ACS Nanoscience Au最新文献

{"title":"Tuning Chemical DNA Ligation within DNA Crystals and Protein–DNA Cocrystals","authors":"Abigail R. Orun, Caroline K. Slaughter, Ethan T. Shields, Ananya Vajapayajula, Sara Jones, Rojina Shrestha, Christopher D. Snow","doi":"10.1021/acsnanoscienceau.4c00013","DOIUrl":"https://doi.org/10.1021/acsnanoscienceau.4c00013","url":null,"abstract":"Biomolecular crystals can serve as materials for a plethora of applications including precise guest entrapment. However, as grown, biomolecular crystals are fragile in solutions other than their growth conditions. For crystals to achieve their full potential as hosts for other molecules, crystals can be made stronger with bioconjugation. Building on our previous work using carbodiimide 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) for chemical ligation, here, we investigate DNA junction architecture through sticky base overhang lengths and the role of scaffold proteins in cross-linking within two classes of biomolecular crystals: cocrystals of DNA-binding proteins and pure DNA crystals. Both crystal classes contain DNA junctions where DNA strands stack up end-to-end. Ligation yields were studied as a function of sticky base overhang length and terminal phosphorylation status. The best ligation performance for both crystal classes was achieved with longer sticky overhangs and terminal 3′phosphates. Notably, EDC chemical ligation was achieved in crystals with pore sizes too small for intracrystal transport of ligase enzyme. Postassembly cross-linking produced dramatic stability improvements for both DNA crystals and cocrystals in water and blood serum. The results presented may help crystals containing DNA achieve broader application utility, including as structural biology scaffolds.","PeriodicalId":29799,"journal":{"name":"ACS Nanoscience Au","volume":"25 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141530001","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":"Light-Responsive and Antibacterial Graphenic Materials as a Holistic Approach to Tissue Engineering","authors":"Andrea Ferreras,&nbsp;Ana Matesanz,&nbsp;Jabier Mendizabal,&nbsp;Koldo Artola,&nbsp;Yuta Nishina,&nbsp;Pablo Acedo,&nbsp;José L. Jorcano,&nbsp;Amalia Ruiz*,&nbsp;Giacomo Reina* and Cristina Martín*,&nbsp;","doi":"10.1021/acsnanoscienceau.4c0000610.1021/acsnanoscienceau.4c00006","DOIUrl":"https://doi.org/10.1021/acsnanoscienceau.4c00006https://doi.org/10.1021/acsnanoscienceau.4c00006","url":null,"abstract":"<p >While the continuous development of advanced bioprinting technologies is under fervent study, enhancing the regenerative potential of hydrogel-based constructs using external stimuli for wound dressing has yet to be tackled. Fibroblasts play a significant role in wound healing and tissue implants at different stages, including extracellular matrix production, collagen synthesis, and wound and tissue remodeling. This study explores the synergistic interplay between photothermal activity and nanomaterial-mediated cell proliferation. The use of different graphene-based materials (GBM) in the development of photoactive bioinks is investigated. In particular, we report the creation of a skin-inspired dressing for wound healing and regenerative medicine. Three distinct GBM, namely, graphene oxide (GO), reduced graphene oxide (rGO), and graphene platelets (GP), were rigorously characterized, and their photothermal capabilities were elucidated. Our investigations revealed that rGO exhibited the highest photothermal efficiency and antibacterial properties when irradiated, even at a concentration as low as 0.05 mg/mL, without compromising human fibroblast viability. Alginate-based bioinks alongside human fibroblasts were employed for the bioprinting with rGO. The scaffold did not affect the survival of fibroblasts for 3 days after bioprinting, as cell viability was not affected. Remarkably, the inclusion of rGO did not compromise the printability of the hydrogel, ensuring the successful fabrication of complex constructs. Furthermore, the presence of rGO in the final scaffold continued to provide the benefits of photothermal antimicrobial therapy without detrimentally affecting fibroblast growth. This outcome underscores the potential of rGO-enhanced hydrogels in tissue engineering and regenerative medicine applications. Our findings hold promise for developing game-changer strategies in 4D bioprinting to create smart and functional tissue constructs with high fibroblast proliferation and promising therapeutic capabilities in drug delivery and bactericidal skin-inspired dressings.