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Introduction

The blindness rate due to cataracts is decreasing every year; however, cataracts remain the leading cause of blindness worldwide, with a high prevalence rate from 1990 to 2010, especially in developing countries (1). Surgery is the only effective cure for cataracts (2). Although the cataract surgery rate is increasing, it needs more cost to achieve improved visual quality through the multifocal lens, and more ophthalmologists are required to perform cataract surgery (3-5). Studies are ongoing, but there are currently no drugs to treat or reverse the formation of cataracts. Therefore, it is urgent and necessary to study the pathogenesis of cataracts and seek effective preventive measures.

Human lens epithelial cells (HLECs) consist of a single layer of epithelial cells located on the anterior surface of the lens (6). HLECs are widely used to investigate the pathogenesis of cataracts (7,8) and were therefore used in the present study. Ion channels are involved in LEC processes, including proliferation, differentiation and apoptosis (9-11). Chloride channels have been widely studied in the apoptosis of various types of cells, such as cardiomyocytes and nasopharyngeal carcinoma cells (12,13). Chloride channels are associated with ER stress and oxidative stress. For example, chloride channels promote ER stress and induce apoptosis in cardiomyocytes and preadipocytes (14,15). Additionally, chloride channels promote oxidative stress and induce apoptosis in nasopharyngeal carcinoma cells and cardiomyocytes (12,16). However, to the best of our knowledge, there are no studies involving HLECs. Therefore, the role of chloride channels in LECs requires further study. Endoplasmic reticulum (ER) stress induces epithelial-to-mesenchymal transition, oxidative stress, apoptosis and autophagy in HLECs, and is an important mechanism of cataract formation (17-19). Additionally, oxidative stress is a known causative factor in cataract formation (20). On one hand, ER stressors, such as thapsigargin and tunicamycin, produce significant levels of ROS and promote apoptosis in colon carcinoma CT26, breast cancer MDA-MB468 cells and neuronal HT22 cells (21,22). On the other hand, ROS induces apoptosis by activating ER stress in acute myeloid leukemia cells (23). ER stress and oxidative stress interact with each other. However, whether chloride channels affect HLEC apoptosis and the corresponding mechanism have not been investigated. 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) is a non-specific chloride channel inhibitor, which was used in the present study to observe the effects and possible mechanisms of chloride channels in HLECs apoptosis.

Materials and methods

Materials

HLECs were purchased from the American Type Culture Collection. The following additional reagents were used in the present study: NPPB (Abmole Bioscience Inc.), DMSO, 4-phenylbutyric acid (4-PBA; Sigma-Aldrich; Merck KGaA), low-glucose DMEM (HyClone; Cytiva), FBS (Gibco; Thermo Fisher Scientific, Inc.), Cell Counting Kit-8 (CCK-8) assays (cat. no. C0040), RIPA lysis buffer (cat. no. P0013C), SDS-PAGE Sample Loading Buffer 5X (cat. no. P0015), Ca2+ indicator dye Fluo-4 AM kit (cat. no. S1060), reactive oxygen species (ROS) Assay kit (cat. no. S0033M), N-acetylcysteine (NAC; cat. no. ST1546-10g), enhanced bicinchoninic acid (BCA) protein assay kits (cat. no. P0009), JC-1 probes (cat. no. C2006) and antibodies against caspase-3 (1:1,000; cat. no. AC030) (all from Beyotime Institute of Biotechnology), primary antibodies against protein kinase R-like endoplasmic reticulum kinase (PERK; 1:2,000; cat. no. 33247), phosphorylated (p)-PERK (1:1,000; cat. no. 12814), B-cell lymphoma-2 (Bcl-2; 1:500; cat. no. 48496), Bcl-2-associated X (BAX; 1:1,000; cat. no. 48690), C/EBP homologous protein (CHOP; 1:1,000; cat. no. 49418), activating transcription factor 6 (ATF6; 1:1,000; cat. no. 32008), JNK (1:3,000; cat. no. 48615), caspase-12 (1:1,000; cat. no. 48277), β-actin (1:10,000; cat. no. 21338), HRP-conjugated goat anti-mouse IgG secondary antibody (1:10,000; cat. no. L3032) and HRP-conjugated goat anti-rabbit IgG secondary antibody (1:10,000; cat. no. L3042) (all from Signalway Antibody LLC), immobilon western chemiluminescent HRP substrate (cat. no. WBKLS0500; EMD Millipore).

