Pub Date : 2025-12-01Epub Date: 2024-09-06DOI: 10.4103/NRR.NRR-D-24-00129
Zhijian Zheng, Xiaohui Lin, Zijun Zhao, Qiang Lin, Ji Liu, Manli Chen, Wenwen Wu, Zhiyun Wu, Nan Liu, Hongbin Chen
JOURNAL/nrgr/04.03/01300535-202512000-00028/figure1/v/2025-01-31T122243Z/r/image-tiff Adipose-derived stem cell, one type of mesenchymal stem cells, is a promising approach in treating ischemia-reperfusion injury caused by occlusion of the middle cerebral artery. However, its application has been limited by the complexities of the ischemic microenvironment. Hydrogel scaffolds, which are composed of hyaluronic acid and chitosan, exhibit excellent biocompatibility and biodegradability, making them promising candidates as cell carriers. Vascular endothelial growth factor is a crucial regulatory factor for stem cells. Both hyaluronic acid and chitosan have the potential to make the microenvironment more hospitable to transplanted stem cells, thereby enhancing the therapeutic effect of mesenchymal stem cell transplantation in the context of stroke. Here, we found that vascular endothelial growth factor significantly improved the activity and paracrine function of adipose-derived stem cells. Subsequently, we developed a chitosan-hyaluronic acid hydrogel scaffold that incorporated vascular endothelial growth factor and first injected the scaffold into an animal model of cerebral ischemia-reperfusion injury. When loaded with adipose-derived stem cells, this vascular endothelial growth factor-loaded scaffold markedly reduced neuronal apoptosis caused by oxygen-glucose deprivation/reoxygenation and substantially restored mitochondrial membrane potential and axon morphology. Further in vivo experiments revealed that this vascular endothelial growth factor-loaded hydrogel scaffold facilitated the transplantation of adipose-derived stem cells, leading to a reduction in infarct volume and neuronal apoptosis in a rat model of stroke induced by transient middle cerebral artery occlusion. It also helped maintain mitochondrial integrity and axonal morphology, greatly improving rat motor function and angiogenesis. Therefore, utilizing a hydrogel scaffold loaded with vascular endothelial growth factor as a stem cell delivery system can mitigate the adverse effects of ischemic microenvironment on transplanted stem cells and enhance the therapeutic effect of stem cells in the context of stroke.
{"title":"A vascular endothelial growth factor-loaded chitosan-hyaluronic acid hydrogel scaffold enhances the therapeutic effect of adipose-derived stem cells in the context of stroke.","authors":"Zhijian Zheng, Xiaohui Lin, Zijun Zhao, Qiang Lin, Ji Liu, Manli Chen, Wenwen Wu, Zhiyun Wu, Nan Liu, Hongbin Chen","doi":"10.4103/NRR.NRR-D-24-00129","DOIUrl":"10.4103/NRR.NRR-D-24-00129","url":null,"abstract":"<p><p>JOURNAL/nrgr/04.03/01300535-202512000-00028/figure1/v/2025-01-31T122243Z/r/image-tiff Adipose-derived stem cell, one type of mesenchymal stem cells, is a promising approach in treating ischemia-reperfusion injury caused by occlusion of the middle cerebral artery. However, its application has been limited by the complexities of the ischemic microenvironment. Hydrogel scaffolds, which are composed of hyaluronic acid and chitosan, exhibit excellent biocompatibility and biodegradability, making them promising candidates as cell carriers. Vascular endothelial growth factor is a crucial regulatory factor for stem cells. Both hyaluronic acid and chitosan have the potential to make the microenvironment more hospitable to transplanted stem cells, thereby enhancing the therapeutic effect of mesenchymal stem cell transplantation in the context of stroke. Here, we found that vascular endothelial growth factor significantly improved the activity and paracrine function of adipose-derived stem cells. Subsequently, we developed a chitosan-hyaluronic acid hydrogel scaffold that incorporated vascular endothelial growth factor and first injected the scaffold into an animal model of cerebral ischemia-reperfusion injury. When loaded with adipose-derived stem cells, this vascular endothelial growth factor-loaded scaffold markedly reduced neuronal apoptosis caused by oxygen-glucose deprivation/reoxygenation