The Pyroptosis-Related Gene Prognostic Index Associated with Tumor Immune Infiltration for Pancreatic Cancer
Abstract
:1. Introduction
2. Results
2.1. Identification and Functional Enrichment Analysis of Pyroptosis-Related DEGs
2.2. Construction of PRGPI
2.3. Evaluation and Validation of PRGPI
2.4. Analysis of Independent Prognostic Factors and Construction and Validation of Nomogram
2.5. Functional Enrichment and Molecular Characteristics Based on the PRGPI
2.6. PRGPI Was Associated with Immune Signatures of PC
2.7. PRGPI May Predict Chemotherapeutics Response
3. Discussion
4. Materials and Methods
4.1. The Datasets and Samples
4.2. Identification of DEGs
4.3. Identification of Key Prognostic Genes and Establishment of the PRGPI
4.4. Prediction and Validation Analysis
4.5. Prognostic Value Assessment and Nomogram Prediction Model Construction
4.6. Functional Enrichment Analysis for DEGs between Two PRGPI Risk Subgroups
4.7. Analysis of Tumor Immune Signatures for PRGPI Risk Score
4.8. Assessment of the Clinical Drug Response Prediction of PRGPI Risk Score
4.9. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
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]
- Chen, W.; Zheng, R.; Baade, P.D.; Zhang, S.; Zeng, H.; Bray, F.; Jemal, A.; Yu, X.Q.; He, J. Cancer statistics in China, 2015. CA Cancer J. Clin. 2016, 66, 115–132. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Rahib, L.; Smith, B.D.; Aizenberg, R.; Rosenzweig, A.B.; Fleshman, J.M.; Matrisian, L.M. Projecting cancer incidence and deaths to 2030: The unexpected burden of thyroid, liver, and pancreas cancers in the United States. Cancer Res. 2014, 74, 2913–2921. [Google Scholar] [CrossRef] [Green Version]
- Mizrahi, J.D.; Surana, R.; Valle, J.W.; Shroff, R.T. Pancreatic cancer. Lancet 2020, 395, 2008–2020. [Google Scholar] [CrossRef]
- Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2021, 71, 209–249. [Google Scholar] [CrossRef] [PubMed]
- Herbst, B.; Zheng, L. Precision medicine in pancreatic cancer: Treating every patient as an exception. Lancet Gastroenterol. Hepatol. 2019, 4, 805–810. [Google Scholar] [CrossRef]
- Neoptolemos, J.P.; Kleeff, J.; Michl, P.; Costello, E.; Greenhalf, W.; Palmer, D.H. Therapeutic developments in pancreatic cancer: Current and future perspectives. Nat. Rev. Gastroenterol. Hepatol. 2018, 15, 333–348. [Google Scholar] [CrossRef]
- Ho, W.J.; Jaffee, E.M.; Zheng, L. The tumour microenvironment in pancreatic cancer—Clinical challenges and opportunities. Nat. Rev. Clin. Oncol. 2020, 17, 527–540. [Google Scholar] [CrossRef]
- Shi, J.; Zhao, Y.; Wang, K.; Shi, X.; Wang, Y.; Huang, H.; Zhuang, Y.; Cai, T.; Wang, F.; Shao, F. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 2015, 526, 660–665. [Google Scholar] [CrossRef]
- Shi, J.; Gao, W.; Shao, F. Pyroptosis: Gasdermin-Mediated Programmed Necrotic Cell Death. Trends Biochem. Sci. 2017, 42, 245–254. [Google Scholar] [CrossRef]
