{"title":"Potential role of oxytocin in the regulation of memories and treatment of memory disorders","authors":"Vaibhav Walia , Pranay Wal , Shweta Mishra , Ankur Agrawal , Sourabh Kosey , Aditya Dilipkumar Patil","doi":"10.1016/j.peptides.2024.171222","DOIUrl":"https://doi.org/10.1016/j.peptides.2024.171222","url":null,"abstract":"<div><p>Oxytocin (OXT) is an “affiliative” hormone or neurohormone or neuropeptide consists of nine amino acids, synthesized in magnocellular neurons of paraventricular (PVN) and supraoptic nuclei (SON) of hypothalamus. OXT receptors are widely distributed in various region of brain and OXT has been shown to regulate various social and nonsocial behavior. Hippocampus is the main region which regulates the learning and memory. Hippocampus particularly regulates the acquisition of new memories and retention of acquired memories. OXT has been shown to regulate the synaptic plasticity, neurogenesis, and consolidation of memories. Further, findings from both preclinical and clinical studies have suggested that the OXT treatment improves performance in memory related task. Various trials have suggested the positive impact of intranasal OXT in the dementia patients. However, these studies are limited in number. In the present study authors have highlighted the role of OXT in the formation and retrieval of memories. Further, the study demonstrated the outcome of OXT treatment in various memory and related disorders.</p></div>","PeriodicalId":19765,"journal":{"name":"Peptides","volume":"177 ","pages":"Article 171222"},"PeriodicalIF":3.0,"publicationDate":"2024-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140645843","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}
PeptidesPub Date : 2024-04-18DOI: 10.1016/j.peptides.2024.171225
Wenjia Chen , Yu Fu , Yuanyuan Jin , Wanqiu Zheng , Yue Liu
{"title":"Reduced plasma cortistatin is related to clinical parameters in patients with essential hypertension","authors":"Wenjia Chen , Yu Fu , Yuanyuan Jin , Wanqiu Zheng , Yue Liu","doi":"10.1016/j.peptides.2024.171225","DOIUrl":"10.1016/j.peptides.2024.171225","url":null,"abstract":"<div><h3>Background</h3><p>Cortistatin (CST), an endogenous bioactive polypeptide, has been acknowledged for its protective effect against several cardiovascular diseases, but its relationship with hypertension remains unclear. Therefore, we aimed to investigate changes in plasma CST in hypertensive patients and further analyze correlations with blood pressure, metabolic parameters and left ventricular structure and function.</p></div><div><h3>Methods</h3><p>In this hospital-based study, basic information and plasma samples for evaluating clinically relevant indicators such as total cholesterol (TC), triglycerides (TGs), fasting blood glucose (FGB), serum creatinine (Scr) and CST were collected from 81 essential hypertension patients and 75 normotensive subjects. Plasma CST levels were examined by enzyme-linked immunosorbent assay (ELISA).</p></div><div><h3>Results</h3><p>Compared with normotensive subjects, plasma CST was significantly lower in hypertensive patients. Plasma CST levels in hypertensive patients without blood pressure control was significantly lower than those of hypertensive patients with blood pressure control. Plasma CST levels were significantly negatively correlated with SBP and serum creatinine (Scr) in the overall population. Furthermore, multivariate logistic regression analysis showed that the OR of CST for hypertension was 0.64 using the unadjusted model, and there was still statistical significance using the four-adjusted model.</p></div><div><h3>Conclusions</h3><p>The circulating concentration of CST was significantly lower in hypertensive patients and was higher after blood pressure control, suggesting that CST may be a new endogenous protective target for hypertension.</p></div>","PeriodicalId":19765,"journal":{"name":"Peptides","volume":"177 ","pages":"Article 171225"},"PeriodicalIF":3.0,"publicationDate":"2024-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140786423","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}
PeptidesPub Date : 2024-04-16DOI: 10.1016/j.peptides.2024.171216
Giulia Angelini , Sara Russo , Geltrude Mingrone
