bims-ectoca 2022-05-22 papers

bims-ectoca

Biomed News

on Epigenetic control of tolerance in cancer

Issue of 2022‒05‒22
sixteen papers selected by
Ankita Daiya, Birla Institute of Technology and Science

Understanding sarcoma drug resistance one cell at a time.
Complex cross-talk between EZH2 and miRNAs confers hallmark characteristics and shapes the tumor microenvironment.
Histone Modifications, Internucleosome Dynamics, and DNA Stresses: How They Cooperate to "Functionalize" Nucleosomes.
Transcription-coupled H3.3 recycling: A link with chromatin states.
H3-K27M-mutant nucleosomes interact with MLL1 to shape the glioma epigenetic landscape.
Compatibility rules of human enhancer and promoter sequences.
Regulation of protein complex partners as a compensatory mechanism in aneuploid tumors.
Epi-miRNAs: Modern mediators of methylation status in human cancers.
Targeting EZH2 for cancer therapy: From current progress to novel strategies.
KDM3A-mediated SP1 activates PFKFB4 transcription to promote aerobic glycolysis in osteosarcoma and augment tumor development.
Identifying common transcriptome signatures of cancer by interpreting deep learning models.
Epigenetic modulation in sensitizing metastatic sarcomas to therapies and overcoming resistance.
Ezh2 competes with p53 to license lncRNA Neat1 transcription for inflammasome activation.
Spatially resolved transcriptomics provide a new method for cancer research.
Transcriptome analysis reveals high tumor heterogeneity with respect to re-activation of stemness and proliferation programs.
Machine learning phenomics (MLP) combining deep learning with time-lapse-microscopy for monitoring colorectal adenocarcinoma cells gene expression and drug-response.

Cancer Drug Resist. 2022 ;5(1): 90-92

Understanding sarcoma drug resistance one cell at a time.

Salah-Eddine Cherradi-Lamhamedi, Danh Truong, Joseph A Ludwig.

DOI: https://doi.org/10.20517/cdr.2021.87
Epigenomics. 2022 May 16.

Complex cross-talk between EZH2 and miRNAs confers hallmark characteristics and shapes the tumor microenvironment.

Christopher Rt Hillyar, Shivani S Kanabar, Kathrine S Rallis, Jajini S Varghese.

  Cancer epigenetic mechanisms support the acquisition of hallmark characteristics during oncogenesis. EZH2 - an important histone methyltransferase that writes histone H3 lysine 27 trimethylation marks - is known to be dysregulated in cancer cells. However, the interactions between EZH2 and miRNAs that form a complex network of cross-talk and reciprocal regulation that enable cancer cells to acquire hallmark characteristics have been relatively poorly appreciated. The specific functions of EZH2 appear to be regulated by a vast array of miRNAs, which direct EZH2 toward regulation over the development of specific hallmark characteristics. This review discusses recent advances in the understanding of EZH2, focusing on its collaboration with miRNAs to orchestrate oncogenesis. These epigenetic processes promote the evasion of apoptosis/cell cycle arrest, cellular dedifferentiation and the establishment of a tumor microenvironment that facilitates local cancer cell invasion, anti-cancer drug resistance and evasion of the immune response.

Keywords:  DNA methylation; EZH2; epigenetics; histone modification; miRNAs; tumor microenvironment

DOI:  https://doi.org/10.2217/epi-2021-0534
Front Genet. 2022 ;13 873398

Histone Modifications, Internucleosome Dynamics, and DNA Stresses: How They Cooperate to "Functionalize" Nucleosomes.

Wladyslaw A Krajewski.

