A Novel Aging-Related Prognostic lncRNA Signature Correlated with Immune Cell Infiltration and Response to Immunotherapy in Breast Cancer
Abstract
:1. Introduction
2. Results
2.1. Differential Expression of Age-Related lncRNA Identification
2.2. The Construction of Age-Related lncRNA Signature and Verification
2.3. Construction of a Nomogram of BC Patients
2.4. Correlation of the Risk Score with Clinicopathological Features
2.5. Differential Analysis of Signaling Pathway and Immune Functions in the Groups with High Risk and Low Risk
2.6. Analysis of Immune Cell Infiltration
2.7. Genetic Alteration in Aging-Related Genes
2.8. Immunotherapy Effect and Drug Sensitivity
3. Discussion
4. Materials and Methods
4.1. Data and Clinical Information Acquisition and Collation
4.2. Identification and Differential Analysis of Aging-Related lncRNAs
4.3. Construction and Validation of the Aging-Related lncRNA Prognostic Signature
4.4. Development and Evaluation of a Nomogram in BC Patients
4.5. Kyoto Encyclopedia of Genes and Genomes, Gene Ontology, Gene Set Enrichment Analysis and Single-Sample Gene Set Enrichment Analysis
4.6. Immune Cell Infiltration Analysis
4.7. Immunotherapy and Drug Sensitivity Prediction
4.8. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics, 2020. CA Cancer J. Clin. 2020, 70, 7–30. [Google Scholar] [CrossRef] [PubMed]
- Zhao, S.; Liu, X.Y.; Jin, X.; Ma, D.; Xiao, Y.; Shao, Z.M.; Jiang, Y.Z. Molecular portraits and trastuzumab responsivenessofestrogen receptor-positive, progesterone receptor-positive, and HER2-positivebreast cancer. Theranostics 2019, 9, 4935–4945. [Google Scholar] [CrossRef] [PubMed]
- Wang, X.; Sun, C.; Huang, X.; Li, J.; Fu, Z.; Li, W.; Yin, Y. The advancing roles of exosomes in breast cancer. Front. Cell Dev. Biol. 2021, 9, 731062. [Google Scholar] [CrossRef] [PubMed]
- Carroll, J.E.; Van Dyk, K.; Bower, J.E.; Scuric, Z.; Petersen, L.; Schiestl, R.; Irwin, M.; Ganz, P.A. Cognitive performance in survivors of breast cancer and markers of biological aging. Cancer 2018, 125, 298–306. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Smetana, K.; Lacina, L.; Szabo, P.; Dvořánková, B.; Brož, P.; Šedo, A. Ageing as an Important Risk Factor for Cancer. Anticancer. Res. 2016, 36, 5009–5017. [Google Scholar] [CrossRef] [Green Version]
- Gu, X.; Wang, B.; Zhu, H.; Zhou, Y.; Horning, A.M.; Huang, T.H.-M.; Chen, Y.; Houghton, P.; Lai, Z.; Michalek, J.E.; et al. Age-associated genes in human mammary gland drive human breast cancer progression. Breast Cancer Res. 2020, 22, 1–15. [Google Scholar] [CrossRef]
- Ren, S.; Lin, P.; Wang, J.; Yu, H.; Lv, T.; Sun, L.; Du, G. Circular RNAs, Promising Molecular Biomarkers of Human Aging-Related Diseases via Functioning as an miRNA Sponge. Mol. Ther.-Methods Clin. Dev. 2020, 18, 215–229. [Google Scholar] [CrossRef]
- Yue, T.; Chen, S.; Zhu, J.; Guo, S.; Huang, Z.; Wang, P.; Zuo, S.; Liu, Y. The aging-related risk signature in colorectal cancer. Aging 2021, 13, 7330–7349. [Google Scholar] [CrossRef]
