bims-mirbon 2021-11-07 papers

bims-mirbon

Biomed News

on MicroRNAs in bone

Issue of 2021–11–07
eleven papers selected by
Japneet Kaur, Mayo Clinic

Mg2+ -mediated autophagy-dependent polarization of macrophages mediates the osteogenesis of bone marrow stromal stem cells by interfering with macrophage-derived exosomes containing miR-381.
Exosomes from bone marrow mesenchymal stem cells promoted osteogenic differentiation by delivering miR-196a that targeted Dickkopf-1 to activate Wnt/β-catenin pathway.
Circ_0134944 inhibits osteogenesis through miR-127-5p/PDX1/SPHK1 pathway.
lncRNA SNHG1 induced by SP1 regulates bone remodeling and angiogenesis via sponging miR-181c-5p and modulating SFRP1/Wnt signaling pathway.
Investigation of the role of the miR17-92 cluster in BMP9-induced osteoblast lineage commitment.
Exosomal miR-21-5p derived from bone marrow mesenchymal stem cells promote osteosarcoma cell proliferation and invasion by targeting PIK3R1.
m6A-mediated upregulation of AC008 promotes osteoarthritis progression through the miR-328-3p‒AQP1/ANKH axis.
MiR-144-3p induced by SP1 promotes IL-1β-induced pyroptosis in chondrocytes via PTEN/PINK1/Parkin axis.
Bone mesenchymal stem cells-derived miR-223-3p-containing exosomes ameliorate lipopolysaccharide-induced acute uterine injury via interacting with endothelial progenitor cells.
MicroRNA-106a regulates autophagy-related cell death and EMT by targeting TP53INP1 in lung cancer with bone metastasis.
HDAC8-inhibitor PCI-34051-induced exosomes inhibit human bronchial smooth muscle cell proliferation via miR-381-3p mediated TGFB3.

J Orthop Res. 2021 Nov 02.

Mg2+ -mediated autophagy-dependent polarization of macrophages mediates the osteogenesis of bone marrow stromal stem cells by interfering with macrophage-derived exosomes containing miR-381.

Yong Zhu, Shushan Zhao, Liang Cheng, Zhangyuan Lin, Min Zeng, Zhe Ruan, Buhua Sun, Zhongwei Luo, Yifu Tang, Haitao Long.

  Magnesium ion (Mg2+ ) has received increased attention due to the roles it plays in promoting osteogenesis and preventing inflammation. This study was designed to investigate the mechanism by which Mg2+ influences the osteoblastic differentiation of bone marrow stromal stem cells (BMSCs). The polarization of Mø (macrophages) was measured after treatment with Mg2+ . Meanwhile, autophagy in Mø was measured by detecting LC3B expression. Mø-derived exosomes were isolated and cocultured with BMSCs; after which, osteogenic differentiation was evaluated by Alizarin Red staining and detection of alkaline phosphatase (ALP). Our results showed that Mg2+ could induce autophagy in macrophages and modulate the M1/M2 polarization of macrophages. Mg2+ -mediated macrophages could facilitate the osteogenic differentiation of BMSCs by regulating autophagy, and this facilitation by Mg2+ -mediated macrophages was closely related to macrophage-derived exosomes, and especially exosomes containing miR-381. However, miR-381 in macrophages did not influence autophagy or the polarization of Mg2+ -mediated macrophages. Furthermore, macrophage-derived exosomes containing miR-381 mainly determined the osteogenic differentiation of BMSCs. Mg2+ -mediated macrophages were shown to promote the osteogenic differentiation of BMSCs via autophagy through reducing miR-381 in macrophage-derived exosomes. In conclusion, our results suggest Mg2+ -mediated macrophage-derived exosomes containing miR-381 as novel vehicles for promoting the osteogenic differentiation of BMSCs.

Keywords:  autophagy; bone marrow stromal cells; macrophages polarization; magnesium ion; osteogenic differentiation

DOI:  https://doi.org/10.1002/jor.25189
Bioengineered. 2021 Nov 01.

Exosomes from bone marrow mesenchymal stem cells promoted osteogenic differentiation by delivering miR-196a that targeted Dickkopf-1 to activate Wnt/β-catenin pathway.

Zhi Peng, Sheng Lu, Zhenkai Lou, Zhongjie Li, Shaobo Li, Kaishun Yang, Chao Li.