{"title":"Incretin hormones, obesity and gut microbiota","authors":"Giulia Angelini , Sara Russo , Geltrude Mingrone","doi":"10.1016/j.peptides.2024.171216","DOIUrl":"10.1016/j.peptides.2024.171216","url":null,"abstract":"<div><p>Over the past 40 years, the prevalence of obesity has risen dramatically, reaching epidemic proportions. By 2030 the number of people affected by obesity will reach 1.12 billion worldwide. Gastrointestinal hormones, namely incretins, play a vital role in the pathogenesis of obesity and its comorbidities. GIP (glucose-dependent insulinotropic polypeptide) and GLP-1 (glucagon-like peptide-1), which are secreted from the intestine after nutrient intake and stimulate insulin secretion from pancreatic β cells, influence lipid metabolism, gastric empting, appetite and body weight. The gut microbiota plays an important role in various metabolic conditions, including obesity and type 2 diabetes and influences host metabolism through the interaction with enteroendocrine cells that modulate incretins secretion. Gut microbiota metabolites, such as short-chain fatty acids (SCFAs) and indole, directly stimulate the release of incretins from colonic enteroendocrine cells influencing host satiety and food intake. Moreover, bariatric surgery and incretin-based therapies are associated with increase gut bacterial richness and diversity. Understanding the role of incretins, gut microbiota, and their metabolites in regulating metabolic processes is crucial to develop effective strategies for the management of obesity and its associated comorbidities.</p></div>","PeriodicalId":19765,"journal":{"name":"Peptides","volume":"178 ","pages":"Article 171216"},"PeriodicalIF":3.0,"publicationDate":"2024-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S019697812400069X/pdfft?md5=2b030edf305987e46a9561614bbb321e&pid=1-s2.0-S019697812400069X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140766466","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}
PeptidesPub Date : 2024-04-16DOI: 10.1016/j.peptides.2024.171220
Weslei da Silva Oliveira , César Roberto Viana Teixeira , Hilário Cuquetto Mantovani , Silvio Santana Dolabella , Sona Jain , Ana Andréa Teixeira Barbosa
{"title":"Nisin variants: What makes them different and unique?","authors":"Weslei da Silva Oliveira , César Roberto Viana Teixeira , Hilário Cuquetto Mantovani , Silvio Santana Dolabella , Sona Jain , Ana Andréa Teixeira Barbosa","doi":"10.1016/j.peptides.2024.171220","DOIUrl":"10.1016/j.peptides.2024.171220","url":null,"abstract":"<div><p>Nisin A is a lantibiotic bacteriocin typically produced by strains of <em>Lactococcus lactis</em>. This bacteriocin has been approved as a natural food preservative since the late 1980 s and shows antimicrobial activity against a range of food-borne spoilage and pathogenic microorganisms. The therapeutic potential of nisin A has also been explored increasingly both in human and veterinary medicine. Nisin has been shown to be effective in treating bovine mastitis, dental caries, cancer, and skin infections. Recently, it was demonstrated that nisin has an affinity for the same receptor used by SARS-CoV-2 to enter human cells and was proposed as a blocker of the viral infection. Several nisin variants produced by distinct bacterial strains or modified by bioengineering have been described since the discovery of nisin A. These variants present modifications in the peptide structure, biosynthesis, mode of action, and spectrum of activity. Given the importance of nisin for industrial and therapeutic applications, the objective of this study was to describe the characteristics of the nisin variants, highlighting the main differences between these molecules and their potential applications. This review will be useful to researchers interested in studying the specifics of nisin A and its variants.