*Article* **Chronological Gene Expression of Human Gingival Fibroblasts with Low Reactive Level Laser (LLL) Irradiation**

**Yuki Wada <sup>1</sup> , Asami Suzuki 2,\*, Hitomi Ishiguro 1,3, Etsuko Murakashi <sup>1</sup> and Yukihiro Numabe 1,3**


**Abstract:** Though previously studies have reported that Low reactive Level Laser Therapy (LLLT) promotes wound healing, molecular level evidence was uncleared. The purpose of this study is to examine the temporal molecular processes of human immortalized gingival fibroblasts (HGF) by LLLT by the comprehensive analysis of gene expression. HGF was seeded, cultured for 24 h, and then irradiated with a Nd: YAG laser at 0.5 W for 30 s. After that, gene differential expression analysis and functional analysis were performed with DNA microarray at 1, 3, 6 and 12 h after the irradiation. The number of genes with up- and downregulated differentially expression genes (DEGs) compared to the nonirradiated group was large at 6 and 12 h after the irradiation. From the functional analysis results of DEGs, Biological Process (BP) based Gene Ontology (GO), BP 'the defense response' is considered to be an important process with DAVID. Additionally, the results of PPI analysis of DEGs involved in the defense response with STRING, we found that the upregulated DEGs such as CXCL8 and NFKB1, and the downregulated DEGs such as NFKBIA and STAT1 were correlated with multiple genes. We estimate that these genes are key genes on the defense response after LLLT.

**Keywords:** Low reactive Level Laser Therapy (LLLT); human gingival fibroblasts (HGF); microarray; differentially gene expression (DEGs); gene ontology; biological processes (BP); protein–protein interaction (PPI)

### **1. Introduction**

Periodontal disease is a chronic multifactorial inflammatory disease caused by genetic, immune, environmental, microbial factors and lifestyles, with anaerobic bacteria in the oral cavity as the main causative organism. Periodontal disease is generally treated with nonsurgical therapy, that is performed with hand or power-driven instrumentation. In recent years, combination therapies with scaler and laser, or laser alone have attracted attention [1–3].

The laser therapy methods currently used for treatment of periodontal diseases can be broadly divided into two types: High reactive Level Laser Therapy (HLLT) and Low reactive Level Laser Therapy (LLLT).

HLLT is an application of laser intensity that produces an irreversible reaction (photobiological destruction reaction) beyond the cell survival region and is used for tissue incision and transpiration [4–6].

On the other hand, LLLT is a treatment that applies laser intensity to generate a reversible reaction (photobiologically active reaction) within the cell survival threshold. LLLT are expected to have anti-inflammatory effects [7–12]; pain relief [13], improvement/promotion of blood flow [14], activation of cells in tissues, wound healing by proliferation [15] and tissue regeneration without causing tissue degeneration with low-power

**Citation:** Wada, Y.; Suzuki, A.; Ishiguro, H.; Murakashi, E.; Numabe, Y. Chronological Gene Expression of Human Gingival Fibroblasts with Low Reactive Level Laser (LLL) Irradiation. *J. Clin. Med.* **2021**, *10*, 1952. https://doi.org/10.3390/ jcm10091952

Academic Editors: Gianrico Spagnuolo and Yorimasa Ogata

Received: 19 April 2021 Accepted: 29 April 2021 Published: 1 May 2021

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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/).

laser irradiation conditions [16–18]. In recent years, the promotion of wound healing with LLLT has been one of the highlights.

Wound healing is thought to progress in the process of hemorrhagic coagulation phase, inflammatory phase, proliferative phase, reconstruction phase after injury by external stimulus. During the hemorrhagic coagulation phase, blood is coagulated by platelets, which is one of the coagulation factors, and growth factors such as platelet-derived growth factor (PDGF) and cytokines are released from the platelets. During the inflammatory phase, factors such as Nuclear Factor-κB (NF-κB) cause infiltration of inflammatory cells such as neutrophils and macrophages. Then, the release of growth factors and cytokines such as transforming growth factor-β (TGF-β) and fibroblast growth factor (FGF) is observed [19]. During the proliferative phase, it promotes the migration and proliferation of fibroblasts and keratinocytes [20]. Extracellular matrix is synthesized from fibroblasts and serves as a scaffold for cell migration and adhesion. During maturity, scar tissue formation occurs.

