Mastitis-associated production of S100A10 in the mammary gland of goats

Mastitis is the most common inflammatory disease of the mammary gland in dairy ruminants and remains a major challenge in the dairy industry. S100A10 is a calcium-binding protein involved in plasmin generation and immune regulation, however, its relationship with mammary inflammation has not been fully clarified. This study investigated the production of S100A10 in the mammary glands of goats as a ruminant model and quantified temporal changes in its milk concentration following intramammary lipopolysaccharide (LPS) infusion. Milk S100A10 concentrations were measured by ELISA, while S100A10 and plasmin-related gene expressions in milk somatic cells (SCs) were analyzed using RT-PCR. Localization of S100A10 in mammary tissue and milk leukocytes was assessed through immunohistochemistry and immunocytochemistry. S100A10-positive cells were identified in macrophages and neutrophils in both milk and mammary tissue. Intramammary LPS infusion markedly increased S100A10 secretion into milk compared with pre-infusion levels. Moreover, the expression of plasminogen activator–related genes (PLAU and PLAUR) in milk SCs increased significantly after LPS infusion. These findings indicate that S100A10 is produced within the mammary gland and that its production is closely associated with inflammatory responses and plasmin generation.

1 Introduction

Mastitis is the most common inflammatory disease of the mammary gland in dairy ruminants and remains a major challenge for the dairy industry due to its substantial economic impact. Mastitis leads to decreased milk yield, altered milk quality, and increased treatment costs. Therefore, a deeper understanding of the immune defense mechanisms of the mammary gland is essential for developing additional preventive strategies and identifying reliable markers for early detection of mastitis.

The mammary gland possesses its own immune defense system to protect against invading pathogens. Among the key components of this system is the S100 calcium-binding protein family includes S100A7 and S100A8, which play important roles in the innate immunity within the mammary glands. S100A7 is primarily produced by teat epithelial cells (Zhang et al., 2014), while S100A8 is mainly found in leukocytes (Purba et al., 2019), and both exhibit antimicrobial activity against specific bacteria. S100A10 is also a member of the S100 family and is ubiquitously expressed in many cell types, where it typically forms a complex with annexin A2. This complex is involved in cell-surface plasmin generation and is functionally important for leukocyte, particularly macrophage, recruitment during inflammatory responses (O'Connell et al., 2010). These findings suggest that, beyond the classical antimicrobial proteins, other S100 family members, including S100A10, may also contribute to immune defense mechanisms in the mammary gland. However, the expression and function of S100A10 in the ruminant mammary tissue, particularly under inflammatory conditions, have not been reported. This study therefore focuses on the role of S100A10 during mastitis.

Annexin A2 (ANXA2) is a calcium-dependent phospholipid-binding protein that forms a heterotetrameric complex with two S100A10 molecules (Miller et al., 2017). This ANXA2/S100A10 complex, known as AIIt, acts as a co-receptor for plasmin activation and is localized on the extracellular membranes of various cell types, including endothelial cells, monocytes, and macrophages (Bharadwaj et al., 2013). AIIt stimulates the conversion of plasminogen to plasmin via urokinase and tissue plasminogen activators (uPA and tPA), which are essential for coagulation, fibrinolysis, and complement system regulation (Kassam et al., 1998; Heissig et al., 2020). Plasmin, generated on the cell surface, also contributes to immune regulation by modulating inflammatory mediator synthesis, recruiting macrophages, and promoting neutrophil apoptosis, as well as cleaves casein to produce antimicrobial peptides (Heissig et al., 2020; Cox et al., 1995; Perucci et al., 2023). In mammary epithelial cells (MECs) and milk leukocytes, plasminogen is converted to plasmin by plasminogen activators (Heegard et al., 1994), suggesting that S100A10 and ANXA2 complex may enhance immune function in the mammary gland. While annexins have been studied in the udder (Zhang et al., 2018; Gao et al., 2019), the specific production and role of S100A10 in ruminant mammary glands remain underexplored.

