The Phosphoproteomic Response of Rice Seedlings to Cadmium Stress
Abstract
:1. Introduction
2. Results
2.1. The Phenotypic and Physiological Effects of Exposure to Cd2+ Stress
2.2. The Identification of Phosphorylated Proteins and Phosphosites
2.3. Predicted Subcellular Localization of Phosphoproteins
2.4. Peptide Motifs Associated with Phosphorylation
2.5. Differentially Phosphorylated Proteins in Response to Cd2+ Treatment
2.6. Functional Assignment of the Differentially Phosphorylated Proteins
2.7. The Abundance of Transcript from Genes Encoding Differentially Phosphorylated Proteins
2.8. Protein-Protein Interactions Involving Differentially Phosphorylated Proteins
3. Discussion
3.1. Protein Kinases/Phosphatases Participated in Signal Perception and Transduction
3.2. Phosphorylated Transcription Factors Related to Stress Response and Defense
3.3. Phosphoproteins Classified as Stress-Related Proteins
3.4. ROS-Related Phosphoproteins
3.5. Phosphoproteins Involved in Water and Ion Transport
4. Materials and Methods
4.1. Rice Materials and Plant Growth Conditions
4.2. Plant Growth and Chlorophyll Content Analysis
4.3. Determination of Shoot Cd Contents
4.4. RNA Extraction and qRT-PCR Analysis
4.5. Sample Preparation and iTRAQ Labeling
4.6. Enrichment for Phosphorylated Peptides
4.7. Liquid Chromatography Tandem-Mass Spectrometry (LC-MS/MS) Analysis
4.8. Phosphopeptide and Phosphosite Identification
4.9. Bioinformatics
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
- Sanita di Toppi, L.; Gabbrielli, R. Response to cadmium in higher plants. Environ. Exp. Bot. 1999, 41, 105–130. [Google Scholar] [CrossRef]
- Prasad, M.N.V. Cadmium toxicity and tolerance in vascular plants. Environ. Exp. Bot. 1995, 35, 525–545. [Google Scholar] [CrossRef]
- Das, P.; Samantaray, S.; Rout, G.R. Studies on cadmium toxicity in plants: A review. Environ. Pollut. 1997, 98, 29–36. [Google Scholar] [CrossRef]
- Hernandez, L.E.; Carpena-Ruiz, R.; Garate, A. Alterations in the mineral nutrition of pea seedlings exposed to cadmium. J. Plant Nutr. 1996, 19, 1581–1598. [Google Scholar] [CrossRef]
- Barceló, J.; Poschenrieder, C. Plant water relations as affected by heavy metal stress: A review. J. Plant Nutr. 1990, 13, 1–37. [Google Scholar] [CrossRef]
- Kesseler, A.; Brand, M.D. The mechanism of the stimulation of state 4 respiration by cadmium in potato tuber (Solanum tuberosum) mitochondria. Plant Physiol. Biochem. 1995, 33, 519–528. [Google Scholar]
- Obata, H.; Inoue, N.; Umebayashi, M. Effect of Cd on plasma membrane ATPase from plant roots differing in tolerance to Cd. Soil Sci. Plant Nutr. 1996, 42, 361–366. [Google Scholar]
- Hendry, G.A.F.; Baker, A.J.M.; Ewart, C.F. Cadmium tolerance and toxicity, oxygen radical processes and molecular damage in cadmium-tolerant and cadmium-sensitive clones of Holcus lanatus L. Plant Biol. 1992, 41, 271–281. [Google Scholar]
- Hall, J.L. Cellular mechanisms for heavy metal detoxification and tolerance. J. Exp. Bot. 2002, 53, 1–11. [Google Scholar] [CrossRef] [PubMed]
- Cobbett, C.S. Phytochelatin biosynthesis and function in heavy-metal detoxification. Curr. Opin. Plant Biol. 2000, 3, 211–216. [Google Scholar] [CrossRef]
