Transcriptome-wide analysis reveals the progress of Cordyceps militaris subculture degeneration
Transcriptome-wide analysis reveals the progress of Cordyceps militaris subculture degeneration
Juan Yin 1 2
Xiangdong Xin 1 2
Yujie Weng 1 2
Zhongzheng Gui 0 1 2
0 Sericultural Research Institute, Chinese Academy of Agricultural Science , Zhenjiang, Jiangsu , China
1 School of Biotechnology, Jiangsu University of Science and Technology , Zhenjiang, Jiangsu , China
2 Editor: Erjun Ling, Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences , CHINA
The entomopathogenic mushroom Cordyceps militaris is an important medicinal and food resource owing to its various medicinal components and pharmacological effects. However, the high frequency of strain degeneration during subculture seriously restricts the largescale production of C. militaris, and the mechanism underlying strain degeneration remains unclear. In this study, we artificially cultured C. militaris for six generations and compared changes during fruiting body growth. The transcriptome of six generations of C. militaris strains were sequenced with the Illumine Hiseq4000.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This research was funded by the Special
Fund for Agro-scientific Research in the Public
Interest of China (No. 201403064).
Competing interests: The authors have declared
that no competing interests exist.
Abbreviations: C. militaris, Cordyceps militaris; AS,
alternative splicing; DEGs, differentially expressed
The subcultured C. militaris strains degenerated beginning at the third generation, with incom
plete fruiting body growth beginning at the fourth generation. Over 9,015 unigenes and 731
new genes were identified. In addition, 35,323 alternative splicing (AS) events were detected
in all samples, and more AS events occurred in the second, fourth and sixth generations.
Compared with the first generation, the third generation (degenerated strain) included 2,498
differentially expressed genes (DEGs) including 1,729 up-regulated and 769 down-regulated
genes. This number was higher than the number of DEGs in the second (1,892 DEGs), fourth
(2,006 DEGs), fifth (2,273 DEGs) and sixth (2,188 DEGs) generations. Validation of RNA-seq
by qRT-PCR showed that the expression patterns of 51 DEGs were in accordance with the
Our results suggest that the mechanism of C. militaris strain degeneration is associated with gene involved in toxin biosynthesis, energy metabolism, and DNA methylation and chromosome remodeling.
Cordyceps militaris, a tonic herb used in traditional Chinese medicine, is an economically
valuable, edible entomopathogenic mushroom that is used clinically as a substitute of Cordyceps
genes; GO, Gene Ontology; KEGG, Kyoto
Encyclopedia of Genes and Genomes; qRT-PCR,
quantitative real-time PCR; YCC, the strain of C.
militaris used in this study; PDA, potato dextrose
agar; CmGAPDH, C. militaris housekeeping gene,
glyceraldehyde-phosphate dehydrogenase; CHM,
correlation heat map; MAT, mating-type; CYP51,
cytochrome P450 51; CYP61, cytochrome P450
61; ROS, reactive oxygen species.
sinensis owing to its similar chemical components and pharmacological activities [
militaris contains several pharmacologically active ingredients, such as cordycepin, adenosine,
polysaccharide, ergosterol, that exhibit significant biological activities known to protect and
improve lung and kidney function [
], and to immunomodulatory [
], antioxidant ,
], and hypotensive effects [
Because naturally occurring C. militaris is rare, cultivated C. militaris fruiting bodies are
currently commercially available as medicinal materials and health food products in China,
Korea, and Southeast Asia [
]. This artificial cultivation has improved the imbalance between
the supply and the demand of wild C. militaris [
]. However, the high frequency of strain
degeneration during subculture and preservationof C. militaris has limited its large-scale
production and industrial process [
]. Strain degeneration results in the significant fluctuations
in sporulation quantity, deficiencies in fruiting body formation, reductions in secondary
metabolite yields, and other irreversible hereditary phenotypes, leadings to considerable
economic losses [
].The strain degeneration of C. militaris involves complex regulatory processes
and involves many factors [
]. Studies have shown that mating type change, DNA
methylation, genetic mutations, harmful substance accumulation, and virus infections are all
precipitating factors of C. militaris degeneration [
The genome sequence of C. militaris and the transcriptomes of the mycelium and fruiting
body have been analyzed by next-generation sequencing (NGS) [
]. However, the
molecular mechanism of strain degeneration remains unclear. In this study, we cultured C. militaris
for six generations and compared changes in fruiting body growth during subculture. We also
analyzed the transcriptomes of different generations of C. militaris using Illumine Hiseq4000
technology. Subsequently, the differentially expressed genes (DEGs) as determined by
RNA-Seq were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes
(KEGG) databases and were validated by quantitative real-time PCR (qRT-PCR). Our results
provide a better understanding the transcriptomic changes that occur in C. militaris during
subculture and the underlying molecular mechanism of strain degeneration.
