Toxicon最新文献

{"title":"Concerns regarding the study on mangrove pit viper envenomation in Malaysia","authors":"Ahmad Khaldun Ismail, David A. Warrell, Scott A. Weinstein","doi":"10.1016/j.toxicon.2024.108126","DOIUrl":"10.1016/j.toxicon.2024.108126","url":null,"abstract":"","PeriodicalId":23289,"journal":{"name":"Toxicon","volume":"251 ","pages":"Article 108126"},"PeriodicalIF":2.6,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142475676","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deoxynivalenol induces ovarian damage and uterine changes in prepubertal and adult mice","authors":"G.A.A. Lemos ,&nbsp;J.R. Gerez ,&nbsp;J.B. Costa ,&nbsp;E.J. Venâncio ,&nbsp;M. Souza ,&nbsp;P.O. Favaron ,&nbsp;J.R. Greghi ,&nbsp;E.M. Gloria ,&nbsp;L. Staurengo-Ferrari ,&nbsp;W.A. Verri ,&nbsp;A.P.F.R.L. Bracarense","doi":"10.1016/j.toxicon.2024.108123","DOIUrl":"10.1016/j.toxicon.2024.108123","url":null,"abstract":"<div><div>Deoxynivalenol (DON) is associated with reproductive toxicity in animals. The frequent contamination of cereal-based foods with DON and the high intake of these by children raises particular concern about the susceptibility of this subpopulation to adverse effects from this mycotoxin. However, age-related differences in the <em>in vivo</em> reproductive toxicity of DON have not been evaluated. Therefore, the effects of DON on serum follicle-stimulating hormone (FSH) levels, histology, and inflammatory and oxidative stress responses in the ovaries and uteruses of prepubertal and adult mice were investigated. Twenty female prepubertal Swiss mice (21 days old) and 20 young adult mice (65 days old) were fed a control diet or a diet containing 10 mg of DON/kg of feed for 15 days (prepubertal mice) and 28 days (adult mice). In the ovaries, DON induced an increase in the lesional score in both age groups. Ingestion of DON decreased FSH levels in prepubertal females, whereas an increase was observed in adult mice. In prepubertal mice, a reduction in the number of macrophages and increased levels of TNF-α were observed in the ovaries of the DON group, while in adult animals, an increase in the number of macrophages and higher levels of TNF-α were noted. Exposure to DON led to an increase in type I collagen in the uteruses of adult mice, while in prepubertal mice, a decrease in type III collagen was observed. DON exposure also resulted in a decrease in FRAP levels and an increase in ABTS and lipid peroxidation in the uteruses of prepubertal mice. Taken together, the results indicate that the effects of DON on reproductive organs are age-specific, with toxicity established as early as the prepubertal period.</div></div>","PeriodicalId":23289,"journal":{"name":"Toxicon","volume":"251 ","pages":"Article 108123"},"PeriodicalIF":2.6,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142475677","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Using a conditioned place preference assay in fruit flies to examine effects of insecticidal compounds on contextual memory","authors":"Yachen Wang ,&nbsp;Shaodong Guo ,&nbsp;Tomer Ventura ,&nbsp;Volker Herzig","doi":"10.1016/j.toxicon.2024.108128","DOIUrl":"10.1016/j.toxicon.2024.108128","url":null,"abstract":"<div><div>Insecticides are vital for safeguarding agricultural crops against pests, albeit many lack selectivity towards pest species and are poorly bio-degradable. This leads to targeting of beneficial organisms like pollinators and widespread environmental contamination of soil and water. Exposure to insecticides such as neonicotinoids causes insect paralysis and mortality at higher doses, while sublethal doses can disrupt other functions that are crucial for survival such as learning and memory performance. Potent and selective arachnid venom peptides affecting a variety of molecular targets are being explored as bioinsecticide candidates. However, their effect on insect learning is poorly understood. We therefore established a sucrose-induced conditioned place preference (CPP) assay using <em>Drosophila melanogaster</em> fruit flies to provide a means of evaluating how various classes of insecticidal compounds interact with insect memory to assess their broader ecological consequences. Our results confirmed the adverse effect of a sublethal dose of the neonicotinoid insecticide imidacloprid (20 pg/fly) on fly CPP formation upon daily injection during the conditioning phase. However, imidacloprid did not affect CPP retrieval when applied after the conditioning phase. Sublethal doses of the two insecticidal spider venom peptides μ-DGTX-Dc1a (Dc1a; 70 pg/fly) and U1-AGTX-Ta1a (Ta1a; 125 pg/fly) had no effect on either CPP formation or retrieval, underlining their potential as novel and safe bioinsecticide candidates.