Shabnam Mostofi, Dariush Shanehbandi, Seyyed Ali Rahmani, Milad Asadi
{"title":"Anti-migratory effect of curcumin on A-549 lung cancer cells via epigenetic reprogramming of <i>RECK/</i> matrix metalloproteinase axis.","authors":"Shabnam Mostofi, Dariush Shanehbandi, Seyyed Ali Rahmani, Milad Asadi","doi":"10.1515/hmbci-2021-0100","DOIUrl":"https://doi.org/10.1515/hmbci-2021-0100","url":null,"abstract":"<p><strong>Objectives: </strong>The aim of this study was to investigate the effects of curcumin on the viability, migration, and apoptosis of A549 lung cancer cells. Furthermore, <i>RECK</i>/<i>MMPs</i> axis as a probable regulator of cancer cell migration was assessed.</p><p><strong>Methods: </strong>In this study, effect of curcumin on viability changes, cell migration, and percentage of apoptosis of A549 non-small cell lung carcinoma was examined. The methylation status of <i>RECK</i> gene was investigated using MS-HRM technique. Moreover, expression changes of genes involved in apoptosis and migration (including <i>CASP3</i>, <i>CASP8</i>, <i>CASP9</i>, <i>BAX</i>, <i>BCL2</i>, <i>MMP9</i>, <i>MMP2</i>, and <i>RECK</i>) were investigated by quantitative Real-Time PCR.</p><p><strong>Results: </strong>The results of MTT assay showed that the cytotoxic effect of curcumin was in a dose dependent manner. Flow cytometry results demonstrated a significant increase in the percentage of apoptotic cells in curcumin treated group. In addition, curcumin inhibited migration rate in lung cancer cells. qRT-PCR revealed that expression of the candidate genes was in line with suppressed growth and migration. This could be due to, decreased methylation of the <i>RECK</i> gene promoter after curcumin treatment.</p><p><strong>Conclusions: </strong>Curcumin inhibited lung cancer cells through various molecular pathways. <i>RECK</i>/<i>MMPs</i> axis as a regulator of cancer cell migration was modulated after curcumin treatment and invasion of lung cancer cells was decreased.</p>","PeriodicalId":13224,"journal":{"name":"Hormone Molecular Biology and Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10476859","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":"COVID-19 pandemic: transmission of SARS-CoV-2 in animals, a risk for human beings.","authors":"Ashok Kumar Ahirwar, Kirti Kaim, Pradeep Ahirwar, Jitender Prasad","doi":"10.1515/hmbci-2021-0109","DOIUrl":"https://doi.org/10.1515/hmbci-2021-0109","url":null,"abstract":"","PeriodicalId":13224,"journal":{"name":"Hormone Molecular Biology and Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10627137","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}
Zi Ni Ngai, Kian Chung Chok, Khuen Yen Ng, Rhun Yian Koh, Soi Moi Chye
{"title":"Potential role of melatonin in prevention and treatment of lung cancer.","authors":"Zi Ni Ngai, Kian Chung Chok, Khuen Yen Ng, Rhun Yian Koh, Soi Moi Chye","doi":"10.1515/hmbci-2022-0018","DOIUrl":"https://doi.org/10.1515/hmbci-2022-0018","url":null,"abstract":"<p><p>Lung cancer is the second most common cancer and the most lethal cancer worldwide. Melatonin, an indoleamine produced in the pineal gland, shows anticancer effects on a variety of cancers, especially lung cancer. Herein, we clarify the pathophysiology of lung cancer, the association of circadian rhythm with lung, and the relationship between shift work and the incidence of lung cancer. Special focus is placed on the role of melatonin receptors in lung cancer, the relationship between inflammation and lung cancer, control of cell proliferation, apoptosis, autophagy, and immunomodulation in lung cancer by melatonin. A review of the drug synergy of melatonin with other anticancer drugs suggests its usefulness in combination therapy. In summary, the information compiled may serve as a comprehensive reference for the various mechanisms of action of melatonin against lung cancer, as a guide for the design of future experimental research and for advancing melatonin as a therapeutic agent for lung cancer.