Materials and methods
Strain and sample preparation
Wild-type of C. militaris, designated YCC, was collected from Maoshan hilly area (at altitude
267.5 m) without specific permissions, Jiangsu province, China and isolated by tissue
separation in the asexual phase by the Laboratory of Hi-Tech Processing of Sericultural Resources,
Sericultural Research Institute, Chinese Academy of Agricultural Sciences, China.
For the collection of different generations following asexual development, the strain was
inoculated onto potato dextrose agar (PDA) plates (20% potato, 2% dextrose, 1.5% agar and
1% peptone, w/v) and incubated at 23ÊC on a shaker at 150 rpm for 7 d in the dark. Mycelia
were inoculated into rice medium, cultivated in the dark at 23ÊC for 10 d, and then kept at
23ÊC under a 17:7 h dark/light cycle for fruiting body production [
]. The mycelia and
fruiting body of this strain were subcultured for the acquisition of second, third, fourth, fifth, and
sixth generations, designated YCCZ2, YCCZ3, YCCZ4, YCCZ5, and YCCZ6, respectively.
Mycelium from all six generations were collected for DNA and RNA extraction.
RNA extraction and sequencing
Total RNA from each sample was extracted using RNeasy Plant Mini kit (Qiagen Co. LtD.,
Beijing, China) and the residual genomic DNA was digested using RNase-free DNase I
according to the manufacturer's protocol. Poly (A) mRNA was enriched using oligo (dT) beads and
fragmented using fragmentation buffer. Finally, 100 ng purified and enriched mRNA was used
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to construct a cDNA library for each sample using NEBNext1 UltraTM RNA Library Prep Kit
for Illumina (New England Biolabs, Ipswich, MA, USA). cDNA fragments of 200 bps (± 25
bps) were selected and purified by gel-electrophoresis and used as templates for amplification
with PCR for end- repair and poly (A) addition. Purified library products were evaluated using
the Agilent 2200 Tape Station and Qubit 2.0 software (Life Technologies, Carlsbad, CA, USA).
RNA-Seq was performed at Gene Denovo Co., Ltd. (Shenzhen, China) using the HiSeq TM
4000 (Illumina, San Diego, CA, USA).
Transcriptome assembly and function annotation
Prior to sequencing, raw data were filtered to produce high-quality clean data. Clean reads
were then assembled de novo into longer contigs based on overlapping regions using the
Trinity platform (http://trinityrnaseq.sourceforge.net/) [
]. All subsequent analyses were
performed using clean data. For annotation, clean data were mapped to the C. militaris genomic
data (BioProject acession no. PRJNA225510) from the NCBI transcriptome reference
All splice junction sites of the same gene ( 5 reads) were determined and compared with
the reference splice junction sites ( 1 base) using TopHat align to distinguish new alternative
splicing (AS) events. DEGs were filtered and identified according to the edgeR criteria (http://
www.Bioconductor.org/packages/release/bioc/html/edgeR.html). Functional annotation (GO
terms) were downloaded from Uniprot database (http://www.uniprot.org/uniprot). For GO
enrichment analysis, GO annotations for each DEG were retrieved by mapping to GO terms
in the database at http://www.geneontology.org. For KEGG pathway analysis, KEGG
orthology terms for DEGs were retrieved from the KEGG pathway database (http://www.genome.jp/
kegg/). Cluster analysis of gene expression patterns was performed using cluster software [
and Java Treeview software [
]. All expressed genes in the current transcriptomes were
annotated based on BLAST homology searches and searched against the Swiss-Prot and TrEMBL
databases by double-direction BLAST. To further explore the function of DEGs in different
samples, KEGG enrichment analyses were performed using hypergeometric tests with the
Validation of RNA-Seq by quantitative real-time PCR
qRT-PCR was carried out to validate the quality of RNA-Seq data. Total RNA was extracted as
described above. Each RNA sample was treated with RNase-free DNase I (TaKaRa, Shiga,
Japan) following the manufacturer's protocol to remove any residual genomic DNA. DNase
Itreated RNA (2 mg) was subjected to reverse transcriptase using oligo (dT) primer and
PrimeScriptTM Reverse Transcriptase (TaKaRa, Shiga, Japan) according to the manufacturer's
protocol. Total RNA (2 μg) was used to synthesize cDNA with PrimeScript™ RT reagent Kit (Perfect
Real Time, TaKaRa, Japan). The C. militaris housekeeping gene, glyceraldehyde-phosphate
dehydrogenase (Cmgapdh), was used as an internal control for normalization. Primers for the
qRT-PCR of 51 DEGs were designed with Premier 6.0 software and are shown in S1 Table.