- Xue, Y.; Tuipulotu, D.E.; Tan, W.H.; Kay, C.; Man, S.M. Emerging Activators and Regulators of Inflammasomes and Pyroptosis. Trends Immunol. 2019, 40, 1035–1052. [Google Scholar] [CrossRef]
- Ding, J.; Wang, K.; Liu, W.; She, Y.; Sun, Q.; Shi, J.; Sun, H.; Wang, D.C.; Shao, F. Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 2016, 535, 111–116. [Google Scholar] [CrossRef] [PubMed]
- Broz, P.; Pelegrín, P.; Shao, F. The gasdermins, a protein family executing cell death and inflammation. Nat. Rev. Immunol. 2020, 20, 143–157. [Google Scholar] [CrossRef] [PubMed]
- Liu, X.; Xia, S.; Zhang, Z.; Wu, H.; Lieberman, J. Channelling inflammation: Gasdermins in physiology and disease. Nat. Rev. Drug Discov. 2021, 20, 384–405. [Google Scholar] [CrossRef] [PubMed]
- Tamura, M.; Tanaka, S.; Fujii, T.; Aoki, A.; Komiyama, H.; Ezawa, K.; Sumiyama, K.; Sagai, T.; Shiroishi, T. Members of a novel gene family, Gsdm, are expressed exclusively in the epithelium of the skin and gastrointestinal tract in a highly tissue-specific manner. Genomics 2007, 89, 618–629. [Google Scholar] [CrossRef] [Green Version]
- He, W.-T.; Wan, H.; Hu, L.; Chen, P.; Wang, X.; Huang, Z.; Yang, Z.-H.; Zhong, C.-Q.; Han, J. Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion. Cell Res. 2015, 25, 1285–1298. [Google Scholar] [CrossRef] [PubMed]
- Kayagaki, N.; Stowe, I.B.; Lee, B.L.; O’Rourke, K.; Anderson, K.; Warming, S.; Cuellar, T.; Haley, B.; Roose-Girma, M.; Phung, Q.T.; et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 2015, 526, 666–671. [Google Scholar] [CrossRef]
- Shi, J.; Zhao, Y.; Wang, Y.; Gao, W.; Ding, J.; Li, P.; Hu, L.; Shao, F. Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 2014, 514, 187–192. [Google Scholar] [CrossRef]
- Rogers, C.; Fernandes-Alnemri, T.; Mayes, L.; Alnemri, D.; Cingolani, G.; Alnemri, E.S. Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death. Nat. Commun. 2017, 8, 14128. [Google Scholar] [CrossRef] [Green Version]
- Zhou, Z.; He, H.; Wang, K.; Shi, X.; Wang, Y.; Su, Y.; Wang, Y.; Li, D.; Liu, W.; Zhang, Y.; et al. Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells. Science 2020, 368, eaaz7548. [Google Scholar] [CrossRef]
- Xia, X.; Wang, X.; Cheng, Z.; Qin, W.; Lei, L.; Jiang, J.; Hu, J. The role of pyroptosis in cancer: Pro-cancer or pro-“host”? Cell Death Dis. 2019, 10, 650. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Yu, J.; Li, S.; Qi, J.; Chen, Z.; Wu, Y.; Guo, J.; Wang, K.; Sun, X.; Zheng, J. Cleavage of GSDME by caspase-3 determines lobaplatin-induced pyroptosis in colon cancer cells. Cell Death Dis. 2019, 10, 193. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wang, Y.; Gao, W.; Shi, X.; Ding, J.; Liu, W.; He, H.; Wang, K.; Shao, F. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin. Nature 2017, 547, 99–103. [Google Scholar] [CrossRef] [PubMed]
