, provided that the original article is in whole, unmodified, and properly cited.
On the factor structure of the Dissociative Experiences Scale: contribution with an Italian version of the DES-II
Struktura czynnikowa Skali Przeżyć Dysocjacyjnych: włoska wersja skali DES-II
INTRODUCTION
D
issociation encompasses those processes involving a “lack of normal integration of thoughts, feelings, and experiences into the flow of consciousness and memory” (p. 727; Bernstein and Putnam, 1986).
Although they may occasionally occur in the daily life of
healthy people, pathological levels of dissociative experiences have been reported as characteristic or co-occurring
across diverse psychiatric conditions (Dell and O’Neil,
2009; Sar et al., 2007; Schäfer et al., 2010), with the strongest association usually reported between dissociation and
vulnerability to psychotic symptoms (Barker-Collo, 2001;
Simões et al., 2014). Recent evidence also supported the
presence of dissociation in individuals who display violent
behaviour (Moskowitz, 2004; Ruiz et al., 2008).
Notwithstanding the clinical relevance of dissociation
(Holmes et al., 2005; Liotti, 2006), there is a need for
a clearer conceptualization of the dissociation construct
(Dell and O’Neil, 2009). According to the DSM-5 (American Psychiatric Association, 2013), dissociative disorders
(DD) are defined into three broad categories, namely: dissociative identity disorder, dissociative amnesia, and depersonalization/derealization disorder. In the DES literature, dissociation has historically been described with
a three-factor model, respectively encompassing absorption, depersonalization/derealization, and amnesia experiences (e.g., Carlson and Putnam, 1993). Notably, in this
framework identity alteration would not be distinguished
from other experiences. Another conceptualization which
is worth mentioning has been derived from clinical description of dissociation (Allen, 2001; Cardeña, 1994) that subsequently yielded convergent empirical findings (Brown,
2002; Holmes et al., 2005). According to this model (Allen, 2001), two distinct qualitative forms of dissociation
were described, namely: detachment and compartmentalization. Detachment has been depicted as the most pervasive form of dissociative disturbance, which encompasses
depersonalization, derealization, and similar phenomena such as out-of-body experiences. On the other hand,
compartmentalization incorporated the more dramatic and perplexing of dissociative phenomena: amnesia,
fugues, and episodes of dissociative identity disorder (Allen, 2001; Holmes et al., 2005). Finally, another way to
conceptualize dissociation is to distinguish between pathological and non-pathological dissociative experiences (e.g.,
Waller et al., 1996; Watson, 2003). As a result, some authors recently argued that to date there is no agreement on
how to conceptualize dissociation (Dell and O’Neil, 2009).
Notably, factor analytic studies with mainstream measures
of dissociation could help in reaching a clearer picture.
If the Structured Clinical Interview for DSM-IV Dissociative Disorders (SCID-D; Steinberg, 1993) is regarded as the best diagnostic assessment tool for DD, the
Dissociative Experience Scale (DES; Bernstein and Putnam, 1986) is considered the gold-standard instrument to
© PSYCHIATR PSYCHOL KLIN 2015, 15 (1), p. 4–12
quantify the frequency of self-reported dissociative experiences (van IJzendoorn and Schuengel, 1996). It has been
translated into more than 20 languages and it has mostly
been used in its revised version (DES-II; Carlson and Putnam, 1993). The DES can be used as a screening instrument
for DD and for determining the contribution of dissociation
to other psychiatric syndromes as well as with nonclinical
samples (Carlson and Putnam, 1993; Espírito Santo and
Abreu, 2009; van IJzendoorn and Schuengel, 1996).
Unfortunately, studies exploring the factor structure of the
DES-II have to date been unsupportive in deciding among
different conceptual models, yielding conflicting results.
The original three-factor model proposed by Carlson and
Putnam (1993) was confirmed across several studies (Carlson et al., 1993; Fabbri Bombi et al., 1996; Ross et al., 1991;
Ruiz et al., 2008; Stockdale et al., 2002). Nevertheless,
other authors found different factorial patterns for the
DES, ranging from one- (Bernstein et al., 2001; Lipsanen et al., 2003; Mazzotti and Cirrincione, 2001) to seven-factor models (Ray et al., 1992). Moreover, Amdur and
Liberzon’s (1996), and Espírito Santo and Abreu’s (2009)
findings supported a factorial structure composed by four
factors.
Also, a two-factor structure first emerged in the study
by Waller et al. (1996), who adopted a typological model of dissociation. However, Waller et al. (1996) referred to
these factors as respectively resembling pathological (Factor 1) and non-pathological (Factor 2) dissociation. Notably, a categorical distinction between pathological and
non-pathological dissociation has failed to prove its utility
(Leavitt, 1999) and the dimensional conceptualization of
dissociation as a continuum ranging from normal to pathological forms is preferred both in clinical and research settings (Bernstein and Putnam, 1986; Bernstein et al., 2001;
Carlson et al., 1993; Holmes et al., 2005; Ruiz et al., 2008).
Recently, a French version of the DES (Larøi et al., 2013)
replicated the two-factor structure, although proposing
a different interpretation. Indeed, Larøi et al. (2013) described two forms of dissociative experiences. The first one
included both dissociative amnesia episodes and depersonalization/derealization experiences and was described
as resembling dissociation episodes, which may act as
a defensive mechanism. The second factor was depicted
as encompassing dissociative episodes associated with different forms of “cognitive failures,” often concerning memory or attention. They referred to this type as automatic pilot-related dissociation episodes. Notwithstanding the
possible clinical relevance of such an explanation, to the
best of our knowledge this interpretation of the two factors
does not seem consistent with any other empirical evidence
(Brown, 2002), nor with mainstream clinical theories (Dell
and O’Neil, 2009; Holmes et al., 2005). Such inconsistency in the factor structure of the DES fosters at least one
risk, which is the use of the original subscales reported by
Carlson and Putnam (1993) without testing the factorial
structure and the item loadings of the DES with the target
DOI: 10.15557/PiPK.2015.0001
5
Carlo Garofalo, Patrizia Velotti, Giulio Cesare Zavattini, Marco Tommasi, Roberta Romanelli, Helena Espírito Santo, Aristide Saggino
sample, which in turn can lead to wrong-headed theoretical inferences on the dissociation construct.
In the present study, we sought to explore the factorial
structure of an Italian version of the DES-II, using a combined sample of inmates and community-dwelling participants. It is noteworthy that to date there have been no
studies that have investigated the psychometric properties
and the factorial structure of the DES in Italian samples,
let alone used it with Italian offender samples, even though
the Italian translation has been available and widely used
for almost 20 years (Barbasio and Granieri, 2013; Conti,
2000; Fabbri Bombi et al., 1996).
per week). The DES-II total score is then computed averaging the score on each of the 28 items. Participants were
instructed to rate the extent to which they experienced dissociative symptoms, if any, without being under the effects
of alcohol or drugs. The DES-II showed high reliability in
its original version (test-retest = .79 < r < .84; split-half =
.83 < r < .93; Cronbach’s α = .95; Carlson and Putnam,
1993) as well as in its Italian translation (Cronbach’s α
= .91; split-half: r = .92; Fabbri Bombi et al., 1996). In the
present study, we used the Italian translation reported by
Conti (2000), which in our study showed an excellent internal consistency (Cronbach’s α = .96).
MATERIAL AND METHODS
Statistical analyses
Participants
We chose natural logarithmic transformations as the best
technique to reduce the skewness in our raw data (Roberts, 2008). A first way to determine the number of factors to retain was a parallel analysis (Watkins, 2000) using a Monte Carlo PCA, because it is a way of calculating
the average eigenvalues for 100 sets of random data of the
same size as our data (28 variables × 320 participants).
After doing that, each eigenvalue obtained in SPSS was
compared with the corresponding value from the random
results generated by parallel analysis. Then, we performed
an exploratory factor analysis with Maximum Likelihood
(ML) estimator, because it allows the estimation of indices of model fit and permits the computation of model
parameter standard errors, confidence intervals, and significance tests. The Root Means Squared Error of Approximation (RMSEA) is particularly indicated for testing model fit. According to Browne and Cudeck (1993),
a good model should have a RMSEA value lower than .08,
while Hu and Bentler (1999) said that it should be lower
than .06. MacCallum et al. (1996) defined the following
criteria: a model has a close fit if RMSEA < .05; a fair fit
if .05 < RMSEA < .08; a mediocre fit if .08 < RMSEA
< .10; a poor fit if RMSEA > .10. The appropriate number of factors is determined by examining the RMSEA values for the sequence of models. The sequence starts with
a monofactorial model and a new factor is added step by
step until a model with an RMSEA lower than .05 (ideally) is reached (Fabrigar and Wegener, 2012). The RMSEA
difference (∆RMSEA) between two models is also important. According to Fabrigar and Wegener (2012), any difference of .02 or greater can be considered a substantial
difference in fit; differences between .01 and .019 can be
considered marginal differences; differences less than .01
can be considered not meaningful. Therefore, if adding
a new factor, ∆RMSEA is lower than .01 (in absolute values), then the new factor can be considered not meaningful and the previous model determines the appropriate
number of factors. After calculating descriptive statistics for both samples, we tested for significant differences between inmates and controls, by carrying out multiple t-tests for independent samples.
The sample was composed of 320 subjects: 122 inmates
and 198 community participants. Inmates had an average
age of 39.97 years (SD = 11.76) and community participants had an average age of 32.51 years (SD = 10.30);
98% of inmates and 58.6% of individuals from the community were males. All participants were Caucasian, and
all inmates were convicted of violent offenses (i.e., armed
robbery, assault, sexual offenses or abuse, murder or attempted murder). Exclusion criteria for both groups were
the presence of cognitive disability or a diagnosed psychiatric disorder.
Procedures
Participants in the community sample were enrolled using the snowball sampling technique. The inmate sample was recruited from different jails and prisons located
in or around Rome, as part of a larger study on inmates’
psychopathology. Participants completed the measures
anonymously, individually or in small-group sessions settled in the prison library in the presence of a licensed psychologist. Participants from both groups provided written informed consent to voluntarily take part in the study.
The Italian Ministry of Justice and the Sapienza University of Rome Research Ethics Board approved the whole
procedure, which conformed to the principles included in
the Declaration of Helsinki.
Measure
6
The Dissociative Experiences Scale-II (DES-II; Carlson
and Putnam, 1993) consists of 28 statements describing
various dissociative experiences, some common (e.g., missing part of a conversation) and others much more unusual (e.g., standing in front of a mirror without recognizing).
Participants had to rate the percentage of occurrence of
dissociative experiences using a scale from 0% to 100% (by
10s, resulting in an 11-point scale ranging from 0 to 100,
with 0% meaning never and 100% meaning at least once
DOI: 10.15557/PiPK.2015.0001
© PSYCHIATR PSYCHOL KLIN 2015, 15 (1), p. 4–12
On the factor structure of the Dissociative Experiences Scale: contribution with an Italian version of the DES-II
Struktura czynnikowa Skali Przeżyć Dysocjacyjnych: włoska wersja skali DES-II
# factors
χ2
df
p
RMSEA
90% RMSEA
∆RMSEA
1
873.51
350
<.001
.0705
.0627–.0741
2
597.35
323
<.001
.0537
.0450–.0579
–.0168
3
476.72
297
<.001
.0458
.0361–.0506
–.0079
Tab. 1. Goodness of fit indexes of factorial models tested in exploratory factor analysis with ML estimation. ∆RMSEA lower than the
critical value of .01 is in boldface
RESULTS
The Kaiser-Meyer-Olkin Measure of Sampling Adequacy
was .96 (should be ≥ .60) and Bartlett’s test of sphericity
value was significant (χ2 = 4398.643, df = 378, p < .001),
indicating that the data was adequate for factor analysis.
According to the screeplot, an evident leveling off in correspondence of the third factor emerged. Screeplots are criticized because it is often difficult to see a clear drop in the
curve. The first four eigenvalues were: 11.13; 1.15; .61; .49.
Even if in our case the drop is clearly evident, there was
a strong difference between the first and the second eigenvalue. Yet the second eigenvalue is smaller than the criterion
value from parallel analysis, which showed only one component exceeding the corresponding criterion values for a randomly generated data matrix of the same size (eigenvalue
#2 = 1.50; SD = .04). Therefore, we carried out an analysis for testing differences in RMSEA (∆RMSEA) between
models with different number of factors. We limited our
analyses to a model composed by one, two, or three factors.
Tab. 1 shows the goodness of fit indexes (χ2, RMSEA,
90% C.I. of RMSEA and ∆RMSEA) of factorial models.
DES-II items
Factor 1
Factor 2
h2
1. Driving a car and realizing one doesn’t remember part of the trip
.428
.025
.199
2. Missing part of a conversation
.557
–.099
.241
3. Finding oneself in a place but unaware of how one got there
.332
.455
.531
4. Finding oneself dressed in clothes one doesn’t remember putting on
–.007
.752
.558
5. Finding unfamiliar things among one’s belongings
.352
.417
.507
6. Being called with a different name by people one doesn’t know
.447
.250
.421
7. Seeing oneself as if looking at another person
.176
.626
.580
8. Not recognizing friends or family members
–.164
.828
.520
9. Not remembering important events in one’s life
.366
.294
.373
10. Being accused of lying when one is telling the truth
.511
.137
.380
11. Not recognizing one’s reflection in a mirror
.049
.709
.554
12. Other people and objects do not seem real
.155
.549
.447
13. Feeling as though one’s body is not one’s own
.039
.694
.522
14. Remembering past so vividly one seems to be reliving it
.715
–.075
.440
15. Not sure if remembered event happened or was a dream
.584
.079
.413
16. Being in a familiar place but finding it unfamiliar
.505
.214
.454
17. Absorption in television program or movie
.532
.061
.333
18. So involved in fantasy that it seems real
.492
.204
.426
19. Able to ignore pain
.384
.317
.420
20. Staring into space
.712
.027
.535
21. Talking out loud to oneself when alone
.392
.103
.222
22. Feeling as though one were two different people
.471
.255
.457
23.Usually difficult things can be done with ease and spontaneity
.700
–.095
.404
24. Not sure whether one has done something or only thought about it
.743
–.120
.440
25. Finding evidence of having done things one can’t remember doing
.641
.072
.481
26. Finding notes or drawings that one must have done but doesn’t remember doing
.488
.150
.365
27. Hearing voices inside one’s head
.038
.722
.562
28. Looking at the world through a fog
.079
.641
.488
% explained var.
23.73
20.10
Cronbach’s α
.936
.934
Note. DES-II: Dissociative Experiences Scale-II; h2: communalities.
Loadings greater than .30 (in absolute value) are bolded.
Tab. 2. Items content and factor loadings of the DES-II obtained with oblimin rotation (ML estimation), corresponding percentages of
variance explained by each factor and internal consistency coefficients (Cronbach’s α) for items grouped in each factor
© PSYCHIATR PSYCHOL KLIN 2015, 15 (1), p. 4–12
DOI: 10.15557/PiPK.2015.0001
7
Carlo Garofalo, Patrizia Velotti, Giulio Cesare Zavattini, Marco Tommasi, Roberta Romanelli, Helena Espírito Santo, Aristide Saggino
DES-II items
1. Driving a car and realizing one doesn’t remember part of the trip
2. Missing part of a conversation
3. Finding oneself in a place but unaware of how one got there
4. Finding oneself dressed in clothes one doesn’t remember putting on
5. Finding unfamiliar things among one’s belongings
6. Being called with a different name by people one doesn’t know
7. Seeing oneself as if looking at another person
8. Not recognizing friends or family members
9. Not remembering important events in one’s life
10. Being accused of lying when one is telling the truth
11. Not recognizing one’s reflection in a mirror
12. Other people and objects do not seem real
13. Feeling as though one’s body is not one’s own
14. Remembering past so vividly one seems to be reliving it
15. Not sure if remembered event happened or was a dream
16. Being in a familiar place but finding it unfamiliar
17. Absorption in television program or movie
18. So involved in fantasy that it seems real
19. Able to ignore pain
20. Staring into space
21. Talking out loud to oneself when alone
22. Feeling as though one were two different people
23. Usually difficult things can be done with ease and spontaneity
24. Not sure whether one has done something or only thought about it
25. Finding evidence of having done things one can’t remember doing
26. Finding notes or drawings that one must have done but doesn’t remember doing
27. Hearing voices inside one’s head
28. Looking at the world through a fog
Note. DES-II: Dissociative Experiences Scale-II.
Tab. 3. Structure matrix of DES-II obtained with oblimin rotation (ML estimation)
8
The analysis showed that ∆RMSEA was under the critical value of .01 (Fabrigar and Wegener, 2012) between the
model with two and three factors. Therefore, the addition
of a third factor is not meaningful and we can conclude
that DES measures two latent factors.
On the basis of item content, items which were grouped in
Factor 1 seemed to incorporate both amnesia and absorption experiences. Hence, this factor seemed to resemble
compartmentalization, according to Allen’s (2001) model
and Holmes et al.’s (2005) model, whereas items grouped
into Factor 2 encompassed depersonalization and derealization (i.e., detachment). Tab. 2 shows the factor loadings
of each item after oblimin rotation.
Items 3, 5, and 19 loaded on both factors. However, according to their content and in order to preserve the theoretical
coherence of the factors’ composition, items 3 and 5 seemed
more consistent with Factor 2, whereas item 19 with Factor 1. Correlation between factors was high (r = .776).
Tab. 3 shows the structure matrix of the factor analysis.
Tab. 4 shows the descriptive statistics (means and standard deviations) for both inmates and community participants and the multiple t-tests for the DES-II total score
and subscale scores.
DOI: 10.15557/PiPK.2015.0001
Factor 1
.446
.486
.655
.528
.649
.625
.621
.425
.575
.609
.553
.546
.533
.661
.640
.657
.575
.637
.609
.731
.465
.652
.632
.658
.692
.595
.552
.534
Factor 2
.329
.297
.691
.747
.668
.568
.751
.711
.554
.501
.744
.660
.722
.433
.494
.573
.439
.554
.590
.534
.382
.590
.403
.409
.528
.497
.749
.696
Significant differences emerged in all three scores. Interestingly, within both samples, compartmentalization experiences (Factor 1) yielded a relatively higher score than detachment experiences (Factor 2).
DISCUSSION
As a whole, our results provide further support for the use
of the Italian version of the DES-II as the gold-standard
self-report measure to assess dissociation in nonclinical
Inmates
Community
(n = 122)
(n = 198)
Variables Mean
SD
Mean
SD
t
p
DES-II (F1) 22.01 16.12 14.71
14.41
4.094 <.001
DES-II (F2) 11.03 16.33
5.59
12.16
3.177
<.01
DES-II
18.08 15.61 11.45
13.10
3.917 <.001
Note. DES-II (F1): Dissociative Experiences Scale-II Compartmentalization
score; DES-II (F2): Dissociative Experiences Scale-II Detachment score;
DES-II: Dissociative Experiences Scale-II total score.
Tab. 4. Means, standard deviations (SD) and multiple t-test
comparisons for the DES-II total score and subscales
between Italian inmates and community participants
© PSYCHIATR PSYCHOL KLIN 2015, 15 (1), p. 4–12
On the factor structure of the Dissociative Experiences Scale: contribution with an Italian version of the DES-II
Struktura czynnikowa Skali Przeżyć Dysocjacyjnych: włoska wersja skali DES-II
individuals, extending its reliability and validity to the use
with incarcerated offenders. Regarding the factorial structure of the DES-II, our contribution joins the complicated picture reported in the literature. Although we replicated a two-factor structure, our item loadings differed from
those reported by Waller et al. (1996)*, with both a clinical
(DD) and a nonclinical sample, and Larøi et al. (2013) in
a community sample. Interestingly, the factor structures reported in these two studies were also different from each
other. Furthermore, both Waller et al. (1996) and Larøi
et al. (2013) incorporated in a common factor items referring to depersonalization and amnesia, and interpreted the two factors as representing different degrees of severity. This approach is inconsistent with clinical essays
(Allen, 2001; Cardeña, 1994) which have historically considered depersonalization and amnesia as separate forms
of dissociation. Moreover, to our knowledge, all previous
factor analyses showed that they were always reported in
separate factors (e.g., Holmes et al., 2005), and that items
representing different degrees of severity were equally distributed across factors (e.g., Carlson and Putnam, 1993).
In the current Italian version of the DES-II, all the items
reported by Carlson and Putnam (1993) as describing depersonalization or derealization loaded on Factor 2. Similarly, all the items referring to amnesia and absorption
forms of dissociative experiences loaded on a separate factor, being grouped in Factor 1**. Notably, even though the
primary use of a measure such as the DES is to screen individuals with clinically relevant or empirically significant
levels of dissociation, rather than attempting to characterize the nature of dissociation, our factorial structure seems
partly consistent with a qualitative distinction between two
types of dissociative experiences, being compartmentalization (here, Factor 1), and detachment (here, Factor 2).
On the one hand, states involving a compartmentalization involve both amnesia and other phenomena broadly defined as somatoform dissociation, which are not included within the DES-II items (Brown, 2002; Cardeña,
1994; Holmes et al., 2005; Nijenhuis et al., 1996). According to this framework, the distinctive feature of compartmentalization is represented by an inability to intentionally
*
It is worth noting that Waller et al. (1996) used a taxometric approach
in an effort to develop a short measure for the screening of pathological vs. non-pathological dissociation, rather than conducting a factor analytic study per se.
**
It should be acknowledged that there are two exceptions. In fact, item
4 and item 8 loaded on our Factor 2 (i.e., depersonalization), whereas they are usually considered as part of the amnesia domain (Carlson and Putnam, 1993; Ross et al., 1991). However, item 4 had
already been reported as part of the depersonalization construct
(Carlson et al., 1993), suggesting that its loading may depend on the
interpretation people give to its content (i.e., people “having the experience of finding themselves dressed in clothes that they don’t remember putting on,” which may also derive from a depersonalization episode while getting dressed). Similarly, we propose that item 8 (i.e.,
people “being told that they sometimes do not recognize friends or
family members”) may be interpreted as a derealization experience
(e.g., “those do not seem my real friends, or relatives”) rather than an
actual amnesic fragmentation, especially in nonclinical individuals.
© PSYCHIATR PSYCHOL KLIN 2015, 15 (1), p. 4–12
control processes or actions that people are normally able
to deliberately control (Brown, 2002).
On the other hand, following Holmes et al.’s suggestions
(2005), we propose that subjective experiences of an altered
state of consciousness, accompanied by a sense of separation from some aspects of everyday experience are characteristic of the detachment type of dissociation. Consistent with
a dimensional conceptualization of dissociation, it has been
argued that states of detachment lie on a continuum ranging
from temporary experiences in the everyday life of healthy
individuals, to persistent and/or acute conditions associated
with a psychiatric disorder (Holmes et al., 2005). In between
these two extremes, there would certainly be other forms of
detachment states with associated degrees of severity and
levels of functional impairment (Holmes et al., 2005).
The principal distinction between compartmentalization
and detachment states is thought to be the preservation of
apparently disrupted functions, which occurs in the context
of compartmentalization phenomena (Holmes et al., 2005).
Indeed, even if the affected functions (e.g., the ability to
bring usually accessible information into conscious awareness in the case of dissociative amnesia) are no longer
amenable to intentional control, these compartmentalized processes keep operating normally, being in turn able
to influence feelings, thoughts, and actions (Brown, 2002;
Cardeña, 1994). In other words, even though they are reversible in principle (Cardeña, 1994) they work separately so that this compartmentalization cannot be reversed
by a simple act of will (Holmes et al., 2005). Notably, such
distinction has been corroborated by neurobiological evidence showing different dissociative experiences belonging to each component share common underlying mechanisms in the brain (Brown, 2002; Simeon et al., 2003).
Moreover, the existence of a distinction between the two
components seems to be useful in clinical settings, since
these two forms of dissociation are relevant in the context
of different psychopathological syndromes (Allen, 2001).
However, less attention has been paid to the role of phenomena of absorption, defined as the experience of disconnecting oneself from the surroundings and getting involved in one’s thoughts or other imaginative processes
(Waller et al., 1996), which loaded on the same factor as
dissociative amnesia in the present study. According to
our results, we suggest that states of absorption resemble
more a mild form of compartmentalization than one of
detachment, somehow involving a loss of volitional control (e.g., people “finding that sometimes they are listening to someone talk and they suddenly realize that they
did not hear part or all of what was said” could be thought
of as an inability to maintain attentional control, or simply distractibility). Moreover, another item regards people
“having the experience of sometimes remembering a past
event so vividly that they feel as if they were reliving that
event,” partially resembling a sensorial alteration which is
typical of somatoform dissociation (Nijenhuis et al., 1996),
whose kind of experiences are not explicitly reported
DOI: 10.15557/PiPK.2015.0001
9
Carlo Garofalo, Patrizia Velotti, Giulio Cesare Zavattini, Marco Tommasi, Roberta Romanelli, Helena Espírito Santo, Aristide Saggino
10
in the DES-II, yet are thought to be part of the compartmentalization type of dissociation (Holmes et al., 2005).
Supporting our view of absorption being a mild form of
compartmentalization, Bernstein et al. (2001) noticed that
absorption items were the more endorsed ones in nonclinical subjects, compared to those regarding both depersonalization and amnesia, and other authors agreed in considering absorption less pathological than both amnesia
and depersonalization (Waller et al., 1996). However, future studies are needed given that Allen (2001) included
absorption at the low end of the continuum in the detachment domain. It should also be noted that previous works
(e.g. Allen et al., 1999) described a second possible form
of dissociative amnesia which was different from the one
associated with compartmentalization, being an encoding
problem triggered by extreme detachment.
To further corroborate the proposed distinction between
detachment and compartmentalization phenomena,
some authors have reported findings attesting that people can experience detachment states unrelated to and
without also experiencing compartmentalization episodes (Baker et al., 2003; Simeon et al., 2003), and also
the opposite pattern has been reported (Brown et al., 2005;
Holmes et al., 2005). Accordingly, in both samples considered here, a relatively higher degree of compartmentalization (as opposed to detachment) experiences emerged.
However, the proportion of variance shared by the two factors was approximately 60%.
Notwithstanding the potentially relevant clinical impact of
a qualitative distinction between detachment and compartmentalization type of dissociation, further convergent evidence is needed to support a bi-dimensional conceptualization of dissociation, the latter being the assumption that
detachment and compartmentalization states are on two
separate continuums (Allen, 2001; Holmes et al., 2005).
For instance, further studies are warranted in an attempt
to replicate the two-factor solution of the DES-II reported
here, both with other Italian independent samples and in
other countries, as well as in clinical populations. To date,
the inconsistency in its factor structure across studies
and samples, as well as the high degree of shared variance among the factors, lead us to support the use of the
DES-II as measuring a uni-dimensional construct (Bernstein et al., 2001; Holmes et al., 2005; Ruiz et al., 2008;
van IJzendoorn and Schuengel, 1996). This is especially
true for research purposes (i.e., allowing a comparison of
results from a different sample and corresponding factor
structure), whereas in clinical practice it could be useful
to disentangle different dimensions of dissociative experience, yet taking into account the overall degree of severity.
As a whole, our samples’ scores were in line with normative data reported in the literature worldwide with regard to
the presence and severity of dissociation in both community (Carlson and Putnam, 1993; Carlson et al., 1993; Espírito Santo and Abreu, 2009; Spitzer et al., 2006; Stockdale et al., 2002) and inmate samples (Becker-Blease and
DOI: 10.15557/PiPK.2015.0001
Freyd, 2007; Espirito-Santo and Costa, 2013; van IJzendoorn and Schuengel, 1996; Moskowitz et al., 2005;
Ruiz et al., 2008; Snow et al., 1996). As expected, inmates
reported higher rates of dissociative experiences when
compared with community-dwelling individuals. This finding suggests that dissociation may characterize the mental
functioning of incarcerated individuals. Indeed, their rates
of dissociative symptoms seemed comparable with those
of patients with psychiatric disorders (e.g., van IJzendoorn
and Schuengel, 1996). Thus, mental health services in prison should include an evaluation of dissociative symptoms
in their standard assessment for newly incarcerated individuals as well as during the period of incarceration.
Our findings should be considered in light of their limitations, which also represent directions for future research.
First, we did not assess the presence of psychotic symptoms in our inmate sample (although we excluded those
diagnosed with psychiatric disorders, we cannot rule out
the possible presence of transient psychotic episodes),
and this could have influenced the higher degree of dissociative experiences that we found in the inmate sample.
However, the presence of inmates with psychotic symptoms is likely to be constant across other inmate populations (Ruiz et al., 2008). Then, we only used self-report
measure, whereas a multi-method assessment (e.g., an expert-rated measure for assessing psychiatric symptoms, or
an interview to investigate dissociative experiences) could
limit the measurement bias that may affect questionnaires,
even though both measures we used are widely accepted as
valid and reliable instruments. Finally, we did not include
a sample of psychiatric patients, nor a sample of patients
with DD, thus our findings should not be generalized to
those populations prior further explorations.
CONCLUSIONS
With these cautions in mind, this study provides further
evidence to the reliability and validity of the DES-II as
a measure to assess dissociation in nonclinical and inmate populations, representing the first Italian validation
of the scale. Our results also highlight the alarming prevalence of dissociation in prisoners, calling for the inclusion of a proper psychiatric assessment in prison, which
should include an evaluation of dissociative symptoms.
Finally, the two-factor structure of the Italian version of the
DES-II emerged in the present study, representing the first
empirical support deriving from self-report assessment of
dissociation to the dichotomy between detachment and
compartmentalization types of dissociation (Allen, 2001;
Holmes et al., 2005). Nevertheless, further replications are
necessary, possibly with clinical samples and with patients
suffering from DD.
© PSYCHIATR PSYCHOL KLIN 2015, 15 (1), p. 4–12
On the factor structure of the Dissociative Experiences Scale: contribution with an Italian version of the DES-II
Struktura czynnikowa Skali Przeżyć Dysocjacyjnych: włoska wersja skali DES-II
Conflict of interest
The authors do not report any financial or personal connections with
other persons or organizations which might negatively affect the content
of this publication and/or claim authorship rights to this publication.
Acknowledgment
We are indebted to Prof. Jon Allen for his invaluable comments on an
earlier version of the manuscript.
PIŚMIENNICTWO:
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W2749183303.txt | Hindawi
BioMed Research International
Volume 2017, Article ID 8317357, 9 pages
https://doi.org/10.1155/2017/8317357
Research Article
Emotion Recognition from EEG Signals Using Multidimensional
Information in EMD Domain
Ning Zhuang,1 Ying Zeng,1,2 Li Tong,1 Chi Zhang,1 Hanming Zhang,1 and Bin Yan1
1
China National Digital Switching System Engineering and Technological Research Center, Zhengzhou 450002, China
Key Laboratory for NeuroInformation of Ministry of Education, School of Life Science and Technology,
University of Electronic Science and Technology of China, Chengdu, China
2
Correspondence should be addressed to Bin Yan; ybspace@hotmail.com
Received 27 March 2017; Revised 21 June 2017; Accepted 16 July 2017; Published 16 August 2017
Academic Editor: Robertas Damaševičius
Copyright © 2017 Ning Zhuang 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.
This paper introduces a method for feature extraction and emotion recognition based on empirical mode decomposition (EMD).
By using EMD, EEG signals are decomposed into Intrinsic Mode Functions (IMFs) automatically. Multidimensional information of
IMF is utilized as features, the first difference of time series, the first difference of phase, and the normalized energy. The performance
of the proposed method is verified on a publicly available emotional database. The results show that the three features are effective
for emotion recognition. The role of each IMF is inquired and we find that high frequency component IMF1 has significant effect on
different emotional states detection. The informative electrodes based on EMD strategy are analyzed. In addition, the classification
accuracy of the proposed method is compared with several classical techniques, including fractal dimension (FD), sample entropy,
differential entropy, and discrete wavelet transform (DWT). Experiment results on DEAP datasets demonstrate that our method
can improve emotion recognition performance.
1. Introduction
Emotion plays an important role in our daily life and work.
Real-time assessment and regulation of emotion will improve
people’s life and make it better. For example, in the communication of human-machine-interaction, emotion recognition
will make the process more easy and natural. Another
example, in the treatment of patients, especially those with
expression problems, the real emotion state of patients will
help doctors to provide more appropriate medical care. In
recent years, emotion recognition from EEG has gained mass
attention. Also it is a very important factor in brain computer
interface (BCI) systems, which will effectively improve the
communication between human and machines [1].
Various features and extraction methods have been proposed for emotion recognition from EEG signals, including
time domain techniques, frequency domain techniques, joint
time-frequency analysis techniques, and other strategies.
Statistics of EEG series, that is, first and second difference,
mean value, and power are usually used in time domain [2].
Nonlinear features, including fractal dimension (FD) [3, 4],
sample entropy [5], and nonstationary index [6], are utilized
for emotion recognition. Hjorth features [7] had also been
used in EEG studies [8, 9]. Petrantonakis and Hadjileontiadis introduced higher order crossings (HOC) features
to capture the oscillatory pattern of EEG [10]. Wang et
al. extracted frequency domain features for classification
[11]. Time-frequency analysis is based on the spectrum of
EEG signals; then the energy, power, power spectral density
(PSD), and differential entropy [12] of certain subband are
usually utilized as features. Short-time Fourier transform
(STFT) [13, 14], Hilbert-Huang transform (HHT) [15, 16],
and discrete wavelet transform (DWT) [17–19] are the most
commonly used techniques for spectrum calculating. It has
been commonly tested and verified that higher frequency
subband such as Beta (16–32 Hz) and Gamma (32–64 Hz)
bands outperforms lower subband for emotion recognition
[20, 21].
Other features extracted from combination of electrode
are utilized too, such as coherence and asymmetry of electrodes in different brain regions [22–24] and graph-theoretic
features [25]. Jenke et al. had done a research comparing the
2
BioMed Research International
IMF1
IMF2
EEG signals
(5 s)
EMD
IMF3
Feature
extraction
SVM
classifier
Recognition
results
IMF4
IMF5
Figure 1: Block diagram of the proposed method.
performance of different features mentioned above and got a
guiding rule for feature extraction and selection [26].
Some other strategies such as utilizing deep network
to improve the classification performance have also been
researched. Zheng and Lu used deep neural network to
investigate critical frequency bands and channels for emotion
recognition [27]. Yang et al. used hierarchical network with
subnetwork nodes for emotion recognition [28].
EMD is proposed by Huang et al. in 1998 [29]. Unlike
DWT, which needs to predetermine transform base function
and decomposition level, EMD can decompose signals into
IMF automatically. These IMFs represent different frequency
components of original signals, with band-limited characteristic. By applying Hilbert transform to IMF, we can get
instantaneous phase information of IMF. So EMD is suitable
for analysis of nonlinear and nonstationary sequence, such as
neural signals.
EMD is a good choice for EEG signals and we utilize it
for emotion recognition from EEG data. Which feature is
effective for emotion recognition in EMD domain? Which
IMF component is best for classification? Is the performance
based on EMD strategy better compared to time domain
method and time-frequency method or not? All these have
not been researched yet and we investigate them in our
research.
EMD has been widely used for seizure prediction and
detection, but for emotion recognition based on EMD, there
is not so much research. Higher order statistics of IMFs [30],
geometrical properties of the decomposed IMF in complex
plane [31], and the variation and fluctuation of IMF [32]
are used as features for seizure prediction and detection.
For emotion recognition, Mert and Akan extracted entropy,
power, power spectral density, correlation, and asymmetry of
IMF as features and then utilized independent component
analysis (ICA) to reduce dimension of the feature set [33].
The classification accuracy is computed with all the subjects
mixed together.
In this paper, we present an emotion recognition method
based on EMD. We utilize the first difference of IMF time
series, the first difference of the IMF’s phase, and the normalized energy of IMF as features. The motivation of using
these three features is that they depict the characteristics
of IMF in time, frequency, and energy domain, providing
multidimensional information. The first difference of time
series depicts the intensity of signal change in time domain.
The first difference of phase measures the change intensity in
phase and normalized energy describes the weight of current
oscillation component. The three features constitute a feature
vector, which is fed into SVM classifier for emotional state
detection.
The proposed method is studied on a publicly available
emotional database DEAP [20]. The effectiveness of the
three features is investigated. IMF reduction and channel
reduction for feature extraction are both discussed, which
aim at improving the classification accuracy with less computation complexity. The performance is compared with some
other techniques, including fractal dimension (FD), sample
entropy, differential entropy, and time-frequency analysis
DWT.
2. Method
To realize emotional state recognition, the EEG signals are
decomposed into IMFs by EMD. Three features of IMFs, the
fluctuation of the phase, the fluctuation of the time series, and
the normalized energy, are formed as a feature vector, which
is fed into SVM for classification. The whole process of the
algorithm is shown in Figure 1.
2.1. Data and Materials. DEAP is a publicly available dataset
for emotion analysis, which recorded EEG and peripheral
physiological signals of 32 participants as they watched 40
music videos. All the music video clips last for 1 minute,
representing different emotion visual stimuli, with grade
from 1 to 9. Among the 40 music videos, 20 are high
valence visual stimuli and 20 are low valence visual stimuli.
The situation is exactly the same for arousal dimension.
After watching the music video, participants performed a
self-assessment of their levels on arousal, valence, liking,
dominance, and familiarity, with ratings from 1 to 9. EEG
was recorded with 32 electrodes, placing according to the
international 10-20 system. Each electrode recorded 63 s EEG
signal, with 3 s baseline signal before the trial.
In this paper, we used the preprocessed EEG data for
study, with sample rate 128 Hz and band range 4–45 Hz. EOG
artefacts were removed as method in [20]. The data was segmented into 60-second trials and a 3-second pretrial baseline
removed. The binary classifications of valence and arousal
dimension are considered. We utilized the participants’ selfassessment as label. If the participant’s rating was <5, the label
BioMed Research International
3
of valence/arousal is low and if the rating was ≥5, the label of
valence/arousal is high.
Each music video lasts for 1 minute, and 5 s EEG signals
are extracted as a sample. So for each subject who watched 40
music videos, we acquire 480 labeled samples.
2.2. Empirical Mode Decomposition. EMD decomposes EEG
signals into a set of IMFs by an automatic shifting process.
Each IMF represents different frequency components of
original signals and should satisfy two conditions: (1) during
the whole data set, the number of extreme points and the
number of zero crossings must be either equal or differ at
most by one; (2) at each point, the mean value calculated from
the upper and lower envelope must be zero [29]. For input
signal 𝑥(𝑡), the process of EMD is as follows:
(1) Set ℎ(𝑡) = 𝑥(𝑡) and ℎold (𝑡) = ℎ(𝑡).
(2) Get local maximum and minimum of ℎold (𝑡).
(3) Interpolate the local maximum and minimum with
cubic spline function and get the upper envelope
𝑒max (𝑡) and lower envelope 𝑒min (𝑡).
(4) Calculate the mean value of the upper and lower
envelope as
𝑚 (𝑡) =
(𝑒min (𝑡) + 𝑒max (𝑡))
.
2
(1)
(5) Subtract ℎold (𝑡) with 𝑚(𝑡):
ℎnew (𝑡) = ℎold (𝑡) − 𝑚 (𝑡) .
(3)
(6) If imf𝑛 is gotten, set ℎold (𝑡) as
ℎold (𝑡) = ℎold (𝑡) − imf𝑛 .
(4)
Go to step (2) and repeat steps (2)–(5) to get imf𝑛+1 .
By the iterative process described above, 𝑥(𝑡) can be
finally expressed as
𝐿
𝑥 (𝑡) = ∑ imf𝑛 + 𝑟.
2.3.1. First Difference of IMF Time Series. The first difference
of times series 𝐷𝑡 depicts the intensity of signal change in time
domain. Previous research has revealed that the variation of
EEG time series can reflect different emotion states [2]. For an
IMF component with 𝑁 points, IMF{imf1 , imf2 , . . . , imf𝑁},
the definition of 𝐷𝑡 is
𝐷𝑡 =
1 𝑁−1
∑ |imf (𝑛 + 1) − imf (𝑛)| .
𝑁 − 1 𝑛=1
(5)
𝑧 (𝑛) = 𝑥 (𝑛) + 𝑗𝑦 (𝑛) .
(7)
The analytic signal can be further expressed as follows:
𝑧 (𝑛) = 𝐴 (𝑛) 𝑒𝑗𝜑(𝑛) ,
(8)
where 𝐴(𝑛) = √𝑥(𝑛)2 + 𝑦(𝑛)2 is the amplitude of 𝑧(𝑛) and
𝜑(𝑛) = arctan(𝑦(𝑛)/𝑥(𝑛)) is the instantaneous phase.
First difference of phase 𝐷𝑝 is defined as
𝐷𝑝 =
1 𝑁−1
∑ 𝜑 (𝑛 + 1) − 𝜑 (𝑛)
𝑁 − 1 𝑛=1
(9)
which measures the change intensity in phase and represents
the physical meaning of instantaneous frequency.
2.3.3. Normalized Energy of IMF. For an 𝑁-point IMF,
IMF{imf1 , imf2 , . . . , imf𝑁}, the normalized energy 𝐸norm is
defined as follows:
𝑛=1
It is a linear combination of IMF components and the residual
part 𝑟. Figure 2 shows a segment of original EEG signals
corresponding to the first five decomposed IMFs. EMD works
like an adaptive high pass filter. It shifts out the fastest
changing component first and as the level of IMF increases,
the oscillation of IMF becomes smoother. Each component is
band-limited, which can reflect the characteristic of instantaneous frequency.
(6)
2.3.2. First Difference of IMF’s Phase. Based on EMD, EEG
is decomposed into multilevel IMFs, each IMF being bandlimited and representing an oscillation component of original
EEG signals. For an 𝑁-point IMF, IMF{imf1 , imf2 , . . . , imf𝑁},
Hilbert transform is applied to it, obtaining an analytic signal
𝑧(𝑛)
(2)
If ℎnew (𝑡) satisfies the two conditions of IMF, then the first
IMF component imf1 is gotten; otherwise, set ℎold (𝑡) =
ℎnew (𝑡) and go to step (2), repeating steps (2)–(5) until ℎnew (𝑡)
satisfies the two conditions of IMF. Finally imf1 is gotten as
imf1 = ℎnew (𝑡) .
2.3. Feature Extraction. In this paper, three features of IMF
are utilized for emotion recognition, the first difference of
time series, the first difference of phase, and the normalized
energy. The first difference of time series depicts the intensity
of signal change in time domain. The first difference of
phase reveals the change intensity of phase, representing the
physical meaning of instantaneous frequency. Normalized
energy describes the weight of current oscillation component.
The motivation of using these three features is that they depict
the characteristics of IMF in time, frequency, and energy
domain, utilizing multidimensional information.
𝐸norm =
2
∑𝑁
𝑛=1 imf (𝑛)
2
∑𝑁
𝑛=1 𝑠 (𝑛)
,
(10)
where 𝑠(𝑛) is the original EEG signal points. So the numerator
is the energy of IMF and the denominator represents the
energy of original EEG data set. The normalized energy
describes the weight of current oscillation component. When
fed into the classifier, log(𝐸norm ) is taken as an element of the
feature vector according to [26].
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EEG
1
0
−1
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600
700
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1000
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IMF1
0.5
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IMF2
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IMF3
0.5
0
−0.5
IMF4
0.5
0
−0.5
IMF5
0.5
0
−0.5
Points
Figure 2: EEG signals and the corresponding first five IMFs.
2.4. SVM Classifier. The extracted features are fed into SVM
for classification. SVM is widely used for emotion recognition
[34, 35], which has promising property in many fields. In our
study, LIBSVM is implemented for SVM classifier with radial
basis kernel function and default parameters setting [36].
3. Performance Verification
In the following subsections, we test our method on DEAP
emotional dataset. Training and classifying tasks were conducted for each subject independently and we utilized leaveone-trail-out validation to evaluate the classification performance. Each subject watched 40 music video clips, and every
video clips lasted 1 minute. In our experiment, we utilized the
participants’ self-assessment as label. Every 5 s EEG signals
are extracted as a sample, so for each subject we acquire 480
labeled samples.
In leave-one-trail-out validation, for each subject, 468
samples extracted from 39 trails were assigned to training
set, and 12 samples extracted from the remaining one trail
were assigned to test set. So there was no correlation between
samples in the training set and the test set. Among the total
40 trails of one subject, each trail will be assigned to the test
set once as the validation data. The 40 results from the 40 test
trails then can be averaged to produce a general estimation for
each subject. The final mean accuracy is computed among all
the subjects.
3.1. Effectiveness of the Features for Emotion Recognition. In
order to evaluate the effectiveness of the three features for
emotion recognition, we first use only one single feature
for classification each time. All the experiments in this
subsection are under the condition that the first five IMF
components and total 32 electrodes are utilized for feature
extraction. The training and classifying for each subject
were conducted, respectively, and the mean accuracy was
computed among all the subjects.
The mean classification accuracies of three features are
given in Figure 3. It shows that all the three features can
distinguish high level from low level on both valence and
arousal dimension, higher than random probability of 50%.
For valence dimension, the classification accuracy yields
68.27%, 64.46%, and 61.07% with features 𝐷𝑡 , 𝐷𝑝 , and
𝐸norm , respectively. For arousal dimension, the classification
accuracy yields 69.89%, 67.56%, and 63.76% with features 𝐷𝑡 ,
𝐷𝑝 , and 𝐸norm , respectively.
3.2. IMF Reduction for Feature Extraction. In this subsection, we did two experiments to investigate the role of
different IMF components in emotion recognition. In the
BioMed Research International
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Table 1: Comparison of performance for different IMFs selected for feature extraction (32 channels) (standard deviation shown in
parentheses).
Valence
Component
IMF1
IMF2
IMF3
IMF4
IMF5
IMF1-2
IMF1–3
IMF1–4
IMF1–5
Arousal
𝑡-test (IMF1)
𝑝=1
𝑝 = 0.0002
𝑝=0
𝑝=0
𝑝=0
𝑝 = 0.4399
𝑝 = 0.2705
𝑝 = 0.1688
𝑝 = 0.1086
Accuracy (%)
70.41 (7.05)
63.47 (7.10)
61.45 (8.57)
59.55 (8.56)
55.74 (9.20)
69.02 (7.00)
68.47 (6.69)
67.99 (6.58)
67.59 (6.58)
80
Table 2: Performance of 8 channels selected for feature extraction
(Fp1, Fp2, F7, F8, T7, T8, P7, and P8) (standard deviation shown in
parentheses).
70
60
Accuracy (%)
𝑡-test (IMF1)
𝑝=1
𝑝 = 0.0032
𝑝 = 0.0019
𝑝 = 0.0012
𝑝=0
𝑝 = 0.1940
𝑝 = 0.3116
𝑝 = 0.2107
𝑝 = 0.1293
Accuracy (%)
72.10 (7.51)
66.58 (9.36)
64.56 (10.52)
63.99 (10.96)
62.38 (12.23)
70.47 (8.29)
70.08 (8.10)
69.60 (8.08)
69.00 (8.37)
Label
50
Predict
40
30
20
10
0
Valence
Arousal
Dt
Dp
EHILG
Figure 3: Classification accuracies of three single features. For each
subject, one single feature was extracted from the first five IMF
components. “𝐷𝑡 ,” “𝐷𝑝 ,” and “𝐸norm ” in the figure are corresponding
to the three single features, respectively. The mean accuracies for
all circumstances were computed among all the subjects. Error
bars show the standard deviation of the mean accuracies across all
subjects.
first experiment, each time only one IMF component was
utilized for feature extraction and we analyzed which IMF is
effective for emotion recognition. In the second experiment,
we further verified whether the combination of multi-IMFs
would improve the accuracy.
Table 2 gives all the results in detail. Standard deviation
of the mean accuracies across all subjects is shown in
parenthesis. “IMF1,” “IMF2,” “IMF3,” “IMF4,” and “IMF5”
are corresponding to single IMF component. “IMF1–3” in
the table represents the first three IMFs, corresponding to
IMF1, IMF2, and IMF3. Similarly, “IMF1–4” and “IMF1–5”
are corresponding to the first four IMFs and the first five
IMFs, respectively.
It shows that IMF1 yields the best performance, 70.41%
for valence and 72.10% for arousal. As the level increases, the
performance decreases sharply. The performance of IMF5 is
only 55.74% for valence and 62.38% for arousal. We applied
𝑡-test (𝛼 < 0.05) to examine the performance between only
High
Low
𝐹1 score
Accuracy (%)
Valence
High
6664
2024
Arousal
Low
2723
3949
0.7374
69.10 (6.95)
High
7493
1555
Low
2748
3564
0.7769
71.99 (7.77)
IMF1 utilized for feature extraction and other circumstances.
The null hypothesis is “the performance is similar” and if 𝑝
value is larger than 𝛼, the null hypothesis is accepted. The
results of 𝑡-test in Table 1 show that the performance of IMF1
is more splendid than other single components, IMF2, IMF3,
IM4, and IMF5, with 𝑝 far less than 0.05. It also shows that
performance of multi-IMF combinations is similar to only
IMF1 utilized for feature extraction, with 𝑝 larger than 0.05.
IMF1 represents the fastest changing component of EEG
signals, with the highest frequency characteristic. As the level
increases, the oscillation becomes smoother with frequency
becoming lower and lower. So we infer that the valence and
arousal of emotion relate more tightly to high frequency. It is
also coincided with the finding in [26] that Beta (16–32 Hz)
and Gamma (32–64 Hz) bands are successfully selected more
often than other bands. These two bands are higher frequency
subbands of EEG signals.
So combining the results of classification accuracy and 𝑡test, in practical use, we just need to extract features from
IMF1, which will save vast time and relieve computation
burden because only one level of EMD decomposition needed
to be done.
3.3. Channel Reduction for Feature Extraction. Form verification in Section 3.2, we know that using component IMF1
will achieve good performance. In this subsection, we will
investigate which electrodes are informative based on EMD
strategy.
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BioMed Research International
Dt
Fp1
Af3
F7
F3
Fc5
T7
Fc1
Cp1
P3
Po3
O1
C4
Cp2
Pz
Oz
P4
Fp1
Af3
0.02
F8
F7
0.01
Fc6
Fc2
Cz
Cp5
P7
F4
Fz
C3
0.03
Fp2
Af4
Cp6
T7
P7
−0.02
Po4
O2
Cp1
P3
F8
T8
C4
Cp2
Pz
Oz
0.01
Fc6
Fc2
Cz
Po3
O1
−0.03
F4
Fz
C3
0.02
Fp2
Af4
Fc1
Cp5
−0.01
P8
F3
Fc5
0
T8
0.03
Dp
0.04
P4
Po4
O2
0
Cp6
P8
−0.01
−0.02
−0.04
−0.03
(a)
(b)
EHILG
0.015
Fp1
Af3
F7
F3
Fc5
T7
Fc1
Cp1
P3
Po3
O1
F8
0.005
Fc6
Fc2
Cz
Cp5
0.01
F4
Fz
C3
P7
Fp2
Af4
C4
Cp2
0
Cp6
−0.005
Pz
Oz
T8
P4
Po4
O2
P8
−0.01
−0.015
(c)
Figure 4: Fisher distance of different channels with subject 1. Features are extracted from component IMF1. For each channel, Fisher distance
is calculated among features extracted from 480 labeled emotion samples of subject 1. (a) Fisher distance under feature 𝐷𝑡 . (b) Fisher distance
under feature 𝐷𝑝 . (c) Fisher distance under feature 𝐸norm .
Fisher distance is an efficient criterion of divisibility
between two classes, which is broadly used in pattern
recognition. It computes the ratio of between-class scatter
degree and within-class scatter degree between two classes.
Larger ratio means larger divisibility of the two classes. In
our experiment, we used fisher distance to mark important
electrodes under condition that IMF1 is used for feature
extraction. For each channel, fisher distance is calculated
among features extracted from one subject’s total 480 labeled
emotion samples.
Figure 4 gives fisher distance on valence dimension with
subject 1. Figure 4(a) shows that, under feature 𝐷𝑡 , electrodes
Fp1, Fp2, FC6, Cp1, O1, and Oz have larger values. Figure 4(b)
shows that, under feature 𝐷𝑝 , Fp1, FC6, Cp1, Cp2, O1, Oz,
P7, and P8 have larger values. Figure 4(c) shows that, under
feature 𝐸norm , F7, F8, T7, T8, P7, P8, O1, O2, and Oz have larger
values.
Based on the analysis of all the subjects, we selected the
following 8 electrodes Fp1, Fp2, F7, F8, T7, T8, P7, and P8
for channel reduction verification. Table 2 gives 𝐹1 score and
classification accuracy with 8 channels selected for emotion
recognition. We see that 𝐹1 score is 0.7374 for valence and
0.7769 for arousal. The classification accuracy with 8 channels
is 69.10% for valence and 71.99% for arousal, slightly lower
than accuracy with total 32 channels. We also applied 𝑡-test
to examine whether the performance of 8 channels is similar
BioMed Research International
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performance between classical method and our method. The
null hypothesis is “the performance is similar” and if 𝑝 value
is larger than 𝛼, the null hypothesis is accepted. The results
of 𝑡-test in Table 3 show that the performance of our method
is more splendid than fractal dimension, sample entropy, and
differential entropy of Beta band with 𝑝 far less than 0.05. It
also shows that the performance of our method is similar and
better than the differential entropy of Gamma band.
EMD strategy outperforms time domain method, including fractal dimension and sample entropy. This is because
compared to methods in time domain, EMD has the advantage of utilizing more oscillation information. Compared
to time-frequency method DWT, EMD can decompose
EEG signals automatically, getting rid of selecting transform
window first. The classification accuracy is also higher than
DWT. So the experiment results infer that our method based
on EMD strategy is suitable for emotion recognition from
EEG signals.
70
Accuracy (%)
60
50
40
30
20
10
0
Valence
Arousal
4. Discussion
FD
SampEn
DWT + DE (Beta)
DWT + DE (Gamma)
Our method
Figure 5: Classification accuracies of different methods. “FD,”
“SampEn,” and “DE” in the figure are corresponding to fractal
dimension, sample entropy, and differential entropy, respectively.
The mean accuracy was computed among all the subjects. Error
bars show the standard deviation of the mean accuracies across all
subjects.
to total 32 channels. The null hypothesis is “the performance
is similar” and if 𝑝 value is larger than 𝛼, the null hypothesis is
accepted. The 𝑡-test result shows that the performance under
8 channels and 32 channels is similar, with 𝑝 = 0.4194 for
valence and 𝑝 = 0.9521 for arousal.
So in practical use, we just need to extract features from
IMF1 with 8 channels. Our offline experiment used every 5 s
EEG signals as a labeled emotion sample. This infers that our
method may provide a new solution for real-time emotion
recognition in BCI systems.
3.4. Results Comparison with Other Methods. In this subsection, we compared our proposed method with some classical
methods, including fractal dimension (FD), sample entropy,
differential entropy, and time-frequency analysis DWT. We
used box counting for fractal dimension calculating. The
parameter for sample entropy SampEn(𝑚, 𝑟, 𝑁) was set as 𝑟 =
0.2, 𝑚 = 2, and 𝑁 = 128. We used “db4” decomposition to
realize DWT. Then the differential entropy of Beta (16–32 Hz)
and Gamma (32–64 Hz) bands is extracted as features. Our
method used IMF1 for feature extraction of 𝐷𝑡 , 𝐷𝑝 , and 𝐸norm .
For all the methods, 8 selected channels FP1, FP2, F7, F8, T7,
T8, P7, and P8 are used for feature extraction.
From Figure 5 and Table 3, we see that our method
yields the highest accuracy, 69.10% for valence and 71.99%
for arousal. We applied 𝑡-test (𝛼 < 0.05) to examine the
Emotion recognition from EEG signals has achieved significant progress in recent years. Previous methods are usually
conducted in time domain, frequency domain, and timefrequency domain. In this paper, we propose a method of
feature extraction for emotion recognition in EMD domain,
a new aspect of view. By utilizing EMD, EEG signals can
be decomposed into different oscillation components named
IMF automatically. The characteristics of IMF are utilized
as features for emotion recognition, including the first difference of time series, the first difference of phase, and the
normalized energy.
Compared to methods in time domain, EMD has the
advantage of utilizing more frequency information. The
experiment results show that the proposed method outperforms method in time domain, such as fractal dimension in
[3, 4] and sample entropy in [5]. Compared to time-frequency
methods, such as STFT and DWT, EMD can decompose
EEG signals automatically, getting rid of selecting transform
window first. The classification accuracy is also higher than
DWT in [18].
We investigate the role of each IMF in emotion classification. Features extracted from IMF1 yield the highest accuracy.
IMF1 is corresponding to the fastest changing component
of EEG signals, so our study confirms the deduction that
emotion is more relative to high frequency component.
This consists with findings in [26] that Beta (16–32 Hz) and
Gamma (32–64 Hz) bands are successfully selected more
often than other bands.
Finally, we selected 8 informative channels based on EMD
strategy, namely, FP1, FP2, F7, F8, T7, T8, P7, and P8. Our
proposed method just needs to extract features from IMF1
with 8 channels, which will save time and relieve computation
burden. Also in our experiment, every 5 s EEG signals are
extracted as a sample, so it may provide a new solution for
real-time emotion recognition in BCI systems.
Our limitation is that now we just test it on DEAP dataset,
so in the future we want to experiment it on more emotional
datasets to verify the method comprehensively. Also we will
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BioMed Research International
Table 3: The mean accuracy of different kinds of methods (Fp1, Fp2, F7, F8, T7, T8, P7, and P8) (standard deviation shown in parentheses;
statistical analysis shown in column 𝑡-test).
Methods
Fractal dimension
Sample entropy
DWT + differential entropy (Beta)
DWT + differential entropy (Gamma)
Our method
Accuracy (%)
53.08 (19.14)
57.44 (11.66)
60.87 (11.74)
67.36 (6.61)
69.10 (6.95)
Valence
𝑡-test (our method)
𝑝=0
𝑝=0
𝑝 = 0.0013
𝑝 = 0.3185
𝑝=1
utilize more strategies such as feature smoothing and deep
network to improve the classification accuracy.
5. Conclusion
In this paper, an emotion recognition method based on EMD
using three statistics is proposed. An extensive analysis has
been carried out to investigate the effectiveness of the features
for emotion classification. The results show that the three
features are suitable for emotion recognition. Then the effect
of each IMF component is inquired. The results reveal that,
among the multilevel IMFs, the first component IMF1 plays
the most important role in emotion recognition. Also the
informative channels based on EMD strategy are investigated
and 8 channels, namely, FP1, FP2, F7, F8, T7, T8, P7, and
P8, are selected for feature extraction. Finally, the proposed
method is compared with some classical methods and our
method yields the highest accuracy.
Conflicts of Interest
The authors declare that there are no conflicts of interest
regarding the publication of this paper.
Acknowledgments
This work was supported by the grant from the National
Natural Science Foundation of China (Grant no. 61701089).
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W4310896171.txt | Decorum. An Ancient Idea
for Everyday Aesthetics?
Elisabetta Di Stefano
Everyday Aesthetics was born in the 21st Century as a sub-discipline of Anglo-American Aesthetics and
it has spread in the international debate. However, the contribute of historical perspective has not
properly explored yet. Is it possible to trace the history of everyday aesthetics before the official birth of
this discipline? I will try and give an affirmative answer by focusing on an exemplary category: that of
the decorum. Using the history of ideas, I will analyse the Greek concept of prepon and the similar Latin
concepts of decorum which express the idea of ‘convenience’ or ‘fitness to purpose’ in the ethical and
rhetorical sphere. Later I will analyse the evolution of the concept of decorum in the theory of Ancient
and Renaissance architecture (Vitruvius, Leon Battista Alberti). My goal is to demonstrate that in
Ancient and Renaissance culture decorum is a category that refers to the objects and practices of
everyday life but also a principle that regulates appropriate behaviour in the sphere of good manners.
Consequently, given its pervasiveness in the different areas of everyday life, the concept of decorum can
be a paradigmatic example to trace the history of everyday aesthetics. | Keywords: Decorum, Everyday
Aesthetics, Prepon, Good Manners, History of Ideas
1. Introduction
Originated at the start of the new Millennium as a sub-discipline of AngloAmerican aesthetics focusing on art, Everyday Aesthetics has distinguished
itself for its shift towards practices and objects of everyday life. Several fields
and varied aesthetic categories have been included in its inquiries,
nevertheless one line of investigation has received little attention until now,
that is to say, the historical realm. This essay aims to go back to the early
origins of Western culture in the attempt to trace the steps of the history of
everyday aesthetics before its official birth. To this aim I will rely on the
methodology of the history of ideas in the footsteps of the Polish philosopher
Władysław Tatarkiewicz (1980). His method turns out to be particularly fitting
for my inquiry seeing as Tatarkiewicz developed the history of aesthetics
including all ideas affecting aesthetic issues, even when presented under
different names, and belonging to other disciplines not only philosophical.
Vol. 10/2
2021
25
Tatarkiewicz’s historiographical approach allows us to find a way out of the
traditional impasse inherited from the early historians of aesthetics, Robert
Zimmerman (1858) and Benedetto Croce (1902). They firmly held onto
a general framework excluding all contributions not produced by philosophers,
and inevitably got bogged down while attempting to legitimize a history of
aesthetics also predating the eighteenth-century foundation of the discipline.
By separating the history of words from that of concepts, Tatarkiewicz claimed
that aesthetic thinking began in Europe more than two-thousand years before
a name was found for it and an autonomous field of research was established.
Considering that everyday practices and objects had rarely attracted
philosophical interest before, usually being seen as too trivial, this method,
that targets on implicit aesthetics, proves to be suitable above all for everyday
aesthetics. The reason for this is that it aims at including a variety of concepts
touching upon the field of aesthetics by relying not only on philosophical texts
but also on artistic and literary contributions, technical textbooks and private
documents.
Needless to say, a full outline of the history of everyday aesthetics is an
ambitious goal far beyond the limits of this essay. I will therefore limit myself
by outlining the origins of one single aesthetic idea, that of decorum. This
notion has an exemplary value, inasmuch as it cuts across several cultural
realms. While changing its names in the course of different periods, it
preserves a consistent meaning: ‘aptness’, ‘convenience’, ‘fitness to purpose’.
Tatarkiewicz includes this notion among the varieties of beauty and focuses on
its terminological transformations:
From ancient times regarded as a variety of beauty has been aptness,
specifically the aptness of things to the task the things were meant to
fulfil, to the purpose that they served. The Greeks called this quality
πρέπον; the Romans translated the expression as decorum. […] Later, in
Latin, the name aptum was used more frequently, but in the
Renaissance decorum returned. Frenchmen of the ‘Great [17th] Century’
most often called this property bienséance, Poles of the age spoke of
przystojność. Today one speaks rather of suitability, appropriateness,
purposefulness and functionalism as a quality of certain arts and the
cause of the pleasure that we find in them. The terminology has varied,
but the concept itself has persisted.” (Tatarkiewicz, 1980, p. 159)
Among the above listed terminological variations, decorum is in my opinion
the most interesting, inasmuch as it qualifies as a norm regulating the beauty
of both behaviour and architecture, two realms in which the Italian language
still uses the same Latin-derived word decoro.
The notion of decorum was first conceptualized by Cicero as a norm of rhetoric
and as a principle of everyday life (Orat. 21, 70): “In an oration, as in life,
nothing is harder than to determine what is appropriate. The Greeks call it
πρέπον; let us call it decorum or ‘propriety’” Cicero, 1952, p. 357).
Starting from Cicero’s early definition and in reference to the history of ideas,
I will first explore the Greek notion of πρέπον from which the Latin concept
stems; then I will focus on decorum as conveying the idea of ‘convenience’ and
ELISABETTA DI STEFANO
Decorum. An Ancient Idea for Everyday Aesthetics?
26
‘fitness to purpose’ in the rhetorical and ethical sphere. Finally, I will analyse
the evolution of decorum in the Ancient and Renaissance architectural theory
(Vitruvius, Leon Battista Alberti) and in the field of good manners.
My goal is to demonstrate that decorum – and its analogous terms – is
a category that not only refers to the objects and practices of everyday life but
is also a principle that regulates appropriate behaviour. Consequently, given its
pervasiveness in the different areas of everyday life, the concept of decorum can
be a paradigmatic example to trace the history of everyday aesthetical ideas.
2. The concept of πρέπον in Greek culture
As emphasised by Pohlenz (1933, p. 53), the substantivized adjective to prepon
has many meanings. In archaic Greek it designates a shining quality,
a conspicuousness which, like the virtues of Homeric heroes, stands out before
the eyes of the beholders (Homer, Iliad, XII, 104). However, the timelessness of
the myth fades away when the term to prepon is coupled with the noun kairos,
‘occasion’. The temporal determination provides a new meaning to to prepon
and connects it to what Mario Perniola defines as ‘actual beauty’, that is to say,
a type of beauty waiving off its absolute and universal value to adapt to given
circumstances (Perniola, 1982, p. 45).
The connection between ‘convenience’ (prepon) and ‘occasion’ (kairos) is
abundantly documented in Greek literature and it is particularly powerful in
technical writings about medicine, politics and rhetoric. This bond describes
words and actions being effective, since they are placed in a given space-time
configuration (i.e. the decisive place, the crucial moment) and therefore they
are well adapted to the situation (Trédé, 1992).
In the field of rhetoric, the link between prepon and kairos was first established
by Gorgias and then reinforced by Isocrates, according to whom discourses
cannot be beautiful if they are not attuned to the circumstances and befitting
the topic (Soph. 13). Furthermore, Isocrates was the first to confer an
educational and political value to the notion of prepon connecting it to the
issue of paideia, that is to say, the education of youth in relation to which the
rhetor is the life mentor.
In the realm of philosophy, Socrates plays a key role in the understanding of
the idea of prepon. Since he left no autographic writtings, his legacy can be
evinced from the dialogues of Plato and Xenophon, which often feature
Socrates in the main role.
In the Platonic dialogue, Hippias Major (289d-290e), talking about utensils,
Socrates claims that not all materials are well adapted to all shapes, but only to
those for which they are ‘appropriate’. For stirring a bean soup, a fig wooden
spoon is more appropriate than a golden one because “it makes the soup smell
better, and at the same time, […] it won’t break our pot, spill out the soup, put
out the fire, and make us do without a truly noble meal, when we were going to
have a banquet” (Plato, 1982, p. 13).
However, while Plato’s Socrates is mainly concerned with metaphysical
ELISABETTA DI STEFANO
Decorum. An Ancient Idea for Everyday Aesthetics?
27
questions and the quest for beauty in itself (i.e. the idea of beauty),
Xenophon’s Socrates launches the notion of functional beauty. This latter,
which is to be found again in the Latin concept of aptus (appropriateness to
purpose), will survive through Medieval theoretical contributions – as testified
by Agustine’s surviving treatise, De pulchro et apto (Tatarkiewicz, 1980, p. 160)
– until modern functionalism many centuries later.
Socrates’s key role in the elaboration of an aesthetic category pertaining to
everyday life is made clear by his many references to domestic environments
and objects of daily use. In a passage from Xenophon’s Memorabilia (III, 8, 4),
Socrates claims that a golden shield, although beautiful to look at, is not suited
to a battle since gold is too fragile to guarantee safety. The careful choice of
materials then translates into care for the people who are going to use them
and for their needs. This is clearly stated in a later section of the Memorabilia
(III, 10, 9-13). Talking to the armorer Pistias, who is bragging about his
breastplates and how they are well-made because they are well-proportioned,
Socrates claims that an armour should not be perfectly well-proportioned, but
in relation to the wearer. As a matter of fact, unfitting armours, as they hang
from one’s shoulders or burden some other part of the body, are oppressing
and difficult to wear, while those which are fitting should look like “an
accessory rather than an encumbrance” (Xenophon, 1979, pp. 237-239).
Furthermore, the notion of convenience is abundantly discussed by Aristotle in
his contributions both to rhetoric and to philosophy. He defines convenience as
the right correspondence between the language and the character of the rhetor
(Reth. III, 7,1408 a-b) and in the Poetics (13, 1454a) he emphasises how each
character is supposed to use words and perform actions that are consistent
with their character. This line of thought has then been further developed in
Theophrastus’ Characters.
Finally, among the most important Greek theoretical contributions on the
notion of prepon, one should mention Panetius, a member of the middle Stoa.
While breaking with the early Stoa and its idea of the wise life as isolated from
the world, Panetius addresses all people, who while dealing with everyday
affairs have to make choices and perform actions. According to Panetious,
people should use their abilities and gifts to the benefit of society by following
the prepon which guides everybody to do the right thing at the right time (Cicu,
2000, pp. 136-137).
3. Cicero and decorum
The close correspondence between the Greek word prepon and the Latin
decorum is clearly stated by Cicero in the texts Orator (21,70) and De Officiis (I,
93). Although the two expressions are similar, the notion of decorum undergoes
some evolution in meaning in the transition between the text on rhetoric
written in 46 BC to the one on ethics written two years later.
It would actually be wrong to believe that the notion of decorum as presented
in the Orator is simply a tool of rhetoric. This latter is not only an operational
field connected to politics, but also one in which decorum is presented as
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Decorum. An Ancient Idea for Everyday Aesthetics?
28
a guiding principle, whose validity extends beyond the rhetoric to the arts and
life in general (Guérin, 2009, p. 125). Nevertheless, an even stronger connection
between ethics and aesthetics can be found in the De Officiis, inasmuch as
decorum is introduced as the fourth part of the honestum dealing with the
appropriateness of discourses and daily actions performed within
one’s community.
In the rhetorical field, decorum is a virtue of style, harmoniously regulating the
relations between speaker, message, audience, and register of communication
(Orat. 21, 71). Latin rhetoric distinguishes three styles, genus tenue or plain,
genus medium and genus grande or grand. Decorum should see that each genre is
matched with the appropriate arguments, styles and audiences.
Despite appearing at first as a normative principle with codified rules, decorum
is introduced by Cicero when dealing with the difficulties connected to
understanding what is appropriate both in discourses and in life in general
(Orat. 21, 70: “ut […] in vita sic in oratione nihil est difficilius quam quid deceat
videre”; “In an oration, as in life, nothing is harder than to determine what is
appropriate” Cicero, 1952, p. 357).
It should be added that, in the realm of ethics, grasping the appropriateness of
words and actions with respect to circumstances is the result not of rational
intelligence but of a form of sensibility which is able to perceive nuances,
atmospheres and moods. Jumping ahead to a notion engendered within the
milieu of Romanticism, one might say, with André Demouliez (1976, p. 286),
that convenience belongs to the realm of taste, provided it is not taste itself.
This remark is confirmed by Quintilian (Inst. Or. 11, 1, 91); while connecting
decorum to measure and temperance, he states that convenient behaviour
cannot be guided by pre-established rules but only by sensitivity. We might
refer to it as a certain taste, as suggested by Quintilian’s analogy with food,
where, rules are of little value. Inspired by the philosopher Panetius, in De
officiis Cicero emphasises the bond between decorum and moral integrity,
which is applied to interpersonal relations within the community, in the
following terms:
We have next to discuss the one remaining division of moral rectitude.
That is the one in which we find considerateness and self-control,
which give, as it were, a sort of polish to life; it embraces also
temperance, complete subjection of all the passions, and moderation in
all things. Under this head is further included what, in Latin, may be
called decorum (propriety); for in Greek it is called πρέπον. Such is its
essential nature, that it is inseparable from moral goodness; for what is
proper is morally right, and what is morally right is proper. The nature
of the difference between morality and propriety can be more easily felt
than expressed. (Cicero, De off., I, 27, 93; 1928, pp. 95-97)
Self-control, temperance and moderation make up the essence of decorum and
provide aesthetic value to everyday life (quidam ornatus vitae, ‘a sort of polish
to life’), making one’s manners gentle and one’s behaviour refined, while
respecting not only those who belong to the high society but to all citizen.
As it was in Greek culture, also in Latin culture decorum is a form of beauty.
ELISABETTA DI STEFANO
Decorum. An Ancient Idea for Everyday Aesthetics?
29
This is confirmed by Cicero’s analogy between the harmony of the body and the
appropriateness of one’s conduct. However, whereas physical beauty is visible
to the eye, decorum is a ‘relational’ aesthetic category. In other words, it is
a behavioural beauty which shines through words and daily actions and sparks
approval in the community.
For, as physical beauty with harmonious symmetry of the limbs
engages the attention and delights the eye, for the very reason that all
the parts combine in harmony and grace, so this propriety, which
shines out in our conduct, engages the approbation of our fellow-men
by the order, consistency, and self-control it imposes upon every word
and deed. We should, therefore, in our dealings with people show what
I may almost call reverence toward all men – not only toward the men
who are the best, but toward others as well. […] It is the function of
justice not to do wrong to one's fellow-men; of considerateness, not to
wound their feelings; and in this the essence of propriety is best seen.
(Cicero, De off., I, 28, 98-99; 1928, pp. 101-103)
This form of beauty that glows in a correct behaviour is the distinctive feature
of the vir bonus (honest man), whose ideal portrait is painted by Cicero in De
officiis. This would be someone who is active in public and political life, and
who spends their free time (otium) engaging in the arts and philosophical
studies. By nurturing a sense for aesthetics and abiding to the principles of
honesty and convenience, this person achieves elegance in their being and
acting.
As he outlines this ethical and aesthetic model of the ideal person, Cicero
includes in De officiis (I, 126-134) some sort of ‘manual of good manners’,
suggesting hygiene and behavioural norms inspired by the right measure and
avoiding all excess. He recommends bodily hygiene without affectation but also
without negligence; a way of walking neither too fast, nor too slowly; a clear
and fluent way of conversing, which avoids excluding others from the
conversation or falling into tittle-tattle (Cicu, 2000, pp. 150-154).
As a result, Cicero’s ideal person, in close resemblance to Roman models such
as Scipio Aemilianus, is well-read, balanced, never out of place or out of
measure, with a strong sense of attachment to their homeland and
community.
As Guérin (2009, p. 126) points out “the decorum described in Cicero’s De officiis
is […] a principle of coherence between the ethical agent and his actions,
a means to reach a state of general appropriateness, the rational convenientia
by the virtue of which one can make the choices and accomplish the actions
which correspond to his own nature”. As a result, Cicero applies to his vir bonus
model the same criterion of convenience, based on which the poets choose
only those words and actions that are befitting of a character – according to the
previously mentioned Aristotelian principle consolidated by Theophrastus.
Along the same line one can place Quintilian’s contribution on the topic of
convenience in the XI book of his treatise, Institutio oratoria. According to
Quintilian as well, decorum is a norm regulating the choice of content, the
distribution of words, the style and even the performance of the rhetor.
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Decorum. An Ancient Idea for Everyday Aesthetics?
30
However, as it shares its lexical root with the impersonal verb decet (it is
convenient, it is befitting), decorum qualifies also as a moral principle, guiding
choices and behaviours even to the detriment of personal interest. Quintilian
mentions the example of Socrates, who refrained from simple personal defence
in court, which would have saved him from being sentenced to death, since it
was contrary to his moral values. Hence, the rhetor has to refrain from
persuasion if this latter clashes against a higher value (Inst. Or. 11, 1, 11). As
a result, also for Quintilian, decorum is a rhetorical norm regulating the
rhetorical performance as much a moral principle, guiding one’s lifestyle and
the consistency of thinking and acting. Indeed, in the footsteps of Cicero (De
or. 3, 212), he claims that the main virtue of the orator is prudentia (practical
sagacity). This is a moral virtue that can be acquired with experience and its
effects derive from the ability to grasp what is appropriate in each occasion.
In this regard, Latin culture establishes decorum as an ethical and aesthetic
category which describes the ideal of an honest person (Cicero) and of a rhetor
engaged in the rightful and appropriate administration of public affairs
(Quintilian) for the good of the community.
4. The Architectural Theory: Vitruvius and Leon Battista Alberti
The migration of the notion of decorum into the area of art – already
inaugurated by the numerous architectural metaphors employed by Cicero (De
or. III, 152 and Or. 50.) – was further legitimized by Vitruvius, who imported
into architecture many terms and concepts from rhetoric. According to
Vitruvius, architects have to design a building taking into account beauty
(venustas), utility (usus) and appropriateness (decor) (De arch. VI, 10). In De
architectura (IV, I, 7-8) decor is the principle that connects the use of the
various orders of architecture to the character of the divinity to whom a temple
is dedicated: the Doric style, simple and sober, is appropriate to gods who
manifest strength and military valor; the Ionian style is appropriate to female
deities; the Corinthian style with its delicate ornaments and floral motifs to
the youngest and most graceful gods.
Whereas Vitruvius is mainly receptive to the normative aspect of the principle
of convenience, Leon Battista Alberti also picks up on its moral value. In line
with Cicero’s and Quintilian’s teachings, he claims that a good architect must
be able to evaluate ‘what is fitting’ to each building since each of them has its
own specific ‘character’ (Alberti 1988, IX, 10, p. 315: “The greatest glory in the
art of building is to have a good sense of what is appropriate”). Paradigmatic
remarks are then provided concerning the differences between the palace of
a prince and the fortress of a tyrant:
A royal palace should be sited in the city center, should be of easy
access, and should be gracefully decorated, elegant, and refined, rather
than ostentatious. But that of a tyrant, being a fortress rather than
a house, should be positioned where it is neither inside nor outside the
city. Further, whereas a royal dwelling might be sited next to
a showground, a temple, or the houses of noblemen, that of a tyrant
should be set well back on all sides from any buildings. In either case
an appropriate and useful guideline, which will lend the building
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Decorum. An Ancient Idea for Everyday Aesthetics?
31
dignity, will be to construct it in such a way that, if a royal palace, it
should not be so large that it is impossible to throw out any
troublemaker, or, if a fortress, not so constricted that it resembles
a prison more than the apartment of a fine prince.” (Alberti 1988, V, 3,
pp. 121-122, emphasis mine)
Decorum is therefore connected to the idea of dignitas (dignity)1, which
conveys the ethical and aesthetic distinctive features of individuals with
respect to their character and social status. While developing this line in book
VIII of his treatise on architecture, Alberti claims that decorum establishes the
amount and type of ornaments for different buildings taking into account
hosted functions and the social prestige of their inhabitants.2 Alberti then
outlines an ascending scale of aesthetic values from private dwellings to public
and religious buildings. These latter are said to require greater decorative
richness as no house can be more beautiful than the house of God. As a result,
the ornament is no longer just an additional and decorative element, and is
perfectly integrated to the structure, characterizing each building according to
the principle of decorum, understood as both an ethical and aesthetic measure.
Besides the decorum of the rhetorical tradition, Alberti also retrieves –
although just in a quick implicit remark – the Socratic notion of prepon as
functional beauty. Socrates’ criticism of the golden shield, which despite being
aesthetically pleasing is of little use in battle, seems to find an echo in
Alberti’s words, as they criticize the “doors like those about which we read in
historians and poets, so heavily weighed down with gold, ivory, and reliefs that
they could be opened only by a team of men, and would give off a terrifying
creak” (Alberti 1988, VII, 12, p. 226). On the contrary, those less elaborate and
light, “that are easy to open and close”, should be appreciated more because
they are more functional.
The idea of beauty as aptness or fitness to purpose, already launched by the
Socratic prepon, is reborn along the centuries under many other names. As
emphasised by Tatarkiewicz:
During the Enlightenment, the concept of beauty became still more
strongly bound up with the concept of aptness; in that period, aptness
had advocates especially among the philosophers, essayists and
aestheticians of Britain: no longer now in the sense of social aptness,
but once again in the sense of utility, as formerly in Greece. David
Hume wrote (Treatise, 1739, vol. II) that the beauty of many human
works derives from their utility and fitness for the purpose which they
serve. Likewise Adam Smith (Of the Beauty which the Appearance of
Utility Bestows upon all Productions of Art, 1759, part IV, chap. I): The
effectiveness of any system or machine in producing the purpose for
which they were designed, lends beauty to the entire object. And in the
same vein, Archibald Alison (Essays on the Nature and Principles of
1
The connection between the concept of ‘dignity’ (dignitas) and that of ‘convenience’ (decor /
decorum) is found in the treatise De pictura II, 38, (Alberti 1980, p. 67: “Dignity must be
observed in everything. It would not be suitable to dress Venus or Minerva in a servant's hood
nor to dress Mars or Jupiter in female clothes” (my transl.).
2
Alberti (1988 VIII, 3, p. 250) claims: “In these matters I do feel, however, that even when the
dignity of the individual is considered, a sense of measure must be maintained, and that even
kings may be criticized for overexpenditure.”
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Decorum. An Ancient Idea for Everyday Aesthetics?
32
Taste, 1790) said that there is no shape that does not become beautiful
when it is perfectly suited to its purpose. For these writers the field of
beauty continued to be split: some objects possess their own beauty,
others acquire it thanks to their utility. (Tatarkiewicz, 1980, p. 161)
While the Socratic prepon will launch a line of thinking on architecture and
objects of use which will reach modern functionalism, the ethical and aesthetic
notion of decorum will sustain the debate on good manners and what is
befitting of those who wish to be part of ‘high society’.
5. The Good Manners
The ethical notion of dignity (in Italian decoro) recurs in a lot of books on good
manners. Already in The Book of the Courtier written by Baldassare Castiglione
in 1528, one reads that the perfect courtier must display courtesy and most of
all ‘discretion’ – that is to say, the ability to act appropriately according to the
circumstances (Castiglione 1901, par. 7-8, pp. 82-83).
The Courtier enjoyed great literary success in sixteenth-century Europe, and it
would be misleading to take it as just a handbook of good manners, like those
popping up in the following centuries starting with Giovanni Della Casa’s
Galateo. Already bucking the trend of his time and the decline of court values,
Castiglione aims to outline the ideal figure of the courtier, who is able to
bestow beauty on everyday life through an elegant and graceful behaviour. The
distinctive sprezzatura which shapes all of the courtier’s behaviours is a refined
art which avoids all artificiality. Virtue is indeed achieved, according to
Castiglione, when that affectation is kept at bay, that is to say, when each
activity is distinctively natural, simple and modest. However,
Castiglione’s ideal courtier belongs to the already faded Renaissance world. In
the sixteenth century, the courts of the great Italian lords resemble more
a theatre stage, where a role needs to be played, that is to say, one’s behaviour
needs to be adapted to what ensures the prince’s or leader’s benevolence; as
a result, courtesy turns into sterile formalism and etiquette.
The several treatises on good manners of the following centuries bear
testimony to the need to establish a code of norms defining the behaviour of
high society. Social aesthetics is hence developed on the basis of a shared
ceremonial, which is often more a matter of appearance than a real expression
of virtue.
As Tatarkiewicz points out, over time the notion of aptness appears under
other names, particularly in seventeenth-century French classicist theory:
‘convenance’, ‘justesse’, and especially ‘bienséance’.3
The change was not only one of terminology. A fairly significant shift in
thinking had taken place: the concern now was less with qualities of
things fitting them to their use, and more with qualities of a man
fitting him for his social station: a man is pleasing when his appearance
and behaviour match his estate and dignity. (Tatarkiewicz, 1980, p. 161)
3
As Tatarkiewicz (1980, p. 161) points out: “According to Dictionnaire de l'Academie Française
(1787 edition), ‘bienséance’ signifies ‘convenance de ce qui se dit, de ce qui se fait par rapport
a l’age, au sexe, au temps, au lieu etc.”
ELISABETTA DI STEFANO
Decorum. An Ancient Idea for Everyday Aesthetics?
33
The issue of behavioural appropriateness finds ample development in French
and English debates in the seventeenth and eighteenth century, leading to the
origins of the person ‘of good taste’, a refined, elegant person, who is tactful
and able to select appropriate words and perform appropriate actions in each
moment of everyday life.
6. Decorum as a Category for Everyday Aesthetics
This short historiographical journey has shown that the notion of convenience
has crossed several cultural fields (philosophy, rhetoric, art, good manners) and
has received many names (prepon, decorum, decor, biénseance). What remains to
be investigated is whether this notion can be rightfully credited with
exemplary value with regard to everyday aesthetics.
Although Cicero presents prepon/decorum as operating within every aspect of
life, the propounders of Everyday Aesthetics do not seem to be familiar with
the Greek-Latin notion, despite often speaking of fitness for purpose and
convenience in relation to circumstances.
Within the Anglo-American contemporary debate, only Glenn Parson and
Allen Carlson (2008, pp. 2-4) directly refer to the Socratic prepon in order to
provide their theory of functional beauty with a historical foundation.
However, their inquiry focuses on the question of whether beauty results from
‘being fit’ or ‘looking fit’ for function. They thus forget that, according to the
rhetorical theory of prepon/decorum, there is no dyscrasia between the two
options. Both Aristotle and Cicero (De or. III, 45, 179) conceive the perfection of
the discourse on the model of the human body, that is beautiful because each
organ fits its specific function. Following the rhetorical tradition, the architect
Leon Battista Alberti compares buildings to living organisms (“a building is
very like an animal”; Alberti 1988, IX, 5, p. 301), and on this ground points to
the unity of ‘being’ fitting to purpose and ‘looking’ fitting to purpose:
Take the case of a horse: they realized that where the shape of each
member looked suitable for a particular use, so the whole animal itself
would work well in that use. Thus, they found that grace of form could
never be separated or divorced from suitability for use. (Alberti 1988,
VI, 3, p. 158)
Without mentioning rhetoric but staying in the same line, Jane Forsey (2013,
p. 238) connects the idea of function with those of ‘character’ and ‘active use’
that are reminiscent of the Aristotelian ideas of ‘character’ and ‘enèrgheia’4:
“The functional beauty […] marks out its everyday character, which can be
experienced only through active use as demanding singular and specific
attention”.
Closer to Everyday Aesthetics, the contributions of Japanese-American scholar
Yuriko Saito and Finnish philosopher Ossi Naukkarinen deserve particular
attention here.
In her famous book Everyday Aesthetics (2007, p. 7), focusing on “the care and
4
According to Aristotles, (Met. IX, 6, 1048b e) the act (enérgheia) is the existence itself of the
object.
ELISABETTA DI STEFANO
Decorum. An Ancient Idea for Everyday Aesthetics?
34
respect for the materials, users, and dwellers”, Saito echoes, unawarely, the
notion of appropriateness or adaptedness to purpose already developed in the
classical era through the Greek word prepon and the Latin decorum.
Furthermore, while repeatedly mentioning Archibald Alison, Saito mainly
references his association theory and the emotions evoked by natural
environments. She therefore does not seem to grasp the sense of
Alison’s contribution to the development of an idea of beauty as aptness or
fitness to purpose.5 As already discussed elsewhere (Di Stefano 2020), Alison
retrieves, without particular theoretical rigor, the topos of the expressive
qualities of architectural orders6, which is first to be found in Vitruvius’s De
architectura, but then focuses in particular on everyday objects. Concerning
furniture, machines and tools, he claims, “nor is there any form which does not
become beautiful, where it is found to be perfectly adapted to its end.” (Alison
1821, p. 281)
Although Saito (2017, p. 125) discusses the issue of the appropriateness of
clothing to different circumstances and cultures, she fails to acknowledge that
Alison had already developed a wide investigation on this topic. According to
the Scottish philosopher, no garment is beautiful in absolute terms: the colours
of clothes should fit the situation and the person wearing them (Alison 1821,
pp. 176-178). Bright colours suit young people, sober ones the elderly, and the
colours of a prince’s garments are different from those of a farmer: “the dresses
in every particular performance had some relation to the character of that
performance, and to the emotion it is destined to excite in our mind.” (Alison,
1821, p. 247)
Alison’s remarks influenced the American cultural debate of the late nineteenth
century and had an impact on the theoreticians of modern functionalism. One
can indeed hear the echo of Alison’s words in American sculptor Horatio
Greenough, one of the greatest propounders of American functionalism (Ringe,
1960, pp. 314-321), as he says that: “The most beautiful chairs invite you by
a promise of ease.” (Greenough, 1947, p. 122) According to Greenough, beauty is
the ‘promise of function’ and it expresses a proportion in relation to action and
character.7 Alison also influenced Ralph Waldo Emerson, who established an
insoluble connection between beauty and convenience.8 Along the line traced
by Greenough and Emerson, also Louis H. Sullivan developed contributions
which have earned him the title of father of modern functionalism (Di Stefano
2012).
Over the centuries the category of decorum has undergone several
5
See Tatarkiewicz (1980), pp. 159-161, in particular on Alison, p. 161.
6
Alison (1821, p. 256) claims: “The Tuscan is distinguished by its severity; the Doric by its
simplicity; the Ionic by its elegance; the Corinthian and Composite by their lightness and
gaiety. To these characters, their several ornaments are suited with consummate taste.”
7
Greenough (1947, p. 71) points out: “I define Beauty as the promise of Function; Action as the
presence of Function; Character as the record of Function […] but so long as there is yet a
promise of function there is beauty, proportioned to its relation with action or with
character.”
8
See Hopkins (1951, pp. 78-80).
ELISABETTA DI STEFANO
Decorum. An Ancient Idea for Everyday Aesthetics?
35
transformations in relation to its different philosophical backgrounds. In
Saito’s contributions, for instance, the care for the materials, the users, and the
inhabitants is enriched with perceptual and sensorial nuances previously
unheard of, especially in the aesthetic debate predating the eighteenth century.
As a result, Saito’s concept of convenience acquires connotations which are
closer to sensibility than to normativity. The importance given to sensibility is
clearly expressed when Saito attempts to reconcile aesthetic and functional
criteria in the design of objects and buildings, by emphasizing the need to take
into account the physical and psychological effects that objects and
environments will produce in their users: “a design process also engages the
moral capacity of care and respect for other people”, overall people with special
needs, for instance, children, elderly people, patients, people with disabilities
and refugees (Saito 2017, p. 227).
Along the same line Saito polemicizes against the narcissistic and selfreferential trends in contemporary architecture, which make it arrogant if not
alienating, and hopes instead for “an architecture of courtesy and
attention.” (Saito 2007, p. 221)
Connecting the field of architecture and good manners, Saito is unconsciously
in line with Tristan Edward’s teaching (Edward 1944), who wrote a book titled
Good and Bad Manners in Architecture, thus reconciling the two evolutionary
lines of decorum: the architectural theory and the behavioural precepts.
According to the Welsh architectural critic and town planner, there are selfish
and presumptuous buildings, like skyscrapers, or even rude buildings. To
contrast these, he suggested polite and sociable buildings.
As we have seen, courtesy would in origin stand for the respect of given rules of
conduct and the being endowed with certain virtues (kindness, generosity)
which used to be a prerequisite for the members of the court. In a leap from the
Renaissance to the present time, within the line of the notion of
appropriateness and convenience applied to everyday behaviour, the remarks
on tact presented by another supporter of Everyday Aesthetics, the Finnish
scholar Ossi Naukkarinen, find an appropriate collocation. He claims that tact
is to be understood as a behavioural mode which is appropriate to given
circumstances and respectful of others (Naukkarinen 2014). To behave tactfully
is really important in all those sectors where a given behavioural etiquette is in
place (e.g. at work; in politics; in social relations).
In the globalized and multiethnic world of today, it is easy to crash against
customs stemming from different cultural values and behavioural norms. In
this context, to use tact means to follow a form of sensibility related to the
situation, selecting actions based on circumstances.
7. Conclusion
Following Władisław Tatarkiewicz’s example and traced path, I tried to show
that, since Antiquity and through the Renaissance, decorum has been
a category encompassing both objects and practices of everyday life as well as
the principle that regulated appropriate behaviour.
ELISABETTA DI STEFANO
Decorum. An Ancient Idea for Everyday Aesthetics?
36
Although it might at first seem a normative principle, since Antiquity decorum
has had an ancipital meaning: it has both a normative aspect and an aspect
connected to natural instinct. The former appears to be prominent in rhetoric
and architecture, where the fitting ornaments for each discourse (rhetorical
figures) and for each building are established. The notion of aptness regulates
also the design of objects and affects also the realm of behaviour, whenever
strict rules of conduct need to be followed (e.g. etiquette, diplomacy). However,
rules do not always provide the fitting solution in relation to changing
circumstances, and a natural instinct need to take over. In the realm of social
relations, this kind of sensibility is called tact.
By means of this short historical journey, I have tried to demonstrate to what
extent the concept of decorum has pervaded all spheres of everyday life and to
what extent it not only belongs to the categories of everyday aesthetics, but it
also allows us to trace the history of Everyday Aesthetics before its official
birth.
References
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R. Tavernor. Cambridge Massachusetts - London England: The MIT Press.
Alison, A. (1821) Essays on the Nature and Principles of Taste. Artfor: George
Goodwin & Sons. Anastatic reprint Hildescheim: G. Olms, 1968.
Castiglione, B. (1901) The Book of the Courtier. Translated by L. Eckstein Opdycke, New
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Cicero (1928) De Officiis. Translated by W. Miller. London: William Heinemann Ltd- New
York: G.P. Putnam’s Son.
Cicero (1952) Orator. Translated by H.M. Hubbell. London: William Heinemann Ltd/
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Cicu, L. (2000) ‘Cicerone e il prepon’, Paideia, LV, pp. 123-162.
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Demouliez, A. (1976) Cicéron et son goût. Essai sur une définition d'une esthétique romaine
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Di Stefano, E. (2012) ‘The Aesthetic of Louis H. Sullivan: Between Ornament and
Functionality’, in J. Gleiter (ed.) Ornament Today. Bolzano: Bozen University Press, pp.
64-75.
Di Stefano, E. (2020) ‘Archibald Alison e l’estetica del quotidiano? Una prospettiva
neoestetica’, Aesthetica Edizioni Preprint n. 113, pp. 123-132. DOI: 10.7413/03938522018.
Dowling, Ch. (2010) ‘The Aesthetics of Daily Life’, The British Journal of Aesthetics, 50:3,
pp. 225-242.
Edwards, A. T. (1944) Good and Bad Manners in Architecture: An Essay on the Social
Aspects of Civic Design. London: John Tiranti.
Forsey, J. (2013) The Aesthetics of Design. Oxford: Oxford University Press.
Greenough, H. (1947) Form and Function. Remarks on Art, Design and Architecture. Ed. by
H. A. Small Berkeley; Los Angeles; London: University of California Press.
Guérin, Ch. (2009) ‘Philosophical Decorum and the Literarization of Rhetoric’, in F.
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Arabic World. Hildesheim: Georg Olms Verlag, pp. 119-139.
Hopkins, V.C. (1951) Spires of Form: A Study of Emerson's Aesthetic Theory. Cambridge
(Mass.): Harvard University Press.
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Naukkarinen, O. (2014) ‘Everyday Aesthetic Practices. Ethics and Tact’, Aisthesis, 1,
pp. 23-44.
Parsons, G. - Allen, C. (2008) Functional beauty. Oxford: Clarendon Press.
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12, XXII, pp. 44-52.
Plato (1982) Hippias Major. Translated by P. Wooddruff. Oxford: Basil Blackwell.
Pohlenz, M. (1933) ‘To πρεπόν. Ein BeitragzurGeschichte des griechischenGeistes’,
Nachrichten der Gesellschaft der Wiss. zu Göttingen, Philolog.-histor. Kl., 16, pp. 53-92.
Quintilian (2002) The Orator's Education. Edited and translated by D.A. Russell.
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Ringe, D.A. (1960) ‘Horatio Greenough, Archibald Alison: And the Functionalist Theory
of Art’, College Art Journal, 19(4), pp. 314-321.
Saito, Y. (2007) Everyday Aesthetics. Oxford: Oxford University Press.
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Tatarkiewicz, W. (1980) A History of Six Ideas: An Essay in Aesthetics. The Hague; Boston;
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Elisabetta Di Stefano
University of Palermo, Department of Humanities
viale delle Scienze, build. 12
90128 Palermo - Italy
elisabetta.distefano@unipa.it
ELISABETTA DI STEFANO
Decorum. An Ancient Idea for Everyday Aesthetics?
38
| 7,052 | 44,402 |
W1977699486.txt | Hindawi Publishing Corporation
International Journal of Antennas and Propagation
Volume 2012, Article ID 490327, 9 pages
doi:10.1155/2012/490327
Research Article
Modal Resonant Frequencies and Radiation Quality Factors of
Microstrip Antennas
Jan Eichler, Pavel Hazdra, Miloslav Capek, and Milos Mazanek
Department of Electromagnetic Field, Faculty of Electrical Engineering, Czech Technical University in Prague, Technická 2,
166 27, Prague, Czech Republic
Correspondence should be addressed to Pavel Hazdra, hazdrap@fel.cvut.cz
Received 9 August 2011; Revised 10 January 2012; Accepted 13 January 2012
Academic Editor: Charles Bunting
Copyright © 2012 Jan Eichler 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.
The chosen rectangular and fractal microstrip patch antennas above an infinite ground plane are analyzed by the theory of
characteristic modes. The resonant frequencies and radiation Q are evaluated. A novel method by Vandenbosch for rigorous
evaluation of the radiation Q is employed for modal currents on a Rao-Wilton-Glisson (RWG) mesh. It is found that the
resonant frequency of a rectangular patch antenna with a dominant mode presents quite complicated behaviour including having
a minimum at a specific height. Similarly, as predicted from the simple wire model, the radiation Q exhibits a minimum too. It is
observed that the presence of out-of-phase currents flowing along the patch antenna leads to a significant increase of the Q factor.
1. Introduction
Evaluation of the basic properties of microstrip patch
antennas (MPA) has been numerously discussed in literature,
see, for example, [1–3]. The two main MPA attributes are
resonant frequency (or frequencies but we will deal mostly
with the dominant mode) and the radiation Q factor. So far,
only approximate results and semianalytic equations have
been published. To our knowledge, this is the first time
that these important characteristics have been studied in a
rigorous way. The antennas are treated by using a modal
approach (hence we do not a priori consider any feeding
to be connected), namely, by the theory of characteristic
modes (TCM), [4, 5]. Evaluation of the radiation Q is
performed both by the TCM from the eigenvalues slope and
by novel rigorous equations derived by Vandenbosch [6] and
Vandenbosch and Volski [7].
2. The Theory of Characteristic Modes
For completeness, let us formulate the basics of the characteristic modes for perfectly conducting bodies of area S. The
scattered field Es is related to the electric surface currents J by
the electric field integral equation (EFIE) [8]
L(J) − Ei
tan
= 0.
(1)
Equation (1) is usually treated within the method of
moments (MoMs) [8] framework and, due to the structure
discretization, the L operator is known as the “complex
impedance matrix” [Z] = [R] + j[X].
Then the associated Euler’s equation to be solved is
XJn = λn RJn .
(2)
Equation (2) is a standard weighted eigenvalue equation
leading to a set of real characteristic eigencurrents Jn and
associated eigenvalues λn . Properties of eigenvalues are
described in [9], at this moment it is important to note
that λn reflects the amount of net reactive power (thus λn =
0 means resonance). Instead of eigenvalues, the so-called
characteristic angles αn are introduced to show more visible
behavior with frequency [9]. Characteristic currents form a
complete orthogonal set, and hence the total current on a
conducting body may be expressed as a linear combination
of these mode currents [10].
2.1. Implementation of the Characteristic Modes Theory.
Implementation of the modal decomposition process has
been done in the MATLAB [11] environment using Makarov
EFIE codes [12] with the RWG basis functions [13]. This
usage is restricted to arbitrary 3D PEC structures with air
2
International Journal of Antennas and Propagation
dielectrics. Our developed TCM tool [14] has the following
main advantages:
T1 (x,y,z)
(i) Comsol Multiphysics [15]/MATLAB’s PDE TooLbox
mesh import,
(ii) Optional Green’s function for infinite ground plane
simulations,
(iii) Single solver/multicore solver/distributed solver
(within a computer network with installed
MATLAB).
In [6] a novel theory able to rigorously calculate radiated
power and stored energies directly from currents flowing
along the antenna has been presented. The radiation Q factor
is then readily evaluated by the definition [16]:
m , W
e
max W
Pr
.
1
8πωε0
Ω1
Ω2
Ω
J2
y
r2
(0,0)
Q = QZ =
ω0 ∂Z
ω0 ∂R ∂X
.
+
=
2 ∂ω
2 ∂ω ∂ω
Ω1
1
e =
(Ie − IR ),
W
16πω2 ε0
(5)
1
(Im − IR ),
16πω2 ε0
(6)
Ω2
k2 J(r1 )J(r2 ) − ∇ · J(r1 )∇ · J(r2 )
Ie =
Ω1
Im = k 2
Ω1
Ω2
J(r1 )J(r2 )
cos(kr21 )
dΩ1dΩ2,
r21
(7)
ω0 ∂X
ω0 ∂
e
2ω Wm − W
=
2 ∂ω
2 ∂ω
ω0 ∂λ
= Qeig ,
=
2 ∂ω
(8)
(9)
(10)
(13)
(14)
providing that
3.1. The Modal Radiation Q Factor. The modal radiation Q
factor may be evaluated from the slope of modal eigenvalues
[18]:
ω0 dλ
.
2 dω
1
m − W
e
Pr + j2ω W
2
|I |
m − W
e .
λ = 2ω W
where k is a free-space wavenumber, J is the surface current
density, and r21 is the distance between interacting current
elements. The tilde denotes that the radiation contribution IR
has been subtracted from the stored energies at every point
in space [17]. It is assumed that the currents are flowing in a
vacuum.
Qeig =
(12)
valid for lossless antennas [19] and using the fact that Pr = 1,
(12) results in
Q = QX =
× sin(kr21 )dΩ1dΩ2,
cos(kr21 )
∇ · J(r1 )∇ · J(r2 )
dΩ1dΩ2,
r21
Ω2
(11)
Inserting
(4)
where
k
2
For parallel or series RLC circuit (hence, for one mode), the
“impedance QZ ” equals the exact “current Q” [6]:
Z = R + jX =
IR =
T2 (x,y,z)
x
e , W
m .
Q = 2ω max W
k2 J(r1 )J(r2 ) − ∇ · J(r1 )∇ · J(r2 )
sin(kr21 )
×
dΩ1dΩ2,
r21
m =
W
r1
In [18], (10) is supposed to be an approximation of the
radiation Q, but in resonance it is actually exact.
Since characteristic modes are normalized to radiate unit
power Pr = 1 [4], (3) reduces to
(3)
The equations for radiated power Pr and stored electric and
e , W
m are
magnetic energies W
Pr =
r21 = |r2 − r1 |
Figure 1: Distance between nonoverlapping current elements [23].
3. The Radiation Q Factor
Q = 2ω
J1
(15)
It is therefore concluded that the modal Qeig equals the QX by
definition, and it can be proven (using the reactance theorem
[20, 21]) that in resonance it also equals the radiation Q
defined from energies by (11).
3.2. Software Implementation. The above equations were
implemented in MATLAB for the RWG triangular mesh
where two different interaction situations occur:
(a) Distant Elements. When the triangular elements are not
overlapping, current density on triangles may be simply
approximated as point sources located at the centre of
triangles [22], see Figure 1. No actual integration is then
needed. This centroid approach is very fast with satisfactory
accuracy as will be shown later (however it may fail for
patches located very close to the ground plane).
International Journal of Antennas and Propagation
P3 (x3 , y3 )
h13
(0,1)
A
P1 (x1 , y1 )
h12
P2 (x2 , y2 )
r1
x
(0,0)
T1 (α ,β,γ)
0.01
P0
h23
r2
y
z
β
T1 (x,y,z)
P0
3
(0,0)
α
0.005
(1,0)
0
2H
−0.005
(a)
y
(b)
Figure 2: Self-term evaluation. (a) Original problem, (b) simplex
coordinates transformation [23].
x
−0.01
0.015
0.01
(b) Overlapping (Self) Elements. As known from the method
of moments, the so-called “self ” contributions are of great
importance when dealing with calculations on discrete
elements (meshes).
Here, the self-interaction occurs when two triangles are
overlapping each other. Due to the behavior of integral
kernels, only rapidly varying term cos(kr21 )/r21 has to be
carefully treated. Since k0 R21 → 0 (R21 being the longest side
of the triangle T) is satisfied, one needs only to use the first
term in the Taylor series expansion. The dominant singular
static part is 1/r21 and the integral to be worked out is
1
T
T
(x
2
− x )
+ y − y
2 dx d y dx dy ,
−0.01
−0.015
4. Applications: Rectangular Patch Antenna
Let us first concentrate on a rectangular patch antenna of
dimensions L = 50 mm and W = 30 mm (further noted as
R50 × 30) placed in air at a height H above an infinite ground
plane. Only the dominant TM01 mode will be studied. The
reason for choosing a patch with L/W =
/ 1 is that we do not
have to deal with degenerated modes.
Using the image theory, the radiator in the XY plane
at height z = H above an infinite electric ground plane is
modelled as two patches separated by 2H. The total number
of triangular elements is 676. In the TCM analyser, a proper
out-of-phase mode is selected (Figure 3).
The resonant frequency of the dominant mode is shown
as a function of height H, see Figure 4. It has been evaluated
from a modal resonant condition for eigenvalues λ =
e ) = 0 employing an adaptive frequency
m − W
2ω(W
sweep for each height. The behaviour is quite peculiar,
especially for greater heights. For low heights (H < 10 mm or
−0.01
4
Regular
behaviour
3
Min. f res
2.5
Min. Q
2
1.5
1
where A is the triangle area, hi j are the edge lengths (see
Figure 2), and L is the perimeter of the triangle.
−0.02
0.02
4.5
3.5
4
ln(1 − 2h12 /L) ln(1 − 2h13 /L) ln(1 − 2h23 /L)
I = − A2
+
+
,
3
h12
h13
h23
(17)
−0.03
0.01
0
Figure 3: Model of MPA above infinite ground plane for H =
10 mm, dominant mode TM01 shown.
(16)
where T = T is a triangular area. Using simplex coordinates
transformation (Figure 2), the result is [23, 24]
0
−0.005
f res (GHz)
I=
0.005
0
5
10
15
20
25
30
35
40
45
50
55
60
H (mm)
f r (TCM)
f r (analytic equation)
Figure 4: R50 × 30 resonant frequency of the dominant TM01
mode. The dashed red curve is a quasianalytical equation from [1].
H/λres < 0.08), the resonant frequency decreases “regularly,”
and quasianalytical formulas (see, e.g., [1, 3]) based on
the fringing field concept are valid below this range. For
H ∼
= 25 mm (H/λres ∼
= 0.188) there is absolute minimum
of the TM01 resonant frequency. Further on, the resonant
frequency rises to reach its maximum for H ∼
= 40 mm
(H/λres ∼
= 0.51). Around this specific height the patch also
shows the minimum of the radiation Q. The above described
process repeats periodically. It is yet unclear to the authors
as what is the physical background to the resonant frequency
discontinuity around H/λres ∼
= 0.5.
e ) obtained
m , 2ωW
e , and 2ω(W
m − W
The terms 2ωW
from (5)–(9) and eigenvalues λ are plotted at Figure 5 for
4
International Journal of Antennas and Propagation
an approximate analytical solution is available and in [25] we
showed that the Q is led by the function
20
Reactive energies
15
fQ (H) ∼
=
10
f res
tan(k2H) = k2H,
0
1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3
f (GHz)
λ
2ω (Wm)
2ω (We)
2ω (Wm-We)
Figure 5: Reactive energies and their differences for an R50 × 30
patch at height of 25 mm.
H
λ
min
∼ 3 − 1 = 0.358.
=
2
8
6π
(20)
For sinusoidal currents on dipoles the minimum (evaluated
numerically) occurs for H = 0.36λ.
The minimum of the patch under study is obtained at
H ∼
= 0.4λ, a value that is remarkably close to the simple
dipole model.
4.1. Algorithm Convergence. Since no other methods for
calculating modal Q are available, Qeig is taken as a reference,
and the relative error percentage is defined as:
50
45
40
relative error =
35
Q (–)
(19)
and the first nontrivial root of (19) could be approximated
as [25]
−5
30
25
20
15
10
5
0
(18)
After deriving (18), the condition is worked-out
5
−10
2H
.
k2H − sin(k2H)
0
5
10
15
20
25
30 35
H (mm)
40
45
50
55
60
Q eig
QJ
Figure 6: The radiation Q for dominant mode of an R50 × 30 patch
as a function of height H.
H = 25 mm as a function of frequency. There is excellent
agreement between the difference in stored energies and the
eigenvalues, both obtained in a completely different manner.
There is also very good agreement between the exact QJ
and Qeig confirming the validity of the proposed algorithm
via (14), see Figure 6. Note that Qeig in (10) does not require
the currents to be calculated on the structure while QJ is
evaluated in a rigorous way from modal currents (11).
From Figure 6 it is seen that the radiation Q has a
minimum for a specific height. It is deduced that the reason
lies in the cancelling of the radiated power between the two
out-of-phase currents. Similar behaviour has been observed
in the case of two half-wave thin-wire dipoles with opposite
sinusoidal currents, separated by d = 2H, see [25] for details.
Actually these two out-of-phase dipoles may serve as a very
simple model for a patch antenna with a dominant mode.
When the dipoles are reduced to elementary (Hertzian) ones,
QJ − Qeig
Qeig
· 100,
(21)
where QJ is calculated from the currents using (11). Four
different heights H were chosen, H = 1 mm (0.01λ), H =
2 mm (0.0185λ), H = 10 mm (0.0803λ), and H = 20 mm
(0.151λ), and the relative error was evaluated as a function of
total triangular elements (including the mirror), see Figure 7.
All quality factors were evaluated at the resonant frequency
of the dominant mode for the R50 × 30 patch. As discussed
earlier, the centroid approximation became more inaccurate
with low heights H. However, even for the lowest analyzed
value H = 0.01λ, the relative error is in the order of
a few percent for reasonable mesh density (hundreds of
elements). Further improvements to the integration routine
are considered for the future.
4.2. Fractional Bandwidth of the R50 × 30 Patch Antenna. It
is known that the fractional bandwidth (FBW) is related to
the unloaded Q factor and the desired matching VSWR level.
For VSWR < s we have [26]
FBW ∼
=
s−1
√ [%].
Q s
(22)
Using a full-wave simulator CST-MWS [27], an R50 × 30
patch has been simulated and the FBWCST for VSWR < 2 was
calculated as:
FBWCST =
f2 − f1
,
f0
(23)
where f2 and f1 are margins for VSWR < 2 and f0 is
the centre frequency. Only very low heights were studied
since we used a simple probe feed which introduces an
inductance component to the total input impedance. The
comparison in Figure 8 shows good agreement of both
fractional bandwidths.
Relative error (%)
International Journal of Antennas and Propagation
10
9
8
7
6
5
4
3
2
1
0
0
200
400
600
800
1000
Number of triangle elements
H = 1 mm (0.01 λ)
H = 2 mm (0.0185 λ)
1200
5
J1
J2
σ1
σ2
1400
H = 10 mm (0.0803 λ)
H = 20 mm (0.151 λ)
Figure 7: Relative error of the Q factor as a function of triangular
elements (mesh density).
Figure 9: The first two characteristic modes (currents and charges)
for the SAU1 structure.
5
4.5
W
4
FBW (%)
3.5
3
2.5
J1
J2
σ1
σ2
L
2
1.5
1
0.5
0
1
1.5
2
2.5
3
3.5
H (mm)
4
4.5
5
FBW (CST)
FBW (Q eig )
Figure 8: Fractional bandwidth FBW (VSWR < 2) for a R50 × 30
patch.
0
0
Figure 10: The first two characteristic modes (currents and
charges) for the SAU2 structure.
5. Applications: Fractal Antennas
In this section, a bit more complex structures will be studied.
The first one (the “Self Affine U” fractal, SAU), has been
described in [28] and further analyzed in [29]. This kind
of radiating motif is employed as a dual-band radiator
with mutually orthogonal radiation patterns at both bands.
Therefore we are analyzing the first two modes, where the
currents are orthogonal. These are depicted in Figures 9
and 10 for first (SAU1) and second (SAU2) fractal iteration,
respectively. The current of the first (lower) mode J1 has
two out-of-phase components (see Figure 11 for schematic
current paths) while the second mode comprises inphase
currents only. As we know from previous studies, opposite
currents contribute to a rapid increase of the radiation Q,
and it is expected that J1 will have a much higher Q than J2 .
J1
J2
Original
Infinite ground plane
Image
Figure 11: The main current paths for the first two modes of the
SAU1/2 structure.
6
International Journal of Antennas and Propagation
280
260
240
Mode no.3
Mode no.2
200
Mode no.1
180
Q
αn
220
160
140
120
100
1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4
× 109
f (Hz)
100
90
80
70
60
50
40
30
20
10
0
10
Q1
Q2
12
14
16
18
20
22
24
26
28
30
H (mm)
(a)
(b)
Figure 12: Characteristic angles (left) and radiation Q for the SAU2.
0.03
0.03
J1
0.02
0.01
0.01
0
0
−0.03 −0.02
−0.01
0
J2
0.02
0.01
0.02
0.03
−0.03
−0.02
−0.01
(a)
0.01
0.02
0.03
(b)
0.03
0.03
σ1
0.02
0.01
0
0
−0.01
−0.01
−0.02
−0.02
−0.02
−0.01
0
σ2
0.02
0.01
−0.03
−0.03
0
0.01
0.02
0.03
(c)
−0.03
−0.03
−0.02
−0.01
0
0.01
0.02
0.03
(d)
Figure 13: Degenerated dominant mode J1 , and J2 of the FCL2 antenna (currents and charges).
Figure 11 presents a very simple concept showing the
main current paths for the J1 and J2 modes discussed above
including the mirroring effect of the infinite ground plane. It
could be simply stated that more opposing current paths lead
to significant increase in Q.
We show detailed behaviour only for SAU2 (the situation
is similar for SAU1)—see Figure 12 that confirms high Q for
the J1 mode. Characteristic angles are calculated for H =
29 mm, the actual height for which the dual-band antenna
was designed [29].
5.1. The FCL-2 Fractal Antenna. The second presented structure is the so-called fractal clover leaf (FCL) of the second
iteration, [14]. The antenna is fed by an L-probe [30] that
excites its dominant mode and is located at height H =
36 mm. Actually, the dominant mode is composed of two
degenerated modes J1 and J2 (Figure 13). The second higher
mode J3 is shown at Figure 14 for completeness.
Figure 15 shows the main current paths of these modes,
and we can again deduce that the dominant mode will exhibit
International Journal of Antennas and Propagation
7
0.03
0.03
J3
0.02
σ3
0.02
0.01
0.01
0
0
−0.01
−0.02
−0.03
−0.02
−0.01
0
0.01
0.02
0.03
−0.03
−0.03
−0.02
−0.01
(a)
0
0.01
0.02
0.03
(b)
Figure 14: Second higher mode J3 (currents and charges).
Mode 1, 2
J1 + J2
=
8.25
6.87
5.5
4.12
2.75
1.37
0
−3 .6 2
−7 .2 4
−1 0 .8 7
−1 4 .4 9
−1 8 .1 2
−2 1 .7 4
Mode 3
J3
=
7.32
6.1
4.88
3.66
2.44
1.22
0
−3 .7 7
−7 .5 5
−1 1 .3 3
−1 5 .1 1
−1 8 .8 9
−2 2 .6 7
Figure 15: Schematic depiction of the dominant current paths for the dominant (J1 + J2 ) and the second higher J3 modes together with their
modal radiation patterns.
lower Q compared to J3 . This is confirmed by Figure 16—J3
has more than 200x higher radiation Q.
6. Resonant Properties of Studied Antennas
The properties of studied antennas are summarized in this
section. At first we observed that microstrip antenna could
support different kinds of modes regarding their Q factors
(see Figure 17):
(a) low Q modes with the current flowing in one direction and not changing its phase (dominant modes of
simple shapes like rectangular, circular patch, and so
forth.)
(b) high Q modes with part of the currents flowing in the
opposite direction. These modes exist even on simple
“U” shaped patch (Figure 9 left) and on complex
(fractal) geometries.
Secondly, it has been observed that resonant frequency is
quite a complicated function of height. Unfortunately we do
not yet have any physical explanation as to why some modes
present minimum values of fr .
Looking at Figure 18, it is clear (and interesting) that
the resonant frequency behaves quite differently for lowQ and high-Q modes. The resonant frequency of low-Q
modes is much more sensitive to the height, whereas high-Q
modes exhibit almost constant fr when the height is varied.
The proposed explanation is that the opposite currents
(responsible for high Q) keep reactive fields very close to the
radiating structure so the effect of a fringing field coupled to
the ground plane becomes almost negligible.
7. Conclusions
Modal resonant properties of selected microstrip patch
antennas have been studied with the help of characteristic
8
International Journal of Antennas and Propagation
3
260
2.8
240
2.6
220
2.4
αn
200
fr (GHz)
280
Qeig3 = 226.3
180
160
Qeig1 = Qeig2 = 10.5
140
2
High-Q
modes
1.8
1.4
1.2
100
1
1
1.1
1.2
1.3
1.4
1.5 1.6
f (GHz)
1.7
1.8
1.9
10
High-Q
modes
90
15
20
H (mm)
25
30
35
SAU2 mode 2
R50 × 50
R50 × 30
Figure 18: Resonant frequencies for different antennas/modes.
Using proper feeding techniques (like the L-probe) allows us
to design wideband compact antennas.
The theory now puts current distribution and the radiation Q factor into objective context. Whenever the current
mode exhibits opposite components, high Q may appear.
Future work is needed to connect the presented theory
with parameter sweeps or even optimization, so we will be
able to design novel wideband/multimode compact antennas.
100
80
70
60
50
Low-Q
modes
30
5
FCL2
SAU1 mode 1
SAU1 mode 2
SAU2 mode 1
Figure 16: Characteristic angles for the FCL2 structure at H =
29 mm.
40
0
2
× 109
Mode 1
Mode 2
Mode 3
Q eig
2.2
1.6
120
80
Low-Q
modes
20
Acknowledgments
10
0
0
5
10
15
20
25
30
35
H (mm)
FCL2
SAU1 mode 1
SAU1 mode 2
SAU2 mode 1
SAU2 mode 2
R50 × 50
R50 × 30
This work was supported by the Grant Agency of the
Czech Technical University in Prague, grant no. SGS11/065/
OHK3/1T/13 and by the Project COST 1102. The authors
would like to thank professor Vandenbosch for fruitful
discussions, Neil Bell for his comments, and two anonymous
reviewers who suggested some improvements to the paper.
Figure 17: Radiation Qs for different antennas/modes.
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W2533533103.txt | Malaria Journal
Howes et al. Malar J (2016) 15:502
DOI 10.1186/s12936-016-1556-3
Open Access
RESEARCH
Contemporary epidemiological
overview of malaria in Madagascar: operational
utility of reported routine case data for malaria
control planning
Rosalind E. Howes1,2*, Sedera Aurélien Mioramalala3,4, Brune Ramiranirina3, Thierry Franchard3,
Andry Joeliarijaona Rakotorahalahy3, Donal Bisanzio5, Peter W. Gething5, Peter A. Zimmerman1*
and Arsène Ratsimbasoa3,4,6*
Abstract
Background: Malaria remains a major public health problem in Madagascar. Widespread scale-up of intervention
coverage has led to substantial reductions in case numbers since 2000. However, political instability since 2009 has
disrupted these efforts, and a resurgence of malaria has since followed. This paper re-visits the sub-national stratification of malaria transmission across Madagascar to propose a contemporary update, and evaluates the reported
routine case data reported at this sub-national scale.
Methods: Two independent malariometrics were evaluated to re-examine the status of malaria across Madagascar.
First, modelled maps of Plasmodium falciparum infection prevalence (PfPR) from the Malaria Atlas Project were used to
update the sub-national stratification into ‘ecozones’ based on transmission intensity. Second, routine reports of case
data from health facilities were synthesized from 2010 to 2015 to compare the sub-national epidemiology across the
updated ecozones over time. Proxy indicators of data completeness are investigated.
Results: The epidemiology of malaria is highly diverse across the island’s ecological regions, with eight contiguous
ecozones emerging from the transmission intensity PfPR map. East and west coastal areas have highest transmission
year-round, contrasting with the central highlands and desert south where trends appear more closely associated
with epidemic outbreak events. Ecozones have shown steady increases in reported malaria cases since 2010, with a
near doubling of raw reported case numbers from 2014 to 2015. Gauges of data completeness suggest that interpretation of raw reported case numbers will underestimate true caseload as only approximately 60–75 % of health facility
data are reported to the central level each month.
Discussion: A sub-national perspective is essential when monitoring the epidemiology of malaria in Madagascar
and assessing local control needs. A robust assessment of the status of malaria at a time when intervention coverage
efforts are being scaled up provides a platform from which to guide intervention preparedness and assess change in
future periods of transmission.
Keywords: Malaria, Madagascar, Routine health information systems data, Surveillance, Outbreaks
*Correspondence: rosalind.howes@zoo.ox.ac.uk; paz@case.edu;
aratsimbasoa@gmail.com
1
Center for Global Health and Diseases, Case Western Reserve University,
Cleveland, OH 44106‑4983, USA
3
National Malaria Control Programme, Ministry of Health, Androhibe,
Antananarivo, Madagascar
Full list of author information is available at the end of the article
© 2016 The Author(s). 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.
Howes et al. Malar J (2016) 15:502
Background
The Malagasy context is one of protracted political
instability, most recently in the aftermath of a political coup in 2009 which continues to bear severe economic and social impacts [1] (Fig. 1). A long-term lack
of investment in the country’s infrastructure has had
direct repercussions for malaria control efforts as well
as the country’s broader developmental situation [2].
Madagascar has the seventh lowest per capita gross
domestic product (GDP) globally [3] with an estimated
75.3 % of the population living under the national
poverty line in 2010 [3]. This was estimated to have
increased by more than 10 % since the 2009 crisis, with
the World Bank estimating 92 % of the population to be
living under $2 a day in 2013 [1]. The population lives
predominantly in rural environments (66 %), and almost
half (42.4 %) is younger than 15 years, and the median
age is 18.4 years [3]. Malnutrition is rife, affecting over
half of children, with 25 % of children reported to be
severely malnourished [4], exacerbated by periodic famine outbreaks following severe drought [5] and failed
harvests from locust infestation [1]. Childhood underweight is reported to be the primary overall risk factor
of disease in the Malagasy population [6], increasing
vulnerability to infections and development of severe
Page 2 of 18
sequelae. In 2012, top causes of consultations at health
centres among under 5 year olds were, in descending
order, acute respiratory infections, diarrhoea, digestive
disorders, and uncomplicated malaria [7]. In the five
to 14 years age bracket, uncomplicated malaria represented the second-most common cause of health centre
consultations in 2012, while complicated malaria presented the top cause of district-level hospital mortality,
at 10.1 % overall, and 21.6 % among under-fives [7]. All
23.9 million people across Madagascar’s 112 health districts are at risk of malaria exposure [8].
In view of this substantial disease burden, malaria
represents a main priority for the Malagasy Ministry of
Health (MoH) and associated international public healthfocussed partners [9]. Intervention coverage is high, with
some of the highest ownership rates of long-lasting insecticide-treated nets (LLINs) in the African region in 2013
[8, 10]. This was supplemented at the end of 2015 with
the distribution of 12 million LLINs aiming at universal
coverage of one net per two individuals across 92 of the
country’s 112 health districts where seasonal or perennial transmission is considered to occur. In the lower
transmission districts of the highlands, focalized indoor
residual spraying of insecticide (IRS) has been deployed
against the Anopheles vectors [11]. Nevertheless,
Fig. 1 Timeline of milestones in malaria policy, funding, planning and implementation in Madagascar. Malaria cases (2000–2014) are plotted
from data reported by the NMCP published in the WHO’s World Malaria Report 2015 [8]; numbers of RDT confirmed cases for 2015 are from NMCP
directly. Intervals between Global Fund disbursement when no financial resources were available to the NMCP were: Sept 2010–Feb 2011; Sept
2014–March 2015; and July 2016 onwards (NMCP, pers. comm., June 2016)
Howes et al. Malar J (2016) 15:502
increasing numbers of cases have been reported since
2010. This paper analyses the routine reported malaria
rapid diagnostic test (RDT) data from 2010 to 2015 to
evaluate trends of malaria transmission across Madagascar. This analysis provides a benchmark assessment
of malaria endemicity ahead of the anticipated impact of
the large-scale LLIN distribution campaign prior to the
2016 transmission season. December 2015 also represented the end of the Millennium Development Goals era
(2000–2015) and Fig. 1 summarizes the malaria control
activities that have contributed to the malaria-specific
aspects of this initiative. This overview therefore comes
at an opportune time to assess progress against the target
of “reversing the incidence of malaria” in Madagascar [12,
13].
Transmission of malaria is heterogeneous across Madagascar [14]. The island, the fourth largest globally at over
1500 km in length and with a surface area of 587,000 sq
km, similar to California or Sweden, is highly ecologically and climatically diverse [15, 16]. Reflecting these
conditions, the epidemiology of malaria across the island
shows distinct seasonal trends and transmission intensities between regions [17, 18] with different vector species
and behaviours predominant in different areas [19, 20].
Madagascar has a stated aim to reduce the proportion of
malaria-attributable fevers and mortality, and start transitioning towards pre-elimination status by end of 2017,
the end of the country’s current National Strategic Plan
[21] (Fig. 1). The development of spatially specific interventions informed by local transmission characteristics is
therefore required to optimize the efficacy of strategies to
combat malaria.
Mapping the intensity of malaria transmission across
the island has been done since the start of control efforts.
Joncour et al. published district-level endemicity estimates according to splenic rates in the 1950s, identifying
broad categories of malaria transmission [18]. These were
updated by Mouchet et al. in the early 1990s, stratifying
the country according to climatic and transmission patterns (Fig. 2a [22, 23]). These mapping sub-divisions have
subsequently adapted to changing needs, aligning with
contemporary administrative boundaries (Fig. 2b [21,
24, 25]), and recently been further simplified from Mouchet’s five zones to two broad zones representing high
transmission (east and west coasts) and low transmission (highlands and arid south) by control programme
agencies [11, 21, 25]. Here, the boundaries of these subnational divisions are re-evaluated and used to describe
the contemporary epidemiology of malaria in Madagascar more than a decade after the country started receiving substantial external funding to roll-out its control
programme (Fig. 1).
Page 3 of 18
Methods
Two methodological steps were followed to address the
paper’s goal of assessing Malagasy routine health facility data and its application to programmatic planning.
First, regional sub-divisions were reviewed and updated
in accordance with contemporary transmission patterns.
Second, routine health reports were examined within the
updated sub-national stratifications to identify each area’s
key epidemiological characteristics which might impact
on intervention planning. The data sources accessed in
this study are summarized in the Additional file 1.
Sub‑national stratification of malaria transmission
Transmission intensity, quantified as the communitylevel prevalence of blood stage infection, has been a
traditional marker for regional stratification to guide
malaria control [26, 27]. This approach bypasses potential
weaknesses associated with the surveillance and health
reporting systems. In this present study, mean summary
maps of the modelled prevalence of Plasmodium falciparum infection across the 2–10 year old age range (PfPR)
from the Malaria Atlas Project (MAP; [14, 28]) were used
to assess the regional patterns of malaria infection (Additional file 2). These maps are informed by an extensive
range of malariometric (PfPR community surveys from
Malaria Indicator Surveys [24, 25] and all other available
surveys e.g. [29], as well as annual case incidence estimates), intervention coverage [10] and environmental
covariate datasets [30]. For the present paper’s objectives,
the mean of the 2010–2015 annual surfaces was calculated (PfPR2010–2015; Additional file 2; Fig. 2c). This timeframe ensured consistency with the health facility case
data that were available for investigation in this paper
and provided a contemporary picture whilst attenuating
inter-annual fluctuations in the PfPR estimates. For operational utility, the spatially continuous mean PfPR2010–2015
map was aggregated to current sub-national political
boundaries, and then used to sub-divide the country into
ecozones of contiguous districts with similar transmission intensity (Fig. 2d). The aim of the stratification was
to generate an output that was pertinent for operational
purposes and that allowed an intuitive description of
the sub-national epidemiology of malaria. The number
of ecozones was not prior constrained though the aim
was to keep this under ten to ensure programmatic ease
and avoid too much duplication across zones in terms
of overlapping epidemiology. Automated clustering was
attempted using packages in spatial analysis software but
these were not well adapted to this present analysis due to
the range of mean district level PfPR2010–2015 values which
meant that the clustering analysis mainly identified single
isolated outliers without also encompassing the practical
Howes et al. Malar J (2016) 15:502
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Fig. 2 Defining a contemporary sub-national stratification of malaria transmission intensity in Madagascar. a Mouchet et al.’s stratification of malaria
transmission across Madagascar (reproduced with permission [22, 23]); this map was adapted to district-level administrative boundaries and b
shows the currently used adaptation of the sub-national stratification (Source: NMCP National Strategic Plan [21]). c Represents the Malaria Atlas
Project (MAP) modelled map of P. falciparum prevalence in 2–10 year olds (PfPR mean surface from 2010–2015) [14], and d the updated ecozones
based off the PfPR map (regional figures correspond to the mean district-level PfPR value (2010–2015), and the min–max PfPR values from the
districts in each ecozone. e–h Illustrate some of the environmental covariates which informed the PfPR mapping model, and which therefore
underpin the updated sub-national stratifications. e A model of digital elevation (source: Shuttle Radar Topography Mission (SRTM) [59], plotted
at 1 km resolution). f Maps mean total annual rainfall (mm) from 2010 to 2014 (source: NASA Tropical Rainfall Measuring Mission (TRMM) 3B43
algorithm available at 0.25º by 0.25º spatial resolution), while g shows the annual temperature suitability for P. falciparum transmission, reproduced
with permission from Weiss et al. [60]. h Summarizes land-cover use across Madagascar at 500-m spatial resolution (IGBP MODIS annual landcover
product MCD12Q1 [61, 62])
aspects of operational utility. The final stratification
approach was therefore driven by two objective criteria
including (i) spatial contiguity for operational convenience and (ii) transmission intensity as determined by the
district-level mean PfPR2010–2015 map. Some subjective
decision-making was necessary in situations when these
two criterion could not both be met and instead one
needed to be prioritized over the other. Specific examples
are given in the Results.
Routine health facility data
Routine health management information system (HMIS)
data reports from January 2010 to December 2015 were
evaluated to assess the characteristics of the evidencebase of case reports available to inform decision-making
by the Madagascar National Malaria Control Programme
(NMCP). The routine HMIS in Madagascar follows a bottom-up pyramid, multi-step process of data aggregation
and transfer between levels according to a fixed monthly
schedule. Paper-based summaries of all patient consultations at the community-level health centres, either at
health facilities or through community health workers,
are transferred to the 112 health district offices across
Madagascar where data are entered into the database
software GeSIS, which is accessible by the regional and
central MoH, including the NMCP [31, 32]. Private clinics are on the periphery of the HMIS but are intended to
report to the public health system though many do not,
so these are acknowledged as being poorly represented in
the routine data system [21].
The Malagasy NMCP does not distinguish between
species of malaria in its reporting systems. Combination RDTs are used which can distinguish Plasmodium
falciparum from other species [33, 34], but this information is not recorded. The clinical cases described here
therefore refer indiscriminately to any species of human
Howes et al. Malar J (2016) 15:502
Plasmodium infection, though cross-sectional surveys
have indicated that infection is predominantly from P.
falciparum (>99 % by PCR when aggregated nationally
[24]).
Outbreak surveillance and definitions
Alongside the HMIS, a parallel data reporting channel,
the Integrated Diseases Surveillance and Response System, is intended as an outbreak early-warning system
for notifiable diseases of epidemic potential, including
malaria. Paper or electronic reports are sent weekly to
the central NMCP for analysis. In practice, this reporting
channel is weak and poorly developed in most health districts; the US President’s Malaria Initiative in Madagascar
identified only eight of 112 districts as having functional
reporting [11]. Outbreak surveillance is also conducted
directly at the health facility level based on weekly trends
in observed RDT-positive case numbers. Other surveillance institutions also act as channels to alert NMCP of
outbreak situations, such as the Institut Pasteur’s nationally distributed sentinel site network [35, 36].
The NMCP respond to two categories of outbreaks
[21]. In areas considered at very low risk of malaria,
notably the Central highland and desert South ecozones, evidence of autochthonous transmission triggers an
emergency response. In higher transmission zones outbreaks follow epidemic trends, where escalation of case
numbers is commonly associated with mortality. In these
latter areas, two operational definitions of outbreaks are
applied at the health-facility level which are consistent
with approaches followed elsewhere [37, 38]: (i) a weekly
total of RDT-confirmed cases exceeding a pre-defined
weekly health centre-specific threshold (calculated as two
standard deviations from the mean number of confirmed
cases from at least the preceding 3 years of data); or, (ii)
in health centres lacking a defined outbreak threshold, a
weekly doubling of RDT-positive cases over 3 consecutive weeks [21, 39].
Outbreak reports from January 2012 to December 2015
were collated from NMCP surveillance records to assess
district-level temporal and spatial trends in outbreak
occurrence based on these definitions.
Demographic data
To ensure consistency with NMCP estimates, official
demographic data from the Madagascar MoH [40] were
used in incidence calculations. As per the MoH protocols, a fixed annual population growth rate of 2.8 %
was applied cumulatively at the district level to population data collected from the last official national census
in 1993. In the absence of more specific population age
distribution data, the MoH also defines standardized
Page 5 of 18
age categories, with children under 5 years representing
18 % of the population total. This age bracket differs from
the age categories in the malaria case reporting database
which reflects anti-malarial treatment dosing age categories, so includes 5 year olds with under-fives. To account
for this disparity, the proportion of 5 years old within the
5–14 years category was estimated (28.6 % of the overall
population, thus 2.86 %). This therefore corresponded to
an estimated 20.86 % of the total population being under
6 years [40].
Proxy indicators of data reporting completeness
Indicators of reporting completeness, including the numbers of health centre reports received at the central level
and the proportion of distributed RDTs accounted for
in the health centre reports, were obtained from MoH
records through the NMCP. The 2014 database of drug
and RDT stock-outs, which reported the cumulative total
days of consumable stock-outs annually, contained blank
entries. Stock-out reporting required active participation
by each health centre, thus the lack of reporting could
not necessarily be interpreted to indicate ‘zero stockouts events’. To address this characteristic of the dataset,
a conservative approach was followed, whereby health
centres that reported no specific value for RDT stock-out
events but did report on the status of other consumables,
was taken to indicate zero stock-out days of RDTs. Health
centres that did not report at all were excluded from this
analysis.
Related variables, including the proportion of the population resident further than 5 km from a health facility
and district office accessibility from health centres, were
also collated and evaluated.
Results
An updated map to reflect the contemporary stratification
of malaria transmission
The MAP’s PfPR map predictions for 2010–2015
(Fig. 2c; [14]) were summarized to the district level and
examined for regional trends (Additional file 2). Figure 2e–h illustrate a subset of the main environmental covariates used in the PfPR modelling process. The
overall range of district-aggregated mean PfPR values
was relatively narrow, with only 17 districts exceeding
10 % (these summary metrics mask sub-district variation). This therefore limited the applicability of the traditional malaria endemicity categorization, which has
10 % PfPR as the upper threshold of its lowest endemicity category: ‘hypoendemic’ [27]. Instead, narrower
intervals were necessary to distinguish local transmission intensity patterns. Overall, eight distinct transmission zones emerged from the PfPR2010–2015 output map
Howes et al. Malar J (2016) 15:502
(Fig. 2d). Ensuring spatially contiguous zones meant that
some overlap in the ranges of mean PfPR2010–2015 values was necessary. In different areas, it was sometimes
necessary to prioritise differently the two stratification
criteria of (i) spatial contiguity and (ii) local transmission intensity similarities. For example, the east coast
districts of Mananjary and Ifanadiana (approximately
two-thirds down the east coast) had lower PfPR2010–2015
than the neighbouring east coast districts. However, to
ensure spatial contiguity in the ecozones, these two isolated districts were included in the Southeast ecozone.
Moreover, these two districts showed greater similarity
in terms of transmission intensity to the Southeast districts than to the Highland fringe districts. In contrast,
the high altitude central highland areas include small
islands of very low/negligible autochthonous transmission. The unique epidemiology in these areas justified
their amalgamation as a single ecozone despite lacking spatial continuity as they would require distinct
intervention planning and therefore distinct ecozone
classification. Similarly, Maroantsetra district was classified into the Highland fringe west ecozone due to its
transmission characteristics being more similar to those
than to the Northeast ecozone districts with which
Maroantsetra shared north and south borders. The spatial continuity of the Northeast ecozone was not considered broken by this as the resulting Northeast ecozone
included the full northern section of the east coast. The
stratification therefore aimed to be objective but nevertheless included some subjective decision-making to
optimise the final product for operational utility.
Relative to previous stratifications (Fig. 2a, b), the new
PfPR-based stratification (Fig. 2d and tabulated in Additional file 3) uses a larger number of classes (eight versus
four to five in previous versions). The eight zones came
about directly from the dataset, the number was not predetermined. These additional divisions mean that greater
resolution in the epidemiology of each zone can be characterized. Areas at each extreme of the transmission
spectrum are more clearly differentiated from neighbouring zones. For example, the previous single ‘east coast’
zone is now split into two areas of quite distinct transmission characteristics across the northern and southern
east coast districts, allowing the high transmission areas
to be more clearly identified.
Page 6 of 18
Trends in routine malaria case reports
A total of 175,061 health facility reports were available
for analysis which had been reported from Madagascar’s
3924 health facilities to the central NMCP over the 2010–
2015 time frame. During that time, absolute numbers of
reported RDT-confirmed malaria cases almost quadrupled to a maximum in 2015 of 738,996 reported cases,
a steady increase from 2010 when 201,135 cases were
reported to the NMCP (Fig. 3a; Additional file 4A). The
greatest year-on-year increases were between 2011–2012
and 2014–2015, when there were 65 and 97 % increases
(i.e., near doubling) of reported cases, respectively. All
ecozones other than the Central highlands reported
increases of reported cases between 2010 and 2015. In
the Northeast, there was only a 30 % increase (37,884–
78,716 cases), while in other zones the reported increase
ranged from doubling to tenfold increases (Fig. 3a). The
recent 2014–2015 increase in reported cases was evident from all zones, with five zones reporting more than
a doubling of cases in that 1 year period and the South
ecozone reporting a tenfold increase from 4238 reported
cases in 2014 to 47,583 in 2015. The Southeast ecozone had a 41 % increase in reported cases from 2014
to 2015 (218,675–308,192 cases), accounting for just
under half (42 %) the country’s total reported cases for
2015 despite having only 16 % of the country’s population [40]. The Southeast ecozone was the only area to
have also reported a substantial increase in case numbers
from 2013 to 2014 (66 % increase), other areas had seen
a decrease or only very slight increase ahead of the major
increases into 2015 (Fig. 3a). Seasonal trends are largely
consistent through the time window evaluated, peaking in April–May except in the two east coast ecozones
where an earlier February peak is generally observed.
Trends in the lower endemic Central highlands are less
consistent. Additional file 4A–D provides a more detailed
analysis of the temporal structure of the data through
autocorrelation plots of the time series, and linear regression and generalized additive models.
Incidence of reported RDT-confirmed cases nationally peaked for 2010–2015 in April–May 2015 at 3.8–4.0
cases/1000 population/month, having increased significantly since 2010, increasing from annual incidence of
9.65/1000 RDT+ cases in 2010 to 30.87/1000 reported
in 2015 (linear regression model p < 0.001; Additional
(See figure on next page.)
Fig. 3 Descriptive plots of the RDT-confirmed case data available in the NMCP database for 2010–2015. a Raw reported confirmed case numbers
in the NMCP database, aggregated monthly into the eight ecozones. Additional plots including individual ecozone plots are available in Additional
file 2. b Raw reported age-specific monthly incidence of RDT-confirmed cases per 1000 population. Population size is estimated annually based on a
fixed growth rate (see “Methods” section). All-age population estimates for 2015 by ecozone are (Ministry of Health, 2015): A Northwest: 1.907 million;
B Northeast: 4.470 million; C Southwest: 2.083 million; D Southeast: 3.855 million; E Highland fringe west: 2.907 million; F Highland fringe east: 5.759
million; G South: 1.298 million; H Central highlands: 1.659 million. Additional plots and tables relating to this data set are included in Additional file 2
Howes et al. Malar J (2016) 15:502
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Howes et al. Malar J (2016) 15:502
file 4B). As detailed in Additional file 4B, the fastest rates
of increase in incidence were reported from the southern ecozones: the Southeast, Southwest and South (in
descending order) had the sharpest rates of increase.
Negative trends were identified from the Northeast and
Central highland ecozones, indicating a decrease in incidence, though these trends were non-significant (linear
model p > 0.05). Monthly incidence across two age categories (0–5 and >5 years; Fig. 3b) revealed significantly
different age-specific incidence in all ecozones except the
Highland fringe east (Wilcoxon Signed-Rank test with
95 % confidence). The Central highland ecozone was the
only area where the incidence of malaria in over 5 years
olds exceeded that of infants (0–5 years), in all other ecozones where there was a significant difference, the caseload was greater in infants under-five (Fig. 3b). This was
particularly marked in the Southeast, where the underfive incidence was on average 3.7 times greater than in
the older age category (range: 2.3–5.5).
To account for the potential distortion of temporal
trends in raw reported numbers resulting from incomplete reporting or diagnostic kit stock-outs, RDT-confirmed case counts were adjusted to the total reported
number of consultations and the overall number of
reported RDT results (Additional file 4C–D). HMIS
reported consultations increased by 51 % from 4.8 million in 2010 to 7.3 million in 2015. During the same
time period, total reports of diagnostic tests performed
more than doubled from 603,727 to 1,487,745. Trends
in reported RDT use closely reflect the temporal trends
in confirmed case reports (Additional file 4D). Trends in
these adjusted datasets were less apparent than from the
raw reported data, but linear regression models still indicated overall increasing burden in both adjusted metrics
nationally. Across the country, the annual diagnostic test
positivity rate increased steadily from 33 % in 2010 to
50 % in 2015, indicating that despite substantial increases
in the use of diagnostic testing, there was nevertheless an
increase in the proportion of fever cases seeking treatment
that were positive for malaria. The increasing burden of
malaria was particularly apparent in the south where
the change in RDT positivity rate increased significantly
over the 6-year reporting period in the Southwest (35 %
Page 8 of 18
in 2010 to 60 % in 2015), Southeast (39 to 60 %), South
(16 to 58 %), and both Fringe (west: 28 to 41 %; east: 9 to
24 %) ecozones (p < 0.05 by linear regression; Additional
file 4D). Other zones had a much smaller effect size of
change [negative for the Northeast (37 to 35 %) and Central highlands (17 to 15 %)], which was non-significant. In
the Southwest, positive malaria diagnoses were relatively
rare before 2013, with only 3 months reported to have
>10 % of consultations attributable to malaria; in contrast, health centre reports from 2013 to 2015 indicated
25 months to have had >10 % of consultations concluding with an RDT+ diagnosis (Additional file 4C). In the
higher-endemic Southeast, health facility reports noted
only 2 months during 2010 when RDT+ consultations
were greater than 12 % of all consultations; in contrast,
this occurred during all months of 2015. Seasonal trends
from these reporting-adjusted figures are less prominent
than from the raw RDT positive counts (Fig. 3a), but nevertheless apparent.
An annual lag in autocorrelation across the time series
of diagnostic positivity rate was particularly apparent
from the east coast ecozones (Northeast, Southeast and
Fringe areas), indicative of a seasonality effect, though
this was less evident from western areas (Additional
file 4D).
Completeness of the HMIS data
Of the 3924 health facilities intended to contribute
monthly reports to the HMIS in 2015 [41], 214 were hospitals or specialist institutions, 2563 were community-level
public health facilities staffed by either doctors (63 %) or
nurses (37 %), and 1147 were private-sector institutions.
Reporting improved across the 6-year period reviewed,
from a mean monthly reporting rate of just over half of
health facilities (55 %) in 2010, increasing to three-quarters
(76 %) in 2015 (Fig. 4a). Some inter-annual fluctuation in
total facility count is anticipated due to facility closures
and openings, but fluctuations in the numbers of received
reports are likely to be principally attributable to underreporting. An additional source of malaria case data comes
from the network of community health workers (CHW)
active across the country [41, 42]. However, these volunteers report their activities through their health facility, not
(See figure on next page.)
Fig. 4 Indicators of the completeness of routine HMIS reporting. a Monthly health centre reports received by the NMCP. The solid black line indicates
the total number of health facilities in Madagascar (n = 3924 [41]). The right-hand axis refers to the % of overall health centres reporting across all
zones. Data represent a monthly report being submitted, but provide no indication of the completeness of the submitted reports. b Proportion of
distributed RDTs for which a result (either positive or negative) was reported to the HMIS in 2014. Overall, 1.86 million RDTs were distributed to health
districts nationally, and results for 0.94 million (50.5 %) were reported into the HMIS database. c Reported RDT stock-out incidence. The map shows
the district-level mean RDT stock-out days in 2014. Only data from health centres that submitted at least one report for drug/diagnostic availability in
the year were included in the mean calculation. Plots show stock-out status from 2012 to 2014 per ecozone. Points correspond to the mean number
of stock-out days annually (left hand y axis) and bars represent the reporting rate within each ecozone (right hand y axis)
Howes et al. Malar J (2016) 15:502
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Howes et al. Malar J (2016) 15:502
directly to the HMIS. Increasing numbers of active CHW
should not affect the number of monthly reports submitted to the HMIS, though it will impact on the numbers of
fever-related consultations performed.
While Fig. 4a data indicate that a report was received
at the central level from the health facility, it provides no
indication of the quality of the report. A proxy indicator
of reporting completeness may be represented through
evaluation of the proportion of RDTs distributed for
which a result was reported into the NMCP database
(Fig. 4b). Important limitations exist to assuming this
proxy: not necessarily 100 % of RDTs distributed would
be needed within the year, RDT distribution may not be
constant through time (some health facilities may receive
large deliveries some years and carry lower stocks in
other years), and the risk that not all distributed RDTs are
fit for use (some may be discarded due to void test results
or overdue expiry dates). Nevertheless, the broad categories used in Fig. 4b indicate that even making allowance
for distributed RDTs remaining unused in the health
centres, 79 of 112 health districts (71 %) had more than
half of their distributed RDT stock results in 2014 unaccounted for in the HMIS; in 34 districts, reports were
received for fewer than 30 % of the distributed RDTs.
Overall, 50.5 % of the distributed RDTs across Madagascar in 2014 (n = 1.856 million) were accounted for in the
HMIS results. When aggregated to the ecozone scale,
reporting from Southeast ecozone was highest, accounting for 78 % of distributed RDTs, while the South ecozone had poorest reporting, accounting for only 24 % of
distributed RDTs in 2014. Rates in all other ecozones
ranged between 29 and 56 %.
Case management policies in Madagascar require RDT
confirmation for a case to be treated with anti-malarials
and counted as a confirmed case in the reporting system.
RDT stock-outs will therefore impact directly on the numbers of cases reported. Reporting of presumed cases is not
consistently distinguished from confirmed cases in the
monthly reports, introducing uncertainty into the reports
(NMCP, pers. comm.). Nationally in 2014, 51 % of health
facilities sent no reports of any stock inventories (including of drug stocks), while those that did report indicated
a mean number of 30.5 days with RDT stock-outs (range:
0–330 days). Figure 4c summarizes the district stock-out
data for 2014 (map) and reporting characteristics by ecozone from 2012 to 2014 (graphs). Bar plots (right hand
y axis) show the health centre reporting rates over time,
indicating the large numbers of health centres failing to
report, and thus the difficulty with interpreting the data
on numbers of stock-out days, which otherwise indicate
that stock-outs are relatively infrequent events.
Health centre isolation was evaluated as a potential
factor contributing to reporting incompleteness (Fig. 5).
Page 10 of 18
Figure 5a maps population density across the country, while Fig. 5b charts the proportion of each district’s
population living within 1 h walk of their nearest health
facility (5 km). The colour index in Fig. 5c indicates the
relative difficulty for health centre staff to access the
health district office to submit their monthly health
report. Overall, 56 % of the population are reported to
live further than 5 km from their nearest health centre.
This was generally consistent across all ecozones (range:
51–64 %), peaking in the Southwest. Nearly half of health
facilities were situated more than 50 km from their district office (1125 of 2690 health facilities for which location details were available), with 13 % (n = 147) of those
accessible only by foot or river/sea.
Health centre isolation data (quantified at the district
level as the mean travel time from each health centre to
the central district office) were analysed for potential
correlations with 2014 data on: (i) RDT result underreporting; and, (ii) the RDT stock-out reporting rate. No
correlation emerged between these datasets, which were
only available aggregated to the district level.
Outbreaks
The nature of transmission events classified as ‘outbreaks’ differs substantially between affected geographic
areas, as previously defined. Nevertheless, here these
are considered together, irrespective of the particular
outbreak types. This was partly due to data availability on the classifications of outbreak events, but also
because all types of outbreaks require NMCP intervention, irrespective of the circumstances. Overall, therefore, January 2012 to December 2015 yielded a total
of 292 outbreaks reported to the NMCP’s surveillance
system as shown in Fig. 6 (instances of autochthonous
transmission in low transmission ecozones, or an unexpected excess of cases in endemic areas; definitions provided in “Methods” section). Total reported outbreaks
per year were 42 in 2012, 107 in 2013, 60 in 2014, and
83 in 2015. Over half (n = 63) of the country’s 112 districts reported an outbreak during this period; 31 of
these reported more than one outbreak. Overall, half
the total reported outbreaks occurred in just ten districts. At the commune level (administrative sub-division of a district), 193 of 1579 communes were affected
by outbreaks between January 2012 and December
2015 (12.2 %), though 73.1 % of these reported only a
single outbreak. Only 7.3 % (n = 14) of affected communes reported more than two outbreaks during the
4 year reporting period examined. The data suggest
that although districts could be categorized as being
at higher or lower risk of epidemics, at the communelevel outbreaks do not appear to have a strong temporal
association.
Howes et al. Malar J (2016) 15:502
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Fig. 5 District-level summaries of health centre accessibility, a population density in 2015; b health centre accessibility to catchment populations
and b health centre accessibility to the health district headquarters where monthly data must be reported. a Maps population density in 2015
(WorldPop [63]) together with cities estimated to have populations >100,000). b Shows the proportion of the population resident more than 5 km
from their nearest health facility (equivalent to further than approximately one h walk). The underlying colour indicates the total population resident
in the district, while the pie charts show the proportion of the population per district resident further or closer than 5 km from their nearest health
facility. This is based off a subset of 2690 health facilities (69 % of all health facilities) for which data were available. c Maps pie charts representing
different categories of accessibility for each health centre per district. Colours represent the available mode of transport, while colour intensity indicates distance categories between health centres and the health district headquarters. Pie chart sizes indicate the number of health centres needing
to report data per district (range: 3–55). Arrows indicate the increasing difficulty with reporting. Data from the NMCP (Fig. 5b) and the Malagasy
Health Sector Development Plan for 2014-2019 [64] (Fig. 5c)
The occurrence of outbreaks in relation to the overall
burden of malaria is considered in Fig. 7. Records of the
outbreaks were incomplete, with 133 of the 292 outbreak
reports in the database not specifying the affected month.
The outbreaks barplots in Fig. 7 are therefore incomplete,
but nevertheless suggest a relationship with the intensity
of background transmission. Lower endemicity areas, such
as the two Highland fringe ecozones, have peaks in incidence corresponding with months when outbreaks were
reported; while higher incidence areas, such as along the
East coast and the Northwest ecozone, trends in malaria
incidence do not correspond to outbreak periods. For
example, in the Southeast, the year with fewest reported
outbreaks (2015; n = 4) was when incidence was greatest.
Discussion
An updated sub-national stratification of Madagascar is proposed which reflects contemporary transmission intensity. Within these limits, the epidemiology of
malaria is described as evidenced by routinely reported
health facility data made available to the NMCP. From
this evidence base, the National Programme formulates
control policies, oversees epidemiological monitoring,
and assesses policy impacts. The eight ecozones may
not necessarily each require bespoke control schemes,
but these allow greater sub-national epidemiological
insight, and therefore flexibility in targeting of interventions based on local needs. These sub-divisions are not
static, but instead must be periodically updated to reflect
shifts in the current patterns of transmission, though
with a frequency that does not prevent temporal trends
being assessed in the short term. The narrow time window presented here is the product of decades of malaria
control, so the epidemiology described cannot be interpreted as representing the fundamental niche of malaria
in Madagascar.
Malaria is a disease of increasing public health concern in Madagascar. Since 2010, the number of reported
Howes et al. Malar J (2016) 15:502
Fig. 6 Outbreaks documented in the NMCP records for January 2012–December 2015. Outbreak definitions vary according to
expected local transmission characteristics. Colours indicate the total
number of outbreaks reported per district during the period examined. Overlain symbols indicate the number of years affected between
2012 and 2015. Grey background indicates no reported outbreaks
RDT-confirmed cases has quadrupled across the country. When adjusted for numbers of tests performed (thus
adjusting for (i) reporting rates, (ii) the unavailability of
diagnostics, and (iii) the scale-up of access to diagnostic
testing through community health worker programmes
[42]), the RDT positivity rate increased by 50 % between
2010 and 2015, reaching 50 % slide positivity in 2015.
Despite this evidence of recent resurgences, the incidence of malaria in Madagascar remains among the lowest of the sub-Saharan African region, with high levels of
intervention coverage, particularly of LLINs [8, 14]. This
juncture therefore represents an important opportunity
to re-evaluate strategies and reverse the recent negative
trends. For instance, while LLIN coverage may be high,
treatment-seeking and adherence to recommended artemisinin combination therapy (ACT) policies appears
weak [8, 43].
Page 12 of 18
Although the majority of the malaria burden remains
in the Southeast (42 % in 2015), malaria has increased
across all areas in year-round significance, increasing in
intensity during both high (December–April) and low
transmission periods. The Central highlands seem to be
the exception, but the epidemiology differed there, consisting primarily of imported cases (NMCP, pers. comm.),
and with very low incidence compared to other ecozones.
Outbreaks, defined in this context as unexpectedly high
or rapid increases in transmission or evidence of autochthonous transmission in areas considered at low risk,
appear to associate closely with overall burden trends
in lower-transmission ecozones. A more detailed view
of changes in case numbers at health facilities affected
by outbreaks would allow more robust insights into the
relative contribution of outbreak-driven transmission to
overall caseloads across the country.
An important missing piece from this study’s narrative
is the relative contribution of each Plasmodium species
to the regional case burden. This detail is not recorded
in the data. Prevalence surveys indicate a predominance
of P. falciparum across most areas, but regional pockets
of Plasmodium vivax and sporadic cases of Plasmodium
ovale and Plasmodium malariae are also detected [24, 25,
29, 44, 45]. A longitudinal dataset on the contribution of
each species to morbidity from routine reporting would
improve the evidence-base for determining the value
of introducing non-P. falciparum-specific intervention
policies.
The recent trends described in this study stand in
stark contrast to the country’s stated ambitions to start
the transition towards malaria elimination. The country’s current National Strategic Plan (2013–2017) aims
for pre-elimination status by end 2017, with zero malaria
deaths and the reduction of test positivity rates to <5 % in
15 % of districts, and the halving of the positivity rate in
the remaining districts relative to 2013 baseline data [21,
39]. Overall, however, 2013–2015 saw a 34 % increase
in the positivity rate. Acknowledgement of these trends
by the NMCP led to a revision of the National Strategic
Plan in 2015, with adjustment of their initial goals now
more aligned with achieving burden control than elimination [21]. The drive to control and eventually eliminate malaria from Madagascar receives strong financial
backing from the international community, with disbursements in excess of $400 million since 2004 when
collaborative efforts were galvanized and intensified [8,
11, 46]. In 2006–2010, resources for malaria control in
Madagascar corresponded to higher than average percapita funding across the African region [8, 47]. Since
this time though, the country’s political crisis has severely
impacted on the country’s socio-economic status and
Howes et al. Malar J (2016) 15:502
Page 13 of 18
Fig. 7 Temporal correspondence between reported malaria case incidence (left-hand y axis; plotted as points) and outbreak reports (right hand y
axis; plotted as barplots) aggregated by ecozone for 2012–2015. Of the 292 outbreaks reports, nearly half (n = 133) provided no supporting information about the affected month so these could not be included in the monthly outbreak plots. The total annual outbreaks reported per ecozone is
specified in the plots (fraction denominator), with the numerator summarizing the number of outbreaks missing from the bar plots due to incomplete information
associated health system infrastructure and staffing,
impeded external funding to the country thus preventing
intervention roll-out (see the Global Fund disbursement
delays in Fig. 1), and complicated the control programme
management by preventing direct bilateral relationships
between the MoH and donors [21].
The situation in Madagascar therefore reflects what has
been widely reported from other areas, that withdrawal
Howes et al. Malar J (2016) 15:502
of core funding is closely associated with rapid and predictable resurgence of malaria [48, 49] (Fig. 1). Despite
high coverage rates of interventions around 2013, interrupted funding led to delays in sustaining these levels.
Periods of limited funding are when reliable epidemiological data become all the more important to inform
efficient planning and optimize resource allocation. Such
data require robust processing at all stages of the chain
from the probability of patients seeking treatment at a
health facility, through diagnosis, through data reporting to data management and evaluation at the central
level. More than half the Malagasy population live further than 5 km (roughly corresponding to an hour’s walk)
from their nearest health facility. This likely contributes
substantially to the treatment-seeking rate that ranged
in 2013 from 21.7 to 49.1 % across different parts of the
country [24, 43, 50, 51], with many patients suffering
fevers resorting to self-medication instead (including
both pharmaceutical and traditional herbal remedies)
[52]. Therefore, most cases of fever will never interact
with the country’s formal health system. The routine
HMIS had a largely consistent reporting rate from health
facilities across 2010–2014 at approximately 60 %, though
rising to 75 % in 2015 (Fig. 4a), meaning that almost half
of health centre reports were not recorded in the NMCP’s
databases for most of the time period reviewed. Further
reinforcement of the incompleteness of the datasets is the
disparity between distributed diagnostic kits and the test
results reported (Fig. 4b). The received outbreak surveillance data are also incomplete, evidenced by nearly half
of reports not being month-specific (Fig. 7).
Madagascar has a well-documented history of devastating epidemics, with the earliest reported outbreak
around 1887 coinciding with the establishment of rice
farming [21]. Outbreaks both in Madagascar and globally
have primarily occurred in the context of socio-economic
and political upheavals [48] when there has been a breakdown of the control programme structure, including
surveillance and reporting channels. Implicit in the programmatic concept of an outbreak is that the event triggers an emergency response from the responsible public
health institution to curtail the exacerbation of the situation. An efficient surveillance system will allow outbreaks
to be anticipated and intervention resources to be mobilized ahead of transmission developing out of control.
The reporting system in Madagascar was described
in 2015 by the US President’s Malaria Initiative as
“extremely weak”, hindered in part by a critical shortage of health staff at all levels, with human resources
unevenly distributed across the country [11]. To further
investigate this, a nationally representative review of
the health reporting system was commissioned in 2015
to qualitatively and quantitatively assess the system’s
Page 14 of 18
functionality (thus supplanting the need for proxy indictors of completeness). This included detailed interviews
with staff at all levels of the reporting pyramid, alongside comparisons of raw data to assess the completeness and accuracy of reporting from doctors’ records up
to the central database. Preliminary results suggest that
both the quantity and quality of reported health facility
data were weak. Reporting timeliness was particularly
affected, with only 8 of 45 districts reviewed in January
2015 reporting within the MoH’s defined schedule [31].
Reporting completeness varied across the country, being
highest in the central highlands, and lowest in the tropical coastal areas and desert south. A further concern
identified by the surveys related to the lack of health professionals to make use of the data and identify trends specific to their local catchment population: interpretation
of the data was principally limited to the central level, and
not performed in situ. The investigators’ perspectives of
the NMCP’s data in particular, was that the existence of
a database was a strength of their epidemiological monitoring unit. Weaknesses of the data included the complexity of the reporting system, its fragmented nature and
the difficulty of assimilating the data for analysis, logic
checks are not routinely performed on the data, and confusion existed regarding some of the calculations used to
derive the metrics reported [31]. Surveys actively assessing stocks of diagnostic kits in health centres suggest that
the passively reported data on stock-out events that were
available for review here (Fig. 4c) underestimate the true
incidence of stock-out events [49]. This indicates that the
true rate of under-diagnosis due to diagnostic stock-outs
and knock-on impact on reported case numbers may be
even greater than suggested. Stakeholders are aware of
these limitations to the reporting system, and a concerted
effort to address them is ongoing.
Given the need for annual case estimates to assess both
national- and global-level trends in malaria incidence and
to monitor progress towards pre-defined milestones, the
World Health Organization’s (WHO) Global Malaria Programme and collaborators have developed methods to
account for weaknesses in routine HMIS data [8, 14, 53,
54]. Two main approaches exist to estimate malaria incidence [53], each best suited to different countries according to their programme control phase and the strength of
their disease surveillance system. Method 1 is favoured
by the WHO for countries outside Africa, where routine reporting is considered more reliable. This method
uses parameters to adjust MoH routine HMIS data to
account for (i) incomplete case detection by health facilities; (ii) the likelihood of over-diagnosis of malaria among
patients with fevers; and, (iii) public/private treatmentseeking preferences [53, 55]. Method 2 uses a cartographic
approach based on population prevalence surveys to
Howes et al. Malar J (2016) 15:502
develop spatial and temporal models of the incidence of
clinical disease; this has been the main approach followed
by MAP to derive global endemicity maps of P. falciparum
and P. vivax transmission [14, 56, 57], and is used by the
WHO to estimate case numbers from most sub-Saharan
African countries where routine reporting is considered
too weak for Method 1 estimation. A subset of low-transmission African countries is also considered to have sufficiently strong routine reporting data to allow support
for Method 1-based estimates. The implication being that
these countries have a sufficiently functional health system
to have robust surveillance and reporting systems in place
within the context of a functional and effective malaria
control programme. The sub-Saharan African countries
included in this category are Botswana, Cabo Verde, Eritrea, Namibia, South Africa, Swaziland, and Zimbabwe
(all of which are classified as being “on track for >75 %
decrease in incidence 2000–2015”), and Madagascar (“less
than 50 % change in incidence projected 2000–2015”) [8].
Nevertheless, the WHO lists Madagascar among the eight
sub-Saharan African low-transmission countries with sufficiently accurate HMIS data to permit convincing estimates to be made from simple adjustment of the numbers
of reported cases. This study explores the routine HMIS
data available to the Malagasy NMCP which underpins
this perhaps contradictory classification. Of the subset of
African Method 1 countries, Madagascar had the highest
endemicity in 2015 [8, 14] and among the lowest access
to the formal health system [43]. The magnitude of the
uncertainty in the reported case numbers is evident by the
WHO’s case estimate for Madagascar in 2013. Although
387,045 cases were reported by Madagascar to the World
Malaria Report for 2013, once adjusted using Method 1,
the estimated case number for 2013 was 1.2 million (0.75–
2.10 million) [8], therefore corresponding to more than
three times the reported RDT-confirmed numbers. Given
these acknowledged uncertainties and the recent resurgence of transmission with variable sub-national patterns,
a re-consideration of the method applied to case estimation in Madagascar would be worthwhile. Regular nationally representative Malaria Indicator Surveys and other
prevalence surveys take place, which provide a rich source
of malariometric data complimentary to the HMIS data.
If Madagascar is to remain within the Method 1 countries and have its case estimates derived from adjustment
of the routine reporting data, the spatial heterogeneity
of transmission characteristics and of reporting robustness discussed in this paper would be a valuable addition to the methodology instead of relying on a single
set of parameters. The data adjustment parameters show
widespread variation between ecozones, with treatmentseeking rates in the Fringe areas less than half that of
the South (21.7 and 49.1 %, respectively) [24]. Similarly,
Page 15 of 18
health facility reporting rates differ between areas, and,
although no correlation could be identified from the
aggregated district-level data reviewed here, it is likely
that some association between health centre isolation
and reporting quality and timeliness exists. No single set
of national-level summary adjustment parameters, as per
the current WHO Method 1 protocol, could capture this
heterogeneity. The sub-national variation in transmission
intensity is reflected by factors such as age-specific incidence patterns (Fig. 3b). The Central highlands ecozone,
where transmission is lowest, has no significant age-specific patterns with the whole population at similar risk of
infection. In higher endemic areas, younger children suffer the highest burden of disease, likely due to the development of immunity in older age groups from repeated
exposure in childhood. To adequately adjust health facility data from the HMIS, this spatial variability ought to
be taken into account. In parallel to this, large numbers
of prevalence surveys have been performed across the
country as part of three completed MIS surveys from
2011, 2013, and April 2016 (results expected end 2016),
among others [29]. These community-based surveys
have shown the same increase in prevalence as the HMIS
health-facility based case data. While these prevalence
surveys are generally better standardized than HMIS
data, they are much less comprehensive in space and
time. Comparison of the estimates from these two independent methods (routine data-based and cartographic)
and datasets would be valuable.
Conclusion
Madagascar is highly diverse, not least in terms of its
malarial epidemiology, and accordingly requires spatially specific control planning. This paper presents an
overview of the last 5 years of HMIS data, the period
immediately following a political crisis and the nation’s
subsequent exclusion from the international community
until May 2014. Associated with these events, malaria
control efforts have been hindered, resulting in rapid
resurgences of transmission [49], with ecozones previously considered to be low transmission zones, such as
the Southwest and South, now becoming stable endemic
areas, reflected by differential patterns in age-specific
incidence, for example.
No single approach for malaria control can be applied
across the island, or even binary high/low transmission
approach given the peculiarities of transmission in different ecozones. Regular epidemiological assessments
are therefore recommended at the ecozone levels. The
NMCP is frequently restricted to being reactive rather
than proactive and unable to anticipate demands (e.g., in
terms of anticipating commodity needs or anticipating
outbreaks). Closer analysis of data trends in real-time,
Howes et al. Malar J (2016) 15:502
would be valuable to support the NMCP. For instance,
it will be important to closely evaluate the 2016 data
to assess the impact of the 2015–2016 LLIN distribution programme, and determine how rapidly the sharp
increase of cases in 2015 can be reversed to shift the
country in the direction of its elimination goals.
This study comes at the end of the Millennium Development Goal era (2000–2015), which aimed to have “halted by
2015 and begun to reverse the incidence of malaria” (Target 6.C) [12]. In Madagascar this was clearly being achieved
from 2000 to 2009, though the reverse has occurred since
2010 (Figs. 1, 2, 3). Using the year 2000 as a baseline descriptor may not be a very pertinent approach for evaluating the
current status of progress in malaria control. The situation
in 2000 was one of essentially no financial or political commitment to large-scale intervention roll-out. Most reported
cases would have been presumptively diagnosed based on
symptoms (likely over-estimating case numbers), given that
rapid field-based diagnostics were only introduced in 2007.
More pertinent than noting a decrease in caseload since
2000 is the concerning increase in burden since 2010 when
funding sources were substantially increased and malaria
control was widespread across Madagascar.
While it is important to interpret the imperfect health
facility data with caution [42, 58], the available data do
provide detailed insight into the relative differences
in epidemiology sub-nationally. However, given that
between a quarter and half of health facility reports are
missing from the NMCP’s database, the absolute number of cases reported is evidently a major underestimate.
Approaches to quantify estimates of the absolute annual
burden would be useful in attempting to deduce a more
robust estimate of overall burden rather than relying on
an incomplete database as is currently the case by the
NMCP. Nevertheless, the HMIS data present a convincing case for the growing challenge of malaria across all
areas of Madagascar.
Additional files
Additional file 1. Summary of data sources consulted. The table provides
details of the data sources used for each figure in the paper.
Additional file 2. Malaria Atlas Project (MAP) modelled maps of Plasmodium falciparum parasite prevalence (PfPR) in 2010–2015. This document
provides additional information about the modelled prevalence map that
was used to stratify Madagascar into contemporary ecozones.
Additional file 3. Tabulation of regions and districts by ecozone. Administrative regions and districts of Madagascar classified according to the
newly designed ecozones.
Additional file 4. Additional plots of the trends of reported malaria case
data in Madagascar (2010–2015). This file provides additional analysis
of the temporal trends in the reported malaria data. Data are adjusted
for reporting and diagnostic shortages, and trends are examined using
autocorrelation plots, generalized additive models and linear regression
models.
Page 16 of 18
Authors’ contributions
REH wrote the first draft and conceived the study with PAZ and AR. SAM, BR,
TF, and AJR contributed the data. DB and PWG advised on the analysis. RH,
PAZ and AR were responsible for data interpretation. All authors edited the
final version of the manuscript and approved its submission for publication.
All authors read and approved the final manuscript.
Author details
1
Center for Global Health and Diseases, Case Western Reserve University,
Cleveland, OH 44106‑4983, USA. 2 Department of Zoology, University
of Oxford, Oxford OX1 3PS, UK. 3 National Malaria Control Programme, Ministry
of Health, Androhibe, Antananarivo, Madagascar. 4 Department of Public
Health, Faculty of Medicine, University of Antananarivo, Antananarivo, Madagascar. 5 Oxford Big Data Institute, Li Ka Shing Centre for Health Information
and Discovery, University of Oxford, Oxford OX3 7BN, UK. 6 Faculty of Sciences,
University of Antananarivo, Antananarivo, Madagascar.
Acknowledgements
The authors thank two anonymous reviewers for their helpful suggestions
towards improving this work. The authors are also grateful to Harry Gibson, Zhi
Huang, Mike Thorn and Dan Weiss of the Malaria Atlas Project for help with the
temperature suitability index figure, elevation and rainfall raster surfaces.
Competing interests
SAM, AJR, BR, TF and AR are employees of the National Malaria Control
Programme of Madagascar. The authors declare that they have no competing
interests.
Availability of data and materials
Data are available from the National Control Programme of Madagascar.
Consent for publication
All authors approved the manuscript’s submission for publication.
Funding
The authors, except DB and PWG, acknowledge funding from the NIH to PAZ
(R01 AI097366). REH is also funded by a Wellcome Trust Senior Research Fellowship to Simon I Hay (095066). AJR acknowledges funding from the Global
Fund to Fight AIDS, Tuberculosis and Malaria. PWG acknowledges support
from the Bill and Melinda Gates Foundation (#OPP1068048, #OPP1106023),
which also supports DB.
Received: 5 August 2016 Accepted: 5 October 2016
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W2036675065.txt | UC Irvine
UC Irvine Previously Published Works
Title
Evaluation of daily home spirometry for school children with asthma: new insights.
Permalink
https://escholarship.org/uc/item/3db1m0h1
Journal
Pediatric pulmonology, 41(9)
ISSN
8755-6863
Authors
Thompson, Rohan
Delfino, Ralph J
Tjoa, Thomas
et al.
Publication Date
2006-09-01
DOI
10.1002/ppul.20449
Copyright Information
This work is made available under the terms of a Creative Commons Attribution License,
availalbe at https://creativecommons.org/licenses/by/4.0/
Peer reviewed
eScholarship.org
Powered by the California Digital Library
University of California
Pediatric Pulmonology 41:819–828 (2006)
Evaluation of Daily Home Spirometry for School
Children with Asthma: New Insights
Rohan Thompson, MD,1,2 Ralph J. Delfino, MD, PhD,3* Thomas Tjoa, MPH,
Eliezer Nussbaum, MD,1,2 and Dan Cooper, MD2
3
MS,
Summary. Home spirometers are useful for monitoring asthma therapy and for research, but the
validity of maneuvers in children is in question. We evaluated the quality of PEF, FEV1, and FVC
data obtained from 67 children with persistent asthma who self-administered spirometry at home
using the hand-held ndd EasyOne Frontline Spirometer with full expiratory curve data, electronic
measurements of maneuver quality, and on-screen incentives. Half were studied in 2003 in one
region, and half in 2004 in another region of Southern California. Subjects were followed at home
weekly over 2 months and daily over 10 consecutive days. We retained completed spirometry
sessions (9,916) consisting of three of six best maneuvers in the morning, afternoon, and evening.
Percent compliance, software assessed repeatability and acceptability modified from American
Thoracic Society criteria, and visually assessed quality of maneuvers, were compared across daily
and weekly follow-up, study regions, and subject characteristics. Compliance was higher for daily
(>90%) than for weekly follow-up (>84%), but not significantly different, and was consistent across
subject characteristics. The number with two reproducible and acceptable maneuvers was
significantly lower in the first than second region for daily (70 vs. 90%) and weekly follow-up (66 vs.
87%). Of 22,926 software accepted maneuvers, 1,944 (8.5%) were visually rejected (variable
effort, cough, glottic closure). Maneuver quality was significantly lower for subjects age 9–12
versus 13–18 years, for subjects not taking anti-inflammatory medications, and for subjects with
<80% predicted FEV1. Longitudinal data collection is possible in children with asthma by
employing repeated home training and follow-up, and using spirometers with built in quality
assurance and incentive software. Region, age, and multiple indicators of persistent asthma,
predict ability to perform reliable and accurate lung function maneuvers. Pediatr Pulmonol. 2006;
41:819–828. ß 2006 Wiley-Liss, Inc.
Key words: asthma; spirometry; reproducibility of results; patient compliance; data
collection; evaluation research.
INTRODUCTION
Spirometry and its variable peak expiratory flow (PEF)
have been widely used as an index of measurement for
asthma both in the clinical setting and in research trials.
The use of PEF can be relatively useful when a skilled
technician or medical professional directly guides technique and interpretation. However, the validity of selfadministered PEF is questionable. Some previous studies
have even shown that it can be less sensitive than a simple
symptom diary for revealing acute exacerbations in
asthma.1,2 In addition, subject compliance and ability to
perform maneuvers accurately at home, especially among
children is also questionable.3,4 Typical home monitoring
is done with non-electronic devices. Evidence from three
studies proved that around a fourth to a third of nonelectronic PEF data was falsified.3–5 Despite these and
other limitations, daily PEF is still being widely used in
clinical asthma trials due to its simplicity and low cost.
The best standard for measurement of pulmonary
function has always been the laboratory spirometer and
body Plethysmograph. These devices are not used
frequently in clinical trials due to expense of use and lack
ß 2006 Wiley-Liss, Inc.
of portability. Recently more portable hand held devices
that have some similar capabilities in measuring lung
1
Pediatric Pulmonology Division, Miller Children’s Hospital, Long Beach,
California.
2
Department of Pediatrics, School of Medicine, University of California at
Irvine, Irvine, California.
3
Department of Medicine, Epidemiology Division, School of Medicine,
University of California at Irvine, Irvine, California.
Grant sponsor: National Institute of Environmental Health Sciences
(NIEHS); Grant number: ES011615.
*Correspondence to: Ralph J. Delfino, M.D., Ph.D., Department of
Medicine, Epidemiology Division, School of Medicine, University of
California, Irvine, 100 Theory Dr., Suite 100, Irvine, CA 92697-7555.
E-mail: rdelfino@uci.edu
Received 10 February 2006; Revised 23 March 2006; Accepted 7 April
2006.
DOI 10.1002/ppul.20449
Published online 17 July 2006 in Wiley InterScience
(www.interscience.wiley.com).
820
Thompson et al.
function have become available. These devices have the
capability of measuring forced vital capacity (FVC),
forced expiratory volume in one second (FEV1), forced
expiratory flow at 25%–75% of FVC (FEF25–75), and
PEF. They are superior to peak flow meters by their very
ability to measure all of the aforementioned variables as
well as compliance because they store their data until
retrieved. For the investigator, this mode of data collection
in the field could lead to measurements that are more
objective and accurate, and include detailed parameters of
lung function and maneuver quality checks.
This study aims to evaluate the compliance and
technical quality of home spirometry data from school
children with asthma, for use in research studies. We
obtained home spirometry data from subjects who selfadministered spirometry at home using the hand-held ndd
EasyOne Frontline Spirometer (ndd Medical Technologies, Chelmsford, MA). Mortimer et al.6 previously
validated this portable spirometer against an office
spirometer (12 L dry rolling seal). They showed a 1.1%
difference in FEV1 and 0.9% difference in FVC. The
data and subjects used in the present study are part of a
much larger study to evaluate the effects of air pollutants
on school children with asthma in Southern California. We
report results from 67 subjects who completed 9,916
spirometry sessions, with each session consisting of at
least three maneuvers. Each curve was evaluated and
measured independently for acceptability by selected
criteria. These curves were then further evaluated for
visual acceptability by pediatric pulmonology physicians.
MATERIALS AND METHODS
Population and Design
The design of this study involved repeated measures of
health outcomes and environmental exposures in children
with asthma, and it is referred to as a panel study. We
followed 67 school children with asthma in two regions of
ABBREVIATIONS
ATS
BEV
dFEV1
dFVC
dPEF
EOTV
FEF25–75
FET
FEV1
FVC
GCRC
IBD
PEF
PEFT
American Thoracic Society
Back-extrapolated volume
Difference between highest and 2nd highest FEV1 within in
a test session
Difference between highest and 2nd highest FVC from an
acceptable effort
Difference between highest and 2nd highest PEF from an
acceptable effort
End-of-test volume
Forced expiratory flow between 25% and 75% of FVC
Forced expiratory time
Forced expiratory volume in one second
Forced vital capacity
General Clinical Research Center
Inhaled bronchodilators
Peak expiratory flow
Time to peak expiratory flow
Southern California, Riverside City and Whittier. These
two regions were specifically chosen due to their high
levels of air pollution. Although key to the aims of the
overall study, the health impacts of air pollutants measured
is beyond the scope of this article. The Institutional
Review Boards of the University of California, Irvine
approved the study protocol. Informed written consent
was obtained from all subjects and one of their legal
guardians. Subjects were recruited if they were ages 9–
18 years and had an established diagnosis of asthma based
on self-reported physician-diagnosed asthma, including at
least a 1 year history of episodic symptoms involving
wheezing, cough, or dyspnea. The study targeted recruitment of children with mild to moderate persistent asthma
based upon a modified severity scale established by the
National Heart Lung and Blood Institute.7 This included a
history in the last 12 months of exacerbations of asthma
symptoms requiring the use of prescribed as-needed, oral
or inhaled, bronchodilator(s) on 2 or more days per week,
regardless of anti-inflammatory medication use. Subjects
were also recruited if they were taking oral or inhaled antiinflammatory medications, regardless of recent symptom
frequency. Finally, subjects were recruited with <80%
predicted FEV1 from office spirometry at their baseline
visit to the General Clinical Research Center (GCRC),
University of California, Irvine. Subjects were ineligible if
they smoked or if someone smoked in the subject’s home.
Out of 97 eligible subjects evaluated at the GCRC, 13
were unable or unwilling to participate, and did not enter
the panel study. Of the 84 who entered, shortly after entry
10 dropped out and 7 were dropped due to noncompliance. Two subjects were dropped later due to
non-compliance, but had a sufficient amount of spirometry
data for evaluation (66 and 55 sessions) and they were
retained for the present analysis of spirometry data quality.
Another subject withdrew later in the study and data was
retained for the present analysis (59 sessions), leaving 67
subjects.
Prior to entry in the panel study, subjects were
administered a background questionnaire. For 67 subjects
in the panel study, responses showed 5 subjects (7.5%)
reported having been admitted to the hospital in the past
12 months, and 17 subjects (25%) had been seen in the
emergency room at least once in the past 12 months for
their asthma. In addition, 22 subjects (33%) reported the
need for inhaled bronchodilators (IBD) multiple times per
week, 9 subjects (13%) required IBD at least weekly, and
35 subjects (52%) required IBD less frequently or could
not recall usage frequency. Only 11 subjects (17%)
reported not having used some form of inhaled corticosteroid as a controller medication for their asthma in the last
year. Seven of these subjects did not frequently use IBD
either, but five of them had percent predicted FEV1 <80%,
and the other two had several recent asthma exacerbations
requiring physician evaluations.
Evaluation of Daily Home Spirometry for School Children with Asthma
The first site of study was Riverside where two groups
of approximately 16 asthmatics each were followed in
alternating 1-month blocks over a 4-month period in the
late summer through late fall of 2003 with daily measures
of health outcomes from each subject. This was repeated
at a second site in the late summer through late fall 2004 in
Whittier. This time of the year was chosen because of
historically high levels of particulate air pollution in the
Los Angeles basin.
Subjects performed forced expiratory flow maneuvers
first on an office spirometer during their baseline visit to
the GCRC. Then, subjects were trained in home on how to
perform expiratory flow maneuvers with the ndd portable
spirometer during a 5-day run-in period (around an hour
on the first day, then around 15–30 min per day for the next
4 days as needed). That data is not included in the present
analysis. Subjects were instructed to perform spirometry
in the morning, afternoon, and evening, and to complete an
electronic diary every two waking hours reporting health
outcomes, asthma medications, and time place activity
information.
Subjects were followed in two blocks of 1 month each
for a total of 56 days when they performed thrice-daily
spirometry and completed the diary. Subjects further
participated in a more intensive phase of study over a
continuous 10-day period within one of each subject’s
2 months of follow-up. This additionally involved wearing
a personal air sampler to measure exposure to air
pollutants, and providing a daily measurement of exhaled
nitric oxide. Generally, four subjects were followed in
each 10-day period and on-site validity checks were
conducted daily in their homes. During days when
subjects were not wearing the personal air sampler
(approximately 46 days) they were followed once each
week at their home for validity checks to ensure
compliance. Four of the 67 total subjects did not
participate in the 10-day exposure assessment and their
data are only included in the analysis of spirometry data
from the weekly follow-up.
Spirometry Methods and Assessment
of Repeatability and Acceptability
The ndd portable spirometer is a small hand held
device that uses an ultrasonic flow sensor to measure
air flow, thus eliminating many problems with other
techniques because measurements are independent of
temperature, humidity, pressure, and gas composition. It
further uses ndd’s disposable mouthpiece, spiretteTM, with
no sensor elements and thus does not contribute or affect
air flow measurement. It has a large memory capacity
(700 sessions). A graphic display of 64 160 pixels
along with a 14-button rubber keypad allow for subject
visualization of maneuver performance messages and
input of data (see below).
821
The ndd portable spirometer has software that complies
with American Thoracic Society (ATS) criteria for
acceptability and repeatability.8,9 Acceptability parameters are standardized measurements of the technical
quality of a maneuver. Repeatability is a measure of the
repeatability of expiratory parameters from one maneuver
to the next, and is measured by the differences between the
highest and second-highest FVC, FEV1, and PEF (dFVC,
dFEV1, dPEF). Because studies have shown that the ATS
criteria,9 designed primarily for adults, can be difficult for
children to achieve,8,10 we modified ATS criteria for
children with asthma (Table 2).6,8,10 We lowered the ATS9
recommended forced expiratory time (FET) from 6 to
4 sec given the findings of Arets et al.,8 who found only
15% of children with obstructive lung disease who were
experienced in spirometry were able to achieve FET >6
sec for otherwise acceptable maneuvers, similar to the
findings of Desmond et al. in children with obstructive
lung disease (19%).10 Enright et al.11 found a majority of a
general population sample of children achieved FET >6
sec (65%). We also focused evaluation on premature
termination of effort or abrupt ending based on the end-oftest volume (EOTV) (Table 2). To assess accurately EOTV
and thus abrupt ending, we calculated FET and EOTV to
end of the expiratory plateau and did not include negative
(inspiratory) flow. The end of test was marked by NDD
software where inspiration >150 ml occurred or a plateau
was detected (volume change <45 ml over 2 s for
FET <4 s or 60 ml for FET 4 s). We also considered the
fact that children with asthma show considerable
variability in pulmonary function due in part to bronchial
reactivity. Further, ATS criteria are intended for in-clinic
spirometry guided by technicians using a laboratory
spirometer. Therefore, we used a more relaxed goal for
repeatability than that recommended by the ATS,9 by
setting FEV1 and FVC repeatability at 10% instead of 5%,
and PEF at 20% instead of 10%. Because the curves were
also visually evaluated, we were able to reject unacceptable curves that met less stringent repeatability criteria.
When almost the entire second best curve was ‘‘smooth’’
and perfectly acceptable, but a small portion of the curve
showed ‘‘a departure,’’ it was reasonable to accept rather
than reject the test session. We visually determined
variable effort and other quality problems where there
was more than around a 10% deviation from the
expected smoothed curve. Maneuver sessions that met
acceptability and repeatability criteria by the software
were independently reviewed by physicians trained
in pediatric pulmonology. Physicians classified each
maneuver rejected based on the reasons for rejection
(cough, glottic closure, variable effort, or a combination of
errors). Figure 1 gives examples. Quality problems were
defined by reference to available standardized flowvolume curves showing established abnormal or disrupted
curves.
822
Thompson et al.
Acceptability and repeatability criteria were custom
programmed into the software for this study by ndd
technicians. The ndd EasyOne Frontline model stores raw
data for all three best maneuvers in memory. We
developed custom SAS (Cary, NC) programs (available
upon request) to output performance variables and plot
three best curves for analysis and improved visual review
over the ndd printout of the single best curve.
Specific protocols and instructions were given to all
technicians doing field training and follow-up. Each
spirometer was checked for calibration (3%) with a 3 L
syringe according to the manufacturer’s specifications
before the start of each 1-month follow-up and before
each 10-day follow-up. Subjects were instructed to
use nose clips. The software programming on the ndd
portable spirometer automatically stopped after three
Fig. 1. Examples of curves with visual scoring errors. A: Variable effort: intermittent deceleration
and acceleration in flow, which may also be intermittent breaths during expiratory flow maneuver;
(B) Glottic Closure: abrupt interruption in expiratory flow causing a sharp spike downward
indicating an abrupt stop and start in flow; (C) Cough: erratic interruption in expiratory flow, along
with repetitive glottic closures.
Evaluation of Daily Home Spirometry for School Children with Asthma
823
Fig. 1. (Continued )
good maneuvers were obtained, but would give each
participant up to six chances to meet acceptability and
repeatability criteria (Table 2). If the participant were
unable to meet repeatability and acceptability criteria after
six attempts, the spirometer would automatically store the
best three attempts based on calculation of the largest
FEV1 þ FVC. The spirometer also gave intermittent
instructions to subjects to aid them in performing acceptable and reproducible maneuvers based on the error of
each attempt (Table 2). To unmask the effects of air
pollutants, it was emphasized to all subjects the importance of doing sessions prior to the use of an IBD unless
necessary and to wait at least 4 hr after the use of an IBD
before performing a session. Furthermore, subjects were
required to answer yes/no questions generated by the
spirometer after each spirometry session [(1) Did you have
any cough during this test? (2) Did you need to use your
rescue medication in the last hour?]. Answers specific to
each spirometry session were stored in memory.
Subjects received a handbook that contained detailed
instructions on use of their spirometer and in the
appropriate technique for doing spirometry sessions. In
addition, a large and laminated one-sheet guide with
stepwise illustrations was given to subjects and placed in a
prominent place in the home where maneuvers were done.
They were encouraged to refer to the handbook and onesheet guide if needed. During follow-up, research
technicians downloaded data into laptops during home
visits and checked compliance with the three daily
sessions, and the acceptability of maneuvers as generated
by the ndd software (v 2.6). Subjects were retrained as
needed. The data manager using our custom SAS
programs checked data again the next morning. Problems
were reported to field staff and subjects were retrained if
needed that daily follow-up or before the next weekly
follow-up. Compliance was further encouraged by
audio reminder alarms on the ndd portable spirometer,
and by a monetary sliding scale based on the number of
maneuvers performed with each session. Subjects performing most of the maneuvers (17–21 per week)
received a bonus payment as well. The ndd software also
awarded 500 points for sessions with three acceptable and
two reproducible maneuvers, but only 200 points if all six
attempts failed acceptability and repeatability criteria.
This was used as an additional behavioral incentive.
Analysis
We compared the percentage compliance, and repeatability and acceptability of maneuvers between daily and
weekly follow-up, between study regions (Riverside vs.
Whittier), and across key subject characteristics to better
understand the underlying determinants of data quality.
Key subject characteristics included gender, use of
controller medications, age group (9–12 vs. 13–18 years
old) and percent predicted FEV1 <80 versus 80% based
on repeated measurements of acceptable and reproducible
maneuvers. Percent-predicted normal FEV1 were calculated for a given height, age, gender, and race-ethnicity
using normal lung function equations from the National
Health and Nutrition Examination Survey III.12 Tests for
significant differences in data quality were performed
across these binary group characteristics using a two-sided
824
Thompson et al.
TABLE 1— Study Group Characteristics
Subject variables
Data
Subject number
Riverside
Whittier
Age, mean (range)
Gender N (%)
Female
Male
Race N (%)
White
Black
Hispanic
Asian
Mean percent predicted FEV1 (SD)
Riverside
Whittier
33
34
13.5 (9–18 years)
25 (37.3)
42 (62.7)
25 (37.3)
15 (22.4)
24 (35.8)
3 (4.5)
80.8 (16.3)
89.5 (12.7)*
*P < 0.05 for difference between Riverside and Whittier in percent
predicted FEV1.
t-test with Satterthwaite’s method in SAS v 9.1 Proc
TTEST.13
RESULTS
The 67 subjects in this repeated measures study were of
diverse racial and ethnic background, and ranged in age
from 9 to 18 years (mean 13.5 years) (Table 1). There were
more males than females. Mean percent-predicted FEV1
from all valid maneuvers during daily home follow-up was
lower for subjects in Riverside compared with Whittier
(80.8% vs. 89.5%, respectively).
Despite the prolonged period of study, all home
spirometers assigned to subjects for the duration of this
study were returned undamaged. One spirometer was
found to have minimal loss of calibration (4%) after
4 weeks of use and was sent back to the manufacturer for
calibration.
The distribution of performance variables is shown in
Table 3. Evaluation of acceptability and repeatability of
maneuvers is shown in Table 4. Daily versus weekly
follow-up values of acceptability and repeatability did not
differ significantly, although there was a consistent trend
of higher values during daily follow-up. There was no
statistically significant difference between the more strict
criteria of three acceptable, two reproducible maneuvers
versus two acceptable and reproducible maneuvers,
hereafter referred to as criteria A and B, respectively. As
expected, the lowest percentage of acceptability and
repeatability occurred when all three flow-volume criteria
were evaluated simultaneously, but values remained
>75% across all parameters.
Table 5 shows the frequency of subject errors detected
by ndd EasyOne software, which labeled the spirometry
curves as unacceptable. The most common error made by
subjects was abrupt ending and invalid time to peak
expiratory flow (PEFT).
Table 6 shows causes of physician visual rejection of
software accepted maneuvers. Variable effort was the
most common cause of rejection, often in combination
with cough and glottic closure. Glottic closure followed by
cough was the next most common. Overall frequency of
causes for visual rejection was small being 10.4% and 8%
of overall acceptable and reproducible maneuvers for
daily and weekly follow-up, respectively.
Evaluation of compliance showed excellent overall
compliance of >90% for daily follow-up and >84% for
weekly follow-up data (Table 7). This finding remained
consistent across study region and subject characteristics.
However, the quality of maneuvers differed across a
number of factors. Subjects in Riverside performed worse
than subjects in Whittier for all comparisons. There were
also significant differences across subject characteristics
for both daily and weekly follow-up data for criteria A and
B, and for visually acceptable criteria test sessions. These
TABLE 2— Acceptability and Reproducibility Criteria
Acceptability criteria
Instruction to subjects if criterion not met
Back-extrapolated volume (BEV) must be <150 ml or 5% of the FVC
Time to peak expiratory flow (PEFT) must be >40 msec and 160 mseca
a. We later added an additional instruction in the second half of study for maneuvers
where PEFT was 40 msec: ‘‘wait until buzz before blowing out.’’
No abrupt ending, which is when EOTV >100 ml is accumulated in the last 0.5 sec before
the end of test or for age <12 year, 50 ml in the last 0.25 sec before the end of test. EOTV
is calculated from the forced expiratory time (FET) (0.5 or 0.25 sec to end of expiration).
Abrupt ending also occurs if FET is <2 sec [end-of-test volume (EOTV)]
Repeatability Criteria
Difference between the highest PEF and 2nd highest PEF (dPEF) within in a test session
must be within 20%
Difference between the highest FEV1 and 2nd highest FEV1 (dFEV1) within in a test session
must be within 10%
Difference between the highest FVC and 2nd highest FVC (dFVC) within in a test session
must be within 10%
Do not hesitate at the beginning of the maneuver
Blast out faster when starting the maneuver
Blow out longer
Blast out harder when starting the maneuver
Take a deeper breath
Take a deeper breath
Evaluation of Daily Home Spirometry for School Children with Asthma
TABLE 3— Distribution of Variables for the Quality of the Best Maneuver
825
1
Percentiles
BEV (ml)
BEV (% FVC)
PEFT (msec)
EOTV (ml)
FET (sec)
dFVC (%)
dFEV1 (%)
dPEF (%)
5th
10th
25th
50th
75th
90th
95th
18.1
0.7
50.0
2.5
2.5
0.3
0.3
0.5
26.9
1.0
60.0
3.8
2.8
0.6
0.6
1.1
42.5
1.5
60.0
6.9
3.4
1.4
1.6
2.7
62.5
2.1
80.0
12.5
4.6
3.4
3.7
6.2
86.3
2.8
100.0
24.4
6.2
7.0
7.5
12.4
114.4
3.6
120.0
42.5
8.4
14.0
15.5
25.2
133.7
4.1
140.0
55.6
10.0
24.8
26.9
38.9
1
The maneuver with the highest FEV1 þ FVC for each of 9,916 spirometry sessions.
characteristics included age group 9–12 years performing
worse than 13–18 years, and subjects with FEV1 80%
predicted performing significantly better than their
counterparts. Subjects not on controller medications performed worse than those taking them, but this difference
was statistically significant only for daily follow-up.
Performance between daily and weekly follow-up was not
significantly different. Weekly follow-up performance
between months one and two of subject follow-up was not
significantly different.
DISCUSSION
Overview of Findings and Comparison
with the Literature
Mortimer et al.,6 evaluated data from asthmatic children
using the ndd portable spirometer, which had quality
control software with modified ATS acceptability and
repeatability criteria similar to the present study, including
the relaxed goal for repeatability and end-of-test criteria
tailored for asthmatic children. They conducted an asthma
panel study of 92 children with asthma and found overall
compliance was 87% for 2 weeks (2,151 sessions), similar
to our compliance for weekly follow-up over 2 months
(85%–90%). They also found the proportion of sessions
with at least two acceptable and reproducible maneuvers
by software criteria was 62% overall, which is lower than
the present study (76%–80%). However, their study
differs from the present in that subjects were younger
(ages 6–11), and no exclusion of training period data was
reported.
We found a non-significant small increase in the quality of the maneuvers from month 1 to 2, and no change in
compliance. Wensley and Silverman14 analyzed the
quality of data and compliance in 90 asthmatic school
children ages 7–14 years who used a Vitalograph
home spirometer and were followed at home monthly
over 4 months. They found some deterioration of collected
data over time primarily due to decreasing compliance
(from 81% to 70%) rather than any deterioration in
maneuver quality (from 82% to 80%). However, quality
could be only assessed by percent variability of
FVC þ FEV1 for the two best maneuvers.
We previously used the Vitalograph 2110 (Vitalograph,
Inc., Lenexa, KS) in a panel study of 19 children with
asthma each followed daily for 14 days, and found 80%
compliance and 81% of sessions had acceptable and
reproducible FEV1 data.15 Although these results are
roughly comparable to the present study, quality assurance
TABLE 4— Acceptability and Repeatability of Self-Administered Spirometry for Daily
Compared with Weekly Staff Follow-Up*
At least three acceptable, two
reproducible maneuvers
Parameters
FEV1, FVC, PEF
FEV1 only
FVC only
PEF only
Total no. of
spirometry sessions
Daily follow-up
1
78.7
85.6
84.6
89.2
1814
Weekly follow-up
75.2
82.0
81.4
85.4
8102
At least two acceptable,
two reproducible maneuvers
Daily follow-up
79.9
87.1
86.3
91.2
1814
Weekly follow-up
76.4
84.0
83.6
87.8
8102
*There were no P < 0.05 differences between frequencies for daily versus weekly follow-up or between
three acceptable, two reproducible maneuvers versus two acceptable, two reproducible maneuvers.
1
Percent of total spirometry sessions collected (three maneuvers per session).
826
Thompson et al.
TABLE 5— Frequency of Maneuver Errors
Error type1
Abrupt ending
PEFT
PEFT þabrupt ending
BEV
BEV þabrupt ending
BEV þ PEFT
BEV þ PEFT þabrupt ending
Total maneuvers
Best maneuver2
no. (% total)
2nd best maneuver
no. (% total)
3rd best maneuver
no. (% total)
32 (0.32)
18 (0.18)
3 (0.03)
10 (0.10)
2 (0.02)
14 (0.14)
1 (0.01)
9916
76 (0.77)
79 (0.80)
20 (0.20)
26 (0.26)
6 (0.06)
42 (0.42)
8 (0.08)
9916
170 (1.71)
209 (2.11)
32 (0.32)
73 (0.74)
9 (0.09)
111 (1.12)
15 (0.15)
9916
1
See Table 2 for definitions.
Maneuvers are ranked in order of best effort (FEV1 þ FVC).
2
software was limited to PEFT (acceptable at 40–
290 msec) and prompting of subject maneuvers until
PEF readings were within 10%.
Given that subjects in the present study were usually not
coached by technicians, the distribution of performance
variables shows the quality of the data is reasonably good
(Table 3). Enright et al.11 assessed the quality of
technician-administered spirometry done annually for
3 years in a large cohort study of over 4,000 Southern
California school children ages 9–18. The distribution of
our PEFT and Back-extrapolated volume (BEV) variables
in Table 3 are similar to theirs. We show no negative
EOTV in the lower distribution as they did (see Materials
and Methods), but our 95th percentile was only 56 ml. The
FET in the general population sample of Enright et al.11
was longer (median 6.6 s) compared with a median of
4.6 sec for the persistent asthmatics of our study. Our
repeatability results were worse. The median dFEV1 in
Enright et al.11 was 1.2% whereas ours was 3.7%. Further
evaluation of the quality of maneuvers in our study was
made by evaluating the visual acceptability of curves that
met software criteria for at least two acceptable and two
TABLE 6— Causes of Visual Rejection of Maneuvers That
Met Acceptability Criteria
Causes of visual rejection
Cough
Glottic closure2
Variable effort
Cough/variable effort
Cough/glottic closure
Cough/glottic closure/
variable effort
Glottic closure/
variable effort
Total rejected
Total acceptable
1
Daily follow-up
1
Weekly follow-up
45 (1.0)
33 (0.7)
262 (6.0)
65 (1.5)
9 (0.2)
9 (0.2)
152 (0.8)
223 (1.2)
724 (3.93)
71 (0.4)
71 (0.4)
14 (0.1)
31 (0.7)
240 (1.3)
454 (10.4)
4350 (100)
1496 (8.0)
18576 (100)
Number rejected (percent of total acceptable maneuvers for the
follow-up frequency).
2
Includes problems at the end of the curve, which may not have had an
effect on FEV1 or FVC.
reproducible curves. Variable effort was the most frequent
cause of visual rejection (Table 6).
In the present study, we found that both compliance and
quality of curves were higher for subjects followed up
daily as compared with weekly (Tables 4 and 7), but
differences were not statistically significant. This consistency in technique and compliance could be attributed
to the spirometer itself, which as mentioned above,
contains inherent quality assurance measures that do not
require the presence of a field team member to correct
subjects on common errors during maneuver attempts
(Table 2). Subjects were also given a manual and a small
poster size colored pictograph describing and depicting a
subject doing appropriate spirometry technique. In
addition, compliance was regularly encouraged by
monetary incentives, an on-screen point system, and
audio alarms in the ndd software to remind subjects during
selected periods (see Materials and Methods).
We found quality of maneuvers was significantly worse
in younger subjects. This age difference in the quality of
home spirometry was also found by Mortimer et al.,6
Enright et al.,11 and Pelkonen et al.16 Multiple indicators
of persistent asthma also predicted ability to perform
reliable and accurate lung function maneuvers as shown
by significantly better maneuver quality among subjects
with 80% predicted FEV1 and subjects on controller
medication. We speculate that the improved performance
with better lung function is due to the intrinsic nature of
the subjects’ lung disease (asthma). There were no
significant differences in the distribution of controller
medication use by percent predicted FEV1.
The quality of maneuvers was also significantly worse
in Riverside as compared with Whittier. Underlying group
characteristics might explain this. Riverside and Whittier
were significantly different in the number with <80%
predicted FEV1 (42% vs. 18%, respectively, Fisher’s exact
P < 0.05). However, the regional groups were similar in
use of maintenance asthma controller medications (64 vs.
68%). The groups did differ by age sub-group with the
Riverside group having more subjects 9–12 years old than
the Whittier group (48.5% vs. 17.7%). However, there was
Evaluation of Daily Home Spirometry for School Children with Asthma
827
TABLE 7— Compliance, Acceptability and Repeatability of Self-Administered Spirometry: Differences by Panel, Age,
Controller Medication Use and Lung Function
Daily Follow-up
Characteristic
Riverside
Whittier
Age group
9–12
13–18
Controller medication
On
Not on
Mean percent predicted
FEV1
80
<80
Riverside
Whittier
Age Group
9–12
13–18
Controller Medication
On
Not on
Mean percent predicted
FEV1
80
<80
Follow-up
Month 1
Month 2
No. of Subjects
Compliance: No. test
sessions collected
(% expected)
Percent1 with
3 acceptable,
2 reproducible
Percent with
2 acceptable
and 2 reproducible
Percent visually acceptable
with 2 acceptable
and 2 reproducible
31
32
881 (92.8)
933 (95.2)
68.82
88.1
69.72
89.6
54.92
81.7
21
42
607 (95.0)
1207 (93.6)
66.72
84.8
67.92
86.0
56.52
74.8
42
21
1232 (94.6)
582 (92.8)
83.82
68.0
84.82
69.6
72.3
61.0
45
18
84.62
68.6
73.72
56.4
33
34
82.92
68.4
Weekly follow-up
3918 (84.8)
64.72
4184 (89.7)
85.0
65.52
86.6
52.02
78.4
21
42
2801 (87.4)
5301 (87.2)
64.62
80.8
65.72
82.1
54.42
71.5
42
21
5288 (87.4)
2814 (87.0)
78.0
69.9
79.5
70.6
66.9
63.1
45
18
5576 (87.2)
2526 (87.5)
81.52
61.2
82.72
62.6
74.32
46.4
67
62
4210 (87.8)
3892 (86.7)
72.7
77.8
73.8
79.3
64.0
67.3
1289 (93.6)
525 (95.1)
1
Percentages are the number with the stated characteristic divided by the number of spirometry sessions collected.
P < 0.05 t-test with Satterthwaite’s method for differences in performance between region, age, controller medication group, percent predicted
FEV1, and month of follow-up.
2
no difference in maneuver quality by age sub-group in
Whittier, and performance was a significantly better in
Whittier for both 9–12 and 13–18 year old subjects (not
shown). We speculate that unmeasured characteristics led
to this regional difference in quality of maneuvers.
Application of Findings to Research
In field studies of chronic lung diseases that require
frequent measurements of pulmonary function, the use of
peak flows has been one of the most common measurements available to investigators.2 This is largely due to the
ease of use of peak flow meters, portability and low cost.
Unfortunately, measurement of peak flows has been
shown to have many disadvantages for use in research or
clinical management of patients with asthma or other
chronic lung diseases.1–5,17–20 Previously, full measurement of pulmonary function that allows for evaluation of
other critical variables of air flow (FEV1, FVC, FEF25–75)
has been limited only to the office or laboratory, thus
precluding repeated daily measurements.
Portable spirometers such as the one used in this study
demonstrate several advantages over peak flow meters.
First, its ability to generate true flow-volume loops not
only allows for evaluation of various parameters of
expiratory flows (FEV1, FVC, FEF25–75, PEF), but also
allows for evaluation of technique and effort based on
professional standards programmed into the software
(Table 2). The measurement of these parameters allows for
evaluation of both central and peripheral airway function,
thus the potential for better and more expansive evaluation
of clinical status. Finally, quality assurance is enhanced by
the machine’s ability to: (1) give subjects correcting
instructions on the next maneuver attempt based on errors
evaluated on the current maneuver attempt; and (2) store
large volumes of time-date-stamped data.
CONCLUSIONS
Although there were subgroup differences in compliance and maneuver quality in this study, overall
performance with the use of the ndd portable spirometer
828
Thompson et al.
was good, with acceptability and repeatability criteria
being met at its most stringent (three acceptable and two
reproducible maneuvers) more than 61% of the time for all
subgroups. The lack of significant differences in quality of
maneuvers and compliance for daily versus weekly
follow-up supports the effectiveness of quality assurance
measures built into the ndd and the ease of performing
maneuvers for well-trained subjects even as young as age
9 years.
The use of portable spirometers with the specifications
described is feasible and results in a high yield of valid
data for field research. Handheld spirometers with
capabilities such as the ndd EasyOne are superior to
simple peak flow meters in allowing for comprehensive
measures of lung function, quality assurance measures,
compliance markers, and ease of use. This allows
researchers to evaluate the impact of clinical interventions
or environmental exposures on chronic lung diseases such
as asthma.
We found data quality was affected by a variety of
influences, including clinical severity, treatment regimen,
and population differences, all of which should be taken
into account when interpreting data. Despite these impacts
on data quality, results of this large panel study of pediatric
asthma show that durable and portable spirometers offer
quality assurance and longitudinal data collection capabilities. Further studies evaluating portable electronic
spirometry in research and clinical settings are strongly
encouraged.
ACKNOWLEDGMENTS
The authors declare they have no competing financial
interests. The authors thank Dr. Ira B Tager and
Dr. Kathleen M Mortimer for their assistance in custom
software development for the ndd portable spirometer, our
staff Lynn Chuang and Vanessa Phan for their skilled
effort in training subjects and collecting lung function data
for this study, and Victor Gastanaga for data management
and quality control. Most of all we thank our study
participants for their diligent efforts. The project
described was supported by grant number ES011615
from the National Institute of Environmental Health
Sciences (NIEHS), U.S. National Institutes of Health
(NIH), and its contents are solely the responsibility of the
authors and do not necessarily represent the official views
of the NIEHS, NIH.
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JM. Quality of spirometry test performance in children and
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Kleinman MT, Liu L-JS, Bufalino C, Wu C-F, McLaren CE.
Association of FEV1 in asthmatic children with personal and
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| 6,705 | 43,867 |
W3008689296.txt | LENTERA Jurnal Ilmiah Kependidikan
Vol. 14 No. 2 (Edisi Dies Natalis XXXIV 2019) 41 - 47
ISSN : 0216-7433
TANTANGAN PENDIDIK DI ERA DIGITAL
Diplan1
1. Universitas Muhammadiyah Palangkaraya
diplan161181@gmail.com
ABSTRAK
Di era globalisasi dan informasi ini, guru dan dosen mendapatkan tantangan berupa
perilaku siswa yang senang berselancar di dunia maya melalui android, gadget atau
handphone canggih yang mereka miliki. Perilaku siswa ini ditandai oleh adanya
kecendrungan lebih senang membaca melalui handphone ketimbang buku, senang
mencari berbagai informasi sendiri tanpa melakukan recheck lebih dahulu, senang
mencari teman melalui akun media sosial, dan terkesan individualis. Untuk itu, guru dan
dosen dalam pembelajaran harus mampu memanfaatkan Teknologi Informasi,
menggunakan metode yang menyenangkan, memperkaya keilmuan dengan berbagai
sumber bahan bacaan, dan mampu melakukan penelitian. Demikian uraian yang berkaitan
dengan tantangan pendidik di era Millennial dan upaya yang dilakukan untuk mencari
solusi terhadap tantangan yang dihadapi guru.
Kata Kunci: Era Digital, Pendidik, Milennial
PENDAHULUAN
Sejarah revolusi industri dimulai dari industri 1.0, 2.0, 3.0, hingga industri
4.0. Industri 1.0 ditandai dengan mekanisasi produksi untuk menunjang efektifitas
dan efisiensi aktivitas manusia, industri 2.0 dicirikan oleh produksi massal dan
standarisasi mutu, industri 3.0 ditandai dengan penyesuaian massal dan
fleksibilitas berbasis otomasi dan robot. Industri 4.0 selanjutnya hadir
menggantikan industri 3.0 yang ditandai dengan penggabungan mesin, alur kerja,
dan sistem, dengan menerapkan jaringan cerdas. Istilah industri 4.0 berasal dari
sebuah proyek yang diprakarsai oleh pemerintah Jerman untuk mempromosikan
komputerisasi diberbagai bidang.
Lee et al (2013) menjelaskan, industri 4.0 ditandai dengan peningkatan
digitalisasi yang didorong oleh empat faktor: 1) peningkatan volume data,
kekuatan komputasi, dan konektivitas; 2) munculnya analisis, kemampuan, dan
kecerdasan bisnis; 3) terjadinya bentuk interaksi baru antara manusia dengan
mesin; dan 4) perbaikan instruksi transfer digital ke dunia fisik, seperti robotika
dan 3D printing. Lifter dan Tschiener (2013) menambahkan, prinsip dasar industri
4.0 adalah penggabungan mesin, alur kerja, dan sistem, dengan menerapkan
jaringan cerdas di sepanjang rantai dan proses produksi untuk mengendalikan satu
sama lain secara mandiri.
Hermann et al (2016) menambahkan, ada empat desain prinsip industri 4.0.
Pertama, interkoneksi (sambungan) yaitu kemampuan mesin, perangkat, sensor,
dan orang untuk terhubung dan berkomunikasi satu sama lain melalui Internet of
Things (IoT) atau Internet of People (IoP). Prinsip ini membutuhkan kolaborasi,
keamanan, dan standar. Kedua, transparansi informasi merupakan kemampuan
sistem informasi untuk menciptakan salinan virtual dunia fisik dengan
41
Diplan / LENTERA Jurnal Ilmiah Kependidikan
Vol. 14 No.2 (Juli – Desember 2019 Edisi Dies Natalis XXXIV) 41-47
memperkaya model digital dengan data sensor termasuk analisis data dan
penyediaan informasi. Ketiga, bantuan teknis yang meliputi; (a) kemampuan
sistem bantuan untuk mendukung manusia dengan menggabungkan dan
mengevaluasi informasi secara sadar untuk membuat keputusan yang tepat dan
memecahkan masalah mendesak dalam waktu singkat; (b) kemampuan sistem
untuk mendukung manusia dengan melakukan berbagai tugas yang tidak
menyenangkan, terlalu melelahkan, atau tidak aman; (c) meliputi bantuan visual
dan fisik. Keempat, keputusan terdesentralisasi yang merupakan kemampuan
sistem fisik maya untuk membuat keputusan sendiri dan menjalankan tugas
seefektif mungkin.
Industri 4.0 telah memperkenalkan teknologi produksi massal yang fleksibel
(Kagermann et al, 2013). Mesin akan beroperasi secara independen atau
berkoordinasi dengan manusia (Sung, 2017). Industri 4.0 merupakan sebuah
pendekatan untuk mengontrol proses produksi dengan melakukan sinkronisasi
waktu dengan melakukan penyatuan dan penyesuaian produksi (Kohler & Weisz,
2016). Selanjutnya, Zesulka et al (2016) menambahkan, industri 4.0 digunakan
pada tiga faktor yang saling terkait yaitu; 1) digitalisasi dan interaksi ekonomi
dengan teknik sederhana menuju jaringan ekonomi dengan teknik kompleks; 2)
digitalisasi produk dan layanan; dan 3) model pasar baru. Baur dan Wee (2015)
memetakan industri 4.0 dengan istilah “kompas digital”
Kompas Digital merupakan instrumen bagi perusahaan dalam
mengimplementasikan industri 4.0 agar sesuai dengan kebutuhan mereka. Pada
komponen tenaga kerja (labor), harus memenuhi; 1) kolaborasi manusia dengan
robot; 2) kontrol dan kendali jarak jauh; 3) manajemen kinerja digital; dan 4)
otomasi pengetahuan kerja. Demikian pula pada komponen lainnya digunakan
sebagai instrumen implementasi industri 4.0.