Cell culture

HLECs are located in the inner surface of the lens capsule and are characterized by a flat and irregular polygonal-shaped morphology (6). HLECs were maintained and cultured in DMEM with 10% FBS in a humidified incubator with 5% CO2 at 37°C.

Cell viability assay

NPPB was dissolved in DMSO to form a 0.1 M stock solution. The solution was stored at <−20°C in the dark until use. NPPB was diluted to concentrations of 10-200 µM in serum-free DMEM on the day of the experiment. HLECs were transferred to 96-well plates (100 µl/well) at a density of 0.5×104 cells/well. Cells were incubated for 24 h and treated with 10, 50, 100 and 200 µM of NPPB for 24 h at 37°C. CCK-8 solution (10 µl) was added to each well for 30 min to 1 h at 37°C. The absorbance was expressed as the optical density and was measured at 450 nm using an Infinite M200 Pro microplate reader (Tecan Group, Ltd.).

NAC and 4-PBA with NPPB treatment protocol

NAC was dissolved in double-distilled H2O to form a 1 M stock solution. 4-PBA was dissolved in PBS to form a 500 mM stock solution. Cells were incubated for 24 h and were then treated with 50 µM NPPB with NAC (500 µM) or 4-PBA (20 µM) for 24 h at 37°C.

Intracellular reactive oxygen species (ROS) measurement

The intracellular ROS level was determined using a ROS assay kit and dichloro-dihydro-fluorescein diacetate (DCFH-DA). According to the manufacturer's protocol, the cells were cultured in 96-well plates for 24 h at 37°C and were then washed twice with serum-free medium. Medium containing 10 µM DCFH-DA was added. The cells were incubated in a cell incubator for 20 min at 37°C. Light was avoided during procedures and incubation. After incubation, the cells were washed three times with serum-free medium, then observed and photographed using a fluorescence microscope (magnification, ×400; Olympus Corporation). The fluorescence intensity was quantitatively measured using ImageJ software (v1.51; National Institutes of Health).

Mitochondrial membrane potential (ΔΨm) analysis

The JC-1 probe was used to measure apoptosis via mitochondrial depolarization in HLECs. Briefly, cells (80% confluent) were cultured in 96-well plates after treatment with 50 µM NPPB at 37°C for 24 h. The cells were washed three times with PBS, and 50 µl medium and 50 µl JC-1 working solution were added. The cells were incubated at 37°C for 20 min and washed three times with precooled JC-1 solution. The ΔΨm was determined by measuring the fluorescence intensity of red and green fluorescence using a fluorescence microscope (magnification, ×400). Green fluorescence indicated JC-1 monomers. JC-1 monomers appeared in the cytosol after mitochondrial membrane depolarization, which indicated early stage of apoptosis. Red fluorescence indicated JC-1 aggregation and was located on the mitochondria. Mitochondrial depolarization indicated apoptosis, which was reflected by an increase in the green/red fluorescence intensity ratio. The fluorescence intensity was measured using ImageJ software.

Ca2+ measurement

Cytosolic Ca2+ levels were measured using a calcium ion fluorescent probe (Fluo-4 AM) assay kit. Cells were cultured in 96-well plates after treatment with 50 µM NPPB for 24 h at 37°C. The cells were washed three times with PBS, and Fluo-4 AM (final concentration of 1 µM) was added for 1 h at 37°C and washed three times with PBS. To ensure that the Fluo-4 AM was completely converted into Fluo-4 in the cells, another 30-min incubation at 37°C was necessary after washing. The cells were observed and photographed under a fluorescence microscope (magnification, ×400) at 488 nm.

