Histology and histopathology最新文献

{"title":"Blood-brain barrier disruption following brain injury: Implications for clinical practice.","authors":"Ruojing Bai, Xintong Ge","doi":"10.14670/HH-18-740","DOIUrl":"10.14670/HH-18-740","url":null,"abstract":"<p><p>The blood-brain barrier (BBB) plays a critical role in regulating the exchange of substances between peripheral blood and the central nervous system and in maintaining the stability of the neurovascular unit in neurological diseases. To guide clinical treatment and basic research on BBB protection following brain injury, this manuscript reviews how BBB disruption develops and influences neural recovery after stroke and traumatic brain injury (TBI). By summarizing the pathological mechanisms of BBB damage, we underscore the critical role of promoting BBB repair in managing brain injury. We also emphasize the potential for personalized and precise therapeutic strategies and the need for continued research and innovation. From this, broadening insights into the mechanisms of BBB disruption and repair could pave the way for breakthroughs in the treatment of brain injury-related diseases.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"1435-1441"},"PeriodicalIF":2.5,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140868492","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":"\"Vascular tuft sign\" in neuroendocrine tumors of the pancreas.","authors":"L Díaz-Flores, R Gutiérrez, M P García-Suárez, M González-Gómez, J L Carrasco, J F Madrid, L Díaz-Flores","doi":"10.14670/HH-18-787","DOIUrl":"10.14670/HH-18-787","url":null,"abstract":"<p><p>The often well-developed microvasculature in pancreatic neuroendocrine tumors (PanNETs) has been studied from different perspectives. However, some detailed structural findings have received less attention. Our objective is to study an overlooked event in PanNETs: \"enclosed vascular tufts\" (EVTs). For this purpose, 39 cases of PanNETs were examined with conventional (including serial sections) and immunochemistry procedures. In typical EVTs, the results show: 1) an insulated terminal vascular area, with a globular (glomeruloid) aspect, formed by a cluster of coiled microvessels, presenting CD31-, CD34-positive endothelial cells, αSMA-positive pericytes, and perivascular CD34-positive stromal cells/telocytes, separated by a pseudoglandular space from the surrounding trabeculae of tumor neuroendocrine cells; and 2) a pedicle joining the insulated terminal vascular area, with connective tissue tracts around the enclosing tumor trabeculae. EVTs predominate in the trabecular and nested gyriform pattern of PanNETs, with tumor trabeculae that follow a ribbon coil (winding ribbon pattern) around small vessels, which acquire a tufted image. In EVTs, secondary modifications may occur (fibrosis, hyalinization, myxoid changes, and calcification), coinciding or not with those of the connective tracts. In conclusion, the typical characteristics of unnoticed EVTs allow them to be considered as a morphological sign of PanNETs (a vascular tuft sign). Further in-depth studies are required, mainly to assess the molecular pathways that participate in vascular tuft formation and its pathophysiological implications.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"1457-1472"},"PeriodicalIF":2.5,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141723536","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":"Biomechanical and histological analyses of a multilayer stent in a swine model of suprarenal aortic aneurysm.","authors":"Allana Maryel Tobita, Anna Paula Weinhardt Baptista Strazzi, Maria Fernanda Cassino Portugal, Nelson Wolosker, Ricardo Aun, Frederico de Lima Jacy Monteiro, Erasmo Simão da Silva, Igor Rafael Sincos","doi":"10.14670/HH-18-842","DOIUrl":"https://doi.org/10.14670/HH-18-842","url":null,"abstract":"<p><strong>Objectives: </strong>To analyze and compare, in an animal model, the treatment of thoracoabdominal aneurysms with multilayer stents and its hemodynamic effects through the biomechanical and histological analysis of the aortic wall in contact with the stent.</p><p><strong>Methods: </strong>Large White pigs were randomized into two groups: Stent (n=6) and Control (n=5, non-stent). All animals were subjected to the creation of a suprarenal aneurysm with a bovine pericardial patch. In the Stent group, a multilayer stent was implanted immediately after aneurysm formation. After four weeks, all animals were subjected to angiographic assessment and intravascular ultrasound, and the stent was explanted before euthanasia for histological and biomechanical analyses.</p><p><strong>Results: </strong>At histological analysis, the groups did not differ significantly in maximum thickness of the intima (<i>p</i>=0.526), media (<i>p</i>=0.129), or adventitia (<i>p</i>=0.662). Thrombus formation was observed in 100% of the animals on the intima and media layers of the stented aorta vs. none in the Control group (<i>p</i>=0.048). At biomechanical analysis, no statistical differences were observed in aortic wall elasticity (<i>p</i>=0.158), strength (<i>p</i>=0.360), or thickness (<i>p</i>=0.323).