- Xu, Q.; Chen, Y. An Aging-Related Gene Signature-Based Model for Risk Stratification and Prognosis Prediction in Lung Adenocarcinoma. Front. Cell Dev. Biol. 2021, 9, 685379. [Google Scholar] [CrossRef]
- Liu, L.; Zhao, J.; Du, X.; Zhao, Y.; Zou, C.; Zhou, H.; Li, W.; Yan, X. Construction and validation of a novel aging-related gene signature and prognostic nomogram for predicting the overall survival in ovarian cancer. Cancer Med. 2021, 10, 9097–9114. [Google Scholar] [CrossRef]
- Ransohoff, J.D.; Wei, Y.; Khavari, P.A. The functions and unique features of long intergenic non-coding RNA. Nat. Rev. Mol. Cell Biol. 2018, 19, 143–157. [Google Scholar] [CrossRef]
- Du, C.; Zhang, J.-L.; Wang, Y.; Zhang, Y.-Y.; Zhang, J.-H.; Zhang, L.-F.; Li, J.-R. The Long Non-coding RNA LINC01705 Regulates the Development of Breast Cancer by Sponging miR-186-5p to Mediate TPR Expression as a Competitive Endogenous RNA. Front. Genet. 2020, 11, 779. [Google Scholar] [CrossRef]
- Wan, Q.; Tang, M.; Sun, S.-L.; Hu, J.; Sun, Z.-J.; Fang, Y.-T.; He, T.-C.; Zhang, Y. SNHG3 promotes migration, invasion, and epithelial-mesenchymal transition of breast cancer cells through the miR-186-5p/ZEB1 axis. Am. J. Transl. Res. 2021, 13, 585–600. [Google Scholar]
- Huang, Y.; Xie, B.; Cao, M.; Lu, H.; Wu, X.; Hao, Q.; Zhou, X. LncRNA RNA Component of Mitochondrial RNA-Processing Endoribonuclease Promotes AKT-Dependent Breast Cancer Growth and Migration by Trapping MicroRNA-206. Front. Cell Dev. Biol. 2021, 9, 2591. [Google Scholar] [CrossRef]
- D’Amico, S.; Krasnowska, E.; Manni, I.; Toietta, G.; Baldari, S.; Piaggio, G.; Ranalli, M.; Gambacurta, A.; Vernieri, C.; Di Giacinto, F.; et al. DHA Affects Microtubule Dynamics Through Reduction of Phospho-TCTP Levels and Enhances the Antiproliferative Effect of T-DM1 in Trastuzumab-Resistant HER2-Positive Breast Cancer Cell Lines. Cells 2020, 9, 1260. [Google Scholar] [CrossRef]
- Srivastava, S.; Gopal-Srivastava, R. Biomarkers in cancer screening: A public health perspective. J Nutr. 2002, 132, 2471S–2475S. [Google Scholar] [CrossRef] [Green Version]
- Xu, Z.; Peng, B.; Liang, Q.; Chen, X.; Cai, Y.; Zeng, S.; Gao, K.; Wang, X.; Yi, Q.; Gong, Z.; et al. Construction of a Ferroptosis-Related Nine-lncRNA Signature for Predicting Prognosis and Immune Response in Hepatocellular Carcinoma. Front Immunol. 2021, 12, 719175. [Google Scholar] [CrossRef]
- Franzoi, M.A.; Romano, E.; Piccart, M. Immunotherapy for early breast cancer, Too soon, too superficial, or just right? Ann. Oncol. 2021, 32, 323–336. [Google Scholar] [CrossRef]
- Yi, M.; Li, A.; Zhou, L.; Chu, Q.; Luo, S.; Wu, K. Immune signature-based risk stratification and prediction of immune checkpoint inhibitor’s efficacy for lung adenocarcinoma. Cancer Immunol. Immunother. 2021, 70, 1705–1719. [Google Scholar] [CrossRef]
- Siegel, R.L.; Miller, K.D.; Fuchs, H.E.; Jemal, A. Cancer Statistics, 2021. CA Cancer J Clin. 2021, 71, 7–33. [Google Scholar] [CrossRef]