Revolusi digital dan era disrupsi teknologi adalah istilah lain dari industri
4.0. Disebut revolusi digital karena terjadinya proliferasi komputer dan
otomatisasi pencatatan di semua bidang. Industri 4.0 dikatakan era disrupsi
teknologi karena otomatisasi dan konektivitas di sebuah bidang akan membuat
pergerakan dunia industri dan persaingan kerja menjadi tidak linear. Salah satu
karakteristik unik dari industri 4.0 adalah pengaplikasian kecerdasan buatan atau
artificial intelligence (Tjandrawinata, 2016). Salah satu bentuk pengaplikasian
tersebut adalah penggunaan robot untuk menggantikan tenaga manusia sehingga
lebih murah, efektif, dan efisien.
Unsur-unsur pendidikan dalam definisi di atas terdiri dari usaha (kegiatan)
yang bersifat bimbingan dan dilakukan secara sadar, pendidik (pembimbing), anak
didik, dasar dan tujuan, dan alat-alat yang digunakan dalama usaha itu. Dengan
demikian dapatlah dikatakan bahwa pendidikan sebagai sebuah sistem terdiri dari
tujuan, metode, materi (kurikulum), pendidik, anak didik, alat pendidikan, dan
lingkungan. Semua unsur ini saling berkaitan satu sama lain. Pendidikan saat ini
menghadapi berbagai tantangan, antar lain bisa dilihat dari karakter anak.
Karakter anak zaman now semakin nyeleneh dan semaunya sendiri. Anak-anak
cenderung egois, tidak suka bekerja sama. Hal ini disebabkan seringnya mereka
lebih suka bermain game lewat ponsel android daripada permainan tradisional
yang mengajarkan perilaku untuk bekerjasama. Fenomena ini tidak bisa
dipungkiri, baik itu di kota maupun di pelosok desa sekalipun. Karakter anak pada
sebagian generasi millenial memprihatinkan. Mereka kadang tidak menghargai
42
Tantangan Pendidik di Era Digital
orangtua maupun gurunya. Bahkan dari mereka juga terkadang terjebak pada
dunia criminal dan narkoba. Generasi millenial dalam minat belajar juga sebagian
besar mengalami kemunduran. Di era millennial, kecenderungan dunia pendidikan
antara lain: berkembangnya model belajar jarak jauh (Distance Learning),
mudahnya menyelenggarakan pendidikan terbuka, sharing resource bersama antar
lembaga pendidikan, perpustakaan dan instrument pendidikan lainnya (guru,
dosen, laboratorium) berubah fungsi menjadi sumber informasi daripada sekedar
rak buku. Lembaga pendidikan akan menghadapi sebuah perubahan yang
signifikan akibat proses digital ini. Ini menjadi sebuah peluang dan cara untuk
meningkatkan kualitas pendidikan sekaligus tantangan bagi dunia pendidikan di
Indonesia. Pada era millenial, manusia mulai meninggalkan cara-cara
konvensional dalam menjalani kehidupan, digantikan dengan trend dan gaya
hidup yang lebih fresh and youth, atau yang biasa dikenal dengan istilah
“kekinian”. Seorang aktivisi HMI, Muhammad Ridal, dalam bukunya yang
berjudul “HMI Millenial” mengungkapkan bahwa 33% masyarakat Indonesia saat
ini merupakan generasi millenial. Menurut Ridal, era millenial umumnya
didominasi oleh orang-orang kelahiran tahun 1980 sampai tahun 2000an, dan
berusia 15-34 tahun. Usia ini, tentu saja, merupakan usia dimana individu masih
berstatus sebagai pelajar di sekolah.
PEMBAHASAN
A. Karakteristik Generasi Milennial
Generasi millennial memiliki beberapa karakteristik, yaitu: 1. Millennial
tidak percaya lagi kepada distribusi informasi yang bersifat satu arah. Mereka
lebih percaya kepada konten dan informasi yang dibuat oleh perorangan. Dalam
hal pola konsumsi, banyak dari mereka memutuskan untuk membeli produk
setelah melihat review atau testimoni yang dilakukan oleh orang lain di internet.
Mereka juga tak segan-segan membagikan pengalaman buruk mereka terhadap
suatu merek. 2. Millennial lebih memilih ponsel dibanding TV. Televisi bukanlah
prioritas generasi millennial untuk mendapatkan informasi atau melihat iklan.
Generasi millennial lebih suka mendapat informasi dari ponselnya, dengan
mencarinya ke Google atau perbincangan pada forum-forum yang mereka ikuti. 3.
Millennial wajib punya media sosial. Komunikasi di antara generasi millennial
sangatlah lancar. Komunikasi itu tidak selalu terjadi dengan tatap muka. Banyak
dari mereka melakukan semua komunikasinya melalui text messaging atau juga
chatting di dunia maya, dengan membuat akun yang berisikan profil dirinya,
seperti Twitter, Facebook, hingga Line. Akun media sosial juga dapat dijadikan
tempat untuk aktualisasi diri dan ekspresi. Hampir semua generasi millennial
dipastikan memiliki akun media sosial sebagai tempat berkomunikasi dan
berekspresi. 4. Millennial kurang suka membaca secara konvensional. Bagi
generasi ini, tulisan dinilai memusingkan dan membosankan. Generasi millennial
bisa dibilang lebih menyukai melihat gambar, apalagi jika menarik dan berwarna.
Hobi membaca buku masih tetap ada. Mereka lebih memilih membaca buku
online (e-book) untuk tidak perlu repot membawa buku. Sekarang ini, sudah
banyak penerbit yang menyediakan format e-book untuk dijual, agar pembaca
dapat membaca dalam ponsel pintarnya. 5. Millennial lebih tahu teknologi
43
Diplan / LENTERA Jurnal Ilmiah Kependidikan
Vol. 14 No.2 (Juli – Desember 2019 Edisi Dies Natalis XXXIV) 41-47
dibanding orangtua mereka. Kini semua serba digital dan online. Generasi ini
melihat dunia tidak secara langsung, yaitu dengan berselancar di dunia maya.
Generasi millennial adalah generasi yang sangat modern, lebih daripada orang tua
mereka. Mereka sering mengajarkan teknologi pada kalangan orangtua. 6.
Millennial cenderung tidak loyal namun bekerja efektif. Mereka juga tidak loyal
terhadap suatu pekerjaan atau perusahaan, namun lebih loyal terhadap merek.
Millennial hidup di era informasi yang menjadikan mereka tumbuh cerdas.
Banyak perusahaan yang mengalami kenaikan pendapatan karena
memperkerjakan millennial. 7. Millennial mulai banyak melakukan transaksi
secara cashless. Dengan kecanggihan teknologi yang semakin maju ini, generasi
millennial pun mulai melakukan transaksi pembelian yang sudah tidak
menggunakan uang tunai lagi alias cashless. Generasi ini lebih suka membawa
kartu, karena semua pembelian bisa dibayar menggunakan kartu, sehingga lebih
praktis, hanya perlu gesek atau tapping. Tantangan Guru Citra dan konsep tentang
guru dalam masyarakat kontemporer sangat jauh berbeda dengan konsep masa
lampau. Guru masa dahulu berarti orang yang berilmu, yang arif dan bijaksana.
Kini guru dilihat sebagai fungsionaris pendidikan yang bertugas mengajar atas
dasar kualifikasi keilmuan dan akademis tertentu. Dengan tugas tersebut, guru
memperoleh imbalan materi dari negara atau pihak pengelola pendidikan. Dengan
demikian, faktor terpenting dalam profesi guru adalah kualifikasi keilmuan dan
akademis. Sementara kearifan dan kebijaksanaan yang merupakan sikap dan
tingkah laku moral tidak lagi signifikan. Dalam konsep klasik, faktor moral berada
di atas kualifikasi keguruan. Berdasarkan penelitian bahwa mayoritas millennial
mendapatkan berita bersumber dari media sosial seperti facebook dan twitter,
dimana kredibilitas sumber berita sangat sulit untuk diukur.
B. Tantangan Pendidik di Era Millennial
Kualifikasi akademik seorang guru adalah S1 atau diploma IV. Kompetensi
yang harus dimiliki seorang guru adalah kompetensi pedagogik, kompetensi
kepribadian, kompetensi social dan kompetensi professional yang diperoleh
melalui pendidikan profesi. Masing-masing kompetensi tersebut bila dirinci
sebagai berikut: 1. Memiliki kepribadian sebagai pendidik dengan sub kompetensi
a. Memiliki kepribadian mantap dan stabil b. Memiliki kepribadian dewasa c.
Memiliki kepribadian arif d. Memiliki kepribadian yang berwibawa e. Memiliki
akhlak mulia dan dapat menjadi teladan 2. Memiliki kompetensi pedagogik
dengan sub kompetensi a. Memahami peserta didik b. Merancang pembelajaran c.
Melaksanakan pembelajaran d. Evaluasi hasil belajar e. Pengembangan peserta
didik 3. Memiliki kompetensi profesional sebagai pendidik dengan sub
kompetensi: a. Menguasai bidang studi secara luas & mendalam b. Memahami
materi ajar yang ada dalam kurikulum sekolah c. Memahami struktur, konsep, dan
metode keilmuan yang menaungi/koheren materi ajar d. Memahami hubungan
konsep antar mata-pelajaran terkait e. Menerapkan konsep-konsep keilmuan
dalam kehidupan sehari-hari f. Berkomunikasi secara efektif dengan masyarakat
4. Memiliki kompetensi sosial sebagai pendidik.
Tantangan guru di era milenial sangat berat dibanding guru–guru di era
terdahulu. Selain menguasai aspek materi keilmuan yang di ajarkan. Guru dituntut
memahami teknologi dan selalu menjadi pribadi yang kreatif dan inovatif. Guru
harus menjadi role model bagi siswa di generasi millennial, agar siswa memahami
44
Tantangan Pendidik di Era Digital
batasan– batasan teknologi, sehingga terhindar dari pemamfaatan yang salah
dalam menggunakan teknologi. Tantangan bagi guru tidak berhenti disini,
generasi millennial bukan generasi yang bisa dipaksa–paksa, contoh dengan
melarang siswa membawa handphone. Guru di era sekarang harus lebih terbuka
dengan pemikiran–pemikiran baru. Guru dituntut mendidik siswa sesuai dengan
zamanya. Selama tidak bertentangan dengan norma– norma yang ada tentu
hadirnya teknologi tidak perlu dipermasalahkan. Pendekatan persuasif baiknya
lebih prioritas dilakukan ketimbang melakukan kebijakan–kebijakan yang
terkesan otoriter maupun memaksakan kehendak. Guru bisa memberikan
pengertian kepada siswa melalui kebijakan–kebijakan konkret dengan
memanfaatkan teknologi dalam pembelajaran, seperti e-learning, atau
menggunakan sosial media dll. Siswa justru mendapatkan edukasi bahwa
teknologi ketika dimanfaatkan dengan baik justru memiliki dampak yang positif.
Secara umum, guru hendaknya memahami perubahan sosial yang ada di era
sekarang. Dia tidak berhenti belajar mengenai hal–hal baru. Tantangan global di
era sekarang juga beda dengan tantangan global di era dahulu. Apapun langkah
dan metode yang dilakukan di sekolah pastinya bertujuan membentuk karakter
dan menyiapkan SDM yang berkualitas di Indonesia. Masa depan Indonesia ada
didalam ruang kelas yang kita ajar. Tantangan lain pendidikan di era millennial
sekarang ini adalah informasi datang lebih cepat, massif dan meluas, sehingga
tidak bisa diatasi dengan sekedar pergantian kurikulum. Berapa kali pergantian
kurikulum tidak akan pernah mampu mengejar percepatan informasi yang ada.
Karena itu, pendekatan guru dan murid dalam berinteraksi akan lebih penting.
Perlu keteladanan, membangun kehendak, dan menguatkan – dengan bekal ilmu
pengetahuan tentunya – sehingga anak didiknya mampu mengatasi tantangan
hidupnya. Tantangan terakhir bagi seorang guru adalah adanya empat
keterampilan yang diberikan kepada siswa sejak dini, yaitu melahirkan pemikir,
melahirkan komunikator, melahirkan kolaborator, dan melahirkan penemu atau
pencipta.
C. Upaya yang Dilakukan Menghadapi Era Milennial
Ada beberapa upaya yang harus dilakukan oleh seorang guru menghadapi
tantang dalam mendidik generasi millennial ini. 1. Guru-guru yang lahir pada era
revolusi industri ketiga harus mengajar mereka yang lahir pada era berikutnya.
Tidak bisa tidak, setiap guru wajib mengikuti perkembangan teknologi. Guru
tidak boleh lagi gagap teknologi. Komputer dan gawai harus sudah menjadi
keseharian para guru. Media sosial dan berbagai sumber informasi maupun
sosialisasi juga harus dipahami para guru, sehingga dalam menjalankan profesinya
guru akan kaya dengan materi maupun metode pembelajaran. Siswa pun tidak
akan menganggap remeh guru. 2. Selain menguasai perkembangan teknologi,
guru dituntut juga memahami kecenderungan yang terjadi terkait perubahan
teknologi. Dengan mengikuti perkembangan hasil kemajuan teknologi, guru bakal
mampu memberikan sudut Untuk melahirkan pemikir, siswa dilatih
menyampaikan gagasan aktual, seperti kemacetan, banjir, tawuran. Untuk
melahirkan komunikator, siswa dilatih menyampai ide secara lisan dengan baik.
Untuk melahirkan kolaborator, siswa dikelompokan berdasarkan ragam
kecerdasannya. Untuk melahirkan penemu, siswa dilatih menjadi inovator. Jejen
Musfah, Analisis Kebijakan Pendidikan, Prenadamedia, Jakarta, 2016, hal. 84-86.
45
Diplan / LENTERA Jurnal Ilmiah Kependidikan
Vol. 14 No.2 (Juli – Desember 2019 Edisi Dies Natalis XXXIV) 41-47
Revolusi industri pertama ditandai kemunculan mesin menggantikan
tenaga manusia dan hewan. Generasi kedua ditandai dengan kemunculan
pembangkit tenaga listrik dan motor pembakaran, hingga muncullah pesawat
telepon, mobil, pesawat terbang dan sebagainya. Generasi ketiga ditandai dengan
kemunculan teknologi digital dan internet. Pada revolusi industri generasi
keempat ditandai dengan kemunculan superkomputer, robot pintar, rekayasa
genetika dan perkembangan neuroteknologi yang memungkinkan manusia untuk
lebih mengoptimalkan fungsi otak. Muncul pola-pola baru ketika disruptif
teknologi hadir begitu cepat dan mengancam keberadaan pola lama pandang,
alternatif, bahkan solusi kepada para peserta didik. Di sinilah peran guru yang
tidak tergantikan oleh teknologi. Dalam pembelajaran, guru harus dapat
menguatkan kemampuan olah pikir, olah rasa, olah hati, dan olah ragawi setiap
anak. Hal itu penting agar mereka mampu memaknai, mengolah,
menginterpretasi, menyaring berbagai informasi yang ada di sekitar. Guru harus
memberikan pembelajaran yang relevan dengan siswa. Belajar menjadi lebih
berarti ketika mereka memahami aplikasi praktis informasi yang mereka terima.
Konten harus spesifik, ringkas, dan cepat. Generasi millenal harus informasi dan
akan mencarinya sendiri jika guru tidak menyajikan apa yang mereka anggap
relevan. Karena begitu banyak informasi yang selalu tersedia, mereka tidak
merasa perlu belajar setiap hal segera. Sebaliknya, mereka ingin diajari bagaimana
dan di mana mereka dapat menemukan apa yang mereka butuhkan. Dalam
konteks era digital tentu pendidikan karakter sangat penting. Mengingat, internet
adalah belantara yang liar, di mana konten-konten informasi yang positif dan
negatif bercampur jadi satu. Hanya fondasi moral yang kuat di dalam diri, dalam
arti bisa membedakan mana yang baik dan buruklah yang bisa menjamin masa
depan generasi muda Indonesia tetep cerah. Oleh karena itu, seorang guru harus
menjaga karakter kebangsaan yang potensial terkikis oleh berbagai ideologi
radikalisme yang tidak sesuai dengan Pancasila dan Negara Kesatuan Republik
Indonesia (NKRI). Di era globalisasi ini, pendidikan harus melakukan reformasi
dan inovasi dalam proses belajar mengajar secara terus menerus. Untuk itu,
diperlukan dukungan empirik yang dihasilkan oleh kegiatan penelitian. Oleh
karena itu, guru harus paham penelitian.
PENUTUP
Di era globalisasi dan informasi ini, guru dan dosen mendapatkan
tantangan berupa perilaku siswa yang senang berselancar di dunia maya melalui
android, gadget atau handphone canggih yang mereka miliki. Perilaku siswa ini
ditandai oleh adanya kecendrungan lebih senang membaca melalui handphone
ketimbang buku, senang mencari berbagai informasi sendiri tanpa melakukan
recheck lebih dahulu, senang mencari teman melalui akun media sosial, dan
terkesan individualis. Untuk itu, guru dan dosen dalam pembelajaran harus
mampu memanfaatkan Teknologi Informasi, menggunakan metode yang
menyenangkan, memperkaya keilmuan dengan berbagai sumber bahan bacaan,
dan mampu melakukan penelitian. Demikian uraian yang berkaitan dengan
tantangan pendidik di era Millennial dan upaya yang dilakukan untuk mencari
solusi terhadap tantangan yang dihadapi guru.
46
Tantangan Pendidik di Era Digital
DAFTAR PUSTAKA
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Baru, Cet II. Jakarta: Logos.
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Ilmu Pendidikan-Teoritis dan Praktis (p. 1). Jakarta: Balai Pustaka.
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beserta Penjelesannya. Bandung: Fokusmedia.
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Pendidikan. Jakarta: Sedaun.
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| 2,993 | 22,591 |
W2947392485.txt | Multimodal Technologies
and Interaction
Article
Exploring User Perception Challenges in Vibrotactile
Haptic Display Using Resonant Microbeams under
Contact with Skin
Daehan Wi *
and Angela A. Sodemann
The Engineering Department, Arizona State University, Mesa, AZ 85212, USA; angela.sodemann@asu.edu
* Correspondence: Daehan.Wi@asu.edu
Received: 1 April 2019; Accepted: 24 May 2019; Published: 28 May 2019
Abstract: Resonant vibrotactile microbeams use the concept of resonance to excite the vibration of
cantilever beams, which correspond to pixels of an image. The primary benefit of this type of tactile
display is its potential for high resolution. This paper presents the concept of the proposed system
and human skin contact experiments to explore user perception challenges related to beam vibration
during skin contact. The human skin contact experiments can be described in five phases: dried
skin contact to metal beam tips, wet and soaped skin contact to metal beam tips, skin contact with a
constraint, normal force measurement, and skin contact to the tips of silicone rubber beams attached
to metal beam tips. Experimental results are analyzed to determine in what cases of skin contact the
beams stop vibrating. It is found that the addition of silicone rubber beams allows the primary metal
beams to continue vibrating while in contact with skin. Thus, the vibration response of a metal beam
with silicone rubber beams is investigated for the better understanding of the effect of silicone rubber
beams on the metal beam vibration.
Keywords: resonant microbeams; vibrotactile haptic display; microbeam array; tactile substitution
system; skin contact experiment; silicone rubber beams; vibration response; forced response
1. Introduction
Haptic displays have been extensively explored in assistive technology solutions for individuals
with visual impairments. Tactile vision sensory substitution devices use haptic displays to present
visual images to the sense of touch. This ‘haptic display’ area of research can be divided into two
possible approaches: (1) conversion of information about pictures to touch, or (2) conversion of
complete pictures to touch. In the first approach, an image is first processed by a computer to determine
information about the image, such as distance to the nearest object, then vibration or other tactile
signals are used to convey this information to the user [1]. Haptic face recognition [2–4] and the
human-computer interface system that is studied by D. Hong et al. [5] are examples of this approach.
The second approach has the significant benefit of potentially giving the user an effective
replacement for vision. This area of research relies upon an assumption that the brain is capable of a
type of plasticity known as ‘sensory substitution’, in which information provided via one sense can be
interpreted by the brain as information from a different sense [6–8]. In the second approach, there
are two main types of haptic display devices. The first type of display is the electrotactile display,
commonly placed on the tongue. The second main type of display is the vibration display. Brainport is
the popular example of using the electro tactile displays, a device which consists of a video camera
mounted on a sunglass and an electrode array [9–11]. The image captured by the camera is converted
to a black and white image in software, and pixels from the camera are felt on the tongue via an
electrode array [12]. Besides the electro-tactile display, the vibration displays use vibration motors.
Multimodal Technol. Interact. 2019, 3, 38; doi:10.3390/mti3020038
www.mdpi.com/journal/mti
Multimodal Technol. Interact. 2019, 3, 38
Multimodal Technol. Interact. 2019, 3, x FOR PEER REVIEW
2 of 13
2 of 13
Whilehigh
the electrode
approach
is typically
limited can
to placement
tongue
due
to its
wetness
and,
thus,
conductivity,
vibration
motor devices
be placedon
onthe
any
area of
skin.
This
approach
thus,
high
conductivity,
vibration
motor
devices
can
be
placed
on
any
area
of
skin.
This
approach
commonly uses vibration motors, camera, and a processing unit [13,14]. The image captured by the
commonly
uses vibration
motors, camera,
a processing
unitmotors
[13,14].corresponding
The image captured
by the
camera
is processed
into a vibration
level forand
an array
of vibration
to the image.
camera
is
processed
into
a
vibration
level
for
an
array
of
vibration
motors
corresponding
to
the
image.
However, all of the haptic display solutions found in the literature have two primary
However,
all
of
the
haptic
display
solutions
found
in
the
literature
have
two
primary
complications:
complications: The low resolution and the one-pin-per-element problem. Resolution is the number
The
low resolution
one-pin-per-element
Resolution
is the
number of
pixels
contained
of
pixels
contained and
in anthe
image.
A visual digitalproblem.
image commonly
has
a resolution
such
as 640
× 480
in
an
image.
A
visual
digital
image
commonly
has
a
resolution
such
as
640
×
480
or
720
1080
or 720 × 1080 pixels, but the current highest- resolution tactile display has much lower resolution×such
pixels,
but the
highestresolution
tactile display
has much
lower resolution
such
less than
as
less than
20 ×current
20. Since
a visual
image becomes
more difficult
to interpret,
the lower
theas
resolution
20
×
20.
Since
a
visual
image
becomes
more
difficult
to
interpret,
the
lower
the
resolution
is, to
it ais
is, it is expected that the low resolution such as 20 × 20 would not be enough to deliver an image
expected
that
the
low
resolution
such
as
20
×
20
would
not
be
enough
to
deliver
an
image
to
a
viewer.
viewer. Another problem, the one pin per element problem, stems from the need to individually
Anothereach
problem,
one independently
pin per elementofproblem,
stems from
the need
to individually
each
control
tactilethe
pixel
all the others.
In order
to accomplish
thiscontrol
individual
tactile
pixel
independently
of
all
the
others.
In
order
to
accomplish
this
individual
control,
a
single
control, a single pin of a microcontroller is needed for each tactile element. Thus, in order to achieve
of a microcontroller
is needed
for each pins
tactile
element.
Thus, inas
order
a resolution
of
apin
resolution
of even 20 × 20,
400 individual
would
be required,
wellto
asachieve
400 individual
pulseeven 20
× 20, 400 individual
pins
would
be required,clocks.
as well as 400 individual pulse-width modulation
width
modulation
signals and
their
corresponding
signals
and
their
corresponding
clocks.
To solve these problems and achieve high resolution, we have proposed the resonant microbeam
To solve
thesedisplay
problems
and achieve
high resolution,
weof
have
the resonantvibrotactile
microbeam
vibrotactile
haptic
[15,16].
In this paper,
the concept
the proposed
resonant microbeam
vibrotactile
haptic
display
[15,16].
In
this
paper,
the
concept
of
the
resonant
microbeam
vibrotactile
haptic display is briefly reviewed. The following human touch experiments are presented: dried skin
haptic display
is briefly
The skin
following
human
touch experiments
arebeam
presented:
dried skin
contact,
wet and
soapedreviewed.
skin contact,
contact
to a constraint
and metal
tips, maximum
contact,force
wet measurement,
and soaped skin
skin contact
a constraint
metaltobeam
tips, beam
maximum
normal
andcontact,
skin contact
to silicontorubber
beamsand
attached
the metal
tips.
normal
force
measurement,
and
skin
contact
to
silicon
rubber
beams
attached
to
the
metal
beam
tips.
The forced response of a metal beam depending on the silicone rubber beam length is also presented.
The forced response of a metal beam depending on the silicone rubber beam length is also presented.
2. The Concept of the Resonant Microbeam Vibrotactile Haptic Display
2. The Concept of the Resonant Microbeam Vibrotactile Haptic Display
A resonant microbeam vibrotactile array is a mechatronic system that consists of two
A resonant
microbeam
vibrotactile
arraycontroller.
is a mechatronic
system
that
consists
two subsystems:
subsystems:
a beam
array and
a beam array
The beam
array
consists
ofofcantilever
beams
a
beam
array
and
a
beam
array
controller.
The
beam
array
consists
of
cantilever
beams
made
of a
made of a metal material, which has low damping ratio to reduce the crosstalk between each beam
metal
material,
which
has
low
damping
ratio
to
reduce
the
crosstalk
between
each
beam
vibration.
vibration. Each beam in the array is designed to have a unique length and/or cross-sectional area, so
Eacheach
beam
in the
is designed
to have a unique
length
and/or
cross-sectional
that
that
beam
canarray
be controlled
individually
by having
a unique
natural
frequency.area,
Theso
base
ofeach
the
beam
can
be
controlled
individually
by
having
a
unique
natural
frequency.
The
base
of
the
beam
array
beam array is attached to a surface transducer or a speaker that is actuated to vibrate, as shown in
is attached
Figure
1. to a surface transducer or a speaker that is actuated to vibrate, as shown in Figure 1.
Figure1.1.AAsurface
surfacetransducer
transducerproduces
producesaawave
wavew(t)
w(t)equal
equalto
tothe
thesum
sumof
ofindividual
individualamplitudes
amplitudesand
and
Figure
frequencies
for
each
beam
by
using
Fourier
transform.
In
this
example,
five
beams
are
being
actuated:
frequencies for each beam by using Fourier transform. In this example, five beams are being actuated:
threebeams
beamsat
atan
anamplitude
amplitudeof
of200
200and
andtwo
twobeams
beamsat
at an
an amplitude
amplitude of
of 100.
100.
three
A surface transducer is a device that turns almost any surface into a speaker. In a surface
transducer, there is no cone, and a coil is attached to a metal pad so that the vibration transmits the
pad into any subject it is attached to. In the proposed device, the forcing vibration from a surface
Multimodal Technol. Interact. 2019, 3, 38
3 of 13
A surface transducer is a device that turns almost any surface into a speaker. In a surface
transducer, there is no cone, and a coil is attached to a metal pad so that the vibration transmits the
pad into any subject it is attached to. In the proposed device, the forcing vibration from a surface
transducer excites the beam base. This approach uses the concept of ‘resonance’. When the frequency
of the vibration of the surface transducer matches the natural frequency of one of the beams, that beam
‘resonates’—vibrates with large amplitude. Since the natural frequency of each beam is unique, each
beam can represent a different pixel of an image.
For this approach, there are three important criteria: (1) minimizing natural frequency
(2) maximizing vibrational amplitude (3) using material having low damping ratio. Considering that
the general frequency area for people to feel static touch is less than 500 Hz [17], it is generally assumed
that lower frequencies are more perceivable than higher frequencies even though the perceivable
frequency range with dynamic touch is currently unknown. To improve human perception, maximizing
vibrational amplitude is important. Since the natural frequency of a beam is inversely proportional to
the amplitude of the beam in a lumped mass model of a beam, low frequency is required to maximize
human perception [15]. Therefore, the 1st vibration mode of a metal beam is used in this approach to
minimize the natural frequency and maximize the amplitude of a beam. Using material having low
damping ratio can reduce the bandwidth of beam vibration, which can make it theoretically possible
to make more beams in a given frequency range. To achieve the high resolution of the proposed
approach, as many beams as possible will be needed without the crosstalk of vibration between each
beam. In our previous research [15,16], a beam design simulation algorithm was developed with the
consideration of some important criteria mentioned above. It was found that nearly 13,000 beams
(96 × 128 resolution) can be made in a single display manufactured by the wire Electric Discharge
Machining (EDM) method.
Before moving forward for further development of this device, an important problem was found
that beam vibration stops when skin contacts beam tips to perceive beam vibration and no beam
vibration is perceived. This paper investigates the methods to reduce the friction occurred at the
beam tips where skin directly contacts to solve the user perception problem. Diverse skin contact
experiments with resonant tactile microbeams were conducted to identify cases where the metal beams
do not cease to vibrate.
3. Finger Contact Experiment
3.1. Dried Finger Contact Experiment
3.1.1. The Setup for Dry Skin Contact Experiment
In order for the proposed device to be usable for image perception, individual beam vibration must
be perceivable by a user contacting the beams with the skin. The first requirement for perceivability
is that the beams must continue to vibrate, while in contact with the skin. To investigate human
perceivability and determine whether beams continue to vibrate under skin contact, human skin
contact experiments are conducted on a small prototype device. For this experiment, a beam array
prototype having a total of 12 cantilever metal beams is selected, as shown in Figure 2. The beam array
is designed to have less than 1200 Hz as the resonant frequency range for all metal beams. Every beam
has the same length, 29.6 mm, so that the skin of a finger can easily contact all beam tips simultaneously.
The beam array is manufactured by wire EDM at Arizona State University. Beam number 1 represents
the leftmost beam, and beam number 12 represents the rightmost beam.
Multimodal Technol. Interact. 2019, 3, 38
Multimodal Technol. Interact. 2019, 3, x FOR PEER REVIEW
Multimodal Technol. Interact. 2019, 3, x FOR PEER REVIEW
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Figure
EDM.
Figure 2.
2. The
The image
image of
of 12
12 metal
metal beams
beams in
in aa row
row manufactured
manufactured by
by wire
wire EDM.
Figure 2. The image of 12 metal beams in a row manufactured by wire EDM.
The input of the surface transducer
transducer is
is connected
connected to
to the
the output
output of
of an
an amplifier
amplifier by
by USB
USB cable,
cable,
The
input its
of the
surface
is connected
to
the
output
of
an
amplifier
by
USB
cable,
which
receives
input
from transducer
a function generator.
The
amplifier
is
provided
with
a
constant
The amplifier is provided with constant 5 V
which
receives
its
input
from
a
function
generator.
The
amplifier
isaaprovided
with
a constant
5V
power source. The function
wave
specified
frequency’,
function generator is set
set to
to generate
generate aa sine
sine
wave of
of
specified‘starting
‘starting
frequency’,
powerissource.
The function
generator isthe
setnatural
to generate
a sine of
wave
a specified
‘starting frequency’,
determined
by approximating
frequency
the of
beam
from a lumped-mass
model
which
lumped-mass
which
is
determined
by
approximating
the
natural
frequency
of
the
beam
from
a
lumped-mass
model
based on
onthe
theactual
actualbeam
beam
dimensions.
amplitude
the amplifier
is adjusted
to aenough
small
dimensions.
TheThe
amplitude
dial dial
of the amplifier
is adjusted
to a small
based on
thethe
actual
beam
dimensions.
The2nd
amplitude
dial of
the
is adjusted
to A
a highsmall
enough
amount
that
the 1stdoes
beam
does
not
hit
the
2ndwhen
beam
when
the
1st resonates.
beam
resonates.
amount
that
1st beam
not
hit the
beam
the
1st amplifier
beam
A high-speed
enough
amount
that the
1stsobeam
does
not
2nd
beam
1st
beam
resonates.
highspeed
camera
is positioned
that
the
field
ofhit
view
is
parallel
the axis
of
the
metal
beams
toArecord
camera
is
positioned
so that
the
field
of
view
isthe
parallel
to thetowhen
axis
ofthe
the
metal
beams
to record
the
speed
camera
is
positioned
so
that
the
field
of
view
is
parallel
to
the
axis
of
the
metal
beams
to
record
the
beam
vibration
at the
moment
a finger
touches
beam
tips.
The
high-speed
camera
witha
beam
vibration
at the
moment
a finger
touches
thethe
beam
tips.
The
high-speed
camera
is is
fitfit
with
the
beam vibration
atpositioned
the
moment
a finger
touches
the beams
beam
Theinhigh-speed
camera
is camera
fit view
with
amicroscope
microscope
lens
and
positioned
so
that
or three
are focused
inhigh-speed
the high-speed
lens
and
so that
twotwo
or
three
beams
are tips.
focused
the
camera
a
microscope
lens
and
positioned
so
that
two
or
three
beams
are
focused
in
the
high-speed
camera
view
simultaneously.
A
frame
rate
of
8819
frames
per
second
is
used
for
video
recording
to
capture
simultaneously. A frame rate of 8819 frames per second is used for video recording to capture changes
view
simultaneously.
A frame
rate
of 8819touches
frames
per
second
is used
for video
to capture
changes
in
beam vibrations
when
atouches
finger
the
metal
beams.
Figure
3 shows
the experimental
in
beam
vibrations
when
a finger
the
metal
beams.
Figure
3 shows
therecording
experimental
setup
changes
in abeam
vibrations
when
a finger
touches
thepresses
metal
beams.
Figure
3 shows
the to
experimental
setup
high-speed
camera.
afinger
dried
finger
thetips
beam
tips
with
various
forces
see
with
awith
high-speed
camera.
First,
aFirst,
dried
presses
the
beam
with
various
forces
see ifto
there
setup
with
a
high-speed
camera.
First,
a
dried
finger
presses
the
beam
tips
with
various
forces
to
see
if
is a moment
when
thedoes
beam
is there
a moment
when the
beam
notdoes
stop.not stop.
if there is a moment when the beam does not stop.
Figure 3. The experimental setup with a high-speed camera.
Figure
Figure 3.
3. The experimental setup with a high-speed camera.
3.1.2. The Result of Dry Skin Contact Experiment
3.1.2. The Result of Dry Skin Contact Experiment
The recorded video files are checked with eye, and it is found that when the finger touches the
videooffiles
checked
withdecreases,
eye, and and
it is the
found
thatstops
when
the finger
touches
the
beamThe
tip, recorded
the amplitude
the are
vibrating
beam
beam
vibrating.
After
the skin
beam tip,the
thebeam
amplitude
of the
vibrating
beam decreases,
and the
beam
stops vibrating.
Aftervibration
the skin
contacts
tips, the
beam
stops vibrating
within about
6–10
milliseconds.
No beam
contacts the beam tips, the beam stops vibrating within about 6–10 milliseconds. No beam vibration
Multimodal Technol. Interact. 2019, 3, 38
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3.1.2. The Result of Dry Skin Contact Experiment
The recorded video files are checked with eye, and it is found that when the finger touches the
beam tip, the amplitude of the vibrating beam decreases, and the beam stops vibrating. After the skin
Multimodal
Technol.
Interact.
3, x FOR
PEERvibrating
REVIEW within about 6–10 milliseconds. No beam vibration
5 of 13
contacts the
beam
tips, 2019,
the beam
stops
is
perceived under dry skin contact conditions. Figure 4 illustrates the moment when a finger touches
is
perceived
under
skin
contact conditions.
Figure are
4 illustrates
moment
when
a finger
touches
the
beam tips.
Thedry
three
rectangular
parts vibrating
the first the
three
thinnest
metal
beams,
and a
the
beam
tips.
The
three
rectangular
parts
vibrating
are
the
first
three
thinnest
metal
beams,
and
a
white object above the three beams is the finger.
white object above the three beams is the finger.
Figure 4.
4. The
The image
image of
of aa dried
dried finger
finger pressing
pressing the
the beam
beam tips.
tips.
Figure
The reason for the beam vibration stopping may be the friction that occurs at the contact surface
The reason for the beam vibration stopping may be the friction that occurs at the contact surface
of beam tips and the skin. Here, the friction is the only force introduced by the presence of the skin
of beam tips and the skin. Here, the friction is the only force introduced by the presence of the skin
that is in the direction of the beam vibration (perpendicular to the beam axis), provided that the skin
that is in the direction of the beam vibration (perpendicular to the beam axis), provided that the skin
does not protrude below the surface of the beam tips. If the skin does protrude below the surface of
does not protrude below the surface of the beam tips. If the skin does protrude below the surface of
the beam tips, then that skin would exert a normal force in the direction perpendicular to the beam
the beam tips, then that skin would exert a normal force in the direction perpendicular to the beam
axis. However, from the video captured in this experiment, it is shown that the skin actually does
axis. However, from the video captured in this experiment, it is shown that the skin actually does not
not protrude down. This means that the friction is the only force that could stop the beam vibration.
protrude down. This means that the friction is the only force that could stop the beam vibration. The
The two ways to reduce friction are to reduce the coefficient of friction or to reduce the normal force
two ways to reduce friction are to reduce the coefficient of friction or to reduce the normal force
between the skin and the tops of the metal beams.
between the skin and the tops of the metal beams.
3.2. Wet and Soaped Finger Contact Experiment
3.2. Wet and Soaped Finger Contact Experiment
In the previous experiment with dry skin, it is revealed that the friction generated at the contact of
In the previous experiment with dry skin, it is revealed that the friction generated at the contact
beam tips and the skin is the probable reason for the beam vibration damping when the finger touches
of beam tips and the skin is the probable reason for the beam vibration damping when the finger
it. In this experiment, water and soap are used to reduce the coefficient of the friction.
touches it. In this experiment, water and soap are used to reduce the coefficient of the friction.
3.2.1. The Setup for Wet and Soaped Finger Contact Experiment
3.2.1. The Setup for Wet and Soaped Finger Contact Experiment
A previous study by O’Meara and Smith [18] reveals that the simple presence of soap and water is
A previous
study bydecrease
O’Mearathe
and
Smith [18]
the simple
presence
of soap
water
sufficient
to dramatically
coefficient
ofreveals
frictionthat
between
skin and
steel. The
sameand
setup,
as
is
sufficient
to dramatically
decrease
thesoaped
coefficient
oftouch
friction
between skin
and
steel. The
sametouches
setup,
shown
in Figure
3, is used for
wet and
finger
experiment.
First,
a soaped
finger
as
in Figure
3, is used
wet
soaped
finger touch
experiment.
First, a soaped
theshown
beam tips
in a vibrating
state,for
and
theand
vibration
response
is recorded
by a high-speed
camera.finger
Then,
touches
the
beam
tips
in
a
vibrating
state,
and
the
vibration
response
is
recorded
by
high-speed
a wet and soaped finger touches the beam tips, and it is also recorded by the high-speeda camera.
camera. Then, a wet and soaped finger touches the beam tips, and it is also recorded by the high3.2.2. The
result of Wet and Soaped Finger Contact Experiment
speed
camera.
In the observation of the recorded files with eye, it is still found that when the finger that is wet
3.2.2. The result of Wet and Soaped Finger Contact Experiment
or wet and soaped touches the beam tip, the amplitude of the vibrating beam decreases, and the
the observation
of thethe
recorded
files with
it istips,
stillthe
found
that
when
the finger
that is
wet
beamInstops
vibrating. After
skin contacts
theeye,
beam
beam
stops
vibrating
within
about
or
wetmilliseconds.
and soaped touches
beam tip,
the
amplitude
of the
vibrating
beamfinger
decreases,
andbeam
the beam
6–10
Figure 5the
illustrates
the
moment
when
a wet
and soaped
touches
tips.
stops
vibrating.
Afterpart
the isskin
beammetal
tips, the
beam
vibrating
within
aboutabove
6–10
The three
rectangular
the contacts
first threethe
thinnest
beams,
andstops
a white
object with
bubbles
milliseconds.
Figure
illustrates
the moment
the three beams
is the5 wet
and soaped
finger. when a wet and soaped finger touches beam tips. The
three rectangular part is the first three thinnest metal beams, and a white object with bubbles above
the three beams is the wet and soaped finger.
Multimodal Technol. Interact. 2019, 3, 38
Multimodal Technol. Interact. 2019, 3, x FOR PEER REVIEW
6 of 13
6 of 13
Figure 5. The image of a wet and soaped finger pressing the beam tips.
3.3. Finger Contact Experiment with Constraints
In this experiment, two constraints of acrylic panels, which have almost the same height as the
metal beam tips, are manufactured by a laser cutter to reduce the normal force contributing to the
friction. One of the U-shaped acrylic constraints has a height of 29.6 mm, and the other has a height
of 29.7 mm, as shown in Figure 6.
The image
image of
of a wet and soaped finger pressing the beam tips.
Figure 5. The
3.3.1.
The Setup
forExperiment
Finger Contact
Experiment with Constraints Setup
3.3. Finger
Contact
with Constraints
3.3. Finger Contact Experiment with Constraints
The
metal
beams oftwo
a beam
array are
in the channel
of thealmost
U-shaped
acrylic
constraint,
In this
experiment,
constraints
of located
acrylic panels,
which have
the same
height
as the
In this experiment, two constraints of acrylic panels, which have almost the same height as the
as
shown
in
Figure
7.
The
same
high-speed
camera
setup,
as
shown
in
Figure
3,
is
used.
Since
metal beam tips, are manufactured by a laser cutter to reduce the normal force contributing to the
metal beam tips, are manufactured by a laser cutter to reduce the normal force contributing to the
metal
beams
dothe
notU-shaped
vibrate when
the
U-shapedhas
constraint
them,
a the
clearance
is maintained
friction.
One of
acrylic
constraints
a heightcontacts
of 29.6 mm,
and
other has
a height of
friction. One of the U-shaped acrylic constraints has a height of 29.6 mm, and the other has a height
by
tape to6.fix the constraint to the beam base, so that the constraint does not touch
29.7using
mm,double-sided
as shown in Figure
of 29.7 mm, as shown in Figure 6.
the beams.
3.3.1. The Setup for Finger Contact Experiment with Constraints Setup
The metal beams of a beam array are located in the channel of the U-shaped acrylic constraint,
as shown in Figure 7. The same high-speed camera setup, as shown in Figure 3, is used. Since the
metal beams do not vibrate when the U-shaped constraint contacts them, a clearance is maintained
by using double-sided tape to fix the constraint to the beam base, so that the constraint does not touch
the beams.
The image of U-shaped constraint of Acrylic panels.
Figure 6. The
3.3.1. The
The Result
Setup for
Finger Contact
Contact Experiment
Experiment with
with Constraints
Constraints Setup
3.3.2.
of Finger
The beam
metal vibration
beams of is
a beam
arraybyare
located
intip
theofchannel
of the
U-shaped
constraint,
The
perceived
tapping
the
the metal
beams,
but it isacrylic
hard to
perceive
as shown
in beam
Figurevibration
7. The same
high-speed
setup,
shown
in Figure
3, is used. while
Since the
any
distinct
by just
touchingcamera
the beam
tips.asBeam
vibration
is perceived,
the
metal still
beams
do notthrough
vibrate when
the U-shaped
constraint contacts
clearance
is maintained
by
beam
vibrates
the finger
pressing (touching)
the beamthem,
very aweakly.
Even
though beam
using double-sided
tape
to fix the the
constraint
to and
the beam
base, very
so that
the constraint
not
touch
vibration
is perceived
by touching
beam tip
constraints
weakly,
this maydoes
not be
suitable
Figure 6. The image of U-shaped constraint of Acrylic panels.
the
beams.
for use of the device, since it is impossible to maintain the very weak touch with the beams. Another
problem
that there
is always
some
vibration with
transmitted
through the U-shaped constraint, and it
3.3.2.
TheisResult
of Finger
Contact
Experiment
Constraints
The beam vibration is perceived by tapping the tip of the metal beams, but it is hard to perceive
any distinct beam vibration by just touching the beam tips. Beam vibration is perceived, while the
beam still vibrates through the finger pressing (touching) the beam very weakly. Even though beam
vibration is perceived by touching the beam tip and constraints very weakly, this may not be suitable
for use of the device, since it is impossible to maintain the very weak touch with the beams. Another
problem is that there is always some vibration transmitted through the U-shaped constraint, and it
3.4.1. The Setup for Finger Force Measurement
A scale is used to measure the finger force. First, the surface transducer and the beam array with
the Acrylic constraint are placed on the scale to measure their weight without a finger press. Then,
the vibrating beam tip and constraint are pressed by a finger while the targeted beam vibrates, and
Multimodal Technol. Interact. 2019, 3, 38
7 of 13
the weight caused by the finger force including the whole weight of experimental device is measured,
as shown in Figure 7.
(a)
(b)
Figure 7. The
The image
image of
of finger
finger force
force measurement
measurement using a scale: (a)
(a) Total
Total weight of whole experimental
device without a finger press;
press; (b)
(b) Total
Total weight
weightof
ofwhole
wholeexperimental
experimentaldevice
devicewith
withaafinger
fingerpress.
press.
3.3.2.
The Result
Result of
of Finger
Finger Force
Contact
Experiment with Constraints
3.4.2. The
Measurement
The
beam
vibrationitisisperceived
bythe
tapping
the
tip of by
thethe
metal
beams, but
it isforce,
hard which
to perceive
In the
experiment,
found that
weight
caused
maximum
finger
does
any
distinct
beam
vibration
by
just
touching
the
beam
tips.
Beam
vibration
is
perceived,
while
the =beam
not stop beam vibration, is less than 1 gram. 1 gram is equal to 0.001 kg, which is 0.001 × 9.81
0.01
still
vibrates
the finger
(touching)continuously
the beam very
weakly.
Even
though
N. This
forcethrough
is too small
force pressing
to be maintained
when
people
touch
the beam
beam vibration
tips. It is
is
perceived
by touching
beam be
tipconsidered
and constraints
this maybased
not beon
suitable
for use of
found
that other
methodsthe
should
to aidvery
the weakly,
user perception
this experiment
the
device,
since
it
is
impossible
to
maintain
the
very
weak
touch
with
the
beams.
Another
problem
result.
is that there is always some vibration transmitted through the U-shaped constraint, and it interferes
with
recognizing
the beam vibration.
Moreover,
no beam vibration is perceived after the 4th beam,
3.5. Finger
Touch Experiment
with Silicone
Rubber Beams
which the resonant frequency is 612 Hz, even though the finger either presses the beam tip very weakly
It is found that a new method is needed to reduce the normal force generating from the direct
or taps the tip. There might be two possible reasons for the lack of user perception. One is that the
finger touch to the beam tips. For this, a material known as ‘dragon skin 10 very fast’, a type of silicone
amplitude of the 4th beam may be not enough for the user to perceive the vibration. Another reason is
rubber, is selected to make a silicone rubber beam, which is attached to a metal beam tip so that a
that the perceivable frequency range with dynamic touch may be similar to the one with static touch,
finger could only touch the tips of silicone rubber beams and the normal force of the finger would
which is known up to around 500 Hz. To figure out the exact reason, further research will be needed.
not be exerted on the metal beam tips directly.
3.4. Finger Force Measurement
3.5.1. The Setup for Finger Touch Experiment with Silicone Rubber Beams
It is found that with very weak finger touch to the metal beam tips and constraints, the beam
vibration can be perceived. In this experiment, the goal is to measure the maximum normal force that
does not stop beam vibration.
3.4.1. The Setup for Finger Force Measurement
A scale is used to measure the finger force. First, the surface transducer and the beam array with
the Acrylic constraint are placed on the scale to measure their weight without a finger press. Then,
the vibrating beam tip and constraint are pressed by a finger while the targeted beam vibrates, and the
weight caused by the finger force including the whole weight of experimental device is measured, as
shown in Figure 7.
3.4.2. The Result of Finger Force Measurement
In the experiment, it is found that the weight caused by the maximum finger force, which does
not stop beam vibration, is less than 1 gram. 1 gram is equal to 0.001 kg, which is 0.001 × 9.81 = 0.01 N.
This force is too small force to be maintained continuously when people touch the beam tips. It is found
that other methods should be considered to aid the user perception based on this experiment result.
Multimodal Technol. Interact. 2019, 3, 38
8 of 13
3.5. Finger Touch Experiment with Silicone Rubber Beams
It is found that a new method is needed to reduce the normal force generating from the direct
finger touch to the beam tips. For this, a material known as ‘dragon skin 10 very fast’, a type of silicone
rubber, is selected to make a silicone rubber beam, which is attached to a metal beam tip so that a finger
could only touch the tips of silicone rubber beams and the normal force of the finger would not be
exerted on the metal beam tips directly.
3.5.1. The Setup for Finger Touch Experiment with Silicone Rubber Beams
Multimodal Technol. Interact. 2019, 3, x FOR PEER REVIEW
8 of 13
A and B of ‘Dragon Skin 10 very fast’ are mixed and solidified. The solidified dragon skin is cut to
and B of ‘Dragon
10 very
fast’
are mixed
solidified.
solidified
dragon
is cut
makeArectangular
shapedSkin
silicone
rubber
beams.
Two and
silicone
rubber The
beams
are attached
to skin
the tips
of
to
make
rectangular
shaped
silicone
rubber
beams.
Two
silicone
rubber
beams
are
attached
to
the
two beams of the metal prototype with an ethyl cyanoacrylate adhesive. The length of silicone rubber
tips of two
beams
thetheir
metalthicknesses,
prototype with
an ethyl
cyanoacrylate
Thethe
length
of silicone
beams
is 11.7
mm,ofand
around
1.1mm,
are slightlyadhesive.
thicker than
thickness
of a
rubber
beams
is
11.7
mm,
and
their
thicknesses,
around
1.1mm,
are
slightly
thicker
than
the
thickness
metal beam. Since the silicone rubber beams are cut manually, their thickness is not uniform, as shown
of Figure
a metal8.beam.
Sinceexperimental
the silicone rubber
beams
areincut
manually,
their
thicknessthe
is not
uniform,
in
The same
setup as
shown
Figure
3 is used
to resonate
metal
beams as
to
shownsilicone
in Figure
8. The
sameare
experimental
setup
as shown
in Figure
3 is used
to resonate
metal
which
rubber
beams
attached, and
a finger
touches
the silicone
rubber
beam tip the
when
the
beamsvibrates
to whichinsilicone
rubber
beams
attached,
and aOnly
finger
touches
silicone
beam
tip
beam
order to
perceive
theare
beam
vibration.
two
metal the
beams
withrubber
silicone
rubber
when the
beam are
vibrates
in order
to perceive
the beam
vibration.
Onlyvibration
two metalwith
beams
beams
attached
resonated.
After
investigating
the 2nd
metal beam
the with
long silicone
silicone
nd metal beam vibration with the long
rubber beams,
beams attached
resonated.
investigating
rubber
the long are
silicone
rubberAfter
beams
are cut into the
the 2half
to investigate the difference of the
silicone
rubber
beams, the long silicone rubber beams are cut into the half to investigate the difference
2nd
beam
vibration.
nd
of the 2 beam vibration.
Figure
8. The
and and
two two
silicone
rubber
beams
for high-speed
camera
investigation.
Figure
8. prototype
The prototype
silicone
rubber
beams
for high-speed
camera
investigation.
3.5.2.
The Result
Result of
of Finger
Finger Touch
Touch Experiment
3.5.2. The
Experiment with
with Silicone
Silicone Rubber
Rubber Beams
Beams
The
metalbeam
beamvibration
vibrationcan
can be
be perceived
perceived by
by skin
skin contact
contact to
to the
the silicone
The 2nd
2nd metal
silicone rubber
rubber beam
beam
attached
to
the
2nd
metal
beam
after
cutting
the
long
silicone
rubber
beam
into
the
half.
However,
nd
attached to the 2 metal beam after cutting the long silicone rubber beam into the half. However,
when
beam vibration
vibration is
is observed
observed at
at around
around their
their original
original resonant
resonant
when the
the vibration
vibration test
test is
is conducted,
conducted, no
no beam
frequencies,
which were
After the
the silicone
frequencies, which
were used
used in
in the
the previous
previous skin
skin contact
contact experiments.
experiments. After
silicone rubber
rubber
beam
attached
to
the
2nd
metal
beam
(the
metal
beam
being
observed)
is
removed
from
the
beam,
nd
beam attached to the 2 metal beam (the metal beam being observed) is removed from the beam,
the
the
distinct
beam
vibration
is
observed
at
around
the
resonant
frequency.
This
shows
that
the
distinct beam vibration is observed at around the resonant frequency. This shows that the long
long
silicone
thethe
beam
vibration,
and and
the amplitude
of theof
beam
longthe
silicone
silicone rubber
rubberbeams
beamsabsorb
absorb
beam
vibration,
the amplitude
the with
beamthe
with
long
rubber
beams
becomes
smaller.
It
is
also
found
that
the
resonant
frequency
of
the
metal
beams
can
silicone rubber beams becomes smaller. It is also found that the resonant frequency of the metal
shift
bycan
attaching
siliconethe
rubber
beams
to the
beamtotips.
beams
shift bythe
attaching
silicone
rubber
beams
the beam tips.
Even
though
the
mechanical
properties
like
damping
Even though the mechanical properties like damping ratio
ratio between
between the
the silicone
silicone rubber
rubber beam
beam
and
metal
beam
are
different,
since
the
total
length
of
the
beams
increases
due
to
adding
and metal beam are different, since the total length of the beams increases due to adding the
the silicone
silicone
rubber
beams to
to the
the top
top of
of metal
metal beams;
beams; the
the resonant
resonant frequency
frequency range
range of
of the
the metal
metal beams
beams are
are shifted.
shifted.
rubber beams
For
example,
the
frequency
of
the
1st
metal
beam
is
511
Hz,
and
it
is
shifted
to
490
Hz
after
attaching
st
For example, the frequency of the 1 metal beam is 511 Hz, and it is shifted to 490 Hz after attaching
the
beams.
InIn
thethe
same
way,
thethe
resonant
frequency
of the
the silicone
silicone rubber
rubberbeams
beamstotothe
thetip
tipofofthe
themetal
metal
beams.
same
way,
resonant
frequency
of
the 2nd metal beam shifted from 569 to 525 Hz, the 3rd shifted from 612 to 580 Hz, and the 4th shifted
from 664 to 620 Hz.
Unfortunately, the bandwidth becomes broader after attaching silicone rubber beams. It is found
that cutting the silicon rubber with some increments can affect the frequency shift and the bandwidth
of the beam vibration.
Multimodal Technol. Interact. 2019, 3, 38
9 of 13
2nd metal beam shifted from 569 to 525 Hz, the 3rd shifted from 612 to 580 Hz, and the 4th shifted
from 664 to 620 Hz.
Unfortunately, the bandwidth becomes broader after attaching silicone rubber beams. It is found
that cutting the silicon rubber with some increments can affect the frequency shift and the bandwidth
of the beam vibration.
4. Vibration Response Depending on the Decrease of Silicone Rubber Beam Length
To better understand the effect of the silicone rubber beams on the metal beam bandwidth and
amplitude, further experiments are conducted. In this experiment, a total of 46 cases with the silicone
Multimodal Technol. Interact. 2019, 3, x FOR PEER REVIEW
9 of 13
rubber beam length changes are investigated, and 434 iterations of a high-speed camera experiment
are
are recorded
recorded in
in order
order to
to find
find the
the relationship
relationship between
between the
the dimensions
dimensions of
of the
the silicon
silicon rubber
rubber beams
beams and
and
the
the vibrational
vibrational response
response of
of the
the metal
metal beams.
beams.
4.1. The Setup for Vibration Response Depending on the Decrease Of Silicone Rubber Beam Length
4.1 The Setup for Vibration Response Depending on the Decrease Of Silicone Rubber Beam Length
Silicone rubber beams manually cut are attached to the first five thinnest metal beams because
Silicone rubber beams manually cut are attached to the first five thinnest metal beams because
these metal beams have larger vibrational amplitudes than the other metal beams. Only the 2nd
metal
these metal beams have larger vibrational amplitudes than the other metal beams. Only the 2nd metal
beam is resonated, and its vibration response is recorded. Since the resonant frequency of the 2nd metal
beam is resonated, and its vibration response is recorded. Since the resonant frequency of the 2nd
beam is different, depending on the length of the silicone rubber beams, the resonant frequency of the
metal beam is different, depending on the length of the silicone rubber beams, the resonant frequency
beam must be found with eye observation after cutting the silicon rubber beams with an approximate
of the beam must be found with eye observation after cutting the silicon rubber beams with an
1mm increment. Then, about ten frequencies including the resonant frequency of the metal beam alone
approximate 1mm increment. Then, about ten frequencies including the resonant frequency of the
are recorded with the high-speed camera to find the resonant frequency of the metal beam with the
metal beam alone are recorded with the high-speed camera to find the resonant frequency of the
silicone rubber beam attached. Figure 9 shows the silicone rubber beams attached to the first five
metal beam with the silicone rubber beam attached. Figure 9 shows the silicone rubber beams
thinnest metal beams.
attached to the first five thinnest metal beams.
Figure
9. The
silicone
rubber
beams
first five
thinnest
metal beams.
Figure 9. The
silicone
rubber
beams
attached
toattached
first fiveto
thinnest
metal
beams.
4.2. The Result of Vibration Response Depending on the Decrease of Silicone Rubber Beam Length
4.2. The Result of Vibration Response Depending on the Decrease of Silicone Rubber Beam Length
The recorded vibration response of the 2nd
metal beam is analyzed by an optical flow algorithm
The recorded vibration response of the 2nd metal beam is analyzed by an optical flow algorithm
implemented in Python. The vibration response of the 2nd
beam is analyzed depending on the length
implemented in Python. The vibration response of the 2nd beam is analyzed depending on the length
change of the silicone rubber beams. For example, Figure 10 shows the amplitude of the 2nd
metal
change of the silicone rubber beams. For example, Figure 10 shows the amplitude of the 2nd metal
beam depending on the forcing frequency provided by the surface transducer when the silicone rubber
beam depending on the forcing frequency provided by the surface transducer when the silicone
beam is 3.4mm long. It is shown that when the silicone rubber beam length is 3.4 mm, the 2nd beam’s
rubber beam is 3.4mm long. It is shown that when the silicone rubber beam length is 3.4 mm, the 2nd
resonant frequency is 498 Hz.
beam’s resonant frequency is 498 Hz.
The recorded vibration response of the 2nd metal beam is analyzed by an optical flow algorithm
implemented in Python. The vibration response of the 2nd beam is analyzed depending on the length
change of the silicone rubber beams. For example, Figure 10 shows the amplitude of the 2nd metal
beam depending on the forcing frequency provided by the surface transducer when the silicone
rubber
beam
is 3.4mm
long.3,It38is shown that when the silicone rubber beam length is 3.4 mm, the
2nd
Multimodal
Technol.
Interact. 2019,
10 of
13
beam’s resonant frequency is 498 Hz.
Figure 10. Amplitude of the 2nd beam when silicone rubber beam length is 3.4mm.
Figure 10. Amplitude of the 2nd beam when silicone rubber beam length is 3.4mm.
1.
2.
3.
4.
5.
6.
7.
8.
After all video files are analyzed, several observations are noted from this experiment:
After all video files are analyzed, several observations are noted from this experiment:
There are certain lengths of the silicone rubber beams for which the 2nd metal beam vibrates
with much lower amplitude. For example, when the silicone rubber beam length is about
10.3~10.7 mm, 5.6~6.7 mm, or 2.1 mm, the amplitude of the beam vibration is greatly decreased.
These lengths at which the metal beam vibration is decreased will be referred to here as ‘diminished
vibration lengths’.
Between two diminished vibration lengths, the amplitude of metal beam vibration becomes larger
and then becomes smaller after a diminished vibration length. Table 1 shows the amplitude
difference of the 2nd metal beam depending on the silicone rubber beam lengths.
The resonant frequency of the 2nd metal beam trends towards the resonant frequency of the beam
without the silicone rubber beam, as the silicone rubber beam length becomes shorter. However,
when the amplitude of vibration of the metal beam is very small due to the length of the silicone
rubber beam, the resonant frequency becomes very different than it was without the rubber beam.
When the silicone rubber beam length is longer than 10mm, the maximum amplitude and resonant
frequency of the metal beam cannot be determined, and when the silicone rubber beam length is
shorter than 2mm, the vibration of the beam becomes more difficult to perceive by touch.
Silicone rubber beams may absorb the vibration from other metal beams. For example, there
are 4 silicone rubber beams attached to the 2nd to 5th metal beams, and there is no silicone
rubber beam attached to the 1st beam. When a silicone rubber beam is attached to the 1st beam,
the amplitude of 2nd metal beam decreases significantly.
If the length of the 2nd rubber beam is different than the lengths of the other rubber beams, then
the frequency response of the 2nd metal beam is very different than it is when every rubber beam
has the same length.
Three experiment sets are performed to find the silicone rubber beam length that maximizes the
vibrational amplitude of the 2nd metal beam. In these three experiments, the optimal rubber
length is found to be 4.3, 4.6, 3.4 mm.
If the difference in length between the 2nd rubber beam and other rubber beams is small, and the
length of the 2nd rubber beam is around the length with the maximum amplitude, the vibration
result is similar with the case of all silicone rubber beams with the same length. For example,
if the difference in the length between the 2nd rubber beam and other rubber beams is around
0.6mm, and the length of the 2nd rubber beam is 3.4 mm, the maximum amplitude and resonant
frequency of the 2nd metal beam in both cases are about the same. But the bandwidth is smaller
in the case of every beam having same length of silicone rubber beams.
Based on these results, it is hypothesized that the silicone rubber beam has its own resonant
frequency, so when the resonant frequency of the silicone rubber beam is similar to the resonant
frequency of the metal beam, the amplitude of metal beam vibration is smaller. To verify this
Multimodal Technol. Interact. 2019, 3, 38
11 of 13
assumption, the resonant frequencies of the 2nd silicone rubber beam in 1st and 2nd mode vibration
are calculated, as shown in Table 2.
Table 1. The amplitude and resonant frequency of the 2nd metal beam difference depending on the
silicone rubber beam lengths in an experiment set.
Five Rubber Beam Lengths (mm)
Amplitude of the 2nd Metal
Beam (mm)
Resonant Frequency of the 2nd
Metal Beam (Hz)
(12.2, 12.2, 12.2, 12.2, 12.2)
(10.7, 10.7, 10.7, 10.7, 10.7)
(9.6, 9.6, 9.6, 9.6, 9.6)
(9.6, 8.5, 9.6, 9.6, 9.6)
(8.5, 8.5, 8.5, 8.5, 8.5)
(8.5, 7.6, 8.5, 8.5, 8.5)
(7.6, 7.6, 7.6, 7.6, 7.6)
(7.6, 6.7, 7.6, 7.6, 7.6)
(5.6, 5.6, 5.6, 5.6, 5.6)
(5.6, 4.6, 5.6, 5.6, 5.6)
(4.6, 4.6, 4.6, 4.6, 4.6)
(4.6, 4.0, 4.6, 4.6, 4.6)
(3.4, 3.4, 3.4, 3.4, 3.4)
0.0946
0.0276
0.0854
0.2490
0.2518
0.1509
0.1467
0.0437
0.0278
0.1505
0.1332
0.2601
0.2850
494
Not observable
480
496
495
502
502
505
Not observable
485
484
490
498
Table 2. The calculated resonant frequency of the 2nd silicone rubber beam in 1st and 2nd mode
vibration based on the lumped mass approximation.
The 2nd Silicone Beam
Lengths (mm)
The Resonant Frequency in the
1st Mode (Hz)
The Resonant Frequency in the
2nd Mode (Hz)
12.2
10.7
9.6
8.5
7.6
6.7
5.6
4.6
4.0
3.4
83
108
135
172
215
277
396
587
776
1074
523
680
845
1078
1348
1734
2482
3679
4866
6734
By comparison of Tables 1 and 2, it is found that when the resonant frequency of the 2nd silicone
rubber beam in the 1st or 2nd mode vibration is similar with the resonant frequency of the 2nd metal
beam, the metal beam vibration is not observable since the frequencies of two different materials are
compensated and offset. However, the calculated resonant frequency of the silicone rubber beam
is not exactly the same as the resonant frequency of the metal beam, which was obtained from the
experiment, and there is a frequency difference between them. There might be several possible reasons.
First, other silicone rubber beams attached to other metal beams could affect the resonant frequency
of the 2nd silicone rubber beam since all metal beams are connected by the beam base part. Second,
the size effect in micro-mesoscale depending on a silicone rubber beam length difference could affect
the resonant frequency of silicone rubber beams, which causes the frequency difference. In microscale,
the elastic modulus and damping ratio are changeable due to the size effect [19]. The further research
will be needed to figure out the exact reason for the frequency difference between the frequency of
the 2nd metal beam obtained from this experiment and the calculated frequency of the 2nd silicone
rubber beam.
In order to utilize silicone rubber beams as a compliant mechanism to increase beam vibration
during contact between metal beams and skin, the vibrational characteristics of the silicone rubber
beams will need to be incorporated into the metal beam design.
Multimodal Technol. Interact. 2019, 3, 38
12 of 13
5. Conclusion
In this paper, the haptic display using the resonant vibrotactile microbeam is presented. The concept
of the resonant vibrotactile microbeam haptic display is briefly reviewed. All the parameters are
considered under the three criteria: maximizing amplitude, minimizing frequency, and minimizing
damping ratio of the materials. To solve the micro-meso scale beam vibration stop problem by skin
contact, the human skin contact experiments are presented: dry skin contact, wet and soaped skin
contact, skin contact to a constraint and metal beam tips, normal force measurement, and skin contact
to silicone rubber beam tips attached to metal beam tips. In the skin contact experiment with silicone
rubber beams, beam vibration can be perceived when a finger contacts the silicone rubber beams that
is attached to the vibrating metal beam. This shows it is beneficial to attach silicone rubber beams to
the metal beam tips to solve the user perception problem, in which a vibrating beam stops due to the
normal force exerted by a finger when a finger contacts the beam tips. For more information about the
effect of the silicone rubber beams on the metal beam vibration depending on the length of silicone
rubber beams, forced response experiments for the metal beam are conducted by cutting the silicone
rubber beams with some increments.
Further research will be needed to analyze the relationship between beam vibration and silicon
rubber vibration numerically. This result can be used to define the efficiency of silicone rubber beam
attachment to the metal beam tips in order to solve the user perception problem.
Author Contributions: The authors have contributed equally to the research contained in this paper as follows:
conceptualization, D.W. and A.A.S.; methodology, D.W. and A.A.S.; software, D.W. and A.A.S.; validation,
D.W.; formal analysis, D.W.; investigation, D.W.; resources, A.A.S.; data curation, D.W.; writing—original draft
preparation, D.W.; writing—review and editing, D.W. and A.A.S.; visualization, D.W.; supervision, A.A.S.; project
administration, A.A.S..
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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| 9,169 | 56,587 |
W4309603060.txt | Pak Heart J 2022;55(Supplement1)
51st CARDIOCON 2022: ABSTRACT
COMPARATIVE ANALYSIS OF HEPATOTOXIC POTENTIAL OF
SIMVASTATIN WITH ROSUVASTATIN AND REVERSAL VIA
MONTELUKAST & COENZYME Q10 IN BALB/C MICE
Manzoor Khan1, Sarwat Jahan1, Amber Ashraf1
1
Cardiology Division, Department of Medical Specialities, KMC, KTH - MTI, Peshawar, Pakistan
Objectives: Statins cause elevations of AST and ALT, cholestatic hepatotoxicity,
fulminant hepatitis, autoimmune hepatitis, and cirrhosis. (2) Various mechanisms are
hypothesized including reduced levels of Coenzyme Q10, inflammation and oxidative
stress. Coenzyme Q10 is anti-apoptotic and possess antioxidative potential whereas
Montelukast can reduce the inflammation via inhibition of leukotrienepathway. Hence
both these drugs may have a protective effect against statin induced hepatotoxicity.
This study will include the comparison of hepatotoxic potential of simvastat in and
rosuvastatin and assess the hepatoprotective efficacy of montelukast and coenzyme 10.
Methodology: An experimental study of 2 weeks was conducted in the department of
Pharmacology on a sample of 35 mice, randomly divided into 7 groups of 5 mice each for
2 weeks. Group 1 was used as control. Group 2 received simvastatin 50mg/kg/day of
simvastatin intraperitoneally (I/P). Group 3 received I/P 50mg/kg/day rosuvastatin. Group
4 received 50mg/kg/day I/P simvastatin+3mg/kg of Montelukast. Group 5 received
50mg/kg/day of rosuvastatin +3mg/kg of Montelukast. Group 6 received 50mg/kg/day of
simvastatin+10mg/kg of coenzyme Q10. Group 7 received 50mg/kg/day of
rosuvastatin+10mg/kg of coenzyme Q10 I/P.
Results: Both the statins showed hepatotoxicity, however Simvastatin showed higher
liver function derangements. Montelukast did not reduce hepatotoxicity significantly.
A slightly lower rise in liver parameters was however observed. Coenzyme Q10 did
not show any protective potential against the statins, rather higher liver function
derangements were observed.
Conclusion: Simvastatin is more hepatotoxic as compared to Rosuvastatin. Motelukast
does have a slight protective effect but is not significant enough. Coenzyme Q10 was
not only not protective but infact indicated a hepatotoxic rend either by itself or via
potentiating of statin induced hepatotoxicity.
Keywords: Hepaotoxicity, Hepatoprotection, Oxidative stress, Inflammation
Citation: Khan M, Jahan S, Ahmad F, Ashraf A. Comparative Analysis of Hepatotoxic
Potential of Simvastatin with Rosuvastatin and Reversal via Montelukast & Coenzyme Q10 in
BALB/C
MICE.
Pak
Heart
J.
2022;55(Supplement1):S21.
https://doi.org/10.47144/phj.v55iSupplement1.2437
Corresponding Author: Manzoor Khan, Cardiology Division, Department of Medical Specialities, KMC, KTH MTI, Peshawar.
http://www. pakheartjournal.com
S21
| 368 | 2,798 |
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Comparative Study of Groundwater Pollution of M’nasra and
Sfafaa zones (Gharb, Morocco) by Nitrates
Najib EL KHODRANI*1,4, Serine OMRANIA2, Abdrrahim NOUAYTI3, Abdelmajid ZOUAHRI1, Ahmed
DOUAIK1, Hamza IAAICH1, Mbark LAHMAR1, and Mohammed FEKHAOUI4
1
Research Unit on Environment and Conservation of Natural Resources, Regional Center of Rabat, National Institute of
Agricultural Research (INRA),Rabat, Morocco.
2
Laboratory of Spectroscopy, Molecular Modeling, Materials, Nanomaterials, Waters and Environment, CERNE2D, Faculty
of Sciences, Mohammed V University, Rabat, Morocco.
3
Laboratory of Applied Geosciences, Faculty of Sciences, University Mohamed First Oujda 60000, Morocco.
4
Scientific Institute, Mohammed V University, Rabat, Morocco.
Abstract. The quality of water resources in Morocco is degraded by pollution from agriculture, industry and urbanization.
In this work we have realized a comparative study of two groundwater pollution zones: M’nasra (Kenitra) and Sfafaa (Sidi
Slimane). In general, the results of this study showed that most water wells are strongly mineralized as the conductivity far
exceeds the standard of drinking water. Sodium chloride (NaCl) concentration is between 33.9 and 1184.6 mg / l and shows
some of the wells are loaded with salts. The pH is generally basic in all wells. Pollution by nitrates two regions showed that
the ground water is strongly affected M’nasra zone terms of the permeability of the soil of the city. The concentration of
nitrate can reach high values compared to the results obtained on the ground water at Sfafaa zone whose nitrate
concentrations around 20 mg/l. Further pollution of groundwater in the two regions of the shows values of organic matters
that go far beyond the standards of drinking.
1 Introduction
Groundwater contamination by nitrates (NO3-)
represents
a
massive
environmental
problem. From today, NO3- is the most ubiquitous
groundwater
contaminant
in
the
world
and agricultural activities are mainly blamed for
these problems. Groundwater is a strategic water
source for human activities. In addition to being
often of large volumes, they generally provide
better quality water than surface water. These
waters constitute 23% of the planet's freshwater
resources [1] which must be conserved and
protected from all kinds of pollution.
In fact, the natural levels of nitrates in groundwater
are generally very low (typically less than 10 mg/l),
but their concentrations increase with human
activity such as agriculture (nitrogen fertilizers),
domestic effluents, leachates from landfills, etc.
This increase is the main cause of degradation of
groundwater quality and mainly of more vulnerable
water tables.
*
In general, nitrates move relatively slowly in soil
and groundwater: there is a lag time of
approximately 20 years between pollution activity
and detecting the pollutant in groundwater. For this
reason, it is anticipated that routine pollution
activities will continue to affect nitrate
concentrations for several decades. However, if the
pressure in the aquifer is high, transport can be very
fast in the saturation zone.
In the Gharb region of Morocco, nitrate
concentration in some wells reach 130 mg/l due to
intensive agricultural practices [2]. Vulnerable
aquifers are frequently located in zones of high
water demand, such as coastal areas (Algeria, Italy,
France, Morocco, Tunisia, etc). Besides, irrigated
farming accounts for a large share of total water
withdrawals: 83% in Greece, 68% in Spain, 57% in
Italy, and 52% in Portugal [3]. The drip irrigation
system is more and more encouraged by the
Moroccan government via 80% of subvention. The
mismanagement of irrigation can result in water
misuse and also in strong negative impacts on the
environment, such as nitrate pollution and
eutrophication.
Corresponding author: najibelkhodrani@gmail.com
© 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/).
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The objective of this work is to evaluate and
compare the groundwater pollution of two zones of
M'nasra and Sfafaa (plain of Gharb) by nitrates.
This study will subsequently raise the awareness of
leaders to implement a plan of action to minimize
the vulnerability of groundwater in Morocco.
Water samples were collected in 1l clean plastic
bottles and stored in the freezer (-10°C) until their
analysis.
Geology
Concerning the pedo-landscape, our study zone is a
part of the Vertisol domain of the Gharb region,
with alluvial deposits from the Quaternary, mainly
of the Late Pleistocene and Holocene.
In general, the Gharb plain is a depression between
two major structural domains: the Primary Meseta
to the South and the Rif mountains to the North and
the East. This plain is mainly marked by geological
subsidence of the Middle and Late Miocene. The
Gharb load is contemporary to the Rif mountains
uprising, which caused enormous landslides. The
East of the Gharb plain, corresponding to our area
of study, is an accumulation zone of Quaternary
continental sediments, alternately coarse (cobble
and gravel) and fine (silts and clays), following
climate cycles. The distribution of these deposits is
very heterogeneous both laterally and vertically.
Description of the study area
Geographic location
The surface area of the Gharb region is 600 Km2.
The region of M’nasra is dominated by sandy and
clayey soils. The climate is Mediterranean with
annual precipitations ranging between 480 and 600
mm, and the average temperature is 27°C in
summer and 13°C in winter (Office Régional de
Mise en Valeur Agricole du Gharb, 2017). Eighteen
water samples were collected from 18 wells (less
than 50 m depth) installed in 19 sites located on the
coastal zone of M’nasra in Gharb region, Northwest
of Morocco (Figure. 1). The levy has been carried
out in the periods the summer during 2017.
The Sfafaa zone belongs to the Province of Sidi
Slimane, located around the geographic coordinates
34° 15′0'' and 6° 9′36''. It is limited to the North by
the Province of Sidi Kacem , in the South-east by
the rural commune of Boumaiz (Province of Sidi
Slimane), and to the West by the rural commune of
Kecybia (Province of Sidi Slimane). The zone of
Sfafaa extends over approximately 197km2. The
samples of water wells were collected during the
summer2014.The mean
annual precipitation of 386.4 mm. We have
prospected 17 wells, belonging to the Sfafaa zone.
(a) Piezometric map of the M'nasra zone.
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(b) Piezometric map of the Sfafaa zone.
Fig. 1. Piezometric maps of the two regions chosen for the study.
The main crops groun in the study area are field
crops, vegetable crops (potatoes, tomatoes, etc)
wheat etc (M'nasra and Sfafaa).
Table 1 summarizes the main crops and fertilizer
doses applied at the national level [4].
Table 1. Applied Fertilizer Doses for Main Crops
Crops
Area
(Ha)
IRRIGATED
* Sugar Crops
33.423
7.400
+ Sugar Cane
+ Sugar Beet
* Cereals
5.200
2.200
5.556
* Vegetable
8.265
Quantity of fertilizer
(Kg/Ha)
P2O5
K2O
N
Frequency
(%) *
N
P2O5
K2O
5.853,82
2.949,87
3.274,3
175-200
155
67-97
55
126
42-56
35-102
63
21-28
100
-
910-1.040
341
372,2-539
286
277,2
233,4-311,1
182-530,5
138,6
116,6-155,7
216,8-556,8
805-1.631,4
259,8-641,6
99
111,2-138,8
283,4-396,7
136-693,3
162,4-227,4
46,2
97,5-192,4
428-623,5
293-951,6
113,7-251,7
23
153,9-188,1
1.263,1-1.977,1
878,7
103,6-253,3
86,35-103,6
933,6
180,4-184,2
+ Industrial tomato
+Potato
+ Strawberry
+Melon -Watermelon
+ Niora
1.417
2.719
1.624
1.650
8.55
153-400
296-600
160-400
60
130-160
200-250
50-255
100-140
28
114-225
288-440
110-350
70-155
14
180-220
100
-
*Peanut
* Citrus
10.283
1.919
115-180
54-132
0-80
45-54
0-85
94-96
-
RAINFED
99.252
* Sugar Crops
2.075
155
126
63
100
* Cereals
* Leguminous
59.067
7.744
60-67
14
28-42
28
14-21
14
60
10
* Forages
* Vegetable (Melon)
* Sunflower
* Olive tree
9.376
6.092
6.847
8.051
60
60
60
14
28
28
28
28
14
14
14
14
30
60
60
10
Total Area
Total fertilizer quantity
(Tonne)
132.675
(%)*: Frequency of use by farmers.
3
3.227,4
1.841,64
907,24
321,6
261,45
130,72
2.126,4-2.374,5 992,3-1.488,5 496,2-744,2
10,85
21,7
10,85
168
219,3
246
11,2
78,4
102,34
114,8
22,54
39,2
51,17
57,4
11,2
9.131,22
4.791,51
4.181,54
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2 Material and Methods
2.1 Characteristics of groundwater sampling points
The present study is conducted on a network of 35
stations spread over two regions in the Gharb Plain
M'nasra (Kenitra) and Sfafaa (Sidi slimane) (Figure
2). The stations were chosen according to their
representativity in relation to the urban perimeter of
the two cities, agricultural zones (M'nasra and
Sfafaa) and their proximity to the sources of
pollution (industrial zones or Ouled Berjal waste
discharge). The geographical distribution is as
follows:
- PM12, PM13, PM14 and PM15 stations located in
the North East of the M'nasra zone (upstream of
Sebou).
- PS1, PS2, PS3, PS4, PS5, PS6, PS7, PS8, PS9,
PS10 and PS11 stations located in the center of the
Sfafaa zone.
- PS12, PS13 and PS14 stations located upstream of
Oued Bhet (Sidi Slimane).
- PS15, PS16 and PS17 stations
downstream of Oued Bhet (Sidi Slimane).
-PM1, PM2, PM3, PM4, PM5, PM6, PM11 and
PM16 stations located between the Sebou River and
the Atlantic Ocean.
- Stations PM7, PM8, PM9, PM10, PM11, PM17,
PM18 located after the public landfills (Ouled
Berjal).
Fig. 2. Map of the M’nasra and Sfafaa zones with the sampling points.
4
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2.2 Sampling and laboratory analysis
retains. The PCA was performed in this study using
SPSS 20.
The measurement of electrical conductivity (EC)
and hydrogen ion concentration (pH) were
performed in situ using the Jenway 3510 pH-meter.
Chloride (Cl-) was measured by the titrimetric
method. Nitrates (NO3-) was determined using
Jasco V-530 UV/VIS spectrophotometer. Sodium
(Na+) was determined using the Jenway Clinical
PFP 7 flame photometer and oxidizability by
KMnO4.Those measurements were performed
according to the AFNOR standards [5].
3 Results and discussion
3.1 Parameters Physicochemical
Human activities exercise much polluting pressures
on surface water, then deferred to the water table.
This deterioration in the quality of certain nitrate
wells in the aquifer could be attributed to point and
dispersal releases of livestock products and
wastewater that has not been previously treated.
Certain factors seem to favor this contamination
such as the shallow depth, the permeability of the
soil, the speed of infiltration, the proximity of
public landfills, industrial zones, etc. Deep aquifers
indirectly bound to the soil surface are therefore
better protected against nitrate leaching, in contrast
to shallow aquifers, which are more vulnerable
because of their shallow depth which facilitates
contact between leached nitrates and groundwater
[8]. Contamination of the water table could cause
many diseases in the population using groundwater
as a source of drinking water. The effects on human
health of acute poisoning by nitrates are now
known, which is not the case of long-term effects.
2.3 Statistical analysis
First of all, a matrix of Pearson correlation
coefficients between any two variables was
computed and the coefficients were tested for their
statistical significance. We then used Principal
Component Analysis [6], a multivariate analysis
technique that provides an excellent means for
gaining useful information from data sets with
many variables [7]. In particular, PCA can aid in
the compression and classifcation of data. The
purpose is to reduce the dimensionality of a data set
by finding a new set of variables, smaller than the
original set of variables, which nonetheless retains
most of the sample’s variance. Success relies on the
presence of correlations among at least some of the
original variables; otherwise, the number of new
variables will be almost the same as the number of
original variables. The new variables, called
principal components, are uncorrelated, and are
ordered by the fraction of the total variance each
The results obtained showed that the pH varies
between 7.4 and 8.1 for the aquifer of the M'nasra
zone, whereas for the 17 wells in the Sfafaa zone,
the pH admits a minimum value of 6.6 and 7.4 as
the maximum value. The pH values thus measured
for the two zones chosen for the study are safe for
the consumer (Figure 3).
Fig. 3. Variation of pH values.
Electrical conductivity is used to evaluate the rate
of mineralization of the environment. The higher
the conductivity, the more water is strongly
mineralized. The results of this study show that
some wells in the Sfafaa zone (PS16, PS15 and
PS12) are highly mineralized. The values are
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between 370 and 6110 μS / cm. In addition, the
groundwater conductivity at the M'nasra zone
shows a maximum value of 1269.5 μS / cm. At the
Sfafaa zone, well water is mainly used for irrigation
in the region. This is accompanied by a high
salinity, in particular the PS16 well which has a
concentration of 1184.62 mg / l in NaCl (Figure 4)
while the other wells have concentrations varying
between 33.93 and 477.95 mg / l. In addition, the
salinity for the wells in the M'nasra zone varies
between 53.31 and 319.4 mg / l.
Fig. 4. Variation in the concentration of NaCl.
ingestion, could increase the risk of cancer and fetal
malformation (teratogenicity) in humans [1011].The results obtained for the nitrate analysis for
the two study zones showed that the concentrations
for 18 sampling points in the M'nasra vary between
49.6 and 198.4 mg / l whereas those detected at the
wells of the Sfafaa zone range from 0 to 40.9 mg / l
(Figure 5).
3.2 Nitrate pollution
Domestic and urban discharges contribute locally to
nitrate contamination of aquifers, but fertilization
by the application of chemical or organic nitrogen
fertilizers and intensive animal husbandry (animal
waste) constitute the main cause.
Excess nitrates in soil are driven every year a little
deeper, mainly by winter rains. The rate of
migration of nitrates to the aquifers is slow, on
average 1 to 2 m / year, but varies according to the
nature of the subsoil. The excess of nitrogen spread
on the surface therefore takes on average 10 to 20
years to reach a water table 20 m deep [9]. On the
surface of the soil, organic and mineral elements
alter cycles of water, carbon and nitrogen on a
global scale. These transformations involve
multiple risks to human health, aquatic spaces,
biodiversity and agricultural productivity. The
challenge for agriculture is therefore to produce
more respectfully less using water resources, with
respect for biodiversity and greenhouse gas
emissions [10].
For the M'nasra zone, it is noted that the wells near
the Ouled Berjal landfill and the Kenitra industrial
zone are loaded with nitrates with values that go
well beyond drinking water standards. However, all
the wells in the Sfafaa zone, far from pollution
sources, have nitrate concentrations that are below
the WHO standard (50 mg / l) [10].
However, in the highly fissured geological
formations, the rains reach the aquifer in a few
hours to a few months, this is the case of the
M'nasra zone whose soil has cracks, whereas in the
less permeable formations it is will take several tens
or even hundreds of years.
Nitrates and nitrites are not carcinogenic if they do
not exceed WHO standards, but their possible
transformation into N-nitroso compounds in water
or food before ingestion, or in the body after
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Nitrogen inputs, if they are higher than plant
exports, represent surpluses likely to flow to rivers
and aquifers. In 2010, nitrogen surpluses ranged
from 5 to 145 kg / ha of utilized agricultural area
(UAA), with an average of 32 kg / ha [4]. In
addition, it has been shown that the risk of pollution
of the Tadla water table is aggravated, for vegetable
crops, by the regular application of manure at doses
up to 80 T / ha [12]. This shows that the
mismanagement of fertilizer products can
contribute to the increase in groundwater pollution
and subsequently the infiltration of soluble
substances not retained by vegetation: excess cases
in relation to its needs. The regions most affected
by nitrogen pollution were those of intensive
farming with a high load of organic nitrogen due to
animal effluents, those of heavily fertilized field
crops and those with shallow water resources
(basement areas with low permeability basements,
alluvial aquifers, karst aquifers) [13].
Fig. 5 . Variation of nitrate concentration.
Although, the water table in the M'nasra zone has
higher levels compared to the Sfafaa zone, the
nitrate pollution is high this may be due to
contamination by the largely developed industrial
activities in the city of Kenitra, but also by
leachates from the city's uncontrolled Ouled Berjal
landfill[14].
deficit. The city of Larache landfill is a typical
model of deterioration of water resources through a
leachate contamination from the landfill and
agricultural practices [19].
The results obtained on the pollution of the water
table of the city of Taza showed a more or less
strong degradation especially downstream of the
city discharge [20]. The analysis of ground and
surface waters revealed that nitrate ions have a
mean concentration of less than 50 mg / l with
temporal variations of remarkable amplitude
(standard suggested by WHO) [14,21].
The vulnerability of groundwater depends mainly
on the countries and regions because in reality the
essential variable to be considered is the renewable
resource and, that is to say the volume that
recharges the groundwater each year [15]. In
addition, the results of the physicochemical analysis
in the leachate of several discharges have shown a
high contamination of organic salts and heavy
metals at Egypt [16]. In addition, it has been shown
that in Romania landfills can represent potential
sources of pollution for surface water and
groundwater [17, 18]. It is for this reason that the
development of new disposal areas is of utmost
importance. Leachate percolation from landfill
waste is a source of pollution of groundwater
resources by infiltration, thus constituting a threat
as important as that related to the increasing water
Organic matter by oxidizability by KMnO4
This parameter is used to evaluate the organic
matter content in the well water. In fact, the organic
matter present in the wells varies considerably from
one well to another. In the well waters of M'nasra
zone, it varies between 2.2 and 10.6 mg / l whereas
those in the Sfafaa zone have a minimum of 0 mg /
l and a maximum of 9.9 (Figure 6). However, these
values show that 57.1% of well water cannot be
used as a source of drinking water.
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Fig. 6 . Variation of Organic Matter.
3.3 Multivariate statistical analysis of
physicochemical parameters
Physicochemical parameters (variables) and water
points (individuals) were projected separately in the
factorial plane (PC1xPC2) of the principal
component analysis (PCA) to better understand
their representative distributions with respect to the
factorial axes. The interpretation of the statistical
analysis by the PCA has been done only on two
axes PC1 and PC2 because they alone contain more
than 70% of the information. PC1 axis presents
47.8% and the PC2 axis presents 23.4% of the total
inertia (Figure 7). The PCA allowed us to
characterize 2 groups of chemical elements each
corresponding to a set of groundwater samples with
the same characteristics.
A
C
B
Fig. 7 .Loading plot for the principal component analysis: groundwater properties in the PC1–PC2 plan.
Groups A: This group consists essentially of NaCl
and the electrical conductivity (EC) opposed to pH
and nitrate along the PC1 axis. These parameters
correspond to the set of wells consisting of PS12,
PS15 and PS16 (Group 1) and which has a very
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high conductivity and salinity and very low pH,
nitrates (Figure 7).
This group is formed by organic matter. It is placed
in negative position on the PC2 axis. This setting
characterizes the PS1, PS2, PS3, PS4, PS5, PS6,
PS7, PS8, PS9, PS10, PS11, PS13, PS14, and PS17
wells (Group 2).
Groups B:
1
2
Fig. 8 . Score plot for the principal component analysis: water samples in the PC1–PC2 plan.
In order to avoid ecological and health problems, it
is necessary to take concrete measures to limit the
pollution of the Gharb water table (M'nasra) and to
protect the health of the population in rural areas.
4. Conclusion
The results obtained have shown that parameters
such as salinity, conductivity, and nitrates far
exceed drinking water standards. The concentration
of nitrates in the well waters of the M'nasra zone
varies around 150 mg / l whereas those detected in
the wells of the Sfafaa zone are close to 20 mg / l.
Based on investigations and analysis, some
suggestions for solving the problem, such as best
management practices for nitrogen fertiliser and
improving environmental awareness, are discussed.
Groundwater in the M'nasra zone is more
contaminated with nitrates than Sfafaa. This
pollution detected at the level of the wells is mainly
due to their situations in relation to the landfill and
also in relation to the industry. In addition, the
waters of the Sfafaa zone are more saline compared
to the waters of M'nasra, especially the PS16 point
which is downstream of Oued Beht. To avoid
pollution of the water table, it is necessary to strike
a balance between the misuse of nitrates sources
and the total prohibition of them. With regard to
organic matter determined by KMnO4 oxidability,
57.1% of well water cannot be used as a source of
drinking water.
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10
| 4,074 | 25,661 |
W2997281907.txt | International Electronic Journal of Algebra
Volume 27 (2020) 218-236
DOI: 10.24330/ieja.663075
INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH
THREE
Gabriella D’Este
Received: 2 May 2019; Revised: 8 October 2019; Accepted: 21 October 2019
Communicated by Christian Lomp
Abstract. We investigate a particular class of indecomposable modules of
length three, defined over a K–algebra, with a simple socle and two non isomorphic simple factor modules. These modules may have any projective dimension different from zero. On the other hand their composition factors may
have any countable dimension as vector spaces over the underlying field K.
Moreover their endomorphism rings are K–vector spaces of dimension ≤ 2.
Mathematics Subject Classification (2010): 16G20, 16D99
Keywords: Pseudo projective module, quasi projective module, quiver and
representation
1. Introduction
The aim of this note is to construct left R–modules M , where R is a K–algebra
over some field K, with the following properties:
(1) M has exactly three non zero proper submodules N1 , N2 and N1 ∩ N2 .
(2) M /N1 is not isomorphic to M /N2 .
(3) End M /Ni is isomorphic to K for i = 1, 2.
Given a module M , we say that M is quasi projective (resp. pseudo projective) if,
for any submodule X of M , any morphism (resp. any epimorphism) f ∶ M → M /X
is of the form π ○ g, where π ∶ M → M /X is the canonical epimorphism and g is an
endomorphism of M . By [4, Lemma 2.1] any module M satisfying (1) and (2) is
not quasi projective. Moreover, if (1) and (2) holds, then M is pseudo projective if
and only if it satisfies (3) with K = Z2 . In the sequel we shall say, for short, that M
satisfies (123) if M satisfies conditions (1), (2) and (3). If x is a vertex of a quiver Q,
and R is the K–algebra given by Q, then x also denotes the simple module (Vi , fj ),
with Vx = K, Vi = 0 for i ≠ x and fj = 0 for any j. Moreover, P (x) and I(x) denote
the projective and injective module corresponding to the vertex x. In the examples
constructed in [4] R is always a K–algebra given by quivers with at least 2 vertices
and at most 4 vertices, and the modules satisfying (123) are three dimensional
INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE
219
vector spaces. In this note we consider algebras given by quivers with n vertices
for any n ≥ 1. By making one point extensions or coextensions, or by changing the
relations, we obtain modules satisfying (123) of any injective dimension (Proposition
2.1) and of any projective dimension ≥ 1 (Proposition 2.2). We also show that
modules satisfying (123) are very far from being selforthogonal (Examples 2.3, 2.4
and 2.5), and that they cannot be faithfully balanced (Proposition 3.8). Moreover
their endomorphism ring is a rather small commutative algebra (Theorem 3.6).
As we shall see, the free algebra K < x, y > admits modules of dimension three
satisfying (123) (Example 2.5, Proposition 2.6, Corollaries 3.10 and 3.11). On the
other hand, the free algebra K < x, y, z > admits modules satisfying (123) of any
countable dimension ≥ 3 (Theorem 3.2). Finally, suitable matrix algebras admit
modules satisfying (123) of any infinite dimension over K (Proposition 3.4 and
Corollary 3.5).
This paper is organized as follows. In Section 1 we recall some definitions, we
fix some conventions and we describe the main results. In Section 2 we investigate
homological dimensions and extensions of modules satisfying (123). Finally, in Section 3, we describe composition factors and endomorphism rings of these modules.
For more background on generalizations of projective modules we refer to [3] and
[5]. For more background on quivers and their representations we refer to [1] and
[2].
2. Homological dimensions and extensions
We begin with two results on homological dimensions.
Proposition 2.1. Any d ∈ N ∪ {∞} is the injective dimension of a module M
satisfying (123).
Proof. If d = 0, 1, ∞, then the assertion follows from [4, Example 2.3 and Theorem
2.8]. Assume now 2 ≤ d < ∞. Let n = d + 3 and let R be the K-algebra given by the
220
GABRIELLA D’ESTE
following quiver with relations ab = 0 for all arrows a and b.
●
n
●
●
●
●
1
2
4
●
3
Next let M be the module
0
/ M
12
3
. Then the injective coresolution of M is
/ 5
/ 124
3
4
/ ...
Hence n − 3 = d is the injective dimension of M .
/
n
n−1
/ n
/ 0 .
Proposition 2.2. Any d with 1 ≤ d ≤ ∞ is the projective dimension of a module
M satisfying (123).
Proof. The modules
12
3
and
12
2
in [4, Examples 2.3 and 2.4] have projective
dimension 1 and ∞, respectively. Assume now 1 < d < ∞ and let n = d + 2. Next
let R be the K–algebra given by the following quiver with relations ab = 0 for all
INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE
221
arrows a and b.
1
2
●
●
3
●
4
●
●
n
Let M be the module I(3) =
/ n−1
/ n
0
12
3
. Then the projective resolution of M is
/ ...
n
/ 3
4
/ M
/ 1 ⊕ 2
3
3
/ 0 .
Consequently the projective dimension of M is n − 2 = d.
Propositions 2.1 and 2.2 suggest that we investigate the groups of selfextensions
of the modules satisfying (123).
Example 2.3. There is a module M satisfying (123) such that Extn (M, M ) ≃ K
for any n ≥ 1.
Construction. As in [4, Theorem 2.8], let R be the K–algebra given by the quiver
a
●
1
/
●
d
b
with relations ba = 0 and b3 = 0. Next let M be the module
2
Then I(1) = 1 and I(2) =
12
2
.
2
12
are the indecomposable injective modules. From
2
the exact sequence 0 → M → I(2) → 2 → 0 we obtain the long exact sequence
0
0 Ð→ Hom(M, M ) Ð→ Hom(M, I(2)) Ð→ Hom(M, 2) Ð→ Ext1 (M, M ) Ð→ 0 Ð→
≃
≃
. . . Ð→ 0 Ð→ Exti (M, 2) Ð→ Exti+1 (M, M ) Ð→ 0 . . . . . . .
Consequently we have
Ext1 (M, M ) ≃ Hom(M, 2) ≃ K
(1)
and
Extn+1 (M, M ) ≃ Extn (M, 2)
for any
n ≥ 1.
(2)
222
GABRIELLA D’ESTE
2
On the other hand, from the exact sequence 0 Ð→ 2 Ð→ I(2) Ð→ 1 ⊕
2
Ð→
0, we obtain the long exact sequence 0 Ð→ Hom(M, 2) Ð→ Hom(M, I(2)) Ð→
2
Hom (M, 1 ⊕
) Ð→ Ext1 (M, 2) Ð→ 0 . . . Ð→ 0 Ð→ Exti (M, 1 ⊕
2
2
2
) Ð→
Exti+1 (M, 2) Ð→ 0 Ð→ . . . .
Hence, comparing dimensions and using the fact that 1 is an injective module,
we have
Ext1 (M, 2) ≃ K
(3)
and
Extn+1 (M, 2) ≃ Extn (M,
2
2
)
for any
n ≥ 1.
(4)
Combining (2) and (3), we get
Ext2 (M, M ) ≃ K.
Finally, from the exact sequence 0 Ð→
2
2
the long exact sequence 0 Ð→ Hom (M,
2) Ð→ Ext1 (M,
2
2
(5)
Ð→ I(2) Ð→ 1 ⊕ 2 Ð→ 0, we obtain
2
2
) Ð→ Hom(M, I(2)) Ð→ Hom(M, 1 ⊕
) Ð→ 0 Ð→ . . . Ð→ 0 Ð→ Exti (M, 1 ⊕ 2) Ð→ Exti+1 (M,
2
2
) Ð→
0 → . . . . Therefore we have
Ext1 (M,
2
2
)≃K
(6)
and
Extn+1 (M,
2
2
) ≃ Extn (M, 2)
for any
n ≥ 1.
(7)
Putting (2), (4) and (6) together, we get
Ext3 (M, M ) ≃ K.
(8)
Combining (4) and (7), we obtain
Exti+2 (M, 2) ≃ Exti (M, 2)
for any
i ≥ 1.
(9)
This result and (2) imply that
Exti+3 (M, M ) ≃ Exti+1 (M, M )
for any
i ≥ 1.
(10)
Putting (1), (5), (8) and (10) together, we conclude that Extn (M, M ) ≃ K for any
n ≥ 1.
As the next examples show, we may have Ext1 (M, M ) ≠ 0 also when M is defined
over hereditary algebras.
INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE
223
Example 2.4. There is a module M satisfying (123) such that dim Ext1 (M, M ) =
2.
Construction. Let R be the infinite dimensional K–algebra given by the quiver
a
:
●
1
b
/
●
. Let M be the module described by the following picture
2
8
a
v1
v2
●
●
b
b
●
v3
where {v1 , v2 , v3 } is a basis of M and av1 = v1 , av2 = 0, bv1 = v3 = bv2 . Then
Rv1 , Rv2 and Rv1 ∩ Rv2 = Rv3 are the three non zero proper submodules of M .
On the other hand the three composition factors of M are one dimensional. Let
U1 = M /Rv1 an let U2 = M /Rv2 . Then aU1 = 0 and aU2 ≠ 0. Hence M satisfies
(123). From the exact sequence
0 Ð→ Rv3 ≃ P (2) Ð→ M Ð→ U1 ⊕ U2 Ð→ 0
(1)
we get the long exact sequence
0
0 = Hom(U1 ⊕ U2 , M ) Ð→ Hom(M, M ) Ð→ Hom(Rv3 , M ) Ð→
≃
Ð→ Ext1 (U1 ⊕ U2 , M ) Ð→ Ext1 (M, M ) Ð→ 0. (2)
≃
Let e1 be the path of length zero around the vertex 1. Then the definition of U1 and
U2 guarantees the existence of epimorphisms f ∶ P (1) Ð→ U1 and g ∶ P (1) Ð→ U2
such that Ker f = Ra ⊕ Rb and Ker g = R(e1 − a) ⊕ Rb. Consequently, there are
exact sequences of the form
0 Ð→ P (1) ⊕ P (2) Ð→ P (1) Ð→ Ui Ð→ 0
for i = 1, 2.
(3)
Hence also the following long sequences are exact
0 = Hom(Ui , M ) Ð→ Hom(P (1), M ) Ð→ Hom(P (1) ⊕ P (2), M ) Ð→
Ð→ Ext1 (Ui , M ) Ð→ 0
for i = 1, 2. (4)
Since Hom(P (1), M ) ≃ K 2 and Hom(P (1) ⊕ P (2), M ) ≃ K 3 , we deduce from
(4) that Ext1 (Ui , M ) ≃ K for i = 1, 2. Consequently we deduce from (2) that
dim Ext1 (M, M ) = 2.
In the next example we use a quiver with one vertex.
224
GABRIELLA D’ESTE
Example 2.5. The free algebra A = K < x, y > admits a module M satisfying (123)
such that dim Ext1 (M, M ) = 10.
Construction. Let M be the A–module described by the following picture
x
8
v1
v2
●
●
y
f
y
x
●
v3
More precisely, let {v1 , v2 , v3 } be a basis of M and assume that xv1 = v1 , yv2 = v2 ,
yv1 = v3 = xv2 and xv3 = 0 = yv3 . Also in this case Av1 , Av2 and Av1 ∩ Av2 = Av3
are the three non zero proper submodules of M . Moreover the three composition
factors S1 = Av1 /Av3 , S2 = Av2 /Av3 and S3 = Av3 are pairwise non isomorphic.
Hence M satisfies (123). From the exact sequence
0 Ð→ A2 Ð→ A Ð→ Si Ð→ 0
for i = 1, 2, 3,
(1)
we obtain the long exact sequences
0 Ð→ Hom(Si , M ) Ð→ Hom(A, M ) Ð→ Hom(A2 , M ) Ð→
Ð→ Ext1 (Si , M ) Ð→ 0
for any
i.
Since Hom(Si , M ) = 0 for i = 1, 2 and Hom(S3 , M ) ≃ K, we obtain
dim Ext1 (Si , M ) = 3
for i = 1, 2
(2)
and
dim Ext1 (S3 , M ) = 4.
(3)
Since the sequence 0 Ð→ S3 Ð→ M Ð→ S1 ⊕ S2 Ð→ 0 is exact and A is hereditary,
we obtain the long exact sequence
0 = Hom(S1 ⊕ S2 , M ) Ð→ Hom(M, M ) Ð→ Hom(S3 , M ) Ð→
≃
Ð→ Ext1 (S1 ⊕ S2 , M ) Ð→ Ext1 (M, M ) Ð→ Ext1 (S3 , M ) Ð→ 0. (4)
Putting (2), (3) and (4) together, we conclude that dim Ext1 (M, M ) = 10.
In the next statement we compare the dimensions of certain subspaces of small
modules satisfying (123).
Proposition 2.6. Let A be the free K–algebra K ⟨x, y⟩, and let M be a module
satisfying (123) with dim M = 3. Then, among others, the following cases are
possible:
INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE
225
(i) dim xM = dim yM = 2;
(ii) dim xM = dim yM = 1;
(iii) dim xM = 2, dim yM = 1.
Proof.
(i) This follows from Example 2.5, where we have xM = ⟨v1 , v3 ⟩ and
yM = ⟨v2 , v3 ⟩.
(ii) Let M be the A–module, obtained in an obvious way from Example 2.4, with
basis {v1 , v2 , v3 } described by the following picture.
8
x
v1
v2
●
●
y
y
●
v3
Then we have xM = ⟨v1 ⟩ and yM = ⟨v3 ⟩.
(iii) Let M be the A–module with basis {v1 , v2 , v3 } described by the following
picture.
8
x
v1
v2
●
●
y
x
●
v3
then we have xM = ⟨v1 , v3 ⟩ and yM = ⟨v3 ⟩.
3. Composition factors and endomorphism rings
In the sequel we denote by N+ the set of positive integers. In order to construct
modules satisfying (123) of infinite dimension, we need the following lemma.
Lemma 3.1. Let A = K ⟨x, y⟩. Then for any d ∈ N+ ∪{ℵ0 } there is a simple module
S with dim S = d and End S ≃ K.
Proof. If d = 1, the assertion is obvious. Assume d is finite and d > 1. Let S be
the module with basis {v1 , . . . , vd } described in an obvious way by the following
picture.
v1
●
d
x
/
v2
●
●
y
x
/
vd
●
226
GABRIELLA D’ESTE
If 0 ≠ v = ∑di=1 ki vi , then we have yxi v = kd−i v1 for i = 0, . . . , d − 1. Consequently
Av = Av1 = S. Hence S is a simple module of dimension d. Assume now f ∈ End S
and f (v1 ) = ∑di=1 ki vi . Since yxi v1 = 0 for i = 0, . . . , d−2, we have kd−i v1 = yxi f (v1 ) =
f (yxi v1 ) = 0 for i = 0, . . . , d − 2. Hence kd = kd−1 = ⋅ ⋅ ⋅ = k2 = 0, and so f is the
multiplication by k1 . Assume d = ℵ0 . Let S be the module with basis {vn ∣n ≥ 1}
described in obvious way by the following picture.
x
y
v1
x
)
i
●
●
v2
x
)
i
y
i
●
y
v3
)
●
v4
If 0 ≠ v = ∑ni=1 ki vi with kn ≠ 0, then we have y n−1 v = kn v1 . Consequently Av =
Av1 = S, and so S is a simple module of dimension ℵ0 . Assume now f ∈ End S
and f (v1 ) = ∑ni=1 ki vi for some n ≥ 2. Since yv1 = 0, it follows that ∑n−1
i=1 ki+1 vi =
yf (v1 ) = f (yv1 ) = 0. Hence we have k2 = ⋅ ⋅ ⋅ = kn = 0, and so f is the multiplication
by k1 . The lemma is proved.
By dealing with reasonably large free K–algebras it is easy to construct big
modules satisfying (123).
Theorem 3.2. If di ∈ N+ ∪ {ℵ0 } for i = 1, 2, 3 and A = K ⟨x, y, z⟩, then there is an
A–module M , satisfying (123), such that the three composition factors of M have
dimensions d1 , d2 , d3 and d3 = dim soc M .
Proof. Let M be the A–module with basis B1 ∪ B2 ∪ B3 with ∣Bi ∣ = di for any i
described by the following picture.
●
v1
●
i
●
y
u1
●
i
z
y
z
●
ω1
●
i
y
More precisely we have B1 = {vi }i≥1 , B2 = {ui }i≥1 , B3 = {ωi }i≥1 and the following
conditions hold. If d1 = 1, then yv1 = v1 , xv1 = 0 and zv1 = ω1 . If d1 > 1, then
B1 generates the K ⟨x, y⟩–module of dimension d1 constructed in Lemma 3.1, and
we have zv1 = ω1 and zvi = 0 for any i > 1. If d2 = 1, then zu1 = u1 , xu1 = 0 and
yu1 = ω1 . If d2 > 1, then B2 generates the K ⟨x, z⟩–module of dimension d2 (with z
INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE
227
instead of y) constructed in Lemma 3.1, and we have yu1 = ω1 and yui = 0 for any
i > 1. If d3 = 1, then yω1 = ω1 and xω1 = 0 = zω1 . If d3 > 1, then B3 generates the
K ⟨x, y⟩–module of dimension d3 constructed in Lemma 3.1, and we have zωi = 0
for any i. Let now N1 = Av1 , N2 = Au1 . Then we have N1 ∩ N2 = Aω1 , and so
dim N1 ∩N2 = d3 . On the other hand dim M /N1 = d2 and dim M /N2 = d1 . Moreover,
by Lemma 3.1, K is the endomorphism ring of M /N1 , M /N2 and N1 ∩ N2 . To end
the proof, take any 0 ≠ m ∈ M . If m ∈ N1 ∩N2 , then we have Am = Aω1 = N1 ∩N2 . If
m ∈/ N1 , then there exists a ∈ A and k ∈ K such that zam = u1 + kω1 . Consequently
there is some b ∈ A such that zb(u1 + kω1 ) = u1 , and so N2 = Au1 ⊆ Am. Suppose
now m ∈/ N2 . Then there exist a ∈ A and s ∈ N1 ∩ N2 such that yam = v1 + s.
Consequently ω1 = zyam ∈ Am, and so N1 ∩ N2 = Aω1 ⊆ Am. It follows that
v1 = yam − s ∈ Am; hence N1 = Av1 ⊆ Am. Putting things together, we conclude
that Am = N1 if m ∈ N1 ∖ N2 , Am = N2 if m ∈ N2 ∖ N1 , and Am = N1 + N2 = M if
m ∈/ N1 ∪ N2 . Hence M satisfies (123) and the proof is complete.
Corollary 3.3. Let R be the K–algebra given by the quiver
1
a1
:
●
2
d
c
●
d
b2
.
d
a3
:
a2
b1
:
3
●
d
b3
If di ∈ N+ ∪ {ℵ0 } for i = 1, 2, 3, then there is an R–module V = (Vi , fi ) such that V
satisfies (123) and dim Vi = di for any i.
Proof. By Lemma 3.1 we can define a simple K ⟨x, y⟩–module (Vi ; ai , bi ) with
dimension di and endomorphism ring K for any i = 1, 2, 3. Next we fix a non zero
map c ∶ V1 Ð→ V3 and a non zero map d ∶ V2 Ð→ V3 . In this way we obtain an
R–module V = (V1 , V2 , V3 ; fj ) with the following properties:
● The three non zero proper submodules of V are N1 = (V1 , 0, V3 ; fj ), N2 =
(0, V2 , V3 ; fj ) and N1 ∩ N2 = (0, 0, V3 ; fj ).
● V /N1 is not isomorphic to V /N2 .
● End V /Ni is isomorphic to K for i = 1, 2.
Consequently V satisfies (123) and dim Vi = di for any i.
By hypothesis, two non isomorphic composition factors belonging to the top of
a module satisfying (123) have the smallest possible endomorphism ring. However
228
GABRIELLA D’ESTE
it is easy to see that the socle of a module satisfying (123) may have a large
endomorphism ring.
Proposition 3.4. Let E be an extension field of K with [E ∶ K] > 1. Then
there are a K–algebra R and a faithful R–module M , satisfying (123), such that
End(soc M ) ≃ E and M is not injective.
⎛K
⎜
Proof. Let R be the matrix algebra ⎜ 0
⎜
⎝E
0⎞
⎟
. Next let P be the left ideal
K 0⎟
⎟
E E⎠
⎧
⎫
⎪
⎪
⎛K 0 0 ⎞
⎛0 0 0⎞RRRR
⎪
⎪
⎪
⎪
R
⎪
⎪
R
⎪
⎪
⎜
⎟
⎜
⎟RR
⎜ 0 K 0⎟, and let Q be the left ideal ⎨ ⎜0 0 0⎟RR a ∈ E ⎬. Finally let M =
R
⎜
⎟
⎜
⎟
⎪
⎪
RRR
⎪
⎪
⎪
⎪
⎪
⎪
RR
⎝ E E 0⎠
⎝
⎠
a
−a
0
⎪
⎪
R
⎩
⎭
⎛K 0 0⎞
⎛ 0 0 0⎞
⎜
⎟
⎜
⎟
P /Q, L1 = ⎜ 0 0 0⎟, L2 = ⎜ 0 K 0⎟, N1 = L1 /Q and N2 = L2 /Q. Then N1 ,
⎜
⎟
⎜
⎟
⎝ E E 0⎠
⎝E E 0 ⎠
N2 and N1 ∩ N2 = soc M are the unique proper submodules of M . Moreover M /N1
0
and M /N2 have dimension one over K and we have M /N1 ≃/ M /N2 . Hence M
satisfies (123), and we clearly have End(soc M ) ≃ E. Moreover it is easy to check
⎛E 0 0⎞
⎟
⎜
that M is a faithful R–module. Finally, let P̃ be the left R–module ⎜ 0 E 0⎟. It
⎟
⎜
⎝E E 0⎠
remains to check that M = P /Q is an essential submodule of P̃/Q. To see this, take
⎛x
⎜
any p̃ = ⎜ 0
⎜
⎝z
0
y
t
⎛0
0⎞
⎟ ̃
⎜
∈ P ∖ P . Then ⎜0
0⎟
⎟
⎜
⎝1
0⎠
0
0
0
0⎞
⎟
p̃ =
0⎟
⎟
0⎠
⎛0
⎜
⎜0
⎜
⎝x
⎛0
0⎞
⎟
⎜
and ⎜0
0 0⎟
⎟
⎜
⎝0
0 0⎠
0
0
0
1
0⎞
⎟
p̃ =
0⎟
⎟
0⎠
⎛0 0 0 ⎞
⎟
⎜
⎜0 0 0⎟. Consequently R(p̃+Q)∩P /Q ≠ 0, and so P /Q is an essential submodule
⎜
⎟
⎝0 y 0 ⎠
of P̃/Q, as claimed.
Corollary 3.5. Any infinite cardinal d is the dimension over K of a faithful module
satisfying (123).
Proof. If E is an extension field of K with [E ∶ K] = d, then the claim follows from
the proof of Proposition 3.4.
As the next result shows, only two K–algebras occur as the endomorphism ring
of a module satisfying (123) and defined over a K–algebra.
INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE
229
Theorem 3.6. Let M be a module satisfying (123) and defined over a K–algebra.
Let N1 and N2 be the two maximal submodules of M . The following facts hold:
(i) If N1 ∩ N2 ≃/ M /Ni for any i, then End M is isomorphic to K.
(ii) If N1 ∩ N2 ≃ M /Ni for some i, then End M is isomorphic to K[x]/(x2 ).
Proof. Let π ∶ M Ð→ M /(N1 ∩ N2 ) be the canonical morphism. Since N1 ∩ N2 is
fully invariant in M , for any f ∈ End M there is a unique morphism f ⋆ ∈
End (M /(N1 ∩ N2 )) such that π ○f = f ⋆ ○π. Let ρ ∶ End M → End (M /(N1 ∩ N2 )) ≃
K ⊕ K be the morphism sending f to f ⋆ for any f . We first note that
End (M /(N1 ∩ N2 ))
has non trivial idempotents.
(1)
We next observe that
f2 = 0
for every f ∈ Ker ρ.
(2)
Since M is indecomposable and idempotents lift modulo nil ideals, we conclude
that
ρ is not surjective.
(3)
Suppose first N1 ∩ N2 ≃/ M /Ni for any i. In this case ρ is injective. This remark
and (3) imply that End M is isomorphic to K. Hence (i) holds. Assume now
N1 ∩ N2 ≃ M /Ni for some i. In this case there is some 0 ≠ f ∈ Ker ρ. Since
End (M /Ni ) ≃ K, it follows that Ker ρ is the vector space generated by f . Putting
(2) and (3) together, we conclude that End M is isomorphic to K[x]/(x2 ). Thus
also (ii) holds.
Corollary 3.7. Let R be a K–algebra, let M be a module satisfying (123), and let
E = End M . The following facts hold:
(i) R is not commutative and E is commutative.
(ii) M is a cyclic R–module and M is not a cyclic E–module.
Proof.
(i) We know from [4, Remark 2.2] that R is not commutative, while we
know from Theorem 3.6 that E is commutative.
(ii) Let N1 and N2 be the maximal submodules of M . Then any m ∈ M ∖(N1 ∪N2 )
generates M as an R–module. By Theorem 3.6 we have dim E ≤ 2. On the
other hand we clearly have dim M ≥ 3. Hence M is not a cyclic E–module.
As usually, we say that a bimodule
A XB
is faithfully balanced if the canonical
morphisms A Ð→ End XB and B Ð→ EndA X, sending any a ∈ A and b ∈ B to the
corresponding multiplications, are isomorphisms.
230
GABRIELLA D’ESTE
Proposition 3.8. Let R, M, N1 , N2 and E = End M be as in Corollary 3.7. Let
T be the algebra of all endomorphisms of M as an E–module. The following facts
hold:
(i) M is not a faithfully balanced R − E bimodule.
(ii) If R is a finite dimensional algebra, then we may have dim T = dim R + 1.
Proof. We first note that
M is not a simple R–module.
(1)
Assume first E ≃ K. Then T ≃ EndK (M ) and M is a simple T –module. This
remark and (1) prove that M is not faithfully balanced. Assume now E = K[f ] for
some endomorphism f of M such that
f (N1 ) = 0 and f (N2 ) = N1 ∩ N2 .
(2)
Let now B be a basis of M of the form B1 ∪ B2 ∪ B3 such that B1 ∪ B3 and B2 ∪ B3
are bases of N1 and N2 respectively, while B3 is a basis of N1 ∩ N2 . Next let
s ∶ M Ð→ M be a K–linear map such that
s(b) = 0 for any b ∈ B1 ∪ B3 ,
(3)
s(b) ∈ B1 for any b ∈ B2 .
(4)
and
Consequently we deduce from (2), (3) and (4) that (s ○ f )(b) = 0 = (f ○ s)(b) for any
b ∈ B. It follows that s ∈ T . On the other hand we deduce from (4) that s(N2 ) ⊆/ N2 .
Hence s does not act as the multiplication by an element of R, and so (i) holds. To
prove (ii), we choose R and M as in [4, Example 2.4]. More precisely, let R be the
K –algebra given by the quiver
●
1
and let M be the module
12
2
a
/
●
d
b
with relations ba = 0 and b2 = 0,
2
. We first note that
⎛a
⎜
R is isomorphic to the algebra of all matrices over K of the form ⎜0
⎜
⎝c
0⎞
⎟
.
b 0⎟
⎟
d b⎠
0
(5)
INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE
231
Consequently E is isomorphic to the algebra of all matrices over K of the form
⎛x 0 0 ⎞
⎜
⎟
⎜ 0 x 0 ⎟. Therefore it is easy to check that
⎜
⎟
⎝ 0 y x⎠
⎛a
⎜
T is isomorphic to the algebra of all matrices over K of the form ⎜0
⎜
⎝c
0⎞
⎟
.
b 0⎟
⎟
⎠
d b
e
(6)
Hence, by (5) and (6), we have dim R = 4 and dim T = 5. Hence also (ii) holds.
In the next statement we collect some results on dimensions and generators.
Proposition 3.9. Let R be a K–algebra and let M be an R–module satisfying
(123). The following facts hold:
(i) R is generated by at least two elements.
(ii) dim R ≥ 4.
(iii) There is an algebra B, having a B–module satisfying (123), such that B is
generated by two elements and dim B = 4.
Proof.
(i) This follows from the fact that R is not commutative (Corollary 3.7).
(ii) We first note that M is a cyclic R–module which is not projective (Corollary
3.7 and [4, Lemma 2.1]). Consequently either dim R = ∞ or 3 ≤ dim M <
dim R < ∞.
(iii) Let B be the K–algebra given by the quiver
●
a
1
/
●
d
b
with relations
2
ba = 0 and b2 = 0, used to prove condition (ii) of Proposition 3.8. Next let
e1 and e2 be the paths of length zero around 1 and 2. Then {e1 , e2 , a, b} is a
basis of B, and we clearly have e2 = 1 − e1 , a = (a + b)e1 and b = (a + b)e2 =
(a + b)(1 − e1 ). Hence B is generated by e1 and a + b.
The proof of Proposition 2.1 shows that many modules of dimension 3, satisfying
(123), are injective modules over suitable algebras. We finally show that also the
previous K ⟨x, y⟩–modules of dimension 3 have this property.
Corollary 3.10. Let B be the K–algebra used to prove Proposition 3.9 (iii), and
let M be the K ⟨x, y⟩–module used to prove Proposition 2.6 (ii) (resp. (iii)). Then
M is a faithful injective B–module.
232
GABRIELLA D’ESTE
Proof. By hypothesis M is described by the following picture
8
x
v1
v2
●
●
y
⎛
⎜
⎜
⎜
⎜
⎜
⎜resp.
⎜
⎜
⎜
⎜
⎜
⎝
y
●
v3
8
x
v1
v2
●
●
y
x
●
v3
⎞
⎟
⎟
⎟
⎟
⎟
⎟.
⎟
⎟
⎟
⎟
⎟
⎠
Let f and g be the endomorphisms of M induced by x and y. Then we clearly have
f 2 = f , g 2 = 0, f g = 0 and gf ≠ 0 (resp. f 3 = f 2 , g 2 = 0, f g = 0 and gf = g). On the
other hand, by Proposition 3.9, we have dim K ⟨f, g⟩ ≥ 4. Consequently {1, f, g, gf }
(resp. {1, f, f 2 , g}) is a basis of K ⟨f, g⟩. Moreover the K–linear map B Ð→ K ⟨f, g⟩
such that e1 ↦ f , e2 ↦ 1 − f , a ↦ gf , b ↦ g − gf (resp. e1 ↦ f 2 , e2 ↦ 1 − f 2 ,
a ↦ g, b ↦ f − f 2 ) is an algebra isomorphism. Since
1
2
,
2
2
,
12
2
, 1 , 2 , are the
indecomposable B–modules, we conclude that the B–module M is the injective
module
12
2
.
Corollary 3.11. Let C be the K–algebra given by the Dynkin diagram
a
1
/ 3 o
b
2 ,
and let M be the K ⟨x, y⟩–module constructed in Example 2.5. Then M is a faithful
injective C–module.
Proof. By hypothesis M is described by the following picture.
x
8
v1
v2
●
●
y
f
y
x
●
v3
Let f and g be the endomorphisms of M induced by x and y. Then we clearly have
f 3 = f 2 , g 3 = g 2 , f g = f g 2 = f − f 2 , gf = gf 2 = g − g 2 , and f 2 g = g 2 f = 0. Moreover
{1, f, f 2 , g, g 2 } is a basis of K ⟨f, g⟩. As always, let ei be the path of length zero
around the vertex i. Then the K–linear map C Ð→ K ⟨f, g⟩ such that e1 ↦ f 2 ,
e2 ↦ g 2 , e3 ↦ 1 − f 2 − g 2 , a ↦ g − g 2 , b ↦ f − f 2 is an algebra isomorphism. Hence
the C–module M is the injective module
12
3
.
INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE
233
In all the previous examples of faithful modules M , satisfying (123) and defined
over finite dimensional K–algebras R, we always have dim R − dim M ≤ 2. To see
that this is not always true, it suffices to consider the following example.
Example 3.12. There are a K–algebra R and a faithful R–module M , satisfying
(123), such that dim M = 4, End M ≃ K and dim R = 10.
Construction. Let M be the K ⟨x, y, z⟩–module described by the following picture.
y
8
v1
v2
●
●
f
z
y
z
x
i
●
y
v3
)
●
v4
Then we deduce from Theorems 3.2 and 3.6 that M is a K ⟨x, y, z⟩–module satisfying
(123) with End M ≃ K and dim M = 4. Let f, g, h be the endormorphisms of M
induced by x, y, z respectively, and let R = K ⟨f, g, h⟩. Next let e = 1−g 2 −h2 −gf and
` = ge. Then the linear maps g 2 , h2 , hg, gh, gf, `, f h, f gh, f, e are described by ten
different matrices (with entries in K) with one entry equal to 1 and the remaining
ones equal to 0, as suggested by the following matrix
⎛ g2
⎜
⎜0
⎜
⎜
⎜ hg
⎜
⎝f h
0
0
2
h
0
gh
gf
f gh
f
0⎞
⎟
0⎟
⎟.
⎟
`⎟
⎟
e⎠
This remark and the shape of M imply that R is isomorphic to the matrix algebra
⎛K
⎜
⎜0
⎜
⎜
⎜K
⎜
⎝K
0
0
K
0
K
K
K
K
0⎞
⎟
0⎟
⎟.
⎟
K⎟
⎟
K⎠
Consequently we have dim R = 10, as claimed.
As the next example shows, the dimension of a faithful module satisfying (123)
and that of its endomorphism ring do not determine the dimension of the algebra.
Example 3.13. There are a K–algebra A and a faithful A–module L, satisfying
(123), such that dim L = 4, End L ≃ K and dim A = 9.
234
GABRIELLA D’ESTE
Construction. Let L be the K ⟨x, y, z⟩–module described by the following picture.
v1
●
x
)
i
y
v2
v3
●
●
f
z
y
z
●
v4
Then we have dim L = 4. Proceeding as in Theorem 3.2 we conclude that the
K ⟨x, y, z⟩–module L satisfies (123). Hence we deduce from Theorem 3.6 that
End L ≃ K. Next let f, g, h be the endomorphisms of L induced by x, y, z respectively, and let A = K ⟨f, g, h⟩. Finally let e = 1 − gf − f g − h2 . Then the elements
gf, gf g, f, f g, h2 , hgf, hg, gh, e are described by nine different matrices (with entries
in K) with one entry equal to 1 and the remaining ones equal to 0, as suggested by
the following matrix
⎛ gf
⎜
⎜ f
⎜
⎜
⎜ 0
⎜
⎝hgf
0⎞
⎟
0 0⎟
⎟.
⎟
h2 0⎟
⎟
gh e⎠
gf g
0
fg
0
hg
This observation and the shape of M imply that A is isomorphic to the matrix
algebra
⎛K
⎜
⎜K
⎜
⎜
⎜0
⎜
⎝K
K
0
K
0
0
K
K
K
0⎞
⎟
0⎟
⎟.
⎟
0⎟
⎟
K⎠
Hence we have dim A = 9.
Example 3.14. There are a K–algebra B and a faithful B–module U , satisfying
(123), such that dim U = 4, End U ≃ K[x]/(x2 ) and dim B = 8.
Construction. Let U be the K ⟨x, y, z⟩–module described by the following picture.
v1
●
x
)
i
y
v2
v3
●
●
y
z
●
v4
Let f, g, h be the endomorphisms of U induced by x, y, z respectively, and let B =
K ⟨f, g, h⟩. Then U is a faithful B–module with dim U = 4 and, proceeding as in
INDECOMPOSABLE NON UNISERIAL MODULES OF LENGTH THREE
235
Theorem 3.2, it is easy to see that the B–module U satisfies (123). Finally, by
Theorem 3.6, we have End U ≃ K[x]/(x2 ). Let e = 1 − gf − f g and let ` = g − gf g.
Then the elements gf, gf g, f, f g, e, h, hg, ` are described by matrices with one or two
entries equal to 1 and the remaining ones equal to 0, as suggested by the following
matrix
⎛gf
⎜
⎜f
⎜
⎜
⎜0
⎜
⎝h
gf g
0
fg
0
0
e
hg
`
0⎞
⎟
0⎟
⎟.
⎟
0⎟
⎟
e⎠
This remark and the shape of U imply that B is the K–algebra of all matrices (with
entries in K) of the form
⎛⋆
⎜
⎜⋆
⎜
⎜
⎜0
⎜
⎝⋆
⋆
0
⋆
0
0
t
⋆
⋆
0⎞
⎟
0⎟
⎟.
⎟
0⎟
⎟
t⎠
Hence we have dim B = 8, as claimed.
Corollary 3.15. There exist two finite dimensional factor algebras A and B of
K ⟨x, y, z⟩ and two faithful modules A L and B U , satisfying (123), such that dim L =
dim U , dim soc L = dim soc U , dim A + dim End L = dim B + dim End U and dim A ≠
dim B.
Proof. Let A, B, L and U be as in Examples 3.13 and 3.14. Then we have dim L =
dim U = 4, dim soc L = dim soc U = 1, dim A = 9, dim B = 8, dim End L = 1 and
dim End U = 2.
References
[1]
I. Assem, D. Simson and A. Skowronski, Elements of the Representation Theory of Associative Algebras, Vol. 1, London Mathematical Society Student
Texts, 65, Cambridge University Press, Cambridge, 2006.
[2]
M. Auslander, I. Reiten and S. O. Smalø, Representation Theory of Artin Algebras, Cambridge Studies in Advanced Mathematics, 36, Cambridge University
Press, Cambridge, 1995.
[3]
J. Clark, C. Lomp, N. Vanaja and R. Wisbauer, Lifting Modules, Supplements
and Projectivity in Module Theory, Frontiers in Math., Boston, Birkhäuser,
2006.
[4]
G. D’Este and D. Keskin Tütüncü, Pseudo projective modules which are not
quasi projective and quivers, Taiwanese J. Math., 22(5) (2018), 1083-1090.
236
[5]
GABRIELLA D’ESTE
P. A. Guil Asensio, D. Keskin Tütüncü, B. Kalebog̃az and A. K. Srivastava,
Modules which are coinvariant under automorphisms of their projective covers,
J. Algebra, 466 (2016), 147-152.
Gabriella D’Este
Department of Mathematics
University of Milano
Via Saldini 50
20133 Milano, Italy
e-mail: gabriella.deste@unimi.it
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W2559984460.txt | Biodivers Conserv (2018) 27:1619–1638
https://doi.org/10.1007/s10531-016-1268-6
ORIGINAL PAPER
Offshore renewable energy and nature conservation:
the case of marine tidal turbines in Northern Ireland
John R. Haslett1 • Marina Garcia-Llorente2,3 •
Paula A. Harrison4,5 • Sen Li5 • Pam M. Berry5
Received: 30 August 2016 / Revised: 18 November 2016 / Accepted: 24 November 2016 /
Published online: 5 December 2016
Ó The Author(s) 2017. This article is published with open access at Springerlink.com
Abstract The global demand for renewable energy continues to increase rapidly and with
it the necessity to develop and test new technologies to deliver the power. Offshore
renewable energy sources that harness wind, wave or tidal power are of major interest.
Technological advances in these directions have not been matched by a clear understanding of the environmental impacts of the new devices, with most existing research
concentrated on the impacts of offshore wind farms. Decisions often continue to be made
without the support of a clear evidence base. Here we use an underwater tidal turbine,
SeaGen, constructed and operated within the Strangford Lough marine protected area in
Northern Ireland, as a case study to explore the potential impacts of the turbine as points of
concern and argumentation in the decision-making processes. We use information obtained
from official documents and one-to-one interviews with the main stakeholders. Our results
demonstrate that during the construction and operation of the turbine the perceptions and
views of different stakeholders sometimes disagreed but were often surprisingly similar in
relation to both likelihood and intensity of the potential impacts of the turbine on marine
Communicated by Rob Bugter, Paula Harrison, John Haslett and Rob Tinch.
This article is part of the special issue on the BESAFE project.
& John R. Haslett
john.haslett@sbg.ac.at
1
Department of Cell Biology and Physiology, Division of Animal Structure and Function, University
of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria
2
Department of Applied Research and Agricultural Extension, Madrid Institute for Rural,
Agricultural and Food Research and Development (IMIDRA), Ctra. Madrid-Barcelona (N-II), KM.
38.200, 28802 Alcalá De Henares, Madrid, Spain
3
Social-Ecological Systems Laboratory, Department of Ecology, Universidad Autónoma de Madrid,
c. Darwin 2, Biology, 28049 Madrid, Spain
4
Present Address: Centre for Ecology and Hydrology, Lancaster Environment Centre, Library
Avenue, Bailrigg, Lancaster LA1 4AP, UK
5
Environmental Change Institute, University of Oxford, South Parks Road, Oxford OX1 3QY, UK
123
1620
Biodivers Conserv (2018) 27:1619–1638
biodiversity, ecosystem services and human well-being in general. The overall consensus
of views was refined and evolved under an adaptive management approach over the
10 years of the discussions and decision-making processes. The results are discussed in
relation to cumulative gains in knowledge, future arrays of many underwater turbines and
multiple use of oceans within social ecological systems to maintain the conservation of
marine biodiversity.
Keywords Adaptive management Decision-making Ecosystem service Marine
protected area Stakeholder views Trade-off
Introduction
The global demand for renewable energy continues to increase rapidly (most recently
accelerated by the United Nations 2015 Paris Agreement to combat climate change) and
with it the necessity to develop and test new technologies to deliver renewable/clean
power. Offshore renewable energy sources that harness wind, wave or tidal power are of
major interest. Wind farms in the sea are now commonplace in many regions, but advances
in wave and tidal power technology have also been rapid during their relatively short
history (Lawrence et al. 2013). The Ocean Energy status Report of the EC for 2014 cites an
estimate of 100 tidal energy companies worldwide, with more than half located within the
EU and other strong representation in Canada, Australia, USA and eastern Asia. Most of
these deployment activities are single devices or small, pre-commercial arrays (Magagna
and Uihlein 2015). Unfortunately, the technological advances have not been matched by a
clear understanding of the environmental impacts of the new machinery, and this remains a
major concern and focus of research (Inger et al. 2009; Bailey et al. 2014). Most existing
research has concentrated on the impacts of offshore wind farms on biodiversity (e.g.,
Bergström et al. 2014) and/or ecosystem service provision (e.g., Mangi 2013). However,
even for wind farms, our overall knowledge is still very incomplete and the urgent call for
more data and new research by Inger et al. (2009) remains valid, echoed by Bailey et al.
(2014).
Further complications arise from the relatively recent recognition that biodiversity
conservation in Europe and also globally, is embedded within complex, dynamic socialecological systems that encompass the provision of ecosystem services, wider societal
needs and human well-being, all with the need for appropriate and integrated sectoral
policy and environmental management (Haslett et al. 2010; Garcı́a-Llorente et al. 2015). In
the context of marine environments, offshore renewable energy has already been noted as a
new human activity entering the complexity of marine social-ecological systems (Burkhard
and Gee 2012). How these systems respond has now begun to be directly addressed, but
again the focus has been largely on wind farms (Busch et al. 2011; Burkhard and Gee 2012;
Mangi 2013).
Marine tidal turbines that use tidal currents to turn large, submerged rotors have many
parallels with wind turbines; indeed they function rather like ‘‘submerged windmills’’, but
there are also many unique aspects of this still very young family of devices that can
impact upon marine biodiversity and different components of social-ecological systems.
These unique aspects arise mainly from the rotor blades moving under water rather than in
the air above sea level, thus impacting on different types of organisms and parts of marine
123
Biodivers Conserv (2018) 27:1619–1638
1621
social-ecological systems. The large gaps in knowledge that exist create uncertainties in
decision-making processes concerning siting, construction, operation and eventual
decommissioning of the turbine devices (Inger et al. 2009). This is true even at the level of
testing full size operational prototype devices, including ‘‘SeaGen’’, the turbine that is the
subject of this study.
SeaGen is ‘‘the world’s first’’ commercial scale open stream underwater tidal turbine,
constructed and operated in the strong tidal flow at the entrance to Strangford Lough on the
east coast of County Down, Northern Ireland. Strangford Lough is a shallow, tidal sea
lough about 30 km long and up to approximately 8 km wide. The Lough is almost landlocked, but is connected to the open sea by a narrow 8 km long channel known as the
Strangford Narrows, where the SeaGen turbine is sited. The Lough supports a recognised
wealth of marine and coastal biodiversity, and the entire area has many different designations of nature conservation status, at national and EU levels. These include Special
Protection Area (SPA), Special Area of Conservation (SAC), Natura 2000 site and
RAMSAR status and most recently Marine Conservation Zone under the Marine Act
(Northern Ireland) 2013. Also, the area is important for its landscape, recreational use,
some commercial fishing and a diversity of other ecosystem services (Strangford Lough
and Lecale Partnership 2014).
This marine turbine project is important for the Northern Ireland and UK Governments’
requirement to achieve renewable energy targets set by the EU (Department of the
Environment, Northern Ireland 2009), but at the same time, the impacts throughout all
stages of the turbine development were not well understood. The dual importance of the
necessity to increase renewable energy output, with the associated extra benefits of hi-tech
and industrial advances, while at the same time ensuring the continued essential role of the
Protected Area for marine biodiversity conservation and the provision of a wide range of
ecosystem services, created a spectrum of potential conflicts of stakeholder and policy
interests that was new and complex.
Decision-making processes under these potentially antagonistic circumstances were
guided by the adoption of an adaptive management approach, agreed by the stakeholders
during the early stages of the negotiations. In the present context, adaptive management
refers to an iterative process in which uncertainty surrounding the environmental effects of
a human activity is reduced progressively by carefully managed, science-led monitoring of
agreed indicators of environmental impacts. From the very beginning, an adaptive
approach offers a middle way, so that risks and the needs of the different interest groups
are continually re-assessed in the light of new information and balanced within an agreed
management framework. This is clearly explained by Savidge et al. (2014), who also
provide a useful general summary of adaptive management in the environmental context,
with details of how these principles have been applied to the SeaGen situation. These
authors also provide a primarily biological account of the environmental monitoring
studies undertaken in Strangford Lough in relation to the turbine, including information on
the findings, some of the resulting adaptations and also pointing out the lessons learned.
In the present paper we address the stakeholder argumentation and decision-making
aspects of the turbine development, thus complementing more biological or technical
perspectives. Particularly, we explore the use of the potential impacts of the turbine as
points of concern and argumentation. We examine the levels of agreement between the
main stakeholders involved and then follow the temporal dynamics of the discussions as
the turbine development progressed. We differentiate between the negative impacts on
both biodiversity itself and on humans—including ecosystem services, as well as the
positive potential benefits on each of these. We also distinguish between the likelihood and
123
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Biodivers Conserv (2018) 27:1619–1638
intensity of the impacts. The information obtained should aid future argumentation and
decision-making to protect biodiversity in offshore renewable energy development and
also in wider social-ecological system contexts.
Methods
Information on the turbine development and its impacts was obtained from selected official
written reports and from one-to-one meetings and interviews with a variety of the central
stakeholders.
Reports examined and timeline of events
The reports examined represent important strategic events along the timeline of the discussions and decision-making surrounding the turbine development, as depicted in Fig. 1.
They were chosen as significant and clearly documented markers along the entire chain of
events that conveniently bring the different relevant aspects of the study together. They
were:
Event 1 an initial Independent Environmental Statement, which was an impact
assessment undertaken in the period April 2004–June 2005, before any construction
work was undertaken (Royal Haskoning 2005). One of the general conclusions of this
report was that the potential impact of the SeaGen marine current turbine on some of the
designated features was uncertain. Because of this uncertainty of impact, an adaptive
management approach to construction and deployment, with integrated mitigation and
monitoring was proposed by the turbine company and was accepted by the Department
of Environment, Northern Ireland and became the basis for progressing the project.
Event 2 EU Habitats Directive Article 6 Report, covering the period 15 December
2005–2 February 2008 (Department of the Environment (Northern Ireland) 2008). To
enable the turbine to be constructed and allowed to operate, a government Marine
Licence was required. For this, particularly in relation to modifications (variations) to
the conditions of the Licence and Natura 2000 site requirements, an assessment was
necessary under Article 6 of the EU Habitats Directive. A Stage One Screening
Assessment was undertaken. The report was favourable to granting the Licence and
indicated that a Stage two Assessment was not required.
Event 1
Independent
Environment
al Statement
2004
Event 2
Licensing variations
and Habitats
Directive Article 6
Report by the
Department of the
Environment (DOE)
Event 4
Future scenario
2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2015
Fig. 1 Timeline and the four events identified
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Event 3
Result of Final
Report of
Environmental
Monitoring
Programme
Biodivers Conserv (2018) 27:1619–1638
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Event 3 Environmental Monitoring Programme Final Report (Royal Haskoning 2011).
As a condition of the Marine Licence and following from the 2005 Environmental
Impact Assessment report, it was required that a detailed Environmental Monitoring
Programme and associated suite of mitigation measures be established, covering the
entire duration of the turbine development. Monitoring data collection began preinstallation in 2005 and a final report was delivered in January 2011.
Event 4 a future scenario of multiple turbines (arrays) developed at other sites,
considered during the stakeholder interviews. (See ‘‘Methods’’ section on data gathering
from target stakeholders below).
Target stakeholders
The central stakeholders that were consulted for the present study were identified from
information gained from a number of preliminary stakeholder scoping meetings with
governmental and NGO contacts, conducted in the years 2012 and 2013. These preliminary
discussions revealed that over 50 interested parties, covering a wide spectrum of stakeholder types were included in the dialogue of discussion and consultation during the
10 years and more of the turbine project. However, many of the individuals only contributed for a portion of the proceedings due to changes in job situation or political changes
or discontinuities, while others were not actively involved. To provide continuity within
the present study and to ensure the highest possible degree of consistency over the full time
period, we chose to focus on a small, central group of individuals who had been active and
closely aware of all developments and discussions throughout the entire time of the turbine
project. Each of the five persons consequently interviewed represents a major stakeholder
group and each has been involved in the process from its beginning in 2004 to the end of
the present investigation (end 2013). The stakeholders were:
(A)
(B)
(C)
(D)
(E)
Government (Environment)
Government (Agri-Food)
Consortium of conservation NGOs in Northern Ireland
Academic research
Local residents and cultural heritage NGO
It may be noted that the industrial company responsible for the turbine development
initiative relied upon the information provided by the other stakeholders in the decisionmaking processes and so was not included in the design of the present study.
Data gathering from target stakeholders
In a first round of one-to-one discussion interviews with the above-listed key stakeholders,
the interviewees were asked to comment generally on their perspective of the potential
impacts of the turbine and the relative importance of these to the decision-making processes surrounding the turbine development. From these discussions, in combination with
the information available in the official reports examined, a total of 21 potential negative
impacts of the turbine on biodiversity, ecosystem services or directly affecting humans
were clearly identified relevant to the aims of the present study. Each impact was a possible
point of argument to be considered during the negotiations and their usage could be
followed over the turbine development. In addition, direct questioning during the
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discussions with the interviewees identified 10 possible positive impacts of the turbine.
However, these did not appear in the documentation and did not form any part of the
official discussions and decision-making process. All impacts identified, both negative and
positive, are listed in Table 1.
A further round of interviews with the stakeholders centred on a ‘‘questionnaire’’ in which
each of the interviewees was asked to populate a simple table reflecting their perceived
importance attached to the impacts of the turbine. The table questionnaire was presented in
two parts, covering first the negative impacts and then the positive impacts as listed in
Table 1. Both parts had the same structure, so that for each impact listed, two aspects of
importance, ‘‘likelihood to occur’’ and ‘‘intensity of impact’’ were scored using a Likert-style
scale of 1–5, defined respectively as 1 = very unlikely/very weak; 2 = unlikely/weak;
3 = neutral/medium; 4 = likely/strong; 5 = very likely/very strong. This Likert scale is
similar to that used by Jones and Eiser (2010) to investigate wind farm impacts. The interviewees were required to provide separately scored assessments for each of the events defined
along the timeline. Note that the table consisted of only a list with parallel blank cells and did
not include any statements, sentences or other text. As all of the interviewees had been
actively involved with the turbine throughout its history, this provided a strong temporal
continuity and provides a valuable insight into the role of cumulative gains in knowledge in
the discussion process. All selected interviewees had clear recall of the sequence of events
and had no difficulties in differentiating along the timeline.
In addition to the three historical events, a fourth ‘‘event’’ of a future scenario (2013 and
beyond, see the timeline of Fig. 1) was defined for similar scoring within the questionnaire.
This required the interviewed stakeholders to extend their views on the listed potential
impacts of the single turbine to the consideration of future large arrays of many turbines at
other coastal locations. Previous discussions with the stakeholders had revealed that such
arrays were already being planned. It is most important to note that this future scenario
referred only to arrays at other sites and did not include any reference to the future of the
existing turbine. The interviews with the selected stakeholders were all conducted during
the year 2013. The views expressed by all selected interviewees were based on their
professional experience and judgements, though possible influences of personal views
cannot be completely excluded.
The information obtained from the questionnaires was visualised in polar (radial) diagrams and analysed using simple descriptive statistical techniques to provide information
on similarities and differences between stakeholders and also along the temporal axis. The
Kappa statistic (or coefficient) was calculated as a measure of the level of agreement
between different individuals, taking into account that agreement (or disagreement) can
occur by chance (Viera and Garrett 2005).
Results
Differences and similarities in stakeholder perspectives
The likelihood and intensity of occurrence scores were averaged across the four events on
the timeline and analysed separately for each stakeholder (Figs. 2, 3). The results show
considerable variation in stakeholder views on both likelihood and intensity of both negative and positive impacts of the turbine.
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Table 1 Broad classification of the potential environmental impacts of the marine turbine that are considered in the case study, as identified from the reports examined and consultations with stakeholders
Potential negative impacts on biodiversity
Protected habitats (general)
Protected species (general)
Birds
Marine mammals
Sharks/other elasmobranchs
Teleost fish
Shellfish (molluscs, crustaceans)
Benthic communities
Plankton communities
Cabling to land—electric fields, abrasion
Noise/vibration marine animals
Potential negative impacts on humans, including ecosystem services
Regulating
Nutrient cycling/food web dynamics
Water quality (sediment, waste remediation)
Cultural
Nature watching
Recreational boating/fishing
Landscape/seascape quality
Boating navigation/access
Provisioning
Commercial fishing—teleosts
Commercial fishing (pot fishing)
Human well-being
Noise/vibration humans
Tidal hydrodynamics
Potential positive impacts
Regulating
New marine habitat
Improved biodiversity protection from access and fishing prevention
Water quality
Air quality (reduction in CO2, etc.)
Cultural
New biological research initiatives
Educational value
Cultural heritage addition
Tourist attraction
Human well-being
Power economic value
Local employment
Note that some of the categories can overlap
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Fig. 2 Polar diagrams showing the differences between stakeholder groups of the scores for likelihood
(a) and intensity (b) of occurrence of negative impacts, averaged across all four events
Fig. 3 Polar diagrams showing the differences between stakeholder groups of the scores for likelihood
(a) and intensity (b) of occurrence of positive impacts, averaged across all four events
Particularly Stakeholder C, the conservation NGO consortium, tended to give higher
ratings for likelihood and intensity of negative impacts than the other stakeholders. Ratings
by this stakeholder of positive impacts were less extreme. The difference is borne out by
calculation of Kappa coefficients to measure the degree of agreement of each stakeholder
with the others, for all four events combined (Table 2). Again, stakeholder C is shown to
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Table 2 The degree of agreement/disagreement between stakeholders’ scores of likelihood and intensity of
impacts as measured by Kappa coefficients
Stakeholder B
(Government)
Stakeholder C
(NGO)
Stakeholder D
(Academic research)
Stakeholder E
(NGO)
0.111
0.250
0.352
0.372
0.038
0.152
0.114
Likelihood
Stakeholder A
(Government)
Stakeholder B
(Government)
Stakeholder C (NGO)
0.140
Stakeholder D
(Academic research)
0.214
0.229
Intensity
Stakeholder A
(Government)
Stakeholder B
(Government)
Stakeholder C (NGO)
Stakeholder D
(Academic research)
0.208
0.210
0.295
0.403
0.145
0.144
0.198
0.065
0.126
0.34
Data from four different events were combined together. Likelihood/intensity values were categorised as:
low (1); medium (2–3); high (4–5). Kappa Interpretation: \0 poor agreement; 0.0–0.20 slight agreement;
0.21–0.40 fair agreement; 0.41–0.60 moderate agreement; 0.61–0.80 substantial agreement; 0.81–1.00
almost perfect agreement. Highlighted in the table are kappa [0.2 in bold representing a certain degree of
agreement and kappa \0.1 in italics representing more disagreement between stakeholders
disagree most with the other stakeholders for both the likelihood and intensity of impact
occurrence. The Kappa coefficients also highlight that there are various instances of at least
fair agreement between stakeholders, indicating that some general trends do occur at the
level of the entire time span covered by this study.
In Figs. 2 and 3, these areas of general agreement may be seen where the coloured lines
representing the different stakeholders follow similar patterns. For example, the negative
impacts on sharks and other elasmobranch fish, marine mammals, birds, protected species
and protected habitats are rated similarly in relative terms by the stakeholders for both
likelihood and intensity, although the values assigned by each stakeholder may be different. Those impacts that were scored most highly (the ‘‘top three’’) by each stakeholder
for likelihood and intensity of occurrence also reveal general similarities. For instance, the
likelihood of negative impacts on landscape/seascape quality, boating navigation and
access, and the impacts of noise/vibrations on marine animals were scored highly by 3 of
the 5 stakeholders, while the intensity of the effects of noise on marine animals was also
considered to be high by 3 of the stakeholders, and intensity of impact on recreational
boating/fishing was scored highly by 4 of the 5 stakeholders.
Equivalent patterns can be seen for positive impacts, but these are not so clear. For
example, the similarity in the patterns for cultural heritage, educational value and local
employment, and the high scores given by 4 of the 5 stakeholders to the likelihood of
positive impacts from power economic value and to local employment. Also, for intensity,
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higher scores for the benefits through educational value and the creation of new biological
research were given by 3 of the 5 stakeholders.
To examine more closely where areas of greatest and least differences of stakeholder
opinion occur in relation to the different impacts considered, standard deviations of the
means of ‘‘likelihood’’ and ‘‘intensity’’ values were calculated for each impact, pooled over
all four events on the timeline (Fig. 4). Larger standard deviations indicate greater differences in stakeholder rating values. The ranking order for the negative impacts clearly
indicates that opinions on ecosystem services and other human impacts are the most
strongly divided. Conversely, the ratings given to impacts on particular aspects of
Fig. 4 Levels of disagreement between stakeholder ratings of negative (a) and positive (b) impacts using
standard deviations of the means from all stakeholders over all four events and ranked by likelihood of
impact (grey bars)
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biodiversity and conservation issues are much more consistent between stakeholders—they
share similar opinion values. For the positive impacts there is no such clear interpretation.
Opinions on the turbine being a source of providing improved biodiversity protection, or
benefitting air quality or creating new marine habitat are the most variable, but there is
more general consensus on the other positive impacts, particularly in providing local
employment and the education value.
Temporal dynamics: constancy and change in perceived impacts
along the timeline
Examination of the impacts information separately for each of the four events along the
timeline, but with stakeholder views combined, identifies some general patterns in likelihood and intensity of the influences of the turbine on both biodiversity and ecosystem
services/human well-being.
Amongst the negative impacts (Fig. 5), those acting on parts of biodiversity, including
benthic communities, sharks, marine mammals and protected species and habitats in
general, and also impacts from cabling to land and from noise/vibration effects on animals,
are all classified as having medium to strong intensities, as well as being relatively likely to
occur within all or most of the events. Thus these may be regarded as, in the stakeholders‘
combined consensus, the greatest threats to biodiversity, taken over the entire time period.
This is in contrast to the negative impacts on, for example, birds, teleost fish, shellfish,
plankton commercial fishing and nature watching, all of which are considered to be rather
unlikely to occur, and when they do, rather weak in their intensity across most of the
events. These are still considered by the stakeholders to represent potential threats to
biodiversity and ecosystem services, but at lower levels. Note that there appears to be a
Fig. 5 Polar diagrams showing the differences between stakeholder groups of the scores for likelihood
(a) and intensity (b) of occurrence of negative impacts, averaged across all stakeholders
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general positive correlation between likelihood and intensity of impacts, reflected in the
overall similarities of the shapes of the diagrams Fig. 5a, b).
For the positive impacts (Fig. 6), although a number of these were considered likely to
occur within the time periods of most or all of the events, such as educational value, local
employment, power economic value and new biological research, only the last of these,
new research, was considered to be of strong intensity. Again, as noted with the negative
impacts, the results for the positive impacts also appear to exhibit an overall correlation
between likelihood and intensity (Fig. 6a, b).
The similarities in the patterns of likelihood and intensity of impacts are supported by
examining the mean intensity values in the different likelihood categories (1–5), for both
negative and positive impacts. These show that mean intensity generally increases as
likelihood increases (Table 3). These results also show that overall, stakeholders seem
more cautious in rating intensity, especially when they rated likelihood with high scores. In
other words, an impact may be expected to occur, but its impact intensity may not be as
strong as its likelihood. This is also to be seen in both Figs. 5 and 6, where the plots of
intensity tend to show lower scores (closer to the centre of the circles) than the likelihood
scores. This is particularly pronounced among the positive impacts (Fig. 6).
The patterns described so far have referred to general trends exhibited by the events
along the timeline considered overall. However, the results also permit changes in the
importance of the different impacts to be followed more specifically as the turbine project
evolved along the timeline of events, as well as consideration of the future scenario of
arrays of turbines.
In considering the results below, it should be remembered that of the four events defined
in the timeline of Fig. 1, the first three refer to the documented history of the turbine
project and to a single construction in Strangford Lough, while the fourth event refers to a
scenario situation with arrays of many turbines at other locations along the coast of
Northern Ireland, that are already being planned. Thus the future scenario event 4 must be
interpreted rather differently to the other three events.
Fig. 6 Polar diagrams showing the differences between stakeholder groups of the scores for likelihood
(a) and intensity (b) of occurrence of positive impacts, averaged across all stakeholders
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Table 3 Relationship between likelihood and intensity of impacts
Impacts
Mean intensity value (SD)
Likely = 1
Likely = 2
Likely = 3
Likely = 4
Likely = 5
Intensity of negative impacts
1.06 (0.28)
2.09 (0.63)
2.95 (0.57)
3.53 (0.88)
4.18 (0.98)
Intensity of positive impacts
1.13 (0.34)
2.07 (0.36)
2.89 (0.81)
2.91 (0.85)
3.70 (1.22)
Mean intensity values (standard deviation in parenthesis) within different likelihood score categories (1–5)
for both negative and positive impacts
Throughout the consecutive events 1 to 3, there is a clear, strong trend that the negative
impacts were perceived in the early stages (the initial Environmental Statement, event 1) as
more likely to occur and with noticeable intensity, but the assessment of the importance of
the same impacts decreases with time up to and including event 3, when the Environmental
Monitoring Report was completed in 2011. This is indicated by the progressive inward
retreat of the concentric patterns in the polar diagrams of Fig. 5, so that the results from
event 3 are mostly depicted as tighter, inner rings. Impacts that conform to this pattern
include most of the impacts on both biodiversity and ecosystem services/human wellbeing. The only (sometimes only partial) exceptions are influences on plankton communities, teleost fish, commercial fishing for teleosts and impacts of noise/vibration on
humans. Intensity and likelihood patterns are very similar, but not identical.
In the future scenario of event 4, the scores then show a distinct and strong reverse
trend, with values of intensity and likelihood increasing again, usually very strongly, to
attain levels above those of event 3 (the only exception is the impact of noise/vibration on
humans, where a strong negative impact was reported on a house during the operation of
the turbine). This strong reverse trend reflects the differences between the single turbine
situation where there has been concentrated effort to mitigate the negative effects, and the
consideration of arrays of many turbines together, where environmental risks may be
amplified and are not yet understood.
The temporal dynamics of the positive impacts are rather different. Indeed, in many
instances the trend is the opposite to that of the negative impacts—there is often an
increase in likelihood and intensity ratings from event 1 to event 3, and this trend of
increase usually extends to encompass the future scenario event 4 (Fig. 6). Although the
changes in score values between events are generally not as marked as in the negative
impacts, there is still clear indication that the stakeholders give more credence to the
potential positive impacts of the turbine, or even arrays of turbines, as the turbine project
progressed.
These relative changes over time are described qualitatively with reference to the polar
diagrams. A supporting quantitative analysis is provided in Table 4, in which differences
in mean score values between consecutive events are used to quantify the relative changes
between events. These statistics verify the conclusions drawn from the polar diagrams. For
example, the negative impacts are seen initially as more likely to occur and with higher
intensities but the assessments often decline with time over the events 1–3 (highlighted in
italics in Table 4) and for the scenario event 4 the values of likelihood and intensity
increase again (results highlighted in bold in Table 4). Also, the positive impacts often
exhibit a trend for likelihood and intensity ratings to increase over time from events 1 to 3
and this may extend to include the scenario event 4 (highlighted in bold in Table 4).
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Table 4 Differences in mean score values of each impact, compared between consecutive events
Intensity
P0
PA
Likely
PB
PC
P0
PA
PB
PC
Negative impacts on biodiversity
Protected habitats (general)
3.6
-1
-1
1.6
3.8
-0.2
-1.6
1.2
Protected species (general)
4
-0.6
-1.4
1.4
4
-0.2
-2
1.4
Birds
2
-0.2
-0.4
0.4
2.2
-0.2
-0.4
1
Marine mammals
4.4
-0.6
-1.4
1.4
4.2
-0.4
-1.8
1.4
Sharks/other elasmobranchs
4.4
-0.6
-1.4
1.4
4.4
-0.8
-1.2
1.2
Teleost fish
2
-0.4
-0.2
0.8
2.6
0
-0.4
1.2
Shellfish (molluscs, crustaceans)
2
0
-0.6
1
2.2
-0.2
-0.2
1
Benthic communities
3.4
0
-1
0.8
3.6
-0.2
-1.2
1.6
0.4
Plankton communities
1.4
0
0
0.4
1.4
0
0
Cabling to land—electric fields, abrasion
3.2
-1.4
-0.6
2
4.4
-0.8
-0.8
2.6
Noise/vibration marine animals
4.4
0
-0.6
1
4.2
0.4
-2
1.4
Negative impacts on humans (including ecosystem services)
Nutrient cycling/food web dynamics
1.6
0
-0.2
0.8
1.6
0
-0.2
0.4
Water quality (sediment, waste remediation)
2.2
0.4
-0.6
1.2
2.2
0
-0.6
1.2
Nature watching
2.4
-0.2
-0.8
1.2
2.8
0
-0.6
0.8
Recreational boating/fishing
4.4
0
-0.4
0
4.2
-0.2
-0.8
0.2
Landscape/seascape quality
3.6
-0.2
-0.2
0.6
4.2
-0.6
-0.8
1.8
Noise/vibration humans
2.8
-0.2
0.8
-0.2
3
-0.4
1.2
-0.8
1
Tidal hydrodynamics
3.2
-0.4
-0.6
0.8
3.8
-0.2
-0.4
Boating navigation/access
3.6
-0.2
-0.4
0.6
4.4
-0.6
-0.4
1.8
Commercial fishing—teleosts
1.4
0
0
1.6
1.6
0
0
1.4
Commercial fishing (pot fishing)
1.8
0
-0.4
1.6
2
0.2
-0.4
1.2
Positive impacts
Power economic value
3
-0.4
0.2
0.6
4
-0.4
0.4
1.2
Local employment
2.2
0.2
-0.4
0.2
4.2
0.2
-0.2
1.6
Educational value
2.8
0
0.6
0
3.6
0
0
0.4
Cultural heritage addition
2.4
0.2
0.8
-0.4
2.4
0.2
0
0.6
Tourist attraction
1.8
0.6
0.4
-0.8
2.6
0.8
0
-0.6
New marine habitat
2.2
0
0
1.4
2.6
0
0.2
1.4
Improved biodiversity protection from access and
fishing prevention
2.2
0
0
1.2
2.4
0
0
1.4
0.6
Water quality
1.4
0.2
0
0.6
1.4
0.2
0
Air quality (reduction in CO2 etc.)
2.2
0
0
1
3.4
0
0
1.8
New biological research
2.4
1.4
0.2
0.2
2.4
1
0.2
0.4
P0 averaged values in ‘‘2004–2006’’; PA averaged values in ‘‘2005–2008’’ minus averaged values in
‘‘2004–2006’’, PB averaged values in ‘‘2011’’ minus averaged values in ‘‘2005–2008’’; PC averaged values
in ‘‘2013?’’ minus averaged values in ‘‘2011’’. Highlighted in the table values are relative changes [0 in
bold and relative changes \0 in italics
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Discussion
This study has demonstrated that during the construction and operation of the marine tidal
turbine in Strangford Lough, the perceptions and views of different stakeholders sometimes disagreed but were often surprisingly similar in relation to both likelihood and
intensity of the potential impacts of the turbine on marine biodiversity, ecosystem services
and human well-being in general. While stakeholders maintained their own profiles in
having differences in opinion on the overall degree of importance of the impacts (some
being more conservative in their judgements than others), there was a general consensus of
views that was honed/refined and evolved over the entire timeline of the discussions and
decision-making processes. This underlines the paramount importance of establishing and
maintaining effective stakeholder engagement throughout the negotiations, as has been
previously stressed most recently in regard to marine protected area policy in Scotland
(Hopkins et al. 2016a) and also in regard to the development of marine renewable energy
(Inger et al. 2009).
Such evolution of stakeholderś perspectives, specifically their assessments of the negative impacts, is particularly relevant to understanding the full dynamics of this case study.
This is because the progressive changes observed between events 1, 2 and 3 on the timeline
can be understood as reflecting stakeholderś increased knowledge, particularly the
cumulative gain in scientific information that was obtained from the continuous monitoring
programme and associated academic research (Savidge et al. 2014). This cumulative gain
in knowledge also informed on potential points for consideration in the argumentation
processes relating to future arrays of turbines at other sites, as referenced by the scenario of
event 4.
It is interesting that all the different stakeholders displayed an overall appreciation of
the many gains in scientific knowledge as the monitoring results became available. Thus no
large differences were observed between the responses of stakeholder D, representing
academic biological research and the other stakeholders, as might have been otherwise
expected had the new scientific knowledge been appreciated only by this stakeholder group
(Figs. 2, 3). The clearly higher ratings of likelihood and intensity of negative impacts
assigned by stakeholder C, representing the consortium of conservation NGOs, is most
likely to be a reflection of a slightly stronger tendency towards application of the precautionary principle by this stakeholder. These findings conform with the results of Berry
et al. (2016, this issue) who showed that different stakeholder groups share some common
views on biodiversity and its conservation, particularly in relation to moral, intrinsic and
ecological values.
There are clear patterns in the temporal dynamics of the use and perceived importance
and effectiveness of arguments based on the individual negative impacts during the
negotiations and decision-making process surrounding the turbine development. Most of
the listed negative impacts were used as arguments which were repeated in the events over
the entire time course of the turbine development. Some of these exhibited increased and
broadening use along the timeline of events. Particularly, the arguments of risks to marine
mammals, notably seal populations with some focus on the EU Habitats Directive protected species status of the harbour seal, and the risks of any type of large animal collisions
with the turbine increased their status as centres of attention during the course of the case
(Savidge et al. 2014). However, arguments centred on Protected Area status, including
benthic and Habitats Directive listed habitats, and acoustic/vibration disturbance to animals
also maintained higher profiles. Arguments of negative impacts on cetaceans (dolphins and
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whales), sharks and other elasmobranchs (cartilaginous fish), including risks from electromagnetic fields and cables scraping the seabed, showed lost impetus over the timeline.
Risks to birds maintained a low profile throughout the study. All of these temporal patterns
of argumentation using particular impacts are also the same as, or very similar to, those
reflected in the official report documents examined. From the very broad spectrum of
concerns and uncertainties initially identified in the early Environmental Impact statement
(Royal Haskoning 2005), focus on the different specific impacts was progressively narrowed and concentrated through the Habitats Directive Article 6 Report (Department of the
Environment (Northern Ireland) 2008) and the Final Report of the monitoring programme
(Royal Haskoning 2011), in the manner reported above.
One important point here is that the above examples and the present results overall
clearly show that concerns and discussions along the timeline of the decision-making
processes were very strongly oriented to impacts that centred on aspects of biodiversity or
included aspects of biodiversity as a component. The negative impacts of the turbine on
ecosystem services and human well-being are noticeably absent from active argumentation. All stakeholders were aware of the full spectrum of impacts, including those affecting
ecosystem services and human well-being, and they provided judgements on these in the
questionnaire used. Equally, the human-directed influences were identified and considered
within the formal Environmental Impact Assessment (event 1) in the early stages of the
turbine project (Royal Haskoning 2005). However, after this assessment, the primary
documentation up to and including the Monitoring Programme Report of event 3 (Royal
Haskoning 2011), is mainly focussed on the potential threats to biodiversity and biodiversity protection policy affecting the Protected Area. Other impacts appear to have been
regarded as much more peripheral, even though there can be direct positive links between
biodiversity and ecosystem service provision. For example, in the review of biodiversity
and ecosystem service relationships by Harrison et al. (2014), positive relations were found
to be frequently reported between commercial fishing and the attributes of biodiversity of
species richness, abundance and size of individuals. Also, species-based recreation was
shown to have a positive relation with both abundance and size of the species present.
Concerns about negative impacts on the seal populations in the Lough maintained a
particularly high profile throughout the timeline of the project. Seals may appear simply as
charismatic species capable of attracting public attention. However, and most importantly,
in addition to their strong aesthetic appeal, the seals are the subjects of a more complex set
of arguments involving the logistics of combining environmental policy with changing
levels of scientific knowledge. Particularly, the risks of collisions with the rotating blades
of the turbine, together with the fact that the harbour seal is listed in the Annexes of the EU
Habitats Directive, so that protection of the species is legally obligatory. This is in contrast
to all the other organisms present: no other species or community or habitat type offered
such a strong combination of arguments (sometimes referred to as ‘‘bundles’’ of arguments). For example, it might have been expected that the bird fauna and bird habitats,
which have a strong public profile and are commonly used in argumentation in biodiversity
conservation matters, would play a pivotal role in discussions of the turbine development.
However, despite the EU Habitats and Birds Directives listings of a few species and
recognition that diving bird species potentially could be at risk of colliding with the turbine
rotors whilst in operation, arguments involving birds remained at a low profile, becoming
less important as the project progressed. This is because, as research and monitoring
continued and the associated levels of scientific knowledge increased, it became clear that
apart from some early concerns about disturbances to birds caused by the construction of
the turbine (mitigated by undertaking the work outside the overwintering and breeding
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1635
seasons), birds and bird habitats were at negligible risk from the turbine project
(Department of the Environment, Northern Ireland 2008).
The potential positive impacts of the turbine on biodiversity and human well-being,
beyond the provision of renewable energy (including both the economic value of the power
and the inferred improvement of air quality), were not included in the argumentation and
decision-making processes. The positive impacts listed in Table 1 were identified and
obtained from discussions during meetings with the stakeholders for this study and were
not included in the official reports or other documentation. However, a number of these
potential positive impacts have been recently widely discussed in the literature in relation
to offshore wind farms. Two of the more obvious examples that are particularly pertinent
to the present study may be expanded upon here.
First, there is the recognition of the creation of new habitat provided by the anchoring
structures of the turbine (often referred to as ‘‘foundations’’) on the seabed. The advantages
of such new, artificial substrate have been well advertised in the wind farm literature as
being available for colonisation by benthic organisms, to form new reef communities
(Inger et al. 2009; Busch et al. 2011; Lacroix and Pioch 2011; Mangi 2013; Bergström
et al. 2014; Hammar et al. 2016) and also as structures that may encourage the aggregation
of fish (Bergström et al. 2014; Gilbert et al. 2015; Hammar et al. 2016). Taking the idea a
step further, it has been suggested that new designs of the underwater portions of wind
turbines could incorporate structural features specifically intended to enhance colonisation
and shelter opportunities for marine organisms and even to accommodate aquaculture
needs. Thus offshore wind farms could become an element in the creation of a green
infrastructure in marine environments (Lacroix and Pioch 2011).
Second, boating navigation and fishing restriction/exclusion zones designated around
offshore wind farms for reasons of safety and damage to fishing tackle can provide protection for biodiversity. In this context, offshore wind farms have been widely suggested as
capable of fulfilling functions very similar to those of marine protected areas (Inger et al.
2009; Mangi 2013; Hammar et al. 2016). In addition, protection could extend to boosting
commercial fish stocks, with spillover of populations that could be harvested in adjacent
fishing grounds (Busch et al. 2011; Mangi 2013). But all these issues are complex, not
always observable and relationships are still not well understood (Bergström et al. 2014;
Vandendriessche et al. 2014).
The extent to which these and other recognised potential positive influences could apply
to the rather different situation of tidal turbines with their submerged rotors impacting on
the underwater environment remains unclear.
Both of the above examples serve to highlight the importance of considering possibilities for trade-offs between different impacts that may affect biodiversity protection,
provision of various ecosystem services and human well-being, involving different sectors
and stakeholder interests, all within the framework of entire marine social-ecological
systems. Such trade-offs were not addressed or attempted during the tidal turbine negotiations of the present study, even though the potential clearly exists. But with the focus on
a single turbine in the Strangford Lough Protected Area, all such trade-offs are small and of
only minor importance. Throughout the turbine development as documented by events 1–3,
the central concerns of all the major stakeholders were clearly the maintenance of marine
and coastal biodiversity protection, including the integrity of the protected area and local
cultural landscape, so no major trade-off situations arose. However, when considered in the
light of the future scenario of event 4, large arrays of turbines at other locations (with the
parallels to wind farms), the potential importance of various trade-offs between the different impacts begins to take on a new and much greater significance.
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Biodivers Conserv (2018) 27:1619–1638
Indeed, from the social-ecological system perspective, such arrays will be a major new
addition to multi-sectoral ocean use and will further stimulate trade-offs already documented as being partly catalysed by wind farms. For example, trade-offs identified among
offshore wind energy, commercial fishing and whale watching as explored by White et al.
(2012) in Massachusetts coastal waters. This type of thinking has led to a new recognition
of the importance of considering how we use and divide ocean space and the need for
marine spatial planning that must also keep the maintenance of marine environmental
quality and conservation at the forefront (White et al. 2012; Gilbert et al. 2015; Hammar
et al. 2016; Hopkins et al. 2016a).
Of course the confinement of the present study to a single working prototype turbine
within a marine protected area is also a limitation to the generality of the results—the
findings cannot be used to directly extrapolate to situations of arrays of turbines as
envisaged at other coastal sites. Such extrapolation would be potentially extremely dangerous where much larger areas of seabed are involved. Such need for caution is clearly
reflected by the results of the stakeholderś responses to the questionnaire presented here
and future understanding will require continued new research effort with an emphasis on
marine spatial planning that includes biodiversity protection as discussed above. Also,
some may argue that the restriction of the study to only five stakeholders limits the amount
of data obtained and the ensuing analyses. However, focusing on a small number of
reliable experts that had key roles throughout the entire duration of the project ensured that
the information captured was of a consistent and high quality, avoiding the need for more
complicated statistical analysis of a highly patchy albeit larger dataset.
One potential bureaucratic complication to this study is that Northern Ireland has
experienced a complex set of governmental changes over the years. Issues of environmental policy and its governance within Northern Ireland have been the responsibility of a
variety of governmental entities, mainly associated with the Department of the Environment, but under a variety of different names, during the time course of the SeaGen turbine
project. This includes some periods when the local Government, the Northern Ireland
Assembly, was suspended, during which times responsibility was at the national level,
handled directly from the UK Government in London. However, such internal changes do
not appear to have interfered with the decision-making processes related to the development of the turbine project and indeed reflects the reality of politically unstable situations
that could arise in any country.
Undoubtedly, the innovative ‘‘adaptive management approach’’ to the turbine development as reported here and described by Savidge et al. (2014) with integrated mitigation
and monitoring, adopted from the early stages, has played a crucial role in the success of
the entire turbine project. Adaptive management has been acknowledged as a useful
approach in other studies involving marine protected areas in various contexts, including
management for the provision of ecosystem services (Rees et al. 2012) or for resilience to
climate change (Hopkins et al. 2016b). However, even though there is a general perception
of the need for processes to be adaptive, the reality is that currently, adaptive management
has rarely been demonstrated, nor the legal or scientific capability to carry it out (Hopkins
et al. 2016b). The present study joins the work of Savidge et al. (2014) as one of the first to
provide such evidence. The present study has clearly demonstrated the importance of
establishing and maintaining effective stakeholder engagement and that the flexibility
created among the engaged stakeholders permitted a strong adherence to the precautionary
principle of biodiversity protection throughout. It may be hoped that continued high levels
of stakeholder engagement and agreement will allow such an approach to be maintained
when arrays of underwater turbines are developed in the near future.
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Acknowledgements Open access funding provided by Paris Lodron University of Salzburg. We thank all
the stakeholders who took part in this study and gave freely of their time, thoughts and on occasion fast
access to documented information. We also thank two reviewers for providing helpful and constructive
comments. This work was supported by the European Union, under FP7 project BESAFE (FP7ENV.2011.282743). MGL was also funded by a postdoctoral grant from the Spanish National Institute for
Agriculture and Food Research and Technology (INIA) which is co-funded by the European Social Fund.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution,
and reproduction in any medium, provided you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons license, and indicate if changes were made.
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W3092750669.txt | Cellular and Molecular Life Sciences (2021) 78:1233–1261
https://doi.org/10.1007/s00018-020-03656-y
Cellular and Molecular Life Sciences
REVIEW
TLR4 and CD14 trafficking and its influence on LPS‑induced
pro‑inflammatory signaling
Anna Ciesielska1
· Marta Matyjek1 · Katarzyna Kwiatkowska1
Received: 10 June 2020 / Revised: 25 August 2020 / Accepted: 22 September 2020 / Published online: 15 October 2020
© The Author(s) 2020
Abstract
Toll-like receptor (TLR) 4 belongs to the TLR family of receptors inducing pro-inflammatory responses to invading pathogens. TLR4 is activated by lipopolysaccharide (LPS, endotoxin) of Gram-negative bacteria and sequentially triggers two
signaling cascades: the first one involving TIRAP and MyD88 adaptor proteins is induced in the plasma membrane, whereas
the second engaging adaptor proteins TRAM and TRIF begins in early endosomes after endocytosis of the receptor. The
LPS-induced internalization of TLR4 and hence also the activation of the TRIF-dependent pathway is governed by a GPIanchored protein, CD14. The endocytosis of TLR4 terminates the MyD88-dependent signaling, while the following endosome maturation and lysosomal degradation of TLR4 determine the duration and magnitude of the TRIF-dependent one.
Alternatively, TLR4 may return to the plasma membrane, which process is still poorly understood. Therefore, the course
of the LPS-induced pro-inflammatory responses depends strictly on the rates of TLR4 endocytosis and trafficking through
the endo-lysosomal compartment. Notably, prolonged activation of TLR4 is linked with several hereditary human diseases,
neurodegeneration and also with autoimmune diseases and cancer. Recent studies have provided ample data on the role of
diverse proteins regulating the functions of early, late, and recycling endosomes in the TLR4-induced inflammation caused by
LPS or phagocytosis of E. coli. In this review, we focus on the mechanisms of the internalization and intracellular trafficking
of TLR4 and CD14, and also of LPS, in immune cells and discuss how dysregulation of the endo-lysosomal compartment
contributes to the development of diverse human diseases.
Keyword CD14 · Endocytosis · Endotoxin · Endosome · LPS · TLR4
Abbreviations
AD Alzheimer’s disease
ALS Amyotrophic lateral sclerosis
AOHA Acyloxyacyl hydrolase
Arf6 ADP-ribosylation factor 6
BMDC Bone marrow-derived dendritic cells
BMDM Bone marrow-derived macrophages
CF Cystic fibrosis
CFTR Cystic fibrosis transmembrane conductance
regulator
CHS Chédiak–Higashi syndrome
CLIC Clathrin-independent carrier
Dab2 Disabled-2
DAMP Damage-associated molecular pattern
* Anna Ciesielska
a.ciesielska@nencki.edu.pl
1
Laboratory of Molecular Membrane Biology, Nencki
Institute of Experimental Biology of Polish Academy
of Sciences, 3 Pasteur St., 02‑093 Warsaw, Poland
DC Dendritic cells
ERC Endosomal recycling compartment
GAG Glycosaminoglycan
GEEC GPI-AP-enriched early endosomal
compartment
GMFG Glia maturation factor-γ
GPI-AP Glycosylphosphatidylinositol-anchored
proteins
HMGB1 High mobility group box protein 1
IP3 Inositol 1,4,5-trisphosphate
IFN Interferon
IKK IκB kinase
IL Interleukin
IRAK Interleukin-1 receptor-associated kinase
IRF Interferon regulatory factor
LBP LPS-binding protein
LPS Lipopolysaccharide
LSD Lysosomal storage disorders
MHC Major histocompatibility complex
MPS Mucopolysaccharidosis
13
Vol.:(0123456789)
1234
MVB Multivesicular bodies
NPC Niemann–Pick’s type C disease
OMV Outer membrane vesicles
oxPAPC Oxidized 1-palmitoyl-2-arachidonyl-snglycero-3- phosphorylcholine
PAMP Pathogen-associated molecular pattern
PD Parkinson’s disease
PI Phosphatidylinositol
PI(4,5)P2 Phosphatidylinositol 4,5-bisphosphate
PI(3,4,5)P3 Phosphatidylinositol 3,4,5-trisphosphate
PIP5KI Phosphatidylinositol 4-phosphate 5-kinase
type I
PLCγ2 Phospholipase Cγ2
PRR Pattern recognition receptor
PS Phosphatidylserine
RIPK1 Receptor-interacting serine/threonine-protein
kinase 1
sCD14 Soluble form of CD14
TAK1 Transforming growth factor-beta-activated
kinase 1
TBK1 TANK binding kinase 1
TIR Toll/IL-1R homology domain
TLR Toll-like receptor
TNF-α Tumor necrosis factor-α
Vps Vacuolar protein sorting-associated protein
Introduction
The mammalian family of Toll-like receptors (TLR) consists
of thirteen members with TLR4 being the most extensively
studied one. TLRs are representatives of pattern recognition receptors (PRR), so named for their ability to recognize
evolutionarily conserved components of microorganisms,
including bacteria, viruses, fungi and parasites, collectively
called pathogen-associated molecular patterns (PAMPs).
The recognition of a PAMP by a PRR triggers rapid inflammatory reactions essential for the innate immunity, as discussed in several previous exhaustive reviews [1–5]. TLR4
is activated by lipopolysaccharide (LPS, endotoxin), a major
component of the outer membrane of Gram-negative bacteria. During infection, TLR4 responds to the LPS present in
tissues and the bloodstream and triggers pro-inflammatory
reactions facilitating eradication of the invading bacteria [6].
It has been indicated that TLR4 can also be activated by
endogenous compounds called damage-associated molecular patterns (DAMPs), including high mobility group box
protein 1 (HMGB1) and hyaluronic acid. These compounds
are released during tissue injury and can activate TLR4 in
non-infectious conditions to induce tissue repair [7, 8]. Altogether, apart from LPS and its derivatives, up to 30 naturally
occurring agonists of TLR4 with various chemical structures have been postulated. However, only three of them,
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i2+, the plant secondary metabolite paclitaxel, and disulfide
N
HMGB1 have been demonstrated to be direct activators of
TLR4, while the others can act as chaperones for TLR4 or
promoters of LPS internalization [7, 9, 10]. Nevertheless, the
impact of endogenous DAMPs on the TLR4 activity broadens the spectrum of pathophysiological conditions involving
the TLR4-induced pro-inflammatory responses far beyond
infectious diseases.
TLR4 binds LPS with the help of LPS-binding protein
(LBP) and CD14, and an indispensable contribution of the
MD-2 protein stably associated with the extracellular fragment of the receptor (Fig. 1). The requirement for MD-2 for
TLR4 activation by LPS was established shortly after the
identification of TLR4 as the LPS receptor, since virtually
no responses to LPS were detected in macrophages derived
from MD-2−/− mice [11, 12]. The binding of an LPS molecule to the TLR4/MD-2 complex involves acyl chains and
phosphate groups of lipid A, the conserved part of LPS and
the main inducer of pro-inflammatory responses to LPS [13,
14]. Hexa-acylated and diphosphorylated LPS, like Escherichia coli LPS (O111:B4), is one of the most potent agonists
of TLR4 whereas under-acylated LPS and dephosphorylated LPS species have a weaker pro-inflammatory activity
especially in human cells [15]. Structural determinants of
this phenomenon are found in the TLR4/MD-2 complex and
also in CD14 protein [13, 16], as discussed in the following
sections.
Under-acylated and dephosphorylated LPS is synthesized
by commensal bacteria which colonize human intestines,
like Bacteroides thetaiotaomicron, which evade recognition
by PRR [17] and are crucial for the maintenance of the intestinal immune balance [15]. In addition, the impermeability
of the intestine epithelium to LPS is an important factor
preventing its egress into the bloodstream [18]. On the other
hand, during infection, deacylation and dephosphorylation
of bacterial LPS is important for the termination of inflammatory responses, as discussed in detail below in the section
concerning LPS detoxification.
An exaggerated and uncontrolled pro-inflammatory signaling triggered by TLR4 during infection can lead to sepsis,
septic shock, and death [19]. Infections with Gram-negative
bacteria, including E. coli and Pseudomonas aeruginosa,
are the prevailing cause of severe sepsis in humans [20].
In addition, low doses of LPS derived from the gut microbiota can in certain conditions enter the bloodstream and
evoke so-called metabolic endotoxemia leading to a chronic
low-grade TLR4-dependent inflammation which contributes
to the development of metabolic diseases, such as type 2
diabetes [18, 21–23]. Prolonged activation of TLR4 is also
linked with several human hereditary and neurodegenerative
diseases and also with autoimmune diseases and cancer [24,
25]. On the other hand, an experimental exclusion of TLR4triggered signaling during low-grade polymicrobial sepsis
TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling
1235
Fig. 1 Pro-inflammatory signaling pathways of TLR4. TLR4 activates the MyD88-signaling pathway at the plasma membrane and
after a CD14-dependent endocytosis initiates the TRIF-dependent
cascade. Via activated NF-κB TLR4 also contributes to the activation
of the cytosolic NLRP3 inflammasome. See text for details
resulted in an impaired bacterial clearance and thereby
worsened organ injury leading to a higher mortality of mice
[26, 27]. Thus, the cellular level of TLR4 and its signaling
activity have to be tightly regulated to be beneficial rather
than harmful to the host.
TLR4 is expressed in immune cells mainly of myeloid origin, including monocytes, macrophages and dendritic cells
(DC), and also in some non-immune cells, like endothelial
cells [28]. Most myeloid cells express also high amounts of
plasma membrane-anchored CD14 [29, 30], which facilitates
the activation of TLR4 by LPS and controls the subsequent
internalization of the LPS-activated TLR4 important for
receptor signaling and degradation. With the help of CD14,
TLR4, unlike all the other TLRs, triggers two signaling pathways called the MyD88-dependent and the TRIF-dependent
one after the adaptor proteins involved in their induction
[31–34]. These signaling pathways lead to the production
of two sets of pro-inflammatory cytokines which only partially overlap [35–39]. The MyD88- and TRIF-dependent
signaling pathways are triggered consecutively and linked
with the redistribution of the LPS-activated TLR4 from the
plasma membrane to endosomes [40]. The translocation of
TLR4 to the cell interior and its further lysosomal degradation facilitate termination of the inflammatory response [41].
In this review, we discuss the mechanisms of the internalization and intracellular trafficking of TLR4 and CD14, and
also of LPS in immune cells. We emphasize the mechanisms
regulating the trafficking of TLR4 and CD14 and thereby
affecting the TLR4-induced pro-inflammatory signaling,
its magnitude, duration and eventually also TLR4 degradation. Finally, we discuss data pointing to how disturbances
in those processes contribute to the development of several
human diseases.
Signaling pathways triggered by TLR4
Activation of TLR4 by LPS is preceded by a chain of reactions which aim at converting LPS aggregates, derived
from bacteria, into LPS monomers concentrated at the cell
surface in the vicinity of the receptor (Fig. 1). These reactions are initiated by the serum LBP protein which binds
to LPS aggregates (micelles) and, in the most typical scenario, facilitates subsequent extraction of LPS monomers
13
1236
by CD14 and the delivery of the LPS to the TLR4/MD-2
complex [42–44]. In agreement, blocking of LBP with an
anti-LBP antibody inhibited the LPS-induced TLR4 signaling and endocytosis of the receptor [45]. Recently, the whole
process of LPS transfer from micelles via LBP, CD14 to
TLR/MD-2 was visualized at a single-molecule resolution
[46]. That study indicated that one LBP molecule bound to
an LPS micelle mediates several rounds of LPS transfer to
CD14 molecules [46].
CD14 is a glycosylphosphatidylinositol (GPI)-anchored
protein localized in nanodomains of the plasma membrane
enriched in cholesterol and sphingolipids, so-called rafts,
which are, therefore, considered as sites of TLR4 activation [3]. CD14 is detected predominantly on the surface of
myeloid-lineage cells; however, low amounts are also found
in non-myeloid cells, e.g., hepatocytes, adipocytes, corneal,
and intestinal epithelial cells [29, 30, 47–51]. These latter
cells produce mainly a soluble form of CD14 lacking the
GPI anchor (sCD14) [47, 48]. However, mechanisms of
the release of sCD14 were studied mainly in immune cells,
where limited proteolysis of the membrane form of CD14
and also proteolysis-independent sCD14 formation have
been detected [52]. The proteolysis of the membrane-bound
CD14 can be carried out on the cell surface (so-called shedding) or intracellularly, after phagocytosis of bacteria when
a following secretion of a truncated 13-kDa form of sCD14,
called presepsin, has also been detected [53, 54]. The release
of sCD14 that is independent of its membrane-bound form
should not be neglected since patients suffering from paroxysmal nocturnal hemoglobinuria with defects in GPI anchor
synthesis, who do not express membrane-anchored CD14,
have normal levels of serum sCD14 [55, 56].
Both membrane-bound and soluble CD14 can transfer
the LPS molecule to the TLR4/MD-2 complex [44, 46,
57]. LPS is bound in the N-terminal hydrophobic pocket
of CD14 which differs in some details of structure between
human and murine CD14 [16, 58]. The CD14 hydrophobic
pocket probably accommodates up to five acyl chains of the
endotoxin while the remaining one can facilitate the association of the CD14-LPS complex with MD-2 [16]. It has
been found recently that the transfer of LPS from CD14 to
MD-2 in the TLR4/MD-2 complex is facilitated by TLR4
which probably forms a transient intermediate with CD14,
LPS or both [46].
A crystallographic analysis of the human TLR4/MD-2
complex with LPS bound has revealed that five of the six
acyl chains of LPS are buried in the hydrophobic pocket of
MD-2 while the sixth one interacts with TLR4 of another
TLR4/MD-2 complex, and that the dimerization of the
TLR4/MD-2 complexes is strengthened by ionic bonds
between the phosphate group of lipid A and the neighboring TLR4 molecule [13]. Accordingly, removing of
one or two acyl chains from the LPS molecule converts it
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A. Ciesielska et al.
from an agonist to an antagonist of human TLR4, as was
shown using LPS of Neisseria meningitidis H44/76, Pseudomonas aeruginosa, and Yersinia pestis [59–61]. Interestingly, murine TLR4 is activated with similar efficiency by
tetra-, penta- and hexa-acylated LPS from the same bacteria [59–61]. A contrasting ability to activate human and
murine TLR4/MD2 complex has also been shown for tetraacylated lipid IVa which turned out to be an antagonist for
the former and a weak agonist for the latter [62]. The agonistic activity of lipid IVa toward murine TLR4 is due to
its unique ionic interactions at the dimerization interface
of the murine receptor that cannot be formed with human
TLR4 [63]. Moreover, the length of the acyl chains as well
as their saturation also seem important for the pro-inflammatory activity of LPS, as has been postulated based on
studies of the immunomodulatory activity of lipid A and
cardiolipin analogues [64, 65]. Accordingly, penta-acylated
diphosphorylated LPS with one unsaturated acyl chain from
Rhodobacter spheroides and its synthetic tetra-acylated lipid
A derivative, Eritoran, are antagonists for both human and
mouse TLR4 [66–68].
The interaction of TLR4/MD-2 with two molecules of an
agonistic LPS species induces dimerization of the ectodomains of two TLR4 molecules which acquire an “M-shape”
with their intracellular fragments put in juxtaposition [12,
13, 69]. Each intracellular fragment contains a Toll/IL-1R
homology (TIR) domain prone to homotypic interactions
with TIR domains of four adaptor proteins. When in the
plasma membrane, TLR4 interacts with the first adaptor
pair, TIRAP (also called Mal [70]) and MyD88 [71, 72].
Apart from the TIR domain, TIRAP also carries a domain
enriched in basic and aromatic residues that interacts with
phosphatidylinositols (PIs) and phosphatidylserine (PS)
[72–74]. In the plasma membrane, TIRAP can bind both PS
and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) but
only the latter interaction is required for TLR4 signaling [72,
73]. The TLR4-bound TIRAP recruits MyD88 which further
binds interleukin-1 receptor-associated kinase (IRAK) 1 and
2 and a submembrane signaling complex called the myddosome is formed [72, 75, 76]. The assembled myddosome
recruits E3 ubiquitin ligase TRAF6, which triggers a signaling cascade involving TAK1 kinase and leading, through the
phosphorylation and activation of IκB kinases α/β (IKKα/β),
to nuclear translocation of the NF-κB transcription factor. In
addition, downstream of TRAF6 and TAK1, MAP kinases
are phosphorylated to activate transcription factors AP-1
and CREB [77]. The signaling complex of TLR4 containing TIRAP and MyD88 also activates type I PI3-kinase
which phosphorylates PI(4,5)P 2 to phosphatidylinositol
3,4,5-trisphosphate (PI(3,4,5)P3) and triggers activation of
Akt [74, 78]. Collectively, the MyD88-dependent signaling
pathway induces expression of genes encoding pro-inflammatory mediators, such as tumor necrosis factor α (TNF-α),
TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling
interleukin (IL)-6, cyclooxygenase 2, and type III interferons (IFNλ1/2), the latter contributing to epithelial barrier
integrity, which is crucial for host defense [35, 79–81]. In
addition, the MyD88-dependent signaling pathway also participates in the production of anti-inflammatory mediators,
like IL-10 helping to terminate the inflammation [35, 82]. It
is worth mentioning that several factors negatively affect the
MyD88-depenedent signaling cascade to prevent an exaggerated production of pro-inflammatory mediators. These
include A20 ubiquitin-modifying enzyme, the BCAP-PI3kinase–Akt axis, and Lyn tyrosine kinase [83–86].
Recent studies indicate that the LPS-induced MyD88dependent signaling triggers and also modulates the cell
metabolism. It was shown that TRAF6 interacts with TBK1,
the kinase which in turn activates Akt kinase leading to a
rapid enhancement of glycolysis [87, 88]. The LPS-stimulated glycolysis and subsequent synthesis of acetyl-CoA
and de novo synthesis of fatty acids can facilitate, respectively, histone acetylation required for gene transcription and
expansion of the endoplasmic reticulum and Golgi apparatus
necessary for intense production and secretion of cytokines
[87, 89].
The MyD88-dependent signaling is followed by internalization of TLR4. Simultaneously, TIRAP and MyD88
dissociate from the membrane allowing TLR4 to bind in the
endosome a second set of TIR-containing adaptor proteins,
TRAM and TRIF [90]. In this tandem, TRAM is a bridging adaptor facilitating the interaction of TRIF with TLR4
[91]. In unstimulated cells, both TLR4 and TRAM localize
to the plasma membrane, endosomes, endocytic recycling
compartment (ERC), and the Golgi apparatus [40, 92–95].
However, the presence of TRAM and TLR4 in the same
cellular compartment is not sufficient for their interaction.
Detailed studies have revealed that in the plasma membrane, TRAM localizes to regions enriched in CD14 rather
than TLR4 and stimulation of cells with LPS induces the
interaction between TLR4 and TRAM in endosomes [94].
TRAM binds to membranes via a myristoyl residue attached
to Gly2 (after the removal of the N-terminal methionine)
and via a neighboring polybasic domain that mediates its
interaction with various PIs and phosphatidic acid [40].
Both regions are required for the targeting of TRAM to the
plasma membrane; however, only the N-myristoylation governs its location to endosomes [40]. Furthermore, only the
N-myristoylation is indispensible for the TRAM involvement
in the endosomal signaling pathway of TLR4 [40, 93]. Nonmyristoylated Gly2Ala TRAM acted as a dominant negative
mutant interfering with this signaling pathway of TLR4 [93].
The TRIF-dependent signaling pathway of TLR4 includes
activation of the ubiquitin ligase TRAF3 followed by the
activation of non-canonical IKK kinases: TANK binding kinase 1 (TBK1) and IKKε. TBK1 phosphorylates the
pLxIS consensus motif of TRIF that is necessary to recruit
1237
interferon regulatory factor (IRF) 3. IRF3 is also phosphorylated by TBK1 and then dissociates from TRIF, dimerizes
and translocates to the nucleus [96]. Finally, IRF3 and to
a lower extent also TBK1-activated IRF7 induce expression of genes encoding type I IFN, the chemokine CCL5/
RANTES and interferon-regulated genes as that encoding
the chemokine CXCL10/IP-10 [35, 80, 91, 97]. This pathway also participates in the production of the anti-inflammatory interleukin IL-10 [98]. In addition, TRIF induces
late activation of NF-κB via recruitment and activation
of TRAF6 [99] or receptor-interacting serine/threonineprotein kinase 1 (RIPK1) [100]. RIPK1 interacts with the
RIP-homotypic motif (RHIM) present in the C-terminus
of TRIF, which is also recognized by RIPK3 [101]. RIPK1
and RIPK3 are engaged in LPS-induced ERK1/2 activation
and cytokine production but in certain conditions can also
mediate necrotic cell death [102–104]. Ultimately, TLR4 is
degraded in lysosomes [41].
It has recently been established that TLR4 is also involved
in so-called canonical activation of the NRLP3 inflammasome. The LPS-induced activation of this inflammasome
via the canonical and also via a non-canonical, hence not
involving TLR4, pathways has been intensively studied
and discussed in excellent reviews [105–108]. In brief, the
NLRP3 inflammasome is a multimeric complex of NRLP3,
ASC, NEK7 and pro-caspase-1, which ultimately serves to
promote autoproteolysis and activation of caspase-1. In turn,
caspase-1 cleaves pro-IL-1β and pro-IL-18 to their secreted
forms. Caspase-1 also catalyzes the proteolysis of gasdermin
D which binds to the inner surface of the plasma membrane
and forms pores which can lead to cell death via pyroptosis
or release of IL-1β from living cells [109]. Activation of
TLR4 in human and murine macrophages serves as a priming signal inducing expression of NRLP3 and pro-IL-1β and
posttranslational modifications of NRLP3. The assembly of a
fully active inflammasome is triggered by a second stimulus
provided by diverse PAMPs and DAMPs. The exact mechanism leading to the inflammasome formation in response to
these structurally different molecules is not known, but they
all seem to induce cellular stress manifested by, e.g., K+
efflux, mitochondria dysfunction, lysosome damage, which
is then somehow detected by NLRP3. Since the expression
of NRLP3 is triggered in an NF-κB-dependent manner, both
signaling pathways of TLR4 can in principle be involved.
Accordingly, caspase-1 cleavage and activation occurred
in LPS-stimulated macrophages derived from MyD88- or
TRIF-knock-out mice while the double knock-out did not
support it [110, 111].
Activation of the non-canonical inflammasome does
not require TLR4 participation, but is induced by LPS present in the cytosol. LPS directly binds and activates caspase-11 (human caspase-4 and -5) which next activates the
primed NLRP3 inflammasome and induces IL-1 release
13
1238
and pyroptosis. It is unclear how LPS enters the cytosol;
however, recent studies indicate that it can escape from
endosomes which are formed during internalization of
LPS-rich outer membrane vesicles (OMV) derived from
Gram-negative bacteria or from phagosomes enclosing the
bacteria [112]. It can also be internalized by the receptor for
advanced glycation end-products (RAGE) after binding to
HMGB1 [113].
In addition, in human and porcine monocytes (but not
in murine cells), LPS induces NLRP3 activation and IL-1β
release without the second stimulus [114]. Gaidt et al. called
this phenomenon the “alternative inflammasome” and found
that it is induced by TLR4-dependent activation of the TRIFRIPK1-capsase-8 axis upstream of NLRP3 and caspase-1.
While it ultimately leads to IL-1β secretion, no concomitant
pyroptosis of monocytes occurs [115]. Recently, the alternative activation of inflammasome by apolipoprotein C3 and
TLR2/TLR4 heterodimer has been described, pointing to its
involvement in sterile inflammation triggered by triglyceride-rich lipoproteins [116].
In addition to LPS-induced cytokine production, stimulation of macrophages and DC with the endotoxin induces
of adaptive immune responses which are executed by T
and B lymphocytes. The initiation of the adaptive immune
responses follows activation of various TLRs and involves
antigen presentation in the context of major histocompatibility complex (MHC) class I and II, as reviewed by [5,
117]. It has been established that activation of TLR4 by LPS
in both murine DC and macrophages induces upregulation
of costimulatory molecules (CD40, CD80, and CD86) on
the cell surface which are required for antigen presentation
for T lymphocytes. However, significant differences were
found in the signaling pathways leading to this phenomenon.
Namely, in macrophages, the upregulation depends strictly
on the TRIF-dependent pathway, whereas in DC both the
MyD88- and TRIF-dependent ones are involved [118–120].
The increased cell surface presence of the costimulatory
molecules and also of MHC II is a hallmark of DC maturation required for antigen presentation by these cells. LPS
upregulates the cell surface level of MHC II at various steps
of the formation of antigen-MHC II complexes, including
enhanced lysosomal acidification, proteolysis yielding antigens, and transport of the complexes to the surface of DC
[121, 122]. Beside the contribution to the upregulation of
MHC II and costimulatory molecules, the TLR4-triggered
MyD88-dependent signaling in DC also induces production
of cytokines leading to Th1 cell polarization and also facilitates fusion of MHC I-bearing recycling endosomes with
phagosomes to allow cross-presentation of antigens during
infection [123].
The signaling pathways triggered by TLR4 described
above are best characterized in monocytes, macrophages,
and DC; however, it has to be emphasized that certain
13
A. Ciesielska et al.
aspects of the LPS-induced responses vary between DC
and macrophages. In murine DC, LPS induces activation
of nuclear factor of activated T cells (NFAT) and apoptosis
of fully matured DC [124]. Also in these cells, CD11b contributes significantly to TLR4 activation [125]. Additionally,
murine DC vary from human ones since only selected subtypes of the latter expresses TLR4 and CD14, as described
in more detail in the next chapter.
A cell specificity of the LPS-induced responses is also
observed in other immune cells, including human and
murine granulocytes, mast cells, and lymphocytes which
express low amounts of TLR4. Among those cells, CD14
is found in neutrophils and murine basophils [29, 30,
126–129]. Thus, due to a lack of CD14 and low content of
TLR4/MD-2 and TRAM in mast cells, the TRIF-dependent
pathway could not be detected in cells of murine origin
[130]. Therefore, these cells stimulated with LPS produce
TNF-α but not IFN-β [131, 132]. Activation of TLR4 in
neutrophils leads to ROS generation, cytokine production
and other responses which are cell specific, like autophagy
and modulation of cell survival [30, 133–136]. In addition,
in murine neutrophils, LPS induces production and secretion
of histamine in a PI3K-dependent manner [137]. Moreover, LPS can stimulate neutrophils to form NETs (neutrophil extracellular traps), i.e., extracellular fibers composed
of decondensed chromatin and granule proteins which trap
bacteria allowing their extracellular killing [138–140].
In summary, TLR4 activated by LPS triggers two consecutive signaling pathways which are correlated with a redistribution of the receptor: the MyD88-dependent signaling
is triggered by TLR4 localized to the plasma membrane,
while the TRIF-dependent one by the TLR4 internalized in
endosomes. The two pathways lead to a synchronized production of pro- and anti-inflammatory mediators, contribute
to the activation of NLRP3 inflammasome, modulate cell
metabolism, initiate adaptive immune responses and other
cell type-specific reactions; eventually lysosomal degradation of TLR4 takes place. This course of events suggests that
the TLR4-induced pro-inflammatory reactions can be regulated by the rate of its endocytosis and trafficking through
the endo-lysosomal compartment.
Mechanisms controlling internalization
of TLR4
Endocytosis of TLR4 is required for the TRIF-dependent
pro-inflammatory signaling to occur and also for the following degradation of the receptor and termination of the signaling [40, 41]. Somewhat surprising, it has been established
that the LPS-induced internalization of TLR4 is independent of its signaling activity. Thus, a TLR4 mutant lacking
the intracellular part, hence the TIR domain, underwent
TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling
internalization despite being unable to trigger myddosome
formation in murine bone marrow-derived macrophages
(BMDM) stimulated with LPS. The amount of the truncated
receptor in the plasma membrane progressively declined,
similarly as in the case of wild type TLR4 [17]. On the contrary, the extracellular domain of TLR4 was indispensable
for its LPS-induced endocytosis and further studies have
indicated that in fact the interaction of MD-2 with CD14
drives the uptake of the receptor. Thus, the impact of MD-2
on LPS-induced signaling stems from its role in the dimerization of the TLR4/MD-2 complexes during LPS binding
and the contribution to the endocytosis of TLR4 [17].
In most cases, the LPS-induced internalization of TLR4
is controlled by CD14 (Tab. 1) [32, 33]. Exceptions include
TLR4 endocytosis followed by the TRIF-dependent signaling induced by a TLR4/MD-2 agonistic antibody (UT12) or
a synthetic small-molecule TLR4 ligand (1Z105) [141]. And
while phagocytosis of the Gram-negative E. coli did occur
1239
in DC derived from CD14-knock-out mice [33], no LPSinduced endocytosis of TLR4 took place in DC or BMDM
derived from those animals. Accordingly, the TRIF-dependent signaling was abolished, while the MyD88-dependent
one was not affected by the CD14 depletion especially in
cells stimulated with so-called rough chemotype (devoid of
the O-polysaccharide chain) of LPS [31, 32, 34]. In cells
poor in CD14, such as murine splenic B lymphocytes, TLR4
does not undergo endocytosis [33]. The dependence of TLR4
internalization on CD14 has been confirmed by recent studies on CD14 glycosylation. Inhibition of CD14 core fucosylation caused by depletion of α-(1,6)-fucosyltransferase
impaired CD14 and TLR4 endocytosis and thereby the
TRIF-dependent signaling. It was found that the interference
with CD14 fucosylation led to a reduction of its cell surface
level which was considered the main reason of the impaired
TLR4 endocytosis [142, 143]. Conversely, the increase of
Table 1 Effects of down-regulation of selected proteins involved in TLR4 endocytosis and degradation on TLR4 internalization and signaling
Proteina
CD14
TRAM
PLC-γ2/Ca2+
Syk
PI3K p110δ
Arf6
Dynaminb
Dynamin
Clathrinb
Dab2
Hrs
Lystc
Vps33B
GMFGd
Rab7a
Rab7bd
Rab11
Rab10
TMED7
TAG
Effect on
TLR4 internalization
MyD88-dependent
pathway
TRIF-dependent
pathway
↓
↓
↓
↓
↓
↓
↓
↓
↓
nd
↓
↓
↑
nd
–
–
↓
nd
–
nd
↓
nd
nd
↓
nd
↑
–
–
↑
↓
–
↓
↑
↓
–
–
↑
–
↑
↑
nd
↑
–
↓
–
–
↓
↓
↓
↓
↓
↓
↓
↓
↓
nd
↓
↓
↑
nd
↓
↑
↑
↓
↑
↓
↓
↑
↑
Cells
References
macrophages, DC
HEK293, macrophages
macrophages
DC
macrophages
DC
J776, Raw264.7, macrophages, DC
DC, macrophages, Raw264.7
J774, actrocytes
HEK293
Raw264.7, J774, macrophages
astrocytes
Raw264.7
HEK293, monocytes
macrophages, DC
macrophages
macrophages
macrophages
Raw264.7, macrophages
monocytes, HEK293
macrophages, Raw264.7
HEK293, THP-1, macrophages, PBMCs
HEK293, THP-1, PBMCs
[17, 33, 160]
[40, 93]
[33]
[33, 150]
[33]
[74]
[73, 152, 153]
[40, 74, 155]
[159, 160]
[41]
[155, 160, 265]
[159]
[166]
[41]
[183]
[180]
[196]
[183]
[184]
[95]
[179]
[182]
[181]
↓ Down-regulation, ↑ upregulation, nd no data
a
b
c
d
In most cases, proteins were down-regulated by knock-out or knock-down of encoding genes
Inhibition of the protein caused by a drug treatment
Deleterious mutation
Overexpression of the protein had opposite effects on the MyD88- and TRIF-dependent pathways
13
1240
CD14 level during maturation of murine DC accelerates the
LPS-induced endocytosis of TLR4 [33].
Notably, it has been established that CD14 undergos
low-rate constitutive endocytosis also in unstimulated cells
[17]. The decreasing pool of cell-surface CD14 is replenished by the newly synthesied protein. Binding of LPS to
TLR4/MD-2 and the concomitant interaction of MD-2 with
CD14 converts the TLR4/MD-2/LPS complex into a cargo
of CD14. Simultanously, the rate of CD14 internalization is
accelerated [17, 144]. On the basis of those data, CD14 has
earned the name of “transporter associated with the execution of inflammation” (TAXI) [17].
The mechanism of the CD14-dependent internalization
of TLR4 remains to be revealed, but it is likely linked with
the raft localization of CD14, activation of the Syk tyrosine
kinase, and local turnover of PI(4,5)P2 [33, 50, 145]. CD14
triggers biphasic generation of PI(4,5)P2 in LPS-stimulated
macrophages and the newly generated PI(4,5)P2 accumulates
in the raft fraction of these cells. The PI(4,5)P2 generation
correlated with a biphasic activation of NF-κB and was
required for maximal production of cytokines in the both
signaling pathways of TLR4 [145, 146]. An increase of the
PI(4,5)P2 level can be needed for the recruitment of TIRAP
to the plasma membrane, while the following PI(4,5)P2
hydrolysis/phosphorylation is required for the TLR4 endocytosis to occur. The uptake of TLR4 can be initiated by the
Syk kinase which binds to the immunoreceptor tyrosinebased activation motif of DAP12 which is an adaptor of
CD300b receptor, and/or the γ chain of Fcε receptor [147].
Subsequently, Syk activates phospholipase Cγ2 (PLCγ2).
Indeed, inhibition/knock-down of Syk or PLCγ2 abolished
TLR4 internalization in LPS-stimulated murine BMDM and
DC [33, 50, 147]. It deserves to be mentioned that Syk/
PLC-γ2 inhibitors blocked not only the LPS-, but also the
UT12-induced endocytosis of TLR4 that proceeded without
a CD14 involvement [141]. PLCγ2 cleaves PI(4,5)P2 to DAG
and inositol 1,4,5-trisphosphate ( IP3) which induces C
a2+
release from the endoplasmic reticulum. Inhibition of this
process by 2-ABP, an antagonist of IP3 receptor localized
predominantly in the endoplasmic reticulum, blocked TLR4
endocytosis and the TRIF-dependent signaling [148]. PI(4,5)
P2 is also a substrate for class I PI3-kinase which phosphorylates it to PI(3,4,5)P3. Mutation of the p110δ catalytic
subunit of PI3-kinase in murine DC slowed down the rate
of LPS-induced endocytosis of TLR4 while phagocytosis of
E. coli was not affected [74]. In addition, CD14 controls the
influx of extracellular Ca2+ mediated by TRPM7, a plasma
membrane cation channel with kinase activity. A knock-out
of TRPM7 inhibited endocytosis of CD14/TLR4 in murine
BMDM. Furthermore, phosphorylation and nuclear translocation of IRF3 and also of NF-κB were inhibited and the
following production of the TRIF-dependent cytokines was
reduced. These processes were depended on the influx of
13
A. Ciesielska et al.
extracellular Ca2+ rather than on the release of Ca2+ from
intracellular stores, although an input of the latter to the regulation of TLR4-induced signaling was not precluded [149].
Since the downstream effectors of Syk, PLCγ2 and PI3kinase, are involved in a broad spectrum of cellular processes [74, 150, 151], their activity is likely to affect various
other aspects of TLR4-induced signaling in addition to the
receptor endocytosis. Nevertheless, the impaired internalization of TLR4 observed after inhibition/mutation/knockdown of those enzymes correlated with a down-regulation
of the TRIF-dependent signaling reflected by reduced IRF3
phosphorylation and impaired expression of genes encoding ISG54/IFIT-2, TRAIL, CCL5/RANTES, and IFN-β. The
MyD88-dependent production of cytokines was not affected
or was even elevated at the expense of the TRIF-dependent
one [33, 74, 148].
Another important protein involved in PIs turnover taking
part in the LPS-induced TLR4 endocytosis and signaling is
the small GTPase ADP-ribosylation factor 6 (Arf6). Arf6
activates PI4-phosphate 5-kinase type Iα (PIP5KIα) which
phosphorylates PI(4)P to PI(4,5)P2 [73]; therefore, its activity can modulate TLR4 signaling, endocytosis and trafficking. Indeed, PIP5KIα and PIP5KIγ were found to colocalize
with CD14 in murine macrophage-like J774 cells, and their
silencing reduced LPS-dependent production of TNF-α and
CCL5/RANTES [145]. In Arf6-deficient Raw264.7 cells,
the LPS-induced activation of NF-κB was lower than in their
wild type counterparts suggesting a down-regulation of the
MyD88-dependent signaling due to insufficient PI(4,5)P2
generation in the plasma membrane [73, 152]. Moreover, in
these conditions, the cell surface level of TLR4 was also elevated and increased even further after stimulation of the cells
with LPS likely due to an inhibited endocytosis of the receptor [152]. Furthermore, a depletion or inhibition of Arf6
blocked the TRIF-dependent signaling, which was reflected
by a reduced phosphorylation of IRF3 and decreased expression of Ifnβ1 [152, 153].
Recent studies have indicated that the endosomal signaling of TLR4 is negatively regulated by prostaglandin E2
in thioglycollate-elicited peritoneal murine macrophages.
Inhibition of prostaglandin E2 receptor EP4 or inhibition of
prostaglandin E2 synthesis enhanced the LPS-induced internalization of TLR4 and Syk activation, and consequently
augmented the TRIF-dependent signaling [86]. Endocytosis
of TLR4 is also negatively regulated by CD13 metallopeptidase [154]. CD13 associates with TLR4 in resting murine
BMDM and DC and its expression is enhanced in LPSstimulated cells. CD13 undergoes LPS-induced endocytosis
together with TLR4 and CD14 and these proteins colocalizes
in Rab5-positive endosomes. However, a knock-out of CD13
enhanced TLR4 endocytosis in macrophages and increased
IRF3 activation and IFN-β production. Accordingly, mice
lacking CD13 displayed enhanced IFN-β-triggered signaling
TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling
leading to exacerbated ischemic muscle injury. The mechanism of the CD13 involvement in TLR4 endocytosis remains
unknown, but it could be related to the raft localization of
CD13 [154].
The LPS-induced internalization of TLR4 occurs via
clathrin-independent and/or clathrin-dependent pathways,
while E. coli bacteria undergo phagocytosis in which TLR4
is involved [3, 33, 41, 155, 156]. The TLR4 endocytosis
depends on dynamin, a GTPase responsible for pinching off
of clathrin-coated buds, some macro- and micropinosomes,
and phagosomes [157, 158] inhibition of dynamin with
dynasore prevented LPS-induced internalization of TLR4
in several cell types, including macrophages [40, 141, 155,
159]. Accordingly, in such conditions, the TRIF-dependent
signaling was inhibited [40, 74, 95, 155, 159–161]. In addition, dynasore attenuated the LPS-induced cleavage of caspase-3 in murine microglia and almost completely abolished
expression of IL-1β in these cells and in rat astrocytes overexpressing TLR4 [156, 159]. In HEK293 cells expressing
TLR4/CD14 and a dominant negative dynamin II mutant
(Dyn K44A), the NF-κB activation was upregulated in comparison to cells overexpressing wild-type dynamin, suggesting an upregulation of the MyD88-dependent signaling [41].
In contrast to the well-defined role of dynamin in LPSinduced TLR4 internalization, the role of clathrin in this
process is more puzzling. Early electron microscopy studies suggested that endocytosis of LPS and CD14 is mainly
clathrin-independent, yet no direct observation of TLR4
was possible at that time [162, 163] (see below). On the
other hand, at least partial colocalization of TLR4 with
transferrin, a typical cargo of clathrin-coated vesicles, was
observed in HEK293 cells and in Ba/F3 cells transfected
with TLR4/MD-2 and CD14 and stimulated with LPS for
45–60 min [41, 94]. In addition, partial colocalization of
TLR4 with clathrin at the plasma membrane was observed
in the HEK293 transfectants and in U373 glioma cells transfected with TLR4 and CD14 [164]. The authors claimed
that in the spots of TLR4 that were positive for clathrin also
CD14 was accumulated; however, it has to be noticed that
they did not label CD14 directly but only observed Cy5labeled LPS. In addition, in all the studies cited above CD14
and TLR4/MD-2 transfectants were used, which warrants
their cautious interpretation in view of the relatively low
level of TLR4 expression in native macrophages. Nevertheless, a line of studies using inhibitors of clathrin-dependent
endocytosis support its contribution to TLR4 internalization. Monodansylcadaverine and chlorpromazine inhibited
the LPS-induced internalization of TLR4 in Raw264.7
cells and in rat astrocytes overexpressing TLR4 [155, 159,
165]. Chlorpromazine also reduced production of CCL5/
RANTES and IFN-β in those cells, and of IFN-β in murine
DC and macrophages [155, 159, 165]. Similarly, incubation of J774 cells with another clathrin inhibitor, pitstop-2,
1241
reduced LPS-induced TNF-α and CCL5/RANTES production; however, the effect was weaker than that of dynasore
[160]. Similarly, silencing of raftlin, a protein interacting
with the heavy chain of clathrin and with clathrin-related
adaptor protein AP-2 also reduced TLR4 internalization and
negatively regulated the TRIF-dependent signaling pathway
in human and murine cells poor in CD14 [165]. This additionally proves an engagement of clathrin in the internalization of TLR4 and points to the role of raftlin in this process
[165].