Three biological replicates were performed per sample.
Sequence similarity was analyzed using Blast software. Multiple sequence alignment analysis
was carried out with Clustalx W software. All experiments were repeated at least three times,
and the results are expressed as mean ± S.D. Statistical evaluation was done using ANOVA
(SPSS 18.0), with p < 0.05 considered significant.
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Fig 1. Changes in the C. militaris fruiting body at different generations.
Changes in the morphological characteristics of the C. militaris fruiting body in different generations
The growth of C. militaris fruiting body is an important morphological characteristics in
evaluating strain degeneration during subculture. C. militaris was subcultured in rice medium for
six generations, and changes in morphological characteristics of fruiting body were observed
after 40 days of cultivation (Fig 1). In the third generation, the fruiting body became stronger
and shorter, while the size of fruiting body was significantly reduced in the fourth generation.
The strain appeared to be completely degenerated in the fifth generation, and only mycelia
grew in the sixth generation. As shown in Fig 1, the subculture of C. militaris induced strain
degeneration beginning in the third generation.
Transcriptome sequencing and assembly
As shown in Table 1, the average number of clean reads per sample was 53,118,184. After
quality filtering, over 6×109 bps of clean data was obtained across the different samples (S2 Table),
Fig 2. Differentially expressed genes (DEGs) analyses. Note: (A) DEGs statistics of different samples. (B) Scatter plot of YCCZ1-YCCZ2. (C)
Scatter plot of YCCZ1-YCCZ3. (D) Scatter plot of YCCZ1-YCCZ4. (E) Scatter plot of YCCZ1-YCCZ5. (F) Scatter plot of YCCZ1-YCCZ6.
and more than 98% HQ clean reads were identified (S1 Fig). About 90% reads were mapped to
the C. militaris genome sequence (S3 Table). In total, 9,651 genes were detected in all
transcriptomes, including 9,015 known genes and 731 novel genes. The number of new gene in
each generation from YCCZ1 to YCCZ6 was 668, 705, 699, 704, 702 and 699, respectively
(Table 1). The six samples showed similar matching results, with ~90% of reads matching the
predicted genes in the genome and ~70% being unique matches (S2 Fig). The above results
revealed that our sample data were reasonable with good correlations among biological
replicates and that sequencing data could be used for following analyses.
Analyses of differentially expressed genes (DEGs)
Compared with YCCZ1, 1,892 DEGs were identified in YCCZ2 (1,253 up-regulated and 639
down-regulated), 2,498 in YCCZ3 (1,729 up-regulated and 769 down-regulated), 2,006 in YCCZ4
(1,511 up-regulated and 495 down-regulated), 2,273 in YCCZ5 (1,437 up-regulated and 836
down-regulated) and 2,188 in YCCZ6 (1,602 up-regulated and 586 down-regulated) (Fig 2A).