  Osteoporosis is the most common bone metabolic disease. Emerging evidence suggests that exosomes are secreted by diverse cells such as bone marrow mesenchymal stem cells (BMSCs), and play important role in cell-to-cell communication and tissue homeostasis. Recently, the discovery of exosomes has attracted attention in the field of bone remodeling. In this study, the exosomes were extracted from BMSCs and labeled by PKH-67, and then co-cultured with HFOB1.19 cells to investigate the miR-196a function on the osteoblast differentiation of HFOB1.19. The osteoblast differentiation was detected via alizarin red staining and the expression of osteoblast genes were detected by western blotting. The cell apoptosis was detected by flow cytometer. The target relationship of miR-196a and Dickkopf-1 (Dkk1) were verified by luciferase assay and western blotting. Here, we demonstrated that exosomes extracted from BMSCs (BMSC-exo) significantly promoted HFOB1.19 differentiation to osteoblasts. We found that BMSC-exo were enriched with miR-196a and delivered miR-196a to HFOB1.19 cells to inhibit its target Dkk1, which is a negative regulator of Wnt/β-catenin pathway. BMSC-exo activated Wnt/β-catenin pathway to promote osteogenic differentiation, while BMSC-exo failed to exert the effects when miR-196a was deprived. In conclusion, miR-196a delivered by exosomes from BMSCs plays an essential role in enhancing osteoblastic differentiation by targeting Dkk1 to activate Wnt/β-catenin pathway.

Keywords:  Dkk1; bone marrow mesenchymal stem cells; exosomes; microRNA-196a; osteogenic differentiation; osteoporosis

DOI:  https://doi.org/10.1080/21655979.2021.1996015
Regen Ther. 2021 Dec;18 391-400

Circ_0134944 inhibits osteogenesis through miR-127-5p/PDX1/SPHK1 pathway.

Da-Wei Zhang, Tao Chen, Jin-Xiang Li, Hong-Gang Wang, Zong-Wen Huang, Hai Lv.

   Introduction: Osteoporosis, a common skeletal disorder mainly affecting postmenopausal women, is characterized by the imbalance between osteogenesis and osteoclastogenesis. Circ_0134944 has been recently found to be upregulated in postmenopausal osteoporosis (PMOP) patients. However, its role in osteogenesis remains unknown. Here we aimed to explore the role of circ_0134944 in osteogenesis and reveal the underlying mechanism.
Methods: qRT-PCR was used to determine the expression of circ_0134944, miR-127-5p, PDX1 and SPHK1 in the blood mononuclear cells (BMCs) of PMOP patients. Bone marrow mesenchymal stem cells (BMSCs) were used as the cellular model. Western blotting and qRT-PCR were used to determine the expression of osteogenesis-related genes (Runx2, OPN, OCN). ALP and Alizarin Red S staining were performed to evaluate osteogenic differentiation. The interactions between circ_0134944 and miR-127-5p, miR-127-5p and PDX1, PDX1 and SPHK1 were determined by dual-luciferase reporter and ChIP assay.
Results: Circ_0134944, PDX1 and SPHK1 were upregulated while miR-127-5p was downregulated in PMOP patients. Enhanced expression of circ_0134944 suppressed osteogenesis, which was then reversed by miR-127-5p overexpression. The binding between circ_0134944 and miR-127-5p, PDX1 and miR-127-5p were confirmed by dual-luciferase reporter assay. Moreover, PDX1 was enriched in the promoter region of SPHK1, and SPHK1 overexpression prevented the promotion of osteogenesis induced by miR-127-5p overexpression.
Conclusions: Taken together, these results demonstrate that circ_0134944 inhibit osteogenesis via miR-127-5p/PDX1/SPHK1 axis. Thus, the present study offered evidence that circ_0134944/miR-127-5p/PDX1/SPHK1 axis could be a potential therapeutic target for PMOP.

Keywords:  Osteogenesis; PDX1; SPHK1; circ_0134944; miR-127-5p

DOI:  https://doi.org/10.1016/j.reth.2021.09.004
Mol Med. 2021 Nov 03. 27(1): 141

lncRNA SNHG1 induced by SP1 regulates bone remodeling and angiogenesis via sponging miR-181c-5p and modulating SFRP1/Wnt signaling pathway.