and substantially restored mitochondrial membrane potential and axon morphology. Further in vivo experiments revealed that this vascular endothelial growth factor-loaded hydrogel scaffold facilitated the transplantation of adipose-derived stem cells, leading to a reduction in infarct volume and neuronal apoptosis in a rat model of stroke induced by transient middle cerebral artery occlusion. It also helped maintain mitochondrial integrity and axonal morphology, greatly improving rat motor function and angiogenesis. Therefore, utilizing a hydrogel scaffold loaded with vascular endothelial growth factor as a stem cell delivery system can mitigate the adverse effects of ischemic microenvironment on transplanted stem cells and enhance the therapeutic effect of stem cells in the context of stroke.</p>","PeriodicalId":19113,"journal":{"name":"Neural Regeneration Research","volume":" ","pages":"3591-3605"},"PeriodicalIF":5.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142154635","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01Epub Date: 2024-10-22DOI: 10.4103/NRR.NRR-D-24-00793
Edric D Winford, Adam D Bachstetter
{"title":"Alzheimer's disease and the immune system: the emerging role of TEMRA cells.","authors":"Edric D Winford, Adam D Bachstetter","doi":"10.4103/NRR.NRR-D-24-00793","DOIUrl":"https://doi.org/10.4103/NRR.NRR-D-24-00793","url":null,"abstract":"","PeriodicalId":19113,"journal":{"name":"Neural Regeneration Research","volume":"20 12","pages":"3529-3530"},"PeriodicalIF":5.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143648179","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01Epub Date: 2025-01-31DOI: 10.4103/NRR.NRR-D-24-01656
{"title":"Corrigendum: Neuroprotection by cattle encephalon glycoside and ignotin beyond the time window of thrombolysis in ischemic stroke.","authors":"","doi":"10.4103/NRR.NRR-D-24-01656","DOIUrl":"https://doi.org/10.4103/NRR.NRR-D-24-01656","url":null,"abstract":"","PeriodicalId":19113,"journal":{"name":"Neural Regeneration Research","volume":"20 12","pages":"3429"},"PeriodicalIF":5.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143648183","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01Epub Date: 2024-12-07DOI: 10.4103/NRR.NRR-D-24-00851
Jing Chi, Bin Fan, Yulin Li, Qing Jiao, Guang-Yu Li
The retina, a crucial neural tissue, is responsible for transforming light signals into visual information, a process that necessitates a significant amount of energy. Mitochondria, the primary powerhouses of the cell, play an integral role in retinal physiology by fulfilling the high-energy requirements of photoreceptors and secondary neurons through oxidative phosphorylation. In a healthy state, mitochondria ensure proper visual function by facilitating efficient conversion and transduction of visual signals. However, in retinal degenerative diseases, mitochondrial dysfunction significantly contributes to disease progression, involving a decline in membrane potential, the occurrence of DNA mutations, increased oxidative stress, and imbalances in quality-control mechanisms. These abnormalities lead to an inadequate energy supply, the exacerbation of oxidative damage, and the activation of cell death pathways, ultimately resulting in neuronal injury and dysfunction in the retina. Mitochondrial transplantation has emerged as a promising strategy for addressing these challenges. This procedure aims to restore metabolic activity and function in compromised cells through the introduction of healthy mitochondria, thereby enhancing the cellular energy production capacity and offering new strategies for the treatment of retinal degenerative diseases. Although mitochondrial transplantation presents operational and safety challenges that require further investigation, it has demonstrated potential for reviving the vitality of retinal neurons. This review offers a comprehensive examination of the principles and techniques underlying mitochondrial transplantation and its prospects for application in retinal degenerative diseases, while also delving into the associated technical and safety challenges, thereby providing references and insights for future research and treatment.
