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ISSN 0096-3925, Moscow University Biological Sciences Bulletin, 2022, Vol. 77, No. 4, pp. 264–271. © Allerton Press, Inc., 2022.
Russian Text © The Author(s), 2022, published in Vestnik Moskovskogo Universiteta, Seriya 16: Biologiya, 2022, Vol. 77, No. 00000, pp. 00000–00000.
Research on the Effect of Amino Acid Substitution of Cyclosaplin
Peptide in Breast Cancer Cell Line (MDA-MB-231)
and in a Human Leukemia Cell Line (K562)
P. Kadkhodaei Elyaderania,* (ORCID: 0000-0002-8502-716X), A. M. Asgharianb,
and M. Salehi c (ORCID: 0000-0002-6565-0907)
a Medical Genetics Research Center of Genome, Isfahan University of Medical Sciences, Isfahan, Iran
b Department of Cell and Molecular Biology, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran
c Cellular, Molecular and Genetics Research Center, Isfahan University of Medical Sciences, 8175954319, Isfahan, Iran
* e-mail: kadkhodai23@gmail.com
Received July 11, 2022; revised November 1, 2022; accepted November 28, 2022
Abstract—It was the aim of this study to develop Cyclosaplin analogues and assess the anticancer effects of
those peptide analogues on both MDA-MB-231 and K562 cell lines. The analogues of Cyclosaplin peptide
(Cyclosaplin-2A and Cyclosaplin-7G) were designed and then investigated by online web server predictor
AntiCP. The peptide analogues were applied to MDA-MB-231 and K562 cells in various concentrations and
for various periods of time. The anticancer potential was confirmed by the MTT assay. Haemolytic activity
also was assessed. In order to investigate the apoptotic effects of peptides on cancer cells, different tests such
as morphological examination, Giemsa test, and DNA fragmentation were performed. Lactate dehydroge-
nase leakage assay was used to reject peptide-induced necrosis. As a result of computational studies, we dis-
covered that the analogues of peptides also have anticancer properties. However, we have found through our
practical research that analogues had less anticancer properties than their parent peptides. The MTT assay
and morphological study confirmed the anticancer effects. For MD-AMB-231 cells, an IC50 of Cyclosaplin-
2A was 70 μg/ml, and Cyclosaplin-7G was 90 μg/ml. In addition, for K562 cells, an IC50 of Cyclosaplin-2A
was 10 μg/ml, and Cyclosaplin-7G was 15 μg/ml. Other tests also confirmed the anticancer effect of the pep-
tide analogues. According to haemolytic assays, none of the peptide analogues possessed any haemolytic
activity against human erythrocytes, indicating that the compounds are non-toxic to normal cells. There was
evidence that peptide analogues, particularly Cyclosaplin-2A, had anticancer properties against cells derived
from breast (MDA-MB-231) and blood (K562) cancers.
Keywords: anticancer peptide, peptide analogue, Cyclosaplin, MDA-MB-231, K562, AntiCP
Abbreviations: ACP—Anticancer peptide, 2A—Cyclosaplin-2A, 7G—Cyclosaplin-7G, AntiCP—Anticancer
peptides, SVM—Support vector machine, RP-HPLC—Reversed phase high-performance liquid chromatog-
raphy, MALDI-MS—Matrix-assisted laser desorption/ionization-time of flight, NTC—No Template Con-
trol, RBC—Red blood cell, PI—Propidium iodide, LDH—Lactate dehydrogenase
DOI: 10.3103/S00963925220 40101
One of the leading causes of death worldwide has
long been cancer, which is regarded as a severe health
problem [1–3]. Cancer is the result of a mutation that
disrupts the function of proteins encoded by somatic
genes, resulting in uncontrolled cell division and inva-
sion of surrounding tissue [1, 4]. So far, various kinds
of cancer have been identified, the most common of
which are bladder, breast, colon, rectal, leukemia,
lung, lymphoma, and prostate [3, 5, 6]. A number of
factors contribute to the formation of carcinogens,
including lifestyle, chemical exposure, heredity,
genetics, immune system disorders, and environmen-
tal circumstances [4, 7]. A number of treatment meth-
ods are proposed for cancer, such as surgery, chemo-
therapy, radiation therapy, hormonal therapy, and
immunotherapy [8–10]. Each of these methods is
limited in its application, depending on the organs
involved, the stage of the disease progression, and the
patient’s medical condition [11]. Aside from that, each
of them has its own complications and defects [8, 12].