Xiao Yu, Peng-Ze Rong, Meng-Sheng Song, Ze-Wen Shi, Gong Feng, Xian-Jun Chen, Lin Shi, Cheng-Hao Wang, Qing-Jiang Pang.

   BACKGROUND: We aimed to investigate the functions and underlying mechanism of lncRNA SNHG1 in bone differentiation and angiogenesis in the development of osteoporosis.
METHODS: The differential gene or proteins expressions were measured by qPCR or western blot assays, respectively. The targeted relationships among molecular were confirmed through luciferase reporter, RIP and ChIP assays, respectively. Alkaline phosphatase (ALP), alizarin red S (ARS) and TRAP staining were performed to measure the osteoblast/osteoclast differentiation of BMSCs. The viability, migration and angiogenesis in BM-EPCs were validated by CCK-8, clone formation, transwell and tube formation assays, respectively. Western blot and immunofluorescence detected the cytosolic/nuclear localization of β-catenin. Ovariectomized (OVX) mice were established to confirm the findings in vitro.
RESULTS: SNHG1 was enhanced and miR-181c-5p was decreased in serum and femoral tissue from OVX mice. SNHG1 directly inhibited miR-181c-5p to activate Wnt3a/β-catenin signaling by upregulating SFRP1. In addition, knockdown of SNHG1 promoted the osteogenic differentiation of BMSCs by increasing miR-181c-5p. In contrast, SNHG1 overexpression advanced the osteoclast differentiation of BMSCs and inhibited the angiogenesis of BM-EPCs, whereas these effects were all reversed by miR-181c-5p overexpression. In vivo experiments indicated that SNHG1 silencing alleviated osteoporosis through stimulating osteoblastogenesis and inhibiting osteoclastogenesis by modulating miR-181c-5p. Importantly, SNHG1 could be induced by SP1 in BMSCs.
CONCLUSIONS: Collectively, SP1-induced SNHG1 modulated SFRP1/Wnt/β-catenin signaling pathway via sponging miR-181c-5p, thereby inhibiting osteoblast differentiation and angiogenesis while promoting osteoclast formation. Further, SNHG1 silence might provide a potential treatment for osteoporosis.

Keywords:  Angiogenesis; Bone remodeling; LncRNA SNHG1; SFRP1; Wnt signal; miR-181c-5p

DOI:  https://doi.org/10.1186/s10020-021-00392-2
J Orthop Surg Res. 2021 Oct 30. 16(1): 652

Investigation of the role of the miR17-92 cluster in BMP9-induced osteoblast lineage commitment.

Yunyuan Zhang, Xuran Jing, Zhongzhu Li, Qingwu Tian, Qing Wang, Xian Chen.

   BACKGROUND: Bone morphogenetic protein 9 (BMP9) has been identified as a crucial inducer of osteoblastic differentiation in mesenchymal stem cells (MSCs). Although microRNAs (miRNAs) are known to play a role in MSC osteogenesis, the mechanisms of action of miRNAs in BMP9-induced osteoblastic differentiation remain poorly understood.
METHODS: In this study, we investigate the possible role of the miR17-92 cluster in the BMP9-induced osteogenic differentiation of MSCs by using both in vitro and in vivo bone formation assays.
RESULTS: The results show that miR-17, a member of the miR17-92 cluster, significantly impairs BMP9-induced osteogenic differentiation. This impairment is effectively rescued by a miR-17 sponge, an antagomiR sequence against miR-17. Using TargetScan and the 3'-untranslated region luciferase reporter assays, we show that the direct target of miR-17 is the retinoblastoma gene (RB1), a gene that is pivotal to osteoblastic differentiation. We also confirm that RB1 is essential for the miR-17 effects on osteogenesis.
CONCLUSION: Our results indicate that miR-17 expression impairs normal osteogenesis by downregulating RB1 expression and significantly inhibiting the function of BMP9.

Keywords:  BMP9; Osteogenesis; Rb; miR 17-92

DOI:  https://doi.org/10.1186/s13018-021-02804-9
J Cell Mol Med. 2021 Nov 05.

Exosomal miR-21-5p derived from bone marrow mesenchymal stem cells promote osteosarcoma cell proliferation and invasion by targeting PIK3R1.