{"title":"Mitochondrial transplantation: a promising strategy for the treatment of retinal degenerative diseases.","authors":"Jing Chi, Bin Fan, Yulin Li, Qing Jiao, Guang-Yu Li","doi":"10.4103/NRR.NRR-D-24-00851","DOIUrl":"10.4103/NRR.NRR-D-24-00851","url":null,"abstract":"<p><p>The retina, a crucial neural tissue, is responsible for transforming light signals into visual information, a process that necessitates a significant amount of energy. Mitochondria, the primary powerhouses of the cell, play an integral role in retinal physiology by fulfilling the high-energy requirements of photoreceptors and secondary neurons through oxidative phosphorylation. In a healthy state, mitochondria ensure proper visual function by facilitating efficient conversion and transduction of visual signals. However, in retinal degenerative diseases, mitochondrial dysfunction significantly contributes to disease progression, involving a decline in membrane potential, the occurrence of DNA mutations, increased oxidative stress, and imbalances in quality-control mechanisms. These abnormalities lead to an inadequate energy supply, the exacerbation of oxidative damage, and the activation of cell death pathways, ultimately resulting in neuronal injury and dysfunction in the retina. Mitochondrial transplantation has emerged as a promising strategy for addressing these challenges. This procedure aims to restore metabolic activity and function in compromised cells through the introduction of healthy mitochondria, thereby enhancing the cellular energy production capacity and offering new strategies for the treatment of retinal degenerative diseases. Although mitochondrial transplantation presents operational and safety challenges that require further investigation, it has demonstrated potential for reviving the vitality of retinal neurons. This review offers a comprehensive examination of the principles and techniques underlying mitochondrial transplantation and its prospects for application in retinal degenerative diseases, while also delving into the associated technical and safety challenges, thereby providing references and insights for future research and treatment.</p>","PeriodicalId":19113,"journal":{"name":"Neural Regeneration Research","volume":" ","pages":"3370-3387"},"PeriodicalIF":5.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143033759","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01Epub Date: 2025-01-13DOI: 10.4103/NRR.NRR-D-24-00865
Letian Huang, Mingyue Liu, Ze Li, Bing Li, Jiahe Wang, Ke Zhang
Amyloid-beta clearance plays a key role in the pathogenesis of Alzheimer's disease. However, the variation in functional proteins involved in amyloid-beta clearance and their correlation with amyloid-beta levels remain unclear. In this study, we conducted meta-analyses and a systematic review using studies from the PubMed, Embase, Web of Science, and Cochrane Library databases, including journal articles published from inception to June 30, 2023. The inclusion criteria included studies comparing the levels of functional proteins associated with amyloid-beta clearance in the blood, cerebrospinal fluid, and brain of healthy controls, patients with mild cognitive impairment, and patients with Alzheimer's disease. Additionally, we analyzed the correlation between these functional proteins and amyloid-beta levels in patients with Alzheimer's disease. The methodological quality of the studies was assessed via the Newcastle‒Ottawa Scale. Owing to heterogeneity, we utilized either a fixed-effect or random-effect model to assess the 95% confidence interval (CI) of the standard mean difference (SMD) among healthy controls, patients with mild cognitive impairment, and patients with Alzheimer's disease. The findings revealed significant alterations in the levels of insulin-degrading enzymes, neprilysin, matrix metalloproteinase-9, cathepsin D, receptor for advanced glycation end products, and P-glycoprotein in the brains of patients with Alzheimer's disease, patients with mild cognitive impairment, and healthy controls. In cerebrospinal fluid, the levels of triggering receptor expressed on myeloid cells 2 and ubiquitin C-terminal hydrolase L1 are altered, whereas the levels of TREM2, CD40, CD40L, CD14, CD22, cathepsin D, cystatin C, and α2 M in peripheral blood differ. Notably, TREM2 and cathepsin D showed changes in both brain (SMD = 0.31, 95% CI: 0.16-0.47, P < 0.001, I2 = 78.4%; SMD = 1.24, 95% CI: 0.01-2.48, P = 0.048, I2 = 90.1%) and peripheral blood (SMD = 1.01, 95% CI: 0.35-1.66, P = 0.003, I2 = 96.5%; SMD = 7.55, 95% CI: 3.92-11.18, P < 0.001, I2 = 98.2%) samples. Furthermore, correlations were observed between amyloid-beta levels and the levels of TREM2 ( r = 0.16, 95% CI: 0.04-0.28, P = 0.009, I2 = 74.7%), neprilysin ( r = -0.47, 95% CI: -0.80-0.14, P = 0.005, I2 = 76.1%), and P-glycoprotein ( r = -0.31, 95% CI: -0.51-0.11, P = 0.002, I2 = 0.0%) in patients with Alzheimer's disease. These findings suggest that triggering receptor expressed on myeloid cells 2 and cathepsin D could serve as potential diagnostic biomarkers for Alzheimer's disease, whereas triggering receptor expressed on myeloid cells 2, neprilysin, and P-glycoprotein may represent potential therapeutic targets.
