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. 2023 Dec 13;88(2):168–176. doi: 10.1093/bbb/zbad169

Valine improves mitochondrial function and protects against oxidative stress

Shakshi Sharma 1, Xiaomin Zhang 2, Gohar Azhar 3, Pankaj Patyal 4, Ambika Verma 5, Grishma KC 6, Jeanne Y Wei 7,✉
PMCID: PMC10807754  PMID: 38093456

ABSTRACT

Among the branched-chain amino acids, leucine and isoleucine have been well studied for their roles in improving mitochondrial function and reducing oxidative stress. However, role of valine in mitochondrial function regulation and oxidative stress management remains elusive. This study investigated valine effect on mitochondrial function and oxidative stress in vitro. Valine increased expression of genes involved in mitochondrial biogenesis and dynamics. It upregulates mitochondrial function at complexes I, II, and IV levels of electron transport chain. Flow cytometry studies revealed, valine reduced oxidative stress by significantly lowering mitochondrial reactive oxygen species and protein expression of 4-hydroxynonenal. Functional role of valine against oxidative stress was analyzed by XFe96 Analyzer. Valine sustained oxidative phosphorylation and improved ATP generation rates during oxidative stress. In conclusion, our findings shed more light on the critical function of valine in protecting mitochondrial function thereby preventing mitochondrial/cellular damage induced by oxidative stress.

Keywords: valine, mitochondrial function, oxidative stress, reactive oxygen species

Graphical Abstract

Graphical Abstract.

Graphical Abstract

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Valine improves mitochondrial function and protects against oxidative stress. It modulates oxidative phosphorylation, ATP production, and inhibits mitochondrial dysfunction by reducing reactive oxygen species (ROS) production.

Branched-chain amino acids (BCAAs) are among the most prevalent essential amino acids that are pivotal for the health of the human body (Nie et al.2018). Leucine, isoleucine, and valine are α-amino acids with aliphatic side chains that are important for maintaining the body's nitrogen balance (Doi et al.2003) as well as skeletal muscle cell growth and development and have been linked to multiple metabolic processes, that are important in cellular pathways, and are central in the relationship between diet, health, and aging (Wolfe 2017; Green and Lamming 2019; Vanweert et al.2022). The activation of pathways including mammalian target of rapamycin, phosphoinositide 3-kinase-protein kinase B (PI3K-AKT), and maintenance of physiological metabolic health are all influenced by these amino acids (Viana et al.2019; Mann et al.2021). Aside from helping with protein synthesis, they also aid in translation and degradation (Tom and Nair 2006). The potential of BCAAs as dietary supplements for treating various disorders has been studied in both human and mouse models and has been shown to influence metabolic activity, improve mitochondrial bioenergetics, support muscle growth, and maintain mitochondrial health (Lynch and Adams 2014; Holecek 2018; Ruocco et al. 2021). Mitochondria are crucial for producing adenosine triphoshate (ATP) and also participate in fatty acid oxidation, cell survival, and apoptosis (Hewton et al.2021; Yoneshiro et al.2021). The activation of multiple genes that regulate mitochondrial function, including peroxisome proliferator-activated receptor gamma (PPAR-γ) coactivator-1 alpha (PGC-1α), PPAR-gamma coactivator-1 beta (PGC-1β), and the mitofusins, by BCAAs, in turn stimulates mitochondrial biogenesis. According to several studies (Hinkle et al.2022), mitochondrial dysfunction is associated with oxidative stress, cellular damage and is thought to be a common underlying mechanism of metabolic diseases (Rocha et al.2013; Walters et al.2016). Extreme reactive oxygen species (ROS) production can hinder mitochondrial membrane permeability, and produce lipid peroxidation products, which leads to mitochondrial-related apoptosis (Ren et al.2017; Zhao et al.2019). In skeletal muscle, ROS generation induces a signaling cascade that can produce muscle damage (Lee et al.2017; Pan and Chen 2021). It has been demonstrated that adding BCAA supplements lengthens the average lifespan of mice by boosting the ROS defense mechanism in the skeletal and cardiac muscle tissues and improving mitochondrial biogenesis (Valerio et al.2011). BCAAs alone or together can mediate the antioxidant defense system and reduce oxidative stress in skeletal muscles in middle-aged mice (D'Antona et al.2010; Cruzat et al.2014). Valine (Val), a necessary BCAA, can also act as an energetic molecule and is important for protein synthesis and growth of skeletal muscle (Kohlmeier 2003). Further, valine also plays a favorable function in lipid metabolism (Bishop et al.2020) and have inflammatory properties (Gart et al.2022).