</p><p><strong>Conclusion: </strong>We identified thrombosis of the aneurysmal sac through the presence of thrombi on the intima of the aorta in 100% of the animals in the Stent group; as for the biomechanical analysis, this study showed no statistical differences in vessel wall thickness, strength, and elasticity between groups.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"18842"},"PeriodicalIF":2.5,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142647350","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":"Fortunellin attenuates sepsis-induced acute kidney injury by inhibiting inflammation and ferroptosis via the TLR4/NF-κB pathway.","authors":"Yanmin Zhang, Tianzhi Liu, Zhigang Zuo","doi":"10.14670/HH-18-841","DOIUrl":"10.14670/HH-18-841","url":null,"abstract":"<p><strong>Objective: </strong>To investigate the potential protective effect of fortunellin in sepsis-induced acute kidney injury (AKI) and its underlying mechanisms.</p><p><strong>Methods: </strong>Lipopolysaccharide (LPS)-treated human kidney proximal tubular epithelial (HK-2) cells were used as a cell model and sepsis-induced AKI was induced by cecal ligation and puncture (CLP) surgery in mice. Cell Counting Kit-8 (CCK8) assays and flow cytometry analysis were performed to examine the viability of HK-2 cells. Enzyme-linked immunosorbent assay (ELISA) was performed to investigate the content of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) <i>in vivo</i> and <i>in vitro</i>. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and free iron (Fe²⁺) were measured as indicators of ferroptosis. The phosphorylation levels of Interleukin-1 Receptor-Associated Kinase 4 (p-IRAK4), p65 (p-65), and inhibitor of kappa B alpha (p-IκBα) were detected by western blot as an indication of nuclear factor kappa-B (NF-κB) pathway activation.</p><p><strong>Results: </strong>Our cell and animal experiments revealed that fortunellin exhibits significant anti-inflammatory and cytoprotective properties. Fortunellin counteracted LPS-induced cellular damage in HK-2 cells, enhancing cell survival and suppressing the secretion of pro-inflammatory cytokines. Additionally, fortunellin demonstrated potent antioxidant effects, reducing MDA and Fe²⁺ levels while increasing SOD activity and GSH content. The protective effect of fortunellin was further corroborated in the mouse model of sepsis-induced AKI. Notably, fortunellin suppressed activation of the TLR4/NF-κB pathway in the AKI model, as evidenced by decreased levels of p-p65 and p-IκBα proteins.</p><p><strong>Conclusion: </strong>Fortunellin ameliorates inflammation and oxidative stress in sepsis-induced AKI, possibly through the modulation of the TLR4/NF-κB pathway. These findings suggest fortunellin's potential as a therapeutic agent for sepsis-associated AKI.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"18841"},"PeriodicalIF":2.5,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142619271","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":"Time course analysis of changes in neuronal loss, oxidative stress, and excitotoxicity in gerbil hippocampus following ischemia and reperfusion under hyperthermic conditions.","authors":"Tae-Kyeong Lee, Dae Won Kim, Joon Ha Park, Choong-Hyun Lee, Se-Ran Yang, Myoung Cheol Shin, Moo-Ho Won, Jun Hwi Cho, Ji Hyeon Ahn","doi":"10.14670/HH-18-840","DOIUrl":"https://doi.org/10.14670/HH-18-840","url":null,"abstract":"<p><p>Oxidative stress and excitotoxicity are the major causes of neuronal death/loss in the brain following ischemia and reperfusion (IR). Hyperthermia is known to exacerbate ischemic neuronal damage; however, the underlying mechanisms remain unclear. This study investigated the mechanisms underlying neuronal damage caused by IR injury (IRI) under hyperthermic conditions in the gerbil hippocampal CA1 region. Gerbils were controlled at normothermia (37.5±0.2°C) or hyperthermia (39.5±0.2°C). After temperature control for 30 min, the animals received IRI (following 5 min of transient forebrain ischemia) or sham ischemia, and were subsequently sacrificed at 0, 3, 6, 12, 24, 48, and 120h after IRI. Neuronal death was examined using neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence. Oxidative stress was analyzed by immunohistochemistry for 8-Hydroxy-2'-deoxyguanosine (8OHdG) and superoxide dismutase 2 (SOD2). Excitotoxicity was investigated by immunohistochemistry and western blotting for glutamate transporter 1 (GLT1). Immunohistochemical staining for glial fibrillary acidic proteins (GFAP) was performed to detect reactive astrogliosis. Loss of pyramidal neurons was detected earlier (48h post-IRI) in the hyperthermia-IRI group than in the normothermia-IRI group (120h post-IRI). Further, 8OHdG and SOD2 immunoreactivity in the hyperthermia-IRI group was significantly higher than that in the normothermia-IRI group. Changes in GLT1 immunoreactivity in both groups were biphasic, indicating that the immunoreactivity and protein levels were significantly lower in the hyperthermia-IRI group. GFAP immunoreactivity was enhanced following neuronal loss, indicating that the immunoreactivity was significantly higher in the hyperthermia-IRI group. Taken together, these results suggest that brain IR under hyperthermic conditions can aggravate neuronal damage in the hippocampal CA1 region through severe oxidative stress and excitotoxicity.