Sameer Chabhadiya , D.K. Acharya , Amitsinh Mangrola , Rupal Shah , Edwin A. Pithawala
{"title":"Unlocking the potential of biosurfactants: Innovations in metabolic and genetic engineering for sustainable industrial and environmental solutions","authors":"Sameer Chabhadiya , D.K. Acharya , Amitsinh Mangrola , Rupal Shah , Edwin A. Pithawala","doi":"10.1016/j.biotno.2024.07.001","DOIUrl":"10.1016/j.biotno.2024.07.001","url":null,"abstract":"<div><p>Biosurfactants, synthesized by microorganisms, hold potential for various industrial and environmental applications due to their surface-active properties and biodegradability. Metabolic and genetic engineering strategies enhance biosurfactant production by modifying microbial pathways and genetics. Strategies include optimizing biosurfactant biosynthesis pathways, expanding substrate utilization, and improving stress responses. Genetic engineering allows customization of biosurfactant characteristics to meet industrial needs. Notable examples include engineering <em>Pseudomonas aeruginosa</em> for enhanced rhamnolipid production and creating synthetic biosurfactant pathways in non-native hosts like <em>Escherichia coli</em>. CRISPR-Cas9 technology offers precise tools for genetic manipulation, enabling targeted gene disruption and promoter optimization to enhance biosurfactant production efficiency. Synthetic promoters enable precise control over biosurfactant gene expression, contributing to pathway optimization across diverse microbial hosts. The future of biosurfactant research includes sustainable bio-processing, customized biosurfactant engineering, and integration of artificial intelligence and systems biology. Advances in genetic and metabolic engineering will enable tailor-made biosurfactants for diverse applications, with potential for industrial-scale production and commercialization. Exploration of untapped microbial diversity may lead to novel biosurfactants with unique properties, expanding the versatility and sustainability of biosurfactant-based solutions.</p></div>","PeriodicalId":100186,"journal":{"name":"Biotechnology Notes","volume":"5 ","pages":"Pages 111-119"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665906924000114/pdfft?md5=4bf3ad577ced0d7cd38282ad249883ae&pid=1-s2.0-S2665906924000114-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141853412","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"CRISPR/Cas9 improves targeted knock-in efficiency in Aspergillus oryzae","authors":"Takehiko Todokoro, Yoji Hata, Hiroki Ishida","doi":"10.1016/j.biotno.2024.03.002","DOIUrl":"https://doi.org/10.1016/j.biotno.2024.03.002","url":null,"abstract":"<div><p><em>Aspergillus oryzae</em> is an important fungus in food and industrial enzyme production. In <em>A. oryzae</em>, targeted knock-in transformation is primarily limited to homologous recombination (HR)-based systems, in which non-homologous end-joining (NHEJ)-disruptant hosts are required. However, preparation of hosts and transformation templates for such systems is laborious, in addition to other disadvantages. In the present study, we examined alternative targeted knock-in mediated by CRISPR/Cas9, in which a microhomology-mediated end-joining (MMEJ) and single-strand annealing (SSA) repair system was employed. This approach enabled the efficient development of targeted knock-in transformants without host preparation using only a short homology template. We conclude that this new method could be applied to facilitate the transformation of <em>A. oryzae</em>, and will make it easier to acquire targeted knock-in transformants, especially from industrially important non-model strains.</p></div>","PeriodicalId":100186,"journal":{"name":"Biotechnology Notes","volume":"5 ","pages":"Pages 58-63"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665906924000072/pdfft?md5=c3071d5a3a69f15bfd265d3655c1bb84&pid=1-s2.0-S2665906924000072-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140351146","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Muhammad Hamizan Zawawi , Siti Azhani-Amran , Zuraidah Abdullah , Sabreena Safuan
{"title":"Unraveling current breakthroughs in Scorodocarpus borneensis phytochemical therapeutics: A systematic review","authors":"Muhammad Hamizan Zawawi , Siti Azhani-Amran , Zuraidah Abdullah , Sabreena Safuan","doi":"10.1016/j.biotno.2024.05.001","DOIUrl":"10.1016/j.biotno.2024.05.001","url":null,"abstract":"<div><p><em>Scorodocarpus borneensis</em>, also known as the Kulim tree or Garlic tree, has been consumed by the local communities in Southeast Asia as traditional spice using its old leaves, stem bark, and seeds. The locals also used Kulim tree parts as conventional alternative to treat many diseases such as hemorrhoids, leprosy, diabetes, and diarrhea. However, there was limited scientific evidence to support these traditional claims. Therefore, this systematic review aims to present and evaluate a detailed overview of the phytochemical constituents of <em>S. borneensis</em> from previous existing studies, shedding light on their chemical composition, bioactivity, and potential applications. In addition, current studies regarding <em>S. borneensis</em> are still on a fundamental level. Hence, we aim that this review will reveal the research gap from the previous literature and provide an insight to implement a new research approach in the future. A literature search was conducted using four databases: ScienceDirect, Scopus, Web of Science, and PubMed. The articles were screened and data were extracted based on the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guideline. 