Histology and histopathology最新文献

{"title":"Daphnetin alleviates renal inflammation, oxidative stress, and apoptosis in septic rats via the JAK2/STAT3 signaling pathway.","authors":"Zhuo Zhang, Pan Hu, Qingye Li, Yingling Wang, Ruliang Yao","doi":"10.14670/HH-18-978","DOIUrl":"https://doi.org/10.14670/HH-18-978","url":null,"abstract":"<p><p>Acute kidney injury (AKI) induced by sepsis is a critical condition with high morbidity, posing a significant challenge in clinical settings. Daphnetin (DAP), a natural compound, has demonstrated anti-inflammatory and antioxidant properties in various diseases. This study aims to explore the specific role and underlying mechanism of DAP in sepsis-induced AKI. Sepsis was induced in rats using the cecal ligation and puncture (CLP) method. Renal tissue samples were analyzed via hematoxylin and eosin staining for histopathological analysis and TUNEL assay for apoptosis detection. The expression of proteins associated with apoptosis or the JAK2/STAT3 pathway was determined via western blot analysis. Inflammatory cytokines were measured using ELISA kits. Oxidative stress markers were detected via biochemical analysis. Additionally, an <i>in vitro</i> sepsis model induced by lipopolysaccharide (LPS) was established to validate the effects of DAP. Cytotoxicity of DAP to HK-2 cells was determined using the CCK-8 assay, and cell apoptosis was analyzed via flow cytometry analysis. The results showed that DAP remarkably improved renal function in septic rats; it reduced the levels of inflammatory cytokines (TNF-α, IL-1β, IL-6), attenuated oxidative stress, and suppressed cell apoptosis in renal tissues. DAP inhibited the activation of JAK2/STAT3 signaling in both septic rats and LPS-stimulated HK-2 cells. <i>in vitro</i> experiments showed that DAP or AG490 (a JAK2 inhibitor) alleviated LPS-induced apoptosis, inflammation, and oxidative stress. In conclusion, DAP attenuates sepsis-induced AKI by reducing inflammation, oxidative stress, and apoptosis via the inactivation of the JAK2/STAT3 pathway.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"18978"},"PeriodicalIF":2.0,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144952184","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>Radix Actinidiae chinensis</i> inhibits neovascularization in colorectal cancer and its mechanism.","authors":"Ziqi Meng, Minyuan Chen, Jiante Li, Jieyu Li","doi":"10.14670/HH-18-977","DOIUrl":"10.14670/HH-18-977","url":null,"abstract":"<p><strong>Objective: </strong>Colorectal cancer is one of the most common cancers worldwide, and its angiogenesis is a key factor in tumor growth and metastasis. <i>Radix Actinidiae chinensis</i> is considered to have antitumor activity in traditional Chinese medicine, but its effect on neovascularization in colorectal cancer has not been clarified. Herein, we aimed to evaluate the effect of different concentrations of <i>Radix Actinidiae chinensis</i> on the neovascularization of colorectal cancer and explore its possible mechanisms.</p><p><strong>Method: </strong>A mouse model of colorectal cancer was established, and mice were randomly divided into control, low-, and high-concentration groups. Then the mice in the experimental group were treated with <i>Radix Actinidiae chinensis</i>, and its effects on neovascularization and tumor growth were evaluated by tumor growth curve tracking, immunohistochemical analysis, vessel density assessment, RT-qPCR, and protein immunoblotting to explore the underlying mechanisms.</p><p><strong>Results: </strong>It was shown that tumor tissues in the high-concentration group exhibited significantly slower growth in both mass and volume compared with the low-concentration and control groups. Immunohistochemical staining revealed a reduction in the expression of the vascular endothelial marker CD31 in the <i>Radix Actinidiae chinensis</i> treatment group. Moreover, the protein expression levels of vascular markers in tumor tissues showed a slight decrease in the low-concentration group and a marked reduction in the high-concentration group. These findings suggest that angiogenesis in the tumor microenvironment was inhibited in a concentration-dependent manner, with protein expression levels closely mirroring gene expression patterns.</p><p><strong>Conclusion: </strong>The study found that <i>Radix Actinidiae chinensis</i> inhibits neovascularization in a dose-dependent manner in a mouse model of colorectal cancer. These results provide experimental support for its potential use as a therapeutic agent against colorectal cancer, suggesting that it may suppress tumor growth and metastasis by inhibiting angiogenesis.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"18977"},"PeriodicalIF":2.0,"publicationDate":"2025-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144952246","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"HM13 is a predictive biomarker of metastasis and neutrophil infiltration in colorectal cancer.","authors":"Yanbing Ren, Ying Mao, Xiao Yuan","doi":"10.14670/HH-18-857","DOIUrl":"10.14670/HH-18-857","url":null,"abstract":"<p><strong>Background: </strong>High levels of histocompatibility minor 13 (HM13) have been related to the progression of several cancers. Here, we investigated the function of HM13 in colorectal cancer (CRC).