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"18840"},"PeriodicalIF":2.5,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142619272","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":"Ferroptosis: A key regulator and potential target for tissue injury.","authors":"Ruihan Liu, Qing Luo, Guanbin Song","doi":"10.14670/HH-18-838","DOIUrl":"https://doi.org/10.14670/HH-18-838","url":null,"abstract":"<p><p>The maintenance of iron homeostasis is essential for proper body function. A growing body of evidence suggests that iron imbalance is the common denominator in many tissue injuries, including acute, chronic, and reperfusion injuries. Ferroptosis, a novel form of programmed cell death due to metabolic abnormalities, has become increasingly recognized as an important process mediating the pathogenesis and progression of numerous tissue injuries, including cerebral, myocardial, lung, liver, kidney, and intestinal injuries. Therefore, a thorough understanding of the mechanisms involved in the regulation of ferroptosis might contribute to improvements in disease management. In this review, we summarize the importance of ferroptosis in various tissue injuries, discuss the potential targets of ferroptosis in the treatment of tissue injuries, and describe the current limitations and future directions of these novel treatment targets.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"18838"},"PeriodicalIF":2.5,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142619269","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":"Experimental murine models of interstitial cystitis/bladder pain syndrome: A review.","authors":"Tetsuichi Saito, Tomonori Minagawa, Naoki Yoshimura, Yi Luo, Yoshiyuki Akiyama","doi":"10.14670/HH-18-837","DOIUrl":"https://doi.org/10.14670/HH-18-837","url":null,"abstract":"<p><p>Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic enigmatic disease of the urinary bladder characterized by persistent bladder/pelvic pain in conjunction with lower urinary tract symptoms. IC/BPS is categorized as either Hunner-type IC (HIC) or BPS based on the presence/absence of the Hunner lesion, a reddish mucosal lesion in the bladder. HIC and BPS present with similar symptoms, however, the etiologies are completely different. Recent evidence suggests that HIC is an immune-mediated inflammatory disease of the urinary bladder. In contrast, BPS, other forms of HIC lacking Hunner lesions, is a minimally inflamed condition comprising various clinical phenotypes. Based on this evidence, basic research into IC/BPS has shifted to target each subtype of IC/BPS. Today, experimental murine models of autoimmune cystitis are used for HIC research, whereas models related to neurophysiological and psychosocial dysfunctions have been developed for BPS research. This emerging concept of a subtype-tailored approach may contribute to a better understanding of the full picture of IC/BPS, thereby improving current clinical management strategies and the development of novel therapies.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"18837"},"PeriodicalIF":2.5,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142619267","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":"Impact of 6-OHDA injection and microtrauma in the rat <i>substantia nigra</i> on local brain amyloid beta protein concentrations in the affected area.","authors":"Joshua A Roldán-Kalil, Sara E Vendrell-Gonzalez, Natalia Espinosa-Ponce, Jadier Colón-Vasques, Jescelica Ortiz-Rivera, Vassiliy Tsytsarev, Janaina M Alves, Mikhail Inyushin","doi":"10.14670/HH-18-836","DOIUrl":"10.14670/HH-18-836","url":null,"abstract":"<p><p>Amyloid beta peptides (Aβ) are key indicators of Alzheimer's disease and are also linked to cognitive decline in Parkinson's disease (PD) and other neurodegenerative disorders. This study explored the accumulation of Aβ in a standard 6-Hydroxydopamine (6-OHDA) model of PD. We unilaterally injected 6-OHDA into the <i>substantia nigra</i> of Wistar rats to induce dopaminergic cell degeneration and death, a characteristic of PD. The goal was to detect Aβ protein in tissues and blood vessels showing inflammation or degeneration from the 6-OHDA injection. Our results showed that 6-OHDA injection produced a statistically significant rise in Aβ concentration at the injection site 60 minutes after injection, which was slightly reduced 24 hours post-injection but still significantly higher than in controls. We also tried Gp120 injection in the same zone but it only produced effects comparable to control needle trauma. The presence of Aβ in tissues and blood vessel walls after injection was confirmed through ELISA tests and was supported by immunohistochemical staining of injection areas. We found that the increased Aβ concentration was visible in and around blood vessels and inside blood vessel walls, and also, to a lesser extent in some cells, most probably neurons, in the area. This research highlights the connection between dopaminergic cell poisoning and the accumulation of Aβ, offering insights into the progression of PD to cognitive disorders and dementia.