While studies have been conducted on BCAAs, especially leucine and isoleucine on mitochondrial function, cellular metabolism and oxidative stress (Mattick et al.2013; Tamanna and Mahmood 2014), there is a dearth of literature on the cellular and functional importance of valine. In the present study, we evaluated the effect of valine treatment on mitochondrial function and its response to oxidative stress. Our findings demonstrate that valine administration improved oxygen consumption rate (OCR) and overall ATP generation in the mitochondria. Additionally, valine treatment also enhanced the transcriptional activity of PGC-1α, PGC-1β, and other genes that regulate mitochondrial function. Valine significantly reduced ROS production, thereby maintaining the OCR during H2O2-mediated oxidative stress. The findings from this study enhance our understanding of valine as an important nutrient to improve mitochondrial function, thereby protecting cells against oxidative damage.

Materials and methods

Cell culture and cell proliferation assay

The C2C12 mouse cell line used in this study was purchased from the American Type Culture Collection (ATCC, CRL-1772). Cell culture reagents were obtained from Thermo Fischer Scientific as previously described (Rogers et al.2013). Valine was obtained from Sigma–Aldrich, St. Louis, MO, USA, (V0500), and used following the manufacturer's protocol. C2C12 cell viability after valine treatment was determined by using MTS reagent (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Abcam; ab197010). A 200 µL C2C12 cell suspension (5 × 103/well) was seeded in a 96-well plate. At 70%-80% confluency, cells were treated with two different concentrations (1.0 and 1.5 m m) of valine for 24 h. A volume of 20 µL/well MTS reagent was added in each well and incubated for a period of 0.5-4 h at 37°C following the manufactures’ protocol. The absorbance was recorded at 490 nm.

Measurement of ATP and NAD/NADH levels

C2C12 cells (5 × 103 cells/well) were plated in 96-well plate, treated with 1.0 m m concentration of valine for 24 h. Intracellular ATP levels were assessed using the CellTiter-Glo 2.0 assay kit (Promega, Madison, WI, USA; G9242). Further, the NAD/NADH (nicotinamide adenine dinucleotide) levels after valine treatment were determined using a NAD/NADH Glo Assay Kit (Promega, Madison, WI, USA; G9071) following the manufacturer's protocol.

RNA isolation and quantitative reverse-transcriptase PCR

Cells were pre-treated with 1.0 m m concentration of valine for 24 h. Total cellular RNA was extracted from cells utilizing the RNeasy Kit (Qiagen) in accordance with the manufacturer's protocol. Total RNA obtained was then revere transcribed to Complementary DNA (cDNA) using cDNA Reverse Transcription Kit (Applied Biosystems, Thermo-Fischer Scientific, Waltham, MA, USA; 4368814). The real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) amplification was performed using SYBR Green master mix (Applied Biosystems, Thermo Fischer Scientific, Waltham, MA, USA; 4344463) in a QuantStudioTM 3 Real-Time PCR instrument (Applied Biosystems). The relative expression was measured using StepOne software. All experiments were performed as described previously (Rogers et al.2013). Primer set used for qPCR were listed in Table S1.

Western blot analysis

C2C12 cells were cultured and treated with valine as mentioned above and protein concentration was determined by using a BCA protein assay kit (Thermo–Fisher, Waltham, MA, USA). Western blotting was conducted as previously described (Zhang et al.2012). The antibodies used in the study were PGC-1α (sc-518025) and anti-4HNE antibody (ab46545), anti-mouse HRP (Invitrogen, Carlsbad, CA, USA; 62-6520) and anti-rabbit AP (Bio-Rad, Hercules, CA, USA; 64251130). iBright™ CL1500 (Invitrogen) was used to visualized the protein signal and ImageJ software v1.53t (National Institutes of Health, Bethesda, MD, USA) was used for quantification of protein expression.