8 studies satisfied the inclusion criteria that focused on the phytochemicals from <em>S. borneensis</em>. The major phytochemical compound reported was phenolic compound. S. borneensis also exhibits several therapeutic outcomes such as antioxidant, antimicrobial, and anticancer but current studies are not enough to support the claims regarding <em>S. borneensis</em> health benefit. In conclusion, this review highlights the current understanding of <em>S. borneensis’</em> phytochemical composition and its therapeutic applications which are important in preventing human diseases especially non-communicable diseases.</p></div>","PeriodicalId":100186,"journal":{"name":"Biotechnology Notes","volume":"5 ","pages":"Pages 100-110"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665906924000084/pdfft?md5=2778f6f37bfba7a5ed673fe883e37908&pid=1-s2.0-S2665906924000084-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141401655","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Hosam Elhalis , Mohamed Helmy , Sherilyn Ho , Sharon Leow , Yan Liu , Kumar Selvarajoo , Yvonne Chow
{"title":"Identifying Chlorella vulgaris and Chlorella sorokiniana as sustainable organisms to bioconvert glucosamine into valuable biomass","authors":"Hosam Elhalis , Mohamed Helmy , Sherilyn Ho , Sharon Leow , Yan Liu , Kumar Selvarajoo , Yvonne Chow","doi":"10.1016/j.biotno.2024.01.003","DOIUrl":"https://doi.org/10.1016/j.biotno.2024.01.003","url":null,"abstract":"<div><p>Chitin is a major component of various wastes such as crustacean shells, filamentous fungi, and insects. Recently, food-safe biological and chemical processes converting chitin to glucosamine have been developed. Here, we studied microalgae that can uptake glucosamine as vital carbon and nitrogen sources for valuable alternative protein biomass. Utilizing data mining and bioinformatics analysis, we identified 29 species that contain the required enzymes for glucosamine to glucose conversion. The growth performance of the selected strains was examined, and glucosamine was used in different forms and concentrations. Glucose at a concentration of 2.5 g/L was required to initiate glucosamine metabolic degradation by <em>Chlorella vulgaris</em> and <em>Chlorella sorokiniana</em>. Glucosamine HCl and glucosamine phosphate showed maximum cell counts of about 8.5 and 9.0 log/mL for C. <em>sorokiniana</em> and <em>C. vulgaris</em> in 14 days, respectively. Enzymatic hydrolysis of glucosamine increased growth performance with <em>C. sorokiniana</em> by about 3 folds. The adapted strains were fast-growing and could double their dry biomasses during the same incubation time. In addition, adapted <em>C. sorokiniana</em> was able to tolerate three times glucosamine concentration in the medium. The study illustrated possible strategies for employing <em>C. sorokiniana</em> and <em>C. vulgaris</em> to convert glucosamine into valuable biomass in a more sustainable way.</p></div>","PeriodicalId":100186,"journal":{"name":"Biotechnology Notes","volume":"5 ","pages":"Pages 13-22"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665906924000035/pdfft?md5=2d02dd1a924a37191fa8804cbe12d15c&pid=1-s2.0-S2665906924000035-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139494007","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Karthik K Karunakar , Binoy Varghese Cheriyan , Krithikeshvaran R , Gnanisha M , Abinavi B
{"title":"\"Therapeutic advancements in nanomedicine: The multifaceted roles of silver nanoparticles\"","authors":"Karthik K Karunakar , Binoy Varghese Cheriyan , Krithikeshvaran R , Gnanisha M , Abinavi B","doi":"10.1016/j.biotno.2024.05.002","DOIUrl":"10.1016/j.biotno.2024.05.002","url":null,"abstract":"<div><p>Nanotechnology has the advantages of enhanced bioactivity, reduced toxicity, target specificity, and sustained release and NPs can penetrate cell membranes. The small size of silver nanoparticles, AgNPs, large surface area, and unique physicochemical properties contribute to cell lysis and increased permeability of cell membranes used in the field of biomedicine. Functional precursors integrate with phytochemicals to create distinctive therapeutic properties and the stability of the nanoparticles can be enhanced by Surface coatings and encapsulation methods, The current study explores the various synthesis methods and characterization techniques of silver nanoparticles (AgNPs) and highlights their intrinsic activity in therapeutic applications, Anti-cancer activity noted at a concentration range of 5–50 μg/ml and angiogenesis is mitigated at a dosage range of 10–50 μg/ml, Diabetes is controlled within the same concentration. Wound healing is improved at concentrations of 10–50 μg/ml and with a typical range of 10–08 μg/ml for bacteria with antimicrobial capabilities. Advancement of silver nanoparticles with a focus on the future use of AgNPs-coated wound dressings and medical devices to decrease the risk of infection. Chemotherapeutic drugs can be administered by AgNPs, which reduces adverse effects and an improvement in treatment outcomes. AgNPs have been found to improve cell proliferation and differentiation, making them beneficial for tissue engineering and regenerative medicine. Our study highlights emerging patterns and developments in the field of medicine, inferring potential future paths.