For instance, the major problem with conventional
chemotherapy is the inability to separate cancer cells
from normal cells to deliver the cytotoxic agent and kill
not only cancer cells but also normal cells [2, 13, 14].
Several disadvantages of these conventional methods
include high costs, long treatment cycles, drug resis-
tance, altered biodistribution, biotransformation, and
difficulty in eliminating chemicals from the body [14–
RESEARCH ARTICLE
MOSCOW UNIVERSITY BIOLOGICAL SCIENCES BULLETIN Vol. 77 No. 4 2022
RESEARCH ON THE EFFECT OF AMINO ACID SUBSTITUTION 265
17]. The indeterminate effect of current cancer treat-
ment methods and their complications prompted sci-
entists to find another medicine and approach more
effectively to overcome cancer [18, 19].
The discovery of anticancer peptides (ACPs), a
type of short peptides, generally has led to the emer-
gence of a novel alternative treatment for cancer with
more target-specific and fewer side effects over con-
ventional drugs of cancer therapy [16, 18, 20]. A cat-
ionic quality of ACP, due to the negative charge on
cancer cell membranes, makes them unique for bind-
ing and killing the cancer cells compared to others.
Moreover, amphipathic and hydrophobic properties
of anticancer peptides are vital for specific penetration
into cancer cell membranes. These features contribute
destabilization of the membrane integrity. Thus,
according to the specific potentials of ACPs, they can
be considered as a highlighted option for cancer treat-
ment [5, 21, 24].
Our study focused on the amino acid substitution
of an anticancer peptide with a high level of anticancer
potential to study some of its properties and develop a
novel anticancer peptide. Consequently, Cyclosaplin,
a cyclic peptide with eight amino acids, was chosen as
the framework for this study. It is a cyclic peptide that
was purified from somatic seeds of Santalum album L.
(Sandalwood) and tested and confirmed to have cyto-
toxic activity against MDA-MB-231 in a dose- and
time-dependent manner [25]. Due to the high inhibi-
tory concentration (IC50), short length, positive net
charge, several hydrophobic amino acids, herbal ori-
gin, and cyclic form of Cyclosaplin, this peptide was
considered for substitution. The study was conducted
to replace amino acids in Cyclosaplin, and the peptide
analogues were analyzed in silico by AntiCP
(webs.iiitd.edu.in/raghava/anticp/submission.php)
[13]. In order to explore the anticancer properties of
the peptide analogues experimentally, we investigated
their influence on the MDA-MB123 (breast) and
K562 (blood) cancer cell lines. According to the
Mishra et al. study [25], MDA-MB-231 cells were
selected to compare the properties of peptide ana-
logues with the parent peptide. K562 cells were also
selected for screening due to differences in appearance
and structure with MDA-MB-231 cells.
MATERIALS AND METHODS
Bioinformatic Prediction of ACP
In this study, the in silico model, AntiCP server
(webs.iiitd.edu.in/raghava/anticp/submission.php)
was used to predict ACPs before synthesis and study
on certain cancer cells [13]. For this purpose, the
Cyclosaplin sequence (RLGDGCTR) was submitted
as a framework peptide on the AntiCP website. Several
physiochemical features including charge, hydropho-
bicity, hydrophilicity, amphipathicity and molecular
weight were considered for providing the analogues.
Before running the analysis, model 1 (ACP/AMP
Dataset) was selected and the SVM threshold was set
on “zero”. Following pushing the “Run analysis” bot-
tom, the server displayed the result as “ACP” or “non-
ACP” along with the prediction score and phys-
iochemical properties selected before submission.
Finally, based on the calculated results, users can
select different mutations and prediction scores to
change the activity.
Peptide Preparation
Cyclosaplin-2A and Cyclosaplin-7G (2A and 7G)
peptides were designed and then synthesized in linear
format by TAG Copenhagen company (Denmark).