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"18836"},"PeriodicalIF":4.6,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142603915","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":"USP33 promotes pulmonary microvascular endothelial cell pyroptosis by stabilizing TRAF2 through deubiquitination.","authors":"Jianping Liang, Junbo Chen, Pengfei Xu","doi":"10.14670/HH-18-835","DOIUrl":"10.14670/HH-18-835","url":null,"abstract":"<p><strong>Objective: </strong>Inhibiting the pyroptosis of human pulmonary microvascular endothelial cells (HPMECs) is a promising therapeutic modality for acute lung injury (ALI). Given the undefined effect of ubiquitin-specific protease 33 (USP33) and tumor necrosis factor receptor-associated factor 2 (TRAF2) on pyroptosis in lung injury, this study investigates their roles in the pyroptosis of HPMECs during ALI.</p><p><strong>Methods: </strong>The hypoxia/reoxygenation (H/R)-induced model was constructed in HPMECs. Cell viability, cytotoxicity, and cell death were determined by the cell counting kit-8 (CCK-8), Lactate dehydrogenase (LDH), and Hoechst-PI staining, respectively. Western blot and qRT-PCR were used to detect protein and gene expression levels of pyroptosis-related markers, respectively. The TRAF2 ubiquitination level was measured via immunoprecipitation.</p><p><strong>Results: </strong>USP33 and TRAF2 expressions were elevated in H/R-induced HPMECs. Knockdown of USP33 increased cell viability and inhibited cellular pyroptosis, accompanied by decreases in IL-1β, IL-18, and Caspase-1. USP33 stabilized TRAF2 by deubiquitination. TRAF2 overexpression reversed the effect of USP33 silencing on suppressing HPMEC pyroptosis.</p><p><strong>Conclusion: </strong>USP33 stabilizes TRAF2 by deubiquitination to promote HPMEC pyroptosis during ALI.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"18835"},"PeriodicalIF":2.5,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142590648","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":"E2F1-induced upregulation of <i>TROAP</i> contributes to endometrial cancer progression.","authors":"Shanshan Wang, Yidan Sun, Minjing Guo, Ping Zhu, Beibei Xin","doi":"10.14670/HH-18-834","DOIUrl":"https://doi.org/10.14670/HH-18-834","url":null,"abstract":"<p><strong>Purpose: </strong>To investigate the role of Trophinin-associated protein (TROAP) in endometrial cancer (EC) progression and elucidate how the transcription factor E2F transcription factor 1 (E2F1) modulates EC by upregulating <i>TROAP</i> expression.</p><p><strong>Methods: </strong><i>TROAP</i> expression in EC tissues and cell lines was analyzed using bioinformatics databases, quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry. <i>TROAP</i> was knocked down in EC cells to assess its effects on proliferation, migration, invasion, and glycolysis. Potential transcription factors regulating <i>TROAP</i> were identified, and the relationship between E2F1 and <i>TROAP</i> gene regulation was examined using dual luciferase assay. <i>In vivo</i> tumor growth was evaluated using a mouse xenograft model.</p><p><strong>Results: </strong><i>TROAP</i> was overexpressed in EC tissues and cell lines compared with normal controls. High <i>TROAP</i> expression correlated with poor differentiation, advanced stage, lymph node metastasis, and worse overall survival in EC patients. Knockdown of <i>TROAP</i> inhibited the proliferation, migration, invasion, and glycolytic capacity of EC cells. E2F1 was identified as a transcriptional activator of TROAP. E2F1 overexpression enhanced <i>TROAP</i> expression and promoted EC cell proliferation, migration, and glycolysis in a TROAP-dependent manner. <i>TROAP</i> knockdown suppressed tumor growth <i>in vivo</i>.</p><p><strong>Conclusion: </strong><i>TROAP</i> is transcriptionally activated by E2F1 and promotes EC progression by enhancing cell proliferation, metastasis, and glycolysis. The E2F1-TROAP axis may serve as a potential therapeutic target for EC treatment.</p>","PeriodicalId":13164,"journal":{"name":"Histology and histopathology","volume":" ","pages":"18834"},"PeriodicalIF":2.5,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142582756","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}