Recent studies have revealed that disabled-2 (Dab2), an
adaptor protein involved in the recruitment of some transmembrane receptors, e.g., low-density lipoprotein receptor,
to clathrin-coated pits can also affect the TLR4 internalization [166]. Silencing of Dab2 in Raw264.7 cells increased
the surface level of TLR4 in unstimulated cells but accelerated its LPS-induced endocytosis. In accordance, phosphorylation of IRF3 and expression of TRIF-dependent genes,
including Cxcl10, Ifit1, Ccl5 and Ifnβ1, was upregulated
in these cells. Since TLR4 lacks a binding site for Dab2,
the authors postulated that at steady state Dab2 binds and
sequesters clathrin, thereby preventing TLR4 endocytosis.
Stimulation of cells with LPS induces phosphorylation of
Dab2 probably causing its dissociation from the plasma
membrane and the following release of clathrin, and thereby
allowing the clathrin-dependent internalization of TLR4.
Thus, Dab2 can act as a negative regulator of TLR4 internalization [166].
Finally, the difference in the regulation of TLR4 endocytosis between murine macrophages and DC deserves attention. It was mentioned above that murine DC but not macrophages can phagocyte E. coli without a CD14 involvement
[33]. Additionally, due to the lower level of CD14 in DC
than in macrophages only in DC the endocytosis of TLR4 is
facilitated by CD11b integrin [125]. CD11b in DC promotes
both the MyD88-dependent and TRIF-dependent signaling
of TLR4 in addition to its engagement in the receptor endocytosis. CD11b is also crucial for TLR4-triggered activation of the adaptive immunity by T lymphocyte [125]. The
difference in the responses to LPS between macrophages
and DC can be even more pronounced in humans. Only one
human conventional (myeloid) DC subtype, cDC2 (also
called CD1C +), expresses TLR4 among them only subsets
CD5low/high expresses CD14, whereas murine conventional
DC express both Tlr4 and Cd14 [29, 124, 167–172]. In
addition, in human DC and also monocytes, TLR4 seems
to be mainly intracellular [95, 173]. This suggests that in
human DC, details of CD14-TLR4 signaling and trafficking can differ from those in their murine counterparts or in
human macrophages. In addition, recent studies of murine
mast cells have revealed that TLR4 trafficking in these cells
can be regulated by huntingtin [174].
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In summary, although many gaps in the understanding
of the mechanisms controling TLR4 endocytosis remain,
the crucial roles of CD14, PIs turnover, and dynamin in
this process have been firmly established. Notably, modulation of the duration of the TLR4 presence at the plasma
membrane after binding of LPS can affect the duration of
the MyD88-dependent signaling and reciprocally also the
activation of the TRIF-dependent endosomal pathway. The
TLR4-triggered signaling is also modulated by the mechanisms controlling the intracellular trafficking of the receptor.
Intracellular trafficking of TLR4: an overview
TLR4 is synthetized in the endoplasmic reticulum where
gp96, a paralog of Hsp90, is involved in proper folding of
the receptor, while PRAT4A participates in its maturation
(glycosylation). These proteins control folding and maturation of all TLRs [175, 176]. The newly synthetized TLR4
is transported to the Golgi apparatus, where it associates
with MD-2; potentially TLR4 can reach the cell surface
without an MD-2 assistance and then bind secreted MD-2
[177, 178]. In unstimulated cells, TLR4 can be detected not
only in the endoplasmic reticulum, Golgi apparatus and the
plasma membrane but also in Rab11-positive ERC [92, 94,
95, 179]. Latz et al. showed that in HEK293 cells transfected
with TLR4 and MD-2, the receptor cycled between the Golgi
apparatus and the plasma membrane. This suggested that
the TLR4/MD-2 complex is internalized and directed to
the Golgi apparatus to be transported back to the plasma
membrane [92]. Later studies indicated that LPS binding
changes the intracellular trafficking of TLR4 and revealed
the complexity of this process. Thus, LPS-activated TLR4
is internalized and thereby its amount at the cell surface
decreases with the duration of the stimulation [17, 40, 141].
TLR4 internalized by murine immortalized BMDM stimulated with 1 μg/ml LPS is first (after ~ 10 min of stimulation)
detected in submembrane Rab5-positive vesicles related to
early endosomes (Fig. 2, I) [180]. In murine microglia (BV2
cells), the TLR4 translocation to the Rab5-positive vesicles
seems to be slower than in murine macrophages since a colocalization of TLR4 and Rab5 was visible only after 2 h of
stimulation with the same LPS concentration [156], suggesting that the dynamics of TLR4 endocytosis is cell type specific. In the Rab5-positive endosomes TLR4 interacts with
TRAM and activates the TRIF-dependent signaling cascade
leading to the production of type I IFN and expression of
IFN-induced genes. Next (after ~ 30–60 min of stimulation
of immortalized BMDM) TLR4 is found in the membrane
of Rab7-positive endosomes identified as late endosomes,
where it also colocalizes with TRAM (Fig. 2, II) [180]. The
endosomes marked by Rab7a or Rab7b are multifunctional
in the context of TLR4 activity as the TRIF-dependent
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A. Ciesielska et al.
signaling can be maintained in some of them while others
are sites of TLR4 degradation that progresses in lysosomes
[94, 181–184], as discussed in more detail in the section
below.
Ubiquitination is likely a signal targeting TLR4 for degradation and it increases significantly after stimulation of
cells with LPS, but the exact type of this modification has
not been established [41]. Ubiquitinated TLR4 is recognized
by hepatocyte growth factor regulated tyrosine kinase substrate (Hrs), an early endosome-associated component of the
ESCRT-0 complex which, together with other ESCRT complexes, causes clustering of the receptors and targets them
to intraluminal vesicles in multivesicular bodies (MVB) and
consequently to lysosomal degradation (Fig. 2, III and IV)
[41].
Beside the Rab5- and Rab7-positive endo-lysosome
compartment, in Raw264.7 cells, LPS-activated TLR4
(~ 10–20 min) is also found in Rab10-positive vesicles identified as a subtype of recycling endosomes, while prior to the
stimulation TLR4 is located to the Golgi apparatus where
also Rab10 can be detected (Fig. 2, V). Silencing of Rab10
decreased the amount of TLR4 in the plasma membrane
and diminished the activation of both the MyD88- and the
TRIF-dependent signaling pathway. It has been, therefore,
proposed that Rab10 controls the replenishing of the plasma
membrane pool of LPS-activated TLR4 from its reservoir
in the Golgi apparatus [179]. Additionally, recent data have
indicated that in J774 cells stimulated with LPS the plasma
membrane pool of TLR4 can be replenished by de novo
synthetized protein as well as by recycling of previously
internalized receptor. The recycling route of TLR4 probably
engages the ERC and ERC-related vesicles in which internalized TLR4 is transported back to the plasma membrane.
This process depends on the plasma membrane-localized
SNARE proteins syntaxin 11 and its partner SNAP-23 which
can regulate fusion of the TLR4-bearing vesicles with the
plasma membrane. A knock-down of syntaxin 11 or SNAP23 suppressed the LPS-induced transport of TLR4 to the
cell surface and promoted its lysosomal degradation [185].
Recycling of TLR4 can be down-regulated by TRPM7, as
suggested by Granucci’s analysis of the TLR4 reappearance
on the surface of TRPM7-depleted and LPS-stimulated macrophages [186].
Finally, unusual Rab11-dependent trafficking of an intracellular pool of TLR4 bypassing its plasma membrane
localization was detected during phagocytosis of E. coli
by human monocytes (Fig. 2, VI) [95, 187]. Rab11-positive vesicles also carry TRAM to the phagosomes, while
in LPS-stimulated cells they participate in transporting
GOLD domain-containing proteins to TLR4/LPS-harboring endosomes, thereby affecting the duration of the TRIFdependent signaling [181, 182]. Thus, the Rab11-positive
compartment plays an important role in the regulation of
TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling
1243
Fig. 2 Intracellular trafficking pathways of TLR4 and TRAM.
In unstimulated cells, TLR4 is present in the plasma membrane,
Golgi apparatus and the ERC, whereas its adaptor protein TRAM is
located in the ensosomes, the plasma membrane and Golgi apparatus. (I) After activation by LPS, TLR4 is internalized and translocates to Rab5- and EEA1-positive early endosomes where it interacts
with TRAM and TRIF and initiates the TRIF-dependent signaling.
(II) The association of TRAM with TLR4 and the activation of the
TRIF-dependent pathway is sustained during maturation of early
endosomes to Rab7a- or Rab7b-positive late endosomes. In late
endosomes TMED7 and TAG disrupt the TRIF—TRAM interaction
and facilitate termination of the signaling. (III) In early endosomes,
Hrs and ESCRT recognize ubiquitinated TLR4 and sort it for degradation in late endosomes/MVB and lysosomes. (IV) TLR4 degrada-
tion and signaling can be regulated by proteins involved in the functioning of the endo-lysosomal compartment, like Lyst, GMFG, Rab7a
and Rab7b, and Vps33B. (V) Upon stimulation with LPS, the intracellular TLR4 originally associated with the Golgi apparatus is transported via Rab10-positive endosomes to the plasma membrane. (VI)
The intracellular pool of TLR4 derived from the ERC can trigger the
TRIF-dependent signaling at the phagosome membrane. Both TLR4
and TRAM are transported to the phagosome membrane with a contribution from Rab11. (VII) Possible intracellular transport of TRAM
in Rab11-positive vesicles to the early endosomes. It is unknown
whether the well-established protein trafficking pathway from the
early endosomes to the Golgi apparatus (dashed gray arrow) can be
used by TLR4
the TRIF-dependent signaling pathway of TLR4 discussed
in detail in the following section.
The data presented above indicate that activation of
the TRIF-dependent signaling cascade is coupled with
the trafficking of TLR4 through endo-lysosomal compartments. Therefore, the process of transformation of
early endosomes into late endosomes and lysosomes,
which is correlated with changes of the protein and lipid
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composition of their membrane, acidification of the lumen
and enrichment in hydrolytic enzymes, is crucial for both
the signaling and the degradation of TLR4.
Regulation of TLR4 signaling
in endo‑lysosomal compartment
Progressive acidification of the endosome interior provides
an optimal environment for the dissociation of ligands
from their receptors and for the activity of hydrolytic
enzymes. In agreement, acidification of endosomes was
found to induce splitting of TLR4/MD-2 dimers and dissociation of LPS from the receptor [188, 189], albeit the
pH optimum for those processes has not been determined.
In general, activation of endosomal proteases facilitates
degradation of the internalized proteins, but for endosomal
TLRs a limited proteolysis catalyzed by cathepsins and
asparaginyl endopeptidase is necessary for their dimerization and signal transduction [152, 190]. Notably, TLR4
does not undergo such modification and binding of LPS
overcomes the repulsion of two receptor ectodomains
and forces TLR4 dimerization [69]. The acidic pH of
endosomes could facilitate induction of the TRIF-dependent signaling by TLR4. Such hypothesis has been put forward by Ganglioff and co-workers based on their analysis of the crystallographic structure of TLR3, a receptor
residing in endosomes and triggering the TRAM/TRIFdependent signaling pathway. By analogy with TLR3, the
acidic environment of endosomes (pH 5.5) would induce
changes in the position of TLR4 ectodomains which after
internalization face the endosome lumen. Concomitant
conformational changes of the transmembrane and cytosolic regions of the receptor together with specific features
of the endosome membrane, such as its high curvature
and the presence of distinct species of PIs, could facilitate
recruitment of TRAM to TLR4 [191].
Two approaches have been used to examine the role
of endosome acidification in LPS-induced signaling of
TLR4. Treatment of Raw264.7 cells with chloroquine,
which neutralized the pH of the endosome interior,
enhanced the depletion of LPS-activated TLR4 from the
cell surface [155]. In agreement, chloroquine was shown
to induce intracellular accumulation of LPS-activated
TLR4 instead of its degradation, thus the stability of the
TLR4/MD-2 complex was also prolonged [41, 189]. In
chloroquine-treated Raw264.7 cells, the production of the
MyD88- and TRIF-dependent cytokines was decreased,
which was interpreted as a result of impaired recycling of
TLR4 from endosomes to the plasma membrane [155]. On
the other hand, knock-down of a ATP6V0D2, a subunit of
the V-ATPase responsible for acidification of endosomes,
attenuated the TRIF-dependent signaling pathway in
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A. Ciesielska et al.
Raw264 cells as reflected by a decreased expression of
Ifnb1 and upregulated expression of the MyD88-dependent
Tnfa, Il6 and Il12p40 [152]. In this case, the enhancement
of the MyD88-dependent signaling and the reduction of
the TRIF-dependent one correlated with reduced endocytosis of TLR4 and its prolonged maintenance on the
cell surface. At the basis of this effect was an impaired
interaction of the V-ATPase complex with Arf6 caused
by the silencing of ATP6V0D2 [152], indicating that the
regulation of TLR4 signaling via V-ATPase goes beyond
controlling the endosome acidification and also involves
its influence on the Arf6 activity. Ample data indicate that
several other proteins which determine the functionality of
the endo-lysosomal compartment, such as Lyst, Vps33B,
GMFG and Rab7b, affect the TLR4-triggered signaling
and the receptor degradation (Table 1).
Lyst is a lysosomal trafficking regulator contributing to
endo-lysosomal biogenesis and also takes part in terminal
maturation of secretory vesicles [192]. Structurally, Lyst
belongs to BEACH domain-containing proteins which are
large scaffolding proteins associated with cellular membranes due to the interaction of their PH domain with phospholipids, mainly PIs [193]. In murine BMDM and bone
marrow-derived DC (BMDC) bearing a mutation in the Lyst
gene leading to the production of a dysfunctional Lyst, neither the LPS-induced MyD88-dependent activation of MAP
kinases and IκB nor the TLR4 endocytosis were affected;
however, the TRIF-dependent IRF3 phosphorylation and its
translocation to the nucleus were impaired. In agreement,
that deleterious mutation of Lyst decreased the production
of IFN-β, TNFα, and IL-12. Similar effects were observed in
THP-1 cells of human origin. In vivo studies of pulmonary
inflammation in mice showed that the Lyst dysfunction led
to a lower production of TNF-α and IFN-β after administration of LPS, and protected the animals from endotoxininduced lethal shock. This suggests that the dysfunction of
the endo-lysosomal compartment observed in cells expressing the mutated form of Lyst inhibited the TRIF-dependent
signaling. One can only speculate whether this impairment
resulted from a faster degradation of TLR4 or an inefficient
formation of its endosomal signaling complex. Later studies on the phagocytosis of E. coli and LPS-coated beads
by BMDC expressing the mutated Lyst indicated that the
recruitment of Rab7 (also called Rab7a) to maturing phagosomes, but not their acidification, was affected [183].
Vacuolar protein sorting-associated protein (Vps) 33B,
a homolog of yeast Vps33p, is another factor regulating
TLR4 signaling and degradation. Mammalian Vps33B and
closely related Vps33A are soluble proteins of the Sec1/
Munc18 family which, together with SNAREs, allow
fusion of intracellular compartments with lysosomes.
Vps33B interacts with the VIRAP protein and with Rab10,
Rab11, and Rab25 [194, 195]. After silencing of Vps33B
TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling
in murine macrophages, the phagosomal clearance of E.
coli was impaired even though the rate of phagocytosis was
not changed. Furthermore, the trafficking of LPS-activated
TLR4 to Rab5- and further to Rab7-positive endosomes
was also similar to that in wild-type cells. In both Vps33Bdepleted and control macrophages, TLR4 was also present
in Rab11-positive endosomes. However, degradation of
LPS-activated TLR4 was impaired after Vps33B depletion and the receptor accumulated in LPS-stimulated cells,
which resulted in an overproduction of cytokines such as
IL-6, TNF-α and IFN-β. Collectively, these data suggest
that Vps33B has an important function in the maturation of
phagosomes and also in a late step of endosome maturation,
i.e., the fusion with lysosomes. Therefore, Vsp33B can be
viewed as a negative regulator of the TLR4-induced proinflammatory response [180].
Glia maturation factor-γ (GMFG) is a newly discovered
actin depolymerization factor which binds the Arp2/3 complex and inhibits nucleation of actin monomers and also
promotes debranching of actin filaments, processes important for endosome trafficking. GMFG is expressed mainly
in immune cells and microvascular endothelial cells, where
it facilitates recycling of internalized β1-integrin [196].
GMFG also affects the internalization and trafficking of
LPS-activated TLR4 and thereby the TLR4-induced proinflammatory responses. In THP-1 cells, GMFG associates
with early and late endosome markers, including EEA1,
Rab5, and Rab7. Depletion of GMFG in those cells and in
primary human macrophages resulted in a delayed internalization of TLR4 and its abnormal accumulation in the plasma
membrane and Rab5-positive vesicles. This retention of
TLR4 in the plasma membrane and in early endosomes significantly enhanced the LPS-induced activation of NF-κB,
MAP kinases and IRF3, and the production of TNF-α, IL-6
and IFN-β at both the mRNA and protein level. In accordance, GMFG overexpression decreased the production of
pro-inflammatory cytokines. Interestingly, production of
the anti-inflammatory IL-10 was slightly increased in these
conditions [196].
Rab7b is expressed selectively in some cell types, e.g.,
in monocytes and monocyte-derived immature DC, and is
involved in the transport of cargo to late endosomes and
lysosomes [184, 197, 198]. Silencing of Rab7b in Raw264.7
cells increased the overall level of TLR4, including its cellsurface pool [184]. More-detailed studies on TLR4 localization in EEA1- and LAPM-1-positive compartments indicated that silencing of Rab7b caused accumulation of TLR4
in early endosomes, while overexpression of Rab7b led to
an accelerated TLR4 translocation to late endosomes/lysosomes. Those data suggested that Rab7b is required for
lysosomal degradation of TLR4. Indeed, overexpression
of Rab7b in peritoneal macrophages and Raw264.7 cells
decreased TLR4 protein level without affecting its mRNA
1245
level [184]. An increased expression of Rab7b also downregulated TLR4 surface level in human DC treated with a
water-soluble fraction of the worm Trichuris suis [199]. In
addition, studies on the cerebral ischemic stroke in the rat
revealed an increased expression of Rab7b in the brain after
the stroke. In turn, overexpression of Rab7b in rat brain
reduced the TLR4 and NF-κB levels following the stroke and
also reduced the production of pro-inflammatory cytokines
in both signaling cascades of the receptor. Thus, the overexpression of Rab7b down-regulated the activation of MAP
kinases, NF-κB and IRF3 as well as inhibited the production
of IL-6 and IFN-β in the rat brain [200]. Also in other studies, overexpression of Rab7b reduced the TLR4-triggered
cytokine release, including that induced by a water-soluble
fraction of T. suis in human DC [199]. It should be emphasized, however, that the overexpression of Rab7b led to a
down-regulation of NF-κB level in addition to that of TLR4,
suggesting that also this effect contributed to the reduction
of the TLR4-triggered pro-inflammatory responses [199,
200].
Rab7a and Rab7b exhibit 65% sequence similarity [197]
and have partially overlapping cellular localizations and
functions in maturation of endosomes, yet the two proteins
regulate TLR4 signaling differently. Rab7a is required for
the activation of the TRIF-dependent signaling cascade in
LPS-stimulated BMDMs and does not interfere with the
MyD88-dependent one. Also during phagocytosis of LPScoated beads, Rab7a-positive phagosomes recruited TBK1,
the kinase responsible for phosphorylation of IRF3 [183].
Recent studies have indicated that both Rab7a and Rab7b
can be involved in retrograde transport of cargo from late
endosomes to the trans-Golgi network and called into question the involvement of Rab7b in the transport of selected
proteins from early to late endosomes [198, 201]. The retrograde transport is mediated by a multiprotein complex called
retromer which retrieves membrane-bound receptors, e.g.,
cation-independent mannose-6-phosphate receptor, from
endosomes and transports them to the trans-Golgi network
and to the plasma membrane. The core of the retromer is
formed by the heterotrimer of Vps35, 29 and 26. Silencing
of Vps35 in microglia increased their inflammatory response
to LPS, which was linked with an impaired trafficking of
Trem2, an immunomodulatory microglial surface receptor
[202]. Taking into account that Rab7a, Rab7b, and Vps35
alter the LPS-induced inflammatory responses it seems
plausible that retrograde transport can be important for the
recycling/retrieval of TLR4, but further studies are required
to confirm this hypothesis.
Maturation of endosomes is linked with changes
of the lipid composition of their membrane. Our studies also indicated that such changes are important for
the regulation of the intensity of TLR4-dependent proinflammatory responses. We found that accumulation of
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lysobisphosphatidic acid, a phospholipid enriched in late
endosomes/lysosomes, strongly inhibited the TRIF-dependent signaling cascade and decreased LPS-dependent CCL5/
RANTES production [203].
Taken together, the data discussed above indicate that
disturbances in the endocytosis of LPS-activated TLR4
and also dysfunctions of the endosomal/lysosomal machinery ultimately affect TLR4-triggered pro-inflammatory
responses. A proper maturation of endosomes is necessary
for the TRIF-dependent signaling to occur and can also
affect the MyD88-dependent one, while slowing down of the
TLR4 degradation increases the pro-inflammatory response.
Whether a retrograde transport of TLR4 from Rab7-positvie
endosomes via the Golgi to the plasma membrane, which
could sustain activation of the receptor by extracellular LPS,
is a physiological phenomenon remains an intriguing open
question.
Rab11‑positive recycling endosomes
in LPS‑induced trafficking of TLR4 and TRAM
Early endosomes identified by Rab5 presence provide a sorting environment for diverse internalized membrane proteins.
Some of them recycle back to the trans-Golgi network or to
the plasma membrane, others are directed to lysosomes for
degradation. In general, the routes of protein recycling to the
plasma membrane are classified as “fast” and “slow”. Fast
recycling involves Rab4- or Rab35-marked vesicles which
bud off of the early endosomes and next fuse with the plasma
membrane [204]. On the other hand, proteins retrieved by
the slow recycling route are delivered from early endosomes
to the Rab11-positive ERC closely opposing the Golgi apparatus, and then are transported back to the plasma membrane
by Rab11- and EHD1- or Arf6-positive recycling endosomes
[153, 204, 205]. Rab11-poisitive vesicles participate in the
trafficking of the archetypical recycling protein, the transferrin receptor [206].
Rab11-positive compartments are also crucial for the
TLR4 trafficking and activation during phagocytosis of
Gram-negative bacteria. It was found that Rab11-bearing
ERC is a source of TLR4 that is transported in a Rab11dependent manner toward forming phagosomes (Fig. 2, VI).
This intracellular pool of TLR4 omits the plasma membrane
and triggers the TRIF-dependent signaling at the phagosome
membrane. TRAM, required at the onset of this signaling
pathway, is also transported to the phagosome membrane
with a contribution from Rab11. Local accumulation of
IRF3 and production of IFN-β follow [95]. Further studies have revealed that FIP-2, a Rab11 effector protein, is
required for the TRAM recruitment to the forming phagosomes. Rab11, FIP-2, TRAM, and TRIF assembly into a
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A. Ciesielska et al.
complex which is recruited to the TIR domain of activated
TLR4. FIP-2 guides the TRAM accumulation at the forming phagosomes to activate Rac1 and Cdc42, thereby governing actin filament rearrangement and the uptake of the
bacterium, and also to induce the TRAM/TRIF-dependent
signaling of TLR4 [187].
Rab11-positive recycling endosomes can also be involved
in the intracellular trafficking of TLR4 during stimulation of
cells with LPS. Indeed, a FRAP analysis of TLR4 mobility
in LPS-stimulated cells revealed its high dynamics suggesting that TLR4 enters and exits the ERC [164]. Moreover,
silencing of Rab11 or FIP-2 inhibited the TRIF-dependent
signaling of TLR4 triggered by LPS, resembling the effects
seen during phagocytosis [95, 187]. At present there are
no other data showing that during stimulation of cells with
LPS Rab11-positive vesicles can transport TLR4 from its
intracellular reservoir directly to endosomes. More likely is
a contribution of Rab11 to the transport of TRAM toward
the endosomes which acquired LPS-TLR4 from the plasma
membrane following induction of the MyD88-dependent
signaling cascade. Rab11 can determine the localization of
TRAM in unstimulated cells. Overexpression of a Rab11
in HEK293 cells expressing also TLR4, MD-2, CD14, and
TRAM resulted in accumulation of TRAM in the ERC,
which was concomitant with its depletion in the Golgi
apparatus [164]. As mentioned above, the pool of TRAM
involved in the endosomal TLR4 signaling does not traffic
to endosomes from the plasma membrane [40, 182]; therefore, its possible sources include the Golgi apparatus and
the Rab11-positive ERC (Fig. 2, VII). TRAM can be accumulated in the ERC with the help of Arf6-dependent transport from the vicinity of the plasma membrane [153]. Upon
a subsequent stimulation of cells with LPS, the TRAMRab11-FIP-2-TRIF complex can be transported from the
ERC to LPS/TLR4-harboring endosomes where the TRIFdependent signaling is triggered [95, 164]. This Rab11dependent trafficking of TRAM could facilitate activation
of the endosomal signaling pathway of TLR4. Expression
of a dominant negative or constitutively active Arf6 variant
interfered with the endosomal TLR4 signaling indicating
its dependence on the Arf6-mediated intracellular TRAM
trafficking [153].
Rab11 is also involved in the delivery to endosomes of
two other proteins—transmembrane emp24 domain-containing protein (TMED) 7 and TRAM adaptor with GOLD
domain (TAG), both involved in the TRIF-dependent signaling of TLR4. TMED7 and TAG cooperate to promote termination of TLR4 signaling. Silencing of TAG or TMED7
resulted in upregulation of the TRIF-dependent signaling
reflected by an increase of the ISRE reporter gene expression and enhanced production of CCL5/RANTES. Moreover, in cells with a reduced level of TAG or TMED7 degradation of TLR4 was inhibited [181, 182]. At the basis of
TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling
the regulatory function of TMED7 and TAG in TLR4-triggered signaling is their negative influence on the stability
of the TRAM/TRIF complex. These two proteins contain a
GOLD domain allowing their homotypic interaction [181,
182]. TAG, expressed exclusively in humans, is a splicing
variant of TRAM and also carries the TIR domain [181].
Upon binding to the TIR domain of TRAM, TAG displaces
TRIF from the TRAM/TRIF complex. In unstimulated cells,
TAG localizes mainly to the endoplasmic reticulum while
TMED7 is present in the Golgi apparatus. After LPS stimulation, the two proteins traffic to endosomes. The colocalization of TMED7 with TLR4 and TRAM begins in EEA1positve early endosomes and culminates in Rab7-positive
late endosomes where TMED7 facilitates disruption of the
TRAM/TRIF complex by TAG, which eventually leads to
degradation of TLR4 [181, 182].
The above data indicate that Rab11-positive compartments are involved in the LPS-induced trafficking of TRAM
and also of other proteins regulating the LPS-induced
TRAM/TRIF-dependent signaling. In the following sections, we discuss how the TLR4-triggered signaling can be
affected by cellular trafficking of CD14 and internalization
of LPS.
Cellular trafficking of CD14 and its relation
to the trafficking of TLR4
The participation of CD14 in the activation of TLR4 by
LPS and in the endocytosis of LPS-activated TLR4 followed by the TRIF-dependent pro-inflammatory signaling
is well established, as discussed above. Notably, CD14 is
also involved in the internalization of high doses of LPS
which engages scavenger receptors and leads to LPS detoxification by J774 cells [160], as described in the following
section. However, it has to be emphasized that apart from
LPS, CD14 binds a broad spectrum of other molecules,
including PAMPs, like peptidoglycan, lipoteichoic acid,
CpG DNA, and phospholipids [51, 207], and also DAMPs
such as β-amyloid [208]. Thus, CD14 can affect the activity
of several PRRs. The binding of phospholipids by CD14 is
interesting in the context of the LPS-induced pro-inflammatory signaling of TLR4. Among the phospholipids bound
by CD14 are 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero3-phosphorylcholine, two forms of oxidized 1-palmitoyl2-arachidonyl-sn-glycero-3- phosphorylcholine (oxPAPC)
released from dying cells at the site of tissue injury. The
oxPAPC-induced internalization of CD14 depletes its cell
surface pool and as a consequence makes these cells less
sensitive to LPS. However, in LPS-primed murine macrophages and DC, the CD14-dependent delivery of oxPAPC
into the cells leads to the activation of caspase-11 and
1247
caspase-1 followed by IL-1β release. Notably, oxPAPC did
not induce pyroptosis of such cells leaving them hyperactive
to produce IL-1β without the lethal outcome of an inflammatory response and sepsis in mice [144, 209]. These data support the TAXI name given to CD14 as it is able to capture
various cargo in addition to TLR4/MD-2/LPS and deliver
them to signaling-competent locations [17].
Only scarce data allow speculation on the pathway(s)
involved in the internalization of CD14 carrying LPS and
other ligands, like oxPAPC, and also in the constitutivie
endocytosis of CD14 in resting murine macrophages [17].
The latter is of importance since a down-regulation the cell
surace level of CD14 and TLR4 in resting cells should prevent excessive LPS-induced pro-inflammatory signaling of
TLR4 [161, 210]. This mechanism can also contribute to the
antagonistic activity of R. spheroides LPS toward human
and murine TLR4. Studies performed on murine BMDM
showed that this LPS species induced CD14 endocytosis
preventing subsequent binding of the pro-inflammatory E.
coli LPS to CD14 and consequently to TLR4. The ability of
R. spheroides LPS to induce CD14 endocytosis was determined by diphosphorylation of its lipid A [17].
The LPS-induced endocytosis of CD14 is independent of
the signaling activity of TLR4, but it was reduced by a Syk
inhibitor or a knocdown of PLCγ2 [33]. This suggests that,
upon LPS binding, CD14 and TLR4 are internalized by the
same route discussed above for TLR4 endocytosis. Since this
LPS-induced uptake of CD14/TLR4/MD-2 does not require
the signaling activity of TLR4, it has been proposed to be
controled by CD14 itself [17]. However, it is still unknown
whether the endocytic pathway triggered by LPS is the same
as that involved in the constitutive internalization of CD14
in unstimulated cells or in the CD14-mediated uptake of
other ligands.
Studies on the endocytosis of other GPI-anchored proteins (GPI-APs) may shed light on the LPS-independent
internalization of CD14. The majority of GPI-APs are internalized via the dynamin- and clathrin-independent carrier
(CLIC) endocytic pathway which also contributes markedly
to fluid phase uptake. The internalization of CD14 detected
in resting J774 cells displayed properties of a CLIC-mediated uptake; however, the apparent similarities between the
CD14 and TLR4 uptake found in those inhibitor-based studies do not allow unequivocal identification of the endocytic
pathway involved [161]. The CLIC-mediated endocytosis
is sensitive to actin depolymerization and requires activation of the Arf1 and Cdc42 GTPases [211]. Cdc42 interacts
with PS in the inner leaflet of the plasma membrane. Depletion of CDC50, the α-subunit of the flippase complex, and
the following inhibition of PS transport from the outer to
the inner monolayer of the plasma membrane increased the
surface level of CD14 in unstimulated THP-1 macrophages
[212]. These data suggest that CD14 can be internalized by
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CLIC-mediated endocytosis involving Cdc42 and PS. An
attempt has been undertaken to assess whether the CLICmediated endocytosis is also involved in the internalization
of CD14 upon LPS binding. It was found that, depletion of
galectin-3, a protein triggering CLIC-mediated endocytosis via clustering of cell-surface glycosylated proteins with
glycosphingolipids, did not affect the LPS-induced internalization of either CD14 or TLR4 [17, 213]. Those results
spoke against a role of the galectin-3-facilitated endocytosis of CD14 in LPS-stimulated cells; however, a hypothetical involvement of galectin-3-independent CLIC-mediated
pathway(s) cannot be ruled out.
Internalized GPI-APs accumulate in early endosomes
which therfore earn the name of GPI-AP-enriched early
endosomal compartmet (GEEC). The GEEC undergoes
fast but only modest acidification to pH ~ 6, which is above
the average pH of other early endosomes. The acidic pH
of GEEC can facilitate the release of ligands from the
internalized GPI-APs [211, 214]. Inhibition of V-ATPase
reduced the CLIC/GEEC endocytosis of dextran, a fluid
phase marker [211]. As mentioned earlier, a knock-down
of the ATP6V0D2 subunit of V-ATPase also impaired the
LPS-induced endocytosis of TLR4 [152]. It remains to be
established whether in those conditions, the inhibition of the
internalization of TLR4 was a consequence of a disturbed
uptake of CD14.
At present, there are no data indicating that upon LPS
binding CD14 can recycle to the plasma membrane either
via the fast Rab4/Rab35-dependent pathway, the slow one
dependent on Rab11, or via Rab10-enriched recycling
endosomes, as it was shown for TLR4 [179]. On the contrary, it has been established that at least a fraction of the
internalized CD14 is degraded in lysosomes [17]. Even in
cells not stimulated with LPS CD14 was detected in perinuclear structures and in MVB-like vesicles [163]. Stimulation of cells with LPS increased the degradation of CD14 in
lysosomes while chloroqine treatment inhibited this process
[17]. Notably, CD14 internalized by human monocytes during phagocytosis of E. coli or monosodium urate crystals (a
sterile phagocytic stimulus) is cleaved in phagolysosomes by
elastase and relased as presepsin [54] (see chapter “Signaling pathways triggered by TLR4”).
It is an open question whether disturbances in the endolysosome functioning caused, e.g., by mutation/knock-down/
inhibition of endosomal proteins listed in Table 1 can affect
the cellular level and localization of CD14 and thereby the
LPS-induced TLR4 activation. This possibility is usually
neglected in analyses of the impact of endo-lysosomal proteins on TLR4 trafficking and signaling.
13
A. Ciesielska et al.
LPS uptake and detoxification
Clearance of LPS from circulation and its detoxification
help resolve the inflammation induced by bacterial infection.
These processes are also important for the removal of the gut
microbiota-derived LPS which got access to the bloodstream
[215]. There are several mechanisms of LPS inactivation
in the human body acting both extracellularly and intracellularly. The latter follows LPS uptake by hepatocytes which
remove LPS into the bile, and by immune cells capable of
detoxifying the endotoxin enzymatically [216].
The extracellular inactivation of LPS is executed by
several lipid A-neutralizing proteins which circulate in the
bloodstream, like bactericidal permeability-increasing protein, lactoferrin, lysozyme, collectins, and also anti-LPS
antibodies [217]. Also in the blood LPS can be bound by
lipoproteins, such as chylomicrons and high, low, and very
low-density lipoproteins (HDL, LDL, VLDL) to be next
transported to the liver [218, 219]. The binding of LPS to the
lipoproteins is catalyzed by phospholipid transfer protein,
cholesteryl ester transfer protein, LBP, and sCD14. Liver is
the main source of sCD14, also LBP is produced predominantly by the liver and in addition by epithelial cells of lungs
and the gastrointestinal track [220]. LPB and sCD14 are both
acute phase proteins upregulated significantly during sepsis
[45, 221]. High concentrations of LBP attenuate TLR4 activation by inhibiting the transfer of LPS from CD14 to MD-2
while sCD14 can accept LPS form membrane-bound CD14
and accelerate its transfer to HDL and subsequent detoxification in the liver [49, 207, 215, 222, 223].
Liver also plays an essential direct role in the clearance
of LPS from circulation and its excretion into the bile. In
mice injected in the tail vein with 5 μg LPS, 90% of the
endotoxin accumulated in the liver within 60 min [224]. The
mechanism of the LPS detoxification in the liver is not fully
clear but it is known to engage various types of hepatic cells,
including liver sinusoidal endothelial cells, hepatocytes, and
hepatic macrophages—the Kupffer cells. All these cells can
internalize LPS, but mainly the macrophages inactivate the
internalized endotoxin enzymatically [224–227]. If not internalized by hepatocytes directly, LPS is transferred to them
from the other cells and is next secreted into the bile [224].
It is worth emphasizing that the mechanism of LPS endocytosis preceding its intracellular detoxification depends
on its formulation which includes aggregates, OMV, lipoprotein-bound LPS, and LPS monomers. OMV and LPSenriched liposomes are likely internalized in a clathrindependent and CD14-independent way by macrophage. The
binding of LPS to lipoproteins affects the way it is detoxified
in the liver. The endocytosis of HDL-LPS by Kupffer cells
was not as effective as that of free LPS [224], indicating
that the HDL-LPS complexes are rather internalized by cells
TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling
highly expressing scavenger receptors, such as liver sinusoidal endothelial cells and hepatocytes [227]. The role of the
scavenger receptors, e.g., CD36, CLA-1/SRB1, CLA-2 and
SR-A of immune cells, in LPS-induced processes is more
intricate and includes the CD14-dependent uptake of high
doses of LPS but also the activation of immune responses
[160].
It has been found recently that LPS can also be internalized in complex with secretoglobin 3A2 and HMGB1. The
first protein is abundantly expressed in airway epithelial cells
and is internalized via the SDC1 receptor. The endocytosis of HMGB1, in turn, is mediated by the RAGE receptor
expressed in epithelial cells and macrophages. Both proteins facilitate leakage of LPS from endosomes to the cytosol where it is bound by pro-caspase-11 and activates the
NLRP3 inflammasome [113, 228].
TLR4 makes a small contribution to the internalization
of LPS by immune cells [229]. In agreement, blocking of
TLR4 with a neutralizing anti-TLR4 antibody did not affect
the internalization of a relatively high dose of LPS (1000 ng/
ml) [160]. Although TLR4 did not directly participate in the
uptake of high amounts of LPS by macrophages, it could
indirectly reinforce its clearance from plasma by hepatocytes, as a knock-down of hepatocyte TLR4 inhibited this
process [26]. It was also shown that TLR4, CD14, and MD-2
participate in the clustering of CD11b/CD18a integrins
involved in LPS endocytosis in hepatocytes [230]. Moreover,
TLR4 can regulate deacylation of LPS by immune cells. In
TLR4-deficient mice, significantly less LPS was deacylated
at the site of infection than in wild-type littermates [231].
In accordance, LPS and commensal Gram-negative bacteria
increased the expression of acyloxyacyl hydrolase (AOHA)
in macrophages and DC suggesting that activation of TLR4
can activate production of this enzyme [232, 233]. AOHA
is a highly conserved hydrolase which recognizes LPS and
removes secondary fatty acids at positions 3′, 2′ and/or 2 of
lipid A, converting a hexa-acyl (also hepta- or penta-acyl)
structure into one that has only four acyl chains. The resulting tetra-acyl LPS molecule binds to MD-2 but does not
initiate TLR4 signaling and is also inert to caspase-based
detection systems [234]. Similarly, weaker inflammatory
responses are induced by LPS dephosphorylated by host
phosphatases, like the intestinal alkaline phosphatase which
participates in detoxification of LPS in the gut lumen [235].
Deacylation and dephosphorylation of LPS deprived it of a
pro-inflammatory activity especially in human cells [59, 61],
due to a weaker or no activation of human TLR4 by these
LPS species, as discussed above. Notably, the vast majority
of in vivo studies on LPS detoxification have been carried
out on mice the TLR4 of which can be activated by a broader
range of LPS species (see above), but which are much less
sensitive to LPS-induced septic shock than humans [236].
1249
Intestinal alkaline phosphatase is produced in the duodenum, whereas high expression of AOAH was found in
the liver, mainly in Kupffer cells with a minor contribution
of DC [235, 237]. AOHA expression was also detected in
macrophages and DC from the large intestine and in lung
macrophages and neutrophils [232]. When LPS was applied
subcutaneously, mimicking its release from bacteria in a tissue other than blood, up to 70% of the LPS was deacylated
by AOHA before it left the site of injection. This in situ
detoxification of LPS is carried out by immune cells, including neutrophils, DC, and macrophages [231].
Deacylation of LPS is a slow process taking several days.
Therefore, during infection, the activity of AOHA has little
impact on the early response to LPS and cytokine production, but it is necessary to terminate the inflammation and
affects the activation of B lymphocytes [231, 237]. Many
aspects of AOHA activity are unresolved, the most intriguing one being whether the main side of its action are organelles of immune cells or the extracellular space [231]. In
light of the data on the role of metabolic endotoxemia in the
development of metabolic diseases, and a growing number
of fatal sepsis cases worldwide, the mechanisms of endotoxin disposal by immune cells and overall by the human
body deserve close attention.
Contribution of abnormal trafficking
of TLR4 and CD14 to pathogenesis of certain
diseases
The multiple pathways of TLR4 trafficking in diverse cells
require tight regulation to ensure an optimal magnitude and
duration of pro-inflammatory responses induced by LPS
and DAMPs. For this reason, several human hereditary and
neurodegenerative diseases caused by disturbances in the
functioning of the endo-lysosmal compartment are linked
with TLR4 hyperactivation and inflammation contributing
substantially to their pathogenesis.
This fatal relationship is exemplified by lysosomal storage disorders (LSD) caused by hereditary dysfunctions of
lysosomes which are accompanied by an abnormal activation of TLR4 and production of pro-inflammatory cytokines
[238, 239]. These responses can be triggered by an extracellular accumulation of undegraded substances which act as
DAMPs and also by excessive TLR4 stability and persistent
signaling caused by an impaired lysosomal proteolysis in
cells of LSD patients. In particular, TLR4 has been shown
to be involved in the development of mucopolysaccharidosis
(MPS), a heterogenous group of LSD resulting from deficiencies in lysosomal enzymes degrading glycosaminoglycans (GAGs). Various MPS subtypes affect different tissues
and organs, including central nervous system, bones, muscles, and the connective tissue [238]. Under physiological
13
1250
conditions, hyaluronian of the GAG family is transported
to the extracellular matrix while other GAGs, like heparan
sulphate, are exposed on the cell surface and remain bound
to the plasma membrane via a proteoglycan core. Thereby
heparan sulphate of epithelial cells affects the adhesion
of immune cells during inflammation. Several GAGs also
induce production of pro-inflammatory chemokines and
cytokines. Studies conducted on animal models of MPS VI
and/or VII showed an increased level of IL-1β and TNF-α
and also an upregulated expression of several other inflammatory markers in synovial fluid, fibroblast-like synoviocytes and serum. Those changes were linked with TLR4
activity as a knock-down of TLR4 in MPS VII animals normalized the serum level of TNF-α and phosphorylation of
chondrocyte STAT1 and also alleviated some of the pathological effects of the disease in bones and joints [240, 241].
A similar line of data indicated a contribution of TLR4induced pro-inflammatory signaling of microglia to the onset
of neurodegenerative MPS III [242]. The pro-inflammatory
signaling of TLR4 is considered to be triggered by GAG—
DAMPs which accumulate in the extracellular space. These
include hyaluronian which stimulates TLR4 in cooperation
with CD44, and also heparan sulphate and its derivatives
resulting from an incomplete degradation of this GAG in
lysosomes and subsequent exocytosis of its products. However, an increased level of TLR4 has also been detected in
fibroblast-like synoviocytes of MPS VI animals. In addition,
these cells have increased mRNA levels of LBP, CD14, and
MyD88. The upregulation of TLR4 level was caused by its
accumulation in dysfunctional lysosomes with an unchanged
expression of the Tlr4 gene [240, 241]. Collectively, these
results indicate that an impaired lysosomal degradation of
TLR4 contributes to the inflammation in MPS.
An increased activity of TLR4 is also linked with the
pathogenesis of another LSD, Niemann–Pick’s type C
(NPC) disease leading to progressive neurological dysfunctions in children. NPC is caused by mutations in
the NPC1 or NPC2 genes causing malfunctioning of the
encoded proteins, which results in an aberrant endosomal
membrane flow and accumulation of cholesterol and other
lipids in endo-lysosomes. TLR4 level is increased in fibroblasts and glial cells of NPC patients and in Npc1 knock-out
mice, respectively, as a result of its impaired degradation.
This leads to the accumulation of TLR4 in endosomes and
upregulation of its signaling pathways enhancing the production of pro-inflammatory cytokines, such as IL-6, IL-8, and
IFN-β. The increased inflammatory response, particularly
the glial production of IL-6, can contribute to the pathogenesis of NPC via both the autocrine and paracrine effects of
this cytokine [243].
A participation of the TLR4 receptor has also been noted
in the development of cystic fibrosis (CF)—an autosomal
recessive disorder caused by mutations in the CFTR gene
13
A. Ciesielska et al.
encoding cystic fibrosis transmembrane conductance regulator, a chloride channel expressed in airway epithelial cells
and, to a lower extent, also in other cells. The disease is manifested chiefly by a dysfunction of the airway epithelium,
which leads to a dehydrated and acidic airway surface liquid
favoring chronic bacterial infections [244, 245]. A characteristic feature of cystic fibrosis is an excessive inflammatory
response leading to lung damage [244, 245]. Data concerning the role of bronchial epithelial cells in the inflammatory
response in cystic fibrosis are inconsistent. Early studies
reported a decreased or unchanged expression of TLR4 in
those cells in comparison with the cells of healthy donors
[244, 246]. In contrast, a recent study on immortalized bronchial epithelial cell derived from a CF patient showed an
increased level of TLR4 on the cell surface and its impaired
transport to lysosomes likely contributing to an enhanced
inflammatory response to LPS [247]. However, the hyperinflammatory response in CF lungs is mainly attributed to
macrophages. Accordingly, macrophages from CFTR knockout mice had an elevated level of TLR4 compared with the
wild type, with no differences in the amount of TLR4 mRNA
[248]. The deletion of the Cftr gene caused accumulation of
TLR4 in early endosomes in macrophages and compromised
its degradation, thereby leading to an increased activation of
NF-κB, MAPK, and IRF3. The retention of TLR4 in early
endosomes could ensue from an aberrant maturation of
endosomes in CFTR-depleted LPS-stimulated cells, insufficient endosomal acidification, or perturbations in cholesterol trafficking caused by the CFTR deficiency [248]. These
mechanisms can also cause the accumulation of TLR4 and
an excessive pro-inflammatory response to LPS of human
macrophages ultimately leading to the lung disease of cystic
fibrosis patients [248].
The arthrogryposis–renal dysfunction–cholestasis syndrome is a rare fatal disease caused by autosomal recessive
mutations in the VPS33B gene. Characteristic symptoms
of this disease comprise arthrogryposis, renal tubular acidosis, and neonatal cholestatic jaundice. The patients also
suffer from persistent infections and sepsis [180, 249]. As
it was described above, dysfunction of VPS33B leads to an
impaired degradation of TLR4 and enhanced pro-inflammatory response of macrophages to LPS via the MyD88and TRIF-dependent pathways. It has been proposed that
such exaggerated TLR signaling causes so-called immune
paralysis whereupon the immune system loses the ability to
respond to secondary infection [180].
Studies on Chédiak–Higashi syndrome (CHS) have
expanded the list of diseases linked with disturbances in
TLR4 trafficking and signaling. CHS is a rare autosomal
recessive disease characterized, among others, by partial
albinism, neurological problems, and immunodeficiency.
CHS patients suffer from persistent and recurrent infections of the skin, respiratory tract, and mucous membranes.
TLR4 and CD14 trafficking and its influence on LPS‑induced pro‑inflammatory signaling
Human CHS and its corresponding mouse disorder beige are
caused by mutations of the LYST gene [192]. Recent studies indicate that Lyst controls endosomal TRIF-dependent
signaling of TLR4, as described above. In mice, a loss of
a functional Lyst impaired inflammatory responses against
Gram-negative bacteria and enhanced bacterial dissemination in tissues but also lowered the susceptibility to LPSinduced septic shock as a result of a reduced production of
pro-inflammatory cytokines [183].
TLR4 also participates in the development/progression
of neurodegenerative disorders associated with Alzheimer’s
disease (AD), Parkinson’s disease (PD), multiple sclerosis,
and amyotrophic lateral sclerosis (ALS). Due to its engagement in neurodegeneration, TLR4 is considered a therapeutic target in the treatment of these diseases [250]. At the
early stages of the development of neurodegenerative diseases, activation of TLR4 may be beneficial for the clearance
of β-amyloid (AD) and α-synuclein (PD) and can prevent
accumulation of their aggregates characteristic for these
diseases. However, a prolonged overactivation of microglia
may lead to the release of neurotoxic products and damage
neurons [251, 252]. Such increased activity of microglia
can result from their abnormally high content of TLR4. An
increased expression of TLR4 was observed in brains of a
murine AD model (overexpressing amyloid precursor protein) and in AD patients in areas surrounding plaques [251,
253]. Moreover, β-amyloid increases expression of TLR4 in
cortical neurons, which further activates JNK and caspase-3
and contributes to neuron death [254]. An elevated expression of TLR4 was also detected in ALS and PD patients
[255]. In addition to TLR4, also CD14 is important for the
activation of inflammatory responses and phagocytosis upon
exposure of microglia to β-amyloid [251, 252]. In agreement, an elevated level of CD14 was detected in brains from
murine models of AD, PD, and ALS, and from AD patients
[256–258]. However, the outcome of a CD14 engagement
in AD can be both beneficial and detrimental [259]. The
elevation of the amounts of TLR4 and CD14 in microglia
and exaggerated production of pro-inflammatory mediators
can result from disturbances in endo-lysosomal functioning
in analogy to the LSD described above. This assumption is
supported by the fact that among the features of AD, PD and
ALS are defects of endo-lysosomes caused by mutations of
genes encoding proteins involved directly or indirectly in
their functioning [260, 261].
Recent data link TLR4 activation with the inflammatory
process induced by cerebral stroke and leading to ischemic
brain damage. TLR4 was upregulated after ischemia-induced
stroke, and mice deficient in TLR4 exhibited less-severe
cerebral infarction and neurological deficit compared with
wild-type counterparts [200]. TLR4 is also associated with
the development of allergies and some autoimmune diseases,
like Crohn’s disease, asthma, and type 1 diabetes [262–264].
1251
Further studies are required to establish the contribution of
disturbances in TLR4 trafficking to these processes.
In summary, the involvement of TLR4-triggered signaling in the development and progression of multiple diseases is often linked with its disturbed trafficking through
the endo-lysosomal compartment, as demonstrated for LSD
and strongly suggested for neurodegenerative diseases. It
remains an open question whether defects in TLR4 trafficking contribute also to the severity of acute and chronic
inflammation caused, respectively, by infection and metabolic endotoxemia, the latter known to participate in the
development of type 2 diabetes. A comprehensive knowledge of the TLR4 trafficking pathways and the role of CD14
in these processes is key to understanding mechanisms
regulating TLR4-induced inflammatory response in both
infectious and non-infectious diseases. This in turn should
facilitate the design of new therapeutic strategies for treatment of these diseases.
Acknowledgements This work was supported by grant number
2016/23/D/NZ3/02212 from the National Science Centre, Poland to
A. Ciesielska. We thank Dr. Jan Fronk from the Faculty of Biology,
University of Warsaw for helpful comments and critical discussion.
Author contributions A.C. and M.M.: original draft preparation and
editing, K.K.: review and editing.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
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/.
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W2955484006.txt |
www.bioinformation.net
Volume 15(6)
Research Article
Identification of novel inhibitor targeting Fyn kinase
using molecular docking analysis
Dharani Nedunchezhian1, Kulanthaivel Langeswaran2* & Sundar Santhoshkumar3
1Department
of Biotechnology and Bioinformatics, Bishop Heber College, Bharathidasan University, Tiruchirapalli, Tamil Nadu, India;
Genetics & Molecular Biology Laboratory, Department of Bioinformatics, Science Campus, Alagappa University, Karaikudi, Tamil
Nadu, India; 3Department of Computer Science, Science Campus, Alagappa University, Karaikudi, Tamil Nadu, India; Dr. V. K.
Langeswaran - Email: dr.langeswaran@gmail.com; *Corresponding author
2Cancer
Received May 21, 2019; Accepted June 3, 2019; Published June 15, 2019
DOI: 10.6026/97320630015419
Abstract:
Identification of tyrosine Fyn kinase inhibitor is recognized as an effective and feasible therapeutic measure in reducing consequences of
memory loss disorder Alzheimer's. The present investigation has been attempted with an objective to find out a novel potent inhibitor with
similar homological structure to Fyn kinase using structure based in silico screening measure. Such derived structure was compared with
natural data base pool and were systematically analyzed. Ligand based interaction was also tested and evaluated. We applied a molecular
dynamic simulation technique to validate the stability of the identified complexes and to understand the ligand binding mechanism.
Results provide information on the characteristics of novel and potent inhibitor for tyrosinase Fyn kinase protein so as to develop an
innovative strategy to treat AD.
Keywords: Tyrosine fyn kinase, ligand binding, dynamic simulation
Background:
Tyrosine Fyn Kinase is a determined protein factor involved in the
activity of cell signaling pathway. Unusual occurrence in the rate
of activity of Fyn Kinase in signaling, render toxic substances,
resulting chronic disorder such as Alzheimer's [1]. This played a
crucial role in phosphorylation and anti-phosphorylation process
and confers its intra molecular affinity [2], Cell adhesion, synaptic
activity and signaling [3]. Several earlier reports were
demonstrated and evident that Tyrosine Fyn was recognized as
driven force causing AD by interacting Amyloid B and Tau,
conferring a novel therapeutic strategy to address and minimize the
consequences of AD. Regulation of Fyn activity was obviously
studied in several types of signaling pathways [4]. Shirazi et al. [5]
have reported that Fyn Protein showed immune reactivity while
unusual phosphorylated Tau protein. However, several studies
were focused on inter linkage of Tyrosine Fyn Kinase protein with
AD. Participation of Fyn Kinase Protein in inducing and act as
causative agent for chronic disorder. Therefore, it received more
attention on its ability in signaling pathways. Advancement of
docking based virtual screening technique in accordance with
affinity of protein and ligand conferring an ideal way for screening
the lead novel compounds in the field of molecular docking [6].
Identification of macromolecules and validity was the remarkable
factor in targeting the drugs, however, it is still unclear and
debatable [7]. Therefore, in the present investigation, a trial has
been taken up (i) to predict the novel potent inhibitor targeting Fyn
Kinase and (ii) to identify the ligand by comparing natural database
and (iii) to explore protein ligand complex stability and its
behavior.
ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 15(6): 419-424 (2019)
419
©Biomedical Informatics (2019)
Table 1: Virtual screening of ZINC-NPD, NPB and NCI results
S. No
Natural
I.DS
GE
VDW
COUL
GEM
compounds
ZINC-NPD
1
144027
-16.572
-68.639
-37.652
-30.987
-121.633
2
120095
-15.884
-82.871
-46.541
-36.330
-113.640
3
128794
-15.848
-90.119
-58.883
-31.236
-130.885
4
2582
-15.812
-79.383
-52.157
-27.227
-105.107
5
118082
-15.799
-82.457
-55.718
-26.739
-99.998
NPB
1
2455
-9.946
-63.013
-51.890
-11.123
-92.357
2
7898
-9.838
-64.901
-41.363
-23.538
-81.703
3
2407
-9.595
-58.628
-47.704
-10.923
-81.459
4
4794
-9.096
-42.552
-39.816
-2.737
-65.886
5
2786
-8.961
-53.323
42.002
-11.321
-72.928
NCI
1
9129
-10.713
-46.113
-35.774
-10.339
-68.734
2
6152
-10.199
-35.153
-19.122
-16.031
-48.802
3
4036
-10.026
-39.686
-23.145
-16.541
-52.388
4
9037
-9.874
-38.099
-25.992
-12.107
-65.595
5
2608
-9.845
-35.570
-23.836
-11.734
-61.072
(ZINC-NPD, NPB, NCI) Natural compound database; DS-Docking energy; GE- Glide energy; GEVDE-VDE; GECOUL-COUL; GEMODE-GEM.
Table 2: ADME results of the top compounds
S. No
COMPOUND
MW
DONAR HB
01
ZINC_NPD144027
396.463
1
02
NPB: 2455
240.258
3
03
NCI: 9129
478.636
1
ACCEPT HB
9
2.25
10.5
% HOA
88.588
86.594
85.564
Q plogPo/W
-5.76.
2.038
2.919
Materials and Methods:
Using Software packages such as CPU platforms performed
aforementioned tasks. Stipulations were CPU-Supermicro 32Gb
Nvidia Quadro K620 with 2GB included, LLC, 2019 Desmond. Fyn
protein complex code (PDB ID-2DQ7) with a resolution of 2.8
Armstrong was used for virtual screening and molecular docking.
They contain heavy atoms, covering its cofactors. However, to
avoid left out information on connectivity, protein structure is
imported from PDB into Maestro in the protein preparation wizard
with hydrogen polar. Optimization of Hydrogen bond was
employed and rely on clusters of hydrogen-bonded species were
selectively optimized. Minimizing the structure has been
performed to refine the structure, with a restrained minimization.
The RMSD value of the atom was minimized. The partial atomic
charges were also computed and analyzed.
QplogHERG
-5.708
-4.643
-5.399
QPP Caco
0.235
463.328
209.104
QPPMDCK
0.059
215.385
128.805
in the workspace. Van der Waals (vdW) radius scaling of 14 for
closely contacts with the ligand and the receptor (a protein
molecule) is used. Scaling factor default value is 0.30 and the partial
charge cut off default value is 0.25. Scaling of van der Waals radii
was performed on the non-polar atoms. Scale factor default value is
1.0 as the contact by small gaps in the solvent-exposed regions and
also determines where in the scoring grids are located. Energetic
program was used to analyses spectrum to predict the modes of the
protein ligand complexes. Workflow filtering method was used. It
was also evaluated the acceptability of analogues based on
Lipinski’s rule of 5 which was essential to ensure the drug-like
properties of the molecules were assessed. GSAP complexed with
compounds from Zinc-NPD, NPB, Nci-NPD database was
employed by using the Desmond modular. In this present study,
totally three from Zinc-NPD, NPB, Nci-NPD database (144027,
2455, 9219) were chosen, theses three are the top leads from each
database identified based on the docking score. All these three
complexes were simulated for 10 analyzed deviations (RMSF) and
considered as backbone of the protein. The RMSF was assessed to
estimate fluctuation.
The generated structure of a prepared protein showed in an order
such as receptor, site, constraints and rotatable groups. Receptor is
a workspace and the ligandwere identified as a molecule. The green
color marker should be shown after the ligand molecule is picked
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Figure 1: Interaction profile and docking pose of the best hit compounds obtained from screening for FYN 2DQ7 with Zinc NPD(a, b),
NPB(c, d), NCI-NPD(e, f) databases.
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421
©Biomedical Informatics (2019)
Figure 2: Molecular dynamics simulation results of the best hit compounds from three databases
Result and Discussion:
In the present study high throughput virtual screening (HTPS)
method was used to identify potential molecule inhibitors using
compounds obtained from pharmacophore against active FYN
kinase protein of the ligands were further docked flexibly by XP
which generates compounds were tabulated in Table 1 and shown
in Figure 1. Drug likeness was tested and followed by Quikprop
method of Schrodinger ADME properties of identified inhibitors
were recorded systematically and tabulated (Table 1) based on
Lipiski’s rule of 5 [8]. Results of the docking indicated that efficient
inhibitors identified among three database, chief lead compound
was found to be G Plog Po and was promising alternate.
Final results of screened compounds were selected data base as an
input to find matches to hypothesis to screen the best ligands
molecules. In earlier studies, virtual screening molecules against
several proteins related to human disorder were reported. Poli et al.
[9] have demonstrated that few efficient Fyn inhibitors have
identified and proved to be blocked the activities of Fyn kinase.In
our study, three different Natural compounds databases were
chosen namely ZINC NPD (3,76,884 Ligand molecules), NPB
(20,783 Ligand molecules), NCI(56,000 Ligand molecules) were
taken for screening process. Based on the docking score top three
compounds were selected from each database.
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©Biomedical Informatics (2019)
Analysis on ADME of inhibitors:
ADME properties evaluation of identified chief compound, A
Plogpo scored highest range (Table 2). It was observed that
identified compounds with a group of hydrogen bonding donor
and acceptor pair as found This present study results were
matching the findings of Zhu [10]. The biological membrane access
ranges were found to be range from 293.11 to 2288.96 and the range
of QPPMDCK was 96.8 to 100 [11]. Thomas et al. [12] have
established that improvement in ADME properties of identified
compounds are attributed and translate to the pharmacokinetic
properties. Findings were correlated with earlier study results.In
the present investigation two ligands from the ZINC-NPD database
(95914724), NPB database (2455) and NCI-NPD database (6152)
were chosen. 95914724, 2455, 6152 were the top lead identified
based on docking score. All two complexes were calculated and
analyzed RMED value in order to evaluate deviation in the
structure during simulation (RMSD). The stability of protein-ligand
(2DQ7-144027, 2DQ7- 2455, 2DQ7-9219) complexes is shown.
Figure 2 exhibited that the deviations and fluctuations were not
observed much and hence the results were agreeable. These results
inferred that the compounds have slight fluctuations, which are
observed, only in the loop region of the target protein. Zinc
complexes in lead molecules may contribute and involved in
coordination if neurons characterized by fast ligand exchange [13].
In the present study, it was evident that interaction of compound
with zinc was attributed to the selected ligand and it was
agreement with our findings [14]. The results of the RMSD plot
reported that high scored lead compound could be satisfactorily
explored in the present simulation experiment.
simulation have proved and supported our claims to be the useful
tool and the best ligand to block the Fyn kinase. This study has
confirmed that these compounds can be a potent inhibitor to block
the interactions between the targets and inhibit the progression of
Alzheimer’s disease.
Acknowledgement:
The article has been written with the financial support of RUSA –
phase 2.0 grant sanctioned vide Letter No.F.24-51/2014-U policy
(TNMulti – Gen), Dept of Edn. Govt of India Dt, 09.10.2018.
Conflict of Interest
The authors declared that there are no conflicts of interest.
References
[1] Poli G et al. J Chem Inf Model. 2013 53:2538. [PMID: 24001328]
[2] Krämer-Albers EM et al. Cell Mol Life Sci. 2011 68: 2003
[PMID: 21207100]
[3] Resh MD. Int J Biochem Cell Biol. 1998 30:1159.[PMID:
9839441]
[4] Hu JL et al. Mol Brain. 2010 3:20. [PMID: 20569495]
[5] Shirazi SK et al. Neuroreport. 1993 4:435. [PMID: 8388744]
[6] Nguyen TH et al. J Biol Chem. 2002 277:24274. [PMID:
11983687]
[7] Moellering RE et al. Nature 2009 462:182. [PMID: 19907488]
[8] Lipinski CA et al. Adv Drug Deliv Rev. 2001 46:3. [PMID:
11259830]
[9] Goldsmith JF et al. Biochem Biophys Res Commun. 2002298:501.
[PMID: 12408980]
[10] Zhu L et al. Mol Divers. 2018 22:979. [PMID: 30083853]
[11] Thomas SM et al. Annu Rev Cell Dev Biol. 199713:513. [PMID:
9442882]
[12] Thomas R et al. Pediatrics. 2015 135: e994. [PMID: 25733754]
[13] Elena Atrián-Blasco et al. Dalton Trans. 2017 46: 12750.
[PMID: 28424538]
[14] Monika et al. Bioinformation 2013 9: 583. [PMID: 23888100]
Conclusion:
We are interested identifying the inhibitors targeting the Fyn kinase
for the treatment of Alzheimer’s disease. It was concluded that Fyn
kinase inhibitors are, therefore promising novel molecules and in
disrupting a neurodegenerative disorder. Computational studies
such as receptor based screening and molecular dynamics
Edited by P. Kangueane
Citation: Nedunchezhian et al. Bioinformation 15(6): 419-424 (2019)
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the original work is properly credited. This is distributed under the terms of the Creative Commons Attribution License
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Journal
ISSN 0973-2063 (online) 0973-8894 (print)
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| 1,998 | 13,672 |
W4327590804.txt | Aromatic Hydrocarbons as Molecular Propellants
for Electric Propulsion Thrusters
André Nyberg Borrfors
KTH Royal Institute of Technology
Dan J. Harding
KTH Royal Institute of Technology
Jonas Weissenreider
KTH Royal Institute of Technology
Simone Ciaralli
OHB Sweden AB
Ashley Hallock
OHB Sweden AB
Tore Brinck ( tore@kth.se )
KTH Royal Institute of Technology
Research Article
Keywords:
Posted Date: March 16th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-2666479/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
Additional Declarations: No competing interests reported.
Aromatic Hydrocarbons as Molecular Propellants for Electric
Propulsion Thrusters
André Nyberg Borrfors1, Dan J. Harding2, Jonas Weissenreider3, Simone Ciaralli,4 Ashley Hallock,4
and Tore Brinck1*
1Department
of Chemistry, KTH Royal Institute of Technology, SE 100 44 Stockholm, Sweden
2Department
of Chemical Engineering, KTH Royal Institute of Technology, SE 100 44 Stockholm, Sweden
3KTH,
Materials and Nano Physics, Hannes Alfvens väg 12, 11419 Stockholm, Sweden
4OHB
Sweden AB, Viderögatan 6, 164 29 Kista, Sweden
*tore@kth.se
ORCIDs: DJH: 0000-0002-8622-9046, JW: 0000-0003-1631-4293, AH: 0000-0001-6801-5186,
TB: 0000-0003-2673-075X
ABSTRACT
The aromatic hydrocarbons (AHs) fluorobenzene, naphthalene, and 1-fluoronaphthalene are
introduced as promising alternatives to xenon as propellant for in-space electric propulsion (EP).
These storable molecules have similar mass, lower cost, and lower ionization energies compared
to xenon, as well as the critical advantage of low post-ionization fragmentation compared to other
molecular propellant candidates. The ionization characteristics of AHs are compared with those
of xenon and the diamondoid adamantane, previously evaluated as a molecular propellant for EP.
Quantum chemical calculations and BEB theory together with 25 eV electron ionization mass
spectrometry (EI-MS) measurements have been used to predict the fragmentation of the AHs and
adamantane when ionized in a plasma with an electron temperature of 7 eV (a typical electron
temperature in EP plasmas). A high fraction (81 - 86 %) of the detected AH ions originate from
intact molecules, compared to 34 % for adamantane. indicating extraordinarily low
fragmentation for the selected AHs. The ionization potential of the AHs is similar to that of
adamantane but lower compared to xenon (8.14-9.2 eV for the AHs, 9.25 for adamantane and
12.13 eV for xenon). BEB calculations have also been used to predict total ionization cross
sections. The calculated ionization cross section of the AHs is comparable to that of adamantane
but 3-5 times higher than that of xenon, which together with the low ionization potential can
contribute to more efficient ionization. The AHs may have the potential to perform better than
xenon, despite the absence of fragmentation in xenon.
Acknowledgments
Special thanks to Dr. Abdusalam Uheida of the KTH Functional Materials Division for allowing
us access to his lab and equipment, saving us precious time during the early measurements.
Technical assistance by Lea Hohmann at KTH Dep. of Chemical Engineering is gratefully
acknowledged.
Page 1 of 23
DECLARATIONS
Funding
This work was initiated by OHB Sweden through the NFRP 4 program and is supported by the
Swedish National Space Agency. Additional funding to DJH was provided by the Swedish
Foundation for Strategic Research (SSF) (ITM17-0236). Additional funding to JW was provided
by the Swedish Research Council (VR) (2021-87883).
Conflicts of interest
The authors have no competing interests to declare that are relevant to the content of this article.
Data availability
Computational and experimental data are available from the corresponding author upon
reasonable request.
Code availability
Not applicable.
Authors' contributions
André Nyberg Borrfors (ANB), Simone Ciaralli, Ashley Hallock and Tore Brinck (TB) contributed
to the study conception and general design. ANB, Daniel J. Harding and Jonas Weissenrieder
contributed to the experimental design and data acquisition. ANB and TB devised the theoretical
procedure. ANB performed all computations and analysis of experimental data. ANB wrote the
first draft. All authors commented on previous versions of the manuscript, read and approved the
final manuscript.
Page 2 of 23
1. INTRODUCTION
Electric propulsion devices transfer momentum to a spacecraft by ionizing a propellant and
electrically accelerating the resulting plasma to exhaust velocities an order of magnitude greater
than what can be achieved with conventional chemical thrusters, allowing delivery of a higher
delta-v compared to chemical propulsion for the same propellant mass. With the
commercialization of space and the steadily increasing demand for affordable satellites, the
market for more efficient and cost-effective electric propulsion thrusters has grown. Xenon has
historically been the most common propellant for electric propulsion subsystems, but has begun
to be replaced with more affordable and more readily available alternatives such as krypton. A
storable alternative to xenon offers advantages on subsystem level by eliminating the need for
high-pressure propellant storage. In recent years the use of storable atomic propellants like
bismuth and mercury for high thrust applications, and zinc and magnesium for high specific
impulse applications has been investigated [1–4]. Some molecular propellants have been
investigated, such as water (where the thruster operates on hydrogen and oxygen gas created by
electrolysis) [5,6], iodine [7–13], and adamantane [14–16], but were all found to have prohibitive
disadvantages such as significantly lower performance compared to xenon. A paper on the first
in-space demonstration of (low-power) thruster operation on iodine vapor was recently
published in Nature [13]. Iodine offers a high storage density and vapor pressure combined with
low ionization energy; however its reactivity introduces major challenges to development and
implementation, especially for thrusters operating above 100 kW. The goal of this work is to
identify and evaluate potential storable alternative EP propellants that have a mass and
ionization efficiency comparable to that of xenon.
1.1 Ionization Properties of Molecular Propellants
The hydrocarbon molecule adamantane has previously been explored as a propellant for use in
electric propulsion thrusters [14–16]. Adamantane is the smallest diamondoid and has a mass
similar to the xenon atom. Its ionization cross section is five times larger than that of xenon and
its ionization energy (IE) is 9.25 eV compared to 12.13 eV for xenon [17]. It can serve as a scaffold
for further molecular design and it is easily modified with a wide range of possible substituents;
however experiments involving thruster operation on adamantane and a few of its substituted
derivatives show fragmentation levels too high for these molecules to be effectively used as
propellants for electric propulsion [14,15].
In this study we propose aromatic hydrocarbons (AH) for use as molecular propellants as an
alternative to xenon. In particular, we highlight the advantages of the hydro- and fluorocarbons
fluorobenzene, naphthalene, and 1-fluoronaphthalene (Fig. 1). The availability of AHs (especially
the smaller variants) is reflected in their low prices as is evident from Table. 1 of Appendix A.
While these are estimates and based on what was available from resellers at the time of asking,
the AHs are generally around 10-100 times less expensive than xenon and much easier to procure.
Page 3 of 23
Fig. 1 Molar mass and (adiabatic) ionization energies of adamantane and the AHs considered for
use as molecular propellants in electric plasma thrusters
These candidates were selected from a set of 13 different AHs (see Table. 1) by screening for
desirable properties like high vapor pressure, appropriate phase change temperatures, and low
fragmentation during ionization as measured using standard electron ionization mass
spectrometry. An inherent energy sink associated with ionizing molecules is the occurrence of
fragmentation wherein some molecular ions break apart into multiple fragments where only one
fragment remains charged. The additional energy of having to ionize any resulting neutral
fragments can quickly compound because fragments are typically less stable than the original
molecule and can dissociate even further. Therefore, molecular propellants that are stable both
before and after ionization could offer a higher ionization efficiency compared to molecules with
higher rates of fragmentation.
Compared to other molecules, AHs are unique in their stability towards ionization-induced
dissociation because of their unique electronic and nuclear structures. In an AH, the electronic
structure consists of a combination of - and -orbitals, which provides additional stabilization
upon ionization compared to saturated hydrocarbons such as adamantane. The -orbitals can be
divided into lower-energy core orbitals and bonding valence orbitals. The latter make up a
skeleton of single bonds between all atoms in the molecule. The -orbitals are generally higher in
energy and more delocalized in character, and together they contribute a partial double bond
character to the bonds between all atoms of the ring system. Because of their higher energies,
ionization typically leads to expulsion of an electron from a -orbital, and this only partially
reduces bond orders between the ring atoms as the -orbital single-bond skeleton remains intact.
In the more unlikely event of ionization from a -orbital, individual -bonds may be weakened,
but due to the extra bond-stabilization from the -orbitals this is not likely to lead to bond
rupture. In a saturated hydrocarbon, such as adamantane, ionization occurs predominantly from
the valence sigma-orbitals, and this will inevitably weaken the -bond skeleton, and it is highly
likely to lead to bond rupture and fragmentation. The higher stabilities upon ionization of AHs
compared to saturated hydrocarbons can be verified by comparing their electron ionization mass
spectra, which show significantly higher fragmentation for the saturated hydrocarbons.
AHs also possess remarkable resistance towards thermal degradation [18–21], with gas phase
benzene and naphthalene starting to decompose in the range of 600-650 °C [22]. Heavier AHs
than those in Fig. 1 consisting of an increasing number of fused carbon rings can also serve as
viable propellant candidates and have even higher decomposition temperature but their vapor
pressure is lower than that of their smaller counterparts. This can lead to higher required heater
power to maintain the propellant as a gas in the propellant feed system and potential deposition
of propellant from the thruster plume on spacecraft surfaces. Vapor pressure curves for many
AHs can be found in Appendix A, Fig. 6.
Page 4 of 23
The properties of AHs are readily modified by chemical functionalization, i.e. one or more of the
hydrogens can be substituted for another atom or functional group. The substitution of a
hydrogen for a heavier element typically reduces the resistance towards ionization-induced
fragmentation. The only obvious exception is fluorine substitution, which generally has a minor
effect on the fragmentation as long as the number of substitutions is limited. This anomaly can be
traced to the relatively low weight of the fluorine in combination with the relatively high strength
of the C-F bond. Substituting one of the hydrogens in naphthalene with a single fluorine atom
increases the molecular mass by 14 % from 128.2 to 146.2 amu and the aggregation state changes
from solid to liquid at ambient temperature. Depending on the position of the fluorine two
different naphthalene isomers can be constructed, namely 1-fluoronaphthalene and 2fluoronaphthalene. These have slightly different physical properties, e.g. melting point, but their
ionization behaviors and mass spectra are similar. Considering the similarity of the two isomers,
only 1-fluoronaphthalene is considered in this work together with the other AHs.
The ionization cross sections of molecules are generally larger than for atoms and therefore a
shorter plasma ionizing region is sufficient to reach the required degree of ionization for a
molecular propellant. The most important ionization process taking place in typical electric
propulsion thrusters is (e,2e) electron ionization, where a single incident electron with kinetic
energy T collides with a target and results in the expulsion of a bound electron. This creates a
positively charged radical cation, which can be accelerated by an electric field to produce thrust.
If T is lower than the IE of the target atom or molecule, no ionization can occur from a single
collision. If multiple collisions between a target and electrons (or other molecules when the
pressure is high) are allowed, as in an ionizing plasma, less energetic collisions can still
accumulate enough energy in the target to ultimately lead to ionization.
In this article, we will describe the experimental and analytical method of investigation of the
ionization behavior of the molecules in Fig. 1. Results and discussion of ionization cross section
calculations for the AHs and adamantane are followed by results and discussion of measurements
of the fragmentation of the AHs and adamantane, including a comparison of the estimated
ionization efficiency of the AHs and that of xenon. The predicted performance of a thruster
operating on AHs is then discussed, along with a discussion of accommodation, safety and
environmental aspects. The article concludes with a summary of findings regarding the suitability
of the AHs as an alternative EP thruster propellant.
2. METHODOLOGY
2.1 Method of Calculation of Ionization Cross Section
All quantum chemical calculations were performed using the Gaussian 16 software suite [23].
Relaxed molecular geometries were obtained through structure optimization using the hybrid
M06-2X functional and the 6-311g(d,p) basis set. Vertical ionization energies (IE) used for
producing BEB cross sections (see Appendix B, Eq. 3 and [24,25]) were obtained using electron
propagator theory (EPT) based on Hartree-Fock (HF) wavefunctions and the 6-311+g(2df,2p)
basis set. In the cases where relativistic effects had to be accounted for, such as for BEB
calculations involving xenon and iodine, the Def2-TZVPP basis set with effective corepseudopotentials was used. BEB theory is most sensitive to the accuracy of the IE (B in Eq. 3)
associated with the highest energy valence orbitals. Therefore, when used to calculate t=T/B for
valence orbitals, the value of B was taken to be the more accurate IE obtained by applying the P3
propagator in EPT. As EPT often struggles to converge when the IE is too high, only values of B<20
eV were obtained using P3. For the remaining orbitals, and in the rare instances when P3 failed
to converge while below the threshold, the IE was, by applying Koopmans theorem, taken to be
Page 5 of 23
the negative of the orbital binding energy obtained at the HF level of theory. HF was also used in
producing the value of the orbital kinetic energy U, and by extension its reduced form u=U/B.
2.2 Method of Measurement of Ionization-induced Fragmentation
Mass spectra were collected using a Pfeiffer QMA 400 Quadrupole mass spectrometer (MS)
equipped with a Faraday cup detector and mounted on a small vacuum chamber capable of
reaching 1 × 10−8 mbar base pressure. Software for controlling the MS and collecting
spectroscopic data was QUADERA (build 4.62.004). The chamber pressure was measured using
a Balzers IKR-020 Penning Vacuum Gauge placed out of the line of sight of the MS ionizer. The
Penning pressure detector is a cold cathode ionization-type vacuum gauge that uses a high
electron current with a non-discrete and complex energy distribution to ionize gases within the
chamber. The reported pressure is linearly dependent on the resulting ion current, which, in turn,
depends on the appropriate cross sections of the gases. For simple and common gases (e.g., H2,
N2, Ar) at low pressures, the correction factor for determining the real chamber pressure is
known, but this is not the case for the molecular analytes covered in this study.
Initially, spectra were collected using a residual gas analyzer MS of the type RGA200 from
Stanford Research Systems and showed a strong bias towards low mass fragments. Later
measurements (Fig. 4-5 and EIMS in Fig. 7) instead used the Pfeiffer QMA 400 instrument, where
effort was made to ensure that no detection bias was present. Samples were let into the MS with
a variable leak valve, allowing control of the pressure. Before each MS measurement, volatile
atmospheric gases introduced during sample loading were slowly evacuated by a turbopump
until only a trace amount of water could be detected. To protect the thermionic filament from
overheating when running the MS at low electron energies an emission current of 0.1 A was used
for all measurements (one tenth of the standard setting). To compensate for the resulting loss in
signal intensity the chamber pressure was allowed to reach up to 5 × 10−6 mbar during
measurements, as reported by the Penning gauge. To reduce baseline noise the collection of each
spectrum was cycled four times and averaged. Measurements at different electron energies were
performed in quick succession to ensure the chamber pressure did not drift appreciably. Analytes
were purchased from SigmaAldrich (≥99 % purity) and used as received.
3. RESULTS AND DISCUSSION
3.1 Ionization Cross Section Calculation Results and Discussion
The ionization cross section (analogous to the ionization probability) and the ionization energy
(IE) are important properties of a propellant to maximize the performance and energy efficiency
of a plasma thruster [26]. The IE of AHs can easily be found in the literature and are collected in
Table. 1 of Appendix A, but experimental cross sections are only available for some atoms and for
the simplest AHs, and we therefore refer to ab initio quantum chemical calculations instead. In
Fig. 2 the total ionization cross section curves from (e,2e) ionization as calculated using Binary
Encounter Bethe (BEB) theory are shown for a selection of relevant atomic and molecular species.
Through experiment the total cross sections of the noble gases argon and xenon have been
determined to 1.23-3.52 and 2.62-7.31 Å2, respectively, when ionized by 70-75 eV electrons
[27,28]. Through BEB calculations we obtain 2.06 and 4.17 Å2, respectively, which are within the
range of the experimental values. More importantly, BEB has been shown to accurately predict
cross sections of many low mass molecules, including a range of different hydro-and
fluorocarbons [29–31]. BEB cross sections of aromatic ring systems similar to small AHs are also
known to be in excellent agreement with experimental values [32].
Page 6 of 23
Fig. 2 Total ionization cross sections calculated using BEB theory for the molecules in Fig. 1,
argon, xenon, and atomic/molecular iodine. T is the kinetic energy of the incident electron
impinging on the atom/molecule
A clear separation in the cross sections between the included atoms and molecules can be
observed in Fig. 2, where the former all have a cross section maximum in the range of 2 to 5 Å2.
The maxima of the latter lie above 8 Å2 and rapidly ascend in order of increasing number of atoms
of each respective molecule, starting with molecular iodine. For instance, adamantane and the
naphthalenes have over 5 times the cross section of xenon, which would lead them being ionized
after a much shorter time spent in the plasma. The cross-section curves of both atomic and
molecular iodine are included in Fig. 2, as a large fraction of molecular iodine dissociates upon
being ionized and the resulting uncharged iodine atom has to be ionized to produce thrust. The
length L of the effective ionizing region of the plasma in a plasma thruster is proportional to the
ionization cross section of a given propellant averaged over a Maxwellian velocity distribution
[26] by
𝐿 ∝ 〈𝜎𝑣𝑒 〉−1
Where 〈𝜎𝑣𝑒 〉 ≈ 𝜎(𝑇𝑒 )𝑣̅ is the reaction rate coefficient that can be approximated by the product of
the cross section averaged over a Maxwellian distribution of electrons with temperature 𝑇𝑒 and
the average speed 𝑣̅ of those electrons. Increasing L will increase the degree of ionization of the
propellant fed to the thruster by allowing for more collisions with the plasma electrons and more
energy transfer into the propellant. However, increasing energy deposited into the propellant
leads to decreasing energy efficiency of the thruster, and in the case of a molecular plasma
promotes molecular dissociation processes.
3.2 Fragmentation Measurement Results and Discussion
Incident electrons of kinetic energy T can cause ionization of the target atom or molecule through
the (e,2e) mechanism, given that T exceeds their respective vertical IE. Energy deposition
resulting from successful (e,2e) collisions have been calculated based on BEB theory, and the
Page 7 of 23
gaussian-smoothed probability distributions (Eq. 4-7, Appendix B) are displayed in Fig. 3 for
adamantane and the selected AHs. By increasing T, more strongly bound electrons in the
molecules become accessible and can participate in (e,2e), resulting in the noticeable decrease in
relative intensity of the lowest energy peak for each molecule. This decrease is less pronounced
for adamantane in Fig. 3 (A) compared with the AHs (B-D) and should thus contribute to less
variation in the degree of dissociation as T increases. We will see later, however, that the
proclivity of adamantane to undergo ionization induced dissociation is already very high at T =
25 eV, and that the fragment abundance measured using standard 70 eV mass spectrometry is
highly indicative of the stability (and lack thereof) at lower T.
Fig. 3 Computed probability distributions of the energy deposition caused by single collision
(e,2e) ionization for the molecules (A) adamantane, (B) fluorobenzene, (C) naphthalene, (D) 1fluoronaphthalene. The figure illustrates four (e,2e) scenarios: three where the incident electron
kinetic energies T range from 25 to 70 eV, and one where T is dictated by a Maxwell-Boltzmann
distribution of electron energies corresponding to a plasma temperature Te of 7 eV
In electrostatic EP thrusters ions created through collisions with energetic electrons are
accelerated to high velocities to produce thrust. Due to the significant difference in mass between
the electrons and the larger particles within the anode chamber, a heterogeneous plasma can be
sustained where the ionized species are expelled before thermal equilibrium with the electrons
can be reached. The kinetic energy of the electrons in the ionizing plasma is here assumed to be
well approximated by a Maxwell-Boltzmann distribution with a plasma temperature Te=7 eV (see
Appendix B, Eq. 6). This Te was selected to obtain results comparable with those reported by Dietz
et al in their study on plasma ionization of adamantane, xenon, and molecular iodine [15].
The calculated energy deposition caused by a Maxwellian plasma with an electron temperature
Te = 7 eV is also included for the molecules in Fig. 3 (purple curves). These curves are very similar,
but slightly lower, to the (blue) curves resulting from (e,2e) with T = 25 eV. With the assumption
of a low number of electron collisions taking place within the 7 eV plasma, the 25 eV (e,2e)
measurement is well suited for estimating the adamantane fragmentation within such a plasma.
This was verified in the current study where the 25 eV mass spectrum of adamantane agreed well
with measurements made in the 7 eV plasma thruster by Dietz et al (vide infra). Furthermore, the
difference between energy deposition from low energy (e,2e) and plasma ionization for the
selected AHs is more pronounced, indicating that electron energies even lower than T = 25 eV
could be appropriate when simulating their behavior in a 7 eV plasma.
The electron ionization mass spectra (EIMS) of adamantane and the AHs can be seen in Fig. 4. The
intensities observed in the measurements cannot be used to compare absolute ionization cross
Page 8 of 23
sections between the different analytes, as we had no reliable way of measuring the absolute
pressure (see methods). However, the relative change in ion current as a function of T for each
analyte is easily determined and can be found in the right column of the tables in Fig. 4, as well as
in Appendix A, Table 2. In Table 2 we also list the relative cross sections calculated using the BEB
method. Immediately evident from the spectra is the much lower fragmentation of the AHs when
compared with adamantane, and the similarly sized fragmentation peaks of the naphthalenes.
Fig. 4 Mass spectra from the molecules (A) adamantane, (B) fluorobenzene, (C) naphthalene, (D)
1-fluoronaphthalene, collected at different incident electron energies T. The spectra collected at
40 and 25 eV have been horizontally offset by 4 and 8 m/z, respectively. For each molecule is
included a table with the relative ion current of M+ with respect to the total ion current 𝐼𝑀+ /𝐼𝑡𝑜𝑡
(left), and the relative total ion current with respect to the same total ion current measured at
70
𝑇
𝐼𝑡𝑜𝑡
/𝐼𝑡𝑜𝑡
(right)
As mentioned earlier, adamantane has been shown to suffer from a high degree of fragmentation
under conditions commonly found in EP thruster discharge chambers [15,16]. Per Fig. 2 and
Table 2 (Appendix A), the BEB cross section of adamantane decreases by 12.3 and 43.1 % when
electron energy is reduced from 70 eV to 40 and 25 eV, respectively. This is reasonably well
reflected by our measurements in Fig. 4 (A) by a 14.6 and 51.1 % reduction, respectively.
However, as can be seen in the low ratios in the left column of the table in (A), a very large part of
the adamantane ion current stems from fragments of molecular dissociations. Even when using
the low electron energy T = 25 eV only 33.9 % of the signal is due to the adamantane molecular
ion (M+), a figure which rises to 41.3 % (Table. 2) if none of the neutral hydrogen loss from the
parent molecule is counted towards fragmentation (i.e. M+ = [M-HX]+ where X≥0). Each signal in
Fig. 4 caused by the detection of an ionic fragment is associated with at least one additional
neutral fragment which does not appear in the measured spectra. In a plasma thruster all such
Page 9 of 23
neutrals would have to be ionized before undergoing electric or electromagnetic acceleration, and
for adamantane this would result in a very low energy efficiency because of the excessive
fragmentation. The mass spectra measurements performed by Dietz et al on the exhaust plume
of a modified RIT thruster operating on adamantane (see Figure 9 in [15],) show a very high
abundance of fragments. The authors varied the mass flow of adamantane to the thruster as well
as the power of the radio frequency generator (RFG) to estimate the effect of plasma temperature
and electron-neutral collision frequency on the molecular plasma composition. At the lowest RFG
power, and with plasma temperature Te estimated to be 7 ± 1 eV, the relative abundance of [MHX]+ ions exiting the RIT was roughly 36 % of the total ion current. This high fragmentation agrees
well with the value of 41.3 % we obtained for T = 25 eV in the present work.
The behavior of the AHs in Fig. 4 under ionizing conditions is distinctly different from
adamantane, whereas the differences between the individual AHs are rather small. We will
therefore discuss the naphthalene spectra in more detail and compare them to the adamantane
spectra. The mass spectra of naphthalene in Fig. 4 (C) display much less intense fragment ion
signals in relation to M+ for all T. Notably, naphthalene remains intact to a larger degree even at
T=70 eV (IM+=47.8 %) compared with adamantane at T=25 eV (IM+=33.9 %). At 25 eV the
percentage of intact naphthalene cations is 85.3 % of the total ion current (92.5 % for [M-HX]+),
while the remaining 14.7 % (7.5 %) is due to detected fragment ions. The largest difference in
fragmentation behavior is observed when T is increased from 25 to 40 eV, which is consistent
with the corresponding curves in Fig. 3 where the yellow and red lines (40 and 70 eV) overlap to
a large extent. This highlights the importance of optimizing the ionization conditions of the
plasma thruster to prevent excess energy being deposited into the propellant, as all
fragmentation constitutes a loss of thruster efficiency.
To increase legibility, the 25 eV mass spectrum of naphthalene was converted to a histogram bar
graph by integrating the signals about each integer m/z. The result can be seen in Fig. 5 where
the region 20-105 m/z has been scaled up and labels for the most abundant fragments have been
included.
Fig. 4 Histogram mass spectrum of naphthalene obtained at T=25 eV incident electron energy,
where the region 20-105 m/z has been scaled up
From Fig. 5 it is clear that for single collisions with low energy electrons, naphthalene is likely
only to dissociate into a few select fragments. The rightmost peaks (m/z=101,102 amu) in the
+
, is the fragment associated with loss of acetylene, C2 H2 , whose
scaled-up figure, denoted C8 H5−6
signal is detected as the leftmost peak at m/z=26 amu. Together, these constitute the full
naphthalene molecule or in the case of any associated hydrogen loss, some version of [M-Hx]+.
+
The remaining peak, C6 H4−6
, is associated with a fragment that appears with low intensity at
m/z=50 amu.
Page 10 of 23
The situation becomes significantly more complex when a plasma is used for ionization. By
colliding with energetic electrons, the energy of a molecule increases. If the pressure is high, this
energy is rapidly distributed through molecule-molecule collisions, resulting in a rise in
temperature. At higher temperatures, more dissociation pathways become available and,
consequently, more unique fragments are created during ionization and can be detected from
mass spectrometry. We performed high temperature gas chromatography mass spectrometry
(GC-MS) at 250 °C and T = 70 eV electrons on the analytes in Fig. 4 (except fluorobenzene) to
evaluate how their respective fragment abundances change when heated. The superimposed
spectra from GCMS with those from relevant measurements in Fig. 4 can be found in Appendix A,
Fig. 7. In the case of adamantane, GC-MS resulted in a noticeable increase in fragment intensities
while the effect was more subdued for the naphthalenes, almost to the point of being
indistinguishable when compared with the cold EIMS experiment. Similar effects were reported
by Eren et al [33] who noted that “… for small molecules (below 20 atoms) such as benzene (C6H6),
no or very little vibrational cooling effect is observed in their Cold EI mass spectra, while compounds
with 20-40 atoms show noticeable enhancement of their molecular ion abundance.”
In a study by Alliati et al [34] where they measured ionization of naphthalene using a plasma
sourced from a high-frequency RFG, the types of fragments detected was almost identical to those
we found when using T = 40 and 70 eV electrons in (e,2e) of naphthalene. The relative intensities
of fragment peaks following plasma ionization were, however, found to be much lower and on
par with our 25 eV measurements. The authors did not specify an electron temperature
associated with the plasma electrons, or even if they could be considered Maxwellian and have a
well-defined temperature, and therefore it is difficult to draw conclusions beyond these general
observations in relation to the results obtained in this work. Nevertheless, their results are very
encouraging and support our observations of naphthalene’s stability under relevant conditions.
Expanding the discussion to include the rest of the AHs in Fig. 4, the relative ion current at T = 25
eV for naphthalene appears slightly larger than for 1-fluoronaphthalene in accordance with the
BEB cross sections in Fig. 2. Their fragmentation abundances are nearly identical at all T,
confirming that the addition of a fluorine substituent has negligible effect on the molecular
stability and that fluorine substitution is a useful tool which allows for expanding on the AHs
capabilities as propellants. Fluorobenzene is slightly more prone to dissociate at T = 25 eV
compared to the heavier naphthalenes, but the difference is minor and should not exclude
fluorobenzene from being considered a viable propellant candidate.
Treating [M-HX]+ ions as intact molecules despite loss of neutral hydrogen (as was done in the
paper by Dietz et al [15] when analyzing adamantane) increases the intact-to-total-ion ratio of
the T = 25 eV spectra from that of the right columns in Fig. 4 to 0.844, 0.925, and 0.897 for
fluorobenzene, naphthalene, and 1-fluoronaphthalene, respectively. Disregarding hydrogen
fragmentation is not entirely unwarranted, as both molecular and atomic hydrogen obtain
considerable velocity when having undergone dissociation, due to their low mass. The ionization
cross section of molecular hydrogen is around 0.5 Å2 when T=25 eV [35] and its 1st IE is over 15.4
eV, making it much less likely to be ionized by a plasma thruster designed and optimized for much
heavier molecules with large cross sections and low IE. Atomic hydrogen even less so. A more
likely outcome is that the dissociated neutral hydrogen will, to a large degree, exit the thruster
without being ionized. However, the hydrogen could also participate in neutral-neutral and
neutral-ionic collisions with other molecules. Both processes, which are inherent when dealing
with molecular plasmas, contribute to increasing the ionization inefficiency of the thruster by
promoting further dissociation of the propellant.
Page 11 of 23
4. Performance, Accommodation, and Impact of AHs as EP Propellants
4.1 Performance Predictions for AHs
Fluorobenzene has a relatively low molecular mass of 96.1 amu, high vapor pressure and low
melting point, which could lead to low heater power requirements and higher specific impulse. It
could be an attractive candidate for, e.g., power- and volume-constrained satellites, or long-range
missions with low available power and high delta-v requirements. The naphthalenes are heavier
and therefore better suited for high thrust applications. The larger ionization cross section and
lower IE of the AHs compared to that of xenon could result in a comparable or even higher
ionization efficiency compared to that of xenon despite ionization-induced fragmentation of the
AHs. To put it in perspective, the difference in IE between naphthalene and xenon is 4 eV which
translates to about 386 kJ mol-1 or 92.2 kcal mol-1 (1 mol is approximately 0.13 kg in this case).
With an assumed propellant feed rate of 10 mg s-1 to the thruster (and disregarding any energy
costs associated with fragmentation), the 4 eV difference would equate to a 30-W reduction in
power required for total ionization. Furthermore, the mass of naphthalene (128.2 amu) is very
close to that of xenon (131.3 amu), making it an ideal benchmark propellant for future testing.
4.2 Propellant Storage and Delivery
In Table. 1 of Appendix A the boiling point and density of several AHs are listed. The density of
the AHs in Fig. 1 ranges between 1020-1140 kg m-3 and the boiling point is in all cases high enough
to ensure that they all remain non-gaseous at room temperature and under atmospheric pressure
(i.e., at NTP). A propellant stored onboard a satellite can be kept close to NTP, and as the melting
point of 1-fluoronaphthalene and fluorobenzene are negative on the Celsius scale, they will
remain liquid. Naphthalene is solid at NTP, but the vapor pressure is sufficient to facilitate the
transfer rate required to feed a mN-class thruster and the melting point is sufficiently high to
avoid a solid-liquid phase transition. Vapor pressure data for the fluoronaphthalenes was not
available, but the fluoronaphthalenes are assumed to have similar vapor pressure curves as
naphthalene, since benzene and the fluorobenzenes have similar vapor pressure curves as shown
in Fig. 6.
While the AHs cannot be compressed to the density of pressurized gaseous propellants like xenon
at 186 bar, or the density of heavier atomic propellants like iodine or mercury, they do avoid the
added mass and safety considerations of the high-pressure propellant storage equipment
required by the former and many material incompatibilities of the latter.
4.3 Accommodation in electrostatic EP thrusters
Positively charged propellant electrostatically expelled from the thruster must be neutralized to
prevent the accumulation of negative charge in the spacecraft. This is achieved by electron
emission, which in addition to neutralizing the exhaust, also feeds electrons for the ionizing
plasma. For high power thrusters where high electron currents are needed, neutralization is
typically accomplished by thermionic emission in the form of a hollow cathode placed close to the
thruster exhaust. This type of cathode needs to be fed with a significant amount of propellant to
facilitate the outgoing flow of electron current, all while the actively emitting thermionic material
is kept at temperatures normally between 1300-2000 °C. At those temperatures most types of
molecules will completely disintegrate into their highly reactive atomic constituents. The
materials commonly used for thermionic emission are typically fed with very pure and inert
xenon, and the performance is very sensitive to changes in their surface chemical composition.
Extensive exploration of the literature strongly indicates that hydrocarbons are not compatible
with most common types of thermionic emitter materials [36–43]. While research on the
Page 12 of 23
poisoning behavior of most emitters is scarce, emitters are likely to be inoperable in non-inert
atmospheres because of their fragile and easily disrupted emission capacities.
Alternative electron emission technologies, such as field emission (FE) array cathodes, exist. FE
cathodes require no propellant feed or dedicated heating to operate, but they instead rely on a
strong electric field to pull electrons from the surface of the material. The achievable emission
output is typically limited to only a few mA cm-2, whereas high-performance hollow cathodes can
be made to emit tens to hundreds of amperes. Recent advances have been made in operating
resistant FE array cathodes based on carbon nanotubes in the hazardous environment of Low
Earth Orbit, as part of the KITE mission conducted by the Japanese space agency JAXA [44,45].
Their achieved current output was quite limited, but other researchers have demonstrated
optimized CNT array emitters reaching close to 1 A cm-2 [46]. In the future, FE cathodes like these
could become viable for use with thrusters operated on alternative propellants like the AHs
suggested here.
4.4 Safety Considerations and Environmental Impact
AHs have been linked to several kinds of adverse health effects in humans, both acute and chronic
[49], but studies have shown that the toxicity of unsubstituted AHs generally decreases with
molecular weight, except for the highly carcinogenic benzene [50]. Naphthalene is classified as
harmful if swallowed and suspected to be cancerogenic according to the European Chemicals
Agency (ECHA [51]. The ECHA classification for 1-fluoronaphthalene only states that it causes
serious eye irritation, causes skin irritation and may cause respiratory irritation [52].
Fluorobenzene is only classified to cause serious eye damage, although it has been identified that
it may cause damage to organs through prolonged or repeated exposure [53]. Naphthalene is
very toxic to aquatic life with long-lasting effects. The corresponding effects are less severe for
fluorobenzene and 1-fluoronaphthalene as indicated by their ECHA classifications [51-53]. It
would be necessary to implement appropriate safety procedures for the handling of AHs by
ground facility personnel. However, it should be noted that the potential health hazards of the
AHs are much less severe than for the highly toxic and carcinogenic monopropellant hydrazine
and therefore the safety precautions for AHs would be much less restrictive. In addition, the AHs
are not classified as explosives, and handling procedures would be simplified compared to those
for monopropellants or xenon under high pressure.
As for potential environmental impact, whereas halogenated hydrocarbons of the heavier
halogens may have a damaging effect on the ozone layer when introduced into the stratosphere,
it is known that atmospheric fluorine does not lead to depletion of ozone [47]. Fluorocarbons
(such as the fluorinated compounds of this study) can contribute to global warming, mainly by
absorbing 1000-1400 cm-1 IR radiation where the atmosphere is otherwise relatively transparent
[48]. It should be noted that the lifetime of the AHs in this study is expected to be relatively short
at altitudes above the stratosphere due to high levels of UV-radiation. Thus, the AHs are not likely
to contribute to global warming.
5. CONCLUSIONS
We have evaluated the aromatic hydrocarbon (AH) molecules fluorobenzene (C6H5F),
naphthalene (C10H8), and 1-fluoronaphthalene (C10H7F) for use as alternative propellants in
electric propulsion thrusters for in-space propulsion by comparing environmental and safety
aspects, material compatibility, storability, cost and predicted ionization efficiency.
Neither naphthalene nor the fluorinated AHs pose a risk of depleting the ozone layer if they were
to reach the stratosphere. While appropriate safety procedures would have to be implemented
Page 13 of 23
for the handling of AHs, they are not classed as explosives and do not require the safety
precautions associated with high-pressure gas and monopropellants.
A literature survey indicates that AHs are incompatible with available emitter materials used in
high-current hollow cathode neutralizers, limiting the propellant flow rates and thrust achievable
with AH propellants in electrostatic thrusters; however no material compatibility issues are
foreseen for cathodeless electromagnetic thrusters.
The AHs are liquid or solid at common satellite propulsion subsystem pressures and
temperatures, and while their density is lower compared to high-pressure xenon or propellants
like iodine and mercury, they do avoid the added mass and safety considerations of the highpressure propellant storage equipment required by the former and many material
incompatibilities of the latter. The solid/liquid-vapor phase transition properties of the AHs show
that they will be compatible with a low-power sublimation-based propellant feed system.
Ionization efficiency was estimated by calculation of the ionization cross section and
measurement of the ionization-induced fragmentation. Quantum chemical calculations and
Binary Encounter Bethe (BEB) theory were used to calculate the ionization cross section of these
AHs. The calculated ionization cross sections for the naphthalenes are more than five times higher
than that of xenon. BEB calculations further indicate that 25 eV electrons and electrons from a 7
eV Maxwellian plasma deposit similar amounts of energy into the molecules, allowing estimation
of the fragmentation behavior of the molecules in a 7 eV plasma thruster using 25 eV EI-MS. These
measurements for the AHs show very low fragmentation with 81 - 86 % of the observed ions
originating from intact molecules, compared with 34 % for adamantane. Considering that the
ionization energy of the AHs is 4 eV lower than that of xenon, the higher ionization cross section
together with the low degree of post-ionization dissociation indicate that the ionization efficiency
of the AHs could be similar to or greater than that of xenon.
The investigated AHs are considered viable candidates as an alternative propellant to xenon due
to their similar mass, lower storage pressure, lower cost, and potentially comparable or higher
ionization efficiency.
Page 14 of 23
1. APPENDIX A (TABLES AND FIGURES)
Table. 1 aromatic hydrocarbons (AH) identified as potential EV propellant candidates together
with relevant physical properties reported at standard conditions. Adamantane and xenon are
included at the bottom for easy comparison. Mw=molecular weight, TM=melting point, TV=boiling
point, ρ=density, IEa is the adiabatic ionization energy, and T(ṁ) the temperature corresponding
to an estimated evaporation/sublimation rate of ~10 mg cm-2 s-1 into vacuum as calculated with
Eq. 1 and 2 (Appendix B) using Antoine constants found in Table. 3
Chemical
formula
Mw
g mol-1
TM (TV)
°C
ρ
kg m-3
1st IEa
eV
T(ṁ)
°C
Bulk price4
€ kg-1
benzene
C6H6
78.1
5.5 (80.1)
876
9.24
-57*
38.9 (100L)
fluorobenzene
C6H5F
96.1
-42 (85)
1020
9.20
-57*
39.4 (200kg)
cyanobenzene
C6H5CN
103.1
-10 (191)
1000
9.73
11*
47.9
naphthalene
C10H8
128.2
86.3 (217)
1140
8.14
41
46.8
1-fluoronaphthalene
C10H7F
146.2
-13 (215.2)
1132
8.15
-
93.3
2-fluoronaphthalene
C10H7F
146.2
58 (211.6)
1240
8.23
-
-
fluorene1
C13H10
166.2
116 (298)
1200
7.91
86
73.8 (>98%)
phenanthrene
C14H10
178.3
101 (332)
1180
7.89
102
147.4
anthracene
C14H10
178.3
216.4 (341)
1250
7.44
136
83.1
hexafluorobenzene
C 6F 6
186.1
5.2 (80.2)
1610
9.90
-574
333.7
pyrene
C16H10
202.3
146 (404)
1270
7.43
1424
112.1 (>98%)
6.92
2504
12984.6 (>95%)
>2000
Anthanthrene2
C22H12
276.3
261 (556)
1377
coronene
C24H12
300.3
437.3 (525)
1371
7.29
3094
adamantane
xenon
C10H16
Xe
136.2
131.3
279
-111.8 (-108)
1080
>15003
9.25
12.13
-
1Not
fully aromatic but often considered an AH 2Commonly referred to as
Dibenzo[def,mno]chrysene 3Storage density can be increased through pressurization 4Prices
obtained in the middle of June 2021 and correspond to order size of 100 kg and ≥99 % purity
unless otherwise stated (estimates produced by Chemtronica AB, www.chemtronica.com/en)
4Temperature estimated by extrapolation outside the valid range defined for the Antoine
constants
Page 15 of 23
Table. 2 Relative ion intensities for the spectra shown in Fig. 4 together with relative ionization
cross sections calculated using Binary Encounter Bethe theory
T (eV)
𝐓
𝟕𝟎
𝐈𝐌
+ /𝐈𝐌 +
𝐈𝐌+ /𝐈𝐭𝐨𝐭
𝐈∑[𝐌−𝐇𝐱]+ /𝐈𝐭𝐨𝐭
𝟕𝟎
𝐓
/𝐈𝐭𝐨𝐭
𝐈𝐭𝐨𝐭
𝛔𝐓𝐁𝐄𝐁 /𝛔𝟕𝟎
𝐁𝐄𝐁
naphthalene
25
40
70
0.874
0.958
1.000
0.853
0.567
0.478
0.925
0.673
0.559
0.490
0.809
1.000
0.583
0.879
1.000
1-fluoronaphthalene
25
40
70
0.814
0.911
1.000
0.858
0.578
0.474
0.897
0.642
0.523
0.451
0.747
1.000
0.550
0.857
1.000
fluorobenzene
25
40
70
0.769
0.943
1.000
0.808
0.559
0.490
0.844
0.608
0.537
0.467
0.827
1.000
0.523
0.841
1.000
adamantane
25
40
70
0.974
1.066
1.000
0.339
0.212
0.170
0.413
0.263
0.212
0.489
0.854
1.000
0.569
0.877
1.000
Table. 3 Antoine constants for AHs and benzene analogues and the temperature ranges over
which they are defined. No data for 1-fluoronaphthalene could be found in the literature
Temperature range
(K)
(°C)
A
B (K)
C (K)
Benzene1
280-374
7-101
5.98523
1184.236
-55.623
Fluorobenzene2
255-357
-18-84
4.36225
1409.848
-34.792
Cyanobenzene2
301-464
28-191
4.85410
2110.572
-28.331
Naphthalene1
310-353
368-523
37-80
95-250
8.70592
6.13555
2619.91
1733.71
-52.5
-71.291
Fluorene1
307-348
348-388
34-75
75-115
5.33196
8.91036
1547.377
3245.362
-133.083
-45.893
Phenanthrene1
306-321
356-650
33-48
83-377
11.631
6.37081
4873.4
2329.54
0.05
-77.87
Anthracene1
299-430
26-157
10.5899
4903.3
-1.58
Hexafluorobenzene2
278-378
5-105
4.16054
1229.449
-57.503
Pyrene1
298-401
25-128
10.7545
5072.78
0.0
Anthanthrene1
465-495
192-222
12.014
7060
0.0
Coronene1
430-560
157-287
8.80062
5491.689
-48.484
Pressure given in 1kPa [54] 2102 kPa [55]
Page 16 of 23
Fig. 5 Vapor pressures of examined cyclic aromatic hydrocarbons as a function of temperature
obtained using data in Table 2 and Eq. 1. The melting point (measured at 25 °C and 100 kPa) for
each compound is indicated by vertical lines and the points corresponding to an evaporation mass
flow rate of ~10 mg cm-2 s-1 as calculated using Eq. 2 (where α=1 and Pc=0) are marked with a
black cross if they are within their respective Antoine temperature range, or a grey cross if they
are based on extrapolation outside the range of the source data. These temperatures (and vapor
pressures for benzene and the fluorobenzenes) are indicated numerically in the figure
Page 17 of 23
Fig. 6 Mass spectra of adamantane (A) naphthalene (B) 1-fluoronaphthalene (C), collected by
electron ionization mass spectrometry (EIMS) and gas chromatography mass spectrometry
(GCMS) using T=70 eV incident electron kinetic energy. A lower detection limit of m/z=39 amu
was present for the GCMS measurements, therefore the figure above does not include any of the
fragments detected with EIMS below that point
Page 18 of 23
2. APPENDIX B (EQUATIONS)
Eq. 1-2 Relations for calculating vapor pressures and mass flows by sublimation/evaporation
from solid/liquid phase
log10 (𝑃𝑣 ) = 𝐴 −
𝑚̇ =
𝐵
𝑇𝑠 + 𝐶
𝛼𝐴𝑠 (𝑃𝑣 − 𝑃𝑐 )
√2𝜋𝑘𝑏 𝑇𝑠 𝑚𝑝−1
(1)
(2)
where A, B, C are Antoine constants, 𝑚̇ is the mass flow rate (kg m-2s-1), Pv and Pc is the respective
vapor pressure and surrounding gas pressure (Pa), kb is the Boltzmann constant, Ts is the surface
temperature (K), mp is the molecular mass (kg), As is the surface area (m2) and α is a sticking
coefficient.
Eq. 3 Binary Encounter Bethe theory [24,25] used for calculating absolute single-collision (e,2e)
ionization cross sections by taking the sum over all (occupied) orbitals i of the target atom or
molecule
𝜎𝐵𝐸𝐵 = ∑ 𝜎𝑖 = ∑
𝑖
4𝜋𝑎02 𝑁(𝑅/𝐵𝑖 )2 ln 𝑡
1
1
ln 𝑡
(1 − 2 ) + 1 − −
[
]
𝑡
𝑡 𝑡+1
𝑡+𝑢+1
2
𝑖
(3)
where N is the orbital occupancy number, R the Rydberg constant, 𝑎0 the Bohr radius, Bi the
negative orbital binding energy (i.e., the vertical ionization energy to afford a specific electronic
state), Ui the orbital kinetic energy, T the kinetic energy of the incident electron, and u=U/B and
t=T/B are the reduced versions of U and T, respectively. The procedure for calculating B and U is
detailed in the method section of this article. Note that 𝜎𝑖 = 0 if 𝑇 < 𝐵 as there is not enough
energy to eject an electron from the orbital.
Eq. 4 Probability distribution for the energy deposition E into a target molecule or atom resulting
from successful electron ionization through the (e,2e) process, as presented in the paper by K.
Irikura [56]
𝑃(𝐸, 𝑇) =
∑𝑖 𝛿(𝐸 − 𝐵𝑖 )𝜎𝑖 (𝑇)
∑𝑖 𝜎𝑖 (𝑇)
(4)
where Bi and 𝜎𝑖 are defined in Eq. 3, E is the energy deposited into the ionization target, and 𝛿 is
the Dirac delta function defined by 𝛿(𝑥) = 1 when 𝑥 = 0 and 𝛿(𝑥) = 0 when 𝑥 ≠ 0.
Eq. 5 Modified version of Eq. 4 used to estimate the probabilistic energy deposition caused by
(e,2e) ionization by electrons with energies following a Maxwellian distribution PMB
𝑃(𝐸, 𝑇) =
∑𝑖 𝛿(𝐸 − 𝐵𝑖 )𝑃𝑀𝐵 (𝑇)𝜎𝑖 (𝑇)
∑𝑖 𝑃𝑀𝐵 (𝑇)𝜎𝑖 (𝑇)
(5)
Page 19 of 23
Eq. 6 Probabilistic Maxwell-Boltzmann energy distribution derived for massive, non-relativistic
particles
1
𝑇
𝑇 2
𝑃𝑀𝐵 (𝑇) = 2 ( 3 ) exp (− )
𝑇𝑒
𝜋𝑇𝑒
(6)
where Te is the electron temperature (in eV), and T is (same as Eq. 3-5) the electron kinetic energy.
Eq. 7 Gaussian smoothing function
𝐸 − 𝐵𝑖 2
𝐺 = exp((
)
𝜌
(7)
where 𝜌 = 1.5𝑒𝑉 is the variance used for all calculations presented in this work, and E and Bi are
defined in Eq. 3 and 4, respectively.
Page 20 of 23
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| 9,465 | 62,564 |
W2092815384.txt | Selective Akt Inhibitors Synergize with Tyrosine Kinase
Inhibitors and Effectively Override Stroma-Associated
Cytoprotection of Mutant FLT3-Positive AML Cells
Ellen Weisberg1*., Qingsong Liu2., Xin Zhang2, Erik Nelson1, Martin Sattler1, Feiyang Liu3,
Maria Nicolais1, Jianming Zhang2, Constantine Mitsiades1, Robert W. Smith1, Richard Stone1,
Ilene Galinsky1, Atsushi Nonami1, James D. Griffin1*, Nathanael Gray2
1 Department of Medical Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, United States of America, 2 Department of Biological
Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, United States of America, 3 High Magnetic Field Laboratory, Chinese Academy of
Sciences, Hefei, Anhui, P.R. China
Abstract
Objectives: Tyrosine kinase inhibitor (TKI)-treated acute myeloid leukemia (AML) patients commonly show rapid and
significant peripheral blood blast cell reduction, however a marginal decrease in bone marrow blasts. This suggests
a protective environment and highlights the demand for a better understanding of stromal:leukemia cell communication.
As a strategy to improve clinical efficacy, we searched for novel agents capable of potentiating the stroma-diminished
effects of TKI treatment of mutant FLT3-expressing cells.
Methods: We designed a combinatorial high throughput drug screen using well-characterized kinase inhibitor-focused
libraries to identify novel kinase inhibitors capable of overriding stromal-mediated resistance to TKIs, such as PKC412 and
AC220. Standard liquid culture proliferation assays, cell cycle and apoptosis analysis, and immunoblotting were carried out
with cell lines or primary AML to validate putative candidates from the screen and characterize the mechanism(s) underlying
observed synergy.
Results and Conclusions: Our study led to the observation of synergy between selective Akt inhibitors and FLT3 inhibitors
against mutant FLT3-positive AML in either the absence or presence of stroma. Our findings are consistent with evidence
that Akt activation is characteristic of mutant FLT3-transformed cells, as well as observed residual Akt activity following FLT3
inhibitor treatment. In conclusion, our study highlights the potential importance of Akt as a signaling factor in leukemia
survival, and supports the use of the co-culture chemical screen to identify agents able to potentiate TKI anti-leukemia
activity in a cytoprotective microenvironment.
Citation: Weisberg E, Liu Q, Zhang X, Nelson E, Sattler M, et al. (2013) Selective Akt Inhibitors Synergize with Tyrosine Kinase Inhibitors and Effectively Override
Stroma-Associated Cytoprotection of Mutant FLT3-Positive AML Cells. PLoS ONE 8(2): e56473. doi:10.1371/journal.pone.0056473
Editor: Ivan Cruz Moura, Institut national de la santé et de la recherche médicale (INSERM), France
Received November 6, 2012; Accepted January 9, 2013; Published February 21, 2013
Copyright: ß 2013 Weisberg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Grant Support: QL and NG are supported by National Institutes of Health (NIH) Library of Integrated Network-Based Cellular Signatures (LINCS) grant
HG006097. FL is supported by a Chinese Academy of Science High Magnetic Field Laboratory training grant. JDG is supported by NIH grant CA66996. FL is
supported by CAS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: JDG has a financial interest with Novartis Pharma AG. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing
data and materials.
* E-mail: ellen_weisberg@dfci.harvard.edu (EW); james_griffin@dfci.harvard.edu (JDG)
. These authors contributed equally to this work.
concentrations by HS-5 stroma (including SCF, IL-6, IL-8, IL11, M-CSF and GM-CSF), were able to partially protect TKItreated chronic myeloid leukemia (CML) cells and AML cells
[2,3].
A subset of AML cells expresses a mutated form of the class III
receptor tyrosine kinase FLT3 (Fms-Like Tyrosine kinase-3; STK1, human Stem Cell Tyrosine Kinase-1; or FLK-2, Fetal Liver
Kinase-2) [4], which has inspired the development of a number of
small molecule inhibitors of mutant FLT3. However, FLT3
inhibitors tested thus far, including PKC412 (midostaurin) [5],
which is in late stage (Phase III) clinical trials, and the highly
potent and selective FLT3 inhibitor, AC220 (quizartinib) [6],
which is in early phase clinical trials, generally at best induce
Introduction
Resistance to TKIs in leukemia patients presents a significant
clinical challenge. As small numbers of leukemia cells have been
observed to persist in the bone marrow of TKI-treated patients,
despite rapid and dramatic clearance of peripheral blood blasts,
there is growing interest in determining the role of the bone
marrow microenvironment in the long-term survival of leukemic
stem cells. Indeed, the number of existing leukemic stem cells that
exhibit high survival ability on bone marrow stromal layers has
proven to be a significant prognostic indicator [1]. Of relevance,
we have found that media conditioned by human HS-5 stromal
cells, as well as a cocktail of cytokines secreted in high
PLOS ONE | www.plosone.org
1
February 2013 | Volume 8 | Issue 2 | e56473
Akt and FLT3 Inhibition for Treatment of AML
partial and transient clinical responses in patients when used
alone. In addition, we have found that bone marrow-derived
stroma diminishes the activity of both PKC412 and AC220 [7].
There is thus a need for identification and development of novel
therapies that can be effectively combined with TKIs to delay or
suppress leukemia progression, override stroma-associated drug
resistance, and increase patient survival.
We have recently identified the multi-targeted kinase inhibitor,
dasatinib, and dasatinib-like compounds as being able to
potentiate the activity of TKIs PKC412 and AC220 against
mutant FLT3-expressing cells cultured in the presence of
cytoprotective and cytokine-abundant stromal-conditioned media
(SCM) by performing a combinatorial drug screen using the
KIN001 library (Dr. Nathanael Gray) [7]. Our study also
highlighted the potential of Jak inhibitors to synergize with
PKC412 and AC220 as well as enhance their apoptotic activity
against mutant FLT3-expressing cells cultured in the presence of
SCM [7].
While the significance of stromal-derived growth factors in
viability enhancement and cytoprotection of leukemic stem cells
cannot be denied, not all hematologic malignancies can be rescued
from programmed cell death by secreted cytokines in the absence
of direct communication with the stromal cells themselves. As
examples, protection of AML cells and B-lineage ALL cells from
spontaneous and/or drug-induced apoptosis was observed to
depend on direct bone marrow fibroblast cell:leukemic cell
interaction [8–10]. Similarly, protection of CLL cells from
apoptosis depends on adherence of these cells to bone marrow
stromal layers [11], and adhesion between bone marrow stroma
and myeloma cells is necessary for protection of these cells from
drug-induced apoptosis [12]. Thus, the direct interaction between
stromal cells and leukemic cells is important to fully understand
the mechanisms driving stromal-mediated chemoresistance, as
well as for identification of integral signaling molecules as potential
therapeutic targets for overriding drug resistance.
To address this, we used an adherent stroma-based co-culture
system, as opposed to the SCM-based system used previously, as
the basis for a combinatorial drug screen designed to identify novel
kinase inhibitors able to potentiate the apoptosis-inducing effects
of PKC412 against adherent stroma-protected mutant FLT3positive cells (see schematic in Figure S1, which illustrates both the
adherent stroma-based screen used in this study as well as the
SCM-based chemical screen [7]). In parallel to the KIN001 kinase
inhibitor library, we also screened the LINCS kinase inhibitor
library, which is composed of inhibitors characterized as being
relatively potent and selective toward a limited range of kinase
targets.
Here, we identified selective Akt inhibitors, such as MK2206, as
able to effectively combine with FLT3 inhibitors, including
PKC412 and AC220, against mutant FLT3-expressing cell lines
or primary AML cells cultured in a cytoprotective stromal
environment. This synergy occurs both in the absence as well as
the presence of stroma or stromal-derived cytokines, and could
thus potentially be further investigated as a therapeutic for AML
as well as possibly delay/eradicate residual disease. In addition,
p38 MAPK inhibitors also positively combined with PKC412
against mutant FLT3-expressing cells protected by stroma.
Our findings suggest that the combination of kinase inhibitorenriched chemical libraries and the leukemia cell:stromal cell coculture assay could be useful for discovery of novel therapeutic
combinations for AML. This technical approach could also be
employed for identification of protein kinases with potential to be
exploited as novel therapeutic targets.
PLOS ONE | www.plosone.org
Materials and Methods
Kinase Inhibitor Focused Libraries (KIN001 and LINCS)
Two Kinase Inhibitor Focused Libraries (KIN001 and LINCS)
were chosen for screening to identify single agents with little-to-no
appreciable efficacy but that are able to synergize with PKC412
against the human mutant FLT3-expressing AML cell line,
MOLM14-luc+, cultured in the presence of adherent HS-5
stroma. The KIN001 Library was developed by Dr. Nathanael
Gray’s lab and is comprised of 188 commercially-available kinase
inhibitors as well as in-house developed diverse pharmacophorebased kinase inhibitors targeting either active or inactive kinase
conformations. The chemical screening concentration was
660 nM, which is the same screening concentration as was used
previously when this library was used to identify kinase inhibitors
able to synergize with FLT3 inhibitors in the presence of SCM [7].
The LINCS library is available from Harvard Medical School/
NIH LINCS program (https://lincs.hms.harvard.edu/) and contains 202 known selective and potent kinase inhibitors.
Cell Lines and Cell Culture
The human AML-derived, FLT3-ITD-expressing cell lines,
Molm14 [13] and MV4;11 [14], were provided to us by Dr. Scott
Armstrong, Dana Farber Cancer Institute, Boston, MA.
MOLM14 cells were transduced with the FUW-Luc-mCherrypuro lentivirus as previously described [15]. Within the past six
months, mutant FLT3 expression and integrity in this line was
confirmed. The human AML-derived, FLT3-ITD-expressing cell
line, MOLM-13, was obtained from DSMZ (German Resource
Centre for Biological Material). MOLM-13 cells were also
engineered to express luciferase fused to neomycin phosphotransferase (pMMP-LucNeo) by transduction with a VSVG-pseudotyped retrovirus (MOLM13-luc+ cells), as previously described
[16]. The IL-3-dependent murine hematopoietic cell line, Ba/F3,
was transduced with FLT3-ITD-containing MSCV retroviruses
harboring a neomycin selectable marker, and selected for resistance to neomycin [17]. Mutant FLT3-transduced cells were
selected for growth in G418 (1 mg/ml). Within the past six
months, mutant FLT3 expression and integrity in this line was
confirmed. The HS-5 stromal cell line was purchased from
American Type Culture Collection (ATCC) (Manassas, VA,
USA).
All cell lines were cultured with 5% CO2 at 37uC, at
a concentration of 26105 to 56105/mL in RPMI (Mediatech,
Inc., Herndon, VA) with 10% fetal bovine serum and supplemented with 2% L-glutamine and 1% penicillin/streptomycin.
AML Patient Cells
Mononuclear cells were isolated from AML patients (Table S1).
Mononuclear cells were isolated by density gradient centrifugation
through Ficoll-Plaque Plus (Amersham Pharmacia Biotech AB,
Uppsala, Sweden) at 2000 rpm for 30 minutes, followed by two
washes in 1X PBS. Freeze-thawed cells were then cultured in
liquid culture (DMEM, supplemented with 20% FBS) and then
tested in the presence of 50% SCM with different concentrations
of single and combined agents. All blood and bone marrow
samples from AML patients were obtained through written
consent under approval of the Dana Farber Cancer Institute
Institutional Review Board. The ethics committees approved the
consent procedure.
Chemical Compounds and Biologic Reagents
PKC412 was synthesized by Novartis Pharma AG, Basel,
Switzerland, and was dissolved in DMSO to obtain a 10 mM stock
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Akt and FLT3 Inhibition for Treatment of AML
Trypan Blue exclusion assay to quantify cells for cell seeding, and
Cell Titer Glo for proliferation studies. Cell viability was expressed
as the function of growth affected (FA) drug-treated versus control
cells; data were analyzed by Calcusyn software (Biosoft, Ferguson,
MO and Cambridge, UK), using the Chou-Talalay method [18].
The combination index = [D]1 [Dx]1+ [D]2/[Dx]2, where [D]1
and [D]2 are the concentrations required by each drug in
combination to achieve the same effect as concentrations [Dx]1
and [Dx]2 of each drug alone. Values less than one indicate
synergy, whereas values greater than one indicate antagonism.
Calcusyn combination indices can be interpreted as follows: CI
,0.1 indicate very strong synergism; values 0.1–0.3 indicate
strong synergism; values 0.3–0.7 indicate synergism; values 0.7–
0.85 indicate moderate synergism; values 0.85–0.90 indicate slight
synergism; values 0.9–1.1 indicate nearly additive effects; values
1.10–1.20 indicate slight antagonism; values 1.20–1.45 indicate
moderate antagonism; values 1.45–3.3 indicate antagonism; values
3.3–10 indicate strong antagonism; values .10 indicate very
strong antagonism. Note: For some experiments, namely those in
which there was no observed single agent activity due to stromal
protection, combination indices could not be reliably calculated
using the Calcusyn software.
solution. Serial dilutions were then made, to obtain final dilutions
for cellular assays with a final concentration of DMSO not
exceeding 0.1%.
Dasatinib and AC220 were purchased from Haoyuan Chemexpress (Shanghai, China). KIN112 and KIN113 were developed in
Dr. Gray’s lab (DFCI). KIN001 or LINCS library compounds
identified in the screen as able to synergize with PKC412 in the
presence of adherent HS-5 stroma were as follows: HMSL10035
(KIN001-102; Akt inhibitor); KIN001-200 (VX-702; p38 MAPK
inhibitor); HMSL10168 (LG168, VX0745; p38 MAPK inhibitor).
Akt and p38 MAPK inhibitors tested to assess the significance of
drugs identified in the chemical screen were as follows:
HMSL10167 (SB 203580; RWJ 64809, PB 203580; p38 MAPK
inhibitor); HMSL10060 (TAK-715; p38a inhibitor); HMSL10154
(AT7867; Akt inhibitor); HMSL10128 (GSK 690693; Akt inhibitor); and HMSL10057 (MK2206; Akt inhibitor) (Table S2).
The selective Akt inhibitors used in our study are very wellcharacterized research tools that have been widely used in
different contexts (for inhibitor characteristics and background,
please see Table S2).
Cell Proliferation, Viability and Cell Cycle Analysis
The trypan blue exclusion assay (for quantifying cells prior to
seeding), Annexin-V-Fluos Staining Kit (Boehringer Mannheim,
Indianapolis, IN) (for apoptosis), and cell cycle analysis were
carried out as previously described [5]. Due to technical
convenience, SCM was used instead of adherent stroma for the
apoptosis and cell cycle assays. The Cell Titer Glo assay (Promega,
Madison, WI) (for proliferation) was used for proliferation studies,
and carried out according to manufacturer instructions.
Human Adherent Stroma Validation Experiments
HS-5 human stromal cells (10,000/well) were determined in
a pilot study to be sufficient for maximum cytoprotection of
PKC412-treated MOLM14-luc+ cells (Figure S2). Stromal cells
were seeded 24 hours in advance of seeding MOLM14-luc+ cells
(4000/well), followed by drug treatments. The Bright Glo assay
(Promega, Madison, WI) was performed for co-culture assays to
selectively measure leukemia cell viability and was carried out
according to manufacturer’s instructions.
Antibodies and Immunoblotting
For analysis of phospho- and total Akt and phospho- and total
GSK3b, MOLM14-luc+ cells were treated with drugs for two
hours before they were collected at 1100 rpm for 8 minutes. Cells
were lysed using M-PER lysis buffer (Pierce) supplemented with
phosphatase inhibitors and protease inhibitors (Roche) according
to the manufacturers’ instructions. Equivalent amounts of proteins
were loaded. For analysis of phospho- and total STAT5, phosphoand total S6K, and phospho- and total MAPK, immunoblotting
was carried out as previously described [5].
The following primary antibodies were purchased from Cell
Signaling Technology (Danvers, MA): Anti-phospho-Akt (T308),
anti-phospho-Akt (S473), and anti-Akt. The following primary
antibodies were purchased from Sigma-Aldrich (St Louis, MO):
Anti-tubulin, anti-GSK3b, and anti-phospho-GSK3b. All antibodies were used at 1:1000 dilution, except for anti-tubulin, which
was used at 1:5000. The following primary antibodies (for data
shown in supporting data section) were purchased from Cell
Signaling Technology (Danvers, MA): Phospho-STAT5 (Tyr 694)
(C11C5) (rabbit, #9359), used at 1:500, total STAT5 (rabbit,
#9363), used at 1:1000, phospho-p70 S6K (Thr389) (1A5) (mouse,
#9206), used at 1:300, total p70 S6K (49D7) (rabbit, #2708), used
at 1:1000, phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204)
(rabbit, #9101), used at 1:1000, total p44/42 MAPK (Erk1/2)
(3A7) (mouse, #9107), used at 1:1000. HRP conjugated secondary
antibodies were purchased from Promega and were used at
a dilution of 1:5000.
Results
Chemical Screen Identification of Inhibitors able to
Potentiate Effects of PKC412 against Mutant FLT3expressing Cells co-cultured with Adherent Human
Stromal Cells
In the present study, which is a direct and intentional extension
of our previous work [7], we set out to compare the use of SCM
and adherent stroma as the basis for a chemical screen geared
toward identification of drugs capable of overriding drug resistance due to stromal influences. Specifically, we conducted an
unbiased combinatorial screen of 188 compounds comprising the
KIN001 chemical library in an attempt to identify kinase
inhibitors able to synergize with PKC412 against mutant FLT3positive cells co-cultured with adherent stroma. Similar to previous
findings using HS-5 SCM [7], three dual Src/Abl inhibitorsdasatinib, KIN112, and KIN113- were identified as being able to
positively combine with PKC412 against MOLM14-luc+ cocultured with adherent HS-5 stroma cells as a replacement for
SCM (Figure S3). In addition to confirming previously published
findings, these results also validate the use of either SCM or
adherent stroma as part of a chemical screen approach to identify
agents able to override drug resistance due to a cytoprotective
microenvironment.
We also identified library-derived inhibitors of major signaling
pathways, including the allosteric Akt inhibitor, KIN001-102, as
able to positively combine with PKC412 against adherent stromaprotected mutant FLT3-expressing cells (Figure 1A). In order to
validate whether or not Akt as a therapeutic target was important
for the observed higher percentage of killing of stromal-protected
cells when used in combination with PKC412, we tested a panel of
Drug Combination Studies
For drug combination studies, single agents were added
simultaneously at fixed ratios to mutant FLT3-expressing cells
cultured in the presence of adherent HS-5 stroma, 50–80% SCM,
or RPMI+10% FBS. Cell viability was determined using the
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Akt and FLT3 Inhibition for Treatment of AML
selective Akt inhibitor analogs against MOLM14-luc+ cells under
the same co-culture conditions. Similar to KIN001-102, the
selective Akt inhibitors, AT7867, GSK690693, and MK2206
positively combined with PKC412 against MOLM14-luc+ cells
cultured in either the presence of adherent HS-5 stroma
(Figure 1B, C) or HS-5 SCM (Figure 2), with combination indices
at ED75-ED90 suggestive of synergy (Figures 1 and 2).
To further validate the co-culture model for the combination
drug screen, we investigated the effects of single agents and
combination treatments on adherent stromal cells. This would
establish whether or not stromal cell killing (and hence removal of
the source of protective secreted cytokines) played a role in the
observed synergy between PKC412 and Akt inhibitors. To address
this, selective Akt inhibitors were tested against adherent HS-5
stroma directly. Compared to inhibitor effects against MOLM14luc+ cells, inhibitor activity against adherent stroma was considerably weaker (Figure 1B, D). In addition, whereas PKC412
(40 nM) and selective Akt inhibitors (660 nM) were highly effective
alone and combined against Ba/F3 cells expressing mutant FLT3,
the same drugs at the same concentrations displayed little-to-no
appreciable effects against parental Ba/F3 cells and displayed little
activity in the presence of 15% WEHI as a source of IL-3 (Figure
S4). These data, taken together, suggest that drug activity observed
against mutant FLT3-expressing cells is due to on-target effects.
In addition to Akt inhibitors, positive hits from the chemical
library screens also included inhibitors of p38 MAPK inhibitors,
which positively combined with PKC412 against MOLM14-luc+
cells cultured in the presence of adherent HS-5 stroma (Figure S5).
However, the ability of p38 MAPK inhibitors to positively
combine with PKC412 was substantially diminished when mutant
FLT3-expressing cells were cultured in the presence of HS-5 SCM
as opposed to adherent stroma (Figure S5). There exists the
possibility that high levels of stromal-secreted cytokines may
negatively influence the synergizing potential of p38 MAPK
inhibitors with FLT3 inhibitors. Hence, Akt inhibitors may be
superior in terms of their overall combination potential and
general ability to override stromal-mediated drug resistance and
were therefore our main focus in this study.
Figure 1. Selective inhibitors of AKT positively combine with PKC412 in the presence of adherent HS-5 stroma against MOLM14luc+ cells. (A) Approximately two-day proliferation study performed with MOLM14-luc+ cells cultured in the presence of adherent HS-5 stroma
testing the combination of PKC412 and KIN001-102 versus each agent alone. (B) MOLM14-luc+ cells cultured in the presence of adherent HS-5 stroma
for approximately two days: PKC412 (40 nM)+/2 Akt inhibitors (660 nM). (C) Approximately two-day PKC412 treatment of MOLM14-luc+ cells
cultured in the absence and presence of human stroma. (D) Approximately two-day treatment of adherent HS-5 stroma: PKC412 (40 nM) +/2 Akt
inhibitors (660 nM). (E) Calcusyn combination indices derived from 4-point concentration proliferation experiments. The cut-off for nearly additive
effects (C.I.: 1.1) is marked by a dashed line.
doi:10.1371/journal.pone.0056473.g001
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Akt and FLT3 Inhibition for Treatment of AML
Figure 2. Selective inhibitors of AKT positively combine with PKC412 in the presence of SCM against MOLM14-luc+ cells. (A–D)
Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in the presence of HS-5 SCM. (E)
Approximately two-day PKC412 treatment of MOLM14-luc+ cells cultured in the absence or presence of HS-5 SCM (n = 2). (F) Calcusyn combination
indices derived from the 4-point concentration proliferation experiments shown in A-D. The cut-off for nearly additive effects (C.I.: 1.1) is marked by
a dashed line.
doi:10.1371/journal.pone.0056473.g002
cells (Table 1 and Figures S6 Part I and II). These results suggest
that induction of apoptosis, more than cell cycle arrest, contributes
to the observed synergy between PKC412 and KIN001-102
against mutant FLT3-expressing cells cultured in a cytoprotective
stromal environment.
Synergy was observed between PKC412 and selective Akt
inhibitors against MOLM14-luc+ cells cultured in the presence of
RPMI+10% FBS (Figure 3). Synergy was also observed between
selective Akt inhibitors and the highly potent and selective FLT3
inhibitor, AC220, against mutant FLT3-positive leukemia cells
cultured in RPMI+10% FBS (Figure 4). The ability of selective Akt
inhibitors to positively combine with FLT3 inhibitors against
mutant FLT3-positive AML cells in the presence of RPMI+10%
FBS correlated well with induction of apoptosis, as the combination of PKC412 and KIN001-102 showed the highest percentages
of cell killing as compared to single agent effects (Table 2 and
Figure S7 Part I and II). After 48 hrs in RPMI+10% FBS,
however, the combination of PKC412 and KIN001-102 did not
lead to greater G1 arrest than PKC412 alone (40 nM) for
MOLM14-luc+ cells (Table 2).
Synergy between the selective Akt inhibitors and PKC412 was
additionally observed in Ba/F3-FLT3-ITD cells and the two
FLT3 Inhibitor and Akt Inhibitor Combination Effects on
Cell Cycle Progression and Apoptosis of Stromaprotected AML Cells
Synergy observed between PKC412 and KIN001-102 against
MOLM14-luc+ cells cultured in the presence of 50% SCM
correlated with induction of apoptosis, as drug combinationtreated cells showed the highest percentages of apoptotic cells
(Table 1). An increase in the G1 population was observed for
MOLM14-luc+ cells cultured for 24 hr in the presence of 50%
SCM and treated with PKC412 alone (approximately 86% of cells
were in G1/G0). Combination treatments led to approximately
89% of cells in G1/G0 (Table 1), which is a comparatively small
increase in percentage. In contrast, compared to PKC412 alone,
combination treatment of MOLM14-luc+ cells for 48 hr resulted
in substantially increased apoptosis (PKC412 alone: 27.1%
apoptosis, versus combination treatments: 41.3%-48.9% apoptosis) (Table 1 and Figure S6 Part I and II). Stromal protection was
evidenced by the fact that PKC412 treatment of MOLM14-luc+
cells in RPMI+10% FBS led to 47% viable cells (Table 2 and
Figure S7 Part I and II), whereas PKC412 treatment of
MOLM14-luc+ cells in the presence of SCM led to 71% viable
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Akt and FLT3 Inhibition for Treatment of AML
Table 1. Effects of PKC412 and KIN001-102, alone and combined, on MOLM14-luc+ cell cycle progression (following 24 hours of
treatment) and apoptosis (following 48 hours of treatment) when cells are cultured in the presence of 50% HS-5 SCM.
Cell Cycle Progression
Cell Viability
MOLM14-luc+Treatment
% G1/G0
% G2M
%S
% Viable
% Apoptotic
% Necrotic
DMSO Control
67.95
6.270
25.78
84.70
14.00
1.400
PKC412 (40 nM)
86.24
3.070
10.68
71.00
27.10
2.000
KIN001-102 (165 nM)
71.27
5.090
23.64
81.50
17.30
1.200
KIN001-102 (330 nM)
73.81
4.750
21.44
80.90
17.70
1.400
KIN001-102 (660 nM)
77.79
4.910
17.30
77.80
20.30
2.000
PKC412 (40 nM)+KIN001-102 (165 nM)
88.87
2.980
8.150
57.30
41.30
1.400
PKC412 (40 nM)+ KIN001-102 (330 nM)
89.47
2.310
8.220
52.10
45.70
2.200
PKC412 (40 nM)+ KIN001-102 (660 NM)
89.11
3.880
7.010
47.30
48.90
3.900
Details of the assays used for these studies are provided in the Materials and Methods section.
doi:10.1371/journal.pone.0056473.t001
mutant FLT3-expressing human cell lines, MOLM13-luc+ and
MV4,11, cultured in the presence of RPMI+10% FBS (Figures 5
and 6, Table 3, Figure S8). Partial protection of PKC412-treated
MOLM13-luc+ cells was observed when cells were cultured in the
presence of HS-5 SCM (Figure 5), and combinations of selective
Akt inhibitors and PKC412 were synergistic against MOLM13luc+ cells cultured in the presence of HS-5 SCM (Figure 5,
Table 3).
Figure 3. Selective inhibitors of AKT positively combine with PKC412 in RPMI+10% FBS against MOLM14-luc+ cells. (A–D)
Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in RPMI+10% FBS. (E) Calcusyn
combination indices. The cut-off for nearly additive effects (C.I.: 1.1) is marked by a dashed line.
doi:10.1371/journal.pone.0056473.g003
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Akt and FLT3 Inhibition for Treatment of AML
Figure 4. Selective inhibitors of AKT positively combine with AC220 in RPMI+10% FBS against MOLM14-luc+ cells. (A–D)
Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with AC220 in RPMI+10% FBS. (E) Calcusyn
combination indices. The cut-off for nearly additive effects (C.I.: 1.1) is marked by a dashed line.
doi:10.1371/journal.pone.0056473.g004
Table 2. Effects of PKC412 and KIN001-102, alone and combined, on MOLM14-luc+ cell cycle progression (following 24 hours of
treatment) and apoptosis (following 48 hours of treatment) when cells are cultured in the presence of RPMI+10% FBS.
Cell Cycle Progression
MOLM14-luc+ Treatment
% G1/G0
% G2M
Cell Viability
%S
% Viable
% Apoptotic
% Necrotic
DMSO Control
56.10
9.320
34.58
92.50
6.900
0.600
PKC412 (40 nM)
86.94
1.50
11.56
46.90
52.70
0.400
KIN001-102 (165 nM)
59.60
7.360
33.04
92.00
7.800
0.300
KIN001-102 (330 nM)
61.03
9.010
29.96
91.80
8.000
0.200
KIN001-102 (660 nM)
61.93
7.030
31.05
90.90
8.800
0.300
PKC412 (40 nM)+KIN001-102
(165 nM)
85.22
3.430
11.35
31.50
68.30
0.200
PKC412 (40 nM)+ KIN001-102
(330 nM)
86.92
2.760
10.32
18.10
81.70
0.100
PKC412 (40 nM)+ KIN001-102
(660 NM)
81.11
3.160
15.73
16.90
82.80
0.300
Details of the assays used for these studies are provided in the Materials and Methods section.
doi:10.1371/journal.pone.0056473.t002
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Akt and FLT3 Inhibition for Treatment of AML
Figure 5. Selective inhibitors of AKT positively combine with PKC412 in the absence and presence of HS-5 SCM against MOLM13luc+ cells. (A–B) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in the presence of
RPMI+10% FBS. (C–D) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in 80% HS5 SCM. (E) Treatment of MOLM13-luc+ cells with PKC412 in either RPMI+10% FBS or 80% HS-5 SCM.
doi:10.1371/journal.pone.0056473.g005
2MK2206 (165 nM) (Figure S9). Each agent was tested at
respective concentrations that led to a substantial combination
effect in proliferation studies (Figure 3). PKC412 alone and
PKC412 combined with MK2206 decreased phosphorylation of
STAT5 to similar extents in these cells, and no significant changes
were observed in expression of phosphorylated MAPK between
single agent-treated and drug combination-treated cells (Figure
S9).
Phospho-Akt Mediates Synergy Observed between
Allosteric Akt Inhibitor, KIN001-102, and PKC412
In order to verify observed combination effects in terms of
signaling, we examined the phosphorylation status of Akt following
either single agent treatment or combined drug treatment.
Immunoblots demonstrated that pAKT levels were inhibited to
a greater extent in MOLM14-luc+ cells cultured in the presence of
50% HS-5 SCM upon treatment with a combination of KIN001102 and PKC412, as compared to either drug alone (Figure 7).
The expression of GSK3b was additionally investigated as GSK3b
is a direct substrate of protein kinase Akt. These results suggest
that Akt activity is critical for maintaining stromal cytoprotection
under these conditions. In contrast to the robust drug combination
effect observed against phospho-Akt (S473) at lower concentrations of KIN102, there was no apparent combination effect
observed between PKC412 (40 nM) and KIN102 (165 nM)
against phospho-S6K for MOLM14-luc+ cells cultured in the
presence of SCM (data not shown).
Similarly, no significant changes were observed in expression of
phosphorylated S6K in MOLM14-luc+ cells cultured in
RPMI+10% FBS and treated for 1 hr with PKC412 (5 nM)+/
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Ability of Akt Inhibitors to Potentiate the Activity of
PKC412 or AC220 against Primary AML Patient Cells
Cultured in the Presence of Cytoprotective SCM
In order to validate the stromal cell co-culture screening model,
we tested the lead drugs from the screening on primary AML
patient cells cultured in the presence of cytoprotective HS-5derived SCM. Several of these samples were confirmed to express
FLT3-ITD. Combination studies between PKC412 and KIN001102, AT7867, MK2206, and GSK690693, respectively, showed
the highest degree of cell killing in combination-treated, SCMprotected primary AML cells as compared to any single agent (a
representative dose-response experiment for a highly drug-re8
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Akt and FLT3 Inhibition for Treatment of AML
Figure 6. Selective inhibitors of AKT positively combine with PKC412 in RPMI+10% FBS against MV4,11 and Ba/F3-FLT3-ITD cells.
(A–C) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in RPMI+10% FBS against
MV4,11 cells. (D–F) Approximately two-day proliferation studies performed with selective AKT inhibitors in combination with PKC412 in RPMI+10%
FBS against Ba/F3-FLT3-ITD cells.
doi:10.1371/journal.pone.0056473.g006
performed in the presence of SCM, which provides cytoprotective
cytokines and dampens inhibitor efficacy.
sistant AML patient sample (#2) is shown in Figure 8A). Analysis
of the combinatorial effect with Calcusyn revealed synergy
(ED75:0.39202, ED90:0.55992) for PKC412+AT7867 against
mutant FLT3-positive AML#2.
Given the multiple targets of PKC412, we investigated the
ability of AC220, which has a very high selectivity index, to
combine with Akt inhibitors. A positive combination effect was
observed between AC220 and selective inhibitors of Akt against
MOLM14-luc+ cells cultured in the presence of HS-5-derived
SCM (Figure 8B). AC220 similarly combined positively with Akt
inhibitors against MOLM14-luc+ cells cultured in RPMI+10%
FBS with combination indices at ED75 and ED90 suggestive of
synergy (Figure 8C).
Selective Akt inhibitors were thus also tested for their ability to
potentiate the effects of AC220, as compared to PKC412, against
primary AML patient cells cultured in the presence of HS-5derived SCM. As shown in Figures 8D–F, PKC412 and AC220,
which were tested in parallel, resulted in the highest degree of
patient cell killing when either drug was used in combination with
Akt inhibitors as compared to any single agent against a panel of
primary AML samples. Of note, all drug treatments were
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Discussion
Previous studies of ours suggest that TKI-dependent combination therapy likely represents a potentially useful approach to
counteracting both intrinsic and stroma-associated drug resistance
in leukemia patients [7,19,20,21]. With the recent discovery of
numerous FLT3 inhibitor-responsive serine/threonine and tyrosine phosphorylation sites uncovered in primary AML patient
bone marrow samples [22], identification of protein kinase
inhibitors that are able to enhance the potency of FLT3 inhibitors
makes intuitive sense.
Here, selective inhibitors targeting kinases involved in PI3K/
Akt and Ras/MEK/MAPK signaling were identified in a chemical
screen as synergizing with PKC412 against mutant FLT3expressing cells in the presence of adherent stroma. Akt inhibitors
synergized with FLT3 inhibitors in the presence of either SCM or
adherent stroma, as compared to p38 MAPK inhibitors, which
synergized with FLT3 inhibitors only in the presence of adherent
stroma. One possibility for this may be traced to the nature of
9
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Akt and FLT3 Inhibition for Treatment of AML
Table 3. Calcusyn software-derived combination indices.
Drug Combination Cell Line
ED25
ED50
ED75
ED90
PKC412+MK2206
Ba/F3-FLT3- 0.73063
ITD
0.71787
0.70932 0.70100
PKC412+AT7867
Ba/F3-FLT3- 0.62418
ITD
0.65624
0.68996 0.72540
PKC412+GSK690693 Ba/F3-FLT3- 0.64287
ITD
0.72479
0.81965 0.92849
PKC412+MK2206
(2-day)
MOLM13
0.54469
0.52206
0.55200 0.71660
PKC412+MK2206
(3-day)
MOLM13
0.15758
0.22911
0.34332 0.55817
PKC412+AT7867
(2-day)
MOLM13
0.95116
1.02639
1.11056 1.20483
PKC412+AT7867
(3-day)
MOLM13
0.76889
0.26391
0.40983 0.64279
PKC412+GSK690693 MOLM13
(2-day)
0.97412
0.29241
0.33779 0.56141
PKC412+GSK690693 MOLM13
(3-day)
1.11399
0.79787
0.62777 0.57590
PKC412+MK2206
(SCM)
MOLM13
0.75681
0.23956
0.26180 0.29207
PKC412+GSK690693 MOLM13
(SCM)
0.91433
0.39599
0.51463 0.72551
PKC412+MK2206
MV4,11
0.84669
0.39562
0.43775 0.76686
PKC412+AT7867
MV4,11
0.45640
0.67869
1.08529 1.84914
Figure 7. Phospho-Akt mediates synergy observed between
allosteric Akt inhibitor, KIN001-102, and PKC412. Immunoblots
of protein lysates prepared from MOLM14-luc+ cells treated for 2 hours
with PKC412 (40 nM), KIN001-102 (165, 330, 660 nM), or a combination
of the two agents in the presence of 50% SCM. Data shown are
representative of two independent experiments in which similar results
were achieved.
doi:10.1371/journal.pone.0056473.g007
Data shown here correspond to dose-response curves shown in Figures 5 and 6
and Figure S8. Interpretation of combination indices is provided in the Materials
and Methods section.
doi:10.1371/journal.pone.0056473.t003
nib plus the farnesyltransferase inhibitor lonafarnib [28]. Inhibitors of Akt and Erk combined respectively with nilotinib
diminished resistance. In contrast to our findings, however,
inhibition of p38 MAPK in this study increased TKI (nilotinib)
resistance [29].
Importantly, we observed synergy between selective Akt
inhibitors and FLT3 inhibitors in the absence of stroma as well
as its presence, suggesting that this synergy is not specific to
leukemia cells growing in a cytoprotective microenvironment. Of
significance, there are reports that have been and that are
continuing to be published that support the potential clinical
importance of inhibiting components of major signaling pathways
in combination with TKIs as a way to treat AML.
The identification of Akt and p38 MAPK inhibitors as able to
potentiate the effects of FLT3 inhibitors is at least in part
attributable to the use of the LINCS library to identify
comparatively ‘‘clean’’ kinase inhibitors, in contrast to the
chemical library screened previously [7], which included a number
of multi-kinase inhibitors such as dasatinib. A chemical library
composed of relatively selective inhibitors offers a significant
technical advantage in that it translates into easier elucidation of
mechanism of inhibition by a single agent and synergy between
agents as the drug targets are more well-defined and easier to
validate.
Our in vitro findings with cell lines and primary patient
samples, which closely reflect the genetic heterogeneity amongst
AML patients, warrant further testing and validation in preclinical
models of progressive leukemia and minimal residual disease. In
vivo models that reflect stromal cell interactions, however, are
fairly complex and are beyond the scope of this study. We are
planning to address these questions in future studies.
stromal protection by SCM, characterized by highly concentrated
levels of stromal-derived cytokines.
Of relevance, studies have implicated Akt- and MAPKmediated signaling in stromal enhancement of leukemia cell
viability. For instance, co-culture of leukemia cells and bone
marrow-derived stroma has been shown to lead to activation of the
MAPK/ERK pathway and integrin-linked kinase (ILK), which
phosphorylates Akt [22]. ILK/Akt is likely critical for leukemia cell
survival in bone marrow, and thus inhibitors of ILK have been
proposed as an approach to simultaneously target both leukemia
cells and leukemia-activated stromal cells [23]. Additionally, p38
MAPK activation has been found to play a role in stromadependent survival of B-CLL cells [24] and ALL cells [25].
In addition, continuous FLT3 inhibitor treatment leads to the
development of drug-resistant cells characterized by constitutive
activation of parallel downstream PI3K/Akt and/or Ras/MEK/
MAPK signaling pathways, which is believed to compensate for
the loss of FLT3 activity in terms of survival and growth [26]. In
support of this, constitutive activation of ERK/Akt/STAT
pathways has been observed in AML despite small molecule
inhibition of FLT3-ITD activity, suggesting that optimal treatment
of AML may require FLT3 inhibition combined with inhibition of
additional signaling pathways [27]. Dual inhibition of FLT3 and
Akt-mediated signaling, such as that conferred by the multiple
kinase inhibitor, KP372-1, has indeed been found to inhibit
primary AML cell growth with minimal effect on normal
progenitor cells [28].
Consistent with our results is the finding that Akt, p38MAPK,
and Erk activation correlates with development of resistance of
BCR-ABL-positive acute lymphoblastic leukemia (ALL) to nilotiPLOS ONE | www.plosone.org
10
February 2013 | Volume 8 | Issue 2 | e56473
Akt and FLT3 Inhibition for Treatment of AML
Figure 8. Ability of Akt inhibitors to positively combine with PKC412 or AC220 against AML patient samples in the presence of
cytoprotective SCM. (A) Approximately two-day proliferation study performed with a selective Akt inhibitor in combination with PKC412 in the
presence of HS-5 SCM against mutant FLT3-positive AML#2. (B) Approximately two-day combination studies: AC220 (0.4 nM) +/2 selective AKT
inhibitors (660 nM) against MOLM14-luc+ cells in the presence of 50% HS-5 SCM. (C) Approximately two-day combination studies: AC220 (0.4 nM) +/
2 selective AKT inhibitors (660 nM) against MOLM14-luc+ cells in the presence of RPMI+10% FBS. (D) Approximately two-day combination studies:
PKC412 (40 nM)+/2 selective AKT inhibitors (660 nM) against primary AML patient cells in the presence of 50% HS-5 SCM. (E) Approximately two-day
combination studies: AC220 (0.4 nM) +/2 selective AKT inhibitors (660 nM) against primary AML patient cells in the presence of 50% SCM. (F) Ability
of Akt inhibitors to positively combine with PKC412 or AC220 against primary AML cells in the presence of cytoprotective SCM. Patient information is
provided in Table S1.
doi:10.1371/journal.pone.0056473.g008
Figure S2 Co-culture pilot study. Approximately 1500
MOLM14-luc+ cells were tested in a two-day assay in the
presence and absence of HS-5 stroma seeded at 10,000 cells/well,
20,000 cells/well, and 40,000 cells/well.
(TIF)
In conclusion, selective inhibition of kinases such as Akt in
combination with FLT3 inhibitors in mutant FLT3-positive AML
patients may represent a novel approach to improving treatment
effects and patient survival. Findings presented here may provide
novel options for adjunctive therapy.
Figure S3 Coculture chemical screen identification of
KIN001 library compound, dasatinib, and dasatinib-like
compounds, KIN112 and KIN113, as able to synergize
with PKC412 in the presence of adherent HS-5 stroma
against MOLM14-luc+ cells. (A–C) Approximately two-day
assays, validating the combination potential of the KIN001 coculture chemical screen identified agents (dasatinib, KIN112,
KIN113) to synergize with PKC412 against MOLM14-luc+ cells
in the presence of adherent HS-5 stroma. Approximately 5000
MOLM14-luc+ cells were seeded/well; approximately
10,000 HS-5 stromal cells were seeded/well. (D) PKC412
Supporting Information
Figure S1 Schematic of kinase inhibitor-focused chemical screen approaches. Stromal-conditioned media (SCM)- or
adherent stroma-based chemical libraries are used to identify
agents that are able to potentiate the effects of FLT3 inhibitors
against mutant FLT3-expressing cells cultured in a cytoprotective
microenvironment.
(TIF)
PLOS ONE | www.plosone.org
11
February 2013 | Volume 8 | Issue 2 | e56473
Akt and FLT3 Inhibition for Treatment of AML
Figure S8 Selective inhibitors of AKT positively combine with PKC412 in RPMI+10% FBS against MOLM13luc+ cells. (A–C) Approximately three-day proliferation studies
performed with selective AKT inhibitors in combination with
PKC412 in RPMI+10% FBS against MOLM13-luc+ cells.
(TIF)
treatment of MOLM14-luc+ cells cultured in the absence or
presence of adherent HS-5 stroma (n = 2). (E) Calcusyn combination indices. The cut-off for nearly additive effects (C.I.: 1.1) is
marked by a dashed line.
(TIF)
Figure S4 Treatment of parental Ba/F3 cells and Ba/
F3-FLT3-ITD cells with PKC412, alone and in combination with selective inhibitors of Akt. (A) Approximately
three-day drug treatment of parental Ba/F3 cells cultured in the
presence of IL-3 and Ba/F3-FLT3-ITD cells cultured in the
absence of IL-3. (B) Approximately three-day drug treatment of
Ba/F3-FLT3-ITD cells cultured in the presence of IL-3. PKC412
was used at 40 nM and selective AKT inhibitors were each used at
660 nM.
(TIF)
Figure S9 Investigation of phosphorylation of signaling
molecules downstream of FLT3. Immunoblots of protein
lysates prepared from MOLM14-luc+ cells treated for 1 hour with
PKC412 (5 nM), MK2206 (165 nM), or a combination of the two
agents in RPMI+10% FBS.
(TIF)
Table S1 Patient sample information. Patients shown here
were cultured in the presence of 50% HS-5 SCM, and treated
with different combinations of kinase inhibitors. *Patient information for AML patients 2 and 7 has been previously published
(Weisberg et al, 2012a, Leukemia).
(DOC)
Figure S5 Selective inhibitors of p38 MAPK positively
combine with PKC412 against MOLM14-luc+ cells
cultured in the presence of adherent HS-5 stroma,
however not HS-5 SCM. Calcusyn combination indices. The
cut-off for nearly additive effects (C.I.: 1.1) is marked by a dashed
line.
(TIF)
Table S2 Selective AKT and p38 MAPK inhibitors. *Hirai
H, Soontome H, Nakatsuru Y, Miyama K, Taguchi S, Tsujioka K
et al. MK-2206, an allosteric Akt inhibitor, enhances antitumor
efficacy by standard chemotherapeutic agents or molecular
targeted drugs in vitro and in vivo. Mol Cancer Ther
2010;9:1956-67. **Levy DS, Kahana JA, Kumar R. AKT
inhibitor, GSK690693, induces growth inhibition and apoptosis
in acute lymphoblastic leukemia cell lines. Blood 2009;113:1723-9.
***Grimshaw KM, Hunter LJ, Yap TA, Heaton SP, Walton MI,
Woodhead SJ, et al. AT7867 is a potent and oral inhibitor of AKT
and p70 S6 kinase that induces pharmacodynamic changes and
inhibits human tumor xenograft growth. Mol Cancer Ther
2010;9:1100-10.
(DOC)
Figure S6 Part 1. Annexin/pi staining corresponding to data
shown in Table 1: Effects of PKC412 (40 nM) and KIN001-102
(165, 330, 660 nM), alone and combined, on MOLM14-luc+ cell
apoptosis (following 48 hours of treatment) when cells are cultured
in the presence of 50% HS-5 SCM. Cells labeled ‘‘dying’’ are in
early apoptotic phase, and cells labeled ‘‘apoptotic’’ are in late
apoptotic phase. Part 2. Quantitative values corresponding to
data shown in Figure S6 (part 1): Effects of PKC412 (40 nM) and
KIN001-102 (165, 330, 660 nM), alone and combined, on
MOLM14-luc+ cell apoptosis (following 48 hours of treatment)
when cells are cultured in the presence of 50% HS-5 SCM. Cells
labeled ‘‘dying’’ are in early apoptotic phase, and cells labeled
‘‘apoptotic’’ are in late apoptotic phase.
(DOC)
Author Contributions
Responsible for generation of research findings reported in paper (design/
performance of experiments), integrity and analysis of the data, writing of
the manuscript: EW. Responsible for generation of research findings
reported in paper (design/performance of experiments), integrity and
analysis of the data, editing of the manuscript: QL. Performed Akt,
GSKbeta, tubulin immunoblotting experiments: XZ. Assisted with
proliferation studies: EN. Offered valuable scientific feedback and helped
with conception of research reported in paper: MS. Assisted with the
chemical screening: FL MN JZ AN. Offered valuable scientific feedback
and helped with conception of research reported in paper: CM. Carried
out flow cytometry analysis and helped with cell cycle and apoptosis
studies: RWS. Provided AML patient samples used in this study as well as
patient information: RS IG. Responsible for conception of research
reported in paper, integrity and analysis of the data: JDG NG. Conceived
and designed the experiments: EW QL MS CM AN JDG NG. Performed
the experiments: EW XZ EN FL MN. Analyzed the data: EW QL RWS.
Contributed reagents/materials/analysis tools: JZ RS IG. Wrote the paper:
EW QL MS.
Figure S7 Part 1. Annexin/pi staining corresponding to data
shown in Table 2: Effects of PKC412 (40 nM) and KIN001-102
(165, 330, 660 nM), alone and combined, on MOLM14-luc+ cell
apoptosis (following 48 hours of treatment) when cells are cultured
in the presence of RPMI+10% FBS. Cells labeled ‘‘dying’’ are in
early apoptotic phase, and cells labeled ‘‘apoptotic’’ are in late
apoptotic phase. Part 2. Quantitative values corresponding to
data shown in Figure S7 (part 1): Effects of PKC412 (40 nM) and
KIN001-102 (165, 330, 660 nM), alone and combined, on
MOLM14-luc+ cell apoptosis (following 48 hours of treatment)
when cells are cultured in the presence of RPMI+10% FBS. Cells
labeled ‘‘dying’’ are in early apoptotic phase, and cells labeled
‘‘apoptotic’’ are in late apoptotic phase.
(DOC)
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| 8,165 | 55,945 |
W4239368148.txt | Analytical
Technology in
Nutrition Analysis
Edited by
Jose M. Miranda
Printed Edition of the Special Issue Published in Molecules
www.mdpi.com/journal/molecules
Analytical Technology in
Nutrition Analysis
Analytical Technology in
Nutrition Analysis
Special Issue Editor
Jose M. Miranda
MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin
Special Issue Editor
Jose M. Miranda
Universidade de Santiago de Compostela
Spain
Editorial Office
MDPI
St. Alban-Anlage 66
4052 Basel, Switzerland
This is a reprint of articles from the Special Issue published online in the open access journal Molecules
(ISSN 1420-3049) (available at: https://www.mdpi.com/si/molecules/Nutrition analysis).
For citation purposes, cite each article independently as indicated on the article page online and as
indicated below:
LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year, Article Number,
Page Range.
ISBN 978-3-03928-764-2 (Hbk)
ISBN 978-3-03928-765-9 (PDF)
c 2020 by the authors. Articles in this book are Open Access and distributed under the Creative
Commons Attribution (CC BY) license, which allows users to download, copy and build upon
published articles, as long as the author and publisher are properly credited, which ensures maximum
dissemination and a wider impact of our publications.
The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons
license CC BY-NC-ND.
Contents
About the Special Issue Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Jose M. Miranda
Analytical Technology in Nutrition Analysis
Reprinted from: Molecules 2020, 25, 1362, doi:10.3390/molecules25061362
. . . . . . . . . . . . .
1
Larissa Gabrielly Barbosa Lima, Julia Montenegro, Joel Pimentel de Abreu,
Millena Cristina Barros Santos, Talita Pimenta do Nascimento, Maiara da Silva Santos,
Antônio Gilberto Ferreira, Luiz Claudio Cameron, Mariana Simões Larraz Ferreira and
Anderson Junger Teodoro
Metabolite Profiling by UPLC-MSE, NMR, and Antioxidant Properties of Amazonian Fruits:
Mamey Apple (Mammea Americana), Camapu (Physalis Angulata), and Uxi (Endopleura Uchi)
Reprinted from: Molecules 2020, 25, 342, doi:10.3390/molecules25020342 . . . . . . . . . . . . . .
5
Ahmidin Wali, Atikan Wubulikasimu, Sharafitdin Mirzaakhmedov, Yanhua Gao,
Adil Omar, Amina Arken, Abulimiti Yili and Haji Akber Aisa
Optimization of Scorpion Protein Extraction and Characterization of the Proteins’
Functional Properties
Reprinted from: Molecules 2019, 24, 4103, doi:10.3390/molecules24224103 . . . . . . . . . . . . . 23
Emmanuel Martinez, Jose A. Rodriguez, Alicia C. Mondragon, Jose Manuel Lorenzo and
Eva M. Santos
Influence of Potato Crisps Processing Parameters on Acrylamide Formation and Bioaccesibility
Reprinted from: Molecules 2019, 24, 3827, doi:10.3390/molecules24213827 . . . . . . . . . . . . . 39
Yonghang Ma, Zhengcheng Zeng, Lingchang Kong, Yuanxin Chen and Pingli He
Determination of N-Carbamylglutamate in Feeds and Animal Products by High Performance
Liquid Chromatography Tandem Mass Spectrometry
Reprinted from: Molecules 2019, 24, 3172, doi:10.3390/molecules24173172 . . . . . . . . . . . . . 49
Alexis Ayala-Ni ˜no, Gabriela Mariana Rodr´ıguez-Serrano, Luis Guillermo Gonz´alez-Olivares,
Elizabeth Contreras-López, Patricia Regal-López and Alberto Cepeda-Saez
Sequence Identification of Bioactive Peptides from Amaranth Seed Proteins
(Amaranthus hypochondriacus spp.)
Reprinted from: Molecules 2019, 24, 3033, doi:10.3390/molecules24173033
. . . . . . . . . . . . . 63
Li Duan, Chenmeng Zhang, Chenjing Zhang, Zijing Xue, Yuguang Zheng and Long Guo
Green Extraction of Phenolic Acids from Artemisia argyi Leaves by Tailor-Made Ternary Deep
Eutectic Solvents
Reprinted from: Molecules 2019, 24, 2842, doi:10.3390/molecules24152842 . . . . . . . . . . . . . 77
Thomas Rosemary, Abimannan Arulkumar, Sadayan Paramasivam,
Alicia Mondragon-Portocarrero and Jose Manuel Miranda
Biochemical, Micronutrient and Physicochemical Properties of the Dried Red Seaweeds
Gracilaria edulis and Gracilaria corticata
Reprinted from: Molecules 2019, 24, 2225, doi:10.3390/molecules24122225 . . . . . . . . . . . . . . 89
v
Gretel Dovale-Rosabal, Alicia Rodrı́guez, Elyzabeth Contreras, Jaime Ortiz-Viedma,
Marlys Muñoz, Marcos Trigo, Santiago P. Aubourg and Alejandra Espinosa
Concentration of EPA and DHA from Refined Salmon Oil by Optimizing the Urea–Fatty Acid
Adduction Reaction Conditions Using Response Surface Methodology
Reprinted from: Molecules 2019, 24, 1642, doi:10.3390/molecules24091642 . . . . . . . . . . . . . 103
Aroa Lopez-Santamarina, Jose Manuel Miranda, Alicia del Carmen Mondragon,
Alexandre Lamas, Alejandra Cardelle-Cobas, Carlos Manuel Franco and Alberto Cepeda
Potential Use of Marine Seaweeds as Prebiotics: A Review
Reprinted from: Molecules 2020, 25, 1004, doi:10.3390/molecules25041004 . . . . . . . . . . . . . 115
Marcos Flores, Carolina Saravia, Claudia E. Vergara, Felipe Avila, Hugo Valdés and
Jaime Ortiz-Viedma
Avocado Oil: Characteristics, Properties, and Applications
Reprinted from: Molecules 2019, 24, 2172, doi:10.3390/molecules24112172 . . . . . . . . . . . . . 141
vi
About the Special Issue Editor
Jose M. Miranda (Ph.D.) Prior to his academic and scientific activities, he worked for several
years as a health inspector. Currently, he is working as assistant professor in the University of
Santiago de Compostela (Spain). He has published over 100 scientific articles in indexed journals.
His areas of expertise are food safety, food chemical analysis, characterization of compounds with
nutritional interest, and metagenomics.
vii
molecules
Editorial
Analytical Technology in Nutrition Analysis
Jose M. Miranda
Laboratorio de Higiene Inspección y Control de Alimentos, Departamento de Química Analítica, Nutrición y
Bromatología, Universidade de Santiago de Compostela, 27002 Lugo, Spain; josemanuel.miranda@usc.es;
Tel.: +34-982-252-231 (ext. 22407); Fax: +34-982-254-592
Received: 9 March 2020; Accepted: 11 March 2020; Published: 17 March 2020
The great challenge facing humanity in the coming decades is to secure food for the 9.8 billion
people who are expected to inhabit the planet by around 2050 and 11.2 billion in 2100 [1]. To increase
the food production by traditional methods to meet this demand for food is very difficult. Additionally,
traditional methods of food production, both of plant and animal origin, present specific problems that
make it difficult for them to meet such an ambitious increase target.
In this sense, terrestrial agriculture presents a problem because fresh water (an essential resource)
is an increasingly scarce commodity, and progressive desertification of the Earth’s surface is taking
place and will probably be aggravated in the future by global warming. About 45% of the world’s
land surface is currently considered drylands, while 12 million hectares of land are degraded yearly
through a lack of water and related processes. According to the Food and Agriculture Organization of
the United Nations [2], agricultural productivity is persistently declining at over 1% per year.
With respect to food production of animal origin, this also presents specific challenges, such
as the fact that intensive production methods require large amounts of land, water and feed, and
some livestock (such as ruminants) produce high levels of greenhouse gas emissions. Thus, intensive
methods of animal production have serious drawbacks from the point of view of environmental care.
In order to properly feed such a large population, it will be necessary to increase food production
while respecting ecosystems and natural resources. The current high demand for animal proteins
requires that livestock is reared in large numbers over diminishing land resource which is not possible
and, therefore, alternative substitutes for animal proteins needs to be embraced to overcome this
problem [3].
The abovementioned fact means that demand for food produced from non-traditional sources
is expected to rise in the coming decade [4]. Fortunately, nowadays increasing acceptance for novel
foods is also being observed, not only in developing but also in developed countries, which is mainly
influenced by consumer awareness of the nutritional benefits linked to these kinds of foods [3]. As it
can be seen in Figure 1, the investigation about novel foods has experienced a dramatic increase in the
last decade (about three-fold).
In addition to the need for an increase in food production, nowadays, in most countries of the
world, there is a growing prevalence of chronic non-communicable diseases, many of which are
diet-related [5]. As a result, there is widespread consumer demand for foods with a nutritional
composition more in line with current nutritional guidelines, and which include a greater proportion
of the nutrients that have a potential beneficial effect on human health, or fewer of those components
that have a negative effect on human health [6]. Therefore, both because of the need to ensure food
safety in food, especially in those that do not have a history of safe use. In addition, there is also a
need for analytical methodologies to reliably determine both the presence of specific nutrition-related
components in foods, and the effects of these dietary components on human health, in areas as diverse
as lipidomics, proteomics, transcriptomics, genomics, epigenomics, or metagenomics [7,8].
To meet these needs, it is essential that we in the international scientific community work
intensively to ensure safe, effective and honest food production and to protect the health of consumers.
Molecules 2020, 25, 1362; doi:10.3390/molecules25061362
1
www.mdpi.com/journal/molecules
Molecules 2020, 25, 1362
For this reason, from Molecules it was recognized the need to propose the Special Issue “Analytical
Technologies in Nutrition Analysis”. This Special Issue was aimed to offer an appropriate opportunity
to all the contributors to make their results and techniques more visible, and to present the most
recent findings.
Figure 1. Results analysis for Scopus query “novel foods” in title, keywords or abstract section of the
articles between 2009 and 2019.
This Special Issue has received remarkably positive feedback, with many contributions submitted
by numerous geographically diverse scientists, resulting in a collection of 10 publications, including
two exhaustive review articles [9–18]. Among the contributing authors, authors can be found from
Asia (China, India, and Uzbekistan), Europe (Spain), South America (Brazil and Chile) and North
America (Mexico). The published articles include findings related to the comprehensive bioactive
compound profile and antioxidant capacities of mamey apple (Mammea americana), camapu (Physalis
angulata), and uxi (Endopleura uchi) that can contribute to their economic exploitation [9].
Another article described the multifunctional activity of amaranth (Amaranthus hypochondriacus
spp.) proteins, opening the possibility that amaranth hydrolyzed with alcalase and flavourzyme to
be used as a value-added ingredient with multi-functional bioactive properties [10]. Another article
aimed to find an efficient extraction method and investigate some of physical and chemical parameters,
like water solubility, emulsification, foaming properties, and oil-holding capacity of obtained scorpion
proteins. The results obtained suggest that scorpion proteins can be considered as an important
ingredient and raw material for the creation of water-soluble supramolecular complexes for drugs [11].
A fractional factorial design was used to evaluate the effects of temperature, frying time, blanching
treatment and the thickness of potato slices on a very relevant potential toxic compound (acrylamide)
content in crisps. The findings obtained demonstrate that acrylamide concentration remained at 70%
in fried chips, and reductions took place, mainly at the intestinal phase, as a result of reaction with
nucleophilic compounds [12]. N-carbamylglutamate, a synthetic analogue of N-acetylglutamate, is an
activator of blood ammonia conversion and endogenous arginine synthesis. This study will provide a
solid foundation for the evaluation of availability and metabolic mechanism of N-carbamylglutamate
in animals [13].
The Artemisia argyi leaf has been used as a traditional medicine and food supplement in Asian
countries for hundreds of years. Phytochemical studies disclosed that Artemisia argyi leaf contains
various bioactive constituents, mainly phenolic acids, which have great potential as possible alternatives
to those organic solvents in health-related areas such as food and pharmaceuticals [14]. Regarding
the use of seaweeds as alternative dietary fibre sources to terrestrial vegetables, in this Special Issue
2
Molecules 2020, 25, 1362
an article is presented evaluating the nutritional composition and physicochemical properties of two
dried commercially interesting edible red seaweeds, Gracilaria corticata and G. edulis. In view of the
results, both G. corticata and G. edulis contain important nutrients for human health and are possible
natural functional foods [15]. More generally, a wide review about the current knowledge surrounding
the impacts of seaweeds and their derived polysaccharides on the human microbiota is also presented,
in which potential benefits against chronic non-transmissible diseases were discussed [16].
Finally, two articles describing potentially beneficial food sources of fat for humans are presented.
In one on them, it was concluded that refined commercial salmon oil can be transformed into a
profitable source of eicosapentaenoic and docosapentaenoic acids, thus leading to a product with higher
commercial value, and that this process can be optimized by using response surface methodology [17].
The last article of the Special Issue consists of a review article about avocado oil, including discussion
about the extraction methods, chemical composition, and various applications of avocado oil in the
food and medicine industries. Based on the available data, avocado oil has established itself as an
oil that has a very good nutritional value at low and high temperatures, with multiple technological
applications that can be exploited for the benefit of its producers [18].
This Special Issue is accessible thought the following link: https://www.mdpi.com/journal/
molecules/special_issues/Nutrition_analysis.
As Guest Editor for this Special Issue, I would like to thank all the authors and co-authors for their
contributions and all the reviewers for their effort in carefully and rapid evaluating the manuscripts.
Last but not least, I would like to appreciate the hard work done by the editorial office of the Molecules
journal, as well as their kind assistance in preparing this Special Issue.
Funding: This research received no external funds.
Conflicts of Interest: The author declares no conflict of interest.
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© 2020 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
4
molecules
Article
Metabolite Profiling by UPLC-MSE, NMR, and
Antioxidant Properties of Amazonian Fruits: Mamey
Apple (Mammea Americana), Camapu (Physalis
Angulata), and Uxi (Endopleura Uchi)
Larissa Gabrielly Barbosa Lima 1 , Julia Montenegro 1 , Joel Pimentel de Abreu 1 ,
Millena Cristina Barros Santos 2,3 , Talita Pimenta do Nascimento 2,3 , Maiara da Silva Santos 4 ,
Antônio Gilberto Ferreira 5 , Luiz Claudio Cameron 3 , Mariana Simões Larraz Ferreira 2,3
and Anderson Junger Teodoro 1, *
1
2
3
4
5
*
Laboratory of Functional Foods, Nutrition Biochemistry Core, Food and Nutrition Graduate Program,
Federal University of the State of Rio de Janeiro, UNIRIO. Av. Pasteur, 296, Rio de Janeiro 22290-240, Brazil;
larissagabrielly_lima@hotmail.com (L.G.B.L.); juliamontenegro95@gmail.com (J.M.);
pimenabreu@gmail.com (J.P.d.A.)
Laboratory of Bioactives, Nutrition Biochemistry Core, Food and Nutrition Graduate Program,
UNIRIO. Av. Pasteur, 296, Rio de Janeiro 22290-240, Brazil; barrosmillena@gmail.com (M.C.B.S.);
talitapiment@gmail.com (T.P.d.N.); mariana.ferreira@unirio.br (M.S.L.F.)
Center of Innovation in Mass Spectrometry, Laboratory of Protein Biochemistry, UNIRIO. Av. Pasteur, 296,
Rio de Janeiro 22290-240, Brazil; cameron@unirio.br
Fluminense Federal Institute of Education, Science and Technology, IFF, Av. Dário Viêira Borges,
235-Lia Márcia, Bom Jesus do Itabapoana, Rio de Janeiro 28360-000, Brazil; maiarasantos@yahoo.com.br
Laboratory of NMR, Department of Chemistry, Federal University of São Carlos, UFSCar. Washington Luiz,
s/n, São Carlos 13565-905, SP, Brazil; giba_04@yahoo.com.br
Correspondence: atteodoro@gmail.com; Tel.: +55-21-25427236; Fax: +55-21-25427752
Received: 1 October 2019; Accepted: 12 December 2019; Published: 15 January 2020
Abstract: The metabolite profiling associated with the antioxidant potential of Amazonian fruits
represents an important step to the bioactive compound s characterization due to the large biodiversity
in this region. The comprehensive bioactive compounds profile and antioxidant capacities of mamey
apple (Mammea americana), camapu (Physalis angulata), and uxi (Endopleura uchi) was determined for
the first time. Bioactive compounds were characterized by ultra-performance liquid chromatography
coupled to high resolution mass spectrometry (UPLC-MSE ) in aqueous and ethanolic extracts.
Globally, a total of 293 metabolites were tentatively identified in mamey apple, campau, and uxi
extracts. The main classes of compounds in the three species were terpenoids (61), phenolic acids
(58), and flavonoids (53). Ethanolic extracts of fruits showed higher antioxidant activity and total ion
abundance of bioactive compounds than aqueous. Uxi had the highest values of phenolic content
(701.84 mg GAE/100 g), ABTS (1602.7 μmol Trolox g−1 ), and ORAC (15.04 μmol Trolox g−1 ). Mamey
apple had the highest results for DPPH (1168.42 μmol TE g−1 ) and FRAP (1381.13 μmol FSE g−1 ).
Nuclear magnetic resonance (NMR) spectroscopy results showed that sugars and lipids were the
substances with the highest amounts in mamey apple and camapu. Data referring to chemical
characteristics and antioxidant capacity of these fruits can contribute to their economic exploitation.
Keywords: Amazonian fruits; antioxidant; phenolic compounds; UPLC-MSE ; bioactive compounds
1. Introduction
The Amazonian region offers a wide variety of native fruits and most of them are typically obtained
from nature or grown only for the local market supply in form of pulp or in natura. The economic
Molecules 2020, 25, 342; doi:10.3390/molecules25020342
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Molecules 2020, 25, 342
exploitation is potentially of great importance for the region [1]. These native fruits have been studied
as potential bioactive sources and many of them have shown high antioxidant capacity and elevated
phenolic compounds content [2].
The antioxidant properties of Amazonian fruits have been the object of many researches, mainly
due to the presence of natural antioxidants such as carotenoids and phenolic compounds. The group of
antioxidant compounds found in fruits produced in Amazonian can protect the human body against
toxic effects and preventing diseases such as chronic degenerative disorders, cardiovascular diseases,
premature ageing, diabetes, and neurodegenerative diseases [3–5].
The Brazilian Amazonian region has a great biodiversity with approximately about 220 edible
plant species producing fruit, representing 44% of native fruit diversity in Brazil. Some species such as
mamey apple (Mammea americana), camapu (Physalis angulata), and uxi (Endopleura uchi) have widely
appreciated flavors by Brazilian consumers, but still of moderate importance to the economy. They
show potential for commercialization in both domestic and international markets [6].
Mamey apple (Mammea americana) is a fruit with a reddish yellow color, aromatic, and edible
pulp, and is popularly known as abricó-do-pará, abricó, mammey apple or apricot from St. Domingo.
This fruit has an important agroindustrial potential and this pulp can be used to produce different
products such as syrup, juice, sherbet, jam, and pastes [7]. Physalis angulata L. is an herb indigenous of
the Solanaceae family, and dispersed throughout tropical areas, including the Amazonian region [8].
This is popularly known as “camapu” and its juice is used as sedative, depurative, anti-rheumatic,
and for the relief of earache, and is also used as a traditional medicine [9]. Uxi (Endopleura uxi) is an
important fruit distributed in Pará and Amazonas, states of Northern Brazil. The only edible part of
uxi presents a yellow-brownish color pulp with a rough-like texture, containing high content of fat
(mainly oleic acid) and carotenoids, mostly trans-β-carotene, with an unique flavor [10].
Due to their peculiar biodiversity, knowledge of the species and functional property
characterization of native Amazonian fruits such as mamey apple, camapu, and uxi present a
major challenge to their appreciation, as many of these species are unexplored and their chemical
properties remain unknown.
Few studies in the literature were found to provide a comprehensive metabolomic analysis
of the bioactive compounds combined to antioxidant capacity of these fruits from the Amazonian
biome. However, some studies have shown the metabolomic composition of Amazonian native
fruits. Paz et al. [11] studied Clavija lancifolia Desf. using liquid chromatography mass spectrometry
(LC–MS/MS) and found some compounds of flavonoids for which kaempferol was the main compound.
Souza et al. [12] identified the phenolic compounds cyanidin 3-O-rutinoside, chlorogenic acid, and
rutin in Oenocarpus distichus fruits, using high-performance liquid chromatography (HPLC). It has
been reported the presence of catechin, caffeic acid, rutin, orientin, quercetin, apigenin, luteolin, and
kaempferol in Mauritia flexuosa L. f. (Arecaceae), another fruit from Amazonian biome [13].
Concerning the importance of the characterization of bioactive compounds from Amazonian
fruits, the aim of this study was to assess the phytochemical profile and the total antioxidant capacity
of mamey apple (Mammea americana), camapu (Physalis angulata), and uxi (Endopleura uchi).
2. Results and Discussion
2.1. Total Phenolic Compounds Content
Mamey apple (MA), camapu (C), and uxi (U) were submitted for analysis of total phenolic
compounds from extractions submitted with different vehicles solvents (water (W) and ethanol
(E)) to determine the most efficient extraction solvent of the mentioned compounds. The results
showed that the ethanolic extracts (MAE and UE) of the different Amazonian fruits showed a greater
quantity of phenolic compounds when compared to the aqueous extracts (MAW and UW) (Figure 1).
The preparation and extraction from this wide range of samples depends mostly on the nature of the
sample matrix and the chemical properties of the phenolics, including molecular structure, polarity,
6
Molecules 2020, 25, 342
concentration, number of aromatic rings, and hydroxyl groups. Conventional solid-liquid using
organic solvent extraction is the main method used to extract phenolics. The sample preparation,
polarity of the solvent used, the technique employed and temperature are factors that can influence the
extraction and contents of these compounds [14].
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Figure 1. Total phenolic content in aqueous mamey apple (MAW), ethanolic mamey apple (MAE),
aqueous camapu (CW), ethanolic camapu (CE), aqueous uxi (UW), and ethanolic uxi (UE) extracts.
Results are expressed by mean ± SD (n = 3) and were compared by the one-way ANOVA test with
post-test Tukey s (* p < 0.01; ** p < 0.001).
Uxi ethanolic extract (UE) showed the highest phenolic compound content (701.839 mg GAE/100 g),
followed by mamey apple (MAE) (556.105 mg GAE/100 g), and camapu (CE) (237.39 mg GAE/100 g).
The aqueous extracts of mamey apple (MAW) and uxi (UW) showed significant difference (p < 0.05)
when compared to the respective ethanolic extracts. No significant differences (p > 0.05) were observed
in the content of phenolic compounds between the aqueous and ethanolic extracts in the camapu sample.
Aqueous and ethanolic extract showed similar fruits yield extracts, being dependent on the
moisture percentage of each fruit. Uxi yield was significantly higher (UE-28.82% and UW-29.42%),
followed by mamey apple (MAE-14.04% and MAW-13.13%) and camapu (CE-10.50% and CW-11.10%).
Considering these results, the levels of phenolic compounds per mass of fruits for the highest yield
would be 202.27, 78.08, and 24.92 mg GAE/100 g for uxi, mamey apple, and camapu, respectively.
Péroumal et al. [15] studied the pulp of six mamey apple accessions and found values for
total phenolic content between 90 and 143 mg GAE/100 g. It can be observed that the results
obtained for uxi ethanolic extracts in this work are higher than some Amazonian fruits such as
araça-boi (87.2 ± 3.0 mg GAE/100 g) and araça (129.1 ± 9.3 mg GAE/100 g), and lower than camu-camu
(1797.2 ± 37.7 mg GAE/100 g) reported by Genovese et al. [16]. Comparing the results of this study
with fruits from the Brazilian Cerrado biome, the Amazonian fruits present lower phenolic compounds
in relation to sweet passion fruit (245.36 ± 3.70 mg GAE/100 g), soursop (281 ± 5.40 mg GAE/100 g),
murici (334.37 ± 9.07 mg GAE/100 g), and marolo (739.37 ± 7.92 mg GAE/100 g) [17]. According to the
classification proposed by Vasco et al. [18], MAE, uxi extracts were classified as fruits as having with
medium phenolic content (100–500 mg GAE/100 g).
2.2. Phytochemical Profile by UPLC-MSE
For the first time, bioactive compounds of Amazonian fruits like mamey apple, camapu, and uxi
were elucidated by UPLC-MSE metabolomic approach. Globally, 293 compounds were tentatively
identified from aqueous and ethanolic extracts of these fruits (Table S1) and relatively quantified taking
account all extracts based on ion counting. Table 1 shows the number of identified compounds and
their classification into eight chemical classes according to Phenol Explorer database [19]: phenolic
acid, flavonoids, chalcones, coumarins, amino acid related compounds, fatty acid, and terpene related
compounds. The main bioactive compounds in the three species were terpenoids (n = 61; 21%),
7
Molecules 2020, 25, 342
phenolic acids (n = 58; 20%), and flavonoids (n = 53; 18%). Other metabolites were also identified
such as other polyphenols (n = 50; 17%), including lignans, coumarins and tannins, and also other
metabolites (n = 56; 19%) such as amino acid related, alkaloids and polyketides, showing the extraction
and LC-MS methods were suitable to characterize different polarities compounds.
Table 1. Number of bioactive compounds distributed by classes and other compounds identified in
aqueous (W) and ethanolic (E) extracts of mamey apple, camapu, and uxi.
Compounds (%)
Phenolic Acid
Flavonoids
Chalcones
Coumarins
Others phenolic compounds
Amino acid related compounds
Fatty acids related compounds
Terpenoids
Total of compounds
Mamey Apple
Camapu
Uxi
W
E
W
E
W
E
43 (20.57%)
36 (17.22%)
2 (0.96%)
16 (7.66%)
27 (12.92%)
17 (8.13%)
22 (10.53%)
46 (22.01%)
209 (100%)
37 (19.68%)
30 (15.96%)
2 (1.06%)
15 (7.98%)
25 (13.30%)
14 (7.45%)
22 (11.70%)
43 (22.87%)
188 (100%)
44 (24.04%)
33 (18.03%)
2 (1.09%)
14 (7.65%)
23 (12.57%)
17 (9.29%)
11 (6.01%)
39 (21.31%)
183 (100%)
40 (21.51%)
29 (15.59%)
2 (1.08%)
14 (7.53%)
30 (16.13%)
20 (10.75%)
12 (6.45%)
39 (20.97%)
186 (100%)
38 (23.17%)
24 (14.63%)
3 (1.83%)
8 (4.88)
20 (12.20%)
18 (10.98%)
14 (8.54%)
39 (23.78%)
164 (100%)
35 (21.21%)
27 (16.36%)
3 (1.82%)
9 (5.45%)
26 (15.76%)
15 (9.09%)
15 (9.09%)
35 (21.21%)
165 (100%)
The identification of different phenolic compounds in this study makes it relevant, since the
presence of these compounds has a range of bioactivities, as already reported in vitro and in vivo
studies [20]. In a recent study, extracts of phenolic compounds from jatobá-do-cerrado can inhibit
α-amylase and α-glycosidase after in vitro digestion and modulate the glucose metabolism [21].
Mamey apple was characterized by high number of bioactive compounds. Among the identified
metabolites, 209 (71%) and 188 (64%) were found in aqueous and ethanolic extracts of mamey apple,
respectively. Furthermore, 183 (62%) and 186 (63%) compounds were found in the aqueous extract
ethanolic extracts of camapu, respectively. In uxi, 164 (56%) and 165 (56%) compounds were tentatively
identified in the aqueous and ethanolic extracts, respectively. Notably, 50 (17%) compounds were
found only in mamey apple (e.g., droserone, norlichexanthone, procyanidin C1, 3,4-leucopelargonidin
II, and piquerol A). Piquerol A is a sesquiterpene with low stability in nature and has only previously
been tested as an insecticide and as an inhibitor of metabolism in cell cultures [22]. About 43 (15%)
were tentatively identified only in camapu (e.g., myristicin, 11-deoxocucurbitacin, synapic acid I,
and pseudopurpurin). Medina et al. [23] reported the presence of sinapic acid in Passiflora edulis
Sims. Furthermore, about 36 (12%) compounds were found exclusively in the uxi extract (e.g.,
4-coumaroylchiquimate, ferulic acid I, kaempferol II, jacareubin, 6-deoxyjacareubin, vanylactic acid II,
nigakilactone A, and jasmonic acid). Kaempferol is an important flavonol identified in other fruits like
hybrid grapes by Rosso et al. [24].
These metabolites are antioxidant inhibitors of mutagenic and carcinogenic compounds and
are considered neuroprotective agents in neurodegenerative disorders, such as Parkinson s and
Alzheimer s diseases [25].
The acid phenolic profiling was the most abundant subclass of phenolic compounds in all extracts
evaluated, as shown in the Tables 1 and 2. Aqueous and ethanolic camapu extracts (CW and CE)
showed about 24 and 22% of phenolic acids, respectively, followed by mamey apple aqueous (MAW)
and ethanolic (MAE) extracts. In addition to phenolic acids, flavonoids showed high number of
identification as well as elevated abundance relative.
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Molecules 2020, 25, 342
Table 2. Flavonoids tentatively identified in mamey apple, camapu, and uxi extracts.
Possible
Identifications
CAS
m/z Exp
Genistin
529-59-9
431.0978
RT
(min)
Fragment m/z
4.55
133.0294 (10.20)
Error
(ppm)
Mamey
Camapu
W
E
W
E
W
Uxi
E
−1.23
−
−
−
−
X
X
−2.86
X
X
−
−
−
−
Eriodictiol I
552-58-9
287.0552
3.05
81.0338 (5.46);
93.0339 (78.57);
119.0496 (100);
155.0342 (10.19);
163.0395 (60.20)
Hesperidin
520-26-3
609.1875
0.43
79.0188 (0.66);
369.0671 (50.27);
488.1618 (10.04)
8.37
X
X
X
X
X
X
9.66
X
X
X
X
X
X
X
X
X
X
14259-46-2
579.1775
0.43
72.9924 (78.62);
79.0188 (0.58);
117.0187 (26.50);
135.0294 (87.65);
357.1033 (3.92);
369.0671 (44.06);
535.1514 (21.08)
Pomiferin I
572-03-2
419.1505
2.62
nd
1.21
X
X
X
X
Ononin
486-62-4
429.1170
2.02
nd
−4.89
X
X
X
X
Mammeisin
18483-64-2
405.1677
4.01
109.0653 (0.46);
154.0615 (0.72)
−7.30
X
X
Quercitrin
522-12-3
447.0923
3.48
151.0030 (2.20);
285.0393 (5.36)
−2.09
X
X
X
−1.44
X
X
X
Narirutin
Quercetin
3-galactoside
482-36-0
463.0875
3.47
151.0030 (2.97);
255.0291 (5.58);
271.0241 (16.99);
285.0393 (7.25);
300.0266 (31.67)
Kaempferol I
520-18-3
285.0396
4.90
nd
−2.70
X
X
Dihydroquercetin
480-18-2
303.0505
1.42
147.0120 (100)
−1.65
X
X
Luteoforol
24897-98-1
289.0733
0.56
109.0289 (12.30)
5.54
X
X
X
X
X
X
X
X
Terpenes were between the most abundant bioactive classes in all extracts. About 46 and
43 terpenes, representing 22% of identifications, were found in the MAW and MAE extracts,
respectively. In UE extracts, 39 (23%) terpenes were tentatively identified too. Terpenoids are
important secondary metabolites of plants and are extremely chemically diversified, being estimated
at more than 40,000 substances. Its use has been described as flavoring agents, in addition to providing
benefits to human health through its antioxidant potential [26,27].
Although the number of identifications between the extracts of the same fruit was not significantly
different, the ethanolic extracts showed a greater relative abundance of compounds in relation to the
aqueous extracts in all fruits studied (Figure 2). This fact can be explained by the large variability of the
structures of these bioactive compounds and the proportion of organic solvents with water is required
for better extraction.
The principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied
to explain possible difference among extracts (Figures 3 and 4). First, the PCA biplot represented
both loading (metabolites) and scores (extracts). Such parameters distinguished the profile of
bioactive compounds among the fruits evaluated in all extracts. Principal components (PC1 and PC2)
demonstrated that the fruits extracts have different composition of bioactive compounds, but the use
of aqueous and ethanolic extractors did not favor the removal of distinct metabolite profile in the
species. The two major principal components (PC1 and PC2) explained more than 72% of the variance
pattern (Figure 3). Discriminatory metabolites, which showed maximum variance (eigenvalues) among
9
Molecules 2020, 25, 342
extracts, were observed mainly in the PC2, including the following bioactive compounds bryophyllin
A II, 1-O-2 -hydroxy-4 -methoxycinnamoyl-b-d-glucose I, mammeisin II, eriodictiol II, justicidin A,
hallactone B II, 7,2 -dihydroxy-4 -methoxy-isoflavanol, eleganin I, 5-hydroxyferulic acid methyl ester
II, lancerin II, 6-methoxytaxifolin II, zapoterin, salvinorin A, meconic acid, benzoic acid II, auriculoside,
leucocyanidin II, lophophorine, lancerin I, vernodalol, visnagin, sinapic acid II, sinapyl alcohol II,
6-methoxytaxifolin I, isobrucein A, and syringin I.
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0(
&:
&(
8:
8(
Figure 2. Relative abundance based on total ion counting of identified compounds from aqueous
mamey apple (MAW), ethanolic mamey apple (MAE), aqueous camapu (CW), ethanolic camapu (CE),
aqueous uxi (UW), and ethanolic uxi (UE) extracts. Results are expressed by mean ± SD (n = 3) and
were compared by the one-way ANOVA test with post-test Tukey s (* p < 0.05; ** p < 0.01; *** p < 0.001).
Figure 3. Principal component analysis (PCA) biplot (loadings and scores) of the bioactive compounds
tentatively identified (loadings, empty circles) in the mamey (squares), camapu (triangles), and uxi
(diamonds) fruits extracted with aqueous (W) and ethanolic (E) solvents.
Then, HCA was applied to observe the similarity/dissimilarity among the abundance of the
discriminatory polyphenols (Figure 4) in order to understand the profile of composition among
extracts. The heatmap indicated that metabolites (20) found in uxi extracts showed higher abundance
in comparison to other extracts, especially in the ethanol extracts. Mamey also showed a distinguished
bioactive profile, with six compounds found exclusively. Among them, sinapoyl alcohol II is an
exclusive metabolite with high abundance in the mamey apple extracts. One of the metabolites with
highest abundance in these extracts was syringin. This compound was determined and reported from
fresh jaboticaba methanolic extract by Wu et al. [28] and implicated as immunomodulator having an
anti-allergic effect [29].
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Molecules 2020, 25, 342
Figure 4. Hierarchical cluster analysis (HCA) and heatmap of the bioactive compounds in the mamey
apple, camapu, and uxi fruits extracted with aqueous (W) and ethanolic (E) solvents, which showed
maximum variance (eigenvectors) among extracts.
2.3. In Vitro Antioxidant Activity
Due to the multifunctional characteristics of the phenolic compounds found in Amazonian
fruits, the effectiveness of measuring the antioxidant capacity of a pulp extract is better evaluated
when using commonly accepted tests. The ethanolic and aqueous fruits extracts was determined by
four different methods with different action mechanism (ABTS, DPPH, FRAP, and ORAC) (Table 3).
According to Barros et al. [14], a single assay does not accurately account for all of the groups of
antioxidant compounds, because of complexity of fruit matrices, and because of these methodologies
can suffer interferences.
ABTS assay revealed values ranged between 263.67 ± 23.90 and 1602.7 ± 30 μmol Trolox g−1 .
The highest antioxidant capacity was presented in UE samples (1602.7 ± 30.16 μmol Trolox g−1 ).
Freitas et al. [30] analyses fresh pulp of uxi and found 51.6 mg TE/100 g for ABTS. In general, the ethanolic
extracts of fruits showed the higher antioxidant capacity for this methodology. However, the aqueous
extract from camapu fruit was an exception with a value of 432.74 ± 16.17 μmol Trolox g−1 (Table 3).
Schiassi et al. [31] studied the activity antioxidant of methanolic extracts of araça (10.92 ± 0.11 μmol
Trolox g−1 ), buriti (6.03 ± 0.00 μmol Trolox g−1 ), cagaita (29.32 ± 0.69 μmol Trolox g−1 ), yellow mombin
(5.55 ± 0.01 μmol Trolox g−1 ), and marolo (132.16 ± 1.40 μmol Trolox g−1 ) and found values lower than
the present study.
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Table 3. Antioxidant activity of the mamey apple, camapu, and uxi samples by ABTS, DPPH, FRAP,
and ORAC assays.
Assay
ABTS (μmol Trolox g−1 )
DPPH (μmol Trolox g−1)
FRAP (μmol ferrous sulphate g−1 )
ORAC (μmol Trolox g−1 )
Mamey Apple
Camapu
Uxi
Aqueous
Ethanolic
Aqueous
Ethanolic
Aqueous
Ethanolic
263.67 ± 23.90 a
336.60 ± 3.05 a
564.18 ± 18.90 a
5.17 ± 0.56 a
937.66 ± 218.49 b
1168.42 ± 218.56 b
1381.13 ± 189.95 b
8.88 ± 0.52 b
432.74 ± 16.17 c
386.24 ± 116.99 c
970.60 ± 28.92 c
12.30 ± 1.15 c
419.43 ± 18.55 c
705.77 ± 100.74 d
1183.98 c ± 46.62 b
11.15 ± 0.42 c
271.86 ± 22.14 a
46.95 ± 17.17 e
376.66 ± 1.81 d
14.33 ± 1.36 d
1602.7 ± 30.16 d
509.27 ± 26.95 f
448.68 ± 41.97 e
15.04 ± 0.84 d
Results are expressed as mean ± standard deviation. Different letters on the same line show significant difference.
Results were compared by the One-way ANOVA test with Tukey post-test (p < 0.05).
Antioxidant capacity was also evaluated by DPPH• radical scavenging method (Table 3) and
expressed in aqueous and ethanolic extract concentration μmol Trolox g−1 sample. The highest activity
was found in MAE (1168.42 μmol Trolox g−1 ), followed by CE (705.771 μmol Trolox g−1 ) and UE
(509.68 μmol Trolox g−1 ). According to Huchin et al. [32], the DPPH method can analyze hydrophilic
and lipophilic compounds, and ethanol is one of the most used solvents for bioactive compounds
extraction due, because proposes some benefits, such as low toxicity, good extraction produce, it is safe
for human consumption and allows the extracts to be used in the food industry [33].
Antioxidant activity by FRAP method followed the same pattern of the DPPH analysis, where
higher values were found for the ethanolic extracts. It is probable that the antioxidant compounds
of the samples detected by FRAP are the same with those evaluated by DPPH. MAE also presented
the highest activity (1381.13 μmol ferrous sulphate g−1 ) These results was lower than obtained by
Barros et al. [14] for aqueous extracts of achachairu (712.35 ± 6.61 μmol ferrous sulphate g−1 ), araça-boi
(798.92 ± 1.52 μmol ferrous sulphate g−1 ), and bacaba (6567.45 ± 4.25 μmol ferrous sulphate g−1 ).
Regarding to ORAC assay, the results revealed that UE and UW obtained the highest value
(15.04 ± 0.84 and 14.33 ± 1.36 μmol Trolox g−1 ), followed by CE and CW (11.15 ± 0.42 and
12.30 ± 1.15 μmol Trolox g−1 ). No significant differences (p > 0.05) were observed between the aqueous
and ethanolic extracts of uxi and camapu sample. MAE (8.88 ± 0.52 μmol Trolox g−1 ) and MAW (5.17 ±
0.56 μmol Trolox g−1 ) showed the lowest values (p > 0.05) between extracts. Santos et al. [34] reported to
Amazonian fruits such as bacaca (195.00 ± 10.00 μmol Trolox g−1 ), buriti (83.00 ± 6.00 μmol Trolox g−1 ),
inajá (26.00 ± 2.00 μmol Trolox g−1 ), pupunha (94.00 ± 1.00 μmol Trolox g−1 ), and tucumã
(64.00 ± 4.00 μmol Trolox g−1 ) results higher than camapu, mamey apple and uxi extracts.
2.4. Correlation between Total Phenolic Compounds and Antioxidant Capacity
The results of Pearson correlation coefficients (r) between total phenolic compounds and
antioxidant capacity of aqueous and methanolic extracts suggest that the total phenolic compounds
contributed to the in vitro antioxidant capacity of the extracts according to the method used.
The aqueous extract showed a good association of total phenolic compounds with ABTS (r = 0.843) and
ORAC (r = 0.752) for camapu fruit. ORAC assay has been largely applied to the assessment of free radical
scavenging capacity of pure antioxidant compounds and antioxidant plant extracts [35]. The radical
cation ABTS [2,29-]azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) is one of the spectrophotometric
methods used to measure water-soluble as well as lipid-soluble antioxidants, pure compounds, and food
extracts. The pre-formed radical monocation of 2,29-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS•1 ) is generated by oxidation of ABTS with potassium persulfate and is reduced in the presence
of such hydrogen-donating antioxidants [36]. 1-O-2 -Hydroxy-4 -methoxycinnamoyl-b-d-glucose,
a compound present in CW (Figure 4), this compound belongs to the sub class of organic compounds
known as hydroxycinnamic acid glycosides. Hydroxycinnamic acids are natural antioxidants found in
fruits, vegetables, and cereals [37].
Regarding to mamey-apple, the ethanolic extract presented positive correlation with total phenolic
compounds in relation to DPPH assay (r = 0.910). The 2,2-diphenylpicrylhydrazyl (DPPH) assay is
widely used in plant biochemistry to evaluate their properties for scavenging free radicals. The method
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is based on the spectrophotometric measurement of DPPH concentration change resulting from the
reaction with an antioxidant [38].
According to Figure 4, the acids syrigin I and visnagin were components with high relative
abundancy. Visnagin is an antioxidant furanocoumarin derivative and is a furanochromone that is
furo [3,2-g]chromen-5-one which is substituted at positions 4 and 7 by methoxy and methyl groups,
respectively. Syringic acid is a dimethoxybenzene that is 3,5-dimethyl ether derivative of gallic acid.
It has a role as a plant metabolite, a member of benzoic acids, and derives from a gallic acid. Previous
reports described that hydroxybenzoic acids itself and its derivatives showed antioxidant properties
against different type of free radicals and can prevent or decrease overproduction of reactive species.
The main structural feature responsible for the antioxidative and free radical scavenging activity in the
case of phenolic derivatives is the phenolic hydroxyl group. Phenols are able to donate the hydrogen
atom of the phenolic OH to the free radicals, thus stopping the propagation chain during the oxidation
process. This effect is modulated by the ring substituents, so that electron-withdrawing groups increase
the bond-dissociation enthalpy, due to the stabilization of the phenol by a polar structure that leaves a
positive charge on the OH group [39–41].
In uxi ethanolic extract, some bioactive compounds were identified with great relative abundance,
such as auriculoside, that is a flavan glycoside. Most natural flavans are lipid-soluble and prominent in
fruit skin or peel and leaf surfaces, and are usually found at higher concentrations in immature fruits
compared to mature fruits [42]. Lancerin a plant metabolite found in UE is a member of xanthones,
a C-glycosyl compound and a polyphenol. Among the polyphenols, the xanthones derivatives comprise
an important class of the oxygenated heterocycles with a diversity of substitution patterns that have
been described for their antioxidant activity, show to act as metal chela-tors, free radical scavengers,
as well as inhibitors of lipid peroxidation [43]. The presence of these compounds may have contributed
to the strong correlation (r = 0.910) found between total phenolic compounds and ORAC assay.
These results demonstrate the importance of do different methods to available the antioxidant
activity, above all in complex matrices. The positive correlation between compound phenolics and
ORAC, can be explained due to this assay may be considered a more exact method, because it uses a
biologically relevant radical source (peroxyl) and allows the measurement of total antioxidant capacity
through the combination of the antioxidant capacity of hydrophilic and lipophilic fractions [44].
According to the methodology used, these results suggest that phenolic compounds may be one of the
main factors responsible for the antioxidant capacity of mamey apple, camapu, and uxi.
2.5. NMR Profile
Table 4 described the identified compounds for each fruit, with the chemical shift and signal
multiplicity, as well as coupling constant (J), and the quantification in mg/g of freeze-dried fruit.
Camapu had the greatest number of different compounds, indicating that it has a large variety of
nutrients and metabolites, even more than could be extracted and identified. Uxi had the least amount,
probably due to the great quantity of lipophilic compounds that could not be extracted, so this result
does not mean that there are few compounds in uxi.
Sugars were most abundant compounds in those fruits. Camapu showed the highest amount of
sucrose, and mamey apple showed the highest amount of fructose and glucose, both α and β. In the
uxi extracts no sugars were found. This result indicates that mamey apple and camapu are sweeter
than uxi, and probably would be more accepted by consumers. The sugars present in mamey apple
were sucrose (1.50 ± 0.10 mg/g), fructose (0.85 ±0.03 mg/g), α-glucose, and β-glucose (0.49 ± 0.02 and
0.40 ± 0.01 mg/g, respectively). The sugars found in camapu were sucrose (4.09 ± 0.46 mg/g), followed
by fructose, α-glucose, and β-glucose (0.11 ± 0.00, 0.04 ± 0.00, and 0.04 ± 0.00 mg/g respectively).
Fruit and vegetable flavor depend upon taste (given by sweetness, sourness, acidity, and
astringency) and aroma (concentrations of odor-active volatile compounds). Sweetness is determined
by the concentrations of the predominant sugars, which are ranked relative to sucrose (fructose >
sucrose > glucose). Sourness or acidity is determined by the predominant organic acids, which
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are ranked relative to citric acid (citric > malic > tartaric). Some amino acids, such as aspartic and
glutamic, may also contribute to sourness. In general, consumer acceptance is related to soluble solids
concentration (sugars, organic acids, soluble pectin, some phenolic compounds, and ascorbic acid) or
the ratio of soluble solids to titratable acidity [45].
Table 4. NMR fingerprinting identification and quantification of mamey apple, camapu, and uxi.
Fruit
Compound
δ 1 H (ppm)
Multiplicity (J)
Mass (mg/g)
Mamey Apple
Formic acid
Shikimic acid
Sucrose
α-glucose
β-glucose
Fructose
Choline
Ethanol
8.44
6.48
5.42
5.22
4.64
4.10
3.19
1.18
s
m
d (3.88)
d (3.76)
d (7.94)
d (3.42)
s
t (7.09)
0.01 ± 0.00
0.11 ± 0.01
1.51 ± 0.11
0.49 ± 0.02
0.40 ± 0.02
0.85 ± 0.04
0.01 ± 0.00
0.01 ± 0.00
Camapu
Formic acid
Sucrose
α-glucose
β-glucose
Fructose
Choline
Aspartic acid
Acetic acid
GABA
Alanine
Lactic acid
Ethanol
Valine
8.44
5.42
5.23
4.65
4.11
3.20
2.82
1.94
1.89
1.47
1.33
1.18
1.04
s
d (3.91)
d (3.73)
d (7.92)
d (3.42)
s
dd (17.4; 3.78)
S
quin (7.44)
d (7.2)
d (6.68)
t (7.09)
d (7.07)
0.01 ± 0.00
4.09 ± 0.46
0.04 ± 0.01
0.04 ± 0.01
0.12 ± 0.01
0.01 ± 0.00
0.04 ± 0.01
0.01 ± 0.00
0.02 ± 0.01
0.02 ± 0.01
0.01 ± 0.00
0.02 ± 0.001
0.01 ± 0.00
Uxi
Linoleic acid
Acetic acid
Alanine
Ethanol
Valine
5.32
1.94
1.45
1.16
1.05
M
S
d (7.20)
t (7.10)
d (7.05)
1.79 ± 0.11
0.16 ± 0.01
0.04 ± 0.01
0.15 ± 0.02
0.05 ± 0.001
It has been shown that mamay apple has ethanol (0.01 ± 0.00 mg/g), which is possibly a fruit
sugar fermentation product. It was also found choline (0.01 ± 0.00 mg/g) and organic acids, such
as formic acid and shikimic acid (0.01 ± 0.00 and 0.11 ± 0.00 mg/g) in mamey apple. Shikimic
acid (3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid) is natural acid from the plant metabolism and
common in berries fruits. It is a precursor for the biosynthesis of primary metabolites such as aromatic
amino acids and folic acid, and a great many other aromatic compounds. The benzene ring is formed
through the shikimate pathway, and shikimic acid is an extremely essential compound in plants
and microbes. Shikimic acid has been found to occur in many tissues of a variety of plants, with a
sufficiently high percentage. Moreover, its content and accumulation in different tissues depends on
the rate of metabolic processes taking place in them. Shikimic acid is utilized for industrial synthesis
of the oseltamivir (antiviral), zeylenone (employed as a preparation for chemotherapy of cancer), and
monopalmityloxy shikimic acid (anticoagulant activity). In addition, shikimic acid derivatives represent
a great interest for agriculture because many of them are used as herbicides and antibacterial agents [46].
Choline is a natural amine that can be synthesized in human body, but this production usually is
not enough to meet human needs in men and postmenopausal women. Some important functions of
choline are it is a part of the neurotransmitter acetylcholine; it is a part of the predominant phospholipids
in membranes; it forms betaine, which is an important osmolyte in the kidney glomerulus and helps
with the reabsorption of water from the kidney tubule. Eggs and liver are the main sources of choline,
but many other foods contain significant amounts of choline and esters of choline [47].
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Ethanol (0.02 ± 0.00 mg/g) was also present in camapu, indicating fermentation. The organic acids
found in camapu were lactic acid, acetic acid and formic acid (0.01 ± 0.00, 0.01 ± 0.00, and 0.01 ± 0.00).
Besides those organic acids, it was found γ-amino butyric acid (GABA) in camapu (0.02 ± 0.00 mg/g).
GABA is a four-carbon nonprotein amino acid and is widespread in bacteria, animals, and plants. GABA
is naturally present in small quantities in plants, and is produced in response to anaerobic conditions,
γ-radiation, low pH, low or high temperatures, and darkness, and by mechanical manipulation.
Some functions of GABA in plants are: regulation of cytosolic pH, protection against oxidative stress,
defense against insects, and the regulation of pollen tube growth and guidance [48]. In vertebrates is a
neurotransmitter that is deficient in the brain of people with Alzheimer disease [49]. Camapu has been
shown to be effective in delaying the development of this disease and in its treatment, by increasing
the proliferation of neural stem cells in vivo [50] and also shown an anxiolytic effect [51] which the
authors suppose is due to the presence of GABA agonists. Several amino acids such as aspartic acid,
alanine, and valine (0.03 ± 0.00, 0.02 ± 0.00, and 0.01 ± 0.08 mg/g) and choline (0.01 ± 0.00 mg/g) were
also present in camapu in sufficient quantities to be identified.
For uxi, the compound with in the highest amount was linoleic acid (1.79 ± 0.10 mg/g), which
is the main fatty acid of ω-6 group. This result shows that there are fatty acids that are sufficient to
appear in a spectrum of fingerprints, even though other lipids may not have been completely extracted
from the sample. There is probably a low amount of sugar in uxi fruit because there are high amounts
of lipids and usually sugar and lipids in the pulp of fruits are inversely proportional. However, it was
found ethanol (0.15 ± 0.01 mg/g), indicating that some sugar that was present before it was fermented.
It was also found enough amount of acetic acid (0.15 ± 0.00 mg/g) valine and alanine amino acids
(0.05 ± 0.00 and 0.04 ± 0.00 mg/g, respectively).