HPLC (250 × 4.6 mm C18 column) was used to purify
over 95% of the synthesized peptides. The peptides
were eluted in acetonitrile/water mixed with 0.1% tri-
fluoroacetic acid from 5 to 95% at a flow rate of
1 mL/min. The peptides were further characterized by
mass spectrometry.
Cell Cultures
MDA-MB-231, human breast cancer cells, and
K562, human leukemia cells, were provided from the
Royan Institute, Iran, Tehran in February 2018. Cells
were cultured according to the cell culture protocol
[10]. Both cell cultures were seeded in 96-well micro-
titer plates at a density of 5 × 104 cells/well.
MTT Assay and IC50 Determination of 2A and 7G
against MDA-MB-231 and K562 Cells
Cell viability count and cytotoxicity of ACPs were
performed using an MTT assay [26]. Brief ly, cells were
treated to the five concentrations (10, 15, 50, 100,
150 μg/ml in 24, 48, and 72 h) of 2A and 7G in each
plate, followed by incubation for various periods.
Plates were read at 570 nm using an ELISA analyzer
(Elisys Uno, REF 17200, Germany). The results were
expressed as IC50, representing the concentration at
which cell viability was reduced by 50%.
Determination of Cell Morphology
with Light Microscopy
5 × 104 cells/well MDA-MB-231 and K562 cells
were cultured and incubated for 24 h. Then, both cell
cultures were treated with the IC50 concentration of 2A
and 7G for 48 h. After incubation, treated and control
MDA-MB-231 and K562 cells were collected to
observe morphological changes of cells related to the
apoptotic effects under inverted microscopy (Eclipse
Ts2 , Nikon, Japan) .
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ELYADERANI et al.
Giemsa Staining
Giemsa staining was also performed cytologically
to assess the apoptotic morphology to evaluate the
apoptosis of cell death in MDA-MB-231 and K562
cells. Treated and control cells were collected and
washed according to the Giemsa staining method [10].
The morphological changes of apoptosis cells were
observed by light microscopy (SC Binocular, Nikon,
Japan).
Haemolytic Assay
The haemolytic examination was aimed to assess
whether 2A and 7G caused oxidative damages to the
erythrocyte membrane or not. Erythrocyte suspension
was prepared according to the protocol [2]. The first
well served as a negative control containing only sol-
vent and the last served as a positive control containing
20 μl of 0.1% Triton X-100 in 0.85% saline. Finally,
the absorbance of the supernatant was recorded by
spectrophotometry at 560 nm (M550, UV-Vis True
Double Beam, UK). The average value was calculated
from triplicate assays.
Lactate Dehydrogenase (LDH) Leakage Assay
The growth inhibitory effects of 2A and 7G were
further investigated through LDH into the culture
medium upon damage to the plasma membrane.
Treated cells with IC50 concentration of each peptide
and without peptides (negative control) were incu-
bated for 48 hrs according to the protocol [3]. The
absorption of each well at 490 nm was measured by an
ELISA analyzer (LT-4000, Visible EuroClone, Italy).
DNA Laddering Assay
Untreated and treated with 2A and 7G cells of both
lines were extracted using a DNA extraction kit (QIA-
wave DNA Blood and Tissue Kit, Netherlands)
according to the manufacturer’s protocol. Cells’
extracted DNA along with 100 bp DNA ladder was
loaded on to 1.5% agarose gel. The gel was visualized
under the Gel Doc system to determine DNA frag-
mentation.
Statistical Analysis
Each test in th e in vitro anticancer assay was carried
out in triplicate and the results were expressed as mean
± SEM (p < 0.005). Haemolytic test analysis was car-
ried out using one-way ANOVA.
RESULTS AND DISCUSSION
The detection and development of novel ACPs
effects are extremely time-consuming and often
expensive, so computational methods are absolutely
essential before applying them clinically. As a result,
due to the importance of ACPs, we improved the qual-
ity of identification in this study using a strong AntiCP
predictor. Based on amino acid composition and
binary profiles, AntiCP develops support vector
machine models (SVMs) to predict ACPs. The design
of ACPs was mainly done by modifying the physico-
chemical properties of natural peptides; therefore, it
was expected that the designed peptides would display
new properties.