{"title":"Systematic review of amyloid-beta clearance proteins from the brain to the periphery: implications for Alzheimer's disease diagnosis and therapeutic targets.","authors":"Letian Huang, Mingyue Liu, Ze Li, Bing Li, Jiahe Wang, Ke Zhang","doi":"10.4103/NRR.NRR-D-24-00865","DOIUrl":"10.4103/NRR.NRR-D-24-00865","url":null,"abstract":"<p><p>Amyloid-beta clearance plays a key role in the pathogenesis of Alzheimer's disease. However, the variation in functional proteins involved in amyloid-beta clearance and their correlation with amyloid-beta levels remain unclear. In this study, we conducted meta-analyses and a systematic review using studies from the PubMed, Embase, Web of Science, and Cochrane Library databases, including journal articles published from inception to June 30, 2023. The inclusion criteria included studies comparing the levels of functional proteins associated with amyloid-beta clearance in the blood, cerebrospinal fluid, and brain of healthy controls, patients with mild cognitive impairment, and patients with Alzheimer's disease. Additionally, we analyzed the correlation between these functional proteins and amyloid-beta levels in patients with Alzheimer's disease. The methodological quality of the studies was assessed via the Newcastle‒Ottawa Scale. Owing to heterogeneity, we utilized either a fixed-effect or random-effect model to assess the 95% confidence interval (CI) of the standard mean difference (SMD) among healthy controls, patients with mild cognitive impairment, and patients with Alzheimer's disease. The findings revealed significant alterations in the levels of insulin-degrading enzymes, neprilysin, matrix metalloproteinase-9, cathepsin D, receptor for advanced glycation end products, and P-glycoprotein in the brains of patients with Alzheimer's disease, patients with mild cognitive impairment, and healthy controls. In cerebrospinal fluid, the levels of triggering receptor expressed on myeloid cells 2 and ubiquitin C-terminal hydrolase L1 are altered, whereas the levels of TREM2, CD40, CD40L, CD14, CD22, cathepsin D, cystatin C, and α2 M in peripheral blood differ. Notably, TREM2 and cathepsin D showed changes in both brain (SMD = 0.31, 95% CI: 0.16-0.47, P < 0.001, I2 = 78.4%; SMD = 1.24, 95% CI: 0.01-2.48, P = 0.048, I2 = 90.1%) and peripheral blood (SMD = 1.01, 95% CI: 0.35-1.66, P = 0.003, I2 = 96.5%; SMD = 7.55, 95% CI: 3.92-11.18, P < 0.001, I2 = 98.2%) samples. Furthermore, correlations were observed between amyloid-beta levels and the levels of TREM2 ( r = 0.16, 95% CI: 0.04-0.28, P = 0.009, I2 = 74.7%), neprilysin ( r = -0.47, 95% CI: -0.80-0.14, P = 0.005, I2 = 76.1%), and P-glycoprotein ( r = -0.31, 95% CI: -0.51-0.11, P = 0.002, I2 = 0.0%) in patients with Alzheimer's disease. These findings suggest that triggering receptor expressed on myeloid cells 2 and cathepsin D could serve as potential diagnostic biomarkers for Alzheimer's disease, whereas triggering receptor expressed on myeloid cells 2, neprilysin, and P-glycoprotein may represent potential therapeutic targets.</p>","PeriodicalId":19113,"journal":{"name":"Neural Regeneration Research","volume":" ","pages":"3574-3590"},"PeriodicalIF":5.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143009061","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01Epub Date: 2024-10-22DOI: 10.4103/NRR.NRR-D-24-00809
Marília Inês Móvio, Maria Camila Almeida, Sergio T Ferreira, Alexandre Hiroaki Kihara
{"title":"Shedding light on the retina to see healthy and pathological aging.","authors":"Marília Inês Móvio, Maria Camila Almeida, Sergio T Ferreira, Alexandre Hiroaki Kihara","doi":"10.4103/NRR.NRR-D-24-00809","DOIUrl":"https://doi.org/10.4103/NRR.NRR-D-24-00809","url":null,"abstract":"","PeriodicalId":19113,"journal":{"name":"Neural Regeneration Research","volume":"20 12","pages":"3537-3538"},"PeriodicalIF":5.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143648186","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01Epub Date: 2025-01-13DOI: 10.4103/NRR.NRR-D-24-00628
Ya Zheng, Lingyun Cao, Dan Zhao, Qi Yang, Chunya Gu, Yeran Mao, Guangyue Zhu, Yulian Zhu, Jing Zhao, Dongsheng Xu
