Bioconjugate Chemistry最新文献

{"title":"Bioorthogonally Activatable Photosensitizer for NIR Fluorescence Imaging-Guided Highly Selective Elimination of Senescent Tumor Cells and Chemotherapy Enhancement.","authors":"Yun Feng, Zifan Zhu, Shirui Zhao, Xingyu Jiang, Wen Zhang, Zhiai Xu","doi":"10.1021/acs.bioconjchem.5c00109","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.5c00109","url":null,"abstract":"<p><p>Chemotherapy is a primary modality in cancer treatment, but it may induce cellular senescence, which in turn triggers the release of senescence-associated secretory phenotypes (SASPs) that promote tumor growth and metastasis. To selectively identify senescent cells and mitigate their negative impact on cancer therapy, herein, we have developed a β-galactosidase (β-Gal)-activated and self-immobilizing photosensitizer CyGF-DBCO-T. This photosensitizer can be selectively activated and fluorescently label proteins in situ within senescent cells, enabling near-infrared (NIR) fluorescence imaging-guided photodynamic therapy (PDT) for the precise ablation of these cells. First, we developed an activatable NIR fluorescent probe CyGF-N₃ that can specifically in situ label senescent cells. Subsequently, DBCO-T with free radicals underwent a bioorthogonal click reaction with activated CyGF-N₃ in senescent cells to generate the photosensitizer CyO-DBCO-T. Under light irradiation, CyO-DBCO-T generated singlet oxygen (¹O₂) in situ, thereby enabling precise PDT with fluorescence guidance and photoactivation. Both CyGF-N₃ and DBCO-T were encapsulated in biotinylated liposomes (CyGF-N₃@LIP-B and DBCO-T@LIP-B), which enhanced their water solubility, tumor targeting, and in vivo circulation time. This promoted the accumulation of the probes in senescent tumor cells, thus enabling intense fluorescence imaging of tumor senescence regions in mice and enhancing the efficacy of PDT. This dual-module strategy, guided by fluorescence imaging for PDT, has achieved selective identification and precise ablation of senescent tumor cells in a chemotherapy-induced senescence model, effectively alleviating chemotherapy resistance and suppressing tumor growth.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143950737","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transduction of Glycan-Lectin Binding via an Impedimetric Sensor for Glycoprotein Detection.","authors":"Linhui Lv, Ke Qu","doi":"10.1021/acs.bioconjchem.4c00530","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.4c00530","url":null,"abstract":"<p><p>Glycoproteins are produced by glycosylation modification of proteins, and a number of glycoproteins have served as important tumor biomarkers in clinical application. Alpha-fetoprotein (AFP) is one of the representative glycoproteins that has been employed as a useful predictive and prognostic biomarker for hepatocellular carcinoma. Human AFP has an <i>N</i>-glycan portion at the asparagine residue, which includes four <i>N</i>-acetyl-glucosamine and three mannoses. In this work, building upon lectin-glycan interactions, one type of facile and capable impedimetric biosensor was fabricated utilizing microwave-prepared NH₂-MIL-101(Fe) to decorate lectins as a recognition element. Two different lectins of wheat-germ agglutinin (WGA) and concanavalin A (Con A) were employed to target the <i>N</i>-acetyl-glucosamine and mannose of <i>N</i>-glycan in AFP, respectively. This work has not only accomplished the sensitive impedimetric biosensing of the AFP tumor marker (with the limit of detection down to 0.5 pg/mL and linear concentration spanning 5 orders of magnitude from 10^-2 to 10³ ng/mL) but also replied on two kinds of lectins to \"read\" the sugar chain, transducing the minor difference of this process to impedimetric signals for display. The impedimetric data shed some light on the local microenvironment of the lectin-glycan binding event, providing some electrochemical experimental support for the biantennary structure of <i>N</i>-glycan in AFP. The mannoses were \"buried\" in the interior core of the whole <i>N</i>-glycan, increasing steric hindrance for Con A to approach and thus rendering the WGA@MIL-101(Fe)-based biosensor more superior sensing responses.