</p>","PeriodicalId":13224,"journal":{"name":"Hormone Molecular Biology and Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10415211","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":"Reduced irisin levels in patients with acromegaly.","authors":"Suleyman Nahit Sendur, Gokhan Baykal, Busra Firlatan, Busra Aydin, Incilay Lay, Selcuk Dagdelen, Mehmet Alikasifoglu, Tomris Erbas","doi":"10.1515/hmbci-2022-0009","DOIUrl":"https://doi.org/10.1515/hmbci-2022-0009","url":null,"abstract":"<p><strong>Objectives: </strong>Several metabolic disturbances are seen in acromegaly however, data regarding the contribution of irisin to these disturbances is currently insufficient. In a cohort of patients with acromegaly, we measured serum irisin levels in active and controlled cases and determined independent factors that effect serum irisin including fibronectin type III domain-containing protein 5 (FNDC5) genotyping.</p><p><strong>Methods: </strong>A cross-sectional case-control study including 46 patients with acromegaly (28 F/18 M, age: 50.3 ± 12.1 year, BMI: 30.7 ± 5.1 kg/m<sup>2</sup>) and 81 age-, gender-, body mass index- and body composition-matched healthy controls was conducted. 15 acromegalic patients (33%) had active disease. Irisin levels were measured by enzyme-linked immunosorbent assay. Three different regions (rs3480, rs1746661, and rs16835198) of FNDC5 were subjected to polymorphism analyses.</p><p><strong>Results: </strong>Both groups were overweight and had similar body composition. Irisin levels were lower in patients with acromegaly than controls (median [IQR]: 44.8 [41.7-46.7] ng/mL vs. 51.7 [45.5-60.1] ng/mL, p≤0.001, respectively). Active and controlled patients had similar irisin levels. Irisin was not correlated with growth hormone (GH), insulin-like growth factor 1 (IGF-1), and IGF-1 index. In multiple linear regression model, somatostatin receptor ligand use (<i>β</i>=-20.30, 95% CI [-34]-[-6], p=0.006) was determined as the only independent factor that affect serum irisin.</p><p><strong>Conclusions: </strong>Serum irisin levels are low in patients with acromegaly who are on somatostatin receptor ligand therapy. Single nucleotide polymorphisms (SNPs) of FNDC5 have no independent effects on circulating irisin levels under somatostatin ligand action. Endocrine muscle functions also seem to be regulated by somatostatin action, which requires further studies.</p>","PeriodicalId":13224,"journal":{"name":"Hormone Molecular Biology and Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2022-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40518655","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":"Endometriosis at all ages: diagnostic ultrasound","authors":"M. Bäumler, Niko Heiss, R. Druckmann","doi":"10.1515/hmbci-2021-0082","DOIUrl":"https://doi.org/10.1515/hmbci-2021-0082","url":null,"abstract":"Abstract Endometriosis is a gynecological disease affecting about 10% of the female population. The multifactorial hormonal, inflammatory, genetic, mental and behavior pathogenesis can result in chronic pelvic pain, blooding disorders and infertility causing disruption of quality of life. Correct diagnosis of the extension and topography is substantial in defining the adequate therapeutic strategy. In an increasing proportion of the cases, endometriosis is being managed medically and para-medically; diagnostic or therapeutic surgery can often be avoided or delayed. Transvaginal sonography is considered being the first-line imaging method in the diagnosis of pelvic endometriosis. The paradigm shift from the belief that endometriosis only affects women of reproductive age has drawn attention to endometriosis in both premenarchal and postmenopausal patients. This review resumes the actually recommended ultrasound signs in the case of patients in menstrual age. Specific diagnostic approaches in adolescent and menopausal patients are highlighted.","PeriodicalId":13224,"journal":{"name":"Hormone Molecular Biology and Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2022-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48383504","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}