</div></div>","PeriodicalId":23289,"journal":{"name":"Toxicon","volume":"251 ","pages":"Article 108128"},"PeriodicalIF":2.6,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142475694","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Artificial intelligence application for identifying toxic plant species: A case of poisoning with Datura stramonium","authors":"Kamil Kokulu ,&nbsp;Ekrem Taha Sert","doi":"10.1016/j.toxicon.2024.108129","DOIUrl":"10.1016/j.toxicon.2024.108129","url":null,"abstract":"<div><h3>Introduction</h3><div>The management of plant poisonings in the emergency department (ED) presents various challenges. Foremost among these is the identification of the specific botanical species responsible for the toxic effect. In cases of plant poisoning, it is crucial to accurately identify the plant in order to promptly evaluate if it has cardiotoxic, neurotoxic, hepatotoxic, or anticholinergic properties. Furthermore, it is typically not possible to determine the identity of these plants through blood tests conducted in the ED.</div></div><div><h3>Case report</h3><div>An otherwise healthy 23-year-old male patient presented to the ED with symptoms of restlessness, altered mental state, and hallucinations that occurred 2 h after consuming herbal tea. On physical examination, he was tachypneic, tachycardic, and disoriented. The pupils were bilaterally mydriatic. The patient's symptoms were consistent with both sympathomimetic and anticholinergic (antimuscarinic) toxidromes. We were unable to promptly reach a botanist to identify the plant to which the patient had been exposed. Therefore, we employed Google Gemini, an artificial intelligence software, to ascertain the plant's identity. Google Gemini identified the plant we photographed as Datura stramonium, commonly known as jimson weed, which is known to cause anticholinergic toxicity. The botanist we contacted later confirmed that the plant was D. stramonium. The patient's symptoms were alleviated with the use of intravenous diazepam and physostigmine.</div></div><div><h3>Conclusion</h3><div>We propose that the utilization of artificial intelligence applications with visual recognition capabilities could be beneficial for physicians, patients, and foragers of edible wild plants to accurately identify plants and distinguish toxic species.</div></div>","PeriodicalId":23289,"journal":{"name":"Toxicon","volume":"251 ","pages":"Article 108129"},"PeriodicalIF":2.6,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142475695","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Toxicity of the marine glycosidic macrolides, polycavernoside E and akunolides, from Okinawan cyanobacterium Okeania sp. in mice","authors":"Mari Yotsu-Yamashita ,&nbsp;Kairi Umeda ,&nbsp;Arihiro Iwasaki ,&nbsp;Kiyotake Suenaga","doi":"10.1016/j.toxicon.2024.108122","DOIUrl":"10.1016/j.toxicon.2024.108122","url":null,"abstract":"<div><div>The toxicity of polycavernoside E and its structurally related macrolides, akunolides A, B, and C, was evaluated in mice. These compounds were recently isolated from the marine cyanobacterium <em>Okeania</em> sp. collected in Okinawa, Japan. The minimal toxicity of polycavernoside E was estimated as 0.81 mg/kg by intraperitoneal injection, whereas those of akunolides A–C were &gt;13 mg/kg. Owing to the considerable toxicity of polycavernoside E, the occurrence of this cyanobacterium should be considered in seaweed production environments.