The Journal of Clinical Investigation最新文献

{"title":"TSC/mTORC1 mediates mTORC2/AKT1 signaling in c-MYC-induced murine hepatocarcinogenesis via centromere protein M.","authors":"Yi Zhou,Shu Zhang,Guoteng Qiu,Xue Wang,Andrew Yonemura,Hongwei Xu,Guofei Cui,Shanshan Deng,Joanne Chun,Nianyong Chen,Meng Xu,Xinhua Song,Jingwen Wang,Zijing Xu,Youping Deng,Matthias Evert,Diego F Calvisi,Shumei Lin,Haichuan Wang,Xin Chen","doi":"10.1172/jci174415","DOIUrl":"https://doi.org/10.1172/jci174415","url":null,"abstract":"Activated mTORC2/AKT signaling plays a role in hepatocellular carcinoma (HCC). Research has shown that TSC/mTORC1 and FOXO1 are distinct downstream effectors of AKT signaling in liver regeneration and metabolism. However, the mechanisms by which these pathways mediate mTORC2/AKT activation in HCC are not yet fully understood. Amplification and activation of c-MYC is a key molecular event in HCC. In this study, we explored the roles of TSC/mTORC1 and FOXO1 as downstream effectors of mTORC2/AKT1 in c-MYC-induced hepatocarcinogenesis. Using various genetic approaches in mice, we found that manipulating the FOXO pathway had minimal impact on c-MYC-induced HCC. In contrast, loss of mTORC2 inhibited c-MYC-induced HCC, an effect that was completely reversed by ablating TSC2, which activated mTORC1. Additionally, we discovered that p70/RPS6 and 4EBP1/eIF4E act downstream of mTORC1, regulating distinct molecular pathways. Notably, the 4EBP1/eIF4E cascade is crucial for cell proliferation and glycolysis in c-MYC-induced HCC. We also identified centromere protein M (CENPM) as a downstream target of the TSC2/mTORC1 pathway in c-MYC-driven hepatocarcinogenesis, and its ablation entirely inhibited c-MYC-dependent HCC formation. Our findings demonstrate that the TSC/mTORC1/CENPM pathway, rather than the FOXO cascade, is the primary signaling pathway regulating c-MYC-driven hepatocarcinogenesis. Targeting CENPM holds therapeutic potential for treating c-MYC-driven HCC.","PeriodicalId":520097,"journal":{"name":"The Journal of Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142328891","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"YTHDF1 loss in dendritic cells potentiates radiation-induced antitumor immunity via STING-dependent type I IFN production.","authors":"Chuangyu Wen,Liangliang Wang,András Piffkó,Dapeng Chen,Xianbin Yu,Katarzyna Zawieracz,Jason Bugno,Kaiting Yang,Emile Z Naccasha,Fei Ji,Jiaai Wang,Xiaona Huang,Stephen Y Luo,Lei Tan,Bin Shen,Cheng Luo,Megan E McNerney,Steven J Chmura,Ainhoa Arina,Sean P Pitroda,Chuan He,Hua Liang,Ralph R Weichselbaum","doi":"10.1172/jci181612","DOIUrl":"https://doi.org/10.1172/jci181612","url":null,"abstract":"RNA N6-methyladenosine (m6A) reader YTHDF1 is implicated in cancer etiology and progression. We discovered that radiotherapy (RT) increased YTHDF1 expression in dendritic cells (DCs) of PBMCs from cancer patients, but not in other immune cells tested. Elevated YTHDF1 expression of DCs was associated with poor outcomes in patients receiving RT. We found that loss of Ythdf1 in DCs enhanced the antitumor effects of ionizing radiation (IR) via increasing the cross-priming capacity of DCs across multiple murine cancer models. Mechanistically, IR upregulated YTHDF1 expression in DCs through STING-IFN-I signaling. YTHDF1 in turn triggered STING degradation by increasing lysosomal cathepsins, thereby reducing IFN-I production. We created a YTHDF1 deletion/inhibition prototype DC vaccine, significantly improving the therapeutic effect of RT and radio-immunotherapy in a murine melanoma model. Our findings reveal a new layer of regulation between YTHDF1/m6A and STING in response to IR, which opens new paths for the development of YTHDF1-targeting therapies.","PeriodicalId":520097,"journal":{"name":"The Journal of Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142328898","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Stimulation of an entorhinal-hippocampal extinction circuit facilitates fear extinction in a post-traumatic stress disorder model.","authors":"Ze-Jie Lin,Xue Gu,Wan-Kun Gong,Mo Wang,Yan-Jiao Wu,Qi Wang,Xin-Rong Wu,Xin-Yu Zhao,Michael X Zhu,Lu-Yang Wang,Quanying Liu,Ti-Fei Yuan,Wei-Guang Li,Tian-Le Xu","doi":"10.1172/jci181095","DOIUrl":"https://doi.org/10.1172/jci181095","url":null,"abstract":"Effective psychotherapy of post-traumatic stress disorder (PTSD) remains challenging due to the fragile nature of fear extinction, for which ventral hippocampal CA1 (vCA1) region is considered as a central hub. However, neither the core pathway nor the cellular mechanisms involved in implementing extinction are known. Here, we unveil a direct pathway, where layer 2a fan cells in the lateral entorhinal cortex (LEC) target parvalbumin-expressing interneurons (PV-INs) in