P. Diep, M. Chaudry, Adam Dixon, Faisal Chaudry, V. Kasabri
{"title":"Oxytocin, the panacea for long-COVID? a review","authors":"P. Diep, M. Chaudry, Adam Dixon, Faisal Chaudry, V. Kasabri","doi":"10.1515/hmbci-2021-0034","DOIUrl":"https://doi.org/10.1515/hmbci-2021-0034","url":null,"abstract":"Abstract Objectives In this hypothesis paper we explore the underlying mechanisms for long-COVID and how the oxytocinergic neurones could be infected by SARS-CoV-2 leading to a reduction in plasma oxytocin (OXT). Furthermore, we aim to review the relevance of OXT and hypothalamic function in recovery from long-COVID symptoms and pathology, through exploring the pro-health effects of the OXT neuropeptide. Methods A review of published literature was surveyed using Google Scholar and PubMed. Results Numerous experimental data can be shown to correlate with OXT and long-COVID symptoms and conditions, thus providing strong circumstantial evidence to support our hypothesis. It is postulated that the reduction in plasma OXT due to acute and post-viral damage to the hypothalamus and oxytocinergic neurones contributes to the variable multi-system, remitting and relapsing nature of long-COVID. The intranasal route of OXT application was determined to be most appropriate and clinically relevant for the restoration of oxytocinergic function post COVID-19 infection. Conclusions We believe it is imperative to further investigate whether OXT alleviates the prolonged suffering of patients with long-COVID. Succinctly, OXT may be the much-needed post-pandemic panacea. Highlights – A comparison of long-COVID pathology with OXT therapeutic mechanisms was performed – Following advances in neuroendocrinology and proposed use of OXT as an acute antiviral for COVID-19; we elaborate OXT mechanisms in treating long-COVID, via therapeutic relevance and methods of administration. – Further demonstration of OXT levels for different demographic and susceptibility groups, acute levels in COVID-19 patients and periodic monitoring of OXT levels is warranted.","PeriodicalId":13224,"journal":{"name":"Hormone Molecular Biology and Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2022-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46792580","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":"Anti Mullerian hormone as a diagnostic tool for polycystic ovary syndrome in women of reproductive age with morbid obesity.","authors":"Ibtissem Oueslati, Mohamed Bassem Hammami, Seif Boukriba, Hana Ben Hadj Hassen, Meriem Yazidi, Fatma Chaker, Habiba Mizouni, Moncef Feki, Melika Chihaoui","doi":"10.1515/hmbci-2021-0078","DOIUrl":"10.1515/hmbci-2021-0078","url":null,"abstract":"<p><strong>Objectives: </strong>The aim of the study was to analyze the performance of the anti-mullerian hormone (AMH) level for the diagnosis of polycystic ovary syndrome in women with morbid obesity.</p><p><strong>Study design: </strong>A single-centre cross-sectional study was conducted in 50 women of reproductive age with a body mass index (BMI) ≥ 40 kg/m<sup>2</sup>. Each patient underwent a clinical examination, biological and hormonal assays, and an ovarian ultrasound between the third and the fifth day of the menstrual cycle. Polycystic ovary syndrome was diagnosed according to the Rotterdam's criteria.</p><p><strong>Results: </strong>The mean age of participants was 34.2 ± 7.5 years. Polycystic ovary syndrome was diagnosed in 20 women (40%). Age and anthropometric parameters did not differ between women with and without polycystic ovary syndrome. The mean AMH level was significantly higher in women with polycystic ovary syndrome (3.4 ± 3.6 vs 1.3 ± 1.2 ng/ml, p=0.010). It was positively correlated with the Ferriman and Gallwey score (r=0.496, p=0.016), total testosterone level (r=0.524, p < 10<sup>-3</sup>) and the LH/FSH ratio (r=0.290, p=0.046). In women aged between 35 and 45 years, the optimum cut-off level for the diagnosis of polycystic ovary syndrome was 0.81 ng/mL, providing a sensitivity and a specificity of 90 and 71%, respectively with an area under the ROC curve of 0.857.