  Tight packaging of DNA in chromatin severely constrains DNA accessibility and dynamics. In contrast, nucleosomes in active chromatin state are highly flexible, can exchange their histones, and are virtually "transparent" to RNA polymerases, which transcribe through gene bodies at rates comparable to that of naked DNA. Defining mechanisms that revert nucleosome repression, in addition to their value for basic science, is of key importance for the diagnosis and treatment of genetic diseases. Chromatin activity is largely regulated by histone posttranslational modifications, ranging from small chemical groups up to the yet understudied "bulky" ubiquitylation and sumoylation. However, it is to be revealed how histone marks are "translated" to permissive or repressive changes in nucleosomes: it is a general opinion that histone modifications act primarily as "signals" for recruiting the regulatory proteins or as a "neutralizer" of electrostatic shielding of histone tails. Here, we would like to discuss recent evidence suggesting that histone ubiquitylation, in a DNA stress-dependent manner, can directly regulate the dynamics of the nucleosome and their primary structure and can promote nucleosome decomposition to hexasome particles or additionally stabilize nucleosomes against unwrapping. In addition, nucleosome repression/ derepression studies are usually performed with single mononucleosomes as a model. We would like to review and discuss recent findings showing that internucleosomal interactions could strongly modulate the dynamics and rearrangements of nucleosomes. Our hypothesis is that bulky histone modifications, nucleosome inherent dynamics, internucleosome interactions, and DNA torsions could act in cooperation to orchestrate the formation of different dynamic states of arrayed nucleosomes and thus promote chromatin functionality and diversify epigenetic programming methods.

Keywords:  DNA stresses; hexasomes; histone code; histone modifications; histones; nucleosomes; ubiquitylation

DOI:  https://doi.org/10.3389/fgene.2022.873398
Semin Cell Dev Biol. 2022 May 17. pii: S1084-9521(22)00159-8. [Epub ahead of print]

Transcription-coupled H3.3 recycling: A link with chromatin states.

Kamila Delaney, Geneviève Almouzni.

  Histone variant H3.3 is incorporated into chromatin throughout the cell cycle and even in non-cycling cells. This histone variant marks actively transcribed chromatin regions with high nucleosome turnover, as well as silent pericentric and telomeric repetitive regions. In the past few years, significant progress has been made in our understanding of mechanisms involved in the transcription-coupled deposition of H3.3. Here we review how, during transcription, new H3.3 deposition intermingles with the fate of the old H3.3 variant and its recycling. First, we describe pathways enabling the incorporation of newly synthesized vs old H3.3 histones in the context of transcription. We then review the current knowledge concerning differences between these two H3.3 populations, focusing on their PTMs composition. Finally, we discuss the implications of H3.3 recycling for the maintenance of the transcriptional state and underline the emerging importance of H3.3 as a potent epigenetic regulator for both maintaining and switching a transcriptional state.

Keywords:  H3.3 histone variant; H3.3 recycling; HIRA complex; Histone post-translational modifications; New H3.3; Old H3.3; Transcription

DOI:  https://doi.org/10.1016/j.semcdb.2022.05.003
Cell Rep. 2022 May 17. pii: S2211-1247(22)00609-X. [Epub ahead of print]39(7): 110836

H3-K27M-mutant nucleosomes interact with MLL1 to shape the glioma epigenetic landscape.

Noa Furth, Danielle Algranati, Bareket Dassa, Olga Beresh, Vadim Fedyuk, Natasha Morris, Lawryn H Kasper, Dan Jones, Michelle Monje, Suzanne J Baker, Efrat Shema.

  Cancer-associated mutations in genes encoding histones dramatically reshape chromatin and support tumorigenesis. Lysine to methionine substitution of residue 27 on histone H3 (K27M) is a driver mutation in high-grade pediatric gliomas, known to abrogate polycomb repressive complex 2 (PRC2) activity. We applied single-molecule systems to image individual nucleosomes and delineate the combinatorial epigenetic patterns associated with H3-K27M expression. We found that chromatin marks on H3-K27M-mutant nucleosomes are dictated both by their incorporation preferences and by intrinsic properties of the mutation. Mutant nucleosomes not only preferentially bind PRC2 but also directly interact with MLL1, leading to genome-wide redistribution of H3K4me3. H3-K27M-mediated deregulation of repressive and active chromatin marks leads to unbalanced "bivalent" chromatin, which may support a poorly differentiated cellular state. This study provides evidence for a direct effect of H3-K27M oncohistone on the MLL1-H3K4me3 pathway and highlights the capability of single-molecule tools to reveal mechanisms of chromatin deregulation in cancer.

Keywords:  CP: Cancer; CP: Molecular biology; DIPG; H3-K27M; chromatin; histone modifications; oncohistone; single molecule

DOI:  https://doi.org/10.1016/j.celrep.2022.110836
Nature. 2022 May 20.

Compatibility rules of human enhancer and promoter sequences.