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143955921","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Easy Access to Bioorthogonal Click-to-Release Reagent Bishydroxy-<i>trans</i>-cyclooctene (C₂TCO) and Harnessing of Its Rapid Labeling and Dissecting Feature in Multicycle Imaging.","authors":"V Arun, Minju Lee, Hongseo Choi, Sangwoo Lee, Junwon Choi, Tae Hyeon Yoo, Wook Kim, Eunha Kim","doi":"10.1021/acs.bioconjchem.4c00495","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.4c00495","url":null,"abstract":"<p><p>Bifunctional <i>trans</i>-cyclooctene (bTCO) with a carbamate or carbonate at the allylic position and tetrazine provide a promising bioorthogonal click chemistry pair for the click-to-release approach, successfully employed in various biotechnological applications. Herein, we demonstrate a simple and straightforward method to synthesize C₂TCO, a symmetrical bTCO derivative with two hydroxyl groups at the allylic positions. The efficiently synthesized C₂TCO at first was selectively functionalized with a fluorophore (C₂TCO-FL), and the conjugate was labeled onto monoclonal antibodies (Ab-C₂TCO-FL). The fluorophore of Ab-C₂TCO-FL was easily removed from the antibody through the mild treatment of tetrazine, enabling multicycle fluorescent bioimaging. Next, an antibody-drug conjugate targeting PD-L1 was prepared using the linker based on C₂TCO. The cytotoxic payload was efficiently released from the antibody upon tetrazine treatment, which induced cellular cytotoxicity.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143951219","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Redesigning Ibuprofen for Improved Oral Delivery and Reduced Side Effects.","authors":"Szilvia H Toth, Anca D Stoica, Cristian Sevcencu","doi":"10.1021/acs.bioconjchem.4c00558","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.4c00558","url":null,"abstract":"<p><p>Ibuprofen (IBP) is one of the most widely used nonsteroidal anti-inflammatory drugs (NSAIDs). Being well-known for its efficacy, long history of use, and reduced adverse events compared to other NSAIDs, IBP is authorized as an analgesic and antipyretic drug. IBP's mechanism of action consists of inhibiting cyclooxygenases, which are crucial oxidoreductases in prostaglandin synthesis and generation of inflammation and pain. However, despite being effective and relatively safe, IBP can still induce a dose-dependent toxicity which manifests mainly in the gastrointestinal system as ulcerations and altered mucosal blood flow and cytotoxicity characterized by mitochondrial dysfunction and increased membrane permeability in enterocytes and hepatocytes. Therefore, ongoing research is performed to improve the IBP's activity and treatment outcome, and one way to achieve such improvements is through reducing IBP's toxicity by designing less harmful but still effective novel IBP conjugates. The aim of this review is to summarize the latest achievements with IBP conjugation techniques that created such valuable IBP formulations less toxic than but as effective as the parent drug.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143951032","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparative Study of Click Handle Stability in Common Ligation Conditions.","authors":"Caitlin Fawcett, Joe Watson, Stephen Richards, Alfred E Doherty, Hikaru Seki, Elizabeth A Love, Charlotte H Coles, Diane M Coe, Craig Jamieson","doi":"10.1021/acs.bioconjchem.5c00095","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.5c00095","url":null,"abstract":"<p><p>Click chemistry efficiently ligates molecular building blocks in a robust and high-yielding manner and has found major application in the rapid modification of important molecular actors in biological systems. However, the high reactivity of click handles often correlates with decreased stability, which presents a significant challenge in the practical application of these systems. In the current study, we describe a survey of the stability of commonly deployed click manifolds across a range of widely used ligation conditions. Incompatible click handle and ligation condition combinations are identified, with kinetic half-lives and side products of each undesired reaction determined, including the assessment of stability over extended periods and in a protein environment. This data set provides researchers with a roadmap to expediently determine the most appropriate click reaction conditions for any given bioorthogonal application, thus elevating the probability of success of procedures that utilize click chemistry.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143954282","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a Long-Acting Myeloid-Derived Growth Factor via Site-Specific PEGylation.","authors":"Yong-Shan Zheng, Teng Zhang, Ji-Yang Song, Mingchan Liang, Ya-Li Liu, Zeng-Guang Xu, Cheng He, Zhan-Yun Guo","doi":"10.1021/acs.bioconjchem.5c00026","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.5c00026","url":null,"abstract":"<p><p>Extracellular myeloid-derived growth factor (MYDGF) can improve organ repair. However, short <i>in vivo</i> half-life hampers its therapeutic application. Herein, we developed a long-acting MYDGF via site-specific PEGylation at its C-terminus. Bacterially overexpressed human MYDGF carrying a C-terminal Asn-Ala-Leu tripeptide motif was first ligated with a synthetic azido-functionalized