</div></div>","PeriodicalId":23289,"journal":{"name":"Toxicon","volume":"250 ","pages":"Article 108122"},"PeriodicalIF":2.6,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142406935","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deciphering toxico-proteomics of Asiatic medically significant venomous snake species: A systematic review and interactive data dashboard","authors":"Sher Min Ding,&nbsp;Michelle Khai Khun Yap","doi":"10.1016/j.toxicon.2024.108120","DOIUrl":"10.1016/j.toxicon.2024.108120","url":null,"abstract":"<div><div>Snakebite envenomation (SBE) is a neglected tropical disease (NTD) with an approximate 1.8 million cases annually. The tremendous figure is concerning, and the currently available treatment for snakebite envenomation is antivenom. However, the current antivenom has limited cross-neutralisation activity due to the variations in snake venom composition across species and geographical locations. The proteomics of medically important venomous species is essential as they study the venom compositions within and among different species. The advancement of sophisticated proteomic approaches allows intensive investigation of snake venoms. Nevertheless, there is a need to consolidate the venom proteomics profiles and distribution analysis to examine their variability patterns. This review systematically analysed the proteomics and toxicity profiles of medically important venomous species from Asia across different geographical locations. An interactive dashboard - Asiatic Proteomics Interactive Datasets was curated to consolidate the distribution patterns of the venom compositions, serve as a comprehensive directory for large-scale comparative meta-analyses. The population proteomics demonstrate higher diversities in the predominant venom toxins. Besides, inter-regional differences were also observed in <em>Bungarus</em> sp., <em>Naja</em> sp., <em>Calliophis</em> sp., and <em>Ophiophagus hannah</em> venoms. The elapid venoms are predominated with three-finger toxins (3FTX_s) and phospholipase A₂ (PLA₂). Intra-regional variation is only significantly observed in <em>Naja naja</em> venoms. Proteomics diversity is more prominent in viper venoms, with widespread dominance observed in snake venom metalloproteinase (SVMP) and snake venom serine protease (SVSP). Correlations exist between the proteomics profiles and the toxicity (LD₅₀) of the medically important venomous species. Additionally, the predominant toxins, alongside their pathophysiological effects, were highlighted and discussed as well. The insights of interactive toxico-proteomics datasets provide comprehensive frameworks of venom dynamics and contribute to developing antivenoms for snakebite envenomation. This could reduce misdiagnosis of SBE and accelerate the researchers' data mining process.</div></div>","PeriodicalId":23289,"journal":{"name":"Toxicon","volume":"250 ","pages":"Article 108120"},"PeriodicalIF":2.6,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142406934","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protection efficacy and immunogenicity of Clostridium chauvoei proteins as a subunit blackleg vaccine or an adjuvant for Clostridium perfringens epsilon toxoid","authors":"Roxana Hamzavipour ,&nbsp;Azadeh Zahmatkesh ,&nbsp;Alireza Paradise ,&nbsp;Farzaneh Hosseini","doi":"10.1016/j.toxicon.2024.108124","DOIUrl":"10.1016/j.toxicon.2024.108124","url":null,"abstract":"<div><div>Potential application of <em>Clostridium chauvoei</em> proteins was studied as a subunit blackleg vaccine or a biological adjuvant for <em>Clostridium perfringens</em> epsilon toxoid vaccine. Extracellular and cell surface proteins were extracted from <em>C. chauvoei</em> culture, and their protective efficacy was evaluated by potency test in guinea pigs. In order to investigate the effect of cell surface proteins on <em>C. perfringens</em> epsilon toxoid immunogenicity, rabbits were inoculated subcutaneously twice with: <em>C. perfringens</em> type D toxoid supernatant +200 μg C<em>. chauvoei</em> cell surface proteins (PR-200), toxoid supernatant + 400 μg cell surface proteins (PR-400), inactivated <em>C. perfringens</em> type D vaccine (Vac), toxoid supernatant (Tox), or PBS. Isolation of cell surface proteins yielded about 2.5 mg/L culture protein with a sharp band at 43 kDa probably corresponding to flagellin. Potency test demonstrated the protection ability of both cellular and extracellular proteins of <em>C. chauvoei</em>. ELISA showed that the highest antibody titers against epsilon toxoid belonged to PR-400 and Vac groups. The effect of days post immunization on antibody response was not significant. No significant difference was observed between PR-400 and Vac, as well as PR-200 and Tox groups. <em>Clostridium chauvoei</em> cell surface proteins may have the potential for application as a blackleg disease vaccine and an adjuvant for clostridial toxoids.