</p>","PeriodicalId":29799,"journal":{"name":"ACS Nanoscience Au","volume":"4 4","pages":"263–272 263–272"},"PeriodicalIF":4.8,"publicationDate":"2024-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsnanoscienceau.4c00006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142010490","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":"Making the Most of Nothing: One-Class Classification for Single-Molecule Transport Studies","authors":"William Bro-Jørgensen,&nbsp;Joseph M. Hamill,&nbsp;Gréta Mezei,&nbsp;Brent Lawson,&nbsp;Umar Rashid,&nbsp;András Halbritter*,&nbsp;Maria Kamenetska*,&nbsp;Veerabhadrarao Kaliginedi* and Gemma C. Solomon*,&nbsp;","doi":"10.1021/acsnanoscienceau.4c0001510.1021/acsnanoscienceau.4c00015","DOIUrl":"https://doi.org/10.1021/acsnanoscienceau.4c00015https://doi.org/10.1021/acsnanoscienceau.4c00015","url":null,"abstract":"<p >Single-molecule experiments offer a unique means to probe molecular properties of individual molecules–yet they rest upon the successful control of background noise and irrelevant signals. In single-molecule transport studies, large amounts of data that probe a wide range of physical and chemical behaviors are often generated. However, due to the stochasticity of these experiments, a substantial fraction of the data may consist of blank traces where no molecular signal is evident. One-class (OC) classification is a machine learning technique to identify a specific class in a data set that potentially consists of a wide variety of classes. Here, we examine the utility of two different types of OC classification models on four diverse data sets from three different laboratories. Two of these data sets were measured at cryogenic temperatures and two at room temperature. By training the models solely on traces from a blank experiment, we demonstrate the efficacy of OC classification as a powerful and reliable method for filtering out blank traces from a molecular experiment in all four data sets. On a labeled 4,4′-bipyridine data set measured at 4.2 K, we achieve an accuracy of 96.9 ± 0.3 and an area under the receiver operating characteristic curve of 99.5 ± 0.3 as validated over a fivefold cross-validation. Given the wide range of physical and chemical properties that can be probed in single-molecule experiments, the successful application of OC classification to filter out blank traces is a major step forward in our ability to understand and manipulate molecular properties.</p>","PeriodicalId":29799,"journal":{"name":"ACS Nanoscience Au","volume":"4 4","pages":"250–262 250–262"},"PeriodicalIF":4.8,"publicationDate":"2024-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsnanoscienceau.4c00015","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142010489","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":"Nanomaterials Research at a Primarily Undergraduate Institution: Transforming Nanorods, Undergraduate Research Communities, and Infrastructure","authors":"Katherine E. Plass*,&nbsp;J. Kenneth Krebs,&nbsp;Jennifer L. Morford,&nbsp;Raymond E. Schaak,&nbsp;Joshua J. Stapleton and Adri C. T. van Duin,&nbsp;","doi":"10.1021/acsnanoscienceau.4c0000510.1021/acsnanoscienceau.4c00005","DOIUrl":"https://doi.org/10.1021/acsnanoscienceau.4c00005https://doi.org/10.1021/acsnanoscienceau.4c00005","url":null,"abstract":"<p >Undergraduate research transforms student’s conceptions of themselves as scientists and encourages participation and retention in science, technology, engineering, and mathematics (STEM) fields. Many barriers exist to carrying out scientifically impactful undergraduate research in nanomaterials at primarily undergraduate institutions (PUIs). Here, we share several practices and design principles that demonstrate pathways to overcome these barriers. Design of modular research projects with low entry barriers is essential. Postsynthetic transformation of nanoparticles is a field that enables such design and has been used successfully to advance nanoscience research while being achievable within undergraduate laboratories. Relatively large, inclusive research communities can be supported through the creation of opportunities with peer- and near-peer mentoring. We also share emerging strategies for enabling routine undergraduate access to transmission electron microscopy, which is one of the most mainstream characterization techniques in nanoscience yet is frequently absent from the infrastructure at undergraduate-focused institutions.