In this study, as part of the process of designing the
analogues of Cyclosaplin, much attention was paid to
physicochemical properties of the peptides such as
hydrophobicity, hydrophilicity, amphipathicity,
charge, etc. Thus, amino acid changes were performed
to alter these properties. As a result, in one analogue of
the peptide, the amino acid leucine was substituted
with an alanine (the most hydrophobic amino acids)
at position 2 (RAGDGCTR) to increase hydropho-
bicity, whereas, in another analogue, the amino acid
threonine was substituted with the amino acid glycine
(non-hydrophobic change) at position 7 (RLGDG-
CGR). In replacement of amino acids of analogues,
we expected that the properties of peptides would be
altered, especially their anticancer properties. Fur-
thermore, linear peptides were developed instead of
cyclic ones to reduce synthesis costs. All the physico-
chemical properties of the Cyclosaplin and its ana-
logues were evaluated after changing their features
(Table 1). Despite the changes made to the parent
peptide, it was found that the major physicochemical
properties of the parent peptide and its analogues were
very similar based on the results of the AntiCP predic-
tor server. However, it was not possible to conduct a
computer comparison of the anticancer properties of
peptide analogues and their parent compounds due to
software limitations.
TAGC Company in Denmark synthesized the pep-
tide analogues based on the sequences provided. Syn-
thetic peptides were purified by RP-HPLC, and their
molecular weights were confirmed by MALDI-MS.
RP-HPLC was also been used frequently to measure
hydrophobic-hydrophilic balances of amphipathic
peptides. Due to the amphipathic nature of the pep-
tide, the company suggested a 1:1 solution of water
and acetonitrile to dissolve the peptide analogues.
An MTT assay was performed on MDA-MB-231
and K562 cells to measure the cytotoxicity of 2A and
7G peptides. The results first showed that the modifi-
cations had a different effect on the anticancer activity
of the peptide analogues. Based on the MTT assay,
both peptides decreased cell viability when concentra-
tion was increased in both cells lines (Supplementary
Fig. S1). In order to evaluate the anticancer activity of
the peptide analogues, we calculated the IC50 values of
two cancer cell lines after a certain period of incuba-
tion. On MDA-MB-231 cells with a concentration of
70 μg/ml of 2A and 90 μg/ml of 7G, and with a con-
centration of 10 μg/ml of 2A and 15 μg/ml of 7G on
MOSCOW UNIVERSITY BIOLOGICAL SCIENCES BULLETIN Vol. 77 No. 4 2022
RESEARCH ON THE EFFECT OF AMINO ACID SUBSTITUTION 267
K562 cells in 48 h, less than 50% of the cells were via-
ble (±SEM, p < 0.005) (Supplementary Fig. S2).
According to the above results, after IC50 determina-
tion (the value of less than 50% viable cells), other tests
were conducted based on this level of peptide ana-
logues concentration.
On the other hand, it was observed that the parent
peptide had a greater anticancer effect than peptide
analogues on MDA-MB-231 cells [25]. On MDA-
MB -231, t he IC50 values of the parent, 2A, and 7G
peptides were equivalent to 2.06, 70, and 90 μg/ml,
respectively. 2A and 7G had much lower IC50 values
on K562 cells (2A:10 μg/ml, 7G:15 μg/ml). As a
result, the role of amino acid sequences and peptide
structures were highlighted. In various studies, it was
determined that peptide drugs have different effects on
different cancer cells depending on their structure. For
example, in the study of the effect of Buforin IIb and
cyclic iso-DGR on MBA-MB-231 cancer cells, their
IC50 was calculated as 11.3 and 212 μg/ml, respectively
[23, 25]. In other studies, on other cell lines, the effect
of Longicalycinin A peptide on HepG2 immortal
cells, IC50 was calculated as 13.52 μg/ml [25, 29]. But
in the study of the effect of Brevinin-1Ema peptide on
A498 and A549 cell lines, IC50 was calculated as 0.071
and 1.256 μg/ml, respectively, and also the effect of
Ligatoxin B peptide on U-937-GTB cell line, IC50
equal to 1.8 μg/ml was obtained [17, 25].
