Methods最新文献

{"title":"Chitosan-based xerogel film incorporating Nystatin: Synthesis, structural Analysis, and biological evaluation","authors":"Zahra Zareshahrabadi ,&nbsp;Sara Shenavari ,&nbsp;Forough Karami ,&nbsp;Mohammad Hashem Hashempur ,&nbsp;Mohammad Khorram ,&nbsp;Ali Arabimonfard ,&nbsp;Mahboobeh Jafari ,&nbsp;Ali Mohammad Tamaddon ,&nbsp;Gholamhossein Yousefi ,&nbsp;Kamiar Zomorodian","doi":"10.1016/j.ymeth.2025.02.011","DOIUrl":"10.1016/j.ymeth.2025.02.011","url":null,"abstract":"<div><div>Wound infections are challenging to manage, requiring innovative wound dressing systems with prescribed properties. Active wound dressings should provide a moist environment, protect from secondary infections, and remove wound exudate to accelerate tissue regeneration. Hydrogels are encouraging matrices for bioactive compound encapsulation in pharmaceutical applications. The aim of the present study is to design chitosan/gelatin /polyvinyl alcohol-based xerogel film containing nystatin for wound dressing applications with antifungal properties. The xerogel film is developed using a film-casting method and evaluated for its chemical and physical characteristics using FTIR, SEM, AFM, and tensile analysis. Water barrier properties of the film, such as the moisture content (17.84 ± 3.62 %), water solubility (44.50 ± 5.55 %), gel fraction (55.50 ± 5.55 %), and water vapour transmission rate (1912.25 ± 248.12 g m⁻² per day), suggest a humid microenvironment suitable for wound. The xerogel film, characterized by its robust mechanical strength, substantial swelling capacity (∼120–400 %) across various pH levels, and acceptable bio-adhesive properties, reveals as a potential antifungal wound dressing material. <em>In vitro</em> toxicity assessments confirm its biocompatibility towards both RBCs and NIH-3 T3 fibroblast cells. The findings confirm that the film formulation has strong antifungal properties, with a minimum inhibitory concentration of 2–8 μL/mL against <em>Candida</em> species, as well as outstanding antibiofilm effectiveness (∼85 %) and a significant reduction of the fungal colony count (∼100 %). Moreover, its controlled drug release capabilities, along with antifungal properties, offer it as an appealing dressing for the localized treatment of superficial fungal infections. As a result, this xerogel film can be used as a versatile platform for advanced wound care and therapeutic applications.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"237 ","pages":"Pages 19-33"},"PeriodicalIF":4.2,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143530820","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Computational detection, characterization, and clustering of microglial cells in a mouse model of fat-induced postprandial hypothalamic inflammation","authors":"Clara Sanchez ,&nbsp;Morgane Nadal ,&nbsp;Céline Cansell ,&nbsp;Sarah Laroui ,&nbsp;Xavier Descombes ,&nbsp;Carole Rovère ,&nbsp;Éric Debreuve","doi":"10.1016/j.ymeth.2025.02.008","DOIUrl":"10.1016/j.ymeth.2025.02.008","url":null,"abstract":"<div><div>Obesity is associated with brain inflammation, glial reactivity, and immune cells infiltration. Studies in rodents have shown that glial reactivity occurs within 24 h of high-fat diet (HFD) consumption, long before obesity development, and takes place mainly in the hypothalamus (HT), a crucial brain structure for controlling body weight. Understanding more precisely the kinetics of glial activation of two major brain cells (astrocytes and microglia) and their impact on eating behavior could prevent obesity and offer new prospects for therapeutic treatments. To understand the mechanisms pertaining to obesity-related neuroinflammation, we developed a fully automated algorithm, NutriMorph. Although some algorithms were developed in the past decade to detect and segment cells, they are highly specific, not fully automatic, and do not provide the desired morphological analysis. Our algorithm copes with these issues and performs the analysis of cells images (here, microglia of the hypothalamic arcuate nucleus), and the morphological clustering of these cells through statistical analysis and machine learning. Using the k-Means algorithm, it clusters the microglia of the control condition (healthy mice) and the different states of neuroinflammation induced by high-fat diets (obese mice) into subpopulations. This paper is an extension and re-analysis of a first published paper showing that microglial reactivity can already be seen after few hours of high-fat diet (Cansell et al., 2021 [5]). Thanks to NutriMorph algorithm, we unravel the presence of different hypothalamic microglial subpopulations (based on morphology) subject to proportion changes in response to already few hours of high-fat diet in mice.