Drew T Bergman, Thouis R Jones, Vincent Liu, Judhajeet Ray, Evelyn Jagoda, Layla Siraj, Helen Y Kang, Joseph Nasser, Michael Kane, Antonio Rios, Tung H Nguyen, Sharon R Grossman, Charles P Fulco, Eric S Lander, Jesse M Engreitz.

Gene regulation in the human genome is controlled by distal enhancers that activate specific nearby promoters1. One model for this specificity is that promoters might have sequence-encoded preferences for certain enhancers, for example mediated by interacting sets of transcription factors or cofactors2. This "biochemical compatibility" model has been supported by observations at individual human promoters and by genome-wide measurements in Drosophila3-9. However, the degree to which human enhancers and promoters are intrinsically compatible has not been systematically measured, and how their activities combine to control RNA expression remains unclear. Here we designed a high-throughput reporter assay called ExP STARR-seq (enhancer x promoter self-transcribing active regulatory region sequencing) and applied it to examine the combinatorial compatibilities of 1,000 enhancer and 1,000 promoter sequences in human K562 cells. We identify simple rules for enhancer-promoter compatibility: most enhancers activated all promoters by similar amounts, and intrinsic enhancer and promoter activities combine multiplicatively to determine RNA output (R2=0.82). In addition, two classes of enhancers and promoters showed subtle preferential effects. Promoters of housekeeping genes contained built-in activating motifs for factors such as GABPA and YY1, which decreased the responsiveness of promoters to distal enhancers. Promoters of variably expressed genes lacked these motifs and showed stronger responsiveness to enhancers. Together, this systematic assessment of enhancer-promoter compatibility suggests a multiplicative model tuned by enhancer and promoter class to control gene transcription in the human genome.

DOI: https://doi.org/10.1038/s41586-022-04877-w
Elife. 2022 May 16. pii: e75526. [Epub ahead of print]11

Regulation of protein complex partners as a compensatory mechanism in aneuploid tumors.

Gökçe Senger, Stefano Santaguida, Martin H Schaefer.

  Aneuploidy, a state of chromosome imbalance, is a hallmark of human tumors, but its role in cancer still remains to be fully elucidated. To understand the consequences of whole-chromosome-level aneuploidies on the proteome, we integrated aneuploidy, transcriptomic and proteomic data from hundreds of TCGA/CPTAC tumor samples. We found a surprisingly large number of expression changes happened on other, non-aneuploid chromosomes. Moreover, we identified an association between those changes and co-complex members of proteins from aneuploid chromosomes. This co-abundance association is tightly regulated for aggregation-prone aneuploid proteins and those involved in a smaller number of complexes. On the other hand, we observe that complexes of the cellular core machinery are under functional selection to maintain their stoichiometric balance in aneuploid tumors. Ultimately, we provide evidence that those compensatory and functional maintenance mechanisms are established through post-translational control and that the degree of success of a tumor to deal with aneuploidy-induced stoichiometric imbalance impacts the activation of cellular protein degradation programs and patient survival.

Keywords:  cancer biology; computational biology; human; systems biology

DOI:  https://doi.org/10.7554/eLife.75526
Wiley Interdiscip Rev RNA. 2022 May 17. e1735

Epi-miRNAs: Modern mediators of methylation status in human cancers.

Maria-Alexandra Papadimitriou, Konstantina Panoutsopoulou, Katerina-Marina Pilala, Andreas Scorilas, Margaritis Avgeris.

  Methylation of the fundamental macromolecules, DNA/RNA, and proteins, is remarkably abundant, evolutionarily conserved, and functionally significant in cellular homeostasis and normal tissue/organism development. Disrupted methylation imprinting is strongly linked to loss of the physiological equilibrium and numerous human pathologies, and most importantly to carcinogenesis, tumor heterogeneity, and cancer progression. Mounting recent evidence has documented the active implication of miRNAs in the orchestration of the multicomponent cellular methylation machineries and the deregulation of methylation profile in the epigenetic, epitranscriptomic, and epiproteomic levels during cancer onset and progression. The elucidation of such regulatory networks between the miRNome and the cellular methylation machineries has led to the emergence of a novel subclass of miRNAs, namely "epi-miRNAs" or "epi-miRs." Herein, we have summarized the existing knowledge on the functional role of epi-miRs in the methylation dynamic landscape of human cancers and their clinical utility in modern cancer diagnostics and tailored therapeutics. This article is categorized under: RNA in Disease and Development > RNA in Disease.