3. Material and Methods
3.1. Standards and Chemicals
2,2-Diphenyl-1-picrylhydrazyl (DPPH• ), 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)
diammonium salt (ABTS), gallic acid, quercetin, 2 ,7 -dichlorofluorescin diacetate (DCFH-DA),
L-(+)-ascorbic acid (AA). Phenolic standards (caffeic acid, (+)-catechin, ellagic acid, (−)-epicatechin,
gallic acid, gentisic acid, 4-hydroxybenzoic acid, myricetin, pyrogallol, quercetin, and quercetin
3-O-glucoside) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the solvents used for
the UPLC-MSE analysis were of LC-MS purity grade and were also purchased from Sigma-Aldrich
(St. Louis, MO, USA).
3.2. Samples
The fruits in natura of mamey apple (Mammea americana), camapu (Physalis angulata), and uxi
(Endopleura uchi) were obtained from producing regions of Pará State, in the months of January and
February 2017. They were transported in vacuum plastic containers to Federal University of the State
of Rio de Janeiro (UNIRIO, RJ, Brazil) and stored at −18 ◦ C. The part of the fruit used for extraction
was carried out according to their consumption, for mamey apple only pulp, for camapu the whole
fruit and for uxi pulp and bark.
3.3. Sample Preparation
Samples were extracted by means of 2 extraction solvents: (I) ethanol 70% and (II) water, from 5 g
of sample and 100 mL of extraction solvent, followed by homogenization at room temperature (−20 ◦ C)
in a homogenizer TE-420 (Tecnal, São Paulo, Brazil) for 10 min in the absence of light. The samples were
then centrifuged (Thermo Fisher Scientific, Bartlesville, CA, USA) (5000× g, 5 min, 20 ◦ C) and filtered
through analytical filter paper. The extraction solvents were evaporated using a rotary evaporator
under vacuum (Savant, Thermo Scientific) and the extracts were then frozen at −80 ◦ C in an ultra-freezer
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and lyophilized (Terroni® LD 300, São Carlos, SP, Brazil) for 24 h. After this process, extracts were
frozen at −20 ◦ C until use in the experiments.
For UPLC-MSE analysis, the extraction solvents were evaporated using a rotary evaporator
under vacuum (Savant, Thermo Scientific, USA) at 40 ◦ C and thereafter ressuspended in
methanol/acetonitrile/water (2:5:93; v/v). Stock solutions (500 ppm) of 11 standards (caffeic acid,
(+)-catechin, ellagic acid, (−)-epicatechin, gallic acid, gentisic acid, 4-hydroxybenzoic acid, myricetin,
pyrogallol, quercetin and quercetin 3-O-glucoside from Sigma-Aldrich were prepared individually by
dissolving accurately weighed amounts of standards in aqueous methanol an aliquot of each stock
solution was mixed to achieve a mixed standard solution with a final concentration of 10 mg/L for each
compound. Finally, extracts and standards were filtered through a 0.22 μm syringe filter and stored at
−80 ◦ C until UPLC-MSE analysis.
3.4. Quantification of Total Phenolic Compounds
Total phenolic content was determined by the Folin–Ciocalteu method, which was adapted from
Rocchetti et al. [52]. The extract and 2.5 mL Folin-Ciocalteu reagent solution 10% were combined and
then mixed well using a Vortex. The mixture was allowed to react for 5 min then 2 mL of sodium
carbonate 4% solution was added and mixed well the solution was incubated at room temperature
(25 ◦ C) in the dark for 2 h. The absorbance was measured at 750 nm using a spectrophotometer
(Turner® 340, Haverhill, MA, USA) and the results were expressed in mg gallic acid equivalents (GAE)
per 100 g of extract using a gallic acid (2,5–50 μg/μL) standard curve [53].
3.5. Phytochemical Profile Characterization by UPLC-MSE
The UPLC-MSE analyses were carried out according to Santos et al. (2018) [54] with slightly
modifications. Two uL of extracts and standards were injected in triplicate into an Acquity UPLC (Waters,
Milford, MA, USA) coupled to Xevo G2-S QTOF-MS/MS (Waters, Manchester, UK) system equipped with
an electrospray ionization source (ESI) operating in negative ion mode. The column used was a UPLC
HSS T3 C18 (100 mm × 2.1 mm, 1.8 μm) (Waters, Wexford, Ireland). The flow rate was 0.6 mL/min and the
mobile phase gradient elution consisted of acidified water (5 mM ammonium formate and 0.3% formic
acid, v/v) (pump A) and acetonitrile containing 0.3% formic acid (pump B), as follows: 97% A at 0 min,
50% A at 6.8 min, 15% A at 7.4–8.5 min, followed by an additional equilibration step 97% A at 9.1–12 min.
Data were acquired using a multiplexed MS/MS acquisition in the extended mode with alternating low
and high energy acquisition (MSE ) 30–55 eV using ultra-high pure argon (Ar). The capillary and cone
voltage were set at 3.0 kV and 30 V, respectively. The desolvation gas (N2 ) was set at 600 L/h at 450 ◦ C,
the cone gas was set at 50 L/h and the source temperature at 120 ◦ C. Data acquisition was performed by
using MassLynx 4.1 (Waters Corporation, Milford, MA, USA). To ensure accuracy and reproducibility,
all acquisitions were performed by infusing lock mass calibration with leucine-enkephaline (Waters
Corporation, Milford, MA, USA) (m/z 554.2615) at a concentration of 1.0 ng/L in acetonitrile: H2 O (50:50,
v/v) with 0.1% (v/v) formic acid at a flow rate of 10 μL/min.
The raw data of all replicates were processed with Progenesis QI v2.1 (Nonlinear Dynamics, Waters
Corporation, UK) with the following conditions: centroid data, resolution full-width at half maximum
(FWHM) of 50,000, deprotonated molecule [M − H]− . The identification of phenolic compounds was
performed by searching for polyphenols with MetaScope, using a customized database of polyphenol
compounds from PubChemID by using the following parameters: precursor and fragment mass error
tolerance (5 and 10 mg/L, respectively), retention time limit 7.5 min. Target analysis was also applied
for identification of the phenolic compounds by comparing the run parameters of phenolic standards
such as the retention time, exact mass, mass error, and the MS-MS spectra, besides the other above
mentioned parameters. The processed data were exported to XLSTAT software (Addinsoft, Paris,
France), where the values of relative abundance obtained from each compound based on ion mass
spectra counting were used to relative quantification and then to the multivariate statistical evaluation
where Principal Components Analysis (PCA) biplot was elaborated. The Metaboanalyst 3.0 web server
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was used for analysis of multivariate data (HCA, hierarchical cluster analysis and heat map) where
the eigenvectors of the correlation matrix was used to select the discriminatory bioactive compounds
between the samples analyzed.
3.6. Analysis by Nuclear Magnetic Resonance Spectroscopy
For the NMR analyses, samples were prepared in triplicate. It was done by weighing 30.0 (±0.5) mg
of freeze-dried samples (for each repetition), which was subjected during 10 min to shaking in a vortex
with 0.8 mL of extractor solvent (D2 O for abrico and camapu; methanol-d4 for uxi), with TMSP-d4 at
concentration 0.02% (m/v) as internal reference and mass standard and, after, to ultrasonic bath during
10 min. Next, samples were centrifuged (13,000 RPM) at room temperature for more 10 min. A 0.6 mL
supernatant aliquot was analyzed.
The 1 H-NMR spectra, as well as the 2D experiments, were conducted in the Bruker Avance III
9.4 Tesla (400 MHz for hydrogen frequency) spectrometer equipped with a PABBI probe (5 mm) and
with an Automatic Tuning and Matching (ATMA) unit located in NMR Laboratory of the Chemistry
Department at Federal University of São Carlos-UFSCar, Brazil. The TMSP-d4 was used to determine
the 0 point in the chemical shift scale. 1H NMR spectra were registered at temperature of 300 K using
64 K data points and the standard Bruker pulse sequence (zgps).
All spectra were obtained through pre-saturation pulse sequence in order to suppress the residual
water sign in the solvent. Spectra were acquired through 16 scans (NS), spectral window 15.0191 mg/L
with 128,000 data points, receiver gain (RG) 80.6, relation delay of 50 s (D1) and acquisition time of
10.9 s (AQ). The exponential line broadening function value adopted for spectral apodization (LB) was
0.30 Hz.
The NMR data was analyzed in TopSpin Brucker Software. Quantification was performed by
comparison of the compound s signals area and the area of the TMSP-d4 signal to find out the
compound s mass since the TMSP-d4 mass is known. The equation used was: analyte mass = (analyte
area × TMSP-d4 1 H Nº × TMSP-d4 mass × analyte MM)/(TMSP-d4 area × analyte 1 H Nº × TMSP-d4
MM). The quantification is expressed in mg/g of freeze-dried fruit.
3.7. ABTS
For ABTS assay, the procedure followed the method of Thaipong et al. [55]. The stock solutions
included 7.0 mM ABTS solution and 140 mM potassium persulfate solution. The working solution
was then prepared by mixing the two stock solutions, 5 mL ABTS solution in 88 μL potassium
persulfate solution, and allowing them to react for 16 h at room temperature in the dark. The solution
was then diluted by mixing 1 mL ABTS solution with ethanol necessary to obtain an absorbance of
0.709–0.701 units at 734 nm using the spectrophotometer (Turner® 340, Haverhill, MA, USA). The fruits
extracts were allowed to react with 2.5 mL of the ABTS solution for 6 min in a dark condition. The
standard curve was linear between 0 and 2000 μM Trolox equivalents. Results are expressed in μM
Trolox equivalents g−1 of extract.
3.8. DPPH
The antioxidant activity of all fruits extracts in relation to the DPPH (2,2-diphenyl-1-picrilidrazil)
radical was quantified by using a protocol described by Brand-Williams and Berset [56], using
wave 515 nm in spectrophotometer (Turner® 340, Haverhill, MA, USA). The working solution was
obtained by dissolving 2.4 mg DPPH reagent with 100 mL methanol and stored in the dark until
needed. The extracts were allowed to react with 2.5 mL of the DPPH solution for 30 min in the dark.
The standard curve was linear between 0 μM and 2000 μM Trolox. Results are expressed in μmol
Trolox g−1 of extract.
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Molecules 2020, 25, 342
3.9. FRAP
The FRAP assay was done according to Thaipong et al. [55]. The stock solutions included 0.3 M
acetate buffer, pH 3.6, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution, and 20 mM ferric chloride
solution. The fresh working solution was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZ
solution, and 2.5 mL ferric chloride solution. Fruits extracts were allowed to react with 2.7 mL of the
FRAP solution for 30 min in the dark condition and warmed at 37 ◦ C. Readings of the colored product
were then taken at 595 nm in spectrophotometer (Turner® 340, Haverhill, MA, USA). The standard
curve was linear between 500 μM and 2000 μM ferrous sulphate. Results are expressed in μmol ferrous
sulphate g−1 of extract.
3.10. ORAC
The ORAC procedure used an automated plate reader (SpectraMax i3x, Molecular Devices, USA)
with 96-well plates [54–57]. Analyses were conducted in phosphate buffer pH 7.4 at 37 ◦ C. Peroxyl
radical was generated using 2, 2 -azobis (2-amidino-propane) dihydrochloride which was prepared
fresh for each run. Fluorescein was used as the substrate. Fluorescence conditions were as follows:
excitation at 485 nm and emission at 520 nm. The standard curve was linear between 1 μM and 90 μM
Trolox. Results are expressed as μM TE g−1 of extract.
3.11. Statistical
Statistical comparisons were carried out by ANOVA and post hoc Tukey s test using Graph Pad
Prism 5.0 and the differences were considered significant when p < 0.05 in total phenolic content and
relative abundance of identified compounds. The correlation coefficients between different results
of evaluation antioxidant capacity of the samples, ABTS, DPPH, FRAP, ORAC and total phenolic
compounds (PC) were obtained. Principal component analysis (PCA) and heatmap was used to
interpret data, using Stat graphics software.
4. Conclusions
This work highlights the significantly concentration of phenolic compounds and antioxidant
properties of Amazonian fruits. A total of 293 phenolic compounds were tentatively identified in
mamey apple, camapu, and uxi by comparison with standards and by fragmentation patterns. Mamey
apple extracts presented major number of phenolic compounds, being the phenolic acids and terpenoids
the classes more identified. ABTS, DPPH, FRAP, and ORAC assays revealed that the ethanol extract
presents a higher antioxidant activity and aqueous extract presents a high correlation with phenolic
compounds content in Amazonian fruits. The findings of this study highlight the potential of mamey
apple, camapu, and uxi as a valuable source of natural antioxidants.
Supplementary Materials: The following are available online. Table S1: Bioactive compounds detected and
characterized in mamey apple, camapu and uxi fruits extracted with aqueous (W) and ethanolic (E) solvents.
Author Contributions: Conceptualization, L.G.B.L., A.G.F., L.C.C., M.S.L.F., and A.J.T.; methodology, L.G.B.L.,
J.M., J.P.d.A., M.C.B.S., T.P.d.N., M.d.S.S., A.G.F, M.S.L.F., and A.J.T. data curation, L.G.B.L., J.M., J.P.d.A., M.C.B.S.,
T.P.d.N., M.d.S.S., A.G.F, M.S.L.F., and A.J.T. writing—original draft preparation, L.G.B.L., J.M., M.C.B.S., T.P.d.N.,
M.d.S.S., M.S.L.F., and A.J.T. writing—review and editing, L.G.B.L., M.C.B.S., T.P.d.N., M.S.L.F., and A.J.T.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by FAPERJ grant number 248660, 246295, 216954 and 246296.
Acknowledgments: This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à
Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Foundation for Research Support of the State of São
Paulo (FAPESP).
Conflicts of Interest: The authors declare no conflict of interest.
Availability of Data and Materials: All the processed data are presented in the article. Information on raw data
and materials are available from the corresponding authors upon request.
18
Molecules 2020, 25, 342
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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
22
molecules
Article
Optimization of Scorpion Protein Extraction and
Characterization of the Proteins’ Functional Properties
Ahmidin Wali 1,2 , Atikan Wubulikasimu 1,2 , Sharafitdin Mirzaakhmedov 3 , Yanhua Gao 1 ,
Adil Omar 1,2 , Amina Arken 1,2 , Abulimiti Yili 1, * and Haji Akber Aisa 1
1
2
3
*
Key Laboratory of Plant Resources and Chemistry in Arid Regions, Xinjiang Technical,
Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, Xinjiang, China;
ahmidin@ms.xjb.ac.cn (A.W.); obuatikam@sina.com (A.W.); gaoyh@ms.xjb.ac.cn (Y.G.);
adilomar@126.com (A.O.); ak950208@163.com (A.A.); haji@ms.xjb.ac.cn (H.A.A.)
University of Chinese Academy of Science, Beijing 100039, China
Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent 100125, Uzbekistan;
mirzaakhmedov@mail.ru
Correspondence: abu@ms.xjb.ac.cn; Tel.: +86-991-3835679
Academic Editor: Jose M. Miranda
Received: 26 September 2019; Accepted: 6 November 2019; Published: 13 November 2019
Abstract: Scorpion has long been used in traditional Chinese medicine, because whole scorpion
body extract has anti-cancer, analgesic, anti-thrombotic blood anti-coagulation, immune modulating,
anti-epileptic, and other functions. The purpose of this study was to find an efficient extraction method
and investigate some of physical and chemical parameters, like water solubility, emulsification,
foaming properties, and oil-holding capacity of obtained scorpion proteins. Response surface
methodology (RSM) was used for the determination of optimal parameters of ultrasonic extraction
(UE). Based on single factor experiments, three factors (ultrasonic power (w), liquid/solid (mL/g) ratio,
and extraction time (min)) were used for the determination of scorpion proteins (SPs). The order of
the effects of the three factors on the protein content and yield were ultrasonic power > extraction time
> liquid/solid ratio, and the optimum conditions of extraction proteins were as follows: extraction
time = 50.00 min, ultrasonic power = 400.00 w, and liquid/solid ratio = 18.00 mL/g. For the optimal
conditions, the protein content of the ultrasonic extraction and yield were 78.94% and 24.80%,
respectively. The solubility, emulsification and foaming properties, and water and oil holding capacity
of scorpion proteins were investigated. The results of this study suggest that scorpion proteins
can be considered as an important ingredient and raw material for the creation of water-soluble
supramolecular complexes for drugs.
Keywords: scorpion (Buthus martensii Karsch) protein; ultrasonic extraction; response surface
methodology; scanning electron microscopy; functional properties
1. Introduction
Scorpions are Chelicerate arthropods and members of the class Arachnida. There are about 1500
known scorpion species, and only 30 among them produce venom considered potentially dangerous to
humans, which might be lethal without medical treatment [1,2]. Scorpion venoms are rich sources of
complex mixtures of substances, including toxic peptides, free amino acids, enzymes, nucleotides, lipids,
amines, mucoproteins, heterocyclic components, and inorganic salts, which affect the ion channels of
both excitable and non-excitable cells [3,4]; some scorpion venoms also contains bioactive compounds,
like trimethylamine, nucleosides, and betaine. Therefore, it has anti-thrombosis, anticoagulant,
fibrinolysis, analgesic, anti-tumor, anti-epileptic, and several pharmacological effects [5–8]. A number
of studies indicate that scorpion venom is comprised of more than 300 toxins, most of which are less
Molecules 2019, 24, 4103; doi:10.3390/molecules24224103
23
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Molecules 2019, 24, 4103
than 10 kDa in molecular mass [9]. Scorpions have been used in traditional medicine in Asia and Africa
for thousands of years, and large-scale commercial farming has achieved good social and economic
benefits [10,11]. The body parts of scorpions are effective for the treatment of cancer [12–14]. In this
work, our main attention is focused on investigating whole scorpion body extract, not directed to
venomous part.
With the emergence of several novel methods for the study and identification of scorpion body
parts and venom components, several bioactive peptides have been proved effective to treat a variety
of diseases [15–17]. The whole body part of a scorpion is used as medicinal material in traditional
Chinese medicine. Although there are previous studies on the extraction, isolation, and activity of
scorpion toxin [18,19], few reports are available on the extraction and functional evaluation of scorpion
total protein. Scorpion bodies are a main medicinal recourse, but few studies on the bioactive peptides
from scorpion proteins have been mentioned [20].
Most of the functional characteristics of proteins play a significant role in the protein’s physical
and chemical properties in the processing, storage, preparation, and sale phase at different areas of
economy [21,22]. Many factors, such as the protein’s size, pH, temperature, protein’s shape, ionic
strength, structure, amino acid sequence, composition, and charge distribution are very important
factors of protein functionality. The conditions of the proteins at extraction and each stage of purification
and drying are factors that must be taken into consideration [23,24]. Therefore, the aim of this study was
to investigate some of physical and chemical parameters, like water solubility, emulsification, foaming
properties, and oil-holding capacity of scorpion proteins. In order to reach of this goal, response surface
methodology for the optimization of three parameters of ultrasonic extraction (UE)—ultrasonic power,
solid/liquid ratio, and extraction time—are also investigated.
2. Materials and Methods
2.1. Materials and Chemicals
Scorpions (Buthus martensii Karsch) were collected (about 1000 pieces) in April in the Turpan
region, Xinjiang, China, and after killing, they were put into plastic bottles and kept in a refrigerator.
A Pierce BCA Protein Assay Kit was purchased from Thermo Scientific; electrophoresis reagents ware
purchased from Biosharp Corporation (Beijing, China). All other chemicals were purchased from
local suppliers.
The freeze drier was an FDU-1110 (EYELA Company, Tokyo, Japan), the high-speed refrigerated
centrifuge was a CR22N (Hitachi Koki Co., Ltd., Tokyo, Japan), the refrigerated centrifuge was an 5417R
(Eppendorf, Germany), and the large-capacity oscillator was an HY-8A (Millipore, Burlington, MA,
USA). A Spectra Max M5 enzyme labeling analyzer (Bio-Tec Co., Ltd., USA) was also used.
2.2. Total Extraction of Proteins from Scorpion Bodies
Ten grams of dried scorpion were ground and separated through a 40-mesh separator. After that,
separated powders (8.2 g) were subjected to de-oiling by petroleum ether (150 mL) in a continuous
soxhlet extractor, until the solvent became colorless. The final residue was collected and air-dried.
According to the following procedure, scorpion proteins (SPs) were extracted by (1) distilled water,
(2) sodium chloride (0.5 M), (3) phosphate buffer (PBS; 50 mM, pH = 8.0), and (4) isoelectric precipitation
(adjusted pH to 9.0 with 1.0 N NaOH, and adjusted pH to 4.5 with 1.0 N HCl to precipitate SPs)
using an ultrasonic extraction method and a stirring extraction method (as a comparison) for 1 h,
respectively. This method was used for the first time by us. Scorpion proteins were extracted with
an ultrasonic generator, and the ultrasonic power was 200 w. The extraction steps were repeated
three times and dialyzed against distilled water for 48 h at 4 ◦ C (F0136-1 Dialysis Membrane, MWCO
1000Da, United States). The obtained dialysates were lyophilized using a freeze drier (FDU-1110,
EYELA Company, Tokyo, Japan) and kept at −20 ◦ C until use.
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Molecules 2019, 24, 4103
2.3. Methods for the Determination of Protein Extraction Indexes
2.3.1. Determination of Protein Contents (%)
The protein content of lyophilized SPs was determined by the BCA (bicinchoninic acid) method [25].
Bovine serum albumin (BSA) was used as a standard. A total of 1.0 mL of distilled water was used
to dissolve 1.0 g of sample, which was clarified through centrifugation at 4500× g for 15 min at 4 ◦ C.
According to the specification of the BCA protein measuring kit, the absorbance was read by an enzyme
labeling instrument (Spectra Max M5 enzyme labeling analyzer) at 562 nm. The average value of three
parallel measurements was used to calculate the protein content.
2.3.2. Determination of Yields (%)
The yield of SPs was determined using Equation (1):
mSe
× 100%
mSd
Yield (%) =
(1)
where mSe is the weight of scorpion protein extraction (mg), and mSd is the weight of the defatted
scorpion (mg).
2.4. Single-Factor Experiments
The influence of ultrasonic power (w), extraction time (min) and liquid/solid ratio (mL/g) on
protein content and yield of the UE were investigated. Based on the ultrasonic extraction and stirring
extraction experiments, scorpion protein was extracted with 0.5 M NaCl buffer by the ultrasonic
extraction method. The ultrasonic extraction time (20, 30, 40, 50, and 60 min), liquid/solid ratio (5, 10,
15, 20, and 25 mL/g), and ultrasonic power (50, 100, 200, 300, and 400 w) were tested.
2.5. Gel Electrophoresis Analysis
Gel electrophoresis (SDS-PAGE) of the SPs was done using 15% acrylamide gel (Bio-Rad
Mini-PROREAN Tetra System) [26]. Gel electrophoresis was carried out at 10 v/cm constant voltage.
Coomassie Brilliant Blue R-250 dye was used for staining the protein bands for 1.5 h, which were then
de-stained in a decoloring solution for 1.5 h via shaking by large capacity oscillator (HY-8A).
2.6. Box–Behnken of RSM and Statistical Analysis
RSM is a statistical tool for solving multivariable problems by using reasonable experimental
design method and obtaining certain data through experiments, and by analyzing regression equations
to determine the optimal technological parameters [27]. In this study, the RSM and Box–Behnken design
was used to optimize the extraction conditions. Based on the single-factor experiment, the complete
design was made up of 17 runs, and these were done in triplicate to optimize the levels of the selected
variables (extraction time, ultrasonic power, and liquid/solid ratio). For the statistical analysis, the three
independent variables were coded as X1 , X2 , and X3 , respectively using Equation (2):
xi =
Xi − X0
ΔXi
(2)
where xi is the coded value of an independent variable, Xi is the real value of an independent variable,
X0 is the real value of an independent variable at the center point, and Xi is the step change value.
The quadratic equation of the variables is as follows:
Y = β0 +
βi xi +
25
βii x2i +
βij xi x j
(3)
Molecules 2019, 24, 4103
where Y is the predicted response variable; B0 , βi , βii , and βij are the constant regression coefficients of
the model; and xi and xj (i = 1, 3; j = 1, 3, i j) represent the independent variables.
The accuracy and fitness of the above model were evaluated by the coefficient of determination
(R2 ) and the F-value. Based on the above results, the second order polynomial coefficients were
undertaken using Design Expert (Version 8.0.6, USA) software. The model was performed to evaluate
the analysis of variance (ANOVA).
2.7. Functional Properties
The functional properties of de-oiled scorpion flour (DSF), stirring extraction (SE), and ultrasonic
extraction (UE) have been tested at the optimal conditions.
2.7.1. Protein Solubility Analyses
Using the method from [28], with slight modification, the solubility of the protein was determined.
A total of 200 mg of SP was dissolved in 20 mL deionized water, and the pH was adjusted to 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, and 12 by 1.0 mol/L HCl and 1.0 mol/L NaOH. After the pH was stabilized, it was stirred
for 0.5 h at a speed of 8000 rpm/min for 20 min; centrifugation was done to obtain the supernatant.
Using the Bradford method [29], the protein content of the supernatant was determined. The value
of each sample was measured three times, and the average value was taken. Protein solubility was
calculated using the following formula:
Protein Solubility % =
m
mT
× 100%
(4)
where m is the protein content of the supernatant (mg/g), and mT is the content of total protein in
SP (mg/g).
2.7.2. Foaming Properties
The foaming capacity (FC) and foaming stability (FS) were carried out using the methods described
previously, with minor modifications [24,30]. Using these methods, 0.2 g of SP was dissolved in
20mL of distilled water (w/v = 1.0%), and the solution (V 0 ) was stirred for 2.0 min. The mixture was
transferred immediately into a 50 mL tube, the initial foam volume was measured (V 1 ), and the foam
volume after standing for 30 min was also measured (V 3 ). The FC was calculated using Equation (5):
FC (%) =
V1 + V2 − V0
V0
× 100%
(5)
where V 2 is the volume of the liquid remaining just after stirring.
FS is calculated using the following formula:
FS (%) =
V3
× 100%.
V1
(6)
2.7.3. Water Absorption Capacity (WAC)
Using the method from [31], with slight modifications, water absorption capacity (WAC) was
determined. Here, 1 g (W 0 ) of SP was weighed together with the centrifugal tube (W 1 ). Thereafter,
10 mL of distilled water was added in the tube and mixed. After being held at room temperature for
30 min, the emulsion was centrifuged at 2100× g for 15 min. In the end, the supernatant was decanted,
and the tube with sediment was weighed (W 2 ). Using Equation (7), the WAC was calculated:
WAC =
W2 − W1
.
W0
26
(7)
Molecules 2019, 24, 4103
2.7.4. Oil Absorption Capacity (OAC)
The oil absorption capacity (OAC) was determined using the method reported previously [31],
with minor modifications. One gram (F0 ) of SP was weighed together with a centrifugal tube (F1 )
and mixed with 5 mL of corn oil. The emulsion was incubated at room temperature for 30 min,
and then centrifuged (Eppendorf centrifuge 4530R, Germany) at 3500 rpm/min for 15 min under the
same conditions. The tube was reweighed (F2 ) after the removal of the supernatant. The OAC was
determined using Equation (8):
F2 − F 1
(8)
OAC =
F0
2.7.5. Emulsifying Properties
The emulsifying activity (EA) of the UE and SE was determined by the method described by
Wu et al. [32], with slight modification. Here, 1.0 g of each SP was weighed and dissolved in 20 mL
of distilled water and 20 mL of corn oil, and mixed at a high speed for 1 min at room temperature.
The mixture was centrifuged at 4000 rpm for 5 min. The EA was calculated using Equation (9):
EA (%) =
h
× 100%
H
(9)
where h is the height of the emulsified layer and H is the height of the tube contents.
The emulsion stability (ES) of UE and SE were determined using the method from [30], with slight
modifications. One gram of each SP was weighed and dissolved with 20 mL of distilled water and
20 mL of corn oil, and thereafter mixed at a high speed for 2 min at room temperature. The mixture
was centrifuged at 4000 rpm for 5 min. After preparation, 30 mL of emulsions was then transferred
into test tubes, and the emulsions were stored at room temperature; they separated into a top oil layer
and a bottom serum layer over time. The ES was evaluated using Equation (10):
ES (%) =
Hs
× 100%
Ht
(10)
where Hs is the height of bottom serum layer (mm) and Ht is the total height of emulsion in the
tube (mm).
2.8. Scanning Electron Microscopy (SEM) Analysis
To study the effect of scorpion protein extraction technology on protein content and yield,
we analyzed SPs by scanning electron microscopy (SEM; SUPRA 55VP, ZEISS). The morphology and
surface characteristics of the SPs were observed and recorded by SEM after the samples were fixed on
silicon wafers and sputtered by gold.
2.9. Statistical Analysis
The experiments were performed with three independent trials, and all the determinations were
triplicated. The results were represented as mean ± standard deviation. Analysis of variance (ANOVA)
was performed to identify significant differences (p < 0.05).
3. Results and Discussion
3.1. Analysis of Scorpion Protein Extraction Method
In this study, the effects of diverse buffer solutions for the extraction of scorpion protein were
studied. Meanwhile, the effects of ultrasonic extraction (this method is used to increase the yield
of protein), as well as stirring extraction of the yield (%) and protein content (%) were compared.
Figure 1 and Table 1 show that the order of the effects of four buffer solutions on yield and protein
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Molecules 2019, 24, 4103
content was 0.5 M NaCl > 20 mM PBS > 0.02 M NaOH > water. The 0.5 M NaCl buffer solution (yield
14.64 ± 0.08%, protein content 79.06 ± 0.05%) with ultrasonic was better than other buffers for extracting
scorpion protein, followed by 20 mM PBS (yield 18.29 ± 0.05%, protein content 60.98 ± 0.07%).
Figure 1. SDS-PAGE electrophoresis of scorpion proteins with different extractions and buffer conditions.
M refers to the marker; S1, S2, S3, and S4 are scorpion proteins extracted by stirring with water, 0.5 M
NaCl, 20 mM phosphate buffer (PBS), alkali extraction, and isoelectric precipitation, respectively; U1,
U2, U3, and U4 are scorpion proteins extracted by ultrasonic with water, 0.5 M NaCl, 20 mM PBS, alkali
extraction, and isoelectric precipitation, respectively.
Table 1. Effects of ultrasonic and stirring methods on extraction of total proteins from scorpion body.
Yield (%)
Extraction Method
Water
0.5 M NaCl
20 mM PBS
0.02 M NaOH
Protein Content (%)
Ultrasonic Extraction
Stirring Extraction
Ultrasonic Extraction
Stirring Extraction
34.85 ± 0.06
14.64 ± 0.08
18.29 ± 0.05
7.70 ± 0.11
34.15 ± 0.09
11.80 ± 0.03
13.45 ± 0.10
7.57 ± 0.13
31.14 ± 0.04
79.06 ± 0.05
60.98 ± 0.07
50.87 ± 0.07
18.08 ± 0.06
35.26 ± 0.08
37.25 ± 0.09
51.91± 0.17
3.2. Analysis of a Single Factor Results
3.2.1. Effect of Extraction Time on Yield and Protein Content
Extraction time plays a key role in protein extraction [32]. In this experiment, the effect of extraction
time on the yield and protein content is examined. For that, ultrasound power was 200 W, the liquid/solid
ratio was 15, and extraction time was carried out in a range between 20–60 min. The results obtained are
shown in Figure 2A. Obtained data show that with the increase of ultrasonic extraction time, the yield and
protein content rapidly increased and reached a maximum at 50 min, at 17.95% and 66.35%, respectively.
A further increase of time did not affect the yield and amount of protein.
Figure 2. Effects of extraction time (A), ultrasonic power (B), and liquid/solid ratio (C) on yield and
protein content.
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Molecules 2019, 24, 4103
3.2.2. Effect of Ultrasonic Power on Yield and Protein Content
In this experiment, the effect of ultrasonic power on the yield and protein content was examined.
For that, the extraction time was 50 min, the liquid/solid ratio (mL/g) was 15, and the ultrasonic power
was evaluated in the range 50–400 w. The results obtained are shown in Figure 2B. The obtained data
show that with an increase in power, the yield and protein content increased rapidly, and reached their
maximums at 300 w, at 23.60% and 75.30%, respectively; in addition, a further increase of time did not
affect the yield and amount of protein.
3.2.3. Effect of Liquid/Solid Ratio on Yield and Protein Content
In this experiment, the effect of the liquid/solid ratio on the yield and protein content is examined.
For that, ultrasound power was 200 w, extraction time was 50 min, and the liquid/solid ratio was
evaluated in the range between 5–25 mL/g. The results obtained are shown in Figure 2C. The obtained
data show that with the increase of liquid/solid ratio to the 15 mL/g the yield, protein content rapidly
increased, and when the ratio reached to 20 mL/g, a slower increase can be seen, reaching maximums
of 19.21% and 55.32%, respectively. A further increase of time did not affect the yield and amount
of protein.
3.3. Optimization of Extraction Parameters by RSM
Table 2 shows that there was a significant change in protein content and yield at different values
of the selected parameters. Using Equation (3), the results were analyzed. After multiple regressions
fitting with Design-Expert V8.0.6 software, the regression model equation is as follows:
Y1 = 56.43 + 7.25A + 0.066B + 7.69C + 4.80AB + 4.97AC + 4.67BC + 2.14A2 + 1.28B2 − 3.18C2 (11)
Y2 = 19.46 + 0.86A + 0.75B + 1.93C + 1.14AB + 1.80AC + 1.72BC + 0.31A2 − 1.64B2 − 0.26C2 (12)
where Y1 and Y2 are the predicted conversion (%) of protein content and yield, respectively; and A,
B, and C are the extraction time (X1 , min), liquid/solid ratio (X2 , mL/g), and ultrasonic power (X3 ,
w), respectively.
When the factor sign is positive, it shows that the amount of the response variable increases with
an increase in its value, and vice versa. Figure 3A,C can also be used to compare the model predicted
and the experimental results. Figure 3B,D is the normal probability chart indicating that the points
follow a narrow linear pattern. The analysis of variance of the regression equation is shown in Table 3.
F is the value of the regression model, where the protein content and yield were 17.49 and 34.12 and
the p-values were 0.0005 and <0.0001, respectively. These values show that the model obtained was
significant. The F-value and p-value for the lack of fit of the protein content and yield were 1.54 and
2.04, respectively, and 0.3354 and 0.2506, respectively, which shows that the equation has a good fitting
degree and high reliability. In addition, the decision coefficients (R2 ) of the model were 0.9574 and
0.9777, respectively, and the adjusted coefficients of determination (Adj-R2 ) were 0.9027 and 0.9491,
respectively. The values of R2 and adjusted R2 for the models are shown in Table 3, which shows that
the regression equation can predict the result of extracting scorpion protein accurately.
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Molecules 2019, 24, 4103
Figure 3. Standard statistical diagrams for model verification. (A,C) Model-predicted values versus
actual data. This figure shows a comparison between the model-predicted values and the actual
values that have been obtained by the experiments. (B,D) Normal probability plot of the residuals.
Monotonous distribution and linearity of the data, which are obvious from these figures, confirm the
validity of the model and its capability to predict the results.
Table 2. RSM experimental design and results for the three-factor/three-level Box–Behnken design (BBD).
Runs
Extraction
Time (min) X 1
Liquid/Solid
Ratio (mL/g) X 2
Ultrasonic
Power (W) X 3
Protein
Content/%
Yield /%
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
60
40
20
40
40
60
40
40
60
40
40
60
40
20
20
20
40
15
20
10
15
15
10
20
10
15
10
15
20
15
20
15
15
15
400
400
300
300
300
300
200
200
200
400
300
300
300
300
200
400
300
75.23
69.01
59.31
55.98
56.39
64.09
44.01
49.4
50.17
55.70
55.70
70.00
53.58
46.01
45.49
50.66
60.52
24.32
21.41
17.76
19.53
18.89
16.62
14.84
16.25
17.04
17.74
19.31
20.78
19.52
17.37
18.31
18.39
20.04
30
Molecules 2019, 24, 4103
Table 3. Least-squares fit and parameter estimates (significance of regression coefficient).
Source
Model
X1
X2
X3
X1 × 2
X1 × 3
X2 × 3
X1 2
X2 2
X3 2
Lack of Fit
R2
Adj-R2
Protein Content
Yield
F-Value
p-Value
F-Value
p-Value
17.49
53.50
4.46
60.17
11.73
12.57
11.11
2.45
0.88
5.43
1.54
0.9574
0.9027
0.0005
0.0002
0.9486
0.0001
0.0111
0.0094
0.0125
0.1617
0.3800
0.0526
0.3354
34.12
23.98
18.15
118.71
20.67
51.76
25.77
1.67
45.25
1.12
2.04
0.9777
0.9491
< 0.0001
0.0018
0.0037
< 0.0001
0.0026
0.0002
0.0014
0.2378
0.0003
0.3256
0.2506
3.3.1. Analysis of the Influence of Various Factors on the Extraction of Scorpion Proteins
The degree of influence of each factor on the test index can be compared by the F-value (Table 4):
F (X1 ) = 53.50, 23.98; F (X2 ) = 4.46, 18.15; F (X3 ) = 60.17, 118.71. These values indicate that the order of
influence of each factor on the extraction of scorpion protein was ultrasonic power (w) > extraction
time (min) > liquid/solid ratio (mL/g). The response surface can describe the interaction between
variables and predict the optimal conditional values of each factor [33]. Figure 4A–F shows the effect
of operational variables on protein content and yield protein content and yield. The effect of extraction
time on the protein process is greater than that of the ratio of the liquid/solid, and that of ultrasonic
power is greater than that of extraction time. According to the above analysis, the order of the influence
of each variable on the extraction technology of scorpion protein was ultrasonic power > extraction
time > liquid/solid ratio.
3.3.2. Interactions of Variables
Contour plots for each of the fitted models that display the effects of the three variables (to visualize the
combined effects of the three factors on protein content and yield) were generated. Figure 4 illustrates the
two-dimensional (2D) plots of the binary interactions of the variables on protein content and yield via the
contour plots. The interaction between the extraction time and the liquid/solid ratio is shown in Figure 4A,D.
This plot indicates that protein content and yield depend more on X1 than on X2. Figure 4A,D reveals that
at low values of X1, maximum protein content and yield occurs at higher values of X2. However, at higher
values of X1, maximum protein content and yield occurs at lower values of X2. As seen in Figure 4A,D,
the interaction between the two factors is weak. The interaction between the extraction time and ultrasonic
power is shown in Figure 4B,E. This plot indicates that protein content and yield depend more on X3 than
on X1. In Figure 4C,F, the effect of the interaction between the liquid/solid ratio and ultrasonic power is
depicted. Figure 4C,F shows that increasing the liquid/solid ratio at different ultrasonic power levels has no
important effect on the protein content and yield, so the plot shows that protein content and yield depends
more on X3 than on X2. Therefore, the optimum values of X1, X2, and X3, as determined by the software,
are 50 min, 400 w, and 18 mL/g, respectively.
31
Molecules 2019, 24, 4103
Figure 4. Effects of operational variables on protein content and yield. Interaction between (A) liquid/
solid ratio and extraction time, (B) ultrasonic power and extraction time, and (C) ultrasonic power and
liquid/solid ratio to protein content. Also depicted are the interaction between the (D) liquid/solid ratio
and extraction time, (E) ultrasonic power and extraction time, and (F) ultrasonic power and liquid/solid
ratio to yield.
3.3.3. Determination and Validation of Optimal Extraction Conditions
Optimum conditions, which were obtained by Design-Expert V8.0.6 software, were as follows:
extraction time = 47.68 min; ultrasonic power = 395.84 w; and solid/liquid ratios = 18.01 mL/g. In view
of the feasibility of the experiment, the optimum conditions of extraction proteins were adjusted as
follows: extraction time = 50.00 min, ultrasonic power = 400 w, and solid/liquid ratio = 18.00 mL/g.
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Molecules 2019, 24, 4103
After several tests (n > 3), the protein content and yield were 78.94%, and 24.80%, respectively. It is
shown that the regression equation and the optimal conditions obtained by the response surface
method are reliable.
3.4. Functional Properties of Scorpion Proteins
3.4.1. Protein Solubility (PS)
The protein solubility (PS) profiles of de-oiled scorpion flour (DSF), ultrasonic extraction (UE),
and stirring extraction (SE) in the water at different pH ranges (2–12) are shown in Figure 5. The PS of
DSF, UE, and SE were significantly different, and Figure 5 shows the same U-shaped curves. When the
pH value is in the range of 2–4, the solubility of DSF, UE, and SE decreased; however, when in the pH
range of 6–10, the solubility of DSF, UE, and SE significantly increased. The minimum protein solubility
of DSF, UE, and SE were presented at pH 4, with values of 8.05%, 15.25%, and 18.75%, respectively.
The maximum protein solubility was presented at pH 12, with values of 13.5%, 70.15%, and 79.5%,
respectively. Therefore, results indicate that scorpion protein extracted by ultrasonic method shows
exceptional solubility at an alkaline pH.
Figure 5. Protein solubility profiles of de-oiled scorpion flour (DSF), stirring extraction (SE), and ultrasonic
extraction (UE) at different pH levels.
3.4.2. Water and Oil Absorption Capacity
The obtained results for the water absorption capacity (WAC) and oil absorption capacity (OAC)
of proteins extracted by ultrasonic and stirring are presented in Table 4. The WAC and OAC from both
methods were significantly different (p < 0.05), with UE having the higher WAC (40.3 ± 0.28 g/g) and
OAC (27.70 ± 0.14 g/g) than SE (with WAC = 33.45 ± 0.07 g/g and OAC = 18.80 ± 0.15 g/g) at pH 7.0.
33
Molecules 2019, 24, 4103
Table 4. Functional properties of scorpion proteins.
Properties
Ultrasonic Extraction
Stirring Extraction
Water holding capacity (g/g)
Oil holding capacity (g/g)
Emulsifying activity (%)
Emulsion stability (%)
Foam capacity (%)
Foam stability (%)
25.25 ± 0.21
38.50 ± 0.14
45.55 ± 0.64
85.50 ± 0.28
40.30 ± 0.28
21.70 ± 0.14
20.45 ± 0.07
30.65 ± 0.64
40.25 ± 0.07
69.45 ± 0.35
33.45 ± 0.07
18.80 ± 0.15
3.4.3. Emulsifying Properties
Charged or non-charged polar amino acids in proteins can affect the proteins’ emulsifying and
surfactant properties. Results of emulsifying properties of UE and SE are shown in Table 4. At pH 7.0,
the emulsifying properties of UE and SE were significantly different from one another, with values
of 45.55 ± 0.64% and 40.25 ± 0.07%, respectively. However, the emulsifying property of UE was
significantly different and was higher (85.50 ± 0.28%) than that of SE (69.45 ± 0.35%). The UE had
pronounced effects on the emulsifying properties, since it might be exposing more hydrophobic groups
to the water and oil interface, giving rise to increased emulsifying capacity and more stable emulsion.
3.4.4. Foaming Properties
Foam formation is similar to emulsion formation. In foam formation, water molecules surround
air droplets, which is a nonpolar phase. At pH 7, the foam capacity (FC) of UE was considerably
higher than that of SE, with values of 40.30 ± 0.28% and 33.45 ± 0.07%, respectively. The foam stability
(FS) of UE was significantly higher than that of SE, with values of 21.70 ± 0.14% and 18.80 ± 0.15%,
respectively (Table 4). The results obtained show that the UE, compared to SE, has more flexible protein
structure in aqueous solution, and forms more stable foams for stronger interaction on the air–water
interface. The variations in foaming properties of UE and SE might be explained with the difference in
their protein concentrations. A high protein concentration will improve foam capacity and stability,
increasing the viscosity and promoting the formation of the interfacial multilayer membrane [34].
3.5. Scanning Electron Microscopy Analysis
Protein samples obtained using various extraction methods were investigated by the SEM method.
Figure 6 shows the surface states of protein samples extracted from whole-body, de-oiled protein (DSF)
powder, stirred extraction (SE), and ultrasonic extraction (UE). These studies focused on the study of
the surface layer of protein molecules. Accordingly, in Figure 6A, adhesive surface layers of protein
molecules in dried DSF can be seen. This state has a collapsed form, which shows the density of protein
molecules in relation to one other; therefore, these molecules have a higher crystalline degree than
other states, and this will result in lower water solubility. In Figure 6B, on the surface of dried SE
protein layers, a plurality of elements similar to “wood shavings” can be seen, in contrast to Figure 6A.
These elements formed as a result of separation from the collapsed surface. This means that during
drying after SE, from the crystalline state shown in Figure 6A, the protein molecules transition from
a crystalline to amorphous state, as shown in Figure 6B; the outcome of this results in an increase in the
solubility of the protein molecules in water. Also, in Figure 6C, you can see the results of the drying of
protein molecules obtained after UE. This picture clearly shows the increase the number of “wood
shavings” on the surface, compared to Figure 6B, and how the transformation collapse forms into thin
film pieces. Therefore, UE leads to the formation of scorpion protein molecules from a crystalline state
to an amorphous one, which results in an increase of their water solubility.
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Molecules 2019, 24, 4103
Figure 6. Scanning electron microscope images of (A) DSF, (B) SE, and (C) UE. (1.00 KX magnifications,
bar 10 μm).
4. Conclusions
Studies have shown that the sum of proteins extracted from scorpion bodies had good emulsifying
and foaming properties. At the same time, when they were subjected to extraction under different
conditions, UE was found to be the best way to obtain the sum of proteins with improved water
solubility. According to SEM analysis, the improvement of water solubility of the sum of protein
components is accompanied by the transition of these macromolecules from the collapsed and
crystalline form to a more amorphous and friable phase. For the extraction of scorpion body parts,
some simple ways, i.e., cold water, hot water, and ethanol extraction methods used previously [35,36]
were used, and SPs obtained by these methods were physiologically effective for the treatment of
various diseases. However, we think that using hot water or ethanol solvents may generally result in
the denaturation of native compounds and reduce the real activities of the extracted SPs. Therefore,
using UE in an alternative way, at the optimal conditions of UE (i.e., X1 , X2 , and X3 are 50 min, 400 w,
and 18 mL/g, respectively) resulted in increased water solubility and improvement of its emulsifying
and foaming properties. In our opinion, the SPs obtained by the UE way should keep a number of their
above-mentioned activities, and will be planned for use in creating medicines on that basis in TCM.
On the other hand, UE could be the basis for the creation of water-soluble supramolecular complexes
for those drugs that are poorly soluble in water. Obtaining such drugs in this way will contribute in the
35
Molecules 2019, 24, 4103
future to (1) an increase the drugs’ water solubility, (2) a reduction their therapeutic doses, (c) increase
pharmacological action, and (d) reduce toxicity and side effects. Since the sum of proteins from the
scorpion body are not individual components, it might be promising, if created in their base gel drug
forms, that they could be used through the skin.
Author Contributions: Project administration, A.Y., H.A.A.; Investigation, A.W.(Ahmidin Wali), A.A. and A.O.;
Methodology, A.W.(Atikan Wubulikasimu) and Y.G.; Formal analysis, S.M.
Funding: This research was funded by the Program for National Science and Technology Major Project of China,
grant number 2017ZX09301045 and “the light of the western” talent training plan: Planting of Hotan Rose and Its
Polyphenols Application, Standardized Planting Techniques of Repellent Vernonia and Its Collection, Preservation
and Popularization; It is also supported by the PIFI Fund of the Chinese Academy of Sciences for invited scientists,
grant number 2019VBA0013 and Central Asian Drug Research and Development Center of the Chinese Academy
of Sciences.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
© 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/).
38
molecules
Article
Influence of Potato Crisps Processing Parameters on
Acrylamide Formation and Bioaccesibility
Emmanuel Martinez 1 , Jose A. Rodriguez 1 , Alicia C. Mondragon 2 , Jose Manuel Lorenzo 3 and
Eva M. Santos 1, *
1
2
3
*
Area Academica de Quimica, Universidad Autonoma del Estado de Hidalgo, Carr. Pachuca-Tulancingo Km.
4.5, Mineral de la Reforma, 42184 Hidalgo, Mexico; qaemmanuel@gmail.com (E.M.);
josear@uaeh.edu.mx (J.A.R.)
Laboratorio de Higiene, Inspeccion y Control de Alimentos, Departamento de Quimica Analitica, Nutricion
y Bromatologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain;
alicia.mondragon@deinal.es
Meat Technology Centre of Galicia, Rúa Galicia Nº 4, Parque Tecnológico de Galicia, San Cibrao das Viñas,
32900 Ourense, Spain; jmlorenzo@ceteca.net
Correspondence: emsantos@uaeh.edu.mx
Academic Editor: Jose M. Miranda
Received: 24 September 2019; Accepted: 21 October 2019; Published: 23 October 2019
Abstract: A fractional factorial design was used to evaluate the effects of temperature, frying time,
blanching treatment and the thickness of potato slices on acrylamide content in crisps. The design
was used on freshly harvested and four-month stored potatoes. The critical factors found were
temperature and frying time, and the interaction between blanching treatment and slice thickness.
Once frying conditions were selected, an acrylamide content of 725 and 1030 mg kg−1 was found for
non-stored and 4-month stored tubers, with adequate textural parameters in both cases. The difference
in concentration is related to storage conditions, which must be controlled in order to control
acrylamide levels. Bioaccesibility studies demonstrated that acrylamide concentration remained
at 70%, and reductions took place mainly at the intestinal phase, as a result of reaction with
nucleophilic compounds.
Keywords: acrylamide; crisps; temperature; frying time; blanching; thickness
1. Introduction
Acrylamide, a thermal processing contaminant with a low molecular weight, which is soluble
in water, is formed when carbohydrate-rich foods are subjected to temperatures above 120 ◦ C in
low-moisture conditions, such as frying, roasting or baking. Several studies consider Maillard reactions
to be the main pathway for acrylamide, 5 hydroxymethylfurfural, methylglyoxal–lysine dimers,
Nε–carboxymethyl–lysine and pyrraline formation in processed foods, particularly reactions between
the carbonyl group of reducing sugars and amino acids [1,2]. Initial reports showed relatively high
concentrations of acrylamide in high-carbohydrate foodstuffs, such as crispy bread, breakfast cereals,
pastries, coffee, French fries and crisps. In general, potato products present higher acrylamide contents
(250–4000 μg kg−1 ) compared to other food products, because of the higher concentration of asparagine
in potato tubers [3,4].
Acrylamide has been classified by the Agency for Research on Cancer as “probably carcinogenic
to humans” (Group 2A) and, in the latest report from the European Food Safety Authority Expert Panel
on Contaminants in the Food Chain (CONTAM, 2015), the margins of exposure for acrylamide lead to
concerns regarding their neoplastic effects, based on animal evidence [5]. Therefore, the European
Commission recently established “indicative” levels for the presence of acrylamide in food, suggesting
Molecules 2019, 24, 3827; doi:10.3390/molecules24213827
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www.mdpi.com/journal/molecules
Molecules 2019, 24, 3827
a limit of 750 μg kg−1 for crisps [6]. Previously, the European Union and FDA had encouraged food
industries to reduce the presence of this contaminant.
In response to this, the food industry has been forced to apply different strategies to reduce
acrylamide formation, applying processing modifications according to recommendations published by
the European Food and Drink Federation in the document “Acrylamide Toolbox”, which includes the
following variables: temperature and time during frying, blanching treatment, and thickness of potato
slices [7]. However, the main challenge for the food industries is in producing potato chips with low
acrylamide levels without affecting their sensory properties [8].
Although it is important to evaluate the effect of pre-treatments on the frying process on acrylamide
mitigation, it is also necessary to determine their influence on its bioavailability. Potato chips are an
important part of the snack food market in many countries, and few studies have been published about
the bioavailability of acrylamide after crisps have been through the digestion process [8,9]. Acrylamide
intake in humans occurs mainly via food ingestion, where the total amount of this compound does not
necessarily reflect the available amount to the body. There are chemical changes in the food when it
enters the digestive tract, because of pH variations, and the action of several enzymes in every stage of
the digestion process [10–12]. In this sense, bioaccessibility is used to evaluate the amount of a chemical
compound available for absorption after it is released from the food matrix into the gastrointestinal
tract. Several methodologies can be used to assess the bioaccessibility of contaminants or nutrients:
1) in vitro, 2) ex vivo, 3) in situ and 4) in vivo models [13,14]. Among the techniques mentioned before,
in vitro models are more commonly used than in vivo models, because of their simplicity, lower cost,
lack of ethical issues and good reproducibility under controlled conditions.
The present work evaluated some acrylamide mitigation strategies, employing a fractional
factorial design experiment, and their influence on textural properties and bioaccesibility, by an in vitro
simulated digestion assay.
2. Results and Discussion
2.1. Acrylamide Content
Potatoes of the Atlantic variety were selected for crisp production, because of their low levels of
glucose and fructose compared to other varieties, such as the Ranger Russet [15]. According to the
storage period of potato tubers, two sampling points were considered: freshly harvested potatoes
(summer of 2018) and four-month stored potatoes. The main variables described for acrylamide
reduction in potato crisps are [7]: temperature control and time of frying process, blanching, and the
thickness of slices. These variables were selected because they are easily applicable in the industry
and do not represent extra costs in production. A fractional factorial design (24−1 ) was proposed,
to evaluate the influence of frying process parameters in the total amount of acrylamide formed and its
bioaccessibility. The levels selected for the variables were: temperature (T) of 150 and 180 ◦ C; frying
time (t) of 5 and 10 min; blanching treatment (B)—no blanching treatment and 70 ◦ C for 5 min—and
thickness of slice (w), at 1.5 and 2.0 mm. In this sense, eight experiments (in duplicate, a total of
16 experiments) were performed.
Table 1 shows the matrix used and the acrylamide content for the experimental design. According
to the obtained results, the concentration of acrylamide was higher in stored potatoes. This can
be explained by the difference between the content of reducing sugars between samples, since
a concentration of 23 mg kg−1 of reducing sugars was found in freshly harvested potatoes, while,
in samples stored for 4 months, a concentration of 34 mg kg−1 was found. During storage, a phenomenon
called cold sweetening occurs, consisting of a degradation of the potato starch caused by the low
temperatures [16]. Even if appropriate storage conditions of potato tubers are followed, a slight
increase of reducing sugars is expected, and will affect the acrylamide concentration [17].
40
Molecules 2019, 24, 3827
Table 1. Matrix of the experimental fractional factorial design and the average of obtained responses.
Exp
T (◦ C)
t (min)
B (70 ◦ C, min)
w (mm)
1
2
3
4
5
6
7
8
150(−1)
150(−1)
150(−1)
150(−1)
180(+1)
180(+1)
180(+1)
180(+1)
5(−1)
10(+1)
10(+1)
5(−1)
10(+1)
5(−1)
5(−1)
10(+1)
0(−1)
0(−1)
5(+1)
5(+1)
0(−1)
0(−1)
5(+1)
5(+1)
1.5(−1)
2.0(+1)
1.5(−1)
2.0(+1)
1.5(−1)
2.0(+1)
1.5(−1)
2.0(+1)
Acrylamide Content (μg kg−1 ) ± sd
Non-stored
4-month stored
973 ± 71
752 ± 63
825 ± 67
885 ± 62
2590 ± 243
795 ± 92
1138 ± 220
2250 ± 204
1257 ± 171
890 ± 52
1483 ± 58
1097 ± 205
4386 ± 270
1149 ± 82
1812 ± 70
3540 ± 254
A comparison of each variance with respect to the variance of the residual allows the determination
of the the experimental F value (Fexp ), which is then compared with the Fcritical value (5.32, p = 0.05, 1,8).
The F-test indicated that, in both cases (non-stored and stored tubers), the critical factors for acrylamide
formation were (p = 0.05): temperature (T) and frying time (t). The contribution of potato thickness (w)
was also observed for stored samples (Table 2).
Table 2. Analysis of variance of the results obtained from the fractional factorial design, showing the
factors significantly affecting acrylamide concentration.
Factor
T
t
B
w
Residual
Non-Stored
4-month Stored
Effect
Variance
Fexp
Effect
Variance
Fexp
834.5
656.5
−3
−211
2,785,561
1,723,969
36
178,084
43,718
63.72
39.43
8.2 × 10−4
4.07
1540
1246
62.5
−565.5
9,486,400
6,210,064
15,625
1,279,161
56,593.5
216.99
142.05
0.36
29.26
Figure 1 shows the mean effect graph for the effect of evaluated control factors on acrylamide
concentration. Independently of storage time, the mean effect profile is similar in both cases,
demonstrating that the increase in acrylamide concentration is mainly a consequence of the increment
of reducing sugars concentration, rather than the processing variables. Bertuzzi et al. also reported
a strong influence of reducing sugar concentration on acrylamide formation in processed potato
products [18]. A percentage of reducing sugars between 0.15%–0.20% has been suggested as an
indicator of the suitability of potatoes for processing [19]. Despite the lower reducing sugar content
found in potatoes in this study, acrylamide formation reached values over the 750 μg kg−1 limit
established by the European Commission [6].
The results obtained are congruent with others previously described, in which an increment
in acrylamide content in potatoes, in relation to temperature and frying time, was observed [18,20].
In general, the increase in acrylamide content follows a linear function over time, while the relationship
with frying temperature is not linear, although temperatures over 175 ◦ C significantly increased
acrylamide levels [1,18,20–22]. Any temperature–time combination proposed to mitigate acrylamide
content should not affect the overall quality of the fried product, especially its texture.
Although the other processing parameters (blanching and thickness) had a smaller contribution,
their interaction was also significant (Figure 2). As acrylamide is formed on the potato surface,
the size:volume ratio of the potato slice influences the acrylamide content. In general, thinner and
smaller cut sizes result in increased acrylamide formation [22]. Also, the blanching of potato slides
prior to the frying process has been recommended to reduce the acrylamide content, since this helps
remove reducing sugars on the surface [8,23]. In this study, a blanching process was required for
thinner potatoes, to decrease acrylamide formation. However, a lower concentration of acrylamide
41
Molecules 2019, 24, 3827
was observed in thicker potatoes without the blanching treatment. It seems that, in thicker slices,
heat diffusion is slower when the slice has not been blanched and, consequently, a decrement of
acrylamide content can be observed. So, the blanching treatment should not be suitable for potato
slices with a thickness over 2.00 mm. This result is interesting for the industry because the application
of blanching treatments at high temperatures and longer times represents an additional difficulty, since
most industrial frying processes are continuous, and blanching could be unnecessary for certain types
of crisps.
3000
Acrilamide concentration (μg kg-1)
Non-stored
4-month stored
2500
2000
1500
1000
500
T(-1) T (+1)
t (-1) t (+1)
B(-1) B(+1)
w(-1) w(+1)
Factors and Levels
Figure 1. Mean effect graph vs. acrylamide concentration (μg kg−1 ) of the experimental design for
non-stored and 4-month stored potato tubers storage.
Acrylamide concentration (μg kg-1)
3000
2500
w(+)
2000
w(+)
1500
w(-)
1000
w(-)
500
Non-stored
4-month stored
0
B(-1)
B(+1)
B(-1)
B(+1)
Factor and level
Figure 2. Mean effect graph vs. acrylamide concentration (μg kg−1 ) for the interaction between
blanching (B) and thickness (w) in non-stored and 4-month stored tubers.
42
Molecules 2019, 24, 3827
The combination of settings that generated the minimum acrylamide concentration was:
temperature, 150 ◦ C (−1); time, 5 min (−1); no blanching treatment (−1); and a slice thickness
of 2.0 mm (+1). Acrylamide content of 725 and 1030 mg kg−1 was determined at these conditions for
non-stored and 4-month stored tubers, respectively.
2.2. Texture Analysis and Bioaccesibility
Potato chips are consumed as indulgent foods, with a characteristic flavor and crispiness, the latter
evaluated through textural parameters. In order to evaluate it, crisps were prepared according to
the processing parameters detailed above. Figure 3 shows a representative profile of the force (N) vs.
probe displacement (mm). The force displacement curves have a jagged appearance, with several
fracture events, which are typical of crispy food [24]. These graphics have two well-differentiated
regions, the first one starting from the first contact between the potato crisp and the probe, until the
major force is achieved (associated with major structural breakdown and hardness). The second region
starts from the major structural breakdown and continues until the end, where smaller force events
take place. The parameters evaluated were the maximum force applied (N), related to hardness or
firmness, and the gradient (N s−1 ), which is related to stiffness.
9.0
Non-stored
8.0
4-month stored
7.0
Force (N)
6.0
5.0
4.0
3.0
2.0
1.0
0.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Displacement (mm)
Figure 3. Force displacement curves generated from the analysis of potato crisps obtained from
frying non-stored and 4-month stored tubers. Frying conditions: temperature, 150 ◦ C; time, 5 min;
no blanching treatment; and a slice thickness of 2.0 mm.
The maximum force applied was 6.1 and 8.5 N, for non-stored and 4-month stored samples,
respectively. Genovese et al. have reported values below 6 N for firmness [25]. The storage of the tubers
promoted an increase in firmness. On the other hand, samples obtained from frying the non-stored
potato slices presented the highest gradient (8.9 N s−1 ) compared to 4-month stored samples (7.2 N s−1 ),
making them, therefore, the stiffest. Additionally, the number of total force peaks in each case can
be evaluated: seven for non-stored tubers and 13 for 4-month stored tubers. Taking into account
that values higher than six are considered high-sensory crispiness, both samples can be accepted as
adequate snacks [26].
43
Molecules 2019, 24, 3827
Once the effect of the control factors on acrylamide formation had been evaluated, it was important
to know their effect on bioaccesibility. Values of 79.1% ± 1.5% and 76.9% ± 1.5% of bioaccesibility were
obtained for the snacks using potatoes that were freshly harvested and stored for 4 months before
frying, respectively. The results obtained in this study are in agreement with the predicted behavior of
potato [10] and bakery products [12]. The main effect was observed at the end of the intestinal phase
of the digestion process, and could be explained by the formation of Michael adducts, in which the
acrylamide would be involved.
During the gastric phase of the digestion process, pepsin hydrolyzes the proteins present in the
potato, generating small peptide chains and some amino acid residues, such as cysteine and lysine, that
have a nucleophilic character (-SH and -NH2 ). These peptides are able to interact with acrylamide and
form adducts, which consequently causes an apparent reduction in acrylamide during the intestinal
phase [12]. Taking into account the mentioned results, this can be proposed as an alternative method of
reducing acrylamide concentration in the digestion process, through the formation of adducts with thiol
compounds (i.e., glutathione) contained in other foods, such as spinach, avocados or asparagus [27,28]
3. Materials and Methods
3.1. Reagents
Acrylamide, 2-naphthalenethiol, and the following enzymes: pepsin (≥250 U mg−1 solid), from
porcine gastric mucosa, pancreatin (4 × USP), from porcine pancreas, α-amylase, from human saliva
(500 U) and porcine bile salts, were purchased from Sigma–Aldrich (St. Louis, MO, USA). Potassium
chloride, sodium bicarbonate and sodium chloride were purchased from MEYER (Mexico City, Mexico).
Acetonitrile (HPLC grade) and acetic acid were obtained from J.T. Baker (Philisburg, NJ, USA).
3.2. Samples and Frying Conditions
Potato (Atlantic variety) and vegetable oil (palm olein) were the raw materials used, provided by
Fritos Totis (Tizayuca, Hidalgo, Mexico). Samples were collected in two periods, the first with potatos
freshly harvested and the second after four months of storage, at 8 ◦ C and 95% relative humidity. Slices
(with a thickness of 1.5 and 2.0 mm, and a diameter of around 35 mm) were cut using a slicing machine.
Slices were rinsed immediately after cutting for 1 min in deionized water (resistivity of 18.2 MΩ cm),
to eliminate some starch material adhered onto the surface, before frying. Forty grams of slides were
deep-fried in an electrical fryer Blazer FE-3 (Mexico city, Mexico) 1600 W, 127 V and equipped with
a bowl with 3 L of capacity, a static basket and a regulating thermometer.
The main variables evaluated for acrylamide reduction in potato crisps were [6]: control of
temperature and time of frying process, blanching, and slice thickness. These strategies were selected
because they are easily applicable to the industry and do not represent extra costs in production.
A fractional factorial design (24−1 ) was proposed, to evaluate the influence of frying process parameters
in the total amount of acrylamide formed and its bioaccessibility. The factors selected were temperature
(T), frying time (t), blanching treatment (B), and thickness of potato slices (w). Results obtained from
the fractional factorial design were subjected to an analysis of variance study. The mean effect of
each factor on acrylamide content and the variance was estimated through the Yates algorithm [29].
Experimental results were analyzed using MINITAB® version 17 software (Minitab Inc., State College,
PA, USA).
3.3. Acrylamide Determination
Acrylamide determination was carried out by chemical derivatization, using 2-naphthalenethiol
as a derivatization reagent, followed by HPLC separation and quantification with fluorescence
detection [30]. In brief, 3 g of potato crisps were homogenized with 30 mL of deionized water and
then defatted with hexane. Then, the aqueous phase was centrifuged and filtered using a cellulose
membrane (pore size, 0.45 μm). Afterwards, an aliquot of 7.5 mL was mixed with 1 mL of the solution
44
Molecules 2019, 24, 3827
2-naphthalenethiol (62 mM), in a solution of sodium hydroxide (0.1 M). The mixture was heated at
90 ◦ C for 45 min. The reaction was stopped with 1.5 mL of acetic acid (1%) and the solution was
centrifuged at 700 g for 15 min. Acrylamide concentration of samples was calculated by standard
additions, by spiking each sample with known concentrations of acrylamide.
Chromatography separation was performed using an Agilent Technologies 1260 Infinity
chromatography system (Agilent Technologies, Waldbronn, Germany). Samples were manually
injected using a 20-μL loop, and a C-8 ZORBAX eclipse XDB column (5 μm; 150 × 4.6 mm internal
diameter) from Agilent Technologies was used as the stationary phase. The mobile phase was acetic
acid (1.0% v/v) and acetonitrile in a 50:50 ratio (v/v). The flow rate was 0.8 mL min−1 .
3.4. Texture Evaluation
A TA.XTPlus Texture Analyser (Stable Micro Systems Ltd., Surrey, UK), equipped with a 5 kg
load cell, was used for force/displacement measurements, with a stainless spherical probe (P/0.25S) of
a 0.25 inch diameter. The samples were placed on a crisp fracture rig (HDP/CFS). The test settings
were: test speed of 1 mm s−1 , trigger force of 0.406 N, travel distance of 3 mm.
3.5. In Vitro Digestion
Bioaccessibility was estimated using an in vitro digestion model (oral, gastric and intestinal stages)
and the simulated fluids—salivary (SFS), gastric (SFG) and intestinal (SFI)—were prepared according
to an internationally agreed protocol (Table 3) [31]. Samples of potato crisps (5 g) were transferred to
a conical tube and mixed with 5 mL SFS (containing 75 U α-amylase mL−1 ) for 3 min. After that, 5 mL
of a pepsin solution (12.5 mg mL−1 in 0.1 M HCl) and 10 mL of SFG were added. The mixture was
adjusted to pH 2.0 and incubated in a shaking water bath at 37 ◦ C for 2 h, with an agitation speed of
60 strokes per min. After incubation, the pH was adjusted to 7.5, and then bile salts (10 mg mL−1 ),
dissolved in 20 mL of SFI together with a pancreatin solution (5 mL, 10 mg mL−1 in water), were
added to the tube. The mixture was incubated at 37 ◦ C for 2 h, with shaking at an agitation speed
of 60 strokes per min, to simulate the duodenal phase. Digestion products were placed in a dialysis
bag (6–8000 molecular weight cut off; Sigma Aldrich) and dialyzed in 250 mL of sodium bicarbonate
solution (pH = 7.5) for 12 h. Dialysis aliquots were removed and acrylamide dialyzed was determined
by the method described above. Bioaccesibility was reported as the percentage of acrylamide in the
dialyzed sample compared to the value in the original sample.
Table 3. Composition of simulated salivary, gastric and intestinal electrolytes fluids.
Compound
KCl
KH2 PO4
NaHCO3
NaCl
MgCl2 ·6H2 O
(NH4 )2 CO3
CaCl2 ·2H2 O
Simulated Salivary
Fluid (SSF) mmol L−1
Simulated Gastric
Fluid (SGF) mmol L−1
Simulated Intestinal
Fluid (SIF) mmol L−1
15.10
3.70
13.60
0.15
0.06
1.50
6.90
0.90
25.00
47.20
0.10
0.50
0.15
6.80
0.80
85.00
38.40
0.33
0.60
4. Conclusions
The evaluation of potato crisps’ processing parameters was performed by a chemometric approach,
through fractional factorial design. The factors with a higher contribution were temperature and
frying time, and an increment on the acrylamide, with respect to the store time of raw material, was
also observed. Crisps obtained in the most suitable conditions have adequate texture parameters,
while bioaccesibility takes place mainly at the intestinal phase, through reaction with –NH2 and
45
Molecules 2019, 24, 3827
–SH containing compounds. In order to reduce acrylamide concentration in the digestion process,
the consumption of foods which contain compounds with these functional groups is proposed.
Author Contributions: E.M., J.A.R. and E.M.S. performed the experiments; E.M. and A.C.M. performed
bioaccesibility experiments; J.M.L. analyzed the texture data. J.A.R. and E.M.S. are responsible for the writing of
the work.
Funding: This research was funded by Fritos Totis SA de CV and CONACyT-Incentives for Research, Technological
Development and Innovation Program (conv 2016-232271).
Acknowledgments: Programa para el Desarrollo Profesional Docente (PRODEP), Consejo Nacional de Ciencia y
Tecnología (SNI membership), and Programa de Fortalecimiento de la Calidad Educativa (PFCE-2019).
Conflicts of Interest: The authors declare no conflict of interest.
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47
molecules
Article
Determination of N-Carbamylglutamate in Feeds and
Animal Products by High Performance Liquid
Chromatography Tandem Mass Spectrometry
Yonghang Ma, Zhengcheng Zeng, Lingchang Kong, Yuanxin Chen and Pingli He *
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology,
China Agricultural University, Beijing 100193, China
* Correspondence: hepingli@cau.edu.cn; Tel.: +86-10-62733688
Academic Editor: Jose M. Miranda
Received: 18 July 2019; Accepted: 30 August 2019; Published: 31 August 2019
Abstract: N-carbamylglutamate (NCG), a synthetic analogue of N-acetylglutamate, is an activator
of blood ammonia conversion and endogenous arginine synthesis. Here, we established an
accurate quantitative determination of NCG in feeds, animal tissues, and body fluids using the
high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The sample
pretreatment procedures included extraction with 0.5% of formic acid in water/methanol (80/20, v/v),
and purification using an anionic solid phase extraction cartridge. Satisfactory separation of NCG was
achieved in 20 min with the application of an Atlantis T3 column, and a confirmative detection of NCG
was ensured by multiple reaction monitoring of positive ions. NCG spiked in feeds, tissues, and body
fluids were evaluated in regard to linearity, sensitivity, recovery, and repeatability. Recoveries for
different sample matrices were in the range of 88.12% to 110.21% with relative standard deviations
(RSDs) less than 8.8%. Limits of quantification were within the range of 0.012 to 0.073 mg kg−1 and
0.047 to 0.077 μg mL−1 for solid and liquid samples, respectively. This study will provide a solid
foundation for the evaluation of availability and metabolic mechanism of NCG in animals.
Keywords: N-carbamylglutamate; feeds; animal products; milk; HPLC-MS/MS
1. Introduction
N-carbamylglutamate (NCG) is a synthetic analogue of N-acetylglutamate (NAG) (Figure 1),
which is the allosteric stimulator of carbamyl phosphate synthase-1 (CPS1). CPS1 is a key enzyme
functioning in the urea cycle and endogenous arginine synthesis pathway [1]. Like NAG, NCG can
activate CPS1 and lead the conversion of blood ammonia into the mitochondrial carbamoyl-phosphate,
further stimulating the endogenous synthesis of arginine. Due to its good stability and safety [2,3],
NCG was initially used for the clinical treating of hyperammonemia caused by the NAG synthetase
deficiency [4,5], propionic aciduria and methylmalonic aciduria [6,7], and maple syrup urine disease [8].
NCG is also proven to be a novel, effective and low-cost substitute feed additive for arginine. In 2014,
China’s ministry of agriculture approved NCG as a new feed additive to use in the livestock (new
feed additive certificate no. 2014-01). The maximum addition limit of NCG in the compound feed is
800 g t−1 of sow, and 880 g t−1 of dairy cow, respectively. Compared with the sole supplementation
of NAG or arginine, NCG can avoid being catabolized by the deacylase or amino acid metabolism
enzymes, and does not cause nutritional antagonism against other amino acids, especially lysine,
tryptophan, and histidine [1,9]. Previous studies have indicated that oral administration of NCG
50 mg per kg body weight two times a day, increases piglets’ plasma arginine concentration by 68%,
and weight gain by 61% in 10 days [1]. More recently, several results have been reported that gilts
with dietary supplementation of 0.05% and 0.1% of NCG had more pigs born alive, and significantly
Molecules 2019, 24, 3172; doi:10.3390/molecules24173172
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Molecules 2019, 24, 3172
increased the live litter weight [10,11]. A similar effect of NCG on growth promotion and reproductive
performance enhancement have also been found in other species of animals including the rat [12],
cattle [13], sheep [14], goat [15], and yellow-feather broiler [16].
Figure 1. The chemical structure of N-carbamylglutamate.
Although the physiological functions of NCG have been well elucidated, and NCG has long been
shown to be non-toxic and free of side effects [2], its metabolic process in animals remains unclear.
With the promising use of NCG in the livestock, it is necessary to develop an accurate measurement
method for NCG in the feed and animal products. Based on the chemical structure of NCG, the infrared
spectroscopy (IR), nuclear magnetic resonance (NMR) spectroscopy, and high performance liquid
chromatography (HPLC) have been adopted in the analysis of the structure and content of the NCG
additive (purity > 97%) [17]. However, due to the complexity of the feed and animal products matrices,
few studies have been conducted to deal with the determination of NCG in the feeds [18], and to the
best of our knowledge, there is no former report regarding the NCG residue detection in animal tissues
or body fluids.
Since NCG has poor absorption of the UV-Vis spectrum, using the HPLC based on the UV detection
will result in low responsivity and sensitivity. Our group tried to determine the NCG using the beforeor past-column derivation, but it showed a multiple matrix interference leading to unsatisfactory
resolution results, and the procedures were relatively complex. In recent years, the high performance
liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), due to its high selectivity and
sensitivity, has become the preferred method to analyze the trace small molecular compounds in
the complex substrates [19,20]. This study developed a HPLC-MS/MS method to qualitatively and
quantitatively determine NCG in the feed, animal tissues, and body fluids with excellent accuracy
and sensitivity. The proposed method can achieve a fast separation of NCG within a 20 min gradient
elution, and the method validation was achieved by evaluating the linearity, sensitivity, recovery,
as well as the accuracy and repeatability for NCG in the feeds and animal products.
2. Results and Discussion
2.1. Optimization of HPLC-MS/MS Conditions
In order to improve the analytical sensitivity and selectivity, parameters of the mass spectrometry
such as the ionization mode, source temperature, capillary voltage, nebulizer pressure, sheath gas flow,
collision energy, and fragmentor voltage were optimized using the NCG standard solution. The result
showed that the most abundant precursor ion of NCG was the pseudo-molecular ion [M + H]+ at m/z
191.0 (Figure 2A and Figure S1). The MS/MS spectrum of this precursor ion is shown in Figure 2B.
The most abundant product ion (m/z 84.0) was used for quantitation while the two relatively abundant
product ions (m/z 130.0, m/z 148.0) were selected for qualification (Figure 2C–E). With the aim to increase
50
Molecules 2019, 24, 3172
the signal responses of characteristic product ions and their stability, the parameters (fragmentor,
collision energy) in the second mass analyzer were optimized. The optimal fragmentor of the precursor
ion (m/z 191.0) is 50 V. The optimal collision energy of m/z 84.0, m/z 130.0, and m/z 148.0 are 15 V, 10 V,
and 5 V, respectively.
Figure 2. Cont.
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Molecules 2019, 24, 3172
Figure 2. (A) Parent ion chromatogram of N-carbamylglutamate (NCG); (B) the tandem mass
spectrometry (MS/MS) spectrum of the NCG precursor ion; (C–E) typical extracted mutiple reaction
monitoring (MRM) ion chromatograms of the NCG standard conducted under optimal conditions.
52
Molecules 2019, 24, 3172
NCG is a compound with strong polarity. A Waters Atlantis T3 C18 column (4.6 × 250 mm, 5 μm)
was compared with a Waters BEH C18 column (2.1 × 100 mm, 1.7 μm). The T3 column showed a
better retention for polar compounds with reduced peak tailing thus better peak shapes and improved
analytical results than the BEH column. In addition, considering that there are many free amino acids,
fatty acids, and other small molecular metabolites in animal samples, the long analytical column
with strong polarity is more suitable for the separation of NCG without a precolumnar derivation.
Thus, the Atlantis T3 C18 column was chosen as the analysis column. Moreover, the mobile phase of
HPLC is a critical factor influencing the analytical results and sensitivity of the detection. Compared
with acetonitrile, using methanol as the organic mobile solvent can achieve better chromatographic
separation of NCG. Due to the weak acidity of NCG, the addition of a small amount of formic
acid could improve the peak shape and improve the sensitivity. Therefore, 0.1% of the formic acid
aqueous solution and methanol were selected to be the mobile phases for the binary gradient pump
system. Figure 2C showed the typical extracted MRM ion chromatogram of NCG conducted under
optimal conditions.
2.2. Optimization of Extract Condition
An effective pretreatment should serve the purpose that the impurity substance is excluded
while the target analyte stays as much as possible. As NCG is soluble in both water and methanol,
pilot experiments have been conducted to determine the optimal extraction solvent with the finest
extraction result. For the feed samples, five extract solvents including water, water/methanol (80:20,
v/v), water/methanol (50:50, v/v), water/methanol (20:80, v/v) and methanol were compared. The results
show that the recoveries were water > water/methanol (80:20, v/v) > methanol > water/methanol (50:50,
v/v) > water/methanol (20:80, v/v). However, using water as the extraction solvent was subjected
to a greater matrix effect presumably due to the complex water-soluble additive in the feed. Thus,
water/methanol (80:20, v/v) was selected as the extraction solvent. To better improve the extraction
efficiency of NCG in the water/methanol (80/20, v/v) solvent, different concentration levels of formic
acid in the water including 0, 0.1%, 0.5%, and 1% were compared. The results show that 0.5% of
formic acid in water/methanol (80/20, v/v) manifested the best extraction result with a recovery of
99.1%. In addition, the high recovery (>100%) were shown in Figure 3C and D. We speculated that
the matrix effect in the C and D extract may cause the enhancement of the NCG signal in MS, which
leads to the recovery of NCG over 100%. Therefore, 0.5% of formic acid in water/methanol (80/20,
v/v) was applied as the extraction solvent for NCG in the feeds (Figure 3). For the animal samples,
the biggest interference came from the high lipid and protein content in the samples [21]. Similarly,
different proportions of methanol and 0.5% of formic acid solution were also tested for the optimization
of extraction agents. Moreover, the application of ice-cold methanol combined with the subsequent
refrigerated centrifugation step can efficiently precipitate and remove most of the interference proteins
present in milk, serum, and tissue samples [20,22]. Thus, ice-cold methanol (100%) was finalized as the
optimal extraction solvent for NCG in the animal body fluids and tissues due to its precipitation effect
of proteins and relatively good recovery (Figure 3).
2.3. Optimization of Purification Condition
A subsequent purification experiment of the sample extracts was conducted using the solid-phase
extraction (SPE) cartridges to further decrease the matrix interference and enhance sensitivity. NCG
is a weak acid compound. A strong anion exchange SPE cartridge was used in virtue of its strong
adsorption ability for the anionic compound. For the better retentivity of NCG, an appropriate amount
of 5% ammonia solution was added to adjust the pH of the extract and make NCG anionic. The results
show that the pH of the extract that ranged from 8 to 10 was optimal, the NCG retained above 95%
on the SPE (Figure 4). Since the cartridge was a strong anion exchange column, considering the
isoelectric point of the NCG (pI 3.02), a lower pH (<8) will not be able to sufficiently make the NCG
anionic, resulting in lower retentivity. On the other hand, if the pH is too high (>10), other interfering
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Molecules 2019, 24, 3172
compounds may be turned anionic and compete with NCG for the limited retention capacity of the
cartridge, this will also cause lower retentivity of NCG in the column. In addition, the NCG content in
the feeds were very high, a direct application of the sample extracts may cause oversaturation and lead
to unsatisfactory recoveries of NCG due to limited adsorption capacity of the cartridges. Our study has
indicated that a reduction of the NCG concentration in the sample extracts could significantly increase
the NCG recoveries. To avoid oversaturation and improve the peak shape, feed sample extracts were
diluted 10 times with water before loaded onto the cartridges. On the contrary, if the content of the
NCG in the sample is too low, such as the biological sample, the loading volume should be increased
before passing through the column.
Figure 3. Recoveries of NCG from matrices using different solvents: (a) 0.5% of formic acid solution;
(b) 0.5% of formic acid solution/methanol (80:20, v/v); (c) 0.5% of formic acid solution/methanol (50:50,
v/v); (d) 0.5% of formic acid solution/methanol (20:80, v/v); (e) methanol.
Figure 4. Dependence of the retention abilities of the NCG on the solid-phase extraction (SPE) cartridge
with different pH of the loading solvent.
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Molecules 2019, 24, 3172
2.4. Method Validation
2.4.1. Stability and Matrix Effect
For the stability test, the NCG stock solution was diluted into 0.005, 0.1, 2 μg mL−1 to have six
aliquots with concentrations around the linearity range. These aliquots were stored at −4 ◦ C and 20 ◦ C,
respectively. Normally, the criterion for the stability is that degradation is equal to or less than 5%.
The results show that the solution could be stored stably for at least three months and one week at the
−4 ◦ C refrigerator and 20 ◦ C, respectively.
Matrix effects come from various chemical and physical processes during ionization of the analytes
in the ESI mode of mass spectrometry, which causes signal suppression or enhancement. The matrix
effect was evaluated by comparing three NCG concentrations (0.005, 0.1, 2 μg mL−1 ) constructed in the
solvent and fortified sample extract. The matrix effects were calculated by comparing the peak area
of NCG in the solvent with the peak area of NCG in the fortified sample extract. The results show
that the matrix effects ranged from 1.08 to 0.54 for the feed and biological samples. Especially for the
biological samples, the matrix inhibition effects were obvious when a high concentration NCG was
added. Therefore, the quantification of NCG must be performed using the matrix-matched calibrators.
2.4.2. Linearity, Precision, Accuracy, LOQ and LOD
A series of experiments were carried out with the aim to validate the linearity, sensitivity, precision,
and accuracy of the established method. The NCG stock solution was diluted into 0.0016, 0.08, 0.2, 0.5,
1, 2 μg mL−1 working solutions. Excellent linearity was obtained at the NCG concentrations ranging
from 0.0016–2 μg mL−1 with a correlation coefficient of (R2 ) 0.999. To evaluate the sensitivity of this
method, the limits of detection (LOD) and the limits of quantitation (LOQ) were tested. The LOD,
defined as the analyte concentration at three times the signal to noise ratio (S/N), was determined for
NCG in the feed, milk, serum, meat, liver, and kidney samples. The results indicated that the LOD
values were within the range of 0.0035 to 0.022 mg kg−1 and 0.014 to 0.023 μg mL−1 for the solid and
liquid samples, respectively (Tables 1 and 2). The LOQ, defined as the analyte concentration at 10 times
the S/N, were in the range of 0.012 to 0.073 mg kg−1 and 0.047 to 0.077 μg mL−1 for the solid and liquid
samples, respectively (Tables 1 and 2). Here, the compound feed, concentrated feed, and premixed
feed are all called the feed. Since the three matrices are similar, the LOD and LOQ values for the three
feeds were just slightly different. For convenient use, we have integrated the three similar results into
one LOD and one LOQ.
Table 1. Percentage recoveries and relative standard deviations (RSDs) of NCG in different feed
matrices (n = 3).
Matrices
Compound feed
Concentrated feed
Premix
a
Added
Concentration (%)
Recoveries (%)
LOD (mg kg−1 )
LOQ (mg kg−1 )
0.01
0.05
0.1
0.05
0.2
1.0
0.5
2.0
10
96.85 (1.3) a
98.89 (2.9)
99.24 (5.0)
107.0 (3.5)
106.1 (7.1)
97.60 (8.8)
94.27 (6.8)
91.90 (3.9)
100.2 (3.6)
0.022
0.073
Figures in bracket represented the relative standard deviation (%).
55
Molecules 2019, 24, 3172
Table 2. Percentage recoveries and relative standard deviations (RSDs) of NCG in different animal
samples (n = 3).
Matrices
Serum (μg mL−1 )
Milk (μg mL−1 )
Meat (mg kg−1 )
Kidney (mg
kg−1 )
Liver (mg kg−1 )
a
Added
Concentration
Recoveries (%)
0.1
1.0
10
0.1
1.0
10
0.05
0.1
0.05
0.1
0.05
0.1
94.25(3.8) a
110.2 (2.3)
96.34 (5.1)
88.12 (1.8)
102.5 (3.2)
89.58 (4.7)
93.84 (7.6)
101.6 (3.4)
89.14 (3.1)
93.65 (5.6)
94.54 (7.4)
97.36 (2.9)
LOD (μg mL−1 )
LOQ (μg mL−1 )
0.023
0.077
0.014
0.047
0.0038
0.013
0.0069
0.023
0.0035
0.012
Figures in bracket represented the relative standard deviation (%).
The method precision and accuracy were evaluated by the recoveries of NCG in the spiked samples.
Recoveries were determined for the feeds, milk, serum, meat, procine liver, and kidney, three replicates
were used for each sample at different concentrations. The MRM ion chromatograms for a blank
(free of NCG) feed sample and the compound feed sample spiked with NCG were demonstrated in
Figure 5A,B and Figures S2–S19, respectively. The peak intensity ratio of the three product ions of NCG
can be used to confirm the presence of NCG when compared with the peak intensity ratio obtained
from the standard sample. The most abundant product ion of the mass spectrum was selected for the
quantitative determination and evaluation of the recoveries for NCG. Calibration curves developed
using an external standard were used to quantify NCG in the spiked samples. The spiked levels,
spiked recoveries, and coefficients of variation were evaluated. According to the recommended dose
of NCG in the feeds, NCG was spiked in the compound feed at 0.01%, 0.05%, and 0.1%, the premix
feed at 0.05%, 0.2%, and 1%, and the concentrate feed at 0.5%, 2%, and 10%. Three replicates were
analyzed for each concentration. A good consistency was found between the actual spiked amount of
NCG and the estimated concentrations in three different feed matrices (Table 1). The recoveries were
within the range of 91.90% to 107.0% with the coefficients of variation ranging from 1.3% to 8.8%.
Similarly, the above established method was also applied to the detection of the NCG spiked
animal products. The MRM ion chromatograms for the blank and spiked animal products samples
were shown in Figure 5C–G and Figures S20–S49, respectively. NCG was spiked in meat, kidney,
and liver at 0.05, and 0.1 mg kg −1 . For each sample concentration, three replicates were analyzed
to evaluate the spiked levels, spike recoveries, and coefficients of variation. As presented in Table 2,
reliable results were found for most test samples. The recoveries for NCG ranged from 89.14% to
101.56% and the coefficients of variation were less than 7.6%. The NCG were spiked in milk and
serum at 0.1, 1, and 10 μg mL−1 . The recoveries ranged from 88.12% to 110.21% with the coefficients of
variation ranging from 1.8% to 5.1% (Table 2).
Both the intra-assay and inter-assay reproducibility of this method were evaluated. For instance,
five repeats at an intermediate concentration (0.05%) were used to determine the recoveries of
NCG spiked in the compound feed. According to the analytical results, the recoveries of NCG
were within the range of 98.89% to 102.76% and the coefficients of variation were less than 2.9%
and 3.8% for the intra-assay (within a day) and inter-assay (over a period of five consecutive
days) measurements, respectively. For the animal samples, this method also showed satisfactory
reproducibility. For 1 μg mL−1 of NCG spiked in the serum, the recoveries of NCG were within the
range of 93.25% to 104.68% and the coefficients of variation were less than 4.7% and 5.4% for the
intra-assay and inter-assay measurements, respectively. For 0.05 mg kg−1 of NCG spiked in the liver,
56
Molecules 2019, 24, 3172
the recoveries of NCG were within the range of 91.23% to 106.71% and the coefficients of variation
were less than 3.9% and 6.7% for the intra-assay and inter-assay measurements, respectively.
Figure 5. Quantitative product ion chromatograms of the NCG in different matrices: (A) Blank feed
sample; (B) compound feed; (C) milk; (D) serum; (E) meat; (F) liver; (G) kidney.
2.5. The Analysis of Authentic Samples
The method established in this study was applied to determine the NCG content in the authentic
feed samples added with NCG additive. The results showed that NCG was detected in the compound
feed, concentrated feed, and premix feed samples with the average content of 0.098%, 0.95%, and 9.53%,
respectively. The variation between the two parallel samples was less than 10%, while NCG was not
detected in the ordinary swine feed samples (<0.022 mg kg−1 ). According to the actual addition amount,
the accuracy for the determination of the compound, concentrate, and premix feed was 98.0%, 95.0%,
and 95.3%, respectively. The proposed method was also adopted for the determination of NCG in the
authentic serum samples from pigs and milk sample from cows (each concentration has six replicates).
The results showed that the concentration of NCG in the serum samples increased significantly with
the increase of NCG in the diets (Table 3). Except for the control group, the NCG levels in the serum
of four diets ranged from 0.11 to 1.31 μg mL−1 . Moreover, the multiple relationship between the
serum NCG and dietary NCG was basically consistent. The NCG concentration in milk was about
57
Molecules 2019, 24, 3172
0.0647 μg mL−1 when the cow was fed 20 g of NCG per day. Therefore, the developed method proved
both feasible and reliable for the quantitative determination of NCG in the routine analysis.
Table 3. The results of NCG in the authentic samples (n = 6).
Sample
Diets Added (%)
NCG Concentration
(μg mL−1 )
Intra-Day
Precision CV (%)
Inter-Day
Precision CV (%)
0
0.025
0.05
0.1
0.2
0
20 g/day
ND a
0.112
0.253
0.561
1.31
ND
0.0647
6.8
2.4
7.8
12
11
8.7
5.6
11
5.1
7.4
Serum b
Milk
a
Not detected, b Serum from piglets.
3. Materials and Methods
3.1. Materials and Reagents
The NCG standard (purity >99.5%) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
The methanol and formic acid used were of a HPLC grade and were obtained from Fisher Scientific
International (Hampton, NH, USA) and Dikma Technology (Richmond Hill, ON, Canada), respectively.
Other chemicals used were all of the analytical grade. A Milli-Q system (Millipore Corporation,
Bedford, MA, USA) was applied to provide ultrapure water for the preparation of all aqueous solutions.
The NCG stock solution (1 mg mL−1 ) was prepared by dissolving the NCG standard in ultrapure
water, and preserved at 4 ◦ C. The preparation of the NCG working solutions was done daily by diluting
the stock solution to appropriate concentrations with ultrapure water. The extraction solvent, 0.5% of
formic acid in water/methanol (80/20, v/v), was prepared by mixing 4 mL of formic acid with 796 mL of
Milli-Q water, and then mixing with 200 mL of methanol.
3.2. Instruments and Apparatus
The HPLC- MS/MS was performed on an Agilent 1200 UHPLC system coupled with an Agilent 6460
Triple Quadrupole Mass Spectrometer (Agilent Technologies, Fermont, CA, USA). An Ultrasonic Cleaner
(Kunshan, China) was used to promote the sample dissolution and extraction. A low temperature high
speed centrifuge (Eppendorf, Hamburg, German) was used to centrifuge the samples.
3.3. Sample Preparation
Feed samples free of NCG were provided by the Ministry of Agricultural Feed Industry Centre
(Beijing, China). The liver and kidney as blanks were from a porcine, and milk was from a cow. They
were all purchased from local supermarkets. The serum of the pig as blanks were collected from the
animal research station of China Agricultural University (Hebei, China). Prior to the extraction, the feed
samples were crushed into about 60 mesh by a small mill. The meat, liver, and kidney tissues were
homogenized in a homogenizer for five min. The milk and serum were pre-treated by centrifuging at
14,000 rpm for 10 min at 4 ◦ C, then the supernatant was collected.
3.4. Sample Extraction and Purification
Extraction of the feed samples was carried out by adding 20 mL of the extraction solvent (0.5%
of formic acid in water/methanol, 80/20, v/v) into the feeds (2 g for the compound feed, 0.2 g for the
concentrated feed and premix). Usually, the recommended amounts of NCG in the pig compound
feed, premixed feed, and concentrated feed were about 0.05%, 1% and 10%, respectively. Since the
concentration of NCG in the premix or concentrated feed was too high, in order to avoid too much
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Molecules 2019, 24, 3172
dilution before the HPLC-MS/MS analysis, the sample amount in the premix or concentrated feed was
relatively reduced. After the vortex mixing and a 30 min ultrasonic bath, 10 min of centrifugation at
14,000 rpm at 4 ◦ C was completed and the supernatant was collected. For the fluid samples, 2 mL
of pre-centrifuged milk or serum was pipetted into a Corning® 15 mL centrifuge tube, and 8 mL of
an ice-cold extraction solvent was added. After the vortex mixing, the samples were centrifuged at
14,000 rpm for 10 min at 4 ◦ C and the supernatant collection was done. For the tissue analyses, 2 g
of tissues samples (meat, kidney, or liver) were extracted by adding 20 mL of an ice-cold extraction
solvent. After a thorough vortex mixing, the samples were placed on ice for 30 min and centrifuged for
10 min at 14,000 rpm at 4 ◦ C, then the supernatant collection was done.
Before further purification, the above supernatant was diluted 10 times using water and adjusted
by adding an appropriate amount of 5% of ammonia solution to pH 8–10. The strong anion exchange
cartridge (Agilent SampliQ SAX, 200 mg, 3 mL, Agilent Technologies, Fermont, CA, USA) was firstly
preconditioned with 3 mL of methanol following by 3 mL of water. Then, 3 mL of the diluted
supernatant was loaded and slowly passed through the cartridge with a flow rate of approximately
1 mL min−1 . After rinsing with 3 mL 5% of the ammonia solution and 3 mL of methanol, elution
was carried out with 3 mL 2% of formic acid in methanol. The eluate was then collected and gently
evaporated under a nitrogen stream in a 50 ◦ C water bath. The residue was reconstituted with 1 mL
0.1% of an aqueous formic acid solution and filtered by a 0.1 μm Syringe Filter (Tianjin Fuji Science and
Technology, China). 10 μL of the sample was injected by the autosampler for the HPLC-MS/MS analysis.
3.5. HPLC-MS/MS Conditions
A Waters Atlantis T3 column (4.6 × 250 mm, 5 μm) was applied in the chromatographic separation
of NCG. The column temperature was maintained at 35 ◦ C. The mobile phases consisted of solution
A (0.1% of an aqueous formic acid solution) and B (methanol), and were delivered at a flow rate of
0.8 mL min−1 . The total run time was set at 20 min. A gradient elution program was developed
as follows: 90% of solution A (initial), with 90–15% of solution A (from 6 to 6.1 min), 15–90% of
solution A (from 12 to 12.1 min). Before the next injection, a 7.9 min equilibration was necessary.
A calibration curve calculated from the NCG standard solution was used to quantify the NCG content
in the spiked samples.
The electron spray ionization source was operated under an optimized condition: Capillary voltage
3500 V, nebulizing gas temperature 350 ◦ C, nebulizing gas flow 5 L min−1 , sheath gas temperature
350 ◦ C, and sheath gas flow 7 L min−1 . Positive ions were monitored. The multiple reaction monitor
(MRM) mode was applied for the quantitation analysis. Data were collected and processed on the
Agilent MassHunter Workstation software (Version B.04.00).
3.6. The Method Validation
The method validation was performed with the feeds, serum, meat, liver, and kidney samples.
Extracts of the NCG-free samples were used for the preparation of matrix-matched calibration standards.
The artificially prepared samples were spiked with the NCG standard solution of 0.01–10% in the
feeds, 0.1–10 μg mL−1 in fluids, or 0.05–0.1mg kg−1 in tissues. The validity of the method including the
stability, matrix effect, linearity, sensitivity, as well as the precision and accuracy of the method were
evaluated in three replicates.
3.7. The Analysis of Authentic Samples
One hundred kg of the commercially prepared compound feed, concentrated feed and premix
were added with the NCG feed additive product (NCG content ≥ 97%) obtained from Ya Tai XingMu
company (Beijing, China) to the concentrations of 0.1% for the compound feed, 1% for the concentrated
feed and 10% for the premix, respectively.
The animals breeding experiments were carried out at the animal research station of China
Agricultural University (Hebei, China). All piglets used in this study were housed and handled
59
Molecules 2019, 24, 3172
according to the established guidelines of China Agricultural University, which were approved by the
China Agricultural University Animal Care and Use Committee. Thirty weaned piglets (each 30–40 kg
in weight) were randomly assigned to one of the five groups (n = 6 piglets per group). Five pig diets
were prepared with NCG added 0%, 0.025%, 0.05%, 0.1%, and 0.2%, respectively. After 26 fed days,
blood samples from the precaval veins were collected after fasting 12 h and centrifuged (3000 rpm) for
10 min. Then, the supernatant was transferred into new tubes and stored at −20 ◦ C until use. Milk
samples were collected from the cow before and after feeding 20 g of NCG per day.
Thirty weaned piglets (each 30–40 kg in weight) were randomly assigned to one of the five groups
(n = 6 piglets per group).
4. Conclusions
A fast, sensitive and reliable HPLC-MS/MS method was successfully developed and optimized
for the qualitative and quantitative determination of NCG in the feeds, animal tissues, and body
fluids. Based on the high selectivity and satisfactory separation result of the HPLC-MS/MS technique,
this method can achieve confirmative identification and accurate determination of NCG in the complex
matrices including feeds and animal products. Furthermore, the applicability of the established method
has been validated using the real feed, serum and milk samples.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/17/3172/s1.
Author Contributions: Y.M. and Z.Z. designed and performed the experiments; L.K. and Y.C. analyzed the data;
P.H. and Y.M. wrote the paper.
Funding: The financial support from The National Key Research and Development Program of China (2018YFD0500402)
is gratefully acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
© 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/).
61
molecules
Article
Sequence Identification of Bioactive Peptides from
Amaranth Seed Proteins
(Amaranthus hypochondriacus spp.)
Alexis Ayala-Niño 1 , Gabriela Mariana Rodríguez-Serrano 2 ,
Luis Guillermo González-Olivares 1, *, Elizabeth Contreras-López 1 , Patricia Regal-López 3 and
Alberto Cepeda-Saez 3
1
2
3
*
Chemistry Investigation Center, Universidad Autónoma del Estado de Hidalgo,
Carretera Pachuca-Tulancingo km 4.5, Mineral de la Reforma Hidalgo C.P. 46067, Mexico
Biotechnology Department, Universidad Autónoma Metropolitana, Unidad Iztapalapa,
Mexico City C.P. 55355, Mexico
Universidad de Santiago de Compostela, Campus Lugo, 15705 Santiago de Compostela,
27002 A Coruña, Spain
Correspondence: lgonzalez@uaeh.edu.mx; Tel.: +52-771-717-2000
Received: 1 June 2019; Accepted: 3 August 2019; Published: 21 August 2019
Abstract: Amaranthus hypochondriacus spp. is a commonly grown cereal in Latin America, known for
its high protein content. The objective of this study was to separate and identify bioactive peptides
found in amaranth seeds through enzymatically-assisted hydrolysis using alcalase and flavourzyme.
Hydrolysis was carried out for each enzyme separately and compared to two-step continuous process
where both enzymes were combined. The biological activity of the resulting three hydrolysates was
analyzed, finding, in general, higher bioactive potential of the hydrolysate obtained in a continuous
process (combined enzymes). Its fractions were separated by RP-HPLC, and their bioactivity was
analyzed. In particular, two fractions showed the highest biological activity as ACE inhibitors with
IC50 at 0.158 and 0.134, thrombin inhibitors with IC50 of 167 and 155, and antioxidants in ABTS assay
with SC50 at 1.375 and 0.992 mg/L, respectively. Further sequence analysis of the bioactive peptides
was carried out using MALDI-TOF, which identified amino acid chains that have not been reported as
bioactive so far. Bibliographic survey allowed identification of similarities between peptides reported
in amaranth and other proteins. In conclusion, amaranth proteins are a potential source of peptides
with multifunctional activity.
Keywords: amaranth protein; flavourzyme; alcalase; bioactive peptides; hydrolysates
1. Introduction
Many diseases that prevail nowadays could be tackled more efficiently, and even prevented,
by combining a healthy diet with functional foods intake [1]. Among functional food components,
we can find bioactive peptides, which are generally short sequences of amino acids encrypted in food
proteins [2]. Bioactive peptides are released from proteins as a result of microorganisms’ metabolic
activity during fermentation, by proteolytic enzymes, or finally by the action of gastrointestinal
enzymes once proteins are ingested. Application of commercially available enzymes has become
a simple and inexpensive way to access free amino acids and peptides from proteins [3].
Consequently, a vast number of studies using various proteinases target the preparation of
hydrolysates from different protein sources, such as cereals and pseudocereals, in an ongoing effort to
obtain highly active biopeptides [4–7]. One of the most important highly-consumed Mesoamerican
original seeds, with a high content of proteins and an excellent amino acid balance, is amaranth.
Molecules 2019, 24, 3033; doi:10.3390/molecules24173033
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Molecules 2019, 24, 3033
In Mexico, amaranth is consumed in a fresh form, offered by traditional confectionary and in
typical dishes of Mexican cuisine [8]. Bioactive peptides found in amaranth exhibit various biological
activities—such as anticholesterolemic, antihypertensive, antioxidant, and antithrombotic—and they
are released mainly by in vitro digestion [9–12]. However, multiple bioactivities shown by the same
peptide fraction of amaranth have never been studied.
In order to find peptide sequences with different biological activities, our research work focused
on the separation and identification of the peptide sequences from amaranth proteins released during
hydrolysis with two commercial enzymes, alcalase and flavourzyme, in separate and continuous
hydrolysis processes. The multiple biological activities exhibited by peptides released from amaranth
proteins through enzymatically-assisted hydrolysis have not been reported until now.
2. Results
2.1. Free Amine Groups Analysis during Enzymatic Hydrolysis
Once the hydrolitic enzymes were added to the protein sample, the progress of the reaction was
monitored every 20 min by the measurement of free amine groups concentration released during
the reaction course (Figure 1). It was observed that after 120 min of hydrolysis with alcalase (H1),
the concentration of free amine groups equaled to 6170.53 ± 29.5 mg/L NH:− , whereas flavourzyme
hydrolysis (H2) reached the highest concentration of free amine groups (5551.11 ± 33.83 mg/L
NH:− ) after 90 min of reaction. It was concluded that, in terms of enzymatic efficiency, alcasase
overdoes flavourzyme in the release of peptide fractions. Following the analysis of H1 and H2,
the combined two-step hydrolysis (H3) was carried out, and a final free amine groups concentration
of 7468.89 ± 34.79 mg/L NH:− was obtained after 40 min of reaction. This result proves that the
continuous hydrolitic process (H3) was more efficient than using alcalase and flavourzyme separately.
Figure 1. Free amine groups concentration during amaranth protein hydrolysis. * Different letters
indicate statistically significant differences between treatments.
2.2. Hydrolysates Bioactivity Analysis
Table 1 shows the assessment of the different biological activities exhibited by the hydrolysates
H1, H2, and H3, determined during amaranth proteins hydrolysis. For thrombin inhibition essay,
H1 hydrolysate showed the highest inhibitory potential (90%), followed by H2 hydrolysate which
inhibited thrombin activity by approximately 80%. No significant difference was observed for H3 and H1
hydrolysates as thrombin inhibitors. When angiotensin-converting enzyme inhibition was evaluated,
it was observed that H3 hydrolysate showed the highest inhibitory potential (58%), in comparison
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Molecules 2019, 24, 3033
to H1 and H2 hydrolysates (49% and 39%, respectively). Finally, antioxidant activity was measured
by three different methods based on the free radical scavenging principle, such as ABTS, DPPH,
and FRAP. The highest antioxidant activity was observed by the H3 sample in DPPH and FRAP assays
(388.94 μmol TE/100 g and 592.54 μmol Fe2 E/100 g, respectively), while H1 hydrolysate exhibited the
highest antioxidant activity measured by the ABTS method (425.86 ± 0.66 mg TE/100 g). Noteworthy,
the antioxidant potential of H3 hydrolysate increased fivefold in the DPPH assay and ninefold in the
FRAP assay, compared to the control sample of unhydrolyzed amaranth proteins.
Table 1. Bioactivity potential of amaranth protein hydrolysates after treatment with alcalase (H1),
flavourzyme (H2), and in two-step combined hydrolysis (H3).
Hydrolysis
ACE
Inhibition (%)
Thrombin
Inhibition (%)
Amaranth Protein
H1
H2
H3
10.58 ± 1.19 d
49.49 ± 1.47 b
39.77 ± 2.15 c
58.53 ± 2.58 a
11.90 ± 10.10 c
92.85 ± 3.36 a
80.95 ± 13.46 b
92.85 ± 3.36 a
Antioxidant Activity
DPPH (μmol
Trolox E/100 g)
ABTS (mg
Trolox E/100 g)
FRAP (μmol
Fe2 E/100 g)
76.66 ± 1.60 d
340.17 ± 10.95 b
274.03 ± 10.84 c
388.94 ± 2.73 a
115.65 ± 10.30 d
425.86 ± 0.66 a
398.36 ± 3.62 c
404.90 ± 1.52 b
63.37 ± 5.72 c
241.70 ± 9.38 b
226.29 ± 11.20 b
592.54 ± 29.29 a
Values are shown as mean ± standard deviation (n = 3); values in the same column with different superscript letters
are significantly different (p < 0.05).
2.3. RP-HPLC Separation and Fraction Bioactivity
Since the H3 hydrolysate (obtained by application of continuous enzymatic hydrolysis) showed
the highest bioactivity, it was chosen for further analysis in order to separate fractions and identify
peptide sequences. In total, 56 fractions were obtained through RP-HPLC separation (see Appendix A),
of which only 14 could be identified with known proteins. Moreover, protein content was determined
in samples collected after 27 min (Table 2). In the case of the 14 fractions identified with protein,
their bioactivity was evaluated by measurement of the IC50 in the ACE and thrombin inhibition assays,
and SC50 in the ABTS assay. Results are gathered in Table 2.
Table 2. Bioactivities of amaranth protein fractions (Angiotensin I-Converting Enzyme and Thrombin
inhibitory activity (IC50 , mg/L)) and antioxidant activity (ABTS radical scavenging; SC50 , mg/L).
Fraction
2
3
9
18
19
22
23
27
28
32
34
39
40
45
ACE (IC50 )
cd
0.332
0.442 e
NI
0.614 f
0.173 b
0.158 ab
NI
0.808 c
0.346 d
0.192 b
0.317 cd
NI
0.298 c
0.134 a
Thrombin (IC50 )
ABTS (SC50 )
e
i
4.204
NI
NI
2.538 d
NI
1.375 b
2.809 d
1.616 c
1.728 c
6.931 g
2.593 d
5.561 f
4.547 e
0.992 a
38.46
4.36 h
0.426 f
2.65 g
0.183 b
0.167 ab
0.349 e
0.402 f
0.135 a
0.298 d
0.247 c
0.247 c
0.26 cd
0.155 a
Peptide Concentration (mg/L)
0.2125
0.8375
0.9062
0.7125
0.3750
0.4687
0.5625
0.3125
0.0937
0.4687
0.4937
0.4375
0.8375
0.8125
Values for ACE, thrombin, and ABTS (mg peptide/mL) are mean ± SD (n = 3); values in the same column with
different superscript letters are significantly different (p < 0.05). NI: not identified.
For the antihypertensive potential, the IC50 calculated for the test fractions ranged from 0.134 to
0.808 mg/mL (Table 2). These values stay in agreement with the results reported elsewhere for amaranth
protein hydrolysates [10,13]. On the other hand, values obtained in this work were lower than those
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found for other vegetable protein sources [14], or higher in comparison to buffalo milk [15]. Antioxidant
potential based on the ABTS assay and expressed as SC50 varied from 0.992 to 6.931 mg/mL, and was
slightly higher than that obtained for amaranth hydrolysates by in vitro digestion [16]. This may be
explained by the fact that the type of bioactive peptides released depends not only on the protein nature,
but also on the enzymes used in the particular case of hydrolysis [17,18]. Concerning the thrombin
IC50 values, the results obtained were in the range of 0.992 to 38.46 mg/L, which stay in agreement
with the values reported for amaranth hydrolyzed by alcalase and pepsin [12]. Finally, no statistically
important differences were found for thrombin inhibition potential between H3 hydrolysate (alcalase
and flavourzyme) investigated in this study, and amaranth hydrolyzed by alcalase and pepsin as
reported in Sabbione et al. [12], which was also obtained via sequential two-enzyme process.
Based on the obtained results, fractions 22 and 45 were subjected to MALDI-TOF analysis (Figure 2),
which allowed the identification of sequences with interesting multiple bioactivities.
Figure 2. Fraction 22 and 45 MALDI-TOF mass Spectra.
The structure of the aminoacidic chains and the corresponding protein source are presented in
Table 3.
Table 3. Peptides identified in the most active fractions from amaranth hydrolysate and their
corresponding protein source.
Fraction
Mass m/z
Calc MH+
Sequence
34
3
16
1375.6435
573.2252
874.4448
1375.6341
573.3516
874.4813
ITASANEPDENKS
LVRW
NIDMLRL
Protein
Fraction 22
66
Agglutinin
Agglutinin
Granule bound starch synthase I
Molecules 2019, 24, 3033
Table 3. Cont.
Fraction
Mass m/z
Calc MH+
Sequence
Protein
Fraction 22
12
5
3
13
794.3805
686.3414
573.2251
812.3607
794.4203
686.4081
573.3617
812.4265
27
14
14
4
29
26
24
19
13
6
3
2
1
1
853.4330
707.3505
707.3502
547.1921
861.3847
845.4098
841.4242
823.4281
686.3805
619.2805
531.1927
505.1907
503.1730
503.1731
853.4329
707.3212
707.3185
547.2997
861.4299
845.4254
841.3841
823.3696
686.4080
619.3566
531.3042
505.2447
503.2715
503.2823
RPVFEF
DPKLTL
IKEAL
NVEVHKS
Granule bound starch synthase I
Granule bound starch synthase I
Granule bound starch synthase I
Cystatin
Fraction 45
HVQLGHY
SQIDTGS
NWACTL
VRWS
CIHNIVY
EGTESIPL
PRYDQY
MSNIDML
DPKLTL
IPSRF
ARVW
CQAAL
EELL
LGVAGS
Agglutinin
Agglutinin
Agglutinin
Agglutinin
Granule bound starch synthase I
Granule bound starch synthase I
Granule bound starch synthase I
Granule bound starch synthase I
Granule bound starch synthase I
Granule bound starch synthase I
Granule bound starch synthase I
Granule bound starch synthase I
Granule bound starch synthase I
Granule bound starch synthase I
3. Discussion
Results obtained for the individual enzymatic hydrolysis were similar to those reported by
Zhuang et al. [19] and Ma et al. [20], where it was observed that the enzymatic activity of alcalase and
flavourzyme decreased after 90–120 min of the reaction course. The duration of enzymatic activity
depends on the nature of the protein matrix and the amount of enzyme and substrate present in
the medium. Additionally, other studies indicated a higher hydrolytic degree when only alcalase
was used [21]. For H3 results, they are similar to those reported by Cumby et al. [22], who found
that combined enzymatic hydrolysis increased peptide concentration when compared to hydrolysis
performed solely with alcalase. It was also stated that hydrolysis with flavourzyme did not contribute
to reach higher concentrations of free amine groups when alcalase was used in the first step of the
combined enzymatic hydrolysis. On the other hand, the degree of hydrolysis depends on nature of
protein and the specificity of the enzymes used [17,18].
Alcalase and flavourzyme have been used individually and in continuous hydrolytic processes
applied to fragmentation of different food proteins in search of platelet inhibitory peptides [23,24].
In the case of amaranth proteins, the addition of a second enzyme different to flavourzyme, following
hydrolysis with alcalase, has proven to enhance the inhibition potential of thrombin. This finding
implies that the higher degree of hydrolysis, the greater bioactivity achieved [12,25]. In addition,
Sabbione et al. [12] observed that the degree of hydrolysis of amaranth proteins, such as albumin
and globulin, was an important factor in improved thrombin inhibition, reaching approximately 81%,
which matches with the results obtained in present study (80% for H2, and 90% for H1 and H3).
The increase in thrombin inhibition activity may be due to the fact that amaranth protein has peptide
sequences capable of inhibiting fibrinogen. These peptides are released by the action of proteolytic
enzymes. Furthermore, it was observed that not only was the size of the peptide important for thrombin
inhibition, but also the amino acid sequence, which might be homologous to the fibrinopeptides A and
B from human fibrinogen (GGGVR-GP and PPSAR-GH, respectively) [26]. Therefore, antithrombotic
activity is affected by the competition for platelet receptors between casoplatelin and the γ-chain
of human fibrinogen (HHLGGAKQAGDV) [27,28], which is implicated in one of the three steps
in thrombosis cascade reactions. Additionally, alcalase has been reported as an enzyme capable of
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releasing antithrombotic peptides form proteins like peanut [18], but no reports have been available so
far on hydrolytic potential of flavourzyme to release bioactive peptides from amaranth.
In the case of antihypertensive activity, the results obtained in this study are similar to those
reported by Ambigaipalan et al. [29]. They observed that the combination of two or more enzymes
increased the inhibitory activity of angiotensin, converting the enzyme in date seed protein in
a sequential enzymatic process. Many authors have reported that the variation in antihypertensive
activity of the released peptides could be attributed to the differences in composition and hydrophobicity
of the protein primary structure [30,31]. In this sense, hydrophobic residues of amino acids (leucine,
valine, alanine, tryptophan, tyrosine, proline, and phenylalanine) bind at the ACE catalytic sites,
acting as competitive inhibitors [32]. Nevertheless, milk proteins have been reported as a better
source of bioactive peptides, especially those with antihypertensive activity reaching over 80% of
ACE inhibition [33,34]. Furthermore, studies have been focused on peptides released from vegetable
proteins, such as amaranth. In particular, 11S globulin was reported to show IC50 value in the ACE
assay, ranging from 6.32 mM to 175 μM [9,35]. It has been known that the low molecular weight
of peptides is a prerequisite for their antihypertensive activity [36]. In this study, peptides with
the latter feature were identified using SDS-PAGE (data not shown), revealing typical weights of
these biopeptides.
In addition to antihypertensive activity, peptide size (peptide chain length less than 20 amino
acids) has been related to antioxidant capacity, showing that smaller peptides have greater
potential [37]. This observation could explain the higher antioxidant activity obtained for H3 hydrolysate,
which contained a higher concentration of free amine groups. In conclusion, during the course of
enzymatic hydrolysis H1, H2, and H3 carried out in this study, peptides with different biological
properties were released—for example, those able to chelate reactive agents, or donate electrons or
hydrogen [38]. Moreover, in several studies in which sequential hydrolysis of proteins with different
enzymes was performed, it has been observed that the antioxidant activity remained the same or
might be increased by the addition of a second enzyme [39,40]. Thus, the application of two hydrolytic
enzymes allows the release of new bioactive peptides, which could affect the antioxidant capacity of
the hydrolysates. Consequently, it has been observed that protein hydrolysates might present higher
antioxidant properties as they increase their content of small peptides [41].
For RP-HPLC peptide separation, the results obtained in this work are similar to those reported by
Moronta et al. [42], who hydrolyzed amaranth proteins with alcalase. It was found that peptides with
a higher content of polar amino acids showed higher anti-inflammatory activity. Nonetheless, a fraction
found in the non-polar region of the chromatographic analysis (45 min) exhibited higher potential in
terms of antihypertensive, antithrombotic, and antioxidant activity than other polar fractions. In some
studies, it has been demonstrated that peptides containing hydrophobic amino acids might enhance
biological capacities, such as antihypertensive and antioxidant activities [43].
As observed in Table, fraction 22 contained the longest peptide sequence, identified as
ITASANEPDENKS, with a molecular weight of approximately 1.44 kDa, being the highest molecular
weight found in both fractions. In the case of the peptide NIDMLRL, the last part of its sequence (-LRL)
has been identified in silico as an inhibitor of ACE in amaranth. This asseveration may be explained by
the presence of leucine as hydrophobic amino acid interacting with the active site of ACE [44].
Additionally, the LVRW sequence was found in fraction 22. The three amino acid chain LVR was
previously described as a bioactive peptide with antihypertensive activity in fig sap having an IC50
lower than 20 μM [45]. Additionally, W might be also a bonding amino acid in the active site of ECA.
On the other hand, the IC50 of ACE inhibitory potential found for fraction 45 was lower than for fraction
22. Fraction 45 contained VRWS, and the dipeptide VR was described as an antihypertensive agent
by itself, with an IC50 of 52.80 μM [46]. In addition, even though it was bound to other amino acids,
such as Y or SP, it retained its bioactivity [47,48]. Similarly, the presence of Tyr in the C-terminal end
should promote binding to ACE and thus enhance its inhibition [49]. It was proved for the tripeptide
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Molecules 2019, 24, 3033
IVY [50], also present in the sequence CIHNIVY in fraction 45. Finally, in this fraction, various peptides
containing Tyr residue were found.
In previous works on antioxidant peptides, it was stated that peptides with a length between 5 and
16 amino acids showed antiradical activity [51]. The results obtained in the present study support
these findings, since the potential of antioxidant peptides had a molecular weight in the range of
500–1400 Da and contained 4–13 amino acid residues (Table 3).
One of the peptides described in fraction 22 was LVRW, which could play a role as an antioxidant
agent. Haung et al. [52] determined that the presence of the RW amino acid sequence in the C-terminal
end of the polypeptide chain might be accounted for by its high antiradical activity. A similar effect
was observed for the peptide ARVW, were the antioxidant activity of Trp is mainly due to its indole
group [53].
In fraction 45, the DPKLTL sequence was identified, which might have antioxidant capacity owning
to the presence of the DPK fragment (previously described as an antioxidant peptide). Its bioactivity
could be explained by the presence of aspartic acid, which has the ability to donate electrons and
hydrogen. On the other hand, this fragment is bound to hydrophobic amino acids, namely Pro and
Leu, which in turn could enhance the radical scavenging abilities of this peptide [54]. Additionally,
the DPKLTL peptide might exhibit antithrombotic activity due to the presence of three amino DPK
acid residues, previously known for inactivating thrombin in its active site [25].
According to the work of Wang et al. [55], peptides with antithrombotic bioactivity usually contain
3–20 amino acid residues. Table 3 shows that the longest peptide identified in fraction 22 had a 13-amino
acid chain. Moreover, it has been reported that sequences containing Val and Tyr might possess
antithrombotic activity [23]. These amino acids were mainly found among peptide sequences present
in fraction 45. On the other hand, Pro-Arg bonding N-terminal end of polypeptide chains has been
reported as a thrombin inhibitor in its active site [26]. This bond is present in the structure of PRYDQY;
however, more exhaustive studies are necessary on antithrombotic peptides as thrombin contain three
main structural domains (a catalytic site and two exosites (I and II)), and enzyme inhibition can take
place to different extents at any of these sites [26,56].
4. Materials and Methods
4.1. Sample and Treatments
Raw Amaranth hypochondriacus spp. seeds were obtained from Xochimilco, Mexico City, in July 2016.
Seeds were ground in a Chopin mill, segregated according to their molecules size, and only a fraction
with molecule size between 200 and 800 μm was selected for the experiments. Protein extraction was
performed following the methodology described by Martínez and Añón [57] with some modifications.
First, flour was defatted with n-hexane (10% w/v) for 24 h. Then it was suspended in deionized water
10% (w/v), and pH 9 was adjusted with NaOH. The crude was incubated for 30 min at room temperature
and centrifuged during 20 min at 10,000 rpm. The soluble protein fraction found in the supernatant
was precipitated by pH 5 adjustment, using HCl. The sample was centrifuged 20 min at 10,000 rpm,
and the pellet pH was adjusted to 7. Following the lyophilization, the protein extract was stored at
4 ◦ C until further use. This protein was called protein extract.
4.2. Enzymatic Hydrolysis
The enzymatic hydrolysis was performed according to the method described by Tironi and
Añón [58]. Briefly, 5 g of the protein extract was diluted in 100 mL of deionized water (milli Q
18.2 MΩ*cm, Manufacturer, Darmstadt, Germany). For alcalase hydrolysis (H1), prior to the reaction,
the pH of the solution was adjusted to 10, and alcalase (≥2.4 U/g, Anson Units; Sigma-Aldrich (St. Loui,
MO, USA) was added at concentration of 8 μL/100 mg of the sample. For flavourzyme hydrolysis (H2),
the pH of the reaction media was adjusted to 7 and flavourzyme (≥500 U/g; Sigma Aldrich, St. Louis,
MO, USA) was added at concentration of 5 μL/100 mg of the sample. Finally, when both enzymes were
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Molecules 2019, 24, 3033
used in a two-step continuous hydrolytic process (H3), after 2 h of hydrolysis with alcalase, the enzyme
was inactivated by heating to 85 ◦ C for 10 min. The solution was adjusted to pH 7 and hydrolysis
with flavourzyme was carried out. For all three methods, hydrolysis was followed up during 4 h and
progress of the reaction was monitored every 20 min. The aliquots were heated to 85 ◦ C during 10 min
and frozen until further use.
4.3. Free Amine Groups Analysis by TNBS Test
In order to determine free amine groups released during the enzymatic hydrolysis of the amaranth
proteins, the 2,4,6-Trinitrobenzenesulfonic acid (TNBS) test was performed, with some modifications,
according to the protocol described by Sashidhar et al. [59]. Briefly, 5% TNBS solution (Sigma-Aldrich,
(St. Loui, MO, USA) was diluted in 0.21 M phosphate buffer (pH 8.2) to a final concentration of 1%
(v/v). Two milliliters of the prepared substrate was added to 2 mL of phosphate buffer (0.21 M; pH 8.2),
and 0.25 mL of the test sample. The mixture was incubated during 1 h at 50 ◦ C and the reaction was
stopped by the addition of 2 mL of 0.1 N HCl. The absorbance was read at 340 nm. Results were
plotted against a calibration curve prepared by different glycine concentrations (0, 0.05. 0.1, 0.15, 0.2,
and 0.25 mg/mL) using the following equation (R = 0.9972):
y = 0.004x + 0.134
(1)
Results are expressed as milligrams of free amines per liter (mg/L NH:− ).
4.4. Antihypertensive Activity
Inhibitory effect on angiotensin converting enzyme (ACE) was evaluated spectrophotometrically
according to the method of Cushman et al. [60] using Hippuril-Histidyl-Leucine (St. Loui, MO, USA)
as substrate. Briefly, 5 mM substrate solution was prepared in 0.1 M sodium borate buffer pH 8.3
containing 0.3 M sodium chloride. A 5 mM 100 μL aliquot of substrate solution was mixed with
40 μL of the test sample before adding 10 μL of angiotensin converting enzyme (EC 3.4.15.1, 5.1 U/mg;
Sigma-Aldrich). The reaction mixture was incubated during 1 h 15 min at 37 ◦ C and the enzyme
was inactivated with 1 mL of 0.1 M HCl. The hippuric acid formed in this reaction was extracted
with ethyl acetate, concentrated under reduced pressure, and finally re-dissolved in distilled water.
The absorbance was measured at 220 nm in a GENESYS spectrometer.
The inhibitory activity of ACE was calculated using the formula:
% inhibitory activity = (AbsC − AbsM)/(AbsC −AbsB) × 100
(2)
where:
AbsC: Hippuric acid formed during the reaction with ACE without inhibitor.
AbsB: Hippuril-Histidyl-Leucine that did not react and was extracted with ethyl acetate.
AbsM: Hippuric acid formed after the reaction with ACE in the presence of inhibitory substance.
4.5. Antithrombotic Activity
To evaluate antithrombotic activity, the methodology developed by Zhang et al. [61] was followed,
including modifications of sodium chloride concentration proposed by Pérez-Escalante et al. [56].
Absorbance at 405 nm was measured before adding the enzyme to a microplate reader (Power Wave
XS UV-Biotek, software KC Junior, USA) and after 10 min of incubation with the enzyme at 37 ◦ C.
The percentage of inhibition (% inhibition) was calculated following the equation:
% inhibitory activity = [(C − CB) − (S − SB)]/(C − CB)*100
where:
CB (control blank): The initial absorbance of the negative control of inhibition.
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Molecules 2019, 24, 3033
C (control): The absorbance of the negative control after 10 min of incubation with thrombin.
SB (sample blank): The initial absorbance of the sample.
S (sample): The absorbance of the sample after 10 min of incubation with thrombin.
4.6. Antioxidant Activity
Antioxidant activity was evaluated by three different methods.
4.6.1. ABTS test
Radical scavenging capacity was measured using the radical cation 2,2 -azino-bis
3-ethylbenzothiazoline-6-sulphonic acid (ABTS•+), which was produced by mixing 7 mM of ABTS•+
stock solution with 2.45 mM potassium persulfate in the dark at room temperature for 16 h prior to its
usage. The ABTS•+ solution was diluted with deionized water, so that absorbance measured at 754
nm was of 0.70 ± 0.02. An aliquot of 20 μL of test sample was added to 980 μL of the diluted ABTS•+
solution, and after 7 min of incubation at room temperature, the absorbance readings were taken at
754 nm in a microplate reader (Power Wave XS UV-Biotek, soft-ware KC Junior, USA). Antioxidant
capacity was expressed as milligrams Trolox equivalents per liter (mg TE/100 g) [62].
4.6.2. DPPH Test
Radical scavenging activity was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical.
An ethanolic solution (7.4 mg/100 mL) of the stable DPPH• radical was prepared. Then, 100 μL of
the test sample was added to 500 μL of DPPH• solution, and it was left to sit at room temperature
for 1 h. The solution was stirred and centrifuged at 3000 rpm for 10 min. Finally, absorbance of the
supernatant was measured at 520 nm in a microplate reader. Antioxidant activity was expressed as
micromole of Trolox equivalents per liter (TE μmol/100 g) [63].
4.6.3. FRAP Test
FRAP antioxidant activity was evaluated according to the Benzie and Strein method [64], in which
0.3 M sodium acetate buffer pH 3.6, TPTZ, 20 mM FeCl3, and 5M FeSO4 were elaborated. For the
preparation of FRAP buffer, TPTZ and FeCl3 were mixed at 10:1 ratio (v:v). Briefly, 30 μL of test sample
was mixed with 900 μL of FRAP solution and 90 μL of distilled water, and agitated. The mixture
was incubated during 10 min at 37 ◦ C, after which the absorbance was measured at 593 nm in
a microplate reader. Results were compared with a calibration curve constructed for FeSO4 standards
at concentrations ranging from 0 to 1000 mM. Antioxidant activity was expressed as micromole
equivalents of Fe (II) per 100 g (μmolEFeII/100 g).
4.7. Identification of Bioactive Peptides by RP-HPLC
4.7.1. Sample Preparation
For sample preparation, 10 mg/mL of freeze-dried amaranth protein hydrolysates were prepared
with phosphate buffer (pH 7.8). The crude mixture was stirred during 1 h at 37 ◦ C and followed
by centrifugation at 10,000 rpm for 10 min at room temperature. The resulting supernatant with
corresponding soluble fractions was separated and stored.
4.7.2. RP-HPLC Separation
Peptides were separated by reversed-phase chromatography on a HPLC (Waters, USA) system
equipped with a C8 column (250 mm × 4.6 mm × 5 mm; Waters) and photodiode array detector
(Spectra System Thermo Scientific, USA) A gradient elution was applied from 100 to 0% A in 56 min
at flow rate of 1 mL/min. A binary mobile phase consisted of solvent A: 0.065% trifluoroacetic acid
[TFA] in water/acetonitrile [ACN] 98:2, and solvent B: 0.065% TFA in water/ACN 35:65. Detection was
performed at 280 nm and temperature was set at 40 ◦ C. The injection volume was 200 μL. Fractions
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were manually collected every 1 min in Eppendorf tubes and protein content was evaluated by the
Bradford assay. Finally, samples were lyophilized (Labconco DrySystem/freezone 4.5) and bioactivity
analysis was carried out only on the lyophilized samples containing protein.
4.7.3. MALDI-TOF Spectrometry
The collected fractions with the highest bioactivities were filtered through a Minisart RC4 filter
(0.45 μm) and analyzed by a matrix-assisted laser desorption ionization (MALDI) mass spectrometer,
equipped with a delayed extraction source and a 355 nm pulsed nitrogen laser. A MALDI scoutMTPTM
was run in the linear mode. A 100-times diluted sample was mixed with 1 volume of 20 mg/mL of
sinapinic acid in acetonitrile/water 50:50 (v/v). Finally, 0.5 μL of the mixture was deposited onto the
MALDI target plate. All spectra were the result of signal averaging of 200 shots. The MALDI-TOF/TOF
MS/MS was run in the positive refractor mode. The peptide sequencing was performed by processing
the MS/MS spectra using Auto eXecute software. Mascot (Matrix Science Inc., Boston, MA, US) software
was used to identify and characterize peptide structures.
4.8. Statistical Analysis
All data were obtained in triplicate and expressed as mean ± standard deviation (SD). The data
were analyzed by one-way analysis of variance (ANOVA) tests, and the differences among means
were compared using the Tukey test with significance level set at p < 0.05, using the SPSS® System for
WINTM version 15.0 (IBM®Armonk, New York, NY, USA).
5. Conclusions
Amaranth protein hydrolysate, obtained through enzymatic reaction with alcalase and
flavourzyme in a sequential two-step hydrolytic process, may be a source of bioactive peptides.
Sequences were different from those obtained in more commonly performed enzymatic hydrolysis
using pepsin and pancreatin for amaranth proteins digestion. The biological activities identified for
some peptide sequences in this study have been proven in other food sources. Moreover, novel amino
acid chains with possible multi-functional activities were identified. This is the first report of the
multiple bioactivities of peptide fractions derived from hydrolyzed amaranth proteins. Additionally,
this study shows that amaranth hydrolyzed with alcalase and flavourzyme could be used in the
nutraceutical industry, as a value-added ingredient with multi-functional bioactive properties.
Author Contributions: Conceptualization, L.G.G.-O. and A.A.-N.; methodology, L.G.G.-O., A.A.-N. and G.M.R.-S.;
software, E.C.-L., P.R.-L. and A.C.-S.; validation, P.R.-L., A.C.-S. and L.G.G.-O.; formal analysis, L.G.G.-O.,
A.A.-N. and P.R.-L.; investigation, A.A.-N.; resources, A.C.-S. and L.G.G.-O.; data curation, E.C.-L. and A.C.-S.;
writing—original draft preparation, A.A.-N. and L.G.G.-O.; writing—review and editing, A.A.-N. and L.G.G.-O.;
visualization, L.G.G.-O.; supervision, L.G.G.-O. and G.M.R.-S.; project administration, L.G.G.-O.; funding
acquisition, L.G.G.-O., E.C.-L. and G.M.R.-S.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
72
Molecules 2019, 24, 3033
Appendix A
Figure A1. RP-HPLC separation of amaranth hidrolizated fractions.
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Sample Availability: Samples of the compounds are not available from the authors.
© 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/).
76
molecules
Article
Green Extraction of Phenolic Acids from Artemisia
argyi Leaves by Tailor-Made Ternary Deep
Eutectic Solvents
Li Duan 1,† , Chenmeng Zhang 1,† , Chenjing Zhang 1 , Zijing Xue 2 , Yuguang Zheng 2, * and
Long Guo 2, *
1
2
*
†
College of Chemistry and Material Science, Hebei Normal University, Shijiazhuang 050024, China
School of Pharmacy, Hebei University of Chinese Medicine, Shijiazhuang 050200, China
Correspondence: zyg314@163.com (Y.Z.); guo_long11@163.com (L.G.); Tel.: +86-0311-89926316 (Y.Z.);
+86-0311-89926017 (L.G.)
These authors contributed equally to this work.
Academic Editor: Jose M. Miranda
Received: 25 June 2019; Accepted: 3 August 2019; Published: 5 August 2019
Abstract: The Artemisia argyi leaf (AL) has been used as a traditional medicine and food supplement
in China and other Asian countries for hundreds of years. Phytochemical studies disclosed that AL
contains various bioactive constituents. Among bioactive constituents, phenolic acids have been
recognized as the main active compounds in AL. To the best of our knowledge, no research has been
focused on extraction method for the bioactive phenolic acids from AL. Nowadays, deep eutectic
solvents (DESs) are emerging as a new type of green and sustainable solvent for efficient extraction
of bioactive compounds from natural products. In the present study, an environmentally friendly
extraction method based on DESs was established to extract bioactive phenolic acids from ALs. Diverse
tailor-made solvents, including binary and ternary DESs, were explored for simultaneous extraction
of four phenolic acids (3-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic
acid, and 4,5-di-O-caffeoylquinic acid) from AL. The results indicated that the ternary DES composed
of a 2:1:2 molar ratio of choline chloride, malic acid, and urea showed enhanced extraction yields
for phenolic acids compared with conventional organic solvents and other DESs. Subsequently, the
extraction parameters for the four phenolic acids by selected tailor-made DESs, including liquid–solid
ratios, water content (%) in the DESs, and extraction time, were optimized using response surface
methodology and the optimal extraction conditions were: extraction time, 23.5 min; liquid–solid
ratio, 57.5 mL/g (mL of DES/g dry weight of plant material); water content, 54%. The research
indicated that DESs were efficient and sustainable green extraction solvents for extraction of bioactive
phenolic acids from natural products. Compared to the conventional organic solvents, the DESs have
a great potential as possible alternatives to those organic solvents in health-related areas such as food
and pharmaceuticals.
Keywords: deep eutectic solvents; Artemisia argyi leaves; phenolic acids; extraction; response
surface methodology
1. Introduction
The Artemisia argyi leaf (AL), which is widely distributed in China and other Asian countries, has
been used as a traditional medicine or food supplement for hundreds of years [1]. As a traditional
Chinese medicine, AL is reported to possess antioxidant, antibacterial, anti-inflammatory, anticancer,
hemostatic, and analgesic activities and is commonly used for treatment of hemorrhage, pain, eczema,
and menstruation-related symptoms [2–4]. AL is also consumed as a food ingredient because of its
Molecules 2019, 24, 2842; doi:10.3390/molecules24152842
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Molecules 2019, 24, 2842
delicious flavor and distinctive smell. In China, AL is used as a common condiment and colorant for
the traditional Chinese food “Qingtuan”. In Japan, AL is added into food as an additive to enhance the
flavor and nutrition [5].
Although the AL has been used as an herbal medicine and food ingredient for a long time, studies
on its bioactive compositions are still limited. Phytochemical studies disclosed that AL contains various
bioactive constituents, mainly including volatile oils, phenolic acids, flavonoids, and terpenoids [6,7].
Among bioactive constituents, phenolic acids have been recognized as the main active compounds in
AL [8,9]. Therefore, the content of the phenolic acids is an important index for quality analysis and
normal applications of AL. To date, several bioactive phenolic acids, such as 3-caffeoylquinic acid,
3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid, have been
isolated from AL. A number of analytical methods, including high-performance liquid chromatography
(HPLC) and high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS),
have been used for qualitative and quantitative analysis of the main phenolic acids in AL [5,7]. However,
the research focused on extraction methods for bioactive phenolic acids from AL is still limited.
Nowadays, conventional organic solvents, such as alcohols, ethyl acetate, acetone, and chloroform,
are widely used in the extraction of bioactive components from natural sources [10]. However, the
consumption of large amounts of these volatile and hazardous organic solvents may contribute to
environmental pollution and leave unacceptable solvent residues in extracts. Therefore, in analytical
chemistry, green extraction methods which are environmentally friendly and sustainable for sample
preparation have received more and more attention [11]. Since being introduced as a new type of green
solvent, deep eutectic solvents (DESs) have rapidly gained great interest as sustainable alternatives
to conventional organic solvents. DESs are prepared by simply mixing two or more naturally
occurring, inexpensive, and biodegradable components together to obtain a eutectic mixture [12]. The
availability, low cost, biodegradability, and environmental friendliness of the components make the
DESs versatile alternatives to conventional organic solvents [13]. Due to their excellent properties,
including biodegradability, low toxicity, solute stabilization, and low cost, DESs have been widely used
in organic synthesis, separation processes, and biomedical applications [14,15].
Generally, DESs are prepared by simply mixing two or more naturally occurring, inexpensive,
and biodegradable components together to obtain eutectic mixtures. As tunable solvents, diverse
possible combinations of starting components have different targeted functionality, which means that
we can increase the solubility and extraction efficiency of DESs for target compounds by selecting
appropriate combinations of starting components. The tailor-made DESs have tremendous potential
for efficient and simultaneous extraction and separation of compounds which have obvious differences
in nature [16]. Recently, many reports have shown that tailor-made DESs were successfully employed
in the extraction and separation of different kinds of bioactive compounds, such as phenolic acids,
flavonoids, alkaloids, and saponins, from various plant materials [17–22]. Nonetheless, most of the
research used tailor-made binary DESs as extraction solvents for bioactive compound extractions, the
number of reports on the application of tailor-made ternary DESs for extraction is still limited, and the
efficiency of DESs for extraction of bioactive phenolic acids from AL still remains unknown.
In the present study, in order to evaluate DESs for the extraction of phenolic acids, several
tailor-made binary and ternary DESs were used for simultaneous extraction of four bioactive
phenolic acids (3-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and
4,5-di-O-caffeoylquinic acid) from AL, and the extraction efficiency of tailor-made DESs was compared
with that of conventional organic solvents. Moreover, the extraction parameters for phenolic acids by
tailor-made DESs were systematically optimized using response surface methodology (RSM).
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Molecules 2019, 24, 2842
2. Results and Discussion
2.1. Chromatographic Conditions and Method Validation
In order to achieve a rapid and efficient analysis of the four phenolic acids (3-caffeoylquinic acid,
3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid) in AL,
different mobile phases (including water–methanol, water–acetonitrile, formic acid water–methanol,
and formic acid water–acetonitrile), flow rates (0.7 mL/min, 0.8 mL/min, and 1.0 mL/min), as well
as column temperatures (15 ◦ C, 20 ◦ C, 25 ◦ C, and 30 ◦ C, Supplementary Materials Figure S1) were
examined and compared. As a result, the formic acid water–acetonitrile system at 15 ◦ C with a flow
rate of 0.7 mL/min was finally selected for the suitable analysis duration, greater separation ability,
and better peak shapes. The gradient elution was as follows: 0–5 min, 12% B; 5–15 min, 12–22% B;
15–25 min, 22% B; 25–35 min, 22–25% B; 35–40 min, 25–40% B. The typical HPLC chromatograms of the
AL sample and four phenolic acids reference standards are shown in Figure 1.
Figure 1. The typical HPLC chromatograms of (A) Artemisia argyi leaves sample (20 mg/mL) and (B) four
phenolic acids reference standards. (1. 3-caffeoylquinic acid, 4.96 μg/mL; 2. 3,4-di-O-caffeoylquinic acid,
4.24 μg/mL; 3. 3,5-di-O-caffeoylquinic acid, 5.36 μg/mL; 4. 4,5-di-O-caffeoylquinic acid, 4.16 μg/mL).
Method validation of quantitative analysis was performed. The linearity, limit of detections
(LODs), limit of quantifications (LOQs), precision, repeatability, stability, and accuracy for the four
phenolic acids were validated. Each calibration curve was performed with six different concentrations
in triplicate. All calibration curves were of good linearity with high correlation coefficient (R2 > 0.9997)
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Molecules 2019, 24, 2842
over the tested range. The LODs and LOQs of the four analytes were defined by the concentration that
generated peaks with signal-to-noise values of 3 and 10 using standard solutions. The precision of
the developed method was determined by the intra- and interday variations. For the intraday test,
the sample was analyzed six times within the same day, while for the interday test, the sample was
examined in duplicates for three consecutive days. The relative standard deviations (RSDs) of intraday
and interday precisions were less than 1.61% and 2.14%, respectively. For the repeatability test, six
replicates of the same sample were prepared and analyzed, and for the stability test, the same sample
was stored at room temperature and analyzed by replicate injection analysis at 0, 2, 4, 8, 12, and 24 h.
The repeatability presented as RSDs was less than 2.71%, and the stability was less than 2.06%. The
recovery was used to evaluate the accuracy of the method. Known amounts of the four phenolic acids
standard solutions were added into the same samples in sextuplicate, and then extracted and analyzed
with the same procedures. The recovery of each analyte was calculated by the equation: Recovery
(%) = (Detected amount − Original amount)/Spiked amount × 100%. The overall recoveries of the
four analytes were in the range of 101.15–102.86% with RSDs less than 1.67%. The data of method
validation are shown in Supplementary Materials Table S1.
2.2. Screening of DESs for the Extraction of Phenolic Acids from AL
2.2.1. Extraction of Phenolic Acids by Binary DESs
DESs are composed of a mixture consisting of hydrogen bond acceptors (HBAs) with hydrogen
bond donors (HBDs). The composition of DESs determines their physicochemical properties and
consequently greatly influences extraction efficiency of natural compounds [23]. In the present study,
six choline-chloride-based binary DESs, ChCl-Ma, ChCl-Ur, ChCl-Ga, ChCl-Pa, ChCl-Eg, and ChCl-Gl,
were successfully synthesized (Table 1) and selected to test their extraction efficiency for phenolic acids.
The high viscosity of most DESs at room temperature restricted their application due to a slow mass
transfer. To overcome this problem, extraction conditions were adjusted to reduce the viscosity by
increasing extraction temperature and adding a certain amount of water [24]. In the initial screening
experiments, 75% DES solution in water (v/v) was employed, and the extraction conditions were as
follows: extraction time, 30 min; extraction temperature, 50 ◦ C; liquid–solid ratio, 50 mL/g (mL of
DES/g dry weight of plant material).
Table 1. The binary DESs synthesized in this study.
NO.
Abbreviation
Component 1
Component 2
Molar Ratio
BD-1
BD-2
BD-3
BD-4
BD-5
BD-6
ChCl-Ma
ChCl-Ur
ChCl-Ga
ChCl-Pa
ChCl-Eg
ChCl-Gl
choline chloride
choline chloride
choline chloride
choline chloride
choline chloride
choline chloride
dl-malic acid
urea
glutaric acid
propanedioic acid
ethylene glycol
glycerol
1:1
1:2
1:1
1:1
1:3
1:2
Extraction yields of the four phenolic acids (3-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid,
3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid) with different binary DESs are shown in
Figure 2. The initial screening results indicated that the extraction efficiency for phenolic acids was
influenced by the types of DES solvents, and different types of DESs resulted in different extraction
yields. In general, extraction yields of the four phenolic acids followed the order 3,5-di-O-caffeoylquinic
acid > 3-caffeoylquinic acid > 4,5-di-O-caffeoylquinic acid > 3,4-di-O-caffeoylquinic acid. The phenolic
acid, 3,5-di-O-caffeoylquinic acid, showed high extraction efficiency in ChCl-Eg with the concentration
9.35 ± 0.03 mg/g (mg/g dry weight of plant material), followed by ChCl-Ma (9.05 ± 0.05 mg/g) and
ChCl-Pa (8.71 ± 0.03 mg/g). For 3-caffeoylquinic acid, ChCl-Ma (4.45 ± 0.03 mg/g) and ChCl-Ur (4.47 ±
0.03 mg/g) led to higher extraction yields. For 4,5-di-O-caffeoylquinic acid, ChCl-Ma (4.10 ± 0.02 mg/g),
ChCl-Pa (3.90 ± 0.03 mg/g), and ChCl-Eg (3.72 ± 0.02 mg/g) exhibited higher extraction efficiency. For
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Molecules 2019, 24, 2842
3,4-di-O-caffeoylquinic acid, ChCl-Ur (3.32 ± 0.03 mg/g) showed higher extraction efficiency, followed
by ChCl-Ma (3.03 ± 0.02 mg/g) and ChCl-Pa (2.97 ± 0.03 mg/g).In conclusion, the extraction yields of
the total phenolic acids were calculated, and it was clearly shown that ChCl-Ma (BD-1) was the best
binary DES for extraction of four phenolic acids from AL with the extraction yields 20.64 ± 0.08 mg/g.
Figure 2. Extraction yields of different binary DESs and different ratios of methanol for 3-caffeoylquinic
acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, and total
four phenolic acids from Artemisia argyi leaves (n = 3). Numbers on horizontal axis are in accordance
with the numbers in Table 1. Error bars indicate the SD (n = 3). Extraction yields which do not share
the same letter are significantly different (p < 0.05).
In order to comprehensively compare the extraction efficiency of DESs and conventional solvents
for extraction of phenolic acids from AL, different ratios of methanol (25% MeOH, 50% MeOH, 75%
MeOH, and 100% MeOH), efficient solvents commonly used in the extraction of bioactive compounds
from natural products, were selected as reference solvents [7]. As shown in Figure 2, among the
different ratios of MeOH, 75% MeOH displayed the highest extraction efficiency for the four phenolic
acids. It was clear that all the six tailor-made binary DESs exhibited higher extraction efficiency for
the four phenolic acids compared with 100% MeOH (16.56 ± 0.06 mg/g). However, compared to 75%
MeOH (22.41 ± 0.03 mg/g), none of the six binary DESs exhibited a higher extraction yield. Based
on the results above, ChCl-Ma (BD-1) was selected as the best binary DES for extraction of phenolic
acids from AL, and we attempted to synthesize a series of ternary DESs based on ChCl-Ma to enhance
extraction efficiency of phenolic acids in further tests.
2.2.2. Extraction of Phenolic Acids by Ternary DESs
DESs can be synthesized from two or more components. According to the previous report, the
ternary DESs forming with addition of glycerol to the binary DESs show lower melting points and
viscosities [25]. Adding the third component to binary DESs may change the properties of ternary DESs
and thus influence the extraction yields. In this study, based on the best binary DES (ChCl-Ma) selected
above, several tailor-made ternary DESs were designed in order to further enhance the extraction
yields of phenolic acids from AL. Five ternary DESs, including ChCl-Ma-Ur, ChCl-Ma-Ga, ChCl-Ma-Pa,
ChCl-Ma-Eg, and ChCl-Ma-Gl, were successfully synthesized (Table 2) and further used to test their
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Molecules 2019, 24, 2842
extraction efficiency for phenolic acids. The extraction yields of the four phenolic acids employing the
tailor-made ternary DESs are listed in Figure 3. The results indicated that compared to the binary DESs,
some tailor-made ternary DESs could change the extraction efficiency. Almost all of the tailor-made
ternary DESs exhibited higher extraction efficiency for the four phenolic acids compared with the best
binary DES (ChCl-Ma), except ChCl-Ma-Gl (TD-12 and TD-13). It could be speculated that the addition
of the third component might reduce the viscosity of DESs, enhancing the hydrogen bond interactions
between DESs and the target components, thus improving the extraction yields [26].
Table 2. The ternary DESs synthesized in this study.
NO.
Abbreviation
Component 1
Component 2
Component 3
Molar Ratio
Extraction Yield of
Four Phenolic Acids
(mg/g)
TD-1
TD-2
TD-3
TD-4
TD-5
TD-6
TD-7
TD-8
TD-9
TD-10
TD-11
TD-12
TD-13
ChCl-Ma-Ur
ChCl-Ma-Ur
ChCl-Ma-Ur
ChCl-Ma-Ga
ChCl-Ma-Ga
ChCl-Ma-Pa
ChCl-Ma-Pa
ChCl-Ma-Pa
ChCl-Ma-Pa
ChCl-Ma-Eg
ChCl-Ma-Eg
ChCl-Ma-Gl
ChCl-Ma-Gl
choline chloride
choline chloride
choline chloride
choline chloride
choline chloride
choline chloride
choline chloride
choline chloride
choline chloride
choline chloride
choline chloride
choline chloride
choline chloride
DL-malic acid
DL-malic acid
DL-malic acid
DL-malic acid
DL-malic acid
DL-malic acid
DL-malic acid
DL-malic acid
DL-malic acid
DL-malic acid
DL-malic acid
DL-malic acid
DL-malic acid
urea
urea
urea
glutaric acid
glutaric acid
propanedioic acid
propanedioic acid
propanedioic acid
propanedioic acid
ethylene glycol
ethylene glycol
glycerol
glycerol
2:1:1
2:2:1
2:1:2
2:1:1
2:2:1
2:2:1
2:1:1
1:1:1
2:1:2
1:2:0.5
2:2:1
1:2:0.5
2:2:1
21.92 ± 0.04
22.26 ± 0.04
22.43 ± 0.02
21.41 ± 0.04
21.89 ± 0.02
20.81 ± 0.06
21.30 ± 0.05
21.55 ± 0.04
22.08 ± 0.03
21.67 ± 0.04
22.12 ± 0.05
20.02 ± 0.02
20.09 ± 0.04
Figure 3. Extraction yields of different ternary DESs for 3-caffeoylquinic acid, 3,4-di-O-caffeoylquinic
acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, and total four phenolic acids from
Artemisia argyi leaves (n = 3). Numbers on horizontal axis are in accordance with the numbers in
Tables 1 and 2. Error bars indicate the SD (n = 3). Extraction yields which do not share the same letter
are significantly different (p < 0.05).
Several studies have revealed that different molar ratios of DES components result in different
viscosities and surface tensions, thereby affecting the extraction efficiency of target components from
natural biomass [27]. Thus, in the study, the effects of molar ratios of tailor-made ternary DESs were
also investigated. As shown in Figure 3, the optimal molar ratio of ChCl-Ma-Ur was 2:1:2 (TD-3)
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Molecules 2019, 24, 2842
and the extraction yields of the four phenolic acids were 22.43 ± 0.02 mg/g. The optimal molar ratio
of ChCl-Ma-Ga was 2:2:1 (TD-5) and the extraction yields of the four phenolic acids were 21.89 ±
0.02 mg/g. The optimal molar ratio of ChCl-Ma-Pa was 2:1:2 (TD-9) and the extraction yields of the
four phenolic acids were 22.08 ± 0.03 mg/g. The optimal molar ratio of ChCl-Ma-Eg was 2:2:1 (TD-11)
and the extraction yields of the four phenolic acids were 22.12 ± 0.05 mg/g. The optimal molar ratio
of ChCl-Ma-Gl was 2:2:1 (TD-13) and the extraction yields of the four phenolic acids were 20.09 ±
0.04 mg/g. None of the tailor-made ternary DESs with different molar ratios exhibited an extraction
yield higher than 75% MeOH, the best conventional solvent. However, the tailor-made ternary DES,
ChCl-Ma-Ur (2:1:2), produced similar extraction efficiency for the four phenolic acids compared to 75%
MeOH (p > 0.05). Based on the results, the ChCl-Ma-Ur with a molar ratio of 2:1:2 (TD-3) was selected
as the best ternary DES for extraction of phenolic acids from AL.
2.3. Optimization of the Extraction Parameters for Phenolic Acids
The above extraction investigations showcased that the tailor-made ternary DES, ChCl-Ma-Ur
(2:1:2), was selected as the best ternary DES for extraction of phenolic acids from AL. In order to obtain
the optimal extraction efficiency for phenolic acids from AL, several numerical variables that could
affect the extraction efficiencies were optimized by RSM, a valuable statistical technique to determine
the optimal values of the independent variables and to enable the user to effectively investigate the
effects of multiple factors [28,29]. Similar to previous studies [30], three variables of extraction time,
liquid–solid ratios, and water content in DESs were evaluated using Box–Behnken design (BBD). After
determining the range of extraction factors on the basis of preliminary single-factor test, the extraction
time (A), liquid–solid ratios (B), and water content (C) were varied at three levels (−1, 0, +1) as follows:
A, 8.0–40.0 min; B, 17.5–57.5 mL/g; C 20–70%. The total extraction amounts of the four phenolic acids
were taken as the responses of the design experiments. The experimental orders, levels of variables,
and response values are summarized in Table 3.
Table 3. The experimental orders, levels of variables, and response values in Box–Behnken design.
Run
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Factors
Responses
Extraction Time
(A, min)
Liquid–Solid
Ratios (B, mL/g)
Water Content (C,
%)
Total Extraction Amounts
of Four Phenolic Acids
(mg/g)
24.0
40.0
24.0
8.0
24.0
40.0
24.0
24.0
8.0
24.0
24.0
24.0
8.0
40.0
24.0
8.0
40.0
37.5
57.5
17.5
37.5
37.5
17.5
57.5
37.5
57.5
17.5
57.5
37.5
17.5
37.5
37.5
37.5
37.5
45
45
20
20
45
45
70
45
45
70
20
45
45
70
45
70
20
22.60
22.61
18.47
16.13
22.10
21.96
20.85
21.40
22.34
18.86
15.51
22.65
21.27
19.84
22.02
20.06
17.00
Experiments conducted according to the design resulted in a second-order polynomial equation
for total extraction amounts (Y) expressed using coded variables (A, B, and C) as follows:
Y = 22.16 − 0.20A + 0.095B + 1.56C − 0.11AB − 0.27AC + 1.24BC − 0.14A2 + 0.027B2 − 3.76C2
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The model was evaluated in terms of the square of correlation coefficient (R2 ) and the lack of fit
by the analysis of variance (ANOVA) at the 95% confidence level (Table 4). The resulting R2 value
was 0.9765, indicating that the experimental data were in relatively good agreement with predicted
extraction yields. The lack-of-fit value, which evaluates the failure of the model to represent the data in
the experimental domain points, was insignificant for the response with p-value of 0.3789 (p > 0.05).
Table 4. The ANOVA results of the quadratic multiple regression model for phenolic acids.
Source
Sum of Squares
df
Mean Square
F Value
p-Value Prob > F
Significance
Model
A
B
C
AB
AC
BC
A2
B2
C2
Residual
Lack of Fit
Pure Error
R2
86.54
0.32
0.073
19.53
0.045
0.3
6.13
0.08
3.10 × 10−3
59.51
2.08
1.04
1.04
0.9765
9
1
1
1
1
1
1
1
1
1
7
3
4
9.62
0.32
0.073
19.53
0.045
0.3
6.13
0.08
3.10 × 10−3
59.51
0.3
0.35
0.26
32.38
1.08
0.24
65.78
0.15
1.02
20.64
0.27
0.01
200.42
<0.0001
0.3323
0.6359
<0.0001
0.7085
0.3468
0.0027
0.6188
0.9215
<0.0001
significant
1.34
0.3789
not significant
Statistical analysis and 3D response plots (Figure 4) illustrated the significant variables affecting
extraction yields of phenolic acids and the interaction effects between the variables. In the model, the
water content (C) showed significant effects on the extraction efficiency of phenolic acids (p < 0.0001).
Based on the adequate model, the calculated optimum conditions for the extraction of phenolic acids
from AL were as follows: extraction time, 23.5 min; liquid–solid ratio, 57.5 mL/g; water content, 54%.
Triplicate experiments were carried out under the optimal extraction conditions and mean values of
experimental results were compared with the predicted values. Under the optimum conditions, the
total extraction amounts of the four phenolic acids were 22.80 mg/g, which were closed to the predicted
values of 22.79 mg/g. The results obtained through confirmation experiments indicated the model is
adequate for predicting the expected optimization.
Figure 4. Response surface plots of the model for extraction of phenolic acids from Artemisia argyi
leaves. (A. extraction time, min; B. liquid–solid ratios, mL/g; C. water content, %).
3. Materials and Methods
3.1. Materials and Reagents
AL samples were purchased from a Chinese herbal medicine market (Anguo, China). The samples
were dried in the shade and stored in the desiccator. The AL samples were authenticated by Prof.
Yuguang Zheng from Department of Pharmacognosy, Hebei University of Chinese Medicine and the
voucher specimens were deposited in Hebei University of Chinese Medicine, Shijiazhuang, China.
Four phenolic acids reference compounds, 3-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid,
3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid, were purchased from Chengdu Must
Bio-technology Co., Ltd. (Chengdu, China). The purities of the four reference compounds were
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Molecules 2019, 24, 2842
determined to be higher than 98% by high-performance liquid chromatography diode array detection
analysis. The structures of the four phenolic acids are shown in Figure 5.
Figure 5.
Chemical structures of 3-caffeoylquinic acid,
3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid.
3,4-di-O-caffeoylquinic acid,
Chemical compounds for DES preparation including choline chloride (ChCl), DL-malic acid
(Ma), urea (Ur), glutaric acid (Ga), propanedioic acid (Pa), ethylene glycol (Eg), glycerol (Gl) were
obtained from Aladdin Reagent Company (Shanghai, China). Acetonitrile, methanol, and formic acid
(chromatographic grade) were purchased from Merck (Darmstadt, Germany). Deionized water was
prepared by a Milli-Q water purification system (Millipore, Billerica, MA, USA). Other reagents and
chemicals used in this work were of analytical grade.
3.2. HPLC Analysis
HPLC analysis was performed on an Agilent 1260 HPLC system equipped with a quaternary
pump, a degasser, an autosampler, a thermostated column compartment, and a diode array detector
(Agilent Technologies, Palo Alto, CA, USA). Chromatographic separation was achieved on an Agilent
SB C18 column (4.6 × 250 mm, 5 μm). The mobile phase was composed of 0.1% formic acid water (A)
and acetonitrile (B) with a gradient elution as follows: 0–5 min, 12% B; 5–15 min, 12–22% B; 15–25 min,
22% B; 25–35 min, 22–25% B; 35–40 min, 25–40% B. The detection wavelength was 330 nm. The flow
rate was set at 0.7 mL/min and the column temperature was set at 15 ◦ C.
3.3. Preparation of DESs
The DESs were synthesized according to the previous studies [31]. Briefly, different DESs were
obtained by simply mixing hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs)
together in a proper molar ratio with constant stirring at 80 ◦ C until a clear and homogeneous
liquid formed.
3.4. Extraction of Phenolic Acids From AL
The AL samples were powdered and screened through 40 mesh sieves. An accurately weighed
powder (20 mg) was extracted with 1 mL of different solvents (different DESs, 25% MeOH, 50% MeOH,
75% MeOH, and 100% MeOH) in a 2 mL centrifuge tube by ultrasonic cleaner (KQ5200B, 200W, 40 kHz,
Kunshan, China) for 30 min. After extraction, the extracted solution was centrifuged at 13,000 rpm/min
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Molecules 2019, 24, 2842
for 10 min. Then, 0.1 mL of supernatant was sampled and diluted 10-fold with methanol. The diluted
solution (2 μL) was injected into the HPLC instrument for analysis. Three replicates of each sample
were prepared and analyzed (n = 3).
3.5. DESs Tailoring
In order to determine the effect of the DES compositions on the extraction of the four phenolic
acids from AL, six ChCl-based binary DESs, including ChCl-Ma, ChCl-Ur, ChCl-Ga, ChCl-Pa, ChCl-Eg,
and ChCl-Gl, were successfully synthesized in initial screening. Then, the extraction efficiency of the
different binary DESs for the four phenolic acids was investigated and the optimal binary DES was
selected. Next, different efficient components were added into the optimal binary DES obtained in
initial screening to design different ternary DESs. Five tailored ternary DESs with different molar ratios,
including ChCl-Ma-Ur, ChCl-Ma-Ga, ChCl-Ma-Pa, ChCl-Ma-Eg, and ChCl-Ma-Gl, were synthesized
and the extraction efficiencies for the four phenolic acid compounds were tested.
3.6. Optimization of the DES Extraction Parameters for Phenolic Acids
The optimal extraction parameters for the phenolic acids from AL were obtained using RSM.
RSM was performed using the Design-Expert Ver. 8.0.6 (Stat-Ease Inc., Minneapolis, MN, USA). After
determining the range of extraction variables on the basis of preliminary single-factor test, Box–Behnken
design (BBD) was used to find the optimal values for three independent variables: extraction time
(A), liquid–solid ratios (B), and water content (%) in DESs (C). The total extraction amounts of the
four phenolic acids were taken as the response of the design experiments. Regression analysis was
performed according to the experimental data. Subsequently, additional confirmation experiments
were conducted to confirm the validity of the statistical experimental strategies.
4. Conclusions
In this work, a green and efficient extraction method using tailor-made DESs as extraction solvents
was established for extraction of bioactive phenolic acids from AL. Six binary DESs and five ternary
DESs were successfully synthesized and used to extract four phenolic acids from AL. The results
indicated that the tailor-made DESs were efficient solvents for the extraction of phenolic acids. The
ternary DES, ChCl-Ma-Ur with a molar ratio of 2:1:2, proved to be the most efficient solvent for phenolic
acid extraction from AL. Moreover, the optimal extraction conditions for phenolic acids by ChCl-Ma-Ur
(2:1:2) were determined using RSM and the optimal extraction conditions are extraction time, 23.5 min;
liquid–solid ratio, 57.5 mL/g; and water content, 54%. The present study suggests that the DES-based
extraction method is efficient and sustainable for extraction of bioactive phenolic acids from AL. The
DESs are truly designed and efficient solvents which could be used as green solvents for the extraction
of bioactive compounds from natural products and have a great potential as possible alternatives to
those organic solvents in health-related areas such as food and pharmaceuticals.
Supplementary Materials: The Supplementary Materials are available online.
Author Contributions: Conceptualization, L.D. and L.G.; Data curation, C.Z. (Chenmeng Zhang); Investigation,
L.D. and C.Z. (Chenmeng Zhang); Methodology, C.Z. (Chenjing Zhang); Project administration, Y.Z.; Resources,
Z.X.; Writing—original draft, L.D. and L.G.; Writing—review & editing, Y.Z. and L.G.
Funding: This research was funded by National Natural Science Foundation of China (81803697, 81803685),
Natural Science Foundation of Hebei Province (H2018205185, H2018423032), Research Foundation of Hebei
Province Education Department (BJ2019006, QN2017093), Research Foundation of Hebei Provincial Administration
of Traditional Chinese Medicine (2018108), and Innovation Team of Hebei Province Modern Agricultural Industry
Technology System (HBCT2018060205).
Conflicts of Interest: The authors declare no conflict of interest.
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Molecules 2019, 24, 2842
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© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
88
molecules
Article
Biochemical, Micronutrient and Physicochemical
Properties of the Dried Red Seaweeds
Gracilaria edulis and Gracilaria corticata
Thomas Rosemary 1 , Abimannan Arulkumar 1,2 , Sadayan Paramasivam 1, *,
Alicia Mondragon-Portocarrero 3 and Jose Manuel Miranda 3
1
2
3
*
Department of Oceanography and Coastal Area Studies, School of Marine Sciences, Alagappa University,
Thondi Campus, Thondi-623 409, India; rosemarythomas@gmail.com (T.R.); aruul3@gmail.com (A.A.)
Department of Biotechnology, Achariya Arts and Science College, Villianur, Puducherry-60 110, India
Departamento de Química Analítica, Nutrición y Bromatología, Facultade de Veterinaria, Universidade de
Santiago de Compostela, Pabellón 4, Planta Baja, 27002 Lugo, Spain; aliciamondragon@yahoo.com (A.M.-P.);
josemanuel.miranda@usc.es (J.M.M.)
Correspondence: drparamsan@gmail.com; Tel.: +91-9047733300; Fax: +91-4565-225525
Academic Editor: Gavino Sanna
Received: 25 April 2019; Accepted: 11 June 2019; Published: 14 June 2019
Abstract: The present study sought to evaluate the nutritional composition and physicochemical
properties of two dried commercially interesting edible red seaweeds, Gracilaria corticata and G. edulis.
Proximate composition of the dried seaweeds revealed a higher content in carbohydrates (8.30 g/100 g),
total crude protein (22.84 g/100 g) and lipid content (7.07 g/100 g) in G. corticata than in G. edulis.
Fatty acids profile showed that G. corticata samples contain higher concentrations of saturated
fatty acids, such as palmitic and stearic acids, and polyunsaturated ones such as α-linolenic and
docosahexaenoic acids. Contrariwise, G. edulis contained higher amounts of monounsaturated
oleic acid. Total amino acid content was 76.60 mg/g in G. corticata and 65.42 mg/g in G. edulis,
being the essential amino acid content higher in G. edulis (35.55 mg/g) than in G. corticata (22.76 mg/g).
Chlorophyll a was found in significantly higher amounts in G. edulis (17.14 μg/g) than G. corticata,
whereas carotenoid content was significantly higher in G. corticata (12.98 μg/g) than in G. edulis.
With respect to physical properties, both water- and oil-holding capacities were similar in both
seaweeds, whereas swelling capacity was higher in G. edulis. In view of the results, the present study
suggests that G. corticata and G. edulis contains important nutrients for human health and are possible
natural functional foods.
Keywords: water holding capacity; seaweeds; Gracilaria; carbohydrates; fatty acids; vitamins
1. Introduction
Seaweeds are very important natural resources from the oceans that are employed as human
foods and animal feeds in their whole form, and as sources of polysaccharides (mainly alginates,
carrageenans and agar), carotenoids, lipids, vitamins, minerals, dietary fiber, proline and amino acids
for use in food and pharmaceutical industry [1]. Seaweeds have been included for a long time in the
traditional diet of East Asian countries such as Japan, Korea and China; more recently, their presence
in all forms in the diet of Western countries has been progressively increasing [2].
Seaweeds are considered healthy foods because, despite their low caloric content, they are rich
in important nutrients such as protein, essential amino acids, vitamins, minerals and some bioactive
compounds [1]. Seaweeds are also an excellent source of both soluble and insoluble dietary fiber.
Among red algae, the genus Gracilaria contains a broad diversity of valuable contents for human
Molecules 2019, 24, 2225; doi:10.3390/molecules24122225
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nutrition and are one of the world’s most cultivated and valuable marine seaweed [3]. Its lipid content
is low (1–5% dry weight, DW) [1], but it contains docosahexaenoic acid (DHA) which is recognized
as the most important n-3 polyunsaturated fatty acid (PUFA) to reduce the risk of cardiovascular
diseases [4,5]. In particular, n-3 PUFAs act as excellent antioxidants, strengthening the cell membrane,
repairing damaged cells and tissues, improving heart function and fighting against cancer [6]. n-3
PUFAs were also found to prevent the growth of atherosclerotic plaque that affects blood clotting and
blood pressure and improve the immune function, while n-6 PUFAs decrease low-density lipoprotein
cholesterol and may also decrease high-density lipoprotein, cholesterol which reduces heart disease
risk [7].
With respect to their protein content, the most abundant amino acids in Gracilaria species are
aspartic acid, alanine, glutamic acid and glutamine. These amino acids provide the typical flavor
of algae and accumulate in response to stress conditions [8]. Gracilaria is also a good source of both
soluble and insoluble dietary fiber, so it can be employed as a potential alternative to cereal-based fiber
in Western countries [1]. Soluble dietary fiber helps to increase viscosity and reduce glycemic response
and plasma cholesterol in humans [1]. Insoluble dietary fiber improves the bulking effect caused by
water absorption in feces and thus contributes to weight management, improvement of cardiovascular
and gastrointestinal functions and cancer prevention [1]. Polysaccharides isolated from red seaweeds
show potent antibacterial, antiviral, antioxidant, anticoagulant and anti-inflammatory activity [9].
Seaweeds such as Gracilaria can concentrate minerals from seawater and reach a mineral content
10–20 times higher than that of terrestrial plants [10]. Consequently, they are a valuable source of
minerals, with important human nutrition functions [11,12]. Chlorophyll, an important pigment
constituent present in algae, has positive effects on inflammation, oxidation and wound healing [13].
Chlorophyll acts directly as a reducer of free radicals and has the potential to protect lymphocytes
against oxidative DNA damage by free radicals [14]. Moreover, a large number of potentially bioactive
compounds such as phenols, polyphenols, terpenes, steroids, halogenated ketones and alkanes,
fucoxanthin, polyphloroglucinol and bromophenols have been isolated [15–17].
However, the nutrient profile of seaweeds such as Gracilaria is influenced by different factors such
as seaweed species, habitat, maturity stage, season, water temperature and the sampling conditions
and method employed in the determinations [1,2]. Gracilaria edulis and G. corticata is abundantly
available in almost all seasons in Palk Bay, on the southeast coast of India, rather than other Gracilaria sp.
Both G. edulis and G. corticata are commercially important and commonly edible seaweeds in India.
These two algae exhibited in a previous work high biological activities (proximate composition,
antioxidant, antibacterial, and biopreservative effects in seafoods during preservation and extended
shelf life than other Gracilaria species in a previous work [18]. Thus, the present study sought to
evaluate and compare the chemical composition (proximate composition, lipid profile, amino acids,
vitamins and pigments such as chlorophyll and carotenoids) and physicochemical properties of both,
Gracilaria corticata and Gracilaria edulis from the Thondi coast of Palk Bay, southeast India.
2. Results
Proximate, polysaccharide content and fatty acids profile of both G. corticata and G. edulis in a DW
basis are shown in Table 1.
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Table 1. Proximate composition (g/100 g dry weight seaweed) G. corticata and G. edulis.
Parameter
G. corticata
G. edulis
Moisture
Protein
Fat
Carbohydrates
Ash
Polysaccharides
8.40 ± 0.65 b
22.84 ± 0.87 b
7.07 ± 0.33 a
8.30 ± 1.89 a
8.10 ± 0.49
49.64 ± 3.89 a
10.40 ± 0.69 a
25.29 ± 0.67 a
4.76 ± 0.73 b
4.71 ± 0.60 b
7.36 ± 0.39
38.02 ± 4.32 b
Values are mean ± standard deviation, n = 3.
significantly different.
a–b
values with different superscripts within the same line were
The crude polysaccharide content found for G. corticata and G. edulis was 49.64 g/100 g and
38.02 g/100 g, respectively. The moisture content (in dried seaweeds) of G. corticata and G. edulis was
8.40 g/100 g and 10.40 g/100 g, respectively. With respect to proximate composition, important differences
were obtained for the two seaweeds investigated. Carbohydrates and fat content were significantly
higher in G. corticata, whereas protein content was significantly higher in the case of G. edulis.
With respect to fatty acids profile, total fatty acid content, expressed as g fatty acids methyl esters
(FAME)/100 g total fat, of G. corticata and G. edulis was 5.49 ± 0.30 g/100 g and 3.92 ± 0.13 g/100 g,
respectively (Table 2). The main saturated fatty acids (SFAs) found in both G. corticata and G. edulis were
palmitic acid (C16:0), margaric acid (C17:0) and stearic acid (C18:0). With respect to PUFAs, linoleic acid
(C18:2n-6), α-linolenic acid (C18:3n-3), stearidonic acid (C18:4n-3) and DHA (C22:6n-3) were found
in both seaweeds. In the case of monounsaturated fatty acids (MUFAs), only oleic acid (C18:1) was
detected in relevant amounts in both G. corticata and G. edulis. Margaric, linoleic and stearidonic
acids were found in similar amounts in G. corticata and G. edulis. Palmitic, stearic, α-linolenic acid
were found in higher amounts in G. corticata than in G. edulis. Overall, in G. corticata, SFAs accounted
49.4% of total fatty acids, MUFAs accounted a 3.3% and PUFAs accounted a 47.3%, whereas in the case
of G. edulis, SFAs accounted 43.9% of total fatty acids, MUFAs accounted a 27% of total fatty acids,
and PUFAs accounted a 29%.
Table 2. Fatty acids profile (g fatty acid methyl esters/100 g total fat) of G. corticata and G. edulis.
Parameter
G. corticata
G. edulis
Palmitic acid
Margaric acid
Stearic acid
Oleic acid
Linoleic acid
α-Linolenic acid
Stearidonic acid
Docosohexaenoic acid
FA
1.22 ± 0.04
0.16 ± 0.25
1.31 ± 0.03 a
0.18 ± 0.12 b
0.63 ± 0.04
1.26 ± 0.04 a
0.21 ± 0.01
0.48 ± 0.14 a
5.49 ± 0.30 a
0.63 ± 0.09 b
0.15 ± 0.10
0.93 ± 0.05 b
1.05 ± 0.05 a
0.65 ± 0.18
0.14 ± 0.04 b
0.22 ± 0.05
0.12 ± 0.18 b
3.92 ± 0.13 b
a
FA = Total fatty acids. Values are mean ± standard deviation, n = 3.
the same line were significantly different.
a–b
values with different superscripts within
The protein of G. corticata and G. edulis is shown in Table 3. The total amino acid content was higher
in G. corticata (76.60 ± 5.14 mg/g), than in G. edulis (65.42 ± 3.58 mg/g). These values are comparable
to their corresponding crude protein content of 22.84 ± 0.87 and 25.29 ± 0.67 g/100 g, respectively,
indicating that the amount of non-protein nitrogenous materials in these red seaweeds is low.
Nine essential amino acids (EAAs), and 11 non-essential amino acids (NEAAs), were found in both
G. corticata and G. edulis. Total EAAs where significantly higher in G. edulis (35.55 ± 1.75 mg/g) than in
G. corticata (22.76 ± 1.81 mg/g), whereas total NEAAs where higher in G. corticata (36.14 ± 3.33 mg/g)
than in G. edulis (29.86 ± 1.83 mg/g). The EAAs/total amino acid ratio suggests that more than
50% of the amino acids found in G. edulis are EAAs. The results also indicate a good ratio of
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essential amino acids to non-essential amino acids in G. corticata (0.62 ± 0.54 mg/g) and G. edulis
(1.19 ± 0.95 mg/g). It was noted that a much higher concentration of the essential amino acid threonine
(20.57 ± 0.62 mg/g) was found in G. edulis than in G. corticata. Contrariwise, alanine content was much
higher in G. corticata (21.11 ± 0.54 mg/g) than in G. edulis (1.46 ± 0.18 mg/g). Aspartic acid content was
similar in both seaweeds.
The mineral content of G. corticata and G. edulis is shown in Table 4. G. corticata showed a higher
content of Mg (463.23 ± 8.87 mg/kg) and Fe (1072.48 ± 20.97 mg/kg) than G. edulis. Moreover, G. edulis
was found to possess more of trace elements like Zn (42.73 ± 2.12 mg/kg) and Cu (14.61 ± 0.46 mg/kg)
than G. corticata. In view of the present results, both G. corticata and G. edulis contain an adequate
amount of minerals, which suggests that these seaweeds could act as important sources of mineral
supplements which are essential for human nutrition.
Table 3. Protein composition of G. corticata and G. edulis.
Amino Acid
G. corticata
G. edulis
Aspartic acid
Glutamic acid
Asparagine
Serine
Glutamine
Glycine
Theronine *
Arginine *
Alanine
Cysteine
Tyrosine *
Histidine
Valine *
Methionine *
Isoleucine *
Phenylalanine *
Leucine *
Lysine *
Proline
Tryptophan
Total amino acids
Total EAAs
Total NEAAs
EAAs/Total AAs
EAAs/NEAAs
14.37 ± 0.78
2.54 ± 0.06
1.45 ± 0.05 b
2.23 ± 0.18 b
2.01 ± 0.7 b
4.71 ± 0.18 a
1.32 ± 0.09 b
3.41 ± 0.30
21.11 ± 0.54 a
1.49 ± 0.30
1.25 ± 0.15 b
2.46 ± 0.27 a
0.16 ± 0.01
8.73 ± 0.31 a
2.53 ± 0.16 a
1.42 ± 0.17 b
1.58 ± 0.35 a
2.37 ± 0.27 a
0.47 ± 0.20
1.00 ± 0.07 a
76.60 ± 5.14 a
22.76 ± 1.81 b
36.14 ± 3.33 a
0.29 ± 0.35 b
0.62 ± 0.54 b
12.67 ± 0.64
2.77 ± 0.15
1.89 ± 0.15 a
2.73 ± 0.13 a
2.42 ± 0.29 a
3.42 ± 0.27 b
20.57 ± 0.62 a
3.33 ± 0.17
1.46 ± 0.18 b
1.27 ± 0.06
2.50 ± 0.24 b
0.18 ± 0.02 a
0.15 ± 0.02
4.98 ± 0.48 b
1.22 ± 0.07 b
2.20 ± 0.10 a
0.38 ± 0.02 b
0.22 ± 0.03 b
0.46 ± 0.18
0.59 ± 0.16 b
65.42 ± 3.58 b
35.55 ± 1.75 a
29.86 ± 1.83 b
0.54 ± 0.48 a
1.19 ± 0.95 a
Values are mean ± standard deviation, n = 3 expressed as mg/g seaweed in a dry weight basis. * EAAs: Essential
amino acids; NEAAs: Non-essential amino acids. a–b values with different superscripts within the same line are
significantly different.
Table 4. Mineral content (mg/kg) in G. corticata and G. edulis.
Minerals
G. corticata
G. edulis
Zn
Cu
Mg
Fe
31.52 ± 0.69 b
11.42 ± 0.72 b
463.23 ± 8.87 a
1072.48 ± 20.97 a
42.73 ± 2.12 a
14.61 ± 0.46 a
89.56 ± 0.77 b
557.36 ± 0.57 b
Values are mean ± standard deviation, n = 3 on dry weight basis. Macro minerals: Zinc (Zn), copper (Cu),
magnesium (Mg) and iron (Fe). a–b values with different superscripts within the same line are significantly different.
For both G. corticata and G. edulis, the presence of water-soluble vitamins (vitamin B1 , vitamin B2 ,
vitamin B3 , vitamin B6 , vitamin B9 and vitamin C) and fat-soluble vitamins (vitamin A and vitamin E)
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was found, as shown in Table 5. G. corticata had a higher vitamin A (2.67 ± 0.31 mg/g vs. 2.14 ± 0.17 mg/g)
and vitamin B9 contents (1.00 ± 0.07 mg/g vs. 0.45 ± 0.06 mg/g) than G. edulis, whereas G. edulis showed
a significantly higher content of vitamin B2 (1.54 ± 0.39 mg/g vs. 0.05 ± 0.01 mg/g) and vitamin B6
(4.77 ± 0.23 mg/g vs. 3.79 ± 0.30 mg/g) than G. corticata.
Table 5. Vitamin composition (mg/g), chlorophyll and carotenoid content (μg/g) of G. corticata and
G. edulis.
Name of Vitamins/Pigments
G. corticata
G. edulis
Vitamin B1
Vitamin B2
Vitamin B3
Vitamin B6
Vitamin B9
Vitamin C
Vitamin A
Vitamin E
chlorophyll a
chlorophyll b
carotenoid
0.38 ± 0.02
0.05 ± 0.01 b
1.54 ± 0.39
3.79 ± 0.30 b
1.00 ± 0.07 a
14.66 ± 0.23
2.67 ± 0.30 a
1.40 ± 0.10
8.96 ± 0.39 b
7.74 ± 0.33
12.82 ± 0.50 a
0.36 ± 0.02
1.54 ± 0.07 a
1.10 ± 0.29
4.77 ± 0.23 a
0.45 ± 0.06 b
13.41 ± 0.57
2.07 ± 0.06 b
1.49 ± 0.10
17.14 ± 0.55 a
8.44 ± 0.63
2.99 ± 0.56 b
Values are mean ± standard deviation, n = 3 on dry weight basis.
same line are significantly different.
a–b
values with different superscripts within the
The methanolic extracts of G. corticata and G. edulis at 1 mg/mL concentration indicate the presence
of three major compounds, chlorophyll a, chlorophyll b and carotenoids, in G. corticata (Retention factor
(Rf) value = 0.97, 0.92 and 0.95, respectively) and G. edulis (Rf = 0.96, 0.96 0.84, respectively). G. corticata
and G. edulis contained 8.96 ± 0.39 μg/g and 17.14 ± 0.55 μg/g of chlorophyll a and 7.74 ± 0.33 μg/g and
8.44 ± 0.63 μg/g of chlorophyll b, respectively. With respect to the carotenoid content, it was higher for
G. corticata (12.82 ± 0.50 μg/g) than for G. edulis (2.99 ± 0.56 μg/g).
Table 6 shows the swelling capacity (SWC), water-holding capacity (WHC) and oil-holding
capacity (OHC) of G. corticata and G. edulis. In general, as temperature varied, the SWC and WHC of
G. corticata and G. edulis powder varied, due to an increase in the solubility of the dietary fiber and
the presence of protein in G. corticata and G. edulis. However, it also reaches significant differences
for the case of SWC, whereas no statistical differences were obtained for WHC or OHC. The SWC of
G. edulis were higher than G. corticata at both 25 ◦ C and 37 ◦ C (8.66 ± 0.53 mL/g vs. 7.90 ± 0.32 mL/g,
and 7.70 ± 0.60 mL/g vs. 5.70 ± 0.65 mL/g, respectively).
Table 6. Swelling capacity (SWC), water holding capacity (WHC) and oil holding capacity (OHC) of
G. corticata and G. edulis.
Seaweeds
G. corticata
G. edulis
SWC (mL/g)
25 ◦ C
WHC (g/g)
37 ◦ C
7.90 ± 0.32
8.66 ± 0.53 aA
bA
5.70 ± 0.65
7.70 ± 0.6 aB
bB
OHC (g/g)
25 ◦ C
37 ◦ C
25 ◦ C
37 ◦ C
4.03 ± 0.39
4.09 ± 0.32
3.96 ± 0.58
3.64 ± 0.40
2.06 ± 0.24
1.87 ± 0.28
1.84 ± 0.40
1.91 ± 0.18
Values are mean ± standard deviation, n = 3 on dry weight basis. a–b values with different superscripts within the
same column are significantly different between seaweeds. A–B values with different superscripts within the same
line shows significant differences between temperatures.
With respect to the WHC of G. corticata and G. edulis, values of 4.03 ± 0.39 and 4.09 ± 0.28 g/g,
respectively, were obtained at 25 ◦ C, reduced to 3.96 ± 0.58 g/g in G. corticata and 3.64 ± 0.18 g/g in
G. edulis at 37 ◦ C. In this study, both G. corticata and G. edulis exhibited similar OHC values (about
2 g/g) at both 25 ◦ C and 37 ◦ C.
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3. Discussion
With respect to proximate content, the moisture of G. corticata and G. edulis was lower than most
results obtained for Gracilaria sp. in general, such as the 12.15 g/100 g obtained for G. acerosa [6] the
19.2 g/100 g for G. edulis [8], and the 12.86 g/100 g for G. edulis [1], but higher than the 5.32 g/100 g
obtained for G. changii [19]. In this work, G. edulis showed a higher ash content in a DW basis than
G. corticata. Similarly, it was reported an ash content of 8.70 g/100 g in G. edulis [8], whereas other
authors reported a higher ash content (40.30 g/100 g) in G. changii [19] than those found in the present
work. A high ash content shows the presence of appreciable amounts of diverse minerals found in
both seaweeds. A similar observation was for G. changii [19] in which were found an ash content
of 6.40 g/100 g. Interestingly, total dietary fiber is known to have physiological properties for the
prevention and treatment of cancer, obesity and diabetes [20,21]. Therefore, G. corticata and G. edulis
may have the potential to be used as a source of dietary fiber in the nutraceutical industries.
Other authors found much lower crude protein contents in Gracilaria spp. than those found in the
present work. Thus, it was reported a crude protein content of 6.68 g/100 g for G. edulis [8], 0.61 g/100 g
for G. acerosa [6], 12.57 g/100 g in G. changii [19] or 19.70 g/100 g for G. cervicornis [22]. Moreover, the high
protein content of G. corticata and G. edulis indicates that these seaweeds may be considered as potential
marine plant sources of protein [22]. Proteins from seaweeds can have antibacterial, antioxidant,
immunostimulating, antithrombotic and anti-inflammatory activities. Consequently, they can be used
for prevention and treatment of hypertension, diabetes and hepatitis among other positive effects in
the organism [20].
The total carbohydrate content of both G. corticata and G. edulis was markedly lower than that
reported [8] for G. edulis (10.2 g/100 g) or the 29.44 g/100 g reported for G. changii [19]. However,
other authors found lower carbohydrate content in Gracilaria species, such as G. acerosa, for which was
reported a carbohydrate content of 1.05 g/100 g [6]. The wide variation in the carbohydrate content
observed in red and brown seaweed species might be due to the influence of different factors like
salinity, temperature and sunlight intensity [2]. Moreover, carbohydrate content is also influenced by
biomass, which reveals the link between growth and carbohydrate content [23].
In general, seaweeds have a low fat content [23]; that makes seaweeds low-calorie foods and in
the present work both seaweeds contained fat amounts of 7.07 g/100 g DW seaweed (G. corticata) and
4.71 g/100 g DW seaweed (G. edulis). These results are lower than those obtained by other authors [8],
whose reported a crude lipid content for G. edulis of 8.30 g/100 g but significantly that the 0.3% reported
for G. changi [19], or the 1.7–3.6% reported for G. fisheri and G. tenuistipitata [5]. Thus, Gracilaria content
in fat can widely vary depending on the species and source.
Polysaccharides are polymers composed of at least 10 monosaccharides linked by glycosidic
bonds [9]. Recently, seaweed polysaccharides have been given large attention by the scientific
community due to their outstanding bioactivities and correspondingly low toxicity [9]. They have
been shown to have other beneficial health effects, including their prebiotic effect and antioxidant or
anti-inflammatory activity [20]. The polysaccharide content obtained in the present work was higher
than the polysaccharide extracted from Gracilaria species in previous works, such as 29.08 g/100 g [24],
27.20 g/100 g [25], 21.40 g/100 g [26], and 32.80 g/100 g [27]. Contrariwise, it was also reported a
higher polysaccharide content in G. debilis [28], in the range 52–67 g/100 g. A previous work [29]
reported that the polysaccharide yield from Gracilaria species varies due to seasonal variations,
physiochemical factors, environmental conditions and extraction methods. Additionally, the variations
in the polysaccharide content of Gracilaria can vary depending on atmospheric temperature at the
time of extraction [26]. Hence the present study significantly indicates that the crude polysaccharides
present in G. corticata and G. edulis may exert varied biological activity [25].
With respect to the fatty acids composition, those of seaweeds often differ from those of terrestrial
plants whereby seaweeds have a higher proportion of PUFAs than terrestrial vegetables. Red seaweeds
are particularly rich in SFAs and PUFAs which have nutritional applications that lead to their extensive
use in food, feed, cosmetic, biotechnological and pharmaceutical applications [30,31]. Variation in
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fatty acid content may also be due to the season of collection as well as other abiotic factors such as
nutrition, salinity, light and temperature [8,20]. In the present work, total fatty acids were significantly
lower than those obtained by other authors [8], who found 11.41 g/100 g in G. edulis. According to
this work [8], the most abundant fatty acids in both seaweeds were palmitic, stearic and α-linoleic
acid acids. The same fatty acids were also found abundant in G. changii [20]. However, our results
were significantly lower than those obtained in G. changii for DHA content, in which DHA were found
as the most abundant fatty acids, with a 48.36% of total fatty acids. The results of the present study
revealed that both seaweeds are rich in SFAs and especially in PUFAs, which provide important health
benefits. With respect to most commonly found n-3 PUFA, eicosapentaenoic acid (EPA) and DHA, it is
common that their contents vary dramatically from Gracilaria spp. and even into the same species [32].
No EPA presence were found for the seaweeds tested in the present work. The presence of this n-3
fatty acid in Gracilaria spp. is inconstant, because it was found in G. gracilis [20], but it was not detected
in G. changii [19] or G. edulis [8]. Fatty acids overall profile obtained in this work were significantly
different than 57.5% SFAs, 18.3% MUFAs and 18.4% PUFAs reported for Gracilaria sp. [3] or the 7.5%
SFAs, 38.3% MUFAs and 51.2% PUFAs 18.4% reported for Gracilaria changii [19].
The protein composition found in this work for G. corticata and G. edulis was lower than those
found in a previous work [20], which reported an amino acid content of 91.90 mg/g in G. changii.
The EAAs/total amino acid ratio was higher than those previously reported [6,19,26]. Aspartic acid
content, that is important for the organoleptic point of view because it was reported that it is responsible
for the special flavor and taste of seaweeds [33], was in similar contents in both seaweeds.
Seaweeds are one of the richest sources of minerals and trace elements, because the cell-wall
polysaccharides and proteins of seaweed contain sulfate, anionic carboxyl and phosphate groups
which act as binding sites for metal retention [34]. With respect to the mineral content, G. corticata
showed a higher content of Mg (463.23 mg/kg) and Fe (1072.48 mg/kg) than G. edulis. Moreover,
G. edulis was found to possess more of trace elements like Zn (42.73 mg/kg) and Cu (14.61 mg/kg)
than G. corticata. Both seaweeds had a higher or similar content of minerals like Zn, Cu, Mg and Fe
when compared with the content of G. acerosa [6], G. edulis [8], G. fisheri and G. tenuistipidatata [5] or
G. changii [19], with the exception of Mg in G. edulis which were lower than those found for other
previous works as G. changii [19]. The ability of seaweeds to accumulate metals will depend on a variety
of factors such as location, exposure, salinity, temperature, pH, light, nitrogen content, season, plant age,
metabolic processes or the affinity of the plant for each element among others [35]. In view of the
present results, both G. corticata and G. edulis contain an adequate amount of minerals, which suggests
that these seaweeds could act as important sources of mineral supplements which are essential for
human nutrition.
For both G. corticata and G. edulis, the presence of water-soluble vitamins (vitamin B1 , vitamin B2 ,
vitamin B3 , vitamin B6 , vitamin B9 and vitamin C) and fat-soluble vitamins (vitamin A and vitamin E)
was found. G. corticata had a higher vitamin A (2.67 mg/g vs. 2.14 mg/g) and vitamin B9 contents
(1.00 mg/g vs. 0.45 mg/g) than G. edulis, whereas G. edulis showed a significantly higher content
of vitamin B2 (1.54 mg/g vs. 0.05 mg/g) and vitamin B6 (4.77 mg/g vs. 3.79 mg/g) than G. corticata.
With respect to previously published works, the vitamin content reported for Gracilaria species is
widely different between the different authors [1,6,8]. Perhaps the more remarkable difference in
vitamin content is that in the present work both G. corticata and G. edulis showed a significantly higher
vitamin A content (2.67 and 2.07, respectively) than those previously reported for G. acerosa [6] or for
G. edulis [1].
Another important difference was found for the case of vitamin C that showed a higher content than
those previously reported [6,8] for G. edulis or G. acerosa, respectively. The variation in vitamin content
may be due to some environmental factors such as salinity, atmospheric temperature, seasonality and
methods of preservation and processing [6].
G. corticata and G. edulis contained 8.96 μg/g and 17.14 μg/g of chlorophyll a and 7.74 μg/g and
8.44 μg/g of chlorophyll b, respectively. In a previous work describing the composition of several
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seaweeds [36] it was reported that chlorophyll a and b in red seaweeds ranged from 68 to 162 μg/g and
from 25 to 46 μg/g, respectively. Specifically, for Gracilaria spp., it was reported high chlorophyll a of
577.89 μg/g and low chlorophyll b of 1.11 μg/g in G. changii [19].
With respect to carotenoid content, a higher total carotenoid content was reported for
G. changii than in the present study (74.22 μg/g) [19]. Carotenoids such as β-carotene, lutein,
zeaxanthin and antheraxanthin have been identified in red seaweed, including Gracilaria species.
Further, seaweed carotenoids, especially β-carotene, are preferred by the market of natural products,
because they are a mixture of cis and trans isomers, which may possess anticancer activity [37].
The SWC, WHC and OHC properties of seaweeds are generally related to their content and type
of polysaccharides as well as protein which links to the cell wall of polysaccharide [5]. Previous works
described that variations in temperature can widely vary physicochemical properties of seaweeds,
due to increase in the solubility of the dietary fiber and the presence of protein [5,8]. However, in our
work, only were found significant variations in the case of SWC. Previous works [8] reported a SWC of
20 mL/g in G. edulis, higher than those found in the present study. Similarly, a SWC at 37 ◦ C of 7.68 mL/g
in G. changii was reported [19], whereas for G. acerosa [6] an SWC at 37 ◦ C of 5 mL/g was reported.
With respect to the WHC of G. corticata and G. edulis, a similar observation was also made in a
previous work [8], that reported a WHC for G. edulis of 3.08 g/g. Other authors found better WHC
than in the present work for other Gracilaria spp., such as G. fisheri, for which a WHC of 5.53 g/g was
reported [5], and G. changii [26], for which WHC values of 6.15 g/g at 24 ◦ C and 9.93 g/g at 37 ◦ C were
reported. Both SWC and WHC of seaweeds might be attributed due to different protein content and
increases in the number and nature of the water binding sites on the protein molecules [38].
OHC is another functional property of food ingredients used in formulated foods for consumption.
Ingredients with high OHC values allow the stabilization of food emulsions and high-fat food
products [19]. For other Gracilaria spp., it was reported [8] that G. edulis showed an OHC of 1.64 g/g,
which is very similar to the OHC values of the present study. Moreover, for G. changii [19] OHCs of
3.11 g/g at 24 ◦ C and 1.17 g/g at 37 ◦ C were reported. The low oil absorption capacity of red seaweeds
is generally related to the hydrophilic nature of the changed polysaccharides (agar, carrageenan,
fucans and alginates) of soluble dietary fiber [39]. The results of the present study for physicochemical
properties confirmed that G. corticata and G. edulis could be considered as a source of food ingredients
including proteins, dietary and soluble fiber [39].
4. Materials and Methods
4.1. Sample Collection
Samples of the commercially important and commonly edible red seaweeds G. edulis and G. corticata
were collected by hand from the Thondi Coast (Latitude: 9◦ 44 N and Longitude: 79◦ 00 E), Palk Bay,
on the southeast coast of India. Freshly collected seaweeds were washed thoroughly in seawater
and transported to the laboratory immediately. Epiphytes, sediment particles and other debris were
removed by washing thoroughly using potable water and thoroughly washed with distilled water
immediately after washing with potable water. Seaweeds were identified using a standard manual [40].
The voucher specimen was deposited in museum at Department of Oceanography and Coastal Area
Studies, School of Marine Sciences, Alagappa University. Seaweeds were shade dried for 5 days at
constant temperature of 25 ◦ C ± 2 ◦ C. Dried seaweed samples were powdered using a mechanical
blender and stored at room temperature in an airtight container (Tarsons, Kolkatta, India) for further
analysis within a maximum period of one week. Further the remaining powder sample stored at
frozen condition (−20 ◦ C) for future use.
4.2. Proximate Composition
The proximate composition of each Gracilaria species was determined in all cases following
Association of Official Analytical Chemists (AOAC) methods [41]. AOAC methods were employed
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to determine in G. corticata and G. edulis the ash content by heating at 550 ◦ C for 24 h in a muffle
furnace (AOAC, 930.05), moisture content by heating at 105 ◦ C for 24 h (AOAC, 934.01), total fat by
Soxhlet extraction with petroleum ether (AOAC 991.36) and protein by the Kjeldahl method (N × 6.25)
(AOAC 981.10) [41]. The total carbohydrate content of G. corticata and G. edulis was determined by the
phenol–sulfuric acid method [38]. All measurements were performed in triplicate for each seaweed
and expressed as g/100 g seaweeds in a dry weight matter.
4.3. Isolation of Polysaccharides
Polysaccharides were separated from G. corticata and G. edulis using 2 g samples of each seaweed
according as previously described [42]. Powdered G. corticata and G. edulis were dissolved and
homogenized with distilled water under constant stirring for 2 h at 100 ◦ C. The residues obtained
were then removed by centrifuging the sample at 6300 g for 10 min using a CPR 30 plus centrifuge
(Remi Lab World, Mumbai, India). The obtained supernatant was then precipitated by addition
of an ethanol-in-water solution (1:3 v/v), followed by subsequent washing with 30 mL of acetone.
The precipitated polysaccharides were then collected and subsequently air-dried, re-dissolved in
distilled water and washed with acetone. Afterwards, the collected polysaccharides were stored at
−20 ◦ C in a deep freezer (Blue Star, Mumbai, India) for further use. Isolation were performed in
triplicate for each seaweed and results are expressed as g/100 g seaweeds in a dry weight basis.
4.4. Extraction of Crude Lipid and Determination of Fatty Acids Content
Portions of 500 mg each of powdered G. corticata and G. edulis were mixed with 5 mL of
a chloroform:methanol solution (2:1 v/v), tightly covered with aluminum foil and kept at room
temperature for 24 h. After this period, solutions were filtered through 11 μm Whatman No. 1 filter
paper, and the filtered extract was placed in a pre-weighed and oven-dried beaker. The beaker was
weighed with lipids, and the difference in weight was taken as total lipid content and expressed as a
percentage [43]. Afterwards, an aliquot of the total lipids of each sample was used to determine the fatty
acids content, based on a method published [6]. For this purpose, 0.45 g was introduced into a 10 mL
volumetric flask, dissolved in hexane containing 50 mg of butylated hydroxytoluene per L and diluted
to 10 mL with the same solvent. Afterwards, 2 mL of the solution was transferred into a quartz tube
and evaporated by means of a nitrogen flow. Further, 1.5 mL of a 20 g/L solution of sodium hydroxide
in methanol, covered with nitrogen, was added, capped tightly with a polytetrafluoroethylene-lined
cap, mixed and heated in a water bath for 7 min. After the water bath, samples were cooled at
room temperature, and 2 mL of boron trichloride-methanol solution was added; then, they were
blanketed with nitrogen, capped tightly, mixed and heated in a water bath for 30 min. After this period,
samples were cooled to 40–50 ◦ C, and 1 mL of trimethylpentane was added; then, they were capped
and shaken vigorously for at least 30 s. Immediately, 5 mL of saturated sodium chloride solution was
added, then the samples were covered with nitrogen, capped and vortexed or shaken thoroughly for
at least 15 s. The upper layer was allowed to become clear and then transferred to a separate tube.
In the separate tube, the methanol layer was shaken once more with 1 mL of trimethylpentane and
combined with trimethylpentane extracts. The organic solvent was then removed, and FAME were
subjected to gas chromatography (GC), performed on a Perkin Elmer Clarus 580 gas chromatograph
(Perkin Elmer, Gaithersburg, MD, USA) equipped with a flame ionization detector and an HP-5
capillary column (30 m × 0.25 mm). Initial temperature was maintained at 70 ◦ C, then increased to
250 ◦ C (10 ◦ C/min); the injection temperature employed was 225 ◦ C. Helium was used as carrier gas,
with a flow rate of 1 μL/min. FAME peaks were identified by comparison of their retention times
and quantified by comparison with individual calibration curves performed with a standard FAME
mix (Supelco, Sigma-Aldrich, St Louis, MO, USA). Tricosanoic acid (C23; Sigma-Aldrich) was used
as internal standard. Fatty acid composition was determined in triplicate for each seaweed and was
expressed as g FAME/100 g total fat.
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4.5. Protein Composition
Protein composition of G. corticata and G. edulis was determined based on a reversed-phase high
performance liquid chromatography (HPLC) analysis method [6]. Two grams each of powdered
seaweeds was mixed with phosphate buffer (pH 7.0) and centrifuged at 1200 g (Remi Lab World)
for 20 min at 4 ◦ C. The supernatant was collected, and protein content was precipitated by adding
10% v/v trichloroacetic acid (TCA). The protein pellet was resuspended in 1 N NaOH and hydrolyzed
by heating the solution with 6 N HCl in a boiling water bath for 24 h. After incubation, the supernatant
was collected by centrifuging the sample at 3500 g for 15 min. The supernatant was then filtered and
neutralized by the addition of 1 N NaOH. The filtered supernatant was diluted to 1:100 (v/v) with
deionized water. The sample was subjected to reversed-phase HPLC analysis (Lachrome Hitachi,
Tokyo, Japan) with UV and fluorescence detectors. One μl of sample was injected into a Denali C18
5-mm column (4.6 mm × 150 mm) at 23 ◦ C with detection at 254 nm. The mobile phase used was
20 mM sodium acetate/triethylamine (0.018% v/v) in phase A. The pH was adjusted to 7.2 using 1–2%
acetic acid. In phase B, 20% of 100 mM sodium acetate (pH 7.2) with 1–2% acetic acid was used;
40% acetonitrile was used as the mobile phase. The protein composition was determined in triplicate
for each seaweed and expressed as mg of amino acid/g of seaweed in a DW basis.
4.6. Determination of Mineral Content
Zn, Cu, Mg and Fe analysis of G. corticata and G. edulis was performed according to the European
Standards, with minor modifications according to the method previously described [17]. One gram of
homogenized seaweed sample was added to mixed reagent at a ratio of 5:2:1 (nitric acid:perchloric
acid:sulfuric acid). Mineralization was performed on a hot plate at 50 ◦ C for 30 min. After the end
of digestion, 10 mL of 2 N HCL was added; digested solvents were filtered and made up to 25 mL
with distilled water and stored at room temperature for further analysis [44]. Zn, Cu, Mg and Fe were
determined by atomic absorption spectroscopy (AAS; Anton Paar-AAS, Graz, Austria). The metal
standards were prepared and run to check the precision of the instrument throughout the analysis.
Quality assurance and quality control protocols set by the US Environmental Protection Agency [45]
for metal analysis were used. Quality assurance testing relied on the control of blanks and yield for the
chemical procedure. Mineral content was determined in triplicate for each seaweed and expressed as
mg/kg of DW seaweed.
4.7. Determination of Vitamin Content
The vitamin content of G. corticata and G. edulis was determined by the method previously
described [20]; vitamin A or retinol (328 nm), vitamin B1 or thiamine monohydrate (420 nm),
vitamin B2 or riboflavin (254 nm), vitamin B6 or pyridoxine HCl (254 nm), vitamin C or ascorbic acid,
vitamin E or tocopheryl acetate (520 nm) and folic acid (550 nm) were determined by HPLC methods,
and the results were compared with the respective standards retinyl acetate, thiamine monohydrate,
riboflavin, pyridoxine HCl, ascorbic acid, tocopheryl, and folic acid (Sigma Aldrich). Vitamin content
was determined in triplicate for each seaweed and was expressed as mg/g of DW seaweed.
4.8. Determination of Chlorophyll a, b and Carotenoids
Thin-layer chromatography (TLC) was used to screen chlorophyll a and b and carotenoid content
in the G. corticata and G. edulis extracts [8]. The mobile phase contained methanol and chloroform (1:9).
The sample (approximately 1 mg/mL) was spotted onto the TLC plates and air-dried. The spots were
identified under long-wave and short-wave UV light, and also in an iodine chamber. The Rf value,
which is the distance moved by the solute relative to the distance moved by the solvent, was calculated
to find chlorophyll a and b and carotenoid content.
Afterwards, for chlorophyll a and b determination, 1 g each of G. corticata and G. edulis powder was
extracted with 96% CH3 OH, and the supernatant was collected by centrifugation at 1000 g for 1 min.
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After filtration, the supernatant was again filtered with Whatman No. 1 filter paper, centrifuged at
2300 g (Remi Lab World) for 10 min, and the absorbance of the collected supernatant was measured
using a UV–Vis spectrophotometer [8]. The chlorophyll a and b contents were calculated using the
following formulas in triplicate for each seaweed and expressed as μg/g of DW:
Chlorophyll a = 15.65 (A666 ) − 7.340 (A653 )
(1)
Chlorophyll b = 27.05 (A653 ) − 11.21 (A666 )
(2)
where A666 = absorbance at 666 nm; A653 = absorbance at 653 nm. Carotenoid content was determined
according to [46]. One gram each of G. corticata and G. edulis powder was extracted with 5 mL of
acetone and incubated in the dark for 45 min, and the supernatant was collected by centrifugation
at 10,000 g for 5 min. The supernatant was then stored in a refrigerator, and the extraction repeated
with acetone until it became colourless. The supernatant was pooled and made up to 10 mL with
acetone, and the absorbance of the collected supernatant was measured at 450 nm using a UV–Vis
spectrophotometer (Shimadzu, Kyoto, Japan). Carotenoid content was calculated using the following
formula in triplicate for each seaweed and expressed as μg/g of DW:
Carotenoid content = A450/2500
(3)
where 2500 is the extinction coefficient.
4.9. Physicochemical Properties
In order to determine the physicochemical properties of the G. corticata and G. edulis powder,
the SWC, WHC and OHC were determined for each powder. The SWC of G. corticata and G. edulis was
assessed based on a method previously described [38]. Briefly, 500 mg of G. corticata and G. edulis were
taken and mixed with 20 mL of distilled water and stirred vigorously. The influence of temperature on
SWC was determined by maintaining the tubes at 25 ◦ C and 37 ◦ C overnight. The SWC of G. corticata
and G. edulis was calculated using the following formula and expressed as ml of swollen sample per g
of DW:
SWC = Initial volume of water (mL) − Volume of water after incubation (mL)
(4)
The WHC of G. corticata and G. edulis was assessed by a modified method [38]. Briefly, 500 mg
each of G. corticata and G. edulis was put into two sets of centrifuge tubes, and 20 mL of deionized
water was added. The tubes were kept separately in an incubator shaker for 24 h at 25 and 37 ◦ C.
The supernatant was discarded after centrifuging the tubes at 12,000 g for 30 min (Remi Lab World).
The wet weight of G. corticata and G. edulis was noted. The samples were dehydrated by keeping them
in an oven at 160 ◦ C for 2 h, and the dry weight of the sample was noted. The WHC was determined in
triplicate for each seaweed and calculated using the following formula and expressed as the weight in
g of water held by 1 g of DW sample:
WHC = Wet weight of the sample (g) - Dry weight of the sample (g)
(5)
The OHC of G. corticata and G. edulis was assessed according to previous methods [20,38]. About 3 g
of dried G. corticata and G. edulis was taken in a tube and mixed with 10.5 g of corn oil. The tubes were
placed in a shaker at room temperature for 30 min. The oil supernatant was collected by centrifugation
at 3000 g for 30 min (Remi Lab World). The OHC was determined in triplicate for each seaweed and
was determined using the following formula and expressed as the number of g of oil held by 1 g of
DW sample:
OHC = Initial volume of oil (g) − Volume of oil after incubation (g)
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4.10. Statistical Analysis
All results were expressed as mean ± SD. Paired sample t-test was used to compare composition
values between G. edulis and G. corticata. One-way analysis of variance (ANOVA) and Duncan’s test
were used to compare the effects of temperature on the physicochemical properties. All determinations
were performed using SPPS version 14 (SPSS Science, Chicago, IL, USA). A positive significant variation
was defined at the significance level of p < 0.05.
5. Conclusions
The polysaccharide content of the investigated dried red seaweeds (G. corticata and G. edulis) were
found to be rich sources of polysaccharides. Because of the potential as prebiotics, antioxidant and
anti-inflammatory compounds, carbohydrates from seaweeds has are compounds in high demand
by consumers today. The physicochemical properties and proximate composition revealed that both
G. corticata and G. edulis have appreciable levels of ash, protein, carbohydrate, fatty acid, essential and
non-essential amino acid and vitamin content. It is suggested that both the seaweeds tested have
great potential as potential food supplements and may be used in the food industry as a source of
ingredients with an appreciable amount of nutritional value. Since both red seaweeds were found
to be a good source of essential nutrients, their commercial value can be enhanced by marketing
them as value-added products. However, depending on their composition, G. edulis and G. corticata
have important differences that make it more adequate for certain cases. G. edulis showed higher
concentration of essential amino acids, chlorophyll, vitamin B2 and Zn. Thus, it could be a good
nutrient for low-protein diets or people whose need to reduce their oxidative status, because of its
content in chlorophyll and Zn. Contrariwise, G. corticata showed higher PUFA content, carotenoids and
minerals as Fe and Mg. Thus G. corticata is more adequate than G. edulis for people who need to
reinforce their intake of such nutrients.
Author Contributions: Conceptualization, S.P. and A.A.; methodology, T.R. and A.A.; software, A.A. and A.M.-P.;
validation, A.A.; formal analysis, T.R. and A.A.; investigation, A.A. and S.P.; resources, S.P.; data curation, J.M.M.;
writing—original draft preparation, J.M.M.; writing—review and editing, A.M.-P.; visualization, S.P. and J.M.M.;
supervision, S.P.; project administration, S.P.; funding acquisition, S.P.
Funding: This research received external funding by RUSA–2. 0 grant sanctioned vide Letter No. F. 24–51/2014–U,
Policy (TNMulti–Gen), Department of Education, Government of India and by FEADER (The European Agricultural
Fund for Rural Development (EAFRD)), grant number 2018/001B and ED431C 2018/05.
Acknowledgments: The authors thank the authorities of Alagappa University for providing necessary facilities.
Conflicts of Interest: The authors declare no conflict of interest.
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102
molecules
Article
Concentration of EPA and DHA from Refined Salmon
Oil by Optimizing the Urea–Fatty Acid Adduction
Reaction Conditions Using Response
Surface Methodology
Gretel Dovale-Rosabal 1 , Alicia Rodríguez 1, *, Elyzabeth Contreras 1 , Jaime Ortiz-Viedma 1, *,
Marlys Muñoz 1 , Marcos Trigo 2 , Santiago P. Aubourg 2, * and Alejandra Espinosa 3
1
2
3
*
Department of Food Science and Chemical Technology, Faculty of Chemical and Pharmaceutical Sciences,
Santos Dumont 964, University of Chile, Santiago 8380000, Chile; gretel.dovale@ug.uchile.cl (G.D.-R);
ely_tcn@hotmail.com (E.C.); marlys_dayane@hotmail.com (M.M.)
Department of Food Technology, Marine Research Institute (CSIC), Eduardo Cabello, 6, 36208 Vigo, Spain;
mtrigo@iim.csic.es
Department of Medical Technology, School of Medicine, University of Chile, Santiago 8380000, Chile;
bespinosa@med.uchile.cl
Correspondence: arodrigm@uchile.cl (A.R.); jaortiz@uchile.cl (J.O.-V.); saubourg@iim.csic.es (S.P.A.)
Academic Editor: Jose M. Miranda
Received: 19 March 2019; Accepted: 20 April 2019; Published: 26 April 2019
Abstract: This research focused on obtaining eicosapentaenoic acid (EPA, 20:5 n-3) and
docosahexaenoic acid (DHA, 22:6 n-3) (EPA+DHA) concentrates from refined commercial salmon
oil (RCSO). Independent variables of the complexation process were optimized by means of the
application of response surface methodology (RSM) in order to obtain the maximum content of
such fatty acids (FAs). As a result of employing the optimized conditions for all the variables (6.0,
urea:FA content ratio; −18.0 ◦ C, crystallization temperature; 14.80 h, crystallization time; 500 rpm,
stirring speed), high contents of EPA and DHA could be obtained from RCSO, achieving increases
of 4.1 and 7.9 times in the concentrate, with values of 31.20 and 49.31 g/100 g total FA, respectively.
Furthermore, a 5.8-time increase was observed for the EPA + DHA content, which increased from
13.78 to 80.51 g/100 g total FA. It is concluded that RCSO can be transformed into a profitable source
of EPA and DHA (EPA+DHA), thus leading to a product with higher commercial value.
Keywords: refined commercial salmon oil; n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFAs)
concentration; EPA; DHA; EPA+DHA; total FA yield; process variable maximization; response surface
methodology (RSM); multiple response optimization; desirability function
1. Introduction
In recent years, it has been recognized that the consumption of eicosapentaenoic acid (EPA)
is associated with a low prevalence of coronary, circulatory, and inflammatory diseases [1–4].