</p></div>","PeriodicalId":19765,"journal":{"name":"Peptides","volume":"177 ","pages":"Article 171220"},"PeriodicalIF":3.0,"publicationDate":"2024-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140756519","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":"Attachment-like behavioral expressions to humans in puppies are related to oxytocin and cortisol: A comparative study of Akitas and Labrador Retrievers","authors":"Miho Nagasawa, Sakiko Tomori, Kazutaka Mogi, Takefumi Kikusui","doi":"10.1016/j.peptides.2024.171224","DOIUrl":"10.1016/j.peptides.2024.171224","url":null,"abstract":"<div><p>This study investigated the relationship between urinary hormone concentrations and attachment-related behaviors in two dog breeds, the Akitas and Labrador Retrievers, to elucidate the hormonal and behavioral mechanisms underlying domestication and interspecies attachment to humans. By measuring cortisol and oxytocin concentrations, and conducting the Strange Situation Test (SST), we aimed to investigate breed differences in endocrine secretions associated with domestication and how these differences influence dog behavior toward humans. Our results showed significant breed differences in urinary cortisol concentrations, with Akitas exhibiting higher levels than Labrador Retrievers. This suggests a breed-specific stress response related to genetic proximity to wolves. However, oxytocin concentrations did not differ significantly, which suggests a complex interplay between factors influencing the domestication process and the formation of attachment behaviors. Behavioral observations during the SST revealed breed-specific patterns, with Labrador Retrievers showing more playful and attachment-like behaviors and Akitas showing more exploratory and passive behaviors. The study found correlations between hormones and behaviors within breeds, particularly in Labrador Retrievers, where oxytocin concentrations were associated with attachment-like behaviors, and cortisol concentrations reflected individual differences in physical activity rather than stress responses to social situations. These findings contribute to the understanding of the evolutionary and adaptive processes underlying the ability of domestic dogs to form close relationships with humans while highlighting the role of hormonal mechanisms in mediating attachment behaviors and the influence of breed-specific genetic backgrounds on these processes.</p></div>","PeriodicalId":19765,"journal":{"name":"Peptides","volume":"177 ","pages":"Article 171224"},"PeriodicalIF":3.0,"publicationDate":"2024-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140771909","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}
PeptidesPub Date : 2024-04-14DOI: 10.1016/j.peptides.2024.171223
Hong Zhou , Rui Zhu , Yuqing Xia , Xinming Zhang , Zixu Wang , George H. Lorimer , Reza A. Ghiladi , Hasan Bayram , Jun Wang
{"title":"Neuropeptides affecting social behavior in mammals: Oxytocin","authors":"Hong Zhou , Rui Zhu , Yuqing Xia , Xinming Zhang , Zixu Wang , George H. Lorimer , Reza A. Ghiladi , Hasan Bayram , Jun Wang","doi":"10.1016/j.peptides.2024.171223","DOIUrl":"https://doi.org/10.1016/j.peptides.2024.171223","url":null,"abstract":"<div><p>Oxytocin (OXT), a neuropeptide consisting of only nine amino acids, is synthesized in the paraventricular and supraoptic nuclei of the hypothalamus. Although OXT is best known for its role in lactation and parturition, recent research has shown that it also has a significant impact on social behaviors in mammals. However, a comprehensive review of this topic is still lacking. In this paper, we systematically reviewed the effects of OXT on social behavior in mammals. These effects of OXT from the perspective of five key behavioral dimensions were summarized: parental behavior, anxiety, aggression, attachment, and empathy. To date, researchers have agreed that OXT plays a positive regulatory role in a wide range of social behaviors, but there have been controversially reported results. In this review, we have provided a detailed panorama of the role of OXT in social behavior and, for the first time, delved into the underlying regulatory mechanisms, which may help better understand the multifaceted role of OXT. Levels of OXT in previous human studies were also summarized to provide insights for diagnosis of mental disorders.</p></div>","PeriodicalId":19765,"journal":{"name":"Peptides","volume":"177 ","pages":"Article 171223"},"PeriodicalIF":3.0,"publicationDate":"2024-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140650191","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}
PeptidesPub Date : 2024-04-14DOI: 10.1016/j.peptides.2024.171221
Emilie Lahaye, Sergueï O. Fetissov