Western blot analysis

The cells were washed three times with PBS and scraped with a rubber spatula. Cells were collected and centrifuged at 157 × g for 5 min at 4°C. The cells were lysed in an appropriate amount of RIPA lysis buffer containing 1% phosphatase and protease inhibitors. The protein concentration was determined by BCA protein assay kit. A total of 20 µg protein was mixed with SDS-PAGE sample loading buffer. The samples were boiled for 5 min and kept at -20°C until use. The proteins were separated via 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membrane was blocked with a blocking solution composed of 5% non-fat dry milk and TBS with 0.1% Tween-20 (TBS-T) for 1 h at 37°C, then washed in TBS-T three times for 5 min each. The membranes were incubated with primary antibodies against BAX, Bcl-2, caspase-3, ATF6, p-PERK, PERK, JNK, CHOP, caspase-12 and β-actin overnight at 4°C. The membranes were washed three times with TBS-T for 10 min each, and incubated with the corresponding biotinylated secondary antibodies for 1 h at 37°C. The membranes were washed three times with TBS-T. After coating with immobilon western chemiluminescent HRP substrate, the membranes were observed and photographed using a microscope equipped with a CCD camera (Tanon Science & Technology Co., Ltd.). The western blotting results were presented as the ratio of the fluorescence intensity of the target protein to β-actin or PERK (for p-PERK) calculated using ImageJ software.

Statistical analysis

All experiments were repeated 3 times independently. The data are expressed as the mean ± standard deviation. The data were analysed by unpaired Student's t-test and one-way ANOVA followed by Tukey's post-hoc test and Bonferroni's correction. SPSS statistic software 25.0 (IBM Corp.) was used to analyse the data. P<0.05 was considered to indicate a statistically significant difference.

Results

Effect of NPPB on the viability of HLECs

Cells were treated with different concentrations of NPPB (0, 10, 50, 100 and 200 µM) for 24 h. Cell viability was determined via CCK-8 assays. Fig. 1 shows that NPPB significantly inhibited the viability of HLECs in a dose-dependent manner, with an IC50 value of 53.36±0.1% (P<0.01 vs. 0 µM). These results suggested that NPPB decreased cell survival rate and inhibited the viability of HLECs. A concentration of 50 µM NPPB was used in subsequent experiments.

Figure 1 Effect of NPPB on HLEC viability. HLECs were incubated with the indicated concentrations of NPPB for 24 h, and cell viability was determined using Cell Counting Kit-8 assays. The results are presented as the mean ± standard deviation (n=3). **P<0.01; ***P<0.001; ****P<0.0001 vs. vehicle (0 µM). HLEC, human lens epithelial cell; NPPB, 5-nitro-2-(3-phenylpropylamino) benzoic acid.

Effect of NPPB on mitochondrial apoptosis of HLECs

Red fluorescence indicated that JC-1 accumulated in mitochondria and the cells were alive, while green fluorescence indicated apoptosis. Exposure of HLECs to NPPB (50 µM) for 24 h resulted in the dissipation of the ΔΨm. Green fluorescence increased after JC-1 staining, and the ratio of green to red fluorescence increased, indicating the toxicity of NPPB in mitochondria (Fig. 2). JC-1 aggregated in the mitochondria in the control group, with a ratio of 0.15±0.027, while the NPPB-treated group exhibited a significantly higher ratio (0.59±0.080) since the monomeric form of JC-1 was present in the cytosol. (P<0.001; Fig. 2). Additionally, the expression levels of apoptosis-associated proteins were detected by western blot analysis. NPPB-induced apoptosis was accompanied by a decrease in Bcl-2 expression (0.51±0.07 vs. 0.80±0.16; P<0.05) and a significant increase in BAX (0.92±0.19 vs. 0.57±0.08; P<0.05) and cleaved caspase-3 expression (1.77±0.47 vs. 0.49±0.21; P<0.05) compared with the vehicle group (Fig. 3).

Figure 2 Effect of NPPB on mitochondrial apoptosis of HLECs. The ΔΨm of HLECs treated with 50 µM NPPB was evaluated by JC-1 assays, and the number of cells with low ΔΨm was quantified. The relative green/red fluorescence intensity was determined. Scale bar, 50 µm. The results are presented as the mean ± standard deviation (n=3). ***P<0.001. HLEC, human lens epithelial cell; NPPB, 5-nitro-2-(3-phenylpropylamino) benzoic acid; ΔΨm, mitochondrial membrane potential.