- Yamaguchi, H.; Fukuoka, H.; Arao, T.; Ohyama, A.; Nunome, T.; Miyatake, K.; Negoro, S. Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum. J. Exp. Bot. 2010, 61, 423–437. [Google Scholar] [CrossRef] [PubMed]
- Uraguchi, S.; Kamiya, T.; Sakamoto, T.; Kasai, K.; Sato, Y.; Nagamura, Y.; Yoshida, A.; Kyozuka, J.; Ishikawa, S.; Fujiwara, T. Low-affinity cation transporter (OsLCT1) regulates cadmium transport into rice grains. Proc. Natl. Acad. Sci. USA 2011, 108, 20959–20964. [Google Scholar] [CrossRef] [PubMed]
- He, L.; Ma, X.; Li, Z.; Jiao, Z.; Li, Y.; Ow, D.W. Maize OXIDATIVE STRESS2 Homologs Enhance Cadmium Tolerance in Arabidopsis through Activation of a Putative SAM-Dependent Methyltransferase Gene. Plant Physiol. 2016, 171, 1675–1685. [Google Scholar] [CrossRef] [PubMed]
- Zhou, Q.; Guo, J.J.; He, C.T.; Shen, C.; Huang, Y.Y.; Chen, J.X.; Guo, J.H.; Yuan, J.G.; Yang, Z.Y. Comparative Transcriptome Analysis between Low- and High-Cadmium-Accumulating Genotypes of Pakchoi (Brassica chinensis L.) in Response to Cadmium Stress. Environ. Sci. Technol. 2016, 50, 6485–6494. [Google Scholar] [CrossRef] [PubMed]
- Daud, M.K.; Quiling, H.; Lei, M.; Ali, B.; Zhu, S.J. Ultrastructural, metabolic and proteomic changes in leaves of upland cotton in response to cadmium stress. Chemosphere 2015, 120, 309–320. [Google Scholar] [CrossRef] [PubMed]
- Wang, Y.; Xu, L.; Shen, H.; Wang, J.; Liu, W.; Zhu, X.; Wang, R.; Sun, X.; Liu, L. Metabolomic analysis with GC-MS to reveal potential metabolites and biological pathways involved in Pb & Cd stress response of radish roots. Sci. Rep. 2015, 5, 18296. [Google Scholar] [PubMed]
- Reinders, J.; Sickmann, A. State-of-the-art in phosphoproteomics. Proteomics 2005, 5, 4052–4061. [Google Scholar] [CrossRef] [PubMed]
- Reddy, G.N.; Prasad, M.N.V. Cadmium-Induced Protein-Phosphorylation Changes in Rice (Oryza-Sativa L.) Seedlings. J. Plant Physiol. 1995, 145, 67–70. [Google Scholar] [CrossRef]
- Eraso, P.; Martinez-Burgos, M.; Falcon-Perez, J.M.; Portillo, F.; Mazon, M.J. Ycf1-dependent cadmium detoxification by yeast requires phosphorylation of residues Ser908 and Thr911. FEBS Lett. 2004, 577, 322–326. [Google Scholar] [CrossRef] [PubMed]
- Zhu, J.; Wang, W.S.; Ma, D.; Zhang, L.Y.; Ren, F.; Yuan, T.T. A role for CK2 beta subunit 4 in the regulation of plant growth, cadmium accumulation and H2O2 content under cadmium stress in Arabidopsis thaliana. Plant Physiol. Biochem. 2016, 109, 240–247. [Google Scholar] [CrossRef] [PubMed]
- Raichaudhuri, A. Arabidopsis thaliana MRP1 (AtABCC1) nucleotide binding domain contributes to arsenic stress tolerance with serine triad phosphorylation. Plant. Physiol. Biochem. 2016, 108, 109–120. [Google Scholar] [CrossRef] [PubMed]
- Zhang, M.; Lv, D.; Ge, P.; Bian, Y.; Chen, G.; Zhu, G.; Li, X.; Yan, Y. Phosphoproteome analysis reveals new drought response and defense mechanisms of seedling leaves in bread wheat (Triticum aestivum L.). J. Proteom. 2014, 109, 290–308. [Google Scholar] [CrossRef] [PubMed]