- Wang, Q.; Wang, Y.; Ding, J.; Wang, C.; Zhou, X.; Gao, W.; Huang, H.; Shao, F.; Liu, Z. A bioorthogonal system reveals antitumour immune function of pyroptosis. Nature 2020, 579, 421–426. [Google Scholar] [CrossRef]
- Zhang, Z.; Zhang, Y.; Xia, S.; Kong, Q.; Li, S.; Liu, X.; Junqueira, C.; Meza-Sosa, K.F.; Mok, T.M.Y.; Ansara, J.; et al. Gasdermin E suppresses tumour growth by activating anti-tumour immunity. Nature 2020, 579, 415–420. [Google Scholar] [CrossRef] [PubMed]
- Cui, J.; Zhou, Z.; Yang, H.; Jiao, F.; Li, N.; Gao, Y.; Wang, L.; Chen, J.; Quan, M. MST1 Suppresses Pancreatic Cancer Progression via ROS-Induced Pyroptosis. Mol. Cancer Res. 2019, 17, 1316–1325. [Google Scholar] [CrossRef] [Green Version]
- Wang, J.; Zhang, H.; Shao, P.; Zhang, X.; Zhou, B. Bioinformatic Analysis of Prognostic Value of Pyroptosis-Related Genes and Its Effect on Immune Cell Infiltration in Pancreatic Adenocarcinoma. Int. J. Gen. Med. 2022, 15, 2311–2319. [Google Scholar] [CrossRef]
- Tang, R.; Liu, X.; Liang, C.; Hua, J.; Xu, J.; Wang, W.; Meng, Q.; Liu, J.; Zhang, B.; Yu, X.; et al. Deciphering the Prognostic Implications of the Components and Signatures in the Immune Microenvironment of Pancreatic Ductal Adenocarcinoma. Front. Immunol. 2021, 12, 648917. [Google Scholar] [CrossRef]
- Chen, X.; Zeh, H.J.; Kang, R.; Kroemer, G.; Tang, D. Cell death in pancreatic cancer: From pathogenesis to therapy. Nat. Rev. Gastroenterol. Hepatol. 2021, 18, 804–823. [Google Scholar] [CrossRef]
- Bear, A.S.; Vonderheide, R.H.; O’Hara, M.H. Challenges and Opportunities for Pancreatic Cancer Immunotherapy. Cancer Cell 2020, 38, 788–802. [Google Scholar] [CrossRef]
- Fernandes-Alnemri, T.; Yu, J.-W.; Datta, P.; Wu, J.; Alnemri, E.S. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature 2009, 458, 509–513. [Google Scholar] [CrossRef] [Green Version]
- Li, C.; Zhang, Y.; Cheng, X.; Yuan, H.; Zhu, S.; Liu, J.; Wen, Q.; Xie, Y.; Liu, J.; Kroemer, G.; et al. PINK1 and PARK2 Suppress Pancreatic Tumorigenesis through Control of Mitochondrial Iron-Mediated Immunometabolism. Dev. Cell 2018, 46, 441–455.e8. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wilson, J.E.; Petrucelli, A.S.; Chen, L.; Koblansky, A.A.; Truax, A.D.; Oyama, Y.; Rogers, A.B.; Brickey, W.J.; Wang, Y.; Schneider, M.; et al. Inflammasome-independent role of AIM2 in suppressing colon tumorigenesis via DNA-PK and Akt. Nat. Med. 2015, 21, 906–913. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fisch, D.; Bando, H.; Clough, B.; Hornung, V.; Yamamoto, M.; Shenoy, A.R.; Frickel, E. Human GBP 1 is a microbe-specific gatekeeper of macrophage apoptosis and pyroptosis. Embo J. 2019, 38, e100926. [Google Scholar] [CrossRef]