Figure 3 Effect of NPPB on apoptosis in HLECs. HLECs were incubated with 50 µM NPPB for 24 h, and the expression levels of the apoptosis markers BAX, Bcl-2 and cleaved caspase-3 were determined by western blotting. β-actin was used as an internal control. The results are presented as the mean ± standard deviation (n=3). *P<0.05. HLEC, human lens epithelial cell; NPPB, 5-nitro-2-(3-phenylpropylamino) benzoic acid; Bcl-2, B-cell lymphoma-2; BAX, Bcl-2-associated X.

Effect of NPPB on ROS in HLECs

NPPB induced ROS generation in HLECs. NPPB (50 µM) significantly increased intracellular ROS levels (3.33±0.71 vs. 0.88±0.17; P<0.01) compared with the vehicle group (Fig. 4).

Figure 4 Effect of NPPB on intracellular ROS production in HLECs. HLECs were treated with 50 µM NPPB for 24 h. ROS were stained with a dichloro-dihydro-fluorescein diacetate fluorescent probe and examined using ImageJ software. Scale bar, 50 µm. The results are presented as the mean ± standard deviation (n=3). **P<0.01. HLEC, human lens epithelial cell; NPPB, 5-nitro-2-(3-phenylpropylamino) benzoic acid; ROS, reactive oxygen species.

Effect of NPPB on ER stress and caspase-dependent apoptosis in HLECs

Mitochondria and the ER are major reservoirs of intracellular Ca2+. The imbalance of calcium ion is closely associated with mitochondrial apoptosis and ER stress (24). Therefore, whether NPPB induced apoptosis and ER stress by perturbing intracellular Ca2+ homeostasis was further explored. NPPB stimulation significantly increased Ca2+ levels in the cytosol in HLECs (7.0±0.53 vs. 0.46±0.09; P<0.0001) compared with the vehicle group (Fig. 5).

Figure 5 Effect of NPPB on intracellular Ca2+ dysregulation in HLECs. HLECs were treated with the indicated concentrations of NPPB for 24 h, and cytosolic Ca2+ was measured with Fluo-4 AM, a Ca2+ indicator dye, and examined using ImageJ software. Scale bar, 50 µm. The results are presented as the mean ± standard deviation (n=3). ****P<0.0001. HLEC, human lens epithelial cell; NPPB, 5-nitro-2-(3-phenylpropylamino) benzoic acid.

The expression levels of ER stress-associated proteins were detected by western blot analysis, including p-PERK (1.37±0.03 vs. 1.01±0.16; P<0.05), ATF6 (1.09±0.14 vs. 0.86±0.03; P<0.05), JNK (1.13±0.28 vs. 0.63±0.03; P<0.05), CHOP (1.04±0.04 vs. 0.80±0.04; P<0.01) and caspase-12 (1.07±0.02 vs. 0.87±0.08; P<0.05) were upregulated following NPPB (50 µM) treatment compared with the vehicle group (Fig. 6). Quantification of the western blotting results indicated that NPPB significantly induced ER stress and the unfolded protein response (UPR) in HLECs.

Figure 6 Effect of NPPB on ER stress-associated markers. ER stress markers and ER stress-associated caspase-dependent apoptosis markers ATF6, JNK, CHOP, caspase-12, PERK and p-PERK were measured via western blotting. β-actin was used as an internal control. The results are presented as the mean ± standard deviation (n=3). *P<0.05; **P<0.01. HLEC, human lens epithelial cell; NPPB, 5-nitro-2-(3-phenylpropylamino) benzoic acid; ER, endoplasmic reticulum; ATF6, activating transcription factor 6; CHOP, C/EBP homologous protein; p-PERK, phosphorylated protein kinase R-like endoplasmic reticulum kinase.