</p>","PeriodicalId":29799,"journal":{"name":"ACS Nanoscience Au","volume":"4 4","pages":"223–234 223–234"},"PeriodicalIF":4.8,"publicationDate":"2024-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsnanoscienceau.4c00005","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142010446","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":"Anodic Electrodeposition of IrOx Nanoparticles from Aqueous Nanodroplets","authors":"Saptarshi Paul,&nbsp;Joshua Reyes-Morales,&nbsp;Kingshuk Roy and Jeffrey E. Dick*,&nbsp;","doi":"10.1021/acsnanoscienceau.3c00061","DOIUrl":"10.1021/acsnanoscienceau.3c00061","url":null,"abstract":"<p >Electrodeposition has been used for centuries to create new materials. However, synthetic platforms are still necessary to enrich a variety of nanomaterials that can be electrodeposited. For instance, IrO_<i>x</i> is a popular material for the water oxidation reaction, but electrodeposition strategies for the controlled growth of IrO_<i>x</i> nanoparticles are lacking. Here, we demonstrate the anodic electrodeposition of IrO_<i>x</i> nanoparticles from aqueous nanodroplets. Field emission scanning electron microscopy (FESEM) and scanning transmission electron microscopy (STEM) images confirm the macro- and microstructure of the resulting nanoparticles. IrO_<i>x</i> nanoparticles of 43 ± 10 nm in diameter were achieved. X-ray photoelectron spectroscopy (XPS) showed the presence of Ir(III) and Ir(IV) hydrated oxyhydroxide species. The synthesis of IrO_<i>x</i> nanoparticles under anodic conditions using water nanodroplets expands the capabilities of our technique and provides a tunable platform for IrO_<i>x</i> nanoparticle electrodeposition.</p>","PeriodicalId":29799,"journal":{"name":"ACS Nanoscience Au","volume":"4 3","pages":"216–222"},"PeriodicalIF":0.0,"publicationDate":"2024-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsnanoscienceau.3c00061","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140608889","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":"On-the-Fly Monitoring of the Capture and Removal of Nanoplastics with Nanorobots","authors":"Dean I. Velikov, Anna Jancik-Prochazkova, Martin Pumera","doi":"10.1021/acsnanoscienceau.4c00002","DOIUrl":"https://doi.org/10.1021/acsnanoscienceau.4c00002","url":null,"abstract":"Nanoplastics are considered an emerging organic persistent pollutant with possible severe long-term implications for the environment and human health; therefore, their remediation is of paramount importance. However, detecting and determining the concentration of nanoparticles in water is challenging and time-consuming due to their small size. In this work, we present a universal yet simple method for the detection and quantification of nanoplastics to monitor their removal from water using magnetic nanorobots. Nanoplastics were stained with a hydrophobic fluorescent dye to enable the use of photoluminescence techniques for their detection and quantification. Magnetic nanorobotic tools were employed to capture and subsequently remove the nanoplastics from contaminated waters. We demonstrated that nanorobots can capture and remove more than 90% of the nanoplastics from an aqueous solution within 120 min. This work shows that easy-to-use common fluorescent dyes combined with photoluminescence spectroscopy methods can be used as an alternative method for the detection and quantification of nanoplastics in water environments and swarming magnetic nanorobots for efficient capture and removal. These methods hold great potential for future research to improve the quantification and removal of nanoplastics in water, and it will ultimately reduce their