- Wandel, M.P.; Kim, B.-H.; Park, E.-S.; Boyle, K.B.; Nayak, K.; Lagrange, B.; Herod, A.; Henry, T.; Zilbauer, M.; Rohde, J.; et al. Guanylate-binding proteins convert cytosolic bacteria into caspase-4 signaling platforms. Nat. Immunol. 2020, 21, 880–891. [Google Scholar] [CrossRef] [PubMed]
- Deng, M.; Tang, Y.; Li, W.; Wang, X.; Zhang, R.; Zhang, X.; Zhao, X.; Liu, J.; Tang, C.; Liu, Z.; et al. The Endotoxin Delivery Protein HMGB1 Mediates Caspase-11-Dependent Lethality in Sepsis. Immunity 2018, 49, 740–753. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Arend, W.P.; Palmer, G.; Gabay, C. IL-1, IL-18, and IL-33 families of cytokines. Immunol. Rev. 2008, 223, 20–38. [Google Scholar] [CrossRef]
- Carbone, A.; Vizio, B.; Novarino, A.; Mauri, F.A.; Geuna, M.; Robino, C.; Brondino, G.; Prati, A.; Giacobino, A.; Campra, D.; et al. IL-18 Paradox in Pancreatic Carcinoma: Elevated Serum Levels of Free IL-18 are Correlated with Poor Survival. J. Immunother. 2009, 32, 920–931. [Google Scholar] [CrossRef]
- Li, Z.; Yu, X.; Werner, J.; Bazhin, A.V.; D’Haese, J.G. The role of interleukin-18 in pancreatitis and pancreatic cancer. Cytokine Growth Factor Rev. 2019, 50, 1–12. [Google Scholar] [CrossRef]
- Lu, J.; Liu, X.; Zheng, J.; Song, J.; Liu, Y.; Ruan, X.; Shen, S.; Shao, L.; Yang, C.; Wang, D.; et al. Lin28A promotes IRF6-regulated aerobic glycolysis in glioma cells by stabilizing SNHG14. Cell Death Dis. 2020, 11, 447. [Google Scholar] [CrossRef]
- Li, D.; Cheng, P.; Wang, J.; Qiu, X.; Zhang, X.; Xu, L.; Liu, Y.; Qin, S. IRF6 Is Directly Regulated by ZEB1 and ELF3, and Predicts a Favorable Prognosis in Gastric Cancer. Front. Oncol. 2019, 9, 220. [Google Scholar] [CrossRef] [PubMed]
- Chen, X.; Liu, G.; Yuan, Y.; Wu, G.; Wang, S.; Yuan, L. NEK7 interacts with NLRP3 to modulate the pyroptosis in inflammatory bowel disease via NF-κB signaling. Cell Death Dis. 2019, 10, 906. [Google Scholar] [CrossRef] [PubMed]
- Yan, Z.; Qu, J.; Li, Z.; Yi, J.; Su, Y.; Lin, Q.; Yu, G.; Lin, Z.; Yin, W.; Lu, F.; et al. NEK7 Promotes Pancreatic Cancer Progression and Its Expression Is Correlated with Poor Prognosis. Front. Oncol. 2021, 11, 2656. [Google Scholar] [CrossRef] [PubMed]
- Taabazuing, C.Y.; Griswold, A.R.; Bachovchin, D.A. The NLRP1 and CARD8 inflammasomes. Immunol. Rev. 2020, 297, 13–25. [Google Scholar] [CrossRef]
- Kang, R.; Zeng, L.; Zhu, S.; Xie, Y.; Liu, J.; Wen, Q.; Cao, L.; Xie, M.; Ran, Q.; Kroemer, G.; et al. Lipid Peroxidation Drives Gasdermin D-Mediated Pyroptosis in Lethal Polymicrobial Sepsis. Cell Host Microbe 2018, 24, 97–108. [Google Scholar] [CrossRef] [Green Version]
- Kadamur, G.; Ross, E.M. Mammalian Phospholipase C. Annu. Rev. Physiol. 2013, 75, 127–154. [Google Scholar] [CrossRef] [Green Version]
- Jorgensen, I.; Rayamajhi, M.; Miao, E.A. Programmed cell death as a defence against infection. Nat. Rev. Immunol. 2017, 17, 151–164. [Google Scholar] [CrossRef]