Effects of NAC and 4-PBA in NPPB-treated HLECs

The ROS scavenger NAC (500 µM) and the ER stress inhibitor 4-PBA (20 µM) were used to co-treat cells with 50 µM NPPB at 37°C for 24 h. NAC and 4-PBA significantly decreased the levels of the green-to-red fluorescence ratio in JC-1 staining compared with NPPB alone (NAC + NPPB, 4.53±0.70 and 4-PBA + NPPB, 3.47±0.25 vs. NPPB, 8.81±2.10; both P<0.01; Fig. 7A). Additionally, compared with NPPB alone, NAC and 4-PBA with NPPB decreased the expression levels of BAX (NAC + NPPB, 1.34±0.07 and 4-PBA + NPPB, 1.47±0.12 vs. NPPB, 1.75±0.11; P<0.01 and P<0.05, respectively) and cleaved caspase-3 (NAC+NPPB, 0.69±0.20 and 4-PBA + NPPB, 0.69±0.24 vs. NPPB, 1.77±0.47; both P<0.01) and increased the expression levels of Bcl-2 (NAC + NPPB, 1.16±0.11 and 4-PBA + NPPB, 1.14±0.13 vs. NPPB, 0.82±0.12; both P<0.05) compared with the NPPB group (Fig. 7B). Furthermore, NAC and 4-PBA significantly decreased NPPB-induced ROS (NAC + NPPB, 1.21±0.19 and 4-PBA + NPPB, 1.10±0.21 vs. NPPB, 12.32±1.62; both P<0.0001; Fig. 7C), Ca2+ levels (NAC + NPPB, 4.53±0.70 and 4-PBA + NPPB, 3.47±0.25 vs. NPPB, 8.81±2.10; both P<0.05; Fig. 7D) in the cytosol and the level of ER stress, including the levels of p-PERK (NAC + NPPB, 1.19±0.20 and 4-PBA + NPPB, 1.19±0.12 vs. NPPB, 2.00±0.41; both P<0.05), ATF6 (NAC + NPPB, 0.89±0.03 and 4-PBA+NPPB, 0.85±0.11 vs. NPPB, 1.70±0.07; both P<0.0001,), JNK (NAC + NPPB, 2.74±0.15 and 4-PBA + NPPB, 2.80±0.16 vs. NPPB, 3.50±0.17; both P<0.05), CHOP (NAC+NPPB, 1.85±0.13 and 4-PBA + NPPB, 1.72±0.04 vs. NPPB, 2.25±0.19; P<0.05 and P<0.01, respectively) and caspase-12 (NAC + NPPB, 1.91±0.06 and 4-PBA + NPPB, 1.75±0.07 vs. NPPB, 2.29±0.08; P<0.05 and P<0.01, respectively) (Fig. 7E). Therefore, NPPB activated ROS production and induced the UPR pathway, which likely promoted apoptosis.

Figure 7 NAC and 4-PBA inhibit the effects of NPPB in HLECs. NPPB-treated cells were co-treated with NAC (500 µM) and 4-PBA (20 µM). (A) Relative green/red fluorescence intensity was determined using ImageJ software. (B) Expression levels of apoptosis markers BAX, Bcl-2 and cleaved caspase-3 were determined by western blotting. β-actin was used as an internal control. (C) Reactive oxygen species were stained with a dichloro-dihydro-fluorescein diacetate fluorescent probe and examined using ImageJ software. (D) Cytosolic Ca2+ was measured with Fluo-4 AM, a Ca2+ indicator dye, and examined using ImageJ software. (E) ER stress markers and ER stress-associated caspase-dependent apoptosis markers ATF6, JNK, CHOP, PERK and p-PERK were measured by western blotting. β-actin was used as an internal control. Scale bar, 50 µm. The results are presented as the mean ± standard deviation (n=3). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. HLEC, human lens epithelial cell; NPPB, 5-nitro-2-(3-phenylpropylamino) benzoic acid; Bcl-2, B-cell lymphoma-2; BAX, Bcl-2-associated X; ER, endoplasmic reticulum; ATF6, activating transcription factor 6; CHOP, C/EBP homologous protein; p-PERK, phosphorylated protein kinase R-like endoplasmic reticulum kinase; NAC, N-acetylcysteine; 4-PBA, 4-phenylbutyric acid.