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"236 ","pages":"Pages 28-38"},"PeriodicalIF":4.2,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143530823","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A novel interpretability framework for enzyme turnover number prediction boosted by pre-trained enzyme embeddings and adaptive gate network","authors":"Bing-Xue Du ,&nbsp;Haoyang Yu ,&nbsp;Bei Zhu ,&nbsp;Yahui Long ,&nbsp;Min Wu ,&nbsp;Jian-Yu Shi","doi":"10.1016/j.ymeth.2025.02.010","DOIUrl":"10.1016/j.ymeth.2025.02.010","url":null,"abstract":"<div><div>It is a vital step to identify the enzyme turnover number (kcat) in synthetic biology and early-stage drug discovery. Recently, deep learning methods have achieved inspiring process to predict kcat with the development of multi-species enzyme-substrate pairs turnover number data. However, the performance of existing approaches still heavily depends on the effectiveness of feature extraction for enzymes and substrates, as well as the optimal fusion of these two types of features. Furthermore, it is essential to identify the key molecular substructures that significantly impact kcat prediction. To address these issues, we develop a novel end-to-end dual-representation interpretability framework GELKcat by harnessing graph transformers for substrate molecular encoding and CNNs for enzyme word2vec embeddings. We further integrate substrate and enzyme features using the adaptive gate network, which assigns optimal weights to capture the most suitable feature combinations. The comparison with several state-of-the-art methods demonstrates the superiority of our GELKcat and the ablation studies further illuminate the invaluable roles of three main components. Furthermore, case studies illustrate the interpretability of GELKcat by identifying the key functional groups in a substrate, which are significantly associated with turnover number. It is anticipated that this work can bridge current gaps in enzyme-substrate representation, which can give some guidance for drug discovery and synthetic biology.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"237 ","pages":"Pages 45-52"},"PeriodicalIF":4.2,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143531164","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cardiac titin isoforms: Practice in interpreting results of electrophoretic analysis","authors":"Elmira I. Yakupova ,&nbsp;Polina A. Abramicheva ,&nbsp;Vadim V. Rogachevsky ,&nbsp;Elena A. Shishkova ,&nbsp;Alexey D. Bocharnikov ,&nbsp;Egor Y. Plotnikov ,&nbsp;Ivan M. Vikhlyantsev","doi":"10.1016/j.ymeth.2025.02.007","DOIUrl":"10.1016/j.ymeth.2025.02.007","url":null,"abstract":"<div><div>The sarcomeric giant protein titin affects the passive elasticity of the heart muscle and is crucial for proper cardiac function, including diastolic relaxation of the left ventricle. A useful common method for studying titin is electrophoretic analysis which can be used to examine the distribution of its isoforms in the heart. There are 5 titin parameters that can be analyzed: the N2BA/N2B isoforms ratio, the T2/T1 bands ratio, Cronos isoform content, NT isoform content, the total titin-to-myosin heavy chain (TT/MHC) ratio. These parameters can only be assessed through electrophoresis of giant proteins. It is known that these parameters are related to various biomolecular processes in muscle cells, such as providing of elastic properties, turnover, contraction, and maintaining a highly ordered sarcomere structure. In this review, we discuss the diagnostic potential of electrophoretic visualization of cardiac titin changes in various human heart diseases and animal models of physiological adaptations or pathologies.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"236 ","pages":"Pages 17-25"},"PeriodicalIF":4.2,"publicationDate":"2025-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143490308","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A free method for patient-specific 3D-VR anatomical modeling for presurgical planning using DICOM images and open-source software","authors":"Zachary Ells ,&nbsp;Vinicius Ludwig ,&nbsp;Adam B. Weiner ,&nbsp;Koichiro Kimura ,&nbsp;Andrea Farolfi ,&nbsp;Karim Chamie ,&nbsp;Joseph Shirk ,&nbsp;Nicholas M. Donin ,&nbsp;Robert Reiter ,&nbsp;Johannes Czernin ,&nbsp;Jeremie Calais ,&nbsp;Magnus Dahlbom","doi":"10.1016/j.ymeth.2025.02.006","DOIUrl":"10.1016/j.ymeth.2025.02.006","url":null,"abstract":"<div><h3>Introduction</h3><div>Surgeons commonly use cross sectional images to plan and prepare for surgical procedures. However, cognitively translating 2D images to surgical settings can be difficult and lead to sub-optimal resections. Lymph node dissection can be challenging due to the inability to locate small metastatic lesions, and their proximity to at-risk organ(s). 3D volume rendered (3D-VR) patient specific images can help to address these challenges. We created patient-specific 3D-VR images using freely available open-source programs.