</p></div>","PeriodicalId":100186,"journal":{"name":"Biotechnology Notes","volume":"5 ","pages":"Pages 64-79"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665906924000096/pdfft?md5=e8e08526b06a4c8603d010ed4bfdd106&pid=1-s2.0-S2665906924000096-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141229774","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Gazi Sakir Hossain , Yuanmei Liang , Jee Loon Foo , Matthew Wook Chang
{"title":"Engineered microbial consortia for next-generation feedstocks","authors":"Gazi Sakir Hossain , Yuanmei Liang , Jee Loon Foo , Matthew Wook Chang","doi":"10.1016/j.biotno.2024.01.002","DOIUrl":"https://doi.org/10.1016/j.biotno.2024.01.002","url":null,"abstract":"<div><p>Addressing urgent environmental challenges, this commentary emphasizes the need for green, bio-based solutions in chemical production from renewable feedstocks. It highlights advanced metabolic engineering of microbial strains and the use of microbial consortia as innovative approaches for efficient resource recovery. These strategies aim to enhance the conversion of diverse renewable feedstocks, including agricultural residues, industrial by-products, and greenhouse gases, into value-added chemicals. This article discusses cutting-edge techniques in renewable feedstock upcycling, utilizing both engineered unicellular and multicellular systems. It advocates a paradigm shift in sustainable biomanufacturing, focusing on transforming renewable resources into value-added products. This approach is crucial for developing a circular bioeconomy, aligning with global efforts to mitigate environmental impacts.</p></div>","PeriodicalId":100186,"journal":{"name":"Biotechnology Notes","volume":"5 ","pages":"Pages 23-26"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665906924000011/pdfft?md5=03fd156a1c2e8ff07e3f86b7462c1e05&pid=1-s2.0-S2665906924000011-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139549632","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"A comprehensive study on anaerobic digestion of organic solid waste: A review on configurations, operating parameters, techno-economic analysis and current trends","authors":"D.Jaya Prasanna Kumar , Ranjeet Kumar Mishra , Sampath Chinnam , Prakash Binnal , Naveen Dwivedi","doi":"10.1016/j.biotno.2024.02.001","DOIUrl":"https://doi.org/10.1016/j.biotno.2024.02.001","url":null,"abstract":"<div><p>The excessive discharge and accumulation of solid organic waste into the environment is of severe concern across the globe. Thus, an efficient waste management system is important to mitigate health risks to humans, minimize harmful impacts on the environment, and ensure a sustainable ecosystem. The organic waste is converted into value-added products either using microorganisms or heat energy; these methods are commonly known as biochemical and thermochemical techniques. The biochemical process has the advantage of higher selectivity of the products and lower processing temperatures. The principal conversion processes of this category are fermentation and anaerobic digestion (AD). This review article focuses on AD, a potential method for treating organic waste and creating a variety of products with added value. Here we present the digestibility of various organic wastes, the role of microorganisms, the decomposition process, co-substrates, digester designs, biogas yields, by-products, environmental impacts, and overall techno-economical effectiveness of the process. Further, this review offers insights into new directions for AD for waste treatment and future research without compromising the overall feasibility and environmental sustainability.</p></div>","PeriodicalId":100186,"journal":{"name":"Biotechnology Notes","volume":"5 ","pages":"Pages 33-49"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665906924000047/pdfft?md5=697f3bc9004f1454bf22f245f86a3bf8&pid=1-s2.0-S2665906924000047-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140016320","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ke Jiang , Ruoxuan Bai , Xianglian Luo , Ting Gao , Fangxu Xu , Hongxin Zhao , Dianpeng Zhang