the vCA1 region to propel low gamma-band synchronization of the LEC-vCA1 activity during extinction learning. Bidirectional manipulations of either hippocampal PV-INs or LEC fan cells sufficed fear extinction. Gamma entrainment of vCA1 by deep brain stimulation (DBS) or noninvasive transcranial alternating current stimulation (tACS) of LEC persistently enhanced the PV-IN activity in vCA1, thereby promoting fear extinction. These results demonstrate that the LEC-vCA1 pathway forms a top-down motif to empower low gamma-band oscillations that facilitate fear extinction. Finally, application of low gamma DBS and tACS to a mouse model with persistent PTSD showed potent efficacy, suggesting that the dedicated LEC-vCA1 pathway can be stimulated for therapy to remove traumatic memory trace.","PeriodicalId":520097,"journal":{"name":"The Journal of Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142321025","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Modeling primary microcephaly with human brain organoids reveals fundamental roles of CIT kinase activity.","authors":"Gianmarco Pallavicini,Amanda Moccia,Giorgia Iegiani,Roberta Parolisi,Emily R Peirent,Gaia Elena Berto,Martina Lorenzati,Rami Y Tshuva,Alessia Ferraro,Fiorella Balzac,Emilia Turco,Shachi U Salvi,Hedvig F Myklebust,Sophia Wang,Julia Eisenberg,Maushmi Chitale,Navjit S Girgla,Enrica Boda,Orly Reiner,Annalisa Buffo,Ferdinando Di Cunto,Stephanie L Bielas","doi":"10.1172/jci175435","DOIUrl":"https://doi.org/10.1172/jci175435","url":null,"abstract":"Brain size and cellular heterogeneity are tightly regulated by species-specific proliferation and differentiation of multipotent neural progenitor cells (NPCs). Errors in this process are among the mechanisms of primary hereditary microcephaly (MCPH), a group of disorders characterized by reduced brain size and intellectual disability. Biallelic CIT missense variants that disrupt kinase function (CITKI/KI) and frameshift loss-of-function variants (CITFS/FS) are the genetic basis for MCPH17; however, the function of CIT catalytic activity in brain development and NPC cytokinesis is unknown. Therefore, we created the CitKI/KI mouse model and found that it does not phenocopy human microcephaly, unlike biallelic CitFS/FS animals. Nevertheless, both Cit models exhibited binucleation, DNA damage, and apoptosis. To investigate human-specific mechanisms of CIT microcephaly, we generated CITKI/KI and CITFS/FS human forebrain organoids. We found that CITKI/KI and CITFS/FS organoids lose cytoarchitectural complexity, transitioning from pseudostratified to simple neuroepithelium. This change was associated with defects that disrupt polarity of NPC cytokinesis, in addition to elevating apoptosis. Together, our results indicate that both CIT catalytic and scaffolding functions in NPC cytokinesis are critical for human corticogenesis. Species differences in corticogenesis and the dynamic 3D features of NPC mitosis underscore the utility of human forebrain organoid models for understanding human microcephaly.","PeriodicalId":520097,"journal":{"name":"The Journal of Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142321028","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Innate immune cell activation by adjuvant AS01 in human lymph node explants is age-independent.","authors":"Vicki V Stylianou,Kirstie M Bertram,Van Anh Vo,Elizabeth B Dunn,Heeva Baharlou,Darcii J Terre,James Elhindi,Elisabeth Elder,James French,Farid Meybodi,Stéphane T Temmerman,Arnaud M Didierlaurent,Margherita Coccia,Kerrie J Sandgren,Anthony L Cunningham","doi":"10.1172/jci174144","DOIUrl":"https://doi.org/10.1172/jci174144","url":null,"abstract":"Vaccine adjuvants are thought to work by stimulating innate immunity in the draining lymph node (LN), although this has not been proven in humans. To bridge data obtained in animals to humans, we have developed an in situ human LN explant model to investigate how adjuvants initiate immunity. Slices of explanted LNs were exposed to vaccine adjuvants and revealed responses that were not detectable in LN cell suspensions. We used this model to compare the liposome-based AS01 with its components MPL and QS-21, and TLR ligands. Liposomes were predominantly taken up by subcapsular sinus-lining macrophages, monocytes and dendritic cells. AS01 induced dendritic cell maturation and a strong pro-inflammatory cytokine response in intact LN slices but not in dissociated cell cultures, in contrast to R848. This suggests the onset of the immune response to AS01 requires a coordinated activation of LN cells in time and space. Consistent with the robust immune response observed in older adults with AS01-adjuvanted vaccines, the AS01 response in human LNs was independent of age, unlike R848. This human LN explant