Furthermore, docosahexaenoic acid (DHA) has been associated with fetal development, the prevention
of neurodegenerative diseases, and the correct functioning of the nervous system and visual
organs in the fetus [5–10]. According to the Food and Agriculture Organization/World Health
Organization (FAO/WHO) [1], the recommended intake of EPA+DHA is at least 250 mg/day
for adult males and non-pregnant/non-lactating adult females. Interestingly, the optimal brain
development of children would need a 150 mg/day diet of such fatty acids. EPA+DHA concentrates
may be produced by various methods, such as supercritical fluid chromatography, supercritical
fluid fractionation, molecular distillation, silver complexation, enzymatic methods, and urea
Molecules 2019, 24, 1642; doi:10.3390/molecules24091642
103
www.mdpi.com/journal/molecules
Molecules 2019, 24, 1642
complexation [11,12]. Among them, complexation with the urea can be considered as the most
efficient method, since polyunsaturated fatty acids (PUFAs) may be separated from saturated and
monounsaturated ones by means of an economic process at low temperature [13–18]. The present
research was focused on the employment of refined commercial salmon oil (RCSO) as a profitable
source of EPA+DHA concentrates, which in time could lead to a product of higher commercial
value. For it, independent variables of the urea adduction reaction conditions (urea:FA content ratio,
crystallization time and temperature, and crystallization stirring speed) were optimized by response
surface methodology (RSM) in order to achieve the maximum content of EPA, DHA and EPA+DHA.
Additionally, the quality of the starting salmon oil was determined and evaluated.
2. Results
2.1. Characterization of the Initial Refined Commercial Salmon Oil
Results for the composition of RCSO reported that the most abundant fatty acids were 18:1 9c
(29.61%), 18:2 9c, 12c (16.69%), and 16:0 (13.74 %) followed by EPA (7.53%) and DHA (6.25%) (g/100 g
total FA). The total value of saturated fatty acids (SFAs) was 21.28%, which was composed mainly
of palmitic, stearic, and myristic acids (Table 1). Special interest is the confirmation of the absence of
phytanic acid within the lipid composition of RCSO. This fatty acid has been linked to neurological
disorders in some people, but it is also associated with the prevention of metabolic syndrome or type 2
diabetes [19].
Table 1. Composition of fatty acids in (RCSO) and the optimized concentrate from RCSO (g/100 g
total FA) *.
FA or FA Groups
RCSO
RCSO Optimum
12:0
14:0
15:0
16:0
16:1 9t
16:1 7c
16:1 9c
16:1 11c
16:1 13c
17:0
17:1 10c
18:0
18:1 9c
18:1 11c
18:2 9t, 12t
18:2 9c, 12c
18:2 9c, 15c
18:3 6c, 9c, 12c
20:0
18:3 9c, 12c, 15c
20:1 5c
20:1 8c
20:1 11c
18:4 6c, 9c, 12c, 15c
20:2 11c, 14c
20:3 8c, 11c, 14c
20:3 11c, 14c, 17c
20:4 8c, 11c, 14c, 17c
22:1 13c
20:5 5c, 8c, 11c, 14c, 17c
24:1 15c
0.07
3.19
0.20
13.74
0.15
Nd
4.66
Nd
Nd
0.13
0.56
3.69
29.61
3.69
Nd
16.69
Nd
0.22
0.26
3.25
Nd
Nd
1.60
Nd
0.79
0.30
0.12
0.40
0.21
7.53
Nd
Nd
0.12
0.09
Nd
Nd
Nd
0.51
Nd
Nd
0.10
1.28
0.28
0.59
0.04
Nd
7.48
Nd
1.09
Nd
2.60
Nd
Nd
Nd
Nd
0.05
1.16
0.03
1.37
Nd
31.20
Nd
104
Molecules 2019, 24, 1642
Table 1. Cont.
22:5 7c, 10c, 13c, 16c, 19c
22:6 4c, 7c, 10c, 13c, 16c, 19c
Total SFAs
Total MUFAs
Total PUFAs
Total n-3PUFAs
Total n-3LCPUFAs
EPA+DHA
2.69
6.25
21.28
40.48
38.24
20.45
18.08
13.78
2.70
49.31
0.59
2.42
96.99
87.21
85.82
80.51
* Abbreviations employed: DHA (docosahexaenoic acid), EPA (eicosapentaenoic acid), FA (fatty acid), SFAs (saturated
fatty acids), MUFAs (monounsaturated fatty acids), RCSO (refined commercial salmon oil), n-3LCPUFAs (n-3 long
chain polyunsaturated fatty acids), Nd (not detected).
Values obtained for the oxidative stability of RCSO were: peroxide value (PV) = 5.23 ± 0.05 meq.
active oxygen kg−1 oil; p-anisidine value (pAV) = 6.84 ± 0.46; and total oxidation value
(TOTOX) = 17.30 ± 0.40 and free fatty acids (FFA) = 0.30 ± 0.01 g oleic acid/100 g oil. Previous studies
performed on refined salmon oil samples [18] indicated average values of PV= 3.54 ± 0.16 meq active
oxygen kg−1 oil; p-anisidine value (pAV) = 5.14 ± 1.02 and FFA= 0.23 ± 0.00 g oleic acid/100 g oil. and
at and 12.22 ± 1.34, respectively.
2.2. Effect of Process Variables on Total FA Yield, EPA Contents, and DHA Contents of RCSO Concentrate
2.2.1. Refined Commercial Salmon Oil Concentrate
According to the experimental design, showed in Table 2, 28 assays were performed to obtain
different refined commercial salmon oil concentrates. This table reports the experimental values
obtained for the different response variables: R1 (total FA yield; g FA in the non-urea complexing
fraction/100 g initial saponified oil FA), R2 (EPA content; g/100 g total FA), and R3 (DHA content;
g/100 g total FA) of RCSO concentrate. Table 2 also includes the predicted values for the described
corresponding variables, whereas the experimental values were replaced by the application of the
model (R1’, R2’, and R3’ values, respectively). As a result, all the independent variables (A: urea/FA
content ratio, w/w; B: crystallization temperature, ◦ C; C: crystallization time, h; D: stirring speed, rpm)
significantly affected (p < 0.05) the response variables during the urea complexation process. On the
other hand, the enrichment of EPA and DHA in concentrates varied inversely according to total FA
yield, obtaining correlation coefficient values (r) of −0.7756 and −0.7185, respectively.
Table 2. Values obtained for the experimental and predicted response variables of RCSO concentrate
by central composite rotatable design 24 + star based on the response surface methodology 1 .
1
2
3
4
5
6
7
8
9
10
11
12
Response Variables **
Process Variables *
Run
Experimental Values
Predicted Values
A
B
C
D
R1
R2
R3
R1 ’
R2 ’
R3 ’
1.5
4.5
1.5
4.5
1.5
4.5
1.5
4.5
1.5
4.5
1.5
4.5
−15
−15
15
15
−15
−15
15
15
−15
−15
15
15
14.3
14.3
14.3
14.3
36.8
36.8
36.8
36.8
14.3
14.3
14.3
14.3
200
200
200
200
200
200
200
200
600
600
600
600
40.44
12.64
48.71
17.45
36.25
10.44
4.22
18.12
41.74
12.77
78.45
16.11
10.47
28.41
9.22
25.09
10.46
24.42
7.96
24.87
10.23
30.20
9.02
25.16
11.04
44.38
9.52
32.69
11.13
55.91
8.18
30.47
10.89
46.43
9.41
34.34
33.77
12.23
40.35
18.82
29.02
21.55
20.22
12.76
44.89
12.14
68.43
21.40
13.77
28.09
9.52
23.85
13.22
27.55
8.98
23.30
14.52
28.84
10.27
24.60
14.00
45.89
10.23
26.94
15.24
47.14
11.47
28.18
14.71
46.61
10.94
27.65
105
Molecules 2019, 24, 1642
Table 2. Cont.
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
1.5
4.5
1.5
4.5
0
6
3
3
3
3
3
3
3
3
3
3
−15
−15
15
15
0
0
−30
30
0
0
0
0
0
0
0
0
36.8
36.8
36.8
36.8
25.5
25.5
25.5
25.5
3.05
48.0
25.5
25.5
25.5
25.5
25.5
25.5
600
600
600
600
400
400
400
400
400
400
0
800
400
400
400
400
45.49
9.86
58.94
12.64
57.38
15.86
12.21
33.82
12.71
16.31
19.11
16.47
20.77
20.74
17.20
22.74
9.91
27.56
7.84
23.05
6.33
25.45
29.26
13.52
20.41
23.01
22.62
26.07
22.43
21.40
24.00
18.23
10.06
48.55
7.91
28.93
6.39
30.43
44.37
14.65
22.67
28.93
25.72
33.39
25.82
25.20
29.15
20.23
40.14
7.17
48.30
15.33
67.84
13.34
19.61
34.36
25.65
14.83
13.39
27.09
20.24
20.24
20.24
20.24
13.97
28.30
9.73
24.05
-0.77
27.88
24.94
16.45
21.24
20.15
19.95
21.44
20.69
20.69
20.69
20.69
15.96
47.85
12.18
28.89
0.94
49.55
36.61
13.88
24.00
26.49
24.53
25.95
25.24
25.24
25.24
25.24
R2 Adjusted
Equations
Total FA yield = 57.60 − 19.47A + 0.26B − 0.87C + 0.08D + 2.26AA+ 0.01BB + 0.21AC
− 0.02AD − 0.02BC + 0.001BD
EPA = −0.90 + 9.53A − 0.14B − 0.79AA
DHA = −1.19 + 8.10A + 0.17B − 0.19AB
EPA+DHA = 3.52 + 12.88A − 0.52B
0.72(1)
0.84(2)
0.81(3)
0.80(4)
1 Central composite design: 24 + star, which studies the effects of 4 factors in 28 runs based on the RSM. * Independent
variables: A (urea/FA content ratio, w/w), B (crystallization temperature, ◦ C), C (crystallization time, h), and D
(stirring speed, rpm).** Response variables: R1 (total FA yield, g FA in the non-urea complexing fraction/100 g initial
saponified oil FA), R2 (EPA content, g/100 g total FA), and R3 (DHA content, g/100 g total FA). Predicted response
variables: R1 ’, R2 ’, and R3 ’.
2.2.2. Effect of Process Variables on EPA, DHA, and EPA+DHA Content and Total FA Yield:
Pareto Charts and RSM Analysis
Pareto charts (Figure 1) were obtained for the different dependent variables as a function of
the concentrate processing variables from RCSO; furthermore, the linear, quadratic, and interaction
terms in the second-order polynomial were used to generate a three-dimensional response surface
graph. Panel (A) indicates that the total FA yield (R1) of concentrates was dependent (p < 0.05)
on the linear terms of the urea:FA content ratio (A), crystallization temperature (B), crystallization
time (C), stirring speed (D), the quadratic terms AA and BB, and the interactions terms AD, BD, BC,
and AC. Figure 1 (Panel B) shows the response surface of the urea complexation process for the total
FA yield. The total FA yield decreased when the urea:FA content ratio increased and the crystallization
temperature decreased. A similar result was found for the total FA yield effect in concentrate obtained
from a by-product of rainbow trout processing where the total FA yield presented a minimum value
in the response surface analysis when considering high urea:FA content ratios, at low crystallization
temperature levels and stirring speeds, and at intermediate levels of crystallization time [17]. In the
case of the EPA content (Figure 1, Panel C), the Pareto charts showed that certain linear terms—the
urea:FA content ratio (A), crystallization temperature (B), and the quadratic urea:FA content ratio
(AA)—provided a significant effect (p < 0.05). However, linear terms such as the crystallization
time (C) and stirring speed (D) did not produce significant changes (p > 0.05). Figure 1 (Panel D)
exhibits the response surface of the urea complexation process for EPA content. It was found that
the EPA content increased with the urea:FA content ratio while it decreased as the crystallization
temperature increased. For the DHA content (Figure 1, Panel E), the Pareto charts reported that
the linear terms of the urea:FA content ratio (A), crystallization temperature (B), and the interaction
between the urea:FA content ratio and the crystallization temperature (AB) revealed a significant effect
(p < 0.05). The DHA content increased when the urea:FA content ratio increased, while it decreased
as the crystallization temperature increased (Figure 1, Panel F). Finally, concerning the EPA+DHA
106
Molecules 2019, 24, 1642
content, the urea:FA content ratio showed a positive effect, whereas the crystallization temperature
had a negative effect (p < 0.05) (Figure 1, Panel G). These results agree with those obtained by authors
concerning the employment of the Asian catfish (Pangasius bocourti) and by-product of rainbow trout
oil, which reported an inverse relationship between the urea:FA content ratio and the crystallization
temperature on the urea concentrate process [16,17]. When the urea:FA content ratio increased and the
crystallization temperature decreased as result, high values were obtained for DHA in the non-urea
complexing fraction, as well as a great retention of saturated and monounsaturated FA in the urea
crystal adducts. In this case, similar results for the EPA content were found. In all the cases, the EPA,
DHA, and EPA+DHA contents did not significantly varied depending on the stirring speed (p > 0.05).
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Figure 1. Pareto charts and response surfaces for the effects of different process variables: Panels in
total FA yield (%, panels A,B), EPA content (g/100 g total FA, panels C,D), DHA content (g/100 g total
FA, panels E,F), EPA+DHA content (g/100 g total FA, panels G,H). A: urea/FA contents ratio, w/w; B:
crystallization temperature, ◦ C; C: crystallization time, h; and D: stirring speed, rpm).
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Molecules 2019, 24, 1642
2.2.3. Models Obtained for the Concentration of EPA, DHA, and EPA+DHA
Equations obtained for the experimental process variables of the response surface model calculated
by multiple regression are shown in Table 2. According to the equations obtained, all the response
variables were found to be dependent on the same process variables expressed in the Pareto analysis
(Figure 1). The four RCSO concentrated models had an adjusted R2 by degrees of freedom of 72.0% for
total FA yield (Equation (1)), 84.0% for EPA (Equation (2)), 81.0% for DHA (Equation (3)), and 80.0%
for EPA+DHA (Equation (4)). Such values reported that the models adequately represented the
variability of the results. Since the p-values obtained for lack-of-fit in the ANOVA study (0.014, 0.35,
0.19, 0.28; Equation (1) to Equation (4), respectively) was greater or equal to 0.05, except for Equation (1),
the model appears to be adequate for the observed data at the 95.0% confidence level.
2.2.4. Independent Variables and Multiple Response Optimization
Table 3 (part a) shows the optimization of the independent variables for the response variables
(EPA, DHA, and EPA+DHA) of the urea complexation process. The optimum values of dependent
variables for EPA, DHA, and EPA+DHA content were 33.01, 76.81, and 98.85 (g/100 g total FA),
respectively. In all the cases, a tendency toward the same values for the urea:FA content ratio and
crystallization temperature was observed, so that the optimum EPA, DHA, and EPA+DHA contents
were obtained.
Table 3. Process variables * optimization and multiple response optimization of the response variables.
Part a) Optimization of the Process Variables
Dependent Variables
EPA
DHA
EPA+DHA
Process Variables
A
5.99
6.00
6.00
B
−29.79
−29.98
−29.95
C
3.05
48.05
47.79
D
599.00
108.30
271.36
Stationary
Point
Optimum
Value **
Maximum
Maximum
Maximum
33.01
76.81
98.85
Part b) Multiple Response Optimization of the Response Variables
Dependent Variables
EPA
DHA
EPA+DHA
Maximum desirability
Stationary
Point
Process Variables
A
B
C
D
5.84
−17.69
14.83
453.36
Maximum
Predicted
Value **
30.7162.94
90.07
1.0
Part c) Experimental Validation of the Multiple Response Optimization of the Dependent Variables
Dependent Variables
EPA
DHA
EPA+DHA
Process Variables
A
B
C
D
6.00
−18
14.80
500
Stationary
Point
Experimental
Value **
Maximum
31.20
49.31
80.51
* Process variables (A, B, C, and D) as expressed in Table 2. ** Values expressed as g/100 g total FA.
Table 3b shows the levels of factors that maximized the EPA, DHA, and EPA+DHA contents
(g/100 g total FA) by means of multiple response optimization and the stationary point that predicted
a maximum of 30.71, 62.94, and 90.07 (g/100 g total FA) in EPA, DHA, and EPA+DHA, respectively.
Maximum desirability score of 1 (range, 0–1) was attained. A maximum predicted value could be
obtained, provided that the following process conditions were applied: 5.84 (urea: FA content ratio),
−17.69 ◦ C (crystallization temperature), 14.83 h (crystallization time), and 453.00 rpm (stirring speed).
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Molecules 2019, 24, 1642
Figure 2 (panels A, B) shows the contours and estimated response surface of the urea:FA content
ratio and crystallization temperature of the combination of factors levels to maximize the desirability
function for RCSO concentrate. It is observed that the highest desirability values were reached by
taking into account the high values of the urea:FA content ratio and the low crystallization temperature
values. These results were similar to those previously reported for rainbow trout belly oil [17], where a
maximum desirability score of 0.91 was obtained in the multiple response optimization of EPA,
DHA and EPA+DHA contents. Furthermore, the predictive values of 32.50, 37.00, and 67.70 (g/100 g
total FA) in the by-product concentrate of rainbow trout oil, respectively, were obtained. As a result,
the following process conditions were applied in such study: 4.21 (urea:FA contents ratio), −15.00 ◦ C
(crystallization temperature), 24.0 h (crystallization time), and 1000 rpm (stirring speed).
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Figure 2. Combination of factors to maximize the desirability function for RCSO concentrate:
response surface (A) and contour surface (B).
2.2.5. Validation of the Optimized Process and Characterization of the EPA+DHA Content Obtained
Table 3c shows the validation of multiple response optimization after experimentally performing
the process conditions. For EPA content, comparison of the predicted value and the value obtained
experimentally showed that both values were similar (i.e., 30.71 and 31.20 g/100 g total FA, respectively);
however, the experimental value of DHA content was substantially different from the predictive value
(49.31 and 62.94 g/100 g total FA, respectively). Experimental values performed on rainbow trout belly
oil [17] were shown to be similar to the predicted values obtained in the current study for EPA and
DHA (36.10 and 47.70 g/100 g of total FA, respectively). In the experimental validation, an 80.51 g/100 g
total FA value was obtained for the EPA+DHA content, which agrees with the value revealed by other
authors who obtained a stationary point of 89.38% for the EPA+DHA content [14].
2.3. Composition of FA in the RCSO Optimized Concentrate after Validation
Table 1 shows the FA composition of the RCSO compared to the composition of FA from the
validated optimum concentrate. Optimization process validation was performed experimentally by
combining the factors levels in which the optimal EPA+DHA content was attained (Table 3b,c). In these
experiments, the effect of the urea complexation process on the composition of FA and FA groups in
the optimized concentrate (g/100 g FA) after validation can be observed. When compared to the initial
RCSO, there was a marked increase in the concentration of total PUFAs (96.99%) and a substantial
decrease in the concentration of SFAs (0.59%) and MUFAs (2.42%). Furthermore, the predominant FAs
in the optimum concentrate oil were EPA, DHA, and EPA+DHA (31.20, 49.31, and 80.51 g/100 g total FA,
respectively). The urea complexation under the optimization process conditions has revealed a high
efficiency, since a marked increase of total PUFAs from 38.24 to 96.99 g/100 g total FA could be observed;
this increase was 1.21 times higher than that reported by Pando et al. [18]. Additionally, the total
EPA+DHA final content was 2.5 times higher than that reported for refined salmon oil concentrate
without the optimization process [18].
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Molecules 2019, 24, 1642
3. Discussion
In the present research, the distribution of SFAs in RSCO showed that this FA group was mainly
composed of palmitic, stearic, and myristic acids. Similar values have been established for the SFAs
group from crude commercial salmon oil [18]; interestingly, refined commercial salmon oil and Asian
catfish oil also showed these three FA as the major ones, but a higher presence of stearic acid compared
with myristic acid was detected in our study [16,18]. Among the MUFAs, the most abundant in the
present study were 18:1 9c, 16:1 9c, 18:1 11c, and 20:1 11c. The total n-3 long chain polyunsaturated fatty
acids (n-3 PUFAs) content was 1.14 times higher than the content of total n-6 PUFAs, showing values
higher than those previously reported in different kinds of oils obtained crude commercial salmon,
and rainbow trout (Oncorhynchus mykiss) [18,20]. The analysis of process variables shows a high
recovery performance of RCSO concentrate when the urea:FA content ratio has the lowest value,
and the value considered for the crystallization temperature is the highest. The results show a higher
content of EPA and DHA when the total FA yield is lower, indicating that this experiment eliminated
most of the SFAs and MUFAs from the starting oil, only leaving a small fraction of such acids in the urea
non-complexed fraction. Similar values have been observed by other authors [17,21], who reported
major yields with higher stirring speed, corroborating a significant effect of the stirring speed on the
total FA yield in the urea complexation process (p < 0.05). Current results show an inverse relationship
between the urea:FA content ratio and the crystallization temperature on the DHA content, which is a
conclusion that has already been than those reported by several authors, whose employed different
oils from marine origin such as seal blubber oil, tuna oil, Asian catfish oil, and a by-product of rainbow
trout [13,14,16,17]. In this case, results similar for EPA content were found. As an explanation for
this, it could be argued that the urea:FA content ratio has a significant positive effect (p < 0.05) on the
concentration of EPA and DHA, probably as a result of increasing the concentration of urea with respect
to that of FFA; consequently, this would lead to an increase of the number of adducts formed between
the flat structures of SFA and urea molecules, this favoring the formation of hexagonal complexes and
crystals [13], and leading to an increased concentration of EPA and DHA in the non-urea complexing
solution. Previous studies on tuna oil concentrates have revealed that the regression models for total
FA yield and total EPA+DHA content were highly significant with satisfactory R2 coefficients [14,22].
The present data proved that the urea complexation under the optimization process conditions has
shown to be highly efficient, since an increase in the total PUFAs content from 38.24 to 96.99 g/100 g total
FA could be reached, which was 1.2 times higher than that reported by Pando et al. [18]. Additionally,
the total EPA+DHA final content was 2.5 times higher than that reported for refined salmon oil
concentrate without applying an optimization process [18]. On the other hand, the oxidative and
hydrolytic stability parameters (PV, pAV, TOTOX, and FFA) indicate that the quality of the oil used in
this study was included within the acceptable quality limits for edible fats and oils of marine origin
according to the Chilean Food Sanitary Regulations. Government institutions and trade associations,
such as the Council for Responsible Nutrition (CRN) and the Global Organization for EPA and DHA
Omega-3s (GOED), have set strict guidelines for marine oil quality and safety parameters. According to
the previously described recommended values [23,24], the starting RCSO can be considered included in
the 98% of fish oil products compliant with the PV limit of 10 meq. active oxygen kg−1 oil set by British
Pharmacopeia Fish Oil Type I, European Union (EU) Pharmacopeia Fish Oil Type I, and Australian
government guidelines. Furthermore, salmon oil samples were compliant with the pAV limit of
15 set by British and EU Pharmacopeia Fish Oil Type II, as well as the pAV limit of 20 set by the
GOED, Canada Natural Health Product Directorate (NHP), United States Pharmacopeia, and Codex
Alimentarius Commission, Food and Agriculture Organization of the United Nations (CODEX/FAO).
Thus, the primary and secondary lipid oxidation levels of RCSO are not exceeding the regulatory
thresholds in the testing peroxide values and p-anisidine values. Interestingly, such lipid oxidation
scores can be considered similar to those previously obtained for refined salmon oil samples [18] and
salmon oil [24]. On the other hand, the FA composition of the RCSO showed that most abundant FAs
were C18:1 9c, 18:2 9c, 12c, and C16:0, followed by EPA and DHA. The value of the total SFAs was
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Molecules 2019, 24, 1642
found to be 21.28% in RCSO. Concerning PUFAs, the most abundant in RCSO concentrates were DHA,
EPA, and 18:2 9c, 12c.
4. Materials and Methods
4.1. Materials and Chemicals
Refined commercial salmon oil was provided by Fiordo Austral S.A. (Puerto Montt, Chile).
Fatty acid methyl ester (FAME) standards, fatty acid (FA) standards, and C23:0-methyl ester (2COT
N-23M-A29-4 NU-CHECK-PREP-INC) were obtained from NU-CHECK-PREP, INC (Elysian, MN,
USA). All the solvents and chemicals used (including urea, ethanol, α-tocopherol, and n-hexane) were
of analytical grade (Merck, Santiago, Chile).
4.2. Characterization of Refined Commercial Salmon Oil
Initial RCSO characterization was carried out by chemical analyses. For it, the following standard
Association of Official Analytical Chemists (AOAC) and official methods [25] were carried out: peroxide
value (PV; method Cd 8b-90:1-2), p-anisidine value (pAV; method Cd 18-19:1-2), and total oxidation
value (TOTOX; method Cg 3-91) and free fatty acids (FFA) contents (method Ca 5a-40:1).
4.3. FA Composition of the RCSO and n-3 LCPUFA Concentrates
For analyzing the FA composition of the RCSO and the different LCPUFA concentrates,
a methylation process was performed to obtain FAMEs. For it, a two-step process was performed,
according to previous research [18,26]. FAME analysis was carried out on an HP 5890 series II GLC
with a flame ionization detector (FID) with the injection system split. A fused silica capillary column
(100 m length × 0.25 mm × 0.2 μm film thickness) coated with SPTM-2560 (Supelco, Bellefonte, PA,
USA) was used [18,26–29]. DataApex ClarityTM software (DataApex Ltd., Prague, Czech Republic) for
chromatogram analysis was applied. The reference standard NU-CHEK GLC463 was used to identify
the FA profiles. The concentration of the different FAME was determined from the calibration curves
by assessment of the peak/area ratio. The quantification of all the individual FAs (g/100 g total FA) was
achieved by employing C23:0 methyl ester as the internal standard according to the (AOCS Official
Method (Ce 1j-7, 2009) [26].
4.4. n-3 LCPUFA Concentrates from RCSO
The procedure included salmon oil saponification, which was in agreement with other
authors [17,18,30]. Concentrate from RCSO was prepared by FFA collection, the formation of
urea FFA inclusion complexes, and the extraction of free n-3 LCPUFA. For it, the urea complexation
method was carried out by 28 experimental runs with different urea:FA content ratios (0 to 6 w/w),
crystallization temperatures (−30 to 30 ◦ C), crystallization times (0 to 48 h), and stirring speeds (0 to
800 rpm). The FFAs were mixed with urea and 95 % ethanol, and the mixture was subsequently
stirred and heated at 60 ◦ C with magnetic stirring. Then, it was cooled with constant stirring to
different conditions of temperature and time as described in the experimental design. The crystals
formed were separated from the liquid phase by filtration with a Whatman No.1 paper. The non-urea
complexing fraction was diluted with 100 mL of distilled water by each 10 g, acidified to pH 4.5 with
6 N HCl, and washed in a separating funnel with hexane (400 mL). The hexane phase was filtered with
anhydrous sodium sulfate in a Whatman No.1 paper, and the solvent was partially removed using
a rotatory evaporator at 40 ◦ C under vacuum [16,17]. The resulting n-3 LCPUFA concentrates were
stored at −80 ◦ C with 0.5% of α-tocopherol under nitrogen atmosphere until use for further analysis.
4.5. Experimental Design and Optimization Procedure
A rotational central composite design 24 + star, with 4 factors and 5 levels, was carried out, with 28
experimental runs that included 4 repetitions of the central point based on the RSM. The following
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Molecules 2019, 24, 1642
conditions for the independent variables were considered (Table 1): urea:FA content ratio (variable
A: 0 to 6 w/w), crystallization temperature (variable B: −30 to 30 ◦ C), crystallization time (variable C:
3.05 to 48.0 h), and stirring speed (variable D: 0 to 800 rpm). On the basis of the non-urea complexing
fraction, the following response variables (R variables) of the experiment design were chosen: total
FA yield (variable R1: g FA in the non-urea complexing fraction/100 g initial RCSO), EPA content
(variable R2: g/100 g total FA in concentrate), and DHA content (variable R3: g/100 g total FA in
concentrate). Four replicates were carried out at the central point of the experimental design in
order to evaluate the experimental error. All experiments were performed randomly to minimize
the effect of unexplained variability in responses resulting from extraneous factors [31]. In order
to obtain response surfaces, multiple regression equations were fitted to the responses obtained by
discarding non-significant terms (p > 0.05) to obtain response surfaces. To maximize the desirability
function a multiple response optimization was performed to optimize several responses simultaneously,
which maximized the desirability function scores that ranged between 0 and 1 [32]. The RSM was used
to optimize and maximize the response variables, and a quadratic polynomial regression model was
assumed for predicting individual Y variables. The model proposed for each Y value was as according
to Equation (5):
4
4
3
4
βi Xi +
βii Xi2 +
βij Xi X j + ε,
(5)
Yi = β0 +
i=1
i=1
i=1 j=i+1
where β0 , βi , and βii represent the intercept, linear, and quadratic coefficients, respectively;
βij corresponds to the interaction coefficient terms for the interaction of variables i and j; Xi represents
the independent variables; and E denotes to the random error [31,32].
4.6. Statistical Analysis
Multiple regression analysis, ANOVA. canonical, and ridge maximum of data in the response
surface regression (RSREG) procedure was used. The estimated response surface and contours of the
estimated response surface were developed using the fitted quadratic polynomial equations obtained
from the response surface regression (RSREG) analysis, holding the independent variables with the
least effect on the response at a constant value and changing the levels of the other two variables [31,32].
A multiple-response optimization was performed to assess the combination of experimental factors that
simultaneously optimize several responses; as a result, maximization of the desirability function was
obtained, this function ranging from 0 to 1 [32]. Analyses were performed in triplicate considering the
standard deviation of each sample. The lack-of-fit test was carried out by comparison of the variability
of the current model residuals with the variability between observations at replicate settings of the
factors [31,32]. Statgraphics Centurion XV.II (Manugistics Inc., Rockville, USA) was used.
5. Conclusions
The physical–chemical analyses of refined commercial salmon oil indicate that it is a good quality
raw material that complies with the characteristics that are typical of oils of marine origin. A high
concentration of EPA and DHA was obtained from RCSO, achieving an increase of up to 4.1 and 7.9
times in the concentrate, with values of 31.20 and 49.31 g/100 g total FA, respectively. Interestingly
a 5.8-time increase was observed for the EPA+DHA content, from 13.78 to 80.51 g/100 g total FA.
Therefore, it has been proved that it is possible to maximize the EPA and/or DHA content by the
optimization of the variables in the urea inclusion process. The results of this study could serve as a
basis for the food and nutraceutical industry, whose processes with clean technologies can develop new
functional foods enriched with EPA and DHA. Furthermore, the consumption of such new functional
foods would be likely to produce a positive and profitable impact on the health of a wide range of
consumers such as pregnant women, infants, and older adults in general.
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Molecules 2019, 24, 1642
Author Contributions: Conceptualization, A.R., J.O.-V., S.P.A., and M.T.; methodology, E.C., A.R., M.M., and A.E.;
data curation, M.M. and A.R.; writing—original draft preparation, A.R., G.D.-R., and J.O.-V.; writing—review and
editing, A.R., J.O.-V., A.E., and G.D.-R.
Funding: This research was funded by the FONDECYT program (Government of Chile) throughout,
grant number 1181774.
Conflicts of Interest: The authors declare no conflict of interest.
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metabolism in yeast cells. Biol. Trace Elem. Res. 2019, 187, 316–327. [CrossRef]
Berríos, M.; Rodríguez, A.; Rivera, M.; Pando, M.; Valenzuela, M.A.; Aubourg, S.P. Optimization of rancidity
stability in long-chain PUFA concentrates obtained from a rainbow trout (Oncorhynchus mykiss) by-product.
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Sample Availability: Samples of the compounds are not available from the authors.
© 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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Review
Potential Use of Marine Seaweeds as Prebiotics:
A Review
Aroa Lopez-Santamarina, Jose Manuel Miranda *, Alicia del Carmen Mondragon,
Alexandre Lamas, Alejandra Cardelle-Cobas, Carlos Manuel Franco and Alberto Cepeda
Laboratorio de Higiene Inspección y Control de Alimentos, Departamento de Química Analítica, Nutrición y
Bromatología, Universidade de Santiago de Compostela, 27002 Lugo, Spain; a_lo_san@hotmail.com (A.L.-S.);
aliciamondragon@yahoo.com (A.d.C.M.); alexandre.lamas@usc.es (A.L.); alejandra.cardelle@usc.es (A.C.-C.);
carlos.franco@usc.es (C.M.F.); alberto.cepeda@usc.es (A.C.)
* Correspondence: josemanuel.miranda@usc.es; Tel.: +34-982-252-231 (ext. 22407); Fax: +34-982-254-592
Academic Editors: Sylvia Colliec-Jouault and Jesus Simal-Gandara
Received: 16 December 2019; Accepted: 21 February 2020; Published: 24 February 2020
Abstract: Human gut microbiota plays an important role in several metabolic processes and human
diseases. Various dietary factors, including complex carbohydrates, such as polysaccharides, provide
abundant nutrients and substrates for microbial metabolism in the gut, affecting the members and
their functionality. Nowadays, the main sources of complex carbohydrates destined for human
consumption are terrestrial plants. However, fresh water is an increasingly scarce commodity and
world agricultural productivity is in a persistent decline, thus demanding the exploration of other
sources of complex carbohydrates. As an interesting option, marine seaweeds show rapid growth
and do not require arable land, fresh water or fertilizers. The present review offers an objective
perspective of the current knowledge surrounding the impacts of seaweeds and their derived
polysaccharides on the human microbiome and the profound need for more in-depth investigations
into this topic. Animal experiments and in vitro colonic-simulating trials investigating the effects of
seaweed ingestion on human gut microbiota are discussed.
Keywords: prebiotic; polysaccharides; seaweed; Rhodophyceae; Phaeophyceae; Chlorophyceae
1. Introduction
Marine seaweeds have been consumed whole by East Asian populations for centuries, if not
millennia, appearing in traditional recipe books in many countries [1]. Additionally, their human
consumption in Western countries has been increasing in the latest decades because of their association
with improved human health. Some benefits of their consumption include a lower incidence of cancers,
decreased blood pressure and blood sugar, and antiviral, anti-inflammatory, immunomodulatory or
neuroprotective activities [1,2]. A mechanistic link proposed to explain the prevention of such diseases
by seaweed consumption implicates the presence of diverse health-promoting bioactive compounds in
seaweeds, including sulphated polysaccharides, polyphenols, pigments (chlorophylls, fucoxanthins,
phycobilins), carotenoids, omega-3 fatty acids or mycosporine-like amino acids [1,3,4]. In some cases,
these compounds are not produced by terrestrial plants or are not consumed in adequate quantities as
part of a typical Western diet.
Nowadays, with a continuously expanding world population, fresh water is an increasingly scarce
commodity, and the world’s desertification processes continue. About 45% of the world’s land surface
is considered drylands, while 12 million hectares of land are degraded yearly through lack of water and
related processes [5]. According to the Food and Agriculture Organization of the United Nations [6],
agricultural productivity is persistently declining at over 1% per year. It is thus reasonable to expect
that in the next decades there will be a need for increasing algae production to replace or supplement
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the intake of plant foods of terrestrial origin. Seaweeds have numerous advantages over terrestrial
plants, such as rapid growth rates, and they do not require arable land, fresh water or contaminating
fertilizers [7]. Additionally, an increase in seaweed cultivation would provide environmental services
as an added benefit [5].
Marine seaweeds constitute approximately 25,000–30,000 different species, with a great diversity
of forms and sizes [8]. The taxonomic groups, which reflect their pigmentation, include red algae
(Rhodophyceae), brown algae (Phaeophyceae) and green algae [8]. Although they are still produced
on a very modest scale relative to global food production, their worldwide cultivation has increased
rapidly in the last decades, reaching a current yearly production of about 29 million tonnes [8]. Asian
countries dominate world production, with 99% of the total, while most other maritime countries produce
little or none [5]. Polysaccharides account for the majority of seaweed biomass (up to 76% of dry weight
in some species; [8] and together with oligosaccharides have been the key focus of many studies of
seaweed-derived compounds. Phenolic compounds and proteins from seaweeds have also attracted
interest as potential functional ingredients [8]. Besides their consumption as an entire food, seaweeds or
their polysaccharides are considered valuable additives in the food industry because of their rheological
properties as gelling and thickening agents [2]. Additionally, seaweeds were largely employed in the
formulation of animal feed [9], as well as in the formulation of cosmetics, drugs and fertilizers [10].
Regarding specific benefits on human health, seaweeds have demonstrated to exert
preventive effects against several non-transmissible diseases such as cardiovascular diseases [11,12],
antihypertensive [13], anti-obesity effects [14] and anti-diabetic effects [15,16], anti-cancer [17,18] or
antioxidant activities [19].
Regarding cardiovascular diseases, there are large contributing risk factors that overlap and
intertwine, contributing overall to the onset and growth of the disease [11]. Between them, it can be
cited a cascade of mechanisms including vascular inflammation, oxidative stress, hypercoagulability
and activation of the sympathetic and renin-angiotensin systems. Although in some cases the
exact mechanisms through seaweeds can prevent cardiovascular diseases are not always fully
understood, it was demonstrated that seaweed consumption can prevent cardiovascular diseases [11,12].
Functional oligosaccharides from seaweeds are associated with a variety of biological processes
linked to hypoglycaemic and hypolipidaemic activities, although the concrete mechanisms have not
been well studied [15]. With respect to hypertension, various compounds from seaweeds, such as
protein-derived bioactive peptides and phlorotannins, can prevent hypertension by inhibition of
angiotensin-I converting enzyme activity [13].
The potential anti-obesity activity derived from seaweeds consumption may involve a large
variety of mechanisms and alterations in lipid metabolism, suppression of inflammation, suppression
of adipocyte differentiation and delay in gastric emptying [14]. Between then, an important anti-obesity
activity of seaweeds is the inhibition of peroxisome proliferator-activated receptor γ (PPARγ) expression
and activation of the adenosine monophosphate-activated protein kinase (AMPK) phosphorylation [13].
Other important anti-obesity mechanism of seaweeds is related with inhibition of lipases, especially
pancreatic lipase, that is one of the main therapeutic targets of anti-obesity drugs [14] and was recently
demonstrated for various seaweeds species [20]. Additionally, anti-obesity mechanisms related
with concrete seaweed components, such as phlorotannins, that target the inhibition of adipocyte
differentiation or fucosterol, that decreases the expression of the adipocyte marker proteins PPARγ and
CCAAT/enhancer-binding protein alpha were reported. Seaweeds can also prevent obesity by means of
the modification of the relative quantities of phyla in the gut microbiota (GM), and polysaccharides from
seaweeds can reduce obesity also by repairing the intestinal barrier and reducing inflammation [21].
Although abundant signaling pathways have been found to be involved in the process of glucose
metabolism and anti-diabetic effects [15,16], among them, the IRS1/PI3K/JNK/AKT/GLUT4 pathways
are important mechanisms in insulin signal transduction. Some seaweed components were shown
in animal models to ameliorate the hepatic insulin resistance by regulating the cited signaling
pathways [15,16,22]. In addition, seaweeds significantly increased the abundance and diversity of gut
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microbiota in animal models and showed the ability to increase the population of beneficial microbiota
and maintain the homeostasis of the GM [15,22].
With respect to the antioxidant activity of seaweeds, this protective affects depends mainly on
phlorotannins, secondary metabolites that exert their great antioxidant activity via the scavenging
of reactive oxygen species [19]. Secondary metabolites of seaweeds are also responsible for the
anti-cancer activity, whose includes different mechanisms such as repairing the intestinal barrier by
intensifying the expression of the tight junction proteins via increasing the phosphorylation of MAPK
and ERKT/2 genes [18], and activating the caspase cascades. Other potential mechanisms are reducing
the expression of cyclin-dependent kinases and matrix metalloprotease family [17] and inducing
decreased levels of pro-apoptotic metabolic signals [19].
2. Polysaccharides from Marine Seaweeds
Indigestible dietary polysaccharides attract attention as functional food ingredients with health
benefits [7]. Most carbohydrates entering the colon are fermented in the proximal colon, which is
considered a saccharolytic environment. As digesta moves through the distal colon, carbohydrate
availability decreases, and proteins and amino acids become the main metabolic energy sources
for bacteria in this region. The main end-products of saccharolytic fermentation are short chain
fatty acids (SCFA), which contribute towards the host’s daily energy requirements. On the contrary,
the end-products of proteolytic fermentation include metabolites such as phenolic compounds, and
nitrogenous ones like amines and ammonia, some of which are carcinogens. This means that the
GM exerts a key contribution to the human energy balance and nutrition, by extending the host
metabolic capacity to indigestible polysaccharides. In addition, intestinal microorganisms contribute to
develop and maintain the host immune system, defending the host from colonization by opportunistic
pathogens [2]. The effects of polysaccharides on the GM are generally evaluated by the contents of
SCFAs, the composition and the abundance of beneficial intestinal bacteria [20].
Polysaccharides in today’s human diet originate primarily from terrestrial plant cell walls, while
other sources, such as seaweeds, are less represented [8]. Studies indicate that polysaccharides and
oligosaccharides derived from seaweeds can modulate intestinal metabolism, including fermentation,
inhibit pathogen adhesion and evasion, and potentially treat inflammatory bowel disease [8,23]. Some
seaweed polysaccharides also demonstrate anticoagulant [24], antitumor [25], anti-inflammatory [26],
antiviral, antihyperlipidemic [27] or antioxidant activity [28]. Other research has focused on their
use as prebiotics to aid in limiting the occurrence of non-transmissible chronic diseases common
in Western countries, such as obesity, diabetes, cardiovascular diseases or some types of cancer [2].
Nevertheless, many seaweed fibres are high-molecular-weight polymers that need to be transformed
into oligosaccharides to increase their fermentability by the GM [2].
Depending on the taxonomic classification of algae, polysaccharides can vary greatly in their
composition [8]. Seaweeds feature an integrated network of biopolymers in their cell walls, mainly
formed by polysaccharides associated with other compounds, such as proteins, proteoglycans,
polyphenols and some mineral elements, like calcium and potassium [8]. It is because of this
complexity that for most seaweed polysaccharides, the exact structures, constituents and chemistry are
not fully known [29]. Depending on the algal taxa, both structural and storage polysaccharides may
vary. Structural polysaccharides are the most abundant, and their composition can be influenced by
the seaweed species [29], as well as environmental factors, such as salinity, water temperature and
sunlight intensity [30]. Some of the structural polysaccharides are carboxylated or sulphated, which
can affect their fermentability [2].
Green seaweeds contain mostly sulphated structural polysaccharides, like ulvans (the most
abundant, representing 8–29% of dry weight) and sulphated galactans, xylans and mannans. These
polymers are composed mainly of rhamnose, xylose, glucose, glucuronic acid and sulphates, with
smaller amounts of mannose, arabinose and galactose [31,32]. These polysaccharides are not fully
fermented by the human GM [33,34]. Conversely, the main carbohydrate in storage is starch.
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Contrariwise, brown seaweeds contain mainly cellulose, alginic acids, fucoidans and sargassans
as structural polysaccharides, while the storage polysaccharides are alginates [35] (the most abundant
at 17–45% of dry weight), fucoidans and laminarins [2,8,36]. Finally, red seaweeds contain agars,
carrageenans, xylans, sulphated galactans and porphyrins as main structural polysaccharides, while
the main storage polysaccharide is starch [21,37].
Through a long time of co-evolution between GM and host, the intestinal microbes have evolved
diverse strategies for degrading polysaccharides from terrestrial plants [38]. However, because the
consumption of seaweeds polysaccharides was not common over human evolution, the human GM
did not acquire the same efficacy to degrade seaweed polysaccharides. Thus, although humans
possess the enzymes necessaries to degrade some algal polysaccharides, such as starches, they
are unable to digest the most complex polysaccharides [2,38]. An elegant work carried out by
Hehemann et al. [39], showed that specific genes coding for enzymes with potential capacity to degrade
seaweed polysaccharides, as porphyranases or agarases, can be transferred from a member of marine
Bacteroidetes, Zobellia galactanivorans to the GM bacterium Bacteroides plebeius in Japanese individuals.
As a consequence, GM of those subjects acquires the ability to degrade porphyran and agarose, as
compared to the GM from North American individuals, who are incapable of degrading it [39].
De Jesus Raposo et al. [40] suggested that most seaweed polysaccharides can be regarded as
dietary fibre, as they are resistant to digestion by enzymes present in the human gastrointestinal
tract, reaching the distal gut. In this allocation, polysaccharides are fermented and become food for
the commensal bacteria, stimulating their growth. For this reason, great efforts have been placed
on developing efficient methods for seaweed polysaccharides extraction, purification and structural
characteristics elucidation in order to improve their bioavailability, especially for insoluble fibre [2].
It was previously shown that variations in the chemical structure of a prebiotic can impact its
selective fermentation by bacteria [41]. For this reason, there were published large works regarding
the investigation of the potential prebiotic effect of single polysaccharides, often contained in seaweeds,
but employing pure standards [42–44]. However, it should be considered that seaweeds contain other
components than also can affect GM. Consequently, trials employing whole seaweeds are required to
investigate their real effect in human GM. Furthermore, although several works investigated the effects of
seaweeds in livestock gut microbiota, they are more oriented to study the effects of seaweeds in animal
production and welfare. An elegant review was recently published where they can be checked [32].
3. Other Bioactive Compounds from Marine Seaweeds
In addition to polysaccharides, seaweeds also contain other bioactive compounds, called
secondary metabolites much of them with antioxidant activity [45]. Among these, polyoletides (such as
phlorotannins), isoprenoids (such as terpenes, carotenoids and steroids), alkaloids and shkimates (such as
flavonoids) are the main groups of secondary metabolites found in algae [46]. Compared with other
macroalgae, red seaweeds are richer sources of these secondary metabolites [47]. The human health
benefits afforded by these bioactive compounds include anti-inflammatory, antioxidant, anticoagulant,
antiviral, antimicrobial, antidiabetic, antitumor, antihypertensive, antiallergic and immunomodulatory
activities [45–48].
Exceptionally, phlorotannins or polyphenols are recognized as structural classes of polyketides,
found primarily in brown algae. These compounds can also reach the large intestine where GM can
convert them into beneficial bioactive metabolites. Phlorotannins are highly hydrophilic components
formed by polymerization of monomeric units of phloroglucinol (1,3,5-trihydroxybenzene). There
are six main groups: fucols, floretoles, fucofloretols, fuhalols, isofuhalols and eckols, and all display
strong antioxidant properties and act against oxidative stress [46]. Certain polyphenols are used as
prophylactics against problems such as cardiovascular diseases, cancers, arthritis and autoimmune
disorders [47]. In addition, some phlorotannins have been shown to decrease blood glucose levels
after carbohydrate-rich meals. This action is achieved by interfering with the enzymes amylase and
sucrase that intervene in the digestion and assimilation of these carbohydrates. In addition to their
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effects on the metabolic functions of the host, phlorotannins also appear to have some antibacterial
activities [47], which may explain the low production of AGCC derived from seaweeds in which they
are an important part [7]. However, like other phenolic compounds, bacterial growth inhibition occurs
selectively in microbial populations, including some pathogens, and its antibacterial effect is minor
in commensal bacteria [7]. Because of this selective inhibition of bacterial pathogens, large whole
seaweeds and seaweeds ethanolic extracts has been used to extend the shelf life of fresh fishery foods,
such as Fucus spiralis [48,49], Bifurcaria bifurcata [50], Cytoseira compressa [51] or Gracilaria verrucosa [52]
Another of its potentially therapeutic functions is that extracts with high phlorotannins content have
demonstrated a potent inhibitory action on the growth of cancerous cell lines [53,54].
Bromophenols present in marine algae have attracted much attention in the field of antimicrobial
agents [55]. Previous studies indicate that marine bromophenols possess promising antibacterial [56,57]
and antiviral activities [58]. In addition, symphyocladin G, a new bromophenol adduct derived from the
red seaweed Symphyocladia latiuscula, is found to have antifungal activity against Candida albicans [59].
Several bromophenols isolated from the red alga Odonthalia corymbifera, are promising candidates
for antifungal agents in crop protection [56]. These properties are not exclusive to red algae because
compounds, such as bis(2,3-dibromo-4,5-dihydroxybenzyl) ether, isolated from brown algae Leathesia
nana, showed cytotoxic activity against some cancer cells [54], and exhibited antibacterial activity
against several strains of Gram-positive and Gram-negative bacteria [56].
With respect to terpenes, there are about 200 different diterpenoids, of which some have important
cytotoxic and antiviral, antimicrobial and antiparasitic activities (such as against Leishmania) [60,61].
These compounds are found in red and brown algae.
Flavonoids and their glycosides are present in green, brown and red algae. These compounds
possess antioxidant properties and have demonstrated action against arteriosclerosis and cancer [45].
Within this group, fucoxanthin, β-carotene and violaxanthin stand out. Besides its strong anticancer
activity, fucoxanthin has promise in preventing obesity [62]. The correlation between a carotenoid-rich
diet and a low risk of cardiovascular and ophthalmological diseases has been supported by recent
research with different types of carotenoids in cellular systems and human intervention studies [63].
Specifically, flavonoids from Enteromorpha prolifera influenced the GM balance in diabetic mice,
increasing the presence of Alistipes, Lachnospiraceae and Odoribacter genera [63]. Alistipes spp. is one of
the most abundant bacterial genera in the mouse intestine and is capable of fermenting glucose and lactic
acid to produce propionic, acetic and succinic acid, which modulate the release of intestinal hormones,
thereby influencing the release of insulin and appetite. It is perhaps for this reason that E. prolifera is
traditionally used in China as a natural herb to treat diseases associated with inflammation [63]. It has
recently been observed that a polysaccharide of E. prolifera could be used as a novel agent to treat
obesity and hyperlipidaemia [62].
Other important secondary metabolites contained in seaweeds and responsible of important
beneficial effect in human health are peptides, such as lectins [48]. Lectins primarily show antiviral,
antibacterial, and antifungal activities. Specially one type of lectin, griffithsin, showed important
antiviral activity and is nowadays considered a promise antiviral agent, with great potential concerning
the prevention of sexually transmitted infections [48], including HIV [64]. Other important peptides
are renin inhibitor tridecapeptide [65] and dipeptide [66], which demonstrated hypotensive effect
dipeptide. Phycoerythrin [43] and kahalalide F [67] are other important peptidic compounds isolated
from seaweeds than showed antitumor effect.
Besides the so-called secondary metabolites, seaweeds contain other minor nutrients of immense
importance for human health. Phycobiliproteins, responsible for the characteristic bright pink
appearance of red algae, are classified into phycoerythrin (red) and phycocyanin (blue). These
pigments are used commercially in food, nutraceuticals, and for their therapeutic properties, mainly
antimicrobial, antioxidant, anti-inflammatory, neuroprotective, hepatoprotective, immuno-modulatory
and anticancer effects [67–72]. Such compounds may improve the efficacy of standard anticancer drugs,
decrease their side effects, and act as photosensitisers for the treatment of tumour cells [30].
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4. Effects of Seaweed Polysaccharides on Human Health
Compounds with prebiotic activity, such as oligosaccharides, lactulose, fructo-oligosaccharides
(FOS), inulin, galacto-oligosaccharides and arabinoxylano-saccharides are used as functional ingredients
in the food industry [10]. While most of the above compounds are now derived from terrestrial
plants, some studies have shown that polysaccharides and oligosaccharides derived from marine
algae can also modulate intestinal metabolism, including fermentation, inhibit adhesion and invasion
of pathogens, and treat inflammatory bowel disease [23,73]. Furthermore, these compounds have
demonstrated anticoagulant, antioxidant, immunomodulatory, antitumor and antiviral activities [10].
Being much less degradable by enzymes from the human upper gastrointestinal tract than
their terrestrial plant counterparts, polysaccharides from marine algae reach a greater proportion in
the descending colon. For this reason, some authors [42,74] have found that polysaccharides from
marine algae, such as alginate, agarose oligosaccharides and κ-carrageenan oligosaccharides, have
a higher prebiotic activity than FOS in vitro. Specifically, sulphated polysaccharides from marine algae
show anticoagulant, antiviral, antitumor, anti-inflammatory, antibacterial, immunological, antioxidant
and many other biological and physiological activities [8,75]. Sulphated polysaccharides include
fucoidans (l-fucose and sulphated ester groups) from brown seaweeds, agars and carrageenans
(sulphated galactans) from red seaweeds, and ulvans (sulphated glucuronoxylorhamnan) and other
sulphated glycans from green seaweeds [8].
The consumption of these sulphated polysaccharides can block the adhesion of leukocytes to the
epithelium of blood vessels, preventing the migration of these cells to the site of inflammation [76].
These polysaccharides often stimulate the growth and activity of beneficial bacteria by acting as
substrates for fermentation in the large intestine, leading to the production of SCFA, with multiple
functions that help maintain health [8]. As previously mentioned, seaweed polysaccharides differ in
their properties and compositions from one type of algae to another [46], so their effects on the human
GM will also differ.
The GM, especially in its most distal parts, harbors many bacteria, archaea, protozoa and viruses,
which along with their genetic material, is collectively referred to as the gut microbiome (GMB) [77].
This GM is composed of up to 12 different bacterial phyla of which more than 90% belong to the
Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes [78], while the remaining phyla are much
less constant and numerous [79]. The most frequent bacterial species in the colon, which is where
the highest bacterial concentration exists [78], belong mainly to the families Bacteroidaceae, Prevotellaceae,
Rikenellaceae, Lachnospiraceae and Ruminococcaceae [77]. The GM presents a diverse set of functions
important to human health, such as the extraction of energy from a broad spectrum of nutrients,
the production of vitamins, the promotion of immune homeostasis and the prevention of colonization
of the intestine by pathogens [80]. One of the most important functions of the GM is in the prevention
of chronic low-grade inflammation [81]. Host genetics define the chemistry and physics of the GM,
including the availability of nutrients and the threshold of activity required to induce an immune
response. Consequently, intestinal microbial communities are composed of species that have evolved
to occupy specific ecological niches in the gut, including the ability to metabolize specific molecules
available from the host or to evade host defenses [77].
Nutrients can interact directly with the GM to promote or inhibit its growth. In this sense,
the ability of the GM to extract energy from specific components of the diet offers a direct competitive
advantage to specific members of the GM, allowing them to proliferate at the expense of other
members [81]. Thus, diet affects not only the composition and absolute abundance of intestinal bacteria
but also their growth kinetics [82]. In this context, the most influential nutrients are indigestible
carbohydrates, which can be of both terrestrial and marine algae origin [81].
The human genome encodes a limited number of hydrolases capable of hydrolyzing the glycosidic
bonds of polysaccharides in dietary fibre (collectively referred to as CAZymes). Consequently,
many polysaccharides, such as resistant starch, inulin, lignin, pectin, cellulose and FOS, reach the
large intestine undigested. In contrast, the GMB codes tens of thousands of CAZymes. In the
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presence of bacteria harboring key enzymes involved in carbohydrate metabolism, these complex
polysaccharides can thus be degraded and metabolized in vivo [83]. The bacteria able to degrade these
complex polysaccharides are called primary degraders and include members of the genera Bacteroides,
Bifidobacterium and Ruminococcus, Roseburia, Facealibacterium, Anaerostides or Coprococcus. A relative
abundance of these genera in our GM infers that during a food shortage, these bacteria can alternate
between energy sources by using sensors and regulatory mechanisms that control gene expression [21,
81]. Hydrolases act on polysaccharides to generate oligosaccharides and monosaccharides. Secondary
fermentation of these compounds by the GM produces SCFA, specifically acetic, propionic, butyric,
lactic and succinic acids, which initiate a complex metabolic network [81].
The GM of hunter–gatherer, rural and agricultural populations are usually more bacterially diverse
than in modernized urban societies [84] and so require a greater functional repertoire to maximize their
energy intake from dietary fibres. Conversely, the consumption of a diet composed mainly of products
of animal origin causes an enrichment in the GM of genera of bile-tolerant bacteria, such as Alistipes,
Bilophila and Bacteroides, and the almost total exhaustion of bacteria that metabolize polysaccharides,
such as Roseburia, Eubacterium rectale subgroup and Ruminococcus bromii [81].
Clinical studies investigating prebiotic effects have some disadvantages with respect to ethical
constraints, as well as limited sampling possibilities from the colon and limited measurements of in
situ SCFA production. These concerns are commonly avoided by applying an in vivo approach [41],
that are the most common in the investigation of seaweed effects on human GM, as is described below.
Contrariwise, in vitro studies show important limitations because only represent the first step of a long
process, and the results observed in vitro can be magnified, diminished, or totally different in a more
complex and integrated system [48]. An additional limitation is that, due to the short fermentation time
in in vitro studies, they fails to capture the complete picture of cross-feeding interactions between gut
microbes, and which may not fully correlate with the long-term effects of seaweed compounds on GM [41].
4.1. Polysaccharides from Brown Seaweeds
Although the prebiotic and immuno-modulation properties of brown algae have been studied
both in animal models and in vitro, humans intervention studies are also needed to assess whether
there is a direct association between these uses of algae and the human GM, but are currently restricted
due to ethical concerns [78]. The most relevant results obtained from examining the impacts of brown
seaweeds on the GM can be found in Table 1. In this table it were included results about the prebiotic
effect of brown seaweed species from genus Ecklonia [7,85], Sargassum [86–88], Laminaria [82,89–92],
Ascophyllum [93–95], Fucus [23,63], Undaria [90], Saccorhiza [96] or Porphyra [97]. As can be seem in
Table 1, in most cases, the administration of whole brown seaweed or brown seaweed-extracted
polysaccharides resulted in an increase of SCFA production, stimulating of beneficial bacteria grown
such as Lactobacillus [7,82,85,86,95], Bifidobacterium [7,82,85,87,92] or Faecalibacterium [7,58,87]. In some
cases, the brown seaweed or brown seaweed-extracted polysaccharides also inhibited the growth of
potentially pathogen bacteria [73,86]. In some cases, it were reported other beneficial effects not strictly
related with action on GM, such as reducing serum inflammatory markers [23], reducing serum levels
of lipopolysaccharide-binding protein [44], increasing CAZymes [44], reducing activity of fecal bile
salt hydrolase activity [96], or reduced the expression or diabetes-related genes [15].
Laminar storage polysaccharides, typical of brown seaweeds, are low-molecular-weight, linear
polysaccharides composed of glucose units with a low degree of branching [79]. Besides affecting mucin
composition and SCFA concentration, laminins can affect the adherence, translocation and proliferation
of bacteria in the gut [98,99]. At the same time, laminins stimulate the proportion of Bifidobacterium,
which generates a prebiotic potential. In other research, laminarin has been shown to promote an
immune response [98], and could be useful for inhibiting the production of putrefactive substances
from undigested proteins [100]. In vitro batch fermentation of laminarin for 24 h promoted an increase
in Bifidobacterium and Bacteroides, and propionate and butyrate production [42]. Contradicting results
by other researchers indicated that laminarin was not selectively fermented by Lactobacillus and
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Bifidobacterium, but could modify the composition, secretion and metabolism of the jejunal, ileal, caecal
and colonic mucosa to protect against bacterial translocation [32]. In addition, laminarin increased the
presence of Clostridium spp. and Parabacteroides distasonis in rats [101].
An in vitro study conducted with the species Sargassum thunbergii revealed a dramatic increase in
the population of beneficial bacteria (from 17% to 28%), while a group of harmful Firmicutes decreased
from 75% to 64% after 48 h of fermentation [87]. No noticeable changes were found in Proteobacteria or
Actinobacteria. At the genus level, an increase in Lactobacillus, Bifidobacterium, Roseburia, Parasutterella
and Fusicatenibacter appeared after incubation for 24 h, followed by an increase in Faecalibacterium
and Coprococcus at 48 h of incubation [87]. Bifidobacterium, Coprococcus and Parasutterella have been
negatively correlated with non-alcoholic steatohepatitis, hepatocellular carcinoma and diabetes [102],
while Ruminococcus, Roseburia and Faecalibacterium are producers of butyric acid and are facilitate
the degradation of polysaccharides and fibres [103]. Fusicatenibacter was positively associated with
increased serum leptin in obese rats [104], which reduces their appetite. All these findings highlight the
prebiotic potential of S. thunbergii by its modulation of the composition and abundance of beneficial GM.
An in vitro study using S. wightii in MRS broth evaluated their antioxidant activity and prebiotic
score comparing L. plantarum and Salmonella Typhimurium relative growths. The study showed that
the prebiotic activity score was positive, promoting selectively the growth of L. plantarum with respect
to the pathogen S. Typhimurium. Specifically, a prebiotic effect by 1.42-fold more growth stimulation of
L. plantarum than S. Typhymurium [86].
In other work Chen et al. [58] showed an increase in fucoidan from A. nodosum in an in vitro assay
simulating the human digestive tract was due to an increase in Bacteroidetes, Firmicutes and SCFA. At
the genus level, the genera Bacteroides, Phascolarctobacterium, Oscillospira and Faecalibacterium increased,
while the levels of Fusobacterium, Megamonas, Parabacteroides, Clostridium and Dorea decreased relative to
the samples to which the algae A. nodosum had not been added [46]. demonstrated the in vitro prebiotic
activity of a mixture of fucoidans and alginates obtained from A. nodosum, leading to an increase in
the growth rate of L. delbrueckii and L. casei to levels similar to those observed after administration
of inulin, a standard commercial prebiotic [46]. Other authors [93] conducted a study in rats, which
were administered polysaccharides extracted from A. nodosum, and they were seen an increase in both
acetate, propionate and butyrate SCFAs.
According to Zaporozhets et al. [76], fucoidans obtained from F. evanescens stimulate the
colonic growth of beneficial Bifidobacterium species, such as B. longum B379M and B. bifidum
791B. Lean et al. [23] administered F. vesiculosus-derived fucoidan extracts to mice and, interestingly,
found a reduction in markers associated with inflammatory bowel diseases.
When Wister rats were fed with feed enriched with alginates or laminarins, An et al. [101] found a
notable decrease in the number of metabolites resulting from putrefaction, such as indole, H2 S and
phenol. This result was subsequently confirmed in both in vitro and rat models by Nakata et al. [100],
who also found a decrease in ammonium levels with alginate. At the phyla level, alginate increased
the levels of Actinobacteria, while laminarins increased the levels of Proteobacteria. At the genus
level, Bacteroides was markedly more abundant in the group fed with alginate, and B. capillosus was the
most frequent species. In rats fed with laminarin-enriched feed, Parabacteroides, Lachnospiraceae and
Parasutterella bacterium were detected in greater abundance than in control rats. Nguyen et al. [44]
studied laminarin supplementation in a mice high-fat diet. They could see a decrease in Firmicutes
and an increase in the Bacteroidetes phylum, especially the genus Bacteroides.
Ramnani et al. [94] performed in vitro fermentation with A. nodosum-derived alginates, which
increased Bifidobacterium and SCFAs. An increase in the proportion of Bacteroidetes to Firmicutes was
observed as well in fermentations added with sulphated polysaccharides extracted from A. nodosum
versus controls. Increased levels of Bacteroidetes and decreased levels of Firmicutes have been
associated with a reduced risk of obesity in humans [79].
122
Crude polysaccharide contained
carbohydrates 22.7%, sulphate
content of 17.1% and protein content
1.34%. Hydrolysates showed
25.1–26.7% carbohydrates, 25.3–25%
sulphate and 1.7–1.4%
protein contents
Average molecular weight 31.0 and
56.0 kDa
21% sulfate content; 1330 KDa
molecular weight; 7.3% Man, 24.1%
GlcA, 1.5% Glc, 7.2% Gal, 1.3% Xyl,
58.6% Fuc
48.7% total fibre, 16.1% non-digestible
non-starch polysaccharides, 1.3% total
starch, 43% total sugar, 3.8% protein
and 4.5% total phlotorannin
Polysaccharides extracted from 20 g
of Ascophyllum nodosum in a single
dose, compared to blank and
FOS-added samples
0.2 g of polysaccharides extracted
from A. nodosum per rat for 4 days,
comparing crude polysaccharide with
crude polysaccharide hydrolysates,
alginate and hydrolyzed alginate
1% w/v low molecular weight
polysaccharide derivatives extracted
from A. nodosum for 24 h. Inulin was
used as positive control and cellulose
as negative control
100 mg/kg/day of fucoidans obtained
from A. nodosum for 6 weeks.
Control group received saline solution
1.5% w/v of enzyme-assisted extracted
polysaccharides from Ecklonia radiata
for 24 h. Inulin and resistant starch
were used as positive controls
and glucose and cellulose were used
as negative controls
In vitro fermentation system
using fresh fecal samples from
four healthy donors
In vivo trials using 3 Wistar
male rats per sample, comparing
effect of A. nodosum crude
polysaccharide with hydrolyzed
A. nodosum polysaccharides.
SCFA were generated by
fermentation with Lactobacillus
plantarum BCC 5493 and
Enterococcus faecalis BCC 39,179
In vitro fermentation system
using fresh fecal samples from
three healthy donors
In vivo trial using 18 male
C57BL/6 mice. 6 mice received
fucoidans extracted fro
A. nodosum, and 6 acted as
blank group
In vitro fermentation system
using fresh fecal samples from
3 healthy donors
123
Increase of total bacteria,
Bifidobacterium, Lactobacillus
Fucoidans administration resulted in
a much more diverse cecal microbiota,
increase on Lactobacillus and
Talassospira, whereas
No significant changes in GM
Not provided
Increase in SCFA, acetate and
propionate in A. nodosum added
polysaccharides with respect to blank
samples and FOS-added samples
Increase in Bacteroidetes and
Firmicutes. At genus level, increase of
Bacteroides, Phascolarctobacterium,
Oscillospira, Faecalibacterium, while
decreased Fusobacterium, Megamonas,
Parabacteroides, Clostridium, Dorea
Total carbohydrate 42.3%; uronic acid
11%; protein 1.4% and sulfate content
23.9%; Monosaccharides content were
composed of Man, GlcA, Glc, Gal, Xyl,
and Fuc at a molar ratio of 16.65,
20.34, 1.60, 9.69, 3.44, and 48.29
Increase in total SCFA, acetic and
propionic acids
Fucoidans decreased the serum levels
of lipopolysaccharide-binding protein
Increase in total SCFA, acetic and
propionic acids
Increase in both acetic, propionic and
butyric acids, in this order. SCFA
were higher in the case of
polysaccharides with lower molecular
weight
Significant Changes in
Related Metabolites
Significant Changes in
Gut Microbiota
Polysaccharides Characterization
Seaweed, Dosage and Time
of Exposure
Type of Study
Table 1. Prebiotic effect of different species of brown seaweed.
[85]
[95]
[94]
[93]
[58]
Reference
Molecules 2020, 25, 1004
124
Fucoidans extracted from Fucus
vesiculosus intraperitoneally
(10 mg/kg/day) or orally
(10 mg/kg/day for high purity
fucoidan or 400 mg/kg/day for
focus-polyphenol) for 7 days
0.8 g of fucoidans obtained from
Laminaria japonica for 48 h. Blank
samples contained no polysaccharide
In vitro fermentation system
using fresh fecal samples from
three healthy donors
In vitro fermentation system
using fresh fecal samples from
three3 healthy donors
In vivo trail using 10 C57BL6
mice per group, with previously
induced colitis by
supplementing 3% w/v of
dextran sulphate sodium in the
drinking water for 8 days
Crude extract fraction: 14.4%, fibre,
5.6% non-digestible non-starch
polysaccharides, 20.6% sugar, 0.2%
ManA, 0.5% Man, 17.2% Glc, 0.5%
Gal, 0.3% Xyl, 1.8% Fuc, 4.6%
phlorotannin.
Phlorotannin-enriched fraction: 3.4%
fibre, 3.4% sugar, 3.4% Glc, 13.4%
phlorotannin.
Low molecular weight
polysaccharide-enriched fraction:
0.5% fibre, 0.4% starch, 22.7% sugar,
22.7% Glc, 2.5% phlorotannin.
Polysaccharides extracts obtained by
microwave-intensified enzymatic
process from 4.5 g of crude E. radiata
for 24 h. Four different seaweed
fractions were employed (crude
extract fraction,
phlorotannin-enriched fraction, low
molecular weight
polysaccharide-enriched fraction
and high molecular weight
polysaccharide-enriched fraction.
Inulin was used as positive control
and cellulose as negative control
Not provided
Fucus-polyphenol: 40.2% neutral
carbohydrates; 21.8% sulfates; 26.2%
polyphenols; 3.6% uronic acids and
203.1 kDa peak molecular
weight.High purity fucoidan: 59.5%
neutral carbohydrates; 26.6%
sulphates; <0.5% polyphenols; 1.4%
uronic acids; 61.8 kDa
molecular weight.
High molecular weight
polysaccharide-enriched fraction:
62.4% fibre, 22.8% non-digestible
non-starch polysaccharides, 0.3%
starch, 42.1% sugar, 1.9% GulA, 7.2%
ManA, 2.1% Man, 1.1% GlcA, 17.1%
Glc, 1.7% Gal, 1.5% Xyl, 9.4% Fuc,
1.7% phlorotannin.
Polysaccharides Characterization
Seaweed, Dosage and Time
of Exposure
Type of Study
Table 1. Cont.
Decrease in Enterobacter spp. while
increase in beneficial bacteria as
Lactobacillus and Bifidobacterium
Not provided
Increase of Bifidobacterium,
Lactobacillus, Clostridium coccoides in
all tested fractions with respect to
negative controls.
Low molecular weight
polysaccharide-enriched fraction
showed the better fermentative
results, obtaining better counts that
positive controls for Lactobacillus,
Faecalibacterium prausnitzii, C. coccoides
and Firmicutes
Significant Changes in
Gut Microbiota
Decrease in pH and increase in lactic
acid and SCFA, including acetic and
butyic acids
Both oral fucoidan reduced cytokines
associated with inflammatory bowel
disease such as interleukin-1α,
interleukin-1β, interleukin-10,
macrophrage inflammatory
protein-1α, macrophrage
inflammatory protein-1β, granulocyte
colony-stimulating factor
or granulocyte-macrophage
colony-stimulating factor
Total SCFA were higher in crude
fraction than all other fractions after
24 h fermentation. All fractions
except phlorotannin-enriched fraction
significantly increased SCFA
production with respect to
negative controls
Significant Changes in
Related Metabolites
[82]
[23]
[7]
Reference
Molecules 2020, 25, 1004
Not provided
Not provided
Not provided
Not provided
2% w/w of laminarins for 2 weeks.
Black samples received control diet
1 g laminarins from Laminaria digitata
for 24 h. Glucose was used as
negative control and FOS as positive
control
Supplementation with 10% of dried
L. japonica for 4 weeks. Control rats
were fed with basal diet
Mice received normal diet, high-fat
diet or high-fat diet added with
laminarins at 1% w/w in a high-fat
diet ad libitum for 4 weeks. After
finishing, highly-fat diet was
provided for an additional 2 weeks
Polysaccharides isolated from
Laminaria digitata crude or
depolymerized (1% w/v for 48 h).
Cellulose was used as negative
control and FOS as positive control
In vivo trial using six male
Wistar rats per group
In vitro fermentation system
using fresh fecal samples from
three healthy donors and an
in vivo trial using 20 Wistar rats
In vivo trial using 28 female
Sprague-Dawley rats
In vivo trial using six female
BALB/C mice per group
In vitro fermentation system
using fresh fecal samples from
three healthy donors
Not provided
Fucoidans from L. japonica 18.4%
sulfate content; 310 KDa molecular
weight; 11.2% Man, 7.3% GlcA, 5.2%
Glc, 19.3% Gal, 2.9% Xyl, 54.1% Fuc
100 mg/kg/day of fucoidans obtained
L. japonica for 6 weeks. Control group
received saline solution
In vivo trial using 18 male
C57BL/6 mice. Six mice received
fucoidans extracted Laminaria
japonica, and six acted as
blank group
Polysaccharides Characterization
Seaweed, Dosage and Time
of Exposure
Type of Study
Table 1. Cont.
125
Increase in total SCFA, and butyric
acid. Lower production of acetic acid
propionic acids
Reduction in Firmicutes to
Bacteroidetes ratio and decrease of
pathogenic bacteria such as
Clostridium, Escherichia and
Enterobacter
Increase Parabacteroides, Fibrobacter
and Lachnospiracease and decrease in
Streptococcus, Ruminococcus and
Peptostreptococcaceae in
laminarin-added samples
Increase in SCFA with respect to
cellulose-added samples, but similar
SCFA content or even lower with
respect to FOS-added samples
Mice fed with laminarin
supplementation showed
significantly higher CAZyme families
in feces
[89]
Increase in total SCFA in
laminarin-added culture medium
than in glucose-added. Laminarins
supplementation increased the colon
luminal content of mucin, while
decreased luminal mucin in jejunum,
ileum and caecum in rats
No significant differences were
obtained in the in vitro trial for GM
composition.
Decrease in Firmicutes and increase in
Bacteroidetes phylum, especially
the genus Bacteroides in
laminarin-added fed mice with
respect to controls
[101]
Increase organic acids, specially
propionate, whereas decreased cecal
putrefactive compounds (indole,
phenol and H2 S)
Increase of Bacteroides capillosus,
Clostridium ramosum y Parabacteroides
distasonis
[91]
[44]
[90]
[95]
Reference
Decreased in the serum levels of
lipopolysaccharide-binding protein
Significant Changes in
Related Metabolites
Increase in the abundance of
Ruminococcaceae
Significant Changes in
Gut Microbiota
Molecules 2020, 25, 1004
Reduced activity of fecal bile
salt hydrolase activity and secondary
bile acids
S. confussum administration
significantly reduced the gene
expression of JNK1 and JNK2
in hepatic cells and increased
expression of IRS1 and PI3K
Not provided
Increased gut bacterial diversity in
treated hamsters. Significant increase
in Barnesiella, Tannerella, Eubacterium
and Clostridium XIVa, with significant
decrease in Allobaculum, Bacteroides,
and Clostridium IV in the S. confussumadded group.
Not provided
Not provided
Sulfated oligosaccharide
containing galactose,
sulfated galactose, sulfated
anhydrogalactose and methyl
sulfated galactoside
Not provided
Polysaccharides extracted from
Porphyra haitanensis (250 mg/kg) for 2
weeks. Control mice received 0.9%
normal saline at a dose of 20
mL/kg/day. Positive controls received
the same plus combined
Bifidobacterium, Lactobacillus and
Streptococcus thermophilus tables,
500 mg/kg.
5% w/w polysaccharides extracted
from Saccorhiza polyschides
with high-fat diet for 8 months
150 mg/kg body weight of Sargassum
confussum solution once daily for 60
days by intragastric administration
Extracts from Sargassum multicum (1%
w/v for 24 h). FOS was used as
positive control and no carbon source
was added to the negative control
1% w/v of enzymatic-extracted
polysaccharides from Sargassum
wightii in MRS broth for 48 h
In vivo trial using 24 male
C57BL/6J mice
In vivo trial using 16 male
C57BL/6 mice fed with
a high-fat diet
In vivo trial using
Syrian golden hamsters
In vitro fermentation system
using fresh fecal samples from
three healthy donors
126
In vitro test comparing
the growth of L. plantarum
NCIM 2083 with respect to
Salmonella Typhimurium MTCC
3224
53.5% fiber, 13.2% protein, 2.3% fat,
28.9% ash. Content of Cel, Fru and
Gluc (not specific proportions)
Prebiotic effect by 1.42-fold
more growth stimulation of
L. plantarum than Salmonella
Typhimurium
Increase Bacteroides and Prevotella, and
decrease in Clostridium coccoides and
Eubacterium rectale
Increase of Prevotellaceae Rikenellaceae
and Lactobacillus, while decreased
Lachnoclostridium or Lachnospiraceae
Inhibition of Salmonella Typhimurium,
Listeria monocytogenes or Vibrio
parahaemolyticus adhesion and
invasion
Not provided
Increase in SCFA and lactic acid
production with respect to
negative controls
Not provided
Not provided
Increase of SCFA, acetic propionic
and butyric acids and decrease of pH
in a similar way of the other
beta-glucans assayed
Glu residues with degree of
polymerization between 22 and 25
and 5 kDa as average molecular
weight
Increase of all Bifidobacteria with
respect to initial counts in a similar
way of the other beta-glucans assayed
Supplementation with 0.5% w/v and
0.1% w/v of sodium alginate and
laminarins extracted from Eisenia
bicyclis for 18 h.
β-Glucan > 95%, protein 3%;
monosaccharides: 98% Gluc; 2% Man.
6 kDa as average molecular weight
In vitro fermentation system in
cellular lines
using human-enterocyte-like29-Luc cells
Significant Changes in
Related Metabolites
Beta-glucans from L. digitata (0.5% w/v
for 24 h) compared to barley β-glucan,
Curdlan from Alcaligenes faecalis,
mushroom sclerotia from Pleurothus
tuber-regium and inulin
Significant Changes in
Gut Microbiota
In vitro fermentation system
with individual bifidobacteria
including B. infantis JCM 1222;
B. longum JCM 1217 and
B. adolescentis JCM 1275
Polysaccharides Characterization
Seaweed, Dosage and Time
of Exposure
Type of Study
Table 1. Cont.
[86]
[88]
[15]
[96]
[97]
[73]
[92]
Reference
Molecules 2020, 25, 1004
Increase in total SCFA, and butyric
acid. Lower production of acetic acid
propionic acids
Reduction in Firmicutes to
Bacteroidetes ratio and decrease of
pathogenic bacteria such as
Clostridium, Escherichia
and Enterobacter
68.3% carbohydrate; 0.3% protein;
3.5% sulfate: Monosaccharide molar
ratio: 3.9% arabinose; 6.2% Gal, 3.2%
Glc, 15.6% Xyl, 14.8% Man 15.6%
GulA, 40.6% GlcA. Average molecular
weight 4.8 kDa
Not provided
200 mg polysaccharides extracted
from Sargassum thunberguii for 48 h
Supplementation with 10% of dried
Undaria pinnatifida and for 4 weeks.
Control rats were fed with basal diet
[90]
[87]
Reference
BCC: Biotec Culture Collection; BCC: British Culture Collection; Cel: Cellobiose; Gal: galactose; GlcA: galacturonic acid; Glc: glucose; GulA: guluronic acid; FOS: fructooligosaccharides;
Fru: fructose; Fuc: fucose; JCM: Japan Collection of Microorganism; kDa: kilodaltons; ManA: Mannuronic acid; Man: mannose; MRS: Man, Rogosa and Sharpe; MTCC: Microbial Type
Culture Collection and Gene Bank; NCIM: National Centre of Integrative Medicine; SCFA: Short chain fatty acids; Xyl: Xylose.
In vivo trial using 28 female
Sprague-Dawley rats
Decrease of pH and increase in total
SCFA and acetic, propionic, butiric
and n-valeric acids
Decrease of Firmicutes, while increase
of Bacteroidetes and beneficial
bacteria such as Bifidobacterium,
Roseburia, Parasutterella and
Fusicatenibacter after 24-h
fermentation, and increase of
Faecalibacterium and Coprococcus after
48-h fermentation
In vitro fermentation system
using fresh fecal samples from
three healthy donors
Significant Changes in
Related Metabolites
Significant Changes in
Gut Microbiota
Seaweed, Dosage and Time
of Exposure
Type of Study
Polysaccharides Characterization
Table 1. Cont.
Molecules 2020, 25, 1004
127
Molecules 2020, 25, 1004
Evidence that probiotic bacteria in the gastrointestinal tract utilize dietary alginate was reviewed
by Shang et al. [38]. Among the studies, Kuda et al. [73] found that supplementation with sodium
alginate and laminarin of brown algae inhibited the adhesion and invasion of pathogens, such as
S. Typhimurium, Listeria monocytogenes or Vibrio parahaemolyticus. Other authors reported an increase
in Lactobacillus and Ruminococcus in the intestine of mice fed fucoidans from A. nodosum, besides a
reduction in the opportunistic Peptococcus bacteria [95].
In vitro fermentation experiments conducted by Charoensiddhi et al. [85] demonstrated
the growth-promoting effect of E. radiata extracts on beneficial bacteria, such as Bifidobacterium,
Lactobacillus and Clostridium coccoides, and SCFAs production was stimulated as well. Later, the same
authors [7] found increased levels of beneficial bacteria, such as Bifidobacterium, Lactobacillus and C. coccoides
associated with the phlorotannin-enriched fermentation of E. radiata. Higher numbers of Lactobacillus,
Faecalibacterium prausnitzii, C. coccoides, Firmicutes and E. coli were observed for phlorotannin-supplemented
fermentation compared with inulin fermentation [7]. In contrast, the number of Enterococcus in both
fermentations decreased approximately ten-fold relative to the initial counts.
Other authors tested the effects of supplementation of two brown algae (U. pinnatifida and
L. japonica) on the GM and body status of laboratory rats [90]. In both instances, the animals’ body
weight was reduced, which was thought to be mediated by the influence of the seaweed on the
composition of the intestinal microbial communities associated with obesity, reducing the proportion of
Firmicutes with respect to Bacteroidetes, and the populations of pathogenic bacteria, such as Clostridium,
Escherichia and Enterobacter [90]. Similarly, L. japonica increased beneficial bacteria and SCFA, and
decreased the pH level [82], while β-glucans extracted from L. digitata increased Bifidobacterium and
propionic and butyric acids in vitro, in addition to lowering pH [92].
β-Glucans obtained from another Laminaria species (L. digitata) were able in an in vitro test [92]
to increase Bifidobacterium and propionic and butyric acids, in addition to lowering pH. A study
by Strain et al. [91] in vitro investigated the effect of a polysaccharide-rich raw extract obtained
from L. digitata. A significant alteration of the relative abundance of several families, including
Lachnospiraceae and genera such as Streptococcus, Ruminococcus and Parabacteroides of human faecal
bacterial populations was seen. Concentrations of acetic acid, propionic acid, butyric acid and total
SCFA were significantly higher.
Finally, Huebbe et al. [96] conducted a study on mice that were administered polysaccharides
from S. polyschides with a high-fat diet. A metabolic improvement was seen including normalization of
blood glucose, reduction of plasma leptin, reduction of fecal bile salt hydrolase activity and secondary
bile acids in these mice.
4.2. Polysaccharides from Red Seaweeds
The most relevant results obtained from the investigation of red seaweeds effect on GM can be
found in Table 2. In this table, results about the prebiotic effect of red seaweed species from genus
Acanthopora [86], Gracilaria [105,106], Kappaphycus [107], Euchema and Grateloupia [10], Chondrus [57],
Gelidium [94], or Osmundea [88] were included.
As can be seem in Table 2, as was described previously for brown seaweeds, administration
of red seaweeds or seaweed-extracted polysaccharides resulted in an increase of SCFA production,
stimulating of beneficial bacteria grown such as Lactobacillus [86] or Bifidobacterium [57,94,107], whereas
inhibited the growth of potentially pathogen bacteria [57,86]. It was also reported red seaweeds activity
on the prevention of naproxen-induced gastrointestinal damage [106].
Agarose stands out among the polysaccharides isolated from red algae that cannot be digested
by human intestinal enzymes. When seaweed is consumed, whether as an edible food or food
additive, agarose reaches the most distal portions of the gastrointestinal tract, where it is fermented
and metabolized by the GM [108,109]. As described by Ramnani et al. [94], low-molecular-weight
agarose exerted a prebiotic effect in vitro by promoting the growth of Bifidobacterium and increasing
SCFA concentrations in the medium.
128
Molar mass distribution was found to
be within 2.6 × 106 and
3.8 × 105 g/mol, while the soluble
carbohydrate, protein, and sulfate
contents were 85.5%, 2.5%, and
8.4%, respectively
Average molecular weight 923.3 kDa,
sugar content 0.11%
100 mg of sulphated polysaccharides
obtained from Gracilaria rubra for 24 h.
Basal nutrient medium was used for
control negative group and FOS was
used for control positive group
In vivo trial using male Wistar
rats (six per group)
In vitro fermentation system
using fresh fecal samples from
four healthy donors
62.1% sugar; 21.4% sulfate;
Monosaccharides at molar ratio: 0.01
Man, 0.01 GluA, 1% Gal, 0.09% Xyl,
0.01% Fuc, 0.03% Glu
Rats were pretreated with 0.5%
carboxymethylcelloluse (controls) or
0.5% w/v of of sulphated
polysaccharides from Gracilaria birdiae,
twice daily for 2 days. After 1 h,
naproxen (80 mg/kg) was
administered twice a day for 2 days
0.1–0.5% w/v Eucheuma spinosum for
24 h
In vitro test in MRS broth for
Lactobacillus and Bifidofacterium
compared to MHB broth for
Staphylococcus aureus and
Escherichia coli
Not provided
41.9% sugar; 20.6% sulfate;
Monosaccharides at molar ratio: 0.01
Man, 0.02 GluA, 1% Gal, 0.1% Xyl,
0.05% Fuc, 0.07% Glu
Fed added with 0.5–2.5% (w/w) whole
Chondrus crispus for 21 days
In vivo trial using male
Sprague-Dawley rats
(six per group)
45.9% fiber, 10.9% protein, 1.6% fat,
39.4% ash. Only Glu was found as
monosaccharide
0.1–0.4% w/v Grateloupia filicina added
in culture media for 24 h
1% w/v of enzymatic-extracted
polysaccharides from Acanthopora
spicifera in MRS broth for 48 h
In vitro test comparing
the growth of Lactobacillus
plantarum NCIM 2083 with
respect to Salmonella
Typhimurium MTCC 3224
Polysaccharides Characterization
In vitro test in MRS broth for
Lactobacillus and Bifidofacterium
compared to MHB broth for
S. aureus and E. coli
Seaweed and Dosage
Type of Study
129
Prevention of
naproxen-induced gastrointestinal
damage determined by macro- and
microscopic findings
Increase in total SCFA and acetic,
propionic and isobutyric acids
Increase of Bacteroidetes,
Bacteroidaceae, Prevotellaceae,
Ruminococcaceae and propionic acid,
while decrease Fusobacteriaceae and
Lachnospiraceae. At genus level,
increase of Bacteroides, Prevotella and
Phascolarctobacterium
Not provided
Increase in beneficial bacteria at all
concentrations, without significant
differences between 0.4% and 0.5%.
No inhibition was detected
against pathogens
No relevant variation was observed in
GM populations
Not provided
[105]
[106]
[10]
[10]
[57]
Increase in total SCFA and acetic,
propionic and butiric acids in rats fed
with C. crispus at 0.5% and 2.5%.
Higher concentrations of all SCFA
were found in the case of rats fed
added 2.5% of C. crispus with respect
to rats fed added with 0.5% of
C. crispus
Increase of Bifidobacterium brevis and
decrease of pathogens such as
Clostridium septicum and Streptococcus
pneumoniae
Increase in beneficial bacteria with
better results at 0.1% concentration.
No inhibition was detected
against pathogens
[86]
Reference
Not provided
Significant Changes in Metabolites
Prebiotic effect by 0.84-fold
more growth stimulation of
L. plantarum than S. Typhimurium
Significant Changes in
Gut Microbiota
Table 2. Prebiotic effect of different species of red seaweed.
Molecules 2020, 25, 1004
Average molecular weight 143.8 kDa
Average molecular weight 20.1 kDa
and 6.5 kDa, respectively
Not provided
Not provided
1% w/v low molecular weight
polysaccharide derivatives extracted
from Gracilaria spp. for 24 h. Inulin
was used as positive control and
cellulose as negative control
1% w/v low molecular weight
polysaccharide derivatives extracted
from Gelidium sesquipidale for 24 h.
Inulin was used as positive control
and cellulose as negative control
1% w/v Kappaphycus alvarezii for 24 h
Extracts from Osmundea pinnatifida
(1% w/v for 24 h). FOS was used as
positive control and no carbon source
was added to the negative control
In vitro fermentation system
using fresh fecal samples from
three healthy donors
In vitro fermentation system
using fresh fecal samples from
three healthy donors
In vitro fermentation system
In vitro fermentation system
using fresh fecal samples from
threehealthy donors
Increase in SCFA, acetic and
propionic acids
Increase in SCFA
Increase in Bifidobacterium, decrease in
Clostridium coccoides and Eubacterium
rectale
Increase in Bifidobaterium counts
Both G. sesquipidale extracts (20.1 kDa
and 6.5 kDa molecular weight)
significantly increased total SCFA,
acetic and propionic acids
Increase in total SCFA, acetic and
propionic acids
Significant Changes in Metabolites
Only G. sesquipidale of 6.5 kDa
significantly increased
Bifidobaterium counts
No significant changes in GM
Significant Changes in
Gut Microbiota
[88]
[107]
[94]
[94]
Reference
Gal: galactose; Glc: glucose; GulA: guluronic acid; FOS: fructooligosaccharides; Fuc: fucose; GM. Gut microbiota; kDa: kilodaltons; Man: mannose; MRS: Man, Rogosa and Sharpe;
MTCC: Microbial Type Culture Collection and Gene Bank; NCIM: National Centre of Integrative Medicine; SCFA: Short chain fatty acids; Xyl: Xylose,
Polysaccharides Characterization
Seaweed and Dosage
Type of Study
Table 2. Cont.
Molecules 2020, 25, 1004
130
Molecules 2020, 25, 1004
Bajury et al. [107] conducted an in vitro colon model in which they evaluated the prebiotic capacity
of K. alvarezii. This study showed an increase in SCFA (particularly acetate and propionate) and
Bifidobacterium. In the other hand, decrease in C. coccoides and E. rectale. These results suggested that
K. alvarezii might have the potential as a prebiotic ingredient. A study published by Zhang et al. [110]
focused on the beneficial effect of low-melting-point agarose (in the form of neoagaro-oligosaccharides)
on the GM during the relief of intense exercise-induced fatigue in mice. Results showed the abundance
of Bacteroidetes and Proteobacteria increased and decreased, respectively, during the attenuation of
fatigue and its associated gastrointestinal problems. Ladirat et al. [111] found that mice fed 2.5% (w/v)
neoagaro-oligosaccharides for seven consecutive days achieved a much more pronounced increase
in the population of Lactobacillus spp. and Bifidobacterium spp. in their GM relative to those fed 5%
(w/v) FOS for 14 consecutive days. Likewise, it was demonstrated that agaro-oligosaccharides could
be used as a prebiotic to encourage the growth of beneficial strains of bacteria, such as B. adolescentis
ATCC 15703 and B. infantis ATCC 15697. Low-molecular-weight agar has demonstrated a bifidogenic
effect, along with an increase in SCFA acetate and propionate concentrations, after 24 h of in vitro
fermentation with human faeces inoculant [94].
Another type of polysaccharide with prebiotic function found in red algae are the group of
carrageenans, which are derived from D-galactose, and approved as food additives [79]. In rats fed
2.5% C. crispus, of which carrageenan is a major polysaccharide, B. brevis, as well as SCFA, increased
considerably, while pathogens Clostridium septicum and Streptococcus pneumonia noticeably decreased
compared with the basal diet [57]. Elevation of plasma immunoglobulin levels was also found in rats
fed with C. crispus, resulting in improved host immunity. Consistent with the prebiotic activity of
carrageenan, carrageenans isolated from red algae G. filicina and E- spinosum promoted the growth of
Bifidobacterium [10].
Research led by Di et al. [105] found that the polysaccharides of Gracilaria rubra increased
the relative abundances of Bacteroides, Prevotella and Phascolarctobacterium in vitro compared with
the control group. Bacteroides spp. assists the host with degrading polysaccharides and contains
codifying genes of glucosidase enzymes [39]. Prevotella is another beneficial genus with the potential to
participate in the metabolism and utilization of plant polysaccharides. The genus Phascolarctobacterium
is associated with the production of SCFA [110].
Many other bacterial genera, such as Legionella, Sutterella, Blautia, Holdemania, Shewanella and
Agarivorans, were decreased as a consequence of intake of C. crispus supplements in rats [57]. Decreases
in the presence of Streptococcus were also observed. In conclusion, carrageenans from C. crispus
could act as a fermentable substrate for probiotic bacteria present in the gastrointestinal tract,
thereby promoting the growth of probiotic groups, while inhibiting certain groups of pathogenic
bacteria [57]. Another study in chickens described an overall impact of administering whole red algae
(Sarcodiotheca gaudichaudii and C. crispus) on the intestinal mucosa, increasing the height and surface
of the villi in these animals [112]. Moreover, the abundance of beneficial bacteria, such as B. longum
and Streptococcus salivarius increased, while some harmful bacteria species, such as C. perfringens,
decreased [112] Rodrigues et al. [88] used extracts from the red algae O. pinnatifida and S. muticum in
an in vitro fermentation system, which increased the production of acetate and propionate, and the
population of Bifidobacterium. In work published by Silva et al. [106], in which extracts of sulphated
polysaccharides from G. birdiae were administered to laboratory rats, gastrointestinal damage induced
by naproxen was prevented, although it did not produce notable variations in the GM of these rats.
An in vitro study using A. spicifera in MRS broth evaluated their antioxidant activity and prebiotic
score with L. plantarum and S. Typhimurium. The study showed that the prebiotic activity score
was positive, promoting the growth of L. plantarum and suppressing the growth of the pathogen
S. Typhimurium. Specifically, a prebiotic effect by 0.84-fold more growth stimulation of L. plantarum
than S. Typhymurium [86].
131
Molecules 2020, 25, 1004
4.3. Polysaccharides from Green Seaweeds
Unlike brown and red algae, the current evidence for the fermentation capacity of green algae
and their polysaccharides is scarce, partly because their fermentation requires a specific activity of
α-l-rhamnosidase in the gastrointestinal tract, which is infrequent [113]. The most relevant results
obtained from the investigation of green seaweeds effect on GM can be found in Table 3. In this table,
results about the prebiotic effect of green seaweed species from genus Enteromorpha [38,82,86,113–115]
and Ulva [115] were included. Administration of green seaweeds or seaweed-extracted polysaccharides
also resulted in an increase of SCFA production, stimulating of beneficial bacteria grown such as
Lactobacillus [38,86,115], Bifidobacterium [38], or Akkermansia [38] whereas inhibited the growth of
potentially pathogen bacteria [81,114]. Other beneficial actions were reported such as decrease
lipopolysaccharide-binding protein in female mice [38], diminished histopathological lesions of
inflammatory infiltrations in distal colon [114], or modulating diabetes-related genes expression in
diabetic mice [22].
Ulvans are one of the most frequent polysaccharides in green algae. This polysaccharide is a
water-soluble sulphated heteropolysaccharide [79]. Ulvans contains sulphate and uronic acids, and so
produce undigestible ionic colloids, has ion-exchange capacity and can bind to bile acids, consequently
increasing the excretion of bile acids with cholesterol-lowering or antihyperlipidemic effects [2,79].
Antioxidant and immunomodulatory properties are other beneficial actions elicited by ulvans [116,117].
Ulvans has also been studied for its possible prebiotic potential. Kong et al. [82] performed an
in vitro assay using Enteromorpha with a high content of ulvans, but there were no noticeable variations
in the populations of Enterococcus, Lactobacillus and Bifidobacterium compared with controls. In contrast,
in a recent in vitro faecal fermentation analysis, ulvans stimulated the growth of Bifidobacterium and
Lactobacillus populations and promoted the production of SCFA, such as lactic and acetic acids [42].
In a previous study by Ren et al. [114], both whole Enteromorpha and polysaccharides extracted
from Enteromorpha improved inflammation associated with loperamide-induced constipation in mice.
In those mice, the GM showed an increase in Firmicutes and Actinobacteria compared with the control
mice, whereas the relative amounts of Bacteroidetes and Proteobacteria decreased.
In work by Shang et al. [38], an extract of E. clathrata was administered to mice, resulting in
marked decreased concentrations of genera, such as Enterobacter, Staphylococcus and Streptococcus.
Surprisingly, such supplementation also dramatically reduced the population of A. muciniphila in the
intestine. These observations indicate a possible unfavorable effect of these polysaccharides on the GM.
Contrarily, these polysaccharides were reported to increase the abundance of A. muciniphila, Bacteroides,
Alloprevotella, Ruminococcaceae and Blautia in the intestinal tract of mice, and decrease the abundance of
Peptococcus, Rikenellaceae and Alistipes [96,109].
An in vitro faecal fermentation of xylans derived from Palmaria palmata reported that xylose
was fermented after 6 h, and the SCFA content increased simultaneously [32]. This study did not
determine the bacterial composition. Nonetheless, Xylans and xylo-oligosaccharides extracted from
terrestrial plants, such as wheat husks and corn, are considered potential prebiotics due to evidence of
bifidogenesis, improved plasma lipid profile and positive modulation of immune function markers
in healthy adults [118].
132
133
Not provided
Not provided
Not provided
Not provided
Polysaccharides extracted from Ulva
prolifera (250 mg/kg) for 2 weeks.
Control mice received 0.9% normal
saline at a dose of 20 mL/kg/day.
Positive controls received the same
plus combined Bifidobacterium,
Lactobacillus and Streptococcus
thermophilus tables, 500 mg/kg.
0.8 g of fucoidans obtained from
Enteromorpha prolifera for 48 h. Blank
samples contained no polysaccharide
Loperamide at a dosage of
9.6 mg/kg/twice a day via oral gavage
for 2 weeks was provided to mice to
induce slow-transit constipation in
mice. Afterwards, E. prolifera and
polysaccharides extracted from E.
prolifera added in fed at a 1:5 w/v ratio
was administered for 7 days
Treated mice were fed with high
sucrose/high fat diet for 5 weeks.
Next type-2 diabetes was induced by
intraperitoneal administration of
streptozotocin at 45 mg/kg for 3 days.
Diabetic mice were administered with
150 mg/kg E. prolifera extracts or its
flavonoid-rich fractions less than 3
kDa, respectively, for 4 weeks
In vivo trial using 24 male
C57BL/6J mice
In vitro fermentation system
using fresh fecal samples from
three healthy donors
In vivo trial using 24 Kunming
female mice
In vivo trial using 24 Kunming
male mice
Flavonoids from E. prolifera reduced
blood glucose in mice, reduced
mRNA expressions of JNK1/2 gene
and increased the expression of PI3K,
IRS1 and AKT genes in diabetic mice
Both E. prolifera and E. prolifera
extracts diminished histopathological
lesions of inflammatory infiltrations
in distal colon. Both E. prolifera and
E. prolifera extracts reduced serum
levels of nitric oxide (inhibitory
neurotransmitter) and showed
laxative effects
E. prolifera increased bacterial
diversity, Bacteroidales, Firmicutes,
Actinobacteria, and decreased
Bacteroidetes and Proteobacteria.
Extracts from E. prolifera increased
Prevotellaceae, Firmicutes,
Actinobacteria, and decreased
Bacteroidetes and Proteobacteria
E. prolifera extracts increased the
proportion of Alistipes, Lachnospiraceae
and Odoribacter, while both extracts
reduced the proportion of
Ruminiclostridium and Akkermansia in
GM of diabetic mice
Not significant changes
Not provided
Not provided
Decrease in Enterobacter spp. in
E. prolifera fucoidans-added samples
Polysaccharides supplementation
decreased Tenericutes, and
Cyanobacteria. At genus level,
decreased Lachnospiraceae,
Lactobacillus, Mollicutes and
Mucispirillum, while increased
Prevotellaceae and Rickenellaceae
Prebiotic effect by 1.44-fold
more growth stimulation of
Lactobacillus plantarum than Salmonella
Typhimurium
[22]
[114]
[82]
[115]
[86]
[38]
Reference
Gal: galactose; GlcA: glucuronic acid; Glc: glucose; GM: gut microbiota; kDa: kilodaltons; Man: mannose; MRS: man Rogosa and Sharpe; MTCC: Microbial Type Culture Collection and
Gene Bank; NCIM: National Centre of Integrative Medicine; Rha: rhamnose; SCFA: Short chain fatty acids; Xyl: Xylose.
60.6% fiber, 16.9% protein, 1.2% fat,
25.4% ash. Monosaccharide content
included cellobiose, fructose, glucose
and maltose
1% w/v of enzymatic-extracted
polysaccharides from Enteromorpha
compressa in MRS broth for 48 h
In vitro test comparing
the growth of Lactobacillus
plantarum NCIM 2083 with
respect to Salmonella
Typhimurium MTCC 3224
E. clathrata supplementation
decreased lipopolysaccharide-binding
protein in female mice but not in male
mice
Molecular weight 11.67 kDa; 14.7%
sulfate content. Monosaccharide
composition: 1.0% Man, 49.7% Rha,
10.8% GlcA; 29.9% Glc; 1.3% Gal; 7.2%
Xyl
Enteromorpha clathrata, 100 mg/kg/day
or 50 mg mg/kg/day for 4 weeks
Increase of Akkermansia muciniphila,
Bifidobacterium spp., and Lactobacillus
spp. E. clathrata supplementation
induced much less alteration in the
composition of female GM than in
male GM
In vivo trial using 36 C57BL/6J
mice, 18 males and 18 females in
different trials
Significant Changes in Metabolites
Significant Changes in
Gut Microbiota
Seaweed, Dosage and Time
of Exposure
Type of Study
Polysaccharides Characterization
Table 3. Prebiotic effect of different species of green seaweed.
Molecules 2020, 25, 1004
Molecules 2020, 25, 1004
E. clathrata is an edible green seaweed possessing polysaccharides with numerous bioactivities,
including anticoagulant, immunomodulatory, antioxidant, anticancer and anti-obesity effects [38].
It was reported that the polysaccharides of E. clathrata exerted diverse prebiotic effects on A. muciniphila,
Bifidobacterium and Lactobacillus in male and female mice [38]. The results were most evident in the
male mice because of a sex-specific effect on the GM, as sex hormones play a key role in determining
the composition of intestinal microorganisms [109]. In other work from the same authors, male mice
were supplemented with polysaccharides of E. clathrata in the diet, which increased the abundance of
Bacteroides, Prevotella, Alloprevotella, Eubacterium and Peptococcus, and decreased the proportion of the
cancer-related Helicobacter [109]. In the female counterparts, the abundance of Odoribacter, Clostridium
IV, Oscillibacter and Alistipes spp. increased, and the proportions of beta-proteobacteria decreased [38].
An in vitro study using E. compressa in MRS broth evaluated their antioxidant activity and
prebiotic score with L. plantarum and S. Typhimurium. The study showed that the prebiotic activity
score was positive, promoting the growth of L. plantarum and suppressing the growth of the pathogen
S. Typhimurium. This seaweed exhibited the highest score of prebiotic activity (1.44-fold), stimulating
the growth of L. plantarum than S. typhimurium [86].
The natural products of marine macroalgae have shown notable antidiabetic potential by interfering
with carbohydrate metabolism. For example, E. prolifera contains many bioactive compounds, such
as sulphated polysaccharides, which could improve glucose metabolism, in addition to displaying
anti-inflammatory, antiviral and anticoagulant functions [22].
5. Conclusions
Although substantial evidence of the prebiotic effect of seaweed and seaweed extracts has
been published in recent years, these studies have been performed using in vitro digestion systems
simulating the human colon, or in animal models. Animals, such as mice or rats, differ widely
from humans in the GM composition, immune function, diets, metabolism and other key aspects,
so extrapolating the results obtained from animal models to humans may not be valid. In vitro
systems replicate more similarly the human intestinal microbiota, but are less-dynamic systems than
the real human colonic environment. Additionally, other factors should be considered, such as the
possible effect of other secondary compounds contained in seaweeds on the GM composition, or the
potential to transfer genes from marine bacteria to human GM bacteria coding for enzymes that could
degrade seaweed polysaccharides. Thus, not all people will respond equally after seaweed ingestion.
The decrease in terrestrial agriculture and disposable water is likely to increase the consumption
of algae by humans in the near future. Meanwhile, there is a profound need for more in-depth
investigations into the potential prebiotic effects of marine seaweeds and their derived polysaccharides
on the human GM.
Author Contributions: Conceptualization, J.M.M. and A.C. Literature data collection, A.C.A. and
A.L.-S. Writing—original draft, A.L.-S. and A.d.C.-M. Writing—review and editing, A.C.-C., C.M.F., and
A.L. Supervision: J.M.M. and A.C. All authors have read and agreed to the published version of the manuscript.
Funding: The authors thank the European Regional Development Funds (FEDER), grant ED431C 2018/05, and
Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo (CyTED), grant PCI2018-093245 for covering
the cost of publication.
Conflicts of Interest: The authors declare no conflict of interest.
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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/).
140
molecules
Review
Avocado Oil: Characteristics, Properties,
and Applications
Marcos Flores 1, *, Carolina Saravia 2, *, Claudia E. Vergara 1 , Felipe Avila 3 , Hugo Valdés 4 and
Jaime Ortiz-Viedma 5, *
1
2
3
4
5
*
Departamento de Ciencias Básicas, Facultad de Ciencias, Universidad Santo Tomás, Avenida Carlos Schorr
255, Talca 3473620, Chile; claudiavergarava@santotomas.cl
Escuela de Nutrición y Dietética, Facultad de Salud, Universidad Santo Tomás, Avenida Carlos Schorr 255,
Talca 3473620, Chile
Escuela de Nutrición y Dietética, Facultad de Ciencias de la Salud, Universidad de Talca, Talca 3460000,
Chile; favilac@utalca.cl
Centro de Innovación en Ingeniería Aplicada, Departamento de Computación e Industrias,
Universidad Catolica del Maule, Avenida San Miguel 3605, Talca 3480112, Chile; hvaldes@ucm.cl
Department of Food Science and Chemical Technology, Faculty of Chemical and Pharmaceutical Sciences,
University of Chile, Santos Dumont 964, Santiago 8380494, Chile
Correspondence: marcosflores@santotomas.cl (M.F.); csaraviam@santotomas.cl (C.S.);
jaortiz@uchile.cl (J.O.-V.); Tel.: +56-7123-424-18 (M.F.)
Academic Editor: Jose M. Miranda
Received: 9 May 2019; Accepted: 8 June 2019; Published: 10 June 2019
Abstract: Avocado oil has generated growing interest among consumers due to its nutritional and
technological characteristics, which is evidenced by an increase in the number of scientific articles that
have been published on it. The purpose of the present research was to discuss the extraction methods,
chemical composition, and various applications of avocado oil in the food and medicine industries.
Our research was carried out through a systematic search in scientific databases. Even though there
are no international regulations concerning the quality of avocado oil, some authors refer to the
parameters used for olive oil, as stated by the Codex Alimentarius or the International Olive Oil
Council. They indicate that the quality of avocado oil will depend on the quality and maturity of the
fruit and the extraction technique in relation to temperature, solvents, and conservation. While the
avocado fruit has been widely studied, there is a lack of knowledge about avocado oil and the
potential health effects of consuming it. On the basis of the available data, avocado oil has established
itself as an oil that has a very good nutritional value at low and high temperatures, with multiple
technological applications that can be exploited for the benefit of its producers.
Keywords: avocado oil; oil extraction; antioxidants compounds; fatty acid profile
1. Introduction
Avocado (Persea americana Mill.) is a fruit native to Central America, grown in warm temperate
and subtropical climates throughout the world. The pulp of this fruit contains about 60% oil, 7% skin,
and approximately 2% seed [1,2]. The main producers of avocado oil in the world are New Zealand,
Mexico, the United States, South Africa, and Chile [3]. Avocado oil has sparked a growing interest in
human nutrition, food industry, and cosmetics. The lipid content, mainly of monounsaturated fatty
acids, is associated with cardiovascular system benefits and anti-inflammatory effects [4,5].
There are no internationally defined parameters for avocado oil. The values that are commonly
used are those recommended for olive oil. The quality standard for olive oil is available in the Codex
Alimentarius and the International Olive Oil Council (IOC) [6].
Molecules 2019, 24, 2172; doi:10.3390/molecules24112172
141
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Molecules 2019, 24, 2172
Woolf et al. [7] proposed a classification for avocado oil based on its extraction method and fruit
quality. Avocado oil of a higher quality, “extra virgin”, corresponds to that produced from high-quality
fruit, extracted only with mechanical methods, using a temperature below 50 ◦ C and without the use
of chemical solvents. “Virgin” avocado oil is produced with fruit of a lower quality (with small areas of
rot and physical alterations), extracted by mechanical methods, using a temperature below 50 ◦ C and
without the use of chemical solvents. “Pure” avocado oil is a type of oil for the production of which
the quality of the fruit is not important; it is a bleached and deodorized oil, infused with the natural
flavor of herbs or fruits. Finally, “mixed” avocado oil is combined with olive, macadamia, and other
oils. Therefore, it presents sensory and chemical characteristics that are variable.
The Mexican norm [8] states that the “crude oil of avocado” is a slightly amber-colored fatty
liquid, obtained by physical extraction of the pulp and the seed of the fruit (Persea americana). “Pure”
edible avocado oil is a product with at least 98.5% refined avocado oil.
In this work, a systematic review of the literature was carried out to collect, select, evaluate,
and summarize all available evidence regarding the processes and properties of avocado oil.
The research question was: What are the most published topics on avocado oil? The answer to
this question allowed us to include topics, such as: extraction methods (i.e., cold pressed method,
ultrasound-assisted aqueous extraction method, supercritical CO2 method, CO2 subcritical method,
enzymatic extraction, and solvent extraction), procedures of conservation, contamination/adulteration,
technological applications, composition (characteristics according to the variety and origin of the fruit,
physicochemical characterization, avocado seed oil, and comparison with other oils), and biological
effects (human health effects and experimental studies in animals). Figure 1 shows the exponential
increase of scientific interest in avocado oil. On the topic, “avocado oil” (from 1980 to date), 180 and
224 articles have been published in the Web of Science (WoS) and Scopus, respectively.
Figure 1. Graphic of articles (topic: “avocado oil”) from 1980 to 2019 in the Web of Science (WoS) and Scopus.
The purpose of this research is to produce a complete profile on avocado oil, including extraction
methods, physicochemical characteristics, nutritional properties, as well as various applications in
the food and medicine industries. Avocado oil has proven to be a vegetable oil with a composition
of major and minor components that are highly appreciated by the population, either at low or high
temperatures, with multiple technological applications.
2. Extraction Methods for Avocado Oil
The information presented in this section focuses on the process efficiency, improvement of
production performance, and product quality as well as applications in the food industry.
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Considering the high humidity percentage of avocado (around 70 to 80%), the influence of the
pulp drying method prior to oil extraction has been studied [9]. The quality parameters (peroxide value,
iodine value, amount of oleic acid, refractive index, electrical conductivity, content of carotenoids,
chlorophyll, phenolic compounds, and antioxidant activity) have shown better results when the pulp
is dried at 60 ◦ C under vacuum, and the extraction is performed by the Soxhlet method. Meanwhile,
the bioactive compounds were best preserved when the avocado pulp was dried at 60 ◦ C with air
ventilation and mechanical pressing [10]. On the other hand, avocado pulp oil, pressed and dried in a
microwave, presented a better quality—determined by the acidity index, peroxide index, and oxidative
stability—when compared with oil obtained by extraction with ethanol. The composition of fatty acids
did not differ significantly when analyzing oil obtained by drying under microwaves or in a drying
oven with forced air circulation [11]. According to Chimsook and Assawarachan [12], the studied
drying method of the avocado pulp, prior to the extraction of the oil, does not significantly influence the
composition of fatty acids. However, changes were determined in the antioxidant activity and vitamin
E content of cold-pressed avocado oil (from Thailand). Higher antioxidant activities and a higher
vitamin E content were observed in oil, the pulp of which was dried with hot air, when compared
to oils obtained by an air-dried and vacuum process. This study is consistent with the fact that oils
from the fortune avocado variety, obtained by the pulp drying lyophilization method, resulted in lower
concentrations of α-tocopherol, squalene and β-sitosterol, as well as higher relative concentrations of
campesterol and cycloartenol acetate, compared to oils obtained through hot air-drying processes [13].
2.1. Cold Pressed Method
According to the CODEX STAN 19-1981 [14], the method of extraction of edible vegetable oils is
characterized by mechanical procedures, for example, extrusion or pressing, without the application of
heat. In addition, the oil can only be purified by washing, sedimentation, filtration, and centrifugation.
In the cold pressing method, oil recovery is only obtained from the parenchyma cells of the
pulp; its rupture begins in the first stages of grinding and it can be seen that the idioblastic cells
(oil carriers) remain intact during the process of extraction. The extraction yield increases when the
pulp is beaten at 45.5 ◦ C for 2 h [15]. In this method, a lower extraction yield is obtained, although
with higher concentrations of α-tocopherol and squalene, as well as lower contents of campesterol and
cycloartenol acetate, compared to the Soxhlet method [13]. Drying by lyophilization and subsequent
extraction by the Soxhlet method allows for a better extraction performance. However, when drying
by lyophilization and extracting by cold pressing, oils with a greater concentration of antioxidants, and
other bioactive compounds were obtained [13].
2.2. Ultrasound-Assisted Aqueous Extraction Method (UAAE)
This method uses the cavitation forces produced by acoustic waves to break down the cell walls
of the oil-containing cells. This process allows for the generation of an emulsion, which facilitates oil
extraction. This method can be carried out using an ultrasonic bath or an ultrasonic horn transducer [16].
The high frequency ultrasound conditioning (0.4, 0.6, and 2 MHz, 5 min, 90 kJ/kg) of the avocado
puree can improve the oil separation and potentially reduce the beating time in industrial processes,
without affecting the quality of the oil. If this treatment is applied after shaking, the extractability of the
oil increases by between 2% and 5%. The oils obtained from sonicated purees showed free fatty acids
(FFA) and peroxide values below the levels of industrial specification (peroxide less than 20 meqO2 /kg)
and an increase in total phenolic compounds after a 2 MHz treatment [17]. The ultrasound-assisted
aqueous extraction (UAAE) of low virgin avocado oil in FFA, considered as virgin avocado oil,
is that obtained by mechanical or natural means at low temperatures (<50 ◦ C) and without chemical
refining [7]. The optimal UAAE parameters to produce the highest extraction of virgin avocado oil
was 6 mL/g water-dried pulp powder, 30 min of sonication time at 35 ◦ C. The sonicated virgin avocado
oil was lighter and had a higher level of unsaturated fatty acids, compared to the avocado oil extracted
by the Soxhlet method [16].
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2.3. Supercritical CO2 Method
This method of extraction is based on the use of supercritical fluids, substances that are,
in certain circumstances, in a state in which they have intermediate properties between liquid
and gas. Supercritical CO2 (scCO2 ) is a totally innocuous gas, which becomes a powerful solvent under
conditions of pressure and at a temperature above its critical point [18].
Extraction with scCO2 presents a higher performance at a pressure of 400 bar. The use of ethanol
as a co-solvent favors the extraction of residual oil, benefiting the extraction of a fraction enriched in
tocopherols [19].
Some authors [20,21] proposed the combined extraction of avocado oil and active compounds present
in peppers (capsanthin) and tomatoes (lycopene) using scCO2 in order to enrich the avocado oil. For this,
a fixed bed extractor was used, where the lipids and the desired active ingredient were subjected to
the extraction process, simultaneously with scCO2 . First, both the scCO2 and the oil extracted from the
avocado passed through the avocado bed and then through the second bed, where the plant component
to be co-extracted was found. The lipids obtained in the first chamber served as a co-solvent with scCO2
for extraction in the second chamber. In the case of the simultaneous extraction of edible avocado oil and
the capsanthin (carotenoid) of red pepper, the higher concentration of oil improved the extraction yield of
capsanthin. However, a less concentrated extract was obtained, since the carotenoid was diluted in the
product. In the case of the extraction of avocado oil rich in lycopene, the extraction yield of lycopene
increased as the proportion of avocado in the first extraction chamber increased, being the best condition
for the extraction of lycopene present in tomato pomace at 400 bar and 50 ◦ C.
Restrepo et al. [22] evaluated the quality of avocado oil extracted by Soxhlet, cold pressed,
and scCO2 methods, determining the quality of the oil in terms of free fatty acid titration, peroxide
index, iodine index, saponification, and specific gravity, according to the American Oil Chemists’
Society (AOCS) standards. Extraction with supercritical fluids was the technique by which the highest
yields and quality were obtained. Oils extracted by scCO2 were characterized as possessing a lower
acidity index (0.48%), low oxidation of unsaturated fatty acids (16.87 meqO2 /kg) and higher iodine
index (80.18 cgI2/g), when compared with the other methods. In addition, extraction by cold pressing
showed better results in terms of vitamin E content.
Regarding extraction by pressurized fluids, the extraction with the liquefied gas of compressed
oil (LPG), constituted by a mixture of propane, n-butane, isobutane, ethane, and other hydrocarbons,
showed a higher oil extraction performance in less time and with a lower solvent consumption
than the scCO2 method. On the other hand, the oil obtained by compressed LPG presented higher
concentrations of Stigmasterol, licopersene, palmitic acid, oleic acid, and linoleic acid. However, scCO2
provided a higher yield in terms of antioxidant activity, which was determined by means of the
2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical assay [23].
2.4. CO2 Subcritical Method
Extraction with sCO2 operates under the same principle as the scCO2 extraction, but with
a temperature below 31.1 ◦ C and CO2 pressure of 72.9 bar [24].
In this part of the review, a comparison of the physicochemical properties of avocado oil,
extracted through sCO2 , UAAE, and conventional solvent (AOAC 920.39 [25]) will be analyzed. Extraction
with sCO2 was performed at 27 ◦ C and 68 bar CO2 , UAAE was performed with 60 mL of distilled water,
an ultrasonic power of 240 W and a frequency of 40 kHz for 30 min at 35 ◦ C, followed by a final pressing.
Compared to solvent extraction, oils extracted using sCO2 and UAAE had higher iodine index values but
lower melting points, determined by slip, free fatty acid content, and saponification index values. The oils
extracted by sCO2 and UAAE have a clear color and higher levels of unsaturated fatty acids than the oil
extracted with hexane. Regardless of the extraction method, the main fatty acids in avocado oils were
oleic and palmitic acids, while the main triacylglycerols in avocado oils were palmitoyl-dioleoyl-glycerol
(POO; 22.48–23.01%) and palmitoyl- Oleoyl-linoleoyl-glycerol (POL; 17.64–18.23%) [24].
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2.5. Enzymatic Extraction
In order to improve the performance of extraction by centrifugation, the incorporation of enzymes,
such as pectinases, α-amylase, proteases, and cellulase, to avocado paste have been considered.
The yield varies depending on the concentration and type of enzyme used and the reaction time
and percentage of water used. It is emphasized that this method improves oil by up to 25 times,
in comparison with the performance of a non-enzymatic centrifugation [26].
2.6. Solvent Extraction
Reddy et al. [27] compared four extraction methods to produce avocado oil (Hass and Fuerte variety).
They analyzed: (1) the extraction with traditional solvent using Soxhlet (5.0 g of dried avocado sample
with 250 mL of hexane for 24 h); (2) Ultrasonic Soxhlet extraction (5.0 g of dry avocado sample, sonicated
in a water bath at 60 ◦ C, with hexane as the solvent, for 1 h); (3) Soxhlet extraction, combined with a
microwave treatment (avocado paste 5 mm thick, extended in the rotating plate of a domestic microwave
oven, heated to the maximum power for 11 min, with 5 g of the resulting mass subsequently extracted
by means of the Soxhlet method with hexane); and (4) extraction with supercritical fluid (Argon and
scCO2 used as extraction fluids, extractions performed for 2 h, with a fluid flow rate of 2.8–3.5 mL/min).
The traditional Soxhlet extraction method yields the most reproducible results, whereas the microwave
extraction showed a higher extraction yield and higher fatty acid content (69.94%).
Meyer and Terry [28] performed a sequential extraction and quantification of fatty acids
and avocado sugars. The average oil yield using Soxhlet extraction, with ethanol as the solvent,
was significantly higher than the oil obtained by homogenization with hexane, and the fatty acid
profiles for the two methods were similar. As the maturity of the fruit increases, the extraction of oil
is improved. After lipid removal, methanolic extraction was superior in terms of the sucrose and
perseitol obtained, compared to extraction with 80% ethanol (v/v). The extraction of mannoheptulose
was not affected by any of the solvents used.
The yield of avocado oil extraction has been assessed, comparing four extraction methods using
solvents of different polarities. The extraction was performed using the Soxhlet method, with (1)
petroleum ether, (2) homogenization with petroleum ether, (3) homogenization with a mixture of
chloroform/methanol (2:1 v/v), and finally (4) extraction with chlorine-naphthalene and ball milling.
It was determined that methods that only use petroleum ether as an extraction medium presented
lower yields (6–9% less) than the last two methods. Saponifiable residues were lower when the method
using the chloroform/methanol mixture was employed. However, this method did not completely
eliminate the residual oil from the fruit [29].
Ortiz-Moreno et al. [30] analyzed the effect of four extraction methods on the chemical-physical
quality of avocado oil, namely, (1) the method of microwave extraction and manual pressing,
(2) extraction with hexane using the Soxhlet methodology, (3) microwave extraction, combined
with the Soxhlet methodology, using hexane as the solvent, and (4) extraction with acetone. The method
with the highest oil extraction performance was the third method. The amount of trans fatty acids
produced by the first method was the lowest and the latter method is also the one that generates the
least physicochemical alterations.
When analyzing the effect of the drying method and avocado oil extraction process, ripe fruit,
independent of the drying method, presents a higher extraction performance than immature fruit.
This is influenced by the enzymatic degradation of the cell wall of the parenchyma during maturation.
Freeze drying improves the amount of oil extracted for the scCO2 extracts and, to a lesser extent, for the
hexane extracts. Extraction with hexane has been shown to have a higher oil extraction yield than scCO2
due to the lower degree of selectivity of this solvent, which completely penetrates the plant material [31].
Regarding the performance of the avocado oil extraction process, methodologies have been proposed
for developing countries. One is carried out with boiling petroleum ether (30–60 ◦ C) and another extraction
with distilled water (avocado paste, diluted at a ratio of 3:1 and 5:1 (w/w), heated in a water bath of 75
to 98 ◦ C, with subsequent centrifugation. In the extraction methods, calcium chloride, sodium chloride,
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calcium carbonate and calcium sulfate were used as extraction aids. The presence of inorganic salts at a low
concentration improves the extraction performance, provided that it does not exceed 5%; otherwise, it has
an adverse effect. The most efficient extractions were obtained with a water/avocado ratio of 5:1, pH of 5.5
and centrifugal force of 12,300× g, with the addition of 5% calcium carbonate or calcium sulfate. At higher
heating temperatures (75–98 ◦ C), the oil release time decrease. In addition, the gravity sedimentation for
four days at 37 ◦ C, followed by centrifugation, improves the oil extraction performance [32].
Considering the use of organic solvents in avocado oil extraction processes could alter the quality of
fatty acids by inducing the formation of trans isomers. Ariza-Ortega et al. [33] proposed the application
of infrared spectroscopy by Fourier transform (FTIR) to study trans fatty acids in the avocado oils
of the Hass, Fuerte, and Criollo varieties. For this, oil extraction was performed by centrifugation at
40 ◦ C and extraction with hexane at 70 ◦ C for 4 h. The method using centrifugation did not increase
the deterioration of fatty acids. A strong band at 723 cm−1 was documented, which is attributable
to the cis functional groups, where the green color was maintained. On the other hand, the infrared
spectroscopy with Fourier transform (FTIR) analysis identified an absorption band, located at 968 cm−1 ,
which is associated with fatty acids, with trans isomerism for the Fuerte variety extracted with hexane.
3. Procedures for the Conservation of Avocado Oil
The conservation of oils is a necessary issue to address, since it allows for increasing the useful
life of the products. One of the efforts made to improve the conservation of avocado oil has been the
use of physical techniques, such as the electric field.
The electric field (voltage 9 kV cm−1 , frequency 720 Hz, time of 5 and 25 min) allows the polyphenol
oxidase enzyme present in the avocado pulp to be inactivated, preserving the components present in
the avocado oil. The modifications in the quality of the refined oil (established according to the acidity
index, peroxides, and iodine) are minimal, considering the electric field method as an alternative for
the addition of synthetic antioxidants [34].
The oxidative stability (determined by finding the antioxidant activity reducing ferric ion, FRAP),
during the storage of cold-extracted avocado oil in the presence of the oleoresins of Capsicum annuum L.
(vegetable material rich in carotenoids), was assessed. It was determined that the optimal extraction of
carotenoids was at a concentration of 1:3 (w/v: Capsicum annuum L/avocado oil) for 48 h in darkness
at room temperature. The behavior of the oil under stronger conditions (45 ◦ C, 30 days) showed the
following characteristics: (1) the extracts were stable to lipid oxidation, with a Totox index total value
of 27.34, (2) 85.6% of carotenoids were conserved, (3) 80.66% of the antioxidant activity was retained,
and (4) there was a color change (ΔE) of 1.783. The oleoresins obtained by extraction with avocado oil
can be considered as an economic and sustainable alternative for the extraction of carotenoids, with a
good oxidative stability, compared with organic solvents [35].
4. Use of Analytical Techniques in the Quantification, Adulteration, and Contamination of Avocado Oil
There is growing interest among consumers in accessing quality and authentic products.
Vegetable oils can suffer from contamination and/or adulteration, which causes the product to have
components not specific to the oil. It is here that the development, implementation, and application of
analytical technologies are very useful.
The components present in avocado oil, such as fatty acids and phytosterols, have been
quantified, mainly by gas chromatography, coupled with a flame ionization detector (GC-FID).
In addition, techniques, such as ultra-high-performance liquid chromatography (UHPLC), coupled with
mass spectrometry (UHPLC-MS) or a photodiode array detector (UHPLC-PDA), as well as
Inductively Coupled Plasma Mass Spectrometry (ICP-MS), have been used for the identification
and/or quantification of analytes, such as polyphenols, squalene and minerals, respectively [36,37].
Other analytical techniques have been used for the qualitative determination of the components present
in avocado oil, including 13 C nuclear magnetic resonance spectroscopy (NMR), which has been used
for the identification of its major components, including fatty acids [38]. At the same time, 1 H Nuclear
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magnetic resonance spectroscopy (1 H-NMR) has been used for the detection of the minor components
present in other vegetable oils [39]. Therefore, the development of new analytical methodologies for
quantifying the analytes present in avocado oil represents a major challenge.
Rohman et al. [40] studied the purity of avocado oil, adulterated with palm oil and canola oil, through
FTIR, combined with chemometric techniques. FTIR combined with multivariate calibrations can be used
to detect and quantify the adulteration of avocado oil in binary mixtures with palm oil and canola oil.
The adulteration of avocado oil with soybean oil or grape seed oil can be determined using
mid-infrared spectroscopy, combined with the statistical method of partial least squares discriminant
analysis. This methodology allows for a simple and fast discrimination of avocado oil in binary mixtures
and Tertiary oils. The frequency selected for the authentication of avocado oil was 1500–750 cm−1 , with a
precision of 100% for the analysis of the mixture of two oils and 93.3% for the mixture of three oils [41].
Organophosphorus pesticides in samples of commercial avocado oil were determined using
atmospheric pressure microwave-assisted liquid–liquid extraction (APMAE), with solid-phase extraction
or low-temperature precipitation, as the clean-up step. The analysis was carried out by gas
chromatography-flame photometric detection and gas chromatography-tandem mass spectrometry.
Chlorpyrifos residues were detected in one of four samples of commercially packaged avocado oil,
produced in Chile [42]. While spectroscopic techniques have focused on determining the adulteration of
avocado oil with the presence of other types of vegetable oil, according to the literature reviewed here,
there is a research deficiency related to the modification of the composition of avocado oil, including the
study of its major components, such as triacylglycerides and/or fatty acids, in addition to its minority
components, such as phytosterols, alkanes, aliphatic alcohols, polyphenols, and others. This could provide
information for detecting the contamination of avocado oil with other oils of a different quality.
5. Technological Applications of Avocado Oil
At the industrial level, there is a constant demand for the production of healthy foods that can
maintain their nutritional properties over time, as well as environmentally friendly technological
solutions. Avocado oil is mainly sold for direct consumption due to its interesting contribution of
fatty acids, vitamins, antioxidants, among other compounds. Efforts have been made to develop
products based on avocado oil. Arancibia et al. [43] propose the development of O/W nanoemulsions
using the natural emulsifiers, lecithin and synthetic tween 80, systems that improve the characteristics
with respect to traditional emulsions, such as (i) increased dispersibility of water in the encapsulated
oils, which generates slightly turbid emulsions and an easy production, and (ii) a good physical and
chemical stability, as well as a high bioavailability of its lipid components.
Another interesting technological application for avocado oil has been the production of structured
lipids. Caballero et al. [44] propose the elaboration of triacylglycerides of the MLM type, using
regio-specific immobilized commercial lipases sn-1.3, where M corresponds to saturated medium-chain
fatty acids (6–12 carbon atoms) at positions sn1 and sn3 of glycerol. L corresponds to saturated or
unsaturated long-chain fatty acids (14–24 carbon atoms) in the sn2 position. The increased interest
in this type of lipids is due to the low caloric intake (average caloric density for this family of lipids
5 kcal/g). According to the literature, there are no negative effects associated with the ingestion of
MLM lipids for both animals and humans.
Finally, avocado oil has also been used in the production of biodegradable polymers.
Polyhydroxyalkanoates (PHAs) are linear polyesters, produced by a large number of bacteria under
stress conditions, with different thermal and mechanical properties, which depend on their molecular
structure. Flores-Sanchez et al. [45] prepared PHAs through a fermentative process using the bacterium
C. necátor H-16 with avocado oil and fructose, as a carbon source. The highest yield in obtaining
polymers was obtained when the addition of avocado oil was 20% v/v, which demonstrates the
feasibility of using this oil as a renewable carbon source for the PHA production process.
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6. Composition of Avocado Oil
There is a growing interest in avocado oil, including the determination of the composition of
major and minor components. Therefore, for a total understanding of the nutritional and functional
properties that this oil presents, it is important to consider the different varieties and parts of the fruit.
6.1. Characteristics According to the Variety and Origin of the Fruit
Avocado is a fruit grown mainly in warm temperate and subtropical climates throughout the world,
so it is interesting to study how the climate and country of origin can affect the fruit quality and therefore, the
oil. Thus, the oil from the fruit of the Hass variety, originating from crops from Mexico, Australia, the United
States, and New Zealand, was characterized by a high content of 62% lipids, of which oleic (42–51%) and
palmitic (20–25%) lipids were present in a greater proportion. Among the predominant triacylglycerols were
OOO (21–34%) and OOP (19–24%), where O and P denote oleic and palmitic acids, respectively. On the
other hand, Hass avocado oil from New Zealand contained a significant amount of natural pigments and
unsaturated compounds, compared to oils from Mexico, Australia, and the United States [1].
Studies carried out in South America on the analysis and characterization of avocado oil showed
a high content of monounsaturated fatty acids (69.4%) and a lower amount of polyunsaturated and
saturated fatty acids, which were 16.6% and 14%, respectively. These studies indicate that avocado oil
has a thermal stability close to 176 ◦ C and has a lower concentration of total phenolic compounds than
olive oil. Despite this, the antioxidant activity of avocado oil is similar to that of olive oil. Olive oil has
a high concentration of polyphenols, such as tyrosol and hydroxytyrosol [46].
Galvão et al. [47] analyzed the fatty acid composition of the pulp, seed, and skin oil of the
Fortuna, Collinson, and Barker varieties, indicating that there was a small variation in the composition of
monounsaturated fatty acids in the skin oil among the cultivars. However, the seed oil of the Collinson
variety was the best due to the lower SFA content. The SFA content for pulp oil corresponded to 22.3,
29.4 and 41.3% in the Fortuna, Collinson, and Barker varieties, respectively. In this sense, it was possible
to affirm that the pulp oil of the Fortuna and Collinson varieties presented a better quality, in terms of
fatty acid profile, than the Barker variety.
In Mexico, avocado oil from six local creole varieties (BTancitaro, Irapuato, Orgánico, Puerto,
San José, and STancitaro) were analyzed and compared with oil from the Hass variety. It was observed
that the Mexican creole genotypes had a greater thermal stability, properties resistant to oxidation,
and a greater phenolic content, in comparison with the commercial oil from the Hass variety. In addition,
these varieties showed intense fluorescent peaks at 675 and 720 nm, as well as broad absorption bands
centered at 465 and 510 nm, which can be used as an identification parameter for these oils [48].
Yanty et al. [49] indicated that the avocado oil originating from three Malaysian varieties was
found in a significantly lower proportion than in the Australian Hass variety. In addition to being in a
semi-solid form, all these oils had a higher proportion of oleic acid, although they also had different
proportions of palmitic and linoleic acids. Regarding the composition of TAG for local varieties,
the highest was POO, followed by POL, OOO, and PPO, while in the Hass variety, the distribution was
OOO, followed by PPO, OOL, and POL. As a result of these different compositions in TAG, differences
were found in the iodine index, melting point by slip and melting and solidification characteristics.
6.2. Physicochemical Characterization
Table 1 shows the composition of the common fatty acids of the oils from different varieties and
origin of avocados, discussed in this work. However, only some varieties have the C6:0, C7:0, C8:0,
C9:0, C10:0, C11:0, C12:0, C13:0, C14: 0, C14:1, C15:0, C15:1, C16:1, C17:0, C17:1, C19:0, C20:0, C20:1,
C20:3, C20:4, C22:0, C22:1, C22:2, C23:0, and C24:0 fatty acids in a low proportion, ranging from traces
(<0.06%)–3.58%. Dreher and Davenport [50] refer to the fact that the oil coming from the Hass variety
can contain up to 71% of monounsaturated fatty acids (MUFA), 13% of polyunsaturated fatty acids
(PUFA), and 16% of saturated fatty acids (SFA).
148
149
0,49
0.6
0.63 ± 0.0002
0.37 ± 0.00
0.45 ± 0.16
0.24 ± 0.02
0.30 ± 0.01
0.93 ± 0.08
0.48
0.5
0.38 ± 0.01
0.43 ± 0.01
18,62
13.4
17.37 ± 0.0015
18.17 ± 0.02
13.7 ± 1.5
25.63 ± 0.11
22.59 ± 0.23
20.61 ± 0.16
22.24 ± 0.05
10. 75
20.5
12.16 ± 0.04
16.93 ± 0.03
23.66
21.312 ± 0.550
12.37 ± 0.01
19.9–21.3
28.12–34.48
30.37 ± 0.06
18.18
18.87
18.74 ± 0.06
12.87
0.762 ± 0.021
0.51 ± 0.01
0.23–1.07
1.30 ± 0.01
0.40
0.59
0.51 ± 0.00
1.45
Estearic
18:0
Palmitic
16:0
8,47
3.9
7.52 ± 0.0002
4.03 ± 0.01
3.4 ± 0.4
7.29 ± 0.05
11.63 ± 0.13
10.31 ± 0.03
13.14 ± 0.01
3.14
6.8
6.57 ± 0.01
7.33 ± 0.05
3.58
2.391 ± 0.188
7.58 ± 0.00
2.7–7.0
6.64–8.5
5.22±0.02
6.56
4.16
7.88 ± 0.01
3.86
Palmitoleic
16:1 Ω7
60,17
65.3
62.89 ± 0.0019
51.76 ± 0.04
67.4 ± 3.0
42.59 ± 0.16
49.19 ± 0.57
50.97 ± 0.30
47.69 ± 0.03
74.32
60.6
61.72 ± 0.30
57.39 ± 0.18
47.20
64.436 ± 0.666
64.62 ± 0.20
57.1–64.5
40.73–42.72
43.65 ± 0.04
60.25
63.07
54.40 ± 0.10
57.44
Oleic
18:1 Ω9
10,97
15.2
10.64 ± 0.0004
11.12 ± 0.01
14.4 ± 1.8
20.87 ± 0.10
14.72 ± 0.06
16.10 ± 0.11
14.47 ± 0.01
10.03
13.2
8.30 ± 0.02
8.25 ± 0.02
13.46
9.147 ± 0.030
8.46 ± 0.02
10.6–11.0
15.52–18.88
17.45 ± 0.04
13.03
11.83
10.87 ± 0.01
18.70
Linoleic
18:2 Ω6
0,98
1.3
0.72 ± 0.0001
0.59 ± 0.00
1.1 ± 0.01
3.19 ± 0.06
1.63 ± 0.16
1.72 ± 0.02
1.54 ± 0.00
0.85
0.44 ± 0.00
0.56 ± 0.00
1.60
0.467 ± 0.016
0.47 ± 0.00
0.4–0.6
1.51–2.14
2.03 ± 0.01
1.40
1.32
0.61 ± 0.00
0.92
α linolenic
18:3 Ω3
[51]
[47]
[52]
[52]
[54]
[55]
[52]
[10] 1
[24] 2
[49]
[51]
[51]
[3]
[53]
[1]
[51]
[46]
[37]
[52]
[53] 3
Ref.
composition studied under different extraction methods (cold pressed and solvents); 2 composition studied under different extraction methods (solvents, SCO2 , and UAAE); 3 in
conditions similar to natural ripening.
1
BREDA
CRIOLLA MEXICANA
DE MALASIA
REED
ANTILLANA
VARIETY NO
INDICATED
FUERTE
BACON
PINKERTON
MARGARIDA
FORTUNE
Australia
México
New Zealand
United States
HASS
Varieties and/or Country
of Origin
Table 1. Composition (%) of the common fatty acids of avocado oil.
Molecules 2019, 24, 2172
Molecules 2019, 24, 2172
In order to study the fatty acid profile of the avocado oil from the fruits harvested and artificially
ripened, the avocado oil was extracted from preserved fruit at 5 ◦ C in a controlled atmosphere and was
analyzed (4% of O2 and 6% of CO2 ). Postharvest ripening was stimulated in the presence of exogenous
ethylene (0 or 100 ppm) at a temperature of 18 ◦ C for 24 h and then preserved at 15 and 20 ◦ C. It was
concluded that post-harvest conservation and ripening by means of a controlled atmosphere did not
have a detrimental effect on the fatty acid profile or the amount of oil obtained, when compared with
the one commonly applied in the ready-to-eat market [53]. This information is very important for the
fresh product industry, where avocados move long distances at low temperatures.
When carrying out the physicochemical evaluation of two Hass avocado oils sold in Chile and
labeled as extra virgin, it was demonstrated that both oils presented significant differences in the
content of tocopherols, total phenols, oil stability, measured as the induction time, UV absorption
coefficients, peroxide index, free acidity, total chlorophyll, total carotenoids, and polar compounds.
In addition, the presence of 3,5-stigmastadiene in one of the samples, a compound that has been
associated with a high degree of refining or exposure to high temperatures of oils, indicated a disparity
in the quality parameters and a lack of regulation of avocado oil in the local market [37].
On the other hand, monovarietal oils from the Bacon, Fuerte, Hass, and Pinkerton varieties, obtained
in Spain, were compared with commercial oils originating in Brazil, Chile, Ecuador and New Zealand.
The content of triacylglycerols, fatty acids, aliphatic, and terpene alcohols, desmethylmethyl, methyl and
dimethyl sterols, squalene and tocopherols were determined. The main triacylglycerols were those
with ECN48 (48 equivalents of carbon atoms). The oleic, palmitic, and linoleic fatty acids were the
most abundant fatty acids, and the desmethyl sterols were the main quantified minor compounds.
Small amounts of aliphatic and terpene alcohols were observed. The concentrations of squalene were
higher in the oils of the Bacon, Fuerte, and Pinkerton varieties than in the other varieties. The most
abundant tocopherol was α-tocopherol [52].
While the fruit reaches a minimum of 8% fat at the time of its extraction from the tree, during
vegetative ripening, values of 20% or more are reached, depending on the variety. In this sense, it is
recommended that the avocado oil industry ripen the fruit on the tree, since climacteric ripening,
in comparison with commercial ripening, influences not only the increase in oil content, but also
the profile of the fatty acids, increasing the amount of unsaturated fatty acids, such as oleic acid,
and decreasing the amount of saturated fatty acids, such as palmitic and palmitoleic acids [56].
This situation seems controversial with respect to the previously discussed studies. However, it is well
known that the composition of fruits depends on environmental and growth conditions. In addition,
the quantification of the different analytes depends on the conditions of extraction, processing, and
detection limits of the analytical equipment.
Martinez-Nieto and Moreno-Romero [57] have analyzed the sterol composition of the
unsaponifiable matter of avocado oil in 4 varieties (Reed, Bacon, Fuerte, and Hass) by means of
gas chromatography. The results showed that the Fuerte and Reed varieties contained 2% more of
β-sitosterol than the Bacon and Hass varieties. In relation to the cholesterol content, the smallest amount
was present in the Fuerte variety and the largest in the Hass variety. In this sense, the unsaponifiable
sterol composition can be a guiding tool to determine the authenticity of oil.
Martínez-Nieto et al. [51] analyzed the composition of different fractions in an industrial extraction
process using a continuous method, under conditions similar to those used in the extraction of olive oil,
for ripe avocados of the Fuerte, Reed, Hass, and Antillana varieties. They concluded that it is possible
to obtain a good net oil yield with the industrial equipment used prior to specific modifications in
the grinding stage and in the decanter, as well as good quality parameters, such as the acidity index,
peroxide index, and absorbance coefficients, for both virgin oil and refined avocado oil.
When evaluating the physicochemical characteristics of the avocado oil obtained from the Bantul,
Purwokerto, and Garut varieties, originating in Indonesia, by extraction with solvents, the Garut
variety presented a better quality in terms of the iodine index, which is associated with a greater
amount of unsaturated fatty acids. The conjugated dienes and trienes were significantly different
150
Molecules 2019, 24, 2172
between the samples. The p-anisidine index did not have significant differences between the samples.
The saponification index was higher in the Purwokerto variety, and the peroxide index was higher in
the Bantul variety. In addition, the analysis using differential scanning calorimetry (DSC) showed that
the three samples had different melting and crystallization profiles [58].
6.3. Avocado Seed Oil
In relation to avocado seed oil, Barrera-López and Arrubia-Vélez [59] pointed out that the Lorena
variety contained about 8.47% oil, and the unsaponifiable matter was 76.9%. The phytosterols quantified
in a greater proportion were ergosterol, 5α-cholestane and stigmasterol.
Avocado seed presents, in its composition, a large number of extractable polyphenols, which have
attracted attention due to their high antioxidant capacity. It was determined that, with a higher power
of the ultrasound (0–104 W) and increase of the temperature (20–60 ◦ C), the polyphenol content and
antioxidant capacity was increased [60].
When performing a physicochemical analysis of the seed oil of the Hass variety, cultivated in Peru
and obtained by the Soxhlet method, it was found that it had a high fatty acid profile in linoleic acid
(48.77%) and linolenic acid (12.17%). While the antioxidant activity, determined by the DPPH method,
was low, it was higher in the saponifiable fraction than in the unsaponifiable fraction, which was
attributable to the presence of polyphenols and steroids. In addition, it was determined that the quality
parameters, such as acidity, peroxide, saponification, iodine, and specific gravity indexes, were similar
to those for extra virgin olive oil [61].
When comparing the composition of oil from the pulp and seed of the Fuerte variety, cultivated
in the region of Northeastern Brazil, a great difference in the lipid content between the pulp and the
seed can be seen (15.39% v.s. 1.87%, dry base). It was determined that the parameters of oil quality,
refractive index, gravity, and peroxide index were similar for both oils, but the iodine, acid index,
and saponification index were higher in seed oil than in pulp oil. Gas chromatography showed that
seed oil had a greater variety of fatty acids than pulp oil. Additionally, the fatty acid profile of the
pulp was much more concentrated in monounsaturated fatty acids than that of seed, and conversely,
the seed oil is much more concentrated in polyunsaturated fatty acids than pulp oil [55].
6.4. Comparison with Other Oils
Dubois et al. (2007) [62] compared 80 varieties of vegetable oils, including avocado oil, indicating
that it was composed of more than 60% of monounsaturated fatty acids, a characteristic shared with
olive oil, hazelnut, and macadamia nut profiles. In comparison with olive oil, avocado oil possessed
a higher proportion of saturated fatty acids (16.4%), with a predominance of palmitic acid (15.7%),
a lower proportion of monounsaturated fatty acids (67.8%), with a predominance of oleic acid 60.3%
and a higher proportion of polyunsaturated fatty acids (15.2%), the most important of which was
linoleic acid at 13.7%.
Additionally, similar fatty acid profiles have been published by Berasategi et al. [3], who also
showed that avocado oil had a higher PUFA/SFA and higher omega-6/omega-3 ratios than olive oil.
Berasategi et al. [3] showed that the phytosterol content was higher in avocado oil (3.3 g to
4.5 mg/g of oil) than in olive oil, of which the most abundant was β-sitosterol, followed by sitostanol,
cycloartenol, cycloeucalenol and D7-avenasterol. The number of sterols in the avocado oil was higher,
4-demethyl-sterols being the most abundant, reaching 80% of the total fraction of sterols. This greater
proportion was even maintained under drastic conditions of deterioration (180 ◦ C). Moreover, avocado
oil has a lower proportion of vitamin E, compared to olive oil. This study indicates that the thermal
stability of avocado oil is similar to that of olive oil. From Table 2, it is possible to appreciate a summary
of the different antioxidant components present in the avocado oils of the different varieties shown in
this work.
151
PINKERTON
VARIETY NO INDICATED
ANTILLANA
REED
BACON
FUERTE
HASS
Varieties
α-Tocopherol
86.75 ± 0.62
103.11 ± 6.87
51.90 ± 0.04
45.62 ± 0.19
-
β-Sitosterol
91.917 ± 0.027
95.2
82.95 ± 0.06
94.767 ± 0.012
92.9
80.56 ± 0.08
92.189 ± 0.012
82.6 ± 0.03
94.605 ± 0.027
91.2
89.3
84.08 ± 0.08
9.02 ± 0.09
20.35 ± 1.22
71.61 ± 0.57
13.71 ± 0.56
-
γ-Tocopherol
6.63 ± 0.07
8.81 ± 0.03
9.16 ± 0.03
5.86 ± 0.01
9.42 ± 1.69
Δ5-avenasterol
6.091 ± 0.026
4.7
5.88 ± 0.01
5.043 ± 0.012
6.4
4.62 ± 0.02
6.096 ± 0.010
3.71 ± 0.01
5.123 ± 0.021
8.6
10.6
6.00 ± 0.01
18.36 ± 1.44
Campesterol
0.001 ± 0.001
0.13 ± 0.00
0.001 ± 0.001
0.15 ± 0.00
0.011 ± 0.001
0.40 ± 0.01
0.001 ± 0.001
0.11 ± 0.00
1.11 ± 0.12
Estigmasterol
Table 2. Antioxidant compounds present in avocado oil. Concentration [=] mg × kg−1.
0.46 ± 0.01
0.57 ± 0.04
0.58 ± 0.04
0.41 ± 0.03
2.19 ± 0.22
Sitoestanol
0.04 ± 0.00
0.04 ± 0.00
0.05 ± 0.00
0.04 ± 0.00
0.43 ± 0.03
Campestanol
[57]
[51]
[52]
[57]
[51]
[52]
[57]
[52]
[57]
[51]
[51]
[52]
[3]
Ref.
Molecules 2019, 24, 2172
152
Molecules 2019, 24, 2172
7. Biological Effects
The presence of compounds with nutritional interest, such as unsaturated fatty acids (MUFA and
PUFA), as well as compounds with biological activity, such as tocopherols, tocotrienols, phytosterols,
carotenoids, and polyphenols, have made avocado oil of growing interest for research on the possible
biological effects of avocado oil, with the aim of preventing and treating diseases through the diet of
the population.
7.1. Human Health Effects
A study in 13 healthy adults with a habitual hypercaloric and hyperlipidic diet, where butter was
replaced by avocado oil extracted at 35 ◦ C from the pulp alone, was conducted. The incorporation of
avocado oil for a period of six days reflected an improvement in the postprandial profile of insulin,
glycemia, total cholesterol, low-density lipoproteins, triglycerides, and inflammatory parameters,
such as C-reactive protein (CRP) and interleukin-6 [63]. Avocado pulp oil (Mexican creole genotypes)
has shown anti-inflammatory activity by inhibiting the enzymes COX 1 and COX 2 in a similar way
to the drug, ibuprofen, and extra virgin olive oil [48]. Additionally, when avocado oil was added to
vitamin B12 skin cream preparation, it was well tolerated and had the potential for long-term topical
therapy of psoriasis [64].
7.2. Experimental Studies in Animals
When administering avocado oil to Wistar diabetic rats for 90 days (1 gr/250 g weight), it was
observed that it promoted an improvement in the functionality of the electron transport chain and
decreased the generation of free radicals in the liver, attenuating the harmful effects of oxidative
stress [65].
In the brain, an improvement in mitochondrial function has been observed, as well as a decrease
in free radical levels, lipid peroxidation, and an improvement in the reduced/oxidized glutathione ratio.
These results demonstrate that supplementation with avocado oil prevents mitochondrial dysfunction
in the brain and liver of diabetic rats [66].
Ortiz-Avila et al. [67] demonstrated that supplementation with avocado oil in Wistar rats led to a
reduction of the alterations in the electron transport chain at the renal level, attenuating the oxidative
damage, although a protective effect on lipid peroxidation was not evidenced.
Some authors [68–70] studied the relationship between avocado oil and collagen metabolism in
both the skin and liver, finding that oil obtained from intact fruit (pulp and seed), refined with hexane,
was associated with fibrosis in the liver, an increase in liver enzymes and consequently hepatotoxicity.
In addition to an increase in the solubility of collagen, behavior that is attributed to a decrease in
the activity of lysyl oxidase was observed [66]. A similar conclusion was proposed by Lamaud et al. [71],
stating that the mixture of soybean and avocado oils decreased the degree of collagen cross-linking,
a process that has been associated with a delay in wound healing. However, Oliveira et al. [53] indicated
that the healing activity of a semi-solid formulation of avocado oil (50/50 Vaseline) and avocado pulp
oil promoted the increase of collagen synthesis and decreased the number of inflammatory cells during
the process of wound healing.
153
Molecules 2019, 24, 2172
Regarding the effects on cardiovascular health, it is possible to point out that the atherogenic
power of avocado oil could be similar to that of olive oil and lower than that of corn and coconut
oil [72]. On the other hand, an avocado oil-rich diet modifies the fatty acid content in cardiac and renal
membranes in a tissue-specific manner in Wistar rats. The rise in renal arachidonic acid suggests that
diet content can be a key factor in vascular responses [73].
Márquez-Ramírez et al. [74] studied the antihypertensive effect of avocado oil in rats with induced
hypertension. They were subsequently treated with the administration of 1 mL of oil per 250 g of rat
weight or 40 mg of losartan potassium per kg of rat weight. Avocado oil significantly decreased both
systolic and diastolic pressure in hypertensive rats, but not in controls. Avocado oil mimicked the
effects of the drug, losartan, on blood pressure, vascular performance, and oxidative stress.
In rats fed with sucrose, it was observed that the addition of avocado oil in the diet reduced the
levels of triglycerides, VLDL and LDL, without affecting the levels of HDL. It also reduced the level of
ultrasensitive CPR, indicating that the inflammatory processes associated with metabolic syndrome
were partially re-established [4]. Additionally, a diet high in sucrose, which causes liver alteration in
Sprague-Dawley Weaned rats, was partially reversible by the administration of avocado oil, obtained
by centrifugation and extracted by solvents. This effect was similar to that of olive oil, but would not be
extended to the pancreatic level [5]. In relation to insulin resistance induced by a diet high in sucrose
in Wistar rats, it was possible to reduce it through the dietary addition of 5–20% avocado oil [75].
In female Wistar rats exposed to prolonged androgenic stimulation, avocado oil improved triglyceride,
VLDL and HDL levels, generating a direct regulatory effect on the lipid profile [76]. The influence of
avocado oil on cell damage, induced by methyl methanesulfonate (MMS) and doxorubicin, can be
carried out using in vitro (V79 cells) and in vivo models (Swiss mice). Avocado pulp oil had no
genotoxic effects. The oil was effective in reducing the chromosomal damage induced by MMS and
doxorubicin. However, an increase in liver tissue damage was observed when evaluating a high
dose of avocado oil (measured as an increase in the hepatic enzyme aspartate aminotransferase),
a phenomenon attributable to the high concentration of palmitic fatty acid [77]. Table 3 shows a
summary of the biological effects presented in this study.
154
Daily administration of avocado oil was 1 mL/250 g of
weight for a period of 3, 6 and 12 months to Winstar
and Goto-Kakizaki rats.
Swiss mice were given avocado oil in different
concentrations of 250, 500, 1000 and 2000 mg/kg in an
in vivo model.
In vitro model was applied to different concentrations
of avocado oil: 100, 200 and 400 μg/mL.
Administration of a diet with different concentrations
of avocado oil to insulin resistant rats.
Administration of avocado oil (1 g/250 g weight) to
diabetic rats for 90 days.
Diet supplemented with avocado oil to rats exposed to
prolonged androgenic stimulation.
Administration of a diet with olive oil and avocado oil
to rats exposed to a diet rich in sucrose.
Administration of a diet with olive oil and avocado oil
to rats exposed to a diet rich in sucrose.
Diabetic male rats Goto-Kakizaki; the
control rats were Wistar males.
In vitro (Chinese hamster lung
fibroblasts/V79 cells) and in vivo
models (Swiss mice).
Male Wistar rats
Male Wistar rats
155
Wistar rats
Wistar rats
Wistar rats
Protocol Used
Daily administration of 1.0 mL/250 g of avocado oil by
gavage or losartan at 40 mg/kg for 45 days.
Animal Model
Male Wistar rats
Conclusions
In the liver: (a) A diet high in sucrose causes hepatic impairment,
partially reversible by the administration of avocado oil, which
does not occur at the pancreatic level. (b) Avocado oil
(independent of its extraction method) exhibits effects similar to
those of olive oil in the fatty acid profile.
At the cardiovascular level: In rats fed with sucrose, it was
observed that avocado oil reduces the levels of triglycerides, very
low-density lipoprotein (VLDL) and low-density lipoprotein
(LDL), without affecting the levels of high-density lipoprotein
(HDL). It also reduces the level of ultrasensitive CRP, indicating
that the inflammatory processes associated with metabolic
syndrome are partially reestablished.
(a) Avocado oil mimics the effects of losartan. (b) Effects of
avocado oil could be mediated by decreased actions of
Angiotensin-II in mitochondria. (c) The intake of avocado oil
could mitigate the harmful effects of hypertension in kidneys.
In the brain: (a) Improvement of mitochondrial function. (b)
Decreased levels of free radicals and lipid peroxidation. (c)
Improvement of the reduced/oxidized glutathione ratio. (d)
Prevention of mitochondrial dysfunction.
(a) Avocado pulp oil has no genotoxic effects. (b) The oil was
effective in reducing the chromosomal damage induced by methyl
methanesulfonate and doxorubicin. However, an increase in
hepatic enzyme aspartate aminotransferase was found to be a
marker of liver damage.
(a) The dietary addition of 5–20% avocado oil can reduce glucose
tolerance and insulin resistance, induced by the high sucrose diet,
in Wistar rats. (b) The addition of 5–30% avocado oil reduced the
body weight gain induced by the high sucrose diet in Wistar rats.
In the liver: (a) Promoted an improvement in the functionality of
the electron transport chain. (b) Decreased the generation of free
radicals. (c) Diminished the harmful effects of oxidative stress in
the liver.
Avocado oil exerted a direct regulatory effect on the lipid profile.
Table 3. Experimental studies on the biological effects of avocado.
[4]
[5]
[76]
[65]
[75]
[77]
[66]
[74]
Ref.
Molecules 2019, 24, 2172
The control group received a laboratory pellet, while
the treated group received a diet rich in 10% avocado
oil (weight/weight) for a period of two weeks.
Rabbits were fed a semi-purified diet containing 0.2%
cholesterol and 14% oil for 90 days (corn, coconut,
olive and avocado).
Rats and chicks were fed 10% avocado oil (w/w).
Male Wistar rats
Growing rats were fed for eight weeks with 10% (w/w)
refined or unrefined avocado oil or soybean oil, using
different extraction media.
The rats were fed diets containing 10% avocado oil
(w/w) for four weeks.
Application of oil mixtures in the skin of the dorsal
area of rats for 15 days, treatment with a mixture of 2/3
of soybean oil and 1/3 avocado in a 5% solution of
sweet almond oil.
Charles river female rats.
Charles river female rats.
Rats
Female rats and chicks.
Male rabbits of the New Zealand White
strain.
Protocol Used
Avocado oil administration: 1 mL of avocado oil/250 g
of weight daily for a period of 90 days.
Animal Model
Male Wistar rats
Table 3. Cont.
Conclusions
Rats and chickens fed unrefined avocado oil showed a significant
decrease in total collagen solubility in the liver.
Rats fed with unrefined avocado oil extracted with hexane from
intact fruit (unsaponifiables) or avocado seed oil showed
significant increases in the content of soluble collagen in the skin,
although the total collagen content was not affected.
Rats fed refined or unrefined soybean oils showed no effects.
The consumption of avocado oil extracted from intact fruits can
cause changes in metabolism in the liver. Serum alkaline
phosphatase activity increased in rats fed seed oil, oil extracted
with unrefined solvent of intact fruit or unsaponifiables, and
aspartate aminotransferase activity increased significantly in the
group fed avocado seed oil.
The mixture of soybean and avocado oils decreases the degree of
collagen cross-linking, delaying wound healing.
At the renal level: (a) Protection against induced oxidative stress.
(b) ROS generation decrease. (c) Improvement of the activities of
complexes II and III. (d) Lower peroxidizability index in diabetic
mitocondria.
The administration of a diet rich in avocado oil to rats for two
weeks modifies the content of fatty acids in cardiac and renal
membranes.
The atherogenic power of avocado oil is equivalent to that of olive
oil and lower than that of corn and coconut oil.
[71]
[69]
[70]
[68]
[72]
[73]
[67]
Ref.
Molecules 2019, 24, 2172
156
Molecules 2019, 24, 2172
8. Conclusions
While the composition and quality of the avocado oil depends on the origin, weather conditions,
variety, and extraction methods, it is characterized as a mainly monounsaturated oil, with an adequate
proportion of polyunsaturated fatty acids, similar to olive oil. In addition, it contains other bioactive
compounds, present in the unsaponifiable fraction, such as tocopherols, polyphenols, and a remarkable
proportion of phytosterols. This oil has also been shown to perform well at high temperatures. All these
characteristics indicate that avocado oil has nutritional properties that are greatly appreciated by the
population, even for technological applications. This makes it a product of increasing exploitation
interest for producers.
This growing interest also requires a greater development of studies on the adulteration and
contamination of avocado oil, as well as the further assessment of the promising biological effects of
the different components present in the oil, either in animals or humans. Furthermore, considering
the presented information, it is necessary to inquire into the international regulations concerning the
different qualities of this oil.
Author Contributions: Conceptualization, M.F. and J.O.-V.; methodology, C.E.V., C.S. and J.O.-V.; software, H.V.;
validation, C.E.V., F.A., and H.V.; formal analysis, M.F. and F.A.; investigation, M.F., C.S. and J.O.-V.; resources,
M.F.; data curation, C.S. and C.E.V.; writing—original draft preparation, C.S.; writing—review and editing, M.F.,
F.A., H.V. and J.O.-V.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
UAAE
sCO2
scCO2
LPG
FFA
MUFA
PUFA
SFA
TAG
O
P
L
COX
PCR
FTIR
IOC
DPPH
APMAE
PHAs
ECN48
VLDL
LDL
HDL
Ultrasound-assisted aqueous extraction
Subcritical CO2
Supercritical CO2
Liquefied gas of compressed oil
Free fatty acid
Monounsaturated fatty acid
Polyunsaturated fatty acid
Saturated fatty acid
Triacylglycerol
Oleic acid
Palmitic acid
Linoleic acid
Cyclooxygenase enzyme
C reactive protein
Infrared spectroscopy with Fourier transform
International olive council
2,2-Diphenyl-1-picrylhydrazyl
Atmospheric pressure microwave-assisted liquid–liquid extraction
Polyhydroxyalkanoates
48 equivalents of carbon atoms
Very low-density lipoprotein
Low-density lipoprotein
High-density lipoprotein
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© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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| 86,119 | 568,309 |
W1946643744.txt | Indonesian Journal of International Law
Volume 5
Number 3 Treaty and National Law
Article 6
4-30-2008
Kedudukan Hukum Internasional dalam Sistem Hukum Nasional
Melda Kamil Ariadno
Follow this and additional works at: https://scholarhub.ui.ac.id/ijil
Part of the International Law Commons
Recommended Citation
Ariadno, Melda Kamil (2008) "Kedudukan Hukum Internasional dalam Sistem Hukum Nasional,"
Indonesian Journal of International Law: Vol. 5: No. 3, Article 6.
DOI: 10.17304/ijil.vol5.3.179
Available at: https://scholarhub.ui.ac.id/ijil/vol5/iss3/6
This Article is brought to you for free and open access by the Faculty of Law at UI Scholars Hub. It has been
accepted for inclusion in Indonesian Journal of International Law by an authorized editor of UI Scholars Hub.
| 108 | 802 |
W2147374393.txt | Global Journal of Health Science; Vol. 8, No. 5; 2016
ISSN 1916-9736
E-ISSN 1916-9744
Published by Canadian Center of Science and Education
Psychometric Testing of the Greek Version of the Clinical Learning
Environment-Teacher (CLES+T)
Evridiki Papastavrou1, Maria Dimitriadou1 & Haritini Tsangari2
1
School of Health Sciences, Department of Nursing, Cyprus University of Technology, Cyprus
2
School of Business, Department of Economics and Finance, University of Nicosia, Cyprus
Correspondence: Maria Dimitriadou, School of Health Sciences, Department of Nursing, Cyprus University of
Technology, Cyprus. Tel: 357-9967-1898; Fax: 357-25-002-864. E-mail: maria.dimitriadou@cut.ac.cy
Received: June 1, 2015
doi:10.5539/gjhs.v8n5p59
Accepted: June 24, 2015
Online Published: August 31, 2015
URL: http://dx.doi.org/10.5539/gjhs.v8n5p59
Abstract
Clinical practice is an important part of nursing education, and robust instruments are required to evaluate the
effectiveness of the hospital setting as a learning environment. The study aim is the psychometric test of the
Clinical Learning Environment+Teacher (CLES+T) scale-Greek version. 463 students practicing in acute care
hospitals participated in the study. The reliability of the instrument was estimated with Cronbach’s alpha
coefficients. The construct validity was evaluated using exploratory factor analysis (EFA) with Varimax rotation.
Convergent validity was examined by measuring the bivariate correlations between the scale/subscales. Content,
validity and semantic equivalence were examined through reviews by a panel of experts. The total scale showed
high internal consistency (α=0.95). EFA was identical to the original scale, had eigen values larger than one and
explained a total of 67.4% of the variance. The factor with the highest eigen value and the largest percentage of
variance explained was “supervisory relationship”, with an original eigenvalue of 13.1 (6.8 after Varimax
rotation) and an explanation of around 38% of the variance (or 20% after rotation). Convergent validity was
examined by measuring the bivariate correlations between the scale and a question that measured the general
satisfaction. The Greek version of the CLES+T is a valid and reliable instrument that can be used to examine
students’ perceptions of the clinical learning environment.
Keywords: clinical learning environment, nurse education, psychometric testing, validation
1. Introduction
Nurse education is an integral part of any healthcare delivery system as it has a major role to play in the
educational development of graduates who can deliver safe, good quality nursing care. Nursing professionals’
knowledge, attitudes and skills are acquired both through formal education in institutions and through experience
in the clinical area (Gaberson & Oermann, 2010). The clinical area represents an environment where the student
nurse interact emotionally, physically and cognitively with their surroundings and fulfill their learning outcomes.
Under these circumstances the clinical area is referred to as the ‘clinical learning environment’. Clinical learning
environment (CLE) is viewed as an invaluable resource in familiarizing students with the reality of their
professional role (Henderson, Cooke, Creedy, & Walker, 2012). Students being exposed to a range of clinical
experiences (Edwards, Smith, Courtney, Finlayson, & Chapman, 2004) helps to broaden and deepen both
cognitive and psychomotor skills (Souza, Venkatesaperumal, & Radhakrishnan, 2013), develop caring
relationships and aid nurses in their professional evolution, as embodied within the attitudes of the nursing
workforce (Haugan, Sørensen, & Hanssen, 2012).
Interest in the concept of clinical education as a determinant of quality nursing has gained increasing attention
since 1980, leading to various nursing education reforms (Pollard, Ellis, Stringer, & Cockayne, 2007).
Historically, a number of researchers investigated the effectiveness of the clinical learning environment (CLE)
from students’ perspectives, employed quantitative (Saarikoski, Marrow, Abreu, Riklikiene, & Özbicakçi, 2007;
Melender, Jonsén, & Hilli, 2013; Papathanasiou, Tsaras, & Sarafis, 2014; Dimitriadou, Papastavrou, Efstathiou
& Theodorou, 2015), qualitative (Papp, Markkanen, & Von Bonsdorff, 2003; Mattila, Pitkäjärvi, & Eriksson,
2010) and mix methodology (Bisholt, Ohlsson, Engström, Johansson, & Gustafsson, 2014; Ip & Chan, 2005).
The pedagogical atmosphere characterized by respect, acceptance and opportunities for learning with the mentor
and clinical teacher alike have a stake in making clinical learning successful and reliable (Papp et al., 2003;
59
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Global Journal of Health Science
Vol. 8, No. 5; 2016
Saarikoski et al., 2007; Dimitriadou et al., 2015). Also, student satisfaction was consistently identified as the
most reliable index of a “good” clinical learning environment (Chan, 2001; Papastavrou, Lambrinou, Tsangari,
Saarikoski, & Leino-Kilpi, 2010; Dimitriadou et al., 2015). For that reason, a variety of instruments for
evaluating CLE from students’ perspective have been developed such as the CLEI-Clinical Learning
Environment Inventory (Chan, 2001); the CLESDI-Clinical Learning Environment Diagnostic Inventory scale
(Hosoda, 2006); the BES-CPE (Levett-Jones, 2009); the SECEE - Student Evaluation of the Clinical Education
Environment (Sand-Jecklin, 2009); and the CLES - Clinical Learning Environment and Supervision Scale. The
CLES is a 27-item scale (Saarikoski & Leino-Kilpi, 2002) developed to measure both the clinical learning
environment and clinical supervision. Later, this scale was reviewed by the developers and a new sub-dimension
added culminating in a 34-item CLES + T scale (Saarikoski, Isoaho, Warne, & Leino-Kilpi, 2008) and assesses 5
factors. They are as follows- pedagogical atmosphere, leadership style of the ward manager, supervisory
relationship, the premises in the ward, and the role of the nurse teacher. The CLES+T instrument was used
mainly and extensively in Europe in an effort to develop a powerful, multilingual tool for evaluating the quality
of clinical learning. To date, the CLES+T scale has been translated into nine different languages, i.e., English,
Finnish, Italian, Greek, Swedish, Dutch, Norwegian, German and Spanish. It has been demonstrated to be a valid
and reliable tool among different international samples (Saarikoski et al., 2008; Johansson et al., 2010; De Witte,
Labeau, & De Keyser, 2011; Henriksen, Normann, & Skaalvik, 2012; Tomietto et al., 2012; Bergjan & Hertel
2013; Saarikoski et al., 2013; Watson et al., 2014; Vizcaya-Moreno, Pérez-Cañaveras, De Juan, & Saarikoski,
2015). Since the quality of clinical learning depends on how well the practice curriculum is structured, the use of
a reliable tool is helpful in obtaining information regarding the effective organization of clinical practice and the
quality of it’s supervision. With this in mind, CLES+T is selected to test the psychometric properties with the
aim to enhance cross-cultural collaboration.
2. Methods
2.1 Aim
The current study aim was to test the psychometric properties of the Greek version of the Clinical Learning
Environment Scale + Teacher (CLES+T-GR) of Greek Cypriot students.
2.2 Participants and Settings
The target population of the study included all nursing students enrolled at the three Universities offering a
Bachelor’s degree in Nursing within Cyprus. Only students who were practicing in hospitals were recruited, and
those who were practicing in primary health care centers and other community care settings were excluded. 664
students fulfilled the inclusion criteria and were given the questionnaire. 463 questionnaires were returned,
giving a response rate of 70.33%.
2.3 Ethical Considerations and Data Collection
The research proposal was submitted to the National Bioethics Committee, which is responsible for all research
projects in Cyprus according to the Law (3558/2001). The permission for access to the field research was
obtained by the Chairs of Nursing from each university. The aims of and the rationale for the study, and
assurances that the data would be processed anonymously were included in the information letter supplied with
the questionnaire. The questionnaires were given to the students individually immediately after they had
completed their clinical placement, during a nursing laboratory lesson. After completion, each questionnaire was
returned in a closed envelope. The completion of the questionnaire was considered as an informed consent for
participation in the study.
2.4 Research Instrument
As mentioned the CLES+T scale consists of 34 items classified into 5 subscales: (1) pedagogical atmosphere on
the ward; (2) supervisory relationship; (3) leadership style of the ward manager; (4) premises of ward nursing;
and (5) role of the nurse teacher in clinical practice (Saarikoski et al., 2008). Respondents are asked to score their
perception to each item on a 5-point Likert-type scale ranging from “very dissatisfied” to “very satisfied”. Also
demographic data, hospital and ward type, length of clinical placement, number of meeting with the nurse
teacher, motivational level on clinical placement, and level of satisfaction were collected.
2.5 Data Analysis
For demographic data and scale items, descriptive statistics (frequencies, percentages, means and standard
deviations), skewness and kurtosis were used. The internal consistency of the Greek version of the instrument
and each dimension was estimated with Cronbach’s alpha coefficients. Also, item analysis was conducted on the
data, providing item-to-total correlations and Cronbach’s alpha if the item was deleted from the scale. Construct
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validity was evaluated using exploratory factor analysis (EFA) with Varimax rotation. Principal components
analysis (PCA) was implemented as the extraction method in EFA. Convergent validity was examined by
measuring the bivariate correlations between the scale/subscales and the question about the general satisfaction
of nurses. Content, validity and semantic equivalence were examined through review by a panel of experts of the
content of each item, its wording and the meaning of the items, after translation, in the context of the Cypriot
culture (Squires et al., 2013).
3. Results
3.1 Sample Characteristics
The final sample included 463 participants. Among those, 38.7% were males and 61.3% females, with ages
ranging from 18 to 34 years, a mean of 21.08 years and standard deviation 2.23 years. 149 participants studied in
private universities, and 314 in the single public university.
3.2 The Individual Scale Items
Descriptive statistics (mean and standard deviation) for individual items were calculated. In order to examine the
variability of the answers and test for significant deviations from normality, item skewness and kurtosis were
also reported. Acceptable values of skewness and kurtosis, based on George and Mallery (2001), are those
between -1.5 and +1.5, whereas values between -1 and +1 are considered excellent. The highest mean was of an
item on the supervisory relationship subscale (item 18 in the scale - My mentor showed a positive attitude…),
with a mean of 4.3, and a (low) standard deviation of 1.00. This item had marginal values of skewness and
kurtosis as well. This verified that most of the answers were “agree” or “fully agree”, as opposed to negative
attitudes, but the values were within the acceptable range. Although, in most of the items, there was a weak trend
towards the positive attitudes (agree and strongly agree), there were no critical values (high positive or negative)
of kurtosis or skewness for any item with all values being in the acceptable range. Therefore, transformations
were not deemed necessary and all items were included in the analysis. These results are presented in Table 1.
3.3 Internal Consistency and Reliability-Item Analysis
Cronbach’s alpha was used for testing the reliability of the 34-item scale, as well as for the five subscales, where
values close to one are considered satisfactory. Also, item analysis was conducted to provide information about
how well each individual item correlated to other items in the sub-scale where corrected item-to-total
correlations below 0.30 are usually considered unacceptably low (Polit & Beck, 2011). Also, all of the inter-item
correlations were examined with the reliability of each item being considered by finding if the it’s Cronbach’s
alpha was deleted.
The results showed high internal consistency for the total scale (α=0.95). Similarly, the reliability of each
sub-category was found to be high, ranging from 0.81 (“nursing care”) to 0.96 (“supervisory relationship”). The
latter results are presented in table 1. Item analysis showed that when any item was deleted from the scale, the
alpha was slightly lower or approximately the same as compared to when all the items were included, suggesting
that deleting any item does not change the overall reliability significantly, and as such, that all the items
contribute to the high reliability of the scale. A slight increase was seen in items “I felt comfortable going to the
ward at the start of my shift” and “the staff learned how to know the students by their names”, however small
(from 0.949 to 0.950). The items can thus be considered reliable.
Corrected item-to-scale correlations varied from 0.38 to 0.71, showing that all correlations were satisfactory, that
is, above 0.3. Finally, out of more than 250 inter-item correlations between the 34 items (not reported), only two
correlations exceeded 0.80 (in the supervisory relationship factor). Thus, in general, the results suggest that
within the scale no items duplicated each other. The corrected item-to-scale correlations and Cronbach’s alpha if
the item was deleted appear in Table 1.
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Table 1. Descriptives, Internal consistency and Reliability (sequence as presented in the questionnaire)
Item
Mean
Std.
Deviation
Skewness
Kurtosis
Corrected
Item-Total
Correlation
Cronbach
Alpha if item
Deleted
Pedagogical Atmosphere, 9 items, Cronbach’s alpha 0.875
The staff were easy to approach
3.7646
1.06635
-.843
.183
.522
.948
I felt comfortable going to the ward at
the start of my shift
3.0606
1.22013
-.159
-.893
.385
.950
During staff meetings(e.g. before
shifts) I felt comfortable taking part
in the discussions
4.0475
1.03296
-1.160
1.093
.493
.948
There was a positive atmosphere on
the ward
3.8013
1.04210
-.840
.426
.620
.948
The staffs were generally interested in
student supervision
3.2505
1.19427
-.248
-.733
.605
.948
The staff learned to know the students
by their personal names
2.4514
1.31973
.500
-.870
.403
.950
There were sufficient meaningful
learning situations on the ward
3.4190
1.08981
-.356
-.525
.620
.948
The
learning
situations
were
multi-dimensional in terms of content
3.3826
1.08331
-.289
-.449
.613
.948
The ward can be regarded as a good
learning environment
3.6609
1.09773
-.508
-.476
.613
.948
Leadership style in ward management, 4 items, Cronbach’s alpha 0.849
The WM regarded the staff on his/her
ward as a key resource person
3.9391
1.03774
-.970
.620
.546
.948
The WM was a team member
3.5502
1.18464
-.628
-.351
.490
.949
Feedback from the WM could easy be
consider a learning situation
3.3275
1.13437
-.323
-.525
.584
.948
The effort on individual employee
was appreciated
3.3348
1.09000
-.286
-.425
.531
.948
Nursing care, 4 items, Cronbach’s alpha 0.812
The ward nursing philosophy was
clearly defined
3.6811
.98259
-.569
.090
.576
.948
Patients received individual nursing
care
3.8304
1.02769
-.696
-.099
.512
.948
There were no problem in the
information flow related to patients’
care
3.7609
1.01158
-.636
-.015
.471
.949
Documentation of nursing ( e.g.
nursing plans, daily recording of
nursing procedures etc.) was clear
3.8824
.99743
-.730
.129
.589
.948
Supervisory Relationship, 8 items, Cronbach’s alpha 0.849
My supervisor showed a positive
attitude towards supervision
4.2673
1.05593
-1.482
1.504
.658
.947
I felt that I received individual
supervision
3.7733
1.18752
-.734
-.326
.647
.947
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I continuously received feedback
from supervisor
3.9399
1.16111
-.857
-.219
.683
.947
Overall I am satisfied with the
supervision I received
3.9690
1.22435
-1.057
.110
.705
.947
The supervision was based on a
relationship of equality and promoted
my learning
3.9498
1.15256
-.921
-.048
.703
.947
There was a mutual interaction in the
supervisory relationship
3.9569
1.14764
-.987
.251
.714
.947
Mutual
respect
and
approval
prevailed
in
the
supervisory
relationship
4.0597
1.11053
-1.098
.456
.681
.947
The supervisory relationship was
characterized by a sense of trust
4.0072
1.14320
-1.001
.173
.703
.947
Role of nurse teacher, 9 items, Cronbach’s alpha 0.937
In my opinion, the NT was capable of
integrating theoretical knowledge and
everyday practice of nursing
3.8366
1.18799
-.834
-.179
.607
.948
The NT was capable of operational
sing the learning goals of this
placement
3.8293
1.18870
-.855
-.107
.601
.948
The NT helped me to reduce the
theory-practice gap
3.6800
1.22331
-.704
-.389
.543
.948
The NT was like a member of the
nursing team
3.4181
1.27918
-.417
-.804
.501
.949
The NT was able to give his or her
expertise to the clinical team
3.5730
1.19551
-.561
-.455
.550
.948
The NT and the clinical team worked
in supporting my learning
3.4900
1.22629
-.442
-.718
.573
.948
The common meetings between
myself mentor and NT were
comfortable experience
3.8491
1.15296
-.794
-.174
.594
.948
In our common meetings I felt that
we are colleagues
3.8604
1.14160
-.776
-.232
.632
.947
Focus on meetings was in my
learning needs
3.5643
1.25568
-.553
-.699
.552
.948
3.4 Content Validity and Semantic Equivalence
The Greek Version of the CLES-GR was translated and back-translated following a specific step procedure
(Papastavrou et al., 2010) after obtaining consent from the authors. Although the content validity of the Greek
version of the CLES-GR has been established, the questions were reviewed by five experts as the questionnaire
was modified by the designers and re-named to include the nurse teacher. The expert panel agreed that the
CLES+T-GR reflected the situation in the clinical practice environment, i.e., that the items were suitable and
relevant to be tested on Cypriot students and it was of acceptable face validity.
3.5 Construct Validity
Construct validity was evaluated using exploratory factor analysis (EFA) with Varimax rotation. Principal
components analysis (PCA) was implemented as the extraction method in EFA. The procedure used is similar to
the psychometric studies published for the CLES+T (Saarikoski et al., 2008; Johansson et al., 2010; Henriksen et
al., 2012; Bergjan & Hertel, 2013; Vizcaya-Moreno et al., 2015). First, the assumptions regarding the suitability
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of the data for factor analysis were examined, including the sample size, Kaiser–Mayer–Olkin (KMO) measure
of sampling adequacy and Bartlett’s test of sphericity. The sample size was satisfactory, considering the
rule-of-thumb for determining a priori sample size to be a “participant- to- item” ratio of 10:1 (Costello &
Osborne, 2005). The current study, included 34 items and 463 participants, therefore this ratio was satisfied. The
data were found to be appropriate for factor analysis, since the KMO measure was equal to 0.931, larger than 0.5,
indicating high sampling adequacy, whereas Bartlett’s test of sphericity was significant (p<0.001), thus rejecting
the null hypothesis of an identity correlation matrix (Field, 2009).
The criteria for factor selection included eigenvalues being higher than 1, as well as the percentage of variance
explained by the factors, where each factor was expected to explain at least 5% of the variance to be included
(Polit & Beck, 2011; Field, 2009). In the current study, the five-factor structure of the model that was obtained
from EFA was identical to the original scale, where all the items loaded on each component agreed completely
with the five sub-dimensions of the scale in Saariskoski et al. (2008). The five components had eigenvalues
larger than one and explained a total of 67.4% of the variance. The most important factor for the learning
environment of Cypriot nurses, which had the highest eigenvalue and the largest percentage of variance
explained, was “supervisory relationship”, with an original eigenvalue of 13.1 (6.8 after Varimax rotation) and an
explanation of around 38% of the variance (or 20% after rotation). The factor loadings in this component ranged
from 0.78 up to 0.88. The loadings in all the other components were similarly very high and thus satisfactory, in
that all were higher than 0.5. All the results (factor loadings of each item, eigenvalues and % of variance
explained) appear in the Table 2 below:
Table 2. Factor loadings
Items on factor
Supervisory
relationship
(factor 1)
My supervisor showed a positive
attitude towards supervision
.844
I felt that I received individual
supervision
.783
I continuously received feedback
from supervisor
.826
Overall I am satisfied with the
supervision I received
.864
The supervision was based on a
relationship of equality and
promoted my learning
.866
There was a mutual interaction in
the supervisory relationship
.864
Mutual respect and approval
prevailed in the supervisory
relationship
.880
The supervisory relationship was
characterized by a sense of trust
.833
Role
of
nurse
teacher
(factor 2)
The NT was capable of integrating
theoretical knowledge and
everyday practice of nursing
.812
The NT was capable of operational
sing the learning goals of this
placement
.834
64
Pedagogical
atmosphere
(factor 3)
Premises
of
nursing in the
ward (factor 4)
Leadership style
of
the
Ward
manager (factor
5)
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The NT helped me to reduce the
theory-practice cap
.805
The NT was like a member of the
nursing team
.757
The NT was able to give his or her
expertise to the clinical team
.824
The NT and the clinical team
worked in supporting my learning
.820
The common meetings between
myself mentor and NT were
comfortable experience
.695
In our common meetings I felt that
we are colleagues
.698
Focus on meetings was in my
learning needs
.690
The staff was easy to approach
.727
I felt comfortable going to the ward
at the start of my shift
.659
During staff meetings(e.g. before
shifts) I felt comfortable taking part
in the discussions
.725
There was a positive atmosphere
on the ward
.755
The staffs were generally interested
in student supervision
.693
The staff learned to know the
students by their personal names
.609
There were sufficient meaningful
learning situations on the ward
.524
The learning situations were
multi-dimensional in terms of
content
.516
The ward can be regarded as a
good learning environment
.508
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The ward nursing philosophy was
clearly defined
.640
Patients received individual nursing
care
.741
There were no problem in the
information flow related to
patients’ care
.691
Documentation of nursing ( e.g.
nursing plans, daily recording of
nursing procedures etc.) was clear
.684
.685
The WM regarded the staff on
his/her ward as a key resource
person
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The WM was a team member
.843
Feedback from the WM could easy
be consider a learning situation
.794
The effort on individual employee
was appreciated
.616
Eigenvalues
13.065
3.750
3.179
1.654
1.270
% of variance explained
(total=67.405%)
38.427
11.029
9.349
4.864
3.736
Eigenvalues (after rotation)
6.817
5.990
4.435
2.989
2.687
% of variance explained
(total=67.405%) (after rotation)
20.049
17.618
13.045
8.791
7.902
It should be noted that although item analysis did not show any significant deviations from normality, the
principal axis factoring was implemented in EFA since it is a method without any distributional assumptions
(e.g., Saarikoski et al., 2008; Johansson et al., 2010). However, the results from the two methods (principal
components analysis and principal axis factoring) were identical in terms of the factor structure and grouping of
items, although with slightly altered factor loadings, and therefore the results from the principal axis factoring
are not reported.
In addition, it should be mentioned that, similar to Saarikoski et al. (2008), different models also were compared,
namely with four and six factors. The 4-factor results were similar to the results reported by Saarikoski et al.
(2008) where three dimensions were identical to the 5-factor solution and the fourth combined ward management
and nursing care together, but in addition to the concern that one factor included too many items, the 4-factor
model also explained a lower percentage of variance (63.7%). Finally, the 6-factor model, which was originally
obtained, included an extra factor which included items 6-9 from the list of pedagogical atmosphere items, but
the first two items (6 and 7), loaded similarly on the Pedagogical Atmosphere factor, with high loadings on both
factors, therefore only the last three items (7-9), loaded more significantly on the sixth factor. Including this extra
factor, with only these three items did not make sense intuitively. Therefore, the five-factor solution was the
preferred and most appropriate model structure overall.
3.5 Convergent Validity
Convergent validity was examined by measuring the bivariate correlations between the scale/subscales and the
question that measured the general satisfaction of nurses (replies to which optionally ranged from “fully
dissatisfied” to “fully satisfied”). Spearman’s rho correlation coefficient was significant with p<0.001 between
the overall satisfaction and all the scales, showing that the scale and subscales have convergent validity and
indicating that all the subscales are important and need to be included in the scale. These results are presented in
Table 3.
Table 3. Bivariate Correlations between the item on “total satisfaction” with all the scales/subscales.
Total satisfaction
Scale
Pedagogical
Atmosphere
Ward
Management
Nursing
care
Supervisory
relationship
Role of the
Nurse Teacher
Spearman’s Correlation
coefficient
0.610**
0.521**
0.388**
0.385**
0.550**
0.432**
p-value
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
** Correlation is significant at α=0.01.
3.6 Correlations Between the Subscales
Pearson correlation coefficient was used to examine the relation between the subscales. All the scales were
highly significantly positively related, with p-values<0.001. These correlations are presented in Table 4.
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Table 4. Correlations between the CLES-T subscales
Pedagogical Ward
Scale
Nursing
Atmosphere Management care
Supervisory Role of the
relationship Nurse Teacher
PearsonCorrelation
0.794**
0.685**
0.708**
0.791**
0.748**
p-value
p<0.001
p<0.001
p<0.001
p<0.001
p<0.001
PedagogicalAtmosph PearsonCorrelation
ere
p-value
WardManagement
Nursing care
Supervisory
relationship
0.554
**
p<0.001
0.577
**
p<0.001
**
PearsonCorrelation
0.537
p-value
p<0.001
0.472
**
p<0.001
0.431
**
p<0.001
**
PearsonCorrelation
0.459
p-value
p<0.001
0.402**
p<0.001
0.381**
p<0.001
0.378**
p<0.001
PearsonCorrelation
0.460**
p-value
p<0.001
** Correlation is significant at the 0.001 level.
4. Discussion
Examination of the psychometric properties of the CLES+T-GR demonstrated the internal consistency of the
total scale, and its subscales as well, which was similar to results in other languages (Bergjan & Hertel, 2013;
Henriksen et al., 2012; Johansson et al., 2010; Saarikoski et al., 2008; Vizcaya-Moreno et al., 2015). In the
present study, three validation processes were applied: content, construct and convergent validity, indicating the
degree to which scores measure what they claim to measure.
The results have shown that CLES+T-GR is a multidimensional instrument which consists of five factors, and is
very similar to the first scale CLES-GR used without the “teacher-T” section (Papastavrou et al., 2010). In that
study, the construct validity of the CLES-GR was examined using a sample of 350 students with an exploratory
factor analysis. The total percentage of variance that the factor model explained was high (67%) and the
questions loaded on the same factors as the factors in the original questionnaire. The reliability of the CLES-GR
was satisfactory (Cronbach’s alpha =0.95) and the alpha values of the sub-dimensions ranged from 0.79 to 0.95.
The CLES+T is an extension of the CLES-GR that gives more possibilities to evaluate the new role dimensions
of the nurse teacher in the clinical area. As nursing education has moved to the higher education systems so the
CLES+T is thus more suited to the Cyprus Nursing educational system currently relied upon.
The factor model with a total explanation percentage of 67.4% of the variance found in our study was
comparable with factor models of other studies in Europe, namely Germany 73% (Bergjan & Hertel, 2013)
Spain 66.4% (Vizcaya-Moreno et al., 2015). Italy 67% (Tomietto et al., 2012) and Finland 64% (Saarikoski et al.,
2008). The strongest factor was found in the subcategory “supervisory relationship” with high loadings ranging
from 0.833 to 0.844, followed by the “role of the nurse teacher” factors with loadings ranging from 0.690 to
0.834. It is interesting that “supervisory relationship” has been identified as the strongest factor in most of the
validated versions of the CLES+T (Saarikoski et al., 2008; Johansson et al., 2010; Henriksen et al., 2012;
Bergjan & Hertel, 2013; Vizcaya-Moreno et al., 2015). Only in the Italian version (Tomietto et al., 2012) the
strongest factor was the “pedagogical atmosphere” and “supervisory relationship” was the forth in sequence.
Another interesting finding of our study is that “role of the nurse teacher” is the second strongest factor and as
such is similar to the Norwegian sample (Henriksen et al., 2012). However, loadings were weaker than the
Cypriot sample, ranging from 0.498 to 0.816, and also the question related to the nurse teacher cooperation
loaded in another factor, that of the supervisory relationship. The least important factor of the clinical learning
environment in our study was the “leadership style of the ward manager”, which in combination with the high
importance given to the “role of the nurse teacher” in our study may be considered as shifting all of the learning
responsibility to the nurse teacher, in turn minimizing the role of the ward manager. Initially the ward manager
was recognized as a key person in the establishment of the ward as a good learning environment for student
nurses in 1980’s (Orton, 1983), although subsequent studies found that the pedagogical activities of the clinical
nursing staff are more important in the supervision of students (Mattila et al., 2010). Empirical evidence also
gives more emphasis to models of “one to one” supervision rather than the traditional model of group
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supervision, and likewise to the role of the staff nurse in the clinical supervision process (Saarikoski et al., 2007).
The strong support given to the role of the nurse teacher in our study also gives the message that students regard
their teacher as more important in the process of learning and the person who will fulfill their expectations to
reduce the theory-practice gap.
As mentioned, the CLES+T is validated in seven European countries and languages and despite the differences
in examining the psychometric differences, the results show that in general almost all the items of the scale
loaded on the same factor, although the robustness of each factor in each country varies. This is understandable
in terms of the differences and complexities of health care and hospital organization systems that formulate the
practice environment and influence student learning in each country. Also, although the cultural and social
contexts of the learning environments in the countries mentioned is so varied, the advantage of CLES+T lies in
the opportunity to use an instrument that measures all aspects of these diverse environments in a consistent way.
This means that CLES+T is a strong, valid and reliable instrument that can be safely administered to all nurse
students to evaluate their perceptions of various clinical settings as learning environments. Bachelor degrees in
nurse education in Europe are characterized by different structures, standards and approaches to the relationship
between theoretical and practice-based learning (Salminen et al., 2010). Hence, systematically processed
empirical data collected with valid, reliable instruments are needed urgently for national and international
comparisons, and to increase the pressures on policy makers.
4.1 Limitations
The limitations of the study include the issue of different study settings in that students were practicing in five
different hospitals and many dissimilar wards. This means that the results may be different if analyzed at a
hospital or unit level rather than nationally. Also, the comparisons made with validation studies from other
countries need to be viewed with caution, as those studies did not implement any statistical comparative analyses
of the factor loadings and so it is possible that certain results occurred by chance Finally, it would be interesting
to employ other statistical methods of validation, as a confirmatory factor analysis.
5. Conclusions
The Greek version of the CLES+T has been shown to be reliable and valid. The CLES+T-GR evaluates the
learning context of the clinical practice environment taking into consideration the multidimensional nature of the
clinical area which involves aspects of the ward such as the pedagogical atmosphere, nursing care and ward
management as well as supervision aspects and inter-staff relationships. The role of the teacher is central to the
concept of the clinical learning environment, especially in integrating theoretical knowledge and everyday
practice of nursing. CLES+T-GR enables researchers to use a methodological tool to assess the process of
learning in applied science, such as nursing, that relies heavily on practice. Additionally, it facilitates the design
of more effective, targeted interventions for improving the clinical learning environment. The results further
suggest that there is still much to be done to promote improvements in the clinical area that will facilitate student
learning and further research is needed into this area in the Greek context.
Acknowledgments
The authors would like to thank Mikko Saarikoski for the permission given for using CLES+T, the nursing
students that participated in the study and Mr Gerry Barrett for the language editing.
Author Contributions
Study Design: EP, MD
Data Collection and Analysis: MD, EP, HT
Manuscript Writing: EP, MD, HT
Funding
The research was granted by Technological University of Cyprus.
Conflict of Interest
The authors declare that there is no conflict of interests regarding the publication of this paper.
References
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W4206800567.txt | SciPost Physics
Submission
Dual applications of Chebyshev polynomials method:
Efficiently finding thousands of central eigenvalues for
many-spin systems
Haoyu Guan1 , Wenxian Zhang1* ,
1 School of Physics and Technology, Wuhan University, Wuhan, Hubei 430072, China
* wxzhang@whu.edu.cn
June 28, 2021
Abstract
Computation of a large group of interior eigenvalues at the middle spectrum
is an important problem for quantum many-body systems, where the level
statistics provide characteristic signatures of quantum chaos. We propose an
exact numerical method, dual applications of Chebyshev polynomials (DACP),
to simultaneously find thousands of central eigenvalues, which are exponentially close to each other in terms of the system size. To cope with the neardegenerate problem, we employ twice the Chebyshev polynomials, to construct
an exponential semicircle filter as a preconditioning step and to generate a
large set of proper basis states in the desired subspace. Numerical experiments on Ising spin chain and spin glass shards confirm the correctness and
efficiency of DACP. As numerical results demonstrate, DACP is 30 times faster
than the state-of-the-art shift-invert method for the Ising spin chain while 8
times faster for the spin glass shards. In contrast to the shift-invert method,
the computation time of DACP is only weakly influenced by the required number of eigenvalues, which renders it a powerful tool for large scale eigenvalues
computations. Moreover, the consumed memory also remains a small constant
(5.6 GB) for spin-1/2 systems consisting of up to 20 spins, making it desirable
for parallel computing.
Contents
1 Introduction
2
2 Dual applications of Chebyshev polynomials method
2.1 Exp-semicircle filter
2.2 Chebyshev evolution
2.3 Subspace diagonalization
3
4
6
6
3 Numerical results
7
4 Conclusion
12
A Chebyshev polynomials
13
B Detailed deductions of Eq. (9)
14
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C Evaluation of matrix elements and solution of the generalized eigenvalue problem
15
D Comparison with a hybrid method
16
References
17
1 Introduction
Energy level statistics provides an essential characterization of quantum chaos [1, 2]. Integrable systems often imply level clustering and a Poisson distribution of energy level
spacings [2], while chaotic systems exhibit strong level repulsion and a Wigner-Dyson distribution [3]. Other useful statistical tools like the δ3 statistic [4] and the power spectrum
of the δn statistic also depend on the level distribution [5]. Numerical observation of
eigenvalues is important to exactly characterize these level statistics, because direct theoretical derivation is not available. In addition, the need to access individual eigenstates
at the middle of the spectrum, which correspond to the “infinite temperature” limit, is
emphasized in studying the phenomenon of many-body localization (MBL) [6–8]. Numerical simulations remain instrumental in understanding quantitatively many aspects of
the MBL problem. As the many-body problems of interest often involve a huge Hilbert
space dimension growing exponentially in terms of system size, it is rather challenging
to conduct a full diagonalization of the Hamiltonian or to solve the time-independent
Schrödinger equation. Seeking to resolve the eigenvalue problem in a small part of the
spectrum is thus an unavoidable and desirable substitution.
To obtain the interior eigenstates, a hybrid strategy of matrix spectroscopy is often
invoked [9–11]. It aims at computing eigenstates in selected regions of the spectrum and
couples the ground state solvers with a spectral filter, where the filter is designed to
cast the selected interior regions to the edges of the spectrum. Among these filters, the
Green function (H − λI)−1 is an excellent one. After applying the Green function filter,
the cluster of eigenvalues near the test energy λ is mapped to very large positive and
negative values. The level spacings near λ are greatly amplified, which thus improves the
convergence. The Lanczos method [12] has been coupled with such a filter [9, 10], and the
Chebyshev polynomial expansion of the Green function is implemented [13]. In particular,
the shift-invert method [14] essentially utilizes this spectral transformation and has been
widely used in quantum many-spin systems [8, 15–18]. Moreover, it was even proposed to
be the most efficient one for the MBL problem [8]. However, for large systems this method
suffers rapid increases in both computation time and memory consumption, due to the
factorization of (H − λI)−1 . Other spectral filters have been also proposed, including the
Dirac delta function filter δ(H−λI) [19–22] and the rectangular window function filter [23],
both of which are expanded by the Chebyshev polynomial. Note that all the methods
mentioned above are iterative ones, where the computation time is usually proportional
to the required number of eigenvalues, i.e., more eigenvalues means more filtrations and
reorthogonalizations [8, 20, 21].
In this paper, we propose an exact numerical method, DACP, to calculate thousands
of eigenvalues at the middle of the energy band, which is already helpful to reveal the level
statistics [24,25]. For spin systems, such a region usually locates at the peak of the density
of states where the energy levels are so close that they are nearly degenerate. It is extremely
2
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challenging to distinguish these near-degenerate eigenvalues without factorizing H−1 . In
the DACP method, we construct an exponential of semicircle (exp-semicircle) filter to
quickly damp the unwanted part of the spectrum by employing the Chebyshev polynomial
for the first time. The second application of the Chebyshev polynomial is to fast search a
set of states to span the specific subspace consisting of all the eigenstates corresponding
to the desired eigenvalues. The DACP method transforms the original high dimensional
eigenvalue problem to a low dimensional one. For practical problems in many-spin systems,
the DACP method is very efficient, due to its full exploration of several excellent properties
of the Chebyshev polynomial while other methods usually utilize only one of them. For
a large class of many-spin systems, the DACP method exhibits a significant increase in
the computation speed, up to a factor of 30, in comparison with the shift-invert method.
The memory saving is more drastic, up to a factor of 100. Moreover, the DACP method
distinguishes itself from those iterative filtering ones, as its convergence time varies only
slightly when the required number of eigenvalues is vastly changed.
The paper is organized as follows. The detailed formalism of the DACP method,
including the exp-semicircle filtration, Chebyshev evolution, and subspace diagonalization,
are described in Section 2. Each of the processes relies on a particular property of the
Chebyshev polynomial, distinguishing DACP from other filtering methods . In Section 3
we discuss details of two many-spin systems, the Ising model and the spin glass shards,
and present the computational results. We conclude in Section 4.
2 Dual applications of Chebyshev polynomials method
To access the central eigenvalues of large spin systems, naturally we are restricted by
the matrix-free mode, i.e., the matrix of the Hamiltonian must not be explicitly expressed/stored. Instead, we directly treat the Hamiltonian H as an operator whose input
and output are states/vectors. Therefore, we shall only operate with the quantum states:
|ψ⟩ , H |ψ⟩ , H2 |ψ⟩ , · · · , Hk |ψ⟩ ,
(1)
where k is a positive integer, in the original Hilbert space of dimension up to 5 × 105 . Note
that each “matrix-vector product” H |ψ⟩, being a basic operation, consumes considerable
time while only a small amount of memory. Moreover, as illustrated in Subsection 2.3, even
the states in Eq. (1) are not necessarily required to be stored. Therefore, the requirement
of memory in DACP is pretty tiny. In general, we hope to simultaneously find a large
scale of central eigenvalues with a minimum k. In this manuscript, we set our goal to find
5, 000 eigenvalues accurately at the middle of the spectrum for the many-spin systems as
an illustration.
The idea of DACP is fairly straightforward. We first transform a randomly initialized
state into a wave packet in the subspace spanned essentially by at least 5, 000 central
eigenstates. This transformation is realized by an exp-semicircle filter implemented by the
Chebyshev polynomial. Here the Chebyshev polynomial is used to perform an exponential
decay. With this particular state in hand, we then generate a large amount of linearly
independent states, as large as possible, to approximately span the subspace consisting of
the required eigenstates. The Chebyshev polynomial is used again to oscillate (complex
exponential) the states, producing approximately linearly independent states. Once the
generating set for the desired eigenstates is known, one may explicitly calculate H, the
reduced representation of H in this subspace. The remaining operations are then restricted
in the subspace of dimension around 104 . Finally, direct diagonalization of H, which is of
size 104 × 104 , gives the desired eigenvalues. The detailed procedures and discussions are
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20
10
15
k
T (F(x))
10
10
10
5
10
0
10
−0.1
−0.05
0
0.05
0.1
x
Figure 1: The exp-semicircle filters for a = 0.1 (red lines), 0.05 (blue lines), and 0.025
(black lines), with ka = 24. We have set Emax = 1 and Emin
−1. The solid lines
=
2
are for the Chebyshev polynomials Tk (F (x)), where F (x) = 2x − (1 + a2 ) /(1 − a2 ),
√
while the dashed lines are for the approximations y = exp(2k a2 − x2 ) ≃ Tk (F (x)). The
horizontal pink dotted line denotes the half maximum of the logarithm of filters. Note
that |Tk (F (x))| ≤ 1 when x ∈
/ [−a, a], thus the filter graphs are restricted in [−a, a].
given in the subsections below.
2.1
Exp-semicircle filter
We utilize the exponential growth of the Chebyshev polynomials outside the interval [−1, 1]
to efficiently construct an exp-semicircle filter, as shown in Figure 1. The filter drastically
amplifies the components of a desired range of eigenstates for any randomly initialized
states, resulting a new state that sharply localized at the midlle of the spectrum. We note
that the Chebyshev filter explores the same property as well, except that it amplifies the
lower end of the spectrum [26, 27]. A similar idea has been also applied in the quantum
algorithm for finding ground states [28].
For the Hamiltonian H with energy bounded in [Emin , Emax ], where Emin is the minimum energy (Emin < 0) and Emax the maximum energy (Emax > 0), the exp-semicircle
filter is designed to amplify the components of the eigenstates corresponding to eigenvalues in the interval [−a, a] and to simultaneously dampen those in the interval [Emin , −a]
and [a, Emax ], where a is a real positive parameter. Focusing on the spin systems, we have
assumed Emin < −a and a < Emax . After the filtration, a new state mainly consists of
the eigenstates with eigenvalues belonging to the interval [−a, a] is generated. For simplicity, let us denote the subspace spanned by the eigenstates contained in [−a, a] as L.
Consequently, the ratio of eigenstates outside L is almost negligible, as can be seen in
Figure 1.
We now introduce the specific implementation details. Note that we want to amplify
the middle of the spectrum, but the exponential growth of the Chebyshev polynomial
exists only in both ends. To satisfy this goal, we first square the Hamiltonian, obtaining
2 ] (suppose E
H2 with a spectrum ranges [0, Emax
min = −Emax for simplicity). The middle
spectrum [−a, a] of H is transferred to [0, a2 ] for H2 , which lies exactly at the lower end.
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2 ] into [−1, 1] by shift and normalization of
Next is to map the dampening part [a2 , Emax
H2 . We thus define an operator
H2 − Ec
F=
,
(2)
E0
2
2
where Ec = (Emax
+ a2 )/2 and E0 = (Emax
− a2 )/2. One may easily affirm this map’s
2 , and correspondingly one has F (x) =
correctness by replacing H2 with either a2 or Emax
2
(x − Ec )/E0 . Note that F is simply a polynomial expression of H, so is Tk (F).
We then explore the effect of the filtration using Tk (F). As the eigenvalues inside [0, a2 ]
2
of H2 is mapped into [−1 − 2a2 /(Emax
− a2 ), −1] of F, we have Tk (F) = (−1)k cosh(kΘ)
for the lower end of the spectrum, where
2(a2 − H2 )
−1
Θ = cosh
1+ 2
.
(3)
Emax − a2
P
P
Let |ψ⟩ = i ci |ϕi ⟩ + j dj |χj ⟩ be a random initial state, with ci and dj the random
coefficients, |ϕi ⟩ the eigenstates inside L, |χj ⟩ the eigenstates outside L. The filtration by
Tk (F) is
|ψ (k)⟩ = Tk (F) |ψ⟩
c
d
X in
X ikθout
out
in
j
i
=
ekθi + e−kθi
|ϕi ⟩ +
e j + e−ikθj
|χj ⟩
2
2
i
j
1 X kθin
≃
e i ci |ϕi ⟩,
2
(4)
i
2
2
where θiin = cosh−1 (1 + 2(a2 − Ei2 )/(Emax
− a2 )), θjout = cos−1 (2(Ej2 − a2 )/(Emax
− a2 ) − 1),
Ei and Ej are eigenvalues corresponding to |ϕi ⟩ and |χj ⟩, respectively. In writing Eq. (4)
we have ignored (−1)k , as it does not affect the absolute value of coefficients and is a
2 , one may further deduce
global phase
at the end line. When a is tiny, i.e., a2 ≪ Emax
q
θiin ≃ 2 a2 − Ei2 /Emax via Taylor’s expansion of cosh−1 (1 + ε), where ε is a small positive
number. We thus obtain the exp-semicircle filter
2k
Tk (F) ≃ e Emax
√
a2 −H2
(5)
that peaked sharply at Ei = 0 with a large k, for eigenstates satisfying −a ≤ Ei ≤ a. In
Figure 1 the shape of Eq. (5) is presented in dashed lines, which fits pretty well with the
exact form.
With the initial conditions T0 (F) = 1 and T1 (F) = F, the kth order Chebyshev
polynomial can be efficiently determined using the recurrence relation Eq. (17). In this
paper, we set the cut-off order K = 18Emax /a. Such a filter exponentially (the fastest
rate among all polynomials) amplifies the components of eigenstates inside L [29]. After
the normalization, it equivalently dampens those outside L, generating the target state:
X
|ψE ⟩ ≃
c′i |ϕi ⟩,
(6)
i
where c′i = βeKθi ci and β is the normalization constant. Obviously, the state |ψE ⟩
localizes (in the energy representation) at the middle of the spectrum. We input |ψE ⟩ as
the initial state for Subsection 2.2, Chebyshev evolution.
in
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2.2
Submission
Chebyshev evolution
After obtaining a state confined in the small subspace L, we then make use of the oscillation
property of the Chebyshev polynomial to efficiently generate a set of distinct states, as
many as possible, serving as a complete basis to span L. To achieve this goal, it is
necessary to limit Ei ∈ [−1, 1] (corresponding to x in Tk (x)), within which the Chebyshev
polynomial behaves as a cosine-like function. This region contrasts with the requirement
of the exp-semicircle filter, thus one needs a different transformation of H. Below we
describe the specific details for the second application of the Chebyshev polynomial.
The original Hamiltonian H needs to be shifted by Ec′ and be rescaled by E0′ , where
Ec′ = 12 (Emin + Emax ) and E0′ = 12 (−Emin + Emax ). In a similar way to Eq. (2), we
define an operator G = (H − Ec′ )/E0′ , which is definitely bounded by −1 and 1. Assuming
Emin = −Emax again, which does not affect the conclusion, we obtain
G=
H
.
Emax
(7)
At the same time, the parameter a is rescaled to ar = a/Emax as well.
Let us explore the Chebyshev evolution, i.e., the evolution governed by the operator
Tk (G) as k plays the role of time. We input the state Eq. (6) generated by the filtration
as an initial state for the Chebyshev evolution. Since ∥G∥ ≤ 1, from Eq. (16) we have
Tk (G) = cos(kΩ), where Ω = arccos(G). In this sense, the Chebyshev evolution gives
|ψE (k)⟩ = Tk (G) |ψE ⟩
1 X ikωj
+ e−ikωj c′j |ϕj ⟩ ,
e
=
2
(8)
j
where ωj = arccos(Ej /Emax ). Note that both ωj and k are unitless. Certainly, this is not
a physical evolution, and Tk (G) is not even a unitary operator. Actually, this evolution
essentially represents a superposition of both forward and backward propagation. Each
time the polynomial order k increases by 1, the evolution “time” is added by 1 as well.
With the aid of the Chebyshev evolution, we are able to construct a complete basis
that spans the subspace L. In detail, we collect a set of states as follows
n
o
ˆ sin(X̂), · · · , sin(nX̂), cos(X̂), · · · , cos(nX̂) |ψE ⟩ ,
I,
(9)
where X̂ =πG/ar and n is an integer determined by the relation 2n + 1 ≥ d, with d
the dimension of L. In practice, one may need a relation 2n + 1 = 1.5d to ensure the
completeness of Eq. (9). Here k ≃ mπ/ar serves as the time, with m = 1, · · · , n. Similar
to Subsection 2.1, the kth order Chebyshev polynomial Tk (G) can be calculated. The
cut-off order (evolution time) K ′ = ⌊nπ/ar ⌋. More details can be found in Appendix B.
The duality of the Chebyshev polynomials, as being approximately the trigonometry
functions and being the polynomials, plays a vital role in the DACP method. In a certain sense, the Chebyshev evolution is an efficient (possibly the most one among all the
polynomials) simulation of the quantum oscillations [30].
2.3
Subspace diagonalization
Computing the basis {|Ψi ⟩ : i = 1, · · · , 2n + 1} (Eq. (9)) by combination of the exp-semicircle
filter and the Chebyshev evolution represents the most challenging aspect as well as the
most time-consuming part of the DACP method. Once the appropriate basis has been
constructed, the task remained is straightforward, i.e., to compute the eigenpairs of the
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projected Hamiltonian H. This is equivalent to solving the generalized eigenvalue problem
(10)
HB = SBΛ.
Here, H and S denote the projected Hamiltonian in L and the overlap matrices, respectively,
Hij = ⟨Ψi | H |Ψj ⟩ , Sij = ⟨Ψi |Ψj ⟩ .
(11)
Λ is a diagonal matrix with the eigenvalues in [−a, a] and the matrix B transforms the
found basis Eq. (9) to the eigenstates |ϕj ⟩ of H,
|ϕj ⟩ =
2n+1
X
Bij |Ψi ⟩ .
(12)
i=1
All these matrices are of size (2n + 1) × (2n + 1). Because of the small size of it, H can be
explicitly expressed and stored in core memory. The necessary procedures are conveniently
available in the LAPACK library [31].
Importantly, due to the special property of the Chebyshev polynomial, the computation of matrices H and S can even be achieved without an explicit computation and
storage of the states |Ψi ⟩. This feature gives rise to a further improvement for the DACP
method, both in computation time and memory saving. Besides, for an overcomplete basis
Eq. (9), the overlap matrix S is generally singular. We thus employ the singular value
decomposition (SVD) to solve it. After the SVD, the eigenvalues of S are discarded if
their absolute values sit below the cutoff condition ε = 10−12 . The number of remained
eigenvalues effectively counts the linearly independent states in Eq. (9). We present both
the explicit expressions (denoted by Tk (G) and |ψE ⟩ only) of matrices H and S, and the
solution of the generalized eigenvalue problem in Appendix C.
Eigenvalues obtained by the subspace diagonalization may not own the same accuracy
when compared to true eigenvalues of the system, thus it is necessary to conduct an
independent check to estimate the error bounds. To this end, ifqthe eigenstates |ϕj ⟩
were known, the residual norm (variance of the energy) ||rj || = ⟨H2 ⟩ − ⟨H⟩2 , where
H2 = ⟨ϕj | H2 |ϕj ⟩ and ⟨H⟩ = ⟨ϕj | H |ϕj ⟩, is widely used as the parameter measuring the
accuracy of the results. It has been shown that ||rj || gives an upper bound on the true
error (absolute error) of the computed eigenvalue [32].
3 Numerical results
We apply the DACP method to the quantum spin-1/2 systems with two-body interactions.
Such systems are good models for investigating a large class of important problems in
quantum computing, solid state theory, and quantum statistics [33–36]. A large amount
of exact eigenvalues helps us to obtain the statistical properties, to distinguish quantum
chaos from integrability, and serves as a benchmark to evaluate other approximate methods
as well.
Generally speaking, the DACP method can deal with the spin systems consisting of
couplings between any pair of the N spins. Each of the Pauli matrix σ α or the two coupling
Pauli matrices σ α ⊗ σ β , where α, β = x, y, z, is properly represented by a specific function.
We specify the spin model for two physical systems. One is the disordered onedimensional transverse field Ising model [37], where the Hamiltonian is
N −1
N
1X
1X z z
x x
H=
Ji,i+1 σi σi+1 +
Γi σi ,
4
2
i=1
i=1
7
(13)
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with σi the Pauli matrices for the spin i. This system is exactly solvable by Jordan-Wigner
transformation [35], making it an ideal correctness checker for the DACP method. The
nearest neighbor exchange
interaction
constants Ji,i+1 are random numbers that uniformly
√
√
distributed in [−J/ N , J/ N ] with J = 10. The local random magnetic fields are represented by Γzi , which are random numbers that uniformly distributed in the interval [0, Γ]
with Γ = 1.
Another system is the spin glass shards [25], which represents a class of global-range
interacting systems that require relatively large bond dimensions to be tackled by the
DMRG methods [38]. The Hamiltonian describing the system is
X
X
H=
Jij σix σjx +
Γzi σiz .