Jin Qi, Ruihao Zhang, Yapeng Wang.

  Mesenchymal stem cells (MSCs) are a class of pluripotent cells that can release a large number of exosomes which act as paracrine mediators in tumour-associated microenvironment. However, the role of MSC-derived exosomes in pathogenesis and progression of cancer cells especially osteosarcoma has not been thoroughly clarified until now. In this study, we established a co-culture model for human bone marrow-derived MSCs with osteosarcoma cells, then extraction of exosomes from induced MSCs and study the role of MSC-derived exosomes in the progression of osteosarcoma cell. The aim of this study was to address potential cell biological effects between MSCs and osteosarcoma cells. The results showed that MSC-derived exosomes can significantly promote osteosarcoma cells' proliferation and invasion. We also found that miR-21-5p was significantly over-expressed in MSCs and MSC-derived exosomes by quantitative real-time polymerase chain reaction (qRT-PCR), compared with human foetal osteoblastic cells hFOB1.19. MSC-derived exosomes transfected with miR-21-5p could significantly enhance the proliferation and invasion of osteosarcoma cells in vitro and in vivo. Bioinformatics analysis and dual-luciferase reporter gene assays validated the targeted relationship between exosomal miR-21-5p and PIK3R1; we further demonstrated that miR-21-5p-abundant exosomes derived human bone marrow MSCs could activate PI3K/Akt/mTOR pathway by suppressing PIK3R1 expression in osteosarcoma cells. In summary, our study provides new insights into the interaction between human bone marrow MSCs and osteosarcoma cells in tumour-associated microenvironment.

Keywords:  PI3K/Akt/mTOR pathway; PIK3R1; exosomes; mesenchymal stem cells; miR-21-5p; osteosarcoma

DOI:  https://doi.org/10.1111/jcmm.17024
Exp Mol Med. 2021 Nov 04.

m6A-mediated upregulation of AC008 promotes osteoarthritis progression through the miR-328-3p‒AQP1/ANKH axis.

Jiashu Yang, Ming Zhang, Dawei Yang, Yunfei Ma, Yuting Tang, Mengying Xing, Lingyun Li, Li Chen, Yucui Jin, Changyan Ma.

Long noncoding RNAs (lncRNAs) have emerged as important regulators of osteoarthritis (OA), but the biological roles and clinical significance of most lncRNAs in OA are not fully understood. Microarray analysis was performed to identify differentially expressed lncRNAs, mRNAs, and miRNAs between normal and osteoarthritic cartilage. We found that AC008440.5 (abbreviated AC008), as well as AQP1 and ANKH, were highly expressed in osteoarthritic cartilage, whereas miR-328-3p was expressed at a low level in osteoarthritic cartilage. Functional assays showed that ectopic expression of AC008, AQP1, and ANKH significantly decreased chondrocyte viability and promoted chondrocyte apoptosis and extracellular matrix (ECM) degradation, whereas knockdown of AC008, AQP1, and ANKH resulted in the opposite effects. Moreover, miR-328-3p overexpression increased chondrocyte viability and attenuated chondrocyte apoptosis and ECM degradation, whereas inhibition of miR-328-3p resulted in the opposite effects. Bioinformatics analysis, RNA immunoprecipitation (RIP), and luciferase assays revealed that AC008 functioned as a competing endogenous RNA (ceRNA) to regulate miR-328-3p, which specifically targeted the AQP1 and ANKH genes. In addition, miR-328-3p significantly ameliorated MIA-induced OA, whereas AC008 accelerated OA progression in vivo. Furthermore, fat mass and obesity-associated (FTO)-mediated N6-methyladenosine demethylation downregulated AC008 transcription, while lower FTO expression led to upregulation of AC008 transcription in OA. In conclusion, our data reveal that AC008 plays a critical role in OA pathogenesis via the miR-328-3p‒AQP1/ANKH pathway, suggesting that AC008 may be a potential therapeutic target for OA.

DOI: https://doi.org/10.1038/s12276-021-00696-7
Autoimmunity. 2021 Nov 03. 1-11

MiR-144-3p induced by SP1 promotes IL-1β-induced pyroptosis in chondrocytes via PTEN/PINK1/Parkin axis.

Jin-Mei Jiang, Mei-Li Mo, Xiao-Ping Long, Li-Hu Xie.