- Qiu, J.; Hou, Y.; Wang, Y.; Li, Z.; Zhao, J.; Tong, X.; Lin, H.; Wei, X.; Ao, H.; Zhang, J. A Comprehensive Proteomic Survey of ABA-Induced Protein Phosphorylation in Rice (Oryza sativa L.). Int. J. Mol. Sci. 2017, 18, 60. [Google Scholar] [CrossRef] [PubMed]
- Hou, Y.; Qiu, J.; Wang, Y.; Li, Z.; Zhao, J.; Tong, X.; Lin, H.; Zhang, J. A Quantitative Proteomic Analysis of Brassinosteroid-induced Protein Phosphorylation in Rice (Oryza sativa L.). Front. Plant. Sci. 2017, 8, 514. [Google Scholar] [CrossRef] [PubMed]
- Yu, B.; Li, J.; Koh, J.; Dufresne, C.; Yang, N.; Qi, S.; Zhang, Y.; Ma, C.; Duong, B.V.; Chen, S.; et al. Quantitative proteomics and phosphoproteomics of sugar beet monosomic addition line M14 in response to salt stress. J. Proteom. 2016, 143, 286–297. [Google Scholar] [CrossRef] [PubMed]
- Hu, X.; Wu, L.; Zhao, F.; Zhang, D.; Li, N.; Zhu, G.; Li, C.; Wang, W. Phosphoproteomic analysis of the response of maize leaves to drought, heat and their combination stress. Front. Plant Sci. 2015, 6, 298. [Google Scholar] [CrossRef] [PubMed]
- Hou, Y.; Qiu, J.; Tong, X.; Wei, X.; Nallamilli, B.R.; Wu, W.; Huang, S.; Zhang, J. A comprehensive quantitative phosphoproteome analysis of rice in response to bacterial blight. BMC Plant Biol. 2015, 15, 163. [Google Scholar] [CrossRef] [PubMed]
- Lv, D.W.; Li, X.; Zhang, M.; Gu, A.Q.; Zhen, S.M.; Wang, C.; Li, X.H.; Yan, Y.M. Large-scale phosphoproteome analysis in seedling leaves of Brachypodium distachyon L. BMC Genom. 2014, 15, 375. [Google Scholar] [CrossRef] [PubMed]
- Yu, C.S.; Chen, Y.C.; Lu, C.H.; Hwang, J.K. Prediction of protein subcellular localization. Proteins 2006, 64, 643–651. [Google Scholar] [CrossRef] [PubMed]
- Yu, C.S.; Lin, C.J.; Hwang, J.K. Predicting subcellular localization of proteins for Gram-negative bacteria by support vector machines based on n-peptide compositions. Protein Sci. 2004, 13, 1402–1406. [Google Scholar] [CrossRef] [PubMed]
- Van Wijk, K.J.; Friso, G.; Walther, D.; Schulze, W.X. Meta-Analysis of Arabidopsis thaliana Phospho-Proteomics Data Reveals Compartmentalization of Phosphorylation Motifs. Plant Cell 2014, 26, 2367–2389. [Google Scholar] [CrossRef] [PubMed]
- Wang, K.; Zhao, Y.; Li, M.; Gao, F.; Yang, M.K.; Wang, X.; Li, S.; Yang, P. Analysis of phosphoproteome in rice pistil. Proteomics 2014, 14, 2319–2334. [Google Scholar] [CrossRef] [PubMed]
- Zhang, M.; Ma, C.Y.; Lv, D.W.; Zhen, S.M.; Li, X.H.; Yan, Y.M. Comparative phosphoproteome analysis of the developing grains in bread wheat (Triticum aestivum L.) under well-watered and water-deficit conditions. J. Proteome Res. 2014, 13, 4281–4297. [Google Scholar] [CrossRef] [PubMed]
- Chae, M.J.; Lee, J.S.; Nam, M.H.; Cho, K.; Hong, J.Y.; Yi, S.A.; Suh, S.C.; Yoon, I.S. A rice dehydration-inducible SNF1-related protein kinase 2 phosphorylates an abscisic acid responsive element-binding factor and associates with ABA signaling. Plant Mol. Biol. 2007, 63, 151–169. [Google Scholar] [CrossRef] [PubMed]
- Ning, J.; Li, X.; Hicks, L.M.; Xiong, L. A Raf-Like MAPKKK Gene DSM1 Mediates Drought Resistance through Reactive Oxygen Species Scavenging in Rice. Plant Physiol. 2010, 152, 876–890. [Google Scholar] [CrossRef] [PubMed]