In the study of LLLT, TGF-β1 is closely involved in cell differentiation, migration, and adhesion by LLLT. In addition, it is thought to be involved in a wide range of areas such as ontogeny, tissue reconstruction, wound healing, inflammation/immunity, and cancer infiltration/metastasis. It has also been reported that the expression of NF-κB is increased [21].

Previous studies reported that the effects of low-reaction level laser irradiation on periodontium-derived cultured cells have been mainly on cell proliferation and cell transport ability related to wound healing. However, the elucidation of the mechanism at the molecular level leading to the promotion of wound healing by laser irradiation has been insufficient. It is considered that there may be a series of processes related to various wound healing by LLLT by the approach from biological processes (BP). There are few studies on mechanism analysis at the gene level using microarrays by LLLT for HGF, and only a limited number of studies have analyzed gene expression over time [22,23]. In order to clarify the effect of laser irradiation by LLLT, it is important to analyze and consider changes in gene expression and changes in BP of differentially expression genes (DEGs) over time in order to understand the mechanism at the molecular level.

In this study, HGF was irradiated with LLLT, and gene expression fluctuations at 1, 3, 6, and 12 h after irradiation were analyzed using a DNA microarray. In addition, we focused on the defense response, which showed remarkable changes in gene expression over time in relation to wound healing obtained from the results of vast amounts of analytical data, and to investigate for the mechanism from the expression change genes and BP due to the photobiological effects of laser. The aim of this study was to elucidate the changes of gene expression on the wound healing, especially defense response, over time after irradiation.

### **2. Materials and Methods**

### *2.1. Cell Culture*

Human immortalized gingival fibroblasts (HGF; Applied Biological Material, Richmond, BC, Canada) were used, and 10% Fetal Bovine Serum (Moregate, Bulimba, Australia), 50 U/mL Penicilin G, 50 μg/mL Amphotericin. The study was carried out by culturing in D-MEM/F-12 medium (Life Technologies Corporation, Grand Island, NY, USA) under 37 ◦C, and 5% CO2 conditions. The HGF at the time of irradiation was in the logarithmic growth phase.

### *2.2. Dental Laser Device and Laser Irradiation Stent*

A dental Nd: YAG laser: impulse dental laser (Incisive Japan Co., Ltd., Tokyo, Japan) was used as a dental laser device, and an ultrafine fiber with a diameter of 320 nm was used for the laser light guide tip. Stents (Gikousha, Kanagawa, Japan) were prepared to uniformly irradiate cells with laser, attached to a handpiece, and used for research. Irradiation conditions were found to be significantly different in cell proliferation curvature in previous studies, irradiation output conditions 0.5 W (100 mJ, 5 pps), irradiation time

30 s, irradiation distance from the tip of the fiber guide to each well plate. Laser irradiation was performed with a distance of 20 mm to the bottom [24].

### *2.3. Microarray Analysis*

Total RNA was extracted from HGF with RNeasy® Plus Micro Kit (QIAGEN, Valencia, CA, USA) before laser irradiation 1, 3, 6 and 12 h after irradiation on a 96-well plate. cDNA was synthesized from total RNA using the SuperScript™ VILO™ cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA). After synthesis, the cDNA was fragmented and biotin labeled. Biotin-labeled cDNA was added to the GeneChipTM Human Gene 2.0 ST Array (Thermo Fisher Scientific Inc., Waltham, MA, USA) and hybridized with a probe (GeneChipTM Hybridization, Wash, and Stain Kit; Thermo Fisher Scientific Inc., Waltham, MA, USA). Phycoerythrin staining was performed, and the fluorescence signal was measured with a GeneChip scanner (Scanner 3000 7 G; Thermo Fisher Scientific Inc., Waltham, MA, USA). After normalization, Expression Gene was analyzed by SST-RMA algorithm.

### *2.4. Data Analysis of Differentially Expressed Genes (DEGs)*

DEGs were extracted with Affymetrix ® Expression ConsoleTM (Thermo Fisher Scientific Inc. Waltham, MA, USA). The cutoff values were fold change (FC) ≥ |1.5| and *p*-value < 0.05. The nonirradiated group (control), the irradiated group (test) were defined as upregulated DEGs with significantly increased expression, and downregulated DEGs with significantly decreased expression with respect to the control group.