{"title":"Functional role of immunoglobulin G as an oxytocin-carrier protein","authors":"Emilie Lahaye, Sergueï O. Fetissov","doi":"10.1016/j.peptides.2024.171221","DOIUrl":"https://doi.org/10.1016/j.peptides.2024.171221","url":null,"abstract":"<div><p>It has been long-time known that oxytocin in plasma is bound to a carrier protein, a common feature of circulating peptide hormones, however, the nature of such protein was uncertain. A recent study revealed that about 60% of oxytocin present in plasma is bound to immunoglobulin G (IgG) and that oxytocin-binding IgG plays a role of a functional oxytocin carrier protein. Here, we review the historical background and methodology leading to this discovery. Moreover, we review the data showing the functional role of oxytocin-binding IgG in the modulation of oxytocin signaling relevant to the regulation of motivated behavior and several neuropsychiatric disorders. Furthermore, the possible role of gut microbiota in the origin of such IgG is discussed and the relevant new therapeutic strategies for the enhancement of oxytocin signaling are presented.</p></div>","PeriodicalId":19765,"journal":{"name":"Peptides","volume":"177 ","pages":"Article 171221"},"PeriodicalIF":3.0,"publicationDate":"2024-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0196978124000743/pdfft?md5=4d6eaf64cd7bb365d49672e55ed4d8ec&pid=1-s2.0-S0196978124000743-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140644242","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":"Positive interplay between FFAR4/GPR120, DPP-IV inhibition and GLP-1 in beta cell proliferation and glucose homeostasis in obese high fat fed mice","authors":"A.I. Owolabi, R.C. Corbett, P.R. Flatt, A.M. McKillop","doi":"10.1016/j.peptides.2024.171218","DOIUrl":"10.1016/j.peptides.2024.171218","url":null,"abstract":"<div><p>G-protein coupled receptor-120 (GPR120; <em>FFAR4</em>) is a free fatty acid receptor, widely researched for its glucoregulatory and insulin release activities. This study aimed to investigate the metabolic advantage of FFAR4/GPR120 activation using combination therapy. C57BL/6 mice, fed a High Fat Diet (HFD) for 120 days to induce obesity-diabetes, were subsequently treated with a single daily oral dose of FFAR4/GPR120 agonist Compound A (CpdA) (0.1μmol/kg) alone or in combination with sitagliptin (50 mg/kg) for 21 days. After 21-days, glucose homeostasis, islet morphology, plasma hormones and lipids, tissue genes (qPCR) and protein expression (immunocytochemistry) were assessed. Oral administration of CpdA improved glucose tolerance (34% p<0.001) and increased circulating insulin (38% p<0.001). Addition of CpdA with the dipeptidyl peptidase-IV (DPP-IV) inhibitor, sitagliptin, further improved insulin release (44%) compared to sitagliptin alone and reduced fat mass (p<0.05). CpdA alone (50%) and in combination with sitagliptin (89%) induced marked reductions in LDL-cholesterol, with greater effects in combination (p<0.05). All treatment regimens restored pancreatic islet and beta-cell area and mass, complemented with significantly elevated beta-cell proliferation rates. A marked increase in circulating GLP-1 (53%) was observed, with further increases in combination (38%). With treatment, mice presented with increased <em>Gcg</em> (proglucagon) gene expression in the jejunum (130% increase) and ileum (120% increase), indicative of GLP-1 synthesis and secretion. These data highlight the therapeutic promise of FFAR4/GPR120 activation and the potential for combined benefit with incretin enhancing DPP-IV inhibitors in the regulation of beta cell proliferation and diabetes.</p></div>","PeriodicalId":19765,"journal":{"name":"Peptides","volume":"177 ","pages":"Article 171218"},"PeriodicalIF":3.0,"publicationDate":"2024-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0196978124000718/pdfft?md5=00b12a466d31f791dabd1fa8b9a1c076&pid=1-s2.0-S0196978124000718-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140708548","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}
PeptidesPub Date : 2024-04-13DOI: 10.1016/j.peptides.2024.171219
Emma Rose McGlone , Tricia M.-M. Tan