JOURNAL/nrgr/04.03/01300535-202512000-00026/figure1/v/2025-01-31T122243Z/r/image-tiff Promoting synaptic plasticity and inducing functional reorganization of residual nerve fibers hold clinical significance for restoring motor function following spinal cord injury. Neuromagnetic stimulation targeting the nerve roots has been shown to improve motor function by enhancing nerve conduction in the injured spinal cord and restoring the synaptic ultrastructure of both the sensory and motor cortex. However, our understanding of the neurophysiological mechanisms by which nerve root magnetic stimulation facilitates motor function recovery in the spinal cord is limited, and its role in neuroplasticity remains unclear. In this study, we established a model of spinal cord injury in adult male Sprague-Dawley rats by applying moderate compression at the T10 vertebra. We then performed magnetic stimulation on the L5 nerve root for 3 weeks, beginning on day 3 post-injury. At day 22 post-injury, we observed that nerve root magnetic stimulation downregulated the level of interleukin-6 in the injured spinal cord tissue of rats. Additionally, this treatment reduced neuronal damage and glial scar formation, and increased the number of neurons in the injured spinal cord. Furthermore, nerve root magnetic stimulation decreased the levels of acetylcholine, norepinephrine, and dopamine, and increased the expression of synaptic plasticity-related mRNA and proteins PSD95, GAP43, and Synapsin II. Taken together, these results showed that nerve root magnetic stimulation alleviated neuronal damage in the injured spinal cord, regulated synaptic plasticity, and suppressed inflammatory responses. These findings provide laboratory evidence for the clinical application of nerve root magnetic stimulation in the treatment of spinal cord injury.
{"title":"Nerve root magnetic stimulation regulates the synaptic plasticity of injured spinal cord by ascending sensory pathway.","authors":"Ya Zheng, Lingyun Cao, Dan Zhao, Qi Yang, Chunya Gu, Yeran Mao, Guangyue Zhu, Yulian Zhu, Jing Zhao, Dongsheng Xu","doi":"10.4103/NRR.NRR-D-24-00628","DOIUrl":"https://doi.org/10.4103/NRR.NRR-D-24-00628","url":null,"abstract":"<p><p>JOURNAL/nrgr/04.03/01300535-202512000-00026/figure1/v/2025-01-31T122243Z/r/image-tiff Promoting synaptic plasticity and inducing functional reorganization of residual nerve fibers hold clinical significance for restoring motor function following spinal cord injury. Neuromagnetic stimulation targeting the nerve roots has been shown to improve motor function by enhancing nerve conduction in the injured spinal cord and restoring the synaptic ultrastructure of both the sensory and motor cortex. However, our understanding of the neurophysiological mechanisms by which nerve root magnetic stimulation facilitates motor function recovery in the spinal cord is limited, and its role in neuroplasticity remains unclear. In this study, we established a model of spinal cord injury in adult male Sprague-Dawley rats by applying moderate compression at the T10 vertebra. We then performed magnetic stimulation on the L5 nerve root for 3 weeks, beginning on day 3 post-injury. At day 22 post-injury, we observed that nerve root magnetic stimulation downregulated the level of interleukin-6 in the injured spinal cord tissue of rats. Additionally, this treatment reduced neuronal damage and glial scar formation, and increased the number of neurons in the injured spinal cord. Furthermore, nerve root magnetic stimulation decreased the levels of acetylcholine, norepinephrine, and dopamine, and increased the expression of synaptic plasticity-related mRNA and proteins PSD95, GAP43, and Synapsin II. Taken together, these results showed that nerve root magnetic stimulation alleviated neuronal damage in the injured spinal cord, regulated synaptic plasticity, and suppressed inflammatory responses. These findings provide laboratory evidence for the clinical application of nerve root magnetic stimulation in the treatment of spinal cord injury.</p>","PeriodicalId":19113,"journal":{"name":"Neural Regeneration Research","volume":"20 12","pages":"3564-3573"},"PeriodicalIF":5.9,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143648185","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}