</p><p><strong>Methods: </strong>HM13 expression, clinicopathology analysis, and its influence on survival were analyzed in multiple public databases (TCGA, TIMER2.0, and GEPIA). HM13 mRNA and protein levels in CRC cells were examined by qRT-PCR and western blot, respectively. CCK-8, Transwell, wound-healing, and adhesion assays were used to measure cell proliferation, migration, invasion, and adhesion in HM13-overexpressed and -silenced cells. The relationship between HM13 expression and neutrophil infiltration was also analyzed. CRC xenograft mouse models were used for <i>in vivo</i> verification of HM13 function.</p><p><strong>Results: </strong>In this study, TCGA dataset analysis revealed that elevated HM13 levels correlated with malignant progression and worse survival outcomes in CRC. Cell migration, proliferation, invasion, and adhesion were suppressed through the knockdown of sh-HM13 and enhanced through HM13 overexpression. Additionally, HM13 expression significantly correlated with the infiltration level of neutrophils in CRC in TCGA and TIMER analyses. HM13 levels were also positively correlated with myeloperoxidase (MPO) levels. In addition, <i>in vivo</i> studies further confirmed that MPO overexpression partially abolished the inhibition of tumor growth by sh-HM13 in CRC.</p><p><strong>Conclusion: </strong>The results suggested that high HM13 expression in CRC could promote tumor growth and metastasis by reducing neutrophil infiltration and may serve as a useful target in the treatment of metastatic CRC.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"1435-1446"},"PeriodicalIF":2.0,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142864113","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Gentiopicroside alleviates neuroinflammation in Parkinson's disease by mediating microglial pyroptosis via the NF-κB/NLRP3/GSDMD pathway.","authors":"Hong Shen, Hui Song, Qiang Sun","doi":"10.14670/HH-18-879","DOIUrl":"10.14670/HH-18-879","url":null,"abstract":"<p><strong>Objective: </strong>The study aimed to evaluate the therapeutic potential of gentiopicroside (GPS) in Parkinson's disease (PD) through both <i>in vitro</i> and <i>in vivo</i> experiments, focusing on elucidating the underlying mechanisms of its action.</p><p><strong>Methods: </strong>To achieve this, a PD model was established in C57BL6 mice using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), followed by assessment of behavioral changes, pathological alterations, microglial activation, and neuroinflammation. Simultaneously, a cellular PD model was developed in the BV-2 mouse microglia cell line by exposing them to 1-methyl-4-phenyl-pyridinium (MPP⁺). The expression of pro-inflammatory molecules was quantified using enzyme-linked immunosorbent assay (ELISA), while pyroptosis was analyzed by flow cytometry with caspase-1/PI double staining. The expression of key factors in the nuclear factor-kappa B (NF-κB)/NOD-like receptor thermal protein domain-associated protein 3 (NLRP3)/gasdermin D (GSDMD) signaling pathway was determined by immunoblotting.</p><p><strong>Results: </strong>The findings revealed that GPS effectively mitigated motor deficits, neurological impairments, microglial activation, and neuroinflammation in the MPTP-induced mouse model of PD. Additionally, GPS protected BV-2 cells from MPP⁺-induced inflammatory cytokine production and pyroptosis. Mechanistic studies indicated that GPS may exert its neuroprotective effects by inactivating the NF-κB/NLRP3/GSDMD-mediated pyroptotic pathway in both <i>in vivo</i> and <i>in vitro</i> settings.</p><p><strong>Conclusion: </strong>GPS exhibits neuroprotective effects in PD by suppressing microglia-mediated neuroinflammation and pyroptosis, suggesting its potential as a favorable therapeutic agent for PD treatment.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"1467-1478"},"PeriodicalIF":2.0,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143122792","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Use of digital image analysis to improve rigor and efficiency of physeal bone growth measurements.","authors":"Grant Ozaki, Jay Byrd, Bria Foley, Alexander Farell, Garret Williams, Max Twedt, James Sypherd, Ellen Leiferman, Cameron Jeffers, Matthew A Halanski","doi":"10.14670/HH-18-875","DOIUrl":"10.14670/HH-18-875","url":null,"abstract":"<p><p>In larger, translational animal models, manual measurements of longitudinal bone growth using fluorochrome labels is tedious and may be prone to less rigor due to variations in reader experience, sampling differences, and photobleaching that limits the repeatability of measurements. This study assesses the reliability of three different digital methods to assist in measurement of distance between pulsed fluorochrome labels. Forty-five tibial physes from skeletally immature New Zealand White rabbits were pulsed with fluorochrome labels and measured using Fully Manual Technique (FMT), Manual Digital Measurement (MDM), Computer Assisted Image Processing (AIP), and Fully Automated Measurement (FAM). Pearson's correlation coefficient and Bland-Altman analysis were used to analyze the different methods. Intraclass correlation coefficient (ICC) assessed inter- and intra-reader reliability. Regardless of the method, all growth rate measurement techniques exhibited excellent agreement and reliability. The computer assisted methods allowed rapid data acquisition without compromising reliability, thereby improving efficiency in bone growth research. Clinical Significance: Distances between fluorochrome bone labels on large samples can be reliably measured using digital imaging and processing techniques.