model is a valuable tool for studying the mechanism of action of adjuvants in humans and for screening new formulations to streamline vaccine development.","PeriodicalId":520097,"journal":{"name":"The Journal of Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142321026","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pharmacological regeneration of sensory hair cells restores afferent innervation and vestibular function.","authors":"Hanae Lahlou,Hong Zhu,Wu Zhou,Albert Sb Edge","doi":"10.1172/jci181201","DOIUrl":"https://doi.org/10.1172/jci181201","url":null,"abstract":"The sensory cells that transduce the signals for hearing and balance are highly specialized mechanoreceptors called hair cells that reside in the sensory epithelia of the inner ear. Loss of hair cells from toxin exposure and age can cause balance disorders and is essentially irreversible due to the inability of mammalian vestibular organs to regenerate physiologically active hair cells. Here, we show substantial regeneration of hair cells in a mouse model of vestibular damage by treatment with a combination of glycogen synthase kinase 3β and histone deacetylase inhibitors. The drugs stimulated supporting cell proliferation and differentiation into hair cells. The new hair cells were reinnervated by vestibular afferent neurons, rescuing otolith function by restoring head translation-evoked otolith afferent responses and vestibuloocular reflexes. Drugs that regenerate hair cells thus represent a potential therapeutic approach to the treatment of balance disorders.","PeriodicalId":520097,"journal":{"name":"The Journal of Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142321034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proteogenomic analysis integrated with electronic health records data reveals disease-associated variants in Black Americans.","authors":"Usman A Tahir,Jacob L Barber,Daniel E Cruz,Meltem Ece Kars,Shuliang Deng,Bjoernar Tuftin,Madeline G Gillman,Mark D Benson,Jeremy M Robbins,Zsu-Zsu Chen,Prashant Rao,Daniel H Katz,Laurie Farrell,Tamar Sofer,Michael E Hall,Lynette Ekunwe,Russell P Tracy,Peter Durda,Kent D Taylor,Yongmei Liu,W Craig Johnson,Xiuqing Guo,Yii-Der Ida Chen,Ani W Manichaikul,Deepti Jain,Thomas J Wang,Alex P Reiner,Pradeep Natarajan,Yuval Itan,Stephen S Rich,Jerome I Rotter,James G Wilson,Laura M Raffield,Robert E Gerszten","doi":"10.1172/jci181802","DOIUrl":"https://doi.org/10.1172/jci181802","url":null,"abstract":"BACKGROUNDMost genome wide association studies (GWAS) of plasma proteomics have focused on White individuals of European ancestry, limiting biological insight from other ancestry enriched protein quantitative loci (pQTL).METHODSWe conducted a discovery GWAS of ~3,000 plasma proteins measured by the antibody based Olink platform in 1,054 Black adults from the Jackson Heart Study (JHS), and validated our findings in the Multi-Ethnic Study of Atherosclerosis (MESA). The genetic architecture of identified pQTLs were further explored through fine mapping and admixture association analysis. Finally, using our pQTL findings, we performed a phenome wide association study (PheWAS) across two large multi-ethnic electronic health record (EHR) systems in All of Us and BioMe.RESULTSWe identified 1002 pQTLs for 925 proteins. Fine mapping and admixture analyses suggested allelic heterogeneity of the plasma proteome across diverse populations. We identified associations for variants enriched in African ancestry, many in diseases that lack precise biomarkers, including cis-pQTLs for Cathepsin L (CTSL) and Siglec-9 that were linked with sarcoidosis and non-Hodgkin's lymphoma, respectively. We found concordant associations across clinical diagnoses and laboratory measurements, elucidating disease pathways, including a cis-pQTL associated with circulating CD58, white blood cell count, and multiple sclerosis.CONCLUSIONSOur findings emphasize the value of leveraging diverse populations to enhance biological insights from proteomics GWAS, and we have made this resource readily available as an interactive web portal.","PeriodicalId":520097,"journal":{"name":"The Journal of Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142321024","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Long-lived lung megakaryocytes contribute to platelet recovery in thrombocytopenia models.","authors":"Alison C Livada,Kathleen E McGrath,Michael W Malloy,Chen Li,Sara K Ture,Paul D Kingsley,Anne D Koniski,Leah A Vit,Katherine E Nolan,Deanne Mickelsen,Grace E Monette,Preeti Maurya,James Palis,Craig N Morrell","doi":"10.1172/jci181111","DOIUrl":"https://doi.org/10.1172/jci181111","url":null,"abstract":"Lung megakaryocytes (Mks) are largely extravascular with an immune phenotype (1). Because bone marrow (BM) Mks are short-lived