Gly-Ile-Gly-Lys(N3) tetrapeptide linker via catalysis of [G238 V]BmAEP1, an engineered bamboo-derived asparaginyl endopeptidase (AEP)-type peptide ligase. Thereafter, the azido-functionalized MYDGF was efficiently conjugated with a commercially available dibenzocyclooctyne (DBCO)-functionalized linear PEG30000 via copper-free click chemistry. The site-specifically PEGylated MYDGF (PEG-MYDGF) retained high <i>in vitro</i> activity and showed a much longer <i>in vivo</i> half-life in mice compared with unmodified MYDGF. In diabetic mice, PEG-MYDGF significantly promoted wound healing after subcutaneous injection. Thus, PEG-MYDGF represents a long-acting biologic with therapeutic potential. The present enzymatic peptide ligation and copper-free click chemistry-based approach could be applied to other proteins for site-specific conjugation with various functional moieties.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143954284","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mannosamine-Modified Poly(lactic-<i>co</i>-glycolic acid)-Polyethylene Glycol Nanoparticles for the Targeted Delivery of Rifapentine and Isoniazid in Tuberculosis Therapy.","authors":"Cong Peng, Haopeng Luan, Qisong Shang, Wei Xiang, Parhat Yasin, Xinghua Song","doi":"10.1021/acs.bioconjchem.5c00062","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.5c00062","url":null,"abstract":"<p><p>Tuberculosis, caused by <i>Mycobacterium tuberculosis</i>, is the leading cause of mortality attributed to a single infectious agent. Following macrophage invasion, M. tuberculosis uses various mechanisms to evade immune responses and to resist antituberculosis drugs. This study aimed to develop a targeted drug delivery system utilizing mannosamine (MAN)-modified nanoparticles (NPs) composed of poly(lactic-<i>co</i>-glycolic acid)-polyethylene glycol (PLGA-PEG), loaded with rifapentine and isoniazid, to enhance macrophage-directed therapy and enhance bacterial elimination. PLGA-PEG copolymer was modified with mannosamine through an amidation reaction. Rifapentine- and isoniazid-loaded PLGA-PEG-MAN NPs were synthesized by using the double emulsion solvent evaporation technique. The NPs exhibited an average particle size of 117.67 nm and displayed favorable physicochemical properties without evidence of cellular or hemolytic toxicity. The drug loading rates were 11.73% for rifapentine and 5.85% for isoniazid. Sustained drug release was achieved over a period exceeding 72 h, with antibacterial activity remaining intact during encapsulation. Synergistic bactericidal effects were noted. Additionally, mannosamine-modified NPs enhanced the phagocytic activity of macrophages via mannose receptor-mediated endocytosis, thereby improving drug delivery efficiency and significantly boosting the antibacterial efficacy of the NPs within macrophages. Pathological staining and biochemical analysis of rat organs following intravenous injection indicated that the NPs did not cause any significant toxic side effects in vivo. The findings of this study indicate that mannosamine-modified PLGA-PEG NPs loaded with rifapentine and isoniazid represent a promising drug delivery system for targeting macrophages to enhance the efficacy of antitubercular therapy.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143955594","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Tumor Cell Membrane Biomimetic Mesoporous Silicon Materials in Combination with PD-L1 Knockout Achieved through the CRISPR/Cas9 System for Targeted and Immunotherapeutic Purposes.","authors":"Jinjin Zhao, Xiaorou Wang, Haiguang Zhang, Qunmei Zhang, DeYing Bo, Hua Zhong, Luyang Jiao, Hongchang Yuan, Guangjian Lu","doi":"10.1021/acs.bioconjchem.5c00001","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.5c00001","url":null,"abstract":"<p><p>Nanoparticle-based drug delivery systems, which enable the effective and targeted delivery of chemotherapeutic drugs to tumors, have revolutionized cancer therapy. Mesoporous silicon materials (MSN) have emerged as promising candidates for drug delivery due to their unique properties. The therapeutic efficacy can be significantly enhanced when treatments exhibit both targeting and antiphagocytic properties. In this study, cell membranes extracted from B16-F10 cells were used to encapsulate carboplatin (CBP)-loaded MSN via physical extrusion. Additionally, we intratumorally injected a plasmid containing the CRISPR/Cas9 system to achieve PD-L1 knockout, thereby reactivating the immune system. The cell membrane coating endowed the CBP@MSN with excellent slow-release capability and cytocompatibility. Enhanced tumor cell uptake of CBP@MSN@M was observed due to homologous targeting by cancer cell membranes. Moreover, CBP@MSN@M demonstrated enhanced antitumor efficacy in vivo and promoted the proliferation of immune cells. Finally, the antitumor effect was further improved by the knockout of PD-L1 within the tumor microenvironment. These results suggest that the newly prepared CBP@MSN@M, combined with PD-L1 knockout, holds significant potential as an effective therapeutic approach for treating tumors.