The most DEGs were thus observed in the third generation, when the C. militaris fruiting body
began to experience abnormal growth (Fig 1), indicating that these DEGs may play key roles in
strain degeneration. In addition, a scatter plot was protracted based on DEGs analyses. Compared
with YCCZ1, difference in expression levels of the DEGs in YCCZ3 and YCCZ5 was significantly
higher than those in YCCZ1 and YCCZ2 (Fig 2B±Fig 2F). Furthermore, a correlation heat map
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(CHM) was constructed and sample cluster analysis was performed based on DEGs in different
samples. The heat map of significant DEGs shows the relationship of samples YCCZ1 to YCCZ6
(Fig 3). Taken together, all results suggest that these specific DEGs may be involved in the strain
GO and KEGG pathway analyses of AS genes
GO analysis was performed to summarize and explore the functional categories of the genes
that underwent AS in different samples. GO terms were annotated for a total of 5,014 AS genes
in YCCZ1, 6,230 in YCCZ2, 5,638 in YCCZ3, 6,170 in YCCZ4, 5,951 in YCCZ5 and 6,320 in
YCCZ6 (S3 Fig). The Intergenic (10,863) and IR (5,222) AS events predominated, accounting
for 30.8% and 14.8%, respectively, of all AS events. The number of AS genes in YCCZ2,
YCCZ4 and YCCZ6 was higher than those in other samples. The proportions of enriched GO
terms among AS genes were globally similar between YCCZ1 (wild-type) and YCCZ5
(degenerated) based on biological process, molecular function, and cellular component ontolopies
(Fig 4). For the biological processes category, genes with AS events were predominantly
enriched in GO terms that were relevant to cellular process and metabolic process. For the
molecular function category, most AS genes were annotated with the term binding, followed
by the term catalytic activity.
Moreover, 6,944 AS genes were annotated with KEGG pathways information (Table 2, S4
Fig). Enriched pathway analysis showed that these genes were significantly enriched in 17
pathways mainly relating to antigen processing and presentation, cell adhesion molecules,
phagosome, endocytosis, and natural killer cell-mediated cytotoxicity.
Validation of DEGs by qRT-PCR
The expression of 51 DEGs was validated using qPCR, as shown in Fig 5. According to
qRT-PCR, the DEG expression levels were consistent with those of obtained using RNA-Seq.
Among these genes, 32 genes were up-regulated and 13 were down-regulated. Functional
relative analysis shown that 15 of these genes are involved in toxin biosynthesis and detoxification
(Fig 5C), 14 genes are related to carbohydrate and energy metabolism, nine genes are involved
in DNA methylation and chromosome remodeling (Fig 5A), eight genes are involved in
biosynthesis of secondary metabolites (Fig 5B), and six genes are involved in fruiting body
formation, sexual development and light-induced brown film formation (Fig 5D). These results
suggest that C. militaris strain degeneration involves genes related to toxin biosynthesis, energy
metabolism, DNA methylation, and chromosome remodeling.
C. militaris,a model fungus belonging to the Ascomycetes,is an entomogenous fungus that
forms a fruiting body on several types of media, such as silkworm pupa, solid rice medium,
germinated soybean medium, and soybean broth. It also forms mycelia during submerged
fermentation, producing several bioactive substances [
Irreversible C. militaris strain degeneration is a common phenomenon that occurs with
high frequency during the process of subculture and preservation. Such degeneration seriously
affects large-scale industrialized production of C. militaris, leading to considerable economic
]. There are many factors that may lead to fungal degeneration, including gene
mutations, changes in mating type, DNA methylation, and fungal and viral infections [
previous work indicated that mutations in the 18S rRNA gene and mating-type (MAT) region, as
well as down-regulated of CmMAT gene expression levels, may play important roles in C.
militaris degeneration [
]. Quantitative real-time PCR analysis detected no CmMAT1-2-1 gene
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Fig 3. DEG-based correlation heat map (CHM) and sample cluster analysis.