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"1397-1404"},"PeriodicalIF":2.0,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143046664","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cocaine abuse and its impact on the thymus and spleen.","authors":"Toshihiko Aki, Kana Unuma","doi":"10.14670/HH-18-904","DOIUrl":"10.14670/HH-18-904","url":null,"abstract":"<p><p>Cocaine is a psychostimulant abused worldwide. Its pharmacotoxicological properties are derived mainly from its impact on the neurotransmission of sympathomimetic nerves. Cocaine enhances and prolongs the neurotransmission of monoamines, such as dopamine, serotonin, and adrenaline, which are responsible not only for cocaine's euphoric effects, but also its cardiovascular toxicity. In addition to these effects on central as well as peripheral nerves, immunosuppression is also implicated in cocaine toxicity. The thymus and spleen are lymphoid organs that are essential in lymphocyte maturation and erythrocyte homeostasis. Reductions in thymus and spleen size, which are observed under both physiological and pathological conditions, are known as thymic involution and splenic contraction, respectively. These phenomena are also observed in experimental animal models of binge cocaine abuse. In this brief review, we describe the mechanisms of cocaine toxicity, thymic involution, and splenic contraction, followed by discussions about the possible role of the latter two phenomena in cocaine intoxication.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"1339-1346"},"PeriodicalIF":2.0,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143718701","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Value of the combination of intraepithelial tumor-infiltrating lymphocyte density and the heterogeneity of density as a prognostic marker in stage III colorectal cancers.","authors":"Hye-Yeong Jin, Lingyan Jin, Seung Yeon Yoo, Nam-Yun Cho, Jeong Mo Bae, Jung Ho Kim, Hye Seung Lee, Gyeong Hoon Kang","doi":"10.14670/HH-18-864","DOIUrl":"10.14670/HH-18-864","url":null,"abstract":"<p><p>Tumor-infiltrating lymphocyte (TIL) density is both a prognostic and a predictive factor in colorectal cancer (CRC). Whether the heterogeneity of TIL density across the tumor plays an important role in the clinical outcome of CRC is not well known. Adjuvant chemotherapy-treated patients with stage III CRC were analyzed for survival according to TIL density and density heterogeneity, which were determined on CD8-immunostained slides using a machine learning method and by calculating the Simpson evenness index, respectively. High heterogeneity of the intraepithelial TIL density was found to be an independent prognostic factor, with a hazard ratio of 1.970 (1.207-3.215) in the multivariate analysis of recurrence-free survival. High heterogeneity was closely associated with a high T category, venous invasion, perineural invasion, and <i>KRAS</i> mutation. The combination of both intraepithelial TIL density and density heterogeneity was significantly associated with the prognosis of patients: low TIL density/high TIL heterogeneity showed hazard ratios of 3.284 (1.639-6.578) and 4.176 (1.713-10.178) in the discovery and validation cohorts, respectively. Our findings suggest that the heterogeneity status of intraepithelial TIL density might help delineate patients with better vs. worse survival when combined with intraepithelial TIL density.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"1367-1375"},"PeriodicalIF":2.0,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142948191","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rosmarinic acid inhibits the proliferation of ovarian carcinoma cells by activating the p53/BAX signaling pathway.","authors":"İlhan Özdemır, Dilek Doğan Baş, Şamil Öztürk, Özge Karaosmanoğlu, Mehmet Cudi Tuncer","doi":"10.14670/HH-18-883","DOIUrl":"10.14670/HH-18-883","url":null,"abstract":"<p><strong>Objective: </strong>While chemotherapeutic agents stop the development of cancer cells, they also kill healthy cells. This study aimed to increase anticancer effects and reduce side effects by combining a phytotherapeutic compound with a chemotherapeutic drug.</p><p><strong>Methods: </strong>This study examined the effects of nine concentrations of rosmarinic acid (RA) and doxorubicin (DOX) on human ovarian adenocarcinoma (OVCAR3) and skin keratinocyte (HaCaT) cell lines. Their cytotoxic effects were assessed based on cell viability, evaluated using the MTT assay, and apoptotic activity, evaluated using NucBlue staining and the gene and protein expression of tumor protein p53 (TP53) and BCL2 associated X, apoptosis regulator (BAX) quantified by qRT-PCR and western blots, respectively.