Although both ANXA2 and S100A10 are involved in plasmin generation, this study mainly focuses on S100A10 due to its potential as a biomarker for mammary inflammation and its expression in both milk leukocytes and MECs. While several markers for mastitis exist, including lactoferrin, milk amyloid A (MAA), and cathelicidin (Giagu et al., 2022), S100A10 is a compelling candidate because of its direct involvement in immune responses and proteolytic processes inherent to inflammation. Clarifying the role of S100A10 may provide valuable insights into its function in immune regulation and its relevance to mastitis. Therefore, the objectives of this study were to investigate the production of S100A10 in the goat mammary gland as a representative ruminant model and to quantify changes in its production under inflammatory conditions.

2 Materials and methods

2.1 Experimental animals

All experimental procedures were conducted in accordance with the guidelines for animal experiments issued by the Hiroshima University and approved by the Animal Research Committee of Hiroshima University (No. C 19–4). Fifteen lactating goats with body weights ranging from 20 to 30 kg, ages from 1 to 4 y, and parity of 1–4 were housed at the Hiroshima University farm. Goats with low somatic cell counts (SCC; <1,000,000 cells/mL) in their milk and no inflammatory symptoms in the udder, suggesting healthy were selected for the study (Podhorecká et al., 2021). Goats were fed 0.6 kg of hay and 0.2 kg of barley per day and had free access to water and a trace-mineralized salt block.

2.2 LPS infusion

To induce mammary inflammation, 1 µg of lipopolysaccharide (LPS; Escherichia coli O111:B4; Wako Pure Chemical Industries, Osaka, Japan; 1 mg/mL) dissolved in 5 mL saline was infused into one udder half of each goat (n = 15), while the contralateral halves received no infusion and served as non-infused controls (n = 15).

2.3 Sample collection

2.3.1 Milk collection

To evaluate the temporal dynamics of S100A10 production, milk samples were collected by hand at 0, 2, 4, 8, 12, 24, 48, 72, and 120 h after LPS infusion (n = 10). Milk was also collected from the non-infused udder halves at 0 h (n = 12). All milk samples were centrifuged at 2,300 × g for 5 min at 4°C to remove fat. The resulting skim milk was stored at −20°C and subsequently used to measure inflammation-related markers, including S100A10, MAA, and interleukin-1 receptor antagonist (IL-1ra), using enzyme-linked immunosorbent assay (ELISA).

After centrifugation, the milk precipitates containing leukocytes were used to determine the SCC using a Countess Automated Cell Counter (Life Technologies Japan Co. Ltd., Tokyo, Japan). These precipitates were also subjected to immunocytochemistry for detection of S100A10 and to RT-PCR analyses targeting S100A10 and plasmin-related genes following LPS infusion (n = 6).

2.3.2 Mammary tissue collection

Mammary tissue samples were collected from goats in both the LPS-infused (n = 5) and non-infused control groups (n = 5) 24 h after infusion for immunohistochemical analysis. Deep sedation and anesthesia were induced by slow intravenous administration of xylazine (Bayer HealthCare Pharmaceuticals Inc., Leverkusen, Germany) followed by pentobarbital (Somnopentyl; Kyoritsu Seiyaku, Tokyo, Japan). The goats were subsequently euthanized by exsanguination. Mammary gland tissue from deep areas was then collected and processed for immunohistochemistry to investigate S100A10 production.

2.4 Production of the S100A10 antibody

The antibody was produced by immunizing a part of the amino sequence of S100A10 (Cys-FVVHMKQKGKK; SCRUM, Tokyo, Japan) with rabbit. Immunoglobulins were purified from the antiserum using a HiTrap Protein G High Performance Affinity column (Cytiva, Uppsala, Sweden), according to the manufacturer’s instructions. The affinity-purified S100A10 antibody was used for ELISA, immunohistochemistry, and immunocytochemistry.

2.5 ELISA

Concentrations of S100A10, IL-1ra, and MAA in milk were measured using a competitive ELISA, as previously described (Isobe et al., 2009; Marufah et al., 2025). The antibodies against S100A10 (CFVVHMKQKGKK), IL-1ra (CITDLNQNREQDKR), and MAA (CREANYKGADKYFHARGNYD) were generated in rabbits and purified from serum as previously mentioned above.