Keywords:  DNA methylation; RNA methylation; epigenetics; epitranscriptomics; histone methylation; miRNA; non-coding RNAs

DOI:  https://doi.org/10.1002/wrna.1735
Eur J Med Chem. 2022 Apr 30. pii: S0223-5234(22)00321-X. [Epub ahead of print]238 114419

Targeting EZH2 for cancer therapy: From current progress to novel strategies.

Jia Zeng, Jifa Zhang, Ying Sun, Jiaxing Wang, Changyu Ren, Souvik Banerjee, Liang Ouyang, Yuxi Wang.

  EZH2, the catalytic subunit of PRC2, catalyzes histone H3 lysine 27 (H3K27) trimethylation to induce the agglutination of chromosomes and in turn represses the transcription of the target genes. Numerous reports indicate that EZH2 is overexpressed in a variety of malignant tumor tissues. Therefore, targeting EZH2 protein is a promising strategy for cancer treatment. So far, many small molecule EZH2 specific inhibitors have entered clinical trials, but many of them harbored limited clinical efficacy. New technologies and methods are imperative to enhance the anticancer activity of EZH2. In this review, the structure and biological functions of EZH2 protein will be reviewed. The internal relationship between EZH2 and various diseases will be expounded. The development status of specific inhibitors for EZH2, and the latest progress of new strategies such as drug combination, dual-target inhibitors, targeted protein degradation technology and protein-protein interactions (PPI) inhibitors will be emphatically summarized and analyzed.

Keywords:  Drug combination; Dual-target inhibitors; EZH2; Novel strategies

DOI:  https://doi.org/10.1016/j.ejmech.2022.114419
BMC Cancer. 2022 May 19. 22(1): 562

KDM3A-mediated SP1 activates PFKFB4 transcription to promote aerobic glycolysis in osteosarcoma and augment tumor development.

Wei Wang, Bin Wang.

  BACKGROUND: Lysine-specific histone demethylase 3A (KDM3A) is a potent histone modifier that is frequently implicated in the progression of several malignancies. However, its role in aerobic glycolysis of osteosarcoma (OS) remains unclear.METHODS: KDM3A expression in OS tissues was determined by immunohistochemistry, and that in acquired OS cells was determined by RT-qPCR and western blot assays. KDM3A was silenced in OS cells to examine cellular behaviors and the aerobic glycolysis. Stably transfected cells were injected into nude mice for in vivo experiments. The downstream targets of KDM3A were predicted by bioinformatics systems and validated by ChIP-qPCR. Rescue experiments of SP1 and PFKFB4 were performed to examine their roles in the KDM3A-mediated events.
RESULTS: KDM3A was highly expressed in OS tissues and cells. Knockdown of KDM3A weakened OS cell growth and metastasis in vivo and in vitro, and it suppressed the aerobic glycolysis in OS cells. KDM3A enhanced the transcription of SP1 by demethylating H3K9me2 on its promoter. Restoration of SP1 rescued growth and metastasis of OS cells and recovered the glycolytic flux in cells suppressed by knockdown of KDM3A. SP1 bound to the PFKFB4 promoter to activate its transcription and expression. PFKFB4 expression in OS cells was suppressed by KDM3A silencing but increased after SP1 restoration. Overexpression of PFKFB4 significantly promoted OS cell growth and metastasis as well as the glycolytic flux in cells.
CONCLUSION: This paper elucidates that upregulation of PFKFB4 mediated by the KDM3A-SP1 axis promotes aerobic glycolysis in OS and augments tumor development.

Keywords:  Glycolysis; KDM3A; Osteosarcoma; PFKFB4; SP1

DOI:  https://doi.org/10.1186/s12885-022-09636-8
Genome Biol. 2022 May 17. 23(1): 117

Identifying common transcriptome signatures of cancer by interpreting deep learning models.

Anupama Jha, Mathieu Quesnel-Vallières, David Wang, Andrei Thomas-Tikhonenko, Kristen W Lynch, Yoseph Barash.