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Fig 4. GO classification map of YCCZ1 vs. YCCZ5.
expression in the degenerated strain. Expression levels of the CmMAT1-1-1 and CmMAT1-1-2
genes were significantly down-regulated to only 7.5% and 4.4%, respectively, that of the
The C. militaris genome (147 × coverage) is predicted to have a total genome size of 32.2
Mb and over 9,684 genes [
]. In this study, 9,746 genes were detected, including 731 novel
genes, which fulfilled the criterion for it being referred to as the C. militaris genome, suggesting
that the genome size and gene number in C. militaris is similar to those of Metarhizium
1 Antigen processing and
2 Cell adhesion molecules (CAMs)
5 Natural killer cell mediated
6 Complement and coagulation
7 Regulation of actin cytoskeleton
8 Pancreatic secretion
9 Oxidatie phosphorylation
10 Vitamin digestion and absorption
11 Nicotinate and nicotinnamide
12 Fat digestion and absorption
Fig 5. Validation of DEGs expression by qRT-PCR. (A) Genes related to DNA methylation, chromosome remodeling, mitosis, and meiosis. (B)
Genes related to fruiting body formation, sexual development, light-induced brown film formation, and secondary metabolites biosynthesis. (C)
Genes related to toxin biosynthesis and detoxification, and transmembrane transport. (D) Genes related to cell growth and development, cell
apoptosis and autophagy, and macro molecular metabolism.
anisopliae (10,582 genes) [
] and Metarhizium acridum (9,849 genes) [
]. In addition, over
5,000 novel AS events of seven AS types were detected in each of the subcultured strains,
indicating significantly higher rates of AS than in that observed in the wild-type strain. AS is an
important mechanism for regulating gene expression and generating proteome diversity [
]. Previous studies have shown that AS plays a decisive role in the generation of receptor
diversity and regulation of growth and development . Many genetic diseases have been
closely linked to higher than normal rates of AS [
]. Therefore, we speculated the higher AS
rates in filamentous fungi might be involved in the progression of strain degeneration.
Gene mutation [
], methylation modification , and DNA recombination can alter a
strain's genotype and can induce strain degeneration. In this study, we characterized and
manually annotated the functions of four DEGs involved in methylation modification and in DNA
recombination and repair. Methyltransferase type 11 (CCM_00251) and
6-O-methylguanineDNA methyltransferase (CCM_02107) were significantly up-regulated in later generations
and are crucial for genome stability, preventing mismatch during DNA replication and
]. Two DNA repair genes, DNA excision repair protein (CCM_00249) and
DNA double-strand break repair/VJ recombinationXRCC4 (CCM_04567), were also
up-regulated in later generations.
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Degenerated strains exhibit traits that are generally heritable, including significantly
reduced sporulation, inability to form normal fruiting bodies, and significantly reduced
secondary metabolites contents. Fruiting body and light-induced brown film formation areimportant
characteristics that are altered in C. militaris strain degeneration [
]. Here, we analyzed the
DEGs involved in fruiting body and light-induced brown film formation and found that the
expression of α-1,3-mannosyltransferaseAlg3 (CCM_04231) was up-regulated while that of
sexual development activator VeA (CCM_04531)was down-regulated. The expression of
lightinduced brown film formation-related genes phosducin I (CCM_05237) and phosducin II
(CCM_09459) were significantly up-regulated beginning at the second generation.
Cordycepin, adenosine, polysaccharides, and ergosterol are the major bioactive constituents
important in industrial cultivation [
] speculated genes encoding ribonucleotide
reductase, adenosine kinase, AMP deaminase, pyruvate kinase, 5'-nucleotidase among others were
involved in the putative cordycepin metabolism pathway. This study showed that the
expression of the genes encoding s-adenosylhomocysteine nucleosidase (CCM_02372) and
oxidordeuctase (CCM_02761) was significantly down-regulated, and that of genes encoding adenine
nucleotide alpha hydrolases like (CCM_07740) andadenine nucleotide alpha hydrolases like
(CCM_08422) were significantly up-regulated during the degeneration progress. Ergosterol is
one of the main components of the fungal cell membrane. The genes encoding dimethylallyl
tryptophan synthase (CCM_04410), cytochrome P450 51 (CYP51, CCM_05535), cytochrome
P450 61(CYP61, CCM_02962) were down-regulated, while that encoding lanosterol synthase
(CCM_09526) was up-regulated during strain degeneration. Dimethylallyl tryptophan
synthase is a key regulator of ergosterol synthesis [
]. CYP51 and CYP61 are conserved
members of P450 family and play important role in the biosynthesis of ergosterol [
resultssuggested that the biosynthesis of ergosterol reduced with strain degenerations. C.