For the ELISA, 96-well plates were coated with goat anti-rabbit IgG antibody (1 µg/mL) for the measurement of S100A10 and IL-1ra, followed by the addition of rabbit anti-S100A10 (50 ng/mL) and rabbit anti-IL-1ra (1 µg/mL) antibodies. For MAA measurement, plates were directly coated with rabbit anti-MAA antibody (2 µg/mL). Diluted milk samples, synthetic antigen standards (0, 1, 3, 10, 30, 100, 300, and 1000 ng/mL), and horseradish peroxidase (HRP)-labeled antibodies were added sequentially. After washing with phosphate-buffered saline (PBS) containing Tween 20, tetramethylbenzidine (TMB) substrate solution was added, and the reaction was stopped before measuring absorbance at 450 nm using a Thermo Scientific Multiskan FC microplate reader (Thermo Fisher Scientific, USA).

2.6 Immunostaining for S100A10 in mammary tissues and milk leukocytes

2.6.1 Immunohistochemistry and immunocytochemistry

The collected mammary tissues were fixed, dehydrated, and embedded in paraffin. Sections (3-μm thick) were air-dried on MAS-coated slides. After deparaffinization and washing with PBS, antigen retrieval was performed by autoclaving the sections in a citric acid buffer (pH 6.0) for 20 min at 121°C. The sections were washed with PBS for 10 min. Sections and SCs seeded on the glass slides were incubated at 37°C for 3 h with rabbit antibodies against S100A10 diluted in PBS (10 µg/mL). After washing, the slides were incubated with peroxidase-labeled goat anti-rabbit IgG and anti-mouse IgG antibodies (Histofine MAX-PO; Nichirei Bioscience, Tokyo, Japan) for 1 h at room temperature. The immunosignals from the sections were visualized by incubation with a diaminobenzidine reaction mixture. The slides were counterstained with hematoxylin, dehydrated, and covered. Immunohistochemical images were obtained using an Eclipse E400 microscope and a Digital Sight DS-Fi1 camera (NIS-Elements; Nikon, Tokyo, Japan).

2.6.2 Immunofluorescence

The mammary tissue sections were incubated overnight at 4 °C with the rabbit antibody against S100A10 (10 µg/mL) and mouse monoclonal antibodies against CD3 (25 µg/mL; #NBP2-53386; Novus Biologicals, Littleton, CO, USA) or IBA1 (25 µg/mL; #sc-32725; Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in PBS-T containing 2.5% bovine serum albumin. After washing, the sections were incubated with secondary antibodies (Alexa Fluor 488-conjugated goat anti-rabbit, #A32731; Alexa Fluor 555-conjugated goat anti-mouse, #A32727; Thermo Fisher Scientific, Waltham, MA, USA) diluted with PBS-T containing 2.5% bovine serum albumin for 1 h at room temperature. Immunofluorescent images were obtained using a fluorescence microscope (BZ-9000) and processed using analysis software (Keyence, Osaka, Japan).

2.7 Real-time polymerase chain reaction

Total RNA was extracted from the SCs using Sepasol RNA I Super (Nacalai Tesque, Inc., Kyoto, Japan) according to the manufacturer’s instructions. The extracted total RNA samples (500 ng/µL) were dissolved in TE buffer (10 mM Tris-HCl, pH 8.0, with 1 mM EDTA) and stored at −80°C until RT-PCR analysis. RNA samples were reverse-transcribed using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd., Osaka, Japan) on a PTC-100 programmable thermal controller (MJ Research, Waltham, MA, USA), programmed according to the manufacturer’s instructions.

RT-PCR was performed using an Aria MX real-time PCR system (Agilent Technologies, Santa Clara, CA, USA) with Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies). Table 1 lists the primers used for PCR. The cycling parameters used for amplification were as follows: denaturation at 95°C for 5 s and annealing at 60°C for 10 s. Denaturation and annealing were performed for 55 cycles for all the primer sets. The cycle parameters for the melting step were 95°C for 30 s, 65°C for 30 s, and 95°C for 30 s. To calculate the relative levels of gene expression in each sample, RT-PCR data were analyzed using the 2^−ΔΔCT [ΔCt = Ct (target gene) – Ct (housekeeping gene); ΔΔCt = ΔCt (target sample) - ΔCt (control sample)] method (Nii et al., 2023). Expression levels of the target genes were normalized using the expression of Capra hircus ribosomal protein S18 (RPS18), a housekeeping gene for goats.