harmful impact on the environment and human health.","PeriodicalId":29799,"journal":{"name":"ACS Nanoscience Au","volume":"15 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140593270","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":"On-the-Fly Monitoring of the Capture and Removal of Nanoplastics with Nanorobots","authors":"Dean I. Velikov,&nbsp;Anna Jancik-Prochazkova and Martin Pumera*,&nbsp;","doi":"10.1021/acsnanoscienceau.4c0000210.1021/acsnanoscienceau.4c00002","DOIUrl":"https://doi.org/10.1021/acsnanoscienceau.4c00002https://doi.org/10.1021/acsnanoscienceau.4c00002","url":null,"abstract":"<p >Nanoplastics are considered an emerging organic persistent pollutant with possible severe long-term implications for the environment and human health; therefore, their remediation is of paramount importance. However, detecting and determining the concentration of nanoparticles in water is challenging and time-consuming due to their small size. In this work, we present a universal yet simple method for the detection and quantification of nanoplastics to monitor their removal from water using magnetic nanorobots. Nanoplastics were stained with a hydrophobic fluorescent dye to enable the use of photoluminescence techniques for their detection and quantification. Magnetic nanorobotic tools were employed to capture and subsequently remove the nanoplastics from contaminated waters. We demonstrated that nanorobots can capture and remove more than 90% of the nanoplastics from an aqueous solution within 120 min. This work shows that easy-to-use common fluorescent dyes combined with photoluminescence spectroscopy methods can be used as an alternative method for the detection and quantification of nanoplastics in water environments and swarming magnetic nanorobots for efficient capture and removal. These methods hold great potential for future research to improve the quantification and removal of nanoplastics in water, and it will ultimately reduce their harmful impact on the environment and human health.</p>","PeriodicalId":29799,"journal":{"name":"ACS Nanoscience Au","volume":"4 4","pages":"243–249 243–249"},"PeriodicalIF":4.8,"publicationDate":"2024-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsnanoscienceau.4c00002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142010449","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":"The Huge Role of Tiny Impurities in Nanoscale Synthesis","authors":"Angira Roy,&nbsp;Ciaran P. Healey,&nbsp;Nathaniel E. Larm,&nbsp;Piyuni Ishtaweera,&nbsp;Maryuri Roca* and Gary A. Baker*,&nbsp;","doi":"10.1021/acsnanoscienceau.3c00056","DOIUrl":"10.1021/acsnanoscienceau.3c00056","url":null,"abstract":"<p >Nanotechnology is vital to many current industries, including electronics, energy, textiles, agriculture, and theranostics. Understanding the chemical mechanisms of nanomaterial synthesis has contributed to the tunability of their unique properties, although studies frequently overlook the potential impact of impurities. Impurities can show adverse effects, clouding the interpretation of results or limiting the practical utility of the nanomaterial. On the other hand, as successful doping has demonstrated, the intentional introduction of impurities can be a powerful tool for enhancing the properties of a nanomaterial. This Review examines the complex role of impurities, unintentionally or intentionally added, during nanoscale synthesis and their effects on the performance and usefulness of the most common classes of nanomaterials: nanocarbons, noble metal and metal oxide nanoparticles, semiconductor quantum dots, thermoelectrics, and perovskites.