- Aachoui, Y.; Kajiwara, Y.; Leaf, I.A.; Mao, D.; Ting, J.P.-Y.; Coers, J.; Aderem, A.; Buxbaum, J.; Miao, E.A. Canonical Inflammasomes Drive IFN-γ to Prime Caspase-11 in Defense against a Cytosol-Invasive Bacterium. Cell Host Microbe 2015, 18, 320–332. [Google Scholar] [CrossRef] [Green Version]
- Miao, E.A.; Leaf, I.A.; Treuting, P.M.; Mao, D.P.; Dors, M.; Sarkar, A.; Warren, S.E.; Wewers, M.D.; Aderem, A. Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria. Nat. Immunol. 2010, 11, 1136–1142. [Google Scholar] [CrossRef]
- Tang, R.; Xu, J.; Zhang, B.; Liu, J.; Liang, C.; Hua, J.; Meng, Q.; Yu, X.; Shi, S. Ferroptosis, necroptosis, and pyroptosis in anticancer immunity. J. Hematol. Oncol. 2020, 13, 110. [Google Scholar] [CrossRef]
- Raphael, B.J.; Hruban, R.H.; Aguirre, A.J.; Moffitt, R.A.; Yeh, J.J.; Stewart, C.; Robertson, A.G.; Cherniack, A.D.; Gupta, M.; Getz, G.; et al. Integrated Genomic Characterization of Pancreatic Ductal Adenocarcinoma. Cancer Cell 2017, 32, 185–203.e13. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Jones, S.; Zhang, X.; Parsons, D.W.; Lin, J.C.-H.; Leary, R.J.; Angenendt, P.; Mankoo, P.; Carter, H.; Kamiyama, H.; Jimeno, A.; et al. Core Signaling Pathways in Human Pancreatic Cancers Revealed by Global Genomic Analyses. Science 2008, 321, 1801–1806. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Qian, Z.R.; Rubinson, D.A.; Nowak, J.A.; Morales-Oyarvide, V.; Dunne, R.F.; Kozak, M.M.; Welch, M.W.; Brais, L.K.; Da Silva, A.; Li, T.; et al. Association of Alterations in Main Driver Genes with Outcomes of Patients with Resected Pancreatic Ductal Adenocarcinoma. JAMA Oncol. 2018, 4, e173420. [Google Scholar] [CrossRef] [PubMed]
- Locati, M.; Curtale, G.; Mantovani, A. Diversity, Mechanisms, and Significance of Macrophage Plasticity. Annu. Rev. Pathol. Mech. Dis. 2020, 15, 123–147. [Google Scholar] [CrossRef] [Green Version]
- Ruffell, B.; Coussens, L.M. Macrophages and Therapeutic Resistance in Cancer. Cancer Cell 2015, 27, 462–472. [Google Scholar] [CrossRef] [Green Version]
- Storz, P. The crosstalk between acinar cells with Krasmutations and M1-polarized macrophages leads to initiation of pancreatic precancerous lesions. Oncoimmunology 2015, 4, e1008794. [Google Scholar] [CrossRef] [Green Version]
- Rakaee, M.; Busund, L.-T.R.; Jamaly, S.; Paulsen, E.-E.; Richardsen, E.; Andersen, S.; Al-Saad, S.; Bremnes, R.M.; Donnem, T.; Kilvaer, T.K. Prognostic Value of Macrophage Phenotypes in Resectable Non–Small Cell Lung Cancer Assessed by Multiplex Immunohistochemistry. Neoplasia 2019, 21, 282–293. [Google Scholar] [CrossRef]
- Bagchi, S.; Yuan, R.; Engleman, E.G. Immune Checkpoint Inhibitors for the Treatment of Cancer: Clinical Impact and Mechanisms of Response and Resistance. Annu. Rev. Pathol. Mech. Dis. 2021, 16, 223–249. [Google Scholar] [CrossRef]