</p><p><strong>Conclusions: </strong>AMH level was significantly higher in morbid obese women with polycystic ovary syndrome compared with those without polycystic ovary syndrome. Specific thresholds for this population must be assessed to improve the sensitivity and specificity of AMH for the diagnosis of polycystic ovary syndrome.</p>","PeriodicalId":13224,"journal":{"name":"Hormone Molecular Biology and Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":1.1,"publicationDate":"2022-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10450735","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}
H. Heidari, A. Khalaj, S. Khani, Maasoume Abdollahi, H. Farahani, S. Khani
{"title":"Hypoglycemic, hypolipidemic and hepatoprotective effects of Alpinia officinarum on nicotinamide/streptozotocin induced type II diabetic rats","authors":"H. Heidari, A. Khalaj, S. Khani, Maasoume Abdollahi, H. Farahani, S. Khani","doi":"10.1515/hmbci-2021-0050","DOIUrl":"https://doi.org/10.1515/hmbci-2021-0050","url":null,"abstract":"Abstract Objectives Alpinia officinarum Hance, commonly known as lesser galangal, is a member of the ginger family (Zingiberaceae) traditionally used for many decades to treat inflammation, pain, stomach ache and cold. In the present study, the antidiabetic and hypolipidemic potentials of the hydroalcoholic extract of A. officinarum (AO) were investigated in the nicotinamide/streptozotocin induced type II diabetic rats. Methods Male Wistar rats were divided into following six groups: Group I was normal control rats. Group II: normal diabetic control, Group III: Diabetic rats treated with glibenclamide (0.25 mg/kg), IV, V and VI: Diabetic rats treated with 100, 200 and 500 mg/kg AO hydroalcoholic extract by daily gavage for 28 days, respectively. At the end of treatment, biochemical analysis, histological study, phytochemical analysis and acute toxicity tests were carried out. Results The results show significant reduction in blood glucose, serum lipid profiles, and liver enzyme levels in diabetic rats compared with diabetic control in AO treated group. Conclusions In conclusion, the present study demonstrated that AO extract had significant (p<0.05) antidiabetic and anti-hyperlipidemia effects in addition to hepatoprotective effect in type II diabetic rats.","PeriodicalId":13224,"journal":{"name":"Hormone Molecular Biology and Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2022-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49119399","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}
H. Giha, F. Joatar, Dhuha M. B. AlDehaini, Z. Malalla, Muhalab E. Ali, Ali Al Qarni
{"title":"Association of obesity in T2DM with differential polymorphism of ghrelin, growth hormone secretagogue receptor-1 and telomeres maintenance genes","authors":"H. Giha, F. Joatar, Dhuha M. B. AlDehaini, Z. Malalla, Muhalab E. Ali, Ali Al Qarni","doi":"10.1515/hmbci-2021-0063","DOIUrl":"https://doi.org/10.1515/hmbci-2021-0063","url":null,"abstract":"Abstract Background Although obesity and T2DM comorbidity is too frequent, the molecular basis of diabetic obesity is largely unexplained and barely investigated. Materials Cross-sectional studies were conducted in Kingdom of Saudi Arabia (KSA) in 2013 and Kuwait in 2019. Fasting blood samples were obtained from a total of 216 T2DM patients (104 from KSA) and 193 nondiabetic subjects (93 from KSA) after their consents. Eight SNPs in 5 genes known to be associated with both obesity and T2DM, ghrelin (GHRL) and growth hormone secretagogue receptor -GHSR (KSA) and telomeres maintenance genes (Kuwait) were genotyped by rtPCR. Both patients and controls were grouped into obese and non-obese and sub-grouped