{"title":"Characterization and bioactive component analysis of filamentous bacterium XJ-16","authors":"Ke Jiang , Ruoxuan Bai , Xianglian Luo , Ting Gao , Fangxu Xu , Hongxin Zhao , Dianpeng Zhang","doi":"10.1016/j.biotno.2023.03.001","DOIUrl":"https://doi.org/10.1016/j.biotno.2023.03.001","url":null,"abstract":"<div><p>Actinomycetes, which can produce a variety of bioactive compounds in the metabolic process, is one of the important sources of novel drugs, enzymes, anti-tumor drugs and enzyme inhibitors. It has been the focus of researchers to find and develop <em>Actinomycetes</em> with special characters. Strain XJ-16 is a blue alkali-resistant filamentous bacterium with high antimicrobial activity isolated from saline-alkali land of Xinjiang. Based on the classification, the enzyme production, metabolite antibacterial activity, and antibacterial substance isolation of XJ-16 were explored. which showed that XJ-16 belongs to the blue group of <em>Streptomyces</em> sp, and it can secrete cellulase, lipase, urease, protease, catalase and oxidase during metabolism. In addition, the bacteriostatic substance secreted by the strain XJ-16 showed inhibitory effects against both Gram-positive and Gram-negative bacteria, as well as the yeast <em>Candida albicans</em>. Then it was found that the bacteriostasis produced by XJ-16 has strong tolerance to acid, weak tolerance to alkali, and easy to be inactivated. After tested by HPLC, the retention time of antimicrobial substance was 13.261 min. This study provides new research ideas and theoretical support for searching for new antibacterial compounds and further developing the blue alkaline <em>Actinomycete</em> XJ-16.</p></div>","PeriodicalId":100186,"journal":{"name":"Biotechnology Notes","volume":"4 ","pages":"Pages 49-57"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49731575","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":"Suitability evaluation of toehold switch and EXPAR for cell-free MicroRNA biosensor development","authors":"Caroline E. Copeland , Yong-Chan Kwon","doi":"10.1016/j.biotno.2023.11.003","DOIUrl":"https://doi.org/10.1016/j.biotno.2023.11.003","url":null,"abstract":"<div><p>The development of a robust and cost-effective sensing platform for microRNA (miRNA) is of paramount importance in detecting and monitoring various diseases. Current miRNA detection methods are marred by low accuracy, high cost, and instability. The toehold switch riboregulator has shown promising results in detecting viral RNAs integrated with the freeze-dried cell-free system (CFS). This study aimed to leverage the toehold switch technology and portability to detect miRNA in the CFS and to incorporate the exponential amplification reaction (EXPAR) to bring the detection to clinically relevant levels. We assessed various EXPAR DNA templates under different conditions to enhance the accuracy of the sensing platform. Furthermore, different structures of toehold switches were tested with either high-concentration synthetic miRNA or EXPAR product to assess sensitivity. Herein, we elucidated the mechanisms of the toehold switch and EXPAR, presented the findings of these optimizations, and discussed the potential benefits and drawbacks of their combined use.</p></div>","PeriodicalId":100186,"journal":{"name":"Biotechnology Notes","volume":"4 ","pages":"Pages 83-89"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665906923000090/pdfft?md5=61d37cf4a5204994790dfb4c07164302&pid=1-s2.0-S2665906923000090-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138439364","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"2023 The 1st National Competition of “Bloomag Cup” in CO2 capture, conversion, and utilization","authors":"Ziman Chen, Dan Wang, Zhenyi Wu, Yongqin Lv","doi":"10.1016/j.biotno.2023.12.001","DOIUrl":"https://doi.org/10.1016/j.biotno.2023.12.001","url":null,"abstract":"<div><p>The inaugural National Competition for Carbon Dioxide Capture, Conversion and Utilization Innovation (“Bloomag Cup”) was successfully held on July 30, 2023 in Beijing. This competition was initiated by Professor Tianwei Tan and Prof. Yongqin Lv from Beijing University of Chemical Technology (BUCT), and jointly organized by BUCT and Chongqing University. The competition is slated for annual recurrence, with a rotational hosting arrangement involving various academic institutions. The ongoing competition underscores the primacy of pioneering and exploratory facets inherent to technological innovation. Its principal objective is to catalyze the development of foundational and cutting-edge technological competencies within the realm of CO<sub>2</sub> capture, conversion, and utilization. The overarching goals encompass identifying promising technological breakthroughs, fostering emerging talent in scientific and technological innovation, facilitating high-quality sustainable economic growth within China, and actively contributing to global efforts towards peak carbon emissions and achieving sustainable development goals for humanity. This inaugural Bloomag Cup saw wide participation from students and researchers, generating fruitful discussions that advance the nascent field. It is hoped this competition will continue cultivating solutions that help mitigate anthropogenic climate change through innovative carbon dioxide technologies.</p></div>","PeriodicalId":100186,"journal":{"name":"Biotechnology Notes","volume":"4 ","pages":"Pages 112-117"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2665906923000132/pdfft?md5=e434bed0478aec98b2cd4b2862b56c4d&pid=1-s2.0-S2665906923000132-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138564404","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}