- Sachs, N.; de Ligt, J.; Kopper, O.; Gogola, E.; Bounova, G.; Weeber, F.; Balgobind, A.V.; Wind, K.; Gracanin, A.; Begthel, H.; et al. A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity. Cell 2018, 172, 373–386.e10. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ye, R.; Tang, R.; Gan, S.; Li, R.; Cheng, Y.; Guo, L.; Zeng, C.; Sun, Y. New insights into long non-coding RNAs in non-small cell lung cancer. Biomed Pharmacother. 2020, 131, 110775. [Google Scholar] [CrossRef] [PubMed]
- Wang, M.-Q.; Zhu, W.-J.; Gao, P. New insights into long non-coding RNAs in breast cancer, Biological functions and therapeutic prospects. Exp. Mol. Pathol. 2021, 120, 104640. [Google Scholar] [CrossRef] [PubMed]
- Calcinotto, A.; Kohli, J.; Zagato, E.; Pellegrini, L.; Demaria, M.; Alimonti, A. Cellular Senescence, Aging, Cancer, and Injury. Physiol. Rev. 2019, 99, 1047–1078. [Google Scholar] [CrossRef] [PubMed]
- Mosteiro, L.; Pantoja, C.; Alcazar, N.; Marión, R.M.; Chondronasiou, D.; Rovira, M.; Fernandez-Marcos, P.J.; Muñoz-Martin, M.; Blanco-Aparicio, C.; Pastor, J.; et al. Tissue damage and senescence provide critical signals for cellular reprogramming in vivo. Science 2016, 354, aaf4445. [Google Scholar] [CrossRef]
- Lee, S.; Schmitt, C.A. The dynamic nature of senescence in cancer. Nat. Cell Biol. 2019, 21, 94–101. [Google Scholar] [CrossRef]
- Lv, W.; Zhao, C.; Tan, Y.; Hu, W.; Yu, H.; Zeng, N.; Zhang, Q.; Wu, Y. Identification of an Aging-Related Gene Signature in Predicting Prognosis and Indicating Tumor Immune Microenvironment in Breast Cancer. Front. Oncol. 2021, 11, 5320. [Google Scholar] [CrossRef]
- Kong, W.; Li, H.; Xie, L.; Cui, G.; Gu, W.; Zhang, H.; Ma, W.; Zhou, Y. LncRNA MCF2L-AS1 aggravates the malignant development of colorectal cancer via targeting miR-105-5p/RAB22A axis. BMC Cancer 2021, 21, 1069. [Google Scholar] [CrossRef]
- Li, S.; Lin, L. Long noncoding RNA MCF2L-AS1 promotes the cancer stem cell-like traits in non-small cell lung cancer cells through regulating miR-873-5p level. Environ Toxicol. 2021, 36, 1457–1465. [Google Scholar] [CrossRef]
- Wang, N.; Li, J.; Xin, Q.; Xu, N. USP30-AS1 contributes to mitochondrial quality control in glioblastoma cells. Biochem. Biophys. Res. Commun. 2021, 581, 31–37. [Google Scholar] [CrossRef]
- Sun, Z.; Jing, C.; Xiao, C.; Li, T. An autophagy-related long non-coding RNA prognostic signature accurately predicts survival outcomes in bladder urothelial carcinoma patients. Aging 2020, 12, 15624–15637. [Google Scholar] [CrossRef]
- Gao, M.; Wang, X.; Han, D.; Lu, E.; Zhang, J.; Zhang, C.; Wang, L.; Yang, Q.; Jiang, Q.; Wu, J.; et al. A Six-lncRNA Signature for Immunophenotype Prediction of Glioblastoma Multiforme. Front Genet. 2021, 11, 604655. [Google Scholar] [CrossRef]
- Kong, S.; Xue, H.; Li, Y.; Li, P.; Ma, F.; Liu, M.; Li, W. The long noncoding RNA OTUD6B-AS1 enhances cell proliferation and the invasion of hepatocellular carcinoma cells through modulating GSKIP/Wnt/β-catenin signalling via the sequestration of miR-664b-3p. Exp. Cell Res. 2020, 395, 112180. [Google Scholar] [CrossRef]