Table 1

Table 1. Primers used for mRNA expression analysis.

2.8 Statistical analysis

Data are presented as the mean ± standard error (SEM). Statistical analyses were performed using SAS software (version 9.4; SAS Institute Inc., Cary, NC, USA). RT-PCR data were log₁₀-transformed prior to analysis. The Anderson–Darling test was applied to assess data normality. For non-normally distributed data including SCC, S100A10, MAA, and IL-1ra in milk, comparisons were conducted using the Wilcoxon test, and correlations among variables were evaluated using Spearman’s rank correlation test. For normally distributed data, Tukey’s multiple comparison test was used.

A mixed-effects model was employed to evaluate temporal changes, with time points after infusion included as fixed effects. Differences were considered statistically significant at p < 0.05.

3 Results

3.1 Production of S100A10 in leukocytes in milk and mammary gland tissues

Leukocytes from milk were immunostained with the S100A10 antibody, and S100A10-positive cells were identified among macrophages and neutrophils (Figure 1). Moreover, mammary tissues from both healthy (non-infused gland; Figure 2) and mastitis (LPS-infused gland; Figure 3) udders were subjected to immunofluorescent staining for S100A10, IBA (a macrophage marker), and CD3 (a T cell marker). In both healthy and inflamed tissues, double-positive cells for S100A10 and IBA1 were observed, indicating that S100A10-positive cells included macrophages. However, inflamed mammary glands exhibited a markedly higher number of S100A10/IBA1 double-positive cells compared with healthy glands, whereas the number of CD3-positive cells showed no increase.

Figure 1

Figure 1. Representative image of Immunohistochemistry for S100A10 in leukocytes derived from milk. Brown color shows positive immunoreaction for S100A10. Arrows (A) and arrow heads (B) show neutrophils and macrophages, respectively. Scale bar = 50 µm.

Figure 2

Figure 2. Representative localization of S100A10, IBA1 and CD3 in healthy goat mammary gland tissue. The left panel shows double immunofluorescence staining for S100A10 (green; A) and IBA1 (red; B). The right panel shows double staining for S100A10 (green; D) and CD3 (red; E). The merged images (C, F) illustrate co-localization of S100A10 with either IBA1 or CD3. Nuclei are stained blue. S100A10-positive cells were detected within the connective tissue, and a subset of these cells co-localized with IBA1 and CD3. Arrows indicate positive cells. Scale bar = 50 µm.

Figure 3

Figure 3. Representative localization of S100A10, IBA1 and CD3 in lipopolysaccharide (LPS)-infused mammary gland of goats. The left panel shows double immunofluorescence staining for S100A10 (green; A) and IBA1 (red; B). The right panel shows double staining for S100A10 (green; D) and CD3 (red; E). The merged images (C, F) illustrate co-localization of S100A10 with either IBA1 or CD3. Nuclei are stained blue. Many somatic cells were observed within the alveolar lumen; however, these cells did not show positive staining with any of the antibodies used. Arrows indicate positive cells. Scale bar = 50 µm.

3.2 S100A10 concentration in milk determined using ELISA

The concentration of S100A10 in milk, measured using ELISA, showed a significantly increased concentration after intramammary LPS infusion compared to that detected before infusion (Figure 4A). A time-course analysis revealed that S100A10 concentration was significantly higher at 24 h after intramammary LPS infusion than that before infusion; it increased sharply 2 h after LPS infusion and remained elevated until 24 h (Figure 4B).

Figure 4

Figure 4. Changes in S100A10 concentration in goat milk before (0 h; n = 10) and after (24 h; n = 8) intramammary lipopolysaccharide (LPS) infusion (A). Representative comparison of somatic cell count (SCC) and S100A10 concentration in milk (B). Asterisks (*) indicate significant differences between groups (p < 0.05).