Extracellular flux analysis

C2C12 cells were seeded in XFe96 Well plates at a density of 1.2 × 104 cells/well (Seahorse Bioscience, Billerica, MA, USA). The cells were then treated with 1.0 m m concentration of valine for 24 h respectively. After valine treatment, Seahorse XFe96 Extracellular Flux Analyzer was used to measure OCR using XF Cell Mito Stress Test Kit (Agilent, Santa Clara, CA, USA; 103015-100) and extracellular acidification rate (ECAR) using XF Glycolytic Rate Assay Kit (Agilent, Santa Clara, CA, USA; 103344-100). For determination of ATP production rate from mitochondria and glycolysis, XF-Real-time ATP Assay Kit (Agilent, Santa Clara, CA, USA; 103592-100). All metabolic assays were performed following the procedure from the manufacturer.

High-resolution respirometry

The mitochondrial respiration in the intact C2C12 cells (1 × 106/sample) after valine treatment was determined using Oxygraph-O2K high-resolution respirometer (Oroboros Instruments GmbH, Innsbruck, Austria) (Raiteri et al.2021). The mitochondrial respiratory activity at different complexes was analyzed using substrate-uncoupler-inhibitor-titration protocol as described in (Patyal et al.2022). The OCR from all mitochondrial complex was expressed as oxygen flux (pmol/s*Million Cells). DatLab 6.2 software (Innsbruck, Austria) was used to perform the data analysis.

Oxidative stress analysis

C2C12 cells were seeded at a density of 5 × 103 cells/mL (200 µL) in 96-well plate. H2O2 was obtained from Sigma–Aldrich, St. Louis, MO, USA (88 597). A volume of 200-1000 µmol/L treatment of H2O2 generates oxidative stress in C2C12 cells as previously reported (Li et al.2020). Cells were grown overnight and next day exposed to different concentrations of H2O2 (200, 400, 600, and 800 µmol/L) to stimulate oxidative stress for 6 h (Wu et al.2022). A total of 400 µmol/L concentration of H2O2 was selected as the final concentration for treatment. After H2O2 treatment, cells were treated with 1.0 m m concentration of valine for 24 h to assess cell proliferation viability as described above. Four groups used in this and subsequent experiments were control group (without any treatment), valine treated group, H2O2 treated group, H2O2 + valine treatment group.

Flow cytometry analysis

MitoSOXTM Red (Thermo Fisher, Eugene, OR, USA; M3600) was used to detect the mitochondrial ROS production. The C2C12 cells (1 × 106 cells/well), were plated in 6-well culture plate and treated with 400 µmol/L H2O2 for 6 h. Following H2O2 treatment, cells were treated with a final concentration of 1.0 m m of valine for 24 h. After 24 h, cells were incubated with 5 µm of MitoSox Red for 10 min at 37 °C as previously described (Verma et al.2023). Flow cytometer (BD LSRFortessaTM Cell Analyzer, Franklin Lakes, NJ, USA) was used to quantify the fluorescence intensity and data were processed by FlowJo_v10.8.1 software.

Statistical analysis

Results obtained from this study were described as mean ± standard deviation. The 2-tailed Student's t-test was used to determine the statistical significance between two groups. Multiple groups were ascertained by using one-way analysis of variance (ANOVA) followed Tukey's multiple comparison test. All the P values < 0.05 were considered to indicate as statistically significant. GraphPad Prism 9.11 Software Inc. (Dotmatics, Boston, MA, USA) was utilized to process the statistical analysis.

Results

Valine increases ATP and NAD/NADH production

Valine treatment at two different concentrations (1.0 and 1.5 m m) for 24 h, showed no significant effect on C2C12 cells viability as compared to control as shown in Figure S1. We next investigated the effect of valine treatment on ATP and NAD/NADH production rate. The treatment of valine at 1.0 m m concentration for 24 h, resulted in significantly higher ATP and NAD/NADH production rates (Figure S2a and b). Therefore, we selected this concentration for further experiments.