{"title":"Glucagon-based therapy for people with diabetes and obesity: What is the sweet spot?","authors":"Emma Rose McGlone , Tricia M.-M. Tan","doi":"10.1016/j.peptides.2024.171219","DOIUrl":"https://doi.org/10.1016/j.peptides.2024.171219","url":null,"abstract":"<div><p>People with obesity and type 2 diabetes have a high prevalence of metabolic-associated steatotic liver disease, hyperlipidemia and cardiovascular disease. Glucagon increases hepatic glucose production; it also decreases hepatic fat accumulation, improves lipidemia and increases energy expenditure. Pharmaceutical strategies to antagonize the glucagon receptor improve glycemic outcomes in people with diabetes and obesity, but they increase hepatic steatosis and worsen dyslipidemia. Co-agonism of the glucagon and glucagon-like peptide-1 (GLP-1) receptors has emerged as a promising strategy to improve glycemia in people with diabetes and obesity. Addition of glucagon receptor agonism enhances weight loss, reduces liver fat and ameliorates dyslipidemia. Prior to clinical use, however, further studies are needed to investigate the safety and efficacy of glucagon and GLP-1 receptor co-agonists in people with diabetes and obesity and related conditions, with specific concerns regarding a higher prevalence of gastrointestinal side effects, loss of muscle mass and increases in heart rate. Furthermore, co-agonists with differing ratios of glucagon:GLP-1 receptor activity vary in their clinical effect; the optimum balance is yet to be identified.</p></div>","PeriodicalId":19765,"journal":{"name":"Peptides","volume":"176 ","pages":"Article 171219"},"PeriodicalIF":3.0,"publicationDate":"2024-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S019697812400072X/pdfft?md5=0655069d2d8f2cc91f7cd1e467464703&pid=1-s2.0-S019697812400072X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140558083","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":"Negative association between basal oxytocin and oxytocin changes after repetitive transcranial magnetic stimulation in patients with treatment-resistant depression","authors":"Ryohei Kojima , Hiroshi Tateishi , Hiroko Kunitake , Yoshiomi Imamura , Yutaka Kunitake , Toru Murakawa , Chika Nagahama , Takumi Shiraishi , Ken Takada , Masataka Hirano , Airi Fukai , Akira Tomonari , Akira Monji , Yoshito Mizoguchi","doi":"10.1016/j.peptides.2024.171217","DOIUrl":"https://doi.org/10.1016/j.peptides.2024.171217","url":null,"abstract":"<div><p>Repetitive transcranial magnetic stimulation (rTMS) is a neuromodulator effective for treating depressive symptoms in patients with treatment-resistant depression (TRD). One of the multiple mechanisms for its antidepressant effects proposed is related to the hypothalamus. Oxytocin is a neuropeptide synthesized in the hypothalamus that affects human behavior and psychology, including social and affiliative behaviors, stress regulation, and fear and emotion processing. There have been no reports on the relationship between rTMS and oxytocin for the treatment of TRD. Therefore, we aimed to investigate changes in salivary oxytocin concentrations in patients with TRD before and after 6 weeks of rTMS treatment. A total of 28 patients with TRD who received rTMS at Saga University Hospital between August 2013 and August 2020 were included. Although rTMS treatment significantly improved 24-item Hamilton Depression Rating Scale scores, rTMS treatment did not change mean salivary oxytocin after 6 weeks of treatment in patients with TRD. Multiple regression analysis revealed that the change in salivary oxytocin levels after rTMS treatment was negatively associated with basal oxytocin levels before rTMS treatment, suggesting that rTMS treatment tends to decrease oxytocin levels in patients with depression with high basal oxytocin levels while increasing them in those with low basal levels. These findings suggest that rTMS treatment improved depressive symptoms through mechanisms other than the modulatory effect on oxytocin levels in patients with TRD, while there is room for further studies to confirm these findings using a larger patient sample size and/or a sham rTMS procedure.</p></div>","PeriodicalId":19765,"journal":{"name":"Peptides","volume":"177 ","pages":"Article 171217"},"PeriodicalIF":3.0,"publicationDate":"2024-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140548953","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}