into 4-BMI- grades: normal, overweight (OW), obese (OBS) and severely obese (SOBS). Results Showed that the only SNP which was distinguished between all groups/subgroups in all study subjects was the ACYP2 rs6713088G/C, where the common CC genotype was under-expressed in the obese compared to non-obese diabetics (17.8% vs. 40.4%, p 0.01) and between the 4-BMI-grade (p 0.025). Interestingly the same genotype was over-expressed in obese compared to non-obese non-diabetics (50% vs. 27.6%, p 0.04). Furthermore, the GHRL (rs27647C/T), GHSR (rs509030G/C) and TERC (rs12696304G/C) MAFs were significantly low in normal BMI patients; p=0.034, 0.008 and 0.011, respectively. Conclusions This is the first report about the molecular distinction between the obese and non-obese diabetics, it showed the association of rs6713088G/C mutant allele with diabetic obesity, while the GHRL, GHSR and TERC SNPs were differentially expressed based on the BMI-grades.","PeriodicalId":13224,"journal":{"name":"Hormone Molecular Biology and Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2022-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42680625","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":"A study on lipoprotein-a and PAI-1 in women with polycystic ovary syndrome","authors":"A. K. Shah, B. Yadav, A. Suri, A. K. Shah","doi":"10.1515/hmbci-2021-0044","DOIUrl":"https://doi.org/10.1515/hmbci-2021-0044","url":null,"abstract":"Abstract Objectives PCOS is the most common endocrinological disorder amongst women of reproductive age. The consequences of PCOS extend beyond the reproductive axis and may lead to the development of metabolic syndrome leading to a high risk for hypertension and cardiovascular disease. Therefore, a more comprehensive evaluation of biochemical markers that reflect the cardiovascular risk is required for further understanding of pathophysiologic mechanisms, diagnosis and management. Methods In this case-control study, women diagnosed with PCOS (n=100) in the age group (18–35 years) years were taken as cases and age matched healthy controls (n=100) were enrolled. Estimations of fasting plasma Glucose, serum total cholesterol (TC), triglycerides (TG) and High-density lipoprotein (HDL) concentrations were assayed while Low-density lipoprotein (LDL) was calculated by using Fredrickson Friedwald’s formula. Serum Lipoprotein-a (Lp-a) was estimated using ELISA (Enzyme Linked Immunosorbent Assay). The quantitative data were expressed as Mean ± Standard Deviation (SD). Unpaired Student’s t-test was used to compare the values (PCOS vs Controls) and Pearson’s correlation coefficient was used to elucidate the relationship between the variables. Results FBS and all lipid parameters were significantly increased in PCOS patients compared to control subjects. On the other hand, HDL-C was significantly decreased as compared to the control subjects. The hormones TSH, LH, FSH, PRL and LH/FSH ratio were significantly increased in PCOS patients compared to control subjects. Lipoprotein-a and PAI-1 was significantly increased in PCOS patients compared to the control subjects. Upon bivariate correlation analysis, Lp(a) had significant correlations with PAI-1 (r=0.35, p=0.000), WHR (r=0.25, p=0.000), LDL (r=0.52, p=0.000) and TSH (r=0.24, p=0.000). While the correlations with FBS (r=−0.008, p=0.91) and LH/FSH ratio (r=−0.004, p=0.95) were statistically insignificant. Conclusions The evaluation of serum biomarkers such as Lp-a, PAI-1 and lipid profile routinely in PCOS patients may have diagnostic role in the early detection of metabolic abnormalities and endocrine derangements and timely management of comorbid Diabetes and Cardiovascular disease in PCOS females.","PeriodicalId":13224,"journal":{"name":"Hormone Molecular Biology and Clinical Investigation","volume":null,"pages":null},"PeriodicalIF":1.0,"publicationDate":"2022-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44203058","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}