Valine impacts genes that regulate mitochondrial biogenesis and function

The mRNA expression levels of PGC-1α and PGC-1β (Figure 1a) were upregulated following valine treatment. The protein expression of PGC-1α was also found to be significantly higher in the valine-treated cells (Figure 1b). Further, valine treatment also upregulated the expression levels of mitofusin-1 (MFN1), mitofusin-2 (MFN2), and mitochondrial fission 1 (Fis1), whereas no difference was observed on optic atrophy-1 (Opa 1) gene expression (Figure 1c).

Figure 1.

Figure 1.

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Valine treatment upregulated expression of important mitochondrial genes in C2C12 cells (a) The mRNA expression levels of PGC1-α and PGC1-β were determined using Real-time RT-qPCR (b) The protein levels of PGC-1α was determined using western blotting analysis with gleyecelarldehyde 3-phosphate dehydrogenase (GAPDH) being used as loading control (c) Gene expression levels of MFN1, MFN2, and Fis1 were increased with no change in Opa1 levels (n = 3). *P < 0.05, **P < 0.01, ns: P > 0.05.

Valine distinctively regulates mitochondrial bioenergetics

Valine treatment resulted in increased OCR production and, therefore improved the mitochondrial function in C2C12 cells. Valine treatment elevated all of the different domains of OCR such as basal respiration, maximal respiratory capacity, and spare respiratory capacity (Figure 2a). ECAR analysis resulted in reduced basal glycolysis and compensatory glycolysis following valine treatment (Figure 2b). However, valine treatment significantly increased the mitochondrial ATP production rate, due to an increase in the oxidative phosphorylation (OXPHOS) activity (Figure 2c).

Figure 2.

Figure 2.

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Valine treatment increased OCR and total ATP and reduced glycolytic rate (ECAR) in C2C12 cells. (a) Valine upregulated the basal respiration, maximum respiration, and spare respiratory capacity in C2C12 cells. (b) In ECAR, both basal and compensatory glycolysis were significantly reduced following treatment. (c) Total ATP production was significantly upregulated in oxidative phosphorylation (mitochondrial-ATP) but showed no change in glycolysis (glycolysis-ATP) (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, ns: P > 0.05.

Valine specifically regulates the mitochondrial respiratory chain complex levels

Oxygraph-2 K (O2K) was used to measure the mitochondrial function by analyzing the high-resolution respiratory capacity in C2C12 cells. Valine treatment significantly increased the basal respiration and the maximal capacity of mitochondrial electron transport chain (ETC) (Figure 3a) in C2C12 cells. We then further assessed the effect of valine treatment on different complexes of the ETC. The respiration rates of complexes I (NADH/ubiquinone oxidoreductase), II (succinate dehydrogenase), and IV (cytochrome c oxidase) increased after treatment. No change was observed in complex III activity (cytochrome c reductase) (Figure 3b).

Figure 3.

Figure 3.

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High-resolution respiratory analysis showed an increased mitochondrial respiration following valine treatment. (a) C2C12 cells were treated with 0.1 m m concentration of valine for 24 h. Basal respiration and maximum ET capacity were increased following the treatment (b) Measurement of OCR at different complexes of ETC showed increase in complexes I, II, and IV activity after treatment (n = 3). * P < 0.05, **P < 0.01, ***P < 0.001, ns: P > 0.05.

Valine reduced mitochondrial ROS production induced by H2O2

We first examined the effect of H2O2 treatment on the cellular viability of C2C12 cells. The results showed that treatment of H2O2 at different concentrations for 6 h affected cell viability in dose-dependent manner (Figure S3). We selected 400 µmol/L concentration of H2O2 as our final treatment, as the recovery rate at this dose was found to be higher after valine treatment as compared to the other higher concentration used (Figure S4). The results showed that valine treatment significantly recovered the cell viability in C2C12 cells affected by H2O2 treatment (Figure S5). Further, we observed that H2O2 treatment significantly upregulate the ROS production. Our results showed that treatment of valine, along with H2O2, prevented ROS production (Figure 4a). In addition, H2O2 treatment also increased the protein expression level of 4-hydroxynonenal (4-HNE), which was prevented by valine treatment.