The morphological study was conducted by an
inverted microscope which showed many distinct
morphological changes in both 2A and 7G treated cell
lines compared to untreated cells. Apoptotic cell sig-
naling manifested itself in terms of membrane wrin-
kling, cytoplasm condensation, cell degradation
(change from a spindle or round shape to a wrinkled
shape) and slow growth and progression of cells in the
vicinity of the peptide drug. The morphological
changes demonstrated that concentration and time
were likely to affect cell morphology. Phenotypic
changes were more noticeable as peptide concentra-
tions increase over time (Supplementary Figs. S4 and
S5).
Giemsa staining was also conducted in order to
assess apoptotic morphology. In terms of apoptotic
morphology, our results were similar to those of many
other authors who used this method. The results were
consistent with Chen et al. [8] and Srinivas et al. [9]
morphological analysis of apoptotic morphology
using Giemsa staining. In treated cells as compared to
control cells, apoptotic bodies were found to show
morphological changes and high differentiation. This
included nuclei with chromatin condensation. As seen
with Giemsa staining in our finding, in MDA-MB-
231 cells treated with 70 μg/ml 2A and 90 μg/ml 7G
for 48 h and also in K562 cells treated with 10 μg/ml
2A and 15 μg/ml 7G, nuclei with chromatin conden-
sation, cell size depletion, disruption of membrane
integrity, and formation of apoptotic bodies were
detected by a light microscope (Figs. 1 and 2).
In the evaluation of cellular toxicity and drug
safety, haemolysis is often employed as a rapid
method, as confirmed by Róka et al. and Greco et al.
[29, 30]. Thus, in the present survey, an RBC hemoly-
sis test was used to determine if peptide analogues were
capable of damaging membranes. It is well-known
that erythrocytes, the most abundant cells in the
human body [12], have been widely exploited in drug
delivery. Haemolysis, the destruction of red blood
cells, also occurs much more extensively in the pres-
ence of any toxicant like Triton X-100. In our finding,
it was evaluated whether 2A and 7G could cause oxi-
dative damage to erythrocyte membranes in this
experiment. 2A and 7G peptides, Triton X-100 (posi-
tive control) and solvent (negative control) demon-
strated differential anti-haemolytic activity. The
results of the analysis of the haemolytic activity of pep-
tide analogues at their highest IC50 concentrations are
presented in Table 2. Neither 2A nor 7G peptides
exhibited potent anti-haemolytic action in a dose-
dependent manner compared to the solvent. In con-
trast, Triton X-100 as a strong haemolytic substance
illustrated most haemolytic activity (one-way
ANOVA, p < 0.005). As a result, in parallel with deter-
mining the cytotoxic effect of analogues on cancer
Table 1. Characteristics of both analogue peptides as compared to the parent peptide
Peptide Parent Cyclosaplin-2A Cyclosaplin-7G
Sequenc RLGDGCTR RAGDGCTR RLGDGCGR
No of residues 888
Format Cyclo Linear Linear
AntiCP predictor Anticp Anticp Anticp
Hydrophobicity –0.44 –0.48 –0.40
Hydrophilicity 0.73 0.89 0.78
Amphipathicity 0.61 0.61 0.61
Charge +1 +1 +1
Mr(Da) 877.09 835.00 833.04
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ELYADERANI et al.
cells, the haemolytic assay was performed to rule out
the possibility of analogues' damaging normal cells.
In order to measure the integrity of MDA-MB-231
and K562 cells, LDH activity was assessed. Due to the
fact that damaged cells fragment completely after pro-
longed incubation with drug substances, LDH has
been shown to act as an appropriate biomarker of
patient outcome and response to treatment in cancer
patients. When LDH leaks out of the cytoplasm into
the medium, it indicates a change in plasma mem-
brane permeability. A necrotic process could be
responsible for this event. In the present study, it was
demonstrated by the measurement of LDH in cancer
cell lines treated with various concentrations of pep-
Fig. 1. Giemsa staining in MDA-MB-231 cells observed by light microscope (100×). (a) Control cell line (untreated). (b) Treated
cells by IC50 concentration of Cyclosaplin-2A. (c) Treated cells by IC50 concentration of Cyclosaplin-7G. The treated cells exhib-
ited distinct differences, such as chromatin condensation, loss of cell size, disrupted membrane integrity, and apoptotic bodies,
compared to the controls.