- Stroiński, A.; Chadzinikolau, T.; Giżewska, K.; Zielezińska, M. ABA or cadmium induced phytochelatin synthesis in potato tubers. Biol. Plant 2010, 54, 117–120. [Google Scholar] [CrossRef]
- Hsu, Y.T.; Kao, C.H. Role of abscisic acid in cadmium tolerance of rice (Oryza. sativa L.) seedlings. Plant Cell Environ. 2003, 26, 867–874. [Google Scholar] [CrossRef] [PubMed]
- Fediuc, E.; Lips, S.H.; Erdei, L. O-acetylserine (thiol) lyase activity in Phragmites and Typha plants under cadmium and NaCl stress conditions and the involvement of ABA in the stress response. J. Plant Physiol. 2005, 162, 865–872. [Google Scholar] [CrossRef] [PubMed]
- Stroiński, A.; Giżewska, K. Zielezińska, M. Abscisic acid is required in transduction of cadmium signal to potato roots. Biol. Plant. 2013, 57, 121–127. [Google Scholar] [CrossRef]
- Park, S.Y.; Fung, P.; Nishimura, N.; Jensen, D.R.; Fujii, H.; Zhao, Y.; Lumba, S.; Santiago, J.; Rodrigues, A.; Chow, T.F. Abscisic acid inhibits PP2Cs via the PYR/PYL family of ABA-binding START proteins. Science 2009, 324, 1068–1071. [Google Scholar] [PubMed]
- Joo, J.; Lee, Y.H.; Kim, Y.-K.; Nahm, B.H.; Song, S.I. Abiotic stress responsive rice ASR1 and ASR3 exhibit different tissue-dependent sugar and hormone-sensitivities. Mol. Cells 2013, 35, 421–435. [Google Scholar] [CrossRef] [PubMed]
- Arenhart, R.A.; De Lima, J.C.; Pedron, M.; Carvalho, F.E.L.; Da Silveira, J.A.G.; Rosa, S.B.; Caverzan, A.; Andrade, C.M.B.; SchÜNemann, M.; Margis, R.; et al. Involvement of ASR genes in aluminium tolerance mechanisms in rice. Plant Cell Environ. 2013, 36, 52–67. [Google Scholar] [CrossRef] [PubMed]
- Ganguly, M.; Datta, K.; Roychoudhury, A.; Gayen, D.; Sengupta, D.N.; Datta, S.K. Overexpression of Rab16A gene in indica rice variety for generating enhanced salt tolerance. Plant Signal. Behav. 2012, 7, 502–509. [Google Scholar] [CrossRef] [PubMed]
- Abbasi, F.; Onodera, H.; Toki, S.; Tanaka, H.; Komatsu, S. OsCDPK13, a calcium-dependent protein kinase gene from rice, is induced by cold and gibberellin in rice leaf sheath. Plant Mol. Biol. 2004, 55, 541–552. [Google Scholar] [CrossRef] [PubMed]
- Zhao, L.; Li, Y.; Xie, Q.; Wu, Y. Loss of CDKC;2 increases both cell division and drought tolerance in Arabidopsis thaliana. Plant J. 2017, 91, 816–828. [Google Scholar] [CrossRef] [PubMed]
- Jonak, C.; Nakagami, H.; Hirt, H. Heavy metal stress. Activation of distinct mitogen-activated protein kinase pathways by copper and cadmium. Plant Physiol. 2004, 136, 3276–3283. [Google Scholar] [CrossRef] [PubMed]
- Gao, G.; Zhong, Y.; Guo, A.; Zhu, Q.; Tang, W.; Zheng, W.; Gu, X.; Wei, L.; Luo, J. DRTF: A database of rice transcription factors. Bioinformatics 2006, 22, 1286–1287. [Google Scholar] [CrossRef] [PubMed]
- Shen, H.; Liu, C.; Zhang, Y.; Meng, X.; Zhou, X.; Chu, C.; Wang, X. OsWRKY30 is activated by MAP kinases to confer drought tolerance in rice. Plant Mol. Biol. 2012, 80, 241–253. [Google Scholar] [CrossRef] [PubMed]