</p>","PeriodicalId":29799,"journal":{"name":"ACS Nanoscience Au","volume":"4 3","pages":"176–193"},"PeriodicalIF":0.0,"publicationDate":"2024-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsnanoscienceau.3c00056","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140592711","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":"Enhancing the Therapeutic Efficacy of GLP-1 for Hyperglycemia Treatment: Overcoming Barriers of Oral Gene Therapy with Taurocholic Acid-Conjugated Protamine Sulfate and Calcium Phosphate","authors":"S. M. Shatil Shahriar,&nbsp;Jeong Man An,&nbsp;Sachin S. Surwase,&nbsp;Dong Yun Lee and Yong-kyu Lee*,&nbsp;","doi":"10.1021/acsnanoscienceau.3c00035","DOIUrl":"10.1021/acsnanoscienceau.3c00035","url":null,"abstract":"<p >Activating the glucagon-like peptide-1 (GLP-1) receptor by oral nucleic acid delivery would be a promising treatment strategy against hyperglycemia due to its various therapeutic actions. However, GLP-1 receptor agonists are effective only in subcutaneous injections because they face multiple barriers due to harsh gastrointestinal tract (GIT) conditions before reaching the site of action. The apical sodium bile acid transporter (ASBT) pathway at the intestinal site could be an attractive target to overcome the problem. Herein, we used our previously established multimodal carrier system utilizing bile salt, protamine sulfate, and calcium phosphate as excipients (PTCA) and the GLP-1 gene as an active ingredient (GENE) to test the effects of different formulation doses against diabetes and obesity. The carrier system demonstrated the ability to protect the GLP-1 model gene encoded within the plasmid at the GIT and transport it <i>via</i> ASBT at the target site. A single oral dose, regardless of quantity, showed the generation of GLP-1 and insulin from the body and maintained the normoglycemic condition by improving insulin sensitivity and blood sugar tolerance for a prolonged period. This oral gene therapy approach shows significantly higher therapeutic efficacy in preclinical studies than currently available US Food and Drug Administration-approved GLP-1 receptor agonists such as semaglutide and liraglutide. Also, a single oral dose of GENE/PTCA is more effective than 20 insulin injections. Our study suggests that oral GENE/PTCA formulation could be a promising alternative to injection-based therapeutics for diabetics, which is effective in long-term treatment and has been found to be highly safe in all aspects of toxicology.</p>","PeriodicalId":29799,"journal":{"name":"ACS Nanoscience Au","volume":"4 3","pages":"194–204"},"PeriodicalIF":0.0,"publicationDate":"2024-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsnanoscienceau.3c00035","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140592710","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":"Single-Molecule Investigation of the Binding Interface Stability of SARS-CoV-2 Variants with ACE2","authors":"Ankita Ray,&nbsp;Thu Thi Minh Tran,&nbsp;Rita dos Santos Natividade,&nbsp;Rodrigo A. Moreira,&nbsp;Joshua D. Simpson,&nbsp;Danahe Mohammed,&nbsp;Melanie Koehler,&nbsp;Simon J. L Petitjean,&nbsp;Qingrong Zhang,&nbsp;Fabrice Bureau,&nbsp;Laurent Gillet,&nbsp;Adolfo B. Poma* and David Alsteens*,&nbsp;","doi":"10.1021/acsnanoscienceau.3c00060","DOIUrl":"10.1021/acsnanoscienceau.3c00060","url":null,"abstract":"<p >The SARS-CoV-2 pandemic spurred numerous research endeavors to comprehend the virus and mitigate its global severity. Understanding the binding interface between the virus and human receptors is pivotal to these efforts and paramount to curbing infection and transmission. Here we employ atomic force microscopy and steered molecular dynamics simulation to explore SARS-CoV-2 receptor binding domain (RBD) variants and angiotensin-converting enzyme 2 (ACE2), examining the impact of mutations at key residues upon binding affinity. Our results show that the Omicron and Delta variants possess strengthened binding affinity in comparison to the Mu variant. Further, using sera from individuals either vaccinated or with acquired immunity following Delta strain infection, we assess the impact of immunity upon variant RBD/ACE2 complex formation. Single-molecule force spectroscopy analysis suggests that vaccination before infection may provide stronger protection across variants. These results underscore the need to monitor antigenic changes in order to continue developing innovative and effective SARS-CoV-2 abrogation strategies.</p>","PeriodicalId":29799,"journal":{"name":"ACS Nanoscience Au","volume":"4 2","pages":"136–145"},"PeriodicalIF":0.0,"publicationDate":"2024-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acsnanoscienceau.3c00060","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140074872","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}