</div></div><div><h3>Methods</h3><div>This study included patients part of the clinical trial NCT04857502. Patients received a PET/CT prior to radioguided surgery. 3D Slicer was used to segment anatomy of interest (organs and tumor lesion(s)). After segmentation, the data was exported as an .OBJ file with an accompanying .MTL file. Manipulation of the .MTL file to restore model properties to the .OBJ file, were completed and both files were uploaded into Autodesk Viewer. Surgeons then received an email link to access the finished 3D-VR model on their smartphone or laptop for peri-operative preparation and/or guidance.</div></div><div><h3>Results</h3><div>The method was used in a series of 14 patients with prostate cancer undergoing pelvic lymph node dissection with PSMA-radioguided robotic surgery using pre-operative PSMA PET/CT images acquired on average 103 ± 69 days prior to resection. The creation of the 3D-VR models was successfully conducted in all 14 cases. In all cases, the lesions identified on the pre-operative PET/CT imaging 3D-VR models were successfully removed during surgery.</div></div><div><h3>Conclusion</h3><div>We created patient-specific anatomical 3D-VR models that the surgeons can use for pre-surgical planning and intraoperative tumor localization, by applying free, open-source software that could be used in any procedure requiring careful and strategical planning.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"236 ","pages":"Pages 10-16"},"PeriodicalIF":4.2,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143473927","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"AI and machine learning in bioinformatics and biomedicine","authors":"Haiying Wang (Guest Editor),&nbsp;Xiaohua (Tony) Hu (Guest Editor)","doi":"10.1016/j.ymeth.2025.02.005","DOIUrl":"10.1016/j.ymeth.2025.02.005","url":null,"abstract":"","PeriodicalId":390,"journal":{"name":"Methods","volume":"236 ","pages":"Pages 26-27"},"PeriodicalIF":4.2,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143476048","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Production of AFM wedged cantilevers for stress-relaxation experiments: Uniaxial loading of soft, spherical cells","authors":"Riccardo Campanile ,&nbsp;Jonne Helenius ,&nbsp;Cristina Scielzo ,&nbsp;Lydia Scarfò ,&nbsp;Domenico Salerno ,&nbsp;Mario Bossi ,&nbsp;Marta Falappi ,&nbsp;Alessia Saponara ,&nbsp;Daniel J. Müller ,&nbsp;Francesco Mantegazza ,&nbsp;Valeria Cassina","doi":"10.1016/j.ymeth.2025.02.004","DOIUrl":"10.1016/j.ymeth.2025.02.004","url":null,"abstract":"<div><div>The fabrication of wedge-shaped cantilevers for Atomic Force Microscopy (AFM) remains a critical yet challenging task, particularly when precision and efficiency are required. In this study, we present a streamlined protocol for producing these wedges using NOA63 UV-curing polymer, which simplifies the process and eliminates the need for dedicated equipment. Our method reduces preparation time while maintaining the mechanical properties of the cantilevers, in line with the manufacturer's specifications. We demonstrate the effectiveness of our wedged cantilevers in stress-relaxation experiments performed by means of AFM and confocal microscopy on primary Chronic Lymphocytic Leukemia cells and the MEC1 cell line. These experiments highlight the effectiveness of using modified cantilevers to consistently apply precise uniaxial loading to soft, spherical cells. This technique offers a marked improvement in fabrication speed and operational ease compared to traditional methods, without compromising the accuracy or performance of the measurements. This protocol is not only time-saving, but also adaptable for use in a wide range of biological applications, making it a valuable tool for AFM-based research in cellular mechanics.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"236 ","pages":"Pages 1-9"},"PeriodicalIF":4.2,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143453941","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Latest trends & strategies in ocular drug delivery","authors":"Nishant S. Kulkarni ,&nbsp;Alexander Josowitz ,&nbsp;Roshan James ,&nbsp;Yang Liu ,&nbsp;Bindhu Rayaprolu ,&nbsp;Botir Sagdullaev ,&nbsp;Amardeep S. Bhalla ,&nbsp;Mohammed Shameem","doi":"10.1016/j.ymeth.2025.02.003","DOIUrl":"10.1016/j.ymeth.2025.02.003","url":null,"abstract":"<div><div>Ocular drug delivery is one of the most challenging routes of administration, and this may be attributed to the complex interplay of ocular barriers and clearance mechanisms that restrict therapeutic payload residence. Most of the currently approved products that ameliorate ocular disease conditions are topical, i.e., delivering therapeutics to the outside anterior segment of the eye. This site of administration