- Zhao, H.; Wang, S.; Chen, S.; Jiang, J.; Liu, G. Phylogenetic and stress-responsive expression analysis of 20 WRKY genes in Populus simonii x Populus nigra. Gene 2015, 565, 130–139. [Google Scholar] [CrossRef] [PubMed]
- Hong, C.; Cheng, D.; Zhang, G.; Zhu, D.; Chen, Y.; Tan, M. The role of ZmWRKY4 in regulating maize antioxidant defense under cadmium stress. Biochem. Biophys. Res. Commun. 2017, 482, 1504–1510. [Google Scholar] [CrossRef] [PubMed]
- Yang, G.; Wang, C.; Wang, Y.; Guo, Y.; Zhao, Y.; Yang, C.; Gao, C. Overexpression of ThVHAc1 and its potential upstream regulator, ThWRKY7, improved plant tolerance of Cadmium stress. Sci. Rep. 2016, 6, 18752. [Google Scholar] [CrossRef] [PubMed]
- Ashwini, N.; Sajeevan, R.S.; Udayakumar, M.; Nataraja, K.N. Identification and Characterization of OsWRKY72 Variant in Indica Genotypes. Rice Sci. 2016, 23, 297–305. [Google Scholar] [CrossRef]
- Takahashi, Y.; Ebisu, Y.; Kinoshita, T.; Doi, M.; Okuma, E.; Murata, Y.; Shimazaki, K. bHLH transcription factors that facilitate K+ uptake during stomatal opening are repressed by abscisic acid through phosphorylation. Sci. Signal. 2013, 23, 297–305. [Google Scholar] [CrossRef] [PubMed]
- Jan, A.; Maruyama, K.; Todaka, D.; Kidokoro, S.; Abo, M.; Yoshimura, E.; Shinozaki, K.; Nakashima, K.; Yamaguchi-Shinozaki, K. OsTZF1, a CCCH-tandem zinc finger protein, confers delayed senescence and stress tolerance in rice by regulating stress-related genes. Plant Physiol. 2013, 161, 1202–1216. [Google Scholar] [CrossRef] [PubMed]
- Kanneganti, V.; Gupta, A.K. Overexpression of OsiSAP8, a member of stress associated protein (SAP) gene family of rice confers tolerance to salt, drought and cold stress in transgenic tobacco and rice. Plant Mol. Biol. 2008, 66, 445–462. [Google Scholar] [CrossRef] [PubMed]
- Ben Saad, R.; Zouari, N.; Ben Ramdhan, W.; Azaza, J.; Meynard, D.; Guiderdoni, E.; Hassairi, A. Improved drought and salt stress tolerance in transgenic tobacco overexpressing a novel A20/AN1 zinc-finger “AlSAP” gene isolated from the halophyte grass Aeluropus littoralis. Plant Mol. Biol. 2010, 72, 171–190. [Google Scholar] [CrossRef] [PubMed]
- Bao, Y.-M.; Wang, J.-F.; Huang, J.; Zhang, H.-S. Molecular cloning and characterization of a novel SNAP25-type protein gene OsSNAP32 in rice (Oryza sativa L.). Mol. Biol. Rep. 2008, 35, 145–152. [Google Scholar] [CrossRef] [PubMed]
- Martinoia, E.; Klein, M.; Geisler, M.; Bovet, L.; Forestier, C.; Kolukisaoglu, U.; Muller-Rober, B.; Schulz, B. Multifunctionality of plant ABC transporters—More than just detoxifiers. Planta 2002, 214, 345–355. [Google Scholar] [CrossRef] [PubMed]
- Hartl, F.U.; Bracher, A.; Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 2011, 475, 324–332. [Google Scholar] [CrossRef] [PubMed]
- Lv, D.W.; Subburaj, S.; Cao, M.; Yan, X.; Li, X.; Appels, R.; Sun, D.F.; Ma, W.; Yan, Y.M. Proteome and phosphoproteome characterization reveals new response and defense mechanisms of Brachypodium distachyon leaves under salt stress. Mol. Cell. Proteom. 2014, 13, 632–652. [Google Scholar] [CrossRef] [PubMed]
- Lyzenga, W.J.; Stone, S.L. Abiotic stress tolerance mediated by protein ubiquitination. J. Exp. Bot. 2012, 63, 599–616. [Google Scholar] [CrossRef] [PubMed]