  BACKGROUND: Cancer is a set of diseases characterized by unchecked cell proliferation and invasion of surrounding tissues. The many genes that have been genetically associated with cancer or shown to directly contribute to oncogenesis vary widely between tumor types, but common gene signatures that relate to core cancer pathways have also been identified. It is not clear, however, whether there exist additional sets of genes or transcriptomic features that are less well known in cancer biology but that are also commonly deregulated across several cancer types.RESULTS: Here, we agnostically identify transcriptomic features that are commonly shared between cancer types using 13,461 RNA-seq samples from 19 normal tissue types and 18 solid tumor types to train three feed-forward neural networks, based either on protein-coding gene expression, lncRNA expression, or splice junction use, to distinguish between normal and tumor samples. All three models recognize transcriptome signatures that are consistent across tumors. Analysis of attribution values extracted from our models reveals that genes that are commonly altered in cancer by expression or splicing variations are under strong evolutionary and selective constraints. Importantly, we find that genes composing our cancer transcriptome signatures are not frequently affected by mutations or genomic alterations and that their functions differ widely from the genes genetically associated with cancer.
CONCLUSIONS: Our results highlighted that deregulation of RNA-processing genes and aberrant splicing are pervasive features on which core cancer pathways might converge across a large array of solid tumor types.

Keywords:  Cancer genomics; Deep learning; Transcriptomics

DOI:  https://doi.org/10.1186/s13059-022-02681-3
Cancer Drug Resist. 2022 ;5(1): 25-35

Epigenetic modulation in sensitizing metastatic sarcomas to therapies and overcoming resistance.

Jeff Rytlewski, Qierra R Brockman, Rebecca D Dodd, Mohammed Milhem, Varun Monga.

  Sarcomas are a class of rare malignancies of mesenchymal origin with a heterogeneous histological spectrum. They are classically associated with poor outcomes, especially once metastasized. A path to improving clinical outcomes may be made through modifying the epigenome, where a variety of sarcomas demonstrate changes that contribute to their oncogenic phenotypes. This Perspective article identifies and describes changes in the sarcoma genome, while discussing specific epigenetic changes and their effect on clinical outcomes. Clinical attempts at modulating epigenetics in sarcoma are reviewed, as well as potential implications of these studies. Epigenetic targets to reverse and delay chemotherapy resistance are discussed. Future directions with primary next steps are proposed to invigorate the current understanding of epigenetic biomarkers to enact targeted therapies to epigenetic phenotypes of sarcoma subtypes. Modifications to prior studies, as well as proposed clinical steps, are also addressed.

Keywords:  Epigenetics; metastasis; resistance; sarcoma

DOI:  https://doi.org/10.20517/cdr.2021.88
Cell Death Differ. 2022 May 14.

Ezh2 competes with p53 to license lncRNA Neat1 transcription for inflammasome activation.

Jia Yuan, Qingchen Zhu, Xingli Zhang, Zhenzhen Wen, Guiheng Zhang, Ni Li, Yifei Pei, Yan Wang, Siyu Pei, Jing Xu, Pan Jia, Chao Peng, Wei Lu, Jun Qin, Qian Cao, Yichuan Xiao.

Inflammasome contributes to the pathogenesis of various inflammatory diseases, but the epigenetic mechanism controlling its activation remains elusive. Here, we found that the histone methyltransferase Ezh2 mediates the activation of multiple types of inflammasomes in macrophages/microglia independent of its methyltransferase activity and thus promotes inflammasome-related pathologies. Mechanistically, Ezh2 functions through its SANT2 domain to maintain the enrichment of H3K27 acetylation in the promoter region of the long noncoding RNA (lncRNA) Neat1, thereby promoting chromatin accessibility and facilitating p65-mediated transcription of Neat1, which is a critical mediator of inflammasome assembly and activation. In addition, the tumour suppressor protein p53 competes with Ezh2 for the same binding region in the Neat1 promoter and thus antagonises Ezh2-induced Neat1 transcription and inflammasome activation. Therefore, loss of Ezh2 strongly promotes the binding of p53, which recruits the deacetylase SIRT1 for H3K27 deacetylation of the Neat1 promoter and thus suppresses Neat1 transcription and inflammasome activation. Overall, our study demonstrates an epigenetic mechanism involved in modulating inflammasome activation through an Ezh2/p53 competition model and highlights a novel function of Ezh2 in maintaining H3K27 acetylation to support lncRNA Neat1 transcription.

DOI: https://doi.org/10.1038/s41418-022-00992-3
J Exp Clin Cancer Res. 2022 May 19. 41(1): 179

Spatially resolved transcriptomics provide a new method for cancer research.