### *2.5. Functional Analysis of Differentially Expressed Genes*

A functional analysis of DEGs was performed based on Gene Ontology (GO) with the database for annotation, visualization and integrated discovery (DAVID). Enrichment analysis on the BP was performed on the DEGs at 1, 3, 6, and 12 h after irradiation. The cutoff value was a modified Fisher exact *p*-value < 0.1, total count ≤ 2. The DEGs contained in the upregulated and the downregulated regions at each irradiation time were analyzed.

### *2.6. Protein–Protein Interaction (PPI) of Up- or Downregulated DEGs*

We focused on the defense response which is an important response on the wound healing process. PPI analysis of up- or downregulated DEGs, which were involved in defense response and continuously observed as up- or downregulated at 6 and 12 h, and related expression fluctuations were observed at all times after irradiation and Search Tool for the Retrieval of Interacting Genes (STRING) was performed. Furthermore, from the analysis of PPI, variation in the expression of genes correlated with other DEGs over time was analyzed.

### **3. Results**

### *3.1. Extraction of DEGs*

In order to compare the gene expression after laser irradiation on time course, the DEGs were extracted. DEGs were extracted under the condition that the cutoff value was FC ≥ |1.5| and *p*-value < 0.05. Control and test were defined as upregulated DEGs with significantly increased expression and downregulated DEGs with significantly decreased expression with respect to the control group. At 1 h after the irradiation, 83 upregulated and 50 downregulated genes were extracted (Supplementary Tables S1 and S2). At 3 h after, 46 upregulated genes and 32 downregulated genes (Supplementary Tables S3 and S4), at 6 h after, 362 upregulated and 549 downregulated genes (Supplementary Tables S5 and S6) and at 12 h after, 253 upregulated genes and 413 downregulated were extracted (Supplementary Tables S7 and S8).

The number of DEGs was large 6 and 12 h after the irradiation. At 6 h, the number of DEGs was the highest in both the upregulated gene and downregulated gene groups.

### *3.2. Functional Analysis on GO*

From the results of functional analysis with DAVID, the number of BP related with each DEG after the irradiation was 13 BP on upregulated and 35 BP on downregulated DEGs at 1 h after, 6 BP on upregulated and 68 BP on downregulated DEGs at 3 h after, 212 BP on upregulated and 288 BP on downregulated DEGs at 6 h after, and 84 BP on upregulated and 425 BP on downregulated DEGs at 12 h after. The number of BP was small at 1 and 3 h, and the number of BP was large at 6 and 12 h. Tables 1–8 show the top BPs for each irradiation time.

BPs on upregulated DEGs at 1 h after irradiation are, for example, GO: 0050867 ~ positive regulation of cell activation, GO: 0050865 ~ regulation of cell activation (Table 1), BPs on downregulated DEGs are, for example, GO: 0032774 ~ RNA biosynthetic process, GO: 0007267 ~ cell–cell signaling (Table 2).

At 3 h after the irradiation, BPs on upregulated DEGs are, for example, GO: 0055085 ~ transmembrane transport, GO: 0006820 ~ anion transport (Table 3), BPs on down-]regulated DEGs are, for example, GO: 0032774 ~ RNA biosynthetic process, GO: 0016070 ~ RNA metabolic process (Table 4).

At 6 h, BPs on upregulated DEGs are, for example, GO: 0008283 ~ cell proliferation, GO: 0007155 ~ cell adhesion GO: 0022610 ~ biological adhesion, GO: 0042127 ~ regulation of cell proliferation, GO: 0006952 ~ defense response, GO: 0060429 ~ epithelium development (Table 5), BP on downregulated DEGs are, for example, GO: 0034645 ~ cellular macromolecule biosynthetic process, GO: 0019438 ~ aromatic compound biosynthetic process, GO: 0006325 ~ chromatin organization, GO: 0007049 ~ cell cycle (Table 6).

At 12 h, BPs on upregulated DEGs are, for example, GO: 0006955 ~ immune response, GO: 0006952 ~ defense response, GO: 0009605 ~ response to external stimulus, GO: 0048584 ~ positive regulation of response to stimulus (Table 7), BPs on downregulated DEGs are, for example, GO: 0010468 BP such as ~ regulation of gene expression, GO: 0007155 ~ cell adhesion, GO: 0022610 ~ biological adhesion (Table 8).