- Clemens, S. Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants. Biochimie 2006, 88, 1707–1719. [Google Scholar] [CrossRef] [PubMed]
- Gill, S.S.; Tuteja, N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 2010, 48, 909–930. [Google Scholar] [CrossRef] [PubMed]
- Kargul, J.; Barber, J. Photosynthetic acclimation: Structural reorganisation of light harvesting antenna—Role of redox-dependent phosphorylation of major and minor chlorophyll a/b binding proteins. FEBS J. 2008, 275, 1056–1068. [Google Scholar] [CrossRef] [PubMed]
- Ceccarelli, E.A.; Arakaki, A.K.; Cortez, N.; Carrillo, N. Functional plasticity and catalytic efficiency in plant and bacterial ferredoxin-NADP(H) reductases. Biochim. Biophys. Acta 2004, 1698, 155–165. [Google Scholar] [CrossRef] [PubMed]
- Maria, P.B.; Susana, M.G.; Maria, L.T. Cadmium toxicity in plants. Braz. J. Plant Physiol. 2005, 17, 21–34. [Google Scholar]
- Gomes, D.; Agasse, A.; Thiébaud, P.; Delrot, S.; Gerós, H.; Chaumont, F. Aquaporins are multifunctional water and solute transporters highly divergent in living organisms. Biochim. Biophys. Acta Biomembr. 2009, 1788, 1213–1228. [Google Scholar] [CrossRef] [PubMed]
- Hanikenne, M.; Motte, P.; Wu, M.C.S.; Wang, T.; Loppes, R.; Matagne, R.F. A mitochondrial half-size ABC transporter is involved in cadmium tolerance in Chlamydomonas reinhardtii. Plant Cell. Environ. 2010, 28, 863–873. [Google Scholar] [CrossRef]
- Wang, K. Comparative study on Cd phytotoxicity to different genes of rice. Rural Eco Environ. 1996, 12, 18–23. [Google Scholar]
- Gong, J.M.; Lee, D.A.; Schroeder, J.I. Long-distanc root-to-shoot transport of phytochelatins and cadmium in Arabidopsis. Proc. Natl. Acad. Sci. USA 2003, 100, 10118–10123. [Google Scholar] [CrossRef] [PubMed]
- Wang, W.; Scali, M.; Vignani, R.; Spadafora, A.; Sensi, E.; Mazzuca, S.; Cresti, M. Protein extraction for two-dimensional electrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds. Electrophoresis 2003, 24, 2369–2375. [Google Scholar] [CrossRef] [PubMed]
- Du, Z.; Zhou, X.; Ling, Y.; Zhang, Z.; Su, Z. agriGO: A GO analysis toolkit for the agricultural community. Nucleic Acids Res. 2010, 38, W64–W70. [Google Scholar] [CrossRef] [PubMed]
© 2017 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 (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Zhong, M.; Li, S.; Huang, F.; Qiu, J.; Zhang, J.; Sheng, Z.; Tang, S.; Wei, X.; Hu, P. The Phosphoproteomic Response of Rice Seedlings to Cadmium Stress. Int. J. Mol. Sci. 2017, 18, 2055. https://doi.org/10.3390/ijms18102055
Zhong M, Li S, Huang F, Qiu J, Zhang J, Sheng Z, Tang S, Wei X, Hu P. The Phosphoproteomic Response of Rice Seedlings to Cadmium Stress. International Journal of Molecular Sciences. 2017; 18(10):2055. https://doi.org/10.3390/ijms18102055
Chicago/Turabian StyleZhong, Min, Sanfeng Li, Fenglin Huang, Jiehua Qiu, Jian Zhang, Zhonghua Sheng, Shaoqing Tang, Xiangjin Wei, and Peisong Hu. 2017. "The Phosphoproteomic Response of Rice Seedlings to Cadmium Stress" International Journal of Molecular Sciences 18, no. 10: 2055. https://doi.org/10.3390/ijms18102055