- Lv, W.; Wang, Y.; Zhao, C.; Tan, Y.; Xiong, M.; Yi, Y.; He, X.; Ren, Y.; Wu, Y.; Zhang, Q. Identification and Validation of m6A-Related lncRNA Signature as Potential Predictive Biomarkers in Breast Cancer. Front. Oncol. 2021, 11, 745719. [Google Scholar] [CrossRef]
- Ma, W.; Zhao, F.; Yu, X.; Guan, S.; Suo, H.; Tao, Z.; Qiu, Y.; Wu, Y.; Cao, Y.; Jin, F. Immune-related lncRNAs as predictors of survival in breast cancer: A prognostic signature. J. Transl. Med. 2020, 18, 442. [Google Scholar] [CrossRef]
- Pan, Y.; Pan, Y.; Cheng, Y.; Yang, F.; Yao, Z.; Wang, O. Knockdown of LncRNA MAPT-AS1 inhibites proliferation and migration and sensitizes cancer cells to paclitaxel by regulating MAPT expression in ER-negative breast cancers. Cell Biosci. 2018, 8, 7. [Google Scholar] [CrossRef] [Green Version]
- Wang, D.; Li, J.; Cai, F.; Xu, Z.; Li, L.; Zhu, H.; Liu, W.; Xu, Q.; Cao, J.; Sun, J.; et al. Overexpression of MAPT-AS1 is associated with better patient survival in breast cancer. Biochem. Cell Biol. 2019, 97, 158–164. [Google Scholar] [CrossRef]
- Yang, X.; Song, D.; Zhang, J.; Feng, H.; Guo, J. PRR34-AS1 sponges miR-498 to facilitate TOMM20 and ITGA6 mediated tumor progression in HCC. Exp. Mol. Pathol. 2021, 120, 104620. [Google Scholar] [CrossRef]
- Kesherwani, V.; Shukla, M.; Coulter, D.W.; Sharp, J.G.; Joshi, S.S.; Chaturvedi, N.K. Long non-coding RNA profiling of pediatric Medulloblastoma. BMC Med. Genom. 2020, 13, 87. [Google Scholar] [CrossRef]
- Lin, Y.; Jian, Z.; Jin, H.; Wei, X.; Zou, X.; Guan, R.; Huang, J. Long non-coding RNA DLGAP1-AS1 facilitates tumorigenesis and epithelial-mesenchymal transition in hepatocellular carcinoma via the feedback loop of miR-26a/b-5p/IL-6/JAK2/STAT3 and Wnt/β-catenin pathway. Cell Death Dis. 2020, 11, 34. [Google Scholar] [CrossRef] [Green Version]
- Wang, Z.; Han, Y.; Li, Q.; Wang, B.; Ma, J. LncRNA DLGAP1-AS1 accelerates glioblastoma cell proliferation through targeting miR-515-5p/ROCK1/NFE2L1 axis and activating Wnt signaling pathway. Brain Behav. 2021, 11, e2321. [Google Scholar] [CrossRef] [PubMed]
- Zhao, L.; Zhang, Y.; Gao, Y.; Geng, P.; Lu, Y.; Liu, X.; Yao, R.; Hou, P.; Liu, D.; Lu, J.; et al. JMJD3 promotes SAHF formation in senescent WI38 cells by triggering an interplay between demethylation and phosphorylation of RB protein. Cell Death Differ. 2015, 22, 1630–1640. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Taube, J.M.; Galon, J.; Sholl, L.M.; Rodig, S.J.; Cottrell, T.R.; A Giraldo, N.; Baras, A.S.; Patel, S.S.; A Anders, R.; Rimm, D.L.; et al. Implications of the tumor immune microenvironment for staging and therapeutics. Mod. Pathol. 2017, 31, 214–234. [Google Scholar] [CrossRef] [Green Version]
- Sebestyen, Z.; Prinz, I.; Déchanet-Merville, J.; Silva-Santos, B.; Kuball, J. Translating gammadelta (γδ) T cells and their receptors into cancer cell therapies. Nat. Rev. Drug Discov. 2020, 19, 169–184. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Yi, M.; Jiao, D.; Xu, H.; Liu, Q.; Zhao, W.; Han, X.; Wu, K. Biomarkers for predicting efficacy of PD-1/PD-L1 inhibitors. Mol. Cancer 2018, 17, 129. [Google Scholar] [CrossRef]