**Table 1.** The functional analysis of the upregulated genes at 1 h after Low Reactive Level Laser (LLL) irradiation.


Count: genes involved in the term; percentage (%): involved genes/total genes; *p*-value: modified Fisher exact *p*-value.


**Table 2.** The functional analysis of the downregulated genes at 1 h after LLL irradiation.

Count: genes involved in the term; percentage (%): involved genes/total genes; *p*-value: modified Fisher exact *p*-value.



Count: genes involved in the term; percentage (%): involved genes/total genes; *p*-value: modified Fisher exact *p*-value.


**Table 4.** The functional analysis of the downregulated genes at 3 h after LLL irradiation.

Count: genes involved in the term; percentage (%): involved genes/total genes; *p*-value: modified Fisher exact *p*-value.


**Table 5.** The functional analysis of the upregulated genes at 6 h after LLL irradiation.


### **Table 5.** *Cont.*

Count: genes involved in the term; percentage (%): involved genes/total genes; *p*-value: modified Fisher exact *p*-value.




### **Table 6.** *Cont.*

Count: genes involved in the term; percentage (%): involved genes/total genes; *p*-value: modified Fisher exact *p*-value.




### **Table 7.** *Cont.*

Count: genes involved in the term; percentage (%): involved genes/total genes; *p*-value: modified Fisher exact *p*-value.


**Table 8.** The functional analysis of the downregulated genes at 12 h after LLL irradiation.


### **Table 8.** *Cont.*

Count: genes involved in the term; percentage (%): involved genes/total genes; *p*-value: modified Fisher exact *p*-value.

### *3.3. PPI of Up- or Downregulated DEGs*

As for the upregulated DEGs, there were DEGs involved in the defense response at all irradiation times. From the functional analysis of DAVID, we focused on the defense response related to wound healing, which is the BP of the DEGs of the upregulated DEGs at 6 and 12 h after the irradiation. Among the genes involved in the defense response, HCP5, DAPK3, IGLC2 and IGHV1 OR21-1 at 1 h after the irradiation for the upregulated DEGs, HP, IGHE and TPSAB1 at 3 h, CAKM2 B, IGHM, SEMA7 A, CXCL8 and TNFAIP6 at 6 h. IL34, ITGA2, SERPINE1, INHBA, PRDM1, LYZL4, PVR, BMP6, NFKB1, FOXP1, PER1, IGHG1, IGHA1, IGHA2, LDLR, SLC25 A6, PF4 and TGM2. At 12 h, it was IGHM, ITIH4, SEMA7 A, EDN1, HIST1 H2 BJ, KCNJ8, TNFAIP6, CCL21, CARD9, IFI6, SERPINB9, LYZL2, DEFB108 B, ECSIT, IGHG1, IGKC, IGHD, CHRFAM7 A and SLAMF6.

As the results of PPI analysis of DEGs involved in the defense response with STRING, CXCL8 was a gene associated with multiple genes in the upregulated DEGs of the defense response. The relationships with SERPINE1, PF4, NFKB1, TNFAIP6, EDN1, CCL21, ITIH4 and HP were confirmed. In addition, HP, ITIH4, TNFAIP6 and NFKB1 were also associated with multiple genes (Figure 1). In the downregulated DEGs, STAT1 was a gene associated with multiple genes. In particular, it was associated with SMAD3, IL15, CCL22, NAIP, NRIH3, LY96, NFKBIA, PSMB8, PSMB9, PSMB10, OSA2, OAS3, IRF2, BCL6 and DDX58. In addition, NFKBIA, TLR3, IL15 and IRF2 were genes associated with multiple genes (Figure 2).

Figure 3 shows the changes in the expression of major genes related to other genes over time.

**Figure 1.** PPI of upregulated DEGs. Search tool STRING analysis of interacting genes and proteins reveals a protein–protein interaction PPI network in defense response by LLLT. PPI of upregulated DEGs related to 'defense response' Related genes after the irradiation at 3 h; -<sup>3</sup> , 6 h; -<sup>6</sup> 12 h; <sup>ᬐ</sup> Red line: indicates the presence of fusion evidence; green line: neighborhood evidence; blue line: cooccurrence evidence; purple line: experimental evidence; yellow line: text mining evidence; light blue line: database evidence; black line: coexpression evidence.