(14)
i<j
i
All symbols and parameters are the same as that of the above Ising model, except that
the first summation runs over all possible spin pairs. This system is interesting because
it presents two crossovers from integrability to quantum chaos and back to integrability
again. In the limit J/Γ → 0, the ground state is paramagnetic with all spins in the local
field direction and the system is integrable [25]. In the opposite limit J/Γ → ∞, the
ground state is spin glass and the system is also integrable since there are N operators
(σix ) commuting with the Hamiltonian. A quantum chaos region exists between these two
limits. J = 10 Γ is approximately the border from the quantum chaos to the integrable
(the spin glass side) when N = 20 [25].
By employing the upper-bound-estimator, which costs little extra computation and
bounds up the largest absolute eigenvalue E0 , one may estimate Emax = E0 and Emin =
−E0 [39]. For this setting we have utilized the symmetry of the density of states (DOS),
a bell-shape profile centered at zero, in the many-spin systems. Since we require 5, 000
central eigenvalues, we may set n = 4, 000, corresponding to a dimension 8, 001 and being
adequate to span the whole subspace L. The overlap matrix S is generally singular. The
approximate distribution of DOS ρ(E) may be efficiently calculated through the Fourier
transformation of a time evolved wave function or through a better estimation method
given in [40]. The parameter a is appropriately chosen to ensure that the number of
eigenstates contained in [−a, a] is a little less than 8, 000 (as illustrated in Figure 2, the
precision of some converged eigenvalues may be lower than required).
In practice, sometimes there may exist highly near-degenerate eigenvalues, with level
spacings as small as 10−7 Γ while the average spacing is 10−5 Γ. It is still hard (two
magnitudes longer time) for the Chebyshev evolution to discriminate such close pair of
eigenvalues. To circumvent this challenge, we employ the block filter technique [27], which
means a block of states is filtered or evolved “simultaneously”, in programming of the
DACP method. The idea is that two or several random states in the degenerate subspace
are usually linearly independent. For each numerical test, a block of 5 initial trial states
is randomly generated and employed with the parameter n being adjusted to n = 800
accordingly.
By these settings, we perform numerical tests on the above two systems to show the
high exactness and efficiency of DACP method. For this work, we consider only the eigenvalues computations. All the timing information reported in this manuscript is obtained
from calculations on the Intel(R) Xeon(R) CPU E7-8880 v4, using sequential mode.
In Figure 2, we present the relative error η in logarithmic scale versus the system
energy Eexact , for the Ising model with N = 19 and the spin glass shards with N = 17.
We have defined the relative error η of the computed central eigenvalues E as
η=
E − Eexact
.
Eexact
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0
−6
−8
log10( η )
log10( η )
−4
−8
−10
−12
−12
−14
−16
−3
(a)
−2
−1
0
E (10−2Γ)
1
2
−16
−0.12
3
(b)
−0.06
0
E (Γ)
0.06
0.12
Figure 2: The relative error η in logarithmic scale of the calculated eigenvalues, for (a)
Ising model with N = 19 and (b) spin glass shards model with N = 17. The horizontal
axis is the system energy. The number of eigenvalues satisfying η < 10−6 (black dashed
line) is 5, 385 for (a) and 5, 000 for (b). We suppose that such a distribution comes from
the shape of filters in Figure 1.
Exact eigenvalues of both systems have been obtained by other reliable methods. For
the Ising model, we make use of the famous Jordan-Wigner transformation to reduce
the original 2N × 2N matrix to a 2N × 2N one, and restore the full spectrum of the
original Hamiltonian [35]. For the spin glass shards we simply utilize the function eigs
of MATLAB, to find 5, 000 eigenvalues closest to E = 0. As for our numerical tests, the
parameter a in Figure 2 is 0.036Γ and 0.16Γ for (a) and (b), respectively. In computing the
Ising model, the number of eigenvalues satisfying η < 10−6 is not enough (less than 5, 000)
by the settings mentioned above. By adjusting the block size to 10 and the parameter
n to 500, we then collect enough eigenvalues. The number of converged eigenvalues, i.e.,
computed eigenvalues satisfying the condition η < 10−6 is 5, 385 for (a) and 5, 000 (all
the exact eigenvalues we have) for (b), while the total number of computed eigenvalues is
6, 232 and 5, 910, respectively.
The spike around E = 0 for both figures is due to the smallness of the denominator
Eexact . The smallest absolute eigenvalue is about 4.4 × 10−6 Γ for (a) and 2.9 × 10−5 Γ
for (b). Besides, there is a flat plateau at the middle of the figures, indicating that for
those eigenvalues around E = 0 we encounter the numerical error, i.e., the absolute error
reaches the limit of the double precision representation. In Figure 2(a) one may observe
other spikes, which does not appear in (b). Each of those spikes corresponds to a cluster
of eigenvalues lying extremely close to each other. The integrability of the Ising model
implies the level clustering and a Poissonian distribution of energy level spacings. We
speculate that the Chebyshev evolution does not function well for closely lied eigenvalues,
as they give quite similar phase contributions to the final states. To significantly amplify
the phase difference exp(i∆Et), it requires t ≳ 1/∆E, where ∆E is the energy difference.
An alternative solution is to increase the block size to match up with the dimension of
near-degenerate subspace. Ignoring the central plateau and the spikes, the distribution of
η shows an inverted shape of the exp-semicircle filter.
In Figure 3, we compare the computation time T (CPU time in seconds) of the DACP
method with that of the shift-invert approach. The latter is widely used in computing
eigenpairs at the middle of the spectrum for quantum spin systems, to name a few, like
in [8,15–17]. In our tests, it is implemented by the eigs function of Matlab R2019b, which
employs the implicitly restarted Arnoldi method (ARPACK) [41]. Each of the numerical
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6
log10( T )
5
4
3
Matlab,glass
Matlab,Ising
DACP,glass
DACP,Ising
2
1
13
14
15
16
17
18
19
N
Figure 3: Scaling behavior measured by the computation time T (CPU seconds) in
logarithmic scale, versus the system size N , with the DACP method (solid lines with
squares) compared to the shift-invert method (dahed lines with circles) for the Ising model
(red) and the spin glass shards (blue). Each of the numerical tests, represented either by
squares or circles, finds 5, 000 central eigenvalues. All the four lines are obtained via linear
fitting.
Table 1: Scaling constants α by linear fitting of the four curves in Figure 3.
α
DACP method Shift-invert method
Ising
1.49
1.42
Glass
1.50
1.47
tests finds around 5, 000 eigenvalues for either of the two spin systems. For the DACP
method, we have discarded the time consumed during the subspace diagonalization, as it
is a constant and does not affect the scaling behavior. Specifically, the subspace diagonalization spends roughly 700 CPU seconds in each test. Such a constant is completely
negligible for N ≥ 17 systems.
As shown in Figure 3, for spin systems with 13 ≤ N ≤ 17, the DACP method is about
30 times faster than the shift-invert approach for the Ising model and 8 times faster for the
spin glass shards. Since the shift-invert method essentially finds the ground energy of H−1 ,
which is usually not a sparse matrix, its computation time T is not affected by the sparsity
of the Hamiltonian and the two dashed lines nearly coincide. On the contrary, the DACP
method employs the polynomial combination of H acting on the states, its computation
time T is heavily influenced by the number of Pauli operators of the specific Hamiltonian.
For example, when N = 18 the computation time T for the spin glass shards is 5.5 times
that for the Ising model, while the number of Pauli operators is 6.2 times. Considering
this effect, the DACP method is still advantageous over the shift-invert approach in the
worst case where the exchange interactions run over all possible spin pairs.
Furthermore, the scaling among these two methods is comparable. To compare quantitatively, we extract the scaling constants α of the four lines by fitting the numerical
results, where α is defined by T = T0 exp(αN ). The values of α are shown in Table 1.
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1500
6000
Ising, solving time T
Ising, evolution time TE
CPU time (s)
Glass, solving time T
Glass, evolution time TE
1000
500
1
4000
2
3
2000
4
log10(Number of eigenvalues)
Figure 4: Computation time (CPU seconds) required versus number of eigenvalues satisfying the condition η < 10−6 , for the Ising model (red lines with squares, left axis) and
the spin glass shards (blue lines with circles, right axis), using the DACP method. For
both systems, N = 15. The solid lines show the solving time T = TF + TE + TD , including computation time for the exp-semicircle filtration TF , Chebyshev evolution TE and
subspace diagonalization TD . The dashed lines present TE .
Indeed, the four scaling constants are quite close. Suppose that α remains unchanged for
a bigger N , then the DACP method’s advantage in time cost may keep on for rather large
systems. In addition, as illustrated by the numerical tests of the shift-invert method in [8],
the factorization time for finding H−1 dominates other computation steps. Considering
the factorization part only, the execution time in [8] exhibits a scaling constant α ≃ 1.66,
indicating a worse scaling behavior for systems with large spins. The efficiency of the
DACP method is thus confirmed.
In Figure 4, we show the computation time versus the number of converged eigenvalues,
for the two systems of size N = 15. The horizontal axis represents the number of computed
eigenvalues satisfying the condition η(E) < 10−6 in each test. Recall that the DACP
method is divided into three parts: the exp-semicircle filtration, Chebyshev evolution,
and subspace diagonalization. We denote their time consumption as TF , TE , and TD ,
respectively, while solving time is the full computation time T = TF + TE + TD . One
immediately notice that for the two systems, computation time T decreases on the left
side, reaching a plateau at the middle region, while quickly increases on the right side.
Yet the dashed lines TE present a quite small increasing rate for the whole region, as
the horizontal axis is in logarithmic scale. At the middle of the region, where the DACP
method performs the best, the solving time T and the evolution time TE roughly coincide
with each other.
These features are easily understood if one considers the following four facts. (i) Notice
that the DOS ρ(E) is usually a bell-shape profile peaked at zero in spin systems and that a
is typically a tiny parameter (a/Emax ∼ 10−2 for N = 19), thus one may safely take ρ(E)
as a constant ρ̄ in [−a, a]. Consequently, the number of required eigenvalues is R ≃ 2ρ̄a.
(ii) Setting the action of the Hamiltonian on the state, H |ψ⟩, as a basic step, and denoting
the computation time of the basic step as τ , we may count the filtration time as τ times
twice the cut-off order K = 18Emax /a, i.e., TF ≃ 36Emax τ /a ∝ R−1 . (iii) Similarly, we
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find the Chebyshev evolution time is TE ≃ ⌊RπEmax /a⌋τ ∝ R0 since R ∝ a. (iv) It is well
known that the full diagonalization time is proportional to the cube of the matrix size,
i.e., TD ∝ R3 . The combination of these computation times clearly explains the behavior
shown in Figure 4. For the left side, the exp-semicircle filtration dominates, as TF is
inversely proportional to the parameter R. Whereas, when it comes to the intermediate
region where the number of eigenvalues is several hundreds to thousands, the evolution
time TE consists of the majority of the computation time while the other two are negligible.
As shown in Figure 4, the performance of DACP method is approximately the same in
finding 100 to 3, 000 eigenvalues. As R keeps increasing, the subspace diagonalization time
TD eventually consumes the most computation time.
We remark that the plateau at the middle region in Figure 4 distinguishes the DACP
method from those iterative filtering ones [10, 20, 22, 23, 26, 27], although all of them take
use of the Chebyshev polynomials. The iterative methods usually require a larger amount
of filtration and reorthogonalization, thus a longer convergence time, in finding more
eigenvalues [8,20,21]. In fact, as illustrated in Ref. [8], one finds that the computation time
is roughly proportional to the number of eigenvalues required for the shift-invert approach.
The DACP method is thus highly desirable for large scale eigenvalues computations.
Finally, we note that the memory requirements by DACP method are rather small, as
it works in a matrix-vector product mode which avoids an explicit matrix representation
of the Hamiltonian. In addition, as shown in Appendix C, the whole set of states in
Eq. (9) is not preserved in memory at all. The major memory consumption is the storage
of elements Hij and Sij , thus the memory requirements of the DACP method relate only
to the dimension d of subspace L. By the settings of this work, it occupies around 5.6
GB of memory for appropriately calculating 5, 000 eigenvalues in N ≤ 20 systems. On
the contrary, for the shift-invert method, the matrix size of H−1 grows rather fast as the
system size N increases (proportional to 4N ), demanding a large amount of memory. For
example, in our tests it consumes 573 GB of memory for N = 17 systems, which is already
a factor of 100 more than that of the DACP method.
4 Conclusion
We propose the DACP method to efficiently calculate a large scale (at least 5, 000) of
central eigenvalues with high precision, by employing twice the Chebyshev polynomials.
It explores several excellent properties of Chebyshev polynomial, to efficiently filter out
those non-central parts of spectrum and to construct the appropriate basis states for the
middle of the spectrum. The proposed method is specified to solve the middle of the
spectrum, while its efficiency is improved in return. Two key features distinguish DACP
from the polynomial filtering methods: its computation time is almost irrelevant to the
number of required eigenvalues and its memory requirements are irrelevant to the system
sizes. Moreover, as shown in Appendix D, the DACP method is both more stable and
more efficient than the Lanczos method applied with the exp-semicircle filter.
The numerical tests for the Ising model and the spin glass shards confirm the exactness
and efficiency of the DACP method. Compared to the widely used shift-invert method,
the DACP method gives a considerable increase in the speed of computations, for the
Ising model up to a factor of 30 while for the spin glass shards the increase in speed is less
but still considerable (a factor of 8). Besides, the memory requirements are drastically
decreased, up to a factor of 100 for N = 17 spin systems and even more for larger ones. As
a powerful tool for central eigenvalues calculations, the DACP method may find potential
applications in many physical problems, such as many-body localization in condensed
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T3(x)
(a)
0
−1
−1
(b)
0
0
1
(d)
20
0
−1
−1
1
T5(x)
(c)
0
0
1
−1
−1
0
(e)
80
1
(f)
200
0
40
−200
−20
−2
1
T4(x)
0
2
0
−2
0
x
2
−2
0
2
Figure 5: Chebyshev polynomials Tk (x) for k = 3 (red lines), k = 4 (blue lines), k = 5
(black lines). The first row ((a-c) with solid lines) illustrate the oscillations of Tk (x) inside
the interval [−1, 1] while the second row ((d-f) with dashed lines) show the rapid increase
outside [−1, 1] of Chebyshev polynomials.
matter physics [7, 15, 17, 42], and level statistics in quantum chaos [1, 16, 25, 43].
Acknowledgements
We thank X.-H. Deng for discussions. The numerical calculations in this paper have been
done on the supercomputing system in the Supercomputing Center of Wuhan University.
Funding information This work is supported by the National Natural Science Foundation of China (NSFC) Grant No. 91836101, No. U1930201, and No. 11574239.
A
Chebyshev polynomials
In this paper, we limit ourselves to that only the polynomial combinations of H are the
allowed operations. We utilize the Chebyshev polynomials to fulfill the tasks mentioned
above. It is the key to bridge the combinations of Hk with an approximately exponential
function of H, either exp(kH) or exp(ikH). Due to its several remarkable properties, the
Chebyshev polynomials may exhaust the potential of this type of methods.
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The kth order Chebyshev polynomial of the first kind is defined by
−1
|x| ≤ 1
cos k cos (x) ,
−1
cosh
k
cosh
(x)
,
x>1 ,
Tk (x) =
k
−1
(−1) cosh k cosh (−x) , x < −1
(15)
with initial conditions T0 (x) = 1 and T1 (x) = x [44]. It is a piece-wise function containing
two different kinds of expression, while being the polynomial function, it is both continuous
and smooth. For simplicity, let us set θ = cos−1 (x) (cos θ = x) when x ∈ [−1, 1] and set
θ = cosh−1 (x) when x ∈ [1, ∞), the corresponding range of θ is θ ∈ [0, π] and θ ∈ [0, ∞),
respectively. In terms of the variable θ, Eq. (15) becomes
cos (kθ) ,
|x| ≤ 1
Tk (x) =
(16)
cosh (kθ) ,
x > 1.
One may easily observe that Tk (x) is a sine or cosine-like oscillation function bounded
by −1 and 1 inside the interval [−1, 1], as illustrated in Figure 5 (a-c), while it grows
extremely fast outside [−1, 1], as shown in Figure 5 (d-f).
Note that cosh(kθ) = (ekθ + e−kθ )/2. It is natural to expect an exponential growth of
the Chebyshev polynomial outside the interval [−1, 1]. In fact, it is known that among all
polynomials with degree ≤ k, the Chebyshev polynomial Tk (x) grows the fastest outside
the interval [−1, 1] under comparable conditions [29].
Associated with those properties is a practically useful one: Tk+1 (x) can be efficiently
determined by using the 3-term recurrence
Tk+1 (x) = 2xTk (x) − Tk−1 (x).
(17)
All these properties of the Chebyshev polynomial render it a powerful toolbox and are of
great use for the DACP method.
B
Detailed deductions of Eq. (9)
Here we derive explicit expressions showing how to construct the set Eq. (9) via the
Chebyshev evolution. We focus on the case that a ≪ Emax , which is fairly reasonable for
large (N ≥ 15) spin systems.
|ψE (k)⟩ = Tk (G) |ψE ⟩
X
=
cos(kωj )c′j |ϕj ⟩
j
≃
=
X
kEj ′
kπ
)c |ϕj ⟩
−
2
Emax j
j
P
Ej
n
′
(−1)
j cos(k Emax )cj |ϕj ⟩ ,
k = 2n
(−1)n P sin(k Ej )c′ |ϕ ⟩ ,
j
j
Emax j
k = 2n + 1.
cos(
(18)
Using the fact that when x is small, arccos(x) = π/2−x+o(x), we have ωj = arccos(Ej /Emax ) ≃
π/2 − Ej /Emax at the third line of Eq. (18). Therefore, we have the expression
(−1)n cos(kG), k = 2n
(19)
Tk (G) ≃
(−1)n sin(kG), k = 2n + 1.
We then conduct a Chebyshev evolution with a cutoff order K ′ = ⌊nπ/ar ⌋, recording both
Tk−1 (G) |ψE ⟩ and Tk (G) |ψE ⟩ when k = ⌊mπ/ar ⌋ with m = 1, · · · , n. After the evolution
is done, the set of states Eq. (9) is automatically generated.
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Evaluation of matrix elements and solution of the generalized eigenvalue problem
As shown in Appendix B, we may rewrite the basis Eq. (9), or {|Ψi ⟩ : i = 1, · · · , 2n + 1},
using the Chebyshev polynomials:
{I, Tk1 −1 (G), Tk1 (G) · · · , Tkn −1 (G), Tkn (G)} |ψE ⟩ ,
(20)
where km = ⌊mπ/ar ⌋, m = 1, · · · , n, |Ψ1 ⟩ = |ψE ⟩, |Ψ2 ⟩ = Tk1 −1 (G) |ψE ⟩, |Ψ3 ⟩ =
Tk1 (G) |ψE ⟩, etc. For simplicity, one may further assume km is an even number.
The elements Sij = ⟨Ψi |Ψj ⟩ = ⟨ψE | Tx (G)Ty (G) |ψE ⟩, where x and y are directly determined by i and j. The correspondence is one to one. By making use of the relation [44]
1
Ti (H)Tj (H) = (Ti+j (H) + T|i−j| (H)),
(21)
2
one may even find the matrix elements without recording any states during the Chebyshev
evolution. Instead, we simply record the two numbers ⟨ψE | Tk (G) |ψE ⟩ and ⟨ψE | HTk (G) |ψE ⟩
at the appropriate time, i.e., when k = km − 2, k = km − 1, k = km and k = km + 1.
Finally, we arrive at the explicit expressions of matrix elements
1
⟨ψE | Tx+y (G) + T|x−y| (G) |ψE ⟩ ,
2
1
Hij = ⟨ψE | H Tx+y (G) + T|x−y| (G) |ψE ⟩ .
2
Sij =
(22)
(23)
Also note that since Tx+y is needed, where both x and y may reach the maximum value
kn , the cut-off order K is doubled to K = 2kn in this mode.
For the generalized eigenvalue problem Eq. (10), the Hermitian matrix S is first diagonalized as
S = V Λs V † ,
(24)
where V is the eigenvector matrix for S, V V † = I, and Λs is the associated eigenvalue
matrix. Since S is generally singular, we then contract the (2n + 1) × (2n + 1) matrix V by
elimination of the columns associated with eigenvalues with absolute value below a cutoff
ε = 10−12 . Denoting the number of retained eigenvalues as m, the contracted eigenvector
matrix Ve is of order (2n + 1) × m, and
e s Ve † .
Se = Ve Λ
e Since
The next step is to form the contracted Hamiltonian matrix H.
1
−2 †
− 21
e
e
e
e
e
I = Λs V
S V Λs
,
(25)
(26)
−1
e s 2 , the contracted m×m Hamiltonian matrix
denoting the transformation matrix U = Ve Λ
is
e = U † HU.
H
(27)
e of order m × m with m around 103 to 104 is then diagonalized
The Hermitian matrix H
directly
e = Ye Λ
e Ye † ,
H
(28)
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e and Λ
e is a diagonal matrix consisting of those
where Ye is the eigenvector matrix of H
desired eigenvalues of the original Hamiltonian H contained in [−a, a]. The eigenstates of
the projected Hamiltonian H may be obtained through elementary matrix algebra:
2 e
e−
B = U Ye = Ve Λ
s Y.
1
(29)
Denoting the Eq. (20) as a 2N × (2n + 1) matrix A with |χi ⟩ being the i-th column, the
eigenstates of the original Hamiltonian H contained in [−a, a] is
2 e
e−
Φ = AB = AVe Λ
s Y.
1
(30)
e and Φ.
We finally get the answers Λ
D
Comparison with a hybrid method
Since the Lanczos method is widely employed to compute the lowest (or highest) eigenvalues, while the exp-semicircle filter provides a means to transform the central eigenvalues to
the highest ones, naturally one may combine them together to efficiently compute the central eigenvalues, a way far simpler than the DACP method. But there are several reasons
to prefer complicated techniques used in the DACP method to the standard construction
of the Krylov space, especially when large scale computations are required.
First, it is known that the Lanczos method is not numerically stable under practical conditions. Specifically, although the Lanczos algorithm shows perfect properties in
theory, in practice it loses many of its designed features, e.g., global orthogonality and
linear independence among Lanczos recursion states [32, 45, 46]. These defects effectively
limits the number of eigenvalues which can be computed. In addition, it seems that the
emergence of generalized eigenvalue problem (to deal with non-orthogonal base states)
is unavoidable due to the error accumulations [47, 48]. The Chebyshev recursion, on the
other hand, possesses many interesting properties common in both the ideal and practical calculations [45]. In particular, it is accurate and stable for x ∈ [−1, 1], allowing
the propagation in the Chebyshev order domain for tens of thousands of steps without
accumulating significant errors [30].
Second, taking the reorthogonalization step into consideration, the efficiency of the
Lanczos algorithm decreases. The reorthogonalization step is a necessary part in both the
Lanczos and the Arnoldi method. When there is a large amount (e.g., several thousands)
of eigenvalues to be computed, the total cost is actually dominated by the reorthogonalization [26, 27, 49]. Suppose the total required number of eigenvalues is R and the Hilbert
space dimension is D, then the Lanczos-type methods scale as DR2 , as every generated
Ritz vector needs to be reorthogonalized against the existing ones (see also the discussions
in Refs. [27,41]). In comparison, we have shown in Section 3 that TF ∝ DR−1 , TE ∝ DR0 ,
and TD ∝ D0 R3 , where each one of them is better than DR2 (R ≪ D). Although partial
reorthogonalization schemes have been proposed, they result in an increased cost in computations as well as memory traffic [20,50], and they are not guaranteed to succeed when the
accuracy requirement becomes strict [50]. Moreover, the DACP method and the Lanczos
algorithm are different in terms of space complexity, as the former shows max(DR0 , R2 ),
while the latter requires DR once the reorthogonalizations are needed [41]. Therefore, the
DACP method has superiority over the Lanczos algorithm in both the time and the space
complexity.
Finally, ignoring the requirement of reorthogonalizations, we find that the two methods
are comparable in both the time and the space complexity. Recall that the Chebyshev evolution time, being the dominate term in the DACP method, becomes TE ≃ πREmax τ /a,
16
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Submission
where τ is the time for matrix-vector product. We then discuss the time complexity of
a combined version, the Lanczos method with the exp-semicircle filter. As shown in Fig.
1 of the Supplemental Material in Ref. [21], and the discussions in Ref. [26], the number
of Lanczos steps m shall be at least twice the number of requested eigenvalues R, i.e.,
m ≥ 2R. Since 2R iterations are needed, and every iteration requires a filter application with TF ≃ 36Emax τ /a, the computation time reads 2R · 36Emax τ /a = 72REmax τ /a.
Therefore, the DACP method and the combination of Lanczos with the exp-semicircle
filter share a same time complexity.
In summary, the DACP method is both more stable and more efficient than the Lanczos
method applied with the exp-semicircle filter.
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W2892725157.txt | Hu et al. Cell Death and Disease (2018)9:979
DOI 10.1038/s41419-018-1013-z
Cell Death & Disease
ARTICLE
Open Access
Linc02527 promoted autophagy in
Intrahepatic cholestasis of pregnancy
Jianguo Hu1, Li Liu1, Yangyang Gong1, Lei Zhang1, Xiaoling Gan1, Xiaodong Luo1, Tinghe Yu1, Xiaocui Zhong1,
Xinru Deng1, Lina Hu1, Zhanyu Zhang1 and Xiaojing Dong1
1234567890():,;
1234567890():,;
1234567890():,;
1234567890():,;
Abstract
LncRNA plays a crucial role in human disease. However, the expression and function of LncRNA in ICP(Intrahepatic
cholestasis of pregnancy) is still not fully elucidated. In this study, we found Linc02527 was increased expression in
placenta and serum of ICP patients. Ectopically expression of Linc02527 promoted autophagy and proliferate in HTR8
cells. Silencing Linc02527 suppressed the autophagy and proliferate in HTR8 cells. Mechanically study revealed that
Linc02527 regulated the expression of ATG5 and ATG7 by sponging miR-3185. Linc02527 directly binding to YBX1 and
activated P21. The growth of C57 mouse was retarded when autophagy was activated. In normal condition, inhibited
autophagy using chloroquine did not affect the growth of C57 mouse. However, in the condition of autophagy was
activated, inhibited autophagy using chloroquine can improve the growth of C57 mouse. Overall, the results of this
study identified Linc02527 as a candidate biomarker in ICP and a potential target for ICP therapy. Chloroquine was a
potential drug for ICP therapy.
Introduction
Intrahepatic cholestasis of pregnancy (ICP) is an obstetrical complication. It may lead to intrauterine fetal death.
The hazard behind ICP is the excessive accumulation of bile
acids in hepatocytes and the inhibition of bile excretion1.
Overproduction of bile acids or their impaired removal
from hepatocytes may be the reason of this pathology,
which leads to increased bile acid serum concentration1–4.
Autophagy is an evolutionarily conserved process by
which cytosolic components are sequestered into doublemembrane vesicles and consequently fused with lysosomes5,6. Autophagy plays an important role in cellular
pathways, including in growth, development and differentiation, aging and cell death7–9. Previous study reported
that autophagy was involved in the progress of preeclampsia10. The marker of autophagy was abnormally
expression in placenta with hypertensive disorders in
pregnancy11. However, little is known about the function
of autophagy in ICP.
LncRNA has been demonstrated that played an
important role in human disease. LncRNA HOTAIR12,
lncRNA MACC1-AS113, LncRNA-MALAT114 and
LncRNA NEAT115 were involved in cancer migration and
invasion. In human placenta, some LncRNAs was vrefiied.
Misregulation of LOC391533, LOC284100 and CEACAMP8 may contribute to the mechanism underlying
preeclampsia16. lncRHOXF1 is the first lncRNA that
regulates the host response to viral infections in human
placental progenitor cells17. However, little is known
about the expression and function of LncRNA in ICP.
In the present study, we investigated LncRNA expression
and its potential clinical significance in ICP, trying to clarify
the possible function of LncRNA in the pathogenesis.
Materials and methods
Tissue specimens
Correspondence: Xiaojing Dong (xffdoctor@163.com)
1
Department of Obstetrics and Gynecology, Second Affiliated Hospital,
Chongqing Medical University, Chongqing, China
These authors contributed equally: Jianguo Hu, Li Liu, Yangyang Gong.
Edited by G.M. Fimia
The placenta tissue and serum sample was were
obtained from the second affiliated Department of
© The Author(s) 2018
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction
in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if
changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If
material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Official journal of the Cell Death Differentiation Association
Hu et al. Cell Death and Disease (2018)9:979
Obstetrics and Gynecology, Second Affiliated Hospital,
Chongqing Medical University. The Ethics Committee of
the Chongqing Medical University approved the study
documents and the use of archived placenta tissues and
serum sample.
Animal models
Thirty Sprague-Dawley pregnant rats were randomly
divided into two groups: Group NP and Group ICP. The
rats provided by animal experiment center of Chongqing
medical university. The rats were maintained in collective
cages in an appropriate room with controlled temperature
and with a 12-h light cycle and fed with standard rat chow
and water. They were aged at ~150–180 days and weighed
~250–270 g. From day 13 of gestation, rats in group ICP
were injected intraperitoneally with estradiol benzoate
(5 mg/kg, Abcam) for 7 days consecutively. Group NP
(control group, named Group NP) was injected intraperitoneally with normal saline. On day 13 of gestation, the
levels of TBA, alanine aminotransferase (ALT), aspartate
aminotransferase (AST), and total bile acid(TBA) was
detected in serum which was drawn from tail vein of
pregnant rats. On day 21 of gestation, cesarean section was
performed with anesthesia by intraperitoneal injection of
10% hydral with dosage of 3 mL/kg. The placenta was
obtained for detecting the expression of LC3-II.
Forty C57 pregnant mice were randomly divided into four
groups: group NC, group RAPA, group CQ and group
CQ + RAPA. Mice were maintained in collective cages in an
appropriate room with controlled temperature with a 12-h
light cycle, and mice were allowed access to food and water
ad libitum. From day 12 of gestation of gestation, mice in
group NC were injected intraperitoneally with normal saline
for 6 days consecutively. Group RAPA were injected intraperitoneally with rapamycin(5 mg/kg, CST). Group CQ
were injected intraperitoneally with chloroquine (50 mg/kg,
CST). Group RAPA + CQ were injected intraperitoneally
with rapamycin(5 mg/kg, CST) and chloroquine (50 mg/kg,
CST). On day 21 of gestation, the pregnant mice were
sacrificed. Then, the fetal mice was calculated for body
weight, fetus number, bodylenth and placenta weight.
Microarray assay
Human LncRNA Microarray V4.0 (Agilent, USA) is
designed for global profiling of human lncRNAs and
protein-coding transcripts. Human miRNA Microarray
Release 21.0 (Agilent technologies, USA) is designed for
detecting the miRNA expression. The experiments and
data analysis were performed by Beijing Boao Crystal
Code Biotechnology Co. Ltd.
Cell culture, transfection procedure, and reagents
Human HTR8 cells were cultured in RPMI 1640 medium (Sigma-Aldrich, R8758) containing 10% fetal
Official journal of the Cell Death Differentiation Association
Page 2 of 14
bovineserum. The cells were incubated under 5% carbon
dioxide at 37 °C. siRNA corresponding to the target
sequences were synthesized by Genepharma (Shanghai,
China). The following sequences were targeted for human
Linc02527 small interfering RNA (siRNA): Linc02527-1:
5’-GGAGAGGCUAUAAACUUCUTT-3′;
Linc02527-2:
5′-CCGUGCAUAUUGUCUCCAATT-3′; and NC (negative control) siRNA: 5′-UUCUUCGAAGGUGUCACGU
TT-3′. Lentiviral vectors expressing shRNA targeting
Linc02527 (named LV3-lnc02527-1 and LV3-lnc02527-2)
and the Linc02527-lentiviral expression vector (named
LV5-lnc02527) were provided by Genepharma. miR-3185
mimics (sense: 5′-AGAAGAAGGCGGUCGGUCUGCG
G-3′) were synthesized at Ruibo Biotechnology(Guangzhou,
China). AST detecting using AST Activity Assay Kit
(MAK055, Sigma), ALT detecting using ALT Activity Assay
(MAK052, Sigma); TBA detecting using Total Bile Acids
(TBA) Assay Kit (Colorimetric) (BioVision.Inc, America);
CG detecting using CG RIA Kit(YB-10016, Shanghai Zibo
Biotechnology Co., Ltd. China).
Flow cytometric analysis of cell apoptosis
Cells for apoptotic analysis were double stained with
Annexin V–FITC and propidium iodide 48 h after transfection and analyzed using a flow cytometer (FACScan;
BD Biosciences, Shanghai, China) equipped with CellQuest software (BD Biosciences). Each experiment was
performed in triplicate.
RNA extraction and Quantitative real-time polymerase
chain reaction(RT-qPCR)
Total RNA was isolated using a pure high-purity Total
RNA Rapid Extraction Kit (Bioteke Corporation,
RP1201) according to the manufacturer’s instruction.
cDNA was synthesized using the iSCRIPT cDNA
synthesis kit (Bio-Rad Laboratories, 4106228). All of the
primers used to amplify lncRNAs were synthesized by
GenePharma. The real-time PCR kit was purchased from
GeneCopoeia. PCR conditions were 95 °C for 10 s, 60 °C
for 20 s and 72 °C for 10 s. Each sample was analyzed in
triplicate. Relative quantification of mRNA was performed using the comparative threshold cycle (CT)
method. This value was used to plot the gene expression
using the formula 2−Δ ΔCT.
Dual-luciferase reporter gene assay
Luciferase reporter gene assay was performed using the
Dual-Luciferase Reporter Assay System (Promega Corporation, E1910) according to the manufacturer’s
instruction. Luciferase reporter assays for ATG5, ATG7
and Linc02527, wild-type or mutant reporter constructs
(termed WT or Mut; obtained from Genepharma) were
cotransfected into HTR8 cells in 24-well plates with
100 nM MIR3185 or 100 nM miR-NC and Renilla plasmid
Hu et al. Cell Death and Disease (2018)9:979
Page 3 of 14
Table 1 Summary of lncRNA that are differently expressed between placenta from NP and ICP (ICP vs. NP)
Gene symbol
Chromosomal location
Regulation
P-value
LINC02027
Chr3:810430188-81144798(+)
Up
0.02699606
LINC02527
Chr6:112221691-112223054(−)
Up
0.01485026
FAM239A
ChrX: 3771050-3781615(−)
Up
0.025825039
KCNMB2-AS1
Chr3:178447071-178577338(−)
Up
0.028023811
YEATS2-AS1
Chr3:183524244-183526729(−)
Up
0.0014545523
LINC02050
Chr3: 80814047-80838505(+)
Up
0.09413093
TCONS_00005551
Chr3:81143793-81144462(+)
Up
0.02998128
TCONS_00011228
Chr6:112221561-112230600(−)
Up
0.014197185
LINC01436
Chr21: 36005338-36007838(+)
Up
0.010051289
HIT000075265
Chr16:23315293-23360088(+)
Down
0.021368366
uc021wnb.1
Chr22: 24245625-24247864(−)
Down
0.024611032
by using Endofectin-Plus (Gene-Copoeia, Z01010A). All
experiments were performed at least 3 times.
Western blotting
The expressions of LC3, ATG7, ATG5, YBX1, P21 and
actin proteins were analyzed by western blot18,19. The
primary antibodies used include polyclonal rabbit antiLC3 (Cell Signaling Technology, 4108); rabbit monoclonal
to ATG5 (Abcam, ab109490); rabbit monoclonal to ATG7
(Abcam, ab183188); rabbit monoclonal to YBX1 (Abcam,
ab76149); rabbit monoclonal to P21 (Abcam, ab109520);
and monoclonal rabbit anti-beta actin (Abcam,
ab115777). The band density was analyzed using a gel
imaging system and compared with an internal control.
Cell proliferation assay
Cell proliferation was determined using the EdU assay.
It was performed using the Cell-Light TM EdU imaging
detecting kit according to the instructions in the kit
(Ruibo Biotechnology, Guangzhou, China)20,21.
RNA pulldown assay and matrix-assisted laser desorption/
ionization time of flight mass spectrometry (MALDI-TOFMS)
To find the proteins bonding to Linc02527 directly, the
RNA pull-down assay was performed22,23. The specific
operation steps was according to the previous study24.
Using SDS-PAGE gel electrophoresis, Silver stain and
MALDI-TOF-MS, then, the specific proteins were
identified.
Statistical analysis
All statistical analyses were performed using SPSS
software, version 17.0 (Chicago, IL). Each experiment was
performed in triplicate. Statistical analysis was performed
Official journal of the Cell Death Differentiation Association
by Student t test. Pearson correlation analysis was conducted to analyze the association between Linc02527
expression and serum TBA, CG, ALT or AST. Data were
presented as mean ± standard deviation. Statistical significance was defined as a p-value less than 0.05.
Results
Microarray expression profiles of lncRNAs and mRNAs in
placenta of ICP patients
To explore the potential biological functions of
lncRNAs in ICP, we examined the expression patterns of
lncRNAs and mRNAs in placenta of ICP patients and
controls with full term pregnancy. In total, 358 lncRNAs
and 255 mRNAs showed a ≥1.5-fold change (P < 0.05) in
8 samples (Fig. 1a, b). Of these, 100 lncRNAs were upregulated, and 258 were down-regulated; 123 mRNAs
were up-regulated, 132 were down-regulated (Table 1 and
S1). The differential expression lncRNAs were related to
respiratory diseases, hypertensive diseases, cardiovascular
diseases and so on (Fig. 1c). These lncRNAs were involved
in many cellular process including in sodium channel
activity, peptide disulfide oxdoreductase activity and so on
(Fig. 1d). They also participated in aldosterone-regulated
sodium reabsorption, Stimuli-sensing channels, glutathine
metabolism and transmembrane transport of small
molecules (Fig. 1e).
Linc02527 expression was increased in placenta and serum
of ICP
We verified the expression of 11 LncRNA in placenta of
ICP. We found that Linc02527, KCNMB2-AS1, YEATS2AS1 and TCONS-00011228 were significant increased in
placenta of ICP (Fig. 2a, b, Fig. 2S1). We also observed
Linc02527, KCNMB2-AS1, YEATS2-AS1 and TCONS00011228 expression was significantly increased in serum
Hu et al. Cell Death and Disease (2018)9:979
Page 4 of 14
Fig. 1 Hierarchical clustering of a lncRNA and b mRNA differential expression profiles between the ICP group and control group(NP) in 8 placenta
samples. The heat maps are based on expression values of significantly differentially expressed lncRNAs and mRNAs (absolute fold change ≥1.5 and
P < 0.05) detected by microarray probes. “Red” and “Green” indicated expression above and below, respectively, relative expression. c Terms enriched
disease-related LncRNAs(top 30). d Go and Kegg (e) pathway analysis of differentially expressed LncRNAs(top 30)
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Hu et al. Cell Death and Disease (2018)9:979
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Fig. 2 Linc02527 was detected in placenta and serum of ICP. a The LncRNA was validated by qPCR in placenta of ICP (n = 20) and normal control (n
= 20). b Linc02527 was detected in placenta of ICP (n = 20) and normal control (n = 20). c, d The expression of LncRNA was detected in serum of
ICP (n = 20) and normal control (n = 20). e–h The serum TBA, CG, ALT and AST were detected in ICP patients (ICP; n = 20) and normal control (NP;
n = 20). i–l The expression of Linc02527 in placenta was positively correlated with the expression of serum TBA, CG, ALT and AST. Error bars
represented standard error. Asterisks(*) and ** indicated p < 0.05 and 0.001, respectively
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Table 2 Biochemistry (mean ± SD)
Parameter
NP (n = 20)
ICP (n = 20)
P value
TBA (µmol/L)
3.06 ± 2.12
25.04 ± 16.19
<0.01
CG (µmol/L)
0.85 ± 0.49
25.71 ± 26.66
<0.01
ALT (U/L)
11.75 ± 7.30
143.25 ± 118.48
AST (U/L)
19.35 ± 7.55
88.4 ± 66.05
Table 3 Subject demographic and characteristics (mean
± SD)
Parameter
NP (n = 20)
ICP (n = 20)
P value
Age (years)
30.95 ± 3.35
29.20 ± 4.34
NS
<0.01
Height (cm)
160.45 ± 4.84
159.70 ± 2.68
NS
<0.01
Weight (kg)
70.325 ± 7.87
67.25 ± 8.19
NS
14.275 ± 4.31
13.625 ± 2.86
NS
Total bilirubin (µmol/L)
9.10 ± 2.62
11.64 ± 5.39
NS
Weight gain (kg)
Direct bilirubin (µmol/L)
3.11 ± 1.49
4.92 ± 3.64
<0.05
Gravidity
2.30 ± 1.78
2.45 ± 1.76
NS
Indirect bilirubin (µmol/L)
6.00 ± 2.19
6.72 ± 3.15
NS
Parity
1.25 ± 0.44
1.3 ± 0.47
NS
of ICP (Fig. 2c, d and Fig. S2A-C). The serum TBA, CG,
ALT and AST was increased in ICP compared with that
normal pregnant (Fig. 2e–h and Table 2). The Birth body
length of newborn in ICP group was shorter than that of
normal group (Table 3). The serum TBA, CG, ALT and
AST was positively correlated with the expression of
Linc02527 in placenta (Fig. 2i–l). The serum TBA, CG,
ALT and AST was positively correlated with the expression of Linc02527 in serum (Fig. 2 S2D-G).
Linc02527 promoted proliferation and autophagy in HTR8
cells
We next determined the function of Linc02527. We
firstly detected the apoptosis rate after silencing or ectopically expressed Linc02527 in HTR8 cells. The apoptosis
rate is not significant difference between HTR8 cells
infected with LV3-lnc02527-1 or lnc02527-2 and HTR8
cells infected with LV3-NC (Fig. 3a). Also, the apoptosis
rate is not changed in HTR8 cells infected with LV5lnc02527 compared with infected with LV5-NC (Fig. 3c).
The expression of BCL-2 and Bax was not changed when
silencing or ectopically expressed Linc02527 in HTR8
cells (Fig. 3b, d). This data revealed that Linc02527 did not
regulate apoptosis in HTR8 cells. So we detected whether
Linc02527 regulated autophagy in HTR8 cells. As we
expected, the expression of LC3-II was decreased in
HTR8 cells infected LV3-lnc02527-1 or lnc02527-2
compared to that infected LV3-NC (Fig. 3b). The
expression of LC3-II was increased in HTR8 cells infected
LV5-lnc02527 compared to that infected LV5-NC
(Fig. 3d).
To investigate whether Linc02527 regulated autophagic
flux, we quantified the levels of LC3-II, the autophagosome associated form of the protein, in the absence and
presence of the lysosomal inhibitor chloroquine. The ratio
between the LC3-II levels with and without chloroquine
as an index of overall autophagic flux25. We observed a
increase of this ratio in HTR8 cells infected LV5-lnc02527
compared to that infected LV5-NC (Fig. 3e).This data
indicated that Linc02527 promoted autophagic flux. We
Official journal of the Cell Death Differentiation Association
Gestational weeks at delivery
38.39 ± 0.95
37.82 ± 1.03
NS
Birthweight (kg)
3.2 ± 0.32
3.04 ± 0.44
NS
Birth body length (cm)
50.35 ± 1.27
48.85 ± 1.90
<0.01
determined whether autophagy was activated in the placenta of ICP. We found that the expression of LC3-II,
ATG5, ATG7 and Beclin1 was increased in placenta of
ICP compared to normal pregnant (Fig. 3f).
We also investigated whether Linc02527 was involved in
the proliferate ability. We observed that silencing
Linc02527 suppressed cellular proliferate; however, ectopically expressed Linc02527 promoted the cellular proliferate ability in HTR8 cells (Fig. 3g–i). To determine the
mechanism of Linc02527, the location of Linc02527 was
detected. Our results revealed that Linc02527 was located
in the cytoplasm and nucleus (Fig. 3k).
Linc02527 promoted autophagy by sponging miR-3185
The expression of Linc02527 was increased in HTR8
cells infected LV5-lnc02527 compared to that infected
LV5-NC (Fig. 4a). The differential miRNA expression was
screened using miRNA array (S2). We verified six most
significant down-regulated genes using qPCR. Six miRNAs (miR-1323, miR-3185, miR-3935, miR-3187, miR3663 and miR-515) was decreased in HTR8 cells infected
LV5-lnc02527 compared to that infected LV5-NC (Fig. 4b
and S2). Bioinformatics prediction (LncBase Predicted
v.2) that there was a binding site between miR-3185 and
Linc02527 (Fig. 4c). Luciferase reporter gene assay confirmed the binding of miR-3185 with Linc02527 (Fig. 4d).
Over-expression of miR-3185 suppressed the expression
of Linc02527 (Fig. 4e). However, silencing miR-3185
increased the expression of Linc02527(Fig. 4f).
Bioinformatics prediction(microRNA.org) that ATG5
and ATG7 were the potential target of miR-3185. So we
detected the expression of ATG5 and ATG7 through
silencing or over-expression of miR-3185 in HTR8 cells.
The expression of ATG5 and ATG7 was decreased after
over-expression of miR-3185. In contrast, the expression
of ATG5 and ATG7 was increased after silencing
miR-3185 (Fig. 5a–c). The LC3-II also was regulated by
Hu et al. Cell Death and Disease (2018)9:979
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Fig. 3 Linc02527 regulated autophagy and proliferate in HTR8 cells. a The apoptosis rate was detected using flow cytometry when silencing
Linc02527. b The expression of LC3-II, BCL-2 and BAX was detected by western blot after silencing Linc02527.(c)The apoptosis rate was detected
using flow cytometry when ectopically expression of Linc02527. d The expression of LC3-II, BCL-2 and BAX was detected by western blot when
ectopically expression of Linc02527. e The expression of LC3-II was detected by western blot. LV5-NC: HTR8 cells infected by LV5-NC. LV5-NC + CQ:
HTR8 cells infected by LV5-NC, then adding chloroquine at 10uM for 24 h. LV5: HTR8 cells infected by LV5-lnc02527. LV5 + CQ: HTR8 cells infected by
LV5- lnc02527, then adding chloroquine at 10 uM for 24 h. f The expression of LC3 in placenta of ICP and normal group was detected by western
blot. g–j The cellular proliferation was detected by EdU. k The location of Linc02527 was determined hybridization in situ. Lnc02527 probe was
detected the location of Linc02527. NC was negative control. Error bars represented standard error. Asterisks(*) and ** indicated p < 0.05 and 0.001,
respectively
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Hu et al. Cell Death and Disease (2018)9:979
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Fig. 4 The expression of miR-3185 was regulated by Linc02527. a The expression of Linc02527 was determined by qPCR. b The miRNAs expression
was detected by qPCR in HTR8 cells infected LV5-lnc02527 or LV5-NC. c The potential binding sites between Linc02527 and miR-3185 were predicted.
d HTR8 cells were co-transfected with miR-3185 mimic or control RNA (NC) with luciferase reporter plasmids containing either wild-type (pMIRlnc02527) or mutant (pMIR-lnc02527m) of Linc02527 genes. Luciferase expression was measured. The fold changes of the relative luciferase activity in
miR-3185 mimic indicated that plasmid transfected cells were normalized to NC, indicating plasmid transfected cells. e The expression of Linc02527
was detected by transfecting miR-3185 mimic or NC(negative control). f The expression of Linc02527 was detected by transfecting miR-3185 inhibitor
or NC(negative control). Error bars represented standard error. Asterisks(*) and ** indicated p < 0.05 and 0.001, respectively
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Hu et al. Cell Death and Disease (2018)9:979
Fig. 5 (See legend on next page.)
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Hu et al. Cell Death and Disease (2018)9:979
Page 10 of 14
(see figure on previous page)
Fig. 5 The expression of ATG5 and ATG7 was regulated by miR-3185. a The expression of ATG5 mRNA was detected when transfecting with miR3185 mimic, miR-3185 mimic control, miR-3185 inhibitor or miR-3185 inhibitor mimic. b The expression of ATG7 mRNA was detected when
transfecting with miR-3185 mimic, miR-3185 mimic control, miR-3185 inhibitor or miR-3185 inhibitor mimic. c The protein expression of ATG5, ATG7
and LC3-II was determined when transfecting with miR-3185 mimic, miR-3185 mimic control, miR-3185 inhibitor or miR-3185 inhibitor mimic.
d, e The potential binding sites between miR-3185, ATG5 and ATG7 were predicted. f, g HTR8 cells were co-transfected with miR-3185 mimic or
control RNA (NC) with luciferase reporter plasmids containing either wild-type (pMIR-ATG5-3’UTR and pMIR-ATG7-3’UTR) or mutant (pMIR-ATG53’UTRm and pMIR-ATG7-3’UTRm) of ATG5 and ATG7 genes. Luciferase expression was measured. The fold changes of the relative luciferase activity in
miR-3185 mimic indicated that plasmid transfected cells were normalized to NC, indicating plasmid transfected cells. h, i The expression of ATG5 and
ATG7 was determined in HTR8 cells infected with LV5-NC or LV5-lnc02527. j, k The expression of ATG5 and ATG7 was determined in HTR8 cells
infected with LV3-NC, LV2-lnc02527-1 or LV2-lnc02527-2. Error bars represented standard error. Asterisks(*) and **indicated p < 0.05 and 0.001,
respectively
To further explore the mechanism of Linc02527 regulation autophagy. The RNA pull down assay was performed.
Then using SDS-PAGE gel electrophoresis and Silver stain
six differential bands were obtained(Fig. 6a). Using
MALDI-TOF-MS, some protein including YBX1 were
identified(S3). It indicated that YBX1 was directly bond to
Linc02527(Fig. 6a–c).We confirmed that YBX1 was
directly bond to Linc02527 through RNA pull down assay
(Fig. 6f). The expression of YBX1 was increased in HTR8
cells infected LV5-lnc02527 compared to that infected
LV5-NC(Fig. 6d). The expression of YBX1 was decreased
in HTR8 cells infected LV3-lnc02527-1 or LV3-lnc02527-2
compared to that infected LV3-NC(Fig. 6e). Previous study
reported that P21 pathway was activated by YBX126. P21
pathway can promotes autophagy27–29. P21 was elevated
when over-expression of Linc02527. P21 was downregulated when silencing Linc02527 expression(Fig. 6d,
e). So, we guessed Linc02527 bond to and stabilized YBX1,
then activated P21 pathway and prompted autophagy.
group (Fig. 7d). This data revealed that autophagy was
activated in ICP rat. We also observed YBX1 and P21 was
elevated in the placenta of ICP rat compared to normal
group (Fig. 7d). Then, we explore whether activated
autophagy or inhibited autophagy can affect the growth of
C57 mouse. We observed that the growth of C57 mouse
was retarded when autophagy was activated using rapamycin compared to normal group (Fig. 7E1–2). Compared
with the normal group, the growth of C57 mouse is not
different when autophagy was inhibited using chloroquine
(Fig. 7E3). The inhibition of growth by rapamycin is partly
relieved through adding chloroquine (Fig. 7E4). The number of fetuses is no significant difference between normal
control group, rapamycin group, chloroquine group and
rapamycin + chloroquine group (Fig. 7f).The fetuses birth
weight, body length and placenta weight were decreased in
rapamycin group than that of normal control group
(Fig. 7g–i). The fetuses birth weight, body length and placenta weight were decreased in rapamycin group than that
of normal control group (Fig. 7g–i). The fetuses birth
weight, body length and placenta weight were decreased in
rapamycin + chloroquine group than that of rapamycin
group (Fig. 7g–i). There was not significant difference
between chloroquine group and normal control group
(Fig. 7g–i). These data revealed that the growth of C57
mouse was repressed when autophagy was activated. In
normal condition, inhibited autophagy using chloroquine
did not affect the growth of C57 mouse. However, in the
condition of autophagy was activated, inhibited autophagy
using chloroquine can improve the growth of C57 mouse.
Rapamycin caused fetal growth retardation, possibly due to
inhibition of the mTOR pathway.
Autophagy was involved in growth of fetal mouse
Discussion
We have demonstrated Linc02527 promoted autophagy
in vitro. However, we did not find homologous sequence
of Linc02527 in rat and mouse. We observed autophagy
was activated in ICP. So, we detected whether autophagy
was activated in ICP in the rat model. The serum TBA,
ALT and AST was increased in ICP in the rat model
(Fig. 7a–c). The LC3-II, ATG5 and ATG7 expression was
elevated in the placenta of ICP rat compared to normal
In the present research, we found that Linc02527 was
increased expression in serum and placenta of ICP
patients. Linc02527 promoted autophagy in HTR8 cells.
Linc02527 regulated ATG5 and ATG7 though sponging
miR-3185. Linc02527 directly binding to YBX1 and activated P21, then the autophagy was promoted. We also
found that regulation of autophagy was mediated the
growth of fetal mouse.
miR-3185 (Fig. 5c). We predicted that there was a binding
site between miR-3185, ATG5 and ATG7 (Fig. 5d, e).
Luciferase reporter gene assay confirmed the binding of
miR-3185 with ATG5 and ATG7 (Fig. 5f, g). The
expression of ATG5 and ATG7 was increased in HTR8
cells infected LV5-lnc02527 compared to that infected
LV5-NC (Fig. 5h, i). The expression of ATG5 and ATG7
was decreased in HTR8 cells infected LV3-lnc02527-1 or
LV3-lnc02527-2 compared to that infected LV3-NC
(Fig. 5j, k). This data suggested that Linc02527 promoted autophagy by sponging miR-3185.
Linc02527 directly binding to YBX1 and activated P21
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Hu et al. Cell Death and Disease (2018)9:979
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Fig. 6 Linc02527 directly binding to YBX1 and activated P21. a Silver stains for protein gels obtained by Linc02527 RNA Pulldown. Bio-lnc02527
(sense): treatment group; Bio-lnc02527 (antisense). The Linc02527 specific binding protein gels (in red box) was identified by MALDI-TOF-MS. Results
show that YBX1 was a direct binding protein to Linc02527. b, c YBX1 was identified by MALDI-TOF-MS. d The expression of LC3-II,YBX1 and P21 was
determined in HTR8 cells infected with LV5-NC or LV5-lnc02527. e The expression of LC3-II,YBX1 and P21 was determined in HTR8 cells infected with
LV3-NC, LV2-lnc02527-1 or LV2-lnc02527-2. f The Linc02527 specific binding protein gels was identified by RNA pulldown. FL was the biotin
Linc02527. NC was the Linc02527 without biotin label
Linc02527 was located in 6q21. In 27 different human
tissues, it is distinctive high-expression in placenta and
testis30. In this study, we observed Linc02527 expression
was increased in the placenta and serum of ICP patients.
The serum TBA, CG, ALT and AST was elevated in ICP
patients. The serum TBA and CG were diagnostic criteria
of ICP31–33. The expression of Linc02527 was positively
with the serum TBA, CG, ALT and AST. So, Linc02527 is
a potential biomarker of Intrahepatic Cholestasis of
Pregnancy(ICP).
In this research, we noted that Linc02527 was not
involved in apoptosis. However, it promoted autophagy
and proliferate. Many studies have demonstrated that
Official journal of the Cell Death Differentiation Association
LncRNA regulated the target genes by sponging miRNAs34–36. ATG5 and ATG7 are two important moleculars
to promote autophagy37–41. We found miR-3185 inhibited
autophagy by targeting ATG5 and ATG7. Linc02527
directly bond to and down-regulated miR-3185. Our data
revealed Linc02527 regulated ATG5 and ATG7 through
sponging miR-3185. YBX1 regulates tumor growth via
CDC25a pathway in human lung cancer42. lncRNA GAS5
enhances G1 cell cycle arrest via binding to YBX1 to
regulate p21 expression in stomach cancer26. Phosphorylation of YBX1 activates NF-κB in colon cancer. We
found Linc02527 directly bond to and stabilized YBX1. At
the same time, Linc02527 increased the expression of P21.
Hu et al. Cell Death and Disease (2018)9:979
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Fig. 7 Autophagy was involved in growth of fetal mouse. a–c The serum TBA, ALT and AST were detected in in ICP in the rat model. d The expression
of LC3-II was detected in the placenta of ICP rat or NP group(normal control). e–i Forty C57 pregnant mice were randomly divided into four groups:
group NC(E1), group RAPA(E2), group CQ(E3) and group CQ + RAPA(E4). From day 12 of gestation of gestation, mice in group NC were injected
intraperitoneally with normal saline for 6 days consecutively. Group RAPA were injected intraperitoneally with rapamycin (5 mg/kg). Group CQ were
injected intraperitoneally with chloroquine (50 mg/kg). Group RAPA + CQ were injected intraperitoneally with rapamycin (5 mg/kg) and chloroquine
(50 mg/kg). On day 21 of gestation, the pregnant mice were sacrificed. Then, the fetal mice was calculated for body weight, fetus number, bodylenth
and placenta weight. Error bars represented standard error. Asterisks(*) and **indicated p < 0.05 and 0.001, respectively
Official journal of the Cell Death Differentiation Association
Hu et al. Cell Death and Disease (2018)9:979
P21 pathway can promotes autophagy27–29. So, we guessed Linc02527 bond to and stabilized YBX1, then activated P21 pathway and prompted autophagy. These
results indicated that Linc02527 was a potential target for
ICP.
Autophagy is an evolutionarily conserved catalytic
process by which cytoplasmic components including
damaged macromolecules and organelles are degraded43.
An increased autophagosome formation was observed in
placenta from preeclampsia and FGR44. In this study, we
found LC3-II expression was elevated in placenta from
ICP. The expression of LC3-II also was increased in the
placenta of ICP rat. This result indicated that autophagy
was activated in ICP. Autophagy has an important role in
early embryo development. Increased embryonic or fetal
death caused by inactivation of autophagy related genes in
mouse. Atg5 deficient mice were fertilized normally, but
manifested embryonic lethality at last45. Beclin1 deficient
mice embryos also die as early as embryonic day 7.546.
Previous study has demonstrated chloroquine and
hydroxychloroquine inhibit bladder cancer cell growth by
targeting basal autophagy47. Hydroxychloroquine (HCQ)
inhibits autophagy and sensitize some cancer cells to
chemotherapy48. However, as an inhibitor of autophagy,
HCQ reduces neonatal morbidity in women with SLE by
significantly decreasing the rate of prematurity and
intrauterine growth restriction49. We found chloroquine
(CQ) did not increase the amount of fetal mice. But
induced autophagy using rapamycin, the development of
fetal mice was retard and the fetuses birth weight, body
length and placenta weight were decreased. Chloroquine
(CQ) can rescue the retarded development and increase
the fetuses birth weight, body length and placenta weight
at the condition of activated autophagy. Based on these
results, we guessed that activated autophagy impaired the
growth of fetal mice. Inhibited autophagy promoted the
growth of fetus at the condition of activated autophagy.
Chloroquine is a potential drug of ICP.
To our knowledge, this is the first time reported that
autophagy was activated in ICP. The expression of
Linc02527 was increased in placenta and serum in ICP
patients. Linc02527 bond to and stabilized YBX1, then
activated P21 pathway and prompted autophagy. We also
found Chloroquine (CQ) can rescue the retarded development at the condition of activated autophagy. So,
Linc02527 was a potential biomarker and target for ICP.
Chloroquine may be a drug for treating ICP.
Acknowledgements
We thank members of our laboratory for technical help and stimulating
discussions. This work was supported by the Natural Science Foundation of
Chongqing (CSTC 2012JJB10030); the National Science Foundation of China
(81172492); the National Science Foundation of China (81501220); and
Chongqing Municipal Health and Family Planning Commission Medical
Research Fund(2017MSXM031).
Official journal of the Cell Death Differentiation Association
Page 13 of 14
Conflict of interest
The authors declare that they have no conflict of interest.
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Supplementary Information accompanies this paper at (https://doi.org/
10.1038/s41419-018-1013-z).
Received: 25 May 2018 Revised: 28 August 2018 Accepted: 29 August 2018
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RESEARCH ARTICLE
Late middle Miocene caviomorph rodents
from Tarapoto, Peruvian Amazonia
Myriam Boivin ID1*, Laurent Marivaux2, Walter Aguirre-Diaz3, Aldo Benites-Palomino ID3,4,
Guillaume Billet5, François Pujos ID6, Rodolfo Salas-Gismondi3,7, Narla S. Stutz ID2,8, Julia
V. Tejada-Lara2,3, Rafael M. Varas-Malca ID3, Anne H. Walton9, Pierre-Olivier Antoine2
a1111111111
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OPEN ACCESS
Citation: Boivin M, Marivaux L, Aguirre-Diaz W,
Benites-Palomino A, Billet G, Pujos F, et al. (2021)
Late middle Miocene caviomorph rodents from
Tarapoto, Peruvian Amazonia. PLoS ONE 16(11):
e0258455. https://doi.org/10.1371/journal.
pone.0258455
Editor: Jürgen Kriwet, University of Vienna,
AUSTRIA
Received: July 19, 2021
Accepted: September 24, 2021
Published: November 3, 2021
Peer Review History: PLOS recognizes the
benefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0258455
Copyright: © 2021 Boivin et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files (S1 Table and S2 File). The fossil
material is permanently stored in the Vertebrate
1 Laboratorio de Paleontologı́a de Vertebrados, Instituto de Ecorregiones Andinas (INECOA), Universidad
Nacional de Jujuy, CONICET, San Salvador de Jujuy, Jujuy, Argentina, 2 Laboratoire de Paléontologie,
Institut des Sciences de l’Evolution de Montpellier (ISEM, UMR 5554, CNRS/UM/IRD/EPHE), Université de
Montpellier, Montpellier, France, 3 Departamento de Paleontologı́a de Vertebrados, Museo de Historia
Natural—Universidad Nacional Mayor San Marcos (UNMSM, DPV-MUSM), Lima, Peru, 4 Paläontologisches
Institut und Museum, Universität Zürich, Zürich, Switzerland, 5 Département Origine et Evolution, Muséum
national d’Histoire naturelle (MNHN, CR2P-UMR 7207 CNRS/MNHN/Sorbonne Université), Paris, France,
6 Laboratorio de Paleontologı́a, Instituto Argentino de Nivologı́a, Glaciologı́a y Ciencias Ambientales
(IANIGLA), CCT–CONICET–Mendoza, Mendoza, Argentina, 7 BioGeoCiencias Lab, Facultad de Ciencias y
Filosofı́a/CIDIS, Laboratorios de Investigacion y Desarrollo (LID), Centro de Investigación para el Desarrollo
Integral y Sostenible (CIDIS), Universidad Peruana Cayetano Heredia, Lima, Peru, 8 Programa de PósGraduação em Geociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul,
Brazil, 9 Springfield Technical Community College, Springfield, Massachusset, United States of America
* mboivin@idgym.unju.edu.ar
Abstract
Miocene deposits of South America have yielded several species-rich assemblages of caviomorph rodents. They are mostly situated at high and mid- latitudes of the continent, except for
the exceptional Honda Group of La Venta, Colombia, the faunal composition of which allowed
to describe the late middle Miocene Laventan South American Land Mammal Age (SALMA).
In this paper, we describe a new caviomorph assemblage from TAR-31 locality, recently discovered near Tarapoto in Peruvian Amazonia (San Martı́n Department). Based on mammalian
biostratigraphy, this single-phased locality is unambiguously considered to fall within the Laventan SALMA. TAR-31 yielded rodent species found in La Venta, such as the octodontoid Ricardomys longidens Walton, 1990 (nom. nud.), the chinchilloids Microscleromys paradoxalis
Walton, 1990 (nom. nud.) and M. cribriphilus Walton, 1990 (nom. nud.), or closely-related taxa.
Given these strong taxonomic affinities, we further seize the opportunity to review the rodent
dental material from La Venta described in the Ph.D. volume of Walton in 1990 but referred to
as nomina nuda. Here we validate the recognition of these former taxa and provide their formal
description. TAR-31 documents nine distinct rodent species documenting the four extant
superfamilies of Caviomorpha, including a new erethizontoid: Nuyuyomys chinqaska gen. et
sp. nov. These fossils document the most diverse caviomorph fauna for the middle Miocene
interval of Peruvian Amazonia to date. This rodent discovery from Peru extends the geographical ranges of Ricardomys longidens, Microscleromys paradoxalis, and M. cribriphilus, 1,100
km to the south. Only one postcranial element of rodent was unearthed in TAR-31 (astragalus).
This tiny tarsal bone most likely documents one of the two species of Microscleromys and its
morphology indicates terrestrial generalist adaptations for this minute chinchilloid.
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Paleontological collection of the “Museo de Historia
Natural, Universidad Nacional Mayor de San
Marcos” (MUSM), Lima, Peru.
Funding: LM and POA received funding from The
Leakey Foundation and the LabEx CEBA (ANR-10LABX-0025-01). POA received funding from the
National Geographic Society and from the French
‘Agence Nationale de la Recherche’ (ANR) in the
framework of the GAARAnti program (ANR-17CE31-0009). LM received funding from the
CoopIntEER CNRS-CONICET (n˚ 252540). POA and
FP received funding from the ECOS-SUD/FONCyT
(n˚ A-14U01) international collaboration programs.
MB received funding from the ‘Laboratoire de
Planétologie et de Géodynamique de Nantes‘. 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.
Miocene caviomorphs from Tarapoto, Peru
Introduction
Caviomorphs (Caviomorpha Wood, 1955) are hystricognathous rodents originating from
South America, such as guinea pig, New World porcupines, chinchillas, and spiny rats. Nowadays, they show a great taxonomical diversity and a large array of lifestyles (arboreal, fossorial,
semi-aquatic, and terrestrial) [1]. Among caviomorphs, two main clades, each divided into
two superfamilies, are recognised [2–4]: Erethicavioi Boivin, 2019 (comprising Cavioidea
Fischer, 1817 + Erethizontoidea Bonaparte, 1845 + stem-groups) and Octochinchilloi Boivin,
2019 (comprising Chinchilloidea Bennett, 1833 + Octodontoidea Waterhouse, 1839 + stemgroups). Their evolutionary history extends back to the late middle Eocene, with the earliest
representatives of the group so far recorded in Peruvian Amazonia [5–8].
The Miocene record of caviomorphs is particularly well documented. Numerous fossilbearing localities yielded abundant specimens and species-rich assemblages of rodents [9].
Most of them are situated at high and middle latitudes, notably in Argentina (e.g., Pinturas,
Sarmiento, Santa Cruz, Collón Cura, Cerro Azul and Ituzaingo formations) [9–15] and Chile
(e.g., Laguna del Laja and Pampa Castillo) [16, 17]. At low latitudes of South America, two
remarkable and very species-rich caviomorph faunas are known, La Venta in Colombia
(Honda Group; late middle Miocene) [18] and Acre in Brazil (Solimões Fm. [Formation]; late
Miocene; see [19]). Since the early discoveries of fossil-bearing localities in the Honda Group
of the upper Magdalena valley in 1923, considerable field efforts led by petroleum companies
and several scientific teams were performed in this region of Colombia, and notably in the La
Venta area, until 1992 [20, 21]. As a result, 140 localities over 52 different stratigraphic levels,
yielding thousands of fossils of many vertebrate groups were discovered in the Honda Group
dating from the late middle Miocene [22]. In particular, the recognition in 1945 of the first fossil remains of primates in the Magdalena valley [23, 24] has been one of the most significant
discoveries that motivated numerous subsequent field expeditions [20]. The mammal fauna
from La Venta encompasses at least 77 species and 80 genera, representing 29 families [25, 26].
Concerning rodents, around 20 taxa documenting the four main caviomorph superfamilies
have so far been reported (excluding the indeterminate genera and species) [18, 27–30]. In
addition to La Venta, the Peruvian faunas from MD-67 (Madre de Dios) [31], the Fitzcarrald
local fauna (near Atalaya, Ucayali) [32, 33], and CTA-45 (near Contamana, Loreto) [34], also
middle Miocene in age, document representatives of the four caviomorph superfamilies (4, 8,
and 2 taxa, respectively). Finally, a fourth late middle Miocene caviomorph-bearing locality
also occurs in low latitudes, from the Socorro Formation in Venezuela, but so far it has only
yielded a fragmented distal femur of a giant caviomorph [35].
Over the last decade, our yearly paleontological surveys in the Tarapoto area (San Martı́n
Department, Peruvian Amazonia), have allowed the discovery of a new fossil-bearing locality,
TAR-31, in the vicinity of the Juan Guerra village. This locality is considered to be late middle
Miocene in age (Laventan SALMA) based on mammalian biostratigraphy (see [36]; see Material and Methods section of this paper). TAR-31 yielded a diverse assemblage of aquatic and
terrestrial vertebrates, including numerous caviomorph specimens that are the subject of this
paper. The primary purpose of the present work is to describe the new caviomorph materials
found in TAR-31. This locality shares taxa or closely related taxa with La Venta fauna
described by one of us (AHW) in the framework of her PhD [37]: Microsteiromys, Ricardomys,
and Microscleromys. Because Microsteiromys, Ricardomys, Microscleromys, and the species
included within these genera were not formally described (nomina nuda) [18, 37, 38], here we
also revise the material from La Venta attributed to these taxa, in order to clarify their
systematics.
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Material and methods
TAR-31, Tarapoto, Peru
The rodent fossil material from Peruvian Amazonia described in the present work originates
from the TAR-31 locality, situated in the vicinity of the small town Juan Guerra, along the
Mayo River (Tarapoto area, San Martı́n Department, Peru; Fig 1A and 1B). TAR-31 consists of
a 10–15 cm-thick yellow microconglomerate interbedded within a grey cross-stratified and
sandstone-dominated fluvial unit (Fig 2A [36]). The latter is intercalated between thick violingrey variegated paleosols pointing to the existence of a meandering river with sustainable
floodplain [36]. These levels were originally mapped as belonging to the lower member of
Ipururo Formation (middle Miocene in age in the Huallaga basin) [39–42], an assignation in
agreement with the nature of their facies and depositional environment sequence.
The fossil content of the TAR-31 encompasses plants, amber clasts, crabs, chondrichthyans,
osteichthyans, anurans, chelonians, crocodylomorphs, birds, and mammals (including
metatherians, xenarthrans, liptoterns, notungulates, sirenians, chiropterans, primates, and
caviomorph rodents). The TAR-31 mammal assemblage includes a didelphid marsupial (currently under study by one of us, NSS), the interatheriine notoungulate Miocochilius sp., the
didolodontid Megadolodus sp., the platyrrhine primate Neosaimiri aff. fieldsi [36], and the
caviomorph fauna described here (see ‘Age of TAR-31’ section of this present work). This
assemblage recalls some of the Laventan SALMA localities: Quebrada Honda in Bolivia (13.1–
12.2 Ma) [43–46], the Fitzcarrald local fauna in SE Peru [33], and especially the lower part of
the Villavieja Formation in the La Venta area in Colombia (13.8–11.6 Ma; [47] and see above).
Accordingly, the TAR-31 locality most likely documents the late middle Miocene Laventan
SALMA [36].
The material from TAR-31 was collected by excavating and wet screening (2 and 1 mm
meshes) about 550 kg of sediment during our yearly field expeditions (August 2015, 2016, and
Fig 1. Geographic location of TAR-31 (San Martı́n Department, Peru; late middle Miocene, Laventan SALMA). (A) Location map of the
Tarapoto area (star symbol) in Peru. (B) Location map of the TAR-31 (star symbol) in the Tarapoto area. SALMA, South American Land
Mammal Age.
https://doi.org/10.1371/journal.pone.0258455.g001
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Miocene caviomorphs from Tarapoto, Peru
2018). The caviomorph material corresponds to about 400 isolated teeth (complete or fragmentary), one fragment of mandible (MUSM 4643), one fragment of maxilla (MUSM 4375),
and one astragalus (MUSM 4658). No permits were required for the described study, which
complied with all relevant regulations. The field work on TAR-31 was carried out in the framework of the ongoing cooperation agreement between the ‘Museo de Historia Natural de la
Universidad Nacional Mayor San Marcos’ (Lima, Peru) and the ‘Institut des Sciences de l’Evolution de Montpellier-Université de Montpellier’ (France). The TAR-31 fossil specimens are
permanently stored in the palaeontological collection of the ‘Museo de Historia Natural, Universidad Nacional Mayor de San Marcos’, Lima, Peru (MUSM).
La Venta area, Colombia
The fossil rodent material from La Venta re-analysed here (originally studied in the framework
of the Ph.D. of Anne H. Walton) [18, 37] originates from the badland exposures located in the
vicinities of the Villavieja and La Victoria villages, in the upper valley of the Magdalena River
(Huila Department, Colombia). Numerous fossil-bearing localities are known in the La Venta
area (and the Magdalena upper valley in general) [26]. These are assigned to two successive
formations of the Honda Group: La Victoria and Villavieja formations [48]. The studied material from this area was found in: (i) one locality from La Victoria Formation (IGM-DU loc.
075, between the Chunchullo Beds and the Tatcoa Sandstone Beds), and (ii) distinct localities
from the Villavieja Formation, including several localities in the Fish Bed (CVP 5, 8, 9, 10,
10A, and 13B), two localities in the Monkey Bed (IGM-DU loc. 006–1 and IGM-DU loc. 022
or UCMP loc. V4536), and one in the El Cardón Red Beds (IGM-DU loc. 032 and its screenwashed locality 032–5). The Unit between the Chunchullo and Tatacoa Sandstone Beds is
149.3 m-thick, and is composed of very fine sandstones and mudstones alternating with metric
medium-grained sandstone lenses [48]. The Villavieja Formation is divided into the Baraya
Member, including the Fish and Monkey beds, and the Cerro Colorado Member, including
the El Cardón Red Beds [48]. The Baraya Member is very fossiliferous and is composed mainly
of gray mudstones and sandstones with minor layers of red mudstones, while the Cerro Colorado Member is less fossiliferous and consists of thick horizons of red mudstones with a very
small amount of volcanic litharenites and chert litharenites [48]. The La Venta fauna is late
middle Miocene in age (13.8–12.05 Ma) based on 40Ar/39Ar datings realised on volcanic units
from different localities of the Honda Group (on biotites, hornblendes, and plagioclases; 13.8–
12.2 Ma) and magnetostratigraphic correlations of the Honda Group (13.61 [base of C5ABn
chron] –12.05 Ma [top of C5An.1n]) [26, 47, 49]. The La Venta area yielded a very rich vertebrate fauna including chondrichthyans, osteichthyans, amphibians, squamates, chelonians,
crocodylomorphs, birds and mammals (metatherians, xenarthrans, astrapotheres, litopterns,
notoungulates, cetaceans, chiropterans, sirenians, primates and caviomorph rodents) [21, 22].
The caviomorph remains from La Venta included in this work were formerly studied by
one of us [18, 37]. In a doctoral thesis manuscript, Walton [37] described several new genera
and species such as Microsteiromys jacobsi, Ricardomys longidens, Microscleromys paradoxalis,
and Microscleromys cribriphilus. As the latter taxa were not formally described, they should be
considered as not available following the articles 8, 9, 11, 13 of the International Code of Zoological Nomenclature (i.e., nomen nudum; p. 132–143, 246 [38]). As formally recognised for
nomina nuda (“A nomen nudum is not an available name, and therefore the same name may
be made available later for the same or a different concept; in such a case it would take authorship and date [Arts. 21, 50] from that act of establishment, not from any earlier publication as
a nomen nudum” [38], we chose to keep the taxonomic names proposed by Walton [37]. For
reasons related to the covid pandemic and confinement, the original material from La Venta
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could not be directly observed. The description and comparison of this material in the present
work is based on published photos and drawings [18, 37]. Most of these specimens were collected by surface collecting and wet screening (2 and 0.6 or 0.25 mm meshes) in the years 1985
through 1989. This material is stored in the ‘Museo Geológico José Royo y Gómez, Servio Geológico Colombiano’ (IGM; formerly, ‘Instituto Nacional de Investigaciones en Geociencias,
Mineria y Quı́mica’) and the ‘Departamento de Geociencias, Universidad Nacional de Colombia’ (UNC), Bogotá, Colombia, as well as in the Field Museum of Natural History, Chicago
(FMNH), USA. Some specimens do not have collection number (unnumbered specimens).
We used their field number to refer to them (S1 Table).
Imaging
For the largest specimens (mandibular and maxillary fragments) and high-crowned teeth
found at TAR-31, we used X-ray microtomography (μCT-scan) for obtaining three-dimensional digital models (3D surface renderings). The dental/jaw specimens and the astragalus
were scanned with a resolution of 6 μm and 5 μm, respectively, using a μCT-scanning station
EasyTom 150/Rx Solutions (Montpellier RIO Imaging [MRI], ISEM, Montpellier, France).
They have been virtually delimited by manual segmentation under AVIZO 7.1 software (Visualization Sciences Group). The specimens were prepared within a ‘Label Field’ module of
AVIZO, using the segmentation threshold selection tool. The other teeth figured in this paper
were photographed with a Scanning Electron Microscope (SEM) HITACHI S 4000 (Institut
des Neurosciences de Montpellier [INM], France). Some of the pictures of the astragalus are
photographs of the original specimen, which are the result of the fusion of multifocus images
obtained with an optical stereomicroscope (Leica M 205C) connected to a camera (Leica DFC
420C; ISEM).
Nomenclature and comparisons
The main terminology for the rodent dentition follows Boivin & Marivaux [50], and the literature cited therein. Some extant caviomorphs and related extinct forms show a highly derived/
specialised dental pattern with respect to that of the oldest representatives of this group (for
which the aforementioned nomenclature was based on). This is the case of two taxa described
below: (i) Caviidae indet. gen. et sp., for which nomenclatures proposed by several authors
[51–55] were followed (Fig 2A); and (ii) the adelphomyine Ricardomys longidens, for which we
partly followed the nomenclature proposed by Patterson & Pascual [56] (Fig 2B and 2C).
Lower case letters are used for lower dentition (dp, for decidious premolar; p, for premolar; m,
for molars) and upper case letters for upper dentition (dP, for decidious premolar; P, for premolar; M, for molars). The astragalar nomenclature is based on Ginot et al. [57], Rose & Chinnery [58], and Wible & Hughes [59]. The caviomorph taxa cited in the text and used for dental
comparisons are listed in S2 Table. Three isolated teeth from MD-67 locality in Peru (MUSM
1974, 1975, and 4298; early middle Miocene) were originally attributed to cf. Microsteiromys
sp [31]. They are reassigned here in light of the new material from Tarapoto. For the MUSM
4658 astragalus, we used the same comparative sampling as Boivin et al. [60]. In addition, we
consulted supplementary astragalar material attributed to the erethizontid Steiromys duplicatus
(MACN A 10055–78, 10081 and 10082; see [61]), the caviids Dolicavia minuscula (MMP
10055–78) and Galea leucoblephara (INBIAL-CV 00290), the neoepiblemid Neoepiblema sp.
(probably N. acreensis; UFAC 5249 and 61840), the octodontids Abalosia castellanosi (MMP
1439-M) and Pithanotomys columnaris (MACN-Pv 7429–7431), the echimyid Eumysops chapalmalensis (MACN-Pv 17868), and the ctenomyid Actenomys priscus (MMP 367-S and
395-S). When the fossils described in this paper are compared with several taxa, they are
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Fig 2. Dental nomenclature for cavioid (A) and adelphomyine (B, C) rodent teeth in occlusal view. (A) p4; 1, anterior projection of the anterior lobe; 2,
interprismatic furrow; 3, apexes; 4, hypoflexid; 5, enamel; 6, dentine; 7, posterior lobe; 8, longitudinal furrow opposite to the hypoflexid (= primary internal flexid);
9, anterior lobe. (B) Lower molar; 1, metaconid (+ its posterior arm?); 2, first lamella; 3, metalophulid I; 4, protoconid; 5, ectolophid; 6, hypoflexid; 7, anterior
outgrowth of the hypoconid; 8, hypoconid; 9, anterior arm of the hypoconid; 10, posterolophid; 11, third lamella; 12, metaflexid; 13, second lamella; 14, entoconid;
15, hypolophid; 16, confluence between the anteroflexid and the mesoflexid. (C) Upper molar; 1, first lamella; 2, anteroloph; 3, posterior arm of the protocone (=
lingual protoloph); 4, protocone; 5, posterior outgrowth of the protocone; 6 hypoflexus; 7, mure; 8, anterior arm of the hypocone; 9, hypocone; 10, posteroloph; 11,
confluence of the distal mesoflexus with the metaflexus; 12, mesostyle; 13, third lamella; 14, third transverse crest (= mesoloph and/or mesolophule); 15, mesial
mesoflexus; 16, second lamella; 17, paracone; 18, labial protoloph; 19, paraflexus; ant., anterior; ling., lingual. (B, C) Note that the position of the fused structures is
speculative. The dental terminology is modified after several authors [50–56]. Not to scale.
https://doi.org/10.1371/journal.pone.0258455.g002
primarily listed according to their chronostratigraphic order (from the oldest to the most
recent) and then alphabetically.
Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the
nomenclatural acts it contains have been registered in ZooBank, the online registration system
for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated
information viewed through any standard web browser by appending the LSID to the prefix
“http://zoobank.org/”. The LSID for this publication is: urn:lsid:zoobank.org:pub:ECE79D679203-4929-9CCF-7C8D51C8326E. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories:
LOCKSS.
Dental associations
Working on isolated teeth requires the consideration of distinct levels of morphological variation: (i) variation depending on the dental locus; (ii) variation owing to the occlusal wear; (iii)
intra-specific or intra-taxonomic variation or polymorphism; and (iv) inter-specific or intertaxonomic variation. In this study, several taxa from TAR-31 are described. The association of
the material attributed to these taxa is based on several complementary criteria: (i) a similar
occlusal size; (ii) a similar crown height (for a given occlusal size and stage of wear); (iii) a similar proportion of the maximum mesiodistal length and the maximum linguolabial width; (iv)
a similar occlusal outline; (v) a similar occlusal pattern (bunolophodonty/lophodonty; taeniodonty/non-taeniodonty; presence/absence, development, position of cusp[-id]s/styl[e/id]s;
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presence/absence, continuity/discontinuity, length, height and obliquity of loph[-id]s; and
presence/absence and types of fusion of dental structures); and (vi) a compatible occlusal pattern between upper and lower teeth. For teeth at advanced stages of wear, we took into account
the potential impact of occlusal wear on characters aforementioned. Based on a wide array of
closely- or distantly-related rodent taxa, we prefered to use a size criterion for the recognition
of m1s vs m2s and M1s vs M2s (i.e., m1/M1s to be smaller than the m2/M2s in a given species).
The recognition of other loci is based on the occlusal shape of teeth (for more details, see
descriptions in the Systematic Palaeontology section) and on the presence and/or absence of
mesial/distal vertical contact facets due to contiguous teeth. The occlusal shape of M3s is further characterised by a smaller and more labial hypocone with respect to the protocone than
on M1–2s. However, the recognition of molar loci is quite ambiguous for several taxa, especially for the first two upper molars. In that case, we determined these loci with uncertainty
(e.g., lower molar, m1?, m2?, m3?, upper molar, M1 or M2, M1 or M2?, M3?).
Measurements and statistics
For each dental specimen of TAR-31, we measured both the maximum mesiodistal length
(len) and linguolabial width (wid), as well as the maximum lingual crown height (Hg) and
labial crown height (Hb; S1 Table). These measurements were made by M. Boivin with a measuroscope Nikon 10 and a Keyence Digital Microscope VHX-2000F. For Microsteiromys
jacobsi and Ricardomys longidens from La Venta, we used the measurements available in Walton [37] (S1 Table). However, we noted some inconsistencies regarding the m3 of the type of
Ricardomys, IGM 183847, between the measurements reported in Table 2, p. 33 [37] and the
scaled photo of the specimen in Fig 9A, p. 31 [37]. Similarly, the measurements of the type of
M. jacobsi (FMNH PM 54672 in Table 6, p. 58 [37]) do not match with its scaled representation (in Fig 16A, p. 56 [37]). For these two specimens, we chose to use the measurements made
from their figuration in the Figs 9A and 16A [37]. For the Laventan material attributed to
Microscleromys, the measurements made from the figures of the specimens in Walton (Figs
11–13 [37]) and those from Walton (Fig 24.2 [18]) are different. We extracted the measurements from the first study (Ph.D.) because the specimens were photographed (instead of
drawn) with a larger scale and more material is represented. The hypsodonty index of a tooth
(HI) [62] equals its crown height divided by its anteroposterior length (H/ML); teeth with a
HI < 1 are considered as brachydont, those with a HI = 1 are considered as mesodont, and
those with a HI > 1 are considered as hypsodont. The latter are either protohypsodont when
they still have roots, or euhypsodont if they lack roots [51]. Two hypsodonty indexes were calculated in function to the side of the crown considered: from Hb (HIb) and from Hg (HIg).
The HI values mentioned in the descriptions were measured on specimens at the early stages
of wear. The MUSM 4658 astragalus from TAR-31 was measured following the protocol of
Ginot et al. [57] (Table 1).
Table 1. Measurements (mm) of the astragalus from TAR-31, MUSM 4658.
ABW
AmTL
ATL
ATW
EL
EW
HH
HW
LBH
2.265
~1.220
3.088
2.649
1.447
1.231
0.954
1.316
1.069
LTL
MBH
MTL
NL
SL
SW
TW
mTAH
1.860
1.682
2.015
1.382
1.211
1.035
1.832
0.725
ABW, Astragalus Body Width; AmTL, Astragalar-medial Tarsal facet Length; ATL, Astragalus Total Length; ATW, Astragalus Total Width; EL, Ectal facet Length; EW,
Ectal facet Width; HH, Head Height; HW, Head Width; LBH, Lateral Body Height; LTL, Lateral Trochlear Length; MBH, Medial Body Height; MTL, Medial Trochlear
Length; NL, Neck Length; SL, Sustentacular Facet Length; SW, Sustentacular Facet Width; TW, Trochlear Width; mTAH, medial Trochlear Arc Height.
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In order to help identifying the number of species and discriminating which species of
Microscleromys are present in TAR-31 and La Venta, we performed three types of statistical
analyses on the maximum mesiodistal length and linguolabial width by locus (i.e., dp4, p4, m1–
2(?), m3(?), dP4, P4, M1–2(?), M3(?)) with R v.4.0.3. [63] (S1 and S2 Files). First, we tested statistical differences between potential taxonomic groups mentioned by Walton (M. paradoxalis
nom. nud., M. cribriphilus nom. nud., M. ?paradoxalis, and Microscleromys sp.) [37] and Microscleromys from TAR-31 using Kruskal-Wallis, Permutation Analysis of Variance, Student and
Wilcoxon tests, depending to what preconditions were satisfied, with the ‘dunn.test’, ‘RVAideMemoire’, ‘stats’ and ‘trend’ packages [63–66]. Second, partitioning analyses were conducted
until five (dp4), nine (m3) or fifteen (other loci) groups depending the number specimens for
each loci with the ‘cluster’ package [67]. Finally, we performed several Bayesian models on the
most numerous loci including several specimens previously attributed to M. cribriphilus and M.
paradoxalis (i.e., p4, m1–2(?), and M1–2(?)) in order to obtain the most probable partioning
with the ‘glm’ function of ‘MASS’ and ‘stats’ packages [63, 68]. We tested 39 models for the M1–
2(?) and 51 models for the p4 and m1–2(?) including the partionings following the previous systematic hypotheses and those obtained with the partitioning analyses (see S1 File).
Abbreviations
Institutional abbreviations. FMNH, Field Museum of Natural History, Chicago, USA;
IGM, Servicio Geológico Colombiano (before Instituto Nacional de Investigaciones en Geociencias, Mineria y Quı́mica [INGEOMINAS]), Museo Geológico José Royo y Gómez, Bogotá,
Colombia; IGM-DU, Field numbers from expeditions by the INGEOMINAS in cooperation
with Duke University. Specimens deposited at the Museo Geológico José Royo y Gómez,
Bogotá, Colombia; INBIAL-CV, Instituto de Biologı́a de la Altura, San Salvador de Jujuy,
Argentina; ING-KU, Field numbers from expeditions by the INGEOMINAS in cooperation
with Kyoto University. Specimens deposited at the Museo Geológico José Royo y Gómez,
Bogotá, Colombia; LACM, Los Angeles County Museum, Los Angeles, USA; MCZ, Museum
of Comparative Zoology, Cambridge, USA; MACN, Museo Bernardino Rivadavia, Buenos
Aires, Argentina; MLP, Museo de La Plata, La Plata, Argentina; MMP, Museo Municipal De
Ciencias Naturales Lorenzo Scaglia, Mar del Plata, Argentina; MNHN, Museum National
d’Histoire Naturelle, Paris, France; MPEF-PV, Museo Paleontológico Egidio Feruglio, Trelew,
Argentina; MUSM, Museo de Historia Natural de la Universidad Nacional Mayor San Marcos,
Lima, Peru; UCMP, University of California Museum of Paleontology, Berkeley, USA; UFAC,
Coleção de Paleovertebrados do Laboratório de Pesquisas Paleontológicas, Universidade Federal do Acre, Rio Branco-AC, Brazil; UNC, Departamento de Geociencias, Universidad Nacional de Colombia, Bogotá, Colombia.
Other abbreviations. CVP, locality belongs to the Fish Bed in La Venta area collected by
screenwashing during the expeditions by the INGEOMINAS in cooperation with Duke University; IGM-DU loc., fossiliferous locality found during the expeditions by the INGEOMINAS in cooperation with Duke University in La Venta area; UCMP loc., fossiliferous locality
found during the expeditions by the University of California in La Venta area.
Systematic paleontology
Nomenclatural Remark: The new species and genera described below must be referred to Boivin & Walton, 2021, following the article 50.1 and the “recommendation 50A concerning multiple authors” of the International Code of Zoological Nomenclature (p. 182 [38]). This is of
particular interest for nomina nuda that were previously used by Walton [18, 37] and made
available here, following the International Code of Zoological Nomenclature [38].
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Rodentia Bowdich, 1821
Ctenohystrica Huchon, Catzeflis & Douzery, 2000
Hystricognathi Tullberg, 1899
Caviomorpha Wood, 1955
Erethicavioi Boivin, 2019 in Boivin, Marivaux & Antoine, 2019
Erethizontoidea Bonaparte, 1845
Microsteiromys Boivin & Walton gen. nov. urn:lsid:zoobank.org:act:70F9B9F7-51EB-4FFF913C-2E89EBF5F9DB
Type species
Microsteiromys jacobsi gen. et sp. nov.
Species content
Only the type species.
Derivation of name
Based on the original description of the species [37]: ‘In reference to its small size and general
resemblance to the erethizontoid Steiromys Ameghino, 1887’.
Geographical and stratigraphical distribution
La Venta, Baraya and Cerro Colorado members, Villavieja Fm. (Laventan SALMA, late middle
Miocene), Huila Department, Colombia.
Generic diagnosis
As for the type and only known species.
Microsteiromys jacobsi Boivin & Walton sp. nov. urn:lsid:zoobank.org:act:1DF51272-C9494105-BC5C-299F88252206
Fig 3 and S1 Table
Nom. nud. Microsteiromys jacobsi Walton, 1990, Fig 16A–16C, p. 56.
Nom. nud. Microsteiromys jacobsi Walton, 1997, Figs 24.1B and 24.2B, p. 394, 395.
Holotype
FMNH PM 54672, right mandibular fragment bearing dp4–m3 (Walton, 1990: Fig 16A; Walton, 1997: Figs 24.1B and 24.2B; Fig 3A).
Referred material
IGM-DU 89–249, left mandibular fragment bearing p4–m1 (Walton, 1990: Fig 16C; Fig 3B);
IGM-DU 88–034, right posterior fragment of lower tooth (Walton, 1990: Fig 16B; Fig 3C).
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Fig 3. Microsteiromys jacobsi gen. et sp. nov. from La Venta, Colombia (late middle Miocene, Laventan SALMA). (A) FMNH PM 54672��� ,
right mandibular fragment bearing dp4–m3. (B) IGM-DU 89–249, left mandibular fragment bearing p4–m1 (reversed), (C) IGM-DU 88–034,
right posterior fragment of m1 or m2. Occlusal views. The asterisks appoint the holotype material. Based on Walton (Fig 16A-16C [37]). The
illustrations of the fossil specimens are computerised schematic line drawings.
https://doi.org/10.1371/journal.pone.0258455.g003
Derivation of name
Based on the original description of the species [37]: ‘In honour to Dr. Louis L. Jacobs,
palaeontologist and educator’.
Type locality
IGM-DU loc. 022 or UCMP loc. V4536 (only for the type), Monkey Beds, La Venta Badlands,
upper Magdalena Valley, Huila Department, Colombia.
Other localities
CVP 10 (for IGM-DU 88–034), Fish Bed and screewashed locality 032–5 (for IGM-DU 89–
249), El Cardón Red Bed, La Venta Badlands, upper Magdalena Valley, Huila Department,
Colombia.
Formation and age
Baraya and Cerro Colorado members, Villavieja Fm., late middle Miocene (i.e., Laventan
SALMA).
Diagnosis
Rodent characterised by a small size and brachydont teeth. Lower teeth with a tetralophodont
and non-taeniodont pattern, complete metalophulid I, and oblique and complete ectolophid.
dp4s with a mesiodistally short and linguolabially wide trigonid and a curved metalophulid I.
Large p4s with a linguolabially wide trigonid. Mesiodistally elongated lower molars with a narrow mesoflexid and well-extended metaflexid. Differs from Eopululo, Protosteiromys,
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Branisamyopsis, Hypsosteiromys, Parasteiromys, Eosteiromys, Steiromys, and Neosteiromys in
having a smaller size; from Eosteiromys annectens and Steiromys duplicatus in having non-taeniodont lower teeth; from Hypsosteiromys in having brachydont lower teeth with transverse
lophids and a complete metalophulid I, and less elongated dp4s; from Shapajamys, Branisamyopsis, Eosteiromys homogidens, Steiromys, and Coendou insidiosus in having tetralophodont
dp4s; from Eosteiromys segregatus in having a longer p4 with a long anterior arm of the entonconid; from Shapajamys, Palaeosteiromys, Protosteiromys, Branisamyopsis, Eosteiromys annectens, Steiromys, and Neosteiromys in having more rectangular lower molars; from
Palaeosteiromys, Branisamyopsis australis, Branisamyopsis praesigmoides, Steiromys duplicatus,
and Neosteiromys in having always tetralopodont lower molars; from Shapajamys, Branisamyopsis praesigmoides and Steiromys duplicatus in having an ectolophid always complete and
oblique on lower molars; from Shapajamys in having an ectolophid always oblique on lower
molars; from Palaeosteiromys in having lower molars with a metaflexid more extended; from
Protosteiromys, Branisamyopsis, Eosteiromys homogidens, Steiromys detentus, and Neosteiromys
in having a narrower mesoflexid on lower molars.
Description
FMNH PM 54672 (Fig 3A) and IGM-DU 89–249 (Fig 3B) are two mandibular fragments bearing dp4–m3 and p4–m1, respectively. The body of the FMNH PM 54672 mandible is anteriorly broken at the level of the anterior part of the lower diastema, which makes visible the
lower incisor alveola. Posteriorly, the angular apophysis and most of the ascending ramus,
including the mandibular condyle, are missing. The coronoid process is broken at its base.
Labially, a mental foramen is located at the midpoint of the body of the mandible height (i.e.,
dorsoventrally). The notch for the insertion of the tendon of the zygomatico-mandibularis pars
infraorbitalis is medium-sized, below dp4–m1, and centrally situated on the labial edge of the
mandible. The anterior tip of the masseteric crest ends below the m1, and links the notch for
the insertion of the tendon of the zygomatico-mandibularis pars infraorbitalis, at the level of its
posteroventral region. The masseteric crest is posteriorly broken. It is posteroventrally directed
and prominent in its anterior part.
The dp4 of the FMNH PM 54672 mandibular fragment (Fig 3A) is mesially and distolingually broken at the level of the metaconid, metalophulid I, mesostylid, entoconid, and posterolophid. This tooth is more worn and shorter than the molars (m1–3) beared by FMNH
PM 54672. It is characterised by a short trigonid, which has an equal width with the talonid.
This dp4 is brachydont and non-taeniodont. Despite its damaged state, no supplementary
lophids are visible in addition to the metalophulid I, second transverse cristid, hypolophid
and posterolophid, which suggests that the FMNH PM 54672 dp4 was tetralophodont. As
this tooth is at an advanced stage of wear, the cuspids are not well defined to the lophids.
On the lingual margin of the tooth, the anteroflexid and mesoflexid are closed, thereby suggesting that the mesostylid (or its arms) was connected to the metaconid (or its posterior
arm) and its posterior arm reaches the entoconid (or its anterior arm). The metalophulid I,
mesially convex, seems to be complete and strongly connected to the protoconid labially
(hypothesis 1 on Fig 4A1; see the remark below). This cuspid is crestiform and faintly linked
to the second cristid. The latter is slightly oblique (i.e., mesiodistally directed). It bears posteriorly a tiny and horrizontal cristulid, which joins the mesial margin of the hypolophid,
dividing the mesoflexid into two small and round fossettids. The labiodistal aspect of the
second cristid links to a transverse hypolophid via a very short and oblique ectolophid. The
hypoconid is mesiolingually-distolabially compressed. It displays strong anterior arm, anterior outgrowth and posterolophid. The anterior outgrowth is mesiolabially oriented and
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Fig 4. Hypotheses regarding the homology of structures on dp4 of the erethizontoid Microsteiromys jacobsi (A) and Nuyuyomys chinqaska (B). (A1)
Hypothesis 1: protoconid in anterior position, aligned with the metalophulid I (followed here). (A2) Hypothesis 2: protoconid in posterior position, aligned
with the second transverse cristid. (B1–9) Equivalent transversal cristids (and their associated cuspids/stylids) indicated by numbers (1–6) on all dp4s of N.
chinqaska. (B10) Different hypotheses regarding the position of the anterior cuspids/stylids based on the morphology of MUSM 4333. For the transversal
cristids 1 and 2, the position and number of the structures can slightly vary depending the considering specimen (e.g., MUSM 4332). In the case of the
hypotheses 1–4, the neoconid(s) can be present on the cristid 1 or not (correspond solely to thickenings of this cristid). The hypotheses 1 and 3 are the most
consistent with the morphology and development of the dental structures. (A) IGM-DU 89–249, (B1) MUSM 4326, (B2) MUSM 4327, (B3) MUSM 4328,
(B4) MUSM 4329, (B5) MUSM 4325, (B6) MUSM 4332, (B7) MUSM 4331, (B8) MUSM 4330, and (B9) MUSM 4333. Ed, entoconid; Hd, hypoconid; Md,
metaconid; Mld, mesostylid; Nd, neoconid; Pd, protoconid. The illustrations of the specimens are computerised schematic line drawings.
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delimits with the anterior arm of the hypoconid, hypolophid, ectolophid, second transverse
cristid and protoconid, a triangular-shaped hypoflexid, oriented distolingually, and showing a wide labial opening. Although the lingual part of the posterolophid is missing, the
metaflexid appears lingually closed as is the case as well for the anteroflexid and the
mesoflexid.
The p4 of the IGM-DU 89–249 mandibular fragment (Fig 3B) is much longer and moderately worn with respect to the associated m1, which shows a very advanced state of wear
characterised by a fusion all lophids and cuspids/stylids. As the FMNH PM 54672 dp4, it is
brachydont, tetralophodont and non-taeniodont. However, it is much longer than the dp4
and has a mesial margin more straight. The protoconid corresponds to a large dentine plateform on the mesiolabial corner of the tooth. It is mesially connected to a metalophulid I,
reaching the metaconid on the lingual margin, and distally to the second cristid and to an
oblique and long ectolophid. The labial part of the metalophulid I is mesiolingually directed,
while its lingual part is slightly directed distolingually. The metaconid is faintly linked to the
mesostylid, which appears to have an equal size to the former. Due to the moderate state of
wear of the tooth, the presence of a posterior arm of the metaconid cannot be assessed, but
if it would be present, the latter would be short. The mesostylid bears short anterior and
posterior arms. Comparatively, the entoconid has longer arms, especially its anterior arm,
reaching the posterior arm of the mesostylid. The second cristid is complete and slightly
mesiolingually directed, whereas the hypolophid and the posterolophid are transverse.
There is a connection between the latter and the posterior arm of the entoconid, which lingually closes the metaflexid. Thus, all the lingual flexids are closed and form round-oval fossettids. The anterior arm of the hypoconid is strong and mesiolingually oriented, whereas
its anterior outgrowth is long and mesiolabially oriented. These cristids, with the ectolophid
and protoconid, delimit a widely open and retroverse hypoflexid (triangular-shaped and
distolingually directed).
The m1–3 of the FMNH PM 54672 specimen (Fig 3A) have a similar/compatible size
with the unique and very worn molar (m1) borne by the IGM-DU 89–249 mandibular
fragment (Fig 3B). On FMNH PM 54672, all molars are roughly equal in length. The m1
and m2 have a similar width, whereas m3 is narrower. The latter is characterised by a
shorter posterolophid due to a linguolabial compression of its posterior part. The morphology of lower molars has the same pattern than that of dp4 and p4. As dp4 and p4,
molars are brachydont, tetralophodont, non-taeniodont with a strong anterior arm of the
hypoconid, and they have an oblique ectolophid and a triangular-shaped hypoflexid showing a wide labial opening. This flexid has a general distolingual direction, but the latter
varies depending on the locus: it is slight on m3, more oblique on dp4 and m1, and the
most oblique on p4 and m2. As dp4 and p4, m1–2 display all their lingual flexids closed,
whereas m3 has a mesoflexid opened by a shallow and thin furrow, and a posterolophid
well-separated from the entoconid. On m1–2, this cuspid appears to have a long posterior
arm On m1–3, the metalophulid I is complete, transverse to slightly arched. The metaconid bears a long posterior arm strongly connected to the mesostylid (or its anterior arm).
The second cristid and hypolophid are close and separated by a narrow mesoflexid (i.e.,
furrow-like flexid). These two transverse cristids are straight and roughly parallel on m1
and m3. On m2, most part of the second cristid (i.e., lingual part) is slightly distolingually
directed. Because of a thinning on its central part on m1 and its orientation change on m2,
the second cristid may be composed of two cristids on these lower molars: a neomesolophid lingually and a posterior arm of the protoconid labially. On all molars, the posterolophid is curved, especially on m3. The metaflexid/metafossettid is mesiodistally well
extended.
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Remark
Considering the pattern of dp4s of some erethizontoids (Steiromys detentus, MLP 15−339;
Erethizon dorsatum, MCZ 51367) [69], another hypothesis can be proposed with respect to the
position of the protoconid on the FMNH PM 54672 dp4 (hypothesis 2 on Fig 4A2): this cuspid
could be situated more posterolingually than in hypothesis 1 and aligned with the second
transverse cristid. The discovery of a pristine dp4 of this species would make it possible to
decide between these two proposed hypotheses.
Comparisons
Brachydonty, non-taeniodonty, extended and moderately narrow flexids, weak obliquity of
lophids, and p4 with a wide trigonid suggest erethizontoid affinities for these specimens from
La Venta. They are markedly smaller than most of erethizontoid species, except for Kichkasteiromys raimondii, Shapajamys labocensis [70], Palaeosteiromys amazonensis [71], Noamys
hypsodonta [72] and the material from the early middle Miocene of Madre de Dios, Peru formerly attributed to cf. Microsteiromys sp. (MD-67; [31] and see below). Kichkasteiromys raimondii, N. hypsodonta and the taxon from MD-67 being documented only by upper teeth,
direct morphological comparisons with the material of La Venta cannot be made. The lower
molars from La Venta differ from those of Shapajamys and Palaeosteiromys in being more
rectangular. They are characterised by an ectolophid always complete and oblique, contrary to
Shapajamys, and by a metaflexid more extended than in Palaeosteiromys. These specimens
from La Venta are always tetralophodont, contrary to those of Palaeosteiromys, Branisamyopsis
australis, Branisamyopsis praesigmoides, Steiromys duplicatus and Neosteiromys, which can
exhibit a pentalophodont pattern by the addition of a mesial neolophid [71, 73–77]. By its
rounded shape as well as its short and wide trigonid, the FMNH PM 54672 dp4 is close to the
dp4s of the extant species Coendou insidiosus. The FMNH PM 54672 dp4 is tetralophodont, as
dp4s of Hypsoteiromys [74, 78], but the latter are clearly more elongated, with a longer trigonid
and more extended lingual flexids. Contrary to FMNH PM 54672, dp4s of other erethizontoids
are elongated with a pentalophodont (Eosteiromys homogidens, Eosteiromys annectens and C.
insidiosus) or hexalophodont (Shapajamys, Branisamyopsis, ?Eosteiromys sp. nov. sensu Candela [69], and Steiromys) pattern. MNHN 1903-3-18 (Fig 32 [79]) was originally determined as
a m3 of Protosteiromys asmodeophilus. However, its trigonid is clearly narrower than its talonid, a feature better characterising a premolar (p4 or dp4). MNHN 1903-3-18 is tetralophodont and non-taeniodont as p4s and dp4s from La Venta, but has lingual flexids much more
extended. The material from La Venta differs from Eopululo, Protosteiromys, Branisamyopsis,
Hypsosteiromys, Parasteiromys, Eosteiromys, Steiromys and Neosteiromys [73–76, 78–82] in
having a smaller size; from Hypsosteiromys in having brachydont lower teeth with transverse
lophids and a complete metalophulid I; from E. annectens and S. duplicatus in having non-taeniodont lower teeth; from Branisamyopsis, S. duplicatus, Steiromys verzii sensu Candela [74],
and some p4s of S. detentus in having a p4s with tetralophodont pattern; and from Eosteiromys
segregatus in having a longer p4 with a long anterior arm of the entonconid; from Protosteiromys, Branisamyopsis, E. annectens, Steiromys and Neosteiromys in having more rectangular
lower molars; from Protosteiromys, Branisamyopsis, E. homogidens, Steiromys detentus and
Neosteiromys in having a narrower mesoflexid on lower molars; from B. praesigmoides and S.
duplicatus in having an ectolophid always complete on lower molars. To sum up, these few
specimens from La Venta show a set of distinctions (i.e., small size; brachydonty; tetralophodont and non-taeniodont lower teeth; a complete metalophulid I, and an oblique and complete
ectolophid on lower teeth; round dp4s with a short and wide trigonid; mesiodistally elongated
lower molars with narrow mesoflexid and well-extended metaflexid) that allows us to refer
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them to a new genus and new species. Thus, we validate Microsteiromys jacobsi Walton, 1990,
which had remained until now as a nomen nudum.
Nuyuyomys Boivin & Walton gen. nov. urn:lsid:zoobank.org:act:360EBF95-C067-4C81-81F48FAD41067E7E
Type species
Nuyuyomys chinqaska gen. et sp. nov.
Species content
Only the type species.
Derivation of name
From Nuyuy, flooding in Quechua language, referring to the location of both fossil localities
yielding this genus (at the water level only during the driest season and below it most of the
year) and -mys, mouse in Greek, a classical suffix for rodents.
Geographical and stratigraphical distribution
Pilcopata (MD-67), unnamed Fm. (Colloncuran-Laventan, middle Miocene), Madre de Dios
Department, Peru; Tarapoto/Juan Guerra (TAR-31), lower member, Ipururo Fm. (Laventan,
late middle Miocene), San Martı́n Department, Peru.
Generic diagnosis
As for the type and only known species.
Nuyuyomys chinqaska Boivin & Walton sp. nov. urn:lsid:zoobank.org:act:353264FD-EB4C428F-9F6D-EFC6DC1D7A04
Fig 5 and S1 Table
2013 cf. Microsteiromys sp. Antoine et al., Fig 3M, 3N, p. 94.
Holotype
MUSM 4308 (Fig 5R), right M1?.
Referred material
MUSM 4325–4329 (Fig 5F), left dp4s; MUSM 4330–4333 (Fig 5A), right dp4s; MUSM 4334
(Fig 5B), left p4; MUSM 4335–4339, left lower molars; MUSM 4340–4342, right lower molars;
MUSM 4343, 4344, left m1s; MUSM 4345–4348 (Fig 5C and 5G), right m1s; MUSM 4349–
4357 (Fig 5H, 5J and 5K), left m2s; MUSM 4358–4363 (Fig 5D and 5L–5N), right m2s; MUSM
4364 (Fig 5I), left m3; MUSM 4365, 4366 (Fig 5E and 5O), right m3s; MUSM 4284–4288 (Fig
5Z), left dP4s; MUSM 4289–4294 (Fig 5P, 5U, 5C’ and 5D’), right dP4s; MUSM 4295 (Fig 5Q),
left P4; MUSM 4296, 4297, right upper molars; MUSM 4299, right M1 or M2?; MUSM 4300–
4306, left M1?s; MUSM 4307–4314 (Fig 5A’ and 5B’), right M1?s; MUSM 4315, right M2 (or
M3)?; MUSM 4316–4320 (Fig 5S and 5W), left M2?s; MUSM 4321, 4322, right M2?s; MUSM
4323 (Fig 5X), left M3; MUSM 4324 (Fig 5T), right M3.
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Fig 5. Erethizontoid rodents from TAR-31, Peru (late middle Miocene, Laventan SALMA). (A-E’) Nuyuyomys chinqaska gen. et sp. nov. (F’)
Erethizontoidea gen. et sp. indet. 1. (G’) Erethizontoidea gen. et sp. indet. 2. (A) MUSM 4332, right dp4 (reversed). (B) MUSM 4334, left p4. (C)
MUSM 4347, right m1 (reversed). (D) MUSM 4359, right m2 (reversed). (E) MUSM 4366, right m3 (reversed). (F) MUSM 4329, left dp4. (G)
MUSM 4348, right m1 (reversed). (H) MUSM 4355, left m2. (I) MUSM 4364, left m3. (J) MUSM 4356, left m2. (K) MUSM 4352, left m2. (L)
MUSM 4362, right m2 (reversed). (M) MUSM 4363, right m2 (reversed). (N) MUSM 4360, right m2 (reversed). (O) MUSM 4365, right m3
(reversed). (P) MUSM 4290, right dP4 (reversed). (Q) MUSM 4295, left P4. (R) MUSM 4308��� , right M1? (reversed). (S) MUSM 4319, left M2?.
(T) MUSM 4324, right M3 (reversed). (U) MUSM 4292, right dP4 (reversed). (V) MUSM 4306, left M1?. (W) MUSM 4320, left M2?. (X) MUSM
4323, left M3. (Y) MUSM 4303, left M1?. (Z) MUSM 4285, left dP4. (A’) MUSM 4309, right M1? (reversed). (B’) MUSM 4314, right M1?
(reversed). (C’) MUSM 4293, right dP4 (reversed). (D’) MUSM 4294, right dP4 (reversed). (E’) MUSM 4298, M1 or 2?. (F’) MUSM 4367, left p4.
(G’) MUSM 4368, right M1 or M2 (reversed). The asterisks appoint the holotype material. The illustrations of the fossil specimens are scanning
electron photomicrographs.
https://doi.org/10.1371/journal.pone.0258455.g005
Tentitavely referred material
MUSM 1974 (Antoine et al., 2013: Fig 3M, p. 94), left P4; MUSM 1975 (Antoine et al., 2013:
Fig 3N, p. 94), right upper molar; MUSM 4298 (Fig 5E’), left M1 or M2?.
Derivation of name
From chinqaska, vanished in Quechua language, referring to the disappearance of both MD67 and TAR-31 outcrops since their discovery, due to water erosion and paleontological
exploitation, respectively.
Type locality
TAR-31, Tarapoto/Juan Guerra, Mayo River, Shapaja road, San Martı́n Department, Western
Amazonia, Peru.
Other locality
MD-67, Pilcopata, Rinconadera and Madre de Dios rivers, Madre de Dios Department, Western Amazonia, Peru.
Formation and age
Unnamed Fm., middle Miocene (i.e., Colloncuran-Laventan SALMA); lower member, Ipururo Fm., late middle Miocene (i.e., Laventan SALMA).
Diagnosis
Rodent characterised by small size, brachydont lower teeth and dP4s, brachydont-mesodont
P4s and upper molars, and a weak obliquity of loph(-id)s. dp4s, p4s and upper teeth with a
non-taeniodont pattern. Lower molars with a non-taeniodont or pseudo-taeniodont pattern at
early stages of wear, the former pattern being the most frequent. Elongated dp4s with a hexalophodont pattern. p4s are longer than wide, with a large trigonid and tetralophodont pattern.
Lower molars with a tetralophodont or pentalophodont pattern, the latter corresponding to
the addition of a neolophid between the metalophulid I and the second transverse cristid. dP4s
with a pentalophodont or tetralophodont pattern, the former pattern being the most frequent.
The metaloph (when present) can be lingually free or connected to the posteroloph or to both
the third crest and posteroloph on dP4s. Round and tetralophodont P4s with an anterior arm
of the protocone thicker and mesiolabially directed connected to an anteroloph lower, thinner
and roughly transverse, and without endoloph. Pentalophodont upper molars with a metaloph
always long and joining the metacone to the posteroloph on M2?s and complete or reduced on
M1?s and M3?s. Nuyuyomys is markedly smaller than all erethizontoid species except
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Kichkasteiromys, Shapajamys, Palaeosteiromys, Microsteiromys and Noamys. Differs from Eosteiromys homogidens, Eosteiromys annectens, Hypsosteiromys, Microsteiromys and Coendou
insidiosus in having hexalophodont dp4s; from Shapajamys, Protosteiromys, Eosteiromys and
Microsteiromys in having no neolophid on the anteroflexid on lower molars; from Shapajamys,
Branisamyopsis praesigmoides and Steiromys duplicatus in having an ectolophid always complete. Differs from Kichkasteiromys, Shapajamys and Palaeosteiromys in having a metaloph
disconnected labially to the metacone on some upper molars; from Shapajamys in having no
distinct metaloph on P4s, metaloph more developed on ?M2s and an ectolophid always oblique on lower molars; from Kichkasteiromys in having a lingual protoloph less lingually situated
and a posterior outgrowth of the protocone more oblique on upper molars; from Palaeosteiromys in having a mesoflexid more extended on m3s; from Microsteiromys in having more
elongated dp4s with a narrower trigonid, the mesoflexid still lingually open at advanced stage
of wear on m1–2s and less rectangular lower molars. Differs from Hypsosteiromys in having
loph(-id)s more transverse, round P4s, pentalophodont M1–2s, and a complete metalophulid I
on lower teeth; from Noamys in having pentalophodont and sligthly smaller dP4 and M1 without oval-shaped occlusal contour and flexi less extended. Differs from Eosteiromys annectens
and Steiromys duplicatus in having non-taeniodont lower teeth; from Neosteiromys and Parasteiromys in having always a complete mure on dP4s and upper molars; from Eopululo, Protosteiromys pattersoni and Neosteiromys in having a P4 without endoloph; from Eosteiromys
homogidens in having a mesial mesoflexus less mesiodistally extended on P4s; from Eosteiromys annectens, Branisamyopsis and Neosteiromys in having non-taeniodont dP4s and upper
molars; from Eopululo and Protosteiromys pattersoni in having upper molars always with a
metaloph (which is not reduced, far from being a relictual structure); from Protosteiromys,
Parasteiromys, Branisamyopsis, Eosteiromys, Steiromys and Neosteiromys in having some elongated upper molars (potential M1s) and an incomplete metaloph on some upper molars; from
Protosteiromys medianus in having a complete third crest on M3s.
Description
dp4s are brachydont and much longer than wide, with a talonid slightly wider than the trigonid. They are non-taeniodont and hexalophodont. In addition to six complete transverse
lophids, one dp4 (MUSM 4332; Fig 5A) has a tiny low cristulid stemming from the metalophulid I on the anteroflexid. Owing to its low elevation and shortness, it is interpreted here as a
neolophid. The entoconid and hypoconid and their associated lophids (i.e., hypolophid [cristid
5 in Fig 4B1–4B9], anterior outgrowth of the hypoconid, anterior arm of the hypoconid, and
posterior arm of hypoconid/posterolophid [cristid 6 in Fig 4B1–4B9]), distally positioned on
the tooth, are well recognisable. The entoconid is the largest lingual cuspid. It is clearly isolated
on almost all dp4s. On MUSM 4331, its isolation is less important because it is separated from
the posterolophid by a thinner and shallower notch. The hypolophid is transverse or slightly
arcuate (i.e., mesially concave). The crestiform hypoconid displays an anterior arm, which is
mesiolingually oriented, thin and linked to the labiodistal extremity of the hypolophid. On
MUSM 4332 (Fig 5A), the mesialmost part of the anterior arm of the hypoconid is lower than
its distal part, but it is connected to the hypolophid and clearly separates the hypoflexid from
the metaflexid (i.e., non-taeniodont pattern). The hypoconid exhibits a strong and short anterior outgrowth. The posterolophid is particularly long and curved, notably in its lingual part.
On the less worn dp4s, it is composed of two (MUSM 4329; Fig 5F) or three (MUSM 4327 and
4332; Fig 5A) interconnected parts, a posterior arm of the hypoconid and posterolophid
included. Mesially, the recognition of the main cuspids (i.e., protoconid, metaconid, and
mesostylid) and cristids (i.e., posterior arm of the protoconid, metaconid cristid, mesolophid,
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and neomesolophid) is ambiguous by the addition of some neostructures (i.e., neoconids and
neolophids). Several hypotheses of homology can be proposed (Fig 4B10). The mesialmost
cristid (cristid 1 in Fig 4B1–4B9) is curved. It (mainly) corresponds to the metalophulid I. It
shows one or several cuspate thickenings in its central part and/or extremities (MUSM 4325,
4326, 4329, and 4330, 4332, 4333; Figs 4B1, 4B4–4B6, 4B8, 4B9 and 5A and 5F). They can correspond to the main cuspids (i.e., protoconid, metaconid, mesostylid) and/or neoconids (Fig
4B10). The second cristid (cristid 2 in Fig 4B1–4B9) is highly variable. It can be constituted of
one or two linked cristulids, which can be associated to the thickenings of the metalophulid I.
They can be connected to the central part of the cristid 1 (MUSM 4329 and 4333; Fig 4B4 and
4B9) and/or to one (MUSM 4325; Fig 4B5) or two of its extremities (MUSM 4326, 4330, and
4332; Fig 4B1, 4B6 and 4B8). The next transverse cristid (cristid 3 in Fig 4B1–4B9) is thick and
long, stemming from a cuspate structure on the lingual margin of the tooth to connect to a
large another cuspid-shaped structure on the labial margin. These cuspid-like structures are
clearly isolated at early stages of wear (MUSM 4326, 4330, and 4332; Figs 4B1, 4B6, 4B8 and
5A) and can link the structures located mesially (metalophulid I and annexes) at more
advanced staged of wear (MUSM 4329 and 4333; Figs 4B4, 4B9 and 5F). On MUSM 4326 and
4329, this transverse cristid has two parts: a lingual cristid connected to a labial one, equally
sized (MUSM 4326; Fig 4B1) or shorter (MUSM 4329; Figs 4B4 and 5F). Owing to the important development of this cristid and of its cuspate structures on all dp4s, they are probably not
neostructures (hypotheses 1 and 3 in Fig 4B10). The transverse cristid appearing in fourth
position (cristid 4 in Fig 4B1–4B9) is labially connected to the hypolophid via the distal ectolophid on all dp4s. This cristid can be continuous (MUSM 4327, 4328, and 4330–4333; Fig 4B2,
4B3, 4B6–4B9) or divided into two parts, which are joined (4326 and 4329; Fig 4B1 and 4B4).
In addition, it can be high (MUSM 4326 and 4330–4333; Fig 4B1, 4B6–4B9), entirely low
(MUSM 4327; Fig 4B2) or only its labial part can be low (MUSM 4329; Figs 4B4 and 5F). On
MUSM 4327 (Fig 4B2), it is slightly connected to a bulbous cuspid clearly distinct on the lingual margin of the tooth.
MUSM 4334 is an almost pristine p4 (Fig 5B). It is longer than wide, with a trigonid narrower than the talonid. It is brachydont and tetralophodont. On its mesiolingual corner, the
crestiform metaconid is convex forward. Distally, this cuspid displays a very short posterior
arm. There is a tiny spur-like cristulid on the anteroflexid, labially free and stemming from the
distal extremity of the posterior arm of the metaconid. Mesially, the metaconid has a transverse
and long anterior arm. From its central part, a short cristulid runs distally on the anteroflexid.
The anterior arm of the metaconid is linked to a faintly curved anterior arm of the protoconid.
The latter cristid is clearly sloped with a high distal part stemming from the triangular-shaped
protoconid. The second transverse cristid is essentially composed of a long neomesolophid
joining a very short posterior arm of the protoconid. The ectolophid is mainly oblique and Sshaped. It reaches the distal slope of the protoconid. The entoconid is high and it has a sharp
posterior arm. Labially, this cuspid is connected to a hypolophid, strong and distolingually
oblique. On the labiodistal margin of the ectolophid-hypolophid junction, a tiny cristulid is
present. Its top is separate from the anterior arm of the hypoconid by a very shallow notch.
However, the contact between these structures remains high despite the presence of this notch
(i.e., non-taeniodont pattern). The anterior outgrowth of the hypoconid is short and directed
mesiolabially. The posterolophid is transverse with curved lingual and labial extremities. On
the lingual margin, all structures (i.e., metaconid, mesostylid, entoconid and lingual extremity
of the posterolophid) are merged together at their base, but they remain disconnected at their
apex. The mesostylid is discreet and isolated from the posterior arm of the metaconid and the
entoconid by wide and deep furrows. The notch separating the entoconid from the lingual
extremity of the posterolophid is thinner and shallower. The anteroflexid and metaflexid are
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more expanded than the mesoflexid. The hypoflexid is triangular-shaped with a wider labial
openning.
The lower molars are brachydont and rectangular-shaped. m3s (MUSM 4364–4366; Fig 5E,
5I and 5O) show a talonid slightly narrower than the trigonid, notably lingually. m1s and m2s
usually have a trigonid and a talonid roughly of same width and m2s are larger than m1s and
m3s. On all lower molars, the metaconid, protoconid and hypoconid are crestiform, whereas
the mesostylid and entoconid are more bulbous. The metalophulid I is transverse and complete, joining the metaconid to the protoconid. On the lower molars at the earliest stage of
wear (MUSM 4342, 4347, and 4359; Fig 5C and 5D), this cristid corresponds to a long anterior
arm of the metaconid, whose lingual extremity is low and linked a short anterior arm of the
protoconid. On most lower molars, the mesostylid is linked to the posterior arm of the metaconid, this connection can be slight or tight. These two structures can be separated by a wide furrow (e.g., MUSM 4336, 4357, and 4365; Fig 5O) or a thin notch (e.g., MUSM 4356, 4347, and
4348; Fig 5C, 5G and 5J). Several lower molars exhibit two short cristulids on their mesiolingual area: the first cristulid is situated on the distolingual part of the metalophulid I, and the
second is located distally to the metaconid, on its posterior arm (MUSM 4344, 4352, 4355,
4364, and 4362; Fig 5H, 5I, 5K and 5L). On MUSM 4344, 4364 and 4362 (Fig 5I and 5L), these
two cristulids are joined, and form a mesial neolophid on the anteroflexid (i.e., pentalophodont pattern). On MUSM 4352 and 4355, they remain separate (Fig 5H and 5K). On MUSM
4351, there is only the cristulid of the metalophulid I, whereas on MUSM 4348 (Fig 5G), there
is only the cristulid distally to the metaconid. The second transverse cristid is a long neomesolophid directly connected to the protoconid or via its very short posterior arm. It can be
slightly oblique, transverse, or sometimes sinuous (S-shaped). The ectolophid is always oblique, either aligned with the protoconid or a little more longitudinal. The entoconid can display
strong anterior and posterior arms. On MUSM 4336 and 4351, the posterior arm is particularly long. The latter can be connected (e.g., MUSM 4336, 4352, 4364, and 4359; Fig 5D, 5I and
5K) or disconnected (e.g., MUSM 4351, 4354, 4356, 4357, 4347, and 4365; Fig 5C, 5J and 5O)
to the posterolophid. The hypolophid is well marked and often parallel to the second transverse cristid. The anterior arm of the hypoconid is complete and high in most cases (i.e., nontaeniodont pattern). Two lower molars, MUSM 4356 and 4347 (Fig 5C and 5J), show a
pseudo-taeniodont pattern. On MUSM 4356 (Fig 5J), the anterior arm of the hypoconid is
complete, but moderately low. On MUSM 4347 (Fig 5C), there is a very low cristulid stemming
from the hypolophid-ectolophid junction and joining the mesial slope of the hypoconid,
thereby involving a partial confluence between the metaflexid and the hypoflexid.
dP4s are longer than wide with a rectangular or slightly trapezoidal occlusal outline. On all
dP4s (except on MUSM 4293; Fig 5C’; see below), the protocone is somewhat bulbous, a morphology accentuated by a thinning at the level of its connection with the anteroloph. In addition, the lingual extremity of the latter is sometimes thickened (MUSM 4284, 4287, and 4289–
4292; Fig 5P and 5U). The anteroloph is mesially convex, and can have an enamel thickening
on its labial part, that we interpret as a parastyle, associated with a spur-like crestule (MUSM
4285, 4287, 4289, 4290, and 4292; Fig 5P, 5U and 5Z). This crestule remains lingually free on
the paraflexus. On the labial margin of dP4s, the paracone is the largest cusp. It is clearly separated mesially from the labial extremity of the anteroloph and distally from the mesostyle by
wide and deep furrows in most cases. On MUSM 4290 (Fig 5P), the paracone is separate from
the anteroloph by a shallower notch. The labial protoloph can be transverse (MUSM 4291),
oblique (i.e., mesiolabially directed; MUSM 4293; Fig 5C’) or with an oblique lingual part and
a labial part more transverse (on the other dP4s). On MUSM 4287, in its middle, the labial protoloph displays a tiny crestule on its mesial part. Its ends close to the distal extremity of the
crestule stemming from the parastyle, but does not connect to it. The mure is oblique (e.g.,
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MUSM 4290 and 4293; Fig 5P and 5C’) or more longitudinal (i.e., parallel to the mesiodistal
long axis of the tooth; e.g., MUSM 4291 and 4292; Fig 5U). Its junction with the labial protoloph is linked to the protocone via its thin anterior arm (i.e., non-taeniodont pattern). Labially,
the mesostyle is large, whereas the metacone is faintly visible from the posteroloph. On
MUSM 4290–4292 and 4294 (Fig 5P, 5U and 5D’), the mesostyle shows a long posterior arm.
The mesostyle or its posterior arm is either linked to the metacone-posteroloph complex
(MUSM 4287, 4290, 4291, and 4293; Fig 5P and 5C’) or is separated from this cusp by a thin
and shallow notch (MUSM 4285, 4286, 4292, and 4294; Fig 5U, 5Z and 5D’). The third crest is
complete and S-shaped. On MUSM 4285 and 4292 (Fig 5U and 5Z), this crest consists of a
long mesoloph stemming from the mesostyle and joining lingually the mure-anterior arm of
the hypocone junction directly (MUSM 4292; Fig 5U) or via a short mesolophule (MUSM
4285; Fig 5Z). The posteroloph is mesially concave and links the metacone to the hypocone.
The metaloph shows a variable length, direction and lingual connection. On MUSM 4285,
4290 and 4292 (Fig 5P, 5U and 5Z), the metaloph is long, turned backwardly (either curved or
L-shaped), and linked to the posteroloph via (or not) a posteroloph spur. On MUSM 4286, the
metaloph is short, mesiolingually directed and lingually free. On that tooth, there is also a crestule, close to the metaloph and connected lingually to the posteroloph spur and labially to the
posteroloph. Due to its position and configuration, it is probably a rest of metaloph. On some
worn dP4s (MUSM 4287, 4291, and 4293; Fig 5C’), the metacone and metaloph are likely subsumed within the posteroloph (+ its spur), thereby forming a thick platform distally. Neocrestules connect the third transverse crest to the metacone or to the distal platform on MUSM
4289–4291 (Fig 5P). On MUSM 4294 (Fig 5D’), there is no metaloph or distinct metacone
(which is probably fused with the posteroloph). On that tooth, the thin posteroloph bears a
long spur, which runs mesiolabially on the posterior flexus and remains free. The hypoflexus
forms a U-shaped structure, mesiolabially directed.
P4s are round, MUSM 4295 shows an oval outline (Fig 5Q), whereas MUSM 1974 is longer
and more circular-shaped (Fig 3M [31]). MUSM 4295 is higher-crowned with respect of the
other teeth attributed to Nuyuyomys chinqaska (Hling = 0.89; Hlab = 0.62; Fig 5Q). On MUSM
4295, the area of the protocone and that of the hypocone are large and conical, corresponding
to oval regions of dentine delimited by a very thick exterior enamel layer (Fig 5Q). On the two
P4s, the mesialmost crest is divided into two parts, which are connected together. Its lingual
part, the anterior arm of the protocone, is thicker, mesiolabially directed, longer on MUSM
4295 (Fig 5Q), but shorter on MUSM 1974 (Fig 3M [31]). This crest is in the continuity with
the protocone and its short posterior outgrowth. The labial part of the mesialmost crest, the
anteroloph, is thinner, roughly transverse, shorter on MUSM 4295, but longer on MUSM
1974. It should be lower than the anterior arm of the protocone (and the other occlusal structures) at early stage of wear, because it is barely worn contrary to all other structures of the
occlusal surface. The anteroloph is separate from a strong paracone (or the mesial extremity of
its sloped anterior arm on MUSM 4295) by a thin furrow. The labial protoloph is oblique. It is
connected, with the mure, to a strong and complete posterior arm of the protocone. On
MUSM 4295 (Fig 5Q), the mure is very short and longitudinal (i.e., parallel to the mesiodistal
axis of the tooth). On MUSM 1974 (Fig 3M [31]), the mure is absent or reduced to a very tiny
crestule from the third crest-anterior arm of the hypocone jonction that remains mesially free.
The third crest is almost parallel to the oblique labial protoloph, and is composed of a long
mesolophule sligthly linked to a short mesoloph. The mesostyle is strongly connected to a
curved and thick posteroloph. There is no clear distinct metacone or metaloph. Hence, the
posterior flexus is fully closed. However, the posteroloph appears to have a thickenning, which
could correspond to a relictual metaloph, on MUSM 4295 (Fig 5Q). The latter and paraflexus
are long, narrow mesiolabially-distolingually extended and oriented on MUSM 4295 (Fig 5Q),
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whereas they are more mesiodistally expanded on MUSM 1974 (Fig 3M [31]), due to the
higher length of that tooth. The mesial mesoflexus and hypoflexus are clearly open.
M3s of Nuyuyomys chinqaska (MUSM 4323 and 4324; Fig 5T and 5X) are well recognisable
by their distal part, which is labiolingually pinched (notably labially), and their hypocone,
being clearly smaller and with a lingual margin occupying a more labial position than the protocone. Among other upper molars attributed to this taxon, some are small-sized and subsquare or slightly mesiodistally elongated, and other larger can be also subsquare but they are
usually more transverse, being wider than long. We interpret them as possibly M1s and M2s,
respectively (i.e., M1? and M2?). The height of crown is widely variable. Some upper molars at
early stages of wear have a higher crown (e.g., the MUSM 4303 M1? and 4320 M2?;
Hling = 0.74–0.89) than other, which are less worn (e.g., the MUSM 4313 and 4314 M2?s;
Hling = 0.60–0.69). Because there are teeth with intermediary crown height between these two
extremes and no morphological character allowing to differentiate them, we consider that all
the material reported here documents a single taxon. On all upper molars, the crests are thin.
The paracone and the mesostyle are well differentiated, whereas the protocone and hypocone
are more crestiform, as the metacone in most cases. The latter is more cuspate on two upper
molars (MUSM 4309 and 4320; Fig 5W and 5A’). The protocone and hypocone are mesiolingually-distolabially and mesiodistally compressed, respectively. As on the MUSM 4295 P4, the
anterior arm of the protocone is mesiolabially directed and in continuity with the protocone,
its posterior outgrowth and sometimes a part of the anteroloph, like on MUSM 4314 (Fig 5B’).
The anteroloph (or its labial part) is more transverse. At early stages of wear, labially the paraflexus can be either closed (MUSM 4298 and 4303; Fig 5Y and 5E’) or opened (e.g., MUSM
1975, 4306, 4308, 4309, 4311, and 4320; Fig 3N [31]; Fig 5R, 5V, 5W and 5A’), separate from
the paracone by a thin notch (e.g., MUSM 4308, 4309, and 4320; Fig 5R, 5W and 5A’) or a
wider furrow (MUSM 1975, 4306 and 4311; Fig 3N [31]; Fig 5V). The posterior outgrowth of
the protocone can be very short, particularly on M1?s, or moderately longer. The posterior
arm of the protocone is complete and thin. On MUSM 4303, 4307 and 4314 (Fig 5Y and 5B’),
this crest is slightly lower than the protocone, but remains high (i.e., non-taeniodont pattern).
Although the paracone and mesostyle are close in position, they do not share connection in
most cases. On MUSM 4298, 4320 and 4323 (Fig 5W, 5X and 5E’), however they are linked via
their arms (i.e., posterior arm of the paracone and anterior arm of the mesostyle). The labial
protoloph is usually straight and mesiolabially directed, but it can have a lingual part oblique,
whereas its labial part is more transverse such as on MUSM 4298, 4318 and 4322 (Fig 5E’).
Two upper molars (MUSM 4311 and 4317) have one crestule on the mesial margin of the labial
protoloph, which is mesially free. MUSM 4323 does not have that crestule, but it shows a tiny
enamel swelling on the labial part of the paraflexus (Fig 5X). The third crest is often parallel to
the labial protoloph, and is usually composed of a long mesoloph linked to a mesolophular
spur. The mure is short but complete, oblique and linked to a strong and long anterior arm of
the hypocone. As dP4s, some upper molars have a long posterior arm of the mesostyle
(MUSM 4303, 4307, 4319, and 4323; Fig 5S, 5X and 5Y). On other upper molars, this arm is
usually short, but it can also be undeveloped (MUSM 1975 and 4314; Fig 3N [31]; Fig 5B’).
The metacone can be connected to the mesostyle or its posterior arm (e.g., MUSM 4303, 4308,
and 4319; Fig 3N [31]; Fig 5R, 5S and 5Y), or separated from these structures by a very tiny
notch (e.g., MUSM 4306, 4307 and 4320; Fig 5V and 5W), except on MUSM 1975 and 4314,
where the notch is thicker (Fig 3N [31] and Fig 5B’). On all upper molars, a metaloph is present, either long or short. It is often connected lingually to the posteroloph (via a posteroloph
spur on MUSM 4315 and 4320; Fig 5W), but it can also be lingually free (MUSM 4298, 4303,
and 4312; Fig 5Y and 5E’). On MUSM 1975, 4308 and 4307 (Fig 3N [31] and Fig 5R), it is
reduced to a crestule labially disconnected to the metacone, and lingually linked to the
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posteroloph spur. Specifically, the metaloph is always long and joining the metacone to the
posteroloph on teeth identified as M2?s, whereas it is complete or reduced on M1?s and M3?s.
The MUSM 4323 M3 displays a neocrest stemming from the anterior arm of the metacone,
and connecting lingually to the third crest (Fig 5X). The hypocone has a short posterior arm
slightly connected to the posteroloph (MUSM 4308, 4309, 4311, 4315, 4318, 4319, and 4324;
Fig 5R, 5S, 5T and 5A’) or separate from it by a minute and shallow notch (MUSM 4298, 4303,
4306, 4313, 4320, and 4323; Fig 5V, 5W, 5X, 5Y and 5E’). This notch is wider on MUSM 4314
(Fig 5B’). Some upper molars have a mesiodistal constriction at the base of the hypoflexus
(MUSM 4303, 4307, 4312, 4319, and 4324; Fig 5S, 5T and 5Y).
Remark 1
The MUSM 4293 and 4294 (Fig 5C’ and 5D’) dP4s have size, a crown height and global occlusal morphology similar than the rest of dP4s attributed to Nuyuyomys chinqaska (i.e., rectangular or slightly trapezoidal occlusal outline, pentalophodonty-tetralophodonty, nontaeniodonty, and U-shaped hypoflexus). Nevertheless, they differ from other dP4s attributed
to this taxon in some characters. MUSM 4293 (Fig 5C’) has a protocone strongly linked with
the anteroloph without any thinning of this crest. On MUSM 4294 (Fig 5D’), the protocone is
less bulbous than on other dP4s of N. chinqaska, and the anteroloph shows a more sligthly
thinning. Finally, there is no distinct metacone and metaloph on MUSM 4294 (Fig 5D’). We
interpret all these differences between MUSM 4293, 4294, and the rest of dP4s attributed to N.
chinqaska as documenting intra-specific variation.
Remark 2
We tentatively assigned the MUSM 4298 upper molar (Fig 5E’) to Nuyuyomys chinqaska
because it shows a particular pattern, not really shared with other upper molars attributed to
N. chinqaska. On that tooth, the apices of the paracone and mesostyle appear to be lingually
displaced and they bear very long arms with unusual orientations due to a kind of unusual
invagination of the labial crown margin: the anterior arm of the paracone is mesiolabially
directed, its posterior arm is very slightly distolabially directed, and the anterior arm of the
mesostyle is mesiolingually directed. Owing to the orientation of these arms and the connections anteroloph-anterior arm of the paracone, posterior arm of the paracone-anterior arm of
the mesostyle, and mesostyle-metacone, the dental structures of the tooth labially form a “M”.
In addition, the extremity of the posterior outgrowth of the protocone is bulged appearing as a
cusp-like structure. Nevertheless, MUSM 4298 matches with the morphology of the other
upper molars of N. chinqaska by other characters (i.e., size, crown-height, pentalophodonty,
non-taeniodonty, crestules on the paraflexus, oblique mure, long mesoloph connected to a
mesolophular spur, and tiny and shallow furrow separating the posterior arm of the hypocone
from the posteroloph). As far we know, such M-shaped pattern for the labial dental structures
on upper molars is not found in caviomorphs or very rare. Consequently, the peculiar characters of MUSM 4298 may result to some abnomalies or extreme intra-specific variation of this
taxon.
Remark 3
In addition, the specimens of TAR-31 have a small size and general pattern (i.e., non-taeniodonty; complete mure on upper teeth; presence of a metaloph on dP4s and upper molars) similar to those of the material from the early middle Miocene of Madre de Dios formerly
attributed to cf. Microsteiromys sp. (MD-67, Peru) [31]. The latter is documented by only two
upper teeth: MUSM 1974 P4 and MUSM 1975 upper molar (Fig 3M and 3N, p. 94 [31]). By
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their mesiodistally-elongated shape, ?M1s from TAR-31 are close to MUSM 1975. Particularly,
MUSM 4307 and 4308 (Fig 5R) from TAR-31 show a labial protoloph and a third crest slightly
oblique and a short metaloph labially unconnected to the metacone and lingually linked to the
posteroloph spur, as on MUSM 1975. Due to the absence of lower teeth attributed to the taxon
from MD-67 and differences on P4 (i.e., longer; absence or strong reduction of the mure;
flexus shape), we prefer to tentatively refer this material (MUSM 1974 and 1975) to as Nuyuyomys chinqaska for the time being.
Comparisons
By its non-taeniodonty, extended flexi(-ds), weak obliquity of loph(-id)s, p4 with a large trigonid, rounded P4, pentalophodont upper molars, and a well-defined, and slightly oblique
(faintly backwardly oriented) metaloph on upper molars, the material from TAR-31 exhibits
strong erethizontoid affinities. Moreover, like on some lower molars from TAR-31, a subpentalophodont pattern (i.e., presence of a short neolophid in the anteroflexid) is otherwise found
in some erethizontoids (e.g., Palaeosteiromys amazonensis, Branisamyopsis australis, Branisamyopsis praesigmoides, Steiromys duplicatus and Neosteiromys pattoni) [71, 74–77]. The material from TAR-31 shows brachydont lower teeth and submesodont-mesodont upper teeth, a
rather rare crown condition found among the erethizontoids, which are usually brachydont.
The only extinct erethizontoids so far showing high tooth crowns are Hypsosteiromys (Sarmiento Fm., Argentina; early Miocene) [74, 78, 83] and Noamys (site 3 of Rı́o Chico Locality,
Guanaco Fm., Argentina; late Miocene and/or early Pliocene) [72]. However, Nuyuyomys is
clearly distinct from the latter taxa. Hypsosteiromys has a larger size, loph(-id)s more oblique,
square P4s, tetralophodont M1–2s, tetralophodont-pentalophodont dp4s, and a metalophulid
I reduced to an anterior arm of the metaconid disconnected to the protoconid on lower teeth.
Noamys has tetralophodont and sligthly longer dP4 and M1 with oval-shaped occlusal contour
and flexi more extended, especially anteroposteriorly. The specimens from TAR-31 are
markedly smaller than all erethizontoid species except Kichkasteiromys raimondii, Shapajamys
labocensis, Palaeosteiromys amazonensis, Microsteiromys jacobsi gen. et sp. nov., and Noamys
[18, 37, 70–72] (this work). Contrary to Shapajamys, the P4s from TAR-31 and MD-67
(MUSM 4295 and MUSM 1974) do not have any distinct metaloph. Unlike the material from
MD-67 and TAR-31, Kichkasteiromys, Shapajamys and Palaeosteiromys do not display a metaloph disconnected labially to the metacone on upper molars. One M1 of Shapajamys (MUSM
2993) has a reduced metaloph that is lingually free, as on some M1?s from TAR-31. However, ?
M2s from TAR-31 have a metaloph more developed than in Shapajamys. Upper molars from
TAR-31 differ from the MUSM 2925 M1 or M2 of Kichkasteiromys (Fig 2D, p. 92 [70]) in having a lingual protoloph less lingually situated and a posterior outgrowth of the protocone more
oblique. For lower teeth, like on the material from TAR-31, some erethizontoids (Shapajamys,
Branisamyopsis, ?Eosteiromys sp. nov. sensu Candela [69], and Steiromys) show hexalophodont
dp4s with accessory structures. By its elongated shape and tetralophodont pattern, the MUSM
4334 p4 is particularly close to the p4 of M. jacobsi (IGM-DU 89–249 in Fig 16C, p. 56 [37];
Fig 3B), whereas dp4s from TAR-31 are much more elongated, and with a narrower trigonid
than in M. jacobsi (FMNH PM 54672 in Fig 24.2B, p. 395 [18]; Fig 3A). In addition, dp4s from
TAR-31 are hexalophodont, while the FMNH PM 54672 dp4 appears tetralophodont. The
lower molars of M. jacobsi (FMNH PM 54672) and of Shapajamys do not have any neolophid
on their anteroflexid, contrary to several specimens from TAR-31 and Palaeosteiromys. The
material from TAR-31 differs from M. jacobsi in having the mesoflexid still lingually open at
advanced stage of wear on m1–2s and less rectangular lower molars; and from Shapajamys in
having an ectolophid always complete and oblique on lower molars. Compared to the MUSM
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2861 m3 of Palaeosteiromys (Fig 4L, p. 77 [71]), m3s from TAR-31 have no neolophid or have
a shorter one, and show a mesoflexid more extended. The material from TAR-31 differs from
Eosteiromys annectens and Steiromys duplicatus in having non-taeniodont lower teeth; Eosteiromys homogidens, Eosteiromys annectens, and Coendou insidiosus in having hexalophodont
dp4s; from Protosteiromys and Eosteiromys in having no neolophid on the anteroflexid on
lower molars; from Branisamyopsis praesigmoides and Steiromys duplicatus in having an ectolophid always complete; from Neosteiromys and Parasteiromys in always having a complete
mure on dP4s and upper molars; from Eopululo, Protosteiromys pattersoni and Neosteiromys
in having a P4 without endoloph; from Eosteiromys homogidens in having a mesial mesoflexus
less mesiodistally extended on P4s; from Eosteiromys annectens, Eosteiromys kramarzi sensu
Candela [74], Branisamyopsis and Neosteiromys in having non-taeniodont dP4s and upper
molars; from Eopululo, Protosteiromys pattersoni and the three Erethizonthoidea indet. from
Acre region (Brazil; Late Miocene) [84, 85] in having upper molars always with a metaloph
(which is not reduced, far from being a relictual structure); from Protosteiromys, Parasteiromys, Branisamyopsis, Eosteiromys, Steiromys and Neosteiromys in having some elongated
upper molars (potential M1s) and an incomplete metaloph on some upper molars; from Branisamyopsis pardinas sensu Candela [74] in having a complete third crest on M1–2s; from Protosteiromys medianus in having a complete third crest on M3s [74–77, 80–83]. To summarise,
the erethizontoid teeth from TAR-31 are morphologically close to those of Kichkasteiromys,
Shapajamys laboensis, Palaeosteiromys, and M. jacobsi. In having elongated dp4s, with a hexalophodont pattern, the material from TAR-31 clearly differs from that of M. jacobsi. Hence, we
consider the dental material from TAR-31 as documenting a new genus and species: Nuyuyomys chinqaska.
Erethizontoidea gen. et sp. indet. 1
Fig 5F’ and S1 Table
Referred material
MUSM 4367, left p4 (Fig 5F’).
Locality
TAR-31, Tarapoto/Juan Guerra, Mayo River, Shapaja road, San Martı́n Department, Western
Amazonia, Peru.
Formation and age
Lower member, Ipururo Fm., late middle Miocene (i.e., Laventan SALMA).
Description
MUSM 4367 is a pristine p4, with rounded corners and trigonid slightly narrower than the
talonid (Fig 5F’). It is brachydont, tetralophodont and non-taeniodont. The entoconid is the
highest and largest cuspid. The metaconid and hypoconid are mesiodistally compressed,
whereas the mesostylid is more bulbous. On the mesiolabial corner of MUSM 4367, the metalophulid I is connected to a cuspate structure with a size similar to the mesostylid, that we
interpret as the protoconid due to its position and connection with the metalophulid I. The latter is complete and continuous. It has a transverse and long lingual part connecting to the
poorly distinct metaconid, whereas its labial one is shorter and curved. On its distal slope, the
metaconid has a short and quite transverse posterior arm, which is disconnected to the
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mesostylid. The second transverse cristid is straight and well-marked. It appears to be formed
by a long neomesolophid, connecting the mesostylid to a shorter and sloping posterior arm of
the protoconid. The latter is labially linked to a labial cuspid-shaped structure. This structure
is elongated, mesiolabially directed. It is aligned with a cristid stemming from the hypolophidanterior arm of the hypoconid junction (= the ectolophid or a part of the ectolophid), but
remains separate from it, as well as from the protoconid mesiolabially, by notches. This elongated cuspid-shaped structure could correspond to a part of the ectolophid fused with the protoconid cristid (see [4]). The entoconid is somewhat bulbous and displays thick, but faintly
defined, arms. Its anterior arm is clearly separate from the mesostylid, whereas its posterior
arm almost connects to the lingual extremity of the posterolophid, a very thin and shallow
notch separating both cristids. The hypolophid is slightly oblique and delimits, with the entoconid, the curved posterolophid and the strong and high anterior arm of the hypoconid, a
well-extended and oval metafossettid. The anteroflexid and mesoflexid are rectangular-oval
and have a similar extension, smaller than that of the metafossettid. The U-shaped hypoflexid
with a distolingual direction is linguolabially well-expanded.
Comparisons
MUSM 4367 is brachydont with a large trigonid, non-taeniodont pattern, extended flexi(-ds),
and a weak obliquity of loph(-id)s, which are characters found in erethizontoids. In addition to
its size, which is similar to that of p4s of Microsteiromys jacobsi (IGM-DU 89–249) and Nuyuyomys chinqaska (MUSM 4334), MUSM 4367 has a similar morphology (i.e., brachydonty, tetralophodonty, non-taeniodonty, and oblique ectolophid). However, contrary to p4s of M. jacobsi and
N. chinqaska, as well as the MNHN 1903-3-18 premolar of Protosteiromys asmodeophilus (Fig 32
[79] and see above), MUSM 4367 is more round shaped and less elongated, with a shorter trigonid and a straight mesial crown margin. In particular, MUSM 4367 differs from MUSM 4334 of
N. chinqaska in having a wider trigonid comparing to the width of the talonid, a higher labial part
of the metalophulid I, and a protoconid more mesially canted; from MNHN 1903-3-18 of P.
asmodeophilus in having smaller lingual flexid/fossettids and a larger hypoflexid. Unlike p4s of
Hypsosteiromys, MUSM 4367 has more straight lophids and a complete metalophulid I connected
to the protoconid [74, 83]. The MLP 93-XI-18-1 p4 of Eosteiromys homogenidens [74] appears to
have a similar mesiolabial morphology to MUSM 4367 in having a second cristid labially connected to an elongated and oblique structure, which may correspond to a part of the ectolophid
+ protoconid cristid, disconnected to the rest of the ectolophid. This structure is slightly connected to the protoconid on MLP 93-XI-18-1, contrary to MUSM 4367, but it was probably disconnected to this cuspid at an earlier stage of wear. MUSM 4367 differs from p4s of E.
homogenidens, Eosteiromys annectens [77], Branisamyopsis australis [74, 75], Branisamyopsis
praesigmoides [77], and Steiromys detentus [74] in being more square-shaped; of E. homogenidens
in having a straight metalophulid I and less extended anteroflexid; of E. annectens in having a
non-taeniodont pattern and a less oblique hypolophid and posterolophid; of Eosteiromys segregatus [74] in having no posterior arm of the mesostylid and a more extended metaflexid; of Branisamyopsis, Steiromys duplicatus [74], Steiromys verzii sensu Candela [74], and some p4s of S.
detentus in having a tetralophodont pattern; of S. detentus in having a less extended anteroflexid
and more oblique ectolophid; and of S. duplicatus in having a straight metalophulid I. MUSM
4367 is smaller than Hypsosteiromys, Branisamyopsis, Eosteiromys and Steiromys. Given the paucity of the material, we provisionally assign MUSM 4367 to Erethizontoidea gen. et sp. indet. 1.
Erethizontoidea gen. et sp. indet. 2
Fig 5G’ and S1 Table
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Referred material
MUSM 4368, left M1 or M2 (Fig 5G’).
Locality
TAR-31, Tarapoto/Juan Guerra, Mayo River, Shapaja road, San Martı́n Department, Western
Amazonia, Peru.
Formation and age
Lower member, Ipururo Fm., late middle Miocene (i.e., Laventan SALMA).
Description
The unique M1 or 2 (MUSM 4368; Fig 5G’) documenting this taxon is large, brachydont, and
non-taeniodont. It has a subsquare to rounded occlusal outline. The main cusps/styles (paracone, mesostyle, protocone, and hypocone) are large and well defined. The labial cusps/styles
are also well defined, whereas the lingual ones are somewhat compressed labiodistally-linguomesially, especially the protocone, which has a strongly crestiform aspect. The worn part of the
posterior outgrowth of the protocone is very short at this early stage of wear of the tooth. The
anteroloph is long and curved. It is lingually connected to the protocone and labially separated
from the paracone by a thin notch. The lingual protoloph, stemming from the protocone, is
high and reaches the labial protoloph–mure junction (i.e., non-taeniodont pattern). The labial
protoloph and the third crest are faintly oblique. The mure, linking these two transverse crests
lingually, is longitudinal (i.e., on the mesiodistal axis of the tooth). Labially, a large furrow separates the mesostyle from the paracone. The metacone is visible because it does not display tight
connections with the other labial dental structures. A thin and shallow groove separates it from
the mesostyle, and a very slight connection is present between the tiny posterior arm of the
metacone and the posteroloph. The third transverse crest is complete, but labially thinner and
shorter, thereby indicating that it could be formed primarily by a long mesolophule and a short
labial mesoloph. The metacone displays a straight and very short metaloph. At the level of this
lingual part, the metaloph exhibits: (i) a very short crestule mesially, which connects to the labial
extremity of the mesolophule via a longer crestule; and (ii) an alignement of crestules distally,
which remain free. This connection between the metaloph and the third crest generates a small
and narrow flexus labially located. The hypocone has mesially a long anterior arm, which is
strongly linked to the mure-third crest junction, and distally a posterior arm slightly curved and
connected to the straight posteroloph. In addition, a third short crest runs distolingually stemming from the hypocone, and can be recognised as a posterior outgrowth of this cusp. In its
middle part, the posteroloph bears a spur, labially directed towards the distal crestules of the
metaloph but without connection with them. All the main flexi are well extended and deep.
Comparisons
Among the four superfamilies of caviomorphs, MUSM 4368 exhibits strong erethizontoid affinities in being brachydont, non-taeniodont, pentalophodont, and in showing extended flexi and
straight lophs. Erethizontoid molars have usually a better-defined metaloph than MUSM 4368,
but some species can also be characterised by a reduced metaloph (e.g., Protosteiromys pattersoni,
Eosteiromys kramarzi, Parasteiromys uniformis, and Steiromys detentus) or the absence of this
crest (e.g., Eopululo wigmorei, P. pattersoni, Hypsosteiromys, Eosteiromys segregatus, Steiromys
detentus, and Noamys hypsodonta [72, 74, 78, 80–83, 86]. However, these species with a reduced
metaloph do not show a configuration similar to that of MUSM 4368, with crestules on the
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lingual extremity of the metaloph, some of which are connected to the third transverse crest.
MUSM 4368 is smaller than the upper molars of P. pattersoni, Hypsosteiromys, P. uniformis, E.
kramarzi, E. segregatus, and Steiromys. Moreover, MUSM 4368 differs from the upper molar
attributed to E. wigmorei (LACM 143269 in p. 111 [81]) in having a more quadrate shape; from
upper molars of Hypsosteiromys in having lower crown and lophs less oblique; of P. uniformis in
having a complete mure; and of E. kramarzi in having a lingual protoloph. By its size larger than
MUSM 4367 and the material attributed to N. chinqaska, MUSM 4368 probably documents
another erethizontoid at TAR-31, provisionally identified as Erethizontoidea gen. et sp. indet. 2.
Cavioidea Fischer, 1817
Caviidae Fischer, 1817
Caviidae gen. et sp. indet.
Fig 6 and S1 Table
Referred material
MUSM 4283, right p4 (Fig 6).
Locality
TAR-31, Tarapoto/Juan Guerra, Mayo River, Shapaja road, San Martı́n Department, Western
Amazonia, Peru.
Fig 6. Caviidae gen. et sp. indet. from TAR-31, Peru, MUSM 4283, right p4. (A) Occlusal, (B) occluso-labial, (C) crosssectional, (D) posterior, (E) ventral, (F) labial, (G) lingual, (H) anterior views. The illustrations of the fossil specimen are
3D digital models obtained by X-ray μCT surface reconstruction. The image in C is a 2D orthoslice of the tooth (crosssection).
https://doi.org/10.1371/journal.pone.0258455.g006
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Formation and age
Lower member, Ipururo Fm., late middle Miocene (i.e., Laventan SALMA).
Description
The MUSM 4283 p4 is slightly damaged at the level of its mesiolingual margin (Fig 6A, 6G and
6H). It also shows a crack on its labiodistal part. The enamel and dentine are damaged and
eroded in several regions of the crown and occlusal surface. MUSM 4283 is clearly high
crowned (Hling = 1.15; Hlab = 1.27; Fig 6B, 6D, 6F and 6G) and, although the presence of
roots is not directly visible, the closing of the hypostriid suggests the closing of the crown too
(Fig 6E and 6F). Hence, the tooth was seemingly protohypsodont. The cuspids/stylids and
lophids are not visible because they are subsumed within three enlarged lobes, corresponding
(from anterior to posterior) to the anterior projection, anterior lobe and posterior lobe. The
dentine is predominating, surrounded by a continuous and thin enamel layer. The tooth has a
triangular mesial margin. The anterior projection is narrower than the anterior lobe, which
itself is narrower than the posterior lobe. The anterior projection is roughly tear-drop shaped,
with a labial extremity short and distolabially directed. In its lingual part, the projection has an
oval structure, which is hollow and shallow (Fig 6A and 6B). Nevertheless, it is not a fossettid
because it is not surrounded by enamel layer. It is probably a dentine scar owing to an external
impact during the mastication or a taphonomic process. The limit between the anterior projection of p4 and the anterior lobe is marked by the presence of: (i) labially, a flexid almost labially
closed; and (ii) lingually, an isolated fossettid (Fig 6A). This fossettid is small, round and shallow. It has a thinner enamel layer than that of the margins of the tooth. As the mesialmost lobe
of the tooth is determined as the anterior projection, the flexid almost closed would be the
homologous of the interprismatic furrow sensu Pérez [51] separating the anterior projection
from the anterior lobe in some stem-Caviidae (Guiomys and “Prodolichotis pridiana”) [51, 87–
90], and which is variably present in Caviidae [1, 55, 91–98]. The enamel layer of the distal
margin of this flexid is thinner than that of its mesial margin and of the rest of the exterior
enamel layer of the tooth. On the labial margin, the striid corresponding to the opening of the
interprismatic furrow is mesiodistally very thin. Its depth represents less than 20% of the
crown height (Fig 6B and 6C). Due to their position, the fossettid and/or the interprismatic
furrow could be rests of the anteroflexid. The anterior lobe is quite oval and labially separated
from the posterior lobe by a triangular hypoflexid. This flexid is linguolabially extended and its
lingual extremity reaches more than the half of the width of the tooth. It displays a continuous
and homogenous enamel layer. The hypostriid is mesiodistally large and almost reaches the
neck of MUSM 4283. On the lingual margin of the tooth, there is an inward fold faintly developed at the transition between the anterior and posterior lobes. It could correspond to a very
slight longitudinal furrow opposite to the hypoflexid (= primary internal flexid [hpi]). The posterior lobe is tear-drop shaped, with its labial apex short and mesiolabially directed. In its
mesiolingual part, there is a fossettid linguolabially extended, with a thin enamel layer. Its
depth is slightly shallower than the striid corresponding to the opening of the interprismatic
furrow (Fig 6C). This fossettid probably corresponds to the metafossettid. Because the hypoflexid and metafossettid are not confluent, the tooth is non-taeniodont. The lingual fossettids
and the hypoflexid (at its base) show a white deposit, which by its aspect, does not seem to be
coronary cement, but more probably sediments that filled them after the death of the animal.
Comparisons
The protohypsodonty, formation of lobes, linguolabial extension of the hypoflexid, and nontaeniodonty primarily suggest caviid affinities. The very slight development of a possible
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longitudinal furrow opposite to the hypoflexid (= hpi), a slight development of the interprismatic furrow (= secondary external flexid [hse]), and a relatively narrow anterior projection
exclude hydrochoerine affinities for this material from TAR-31. Moreover, MUSM 4283 does
not have any additional flexids as observed in capybaras (i.e., internal flexids, including secondary internal flexids and supernumerary internal flexid; see [99]). MUSM 4283 shares characters, which would be ancestral during the evolution of the caviid family [89, 100], with stemCaviidae: protohypsodonty (Chubutomys, Luantus minor, Luantus propheticus, Luantus toldensis, Phanomys, and Eocardia), presence of lingual fossettids on worn teeth (Asteromys, Chubutomys, Luantus, Phanomys, Eocardia fissa, and Schistomys erro), and a hypoflexid that does
not reach the lingual margin of the tooth (Asteromys, Chubutomys, and Luantus) [52, 73, 79,
86, 100–104]. The presence of an anterior projection, which can be associated with an interprismatic furrow, is otherwise found in some derived stem-Caviidae (Guiomys and “Prodolichotis” pridiana) [51, 87–90] and crown-Caviidae [1, 55, 91–98]. However, Guiomys,
“Prodolichotis” and the crown caviids do not have any lingual fossettids on worn teeth contrary
to MUSM 4283. p4s of L. toldensis (MACN Pv SC4033 in Fig 5F [101]) and Phanomys [100]
have a lingual thickening of the metalophulid I/their mesial margin, which could correspond
to an incipient anterior projection. If the latter is really present in these taxa, it is less developed
than on MUSM 4283. In all Caviidea with an interprismatic furrow associated with an anterior
projection on p4s, the interprismatic furrow is never almost labially close, unlike on MUSM
4283. This tooth differs from p4s of Asteromys in having a hypoflexid more linguolabially
extended; of Asteromys and Luantus initialis in having a higher crown; of Asteromys, Chubutomys, Eocardia, and L. propheticus in having an anterior projection; of Phanomys in having a
thinner enamel; and of Eocardia in having a hypoflexid less linguolabially extended. The stemcaviid Guiomys is euhypsodont unlike MUSM 4283, and shows a p4 with a hypoflexid more
linguolabially extended, a more developed anterior projection and longitudinal furrow opposite to the hypoflexid [51, 105]. MUSM 4283 differs from p4s of Schistomys erro and Microcardiodon in being protohypsodont, and in having an anterior projection and a well-marked
hypoflexid; of Microcardiodon in having fossettids; and of Schistomys erro in having two persistent lobes [52, 87]. The size of MUSM 4283 is similar with that of Chubutomys simpsoni, Chubutomys leucoreios, Luantus minor, and Guimoys unica. As Dolichotinae gen. 2 large from La
Venta (IGM 183425 in Fig 24.7A, p. 401 [18]), MUSM 4283 shows a very slight longitudinal
furrow opposite to the hypoflexid, and a lingual extremity of the hypoflexid exceeding the half
of the width of the tooth, but without reaching its lingual margin. However, MUSM 4283 is
characterised by the presence of fossettids and an anterior projection, contrary to Dolichotinae
gen. 2 large from La Venta. MUSM 4283 differs from p4s of “P.” pridiana, Neocavia, Prodolichotis prisca, Dolicavia, Orthomyctera, and Dolichotis in having a less developed longitudinal
furrow opposite to the hypoflexid; of Paleocavia, Cavia, Galea and Microcavia in having a
clearly less developed longitudinal furrow, which is opposite to the hypoflexid; of Cavia, Galea
and Microcavia in having a hypoflexid without cement; of “P.” pridiana, Neocavia, Paleocavia?
mawka, Paleocavia impar, P. prisca, Dolicavia, Orthomyctera, Cavia, Dolichotis, and Galea in
having a linguolabially narrower hypoflexid; of Cavia in having a more developed anterior
projection [28, 54, 55, 90, 94, 95, 106–113]. In conclusion, the morphology of MUSM 4283
appears particular by a mosaic association of characters, combining primitive characters (i.e.,
protohypsodonty, presence of fossettids on worn teeth, and a hypoflexid, which do not reach
the lingual margin of the tooth) and more derived ones (i.e., development of a relatively large
anterior projection); and also by an unique configuration of the fossettids and the interprismatic furrow. Thus, it is possible that MUSM 4283 documents a new species among the stemCaviidae, but given the scarce available material, we provisionally identified it as Caviidea gen.
et sp. indet.
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Octochinchilloi Boivin, 2019 in Boivin, Marivaux & Antoine, 2019
Octodontoidea Waterhouse, 1839
Adelphomyidae Patterson & Pascual, 1968, nov. stat.
Ricardomys Boivin & Walton gen. nov. urn:lsid:zoobank.org:act:46B7B00E-E14D-4123-BF6D9AB7AFF4AAD4
Type species
Ricardomys longidens gen. et sp. nov.
Species content
Only the type species.
Derivation of name
Based on the original description of the species [37]: ‘In honor of Richard Kay, professor of
Evolutionary Anthropology at Duke University, who discovered the holotype of this species’.
Geographic and stratigraphic distribution
La Venta (IGM-DU loc. 032), Cerro Colorado Member, Villavieja Fm. (Laventan, late middle
Miocene), Huila Department, Colombia; Tarapoto/Juan Guerra (TAR-31), lower member,
Ipururo Fm. (Laventan, late middle Miocene), San Martı́n Department, Peru.
Generic diagnosis
As for the type and only known species.
Ricardomys longidens Boivn & Walton sp. nov. urn:lsid:zoobank.org:act:83062BD3-90064C73-BB02-37BF66BF6663
Fig 7 and S1 Table
Nom. nud. Ricardomys longidens Walton, 1990, Fig 9A, 9B, p. 31.
Nom. nud. Ricardomys longidens Walton, 1997, Figs 24.1C and 24.2D, p. 394, 395.
Holotype
IGM 183847, left mandibular fragment bearing dp4–m2 (Walton, 1990: Fig 9A, 9B; Walton,
1997: Figs 24.1C and 24.2D; Fig 7A).
Referred material
MUSM 4397–4401 (Fig 7B and 7F), left dp4s; MUSM 4402–4406 (Fig 7J), right dp4s; MUSM
4407, right lower molar; MUSM 4408, left m1 or m2?; MUSM 4409 (Fig 7C), left m1?; MUSM
4410 (Fig 7G), right m1?; MUSM 4411–4413 (Fig 7D and 7H), left m2?s; MUSM 4414, 4415
(Fig 7K), right m2?s; MUSM 4416–4419 (Fig 7E and 7I), left m3?s; MUSM 4370–4373, 4391–
4394 (Fig 7U and 7V), left upper teeth; MUSM 4374, 4395, 4396 right upper teeth; MUSM
4375 (Fig 7P–7R), left fragment of maxillary bearing dP4 (or M1)? and M1 (or M2)?; MUSM
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Fig 7. Ricardomys longidens gen. et sp. nov. (A) Material from La Venta, Colombia. (B–V) Material from TAR-31, Peru.
(A) IGM 183847��� , left mandibular fragment bearing dp4–m2. (B) MUSM 4401, left dp4. (C) MUSM 4409, left m1?. (D)
MUSM 4411, left m2?. (E) MUSM 4416, left m3?. (F) MUSM 4400, left dp4. (G) MUSM 4410, right m1? (reversed). (H)
MUSM 4413, left m2?. (I) MUMS 4418, left m3?. (J) MUSM 4406, right dp4 (reversed). (K) MUSM 4415, right m2?
(reversed). (L) MUSM 4377, left dP4 (or M1)?, morph 1. (M) MUSM 4380, right dP4 (or M1)?, morph 1 (reversed). (N)
MUSM 4376, left dP4 (or M1)?, morph 1. (O) MUMS 4384, left M1 or M2?, morph 2. (P–R) MUSM 4375, left maxilla
fragment bearing dP4–M1 (or M1–2)?, morph 1 and 2. (S) MUSM 4388, right M1 or M2?, morph 2 (reversed). (T) MUSM
4390, right M3?, morph 3. (U) MUSM 4392, left fragmentary upper molar, morph 4. (V) MUSM 4393, left fragmentary
upper molar, morph 4. (A-P, S-V) Occlusal, (Q) labial, and (R) lingual views. The asterisks appoint the holotype material.
The illustrations of the fossil specimens are computerized schematic line drawings based on Walton (Fig 9A [37]). (A),
scanning electron photomicrographs (B-O and S-V), and 3D digital models obtained by X-ray μCT surface reconstruction
(P-R).
https://doi.org/10.1371/journal.pone.0258455.g007
4376, 4377 (Fig 7L and 7N), left dP4s (or M1s)?; MUSM 4378–4380 (Fig 7M), right dP4s (or
M1s)?; MUSM 4381, right dP4?; MUSM 4382–4384 (Fig 7O), left M1 or M2?s; MUSM 4385–
4388 (Fig 7S), right M1 or M2?s; MUSM 4389, left M2?; MUSM 4390 (Fig 7T), right M3?.
Derivation of name
Based on the original description of the species [37]: ‘from the latin longidens, in reference to
the length and slenderness of the tooth row of the holotype of this species’.
Type locality
IGM-DU loc. 032 (only for the holotype), El Cardón Red Bed, La Venta Badlands, upper Magdalena Valley, Huila Department, Colombia.
Other locality
TAR-31, Tarapoto/Juan Guerra, Rı́o Mayo, Shapaja road, San Martı́n Department, Western
Amazonia, Peru.
Formation and age
Cerro Colorado Member, Villavieja Fm.; lower member, Ipururo Fm.; late middle Miocene
(i.e., Laventan SALMA).
Diagnosis
Octodontoid rodent characterised by a small size and non-taeniodont teeth with thick and
oblique lamellae. Lower teeth with a trilophodont pattern characterised by the absence of
metaconid cristid, posterior arm of the protoconid, neomesolophid and mesolophid. Thick
lingual parts of the first and second lamellae on lower teeth, and a first lamella divided into
two parts by a thin and shallow furrow on lower molars. Upper teeth with a transversal extension of the hypoflexus, prompt loss of the third transverse crest with wear and the absence of
metaloph. Differs from Eodelphomys in being smaller and in having lower molars with a thinner furrow dividing the first lamella, more expanded lingual flexids, metaflexid always open at
a more advanced stage of wear, less transversely extended hypoflexid, an entoconid area with
thickening and without discernable entoconid arms. Differs from Xylechimys in having less
transversely extended hypoflexid, an ectolophid which is not discernable because fused with
the hypolophid, hypoconid and the protoconid, an indistinct posterior arm of the metaconid,
and no posterior arm of the protoconid on lower molars. Differs from Ethelomys, Paradelphomys, Quebradahondomys, Deseadomys, Adelphomys, Prostichomys, Stichomys, Prospaniomys, and Spaniomys in having a thickening of the lingual parts of the two first lamellae on
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lower teeth; from Ethelomys in having thicker cristids and a less-transversely extended hypoflexid on lower molars; from Paradelphomys in having a non-taeniodont and trilophodont
dp4s with a lingual part of the first lamella less distally extended and the first and second lamellae very close in position, and no furrow-shaped flexids on lower teeth; from Quebradahondomys in having more oblique cristids on lower molars; from Deseadomys, Adelphomys,
Prostichomys, Stichomys, Prospaniomys, and Spaniomys in always having trilophodont dp4s,
m1s, and m2s; from Deseadomys and Prospaniomys in having no recognisable cusp(-id)s and
more oblique and thicker lamellae; from Deseadomys and Spaniomys in having a less transversely extended hypoflexid on lower teeth; from Prospaniomys in having a more transversely
extended hypoflexus on upper teeth; from Adelphomys in having posterior fossette probably
less persistent on upper teeth, a metaflexid always open at a more advanced stage of wear on
lower teeth, and a first lamella which can be divided into two parts on lower molars; from Spaniomys in having more oblique lamellae, and a straight and distolingually directed second
lamella on m3s; and from Prostichomys in having thicker lamellae, posterior fossette less persistent on upper teeth, and no mesostylid and an indistinct posterior arm of the metaconid on
lower molars.
Description
IGM 183847 is a left mandibular fragment preserving dp4–m2 (Fig 7A). The body of the mandible is anteriorly broken at the level of the anterior root of the dp4. Posteriorly, the angular
apophysis and all the ascending ramus, including the mandibular condyle, are missing. The
coronoid process is broken at its base. Labially, the notch for the insertion of the tendon of the
zygomatico-mandibularis pars infraorbitalis is small, below m1, and situated ventrally on the
labial edge of the mandible. The anterior tip of the masseteric crest and that of the lateral crest
end below the m1, and they appear to be not linked to the notch for the insertion of the tendon
of the zygomatico-mandibularis pars infraorbitalis, at the level of its posteroventral and posterodorsal regions, respectively. The masseteric crest is posteriorly broken. It is posteroventrally
directed and poorly prominent in its anterior part. The lateral crest is posterodorsally directed
and almost undistinct.
The teeth referred to this taxon are brachydont. The less worn tooth from TAR-31 (MUSM
4377) presents a fairly high crown, but its hypsodonty indexes stay below 1 (Hling = 0.62;
Hlab = 0.63). This tooth was probably submesodont at earlier wear stages. All lower and upper
teeth are non-taeniodont. The cusp(-id)s and styles (stylids) are not visible even on the tooth
at the earliest stage of wear (MUSM 4377). They are subsumed within loph(-id)s, thereby
forming flat-topped and thick crestiform structures (lamellae sensu Patterson & Pascual [56]).
Each lamella displays a continuous enamel layer, coating a dentine layer, which is increasingly
thicker with the dental wear.
The dp4s are longer than wide (Fig 7A, 7B, 7F and 7J). Their trigonid is wide but narrower
than the talonid. These teeth are characterised by two roots and a trilophodont pattern with
oblique lamellae. Mesiolabially, the area of the protoconid is well expanded and usually triangular-shaped. From this area, emerge mesially the first lamella and distally the second lamella.
The first lamella is mesially convex. Its lingual part is short and would correspond to the metaconid area (without its posterior arm?). It can be thick with respect of the thin central part of
the first lamella (metalophulid I; IGM 183847, MUSM 4400, 4401, 4405, and 4406; Fig 7A, 7B,
7F and 7J). On the worn MUSM 4397 dp4, the protoconid area is not recognisable from the
rest of the first lamella, these structures are fully fused, forming a round and large structure of
dentine at the mesial part of the tooth. On all dp4s, the second lamella is a central cristid distolingually directed. It would result of the fusion between the entoconid + the hypolophid + the
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mesial and distal ectolophids + a part of the protoconid. As the lingual part of the first lamella
on some specimens, the lingual part of the second lamella (the entoconid area), by its thickness, is well discernable from the other structures. There would be neither metaconid cristid,
neomesolophid, posterior arm of the protoconid, nor mesolophid. The third lamella is cristiform on the dp4 at the earliest stage of wear (MUSM 4401; Fig 7B) and diamond/parallelogram-shaped on other dp4s. It would include the hypoconid + its anterior outgrowth + the
posterolophid. On MUSM 4400 and 4401, it bears a small spur in its mesiolabial aspect (i.e.,
anterior outgrowth; Fig 7B and 7F). It is linked to the second lamella by a complete and thin
anterior arm of the hypoconid. On IGM 183847, the anterior arm of the hypoconid reaches a
short cristulid stemming from the second lamella (Fig 7A). The anterior arm of the hypoconid
is mesiolingually directed, strongly oblique on IGM 183847 and MUSM 4399–4401 (Fig 7B
and 7F), and more longitudinal on MUSM 4404–4406 (Fig 7J). Owing to the absence of the
metaconid cristid, posterior arm of the protoconid, neomesolophid and mesolophid, the anterolingual flexid corresponds to the confluence of the anteroflexid with the mesial and distal
mesoflexids. This flexid and the metaflexid are widely open lingually and more extended than
the U-shaped hypoflexid.
Lower molars have a pattern similar to that characterising dp4s (i.e., non-taeniodonty, trilophodonty, and obliquity, global shape and composition of lamellae; Fig 7A, 7C–7E, 7G–7I and
7K). Comparatively to dp4s, lower molars have three roots (i.e., two mesial and one distal) and
a more rectangular shape with a transverse mesial margin. Four lower molars (MUSM 4416–
4419; Fig 7E and 7I) show a distal part of the talonid labiolingually pinched, and as such they
are probably m3s (determined as m3?s). MUSM 4416 (Fig 7E) also has a posterior root
directed distally, a character typically found on m3s. Among the isolated lower molars without
distal pinch, some are small-sized, and as such could be m1s (determined as m1?s; MUSM
4409, and 4410; Fig 7C and 7G). On IGM 183847 and MUSM 4417, 4419 and 4415 (Fig 7K),
the lingual part of the first lamella is separated from the protoconid area by a thin and shallow
furrow open on the mesial margin of the tooth (MUSM 4415 is broken at the level of the furrow giving the impression that it is larger and deeper; Fig 7K). On MUSM 4417, the margins
of the furrow are composed of two sloped cristulids, one lingual and one labial. The lingual
cristulid stems from the metaconid area (the ?anterior arm of the metaconid), while the labial
one stems from the protoconid area (the ?anterior arm of the protoconid). They are connected
at the base of the furrow. On molars with a more advanced stage of wear (MUSM 4408–4414
and 4418; Fig 7C–7E and 7G–7I), this furrow is fully closed. The morphology of the second
and third lamellae is very similar of that of dp4s, except they are longer.
Regarding upper teeth (Fig 7L–7V), the absence of complete tooth row and a documentation of the whole stages of wear make it difficult to recognise the dental intra-specific variation
related to the difference of loci, wear, or polymorphism. Four morphs can be distinct. The
unique fragment of maxillary found at TAR-31 and attributed to this species, MUSM 4375
(Fig 7P–7R), bears two upper teeth. This specimen and the comparison with upper teeth of
other adelphomyines or closely related forms (e.g., Deseadomys, Adelphomys, Prostichomys,
and Spaniomys) allow us to propose hypotheses regarding the locus determination for these
morphs:
• Morph 1: small upper teeth longer than wide or subsquare, and with a quadrangular occlusal
outline and three transverse lophs (MUSM 4375–4381; Fig 7L–7N and 7P–7R). MUSM 4381
seems to have three roots (i.e., two labial and one large lingual), whereas other teeth have not
conserved their roots. This morph could correspond to the morphology of dP4s (or M1s);
• Morph 2: upper teeth larger than those of the morph 1, either subsquare or rectangular, and
with quadrangular occlusal outlines, three transverse lophs and four roots (MUSM 4375 and
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4382–4389; Fig 7O–7S). This morph could correspond to the morphology of M1s or M2s.
MUSM 4389 has a very short posterior crest as MUSM 4390 (see below morph 3). This
shortness may be related to a strong labial displacement of the hypocone. Nevertheless,
MUSM 4389 shows typical characters of morph 2: large size, quadrangular occlusal outline
with a straight distal margin and four roots. It could correspond to a M2;
• Morph 3: this morph is represented by one small upper tooth (MUSM 4390; Fig 7T) characterised by three roots, a more round outline than teeth of the morphs 1 and 2, distal pinch,
and three transverse lophs. This morph could correspond to the morphology of M3s;
• Morph 4: this morph is represented by six mesially-broken upper teeth (MUSM 4391–4396;
Fig 7U and 7V). These fragments of teeth have different size, some are small-sized (MUSM
4391 and 4392; Fig 7U), medium-sized (MUSM 4393 and 4395; Fig 7V), while other are
larger (MUSM 4396). They have a more rounded occlusal outline than teeth of morphs 1
and 2, and a distal part slightly labiolingually narrower than the mesial part. They seem to be
mesiodistally elongated. Because they are broken, the hypsodonty index cannot be calculated. However, they have thinner crests and deeper flexi than teeth of other morphs, and
three (MUSM 4392 and 4395; Fig 7U) or four (MUSM 4391, 4393, 4394, and 4396; Fig 7V)
transverse lophs. Owing to the latter characters, this morph likely clusters upper molars at
less advanced wear stages than those of morphs 2 and 3.
The first lamella is diamond/parallelogram-shaped with a large and rounded apex at the
mesiolingual corner of the tooth. Two other apices, the first at the mesiolabial corner of the
tooth and the second in the center of its lingual edge, are usually narrower and tapered. This
lamella would include the protocone + the posterior outgrowth of the protocone + the anteroloph. The second lamella, which would correspond to the paracone + the labial protoloph, is
either transverse or oblique (i.e., mesiolabially directed) or lingually oblique and labially transverse. It is short due to a strong transversal extension of the hypoflexus. The lingual protoloph
is thin, distolabially directed and complete, thus linking the first lamella to the second (i.e.,
non-taeniodont pattern). On the trilophodont upper teeth (MUSM 4373, 4375–4389, 4390,
and 4395; Fig 7L–7U), the third lamella is usually bean-shaped, but can be suboval (MUSM
4389 and 4390; Fig 7T). On the tetralophodont MUSM 4394, this third lamella presents a
round flexus still distolabially open. On other tetralophodont upper teeth (MUSM 4391–4393
and 4396; Fig 7V), this flexus is fully closed, thereby forming a round fossette. This flexus/fossette separates the third transverse crest from the posteroloph. On all tetralophodont upper
teeth, these two transverse lophs are very thin and short, except on MUSM 4394, in which the
third transverse crest is thicker. On MUSM 4391 and 4393 (Fig 7V), the third crest is composed of a very short mesoloph, whose lingual extremity is connected to a mesolophular spur.
The third lamella on trilophodont upper teeth would include the mesostyle and/or the metacone (see [50]) + the third transverse crest + the posteroloph + the hypocone + its anterior
arm. Near the center of the tooth, the second lamella and the third lamella are connected via a
mure mesiolabially directed (strongly oblique).
Comparisons
A small size, non-taeniodont teeth with thick and oblique lamellae, and a transverse extension
of the hypoflexus, the prompt loss of the third transverse crest with wear and the absence of
metaloph on upper teeth are dental traits found in adelphomyine octodontoids [4, 56], thereby
suggesting adelphomyine affinities for this material from La Venta and TAR-31. By their size
and morphology, the lower teeth from TAR-31 are close to those of IGM 183847 from La
Venta, previously attributed to Ricardomys longidens nom. nud. (Figs 24.1C and 24.2D [18];
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Fig 9A, 9B [37]; Fig 7A). As lower teeth from TAR-31, the IGM 183847 lower teeth have a trilophodont pattern characterised by the absence of metaconid cristid, posterior arm of the protoconid, neomesolophid and mesolophid. These lower teeth also show oblique lamellae, thick
lingual parts of the first and second lamellae, and a first lamella divided into two parts by a
thin and shallow furrow on lower molars. However, the lower molars from TAR-31 slightly
differ from IGM 183847 in having the first lamella more oblique. A similar pattern to dp4s
from La Venta and TAR-31 is also found on the MUSM 2856 dp4 attributed to Adelphomyinae indet. 2 from CTA-61 (Peru, late Oligocene) [71]. Unlike dp4s from La Venta and TAR31, MUSM 2856 does not have a hypoflexid transversely oriented, but the latter is distolingually oriented. Other adelphomyines or closely related forms have strictly trilophodont lower
molars: Eodelphomys (Santa Rosa, Peru; ?late Eocene/early Oligocene) [81], Ethelomys (Cabeza
Blanca, Argentina; late Oligocene) [114], Paradelphomys (Sarmiento Fm., Argentina; early
Miocene) [56], and Quebradahondomys (Quebrada Honda, Bolivia; late middle Miocene)
[105]. Eodelphomys is only known from a single m3 (LACM 144298 in p. 130 [81]), which
shows a first lamella divided into two parts by a furrow. However, the latter is larger than that
on lower molars from La Venta and TAR-31 (when the furrow is present). The lower molars
from La Venta and TAR-31 also differ from LACM 144298 in having lingual flexids more
expanded mesiodistally, always open at a more advanced stage of wear, less transversely
extended hypoflexid, and an entoconid area with thickening and without discernable entoconid arms. In comparison with the material form La Venta and TAR-31, Ethelomys, Paradelphomys, and Quebradahondomys do not have any thickening of the lingual parts of the two
first lamellae (or at least of the second lamella) on lower teeth; Ethelomys has thinner cristids
and a more transversely extended hypoflexid on lower molars; Paradelphomys has furrowshaped flexids on lower teeth, a taeniodont and tetralophodont dp4 with a lingual part of the
first lamella more distally extended (with posterior arm of the metaconid?) and the first and
second lamellae very close in position; and Quebradahondomys has less oblique lamellae on
lower molars. As the lower teeth from La Venta and TAR-31, Xylechimys (Laguna de los
Machos, Argentina; late Oligocene) [56] appears to develop a lingual thickening of the second
lamella on lower molars with the dental wear (visible on the m1 of the holotype). Nevertheless,
Xylechimys has a trigonid of lower molars clearly different characterised by: (i) a posterior arm
of the protoconid linked to the metaconid area, thus delimiting a small and rounded anterofossettid, (ii) discernable ectolophid and posterior arm of the metaconid, and (iii) a more transversely extended hypoflexid. The general pattern of the upper teeth from TAR-31 is similar to
that of adelphomyines (or closely-related forms) documented by upper teeth: Deseadomys
(Cabeza Blanca and La Flecha, Argentina; late Oligocene) [79], Adelphomys (Pinturas Fm. and
Santa Cruz Fm., Argentina; early Miocene) [73, 77], Prostichomys (Sarmiento Fm. and Pinturas Fm., Argentina; early Miocene) [77, 115, 116], Stichomys (Santa Cruz Fm.; late early Miocene) [73], Prospaniomys (Sarmiento Fm.; early Miocene) [11, 78, 117], and Spaniomys
(Pinturas Fm. and Santa Cruz Fm.; Pampa Castillo, Chile; late early Miocene) [16, 73, 77, 118,
119]. Contrary to the material from La Venta and TAR-31, all these taxa display no thickening
of the lingual parts of the two first lamellae (or at least of the second lamella) on lower teeth,
and can have tetralophodont lower teeth (usually dp4s, m1s or m2s). The tetralophodont pattern of these lower teeth is characterised by the presence of a posterior arm of the protoconid
+ metaconid cristid and/or neomesolophid (and mesolophid on some dp4 of Prostichomys).
The material from La Venta and TAR-31 also differs from Deseadomys and Prospaniomys in
having no recognisable cusp(-id)s and more oblique and thicker lamellae; from Deseadomys
and Spaniomys in having a less transversely extended hypoflexid on lower teeth; from Prospaniomys in having a more transversely extended hypoflexus on upper teeth; from Adelphomys
in having a posterior fossette probably less persistent on upper teeth, always open at a more
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Fig 8. Octodontoidea gen. et sp. indet. from TAR-31, Peru (A–C) and Microscleromys sp. 1 from La Venta, Colombia (D, E). (A)
MUSM 4662, left m1 or m2. (B) MUSM 4660, left dP4. (C) MUSM 4661, right M3 (reversed). (D) UNC unnumbered specimen, left P4.
(E) IGM-DU 85–411, left P4. Occlusal views. The illustrations of the fossil specimens are scanning electron photomicrographs (A-C)
and computerized schematic line drawings based on Walton (Fig 15 [37]) (D-E).
https://doi.org/10.1371/journal.pone.0258455.g008
advanced stage of wear on lower teeth, and a first lamella which can be divided into two parts
on lower molars; from Spaniomys in having more oblique lamellae, and a straight and distolingually directed second lamella on m3s; and from Prostichomys in having thicker lamellae, a
posterior fossette less persistent on upper teeth, and no mesostylid and no discernable posterior arm of the metaconid on lower molars. To summarize, due to the morphological similarity
between the lower teeth from La Venta and TAR-31, we assign them to the same taxon. By its
particular set of characters, the entire material from these both areas is determinable as a new
species, hence we validate Ricardomys longidens.
Octodontoidea gen. et sp. indet.
Fig 8 and S1 Table
Referred material
MUSM 4662, left m1 or m2 (Fig 8A); MUSM 4660, left dP4 (Fig 8B); MUSM 4661, right M3
(Fig 8C).
Locality
TAR-31, Tarapoto/Juan Guerra, Mayo River, Shapaja road, San Martı́n Department, Western
Amazonia, Peru.
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Formation and age
Lower member, Ipururo Fm., late middle Miocene (i.e., Laventan SALMA).
Description
Due to the very advanced stage of wear of the MUSM 4662 m1 or m2 (Fig 8A), its occlusal pattern is faintly discernable, especially on the mesial area of the tooth, where a broad dentine
platform is present. There is a thick, continuous enamel layer on the entire periphery of the
crown. It is thinner at the level of the lingual parts of the hypoflexid and of the mesoflexid.
Only three transverse lophids are visible, delimited by a very thin enamel layer, which is absent
in some regions. The mesialmost lophid is transverse and connects the area of the posterior
arm of the metaconid/mesostylid to the junction of the protoconid (or the protoconid cristid)
and the ectolophid. This lophid is recognised as a second transverse cristid (i.e., neomesolophid and/or posterior arm of the protoconid). Mesially, it is partially fused with the mesial
dentine platform, which probably corresponds to the fusion at least of the metalophulid I with
the anteroflexid. The transverse lophid that is second in position, is the hypolophid, running
slightly labiomesially from the area of the entoconid to link the posterior part of the ectolophid.
The hypolophid is almost parallel to the second cristid. The ectolophid is short and gently oblique. The transverse lophid appearing in third position is a curved posterolophid, lingually connected to a strong posterior arm of the entoconid. The area of the hypoconid is enlarged and
oval, formed by the fusion of the hypoconid, its anterior outgrowth and its posterior arm (if
present) and/or the labial part of the posterolophid. There is a thick and complete anterior
arm of the hypoconid (i.e., non-taeniodont pattern). The mesoflexid and metaflexid are lingually closed, whereas the hypoflexid remains labially open. The metafossettid is oval and
smaller than the mesofossettid.
The MUSM 4660 dP4 (Fig 8B) has a trapezoidal crown outline in occlusal view (i.e., labial
marging longer than the lingual margin). This tooth shows an advanced stage of wear. Labially,
the dental structures have a thick enamel layer, while the lingual structures are surrounded by
a clearly thinner enamel layer. The posteroloph area is very enlarged due to the wear, and
forms a dentine platform, in which a metacone, a metaloph or a rest of this loph may be
included. The hypoflexus is triangular and poorly extended linguolabially. It separates lingually
the area of the protocone from that of the hypocone. The areas of the protocone, mure and of
the hypocone are also strongly enlarged and broadly merged together, as well as with the platform of the posteroloph, forming a S-shape dentine structure on the distolingual part of the
tooth. From this dentine structure, three main lophs run labially: the anteroloph, the labial
protoloph and the third transverse crest. The anteroloph is curved with a lingual part thicker
than its labial part. The labial protoloph and third crest are transverse and parallel. The paraflexus is still open labially, separating the anteroloph from the labial protoloph. In contrast, the
other transverse lophs are connected labially, forming a small oval mesial mesofosette and a
more linguolabially extended distal mesofosette.
As MUSM 4662, the MUSM 4661 M3 (Fig 8C) is much worn, but it documents a slightly
earlier stage of wear. A thick enamel layer surrounds the tooth as well, but the latter is internal
at the level of the mesial mesoflexus, which is still opened labially contrary to MUSM 4662.
Moreover, all the transverse lophs and flexi are still discernable. The hypocone is clearly more
labially situated than the protocone, which indicates that the specimen would be a M3. MUSM
4661 is tetralophodont and non-taeniodont. The area of the protocone corresponds to a broad
dentine platform, including the protocone, its posterior outgrowth and its anterior arm (if
present) and/or the lingual part of the anteroloph. From this platform, the anteroloph runs
labially and links the paracone. The lingual part of the anteroloph is transverse, whereas its
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labial part is curved. Due to the connection between the anteroloph and the paracone, a parafossette is formed and reduced to a thin and straight structure. The enamel layer that surrounds
it is thick, notably on its mesial margin. On the labial margin of the tooth, the paracone and
mesostyle are strong and of similar size. The posterior arm of the protocone is thick and oblique. It connects to the mure, which is linguodistally oriented and in the continuity with the
anterior arm of the hypocone. The labial protoloph and the third transverse crest (i.e., mesoloph and/or mesolophule) are roughly straight and parallel. There is neither metaloph nor
discernable metacone. A well-curved posteroloph is labially connected to the mesostyle and
lingually to the area of the hypocone, which is triangular-shaped. The mesial mesoflexus is
very thin. The posterior fossette (i.e., confluence of the distal mesoflexus and the metaflexus) is
reduced to a very small and rounded structure. As the parafossette, the mesial mesoflexus and
the posterior fossette are surrounded by a thick enamel layer.
Comparisons
By its small size, weak loph(-id) obliquity, non-taeniodonty, tetralophodont upper molars (i.e.,
absence of metaloph), and a strong mesostyle on upper molars, these three teeth from TAR-31
likely document a single and distinctive octodontoid. Indeed, many Palaeogene and Neogene
octodontoids also display these characters. They are usually characterised by a reduction or the
absence of the second transverse cristid, and a reduction of the posterior arm of the metaconid
on lower molars, and a metacone more distally positioned on upper molars (e.g., Acarechimys,
Deseadomys, Dudumus, and Caviocricetus) [30, 79, 120, 121], characters absent on the specimens from TAR-31. However, Paulacoutomys and some lower molars of Platypittamys, Sciamys principalis, Prostichomys and Plesiacarechimys show a complete second transverse cristid
and a well-developed posterior arm of the metaconid [77, 115, 116, 122–126], as it is observed
on the material from TAR-31. Among the latter taxa, the material from TAR-31 is closer to
Paulacoutomys and Plesiacarechimys in having transverse loph(-id)s and a similar extension of
the flexi(-ds). In addition, these genera can have an ectolophid slightly oblique on lower
molars, as it does on MUSM 4662. The material from TAR-31 differs from Paulacoutomys and
Plesiacarechimys in having a smaller size; from Platypittamys and Prostichomys in having a
more longitudinal ectolophid on lower molars; from Platypittamys in having M3 with third
crest that is not curved, a mesial mesoflexus less extended, a posterior flexus/fossette more
extended; from Prostichomys and S. principalis in having a mesostyle less connected to the posteroloph on dP4s and upper molars; from Prostichomys in having a more transverse hypolophid on lower molars, a more transverse labial protoloph on dP4s and upper molars, and more
transverse M3 with a wider distal part; and from S. principalis in having a mesial part of the
lower molars (= fusion of the metalophulid I with the second cristid) more mesiodistally long
at its lingual and labial extremities, a hypoflexid less extended labiolingually, and a mesoflexid
which is lingually closed although a metafossettid is still present on lower molars, and a hypoflexus less extended labiolingually and a mesial mesoflexus which is strongly reduced although
the posterior fossette is still present on upper molars. In sum, given the condition and scarcity
of the material, we provisionally refer these teeth to an octodontoid indet.
Chinchilloidea Bennett, 1833
Microscleromys Boivin & Walton gen. nov. urn:lsid:zoobank.org:act:785C5F6A-DCCD4C02-B136-A2B49702E6DA
Type species
Microscleromys paradoxalis gen. et sp. nov.
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Species content
The type species and Microscleromys cribriphilus gen. et sp. nov.
Derivation of name
Based on the original description of the species [37]: ‘In reference to its small size and general
resemblance to Scleromys Ameghino, 1887’.
Geographic and stratigraphic distribution
La Victoria Fm. and Baraya Member of the Villavieja Fm. (Laventan SALMA, late middle Miocene), Huila Department, Colombia; Tarapoto/Juan Guerra (TAR-31), lower member, Ipururo
Fm., (Laventan SALMA, late middle Miocene), San Martı́n Department, Peru.
Generic diagnosis
Chinchilloid rodent characterised by a small or moderate size. Relative size of m3s/M3s
smaller in average than that of m1–2s/M1–2s. Brachyodont to mesodont teeth. P4s and upper
molars with noticeable high crowns. Upper teeth with a higher lingual crown and lower molars
with a higher distolabially crown. Absence of cement or in small quantity. Homogeneous
thickness of the enamel layer of dental strutures. Poorly distinct cusp(-id)s from the loph(-id)s,
which do not form lamina. At early stages of wear, lower teeth with pseudo-taeniodont or taeniodont pattern, lower molars with a pseudo-taeniodont pattern more frequently present, taeniodont P4s, dP4s and upper molars with a taeniodont pattern more frequently present. dp4s
with a metalophulid I not connected to the metaconid and protoconid at early stages of wear, a
posterolophid well-separated from the entoconid, a mesostylid mesially and distally isolated in
most cases, and a tendency to the reduction or absence of the third crisitid. Lower molars with
usually a groove on the metalophulid I at early stages of wear. Suboval dP4s, with well-arcuate
anteroloph and posteroloph as well as a bulbous protocone slightly or strongly separate from
the anteroloph. Round/oval P4s with a connection between the posterior outgrowth of the protocone and the hypocone. Upper molars with usually a mure oblique and aligned with the
anterior arm of the hypocone. Important variation in terms of development, configuration
and orientation of the second transverse cristid on p4s and lower molars, and of the third
transverse crest and metaloph on upper teeth, but with a tendency to their reduction and
absence. Trilophodont lower molars in most cases. Among upper teeth, P4s with stronger
reduction of the third crest and metaloph. Upper molars with a third crest more frequently
reduced to a mesoloph and without metaloph. Persistent flexids/fossettids on lower molars
and flexi/fossettes except the posterior ones (the distal mesoflexus and metaflexus if present)
on upper molars. Differs from Chambiramys in having slightly higher crowns, and more
quadrangular upper molars; from Eoincamys, Incamys and Scleromys/“Scleromys” in having
more square-shaped upper molars and a more important reduction of the third transverse
crest on upper molars (i.e., more frequent trilophodont pattern than in these genera); from
Eoincamys ameghinoi, Eoincamys pascuali and Incamys in having a second transverse cristid
usually more reduced on lower molars; from Eoincamys pascuali in having a thinner furrow
(when it is present) on the metalophulid I, which can be situated at the middle of this lophid;
from Saremmys in having lower-crowned teeth, thinner loph(-id)s, a metaconid postionned
more distally and strongly connected to the protoconid by a thick second cristid (metaconid
cristid and/or posterior arm of the protoconid) on dp4s, a less oblique hypolophid on p4s and
lower molars, a more bulbous protocone and a hypocone mesiolabially-distolingually compressed on dP4s, and some upper molars with a distinct metacone and a long metaloph and
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mesoloph; from Saremmys, Scleromys/“Scleromys”, Garridomys and Drytomomys in having an
anterior arm of the hypoconid, which can be present on p4s and lower molars, and in a more
curved posterolophid on lower molars; from Incamys, Saremmys, Scleromys/“Scleromys”, Garridomys and Drytomomys in having a smaller size; from Garridomys and Drytomomys in having a second transverse cristid usually more reduced on lower molars; from Garridomys in
having a homogeneous thickness of the enamel layer, thinner loph(-id)s, and strong and high
protoconid and ectolophid on p4s; from “Scleromys” colombianus and Drytomomys aequatorialis in having a separation between the hypocone and the posteroloph thinner and less persistent at early wear stages on upper molars; and from Drytomomys in having a less oblique
hypolophid on lower molars.
Microscleromys paradoxalis Boivin & Walton sp. nov. urn:lsid:zoobank.org:act:38631BE932E6-4E5D-B5CE-469C37B98D8C
Fig 9 and S1 Table
Nom. nud. Microscleromys cribriphilus Walton, 1990, Fig 12F, 12G-12O, p. 45.
Nom. nud. Microscleromys paradoxalis Walton, 1990, Fig 11A-11J, p. 39.
Nom. nud. Microscleromys ?paradoxalis Walton, 1990, Fig 13B, 13C p. 49.
Microscleromys sp. Walton, 1990, Fig 13A, p. 49.
Nom. nud. Microscleromys cribriphilus Walton, 1997, Fig 24.2.N, p. 295.
Nom. nud. Microscleromys paradoxalis Walton, 1997, Fig 24.2.G-I, p. 295.
Holotype
IGM 250321, right M1 or M2? (Fig 9P).
Referred material
MUSM 4643 (Fig 9A–9E), left fragment of mandible bearing incisor and dp4–m1; ING-KU
8604, right fragment of mandible bearing p4–m2; MUSM 4640–4642 (Fig 9F), left dp4s; IGM
250306 (Fig 9G), left p4; IGM 250305 and 250308 (Fig 9K), right p4s; MUSM 4644, left lower
molar; IGM 250265, MUSM 4645, right lower molar; IGM 250288, 250307, IGM-DU 88–814,
89–675, MUSM 4646–4650 (Fig 9H, 9I, 9M and 9N), left m1 or m2; IGM 250319, MUSM
4651–4656 (Fig 9L), right m1 or m2s; IGM 250274, 250285, 250304, MUSM 4657 (Fig 9J), left
m3s; IGM 251020 (Fig 9O), right m3; IGM 250302, IGM-DU 88–815 (Fig 9Z), ING-KU
unnumbered specimen (Fig 9U), MUSM 4623 (Fig 9Q), left dP4s; MUSM 4624 (Fig 9A’), left
P4; MUSM 4625 (Fig 9V), right P4; MUSM 4626, left upper molar; MUSM 4627, right upper
molar; IGM 250283, IGM-DU 89–673, MUSM 4628–4631 (Fig 9X, 9B’ and 9C’), left M1 or
M2?s; MUSM 4632–4636 (Fig 9R, 9S and 9W), right M1 or M2?s; IGM 250284, MUSM 4637
(Fig 9T), left M3?s; IGM-DU 85–410, MUSM 4638, 4639 (Fig 9Y and 9D’), right M3?s.
Derivation of name
Based on the original description of the species [37]: ‘from the latin paradoxalis, in reference
to the strange and confusing morphology of this species’. In light of the last discoveries of
caviomorph faunas in Peru, the morphology of this species does not appear so strange or confusing, but we prefer maintaining this name for the sake of both usage and courtesy.
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Fig 9. Microscleromys paradoxalis gen. et sp. nov. Mateial from TAR-31, Peru (black background) and from La Venta,
Colombia (white background). (A–E) MUSM 4643, left mandibular fragment bearing dp4–m1. (F) MUSM 4641, left dp4. (G)
IGM 250306, left p4. (H) MUSM 4647, left m1 or m2. (I) MUSM 4648, left m1 or m2. (J) MUSM 4657, m3. (K) IGM 250308,
right p4 (reversed). (L) MUSM 4654, right m1 or m2 (reversed). (M) MUSM 4650, left m1 or m2. (N) MUSM 4649, left m1 or
m2. (O) IGM 251020, right m3 (reversed). (P) IGM 250321��� , right M1 or M2? (reversed). (Q) MUSM 4623, left dP4. (R)
MUSM 4634, right M1 or M2? (reversed). (S) MUSM 4633, right M1 or M2? (reversed). (T) MUSM 4637, left M3?. (U)
ING-KU unnumbered specimen, left dP4. (V) MUSM 4625, right P4 (reversed). (W) MUSM 4632, right M1 or M2? (reversed).
(X) MUSM 4631, left M1 or M2?. (Y) MUSM 4638, right M3? (reversed). (Z) IGM-DU 88–815, left dP4. (A’) MUSM 4624, left
P4. (B’) MUSM 4630, left M1 or M2?. (C’) MUSM 4629, left M1 or M2?. (D’) MUSM 4639, right M3? (reversed). (A, F-D’)
Occlusal, (B) anterior, (C) posterior, (D) lingual, and (E) labial views. The asterisks appoint the holotype material. The
illustrations of the fossil specimens are 3D digital models obtained by X-ray μCT surface reconstruction (A-E), scanning
electron photomicrographs (F, H-J, L-N, Q-T, V-Y, and A’-D’), and computerized schematic line drawings based on Walton
(Fig 24.2 [18]; Fig 13 [37]) (G, K, O, P, U, and Z).
https://doi.org/10.1371/journal.pone.0258455.g009
Type locality
CVP 13B locality, Fish Bed near Casa Cuzco, La Venta Badlands, upper Magdalena Valley,
Huila Department, Colombia.
Other localities
localities unknown (for ING-KU unnumbered specimen and ING-KU 8604), IGM-DU loc.
075, between the Chunchullo and the Tatcoa Sandstone Beds (only for IGM 250265), and
localities of the Fish Bed (including CVP 5, 8, 9, 10, and 10A) and IGM-DU loc. 006–1, Monkey Beds (only for IGM-DU 88–814), La Venta Badlands, upper Magdalena Valley, Huila
Department, Colombia; TAR-31, Tarapoto/Juan Guerra, Rı́o Mayo, Shapaja road, San Martı́n
Department, Western Amazonia, Peru.
Formation and age
La Victoria Fm. and Baraya Member of the Villavieja Fm.; lower member, Ipururo Fm.; late
middle Miocene (i.e., Laventan SALMA).
Diagnosis
Species with teeth approximately 25–30 per cent larger than those of M. cribriphilus. dP4s can
have a pseudo-taeniodont or taeniodont pattern.
Description
MUSM 4643 is a right fragment of mandible bearing a part of the lower incisor and dp4–m1
(Fig 9A–9E). The incisor is mesially and distally broken, but the preserved portion testifies to a
long radius of curvature of this tooth. The preserved part of the dentary corresponds to the
ventrally broken body below the dp4–m1. It is very damaged, showing the base of the roots of
the cheek teeth and the entire surface of the preserved fragment of incisor. The anterior root of
dp4 is mesiodistally large, perpendicular to the plan of the neck, but clearly oriented mesioventrally in the dentary. Conversely, the posterior root of m1 is slightly oriented distoventrally.
The morphology of the notch for the insertion of the tendon of the zygomatico-mandibularis
pars infraorbitalis, masseteric crest or lateral crest is not accessible.
In addition to the dp4 preserved on MUSM 4643 (Fig 9A), three other dp4s are attributed
to Microscleromys paradoxalis (Fig 9F). MUSM 4641 is complete (Fig 9F), whereas MUSM
4642 is a posterior fragment of dp4, in which the metaconid and metalophulid I are missing.
MUSM 4640 is worn and abraded especially at the level of the metalophulid I, which is reduced
to its distal part. The two complete dp4s are longer than wide, with a trigonid narrower than
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the talonid. The metalophulid I is distally well arcued, forming the mesial edge of the tooth. It
is separate from the metaconid and protoconid by narrow notches on the MUSM 4643 dp4
(Fig 9A). On MUSM 4641 (Fig 9F), a similar configuration is present for its lingual extremity,
whereas its labial extremity reaches the base of the protoconid. A continuous second cristid
links the metaconid to the protoconid. Hence, it would be formed by a metaconid cristid and/
or posterior arm of the protoconid. It is mesiolingually directed and faintly curved on MUSM
4641 (Fig 9F) and MUSM 4643 dp4 (Fig 9A). It appears more transverse on MUSM 4640. On
the MUSM 4643 dp4, MUSM 4641 and 4642 (Fig 9A and 9F), a mesostylid is present on the
lingual margin. It can be mesially and distally isolated (MUSM 4641; Fig 9F), mesially isolated
(MUSM 4643 dp4; Fig 9A) or connected to both the metaconid and entoconid (MUSM 4642).
From that stylid, a neomesolophid runs labially and connects to a mesolophid reduced to a
low spur. The two complete dp4s show neither neomesolophid nor mesolophid. The mesial
and distal ectolophids are distolingually directed and aligned. They are not discernable from
each other on the MUSM 4640, 4642 and 4643 dp4 (Fig 9A). On MUSM 4641 (Fig 9F), they
are constituted of a short and thin mesial part (?mesial ectolophid) connecting to a longer and
thicker distal part (?distal ectolophid). The labial part of the hypolophid is slightly oblique (i.e.,
distolingually directed), whereas its lingual part is more transverse. At early stages of wear
(MUSM 4641; Fig 9F), the hypoconid displays a very short anterior outgrowth, which becomes
longer with wear (MUSM 4642 and 4643 dp4; Fig 9A). The dp4s at advanced stages of wear
have a non-taeniodont pattern, whereas MUSM 4641 shows a low and thin anterior arm of the
hypoconid (i.e., pseudo-taeniodont pattern). The posterolophid is strong, well curved and separate from the entoconid by a large furrow. The hypoflexid is V-shaped with an anterior margin longer than the distal one.
The p4s are slightly longer than wide with the trigonid smaller than the talonid (Fig 9G and
9K). They are tetralophodont. Due to the moderate-advanced stage of wear of all p4s, the conids and stylids are not discernable from the lophids. The mesial margin of p4s is constituted by
a transverse metalophulid I. Although it cannot be confirmed, a mesostylid may be present
because the area of the posterior arm of the metaconid is long, especially on IGM 250306 (Fig
9G) and 250308 (Fig 9K), and could be fused with this stylid distally. The second cristid is
complete on IGM 250306 (Fig 9G) and 250308 (Fig 9K), transverse on the former and mesiolingually directed on the latter. Moreover, IGM 250306 has a tiny spur on its lingual margin
between the second cristid and the entonconid (Fig 9G). This spur could be a very short neomesolophid. On IGM 250305, the second cristid is reduced to a lingual cristid, labially free. It
woud be a metaconid cristid or a neomesolophid depending on if it is stemming from the
metaconid or the mesostylid, respectively. The metaconid area is thick on the mesiolingual
corner of the p4 of the mandibular fragment ING-KU 8604, and could include a metaconid
cristid. On that tooth, the protoconid has a very short posterior arm, and posteriorly to the
metaconid area, there is a spur similar to that of IGM 250306 (Fig 9G), which is probably a rest
of neomesolophid. The posterior arm of the metaconid (or mesostylid) is slightly connected to
the entoconid on IGM 250305, 250306 (Fig 9G) and 250308 (Fig 9K). It is strongly linked to
this cuspid on the most worn p4 (ING-KU 8604 p4). The ectolophid is oblique and connected
mesially to the protoconid on all p4s. It connects distally to the hypolophid on IGM 250305,
250306 (Fig 9G) and 250308 (Fig 9K). Contrary to the other specimens, the ING-KU 8604 p4
shows the labial extremity of the hypolophid linked to the posterolophid instead of the ectolophid. The hypolophid is roughly transverse on all p4s. The hypoconid is fused with its anterior
outgrowth, and the posterolophid is curved, with its lingual extremity reaching the entoconid.
The anterior arm of the hypoconid is strong and complete on all p4s, except on IGM 250308
in which this arm is absent and the hypoflexid is fully confluent with the metaflexid (Fig 9K).
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Lower molars are brachydont (Hling = 0.47–0.57; Hlab = 0.49–0.72; Fig 9A, 9H–9J and 9L–
9O). The talonid of m3s is slightly narrower than the trigonid and with a distal part pinched
labiolingually. The cuspids are very cristiform and thus, faintly differentiated from the cristids.
At early stages of wear, the lower molars display a groove at the level of the metalophulid I separating it into an anterior arm of the metaconid and an anterior arm of the protoconid. The
position of this groove varies: either in the middle part of the metalophulid I (MUSM 4647 and
4648; Fig 9H and 9I), lingual to the protoconid (IGM 250319, IGM-DU 88–814, 89–675,
MUSM 4650 and 4655; Fig 9M), or closer to the metaconid (MUSM 4654; Fig 9L). On some
other lower molars, the anterior arms of the metaconid and protoconid are slightly linked,
either equally-sized (MUSM 4649–4653; Fig 9N) or the anterior arm of the protoconid can be
shorter (MUSM 4657; Fig 9J). The posterior arm of the metaconid is strong and long (MUSM
4647, 4650, and 4655; Fig 9H and 9M), except on MUSM 4654 in which it is short (Fig 9L). On
that tooth, there is a short posterior arm of the protoconid and metaconid cristid, which are
both labially free. IGM 250304 has a metaconid cristid, which runs distolabially and remains
free. On some lower molars at an advanced stage of wear (e.g., IGM 250274, 250319, 251020,
IGM-DU 88–814, 89–675, ING-KU 8604, MUSM 4652, and 4653; Fig 9O), there is a dentine
plateform at their mesiolingual corner, formed by the fusion of the metaconid, its arms, and
possibly a metaconid cristid (+ ?neomesolophid + ?mesostylid). On MUSM 4644 and 4646,
the ectolophid shows a small expansion, which could be a relic of the posterior arm of the protoconid. The ectolophid is oblique, aligned with the crestiform protoconid in most cases, and
sometimes with the labial part of the hypolophid, its lingual part being more transverse
(IGM-DU 88–814, 89–675, IGM 250319, 251020, and MUSM 4657; Fig 9J and 9O). The entoconid occupies a mesial position with respect to the hypoconid. The former usually has long
and thick anterior and posterior arms, which are separated mesially to the posterior arm of the
metaconid and distally to the posterolophid by tiny furrows (MUSM 4647, 4652, and 4653; Fig
9H). On MUSM 4650, the entoconid, having shorter and thinner arms, is mesially and distally
isolated from these structures by larger notches (Fig 9M). The anterior arm of the hypoconid
is either incomplete (MUSM 4654 and 4657; Fig 9J and 9L), notched (MUSM 4651 and 4653)
or absent (e.g., IGM 250274, 250285, 251020, ING-KU 8604, MUSM 4643, and 4655; Fig 9A
and 9O). On some lower molars at advanced stage of wear, the anterior arm of the hypoconid
is, however, connected to the hypolophid-ectolophid junction (i.e., non-taeniodont pattern).
The anterior outgrowth of the hypoconid is thin and becomes longer with wear. In addition,
the hypoconid is strongly connected to the posterolophid. The latter is thick and C-shaped or
hook-shaped.