(a) (b) (c)
Fig. 2. Giemsa staining in K562 cells observed by light microscope (100×). (a) Control cell line (untreated). (b) Treated cells by
IC50 concentration of Cyclosaplin-2A. (c) Treated cells by IC50 concentration of Cyclosaplin-7G. These cells also showed dis-
tinct differences, such as chromatin condensation, loss of cell size, disrupted membrane integrity, and apoptotic bodies, when
compared to controls.
(a) (b) (c)
Table 2. Hemolytic activity (absorbance) of Triton X-100 (one-way ANOVA, P < 0.005), solvent, Cyclosaplin-2A and
Cyclosaplin-7G on RBC. Three independent experiments were performed
RBC+Solvent RBC+Triton X-100 (20μL) RBC+2A (90 μg/mL) RBC+7G (15 μg/mL)
Absorbance (Mean) 0.454 0.771 0.466 0.463
MOSCOW UNIVERSITY BIOLOGICAL SCIENCES BULLETIN Vol. 77 No. 4 2022
RESEARCH ON THE EFFECT OF AMINO ACID SUBSTITUTION 269
tides that the percentage of LDH leakage from the
treated cells did not increase as a function of dose
compared to the control cells. After 48 hrs of treat-
ment, no increase in LDH levels was observed in both
MDA-MB-231 and K562 cells exposed to IC50 and
higher concentrations of 2A and 7G (Supplementary
Fig. S3). The amount of LDH produced by MDA-
MB-231 cells treated with 2A at IC50 concentration
was 25.9%, and 7G was 24.91%. While for K562 cells
treated with 2A at IC50 concentration was 23.32% and
7G was 25.81% (means ± SEM, p < 0.005). An
unchanged LDH level could be representative that
these cell lines did not experience a necrotic shock.
This technique was widely considered to be a more
reliable and accurate cytotoxicity indicator by
Gurunathan et al. and Asirvatham et al. in their
research [34, 35]. Based on studies of van Zoggel et al.
[15], increased LDH release, positive PI staining as
well as confocal microscopy indicate necrotic mecha-
nisms. Upon binding the Dermaseptin B2 peptide and
disruption of the plasma membrane, they claim these
necrotic mechanisms might be induced.
Further investigation into the apoptotic pathway
was carried out by determining the DNA fragmenta-
tion assay. This method was widely used by researchers
such as Mahassni et al., Mishra et al., and Fani et al. as
a characteristic of apoptosis [15, 25, 28]. The results of
our survey in our study revealed that MDA-MB-231
and K562 cells samples treated with 2A or 7G demon-
strated significant internucleosomal fragmentation. In
Fig. 3, it can be seen that the DNA smear (frag-
mented) was created following treatment with 2A and
7G at IC50 concentration over 48 h. In this study,
inducing apoptosis by analogue peptides was evaluated
and proved by Giemsa staining and DNA fragmenta-
tion. Apoptosis induced by the parent peptide used in
our study has been reported previously by Mishra et al.
They used scanning electron microscopy and DNA
fragmentation techniques in their study [25].
In summary, our present findings indicated that
analogue Cyclosaplin peptides could induce cytotox-
icity to MDA-MB-123 and K562 cells without any
toxicity to normal cells, as evidenced by haemolytic
assay. In MDA-MB-231 cells, IC50 was recorded at
70 µg/mL of 2A and 90 µg/ml of 7G, while in K562
cells, IC50 was recorded at 10 µg/ml of 2A and
15 µg/mL of 7G through the MTT test. It was demon-
strated that the 2A analogue had a greater cytotoxic
effect than 7G on both cancer cell lines. Additionally,
MDA-MB-123 and K562 cells treated by 2A and 7G
represented no changes in LDH leakage levels, and
this issue proves neither type of cell undergone a
necrotic shock. Further enhancement of analogues'
anticancer activity may be achieved by modifying pep-
tide properties such as their hydrophobicity, structure,
or net charge in a specific time and concentration.
ACKNOWLEDGMENTS
Not applicable.
FUNDING
This research received no specific grant from any fund-
ing agency.
COMPLIANCE WITH ETHICAL STANDARDS
Statement on the Welfare of Humans or Animals. This
article does not contain any studies involving humans or
animals performed by the author.
CONFLICT OF INTEREST
The authors declare that they have no conflicts of inter-
est.
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