- Charoentong, P.; Finotello, F.; Angelova, M.; Mayer, C.; Efremova, M.; Rieder, D.; Hackl, H.; Trajanoski, Z. Pan-cancer Immunogenomic Analyses Reveal Genotype-Immunophenotype Relationships and Predictors of Response to Checkpoint Blockade. Cell Rep. 2017, 18, 248–262. [Google Scholar] [CrossRef] [Green Version]
- Yates, A.D.; Achuthan, P.; Akanni, W.; Allen, J.; Allen, J.; Alvarez-Jarreta, J.; Amode, M.R.; Armean, I.M.; Azov, A.G.; Bennett, R.; et al. Ensembl 2020. Nucleic Acids Res. 2020, 48, D682–D688. [Google Scholar] [CrossRef]
- Schober, P.; Boer, C.; Schwarte, L.A. Correlation Coefficients, Appropriate Use and Interpretation. Anesth. Analg. 2018, 126, 1763–1768. [Google Scholar] [CrossRef]
- Bian, Z.; Fan, R.; Xie, L. A Novel Cuproptosis-Related Prognostic Gene Signature and Validation of Differential Expression in Clear Cell Renal Cell Carcinoma. Genes 2022, 13, 851. [Google Scholar] [CrossRef]
- Van De Vijver, M.J.; He, Y.D.; Van’t Veer, L.J.; Dai, H.; Hart, A.A.; Voskuil, D.W.; Schreiber, G.J.; Peterse, J.L.; Roberts, C.; Marton, M.J.; et al. A gene-expression signature as a predictor of survival in breast cancer. N. Engl. J. Med. 2002, 347, 1999–2009. [Google Scholar] [CrossRef] [Green Version]
- Liu, G.-M.; Zeng, H.-D.; Zhang, C.-Y.; Xu, J.-W. Identification of a six-gene signature predicting overall survival for hepatocellular carcinoma. Cancer Cell Int. 2019, 19, 138. [Google Scholar] [CrossRef] [Green Version]
- Du, J.-X.; Chen, C.; Luo, Y.-H.; Cai, J.-L.; Cai, C.-Z.; Xu, J.; Ni, X.-J.; Zhu, W. Establishment and validation of a novel autophagy-related gene signature for patients with breast cancer. Gene 2020, 762, 144974. [Google Scholar] [CrossRef]
- Drevets, W.C.; Wittenberg, G.M.; Bullmore, E.T.; Manji, H.K. Immune targets for therapeutic development in depression: Towards precision medicine. Nat. Rev. Drug Discov. 2022, 21, 224–244. [Google Scholar] [CrossRef]
- Lu, T.; Xu, R.; Li, Q.; Zhao, J.-Y.; Peng, B.; Zhang, H.; Guo, J.-D.; Zhang, S.-Q.; Li, H.-W.; Wang, J.; et al. Systematic profiling of ferroptosis gene signatures predicts prognostic factors in esophageal squamous cell carcinoma. Mol. Ther.-Oncolytics 2021, 21, 134–143. [Google Scholar] [CrossRef]
- Chen, S.; Yang, D.; Lei, C.; Li, Y.; Sun, X.; Chen, M.; Wu, X.; Zheng, Y. Identification of crucial genes in abdominal aortic aneurysm by WGCNA. PeerJ 2019, 7, e7873. [Google Scholar] [CrossRef] [Green Version]
- Ogrodnik, M. Cellular aging beyond cellular senescence, Markers of senescence prior to cell cycle arrest in vitro and in vivo. Aging Cell 2021, 20, e13338. [Google Scholar] [CrossRef]
- Iasonos, A.; Schrag, D.; Raj, G.V.; Panageas, K.S. How to Build and Interpret a Nomogram for Cancer Prognosis. J. Clin. Oncol. 2008, 26, 1364–1370. [Google Scholar] [CrossRef]
- Van Calster, B.; Wynants, L.; Verbeek, J.F.; Verbakel, J.Y.; Christodoulou, E.; Vickers, A.J.; Roobol, M.J.; Steyerberg, E.W. Reporting and Interpreting Decision Curve Analysis, A Guide for Investigators. Eur. Urol. 2018, 74, 796–804. [Google Scholar] [CrossRef]