The dP4s have a suboval/subtrapezoidal occlusal outline, with rounded corners (Fig 9Q, 9U
and 9Z). The cusps are quite bulbous. Mesially, the anteroloph is curved and separate labially
from the paracone. Lingually, it is strongly (IGM-DU 88–815 and ING-KU unnumbered specimen; Fig 9U and 9Z) or slightly (IGM 250302 and MUSM 4623; Fig 9Q) connected to a massive protocone or its anterior arm. The posterior outgrowth of the protocone is very short. On
IGM 250302 and ING-KU unnumbered specimen (Fig 9U), a short crestule (i.e., neoformation) runs distally from the anteroloph and remains free. On all dP4s, the lingual protoloph is
absent (i.e., taeniodont pattern). The labial protoloph is strong and either transverse (IGM
250302, ING-KU unnumbered specimen, and MUSM 4623; Fig 9Q and 9U) or oblique
(IGM-DU 88–815; Fig 9Z). The mure is complete, oblique, and tends to be aligned with the
anterior arm of the hypocone. On the labial margin, the paracone and mesostyle have a similar
size, whereas the metacone remains poorly distinct (not well-defined) as it is fused with the
curved posterolophid. The mesostyle is connected to the metacone solely (or its anterior arm)
on IGM-DU 88–815 (Fig 9Z), whereas it joins the paracone and metacone or its anterior arm
on ING-KU unnumbered specimen and MUSM 4623 (Fig 9Q and 9U). On IGM 250302, the
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mesostyle is mesially and distally isolated. The third transverse crest corresponds to a mesoloph, which is lingually free (IGM 250302) or slightly connected to a mesolophular spur stemming from the anterior arm of the hypocone (MUSM 4623; Fig 9Q), or directly to the anterior
arm of the hypocone (ING-KU unnumbered specimen and IGM-DU 88–815; Fig 9U and 9Z).
Most of dP4s have a short metaloph backwardly connected to the posteroloph via a longer posteroloph spur or not, delimiting a small and oval metafossette. However, it can be more slightly
connected to the posteroloph spur (MUSM 4623; Fig 9Q). On IGM-DU 88–815, the metacone
does not seem to bear a metaloph (Fig 9Z). On that tooth, a long crest slightly linked to the
posteroloph in its lingual part is present, which could be a rest of metaloph and/or the posteroloph spur. In addition, there is a tiny crestule stemming from the labial part of the posteroloph,
which connects to the labial extremity of the metaloph/posteroloph spur, thus delimiting a
small and oval fossette on IGM-DU 88–815 (Fig 9Z).
MUSM 4624 (Fig 9A’) and 4625 (Fig 9V) are two complete P4s at moderate stages of wear.
They preserve a part of their roots: a lingual one very massive and two labial much smaller,
which externally diverge. These two specimens are oval and high-crowned, especially at the
level of their lingual margin, their labial margin being lower (Hling = 0.80–0.87; Hlab = 0.62–
0.70). On the labial margin, the paracone is large and connected to a strong and oblique labial
protoloph. On MUSM 4625 (Fig 9V), the mure strongly links the labial protoloph to the short
anterior arm of the hypocone. On that tooth, the mure is aligned with the anterior arm of the
hypocone. On MUSM 4624 (Fig 9A’), the mure is slightly connected to the anterior arm of the
hypocone by a thin and short crestule. In addition to the paracone, the rest of the occlusal outline is composed of a curved and long crest formed by the fusion of the anteroloph, protocone,
its anterior arm and its posterior outgrowth, the mesostyle, posteroloph, and hypocone. On
MUSM 4624, the paracone is mesially and distally separated from this long crest by a thin furrow (Fig 9A’), while the anteroloph reaches this cusp on MUSM 4625 (Fig 9V). On MUSM
4625, there are two connected crestules, one stemming from the anteroloph and the other
from the labial protoloph (Fig 9V). The lingual protoloph is absent (i.e., taeniodont pattern).
On MUSM 4624, the mesostyle is smaller than the paracone, whithout any mesoloph, and it
joins the posteroloph (Fig 9A’). On that tooth, a metaloph stemming from a metacone entirely
subsumed within the posteroloph, runs mesiolingually. Its lingual extremity is distally connected to a spur of the posteroloph, and ends close to a longer mesolophule, without connection to the latter. On MUSM 4625, the posteroloph exhibits a thickening in its labial part,
which may correspond to its fusion with the mesostyle/mesoloph and/or metacone/metaloph
(Fig 9V).
The upper molars determined as M1 or M2?s are clearly mesiodistally elongated (IGM
250283, MUSM 4633, and 4634; (Fig 9R and 9S) or subsquare (IGM 250321, IGM-DU 89–673,
MUSM 4629–4632, and 4635; Fig 9W, 9X, 9B’ and 9C’). It seems that these upper molars show
a tendency of shape variation, ranging from mesiodistally elongated at early stages of wear to
squarer configuration at later stages of wear. On these upper molars, the hypocone is either
more labially situated than the protocone, or situated at the same level of this cusp. Other
upper molars have a hypocone clearly positioned labially with respect to the protocone, and
thus they could be M3s (determined as M3?s). All upper molars have a taeniodont pattern
characterised by a paraflexus and hypoflexus fully confluent. On the two upper molars at the
earliest stage of wear (MUSM 4634 and 4637; Fig 9R and 9T), there is a short anteroloph
(+ parastyle?), which is separate from the long and curved anterior arm of the protocone by a
thin (MUSM 4634; Fig 9R) or wider (MUSM 4637; Fig 9T) furrow. The anteroloph is either
not connected to the paracone (e.g., IGM-DU 89–673, MUSM 4629, 4632, 4634; Fig 9R, 9W
and 9C’), slightly (MUSM 4633, 4638; Fig 9S and 9Y) or more strongly (MUSM 4637, 4639;
Fig 9T and 9D’) connected to this cuspid. The strong labial protoloph is usually oblique but it
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can also be transverse (IGM 250321 and MUSM 4629; Fig 9C’). The anterior arm of the hypocone is oriented mesiolabially, except on MUSM 4634, in which it is more longitudinal. It
tends to be aligned with a short mure. The labial protoloph can be in continuity to both the
mure and anterior arm of the hypocone with roughly the same orientation (MUSM 4632,
4635, 4637, and 4639; Fig 9T, 9W and 9D’). On MUSM 4635, the labial protoloph bears on its
mesial part a short crestule, which remains free on the paraflexus. It likely corresponds to a
neocrest. Some upper molars have a well-defined mesostyle (MUSM 4626, 4632, 4634, 4637,
and 4639; Fig 9R, 9T, 9W and 9D’). At early stages of wear, a thin and shallow notch separates
the paracone from the mesostyle. A mesoloph, stemming from this style, is usually presents
with a medium or short length, but it can also be more reduced as on MUSM 4637, in which it
is incipient and poorly distinct from the mesostyle (Fig 9T). It can be transverse, either lingually free (IGM 250321 and MUSM 4637; Fig 9T), connected to a short mesolophule (MUSM
4633; Fig 9S), or directly connected to the anterior arm of the hypocone (MUSM 4631; Fig
9X). Some upper molars (IGM 250283, 250284, IGM-DU 89–673, MUSM 4630, and 4634; Fig
9R and 9B’) have a mesoloph backarldly directed and linked to the posteroloph (or its spur),
thereby delimiting an oval and small posterior fossette. On MUSM 4626, 4632, and 4639 (Fig
9W and 9D’), the third crest is absent (neither mesoloph nor mesolophule). There is no clear
metacone on all upper molars, but a metaloph connected lingually to the posteroloph is present on MUSM 4631 (Fig 9X). It is faintly linked to the posterior arm of the mesostyle on that
tooth. One upper molar at an early stage of wear, MUSM 4639 (Fig 9D’), has a trilophodont
pattern without neither third crest nor metaloph. At more advanced stages of wear, some
molars are trilophodont but with a thick posteroloph, which may include a mesoloph and/or a
metaloph and their associated cusps/styles (MUSM 4627–4629, 4632, 4635, and 4638; Fig 9W,
9Y and 9C’).
Microscleromys cribriphilus Boivin & Walton sp. nov. urn:lsid:zoobank.org:act:74B60D5F-A8B3-4A2C-9483-D22739400EC8
Fig 10 and S1 Table
Nom. nud. Microscleromys cribriphilus Walton, 1990, Fig 12A-12E, p. 45.
Nom. nud. Microscleromys cribriphilus Walton, 1997, Fig 24.2.K-M,O, p. 295.
Nom. nud. Microscleromys paradoxalis Walton, 1997, Fig 24.2.J, p. 295.
Holotype
MUSM 4491, left M1 or M2? (Fig 10H’).
Referred material
MUSM 4545, right fragmentary dp4 or p4; MUSM 4532–4541 (Fig 10A, 10F and 10K), left
dp4s; MUSM 4542–4544, right dp4s; IGM-DU 88–818, MUSM 4546–4550 (Fig 10G, 10L and
10P), left p4s; IGM 251018 (Fig 10Q), MUSM 4551–4558 (Fig 10B), right p4s; IGM-DU 89–
676, left m1 or m2 (or p4); MUSM 4558–4561, left lower molars; MUSM 4562–4565, right
lower molars; MUSM 4566, left m1 or m2?; IGM 250309, right m1 or m2?; MUSM 4567–4588
(Fig 10H and 10M), left m1?s; MUSM 4589–4600 (Fig 10C), right m1?s; MUSM 4601–4608
(Fig 10D, 10I, 10N and 10S), left m2?s; IGM 250303 (Fig 10R), MUSM 4609–4615, right m2s;
MUSM 4621, left m3?; MUSM 4616–4620 (Fig 10E, 10O and 10T), left m3s; MUSM 4622 (Fig
10J), right m3; MUSM 4420–4433 (Fig 10U and 10E’), left dP4s; IGM 250320 (Fig 10J’),
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Fig 10. Microscleromys cribriphilus gen. et sp. nov. Material from TAR-31, Peru (black background) and from La Venta, Colombia (white
background). (A) MUSM 4532, left dp4. (B) MUSM 4553, right p4 (reversed). (C) MUSM 4599, right m1? (reversed). (D) MUSM 4603, left
m2?. (E) MUSM 4619, left m3. (F) MUSM 4540, left dp4. (G) MUSM 4548, left p4. (H) MUSM 4581, left m1?. (I) MUSM 4606, left m2?. (J)
MUSM 4622, right m3 (reversed). (K) MUSM 4536, left dp4. (L) MUSM 4546, left p4. (M) MUSM 4583, left m1?. (N) MUSM 4605, left m2?.
(O) MUSM 4616, left m3. (P) MUSM 4550, left p4. (Q) IGM 251018, right p4 (reversed). (R) IGM 250303, right m2? (reversed). (S) MUSM
4602, left m2?. (T) MUSM 4620, left m3. (U) MUSM 4425, left dP4. (V) MUSM 4453, left P4. (W) MUSM 4505, right M1 or M2? (reversed).
(X) MUSM 4514, right M2? (reversed). (Y) MUSM 4521, left M3?. (Z) MUSM 4445, right dP4 (reversed). (A’) MUSM 4466, right P4
(reversed). (B’) MUSM 4489, left M1 or M2?. (C’) MUSM 4511, left M2?. (D’) MUSM 4530, right M3? (reversed). (E’) MUSM 4428, left dP4.
(F’) MUSM 4452, left P4. (G’) MUSM 4502, right M1 or M2? (reversed). (H’) MUSM 4491��� , left M1 or M2?. (I’) MUSM 4525, right M3?
(reversed). (J’) IGM 250320, right dP4 (reversed). (K’) IGM 251040, right P4 (reversed). (L’) MUSM 4508, right M1 or M2? (reversed). (M’)
MUSM 4513, left M2?. (N’) MUSM 4517, left M3?. Occlusal views. The asterisks appoint the holotype material. The illustrations of the fossil
specimens are scanning electron photomicrographs (A-P, S-I’, and L’-N’) and computerized schematic line drawings based on Walton (Fig
24.2 [18]; Fig 14 [37]) (Q, R, J’, and K’).
https://doi.org/10.1371/journal.pone.0258455.g010
MUSM 4434–4447 (Fig 10Z), right dP4s; MUSM 4469, right P4 or M3?; MUSM 4448–4458
(Fig 10V and 10F’), left P4s; IGM 251040 (Fig 10K’), MUSM 4459–4468 (Fig 10A’), right P4s;
MUSM 4470, 4471, left upper molars; MUSM 4472–4478, right upper molars; MUSM 4479–
4497 (Fig 10B’ and 10H’), left M1 or M2?s; MUSM 4498–4510 (Fig 10W, 10G’ and 10L’), right
M1 or M2?s; MUSM 4511–4513 (Fig 10C’ and 10M’), left M2?s; MUSM 4514–4516 (Fig 10X),
right M2?s; MUSM 4517–4524 (Fig 10Y and 10N’), left M3?s; MUSM 4525–4531 (Fig 10D’
and 10I’), right M3?s.
Derivation of name
Based on the original description of the species [37]: from the latin cribri (sieve) and philus
(liking to or attracted to) ‘because this rodent is so far only known from isolated teeth recovered by screenwashing’.
Type locality
TAR-31, Tarapoto/Juan Guerra, Mayo River, Shapaja road, San Martı́n Department, Western
Amazonia, Peru.
Other localities
Localities of the Fish Bed (including CVP 9, 10A, and 13B), La Venta Badlands, upper Magdalena Valley, Huila Department, Colombia.
Formation and age
Lower member, Ipururo Fm.; Baraya Member of the Villavieja Fm.; late middle Miocene (i.e.,
Laventan SALMA).
Diagnosis
Species with teeth approximately 25–30 per cent smaller than those of M. paradoxalis. dP4s
can have a non-taeniodont pattern characterised by a complete and high lingual protoloph in
continuity with the labial protoloph.
Description
The pattern of teeth usually follows that of M. paradoxalis described above, with few exceptions, but mostly the smaller size allows for a clear distinction of the two species (see S1 File).
dp4s are longer than wide, with a trigonid narrower than the talonid. The protoconid
appears to be the largest cuspid. On the dp4 at the earliest stage of wear (MUSM 4532; Fig
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10A), the metaconid is the most cristiform cuspid, which is linguolabially pinched. On worn
dp4s (MUSM 4539, 4540, and 4544; Fig 10F), the protoconid displays three connections: (i) a
mesiolingual one with a curved metalophulid I, which extends lingually to the metaconid; (ii)
a distolingual one with the second transverse cristid, which connects labially to the metaconid;
and (iii) a distal one with the mesial extremity of the mesial ectolophid. On the dp4s at the earliest stage of wear (MUSM 4532 and 4536; Fig 10A and 10K), the protoconid is separated from
the metalophulid I and from the mesial ectolophid by two notches. The notch between the protoconid and the metalophulid I is the deepest and thus, it closes at a latter stage of wear than
that between the protoconid and the mesial ectolophid (MUSM 4534). Conservely to MUSM
4532, the protoconid is also isolated from the second transverse cristid, which is reduced to a
medium-sized metaconid cristid, on MUSM 4536 (Fig 10K). However, both structures are
connected at their base. On MUSM 4532, 4534, and 4536 (Fig 10K), the metalophulid I is not
connected to the metaconid (Fig 10A). On all dp4s, the second transverse cristid is slightly
directed mesiolingually. The mesial and distal ectolophids are thin, distolingually directed,
aligned and often not discernable from each other. A small mesostylid is often present
(MUSM 4534, 4536, 4539, 4540, and 4541; Fig 10F and 10K). It is isolated from the entoconid
on all dp4s. It is connected to the metaconid on MUSM 4534, 4539 and 4540 (Fig 10F). On
MUSM 4532 (Fig 10A), a mesostyle may be present, fused with the long posterior arm of the
metaconid. On MUSM 4539, a complete third transverse cristid links the mesostylid with the
ectolophids. The other dp4s do not show any development of the third cristid. On all dp4s, a
well-marked hypolophid links the large entoconid to the distal ectolophid. In most cases, the
labial part of the hypolophid is slightly oblique (i.e., distolingually directed), whereas its lingual
part is more transverse. The hypoconid always displays a short anterior outgrowth and a low,
but complete anterior arm, reaching the hypolophid-distal ectolophid junction (i.e., pseudotaeniodont pattern). Besides, the hypoconid has lingually a very short posterior arm, connecting to a strong and well-curved posterolophid on MUSM 4532 and 4534 (Fig 10A). At early
stages of wear, the posterolophid is separate from the entoconid or its posterior arm by a large
furrow. The three lingual flexids show a similar large expansion. The hypoflexid is less
extended, distolingually oriented and V- or U- shaped. Its anterior margin is longer than its
distal one, except on MUSM 4533.
p4s are brachydont-submesodont (Hling = 0.45–0.77; Hlab = 0.54–0.81; Fig 10B, 10G, 10L,
10P and 10Q). They are slightly longer than wide, with a talonid clearly wider than the trigonid. By their high elevation and large surface, the protoconid and hypoconid are the most distinct cuspids of the crown at the earliest stage of wear (MUSM 4548; Fig 10G). The protoconid
is labiolingually pinched, whereas the hypoconid is mesiodistally compressed. At the mesiolingual corner, the metaconid is well defined during the first stages of wear (MUSM 4548 and
4553; Fig 10B and 10G). It displays a short or moderate posterior arm (MUSM 4548, 4550, and
4553; Fig 10B, 10G and 10P). On MUSM 4548 (Fig 10G), the metalophulid I is continuous and
high, joining the protoconid to the metaconid. On MUSM 4546 and 4553 (Fig 10B), the anterior arms of the protoconid and metaconid are short, strongly sloped and separated by a large
furrow, which is thinner on MUSM 4547 and 4550 (Fig 10P). The protoconid frequently has a
short posterior arm, which can be very short (MUSM 4546–4548; Fig 10G and 10L) or reduced
to a spur (IGM-DU 88–818, MUSM 4550, 4551, and 4557; Fig 10P). On IGM-DU 88–818,
MUSM 4547 and 4557, it is lingually free. The posterior arm of the protoconid is absent on
IGM 251018, MUSM 4553 and 4558 (Fig 10B). A thin and short metaconid cristid can be present (MUSM 4546 and 4548; Fig 10G and 10L). On MUSM 4546 (Fig 10L), it is slightly connected to the posterior arm of the protoconid. On MUSM 4548 (Fig 10G), these two cristids
are linked at their base but not connected at their apex. The worn MUSM 4554 p4 shows an
oblique second transverse cristid, linking the metaconid to the protoconid. Thus, this cristid
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may be formed here by the fusion of a metaconid cristid with a posterior arm of the protoconid. On MUSM 4551, a round and small mesostylid is clearly distinct, separated from the
metaconid (or its posterior arm) and entoconid by tiny notches. On that tooth, a cristid, probably a neomesolophid, is faintly connected to the mesostylid and to a very tiny posterior arm
of protoconid. In addition, it shares a very slight connection mesially with the mesialmost
lophid and distally with the hypolophid. On MUSM 4546, the distal extremity of the posterior
arm of the metaconid displays a tiny spur that may be a neomesolophid that would be connected to a mesostylid fused with the posterior arm of the metaconid (Fig 10L). At some
advanced stage of wear, the metaconid, its anterior and posterior arms, metaconid cristid, as
well as possibly the mesostylid and neomesolophid (if present), fuse and form a small platform
of dentine on the mesiolingual corner of the teeth. If the entoconid has anterior and posterior
arms, the latter is longer than the former (MUSM 4547, 4548, 4550, and 4556; Fig 10G and
10P), except on IGM 251018 and MUSM 4546, in which they are equally-sized (Fig 10L and
10Q). The entoconid (or its anterior arm) is separated from the mesostylid-posterior arm of
the metaconid by a large furrow (MUSM 4548 and 4553; Fig 10B and 10G) or a very thin
notch (MUSM 4546, 4547, 4550, and 4556; Fig 10L and 10P). On p4s at a more advanced stage
of wear, these structures are connected. Stemming from the entoconid, a hypolophid runs
labiomesially (i.e., oblique) and slightly connects to the ectolophid (IGM-DU 88–818, MUSM
4546, and 4548; Fig 10L). The anterior arm of the hypoconid usually reaches the hypolophidectolophid junction (i.e., non-taeniodont pattern). On p4s at early stages of wear (MUSM
4546, 4548, and 4553; Fig 10B, 10G and 10L), this cristid is more or less notched but remains
elevated. However, on MUSM 4550 (Fig 10P), the notch of the anterior arm of the hypoconid
is more pronounced, involving a partial confluence between the metaflexid and the retroverse
hypoflexid (i.e., pseudo-taeniodont pattern). IGM 251018 appears to have a taeniodont pattern
(Fig 10Q). The posterior arm of the hypoconid is fused with the strong and curved posterolophid, the latter extending lingually and ending directly distal to the entoconid, connecting to
its base. The hypoconid displays a long and thin anterior outgrowth.
Lower molars are brachydont-mesodont (Hling = 0.50–0.79; Hlab = 0.54–0.91; Fig 10C–
10E, 10H–10J, 10M–10O and 10R–10T). The m3s have a distal part of the talonid labiolingually pinched, and thus are more recognisable than m1s and m2s. Some lower molars appear
smaller, and could be m1s (identified as m1?s). The m3s are either roughly equal in size to the
lower molars determined as m1?s or smaller. Lower molars differ from p4s in having a wider
trigonid comparatively to the talonid. The cristids are thick and incorporate in their ends the
cuspids, which are thus faintly differentiated (except for the entoconid). The metalophulid I
can show a thin interruption (groove) either in its middle part, separating equally-sized anterior arms of the metaconid and of the protoconid (MUSM 4611), or directly lingual to the protoconid, separating a long anterior arm of the metaconid from a very short and low anterior
arm of the protoconid (MUSM 4588 and 4616; Fig 10O). On some other lower molars, both
arms are slightly connected (e.g., IGM 250303, MUSM 4573, 4597, 4599, and 4620; Fig 10C,
10R and 10T). MUSM 4586 shows an additional cristid stemming from the metalophulid I,
running distally and ending freely. The metaconid has a strong posterior arm, which can be
long (e.g., MUSM 4592) or short (e.g., MUSM 4580, 4588, and 4599; Fig 10C). This cristid connects to a distinct mesostylid on some lower molars (MUSM 4580, 4582, 4584, 4588, 4597,
4599, 4601, and 4605; Fig 10C and 10N). A neomesolophid stemming from the mesostylid can
be present (e.g., MUSM 4580, 4588, and 4605; Fig 10N) or absent (e.g., MUSM 4582, 4584,
4597, 4599, and 4601; Fig 10C). The neomesolophid is either limited to a very short enamel
spur or short cristulid. It is labially free on all specimens. Two lower molars (MUSM 4597 and
4613) exhibit a short lingual cristulid from the metaconid, recognised as a metaconid cristid,
which remains labially free. As p4s, some lower molars at an advanced stage of wear have a
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mesiolingual dentine platform, formed by the fusion of the metaconid, its arms, and possibly
the metaconid cristid and/or neomesolophid + mesostylid. On several lower molars (e.g.,
MUSM 4560, 4563, 4568, 4571, 4588, 4613, and 4622; Fig 10J), there is a small lingual expansion on the ectolophid that is likely to be a relic of the posterior arm of the protoconid. With
the reduction or absence of the neomesolophid and the posterior arm of the protoconid, the
mesoflexid is partly or entirely confluent with the anteroflexid. The ectolophid is oblique, frequently aligned with the crestiform protoconid and the hypolophid, thereby forming a long
mesiocentral (diagonal) cristid. Lingually, the entoconid is the most differentiated cuspid. It is
frequently mesiodistally elongated by developing strong anterior and posterior arms, which
are particularly long on MUSM 4582. They tend to be mesially connected with wear to the
mesostylid-posterior arm of the metaconid, and distally to the posterolophid. On lower molars
at earliest stages of wear, thin furrows usually isolate the entoconid from these other structures
mesially and distally. A short but strong hypolophid extends from the entoconid to the distal
extremity of the ectolophid. As on p4s, the hypoconid on lower molars is mesiodistally compressed and displays a thin anterior outgrowth, which becomes long with wear. The hypoconid
is distal in position to the entoconid. The anterior arm of the hypoconid can be present or
absent (e.g., IGM 250303, MUSM 4600, and 4616; Fig 10O and 10R). When it is present, it is
faintly developed and runs to the ectolophid–hypolophid junction. It is grooved (e.g., MUSM
4579, 4605, and 4620; Fig 10N and 10T) or deeply notched (e.g., MUSM 4569 and 4602; Fig
10S), showing a suite of intermediate sizes of the notch. Hence, the metaflexid is more or less
confluent with the hypoflexid (i.e., from pseudo-taeniodont pattern to taeniodont pattern).
However, many lower molars at an advanced stage of wear show a complete anterior arm of
the hypoconid, which can be thick (i.e., non-taeniodont pattern; e.g., MUSM 4581, 4606, and
4622; Fig 10H–10J). A massive and hook-shaped posterolophid strongly connects to the hypoconid, both structures being fused and in perfect continuity.
dP4s are longer than wide and with rounded corners (Fig 10U, 10Z, 10E’ and 10J’). They
have a suboval occlusal outline, with a well-arcuate anteroloph (i.e., mesially convex) and posteroloph (i.e., distally convex). All dP4s are brachydont. The cusps are usually moderately
inflated. The anteroloph is curved, short, and located far mesially. At early stages of wear, two
notchs separate the anteroloph labially from the paracone (except on MUSM 4445; Fig 10Z)
and lingually from the strong and bulbous protocone or its anterior arm. The first notch is
wide, while the second is highly variable in terms of development. It corresponds to a very
large notch on some dP4s (MUSM 4425, 4427, 4428, 4431, 4432, and 4435; Fig 10U and 10E’),
whereas, on other teeth, it is a thinner and shallower furrow (MUSM 4429, 4439, 4440, and
4443) or a tiny groove (MUSM 4430, 4437, 4442, 4445, and 4446; Fig 10Z). On MUSM 4438,
the protocone is mesiolingually-distolabially pinched, developing a short anterior arm, which
is strongly connected to the anteroloph. The posterior outgrowth of the protocone is very
short on the whole dP4s. On MUSM 4422 and 4428 (Fig 10E’), a well-cuspate parastyle is
discernable on the mesiolabial corner of the tooth. This style bears short, but strong, anterior
and posterior arms, separate by deep furrows from the anteroloph and paracone, respectively.
On MUSM 4422, a medium-sized loph (i.e., neoformation) runs lingually from the parastyle,
slightly directed distolingually, but with its lingual extremity faintly linked to the anteroloph
via a very short and thin crestule. On MUSM 4428 (Fig 10E’), this loph is present but reduced
to a short spur, whose base is connected to a peculiar anteroloph, short and labially concave.
Hence, the parastyle, its anterior arm, its spur and the anteroloph delimit a small and round
fossette. On the labial margin, the paracone and mesostyle are equally sized, and the metacone
is smaller and usually still visible. The mesostyle is separate mesially from the paracone by a
large furrow, that closes with wear. This style can be connected (MUSM 4422, 4431, and 4438)
or disconnected (MUSM 4424, 4425, 4427, 4428, 4430, 4439, and 4445; Fig 10U, 10Z and 10E’)
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to the metacone (or its anterior arm). Posteriorly, the metacone is strongly connected to the
well-curved posteroloph. On dP4s at early stages of wear, the lingual protoloph can be absent
(i.e., taeniodont pattern; e.g., MUSM 4422, 4427, and 4428; Fig 10E’) or present, either thin
and low (i.e., pseudo-taeniodont pattern; MUSM 4437, 4438, and 4445; Fig 10Z) or high (i.e.,
non-taeniodont pattern; MUSM 4425, 4431, and 4446; Fig 10U). The labial protoloph is thick
and usually straight, but it can also be slightly oblique. On IGM 250320, MUSM 4420, 4427,
4428, 4444, and 4445 (Fig 10Z, 10E’ and 10J’), it is particularly strongly oblique. On MUSM
4425, 4429, 4431, 4432, 4439, 4440, 4442 and 4446, the labial and lingual protolophs are aligned
and form a transverse crest linking the paracone to the protocone (Fig 10U). The mure is complete and high, except on MUSM 4429 in which it is low, involving a partial confluence of the
mesial mesoflexus with the hypoflexus. It is usually longitudinal (i.e., parallel to the mesiodistal
axis of the tooth), but can be more oblique, aligned with the anterior arm of the hypocone
(IGM 250320, MUSM 4420, 4421, 4422, 4427, and 4443; Fig 10J’), and sometimes with an oblique protoloph (MUSM 4420 and 4427). The third transverse crest is parallel with the labial
protoloph. It is composed of two crests: a short and thick mesoloph stemming from the mesostyle and a mesolophular spur stemming from the anterior arm of the hypocone. On some dP4s
(MUSM 4425, 4438, and 4446; Fig 10U), the third crest is complete, with the mesoloph linked
to a short mesolophule, whereas they can be unconnected or faintly connected together on
other teeth (MUSM 4420, 4427, 4428 and 4443–4445; Fig 10Z and 10E’). On IGM 250320,
MUSM 4428 and 4444 (Fig 10E’ and 10J’), the mesoloph seems to be absent or strongly
reduced. IGM 250320 appears to not have any mesolophule (Fig 10J’). Most of dP4s have a
short metaloph backwardly connected to the posteroloph via a longer posteroloph spur or not,
delimiting a small and oval metafossette. However, it can be lingually free (MUSM 4427) or
absent (MUSM 4425, 4428, and 4431; Fig 10U and 10E’). On MUSM 4442, there is a short posteroloph spur and a cusp-like structure on the distalmost flexus, which is probably a relic of
the metaloph. The latter structures are not linked. On dP4s at the earliest stages of wear, the
posteroloph displays a shallow (MUSM 4425, 4430, 4431, 4442; Fig 10U) or deep (MUSM
4427, 4428, 4446; Fig 10E’) furrow, which separates it from the hypocone.
P4s are high-crowned, especially at the level of their lingual crown, their labial crown being
lower (Hling = 0.81–0.96; Hlab = 0.45–0.79; Fig 10V, 10A’, 10F’ and 10K’). In occlusal view,
their crown is oval, labiolingually wider than longer. Mesiolingually, the anteroloph, protocone, its anterior arm and its posterior outgrowth form a long crest, which is separate from the
large and well-cuspate paracone by a thin furrow. Distally, the paracone remains free and separate from a long distal crest including the mesostyle, posteroloph, hypocone and its long anterior outgrowth. Therefore, a usually wider and persistant furrow occurs distal to the paracone.
The mesial extremity of the anterior outgrowth of the hypocone is connected to the posterior
outgrowth of the protocone, and to the whole of the aforementioned structures, thereby forming most of the peripheral margin of the tooth. There is no lingual protoloph on the whole
specimens. Accordingly, the most anterior flexus is a fossette, corresponding to the confluence
of the paraflexus with the hypoflexus (i.e., taeniodont pattern). In mostcases, the mure and the
short anterior arm of the hypocone are thin, linked, and aligned, forming a longitudinal or
oblique crest. On MUSM 4450 and 4455, these two structures are not aligned, the mure being
more longitudinal than the anterior arm of the hypocone. On MUSM 4456, the mure is not
linked to the anterior arm of the hypocone or to the posteroloph. The mure is connected to the
lingual extremity of the labial protoloph, which is almost transverse (IGM 251040, MUSM
4449, 4455, 4457, 4459, and 4466; Fig 10A’ and 10K’) or oblique (MUSM 4450–4454, 4456,
4461–4464, 4467, and 4468). The mure is absent on MUSM 4463, in which the labial protoloph
is not connected to the anterior arm of the hypocone. On MUSM 4449, a longitudinal crest
connects the anteroloph to the labial protoloph. Hence, these strutures and the paracone
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delimits a very small rounded fossette. The mesostyle is small and crestiform, poorly distinct
from the posteroloph. On MUSM 4452 and 4466 (Fig 10A’ and 10F’), it displays a very short
mesoloph lingually free, longer on MUSM 4466 (Fig 10A’) than on MUSM 4452 (Fig 10F’).
On P4s in a more advanced stage of wear (MUSM 4451, 4456, and 4468), the mesoloph is longer. On MUSM 4451, its lingual extremity is slightly connected to the mure-anterior arm of
the hypocone and to the posteroloph. One P4, MUSM 4453 (Fig 10V), do not show any mesoloph. MUSM 4466 has a very short metalophular spur labially free, as well as a tiny crestule
from the posteroloph, which could be a posteroloph spur or a metaloph (Fig 10A’). With wear,
the posteroloph often exhibits a thickening in its labial part, which may correspond to its
fusion with the mesostyle/mesoloph, metacone/metaloph and/or posteroloph spur (IGM
251040, MUSM 4450, 4455, 4458, 4459, 4463, and 4467; Fig 10K’). This plateform can be free,
joined lingually to a tiny mesolophular spur (MUSM 4450) or directly to the mure (MUSM
4455).
The different loci documenting upper molars (i.e., M1 vs M2 vs M3) are not easily recognisable (Fig 10W–10Y, 10B’–10D’, 10G’–10I’ and 10L’–10N’). Some upper molars are small,
with a rounded occlusal outline and a hypocone clearly labially positioned with respect to the
protocone, and thus they could be M3s (determined as M3?s; Fig 10Y, 10D’, 10I’ and 10N’).
Other upper molars are square, mesiodistally elongated or transverse. On these upper molars,
the hypocone is either more labially situated than the protocone or situated at the level of this
cusp (i.e., distal). As for P4s, upper molars are labiolingually asymmetrical with a higher lingual crown (Hling = 0.71–1.07; Hlab = 0.42–0.65). In general, the cusps/styles are crestiform,
and not well defined. In most cases (MUSM 4487, 4493, 4502, 4508, 4511, 4525, and 4527; Fig
10C’, 10G’, 10I’ and 10L’), the protocone shows a long and curved anterior arm, which is
slightly connected to a shorter and straight anteroloph (+ ?parastyle). These two crests can be
also strongly connected (MUSM 4489 and 4520; Fig 10B’) or separated by a narrow (MUSM
4491; Fig 10H’) or wide (MUSM 4505; Fig 10W) furrow. The anteroloph is connected to the
mesial aspect of the paracone on some M1s or M2s (MUSM 4483, 4487, 4489, 4495, and 4502;
Fig 10B’ and 10G’) and all M3?s (MUSM 4520, 4526, and 4527), whereas it remains separate
from the paracone by a narrow furrow such as on MUSM 4482, 4486, 4491, 4494, 4499, 4505,
4513, and 4514 (Fig 10W, 10X, 10H’ and 10M’). On the MUSM 4525 M3?, the paracone is
fully fused with the anteroloph (Fig 10I’). On MUSM 4511, the anteroloph bears a short but
well-marked crestule lingually free on the paraflexus (Fig 10C’). The lingual protoloph is
absent on all upper molars, thereby the paraflexus and hypoflexus are confluent (i.e., taeniodont pattern). The protocone displays a long and strong posterior outgrowth that can end
close to the hypocone (MUSM 4495, 4498, 4517, 4519, 4520, 4524, and 4527–4530; Fig 10D’
and 10N’), or can connect to this cusp with wear (MUSM 4521; Fig 10Y). On all upper molars,
the hypocone is usually smaller than the other main cusps. It displays a strong and oblique
anterior arm, which connects to the mure and tends to be aligned with it. A labial protoloph,
strong, short and slightly (e.g., MUSM 4486 and 4529) or strongly (e.g., MUSM 4483, 4489,
and 4520; Fig 10B’) oblique, links the paracone to the mure. The anterior arm of the hypocone
and the mure are in continuity with the labial protoloph, forming a long and diagonal central
crest with a strong obliquity on some upper teeth (e.g., MUSM 4481, 4485, 4489, 4493, 4520;
Fig 10B’). The labial protoloph can bear a short crestule mesially free on the paraflexus
(MUSM 4498 and 4511; Fig 10C’). Labially, a strong mesostyle is present. It is clearly distinct
on some upper molars (MUSM 4482, 4505, 4514, 4516, 4527, and 4530; Fig 10W, 10X and
10D’). It is separated from the paracone by a thin and shallow notch (e.g., MUSM 4483, 4487,
4505, and 4529; Fig 10W) or connected to this cusp (or to the posterior arm of paracone;
MUSM 4489, 4516, and 4520; Fig 10B’), thereby forming a labial wall that closes the mesial
mesoflexus. Distolabially, a metacone is either well separate from the posterior arm of the
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mesostyle by a large but shallow notch (MUSM 4505 and 4514; Fig 10W and 10X), faintly connected to the posterior arm of the mesostyle (MUSM 4482, 4487, 4508, and 4522; Fig 10L’) or
more strongly linked to it (MUSM 4516 and 4521; Fig 10Y). The upper molars show an important variation in the development, configuration and orientation of the third crest and metaloph. From the mesostyle, a strong but short mesoloph runs either (i) lingually: it is free
(MUSM 4510, 4512, 4514, 4516, 4522, and 4525; Fig 10X and 10I’) or connects to a short mesolophule (MUSM 4471, 4487, 4494, 4513, 4515, and 4520; Fig 10M’); or (ii) backwardly: it is
free (MUSM 4483 and 4499), or connects to the metaloph (MUSM 4508 and 4521; Fig 10Y
and 10L’), to the posteroloph (via a spur or not; MUSM 4481, 4489, 4491, 4501, 4504, 4511,
and 4528; Fig 10B’, 10C’ and 10H’), or seemingly to both crests (MUSM 4482). On MUSM
4521, the mesolophule is not connected to the mesoloph (Fig 10Y). On MUSM 4505, the mesoloph is slightly joined to a tiny mesolophule as well as to the metaloph (Fig 10W). Some upper
molars show a distinct metacone, but without metaloph (or almost absent; MUSM 4491; Fig
10H’). If the metaloph is present and not linked to the mesoloph and/or to the posteroloph
(via a spur or not; MUSM 4482, 4505, 4508, and 4521; Fig 10W, 10Y and 10L’), it is lingually
free (MUSM 4487, 4514, 4516, and 4522; Fig 10X). On MUSM 4502, 4529 and 4530 (Fig 10D’
and 10G’), there is neither the third crest nor the metaloph (i.e., trilophodont pattern). Most of
molars at more advanced stages of wear are trilophodont (MUSM 4470, 4476, 4481, 4484,
4492, 4495, 4496, 4498, 4518, 4519, and 4528), but have a enlarged posteroloph, which may
include a mesoloph and/or a metaloph and their associated cusps/styles (entirely subsumed
within the posteroloph). The flexi are furrow-shaped, especially the anterior flexus (i.e., paraflexus + hypoflexus).
Remark
The specimens attributed to Microscleromys cribriphilus show a great variation of size. For
example, the size range of the lower molars extends from 1.08 mm to 1.78 mm for the length,
and from 1.01 mm to 1.57 mm for the width. Likewise, the size range of upper molars is 1.17–
1.71 mm for the length and 1.07–1.66 mm for the width. Due to the sample size that is distributed quite uniformly for the minimum range and without shape variation, we consider that all
the material reported here documents a single taxon. Accordingly, we consider the smaller
specimens to document smaller individuals. Moreover, it is worth noting that the smallest
specimens are often teeth determined as m3?s and M3?s. We attribute the observed size variation between specimens partly resulting of size variation between loci.
Common comparisons
Basically, among caviomorph rodents, (i) the presence of high-crowned teeth with a taeniodont pattern and oblique loph(-id)s, (ii) the absence or reduction of second transverse cristid
on lower molars, and (iii) the absence of metaloph and third transverse crest, or the reduction
of the latter (mesoloph) on upper molars, are dental features characterising Chinchilloidea.
The specimens described here do not show any heterogeneous thickness of the enamel layer,
usually found in most advanced chinchilloid forms (including Incamys) [114, 127, 128]. There
is also no heterogeneous thickness of the enamel layer in the alleged chinchilloid Branisamys
(Salla, Bolivia, late early Oligocene–late Oligocene; [129–131], but see [4]), Saremmys [132],
and in taxa recognised as stem-Chinchilloidea (Eoincamys and Chambiramys) [4]. Furthermore, this character is only insinuated in Scleromys/“Scleromys” [127]. By having highcrowned teeth with a tendency to show oblique loph(-id)s, not forming lamina, and with flexi
(-ds) not filled with cement; p4s with a complete tetralophodont pattern or with a more trilophodont pattern; lower molars with a groove on the metalophulid I and without second
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transverse cristid (or the latter if present, being strongly reduced); upper teeth with a metaloph
reduced or absent, a third transverse crest absent or limited usually to a short mesoloph lingually free or backwardly connected to the posteroloph, the concerned dental specimens from
TAR-31 have a morphology matching that of Microscleromys nom. nud. from La Venta
(Colombia; late middle Miocene; Fig 24.2G-24.2O, p. 395 [18]; Figs 11–13 [37]). In particular,
some specimens from TAR-31 are larger and attribuable to M. paradoxalis, whereas other,
smaller, are determinable as M. cribriphilus (S1 File) [18, 37]. In addition, M. cribriphilus differs from M. paradoxalis in having some dP4s showing a non-taeniodont pattern characterised
by a complete and high lingual protoloph in continuity to the labial protoloph (this study).
Several other chinchilloid genera (Eoincamys, Chambiramys, Incamys, Saremmys, and Scleromys/“Scleromys”) [28, 70, 71, 73, 81, 114, 118, 130–134] have a similar pattern (i.e., highcrowned teeth, homogeneous thickness of the enamel layer; taeniodonty, obliquity of loph(-id)
s, no lamina and no cement). Like Microscleromys, Eoincamys, Chambiramys, Incamys and
Scleromys/“Scleromys” also show a great variation of the morphology of the second transverse
cristid on lower molars, and third transverse crest and metaloph on upper molars in terms of
presence/absence, length, orientation and connections. No lower molar from TAR-31 and La
Venta has a neomesolophid as long as or longer than on the MUSM 2875 m2 of Chambiramys
sylvaticus (Fig 5G, p. 80 [71]). Microscleromys differs from Chambiramys in being slightly
higher-crowned, and with more quadrangular upper molars; from Eoincamys, Incamys and
Scleromys/“Scleromys” in having upper molars more square-shaped and a reduction more
important of the third transverse crest on upper molars (i.e., more frequent trilophodont pattern than in these genera); from Eoincamys ameghinoi, Eoincamys pascuali and Incamys in
having a second transverse cristid usually more reduced on lower molars; from Eoincamys pascuali in having a thinner furrow (when it is present) on the metalophulid I, which can be situated in the middle of this lophid; from Saremmys in having lower-crowned teeth, thinner loph
(-id)s, a less oblique hypolophid on p4s and lower molars, and some upper molars with a distinct metacone, and long metaloph and mesoloph; from Saremmys and Scleromys/“Scleromys”
in having an anterior arm of the hypoconid, which can be present on p4s and lower molars,
and with a more curved posterolophid on lower molars; and from Incamys, Saremmys, and
Scleromys/“Scleromys” in having a smaller size. Saremmys is documented by two dp4s at early
stages of wear (MPEF-PV 11348b and 11349a in Fig 6A and 6B [132]). However, in our opinion, MPEF-PV 11348b would be a dP4 by the proportions between its mesial and distal parts,
shape of its outline margin and a pentalophodont pattern closer to an occlusal morphology of
a dP4 than dp4. Comparatively to MPEF-PV 11349a, dp4s from La Venta and TAR-31 show a
metaconid postionned more distally and strongly connected to the protoconid by a thick second cristid, which corresponds to a metaconid cristid and/or a posterior arm of the protoconid. Unlike dP4s of Microscleromys, MPEF-PV 11348b has a hypocone compressed
distolabially-mesiolingually. Moreover, MPEF-PV 11348b displays a more crestiform protocone. Microscleromys differs from Garridomys and Drytomomys in having a smaller size, an
anterior arm of the hypoconid that can be present on p4s and lower molars, and a second
transverse cristid usually more reduced, and a more curved posterolophid on lower molars;
from Garridomys in having a homogeneous thickness of the enamel layer, thinner loph(-id)s,
and strong and high protoconid and ectolophid on p4s; from “Scleromys” colombianus and
Drytomomys aequatorialis in having a separation between the hypocone and the posteroloph,
which is narrower and less persistent at early wear stages on upper molars [28, 29, 127, 135];
and from Drytomomys in having a less oblique hypolophid on lower molars. To sum up, the
material from TAR-31 is closer to Microscleromys. After revison, we validate Microscleromys
paradoxalis and M. cribriphilus described by Walton [37] from La Venta, and identify both
species at TAR-31.
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Microscleromys sp. 1
Fig 8D and 8E and S1 Table
Nom. nud. Microscleromys ?paradoxalis Walton, 1990, Fig 13E, 13D, p. 49.
Referred material
UNC unnumbered specimen, left P4 (Fig 8D); IGM-DU 85–411, left P4 (Fig 8E).
Localities
Locality unknown (for UNC unnumbered specimen) and CVP locality of the Fish Bed (for
IGM-DU 85–411), La Venta Badlands, upper Magdalena Valley, Huila Department,
Colombia.
Formation and age
Baraya Member of the Villavieja Fm.; late middle Miocene (i.e., Laventan SALMA).
Description and comparison
UNC unnumbered specimen (Fig 8D) and IGM-DU 85–411 (Fig 8E) are two P4s at advanced
states of wear. They have a morphology very similar to those of Microscleromys paradoxalis
and M. cribriphilus characterised by a circular/oval outline, a taeniodont pattern, the reduction/absence of third crest and metaloph, and a hypoflexus lingually close due to a connection
between the posterior outgrowth of the protocone and the hypocone. The latter structures, as
well as the paracone, anteroloph, protocone, posteroloph and mesostyle (and metacone if present) are fused, and form a circular peripheral wall. The paracone is distally separate from this
circular wall by a very thin furrow on UNC unnumbered specimen (Fig 8D), whereas both
structures are slightly linked together on IGM-DU 85–411 (Fig 8E). The labial protoloph and
the mure are mesiolabially oriented on IGM-DU 85–411 (Fig 8E). On UNC unnumbered specimen, the labial protoloph is transverse and the mure is longitudinal (i.e., parallel to the mesiodistal plan of the tooth; Fig 8D). On that tooth, the anterior arm of the hypocone is tiny,
oblique, and links the mure to the hypocone. From the mesostyle area runs a short crest, corresponding to a mesoloph, which remains labially free on UNC unnumbered specimen (Fig 8D).
On the two P4s, there is no apparent metaloph. In addition, IGM-DU 85–411 does not have
any distinct mesoloph (Fig 8E). However, this tooth shows a more advanced stage of wear and
a thick labial part of the posteroloph, which could encorporate a mesostyle, the metacone, and/
or the rest of a mesoloph and/or metaloph. These two teeth differ from their counterparts in
M. paradoxalis and M. cribriphilus in being clearly larger (S1 File). By their taeniodonty, complete and elevated mure, and the connection between the posterior outgrowth of the procone
and hypocone, the pattern of these P4s recalls the morphology of P4s of certain stem-caviomorphs (sensu Boivin et al. [4]; such as Cachiyacuy contamanensis, Cachiyacuy kummeli, Pozomys, Eoespina, and Tarapotomys), the chinchilloid Eoincamys parvus, and some cavioids
(Australopracta, Luantus, and Neoreomys) [6, 11, 16, 70, 81, 86, 101, 136, 137]. The material
from La Venta differs from Cachiyacuy, Pozomys, E. parvus and Tarapotomys in having a
larger size; from C. contamanensis and some P4s of Tarapotomys subandinus in having a more
reduced third crest; from C. kummeli in having an anterior arm of the hypocone and third
crest (when present) reduced to a mesoloph instead of a mesolophule; from Pozomys in having
a complete anteroloph; from Australoprocta, Luantus initialis and L. propheticus in having a
slightly larger size; from Australoprocta in having no (or more reduced) metaloph; from L.
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initialis and L. propheticus in having a curved lingual edge of the hypocone; from L. propheticus
in having a lower crown and a less quick closure of the lingual mesoflexus; from Neoreomys in
having a lower crown, thinner crests, more expanded flexi, and a mesoloph not connected to
the posteroloph. In summary, these two P4s from La Venta are clearly closer to those of Microscleromys. Their large size is not compatible with that of two species of the genus. Thus, we
attribute them to Microscleromys sp. 1.
Microscleromys sp. 2
Fig 11 and Table 1
Referred material
MUSM 4658, left astragalus.
Fig 11. Microscleromys sp. 2 from TAR-31, Peru, MUSM 4658, left astragalus. (A-C) Dorsal, (D, E) lateral, (F, G) plantar, (H, I) medial, (J, K) distal, and (L, M)
proximal views. (B, C) Stereomicroscopic views. The illustrations of the fossil specimen are the result of the fusion of numerical multi-focus images obtained with an
optical stereomicroscope (A, D, F, H, J, and L) and 3D digital models obtained by X-ray μCT surface reconstruction (B-C, E, G, I, K, and M).
https://doi.org/10.1371/journal.pone.0258455.g011
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Locality
TAR-31, Tarapoto/Juan Guerra, Mayo River, Shapaja road, San Martı́n Department, Western
Amazonia, Peru.
Formation and age
Lower member, Ipururo Fm., late middle Miocene (i.e., Laventan SALMA).
Description
MUSM 4658 from TAR-31 is a well preserved left astragalus (Fig 11). It is slightly damaged at
the level of the lateral ridge of the trochlea (= lateral tibial and lateral fibular facets) and the
ectal facet. In dorsal view, the astragalar body is slightly longer (proximodistally) than wide
(lateromedially; Fig 11A–11C). The combined length of the neck and head is shorter than that
of the trochlea. The latter is characterised by steep-sided lateral and medial margins (fibular
facet and medial tibial facet, respectively). It is moderately grooved with slightly asymmetric
internal facets. The internal lateral facet of the trochlea (= lateral tibial facet) is extensive and
slopes gently, whereas the medial one is more inclined, taller, narrower, and barely longer (Fig
11A–11C and 11J–11M). No astragalar foramen (= superior astragalar foramen sensu Cifelli
[138]) is evident. The neck is thick, short and very deflected medially (36.2˚; Fig 11A–11C).
The head is rounded and bears a navicular facet wider than long. In medial view (Fig 11H and
11I), the navicular and medial tarsal facets are slightly proximally distinct, but distally continuous. The medial tarsal facet is particularly extensive proximally on the medial aspect of the
neck (Fig 11H and 11I). In lateral and medial views, the rims of the trochlea are circular, with
a moderate radius of curvature (Fig 11D, 11E, 11H and 11I). In medial view, seven large
foramina are recognisable (Fig 11H and 11I). Three foramina are present distally to the area
along which the tendons of m. peroneus longus and brevis slide and ventroproximally situated
to the most distalmost rim of the medial ridge of the trochlea. They are ventroproximally
aligned, the smallest foramen being the most proximal, whereas the largest is in the middle.
There are three other foramina on the dorsodistal part of the area along which the tendons of
m. peroneus longus and brevis slide, which are dorsoproximally aligned. The last foramen is
located proximally to the area along which the tendons of m. peroneus longus and brevis slide.
In plantar view (Fig 11F and 11G), the ectal (= calcaneo-astragalar) facet is oval and faintly
curved (very slightly concave; i.e., high radius of curvature). Its distal part is not markedly projected laterally (i.e., absence of a protruding lateral process). It is clearly separated from the sustentacular facet by a deep and wide sulcus calcaneus, which is oblique (proximomediallydistolaterally) and divergent distally. As the proximolateral side of the sustentacular facet is
gently sloping, the sulcus appears particularly wide. Three main foramina are situated on the
sulcus, the largest being the most proximal, close to the medial plantar tuberosity. It might correspond to the plantar astragalar foramen (= inferior astragalar foramen sensu Cifelli [138]).
Another foramen is present at the base of the proximolateral slope of the sustentacular facet.
This tuberosity is well marked. The sustentacular facet is triangular-shaped and with a major
axis proximodistally oriented. It is well extended distally toward the head, and reaches the
navicular facet. The distal extremity of the sustentacular facet is wide and concave, whereas its
proximal one is narrower and convex. The sustentacular and ectal facets have similar dimensions (i.e., length and width). The navicular and medial tarsal facets are poorly distinct over
their entire contact. In distal view (Fig 11J and 11K), two large foramina are present on the distal extremity of the lateral ridge of the trochlea.
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Comparisons
The trochlea of MUSM 4658 is shorter than in Euryzygomatomys, narrower than in Steiromys,
Neoepiblema and Coendou, and less grooved than in Cavia, Dolichotis and Proechimys. It has a
lateral ridge narrower and less distally positioned than Chinchilla and Dactylomys, and wider
(but somewhat sloping) than the medial one, a feature observed on astragali of several other
taxa (Platypittamys, Drytomomys, Phoberomys, Abalosia, Actenomys, Dolicavia, Eucelophorus,
Eumysops, Pithanotomys, Praectenomys, Dactylomys, Euryzygomatomys, Lagostomus, Proechimys, and Octodon) and particularly accentuated on astragali of Steiromys, Neoepiblema and
Coendou. The neck of MUSM 4658 is well differentiated from the body, and is strongly
deflected medially as on astragali of Steiromys, Drytomomys, Neoepiblema, Phoberomys, Abalosia, Eumysops, Coendou, Dactylomys, Euryzygomatomys, Myocastor and Proechimys. Particularly, the deflection angle of MUSM 4658 is similar to that of astragali of Abalosia, Eumysops,
Myocastor and Proechimys (35–37˚). Conversely, astragali of Platypittamys, Neoreomys, “Scleromys”, Actenomys, Dolicavia, Eucelophorus, Pithanotomys, Praectenomys, Cavia, Chinchilla,
Clidomys, Ctenomys, Dinomys, Dolichotis, Galea, Lagostomus and Octodon have their neck
being clearly less or not deflected medially. On MUSM 4658, the head is rounded with the
medial tarsal facet strongly developed on the medial aspect of the neck in dorsal view as on
astragali of Steiromys, Drytomomys, Actenomys, Eucelophorus, Eumysops, Pithanotomys, Coendou, Dactylomys, Octodon, and Proechimys. MUSM 4658 displays an ectal facet narrower than
that of astragali of Neoepiblema, Actenomys and Eumysops; shorter than in Pithanotomys; narrower and longer than in Coendou. Contrary to the astragali of Neoreomys, “Scleromys” colombianus, Drytomomys, Neoepiblema, Eumysops, Pithanotomys, Cavia, Chinchilla, Dinomys,
Dolichotis, Galea, Myocastor, Octodon and Proechimys cuvieri, there is no well marked lateral
process of the ectal facet on MUSM 4658. The ectal facet is less concave than in Steiromys,
Neoepiblema, Pithanotomys, Cavia porcellus, Dolichotis and Galea. Several taxa (Steiromys,
“Scleromys”, Drytomomys, Neoepiblema, Phoberomys, Abalosia, Actenomys, Dolicavia, Eucelophorus, Eumysops, Pithanotomys, Cavia, Chinchilla, Dactylomys, Dinomys, Dolichotis, Euryzygomatomys, Galea, Lagostomus, Myocastor, Octodon, and Proechimys) have a wide sulcus
calcaneus distally divergent like on MUSM 4658, but none seem to have a similar configuration of this sulcus and the proximolateral side of the sustentacular facet, which is gently sloping. In addition, MUSM 4658 differs from astragali of all aforementioned taxa in having a
particular triangular-shaped sustentacular facet; from Steiromys, Neoepiblema, Coendou,
Lagostomus, and Myocastor in having a sustentacular facet oriented proximodistally; from
Dolicavia, Cavia, Chinchilla, Dolichotis, Euryzygomatomys, Galea and Myocastor in having a
sustentacular facet not strongly fused with the navicular facet and/or the medial tarsal facet of
the neck; and from Steiromys, Abalosia, Eumysops, Pithanotomys and Dactylomys in having no
clear separation between the sustentacular facet and the navicular and medial tarsal facets. At
first order, the MUSM 4658 astragalus matches very small body-sized extant and fossil rodents.
Regression of the body mass in function of trochlear width in living mammals [139] shows
that MUSM 4658 belonged to a small rodent having a body mass of about 43 g (Table 2). The
smallest body-sized species from TAR-31, Ricardomys longidens, Microscleromys paradoxalis
and M. cribriphilus, could be the best candidates. Considering the least-squares regression
equation of [140] based on the m1 area in mammals, the body mass of R. longidens is estimated
to be ~40 g, that of M. paradoxalis to be ~51 g and that of M. cribriphilus to be ~21 g (Table 2).
The values estimated with equation proposed by the same author for rodents are clearly higher
(~132, ~161, and ~76 g; Table 2), which makes it difficult to conclude. Given that the dental
material from TAR-31 referred to M. cribriphilus is more abundant than that of R. longidens
and M. paradoxalis, referral of MUSM 4658 to M. cribriphilus could be a plausible taxonomic
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Table 2. Metric feature of the MUSM 4658 astragalus (mm) and m1s (mm2) from TAR-31 and La Venta and body mass estimates (g).
Material
Trochlear Width
(mm)
MUSM 4658
1.832
Mean area m1
(mm2)
Dental locus used N
Body mass estimates
(g)
Regression equation
for:
Reference
1
43.00
mammals
Tsubamoto,
2014
243.26
mammals
Legendre, 1986
614.50
rodents
89.98
mammals
262.28
rodents
40.49
mammals
132.40
rodents
Microsteiromys
jacobsi
7.51
m1
1
Nuyuyomys
chinqaska
4.36
m1
6
Ricardomys longidens
2.82
m1?
2
Microscleromys cribriphilus
Microscleromys paradoxalis
“Scleromys” sp.
�
1.97
3.19
14.67
m1?
m1�
m
34 21.15
2
2
mammals
75.94
rodents
50.90
mammals
161.06
rodents
826.65
mammals
1751.02
rodents
based on MUSM 4643 from TAR-31 and IGM unnumbered specimen from La Venta.
https://doi.org/10.1371/journal.pone.0258455.t002
option. Nevertheless, as the size ranges of two co-occuring species of Microscleromys are overlapping, we prefer to keep it in open nomenclature as Microscleromys sp. 2.
“Scleromys” Ameghino, 1887
“Scleromys” sp.
Fig 12 and S1 Table
Referred material
MUSM 4277, left dp4; MUSM 4278, 4279, right fragmentary dp4s; MUSM 4280, left p4;
MUSM 4281, 4282, right lower molars; MUSM 4270–4272, left fragmentary upper molars;
MUSM 4273, 4274, left upper molars; MUSM 4275, 4276 right upper molars.
Locality
TAR-31, Tarapoto/Juan Guerra, Mayo River, Shapaja road, San Martı́n Department, Western
Amazonia, Peru.
Formation and age
Lower member, Ipururo Fm., late middle Miocene (i.e., Laventan SALMA).
Description
MUSM 4278 is a distal fragment of dp4 preserving only the posterolophid, the hypoconid, its
anterior outgrowth, and the distal margin of the ectolophid, hypolophid, and of the entoconid.
MUSM 4279 is a mesial fragment of dp4, broken at the level of the ectolophids, the hypolophid
and the entoconid (Fig 12C). This tooth also shows several damaged areas, especially at the
level of the protoconid and the distal part of the metalophulid I. MUSM 4277 (Fig 12A) is a
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Fig 12. “Scleromys” sp. indet. from TAR-31, Peru. (A) MUSM 4277, left dp4. (B) MUSM 4281, right lower molar (reversed). (C) MUSM 4279,
fragment of right dp4 (reversed). (D) MUSM 4282, right lower molar (reversed). (E–G) MUSM 4280, left p4. (H) MUSM 4273, left upper molar. (I)
MUSM 4275, right upper molar (reversed). (J) MUSM 4272, left upper molar. (K–O) MUSM 4276, right upper molar (reversed). (A-E, H-K) Occlusal,
(F, N) labial, (G, L) lingual, (M) anterior, and (O) posterior views. The illustrations of the fossil specimens are 3D digital models obtained by X-ray μCT
surface reconstruction.
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complete dp4 at a more advanced wear stage than MUSM 4278 and 4279. These three brachydont dp4s are characterised by thick lophids. MUSM 4277 is pentalophodont, probably like
MUSM 4279. Despite its degree of wear, MUSM 4279 seemingly displays bulbous lingual cuspids. On this tooth, the metalophulid I is mesially convex and shows a mesiodistal thickening
at its centre. The metaconid and protoconid are distally linked by a second cristid, mesially
concave, corresponding to a posterior arm of the protoconid and/or a metaconid cristid. The
metalophulid I, the second cristid, the metaconid and protoconid delimit a C-shaped anterofossettid. The mesostylid is large and displays a short and strong neomesolophid mesiolabially
directed and lingually free. The mesostylid is well separated from the metaconid and entoconid. This cuspid is higher than the mesostylid and metaconid. The hypolophid, distal ectolophid and mesial ectolophid are fused, and form a long cristid stemming from the entoconid
and directed toward the protoconid, but without connection to it. This cristid is mesiolabially
oriented, but its labial part is more longitudinal than its lingual part. The labial extremity of
the neomesolophid is close to the mesial margin of the cristid combining the hypolophid and
ectolophids. MUSM 4277 (Fig 12A) shows a similar pattern to that of MUSM 4279 (Fig 12C),
but with features deriving from a more advanced stage of wear: loss of relief, lingual closure of
the mesial mesoflexid and metaflexid, lingual and labial closure of the distal mesoflexid, and
reduction of the anterofossettid and of the mesial mesofossettid to two small and circular fossettids. MUSM 4279 displays a heterogeneous thickness of the enamel layer, which is thicker
on the trailing edges and thinner on the leading edges. On MUSM 4277 and 4278, the distalmost cristid (posterolophid + hypoconid + its anterior outgrowth) is curved and there is no
anterior arm of the hypoconid, thereby involving the confluence of the metaflexid with the
hypoflexid (i.e., taeniodont pattern).
The MUSM 4280 p4 (Fig 12E) is slightly damaged at the level of the protoconid, the hypoconid and its anterior outgrowth. MUSM 4280 is hypsodont (Hling = 1.13; Hlab = 1.52; Fig
12F and 12G). Compared to dp4s, MUSM 4280 is tetralophodont, more square-shaped, and
with a trigonid clearly narrower than the talonid. The cuspids are crestiform and faintly differentiated from the lophids. On the lingual margin, the metaconid and mesostylid are equally
sized and separated by the anterostriid. The metaconid is connected to a metalophulid I,
which is very slightly concave and labially free. This crest corresponds to an anterior arm of
the metaconid, the anterior arm of the protoconid being absent. Its labial extremity is thin and
separated from the protoconid by a narrow and deep furrow, which forms a striid. The second
transverse cristid is reduced to a short neomesolophid stemming from the mesostylid. The
neomesolophid is mesiolabially directed and labially free. The protoconid is fused with the
ectolophid, forming a cristid distolingually oriented. The hypolophid is parallel to the neomesolophid and in continuity of the cristid composed of the protoconid + ectolophid (but the latter being more longitudinal). The hypoconid has no anterior arm (i.e., taeniodont pattern)
and it is not distinct from its anterior outgrowth and the posterolophid. The main direction of
the posterolophid is the same that those of the neomesolophid and hypolophid. The flexids are
furrow-shaped and form striids. On the labial margin, the hypostriid is deep, but ends far from
the neck of the crow. On the lingual margin, the anterostriid and mesostriid have a similar
depth, which represents more than the double that of the short metastriid.
As for p4, the lower molars, MUSM 4281 and 4282, are high crowned (but retaining roots),
tetralophodont and taeniodont, with deep furrow-shaped flexids, forming striids, dental structures that fused with wear, forming thick and oblique cristids, and a second transverse cristid
reduced to a neomesolophid (Fig 12B and 12D). Contrary to the MUSM 4280 p4, their trigonid and talonid are equally wide. They present a more advanced stage of wear with respect to
MUSM 4280: their crown is lower (Hling = 0.82–0.83; Hlab = 0.82–1.19) and their lingual flexids are closed except the metaflexid on MUSM 4281. The metalophulid I is complete on both
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molars. On MUSM 4281 (Fig 12B), the neomesolophid is very short and labially free. Consequently, the Y-shaped anterior fossettid corresponds to the confluence between the anteroflexid with the mesoflexid. Conversely, on MUSM 4282 (Fig 12D), the neomesolophid is
longer and labially connected to the metalophulid I, thereby delimiting a small and circular
anterofossettid. Like on MUSM 4280, the hypolophid, ectolophid and protoconid are in continuity, forming a central cristid. The latter is straight and oblique (i.e., mesiolabially directed)
on MUSM 4281. On MUSM 4282, its labial part is mesiolabially directed, while the hypolophid
is more transverse. Due to the closure of the anteroflexid and mesoflexus on MUSM 4281 and
4282, the trigonid is delimited by a triangular-shaped structure formed by the metalophulid I,
metaconid, mesostylid, entoconid, hypolophid, ectolophid and protoconid. This triangularshaped structure is separate from the distalmost cristid by a very thin furrow-shaped flexid
(i.e., confluence of the metaflexid with the hypoflexid). The distalmost cristid, formed by the
fusion of the posterolophid, hypoconid and its anterior outgrouwth, is mesially concave. The
thickness of the enamel layer is homogenous on MUSM 4282 (Fig 12D). MUSM 4281 displays
a homogenous thickness of the enamel layer at the level of the metaflexid but a heterogeneous
one (thicker on the trailing edges and thinner on the leading edges) at the level of its mesial
and distal margins and those of the hypoflexid (Fig 12B). The hypostriid has a height similar to
that of the MUSM 4280 p4.
Upper molars have an occlusal pattern similar to that characterising p4 and lower molars
(i.e., tetralophodonty, taeniodonty, deep furrow-shaped flexi, forming stria, and dental structures merging with wear, forming thick and oblique crests), which can contribute to confuse
them. They show a unilateral hypsodonty characterised by a lingual crown higher that the
labial one (Hling = 1.00–1.69; Hlab = 0.51–0.92), whereas the distolabial crown is the highest
part of crown on p4 and lower molars. Upper molars are labiolingually wider than lower
molars. Upper molars have three roots: a large lingual one and two small labial ones. The
mesial margin of the crown is formed by a curved crest, which corresponds to the fusion of the
anteroloph, protocone and its posterior outgrowth. On all complete upper molars, this crest is
labially connected to the paracone. The labial protoloph is roughly transverse or slightly oblique and in continuity with the mure and anterior arm of the hypocone, which are more distolingually directed. The mesostyle is faintly isolated from the metacone on MUSM 4273 (Fig
12H). On the other upper molars, it is slightly (MUSM 4270 and 4272; Fig 12J) or strongly
(MUSM 4274–4276; Fig 12I and 12K) connected to the this cusp and to the paracone. As for
the second transverse cristid on p4 and lower molars, the third transverse crest is reduced to a
mesoloph stemming from the mesostyle, either lingually free (MUSM 4270 and 4272) or connected to the posteroloph (MUSM 4273 and 4275; Fig 12H and 12I). On MUSM 4272 (Fig
12J), the posteroloph presents, on its mesial margin, a very short crestule (i.e., posteroloph
spur), mesially free. On MUSM 4273 (Fig 12H) and 4275 (Fig 12I), the mesoloph and posteroloph (+ posteroloph spur?) delimit a small and circular fossette. On upper molars at a more
advanced stage of wear (MUSM 4274 and 4276; Fig 12K), the small posterior fossette disappears due to the fusion between the mesoloph, mesostyle and posteroloph (+ posteroloph
spur?). On MUSM 4270 and 4272 (Fig 12J), a tiny notch separtes the hypocone from the posteroloph. On the other upper molars, which show a more advanced stage of wear, the hypocone is strongly linked to the posteroloph. On the most worn upper molar, MUSM 4274, the
two remaining flexi (the mesialmost) are fully closed and divided into two parts. Most upper
molars (MUSM 4270, 4272 and 4274–4276; Fig 12J, 12I and 12K) display a homogenous thickness of the enamel layer, while MUSM 4273 (Fig 12H) has a slight heterogeneous thickness of
the enamel layer (thicker on the trailing edges and thinner on the leading edges) at the level of
its mesial and distal margins, and the mesial and distal margins of the anterior flexus.
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Comparisons
These large dental specimens from TAR-31 described here show typical chinchilloid affinities,
notably characters documenting Scleromys/“Scleromys”: (i) medium-sized and protohypsodont
teeth, (ii) taeniodonty, (iii) dental structures merging with wear, forming thick and oblique
crests/cristids, (iv) deep furrow-shaped flexi(-ds), forming striia/striids, (v) upper molars tetralophodont at early stages of wear, becoming trilophodont at more advanced stages of wear, (vi)
a metaflexid as being the most persistent lingual flexid on lower teeth, and (vii) an enamel
layer with a thickness homogeneous or slightly heterogeneous (i.e., insinuated heterogeneous
thickness sensu Kramarz, Vucetich & Arnal [127]; see also [128]). The dental remains from
TAR-31 are similar in size to those from La Venta (Colombia; late middle Miocene) attributed
to either “Scleromys” schurmanni Stehlin, 1940 [18, 27, 28] or “Scleromys” cf. schurmanni [18].
By its short and labially-free metalophulid I and neomesolophid, the pattern of the MUSM
4280 p4 is particularly close to that of the p4 borne on the IGM 184233 specimen from La
Venta attributed to “Scleromys” cf. schurmanni (Fig 24.3E, p. 397 [18]). However, it differs
from the latter in having no mesiolingual protuberance of crown, and in showing separated
metaconid and mesostylid. As for the material from TAR-31, the lower molars of “Scleromys”
cf. schurmanni from La Venta can have a neomesolophid labially free or connected to the
metalophulid I. The figured p4 of “Scleromys” schurmanni, also from La Venta (UCMP 38967
in Fig 24.3D, p. 397 [18]), has a complete second transverse cristid composed of a neomesolophid and a shorter posterior arm of the protoconid, contrary to MUSM 4280. The p4s of
“Scleromys” schurmanni described by Field [28] show a complete second transverse cristid as
well (UCMP 38987 in Figs 4A and 5D, p. 285 [28]) or a second cristid reduced to a neomesolophid linked to the metalophulid I (UCMP 37931 in Figs 4D and 5C, p. 285 [28]). The dp4s of
“S”. schurmanni (UCMP 39902 and 38974 in Fig 5A and 5B, p. 285 [28]) seem to be hexalophodont, having more anterior flexids than on dp4s from TAR-31. The TAR-31 teeth are
smaller than those of S. quadrangulatus (Pinturas Fm., Argentina; late early Miocene) [133], S.
angustus (Santa Cruz Fm., Argentina; late early Miocene) [73] and S. osbornianus (Pinturas
and Santa Cruz Fm.; late early Miocene) [118, 133]. Comparatively to S. quadrangulatus, teeth
from TAR-31 have a slender closure of the flexi(-ds), a longer metalophulid I on p4, and a
paracone area smaller on upper molars at early stages of wear. The MUSM 4280 p4 has a lingual margin more parallel to the mesiodistal axis of the tooth than p4s of S. quadrangulatus
and S. angustus. In addition, MUSM 4280 has a neomesolophid, contrary to those species of
Scleromys. At early stages of wear, molars of S. angustus and S. osbornianus display a trilophodont pattern [73, 133], contrary to molars from TAR-31, which remain tetralophodont. The
dp4s from TAR-31 share a similar pattern in terms of number, types and morphology of the
main cristids with the two dp4s from Madre de Dios, Peru: dp4 referred to S. quadrangulatus
(MD-61, early Miocene) [141] and dp4 referred to “Scleromys” sp., gr. quadrangulatus-schurmanni-colombianus (MD-67, early middle Miocene) [31]. Nevertheless, they have a metalophulid I connected to the metaconid and protoconid, contrary to dp4s from MD-61 and -67.
Unlike dp4s from TAR-31, dp4s from MD-61 and -67 have supernumerary structures on the
anteroflexid: a cuspid-shaped structure on the dp4 from MD-67, and a low cristulid connected
to the lingual and labial extremity of the metalophulid I on the dp4 from MD-61. The dp4s
from TAR-31 display more opened lingual flexids than the dp4 from MD-61. They show other
distinctive characters with respect to the dp4 from MD-67: a metaconid isolated from the
mesostylid, and a second cristid and a hypolophid-ectolophid cristid, which are more curved.
“Scleromys” colombianus and Drytomomys aequatorialis, both from La Venta (and from
Nabón region, Ecuador, for D. aequatorialis; late middle Miocene) [28, 29, 135] are recognisable in having larger-sized teeth and a separation between the hypocone and the posteroloph,
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wider and more persistent at early stages of wear on upper molars, than on the TAR-31 material. The material from TAR-31 differs from “S.” colombianus [18, 28] and “Scleromys” cf.
colombianus [18] in having p4s with a transverse mesial margin and a shorter neomesolophid
labially free, and upper molars with a more persistent posterior fossette. It differs from Drytomomys in having a quicker reduction of the anterofossettid on lower molars; from D. aequatorialis in having shorter neomesolophid on p4s; from D. typicus (Ituzaingo Fm., Argentina;
late Miocene) [29, 106] in having different morphology of dp4s with a third cristid reduced to
a short neomesolophid and without neolophid on the anterofossettid. Comparatively to
MUSM 4280, “Drytomomys” sp. (small) and “Drytomomys” sp. (large) from La Venta (“Olenopsis” sp. sensu Walton [18], but see [29]) show a longer neomesolophid labially connected to
the metalophulid I on p4s. Moreover, p4s of “Drytomomys” sp. (large) are pentalophodont
with a short metaconid cristid, contrary to MUSM 4280. The moderate size, marked hypsodonty and thickness of the crests of these teeth from TAR-31 exclude affinities with Eoincamys
(Brazil and Peru; ?late Eocene–early Oligocene) [8, 19, 70, 81], Microscleromys (La Venta,
TAR-31; this study) [18] and with the octodontoid Protadelphomys (Sarmiento Fm., Argentina; early Miocene) [142–144]. They are also too high-crowned and slightly larger for Maquiamys (CTA-61, Peru; late Oligocene) [4, 71]. To sum up, the material from TAR-31 has the
closest affinities with the specimens of “Scleromys” found in northern South America (La
Venta, Colombia and MD-61 and MD-67, Peru). As it would be necessary to deeply revise
Scleromys and notably the northern forms (recognized as “Scleromys” [18, 71]), we prefer keeping the material from TAR-31 in open nomenclature as “Scleromys” sp.
Discussion
The caviomorph fauna from La Venta: Revision and validation of Walton’s
nomina nuda
Microsteiromys jacobsi, Ricardomys longidens, Microscleromys paradoxalis and Microscleromys
cribriphilus, originally described in La Venta in the framework of the Anne H. Walton’s Ph.D
[37], have remained nomina nuda given that they were not formally described so far. According to the International Code of Zoological Nomenclature (p. 246 [38]), a nomen nudum is not
available but the same name can be made available subsequently for the same concept or for a
different concept, in a way meeting the criteria of availability (chapter 4 [38]). Our revision of
the original material of these species, combined with the study of the new specimens from
TAR-31, allowed us to validate all the concerned taxa. It is worth noting that we have usually
not followed the unpublished diagnoses proposed by Walton [37]. In the case of Microscleromys, a classical descriptive and comparative approach combined with statistical analyses on
dental dimensions (length and width; S1 File) allowed us to test the taxonomic groups proposed by Walton [18, 37]. Our results (i.e., statistical differences) suggest an inappropriate taxonomic delimitation of these potential groups (S1 File). Thus, we propose a new delimitation
of the two Microscleromys species implying changes in the assignation of some specimens (i.e.,
from M. cribriphilus to M. paradoxalis, and vice versa). In addition, we revised the suprageneric status of this taxon, which was previously assigned to the cavioid family Dasyproctidae [18,
37]. Like Microscleromys, other fossil taxa such as Eoincamys, Incamys and Scleromys, were
previously attributed to the dasyproctids [81, 130, 131]. However, Eoincamys, Incamys and
Scleromys have then been related to the superfamily Chinchilloidea [70, 114, 133]. As noted by
Walton [18], Microscleromys shows an occlusal pattern similar to Scleromys, as well as to
chinchilloids in general (i.e., high-crowned teeth with a taeniodont pattern and oblique loph
[-id]s, the absence of the second transverse cristid or its reduction on lower molars, and the
absence of the metaloph and third transverse crest, or the reduction of the latter to notably a
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mesoloph on upper molars). According to the recent phylogenetic analyses of Boivin et al. [4],
the phylogenetic position of Microscleromys, but also those of Eoincamys, Incamys and Scleromys were retrieved as stem representatives of Chinchilloidea (but see [127, 145, 146]). Finally,
as noted by Walton [18, 37], Ricardomys shows an occlusal morphology similar to Adelphomys
and other adelphomyine octodontoids. Here, we assign it to the family Adelphomyidae, following the results of Boivin et al. [4].
The caviomorph assemblage from TAR-31
In being restricted to a single 5–20 cm-thick channelised lens in a very small area (~5 square
metres in one piece), TAR-31 records a single attritional accumulation [36], which strongly
contrasts with La Venta and Fitzcarrald local faunas (several levels from distinct outcrops
spanning a long or short interval, respectively) [26, 33]. Nevertheless, TAR-31 yields a total of
nine rodent taxa, with the co-occurrence of the four extant superfamilies: Erethizontoidea
(three taxa), Cavioidea (one taxon), Octodontoidea (two taxa) and Chinchilloidea (three taxa).
Compared with other Laventan caviomorph assemblages from low latitudes of South America,
TAR-31 ranks second just in front to the fauna from Fitzcarrald Arch (eight taxa) [32, 33], but
far from La Venta, which includes 28 taxa (21 excluding the indeterminate genera and specie;
La Victoria and Villavieja formations, Colombia; see [18, 28] and this work). Like TAR-31, the
faunas from La Venta and Fitzcarrald Arch, as well as that from MD-67 (Madre de Dios; similar outcrop conditions as in TAR-31) [31], show representatives of the four caviomorph superfamilies. Without considering the validation of the nomina nuda from La Venta, TAR-31
records a new erethizontoid (Nuyuyomys chinqaska gen. et sp. nov.) and perhaps a new stemcaviid, here described as Caviidae gen. et sp. indet. (see Systematic Palaeontology section). The
single specimen assigned to this taxon does not allow for its formal description as a new species. TAR-31 shares two taxa or closely related taxa with MD-67 (“Scleromys” sp. and Nuyuyomys chinqaska) and one with the assemblages from Fitzcarrald local fauna and La Victoria
Fm. of La Venta (“Scleromys”). TAR-31 shares with the Villavieja Fm. of La Venta a representative of “Scleromys”, as well as three species only recorded in La Venta so far: Microscleromys
paradoxalis, M. cribriphilus and Ricardomys longidens. In striking contrast, there is no taxon in
common between TAR-31 and most middle Miocene assemblages at mid- and high latitudes
in South America (Argentina, Bolivia; Chile) [10, 30, 51, 87, 105, 123, 126, 147–152]. The only
assemblage showing similarities with TAR-31 is that from the Marino Fm. (Divisadero Largo
area, Mendoza, Argentina) [153], in yielding Scleromys sp. Nevertheless, Scleromys (and “Scleromys”) had a wide geographic (Argentina, Brazil, Chile, Colombia, and Peru) and a long stratigraphical range (late Oligocene–late middle Miocene) [15–19, 27, 28, 31, 33, 34, 73, 133, 141,
154]. Moreover, some doubts were expressed about the cogeneric afinities between Scleromys
spp. from northern versus southern South America [18, 71].
Astragalus of Microscleromys from TAR-31: Morpho-functional
implications
The trochlea of MUSM 4658 does not have a mediolateral widening nor a high degree of asymmetry between its medial and lateral rims, thereby suggesting that parasagittal movements
would be more favoured during plantar- or dorsi- flexion of the foot [57, 61, 155]. The trochlea
is however moderately grooved with shallower and less sharp crests than those of astragali of
cursorial-jumping taxa in which lateral movements are strongly restricted (cavioids: Cavia,
Dasyprocta, Dolichotis, Galea) [61]. The ectal facet is long with a high radius of curvature and
not projected laterally, which would give proximodistal and mediolateral mobility with the calcaneus (astragalo-calcaneal joint = sub-astragalar joint) [57]. The narrowing and the
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proximodistal orientation of the sustentacular facet (here long reaching the navicular facet)
would indicate parasagittal movements at the astragalo-calcaneal joint, limiting the inversion
of the foot [57, 61]. In addition, the sulcus calcaneus is particularly wide, thereby suggesting
the presence of a strong ligament linked to the dorsal side of the calcaneus [57]. This character
is found in numerous caviomorphs, but associated with a proximodistal sustentacular facet, it
would confer more resistance of the ankle by limiting the general mobility of the foot with
respect to the leg [156, 157]. The neck is long and medially deflected with a medial tarsal facet
on its dorsal and medial aspects, and a round head, characters typically found in arboreal taxa
such as Coendou or Sciurus, but also in some terrestrial echimyids (Proechimys) [57, 61]. In
addition to the increase of the articular surface of the sustentacular facet with the calcaneus
[57], these featurs would therefore be facilitated lateral and rotator movements of the foot at
the astragalo-navicular joint and at the contact between the astragalus and the medial tarsal
bone (= first sesamoid) [61, 155]. Moreover, the extension of the medial tarsal facet on the
neck would permit to stabilize the astragalus during inversion of the foot [57, 155, 158]. In
summary, MUSM 4658 shows an intermediary morphology between morphotypes of the cursorial-jumping and climbing locomotory groups recognised by Ginot et al. [57]. Some of its
characters (i.e., ectal facet slightly concave and not projected laterally, neck medially deflected,
round head, medial tarsal facet extended on the dorsal and medial aspects of the neck) would
enhance mediolateral movements particularly at the level of the distal joints with the navicular
and medial tarsal bone, whereas others (i.e., moderately groveed and slightly asymmetric
trochlea with no mediolateral widening, narrowing and the proximodistal orientation of the
sustentacular facet, wide sulcus calcaneus) would tend to favor parasagittal movements especially of the astragalo-tibial joint (= cruro-astragalar joint). Such an astragalar morphology
matches that of terrestrial generalists with a possible higher trend toward arboreal locomotion.
Age of TAR-31 and biostratigraphical implications
The caviomorph fauna from TAR-31 shares four taxa with the assemblage characterising the
lower part of the Villavieja Fm. at La Venta (see above). Similar affinities are observed on their
non-rodent mammalian content (e.g., Miocochilius anomopodus, Neosaimiri fieldsi and Megadolodus molariformis; Fig 29.5, p. 508 [26]; see also [36] and the section Material and methods
of this paper). An age ranging between 13.18–12.47 Ma can therefore be hypothesised for
TAR-31, corresponding with the timespan of the magnetostratigraphically-constrained lower
part of the Villavieja Fm. (C5AAn–C5AR.1R chrons) [26, 49].
The MUSM 4283 p4 of Caviidae gen. et sp. indet. from TAR-31 is particular in having more
primitive characters (i.e., protohypsodonty, presence of flexids/fossettids, and a hypoflexid not
reaching the lingual margin of the tooth) than the caviids found in other caviomorph faunas at
low latitudes of South America from Laventan deposits such as Prodolichotis pridiana (La Venta
and Fitzcarrald Arch), Dolichotinae gen. 2 large and Dolichotinae gen. 2 small (La Venta), and
Guiomys sp. (MD-67) [18, 31, 33] or from sligthly younger deposits (Mayoan SALMA; CTA-44
Top, CTA-43) [34]. Similarly, it differs from middle Miocene caviids from southern South
America (Cardiomys? andinus, Cardiomys? buemulensis, Eocardia robusta, Eocardia robertoi,
Guiomys unica, Microcardiodon huemulensis and Microcardiodon williensis) [51, 87, 105]; in
having of flexids/fossettids, and a hypoflexid, which does not reach the lingual margin of the
tooth, characters otherwise found only in early Miocene stem-caviids such as Chubutomys or
Luantus [52, 101, 103]. Thus, the discovery of a tooth with such characters suggests that this
kind of morphotype may have subsisted until the late middle Miocene, at least in some representatives of the superfamily at low latitudes of the continent. Occurring in both MD-67 and
TAR-31 localities, Nuyuyomys chinqaska likely spans the entire middle Miocene interval.
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Palaeobiogeographical implications
The Miocene epoch is marked by important environmental changes, particularly in low-latitude regions of South America [159]. During the latest Oligocene–early Miocene interval,
Andean deformations are generalised, in particular by the Eastern Cordillera of the Northern
and Central Andes (beginning of the “Quechua phase” as defined by Steinmann [160]; [161–
165]). However, the Eastern Cordillera of Northern Andes had not reached its full elevation at
that time, with a major uplift episode during late Miocene–Pliocene times [166–168]. In relation with the uplift of this region, the Solimões Basin in Brazil is occupied by swamps and shallow lakes [169–173]. These bodies of water belonged to a vast wet system (mega-wetland),
which also covered the Andean foreland retroarc basins in Peru and Colombia, and had a
north connection with the Caribbean Sea [174]. This system reaches its maximal extension
from ~16.0 to 11.3 Ma and forms the Pebas System, which corresponds to a dynamic mosaic
of swampy, lake, fluvial and fluvio-marine environments [174, 175].
With our results, the geographical ranges of Ricardomys longidens, Microscleromys paradoxalis and M. cribriphilus are now extended southerly to Peruvian Amazonia for the Laventan
interval. More generally, strong taxonomic affinities between the vertebrate assemblages of La
Venta (Colombia) and Fitzcarrald, Madre de Dios (MD-67), Iquitos, Contamana, and TAR-31
in Peruvian Amazonia support the existence of a single “Pebasian” biome throughout the middle–earliest late Miocene, and more specifically during the Laventan SALMA, as shown by
their terrestrial and aquatic components (crocodylomorphs, chelonians, marsupials, rodents,
primates, notoungulates, astrapotheres, litopterns, sirenians, and/or cetaceans; [21, 26, 31–34,
36, 176–178] and see this work). These moderate or strong faunal similarities suggest that the
Eastern Cordillera of the North Andes and Pebas System would have not constituted barriers
for mammalian dispersals during this interval at least for a region close to the Andes (Western
Corridor) [36, 44].
Conclusions
The TAR-31 locality in Tarapoto area, Peru, documents nine caviomorph taxa encompassing
all four living superfamilies. This assemblage encompasses: Microsteiromys jacobsi gen. et sp.
nov., Nuyuyomys chinqaska gen. et sp. nov., Erethizontoidea gen. et sp. indet. 1 and 2 (Erethizontoidea), Caviidae gen. et sp. indet. (Cavioidea), Ricardomys longidens gen. et sp. nov. and
Octodontoidea gen. et sp. indet. (Octodontoidea), plus Microscleromys paradoxalis gen. et sp.
nov., M. cribriphilus gen. et sp. nov., and “Scleromys” sp. (Chinchilloidea). The study of an
astragalus suggests that at least a representative of Microscleromys was a terrestrial generalist
rodent.
Although very restricted in terms of area (5 square meters) and volume investigated (~550
kg wet-screened) with respect to other assemblages, this locality yields the most diverse middle
Miocene caviomorph fauna recorded in Western Amazonia. In addition, it is the second most
diverse for this time window in low-latitude regions of South America, well after La Venta.
The geographical range of Ricardomys longidens, Microscleromys paradoxalis and M. cribriphilus is now expanded 1,100 km to the south, during the Laventan SALMA, as a further testimony of the extent of the Pebasian biome at those times.
Supporting information
S1 File. Exploring the potential species number of Microscleromys from TAR-31 and La
Venta.
(DOCX)
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S2 File. Data and corresponding script(s) for each dental locus used for statistical analyses.
(ZIP)
S1 Table. Dental measurements (in millimeters) of rodent material from TAR-31 (Tarapoto, Peru; late middle Miocene) and La Venta (Colombia; late middle Miocene). The previous assignation of the material from La Venta are specified (Walton, 1990, 1997). � indicates
the specimens selected by Walton (1990) as holotypes, ��� indicates the specimens selected in
this study as holotypes.
(XLSX)
S2 Table. Extinct and extant caviomorph taxa used for dental comparisons in this study.
For the references, see the manuscript. Fm., Formation; INGEMMET, Instituto Geológico
Minero y Metalúrgico, Lima; MACN, Museo Argentino de Ciencias Naturales, Buenos Aires;
MLP, Museo de Ciencias Naturales de La Plata; MMP, Museo Municipal De Ciencias Naturales Lorenzo Scaglia, Mar del Plata; MNHN-Bol, Museo Nacional de Historia Natural, La Paz,
Bolivia; MNHN, Musée National d’Histoire Naturelle, Paris; MUSM, Museo de Historia Natural de la Universidad Nacional Mayor San Marcos, Lima; UM, Université de Montpellier.
(XLSX)
Acknowledgments
Many thanks to M. Roddaz (GET, Toulouse, France), M.A. Custódio and R. Ventura (GEOCRON/IG, Brası́lia, Brazil) and whoever helped us in the field and in the lab. We are particularly grateful to our drivers Giancarlo and Manuel, for their long standing help during the
yearly field seasons. We warmly thank M.R. Ruiz-Monachesi (IBIGEO, Salta, Argentina) for
discussing on statistics, his relevant suggestions and aid with respect to the Bayesian models
and the osteological preparation of the Galea. We are much indebted to M.C. Madozzo-Jaén
(Lillo, San Miguel de Tucumán, Argentina) and to M.E. Pérez (MEF, Trelew, Argentina) for
discussing on phylogeny and homology of dental structures in cavioids. Many thanks to A.
Álvarez and M.D. Ercoli (INECOA, San Salvador de Jujuy, Argentina) for providing us photos
of collection material for dental comparisons. Christine Argot (MNHN, Paris, France), M.
Reguero (MLP, La Plata, Argentina), L. Chornogubsky and A.G. Kramarz (MACN, Buenos
Aires, Argentina), and M. Taglioretti (MMP, Mar del Plata, Argentina) kindly granted access
to the collections under their care. We are much indebted to M. Gómez Pérez, the team of
Museo Geológico Nacional—Servicio Geológico Colombiano and J.D. Carrillo (MNHN) for
the correspondance between the field numbers and the collection numbers for the material
from La Venta. We warmly thank B. Rondeau and L. Lena (LPG, Nantes, France) for access to
a Keyence Digital Microscope facility; C. Cazeveille (Institut des Neurosciences de Montpellier
[INM], France) for access to a electron microscope scanning facility; and A.-L. Charruault and
R. Lebrun (ISEM, Montpellier, France) for μCT scan acquisitions and treatments. We thank
the MRI and the LabEx CeMEB for access to the electron microscope and μCT scanning facilities (ISEM, Montpellier). We are indebted to A.-L. Charruault (ISEM, Montpellier, France) for
the cast preparation of the rodent material from TAR-31. We gratefully thank the E. Beucler
and O. Bourgeois (LPG, Nantes, France) in order to have allowed MB realizing a journey in
Montpellier. This is ISEM publication 2021-225-Sud.
Author Contributions
Conceptualization: Myriam Boivin, Laurent Marivaux, Pierre-Olivier Antoine.
Formal analysis: Myriam Boivin.
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Funding acquisition: Myriam Boivin, Laurent Marivaux, François Pujos, Pierre-Olivier
Antoine.
Investigation: Myriam Boivin, Laurent Marivaux, Walter Aguirre-Diaz, Aldo Benites-Palomino, Guillaume Billet, François Pujos, Rodolfo Salas-Gismondi, Narla S. Stutz, Julia V.
Tejada-Lara, Rafael M. Varas-Malca, Anne H. Walton, Pierre-Olivier Antoine.
Methodology: Myriam Boivin.
Resources: Laurent Marivaux, Pierre-Olivier Antoine.
Visualization: Myriam Boivin, Laurent Marivaux, Narla S. Stutz.
Writing – original draft: Myriam Boivin.
Writing – review & editing: Myriam Boivin, Laurent Marivaux, Guillaume Billet, Narla S.
Stutz, Julia V. Tejada-Lara, Anne H. Walton, Pierre-Olivier Antoine.
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W4393570792.txt | Análise de Canais do YouTube como Objetos de
Aprendizagem de Suporte ao Estudo de Cálculo I em Cursos
da Área de Ciências Exatas
Analyzing YouTube Channels as Objects of Learning to Support the
Study of Calculus I in Exact Sciences Courses
O
Isabela Aparecida DIVINO1*
Mariele Rodrigues MOREIRA1
EL
Daniel Alfonso SIERRA1
Rodrigo Naoto SHIRAISHI1
Victoria Alejandra Salazar HERRERA2
1
Universidade Estadual de Campinas, Campinas - SP, BRASIL
CECS -Universidade Federal do ABC, Santo André – SP, BRASIL
*
i265342@dac.unicamp.br
PR
2
Resumo. A videoaula deixou de ser apenas uma coadjuvante da aprendizagem, e tem sido cada
vez mais agregada à rotina de estudos de alunos em diferentes níveis educacionais. O uso de
videoaulas do YouTube como ferramenta complementar é cada vez mais crescente,
independente da área de estudo. Entretanto, é escassa a literatura em relação à eficácia dos
modelos de ensino utilizados nestas videoaulas. Tendo em vista a dificuldade por parte dos
alunos na aprendizagem em matemática, o objetivo deste trabalho foi realizar o levantamento e
análise dos principais canais de Matemática do YouTube assistidos por alunos dos cursos da área
de Ciências Exatas da Universidade Virtual do Estado de São Paulo como complementaridade aos
estudos. Para isso, aplicaram-se os doze princípios da aprendizagem multimídia formulada por
Richard Mayer: Coerência, Sinalização, Redundância, Contiguidade espacial, Contiguidade
temporal, Segmentação, Pré-treinamento, Modalidade, Multimídia, Personalização, Voz e
Imagem. Foram coletadas informações dos canais acessados pelos estudantes por meio de um
formulário e selecionados os cinco canais com maior número de visualizações na plataforma
YouTube. Após a análise constatou-se que boa parte dos princípios está presente na didática dos
professores que dirigem os canais, sendo os que mais apareceram: redundância, contiguidade
temporal, modalidade, personalização, voz e imagem. Ainda assim, detalhes relacionados à
questões visuais e de acessibilidade na reprodução dos vídeos podem ser melhorados.
Palavras-chave: Objetos de aprendizagem. Tecnologias da informação e comunicação. Ensino
superior. YouTube.
EL
O
Abstract. Video lessons are no longer just an adjunct to learning, they have been increasingly
added to the study routine of students at different educational levels. The use of YouTube video
lessons as a complementary tool is increasingly growing, regardless of the area of study.
However, there is little literature regarding the effectiveness of the teaching models used in
these videos. Considering the difficulties of students in learning mathematics, the objective of
this work is to carry out a survey and analysis of the main Mathematics channels on YouTube
watched by students, as complementarity to studies, from courses in the STEAM area of the
Virtual University of the State of São Paulo. To achieve such goal the twelve principles of
multimedia learning formulated by Richard Mayer were applied: Coherence, Signaling,
Redundancy, Spatial Contiguity, Temporal Contiguity, Segmentation, Pre-training, Modality,
Multimedia, Personalization, Voice, and Image. Information was collected from the channels
accessed by students using forms and the five channels with the highest number of views on the
YouTube platform were selected. After the analysis, it was found that a most of the principles are
present in teaching strategies of the teachers who manage the channels, highlighting
redundancy, temporal contiguity, modality, personalization, voice and image. Still, details related
to visual issues and accessibility when playing videos can be improved.
PR
Keywords: Learning objects. Information and communication technologies. University. teaching,
YouTube.
1. Introdução
A internet tem aumentado de forma exponencial sua influência no dia a dia das pessoas em
todos os âmbitos, no Brasil o número de usuários chegou a representar 81% da população em
2020 (FOLHA, 2021) se constituindo numa ferramenta de democratização do conhecimento e da
informação. Em todos os planos de ensino, formais ou não formais, se usa este canal de
comunicação, seja para organizar as plataformas de estudo ou para pesquisas aprofundadas em
tópicos específicos. A metodologia tradicional de estudos foi eficiente numa era onde o acesso à
informação era restrito. Atualmente, com uma sociedade conectada o tempo todo, as pessoas
buscam aprendizado online em sites e aplicativos sociais. Essa mudança progressiva faz com que
as escolas tenham que se adaptar, buscando estratégias de ensino baseadas em materiais de
aprendizagem digitais (SOUZA e MORALES, 2015).
EaD em Foco,2024, 14(1): e2090
|2
O
Algumas ferramentas de aprendizagem virtual vêm sendo utilizadas para enriquecer o ensino.
Entre elas podem ser citados os objetos de aprendizagem. A IEEE Standard for Learning Object
Metadata (2020) define Objetos de Aprendizagem (OAs) como: “qualquer entidade digital ou não
digital que possa ser usada, reutilizada ou referenciada durante o uso de tecnologias que
suportam o ensino”. Como mencionado por Coradini (2009), os objetos de aprendizagem fazem
referência a conteúdo multimídia, conteúdos instrucionais, software em geral entre outros. Do
mesmo modo, o site Younder (2021) aponta os objetos de aprendizagem como recursos
educacionais utilizados para potencializar a transmissão dos conteúdos, citando como exemplos
podcasts, vídeos, fóruns online, infográficos e e-books. Neste universo de OAs, o vídeo foi
escolhido pelo fato de juntar o meio visual, texto, animação e áudio e ser uma ferramenta que as
pessoas utilizam com frequência. Dessa forma é importante citar a plataforma YouTube, que é o
maior site de compartilhamento de vídeos do mundo, e entre outros usos, é utilizado para
difundir informação educativa. Além de livre acesso mundial, as pessoas conseguem interagir,
dar feedbacks e curtidas (CURRAN et al., 2020).
PR
EL
Existem diversos canais de YouTube, podendo ser caracterizados como repositórios de OAs, que
oferecem um conteúdo atraente, rigoroso, embasado cientificamente e que auxilia nos planos de
estudo. Exemplos conhecidos como o canal alemão “Kurzgesagt – In a Nutshell” com tradução
para o inglês e o português, oferecem vídeos com os mais diversos temas como Astrologia,
História ou Matemática. No Brasil podemos ressaltar os canais associados a ScienceBlogs Brasil
(2021) como BlaBlalogia, Minuto da Física e Marcelo Gleiser, os quais são revisados por
profissionais especialistas que verificam a rigorosidade científica do seu conteúdo e dão o selo de
aprovação. Levando estas definições em consideração, é possível concluir que vídeos de
YouTube, usados como suporte nos processos de ensino-aprendizado, são Objetos de
Aprendizagem digital e, portanto, é possível analisar sua qualidade usando a teoria da
aprendizagem multimídia.
A aprendizagem multimídia resume-se em uma representação mental construída pelo aluno, a
partir da junção de falas e imagens. Alguns exemplos dessa abordagem incluem gráficos, vídeos,
mapas e animações. Um recurso multimídia não trabalha apenas conteúdos determinados como
por exemplo livros e computadores, mas sim abrange diversas características como palavras
dentro de textos escritos ou falados, além de informações gráficas como figuras, fotos,
animações, gráficos e mapas. Nesse sentido, Mayer (1999) acredita que o meio não tem relação
direta com a aprendizagem, então a questão a ser resolvida passa a ser como desenvolver
recursos multimídia para aperfeiçoar os conteúdos e mensagens abordados. Para isso, o
professor divide dois tipos de abordagem para produção dos recursos multimídia: a centrada na
tecnologia e a centrada nos alunos/aprendizes. A primeira abordagem possibilita o acesso a
novas tecnologias, assim é necessário que os alunos/aprendizes se adaptem a elas, a segunda
abordagem é o contrário, são as novas tecnologias que devem se adaptar às necessidades das
pessoas facilitando o entendimento do conteúdo (MIRANDA, 2009).
EaD em Foco,2024, 14(1): e2090
|3
Para que a aprendizagem multimídia aconteça de fato é necessário que alguns processos
aconteçam: seleção de palavras para o trabalho verbal sobre o assunto; seleção de imagens
relevantes para o trabalho visual; organizar as palavras de forma que mantenham coerência
dentro do modelo mental verbal; organizar as imagens de forma mantenham coerência dentro
do modelo mental visual; e juntar todas as informações de forma que tenha harmonia entre as
representações verbais visuais e conhecimento prévio (MAYER et al., 2004).
O
Para Mayer (2009) a cognição humana pode ser organizada em: Pressuposto do canal duplo:
existem dois sistemas não-equivalentes de processamento de informação verbal/auditivo e
visual/pictórico; pressuposto da capacidade cognitiva: a quantidade de informação processada
simultaneamente em cada canal é limitada: pessoas aprendem melhor com a utilização de
palavras e imagens do que somente com palavras (MAYER,2001). Esta teoria se aplica em
recursos de aprendizagem multimídia como vídeos, animações e jogos.
EL
Existem diversos trabalhos de pesquisa que avaliam objetos de aprendizagem aplicando os
princípios da Teoria de Aprendizagem Multimídia formulada por Mayer (2009). Silva e Montané
(2017) avaliam e aprimoram dois Objetos de Aprendizagem da temática Modelos Atômicos
consultando a professores de ciências exatas e aplicando os princípios de Mayer (2009). Por
outra parte, Almeida et al. (2014) analisam Objetos de Aprendizagem da temática Sistema
Digestivo incluindo vídeos de YouTube e materiais audiovisuais do Banco Internacional de
Objetivos Educacionais (objetoseducacionais2.mec.gov.br) entre outras fontes.
PR
O Youtube não é uma ferramenta desenvolvida para fins educacionais e pedagógicos, apesar de
ser atualmente usado também para divulgar aulas. E caso o educador escolha este repositório
para suas práticas educacionais, é importante que acompanhe e aprove o conteúdo a ser
estudado por seus alunos na plataforma, para que de fato, a aprendizagem seja relevante.
Segundo Cortez (2010), a recomendação é que o professor assista os vídeos com antecedência e
proponha a partir dele algum exercício, para que o vídeo não substitua o ensino em aula, mas
sim, complemente. Mesmo com essa recomendação, a literatura atual ainda é escassa com
relação à avaliação dos canais de Youtube voltados à aprendizagem, bem como as metodologias
necessárias para se realizar essa avaliação. É necessário que se hajam estudos nesse sentido, a
fim de que se padronize meios para dizer sobre a qualidade do vídeo, e por fim a recomendação
de utilizá-lo ou não em complementaridade ao conteúdo lecionado em sala de aula.
Estrategicamente, o professor pode recomendar o Youtube para fins de aprendizagem,
sobretudo em conteúdos em que os alunos apresentam mais dificuldade. Segundo o Programa
Internacional de avaliação de estudantes (PISA), matemática e ciências são as disciplinas com
pior desempenho entre os estudantes. No Brasil, apenas 27% dos estudantes alcançaram em
2022 o nível 2 de proficiência, que é o patamar mínimo considerado, enquanto em outros países,
a média é de 69% dos estudantes atingindo esse nível. (Pimentel, 2023). São inúmeros os canais
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no Youtube que trazem a proposta de ensino em matemática, mas ainda não se sabe sobre a
preferência dos alunos e a qualidade do conteúdo disponível.
EL
O
Diante disso este trabalho tem como objetivo principal a análise de vídeos em YouTube da
temática Cálculo I (Cálculo Diferencial e Cálculo Integral) segundo a ementa do plano de Estudos
dos cursos de exatas da Universidade Virtual do Estado de São Paulo (UNIVESP), aplicando os
princípios da teoria de Aprendizagem Multimídia formulada por Mayer (2009). Para atingir este
objetivo foi realizado um levantamento dos canais utilizados pelos alunos em
complementaridade a aprendizagem de Cálculo, e posteriormente, a análise desses canais. O
levantamento foi feito usando um formulário on-line para consultar tutores e estudantes sobre
seus canais preferidos e mais usados e após esta contribuição foram escolhidos os canais com
maior quantidade de subscrições. A análise feita seguiu a teoria de Aprendizagem Multimídia de
Mayer (2009), observando vídeos da plataforma YouTube sobre os conteúdos da disciplina
Cálculo I, com o intuito de revisar sua qualidade e capacidade de complementar o aprendizado
de Cálculo por estudantes de ensino superior. Este estudo é relevante vista a ausência de estudos
que validem desde o ponto de vista pedagógico a qualidade de OAs desse tipo sendo que são os
mais comuns entre os estudantes.
1.1. Princípios de Aprendizagem Multimídia de Mayer
Nesta seção serão descritos os princípios de aprendizagem multimídia (MAYER, 1999 e MAYER,
2014) utilizados neste trabalho para analisar os canais de YouTube que contém vídeos
relacionados com Cálculo I. Eles estão separados de acordo com objetivos específicos.
PR
Inicialmente apresentam-se os princípios que visam reduzir o processamento desnecessário
(evitar sobrecarga cognitiva), eles são: Coerência, Sinalização, Redundância, Contiguidade
espacial e Contiguidade temporal.
Coerência: se aprende melhor quando informações desnecessárias (palavras, figuras, símbolos,
sons etc.) são excluídas, é importante que a apresentação seja o mais simples possível.
Sinalização: se aprende melhor quando a organização do conteúdo é explicada, para que o
aprendiz possa se focar no que é essencial, favorecendo a organização mental. Um exemplo
desse princípio são vídeos que apresentem um menu de conteúdos e minutos na legenda. Este
princípio também é válido para destacar elementos durante a apresentação do material como
setas, cores destaque, grifos, letras maiores etc.
Redundância: se aprende melhor com imagens e narração, mas não com imagens, narração e
legenda (do que está sendo narrado), pois isto ocasiona sobrecarga do canal visual
Contiguidade espacial: se aprende melhor quando as palavras (informação verbal) e imagens
(informação visual) correspondentes estão espacialmente próximas, gastando menos recursos
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cognitivos para estabelecer estas conexões sem necessidade de buscá-las na tela. Aqui é
importante esclarecer que que as imagens não devem incluir palavras descritivas (MAYER, 2014)
Contiguidade temporal: se aprende melhor quando palavras (informação verbal) e imagens
(informação visual) correspondentes aparecem ao mesmo tempo, gastando menos recursos
cognitivos estabelecendo estas conexões
Existem também, princípios que visam favorecer a administração do processamento essencial (a
representação do material na memória) em que a sobrecarga diminui os recursos cognitivos para
realizar o processamento gerador (organização e integração das representações mentais
produzidas), eles são: Segmentação, Pré-treinamento e Modalidade.
O
Segmentação: se aprende melhor quando o recurso é apresentado em partes, nas quais o
usuário pode definir o ritmo (cada um tem um tempo diferente de processamento). Por exemplo,
dividir o conteúdo que será apresentado em partes dentro do próprio vídeo, ou em vários
vídeos. Desta maneira o conteúdo é processado em um ritmo adequado, diminuindo a
sobrecarga cognitiva e permitindo maior controle sobre o conteúdo (MAYER, 2014).
EL
Pré-treinamento: se aprende melhor quando já se sabe os nomes e as características dos
principais conceitos antes de entrar em mais detalhes. Aqui seria interessante observar se a
videoaula parte do básico para o avançado, ou se já parte do avançado, mas deixa alguma
sugestão como: “clique no card no topo do vídeo (ou no link da legenda) e assista um vídeo
sobre X antes de assistir a esta aula.”
PR
Modalidade: se aprende melhor com imagens e textos falados do que com figuras e textos
escritos (competem no canal visual).
Outros princípios visam promover o princípio gerador (organização e integração das
representações mentais produzidas), eles são: Multimídia, Personalização, Voz e Imagem.
Multimídia: se aprende melhor com palavras e imagens do que só com palavras.
Personalização: o estilo formal de conversação não favorece a aprendizagem, portanto, se
aprende melhor quando as palavras estão em estilo informal, sendo recomendado o uso de
comentários em primeira e segunda pessoa (MAYER, 2014).
Voz: se aprende melhor quando a voz da narração é humana do que quando a voz é de máquina.
A imagem de quem fala pode trazer um sentido de presença social, mas não necessariamente se
aprende melhor quando a imagem de quem está falando está na tela, pois dependendo da
quantidade de informações fornecidas com a imagem pode favorecer sobrecarga mental.
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2. Metodologia
Este trabalho é uma pesquisa exploratória, pois utilizou um questionário para levantamento de
canais utilizados por estudantes e após esse levantamento foi aplicada uma análise de canais
com abordagem qualitativa e mediante o método indutivo baseadas nos princípios de Mayer. O
público-alvo escolhido foram os estudantes de cálculo por ser uma disciplina considerada como
complexa pelos estudantes de vários cursos, e sendo que o contexto é numa instituição que
trabalha só na modalidade EaD as videoaulas estão todas disponíveis no YouTube.
EL
O
O questionário contou com 14 questões e foi disponibilizado por 21 dias. A escolha dos canais de
Cálculo Matemático I (Cálculo Diferencial e Cálculo Integral) da plataforma YouTube foi feita a
partir da indicação dos alunos dos cursos da área de Ciências Exatas que estão cursando ou já
cursaram a disciplina de Cálculo Matemático I na UNIVESP através de um formulário de pesquisa
que foi dividido em três partes: 1- Termo de Consentimento Livre e Esclarecido; 2- Questões de
rastreamento do perfil socioeconômico dos participantes para que possamos entender um
pouco sobre os motivos que os levam a acessar estes conteúdos; 3- Questões que irão mostrar
quais canais eles utilizam e por qual motivo fazem uso deste objeto de aprendizagem. Caso o
participante tenha interesse ele poderá deixar o contato de e-mail ao final do questionário para
que possamos enviar o artigo ao final da pesquisa.
PR
Da coleta de dados foram selecionados cinco canais de Cálculo Matemático da Plataforma
YouTube dentre os citados pelos alunos no formulário mostrado no. O critério de escolha foi a
quantidade de visualizações de cada canal. Foram assistidas as cinco últimas videoaulas
postadas. Os canais foram analisados pela apresentação de seus conteúdos, se estavam de forma
a facilitar a aprendizagem ou gerar sobrecarga mental - dificultando a aprendizagem - a partir
dos 12 princípios da aprendizagem multimídia de MAYER (2009). A qualidade do conteúdo
apresentado não foi levada em consideração, mas sim como ele foi transmitido e em como foi
feita a produção midiática do material.
3. Resultados e Discussão
Os últimos dados levantados pela UNIVESP (2021), mostram que há um total de 48.3131 alunos
matriculados nas diferentes graduações ofertadas, e que 50% desses alunos são mulheres. Têm
faixa etária entre 17 - 80 anos e a grande maioria (72%) vieram de escola pública. Em
complemento, em (UNIVESP, 2020) verifica-se que 79% dos alunos são responsáveis ou
contribuem com a renda familiar e 85% recebem entre 2 a 4 salários mínimos, a “grande maioria
dos alunos já atua no mercado de trabalho e compõe a renda da família, que optam pelo EaD por
ter um tempo reduzido para dedicação aos estudos” (UNIVESP, 2020). Nos cursos atingidos por
nosso questionário (ciência de dados, Licenciatura em Matemática e Engenharia da
computação), há um total de 15.656 estudantes, e, apesar de nossa amostra corresponder a uma
pequena parte desses estudantes, conseguimos atingir pessoas de 20 a 67 anos de idade, das
quais a maioria trabalha e não tem mais de 4 horas diárias para estudar.
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Como um dos objetivos deste trabalho era coletar um conjunto de canais que fossem acessados
pelos alunos para poder analisá-los mediante os princípios de Mayer, a coleta de informações via
formulário não focou na quantidade de respondentes, e sim na quantidade de canais sugeridos.
Obteve-se um total de 27 respostas ao formulário, sendo todos alunos da UNIVESP. Apesar de ser
uma amostra pequena, serão detalhadas algumas características sociocomportamentais,
lembrando que o critério da escolha dos canais foi a maior quantidade de subscrições e
visualizações dos canais mencionados nas respostas ao questionário, isto é o questionário foi
aplicado no intuito de saber quais canais são comumente acessados pelos alunos, mas a escolha
dos canais foi um critério externo às respostas dos respondentes do questionário.
O
Dos respondentes, 18 foram do sexo masculino e 9 do sexo feminino, com a faixa etária de 20 a
67 anos, pertencentes aos cursos de: Ciências de dados, Licenciatura em Matemática e
Engenharia da Computação, apesar de ter sido disponibilizado para todos os cursos da área de
exatas da universidade. Todos os cursos descritos possuem a disciplina de Cálculo.
EL
Segundo análises do perfil socioeconômico dos entrevistados, 23 dos 27 estudantes possuem
ocupação remunerada, enquanto 4 dedicam-se exclusivamente à UNIVESP. O que corrobora com
estudos recentes onde 48% dos universitários possuem jornada dupla, sendo 50% destes com
mais de 8 horas de dedicação no trabalho, precisando enfrentar desgaste físico, mental e
acúmulo excessivo de tarefas diariamente. A renda mensal encontra-se acima de 1,5
salário-mínimo per capita familiar para 85% dos estudantes, o que representa cerca de
R$1818,00 reais, enquanto 15% possuem menos de 1,5 salário mínimo de renda per capita
familiar por mês. Esse último dado é semelhante à amostra geral apresentada em (UNIVESP,
2020) onde 85% dos alunos têm uma renda entre 2 e 4 salários mínimos.
PR
Com relação ao tempo de dedicação aos estudos, 11 relataram que estudam até 2 horas por dia,
11 relataram que estudam mais de 2 horas por dia, enquanto 5 relataram que não conseguem
estudar diariamente. O tempo diário utilizando internet para atividades acadêmicas foi relatado
em 11% dos estudantes sendo acima de 5 horas por dia, os outros 89% usam até 4 horas diárias.
Sobre a utilização de aulas do YouTube como apoio às aulas institucionais, 15 estudantes
relataram utilizar destes recursos frequentemente, representando mais da metade dos
entrevistados. Ao serem questionados sobre os motivos que os levaram a buscar outros recursos
educacionais para aprenderem a disciplina de Cálculo I, 29,6% responderam que as aulas no
ambiente virtual não eram didáticas, 44,5% responderam que as aulas eram didáticas, porém
não eram suficientes para compreensão, já 18,5% justificaram o uso para reforçar o aprendizado.
Houve ainda justificativa com relação à duração das aulas institucionais, mais longas, segundo
um entrevistado, enquanto as aulas encontradas no YouTube eram sucintas e de fácil
compreensão. Além do YouTube, recursos como livros, artigos e programas interativos também
são utilizados para estudo.
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Os canais utilizados pelos alunos como recurso de apoio aos estudos em Cálculo I encontram-se
listados no Quadro 1, sendo 17 no total. Os canais com maior número de inscritos e de acessos,
bem como seus vídeos mais recentes, foram selecionados para análise, são eles: Ferreto
matemática, Me salva!, Matemática Rio, Equaciona e Marcos Aba.
A partir da escolha dos canais mais visualizados, foi realizada uma análise baseada nos 12
princípios de Mayer (2009) nos quatro últimos vídeos postados nos canais. No Quadro 2,
apresentam-se as informações recolhidas, as quais se dispõem da seguinte maneira: quando o
canal cumpre o tópico, é colocado “sim” e quando não cumpre, coloca-se “não”.
Quadro 1: Canais do YouTube utilizados pelos estudantes como apoio nos estudos para a disciplina de
Cálculo I.
Professor Douglas Maioli
Acesse aqui
O
Canal do YouTube
professordouglasmaioli
Professor Murakami – Matemática rapidola rapidola
Ferreto Matemática
professorferretto
equacionamatematica
EL
Equaciona com Paulo Pereira
respondeaí
Me salva!
mesalva
Canal Univesp
univesptv
Matemática para todos
marciorafaelbarros
Omatematico.com
mmatematicogrings
Matemática - Ester Velasquez
matemateca
Help engenharia com Denis Rodrigues
helpengenharia
Matemática Rio com Rafael Procopio
matematicario
Marcos Aba
marcosaba
Matemática da Tamires
matematicadatamires
Universo narrado
universonarrado
Cláudio Possani
claudiopossani
PR
Responde aí
Dicasdemat Sandro Curió
dicasdemat
Fonte: Elaborado pelos autores (2022)
Quadro 2: Canais do YouTube escolhidos e analisados segundo os tópicos da Teoria de aprendizagem e
multimídia de Mayer (2009).
Ferreto
Matemática
Me
salva!
Matemática com
Rafael Procopio
Equaciona com
Paulo Pereira
Marcos
Aba
Coerência
Sim
Não
Sim
Sim
Sim
Sinalização
Sim
Sim
Não
Sim
Não
Redundância
Sim
Sim
Sim
Sim
Sim
Critérios
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Não
Não
Não
Sim
Sim
Contiguidade
temporal
Sim
Sim
Sim
Sim
Sim
Segmentação
Sim
Sim
Não
Sim
Não
Pré treinamento
Sim
Não
Sim
Não
Sim
Modalidade
Sim
Sim
Sim
Sim
Sim
Multimídia
Não
Sim
Não
Não
Sim
Personalização
Sim
Sim
Sim
Sim
Sim
Voz
Sim
Sim
Sim
Sim
Sim
Imagem
Sim
Sim
O
Contiguidade
espacial
Sim
Sim
Sim
Fonte: Elaborado pelos autores (2022)
Uma descrição mais detalhada de cada canal é apresentada nas seguintes subseções.
EL
3.1. Ferreto Matemática
O canal Ferreto matemática possui quase 3 milhões de inscritos e milhares de visualizações em
vídeos. Algo interessante no canal, é a presença de aulas ao vivo. Observamos que existem
inúmeras aulas temáticas fracionadas, de maneira que um conteúdo todo, por exemplo de razão,
fica disposto em diversos vídeos, o que ganha muitos pontos no tópico segmentação. Voz e
personalização também são pontos fortes, bem como coerência e redundância.
PR
Outro aspecto muito atrativo são vídeos de revisão para ENEM, com resolução de problemas.
São, no geral, os vídeos mais assistidos. O canal cumpre com a maioria dos princípios, porém
poderia melhorar em quesitos de apresentação de figuras, o que faz com que não seja cumprida
a contiguidade espacial juntamente com a multimídia: o professor sempre mostra lousas e
cartazes na tela, mas há baixa presença de figuras propriamente ditas, por exemplo, ao explicar
fração, poder-se-ia utilizar uma figura e fracioná-la, ao invés de somente apresentar uma imagem
com texto escrito.
3.2. Me salva!
O canal “Me salva” é constituído por vários professores e aborda inúmeras disciplinas, mas para
elaboração dessa tabela, foram vistos somente os conteúdos de matemática. As aulas são
sempre de revisão para ENEM, resumos ou resolução de problemas. Utilizam métodos de
“escrita à mão”, algo parecido com o caderno, onde o vídeo se baseia no professor filmando a
própria mão num quadro branco resolvendo os problemas utilizando-se muitas setas, rabiscos,
esquemas, coisas que ensinam bem sem poluir a imagem. A fala é sempre informal, mas, muito
compreensível, humana e o conteúdo é interessante. O que podemos observar de ponto
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negativo, é principalmente a extensão dos vídeos: mais de uma hora, outras quase 2 horas. Isso
porque os professores fazem coletâneas de aulas, abordando muitos conteúdos num vídeo só. E
mesmo vídeos que enfatizam apenas um material, acabam sendo bem extensos, e não há
legendas de minutos, o que dificulta e torna ainda mais maçante a visualização. Segundo Mayer
(2014) “quanto mais simples e objetiva for a apresentação do conteúdo, mais livre ficará a
memória operacional para processar um número maior de conhecimento”.
EL
3.3. Matemática com Rafael Procópio
O
A contiguidade espacial também acaba sendo insuficiente neste canal. Recursos de sinalização
são bem utilizados, porém, figuras e imagens não estão presentes. Obviamente, em disciplinas
de cálculo, a numeração acaba por ser mais presente, mas a utilização de imagens e figuras,
vinculada a palavras, é mais eficiente, o que se pode ver no item multimídia. Além disso, como as
aulas são filmagens de resolução de problemas num papel, há um certo momento em que ocorre
uma “poluição visual”, pois na folha utilizada, espalham-se números e setas para todos os lados,
às vezes rabiscos e rasuras, o que acaba deixando confuso.
PR
O canal do Rafael Procópio apresenta os conceitos de uma forma limpa e organizada. Levando
em conta o princípio da coerência, ele desenvolve exercícios mostrando o passo a passo numa
lousa tirando as informações que não aportam na solução do problema e sendo simples e direto.
Pode-se classificar os vídeos em dois grupos: 1) vídeos curtos (de 2 a 5 minutos) apresentando
dicas de cálculos simples e curiosidades e 2) vídeos maiores (10 a 40 minutos) com conteúdo
formal e completo. Nota-se que não há inclusão de divisão dos temas em barra de reprodução
ou tempo sendo insuficiente em sinalização, em compensação mostra no começo do vídeo o
desenvolvimento da aula e a duração.
As palavras e as imagens estão espacialmente próximas, quando se fala de um mesmo conceito.
Quando apresenta uma imagem fala dela e não deixa um texto explicativo na tela (Contiguidade
Espacial e Temporal). A narração é muito clara com um tom de voz calmo, mas não monótono,
além de ser informal, mas ao mesmo tempo, sem perder rigorosidade. As aulas sempre vão do
básico ao avançado, seguindo o critério de Pré-treinamento. Baseados nos critérios de Meyer,
podemos sugerir algumas melhorias: Indicar na barra de tempo à divisão dos temas do vídeo
(sinalização e segmentação), usar software gráfico que aprimore os traços a mão alçada, linhas
retas (Contiguidade Espacial) e melhorar a qualidade das imagens, levando em conta que
imagens já feitas em programas de design ficam mais claras que feitas a mão durante a aula.
(Multimídia).
3.4. Equaciona com Paulo Pereira
Canal de um professor e mestre em Matemática, oferece videoaulas curtas e descontraídas.
Dentre os princípios da aprendizagem multimídia que o canal apresenta estão: coerência - a
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apresentação dos conteúdos é bastante simples, somente a equação sendo resolvida na tela;
sinalização - como o canal se dedica a resolução de exercícios em vídeos curtos, não tem menu
de minutos, mas o professor utiliza bastante a mão e caneta de cor vermelha para destacar
informações em azul; redundância - os vídeos consistem em uma equação na tela (imagem) e a
fala do professor (narração); contiguidade temporal - a resolução do exercício se dá de maneira
simultânea; segmentação - antes de iniciar a resolução dos exercícios, ele apresenta aos alunos
qual a estratégia para resolução da atividade, ou seja, quais etapas serão cumpridas para
resolução do exercício; personalização - utiliza linguagem informal e comentários em primeira
pessoa, aproximando seu público-alvo usando termos “engraçados e chamativos”. Outros
princípios que também são cumpridos são o da contiguidade espacial e modalidade.
O
Idealmente as ferramentas multimídia devem utilizar ambos os canais (visual e auditivo), para
favorecer a aprendizagem evitando sobrecarga cognitiva (BRAGA et al., 2019) o que pode ser
apreciado em vídeo aulas oferecidas no canal, onde não há uso simultâneo de texto escrito e
imagens, ou de informações excessivas na tela.
PR
3.5. Canal Marcos ABA
EL
Dentre os princípios que não apresenta estão: pré-treinamento - o canal parte do avançado,
porém explica conceitos básicos de matemática para esclarecer melhor o porquê de cada passo
do exercício e o princípio multimídia - utiliza somente equações. Como sugestões para o canal
adequar-se aos princípios que ainda não apresenta, sugerimos o uso de links de conteúdos
relacionados aos temas na descrição dos vídeos para auxiliar e complementar o entendimento
dos alunos e utilizar, quando possível, imagens ou desenhos para ilustrar o raciocínio
desenvolvido nas equações.
Fundado pelo professor Marcos com mais de 3,48 mil inscritos e 3,1 mil vídeos, o canal aborda
diversos temas dentro da matemática e da física, desde assuntos mais básicos até matérias
avançadas de nível universitário (para este estudo avaliamos apenas aulas de matemática). Uma
característica forte do canal é que cada tema principal é dividido em várias aulas explicativas
respeitando o tempo de raciocínio dos diferentes alunos.
É notável que o professor gosta do método tradicional usando caneta e papel ou caneta e lousa
para resolução e explicação dos exercícios, facilitando a compreensão e visualização das contas
matemáticas de forma simples sem excesso de informações. O canal vem crescendo
exponencialmente e novos professores têm se juntado à equipe, atendendo assim diversos
pedidos específicos de seguidores resolvendo suas dúvidas em vídeos explicativos rápidos em
média de 20 minutos. Para melhorar a sinalização seria ideal utilizar canetas de cores diferentes
durante a resolução dos exercícios. O professor, na maioria dos vídeos, utiliza apenas uma cor de
caneta. E para melhorar a segmentação de alguns vídeos que é feito uma explicação prévia sobre
o tema abordado antes do exercício seria o ideal dividir em 2 blocos dentro do mesmo vídeo
separando melhor as etapas introdução e exercício.
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3.6. Aulas Cálculo - Univesp
3.7. Considerações gerais sobre os canais
O
A fim de compreender melhor sobre a necessidade dos alunos de buscarem conteúdo
complementar àqueles que lhes são oferecidos pela UNIVESP, decidimos analisar as aulas de
Cálculo disponíveis no canal da UNIVESP no YouTube, Observando as 4 últimas aulas postadas,
notamos que alguns dos tópicos de Mayer (2009) deixam a desejar naquilo que é proposto: se
usam poucos acessórios de sinalização, bem como não há nenhum menu de conteúdos que
facilite o aluno a se localizar. O pré-treinamento não é aplicado, uma vez que os vídeos tratam de
conteúdos diferentes, mas não há um direcionamento sobre conteúdos anteriores, nem
introdução na fala da professora. A modalidade pode ser melhorada também, pois usa-se muitos
textos e poucas figuras na narração. A imagem da educadora o tempo todo na tela, acaba em
uma sobrecarga visual desnecessária também. Nota-se que há muita propriedade e um excelente
conteúdo nas aulas, mas esses tópicos, se melhorados, poderiam influenciar na maior afinidade
dos alunos para com o Canal UNIVESP.
PR
EL
Após o levantamento detalhado para cada canal, considera-se importante ressaltar que a
apresentação de passo a passo à mão faz com que se desenhem figuras com baixa qualidade. Em
todos os canais faz falta o uso de imagens pré-desenhadas ou prontas, que facilitem a
visualização apoiadas pelas explicações feitas pelo narrador. Também é preciso um uso maior da
segmentação: dividir a barra de tempo (ou reprodução) no YouTube indicando as partes dos
vídeos e os temas e indicar ao começo do vídeo como vai ser organizado (simular a um
planejamento de aula), atualmente existem ferramentas de inteligência artificial que ajudam
com a segmentação de vídeos. Em alguns canais faltou uma melhor organização indicando o
avanço dos temas e os links com os conteúdos prévios. Pensando em aproveitar as vantagens
observadas dos canais do YouTube, seria de grande valia que instituições de ensino, como é o
caso da UNIVESP, adotassem técnicas de engajamento, modulando o tom de voz e fazendo
vídeos curtos ou técnicas como o uso de nomes “engraçados” para as equações ou o uso de
desenhos que provoquem emoções e sentimento de pertinência também podem ajudar na
melhora da qualidade das aulas, (MAYER, 2011). É importante ressaltar que é possível que os
materiais da UNIVESP contenham resolução de exercícios em outros médios no AVA utilizado
pela instituição, mas aqui foi analisado apenas o elemento videoaula.
4. Conclusão
Segundo Schwartz (2013), os vídeos publicados no YouTube se caracterizam pela facilidade em
assistir, manobrar e absorver o conteúdo. Porém, o conteúdo assistido é apenas superficial, uma
vez que é possível compreender conhecimentos contextuais ou mais profundos em situações
verídicas do cotidiano. Esse pensamento reforça o fato de que é necessária uma avaliação do
material disponível no YouTube e uma reflexão por parte dos professores que se propõem a
construção desses canais, no sentido de sempre evoluir e simplificar seus ensinamentos.
EaD em Foco,2024, 14(1): e2090
| 13
Também pode ser um dos motivos pelos quais os estudantes procuram este tipo de ferramenta
para o seu aprendizado, visto que podem usar menor quantidade de tempo.
É importante considerar que tais canais têm como objetivo realizar o suporte à aprendizagem
informalmente, e não estão associados a uma instituição de ensino. O que ocorre de maneira
diferente da UNIVESP, a qual oferece graduação em ensino superior e por este motivo deve
seguir uma ementa, de acordo com os requisitos do Ministério da Educação, situação que traz
maior responsabilidade na elaboração dos conteúdos apresentados.
EL
O
Através do levantamento dos canais observou-se, adicionalmente, a busca dos alunos por um
conteúdo mais dinâmico e ilustrativo, os levando à procura por conteúdos além dos
disponibilizados pela UNIVESP. O objetivo inicial foi fazer um levantamento real dos canais mais
assistidos pelos alunos que passam pela disciplina de Cálculo, e a partir desse levantamento
caminhar para o objetivo principal, isto é a realização de uma análise pedagógica dos canais
listados, o que permitiu trazer apontamentos importantes sobre o perfil de ensino que os alunos
têm preferência. Em todos os canais analisados, o aprendizado é focado na resolução de
exercícios, com uso de caneta ou lousa, deixando ver os cálculos mais ilustrativos e práticos.
Também ressalta o bom uso da voz, usando uma narrativa fluida e descontraída sem perder
rigorosidade, criando uma conexão com os alunos e ajudando a perder o medo das matemáticas.
Estes dois fatos (solução passo a passo e voz informal) normalmente não são aplicados nas aulas
da UNIVESP, vista a formalidade por ser uma IES, e poderiam se implementar considerando que
podem ajudar a quebrar barreiras de comunicação na EaD e fazer as aulas mais atraentes para os
estudantes.
PR
Diante dos dados apresentados podemos concluir que os doze princípios da teoria de
aprendizagem de Mayer (2009) são eficientes num processo de ensino virtual, e que a adesão
dos alunos a materiais que seguem esse pressuposto (ou boa parte dele) é alta. Ainda assim, há
muito o que ser implementado nos canais gratuitos disponíveis no YouTube, para que possam
atingir todos os princípios. Além disso, a crítica construtiva às videoaulas apresentadas pela
UNIVESP na disciplina de cálculo foi feita de forma isolada, sem considerar o material completo
que a instituição disponibiliza para os estudantes.
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Biodados e contatos dos autores
Isabela DIVINO é nutricionista pelo Centro Universitário de Jaguariúna
(2017) e atuou na prática clínica durante 5 anos. Durante o mestrado foi
bolsista
no
programa
de
especialização
em
Processos
didáticos-pedagógicos para cursos de modalidade a distância. Doutoranda
e Mestra em Ciências da Nutrição e do esporte e metabolismo pela da
Faculdade de Ciências Aplicadas da UNICAMP. Trabalha no Laboratório de
Biologia Celular e Tecidual, desenvolve pesquisas com ênfase no Músculo
esquelético e regeneração. Atualmente bolsista CAPES.
ORCID: https://orcid.org/0000-0002-7007-7842
O
Contato: +55 (19) 99494-0084
EL
Rodrigo Naoto SHIRAISHI é doutorando na Faculdade de Ciências Médicas da
UNICAMP pelo programa de Fisiopatologia Médica e técnico em laboratório
no Hemocentro da mesma universidade. Graduado em Ciências Biológicas
pela UNICAMP, destacou-se em pesquisas sobre leucemia promielocítica
aguda, com foco nos efeitos do extrato de Chá Verde em modelos animais.
Possui experiência como facilitador EAD na UNIVESP, estagiou na empresa
de biotecnologia Helixxa e no Instituto Nacional de Pesquisa e Conservação
de Peixes Continentais. Seus conhecimentos incluem análise química e
bioquímica, obtidos durante seu curso técnico na ETECAP e estágio no
Instituto Agronômico de Campinas.
PR
ORCID: https://orcid.org/0000-0001-6778-6886
Contato: +55 (19) 99825-2442
Mariele Rodrigues MOREIRA é doutoranda e Mestra em Nutrição e
Metabolismo pelo Programa de Ciências da Nutrição e do Esporte e
Metabolismo da Faculdade de Ciências Aplicadas da UNICAMP em que
trabalhou com desenvolvimento e análise de um alimento clean label.
Durante o mestrado foi bolsista no programa de especialização em Processos
didáticos-pedagógicos para cursos de modalidade a distância da UNIVESP
onde além da formação teórica teve a prática atuando como tutora nos
cursos de graduação. Graduada em Ciências com habilitação plena em
Química que permitiu atar como professora por dez anos em escolas
públicas, particulares e cursinho pré-vestibular. Seus interesses de pesquisa
atuais incluem educação alimentar e nutricional com ênfase em medicina
culinária.
ORCID: https://orcid.org/0000-0001-8056-8269
Daniel Alfonso SIERRA é doutorando na Faculdade de Engenharia Civil, Arquitetura
e Urbanismo da UNICAMP. Completou o seu mestrado na mesma faculdade, na
EaD em Foco,2024, 14(1): e2090
| 15
área de Recursos Hídricos, Energéticos e Ambientais. Seus interesses de pesquisa incluem redes de
abastecimento de água, adutoras, sistema de proteção contra transitórios hidráulicos, com destaque para a
modelação de transitórios hidráulicos e na otimização do dimensionamento de dispositivos de proteção.
ORCID: https://orcid.org/0000-0003-0699-4512
Contato: +55 19992196674
O
Victoria HERRERA é professora do Centro de Engenharia Modelagem e
Ciências sociais Aplicadas da Universidade Federal do ABC (UFABC).
Formada em engenharia eletrônica pela Universidad Mayor de San Andrés,
mestre e doutora em Energia pela UFABC. Atua em elaboração de objetos
de aprendizagem para engenharias, modelagem de sistemas dinâmicos,
diagnóstico de falhas e educação a distância.
EL
ORCID: https://orcid.org/0000-0001-8056-8269
Referências Bibliográficas
PR
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BRAGA, A., et al. A teoria cognitiva da aprendizagem multimídia no desenvolvimento de atividades de
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CORTEZ, L.C. OS VÍDEOS DO YOUTUBE COMO RECURSO DIDÁTICO. 2010. 52 f. Monografia
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Federal do Paraná, Curitiba, 2010.
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PIMENTEL, C. Repórter da Agência Brasil. Pisa: menos de 50% dos alunos sabem o básico em
matemática e ciências. 2023. Disponível em: https://rb.gy/iegouv Acesso em: 13 mar. 2024.
SILVA, A.M.; MONTANÉ, F. Objetos de Aprendizagem baseados na teoria da Aprendizagem Multimídia.
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SCHWARTZ, M. Khan academy: The illusion funder standing. Online Learning Journal, v. 17, n. 4, 2013.
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Disponível em: https://rb.gy/d0wb63 - Acesso em: 09 ago, 2022
UNIVESP. GUIA GERAL Produção de Disciplinas. São Paulo: Governo do Estado de São Paulo, 2020. 19 p.
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YOUNDER.
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EaD em Foco,2024, 14(1): e2090
os
treinamentos.
Disponível
| 17
COMO CITAR ESTE TRABALHO
PR
EL
O
ABNT: DIVINO, I. A. et al. Análise de Canais do YouTube como Objetos de
Aprendizagem de Suporte ao Estudo de Cálculo I em Cursos da Área de Ciências
Exatas.
EaD
em
Foco,
v.
14,
n.
1,
e2090,
2024.
doi:
https://doi.org/10.18264/eadf.v14i1.2090
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| 6,953 | 46,097 |
W2109539792.txt | Borrelia lusitaniae and Green
Lizards (Lacerta viridis), Karst
Region, Slovakia
Viktória Majláthová,* Igor Majláth,† Marketa Derdáková,*‡ Bronislava Víchová,*
and Branislav Pet’ko*
In Europe, spirochetes within the Borrelia burgdorferi
sensu lato complex are transmitted by Ixodes ricinus ticks.
Specific associations are described between reservoir
hosts and individual genospecies. We focused on green
lizard (Lacerta viridis) as a host for ticks and potential host
for borreliae. In 2004 and 2005, a total of 146 green lizards
infested by ticks were captured, and 469 I. ricinus ticks
were removed. Borrelial infection was detected in 16.6% of
ticks from lizards. Of 102 skin biopsy specimens collected
from lizards, 18.6% tested positive. The most frequently
detected genospecies was B. lusitaniae (77.9%–94.7%).
More than 19% of questing I. ricinus collected in areas
where lizards were sampled tested positive for borreliae. B.
garinii was the dominant species, and B. lusitaniae represented 11.1%. The presence of B. lusitaniae in skin biopsy
specimens and in ticks that had fed on green lizards implicates this species in the transmission cycle of B. lusitaniae.
he causative agents of Lyme borreliosis, spirochetes of
the Borrelia burgdorferi sensu lato complex, are maintained in natural foci by circulation between the vector
ticks in the Ixodes ricinus complex and reservoir hosts of
various vertebrate taxa. The B. burgdorferi s.l. complex
encompasses 12 species (1–3); 4 species have been clearly
established as pathogenic to humans: B. afzelii, B. garinii,
B. burgdorferi s. s., and B. spielmanii (4–6). B. valaisiana
and B. lusitaniae, which were previously considered nonpathogenic, may cause disease as well (7,8). Different
species are associated with distinct ecologic features, levels of pathogenicity, and clinical symptoms in patients.
In Europe, I. ricinus ticks infest a wide variety of vertebrate hosts, such as mammals, birds, and lizards. The verte-
T
*Parasitological Institute of Slovak Academy of Sciences, Košice,
Slovakia; †University of P.J. Šafárik in Košice, Košice, Slovakia;
and ‡Institute of Zoology of Slovak Academy of Sciences, Košice,
Slovakia
brate hosts are necessary to maintain the tick population
and may also serve as reservoirs for the pathogen.
Therefore, the identification of reservoir host species is
essential to clarify the transmission patterns of B. burgdorferi s.l. in natural foci. The importance of rodents for maintaining B. afzelii (9), and of birds for B. garinii and B.
valaisiana (10), in endemic regions of Slovakia is now
indisputable. The National Park Slovak Karst is within the
region in which B. burgdorferi s.l. in questing ticks and
birds has been reported (V. Taragel'ová, unpub. data). In
this area, 2 lizard species occur sympatrically, the common
wall lizard (Podarcis muralis) and the green lizard (Lacerta
viridis). The green lizard, the dominant species, is frequently infested by immature stages of I. ricinus ticks (11).
The importance of lizards in the maintenance cycles of
B. burgdorferi s.l. spirochetes is still controversial. In Italy,
B. lusitaniae was detected in blood and tissue samples of
P. muralis (12). Furthermore, Psammodromus algirus, the
most abundant lizard species in North Tunisia, was found
to be the primary host for immature stages of I. ricinus.
Thus, it could play a role in the circulation of borreliae
(13). B. burgdorferi s.s., B. andersonii, and B. bisettii were
detected in the blood of 9 lizard species in the southeastern
United States (14). On the other hand, several other studies in the United States have shown that the lizards
Sceloporus occidentalis and Elgaria multicarinata are
reservoir-incompetent for borreliae because they possess
borreliacidal factor in their blood (15,16). However, 2
lizard species, Eumeces inexpectatus and Anolis carolinensis, can sustain B. burgdorferi s. s. infection (17).
In the Slovak Karst (southeastern part of Slovakia), the
green lizard is the major host for immature stages of I. ricinus ticks (11). Therefore, the main aim of this study was to
find out whether green lizards can participate in the maintenance cycles of B. burgdorferi s.l. in natural foci and
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 12, December 2006
1895
RESEARCH
whether an association with specific borrelial genospecies
exists.
Materials and Methods
Study Area
The study was conducted in the National Park Slovak
Karst. This area represents a part of the Inner Carpathians
in southeastern Slovakia (48°36′ N, 20°52′ E). The climate
is warm with low humidity and average temperatures of −
4°C in January and 18°C in July. The average rainfall is
700 mm/year.
merase (5 U/µL), 0.5 µL deoxynucleoside triphosphate
(dNTP) mix (10 mmol/L) (Fermentas, Vilnius, Lithuania),
1.25 µL of each primer (10 pmol/µL) (Invitrogen, Paisley,
Scotland), and 2.5 µL DNA template.
To verify that DNA had been successfully isolated from
each tick, primers for the fragment of the tick’s mitochondrial cytochrome b gene (620 bp) were used (19). Negative
samples were excluded from the further analysis. Positive
samples were examined for the presence of B. burgdorferi
s. l. by amplifying a portion of the 5S (rrfA)-23S (rrlB)
rDNA intergenic spacer (20). PCR products were subjected
to electrophoresis on a 1% agarose gel, stained with ethidium bromide, and visualized with a UV transilluminator.
Tick and Lizard Collection
This survey was conducted in 2004–2005, from May to
September, when lizards and ticks are active. Questing I.
ricinus nymphs and adults were collected by flagging the
vegetation in areas where lizards were sampled. Ticks
were immediately stored in 70% ethanol.
Green lizards were captured along hiking paths by hand
or by noosing, in which a loop made from fishing nylon
was attached to the end of a wooden stick and dangled in
front of a lizard, which would be captured as it walked
through the loop. Animals were characterized by sex and
age (adult, subadult, juvenile) and examined for ticks.
Ticks were removed with forceps immediately after
capture and stored in 70% ethanol. Biopsy specimens (a
2-cm distal part of the tail and a 1-mm × 1.5-mm piece of
skin from collar scales) were taken from each lizard with
sterile scissors and put in separate vials with 70% ethanol.
Ticks were identified to the species and sex. Only
I. ricinus ticks were further examined for B. burgdorferi
sensu lato.
DNA Isolation
Immediately before extraction, ticks and tissues were
dried for 30 min to evaporate the ethanol. Each sample was
cut with a disposable sterile scalpel. Tissue DNA from
lizards’ tails and scales was extracted by using DNeasy tissue kit (Qiagen, Hilden, Germany). Extraction steps were
conducted according to the manufacturer’s protocol.
Genomic DNA from ticks was isolated by alkaline hydrolysis (18). Incubation time was extended from 5 to 30 min.
Isolated DNA was stored at −20°C.
PCR
PCR amplification was performed in a 25-µL reaction
mixture from the MasterTaq DNA polymerase kit
(Eppendorf AG, Hamburg, Germany) containing 10.4 µL
deionized water, 5 µL 5× TaqMaster PCR Enhancer, 2.5
µL 10× Taq buffer (with 15 mmol/L Mg2+), 1.5 µL 25mmol/L solution of Mg (OAc)2, 0.1 µL Taq DNA poly-
1896
RFLP Analysis
The positive PCR products of the 5S-23S rDNA intergenic spacer regions were further analyzed by restriction
fragment length polymorphism (RFLP). Previously extracted DNA of B. afzelii, B. garinii, B. valaisiana, and B.
burgdorferi s.s. were used as positive controls. For each
positive sample, 13 µL amplified DNA was digested at
65°C overnight in a solution containing 5 U of Tru1I (300
U/mL) and 1× Buffer R (Fermentas). Electrophoresis was
conducted in 16% polyacrylamide gel at 150 V for 3 h. The
gels were stained with SYBR gold nucleic acid gel stain
(Molecular Probes, Leiden, the Netherlands) for 20 min,
and bands were visualized with a UV transilluminator.
RFLP profiles that differed from the known profiles of positive controls were further analyzed by sequence analysis.
DNA Sequencing of PCR Products
Sequencing was performed at the Department of
Molecular Biology (Faculty of Natural Sciences
Commenius University, Bratislava, Slovak Republic).
PCR of the 5S-23S rDNA intergenic spacer was conducted according to the protocol described above. For the fla
gene amplicons, DNA strands were sequenced as
described previously (21). PCR products were purified by
using a QIAquick PCR purification kit (Qiagen). The complementary strands of each sequenced product were manually assembled. Sequences were compared with GenBank
entries by Blast N2.2.13 (22). Homologous sequences
were aligned by using the CLUSTAL W Multiple
Sequence Alignment Program (version 1.81) (23).
Sequence similarity among the sequences were calculated
by EMBOSS Align, a pairwise alignment algorithm
(http://www.ebi.ac.uk/emboss/align).
The accession numbers of 5S-23S rDNA intergenic
spacer sequences obtained in this study are DQ539339 and
DQ539340. Accession numbers of flagellin sequences
obtained in this study are DQ788618, DQ788619, and
DQ788620.
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 12, December 2006
Borrelia lusitaniae and Green Lizards, Slovakia
Data Analysis and Statistics
To estimate the probability of a tick’s becoming infected after engorging on a green lizard and to measure the
degree of infectiousness of infected animals, specific
infectivity Is (24) and transmission coeficient βH-T (9) were
calculated. Individual infectivity (i) is defined as the proportion of larvae derived from an individual lizard that are
infected (i = li/lh, li is the number of larvae that become
infected, and lh is the total number of larvae derived from
that host). The specific infectivity (Is) of a reservoir host
species is defined as the sum of individual infectivities and
number of individual lizards sampled (Is = ΣiS/nS, n is the
number of individual ticks captured). The host-to-tick
transmission coefficient (βH–T) is defined as the portion of
the sum of individual infectivities and the number of
lizards that infected >1 larva (βH–T = ΣiS/niS (ni is the number of individual hosts that gave rise to at least 1 infected
tick). Differences in the prevalence of B. burgdorferi s.l. in
I. ricinus were evaluated statistically with the 2-tailed χ2
test (degrees of freedom [df] = 1). A value of p<0.05 was
considered statistically significant.
Results
Lizards and Infestation with Ticks
One hundred forty-six (84 male, 52 female, and 10
subadult) of 165 (89 male, 61 female, and 15 subadult)
captured green lizards were infested by ticks during the
study period. In total, 469 (199 larvae and 270 nymphs)
ticks were removed and further identified as I. ricinus.
Male lizards were infested with 363 ticks (131 larvae and
232 nymphs), which represented 77.4% of all collected
ticks. Moreover, 53 tails and 102 skin biopsy specimens
were taken from the captured lizards.
B. burgdorferi Prevalence in Ticks
Collected from Lizards
DNA isolation was successful in 464 ticks (197 larvae
and 267 nymphs), from which the fragment of cytochrome
b gene was amplified. These ticks were further analyzed
for the presence of B. burgdorferi s.l. In total, 77 (16.6%)
ticks carried borreliae. The infection prevalence between
nymphs (15.2%) and larvae (17.6%) did not differ significantly (p = 0.49669, df = 1) (Table 1). Twenty-nine percent
of tick-infested lizards carried >1 infected tick. Infected
lizards yielded ≈2 infected larvae per host.
Genotyping with PCR-RFLP identified the following
species: B. lusitaniae, B. afzelii, B. garinii,B. burgdorferi
s.s., and B. valaisiana. Of the B. burgdorferi–positive
ticks, most (77.9%) were infected with B. lusitaniae. The
presence of this species was significantly higher than that
of other species (p<0.001). B. lusitaniae was detected in 26
(86.7%) larvae. B. afzelii, B. garinii, and B. burgdorferi s.
s. each were found in 1 larva. Of the 47 B.
burgdorferi–infected nymphs, 34 (72.3%) were infected
with B. lusitaniae, 5 (10.6%) with B. afzelii, 2 (4.3%) with
B. burgdorferi s. s., and 1 (2.1%) with B. garinii. A mixed
infection of B. lusitaniae and B. burgdorferi sensu stricto
was found in 5 (10.6%) nymphs (Table 1). Nymphs and
larvae did not differ significantly in the prevalence of B.
lusitaniae.
Male lizards were parasitized by 61 (79.2%) of 77
infected ticks. Variability of detected genospecies was
higher in ticks collected from male than from female
lizards. Larvae that fed on female lizards were only infected with B. lusitaniae. Out of 7 infected nymphs collected
from females, B. lusitaniae was present in 5 and B. afzelii
in 1 tick; B. lusitaniae and B. burgdorferi s.s. were detected as mixed infection in 1 nymph. The specific infectivity
from lizards to larval ticks was highest for B. lusitaniae.
The specific infectivity of female lizards was slightly higher than that of males (Table 2).
B. burgdorferi Prevalence in Lizards
Isolated genomic DNA from tails and skin biopsy specimens from collar scales was tested for the presence of B.
burgdorferi sensu lato. None of 53 tested tail samples was
positive. Of 102 skin biopsy specimens collected from
green lizards, 19 (18.6%) tested positive. Differences in
infection prevalence between sexes (18.2% in males vs.
23.7% in females) were not significant. Of 9 skin biopsy
specimens from subadult individual lizards, 2 (22.2%)
were borrelia positive. The most frequently detected
genospecies was B. lusitaniae (94.7%), which was present
in 18 samples. One lizard was infected with B. afzelii.
B. burgdorferi Prevalence in Questing Ticks
Cytochrome b was amplified in 325 of 331 (71 female,
73 male, and 187 nymph) questing ticks. Therefore, only
these 325 ticks (71 female, 71 male, and 183 nymph) were
analyzed further for the presence of B. burgdorferi s.l.
Sixty-three (19.3%) ticks tested positive. B. burgdorferi
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 12, December 2006
1897
RESEARCH
prevalence in female ticks was the same as in male ticks
(22.5%), and it was lower in nymphs (16.9%).
RFLP analysis of the amplified products resulted in 5
distinct profiles. Of the 63 positive ticks, 21 (33.3%) were
infected with B. garinii, 19 (30.2%) were infected with B.
afzelii, 8 (12.7%) were infected with B. burgdorferi s. s., 7
(11.1%) were infected with B. lusitaniae, and 7 (11.1%)
were infected with B. valaisiana. One nymph was infected
simultaneously with B. garinii and B. valaisiana (Table 3).
Sequence Analysis
Representative samples of RFLP profiles that were different from the known profiles of positive controls were
sequenced. The fragment of the 5S-23S rDNA intergenic
spacer obtained from the B. burgdorferi s.l.–positive
nymph (538N) from lizard belonged to B. lusitaniae. It
was 100% identical to a Borrelia-positive skin biopsy
specimen (277S) sampled from a lizard. Both obtained
sequences were 100% identical with the Turkish B. lusitaniae strain Tr213 (AB 091802) and 98.9%, 98.4%, and
94.5% similar to PotiBL37 (AY 463167), PotiB2
(L30131), and PotiB3 (L30132) strains from Portugal,
respectively. To better characterize B. lusitaniae circulating in ticks and lizards from Slovak Karst, the fla gene
from a B. burgdorferi s. l.–positive questing adult tick and
skin biopsy specimen from collar scale was amplified and
sequenced. The flagellin sequence of B. lusitaniae detected in a skin biopsy specimen (277 S) was 100% identical
and 99.6% similar to B. lusitaniae detected in questing
adult ticks (43 ZLIF, 47 ZMLIM), respectively. Genotypes
277S and 43ZLIF were 100% identical with the Turkish B.
lusitaniae strain Tr213 (AB091812) as well as with the
Polish strain D23–04 (DQ 016623). Genotype 47ZLIM
was 99.6%, 99.6%, and 99.4% similar to Tr213, D23–04,
and PotiB2 (DQ111036), respectively.
1898
Discussion
The role of lizard species in maintaining B. burgdorferi
s.l. has not been clearly elucidated yet. In United States,
some lizard species have sustained borrelial infection
(14,17); however, other species are incompetent reservoir
hosts (15,16). The reservoir competence of lizards seems
to be species specific. Therefore the aim of our study was
to establish whether a relationship exists between green
lizards, the dominant lizard species in the Slovak Karst,
and B. burgdorferi s.l., which circulates in this area.
Seventeen percent of ticks that fed on lizards were
infected with B. burgdorferi s.l. Seventy-eight percent of
all infected ticks were infected with B. lusitaniae.
Moreover, 18.6% of skin biopsy specimens from lizards
were positive for B. burgdorferi s.l., and almost all
(94.7%) were infected with B. lusitaniae. Similarly, B.
lusitaniae have been detected in blood and tissue samples
of Podarcis muralis in Tuscany in Italy, where borreliae
were detected in 2 of 14 tested whole tails from lizards
(12). At the beginning of our study, we also collected the
distal tip of a lizard’s tail because this method is minimally invasive and convenient for obtaining a tissue sample.
The tissue at the tail, however, is squamous and keratinized, and none of the collected samples was borreliae
positive. Therefore, we also obtained skin biopsy specimens from collar scales. These are elongated and extend
from the skin on the ventral side, so collecting them is minimally invasive and perhaps more likely to detect infection
with B. burgdorferi s.l. because most of the immature I.
ricinus ticks parasitize at the dorsal area (pers. observation). Furthermore, collar scales were chosen to avoid
detecting the borreliae that persist in the skin after feeding
of the infected ticks, which may enable infection of ticks
by “extended co-feeding” (25). In this manner, incompetent host species may contribute to the circulation of
B. burgdorferi s.l. in nature. For example, in England,
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 12, December 2006
Borrelia lusitaniae and Green Lizards, Slovakia
I. ricinus ticks cofeeding on sheep become infected with B.
burgdorferi, although sheep themselves are refractory to
infection (26). In Europe the principal importance of
cofeeding to Lyme disease ecology has been suggested to
be the extent of the range of vertebrate host species that
contribute significantly to the maintenance of B, burgdorferi s.l. spirochetes in nature (27). Therefore, cofeeding
transmission could also be responsible for B. afzelii, B.
garinii, and B. burgdorferi s.s. infection in larvae that fed
on lizards collected in our study, even though skin biopsy
results yielded mostly B. lusitaniae. Another possible
explanation for the presence of non–B. lusitaniae spirochetes is that these larvae may have been infected transovarially (28). Cofeeding transmission might explain why
individual lizards with borreliae negative skin biopsy specimens carry borreliae-positive larvae. Because the quantity of borreliae is low in the vertebrate host and may lodge
in deeper organs, detecting them in skin biopsy specimens
may not always be possible (29). Thus, a negative skin
biopsy result does not prove conclusively that the lizard is
not infected.
Despite the fact that male lizards hosted >75% of all
host-feeding ticks, as well as 79.2% of all infected ticks,
the specific infectivity and host-to-tick transmission coefficient were almost the same for male and female lizards.
The seasonal activity of green lizards and different patterns
in male and female behavior were monitored in the Slovak
Karst (I. Majlath, unpub. data). Larger numbers of ticks
feeding on male lizards are associated with higher male
activity in spring months, when tick activity peaks as well.
Male lizards end hibernation first and are active when the
air temperature reaches 10°C–12°C. They need to restock
the energy that was depleted during winter and to gain
energy for fighting other male lizards to compete for territory and females, for seeking female lizards, and for mating. Female activity increases in summer months when
they are incubating eggs.
As determined by PCR, the overall prevalence of infection in our sample of questing ticks (19.3%) is consistent
with 20.5% found in southern Czech Republic (20) but
lower than that reported for a geographically close area in
western Slovakia (40%–49%) (30). The total prevalence
was higher in adults (22.5%) than in nymphs (19.6%),
which is in agreement with the general pattern of increasing Borrelia prevalence through the life stages of ticks as
their adults feed on a multiple hosts (31). The total prevalence of borreliae in male and female ticks was identical,
but the distribution of genospecies was different. B. garinii
was the predominant genospecies in this locality. B. garinii
and B. valaisiana are the most commonly reported species
in central Europe (32).
The high prevalence of B. lusitaniae in borrelia-positive larvae and nymphs as well as skin biopsy specimens
from lizards suggests that green lizards are susceptible and
transmission competent for B. lusitaniae. On the other
hand, a lack or low prevalence of other genospecies in
ticks that had fed on lizards may suggest that these
genospecies could be negatively selected against by green
lizards. A similar suppressive effect of Madeiran wall
lizard (Podarcis dugesii) on the transmission of spirochetes was observed (33). Borreliacidal activity against B.
burgdorferi s.s. was observed in the lizards S. occidentalis
and E. multicarinata in North America (15,16). These
findings add to the growing support for the hypothesis that
there are Borrelia species-specific associations with specific reservoir host species that result from Borrelia
species–specific interactions with host serum complement
(29).
Significant differences were found in B. lusitaniae
prevalence in fed larvae compared with questing nymphs
(p<0.001, df = 1); none of 183 examined nymphs was
infected by this genospecies. This finding raises the questions of whether borreliae are eliminated during molting
and thus do not contribute to the transmission cycle or
whether we were just unable to detect it. Significant differences were found in B. lusitaniae prevalence also in fed
nymphs compared with questing adults (p<0.01, df = 1).
The infection prevalence decreased from 74.5% in fed
nymphs to 5% in questing adults. Reduction of infection
prevalence has been observed in B. afzelii from 47% in
nymphs engorged on the rodents to 7% in questing nymphs
(9).
The occurrence of B. lusitaniae in ticks is frequent in
some areas of the Iberian Peninsula and North Africa,
where the organism often represents the only species of B.
burgdorferi s.l. complex (13,34). In the rest of the Europe,
it has been isolated or detected less frequently, with low
prevalence in ticks (30,35,36). The prevalence of B. lusitaniae is the highest in southern Europe and can be exported
to other areas by hosts such as birds (37). The 5S-23S
rDNA and flagellin sequences of B. lusitaniae–positive
ticks and skin biopsy specimens in our study were 100%
identical to the B. lusitaniae strain Tr213 from a tick in
Turkey (38). The distribution of this borrelial species may
be associated with the distribution range of reservoir hosts,
including lizards, that inhabit drier and warmer areas.
These ecosystems are less abundant in central Europe than
in the Mediterranean. Thus, lizards may influence the
transmission cycle of borreliae in some localities in which
they are the predominant host for ticks. In our study, we
found B. lusitaniae in skin biopsy specimens and ticks that
fed on green lizards. These findings implicate this species
of lizard in the transmission cycle of B. lusitaniae. The
competence of other lizard species that feed ticks should
be also investigated. The low prevalence of B. lusitaniae in
questing ticks, however, indicates that the ecology of
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 12, December 2006
1899
RESEARCH
B. lusitaniae in endemic foci of central Europe is more
complex. Further studies that analyze the circulation of B.
burgdorferi s.l. among a broader spectrum of host species
should be undertaken.
Acknowledgments
We thank Jean Tsao and Slavka Barlakova for their critical
reading of the manuscript and helpful comments and Marcela
Mirekova and Renata Ivanova for technical assistance.
This work was partly supported by the Slovak Agency of
Research and Development, APVV-51-009205 project (M.D.),
VEGA 2/6163/26 to (B.P.), and VEGA 1/1284/04 (I.M.).
Mrs Majláthová is a doctoral student at the Parasitological
Institute of the Slovak Academy of Sciences. Her research interests lie in ecology and epidemiology of tickborne pathogens.
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Address for correspondence: Viktória Majláthová, Parasitological
Institute, Slovak Academy of Sciences, Hlinkova 3, 040 01 Košice,
Slovakia; email: majlat@saske.sk
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W3181906828.txt | Manuscript to be reviewed
Short-chain soluble polyphosphate fertilizers increased soil P
availability and mobility by reducing P fixation in two
contrasting calcareous soils
Jawad Ali Shah 1 , Guixin Chu Corresp. 1
1
Department of Envirnmental Science and Engineering, College of Life Science, Shaoxing University, Shaoxing, Zhejiang, P. R. China
Corresponding Author: Guixin Chu
Email address: chuguixin@usx.edu.cn
Short-chain polyphosphate fertilizers have been increasingly applied in agriculture, but
little is known about the chemical behaviors of polyphosphate in soils. Herein, a cylinder
experiment was carried out to investigate the influences of different P types (i.e. mono-
ammonium phosphate (MAP), phosphoric acid (PA) and ammonium polyphosphate (poly-P)
and their application methods (single vs split) on the mobility and availability of P in soil
through a column millimeter-scale slice cutting method; meanwhile a soil microcosm
experiment (560-day) was conducted to investigate the effects of different P types on
phosphorus dynamic transformation. Polyphosphate addition significantly increased P
mobility. The average distance of P downward movement (81.5 mm) in soil profile in the
poly-P application treatment increased by 33.6% and 81.1%, respectively, compared to the
MAP and PA treatments. Different P application methods also markedly influenced
phosphorus mobility. For instance, the average distance of P vertical movement in the split
P application treatment was 21.2% higher than in the single application treatment,
indicating that split P addition significantly increased P downward movement. Moreover,
polyphosphate application decreased soil P fixation by blocking the transformation of the
applied-P from labile to recalcitrant forms (HCl-P and residual-P). Overall, our findings
provide meaningful information to current phosphorus fertilization practice in increasing
soil P mobility and bioavailability. We suggest that polyphosphate could be regarded as an
alternative P source used in agriculture, and split polyphosphate application is
recommended as an effective P fertilization strategy.
PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021)
Manuscript to be reviewed
1
Type of contribution: Research paper
2
Number of text pages: 20
3
Number of tables: 05
4
Number of figures: 04
5
6
Title:
7
Short-chain soluble polyphosphate fertilizers increased soil P availability and mobility by
8
reducing P fixation in two contrasting calcareous soils
9
Authors: Jawad Ali SHAH1 and Gui-xin CHU 1*
10
Affiliations:
11
1.
12
China.
13
* Corresponding author: Prof. CHU Gui-xin
14
Address:Department of Environmental Science and Engineering, School of Life Science,
15
Shaoxing University, Huancheng West Road 508, Shaoxing 312000, P. R. China.
16
Tel: +86 17857505869; Fax: +86 575 8834 1905
17
Email: chuguixin@usx.edu.cn, ali_jawad009@yahoo.com
College of Life Science, Shaoxing University, Shaoxing City, 312000, Zhejiang Province,
18
19
20
21
PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021)
Manuscript to be reviewed
22
23
24
ABSTRACT
25
Short-chain polyphosphate fertilizers have been increasingly applied in agriculture, but little is
26
known about the chemical behaviors of polyphosphate in soils. Herein, a cylinder experiment
27
was carried out to investigate the influences of different P types (i.e. mono- ammonium
28
phosphate (MAP), phosphoric acid (PA) and ammonium polyphosphate (poly-P) and their
29
application methods (single vs split) on the mobility and availability of P in soil through a
30
column millimeter-scale slice cutting method; meanwhile a soil microcosm experiment (560-day)
31
was conducted to investigate the effects of different P types on phosphorus dynamic
32
transformation. Polyphosphate addition significantly increased P mobility. The average distance
33
of P downward movement (81.5 mm) in soil profile in the poly-P application treatment increased
34
by 33.6% and 81.1%, respectively, compared to the MAP and PA treatments. Different P
35
application methods also markedly influenced phosphorus mobility. For instance, the average
36
distance of P vertical movement in the split P application treatment was 21.2% higher than in the
37
single application treatment, indicating that split P addition significantly increased P downward
38
movement. Moreover, polyphosphate application decreased soil P fixation by blocking the
39
transformation of the applied-P from labile to recalcitrant forms (HCl-P and residual-P). Overall,
40
our findings provide meaningful information to current phosphorus fertilization practice in
41
increasing soil P mobility and bioavailability. We suggest that polyphosphate could be regarded
42
as an alternative P source used in agriculture, and split polyphosphate application is
PeerJ reviewing PDF | (2020:08:52284:1:0:NEW 26 Mar 2021)
Manuscript to be reviewed
43
recommended as an effective P fertilization strategy.
44
Keywords: Phosphorus; Ammonium polyphosphate; Mobility; Availability; P transformation;
45
Calcareous soil
46
1. Introduction
47
Phosphorus (P) is an essential and yield-limiting macro-element for higher plants. However,
48
about two-thirds of the total cultivated soil belongs to P-deficient soil (e.g. plant available P <10
49
mg P kg-1) in China (Cao et al., 2012; Zhang et al., 2019), and approximately 5.7 billion ha of
50
area was deficient of soil available P on global scale (Hinsinger, 2001). Chemical synthesized
51
orthophosphate fertilizers (ortho-P) have been excessively applied in agriculture in the past
52
several decades (Chowdhury et al., 2017). MacDonald et al. (2012) addressed that P input was
53
23.8 Tg P·y−1 globally in 2000, resulted in an annual P surplus of 11.5 Tg of P·y−1. However,
54
only a small fraction of the applied P was absorbed by plants (Mueller et al., 2012), and plant-
55
accessible P only accounted for 6 % (range 1.5 to 11%) of total P in soils (Stutter et al., 2012).
56
On the other hand, P is a non-renewable resource, the exploitable P ore will be depleted within
57
60-100 yr, because ca 31 million tons of P was exploited annually (Cordell et al., 2009).
58
Therefore, set up an effective P management strategy is imperative to recycle P more efficient
59
and avoid running out of the finite P resource.
60
The applied P in soils undergoes a sequence of intricate transformation processes. In neutral or
61
calcareous soil, the freshly added P easily transformed from dibasic calcium phosphate (DCP) to
62
brushite dicalcium phosphate dihydrate, (DCPD), then to octacalcium phosphate (OCP), finally
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to hydroxyapatite (HAP) (Weihrauch & Opp, 2018; Ge et al., 2019). When P is applied through
64
basal application method, a relative high P concentration appears at P application site (Chien et
65
al., 2010; Li et al., 2019). In this way, the added-P progressively converts to octa-calcium
66
phosphate (Ca8H2(PO4)6·5H2O, OCP) within 3 to 5 months, following by constitutes β-tri-
67
calcium phosphate (β-TCP) after 8 to 10 months, finally turns into hydroxyapatite
68
Ca10(PO4)6(OH)2. In contrast, more added P remains in available form when P fertilizer is split
69
repeated applied at low application dose in crop growing season (Khatiwada et al., 2014;
70
González-Jimenez et al. 2019). Consequently, a high crop yield and phosphorus use efficiency
71
(PUE) can be achieved (Saarela et al., 2006). Unfortunately, at present, basal application of P
72
fertilizer remains used as main P fertilization technology in most agriculture areas.
73
Different P sources (ortho-P vs poly-P) significantly influenced phosphorus availability and
74
transformation (McBeath et al., 2005; Wang & Chu, 2015; Gao et al., 2019). Polyphosphates
75
(poly-P) are superior over ortho-P fertilizers (Hamilton et al., 2018). This may largely be
76
ascribed to its slow release characteristic (Kulakovskaya et al., 2012; Hamilton et al., 2016).
77
Following this, polyphosphate application significantly increased soil available P (Gao et al.,
78
2019) and improved crop yield (Holloway et al., 2004). However, other studies showed that
79
polyphosphate (ammonium polyphosphate, APP) had no obvious advantage or even less than
80
ortho-P fertilizer (granular monoammonium phosphate, MAP) in increasing soil P availability
81
and crop yield (Ottman et al., 2006). These controversial or paradoxical results implied that the
82
physiochemical behaviors (i.e. mobility, availability, and transformation) of polyphosphate in
83
soils need to be further explored.
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In this study, a soil cylinder experiment and a soil microcosm experiment were carried out in
85
contrasting calcareous soils. We hypothesize that split repeated polyphosphate application
86
outcompetes conventional orthophosphate in increasing soil P availability and mobility.
87
Therefore, the objectives of our study were to (i) compare the effects of different P types and
88
their application methods on soil P mobility; (ii) clarify the response of soil P availability and
89
transformation to different P addition, (iii) finally put forward an appropriate P fertilization
90
strategy to increase soil P availability and improve PUE in calcareous soil.
91
2. Materials and Methods
92
2.1 Soils sampling and soil description
93
Experiments were conducted using two calcareous soils (loam and clay soils). According to
94
FAO/WRB soil taxonomy, both were classified as Calcisol Fluvisols. Loam soil was taken from
95
experimental station of Shihezi University (44°18′ N, 86°02′ E) and clay soil was collected from
96
147 State Farm (44°37′ N, 86°10′ E) in Shihezi region. Soil samples were air dried, stones and
97
small visible plant residues were manually removed. All samples were ground to pass through a
98
2 mm sieve prior to measurement of soil properties. The selected soil physical and chemical
99
properties are presented in (Table 1).
100
Insert “(Table 1)”
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2.2 Different P sources Description
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Three types of P sources including (i) mono-ammonium phosphate (powder MAP, NH4H2PO4)
103
with purity of 99% and density of 1.803 g cm-3 and 26.9% of P (Shengao chemical reagent co.,
104
LTD, China); (ii) phosphoric acid (fluid PA, H3PO4) with purity of 85% and density of 1.685 g
105
cm-3 and 27.1% of P (Fuyu special chemicals co., LTD, China), and (iii) ammonium
106
polyphosphate (fluid APP, H6P4O13, n=4) with purity of 85% and density of 2.1g cm-3 and 31.3%
107
of P (Aladdin Industrial Corporation, U.S.A) were employed in this study.
108
2.3 Experiment Ⅰ Soil cylinder experiment
109
Soil cylinder experiment was carried out in a transparency Plexi glass cylinders (350 mm height,
110
64 mm internal diameter). Five holes (2 mm diameter) were drilled at bottom of the cylinder to
111
maintain aerobic condition. A 10-mm-thick layer of fine sand was placed at the bottom of the
112
cylinder and placed two pieces of filter paper on the surface of the sand layer. A totally of 1302 g
113
loam soil and 1331 g clay soil were placed into each cylinder. Another two pieces of filter paper
114
was placed on the top of the cylinder. Soil bulk densities of 1.35 g cm-3 and 1.38 g cm-3 were
115
designed for loam and clay soil, respectively. Three P types (i.e., powder MAP, fluid PA, and
116
fluid APP) were applied to the each cylinder following the methods of: (i) Single application, the
117
total amount of P fertilizer was uniformly mixed into the upper 20 mm of soil layer and watered
118
four times over the four-week-long incubation period; (ii) split repeated application, the fertilizer
119
was initially dissolved with 50 ml milli-Q water in a 250 ml beaker individually except MAP
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which was applied directly and then fertilizer solution was injected into soil at the center site of
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the cylinder using a syringe, P fertilizer was four times evenly added at equivalent ratio during
122
entire 28-days period. The irrigation water was poured into a Markov bottle, and hanged the
123
bottle 1.5 m above each cylinder (Fig. 1). The flow rate of the emitter was controlled at 6 drops
124
per second by connecting each emitter to individual Markov bottle. The total water column
125
irrigated were 401.6 mL and 515.8 mL cylinder-1 for loam and clay soils, respectively. P
126
application rates were 58.5 mg P cylinder-1 for loam and 59.8 mg P cylinder-1 for clay soils,
127
respectively. The P fertilizer application rate was two to three times higher than in normal
128
farmland (Du et al., 2013; Wang & Chu, 2015). Each treatment replicated three times giving a
129
total of 42 cylinders (containing 6 CK treatments with no fertilizer either in loam or clay soil, 18
130
cylinders for single application of MAP, PA and APP either in loam or clay soil and another 18
131
cylinders for split application of MAP, PA and APP either in loam or clay soil). All cylinders
132
were covered with a piece of parafilm, and incubated at room temperature during incubation
133
period.
134
After 28-day incubation, all cylinders were placed into a -80℃ freezer for 12 hours to keep
135
soil column hard enough. The frozen cylinders were cut into the given thick soil slices by placing
136
the cylinder to a high-speed spindle of the lathe with a sharp blade at millimeter-scale (Type:
137
CW6163C, made in Dalian, China) (Supplementary Fig). Each cylinder was vertically cut into
138
20 slices with a 5-mm thickness for each slice, then another 10 slices with a 20-mm-thickness
139
were cut. Each slice was immediately placed in a plastic bag and transferred to fridge.
140
All collected soil slices were used to determine soil moisture, soil WE-P (water extracted
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phosphorus) and Olsen-P. Briefly, to determine WE-P, an aliquot 2 g soil was taken in a 100 mL
142
centrifuge tube with 50 mL triple de-ionized (TDI) water. Shaking the soil sample for 1 hour at
143
25℃, following centrifugation for 15 min at 900 rpm, decant supernatant was separated and WE-
144
P was measured using malachite green colorimetric method at an absorbance of 610nm (Masson
145
et al., 2001). Olsen-P was determined in 0.5 M NaHCO3 extracts (pH 8.5), using the
146
molybdenum blue method (Olsen et al., 1954).
147
148
An exponential decay equation model was employed to describe the P downward P movement
in soil cylinder.
149
Y = (Y0 - Plateau) ×exp (-K × X) + Plateau
150
Where X represents the depth of the soil column expressed as mm. Y indicates the content of
151
phosphorus in soil column in mg/kg. Y0 is the Y value when X (depth) is zero. It is expressed in
152
the same units as Y; Plateau is the Y value at infinite depths, expressed in the same units as Y; K
153
is the rate constant, expressed in reciprocal of the X axis depth units (mm-1); Half-depth is in the
154
depth units of the X axis. It is computed as ln(2)/K, which means the movement depth when the
155
concentration of P drop down to the half.
156
(1)
Insert “(Figure 1)”
157
2.4 Experiment Ⅱ Soil incubation experiment
158
To explore the influences of different P types on phosphorus transformation, a 560-day
159
microcosm incubation experiment was set up. Herein only loam soil was used as described in the
160
previous section. In brief, a total of 253 g soil was thoroughly mixed with different P fertilizers
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(MAP, PA and APP) at the addition rate of 40.3 mg P pot-1. The mixed soil was placed in a
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plastic pot (5.7 cm in height, 6.6 cm in diameter) with soil bulk density of 1.30 g cm-3, all pots
163
were incubated under room condition for 560 days. Tap water was added periodically per week
164
to keep water holding capacity of 60% (WHC). Each treatment was replicated four times giving
165
a total of 16 pots (including 4 CK treatments having no fertilizer).
166
After 65-, 140-, 230-, 320-, 560-day incubation, soil samples were collected from each pot. All
167
soil samples were air dried and grounded for determination of different soil inorganic P fractions
168
according to Guppy et al. (2000) and modified by Aulakh et al. (2003). Briefly, soil P sequential
169
extraction followed: (1) resin-P: an aliquot 0.500g soil sample was mixed with 30 mL deionized
170
water in a 500 mL centrifuge tube, two Cl-saturated anion exchange resin strips were added to
171
remove resin-P, the sample was shaken for 16 h. After shaking the two Cl-saturated anion
172
exchange resin strips were taken in a 100 mL centrifuge tube and 30 mL 0.7 moL/L NaCl was
173
added subsequently, shaking the sample for 1 h. (2) NaHCO3-P: the soil residue in the previous
174
step was treated with 30 mL 0.5 moL/L NaHCO3 to remove NaHCO3-P, shaking the sample for
175
stay overnight, and centrifugation for 30 min. (3) NaOH-P: the soil residue was treated with 30
176
mL 0.1 M NaOH and 1 mL of 4 M NaCl to remove NaOH-P, shaking the sample for 16 h, and
177
subsequently centrifugation for 30 min. (4) HCl-P: the soil residue was treated with 30 mL
178
1moL/L HCl, shaking stay over. (5) Residue-P: aliquot 0.5 g anhydrous MgSO4 and 5 mL of
179
H2SO4: HClO4 (20:1) acid solution to remove residue-P. Inorganic P concentration in each of the
180
extracts was determined using malachite green colorimetric method at an absorbance of 610 nm
181
(Masson et al., 2001). The total P content of soil samples was determined using molybdenum
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183
blue spectrophotometry method after digestion with HClO4-H2SO4 (Masson et al., 2001).
The proportion of applied P fertilizer transformed to different P fractions in soils was figured
184
out referring to Aulakh et al. (2003). Individual parameters were calculated as
185
Amount of fertilizer P recovered as Pi in soil (kg P ha-1)
186
= [amount of Pi in P-fertilized treatment (kg P ha-1)] - [amount of Pi in no-P control (kg P ha-1)]
187
(2)
188
Total P recovered in soil (kg P ha-1) = [sum of P recovered in different fractions in the soils (kg P
189
ha-1) × (2.325×106 kg ha-1)
190
(3)
Where 2.325×106 kg P ha-1 is soil mass of 0-150 mm soil layer computed using field bulk
191
density of 1.55 g cm-3.
192
Total fertilizer P recovered in soil (kg P ha-1)
193
= [total P recovered in P-fertilized treatment (kg P ha-1)] – [total P recovered in no-P control (kg
194
P ha-1)]
195
Fertilizer P present in any specific soil fraction (%)
196
=
[(Pi in P - fertilized treatment ) - (Pi in no - P control )](kg P ha -1 )
Total fertilizer P recovered in soil (kg P ha -1 )
(4)
(5)
197
2.5 Statistical analyses
198
Data were analyzed using the SPSS 11.5 statistical program (SPSS Inc., Chicago, USA) with
199
two-way ANOVA at a significance level of p < 0.05. A Duncan multiple range test was carried
200
out to test the significant differences between different treatments. Microsoft Excel 2003 and
201
Graphpad Prism 5.0 software (Graphpad software, Inc., USA) were used for data processing and
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images making. All results in figures and tables were presented as mean of three or four
203
replicates with a standard deviation (SD), a statistical significance level of p < 0.05 was used for
204
all analyses.
205
3. Results
206
3.1 Different P application methods on P mobility and availability
207
Soil Olsen-P decreased with soil depth (0-100 mm) increasing across all P sources (Fig. 2).
208
Compared to the single application treatment, the significant higher values of Olsen-P were
209
always appeared in the repeated PA and APP application at 0-40 mm depth (p < 0.05). The
210
vertical movement of Olsen-P reached the same value with CK (baseline) were 80 mm and 95
211
mm, respectively, in the PA and APP repeated application treatment, while those were merely 42
212
mm and 50 mm in the single application treatment in loam soil. Similar tendency was also
213
observed in clay soil, indicating that repeated P application significantly promoted P vertical
214
migration.
215
An exponential decay model was employed to describe Olsen-P movement in soil cylinder
216
(Table 2). Rate constant (K) and half-depth were key parameters to reflect the effects of different
217
application methods on P movement. The steeper the response curve (higher K value) and lower
218
half-depth, the shorter distance the added-P moved, and vice versa. In clay soil, K value (Rate
219
constant) in the repeated P application was lower than that in the single application treatment
220
across all treatments. The values of half-depth increased by 39.7%, 49.8%, 158.6% in the
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repeated application treatment, respectively, relative to the single MAP, PA and APP application
222
treatments.
223
Insert “(Figure 2, and Table 2)”
224
3.2 Different P types on P mobility and availability
225
Compared to the treatments of MAP and PA application, soil P mobility was significantly
226
increased by APP application (Fig. 3). The distance of WE-P downward movement in the single
227
P application treatment in loam soil followed order of APP (80 mm) > MAP (60 mm) > PA (35
228
mm). Similarly, the distance of WE-P downward movement in clay soil followed the order of
229
APP (83 mm) > MAP (62 mm) > PA (55 mm). Moreover, the exponential decay equation model
230
showed that K value in loam soil followed the order of APP (0.054 mm-1) < MAP (0.065 mm-1)
231
< PA (0.115 mm-1), and the half-depth followed the order of APP (12.70 mm) > MAP (10.66
232
mm) > PA (6.02 mm) when P fertilizer applied as single application method (Table 3). Likewise,
233
K value in the repeated application treatment (loam soil) followed order of APP (0.039 mm-1) <
234
MAP (0.044 mm-1) < PA (0.062 mm-1), the half-depth followed the sequence of APP (17.92 mm)
235
> MAP (15.88 mm) > PA (11.24 mm). The half-depth in the APP treatment were 4.84% and
236
31.5%, respectively, greater than the MAP and PA treated clay soils. These results indicate that
237
APP fertilization significantly improved P mobility and availability.
238
Insert “(Figure 3, and Table 3)”
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3.3 Inorganic P transformation
240
During whole incubation time, resin-P and NaHCO3-P (labile P) notably decreased, while
241
NaOH-P (moderately labile P) and HCl-P (recalcitrant soil P) showed an increasing tendency,
242
(Fig. 4). Furthermore, soil resin-P, NaHCO3-P and NaOH-P in the APP added treatment were
243
significantly higher than in the PA and MAP treatments. For instance, at the end of incubation
244
(560 d), resin-P (5.33 mg kg-1) and NaHCO3-P (73.7 mg kg-1) in the APP addition treatment
245
increased by 51.6% and 191% respectively, relative to the MAP and PA application treatments
246
(on average). In addition, When P fertilizer was added as polyphosphate, NaOH-P was increased
247
by 24.6% and 39.8%, respectively, relative to the MAP and PA treatments. The influence of the
248
different P types on non-labile P distribution proportion followed order of MAP (87.8%) > PA
249
(73.7%) > APP (54.5%) (Table 4), indicating that polyphosphate addition significantly retarded
250
the transformation of the added-P from labile to non-labile P forms, thus reduced P fixation
Insert “(Figure 4, and Table 4)”
251
252
4. Discussion
253
Generally, short-chain soluble polyphosphate fertilizers are made up from different inorganic P
254
components at a certain proportion (i.e. ortho-P, pyro-P, triple-P, tetra-P). Therefore, poly-P
255
can’t be directly absorbed by plants unless it be hydrolyzed into orthophosphate (H2PO4- or
256
HPO42-) (Kulakovskaya et al., 2012). In this study, phosphorus downward movement in soil
257
column was significantly influenced by different types of P fertilizers (Table 2, Fig. 3).
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Application of slow release fertilizers (poly-P) significantly increased P movement. We found
259
that soil P vertical movement well-fitted the nonlinear exponential regression (0.81 < R2 < 0.99),
260
which provides a reliable evidence to our hypothesis that split repeated polyphosphate
261
application was superior over the single application method in increasing P mobility. Following
262
this, when P fertilizer is applied in single basal method, the freshly added-P is easily fixed due to
263
high P concentration in fertilization placement (Yang et al., 2012). A in situ imaging of liquid-
264
cell atomic force microscopy (AFM) showed that the formation of calcium phosphates (Ca-P)
265
nanoclusters was markedly increased as P presented at high concentration [50 mM (NH4)2HPO4]
266
(Wang and Putnis, 2020). Besides, the direction of P diffusion from P application site to outside
267
is opposite to the direction of water movement caused by low soil water potential (Hedley &
268
McLaughlin, 2005). Therefore, the diffusion of H2PO4-/HPO42- would inevitably be blocked by
269
water movement (Hinsinger, 2001). In contrast, when P fertilizer is applied though split repeated
270
method, the concentration of P in P application site is relative low, resulted in the distribution P
271
in soil was scattering and dispersive (Hettiarachchi et al., 2006; McLaughlin et al., 2011). Our
272
results were consistent with the findings by Lombi et al. (2004) who noted that fluid sources of P
273
enhanced phosphorus mobility and liability compared with granular P fertilizers. The reasons
274
may likely attributed to: (і) poly-P fertilizers belong to slow-release fertilizer, they move in
275
condensed form in soil before it be hydrolyzed (McBeath et al., 2007), and it hardly interacted
276
with soil mineral particles of Fe2O3/Al2O3 (Hamilton et al., 2016; Hamilton et al., 2018); (іі)
277
poly-P may mobilize soil native P via chelating reaction. In this way, Mcbeath et al. (2007)
278
showed that poly-P activated recalcitrant oxidation forms of soil P (Fe2O3 and MnO2). Similarly,
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Wang et al. (2019) found that poly-P fertilizer treatment significantly increased P availability in
280
calcareous soil. Besides, poly-P fertilizer application decrease soil rhizosphere pH value at 0.1-
281
0.5 unit (Hamilton et al., 2018; Rahmatullah et al., 2006; Mcbeath et al., 2007: Du et al., 2013).
282
Likewise, our data showed that the poly-P fertilizer application treatment decreased solution pH
283
by ~0.34 unit than MAP and PA fertilizer (Table 5), which might be another possible reason for
284
polyphosphate mobilizing native soil P. Although the effects of poly-P applied to agricultural
285
soils on increasing soil P availability and reducing its fixation is promising, nevertheless further
286
study is needed by using edge-cutting technologies to fully elucidate the mechanisms of
287
mobilizing effect by poly-P application and their interaction with soil particles in a wide-range of
288
soil conditions.
Insert “(Table 5)”
289
290
5. Conclusion
291
The mobility, effectiveness, and availability of fertilizer P were significantly increased with
292
ammonium-polyphosphate application compared with MAP and PA application. Compared to P
293
fertilizer basal application, split repeated P application markedly promoted soil P movement.
294
Moreover, the added-P transformation from labile (resin-P and NaHCO3-P) to non-labile forms
295
(HCl-P and residue-P) in the poly-P treated soil was significantly retarded, in contrast to MAP
296
and PA application treatments. Therefore, considering the scarcity of P resource and low P
297
fertilizer use efficiency, polyphosphate fertilizers coupled with split repeated application method
298
is recommended as an effective P management strategy in increasing soil available P decreasing
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P fixation in calcareous soil.
300
301
Funding
302
This work was jointly supported by National Natural Science Foundation of China (No.
303
41161047), and the scientific development and technology innovation project of Xinjiang
304
Production and Construction Group (XPCG), China (No. 2017BA041) and the Shenzhen Batian
305
ecological engineering Co., Ltd.
306
307
Competing Interests
308
The authors declare that they have no competing interests.
309
310
Acknowledgement
311
Thanks are given to Dr. & Prof. Wakelin A Steve from New Zealand for his helpful comments
312
and revision.
313
314
Author Contributions
315
Jawad Ali SHAH: methodology, sampling, measurement, data collection and analysis writing-
316
original draft, interpretation, and approved the final draft.
317
CHU Gui-xin: project administration, conceptualization, experiment-design, supervision,
318
funding acquisition, resources, critical manuscript revision, and approved the final draft.
319
Data Availability
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The following information was supplied regarding data availability:
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The raw data is available as a Supplemental File.
322
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Table 1(on next page)
Selected physical and chemical properties of the tested soils
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Table 1.
1
2
3
4
5
6
7
8
9
Soil properties
Texture a (<0.01mm %)
pH b
EC c
OM d (g kg-1)
Total-N e(g kg-1)
Olsen-P f (mg kg-1)
Water-P g (mg kg-1)
Total-P h (g kg-1)
Olsen-K i (mg kg-1)
CaCO3 j (%)
Loam
37.0±0.02
7.83±0.03
0.53±0.01
21.0±0.23
0.85±0.05
24.2±3.52
3.54±0.39
1.22±0.12
518±2.07
16.1±0.00
Clay
71.4±0.02
8.15±0.05
1.22±0.02
15.6±0.03
0.71±0.02
11.9±2.18
3.58±0.01
0.95±0.01
291±5.53
15.0±0.00
10
the mean ± standard deviation (SD), n=3 at a significance level of p < 0.05.
11
aTexture
12
bpH
13
cSoil
14
dOrganic
15
eSoil
16
fOlsen
17
gWater-P
18
hTotal
P was measured by the perchloric acid digestion method (Olsen et al. 1954).
19
iOlsen
K was measured by the flame photometry method (Bao 2000).
20
jCaCO
3
Selected
physical and
chemical
properties of
the tested
soils
Data
presented
was determined by the Bouyoucos hydrometer method and Katschinski classification System (Bouyoucos 1962).
was determined at soil to milli-Q water ratio of 1:2.5 w/v using pH meter
EC was measured using a 1:2.5 ratio of soil to Milli-Q water.
C was measured by the wet-oxidation technique (Shaw 1959).
total N was measured by the semimicro-Kjeldahl method; (Bao 2000).
P was measured by the Olsen method (Olsen et al. 1954).
was measured using a 1:25 ratio of soil to milli-Q water.
was measured by neutral titration method (Bao 2000).
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were
as
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21
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Table 2(on next page)
Exponential equations simulating the influences of different P fertilization methods on
soil P downward movement (Olsen-P, mg kg-1)
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Table 2. Exponential equations simulating the influences of different P fertilization methods on soil P downward movement (Olsen-P, mg kg-1)
1
P form
MAP
Application
method
Single
Repeated
Single
Repeated
PA
Loam soil
Clay soil
Equation
y = 192.03e-0.059x+19.07
K (mm-1)
0.059
Half-depth (mm)
11.8
R2
0.97
Equation
y = 216.05e-0.056x+10.45
K (mm-1)
0.056
Half-depth (mm)
12.49
R2
0.98
y = 144.70e-0.053x+24.70
y = 152.68e-0.105x+17.12
0.053
0.105
13.1
6.57
0.94
0.87
y = 155.72e-0.040x+7.38
y = 153.98e-0.059x+13.12
0.040
0.059
17.45
11.72
0.96
0.98
y = 181.36e-0.063x+20.94
y = 187.05e-0.056x+17.25
0.063
0.056
10.9
12.3
0.90
0.97
y = 151.40e-0.040x+8.10
y = 286.92e-0.060x+10.58
0.040
0.060
17.56
11.51
0.97
0.81
0.023
29.77
0.94
Single
Repeated
y = 785.95e-0.044x+12.65
0.044
15.9
0.99
y =471.57e-0.023x-40.17
Data were presented as the mean value of three replicates and standard deviation (SD) at a significance level of p < 0.05.
APP
2
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Table 3(on next page)
Exponential equations simulating the influences different P sources on phosphorus
downward movement (water-P, mg kg-1)
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1
Table 3. Exponential equations simulating the influences different P sources on phosphorus downward movement (water-P, mg kg-1)
Application
method
P
form
Loam soil
Clay soil
Equation
y = 192.21e-0.065x+12.79
K (mm-1)
0.065
Half-depth (mm)
10.7
R2
0.97
Equation
y = 104.94e-0.041x+9.56
K (mm-1)
0.041
Half-depth (mm)
16.72
R2
0.94
y =163.38e-0.115x+12.02
y = 410.54e-0.054 x +12.96
y = 95.8e-0.044x+15.30
0.115
0.054
0.044
6.02
12.7
15.9
0.92
0.95
0.92
y = 89.25e-0.052x+14.25
y = 182.075e-0.040x+8.53
y =98.48e-0.031x+6.02
0.052
0.040
0.031
13.33
17.53
22.43
0.93
0.96
0.93
PA
y = 153.71e-0.062x+14.29
0.062
11.2
0.93
y = 90.86e-0.031x+8.94
APP
y = 496.43e-0.039x+5.87
0.039
17.9
0.97
y =334.01e-0.014x-80.51
Data were presented as the mean value of three replicates and standard deviation (SD) at a significance level of p < 0.05.
0.031
0.014
22.47
50.93
0.91
0.96
MAP
Single
PA
APP
MAP
Repeated
2
3
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Table 4(on next page)
The influences of MAP, PA and APP applications on different inorganic P fractionations in
soil (after 560-day incubation)
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Table 4. The influences of MAP, PA and APP applications on different inorganic P fractionations in soil (after
2
560-day incubation)
3
4
5
Treatment
Resin-P (%)
NaHCO3-P (%)
NaOH-P (%)
HCl-P (%)
Residue-P (%)
MAP
PA
APP
0.23±0.06
1.07±0.03
1.64±0.15
6.13±0.2
20.0±0.1
36.7±0.4
5.82±0.8
5.19±0.1
7.25±0.9
21.7±0.6
58.2±0.9
45.4±0.5
66.1±1.0
15.5±0.8
9.10±0.1
Abbreviation: MAP, mono-ammonium phosphate; PA, phosphoric acid; APP, ammonium polyphosphate.
Data were presented as the mean value of four replicates and standard deviation (SD) at a significance level of p < 0.05.
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Table 5(on next page)
pHs in MAP, PA and APP fertilizer and mixed with soils.
pHs in MAP, PA and APP fertilizer and mixed with soils.
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1
2
3
4
5
6
7
8
9
Table 5. pHs in MAP, PA and APP fertilizer and mixed with soils.
Treatment
pH (fertilizer)
pH (fertilizer mixed in loam soil)
pH (fertilizer mixed in clay soil)
MAP
PA
APP
5.15±0.08
2.01±0.05
1.36±0.02
7.81±0.07
7.99±0.09
7.55±0.06
8.37±0.03
8.38±0.09
8.06±0.05
Abbreviation: MAP, mono-Ammonium phosphate; PA, phosphoric acid; APP, ammonium polyphosphate.
10
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Figure 1
Schematic diagram of soil cylinder experiment.
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Figure 2
The effect of different P application methods on phosphorus mobility and availability.
Data are presented as mean ± standard deviation (SD) of three replicates at a
significance level of p < 0.05. The three lines represent MAP, PA and APP, respectively.
T
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Figure 3
The influences of different P sources on soil phosphorus mobility and availability. Data
are presented as mean ± standard deviation (SD) of three replicates at a significance
level of p < 0.05. The upper and lower rows represent single application and rep
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Figure 4(on next page)
The effect of different P sources and their application methods on the changes of soil
resin-P, NaHCO3-P, NaOH-P, HCl-P and residue-P. Data are presented as mean ±
standard deviation (SD) of four replicates at a significance level of p < 0.05 (n
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Fig. 4
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| 7,305 | 48,008 |
W3166842837.txt | Journal of Learning Development in Higher Education
ISSN: 1759-667X
Special Issue 22: Compendium of Innovative Practice
October 2021
________________________________________________________________________
Watch party lectures: synchronous delivery of asynchronous
material
Carolina E. Kuepper-Tetzel
University of Glasgow
Emily Nordmann
University of Glasgow
Keywords: instruction; lectures; synchronous and asynchronous learning; student
engagement; Covid-19
The challenge
When the Covid-19 pandemic hit in early 2020, the majority of university activities on campus
were suspended across the world, and teaching pivoted online (Nordmann et al., 2020). The
challenge was to provide students who were enrolled and expecting a face-to-face, oncampus learning experience with a similar university experience that would encourage them
to engage with learning and to develop a sense of belonging (Peacock et al., 2020). As year
leads for first- and second-year Psychology at our university, our cohorts were particularly
large with approximately 600 first-year students and 250 second-year students. Each week
students attend three to five lectures and one practical lab session, and previously all
teaching was delivered in person on campus.
In the process of planning our move online, we realised the need to develop an approach that
offered both asynchronous and synchronous elements. Synchronous sessions are vital, not
only for community building, but there is also a positive association between scheduled
teaching of lectures and practicals and academic achievement (Torenbeek et al., 2012).
However, with students studying from home in different countries, under a variety of
circumstances, and with differential access to internet, study space and technology, it was
important to offer all essential teaching material asynchronously too (Nordmann et al., 2020).
Kuepper-Tetzel and
Nordmann
Watch party lectures: synchronous delivery of asynchronous material
In the initial period of emergency remote teaching directly following the first lockdown, we kept
in touch with students through online remote social activities such as meeting online for a
watch party of psychology-related movies and used the integrated chat box function to write
comments or thoughts. This was when the concept of ‘watch party lectures’ was born.
The response
Watch party lectures combine asynchronous and synchronous teaching approaches.
Lecturers record short videos of directly instructed content; for one-hour lectures, lecturers
aim to record 2-3 short videos for a maximum of 40-45 minutes. These pre-recorded videos
are uploaded onto the virtual learning environment (VLE). However, the crucial element of
watch party lectures is the scheduled, synchronous time during which these pre-recordings
are watched together via screen sharing. Watch parties purposely differ from a flipped
classroom approach in which students are required to complete the pre-recordings in their
own time before coming to class. While, in general, the flipped classroom approach has
shown positive effects on student performance across educational sectors and disciplines
(Strelan et al., 2020), students, particularly younger cohorts, may become overwhelmed by
the added requirement of independent learning and, consequently, not complete the learning
activities prior to class (Tomas et al., 2019). This may be more pronounced now as students
report additional strains on mental health and wellbeing during the pandemic (e.g., Chirikov et
al., 2020; Mechili et al., 2020; Wang et al., 2020). Thus, watch party lectures offer a
scheduled time during which the core teaching content is delivered.
Perhaps more importantly, watch party sessions allowed for meaningful interactions between
lecturers and students while watching the videos. Students would post questions or
comments in the chat box, and the lecturer could address them immediately, for example, by
providing links to additional resources. We observed more engagement during watch party
lectures from a more diverse body of students than we have experienced in face-to-face
lectures, which reflects students’ preference for using the chat box during instruction (see Vu
and Fadde, 2013). Collaborative co-creation of knowledge that combines social and cognitive
Journal of Learning Development in Higher Education, Issue 22: Compendium October 2021
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Kuepper-Tetzel and
Nordmann
Watch party lectures: synchronous delivery of asynchronous material
processes aligns well with the theoretical framework of Community of Inquiry (Garrison et al.,
2001; Cleveland-Innes et al., 2019); and is an essential aspect of watch party lectures.
Discussion took place not only between lecturers and students, but also between peers who
would frequently answer questions or provide follow-up opinions and thoughts to points raised
in the chat. Besides the ongoing discussions, lecturers enriched the watch party sessions with
short quiz activities, either to review previously taught material or to prompt upcoming
content. Quizzing during lectures not only provides students with a short break from watching
the videos, but is also a beneficial strategy to increase retention and transfer of knowledge
(Thomas et al., 2020).
Although multiple platforms would be capable of presenting watch parties, we used Zoom
(Zoom Video Communications Inc, 2016). In addition to being adopted as a core technology
by our institution, Zoom had no known restrictions in terms of international access and was
also available for students on mobile devices, and we were not made aware of any cases in
which students who wished to engage with the sessions were not able to do so. Recordings
of the watch party sessions including the chat conversations were also made available to
students for asynchronous engagement, although the community and active learning benefits
of the sessions would have been limited.
The feedback from students on the watch party lectures has been overwhelmingly positive,
for example:
I like that all lectures are watch party style, it makes it feel more interactive and
'normal’. None of my other subjects have done this so every week I look forward to my
psychology lectures. (Anonymous, 2020a)
Online live lecture watch parties are amazing because it feels a lot more engaging than
just having to watch the already recorded lecture alone. (Anonymous, 2020b)
In student evaluations, our approach was praised as an effective way to engage with the
material, ask questions during the session, and catch-up afterwards. Students appreciated
Journal of Learning Development in Higher Education, Issue 22: Compendium October 2021
3
Kuepper-Tetzel and
Nordmann
Watch party lectures: synchronous delivery of asynchronous material
the different activities during the watch party sessions, citing that these kept them focused
and encouraged further elaboration on the topic. As expected, students highlighted the chat
box conversations as a positive feature of watch party lectures. Student feedback was so
positive that the format was adopted by other lecturers and departments at both our own and
other institutions.
Recommendations
Lecturers interested in implementing this approach may experience an initial hesitation about
watching themselves during the watch party session, but from our own experiences and from
speaking to our colleagues, this was not perceived as an issue, or was one that quickly
became normal. Lecturers enjoyed the interactions in the chat box, which allowed them time
to construct more elaborate answers to student questions. When moving back to face-to-face
lectures, we recommend integrating text-based chats during lectures. Lecturers could
encourage students to post comments and questions during the lecture using existing tools
(e.g., Teams, forums on the VLE) and then take brief Q&A breaks during the lecture. After the
lecture, the lecturer could reply to questions that require more elaborate answers or additional
resources.
In conclusion, watch party lectures demonstrate the benefits of strategically combining
asynchronous and synchronous approaches. When considering what the new normal of
lectures will look like, it is crucial to consider their social and community aspects (Nordmann
et al., 2021), and we strongly recommend integrating both asynchronous and synchronous
elements regardless of the modality or format of teaching delivery.
References
Chirikov, I., Soria, K.M., Horgos, B, and Jones-White, D. (2020) ‘Undergraduate and graduate
students’ mental health during the COVID-19 pandemic’, UC Berkeley: Center for
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Nordmann
Watch party lectures: synchronous delivery of asynchronous material
Studies in Higher Education, viewed 3 June 2021. Available at:
https://escholarship.org/uc/item/80k5d5hw (Accessed: 30 June 2021).
Cleveland-Innes, M., Gauvreau, S., Richardson, G., Mishra, S. and Ostashewski, N. (2019)
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of Inquiry theoretical framework’, International Journal of E-Learning & Distance
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Nordmann, E., Hutchison, J. and MacKay, J.R.D. (2021) ‘Lecture rapture: the place and case
for lectures in the new normal’, EdArXiv. Available at:
https://doi.org/10.35542/osf.io/h8zt6 (Accessed: 5 July 2021).
Peacock, S., Cowan, J., Irvine, L. and Williams, J. (2020) ‘An exploration into the importance
of a sense of belonging for online learners’, The International Review of Research in
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Open and Distributed Learning, 21(2), pp. 18-35. Available at:
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achievement: the role of the curriculum, time investment and self-regulated learning’,
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Strelan, P., Osborn, A. and Palmer, E. (2020) ‘The flipped classroom: a meta-analysis of
effects on student performance across disciplines and education levels’, Educational
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https://doi.org/10.1016/j.edurev.2020.100314 (Accessed: 5 July 2021).
Thomas, A.K., Smith, A.M., Kamal, K. and Gordon, L.T. (2020) ‘Should you use frequent
quizzing in your college course? Giving up 20 minutes of lecture time may pay off’,
Journal of Applied Research in Memory and Cognition, 9(1), pp. 83-95. Available at:
https://doi.org/10.1016/j.jarmac.2019.12.005 (Accessed: 5 July 2021).
Tomas, L., Doyle, T. and Skamp, K. (2019) ‘Are first year students ready for a flipped
classroom? A case for a flipped learning continuum’, International Journal of
Educational Technology in Higher Education, 16(1), pp.1-22. Available at:
https://doi.org/10.1186/s41239-019-0135-4 (Accessed: 5 July 2021).
Wang, X., Hegde, S., Son, C., Keller, B., Smith, A. and Sasangohar, F. (2020) ‘Investigating
mental health of US college students during the COVID-19 pandemic: cross-sectional
survey study’, Journal of Medical Internet Research, 22(9), pp. e22817. Available at:
https://doi.org/10.2196/22817 (Accessed: 5 July 2021).
Vu, P. and Fadde, P.J. (2013) ‘When to talk, when to chat: student interactions in live virtual
classrooms’, Journal of Interactive Online Learning, 12(2), pp.41-52. Available at:
http://www.ncolr.org/jiol/issues/pdf/12.2.1.pdf (Accessed: 5 July 2021).
Journal of Learning Development in Higher Education, Issue 22: Compendium October 2021
6
Kuepper-Tetzel and
Nordmann
Watch party lectures: synchronous delivery of asynchronous material
Zoom Video Communications Inc. (2016) Security guide. Available at:
https://d24cgw3uvb9a9h.cloudfront.net/static/81625/doc/Zoom-Security-WhitePaper.pdf (Accessed: 30 June 2021).
Author details
Carolina E. Kuepper-Tetzel is a Senior Lecturer in Psychology at the University of Glasgow
and a Senior Fellow of the HEA. She is an expert in applying findings from cognitive science
to education and an enthusiastic science communicator. She is a member of The Learning
Scientists and the founder of the Teaching Innovation & Learning Enhancement (TILE)
network. You can follow her work on Twitter: @pimpmymemory.
Emily Nordmann is a Senior Lecturer in Psychology at the University of Glasgow and was
awarded the British Psychological Society HE Psychology Teacher of the Year in 2020. She
is an expert in lecture capture and a strong proponent of open educational resources and the
teaching of reproducible research methods. You can follow her work on Twitter:
@emilynordmann.
Journal of Learning Development in Higher Education, Issue 22: Compendium October 2021
7
| 1,964 | 13,999 |
W2996636121.txt | materials
Article
A Parameter Study for 3D-Printing Organized
Nanofibrous Collagen Scaffolds Using
Direct-Write Electrospinning
Frank A. Alexander Jr. 1, *, Lee Johnson 2, *, Krystaufeux Williams 3 and Kyle Packer 4
1
2
3
4
*
The Geneva Foundation, Tacoma, WA 98402, USA
Meadowave LLC, NE Washington, DC 20012, USA
Chemistry Division, Center for Corrosion Science and Engineering (CCSE), US Naval Research Laboratory,
SW Washington, DC 20375, USA; krystaufeux.williams@nrl.navy.mil
Womack Army Medical Center, Fort Bragg, NC 28310, USA; kyle.t.packer.mil@mail.mil
Correspondence: falexander@genevausa.org (F.A.A.J.); ljohnson@meadowave.com (L.J.)
Received: 1 November 2019; Accepted: 27 November 2019; Published: 10 December 2019
Abstract: Collagen-based scaffolds are gaining more prominence in the field of tissue engineering.
However, readily available collagen scaffolds either lack the rigid structure (hydrogels) and/or the
organization (biopapers) seen in many organ tissues, such as the cornea and meniscus. Direct-write
electrospinning is a promising potential additive manufacturing technique for constructing highly
ordered fibrous scaffolds for tissue engineering and foundational studies in cellular behavior, but
requires specific process parameters (voltage, relative humidity, solvent) in order to produce organized
structures depending on the polymer chosen. To date, no work has been done to optimize direct-write
electrospinning parameters for use with pure collagen. In this work, a custom electrospinning 3D
printer was constructed to derive optimal direct write electrospinning parameters (voltage, relative
humidity and acetic acid concentrations) for pure collagen. A LabVIEW program was built to
automate control of the print stage. Relative humidity and electrospinning current were monitored in
real-time to determine the impact on fiber morphology. Fiber orientation was analyzed via a newly
defined parameter (spin quality ratio (SQR)). Finally, tensile tests were performed on electrospun
fibrous mats as a proof of concept.
Keywords: electrospinning; collagen; direct-write electrospinning; scaffolds
1. Introduction
Electrospinning is a well-established technique for creating nanofibers that can be used for a
variety of applications. The core technique was first demonstrated more than a century ago [1];
however, recent interest in biomaterials has caused a resurgent interest in it as a method for developing
scaffolds for regenerative medicine and wound dressings [2]. In a classic far-field electrospinning
(FFES) setup, a syringe pump perfuses a polymer solution through a conductive syringe tip to a
grounded collector with a high voltage power supply. When enough voltage is applied between the
syringe and collector circuit, charge buildup in the viscoelastic fluid pumped through the syringe
deforms the meniscus into a “Taylor cone” and a nanofiber jet erupts from the tip of the cone when
the electrical force overcomes surface tension. As the nanofiber jet is drawn closer to the surface, the
carrier liquid evaporates and nanofibers form. Dried fibers accumulate onto the grounded collector
electrode surface located below the syringe.
Typically, in FFES the syringe tip is held a large distance (centimeters) from a target. This results in
nanofibers collecting in random “pile of spaghetti”-like orientations. Alongside bioprinted hydrogels [3],
Materials 2019, 12, 4131; doi:10.3390/ma12244131
www.mdpi.com/journal/materials
Materials 2019, 12, 4131
2 of 14
unorganized “biopaper” scaffolds [4] have been popular due to their ease of production and ability
to recapitulate biochemical cues seen in vivo by using natural polymers and a nanoporous structure.
However, these systems still lack critical microarchitecture especially when creating tissue analogues
of more ordered nanofibrous structures [5]. The lack of systematically organized fibrils may result in
insufficient mechanical properties of these artificial tissues. Some groups have designed bioreactors to
physically stimulate cellular production of organized collagen structures but production times can be
extensive [6]. Leveraging additive manufacturing techniques to produce more similar tissue-specific
analogues may increase utility.
Various groups have developed methods to finely pattern electrospun nanofibers into organized
structures. Fibers have been directed onto spinning mandrel collectors to create fiber spools [7],
micropatterned via insulator-collector geometries to induce specific shapes [8] and positioned in precise
locations by rapidly translating the collector with an X-Y stage [9–12]. The latter technique, sometimes
termed direct-write electrospinning (DWES) is a relatively new additive manufacturing approach to
electrospinning into highly organized confirmations.
DWES significantly lowers the required voltage to induce Taylor cone formation by decreasing
the syringe tip to collector distance and incorporates a translating X-Y stage to pattern collected
nanofibers. Li et al. successfully lowered spinning voltages to thresholds as small as 50 V, enabling
them to electrospin living bacteria immersed in a polyethylene oxide-water solution [10]. Furthermore,
they demonstrated the ability to electrospin directly onto hydrogels and PDMS films as well as
electrospin freestanding fibers across two spin “initiators.” [8]. Polymer melts have been elecrospun
in melt electro-writing (MEW) to create organized fibrous scaffolds, but they are incompatible with
heat sensitive polymers like collagen [13–18]. Additionally, varying polymer solutions and custom
equipment make it is necessary to re-optimize methods across laboratories. This drawback is the
primary disadvantage preventing DWES from being a scalable method for scaffold manufacturing.
Natural, synthetic, and even blended polymer biomaterials [2] have been employed in
electrospinning to generate nanofiber constructs that imitate native extracellular matrix (ECM) for
tissue regeneration [2]. Collagen is particularly interesting as a scaffolding material because it is highly
abundant within mammalian ECM. Of the 29 types of native collagen, Type I makes up 70–90% of
collagen within the human body and can be found in tightly organized fiber networks in tissues like
the meniscus [19,20]. Despite this, instead of electrospinning pure collagen into organized nanofibrous
collagen scaffolds, the majority of researchers have opted for hydrogels or nanofibrous scaffolds with
random orientation. These scaffolds are suitable in many applications, however, in many tissues
precise fiber organization results in unique in vivo properties like transparency in the cornea [21].
For this reason, DWES is a superior method for producing organized collagen scaffolds.
Despite the extensive work to optimize parameters for traditional collagen electrospinning,
organized collagen scaffolds have yet to be produced via DWES. Leveraging DWES as a 3D printing
approach can help to further develop more complex and biomimetic scaffolds that mimic the native
environment of cells or introduce bio-bandages that accelerate wound healing. A critical and often
overlooked step in electrospinning is determining optimal environmental parameters for nanofiber
production, as process temperature and relative humidity can directly impact fiber morphology.
For this reason, it is critical to optimize a consistent production environment for repeatable fiber
making in order to scale for large-scale manufacturing of scaffolds. Many prior groups have done
exhaustive studies on solution characteristics such as solvent composition [22] but relatively few
have done exhaustive studies on environmental parameters. To our knowledge, no such studies on
environmental parameters have been performed for direct-write electrospinning of pure collagen.
Here, we present a systematic approach to optimizing critical parameters for DWES of
collagen employing an environmental chamber for strict monitoring of relative humidity during
the electrospinning process. Our printer, capable of manufacturing highly ordered 3D printed scaffolds
from nanofibrous collagen, was evaluated by optimizing electrospinning characteristics of collagen
across an array of varied process parameters including spin voltage, relative humidity and acetic acid
Materials 2019, 12, 4131
3 of 14
concentration. The print procedure was automated using custom LabVIEW software built to control
translation of the print stage and import common G-code files. Additionally, process parameters
were monitored in real-time to assess the impacts of small-scale voltage and humidity fluctuations
effect fiber morphology and placement. Electrospun collagen fibers were examined to ascertain the
morphology
of2019,
nanofibers
produced
Materials
12, x FOR PEER
REVIEW and the quality of fiber placement which we have defined
3 of 14 as the
spin quality ratio (SQR). Finally, to demonstrate their mechanical properties we have tensile tested the
96
monitored
in real-timefiber
to assess
the impacts of small-scale voltage and humidity fluctuations effect
manufactured
nanofibrous
mats.
97
fiber morphology and placement. Electrospun collagen fibers were examined to ascertain the
morphology
of nanofibers produced and the quality of fiber placement which we have defined as the
2. 98
Materials
and Methods
99
spin quality ratio (SQR). Finally, to demonstrate their mechanical properties we have tensile tested
100Electrospinning
the manufactured
nanofibrous
2.1.
Printer
Setup fiber mats.
101An 2.
Materials and Methods
electrospinning
3D printer was designed for all collagen fiber production experiments. The
electrospinning printer contains four main components: an X-Y stage, a syringe pump, a high voltage
102
2.1. Electrospinning Printer Setup
DC power supply and a Z-positioner. Two DC servo linear translation stages (ThorLabs, Newton, NJ,
103 were bolted
An electrospinning
3D printer
was
designed
for all collagen
fiber production
experiments.
USA)
together to serve
as the
main
X-Y positioner.
Attached
to the X-Y
positioner,The
a custom
104
electrospinning printer contains four main components: an X-Y stage, a syringe pump, a high voltage
stage (3D printed with a Form2 from FormLabs, Somerville, MA, USA) houses an integrated ground
105
DC power supply and a Z-positioner. Two DC servo linear translation stages (ThorLabs, Newton NJ)
plane
gold-coated
Thepositioner.
ground plane
is to
connected
to a metal
microscope
106 made
were from
boltedatogether
to serveglass
as theslide.
main X-Y
Attached
the X-Y positioner,
a custom
slide
is clipped
the negative
terminal
of a PS300
voltage
powerground
supply
(Stanford
107 holder
stagethat
(3D printed
withto
a Form2
from FormLabs,
Somerville
MA)high
houses
an integrated
plane
108
made
from Sunnyvale,
a gold-coatedCA,
glass
slide.An
TheNE-1000
ground plane
is connected
to a Era,
metalFarmingdale,
microscope slide
Research
Systems,
USA).
syringe
pump (New
NY, USA)
109
holder
that
is
clipped
to
the
negative
terminal
of
a
PS300
high
voltage
power
supply
(Stanford
was used to drive collagen solution from a 3 mL syringe to a 27-gauge needle tip mounted to a third
110
Research
Systems, Sunnyvale
An NE-1000 A
syringe
pumpInstruments
(New Era, Farmingdale
was used with
screw-driven
translation
stage for CA).