</p><p><strong>Results: </strong>The half-maximal inhibitory concentration after 48 hours was 880.4 μM for RA and 2.26 μM for DOX. The cytotoxicity analysis revealed that cell viability decreased with the RA concentration. RA increased apoptosis in OVCAR3 cells by activating the p53/BAX pathway. Western blots showed that RA and DOX upregulated p53 and BAX protein levels in OVCAR3 cells.</p><p><strong>Conclusions: </strong>The RA and DOX combination inhibited cell proliferation by inducing apoptosis in OVCAR3 cells. These results suggest that RA may reduce the side effects of DOX toxicity.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"1415-1423"},"PeriodicalIF":2.0,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143407355","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessing acute pancreatitis: A novel method combining live cell imaging with tissue damage evaluation.","authors":"Polona Kovačič, Maša Skelin Klemen, Eva Paradiž Leitgeb, Viktória Venglovecz, Lóránd Kiss, Gabriella Mihalekné Fűr, Andraž Stožer, Jurij Dolenšek","doi":"10.14670/HH-18-882","DOIUrl":"10.14670/HH-18-882","url":null,"abstract":"<p><p>Acute pancreatitis (AP) is a sudden inflammation of the exocrine part of the pancreas, resulting in self-digestion and destruction of exocrine tissue. The intricate relationship between exocrine and endocrine functions is pivotal, as damage to acinar cells can affect endocrine cell function and <i>vice versa</i>. However, our understanding of these interactions remains limited. An effective strategy for investigating pancreatic cells involves the utilization of live <i>in-situ</i> acute mouse pancreas tissue slice preparations, combined with noninvasive fluorescent calcium labeling of endocrine or exocrine cells, and subsequent analysis using confocal laser scanning microscopy. Nevertheless, this approach encounters inherent conflicts with conventional methodologies employed to histologically assess the severity of tissue damage due to AP in the model. Traditional methods involve fixing and staining tissue samples with hematoxylin and eosin, thereby precluding live-cell imaging. In this study, our objective was to introduce an innovative method utilizing a commercial fluorescence Live/Dead assay that enables calcium imaging and tissue damage assessment in the same sample. This approach was validated against the classical histological grading of AP severity, and we found a good correlation between the classical histological grading method and the <i>in-situ</i> approach employing the Live/Dead assay. The primary advantage of our novel approach lies in its capacity to enable timely and efficient live-cell imaging together with damage assessment in the same tissue, thereby enabling the study of functional consequences of structural damage at the cellular level and reducing the number of animals required for experimentation.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"1405-1413"},"PeriodicalIF":2.0,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143399114","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The synergetic effect of edaravone and scutellarin in the MPP(+)-induced cell model of Parkinson's disease.","authors":"Wei Wei, Jing Wu, Dandan Zhang, Shoucheng Xu, Yi Wang, Xuezhong Li, Ming Yu, Xiaopeng Chen","doi":"10.14670/HH-18-874","DOIUrl":"10.14670/HH-18-874","url":null,"abstract":"<p><p>Parkinson's disease (PD) is a limb movement disorder caused by the degeneration of brain neurons and seriously affects the quality of life of the elderly. However, the current drugs are symptomatic treatments that cannot prevent or delay the development of the disease. Targeted therapy for pathogenesis may be the direction of development in the future. Oxidative stress and the inflammatory response are involved in the pathogenesis of PD. Edaravone and scutellarin have been studied in antioxidant and anti-inflammatory reactions. The focus of this study is whether there is synergy between the two and its mechanism. Through research, we found that edaravone and scutellarin at different dose combinations have synergistic effects in 1-methyl-4-phenylpyridinium (MPP+)-induced PC12 cells using the Chou-Talalay joint index method. According to the CompuSyn software calculation, the results showed that the combination index (CI) of the combined application of 15 µM edaravone and 15 µM scutellarin was the lowest, indicating that the synergistic effect was the best. Compared with the single drug, the synergy increased superoxide dismutase (SOD) and reduced glutathione (GSH) levels, reduced the levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and enhanced the anti-apoptosis ability in the MPP(+) induced cell model of PD.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"1457-1466"},"PeriodicalIF":2.0,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143028654","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}