**Figure 2.** PPI of downregulated DEGs. Search tool STRING analysis of interacting genes and proteins reveals a protein–protein interaction network between proteins in defense response by LLLT. PPI of downregulated DEGs related to 'defense response.' Related genes after the irradiation at 3 h; -<sup>3</sup> , 6 h; -<sup>6</sup> 12 h; <sup>ᬐ</sup>.

**Figure 3.** The FC of the key genes.

### **4. Discussion**

Laser treatment has been studied for clinical application in many fields including medical and dentistry. In the field of dentistry, lasers are used for various purposes such as promoting wound healing, gingival incision, caries removal and sterilization/disinfection effect [25]. It has been reported that LLLT promotes wound healing and bone formation in the oral cavity by utilizing the bioactivating effect of chronic periodontitis.

In addition, at the cellular and tissue levels, there are research reports related to promotion of wound healing, such as cell proliferation of fibroblasts [15,20], osteoblasts [22,26] and vascular endothelial cells [18,27] by LLLT. However, clinical application in medical and dental treatments has not been carried out much. This is because the molecular biological findings are not clear.

Intracellular biological effects of LLLT include physiological activity by photoreceptors, changes in intracellular signal cascades, and changes in genes. The intracellular photoreceptor of LLLT is cytochrome c oxidase, which is a transmembrane protein complex that is an electron transport chain enzyme found in mitochondria. Promotion of cell proliferation by increasing cytochrome c oxidase activity and ATP is one of the representative mechanisms in LLLT research [28–31].

As a method for elucidating the mechanism of wound healing by LLLT, DNA microarrays are considered to be useful because they can examine thousands to tens of thousands of gene expressions at a time. From the obtained gene expression data, DEGs are extracted using bioinformatics analysis tools [32,33]. Furthermore, based on GO, by analyzing the biological process (BP) of DEGs, it is possible to estimate what is happening at each time by knowing the known functions of the gene contained in DEGs. It is also possible to infer what is about to happen at that time by performing chronological analysis. In addition, by searching for protein–protein interaction (PPI), it is possible to search for relationships at the molecular level [34,35]. The method plays a major role in elucidating the effects of proteins controlled by LLLT-stimulated genes at the molecular level.

In this study, we focused on the defense response from a huge amount of microarray data and analyzed the chronological changes in gene expression and the function of the genes after LLLT to HGF and the function of the genes.

The gene expression reactions related to wound healing with LLLT were remarkable 6–12 h after the irradiation. Analysis of the gene expression changes within these times were considered important for investigating the molecular mechanism of effects on HGF with LLLT. In particular, among the DEGs, those that are common over time and those with a significantly large expression fluctuation amount were considered largely affected by LLLT.

Analysis of BP over time revealed that upregulated were activated from 1 to 3 h after the initial irradiation, and downregulated BP was involved in RNA metabolism and activity. In both cases, the number of applicable DEGs for BP was 10 or less. It was suggested that there was no cohesive expression fluctuation as a function.

At 6 h after the irradiation, upregulated DEGs were observed related to cell proliferation, adhesion and defense reaction. Additionally, downregulated DEGs were observed in many BPs involved in RNA metabolism, activity, cell polymer production, and metabolism.

At 12 h after the irradiation, upregulated DEGs were observed with many BPs involved in defense reaction, immune reaction and response to external stimuli, and downregulated DEGs were observed with many BPs involved in RNA metabolism, activity, cell polymer production and metabolism. In the upregulated group, many BPs associated with wound healing were observed at 6–12 h after irradiation. Additionally, in downregulated, similar BP such as RNA metabolism were observed from 1 to 12 h after irradiation.

The BP 'the defense response' focused on in this study belongs to the BP 'the response to stress of response to stimulus' in GO. The response to stimulus is the process by which the state or activity of a cell or organism changes as a result of stimulation. The response to stress also causes motility, secretion, enzyme production, gene expression, etc., as a result of impaired homeostasis of the organism or cell due to extrinsic factors (temperature, humidity, ionizing radiation). The defense response, which belongs to response to stress, is a reaction caused in response to the presence of foreign substances or the occurrence of injuries, and is an important BP involved in the restriction, prevention/recovery of damage to living organisms.