it has been assumed that extravascular lung Mks are constantly 'seeded' from the BM. To investigate lung Mk origins and how that impacts their functions, we developed methods to specifically label lung Mks using CFSE dye and biotin delivered oropharyngeal. Labeled lung Mks were present for up to four months, while BM Mks had a <1 week lifespan. In a parabiosis model, lung Mks were partially replaced over 1-month from a circulating source. Unlike tissue-resident macrophages, using MDS1-Cre-ERT2 TdTomato mice, we found that lung Mks arise from hematopoietic stem cells. However, studies with FlkSwitch mTmG mice showed that lung Mks are derived from a Flt3-independent lineage that does not go through a multipotent progenitor. CFSE labeling to track lung Mk-derived platelets showed that about 10% of circulating platelets are lung resident Mk-derived at steady state, but in sterile thrombocytopenia this was doubled (about 20%). Lung-derived platelets were similarly increased in a malaria infection model (Plasmodium yoelii) typified by thrombocytopenia. These studies indicate that lung Mks arise from a Flt3-negative BM source, are long-lived, and contribute more platelets during thrombocytopenia.","PeriodicalId":520097,"journal":{"name":"The Journal of Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142273500","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PAC1 constrains type II inflammation through promotion of CGRP signaling in ILC2s.","authors":"Yuan Jin,Bowen Liu,Qiuyu Li,Xiangyan Meng,Xiaowei Tang,Yan Jin,Yuxin Yin","doi":"10.1172/jci180109","DOIUrl":"https://doi.org/10.1172/jci180109","url":null,"abstract":"Dysfunction of group II innate lymphoid cells (ILC2s) plays an important role in the development of type II inflammation-related diseases such as asthma and pulmonary fibrosis. Notably, neural signals are increasingly recognized as pivotal regulators of ILC2s. However, how ILC2s intrinsically modulate their responsiveness to these neural signals is still largely unknown. Here, using single-cell RNA sequencing, we found that the immune regulatory molecule PAC1 (phosphatase of activated cells 1) selectively promotes the signaling of neuropeptide CGRP (calcitonin gene-related peptide) in ILC2s through a cell-intrinsic manner. Genetic ablation of PAC1 in ILC2s substantially impaired the inhibitory effect of CGRP on proliferation and IL-13 secretion. PAC1 deficiency significantly exacerbated allergic airway inflammation induced by Alternaria alternata or papain in mice. Moreover, in human circulating ILC2s, the expression level of PAC1 was also significantly negatively correlated with the cell amount and the expression level of IL13. Mechanistically, PAC1 was necessary for ensuring the expression of CGRP-response genes by influencing chromatin accessibility. In summary, our study demonstrated that PAC1 is an important regulator of ILC2 responses and we proposed that PAC1 is a potential target for therapeutic interventions of type II inflammation-related diseases.","PeriodicalId":520097,"journal":{"name":"The Journal of Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142245512","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CRISPR-Cas13d targeting suppresses repeat-associated non-AUG translation of C9orf72 hexanucleotide repeat RNA.","authors":"Honghe Liu,Xiao-Feng Zhao,Yu-Ning Lu,Lindsey R Hayes,Jiou Wang","doi":"10.1172/jci179016","DOIUrl":"https://doi.org/10.1172/jci179016","url":null,"abstract":"A hexanucleotide GGGGCC repeat expansion in the non-coding region of C9orf72 gene is the most common genetic mutation identified in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The resulting repeat RNA and dipeptide repeat proteins from non-conventional repeat translation have been recognized as important markers associated with the diseases. CRISPR-Cas13d, a powerful RNA targeting tool, has faced challenges in effectively targeting RNA with stable secondary structures. Here we report that CRISPR-Cas13d can be optimized to specifically target GGGGCC repeat RNA. Our results demonstrate that the CRISPR-Cas13d system can be harnessed to significantly diminish the translation of poly-dipeptides originating from the GGGGCC repeat RNA. This efficacy has been validated in various cell types, including induced pluripotent stem cells and differentiated motor neurons originating from C9orf72-ALS patients, as well as in C9orf72 repeat transgenic mice. These findings demonstrate the application of CRISPR-Cas13d in targeting RNA with intricate higher-order structures and suggest a potential therapeutic approach for ALS and FTD.","PeriodicalId":520097,"journal":{"name":"The Journal of Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142245511","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}