works well for certain conditions such as local infections but due to the presence of numerous ocular barriers, the permeation of therapeutics to the posterior segment of the eye is limited. Conditions such as age-related macular degeneration and diabetic retinopathy that contribute to an extreme deterioration of vision acuity require therapeutic interventions at the posterior segment of the eye. This necessitates development of intraocular delivery systems such as intravitreal injections, implants, and specialized devices that deliver therapeutics to the posterior segment of the eye. Frequent dosing regimens and high concentration formulations have been strategized and developed to achieve desired therapeutic outcomes by overcoming some of the challenges of drug clearance and efficacy. Correspondingly, development of suitable delivery platforms such as biodegradable and non-biodegradable implants, nano delivery systems, and implantable devices have been explored. This article provides an overview of the current trends in the development of suitable formulations &amp; delivery systems for ocular drug delivery with an emphasis on late-stage clinical and approved product. Moreover, this work aims to summarize current challenges and highlights exciting pre-clinical developments, and future opportunities in cell and gene therapies that may be explored for effective ocular therapeutic outcomes.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"235 ","pages":"Pages 100-117"},"PeriodicalIF":4.2,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143403229","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A high sensitivity assay of UBE3A ubiquitin ligase activity","authors":"Linna Han,&nbsp;Z. Begum Yagci,&nbsp;Albert J. Keung","doi":"10.1016/j.ymeth.2025.02.002","DOIUrl":"10.1016/j.ymeth.2025.02.002","url":null,"abstract":"<div><div>UBE3A is an E3 ubiquitin ligase associated with several neurodevelopmental disorders. The development of several preclinical therapeutic approaches involving UBE3A, such as gene therapy, enzyme replacement therapy, and epigenetic reactivation, require the detection of its ubiquitin ligase activity. Prior commercial assays leveraged Western Blotting to detect shifts in substrate size due to ubiquitination, but these suffered from long assay times and have also been discontinued. Here we develop a new assay that quantifies UBE3A activity. It measures the fluorescence intensity of ubiquitinated p53 substrates with a microplate reader, eliminating the need for Western Blot antibodies and instruments, and enables detection in just 1 h. The assay is fast, cost-effective, low noise, and uses components with long shelf lives. Importantly, it is also highly sensitive, detecting UBE3A levels as low as 1 nM, similar to that observed in human and mouse cerebrospinal fluid. It also differentiates the activity of wild-type UBE3A and catalytic mutants. We also design a p53 substrate with a triple-epitope tag HIS-HA-CMYC on the N terminus, which allows for versatile detection of UBE3A activity from diverse natural and engineered sources. This new assay provides a timely solution for growing needs in preclinical validation, quality control, endpoint measurements for clinical trials, and downstream manufacturing testing and validation.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"235 ","pages":"Pages 92-99"},"PeriodicalIF":4.2,"publicationDate":"2025-02-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143394412","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"HybProm: An attention-assisted hybrid CNN-BiLSTM model for the interpretable prediction of DNA promoter","authors":"Rentao Luo,&nbsp;Jiawei Liu,&nbsp;Lixin Guan,&nbsp;Mengshan Li","doi":"10.1016/j.ymeth.2025.02.001","DOIUrl":"10.1016/j.ymeth.2025.02.001","url":null,"abstract":"<div><div>Promoter prediction is essential for analyzing gene structures, understanding regulatory networks, transcription mechanisms, and precisely controlling gene expression. Recently, computational and deep learning methods for promoter prediction have gained attention. However, there is still room to improve their accuracy. To address this, we propose the HybProm model, which uses DNA2Vec to transform DNA sequences into low-dimensional vectors, followed by a CNN-BiLSTM-Attention architecture to extract features and predict promoters across species, including E. coli, humans, mice, and plants. Experiments show that HybProm consistently achieves high accuracy (90%-99%) and offers good interpretability by identifying key sequence patterns and positions that drive predictions.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"235 ","pages":"Pages 71-80"},"PeriodicalIF":4.2,"publicationDate":"2025-02-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143377724","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}