Bowen Zheng, Lin Fang.

  A major feature of cancer is the heterogeneity, both intratumoral and intertumoral. Traditional single-cell techniques have given us a comprehensive understanding of the biological characteristics of individual tumor cells, but the lack of spatial context of the transcriptome has limited the study of cell-to-cell interaction patterns and hindered further exploration of tumor heterogeneity. In recent years, the advent of spatially resolved transcriptomics (SRT) technology has made possible the multidimensional analysis of the tumor microenvironment in the context of intact tissues. Different SRT methods are applicable to different working ranges due to different working principles. In this paper, we review the advantages and disadvantages of various current SRT methods and the overall idea of applying these techniques to oncology studies, hoping to help researchers find breakthroughs. Finally, we discussed the future direction of SRT technology, and deeper investigation into the complex mechanisms of tumor development from different perspectives through multi-omics fusion, paving the way for precisely targeted tumor therapy.

Keywords:  Cancer; Research Progress; Spatially resolved transcriptomics

DOI:  https://doi.org/10.1186/s13046-022-02385-3
PLoS One. 2022 ;17(5): e0268626

Transcriptome analysis reveals high tumor heterogeneity with respect to re-activation of stemness and proliferation programs.

Artem Baranovsky, Timofei Ivanov, Marina Granovskaya, Dmitri Papatsenko, Dmitri D Pervouchine.

Significant alterations in signaling pathways and transcriptional regulatory programs together represent major hallmarks of many cancers. These, among all, include the reactivation of stemness, which is registered by the expression of pathways that are active in the embryonic stem cells (ESCs). Here, we assembled gene sets that reflect the stemness and proliferation signatures and used them to analyze a large panel of RNA-seq data from The Cancer Genome Atlas (TCGA) Consortium in order to specifically assess the expression of stemness-related and proliferation-related genes across a collection of different tumor types. We introduced a metric that captures the collective similarity of the expression profile of a tumor to that of ESCs, which showed that stemness and proliferation signatures vary greatly between different tumor types. We also observed a high degree of intertumoral heterogeneity in the expression of stemness- and proliferation-related genes, which was associated with increased hazard ratios in a fraction of tumors and mirrored by high intratumoral heterogeneity and a remarkable stemness capacity in metastatic lesions across cancer cells in single cell RNA-seq datasets. Taken together, these results indicate that the expression of stemness signatures is highly heterogeneous and cannot be used as a universal determinant of cancer. This calls into question the universal validity of diagnostic tests that are based on stem cell markers.

DOI: https://doi.org/10.1371/journal.pone.0268626
Sci Rep. 2022 May 20. 12(1): 8545

Machine learning phenomics (MLP) combining deep learning with time-lapse-microscopy for monitoring colorectal adenocarcinoma cells gene expression and drug-response.

M D'Orazio, M Murdocca, A Mencattini, P Casti, J Filippi, G Antonelli, D Di Giuseppe, M C Comes, C Di Natale, F Sangiuolo, E Martinelli.

High-throughput phenotyping is becoming increasingly available thanks to analytical and bioinformatics approaches that enable the use of very high-dimensional data and to the availability of dynamic models that link phenomena across levels: from genes to cells, from cells to organs, and through the whole organism. The combination of phenomics, deep learning, and machine learning represents a strong potential for the phenotypical investigation, leading the way to a more embracing approach, called machine learning phenomics (MLP). In particular, in this work we present a novel MLP platform for phenomics investigation of cancer-cells response to therapy, exploiting and combining the potential of time-lapse microscopy for cell behavior data acquisition and robust deep learning software architectures for the latent phenotypes extraction. A two-step proof of concepts is designed. First, we demonstrate a strict correlation among gene expression and cell phenotype with the aim to identify new biomarkers and targets for tailored therapy in human colorectal cancer onset and progression. Experiments were conducted on human colorectal adenocarcinoma cells (DLD-1) and their profile was compared with an isogenic line in which the expression of LOX-1 transcript was knocked down. In addition, we also evaluate the phenotypic impact of the administration of different doses of an antineoplastic drug over DLD-1 cells. Under the omics paradigm, proteomics results are used to confirm the findings of the experiments.

DOI: https://doi.org/10.1038/s41598-022-12364-5