Figure 4.

Figure 4.

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Valine reduced mitochondrial ROS production in C2C12 cells. (a) ROS generation was evaluated using a flow cytometer. H2O2 treatment caused ROS production in 55.6% of cells, whereas, when H2O2 was used along with valine, recovery from oxidative stress was improved and only 28.8% of cells were ROS positive. (b) The protein expression of 4-HNE antibody was assessed by western blotting technique with β-actin as a loading control (n = 3). ****P < 0.0001.

Valine protects against oxidative stress and metabolic alterations

To confirm the protective role of valine against oxidative stress, we next assessed its impact on oxidative phosphorylation. Data were normalized to remove variation due to cell density before the experiment. H2O2 treatment initially repressed basal respiration, maximal respiratory capacity and spare respiratory capacity (Figure 5a). Valine treatment alone and in the presence of H2O2 significantly improved the maximal respiratory capacity and spare respiratory capacity. Additionally, we also determined the effect of H2O2 on total ATP production rate. H2O2 treatment significantly reduced the mitochondrial ATP rate, which was restored and improved by valine treatment (Figure 5b).

Figure 5.

Figure 5.

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Protective role of valine against oxidative stress. (a) OCR, Maximum respiration and spare respiratory capacity was repressed due to oxidative stress after H2O2 treatment, which was restored after valine treatment. (b) Total ATP production was decreased in both oxidative phosphorylation and glycolysis, which was recovered after valine treatment (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001, ns: P > 0.05.

Discussion

This study has three major findings: first, valine plays a crucial role in improvement of cellular mitochondrial function; second, valine has a protective effect against oxidative stress by minimizing the production of mitochondrial ROS; and third, valine maintains oxidative phosphorylation and ATP rate during oxidative stress. Clinically, BCAAs intake have been suggested to reduce sarcopenia in older adults and possibly improve glucose metabolism, potentially by increasing mitochondrial biogenesis and muscle cellular function (Yoon 2016; Kitajima et al.2018). Despite many studies demonstrating the correlation between BCAAs and insulin resistance (Schnuck et al.2016), valine was reported not to cause insulin sensitivity or worsen insulin resistance (Rivera et al.2020; Mao et al.2022). We are among the first to report on the key role of the BCAA, valine, in enhancing mitochondrial function and reducing oxidative stress in a skeletal muscle cell line. Our study focused on exploring whether valine could be utilized to improve mitochondrial function. We analyzed the effect of valine treatment on cell viability of C2C12 cells. Valine treatment showed no change in cell viability. Our observation is consistent with no effect of varying concentrations (0.5 m m–2 m m) of valine for up to 48 h of treatment in C2C12 cells (Rivera et al.2020).

Our study suggests that mitochondrial biogenesis is improved with valine treatment. PGC-1α and PGC-1β are the main transcriptional coactivators of oxidative phosphorylation in mitochondria and also regulate mitochondrial biogenesis and function (Kang and Li 2012). Other studies have shown that leucine stimulates expression of PGC-1α and PGC-1β to promote mitochondrial biogenesis (Liang et al.2014; Johnson et al.2018; Rivera et al.2020; Ye et al.2020). Similarly, we found that valine regulates mitochondrial biogenesis and dynamics by increasing the expression levels of PGC-1α, PGC-1β, MFN1, MFN2, and Fis1. Mitochondria play a predominant role in regulating energy metabolism, ATP production and oxygen consumption and for the maintenance of cellular functions (Chu et al.2021). The increased OCR is an important indicator, which reflects the mitochondrial respiratory chain complex activity (du Plessis et al.2015). Our data showed increased OCR and reduced ECAR in C2C12 cells following valine treatment. This reduction in ECAR could be due to extreme oxidation of fatty acids, which inactivate pyruvate dehydrogenase and obstruct the normal process of glycolysis (Savage et al.2007). Others have reported that valine did not alter mitochondrial biogenesis or glycolysis (Rivera et al.2020). We also observed an increase in total ATP production in C2C12 cells following valine treatment, which emphasized the crucial role mitochondrial oxidative phosphorylation plays in sustaining energy status (Zheng 2012). Interestingly, our findings highlighted the important part that valine plays in improving mitochondrial activity at electron transport complex levels, including complex I, II, and IV. Recent studies have focused on inhibiting Complex I to target the Warburg effect and metabolic plasticity of cancer cells (Chaube et al.2015). The role of valine in regulating complex I is well demonstrated in T-cell acute lymphoblastic leukemia (Thandapani et al.2022). It is possible that valine could act as an anaplerotic metabolite and drive the tricarboxylic acid cycle to sustain mitochondrial ATP production.