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143952149","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>E. coli</i> as a Smart Thermo-Vector for Combating Solid Tumors: A Synergistic Heat-Induced Cancer Therapy Approach.","authors":"Tashmeen Kaur, Neeta Devi, Deepika Sharma","doi":"10.1021/acs.bioconjchem.5c00102","DOIUrl":"10.1021/acs.bioconjchem.5c00102","url":null,"abstract":"<p><p>Heat-induced cancer therapies such as magnetic hyperthermia-based cancer therapy (MHCT) and photothermal tumor ablation (PTT) have garnered significant attention as minimally invasive new-generation cancer therapy modalities. However, solid tumors associated with hypoxia present a considerable challenge to effective cancer therapy. In this study, we took up the challenge of mitigating the limiting penetration ability of nanoparticles by integrating polydopamine-coated magnetic nanoparticles and motile anaerobic bacteria (PDBs) to function as a smart thermo-vector. The developed PDBs are capable of self-navigating hypoxic tumors and as thermo-therapy agents with the ability to induce heat through exposure to an alternating magnetic field or near-infrared laser light. The thermo-vector system exhibited a dual-functioning synergistic antitumor effect of MHCT and PTT and an outstanding tumor targeting efficiency, outperforming the conventional 'nanoparticles only' approach. The heat-induced cellular oxidative stress and disrupted mitochondrial function led to 80% cellular cytotoxicity within 24 h of treatment. The PDB-based approach led to complete tumor regression in c57BL/6 mice within 21 days of treatment and a tumor-free survival for 60 days without recurrence, proving the capability of the developed PDBs in combatting solid tumors.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":"867-880"},"PeriodicalIF":4.0,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143655445","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mechanistic Characterization of the Potency of THIOMAB Antibody-Drug Conjugates Targeting <i>Staphylococcus aureus</i> and ETbR-Expressing Tumor Cells Using Quantitative LC-MS/MS Analysis of Intracellular Drug Accumulation.","authors":"Hilda Hernandez-Barry, Josefa Dela Cruz-Chuh, Kimberly K Kajihara, Jyoti Asundi, Richard Vandlen, Donglu Zhang, Wouter L W Hazenbos, Thomas Pillow, Yichin Liu, Cong Wu, Katherine R Kozak, Kelly M Loyet","doi":"10.1021/acs.bioconjchem.4c00533","DOIUrl":"10.1021/acs.bioconjchem.4c00533","url":null,"abstract":"<p><p>THIOMAB drug conjugate (TDC) technology provides site-specific conjugation of linker drugs to antibodies, allowing for targeted delivery of the payload. While a direct measurement of TDC cytotoxic potency allows efficient screening and confirmation that new drugs conjugated to antibodies result in proper processing in cells, additional mechanistic characterization is often needed to provide information-rich data to guide further optimization of TDC design. For example, a quantitative understanding of how TDCs are processed intracellularly can help determine which processing step is impacting payload delivery and thereby inform the basis of the TDC efficacy. Here, we measure the cellular accumulation of two different TDC drug payloads: MAPK (mitogen-activated protein kinase) pathway inhibitor targeting ETbR-expressing tumor cells and an antibiotic active against <i>Staphylococcus aureus</i> with an <i>in vitro</i> cell-based drug release LC-MS/MS assay in a 96-well format. This assay allowed us to correlate the cellular potency of each unconjugated molecule with the amount of payload that accumulated inside the cell. In the case of the pathway inhibitor drug, the biochemical characterization of TDC processing by cathepsin B and purified human liver enzyme extract demonstrated a correlation between the efficiency of the linker drug cleavage and intracellular payload accumulation. For the antibody-antibiotic conjugate, kinetic analysis of intracellular free drug retention provided valuable insight into the chemistry modifications needed for an efficient TDC. Taken together, we demonstrated the utility of quantitative LC-MS/MS assays as one tool in guiding the design of more effective TDCs via the mechanistic release characterization of two distinct payloads.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":"652-661"},"PeriodicalIF":4.0,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12007502/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143778586","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}