militaris polysaccharidesare not only important immune regulators, but also exhibit antioxidant,
antibacterial, antitumor, and antivirus activities in addition to other biological activities [
Several genes are related to the metabolism of C. militaris polysaccharide,
including1,4-α-glucan branching enzyme(CCM_03369), α-N-acetylglucosaminidase (CCM_04090), mannan
endo-1,6-α-mannosidase (CCM_05709), sorbitol dehydrogenase (CCM_06561), mannosidase
MsdS (CCM_08733), and sorbitol utilization protein (CCM_09007)[
Toxic substances such as mycotoxins, active oxygen and other nonnutritional xenobiotic
compounds, naturally exist in the environment and can be harmful to organisms [
scholars have speculated that accumulation of toxin and active oxygen molecules may cause
fungal strain degeneration [
]. Here, the expression of several genes involved in
detoxification was up-regulated during strain degeneration, including that encoding
streptothricinacetyltransferase (CCM_00152), which degrades streptothricin [
gamma-glutamyltranspeptidase (CCM_02065), which reduces glutathione [
]; MFS multidrug transporter (CCM_
02974), which confers resistance to antibiotics [
]; glutathione S-transferase (CCM_03461),
which processes pesticides, heavy metals, fluoride and eliminating reactive oxygen species
]; 30 kDa heat shock protein (CCM_06821), which is involved in stress response,
signal transduction, and xenobiotic compounds metabolism ; and alcohol dehydrogenase
(CCM_09345), which ameliorates the harmful effects of high concentration of ethanol [
However, expression of the gene encoding heavy metal tolerance protein (CCM_06182),
which binds heavy metals ions, was down-regulated during strain degeneration [
addition, levels of non-nutritional xenobiotic compounds increased during strain degeneration
induced by subculture.
In contrast, expression of genes related to the metabolism of carbohydrate, lipid, protein,
amino acid, nucleic acid, and nucleotide was significantly up-regulated during strain
degeneration. These included the genes encoding mitochondrial hypoxia responsive protein
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(CCM_05400), mitochondrial co-chaperone GrpE (CCM_00863), glycoside hydrolase, family
2 (CCM_09220), trypsin-like serine protease (CCM_03489), metalloprotease 1 (CCM_07917),
acetate transporter (CCM_07990), SGT1 and CS protein (CCM_09524),
formyltetrahydrofolate deformylase (CCM_09394), nucleoside triphosphate hydrolases like (CCM_01902), and
uracil phosphoribosyltransferase (CCM_07057).
The subcultured C. militaris strains degenerated beginning at the third generation, with
incomplete fruiting body growth beginning at the fourth generation. Transcriptome analysis
on the progress of C. militaris subculture degeneration showed that over 9,015 unigenes and
731 new genes were identified, and 35,323 alternative splicing (AS) events were detected.
Compared with the first generation, the third generation (degenerated strain) included 2,498
differentially expressed genes (DEGs) including 1,729 up-regulated and 769 down-regulated genes.
Validation of RNA-seq by qRT-PCR showed that the expression patterns of 51 DEGs were in
accordance with the transcriptome data. In summary, our results indicated the mechanism of
C. militaris strain degeneration is associated with gene involved in toxin biosynthesis, energy
metabolism, DNA methylation, and chromosome remodeling.
S1 Fig. Classification of clean reads.
S2 Fig. Distribution of gene coverage.
S3 Fig. Alternative splicing (AS) distribution of the six samples.
S4 Fig. KEGG pathway enrichment analysis.
S1 Table. Primers used for qRT-PCR.
S2 Table. Sequence numbers of the clean data.
S3 Table. Sequence numbers of the read data.
Conceptualization: Zhongzheng Gui.
Data curation: Juan Yin, Xiangdong Xin.
Methodology: Yujie Weng.
Validation: Juan Yin, Yujie Weng.
Writing ± original draft: Zhongzheng Gui.
Writing ± review & editing: Zhongzheng Gui.
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