- Brahmer, J.R.; Tykodi, S.S.; Chow, L.Q.M.; Hwu, W.-J.; Topalian, S.L.; Hwu, P.; Drake, C.G.; Camacho, L.H.; Kauh, J.; Odunsi, K.; et al. Safety and Activity of Anti-PD-L1 Antibody in Patients with Advanced Cancer. N. Engl. J. Med. 2012, 366, 2455–2465. [Google Scholar] [CrossRef] [Green Version]
- Mace, T.A.; Shakya, R.; Pitarresi, J.R.; Swanson, B.; McQuinn, C.W.; Loftus, S.; Nordquist, E.; Cruz-Monserrate, Z.; Yu, L.; Young, G.; et al. IL-6 and PD-L1 antibody blockade combination therapy reduces tumour progression in murine models of pancreatic cancer. Gut 2016, 67, 320–332. [Google Scholar] [CrossRef] [Green Version]
- O’Reilly, E.M.; Oh, D.-Y.; Dhani, N.; Renouf, D.J.; Lee, M.A.; Sun, W.; Fisher, G.; Hezel, A.; Chang, S.-C.; Vlahovic, G.; et al. Durvalumab with or without Tremelimumab for Patients with Metastatic Pancreatic Ductal Adenocarcinoma: A Phase 2 Randomized Clinical Trial. JAMA Oncol. 2019, 5, 1431–1438. [Google Scholar] [CrossRef] [PubMed]
- Chin, V.; Nagrial, A.; Sjoquist, K.; O’Connor, C.A.; Chantrill, L.; Biankin, A.V.; Scholten, R.J.; Yip, D. Chemotherapy and radiotherapy for advanced pancreatic cancer. Cochrane Database Syst. Rev. 2018, 3, CD011044. [Google Scholar] [CrossRef] [PubMed]
- Wu, J.; Li, L.; Zhang, H.; Zhao, Y.; Zhang, H.; Wu, S.; Xu, B. A risk model developed based on tumor microenvironment predicts overall survival and associates with tumor immunity of patients with lung adenocarcinoma. Oncogene 2021, 40, 4413–4424. [Google Scholar] [CrossRef] [PubMed]
Characteristics | Variable | TCGA Dataset (n = 177) |
---|---|---|
Age, years | ≤65 | 94 |
>65 | 83 | |
Gender | Female | 80 |
Male | 97 | |
Grade | G1 | 30 |
G2 | 95 | |
G3 | 48 | |
G4 | 2 | |
GX | 2 | |
Primary site | Head | 131 |
Body & Tail | 32 | |
Unknown | 14 | |
pT | T1 | 10 |
T2 | 96 | |
T3 | 55 | |
T4 | 3 | |
TX | 13 | |
pN | N0 | 51 |
N1 | 73 | |
N2 | 52 | |
NX | 1 | |
TNM Stage | I | 32 |
II | 83 | |
III | 54 | |
IV | 4 | |
Unknown | 4 | |
Radiotherapy | Yes | 37 |
No | 101 | |
Unknown | 39 |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 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
Xie, W.; Li, X.; Yang, C.; Li, J.; Shen, G.; Chen, H.; Xiao, S.-Y.; Li, Y. The Pyroptosis-Related Gene Prognostic Index Associated with Tumor Immune Infiltration for Pancreatic Cancer. Int. J. Mol. Sci. 2022, 23, 6178. https://doi.org/10.3390/ijms23116178
Xie W, Li X, Yang C, Li J, Shen G, Chen H, Xiao S-Y, Li Y. The Pyroptosis-Related Gene Prognostic Index Associated with Tumor Immune Infiltration for Pancreatic Cancer. International Journal of Molecular Sciences. 2022; 23(11):6178. https://doi.org/10.3390/ijms23116178
Chicago/Turabian StyleXie, Wen, Xiaoyi Li, Chunxiu Yang, Jiahao Li, Guoyan Shen, Hongshan Chen, Shu-Yuan Xiao, and Yueying Li. 2022. "The Pyroptosis-Related Gene Prognostic Index Associated with Tumor Immune Infiltration for Pancreatic Cancer" International Journal of Molecular Sciences 23, no. 11: 6178. https://doi.org/10.3390/ijms23116178