Discussion

Investigation of LEC apoptosis is the main method to study the pathogenesis of cataracts (25). Chloride channels serve important roles in numerous cellular aspects, especially apoptosis (26-29). The non-specific inhibitor of chloride channels NPPB is commonly used to inhibit the chloride current and to study the function of voltage-gated chloride channels (30). Voltage-gated chloride channels are involved in the occurrence of some diseases, such as cardiovascular and nervous system diseases and nasopharyngeal carcinoma (29,31,32). However, their role in cataracts has not been widely discussed. Therefore, NPPB was used in the present study to investigate whether chloride channels were involved in the pathogenesis of cataracts. If NPPB was associated with the apoptosis of LECs, further studies may examine which voltage-gated chloride channels may be involved in the formation of cataracts. NPPB is widely used to study apoptosis mechanisms (30,33,34). Souktani et al (35) demonstrated that NPPB promoted cardiomyocyte apoptosis. However, Malekova et al (30) indicated that NPPB served a protective role in H2O2-induced cardiomyocyte apoptosis. To the best of our knowledge, the mechanism of chloride channel mediation of LEC apoptosis has not been previously studied. Therefore, NPPB was used in the present study to investigate the role of chloride channels in LEC apoptosis. The current results revealed that NPPB inhibited cell viability in a concentration- dependent manner. The results of CCK-8 assays indicated that the IC50 of NPPB in LECs at 24 h was ~50 µM. Therefore, a concentration of 50 µM was used in subsequent experiments. NPPB increased the number of JC-1 monomers and the expression levels of BAX and cleaved caspase-3, and decreased Bcl-2 expression. Therefore, it was concluded that NPPB induced HLEC apoptosis via a mitochondrial-dependent pathway. Similar results were obtained in basilar artery smooth muscle cells and human bronchial epithelium (36,37). The effects of chloride channels on apoptosis are distinct in different cell types. Chloride channels have been shown to be cardioprotective in ischemic preconditioning of isolated hearts; however, the inhibitor of chloride channels NPPB prevented the appearance of H2O2-induced apoptosis of pheochromocytoma cells (38,39). Chloride channel activation in intestinal epithelial cells leads to apoptosis via activation of caspase 3 (40). The reason for the different effects may be associated with the various experimental conditions and models used.

Cataract pathogenesis is closely associated with oxidative stress (41). To the best of our knowledge, the present study revealed for the first time that NPPB induced oxidative stress in LECs and increased the production of intracellular ROS, and that NAC inhibited NPPB-induced production of ROS. ROS promote apoptosis in a variety of ways, such as via ER stress, PI3K/AKT signalling, the Foxo3a/TRIM69/p53 regulatory network and the calmodulin-like-protein 3-dependent JNK1/2 and ERK1/2 signalling pathways (17,42-44). In the present study, 4-PBA also inhibited the production of NPPB-induced ROS, suggesting that oxidative stress and ER stress interacted and influenced each other.

The ER is a central organelle used in a series of important biological processes to maintain the stability of the intracellular environment. The present study revealed that NPPB increased intracellular Ca2+ and the levels of ER stress-associated proteins, such as p-PERK, PERK, ATF6, JNK and CHOP, which indicated that NPPB significantly induced ER stress in HLECs. Additionally, NPPB increased the expression levels of caspase-12, which indicated that NPPB significantly induced apoptosis via ER stress and the mitochondrial pathway. NAC and 4-PBA decreased intracellular Ca2+ and the expression levels of ER stress-associated proteins, therefore attenuating NPPB-induced ER stress in HLECs. The homeostasis of Ca2+ in HLECs is very important since its balance maintains clarity of the lens (45). When ER stress occurs, the ER releases Ca2+, increasing the cytosolic Ca2+ levels (46). Ca2+ dysregulation further promotes apoptotic cell death (47,48). Therefore, chloride channels serve important roles in maintaining lens transparency, inhibiting ER stress and protecting lens cells.

In conclusion, the present study demonstrated that NPPB induced HLEC apoptosis. NPPB inhibited cell viability and induced ROS, ER stress and apoptosis. The current findings provide strong evidence that chloride channels may serve an important role in the pathogenesis of cataracts.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

All authors designed the present study. LN, JZ, KL and YW performed the experiments. LN, XL, JZ and YZ confirmed the authenticity of the data. LN, YW, XL, KL and YL analysed the data and prepared the figures. LN, XL, JZ and YS drafted the initial manuscript. XL, JZ and YZ reviewed and revised the manuscript. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

Not applicable.

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