Muscle tissues are most vulnerable to a high risk of oxidative stress (Li et al.2020). Oxidative stress can impair mitochondrial oxidation and worsen metabolic disorders (Lian et al.2022). Extreme levels of ROS production can impair skeletal muscle function by harming the cells through oxidation (Zhang et al.2001; Zhao et al.2021). Our results indicate that valine significantly reduced mitochondrial ROS production and protein expression of 4-HNE in C2C12 cells, and thus can help in promoting the growth of muscle and the healing of damaged tissue. Interestingly, valine also protects the cell by sustaining oxidative phosphorylation and generating more ATP for energy supplementation to counter the H2O2-induced oxidative stress (Jeong et al.2022). In accordance with our findings, cysteine was reported to reduce the H2O2-induced mitochondrial stress and restore mitochondrial function in C2C12 myoblasts (Mizugaki et al.2022). Valine could potentially be used therapeutically to support good mitochondrial activity and assist in reducing cellular and mitochondrial damage induced by ROS-driven oxidative stress.

Limitations

This study primarily focused on determining the effect of valine treatment on one cell line. Further research is required to completely delineate the mechanism (s) by which valine might help in cell survival during stress in different cell lines and in vivo.

Conclusion

In conclusion, we found that valine treatment does improve mitochondrial function, by enhancing mitochondrial oxidative phosphorylation and total ATP production. This could be due to its role in the improvement of the gene expression of PGC-1α, PGC-1β, and those involved in biogenesis and function. By lowering the H2O2-driven oxidative stress, valine apparently preserves cell viability, maximizes respiratory capacity, and lowers the formation of mitochondrial ROS. The findings of this study provide insight into valine's role in providing mitochondrial protection against oxidative stress, and suggest that it may be a future target for maintaining cellular function under stress.

Supplementary Material

zbad169_Supplemental_File
Click here for additional data file. (48.3KB, docx)

Acknowledgments

We thank Amanda Pangle and Yingni Chi for their assistance. We are also thankful to the UAMS digital flow cytometry core facility for the expertise and equipment access.

Contributor Information

Shakshi Sharma, Donald W. Reynolds Department of Geriatrics, Institute on Aging, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

Xiaomin Zhang, Donald W. Reynolds Department of Geriatrics, Institute on Aging, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

Gohar Azhar, Donald W. Reynolds Department of Geriatrics, Institute on Aging, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

Pankaj Patyal, Donald W. Reynolds Department of Geriatrics, Institute on Aging, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

Ambika Verma, Donald W. Reynolds Department of Geriatrics, Institute on Aging, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

Grishma KC, Donald W. Reynolds Department of Geriatrics, Institute on Aging, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

Jeanne Y Wei, Donald W. Reynolds Department of Geriatrics, Institute on Aging, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

Data Availability

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

Author contribution

Conceived and designed the experiments: S.S., G.A., X.Z., and J.Y.W. Performed the experiments: S.S., P.P., A.V. and G.K. Analyzed the data: S.S., X.Z. and G.A. Wrote the paper: S.S., X.Z., G.A. and J.Y.W. Statistical analysis: S.S., X.Z. and G.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported in part by the Claude D. Pepper Older American Independence Center Grant (P30AG28718) from National Institute on Aging (NIA).

Disclosure statement

The authors declare no conflict of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

zbad169_Supplemental_File
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Data Availability Statement

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

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