</div></div>","PeriodicalId":23289,"journal":{"name":"Toxicon","volume":"251 ","pages":"Article 108124"},"PeriodicalIF":2.6,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142475691","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Blood levels of zearalenone, thyroid-stimulating hormone, and thyroid hormones in patients with colorectal cancer","authors":"Sylwia Lisieska-Żołnierczyk ,&nbsp;Magdalena Gajęcka ,&nbsp;Łukasz Zielonka ,&nbsp;Michał Dąbrowski ,&nbsp;Maciej T. Gajęcki","doi":"10.1016/j.toxicon.2024.108125","DOIUrl":"10.1016/j.toxicon.2024.108125","url":null,"abstract":"<div><div>Mycotoxins are secondary metabolites produced by various species of mold fungi commonly found in plant materials. Zearalenone (ZEN) adversely affects the endocrine system. This study aimed to determine whether thyroid-stimulating hormone (TSH), procalcitonin (PCT), free triiodothyronine (fT3), and free thyroxine (fT4) levels are altered during natural zearalenone mycotoxicosis in patients diagnosed with sigmoid colon cancer (SCC) or colorectal cancer (CRC). A study was conducted on women and men diagnosed with SCC or CRC accompanied by the presence or absence (Patients Without ZEN – PWZ group) of ZEN in the blood. The PWZ group consisted of 17 patients with symptoms of SCC and CRC in whom ZEN and its metabolites were not detected in peripheral blood. The experimental (empirical) groups included a total of 16 SCC and CRC patients who tested positive for ZEN, but not its metabolites. TSH values in both sexes were within the upper limit of the reference range (0.27–4.2 μIU/mL) adopted by the hospital laboratory and corresponded to the upper second tertile and the lower third tertile. PCT values demonstrated that SCC and CRC were accompanied by a systemic or local bacterial infection. All mean values of fT3 were in the middle of the reference range, and the mean values of fT4 were within the upper reference limit. The fT3/fT4 prognostic marker was somewhat above the cut-off point of 0.22. These results indicate that in postmenopausal women and andropausal men who were diagnosed with SCC and CRC and were exposed to food-borne ZEN, higher values of the prognostic marker (fT3/fT4) were associated with an unfavorable prognosis. The study also revealed that the more distal the neoplastic lesions in the colon, the higher the percentage of both thyroid hormones, regardless of the patient's sex. The presence of ZEN in the diet alters thyroid activity in patients diagnosed with SCC and CRC.</div></div>","PeriodicalId":23289,"journal":{"name":"Toxicon","volume":"251 ","pages":"Article 108125"},"PeriodicalIF":2.6,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142475674","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Aflatoxin M1 level and risk assessment in milk, yogurt, and cheese in Tabriz, Iran","authors":"Peyman Behtarin ,&nbsp;Mohammadhossein Movassaghghazani","doi":"10.1016/j.toxicon.2024.108119","DOIUrl":"10.1016/j.toxicon.2024.108119","url":null,"abstract":"<div><h3>Aim and background</h3><div>The objective of this study was to investigate the presence of aflatoxin M₁ (AFM₁) in milk, yogurt, and cheese samples collected from Tabriz, Iran. Additionally, the study conducted a risk assessment related to the consumption of milk and dairy products within Tabriz city.</div></div><div><h3>Study method</h3><div>For this study, 56 samples (raw milk, pasteurized milk, ultra-high temperature milk (UHT), traditional yogurt, pasteurized yogurt, traditional cheese, and pasteurized cheese) were collected randomly in Tabriz from December 2021 to March 2022. The analysis was carried out using liquid chromatography, which was equipped with a fluorescence detector. The estimated dietary intake (EDI) and the hazard index (HI) were calculated for the risk assessment.