  Rheumatoid arthritis (RA) often leads to functional disabilities and deformities. MiRNA plays a vital role in cell pyroptosis. Nevertheless, the function and underlying mechanism of miR-144-3p in pyroptosis during the progression of RA remains unclear. In this study, N1511 cells were stimulated with IL-1β to construct a RA model. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was performed to assess the cell viability. Cell pyroptosis was detected by flow cytometry. The levels of inflammatory cytokines (TNF-α, IL-6, and IL-18) were assessed by enzyme-linked immunosorbent assay (ELISA). The relationship among specific protein 1 (SP1), microRNA-144-3p (miR-144-3p), and phosphatase and tensin homolog (PTEN) was explored by dual-luciferase reporter assay, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP), respectively. The level of miR-144-3p in N1511 cells was upregulated by IL-1β. MiR-144-3p knockdown inhibited IL-1β-induced pyroptosis in N1511 cells, and the expressions of NOD-like receptor family pyrin domain containing 3 (NLRP3), Cleaved caspase-1, Gasdermin D (GSDMD), and Cleaved caspase-3 in IL-1β-stimulated N1511 cells were increased. The levels of inflammatory cytokines in N1511 cells were increased by IL-1β, which were restored by miR-144-3p knockdown. MiR-144-3p knockdown abolished IL-1β-induced inactivation of putative kinase 1 (PINK1)/Parkin RBR E3 ubiquitin-protein (Parkin) signalling. Moreover, transcription factor SP1 could upregulate miR-144-3p expression and miR-144-3p negatively regulated PTEN expression. In summary, MiR-144-3p induced by SP1 could promote IL-1β-induced chondrocyte pyroptosis via inhibiting PTEN expression and suppressing the activation of PINK1/Parkin signalling, which provided a new strategy against RA.

Keywords:  Rheumatoid arthritis; SP1; inflammatory responses; miR-144-3p; pyroptosis

DOI:  https://doi.org/10.1080/08916934.2021.1983802
Bioengineered. 2021 Nov 05.

Bone mesenchymal stem cells-derived miR-223-3p-containing exosomes ameliorate lipopolysaccharide-induced acute uterine injury via interacting with endothelial progenitor cells.

Yana Liu, Shihong Zhang, Zhiwei Xue, Xiaoxia Zhou, Lin Tong, Jiachen Liao, Huan Pan, Shu Zhou.

  Bone mesenchymal stem cells (BMSCs) have been used for the treatment of acute uterine injury (AUI)-induced intrauterine adhesion (IUA) via interacting with the endothelial progenitor cells (EPCs), and BMSCs-derived exosomes (BMSCs-exo) may be the key regulators for this process. However, the underlying mechanisms have not been studied. Based on the existed literatures, lipopolysaccharide (LPS) was used to induce AUI in mice models and EPCs to mimic the realistic pathogenesis of IUA in vivo and in vitro. Our data suggested that LPS induced apoptotic and pyroptotic cell death in mice uterine horn tissues and EPCs, and the clinical data supported that increased levels of pro-inflammatory cytokines IL-18 and IL-1β were also observed in IUA patients' serum samples, and silencing of NLRP3 rescued cell viability in LPS-treated EPCs. Next, the LPS-treated EPCs were respectively co-cultured with BMSCs in the Transwell system and BMSCs-exo, and the results hinted that both BMSCs and BMSCs-exo reversed the promoting effects of LPS treatment-induced cell death in EPCs. Then, we screened out miR-223-3p, as the upstream regulator for NLRP3, was enriched in BMSCs-exo, and BMSCs-exo inactivated NLRP3-mediated cell pyroptosis in EPCs via delivering miR-223-3p. Interestingly, upregulation of miR-223-3p attenuated LPS-induced cell death in EPCs. Collectively, we concluded that BMSCs-exo upregulated miR-223-3p to degrade NLRP3 in EPCs, which further reversed the cytotoxic effects of LPS treatment on EPCs to ameliorate LPS-induced AUI.

Keywords:  NLRP3-mediated pyroptotic cell death; bone mesenchymal stem cells; endothelial progenitor cells; intrauterine adhesion; miR-223-3p

DOI:  https://doi.org/10.1080/21655979.2021.2001185
Cell Death Dis. 2021 Oct 30. 12(11): 1037

MicroRNA-106a regulates autophagy-related cell death and EMT by targeting TP53INP1 in lung cancer with bone metastasis.