In the BP 'defense response', the protein encoded by CXCL8, which is a downregulated DEG, is called interleukin-8 (IL-8). IL-8 is secreted by mononuclear macrophages, neutrophils, eosinophils, T lymphocytes, epithelial cells, and fibroblasts. IL-8 is also known as a neutrophil chemotactic factor with two major functions. It induces chemotaxis of target cells to the infected site. IL-8 is also known as a strong promoter of angiogenesis. IL-8 expression is regulated by the transcription factor NF-κB [36–42].

In particular, the upregulated DEGs NFKB1 and the downregulated DEGs NFKBIA are genes involved in NF-κB. These are one of the genes involved in the existing mechanism. NFKB1 is a transcriptional regulator that is activated by various intracellular and extracellular stimuli such as cytokines, oxidant free radicals, UV irradiation and bacterial or viral products. Activated NFKB stimulates the expression of genes involved in biological functions associated with many biological processes such as inflammation, immunity, differentiation and cells [43]. NFKBIA is a member of a family of cellular proteins that function to inhibit NF-κB transcription factors and IκBα masks the nuclear localization signals (NLS) of NF-κB proteins and inactivates them in the cytoplasm. It inhibits NF-κB by isolating it into a state [44]. From this result, it can be seen that NFKB1 increases and NFKBIA decreases when irradiation is performed, so that the activity of the pathway containing NF-κB occurs. Additionally, the defense response of BP is considered to be strongly related to the NF-κB pathway.

In a study by Chen et al., NF-κB activity was observed 1–10 h after irradiation [21].

In this study as well, changes in the expression of NFKB1 and NFKBIA were observed 6 h after irradiation, and the results of analysis from the viewpoint of BP also suggest that the movement of NF-κB due to irradiation is an important process in the mechanism of LLLT. In this study, we focused on the defense response. Further, we also will need to focus on wound healing-related processes such as BP 'the immune response'.

By adding analysis more time points, it is possible to analyze detailed time-series processes after LLL irradiation. As future developments, we plan to study other BPs, collect LLLT microarray data at different time points, and analyze the effects of laser irradiation on fibroblasts and molecular-level processes during the healing process. We will lead to the elucidation of molecular evidence in 'the response to stress of response to stimulus' by LLLT.

### **5. Conclusions**

The time points of 1, 3, 6 and 12 h after LLL irradiation were compared over time. The most DEGs after the LLL irradiation on HGF were showed at 6 h upregulated gene. The number of DEGs peaked 6 h after irradiation and slightly decreased at 12 h after irradiation. From the time-dependent functional analysis, the upregulated DEGs were involved in BPs of cell proliferation, adhesion, and defense response related to wound healing from 6 h. In addition, defense response is one of the important mechanisms in BP after the irradiation. We found that the upregulated DEGs such as CXCL8 and NFKB1, and the downregulated DEGs such as NFKBIA and STAT1 were correlated with multiple genes from these PPI. From these results, irradiation of LLLT showed fluctuations in the expression of genes related to BP defense response.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/jcm10091952/s1, Table S1: DEGs of the up-regulated genes at 1 h after LLL irradiation, Table S2: DEGs of the down-regulated genes at 1 h after LLL irradiation, Table S3: DEGs of the up-regulated genes at 3 h after LLL irradiation, Table S4: DEGs of the down-regulated genes at 3 h after LLL irradiation, Table S5: DEGs of the up-regulated genes at 6 h after LLL irradiation, Table S6: DEGs of the down-regulated genes at 6 h after LLL irradiation, Table S7: DEGs of the up-regulated genes at 12 h after LLL irradiation, Table S8: DEGs of the down-regulated genes at 12 h after LLL irradiation.

**Author Contributions:** Conceptualization, Y.W., A.S., H.I., E.M. and Y.N.; methodology, Y.W., A.S., H.I., E.M. and E.M.; software, Y.W. and A.S.; validation, Y.W., A.S., H.I., and E.M.; formal analysis, Y.W., A.S. and H.I.; investigation, Y.W., A.S., H.I., and E.M.; resources, Y.W., A.S., H.I., and E.M.; data curation, Y.W., A.S., H.I., and E.M.; writing—original draft preparation, Y.W., A.S., H.I., E.M. and Y.N.; writing—review and editing, Y.W., A.S., H.I., E.M. and Y.N.; visualization, Y.W. and A.S.; supervision, Y.N.; project administration, A.S., H.I., and E.M.; funding acquisition, H.I. and E.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by grants from the JSPS KAKENHI (grant numbers: JP18 K09585).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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