Z-positioning.
National
PXIe-1071NY)
workstation
111
to drive collagen solution from a 3 mL syringe to a 27-gauge needle tip mounted to a third screwa custom LabVIEW program was used to synchronize the voltage supply, syringe pump and stage into
112
driven translation stage for Z-positioning. A National Instruments PXIe-1071 workstation with a
a 113
coordinated
routine.
Within
G-code
can be supply,
uploaded
to print
customprint
LabVIEW
program
was the
usedprogram,
to synchronize
the voltage
syringe
pumpcomplex
and stagegeometries.
into
The
entire
print
setup
is
housed
in
an
ETS-5532
environmental
chamber
to
control
temperature
and
114
a coordinated print routine. Within the program, G-code can be uploaded to print
complex
115
geometries.
The
entire
print
setup
is
housed
in
an
ETS-5532
environmental
chamber
to
control
humidity (Figure 1).
116
temperature and humidity (Figure 1).
(A) A1.schematic
depiction
of the
complete
printer
setup.
(B)direct-write
The direct-write
117Figure 1.Figure
(A) A schematic
depiction
of the
complete electrospinning
electrospinning printer
setup.
(B) The
118printhead
printhead
and collector
stage.
Detail
imageshowing
showing single
erupting
fromfrom
TaylorTaylor
cone on
and collector
stage.
(C) (C)
Detail
image
singlefiber
fiber
erupting
cone on
119surface of
surface
of the droplet
meniscus
depositingon
onsubstrate
substrate collector.
the droplet
meniscus
andand
depositing
collector.
Materials 2019, 12, 4131
4 of 14
2.2. Collagen Solution Formula
Collagen solutions were made with acetic acid and evaluated for their ability to form aligned
fibers during the electrospinning process. 500 mg of type I bovine dermis derived atelocollagen powder
(Cosmo Bio USA, Carlsbad, CA) was vortexed in 2 mL of 40, 50 and 60% acetic acid (Sigma-Aldrich,
St. Louise, MO, USA) diluted in deionized water until completely dissolved (approximately 30 min)
to a concentration of 250 mg/mL. The solution was then preloaded into a 3 mL syringe and placed
onto the syringe pump. From there, it was coupled to the dispensing needle using 24 Gauge
polytetrafluoroethylene (PTFE) tubing with luer lock hubs from Hamilton (Reno, NV, USA).
2.3. Electrospinning Process
Collagen fibers were electrospun directly onto glass slides pre-treated with dry film Teflon spray
(Dupont, Midland, MI, USA). Up to 600 lines of collagen were spun onto samples 30 lines at a time with
a pitch of 15 microns and an approximate syringe tip to substrate distance of ~0.580 mm. A two-step
voltage application was employed where 1600 V was initially applied to induce fiber formation and
the voltage was then lowered to a preset maintenance voltage after three seconds and held there for
the duration of the run. Temperature, relative humidity, and maintenance voltage were all controlled
via the custom LabVIEW software and parameters were varied to determine optimal spin parameters.
Table 1 shows the experimental parameters tested. Optimal spin quality was determined by counting
the number of organized, straight fibers drawn on the sample using image processing in ImageJ
and Matlab.
Table 1. The list of parameters surveyed for optimizing collagen nanofiber formation.
Parameters
[Acetic Acid] (%)
Relative Humidity (%)
Voltage (V)
40
20
1000
50
30
1100
60
40
1200
50
1300
1400
1500
2.4. Scanning Electron Microscopy (SEM)
Electrospun collagen fibers were examined with scanning electron microscopy to ensure a fibrous
morphology and to compare microstructural differences across multiple spinning parameters. Samples
to be imaged were sputter coated with a thin layer of carbon (~30 nm) prior to loading in the
SEM. Scanning electron microscopy images were acquired using a Hitachi SU-70 SEM (Tokyo, Japan).
All electron microscopy was performed at the Advanced Imaging and Microscopy Laboratory (AIMLab)
at the University of Maryland Nanocenter.
2.5. Definition of High-Quality Fibers
The quality of fibers produced can vary drastically given the combination of chamber humidity,
spin voltage, and acetic acid concentration. For this reason, a quantitative measure of spin quality
was defined after preliminary investigations of direct-write electrospinning parameters. During each
electrospinning experiment, a target number of straight collagen fibers was recorded along with the
resultant number of straight fibers produced. Primarily, our use of direct-write electrospinning is as
an additive manufacturing technique capable of producing highly organized electrospun scaffolds.
As such, production of wavy or curly fibers reduce placement accuracy and predictability of the print
technique, and hence, these fibers were excluded from the line counts (Figure 2). Here, we define the
spin quality ratio (SQR) as Equation (1):
SQR = (# of straight lines produced)/(# of lines attempted),
(1)
Materials 2019, 12, 4131
5 of 14
The SQR can be used as a plotted metric to estimate quantitatively the linearity of lines produced
during
process.
Materials the
2019,electrospin
12, x FOR PEER
REVIEW
5 of 14
159
160
161
Figure 2. A light microscopy image comparing straight fibers and curly fibers produced during the
electrospinning
fibers
were
detected
usingusing
an image
processing
algorithm
and counted
electrospinningprocess.
process.Straight
Straight
fibers
were
detected
an image
processing
algorithm
and
and
curlyand
fibers
were
filtered
counts.
counted
curly
fibers
werefrom
filtered
from counts.
162
2.6. Fiber Counting Algorithm
2.6. Fiber Counting Algorithm
Samples were imaged with a Nikon Eclipse Upright microscope (Tokyo, Japan) with a motorized
Samples were imaged with a Nikon Eclipse Upright microscope (Tokyo, Japan) with a motorized
X-Y stage and a composite image of the entire slide was constructed using Nikon Elements BR (Basic
X-Y stage and a composite image of the entire slide was constructed using Nikon Elements BR (Basic
Research) software (version 4.13.04, Tokyo, Japan). An ImageJ pre-processing macro was created
Research) software (version 4.13.04, Tokyo, Japan). An ImageJ pre-processing macro was created to
to enhance the contrast between the background and fibers in order to binarize images. A Matlab
enhance the contrast between the background and fibers in order to binarize images. A Matlab
algorithm was formulated to count the number of high-quality fibers deposited onto sample slides.
algorithm was formulated to count the number of high-quality fibers deposited onto sample slides.
The block diagram in Figure 3 outlines the procedures for (a) pre-processing images and (b) fiber
The block diagram in Figure 3 outlines the procedures for (a) pre-processing images and (b) fiber
calculations. First a single slide image was converted into an 8-bit format within ImageJ. Brightness
calculations. First a single slide image was converted into an 8-bit format within ImageJ. Brightness
and contrast were adjusted to 80 and 167, respectively. A rolling ball image background subtraction
and contrast were adjusted to 80 and 167, respectively. A rolling ball image background subtraction
was performed to improve contrast further (rolling ball = 5 light disable) and the image threshold was
was performed to improve contrast further (rolling ball = 5 light disable) and the image threshold
set to 230. Finally, the image was exported as a binary image file into Matlab.
was set to 230. Finally, the image was exported as a binary image file into Matlab.
In Matlab, a Hough Transform routine was used to approximate the number of organized
In Matlab, a Hough Transform routine was used to approximate the number of organized
horizontal fibers in the image. A 1000 × 1000-pixel subsection was first extracted along the centerline
horizontal fibers in the image. A 1000 × 1000-pixel subsection was first extracted along the centerline
of the parent image and the Hough transform was calculated for that sub-image. Peaks were found
of the parent image and the Hough transform was calculated for that sub-image. Peaks were found
within the transform image and used to identify horizontal lines within a given angular threshold.
within the transform image and used to identify horizontal lines within a given angular threshold.
The entire sequence was repeated for the entire parent image. The algorithm was refined and validated
The entire sequence was repeated for the entire parent image. The algorithm was refined and
via manual counts across several sample images.
validated via manual counts across several sample images.
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
Materials 2019, 12, 4131
Materials 2019, 12, x FOR PEER REVIEW
6 of 14
6 of 14
179
180
181
182
Figure 3. Block diagrams of (A) an image pre-processing algorithm for binarizing composite images of
Figure 3. Block diagrams of (A) an image pre-processing algorithm for binarizing composite images
electrospun fibers and (B) a nanofiber counting algorithm that quantifies the number of straight fibers
of electrospun fibers and (B) a nanofiber counting algorithm that quantifies the number of straight
crossing the center of the imaged sample. Manual counting was performed to validate automated counts.
fibers crossing the center of the imaged sample. Manual counting was performed to validate
automated
counts.
2.7. Tensile
Testing
Procedures
183
184
185
186
187
188
189
190
191
192
193
194
showcase
ability to build collagen fiber constructs, 30 layers of fibers were electrospun
2.7. To
Tensile
Testingour
Procedures
(1600 V, 30% RH, and 28 ◦ C) with 15 µm spacing into 2.1 mm × 1.8 mm fiber mats and tensile tested.
To showcase
ourmats
ability
to build
collagen
constructs,
30 layers of fibers were
electrospun
For comparison,
fibers
were
also spun
from fiber
a solution
of hexafluoro-2-propranol
(HFIP),
another
(1600V,
30%
RH,
and
28C)
with
15
µm
spacing
into
2.1
mm
x
1.8
mm
fiber
mats
and
tensile
tested.
solvent commonly used for collagen fiber spinning [22]. HFIP-collagen solutions were mixed
to a
For
comparison,
fibers
mats
were
also
spun
from
a
solution
of
hexafluoro-2-propranol
(HFIP),
concentration of 8% w/w (138 mg/mL) and electrospun with the same parameters described above. Fiber
another
commonly
used
for collagen
fiber spinning
mixed
mats
weresolvent
removed
from Teflon
coated
glass substrates
with a[29].
razorHFIP-collagen
blade and the solutions
thickness were
of samples
to
a
concentration
of
8%
w/w
(138
mg/ml)
and
electrospun
with
the
same
parameters
described
was measured before loading into an Electrodynamic Testing System from Test Resources (Shakopee,
above.
Fiber
mats were
Teflon
substrates
with(3M,
a razor
blade and
the
MN,
USA).
Samples
wereremoved
secured from
between
twocoated
pieces glass
of P400
sandpaper
Maplewood,
MN,
thickness
of samples
measured
before loading
into antoElectrodynamic
Testing
System
fromand
Test
USA)
and stretched
at was
a rate
of 1.0 mm/minute
until failure
determine ultimate
tensile
strength
Resources
(Shakopee,
MN).
Samples
were
secured
between
two
pieces
of
P400
sandpaper
(3M,
Young’s modulus. Analysis of the results was performed using Monotonic software (Test Resources)
Maplewood
MN)
and stretched at a rate of 1.0 mm/minute until failure to determine ultimate tensile
and
plotted with
Excel.
strength and Young’s modulus. Analysis of the results was performed using Monotonic software
3.(Test
Results
Resources) and plotted with Excel.
195
3.1.
Initial Characterization of Electrospun Collagen Fibers
3. Results
196
Collagen fibers were spun directly onto Teflon-coated glass slides to evaluate spin quality
3.1. Initial Characterization of Electrospun Collagen Fibers
for a given set of critical electrospinning parameters. Voltage, relative humidity, and acetic acid
concentration
to establish
parametersglass
for direct-write
electrospinning
asfor
ana
Collagenwere
fibersvaried
were spun
directly optimal
onto Teflon-coated
slides to evaluate
spin quality
additive
manufacturing
30% acetic
acid solution
were also
prepared
and electrospun
but was
given set
of critical method.
electrospinning
parameters.
Voltage,
relative
humidity,
and acetic
acid
not
spinnable atwere
relative
humidity
lower than
50% (results
not shown).
SEM images
of fibers produced
concentration
varied
to establish
optimal
parameters
for direct-write
electrospinning
as an
with
40% acetic
acid display
most fibers
light
in color,
withwere
clearalso
shading.
This and
hintselectrospun
at fibers being
additive
manufacturing
method.
30% are
acetic
acid
solution
prepared
but
solid
cylindricalat(Figure
4A,C).
Figurelower
4B shows
electrospun
suboptimal
was and
not spinnable
relative
humidity
than low
50%quality
(resultsfibers
not shown).
SEMwith
images
of fibers
parameters.
Fibers
here
appear
darkfibers
in color
appearing
on the background.
produced with
40%
acetic
aciduniformly
display most
arewith
lightno
in shading
color, with
clear shading.
This hints at
Closer
higher
magnifications
Figure
4D) reveal
fibers seem
to havefibers
a flat electrospun
and planar
fibers inspection
being solidatand
cylindrical
(Figure(inset
4A and
C). Figure
4B shows
low quality
morphology.
Here,
it is shownFibers
that fiber
can vary
the
electrospinning
process.
with suboptimal
parameters.
here morphology
appear uniformly
darkduring
in color
with
no shading appearing
on the background. Closer inspection at higher magnifications (inset 4D) reveal fibers seem to have
a flat and planar morphology. Here, it is shown that fiber morphology can vary during the
197
198
199
200
201
202
203
204
205
206
Materials 2019, 12, 4131
Materials 2019, 12, x FOR PEER REVIEW
7 of 14
7 of 14
207
208
Organized fibers appear in linear bundles of vertical lines with minimal distortion while unorganized
electrospinning
process.
Organized
fibers
in linear bundles of vertical lines with minimal
fibers
appear in loops
that crowd
the base
of appear
the substrate.
distortion while unorganized fibers appear in loops that crowd the base of the substrate.
209
210
211
212
213
214
Figure4.4.Scanning
Scanningelectron
electronmicroscopy
microscopy image
image of
of electrospun
electrospun collagen
Figure
collagen nanofibers.
nanofibers. Example
Exampleimage
imageof
morphology and
and fiber
fiber alignment
alignmentatatlow
low(A)
andhigh
high
ofelectrospun
electrospunfibers
fibers with
with good
good cylindrical
cylindrical morphology
(A) and
magnification
(C).
(Inset
C)
Fibers
appear
three
dimensional
and
are
tightly
packed
into
stacks.
magnification (C). (Inset C) Fibers appear three dimensional and are tightly packed into stacks.
Exampleimage
imageofofelectrospun
electrospunfibers
fiberswith
withpoor
poormorphology
morphologyand
andrandom
randomorientation
orientationatatlow
low(B)
(B)and
and
Example
highmagnification
magnification
(D).
(Inset
Fibers
appear
with
a flat
morphology
randomly
arranged
high
(D).
(Inset
D) D)
Fibers
appear
with
a flat
morphology
and and
randomly
arranged
on theon
the collector
substrate.
collector
substrate.
215
3.2.
3.2.Optimization
OptimizationofofCollagen
CollagenElectrospinning
ElectrospinningParameters
ParametersBased
BasedononSQR
SQR
Composite
216
Compositeimages
imagesofofelectrospun
electrospunsamples
sampleswere
wereanalyzed
analyzedwith
witha aMatlab
Matlabimage
imageprocessing
processing
sequence
to
detect
fiber
linearity.
Spin
quality
ratio
(SQR)
was
defined
to
quantitatively
determine
217
sequence to detect fiber linearity. Spin quality ratio (SQR) was defined to quantitatively determine
218 the
theorganization
organizationofoffibers
fibersproduced
producedvia
viadirect-write
direct-writeelectrospinning.
electrospinning.Figure
Figure55shows
showsthe
theresulting
resulting
219 data
dataset
setinina aseries
seriesofofplots
plotsofofRH
RHvsvsSQR
SQRfor
forall
allacetic
aceticacid
acidconcentrations
concentrations(vertical
(verticalaxis)
axis)and
andvoltages
voltages
(horizontal
axes).
Generally,
as
RH
increased,
SQR
decreased
for
all
data
sets,
resulting
in
an
increase
220
(horizontal axes). Generally, as RH increased, SQR decreased for all data sets, resulting in an increase
of fiber
fiber linearity.
linearity. 40%and
40% and
50%
2nd rows)
221 ofofdisorganized
disorganizedfiber
fiberspinning
spinning and
and aa decrease
decrease of
50%
(1st(1st
andand
2nd rows)
acetic
acid produced
the highest
SQRs,
peaking
at 0.66
Fibers
produced
222 acetic
acid produced
fibersfibers
with with
the highest
SQRs,
peaking
at 0.66
and and
0.63.0.63.
Fibers
produced
withwith
these
these
conditions
showed
strong
linearity
when
examined
under
light
microscopy.
High
SQR
indicates
223
conditions showed strong linearity when examined under light microscopy. High SQR indicates
224 well-controlled
well-controlledfiber
fiberformation
formationwith
withsignificant
significantlinearity
linearitymaking
makingthese
thesetwo
twoconcentrations
concentrationspreferable
preferable
225 for
forcontrolled
controlledelectrospinning.
electrospinning.
226
Further inspection of fiber spinning with 40% acetic acid shows that a lower relative humidity
227
produces fibers with the best linearity peaking at 20 and 30%. Notably, as the voltage reaches 1300
228
and 1500 the SQR peaks at 30% RH for 40% acetic acid. For 50% acetic acid, a more consistent trend
229
of peak SQR is found at 30% RH. 60% acetic acid consistently produced random and unorganized
230
fibers with lower SQR values that rarely surpassed 0.3. Interestingly, for 50% relative humidity the
231
calculated SQR is comparable for lower acetic acid concentrations. Generally, once the acetic acid
232
concentration reaches 60%, SQR decreases significantly and fiber formation becomes more chaotic
233
and less controllable.
234
For 40% acetic acid, as the voltage increases there is a slight increase in SQR, particularly at a
235
relative humidity of 30%, while no apparent trends seem to be present for 50 and 60% concentrations.
236
Microscopic inspection of samples (not shown) showed an increased presence of electrospun material
Materials 2019, 12, x FOR PEER REVIEW
237
238
8 of 14
at higher humidity. As the RH increases, the fiber production rate also increases, resulting in fibers
8 of 14
piling up in an organized fashion as they exceed the XY motor translation speed.
Materials 2019, 12, 4131
239
240
241
Figure 5. Data plot summary for relative humidity (RH) vs. spin quality ratio (SQR). The horizontal
Figure
5. Data
summary
forincreasing
relative humidity
(RH)the
vs.vertical
spin quality
ratio data
(SQR).
The horizontal
axes
show
data plot
electrospun
with
voltage and
axes show
electrospun
with
axes
show
data
electrospun
with
increasing
voltage
and
the
vertical
axes
show
data
electrospun
with
increasing acetic acid concentration.
increasing acetic acid concentration.
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
Further inspection of fiber spinning with 40% acetic acid shows that a lower relative humidity
While
lightwith
microscopy
readily identifies
linearity,
SEM images
recorded
order
to
produces
fibers
the best linearity
peaking atfiber
20 and
30%. Notably,
as thewere
voltage
reachesin1300
and
closely
fiber
morphology
as acetic
the voltage
and50%
relative
wasconsistent
manipulated.
1500
the examine
SQR peaks
at 30%
RH for 40%
acid. For
acetichumidity
acid, a more
trendFigure
of peak6
illustrates
the
morphology
for
collagen
fibers
produced
with
40%
acetic
acid
solution
and
30%RH
SQR is found at 30% RH. 60% acetic acid consistently produced random and unorganized fibers
with
over
voltages
covering
the
entire
investigated
range.
Fibers
were
solid
with
cylindrical
morphology
lower SQR values that rarely surpassed 0.3. Interestingly, for 50% relative humidity the calculated SQR
high aspect
ratio acetic
acrossacid
theconcentrations.
range of attempted
voltages.
Collagen
fiber
diameter remained
isand
comparable
for lower
Generally,
once the
acetic acid
concentration
reaches
consistent,
measuring
~1–2
microns
across
the
voltage
range.
Small
cracks
and
surface
defects
60%, SQR decreases significantly and fiber formation becomes more chaotic and less controllable.seen
on images
can
be attributed
to voltage
defects in
the carbon
coating
usedincrease
for SEMininspection.
These fibers
For 40%
acetic
acid, as the
increases
there
is a slight
SQR, particularly
at a
exhibited
both good
morphology
high SQR,
demonstrating
highlyfor
organized
fiber
placement.
relative
humidity
of 30%,
while noand
apparent
trends
seem to be present
50 and 60%
concentrations.
SEM images
of samples
electrospun
at 1500V
across
all investigated
humidities
and acetic
acid
Microscopic
inspection
of samples
(not shown)
showed
an increased
presence
of electrospun
material
concentrations
are As
shown
in increases,
Figure 7 the
in fiber
a more
expansive
of fiber resulting
morphology
and
at
higher humidity.
the RH
production
ratestudy
also increases,
in fibers
organization.
At
the
lowest
humidity
(20RH)
collagen
fiber
morphology
generally
appeared
solid,
piling up in an organized fashion as they exceed the XY motor translation speed.
withWhile
few flat
and
planar fibers
present
on thefiber
surface.
Fibers
were
well-organized,
consistent
with
light
microscopy
readily
identifies
linearity,
SEM
images
were recorded
in order
to
the SQR
values fiber
foundmorphology
previously.as
30RH
revealed
similar
morphology
and manipulated.
arrangement for
fibers
closely
examine
the voltage
and
relative
humidity was
Figure
6
formed
from
40
and
50%
acetic
acid
solutions,
while
60%
acetic
acid
solutions
produced
illustrates the morphology for collagen fibers produced with 40% acetic acid solution and 30% RH overa
combination
of solid
and flat
fibers. Additionally,
there
is solid
a significant
increase morphology
in the volume
of
voltages
covering
the entire
investigated
range. Fibers
were
with cylindrical
and
unorganized
fibers.
high
aspect ratio
across the range of attempted voltages. Collagen fiber diameter remained consistent,
Once
the
relative
humidity
exceeded
30%,
fiber
morphology
and
organization
degraded
measuring ~1–2 microns across
the voltage
range.
Small
cracks
and surface
defects
seen on images
can
significantly
all acetic
concentrations.
Atfor
40RH,
the acetic acid
concentration
increased
be
attributed across
to defects
in theacid
carbon
coating used
SEMas
inspection.
These
fibers exhibited
both
fibersmorphology
became flatter
morphology.
At the highest
tested
relativefiber
humidity,
all fibers viewed took
good
andinhigh
SQR, demonstrating
highly
organized
placement.
on a planar morphology. Under high magnification fibers appeared almost indistinguishable with
the background and are distributed randomly across the sample space. This finding is consistent with
the steep drop-offs in SQR found in the previous section.
Materials 2019, 12, 4131
Materials 2019, 12, x FOR PEER REVIEW
264
265
266
267
9 of 14
9 of 14
Figure
6. Scanning
electron
microscopy
images
of collagen
nanofibers.
Fibers
were
electrospun
from
Figure
6. Scanning
electron
microscopy
images
of collagen
nanofibers.
Fibers
were
electrospun
from
40%
acetic
acid
at
30%
relative
humidity
with
varying
voltages
of
(A)
1100
V,
(B)1300
V,
and
(C)
1500
40% acetic acid at 30% relative humidity with varying voltages of (A) 1100V, (B)1300V, and (C) 1500V. V.
Fibers
have
a clear
cylindrical
morphology
and
appear
to have
a high
degree
of fiber
alignment
Fibers
all all
have
a clear
cylindrical
morphology
and
appear
to have
a high
degree
of fiber
alignment
on on
substrate.
thethe
substrate.
SEM images of samples electrospun at 1500 V across all investigated humidities and acetic acid
concentrations are shown in Figure 7 in a more expansive study of fiber morphology and organization.
At the lowest humidity (20 RH) collagen fiber morphology generally appeared solid, with few flat and
planar fibers present on the surface. Fibers were well-organized, consistent with the SQR values found
previously. 30 RH revealed similar morphology and arrangement for fibers formed from 40 and 50%
acetic acid solutions, while 60% acetic acid solutions produced a combination of solid and flat fibers.
Additionally, there is a significant increase in the volume of unorganized fibers.
Once the relative humidity exceeded 30%, fiber morphology and organization degraded
significantly across all acetic acid concentrations. At 40 RH, as the acetic acid concentration increased
fibers became flatter in morphology. At the highest tested relative humidity, all fibers viewed took on
a planar morphology. Under high magnification fibers appeared almost indistinguishable with the
background and are distributed randomly across the sample space. This finding is consistent with the
steep drop-offs in SQR found in the previous section.
Materials 2019, 12, 4131
Materials 2019, 12, x FOR PEER REVIEW
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
10 of 14
10 of 14
Figure 7. SEM images of fibers electrospun at 1500 V for various relative humidity (shown in upper
right
of each
image)
and of
acetic
acid
concentrations
of (A)
(B) 50%,
andhumidity
(C) 60%. (shown in upper
Figure
7. SEM
images
fibers
electrospun
at 1500V
for40%,
various
relative
right of each image) and acetic acid concentrations of (A) 40%, (B) 50%, and (C) 60%.
3.3. Peak Loadand Young’s Modulus of Uncrosslinked Fibers
3.3. As
Peak
Loadand
Young’s Modulus
of Uncrosslinked
a proof
of concept,
thick collagen
nanofiberFibers
mats were electrospun at 30% relative humidity
from 40%
(250 mg/mL
collagen)
and HFIP
solutions
(138 mg/mL
collagen)
to humidity
form 30
As a acetic
proof acid
of concept,
thick collagen
nanofiber
mats
were electrospun
at 30%
relative
layers
of
fibers.
These
nanofiber
mats
were
mechanically
tested
to
measure
ultimate
tensile
strength
from 40% acetic acid (250mg/ml collagen) and HFIP solutions (138 mg/ml collagen) to form 30 layers
and
Young’s
modulus.
Fiber
mats
were
electrospun tested
to a thickness
of 30
layers. tensile
Figure strength
8A,B show
of fibers.
These
nanofiber
mats
were
mechanically
to measure
ultimate
and
the
resultant
fiber
mat
loaded
into
the
tensile
testing
setup
at
the
beginning
of
an
experiment.
is
Young’s modulus. Fiber mats were electrospun to a thickness of 30 layers. Figure 8A,B showIt the
apparent
from
the
image
that
fiber
mats
spun
from
HFIP
solution
are
thicker
than
mats
from
acetic
resultant fiber mat loaded into the tensile testing setup at the beginning of an experiment. It is
acid
solution.
This
consistent
work
showing
electrospun
frommats
HFIPfrom
solutions
apparent
from
theis image
thatwith
fiberprevious
mats spun
from
HFIP solution
are collagen
thicker than
acetic
having
larger
fiber
diameters
than
acetic
acid
solutions
[23–25].
Figure
8C,D
compare
the
average
acid solution. This is consistent with previous work showing electrospun collagen from
HFIP
peak
load
and
young’s
modulus
with
standard
deviation.
Although
the
peak
load
for
HFIP-based
solutions having larger fiber diameters than acetic acid solutions [23–25]. Figure 8C,D compare the
fiber
matspeak
is significantly
higher (p
= 0.006)with
thanstandard
AA-based
fiber mats,
the young’s
modulus
no
average
load and young’s
modulus
deviation.
Although
the peak
load show
for HFIPstatistically
significant
difference
(p
=
0.268).
Future
work
and
studies
will
look
at
further
categorizing
based fiber mats is significantly higher (p = 0.006) than AA-based fiber mats, the young’s modulus
the
influence
of crosslinking,
layerdifference
amount, and
solventwork
on material
properties.
show
no statistically
significant
(p =spinning
0.268). Future
and studies
will look at further
categorizing the influence of crosslinking, layer amount, and spinning solvent on material properties.
Materials 2019, 12, 4131
Materials 2019, 12, x FOR PEER REVIEW
11 of 14
11 of 14
Figure 8. Example image of tensile testing system with collagen fiber mats made with (A) HFIP and (B)
40%
acetic
acid. Collagen
mats testing
were stretched
in thecollagen
above configuration
untilwith
failure
theand
(C)
Figure
8. Example
image fiber
of tensile
system with
fiber mats made
(A)and
HFIP
peak
load
(n
=
4,
p
=
0.006)
and
(D)
young’s
modulus
(n
=
4,
p
=
0.268)
were
recorded
for
samples
and
(B) 40% acetic acid. Collagen fiber mats were stretched in the above configuration until failure and
the
were
plotted.
the averages
(C) peak and
loadstandard
(n = 4, pdeviations
= 0.006) and
(D)
young’s modulus (n = 4, p = 0.268) were recorded for
283
284
285
286
samples and the averages and standard deviations were plotted.
4. Discussion
287
4. Discussion
Relative
Humidity, Acetic Acid Concentration and Fiber Morphology
288
Direct-write
exhibits potential
as a method to produce nanofibrous scaffolds.
Relative
Humidity, electrospinning
Acetic Acid Concentration
and Fiber Morphology
Precise control over fiber placement in the future will allow researchers to build precision matrix scaffolds
electrospinning
exhibits
potential
as a method
produce
scaffolds.
muchDirect-write
like native ECM.
Furthermore,
bioanalogs
of ordered
tissuetolike
corneananofibrous
and meniscus
can be
Precise
control
over
fiber
placement
in
the
future
will
allow
researchers
to
build
precision
matrix
produced for regenerative medicine and transplantation [26,27]. To effectively incorporate DWES as
a 3D
scaffoldsmodality,
much likeenvironmental
native ECM. Furthermore,
bioanalogs
ordered
tissue
cornea and meniscus
printing
and process settings
mustofbe
optimized
forlike
reproducibility.
Relative
can be produced
forenvironmental
regenerative medicine
[26,27]. To effectively
incorporate
humidity
is a critical
parameterand
thattransplantation
contributes to systematically
electrospinning
fibers
DWES
as
a
3D
printing
modality,
environmental
and
process
settings
must
be
optimized
for
into an ordered structure. In addition, selecting the correct solvent is paramount to maintaining high
reproducibility.
RelativeFor
humidity
is aancritical
environmental
parametertothat
contributes
to
quality
electrospinning.
this reason,
exhaustive
study was performed
extract
the optimal
systematically
electrospinning
fibers
into
an
ordered
structure.
In
addition,
selecting
the
correct
relative humidity and acetic acid concentration for direct-write electrospinning of collagen nanofibers.
solvent
is paramount
to maintaining
electrospinning.
exhaustive
Based
on the presented
study, 40%high
aceticquality
acid was
confirmed to beFor
thethis
bestreason,
solventan
concentration
study
was performedcollagen
to extractfibers.
the optimal
humidity
and acetic acid
for directfor
electrospinning
Purerelative
acetic acid
is well-known
for concentration
its ability to solubilize
write
electrospinning
of
collagen
nanofibers.
collagen but initially was unsuitable as a solvent for electrospinning due to its high affinity to
Based
onslow
the presented
study,
acetic
aciddemonstrations
was confirmed of
to collagen
be the best
solvent concentration
collagen
and
evaporation
rate 40%
[28–30].
Early
electrospinning
utilized
for electrospinning collagen fibers. Pure acetic acid is well-known for its ability to solubilize collagen
but initially was unsuitable as a solvent for electrospinning due to its high affinity to collagen and
slow evaporation rate [28–30]. Early demonstrations of collagen electrospinning utilized
289
290
291
292
293
294
295
296
297
298
299
300
301
302
Materials 2019, 12, 4131
12 of 14
hexafluoroisopropanol (HFIP) or trifluro-ethanol (TFE) as solvents to achieve a spinnable viscosity,
but high toxicity of these fluoro-organic solvents make them unsuitable for everyday use [31].
Additionally, there are conflicting reports on how similar electrospun collagen produced from these
solvents is to native collagen on a molecular level [32].
Acetic acid still shows promise as a solvent because it does not completely denature collagen
in solution like HFIP and TFE [22]. Here, we analyzed the impact of acetic acid concentration on
fiber morphology and organization. The highest concentration tested (60%) resulted in consistently
poor fiber morphology and low control on fiber placement across all relative humidities. The lower
concentrations (40 and 50%) resulted in solid, cylindrical fiber morphology and improved fiber
placement, especially at relative humidities below 40%. From this we can deduce that diluting the
acetic acid concentration with water increases the evaporation rate of the carrier solvent when the
humidity is sufficiently low allowing fibers to dry prior to deposition on the collector.
Evaporation of a solution is regulated by the temperature and humidity of the surrounding
environment as well as the vapor pressure of the solution. Because the vapor pressure of water and
acetic acid are very similar (2.5 kPa and 3.76 kPa, at 28 ◦ C respectively) [33] the relative humidity
seems to highly impact the rate of spinning and fiber morphology. Liu et al. demonstrated a diluted
40% acetic acid solution as a suitable electrospinning solvent for type I collagen, however relative
humidity and other acetic acid concentrations were left unreported. Polk et al. reported dissolving
collagen in acetic acid solutions of 20–50% for compressed air-driven pneumatospinning but do not
report environmental parameters or results for solutions other than 40% [25]. Despite its critical role in
determining fiber morphology, few researchers have reported on the relative humidity required to
produce electrospun collagen. This may be due to the prevalence of fluoro-alcohol use as a solvent.
Our work fills a void in the literature showing the relationship between collagen electrospinning
solvent, relative humidity and fiber morphology and organization.
5. Conclusions
In summary, an exhaustive study was performed to ascertain ideal acetic acid concentration,
relative humidity and voltage parameters for direct-write electrospinning of highly organized collagen
nanofibers for scaffold production. A custom electrospinning printer was designed and built in
order to produce organized single lines of collagen nanofibers with cylindrical morphology. Acetic
acid concentration, relative humidity and spin voltage were all varied to establish best practices for
producing single fibers of collagen with high linearity and control of fiber placement.
An acetic acid concentration of 60% produced wet fibers that absorbed onto the substrate with
an increasingly planar morphology with increasing humidity, despite showing a degree of control
over fiber placement for 20% relative humidity. Acetic acid concentrations of 40 and 50% were found
to consistently produce solid fibers with cylindrical morphology at relative humidity lower than
40% making them optimal concentrations for direct-write electrospinning of collagen. As a rule of
thumb, the relative humidity should be kept ≤30% in order to maintain a solid fiber morphology.
Additionally, SQR most consistently peaked at 20% and 30% relative humidity for 40% and 50% acetic
acid, respectively. Finally, the mechanical properties of fiber mats electrospun with 40% acetic acid and
HFIP as a solvent were tested to show the ability to electrospin large scaffolds of materials. The process
parameters identified in this work can enable researchers to direct-write electrospin pure collagen
scaffolds with specific morphologies that more accurately mimic native ECM in specific tissues.
Author Contributions: Conceptualization, F.A.A.J., L.J., and K.P.; methodology, F.A.A.J. and K.W.; software,
F.A.A.J. and L.J.; validation, F.A.A.J., L.J.; formal analysis, F.A.A.J.; investigation, F.A.A.J. and L.J.; resources, L.J.;
data curation, F.A.A.J.; writing—original draft preparation, F.A.A.J.; writing—review and editing, K.W. and K.P.;
visualization, L.J.; supervision, K.P.; project administration, K.P.; funding acquisition, L.J. and K.P.
Funding: This research was funded by US Army Medical Research and Materiel Command (USAMRMC), award
number W81XWH-17-C-003. The views, opinions and/or findings contained in this report are those of the author(s)
and should not be construed as an official Department of the Army position, policy or decision unless so designated
by other documentation.
Materials 2019, 12, 4131
13 of 14
Acknowledgments: The authors would like to acknowledge Linzie Wagner and Carolynn Nofsinger for their
administrative support of this project.
Conflicts of Interest: The authors declare no conflict of interest
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| 7,862 | 54,656 |
W4306924819.txt | Original Research
20 October 2022
DOI 10.3389/fmars.2022.873548
TYPE
PUBLISHED
OPEN ACCESS
EDITED BY
Jeremy Kiszka,
Florida International University,
United States
REVIEWED BY
Roberto Carlucci,
University of Bari Aldo Moro, Italy
Tiago Andre Marques,
University of St. Andrews,
United Kingdom
Movements and diving behavior
of Risso’s dolphins in the
Southern California Bight
Brenda K. Rone 1*, David A. Sweeney 1, Erin A. Falcone 1,
Stephanie L. Watwood 2 and Gregory S. Schorr 1
Foundation for Marine Ecology and Telemetry Research, Seabeck, WA, United States,
Ranges, Engineering and Analysis Department, Naval Undersea Warfare Center, Newport,
RI, United States
1
2
*CORRESPONDENCE
Brenda K. Rone
brenda.rone@marecotel.org
SPECIALTY SECTION
This article was submitted to
Marine Megafauna,
a section of the journal
Frontiers in Marine Science
10 February 2022
30 August 2022
PUBLISHED 20 October 2022
RECEIVED
ACCEPTED
CITATION
Rone BK, Sweeney DA, Falcone EA,
Watwood SL and Schorr GS (2022)
Movements and diving behavior of
Risso’s dolphins in the Southern
California Bight.
Front. Mar. Sci. 9:873548.
doi: 10.3389/fmars.2022.873548
COPYRIGHT
© 2022 Rone, Sweeney, Falcone,
Watwood and Schorr. This is an openaccess 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.
Risso’s dolphins (Grampus griseus), uncommon prior to the 1970’s, are now
regularly observed within the Southern California Bight. During long-term
cetacean monitoring programs on United States Navy range areas in the
Southern California Bight from 2009–2019, we deployed 16 Argos-linked
satellite tags on Risso’s to acquire objective, detailed depictions of their
movements and behaviors. Individuals were tracked for a median of 10.7
days (range = 0.8 – 19.7). Kernel density estimation suggested individuals
utilized the entire Southern California Bight with the 50% core use area
centered around San Clemente and Santa Catalina Islands where most of the
tag deployments occurred. Grand median dive depth was 101 m (max = 528)
and dive duration was 5.6 min (max = 11.1). We used generalized mixed models
to assess seasonal and environmental effects on distribution and diving
behavior including month, distance to shore, time of day, lunar phase, sea
surface temperature, and chlorophyll-a residuals. Animals were further from
shore (including islands) during a full versus new moon and from the mainland
during the last versus first quarter moon. Animals also tended to be closer to
land in the fall and early winter months. Dives were deeper yet shorter during
the night, during a full moon, and when animals were further offshore. Animals
conducted nearly twice as many dives at night compared to day, though deep
dives (> 500 m) occurred at all times of day. This study provides insights into
Risso’s distribution and behavioral trends while identifying priorities for
future research.
KEYWORDS
Risso’s dolphin, Grampus griseus, satellite telemetry, generalized linear mixed models,
Southern California Bight
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Risso’s have a primarily teuthophagus diet consisting of both
squid and octopus (Clarke, 1996; Baird, 2002; Blanco et al., 2006;
Jefferson et al., 2006; Bearzi et al., 2011; Yates and PalavecinoSepú lveda, 2011; Bloch, 2012; Milani et al., 2017; Luna et al.,
2021), though they also feed on fish and crustaceans (Reeves
et al., 2002; Bloch, 2012). With the exception of octopus in the
western Mediterranean (Blanco et al., 2006), squid appear to be
their preferred cephalopod prey (Clarke, 1996; Bearzi et al., 2011;
Yates and Palavecino-Sepú lveda, 2011; Bloch, 2012). In
California waters, some species of squid that have been
identified as important prey for Risso’s, including jumbo squid
(Dosidicus gigas) (Orr, 1966; Gilly et al., 2006) and market squid
(Loligo opalescens) (Kruse, 1989), perform diurnal vertical
migrations (Watanabe et al., 2006). These species serve as both
prey and predators within the ecosystem, feeding on deep-, mid-,
and shallow-water organisms such as krill and other crustaceans,
small fish, and even other cephalopods, with observations
suggesting they may feed at the surface in the early evening
(Markaida and Sosa-Nishizaki, 2003). Jumbo squid in particular
have demonstrated daytime depths exceeding 250 m, nearsurface depths at dusk, and variable depths throughout the
night (Gilly et al., 2006).
Our understanding of foraging behavior of Risso’s has
advanced markedly in recent years. Initial observational
studies suggested Risso’s rested or traveled during the day and
foraged at night (Shane, 1995b). An increase in both click rates
and bouts at night suggested Risso’s foraging activity is related to
diurnal patterns in squid behavior (Soldevilla et al., 2010). Most
recent work indicates that Risso’s perform foraging dives during
the day as well as at night (Benoit-Bird et al., 2017; Arranz et al.,
2018; Benoit-Bird et al., 2019) with maximum depths exceeding
500 m particularly common during the day (Benoit-Bird et al.,
2019). They regularly switch predation tactics between a nearsurface generalist and deep-water squid specialist even within a
single dive (Benoit-Bird et al., 2019). They plan foraging dives
based on knowledge attained on previous dives (Arranz et al.,
2018, Visser et al., 2021) and use a spin dive strategy to decrease
transit time to exploit the diurnal mesopelagic layer (Visser
et al., 2021).
Reliable assessments of Risso’s movements and diving
behavior are challenging yet essential for understanding
population structure, stock boundaries, habitat use, and
potential exposure to anthropogenic impacts. Satellite tags can
provide a more objective, detailed depiction of individual
movements and behavior than visual or acoustic surveys alone.
As part of a long-term research effort to examine cetacean
movements, habitat use, and ecology within the Southern
Introduction
Risso’s dolphins (Grampus griseus, hereafter referred to as
“Risso’s”) are a medium-sized, cosmopolitan odontocete found in
temperate and tropical waters. They typically occur between
latitudes 60°N to 60°S where sea surface temperatures are
greater than 10°C; however, the highest numbers of individuals
are found between 30° and 45° latitudes, in waters 15–20°C (Kruse
et al., 1999; Baird, 2002; Jefferson et al., 2014b). In the eastern
Pacific, Risso’s are abundant and widely dispersed year-round
(Leatherwood et al., 1980). They can be found as far north as the
Gulf of Alaska and south to Tierra del Fuego (Baird, 2002;
Jefferson et al., 2014b). Risso’s are most often associated with
shelf-edge and upper continental slope habitat, in areas of steep
bottom topography with average depths ranging from 100 to 1000
m (Kruse, 1989; Baumgartner, 1997; Kruse et al., 1999; Baird,
2002; Cañadas et al., 2002; Jefferson et al., 2006; Azzellino et al.,
2008). Although highest densities are found along this shelf/slope
region, they utilize a wide range of habitats, from shallow, coastal
areas (Boer et al., 2013; Jefferson et al., 2014b) to offshore pelagic
waters (Baird, 2016). Group size is typically 10–50, although
singletons and groups upwards of 4,000 individuals have been
documented (Leatherwood et al., 1980; Baird, 2002; Baird, 2016).
Currently, the population abundance of Risso’s along the
coasts of Washington, Oregon, and California is estimated at
6,336 (CV = 0.32) individuals (Carretta et al., 2020). Movement
patterns within this population have been discerned through
sightings and acoustic detections. It is thought that Risso’s in this
region demonstrate broadscale seasonal shifts northward from
California into Oregon/Washington in the spring/summer
months coincident with rising water temperatures (Green
et al., 1992). High inter-annual seasonal variability north of
(Dohl et al., 1983; Kruse, 1989; Soldevilla et al., 2010), and yearround presence within, the Southern California Bight (Soldevilla
et al., 2010), a productive oceanographic region off the coast of
the southwestern United States (U.S.), have been documented.
Long–term changes in the abundance and distribution of Risso’s
have also been documented in the Southern California Bight. In
the late 1950s, they were rarely encountered there (Norris and
Prescott, 1961). Between 1972 and 1975, sightings increased in
frequency as their distribution shifted inshore (Leatherwood
et al., 1980). Starting in the El Niño years of 1982–83,
occurrence further increased in the Bight and Santa Catalina
Island was identified as particularly important habitat (Shane,
1995b). They continue to be regularly documented there both
visually and acoustically (Soldevilla et al., 2010; Jefferson et al.,
2014a; Bacon et al., 2017).
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SPLASH10-A tags transmitted a record of diving behavior in
the form of a behavior log (BL). BL data were received via the
Argos satellite system and from a land-based Argos receiving
station (Wildlife Computers Mote) installed on San Clemente
Island in 2014 (Jeanniard-du-Dot et al., 2017). BL data consisted
of summarized dive and surfacing events and included event
start and end times and the maximum depth of dives. As the
number of tag deployments grew, our understanding of Risso’s
dive behavior improved. Thus, adjustments to tag programming
were made during the course of study to maximize the quantity
and relevance of BL data captured while minimizing data gaps.
Dive events were defined as any submergence that exceeded
either 30 m (6 tags) or 50 m (2 tags) depth and lasted more than
30 s (3 tags) or 1 min (5 tags) (Table 1). Depth accuracy for the
pressure transducers in these tags was independently verified in
a pressure chamber to 3,000 m, resulting in a maximum error of
<+/- 2.5% of the recorded value (Schorr et al., 2014). Initially,
start and end times were determined by crossing the 5 m depth
threshold upon descent or ascent of a qualifying dive for the first
two tags. After assessment of tag performance, the next six tags
were programmed with start and end times determined by the
transition from wet to dry when the tag broke the surface during
a typical breath, and therefore this programming regimen
California Bight, we remotely deployed 16 Argos-linked satellite
tags on Risso’s between 2009 and 2019, and here report on habitat
use, dive behavior, and movements of these tagged individuals.
Materials and methods
Data collection
Tag data collection and processing
Fieldwork was undertaken in the Southern California Bight,
primarily off San Clemente and Santa Catalina Islands, from
2009 to 2019 (Figure 1). Satellite tags in the Low Impact
Minimally Percutaneous External-electronics Transmitter
(LIMPET) configuration were deployed on or near the dorsal
fin using a Dan-Inject modified air rifle. Tags were affixed by two
3.4 cm medical-grade titanium barbed darts which were gas
sterilized prior to attachment (Andrews et al., 2019). The tags
contained either Wildlife Computers’ (Redmond, WA) SPOT5
location-only, or SPLASH10-A or SPLASH10-F location and
dive reporting transmitters. The age class of each tagged
individual was estimated based on the relative size and level of
scarring (Kruse et al., 1999; Hartman et al., 2016).
FIGURE 1
Maps of the study area with filtered locations of Risso’s dolphins tagged within the Southern California Bight annually. The inset shows the study
area in a broader geographic context of the state of California. Range Polygons: Blue = Southern California Range Complex; Purple = Point
Mugu Sea Range; Orange = Southern California Anti-submarine Warfare Range; Pink = Shore Bombardment Area.
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TABLE 1 Details of Risso’s dolphin satellite tag deployments by individual.
Tag ID
Age
class
Tag
type
GgTag001
A
GgTag002
Event Start/End
time by
Defined dive
threshold
Deployment
date
Locality
Transmission
duration (days)
No. locations for
analyses
SPOT5
19Jul2009
East side of
SCI
13.6
100
J
SPOT5
11Nov2009
Santa Cruz
I.
19.3
221
GgTag003*
A
SPOT5
24Jun2010
SOAR
19.7
239
GgTag004*
A
SPOT5
24Jun2010
SOAR
11.2
127
GgTag005
A
SPOT5
08Jan2011
Catalina I.
6.7
82
GgTag006
A
SP10-A
18Jun2011
San Diego
8.3
21
GgTag008
A
SP10-A
5 m Depth
50m/1min
24Jul2011
East side of
SCI
5.3
76
GgTag009
A
SP10-A
5 m Depth
30m/1min
20Sep2011
Catalina I.
0.3
7
GgTag010
A
SPOT5
20Jan2012
Catalina I.
10.1
175
GgTag011
A
SP10-A
Wet/Dry
30m/30s
26Mar2013
Catalina
Basin
13.9
204
GgTag012
A
SP10-A
Wet/Dry
30m/30s
21May2013
Catalina
Basin
8.0
100
GgTag015
A
SP10-A
Wet/Dry
30m/30s
26Feb2016
East side of
SCI
11.7
167
GgTag016
S
SP10-F
11Apr2016
Catalina I.
7.3
109
GgTag018
A
SP10-A
Wet/Dry
50m/1min
04Apr2017
San Nicolas
Basin
18.6
354
Gg-2018112098368
A
SP10-A
Wet/Dry
30m/1min
20Nov2018
SOAR
7.6
120
Gg-20190719102468
A
SP10-A
Wet/Dry
30m/1min
19Jul2019
Catalina
Basin
11.3
197
*Tagged within the same group.
SCI, San Clemente Island; SOAR, Southern California Anti-submarine Warfare Range; For age classes, A, Adult; S, Sub-adult; J, Juvenile; for tag type, SP, SPLASH; A, Argos; F, Fastloc GPS.
removed) were combined with the tag’s Douglas-filtered Argos data
to provide the highest resolution location data available for this
individual. The cumulative distance traveled during each tag
transmission and the straight-line distance from the deployment
location were calculated using all locations that passed the filter,
regardless of quality.
For dive behavior analyses, Douglas-filtered locations were
fit by a continuous-time correlated random walk model
(Johnson et al., 2008) that incorporated Argos ellipse errors
(McClintock et al., 2015) using the crawl (v2.2.1) R package
(Johnson and London, 2018; R Core Team, 2021) to estimate the
animal’s position at the start time of every dive. Although
standard errors ranged from 0 to 26 km for model-estimated
locations, a function of Argos location qualities and time
between consecutive Douglas-filtered locations, they were
considered the best estimate of the animal’s position at the
start time of each dive. Given these inherent sources of error or
uncertainty in our spatial data, we exercised caution when
interpreting results associated with data derived from both
model-estimated and Douglas-filtered locations.
allowed for a more accurate assessment of dive and surfacing
times (Table 1). The single SPLASH10-F tag that was deployed
during this study was programmed to collect only maximum
dive depth histogram data (https://wildlifecomputers.com/ourtags/splash-archiving-tags/splash10-f/) to prioritize the
collection of higher resolution Fastloc GPS location data in
addition to Argos location data, and thus was excluded from
dive analyses.
Locations received via Argos satellite were re-processed by
Argos using the Kalman filter to improve location accuracy
(Lopez et al., 2014), uploaded to Movebank (www.movebank.
org), and then processed using the Douglas Argos-Filter (v10.5.2)
with the ‘distance-angle-rate’ filter (Douglas et al., 2012). Filtering
parameters were set as follows: maximum-redundant-distance at 3
km, maximum sustainable rate at 15 km h-1, and the rate coefficient
(the angle created by three subsequent points, Ratecoef) at the
default of 25. Argos location classes 2 and 3 have an estimated error
from Argos of < 500 m (Argos User’s Manual) and were
automatically retained. Filtered SPLASH-10F tag Fastloc GPS
locations (locations with residuals > 35 or time error > +/- 3 min
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the cosine term describe phase shifts near new and full moons,
while effects from the sine term describe phase shifts near the
first and last quarters. Seasonal influence was investigated using
month (for movement models) and sea surface temperature and
residuals from the linear regression of chlorophyll-a against sea
surface temperature (for dive models), referred to as
chlorophyll-a residuals (Soldevilla et al., 2011). The calculation
of chlorophyll-a residuals was performed using the glmmTMB
(v1.1.3) R package (Brooks et al., 2017) and included a random
effect to account for differing oceanographic measurements
across tagged individuals due to different deployment dates
and movements for each individual.
Environmental data collection
A series of ArcGIS layers were created containing bathymetric,
remote sensing, land, and Navy range boundaries. Depth, slope,
distances to nearest shore (either islands or mainland) and to the
mainland, and assessment of locations within range boundaries
were extracted by overlaying tag location data onto raster surfaces
or in relation to polygons in ArcGIS v10.7.1 (NAD 1983 UTM Zone
11N) (ESRI, 2019). Less than 1% of location estimates were on land
according to the raster surfaces—a result of poor-quality locations
and Risso’s use of steep near-shore bathymetry; these were removed
from subsequent movement and dive behavioral analyses. For
analyses of depth and slope, only Douglas-filtered locations of
qualities 2 and 3 were included, given the size of error ellipses
associated with lesser quality Argos positions (Lopez et al., 2014)
and the variable nature of depth and slope over a limited geographic
area. Bathymetric data were obtained from the 2-min Gridded
Global Relief Data (ETOPO2v2) (National Centers for
Environmental Information, 2006). The slope raster was created
using the slope surface tool available in ArcGIS Spatial Analyst.
Remote sensing data from the CoastWatch ERDAP server
(https://coastwatch.pfeg.noaa.gov/erddap) were extracted using
the xtractomatic (v3.3.2) package (Mendelssohn, 2017) in R, and
associated with each Douglas-filtered location for general habitat
description and with each dive’s crawl-modeled location for dive
models. Mean sea surface temperature (°C) was extracted from
the National Oceanic and Atmospheric Administration
(Optimum Interpolation Sea Surface Temperature 0.25˚
resolution) with the Advanced Very High Resolution
Radiometer sensor which combines both satellite and in-situ
data to produce daily gap-free composites (Reynolds et al.,
2007). Mean chlorophyll-a concentration (mg m -3 ), an
indicator of primary productivity, was obtained from the
Moderate Resolution Imaging Spectroradiometer on board the
Aqua satellite (West U.S., 0.0125˚ resolution); eight-day
composites were used to fill in data gaps due to cloud cover.
Median values are reported to minimize the effects of outliers.
Solar elevation and lunar phase were calculated for Douglasfiltered and crawl-modeled locations using the oce (v1.4-0)
package (Kelley and Richards, 2020) in R. A time-of-day
classifier was assigned to each record: day (solar elevation ≥
-12˚ below the horizon, inclusive of nautical dawn and twilight)
and night (< -12˚ below the horizon). Lunar phase was
incorporated into each model as a pair of cyclical predictors
using the sine and cosine of the calculated lunar phase to
estimate the amplitude and phase angle of lunar effects
(DeBruyn and Meeuwig, 2001). To do this, calculated lunar
phases were converted into radians where a lunar phase of 0 was
0 radians, 0.5 was p radians, and 1 was 2p radians. Effects from
Frontiers in Marine Science
Data analysis
Movements
Areas of common use were approximated using 50% and
95% kernel density estimation based on the Douglas-filtered
locations from all tags using the adehabitatHR (v0.4.19) package
(Calenge, 2006) in R. The adehabitatHR ad hoc method
(function input h = “href”) for computing the smoothing
parameter was used, along with all other default function
inputs. For days with more than one filtered location per
individual, an average daily position was used to reduce the
influence of autocorrelation between multiple locations in a day
(Heide-Jørgensen et al., 2002). These kernel density isopleths
were plotted in ArcGIS v10.7.1.
Inshore/offshore movements were evaluated using two
metrics: distance to shore (included mainland and islands) and
distance to mainland (west coast only, excluded islands). These
movements were modeled as a function of cyclical lunar phase,
month, and time of day using generalized linear mixed models
from the glmmTMB package (Brooks et al., 2017). Models were
fit as gamma distributions with log link functions and used the
maximum likelihood parameter estimation method. Two
movement models were created: one with distance to shore as
the response variable and the other with distance to mainland as
the response variable. To account for autocorrelation within
individuals, a random effect was included in all models
specifying the tagged individual (or pair of tagged individuals
if from the same group). We tagged two individuals within the
same encounter, although these individuals were not observed to
be closely associated at the time of tagging (tagged 32 min apart,
group size = 45, group spread = 300 x 400 m). Following
inspection of the animals’ movements, we used a random
effect value indicating periods of associated movements to
minimize autocorrelation within this apparent social group.
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Inspection of a plot of the residual autocorrelation function
indicated that this random effect did not sufficiently reduce
temporal residual autocorrelation. Thus, we incorporated
secondary, nested random effects of different time increments
(1, 2, 3, 4, 6, 8, and 12 h) within individuals to account for
temporal autocorrelation, selecting the time period that
minimized Akaike’s information criterion (AIC) for each
model. After determining the nested effect that minimized
AIC, separate models were created for each possible
combination of lunar phase predictors (only cosine, only sine,
both cosine and sine), and the model that minimized AIC was
chosen as the best-fit model that most accurately described the
lunar phase shift.
Within each best-fit model, predictor significance was
determined using a Type II Wald Chi-Square Test (a < 0.05)
using the ANOVA function from the car (v3.0-12) R package (Fox
and Weisberg, 2019). Model predictions were created for the
average individual by setting all random effects to zero using the
predict.glmmTMB function from the glmmTMB package (Brooks
et al., 2017) in R. Prediction plots were created to display the
effects of each significant predictor on the response variables using
the patchwork (v1.1.1), ggplot2 (v3.3.5) and ggformula (v0.10.1)
packages (Wickham, 2016; Kaplan and Pruim, 2020; Pedersen,
2020) in R. For these plots, as stated in each figure’s caption, the
values for other predictors in each model were fixed at the median
(continuous variables) or modal value (categorical variables).
Dives
Dive rates were calculated within each day and night period
by dividing the number of dives recorded by the duration of BL
messages received during the respective day/night periods. We
used the maptools (v1.1-4) R package (Bivand and Lewin-Koh,
2022) to determine the times at which the sun crossed the -12
degrees threshold to separate the day and night periods. We then
created a generalized linear mixed model of dive rates as a
function of time of day (day or night) using the maximum
likelihood parameter estimation method from the glmmTMB
package (Brooks et al., 2017). This model included a random
effect of tagged individual to account for autocorrelation in dive
rates, and modeled data were limited to day/night periods during
which less than 50% of the period included BL data gaps (n = 25
day and 32 night periods modeled).
Dive duration and maximum depth were each modeled
separately using generalized additive mixed models fit as
gamma distributions with log link functions using the gamm4
(v0.2-6) and MuMIn (v1.43.17) R packages (Barton, 2020; Wood
and Scheipl, 2020) and the maximum likelihood parameter
estimation method. Dive duration and depth were both
modeled as a function of time of day, cyclical lunar phase (as
used in the movement models), distance to shore (including
islands), sea surface temperature, chlorophyll-a residuals, and a
smoothed term of either dive depth or duration, respectively,
formed via shrinkage cubic regression splines with four
A
B
FIGURE 2
Distribution of filtered locations (A, from 16 tags) and dives (B, from 8 tags) across months from satellite tags deployed on Risso’s dolphins in the
Southern California Bight between 2009 – 2019. The number of tags that contribute to monthly data are denoted above each bar.
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tagged near San Clemente or Santa Catalina Islands. Of the
two remaining tags, one was deployed near San Diego and one
south of Santa Cruz Island (Table 1; Figure 1). Individuals of
three age classes were tagged: adult (n = 14), sub-adult (n = 1),
and juvenile (n = 1). Animals were tracked for a median
duration of 10.7 days (range = 0.3–19.7) with a combined total
of 2,299 locations after Douglas filtering (Table 1). All Risso’s
remained within the Southern California Bight throughout
the duration of tag deployments (Figure 1). Cumulative
straight-line distances between consecutive locations per tag
ranged from 14.5 km with an average speed of 0.5 km h-1 to
1,836.2 km at 3.9 km h-1 (Table 2).
Movements revealed by satellite tracking showed individual
variation within the Southern California Bight: some animals
ranged broadly throughout the Bight, while others remained
more localized (Figure 1). Risso’s utilized most of the Bight with
the 50% core use centered around San Clemente and Santa
Catalina Islands and the surrounding basins (Figure 3), a pattern
likely influenced by tagging location. Tagged dolphins utilized
habitat within the boundaries of all four U.S. Navy ranges
dimensions of the basis. Dive depth and duration models
included the same implementation of nested random effects,
and we used the same AIC selection strategy for detecting lunar
phase shifts as was used for the movement models described
above. Predictor significance was determined using Wald F Tests
(a < 0.05) from the mgcv (v1.8-38) R package (Wood, 2011) and
model prediction plots were created for the average individual by
excluding all random effects using the predict.gam function from
the mgcv package (Wood, 2011).
Results
Movements
Location data were obtained from 16 Risso’s tagged within
the Southern California Bight (Table 1; Figure 1). Location data
were collected across nine years with data collected from every
month except October (Figure 2). Fourteen individuals were
TABLE 2 Summary of habitat characteristics at filtered Argos locations for individual Risso’s dolphins.
Tag ID
Distance from Cumulative
deployment
distance
location (km) moved (km)
Average
daily
distance
traveled
(km/day)
median(range)
Distance to
shore (km)
Distance
to
mainland
(km)
Bottom
depth (m)
Slope
(˚)
Chl
(mg m-3)
SST
(˚C)
median
(range)
median
(range)
median
(range)
median
(range)
median
(range)
median
(range)
GgTag001
57.0 (9.1–138.7)
767.3
56
16.8 (1.2–39.0)
82.6 (50.0–112.2)
948 (31–1,944)
4 (0.1–11)
0.4 (0.2–1.4)
19 (17–21)
GgTag002
28.7 (1.4–80.4)
1,301.0
67
17.2 (3.4–35.6)
44.1 (11.9–85.0)
895 (150–1,944)
4 (0.1–11)
0.7 (0.2–2.3)
16 (15–17)
957 (57–1,969)
GgTag003*
62.4 (6.9–156.0)
1,836.2
93
28.2 (2.2–72.3)
87.3 (13.4–154.9)
GgTag004*
78.5 (7.4–132.6)
1,060.4
94
27.9 (5.9–47.9)
110.3 (43.3–150.1) 1082 (103–1,900)
2 (0–12)
0.4 (0.1–1.4)
16 (14–18)
3 (0.1–12)
0.6 (0.2–1.0)
14 (13–17)
GgTag005
40.3 (2.8–63.9)
660.4
98
20.1 (1.1–46.6)
63.9 (30.2–100.3)
918 (135–1,257)
2 (0.1–7)
0.3 (0.2–18)
14 (14–15)
GgTag006
41.2 (11.9–118.8)
204.2
24
39.9 (3.7–46.8)
47.6 (19.4–61.8)
893 (193–1,185)
2 (0.2–12)
0.3 (0.2–0.4)
18 (17–19)
GgTag008
27.8 (3.2–78.9)
396.2
74
7.1 (1.05–26.7)
77.6 (34.6–95.7)
923 (24–1,307)
4 (0.1–13)
0.3 (0.2–0.4)
19 (18–20)
GgTag009
6.8 (3.3–8.2)
14.5
NA
3.7 (1.6–6.5)
33.1 (27.0–34.5)
233 (129–518)
5 (2–7)
0.3 (0.2–0.4)
19 (18–19)
GgTag010
19.2 (0.4–113.4)
615.3
61
1.8 (0.02–35.3)
44.4 (28.6–52.4)
114 (9–1,296)
3 (0–13)
0.7 (0–73)
15 (15–16)
GgTag011
54.6 (2.0–95.9)
1,153.0
83
38.0 (8.5–68.2)
86.0 (27.7–131.7)
1,367 (363–2,006)
3 (0.1–14)
0.3 (0.2–0.8)
15 (14–16)
GgTag012
43.2 (4.6–66.9)
590.2
74
16.4 (1.58–27.7)
26.1 (7.5–55.9)
714 (155–1,295)
2 (0–11)
0.3 (0.2–0.4)
18 (18–19)
GgTag015
36.0 (7.1–73.5)
1,031.2
88
18.1 (1.2–52.1)
90.6 (22.6–130.0)
1,004 (108–1,914)
2 (0.1–11)
0.3 (0.2–0.7)
17 (15–18)
17 (15–18)
GgTag016
33.8 (0.5–83.4)
804.5
110
18.8 (1.4–39.4)
39.3 (22.9–82.0)
711 (31–1,906)
3 (0–7)
0.9 (0.4–3.6)
GgTag018
48.5 (2.0–108.0)
1,615.4
87
42.9 (0.3–93.0)
122.4 (44.3–178.0)
1,078 (71–1,890)
3 (0–13)
0.9 (0.3–6.6)
16 (16–18)
Gg2018112098368
72.9 (6.9–83.6)
489.8
64
2.3 (0.09–41.0)
39.5 (29.5–106.3)
898 (9–1,715)
4 (0.3–12)
0.2 (0.1–1.3)
18 (18–19)
Gg20190719102468
24.6 (2.5–60.9)
826.7
73
11.8 (0.37–27.8)
52.5 (5.0–95.8)
795 (63–1,323)
3 (0.1–12)
0.5 (0.3–1.0)
21 (18–22)
*Tagged within the same group.
Cumulative horizontal distances were calculated using straight-line distances between filtered locations. Positions that were located on land were removed for habitat values but retained for
distance calculations. Only locations with Argos quality 2 and 3 were used for quantifying depth and slope. Chl, Chlorophyll-a; NA, not applicable; SST, Sea Surface Temperature.
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FIGURE 3
Core use areas for 16 Risso’s dolphins tagged in the Southern California Bight from 2009–2019 as indicated by 50% (gray line) and 95% (black
line) kernel density estimates of the combined location data across tags. Stars represent deployment locations. Kernel density isopleths were
derived from one average daily position to minimize autocorrelation bias, and are overlayed on high resolution ETOPOv2 bathymetry in meters.
Military training ranges are indicated by colored polygons as follows: Blue = Southern California Range Complex; Purple = Point Mugu Sea
Range; Orange = Southern California Anti-submarine Warfare Range; Pink = Shore Bombardment Area.
early winter (Figures 4B, C), with closest distances occurring near
the islands during that time (Figure 4C).
considered (Figure 1). All tags but one were deployed within
either the Southern California Range Complex or Point Mugu
Sea Range training areas, and 89% of locations fell within these
areas, spanning all 11 months that tags transmitted. Fourteen
percent of locations fell within the Southern California Antisubmarine Warfare Range and the Shore Bombardment Areas
specifically, and dolphins utilized these areas during seven
different months of the year.
Details of movement model parameter estimation and
significance testing are provided in Table 3. Distance from
mainland was significantly predicted by time of day, month, and
sine of lunar phase (Figures 4A, B, D), while distance from shore
was significantly predicted by month and cosine of lunar phase
(Figures 4C, E). Animals were found closer to the mainland during
first quarter lunar phase and furthest during the last quarter phase
(Figure 4D). Animals were predicted to be farther from shore
during a full moon compared to during a new moon (Figure 4E),
although 95% confidence intervals were large across all lunar
phases. Time of day helped explain differences in distance to
mainland, although minimally, with animals closer to mainland
during the daylight hours versus night (Figure 4A). Seasonality
influenced movements with some variability. Animals were
generally closer to land (both mainland and shore) in fall and
Frontiers in Marine Science
Dives
A total of 1,469 h of BL data were recorded (n = 8 tags, range =
0.87–428.3 h per tag) (Table 4). Dive data were collected in seven
different months across five years from eight of the 16 tagged
individuals (Table 1; Figure 2). These data were comprised of 3,071
dives with a grand median (median of all tag medians) depth of 101
m (max = 528 m) and a grand median dive duration of 5.6 min
(max = 11.1 min) (Table 4). The maximum dive depth recorded for
each dolphin ranged from 148–656 m (Table 4); the longest dive,
16.1 min, was down to 64 m at night. Dives beyond 500 m depth
were conducted during both day and night hours, as demonstrated
by a sample two-day dive trace (Figure 5A), with comparable
frequency (7% of total dives in each period). Animals dove more
frequently at night than during the day (p = 8.14e-07), with night
and day dive rates at 5.21 (SE = 0.5) and 2.73 (SE = 0.5) dives per
hour, respectively. Shallow dives (< 100 m) were more frequent
during the day (63%, Figure 5B) versus night (35%, Figure 5B).
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TABLE 3 Model results including type II Wald chi-square (movement models) or Wald F (dive models) testing and parameter estimation with
associated significance testing.
Response variable Explanatory variable
Chi-Square or F testing
Model parameter estimation and Significance testing
Chisq of F Value p Value Parameter Variable
Distance to Mainland
Time of Day
18.250
0.000
Month
36.492
0.000
sin (Lunar Phase)
18.748
0.000
Distance to Shore
Time of Day
0.215
0.643
Month
22.855
0.011
cos (Lunar Phase)
9.060
0.003
Dive Depth
Time of Day
SST
b
b Std. Error z Value p Value
Intercept [Day, April]
4.407
0.133
33.220
0.000
Night
0.022
0.005
4.270
0.000
January
-3.550
0.227
-1.560
0.118
February
-1.690
0.156
-1.090
0.276
March
-0.290
0.100
-2.900
0.004
May
-1.163
0.291
-4.000
0.000
June
-0.406
0.195
-2.080
0.038
July
-0.209
0.170
-1.230
0.218
August
-0.355
0.230
-1.540
0.122
September
-1.154
0.367
-3.140
0.002
November
-0.686
0.225
-3.050
0.002
December
-0.884
0.324
-2.730
0.006
sin (Lunar Phase)
-0.144
0.033
-4.330
0.000
Intercept [Day, April]
3.311
0.291
11.375
0.000
Night
0.009
0.020
0.463
0.643
January
-1.354
0.496
-2.731
0.006
February
-0.621
0.324
-1.920
0.055
March
-0.082
0.205
-0.399
0.690
May
-0.835
0.643
-1.298
0.194
June
-0.412
0.423
-0.974
0.330
July
-0.506
-0.370
-1.367
0.172
August
0.083
0.483
0.172
0.863
September
-1.716
0.778
-2.206
0.027
November
-1.383
0.496
-2.791
0.005
December
-1.398
0.692
-2.019
0.043
cos (Lunar Phase)
-0.185
0.062
-3.010
0.003
Intercept [Day]
5.050
0.295
17.131
0.000
103.949
0.000
Night
0.242
0.024
10.196
0.000
3.069
0.080
SST
-0.029
0.016
-1.752
0.080
Chlorophyll-a Residuals
0.007
0.935
Chlorophyll-a Residuals
0.002
0.027
0.081
0.935
Distance to Shore
11.793
0.001
Distance to Shore
0.003
0.001
3.434
0.001
cos (Lunar Phase)
20.951
0.000
cos (Lunar Phase)
-0.112
0.025
-4.577
0.000
Dive Duration
22255
0.000
Dive Duration
Dive Duration
Effective degrees of freedom = 2.687
0.185
8.284
119.230
0.000
Night
-0.142
0.013
-10.919
0.000
SST
2.426
0.119
SST
0.016
0.010
1.558
0.119
0.559
Time of Day
Intercept [Day]
1.534
0.000
Chlorophyll-a Residuals
0.341
0.559
Chlorophyll-a Residuals
0.008
0.014
0.584
Distance to Shore
8.556
0.003
Distance to Shore
-0.001
0.001
-2.925
0.003
cos (Lunar Phase)
8.405
0.004
cos (Lunar Phase)
0.038
0.013
2.899
0.004
Dive Depth
38290
0.000
Dive Depth
Effective degrees of freedom = 2.898
Parameter information for categorical variables is relative to the categories listed in brackets next to each model’s intercept term. For the dive models, smooth term effective degrees of
freedom are listed in place of model parameter information.
m-3 (range = 0–73) with the lowest and highest concentrations in
January 2012 (Table 2).
Details of dive model parameter estimation and significance
testing are provided in Table 3. Dive depth was significantly
Across all years and months, Risso’s were documented in median
sea surface temperatures of 17˚C (range = 13–22˚C), with the
coolest temperatures in June 2010 and warmest in July 2019
(Table 2). The median chlorophyll-a concentration was 0.4 mg
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A
B
C
D
E
FIGURE 4
Prediction plots from the fitted models for distance to mainland (A, B, D) and distance to shore (C, E). Solid black lines/dots represent mean
predicted values with shaded areas/error bars representing the 95% CI. Hash marks along the x-axis shows the spread of data. For the creation
of these plots, values for other predictors in each model were set at the median or modal value as follows: time of day = day, month = April,
sine of lunar phase = -0.370, cosine of lunar phase = -0.010.
TABLE 4 Summary of dive parameters by individual of eight Risso’s dolphins satellite-tagged within the Southern California Bight.
Whale ID
Total behavior log data (h)
Dives
No. dives for analyses
Duration (min.)
median (range)
Depth (m)
median (range)
GgTag008
81.8
116
3.7 (1.1–9.3)
59 (30–608)
GgTag009
0.8
5
5.3 (4.4–5.9)
124 (42–132)
GgTag011
299.5
405
6.2 (0.9–13.8)
152 (30–624)
GgTag012
0.9
5
11.1 (5.4–11.4)
528 (90–560)
GgTag015
259.8
195
5.8 (1.0–16.1)
74 (30–576)
232 (50–656)
GgTag018
428.3
1285
6.4 (1.4–11.1)
Gg-20181120-98368
169.5
470
4.0 (1.1–11.0)
72 (30–592)
Gg-20190719-102468
228.4
590
5.2 (1.1–11.1)
78 (30–608)
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A
B
FIGURE 5
(A) Sample two-day dive trace from a Risso’s dolphin in the Southern California Bight with the time of day denoted by dots for day (yellow) and
night (blue). Blanks indicate periods where there are gaps in the data. Extended surface time is indicated by a straight horizontal line along 0 m.
(B) Distribution of dive depth estimates for all dive tags during the day and night. For both plots, time of day was defined by a solar altitude
(≥ -12 degrees for day and < -12 degrees for night), which was calculated for every dive start at the modeled location estimate closest in time.
characteristic of these steep topographic areas (Kruse et al.,
1999). Outside of the Southern California Bight, they are
documented in various habitats ranging from shallow waters
such as 2–5 m in Monterey Bay, CA (Jefferson et al., 2014b) to
offshore pelagic waters of Hawai'i in depths exceeding 3,000 m
(Baird, 2016). In the Azores around Pico Island, they can be
found from 0–10 km from shore (Hartman et al., 2016) where
steeply sloped nearshore topography results in water depths
increasing from 200 m to 1000 m within 5 km (Hartman
et al., 2008).
While relatively short deployment durations of this study
preclude any meaningful discussion of large-scale movements
into and out of the Southern California Bight, they do provide
new insights into localized movements. Changes in Risso’s
density indicate latitudinal seasonal shifts occur along the U.S.
west coast (Green et al., 1992; Forney and Barlow, 1998). In the
Bight, Santa Catalina Island has repeatedly been identified as
important habitat for Risso’s (Shane, 1995a; Shane, 1995b;
Soldevilla et al., 2010), with acoustic detections occurring on
75% of the days at a site on the south end of the island (Soldevilla
et al., 2010). The near year-round acoustic presence at Santa
Catalina Island may suggest that an island-associated population
exists and/or a greater Southern California Bight resident
population, with possible seasonal influx from a larger
migratory population (Soldevilla et al., 2010). In this study, all
Risso’s were tagged in the outer waters (defined as waters
offshore of the mainland coast) of the Southern California
Bight, mainly in the greater surrounding waters of the outer
predicted by dive duration, time of day, distance to shore, and
cosine of the lunar phase (Figure 6). Dive duration was
significantly predicted by dive depth, time of day, distance to
shore, and cosine of lunar phase (Figure 7). Dives were deeper
and shorter at night than during the day (Figures 6B, 7B), when
dolphins were further offshore (Figures 6C, 7C), and during a
full versus a new moon, respectively (Figures 6D, 7D). Wide 95%
confidence intervals for dive depth and duration as a function of
lunar phase reflect low sample sizes at some lunar phases (see
hash marks along x-axis of (Figures 6D, 7D).
Discussion
As regular inhabitants of the Southern California Bight,
Risso’s have been frequently documented on line-transect
surveys throughout the region (e.g., Barlow and Forney, 2007;
Jefferson et al., 2014a) and across seasons in recent years (e.g.,
Schorr et al., 2019). Habitat used by tagged dolphins in this study
ranged from the deep waters of the numerous basins to the
shallow waters inshore of San Clemente and Santa Catalina
Islands along the mainland coast (9–2,006 m), with Argos
locations sometimes clustered around steep slope features
(Figure 1). Globally, densities of Risso’s appear higher over the
outer continental shelf and upper regions of the continental
slope (Jefferson et al., 2014b). The patchy distribution and local
abundance of this species may be driven by enhanced
productivity associated with currents and upwellings
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A
B
C
D
FIGURE 6
Prediction plots of significant variables (A–D) from the models of dive depth. Solid black lines/dots represent mean predicted values with shaded
areas/error bars representing the 95% CI. Hash marks along the x-axis shows the spread of data. For the creation of these plots, values for other
predictors in each model were set at the median or modal value as follows: time of day = night, Chl residuals = -0.008, SST = 15.81, distance to
shore = 24.68 km, sine of lunar phase = -0.736, cosine of lunar phase = -0.435, dive duration = 5.6 min. Chl, Chlorophyll-a (mg m-3); SST, Sea
Surface Temperature (°C).
despite demonstrating varied and often highly mobile
movement patterns, and most remained relatively local to
their tagging location (Figure 3), suggesting year-round
residence within the Bight is plausible. Alternatively, if some
of these individuals were members of the larger WashingtonOregon-California migratory stock, it is entirely feasible that the
limited number of tags and their generally short durations
simply did not capture movements out of this region.
Unfortunately, durations like these are not uncommon for
remotely deployed tags on small cetaceans, where factors
including surface activity, interactions with conspecifics, and
the challenge of achieving optimal tag placement often lead to
premature detachments.
The seasonality of inshore/offshore movements were most
apparent when considering distance to any landmass, rather
than to the mainland coast. According to our models, Risso’s
were significantly closer to land, though this was typically an
Channel Islands. Nearly all individuals spent at least some of
their time within the shallower nearshore waters of these islands
(Figure 1). Of the 16 Risso’s, 12 approached within 20 km of
Santa Catalina Island (Figure 1), in all seasons and in eight
different years. Of these 12, only half were tagged within 20 km
of this island (min = 3 km). All but one of these six tags also left
this island; the brief transmission for the sixth tag prevents
further discussion of its movements. In fact, the only animal out
of the 16 that demonstrated close association with Santa Catalina
Island for most of its tag transmission was actually tagged in the
San Nicolas Basin on the west side of San Clemente Island.
While these tags may not confirm or refute the presence of
island-associated individuals or populations, they do further
demonstrate the importance of Santa Catalina Island for the
broader, regional population throughout the year.
While animals in this study were tagged in all but one month
of the year, all remained within the Southern California Bight
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A
B
C
D
FIGURE 7
Prediction plots of significant variables (A–D) from the models of dive duration. Solid black lines/dots represent mean predicted values with
shaded areas/error bars representing the 95% CI. Hash marks along the x-axis shows the spread of data. For the creation of these plots, values
for other predictors in each model were set at the median or modal value as follows: time of day = night, Chl residuals = -0.008, SST = 15.81,
distance to shore = 24.68 km, cosine of lunar phase = -0. 435, dive depth = 123.5 m. Chl, Chlorophyll-a (mg m-3); SST, Sea Surface
Temperature (°C).
While earlier research in the Bight suggested that Risso’s
foraged at night (Shane 1995a; Soldevilla et al., 2010) with slow
travel/rest the predominant daytime behaviors (Shane, 1995b;
Smultea et al., 2018), more recent studies have documented
daytime foraging excursions (Arranz et al., 2018; Arranz et al.,
2019; Benoit-Bird et al., 2019). Dives in excess of 500 m recorded
on these tags occurred across all times of day (max = 607 m day,
656 m night) (Figure 5B) and are comparable to results from
Benoit-Bird et al. (2019). Dives were predicted to be 1.27 times
deeper (eb; b = 0.24, SE = 0.02, p-value < 2e-16) and 4.70 times
shorter (eb; b = -0.14, SE = 0.01, p-value < 2e-16) at night versus
day (Figures 6B, 7B), and to occur nearly twice as often. This
depth difference is potentially driven by both a greater number
of dives at < 100 m depth during the day (63% day versus 35%
night) and > 100 m at night (65% night versus 37% day)
(Figure 5B). These results would align with increased
nighttime foraging activities that have been described
island, from September through January (p < 0.05). Although
market squid spawn in the Southern California Bight year-round
(Navarro et al., 2018), concentrated spawning aggregations
occur nearshore over shallow sandy substrate in southern
California along the mainland coast and the Channel Islands
(Zeidberg et al., 2012; Van Noord, 2017) from October through
April/May (Fields, 1965). Past observational research suggested
that both Risso’s and short-finned pilot whales (Globicephala
macrorhynchus), another squid-eating species common within
the Southern California Bight prior to the 1982–83 El Niño
event, shift into the Channel Islands during the winter months to
feed on spawning market squid (Sinclair, 1992; Shane, 1995b).
Our data suggest this seasonal, prey-driven shift has continued
in the decades since. These may be fine-scale, localized shifts by
animals that remain within the greater Southern California Bight
throughout the year, those of migratory animals shifting into the
Bight, or both.
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(Clarke and Denton, 1962). Deep scattering layer species seek
lower light levels to avoid predators, and are thus found at
deeper depths during brightly moonlit nights (Boden and
Kampa, 1967), increasing use of near-surface waters at night
as the moon wanes (Herná ndez-Leó n et al., 2002). It may be a
cost-effective strategy for Risso’s to pursue prey at deeper depths
around a full moon—as suggested by our model—when layers
may provide additional biomass to target. The question also
should be raised whether changes in dive behavior associated
with lunar phases are driven by illumination levels themselves,
or whether they are the result of an endogenous signal that is
associated with the 29.5 day lunar cycle, or a combination of
both. It seems plausible that light may have significant biological
influence on marine systems with predominantly clear water and
skies but in geographic locations where waters are more turbid
and skies are obscured by clouds for at least part of the year, as
they are in the Southern California Bight (e.g., Rastogi et al.,
2016), an endogenous cue may play a more significant role. In
(Benoit-Bird et al. 2009), lunar phase accounted for more
variability in micronekton migration than surface irradiance,
suggesting that lunar effects other than light levels effect these
prey, and in turn, the predators that pursue them.
Results from this study suggest seasonal trends in
movements, demonstrate year-round presence within the
Bight, and further describes lunar and diel influences on
behavior. We have presented our statistical findings and
suggest biological explanations based on findings in the
literature. However, we must acknowledge that statistical
significance according to model output should be interpreted
with caution from a biological perspective, as these data have
limitations. Although several of these tags lasted nearly three
weeks, the kernel density analysis was likely biased by
deployment location. In addition to sample size limitations,
some of these data included gaps from failed transmission and
from short tag durations that didn’t span across seasons and
lunar phases. The effects of Argos location quality and
resolution, and the resulting prediction errors from modeled
animal tracks based on Argos data, further limit interpretation of
these results. While we filtered Argos locations to reduce error
before using them in standardized movement models, predicted
locations had standard errors ranging from 0 to 26 km. There
were occasional adjustments to tag programming to optimize
data collection that may have affected some statistics. When
looking at environmental variables such as sea surface
temperature and chlorophyll-a, in-situ measurements collected
around an animal is the only way to provide accurate, real-time
representation of that variable with high resolution, but this was
not available for these tags. Remote sensing can capture patterns
in these data over a broad spatial region, but cloud cover is an
inherent limitation. Although we were able to combine a remote
previously (Soldevilla et al., 2010; Arranz et al., 2019; BenoitBird et al., 2019). While Benoit-Bird et al. (2019) documented
elevated Risso’s echolocation in shallow waters at night, we are
unable to make further comparisons due to the limited temporal
and spatial coverage of acoustic data and the lack of nighttime
tag data from that study.
Described as dynamic foragers, Risso’s target different prey
layers, switching between shallow water generalist and deep
water specialist strategies (Benoit-Bird et al., 2019) and appear
to consider optimal energy investments and tradeoffs prior to the
next dive (Arranz et al., 2018; Visser et al., 2021). Jumbo squid
are at depths > 250 m during the day, travel to near-surface
waters at dusk, and then demonstrate highly variable dive
behavior until they presumably find a zone to exploit for food
(Gilly et al., 2006). In Hawai'i, the mesopelagic layer migrates
from the 400–700 m depth during daylight hours to 0–400 m at
night, though pilot whales appear to target the deeper 400 m
layer at night, potentially in search of higher caloric prey (Owen
et al., 2019). In the Azores, Risso’s target two deep scattering
layers, a broad one at 500–700 m and a narrow one at 400 m,
with both layers performing diel vertical migration (Visser et al.,
2021). Off Santa Catalina Island, Risso’s were detected in three
layers: a deep static layer at 450 m, a mid-water migrating layer
at 100–300 m, and an intermittent shallow layer at 50 m along
with scattered patches consisting of fish and squid (Benoit-Bird
et al., 2019). It’s possible that Risso’s utilize the photic advantage
of daylight to capitalize on the low energetic costs of shallow
layer exploitation in between bouts of deep layer explorations
(the switching behavior documented in Benoit-Bird et al., 2019)
and thereby conserve energy for nighttime hunts that are
directed at preferential prey, such as jumbo squid, that while
vertically migratory, are not necessarily reaching shallow waters
throughout the nighttime hours.
Lunar phase has been linked to changes in zooplankton
biomass (Herná ndez-Leó n et al., 2002) and depth (Boden and
Kampa, 1967) and horizontal displacement in the mesopelagic
layer in Hawaiʻi (Benoit-Bird et al., 2009). In this study, lunar
phase predicted both horizontal and vertical shifts, albeit with
large confidence intervals. Risso’s were located 1.45 times further
away from shore (including islands) (Figure 4E) during a full
moon than a new moon, a pattern similar to that observed in
Hawaiian pilot whales (Owen et al., 2019). Lunar phase has been
associated with changing use of the water column in Cuvier’s
beaked whales (Ziphius cavirostris) (Barlow et al., 2020) and
jumbo squid (Gilly et al., 2006), and like the Risso’s tagged in this
study, pilot whales (Owen et al., 2019), Galá pagos fur seals
(Arctocephalus galapagoensis) (Horning and Trillmich, 1999),
and bigeye tuna (Thunnus obesus) (Musyl et al., 2003) all appear
to use deeper waters during a full moon. Full moon light levels at
100–150 m are equivalent to mid-day light levels at ~500 m
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10.3389/fmars.2022.873548
sensing and an in-situ dataset to derive sea surface temperature
at modeled locations, we had to create 8-day averaged
composites for chlorophyll-a to create gap free composites.
This mismatch in resolution between animal location
estimates and environmental variables is another potential
source of error. It should be noted that remote sensing can
underestimate values for chlorophyll-a and sea surface
temperature (Gomez et al., 2020; Keates et al., 2020), and
absolute values should be interpreted with caution. These
aforementioned factors may have influenced the significance of
these predictors in the dive models.
We believe one of the greatest values of these analyses are their
ability to aid formulation of new hypotheses and research
directions. Additional targeted data collection will strengthen
model results and allow for more concrete biological
interpretations of these findings. Deployments in other regions
of the Southern California Bight as well as during specific periods
where data gaps currently exist [e.g., gaps in month (Figure 2) and
lunar phase (Figure 6)] would provide a more comprehensive
regional dataset. The short duration of these tags highlights the
need for more tag development to improve attachment while
continuing to minimize impact. A regional photo-identification
catalog, genetic analyses, and the use of medium-duration, dart
attached archival acoustic tags (e.g., Sweeney et al., 2022) along
with prey mapping would provide meaningful connections
between the existing research within this region. It is our hope
that this work will foster purposeful discussions and ideas for
future work to manage and protect this species.
Funding
Funding for fieldwork and tags was provided by the U.S. Navy,
Pacific Fleet (N66604-14-C-0145, N66604-18-P-2187) and via the
Californian Cooperative Ecosystems Studies Unit (N62473-19-20025), the Chief of Naval Operations Environmental Readiness
Division (N45) via a grant from the Naval Postgraduate School
(Grant No. N000244-10-1-0050) and the Environmental Security
Technology Certification Program (N66604-14-C-2438).
Additional support was provided by the Office of Naval Research
and the Living Marine Resources program.
Acknowledgments
We would like to thank the U.S. Navy, Pacific Fleet, N45, the
Environmental Security Technology Certification Program, the
Living Marine Resources program, and the Office of Naval
Research for support of this research. We thank all
participants from the Marine Mammal Monitoring on Navy
Ranges program at the Naval Undersea Warfare Center, Jeff
Foster for contributions in the field, and the Naval Postgraduate
School and Californian Cooperative Ecosystems Studies Unit for
contract facilitation. We thank Stacy DeRuiter for assistance
with analyses and Chip Johnson, Jessica Chen, and Shannon
Coates and three reviewers for their thoughtful comments and
suggestions. This study was conducted under U.S. National
Marine Fisheries permits No. 540-1811, 16111, 15330, 20605.
Tagging was approved by the Cascadia Research Collective
Institutional Animal Care and Use Committee.
Data availability statement
Conflict of interest
The raw data supporting the conclusions of this article will
be made available by the authors, without undue reservation.
Ethics statement
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
The animal study was reviewed and approved by Cascadia
Research Collective Institutional Animal Care and Use Committee.
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 manufacturer, is not guaranteed
or endorsed by the publisher.
Author contributions
BR, EF, GS, and SW conducted the fieldwork. BR, GS, and
DS conducted the data analysis. All authors contributed to the
article and approved the submitted version.
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10.3389/fmars.2022.873548
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W4297486116.txt | Review
The Relationship between the Gut Microbiota and Exercise:
A Narrative Review
Diego Domínguez-Balmaseda
and Guillermo García-Pérez-de-Sevilla *
Faculty of Sport Sciences, Universidad Europea de Madrid, 28270 Madrid, Spain
* Correspondence: guillermo.garcia@universidadeuropea.es
Abstract: The intestinal microbiota plays an important role in the homeostasis of the intestinal
tract, and the fact that exercise could have an impact on its functionality is currently the subject
of various studies. Exercise is used as a strong preventive and treatment strategy in numerous
chronic diseases, influencing metabolism, immunity, and physiological functions. Some recent
studies provide information on exercise as a factor that could favor the growth of beneficial species
and increase the diversity of the intestinal microbiota. However, the difficulty in determining the true
effect of exercise is only due to the confounding influence of lifestyle and dietary habits. Regardless,
there is still interest in the implications of the gut microbiota for elite sports because the details are
considered crucial for success in different competitions. This narrative review tries to collect the
information available in the literature on the role that exercise plays in the modulation of the balance
of the intestinal microbiota.
Keywords: gut microbiota; exercise; health; athletes; sports
1. Gut Microbiota
Citation: Domínguez-Balmaseda, D.;
García-Pérez-de-Sevilla, G. The
Relationship between the Gut
Microbiota and Exercise: A Narrative
Review. Hygiene 2022, 2, 152–162.
https://doi.org/10.3390/
hygiene2040014
Academic Editor: Daniela Haluza
Received: 29 August 2022
Accepted: 22 September 2022
Published: 28 September 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affiliations.
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/).
The main objective of the narrative is to analyze the association between the intestinal
microbiota and exercise and to conclude if there is sufficient evidence to consider new
approaches for intestinal recovery and protection as a new important factor in performance.
Joshua Lederberg, an American biologist who received the Nobel Prize in Medicine in
1958, stated that the microorganisms found in humans protect us symbiotically in our own
body. These communities of microorganisms, known today under the term microbiota,
consist of a variety of microorganisms that includes eukaryotes, archaea, bacteria, and
viruses, constituting about 1.3% of the body mass, and are essential to maintain optimal
health. These microorganisms inhabit different parts of the body, colonizing the mucosa
within a given anatomical niche, such as the respiratory system, the walls of the digestive
system or also the urogenital system, among other surfaces [1]. More diversity of the
microbiota is found in the intestinal tract and mouth, while the skin contains less diversity,
followed by the vagina, although a consensus has not yet been reached on the number of
species that inhabit it [2].
The microbiota is known as the set of microorganisms residing in each ecosystem,
with a symbiotic relationship and with adaptive properties and rapid renewal, forming
a large metabolic unit. The intestinal microbiota is a set of microorganisms made up
of approximately 100,000 million bacteria that live in our intestine. The microbiota is
responsible, among other functions, for maintaining the well-being of the intestinal mucosa,
helping us digest food and converting harmful elements into less toxic substances.
In 2014, the term “intestinal flora” was coined to describe the intestinal microbiota,
although later, it was changed to gut microbiota [3].
Some gut microbiota bacteria attach to the cells’ external wall and to specific receptors
called adhesins. They can adapt to the specific conditions of the environment, such as
humidity, temperature, or pH, being able to activate defense mechanisms in case of interaction, for example, with a harmful virus. The homeostasis of the intestinal tract depends
Hygiene 2022, 2, 152–162. https://doi.org/10.3390/hygiene2040014
https://www.mdpi.com/journal/hygiene
Hygiene 2022, 2
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on the balance between the microbiota, intestinal permeability, and local immunity; in
the event of any non-adaptive alteration, it can produce negative effects on the host itself.
When these alterations occur, it is known as intestinal dysbiosis, which can affect digestion,
nutrient absorption, vitamin production, and the control of harmful microorganisms [3].
Most of the bacteria that make up the gut microbiota live in the colon [4]. The colon is
described as a potentially active organ due to the extensive activity of the gut microbiota
and has been compared to the liver due to its high metabolic capacity [5]. The microbiota is
dominated by four main phyla: Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria [6].
Although there are also bacteria in smaller proportions that are vital for the function of the
microbiota, Firmicutes and Bacteriodetes stand out due to their proportion throughout the
intestinal tract, as they represent 60% and 20%, respectively, of all the gut microbiota, while
the Proteobacteria phylum covers 5% and Actinobacteria 3%, approximately [7].
2. The Gut Microbiota Function
The functions of the gut microbiota include the following:
2.1. Protection Function in the Intestinal Barrier
The gut microbiota plays an important role in helping to maintain the integrity of
the intestinal barrier, thus protecting the host from pathogens. In turn, the microbiota
develops defense mechanisms together with the epithelial cells, which are responsible
for establishing an effective and harmonious intestinal barrier, preventing the passage of
harmful substances, antigens, toxins, and microbial products and, on the other hand, the
absorption of nutrients benign [8].
The main elements that form the intestinal barrier are the mucus, the tight junctions
between enterocytes, antimicrobial peptides, and immunoglobulin A (IgA) secretion [9].
The intercellular junctions of the intestinal barrier separate the internal environment from
the external environment, making it impermeable to certain pathogenic substances and
permeable to nutrients [10].
The mucosa interacts with the microbiota to maintain intestinal homeostasis; in addition, the intestinal microbiota induces the biosynthesis of antimicrobial peptides by mucosal
Paneth cells, such as cathelicidins, lectins, and defensins, by activating their membrane
receptors. Bacteriodetes thetaiotaomicron and Lactobacillus innocua are two of the main agents
that stimulate Paneth cells [11].
Another reinforcement of this barrier function by the microbiota of the intestinal
lumen is the production of lactic acid by Lactobacillus, which reinforces the ability of human
lysozyme released in the lumen to destroy peptidoglycan [12]. Short-chain fatty acids (SCFAs) released by bacterial fermentation also stimulate the production of the antimicrobial
peptide cathelicidin. These SCFAs acidify the intestinal lumen and prevent colonization by
pathogens sensitive to this pH, such as Salmonella, Escherichia, and Clostridium difficile [9].
The homeostasis of the barrier function would result in an optimal symbiosis; otherwise, if the function of the intestinal barrier does not favor an adequate mechanism, it
will have an exaggerated or abnormal immune response that would lead to poor control of
intestinal permeability, leading to what we know as intestinal dysbiosis [13,14].
2.2. Function on the Development of the Immune System
The gut microbiota contributes to gut immunomodulation in conjunction with the
innate and adaptive immune systems [9]. In line with the previous protection function,
the immune system has co-evolved to maintain a symbiotic relationship with the commensal species of the microbiota while keeping other species considered pathogenic in this
intestinal ecosystem under control. About 70% of the lymphocytes in the human body live
in the digestive system, which gives us an idea of the importance of this function in the
development [15].
Components and cell types of the immune system involved in the immunomodulatory
process include gut-associated lymphoid tissues (GALT), effector and regulatory T cells, IgA-
Hygiene 2022, 2
154
producing B (plasma) cells, Group 3 innate lymphoid cells, resident macrophages, and
dendritic cells in the lamina itself [16].
The role of the gut microbiota in forming a functional GALT is implicit in the poor
development of Peyer’s patches and isolated lymphoid follicles that are marked by an
abundance of IgE B cells rather than IgA B cells [17]. The absence of the intestinal microbiota
causes the plasma cell populations of the intestinal mucosa to differentiate mainly towards
greater production of IgE, instead of IgA, which is associated with an increased risk of
allergies [14].
Effector T cells are regulated in the mucosa by Th17 cells. Some bacteria, such as Bacillus fragilis, belonging to the Bifidobacterium or Clostridium genera, activate Treg lymphocytes
via Toll-like receptors (TLR) that are part of the innate immune system, inducing systemic
anti-inflammatory effects, which helps to develop tolerance to antigens present in the diet
and some bacteria in the lumen [16].
This cross-communication between the microbiota and the epithelial cells is also
responsible for the release of IgA and for stimulating the production of glycoproteins to
form more mucus. The main stimulators of epithelial IgA production are Gram-negative
species such as Bacteroides, which activate dendritic cells in the mucosa so that they induce
IgA production by plasma cells (activated lymphocytes) in the submucosa. This IgA
prevents microorganisms from activating pro-inflammatory pathways, as they cannot bind
when coated by these antibodies, to receptors such as TLRs. In turn, IgA collaborates in
capturing antigens and prevents their penetration through the intestinal barrier, limiting the
movement of pathogens and neutralizing toxins throughout the lumen [18]. The presence
of high populations of Suturella, on the other hand, reduces IgA production in the lumen,
since this bacterium releases IgA-degrading proteases [15]. The complexity of the intestinal
microbiota, per se, is a defense against the colonization of this ecosystem by other types of
microorganisms [18]. This is especially important in adults who have a much more stable
intestinal microbiota than children or subjects with intestinal dysbiosis. The production
of bacteriocins by the resistant microbiota is responsible for this resistance to colonization
of the lumen by other bacteria. Similarly, the bacteriophages in the intestinal lumen can
prevent the establishment of susceptible bacterial species. All these mechanisms prevent
the bacterial translocation factor through the intestinal epithelium and therefore prevent a
systemic immune response to a possible infection [19].
2.3. The Intestinal Microbiota Performs a Metabolic Function
Contributing to the digestion of nutrients from the diet [9]. The gut microbiota obtains
its nutrients mainly from carbohydrates in the diet. Fermentation of carbohydrates that
escape proximal digestion and indigestible oligosaccharides are metabolized by bacteria
such as Bacteroides, Roseburia, Bifidobacterium, Faecalibacterium, and Enterobacteria, resulting
in the synthesis of SCFAs such as butyrate, propionate, and acetate, which are rich sources
of energy for the host [20].
This metabolic process is favored by the more acidic pH of the right colon. In contrast,
in the left colon, microorganisms grow more slowly due to the low arrival of food with a
more neutral pH [21].
Members of the genus Bacteroides are the predominant organisms involved in carbohydrate metabolism, achieved by expressing enzymes such as glycosyltransferases,
glycoside hydrolases, and polysaccharide lyases. The best example among these organisms is Bacteroides thetaiotaomicron, which is endowed with a genome encoding more than
260 hydrolases [22].
The gut microbiota also has an efficient protein metabolizing machinery that functions
through microbial proteinases and peptidases in conjunction with human proteinases.
Various amino acid transporters in the bacterial cell wall facilitate the entry of amino acids
from the intestinal lumen into the bacteria, where they convert the amino acids into small
signaling molecules and antimicrobial peptides (bacteriocins) [23].
Hygiene 2022, 2
is Bacteroides thetaiotaomicron, which is endowed with a genome encoding more than 260
hydrolases [22].
The gut microbiota also has an efficient protein metabolizing machinery that functions through microbial proteinases and peptidases in conjunction with human proteinases. Various amino acid transporters in the bacterial cell wall facilitate the entry of amino 155
acids from the intestinal lumen into the bacteria, where they convert the amino acids into
small signaling molecules and antimicrobial peptides (bacteriocins) [23].
Another metabolic function of great importance is described in the literature, such as
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described
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as the synthesis of vitamins such as vitamin K and vitamin B, and the function of lipid
lation. In addition, members of the genus Bacteroides synthesize conjugated linoleic acid
regulation. In addition, members of the genus Bacteroides synthesize conjugated linoleic
(CLA), which has antidiabetic, antiatherogenic, antiobesogenic, hypolipidemic, and imacid (CLA), which has antidiabetic, antiatherogenic, antiobesogenic, hypolipidemic, and
munomodulatory properties [24]. The gut microbiota has also been shown to positively
immunomodulatory properties [24]. The gut microbiota has also been shown to positively
impact lipid metabolism by suppressing the inhibition of lipoprotein lipase (LPL) activity
impact lipid metabolism by suppressing the inhibition of lipoprotein lipase (LPL) activity
in adipocytes [25].
in adipocytes [25].
3. Factors
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Exercise and Gut Microbiota
There are several potential mechanisms by which exercise could modify the gut microbiota. Several studies by Hoffman Goetz and colleagues [27,28] have reported that exercise
alters intraepithelial lymphocyte gene expression, downregulating proinflammatory cytokines and up-regulating anti-inflammatory cytokines and antioxidant enzymes. These
immune cells reside close to microbial communities and produce antimicrobial factors that
are essential for mediating host-microbial homeostasis [29].
Similarly, exercise can affect the integrity of the intestinal mucus layer, which plays
an important role in preventing pathogens from adhering to the intestinal epithelium
and serves as an important substrate for certain mucosa-associated bacteria, such as A.
Hygiene 2022, 2
156
muciniphila. Exercise raises core temperature and causes heat stress, particularly when
performed for prolonged periods or in a hot environment [27]. Exercise can also reduce
intestinal blood flow by more than 50%, with significant intestinal ischemia occurring
within 10 min of high-intensity exercise [28]. At rest, the splanchnic bed undergoes rapid
reperfusion. Although the intestine is an anaerobic environment, intestinal epithelial
cells primarily use oxidative metabolism, and high-intensity exercise is known to transiently impair intestinal barrier function [28]. Therefore, exercise-induced heat stress and
ischemia may produce interactions between the intestinal mucosal immune system and the
microbiota residing in the intestinal lumen.
Evidence for the role of exercise in modifying the human gut microbiota first emerged
from cross-sectional studies in 2014 [29]. Clarke et al. found that the gut microbiota of
professional rugby players had a higher alpha diversity and a higher relative abundance
of 40 different bacterial taxa than the gut microbiota of the control group, being sedentary
subjects [29]. More recently, Bressa et al. compared active women with sedentary controls
and found that women who engaged in at least 3 h of exercise per week had increased
levels of Faecalibacterium prausnitzii, Roseburia hominis, and Akkermansia muciniphila [30],
which are producers of butyrate [31], while A. muciniphila has been associated with a lean
body mass index (BMI) and better metabolic health [32].
Several studies have also attempted to correlate the composition and metabolic capacity of the microbiota with cardiorespiratory fitness, with Durk et al. showing a higher ratio
of Firmicutes to Bacteroidetes in subjects with better metabolic health, and was significantly
correlated with maximal oxygen consumption (VO2max ) [33]. Estaki et al. found in younger
adults that microbial diversity and abundance of butyrate-producing bacterial taxa were
positively correlated with cardiorespiratory fitness [34], while Barton et al. found, through
metagenomic analyses differences in fecal microbiota between athletes and sedentary controls. Athletes showed relative increases in different metabolic pathways, such as amino
acid biosynthesis and carbohydrate metabolism, as well as increased production of SCFAs,
such as acetate, propionate, and butyrate produced by the microbiota [35].
However, all these studies were limited by their cross-sectional design and their
difficulty in controlling diet effects on the gut microbiota. There is considerable interindividual variability in the microbiota composition, and active individuals tend to eat
differently from sedentary ones [36]. For example, Clarke and colleagues found that
increased protein intake by elite rugby players explained many of the observed differences
in gut microbiota [29]. These limitations suggested the need to analyze longitudinal studies
to determine whether exercise independently alters the gut microbiota in humans.
Recently, Allen and colleagues obtained results in the first controlled longitudinal
study with a strict diet to evaluate the effects of exercise on the gut microbiota. Various
bacterial taxa were differentially altered by exercise depending on the subjects’ BMI. For
example, exercise increased Faecalibacterium species in lean subjects but reduced their
abundance in obese subjects, Bacteroides species decreased in lean subjects and increased in
obese subjects. Six weeks of exercise also increased the abundance of butyrate-producing
taxa and fecal acetate concentrations, but only in lean subjects. Interestingly, most bacterial
taxa and SCFAs that increased with exercise subsequently decreased during the 6-week
sedentary rest period, indicating that the effects of exercise on the microbiota were transient
and reversible [37].
Similarly, Cronin and colleagues sought to determine whether a short-term exercise
regimen, with or without whey protein supplementation, could alter gut microbial composition and function in predominantly overweight or obese adult men and women. The
post-intervention evaluation revealed no significant changes in taxonomic composition
or metabolic pathways in either exercise group compared to baseline. However, a trend
for increased bacterial diversity was observed in the exercise and exercise plus whey
protein groups compared to the group receiving whey protein alone. Metagenomic and
metabolomic analyzes revealed only modest alterations in microbial metabolism. Although
the study had a large sample size, the authors note that self-reported maintenance of usual
Hygiene 2022, 2
position and function in predominantly overweight or obese adult men and women. The
post-intervention evaluation revealed no significant changes in taxonomic composition or
metabolic pathways in either exercise group compared to baseline. However, a trend for
increased bacterial diversity was observed in the exercise and exercise plus whey protein
groups compared to the group receiving whey protein alone. Metagenomic and metabolomic analyzes revealed only modest alterations in microbial metabolism. Although 157
the
study had a large sample size, the authors note that self-reported maintenance of usual
dietary intake and a wide BMI range may have prevented the detection of more significant
changes
[38]. and a wide BMI range may have prevented the detection of more significant
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reduces transit time in the large intestine and has been shown to speed the movement of
gas through the gastrointestinal tract [42,43].
Exercise impacts the autonomic nervous system by increasing general vagal and
sympathetic tone [44]. However, regional, or global changes in gastrointestinal transit are
likely to deeply affect the intestinal pH, mucus secretion, biofilm formation, and nutrient
availability to the microbiota. Mechanical forces increase in the abdomen during most
types of aerobic exercise, which could potentially influence intestinal motility or increase
the mixing of intestinal contents [44].
Physical training can also alter the enterohepatic circulation of bile acids [45]. Bile
acids are potent regulators of gut microbiota community structure, and the absence of these
molecules is associated with significant alterations in gut microbial communities [46].
Hygiene 2022, 2
158
We have seen how physical exercise can modulate the intestinal microbiota, we also
see that adaptations to exercise can also be influenced by the intestinal microbiota, playing
an important role in the production, storage, and expenditure of energy obtained from the
diet, as well as in inflammation, oxidative reactions, and hydration status [47].
The intestinal microbiota promotes the digestion and absorption of food to produce
energy by the host, in this process the colon metabolizes complex carbohydrates and later
they are fermented into SCFA such as butyrate, acetate and propionate [48]. Propionate and
acetate are transported in the bloodstream to different organs in the human body where
they are used as substrates for energy metabolism, particularly by hepatocyte cells, which
use propionate for gluconeogenesis [49]. Given the energy requirements during physical
exercise, the intestinal microbiota exerts positive effects on the performance of athletes by
providing energy substrate to the muscle through the production of SCFA [48,50].
One of the main physiological adaptations to physical exercise is the modulation of
oxidative and nitrosativo stress to avoid tissue damage from intestinal permeability and
bacterial translocation [51]. It has been described that endurance athletes have a high
prevalence of upper respiratory tract infections and gastrointestinal problems, including
increased permeability of the gastrointestinal epithelial wall, also called “leaky gut”, altered
mucosal thickness, and higher bacterial translocation rates. Athletes exposed to highintensity exercise show a higher incidence of gastrointestinal problems, symptoms such as
diarrhea, bloating, nausea, and even bleeding [52].
Ismaeel et al. recently described that ROS can be decisive for the cell adaptation
process, acting as redox signaling molecules. The intestinal microbiota regulates the
maintenance of the gastrointestinal redox state and mitochondrial morphology, through
the production of SCFAs and secondary bile acids, which will improve the functioning of
the intestinal barrier and the immune response of the mucosa [53].
Recently, the intestinal microbiota has been related to the maintenance of adequate
hydration during exercise. The phylum Bacteroidetes has been shown to reduce plasma
sodium levels, while the Clostridium genus reduced plasma osmolality levels in healthy
young individuals. These results suggested that the microbiota influences cellular solute
transport across the intestinal mucosa and contributes to hydration status, while reducing
plasma osmolality. In the same experiment, high numbers of Bifidobacterium influenced
T cell levels, reflecting interaction with the host’s immune response. Because good hydration status and a properly functioning protective gut barrier are essential for athletic
performance, and because some athletes do not meet their fluid needs during exercise, it
is important to understand the role of the microbiota in the water transport and changes
associated with the intestinal mucosa [54].
4. Methods Used to Study the Microbiota
Currently, the analysis of the intestinal microbiota is carried out with the stool samples
of the subjects participating in the studies, before DNA extraction, it can be considered
to lyophilize the samples, collected in the sterile container, in which, in this way, the
percentage of water can be obtained. Next, the most widely used standardized technique is
DNA extraction using the IHMS (International Human Microbiota Standards) standard
operating protocol. The standardized IHMS “Q” protocol is the one recommended by
the International Microbiome Consortium [55]. The homogenizer (Bullet Blender Storm,
Next Advance, NY, USA) was used using glass balls. DNA quantification was performed
with the PicoGreen dsDNA Quant iT kit (ThermoFisher Scientific, Waltham, MA, USA)
and the FP 8300 spectrofluorometer (Jasco, Tokyo, Japan) and DNA purity estimation
was performed with a spectrophotometer, using Nanodrop 1100 (Thermofisher Scientific,
Waltham, MA, USA).
The product of the extraction is used for massive sequencing. For sequencing, libraries
are generated by amplifying the V3 and V4 hypervariable regions of the 16S rRNA gene,
which are present in all bacterial species.
Hygiene 2022, 2
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The primers used for library generation included the double index sequence needed
for multiplexing along with heterogeneity spacers to increase library complexity. The
product was a 459 bp amplification which is visualized on a Gelred stained 0.8% agarose
gel (Merck KGaA, Darmstadt, Germany). DNA amplifications are typically sequenced on
a MiSeq Illumina platform (Illumina, San Diego, CA). Sequence results can be analyzed
using Quantitative Insights into Microbial Ecology (QIIME2), v. 18.2, [56].
Reads are assigned to an Organizational Taxonomic Unit (OTU) when sequence
identity was greater than or equal to 97%. Sequences that did not match any reference
are discarded. The analysis of beta diversity allows us to know the distribution of each
microbiota sample in space, considering the distance matrix that exists between the different
OTUs that make up the community, so that the microbiota samples that are close in space
they are more like each other. Four indices are commonly used to determine beta diversity:
•
•
•
Bray Curtis: this index is not affected by null values (when a species is not detected, or
its value is 0). It is a quantitative index because it considers the abundance of species
and does not consider phylogenetic distances. This index is between 0 and 1.
Jaccard: measures the degree of similarity, its range is from 0 to 1, it does not consider
the abundance of the species but only their presence or absence, so it is affected by
null values.
Weighted Unifrac and Unweighted Unifrac: Unifrac methods consider information
about the abundance of species and their phylogenetic distance. The Unweighted parameter is qualitative because it only considers the presence or absence of individuals,
while the Weighted parameter considers the abundances.
The analysis of alpha diversity allows us to know quantitatively, how diverse the
microbiota is, how many different species there are. Four indices are commonly used to
terminate alpha diversity:
•
•
•
•
Observed OTUs: this index allows us to know at a quantitative level the number of
OTUs in each sample.
Pielou index (evenness): this index is indicative of how the species are distributed,
of their uniformity in the microbiota, if there are a few species that dominate the
microbiota (low evenness) or the abundance of the species is similar (high evenness).
Shannon index: the Shannon index is an index that considers the richness of species
(richness) and the scale considering the distribution of abundance of each species
(evenness).
Faith’s index: it is an index that takes taxonomy into account, that is, it is a phylogenetic
index that measures the distances of the branches that separate one OTU from another,
the more different the 2 OTUS are phylogenetically, the higher the Faith’s index, the
greater the Phylogenetic separation of all OTUS within Faith’s largest index sample.
5. Conclusions
The key component that determines the correct function of the intestinal barrier is the
gut microbiota. However, more studies are needed to understand how the gut microbiota
composition can influence physical performance. Given that details and small gains are
recognized as crucial for competitive success in athletes, it is reasonable to consider the gut
microbiota as a potential factor that could modulate physical efficiency and would need
further study to determine its importance and to find strategies that can mediate the effects
of exercise.
Author Contributions: D.D.-B.: writing and review of the drafts of the manuscript and acquisition,
analysis, and interpretation of data. G.G.-P.-d.-S.: design and conceptualization of the study, writing
and review of the drafts of the manuscript, and supervision. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Hygiene 2022, 2
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Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no potential conflict of interest with respect to the research,
authorship, and/or publication of this article.
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W2521344850.txt | Chan et al. Trials (2016) 17:460
DOI 10.1186/s13063-016-1582-z
STUDY PROTOCOL
Open Access
A home-based exercise intervention for
caregivers of persons with dementia: study
protocol for a randomised controlled trial
Wai Chi Chan1* , Nicola Lautenschlager2,3,4, Briony Dow5, Suk Ling Ma6, Corine Sau Man Wong1
and Linda Chiu Wa Lam6
Abstract
Background: Family members, who provide the majority of care for persons with dementia, are especially
vulnerable to developing depression. Interventions targeting their depressive symptoms have been proposed but
their efficacies vary considerably. It has been suggested that interventions carried out in the home setting and
involving both caregivers and care recipients are more efficacious. This study aims to compare the efficacy of a
home-based structured exercise programme involving both persons with dementia and their caregivers with
nonexercise social contact control in treating depression among caregivers.
Methods/design: This is a parallel-group, assessor-blind, randomised controlled trial. A total of 136 caregiver-carerecipient dyads (i.e. 272 participants in total) will be recruited and randomly allocated to either a home-based
structured exercise (sitting Tai Chi) group or a social contact control group. The trial comprises a 3-month
intervention phase followed by an extended observation phase of another 3 months. All participants will be
assessed at baseline, 6th week, 12th week and 24th week. The primary outcome will be the reduction in depression
among caregivers as measured by the Hamilton Rating Scale for Depression. The secondary outcomes will be
burden, quality of life, cognitive performance and balance ability of the caregivers, as well as the neuropsychiatric
symptoms, cognitive function, balance and functional abilities of the persons with dementia. We will also examine
whether the brain-derived neurotrophic factor gene modulates mood changes in response to exercise.
Discussion: The findings offer a potential avenue of intervention by providing a low-cost, safe and effective
treatment for depression among dementia caregivers, which may in turn also benefit the care recipients.
Trial registration: ClinicalTrials.gov Identifier: NCT02132039, registered on 28 April 2014.
Keywords: Home-based exercise, Tai Chi, Caregivers, Dementia, Depression, Randomised controlled trial
Background
Dementia affects over 47 million people worldwide [1].
It causes decline in cognitive function, impairment in activities of daily living, and is associated with significant
behavioural and psychological symptoms among the persons affected. Since the majority of dementia care is provided by family members [2], it is not surprising that
they suffer from a high level of psychological distress
* Correspondence: waicchan@hku.hk
1
Department of Psychiatry, The University of Hong Kong, Queen Mary
Hospital, Pokfulam, Hong Kong
Full list of author information is available at the end of the article
and burden. It is now evident that informal caregivers of
persons with dementia are at an elevated risk of developing depression and anxiety, and report poorer quality of
life [3–5]. They also experience more stress than those
who care for someone with physical disorders [6]. In a
local study interviewing family caregivers of older adults
with dementia in Hong Kong, they suffered significant
stress and reported various negative emotions, conflicts
between social roles, and difficulty in providing daily
care and managing the care recipients’ behaviours [7].
Dementia caregiving has, therefore, emerged as a pressing health care issue.
© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Chan et al. Trials (2016) 17:460
In view of this, different interventions have been developed to alleviate depressive symptoms among caregivers of dementia persons. Recent reviews have
analysed the efficacies of educational [8] and social support (e.g. befriending and peer support, family support
and social network interventions, and support groups) [9],
and psychological [10] and Internet-based interventions
[11] for caregivers of persons with dementia. Though clinically meaningful, these interventions achieved only
small effects [8, 11] or inconsistent results [9]. This
may be partly because the programme goals and target groups were not clearly defined. Therefore, it has
been suggested that research should focus on subgroups of caregivers such as depressed caregivers
[12].
Among interventions for depression, exercise therapy
has drawn much attention from researchers and practitioners. Exercise is a subset of physical activity that is
planned, structured, repetitive and purposeful, and regular exercise compares favourably to antidepressants as
treatment for milder depression [13]. Though its antidepressant mechanism is not yet clear, evidence suggests
that exercise improves mood by normalising brainderived neurotrophic factor levels [14], modifying serotonin function and releasing endogenous opioids [15].
More recently, exercise is reported to reduce atrial natriuretic peptide and brain natriuretic peptide, and to
enhance copepetin and growth hormone among depressed individuals [16]. In addition, exercise improves
one’s psychological and social sense of wellbeing [15]. It
is, therefore, not surprising that a recent meta-analysis
showed that exercise had a large and significant effect on
depression (standardised mean deviation adjusted for publication bias = 1.11) with a fail-safe number of 1057 [17].
There has been emerging evidence supporting the
effectiveness of exercise in treating depression in dementia
caregivers. In a recent study, 17 caregivers participating in
regular exercise of moderate intensity experienced less
sense of burden and fatigue, and reported better sleep
when compared with those in the control group [18]. Encouraging results have also been reported with endurance
exercise [19], yoga [20], walking [21], and a combination
of various physical activities [22].
However, the number of exercise studies for caregivers
remains limited, and the sample sizes involved are usually
small. This is probably related to the difficulty for dementia caregivers of participating in centre- or communitybased exercise interventions because they are unable to
find someone to take care of the care recipients, and lack
time for additional activities [23]. In some previous studies, caregivers also voiced a preference for a programme
that both caregiver and care recipient can participate in
[22]. Home-based programmes involving both caregivers
and care recipients may offer a possible solution [24]. Not
Page 2 of 8
only does the home-based dyadic intervention improve
treatment adherence [25], it is also more successful in
enhancing care-recipient function and reducing caregiver
sense of burden [26].
We therefore propose to conduct the following
assessor-blind randomised controlled trial of a homebased exercise intervention for both dementia caregivers
and their care recipients.
Objectives
The objectives of the study are as follows:
1. To evaluate the effect of a home-based exercise
programme on:
(a). caregivers’ depression
(b).caregivers’ burden, quality of life, cognitive
performance and balance ability
(c).neuropsychiatric symptoms, cognitive function,
and balance and functional abilities among
persons with dementia
2. To assess whether the brain-derived neurotrophic
factor (BDNF) gene modulates mood changes in
response to exercise intervention
Methods/design
Design
This is a parallel-group, assessor-blind, randomised
controlled trial, which comprises a 3-month intervention phase followed by an extended observation phase
lasting for 3 months. Participants will be randomly
assigned to either the home-based structured exercise
(intervention) or the standard nonexercise social contact (control) group. The study is registered with
ClinicalTrials.gov (NCT02132039), and conducted in
accordance with the Declaration of Helsinki. Ethics
approval has been sought from respective Institutional
Review Boards. All results will be reported according
to the Consolidated Standards of Reporting Trials
(CONSORT) 2010 Statement [27]. A CONSORT flow
diagram of the study is depicted in Fig. 1.
Participants
Caregiver-care-recipient dyads will be recruited from
psychogeriatric and geriatric outpatient clinics and
various community centres for elders in Hong Kong
by convenience sampling. Potential participants will
be identified by the research team and screened for
eligibility based on the inclusion and exclusion
criteria. Informed consent will be obtained from all
eligible participants before enrollment.
Inclusion criteria
Caregivers:
Chan et al. Trials (2016) 17:460
Page 3 of 8
Fig. 1 Consolidated Standards of Reporting Trials (CONSORT) flow diagram of the home-based exercise trial
Aged 50 years or above
Informal caregivers, i.e. unpaid family members who
offer a substantial amount of care for the persons
with dementia
Geriatric Depression Scale (GDS) [28] score >0 but
<8 (i.e. the local cut-off score for clinically significant
depression)
Understand Chinese
Receiving antidepressant treatment on a steady dose
for at least 3 months (if applicable)
Care recipients:
Aged 60 years or above
Dementia diagnosis confirmed by a physician
Dependence in at least one of the activities of daily
living (ADL)
Mini Mental State Examination (MMSE) [29] score >10
Exclusion criteria
Both caregivers and care recipients:
No regular (i.e. at least three times/week) Tai Chi
practice or other forms of mind-body exercise such
as yoga, qigong, or mindfulness training in the past
6 months
Presence of any condition which renders
participants unsuitable for physical training, for
example, severe psychotic symptoms, imminently
suicidal, significant orthopaedic problems, or
unstable medical conditions
Randomisation and blinding
Participants will be randomly assigned to either the
intervention or the control group using a computergenerated block randomisation sequence (block sizes of
4). Randomisation will be performed by an independent
Chan et al. Trials (2016) 17:460
team member who will not be involved in the recruitment, assessment, or delivery of the intervention. The
allocation sequence will be concealed from the study
investigators and research staff in sequentially numbered, opaque, and sealed envelopes. The assessor for
clinical parameters will be blinded to the randomisation
status, and the trainer who conducts the intervention
will be blinded to the assessment results.
Intervention
The intervention consists of eight home-based sessions
of sitting Tai Chi. Tai Chi is a traditional Chinese exercise that combines rhythmic and flowing patterns of
bodily movements with coordinated breathing and
mindful meditation [30]. It is a moderate-intensity aerobic exercise at about 3 to 6 metabolic equivalents [31].
It has been successfully implemented in the local older
population [32] and, as a low impact exercise, it is associated with a lower risk of musculoskeletal injury making
it a suitable form of exercise for older adults. Since the
current study includes persons with significant cognitive
impairment, we adopt sitting Tai Chi which is a simpler
version of Tai Chi developed by Hong Kong Polytechnic
University [33]. The exercise consists of 12 steps, which
include weight shifting in different sitting positions,
trunk and upper limb movements, and alternate thigh
lift in a smooth and coordinated manner [33].
Four weekly exercise sessions will be arranged over the
first 4 weeks, and four more biweekly sessions over the
next 8 weeks. Each session will last for 1 h, which starts
with 10 min of warm-up activities followed by 40 min of
sitting Tai Chi practice and, lastly, 10 min of cool-down
exercises. The caregiver-care-recipient dyads will practise Tai Chi in their home under the instruction of a Tai
Chi teacher with more than 20 years of teaching experience, and a trained research assistant. In order to monitor the progress and address participants’ concerns,
biweekly phone contacts will be made during the study
period (12 scheduled phone contacts in 24 weeks). On
completion of the eight home-based Tai Chi sessions,
participants will be given a manual and training video
with detailed instructions of the Tai Chi poses to facilitate their continuation of Tai Chi practices.
A standard nonexercise social contact will be provided
to the participants in the control group. Since social
contact may be beneficial to one’s mood and may contribute to any improvement observed in the intervention
group, we provide participants in the control group with
a level of social contact equivalent to the intervention
group. They will be visited by the research assistant eight
times over 12 weeks. The visits involve a series of conversations related to neutral topics that are designed
according to the principles of a befriending programme
[34]. These topics are structured and standardised, and
Page 4 of 8
do not involve any topic concerning physical activity.
Similar to the intervention group, 12 biweekly phone
calls will be made during the study period to offer a
comparable level of social contact.
Primary outcome
Participants will be assessed at baseline, 6th week, 12th
week and 24th week for their responses to intervention.
The assessment schedules for caregivers and care recipients are described in Table 1. The primary outcome of
the study is the proportion of participants who are classified as responders by the Hamilton Rating Scale for
Depression (HAM-D-17) [35]. HAM-D-17 is a widely
used and reliable measure of depressive symptoms. The
scores range from 0 to 52, with higher scores indicating
greater depression severity. Response to the intervention
is defined as a reduction of the HAM-D-17 total score
by ≥50 % from baseline to endpoint. Assessors will follow the structured interview guide for the Hamilton
Depression Rating Scale (SIGH-D) when administering
the HAM-D-17 [36].
Assessment for caregivers only:
1. The Zarit Burden Interview (ZBI) [37] is a 22-item
self-administrated questionnaire specially designed
to measure the stresses experienced by caregivers of
persons with dementia. Caregivers will assess the
impact of the patient’s disabilities on their life
2. The SF-12 Health Survey (SF-12) [38] measures the
health-related quality of life and gives two component scores: a physical component summary (PCS)
and a mental component summary (MCS)
3. The Executive Interview (EXIT25) [39] is a 25-item
screening instrument developed to assess executive
cognitive dysfunction
4. The International Personality Item Pool (IPIP) [40]
consists of 50 items assessing the ‘Big Five’
personality traits: openness to experience,
conscientiousness, extraversion, agreeableness, and
neuroticism
Assessment for care recipients only:
1. The Cornell Scale for Depression in Dementia
(CSDD) [41] is a clinician-rated instrument measuring depressive symptoms in persons with cognitive
impairment
2. The Neuropsychiatric Inventory (NPI) [42] assesses
the frequency and severity of neuropsychiatric
symptoms across 12 domains including psychotic
features, mood changes, activities disturbances and
vegetative symptoms
3. The Disability Assessment for Dementia (DAD) [43]
evaluates functional abilities in both basic and
Chan et al. Trials (2016) 17:460
Page 5 of 8
Table 1 Assessment schedule for caregivers and care recipients
Baseline
assessment
Week
6
Week
12
Week
24
Caregiver only
GDS (screening only)
√
HAM-D-17
√
√
√
√
ZBI
√
√
√
√
SF-12
√
√
√
EXIT25
√
√
√
IPIP
√
√
√
Care recipients only
CSDD
√
√
NPI
√
√
√
DAD
√
√
√
MBI
√
√
√
MMSE
√
√
√
Digit span
√
√
√
Delayed recall
√
√
√
CVFT
√
√
√
BBS
√
√
√
FR
√
√
√
TUG
√
√
√
Logbook
√
√
√
√
Pedometer
√
√
√
√
BDNF genotype
√
Body weight and
height
√
√
√
Blood pressure and
pulse
√
√
√
Caregivers and care
recipients
BBS Berg Balance Scale, BDNF brain-derived neurotrophic factor, CSDD Cornell
Scale for Depression in Dementia, CVFT Category Verbal Fluency Test, DAD
Disability Assessment for Dementia, EXIT25 Executive Interview, FR Functional
Reach, GDS Geriatric Depression Scale, HAM-D-17 Hamilton Rating Scale for
Depression, IPIP International Personality Item Pool, MBI Modified Barthel
Index, MMSE Mini Mental State Examination, NPI Neuropsychiatric Inventory,
TUG Timed Up and Go, ZBI Zarit Burden Interview
instrumental activities of daily living in people with
dementia
4. The Modified Barthel Index (MBI) [44] measures
individuals’ performance on 10 activities of daily
living: eating, personal hygiene, bathing, toileting,
dressing, bowel control, bladder control, transfers,
walking, and stair climbing
Assessment for both caregivers and care recipients:
1. Sociodemographic information includes age, gender,
education level and marital status
2. Lifestyle factors such as smoking, drinking, and
sleeping habits. Health-related information like
chronic health problems and medications will also
be recorded. Resting blood pressure and heart rate
will be taken. Body height and weight will also be
measured to calculate Body Mass Index
3. Global cognitive function will be measured by the
MMSE [29], digit span, delayed recall, and the
Category Verbal Fluency Test (CVFT) [45]
4. Balance ability and functional mobility will be
measured by the Berg Balance Scale (BBS) [46], the
Functional Reach (FR) test [47], and the Timed Up
and Go (TUG) test [48]
5. Physical activity and programme adherence will be
assessed using logbook recordings. Before each
assessment, participants will be asked to wear a
pedometer for 1 week. Number of walking steps,
history of falls, frequency, duration, and type of
exercises during the week will be recorded in the
logbook
6. Physiological markers – to examine the role of the
BDNF Val66Met polymorphism in the
antidepressant effect of exercise, buccal swabs will
be collected at the baseline assessment
Sample size calculation
The sample size is calculated by the proportion of caregivers who respond to the intervention as indicated by
the HAM-D-17. Taking reference of the previous study
[49], we expect a 30 % reduction in the proportion of
participants rated as depressed in the control group with
social contact only. Assuming that a further 30 % reduction is achieved by sitting Tai Chi, we need 56 caregivercare-recipient dyads (i.e. 112 participants) in each arm
to identify a significant effect at a two-tailed alpha of
0.05 and 80 % power. Allowing for 20 % dropout, the
number of dyads in each arm will rise to 68. A total
number of participants of 272 will be recruited.
Statistical analyses
Baseline differences in demographic and clinical characteristics between the intervention and control
groups will be evaluated by chi-square and independent sample t tests. All data will be analysed using the
intent-to-treat principle. For outcome measures that
are not normally distributed (e.g. the HAM-D-17), log
transformation will be applied to perform the analyses. Mixed-effects models will be fitted for each outcome measure using baseline, 6th, 12th and 24th
week follow-up data. Possible confounding factors
such as sex, age, and use of antidepressant medication
will be treated as time-dependent covariate in the
mixed-effects models. If applicable, subgroup analysis
will be carried out to estimate whether there is any
Chan et al. Trials (2016) 17:460
Page 6 of 8
gender difference (female versus male caregivers)
regarding the effect of the intervention in alleviating
depressive symptoms. Fixed effects for time, intervention group, and their interactions will be examined
and unstructured covariance structure will be employed
to account for the within-subject correlation over time.
Levels of significance will be set at p < 0.05. All analyses
will be performed using SPSS version 20.0 (SPSS Inc.,
Chicago, IL, USA).
Abbreviations
ADL: Activities of daily living; BBS: Berg Balance Scale; BDNF: brain-derived
neurotrophic factor; CONSORT: Consolidated Standards of Reporting Trials;
CSDD: Cornell Scale for Depression in Dementia; CVFT: Category Verbal
Fluency Test; DAD: Disability Assessment for Dementia; EXIT25: Executive
Interview; FR: Functional Reach; GDS: Geriatric Depression Scale; HAM-D17: Hamilton Rating Scale for Depression; IPIP: International Personality Item
Pool; MBI: Modified Barthel Index; MCS: Mental component summary;
MMSE: Mini Mental State Examination; NPI: Neuropsychiatric Inventory;
PCS: Physical component summary; REC: Research Ethics Committee;
SF-12: SF-12 Health Survey; SIGH-D: Structured interview guide for the
Hamilton Depression Rating Scale; TUG: Timed Up and Go; ZBI: Zarit Burden
Interview
Discussion
Since the prevalence of dementia doubles every 5 years
after the age of 60, population ageing inevitably
increases the number of persons with dementia. Hong
Kong is no exception to this trend. It is estimated that
around one in 10 local older persons are now suffering
from dementia [50].
Caregiving for persons with dementia is demanding
and challenging. Evidence shows that it adversely
affects caregivers’ physical [51] as well as mental wellbeing [3–5], including a vulnerability to developing
depression. In a review of 10 studies involving 790
caregivers of persons with dementia, more than one
fifth of them suffered from a depressive disorder [3].
The prevalence of depressive symptoms among caregivers is even higher [14].
This study will investigate the efficacy of a structured
exercise programme in alleviating depressive symptoms
among caregivers of persons with dementia. We focus
on caregivers with depressive symptoms instead of
depressive disorders as the former constitute the largest
group of depressed caregivers. Besides, because of ethical
concerns, we will refer those who suffer from clinically
significant depression to appropriate agencies for psychiatric assessment and treatment.
The intervention is tailor-made for family caregivers
in three ways. First, the programme is delivered at
participants’ homes to facilitate their participation.
Second, both caregivers and care recipients will be
involved in the study. Third, a moderate-intensity aerobic exercise that has been successfully implemented
in the local older population (i.e. sitting Tai Chi) is
chosen as the intervention.
Sitting Tai Chi is a safe, low-impact and low-cost exercise intervention. If it is shown to improve caregivers’
mood, it will offer a potentially effective treatment alternative for depression among caregivers of persons with
dementia. In addition, we expect that home-based exercise will also benefit the care recipients.
Acknowledgements
We would like to express our sincere thanks to our Tai Chi teacher Mr.
Charles Lee for agreeing to take part in the study. We would also like to
thank the social centres and clinics for their valuable support in participant
recruitment. In addition, we are grateful to Mrs Eleanor Chan and Mrs
Miranda Tung for their advice on the assessment methods.
Trial status
The study is still recruiting at the time of submission.
Funding
The study is supported by the Health and Medical Research Fund of Food
and Health Bureau of the Hong Kong Special Administrative Region
Government (Ref: 11121441).
Availability of data and materials
Not applicable.
Authors’ contributions
WCC conceived the idea for the study, obtained funding, and took primary
responsibility for study coordination and manuscript preparation. NL, BD,
SLM and LCWL obtained funding and contributed to the concept and
design of the study, intervention and outcome assessment. CSMW
contributed to the coordination of the study, statistical analysis, and
manuscript preparation. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
Ethics approval has been sought from the Institutional Review Board of the
University of Hong Kong/Hospital Authority Hong Kong West Cluster
(UW 13-453), Joint Chinese University of Hong Kong-New Territories East
Cluster Clinical Research Ethics Committee (2014.254-T), New Territories
West Cluster Clinical and Research Ethics Committee (REC) (NTWC/CREC/
1302/14), Kowloon West Cluster REC (KW-EX-15-076-86-12TCM), and
Hong Kong East Cluster REC (HKEC-2015-045). Participation in the study
is completely voluntary. Participants will be able to withdraw from it at
any time. The decision to participate or withdraw will not affect their
existing treatment or service received. Caregivers who are found to have
significant depressive symptoms or other mental health problems will be
referred to appropriate health services for follow-up. All personal information
collected will be kept strictly confidential and used for research purposes only.
Research team members will be responsible for safekeeping the personal data.
Only the principal investigator and designated research staff will have access to
the interim results and final trial dataset. Informed consent will be obtained
from all eligible participants before enrollment.
Author details
1
Department of Psychiatry, The University of Hong Kong, Queen Mary
Hospital, Pokfulam, Hong Kong. 2Academic Unit for Psychiatry of Old Age,
Department of Psychiatry, University of Melbourne, Melbourne, VIC, Australia.
3
NorthWestern Aged Mental Health, Royal Park Campus, Parkville, VIC,
Australia. 4School of Clinical Neurosciences and the Western Australia Centre
and Health and Ageing, University of Western Australia, Perth, WA, Australia.
5
National Ageing Research Institute, The University of Melbourne, Melbourne,
Chan et al. Trials (2016) 17:460
VIC, Australia. 6Department of Psychiatry, The Chinese University of Hong
Kong, Tai Po Hospital, Tai Po, Hong Kong.
Received: 7 May 2016 Accepted: 27 August 2016
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W4296115647.txt | Academic Journal of Science and Technology
ISSN: 2771-3032 | Vol. 2, No. 3, 2022
The Application of New Energy Materials in New‐energy
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Yuting Qin1, a, Junying Fang2
1Xiamen
University Malaysia, Jalan Sunsuria, Bandar Sunsuria, 43900, Sepang, Selangor Darul Ehsan, Malaysia
University of Technology.69 Hongguang Dadao, Banan District, Chongqing, China
aCorresponding Author’s Email: ege1909292@xmu.edu.my
2Chongqing
Abstract: Climate change is becoming one of the biggest environmental challenges in the 21st century. New-energy vehicle
produce less greenhouse gases as compared to the traditional vehicles with internal combustion engines. New energy vehicles
have great potential to successfully address the issue of climate change. Additionally, new energy materials can also be used in
automotive lightweight technology. This paper discussed the currently lightweight materials including high-strength low-alloy
steel (HSLA), carbon fiber composite material, and modified plastics. According to the discussion in this paper, high-strength
low-alloy steel can be used in automotive safety parts, chassis, and body. Carbon fiber composite materials can be used in car
bodies, chassis, roofs, doors, head covers, hoods, rear wings, center consoles, trim strips, drive shafts, leaf springs, frames, brake
pads, interior and exterior accessories. Modified plastics can be mainly used in exterior decorative parts, interior decorative parts,
functional parts, and structural parts. However, three are still many drawbacks of application of new energy materials in newenergy vehicle. The current new energy materials used in automotive lightweight technology are usually costly and timeconsuming. Moreover, the characteristics of the new energy materials are still not clear. It could have a negative impact on the
environment. Before this technology can be widely used in new-energy vehicles, more research is needed regarding the new
energy materials.
Keywords: New energy material, New-energy vehicle, Climate change, High-strength low-alloy steel (HSLA), Carbon fiber
composite material, Modified plastic, Greenhouse gases.
scale of the auto parts market continues to increase. The scale
of China's auto parts market has grown from 3.46 trillion yuan
in 2016 to 4.57 trillion yuan in 2020, with an average annual
compound growth rate of 7.2%; the market size is expected to
reach 4.9 trillion yuan in 2021.
In recent years, new energy materials have wide
applications in a variety of technological fields such as thinfilm solar cells, light-emitting device, and photodetector.
Many new energy materials have been introduced to the newenergy vehicle industry and has produced some promising
outcomes. Clean energy technologies of new-energy vehicles
usually require a wide range of minerals1 and metals. The
type and volume of mineral needs are very different based on
the clean energy technologies (Table 1) [5]. In the current
new-energy vehicle industry, reduction in weight is one of the
development directions of the automotive industry in the
future. The reduction in weight directly means an increase in
cruising range. It is found that for the pure electric vehicles,
the vehicle weight can be reduced by 10 kg, and the driving
range can be increased by 2.5 km. As a result, the GHG
emissions could be significantly reduced in the same diving
range. In the lightweight industry chain of new-energy
vehicles, the main raw materials for lightweight include highstrength steel, aluminum alloy, magnesium alloy, carbon fiber
composite materials and modified plastics. These materials
could constitute chassis, engine, seat, luggage rack, gear lever,
interior, exterior and steering wheel, etc.
Few studies have been conducted in this field due to the
rapid growth of new energy material technology. The
objectives of this paper are to: 1) present the classification of
new energy materials and the application in new-energy
vehicle; 2) discuss the future vision of new energy materials
applied in new-energy vehicle.
1. Introduction
Climate change is one of the biggest challenges of our
times[1]. It is believed that one of the major reasons of climate
change is human activity such as fossil fuel combustion.
When these fossil fuels are combusted, greenhouse gases are
released into the Earth's atmosphere. As a result, the heat from
the sun was trapped and global temperatures to rise. Based on
climate models, the Intergovernmental Panel on Climate
Change (IPCC) predicts that the global temperatures will rise
by about 1.4-5.8 degrees Celsius (2.5-10.4 degrees Fahrenheit)
by 2100 [2]. According to this forecast, global temperatures
will undergo dramatic changes not seen in the past 10,000
years, with potentially significant impacts on the global
environment. To reduce global warming, many countries have
agreed to reduce their greenhouse gas (GHG) emissions into
the atmosphere to 1990 levels.
Generally speaking, transportation sector produces one of
the largest proportion of GHG emissions. It also has a
negative impact on public health and the environment.
Vehicle electrification has great potential to reduce air
pollutions and tackle climate change [3]. Compared with the
internal combustion engine vehicles, new-energy vehicles
hardly generate greenhouse gases during driving. If it can
completely replace internal combustion engine, new energy
vehicles could effectively solve the climate change problem.
According to data released by the China Association of
Automobile Manufacturers, the production and sales of newenergy vehicles reached 397,000 and 383,000 respectively in
October 2021, an increase of 1.3 times year-on-year. From
January to October, the production and sales of new-energy
vehicles reached 2.566 million and 2.542 million respectively,
a year-on-year increase of 1.8 times [4]. At the same time, the
111
Table 1. Critical mineral needs for clean energy technologies
Copper
Nickel
Chromium
Solar photovoltaic
High
Low
Low
Bioenergy
High
Low
Low
Nuclear
Moderate
Moderate
Moderate
Electricity networks
High
Low
Low
New-energy vehicle and battery storage
High
High
Low
Hydrogen
Low
High
Low
Aluminium
High
Moderate
Low
High
High
Moderate
Automotive lightweight technology is mainly divided into
three aspects: structural optimization design, lightweight
material application and advanced manufacturing technology.
Among them, the use of lightweight alternative materials is
generally recognized in the industry. The currently used
lightweight materials mainly include high-strength low-alloy
steel (HSLA), carbon fiber composite material, and modified
plastics. Table 2 shows the mass reduction for different new
energy materials.
2. New Energy Materials and the
Application in New-energy Vehicle
2.1. Classification of New Energy Materials
At present, new energy materials has been widely
implemented in automotive lightweight technology. With the
development of this technology in the last decade, research
has begun to utilize new energy materials' properties.
Table 2. Mass reduction for different new energy materials
Lightweight materials
Mass reduction
Magnesium
30-70%
Carbon fiber composites
50-70%
Aluminum
30-60%
Titanium
40-55%
Advanced high strength steel
15-25%
High-strength low-alloy steel (HSLA) refers to steel with
cold rolling of 340 MPa and hot rolling of 490 MPa or more
[6]. High strength steel has a better weight reduction effect.
Compared with the traditional 340 MPa material, the
theoretical weight reduction potential of the 600 MPa grade
steel is about 20%, and the weight reduction potential of the
800 MPa material will increase to more than 30%. At present,
high-strength steel is mainly used in automotive safety parts,
chassis, and body.
Carbon fiber composite materials can be used in car bodies,
chassis, roofs, doors, head covers, hoods, rear wings, center
consoles, trim strips, drive shafts, leaf springs, frames, brake
pads, interior and exterior accessories etc. In 2019, the
domestic demand of carbon fiber composite materials in
China was 38000 tons (Figure 1) [7]. In the future, with the
increase of China's scientific research capabilities and the
steady development of the carbon fiber composite material
industry, the domestic demand will continuously increase.
Figure 1. China’s domestic demand of carbon fiber composite materials in in past decades
Modified plastics are the plastic products that are processed
and modified by filling, blending, strengthening and other
methods based on general-purpose plastics and engineering
plastics to improve the properties of flame retardancy,
strength, impact resistance, toughness, etc. At present,
modified plastics are mainly used in exterior decorative parts,
interior decorative parts, functional parts, and structural parts.
In 2019, the output of modified plastics of industrial
enterprises above designated size in China reached 19.55
million tons. In 2021, China's domestic demand for modifies
112
roofs, doors, head covers, hoods, rear wings, center consoles,
trim strips, drive shafts, leaf springs, frames, brake pads,
interior and exterior accessories. Modified plastics can be
mainly used in exterior decorative parts, interior decorative
parts, functional parts, and structural parts. However, three
are still many drawbacks of application of new energy
materials in new-energy vehicle. The current new energy
materials used in automotive lightweight technology are
usually costly and time-consuming. Moreover, the
characteristics of the new energy materials are still not clear.
It could have a negative impact on the environment. Before
this technology can be widely used in new-energy vehicles,
more research is needed regarding the new energy materials.
It can be seen that China's new-energy vehicle industry is
splendid. But as far as the future is concerned, if new-energy
vehicle is a solution for reduction of air pollutions and climate
change, then automotive lightweight technology is the core
technology. The new cutting-edge materials continuously
incubated by research and development institute will become
the basis and source of inspiration for the vigorous
development of energy innovation and intelligent technology.
It can provide infinite possibilities for the future of the newenergy vehicle industry.
plastics of industrial enterprises above designated size is 21.1
million tons and the sales scale exceed three trillion RMB.
2.2. Future Vision of New Energy Materials
applied in New-Energy Vehicle
In December 2017, the Ministry of Industry and
Information Technology issued the "Three-Year Action Plan
for Promoting the Development of the New-Generation
Artificial Intelligence Industry (2018-2020)", which pointed
out to establish an architecture that supports vehicle
intelligent computing platform, vehicle intelligent chips, and
automatic driving operations. Research and development of
key technologies and products such as systems and vehicle
intelligent algorithms, and build a vehicle intelligent platform
that integrates software, hardware, and algorithms. Electric
energy, electronic control systems, and lightweight vehicle
bodies play an important role in sensor interconnection,
automated driving, and safety assurance of new-energy
vehicle which will be the development trend of future
transportation.
The development of the new energy material industry will
become the infrastructure supporting this trend. For example,
the new-energy vehicle bodies can be built by plexiglas glass,
which could be a god alternative for automotive glass. It has
many advantages such as high transparency and high strength
compared to the currently widely used metal and glass
materials. The current new energy materials used in newenergy vehicle can be implanted with sensors. The
manufacturing of special functional parts can be completed
through 3D printing. The application of these new materials
in the automotive industry will also be the development trend
of future transportation. Last but not least, the existence of 5G
signals will serve for truly intelligent urban transportation,
and new-energy vehicle will become a new entrance to the
Internet in the era of everything connected.
References
[1] Seymour, F., & Busch, J. (2016). Why forests? Why now?: The
science, economics, and politics of tropical forests and climate
change. Brookings Institution Press.
[2] Milcarek, R. J., Turner, S., Zhang, R., Ahn, J., & Zhang, J.
(2017). Predicting envelope and micro cogeneration design
conditions for future climates. ASHRAE Transactions, 123(1).
[3] Jacobson, M. Z. (2009). Review of solutions to global warming,
air pollution, and energy security. Energy & Environmental
Science, 2(2), 148-173.
[4] Laitimes. (2021). China's New Energy Vehicle Production and
sales lead the World (Expert Interpretation). laitimes. Retrieved
July
10,
2022,
from
https://www.laitimes.com/en/article/18udh_1a483.html
3. Conclusion
New energy material is being developed at an exponential
rate, and it will revolutionize the new-energy vehicle industry.
Compared with the internal combustion engine vehicles, newenergy vehicles hardly generate greenhouse gases during
driving. If it can completely replace internal combustion
engine, new energy vehicles could effectively solve the
climate change problem. In addition, new energy materials
have a great potential to implement in automotive lightweight
technology due to their properties. According to the
discussion in this paper, high-strength low-alloy steel can be
used in automotive safety parts, chassis, and body. Carbon
fiber composite materials can be used in car bodies, chassis,
[5] IEA (2021), The Role of Critical Minerals in Clean Energy
Transitions, IEA, Paris, Retrieved July 10, 2022, from
https://www.iea.org/reports/the-role-of-critical-minerals-inclean-energy-transitions.
[6] Yan, X., Xia, Y., Blum, H. B., & Gernay, T. (2020). Elevated
temperature material properties of advanced high strength steel
alloys. Journal of Constructional Steel Research, 174, 106299.
[7] Li, Z., Feng, Z., Xu, L., Lv, C., Li, L., & Zhu, S. (2020)
Research on the development strategy of high-performance
fibers and their composite materials in my country[J]. China
Engineering Science, 22(5):28-36.
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W3006149597.txt | International Orthopaedics (2020) 44:911–918
https://doi.org/10.1007/s00264-020-04494-0
ORIGINAL PAPER
The column procedure preserves elbow stability
on biomechanical testing
Andrzej P. Podgórski 1,2
&
Bartłomiej Kordasiewicz 1 & Stanisław Pomianowski 1
Received: 1 June 2019 / Accepted: 30 January 2020 / Published online: 12 February 2020
# The Author(s) 2020
Abstract
Purpose The effect of open release of a post-traumatic elbow contracture on the stability of the joint has not been so far studied
in vivo. Resection of elbow joint capsule, the key element of surgery, was reported to have no effect on the stability of cadaveric
elbows. The joint capsule is yet known to participate in maintaining elbow stability as one of secondary stabilizers.
Methods We assessed elbow joint laxity in 39 patients who underwent an open contracture release via the ‘column procedure’
described by B. Morrey and P. Mansat within the preceeding three to nine months. The measurements were taken with an
apparatus designed particularly for this experiment according to the predetermined protocol. A preliminary part of the experiment
showed that there was no significant difference between laxity of two elbow joints in healthy volunteers. Laxity of the operated
elbows could be then compared with the contralateral joints.
Results Mean absolute difference of laxity between healthy and operated elbows was 1.55° (0.1°–4.1°, SD = 1.1) being significantly lower than 2°, p = 0.0056. The difference of the joint laxity between the operated and healthy elbows did not differ
statistically significantly by more than 0.6° from the difference of the laxity of two healthy elbows and, therefore, is not clinically
noticeable.
Conclusions Our experiment confirmed that the ‘column procedure’ is a safe procedure which does not compromise the stability
of the elbow joint.
Keywords Elbow stability . Elbow trauma . Elbow contracture . Column procedure
Introduction
Elbow stability results primarily from the integrity of relevant
anatomical structures which maintain physiological laxity of
the joint. Laxity can be defined as range of motion of the joint
in the coronal plane and differs broadly between individuals
[1, 2]. Physiological amounts of elbow laxity provide stability
of the joint, which is a clinically assessed feature and implies
This article does not contain any studies with animals performed by any
of the authors.
* Andrzej P. Podgórski
apodgors@poczta.onet.pl; andrzej.podgorski@wim.mil.pl
1
Department of Traumatology and Orthopaedic Surgery, Adam Gruca
Clinical Hospital, Center for Postgraduate Medical Education,
Otwock, Poland
2
Department of Neurosurgery, Military Institute of Medicine, ul.
Szaserów 128, 04-141 Warsaw, Poland
correct biomechanics of the joint. Following biomechanical
testing of cadaver elbows, Nielsen [3] and Dos Remedios [4]
independently concluded that resection of elbow joint capsule,
which is the key element of surgery for a post-traumatic contracture, does not affect the joint laxity, hence would not lead
to instability in clinical setting. Those findings have not been
confirmed in vivo even though the joint capsule is reported to
participate in maintaining the stability of the elbow [2]. In our
study, we assessed elbow joint laxity in 39 patients who had
undergone an open release of a post-traumatic contracture.
The measurements were taken using a specially constructed
apparatus which allowed full and safe immobilization of the
upper limb and precise biomechanical testing.
Elbow stability derives from the congruence of the articular
surfaces in roughly 50%, while the remaining half depends on
the integrity of ligaments, capsule, interosseous membrane
and, to a lesser degree, muscles of the arm and forearm which
act as dynamic stabilizers [2, 5, 6]. Those structures are also
classified as either primary or secondary stabilizers; the former
are those whose injury leads directly to increased laxity of the
912
International Orthopaedics (SICOT) (2020) 44:911–918
joint, and the latter are the structures whose damage would
increase laxity only after the relevant primary stabilizers had
also been injured. Primary stabilizers include the anterior bundle of the medial collateral ligament, lateral collateral ligament
complex and the congruence of the ulnohumeral joint [1, 7–9].
Important secondary stabilizers comprise the congruence of
the radiohumeral joint, joint capsule and muscular attachments of the pronator and flexor muscles of the forearm and
wrist to the medial humeral epicondyle and extensor muscles
to the lateral epicondyle, respectively. An important part of the
lateral collateral ligament complex, the lateral ulnar collateral
ligament, serves as a primary stabilizer acting against posterolateral rotatory instability of the elbow [10].
Morrey and An in their biomechanical experiments examined the amount of contribution of the particular anatomic
structures to the joint stability depending on the position of
the elbow [2]. They noted that the articular capsule is responsible for 30% of valgus stability in full extension and that its
importance diminishes with the flexion of the joint, dropping
to near-zero values in 90° of flexion. The capsule also provides 32% and 13% of varus stability in elbow extension and
90° of flexion, respectively. It serves as the main stabilizer
against joint distraction forces, providing 85% of resistance
in extension and 8% in 90° of flexion. There is also an important relation between forearm rotation and elbow laxity, which
is minimal in full supination, as demonstrated by
Pomianowski [11].
The post-traumatic contracture of the elbow is a common
complication of fractures and dislocations around the elbow
caused by thickening and scarring of the articular capsule,
altered shape and incongruence of bony surfaces, osteophyte
formation, presence of intra-articular loose bodies and heterotopic ossification. Its occurrence is related to initial damage to
the joint structures, intra-articular haematoma formation and
individual propensity but can usually be effectively prevented
by correct treatment and particularly by avoiding immobilization of the joint for longer than three weeks. Indications for
Table 1 Initial lesion of the
elbow in patients who
subsequently underwent an open
release of its contracture in our
series
surgical release include failure of physiotherapy after four to
six months or presence of a surgical pathology, e.g. loose
bodies within the joint, ossifications or a radioulnar synostosis. There are also patients with the so-called ‘stiff elbow’ who
do not benefit from physiotherapy and who should be treated
surgically without the usual delay.
Our experience involves 279 patients who have undergone
an open release of post-traumatic elbow contracture in our
centre since 2003 (Table 1). The relatively high percentage
of patients who developed a contracture following a radial
head fracture was mainly due to prolonged immobilization
of the joint and lack of adequate physiotherapy. Our surgical
technique follows the outline of the ‘column procedure’ described by B. Morrey and P. Mansat [12]. The joint is
approached via the anterolateral route between the extensor
carpi radialis longus and brevis and the extensor digitorum
communis anteriorly to the lateral collateral ligament. The
articular capsule is bluntly dissected and excised until the
anterior aspect of the ulna is visualized. The posterior compartment of the joint can be opened through the same incision
if needed, and posterior capsulectomy can also be performed.
Scar tissue, osteophytes and intra-articular loose bodies are
removed. If deemed necessary, the procedure can involve other steps, e.g. radial head excision, interpositional arthroplasty,
excision of synostosis or ulnar nerve transposition via a medial approach. Although there is no agreement on the superiority of the radial head replacement over simple excision [13],
we always replaced the radial head with a prosthesis (KPS)
designed by the senior author. Release of the ulnar nerve was
consistently combined with its anterior transposition and was
never performed prophylactically despite recent reports
underlining its benefit [14–16]. Arthrolysis of the elbow can
be performed using other approaches, most often Kocher approach, as well as combined with exposing the joint from the
medial or posterior aspect [17–20] depending on the underlying pathology [21]. Sequelae of complex injuries, e.g. terrible
triad of the elbow, are particularly demanding. Protocols
Lesion
Number of cases
Percentage
Distal humeral fracture
Radial head fracture
Proximal radius and ulna fractures
Elbow dislocation
Elbow dislocation and radial head fracture
Elbow dislocation and other or multiple fractures
Olecranon fracture
Radius and ulna shaft fractures
Multiple or other fractures
Trauma to elbow with no fracture
All
58
62
13
18
19
20
16
2
35
36
279
20.8
22.2
4.7
6.4
6.8
7.2
5.7
0.7
12.5
13.0
International Orthopaedics (SICOT) (2020) 44:911–918
913
Table 2 Additional procedures performed during the surgical release of
elbow contracture in our patients
Materials and methods
Radial head replacement (KPS bipolar endoprosthesis)
Radioulnar synostosis resection
Lateral collateral ligament repair
Assessment of elbow joint stability was performed using an
apparatus designed particularly for this experiment, which
measured the laxity of the joint by pivoting the forearm of
the immobilized upper limb into valgus and varus alignment.
The combined deviation angle was subsequently calculated by
the device and was established as the physiological laxity of
the examined elbow joint.
The apparatus consisted of a chair equipped with an extending arm and a moveable frame containing the elbow and
wrist immobilizers, the measuring module and the control
panel (Figs. 1 and 2). The examined limb was immobilized
in a padded two-part holder covering the distal part of the arm
above the humeral epicondyles, which was tightened after the
limb had been placed in the correct position. Padding increased the comfort of the patient and prevented the arm from
rotating in the holder. The axilla was additionally fixed by an
adjustable-length strap. The tested limb was positioned in 30°
of elbow flexion to increase the contribution of the joint capsule to the stability of the elbow. The distal part of the forearm
and the wrist was blocked in full supination to reduce the
inherent laxity of the joint, as we assumed that any increase
in the laxity would be more evident in this position (Fig. 3).
The maximum valgus and varus forearm deviation was
recorded based on the analysis of the increase in the moment
of resistance. The measuring module recorded the current angle of the forearm deviation and the instantaneous torque to
monitor the value of the torque derivative (Fig. 4). Deflection
of the forearm would be stopped when either the torque itself
9
6
12
involving extensive arthrolysis, radial head excision and temporary external fixation have been elaborated [22, 23]. Due to
the complexity of elbow injuries, optimal treatment of their
sequelae varies among different centres according to the individual experience [23–25].
The results of the operative treatment vary from excellent
to poor being closely dependent on the pre-operative status of
the elbow, namely, on bone alignment and articular incongruence, as reported in other series [26–28]. An analysis of the
213 cases in which full medical record was available including
the final assessment 12 months after the operation showed that
mean MEPI score following surgery was 86.3 points compared to 63.2 points preoperatively (mean gain 22.9 points).
If the articular surfaces were intact or healed in anatomic
alignment, the results were usually spectacular with a significant operative gain regarding both the range of motion and the
antalgic effect. Mean gain in flexion-extension range of movement after 12 months was 29° (− 10 to 95°), and mean improvement of forearm rotation was 26.8° (0 to 140°). In 27
cases, the surgical procedure involved other steps listed in
Table 2. Seven patients required re-operation due to recurrence of the contracture, and two patients developed a deep
infection.
Fig. 1 Technique of ‘column
procedure’. a Development of
interval between the extensor
carpi radialis longus and brevis
and the extensor digitorum
communis. b Blunt retractor is
passed exposing anterior surface
of distal humerus. c Exposure and
incision of lateral capsule. d Final
operative view after anterior
capsulectomy and exposure of
coronoid process
914
Fig. 2 The adjustable frame of the measuring device was equipped with a
padded arm holder (1), elbow support (2), wrist immobilizer (3),
electronic module (4) and safety switch (5) allowing steady and comfortable positioning of the examined limb. See text for details
or torque derivative reached the maximum value defined by
the investigators. These values had been predetermined experimentally to ensure that the forearm was deflected strongly
enough, but the applied force did not cause the pain or arm
movement in the immobilizing holder (Fig. 3). Minimum value of the torque derivative was also adjusted, which prevented
the position of the maximum deviation from being recorded
too early, e.g. due to muscle tightening by the examined patient. The selected parameters were identical for all measurements in the entire study.
According to the established protocol, the measuring apparatus performed a cycle consisting of six transitions from maximum valgus to maximum varus deviation. At each extreme
position, the angular value was recorded for the difference
between neutral position and extreme deviation. Two results
– the highest valgus and varus deviations – were rejected. The
remaining values were used to calculate the mean joint laxity,
being the sum of the mean valgus and varus deviations. High
reproducibility of results was obtained; the difference between
International Orthopaedics (SICOT) (2020) 44:911–918
the results rarely exceeded 0.5° for individual deviations in the
same direction.
The aim of our study was to determine if a correctly performed ‘column procedure’ leads to an increase in the physiological laxity of the joint, which in turn could result in instability of the elbow. In our reasoning, we assumed that the
laxity of the joint following the operation could be assessed
by comparing it with the contralateral normal elbow. The goal
of the preliminary part of the experiment was therefore to
define if there is any difference in laxity between the two
elbow joints in a healthy individual. A series of measurements
was taken on a group of volunteers recruited from 52 healthy
individuals (29 males, 23 females) of mean age of 34 years (24
to 68) [29]. The selection criteria were normal anatomy and
function of both elbow joints with no history of trauma to
upper limbs or rheumatologic disease. Owing to the construction of the measuring apparatus, also individuals of exceptionally sturdy or slender build as well as those in whom their
natural valgus angle of elbows exceeded 20° could not participate in the study. The results led to a conclusion that although
the range of physiological elbow laxity defined as the deviation from the maximal valgus to maximal varus position varies
significantly (10.6°–26.5°, mean value 17.8°), the difference
in the laxity between two elbows of the same volunteer was
only slight, amounting to a mean value of 1.19° (0.1°–3.8°).
Moreover, there was no correlation between the side of the
elbow with greater laxity and the dominant limb. We therefore
concluded that the contralateral elbow could be used as a
reference to assess a possible change of the joint laxity following surgery.
The main part of the experiment consisted of examination
of 39 patients who had undergone a standard open release of a
post-traumatic elbow contracture in our centre during the
preceeding 12 months. The patients were recruited according
to precise criteria (Table 3).
The measurements took place after completion of the physiotherapy and reaching the optimal range of motion of the
elbow. The proposed time frame of three to nine months between the surgery and the examination was short enough to
ensure that the results would not be influenced by any degenerative changes in the joint.
There were 39 patients of mean age of 33 years included in
the study, 25 (64.1%) of whom were males and 14 (33.9%)
females. Thirty-six patients (92.4%) were right-handed, and
three patients were left-handed (7.6%). The most prevalent
injuries to the elbow were radial head fracture (11 cases –
28.2%) and distal humeral fracture (9 cases – 23%), which
was in accordance with our overall experience.
The mean flexion and extension of the elbow joint before
the surgery were 117.4° and 44°, respectively, compared to
125.7° and 19.4° after the surgery. The resulting mean increase in the range of motion with respect to the flexion and
extension arc was 32.8°. The mean increase in the rotational
International Orthopaedics (SICOT) (2020) 44:911–918
915
Fig. 3 The measuring device
conducted a cycle of transitions of
the forearm between maximal
valgus and varus deviations
according to the predetermined
protocol
movements of the forearm was 13°. The average MEPI score
was 67 points pre-operatively and 91 points post-operatively.
In 24 patients (61.5%), the greater range of physiological laxity was found in the intact elbow joint and in 15 (38.5%)
patients in the operated joint.
Results
Fig. 4 Illustrative graph showing mean relation between torque values
exerted by the measuring device and the forearm deviation. Two dotted
horizontal lines represent minimal (lower) and maximal (upper line)
torque values predetermined by the investigators. Vertical arrow shows
range of torque values within which measurements could be registered.
Two coloured dots show mean valgus and varus deviation value. It can be
appreciated that the valgus dot is located on the maximal torque line,
while the varus dot is slightly lower. This shows that whereas the valgus
deviation was registered upon reaching maximal torque value, the varus
deviation was recorded when maximal torque derivative was reached
For each patient, total range of laxity was calculated separately
for the healthy and operated elbow. The absolute difference
between the laxity ranges of both elbows was then calculated.
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Table 3 Inclusion and exclusion criteria for biomechanical testing of
the elbow stability in operated patients
Inclusion criteria
1. Standard open release of a posttraumatic elbow contracture (‘column
procedure’)
2. Surgery within the preceeding 3 to 9 months
Exclusion criteria
1. Permanent damage to articular surfaces of the elbow
a. Malunited intraartricular fractures
b. Defect of an articular surface, e.g. excision of the radial head
c. Radial head replacement
2. Surgical repair of a collateral ligament
3. Patients undergoing multiple surgeries for a contracture
4. Injury or other pathologies involving the contralateral elbow (except
nerve entrapment or asymptomatic tendinopathy)
5. Technical limitations or problems with adhering to the protocol
a. Patients of particularly sturdy or slender build
b. Persistent flexion contracture limiting elbow extension to more than
30°
c. Individual valgus alignment of elbow of more than 20°
International Orthopaedics (SICOT) (2020) 44:911–918
The comparison between the two groups showed that the
difference in the elbow laxity range between the operated and
healthy elbows in the operated patients did not differ statistically significantly from the difference in the elbow laxity
range between two healthy elbows in the healthy group
(p > 0.1).
The collected data allowed for detection of a difference of
0.3° in the healthy group and 0.46° in the group of operated
patients with a statistical power of 80%. With 52 and 39 observations in the healthy and operated groups respectively, the
mean value of 1.19° in a group of 52 healthy volunteers and
the standard deviation in both groups of 1°, a difference between means of 0.6° could be considered as statistically significant. It has been found that the difference in the total deviation between the operated and healthy elbows does not
differ statistically significantly by more than 0.6 from the difference in the total deviation of the two healthy elbows and,
therefore, is not clinically noticeable.
Discussion
As stated earlier, analogous data had been previously obtained
in healthy volunteers.
Wilcoxon signed-rank test was used to verify the hypotheses concerning mean values of absolute differences between
healthy elbows and between healthy and operated elbows by
comparing the mean differences with the values of 1° and 2°.
The equality of the absolute differences in the laxity of elbows
between the healthy and operated groups was checked using
the Mann-Whitney U test. Statistical significance was assumed at p < 0.05. The difference of 1° between laxity of the
two elbows was considered as clinically noticeable.
The results of previously performed examinations
assessing range of laxity in healthy subjects showed that the
mean elbow valgus deviation was 11.2° (6.4°–16.1°) and
mean elbow varus deviation was 6.6° (3°–10.7°). The laxity
range of the elbow was 17.8° (10.6°–26.5°). The mean difference in laxity between the two opposite elbows in the same
person was 1.19° (0.1°–3.8°, SD = 0.84) and was significantly
lower than 2° (p < 0.0001). It should be noted that there is no
correlation between the side with greater elbow laxity and the
dominant side. Although there were 94.2% right-handed patients in the healthy group, only in 57.6% of the volunteers the
right elbow was the joint with a greater range of motion in the
coronal plane than the left one.
The results of the study in patients after surgical release of
elbow joint contraction indicated that the mean absolute difference in the laxity range between healthy and diseased elbows was 1.55° (0.1°–4.1°, SD = 1.1) and was also significantly lower than 2°, p = 0.0056.
Column procedure is a well-established and efficient technique in elbow surgery associated with favourable results.
Although joint instability is not recognized as a common complication of this type of surgery, this issue has received little
attention. The aim of our study was to analyse in an objective
manner whether column procedure affects elbow laxity, which
could in turn lead to joint instability. The vast experience of
our centre allowed us to operate in a reproducible manner and
to obtain surgical results concomitant with those of other authors, which additionally validates our conclusions.
According to our knowledge and thorough search through
the available medical databases, there were only two reports
involving similar studies conducted in cadaver labs. Nielsen
et al. examined the effect of anterior and posterior
capsulectomy on elbow laxity in seven cadaver specimens,
finding no convincing influence. Dos Remedios et al. in another study came to the same conclusions. These experiments
were not, however, continued in vivo, and we suppose that the
clinical value of these studies was limited as some of the
biomechanical aspects, e.g. dynamic forces exerted by muscles around the elbow, could not have been well reproduced.
As far as we are concerned, our experiment was the first biomechanical assessment of elbow laxity following column procedure with the use of a dedicated measuring device.
The main limitation of our study is its narrow field. The
study was not designed to analyse operative results of a widely
used procedure nor to present improvements to the surgical
technique but to prove using an impartial approach and sophisticated equipment that joint laxity is not violated.
International Orthopaedics (SICOT) (2020) 44:911–918
Conclusion
Our experiment confirmed that the ‘column procedure’ is a
safe procedure which does not compromise the stability of the
elbow joint.
Acknowledgements We would like to acknowledge the technical help of
Hugon Rudnicki, a design engineer and a programmer who had previously collaborated with our institution on the development and construction of the measuring device used during the experiment.
Funding information There were no funding nor other sources of support
to be disclosed. Neither the authors nor any members of their families
have received any financial remuneration related to the subject of the
article.
Compliance with ethical standards
Conflict of interest There was no conflict of interest.
Ethical approval All procedures were in accordance with the ethical
standards of the institutional research committee (Centre of
Postgraduate Medical Education) and with the 1964 Helsinki declaration
and its later amendments or comparable ethical standards. The measuring
apparatus had been approved as a diagnostic device in living subjects by
the Polish Ministry of Health (decision number 2/10/PLW). Informed
consent, as stated above, was obtained from all individual participants
included in the study.
Statement of Informed consent Informed consent was obtained from
all individual participants included in the study.
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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
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statutory regulation or exceeds the permitted use, you will need to obtain
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licence, visit http://creativecommons.org/licenses/by/4.0/.
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W1660749205.txt | Hindawi Publishing Corporation
BioMed Research International
Volume 2015, Article ID 457326, 2 pages
http://dx.doi.org/10.1155/2015/457326
Editorial
Chemotherapy-Induced Nausea and Vomiting
Bernardo Leon Rapoport,1 Alexander Molasiotis,2
Haralambos Raftopoulos,3 and Fausto Roila4
1
The Medical Oncology Center of Rosebank, Saxonwold, Johannesburg 2196, South Africa
School of Nursing, The Hong Kong Polytechnic University, Hong Kong
3
Merck & Co., Rahway, NJ 07065, USA
4
Medical Oncology, Santa Maria Hospital, Terni, Italy
2
Correspondence should be addressed to Bernardo Leon Rapoport; brapoport@icon.co.za
Received 15 June 2015; Accepted 15 June 2015
Copyright © 2015 Bernardo Leon Rapoport 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.
In the past two decades, significant advances have been made
in the management of chemotherapy-induced nausea and
vomiting (CINV). These advances are primarily due to a
greater understanding of the physiological and molecular
pathways underlying CINV, which resulted in major progress
in the management of patients with CINV.
In the early 1990s, CINV treatment consisted of dexamethasone [1]. Improvements in the management of
CINV control were achieved with the discovery of 5hydroxytryptamine (5HT3 ) receptor and the development of
5HT3 receptor antagonists (RA). This pathway is primarily
involved in the acute phase of CINV. Subsequent studies
demonstrated that the usage of the combination of 5HT3 RA
and dexamethasone resulted in additional improvements in
CINV control [2, 3].
Over the last decade, the discovery of the neurokinin-1
receptor antagonists (NK1 -RA) and its role in the pathogenesis of delayed phase of CINV has led to significant developments in the management of this complication of anticancer
treatment. More importantly, these milestone achievements
are significant and resulted in an improvement in anticancer
treatment compliance, as well as an improvement in the
quality of life of patients diagnosed with cancer.
Despite these achievements, nausea, in particular, and
vomiting remain a clinically significant problem for patients
receiving both highly emetogenic chemotherapy (HEC) and
moderately emetogenic chemotherapy (MEC). Seventy percent of patients treated with cisplatin-based HEC will achieve
an overall antiemetic complete response when managed with
a triple therapy consisting of a NK1 RA aprepitant in combination with a 5HT3 RA and corticosteroids prophylaxis [4, 5].
The current antiemetic guidelines (MASCC/ESMO, ASCO,
and NCCN) endorse triple therapy treatment for patients
receiving cisplatin- and AC-based chemotherapy regimes [6–
8].
The current special issue includes several reviews including the biology and pharmacology of the NK1 receptor and
substance P, the antiemetic management of germ cell tumor
patients undergoing multiple days’ chemotherapy treatment,
radiotherapy induced nausea and vomiting (RINV), CINV
induced by oral cytotoxic agents and targeted therapies in
patients undergoing treatment for solid tumors, adherence to
CINV guidelines and the benefits of NEPA (a new agent consisting of a combination of netupitant and palonosetron), and
the treatment of breakthrough and refractory chemotherapyinduced nausea and vomiting. Additionally, 2 original clinical
papers are presented investigating the ramosetron and olanzapine in the management of CINV.
The review article by S. Garcia and P. Gascon provides an
extensive overview of the basic knowledge of the NK1 receptor and substance P biology and the pharmacological basis of
the usage of NK1 receptor antagonists in the management of
delayed phase of CINV.
The 5-HT3 receptor antagonists play an important role
in the pathogenesis of the acute phase of CINV. K.-R.
Kim et al. presented a pilot study with the potential usage
of ramosetron, a tetrahydrobenzimidazole derivative structurally independent of the previously developed 5-HT3
receptor antagonists, such as ondansetron, granisetron, and
tropisetron.
Patients with germ cell tumor undergoing 5 days of
cisplatin-based chemotherapy have a different mechanism
2
BioMed Research International
and pattern of CINV compared to those receiving single day
chemotherapy. The efficacy of antiemetic drugs, as observed
in single day chemotherapy, is therefore not applicable. In
this issue, P. Ranganath et al. from Indiana University discuss
current recommendations and future directions for patients
undergoing multiple day treatment.
Depending on the site of irradiation, dosing, fractionation, irradiated volume, and radiotherapy techniques, the
incidence of nausea and vomiting after radiotherapy is
approximately 50–80%. RINV is a very important and not
well researched area often underestimated by physicians.
K. Jordan et al. include an overview of RINV and current
guidelines recommendations as well as future directions.
The treatment of nausea and vomiting caused by oral
antineoplastic agents is primarily empirical, consisting of the
administration of daily oral antiemetic therapy. The level of
evidence of prophylactic antiemetics recommended for these
agents is low. A. L. Costa et al. discuss the management
of CINV induced by oral cytotoxic agents and targeted
therapies in patients undergoing treatment for solid tumors.
This article highlights the differences in the classification of
emetogenic potential of oral antineoplastic agents between
the different international guidelines, as well as different
recommendations for prophylactic antiemetic treatments.
NEPA is a new oral single fixed combination agent,
containing a highly selective NK1 RA, netupitant with
palonosetron. This agent is a pharmacologically and clinically
distinct 5-HT3 RA. Palonosetron has a longer half-life compared with older 5-HT3 RAs. Palonosetron works synergistically with netupitant and has the potential to improve the
efficacy in the prevention of the delayed phase of CINV when
used in combination. P. J. Hesketh et al. discuss the use of
NEPA in the context of how NEPA may overcome some of the
barriers interfering with adherence to the various antiemetic
guidelines.
Several studies have shown that the antipsychotic agent
olanzapine is effective in the management of CINV. The
pharmacological mechanism of action consists of the blocking of neurotransmitter receptors including dopaminergic at
D1 , D2 , D3 , and D4 brain receptors, serotonergic at 5-HT2a ,
5-HT2c , 5-HT3 , and 5-HT6 receptors, catecholamines at
alpha1 adrenergic receptors, acetylcholine at muscarinic
receptors, and histamine at H1 receptors. In this special issue,
R. M. Navari discusses the treatment of breakthrough and
refractory chemotherapy-induced nausea and vomiting with
special reference to olanzapine in this setting. In another
paper, M. Abe et al. retrospectively analyze the role of olanzapine in 50 gynecologic cancer patients receiving cisplatinbased chemotherapy who had nausea despite the use of
standard therapy.
The treatment of CINV is evolving and this special issue
highlights some of the recent developments as well as some
of the controversies in this important field of oncology.
Bernardo Leon Rapoport
Alexander Molasiotis
Haralambos Raftopoulos
Fausto Roila
References
[1] M. S. Aapro and D. S. Alberts, “Dexamethasone as an antiemetic
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[3] D. B. Smith, E. S. Newlands, O. W. Spruyt et al., “Ondansetron
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[4] P. J. Hesketh, S. M. Grunberg, R. J. Gralla et al., “The
oral neurokinin-1 antagonist aprepitant for the prevention of
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receiving high-dose cisplatin—the aprepitant protocol 052
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4112–4119, 2003.
[5] S. Poli-Bigelli, J. Rodrigues-Pereira, A. D. Carides et al., “Addition of the neurokinin 1 receptor antagonist aprepitant to standard antiemetic therapy improves control of chemotherapyinduced nausea and vomiting: results from a randomized,
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[6] National Comprehensive Cancer Network, “NCNN Guidelines
1.2012: antiemesis 1.2015,” Tech. Rep. 1.2015, 2015.
[7] F. Roila, J. Herrstedt, M. Aapro et al., “Guideline update
for MASCC and ESMO in the prevention of chemotherapyand radiotherapy-induced nausea and vomiting: results of the
Perugia consensus conference,” Annals of Oncology, vol. 21,
supplement 5, pp. v232–v243, 2010.
[8] E. Basch, A. A. Prestrud, P. J. Hesketh et al., “Antiemetics: American Society of Clinical Oncology clinical practice guideline
update,” Journal of Clinical Oncology, vol. 29, no. 31, pp. 4189–
4198, 2011.
| 1,348 | 9,367 |
W4387198042.txt | Zulfadli Abdul Aziz, Masrizal Mahmud, Dista Nurhasanah: Women’s Languange…
AL-TA’LIM JOURNAL, 29 (3), 2022, (253-266)
(Print ISSN 1410-7546 Online ISSN 2355-7893)
Available online at http://journal.tarbiyahiainib.ac.id/index.php/attalim
Women’s Language Used in ‘Birds of Prey and the Fantabulous
Emancipation of One Harley Quinn’ movie
Received: 05th April 2022; Revised: 06th November 2022; Accepted: 19 th November 2022
Permalink/DOI: http://dx.doi.org/10.15548/jt.v29i3.821
Zulfadli Abdul Aziz *)
Universitas Syiah Kuala, Banda Aceh,
Indonesia.
E-mail: zulfadli.aziz@unsyiah.ac.id
Masrizal Mahmud
Universitas Syiah Kuala, Banda Aceh,
Indonesia.
E-mail: masrizal@unsyiah.ac.id
Dista Nurhasanah
Universitas Syiah Kuala, Banda Aceh,
Indonesia.
E-mail: distaanurhasanah@gmail.com
Abstract: The purpose of this study is to analyse the major
features of women’s language used by the female characters in
“Birds of Prey and the Fantabulous Emancipation of One
Harley Quinn” movie. This study investigates the types of
women speech features found in the movie. A qualitative
approach employing a content analysis method was used in this
study. The data were taken from female characters’ utterances.
The data were then identified and analysed based on the
classifications of women’s speech features proposed by Lakoff.
Findings of this study indicate that six of the features are used
by the female characters: lexical hedges/fillers, empty
adjectives, tag questions, super polite forms, avoiding strong
words, and emphatic stress. Rising intonation, precise colour
terms, intensifiers, and hypercorrect grammar have not been
found in the current research data.
Keywords: Language and gender; women’s language; women’s speech features.
How to Cite: Aziz, Z., Mahmud, M., & Nurhasanah, D. (2022). Women’s Language Used in ‘Birds of Prey and
the Fantabulous Emancipation of One Harley Quinn’ movie. Al-Ta lim Journal, 29(3).
doi:https://doi.org/10.15548/jt.v29i3.821
INTRODUCTION
empirical research, women's language based
on the definition of Lakoff is used to describe
women and the language they use (Bell &
Gibson, 2011; Pourkalhor & Esfandiari, 2017;
Swann & Deumert, 2018). Gender differences
in language are often just one aspect of more
persuasive linguistic differences in the society
reflecting social status or power differences, if
a community is very hierarchical, for instance,
and within each level of the hierarchy men are
more powerful than women, then linguistic
differences between the speech of women and
men may be just one dimension of more
extensive differences reflecting the social
hierarchy as a whole (Campbell-Kibler, 2010;
Holmes, 2013; Jinyu, 2014). For example, men
always try to take control in every
conversation, sort out the topic, and interrupt
Many people still believe in
stereotypes about the difference in speech
between female and male that are always
related to power and status (Holmes, 2013;
Holmes & Wilson, 2001; Kafi &
Motallebzadeh, 2016). Montgomery (1995)
however, suggests that even though there are
some variations in speech differences between
males and females, 'the differences of speech
are not clear-cut and that a series of universal
differences does not exist.’ Early studies that
regard power as a main factor in speech
differences between different genders can be
attributed to Lakoff’s (2004, 2017). Although
relying heavily on personal observations, and
later Lakoff's definition of 'women's language'
was criticized for feminist bias and a lack of
253
© 2022 by Al-Ta’lim. This work is licensed under (CC-BY-SA)
254
Volume 29, Number 3, November, 2022, Page 253-266
when they are talking. While women are more
careful in choosing the language they use, they
don’t have power to interrupt conversation,
and also because the status of women is lower
than men (Fishman, 2019; Sandberg, 2015).
This present research aims to find out
the types of women’s speech feature found in
two movies: “Birds of Prey and the
Fantabulous Emancipation of One Harley
Quinn”. In these movies, the female characters
are depicted as strong characters which had
super powers and not the type of women who
are just purely obedient to men. This movie is
interesting to analyse because the female
characters sometimes show their power
towards the male characters, attracting us to
further investigate the women’s language used
by the female characters.
This research seeks the answer types of
women’s speech features found in “Birds of
Prey and the Fantabulous Emancipation of One
Harley Quinn” movie”. The researchers use
Lakoff’s (1975) theory about the types of
women’s language features.
A language that signifies the
characteristics of a woman's speaking style can
be seen, for example, from relying more on an
adjustment that conveys uncertainty and
avoids bold and direct speech. A woman will
question themselves if they feel unsure of their
own beliefs, this can happen if they are unsure
about a problem, that's why many problems
faced by women appear with question marks
(Chalupnik et al., 2017; Garcés-Conejos
Blitvich & Sifianou, 2017). When women use
more standard forms than men, it can be seen
as women’s linguistic behaviour (Holmes,
2001). In many speech communities, when
women use more of a linguistic form than men,
it is generally the standard form which overtly
prestigious form that women favour. Holmes
also adds that the reasons women use more
standard form because of the social class that
is related to their status which is wrongly
regarded as a subordinate group and their role
in society relating to the function of speech
expressing masculinity. As women are serving
as models for children’s speech, the society
expects that women use standard form
commonly associated with more formal and
less personal interactions. The role of
modelling correct behaviour in the community
is usually designated for women.
Women may use different linguistic
forms such as word-shape (morphology), for
example, brunch or travelogue, pronunciation
which women tend to pronounce more clearly,
and different affixes due to the fact that women
and men speak differently. Women also use
more formal variant in the pronoun system, for
example the use of the pronoun I do we, and
they use vocabulary items differently (Holmes,
2001).
Lakoff (1975) has proposed that there
are linguistic differences between men and
women. He strengthens that there are ten
linguistic features which characterize women's
speech: lexical hedges or fillers, tag questions,
rising intonation on declaratives, 'empty'
adjectives, precise colour terms, intensifiers
such as just and so, hypercorrect grammar,
super polite forms, avoidance of strong swear
words, and emphatic stress.
Holmes (2001) asserts that there is a
distinction between ‘fillers’ and ‘hedges’, with
sort of classified as a hedge, while ‘well’ and
‘you see’ are described as ‘meaningless
particles’ and assigned to the same category as
‘pause fillers’ such as uh, um, and ah. Women
tend to use this feature more than the men do.
They use this feature to keep their femininity
and as a protection when they are making a
certain statement but they are not sure about
their statement.
When a woman attempts to describe a
book for her friend, for example, she doubts
that the book is a kind of book that is either
interesting to read or boring for some people
because she is unsure what kind of book is
preferred by her friend. For this reason, a
hedge is used to mitigate the meaning, as in the
following examples.
(a) The book is kind of well to read.
(b) You know, this novel is good.
The examples illustrate the speaker’s
uncertainty about what is being uttered. She is
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using a lexical hedge to weaken the strength of
an assertion that may cause offense.
Furthermore, women also use tag
question to express uncertainty (Lakoff, 1975).
Holmes (2001) says that “tag may also express
affective meaning. They may function as
facilitative or positive politeness devices,
providing an addressee with an easy entrée
into a conversation”. For women, using tag
question helps them overcome their
uncertainty on a subject when they were trying
to start a conversation (a). In this case, they
demand a confirmation of their statement from
others. However, in some cases, it is just a tool
to start a small conversation that emphasizes a
reality (b).
(a) She’s very wonderful, isn’t she?
(b) Today is very hot, isn’t it?
Lakoff (1975) also argues that there is
a special use of syntactic rules in female
intonation patterns. They tend to use over-thetop expression because they anticipate to not
being taken seriously by their counterparts,
e.g., it’s really good? When women have to
give a definite answer on a question, they will
say it in a declarative tone by raising their
intonation.
(a) Who will you marry?
(b) Uhm ... my bias, Yoon Dowoon ...?
The example shows lack of specific
answer to the question and uncertainty thus
reflects on doubt of the character hence why
women words are not treated seriously. In
addition, women incline to use empty
adjective. It is an adjective only used by
women as opposed to that natural adjective
commonly used by men. It is used to express
a remarkable state of circumstances (Hall &
Bucholtz, 2012). For example, men will say
‘great’ for something they think is really good,
but women will say ‘adorable’ instead. There
is, for instance, a group of adjectives that do
not only have specific and literal meaning, but
also have another use that indicating the
speaker’s admiration for something. Some of
these adjectives are neutral to both men and
women, but another set seems to be confined
to women’s speech. Representative lists of
both types are below:
Neutral
Great
Terrific
Cool
Neat
women only
adorable
charming
sweet
lovely
Men barely used some words listed in
the right column because they believe it will
damage their reputation, while women are free
to use both. In expressing colour, women have
more colour vocabulary than men because
women's words are related to their interests.
Women have a special term for colours like
baby blue, maroon, navy etc (Romaine, 2003,
2017). (Blommaert, 2015) said that such a
discrimination is absent from that of most men.
As an example of precise colour terms, a
woman may say: ‘The door is navy’ instead of
blue. Women make the distinction of colour
terms to indicate the level of importance
whereas men use it to imitate women in a
sarcastic way.
Women also use intensifier when they
want to express and emphasize what they want
to say. Intensifier is a word that can emphasize
a statement in order to make the listener surer
of what the speaker is saying. When a woman
use intensifier, it means she wants to express
her opinion with a felling and to convey the
emotional message as opposed to accurately
phrasing a cognitive statement (Carli &
Bukatko, 2012; Edwards & Hamilton, 2004).
For example:
(a) I feel so unhappy
(b) That movie made me so sick
Furthermore, women do not speak
harshly and use hypercorrect grammar. They
use subtle speech with standard verb to sound
more polite (Lakoff, 1975). The standard verb
form is used to avoid words or sentences that
are not good and to reinforce politeness, e.g. I
would be very thankful if you can show the
way. The reason on why women use more
correct grammar in their speech is that they
tend to be more status conscious. Women
adhere to social norms because they’re afraid
to be judged when they speak so they aspire to
exhibit “better” social behaviour than men. For
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example, women use more -ing pronunciations
and fewer -in pronunciations than men, e.g.
women usually use singing rather than singin’
(Garimella et al., 2019; Wolfson, 2014).
To avoid an unpleasing situation,
women use super polite form or speak
indirectly so that to the audience do not feel
offended or embarrassed by what they are
saying and to sound more polite, e.g., she
passed away (she died) (Lorenzo-Dus & BouFranch, 2003). Svendsen (2019) also argues
that women tend to avoid offensive
expressions because they are believed to be the
experts of euphemism. Women also required
to use particular polite forms such as” please”
and “thank you”. Here is an example:
(a) Come here.
(b) Will you please come here?
From the example above, (a) shows a
less polite than a request “will you please come
here?” because the addressee is not being
forced to respond a command. Using an order
as a question by suggesting the addressee
seems more polite than an overt order.
Additionally, unlike men, women avoid using
swear words as they do not want to be seen as
rude and disrespectful (Holmes, 2001). They
prefer to use the word "oh dear" rather than
“shit”. For the example:
(a) oh goodness you take those things
in wrong way again!
(b) Damn it you take those things in
wrong way again!
The expression in (a) uses weaker
swear word while expression (b) uses strong
swear word. Because women tend to speak in
more polite way, people will expect that
expression (a) is spoken by a woman and
expression (b) is spoken by a man. However,
women use strong swear words more and more
nowadays, but women’s language is not
adopted by men. The differences between
expression (a) and (b) are in how strong those
words evoke the negative feeling on the
addressee.
In addition, women typically use a
greater range of pitch than those used by men.
For example, “what a beautiful dress!” and “it
was brilliant performance”. When the speaker
says those sentences, the speaker verbally
highlights the words beautiful and brilliant by
using pitch (usually higher), elongation
(making the vowels last longer) or variation in
dynamic volume (loudness). The speaker uses
emphatics stress to emphasize the most
important word in a sentence.
METHOD
The present study qualitatively
analysed and examined the types of women’s
women speech features found in the Birds of
Prey and the Fantabulous Emancipation of One
Harley Quinn movie. The subject of this
research was the Harley Quinn’s Birds of Prey
film, whereas the object of the research was the
women's languages used in the movie.
The data were gathered from the
female characters’ utterances. They include
words, phrases, and sentences that contain
types of female speech features related to the
topic. In the present study, the researchers used
Miles et al. (2014) framework for data
collection and analysis, which are commonly
known as data reduction, data display, and
drawing conclusion/ verification.
There are nine steps of data reductions
introduced by Miles and Huberman (2014), but
the researchers only used two steps; sorting out
the data and coding. The first step was used to
collect the data and the second was used to
analyse the data. Three steps of data collection
were involved in the present study; The first
has to do with identifying the conversations
carried out by all the female characters in the
movie. In the next step, we compare the
transcript-data with subtitles. At the final
stage, we organize the speech, particularly all
utterances collected from the female
characters. This made it easier for the
researchers to analyse the language used by the
female characters in the film.
We also
eliminated similar data that were considered
redundant, so there is no repetition of data. The
researchers selected the main points, and only
focused on the important points while
simplifying the data.
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Zulfadli Abdul Aziz, Masrizal Mahmud, Dista Nurhasanah: Women’s Languange…
Finally, the data were analysed based
on the classifications of women’s speech
features originally proposed by Lakoff’s
(2004) and re-introduced by Holmes (2013):
namely lexical hedges or fillers, tag questions,
rising intonation on declaratives, ‘empty'
adjectives, precise colour terms, intensifiers
such as just and so, hypercorrect grammar,
super polite forms, avoidance of strong swear
words, and emphatic stress.
The researchers used an observation
checklist to record utterances containing
particular language features currently being
investigated. During this stage, the researchers
underline and change the colours of the
selected utterances based on Lakoff's (2004)
theory of women's language. The researchers
also used different letter symbols to distinguish
each character to help with the analysis
process.
Techniques of Data Analysis
After collecting the data, the
researchers analysed the data using content
analysis techniques. First of all, the types of
previously mentioned women's speech
features used by the female characters were
identified. To ease the data analysis process,
the following initials were used for each
character; Haley Quinn as (HQ), Huntress as
(H), Black Canary as (BC), Rene Montoya as
(RM) and Casandra Cain as (CC). These were
used in the observation checklist, as seen in the
following table.
Table 1. The observation checklist
Features
Lexical Hedges/Filler
Tag Questions
Empty' Adjectives
Precise Color Terms
Intensifiers
Hypercorrect'
Grammar
Super polite' Forms
Avoidance of Swear
Words
Emphatic Stress
HQ
H
BC
RM
CC
Secondly, the researchers determined
which data were considered the dominant
types and the dominant patterns of the
women's language for each character.
Afterwards, each of women’s speech features
was investigated in order to find its functions.
The
results
were
analysed
pragmatically based on the speaker’s meaning
that is related to the context. At this stage, we
provide an explanation of how the characters
in the movie used women's language in their
speech. The researchers then discussed the use
of women's language by the characters based
on social conditions. The final step is drawing
conclusions, where the researchers use data
display technique proposed by Miles et. al.
(2014).
RESULT AND DISCUSSION
The researchers used Lakoff's (2004)
theory to determine the types of women's
speech features found in Birds of Prey and The
Fantabulous Emancipation of One Harley
Quinn movie. This theory was used as the
reference in analysing the findings in which
each classification was classified in the
observation checklist.
After carefully
evaluating the movie conversations and its
transcripts, the speech features used by women
characters in the movie can be seen in the
following table.
Table 2. Women’s Speech Features in the Movie
Features
Lexical
Hedges/Filler
Tag Questions
Rising Intonation on
Declarative
Empty Adjectives
Precise Color Terms
Intensifiers
Hypercorrect
Grammar
Super polite Forms
Avoidance of Swear
Words
Emphatic Stress
Total Features
HQ
H
3
1
RM
C
2
1
5
1
1
3
2
3
2
2
1
17
3
6
BC
1
2
1
1
2
3
11
2
From the data, we can see that there are
eleven features spoken by Harley Quinn. She
uses three lexical hedges/fillers (‘kinda hard’,
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‘kinda nice’, ‘you know’), five tag questions
(aren’t you), three empty adjectives (‘nice
baby’, ‘sentimental value’, ‘pretty great’),
three super polite form (‘gone’), two
avoidance swear words (‘oh god’, ‘Jesus
Christ’) and one emphatic stress (‘whatever’).
Hunter uses six utterances of women’s
speech features. She uses one filler (‘mhmm’),
two super polite forms (‘I need your help’,
‘gone’), three emphatic stresses (‘it is not’, ‘a
fucking’, and ‘I don’t have’).
Black Canary uses one super polite
from (‘lady’), and one avoidance swear word
(‘thank God’). Rene Montaya uses 11
utterances of women’s speech features. She
uses two lexical hedges/fillers (‘hmm’, ‘well
you know’), one tag questions, (‘isn’t it’), two
empty adjectives (‘sweetheart’ ‘amazing’),
two super polite forms (‘loss’, ‘gone’), one
avoidance swear word (‘Jesus’), and three
emphatic stresses when she verbally highlights
the words ‘spot’, ‘relationship’ and on
‘herself’. Casandra Cain only uses one filler
which (‘mhmm’) and one tag question (‘aren’t
they’).
Lexical hedges/filler
Hedges/filler is an expression that is
used as a protection to the lack of confidence
about the speaker’s statement. There are seven
hedges/fillers found in the movie:
HQ: “…. like out of a plane without a
parachute, right in your fucking dumb
face kinda hard.”
HQ: “It was kinda nice having the kid
around.”
Harley utters the word "kinda" two
times in different ways. The first one is when
she talks about how she feels when she falls in
love with Mr. J. She describes her emotions by
saying that falling in love with Mr. J is like
falling from a plane without a parachute. The
second statement shows that Harley is not sure
about her feelings for the kid. She is not sure
whether the kid is really nice or bad. In both
utterances, she uses "kinda" to express her
uncertainty about her statement. To cover it up,
she uses the word as protection.
RM: “Well you know, the DA office is
building a case against him too.”
The use of hedges in Rene's utterance
is a form of uncertainty about her statement.
When Rene talks with her boss, she uses the
phrase "well you know" as a meaningless
particle. She uses it to cover up her uncertainty
about the DA's office, who built a case against
Sionis. She is not sure about her statement, but
she still wants her boss to believe that the DA's
office would build a case against Sionis.
H: “Mhmm… I really like how you
were able to kick so high in those tight
pants.”
RM: “Yes he is uhmm.. I uhmm he no
longer there but-.”
CC: “Uhmm thanks for the car.”
Hunter, Rene, and Cassadra use fillers
in their speech when they are uncertain about
their statement. All of them use the fillers
"Mhmm" and "Uhmm". When Hunter uses
filler in her utterance, she is not sure if she
could praise Black Canary or not. She says her
compliments after using the filler, so she will
feel more protected because of the pause filler.
The same is applied to Rene and Cassandra’s
utterances. They both use pause filler to cover
up their lack of confidence when talking to
someone who has a higher position than they
do. Rene uses the filler when she is not sure if
her partner is still in the Sionis’ army or not.
For Cassandra’s utterance, the use of
pause filler is also as a protection for her lack
of confidence. When the toy car that Hunter
lent her is able to relive her fear, she was very
grateful. Cassandra is shy to say "thank you"
to Hunter, so she uses the filler "Uhmm" before
it to cover up her uncertainty.
Tag Questions
When they’re uncertain about
something, women use tag questions to make
the addressee agree with their statement. Here
are the examples of tag question used in the
movie:
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Zulfadli Abdul Aziz, Masrizal Mahmud, Dista Nurhasanah: Women’s Languange…
RM: “Isn’t Ms Yee?”
HQ: “You are not gonna run, aren’t
you?”
CC: “They are all here for me, aren’t
they?”
Rene, Cassandra and Harley use tag
questions for confirmation. Rene uses the word
"isn't" because she wants an agreement from
Ms. Yee. She also wants Ms. Yee to support
her statement. Rene uses "isn't" because she is
not sure if the spy is still in the building or not.
She also wants Ms. Yee to help her convince
their boss about her statement.
The word "aren't you" is used in
Harley's utterance when she talks with
Cassandra in a shopping mall. "You're not
gonna run, aren't you?" the tag question "aren't
you" is used because she is not sure about what
she is saying. She knows Cassandra prefers to
be with her, but she is not really sure about it.
She pauses for a moment after saying, "you are
not gonna run," after she realizes that she is
noy sure about what she is saying. She uses the
tag question as a form of confirmation from
Cassandra because she is not confident with
her words.
When Harley uses the words "nice
baby", "sentimental value", and "pretty great",
she wants the addressee to know about her
feelings. She uses the word "nice baby" when
she gives her pet a treat and talks with her. She
uses the word "nice baby" as a form of
admiration towards her pet. Harley knows her
pet is not a baby, but calls it as such because
she wants the pet to know that she loves her
very much even though her pet does not
understand what she is saying.
Haley also uses two empty adjectives
in her speech, "Oh no" and "pretty great". She
uses the phrase "sentimental value" when Rene
wants to use her baseball bat. She wants the
addressee to know that the baseball bat meant
a lot to her, which is why she uses an empty
adjective to convey her feelings to the
addressee. The phrase "pretty great" is used to
express her feelings, that she feels pretty good
despite the unexpected bad luck.
RM: “Woahh.. I hate to be the one to
break your heart sweetheart, but you
are not done.”
RM: “I mean we are amazing there.”
Cassandra also uses tag question to
make sure that the addressee agrees with her
statement. "They are all here for me, aren't
they?" when she is saying that, she knows for
sure that Sionis's army is there to catch her
because of the diamond in his stomach. She is
not questioning the situation, but more of a
confirmation from others.
She is calling Hunter "sweetheart" so
that Hunter knows that she feels sorry for
Hunter. The word "sweetheart" is usually used
to address children, but Rene calls Hunter
"sweetheart" in her utterance because she
wants to. Hunter knows that Rene loves her
and that she is not alone in dealing with Sionis.
The word "amazing" is used when Rene
reveals her admiration to themselves when
they are able to beat a dozen of men.
Empty adjectives
Super polite forms
The use of empty adjectives in
women’s language indicates that the speaker
wants to show the relationship of her
emotional expression with her addressee. In
this movie, the female characters use five
words of empty adjective.
Women use the super polite form or
speak indirectly so that people they are talking
to do not feel offended and to sound more
polite. Thus, women also use tag orders, tag
questions, and other requests to state polite
commands.
HQ: “Hello nice baby.”
HQ: “but still, that felt pretty great.”
HQ: “Oh no no not that one,
sentimental value.”
HQ: “You heard the cop said, Sionis is
gone.”
H: “He’s gone,”
RM: “Sionis is gone “
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When they finally beat Sionis, Harley,
Hunter, and Rene express their joy by saying
"he's gone?" and "Sionis is gone". Instead of
using the word ‘death’, they use "gone"
because they do not want to sound harsh on
Sionis deaths.
H: “I need
someone.”
your
help
locating
When Hunter meets Sir Kebab for the
first time, she asks him for help to find
someone. She says, "I need your help."
because she is trying to look polite and does
not want the addressee to feel like he is being
given orders.
BC: “Yeah, I do not know who you
think I am lady, but I’m not her.”
Black Canary and Harley both use the
word "lady" in their speech. "Lady" is a term
used to refer to a woman who has a high social
status. They use the word "lady" to honour the
woman they are talking to. Canary uses it
because she respects Harley even though
Harley has broken up with Joker. Harley uses
it to honour every girl she met. She uses "lady"
to make the addressee feel less offended.
RM: “I’m sorry for your loss.”
Rene uses super polite form when she
talks to Black Canary to express her
condolences even though it is too late. Rene
still says it so that the addressee does not feel
offended by what Rene says earlier about
Black Canary's mother. Rene does not want
Black Canary to think that she does not care
about her mother's death. She expresses her
condolence so that Black Canary would not
feel offended and to reduce her embarrassment
for not being able to help Black Canary's
mother.
Avoidance of Strong Swear Words
HQ: “Oh god, stop.”
HQ: “oh Jesus Christ.”
Harley prefers to use the words "oh
god" and "Jesus Christ" instead of swearing
when talking to Sionis. Harley usually prefers
to swear rather than avoid using swear words,
but when dealing with Sionis under threat, she
prefers to use the words "Oh God" and "Jesus
Christ".
According to Allan & Burridge (2006),
the use of swear words is closely related to
one's emotions because using swear words can
diverge someone's feelings effectively. Even
though Harley is threatened and upset, she
chooses to avoid using swear words because
she does not want Sionis to feel that he does
not respect him.
BC: “Oh Thank god, don’t worry I’m
gonna get you outta here.”
Black Canary avoids using swear
words when she finds Cassandra tied up in the
toilet of an abandoned building. She feels
grateful that Cassandra is still alive. The
difference between using swear words and
avoiding using swear words can be seen from
the speaker's feelings. When she find
Cassandra, Black Canary feel more grateful
than angry, and that is why she is saying "thank
God" instead of swearing.
RM: “Jesus who the fuck is this?”
When Rene gets multiple phone calls,
she is annoyed because the calls interfere with
her meeting with her boss. She says "Jesus"
after saying "who the fuck is this" to relieve her
annoyance because she still wants to sound
polite even though she cursed afterward.
Emphatic Stress
Women typically use a greater range of
pitch than those used by men.
HQ: “I could do whatever I want.”
Harley uses empathic stress in her
utterances to emphasize words he thinks are
important. She adds the word "whatever" to
emphasize that she really can do whatever she
wants. In this context, the use elongation is
obvious so that the listener focuses on the word
she is emphasizing.
H: “It’s not, a fucking, bow and arrow.”
Hunter convinces the addressee that the
weapon she is using was not a bow and arrow
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Zulfadli Abdul Aziz, Masrizal Mahmud, Dista Nurhasanah: Women’s Languange…
but a crossbow by emphasizing the words "it is
not" and "a fucking". Hunter uses pitch when
saying "it is not" and makes the vowels last
longer when saying "a fucking". She does not
want the addressee to take her words as a joke.
H: “I don’t have, rage issue!”
When she is talking with hunter about
the crossbow, Black Canary teases hunter by
saying that she has rage issues. Hunter does
not accept that and says, "I don't have a range
issue." She emphasizes the word "I don't" to
make black canary stop teasing her and take
her words seriously.
RM: “This was their spot. She just
publicly updated her relationship
status. Harley Quinn just called open
season on herself.”
Just like Harley and Hunter, Rene
emphasizes a few words in her speech so that
the addressee does not take her words lightly.
Rene emphasizes the word "spot.",
"relationship status", and "herself" as she
explains why Harley blow up a building and
throw away the necklace he used to wear. Rene
pauses between each word at the end of her
sentence to further clarify her statement.
Based on the findings, out of Lakoff’s
(2004) ten linguistic features which
characterize women's speech, the researchers
only found six in the movie; lexical
hedges/filler, tag question, empty adjective,
super polite form, avoidance of swear words,
and emphatic stress.
Super polite form is a feature that is
mostly used by the female characters. There
are eight super polite forms used by all female
characters except Cassandra. The female
characters do not use rising intonation, precise
colour terms, intensifiers, and hypercorrect
grammar. In contrast to the current findings,
Leaper & Robnett (2011) only found one
utterance related to super polite form. The
most used feature in her study is lexical
hedges/fillers, which are found in 19
utterances. Similar to Amanda (2017), she
found that lexical hedges/fillers were the most
used form (41.54%) by Hillary Clinton in her
Presidential Debates.
The use of female language by female
characters also varies. Harley uses the most
women's speech features in the movie. She
uses 15 words for each different feature. Her
use of female language is as a form of lack of
confidence and friendliness when talking to
others. This can be seen from the many uses of
question tags, super polite forms, lexical
hedges/fillers, and empty adjectives in her
speech. Despite being a female criminal,
Harley is a character who uses the most female
language in the movie. She often uses women's
language due to her past as a psychiatrist where
she was required to speak politely and gently
to her patients. The inharmonious environment
and her untrustworthy relationship with the
surrounding community are one of the reasons
she uses women's language a lot in her
speeches. The use of women's language in
Harley's speech is a form of femininity and
also covers her lack of confidence.
Hunter and Rene also use women's
language features as a form of respect to the
addressee and lack of confidence. They use
emphatic stress in their speech so the addressee
does not take their words lightly and to
emphasise the serious tone of the situation. It
is used to intensify the speaker’s utterances
and emotional message (Bylund & Makoul,
2002; Palczewski et al., 2023).
Black Canary tend to avoid swear
words because she doesn't want to be
considered rude by the addressee. She used this
feature when talking to Cassandra who is much
younger than her (Lakoff, 2004) notes that
women tend to avoid using swear words
because they will consider as unladylike.
The linguistic devices that deeply
distinguish women's language from that of
men are lexical hedges or fillers. It is an
utterance constructed with one or more words
that expresses uncertainty from a lack of
confidence (Purnata et al., 2021). Cassandra
Cain used lexical hedge/filler and tag question
to expresses her uncertainty and as a form of
lack of confidence.
The results suggest that the female
characters used women's language features to
keep their femininity. Women should talk like
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women; if they refuse, they are ridiculed and
subjected to criticism as unfeminine (Lakoff,
2004). Men's social roles are designed to
support the masculine concept, while women's
social roles are to be feminine (Sun, 2013). The
movie implies that the female characters use
the women’s language features because they
want to adhere to their social roles and didn’t
want to be considered rude when talking to
others.
The Use of the Opposite of Women’s
Language
Unlike previous studies that are
focusing on women’s language used only by
women, the researcher found contrary
evidences in this movie. The female characters
freely use the opposite of women’s speech
features more than the women’s speech
features described by Lakoff (2004). The
researcher found two uses of the word “cool”,
which according to Lakoff (2004) is used more
often by men than women.
According to Aini (2016), women are
free to choose between neutral words and
feminine words in empty adjectives. Both
genders can also use adverbs to their liking
(Hanafiyeh & Afghari, 2014). However, it is
also wrong to say that there is no risk for
women to freely use neutral or women's words,
because they will be considered as being
unable to express their feelings by doing so.
Chen (2021) claims that women use
more adjectives than men do, although the
qualitative differences are not significant. For
this reason, we can conclude that the empty
adjective feature is not gender specific. Both
women and men use empty adjectives in their
speech. The number of uses depends on the
speaker’s social background or the topic being
discussed.
The researchers also found that the
female characters use impolite forms of speech
and offensive expressions to strengthen and
emphasize their words so as not to be
underestimated by others as well as to express
their annoyance. This finding is not in line with
Lakoff's (2004) theory suggesting that women
use the super polite form more in their speech
to avoid offending others. To explain this
phenomenon, Cameron and Coates (1985)
explain that the amount of discourse we
produce is influenced by who we are with and
what we are doing.
Omen (2017) suggests that Linguistic
politeness is the use of language in
conversation to show consideration for the
feelings and desires of one's interlocutors, to
create and maintain interpersonal relationships
(so-called political behaviour), and to follow
the rules for what society or one's culture
considers appropriate behaviour. Hobbs
(2003) mentions that the frequency of negative
politeness markers of male speakers is about
the same as that of women, whereas men prefer
to use more positive politeness techniques than
women when they are in legal settings. It is
suggested that women are often more polite
than men is not true.
According to Lakoff (2004), women
tend to avoid the use of swear words, however
the researchers found that all female characters
use many swears words in their speech. There
were
nine
‘fucking’
words,
eighteen ‘fuck’ words, four ‘motherfucking’ or
‘motherfucker’ words, fifteen ‘shit´ words,
five ‘hell’ words, one ‘fuckbag’ word, two
‘damn’ words, two ‘asshole’ words, one
‘stupid-ass’ word, one ‘dick’ word, and
one ‘crap’ word. The female characters used
swear words as an affix. It was not used for
swearing but to emphasize their subsequent
words.
In contrast to our findings, Walsh
(2016) assert that male speakers use swear
words more frequently than females.
Interestingly, the female characters in the
movie used more swear words than men, either
when they were stress, in hurry, frustrated,
habit, or in a precarious situation. Swearing
can also be associated with their educational
background. In the movie, the female
characters grow up in a high crime rate
environment. They live in a city where the
average people are criminals and mafia.
Furthermore, work-environments can
also be the reason behind vulgar languages.
The female characters, except Cassandra, work
© 2022 by Al-Ta’lim. This work is licensed under (CC-BY-SA)
Zulfadli Abdul Aziz, Masrizal Mahmud, Dista Nurhasanah: Women’s Languange…
in a place that was closely related to criminals.
Harley is unemployed, Hunter is an assassin,
Rene is a low-ranking police officer, Black
Canary is a club-singer, while Cassandra is just
a child whose parents are always fighting.
Based on social classes, the low-class people
use swear words more often than those of the
high class in their conversation.
In other situation, people use swear
words to create or show intimacy Wiranty
(2015). Women use swear words more when
they are communicating with their own gender
(Varma & Tan, 2015). The words they use are
mostly related to religion, bodily functions,
and body parts, such as shit, fuck, hell, and
God. It can be said that women use swear
words in their speech because they want to
express their feelings. Their low social status
and living environment also motivate the use
of swear words in their speech.
Hay (2000) says that uncertainty and
lack of confidence is not the only reason why
women use women's speech features. She
agrees that there are several factors aside from
gender, topic and social status that influence
the use of women's language. These factors
include practice community, educational
background, and social psychological
perceptions (Arisandy, 2018). Nowadays, not
only women, men also practice Lakoff's (2004)
women’s language features in their daily
conversations.
Many linguists and researchers have
also criticized and expressed opposing views
on Lakoff's theory. Hall & Bucholtz (2012)
point out that Lakoff's claims are not supported
by empirical evidence. The majority of her
findings were made by researchers in the
empirically
oriented
social
sciences
(anthropology, psychology, sociology, and
speech communication). Likewise, Holmes
(2001) notes that Lakoff's assertions were
based on her intuitions and observations, and
that much of her initial research was
methodologically unsatisfactory. Anderson et
al. (2011) argued that Lakoff's description of
women's
linguistic
characteristics
is
inaccurate. They demonstrate that the
frequency of women's language features in
speech is correlated with two other factors: the
speaker's social status and the speaker's
experiences.
In her study, Aini (2016) found male
characters in the movie used five types of
women's language features. They were: raising
intonation, super polite form, lexical hedge,
intensifier, and tag questions. In the "I' Tonya"
movie, three women's language features were
used by the male characters, which are lexical
hedges/fillers, intensifiers, and avoidance of
swear words (Purnata et al., 2021). From the
previous linguistic and studies above, we can
conclude that women's language features can
also be used by men.
Furthermore, after analysing the use of
women's language features used by female
characters in the movie, the researchers
concludes that the use of women's language in
this movie does not always reflect selfconfidence, difficulty in making decisions, and
uncertainty about their words, but there are
several social factors and dimensions that can
be the reason behind the use of women's
language, such as educational background,
social
status,
friendship,
topics
of
conversation, formal settings, and work.
Nowadays, many women have started using
the male language as well as many men. Men
have also started using some features of the
female language. Women and men can interact
with each other without having to think about
their gender status (Pebrianti, 2013). We can
observe that in social and work relationships,
women and men can communicate without
having to think about gender differences or
language styles.
CONCLUSION AND
RECOMMENDATION
The female characters use different
women’s language features in their speech to
express specific purposes. In the Birds of Prey
and the Fantabulous Emancipation of One
Harley Quinn movie, the researchers found six
features used by the female characters, they are
lexical hedges/fillers, empty adjectives, tag
questions, super polite forms, avoiding strong
words, and emphatic stress.
The female characters use female
language features to express their femininity.
© 2022 by Al-Ta’lim. This work is licensed under (CC-BY-SA)
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Volume 29, Number 3, November, 2022, Page 253-266
They do not seem to be perceived as impolite
when conversing with people in higher
positions. They also use female language
features to express their emotions when they
speak. It does not always reflect selfconfidence, difficulty in making decisions, and
uncertainty about their words, but there are
several social factors and dimensions which
can be considered as a reflection of the use of
women's language, such as educational
background, social status, friendship, topics of
conversation, formal settings, and work.
Arisandy, A. (2018). Women’s Language
Features Found In Nora Dominguez’s
And Mary Dominguez’s Utterances In
From Prada To Nada Movie [PhD
Thesis]. Sanata Dharma University.
The researchers also found the use of
neutral words, swear words, offensive words,
and impolite requests by the female characters
in the movie. This provides evidence that
women also began to use the opposite of
women's language. When women make fast
decisions, they often express their annoyance
using harsh words like men do and become
more confident in speaking.
Blommaert, J. (2015). Chronotopes, scales,
and complexity in the study of
language in society. Annual Review of
Anthropology, 44, 105–116.
Since the scope of this study was
limited to analysing the use of women's
language features based on Lakoff's theory, it
would be interesting to see how the results may
response if it was analysed using the most
recent theory from other experts. Future
researchers can also compare the use of
women's language proposed by Lakoff with
that proposed by other expert to find out the
differences between the two theories.
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