To determine whether S100A10 was linked to inflammation, its correlation with other inflammatory markers (SCC and MAA) and the anti-inflammatory cytokine (IL-1ra) were analyzed (Figure 5). S100A10 showed a significant positive correlation with SCC (r = 0.654, p = 0.021) and MAA (r = 0.790, p = 0.002) and a significant negative correlation with IL-1ra (r = ­0.603, p = 0.038).

Figure 5

Figure 5. Correlation analyses of S100A10 and inflammatory indicators in goat milk. Correlation between S100A10 and (A) somatic cell count (SCC), (B) milk amyloid A (MAA), and (C) interleukin-1 receptor antagonist (IL-1ra) in milk sample from non-infused udder halves (n = 12).

3.3 Expression of genes related to S100A10

RT-PCR analysis was performed to evaluate the relationship between S100A10 and plasminogen-associated molecules (Figure 6). The results showed that S100A10 mRNA expression decreased significantly at 8 h after LPS infusion, whereas the expression levels of AnxA2 and PLGRKT remained unchanged compared with those at 0 h. In contrast, both PLAU and PLAUR mRNA expression increased significantly at 8 h post-infusion but subsequently declined over time.

Figure 6

Figure 6. mRNA expression of S100A10 and plasmin-related genes in the leukocytes (A–E) and somatic cell count (SCC; F) after lipopolysaccharide (LPS) intramammary infusion (n = 6). Asterisks (*) indicate significant differences between groups (p < 0.05). PLGRKT, Plasminogen receptor with C-terminal lysine; PLAU, Plasminogen activator urokinase; PLAUR, Plasminogen activator urokinase receptor. AnxA2: Annexin A2.

4 Discussion

Immunostaining of goat mammary glands was carried out to investigate the localization of S100A10. The results showed that S100A10 was localized in the connective tissue between the mammary alveoli. Furthermore, this positive reaction colocalized with a macrophage marker (IBA1) on double staining. In addition, S100A10 immunopositive cells were identified among macrophages and neutrophils in both milk and mammary tissue. However, S100A10 expression was not detected in MECs in this study, suggesting that the expression of S100A10 in milk mainly comes from leukocytes.

The S100A10 concentration in milk was below 1 µg/mL prior to LPS infusion but increased markedly to 1–4 µg/mL at 24 h post-infusion. A detailed time-course analysis revealed that S100A10 concentration in milk rose rapidly within a few hours after intramammary LPS infusion and began to decline after approximately 48 h. When compared with the universal mastitis indicator, somatic cell count (SCC), the temporal pattern of S100A10 closely mirrored the changes observed in SCC, indicating a rapid responsiveness of S100A10 to inflammatory stimuli. In the present study, S100A10 showed significant positive correlations with SCC and MAA, as well as a significant negative correlation with the anti-inflammatory cytokine IL-1ra, further supporting its close association with mammary inflammatory status. MAA is a well-established acute-phase protein in milk, and its increased concentration reflects activation of the innate immune response during infection or inflammation. Jaeger et al. (2017) reported that cows with intramammary infection caused by either Gram-positive or Gram -negative bacteria exhibited 3–8-fold higher MAA concentrations compared with no growth quarters. In contrast, the present study demonstrated substantially larger differences in S100A10 concentrations between inflamed and non-inflamed mammary glands (2–70-fold), suggesting that S100A10 may provide greater sensitivity for detecting mammary inflammation. In addition to MAA, several milk components, including lactoferrin (Ali et al., 2025), N-acetyl-β-D-glucosaminidase (NAGase) (Hovinen et al., 2016), lactoperoxidase (Yamasaki et al., 2017), cathelicidin (Addis et al., 2016), and defensins (Kawai et al., 2013), have been proposed as mastitis biomarkers. Compared with these established indicators, S100A10 exhibits rapid responsiveness, strong correlations with inflammatory markers, and a wide concentration range during inflammation, suggesting that S100A10 may serve as a sensitive and reliable biomarker for mastitis.