</div></div><div><h3>Results and discussion</h3><div>AFM₁ was detected in all samples. The highest concentration of AFM₁ was observed in traditional cheese (<em>P</em> &lt; 0.05). The lowest concentration was observed in UHT milk and there were no significant differences between the various milk types and pasteurized yogurt (<em>P</em> &gt; 0.05). Moreover, in all samples, the levels of AFM₁ were below the maximum limit permitted by Iranian national standards. AFM₁ levels in traditional yogurt exceeded the European Union's permissible limit in 25% of the samples. The HI in all samples was less than one for both adult and child consumers, except for milk samples for children, which were more than one and indicated a medium risk. In conclusion, the levels of contamination in milk and dairy products and risk assessment appear not to pose a public health risk to Tabriz consumers.</div></div>","PeriodicalId":23289,"journal":{"name":"Toxicon","volume":"250 ","pages":"Article 108119"},"PeriodicalIF":2.6,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142401429","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The impact of lethal and sub-lethal exposure of emamectin benzoate on populations of Spodoptera litura (Lepidoptera: Noctuidae) under laboratory conditions","authors":"Meena Devi ,&nbsp;Arushi Mahajan ,&nbsp;Harvinder Singh Saini ,&nbsp;Sanehdeep Kaur","doi":"10.1016/j.toxicon.2024.108121","DOIUrl":"10.1016/j.toxicon.2024.108121","url":null,"abstract":"<div><div>Emamectin benzoate is an avermectin bio-insecticide commonly used for managing several insect pests including <em>Spodoptera litura</em> (Fabricius) (Lepidoptera: Noctuidae), a major polyphagous pest of many cultivated crops. The current study was conducted to evaluate the effects of emamectin benzoate on the fitness of <em>S. litura</em> populations exhibiting differential susceptibility to insecticide. The selection process and all the bioassays were carried out using 6-day-old 2nd instar larvae of <em>S. litura</em>. A field-collected population of <em>S. litura</em> was divided into two groups: one selected with emamectin benzoate for eight generations (EB-Sel) and the other kept unexposed (Unsel-Lab) to insecticide in the laboratory. An increase in resistance ratio from 1.71-fold in the F₁ generation to 22.54-fold in the F₈ generation of the EB-Sel population was observed compared to the Unsel-Lab (F₈) population. The EB-Sel and Unsel-Lab populations were treated with their respective lethal and sub-lethal concentrations which resulted in an extended development period, decreased larval survival, and adult emergence along with increased morphological abnormalities in adults. Significant reductions were observed in both male and female longevity, fecundity, egg hatching, net reproductive rate (<em>R</em>_<em>0</em>), intrinsic rate of increase (<em>r</em>_<em>m</em>), and finite rate of increase (<em>λ</em>) in EB-Sel and Unsel-Lab populations. Higher concentrations of the insecticide also reduced the relative fitness (<em>R</em>_<em>f</em>) of <em>S. litura</em> larvae, with maximum effect at LC₅₀ of the EB-Sel population where the <em>R</em>_<em>f</em> value was 0.32 compared to the Unsel-Lab population. Both populations have been affected by emamectin benzoate exposure, however, the impact was more pronounced in the EB-Sel population indicating fitness costs. Our results suggested the fitness cost linked to emamectin benzoate resistance in <em>S. litura</em> which might favor managing insecticide resistance by reducing the frequency of resistant alleles by removing selection pressure. Consequently, our research provides significant insights to devise better pest management strategies for <em>S. litura</em>.</div></div>","PeriodicalId":23289,"journal":{"name":"Toxicon","volume":"250 ","pages":"Article 108121"},"PeriodicalIF":2.6,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142401430","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}