Lei Han, Zeyong Huang, Yan Liu, Lijuan Ye, Dongqi Li, Zhihong Yao, Cao Wang, Ya Zhang, Hang Yang, Zunxian Tan, Jiadai Tang, Zuozhang Yang.

Bone metastasis is one of the most serious complications in lung cancer patients. MicroRNAs (miRNAs) play important roles in tumour development, progression and metastasis. A previous study showed that miR-106a is highly expressed in the tissues of lung adenocarcinoma with bone metastasis, but its mechanism remains unclear. In this study, we showed that miR-106a expression is dramatically increased in lung cancer patients with bone metastasis (BM) by immunohistochemical analysis. MiR-106a promoted A549 and SPC-A1 cell proliferation, migration and invasion in vitro. The results of bioluminescence imaging (BLI), micro-CT and X-ray demonstrated that miR-106a promoted bone metastasis of lung adenocarcinoma in vivo. Mechanistic investigations revealed that miR-106a upregulation promoted metastasis by targeting tumour protein 53-induced nuclear protein 1 (TP53INP1)-mediated metastatic progression, including cell migration, autophagy-dependent death and epithelial-mesenchymal transition (EMT). Notably, autophagy partially attenuated the effects of miR-106a on promoting bone metastasis in lung adenocarcinoma. These findings demonstrated that restoring the expression of TP53INP1 by silencing miR-106a may be a novel therapeutic strategy for bone metastatic in lung adenocarcinoma.

DOI: https://doi.org/10.1038/s41419-021-04324-0
Pulm Pharmacol Ther. 2021 Nov 02. pii: S1094-5539(21)00108-5. [Epub ahead of print] 102096

HDAC8-inhibitor PCI-34051-induced exosomes inhibit human bronchial smooth muscle cell proliferation via miR-381-3p mediated TGFB3.

Shi-Yao Bai, Meng-Lu Li, Yuan Ren, Xin-Ming Su.

  The present study aimed to investigate the effects of PCI-34051-induced human bronchial epithelial cells (HBECs)-derived exosomes (PCI-Exo) on human bronchial smooth muscle cells (HBSMCs) and the key exosomal miRNAs involved in this process. Blank exosomes (Exo) and PCI-Exo were extracted from HBECs treated with PBS and PCI-34051, respectively. RNA-sequencing was performed to uncover the miRNA expression profile affected by PCI-Exo. The MTT, flow cytometry and TUNEL assays were performed to reveal the effect of PCI-34051 and PCI-Exo on the proliferation and apoptosis of HBSMCs. Western blotting and qRT-PCR were used for detecting protein and mRNA expression. A total of 25 exosomal miRNAs consisted of 17 down-regulated and eight up-regulated miRNAs were differentially expressed among PCI-Exo and Exo. Target genes of the exosomal miRNAs were mainly associated with signal transduction, cell adhesion, microRNAs in cancer, and ECM receptor interaction. miR-381-3p was identified as the most significant upregulated differential miRNA in PCI-Exo after qRT-PCR validation and could be transferred to HBSMCs by PCI-Exo. PCI-Exo treatment inhibited the proliferation but induced the apoptosis of HBSMCs. TGFβ3 was identified as a target gene of miR-381-3p which could directly bind to the 3'UTR of TGFβ3 mRNA. After transfecting the miR-381-3p mimic into HBSMCs, the proliferation inhibition and apoptosis rate of HBSMCs was significantly increased, and siTGFβ3 transfection showed similar effects. Moreover, miR-381-3p overexpression could not only decrease the expression of α-SMA, FN1 and collagen I but also increase that of E-cadherin in HBSMCs. Our findings suggested that PCI-Exo could hinder the proliferation and obviously induce the apoptosis of HBSMCs, and its mechanisms might partly be attributable to the reduction of TGFβ3 level by up-regulating exosomal miR-381-3p expression. These results may be vital for the treatment of lung related-diseases, especially asthma.

Keywords:  Asthma; PCI-34051; TGFβ3; exosomes; miR-381-3p

DOI:  https://doi.org/10.1016/j.pupt.2021.102096