Furthermore, an in vivo study demonstrated that knockdown of S100A10 in LPS-stimulated human chondrocytes inhibited the secretion of inflammatory cytokines, including tumor necrosis factor (TNF)-α, IL-1β, and IL-10, an effect mediated through inhibition of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB (Song et al., 2012). Indicating that S100A10 not only serves as an inflammation-responsive protein but may also actively modulate inflammatory signaling during inflammation. S100A10 is a cell-surface scaffold that facilitates the rapid and localized conversion of the inactive precursor plasminogen into active plasmin, thereby promoting extracellular matrix degradation and the proteolytic activation of several enzymes, including matrix metalloproteinases (MMPs), procathepsin B, and pro-uPA (Aisina and Mukhametova, 2014; Saiki and Horii, 2019; Mai et al., 2000). These processes contribute to tissue remodeling and immune cell migration during inflammation. Supporting this role, O'Connell et al. (2010) demonstrated that S100A10-deficient mice show reduced macrophage recruitment and impaired plasmin generation in peritoneal inflammation model, highlighting its importance of S100A10 in inflammatory cell migration. In the present study, intramammary LPS infusion significantly increased S100A10 concentration in milk, corresponding with an elevated SCC. This indicates enhanced leukocyte trafficking into the mammary gland and supports the involvement of S100A10 in the inflammatory response.

Notably, this increase in S100A10 protein occurred without a corresponding rise in S100A10 mRNA expression in milk leukocytes. In contrast, mRNA expression of PLAU and PLAUR increased significantly at 8 h post-infusion, suggesting active synthesis of uPA and uPAR and a coordinated upregulation of the plasminogen activation system on the leukocyte surface. S100A10 is typically localized to the cell surface as part of a the heterotetrameric annexin A2–S100A10 complex (AIIt), composed of two molecules each of annexin A2 and S100A10. Within this complex, annexin A2 anchors S100A10 to the plasma membrane and stabilizes the protein by preventing its rapid degradation (Ma et al., 2022). However, the lack of change in AnxA2 expression suggests that S100A10 is likely acting independently or with low assistance from annexin A2, relying purely on its C-terminal lysine residues to recruit plasminogen to the site of uPA/uPAR activity (Madureira et al., 2012). Functionally, S100A10 serves a dual role by directly binding circulating plasminogen and colocalizing it with the uPA/uPAR complex at the cell surface. These binding places plasminogen in close proximity to uPA, thereby markedly enhancing the efficiency and localization of plasmin generation. The discordance between increased S100A10 protein levels and decreased S100A10 mRNA expression further indicates that the elevated milk S100A10 is unlikely to result from de novo transcription in leukocytes. Instead, it may reflect post-transcriptional regulation, release from pre-existing intracellular stores, secretion from damaged or dying cells, or contributions from alternative cellular sources.

In the present study, we suggested that S100A10 is synthesized primarily by leukocytes, particularly macrophages and neutrophils. Regardless of its cellular origin, once present on the cell surface, S100A10 cooperates with newly synthesized uPA/uPAR to promote efficient plasmin generation, a process essential for extracellular matrix degradation and leukocyte migration during mammary inflammation. Collectively, these findings suggest that plasminogen activation in mammary leukocytes is predominantly driven by the uPA/uPAR system, supported by available S100A10 protein. Further studies are warranted to identify the precise cellular sources of S100A10 and to elucidate the regulatory mechanisms governing its expression and release in inflamed mammary tissue.

5 Conclusion

In conclusion, S100A10 was synthesized by milk leukocytes, particularly macrophages and neutrophils. Intramammary LPS infusion markedly increases the secretion of S100A10 into milk, resulting in substantial differences between inflamed and non-inflamed mammary glands. The strong correlation of S100A10 with SCC and MAA, along with its inverse relationship with IL-1ra, underscores its role as an inflammation-responsive protein. Additionally, the upregulation of plasminogen activator-related genes highlights its involvement in plasmin generation and immune modulation. These findings highlight S100A10 as a promising biomarker for mastitis.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement

The animal study was approved by Animal Research Committee of Hiroshima University (No. C 24–9). The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

JJ: Investigation, Writing – original draft, Writing – review & editing. Z-LL: Formal analysis, Investigation, Writing – original draft. NM: Investigation, Writing – original draft. YT: Investigation, Methodology, Writing – original draft. TN: Methodology, Resources, Writing – review & editing. NI: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS Kakenhi; Grant Numbers 21K05893 and 24K09211).

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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