- Chen, Z.-A.; Tian, H.; Yao, D.-M.; Zhang, Y.; Feng, Z.-J.; Yang, C.-J. Identification of a Ferroptosis-Related Signature Model Including mRNAs and lncRNAs for Predicting Prognosis and Immune Activity in Hepatocellular Carcinoma. Front. Oncol. 2021, 11, 738477. [Google Scholar] [CrossRef]
- Wang, Y.; Chen, L.; Ju, L.; Qian, K.; Liu, X.; Wang, X.; Xiao, Y. Novel Biomarkers Associated With Progression and Prognosis of Bladder Cancer Identified by Co-expression Analysis. Front. Oncol. 2019, 9, 1030. [Google Scholar] [CrossRef] [Green Version]
- Chen, X.; Lan, H.; He, D.; Xu, R.; Zhang, Y.; Cheng, Y.; Chen, H.; Xiao, S.; Cao, K. Multi-Omics Profiling Identifies Risk Hypoxia-Related Signatures for Ovarian Cancer Prognosis. Front. Immunol. 2021, 12, 645839. [Google Scholar] [CrossRef] [PubMed]
- Fu, Y.; Bao, Q.; Liu, Z.; He, G.; Wen, J.; Liu, Q.; Xu, Y.; Jin, Z.; Zhang, W. Development and Validation of a Hypoxia-Associated Prognostic Signature Related to Osteosarcoma Metastasis and Immune Infiltration. Front. Cell Dev. Biol. 2021, 9, 633607. [Google Scholar] [CrossRef] [PubMed]
- Newman, A.M.; Liu, C.L.; Green, M.R.; Gentles, A.J.; Feng, W.; Xu, Y.; Hoang, C.D.; Diehn, M.; Alizadeh, A.A. Robust enumeration of cell subsets from tissue expression profiles. Nat. Methods 2015, 12, 453–457. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Aslam, M. Clinical laboratory medicine measurements correlation analysis under uncertainty. Ann. Clin. Biochem. Int. J. Biochem. Lab. Med. 2021, 58, 377–383. [Google Scholar] [CrossRef]
- Mayakonda, A.; Lin, D.-C.; Assenov, Y.; Plass, C.; Koeffler, H.P. Maftools: Efficient and comprehensive analysis of somatic variants in cancer. Genome Res. 2018, 28, 1747–1756. [Google Scholar] [CrossRef] [Green Version]
- Geeleher, P.; Cox, N.J.; Huang, R.S. Clinical drug response can be predicted using baseline gene expression levels and in vitro drug sensitivity in cell lines. Genome Biol. 2014, 15, R47. [Google Scholar] [CrossRef] [Green Version]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Liu, Z.; Ren, C.; Cai, J.; Yin, B.; Yuan, J.; Ding, R.; Ming, W.; Sun, Y.; Li, Y. A Novel Aging-Related Prognostic lncRNA Signature Correlated with Immune Cell Infiltration and Response to Immunotherapy in Breast Cancer. Molecules 2023, 28, 3283. https://doi.org/10.3390/molecules28083283
Liu Z, Ren C, Cai J, Yin B, Yuan J, Ding R, Ming W, Sun Y, Li Y. A Novel Aging-Related Prognostic lncRNA Signature Correlated with Immune Cell Infiltration and Response to Immunotherapy in Breast Cancer. Molecules. 2023; 28(8):3283. https://doi.org/10.3390/molecules28083283
Chicago/Turabian StyleLiu, Zhixin, Chongkang Ren, Jinyi Cai, Baohui Yin, Jingjie Yuan, Rongjuan Ding, Wenzhuo Ming, Yunxiao Sun, and Youjie Li. 2023. "A Novel Aging-Related Prognostic lncRNA Signature Correlated with Immune Cell Infiltration and Response to Immunotherapy in Breast Cancer" Molecules 28, no. 8: 3283. https://doi.org/10.3390/molecules28083283